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  1. Up-regulated BAFF and BAFF receptor expression in patients with intractable temporal lobe epilepsy and a pilocarpine-induced epilepsy rat model.

    Science.gov (United States)

    Ma, Limin; Li, Ruohan; Huang, Hao; Yuan, Jinxian; Ou, Shu; Xu, Tao; Yu, Xinyuan; Liu, Xi; Chen, Yangmei

    2017-05-01

    Some studies have suggested that BAFF and BAFFR are highly expressed in the central nervous system (CNS) and participate in inflammatory and immune associated diseases. However, whether BAFF and BAFFR are involved in the pathogenesis of epilepsy remains unknown. This study aimed to investigate the expression of BAFF and BAFFR proteins in the brains of patients with temporal lobe epilepsy (TLE) and in a pilocarpine-induced rat model of TLE to identify possible roles of the BAFF-BAFFR signaling pathway in epileptogenesis. Real-time quantitative polymerase chain reaction (RT-qPCR), western blot, immunohistochemistry, and double-immunofluorescence were performed in this study. The results showed that BAFF and BAFFR expression levels were markedly up-regulated in intractable TLE patients and TLE rats. Moreover, BAFF and BAFFR proteins mainly highly expressed in the membranes and cytoplasms of the dendritic marker MAP2 in the cortex and hippocampus. Therefore, the significant increased in BAFF and BAFFR protein expression in both TLE patients and rats suggest that BAFF and BAFFR may play important roles in regulating the pathogenesis of epilepsy. Copyright © 2017 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

  2. Characterisation and expression analysis of B-cell activating factor (BAFF) in spiny dogfish (Squalus acanthias): cartilaginous fish BAFF has a unique extra exon that may impact receptor binding.

    Science.gov (United States)

    Li, Ronggai; Dooley, Helen; Wang, Tiehui; Secombes, Christopher J; Bird, Steve

    2012-04-01

    B-cell activating factor (BAFF), also known as tumour necrosis factor (TNF) ligand superfamily member 13B, is an important immune regulator with critical roles in B-cell survival, proliferation, differentiation and immunoglobulin secretion. A BAFF gene has been cloned from spiny dogfish (Squalus acanthias) and its expression studied. The dogfish BAFF encodes for an anchored type-II transmembrane protein of 288 aa with a putative furin protease cleavage site and TNF family signature as seen in BAFFs from other species. The identity of dogfish BAFF has also been confirmed by conserved cysteine residues, and phylogenetic tree analysis. The dogfish BAFF gene has an extra exon not seen in teleost fish, birds and mammals that encodes for 29 aa and may impact on receptor binding. The dogfish BAFF is highly expressed in immune tissues, such as spleen, and is up-regulated by PWM in peripheral blood leucocytes, suggesting a potentially important role in the immune system. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Transgenic overexpression of BAFF regulates the expression of ...

    Indian Academy of Sciences (India)

    To investigate whether transgenic overexpression of the zebrafish BAFF leads to ... and BAFF proteins were expressed separately and confirmed in HeLa cells. ... body homogenate of zebrafish and demonstrated a significant increase in ...

  4. Molecular structure, bioinformatics analysis, expression and bioactivity of BAFF (TNF13B) in dog (Canis familiaris).

    Science.gov (United States)

    Shen, Yuefen; Wang, Shule; Ai, Hongxin; Min, Cui; Chen, Yuqing; Zhang, Shuangquan

    2011-07-15

    B cell activating factor (BAFF), belonging to the tumor necrosis factor (TNF) family, plays an important role in B cell survival, proliferation and differentiation. In the present study, we amplified the cDNA of dog (Canis familiaris) BAFF (designated doBAFF) from spleen by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) strategies. The open reading frame (ORF) of doBAFF covers 879 bp encoding 292 amino acids, with a 152-aa mature peptide. The soluble mature part of doBAFF (dosBAFF) shares 91.5%, 72.1%, 96.7%, 94.0% and 91.5% identity with the human, mouse, cattle, pig and rabbit counterparts, respectively. The predicted three-dimensional (3D) structural analysis of dosBAFF analyzed by "comparative protein modeling" revealed that it was very similar to its human counterpart. Real-time quantitative PCR analysis revealed that doBAFF was mainly expressed in spleen. Two fusion proteins SUMO-dosBAFF and GFP/dosBAFF were efficiently expressed in Escherichia coli BL21 (DE3) and purified using metal chelate affinity chromatography (Ni-NTA). Laser scanning confocal microscopy analysis showed that GFP/dosBAFF could bind to the mouse splenic B-cell. In vitro, SUMO-dosBAFF and GFP/dosBAFF were able to promote the survival/proliferation of dog lymphocytes or mouse splenic B cells with/without anti-IgM. Therefore, BAFF may be a potential immunologic factor for enhancing immunological efficacy in dog. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents

    Directory of Open Access Journals (Sweden)

    Feng Zhang

    2017-12-01

    Full Text Available Paeoniflorin-6′-O-benzene sulfonate (code: CP-25 was the chemistry structural modifications of Paeoniflorin (Pae. CP-25 inhibited B cells proliferation stimulated by B cell activating factor belonging to the TNF family (BAFF or Tumor necrosis factor alpha (TNF-alpha. CP-25, Rituximab and Etanercept reduced the percentage and numbers of CD19+ B cells, CD19+CD20+ B cells, CD19+CD27+ B cells and CD19+CD20+CD27+ B cells induced by BAFF or TNF-alpha. There was significant difference between CP-25 and Rituximab or CP-25 and Etanercept. CP-25 down-regulated the high expression of BAFFR, BCMA, and TACI stimulated by BAFF or TNF-alpha. The effects of Rituximab and Etanercept on BAFFR or BCMA were stronger than that of CP-25. CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38, P-p65, TRAF2, and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha. These results suggest that CP-25 regulated moderately activated B cells function by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.

  6. CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents.

    Science.gov (United States)

    Zhang, Feng; Shu, Jin-Ling; Li, Ying; Wu, Yu-Jing; Zhang, Xian-Zheng; Han, Le; Tang, Xiao-Yu; Wang, Chen; Wang, Qing-Tong; Chen, Jing-Yu; Chang, Yan; Wu, Hua-Xun; Zhang, Ling-Ling; Wei, Wei

    2017-01-01

    Paeoniflorin-6'- O -benzene sulfonate (code: CP-25) was the chemistry structural modifications of Paeoniflorin (Pae). CP-25 inhibited B cells proliferation stimulated by B cell activating factor belonging to the TNF family (BAFF) or Tumor necrosis factor alpha (TNF-alpha). CP-25, Rituximab and Etanercept reduced the percentage and numbers of CD19 + B cells, CD19 + CD20 + B cells, CD19 + CD27 + B cells and CD19 + CD20 + CD27 + B cells induced by BAFF or TNF-alpha. There was significant difference between CP-25 and Rituximab or CP-25 and Etanercept. CP-25 down-regulated the high expression of BAFFR, BCMA, and TACI stimulated by BAFF or TNF-alpha. The effects of Rituximab and Etanercept on BAFFR or BCMA were stronger than that of CP-25. CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38, P-p65, TRAF2, and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha. These results suggest that CP-25 regulated moderately activated B cells function by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.

  7. Transgenic overexpression of BAFF regulates the expression of ...

    Indian Academy of Sciences (India)

    LI ZHANG

    Hubei 430030, People's Republic of China. 3Second ... body homogenate of zebrafish and demonstrated a significant increase in BAFF-transgenic group. Therefore, our ... diet and brine shrimp according to the conditions in our sys- tem (Li et al. 2014). ... images were captured using a Leica DM3000B microscope. Embryos ...

  8. BAFF induces spleen CD4+ T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

    International Nuclear Information System (INIS)

    Ji, Fang; Chen, Rongjing; Liu, Baojun; Zhang, Xiaoping; Han, Junli; Wang, Haining; Shen, Gang; Tao, Jiang

    2012-01-01

    Highlights: ► Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4 + T cells. ► Carrying out siRNA technology to study FOXO3A protein function. ► Helpful to understand the T cell especially CD4 + T cell‘s role in immunological reaction. -- Abstract: The TNF ligand family member “B cell-activating factor belonging to the TNF family” (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4 + spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4 + T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4 + spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4 + T cell proliferation.

  9. Curcumin protects against collagen-induced arthritis via suppression of BAFF production.

    Science.gov (United States)

    Huang, Gang; Xu, Zhizhen; Huang, Yan; Duan, Xiaojun; Gong, Wei; Zhang, Yan; Fan, Jishan; He, Fengtian

    2013-04-01

    The aim of the present study was to evaluate whether the anti-Rheumatoid arthritis (RA) effect of curcumin is associated with the regulation of B cell-activating factor belonging to the TNF family (BAFF) production. Collagen-induced arthritis (CIA) was induced in DBA/1 J mice by immunization with bovine type II collagen. To investigate the anti-arthritic effect of curcumin in the CIA model, mice were injected intraperitoneally with curcumin (50 mg/kg) on every other day either from day 1 or from day 28 after the first immunization. The clinical severity of arthritis was monitored. BAFF, interleukin-6 (IL-6) and interferon-γ (IFNγ) production in serum were measured. Furthermore, the effect of curcumin on IFNγ-induced BAFF expression and transcriptional activation in B lymphocytes was determined by qPCR, Western Blot, and luciferase assay. Finally, IFNγ related signal transducers and activators of transcription 1 (STAT1) signaling in B lymphocytes were studied using Western Blot. Curcumin dramatically attenuated the progression and severity of CIA in DBA/1 J mice, accompanied with decrease of BAFF production in serum and spleen cells as well as decrease of serum IFNγ and IL-6. Treatment of B lymphocytes with curcumin suppressed IFNγ-induced BAFF expression, STAT1 phosphorylation and nuclear translocation, suggesting that curcumin may repress IFNγ-induced BAFF expression via negatively interfering with STAT1 signaling. The results of the present study suggest that suppression of BAFF production may be a novel mechanism by which curcumin improves RA.

  10. BAFF induces spleen CD4{sup +} T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Fang; Chen, Rongjing [Department of Orthodontics, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Liu, Baojun [Laboratory of Lung, Inflammation and Cancers, Huashan Hospital, Fudan University, Shanghai (China); Zhang, Xiaoping [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Han, Junli; Wang, Haining [Department of General Dentistry, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Shen, Gang [Department of Orthodontics, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Tao, Jiang, E-mail: taojiang2012@yahoo.cn [Department of General Dentistry, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4{sup +} T cells. Black-Right-Pointing-Pointer Carrying out siRNA technology to study FOXO3A protein function. Black-Right-Pointing-Pointer Helpful to understand the T cell especially CD4{sup +} T cell's role in immunological reaction. -- Abstract: The TNF ligand family member 'B cell-activating factor belonging to the TNF family' (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4{sup +} spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4{sup +} T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4{sup +} spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4{sup +} T cell proliferation.

  11. Molecular and functional characterization of BAFF from the Yangtze alligator (Alligator sinensis, Alligatoridae).

    Science.gov (United States)

    Zhang, Jia-Xin; Song, Ren; Sang, Ming; Sun, Si-Qing; Ma, Lei; Zhang, Jie; Zhang, Shuang-Quan

    2015-10-01

    B-cell activating factor (BAFF) from the TNF family is critical for B-cell survival and maturation. In this study, we identified a Yangtze alligator (Alligator sinensis, Alligatoridae) BAFF cDNA, designated as asBAFF, using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The open reading frame of this cDNA encodes a 287-amino acid protein containing a predicted transmembrane domain and a furin protease cleavage site, similar to mammalian and avian BAFF. The amino acid identity between biologically soluble asBAFF (assBAFF) and csBAFF, hsBAFF, and msBAFF is 94, 76, and 71%, respectively. Real-time quantitative PCR analysis showed that the asBAFF gene is strongly expressed in the spleen. Since BAFF is always expressed as inclusion bodies in bacteria, it is difficult to purify. To enhance the soluble expression of assBAFF in Escherichia coli, we fused the extracellular region of the asBAFF gene to a small ubiquitin-related modifier gene (SUMO). Purified assBAFF was able to promote the survival of splenic lymphocytes and co-stimulate the proliferation of mouse B cells with anti-mouse IgM. These findings suggest that asBAFF plays an important role in the survival and proliferation of Yangtze alligator B cells, and because it is evolutionarily highly conserved, functional cross-reactivity exists between mammalian and Yangtze alligator BAFF. Copyright © 2015 Elsevier GmbH. All rights reserved.

  12. BAFF promotes regulatory T-cell apoptosis and blocks cytokine production by activating B cells in primary biliary cirrhosis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bo; Hu, Mintao [Department of Hepatology, Wuxi Infectious Diseases Hospital, Wuxi, Jiangsu (China); Zhang, Peng [Nanjing Medical University, Nanjing, Jiangsu (China); Cao, Hong [Department of Hepatology, Wuxi Infectious Diseases Hospital, Wuxi, Jiangsu (China); Wang, Yongzhen [The Second Hospital of Nanjing, Nanjing, Jiangsu (China); Wang, Zheng; Su, Tingting [Department of Hepatology, Wuxi Infectious Diseases Hospital, Wuxi, Jiangsu (China)

    2013-05-10

    Primary biliary cirrhosis (PBC) is a chronic and slowly progressive cholestatic liver disease of autoimmune etiology. A number of questions regarding its etiology are unclear. CD4+CD25+ regulatory T cells (Tregs) play a critical role in self-tolerance and, for unknown reasons, their relative number is reduced in PBC patients. B-cell-activating factor (BAFF) is a key survival factor during B-cell maturation and its concentration is increased in peripheral blood of PBC patients. It has been reported that activated B cells inhibit Treg cell proliferation and there are no BAFF receptors on Tregs. Therefore, we speculated that excessive BAFF may result in Treg reduction via B cells. To prove our hypothesis, we isolated Tregs and B cells from PBC and healthy donors. BAFF and IgM concentrations were then analyzed by ELISA and CD40, CD80, CD86, IL-10, and TGF-β expression in B cells and Tregs were measured by flow cytometry. BAFF up-regulated CD40, CD80, CD86, and IgM expression in B cells. However, BAFF had no direct effect on Treg cell apoptosis and cytokine secretion. Nonetheless, we observed that BAFF-activated B cells could induce Treg cell apoptosis and reduce IL-10 and TGF-β expression. We also showed that BAFF-activated CD4+ T cells had no effect on Treg apoptosis. Furthermore, we verified that bezafibrate, a hypolipidemic drug, can inhibit BAFF-induced Treg cell apoptosis. In conclusion, BAFF promotes Treg cell apoptosis and inhibits cytokine production by activating B cells in PBC patients. The results of this study suggest that inhibition of BAFF activation is a strategy for PBC treatment.

  13. Expression of BAFF and BR3 in patients with systemic lupus erythematosus

    Directory of Open Access Journals (Sweden)

    J.H. Duan

    2016-03-01

    Full Text Available The objective of this study was to examine the relationship between the expression of B cell activating factor (BAFF and BAFF receptor in patients with disease activity of systemic lupus erythematosus (SLE. Real-time RT-PCR was used to examine BAFF mRNA expression in peripheral blood monocytes of active and stable SLE patients and healthy controls. The percentage of BAFF receptor 3 (BR3 on B lymphocytes was measured by flow cytometry. Soluble BAFF levels in serum were assayed by ELISA. Microalbumin levels were assayed by an automatic immune analysis machine. BAFF mRNA and soluble BAFF levels were highest in the active SLE group, followed by the stable SLE group, and controls (P<0.01. The percentage of BR3 on B lymphocytes was downregulated in the active SLE group compared with the stable SLE group and controls (P<0.01. BAFF mRNA levels and soluble BAFF levels were higher in patients who were positive for proteinuria than in those who were negative (P<0.01. The percentage of BR3 on B lymphocytes was lower in patients who were positive for proteinuria than in those who were negative (P<0.01. The BAFF/BR3 axis may be over-activated in SLE patients. BAFF and BR3 levels may be useful parameters for evaluating treatment.

  14. The BAFF/APRIL system: emerging functions beyond B cell biology and autoimmunity.

    Science.gov (United States)

    Vincent, Fabien B; Saulep-Easton, Damien; Figgett, William A; Fairfax, Kirsten A; Mackay, Fabienne

    2013-06-01

    The BAFF system plays a key role in the development of autoimmunity, especially in systemic lupus erythematosus (SLE). This often leads to the assumption that BAFF is mostly a B cell factor with a specific role in autoimmunity. Focus on BAFF and autoimmunity, driven by pharmaceutical successes with the recent approval of a novel targeted therapy Belimumab, has relegated other potential roles of BAFF to the background. Far from being SLE-specific, the BAFF system has a much broader relevance in infection, cancer and allergy. In this review, we provide the latest views on additional roles of the BAFF system in health and diseases, as well as an update on BAFF and autoimmunity, with particular focus on current clinical trials. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Overexpression of the Cytokine BAFF and Autoimmunity Risk.

    Science.gov (United States)

    Steri, Maristella; Orrù, Valeria; Idda, M Laura; Pitzalis, Maristella; Pala, Mauro; Zara, Ilenia; Sidore, Carlo; Faà, Valeria; Floris, Matteo; Deiana, Manila; Asunis, Isadora; Porcu, Eleonora; Mulas, Antonella; Piras, Maria G; Lobina, Monia; Lai, Sandra; Marongiu, Mara; Serra, Valentina; Marongiu, Michele; Sole, Gabriella; Busonero, Fabio; Maschio, Andrea; Cusano, Roberto; Cuccuru, Gianmauro; Deidda, Francesca; Poddie, Fausto; Farina, Gabriele; Dei, Mariano; Virdis, Francesca; Olla, Stefania; Satta, Maria A; Pani, Mario; Delitala, Alessandro; Cocco, Eleonora; Frau, Jessica; Coghe, Giancarlo; Lorefice, Lorena; Fenu, Giuseppe; Ferrigno, Paola; Ban, Maria; Barizzone, Nadia; Leone, Maurizio; Guerini, Franca R; Piga, Matteo; Firinu, Davide; Kockum, Ingrid; Lima Bomfim, Izaura; Olsson, Tomas; Alfredsson, Lars; Suarez, Ana; Carreira, Patricia E; Castillo-Palma, Maria J; Marcus, Joseph H; Congia, Mauro; Angius, Andrea; Melis, Maurizio; Gonzalez, Antonio; Alarcón Riquelme, Marta E; da Silva, Berta M; Marchini, Maurizio; Danieli, Maria G; Del Giacco, Stefano; Mathieu, Alessandro; Pani, Antonello; Montgomery, Stephen B; Rosati, Giulio; Hillert, Jan; Sawcer, Stephen; D'Alfonso, Sandra; Todd, John A; Novembre, John; Abecasis, Gonçalo R; Whalen, Michael B; Marrosu, Maria G; Meloni, Alessandra; Sanna, Serena; Gorospe, Myriam; Schlessinger, David; Fiorillo, Edoardo; Zoledziewska, Magdalena; Cucca, Francesco

    2017-04-27

    Genomewide association studies of autoimmune diseases have mapped hundreds of susceptibility regions in the genome. However, only for a few association signals has the causal gene been identified, and for even fewer have the causal variant and underlying mechanism been defined. Coincident associations of DNA variants affecting both the risk of autoimmune disease and quantitative immune variables provide an informative route to explore disease mechanisms and drug-targetable pathways. Using case-control samples from Sardinia, Italy, we performed a genomewide association study in multiple sclerosis followed by TNFSF13B locus-specific association testing in systemic lupus erythematosus (SLE). Extensive phenotyping of quantitative immune variables, sequence-based fine mapping, cross-population and cross-phenotype analyses, and gene-expression studies were used to identify the causal variant and elucidate its mechanism of action. Signatures of positive selection were also investigated. A variant in TNFSF13B, encoding the cytokine and drug target B-cell activating factor (BAFF), was associated with multiple sclerosis as well as SLE. The disease-risk allele was also associated with up-regulated humoral immunity through increased levels of soluble BAFF, B lymphocytes, and immunoglobulins. The causal variant was identified: an insertion-deletion variant, GCTGT→A (in which A is the risk allele), yielded a shorter transcript that escaped microRNA inhibition and increased production of soluble BAFF, which in turn up-regulated humoral immunity. Population genetic signatures indicated that this autoimmunity variant has been evolutionarily advantageous, most likely by augmenting resistance to malaria. A TNFSF13B variant was associated with multiple sclerosis and SLE, and its effects were clarified at the population, cellular, and molecular levels. (Funded by the Italian Foundation for Multiple Sclerosis and others.).

  16. BAFF/APRIL system in pediatric OMS: relation to severity, neuroinflammation, and immunotherapy.

    Science.gov (United States)

    Pranzatelli, Michael R; Tate, Elizabeth D; McGee, Nathan R; Travelstead, Anna L; Colliver, Jerry A; Ness, Jayne M; Ransohoff, Richard M

    2013-01-16

    B-cell dysregulation has been implicated but not fully characterized in pediatric opsoclonus-myoclonus syndrome (OMS), a neuroblastoma-associated neuroinflammatory disorder. To assess the role of B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), two critical B cell-modulating cytokines, as potential biomarkers of disease activity and treatment biomarkers in OMS. Soluble BAFF and APRIL were measured in cerebrospinal fluid (CSF) and serum by ELISA in 433 children (296 OMS, 109 controls, 28 other inflammatory neurological disorders (OIND)). BAFF-R receptors on circulating CD19+ B cells were measured by flow cytometry. A blinded scorer rated motor severity on the OMS Evaluation Scale. Immunotherapies were evaluated cross-sectionally and longitudinally. The mean CSF BAFF concentration, which was elevated in untreated OMS and OIND, correlated with OMS severity category (P = 0.006), and reduction by adrenocorticotropic hormone or corticotropin (ACTH) (-61%) or corticosteroids (-38%) was seen at each level of severity. In contrast, CSF APRIL was normal in OMS and OIND and unaffected by immunotherapy. When the entire OMS dataset was dichotomized into 'high' versus 'normal' CSF BAFF concentration, the phenotype of the high group included greater motor severity and number of CSF oligoclonal bands, and a higher concentration of inflammatory chemokines CXCL13 and CXCL10 in CSF and CXCL9 and CCL21 in serum. Serum APRIL was 6.7-fold higher in the intravenous immunoglobulins (IVIg) group, whereas serum BAFF was 2.6-fold higher in the rituximab group. The frequency of B cell BAFF-R expression was similar in untreated and treated OMS. Longitudinal studies of CSF BAFF revealed a significant decline in ACTH-treated patients (with or without rituximab) (P OMS displayed heterogeneity in CSF BAFF expression, which met many but not all criteria as a potential biomarker of disease activity. We speculate that CSF BAFF may have more utility in a biomarker panel

  17. CLINICAL VALUE OF BAFF AND APRIL CONCENTRATIONS IN SYSTEMIC LUPUS ERYTHEMATOSUS

    Directory of Open Access Journals (Sweden)

    T. A. Panafidina

    2016-01-01

    Full Text Available Systemic lupus erythematosus (SLE is characterized by the pathological activation and differentiation of B lymphocytes. The B-lymphocyte stimulator (BLyS, also known as B cell-activating factor of the tumor necrosis factor family (BAFF, and its homologue, a proliferation-inducing ligand (APRIL, belong to the ligands of the tumor necrosis factor (TNF family and play a key role in B-lymphocyte selection and survival.Objective: to determine serum BAFF and APRIL concentrations in patients with SLE and a relationship of the clinical and laboratory parameters of the disease to the level of these cytokines.Subjects and methods. The investigation enrolled 73 patients (62 women and 11 men; median age, 30.0 [28.0; 46.0] years with SLE (disease duration, 5.0 [1.5; 11.0] years and its high activity (the median SLEDAI-2K scores of 8 [2; 13].Involvement of the kidneys and joints were found in 40 and 36% of cases, respectively; there were hematologic disorders in 38%, antinuclear factor (ANF in 94.5%, and anti-double stranded DNA antibodies in 77%. The concurrent antiphospholipid syndrome was detected in 15% of the patients. 66% of the patients took glucocorticoids (GCs (the median dose was 10 [0; 15] mg/day, calculated with reference to prednisolone, 42% received cytotoxic drugs (cyclophosphamide, mycophenolate mofetil, azathioprine; 12% used biological agents (BAs; 31.5% received no therapy at enrolment in the investigation. Serum BAFF and APRIL concentrations were estimated using an enzyme immunoassay.Results and discussion. The concentrations of BAFF and APRIL did not differ essentially in the patients with SLE and in the controls: the median level of BAFF was 0.02 [0.01; 0.64] and 0.02 [0.01; 0.03] ng/ml; that of APRIL was 2.09 [0.01; 3.80] and 0.01 [0.01; 4.16] ng/ml, respectively. The elevated concentration of BAFF (>0.82 ng/ml was revealed in 5.5% of the patients with SLE and that of APRIL (>5.96 ng/ml in 4.1%. There was a positive correlation between

  18. A benzenediamine derivate FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting myeloid dendritic cell-secreted BAFF.

    Science.gov (United States)

    Ji, Jianjian; Xu, Jingjing; Li, Fanlin; Li, Xiaojing; Gong, Wei; Song, Yuxian; Dou, Huan; Hou, Yayi

    2016-05-01

    Myeloid dendritic cells (DCs) can produce B-cell-activating factor (BAFF) that modulates survival and differentiation of B cells and plays a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). Toll-like receptor 4 (TLR4) signaling has important functions in the process of BAFF production. Our previous study showed that a benzenediamine derivate FC-99 possesses anti-inflammation activity and directly interacts with interleukin-1 receptor-associated kinase 4 (IRAK4), which was a pivotal molecule in TLR4 signaling. In this study, we demonstrated that FC-99 attenuated lupus nephritis in the MRL/lpr mice. FC-99 also decreased the levels of total immunoglobulin G (IgG), total IgG2a and IgM in sera, as well as the activation of B cells in the spleens of MRL/lpr mice. Moreover, FC-99 inhibited abnormal activation of myeloid DCs in spleens and reduced the levels of BAFF in sera, spleens, and kidneys of MRL/lpr mice. Furthermore, upon TLR4 stimulation with lipopolysaccharide in vitro, FC-99 inhibited IRAK4 phosphorylation, as well as the activation and BAFF production in murine bone marrow-derived DCs. These data indicate that FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting DC-secreted BAFF, suggesting that FC-99 may be a potential therapeutic candidate for the treatment of SLE. © The Author 2016. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

  19. Elevated serum BAFF levels in patients with localized scleroderma in contrast to other organ-specific autoimmune diseases.

    Science.gov (United States)

    Matsushita, Takashi; Hasegawa, Minoru; Matsushita, Yukiyo; Echigo, Takeshi; Wayaku, Takamasa; Horikawa, Mayuka; Ogawa, Fumihide; Takehara, Kazuhiko; Sato, Shinichi

    2007-02-01

    Serum levels of B-cell activating factor belonging to the tumor necrosis factor family (BAFF), a potent B-cell survival factor, are elevated in patients with systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis and systemic sclerosis (SSc). The objective of this study was to determine serum BAFF levels and relate the results to the clinical features in patients with organ-specific autoimmune diseases of the skin, such as localized scleroderma and autoimmune bullous diseases. Serum BAFF levels were examined by enzyme-linked immunosorbent assay in 44 patients with localized scleroderma, 20 with pemphigus vulgaris/pemphigus foliaceus, 20 with bullous pemphigoid and 30 healthy controls. Twenty patients with SSc and 20 with SLE were also examined as disease controls. Serum BAFF levels were elevated in localized scleroderma patients compared with healthy controls. Concerning localized scleroderma subgroups, patients with generalized morphea, the severest form of localized scleroderma, had higher serum BAFF levels than linear scleroderma or morphea patients. The BAFF levels of generalized morphea were comparable with those of SSc or SLE. Furthermore, serum BAFF levels correlated positively with antihistone antibody levels and the severity of skin lesion as well as the number of skin lesions. By contrast, serum BAFF levels were not significantly elevated in patients with pemphigus or pemphigoid. These results suggest that BAFF may be contributing to autoimmunity and disease development in localized scleroderma.

  20. Investigations of a rabbit (Oryctolagus cuniculus model of systemic lupus erythematosus (SLE, BAFF and its receptors.

    Directory of Open Access Journals (Sweden)

    Jiahui Yang

    2009-12-01

    Full Text Available B-cell activation factor belonging to the tumor necrosis factor family (BAFF is a major contributor to survival of B lymphocytes during development and maturation. A relationship between circulating BAFF levels and disease activity has been reported in patients with the autoimmune disease Systemic Lupus Erythematosus (SLE. Clinical trials targeting BAFF or its receptors are currently in progress. In order to further characterize a rabbit (Oryctolagus cuniculus model of SLE, we investigated the expression of BAFF and its receptors in non-inbred, pedigreed rabbits derived from breeding and selection based on autoantibody responses. We immunized rabbits related to previous groups that developed autoantibodies and inflammatory responses after immunizations with peptides synthesized on multiple antigen-branched polylysine backbones. Blood and sera collected before immunization and after boosts were used for health monitoring, analyses of serum autoantibody responses by ELISA and immunofluorescence. Peripheral blood mononuclear cells (PBMC were studied by flow cytometry and were the source of mRNA for quantitative PCR analyses. We hypothesized that BAFF mRNA expression and serum BAFF levels measured indirectly through BAFF receptor binding might increase in autoantibody-producing rabbits. Immunized rabbits developed elevated levels of leucocyte populations, anti-nuclear, anti-dsDNA and other autoantibodies. BR3 mRNA levels in total PBMC decreased and BAFF levels remained low and unchanged in most immunized rabbits. By flow cytometry, percentages of BAFF positive cells decreased. Percentages of transmembrane activator and CAML interactor (TACI decreased in most rabbits from all the immunized groups. The rabbit is an important model for human autoimmune and infectious diseases, and a high quality draft rabbit genome assembly was recently completed. Human disease models developed in non-inbred pedigreed animals are better able to reflect the complexities

  1. CP-25 attenuates the inflammatory response of fibroblast-like synoviocytes co-cultured with BAFF-activated CD4(+) T cells.

    Science.gov (United States)

    Jia, Xiaoyi; Wei, Fang; Sun, Xiaojing; Chang, Yan; Xu, Shu; Yang, Xuezhi; Wang, Chun; Wei, Wei

    2016-08-02

    Total glucosides of paeony (TGP) is the first anti-inflammatory immune regulatory drug approved for the treatment of rheumatoid arthritis in China. A novel compound, paeoniflorin-6'-O-benzene sulfonate (code CP-25), comes from the structural modification of paeoniflorin (Pae), which is the effective active ingredient of TGP. The aim of the present study is to investigate the effect of CP-25 on adjuvant arthritis (AA) fibroblast-like synoviocytes (FLS) co-cultured with BAFF-activated CD4(+) T cells and the expression of BAFF-R in CD4(+) T cells. The mRNA expression of BAFF and its receptors was assessed by qPCR. The expression of BAFF receptors in CD4(+) T cells was analyzed by flow cytometry. The effect of CP-25 on AA rats was evaluated by their joint histopathology. The cell culture growth of thymocytes and FLS was detected by cell counting kit (CCK-8). The concentrations of IL-1β, TNF-α, and IL-6 were measured by Enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of BAFF and BAFF-R were enhanced in the mesenteric lymph nodes of AA rats, TACI expression was reduced, and BCMA had no change. The expression of BAFF-R in CD4(+) T cells was also enhanced. CP-25 alleviated the joint histopathology and decreased the expression of BAFF-R in CD4(+) T cells from AA rats in vivo. In vitro, CP-25 inhibited the abnormal cell culture growth of BAFF-stimulated thymocytes and FLS. In the co-culture system, IL-1β, IL-6 and TNF-α production was enhanced by FLS co-cultured with BAFF-activated CD4(+) T cells. Moreover, BAFF-stimulated CD4(+) T cells promoted the cell culture growth of FLS. The addition of CP-25 decreased the expression of BAFF-R in CD4(+) T cells and inhibited the cell culture growth and cytokine secretion ability of FLS co-cultured with BAFF-activated CD4(+) T cells. The present study indicates that CP-25 may repress the cell culture growth and cytokine secretion ability of FLS, and its inhibitory effects might be associated with its ability

  2. Expression of BAFF receptors in muscle tissue of myositis patients with anti-Jo-1 or anti-Ro52/anti-Ro60 autoantibodies.

    Science.gov (United States)

    Kryštůfková, Olga; Barbasso Helmers, Sevim; Venalis, Paulius; Malmström, Vivianne; Lindroos, Eva; Vencovský, Jiří; Lundberg, Ingrid E

    2014-10-10

    Anti-Jo-1 and anti-Ro52 autoantibodies are common in patients with myositis, but the mechanisms behind their production are not known. Survival of autoantibody-producing cells is dependent on B-cell-activating factor of the tumour necrosis factor family (BAFF). BAFF levels are elevated in serum of anti-Jo-1-positive myositis patients and are influenced by type-I interferon (IFN). IFN-producing cells and BAFF mRNA expression are present in myositis muscle. We investigated expression of the receptors for BAFF in muscle tissue in relation to anti-Jo-1 and anti-Ro52/anti-Ro60 autoantibodies and type-I IFN markers. Muscle biopsies from 23 patients with myositis selected based on autoantibody profile and 7 healthy controls were investigated for expression of BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). Nineteen samples were assessed for plasma (CD138) and B-cell (CD19) markers. The numbers of positive cells per area were compared with the expression of plasmacytoid dendritic cell (pDC) marker blood dendritic cell antigen-2 (BDCA-2) and IFNα/β-inducible myxovirus resistance-1 protein (MX-1). BAFF-R, BCMA and TACI were expressed in five, seven and seven patients, respectively, and more frequently in anti-Jo-1-positive and/or anti-Ro52/anti-Ro60-positive patients compared to controls and to patients without these autoantibodies (P = BAFF-R: 0.007, BCMA: 0.03 and TACI: 0.07). A local association of receptors with B and plasma cells was confirmed by confocal microscopy. The numbers of CD138-positive and BCMA-positive cells were correlated (r = 0.79; P = 0.001). Expression of BDCA-2 correlated with numbers of CD138-positive cells and marginally with BCMA-positive cells (r = 0.54 and 0.42, respectively; P = 0.04 and 0.06, respectively). There was a borderline correlation between the numbers of positively stained TACI cells and MX-1 areas (r = 0.38, P = 0.08). The expression

  3. Chemokine/cytokine profiling after rituximab: reciprocal expression of BCA-1/CXCL13 and BAFF in childhood OMS.

    Science.gov (United States)

    Pranzatelli, Michael R; Tate, Elizabeth D; Travelstead, Anna L; Verhulst, Steven J

    2011-03-01

    The aim of the study was to test the hypothesis that B-cell repopulation following rituximab (anti-CD20) therapy is orchestrated by chemokines and non-chemokine cytokines. Twenty-five children with opsoclonus-myoclonus syndrome (OMS) received rituximab with or without conventional agents. A comprehensive panel of 40 chemokines and other cytokines were measured in serum by ELISA and multiplexed fluorescent bead-based immunoassay. Serum BAFF concentration changed dramatically (even after first infusion) and inversely with B-cell depletion/repopulation and CXCL13 concentration at 1, 3, and 6 months. Negative correlations were found for BAFF concentration vs blood B cell percentage and serum CXCL13 concentration; positive correlations with serum rituximab concentrations. Six months after initiation of therapy, no significant difference in the levels of APRIL, CXCL10, IL-6, or 17 other cytokines/chemokines were detected. These data reveal a major role for BAFF in peripheral B cell repopulation following rituximab-induced B-cell depletion, and novel changes in CXCL13. ClinicalTrials.gov NCT0024436. Copyright © 2010 Elsevier Ltd. All rights reserved.

  4. Outgroup, alignment and modelling improvements indicate that two TNFSF13-like genes existed in the vertebrate ancestor.

    Science.gov (United States)

    Redmond, Anthony K; Pettinello, Rita; Dooley, Helen

    2017-03-01

    The molecular machinery required for lymphocyte development and differentiation appears to have emerged concomitantly with distinct B- and T-like lymphocyte subsets in the ancestor of all vertebrates. The TNFSF superfamily (TNFSF) members BAFF (TNFSF13/Blys) and APRIL (TNFSF13) are key regulators of B cell development survival, and activation in mammals, but the temporal emergence of these molecules, and their precise relationship to the newly identified TNFSF gene BALM (BAFF and APRIL-like molecule), have not yet been elucidated. Here, to resolve the early evolutionary history of this family, we improved outgroup sampling and alignment quality, and applied better fitting substitution models compared to past studies. Our analyses reveal that BALM is a definitive TNFSF13 family member, which split from BAFF in the gnathostome (jawed vertebrate) ancestor. Most importantly, however, we show that both the APRIL and BAFF lineages existed in the ancestors of all extant vertebrates. This implies that APRIL has been lost, or is yet to be found, in cyclostomes (jawless vertebrates). Our results suggest that lineage-specific gene duplication and loss events have caused lymphocyte regulation, despite shared origins, to become secondarily distinct between gnathostomes and cyclostomes. Finally, the structure of lamprey BAFF-like, and its phylogenetic placement as sister to BAFF and BALM, but not the more slowly evolving APRIL, indicates that the primordial lymphocyte regulator was more APRIL-like than BAFF-like.

  5. Increased expression of TACI on NOD B cells results in germinal centre reaction anomalies, enhanced plasma cell differentiation and immunoglobulin production.

    Science.gov (United States)

    Banday, Viqar S; Thyagarajan, Radha; Sundström, Mia; Lejon, Kristina

    2016-11-01

    B cells have an important pathogenic role in the development of type 1 diabetes in the non-obese diabetic (NOD) mouse. We have previously reported that NOD mice display an increased percentage of TACI high -expressing B cells compared with C57BL/6 mice and this trait is linked to chromosomes 1 and 8. In this paper the genetic association of the transmembrane activator, calcium modulator and cyclophilin ligand interactor (TACI) trait was confirmed using double congenic NOD.B6C1/Idd22 mice. TACI ligation by a proliferation-inducing ligand (APRIL) has been shown to influence plasma cell differentiation, immunoglobulin production and isotype switch. Hence, the functional consequence of the up-regulation of TACI on NOD B cells was analysed both in vitro and in vivo. NOD B cells stimulated with APRIL showed an enhanced plasma cell differentiation and class switch to IgG and IgA compared with B cells from C57BL/6 mice. Moreover, flow cytometry analyses revealed that germinal centre B cells in NOD failed to down-regulate TACI. Availability of the TACI ligand B-cell activating factor (BAFF) has been shown to be a limiting factor in the germinal centre reaction. In line with this, upon immunization with 4-hydroxy-3-nitrophenylacetyl hapten-conjugated hen egg lysozyme, NOD mice produced higher titres of low-affinity antibodies compared with C57BL/6 mice. This observation was supported by the detection of increased levels of BAFF in NOD germinal centres after immunization compared with C57BL/6 by immunofluorescence. Our results support the hypothesis that increased TACI expression on NOD B cells contributes to the pathogenesis of type 1 diabetes in the NOD mouse. © 2016 John Wiley & Sons Ltd.

  6. The challenge of legitimizing spatially differentiated regulation

    DEFF Research Database (Denmark)

    Thorsøe, Martin Hvarregaard; Graversgaard, Morten; Noe, Egon

    2017-01-01

    Differentiating regulation is a promising approach to agri-environmental regulation that may potentially reduce the environmental impact of agriculture at the lowest possible costs for the farmers and society, but also possesses a number of challenges. In this article, we explore the challenges...... to the legitimacy of agri-environmental regulation that occurs when the regulatory regime changes from general regulation to differentiated regulation. The analysis is based on a case study of the implementation of the Buffer zone act in Denmark – a regulation that prevents agricultural production in a 10 (later 9......) meter fringe around selected waterbodies. We distinguish between two different ways of legitimizing: Producing knowledge and participation. We conclude that to harvest some of the obvious benefits of differentiated regulation a number of challenges must be resolved, 1) ensuring legitimacy...

  7. B cell activating factor is central to bleomycin- and IL-17-mediated experimental pulmonary fibrosis.

    Science.gov (United States)

    François, Antoine; Gombault, Aurélie; Villeret, Bérengère; Alsaleh, Ghada; Fanny, Manoussa; Gasse, Paméla; Adam, Sylvain Marchand; Crestani, Bruno; Sibilia, Jean; Schneider, Pascal; Bahram, Seiamak; Quesniaux, Valérie; Ryffel, Bernhard; Wachsmann, Dominique; Gottenberg, Jacques-Eric; Couillin, Isabelle

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive devastating, yet untreatable fibrotic disease of unknown origin. We investigated the contribution of the B-cell activating factor (BAFF), a TNF family member recently implicated in the regulation of pathogenic IL-17-producing cells in autoimmune diseases. The contribution of BAFF was assessed in a murine model of lung fibrosis induced by airway administered bleomycin. We show that murine BAFF levels were strongly increased in the bronchoalveolar space and lungs after bleomycin exposure. We identified Gr1(+) neutrophils as an important source of BAFF upon BLM-induced lung inflammation and fibrosis. Genetic ablation of BAFF or BAFF neutralization by a soluble receptor significantly attenuated pulmonary fibrosis and IL-1β levels. We further demonstrate that bleomycin-induced BAFF expression and lung fibrosis were IL-1β and IL-17A dependent. BAFF was required for rIL-17A-induced lung fibrosis and augmented IL-17A production by CD3(+) T cells from murine fibrotic lungs ex vivo. Finally we report elevated levels of BAFF in bronchoalveolar lavages from IPF patients. Our data therefore support a role for BAFF in the establishment of pulmonary fibrosis and a crosstalk between IL-1β, BAFF and IL-17A. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Biophysical regulation of stem cell differentiation.

    Science.gov (United States)

    Govey, Peter M; Loiselle, Alayna E; Donahue, Henry J

    2013-06-01

    Bone adaptation to its mechanical environment, from embryonic through adult life, is thought to be the product of increased osteoblastic differentiation from mesenchymal stem cells. In parallel with tissue-scale loading, these heterogeneous populations of multipotent stem cells are subject to a variety of biophysical cues within their native microenvironments. Bone marrow-derived mesenchymal stem cells-the most broadly studied source of osteoblastic progenitors-undergo osteoblastic differentiation in vitro in response to biophysical signals, including hydrostatic pressure, fluid flow and accompanying shear stress, substrate strain and stiffness, substrate topography, and electromagnetic fields. Furthermore, stem cells may be subject to indirect regulation by mechano-sensing osteocytes positioned to more readily detect these same loading-induced signals within the bone matrix. Such paracrine and juxtacrine regulation of differentiation by osteocytes occurs in vitro. Further studies are needed to confirm both direct and indirect mechanisms of biophysical regulation within the in vivo stem cell niche.

  9. Interleukin (IL)‐39 [IL‐23p19/Epstein–Barr virus‐induced 3 (Ebi3)] induces differentiation/expansion of neutrophils in lupus‐prone mice

    Science.gov (United States)

    Liu, X.; Zhang, Y.; Wang, Z.; Zhu, G.; Han, G.; Chen, G.; Hou, C.; Wang, T.; Ma, N.; Shen, B.; Li, Y.; Wang, R.

    2016-01-01

    Summary Interleukin (IL)‐12 family cytokines play critical roles in autoimmune diseases. Our previous study has shown that IL‐23p19 and Epstein–Barr virus‐induced 3 (Ebi3) form a new IL‐12 family heterodimer, IL‐23p19/Ebi3, termed IL‐39, and knock‐down of p19 or Ebi3 reduced diseases by transferred GL7+ B cells in lupus‐prone mice. In the present study, we explore further the possible effect of IL‐39 on murine lupus. We found that IL‐39 in vitro and in vivo induces differentiation and/or expansion of neutrophils. GL7+ B cells up‐regulated neutrophils by secreting IL‐39, whereas IL‐39‐deficient GL7+ B cells lost the capacity to up‐regulate neutrophils in lupus‐prone mice and homozygous CD19cre (CD19‐deficient) mice. Finally, we found that IL‐39‐induced neutrophils had a positive feedback on IL‐39 expression in activated B cells by secreting B cell activation factor (BAFF). Taken together, our results suggest that IL‐39 induces differentiation and/or expansion of neutrophils in lupus‐prone mice. PMID:27400195

  10. YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway

    International Nuclear Information System (INIS)

    Lin, Yi-Ting; Ding, Jing-Ya; Li, Ming-Yang; Yeh, Tien-Shun; Wang, Tsu-Wei; Yu, Jenn-Yah

    2012-01-01

    Tight regulation of cell numbers by controlling cell proliferation and apoptosis is important during development. Recently, the Hippo pathway has been shown to regulate tissue growth and organ size in Drosophila. In mammalian cells, it also affects cell proliferation and differentiation in various tissues, including the nervous system. Interplay of several signaling cascades, such as Notch, Wnt, and Sonic Hedgehog (Shh) pathways, control cell proliferation during neuronal differentiation. However, it remains unclear whether the Hippo pathway coordinates with other signaling cascades in regulating neuronal differentiation. Here, we used P19 cells, a mouse embryonic carcinoma cell line, as a model to study roles of YAP, a core component of the Hippo pathway, in neuronal differentiation. P19 cells can be induced to differentiate into neurons by expressing a neural bHLH transcription factor gene Ascl1. Our results showed that YAP promoted cell proliferation and inhibited neuronal differentiation. Expression of Yap activated Shh but not Wnt or Notch signaling activity during neuronal differentiation. Furthermore, expression of Yap increased the expression of Patched homolog 1 (Ptch1), a downstream target of the Shh signaling. Knockdown of Gli2, a transcription factor of the Shh pathway, promoted neuronal differentiation even when Yap was over-expressed. We further demonstrated that over-expression of Yap inhibited neuronal differentiation in primary mouse cortical progenitors and Gli2 knockdown rescued the differentiation defect in Yap over-expressing cells. In conclusion, our study reveals that Shh signaling acts downstream of YAP in regulating neuronal differentiation. -- Highlights: ► YAP promotes cell proliferation and inhibits neuronal differentiation in P19 cells. ► YAP promotes Sonic hedgehog signaling activity during neuronal differentiation. ► Knockdown of Gli2 rescues the Yap-overexpression phenotype in P19 cells. ► Knockdown of Gli2 rescues the Yap

  11. YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yi-Ting; Ding, Jing-Ya [Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Li, Ming-Yang [Department of Life Science, National Taiwan Normal University, Taipei 116, Taiwan (China); Yeh, Tien-Shun [Department of Anatomy and Cell Biology, National Yang-Ming University, Taipei 112, Taiwan (China); Wang, Tsu-Wei [Department of Life Science, National Taiwan Normal University, Taipei 116, Taiwan (China); Yu, Jenn-Yah [Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Brain Research Center, National Yang-Ming University, Taipei 112, Taiwan (China)

    2012-09-10

    Tight regulation of cell numbers by controlling cell proliferation and apoptosis is important during development. Recently, the Hippo pathway has been shown to regulate tissue growth and organ size in Drosophila. In mammalian cells, it also affects cell proliferation and differentiation in various tissues, including the nervous system. Interplay of several signaling cascades, such as Notch, Wnt, and Sonic Hedgehog (Shh) pathways, control cell proliferation during neuronal differentiation. However, it remains unclear whether the Hippo pathway coordinates with other signaling cascades in regulating neuronal differentiation. Here, we used P19 cells, a mouse embryonic carcinoma cell line, as a model to study roles of YAP, a core component of the Hippo pathway, in neuronal differentiation. P19 cells can be induced to differentiate into neurons by expressing a neural bHLH transcription factor gene Ascl1. Our results showed that YAP promoted cell proliferation and inhibited neuronal differentiation. Expression of Yap activated Shh but not Wnt or Notch signaling activity during neuronal differentiation. Furthermore, expression of Yap increased the expression of Patched homolog 1 (Ptch1), a downstream target of the Shh signaling. Knockdown of Gli2, a transcription factor of the Shh pathway, promoted neuronal differentiation even when Yap was over-expressed. We further demonstrated that over-expression of Yap inhibited neuronal differentiation in primary mouse cortical progenitors and Gli2 knockdown rescued the differentiation defect in Yap over-expressing cells. In conclusion, our study reveals that Shh signaling acts downstream of YAP in regulating neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer YAP promotes cell proliferation and inhibits neuronal differentiation in P19 cells. Black-Right-Pointing-Pointer YAP promotes Sonic hedgehog signaling activity during neuronal differentiation. Black-Right-Pointing-Pointer Knockdown of Gli2 rescues the Yap

  12. Forkhead Box C1 Regulates Human Primary Keratinocyte Terminal Differentiation.

    Directory of Open Access Journals (Sweden)

    Lianghua Bin

    Full Text Available The epidermis serves as a critical protective barrier between the internal and external environment of the human body. Its remarkable barrier function is established through the keratinocyte (KC terminal differentiation program. The transcription factors specifically regulating terminal differentiation remain largely unknown. Using a RNA-sequencing (RNA-seq profiling approach, we found that forkhead box c 1 (FOXC1 was significantly up-regulated in human normal primary KC during the course of differentiation. This observation was validated in human normal primary KC from several different donors and human skin biopsies. Silencing FOXC1 in human normal primary KC undergoing differentiation led to significant down-regulation of late terminal differentiation genes markers including epidermal differentiation complex genes, keratinization genes, sphingolipid/ceramide metabolic process genes and epidermal specific cell-cell adhesion genes. We further demonstrated that FOXC1 works down-stream of ZNF750 and KLF4, and upstream of GRHL3. Thus, this study defines FOXC1 as a regulator specific for KC terminal differentiation and establishes its potential position in the genetic regulatory network.

  13. Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation.

    Science.gov (United States)

    Martin, E C; Qureshi, A T; Llamas, C B; Burow, M E; King, A G; Lee, O C; Dasa, V; Freitas, M A; Forsberg, J A; Elster, E A; Davis, T A; Gimble, J M

    2018-02-07

    Stromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.

  14. Protective Effects of Total Glucosides of Paeony on N-nitrosodiethylamine-induced Hepatocellular Carcinoma in Rats via Down-regulation of Regulatory B Cells.

    Science.gov (United States)

    Song, S S; Yuan, P F; Li, P P; Wu, H X; Ni, W J; Lu, J T; Wei, W

    2015-01-01

    Total glucoside of paeony (TGP), extracted from the root of Paeonia Lactiflora, has been known to show anti-inflammatory, anti-oxidative, hepato-protective and immuno-regulatory activities. The aim of this present study was to determine the anti-tumor effect of TGP against N-nitrosodiethylamine (DEN)-induced hepatocellular carcinoma (HCC) in rats, and to find the related mechanisms. Rat HCC model was established by intragastrically administrating with DEN (8 mg/kg). We found the number of tumor nodules and the index of liver and spleen were increased in the model group compared with the normal group, and was significantly decreased by TGP. Additionally, TGP obviously improved the hepatic pathological lesions induced by DEN, and decreased the elevated levels of serum alanine aminotransferase (ALT), glutamic oxalacetic transaminase (AST), alkaline phosphatase (ALP) and alpha fetoprotein (AFP) by DEN. Moreover, TGP decreased the level of B cell-activating factor (BAFF) and the proportion of IL-10-producing regulatory B cells (Bregs), and the decrease of BAFF by TGP is positively correlated to the decrease of IL-10-producing Bregs by TGP. These results suggest that TGP had a good therapeutic action on DEN-induced HCC rats, which might be due to its down-regulation of Bregs through reducing the level of BAFF.

  15. Biophysical Regulation of Vascular Differentiation and Assembly

    CERN Document Server

    Gerecht, Sharon

    2011-01-01

    The ability to grow stem cells in the laboratory and to guide their maturation to functional cells allows us to study the underlying mechanisms that govern vasculature differentiation and assembly in health and disease. Accumulating evidence suggests that early stages of vascular growth are exquisitely tuned by biophysical cues from the microenvironment, yet the scientific understanding of such cellular environments is still in its infancy. Comprehending these processes sufficiently to manipulate them would pave the way to controlling blood vessel growth in therapeutic applications. This book assembles the works and views of experts from various disciplines to provide a unique perspective on how different aspects of its microenvironment regulate the differentiation and assembly of the vasculature. In particular, it describes recent efforts to exploit modern engineering techniques to study and manipulate various biophysical cues. Biophysical Regulation of Vascular Differentiation and Assembly provides an inter...

  16. PPARγ isoforms differentially regulate metabolic networks to mediate mouse prostatic epithelial differentiation.

    Science.gov (United States)

    Strand, D W; Jiang, M; Murphy, T A; Yi, Y; Konvinse, K C; Franco, O E; Wang, Y; Young, J D; Hayward, S W

    2012-08-09

    Recent observations indicate prostatic diseases are comorbidities of systemic metabolic dysfunction. These discoveries revealed fundamental questions regarding the nature of prostate metabolism. We previously showed that prostate-specific ablation of PPARγ in mice resulted in tumorigenesis and active autophagy. Here, we demonstrate control of overlapping and distinct aspects of prostate epithelial metabolism by ectopic expression of individual PPARγ isoforms in PPARγ knockout prostate epithelial cells. Expression and activation of either PPARγ 1 or 2 reduced de novo lipogenesis and oxidative stress and mediated a switch from glucose to fatty acid oxidation through regulation of genes including Pdk4, Fabp4, Lpl, Acot1 and Cd36. Differential effects of PPARγ isoforms included decreased basal cell differentiation, Scd1 expression and triglyceride fatty acid desaturation and increased tumorigenicity by PPARγ1. In contrast, PPARγ2 expression significantly increased basal cell differentiation, Scd1 expression and AR expression and responsiveness. Finally, in confirmation of in vitro data, a PPARγ agonist versus high-fat diet (HFD) regimen in vivo confirmed that PPARγ agonization increased prostatic differentiation markers, whereas HFD downregulated PPARγ-regulated genes and decreased prostate differentiation. These data provide a rationale for pursuing a fundamental metabolic understanding of changes to glucose and fatty acid metabolism in benign and malignant prostatic diseases associated with systemic metabolic stress.

  17. Alternative Splicing of G9a Regulates Neuronal Differentiation

    Directory of Open Access Journals (Sweden)

    Ana Fiszbein

    2016-03-01

    Full Text Available Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10 through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10+ isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.

  18. New insights into redox regulation of stem cell self-renewal and differentiation.

    Science.gov (United States)

    Ren, Fenglian; Wang, Kui; Zhang, Tao; Jiang, Jingwen; Nice, Edouard Collins; Huang, Canhua

    2015-08-01

    Reactive oxygen species (ROS), the natural byproducts of aerobic metabolism, are precisely orchestrated to evoke diverse signaling pathways. To date, studies have focused mainly on the detrimental effects of ROS in stem cells. Recently, accumulating evidence has suggested that ROS also function as second messengers that modulate stem cell self-renewal and differentiation by regulating intricate signaling networks. Although many efforts have been made to clarify the general effects of ROS on signal transduction in stem cells, less is known about the initial and direct executors of ROS signaling, which are known as 'redox sensors'. Modifications of cysteine residues in redox sensors are of significant importance in the modulation of protein function in response to different redox conditions. Intriguingly, most key molecules in ROS signaling and cell cycle regulation (including transcriptional factors and kinases) that are crucial in the regulation of stem cell self-renewal and differentiation have the potential to be redox sensors. We highlight herein the importance of redox regulation of these key regulators in stem cell self-renewal and differentiation. Understanding the mechanisms of redox regulation in stem cell self-renewal and differentiation will open exciting new perspectives for stem cell biology. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Regulators of Tfh cell differentiation

    Directory of Open Access Journals (Sweden)

    Gajendra Motiram Jogdand

    2016-11-01

    Full Text Available The follicular helper T (Tfh cells help is critical for activation of B cells, antibody class switching and germinal center formation. The Tfh cells are characterized by the expression of CXCR5, ICOS, PD-1, Bcl-6, and IL-21. They are involved in clearing infections and are adversely linked with autoimmune diseases and also have a role in viral replication as well as clearance. Tfh cells are generated from naïve CD4 T cells with sequential steps involving cytokine signaling (IL-21, IL-6, IL-12, activin A, migration and positioning in the germinal center by CXCR5, surface receptors (ICOS/ICOSL, SAP/SLAM as well as transcription factor (Bcl-6, c-Maf, STAT3 signaling and repressor miR155. On the other hand Tfh generation is negatively regulated at specific steps of Tfh generation by specific cytokine (IL-2, IL-7, surface receptor (PD-1, CTLA-4, transcription factors Blimp-1, STAT5, T-bet, KLF-2 signaling and repressor miR 146a. Interestingly, miR 17-92 and FOXO1 acts as a positive as well as a negative regulator of Tfh differentiation depending on the time of expression and disease specificity. Tfh cells are also generated from the conversion of other effector T cells as exemplified by Th1 cells converting into Tfh during viral infection. The mechanistic details of effector T cells conversion into Tfh are yet to be clear. To manipulate Tfh cells for therapeutic implication and or for effective vaccination strategies, it is important to know positive and negative regulators of Tfh generation. Hence, in this review we have highlighted and interlinked molecular signaling from cytokines, surface receptors, transcription factors, ubiquitin Ligase and miRNA as positive and negative regulators for Tfh differentiation.

  20. EZH2: a pivotal regulator in controlling cell differentiation.

    Science.gov (United States)

    Chen, Ya-Huey; Hung, Mien-Chie; Li, Long-Yuan

    2012-01-01

    Epigenetic regulation plays an important role in stem cell self-renewal, maintenance and lineage differentiation. The epigenetic profiles of stem cells are related to their transcriptional signature. Enhancer of Zeste homlog 2 (EZH2), a catalytic subunit of epigenetic regulator Polycomb repressive complex 2 (PRC2), has been shown to be a key regulator in controlling cellular differentiation. EZH2 is a histone methyltransferase that not only methylates histone H3 on Lys 27 (H3K27me3) but also interacts with and recruits DNA methyltransferases to methylate CpG at certain EZH2 target genes to establish firm repressive chromatin structures, contributing to tumor progression and the regulation of development and lineage commitment both in embryonic stem cells (ESCs) and adult stem cells. In addition to its well-recognized epigenetic gene silencing function, EZH2 also directly methylates nonhistone targets such as the cardiac transcription factor, GATA4, resulting in attenuated GATA4 transcriptional activity and gene repression. This review addresses recent progress toward the understanding of the biological functions and regulatory mechanisms of EZH2 and its targets as well as their roles in stem cell maintenance and cell differentiation.

  1. Mitochondrial activity in the regulation of stem cell self-renewal and differentiation.

    Science.gov (United States)

    Khacho, Mireille; Slack, Ruth S

    2017-12-01

    Mitochondria are classically known as the essential energy producers in cells. As such, the activation of mitochondrial metabolism upon cellular differentiation was deemed a necessity to fuel the high metabolic needs of differentiated cells. However, recent studies have revealed a direct role for mitochondrial activity in the regulation of stem cell fate and differentiation. Several components of mitochondrial metabolism and respiration have now been shown to regulate different aspects of stem cell differentiation through signaling, transcriptional, proteomic and epigenetic modulations. In light of these findings mitochondrial metabolism is no longer considered a consequence of cellular differentiation, but rather a key regulatory mechanism of this process. This review will focus on recent progress that defines mitochondria as the epicenters for the regulation of stem cell fate decisions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Towards spatially differentiated regulation of nitrogen

    DEFF Research Database (Denmark)

    Lajer Højberg, Anker; Refsgaard, Jens Christian; Jørgensen, Lisbeth Flindt

    EU member states are challenged by nitrogen loads to estuaries and inland freshwater systems impeding the achievement of good ecological status as required by the Water Framework Directive (WFD). In Denmark nitrate leaching from the root zone has been reduced by 50% since 1987, but additional...... reductions of 30-50% are required to meet the objectives of the WFD. Achieving such abatements by uniform restrictions for all areas, would be very costly and inefficient as studies have shown that reduction varies spatially depending on the local hydrogeological conditions, the presence and dynamics...... of drains and hydro-biogeochemical conditions in associated riparian lowlands. Hence, a shift of paradigm in regulation practice is needed, whit a cost-effective regulation accounting for this variability and differentiate the regulations/restrictions between resilient and vulnerable areas. However...

  3. Non-coding RNAs as epigenetic regulator of glioma stem-like cell differentiation

    Directory of Open Access Journals (Sweden)

    Keisuke eKatsushima

    2014-02-01

    Full Text Available Glioblastomas show heterogeneous histological features. These distinct phenotypic states are thought to be associated with the presence of glioma stem cells (GSCs, which are highly tumorigenic and self-renewing sub-population of tumor cells that have different functional characteristics. Differentiation of GSCs may be regulated by multi-tiered epigenetic mechanisms that orchestrate the expression of thousands of genes. One such regulatory mechanism involves functional non-coding RNAs (ncRNAs, such as microRNAs (miRNAs; a large number of ncRNAs have been identified and shown to regulate the expression of genes associated with cell differentiation programs. Given the roles of miRNAs in cell differentiation, it is possible they are involved in the regulation of gene expression networks in GSCs that are important for the maintenance of the pluripotent state and for directing differentiation. Here, we review recent findings on ncRNAs associated with GSC differentiation and discuss how these ncRNAs contribute to the establishment of tissue heterogeneity during glioblastoma tumor formation.

  4. Human Long Noncoding RNA Regulation of Stem Cell Potency and Differentiation

    Directory of Open Access Journals (Sweden)

    Seahyoung Lee

    2017-01-01

    Full Text Available Because of their capability of differentiation into lineage-specific cells, stem cells are an attractive therapeutic modality in regenerative medicine. To develop an effective stem cell-based therapeutic strategy with predictable results, deeper understanding of the underlying molecular mechanisms of stem cell differentiation and/or pluripotency maintenance is required. Thus, reviewing the key factors involved in the transcriptional and epigenetic regulation of stem cell differentiation and maintenance is important. Accumulating data indicate that long noncoding RNAs (lncRNAs mediate numerous biological processes, including stem cell differentiation and maintenance. Here, we review recent findings on the human lncRNA regulation of stem cell potency and differentiation. Although the clinical implication of these lncRNAs is only beginning to be elucidated, it is anticipated that lncRNAs will become important therapeutic targets in the near future.

  5. Increased visfatin levels are associated with higher disease activity in anti-Jo-1-positive myositis patients.

    Science.gov (United States)

    Hulejová, Hana; Kryštůfková, Olga; Mann, Heřman; Klein, Martin; Pavlíčková, Klára; Zámečník, Josef; Vencovský, Jiří; Šenolt, Ladislav

    2016-01-01

    The aim of this study was to evaluate serum levels of visfatin in anti-Jo-1-positive myositis patients, its expression in muscle tissue and to investigate potential relationships between visfatin, B-cell activating factor of the TNF family (BAFF), disease activity and anti-Jo-1 autoantibody levels. Serum levels of visfatin and BAFF were measured in 38 anti-Jo-1 positive myositis patients and 35 healthy subjects. Disease activity was evaluated by myositis disease activity assessment tool (MYOACT) using visual analogue scales (VAS) and by serum muscle enzymes. Visfatin expression was evaluated by immunohistochemistry in muscle tissue of myositis patients (n=10) and compared with non-inflammatory control muscle tissue samples from patients with myasthenia gravis (n=5). Serum visfatin and BAFF levels were significantly higher in myositis patients compared to healthy subjects and were associated with clinical muscle activity assessed by VAS. Only serum BAFF levels, but not visfatin levels, positively correlated with muscle enzyme concentrations and anti-Jo1 antibody levels. There was a positive correlation between visfatin and BAFF serum levels in myositis patients but a negative correlation was observed in healthy subjects. Visfatin expression was up-regulated in endomysial and perimysial inflammatory infiltrates of muscle tissue from myositis patients. Up-regulation of visfatin in myositis muscle tissue and an association between increased visfatin levels and muscle disease activity evaluated by MYOACT in anti-Jo-1 positive myositis patients could support possible role of visfatin in the pathogenesis of myositis.

  6. Regulation of T Cell Differentiation and Function by EZH2

    Science.gov (United States)

    Karantanos, Theodoros; Christofides, Anthos; Bardhan, Kankana; Li, Lequn; Boussiotis, Vassiliki A.

    2016-01-01

    The enhancer of zeste homolog 2 (EZH2), one of the polycomb-group proteins, is the catalytic subunit of Polycomb-repressive complex 2 (PRC2) and induces the trimethylation of the histone H3 lysine 27 (H3K27me3) promoting epigenetic gene silencing. EZH2 contains a SET domain promoting the methyltransferase activity, while the three other protein components of PRC2, namely EED, SUZ12, and RpAp46/48, induce compaction of the chromatin permitting EZH2 enzymatic activity. Numerous studies highlight the role of this evolutionary conserved protein as a master regulator of differentiation in humans involved in the repression of the homeotic gene and the inactivation of X-chromosome. Through its effects in the epigenetic regulation of critical genes, EZH2 has been strongly linked to cell cycle progression, stem cell pluripotency, and cancer biology, being currently at the cutting edge of research. Most recently, EZH2 has been associated with hematopoietic stem cell proliferation and differentiation, thymopoiesis and lymphopoiesis. Several studies have evaluated the role of EZH2 in the regulation of T cell differentiation and plasticity as well as its implications in the development of autoimmune diseases and graft-versus-host disease (GVHD). The aim of this review is to summarize the current knowledge regarding the role of EZH2 in the regulation of the differentiation and function of T cells focusing on possible applications in various immune-mediated conditions, including autoimmune disorders and GVHD. PMID:27199994

  7. Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-α

    International Nuclear Information System (INIS)

    Tsukasaki, Masayuki; Yamada, Atsushi; Suzuki, Dai; Aizawa, Ryo; Miyazono, Agasa; Miyamoto, Yoichi; Suzawa, Tetsuo; Takami, Masamichi; Yoshimura, Kentaro; Morimura, Naoko; Yamamoto, Matsuo; Kamijo, Ryutaro

    2011-01-01

    Highlights: → TNF-α inhibits POEM gene expression. → Inhibition of POEM gene expression is caused by NF-κB activation by TNF-α. → Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-α. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-α (TNF-α), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-κB) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-α in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-α-induced inhibition of osteoblast differentiation. These results suggest that TNF-α inhibits POEM expression through the NF-κB signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-α.

  8. Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Tsukasaki, Masayuki [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Suzuki, Dai [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Aizawa, Ryo [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Department of Periodontology, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta, Tokyo 145-8515 (Japan); Miyazono, Agasa [Department of Periodontology, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta, Tokyo 145-8515 (Japan); Miyamoto, Yoichi; Suzawa, Tetsuo; Takami, Masamichi; Yoshimura, Kentaro [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Morimura, Naoko [Laboratory for Comparative Neurogenesis, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Yamamoto, Matsuo [Department of Periodontology, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta, Tokyo 145-8515 (Japan); Kamijo, Ryutaro [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan)

    2011-07-15

    Highlights: {yields} TNF-{alpha} inhibits POEM gene expression. {yields} Inhibition of POEM gene expression is caused by NF-{kappa}B activation by TNF-{alpha}. {yields} Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-{alpha}. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-{alpha} (TNF-{alpha}), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-{alpha}-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-{kappa}B) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-{alpha} in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-{alpha}-induced inhibition of osteoblast differentiation. These results suggest that TNF-{alpha} inhibits POEM expression through the NF-{kappa}B signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-{alpha}.

  9. TGFβ activated kinase 1 (TAK1 at the crossroad of B cell receptor and Toll-like receptor 9 signaling pathways in human B cells.

    Directory of Open Access Journals (Sweden)

    Dániel Szili

    Full Text Available B cell development and activation are regulated by combined signals mediated by the B cell receptor (BCR, receptors for the B-cell activating factor of the tumor necrosis factor family (BAFF-R and the innate receptor, Toll-like receptor 9 (TLR9. However, the underlying mechanisms by which these signals cooperate in human B cells remain unclear. Our aim was to elucidate the key signaling molecules at the crossroads of BCR, BAFF-R and TLR9 mediated pathways and to follow the functional consequences of costimulation.Therefore we stimulated purified human B cells by combinations of anti-Ig, B-cell activating factor of the tumor necrosis factor family (BAFF and the TLR9 agonist, CpG oligodeoxynucleotide. Phosphorylation status of various signaling molecules, B cell proliferation, cytokine secretion, plasma blast generation and the frequency of IgG producing cells were investigated. We have found that BCR induced signals cooperate with BAFF-R- and TLR9-mediated signals at different levels of cell activation. BCR and BAFF- as well as TLR9 and BAFF-mediated signals cooperate at NFκB activation, while BCR and TLR9 synergistically costimulate mitogen activated protein kinases (MAPKs, ERK, JNK and p38. We show here for the first time that the MAP3K7 (TGF beta activated kinase, TAK1 is responsible for the synergistic costimulation of B cells by BCR and TLR9, resulting in an enhanced cell proliferation, plasma blast generation, cytokine and antibody production. Specific inhibitor of TAK1 as well as knocking down TAK1 by siRNA abrogates the synergistic signals. We conclude that TAK1 is a key regulator of receptor crosstalk between BCR and TLR9, thus plays a critical role in B cell development and activation.

  10. Regulation of T cell differentiation and function by EZH2

    Directory of Open Access Journals (Sweden)

    THEODOROS KARANTANOS

    2016-05-01

    Full Text Available The enhancer of zeste homologue 2 (EZH2, one of the polycomb group (PcG proteins, is the catalytic subunit of Polycomb-repressive complex 2 (PRC2 and induces the trimethylation of the histone H3 lysine 27 (H3K27me3 promoting epigenetic gene silencing. EZH2 contains a SET domain promoting the methyltransferase activity while the three other protein components of PRC2, namely EED, SUZ12 and RpAp46/48 induce compaction of the chromatin permitting EZH2 enzymatic activity. Numerous studies highlight the role of this evolutionary conserved protein as a master regulator of differentiation in humans involved in the repression of the homeotic (Hox gene and the inactivation of X-chromosome. Through its effects in the epigenetic regulation of critical genes, EZH2 has been strongly linked to cell cycle progression, stem cell pluripotency and cancer biology. Most recently, EZH2 has been associated with hematopoietic stem cell proliferation and differentiation, thymopoiesis and lymphopoiesis. Several studies have evaluated the role of EZH2 in the regulation of T cell differentiation and plasticity as well as its implications in the development of autoimmune diseases and graft versus host disease (GvHD. In this review we will briefly summarize the current knowledge regarding the role of EZH2 in the regulation of T cell differentiation, effector function and homing in the tumor microenvironment and we will discuss possible therapeutic targeting of EZH2 in order to alter T cell immune functions.

  11. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

    DEFF Research Database (Denmark)

    Eskildsen, Tilde; Taipaleenmäki, Hanna; Stenvang, Jan

    2011-01-01

    Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators......-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore...

  12. Palmitoylation regulates epidermal homeostasis and hair follicle differentiation.

    Directory of Open Access Journals (Sweden)

    Pleasantine Mill

    2009-11-01

    Full Text Available Palmitoylation is a key post-translational modification mediated by a family of DHHC-containing palmitoyl acyl-transferases (PATs. Unlike other lipid modifications, palmitoylation is reversible and thus often regulates dynamic protein interactions. We find that the mouse hair loss mutant, depilated, (dep is due to a single amino acid deletion in the PAT, Zdhhc21, resulting in protein mislocalization and loss of palmitoylation activity. We examined expression of Zdhhc21 protein in skin and find it restricted to specific hair lineages. Loss of Zdhhc21 function results in delayed hair shaft differentiation, at the site of expression of the gene, but also leads to hyperplasia of the interfollicular epidermis (IFE and sebaceous glands, distant from the expression site. The specific delay in follicle differentiation is associated with attenuated anagen propagation and is reflected by decreased levels of Lef1, nuclear beta-catenin, and Foxn1 in hair shaft progenitors. In the thickened basal compartment of mutant IFE, phospho-ERK and cell proliferation are increased, suggesting increased signaling through EGFR or integrin-related receptors, with a parallel reduction in expression of the key differentiation factor Gata3. We show that the Src-family kinase, Fyn, involved in keratinocyte differentiation, is a direct palmitoylation target of Zdhhc21 and is mislocalized in mutant follicles. This study is the first to demonstrate a key role for palmitoylation in regulating developmental signals in mammalian tissue homeostasis.

  13. Perturbation-expression analysis identifies RUNX1 as a regulator of human mammary stem cell differentiation.

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    Ethan S Sokol

    2015-04-01

    Full Text Available The search for genes that regulate stem cell self-renewal and differentiation has been hindered by a paucity of markers that uniquely label stem cells and early progenitors. To circumvent this difficulty we have developed a method that identifies cell-state regulators without requiring any markers of differentiation, termed Perturbation-Expression Analysis of Cell States (PEACS. We have applied this marker-free approach to screen for transcription factors that regulate mammary stem cell differentiation in a 3D model of tissue morphogenesis and identified RUNX1 as a stem cell regulator. Inhibition of RUNX1 expanded bipotent stem cells and blocked their differentiation into ductal and lobular tissue rudiments. Reactivation of RUNX1 allowed exit from the bipotent state and subsequent differentiation and mammary morphogenesis. Collectively, our findings show that RUNX1 is required for mammary stem cells to exit a bipotent state, and provide a new method for discovering cell-state regulators when markers are not available.

  14. Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors

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    Lamblin Anne-Francoise

    2007-10-01

    Full Text Available Abstract Background Osteoblast differentiation requires the coordinated stepwise expression of multiple genes. Histone deacetylase inhibitors (HDIs accelerate the osteoblast differentiation process by blocking the activity of histone deacetylases (HDACs, which alter gene expression by modifying chromatin structure. We previously demonstrated that HDIs and HDAC3 shRNAs accelerate matrix mineralization and the expression of osteoblast maturation genes (e.g. alkaline phosphatase, osteocalcin. Identifying other genes that are differentially regulated by HDIs might identify new pathways that contribute to osteoblast differentiation. Results To identify other osteoblast genes that are altered early by HDIs, we incubated MC3T3-E1 preosteoblasts with HDIs (trichostatin A, MS-275, or valproic acid for 18 hours in osteogenic conditions. The promotion of osteoblast differentiation by HDIs in this experiment was confirmed by osteogenic assays. Gene expression profiles relative to vehicle-treated cells were assessed by microarray analysis with Affymetrix GeneChip 430 2.0 arrays. The regulation of several genes by HDIs in MC3T3-E1 cells and primary osteoblasts was verified by quantitative real-time PCR. Nine genes were differentially regulated by at least two-fold after exposure to each of the three HDIs and six were verified by PCR in osteoblasts. Four of the verified genes (solute carrier family 9 isoform 3 regulator 1 (Slc9a3r1, sorbitol dehydrogenase 1, a kinase anchor protein, and glutathione S-transferase alpha 4 were induced. Two genes (proteasome subunit, beta type 10 and adaptor-related protein complex AP-4 sigma 1 were suppressed. We also identified eight growth factors and growth factor receptor genes that are significantly altered by each of the HDIs, including Frizzled related proteins 1 and 4, which modulate the Wnt signaling pathway. Conclusion This study identifies osteoblast genes that are regulated early by HDIs and indicates pathways that

  15. Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor

    Directory of Open Access Journals (Sweden)

    Manetta J

    2014-08-01

    Full Text Available Joseph Manetta, Holly Bina, Paul Ryan, Niles Fox, Derrick R Witcher, Kristine Kikly Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Abstract: B-cell activating factor (BAFF is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. Keywords: autoimmunity, B-cell malignancies, B-cell survival factor, BAFF

  16. Global regulator SATB1 recruits beta-catenin and regulates T(H2 differentiation in Wnt-dependent manner.

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    Dimple Notani

    2010-01-01

    Full Text Available In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of beta-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs to activate target genes. Wnt/beta -catenin signalling is essential for T cell development and differentiation. Here we show that special AT-rich binding protein 1 (SATB1, the T lineage-enriched chromatin organizer and global regulator, interacts with beta-catenin and recruits it to SATB1's genomic binding sites. Gene expression profiling revealed that the genes repressed by SATB1 are upregulated upon Wnt signalling. Competition between SATB1 and TCF affects the transcription of TCF-regulated genes upon beta-catenin signalling. GATA-3 is a T helper type 2 (T(H2 specific transcription factor that regulates production of T(H2 cytokines and functions as T(H2 lineage determinant. SATB1 positively regulated GATA-3 and siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4(+ T cells, suggesting that SATB1 influences T(H2 lineage commitment by reprogramming gene expression. In the presence of Dickkopf 1 (Dkk1, an inhibitor of Wnt signalling, GATA-3 is downregulated and the expression of signature T(H2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for T(H2 differentiation. Knockdown of beta-catenin also produced similar results, confirming the role of Wnt/beta-catenin signalling in T(H2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits beta-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating T(H2 cells in a Wnt-dependent manner. SATB1 coordinates T(H2 lineage commitment by reprogramming gene expression. The SATB1:beta-catenin complex activates a number of SATB1 regulated genes, and hence this study has potential to find novel Wnt responsive genes. These results demonstrate that SATB1

  17. Regulation of white and brown adipocyte differentiation by RhoGAP DLC1.

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    Choon Kiat Sim

    Full Text Available Adipose tissues constitute an important component of metabolism, the dysfunction of which can cause obesity and type II diabetes. Here we show that differentiation of white and brown adipocytes requires Deleted in Liver Cancer 1 (DLC1, a Rho GTPase Activating Protein (RhoGAP previously studied for its function in liver cancer. We identified Dlc1 as a super-enhancer associated gene in both white and brown adipocytes through analyzing the genome-wide binding profiles of PPARγ, the master regulator of adipogenesis. We further observed that Dlc1 expression increases during differentiation, and knockdown of Dlc1 by siRNA in white adipocytes reduces the formation of lipid droplets and the expression of fat marker genes. Moreover, knockdown of Dlc1 in brown adipocytes reduces expression of brown fat-specific genes and diminishes mitochondrial respiration. Dlc1-/- knockout mouse embryonic fibroblasts show a complete inability to differentiate into adipocytes, but this phenotype can be rescued by inhibitors of Rho-associated kinase (ROCK and filamentous actin (F-actin, suggesting the involvement of Rho pathway in DLC1-regulated adipocyte differentiation. Furthermore, PPARγ binds to the promoter of Dlc1 gene to regulate its expression during both white and brown adipocyte differentiation. These results identify DLC1 as an activator of white and brown adipocyte differentiation, and provide a molecular link between PPARγ and Rho pathways.

  18. Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Erica M. [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Niu, MengMeng; Bergholz, Johann [Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China); Jim Xiao, Zhi-Xiong, E-mail: jxiao@bu.edu [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China)

    2015-05-29

    The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.

  19. RAGE, receptor of advanced glycation endoproducts, negatively regulates chondrocytes differentiation.

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    Tatsuya Kosaka

    Full Text Available RAGE, receptor for advanced glycation endoproducts (AGE, has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms.

  20. [Comprehensive regulation effect of traditional Chinese medicine on proliferation and differentiation of neural stem cells].

    Science.gov (United States)

    Wang, Hong-Jin; Li, Jing-Jing; Ke, Hui; Xu, Xiao-Yu

    2017-11-01

    Since the discovery of neural stem cells(NSCs) in embryonic and adult mammalian central nervous systems, new approaches for proliferation and differentiation of NSCs have been put forward. One of the approaches to promote the clinical application of NSCs is to search effective methods to regulate the proliferation and differentiation. This problem is urgently to be solved in the medical field. Previous studies have shown that traditional Chinese medicine could promote the proliferation and differentiation of NSCs by regulating the relevant signaling pathway in vivo and in vitro. Domestic and foreign literatures for regulating the proliferation and differentiation of neural stem cells in recent 10 years and the reports for their target and signaling pathways were analyzed in this paper. Traditional Chinese medicine could regulate the proliferation and differentiation of NSCs through signaling pathways of Notch, PI3K/Akt, Wnt/β-catenin and GFs. However, studies about NSCs and traditional Chinese medicine should be further deepened; the mechanism of multiple targets and the comprehensive regulation function of traditional Chinese medicine should be clarified. Copyright© by the Chinese Pharmaceutical Association.

  1. The regulation and role of c-FLIP in human Th cell differentiation.

    Science.gov (United States)

    Kyläniemi, Minna K; Kaukonen, Riina; Myllyviita, Johanna; Rasool, Omid; Lahesmaa, Riitta

    2014-01-01

    The early differentiation of T helper (Th) cells is a tightly controlled and finely balanced process, which involves several factors including cytokines, transcription factors and co-stimulatory molecules. Recent studies have shown that in addition to the regulation of apoptosis, caspase activity is also needed for Th cell proliferation and activation and it might play a role in Th cell differentiation. The isoforms of the cellular FLICE inhibitory protein (c-FLIP) are regulators of CASPASE-8 activity and the short isoform, c-FLIPS, has been shown to be up-regulated by IL-4, the Th2 driving cytokine. In this work, we have studied the expression and functional role of three c-FLIP isoforms during the early Th cell differentiation. Only two of the isoforms, c-FLIPS and c-FLIPL, were detected at the protein level although c-FLIPR was expressed at the mRNA level. The knockdown of c-FLIPL led to enhanced Th1 differentiation and elevated IL-4 production by Th2 cells, whereas the knockdown of c-FLIPS diminished GATA3 expression and IL-4 production by Th2 cells. In summary, our results provide new insight into the role of c-FLIP proteins in the early differentiation of human Th cells.

  2. Ihh/Gli2 signaling promotes osteoblast differentiation by regulating Runx2 expression and function.

    Science.gov (United States)

    Shimoyama, Atsuko; Wada, Masahiro; Ikeda, Fumiyo; Hata, Kenji; Matsubara, Takuma; Nifuji, Akira; Noda, Masaki; Amano, Katsuhiko; Yamaguchi, Akira; Nishimura, Riko; Yoneda, Toshiyuki

    2007-07-01

    Genetic and cell biological studies have indicated that Indian hedgehog (Ihh) plays an important role in bone development and osteoblast differentiation. However, the molecular mechanism by which Ihh regulates osteoblast differentiation is complex and remains to be fully elucidated. In this study, we investigated the role of Ihh signaling in osteoblast differentiation using mesenchymal cells and primary osteoblasts. We observed that Ihh stimulated alkaline phosphatase (ALP) activity, osteocalcin expression, and calcification. Overexpression of Gli2- but not Gli3-induced ALP, osteocalcin expression, and calcification of these cells. In contrast, dominant-negative Gli2 markedly inhibited Ihh-dependent osteoblast differentiation. Ihh treatment or Gli2 overexpression also up-regulated the expression of Runx2, an essential transcription factor for osteoblastogenesis, and enhanced the transcriptional activity and osteogenic action of Runx2. Coimmunoprecipitation analysis demonstrated a physical interaction between Gli2 and Runx2. Moreover, Ihh or Gli2 overexpression failed to increase ALP activity in Runx2-deficient mesenchymal cells. Collectively, these results suggest that Ihh regulates osteoblast differentiation of mesenchymal cells through up-regulation of the expression and function of Runx2 by Gli2.

  3. MicroRNA-378 regulates neural stem cell proliferation and differentiation in vitro by modulating Tailless expression

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yanxia [Department of Psychology and Psychiatry, The Second Affiliated Hospital of Xi' an Jiaotong University, Xi' an 710004 (China); Department of Rehabilitation, Xi' an Children' s Hospital, Xi' an 710003 (China); Liu, Xiaoguai [The 3rd Department of Infectious Diseases, Xi' an Children' s Hospital, Xi' an 710003 (China); Wang, Yaping, E-mail: yapwangyy@163.com [Department of Psychology and Psychiatry, The Second Affiliated Hospital of Xi' an Jiaotong University, Xi' an 710004 (China)

    2015-10-16

    Previous studies have suggested that microRNAs (miRNAs) play an important role in regulating neural stem cell (NSC) proliferation and differentiation. However, the precise role of miRNAs in NSC remains largely unexplored. In this study, we showed that miR-378 can target Tailless (TLX), a critical regulator of NSC, to regulate NSC proliferation and differentiation. By bioinformatic algorithms, miR-378 was found to have a predicted target site in the 3′-untranslated region of TLX, which was verified by a dual-luciferase reporter assay. The expression of miR-378 was increased during NSC differentiation and inversely correlated with TLX expression. qPCR and Western blot analysis also showed that miR-378 negatively regulated TLX mRNA and protein expression in neural stem cells (NSCs). Intriguingly, overexpression of miR-378 increased NSC differentiation and reduced NSC proliferation, whereas suppression of miR-378 led to decreased NSC differentiation and increased NSC proliferation. Moreover, the downstream targets of TLX, including p21, PTEN and Wnt/β-catenin were also found to be regulated by miR-378. Additionally, overexpression of TLX rescued the NSC proliferation deficiency induced by miR-378 overexpression and abolished miR-378-promoted NSC differentiation. Taken together, our data suggest that miR-378 is a novel miRNA that regulates NSC proliferation and differentiation via targeting TLX. Therefore, manipulating miR-378 in NSCs could be a novel strategy to develop novel interventions for the treatment of relevant neurological disorders. - Highlights: • miR-378 targeted and regulated TLX. • miR-378 was increased during NSC differentiation. • miR-378 regulated NSC proliferation and differentiation. • miR-378 regulated NSC self-renew through TLX.

  4. MicroRNA-378 regulates neural stem cell proliferation and differentiation in vitro by modulating Tailless expression

    International Nuclear Information System (INIS)

    Huang, Yanxia; Liu, Xiaoguai; Wang, Yaping

    2015-01-01

    Previous studies have suggested that microRNAs (miRNAs) play an important role in regulating neural stem cell (NSC) proliferation and differentiation. However, the precise role of miRNAs in NSC remains largely unexplored. In this study, we showed that miR-378 can target Tailless (TLX), a critical regulator of NSC, to regulate NSC proliferation and differentiation. By bioinformatic algorithms, miR-378 was found to have a predicted target site in the 3′-untranslated region of TLX, which was verified by a dual-luciferase reporter assay. The expression of miR-378 was increased during NSC differentiation and inversely correlated with TLX expression. qPCR and Western blot analysis also showed that miR-378 negatively regulated TLX mRNA and protein expression in neural stem cells (NSCs). Intriguingly, overexpression of miR-378 increased NSC differentiation and reduced NSC proliferation, whereas suppression of miR-378 led to decreased NSC differentiation and increased NSC proliferation. Moreover, the downstream targets of TLX, including p21, PTEN and Wnt/β-catenin were also found to be regulated by miR-378. Additionally, overexpression of TLX rescued the NSC proliferation deficiency induced by miR-378 overexpression and abolished miR-378-promoted NSC differentiation. Taken together, our data suggest that miR-378 is a novel miRNA that regulates NSC proliferation and differentiation via targeting TLX. Therefore, manipulating miR-378 in NSCs could be a novel strategy to develop novel interventions for the treatment of relevant neurological disorders. - Highlights: • miR-378 targeted and regulated TLX. • miR-378 was increased during NSC differentiation. • miR-378 regulated NSC proliferation and differentiation. • miR-378 regulated NSC self-renew through TLX.

  5. Novel function of the retinoblastoma protein in fat: regulation of white versus brown adipocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Jacob B; te Riele, Hein; Kristiansen, Karsten

    2004-01-01

    the major energy store and brown adipocytes being potent energy-dissipaters through thermogenesis. Yet, little is known about factors differentially regulating the formation of white and brown fat cells. Members of the retinoblastoma protein family (pRB, p107, p130) have been implicated in the regulation...... of adipocyte differentiation, and expression and phosphorylation of the three retinoblastoma family proteins oscillate in a characteristic manner during differentiation of the white preadipocyte cell line 3T3-L1. We have recently demonstrated a surprising function of the retinoblastoma protein...... in the regulation of white versus brown adipocyte differentiation in vitro and possibly in vivo. Here we summarize the current knowledge on the retinoblastoma protein in fat cells, with particular emphasis on its potential role in adipocyte lineage commitment and differentiation....

  6. Gli3 acts as a repressor downstream of Ihh in regulating two distinct steps of chondrocyte differentiation.

    Science.gov (United States)

    Koziel, Lydia; Wuelling, Manuela; Schneider, Sabine; Vortkamp, Andrea

    2005-12-01

    During endochondral ossification, the secreted growth factor Indian hedgehog (Ihh) regulates several differentiation steps. It interacts with a second secreted factor, parathyroid hormone-related protein (PTHrP), to regulate the onset of hypertrophic differentiation, and it regulates chondrocyte proliferation and ossification of the perichondrium independently of PTHrP. To investigate how the Ihh signal is translated in the different target tissues, we analyzed the role of the zinc-finger transcription factor Gli3, which acts downstream of hedgehog signals in other organs. Loss of Gli3 in Ihh mutants restores chondrocyte proliferation and delays the accelerated onset of hypertrophic differentiation observed in Ihh-/- mutants. Furthermore the expression of the Ihh target genes patched (Ptch) and PTHrP is reactivated in Ihh-/-;Gli3-/- mutants. Gli3 seems thus to act as a strong repressor of Ihh signals in regulating chondrocyte differentiation. In addition, loss of Gli3 in mice that overexpress Ihh in chondrocytes accelerates the onset of hypertrophic differentiation by reducing the domain and possibly the level of PTHrP expression. Careful analysis of chondrocyte differentiation in Gli3-/- mutants revealed that Gli3 negatively regulates the differentiation of distal, low proliferating chondrocytes into columnar, high proliferating cells. Our results suggest a model in which the Ihh/Gli3 system regulates two distinct steps of chondrocyte differentiation: (1) the switch from distal into columnar chondrocytes is repressed by Gli3 in a PTHrP-independent mechanism; (2) the transition from proliferating into hypertrophic chondrocytes is regulated by Gli3-dependent expression of PTHrP. Furthermore, by regulating distal chondrocyte differentiation, Gli3 seems to position the domain of PTHrP expression.

  7. EZH2 regulates neuroblastoma cell differentiation via NTRK1 promoter epigenetic modifications.

    Science.gov (United States)

    Li, Zhenghao; Takenobu, Hisanori; Setyawati, Amallia Nuggetsiana; Akita, Nobuhiro; Haruta, Masayuki; Satoh, Shunpei; Shinno, Yoshitaka; Chikaraishi, Koji; Mukae, Kyosuke; Akter, Jesmin; Sugino, Ryuichi P; Nakazawa, Atsuko; Nakagawara, Akira; Aburatani, Hiroyuki; Ohira, Miki; Kamijo, Takehiko

    2018-05-01

    The polycomb repressor complex 2 molecule EZH2 is now known to play a role in essential cellular processes, namely, cell fate decisions, cell cycle regulation, senescence, cell differentiation, and cancer development/progression. EZH2 inhibitors have recently been developed; however, their effectiveness and underlying molecular mechanisms in many malignancies have not yet been elucidated in detail. Although the functional role of EZH2 in tumorigenesis in neuroblastoma (NB) has been investigated, mutations of EZH2 have not been reported. A Kaplan-Meier analysis on the event free survival and overall survival of NB patients indicated that the high expression of EZH2 correlated with an unfavorable prognosis. In order to elucidate the functional roles of EZH2 in NB tumorigenesis and its aggressiveness, we knocked down EZH2 in NB cell lines using lentivirus systems. The knockdown of EZH2 significantly induced NB cell differentiation, e.g., neurite extension, and the neuronal differentiation markers, NF68 and GAP43. EZH2 inhibitors also induced NB cell differentiation. We performed a comprehensive transcriptome analysis using Human Gene Expression Microarrays and found that NTRK1 (TrkA) is one of the EZH2-related suppression targets. The depletion of NTRK1 canceled EZH2 knockdown-induced NB cell differentiation. Our integrative methylome, transcriptome, and chromatin immunoprecipitation assays using NB cell lines and clinical samples clarified that the NTRK1 P1 and P2 promoter regions were regulated differently by DNA methylation and EZH2-related histone modifications. The NTRK1 transcript variants 1/2, which were regulated by EZH2-related H3K27me3 modifications at the P1 promoter region, were strongly expressed in favorable, but not unfavorable NB. The depletion and inhibition of EZH2 successfully induced NTRK1 transcripts and functional proteins. Collectively, these results indicate that EZH2 plays important roles in preventing the differentiation of NB cells and also

  8. Developmental regulation of nucleolus size during Drosophila eye differentiation.

    Directory of Open Access Journals (Sweden)

    Nicholas E Baker

    Full Text Available When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.

  9. Developmental regulation of nucleolus size during Drosophila eye differentiation.

    Science.gov (United States)

    Baker, Nicholas E

    2013-01-01

    When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.

  10. The H3K27 demethylase, Utx, regulates adipogenesis in a differentiation stage-dependent manner.

    Directory of Open Access Journals (Sweden)

    Kazushige Ota

    Full Text Available Understanding the molecular mechanisms that drive adipogenesis is important in developing new treatments for obesity and diabetes. Epigenetic regulations determine the capacity of adipogenesis. In this study, we examined the role of a histone H3 lysine 27 demethylase, the ubiquitously transcribed tetratricopeptide repeat protein on the X chromosome (Utx, in the differentiation of mouse embryonic stem cells (mESCs to adipocytes. Using gene trapping, we examined Utx-deficient male mESCs to determine whether loss of Utx would enhance or inhibit the differentiation of mESCs to adipocytes. Utx-deficient mESCs showed diminished potential to differentiate to adipocytes compared to that of controls. In contrast, Utx-deficient preadipocytes showed enhanced differentiation to adipocytes. Microarray analyses indicated that the β-catenin/c-Myc signaling pathway was differentially regulated in Utx-deficient cells during adipocyte differentiation. Therefore, our data suggest that Utx governs adipogenesis by regulating c-Myc in a differentiation stage-specific manner and that targeting the Utx signaling pathway could be beneficial for the treatment of obesity, diabetes, and congenital utx-deficiency disorders.

  11. Regulation of TFIIIB during F9 cell differentiation.

    Science.gov (United States)

    Athineos, Dimitris; Marshall, Lynne; White, Robert J

    2010-03-12

    Differentiation of F9 embryonal carcinoma (EC) cells into parietal endoderm (PE) provides a tractable model system for studying molecular events during early and inaccessible stages of murine development. PE formation is accompanied by extensive changes in gene expression both in vivo and in culture. One of the most dramatic is the ~10-fold decrease in transcriptional output by RNA polymerase (pol) III. This has been attributed to changes in activity of TFIIIB, a factor that is necessary and sufficient to recruit pol III to promoters. The goal of this study was to identify molecular changes that can account for the low activity of TFIIIB following F9 cell differentiation. Three essential subunits of TFIIIB decrease in abundance as F9 cells differentiate; these are Brf1 and Bdp1, which are pol III-specific, and TBP, which is also used by pols I and II. The decreased levels of Brf1 and Bdp1 proteins can be explained by reduced expression of the corresponding mRNAs. However, this is not the case for TBP, which is regulated post-transcriptionally. In proliferating cells, pol III transcription is stimulated by the proto-oncogene product c-Myc and the mitogen-activated protein kinase Erk, both of which bind to TFIIIB. However, c-Myc levels fall during differentiation and Erk becomes inactive through dephosphorylation. The diminished abundance of TFIIIB is therefore likely to be compounded by changes to these positive regulators that are required for its full activity. In addition, PE cells have elevated levels of the retinoblastoma protein RB, which is known to bind and repress TFIIIB. The low activity of TFIIIB in PE can be attributed to a combination of changes, any one of which could be sufficient to inhibit pol III transcription. Declining levels of essential TFIIIB subunits and of activators that are required for maximal TFIIIB activity are accompanied by an increase in a potent repressor of TFIIIB. These events provide fail-safe guarantees to ensure that pol III

  12. MiR-217 is down-regulated in psoriasis and promotes keratinocyte differentiation via targeting GRHL2

    International Nuclear Information System (INIS)

    Zhu, Haigang; Hou, Liyue; Liu, Jingjing; Li, Zhiming

    2016-01-01

    MiR-217 is a well-known tumor suppressor, and its down-regulation has been shown in a wide range of solid and leukaemic cancers. However, the biological role of miR-217 in psoriasis pathogenesis, especially in keratinocyte hyperproliferation and differentiation, is not clearly understood. In this study, we found the expression of miR-217 was markedly down-regulated in psoriasis keratinocytes of psoriatic patients. In addition, overexpression of miR-217 inhibited the proliferation and promoted the differentiation of primary human keratinocytes. On the contrary, inhibition of endogenous miR-217 increased cell proliferation and delayed differentiation. Furthermore, Grainyhead-like 2 (GRHL2) was identified as a direct target of miR-217 by luciferase reporter assay. The expression of miR-217 and GRHL2 was inversely correlated in both transfected keratinocytes and in psoriasis lesional skin. Moreover, knocking down GRHL2 expression by siRNA enhanced keratinocyte differentiation. Taken together, our results demonstrate a role for miR-217 in the regulation of keratinocyte differentiation, partially through the regulation of GRHL2. - Highlights: • miR-217 is down-regulated in psoriasis skin lesions. • miR-217 inhibits the proliferation and promotes differentiation of keratinocytes. • GRHL2 is a novel target of miR-217 in keratinocytes. • GRHL2 is up-regulated and inversely correlated with miR-217 in psoriasis skin lesions.

  13. MiR-217 is down-regulated in psoriasis and promotes keratinocyte differentiation via targeting GRHL2

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Haigang; Hou, Liyue; Liu, Jingjing; Li, Zhiming, E-mail: lizm_1001@sina.com

    2016-02-26

    MiR-217 is a well-known tumor suppressor, and its down-regulation has been shown in a wide range of solid and leukaemic cancers. However, the biological role of miR-217 in psoriasis pathogenesis, especially in keratinocyte hyperproliferation and differentiation, is not clearly understood. In this study, we found the expression of miR-217 was markedly down-regulated in psoriasis keratinocytes of psoriatic patients. In addition, overexpression of miR-217 inhibited the proliferation and promoted the differentiation of primary human keratinocytes. On the contrary, inhibition of endogenous miR-217 increased cell proliferation and delayed differentiation. Furthermore, Grainyhead-like 2 (GRHL2) was identified as a direct target of miR-217 by luciferase reporter assay. The expression of miR-217 and GRHL2 was inversely correlated in both transfected keratinocytes and in psoriasis lesional skin. Moreover, knocking down GRHL2 expression by siRNA enhanced keratinocyte differentiation. Taken together, our results demonstrate a role for miR-217 in the regulation of keratinocyte differentiation, partially through the regulation of GRHL2. - Highlights: • miR-217 is down-regulated in psoriasis skin lesions. • miR-217 inhibits the proliferation and promotes differentiation of keratinocytes. • GRHL2 is a novel target of miR-217 in keratinocytes. • GRHL2 is up-regulated and inversely correlated with miR-217 in psoriasis skin lesions.

  14. Activated Fps/Fes tyrosine kinase regulates erythroid differentiation and survival.

    Science.gov (United States)

    Sangrar, Waheed; Gao, Yan; Bates, Barbara; Zirngibl, Ralph; Greer, Peter A

    2004-10-01

    A substantial body of evidence implicates the cytoplasmic protein tyrosine kinase Fps/Fes in regulation of myeloid differentiation and survival. In this study we wished to determine if Fps/Fes also plays a role in the regulation of erythropoiesis. Mice tissue-specifically expressing a "gain-of-function" mutant fps/fes transgene (fps(MF)) encoding an activated variant of Fps/Fes (MFps), were used to explore the in vivo biological role of Fps/Fes. Erythropoiesis in these mice was assessed by hematological analysis, lineage marker analysis, bone-marrow colony assays, and biochemical approaches. fps(MF) mice displayed reductions in peripheral red cell counts. However, there was an accumulation of immature erythroid precursors, which displayed increased survival. Fps/Fes and the related Fer kinase were both detected in early erythroid progenitors/blasts and in mature red cells. Fps/Fes was also activated in response to erythropoietin (EPO) and stem cell factor (SCF), two critical factors in erythroid development. In addition, increased Stat5A/B activation and reduced Erk1/2 phosphorylation was observed in fps(MF) primary erythroid cells in response to EPO or SCF, respectively. These data support a role for Fps/Fes in regulating the survival and differentiation of erythroid cells through modulation of Stat5A/B and Erk kinase pathways induced by EPO and SCF. The increased numbers and survival of erythroid progenitors from fps(MF) mice, and their differential responsiveness to SCF and EPO, implicates Fps/Fes in the commitment of multilineage progenitors to the erythroid lineage. The anemic phenotype in fps(MF) mice suggests that downregulation of Fps/Fes activity might be required for terminal erythroid differentiation.

  15. Suppression of MicroRNA let-7a Expression by Agmatine Regulates Neural Stem Cell Differentiation.

    Science.gov (United States)

    Song, Juhyun; Oh, Yumi; Kim, Jong Youl; Cho, Kyoung Joo; Lee, Jong Eun

    2016-11-01

    Neural stem cells (NSCs) effectively reverse some severe central nervous system (CNS) disorders, due to their ability to differentiate into neurons. Agmatine, a biogenic amine, has cellular protective effects and contributes to cellular proliferation and differentiation in the CNS. Recent studies have elucidated the function of microRNA let-7a (let-7a) as a regulator of cell differentiation with roles in regulating genes associated with CNS neurogenesis. This study aimed to investigate whether agmatine modulates the expression of crucial regulators of NSC differentiation including DCX, TLX, c-Myc, and ERK by controlling let-7a expression. Our data suggest that high levels of let-7a promoted the expression of TLX and c-Myc, as well as repressed DCX and ERK expression. In addition, agmatine attenuated expression of TLX and increased expression of ERK by negatively regulating let-7a. Our study therefore enhances the present understanding of the therapeutic potential of NSCs in CNS disorders.

  16. Flow-regulated versus differential pressure-regulated shunt valves for adult patients with normal pressure hydrocephalus

    DEFF Research Database (Denmark)

    Ziebell, Morten; Wetterslev, Jørn; Tisell, Magnus

    2013-01-01

    Since 1965 many ventriculo-peritoneal shunt systems have been inserted worldwide to treat hydrocephalus. The most frequent indication in adults is normal pressure hydrocephalus (NPH), a condition that can be difficult to diagnose precisely. Surgical intervention with flow-regulated and differential...

  17. Emergence of differentially regulated pathways associated with the development of regional specificity in chicken skin.

    Science.gov (United States)

    Chang, Kai-Wei; Huang, Nancy A; Liu, I-Hsuan; Wang, Yi-Hui; Wu, Ping; Tseng, Yen-Tzu; Hughes, Michael W; Jiang, Ting Xin; Tsai, Mong-Hsun; Chen, Chien-Yu; Oyang, Yen-Jen; Lin, En-Chung; Chuong, Cheng-Ming; Lin, Shau-Ping

    2015-01-23

    Regional specificity allows different skin regions to exhibit different characteristics, enabling complementary functions to make effective use of the integumentary surface. Chickens exhibit a high degree of regional specificity in the skin and can serve as a good model for when and how these regional differences begin to emerge. We used developing feather and scale regions in embryonic chickens as a model to gauge the differences in their molecular pathways. We employed cosine similarity analysis to identify the differentially regulated and co-regulated genes. We applied low cell techniques for expression validation and chromatin immunoprecipitation (ChIP)-based enhancer identification to overcome limited cell availabilities from embryonic chicken skin. We identified a specific set of genes demonstrating a high correlation as being differentially expressed during feather and scale development and maturation. Some members of the WNT, TGF-beta/BMP, and Notch family known to be involved in feathering skin differentiation were found to be differentially regulated. Interestingly, we also found genes along calcium channel pathways that are differentially regulated. From the analysis of differentially regulated pathways, we used calcium signaling pathways as an example for further verification. Some voltage-gated calcium channel subunits, particularly CACNA1D, are expressed spatio-temporally in the skin epithelium. These calcium signaling pathway members may be involved in developmental decisions, morphogenesis, or epithelial maturation. We further characterized enhancers associated with histone modifications, including H3K4me1, H3K27ac, and H3K27me3, near calcium channel-related genes and identified signature intensive hotspots that may be correlated with certain voltage-gated calcium channel genes. We demonstrated the applicability of cosine similarity analysis for identifying novel regulatory pathways that are differentially regulated during development. Our study

  18. Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Jensen, Charlotte H; Gutierrez, Gloria

    2004-01-01

    Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. INTRODUCTION......: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. MATERIALS AND METHODS: As a model for hMSCs, we have...... was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. RESULTS: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high...

  19. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells.

    Science.gov (United States)

    Gao, Fei; Kishida, Tsunao; Ejima, Akika; Gojo, Satoshi; Mazda, Osam

    2013-02-08

    Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Unkempt is negatively regulated by mTOR and uncouples neuronal differentiation from growth control.

    Directory of Open Access Journals (Sweden)

    Amélie Avet-Rochex

    2014-09-01

    Full Text Available Neuronal differentiation is exquisitely controlled both spatially and temporally during nervous system development. Defects in the spatiotemporal control of neurogenesis cause incorrect formation of neural networks and lead to neurological disorders such as epilepsy and autism. The mTOR kinase integrates signals from mitogens, nutrients and energy levels to regulate growth, autophagy and metabolism. We previously identified the insulin receptor (InR/mTOR pathway as a critical regulator of the timing of neuronal differentiation in the Drosophila melanogaster eye. Subsequently, this pathway has been shown to play a conserved role in regulating neurogenesis in vertebrates. However, the factors that mediate the neurogenic role of this pathway are completely unknown. To identify downstream effectors of the InR/mTOR pathway we screened transcriptional targets of mTOR for neuronal differentiation phenotypes in photoreceptor neurons. We identified the conserved gene unkempt (unk, which encodes a zinc finger/RING domain containing protein, as a negative regulator of the timing of photoreceptor differentiation. Loss of unk phenocopies InR/mTOR pathway activation and unk acts downstream of this pathway to regulate neurogenesis. In contrast to InR/mTOR signalling, unk does not regulate growth. unk therefore uncouples the role of the InR/mTOR pathway in neurogenesis from its role in growth control. We also identified the gene headcase (hdc as a second downstream regulator of the InR/mTOR pathway controlling the timing of neurogenesis. Unk forms a complex with Hdc, and Hdc expression is regulated by unk and InR/mTOR signalling. Co-overexpression of unk and hdc completely suppresses the precocious neuronal differentiation phenotype caused by loss of Tsc1. Thus, Unk and Hdc are the first neurogenic components of the InR/mTOR pathway to be identified. Finally, we show that Unkempt-like is expressed in the developing mouse retina and in neural stem

  1. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Fei; Kishida, Tsunao; Ejima, Akika [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Gojo, Satoshi [Department of Cardiac Support, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, Osam, E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2013-02-08

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases.

  2. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Gao, Fei; Kishida, Tsunao; Ejima, Akika; Gojo, Satoshi; Mazda, Osam

    2013-01-01

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases

  3. NFIX Regulates Neural Progenitor Cell Differentiation During Hippocampal Morphogenesis

    Science.gov (United States)

    Heng, Yee Hsieh Evelyn; McLeay, Robert C.; Harvey, Tracey J.; Smith, Aaron G.; Barry, Guy; Cato, Kathleen; Plachez, Céline; Little, Erica; Mason, Sharon; Dixon, Chantelle; Gronostajski, Richard M.; Bailey, Timothy L.; Richards, Linda J.; Piper, Michael

    2014-01-01

    Neural progenitor cells have the ability to give rise to neurons and glia in the embryonic, postnatal and adult brain. During development, the program regulating whether these cells divide and self-renew or exit the cell cycle and differentiate is tightly controlled, and imbalances to the normal trajectory of this process can lead to severe functional consequences. However, our understanding of the molecular regulation of these fundamental events remains limited. Moreover, processes underpinning development of the postnatal neurogenic niches within the cortex remain poorly defined. Here, we demonstrate that Nuclear factor one X (NFIX) is expressed by neural progenitor cells within the embryonic hippocampus, and that progenitor cell differentiation is delayed within Nfix−/− mice. Moreover, we reveal that the morphology of the dentate gyrus in postnatal Nfix−/− mice is abnormal, with fewer subgranular zone neural progenitor cells being generated in the absence of this transcription factor. Mechanistically, we demonstrate that the progenitor cell maintenance factor Sry-related HMG box 9 (SOX9) is upregulated in the hippocampus of Nfix−/− mice and demonstrate that NFIX can repress Sox9 promoter-driven transcription. Collectively, our findings demonstrate that NFIX plays a central role in hippocampal morphogenesis, regulating the formation of neuronal and glial populations within this structure. PMID:23042739

  4. microRNAs as regulators of adipogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Hamam, Dana; Ali, Dalia; Kassem, Moustapha; Aldahmash, Abdullah; Alajez, Nehad M

    2015-02-15

    microRNAs (miRNAs) constitute complex regulatory network, fine tuning the expression of a myriad of genes involved in different biological and physiological processes, including stem cell differentiation. Mesenchymal stem cells (MSCs) are multipotent stem cells present in the bone marrow stroma, and the stroma of many other tissues, and can give rise to a number of mesoderm-type cells including adipocytes and osteoblasts, which form medullary fat and bone tissues, respectively. The role of bone marrow fat in bone mass homeostasis is an area of intensive investigation with the aim of developing novel approaches for enhancing osteoblastic bone formation through inhibition of bone marrow fat formation. A number of recent studies have reported several miRNAs that enhance or inhibit adipogenic differentiation of MSCs and with potential use in microRNA-based therapy to regulate adipogenesis in the context of treating bone diseases and metabolic disorders. The current review focuses on miRNAs and their role in regulating adipogenic differentiation of MSCs.

  5. Vitamin D receptor–retinoid X receptor heterodimer signaling regulates oligodendrocyte progenitor cell differentiation

    Science.gov (United States)

    de la Fuente, Alerie Guzman; Errea, Oihana; van Wijngaarden, Peter; Gonzalez, Ginez A.; Kerninon, Christophe; Jarjour, Andrew A.; Lewis, Hilary J.; Jones, Clare A.; Nait-Oumesmar, Brahim; Zhao, Chao; Huang, Jeffrey K.; ffrench-Constant, Charles

    2015-01-01

    The mechanisms regulating differentiation of oligodendrocyte (OLG) progenitor cells (OPCs) into mature OLGs are key to understanding myelination and remyelination. Signaling via the retinoid X receptor γ (RXR-γ) has been shown to be a positive regulator of OPC differentiation. However, the nuclear receptor (NR) binding partner of RXR-γ has not been established. In this study we show that RXR-γ binds to several NRs in OPCs and OLGs, one of which is vitamin D receptor (VDR). Using pharmacological and knockdown approaches we show that RXR–VDR signaling induces OPC differentiation and that VDR agonist vitamin D enhances OPC differentiation. We also show expression of VDR in OLG lineage cells in multiple sclerosis. Our data reveal a role for vitamin D in the regenerative component of demyelinating disease and identify a new target for remyelination medicines. PMID:26644513

  6. Negative regulation of neuronal cell differentiation by INHAT subunit SET/TAF-Iβ.

    Science.gov (United States)

    Kim, Dong-Wook; Kim, Kee-Beom; Kim, Ji-Young; Lee, Kyu-Sun; Seo, Sang-Beom

    2010-09-24

    Epigenetic modification plays an important role in transcriptional regulation. As a subunit of the INHAT (inhibitor of histone acetyltransferases) complex, SET/TAF-Iβ evidences transcriptional repression activity. In this study, we demonstrate that SET/TAF-Iβ is abundantly expressed in neuronal tissues of Drosophila embryos. It is expressed at high levels prior to and in early stages of neuronal development, and gradually reduced as differentiation proceeds. SET/TAF-Iβ binds to the promoters of a subset of neuronal development markers and negatively regulates the transcription of these genes. The results of this study show that the knockdown of SET/TAF-Iβ by si-RNA induces neuronal cell differentiation, thus implicating SET/TAF-Iβ as a negative regulator of neuronal development. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. Gαq Regulates the Development of Rheumatoid Arthritis by Modulating Th1 Differentiation.

    Science.gov (United States)

    Wang, Dashan; Liu, Yuan; Li, Yan; He, Yan; Zhang, Jiyun; Shi, Guixiu

    2017-01-01

    The G α q-containing G protein, an important member of G q/11 class, is ubiquitously expressed in mammalian cells. G α q has been found to play an important role in immune regulation and development of autoimmune disease such as rheumatoid arthritis (RA). However, how G α q participates in the pathogenesis of RA is still not fully understood. In the present study, we aimed to find out whether G α q controls RA via regulation of Th1 differentiation. We observed that the expression of G α q was negatively correlated with the expression of signature Th1 cytokine (IFN- γ ) in RA patients, which suggests a negative role of G α q in differentiation of Th1 cells. By using G α q knockout ( Gnaq-/- ) mice, we demonstrated that loss of G α q led to enhanced Th1 cell differentiation. G α q negative regulated the differentiation of Th1 cell by modulating the expression of T-bet and the activity of STAT4. Furthermore, we detected the increased ratio of Th1 cells in Gnaq-/- bone marrow (BM) chimeras spontaneously developing inflammatory arthritis. In conclusion, results presented in the study demonstrate that loss of G α q promotes the differentiation of Th1 cells and contributes to the pathogenesis of RA.

  8. Substrate stiffness and oxygen as regulators of stem cell differentiation during skeletal tissue regeneration: a mechanobiological model.

    Directory of Open Access Journals (Sweden)

    Darren Paul Burke

    Full Text Available Extrinsic mechanical signals have been implicated as key regulators of mesenchymal stem cell (MSC differentiation. It has been possible to test different hypotheses for mechano-regulated MSC differentiation by attempting to simulate regenerative events such as bone fracture repair, where repeatable spatial and temporal patterns of tissue differentiation occur. More recently, in vitro studies have identified other environmental cues such as substrate stiffness and oxygen tension as key regulators of MSC differentiation; however it remains unclear if and how such cues determine stem cell fate in vivo. As part of this study, a computational model was developed to test the hypothesis that substrate stiffness and oxygen tension regulate stem cell differentiation during fracture healing. Rather than assuming mechanical signals act directly on stem cells to determine their differentiation pathway, it is postulated that they act indirectly to regulate angiogenesis and hence partially determine the local oxygen environment within a regenerating tissue. Chondrogenesis of MSCs was hypothesized to occur in low oxygen regions, while in well vascularised regions of the regenerating tissue a soft local substrate was hypothesised to facilitate adipogenesis while a stiff substrate facilitated osteogenesis. Predictions from the model were compared to both experimental data and to predictions of a well established computational mechanobiological model where tissue differentiation is assumed to be regulated directly by the local mechanical environment. The model predicted all the major events of fracture repair, including cartilaginous bridging, endosteal and periosteal bony bridging and bone remodelling. It therefore provides support for the hypothesis that substrate stiffness and oxygen play a key role in regulating MSC fate during regenerative events such as fracture healing.

  9. Plant GSK3 proteins regulate xylem cell differentiation downstream of TDIF-TDR signalling

    Science.gov (United States)

    Kondo, Yuki; Ito, Tasuku; Nakagami, Hirofumi; Hirakawa, Yuki; Saito, Masato; Tamaki, Takayuki; Shirasu, Ken; Fukuda, Hiroo

    2014-03-01

    During plant radial growth typically seen in trees, procambial and cambial cells act as meristematic cells in the vascular system to self-proliferate and differentiate into xylem cells. These two processes are regulated by a signalling pathway composed of a peptide ligand and its receptor; tracheary element differentiation inhibitory factor (TDIF) and TDIF RECEPTOR (TDR). Here we show that glycogen synthase kinase 3 proteins (GSK3s) are crucial downstream components of the TDIF signalling pathway suppressing xylem differentiation from procambial cells. TDR interacts with GSK3s at the plasma membrane and activates GSK3s in a TDIF-dependent fashion. Consistently, a specific inhibitor of plant GSK3s strongly induces xylem cell differentiation through BRI1-EMS SUPPRESSOR 1 (BES1), a well-known target transcription factor of GSK3s. Our findings provide insight into the regulation of cell fate determination in meristem maintenance.

  10. Zfp206 regulates ES cell gene expression and differentiation.

    Science.gov (United States)

    Zhang, Wen; Walker, Emily; Tamplin, Owen J; Rossant, Janet; Stanford, William L; Hughes, Timothy R

    2006-01-01

    Understanding transcriptional regulation in early developmental stages is fundamental to understanding mammalian development and embryonic stem (ES) cell properties. Expression surveys suggest that the putative SCAN-Zinc finger transcription factor Zfp206 is expressed specifically in ES cells [Zhang,W., Morris,Q.D., Chang,R., Shai,O., Bakowski,M.A., Mitsakakis,N., Mohammad,N., Robinson,M.D., Zirngibl,R., Somogyi,E. et al., (2004) J. Biol., 3, 21; Brandenberger,R., Wei,H., Zhang,S., Lei,S., Murage,J., Fisk,G.J., Li,Y., Xu,C., Fang,R., Guegler,K. et al., (2004) Nat. Biotechnol., 22, 707-716]. Here, we confirm this observation, and we show that ZFP206 expression decreases rapidly upon differentiation of cultured mouse ES cells, and during development of mouse embryos. We find that there are at least six isoforms of the ZFP206 transcript, the longest being predominant. Overexpression and depletion experiments show that Zfp206 promotes formation of undifferentiated ES cell clones, and positively regulates abundance of a very small set of transcripts whose expression is also specific to ES cells and the two- to four-cell stages of preimplantation embryos. This set includes members of the Zscan4, Thoc4, Tcstv1 and eIF-1A gene families, none of which have been functionally characterized in vivo but whose members include apparent transcription factors, RNA-binding proteins and translation factors. Together, these data indicate that Zfp206 is a regulator of ES cell differentiation that controls a set of genes expressed very early in development, most of which themselves appear to be regulators.

  11. N-Myc Differentially Regulates Expression of MXI1 Isoforms in Neuroblastoma

    Directory of Open Access Journals (Sweden)

    Michael B. Armstrong

    2013-12-01

    Full Text Available Amplification of the MYCN proto-oncogene is associated with a poor prognosis in patients with metastatic neuroblastoma (NB. MYCN encodes the N-Myc protein, a transcriptional regulator that dimerizes with the Max transcription factor, binds to E-box DNA sequences, and regulates genes involved in cell growth and apoptosis. Overexpression of N-Myc leads to transcriptional activation and an increase in NB cell proliferation. Mxi1, a member of the Myc family of transcriptional regulators, also binds to Max. However, Mxi1 is a transcriptional repressor and inhibits proliferation of NB cells, suggesting that Mxi1 functions as an N-Myc antagonist. Our laboratory previously identified Mxi1-0, an alternatively transcribed Mxi1 isoform. Mxi1-0 has properties distinct from those of Mxi1; in contrast to Mxi1, Mxi1-0 is unable to suppress c-Myc-dependent transcription. We now show that Mxi1-0 expression increases in response to MYCN overexpression in NB cells, with a positive correlation between MYCN and MXI1-0 RNA levels. We also show that N-Myc expression differentially regulates the MXI1 and MXI1-0 promoters: Increased MYCN expression suppresses MXI1 promoter activity while enhancing transcription through the MXI1-0 promoter. Finally, induction of Mxi1-0 leads to increased proliferation, whereas expression of Mxi1 inhibits cell growth, indicating differential roles for these two proteins. These data suggest that N-Myc differentially regulates the expression of MXI1 and MXI1-0 and can alter the balance between the two transcription factors. Furthermore, MXI1-0 appears to be a downstream target of MYCN-dependent signaling pathways and may contribute to N-Myc-dependent cell growth and proliferation.

  12. The regulation of health care providers' payments when horizontal and vertical differentiation matter.

    Science.gov (United States)

    Bardey, David; Canta, Chiara; Lozachmeur, Jean-Marie

    2012-09-01

    This paper analyzes the regulation of payment schemes for health care providers competing in both quality and product differentiation of their services. The regulator uses two instruments: a prospective payment per patient and a cost reimbursement rate. When the regulator can only use a prospective payment, the optimal price involves a trade-off between the level of quality provision and the level of horizontal differentiation. If this pure prospective payment leads to underprovision of quality and overdifferentiation, a mixed reimbursement scheme allows the regulator to improve the allocation efficiency. This is true for a relatively low level of patients' transportation costs. We also show that if the regulator cannot commit to the level of the cost reimbursement rate, the resulting allocation can dominate the one with full commitment. This occurs when the transportation cost is low or high enough, and the full commitment solution either implies full or zero cost reimbursement. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. MicroRNA-378 regulates neural stem cell proliferation and differentiation in vitro by modulating Tailless expression.

    Science.gov (United States)

    Huang, Yanxia; Liu, Xiaoguai; Wang, Yaping

    2015-10-16

    Previous studies have suggested that microRNAs (miRNAs) play an important role in regulating neural stem cell (NSC) proliferation and differentiation. However, the precise role of miRNAs in NSC remains largely unexplored. In this study, we showed that miR-378 can target Tailless (TLX), a critical regulator of NSC, to regulate NSC proliferation and differentiation. By bioinformatic algorithms, miR-378 was found to have a predicted target site in the 3'-untranslated region of TLX, which was verified by a dual-luciferase reporter assay. The expression of miR-378 was increased during NSC differentiation and inversely correlated with TLX expression. qPCR and Western blot analysis also showed that miR-378 negatively regulated TLX mRNA and protein expression in neural stem cells (NSCs). Intriguingly, overexpression of miR-378 increased NSC differentiation and reduced NSC proliferation, whereas suppression of miR-378 led to decreased NSC differentiation and increased NSC proliferation. Moreover, the downstream targets of TLX, including p21, PTEN and Wnt/β-catenin were also found to be regulated by miR-378. Additionally, overexpression of TLX rescued the NSC proliferation deficiency induced by miR-378 overexpression and abolished miR-378-promoted NSC differentiation. Taken together, our data suggest that miR-378 is a novel miRNA that regulates NSC proliferation and differentiation via targeting TLX. Therefore, manipulating miR-378 in NSCs could be a novel strategy to develop novel interventions for the treatment of relevant neurological disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Withaferin A Associated Differential Regulation of Inflammatory Cytokines

    Directory of Open Access Journals (Sweden)

    Seema Dubey

    2018-02-01

    Full Text Available A role of inflammation-associated cytokines/chemokines has been implicated in a wide variety of human diseases. Here, we investigated the regulation of inflammatory cytokines released by monocyte-derived THP-1 cells following treatment with the dietary agent withaferin A (WFA. Membrane-based cytokine array profiling of the culture supernatant from adenosine triphosphate-stimulated WFA-treated THP-1 cells showed differential regulation of multiple cytokines/chemokines. A selected group of cytokines/chemokines [interleukin-1 beta (IL-1β, CCL2/MCP-1, granulocyte-macrophage colony stimulating factor, PDGF-AA, PTX3, cystatin-3, relaxin-2, TNFRSF8/CD30, and ACRP30] was validated at the transcription level using qPCR. In silico analysis for transcriptional binding factors revealed the presence of nuclear factor-kappa B (NF-κB in a group of downregulated cytokine gene promoters. WFA treatment of THP-1 cells blocks the nuclear translocation of NF-kB and corresponds with the reduced levels of cytokine secretion. To further understand the differential expression of cytokines/chemokines, we showed that WFA alters the nigericin-induced co-localization of NLRP3 and ASC proteins, thereby inhibiting caspase-1 activation, which is responsible for the cleavage and maturation of pro-inflammatory cytokines IL-1β and IL-18. These data suggest that dietary agent WFA concurrently targets NF-κB and the inflammasome complex, leading to inhibition of IL-1β and IL-18, respectively, in addition to differential expression of multiple cytokines/chemokines. Taken together, these results provide a rationale for using WFA to further explore the anti-inflammatory mechanism of cytokines/chemokines associated with inflammatory diseases.

  15. Nicotine induces cell proliferation in association with cyclin D1 up-regulation and inhibits cell differentiation in association with p53 regulation in a murine pre-osteoblastic cell line

    International Nuclear Information System (INIS)

    Sato, Tsuyoshi; Abe, Takahiro; Nakamoto, Norimichi; Tomaru, Yasuhisa; Koshikiya, Noboru; Nojima, Junya; Kokabu, Shoichiro; Sakata, Yasuaki; Kobayashi, Akio; Yoda, Tetsuya

    2008-01-01

    Recent studies have suggested that nicotine critically affects bone metabolism. Many studies have examined the effects of nicotine on proliferation and differentiation, but the underlying molecular mechanisms remain unclear. We examined cell cycle regulators involved in the proliferation and differentiation of MC3T3-E1 cells. Nicotine induced cell proliferation in association with p53 down-regulation and cyclin D1 up-regulation. In differentiated cells, nicotine reduced alkaline phosphatase activity and mineralized nodule formation in dose-dependent manners. Furthermore, p53 expression was sustained in nicotine-treated cells during differentiation. These findings indicate that nicotine promotes the cell cycle and inhibits differentiation in association with p53 regulation in pre-osteoblastic cells

  16. B-cell activating factor in the pathophysiology of multiple myeloma: a target for therapy?

    International Nuclear Information System (INIS)

    Hengeveld, P J; Kersten, M J

    2015-01-01

    Multiple myeloma (MM) is a currently incurable malignancy of plasma cells. Malignant myeloma cells (MMCs) are heavily dependent upon the bone marrow (BM) microenvironment for their survival. One component of this tumor microenvironment, B-Cell Activating Factor (BAFF), has been implicated as a key player in this interaction. This review discusses the role of BAFF in the pathophysiology of MM, and the potential of BAFF-inhibitory therapy for the treatment of MM. Multiple studies have shown that BAFF functions as a survival factor for MMCs. Furthermore, MMCs express several BAFF-binding receptors. Of these, only Transmembrane Activator and CAML Interactor (TACI) correlates with the MMC's capability to ligate BAFF. Additionally, the level of expression of TACI correlates with the level of the MMC's BM dependency. Ligation of BAFF receptors on MMCs causes activation of the Nuclear Factor of κ-B (NF-κB) pathway, a crucial pathway for the pathogenesis of many B-cell malignancies. Serum BAFF levels are significantly elevated in MM patients when compared to healthy controls, and correlate inversely with overall survival. BAFF signaling is thus an interesting target for the treatment of MM. Several BAFF-inhibitory drugs are currently under evaluation for the treatment of MM. These include BAFF-monoclonal antibodies (tabalumab) and antibody-drug conjugates (GSK2857916)

  17. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    Directory of Open Access Journals (Sweden)

    Alberto Miranda

    2011-04-01

    Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  18. A novel crosstalk between Alk7 and cGMP signaling differentially regulates brown adipocyte function

    Directory of Open Access Journals (Sweden)

    Aileen Balkow

    2015-08-01

    Conclusions: We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes.

  19. Tracking differentiating neural progenitors in pluripotent cultures using microRNA-regulated lentiviral vectors.

    Science.gov (United States)

    Sachdeva, Rohit; Jönsson, Marie E; Nelander, Jenny; Kirkeby, Agnete; Guibentif, Carolina; Gentner, Bernhard; Naldini, Luigi; Björklund, Anders; Parmar, Malin; Jakobsson, Johan

    2010-06-22

    In this study, we have used a microRNA-regulated lentiviral reporter system to visualize and segregate differentiating neuronal cells in pluripotent cultures. Efficient suppression of transgene expression, specifically in undifferentiated pluripotent cells, was achieved by using a lentiviral vector expressing a fluorescent reporter gene regulated by microRNA-292. Using this strategy, it was possible to track progeny from murine ES, human ES cells, and induced pluripotent stem cells as they differentiated toward the neural lineage. In addition, this strategy was successfully used to FACS purify neuronal progenitors for molecular analysis and transplantation. FACS enrichment reduced tumor formation and increased survival of ES cell-derived neuronal progenitors after transplantation. The properties and versatility of the microRNA-regulated vectors allows broad use of these vectors in stem cell applications.

  20. TET2 Regulates Mast Cell Differentiation and Proliferation through Catalytic and Non-catalytic Activities

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    Sara Montagner

    2016-05-01

    Full Text Available Summary: Dioxygenases of the TET family impact genome functions by converting 5-methylcytosine (5mC in DNA to 5-hydroxymethylcytosine (5hmC. Here, we identified TET2 as a crucial regulator of mast cell differentiation and proliferation. In the absence of TET2, mast cells showed disrupted gene expression and altered genome-wide 5hmC deposition, especially at enhancers and in the proximity of downregulated genes. Impaired differentiation of Tet2-ablated cells could be relieved or further exacerbated by modulating the activity of other TET family members, and mechanistically it could be linked to the dysregulated expression of C/EBP family transcription factors. Conversely, the marked increase in proliferation induced by the loss of TET2 could be rescued exclusively by re-expression of wild-type or catalytically inactive TET2. Our data indicate that, in the absence of TET2, mast cell differentiation is under the control of compensatory mechanisms mediated by other TET family members, while proliferation is strictly dependent on TET2 expression. : The impact of TET enzymes on gene expression and cell function is incompletely understood. Montagner et al. investigate the TET-mediated regulation of mast cell differentiation and function, uncover transcriptional pathways regulated by TET2, and identify both enzymatic activity-dependent and -independent functions of TET2. Keywords: differentiation, DNA hydroxymethylation, epigenetics, mast cells, proliferation, TET

  1. Meis1 regulates Foxn4 expression during retinal progenitor cell differentiation

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    Mohammed M. Islam

    2013-09-01

    The transcription factor forkhead box N4 (Foxn4 is a key regulator in a variety of biological processes during development. In particular, Foxn4 plays an essential role in the genesis of horizontal and amacrine neurons from neural progenitors in the vertebrate retina. Although the functions of Foxn4 have been well established, the transcriptional regulation of Foxn4 expression during progenitor cell differentiation remains unclear. Here, we report that an evolutionarily conserved 129 bp noncoding DNA fragment (Foxn4CR4.2 or CR4.2, located ∼26 kb upstream of Foxn4 transcription start site, functions as a cis-element for Foxn4 regulation. CR4.2 directs gene expression in Foxn4-positive cells, primarily in progenitors, differentiating horizontal and amacrine cells. We further determined that the gene regulatory activity of CR4.2 is modulated by Meis1 binding motif, which is bound and activated by Meis1 transcription factor. Deletion of the Meis1 binding motif or knockdown of Meis1 expression abolishes the gene regulatory activity of CR4.2. In addition, knockdown of Meis1 expression diminishes the endogenous Foxn4 expression and affects cell lineage development. Together, we demonstrate that CR4.2 and its interacting Meis1 transcription factor play important roles in regulating Foxn4 expression during early retinogenesis. These findings provide new insights into molecular mechanisms that govern gene regulation in retinal progenitors and specific cell lineage development.

  2. Epigenetic landscapes reveal transcription factors regulating CD8+ T cell differentiation

    Science.gov (United States)

    Yu, Bingfei; Zhang, Kai; Milner, J. Justin; Toma, Clara; Chen, Runqiang; Scott-Browne, James P.; Pereira, Renata M.; Crotty, Shane; Chang, John T.; Pipkin, Matthew E.; Wang, Wei; Goldrath, Ananda W.

    2017-01-01

    Dynamic changes in the expression of transcription factors (TFs) can influence specification of distinct CD8+ T cell fates, but the observation of equivalent expression of TF among differentially-fated precursor cells suggests additional underlying mechanisms. Here, we profiled genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8+ T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that TF expression and binding contributed to establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal novel TFs influencing the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8+ T cell differentiation, regulated the formation of terminal-effector and memory-precursor cell-fates, respectively. Our data define the epigenetic landscape of differentiation intermediates, facilitating identification of TFs with previously unappreciated roles in CD8+ T cell differentiation. PMID:28288100

  3. The heterocyst differentiation transcriptional regulator HetR of the filamentous cyanobacterium Anabaena forms tetramers and can be regulated by phosphorylation.

    Science.gov (United States)

    Valladares, Ana; Flores, Enrique; Herrero, Antonia

    2016-02-01

    Many filamentous cyanobacteria respond to the external cue of nitrogen scarcity by the differentiation of heterocysts, cells specialized in the fixation of atmospheric nitrogen in oxic environments. Heterocysts follow a spatial pattern along the filament of two heterocysts separated by ca. 10-15 vegetative cells performing oxygenic photosynthesis. HetR is a transcriptional regulator that directs heterocyst differentiation. In the model strain Anabaena sp. PCC 7120, the HetR protein was observed in various oligomeric forms in vivo, including a tetramer that peaked with maximal hetR expression during differentiation. Tetramers were not detected in a hetR point mutant incapable of differentiation, but were conspicuous in an over-differentiating strain lacking the PatS inhibitor. In differentiated filaments the HetR tetramer was restricted to heterocysts, being undetectable in vegetative cells. HetR co-purified with RNA polymerase from Anabaena mainly as a tetramer. In vitro, purified recombinant HetR was distributed between monomers, dimers, trimers and tetramers, and it was phosphorylated when incubated with (γ-(32)P)ATP. Phosphorylation and PatS hampered the accumulation of HetR tetramers and impaired HetR binding to DNA. In summary, tetrameric HetR appears to represent a functionally relevant form of HetR, whose abundance in the Anabaena filament could be negatively regulated by phosphorylation and by PatS. © 2015 John Wiley & Sons Ltd.

  4. N-Myc Differentially Regulates Expression of MXI1 Isoforms in Neuroblastoma1

    Science.gov (United States)

    Armstrong, Michael B; Mody, Rajen J; Ellis, D Christian; Hill, Adam B; Erichsen, David A; Wechsler, Daniel S

    2013-01-01

    Amplification of the MYCN proto-oncogene is associated with a poor prognosis in patients with metastatic neuroblastoma (NB). MYCN encodes the N-Myc protein, a transcriptional regulator that dimerizes with the Max transcription factor, binds to E-box DNA sequences, and regulates genes involved in cell growth and apoptosis. Overexpression of N-Myc leads to transcriptional activation and an increase in NB cell proliferation. Mxi1, a member of the Myc family of transcriptional regulators, also binds to Max. However, Mxi1 is a transcriptional repressor and inhibits proliferation of NB cells, suggesting that Mxi1 functions as an N-Myc antagonist. Our laboratory previously identified Mxi1-0, an alternatively transcribed Mxi1 isoform. Mxi1-0 has properties distinct from those of Mxi1; in contrast to Mxi1, Mxi1-0 is unable to suppress c-Myc-dependent transcription. We now show that Mxi1-0 expression increases in response to MYCN overexpression in NB cells, with a positive correlation between MYCN and MXI1-0 RNA levels. We also show that N-Myc expression differentially regulates the MXI1 and MXI1-0 promoters: Increased MYCN expression suppresses MXI1 promoter activity while enhancing transcription through the MXI1-0 promoter. Finally, induction of Mxi1-0 leads to increased proliferation, whereas expression of Mxi1 inhibits cell growth, indicating differential roles for these two proteins. These data suggest that N-Myc differentially regulates the expression of MXI1 and MXI1-0 and can alter the balance between the two transcription factors. Furthermore, MXI1-0 appears to be a downstream target of MYCN-dependent signaling pathways and may contribute to N-Myc-dependent cell growth and proliferation. PMID:24403858

  5. FGF signalling regulates chromatin organisation during neural differentiation via mechanisms that can be uncoupled from transcription.

    Directory of Open Access Journals (Sweden)

    Nishal S Patel

    Full Text Available Changes in higher order chromatin organisation have been linked to transcriptional regulation; however, little is known about how such organisation alters during embryonic development or how it is regulated by extrinsic signals. Here we analyse changes in chromatin organisation as neural differentiation progresses, exploiting the clear spatial separation of the temporal events of differentiation along the elongating body axis of the mouse embryo. Combining fluorescence in situ hybridisation with super-resolution structured illumination microscopy, we show that chromatin around key differentiation gene loci Pax6 and Irx3 undergoes both decompaction and displacement towards the nuclear centre coincident with transcriptional onset. Conversely, down-regulation of Fgf8 as neural differentiation commences correlates with a more peripheral nuclear position of this locus. During normal neural differentiation, fibroblast growth factor (FGF signalling is repressed by retinoic acid, and this vitamin A derivative is further required for transcription of neural genes. We show here that exposure to retinoic acid or inhibition of FGF signalling promotes precocious decompaction and central nuclear positioning of differentiation gene loci. Using the Raldh2 mutant as a model for retinoid deficiency, we further find that such changes in higher order chromatin organisation are dependent on retinoid signalling. In this retinoid deficient condition, FGF signalling persists ectopically in the elongating body, and importantly, we find that inhibiting FGF receptor (FGFR signalling in Raldh2-/- embryos does not rescue differentiation gene transcription, but does elicit both chromatin decompaction and nuclear position change. These findings demonstrate that regulation of higher order chromatin organisation during differentiation in the embryo can be uncoupled from the machinery that promotes transcription and, for the first time, identify FGF as an extrinsic signal that

  6. Regulation of caste differentiation in the honey bee (Apis mellifera L.)

    NARCIS (Netherlands)

    Goewie, E.A.

    1978-01-01

    The nutritional environment of honey-bee larvae affects the juvenile hormone (JH) titre of larval haemolymph and tissues. In this investigation the mechanism for the regulation of caste differentiation has been studied.

    Chemo- and mechanoreceptors are found on larval mouthparts.

  7. The Satellite Cell Niche Regulates the Balance between Myoblast Differentiation and Self-Renewal via p53.

    Science.gov (United States)

    Flamini, Valentina; Ghadiali, Rachel S; Antczak, Philipp; Rothwell, Amy; Turnbull, Jeremy E; Pisconti, Addolorata

    2018-03-13

    Satellite cells are adult muscle stem cells residing in a specialized niche that regulates their homeostasis. How niche-generated signals integrate to regulate gene expression in satellite cell-derived myoblasts is poorly understood. We undertook an unbiased approach to study the effect of the satellite cell niche on satellite cell-derived myoblast transcriptional regulation and identified the tumor suppressor p53 as a key player in the regulation of myoblast quiescence. After activation and proliferation, a subpopulation of myoblasts cultured in the presence of the niche upregulates p53 and fails to differentiate. When satellite cell self-renewal is modeled ex vivo in a reserve cell assay, myoblasts treated with Nutlin-3, which increases p53 levels in the cell, fail to differentiate and instead become quiescent. Since both these Nutlin-3 effects are rescued by small interfering RNA-mediated p53 knockdown, we conclude that a tight control of p53 levels in myoblasts regulates the balance between differentiation and return to quiescence. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  8. microRNA-145 promotes differentiation in human urothelial carcinoma through down-regulation of syndecan-1

    International Nuclear Information System (INIS)

    Fujii, Tomomi; Shimada, Keiji; Tatsumi, Yoshihiro; Hatakeyama, Kinta; Obayashi, Chiho; Fujimoto, Kiyohide; Konishi, Noboru

    2015-01-01

    A new molecular marker of carcinoma in the urinary bladder is needed as a diagnostic tool or as a therapeutic target. Potential markers include microRNAs (miRNAs), which are short, low molecular weight RNAs 19–24 nt long that regulate genes associated with cell proliferation, differentiation, and development in various cancers. In this study, we investigated the molecular mechanisms by which miR-145 promotes survival of urothelial carcinoma cells and differentiation into multiple lineages. We found miR-145 to regulate expression of syndecan-1, a heparin sulfate proteoglycan. Cell proliferation in the human urothelial carcinoma cell lines T24 and KU7 was assessed by MTS assay. Cellular senescence and apoptosis were measured by senescence-associated β-galactosidase (SA-β-gal) and TUNEL assay, respectively. Quantitative RT-PCR was used to measure mRNA expression of various genes, including syndecan-1, stem cell factors, and markers of differentiation into squamous, glandular, or neuroendocrine cells. Overexpression of miR-145 induced cell senescence, and thus significantly inhibited cell proliferation in T24 and KU7 cells. Syndecan-1 expression diminished, whereas stem cell markers such as SOX2, NANOG, OCT4, and E2F3 increased. miR-145 also up-regulated markers of differentiation into squamous (p63, TP63, and CK5), glandular (MUC-1, MUC-2, and MUC-5 AC), and neuroendocrine cells (NSE and UCHL-1). Finally, expression of miR-145 was down-regulated in high-grade urothelial carcinomas, but not in low-grade tumors. Results indicate that miR-145 suppresses syndecan-1 and, by this mechanism, up-regulates stem cell factors and induces cell senescence and differentiation. We propose that miR-145 may confer stem cell-like properties on urothelial carcinoma cells and thus facilitate differentiation into multiple cell types. The online version of this article (doi:10.1186/s12885-015-1846-0) contains supplementary material, which is available to authorized users

  9. Identification of novel regulators in T-cell differentiation of aplastic anemia patients

    Directory of Open Access Journals (Sweden)

    Probst-Kepper Michael

    2006-10-01

    Full Text Available Abstract Background Aplastic anemia (AA is a bone marrow failure syndrome mostly characterized by an immune-mediated destruction of marrow hematopoietic progenitor/stem cells. The resulting hypocellularity limits a detailed analysis of the cellular immune response. To overcome this technical problem we performed a microarray analysis of CD3+ T-cells derived from bone marrow aspirates and peripheral blood samples of newly diagnosed AA patients and healthy volunteers. Two AA patients were additionally analyzed after achieving a partial remission following immunosuppression. The regulation of selected candidate genes was confirmed by real-time RT-PCR. Results Among more than 22.200 transcripts, 583 genes were differentially expressed in the bone marrow of AA patients compared to healthy controls. Dysregulated genes are involved in T-cell mediated cytotoxicity, immune response of Th1 differentiated T-cells, and major regulators of immune function. In hematological remission the expression levels of several candidate genes tend to normalize, such as immune regulators and genes involved in proinflammatory immune response. Conclusion Our study suggests a pivotal role of Th1/Tc1 differentiated T-cells in immune-mediated marrow destruction of AA patients. Most importantly, immune regulatory genes could be identified, which are likely involved in the recovery of hematopoiesis and may help to design new therapeutic strategies in bone marrow failure syndromes.

  10. Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Green, A.C. [St Vincent' s Institute, Fitzroy, Victoria 3065 (Australia); Department of Medicine at St. Vincent' s Hospital, The University of Melbourne, Victoria 3065 (Australia); Kocovski, P.; Jovic, T.; Walia, M.K. [St Vincent' s Institute, Fitzroy, Victoria 3065 (Australia); Chandraratna, R.A.S. [IO Therapeutics, Inc., Santa Ana, CA 92705 (United States); Martin, T.J.; Baker, E.K. [St Vincent' s Institute, Fitzroy, Victoria 3065 (Australia); Department of Medicine at St. Vincent' s Hospital, The University of Melbourne, Victoria 3065 (Australia); Purton, L.E., E-mail: lpurton@svi.edu.au [St Vincent' s Institute, Fitzroy, Victoria 3065 (Australia); Department of Medicine at St. Vincent' s Hospital, The University of Melbourne, Victoria 3065 (Australia)

    2017-01-01

    Low and high serum retinol levels are associated with increased fracture risk and poor bone health. We recently showed retinoic acid receptors (RARs) are negative regulators of osteoclastogenesis. Here we show RARs are also negative regulators of osteoblast and adipocyte differentiation. The pan-RAR agonist, all-trans retinoic acid (ATRA), directly inhibited differentiation and mineralisation of early osteoprogenitors and impaired the differentiation of more mature osteoblast populations. In contrast, the pan-RAR antagonist, IRX4310, accelerated differentiation of early osteoprogenitors. These effects predominantly occurred via RARγ and were further enhanced by an RARα agonist or antagonist, respectively. RAR agonists similarly impaired adipogenesis in osteogenic cultures. RAR agonist treatment resulted in significant upregulation of the Wnt antagonist, Sfrp4. This accompanied reduced nuclear and cytosolic β-catenin protein and reduced expression of the Wnt target gene Axin2, suggesting impaired Wnt/β-catenin signalling. To determine the effect of RAR inhibition in post-natal mice, IRX4310 was administered to male mice for 10 days and bones were assessed by µCT. No change to trabecular bone volume was observed, however, radial bone growth was impaired. These studies show RARs directly influence osteoblast and adipocyte formation from mesenchymal cells, and inhibition of RAR signalling in vivo impairs radial bone growth in post-natal mice. - Graphical abstract: Schematic shows RAR ligand regulation of osteoblast differentiation in vitro. RARγ antagonists±RARα antagonists promote osteoblast differentiation. RARγ and RARα agonists alone or in combination block osteoblast differentiation, which correlates with upregulation of Sfrp4, and downregulation of nuclear and cytosolic β-catenin and reduced expression of the Wnt target gene Axin2. Red arrows indicate effects of RAR agonists on mediators of Wnt signalling.

  11. SNF5 is an essential executor of epigenetic regulation during differentiation.

    Science.gov (United States)

    You, Jueng Soo; De Carvalho, Daniel D; Dai, Chao; Liu, Minmin; Pandiyan, Kurinji; Zhou, Xianghong J; Liang, Gangning; Jones, Peter A

    2013-04-01

    Nucleosome occupancy controls the accessibility of the transcription machinery to DNA regulatory regions and serves an instructive role for gene expression. Chromatin remodelers, such as the BAF complexes, are responsible for establishing nucleosome occupancy patterns, which are key to epigenetic regulation along with DNA methylation and histone modifications. Some reports have assessed the roles of the BAF complex subunits and stemness in murine embryonic stem cells. However, the details of the relationships between remodelers and transcription factors in altering chromatin configuration, which ultimately affects gene expression during cell differentiation, remain unclear. Here for the first time we demonstrate that SNF5, a core subunit of the BAF complex, negatively regulates OCT4 levels in pluripotent cells and is essential for cell survival during differentiation. SNF5 is responsible for generating nucleosome-depleted regions (NDRs) at the regulatory sites of OCT4 repressed target genes such as PAX6 and NEUROG1, which are crucial for cell fate determination. Concurrently, SNF5 closes the NDRs at the regulatory regions of OCT4-activated target genes such as OCT4 itself and NANOG. Furthermore, using loss- and gain-of-function experiments followed by extensive genome-wide analyses including gene expression microarrays and ChIP-sequencing, we highlight that SNF5 plays dual roles during differentiation by antagonizing the expression of genes that were either activated or repressed by OCT4, respectively. Together, we demonstrate that SNF5 executes the switch between pluripotency and differentiation.

  12. SNF5 is an essential executor of epigenetic regulation during differentiation.

    Directory of Open Access Journals (Sweden)

    Jueng Soo You

    2013-04-01

    Full Text Available Nucleosome occupancy controls the accessibility of the transcription machinery to DNA regulatory regions and serves an instructive role for gene expression. Chromatin remodelers, such as the BAF complexes, are responsible for establishing nucleosome occupancy patterns, which are key to epigenetic regulation along with DNA methylation and histone modifications. Some reports have assessed the roles of the BAF complex subunits and stemness in murine embryonic stem cells. However, the details of the relationships between remodelers and transcription factors in altering chromatin configuration, which ultimately affects gene expression during cell differentiation, remain unclear. Here for the first time we demonstrate that SNF5, a core subunit of the BAF complex, negatively regulates OCT4 levels in pluripotent cells and is essential for cell survival during differentiation. SNF5 is responsible for generating nucleosome-depleted regions (NDRs at the regulatory sites of OCT4 repressed target genes such as PAX6 and NEUROG1, which are crucial for cell fate determination. Concurrently, SNF5 closes the NDRs at the regulatory regions of OCT4-activated target genes such as OCT4 itself and NANOG. Furthermore, using loss- and gain-of-function experiments followed by extensive genome-wide analyses including gene expression microarrays and ChIP-sequencing, we highlight that SNF5 plays dual roles during differentiation by antagonizing the expression of genes that were either activated or repressed by OCT4, respectively. Together, we demonstrate that SNF5 executes the switch between pluripotency and differentiation.

  13. Convergent differential regulation of parvalbumin in the brains of vocal learners.

    Directory of Open Access Journals (Sweden)

    Erina Hara

    Full Text Available Spoken language and learned song are complex communication behaviors found in only a few species, including humans and three groups of distantly related birds--songbirds, parrots, and hummingbirds. Despite their large phylogenetic distances, these vocal learners show convergent behaviors and associated brain pathways for vocal communication. However, it is not clear whether this behavioral and anatomical convergence is associated with molecular convergence. Here we used oligo microarrays to screen for genes differentially regulated in brain nuclei necessary for producing learned vocalizations relative to adjacent brain areas that control other behaviors in avian vocal learners versus vocal non-learners. A top candidate gene in our screen was a calcium-binding protein, parvalbumin (PV. In situ hybridization verification revealed that PV was expressed significantly higher throughout the song motor pathway, including brainstem vocal motor neurons relative to the surrounding brain regions of all distantly related avian vocal learners. This differential expression was specific to PV and vocal learners, as it was not found in avian vocal non-learners nor for control genes in learners and non-learners. Similar to the vocal learning birds, higher PV up-regulation was found in the brainstem tongue motor neurons used for speech production in humans relative to a non-human primate, macaques. These results suggest repeated convergent evolution of differential PV up-regulation in the brains of vocal learners separated by more than 65-300 million years from a common ancestor and that the specialized behaviors of learned song and speech may require extra calcium buffering and signaling.

  14. miR-24-mediated down-regulation of H2AX suppresses DNA repair in terminally differentiated blood cells

    Science.gov (United States)

    Lal, Ashish; Pan, Yunfeng; Navarro, Francisco; Dykxhoorn, Derek M.; Moreau, Lisa; Meire, Eti; Bentwich, Zvi; Lieberman, Judy; Chowdhury, Dipanjan

    2010-01-01

    Terminally differentiated cells have reduced capacity to repair double strand breaks (DSB), but the molecular mechanism behind this down-regulation is unclear. Here we find that miR-24 is consistently up-regulated during post-mitotic differentiation of hematopoietic cell lines and regulates the histone variant H2AX, a key DSB repair protein that activates cell cycle checkpoint proteins and retains DSB repair factors at DSB foci. The H2AX 3’UTR contains conserved miR-24 binding sites regulated by miR-24. Both H2AX mRNA and protein are substantially reduced during hematopoietic cell terminal differentiation by miR-24 up-regulation both in in vitro differentiated cells and primary human blood cells. miR-24 suppression of H2AX renders cells hypersensitive to γ-irradiation and genotoxic drugs. Antagonizing miR-24 in differentiating cells protects them from DNA damage-induced cell death, while transfecting miR-24 mimics in dividing cells increases chromosomal breaks and unrepaired DNA damage and reduces viability in response to DNA damage. This DNA repair phenotype can be fully rescued by over-expressing miR-24-insensitive H2AX. Therefore, miR-24 up-regulation in post-replicative cells reduces H2AX and thereby renders them highly vulnerable to DNA damage. PMID:19377482

  15. SIRT3/SOD2 maintains osteoblast differentiation and bone formation by regulating mitochondrial stress

    OpenAIRE

    Gao, Jing; Feng, Zhihui; Wang, Xueqiang; Zeng, Mengqi; Liu, Jing; Han, Shujun; Xu, Jie; Chen, Lei; Cao, Ke; Long, Jiangang; Li, Zongfang; Shen, Weili; Liu, Jiankang

    2017-01-01

    Recent studies have revealed robust metabolic changes during cell differentiation. Mitochondria, the organelles where many vital metabolic reactions occur, may play an important role. Here, we report the involvement of SIRT3-regulated mitochondrial stress in osteoblast differentiation and bone formation. In both the osteoblast cell line MC3T3-E1 and primary calvarial osteoblasts, robust mitochondrial biogenesis and supercomplex formation were observed during differentiation, accompanied by in...

  16. Regulation of mat responses by a differentiation MAPK pathway in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Sheelarani Karunanithi

    Full Text Available Fungal species exhibit diverse behaviors when presented with extracellular challenges. Pathogenic fungi can undergo cell differentiation and biofilm formation in response to fluctuating nutrient levels, and these responses are required for virulence. In the model fungal eukaryote Saccharomyces cerevisiae, nutrient limitation induces filamentous growth and biofilm/mat formation. Both responses require the same signal transduction (MAPK pathway and the same cell adhesion molecule (Flo11 but have been studied under different conditions. We found that filamentous growth and mat formation are aspects of a related response that is regulated by the MAPK pathway. Cells in yeast-form mats differentiated into pseudohyphae in response to nutrient limitation. The MAPK pathway regulated mat expansion (in the plane of the XY-axis and substrate invasion (downward in the plane of the Z-axis, which optimized the mat's response to extracellular nutrient levels. The MAPK pathway also regulated an upward growth pattern (in the plane of the Z-axis in response to nutrient limitation and changes in surface rigidity. Upward growth allowed for another level of mat responsiveness and resembled a type of colonial chemorepulsion. Together our results show that signaling pathways play critical roles in regulating social behaviors in which fungal cells participate. Signaling pathways may regulate similar processes in pathogens, whose highly nuanced responses are required for virulence.

  17. B-cell activating factor detected on both naïve and memory B cells in bullous pemphigoid.

    Science.gov (United States)

    Qian, Hua; Kusuhara, Masahiro; Li, Xiaoguang; Tsuruta, Daisuke; Tsuchisaka, Atsunari; Ishii, Norito; Koga, Hiroshi; Hayakawa, Taihei; Ohara, Koji; Karashima, Tadashi; Ohyama, Bungo; Ohata, Chika; Furumura, Minao; Hashimoto, Takashi

    2014-08-01

    B-cell activating factor (BAFF), an important immune regulatory cytokine, is involved in development of autoimmune diseases. Although BAFF is expressed in various cells, including dendritic cells (DCs) and monocytes, BAFF expression on B cells has not been well documented. In the present study, BAFF molecules on DCs and naïve and memory B cells in autoimmune bullous diseases, including pemphigus vulgaris, pemphigus foliaceus and bullous pemphigoid (BP), were analysed by flow cytometry. Compared with healthy controls (HC), BAFF expression on naïve and memory B cells increased significantly in BP. No difference in BAFF receptor expression in naïve and memory B cells was shown among all study groups. Furthermore, BAFF expression in both naïve and memory B cells of BP, but not HC, was detected by confocal microscopic analysis. These results implied that BAFF expressed by B cells may play a pathogenic role in autoimmune bullous diseases, particularly BP. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Glucose metabolism regulates T cell activation, differentiation and functions

    Directory of Open Access Journals (Sweden)

    Clovis Steve Palmer

    2015-01-01

    Full Text Available The adaptive immune system is equipped to eliminate both tumors and pathogenic microorganisms. It requires a series of complex and coordinated signals to drive the activation, proliferation and differentiation of appropriate T cell subsets. It is now established that changes in cellular activation are coupled to profound changes in cellular metabolism. In addition, emerging evidence now suggest that specific metabolic alterations associated with distinct T cell subsets may be ancillary to their differentiation and influential in their immune functions. The Warburg effect originally used to describe a phenomenon in which most cancer cells relied on aerobic glycolysis for their growth is a key process that sustain T cell activation and differentiation. Here we review how different aspects of metabolism in T cells influence their functions, focusing on the emerging role of key regulators of glucose metabolism such as HIF-1α. A thorough understanding of the role of metabolism in T cell function could provide insights into mechanisms involved in inflammatory-mediated conditions, with the potential for developing novel therapeutic approaches to treat these diseases.

  19. MicroRNA let-7b regulates neural stem cell proliferation and differentiation by targeting nuclear receptor TLX signaling.

    Science.gov (United States)

    Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Lang, Ming-Fei; Yang, Su; Li, Wendong; Shi, Yanhong

    2010-02-02

    Neural stem cell self-renewal and differentiation is orchestrated by precise control of gene expression involving nuclear receptor TLX. Let-7b, a member of the let-7 microRNA family, is expressed in mammalian brains and exhibits increased expression during neural differentiation. However, the role of let-7b in neural stem cell proliferation and differentiation remains unknown. Here we show that let-7b regulates neural stem cell proliferation and differentiation by targeting the stem cell regulator TLX and the cell cycle regulator cyclin D1. Overexpression of let-7b led to reduced neural stem cell proliferation and increased neural differentiation, whereas antisense knockdown of let-7b resulted in enhanced proliferation of neural stem cells. Moreover, in utero electroporation of let-7b to embryonic mouse brains led to reduced cell cycle progression in neural stem cells. Introducing an expression vector of Tlx or cyclin D1 that lacks the let-7b recognition site rescued let-7b-induced proliferation deficiency, suggesting that both TLX and cyclin D1 are important targets for let-7b-mediated regulation of neural stem cell proliferation. Let-7b, by targeting TLX and cyclin D1, establishes an efficient strategy to control neural stem cell proliferation and differentiation.

  20. APRIL is overexpressed in cancer: link with tumor progression

    International Nuclear Information System (INIS)

    Moreaux, Jérôme; Veyrune, Jean-Luc; De Vos, John; Klein, Bernard

    2009-01-01

    BAFF and APRIL share two receptors – TACI and BCMA – and BAFF binds to a third receptor, BAFF-R. Increased expression of BAFF and APRIL is noted in hematological malignancies. BAFF and APRIL are essential for the survival of normal and malignant B lymphocytes, and altered expression of BAFF or APRIL or of their receptors (BCMA, TACI, or BAFF-R) have been reported in various B-cell malignancies including B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's lymphoma, multiple myeloma, and Waldenstrom's macroglobulinemia. We compared the expression of BAFF, APRIL, TACI and BAFF-R gene expression in 40 human tumor types – brain, epithelial, lymphoid, germ cells – to that of their normal tissue counterparts using publicly available gene expression data, including the Oncomine Cancer Microarray database. We found significant overexpression of TACI in multiple myeloma and thyroid carcinoma and an association between TACI expression and prognosis in lymphoma. Furthermore, BAFF and APRIL are overexpressed in many cancers and we show that APRIL expression is associated with tumor progression. We also found overexpression of at least one proteoglycan with heparan sulfate chains (HS), which are coreceptors for APRIL and TACI, in tumors where APRIL is either overexpressed or is a prognostic factor. APRIL could induce survival or proliferation directly through HS proteoglycans. Taken together, these data suggest that APRIL is a potential prognostic factor for a large array of malignancies

  1. Pbx and Prdm1a transcription factors differentially regulate subsets of the fast skeletal muscle program in zebrafish

    Directory of Open Access Journals (Sweden)

    Zizhen Yao

    2013-04-01

    The basic helix–loop–helix factor Myod initiates skeletal muscle differentiation by directly and sequentially activating sets of muscle differentiation genes, including those encoding muscle contractile proteins. We hypothesize that Pbx homeodomain proteins direct Myod to a subset of its transcriptional targets, in particular fast-twitch muscle differentiation genes, thereby regulating the competence of muscle precursor cells to differentiate. We have previously shown that Pbx proteins bind with Myod on the promoter of the zebrafish fast muscle gene mylpfa and that Pbx proteins are required for Myod to activate mylpfa expression and the fast-twitch muscle-specific differentiation program in zebrafish embryos. Here we have investigated the interactions of Pbx with another muscle fiber-type regulator, Prdm1a, a SET-domain DNA-binding factor that directly represses mylpfa expression and fast muscle differentiation. The prdm1a mutant phenotype, early and increased fast muscle differentiation, is the opposite of the Pbx-null phenotype, delayed and reduced fast muscle differentiation. To determine whether Pbx and Prdm1a have opposing activities on a common set of genes, we used RNA-seq analysis to globally assess gene expression in zebrafish embryos with single- and double-losses-of-function for Pbx and Prdm1a. We find that the levels of expression of certain fast muscle genes are increased or approximately wild type in pbx2/4-MO;prdm1a−/− embryos, suggesting that Pbx activity normally counters the repressive action of Prdm1a for a subset of the fast muscle program. However, other fast muscle genes require Pbx but are not regulated by Prdm1a. Thus, our findings reveal that subsets of the fast muscle program are differentially regulated by Pbx and Prdm1a. Our findings provide an example of how Pbx homeodomain proteins act in a balance with other transcription factors to regulate subsets of a cellular differentiation program.

  2. Expression profiling of genes regulated by TGF-beta: Differential regulation in normal and tumour cells

    Directory of Open Access Journals (Sweden)

    Takahashi Takashi

    2007-04-01

    Full Text Available Abstract Background TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells. Results Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated by TGF-beta in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell-lines. Semi-quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. In order to identify the signalling pathways that influence TGF-beta mediated gene regulation, we used specific inhibitors of p38 MAP kinase, ERK kinase, JNK kinase and integrin signalling pathways. The data suggest that regulation of majority of the selected genes is dependent on at least one of these pathways and this dependence is cell-type specific. Interestingly, an integrin pathway inhibitor, RGD peptide, significantly affected TGF-beta regulation of Thrombospondin 1 in A549 cells. Conclusion These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells and significant role of non-canonical TGF-beta pathways in the regulation of many genes by TGF-beta.

  3. Higher B-cell activating factor levels at birth are positively associated with maternal dairy farm exposure and negatively related to allergy development.

    Science.gov (United States)

    Lundell, Anna-Carin; Hesselmar, Bill; Nordström, Inger; Adlerberth, Ingegerd; Wold, Agnes E; Rudin, Anna

    2015-10-01

    A high proportion of circulating immature/naive CD5(+) B cells during early infancy is a risk factor for allergy development. B-cell activating factor (BAFF) is an important cytokine for B-cell maturation. We sought to investigate whether BAFF levels are related to environmental exposures during pregnancy and early childhood and whether BAFF levels are associated with postnatal B-cell maturation and allergic disease. In the FARMFLORA study, including both farming and nonfarming families, we measured BAFF levels in plasma from mothers and their children at birth and at 1, 4, 18, and 36 months of age. Infants' blood samples were also analyzed for B-cell numbers and proportions of CD5(+) and CD27(+) B cells. Allergic disease was clinically evaluated at 18 and 36 months of age. Circulating BAFF levels were maximal at birth, and farmers' children had higher BAFF levels than nonfarmers' children. Higher BAFF levels at birth were positively associated with proportions of CD27(+) memory B cells among farmers' children and inversely related to proportions of CD5(+) immature/naive B cells among nonfarmers' children. Children with allergic disease at 18 months of age had lower cord blood BAFF levels than nonallergic children. At birth, girls had higher BAFF levels and lower proportions of CD5(+) B cells than boys. Farm exposure during pregnancy appears to induce BAFF production in the newborn child, and high neonatal BAFF levels were associated with more accelerated postnatal B-cell maturation, which lend further strength to the role of B cells in the hygiene hypothesis. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  4. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

    Science.gov (United States)

    Eskildsen, Tilde; Taipaleenmäki, Hanna; Stenvang, Jan; Abdallah, Basem M.; Ditzel, Nicholas; Nossent, Anne Yael; Bak, Mads; Kauppinen, Sakari; Kassem, Moustapha

    2011-01-01

    Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3′ UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central role in promoting osteoblast differentiation, as a bona fide target of miR-138. We show that miR-138 attenuates bone formation in vivo, at least in part by inhibiting the focal adhesion kinase signaling pathway. Our findings suggest that pharmacological inhibition of miR-138 by antimiR-138 could represent a therapeutic strategy for enhancing bone formation in vivo. PMID:21444814

  5. Can green consumerism replace environmental regulation? A differentiated-products example

    Energy Technology Data Exchange (ETDEWEB)

    Eriksson, Clas [Department of Economics, Swedish University of Agricultural Sciences, P.O. Box 7013, S-750 07 Uppsala (Sweden)

    2004-09-01

    This paper assumes that consumers are willing to pay an extra premium for a good if it has a low impact on the environment. We examine if a little dose of such idealistic behavior has a large impact on the market equilibrium, and to what extent it can replace the environmental regulation. The analysis is carried out in a model with product differentiation, where consumers differ in their preferences for product quality. Consumers' willingness to pay the environmental premium may be uniformly or non-uniformly distributed. Green consumerism will only be modestly influential in both cases, despite the fact that product differentiation leads to relaxed competition and increased profits, and thereby creates leverage.

  6. Can green consumerism replace environmental regulation? A differentiated-products example

    International Nuclear Information System (INIS)

    Eriksson, Clas

    2004-01-01

    This paper assumes that consumers are willing to pay an extra premium for a good if it has a low impact on the environment. We examine if a little dose of such idealistic behavior has a large impact on the market equilibrium, and to what extent it can replace the environmental regulation. The analysis is carried out in a model with product differentiation, where consumers differ in their preferences for product quality. Consumers' willingness to pay the environmental premium may be uniformly or non-uniformly distributed. Green consumerism will only be modestly influential in both cases, despite the fact that product differentiation leads to relaxed competition and increased profits, and thereby creates leverage

  7. Ubx regulates differential enlargement and diversification of insect hind legs.

    Directory of Open Access Journals (Sweden)

    Najmus Mahfooz

    2007-09-01

    Full Text Available Differential enlargement of hind (T3 legs represents one of the hallmarks of insect evolution. However, the actual mechanism(s responsible are yet to be determined. To address this issue, we have now studied the molecular basis of T3 leg enlargement in Oncopeltus fasciatus (milkweed bug and Acheta domesticus (house cricket. In Oncopeltus, the T3 tibia displays a moderate increase in size, whereas in Acheta, the T3 femur, tibia, and tarsus are all greatly enlarged. Here, we show that the hox gene Ultrabithorax (Ubx is expressed in the enlarged segments of hind legs. Furthermore, we demonstrate that depletion of Ubx during embryogenesis has a primary effect in T3 legs and causes shortening of leg segments that are enlarged in a wild type. This result shows that Ubx is regulating the differential growth and enlargement of T3 legs in both Oncopeltus and Acheta. The emerging view suggests that Ubx was co-opted for a novel role in regulating leg growth and that the transcriptional modification of its expression may be a universal mechanism for the evolutionary diversification of insect hind legs.

  8. Biphasic regulation of intracellular calcium by gemfibrozil contributes to inhibiting L6 myoblast differentiation: implications for clinical myotoxicity.

    Science.gov (United States)

    Liu, Aiming; Yang, Julin; Gonzalez, Frank J; Cheng, Gary Q; Dai, Renke

    2011-02-18

    Gemfibrozil is the most myotoxic fibrate drug commonly used for dyslipidemia, but the mechanism is poorly understood. The current study revealed that gemfibrozil inhibits myoblast differentiation through the regulation of intracellular calcium ([Ca(2+)]i) as revealed in L6 myoblasts by use of laser scan confocal microscopy and flow cytometry using Fluo-4 AM as a probe. Gemfibrozil at 20-400 μM, could regulate [Ca(2+)]i in L6 cells in a biphasic manner, and sustained reduction was observed when the concentration reached 200 μM. Inhibition of L6 differentiation by gemfibrozil was concentration-dependent with maximal effect noted between 200 and 400 μM, as indicated by creatine kinase activities and the differentiation index, respectively. In differentiating L6 myoblasts, gemfibrozil at concentrations below 400 μM led to no significant signs of apoptosis or cytotoxicity, whereas differentiation, inhibited by 200 μM gemfibrozil, was only partially recovered. A good correlation was noted between gemfibrozil concentrations that regulate [Ca(2+)]i and inhibit L6 myoblasts differentiation, and both are within the range of total serum concentrations found in the clinic. These data suggest a potential pharmacodynamic effect of gemfibrozil on myogenesis as a warning sign, in addition to the complex pharmacokinetic interactions. It is also noteworthy that mobilization of [Ca(2+)]i by gemfibrozil may trigger complex biological responses besides myocyte differentiation. Information revealed in this study explores the mechanism of gemfibrozil-induced myotoxicity through the regulation of intracellular calcium.

  9. MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Zhiyuan; Sun, Xiaorui; Xu, Dequan; Xiong, Yuanzhu; Zuo, Bo, E-mail: zuobo@mail.hzau.edu.cn

    2015-11-27

    Myogenesis is a complex process including myoblast proliferation, differentiation and myotube formation and is controlled by myogenic regulatory factors (MRFs), MyoD, MyoG, Myf5 and Myf6 (also known as MRF4). MicroRNA is a kind of ∼22 nt-long non-coding small RNAs, and act as key transcriptional or post-transcriptional regulators of gene expression. Identification of miRNAs involved in the regulation of muscle genes could improve our understanding of myogenesis process. In this study, we investigated the regulation of Myf6 gene by miRNAs. We showed that miR-374b specifically bound to the 3'untranslated region (UTR) of Myf6 and down-regulated the expression of Myf6 gene at both mRNA and protein level. Furthermore, miR-374b is ubiquitously expressed in the tissues of adult C57BL6 mouse, and the mRNA abundance increases first and then decreases during C2C12 myoblasts differentiation. Over-expression of miR-374b impaired C2C12 cell differentiation, while inhibiting miR-374b expression by 2′-O-methyl antisense oligonucleotides promoted C2C12 cell differentiation. Taken together, our findings identified miR-374b directly targets Myf6 and negatively regulates myogenesis. - Highlights: • MiR-374b directly targets 3′UTR of Myf6. • MiR-374b negatively regulates Myf6 in C2C12 cells. • MiR-374b abundance significiently changes during C2C12 cells differentiation. • MiR-374b negatively regulates C2C12 cells differentiation.

  10. Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ying [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023 (China); Huang, Xiaohua [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Clinical Biochemistry, College of Laboratory Medicine, Dalian Medical University, Dalian 116044 (China); An, Yue [Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023 (China); Ren, Feng [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Yang, Zara Zhuyun; Zhu, Hongmei; Zhou, Lei [The Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650228 (China); Department of Anatomy and Developmental Biology, Monash University, Clayton 3800 (Australia); He, Xiaowen; Schachner, Melitta [Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, New Brunswick, NJ (United States); Xiao, Zhicheng, E-mail: zhicheng.xiao@monash.edu [The Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650228 (China); Department of Anatomy and Developmental Biology, Monash University, Clayton 3800 (Australia); Ma, Keli, E-mail: makeli666@aliyun.com [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Li, Yali, E-mail: yalilipaper@gmail.com [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Anatomy, National University of Singapore, Singapore 119078 (Singapore)

    2013-10-25

    Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.

  11. Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

    International Nuclear Information System (INIS)

    Li, Ying; Huang, Xiaohua; An, Yue; Ren, Feng; Yang, Zara Zhuyun; Zhu, Hongmei; Zhou, Lei; He, Xiaowen; Schachner, Melitta; Xiao, Zhicheng; Ma, Keli; Li, Yali

    2013-01-01

    Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression

  12. Distinct bone marrow blood vessels differentially regulate haematopoiesis.

    Science.gov (United States)

    Itkin, Tomer; Gur-Cohen, Shiri; Spencer, Joel A; Schajnovitz, Amir; Ramasamy, Saravana K; Kusumbe, Anjali P; Ledergor, Guy; Jung, Yookyung; Milo, Idan; Poulos, Michael G; Kalinkovich, Alexander; Ludin, Aya; Kollet, Orit; Shakhar, Guy; Butler, Jason M; Rafii, Shahin; Adams, Ralf H; Scadden, David T; Lin, Charles P; Lapidot, Tsvee

    2016-04-21

    Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.

  13. UVA and UVB Irradiation Differentially Regulate microRNA Expression in Human Primary Keratinocytes

    Science.gov (United States)

    Kraemer, Anne; Chen, I-Peng; Henning, Stefan; Faust, Alexandra; Volkmer, Beate; Atkinson, Michael J.; Moertl, Simone; Greinert, Ruediger

    2013-01-01

    MicroRNA (miRNA)-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2), which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis. PMID:24391759

  14. UVA and UVB irradiation differentially regulate microRNA expression in human primary keratinocytes.

    Directory of Open Access Journals (Sweden)

    Anne Kraemer

    Full Text Available MicroRNA (miRNA-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2, which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis.

  15. DISC1 (disrupted-in-schizophrenia-1 regulates differentiation of oligodendrocytes.

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Hattori

    Full Text Available Disrupted-in-schizophrenia 1 (DISC1 is a gene disrupted by a translocation, t(1;11 (q42.1;q14.3, that segregates with major psychiatric disorders, including schizophrenia, recurrent major depression and bipolar affective disorder, in a Scottish family. Here we report that mammalian DISC1 endogenously expressed in oligodendroglial lineage cells negatively regulates differentiation of oligodendrocyte precursor cells into oligodendrocytes. DISC1 expression was detected in oligodendrocytes of the mouse corpus callosum at P14 and P70. DISC1 mRNA was expressed in primary cultured rat cortical oligodendrocyte precursor cells and decreased when oligodendrocyte precursor cells were induced to differentiate by PDGF deprivation. Immunocytochemical analysis showed that overexpressed DISC1 was localized in the cell bodies and processes of oligodendrocyte precursor cells and oligodendrocytes. We show that expression of the myelin related markers, CNPase and MBP, as well as the number of cells with a matured oligodendrocyte morphology, were decreased following full length DISC1 overexpression. Conversely, both expression of CNPase and the number of oligodendrocytes with a mature morphology were increased following knockdown of endogenous DISC1 by RNA interference. Overexpression of a truncated form of DISC1 also resulted in an increase in expression of myelin related proteins and the number of mature oligodendrocytes, potentially acting via a dominant negative mechanism. We also identified involvement of Sox10 and Nkx2.2 in the DISC1 regulatory pathway of oligodendrocyte differentiation, both well-known transcription factors involved in the regulation of myelin genes.

  16. Role of RHEB in Regulating Differentiation Fate of Mesenchymal Stem Cells for Cartilage and Bone Regeneration

    Directory of Open Access Journals (Sweden)

    Sajjad Ashraf

    2017-04-01

    Full Text Available Advances in mesenchymal stem cells (MSCs and cell replacement therapies are promising approaches to treat cartilage and bone defects since substantial differentiation capacities of MSCs match the demands of tissue regeneration. Our understanding of the dynamic process requiring indispensable differentiation of MSCs remains limited. Herein, we describe the role of RHEB (Ras homolog enriched in brain regulating gene signature for differentiation of human adipose derived mesenchymal stem cells (ASCs into chondrogenic, osteogenic, and adipogenic lineages. RHEB-overexpression increases the proliferation of the ASCs. RHEB enhances the chondrogenic differentiation of ASCs in 3D culture via upregulation of SOX9 with concomitant increase in glycosaminoglycans (GAGs, and type II collagen (COL2. RHEB increases the osteogenesis via upregulation of runt related transcription factor 2 (RUNX2 with an increase in the calcium and phosphate contents. RHEB also increases the expression of osteogenic markers, osteonectin and osteopontin. RHEB knockdown ASCs were incapable of expressing sufficient SRY (Sex determining region Y-box 9 (SOX9 and RUNX2, and therefore had decreased chondrogenic and osteogenic differentiation. RHEB-overexpression impaired ASCs differentiation into adipogenic lineage, through downregulation of CCAAT/enhancer binding protein beta (C/EBPβ. Conversely, RHEB knockdown abolished the negative regulation of adipogenesis. We demonstrate that RHEB is a novel regulator, with a critical role in ASCs lineage determination, and RHEB-modulated ASCs may be useful as a cell therapy for cartilage and bone defect treatments.

  17. Angiotensin II Regulation of Proliferation, Differentiation, and Engraftment of Hematopoietic Stem Cells.

    Science.gov (United States)

    Kim, Seungbum; Zingler, Michael; Harrison, Jeffrey K; Scott, Edward W; Cogle, Christopher R; Luo, Defang; Raizada, Mohan K

    2016-03-01

    Emerging evidence indicates that differentiation and mobilization of hematopoietic cell are critical in the development and establishment of hypertension and hypertension-linked vascular pathophysiology. This, coupled with the intimate involvement of the hyperactive renin-angiotensin system in hypertension, led us to investigate the hypothesis that chronic angiotensin II (Ang II) infusion affects hematopoietic stem cell (HSC) regulation at the level of the bone marrow. Ang II infusion resulted in increases in hematopoietic stem/progenitor cells (83%) and long-term HSC (207%) in the bone marrow. Interestingly, increases of HSCs and long-term HSCs were more pronounced in the spleen (228% and 1117%, respectively). Furthermore, we observed higher expression of C-C chemokine receptor type 2 in these HSCs, indicating there was increased myeloid differentiation in Ang II-infused mice. This was associated with accumulation of C-C chemokine receptor type 2(+) proinflammatory monocytes in the spleen. In contrast, decreased engraftment efficiency of GFP(+) HSC was observed after Ang II infusion. Time-lapse in vivo imaging and in vitro Ang II pretreatment demonstrated that Ang II induces untimely proliferation and differentiation of the donor HSC resulting in diminished HSC engraftment and bone marrow reconstitution. We conclude that (1) chronic Ang II infusion regulates HSC proliferation, mediated by angiotensin receptor type 1a, (2) Ang II accelerates HSC to myeloid differentiation resulting in accumulation of C-C chemokine receptor type 2(+) HSCs and inflammatory monocytes in the spleen, and (3) Ang II impairs homing and reconstitution potentials of the donor HSCs. These observations highlight the important regulatory roles of Ang II on HSC proliferation, differentiation, and engraftment. © 2016 American Heart Association, Inc.

  18. CCAAT/Enhancer Binding Protein β Regulates Expression of Indian Hedgehog during Chondrocytes Differentiation

    Science.gov (United States)

    Ushijima, Takahiro; Okazaki, Ken; Tsushima, Hidetoshi; Ishihara, Kohei; Doi, Toshio; Iwamoto, Yukihide

    2014-01-01

    Background CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that promotes hypertrophic differentiation of chondrocytes. Indian hedgehog (Ihh) also stimulates the hypertrophic transition of chondrocytes. Furthermore, runt-related transcription factor-2 (RUNX2) was reported to regulate chondrocyte maturation during skeletal development and to directly regulate transcriptional activity of Ihh. In this study, we investigated whether the interaction of C/EBPβ and RUNX2 regulates the expression of Ihh during chondrocyte differentiation. Methodology/Results Immunohistochemistry of embryonic growth plate revealed that both C/EBPβ and Ihh were strongly expressed in pre-hypertrophic and hypertrophic chondrocytes. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked stimulation of Ihh and Runx2. Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells. A reporter assay revealed that C/EBPβ stimulated transcriptional activity of Ihh. Deletion and mutation analysis showed that the C/EBPβ responsive element was located between −214 and −210 bp in the Ihh promoter. An electrophoretic mobility shift assay (EMSA) and a chromatin immunoprecipitation (ChIP) assay also revealed the direct binding of C/EBPβ to this region. Moreover, reporter assays demonstrated that RUNX2 failed to stimulate the transcriptional activity of the Ihh promoter harboring a mutation at the C/EBPβ binding site. EMSA and ChIP assays showed that RUNX2 interacted to this element with C/EBPβ. Immunoprecipitation revealed that RUNX2 and C/EBPβ formed heterodimer complex with each other in the nuclei of chondrocytes. These data suggested that the C/EBPβ binding element is also important for RUNX2 to regulate the expression of Ihh. Ex vivo organ culture of mouse limbs transfected with C/EBPβ showed that the expression of Ihh and RUNX2 was increased upon ectopic C/EBPβ expression. Conclusions C

  19. CCAAT/enhancer binding protein β regulates expression of Indian hedgehog during chondrocytes differentiation.

    Directory of Open Access Journals (Sweden)

    Takahiro Ushijima

    Full Text Available CCAAT/enhancer binding protein β (C/EBPβ is a transcription factor that promotes hypertrophic differentiation of chondrocytes. Indian hedgehog (Ihh also stimulates the hypertrophic transition of chondrocytes. Furthermore, runt-related transcription factor-2 (RUNX2 was reported to regulate chondrocyte maturation during skeletal development and to directly regulate transcriptional activity of Ihh. In this study, we investigated whether the interaction of C/EBPβ and RUNX2 regulates the expression of Ihh during chondrocyte differentiation.Immunohistochemistry of embryonic growth plate revealed that both C/EBPβ and Ihh were strongly expressed in pre-hypertrophic and hypertrophic chondrocytes. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked stimulation of Ihh and Runx2. Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells. A reporter assay revealed that C/EBPβ stimulated transcriptional activity of Ihh. Deletion and mutation analysis showed that the C/EBPβ responsive element was located between -214 and -210 bp in the Ihh promoter. An electrophoretic mobility shift assay (EMSA and a chromatin immunoprecipitation (ChIP assay also revealed the direct binding of C/EBPβ to this region. Moreover, reporter assays demonstrated that RUNX2 failed to stimulate the transcriptional activity of the Ihh promoter harboring a mutation at the C/EBPβ binding site. EMSA and ChIP assays showed that RUNX2 interacted to this element with C/EBPβ. Immunoprecipitation revealed that RUNX2 and C/EBPβ formed heterodimer complex with each other in the nuclei of chondrocytes. These data suggested that the C/EBPβ binding element is also important for RUNX2 to regulate the expression of Ihh. Ex vivo organ culture of mouse limbs transfected with C/EBPβ showed that the expression of Ihh and RUNX2 was increased upon ectopic C/EBPβ expression.C/EBPβ and RUNX2 cooperatively stimulate

  20. SOX17 Regulates Cholangiocyte Differentiation and Acts as a Tumor Suppressor in Cholangiocarcinoma

    DEFF Research Database (Denmark)

    Merino-Azpitarte, M; Lozano, E; Perugorria, M J

    2017-01-01

    /function was evaluated along the differentiation of human induced pluripotent stem cells (iPSC) into cholangiocytes, in the dedifferentiation process of normal human cholangiocytes (NHC) in culture and in cholangiocarcinogenesis. Lentiviruses for SOX17 overexpression or knock-down were used. Gene expression and DNA......BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a biliary malignancy linked to genetic and epigenetic abnormalities, such as hypermethylation of SOX17 promoter. Here, the role of SOX17 in cholangiocyte differentiation and cholangiocarcinogenesis was studied. METHODS: SOX17 expression...... methylation profiling were performed. RESULTS: SOX17 expression is induced in the last stage of cholangiocyte differentiation from iPSC and regulates the acquisition of biliary markers. SOX17 becomes downregulated in NHC undergoing dedifferentiation; experimental SOX17 knock-down in differentiated NHC...

  1. Clinical and pathogenetic interrelation between molecular regulation of apoptosis and cell differentiation in osteoarthritis

    Directory of Open Access Journals (Sweden)

    M A Kabalyk

    2018-02-01

    Full Text Available Aim. To determine clinical and pathogenetic relationship between the levels of apoptosis and growth and differentiation regulation (growth inhibitor 1 induced by oxidative stress, growth/differentiation factor 5 in osteoarthritis. Methods. In a rheumatology office of Vladivostok polyclinic №3 65 patients with knee osteoarthritis Kellgren grade 1-4 aged 66.5±8.0 years were examined. 25 healthy volunteers matched by sex and age without clinical and radiologic manifestations of osteoarthritis were included into control group. To measure concentration of the studied molecules in study patients’ blood, ELISA method was used. Results. Patients with osteoarthritis compared to control group had statistically significantly increased levels of Fas, growth/differentiation factor 5 and ratio of growth/differentiation factor 5/growth inhibitor 1 induced by oxidative stress. Fas levels were significantly lower in late stages 2-4 of osteoarthritis compared to stages 1 and 2. Growth/differentiation factor 5 level was lower in patients with stage 3-4 of osteoarthritis compared to stages 1 and 2. As radiologic signs of osteoarthritis progressed, decrease of the ratio of growth/differentiation factor 5/growth inhibitor 1 induced by oxidative stress, was registered which was significantly lower in stages 2 and 3 compared to stage 1. Conclusion. Extrinsic pathway of apoptosis plays a big role in forming pain syndrome in osteoarthritis, and its maintenance is provided by other mechanisms which include influence of oxidative stress via inhibition of cell cycle mediated by growth inhibitor 1 induced by oxidative stress, reduced involvement of growth/differentiation factor 5 in differentiation processes and regulation of protein synthesis of extracellular cartilaginous tissue matrix.

  2. Transgenic overexpression of BAFF regulates the expression of ...

    Indian Academy of Sciences (India)

    LI ZHANG

    animal models could effectively remove B cells, delay the. Keywords. .... sues were crushed in a mortar filled with liquid nitrogen ... The whole body of zebrafish was collected and homoge- ... (Yang et al. ... ating a soluble protein fragment with a molecular weight of ... showed traits of prematurity with a distinct tail and a yolk.

  3. BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

    Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

  4. Sialylation regulates myofibroblast differentiation of human skin fibroblasts.

    Science.gov (United States)

    Sasaki, Norihiko; Itakura, Yoko; Toyoda, Masashi

    2017-04-18

    differentiation in LP fibroblasts was restored by a sialidase inhibitor. Desialylation of CD44 with increased sialidase during the process to senescence reduced the localization of CD44 in lipid rafts after TGF-β1 stimulation, leading to the inhibition of myofibroblast differentiation. Thus, regulation of sialylation may be an attractive strategy for the prevention and regenerative therapy of age-related skin diseases, cosmetic skin alterations, and chronic wounds caused by delayed healing in elderly people.

  5. Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Qiao, Lan [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Wang, Yongsheng [Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2013-05-31

    Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.

  6. Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

    International Nuclear Information System (INIS)

    Qiao, Lan; Paul, Pritha; Lee, Sora; Qiao, Jingbo; Wang, Yongsheng; Chung, Dai H.

    2013-01-01

    Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma

  7. MiRNA-20a promotes osteogenic differentiation of human mesenchymal stem cells by co-regulating BMP signaling.

    Science.gov (United States)

    Zhang, Jin-fang; Fu, Wei-ming; He, Ming-liang; Xie, Wei-dong; Lv, Qing; Wan, Gang; Li, Guo; Wang, Hua; Lu, Gang; Hu, Xiang; Jiang, Su; Li, Jian-na; Lin, Marie C M; Zhang, Ya-ou; Kung, Hsiang-fu

    2011-01-01

    Osteogenic differentiation of mesenchymal stem cells (MSCs) is a complex process, which is regulated by various factors including microRNAs. Our preliminary data showed that the expression of endogenous miR-20a was increased during the course of osteogenic differentiation. Simultaneously, the expression of osteoblast markers and regulators BMP2, BMP4, Runx2, Osx, OCN and OPN was also elevated whereas adipocyte markers PPARγ and osteoblast antagonist, Bambi and Crim1, were downregulated, thereby suggesting that miR-20a plays an important role in regulating osteoblast differentiation. To validate this hypothesis, we tested its effects on osteogenic differentiation by introducing miR-20a mimics and lentiviral-miR20a-expression vectors into hMSCs. We showed that miR-20a promoted osteogenic differentiation by the upregulation of BMP/Runx2 signaling. We performed bioinformatics analysis and predicted that PPARγ, Bambi and Crim1 would be potential targets of miR-20a. PPARγ is a negative regulator of BMP/Runx2 signaling whereas Bambi or Crim1 are antagonists of the BMP pathway. Furthermore, we confirmed that all these molecules were indeed the targets of miR-20a by luciferase reporter, quantitative RT-PCR and western blot assays. Similarly to miR-20a overexpression, the osteogenesis was enhanced by the silence of PPARγ, Bambi or Crim1 by specific siRNAs. Taken together, for the first time, we demonstrated that miR-20a promoted the osteogenesis of hMSCs in a co-regulatory pattern by targeting PPARγ, Bambi and Crim1, the negative regulators of BMP signaling.

  8. Histone Methylation and microRNA-dependent Regulation of Epigenetic Activities in Neural Progenitor Self-Renewal and Differentiation.

    Science.gov (United States)

    Cacci, Emanuele; Negri, Rodolfo; Biagioni, Stefano; Lupo, Giuseppe

    2017-01-01

    Neural stem/progenitor cell (NSPC) self-renewal and differentiation in the developing and the adult brain are controlled by extra-cellular signals and by the inherent competence of NSPCs to produce appropriate responses. Stage-dependent responsiveness of NSPCs to extrinsic cues is orchestrated at the epigenetic level. Epigenetic mechanisms such as DNA methylation, histone modifications and non-coding RNA-mediated regulation control crucial aspects of NSPC development and function, and are also implicated in pathological conditions. While their roles in the regulation of stem cell fate have been largely explored in pluripotent stem cell models, the epigenetic signature of NSPCs is also key to determine their multipotency as well as their progressive bias towards specific differentiation outcomes. Here we review recent developments in this field, focusing on the roles of histone methylation marks and the protein complexes controlling their deposition in NSPCs of the developing cerebral cortex and the adult subventricular zone. In this context, we describe how bivalent promoters, carrying antagonistic epigenetic modifications, feature during multiple steps of neural development, from neural lineage specification to neuronal differentiation. Furthermore, we discuss the emerging cross-talk between epigenetic regulators and microRNAs, and how the interplay between these different layers of regulation can finely tune the expression of genes controlling NSPC maintenance and differentiation. In particular, we highlight recent advances in the identification of astrocyte-enriched microRNAs and their function in cell fate choices of NSPCs differentiating towards glial lineages.

  9. Proximity-based differential single cell analysis of the niche to identify stem/progenitor cell regulators

    Science.gov (United States)

    Silberstein, Lev; Goncalves, Kevin A; Kharchenko, Peter V; Turcotte, Raphael; Kfoury, Youmna; Mercier, Francois; Baryawno, Ninib; Severe, Nicolas; Bachand, Jacqueline; Spencer, Joel; Papazian, Ani; Lee, Dongjun; Chitteti, Brahmananda Reddy; Srour, Edward F; Hoggatt, Jonathan; Tate, Tiffany; Celso, Cristina Lo; Ono, Noriaki; Nutt, Stephen; Heino, Jyrki; Sipilä, Kalle; Shioda, Toshihiro; Osawa, Masatake; Lin, Charles P; Hu, Guo-fu; Scadden, David T

    2016-01-01

    SUMMARY Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on differential single-cell gene expression analysis of mesenchymal osteolineage cells close to and further removed from hematopoietic stem/progenitor cells to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. Amongst the genes which were preferentially expressed in proximal cells, we functionally examined three secreted or cell surface molecules not previously connected to HSPC biology: the secreted RNase Angiogenin, the cytokine IL18 and the adhesion molecule Embigin and discovered that all of these factors are HSPC quiescence regulators. Our proximity-based differential single cell approach therefore reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance understanding of microenvironmental regulation of stem cell function. PMID:27524439

  10. Molecular and Genetic Analysis of Hormone-Regulated Differential Cell Elongation in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Ecker, Joseph R.

    2005-09-15

    We have utilized the response of Arabidopsis seedlings to the plant hormone ethylene to identify new genes involved in the regulation of ethylene biosynthesis, perception, signal transduction and differential cell growth. In building a genetic framework for the action of these genes, we have developed a molecular model that has facilitated our understanding of the molecular requirements of ethylene for cell elongation processes. The ethylene response pathway in Arabidopsis appears to be primarily linear and is defined by the genes: ETR1, ETR2, ERS1, ERS2, EIN4, CTR1, EIN2, EIN3, EIN5, EIN6, and EIN. Downstream branches identified by the HLS1, EIR1, and AUX1 genes involve interactions with other hormonal (auxin) signals in the process of differential cell elongation in the hypocotyl hook. Cloning and characterization of HLS1 (and three HLL genes) and ETO1 (and ETOL genes) in my laboratory has been supported under this award. HLS1 is required for differential elongation of cells in the hypocotyl and may act in the establishment of hormone gradients. Also during the previous period, we have identified and characterized a gene that genetically acts upstream of the ethylene receptors. ETO1 encodes negative regulators of ethylene biosynthesis.

  11. A VESICLE TRAFFICKING PROTEIN αSNAP REGULATES PANETH CELL DIFFERENTIATION IN VIVO

    Science.gov (United States)

    Lechuga, Susana; Naydenov, Nayden G.; Feygin, Alex; Jimenez, Antonio J.; Ivanov, Andrei I.

    2017-01-01

    A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. PMID:28359759

  12. Regulation of eukaryotic initiation factor 4AII by MyoD during murine myogenic cell differentiation.

    Directory of Open Access Journals (Sweden)

    Gabriela Galicia-Vázquez

    Full Text Available Gene expression during muscle cell differentiation is tightly regulated at multiple levels, including translation initiation. The PI3K/mTOR signalling pathway exerts control over protein synthesis by regulating assembly of eukaryotic initiation factor (eIF 4F, a heterotrimeric complex that stimulates recruitment of ribosomes to mRNA templates. One of the subunits of eIF4F, eIF4A, supplies essential helicase function during this phase of translation. The presence of two cellular eIF4A isoforms, eIF4AI and eIF4AII, has long thought to impart equivalent functions to eIF4F. However, recent experiments have alluded to distinct activities between them. Herein, we characterize distinct regulatory mechanisms between the eIF4A isoforms during muscle cell differentiation. We find that eIF4AI levels decrease during differentiation whereas eIF4AII levels increase during myofiber formation in a MyoD-dependent manner. This study characterizes a previously undefined mechanism for eIF4AII regulation in differentiation and highlights functional differences between eIF4AI and eIF4AII. Finally, RNAi-mediated alterations in eIF4AI and eIF4AII levels indicate that the myogenic process can tolerate short term reductions in eIF4AI or eIF4AII levels, but not both.

  13. Epigenetic landscapes reveal transcription factors that regulate CD8+ T cell differentiation.

    Science.gov (United States)

    Yu, Bingfei; Zhang, Kai; Milner, J Justin; Toma, Clara; Chen, Runqiang; Scott-Browne, James P; Pereira, Renata M; Crotty, Shane; Chang, John T; Pipkin, Matthew E; Wang, Wei; Goldrath, Ananda W

    2017-05-01

    Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8 + T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8 + T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8 + T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8 + T cell differentiation.

  14. VMAT2 identified as a regulator of late-stage β-cell differentiation.

    Science.gov (United States)

    Sakano, Daisuke; Shiraki, Nobuaki; Kikawa, Kazuhide; Yamazoe, Taiji; Kataoka, Masateru; Umeda, Kahoko; Araki, Kimi; Mao, Di; Matsumoto, Shirou; Nakagata, Naomi; Andersson, Olov; Stainier, Didier; Endo, Fumio; Kume, Kazuhiko; Uesugi, Motonari; Kume, Shoen

    2014-02-01

    Cell replacement therapy for diabetes mellitus requires cost-effective generation of high-quality, insulin-producing, pancreatic β cells from pluripotent stem cells. Development of this technique has been hampered by a lack of knowledge of the molecular mechanisms underlying β-cell differentiation. The present study identified reserpine and tetrabenazine (TBZ), both vesicular monoamine transporter 2 (VMAT2) inhibitors, as promoters of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Neurog3 (referred to henceforth as Ngn3)-positive endocrine precursors. VMAT2-controlled monoamines, such as dopamine, histamine and serotonin, negatively regulated β-cell differentiation. Reserpine or TBZ acted additively with dibutyryl adenosine 3',5'-cyclic AMP, a cell-permeable cAMP analog, to potentiate differentiation of embryonic stem (ES) cells into β cells that exhibited glucose-stimulated insulin secretion. When ES cell-derived β cells were transplanted into AKITA diabetic mice, the cells reversed hyperglycemia. Our protocol provides a basis for the understanding of β-cell differentiation and its application to a cost-effective production of functional β cells for cell therapy.

  15. Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

    Science.gov (United States)

    Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

    2016-01-01

    Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

  16. PDK1 Is a Regulator of Epidermal Differentiation that Activates and Organizes Asymmetric Cell Division

    Directory of Open Access Journals (Sweden)

    Teruki Dainichi

    2016-05-01

    Full Text Available Asymmetric cell division (ACD in a perpendicular orientation promotes cell differentiation and organizes the stratified epithelium. However, the upstream cues regulating ACD have not been identified. Here, we report that phosphoinositide-dependent kinase 1 (PDK1 plays a critical role in establishing ACD in the epithelium. Production of phosphatidyl inositol triphosphate (PIP3 is localized to the apical side of basal cells. Asymmetric recruitment of atypical protein kinase C (aPKC and partitioning defective (PAR 3 is impaired in PDK1 conditional knockout (CKO epidermis. PDK1CKO keratinocytes do not undergo calcium-induced activation of aPKC or IGF1-induced activation of AKT and fail to differentiate. PDK1CKO epidermis shows decreased expression of Notch, a downstream effector of ACD, and restoration of Notch rescues defective expression of differentiation-induced Notch targets in vitro. We therefore propose that PDK1 signaling regulates the basal-to-suprabasal switch in developing epidermis by acting as both an activator and organizer of ACD and the Notch-dependent differentiation program.

  17. LIX1 regulates YAP1 activity and controls the proliferation and differentiation of stomach mesenchymal progenitors.

    Science.gov (United States)

    McKey, Jennifer; Martire, Delphine; de Santa Barbara, Pascal; Faure, Sandrine

    2016-04-28

    Smooth muscle cell (SMC) plasticity maintains the balance between differentiated SMCs and proliferative mesenchymal progenitors, crucial for muscular tissue homeostasis. Studies on the development of mesenchymal progenitors into SMCs have proven useful in identifying molecular mechanisms involved in digestive musculature plasticity in physiological and pathological conditions. Here, we show that Limb Expression 1 (LIX1) molecularly defines the population of mesenchymal progenitors in the developing stomach. Using in vivo functional approaches in the chick embryo, we demonstrate that LIX1 is a key regulator of stomach SMC development. We show that LIX1 is required for stomach SMC determination to regulate the expression of the pro-proliferative gene YAP1 and mesenchymal cell proliferation. However, as stomach development proceeds, sustained LIX1 expression has a negative impact on further SMC differentiation and this is associated with a decrease in YAP1 activity. We demonstrate that expression of LIX1 must be tightly regulated to allow fine-tuning of the transcript levels and state of activation of the pro-proliferative transcriptional coactivator YAP1 to regulate proliferation rates of stomach mesenchymal progenitors and their differentiation. Our data highlight dual roles for LIX1 and YAP1 and provide new insights into the regulation of cell density-dependent proliferation, which is essential for the development and homeostasis of organs.

  18. Invariant NKT cells regulate experimental autoimmune uveitis through inhibition of Th17 differentiation.

    Science.gov (United States)

    Oh, Keunhee; Byoun, Ok-Jin; Ham, Don-Il; Kim, Yon Su; Lee, Dong-Sup

    2011-02-01

    Although NKT cells have been implicated in diverse immunomodulatory responses, the effector mechanisms underlying the NKT cell-mediated regulation of pathogenic T helper cells are not well understood. Here, we show that invariant NKT cells inhibited the differentiation of CD4(+) T cells into Th17 cells both in vitro and in vivo. The number of IL-17-producing CD4(+) T cells was reduced following co-culture with purified NK1.1(+) TCR(+) cells from WT, but not from CD1d(-/-) or Jα18(-/-) , mice. Co-cultured NKT cells from either cytokine-deficient (IL-4(-/-) , IL-10(-/-) , or IFN-γ(-/-) ) or WT mice efficiently inhibited Th17 differentiation. The contact-dependent mechanisms of NKT cell-mediated regulation of Th17 differentiation were confirmed using transwell co-culture experiments. On the contrary, the suppression of Th1 differentiation was dependent on IL-4 derived from the NKT cells. The in vivo regulatory capacity of NKT cells on Th17 cells was confirmed using an experimental autoimmune uveitis model induced with human IRBP(1-20) (IRBP, interphotoreceptor retinoid-binding protein) peptide. NKT cell-deficient mice (CD1d(-/-) or Jα18(-/-) ) demonstrated an increased disease severity, which was reversed by the transfer of WT or cytokine-deficient (IL-4(-/-) , IL-10(-/-) , or IFN-γ(-/-) ) NKT cells. Our results indicate that invariant NKT cells inhibited autoimmune uveitis predominantly through the cytokine-independent inhibition of Th17 differentiation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. 17β-estradiol regulates the differentiation of cementoblasts via Notch signaling cascade

    Energy Technology Data Exchange (ETDEWEB)

    Liao, Jing; Zhou, Zeyuan; Huang, Li; Li, Yuyu [Department of Orthodontics, The State Key Laboratory of Oral Disease, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan Province (China); Li, Jingtao [Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan Province (China); Zou, Shujuan, E-mail: drzsj@scu.edu.cn [Department of Orthodontics, The State Key Laboratory of Oral Disease, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan Province (China)

    2016-08-12

    Estrogen has been well recognized as a key factor in the homeostasis of bone and periodontal tissue, but the way it regulates the activities of cementoblasts, the cell population maintaining cementum has not been fully understood. In this study, we examined the expression of estrogen receptor in OCCM-30 cells and the effect of 17β-estradiol (E2) on the proliferation and differentiation of OCCM-30 cells. We found that both estrogen receptor α and β were expressed in OCCM-30 cells. E2 exerted no significant influence on the proliferation of OCCM-30 cells, but inhibited the transcription and translation of BSP and Runx2 in the early phase of osteogenic induction except the BSP mRNA. Afterwards in the late phase of osteogenic induction, E2 enhanced the transcription and translation of BSP and Runx2 and promoted the calcium deposition. In addition, the expression level of Notch1, NICD and Hey1 mRNAs responded to exogenous E2 in a pattern similar to that of the osteoblastic markers. DAPT could attenuate the effect of E2 on the expression of osteoblastic markers. These findings indicated that E2 might regulate the differentiation of cementoblasts via Notch signaling. - Highlights: • 17β-estradiol showed no significant effect on the proliferation of cementoblasts. • 17β-estradiol promoted the osteoblastic differentiation of cementoblasts despite of an early transient inhibition. • Notch signaling was regulated by 17β-estradiol and was responsible for mediating the effect of E2 on cementoblasts. • Hey1 might display an opposite expression pattern to Notch signaling in certain circumstances.

  20. CUDC-907 Promotes Bone Marrow Adipocytic Differentiation Through Inhibition of Histone Deacetylase and Regulation of Cell Cycle.

    Science.gov (United States)

    Ali, Dalia; Alshammari, Hassan; Vishnubalaji, Radhakrishnan; Chalisserry, Elna Paul; Hamam, Rimi; Alfayez, Musaad; Kassem, Moustapha; Aldahmash, Abdullah; Alajez, Nehad M

    2017-03-01

    The role of bone marrow adipocytes (BMAs) in overall energy metabolism and their effects on bone mass are currently areas of intensive investigation. BMAs differentiate from bone marrow stromal cells (BMSCs); however, the molecular mechanisms regulating BMA differentiation are not fully understood. In this study, we investigated the effect of CUDC-907, identified by screening an epigenetic small-molecule library, on adipocytic differentiation of human BMSCs (hBMSCs) and determined its molecular mechanism of action. Human bone marrow stromal cells exposed to CUDC-907 (500 nM) exhibited enhanced adipocytic differentiation (∼2.9-fold increase, P < 0.005) compared with that of control cells. Global gene expression and signaling pathway analyses of differentially expressed genes revealed a strong enrichment of genes involved in adipogenesis, cell cycle, and DNA replication. Chromatin immune precipitation combined with quantitative polymerase chain reaction showed significant increase in H3K9ac epigenetic marker in the promoter regions of AdipoQ, FABP4, PPARγ, KLF15, and CEBPA in CUDC-907-treated hBMSCs. Follow-up experiments corroborated that the inhibition of histone deacetylase (HDAC) activity enhanced adipocytic differentiation, while the inhibition of PI3K decreased adipocytic differentiation. In addition, CUDC-907 arrested hBMSCs in the G0-G1 phase of the cell cycle and reduced the number of S-phase cells. Our data reveal that HDAC, PI3K, and cell cycle genes are important regulators of BMA formation and demonstrate that adipocyte differentiation of hBMSCs is associated with complex changes in a number of epigenetic and genetic pathways, which can be targeted to regulate BMA formation.

  1. Pupils' Self-Regulation in Physical Education: The Role of Motivational Climates and Differential Achievement Goals

    Science.gov (United States)

    Ommundsen, Yngvar

    2006-01-01

    This study examined the relationship of motivational climate and differential achievement goals to meta-cognitive self-regulation, regulation of effort, help-seeking and self-handicapping in physical education (PE). The sample consisted of 273 pupils (boys n = 125; girls n = 148) attending 10th grade PE classes in Norway. Both motivational…

  2. Theories of estimation of differentiation for regulation of social-economic development of the city agglomeration

    OpenAIRE

    Anikina, Yu; Litovchenko, V.

    2009-01-01

    Theories of estimation of differentiation of social-economic development of territorial units in city agglomeration are discussed in the article. Approbation of the given methods helped find out successfulness of the regulation of municipal development of administrative-territorial units in Krasnoyarsk agglomeration, set the goals of regional policy on peculiarities of development of the phenomenon of differentiation.

  3. Distinctive features and differential regulation of the DRTS genes of Arabidopsis thaliana.

    Science.gov (United States)

    Maniga, Antonio; Ghisaura, Stefania; Perrotta, Lara; Marche, Maria Giovanna; Cella, Rino; Albani, Diego

    2017-01-01

    In plants and protists, dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are part of a bifunctional enzyme (DRTS) that allows efficient recycling of the dihydrofolate resulting from TS activity. Arabidopsis thaliana possesses three DRTS genes, called AtDRTS1, AtDRTS2 and AtDRTS3, that are located downstream of three members of the sec14-like SFH gene family. In this study, a characterization of the AtDRTS genes identified alternatively spliced transcripts coding for AtDRTS isoforms which may account for monofunctional DHFR enzymes supporting pathways unrelated to DNA synthesis. Moreover, we discovered a complex differential regulation of the AtDRTS genes that confirms the expected involvement of the AtDRTS genes in cell proliferation and endoreduplication, but indicates also functions related to other cellular activities. AtDRTS1 is widely expressed in both meristematic and differentiated tissues, whereas AtDRTS2 expression is almost exclusively limited to the apical meristems and AtDRTS3 is preferentially expressed in the shoot apex, in stipules and in root cap cells. The differential regulation of the AtDRTS genes is associated to distinctive promoter architectures and the expression of AtDRTS1 in the apical meristems is strictly dependent on the presence of an intragenic region that includes the second intron of the gene. Upon activation of cell proliferation in germinating seeds, the activity of the AtDRTS1 and AtDRTS2 promoters in meristematic cells appears to be maximal at the G1/S phase of the cell cycle. In addition, the promoters of AtDRTS2 and AtDRTS3 are negatively regulated through E2F cis-acting elements and both genes, but not AtDRTS1, are downregulated in plants overexpressing the AtE2Fa factor. Our study provides new information concerning the function and the regulation of plant DRTS genes and opens the way to further investigations addressing the importance of folate synthesis with respect to specific cellular activities.

  4. Rac1 Regulates the Proliferation, Adhesion, Migration, and Differentiation of MDPC-23 Cells.

    Science.gov (United States)

    Ren, Jing; Liang, Guobin; Gong, Li; Guo, Bing; Jiang, Hongwei

    2017-04-01

    Stem cells are responsible for replacing damaged pulp tissue; therefore, promoting their survival and inducing their adhesion to dentin are vital. As a member of the Rho family of guanosine triphosphatases, Rac1 is an important regulator of osteoblast functions. However, little is known about its role in regenerative endodontic procedures. The current study examined the role of Rac1 in the proliferation, migration, and odontoblastic differentiation of MDPC-23 cells. MDPC-23 cells were transfected with small interfering RNA to knock down Rac1 expression, and then their proliferation, migration, adhesion, and odontoblastic differentiation were examined in vitro. MDPC-23 cells transfected with si-Rac1 exhibited the increased expression of several key odontogenic protein markers, including Dmp1, Dspp, Runx2, and alkaline phosphatase, as well as decreased proliferation and migration in vitro. The results suggest that Rac1 might regulate nuclear factor kappa B signaling in MDPC-23 cells. Rac1 may have vital roles in the proliferation, migration, adhesion, and odontoblastic differentiation of MDPC-23 cells. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  5. Regulation of B cell differentiation by intracellular membrane associated proteins and microRNAs: role in the antibody response

    Directory of Open Access Journals (Sweden)

    Zheng eLou

    2015-10-01

    Full Text Available B cells are central to adaptive immunity and their functions in antibody responses are exquisitely regulated. As suggested by recent findings, B cell differentiation is mediated by intracellular membrane structures (including endosomes, lysosomes and autophagosomes and protein factors specifically associated with these membranes, including Rab7, Atg5 and Atg7. These factors participate in vesicle formation/trafficking, signal transduction and induction of gene expression to promote antigen presentation, CSR/SHM, and generation/maintenance of plasma cells and memory B cells. Their expression is induced in B cells activated to differentiate and further fine-tuned by immune-modulating microRNAs, which coordinates CSR/SHM, plasma cell differentiation and memory B cell differentiation. These short non-coding RNAs would individually target multiple factors associated with the same intracellular membrane compartments and collaboratively target a single factor in addition to regulate AID and Blimp-1. These, together with regulation of microRNA biogenesis and activities by endosomes and autophagosomes, show that intracellular membranes and microRNAs, two broadly relevant cell constituents, play important roles in balancing gene expression to specify B cell differentiation processes for optimal antibody responses.

  6. Uhrf1 controls the self-renewal versus differentiation of hematopoietic stem cells by epigenetically regulating the cell-division modes.

    Science.gov (United States)

    Zhao, Jingyao; Chen, Xufeng; Song, Guangrong; Zhang, Jiali; Liu, Haifeng; Liu, Xiaolong

    2017-01-10

    Hematopoietic stem cells (HSCs) are able to both self-renew and differentiate. However, how individual HSC makes the decision between self-renewal and differentiation remains largely unknown. Here we report that ablation of the key epigenetic regulator Uhrf1 in the hematopoietic system depletes the HSC pool, leading to hematopoietic failure and lethality. Uhrf1-deficient HSCs display normal survival and proliferation, yet undergo erythroid-biased differentiation at the expense of self-renewal capacity. Notably, Uhrf1 is required for the establishment of DNA methylation patterns of erythroid-specific genes during HSC division. The expression of these genes is enhanced in the absence of Uhrf1, which disrupts the HSC-division modes by promoting the symmetric differentiation and suppressing the symmetric self-renewal. Moreover, overexpression of one of the up-regulated genes, Gata1, in HSCs is sufficient to phenocopy Uhrf1-deficient HSCs, which show impaired HSC symmetric self-renewal and increased differentiation commitment. Taken together, our findings suggest that Uhrf1 controls the self-renewal versus differentiation of HSC through epigenetically regulating the cell-division modes, thus providing unique insights into the relationship among Uhrf1-mediated DNA methylation, cell-division mode, and HSC fate decision.

  7. Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.

    Science.gov (United States)

    Chen, Wan-Chun; Lin, Hsi-Hui; Tang, Ming-Jer

    2014-09-15

    To explore whether matrix stiffness affects cell differentiation, proliferation, and transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in primary cultures of mouse proximal tubular epithelial cells (mPTECs), we used a soft matrix made from monomeric collagen type I-coated polyacrylamide gel or matrigel (MG). Both kinds of soft matrix benefited primary mPTECs to retain tubular-like morphology with differentiation and growth arrest and to evade TGF-β1-induced EMT. However, the potent effect of MG on mPTEC differentiation was suppressed by glutaraldehyde-induced cross-linking and subsequently stiffening MG or by an increasing ratio of collagen in the soft mixed gel. Culture media supplemented with MG also helped mPTECs to retain tubular-like morphology and a differentiated phenotype on stiff culture dishes as soft MG did. We further found that the protein level and activity of ERK were scaled with the matrix stiffness. U-0126, a MEK inhibitor, abolished the stiff matrix-induced dedifferentiation and proliferation. These data suggest that the ERK signaling pathway plays a vital role in matrix stiffness-regulated cell growth and differentiation. Taken together, both compliant property and specific MG signals from the matrix are required for the regulation of epithelial differentiation and proliferation. This study provides a basic understanding of how physical and chemical cues derived from the extracellular matrix regulate the physiological function of proximal tubules and the pathological development of renal fibrosis. Copyright © 2014 the American Physiological Society.

  8. The regulation of autophagy differentially affects Trypanosoma cruzi metacyclogenesis.

    Directory of Open Access Journals (Sweden)

    María Cristina Vanrell

    2017-11-01

    Full Text Available Autophagy is a cellular process required for the removal of aged organelles and cytosolic components through lysosomal degradation. All types of eukaryotic cells from yeasts to mammalian cells have the machinery to activate autophagy as a result of many physiological and pathological situations. The most frequent stimulus of autophagy is starvation and the result, in this case, is the fast generation of utilizable food (e.g. amino acids and basic nutrients to maintain the vital biological processes. In some organisms, starvation also triggers other associated processes such as differentiation. The protozoan parasite Trypanosoma cruzi undergoes a series of differentiation processes throughout its complex life cycle. Although not all autophagic genes have been identified in the T. cruzi genome, previous works have demonstrated the presence of essential autophagic-related proteins. Under starvation conditions, TcAtg8, which is the parasite homolog of Atg8/LC3 in other organisms, is located in autophagosome-like vesicles. In this work, we have characterized the autophagic pathway during T. cruzi differentiation from the epimastigote to metacyclic trypomastigote form, a process called metacyclogenesis. We demonstrated that autophagy is stimulated during metacyclogenesis and that the induction of autophagy promotes this process. Moreover, with exception of bafilomycin, other classical autophagy modulators have similar effects on T. cruzi autophagy. We also showed that spermidine and related polyamines can positively regulate parasite autophagy and differentiation. We concluded that both polyamine metabolism and autophagy are key processes during T. cruzi metacyclogenesis that could be exploited as drug targets to avoid the parasite cycle progression.

  9. Syndecan-4 Regulates Muscle Differentiation and Is Internalized from the Plasma Membrane during Myogenesis.

    Science.gov (United States)

    Rønning, Sissel B; Carlson, Cathrine R; Stang, Espen; Kolset, Svein O; Hollung, Kristin; Pedersen, Mona E

    2015-01-01

    The cell surface proteoglycan syndecan-4 has been reported to be crucial for muscle differentiation, but the molecular mechanisms still remain to be fully understood. During in vitro differentiation of bovine muscle cells immunocytochemical analyses showed strong labelling of syndecan-4 intracellularly, in close proximity with Golgi structures, in membranes of intracellular vesicles and finally, in the nuclear area including the nuclear envelope. Chase experiments showed that syndecan-4 was internalized from the plasma membrane during this process. Furthermore, when syndecan-4 was knocked down by siRNA more myotubes were formed, and the expression of myogenic transcription factors, β1-integrin and actin was influenced. However, when bovine muscle cells were treated with a cell-penetrating peptide containing the cytoplasmic region of syndecan-4, myoblast fusion and thus myotube formation was blocked, both in normal cells and in syndecan-4 knock down cells. Altogether this suggests that the cytoplasmic domain of syndecan-4 is important in regulation of myogenesis. The internalization of syndecan-4 from the plasma membrane during muscle differentiation and the nuclear localization of syndecan-4 in differentiated muscle cells may be part of this regulation, and is a novel aspect of syndecan biology which merits further studies.

  10. Syndecan-4 Regulates Muscle Differentiation and Is Internalized from the Plasma Membrane during Myogenesis.

    Directory of Open Access Journals (Sweden)

    Sissel B Rønning

    Full Text Available The cell surface proteoglycan syndecan-4 has been reported to be crucial for muscle differentiation, but the molecular mechanisms still remain to be fully understood. During in vitro differentiation of bovine muscle cells immunocytochemical analyses showed strong labelling of syndecan-4 intracellularly, in close proximity with Golgi structures, in membranes of intracellular vesicles and finally, in the nuclear area including the nuclear envelope. Chase experiments showed that syndecan-4 was internalized from the plasma membrane during this process. Furthermore, when syndecan-4 was knocked down by siRNA more myotubes were formed, and the expression of myogenic transcription factors, β1-integrin and actin was influenced. However, when bovine muscle cells were treated with a cell-penetrating peptide containing the cytoplasmic region of syndecan-4, myoblast fusion and thus myotube formation was blocked, both in normal cells and in syndecan-4 knock down cells. Altogether this suggests that the cytoplasmic domain of syndecan-4 is important in regulation of myogenesis. The internalization of syndecan-4 from the plasma membrane during muscle differentiation and the nuclear localization of syndecan-4 in differentiated muscle cells may be part of this regulation, and is a novel aspect of syndecan biology which merits further studies.

  11. Molecular and Genetic Analysis of Hormone-Regulated Differential Cell Elongation in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Ecker, Joseph R.

    2002-12-03

    The authors have utilized the response of Arabidopsis seedlings to the plant hormone ethylene to identify new genes involved in the regulation of ethylene biosynthesis, perception, signal transduction and differential cell growth. In building a genetic framework for the action of these genes, they developed a molecular model that has facilitated the understanding of the molecular requirements of ethylene for cell elongation processes. The ethylene response pathway in Arabidopsis appears to be primarily linear and is defined by the genes: ETR1, ETR2, ERS1, ERS2, EIN4, CTR1, EIN2, EIN3, EIN5 EIN6, and EIN. Downstream branches identified by the HLS1, EIR1, and AUX1 genes involve interactions with other hormonal (auxin) signals in the process of differential cell elongation in the hypocotyl hook. Cloning and characterization of HLS1 and three HLS1-LIKE genes in the laboratory has been supported under this award. HLS1 is required for differential elongation of cells in the hypocotyl and may act in the establishment of hormone gradients. Also during the award period, they have identified and begun preliminary characterization of two genes that genetically act upstream of the ethylene receptors. ETO1 and RAN1 encode negative regulators of ethylene biosynthesis and signaling respectively. Progress on the analysis of these genes along with HOOKLESS1 is described.

  12. miR-150-Mediated Foxo1 Regulation Programs CD8+ T Cell Differentiation.

    Science.gov (United States)

    Ban, Young Ho; Oh, Se-Chan; Seo, Sang-Hwan; Kim, Seok-Min; Choi, In-Pyo; Greenberg, Philip D; Chang, Jun; Kim, Tae-Don; Ha, Sang-Jun

    2017-09-12

    MicroRNA (miR)-150 is a developmental regulator of several immune-cell types, but its role in CD8 + T cells is largely unexplored. Here, we show that miR-150 regulates the generation of memory CD8 + T cells. After acute virus infection, miR-150 knockout (KO) mice exhibited an accelerated differentiation of CD8 + T cells into memory cells and improved production of effector cytokines. Additionally, miR-150 KO CD8 + T cells displayed an enhanced recall response and improved protection against infections with another virus and bacteria. We found that forkhead box O1 (Foxo1) and T cell-specific transcription factor 1 (TCF1) are upregulated during the early activation phase in miR-150 KO CD8 + T cells and that miR-150 directly targets and suppresses Foxo1. These results suggest that miR-150-mediated suppression of Foxo1 regulates the balance between effector and memory cell differentiation, which might aid in the development of improved vaccines and T cell therapeutics. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators.

    Science.gov (United States)

    Silberstein, Lev; Goncalves, Kevin A; Kharchenko, Peter V; Turcotte, Raphael; Kfoury, Youmna; Mercier, Francois; Baryawno, Ninib; Severe, Nicolas; Bachand, Jacqueline; Spencer, Joel A; Papazian, Ani; Lee, Dongjun; Chitteti, Brahmananda Reddy; Srour, Edward F; Hoggatt, Jonathan; Tate, Tiffany; Lo Celso, Cristina; Ono, Noriaki; Nutt, Stephen; Heino, Jyrki; Sipilä, Kalle; Shioda, Toshihiro; Osawa, Masatake; Lin, Charles P; Hu, Guo-Fu; Scadden, David T

    2016-10-06

    Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on the differential single-cell gene expression analysis of mesenchymal osteolineage cells close to, and further removed from, hematopoietic stem/progenitor cells (HSPCs) to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. We functionally examined, among the genes that were preferentially expressed in proximal cells, three secreted or cell-surface molecules not previously connected to HSPC biology-the secreted RNase angiogenin, the cytokine IL18, and the adhesion molecule Embigin-and discovered that all of these factors are HSPC quiescence regulators. Therefore, our proximity-based differential single-cell approach reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance the understanding of microenvironmental regulation of stem cell function. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Regulation of granulocyte colony-stimulating factor receptor-mediated granulocytic differentiation by C-mannosylation.

    Science.gov (United States)

    Otani, Kei; Niwa, Yuki; Suzuki, Takehiro; Sato, Natsumi; Sasazawa, Yukiko; Dohmae, Naoshi; Simizu, Siro

    2018-04-06

    Granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is a type I cytokine receptor which is involved in hematopoietic cell maturation. G-CSFR has three putative C-mannosylation sites at W253, W318, and W446; however, it is not elucidated whether G-CSFR is C-mannosylated or not. In this study, we first demonstrated that G-CSFR was C-mannosylated at only W318. We also revealed that C-mannosylation of G-CSFR affects G-CSF-dependent downstream signaling through changing ligand binding capability but not cell surface localization. Moreover, C-mannosylation of G-CSFR was functional and regulated granulocytic differentiation in myeloid 32D cells. In conclusion, we found that G-CSFR is C-mannosylated at W318 and that this C-mannosylation has role(s) for myeloid cell differentiation through regulating downstream signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Intracellular glutathione status regulates mouse bone marrow monocyte-derived macrophage differentiation and phagocytic activity

    International Nuclear Information System (INIS)

    Kim, Jin-Man; Kim, Hyunsoo; Kwon, Soon Bok; Lee, Soo Young; Chung, Sung-Chang; Jeong, Dae-Won; Min, Byung-Moo

    2004-01-01

    Although a redox shift can regulate the development of cells, including proliferation, differentiation, and survival, the role of the glutathione (GSH) redox status in macrophage differentiation remains unclear. In order to elucidate the role of a redox shift, macrophage-like cells were differentiated from the bone marrow-derived monocytes that were treated with a macrophage colony stimulating factor (M-CSF or CSF-1) for 3 days. The macrophagic cells were characterized by a time-dependent increase in three major symptoms: the number of phagocytic cells, the number of adherent cells, and the mRNA expression of c-fms, a M-CSF receptor that is one of the macrophage-specific markers and mediates development signals. Upon M-CSF-driven macrophage differentiation, the GSH/GSSG ratio was significantly lower on day 1 than that observed on day 0 but was constant on days 1-3. To assess the effect of the GSH-depleted and -repleted status on the differentiation and phagocytosis of the macrophages, GSH depletion by BSO, a specific inhibitor of the de novo GSH synthesis, inhibited the formation of the adherent macrophagic cells by the down-regulation of c-fms, but did not affect the phagocytic activity of the macrophages. To the contrary, GSH repletion by the addition of NAC, which is a GSH precursor, or reduced GSH in media had no effect on macrophage differentiation, and led to a decrease in the phagocytic activity. Furthermore, we observed that there is checkpoint that is capable of releasing from the inhibition of the formation of the adherent macrophagic cells according to GSH depletion by BSO. Summarizing, these results indicate that the intracellular GSH status plays an important role in the differentiation and phagocytosis of macrophages

  16. Sox9-regulated miRNA-574-3p inhibits chondrogenic differentiation of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    David Guérit

    Full Text Available The aim of this study was to identify new microRNAs (miRNAs that are modulated during the differentiation of mesenchymal stem cells (MSCs toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0 and at early time points (day 0.5 and 3 after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXRα is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.

  17. The regulation of mitochondrial transcription factor A (Tfam) expression during skeletal muscle cell differentiation.

    Science.gov (United States)

    Collu-Marchese, Melania; Shuen, Michael; Pauly, Marion; Saleem, Ayesha; Hood, David A

    2015-05-19

    The ATP demand required for muscle development is accommodated by elevations in mitochondrial biogenesis, through the co-ordinated activities of the nuclear and mitochondrial genomes. The most important transcriptional activator of the mitochondrial genome is mitochondrial transcription factor A (Tfam); however, the regulation of Tfam expression during muscle differentiation is not known. Thus, we measured Tfam mRNA levels, mRNA stability, protein expression and localization and Tfam transcription during the progression of muscle differentiation. Parallel 2-fold increases in Tfam protein and mRNA were observed, corresponding with 2-3-fold increases in mitochondrial content. Transcriptional activity of a 2051 bp promoter increased during this differentiation period and this was accompanied by a 3-fold greater Tfam mRNA stabilization. Interestingly, truncations of the promoter at 1706 bp, 978 bp and 393 bp promoter all exhibited 2-3-fold higher transcriptional activity than the 2051 bp construct, indicating the presence of negative regulatory elements within the distal 350 bp of the promoter. Activation of AMP kinase augmented Tfam transcription within the proximal promoter, suggesting the presence of binding sites for transcription factors that are responsive to cellular energy state. During differentiation, the accumulating Tfam protein was progressively distributed to the mitochondrial matrix where it augmented the expression of mtDNA and COX (cytochrome c oxidase) subunit I, an mtDNA gene product. Our data suggest that, during muscle differentiation, Tfam protein levels are regulated by the availability of Tfam mRNA, which is controlled by both transcription and mRNA stability. Changes in energy state and Tfam localization also affect Tfam expression and action in differentiating myotubes. © 2015 Authors.

  18. Leptin differentially regulate STAT3 activation in ob/ob mouse adipose mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Zhou Zhou

    2012-12-01

    Full Text Available Abstract Background Leptin-deficient ob/ob mice exhibit adipocyte hypertrophy and hyperplasia as well as elevated adipose tissue and systemic inflammation. Multipotent stem cells isolated from adult adipose tissue can differentiate into adipocytes ex vivo and thereby contribute toward increased adipocyte cell numbers, obesity, and inflamm ation. Currently, information is lacking regarding regulation of adipose stem cell numbers as well as leptin-induced inflammation and its signaling pathway in ob/ob mice. Methods Using leptin deficient ob/ob mice, we investigated whether leptin injection into ob/ob mice increases adipose stem cell numbers and adipose tissue inflammatory marker MCP-1 mRNA and secretion levels. We also determined leptin mediated signaling pathways in the adipose stem cells. Results We report here that adipose stem cell number is significantly increased following leptin injection in ob/ob mice and with treatment of isolated stem cells with leptin in vitro. Leptin also up-regulated MCP-1 secretion in a dose- and time-dependent manner. We further showed that increased MCP-1 mRNA levels were due to increased phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3 Ser727 but not STAT3 Tyr705 phosphorylation, suggesting differential regulation of MCP-1 gene expression under basal and leptin-stimulated conditions in adipose stem cells. Conclusions Taken together, these studies demonstrate that leptin increases adipose stem cell number and differentially activates STAT3 protein resulting in up-regulation of MCP-1 gene expression. Further studies of mechanisms mediating adipose stem cell hyperplasia and leptin signaling in obesity are warranted and may help identify novel anti-obesity target strategies.

  19. GCN5 Regulates FGF Signaling and Activates Selective MYC Target Genes during Early Embryoid Body Differentiation

    Directory of Open Access Journals (Sweden)

    Li Wang

    2018-01-01

    Full Text Available Precise control of gene expression during development is orchestrated by transcription factors and co-regulators including chromatin modifiers. How particular chromatin-modifying enzymes affect specific developmental processes is not well defined. Here, we report that GCN5, a histone acetyltransferase essential for embryonic development, is required for proper expression of multiple genes encoding components of the fibroblast growth factor (FGF signaling pathway in early embryoid bodies (EBs. Gcn5−/− EBs display deficient activation of ERK and p38, mislocalization of cytoskeletal components, and compromised capacity to differentiate toward mesodermal lineage. Genomic analyses identified seven genes as putative direct targets of GCN5 during early differentiation, four of which are cMYC targets. These findings established a link between GCN5 and the FGF signaling pathway and highlighted specific GCN5-MYC partnerships in gene regulation during early differentiation.

  20. Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum.

    Science.gov (United States)

    Brancucci, Nicolas M B; Gerdt, Joseph P; Wang, ChengQi; De Niz, Mariana; Philip, Nisha; Adapa, Swamy R; Zhang, Min; Hitz, Eva; Niederwieser, Igor; Boltryk, Sylwia D; Laffitte, Marie-Claude; Clark, Martha A; Grüring, Christof; Ravel, Deepali; Blancke Soares, Alexandra; Demas, Allison; Bopp, Selina; Rubio-Ruiz, Belén; Conejo-Garcia, Ana; Wirth, Dyann F; Gendaszewska-Darmach, Edyta; Duraisingh, Manoj T; Adams, John H; Voss, Till S; Waters, Andrew P; Jiang, Rays H Y; Clardy, Jon; Marti, Matthias

    2017-12-14

    Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. Environmental sensing by mature B cells is controlled by the transcription factors PU.1 and SpiB.

    Science.gov (United States)

    Willis, Simon N; Tellier, Julie; Liao, Yang; Trezise, Stephanie; Light, Amanda; O'Donnell, Kristy; Garrett-Sinha, Lee Ann; Shi, Wei; Tarlinton, David M; Nutt, Stephen L

    2017-11-10

    Humoral immunity requires B cells to respond to multiple stimuli, including antigen, membrane and soluble ligands, and microbial products. Ets family transcription factors regulate many aspects of haematopoiesis, although their functions in humoral immunity are difficult to decipher as a result of redundancy between the family members. Here we show that mice lacking both PU.1 and SpiB in mature B cells do not generate germinal centers and high-affinity antibody after protein immunization. PU.1 and SpiB double-deficient B cells have a survival defect after engagement of CD40 or Toll-like receptors (TLR), despite paradoxically enhanced plasma cell differentiation. PU.1 and SpiB regulate the expression of many components of the B cell receptor signaling pathway and the receptors for CD40L, BAFF and TLR ligands. Thus, PU.1 and SpiB enable B cells to appropriately respond to environmental cues.

  2. A vesicle trafficking protein αSNAP regulates Paneth cell differentiation in vivo.

    Science.gov (United States)

    Lechuga, Susana; Naydenov, Nayden G; Feygin, Alex; Jimenez, Antonio J; Ivanov, Andrei I

    2017-05-13

    A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation.

    Science.gov (United States)

    Deng, Tao; Postnikov, Yuri; Zhang, Shaofei; Garrett, Lillian; Becker, Lore; Rácz, Ildikó; Hölter, Sabine M; Wurst, Wolfgang; Fuchs, Helmut; Gailus-Durner, Valerie; de Angelis, Martin Hrabe; Bustin, Michael

    2017-04-07

    An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior. Published by Oxford University Press on behalf of Nucleic Acids Research 2016.

  4. HDAC4 regulates satellite cell proliferation and differentiation by targeting P21 and Sharp1 genes.

    Science.gov (United States)

    Marroncelli, Nicoletta; Bianchi, Marzia; Bertin, Marco; Consalvi, Silvia; Saccone, Valentina; De Bardi, Marco; Puri, Pier Lorenzo; Palacios, Daniela; Adamo, Sergio; Moresi, Viviana

    2018-02-22

    Skeletal muscle exhibits a high regenerative capacity, mainly due to the ability of satellite cells to replicate and differentiate in response to appropriate stimuli. Epigenetic control is effective at different stages of this process. It has been shown that the chromatin-remodeling factor HDAC4 is able to regulate satellite cell proliferation and commitment. However, its molecular targets are still uncovered. To explain the signaling pathways regulated by HDAC4 in satellite cells, we generated tamoxifen-inducible mice with conditional inactivation of HDAC4 in Pax7 + cells (HDAC4 KO mice). We found that the proliferation and differentiation of HDAC4 KO satellite cells were compromised, although similar amounts of satellite cells were found in mice. Moreover, we found that the inhibition of HDAC4 in satellite cells was sufficient to block the differentiation process. By RNA-sequencing analysis we identified P21 and Sharp1 as HDAC4 target genes. Reducing the expression of these target genes in HDAC4 KO satellite cells, we also defined the molecular pathways regulated by HDAC4 in the epigenetic control of satellite cell expansion and fusion.

  5. MicroRNAs regulate T-cell production of interleukin-9 and identify hypoxia-inducible factor-2α as an important regulator of T helper 9 and regulatory T-cell differentiation.

    Science.gov (United States)

    Singh, Yogesh; Garden, Oliver A; Lang, Florian; Cobb, Bradley S

    2016-09-01

    MicroRNAs (miRNAs) regulate many aspects of helper T cell (Th) development and function. Here we found that they are required for the suppression of interleukin-9 (IL-9) expression in Th9 cells and other Th subsets. Two highly related miRNAs (miR-15b and miR-16) that we previously found to play an important role in regulatory T (Treg) cell differentiation were capable of suppressing IL-9 expression when they were over-expressed in Th9 cells. We used these miRNAs as tools to identify novel regulators of IL-9 expression and found that they could regulate the expression of Epas1, which encodes hypoxia-inducible factor (HIF)-2α. HIF proteins regulate metabolic pathway usage that is important in determining appropriate Th differentiation. The related protein, HIF-1α enhances Th17 differentiation and inhibits Treg cell differentiation. Here we found that HIF-2α was required for IL-9 expression in Th9 cells, but its expression was not sufficient in other Th subsets. Furthermore, HIF-2α suppressed Treg cell differentiation like HIF-1α, demonstrating both similar and distinct roles of the HIF proteins in Th differentiation and adding a further dimension to their function. Ironically, even though miR-15b and miR-16 suppressed HIF-2α expression in Treg cells, inhibiting their function in Treg cells did not lead to an increase in IL-9 expression. Therefore, the physiologically relevant miRNAs that regulate IL-9 expression in Treg cells and other subsets remain unknown. Nevertheless, the analysis of miR-15b and miR-16 function led to the discovery of the importance of HIF-2α so this work demonstrated the utility of studying miRNA function to identify novel regulatory pathways in helper T-cell development. © 2016 John Wiley & Sons Ltd.

  6. Ly49Q, an ITIM-bearing NK receptor, positively regulates osteoclast differentiation

    International Nuclear Information System (INIS)

    Hayashi, Mikihito; Nakashima, Tomoki; Kodama, Tatsuhiko; Makrigiannis, Andrew P.; Toyama-Sorimachi, Noriko; Takayanagi, Hiroshi

    2010-01-01

    Osteoclasts, multinucleated cells that resorb bone, play a key role in bone remodeling. Although immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling is critical for osteoclast differentiation, the significance of immunoreceptor tyrosine-based inhibitory motif (ITIM) has not been well understood. Here we report the function of Ly49Q, an Ly49 family member possessing an ITIM motif, in osteoclastogenesis. Ly49Q is selectively induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) stimulation in bone marrow-derived monocyte/macrophage precursor cells (BMMs) among the Ly49 family of NK receptors. The knockdown of Ly49Q resulted in a significant reduction in the RANKL-induced formation of tartrate-resistance acid phosphatase (TRAP)-positive multinucleated cells, accompanied by a decreased expression of osteoclast-specific genes such as Nfatc1, Tm7sf4, Oscar, Ctsk, and Acp5. Osteoclastogenesis was also significantly impaired in Ly49Q-deficient cells in vitro. The inhibitory effect of Ly49Q-deficiency may be explained by the finding that Ly49Q competed for the association of Src-homology domain-2 phosphatase-1 (SHP-1) with paired immunoglobulin-like receptor-B (PIR-B), an ITIM-bearing receptor which negatively regulates osteoclast differentiation. Unexpectedly, Ly49Q deficiency did not lead to impaired osteoclast formation in vivo, suggesting the existence of a compensatory mechanism. This study provides an example in which an ITIM-bearing receptor functions as a positive regulator of osteoclast differentiation.

  7. Mechanical regulation of stem-cell differentiation by the stretch-activated Piezo channel.

    Science.gov (United States)

    He, Li; Si, Guangwei; Huang, Jiuhong; Samuel, Aravinthan D T; Perrimon, Norbert

    2018-03-01

    Somatic stem cells constantly adjust their self-renewal and lineage commitment by integrating various environmental cues to maintain tissue homeostasis. Although numerous chemical and biological signals have been identified that regulate stem-cell behaviour, whether stem cells can directly sense mechanical signals in vivo remains unclear. Here we show that mechanical stress regulates stem-cell differentiation in the adult Drosophila midgut through the stretch-activated ion channel Piezo. We find that Piezo is specifically expressed in previously unidentified enteroendocrine precursor cells, which have reduced proliferation ability and are destined to become enteroendocrine cells. Loss of Piezo activity reduces the generation of enteroendocrine cells in the adult midgut. In addition, ectopic expression of Piezo in all stem cells triggers both cell proliferation and enteroendocrine cell differentiation. Both the Piezo mutant and overexpression phenotypes can be rescued by manipulation of cytosolic Ca 2+ levels, and increases in cytosolic Ca 2+ resemble the Piezo overexpression phenotype, suggesting that Piezo functions through Ca 2+ signalling. Further studies suggest that Ca 2+ signalling promotes stem-cell proliferation and differentiation through separate pathways. Finally, Piezo is required for both mechanical activation of stem cells in a gut expansion assay and the increase of cytosolic Ca 2+ in response to direct mechanical stimulus in a gut compression assay. Thus, our study demonstrates the existence of a specific group of stem cells in the fly midgut that can directly sense mechanical signals through Piezo.

  8. Tissue-specific 5' heterogeneity of PPARα transcripts and their differential regulation by leptin.

    Directory of Open Access Journals (Sweden)

    Emma S Garratt

    Full Text Available The genes encoding nuclear receptors comprise multiple 5'untranslated exons, which give rise to several transcripts encoding the same protein, allowing tissue-specific regulation of expression. Both human and mouse peroxisome proliferator activated receptor (PPAR α genes have multiple promoters, although their function is unknown. Here we have characterised the rat PPARα promoter region and have identified three alternative PPARα transcripts, which have different transcription start sites owing to the utilisation of distinct first exons. Moreover these alternative PPARα transcripts were differentially expressed between adipose tissue and liver. We show that while the major adipose (P1 and liver (P2 transcripts were both induced by dexamethasone, they were differentially regulated by the PPARα agonist, clofibric acid, and leptin. Leptin had no effect on the adipose-specific P1 transcript, but induced liver-specific P2 promoter activity via a STAT3/Sp1 mechanism. Moreover in Wistar rats, leptin treatment between postnatal day 3-13 led to an increase in P2 but not P1 transcription in adipose tissue which was sustained into adulthood. This suggests that the expression of the alternative PPARα transcripts are in part programmed by early life exposure to leptin leading to persistent change in adipose tissue fatty acid metabolism through specific activation of a quiescent PPARα promoter. Such complexity in the regulation of PPARα may allow the expression of PPARα to be finely regulated in response to environmental factors.

  9. LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration

    Science.gov (United States)

    Wang, Lijun; Zhao, Yu; Bao, Xichen; Zhu, Xihua; Kwok, Yvonne Ka-yin; Sun, Kun; Chen, Xiaona; Huang, Yongheng; Jauch, Ralf; Esteban, Miguel A; Sun, Hao; Wang, Huating

    2015-01-01

    Emerging studies document the roles of long non-coding RNAs (LncRNAs) in regulating gene expression at chromatin level but relatively less is known how they regulate DNA methylation. Here we identify an lncRNA, Dum (developmental pluripotency-associated 2 (Dppa2) Upstream binding Muscle lncRNA) in skeletal myoblast cells. The expression of Dum is dynamically regulated during myogenesis in vitro and in vivo. It is also transcriptionally induced by MyoD binding upon myoblast differentiation. Functional analyses show that it promotes myoblast differentiation and damage-induced muscle regeneration. Mechanistically, Dum was found to silence its neighboring gene, Dppa2, in cis through recruiting Dnmt1, Dnmt3a and Dnmt3b. Furthermore, intrachromosomal looping between Dum locus and Dppa2 promoter is necessary for Dum/Dppa2 interaction. Collectively, we have identified a novel lncRNA that interacts with Dnmts to regulate myogenesis. PMID:25686699

  10. Differential regulation of cystic fibrosis transmembrane conductance regulator and Na+,K+ -ATPase in gills of striped bass, Morone saxatilis: effect of salinity and hormones

    DEFF Research Database (Denmark)

    Madsen, Steffen; Jensen, Lars Nørholm; Tipsmark, Christian Kølbaek

    2007-01-01

    -regulated kinase (ERK) 1/2 was stimulated by EGF but not affected by IGF-I. This study is the first to report a branchial EGF response and to demonstrate a functional ERK 1/2 pathway in the teleost gill. In conclusion, CFTR and Na(+),K(+) -ATPase are differentially regulated by salinity and hormones in gills...

  11. DNER, an epigenetically modulated gene, regulates glioblastoma-derived neurosphere cell differentiation and tumor propagation.

    Science.gov (United States)

    Sun, Peng; Xia, Shuli; Lal, Bachchu; Eberhart, Charles G; Quinones-Hinojosa, Alfredo; Maciaczyk, Jarek; Matsui, William; Dimeco, Francesco; Piccirillo, Sara M; Vescovi, Angelo L; Laterra, John

    2009-07-01

    Neurospheres derived from glioblastoma (GBM) and other solid malignancies contain neoplastic stem-like cells that efficiently propagate tumor growth and resist cytotoxic therapeutics. The primary objective of this study was to use histone-modifying agents to elucidate mechanisms by which the phenotype and tumor-promoting capacity of GBM-derived neoplastic stem-like cells are regulated. Using established GBM-derived neurosphere lines and low passage primary GBM-derived neurospheres, we show that histone deacetylase (HDAC) inhibitors inhibit growth, induce differentiation, and induce apoptosis of neoplastic neurosphere cells. A specific gene product induced by HDAC inhibition, Delta/Notch-like epidermal growth factor-related receptor (DNER), inhibited the growth of GBM-derived neurospheres, induced their differentiation in vivo and in vitro, and inhibited their engraftment and growth as tumor xenografts. The differentiating and tumor suppressive effects of DNER, a noncanonical Notch ligand, contrast with the previously established tumor-promoting effects of canonical Notch signaling in brain cancer stem-like cells. Our findings are the first to implicate noncanonical Notch signaling in the regulation of neoplastic stem-like cells and suggest novel neoplastic stem cell targeting treatment strategies for GBM and potentially other solid malignancies.

  12. RNase L controls terminal adipocyte differentiation, lipids storage and insulin sensitivity via CHOP10 mRNA regulation

    DEFF Research Database (Denmark)

    Fabre, Odile Martine Julie; Salehzada, T; Lambert, K

    2012-01-01

    Adipose tissue structure is altered during obesity, leading to deregulation of whole-body metabolism. Its function depends on its structure, in particular adipocytes number and differentiation stage. To better understand the mechanisms regulating adipogenesis, we have investigated the role...... is associated with CHOP10 mRNA and regulates its stability. CHOP10 expression is conserved in RNase L(-/-)-MEFs, maintaining preadipocyte state while impairing their terminal differentiation. RNase L(-/-)-MEFs have decreased lipids storage capacity, insulin sensitivity and glucose uptake. Expression of ectopic...... RNase L in RNase L(-/-)-MEFs triggers CHOP10 mRNA instability, allowing increased lipids storage, insulin response and glucose uptake. Similarly, downregulation of CHOP10 mRNA with CHOP10 siRNA in RNase L(-/-)-MEFs improves their differentiation in adipocyte. In vivo, aged RNase L(-)/(-) mice present...

  13. The ROCK isoforms differentially regulate the morphological characteristics of carcinoma cells.

    Science.gov (United States)

    Jerrell, Rachel J; Leih, Mitchell J; Parekh, Aron

    2017-06-26

    Rho-associated kinase (ROCK) activity drives cell migration via actomyosin contractility. During invasion, individual cancer cells can transition between 2 modes of migration, mesenchymal and amoeboid. Changes in ROCK activity can cause a switch between these migration phenotypes which are defined by distinct morphologies. However, recent studies have shown that the ROCK isoforms are not functionally redundant as previously thought. Therefore, it is unclear whether the ROCK isoforms play different roles in regulating migration phenotypes. Here, we found that ROCK1 and ROCK2 differentially regulate carcinoma cell morphology resulting in intermediate phenotypes that share some mesenchymal and amoeboid characteristics. These findings suggest that the ROCK isoforms play unique roles in the phenotypic plasticity of mesenchymal carcinoma cells which may have therapeutic implications.

  14. Differential co-expression and regulation analyses reveal different mechanisms underlying major depressive disorder and subsyndromal symptomatic depression.

    Science.gov (United States)

    Xu, Fan; Yang, Jing; Chen, Jin; Wu, Qingyuan; Gong, Wei; Zhang, Jianguo; Shao, Weihua; Mu, Jun; Yang, Deyu; Yang, Yongtao; Li, Zhiwei; Xie, Peng

    2015-04-03

    Recent depression research has revealed a growing awareness of how to best classify depression into depressive subtypes. Appropriately subtyping depression can lead to identification of subtypes that are more responsive to current pharmacological treatment and aid in separating out depressed patients in which current antidepressants are not particularly effective. Differential co-expression analysis (DCEA) and differential regulation analysis (DRA) were applied to compare the transcriptomic profiles of peripheral blood lymphocytes from patients with two depressive subtypes: major depressive disorder (MDD) and subsyndromal symptomatic depression (SSD). Six differentially regulated genes (DRGs) (FOSL1, SRF, JUN, TFAP4, SOX9, and HLF) and 16 transcription factor-to-target differentially co-expressed gene links or pairs (TF2target DCLs) appear to be the key differential factors in MDD; in contrast, one DRG (PATZ1) and eight TF2target DCLs appear to be the key differential factors in SSD. There was no overlap between the MDD target genes and SSD target genes. Venlafaxine (Efexor™, Effexor™) appears to have a significant effect on the gene expression profile of MDD patients but no significant effect on the gene expression profile of SSD patients. DCEA and DRA revealed no apparent similarities between the differential regulatory processes underlying MDD and SSD. This bioinformatic analysis may provide novel insights that can support future antidepressant R&D efforts.

  15. Biosynthesis of ribosomal RNA in nucleoli regulates pluripotency and differentiation ability of pluripotent stem cells.

    Science.gov (United States)

    Watanabe-Susaki, Kanako; Takada, Hitomi; Enomoto, Kei; Miwata, Kyoko; Ishimine, Hisako; Intoh, Atsushi; Ohtaka, Manami; Nakanishi, Mahito; Sugino, Hiromu; Asashima, Makoto; Kurisaki, Akira

    2014-12-01

    Pluripotent stem cells have been shown to have unique nuclear properties, for example, hyperdynamic chromatin and large, condensed nucleoli. However, the contribution of the latter unique nucleolar character to pluripotency has not been well understood. Here, we show that fibrillarin (FBL), a critical methyltransferase for ribosomal RNA (rRNA) processing in nucleoli, is one of the proteins highly expressed in pluripotent embryonic stem (ES) cells. Stable expression of FBL in ES cells prolonged the pluripotent state of mouse ES cells cultured in the absence of leukemia inhibitory factor (LIF). Analyses using deletion mutants and a point mutant revealed that the methyltransferase activity of FBL regulates stem cell pluripotency. Knockdown of this gene led to significant delays in rRNA processing, growth inhibition, and apoptosis in mouse ES cells. Interestingly, both partial knockdown of FBL and treatment with actinomycin D, an inhibitor of rRNA synthesis, induced the expression of differentiation markers in the presence of LIF and promoted stem cell differentiation into neuronal lineages. Moreover, we identified p53 signaling as the regulatory pathway for pluripotency and differentiation of ES cells. These results suggest that proper activity of rRNA production in nucleoli is a novel factor for the regulation of pluripotency and differentiation ability of ES cells. © 2014 AlphaMed Press.

  16. Local Inflammatory Cues Regulate Differentiation and Persistence of CD8+ Tissue-Resident Memory T Cells

    Directory of Open Access Journals (Sweden)

    Tessa Bergsbaken

    2017-04-01

    Full Text Available Many pathogens initiate infection at mucosal surfaces, and tissue-resident memory T (Trm cells play an important role in protective immunity, yet the tissue-specific signals that regulate Trm differentiation are poorly defined. During Yersinia infection, CD8+ T cell recruitment to areas of inflammation within the intestine is required for differentiation of the CD103−CD69+ Trm subset. Intestinal proinflammatory microenvironments have elevated interferon (IFN-β and interleukin-12 (IL-12, which regulated Trm markers, including CD103. Type I interferon-receptor- or IL-12-receptor-deficient T cells functioned similarly to wild-type (WT cells during infection; however, the inability of T cells to respond to inflammation resulted in defective differentiation of CD103−CD69+ Trm cells and reduced Trm persistence. Intestinal macrophages were the main producers of IFN-β and IL-12 during infection, and deletion of CCR2+ IL-12-producing cells reduced the size of the CD103− Trm population. These data indicate that intestinal inflammation drives phenotypic diversity and abundance of Trm cells for optimal tissue-specific immunity.

  17. Roles of Retinoids and Retinoic Acid Receptors in the Regulation of Hematopoietic Stem Cell Self-Renewal and Differentiation

    Directory of Open Access Journals (Sweden)

    Louise E. Purton

    2007-01-01

    Full Text Available Multipotent hematopoietic stem cells (HSCs sustain blood cell production throughout an individual's lifespan through complex processes ultimately leading to fates of self-renewal, differentiation or cell death decisions. A fine balance between these decisions in vivo allows for the size of the HSC pool to be maintained. While many key factors involved in regulating HSC/progenitor cell differentiation and cell death are known, the critical regulators of HSC self-renewal are largely unknown. In recent years, however, a number of studies describing methods of increasing or decreasing the numbers of HSCs in a given population have emerged. Of major interest here are the emerging roles of retinoids in the regulation of HSCs.

  18. Roquin Paralogs Differentially Regulate Functional NKT Cell Subsets.

    Science.gov (United States)

    Drees, Christoph; Vahl, J Christoph; Bortoluzzi, Sabrina; Heger, Klaus D; Fischer, Julius C; Wunderlich, F Thomas; Peschel, Christian; Schmidt-Supprian, Marc

    2017-04-01

    NKT cells represent a small subset of glycolipid-recognizing T cells that are heavily implicated in human allergic, autoimmune, and malignant diseases. In the thymus, precursor cells recognize self-glycolipids by virtue of their semi-invariant TCR, which triggers NKT cell lineage commitment and maturation. During their development, NKT cells are polarized into the NKT1, NKT2, and NKT17 subsets, defined through their cytokine-secretion patterns and the expression of key transcription factors. However, we have largely ignored how the differentiation into the NKT cell subsets is regulated. In this article, we describe the mRNA-binding Roquin-1 and -2 proteins as central regulators of murine NKT cell fate decisions. In the thymus, T cell-specific ablation of the Roquin paralogs leads to a dramatic expansion of NKT17 cells, whereas peripheral mature NKT cells are essentially absent. Roquin-1/2-deficient NKT17 cells show exaggerated lineage-specific expression of nearly all NKT17-defining proteins tested. We show through mixed bone marrow chimera experiments that NKT17 polarization is mediated through cell-intrinsic mechanisms early during NKT cell development. In contrast, the loss of peripheral NKT cells is due to cell-extrinsic factors. Surprisingly, Roquin paralog-deficient NKT cells are, in striking contrast to conventional T cells, compromised in their ability to secrete cytokines. Altogether, we show that Roquin paralogs regulate the development and function of NKT cell subsets in the thymus and periphery. Copyright © 2017 by The American Association of Immunologists, Inc.

  19. Semipermeable Capsules Wrapping a Multifunctional and Self-regulated Co-culture Microenvironment for Osteogenic Differentiation

    Science.gov (United States)

    Correia, Clara R.; Pirraco, Rogério P.; Cerqueira, Mariana T.; Marques, Alexandra P.; Reis, Rui L.; Mano, João F.

    2016-02-01

    A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the “stem cell niche”, the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

  20. Erk1 positively regulates osteoclast differentiation and bone resorptive activity.

    Directory of Open Access Journals (Sweden)

    Yongzheng He

    Full Text Available The extracellular signal-regulated kinases (ERK1 and 2 are widely-expressed and they modulate proliferation, survival, differentiation, and protein synthesis in multiple cell lineages. Altered ERK1/2 signaling is found in several genetic diseases with skeletal phenotypes, including Noonan syndrome, Neurofibromatosis type 1, and Cardio-facio-cutaneous syndrome, suggesting that MEK-ERK signals regulate human skeletal development. Here, we examine the consequence of Erk1 and Erk2 disruption in multiple functions of osteoclasts, specialized macrophage/monocyte lineage-derived cells that resorb bone. We demonstrate that Erk1 positively regulates osteoclast development and bone resorptive activity, as genetic disruption of Erk1 reduced osteoclast progenitor cell numbers, compromised pit formation, and diminished M-CSF-mediated adhesion and migration. Moreover, WT mice reconstituted long-term with Erk1(-/- bone marrow mononuclear cells (BMMNCs demonstrated increased bone mineral density as compared to recipients transplanted with WT and Erk2(-/- BMMNCs, implicating marrow autonomous, Erk1-dependent osteoclast function. These data demonstrate Erk1 plays an important role in osteoclast functions while providing rationale for the development of Erk1-specific inhibitors for experimental investigation and/or therapeutic modulation of aberrant osteoclast function.

  1. Enteric neural crest cells regulate vertebrate stomach patterning and differentiation.

    Science.gov (United States)

    Faure, Sandrine; McKey, Jennifer; Sagnol, Sébastien; de Santa Barbara, Pascal

    2015-01-15

    In vertebrates, the digestive tract develops from a uniform structure where reciprocal epithelial-mesenchymal interactions pattern this complex organ into regions with specific morphologies and functions. Concomitant with these early patterning events, the primitive GI tract is colonized by the vagal enteric neural crest cells (vENCCs), a population of cells that will give rise to the enteric nervous system (ENS), the intrinsic innervation of the GI tract. The influence of vENCCs on early patterning and differentiation of the GI tract has never been evaluated. In this study, we report that a crucial number of vENCCs is required for proper chick stomach development, patterning and differentiation. We show that reducing the number of vENCCs by performing vENCC ablations induces sustained activation of the BMP and Notch pathways in the stomach mesenchyme and impairs smooth muscle development. A reduction in vENCCs also leads to the transdifferentiation of the stomach into a stomach-intestinal mixed phenotype. In addition, sustained Notch signaling activity in the stomach mesenchyme phenocopies the defects observed in vENCC-ablated stomachs, indicating that inhibition of the Notch signaling pathway is essential for stomach patterning and differentiation. Finally, we report that a crucial number of vENCCs is also required for maintenance of stomach identity and differentiation through inhibition of the Notch signaling pathway. Altogether, our data reveal that, through the regulation of mesenchyme identity, vENCCs act as a new mediator in the mesenchymal-epithelial interactions that control stomach development. © 2015. Published by The Company of Biologists Ltd.

  2. 2-Bromopalmitate modulates neuronal differentiation through the regulation of histone acetylation

    Directory of Open Access Journals (Sweden)

    Xueran Chen

    2014-03-01

    Full Text Available In order to evaluate the functional significance of palmitoylation during multi-potent neural stem/progenitor cell proliferation and differentiation, retinoic acid-induced P19 cells were used in this study as a model system. Cell behaviour was monitored in the presence of the protein palmitoylation inhibitor 2-bromopalmitate (2BP. Here, we observed a significant reduction in neuronal differentiation in the 2BP-treated cell model. We further explored the underlying mechanisms and found that 2BP resulted in the decreased acetylation of histones H3 and H4 and interfered with cell cycle withdrawal and neural stem/progenitor cells' renewal. Our results established a direct link between palmitoylation and the regulation of neural cell fate specification and revealed the epigenetic regulatory mechanisms that are involved in the effects of palmitoylation during neural development.

  3. Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway

    OpenAIRE

    Hatori, Yuta; Yan, Ye; Schmidt, Katharina; Furukawa, Eri; Hasan, Nesrin M.; Yang, Nan; Liu, Chin-Nung; Sockanathan, Shanthini; Lutsenko, Svetlana

    2016-01-01

    Brain development requires a fine-tuned copper homoeostasis. Copper deficiency or excess results in severe neuro-pathologies. We demonstrate that upon neuronal differentiation, cellular demand for copper increases, especially within the secretory pathway. Copper flow to this compartment is facilitated through transcriptional and metabolic regulation. Quantitative real-time imaging revealed a gradual change in the oxidation state of cytosolic glutathione upon neuronal differentiation. Transiti...

  4. Distinct roles of the receptor tyrosine kinases c-ErbB and c-Kit in regulating the balance between erythroid cell proliferation and differentiation

    NARCIS (Netherlands)

    Wessely, O.; Mellitzer, G.; von Lindern, M.; Levitzki, A.; Gazit, A.; Ischenko, I.; Hayman, M. J.; Beug, H.

    1997-01-01

    In the bone marrow, multipotent and committed hematopoietic progenitors have to closely regulate their balance between sustained proliferation without differentiation (self renewal) and entering a terminal differentiation pathway. A useful model to analyze this regulation at the molecular level is

  5. PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Lan; Josifi, Erlena; Tiao, Joshua R. [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Retinoic acid (RA) induces neuroblastoma cells differentiation, which is accompanied by G0/G1 cell cycle arrest. Black-Right-Pointing-Pointer RA resulted in neuroblastoma cell survival and inhibition of DNA fragmentation; this is regulated by PI3K pathway. Black-Right-Pointing-Pointer RA activates PI3K and ERK1/2 pathway; PI3K pathway mediates RA-induced neuroblastoma cell differentiation. Black-Right-Pointing-Pointer Upregulation of p21 is necessary for RA-induced neuroblastoma cell differentiation. -- Abstract: Neuroblastoma, the most common extra-cranial solid tumor in infants and children, is characterized by a high rate of spontaneous remissions in infancy. Retinoic acid (RA) has been known to induce neuroblastoma differentiation; however, the molecular mechanisms and signaling pathways that are responsible for RA-mediated neuroblastoma cell differentiation remain unclear. Here, we sought to determine the cell signaling processes involved in RA-induced cellular differentiation. Upon RA administration, human neuroblastoma cell lines, SK-N-SH and BE(2)-C, demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Moreover, cell cycle arrest occurred in G1/G0 phase. The protein levels of cyclin-dependent kinase inhibitors, p21 and p27{sup Kip}, which inhibit cell proliferation by blocking cell cycle progression at G1/S phase, increased after RA treatment. Interestingly, RA promoted cell survival during the differentiation process, hence suggesting a potential mechanism for neuroblastoma resistance to RA therapy. Importantly, we found that the PI3K/AKT pathway is required for RA-induced neuroblastoma cell differentiation. Our results elucidated the molecular mechanism of RA-induced neuroblastoma cellular differentiation, which may be important for developing novel therapeutic strategy against poorly differentiated neuroblastoma.

  6. The T-ALL related gene BCL11B regulates the initial stages of human T-cell differentiation.

    Science.gov (United States)

    Ha, V L; Luong, A; Li, F; Casero, D; Malvar, J; Kim, Y M; Bhatia, R; Crooks, G M; Parekh, C

    2017-11-01

    The initial stages of T-cell differentiation are characterized by a progressive commitment to the T-cell lineage, a process that involves the loss of alternative (myelo-erythroid, NK, B) lineage potentials. Aberrant differentiation during these stages can result in T-cell acute lymphoblastic leukemia (T-ALL). However, the mechanisms regulating the initial stages of human T-cell differentiation are obscure. Through loss of function studies, we showed BCL11B, a transcription factor recurrently mutated T-ALL, is essential for T-lineage commitment, particularly the repression of NK and myeloid potentials, and the induction of T-lineage genes, during the initial stages of human T-cell differentiation. In gain of function studies, BCL11B inhibited growth of and induced a T-lineage transcriptional program in T-ALL cells. We found previously unknown differentiation stage-specific DNA binding of BCL11B at multiple T-lineage genes; target genes showed BCL11B-dependent expression, suggesting a transcriptional activator role for BCL11B at these genes. Transcriptional analyses revealed differences in the regulatory actions of BCL11B between human and murine thymopoiesis. Our studies show BCL11B is a key regulator of the initial stages of human T-cell differentiation and delineate the BCL11B transcriptional program, enabling the dissection of the underpinnings of normal T-cell differentiation and providing a resource for understanding dysregulations in T-ALL.

  7. RSPO1/β-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary

    NARCIS (Netherlands)

    Chassot, Anne-Amandine; Gregoire, Elodie P.; Lavery, Rowena; Taketo, Makoto M.; de Rooij, Dirk G.; Adams, Ian R.; Chaboissier, Marie-Christine

    2011-01-01

    Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog

  8. RSPO1/beta-Catenin Signaling Pathway Regulates Oogonia Differentiation and Entry into Meiosis in the Mouse Fetal Ovary

    NARCIS (Netherlands)

    Chassot, A.A.; Gregoire, E.P.; Lavery, R.; Taketo, M.M.; de Rooij, D.G.; Adams, I.R.; Chaboissier, M.C.

    2011-01-01

    Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog

  9. Regulator of calcineurin 1 differentially regulates TLR-dependent MyD88 and TRIF signaling pathways.

    Directory of Open Access Journals (Sweden)

    Zheng Pang

    Full Text Available Toll-like receptors (TLRs recognize the conserved molecular patterns in microorganisms and trigger myeloid differentiation primary response 88 (MyD88 and/or TIR-domain-containing adapter-inducing interferon-β (TRIF pathways that are critical for host defense against microbial infection. However, the molecular mechanisms that govern TLR signaling remain incompletely understood. Regulator of calcineurin-1 (RCAN1, a small evolutionarily conserved protein that inhibits calcineurin phosphatase activity, suppresses inflammation during Pseudomonas aeruginosa infection. Here, we define the roles for RCAN1 in P. aeruginosa lipopolysaccharide (LPS-activated TLR4 signaling. We compared the effects of P. aeruginosa LPS challenge on bone marrow-derived macrophages from both wild-type and RCAN1-deficient mice and found that RCAN1 deficiency increased the MyD88-NF-κB-mediated cytokine production (IL-6, TNF and MIP-2, whereas TRIF-interferon-stimulated response elements (ISRE-mediated cytokine production (IFNβ, RANTES and IP-10 was suppressed. RCAN1 deficiency caused increased IκBα phosphorylation and NF-κB activity in the MyD88-dependent pathway, but impaired ISRE activation and reduced IRF7 expression in the TRIF-dependent pathway. Complementary studies of a mouse model of P. aeruginosa LPS-induced acute pneumonia confirmed that RCAN1-deficient mice displayed greatly enhanced NF-κB activity and MyD88-NF-κB-mediated cytokine production, which correlated with enhanced pulmonary infiltration of neutrophils. By contrast, RCAN1 deficiency had little effect on the TRIF pathway in vivo. These findings demonstrate a novel regulatory role of RCAN1 in TLR signaling, which differentially regulates MyD88 and TRIF pathways.

  10. MicroRNAs define distinct human neuroblastoma cell phenotypes and regulate their differentiation and tumorigenicity

    International Nuclear Information System (INIS)

    Samaraweera, Leleesha; Grandinetti, Kathryn B; Huang, Ruojun; Spengler, Barbara A; Ross, Robert A

    2014-01-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in children. NB tumors and derived cell lines are phenotypically heterogeneous. Cell lines are classified by phenotype, each having distinct differentiation and tumorigenic properties. The neuroblastic phenotype is tumorigenic, has neuronal features and includes stem cells (I-cells) and neuronal cells (N-cells). The non-neuronal phenotype (S-cell) comprises cells that are non-tumorigenic with features of glial/smooth muscle precursor cells. This study identified miRNAs associated with each distinct cell phenotypes and investigated their role in regulating associated differentiation and tumorigenic properties. A miRNA microarray was performed on the three cell phenotypes and expression verified by qRT-PCR. miRNAs specific for certain cell phenotypes were modulated using miRNA inhibitors or stable transfection. Neuronal differentiation was induced by RA; non-neuronal differentiation by BrdU. Changes in tumorigenicity were assayed by soft agar colony forming ability. N-myc binding to miR-375 promoter was assayed by chromatin-immunoprecipitation. Unsupervised hierarchical clustering of miRNA microarray data segregated neuroblastic and non-neuronal cell lines and showed that specific miRNAs define each phenotype. qRT-PCR validation confirmed that increased levels of miR-21, miR-221 and miR-335 are associated with the non-neuronal phenotype, whereas increased levels of miR-124 and miR-375 are exclusive to neuroblastic cells. Downregulation of miR-335 in non-neuronal cells modulates expression levels of HAND1 and JAG1, known modulators of neuronal differentiation. Overexpression of miR-124 in stem cells induces terminal neuronal differentiation with reduced malignancy. Expression of miR-375 is exclusive for N-myc-expressing neuroblastic cells and is regulated by N-myc. Moreover, miR-375 downregulates expression of the neuronal-specific RNA binding protein HuD. Thus, miRNAs define distinct NB cell phenotypes

  11. RNF20 and USP44 regulate stem cell differentiation by modulating H2B monoubiquitylation

    Science.gov (United States)

    Fuchs, Gilad; Shema, Efrat; Vesterman, Rita; Kotler, Eran; Wolchinsky, Zohar; Wilder, Sylvia; Golomb, Lior; Pribluda, Ariel; Zhang, Feng; Haj-Yahya, Mahmood; Feldmesser, Ester; Brik, Ashraf; Yu, Xiaochun; Hanna, Jacob; Aberdam, Daniel; Domany, Eytan; Oren, Moshe

    2012-01-01

    Summary Embryonic stem cells (ESC) maintain high genomic plasticity, essential for their capacity to enter diverse differentiation pathways. Post-transcriptional modifications of chromatin histones play a pivotal role in maintaining this plasticity. We now report that one such modification, monoubiquitylation of histone H2B on lysine 120 (H2Bub1), catalyzed by the E3 ligase RNF20, increases during ESC differentiation and is required for efficient execution of this process. This increase is particularly important for the transcriptional induction of relatively long genes during ESC differentiation. Furthermore, we identify the deubiquitinase USP44 as a negative regulator of H2B ubiquitylation, whose downregulation during ESC differentiation contributes to the increase in H2Bub1. Our findings suggest that optimal ESC differentiation requires dynamic changes in H2B ubiquitylation patterns, which must occur in a timely and well-coordinated manner. PMID:22681888

  12. HIF-1α as a Regulator of BMP2-Induced Chondrogenic Differentiation, Osteogenic Differentiation, and Endochondral Ossification in Stem Cells

    Directory of Open Access Journals (Sweden)

    Nian Zhou

    2015-04-01

    Full Text Available Background/Aims: Joint cartilage defects are difficult to treat due to the limited self-repair capacities of cartilage. Cartilage tissue engineering based on stem cells and gene enhancement is a potential alternative for cartilage repair. Bone morphogenetic protein 2 (BMP2 has been shown to induce chondrogenic differentiation in mesenchymal stem cells (MSCs; however, maintaining the phenotypes of MSCs during cartilage repair since differentiation occurs along the endochondral ossification pathway. In this study, hypoxia inducible factor, or (HIF-1α, was determined to be a regulator of BMP2-induced chondrogenic differentiation, osteogenic differentiation, and endochondral bone formation. Methods: BMP2 was used to induce chondrogenic and osteogenic differentiation in stem cells and fetal limb development. After HIF-1α was added to the inducing system, any changes in the differentiation markers were assessed. Results: HIF-1α was found to potentiate BMP2-induced Sox9 and the expression of chondrogenesis by downstream markers, and inhibit Runx2 and the expression of osteogenesis by downstream markers in vitro. In subcutaneous stem cell implantation studies, HIF-1α was shown to potentiate BMP2-induced cartilage formation and inhibit endochondral ossification during ectopic bone/cartilage formation. In the fetal limb culture, HIF-1α and BMP2 synergistically promoted the expansion of the proliferating chondrocyte zone and inhibited chondrocyte hypertrophy and endochondral ossification. Conclusion: The results of this study indicated that, when combined with BMP2, HIF-1α induced MSC differentiation could become a new method of maintaining cartilage phenotypes during cartilage tissue engineering.

  13. Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis.

    Science.gov (United States)

    Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K

    2012-06-12

    Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior.

  14. Identification of genes differentially regulated in rat alveolar bone wound healing by subtractive hybridization.

    Science.gov (United States)

    Ohira, T; Myokai, F; Shiomi, N; Yamashiro, K; Yamamoto, T; Murayama, Y; Arai, H; Nishimura, F; Takashiba, S

    2004-07-01

    Periodontal healing requires the participation of regulatory molecules, cells, and scaffold or matrix. Here, we hypothesized that a certain set of genes is expressed in alveolar bone wound healing. Reciprocal subtraction gave 400 clones from the injured alveolar bone of Wistar rats. Identification of 34 genes and analysis of their expression in injured tissue revealed several clusters of unique gene regulation patterns, including the up-regulation at 1 wk of cytochrome c oxidase regulating electron transfer and energy metabolism, presumably occurring at the site of inflammation; up-regulation at 2.5 wks of pro-alpha-2 type I collagen involving the formation of a connective tissue structure; and up-regulation at 1 and 2 wks and down-regulation at 2.5 and 4 wks of ubiquitin carboxyl-terminal hydrolase l3 involving cell cycle, DNA repair, and stress response. The differential expression of genes may be associated with the processes of inflammation, wound contraction, and formation of a connective tissue structure.

  15. Regulation of differentiation flux by Notch signalling influences the number of dopaminergic neurons in the adult brain

    Directory of Open Access Journals (Sweden)

    Niurka Trujillo-Paredes

    2016-03-01

    Full Text Available Notch signalling is a well-established pathway that regulates neurogenesis. However, little is known about the role of Notch signalling in specific neuronal differentiation. Using Dll1 null mice, we found that Notch signalling has no function in the specification of mesencephalic dopaminergic neural precursor cells (NPCs, but plays an important role in regulating their expansion and differentiation into neurons. Premature neuronal differentiation was observed in mesencephalons of Dll1-deficient mice or after treatment with a Notch signalling inhibitor. Coupling between neurogenesis and dopaminergic differentiation was indicated from the coincident emergence of neuronal and dopaminergic markers. Early in differentiation, decreasing Notch signalling caused a reduction in NPCs and an increase in dopaminergic neurons in association with dynamic changes in the proportion of sequentially-linked dopaminergic NPCs (Msx1/2+, Ngn2+, Nurr1+. These effects in differentiation caused a significant reduction in the number of dopaminergic neurons produced. Accordingly, Dll1 haploinsufficient adult mice, in comparison with their wild-type littermates, have a consistent reduction in neuronal density that was particularly evident in the substantia nigra pars compacta. Our results are in agreement with a mathematical model based on a Dll1-mediated regulatory feedback loop between early progenitors and their dividing precursors that controls the emergence and number of dopaminergic neurons.

  16. Mof-associated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation

    Science.gov (United States)

    Ravens, Sarina; Fournier, Marjorie; Ye, Tao; Stierle, Matthieu; Dembele, Doulaye; Chavant, Virginie; Tora, Làszlò

    2014-01-01

    The histone acetyltransferase (HAT) Mof is essential for mouse embryonic stem cell (mESC) pluripotency and early development. Mof is the enzymatic subunit of two different HAT complexes, MSL and NSL. The individual contribution of MSL and NSL to transcription regulation in mESCs is not well understood. Our genome-wide analysis show that i) MSL and NSL bind to specific and common sets of expressed genes, ii) NSL binds exclusively at promoters, iii) while MSL binds in gene bodies. Nsl1 regulates proliferation and cellular homeostasis of mESCs. MSL is the main HAT acetylating H4K16 in mESCs, is enriched at many mESC-specific and bivalent genes. MSL is important to keep a subset of bivalent genes silent in mESCs, while developmental genes require MSL for expression during differentiation. Thus, NSL and MSL HAT complexes differentially regulate specific sets of expressed genes in mESCs and during differentiation. DOI: http://dx.doi.org/10.7554/eLife.02104.001 PMID:24898753

  17. Mediator complex cooperatively regulates transcription of retinoic acid target genes with Polycomb Repressive Complex 2 during neuronal differentiation.

    Science.gov (United States)

    Fukasawa, Rikiya; Iida, Satoshi; Tsutsui, Taiki; Hirose, Yutaka; Ohkuma, Yoshiaki

    2015-11-01

    The Mediator complex (Mediator) plays key roles in transcription and functions as the nexus for integration of various transcriptional signals. Previously, we screened for Mediator cyclin-dependent kinase (CDK)-interacting factors and identified three proteins related to chromatin regulation. One of them, SUZ12 is required for both stability and activity of Polycomb Repressive Complex 2 (PRC2). PRC2 primarily suppresses gene expression through histone H3 lysine 27 trimethylation, resulting in stem cell maintenance and differentiation; perturbation of this process leads to oncogenesis. Recent work showed that Mediator contributes to the embryonic stem cell state through DNA loop formation, which is strongly associated with chromatin architecture; however, it remains unclear how Mediator regulates gene expression in cooperation with chromatin regulators (i.e. writers, readers and remodelers). We found that Mediator CDKs interact directly with the PRC2 subunit EZH2, as well as SUZ12. Known PRC2 target genes were deregulated by Mediator CDK knockdown during neuronal differentiation, and both Mediator and PRC2 complexes co-occupied the promoters of developmental genes regulated by retinoic acid. Our results provide a mechanistic link between Mediator and PRC2 during neuronal differentiation. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  18. NAD+ protects against EAE by regulating CD4+ T-cell differentiation

    Science.gov (United States)

    Tullius, Stefan G.; Biefer, Hector Rodriguez Cetina; Li, Suyan; Trachtenberg, Alexander J.; Edtinger, Karoline; Quante, Markus; Krenzien, Felix; Uehara, Hirofumi; Yang, Xiaoyong; Kissick, Haydn T.; Kuo, Winston P.; Ghiran, Ionita; de la Fuente, Miguel A.; Arredouani, Mohamed S.; Camacho, Virginia; Tigges, John C.; Toxavidis, Vasilis; El Fatimy, Rachid; Smith, Brian D.; Vasudevan, Anju; ElKhal, Abdallah

    2014-01-01

    CD4+ T cells are involved in the development of autoimmunity, including multiple sclerosis (MS). Here we show that nicotinamide adenine dinucleotide (NAD+) blocks experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by inducing immune homeostasis through CD4+IFNγ+IL-10+ T cells and reverses disease progression by restoring tissue integrity via remyelination and neuroregeneration. We show that NAD+ regulates CD4+ T-cell differentiation through tryptophan hydroxylase-1 (Tph1), independently of well-established transcription factors. In the presence of NAD+, the frequency of T-bet−/− CD4+IFNγ+ T cells was twofold higher than wild-type CD4+ T cells cultured in conventional T helper 1 polarizing conditions. Our findings unravel a new pathway orchestrating CD4+ T-cell differentiation and demonstrate that NAD+ may serve as a powerful therapeutic agent for the treatment of autoimmune and other diseases. PMID:25290058

  19. Characterization of the Methylation Status of Pax7 and Myogenic Regulator Factors in Cell Myogenic Differentiation.

    Science.gov (United States)

    Chao, Zhe; Zheng, Xin-Li; Sun, Rui-Ping; Liu, Hai-Long; Huang, Li-Li; Cao, Zong-Xi; Deng, Chang-Yan; Wang, Feng

    2016-07-01

    Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

  20. The hippo pathway promotes Notch signaling in regulation of cell differentiation, proliferation, and oocyte polarity.

    Directory of Open Access Journals (Sweden)

    Jianzhong Yu

    2008-03-01

    Full Text Available Specification of the anterior-posterior axis in Drosophila oocytes requires proper communication between the germ-line cells and the somatically derived follicular epithelial cells. Multiple signaling pathways, including Notch, contribute to oocyte polarity formation by controlling the temporal and spatial pattern of follicle cell differentiation and proliferation. Here we show that the newly identified Hippo tumor-suppressor pathway plays a crucial role in the posterior follicle cells in the regulation of oocyte polarity. Disruption of the Hippo pathway, including major components Hippo, Salvador, and Warts, results in aberrant follicle-cell differentiation and proliferation and dramatic disruption of the oocyte anterior-posterior axis. These phenotypes are related to defective Notch signaling in follicle cells, because misexpression of a constitutively active form of Notch alleviates the oocyte polarity defects. We also find that follicle cells defective in Hippo signaling accumulate the Notch receptor and display defects in endocytosis markers. Our findings suggest that the interaction between Hippo and classic developmental pathways such as Notch is critical to spatial and temporal regulation of differentiation and proliferation and is essential for development of the body axes in Drosophila.

  1. Tissue-Specific 5′ Heterogeneity of PPARα Transcripts and Their Differential Regulation by Leptin

    Science.gov (United States)

    Garratt, Emma S.; Vickers, Mark H.; Gluckman, Peter D.; Hanson, Mark A.

    2013-01-01

    The genes encoding nuclear receptors comprise multiple 5′untranslated exons, which give rise to several transcripts encoding the same protein, allowing tissue-specific regulation of expression. Both human and mouse peroxisome proliferator activated receptor (PPAR) α genes have multiple promoters, although their function is unknown. Here we have characterised the rat PPARα promoter region and have identified three alternative PPARα transcripts, which have different transcription start sites owing to the utilisation of distinct first exons. Moreover these alternative PPARα transcripts were differentially expressed between adipose tissue and liver. We show that while the major adipose (P1) and liver (P2) transcripts were both induced by dexamethasone, they were differentially regulated by the PPARα agonist, clofibric acid, and leptin. Leptin had no effect on the adipose-specific P1 transcript, but induced liver-specific P2 promoter activity via a STAT3/Sp1 mechanism. Moreover in Wistar rats, leptin treatment between postnatal day 3–13 led to an increase in P2 but not P1 transcription in adipose tissue which was sustained into adulthood. This suggests that the expression of the alternative PPARα transcripts are in part programmed by early life exposure to leptin leading to persistent change in adipose tissue fatty acid metabolism through specific activation of a quiescent PPARα promoter. Such complexity in the regulation of PPARα may allow the expression of PPARα to be finely regulated in response to environmental factors. PMID:23825665

  2. MiR-132 regulates osteogenic differentiation via downregulating Sirtuin1 in a peroxisome proliferator-activated receptor β/δ–dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Kai; Qu, Bo; Liao, Dongfa; Liu, Da; Wang, Cairu; Zhou, Jingsong; Pan, Xianming, E-mail: xianmingpanxj@163.com

    2016-09-09

    MicroRNAs (miRNAs) play significant roles in multiple diseases by regulating the expression of their target genes. Type 2 diabetes mellitus (T2DM) is a chronic endocrine and metabolic disease with complex mechanisms. T2DM can result in diabetic osteoporosis (DO), which is characterized by bone loss, decreased bone mineral density and increased bone fractures. The promotion of osteogenic differentiation of osteoblasts is an effective way to treat osteoporosis. In the present study, high glucose (HG) and free fatty acids (FFA) were employed to mimic T2DM in MC3T3-E1 cells. To induce osteogenic differentiation, MC3T3-E1 cells were cultured in osteogenic medium. The results showed that osteogenic differentiation was significantly suppressed by HG and FFA. We found that miR-132 expression was significantly upregulated and much higher in HG-FFA–induced cells than other selected miRNAs, indicating that miR-132 might play an important role in DO. Furthermore, overexpression of miR-132 markedly inhibited the expression of key markers of osteogenic differentiation and alkaline phosphatase (ALP) activity. Reciprocally, inhibition of miR-132 restored osteogenic differentiation, even under treatment with HG-FFA. We also showed that Sirtuin 1 (Sirt1) was one of the target genes of miR-132, whose expression was controlled by miR-132. Ectopic expression of Sirt1 reversed the decrease in osteogenic differentiation caused by miR-132 and HG-FFA. These results demonstrated the direct role of miR-132 in suppressing osteogenic differentiation through downregulating Sirt1. Moreover, we demonstrated that peroxisome proliferator-activated receptor β/δ (PPARβ/δ) was a downstream molecule of Sirt1, and its knockout by PPARβ/δ siRNA significantly abolished the promotive effects of Sirt1 on osteogenic differentiation, indicating that Sirt1 functioned in a PPARβ/δ–dependent manner. Taken together, we provide crucial evidence that miR-132 plays a key role in regulating osteogenic

  3. MiR-132 regulates osteogenic differentiation via downregulating Sirtuin1 in a peroxisome proliferator-activated receptor β/δ–dependent manner

    International Nuclear Information System (INIS)

    Gong, Kai; Qu, Bo; Liao, Dongfa; Liu, Da; Wang, Cairu; Zhou, Jingsong; Pan, Xianming

    2016-01-01

    MicroRNAs (miRNAs) play significant roles in multiple diseases by regulating the expression of their target genes. Type 2 diabetes mellitus (T2DM) is a chronic endocrine and metabolic disease with complex mechanisms. T2DM can result in diabetic osteoporosis (DO), which is characterized by bone loss, decreased bone mineral density and increased bone fractures. The promotion of osteogenic differentiation of osteoblasts is an effective way to treat osteoporosis. In the present study, high glucose (HG) and free fatty acids (FFA) were employed to mimic T2DM in MC3T3-E1 cells. To induce osteogenic differentiation, MC3T3-E1 cells were cultured in osteogenic medium. The results showed that osteogenic differentiation was significantly suppressed by HG and FFA. We found that miR-132 expression was significantly upregulated and much higher in HG-FFA–induced cells than other selected miRNAs, indicating that miR-132 might play an important role in DO. Furthermore, overexpression of miR-132 markedly inhibited the expression of key markers of osteogenic differentiation and alkaline phosphatase (ALP) activity. Reciprocally, inhibition of miR-132 restored osteogenic differentiation, even under treatment with HG-FFA. We also showed that Sirtuin 1 (Sirt1) was one of the target genes of miR-132, whose expression was controlled by miR-132. Ectopic expression of Sirt1 reversed the decrease in osteogenic differentiation caused by miR-132 and HG-FFA. These results demonstrated the direct role of miR-132 in suppressing osteogenic differentiation through downregulating Sirt1. Moreover, we demonstrated that peroxisome proliferator-activated receptor β/δ (PPARβ/δ) was a downstream molecule of Sirt1, and its knockout by PPARβ/δ siRNA significantly abolished the promotive effects of Sirt1 on osteogenic differentiation, indicating that Sirt1 functioned in a PPARβ/δ–dependent manner. Taken together, we provide crucial evidence that miR-132 plays a key role in regulating osteogenic

  4. 4E-BP1 regulates the differentiation of white adipose tissue.

    Science.gov (United States)

    Tsukiyama-Kohara, Kyoko; Katsume, Asao; Kimura, Kazuhiro; Saito, Masayuki; Kohara, Michinori

    2013-07-01

    4E Binding protein 1 (4E-BP1) suppresses translation initiation. The absence of 4E-BP1 drastically reduces the amount of adipose tissue in mice. To address the role of 4E-BP1 in adipocyte differentiation, we characterized 4E-BP1(-/-) mice in this study. The lack of 4E-BP1 decreased the amount of white adipose tissue and increased the amount of brown adipose tissue. In 4E-BP1(-/-) MEF cells, PPARγ coactivator 1 alpha (PGC-1α) expression increased and exogenous 4E-BP1 expression suppressed PGC-1α expression. The level of 4E-BP1 expression was higher in white adipocytes than in brown adipocytes and showed significantly greater up-regulation in white adipocytes than in brown adipocytes during preadipocyte differentiation into mature adipocytes. The amount of PGC-1α was consistently higher in HB cells (a brown preadipocyte cell line) than in HW cells (a white preadipocyte cell line) during differentiation. Moreover, the ectopic over-expression of 4E-BP1 suppressed PGC-1α expression in white adipocytes, but not in brown adipocytes. Thus, the results of our study indicate that 4E-BP1 may suppress brown adipocyte differentiation and PGC-1α expression in white adipose tissues. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  5. Differential expression of genes regulated in response to drought stress in diploid cotton (Gossypium arboreum) (abstract)

    International Nuclear Information System (INIS)

    Hussain, T.; Majeed, A.; Maqbool, A.; Hussain, S.S.; Ali, T.; Riazuddin, S.

    2005-01-01

    Negative effects on the Water status of plants is one of the most common and deleterious stresses experienced by wild and cultivated plants throughout the World. Our project is designed to identify, clone and characterize gene sequences regulated in response to Water stress (e.g., drought). We used the differential-display reverse transcriptase polymerase chain reaction (DD-RT- PCA) methodology to accomplish our Objectives. Structural and functional characterization of environmental stress-induced genes has contributed to a better understanding of how plants respond and adapt to different abiotic stresses. Differential display was used to compare overall difference in gene expression between draught stressed and unstressed (control) plants of diploid Cotton (Gossypium arboreum). DDRT-PCR product from stressed and unstressed samples resolved side by side on 6% PAGE to compare qualitative and quantitative difference in mRNA expression. A total of 81 primer combinations were tested. DDRT -PCR enabled us to identify differentially expressed transcripts between water stressed and non-stressed cotton seedlings. PAGE revealed a total of 347 DNA transcripts in stressed samples (New Transcripts) while 110 down regulated and 209 up regulated DNA transcripts were also recorded. Similarly. 22 DNA transcripts were identified based on the comparative study of PAGE and Agarose gel electrophoresis. These sequences showed various degree homology With draught tolerant genes in the gene bank. (author)

  6. Sox2 Promotes Malignancy in Glioblastoma by Regulating Plasticity and Astrocytic Differentiation

    Directory of Open Access Journals (Sweden)

    Artem D. Berezovsky

    2014-03-01

    Full Text Available The high-mobility group–box transcription factor sex-determining region Y–box 2 (Sox2 is essential for the maintenance of stem cells from early development to adult tissues. Sox2 can reprogram differentiated cells into pluripotent cells in concert with other factors and is overexpressed in various cancers. In glioblastoma (GBM, Sox2 is a marker of cancer stemlike cells (CSCs in neurosphere cultures and is associated with the proneural molecular subtype. Here, we report that Sox2 expression pattern in GBM tumors and patient-derived mouse xenografts is not restricted to a small percentage of cells and is coexpressed with various lineage markers, suggesting that its expression extends beyond CSCs to encompass more differentiated neoplastic cells across molecular subtypes. Employing a CSC derived from a patient with GBM and isogenic differentiated cell model, we show that Sox2 knockdown in the differentiated state abolished dedifferentiation and acquisition of CSC phenotype. Furthermore, Sox2 deficiency specifically impaired the astrocytic component of a biphasic gliosarcoma xenograft model while allowing the formation of tumors with sarcomatous phenotype. The expression of genes associated with stem cells and malignancy were commonly downregulated in both CSCs and serum-differentiated cells on Sox2 knockdown. Genes previously shown to be associated with pluripontency and CSCs were only affected in the CSC state, whereas embryonic stem cell self-renewal genes and cytokine signaling were downregulated, and the Wnt pathway activated in differentiated Sox2-deficient cells. Our results indicate that Sox2 regulates the expression of key genes and pathways involved in GBM malignancy, in both cancer stemlike and differentiated cells, and maintains plasticity for bidirectional conversion between the two states, with significant clinical implications.

  7. Tissue transglutaminase (TG2 activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line

    Directory of Open Access Journals (Sweden)

    Xiaoxue Yin

    2012-08-01

    Full Text Available Tissue transglutaminase (type II, TG2 has long been postulated to directly promote skeletal matrix calcification and play an important role in ossification. However, limited information is available on the expression, function and modulating mechanism of TG2 during osteoblast differentiation and mineralization. To address these issues, we cultured the well-established human osteosarcoma cell line SAOS-2 with osteo-inductive conditioned medium and set up three time points (culture days 4, 7, and 14 to represent different stages of SAOS-2 differentiation. Osteoblast markers, mineralization, as well as TG2 expression and activity, were then assayed in each stage. Furthermore, we inhibited TG activity with cystamine and then checked SAOS-2 differentiation and mineralization in each stage. The results showed that during the progression of osteoblast differentiation SAOS-2 cells presented significantly high levels of osteocalcin (OC mRNA, bone morphogenetic protein-2 (BMP-2 and collagen I, significantly high alkaline phosphatase (ALP activity, and the increased formation of calcified matrix. With the same tendency, TG2 expression and activity were up-regulated. Furthermore, inhibition of TG activity resulted in a significant decrease of OC, collagen I, and BMP-2 mRNA and of ALP activity and mineralization. This study demonstrated that TG2 is involved in osteoblast differentiation and may play a role in the initiation and regulation of the mineralization processes. Moreover, the modulating effects of TG2 on osteoblasts may be related to BMP-2.

  8. The transcription factor Jdp2 controls bone homeostasis and antibacterial immunity by regulating osteoclast and neutrophil differentiation.

    Science.gov (United States)

    Maruyama, Kenta; Fukasaka, Masahiro; Vandenbon, Alexis; Saitoh, Tatsuya; Kawasaki, Takumi; Kondo, Takeshi; Yokoyama, Kazunari K; Kidoya, Hiroyasu; Takakura, Nobuyuki; Standley, Daron; Takeuchi, Osamu; Akira, Shizuo

    2012-12-14

    Jdp2 is an AP-1 family transcription factor that regulates the epigenetic status of histones. Previous in vitro studies revealed that Jdp2 is involved in osteoclastogenesis. However, the roles of Jdp2 in vivo and its pleiotropic functions are largely unknown. Here we generated Jdp2(-/-) mice and discovered its crucial roles not only in bone metabolism but also in differentiation of neutrophils. Jdp2(-/-) mice exhibited osteopetrosis resulting from impaired osteoclastogenesis. Jdp2(-/-) neutrophils were morphologically normal but had impaired surface expression of Ly6G, bactericidal function, and apoptosis. We also found that ATF3 was an inhibitor of neutrophil differentiation and that Jdp2 directly suppresses its expression via inhibition of histone acetylation. Strikingly, Jdp2(-/-) mice were highly susceptible to Staphylococcus aureus and Candida albicans infection. Thus, Jdp2 plays pivotal roles in in vivo bone homeostasis and host defense by regulating osteoclast and neutrophil differentiation. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Bone morphogenic protein-2 regulates the myogenic differentiation of PMVECs in CBDL rat serum-induced pulmonary microvascular remodeling

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chang; Chen, Lin; Zeng, Jing; Cui, Jian; Ning, Jiao-nin [Department of Anesthesia, Southwest Hospital, The Third Military Medical University, Chongqing 400038 (China); Wang, Guan-song [Institute of Respiratory Disease, Xinqiao Hospital, The Third Military Medical University, Chongqing 400037 (China); Belguise, Karine; Wang, Xiaobo [Université P. Sabatier Toulouse III and CNRS, LBCMCP, 31062 Toulouse Cedex 9 (France); Qian, Gui-sheng [Institute of Respiratory Disease, Xinqiao Hospital, The Third Military Medical University, Chongqing 400037 (China); Lu, Kai-zhi [Department of Anesthesia, Southwest Hospital, The Third Military Medical University, Chongqing 400038 (China); Yi, Bin, E-mail: yibin1974@163.com [Department of Anesthesia, Southwest Hospital, The Third Military Medical University, Chongqing 400038 (China)

    2015-08-01

    Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observed that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling. - Highlights: • CBDL-rat serum promotes the myogenic

  10. Regulation of intracellular free calcium concentration during heterocyst differentiation by HetR and NtcA in Anabaena sp. PCC 7120.

    Science.gov (United States)

    Shi, Yunming; Zhao, Weixing; Zhang, Wei; Ye, Zi; Zhao, Jindong

    2006-07-25

    Calcium ions are important to some prokaryotic cellular processes, such as heterocyst differentiation of cyanobacteria. Intracellular free Ca(2+)concentration, [Ca(2+)](i), increases several fold in heterocysts and is regulated by CcbP, a Ca(2+)-binding protein found in heterocyst-forming cyanobacteria. We demonstrate here that CcbP is degraded by HetR, a serine-type protease that controls heterocyst differentiation. The degradation depends on Ca(2+) and appears to be specific because HetR did not digest other tested proteins. CcbP was found to bind two Ca(2+) per molecule with K(D) values of 200 nM and 12.8 microM. Degradation of CcbP releases bound Ca(2+) that contributes significantly to the increase of [Ca(2+)](i) during the process of heterocyst differentiation in Anabaena sp. strain PCC 7120. We suggest that degradation of CcbP is a mechanism of positive autoregulation of HetR. The down-regulation of ccbP in differentiating cells and mature heterocysts, which also is critical to the regulation of [Ca(2+)](i), depends on NtcA. Coexpression of ntcA and a ccbP promoter-controlled gfp in Escherichia coli diminished production of GFP, and the decrease is enhanced by alpha-ketoglutarate. It was also found that NtcA could bind a fragment of the ccbP promoter containing an NtcA-binding sequence in a alpha-ketoglutarate-dependent fashion. Therefore, [Ca(2+)](i) is regulated by a collaboration of HetR and NtcA in heterocyst differentiation in Anabaena sp. strain PCC 7120.

  11. Identification of key factors regulating self-renewal and differentiation in EML hematopoietic precursor cells by RNA-sequencing analysis.

    Science.gov (United States)

    Zong, Shan; Deng, Shuyun; Chen, Kenian; Wu, Jia Qian

    2014-11-11

    Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient's hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study. RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment. In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

  12. Transiently truncated and differentially regulated expression of midkine during mouse embryogenesis

    International Nuclear Information System (INIS)

    Chen Qin; Yuan Yuanyang; Lin Shuibin; Chang Youde; Zhuo Xinming; Wei Wei; Tao Ping; Ruan Lingjuan; Li Qifu; Li Zhixing

    2005-01-01

    Midkine (MK) is a retinoic acid response cytokine, mostly expressed in embryonic tissues. Aberrant expression of MK was found in numerous cancers. In human, a truncated MK was expressed specifically in tumor/cancer tissues. Here we report the discovery of a novel truncated form of MK transiently expressed during normal mouse embryonic development. In addition, MK is concentrated at the interface between developing epithelium and mesenchyme as well as highly proliferating cells. Its expression, which is closely coordinated with angiogenesis and vasculogenesis, is spatiotemporally regulated with peaks in extensive organogenesis period and undifferentiated cells tailing off in maturing cells, implying its role in nascent blood vessel (endothelial) signaling of tissue differentiation and stem cell renewal/differentiation.. Cloning and sequencing analysis revealed that the embryonic truncated MK, in which the conserved domain is in-frame deleted, presumably producing a novel secreted small peptide, is different from the truncated form in human cancer tissues, whose deletion results in a frame-shift mutation. Our data suggest that MK may play a role in epithelium-mesenchyme interactions, blood vessel signaling, and the decision of proliferation vs differentiation. Detection of the transiently expressed truncated MK reveals its novel function in development and sheds light on its role in carcinogenesis

  13. Differential Regulation of Elastic Fiber Formation by Fibulin-4 and -5*

    Science.gov (United States)

    Choudhury, Rawshan; McGovern, Amanda; Ridley, Caroline; Cain, Stuart A.; Baldwin, Andrew; Wang, Ming-Chuan; Guo, Chun; Mironov, Aleksandr; Drymoussi, Zoe; Trump, Dorothy; Shuttleworth, Adrian; Baldock, Clair; Kielty, Cay M.

    2009-01-01

    Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils. PMID:19570982

  14. SAM pointed domain ETS factor (SPDEF) regulates terminal differentiation and maturation of intestinal goblet cells

    Energy Technology Data Exchange (ETDEWEB)

    Noah, Taeko K.; Kazanjian, Avedis [Gastroenterology, Hepatology and Nutrition, Cincinnati Children' s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH (United States); Whitsett, Jeffrey [Developmental Biology, Cincinnati Children' s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH (United States); Neonatology and Pulmonary Biology, Cincinnati Children' s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH (United States); Shroyer, Noah F., E-mail: noah.shroyer@cchmc.org [Gastroenterology, Hepatology and Nutrition, Cincinnati Children' s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH (United States); Developmental Biology, Cincinnati Children' s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH (United States)

    2010-02-01

    Background and Aims: SPDEF (also termed PDEF or PSE) is an ETS family transcription factor that regulates gene expression in the prostate and goblet cell hyperplasia in the lung. Spdef has been reported to be expressed in the intestine. In this paper, we identify an important role for Spdef in regulating intestinal epithelial cell homeostasis and differentiation. Methods: SPDEF expression was inhibited in colon cancer cells to determine its ability to control goblet cell gene activation. The effects of transgenic expression of Spdef on intestinal differentiation and homeostasis were determined. Results: In LS174T colon cancer cells treated with Notch/{gamma}-secretase inhibitor to activate goblet cell gene expression, shRNAs that inhibited SPDEF also repressed expression of goblet cell genes AGR2, MUC2, RETLNB, and SPINK4. Transgenic expression of Spdef caused the expansion of intestinal goblet cells and corresponding reduction in Paneth, enteroendocrine, and absorptive enterocytes. Spdef inhibited proliferation of intestinal crypt cells without induction of apoptosis. Prolonged expression of the Spdef transgene caused a progressive reduction in the number of crypts that expressed Spdef, consistent with its inhibitory effects on cell proliferation. Conclusions: Spdef was sufficient to inhibit proliferation of intestinal progenitors and induce differentiation into goblet cells; SPDEF was required for activation of goblet cell associated genes in vitro. These data support a model in which Spdef promotes terminal differentiation into goblet cells of a common goblet/Paneth progenitor.

  15. TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell Culture Model of the Corneal Epithelium.

    Science.gov (United States)

    Martínez-Rendón, Jacqueline; Sánchez-Guzmán, Erika; Rueda, Angélica; González, James; Gulias-Cañizo, Rosario; Aquino-Jarquín, Guillermo; Castro-Muñozledo, Federico; García-Villegas, Refugio

    2017-07-01

    TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca 2+ imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. J. Cell. Physiol. 232: 1794-1807, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.

    Science.gov (United States)

    Saito, Emi; Suzuki, Dai; Kurotaki, Daisuke; Mochizuki, Ayako; Manome, Yoko; Suzawa, Tetsuo; Toyoshima, Yoichi; Ichikawa, Takahiro; Funatsu, Takahiro; Inoue, Tomio; Takami, Masamichi; Tamura, Tomohiko; Inagaki, Katsunori; Kamijo, Ryutaro

    2017-06-01

    Interferon regulatory factor 8 (Irf8) is a transcription factor that negatively regulates osteoclast differentiation and Irf8 global knockout (Irf8 -/- ) mice have been shown to have reduced bone volume resulting from increased osteoclast numbers. However, detailed analysis of the functions of Irf8 in osteoclast precursors with a monocyte/macrophage linage is difficult, because the population and properties of hematopoietic cells in Irf8 -/- mice are severely altered. Therefore, to clearly elucidate the functions of Irf8 during osteoclastogenesis, we established myeloid cell-specific Irf8 conditional knockout (Irf8 fl/fl ;Lyz2 cre/+ ) mice. We found that trabecular bone volume in the Irf8 fl/fl ;Lyz2 cre/+ mice was not significantly affected, while exposure to M-CSF and RANKL significantly increased TRAP activity in vitro in osteoclasts that underwent osteoclastogenesis from bone marrow-derived macrophages (BMMs) induced from bone marrow cells (BMCs) of those mice by addition of M-CSF. Our results also showed that expression of Irf8 mRNA and protein in BMMs obtained from Irf8 fl/fl ;Lyz2 cre/+ mice and cultured with M-CSF was reduced. These findings predicted that Lyz2/Lyz2-cre expression is induced when BMCs differentiate into BMMs in cultures with M-CSF. In osteoclast differentiation cultures, Lyz2 was gradually increased by M-CSF during the first 3 days of culture, then rapidly decreased by the addition of RANKL with M-CSF during the next 3 days. Furthermore, BMCs differentiated into osteoclasts while maintaining a low level of Lyz2 expression when cultured simultaneously with both M-CSF and RANKL from the initiation of culture. These findings suggest that Lyz2-cre expression is induced along with differentiation to BMMs by BMCs obtained from Irf8 fl/fl ;Lyz2 cre/+ mice and cultured with M-CSF. In addition, Irf8 was down-regulated by activation of the cre/loxP recombination system in BMMs and osteoclastogenesis was accelerated. Based on our results, we propose

  17. Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche

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    LM McNamara

    2012-01-01

    Full Text Available Mesenchymal stem cells (MSCs within their native environment of the stem cell niche in bone receive biochemical stimuli from surrounding cells. These stimuli likely influence how MSCs differentiate to become bone precursors. The ability of MSCs to undergo osteogenic differentiation is well established in vitro;however, the role of the natural cues from bone’s regulatory cells, osteocytes and osteoblasts in regulating the osteogenic differentiation of MSCs in vivo are unclear. In this study we delineate the role of biochemical signalling from osteocytes and osteoblasts, using conditioned media and co-culture experiments, to understand how they direct osteogenic differentiation of MSCs. Furthermore, the synergistic relationship between osteocytes and osteoblasts is examined by transwell co-culturing of MSCs with both simultaneously. Osteogenic differentiation of MSCs was quantified by monitoring alkaline phosphatase (ALP activity, calcium deposition and cell number. Intracellular ALP was found to peak earlier and there was greater calcium deposition when MSCs were co-cultured with osteocytes rather than osteoblasts, suggesting that osteocytes are more influential than osteoblasts in stimulating osteogenesis in MSCs. Osteoblasts initially stimulated an increase in the number of MSCs, but ultimately regulated MSC differentiation down the same pathway. Our novel co-culture system confirmed a synergistic relationship between osteocytes and osteoblasts in producing biochemical signals to stimulate the osteogenic differentiation of MSCs. This study provides important insights into the mechanisms at work within the native stem cell niche to stimulate osteogenic differentiation and outlines a possible role for the use of co-culture or conditioned media methodologies for tissue engineering applications.

  18. Down-regulation of E protein activity augments an ILC2 differentiation program in the thymus

    Science.gov (United States)

    Innate lymphoid cells (ILCs) are important regulators in various immune responses. Current paradigm states that all newly-made ILCs originate from common lymphoid progenitors (CLP) in the bone marrow. Id2, an inhibitor of E protein transcription factors, is indispensable for ILC differentiation. Une...

  19. Differential regulation of EGFR-MAPK signaling by deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) in colon cancer.

    Science.gov (United States)

    Centuori, Sara M; Martinez, Jesse D

    2014-10-01

    A high-fat diet coincides with increased levels of bile acids. This increase in bile acids, particularly deoxycholic acid (DCA), has been strongly associated with the development of colon cancer. Conversely, ursodeoxycholic acid (UDCA) may have chemopreventive properties. Although structurally similar, DCA and UDCA present different biological and pathological effects in colon cancer progression. The differential regulation of cancer by these two bile acids is not yet fully understood. However, one possible explanation for their diverging effects is their ability to differentially regulate signaling pathways involved in the multistep progression of colon cancer, such as the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway. This review will examine the biological effects of DCA and UDCA on colon cancer development, as well as the diverging effects of these bile acids on the oncogenic signaling pathways that play a role in colon cancer development, with a particular emphasis on bile acid regulation of the EGFR-MAPK pathway.

  20. Ethanol negatively regulates hepatic differentiation of hESC by inhibition of the MAPK/ERK signaling pathway in vitro.

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    Wei Gao

    Full Text Available Alcohol insult triggers complex events in the liver, promoting fibrogenic/inflammatory signals and in more advanced cases, aberrant matrix deposition. It is well accepted that the regenerative capacity of the adult liver is impaired during alcohol injury. The liver progenitor/stem cells have been shown to play an important role in liver regeneration -in response to various chronic injuries; however, the effects of alcohol on stem cell differentiation in the liver are not well understood.We employed hepatic progenitor cells derived from hESCs to study the impact of ethanol on hepatocyte differentiation by exposure of these progenitor cells to ethanol during hepatocyte differentiation.We found that ethanol negatively regulated hepatic differentiation of hESC-derived hepatic progenitor cells in a dose-dependent manner. There was also a moderate cell cycle arrest at G1/S checkpoint in the ethanol treated cells, which is associated with a reduced level of cyclin D1 in these cells. Ethanol treatment specifically inhibited the activation of the ERK but not JNK nor the p38 MAP signaling pathway. At the same time, the WNT signaling pathway was also reduced in the cells exposed to ethanol. Upon evaluating the effects of the inhibitors of these two signaling pathways, we determined that the Erk inhibitor replicated the effects of ethanol on the hepatocyte differentiation and attenuated the WNT/β-catenin signaling, however, inhibitors of WNT only partially replicated the effects of ethanol on the hepatocyte differentiation.Our results demonstrated that ethanol negatively regulated hepatic differentiation of hESC-derived hepatic progenitors through inhibiting the MAPK/ERK signaling pathway, and subsequently attenuating the WNT signaling pathway. Thus, our finding provides a novel insight into the mechanism by which alcohol regulates cell fate selection of hESC-derived hepatic progenitor cells, and the identified pathways may provide therapeutic targets

  1. Post-Transcriptional Regulation of KLF4 by High-Risk Human Papillomaviruses Is Necessary for the Differentiation-Dependent Viral Life Cycle.

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    Vignesh Kumar Gunasekharan

    2016-07-01

    Full Text Available Human papillomaviruses (HPVs are epithelial tropic viruses that link their productive life cycles to the differentiation of infected host keratinocytes. A subset of the over 200 HPV types, referred to as high-risk, are the causative agents of most anogenital malignancies. HPVs infect cells in the basal layer, but restrict viral genome amplification, late gene expression, and capsid assembly to highly differentiated cells that are active in the cell cycle. In this study, we demonstrate that HPV proteins regulate the expression and activities of a critical cellular transcription factor, KLF4, through post-transcriptional and post-translational mechanisms. Our studies show that KLF4 regulates differentiation as well as cell cycle progression, and binds to sequences in the upstream regulatory region (URR to regulate viral transcription in cooperation with Blimp1. KLF4 levels are increased in HPV-positive cells through a post-transcriptional mechanism involving E7-mediated suppression of cellular miR-145, as well as at the post-translational level by E6-directed inhibition of its sumoylation and phosphorylation. The alterations in KLF4 levels and functions results in activation and suppression of a subset of KLF4 target genes, including TCHHL1, VIM, ACTN1, and POT1, that is distinct from that seen in normal keratinocytes. Knockdown of KLF4 with shRNAs in cells that maintain HPV episomes blocked genome amplification and abolished late gene expression upon differentiation. While KLF4 is indispensable for the proliferation and differentiation of normal keratinocytes, it is necessary only for differentiation-associated functions of HPV-positive keratinocytes. Increases in KLF4 levels alone do not appear to be sufficient to explain the effects on proliferation and differentiation of HPV-positive cells indicating that additional modifications are important. KLF4 has also been shown to be a critical regulator of lytic Epstein Barr virus (EBV replication

  2. Hyperthyroidism differentially regulates neuropeptide S system in the rat brain.

    Science.gov (United States)

    González, Carmen R; Martínez de Morentin, Pablo B; Martínez-Sánchez, Noelia; Gómez-Díaz, Consuelo; Lage, Ricardo; Varela, Luis; Diéguez, Carlos; Nogueiras, Rubén; Castaño, Justo P; López, Miguel

    2012-04-23

    Thyroid hormones play an important role in the regulation of energy balance, sleep and emotional behaviors. Neuropeptide S (NPS) is a recently discovered neuropeptide, regulating feeding, sleep and anxiety. Here, we examined the effect of hyperthyroidism on the gene and protein expression of neuropeptide S and its receptor (NPS-R) in the hypothalamus, brainstem and amygdala of rats. Our results showed that the expression of NPS and NPS-R was differentially modulated by hyperthyroidism in the rat brain. NPS and NPS-R mRNA and protein levels were decreased in the hypothalamus of hyperthyroid rats. Conversely NPS-R expression was highly increased in the brainstem and NPS and NPS-R expression were unchanged in the amygdala of these rats. These data suggest that changes in anxiety and food intake patterns observed in hyperthyroidism could be associated with changes in the expression of NPS and NPS-R. Thus, the NPS/NPS-R system may be involved in several hyperthyroidism-associated comorbidities. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Leptin differentially regulates chondrogenesis in mouse vertebral and tibial growth plates.

    Science.gov (United States)

    Yu, Bo; Jiang, Kaibiao; Chen, Bin; Wang, Hantao; Li, Xinfeng; Liu, Zude

    2017-05-31

    Leptin plays an important role in mediating chondrogenesis of limb growth plate. Previous studies suggest that bone structures and development of spine and limb are different. The expression of Ob-Rb, the gene that encodes leptin receptors, is vertebral and appendicular region-specific, suggesting the regulation of leptin on VGP and TGP chondrogenesis may be very different. The aim of the present study was to investigate the differential regulation of leptin on the chondrogenesis of vertebral growth plate (VGP) and tibial growth plate (TGP). We compared the VGP and TGP from wild type (C57BL/6) and leptin-deficient (ob/ob) mice. We then generated primary cultures of TGP and VGP chondrocytes. By treating the primary cells with different concentrations of leptin in vitro, we analyzed proliferation and apoptosis of the primary chondrocytes from TGP and VGP. We further measured expression of chondrogenic-related genes in these cells that had been incubated with different doses of leptin. Leptin-deficient mice of 8-week-old had shorter tibial and longer vertebral lengths than the wide type mice. Disturbed columnar structure was observed for TGP but not for VGP. In primary chondrocyte cultures, leptin inhibited VGP chondrocyte proliferation but promoted their apoptosis. Collagen IIA and aggrecan mRNA, and the protein levels of proliferation- and chondrogenesis-related markers, including PCNA, Sox9, and Smad4, were downregulated by leptin in a dose-dependent manner. In contrast, leptin stimulated the proliferation and chondrogenic differentiation of TGP chondrocytes at physiological levels (i.e., 10 and 50 ng/mL) but not at high levels (i.e., 100 and 1000 ng/mL). Leptin exerts a stimulatory effect on the proliferation and chondrogenic differentiation of the long bone growth plate but an inhibitory effect on the spine growth plate. The ongoing study will shed light on the regulatory mechanisms of leptin in bone development and metabolism.

  4. The gga-let-7 family post-transcriptionally regulates TGFBR1 and LIN28B during the differentiation process in early chick development.

    Science.gov (United States)

    Lee, Sang In; Jeon, Mi-Hyang; Kim, Jeom Sun; Jeon, Ik-Soo; Byun, Sung June

    2015-12-01

    Early chick embryogenesis is governed by a complex mechanism involving transcriptional and post-transcriptional regulation, although how post-transcriptional processes influence the balance between pluripotency and differentiation during early chick development have not been previously investigated. Here, we characterized the microRNA (miRNA) signature associated with differentiation in the chick embryo, and found that as expression of the gga-let-7 family increases through early development, expression of their direct targets, TGFBR1 and LIN28B, decreases; indeed, gga-let-7a-5p and gga-let-7b miRNAs directly bind to TGFBR1 and LIN28B transcripts. Our data further indicate that TGFBR1 and LIN28B maintain pluripotency by regulating POUV, NANOG, and CRIPTO. Therefore, gga-let-7 miRNAs act as post-transcriptional regulators of differentiation in blastodermal cells by repressing the expression of the TGFBR1 and LIN28B, which intrinsically controls blastodermal cell differentiation in early chick development. © 2015 Wiley Periodicals, Inc.

  5. YB1/p32, a nuclear Y-box binding protein 1, is a novel regulator of myoblast differentiation that interacts with Msx1 homeoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Song, Young Joon [Department of Biological Sciences, College of Natural Science, Inha University, 253 Yonghyun-dong, Nam-Gu, Incheon, Korea, 402-751 (Korea, Republic of); Lee, Hansol, E-mail: hlee@inha.ac.kr [Department of Biological Sciences, College of Natural Science, Inha University, 253 Yonghyun-dong, Nam-Gu, Incheon, Korea, 402-751 (Korea, Republic of)

    2010-02-15

    Precisely controlled cellular differentiation is essential for the proper development of vertebrate embryo and deregulated differentiation is a major cause of many human congenital diseases as well as cancer. Msx1 is a member of the homeoprotein family implicated in these processes, which inhibits the differentiation of skeletal muscle and other cell types, presumably by regulating transcription of target genes through interaction with other cellular factors. We presently show that YB1/p32, a nuclear Y-box binding protein 1, interacts with Msx1 homeoprotein and functions as a regulator of C2C12 myoblast differentiation. We demonstrate that YB1/p32 functionally interacts with Msx1 through its N-terminal region and colocalizes with Msx1 at the nuclear periphery. Moreover, we find that YB1/p32 is competent for inhibition of C2C12 myoblast differentiation, which is correlated with its activity as a negative regulator of MyoD gene expression and binding to the MyoD core enhancer region (CER). Furthermore, YB1/p32 cooperates with Msx1 in transcriptional repression and knocking down the expression of endogenous YB1 attenuates the effects of Msx1. Taken together, our study has uncovered a new function of YB1/p32, a regulator of skeletal muscle differentiation.

  6. YB1/p32, a nuclear Y-box binding protein 1, is a novel regulator of myoblast differentiation that interacts with Msx1 homeoprotein

    International Nuclear Information System (INIS)

    Song, Young Joon; Lee, Hansol

    2010-01-01

    Precisely controlled cellular differentiation is essential for the proper development of vertebrate embryo and deregulated differentiation is a major cause of many human congenital diseases as well as cancer. Msx1 is a member of the homeoprotein family implicated in these processes, which inhibits the differentiation of skeletal muscle and other cell types, presumably by regulating transcription of target genes through interaction with other cellular factors. We presently show that YB1/p32, a nuclear Y-box binding protein 1, interacts with Msx1 homeoprotein and functions as a regulator of C2C12 myoblast differentiation. We demonstrate that YB1/p32 functionally interacts with Msx1 through its N-terminal region and colocalizes with Msx1 at the nuclear periphery. Moreover, we find that YB1/p32 is competent for inhibition of C2C12 myoblast differentiation, which is correlated with its activity as a negative regulator of MyoD gene expression and binding to the MyoD core enhancer region (CER). Furthermore, YB1/p32 cooperates with Msx1 in transcriptional repression and knocking down the expression of endogenous YB1 attenuates the effects of Msx1. Taken together, our study has uncovered a new function of YB1/p32, a regulator of skeletal muscle differentiation.

  7. Cholesterol and phytosterols differentially regulate the expression of caveolin 1 and a downstream prostate cell growth-suppressor gene

    Science.gov (United States)

    Ifere, Godwin O.; Equan, Anita; Gordon, Kereen; Nagappan, Peri; Igietseme, Joseph U.; Ananaba, Godwin A.

    2010-01-01

    Background The purpose of our study was to show the distinction between the apoptotic and anti-proliferative signaling of phytosterols and cholesterol enrichment in prostate cancer cell lines, mediated by the differential transcription of caveolin-1, and N-myc downstream regulated gene1 (NDRG1), a pro-apoptotic androgen-regulated tumor suppressor. Methods PC-3 and DU145 cells were treated with sterols (cholesterol and phytosterols) for 72 h, followed by trypan blue dye exclusion measurement of necrosis and cell growth measured with a Coulter counter. Sterol induction of cell growth-suppressor gene expression was evaluated by mRNA transcription using RT-PCR, while cell cycle analysis was performed by FACS analysis. Altered expression of Ndrg1 protein was confirmed by Western blot analysis. Apoptosis was evaluated by real time RT-PCR amplification of P53, Bcl-2 gene and its related pro- and anti-apoptotic family members. Results Physiological doses (16 µM) of cholesterol and phytosterols were not cytotoxic in these cells. Cholesterol enrichment promoted cell growth (Pphytosterols significantly induced growth-suppression (Pphytosterols decreased mitotic subpopulations. We demonstrated for the first time that cholesterols concertedly attenuated the expression of caveolin-1(cav-1) and NDRG1 genes in both prostate cancer cell lines. Phytosterols had the opposite effect by inducing overexpression of cav-1, a known mediator of androgen-dependent signals that presumably control cell growth or apoptosis. Conclusions Cholesterol and phytosterol treatment differentially regulated the growth of prostate cancer cells and the expression of p53 and cav-1, a gene that regulates androgen-regulated signals. These sterols also differentially regulated cell cycle arrest, downstream pro-apoptotic androgen-regulated tumor-suppressor, NDRG1 suggesting that cav-1 may mediate pro-apoptotic NDRG1 signals. Elucidation of the mechanism for sterol modulation of growth and apoptosis signaling

  8. c-Myb Regulates the T-Bet-Dependent Differentiation Program in B Cells to Coordinate Antibody Responses

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    Dana Piovesan

    2017-04-01

    Full Text Available Summary: Humoral immune responses are tailored to the invading pathogen through regulation of key transcription factors and their networks. This is critical to establishing effective antibody-mediated responses, yet it is unknown how B cells integrate pathogen-induced signals to drive or suppress transcriptional programs specialized for each class of pathogen. Here, we detail the key role of the transcription factor c-Myb in regulating the T-bet-mediated anti-viral program. Deletion of c-Myb in mature B cells significantly increased serum IgG2c and CXCR3 expression by upregulating T-bet, normally suppressed during Th2-cell-mediated responses. Enhanced expression of T-bet resulted in aberrant plasma cell differentiation within the germinal center, mediated by CXCR3 expression. These findings identify a dual role for c-Myb in limiting inappropriate effector responses while coordinating plasma cell differentiation with germinal center egress. Identifying such intrinsic regulators of specialized antibody responses can assist in vaccine design and therapeutic intervention in B-cell-mediated immune disorders. : Piovesan et al. examine how B cells establish transcriptional programs that result in tailored responses to invading pathogens. The authors find that the transcription factor c-Myb represses the T-bet-mediated anti-viral program in B cells. c-Myb limits inappropriate effector responses while coordinating plasma cell differentiation with germinal center egress. Keywords: B cells, c-Myb, T-bet, immunoglobulin, CXCR3, plasma cell, germinal center

  9. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

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    Chen-Shuang Li

    2016-01-01

    Full Text Available Human umbilical cord mesenchymal stem cells (hUCMSCs are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering.

  10. Transcription factor KLF7 regulates differentiation of neuroectodermal and mesodermal cell lineages

    International Nuclear Information System (INIS)

    Caiazzo, Massimiliano; Colucci-D'Amato, Luca; Esposito, Maria T.; Parisi, Silvia; Stifani, Stefano; Ramirez, Francesco; Porzio, Umberto di

    2010-01-01

    Previous gene targeting studies in mice have implicated the nuclear protein Krueppel-like factor 7 (KLF7) in nervous system development while cell culture assays have documented its involvement in cell cycle regulation. By employing short hairpin RNA (shRNA)-mediated gene silencing, here we demonstrate that murine Klf7 gene expression is required for in vitro differentiation of neuroectodermal and mesodermal cells. Specifically, we show a correlation of Klf7 silencing with down-regulation of the neuronal marker microtubule-associated protein 2 (Map2) and the nerve growth factor (NGF) tyrosine kinase receptor A (TrkA) using the PC12 neuronal cell line. Similarly, KLF7 inactivation in Klf7-null mice decreases the expression of the neurogenic marker brain lipid-binding protein/fatty acid-binding protein 7 (BLBP/FABP7) in neural stem cells (NSCs). We also report that Klf7 silencing is detrimental to neuronal and cardiomyocytic differentiation of embryonic stem cells (ESCs), in addition to altering the adipogenic and osteogenic potential of mouse embryonic fibroblasts (MEFs). Finally, our results suggest that genes that are key for self-renewal of undifferentiated ESCs repress Klf7 expression in ESCs. Together with previous findings, these results provide evidence that KLF7 has a broad spectrum of regulatory functions, which reflect the discrete cellular and molecular contexts in which this transcription factor operates.

  11. Transcription factor KLF7 regulates differentiation of neuroectodermal and mesodermal cell lineages

    Energy Technology Data Exchange (ETDEWEB)

    Caiazzo, Massimiliano, E-mail: caiazzo@igb.cnr.it [Institute of Genetics and Biophysics ' A. Buzzati-Traverso,' CNR, 80131 Naples (Italy); Istituto di diagnosi e cura ' Hermitage Capodimonte,' 80131 Naples (Italy); Colucci-D' Amato, Luca, E-mail: luca.colucci@unina2.it [Institute of Genetics and Biophysics ' A. Buzzati-Traverso,' CNR, 80131 Naples (Italy); Dipartimento di Scienze della Vita, Seconda Universita di Napoli, 81100 Caserta (Italy); Esposito, Maria T., E-mail: maria_teresa.esposito@kcl.ac.uk [CEINGE Biotecnologie Avanzate, 80145 Naples (Italy); Parisi, Silvia, E-mail: parisi@ceinge.unina.it [CEINGE Biotecnologie Avanzate, 80145 Naples (Italy); Stifani, Stefano, E-mail: stefano.stifani@mcgill.ca [Centre for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4 (Canada); Ramirez, Francesco, E-mail: francesco.ramirez@mssm.edu [Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029 (United States); Porzio, Umberto di, E-mail: diporzio@igb.cnr.it [Institute of Genetics and Biophysics ' A. Buzzati-Traverso,' CNR, 80131 Naples (Italy)

    2010-08-15

    Previous gene targeting studies in mice have implicated the nuclear protein Krueppel-like factor 7 (KLF7) in nervous system development while cell culture assays have documented its involvement in cell cycle regulation. By employing short hairpin RNA (shRNA)-mediated gene silencing, here we demonstrate that murine Klf7 gene expression is required for in vitro differentiation of neuroectodermal and mesodermal cells. Specifically, we show a correlation of Klf7 silencing with down-regulation of the neuronal marker microtubule-associated protein 2 (Map2) and the nerve growth factor (NGF) tyrosine kinase receptor A (TrkA) using the PC12 neuronal cell line. Similarly, KLF7 inactivation in Klf7-null mice decreases the expression of the neurogenic marker brain lipid-binding protein/fatty acid-binding protein 7 (BLBP/FABP7) in neural stem cells (NSCs). We also report that Klf7 silencing is detrimental to neuronal and cardiomyocytic differentiation of embryonic stem cells (ESCs), in addition to altering the adipogenic and osteogenic potential of mouse embryonic fibroblasts (MEFs). Finally, our results suggest that genes that are key for self-renewal of undifferentiated ESCs repress Klf7 expression in ESCs. Together with previous findings, these results provide evidence that KLF7 has a broad spectrum of regulatory functions, which reflect the discrete cellular and molecular contexts in which this transcription factor operates.

  12. IL-13 regulates human nasal epithelial cell differentiation via H3K4me3 modification

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    Yu L

    2018-01-01

    Full Text Available Lei Yu,1 Na Li,1 Jisheng Zhang,2 Yan Jiang1 1Department of Otorhinolaryngology, 2Key Laboratory of Otolaryngology-Head and Neck Surgery, Affiliated Hospital of Qingdao University, Qingdao, China Introduction: Epigenetic regulation has been shown to play an important role in the development of inflammatory diseases, including chronic rhinosinusitis and nasal polyps. The latter are characterized by epithelial mis-differentiation and infiltration of inflammatory cytokines. H3K4me3 has been shown to be involved in regulating lineage commitment. However, the underlying mechanisms, especially in human nasal epithelial cells (HNEpC, remain underexplored. The objective of this study was to investigate the role of H3K4me3 in HNEpC differentiation treated with the Th2 cytokine IL-13. Patients and methods: The expression levels of mRNA and proteins were investigated using reverse transcription-polymerase chain reaction (RT-PCR assays and Western blot in nasal polyp tissues and human nasal epithelial cells respectively. We measured these levels of H3K4me3, MLL1 and targeted genes compared with control subjects.Results: We demonstrate that expression of H3K4me3 and its methyltransferase MLL1 was significantly upregulated in IL-13-treated HNEpC. This elevation was also observed in nasal polyps. Expression of cilia-related transcription factors FOXJ1 and DNAI2 decreased, while goblet cell-derived genes CLCA1 and MUC5a increased upon IL-13 treatment. Mechanistically, knockdown of MLL1 restored expression of these four genes induced by IL-13. Conclusion: These findings suggest that H3K4me3 is a critical regulator in control of nasal epithelial cell differentiation. MLL1 may be a potential therapeutic target for nasal inflammatory diseases. Keywords: IL-13, H3K4me3 modification, nasal epithelial cell, differentiation 

  13. SEPTIN2 and STATHMIN Regulate CD99-Mediated Cellular Differentiation in Hodgkin's Lymphoma.

    Directory of Open Access Journals (Sweden)

    Wenjing Jian

    Full Text Available Hodgkin's lymphoma (HL is a lymphoid neoplasm characterized by Hodgkin's and Reed-Sternberg (H/RS cells, which is regulated by CD99. We previously reported that CD99 downregulation led to the transformation of murine B lymphoma cells (A20 into cells with an H/RS phenotype, while CD99 upregulation induced differentiation of classical Hodgkin's lymphoma (cHL cells (L428 into terminal B-cells. However, the molecular mechanism remains unclear. In this study, using fluorescence two-dimensional differential in-gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS, we have analyzed the alteration of protein expression following CD99 upregulation in L428 cells as well as downregulation of mouse CD99 antigen-like 2 (mCD99L2 in A20 cells. Bioinformatics analysis showed that SEPTIN2 and STATHMIN, which are cytoskeleton proteins, were significantly differentially expressed, and chosen for further validation and functional analysis. Differential expression of SEPTIN2 was found in both models and was inversely correlated with CD99 expression. STATHMIN was identified in the A20 cell line model and its expression was positively correlated with that of CD99. Importantly, silencing of SEPTIN2 with siRNA substantially altered the cellular cytoskeleton in L428 cells. The downregulation of STATHMIN by siRNA promoted the differentiation of H/RS cells toward terminal B-cells. These results suggest that SEPTIN2-mediated cytoskeletal rearrangement and STATHMIN-mediated differentiation may contribute to changes in cell morphology and differentiation of H/RS cells with CD99 upregulation in HL.

  14. Peroxisome Proliferator-Activated Receptor Gamma Negatively Regulates the Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells Toward Myofibroblasts in Liver Fibrogenesis

    Directory of Open Access Journals (Sweden)

    Shuangshuang Jia

    2015-11-01

    Full Text Available Background/Aims: Bone marrow-derived mesenchymal stem cells (BMSCs have been confirmed to have capacity to differentiate toward hepatic myofibroblasts, which contribute to fibrogenesis in chronic liver diseases. Peroxisome proliferator-activated receptor gamma (PPARγ, a ligand-activated transcription factor, has gained a great deal of recent attention as it is involved in fibrosis and cell differentiation. However, whether it regulates the differentiation of BMSCs toward myofibroblasts remains to be defined. Methods: Carbon tetrachloride or bile duct ligation was used to induce mouse liver fibrosis. Expressions of PPARγ, α-smooth muscle actin, collagen α1 (I and collagen α1 (III were detected by real-time RT-PCR and Western blot or immunofluorescence assay. Results: PPARγ expression was decreased in mouse fibrotic liver. In addition, PPARγ was declined during the differentiation of BMSCs toward myofibroblasts induced by transforming growth factor β1. Activation of PPARγ stimulated by natural or synthetic ligands suppressed the differentiation of BMSCs. Additionally, knock down of PPARγ by siRNA contributed to BMSC differentiation toward myofibroblasts. Furthermore, PPARγ activation by natural ligand significantly inhibited the differentiation of BMSCs toward myofibroblasts in liver fibrogenesis and alleviated liver fibrosis. Conclusions: PPARγ negatively regulates the differentiation of BMSCs toward myofibroblasts, which highlights a further mechanism implicated in the BMSC differentiation.

  15. MLL5, a trithorax homolog, indirectly regulates H3K4 methylation, represses cyclin A2 expression, and promotes myogenic differentiation

    Science.gov (United States)

    Sebastian, Soji; Sreenivas, Prethish; Sambasivan, Ramkumar; Cheedipudi, Sirisha; Kandalla, Prashanth; Pavlath, Grace K.; Dhawan, Jyotsna

    2009-01-01

    Most cells in adult tissues are nondividing. In skeletal muscle, differentiated myofibers have exited the cell cycle permanently, whereas satellite stem cells withdraw transiently, returning to active proliferation to repair damaged myofibers. We have examined the epigenetic mechanisms operating in conditional quiescence by analyzing the function of a predicted chromatin regulator mixed lineage leukemia 5 (MLL5) in a culture model of reversible arrest. MLL5 is induced in quiescent myoblasts and regulates both the cell cycle and differentiation via a hierarchy of chromatin and transcriptional regulators. Knocking down MLL5 delays entry of quiescent myoblasts into S phase, but hastens S-phase completion. Cyclin A2 (CycA) mRNA is no longer restricted to S phase, but is induced throughout G0/G1, with activation of the cell cycle regulated element (CCRE) in the CycA promoter. Overexpressed MLL5 physically associates with the CCRE and impairs its activity. MLL5 also regulates CycA indirectly: Cux, an activator of CycA promoter and S phase is induced in RNAi cells, and Brm/Brg1, CCRE-binding repressors that promote differentiation are repressed. In knockdown cells, H3K4 methylation at the CCRE is reduced, reflecting quantitative global changes in methylation. MLL5 appears to lack intrinsic histone methyl transferase activity, but regulates expression of histone-modifying enzymes LSD1 and SET7/9, suggesting an indirect mechanism. Finally, expression of muscle regulators Pax7, Myf5, and myogenin is impaired in MLL5 knockdown cells, which are profoundly differentiation defective. Collectively, our results suggest that MLL5 plays an integral role in novel chromatin regulatory mechanisms that suppress inappropriate expression of S-phase-promoting genes and maintain expression of determination genes in quiescent cells. PMID:19264965

  16. Chondrocytic Atf4 regulates osteoblast differentiation and function via Ihh.

    Science.gov (United States)

    Wang, Weiguang; Lian, Na; Ma, Yun; Li, Lingzhen; Gallant, Richard C; Elefteriou, Florent; Yang, Xiangli

    2012-02-01

    Atf4 is a leucine zipper-containing transcription factor that activates osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation of skeletal development and bone formation is poorly understood. Investigations of the Atf4(-/-);Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteogenesis during development and bone remodeling postnatally. Atf4 overexpression in chondrocytes of the Atf4(-/-);Col2a1-Atf4 double mutants corrects the reduction in stature and limb in Atf4(-/-) embryos and rectifies the decrease in Ihh expression, Hh signaling, proliferation and accelerated hypertrophy that characterize the Atf4(-/-) developing growth plate cartilages. Unexpectedly, this genetic manipulation also restores the expression of osteoblastic marker genes, namely Ocn and bone sialoprotein, in Atf4(-/-) developing bones. In Atf4(-/-);Col2a1-Atf4 adult mice, all the defective bone parameters found in Atf4(-/-) mice, including bone volume, trabecular number and thickness, and bone formation rate, are rescued. In addition, the conditioned media of ex vivo cultures from wild-type or Atf4(-/-);Col2a1-Atf4, but not Atf4(-/-) cartilage, corrects the differentiation defects of Atf4(-/-) bone marrow stromal cells and Ihh-blocking antibody eliminates this effect. Together, these data indicate that Atf4 in chondrocytes is required for normal Ihh expression and for its paracrine effect on osteoblast differentiation. Therefore, the cell-autonomous role of Atf4 in chondrocytes dominates the role of Atf4 in osteoblasts during development for the control of early osteogenesis and skeletal growth.

  17. Regulation of proliferation and differentiation of adipocyte precursor cells in rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Bouraoui, L; Gutiérrez, J; Navarro, I

    2008-09-01

    Here, we describe optimal conditions for the culture of rainbow trout (Oncorhynchus mykiss) pre-adipocytes obtained from adipose tissue and their differentiation into mature adipocytes, in order to study the endocrine control of adipogenesis. Pre-adipocytes were isolated by collagenase digestion and cultured on laminin or 1% gelatin substrate. The expression of proliferating cell nuclear antigen was used as a marker of cell proliferation on various days of culture. Insulin growth factor-I stimulated cell proliferation especially on days 5 and 7 of culture. Tumor necrosis factor alpha (TNFalpha) slightly enhanced cell proliferation only at a low dose. We verified the differentiation of cells grown in specific medium into mature adipocytes by oil red O (ORO) staining. Quantification of ORO showed an increase in triglycerides throughout culture. Immunofluorescence staining of cells at day 11 revealed the expression of CCAAT/enhancer-binding protein and peroxisome proliferator-activator receptor gamma, suggesting that these transcriptional factors are involved in adipocyte differentiation in trout. We also examined the effect of TNFalpha on the differentiation of these adipocytes in primary culture. TNFalpha inhibited the differentiation of these cells, as indicated by a decrease in glycerol-3-phosphate dehydrogenase activity, an established marker of adipocyte differentiation. In conclusion, the culture system described here for trout pre-adipocytes is a powerful tool to study the endocrine regulation of adipogenesis in this species.

  18. PPARγ and MyoD are differentially regulated by myostatin in adipose-derived stem cells and muscle satellite cells

    International Nuclear Information System (INIS)

    Zhang, Feng; Deng, Bing; Wen, Jianghui; Chen, Kun; Liu, Wu; Ye, Shengqiang; Huang, Haijun; Jiang, Siwen; Xiong, Yuanzhu

    2015-01-01

    Myostatin (MSTN) is a secreted protein belonging to the transforming growth factor-β (TGF-β) family that is primarily expressed in skeletal muscle and also functions in adipocyte maturation. Studies have shown that MSTN can inhibit adipogenesis in muscle satellite cells (MSCs) but not in adipose-derived stem cells (ADSCs). However, the mechanism by which MSTN differently regulates adipogenesis in these two cell types remains unknown. Peroxisome proliferator-activated receptor-γ (PPARγ) and myogenic differentiation factor (MyoD) are two key transcription factors in fat and muscle cell development that influence adipogenesis. To investigate whether MSTN differentially regulates PPARγ and MyoD, we analyzed PPARγ and MyoD expression by assessing mRNA, protein and methylation levels in ADSCs and MSCs after treatment with 100 ng/mL MSTN for 0, 24, and 48 h. PPARγ mRNA levels were downregulated after 24 h and upregulated after 48 h of treatment in ADSCs, whereas in MSCs, PPARγ levels were downregulated at both time points. MyoD expression was significantly increased in ADSCs and decreased in MSCs. PPARγ and MyoD protein levels were upregulated in ADSCs and downregulated in MSCs. The CpG methylation levels of the PPARγ and MyoD promoters were decreased in ADSCs and increased in MSCs. Therefore, this study demonstrated that the different regulatory adipogenic roles of MSTN in ADSCs and MSCs act by differentially regulating PPARγ and MyoD expression. - Highlights: • PPARγ and MyoD mRNA and protein levels are upregulated by myostatin in ADSCs. • PPARγ and MyoD mRNA and protein levels are downregulated by myostatin in MSCs. • PPARγ exhibited different methylation levels in myostatin-treated ADSCs and MSCs. • MyoD exhibited different methylation levels in myostatin-treated ADSCs and MSCs. • PPARγ and MyoD are differentially regulated by myostatin in ADSCs and MSCs

  19. PPARγ and MyoD are differentially regulated by myostatin in adipose-derived stem cells and muscle satellite cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Feng [Key Laboratory of Swine Genetics and Breeding of the Agricultural Ministry and Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070 (China); Deng, Bing [Wuhan Institute of Animal Science and Veterinary Medicine, Wuhan Academy of Agricultural Science and Technology, Wuhan, Hubei, 430208 (China); Wen, Jianghui [Wu Han University of Technology, Wuhan 430074 (China); Chen, Kun [Key Laboratory of Swine Genetics and Breeding of the Agricultural Ministry and Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070 (China); Liu, Wu; Ye, Shengqiang; Huang, Haijun [Wuhan Institute of Animal Science and Veterinary Medicine, Wuhan Academy of Agricultural Science and Technology, Wuhan, Hubei, 430208 (China); Jiang, Siwen, E-mail: jiangsiwen@mail.hzau.edu.cn [Key Laboratory of Swine Genetics and Breeding of the Agricultural Ministry and Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070 (China); Xiong, Yuanzhu, E-mail: xiongyzhu@163.com [Key Laboratory of Swine Genetics and Breeding of the Agricultural Ministry and Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070 (China)

    2015-03-06

    Myostatin (MSTN) is a secreted protein belonging to the transforming growth factor-β (TGF-β) family that is primarily expressed in skeletal muscle and also functions in adipocyte maturation. Studies have shown that MSTN can inhibit adipogenesis in muscle satellite cells (MSCs) but not in adipose-derived stem cells (ADSCs). However, the mechanism by which MSTN differently regulates adipogenesis in these two cell types remains unknown. Peroxisome proliferator-activated receptor-γ (PPARγ) and myogenic differentiation factor (MyoD) are two key transcription factors in fat and muscle cell development that influence adipogenesis. To investigate whether MSTN differentially regulates PPARγ and MyoD, we analyzed PPARγ and MyoD expression by assessing mRNA, protein and methylation levels in ADSCs and MSCs after treatment with 100 ng/mL MSTN for 0, 24, and 48 h. PPARγ mRNA levels were downregulated after 24 h and upregulated after 48 h of treatment in ADSCs, whereas in MSCs, PPARγ levels were downregulated at both time points. MyoD expression was significantly increased in ADSCs and decreased in MSCs. PPARγ and MyoD protein levels were upregulated in ADSCs and downregulated in MSCs. The CpG methylation levels of the PPARγ and MyoD promoters were decreased in ADSCs and increased in MSCs. Therefore, this study demonstrated that the different regulatory adipogenic roles of MSTN in ADSCs and MSCs act by differentially regulating PPARγ and MyoD expression. - Highlights: • PPARγ and MyoD mRNA and protein levels are upregulated by myostatin in ADSCs. • PPARγ and MyoD mRNA and protein levels are downregulated by myostatin in MSCs. • PPARγ exhibited different methylation levels in myostatin-treated ADSCs and MSCs. • MyoD exhibited different methylation levels in myostatin-treated ADSCs and MSCs. • PPARγ and MyoD are differentially regulated by myostatin in ADSCs and MSCs.

  20. The Onecut Transcription Factors Regulate Differentiation and Distribution of Dorsal Interneurons during Spinal Cord Development

    Directory of Open Access Journals (Sweden)

    Karolina U. Kabayiza

    2017-05-01

    Full Text Available During embryonic development, the dorsal spinal cord generates numerous interneuron populations eventually involved in motor circuits or in sensory networks that integrate and transmit sensory inputs from the periphery. The molecular mechanisms that regulate the specification of these multiple dorsal neuronal populations have been extensively characterized. In contrast, the factors that contribute to their diversification into smaller specialized subsets and those that control the specific distribution of each population in the developing spinal cord remain unknown. Here, we demonstrate that the Onecut transcription factors, namely Hepatocyte Nuclear Factor-6 (HNF-6 (or OC-1, OC-2 and OC-3, regulate the diversification and the distribution of spinal dorsal interneuron (dINs. Onecut proteins are dynamically and differentially distributed in spinal dINs during differentiation and migration. Analyzes of mutant embryos devoid of Onecut factors in the developing spinal cord evidenced a requirement in Onecut proteins for proper production of a specific subset of dI5 interneurons. In addition, the distribution of dI3, dI5 and dI6 interneuron populations was altered. Hence, Onecut transcription factors control genetic programs that contribute to the regulation of spinal dIN diversification and distribution during embryonic development.

  1. MicroRNA-140 Provides Robustness to the Regulation of Hypertrophic Chondrocyte Differentiation by the PTHrP-HDAC4 Pathway.

    Science.gov (United States)

    Papaioannou, Garyfallia; Mirzamohammadi, Fatemeh; Lisse, Thomas S; Nishimori, Shigeki; Wein, Marc N; Kobayashi, Tatsuya

    2015-06-01

    Growth plate chondrocytes go through multiple differentiation steps and eventually become hypertrophic chondrocytes. The parathyroid hormone (PTH)-related peptide (PTHrP) signaling pathway plays a central role in regulation of hypertrophic differentiation, at least in part, through enhancing activity of histone deacetylase 4 (HDAC4), a negative regulator of MEF2 transcription factors that drive hypertrophy. We have previously shown that loss of the chondrocyte-specific microRNA (miRNA), miR-140, alters chondrocyte differentiation including mild acceleration of hypertrophic differentiation. Here, we provide evidence that miR-140 interacts with the PTHrP-HDAC4 pathway to control chondrocyte differentiation. Heterozygosity of PTHrP or HDAC4 substantially impaired animal growth in miR-140 deficiency, whereas these mutations had no effect in the presence of miR-140. miR-140-deficient chondrocytes showed increased MEF2C expression with normal levels of total and phosphorylated HDAC4, indicating that the miR-140 pathway merges with the PTHrP-HDAC4 pathway at the level of MEF2C. miR-140 negatively regulated p38 mitogen-activated protein kinase (MAPK) signaling, and inhibition of p38 MAPK signaling reduced MEF2C expression. These results demonstrate that miR-140 ensures the robustness of the PTHrP/HDAC4 regulatory system by suppressing MEF2C-inducing stimuli. © 2014 American Society for Bone and Mineral Research © 2015 American Society for Bone and Mineral Research. © 2014 American Society for Bone and Mineral Research.

  2. SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production.

    Science.gov (United States)

    Chen, Gang; Korfhagen, Thomas R; Xu, Yan; Kitzmiller, Joseph; Wert, Susan E; Maeda, Yutaka; Gregorieff, Alexander; Clevers, Hans; Whitsett, Jeffrey A

    2009-10-01

    Various acute and chronic inflammatory stimuli increase the number and activity of pulmonary mucus-producing goblet cells, and goblet cell hyperplasia and excess mucus production are central to the pathogenesis of chronic pulmonary diseases. However, little is known about the transcriptional programs that regulate goblet cell differentiation. Here, we show that SAM-pointed domain-containing Ets-like factor (SPDEF) controls a transcriptional program critical for pulmonary goblet cell differentiation in mice. Initial cell-lineage-tracing analysis identified nonciliated secretory epithelial cells, known as Clara cells, as the progenitors of goblet cells induced by pulmonary allergen exposure in vivo. Furthermore, in vivo expression of SPDEF in Clara cells caused rapid and reversible goblet cell differentiation in the absence of cell proliferation. This was associated with enhanced expression of genes regulating goblet cell differentiation and protein glycosylation, including forkhead box A3 (Foxa3), anterior gradient 2 (Agr2), and glucosaminyl (N-acetyl) transferase 3, mucin type (Gcnt3). Consistent with these findings, levels of SPDEF and FOXA3 were increased in mouse goblet cells after sensitization with pulmonary allergen, and the proteins were colocalized in goblet cells lining the airways of patients with chronic lung diseases. Deletion of the mouse Spdef gene resulted in the absence of goblet cells in tracheal/laryngeal submucosal glands and in the conducting airway epithelium after pulmonary allergen exposure in vivo. These data show that SPDEF plays a critical role in regulating a transcriptional network mediating the goblet cell differentiation and mucus hyperproduction associated with chronic pulmonary disorders.

  3. Transcriptional up-regulation of restin by all-trans retinoic acid through STAT1 in cancer cell differentiation process

    International Nuclear Information System (INIS)

    Fu Haiyan; Yang Guodong; Lu Fan; Wang Ruihua; Yao Libo; Lu Zifan

    2006-01-01

    RESTIN, a member of the melanoma-associated antigen superfamily, is a nuclear protein induced by atRA (all-trans retinoic acid) in HL60 cells. HeLa cells stably transfected with restin results in G1 cell cycle arrest. How this gene is regulated by atRA in the cell differentiation process is still unclear. In this study, we observed that up-regulation of restin was present during the atRA-induced HL60 cell differentiation process, suggesting the functional relevance between RESTIN and atRA-induced cellular effects. In order to further define the transcriptional regulation of restin by atRA, we analyzed the promoter region of restin. About 2.1 kb 5' flanking sequence of this gene was cloned into vector pGL3 and its core promoter region was identified through systemic deletions. Interestingly, restin promoter containing several potential consensus-binding sites of STAT-1α was activated by atRA in ER + MCF-7 cells but not in ER - MDA-MB-231 cells, over-expression of STAT-1α in latter rescued the activation effect of restin promoter in response to atRA and IFNγ. Our evidence supported that STAT-1α plays an important role in the atRA-induced transcriptional up-regulation of restin, which was associated with the atRA-induced HL60 cell differentiation and potentially mediated the downstream effects of atRA signal pathway via STAT-1α in some cancer cells

  4. Gammaherpesvirus-driven plasma cell differentiation regulates virus reactivation from latently infected B lymphocytes.

    Directory of Open Access Journals (Sweden)

    Xiaozhen Liang

    2009-11-01

    Full Text Available Gammaherpesviruses chronically infect their host and are tightly associated with the development of lymphoproliferative diseases and lymphomas, as well as several other types of cancer. Mechanisms involved in maintaining chronic gammaherpesvirus infections are poorly understood and, in particular, little is known about the mechanisms involved in controlling gammaherpesvirus reactivation from latently infected B cells in vivo. Recent evidence has linked plasma cell differentiation with reactivation of the human gammaherpesviruses EBV and KSHV through induction of the immediate-early viral transcriptional activators by the plasma cell-specific transcription factor XBP-1s. We now extend those findings to document a role for a gammaherpesvirus gene product in regulating plasma cell differentiation and thus virus reactivation. We have previously shown that the murine gammaherpesvirus 68 (MHV68 gene product M2 is dispensable for virus replication in permissive cells, but plays a critical role in virus reactivation from latently infected B cells. Here we show that in mice infected with wild type MHV68, virus infected plasma cells (ca. 8% of virus infected splenocytes at the peak of viral latency account for the majority of reactivation observed upon explant of splenocytes. In contrast, there is an absence of virus infected plasma cells at the peak of latency in mice infected with a M2 null MHV68. Furthermore, we show that the M2 protein can drive plasma cell differentiation in a B lymphoma cell line in the absence of any other MHV68 gene products. Thus, the role of M2 in MHV68 reactivation can be attributed to its ability to manipulate plasma cell differentiation, providing a novel viral strategy to regulate gammaherpesvirus reactivation from latently infected B cells. We postulate that M2 represents a new class of herpesvirus gene products (reactivation conditioners that do not directly participate in virus replication, but rather facilitate virus

  5. Gammaherpesvirus-driven plasma cell differentiation regulates virus reactivation from latently infected B lymphocytes.

    Science.gov (United States)

    Liang, Xiaozhen; Collins, Christopher M; Mendel, Justin B; Iwakoshi, Neal N; Speck, Samuel H

    2009-11-01

    Gammaherpesviruses chronically infect their host and are tightly associated with the development of lymphoproliferative diseases and lymphomas, as well as several other types of cancer. Mechanisms involved in maintaining chronic gammaherpesvirus infections are poorly understood and, in particular, little is known about the mechanisms involved in controlling gammaherpesvirus reactivation from latently infected B cells in vivo. Recent evidence has linked plasma cell differentiation with reactivation of the human gammaherpesviruses EBV and KSHV through induction of the immediate-early viral transcriptional activators by the plasma cell-specific transcription factor XBP-1s. We now extend those findings to document a role for a gammaherpesvirus gene product in regulating plasma cell differentiation and thus virus reactivation. We have previously shown that the murine gammaherpesvirus 68 (MHV68) gene product M2 is dispensable for virus replication in permissive cells, but plays a critical role in virus reactivation from latently infected B cells. Here we show that in mice infected with wild type MHV68, virus infected plasma cells (ca. 8% of virus infected splenocytes at the peak of viral latency) account for the majority of reactivation observed upon explant of splenocytes. In contrast, there is an absence of virus infected plasma cells at the peak of latency in mice infected with a M2 null MHV68. Furthermore, we show that the M2 protein can drive plasma cell differentiation in a B lymphoma cell line in the absence of any other MHV68 gene products. Thus, the role of M2 in MHV68 reactivation can be attributed to its ability to manipulate plasma cell differentiation, providing a novel viral strategy to regulate gammaherpesvirus reactivation from latently infected B cells. We postulate that M2 represents a new class of herpesvirus gene products (reactivation conditioners) that do not directly participate in virus replication, but rather facilitate virus reactivation by

  6. A supramolecular look at microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny

    Directory of Open Access Journals (Sweden)

    Marcela Aldrovani

    Full Text Available ABSTRACT Various approaches have been taken to improve our knowledge of the microenvironmental regulation of limbal epithelial stem cells. Researchers have extensively investigated the roles of growth factors, survival factors, cytokines, enzymes, and permeable molecules secreted by the limbal cells. However, recent evidence suggests that stem cell fate (i.e., self-renewal or differentiation can also be influenced by biophysical and mechanical cues related to the supramolecular organization and the liquid crystalline (mesophase nature of the stromal extracellular matrix. These cues can be sensed by stem cells and transduced into intracellular biochemical and functional responses, a process known as mechanotransduction. The objective of this review is to offer perspectives on the supramolecular microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny.

  7. Dystroglycan and mitochondrial ribosomal protein L34 regulate differentiation in the Drosophila eye.

    Directory of Open Access Journals (Sweden)

    Yougen Zhan

    2010-05-01

    Full Text Available Mutations that diminish the function of the extracellular matrix receptor Dystroglycan (DG result in muscular dystrophies, with associated neuronal migration defects in the brain and mental retardation e.g. Muscle Eye Brain Disease. To gain insight into the function of DG in the nervous system we initiated a study to examine its contribution to development of the eye of Drosophila melanogaster. Immuno-histochemistry showed that DG is concentrated on the apical surface of photoreceptors (R cells during specification of cell-fate in the third instar larva and is maintained at this location through early pupal stages. In point mutations that are null for DG we see abortive R cell elongation during differentiation that first appears in the pupa and results in stunted R cells in the adult. Overexpression of DG in R cells results in a small but significant increase in their size. R cell differentiation defects appear at the same stage in a deficiency line Df(2RDg(248 that affects Dg and the neighboring mitochondrial ribosomal gene, mRpL34. In the adult, these flies have severely disrupted R cells as well as defects in the lens and ommatidia. Expression of an mRpL34 transgene rescues much of this phenotype. We conclude that DG does not affect neuronal commitment but functions R cell autonomously to regulate neuronal elongation during differentiation in the pupa. We discuss these findings in view of recent work implicating DG as a regulator of cell metabolism and its genetic interaction with mRpL34, a member of a class of mitochondrial genes essential for normal metabolic function.

  8. SHP1 Regulates Bone Mass by Directing Mesenchymal Stem Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Menghui Jiang

    2016-07-01

    Full Text Available Osteoblasts and adipocytes are derived from a common precursor, mesenchymal stem cells (MSCs. Alterations in the normal fate of differentiating MSCs are involved in the development of obesity and osteoporosis. Here, we report that viable motheaten (mev mice, which are deficient in the SH2-domain-containing phosphatase-1 (SHP1, develop osteoporosis spontaneously. Consistently, MSCs from mev/mev mice exhibit significantly reduced osteogenic potential and greatly increased adipogenic potential. When MSCs were transplanted into nude mice, SHP1-deficient MSCs resulted in diminished bone formation compared with wild-type MSCs. SHP1 was found to bind to GSK3β and suppress its kinase activity by dephosphorylating pY216, thus resulting in β-catenin stabilization. Mice, in which SHP1 was deleted in MSCs using SHP1fl/flDermo1-cre, displayed significantly decreased bone mass and increased adipose tissue. Taken together, these results suggest a possible role for SHP1 in controlling tissue homeostasis through modulation of MSC differentiation via Wnt signaling regulation.

  9. KDM1A/LSD1 regulates the differentiation and maintenance of spermatogonia in mice.

    Directory of Open Access Journals (Sweden)

    Dexter A Myrick

    Full Text Available The proper regulation of spermatogenesis is crucial to ensure the continued production of sperm and fertility. Here, we investigated the function of the H3K4me2 demethylase KDM1A/LSD1 during spermatogenesis in developing and adult mice. Conditional deletion of Kdm1a in the testis just prior to birth leads to fewer spermatogonia and germ cell loss before 3 weeks of age. These results demonstrate that KDM1A is required for spermatogonial differentiation, as well as germ cell survival, in the developing testis. In addition, inducible deletion of Kdm1a in the adult testis results in the abnormal accumulation of meiotic spermatocytes, as well as apoptosis and progressive germ cell loss. These results demonstrate that KDM1A is also required during adult spermatogenesis. Furthermore, without KDM1A, the stem cell factor OCT4 is ectopically maintained in differentiating germ cells. This requirement for KDM1A is similar to what has been observed in other stem cell populations, suggesting a common function. Taken together, we propose that KDM1A is a key regulator of spermatogenesis and germ cell maintenance in the mouse.

  10. DIDO as a Switchboard that Regulates Self-Renewal and Differentiation in Embryonic Stem Cells.

    Science.gov (United States)

    Fütterer, Agnes; de Celis, Jésus; Navajas, Rosana; Almonacid, Luis; Gutiérrez, Julio; Talavera-Gutiérrez, Amaia; Pacios-Bras, Cristina; Bernascone, Ilenia; Martin-Belmonte, Fernando; Martinéz-A, Carlos

    2017-04-11

    Transition from symmetric to asymmetric cell division requires precise coordination of differential gene expression. We show that embryonic stem cells (ESCs) mainly express DIDO3 and that their differentiation after leukemia inhibitory factor withdrawal requires DIDO1 expression. C-terminal truncation of DIDO3 (Dido3ΔCT) impedes ESC differentiation while retaining self-renewal; small hairpin RNA-Dido1 ESCs have the same phenotype. Dido3ΔCT ESC differentiation is rescued by ectopic expression of DIDO3, which binds the Dido locus via H3K4me3 and RNA POL II and induces DIDO1 expression. DIDO1, which is exported to cytoplasm, associates with, and is N-terminally phosphorylated by PKCiota. It binds the E3 ubiquitin ligase WWP2, which contributes to cell fate by OCT4 degradation, to allow expression of primitive endoderm (PE) markers. PE formation also depends on phosphorylated DIDO3 localization to centrosomes, which ensures their correct positioning for PE cell polarization. We propose that DIDO isoforms act as a switchboard that regulates genetic programs for ESC transition from pluripotency maintenance to promotion of differentiation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Differential regulation of c-di-GMP metabolic enzymes by environmental signals modulates biofilm formation in Yersinia pestis

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    Gai-Xian eRen

    2016-06-01

    Full Text Available Cyclic diguanylate (c-di-GMP is essential for Yersinia pestis biofilm formation, which is important for flea-borne blockage-dependent plague transmission. Two diguanylate cyclases (DGCs, HmsT and HmsD and one phosphodiesterase (PDE, HmsP are responsible for the synthesis and degradation of c-di-GMP in Y. pestis. Here, we systematically analyzed the effect of various environmental signals on regulation of the biofilm phenotype, the c-di-GMP levels, and expression of HmsT, HmsD and HmsP in Y. pestis. Biofilm formation was higher in the presence of nonlethal high concentration of CaCl2, MgCl2, CuSO4, sucrose, sodium dodecyl sulfonate, or dithiothreitol, and was lower in the presence of FeCl2 or NaCl. In addition, we found that HmsD plays a major role in biofilm formation in acidic or redox environments. These environmental signals differentially regulated expression of HmsT, HmsP and HmsD, resulting in changes in the intracellular levels of c-di-GMP in Y. pestis. Our results suggest that bacteria can sense various environmental signals, and differentially regulates their DGCs and PDEs to coordinately regulate and adapt metabolism of c-di-GMP and biofilm formation to changing environments.

  12. Selective phosphorylation during early macrophage differentiation

    KAUST Repository

    Zhang, Huoming

    2015-08-26

    The differentiation of macrophages from monocytes is a tightly controlled and complex biological process. Although numerous studies have been conducted using biochemical approaches or global gene/gene profiling, the mechanisms of the early stages of differentiation remain unclear. Here we used SILAC-based quantitative proteomics approach to perform temporal phosphoproteome profiling of early macrophage differentiation. We identified a large set of phosphoproteins and grouped them as PMA-regulated and non-regulated phosphoproteins in the early stages of differentiation. Further analysis of the PMA-regulated phosphoproteins revealed that transcriptional suppression, cytoskeletal reorganization and cell adhesion were among the most significantly activated pathways. Some key involved regulators of these pathways are mTOR, MYB, STAT1 and CTNNB. Moreover, we were able to classify the roles and activities of several transcriptional factors during different differentiation stages and found that E2F is likely to be an important regulator during the relatively late stages of differentiation. This study provides the first comprehensive picture of the dynamic phosphoproteome during myeloid cells differentiation, and identifies potential molecular targets in leukemic cells.

  13. Gastrointestinal differentiation marker Cytokeratin 20 is regulated by homeobox gene CDX1

    DEFF Research Database (Denmark)

    Chan, Carol W M; Wong, Newton A; Liu, Ying

    2009-01-01

    colorectal cancer cell lines. Deletion and mutation analysis of the KRT20 promoter showed that the minimum regulatory region for the control of KRT20 expression by CDX1 is within 246 bp upstream of the KRT20 transcription start site. ChIP analysis confirmed that CDX1 binds to the predicted CDX elements...... in this region of the KRT20 promoter in vivo. In addition, immunohistochemistry showed expression of CDX1 parallels that of KRT20 in the normal crypt, which further supports their close relationship. In summary, our observations strongly imply that KRT20 is directly regulated by CDX1, and therefore suggest...... a role for CDX1 in maintaining differentiation in intestinal epithelial cells. Because a key feature of the development of a cancer is an unbalanced program of proliferation and differentiation, dysregulation of CDX1 may be an advantage for the development of a colorectal carcinoma. This could, therefore...

  14. Adhesive and mechanical regulation of mesenchymal stem cell differentiation in human bone marrow and periosteum-derived progenitor cells

    Directory of Open Access Journals (Sweden)

    Jeroen Eyckmans

    2012-08-01

    It has previously been demonstrated that cell shape can influence commitment of human bone marrow-derived mesenchymal stem cells (hBMCs to adipogenic, osteogenic, chondrogenic, and other lineages. Human periosteum-derived cells (hPDCs exhibit multipotency similar to hBMCs, but hPDCs may offer enhanced potential for osteogenesis and chondrogenesis given their apparent endogenous role in bone and cartilage repair in vivo. Here, we examined whether hPDC differentiation is regulated by adhesive and mechanical cues comparable to that reported for hBMC differentiation. When cultured in the appropriate induction media, hPDCs at high cell seeding density demonstrated enhanced levels of adipogenic or chondrogenic markers as compared with hPDCs at low cell seeding density. Cell seeding density correlated inversely with projected area of cell spreading, and directly limiting cell spreading with micropatterned substrates promoted adipogenesis or chondrogenesis while substrates promoting cell spreading supported osteogenesis. Interestingly, cell seeding density influenced differentiation through both changes in cell shape and non-shape-mediated effects: density-dependent adipogenesis and chondrogenesis were regulated primarily by cell shape whereas non-shape effects strongly influenced osteogenic potential. Inhibition of cytoskeletal contractility by adding the Rho kinase inhibitor Y27632 further enhanced adipogenic differentiation and discouraged osteogenic differentiation of hPDCs. Together, our results suggest that multipotent lineage decisions of hPDCs are impacted by cell adhesive and mechanical cues, though to different extents than hBMCs. Thus, future studies of hPDCs and other primary stem cell populations with clinical potential should consider varying biophysical metrics for more thorough optimization of stem cell differentiation.

  15. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David  S.; Poulin, Benoit; Ramaprasad, Abhinay; Wall, Richard  J.; Ferguson, David  J.P.; Brady, Declan; Patzewitz, Eva-Maria; Whipple, Sarah; Straschil, Ursula; Wright, Megan  H.; Mohamed, Alyaa  M.A.H.; Radhakrishnan, Anand; Arold, Stefan T.; Tate, Edward  W.; Holder, Anthony  A.; Wickstead, Bill; Pain, Arnab; Tewari, Rita

    2014-01-01

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  16. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David S.

    2014-07-09

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  17. CSK negatively regulates nerve growth factor induced neural differentiation and augments AKT kinase activity

    International Nuclear Information System (INIS)

    Dey, Nandini; Howell, Brian W.; De, Pradip K.; Durden, Donald L.

    2005-01-01

    Src family kinases are involved in transducing growth factor signals for cellular differentiation and proliferation in a variety of cell types. The activity of all Src family kinases (SFKs) is controlled by phosphorylation at their C-terminal 527-tyrosine residue by C-terminal SRC kinase, CSK. There is a paucity of information regarding the role of CSK and/or specific Src family kinases in neuronal differentiation. Pretreatment of PC12 cells with the Src family kinase inhibitor, PP1, blocked NGF-induced activation of SFKs and obliterated neurite outgrowth. To confirm a role for CSK and specific isoforms of SFKs in neuronal differentiation, we overexpressed active and catalytically dead CSK in the rat pheochromocytoma cell line, PC12. CSK overexpression caused a profound inhibition of NGF-induced activation of FYN, YES, RAS, and ERK and inhibited neurite outgrowth, NGF-stimulated integrin-directed migration and blocked the NGF-induced conversion of GDP-RAC to its GTP-bound active state. CSK overexpression markedly augmented the activation state of AKT following NGF stimulation. In contrast, kinase-dead CSK augmented the activation of FYN, RAS, and ERK and increased neurite outgrowth. These data suggest a distinct requirement for CSK in the regulation of NGF/TrkA activation of RAS, RAC, ERK, and AKT via the differential control of SFKs in the orchestration of neuronal differentiation

  18. MicroRNA-1 Regulates the Differentiation of Adipose-Derived Stem Cells into Cardiomyocyte-Like Cells

    Directory of Open Access Journals (Sweden)

    Can Chen

    2018-01-01

    Full Text Available Stem cell transplantation is one of most valuable methods in the treatment of myocardial infarction, and adipose-derived stem cells (ASCs are becoming a hot topic in medical research. Previous studies have shown that ASCs can be differentiated into cardiomyocyte-like cells, but the efficiency and survival rates are low. We investigated the role and mechanism of microRNA-1 (miR-1 in the differentiation of ASCs into cardiomyocyte-like cells. ASCs and cardiomyocytes were isolated from neonatal rats. We constructed lentivirus for overexpressing miR-1 and used DAPT, an antagonist of the Notch1 pathway, for in vitro analyses. We performed cocultures with ASCs and cardiomyocytes. The differentiation efficiency of ASCs was detected by cell-specific surface antigens. Our results showed that miR-1 can promote the expression of Notch1 and reduce the expression of Hes1, a Notch pathway factor, and overexpression of miR-1 can promote the differentiation of ASCs into cardiomyocyte-like cells, which may occur by regulating Notch1 and Hes1.

  19. The transition from proliferation to differentiation in colorectal cancer is regulated by the calcium activated chloride channel A1.

    Directory of Open Access Journals (Sweden)

    Bo Yang

    Full Text Available Breaking the balance between proliferation and differentiation in animal cells can lead to cancer, but the mechanisms maintaining this balance remain largely undefined. The calcium activated chloride channel A1 (CLCA1 is a member of the calcium sensitive chloride conductance family of proteins and is expressed mainly in the colon, small intestine and appendix. We show that CLCA1 plays a functional role in differentiation and proliferation of Caco-2 cells and of intestinal tissue. Caco-2 cells spontaneously differentiate either in confluent culture or when treated with butyrate, a molecule present naturally in the diet. Here, we compared CLCA1 expressional levels between patients with and without colorectal cancer (CRC and determined the functional role of CLCA1 in differentiation and proliferation of Caco-2 cells. We showed that: 1 CLCA1 and CLCA4 expression were down-regulated significantly in CRC patients; 2 CLCA1 expression was up-regulated in Caco-2 cells induced to differentiate by confluent culture or by treatment with sodium butyrate (NaBT; 3 Knockdown of CLCA1 with siRNA significantly inhibited cell differentiation and promoted cell proliferation in Caco-2 confluent cultures, and 4 In Caco-2 3D culture, suppression of CLCA1 significantly increased cell proliferation and compromised NaBT-induced inhibition of proliferation. In conclusion, CLCA1 may contribute to promoting spontaneous differentiation and reducing proliferation of Caco-2 cells and may be a target of NaBT-induced inhibition of proliferation and therefore a potential diagnostic marker for CRC prognosis.

  20. miR-140-5p regulates hypoxia-mediated human pulmonary artery smooth muscle cell proliferation, apoptosis and differentiation by targeting Dnmt1 and promoting SOD2 expression

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanwei; Xu, Jing, E-mail: xujingdoc@163.com

    2016-04-22

    miR-140-5p is down-regulated in patients with pulmonary arterial hypertension (PAH) and experimental models of PAH, and inhibits hypoxia-mediated pulmonary artery smooth muscle cell (PASMC) proliferation in vitro. Delivery of synthetic miR-140-5p prevents and treats established, experimental PAH. DNA methyltransferase 1 (Dnmt1) is up-regulated in PAH associated human PASMCs (HPASMCs), which promotes the development of PAH by hypermethylation of CpG islands within the promoter for superoxide dismutase 2 (SOD2) and down-regulating SOD2 expression. We searched for miR-140-5p targets using TargetScan, PicTar and MiRanda tools, and found that Dnmt1 is a potential target of miR-140-5p. Based on these findings, we speculated that miR-140-5p might target Dnmt1 and regulate SOD2 expression to regulate hypoxia-mediated HPASMC proliferation, apoptosis and differentiation. We detected the expression of miR-140-5p, Dnmt1 and SOD2 by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assays, respectively, and found down-regulation of miR-140-5p and SOD2 and up-regulation of Dnmt1 exist in PAH tissues and hypoxia-mediated HPASMCs. Cell proliferation, apoptosis and differentiation detection showed that miR-140-5p inhibits proliferation and promotes apoptosis and differentiation of HPASMCs in hypoxia, while the effect of Dnmt1 on hypoxia-mediated HPASMCs is reversed. Luciferase assay confirmed that miR-140-5p targets Dnmt1 directly. An inverse correlation is also found between miR-140-5p and Dnmt1 in HPASMCs. In addition, we further investigated whether miR-140-5p and Dnmt1 regulate HPASMC proliferation, apoptosis and differentiation by regulating SOD2 expression, and the results confirmed our speculation. Taken together, these results indicated that miR-140-5p at least partly targets Dnmt1 and regulates SOD2 expression to inhibit proliferation and promote apoptosis and differentiation of HPASMCs in hypoxia. - Highlights: • miR-140-5p and SOD2 are down-regulated

  1. miR-342-5p Regulates Neural Stem Cell Proliferation and Differentiation Downstream to Notch Signaling in Mice

    Directory of Open Access Journals (Sweden)

    Fang Gao

    2017-04-01

    Full Text Available Summary: Notch signaling is critically involved in neural development, but the downstream effectors remain incompletely understood. In this study, we cultured neurospheres from Nestin-Cre-mediated conditional Rbp-j knockout (Rbp-j cKO and control embryos and compared their miRNA expression profiles using microarray. Among differentially expressed miRNAs, miR-342-5p showed upregulated expression as Notch signaling was genetically or pharmaceutically interrupted. Consistently, the promoter of the miR-342-5p host gene, the Ena-vasodilator stimulated phosphoprotein-like (Evl, was negatively regulated by Notch signaling, probably through HES5. Transfection of miR-342-5p promoted the differentiation of neural stem cells (NSCs into intermediate neural progenitors (INPs in vitro and reduced the stemness of NSCs in vivo. Furthermore, miR-342-5p inhibited the differentiation of neural stem/intermediate progenitor cells into astrocytes, likely mediated by targeting GFAP directly. Our results indicated that miR-342-5p could function as a downstream effector of Notch signaling to regulate the differentiation of NSCs into INPs and astrocytes commitment. : In this article, Han and colleagues show that miR-342-5p acts as a downstream effector of Notch signaling in the mouse CNS. Notch signal inhibits miR-342-5p expression by regulating its host gene Evl. And with attenuated Notch signal in NSCs, miR-342-5p is upregulated to promote NSCs transition into INPs, and to inhibit astrocyte commitment by targeting GFAP. Keywords: neural stem cells, intermediate neural progenitors, Notch, RBP-J, neuron, glia, miR-342-5p

  2. The NO signaling pathway differentially regulates KCC3a and KCC3b mRNA expression.

    Science.gov (United States)

    Di Fulvio, Mauricio; Lauf, Peter K; Adragna, Norma C

    2003-11-01

    Nitric oxide (NO) donors and protein kinase G (PKG) acutely up-regulate K-Cl cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in vascular smooth muscle cells (VSMCs). Here, we report the presence, relative abundance, and regulation by sodium nitroprusside (SNP) of the novel KCC3a and KCC3b mRNAs, in primary cultures of rat VSMCs. KCC3a and KCC3b mRNAs were expressed in an approximate 3:1 ratio, as determined by semiquantitative RT-PCR analysis. SNP as well as YC-1 and 8-Br-cGMP, a NO-independent stimulator of soluble guanylyl cyclase (sGC) and PKG, respectively, increased KCC3a and KCC3b mRNA expression by 2.5-fold and 8.1-fold in a time-dependent manner, following a differential kinetics. Stimulation of the NO/sGC/PKG signaling pathway with either SNP, YC-1, or 8-Br-cGMP decreased the KCC3a/KCC3b ratio from 3.0+/-0.4 to 0.9+/-0.1. This is the first report on a differential regulation by the NO/sGC/PKG signaling pathway of a cotransporter and of KCC3a and KCC3b mRNA expression.

  3. An automated high throughput screening-compatible assay to identify regulators of stem cell neural differentiation.

    Science.gov (United States)

    Casalino, Laura; Magnani, Dario; De Falco, Sandro; Filosa, Stefania; Minchiotti, Gabriella; Patriarca, Eduardo J; De Cesare, Dario

    2012-03-01

    The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable ESC neural differentiation assay by exploiting the Cell(maker) robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the identification of regulators of ESC neural differentiation in full automation.

  4. Differential trypanosome surface coat regulation by a CCCH protein that co-associates with procyclin mRNA cis-elements.

    Directory of Open Access Journals (Sweden)

    Pegine Walrad

    2009-02-01

    Full Text Available The genome of Trypanosoma brucei is unusual in being regulated almost entirely at the post-transcriptional level. In terms of regulation, the best-studied genes are procyclins, which encode a family of major surface GPI-anchored glycoproteins (EP1, EP2, EP3, GPEET that show differential expression in the parasite's tsetse-fly vector. Although procyclin mRNA cis-regulatory sequences have provided the paradigm for post-transcriptional control in kinetoplastid parasites, trans-acting regulators of procyclin mRNAs are unidentified, despite intensive effort over 15 years. Here we identify the developmental regulator, TbZFP3, a CCCH-class predicted RNA binding protein, as an isoform-specific regulator of Procyclin surface coat expression in trypanosomes. We demonstrate (i that endogenous TbZFP3 shows sequence-specific co-precipitation of EP1 and GPEET, but not EP2 and EP3, procyclin mRNA isoforms, (ii that ectopic overexpression of TbZFP3 does not perturb the mRNA abundance of procyclin transcripts, but rather that (iii their protein expression is regulated in an isoform-specific manner, as evidenced by mass spectrometric analysis of the Procyclin expression signature in the transgenic cell lines. The TbZFP3 mRNA-protein complex (TbZFP3mRNP is identified as a trans-regulator of differential surface protein expression in trypanosomes. Moreover, its sequence-specific interactions with procyclin mRNAs are compatible with long-established predictions for Procyclin regulation. Combined with the known association of TbZFP3 with the translational apparatus, this study provides a long-sought missing link between surface protein cis-regulatory signals and the gene expression machinery in trypanosomes.

  5. Wnt and Hedgehog Signaling Regulate the Differentiation of F9 Cells into Extraembryonic Endoderm

    Directory of Open Access Journals (Sweden)

    Gurjoth S. J. Deol

    2017-10-01

    Full Text Available Mouse F9 cells differentiate into primitive extraembryonic endoderm (PrE when treated with retinoic acid (RA, and this is accompanied by an up-regulation of Gata6. The role of the GATA6 network in PrE differentiation is known, and we have shown it directly activates Wnt6. Canonical Wnt/β-catenin signaling is required by F9 cells to differentiate to PrE, and this, like most developmental processes, requires input from one or more additional pathways. We found both RA and Gata6 overexpression, can induce the expression of Indian Hedgehog (Ihh and a subset of its target genes through Gli activation during PrE induction. Chemical activation of the Hh pathway using a Smoothened agonist (SAG also increased Gli reporter activity, and as expected, when Hh signaling was blocked with a Smoothened antagonist, cyclopamine, this RA-induced reporter activity was reduced. Interestingly, SAG alone failed to induce markers of PrE differentiation, and had no effect on Wnt/β-catenin-dependent TCF-LEF reporter activity. The expected increase in Wnt/β-catenin-dependent TCF-LEF reporter activity and PrE markers induced by RA was, however, blocked by cyclopamine. Finally, inhibiting GSK3 activity with BIO increased both TCF-LEF and Gli reporter activities. Together, we demonstrate the involvement of Hh signaling in the RA-induced differentiation of F9 cells into PrE, and while the activation of the Hh pathway itself is not sufficient, it as well as active Wnt/β-catenin are necessary for F9 cell differentiation.

  6. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

    International Nuclear Information System (INIS)

    Brady, Robert T.; O'Brien, Fergal J.; Hoey, David A.

    2015-01-01

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting

  7. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Brady, Robert T. [Tissue Engineering Research Group, Dept. of Anatomy, Royal College of Surgeons in Ireland (Ireland); Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Advanced Materials and BioEngineering Research Centre (AMBER), Trinity College Dublin & Royal College of Surgeons in Ireland (Ireland); Dept. of Mechanical, Aeronautical and Biomedical Engineering, University of Limerick (Ireland); O' Brien, Fergal J. [Tissue Engineering Research Group, Dept. of Anatomy, Royal College of Surgeons in Ireland (Ireland); Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Advanced Materials and BioEngineering Research Centre (AMBER), Trinity College Dublin & Royal College of Surgeons in Ireland (Ireland); Hoey, David A., E-mail: david.hoey@ul.ie [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Dept. of Mechanical, Aeronautical and Biomedical Engineering, University of Limerick (Ireland); The Centre for Applied Biomedical Engineering Research, University of Limerick (Ireland); Materials & Surface Science Institute, University of Limerick (Ireland)

    2015-03-27

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting

  8. Arginine Methylation Regulates MEIS2 Nuclear Localization to Promote Neuronal Differentiation of Adult SVZ Progenitors.

    Science.gov (United States)

    Kolb, Jasmine; Anders-Maurer, Marie; Müller, Tanja; Hau, Ann-Christin; Grebbin, Britta Moyo; Kallenborn-Gerhardt, Wiebke; Behrends, Christian; Schulte, Dorothea

    2018-04-10

    Adult neurogenesis is regulated by stem cell niche-derived extrinsic factors and cell-intrinsic regulators, yet the mechanisms by which niche signals impinge on the activity of intrinsic neurogenic transcription factors remain poorly defined. Here, we report that MEIS2, an essential regulator of adult SVZ neurogenesis, is subject to posttranslational regulation in the SVZ olfactory bulb neurogenic system. Nuclear accumulation of MEIS2 in adult SVZ-derived progenitor cells follows downregulation of EGFR signaling and is modulated by methylation of MEIS2 on a conserved arginine, which lies in close proximity to nested binding sites for the nuclear export receptor CRM1 and the MEIS dimerization partner PBX1. Methylation impairs interaction with CRM1 without affecting PBX1 dimerization and thereby allows MEIS2 nuclear accumulation, a prerequisite for neuronal differentiation. Our results describe a form of posttranscriptional modulation of adult SVZ neurogenesis whereby an extrinsic signal fine-tunes neurogenesis through posttranslational modification of a transcriptional regulator of cell fate. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Regulator of differentiation 1 (ROD1) binds to the amphipathic C-terminal peptide of thrombospondin-4 and is involved in its mitogenic activity.

    Science.gov (United States)

    Sadvakassova, Gulzhakhan; Dobocan, Monica C; Difalco, Marcos R; Congote, Luis F

    2009-09-01

    The matrix protein thrombospondin-4 has an acidic amphipathic C-terminal peptide (C21) which stimulates erythroid cell proliferation. Here we show that C21 stimulates red cell formation in anemic mice in vivo. In vitro experiments indicated that the peptide-mediated increase of erythroid colony formation in cultures of human CD34+ hematopoietic progenitor cells was possible only under continuous presence of erythropoietin. In the absence of this cytokine, C21 stimulated exclusively myeloid colony formation. Therefore, the peptide is not a specific erythroid differentiation factor. In fact, it is mitogenic in non-erythroid cells, such as skin fibroblasts and kidney epithelial cells. In erythroleukemic TF-1 cells, it actually decreased the production of the erythroid differentiation marker glycophorin A. C21-affinity chromatography revealed regulator of differentiation 1 (ROD1) as a major C21-binding protein. ROD1 is the hematopoietic cell paralog of polypyrimidine tract binding proteins (PTBs), RNA splice regulators which regulate differentiation by repressing tissue-specific exons. ROD1 binding to C21 was strongly inhibited by synthetic RNAs in the order poly A > poly U > poly G = poly C and was weakly inhibited by a synthetic phosphorylated peptide mimicking the C-terminal domain of RNA polymerase II. Cellular overexpression or knockdown experiments of ROD1 suggest a role for this protein in the mitogenic activity of C21. Since the nuclear proteins ROD1 and PTBs regulate differentiation at a posttranscriptional level and there is a fast nuclear uptake of C21, we put forward the idea that the peptide is internalized, goes to the nucleus and maintains cells in a proliferative state by supporting ROD1-mediated inhibition of differentiation.

  10. Heterokaryon analysis of muscle differentiation: regulation of the postmitotic state.

    Science.gov (United States)

    Clegg, C H; Hauschka, S D

    1987-08-01

    MM14 mouse myoblasts withdraw irreversibly from the cell cycle and become postmitotic within a few hours of being deprived of fibroblast growth factor (Clegg, C. H., T. A. Linkhart, B. B. Olwin, and S. D. Hauschka, 1987, J. Cell Biol., 105:949-956). To examine the mechanisms that may regulate this developmental state of skeletal muscle, we tested the mitogen responsiveness of various cell types after their polyethylene glycol-mediated fusion with post-mitotic myocytes. Heterokaryons containing myocytes and quiescent nonmyogenic cells such as 3T3, L cell, and a differentiation-defective myoblast line (DD-1) responded to mitogen-rich medium by initiating DNA synthesis. Myonuclei replicated DNA and reexpressed thymidine kinase. In contrast, (myocyte x G1 myoblast) heterokaryons failed to replicate DNA in mitogen-rich medium and became postmitotic. This included cells with a nuclear ratio of three myoblasts to one myocyte. Proliferation dominance in (myocyte x 3T3 cell) and (myocyte x DD-1) heterokaryons was conditionally regulated by the timing of mitogen treatment; such cells became postmitotic when mitogen exposure was delayed for as little as 6 h after cell fusion. In addition, (myocyte x DD-1) heterokaryons expressed a muscle-specific trait and lost epidermal growth factor receptors when they became postmitotic. These results demonstrate that DNA synthesis is not irreversibly blocked in skeletal muscle; myonuclei readily express proliferation-related functions when provided with a mitogenic signal. Rather, myocyte-specific repression of DNA synthesis in heterokaryons argues that the postmitotic state of skeletal muscle is regulated by diffusible factors that inhibit processes of cellular mitogenesis.

  11. Inhibition of Rho kinase regulates specification of early differentiation events in P19 embryonal carcinoma stem cells.

    Directory of Open Access Journals (Sweden)

    Roman J Krawetz

    Full Text Available The Rho kinase pathway plays a key role in many early cell/tissue determination events that take place in embryogenesis. Rho and its downstream effector Rho kinase (ROCK play pivotal roles in cell migration, apoptosis (membrane blebbing, cell proliferation/cell cycle, cell-cell adhesion and gene regulation. We and others have previously demonstrated that inhibition of ROCK blocks endoderm differentiation in embryonal carcinoma stem cells, however, the effect of ROCK inhibition on mesoderm and ectoderm specification has not been fully examined. In this study, the role of ROCK within the specification and differentiation of all three germ layers was examined.P19 cells were treated with the specific ROCK inhibitor Y-27623, and increase in differentiation efficiency into neuro-ectodermal and mesodermal lineages was observed. However, as expected a dramatic decrease in early endodermal markers was observed when ROCK was inhibited. Interestingly, within these ROCK-inhibited RA treated cultures, increased levels of mesodermal or ectodermal markers were not observed, instead it was found that the pluripotent markers SSEA-1 and Oct-4 remained up-regulated similar to that seen in undifferentiated cultures. Using standard and widely accepted methods for reproducible P19 differentiation into all three germ layers, an enhancement of mesoderm and ectoderm differentiation with a concurrent loss of endoderm lineage specification was observed with Y-27632 treatment. Evidence would suggest that this effect is in part mediated through TGF-β and SMAD signaling as ROCK-inhibited cells displayed aberrant SMAD activation and did not return to a 'ground' state after the inhibition had been removed.Given this data and the fact that only a partial rescue of normal differentiation capacity occurred when ROCK inhibition was alleviated, the effect of ROCK inhibition on the differentiation capacity of pluripotent cell populations should be further examined to elucidate the

  12. Erythroid Differentiation Regulator 1 as a Novel Biomarker for Hair Loss Disorders.

    Science.gov (United States)

    Woo, Yu Ri; Hwang, Sewon; Jeong, Seo Won; Cho, Dae Ho; Park, Hyun Jeong

    2017-02-03

    Erythroid differentiation regulator 1 (Erdr1) is known to be involved in the inflammatory process via regulating the immune system in many cutaneous disorders, such as psoriasis and rosacea. However, the role of Erdr1 in various hair loss disorders remains unclear. The aim of this study was to investigate the putative role of Erdr1 in alopecias. Skin samples from 21 patients with hair loss disorders and five control subjects were retrieved, in order to assess their expression levels of Erdr1. Results revealed that expression of Erdr1 was significantly downregulated in the epidermis and hair follicles of patients with hair loss disorders, when compared to that in the control group. In particular, the expression of Erdr1 was significantly decreased in patients with alopecia areata. We propose that Erdr1 downregulation might be involved in the pathogenesis of hair loss, and could be considered as a novel biomarker for hair loss disorders.

  13. Low-intensity pulsed ultrasound regulates proliferation and differentiation of osteoblasts through osteocytes

    International Nuclear Information System (INIS)

    Li, Lei; Yang, Zheng; Zhang, Hai; Chen, Wenchuan; Chen, Mengshi; Zhu, Zhimin

    2012-01-01

    Highlights: ► CM from LIPUS-stimulated osteocytes inhibits proliferation of osteoblasts. ► CM from LIPUS-stimulated osteocytes enhances differentiation of osteoblasts. ► LIPUS stimulates MLO-Y4 cells to secrete PGE 2 and NO. -- Abstract: Low-intensity pulsed ultrasound (LIPUS) has been used as a safe and effective modality to enhance fracture healing. As the most abundant cells in bone, osteocytes orchestrate biological activities of effector cells via direct cell-to-cell contacts and by soluble factors. In this study, we have used the osteocytic MLO-Y4 cells to study the effects of conditioned medium from LIPUS-stimulated MLO-Y4 cells on proliferation and differentiation of osteoblastic MC3T3-E1 cells. Conditioned media from LIPUS-stimulated MLO-Y4 cells (LIPUS-Osteocyte-CM) were collected and added on MC3T3-E1 cell cultures. MC3T3-E1 cells cultured in LIPUS-Osteocyte-CM demonstrated a significant inhibition of proliferation and an increased alkaline phosphatase activity. The results of PGE 2 and NO assay showed that LIPUS could enhance PGE 2 and NO secretion from MLO-Y4 cells at all time points within 24 h after LIPUS stimulation. We conclude that LIPUS regulates proliferation and differentiation of osteoblasts through osteocytes in vitro. Increased secretion of PGE 2 from osteocytes may play a role in this effect.

  14. The transcription factor Th-POK negatively regulates Th17 differentiation in Vα14i NKT cells

    Science.gov (United States)

    Engel, Isaac; Zhao, Meng; Kappes, Dietmar; Taniuchi, Ichiro

    2012-01-01

    The majority of mouse Vα14 invariant natural killer T (Vα14i NKT) cells produce several cytokines, including IFNγ and IL-4, very rapidly after activation. A subset of these cells, known as NKT17 cells, however, differentiates in the thymus to preferentially produce IL-17. Here, we show that the transcription factor—known as T helper, Poxviruses, and Zinc-finger and Krüppel family, (Th-POK)—represses the formation of NKT17 cells. Vα14i NKT cells from Th-POK–mutant helper deficient (hd/hd) mice have increased transcripts of genes normally expressed by Th17 and NKT17 cells, and even heterozygosity for this mutation leads to dramatically increased numbers of Vα14i NKT cells that are poised to express IL-17, especially in the thymus and lymph nodes. In addition, using gene reporter mice, we demonstrate that NKT17 cells from wild-type mice express lower amounts of Th-POK than the majority population of Vα14i NKT cells. We also show that retroviral transduction of Th-POK represses the expression of the Th17 master regulator RORγT in Vα14i NKT-cell lines. Our data suggest that NKT17-cell differentiation is intrinsically regulated by Th-POK activity, with only low levels of Th-POK permissive for the differentiation of NKT17 cells. PMID:23034280

  15. Expression of osterix Is Regulated by FGF and Wnt/β-Catenin Signalling during Osteoblast Differentiation.

    Directory of Open Access Journals (Sweden)

    Katharina Felber

    Full Text Available Osteoblast differentiation from mesenchymal cells is regulated by multiple signalling pathways. Here we have analysed the roles of Fibroblast Growth Factor (FGF and canonical Wingless-type MMTV integration site (Wnt/β-Catenin signalling pathways on zebrafish osteogenesis. We have used transgenic and chemical interference approaches to manipulate these pathways and have found that both pathways are required for osteoblast differentiation in vivo. Our analysis of bone markers suggests that these pathways act at the same stage of differentiation to initiate expression of the osteoblast master regulatory gene osterix (osx. We use two independent approaches that suggest that osx is a direct target of these pathways. Firstly, we manipulate signalling and show that osx gene expression responds with similar kinetics to that of known transcriptional targets of the FGF and Wnt pathways. Secondly, we have performed ChIP with transcription factors for both pathways and our data suggest that a genomic region in the first intron of osx mediates transcriptional activation. Based upon these data, we propose that FGF and Wnt/β-Catenin pathways act in part by directing transcription of osx to promote osteoblast differentiation at sites of bone formation.

  16. Differential regulation of the foraging gene associated with task behaviors in harvester ants

    Directory of Open Access Journals (Sweden)

    Kleeman Lindsay

    2011-08-01

    Full Text Available Abstract Background The division of labor in social insect colonies involves transitions by workers from one task to another and is critical to the organization and ecological success of colonies. The differential regulation of genetic pathways is likely to be a key mechanism involved in plasticity of social insect task behavior. One of the few pathways implicated in social organization involves the cGMP-activated protein kinase gene, foraging, a gene associated with foraging behavior in social insect species. The association of the foraging gene with behavior is conserved across diverse species, but the observed expression patterns and proposed functions of this gene vary across taxa. We compared the protein sequence of foraging across social insects and explored whether the differential regulation of this gene is associated with task behaviors in the harvester ant, Pogonomyrmex occidentalis. Results Phylogenetic analysis of the coding region of the foraging gene reveals considerable conservation in protein sequence across insects, particularly among hymenopteran species. The absence of amino acid variation in key active and binding sites suggests that differences in behaviors associated with this gene among species may be the result of changes in gene expression rather than gene divergence. Using real time qPCR analyses with a harvester ant ortholog to foraging (Pofor, we found that the brains of harvester ant foragers have a daily fluctuation in expression of foraging with mRNA levels peaking at midday. In contrast, young workers inside the nest have low levels of Pofor mRNA with no evidence of daily fluctuations in expression. As a result, the association of foraging expression with task behavior within a species changes depending on the time of day the individuals are sampled. Conclusions The amino acid protein sequence of foraging is highly conserved across social insects. Differences in foraging behaviors associated with this gene among

  17. TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs.

    Science.gov (United States)

    Hawse, William F; Boggess, William C; Morel, Penelope A

    2017-07-15

    The Akt/mTOR pathway is a key driver of murine CD4 + T cell differentiation, and induction of regulatory T (Treg) cells results from low TCR signal strength and low Akt/mTOR signaling. However, strong TCR signals induce high Akt activity that promotes Th cell induction. Yet, it is unclear how Akt controls alternate T cell fate decisions. We find that the strength of the TCR signal results in differential Akt enzymatic activity. Surprisingly, the Akt substrate networks associated with T cell fate decisions are qualitatively different. Proteomic profiling of Akt signaling networks during Treg versus Th induction demonstrates that Akt differentially regulates RNA processing and splicing factors to drive T cell differentiation. Interestingly, heterogeneous nuclear ribonucleoprotein (hnRNP) L or hnRNP A1 are Akt substrates during Treg induction and have known roles in regulating the stability and splicing of key mRNAs that code for proteins in the canonical TCR signaling pathway, including CD3ζ and CD45. Functionally, inhibition of Akt enzymatic activity results in the dysregulation of splicing during T cell differentiation, and knockdown of hnRNP L or hnRNP A1 results in the lower induction of Treg cells. Together, this work suggests that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates and demonstrates that proteins involved with alternative splicing are important factors in T cell fate decisions. Copyright © 2017 by The American Association of Immunologists, Inc.

  18. Bacterial competition reveals differential regulation of the pks genes by Bacillus subtilis.

    Science.gov (United States)

    Vargas-Bautista, Carol; Rahlwes, Kathryn; Straight, Paul

    2014-02-01

    Bacillus subtilis is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The pks genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of pks genes appear to be organized as a giant operon (>74 kb from pksC-pksR). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305-310, 2007, doi:10.1073/pnas.0609073103), a deletion of the pks operon in B. subtilis was found to induce prodiginine production by Streptomyces coelicolor. Here, colonies of wild-type B. subtilis formed a spreading population that induced prodiginine production from Streptomyces lividans, suggesting differential regulation of pks genes and, as a result, bacillaene. While the parent colony showed widespread induction of pks expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the pks genes. To identify regulators that control pks genes, we first determined the pattern of pks gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of pks gene expression. We found that expression of the pks genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of degU, comA, and scoC had moderate effects, disrupting the timing and level of pks gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for B. subtilis.

  19. Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues.

    Science.gov (United States)

    Amoyel, Marc; Hillion, Kenzo-Hugo; Margolis, Shally R; Bach, Erika A

    2016-11-01

    Stem cells reside in niches that provide signals to maintain self-renewal, and differentiation is viewed as a passive process that depends on loss of access to these signals. Here, we demonstrate that the differentiation of somatic cyst stem cells (CySCs) in the Drosophila testis is actively promoted by PI3K/Tor signaling, as CySCs lacking PI3K/Tor activity cannot differentiate properly. We find that an insulin peptide produced by somatic cells immediately outside of the stem cell niche acts locally to promote somatic differentiation through Insulin-like receptor (InR) activation. These results indicate that there is a local 'differentiation' niche that upregulates PI3K/Tor signaling in the early daughters of CySCs. Finally, we demonstrate that CySCs secrete the Dilp-binding protein ImpL2, the Drosophila homolog of IGFBP7, into the stem cell niche, which blocks InR activation in CySCs. Thus, we show that somatic cell differentiation is controlled by PI3K/Tor signaling downstream of InR and that the local production of positive and negative InR signals regulates the differentiation niche. These results support a model in which leaving the stem cell niche and initiating differentiation are actively induced by signaling. © 2016. Published by The Company of Biologists Ltd.

  20. Insight into podocyte differentiation from the study of human genetic disease: nail-patella syndrome and transcriptional regulation in podocytes.

    Science.gov (United States)

    Morello, Roy; Lee, Brendan

    2002-05-01

    In recent years, our understanding of the molecular basis of kidney development has benefited from the study of rare genetic diseases affecting renal function. This has especially been the case with the differentiation of the highly specialized podocyte in the pathogenesis of human disorders and mouse phenotypes affecting the renal filtration barrier. This filtration barrier represents the end product of a complex series of signaling events that produce a tripartite structure consisting of interdigitating podocyte foot processes with intervening slit diaphragms, the glomerular basement membrane, and the fenestrated endothelial cell. Dysregulation of unique cytoskeletal and extracellular matrix proteins in genetic forms of nephrotic syndrome has shown how specific structural proteins contribute to podocyte function and differentiation. However, much less is known about the transcriptional determinants that both specify and maintain this differentiated cell. Our studies of a skeletal malformation syndrome, nail-patella syndrome, have shown how the LIM homeodomain transcription factor, Lmx1b, contributes to transcriptional regulation of glomerular basement membrane collagen expression by podocytes. Moreover, they raise intriguing questions about more global transcriptional regulation of podocyte morphogenesis.

  1. Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation*

    Science.gov (United States)

    Hao, Wu; Tashiro, Syoichi; Hasegawa, Tomoka; Sato, Yuiko; Kobayashi, Tami; Tando, Toshimi; Katsuyama, Eri; Fujie, Atsuhiro; Watanabe, Ryuichi; Morita, Mayu; Miyamoto, Kana; Morioka, Hideo; Nakamura, Masaya; Matsumoto, Morio; Amizuka, Norio; Toyama, Yoshiaki; Miyamoto, Takeshi

    2015-01-01

    Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that de-differentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D3, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheral nerve de-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D3 administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition. PMID:25998127

  2. Yeast three-hybrid screen identifies TgBRADIN/GRA24 as a negative regulator of Toxoplasma gondii bradyzoite differentiation.

    Directory of Open Access Journals (Sweden)

    Anahi V Odell

    Full Text Available Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite's life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.

  3. Characterization of a type-A response regulator differentially expressed during adventitious caulogenesis in Pinus pinaster.

    Science.gov (United States)

    Alvarez, José M; Cortizo, Millán; Ordás, Ricardo J

    2012-12-15

    The molecular cloning and characterization of PipsRR1, a type-A response regulator in Pinus pinaster, is reported here. Type-A response regulators mediate downstream responses to cytokinin and act as negative feedback regulators of the signal transduction pathway. Some type-A response regulators in Arabidopsis have been related to de novo meristem formation. However, little information exists in Pinus spp. The PipsRR1 gene contains 5 exons, as do all type-A response regulators in Arabidopsis, and the deduced protein contains a receiver domain with the conserved DDK residues and a short C terminal extension. Expression analysis showed that the PipsRR1 gene is differentially expressed during the first phases of adventitious caulogenesis induced by benzyladenine in P. pinaster cotyledons, suggesting that PipsRR1 plays a role in caulogenesis in conifers. Additionally, a binary vector carrying the PipsRR1 promoter driving GFP:GUS expression was constructed to analyze the promoter activity in P. pinaster somatic embryos. The results of genetic transformation showed GUS activity during somatic embryo mass proliferation and embryo maturation. Copyright © 2012 Elsevier GmbH. All rights reserved.

  4. RSPO1/β-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary.

    Directory of Open Access Journals (Sweden)

    Anne-Amandine Chassot

    Full Text Available Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog gene and factors synthesized by the somatic Sertoli cells promote gonocyte differentiation. In the female, ovarian differentiation requires activation of the WNT/β-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/β-catenin signaling pathway in germ cells. In XX Rspo1(-/- gonads, germ cell proliferation, expression of the early meiotic marker Stra8, and entry into meiosis are all impaired. In these gonads, impaired entry into meiosis and germ cell sex reversal occur prior to detectable Sertoli cell differentiation, suggesting that β-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/β-catenin signaling is involved in meiosis in fetal germ cells and contributes to the cellular decision of germ cells to differentiate into oocyte or sperm.

  5. Molecular and biophysical mechanisms regulating hypertrophic differentiation in chondrocytes and mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    D Studer

    2012-07-01

    Full Text Available Chondrocyte hypertrophy is one of the key physiological processes involved in the longitudinal growth of long bones, yet the regulation of hypertrophy is also becoming increasingly relevant for clinical application of mesenchymal stem cells (MSCs and screening for drugs to treat hypertrophic osteoarthritis. The extraordinary cell volume increase during hypertrophy is accompanied by an up-regulation of collagen X, matrix metalloproteinases (MMPs, and vascular endothelial growth factor (VEGF, all which are targets of the runt-related transcription factor 2 (Runx2. Many pathways, including parathyroid hormone-related protein (PTHrP/Indian Hedgehog, Wingless/Int (Wnt/β-catenin, and transforming growth factor beta (TGF-β/Sma and Mad Related Family (Smad pathways, can regulate hypertrophy, but factors as diverse as hypoxia, co-culture, epigenetics and biomaterial composition can also potently affect Runx2 expression. Control of hypertrophic differentiation can be exploited both for cartilage repair, where a stable phenotype is desired, but also in bone regeneration, where hypertrophic cartilage could act as a template for endochondral bone formation. We hope this review will motivate the design of novel engineered microenvironments for skeletal regeneration applications.

  6. 1α,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells.

    Science.gov (United States)

    Ma, Yingyu; Hu, Qiang; Luo, Wei; Pratt, Rachel N; Glenn, Sean T; Liu, Song; Trump, Donald L; Johnson, Candace S

    2015-04-01

    Bladder cancer is the fourth most commonly diagnosed cancer in men and eighth leading cause of cancer-related death in the US. Epidemiological and experimental studies strongly suggest a role for 1α,25(OH)2D3 in cancer prevention and treatment. The antitumor activities of 1α,25(OH)2D3 are mediated by the induction of cell cycle arrest, apoptosis, differentiation and the inhibition of angiogenesis and metastasis. miRNAs play important regulatory roles in cancer development and progression. However, the role of 1α,25(OH)2D3 in the regulation of miRNA expression and the potential impact in bladder cancer has not been investigated. Therefore, we studied 1α,25(OH)2D3-regulated miRNA expression profiles in human bladder cancer cell line 253J and the highly tumorigenic and metastatic derivative line 253J-BV by miRNA qPCR panels. 253J and 253J-BV cells express endogenous vitamin D receptor (VDR), which can be further induced by 1α,25(OH)2D3. VDR target gene 24-hydroxylase was induced by 1α,25(OH)2D3 in both cell lines, indicating functional 1α,25(OH)2D3 signaling. The miRNA qPCR panel assay results showed that 253J and 253J-BV cells have distinct miRNA expression profiles. Further, 1α,25(OH)2D3 differentially regulated miRNA expression profiles in 253J and 253J-BV cells in a dynamic manner. Pathway analysis of the miRNA target genes revealed distinct patterns of contribution to the molecular functions and biological processes in the two cell lines. In conclusion, 1α,25(OH)2D3 differentially regulates the expression of miRNAs, which may contribute to distinct biological functions, in human bladder 253J and 253J-BV cells. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Low-intensity pulsed ultrasound regulates proliferation and differentiation of osteoblasts through osteocytes

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lei, E-mail: geraldleelei@163.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Yang, Zheng [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Zhang, Hai [Department of Restorative Dentistry, School of Dentistry, University of Washington, Seattle, WA (United States); Chen, Wenchuan [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China); Chen, Mengshi [Department of Biomechanics, Sichuan University, Chengdu (China); Zhu, Zhimin, E-mail: hxzhimin@163.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu (China)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer CM from LIPUS-stimulated osteocytes inhibits proliferation of osteoblasts. Black-Right-Pointing-Pointer CM from LIPUS-stimulated osteocytes enhances differentiation of osteoblasts. Black-Right-Pointing-Pointer LIPUS stimulates MLO-Y4 cells to secrete PGE{sub 2} and NO. -- Abstract: Low-intensity pulsed ultrasound (LIPUS) has been used as a safe and effective modality to enhance fracture healing. As the most abundant cells in bone, osteocytes orchestrate biological activities of effector cells via direct cell-to-cell contacts and by soluble factors. In this study, we have used the osteocytic MLO-Y4 cells to study the effects of conditioned medium from LIPUS-stimulated MLO-Y4 cells on proliferation and differentiation of osteoblastic MC3T3-E1 cells. Conditioned media from LIPUS-stimulated MLO-Y4 cells (LIPUS-Osteocyte-CM) were collected and added on MC3T3-E1 cell cultures. MC3T3-E1 cells cultured in LIPUS-Osteocyte-CM demonstrated a significant inhibition of proliferation and an increased alkaline phosphatase activity. The results of PGE{sub 2} and NO assay showed that LIPUS could enhance PGE{sub 2} and NO secretion from MLO-Y4 cells at all time points within 24 h after LIPUS stimulation. We conclude that LIPUS regulates proliferation and differentiation of osteoblasts through osteocytes in vitro. Increased secretion of PGE{sub 2} from osteocytes may play a role in this effect.

  8. Slit/Robo1 signaling regulates neural tube development by balancing neuroepithelial cell proliferation and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Guang; Li, Yan; Wang, Xiao-yu [Key Laboratory for Regenerative Medicine of The Ministry of Education, Department of Histology and Embryology, School of Medicine, Jinan University, Guangzhou 510632 (China); Han, Zhe [Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510224 (China); Chuai, Manli [College of Life Sciences Biocentre, University of Dundee, Dundee DD1 5EH (United Kingdom); Wang, Li-jing [Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510224 (China); Ho Lee, Kenneth Ka [Stem Cell and Regeneration Thematic Research Programme, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin (Hong Kong); Geng, Jian-guo, E-mail: jgeng@umich.edu [Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510224 (China); Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109 (United States); Yang, Xuesong, E-mail: yang_xuesong@126.com [Key Laboratory for Regenerative Medicine of The Ministry of Education, Department of Histology and Embryology, School of Medicine, Jinan University, Guangzhou 510632 (China)

    2013-05-01

    development by tightly coordinating cell proliferation and differentiation during neurulation. - Highlights: ► The role of Slit/Robo1 signaling was investigated with chick and mouse models. ► Disturbance of Slit/Robo1 signaling resulted in neural tube defects. ► Slit/Robo1 signaling regulated the proliferation of neural tube cells. ► Slit/Robo1 signaling modulated the differentiation of neural tube cells. ► Slit/Robo1 signaling balanced the proliferation and differentiation of neural tube.

  9. Financial Incentives Differentially Regulate Neural Processing of Positive and Negative Emotions during Value-Based Decision-Making

    Directory of Open Access Journals (Sweden)

    Anne M. Farrell

    2018-02-01

    Full Text Available Emotional and economic incentives often conflict in decision environments. To make economically desirable decisions then, deliberative neural processes must be engaged to regulate automatic emotional reactions. In this functional magnetic resonance imaging (fMRI study, we evaluated how fixed wage (FW incentives and performance-based (PB financial incentives, in which pay is proportional to outcome, differentially regulate positive and negative emotional reactions to hypothetical colleagues that conflicted with the economics of available alternatives. Neural activity from FW to PB incentive contexts decreased for positive emotional stimuli but increased for negative stimuli in middle temporal, insula, and medial prefrontal regions. In addition, PB incentives further induced greater responses to negative than positive emotional decisions in the frontal and anterior cingulate regions involved in emotion regulation. Greater response to positive than negative emotional features in these regions also correlated with lower frequencies of economically desirable choices. Our findings suggest that whereas positive emotion regulation involves a reduction of responses in valence representation regions, negative emotion regulation additionally engages brain regions for deliberative processing and signaling of incongruous events.

  10. Financial Incentives Differentially Regulate Neural Processing of Positive and Negative Emotions during Value-Based Decision-Making.

    Science.gov (United States)

    Farrell, Anne M; Goh, Joshua O S; White, Brian J

    2018-01-01

    Emotional and economic incentives often conflict in decision environments. To make economically desirable decisions then, deliberative neural processes must be engaged to regulate automatic emotional reactions. In this functional magnetic resonance imaging (fMRI) study, we evaluated how fixed wage (FW) incentives and performance-based (PB) financial incentives, in which pay is proportional to outcome, differentially regulate positive and negative emotional reactions to hypothetical colleagues that conflicted with the economics of available alternatives. Neural activity from FW to PB incentive contexts decreased for positive emotional stimuli but increased for negative stimuli in middle temporal, insula, and medial prefrontal regions. In addition, PB incentives further induced greater responses to negative than positive emotional decisions in the frontal and anterior cingulate regions involved in emotion regulation. Greater response to positive than negative emotional features in these regions also correlated with lower frequencies of economically desirable choices. Our findings suggest that whereas positive emotion regulation involves a reduction of responses in valence representation regions, negative emotion regulation additionally engages brain regions for deliberative processing and signaling of incongruous events.

  11. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Qiang [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhao, Zhi-Ning [Clinical Laboratory, 451 Hospital of Chinese PLA, Xi' an 710054 (China); Cheng, Jing-Tao [Department of Special Dentistry, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhang, Bin [Department of Orthodontics, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Xu, Jie [Department of Periodontology, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Huang, Fei; Zhao, Rui-Ni [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Chen, Yong-Jin, E-mail: cyj1229@fmmu.edu.cn [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China)

    2011-01-07

    Research highlights: {yields} Ibandronate significantly promote the proliferation of PDLSC cells. {yields} Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. {yields} The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. {yields} Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. {yields} Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation

  12. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    International Nuclear Information System (INIS)

    Zhou, Qiang; Zhao, Zhi-Ning; Cheng, Jing-Tao; Zhang, Bin; Xu, Jie; Huang, Fei; Zhao, Rui-Ni; Chen, Yong-Jin

    2011-01-01

    Research highlights: → Ibandronate significantly promote the proliferation of PDLSC cells. → Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. → The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. → Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. → Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation-related genes via miRNAs. The exact

  13. miR-146a modulates autoreactive Th17 cell differentiation and regulates organ-specific autoimmunity.

    Science.gov (United States)

    Li, Bo; Wang, Xi; Choi, In Young; Wang, Yu-Chen; Liu, Siyuan; Pham, Alexander T; Moon, Heesung; Smith, Drake J; Rao, Dinesh S; Boldin, Mark P; Yang, Lili

    2017-10-02

    Autoreactive CD4 T cells that differentiate into pathogenic Th17 cells can trigger autoimmune diseases. Therefore, investigating the regulatory network that modulates Th17 differentiation may yield important therapeutic insights. miR-146a has emerged as a critical modulator of immune reactions, but its role in regulating autoreactive Th17 cells and organ-specific autoimmunity remains largely unknown. Here, we have reported that miR-146a-deficient mice developed more severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS). We bred miR-146a-deficient mice with 2D2 T cell receptor-Tg mice to generate 2D2 CD4 T cells that are deficient in miR-146a and specific for myelin oligodendrocyte glycoprotein (MOG), an autoantigen in the EAE model. miR-146a-deficient 2D2 T cells induced more severe EAE and were more prone to differentiate into Th17 cells. Microarray analysis revealed enhancements in IL-6- and IL-21-induced Th17 differentiation pathways in these T cells. Further study showed that miR-146a inhibited the production of autocrine IL-6 and IL-21 in 2D2 T cells, which in turn reduced their Th17 differentiation. Thus, our study identifies miR-146a as an important molecular brake that blocks the autocrine IL-6- and IL-21-induced Th17 differentiation pathways in autoreactive CD4 T cells, highlighting its potential as a therapeutic target for treating autoimmune diseases.

  14. MxiN Differentially Regulates Monomeric and Oligomeric Species of the Shigella Type Three Secretion System ATPase Spa47.

    Science.gov (United States)

    Case, Heather B; Dickenson, Nicholas E

    2018-04-17

    Shigella rely entirely on the action of a single type three secretion system (T3SS) to support cellular invasion of colonic epithelial cells and to circumvent host immune responses. The ATPase Spa47 resides at the base of the Shigella needle-like type three secretion apparatus (T3SA), supporting protein secretion through the apparatus and providing a likely means for native virulence regulation by Shigella and a much needed target for non-antibiotic therapeutics to treat Shigella infections. Here, we show that MxiN is a differential regulator of Spa47 and that its regulatory impact is determined by the oligomeric state of the Spa47 ATPase, with which it interacts. In vitro and in vivo characterization shows that interaction of MxiN with Spa47 requires the six N-terminal residues of Spa47 that are also necessary for stable Spa47 oligomer formation and activation. This interaction with MxiN negatively influences the activity of Spa47 oligomers while upregulating the ATPase activity of monomeric Spa47. Detailed kinetic analyses of monomeric and oligomeric Spa47 in the presence and absence of MxiN uncover additional mechanistic insights into the regulation of Spa47 by MxiN, suggesting that the MxiN/Spa47 species resulting from interaction with monomeric and oligomeric Spa47 are functionally distinct and that both could be involved in Shigella T3SS regulation. Uncovering regulation of Spa47 by MxiN addresses an important gap in the current understanding of how Shigella controls T3SA activity and provides the first description of differential T3SS ATPase regulation by a native T3SS protein.

  15. The Signaling Pathways Involved in Chondrocyte Differentiation and Hypertrophic Differentiation

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    Jianmei Li

    2016-01-01

    Full Text Available Chondrocytes communicate with each other mainly via diffusible signals rather than direct cell-to-cell contact. The chondrogenic differentiation of mesenchymal stem cells (MSCs is well regulated by the interactions of varieties of growth factors, cytokines, and signaling molecules. A number of critical signaling molecules have been identified to regulate the differentiation of chondrocyte from mesenchymal progenitor cells to their terminal maturation of hypertrophic chondrocytes, including bone morphogenetic proteins (BMPs, SRY-related high-mobility group-box gene 9 (Sox9, parathyroid hormone-related peptide (PTHrP, Indian hedgehog (Ihh, fibroblast growth factor receptor 3 (FGFR3, and β-catenin. Except for these molecules, other factors such as adenosine, O2 tension, and reactive oxygen species (ROS also have a vital role in cartilage formation and chondrocyte maturation. Here, we outlined the complex transcriptional network and the function of key factors in this network that determine and regulate the genetic program of chondrogenesis and chondrocyte differentiation.

  16. FOXO1-suppressed miR-424 regulates the proliferation and osteogenic differentiation of MSCs by targeting FGF2 under oxidative stress

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    Li, Liangping; Qi, Qihua; Luo, Jiaquan; Huang, Sheng; Ling, Zemin; Gao, Manman; Zhou, Zhiyu; Stiehler, Maik; Zou, Xuenong

    2017-02-01

    Recently, microRNAs (miRNAs) have been identified as key regulators of the proliferation and differentiation of mesenchymal stem cells (MSCs). Our previous in vivo study and other in vitro studies using miRNA microarrays suggest that miR-424 is involved in the regulation of bone formation. However, the role and mechanism of miR-424 in bone formation still remain unknown. Here, we identified that the downregulation of miR-424 mediates bone formation under oxidative stress, and we explored its underlying mechanism. Our results showed that miR-424 was significantly downregulated in an anterior lumbar interbody fusion model of pigs and in a cell model of oxidative stress induced by H2O2. The overexpression of miR-424 inhibited proliferation and osteogenic differentiation shown by a decrease in alkaline phosphatase (ALP) activity, mineralization and osteogenic markers, including RUNX2 and ALP, whereas the knockdown of miR-424 led to the opposite results. Moreover, miR-424 exerts its effects by targeting FGF2. Furthermore, we found that FOXO1 suppressed miR-424 expression and bound to its promoter region. FOXO1 enhanced proliferation and osteogenic differentiation in part through the miR-424/FGF2 pathway. These results indicated that FOXO1-suppressed miR-424 regulates both the proliferation and osteogenic differentiation of MSCs via targeting FGF2, suggesting that miR-424 might be a potential novel therapeutic strategy for promoting bone formation.

  17. Bacterial Competition Reveals Differential Regulation of the pks Genes by Bacillus subtilis

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    Vargas-Bautista, Carol; Rahlwes, Kathryn

    2014-01-01

    Bacillus subtilis is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The pks genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of pks genes appear to be organized as a giant operon (>74 kb from pksC-pksR). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305–310, 2007, doi:10.1073/pnas.0609073103), a deletion of the pks operon in B. subtilis was found to induce prodiginine production by Streptomyces coelicolor. Here, colonies of wild-type B. subtilis formed a spreading population that induced prodiginine production from Streptomyces lividans, suggesting differential regulation of pks genes and, as a result, bacillaene. While the parent colony showed widespread induction of pks expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the pks genes. To identify regulators that control pks genes, we first determined the pattern of pks gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of pks gene expression. We found that expression of the pks genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of degU, comA, and scoC had moderate effects, disrupting the timing and level of pks gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for B. subtilis. PMID:24187085

  18. Gonad Transcriptome Analysis of the Pacific Oyster Crassostrea gigas Identifies Potential Genes Regulating the Sex Determination and Differentiation Process.

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    Yue, Chenyang; Li, Qi; Yu, Hong

    2018-04-01

    The Pacific oyster Crassostrea gigas is a commercially important bivalve in aquaculture worldwide. C. gigas has a fascinating sexual reproduction system consisting of dioecism, sex change, and occasional hermaphroditism, while knowledge of the molecular mechanisms of sex determination and differentiation is still limited. In this study, the transcriptomes of male and female gonads at different gametogenesis stages were characterized by RNA-seq. Hierarchical clustering based on genes differentially expressed revealed that 1269 genes were expressed specifically in female gonads and 817 genes were expressed increasingly over the course of spermatogenesis. Besides, we identified two and one gene modules related to female and male gonad development, respectively, using weighted gene correlation network analysis (WGCNA). Interestingly, GO and KEGG enrichment analysis showed that neurotransmitter-related terms were significantly enriched in genes related to ovary development, suggesting that the neurotransmitters were likely to regulate female sex differentiation. In addition, two hub genes related to testis development, lncRNA LOC105321313 and Cg-Sh3kbp1, and one hub gene related to ovary development, Cg-Malrd1-like, were firstly investigated. This study points out the role of neurotransmitter and non-coding RNA regulation during gonad development and produces lists of novel relevant candidate genes for further studies. All of these provided valuable information to understand the molecular mechanisms of C. gigas sex determination and differentiation.

  19. Regulation of epithelial differentiation in rat intestine by intraluminal delivery of an adenoviral vector or silencing RNA coding for Schlafen 3.

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    Pavlo L Kovalenko

    Full Text Available Although we stimulate enterocytic proliferation to ameliorate short gut syndrome or mucosal atrophy, less effort has been directed at enterocytic differentiation. Schlafen 3 (Slfn3 is a poorly understood protein induced during IEC-6 enterocytic differentiation. We hypothesized that exogenous manipulation of Slfn3 would regulate enterocytic differentiation in vivo. Adenoviral vector coding for Slfn3 cDNA (Ad-GFP-Slfn3 or silencing RNA for Slfn3 (siSlfn3 was introduced intraluminally into rat intestine. We assessed Slfn3, villin, sucrase-isomaltase (SI, Dpp4, and Glut2 by qRT-PCR, Western blot, and immunohistochemistry. We also studied Slfn3 and these differentiation markers in atrophic defunctionalized jejunal mucosa and the crypt-villus axis of normal jejunum. Ad-GFP-Slfn3 but not Ad-GFP increased Slfn3, villin and Dpp4 expression in human Caco-2 intestinal epithelial cells. Injecting Ad-GFP-Slfn3 into rat jejunum in vivo increased mucosal Slfn3 mRNA three days later vs. intraluminal Ad-GFP. This Slfn3 overexpression was associated with increases in all four differentiation markers. Injecting siSlfn3 into rat jejunum in vivo substantially reduced Slfn3 and all four intestinal mucosal differentiation markers three days later, as well as Dpp4 specific activity. Endogenous Slfn3 was reduced in atrophic mucosa from a blind-end Roux-en-Y anastomosis in parallel with differentiation marker expression together with AKT and p38 signaling. Slfn3 was more highly expressed in the villi than the crypts, paralleling Glut2, SI and Dpp4. Slfn3 is a key intracellular regulator of rat enterocytic differentiation. Understanding how Slfn3 works may identify targets to promote enterocytic differentiation and maintain mucosal function in vivo, facilitating enteral nutrition and improving survival in patients with mucosal atrophy or short gut syndrome.

  20. VEGF-C and TGF-β reciprocally regulate mesenchymal stem cell commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes.

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    Igarashi, Yasuyuki; Chosa, Naoyuki; Sawada, Shunsuke; Kondo, Hisatomo; Yaegashi, Takashi; Ishisaki, Akira

    2016-04-01

    The direction of mesenchymal stem cell (MSC) differentiation is regulated by stimulation with various growth factors and cytokines. We recently established MSC lines, [transforming growth factor-β (TGF-β)-responsive SG‑2 cells, bone morphogenetic protein (BMP)-responsive SG‑3 cells, and TGF-β/BMP-non-responsive SG‑5 cells], derived from the bone marrow of green fluorescent protein-transgenic mice. In this study, to compare gene expression profiles in these MSC lines, we used DNA microarray analysis to characterize the specific gene expression profiles observed in the TGF-β-responsive SG‑2 cells. Among the genes that were highly expressed in the SG‑2 cells, we focused on vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3), the gene product of FMS-like tyrosine kinase 4 (Flt4). We found that VEGF-C, a specific ligand of VEGFR3, significantly induced the cell proliferative activity, migratory ability (as shown by Transwell migration assay), as well as the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the SG‑2 cells. Additionally, VEGF-C significantly increased the expression of prospero homeobox 1 (Prox1) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), which are lymphatic endothelial cell markers, and decreased the expression of osteogenic differentiation marker genes in these cells. By contrast, TGF-β significantly increased the expression of early-phase osteogenic differentiation marker genes in the SG‑2 cells and markedly decreased the expression of lymphatic endothelial cell markers. The findings of our study strongly suggest the following: i) that VEGF-C promotes the proliferative activity and migratory ability of MSCs; and ii) VEGF-C and TGF-β reciprocally regulate MSC commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes, respectively. Our findings provide new insight into the molecular mechanisms underlying the regenerative ability of MSCs.

  1. Differential regulation of BACE1 expression by oxidative and nitrosative signals

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    Xu Huaxi

    2011-03-01

    Full Text Available Abstract Background It is well established that both cerebral hypoperfusion/stroke and type 2 diabetes are risk factors for Alzheimer's disease (AD. Recently, the molecular link between ischemia/hypoxia and amyloid precursor protein (APP processing has begun to be established. However, the role of the key common denominator, namely nitric oxide (NO, in AD is largely unknown. In this study, we investigated redox regulation of BACE1, the rate-limiting enzyme responsible for the β-cleavage of APP to Aβ peptides. Results Herein, we studied events such as S-nitrosylation, a covalent modification of cysteine residues by NO, and H2O2-mediated oxidation. We found that NO and H2O2 differentially modulate BACE1 expression and enzymatic activity: NO at low concentrations (2O2 (1-10 μM induces BACE1 expression via transcriptional activation, resulting in increased enzymatic activity. The differential effects of NO and H2O2 on BACE1 expression and activity are also reflected in their opposing effects on Aβ generation in cultured neurons in a dose-dependent manner. Furthermore, we found that BACE1 is highly S-nitrosylated in normal aging brains while S-nitrosylation is markedly reduced in AD brains. Conclusion This study demonstrates for the first time that BACE1 is highly modified by NO via multiple mechanisms: low and high levels of NO suppress BACE1 via transcriptional and post translational regulation, in contrast with the upregulation of BACE1 by H2O2-mediated oxidation. These novel NO-mediated regulatory mechanisms likely protect BACE1 from being further oxidized by excessive oxidative stress, as from H2O2 and peroxynitrite which are known to upregulate BACE1 and activate the enzyme, resulting in excessive cleavage of APP and Aβ generation; they likely represent the crucial house-keeping mechanism for BACE1 expression/activation under physiological conditions.

  2. Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway.

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    Hatori, Yuta; Yan, Ye; Schmidt, Katharina; Furukawa, Eri; Hasan, Nesrin M; Yang, Nan; Liu, Chin-Nung; Sockanathan, Shanthini; Lutsenko, Svetlana

    2016-02-16

    Brain development requires a fine-tuned copper homoeostasis. Copper deficiency or excess results in severe neuro-pathologies. We demonstrate that upon neuronal differentiation, cellular demand for copper increases, especially within the secretory pathway. Copper flow to this compartment is facilitated through transcriptional and metabolic regulation. Quantitative real-time imaging revealed a gradual change in the oxidation state of cytosolic glutathione upon neuronal differentiation. Transition from a broad range of redox states to a uniformly reducing cytosol facilitates reduction of the copper chaperone Atox1, liberating its metal-binding site. Concomitantly, expression of Atox1 and its partner, a copper transporter ATP7A, is upregulated. These events produce a higher flux of copper through the secretory pathway that balances copper in the cytosol and increases supply of the cofactor to copper-dependent enzymes, expression of which is elevated in differentiated neurons. Direct link between glutathione oxidation and copper compartmentalization allows for rapid metabolic adjustments essential for normal neuronal function.

  3. miR-96 regulates the progression of differentiation in mammalian cochlear inner and outer hair cells.

    Science.gov (United States)

    Kuhn, Stephanie; Johnson, Stuart L; Furness, David N; Chen, Jing; Ingham, Neil; Hilton, Jennifer M; Steffes, Georg; Lewis, Morag A; Zampini, Valeria; Hackney, Carole M; Masetto, Sergio; Holley, Matthew C; Steel, Karen P; Marcotti, Walter

    2011-02-08

    MicroRNAs (miRNAs) are small noncoding RNAs able to regulate a broad range of protein-coding genes involved in many biological processes. miR-96 is a sensory organ-specific miRNA expressed in the mammalian cochlea during development. Mutations in miR-96 cause nonsyndromic progressive hearing loss in humans and mice. The mouse mutant diminuendo has a single base change in the seed region of the Mir96 gene leading to widespread changes in the expression of many genes. We have used this mutant to explore the role of miR-96 in the maturation of the auditory organ. We found that the physiological development of mutant sensory hair cells is arrested at around the day of birth, before their biophysical differentiation into inner and outer hair cells. Moreover, maturation of the hair cell stereocilia bundle and remodelling of auditory nerve connections within the cochlea fail to occur in miR-96 mutants. We conclude that miR-96 regulates the progression of the physiological and morphological differentiation of cochlear hair cells and, as such, coordinates one of the most distinctive functional refinements of the mammalian auditory system.

  4. MiRNA-199a-3p Regulates C2C12 Myoblast Differentiation through IGF-1/AKT/mTOR Signal Pathway

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    Long Jia

    2013-12-01

    Full Text Available MicroRNAs constitute a class of ~22-nucleotide non-coding RNAs. They modulate gene expression by associating with the 3' untranslated regions (3' UTRs of messenger RNAs (mRNAs. Although multiple miRNAs are known to be regulated during myoblast differentiation, their individual roles in muscle development are still not fully understood. In this study, we showed that miR-199a-3p was highly expressed in skeletal muscle and was induced during C2C12 myoblasts differentiation. We also identified and confirmed several genes of the IGF-1/AKT/mTOR signal pathway, including IGF-1, mTOR, and RPS6KA6, as important cellular targets of miR-199a-3p in myoblasts. Overexpression of miR-199a-3p partially blocked C2C12 myoblast differentiation and the activation of AKT/mTOR signal pathway, while interference of miR-199a-3p by antisense oligonucleotides promoted C2C12 differentiation and myotube hypertrophy. Thus, our studies have established miR-199a-3p as a potential regulator of myogenesis through the suppression of IGF-1/AKT/mTOR signal pathway.

  5. Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation.

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    Hao, Wu; Tashiro, Syoichi; Hasegawa, Tomoka; Sato, Yuiko; Kobayashi, Tami; Tando, Toshimi; Katsuyama, Eri; Fujie, Atsuhiro; Watanabe, Ryuichi; Morita, Mayu; Miyamoto, Kana; Morioka, Hideo; Nakamura, Masaya; Matsumoto, Morio; Amizuka, Norio; Toyama, Yoshiaki; Miyamoto, Takeshi

    2015-07-10

    Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that de-differentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D3, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheral nerve de-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D3 administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. MicroRNA-4739 regulates osteogenic and adipocytic differentiation of immortalized human bone marrow stromal cells via targeting LRP3

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    Mona Elsafadi

    2017-04-01

    Full Text Available Understanding the regulatory networks underlying lineage differentiation and fate determination of human bone marrow stromal cells (hBMSC is a prerequisite for their therapeutic use. The goal of the current study was to unravel the novel role of the low-density lipoprotein receptor-related protein 3 (LRP3 in regulating the osteogenic and adipogenic differentiation of immortalized hBMSCs. Gene expression profiling revealed significantly higher LRP3 levels in the highly osteogenic hBMSC clone imCL1 than in the less osteogenic clone imCL2, as well as a significant upregulation of LRP3 during the osteogenic induction of the imCL1 clone. Data from functional and gene expression assays demonstrated the role of LRP3 as a molecular switch promoting hBMSC lineage differentiation into osteoblasts and inhibiting differentiation into adipocytes. Interestingly, microRNA (miRNA expression profiling identified miR-4739 as the most under-represented miRNA (−36.11 fold in imCL1 compared to imCL2. The TargetScan prediction algorithm, combined with functional and biochemical assays, identified LRP3 mRNA as a novel target of miR-4739, with a single potential binding site for miR-4739 located in the LRP3 3′ UTR. Regulation of LRP3 expression by miR-4739 was subsequently confirmed by qRT-PCR, western blotting, and luciferase assays. Over-expression of miR-4739 mimicked the effects of LRP3 knockdown on promoting adipogenic and suppressing osteogenic differentiation of hBMSCs. Hence, we report for the first time a novel biological role for the LRP3/hsa-miR-4739 axis in balancing osteogenic and adipocytic differentiation of hBMSCs. Our data support the potential utilization of miRNA-based therapies in regenerative medicine.

  7. Itga2b regulation at the onset of definitive hematopoiesis and commitment to differentiation.

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    Stephanie Dumon

    Full Text Available Product of the Itga2b gene, CD41 contributes to hematopoietic stem cell (HSC and megakaryocyte/platelet functions. CD41 expression marks the onset of definitive hematopoiesis in the embryo where it participates in regulating the numbers of multipotential progenitors. Key to platelet aggregation, CD41 expression also characterises their precursor, the megakaryocyte, and is specifically up regulated during megakaryopoiesis. Though phenotypically unique, megakaryocytes and HSC share numerous features, including key transcription factors, which could indicate common sub-regulatory networks. In these respects, Itga2b can serve as a paradigm to study features of both developmental-stage and HSC- versus megakaryocyte-specific regulations. By comparing different cellular contexts, we highlight a mechanism by which internal promoters participate in Itga2b regulation. A developmental process connects epigenetic regulation and promoter switching leading to CD41 expression in HSC. Interestingly, a similar process can be observed at the Mpl locus, which codes for another receptor that defines both HSC and megakaryocyte identities. Our study shows that Itga2b expression is controlled by lineage-specific networks and associates with H4K8ac in megakaryocyte or H3K27me3 in the multipotential hematopoietic cell line HPC7. Correlating with the decrease in H3K27me3 at the Itga2b Iocus, we find that following commitment to megakaryocyte differentiation, the H3K27 demethylase Jmjd3 up-regulation influences both Itga2b and Mpl expression.

  8. Modeling endocrine regulation of the menstrual cycle using delay differential equations.

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    Harris, Leona A; Selgrade, James F

    2014-11-01

    This article reviews an effective mathematical procedure for modeling hormonal regulation of the menstrual cycle of adult women. The procedure captures the effects of hormones secreted by several glands over multiple time scales. The specific model described here consists of 13 nonlinear, delay, differential equations with 44 parameters and correctly predicts blood levels of ovarian and pituitary hormones found in the biological literature for normally cycling women. In addition to this normal cycle, the model exhibits another stable cycle which may describe a biologically feasible "abnormal" condition such as polycystic ovarian syndrome. Model simulations illustrate how one cycle can be perturbed to the other cycle. Perturbations due to the exogenous administration of each ovarian hormone are examined. This model may be used to test the effects of hormone therapies on abnormally cycling women as well as the effects of exogenous compounds on normally cycling women. Sensitive parameters are identified and bifurcations in model behavior with respect to parameter changes are discussed. Modeling various aspects of menstrual cycle regulation should be helpful in predicting successful hormone therapies, in studying the phenomenon of cycle synchronization and in understanding many factors affecting the aging of the female reproductive endocrine system. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Macrophage activation and differentiation signals regulate schlafen-4 gene expression: evidence for Schlafen-4 as a modulator of myelopoiesis.

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    Wendy J van Zuylen

    Full Text Available BACKGROUND: The ten mouse and six human members of the Schlafen (Slfn gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity. METHODOLOGY/PRINCIPAL FINDINGS: Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM by the Toll-like Receptor (TLR4 agonist lipopolysaccharide (LPS, the TLR3 agonist Poly(I∶C, and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1⁻/⁻ BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens. CONCLUSIONS: Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the

  10. NFAT5 regulates the canonical Wnt pathway and is required for cardiomyogenic differentiation

    International Nuclear Information System (INIS)

    Adachi, Atsuo; Takahashi, Tomosaburo; Ogata, Takehiro; Imoto-Tsubakimoto, Hiroko; Nakanishi, Naohiko; Ueyama, Tomomi; Matsubara, Hiroaki

    2012-01-01

    Highlights: ► NFAT5 protein expression is downregulated during cardiomyogenesis. ► Inhibition of NFAT5 function suppresses canonical Wnt signaling. ► Inhibition of NFAT5 function attenuates mesodermal induction. ► NFAT5 function is required for cardiomyogenesis. -- Abstract: While nuclear factor of activated T cells 5 (NFAT5), a transcription factor implicated in osmotic stress response, is suggested to be involved in other processes such as migration and proliferation, its role in cardiomyogenesis is largely unknown. Here, we examined the role of NFAT5 in cardiac differentiation of P19CL6 cells, and observed that it was abundantly expressed in undifferentiated P19CL6 cells, and its protein expression was significantly downregulated by enhanced proteasomal degradation during DMSO-induced cardiomyogenesis. Expression of a dominant negative mutant of NFAT5 markedly attenuated cardiomyogenesis, which was associated with the inhibition of mesodermal differentiation. TOPflash reporter assay revealed that the transcriptional activity of canonical Wnt signaling was activated prior to mesodermal differentiation, and this activation was markedly attenuated by NFAT5 inhibition. Pharmacological activation of canonical Wnt signaling by [2′Z, 3′E]-6-bromoindirubin-3′-oxime (BIO) restored Brachyury expression in NFAT5DN-expressing cells. Inhibition of NFAT5 markedly attenuated Wnt3 and Wnt3a induction. Expression of Dkk1 and Cerberus1, which are secreted Wnt antagonists, was also inhibited by NFAT5 inhibition. Thus, endogenous NFAT5 regulates the coordinated expression of Wnt ligands and antagonists, which are essential for cardiomyogenesis through the canonical Wnt pathway. These results demonstrated a novel role of NFAT5 in cardiac differentiation of stem cells.

  11. Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

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    Ocampo, J.; McCormack, B.; Navarro, E.; Moreno, S.; Garre, V.

    2012-01-01

    The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. PMID:22635921

  12. Differential regulation of histamine- and bradykinin-stimulated phospholipase C in adrenal chromaffin cells: evidence for involvement of different protein kinase C isoforms.

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    Sena, C M; Rosário, L M; Parker, P J; Patel, V; Boarder, M R

    1996-03-01

    In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC-alpha, epsilon, and zeta were identified. To characterize down-regulation of these enzymes, cells were incubated for 6-48 h with 1 microM phorbol myristate acetate (PMA). PKC-epsilon down-regulated more rapidly than PKC-alpha. At 12 h, PMA pretreatment, for example, PKC-epsilon was maximally down-regulated (23 +/- 4% of controls), whereas PKC-alpha was unchanged. PKC-alpha showed partial down-regulation by 24 h of PMA pretreatment. PKC-zeta did not down-regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC-epsilon than for PKC-alpha. The accumulation of total 3H-inositol (poly) phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10-min PMA treatment (1 microM). However, with 12-h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC-epsilon, whereas the restoration of the histamine response corresponds to the loss of PKC-alpha. This picture was confirmed with further studies on cytosolic Ca2+. The results show that chromaffin cells exhibit an unusual pattern of down-regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC-linked receptors in chromafin

  13. Keratin 5/14‑mediated cell differentiation and transformation are regulated by TAp63 and Notch‑1 in oral squamous cell carcinoma‑derived cells.

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    Srivastava, Saumya S; Alam, Hunain; Patil, Sonam J; Shrinivasan, Rashmi; Raikundalia, Sweta; Chaudhari, Pratik Rajeev; Vaidya, Milind M

    2018-05-01

    Keratins 5/14 (K5/14) are intermediate filament proteins expressed in the basal layer of stratified epithelial cells and are known targets of p63. Previous research in our laboratory showed that upon K5/14 downregulation in oral squamous cell carcinoma (OSCC)‑derived cells, there was an increase in intracellular Notch‑1 levels and differentiation markers such as involucrin, keratin 1 and a decrease in tumorigenic potential in vivo. However, the molecules involved in the K14 regulated cell differentiation and transformation are not known to date. In order to understand the possible role of TAp63, we downregulated TAp63 in a K14‑knockdown background. We observed that there was a decrease in the expression of Notch‑1. Expression levels of differentiation markers such as involucrin, K1, loricrin and filaggrin were also decreased. Furthermore, TAp63 downregulation led to an increase in invasion, migration and in vivo tumorigenic potential of these cells. We observed a decrease in β‑catenin signaling in K14‑downregulated cells. Notably, when TAp63 was downregulated in K14‑knockdown cells, there was increase in non‑phospho β‑catenin levels. Hence, this study indicates that TAp63 plays an important role in K14‑downregulated cells possibly by regulating the Notch‑1 expression. K14 regulates the expression of TAp63 which in turn regulates expression of Notch‑1. The present study is a step forward in our quest to understand the functional significance of molecules that regulate the process of differentiation and tumorigenesis in stratified epithelial cells.

  14. Fibroblast growth factor 2 inhibits up-regulation of bone morphogenic proteins and their receptors during osteoblastic differentiation of human mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Biver, Emmanuel, E-mail: ebiver@yahoo.fr [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France); Department of Rheumatology, Lille University Hospital, Roger Salengro Hospital, 59037 Lille cedex (France); Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14 (Switzerland); Soubrier, Anne-Sophie [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France); Department of Rheumatology, Lille University Hospital, Roger Salengro Hospital, 59037 Lille cedex (France); Thouverey, Cyril [Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14 (Switzerland); Cortet, Bernard [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France); Department of Rheumatology, Lille University Hospital, Roger Salengro Hospital, 59037 Lille cedex (France); Broux, Odile [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France); Caverzasio, Joseph [Service of Bone Diseases, Department of Internal Medicine Specialties, University Hospital of Geneva, CH-1211 Geneva 14 (Switzerland); Hardouin, Pierre [Physiopathology of Inflammatory Bone Diseases, EA 4490, University Lille North of France, Quai Masset, Bassin Napoleon, BP120, 62327 Boulogne sur Mer (France)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer FGF modulates BMPs pathway in HMSCs by down-regulating BMP/BMPR expression. Black-Right-Pointing-Pointer This effect is mediated by ERK and JNK MAPKs pathways. Black-Right-Pointing-Pointer Crosstalk between FGF and BMPs must be taken into account in skeletal bioengineering. Black-Right-Pointing-Pointer It must also be considered in the use of recombinant BMPs in orthopedic and spine surgeries. -- Abstract: Understanding the interactions between growth factors and bone morphogenic proteins (BMPs) signaling remains a crucial issue to optimize the use of human mesenchymal stem cells (HMSCs) and BMPs in therapeutic perspectives and bone tissue engineering. BMPs are potent inducers of osteoblastic differentiation. They exert their actions via BMP receptors (BMPR), including BMPR1A, BMPR1B and BMPR2. Fibroblast growth factor 2 (FGF2) is expressed by cells of the osteoblastic lineage, increases their proliferation and is secreted during the healing process of fractures or in surgery bone sites. We hypothesized that FGF2 might influence HMSC osteoblastic differentiation by modulating expressions of BMPs and their receptors. BMP2, BMP4, BMPR1A and mainly BMPR1B expressions were up-regulated during this differentiation. FGF2 inhibited HMSCs osteoblastic differentiation and the up-regulation of BMPs and BMPR. This effect was prevented by inhibiting the ERK or JNK mitogen-activated protein kinases which are known to be activated by FGF2. These data provide a mechanism explaining the inhibitory effect of FGF2 on osteoblastic differentiation of HMSCs. These crosstalks between growth and osteogenic factors should be considered in the use of recombinant BMPs in therapeutic purpose of fracture repair or skeletal bioengineering.

  15. Tiam1 Regulates the Wnt/Dvl/Rac1 Signaling Pathway and the Differentiation of Midbrain Dopaminergic Neurons

    Science.gov (United States)

    Čajánek, Lukáš; Ganji, Ranjani Sri; Henriques-Oliveira, Catarina; Theofilopoulos, Spyridon; Koník, Peter

    2013-01-01

    Understanding the mechanisms that drive the differentiation of dopaminergic (DA) neurons is crucial for successful development of novel therapies for Parkinson's disease, in which DA neurons progressively degenerate. However, the mechanisms underlying the differentiation-promoting effects of Wnt5a on DA precursors are poorly understood. Here, we present the molecular and functional characterization of a signaling pathway downstream of Wnt5a, the Wnt/Dvl/Rac1 pathway. First, we characterize the interaction between Rac1 and Dvl and identify the N-terminal part of Dvl3 as necessary for Rac1 binding. Next, we show that Tiam1, a Rac1 guanosine exchange factor (GEF), is expressed in the ventral midbrain, interacts with Dvl, facilitates Dvl-Rac1 interaction, and is required for Dvl- or Wnt5a-induced activation of Rac1. Moreover, we show that Wnt5a promotes whereas casein kinase 1 (CK1), a negative regulator of the Wnt/Dvl/Rac1 pathway, abolishes the interactions between Dvl and Tiam1. Finally, using ventral midbrain neurosphere cultures, we demonstrate that the generation of DA neurons in culture is impaired after Tiam1 knockdown, indicating that Tiam1 is required for midbrain DA differentiation. In summary, our data identify Tiam1 as a novel regulator of DA neuron development and as a Dvl-associated and Rac1-specific GEF acting in the Wnt/Dvl/Rac1 pathway. PMID:23109420

  16. Extremely Low-Frequency Electromagnetic Fields Promote In Vitro Neuronal Differentiation and Neurite Outgrowth of Embryonic Neural Stem Cells via Up-Regulating TRPC1

    Science.gov (United States)

    Ma, Qinlong; Chen, Chunhai; Deng, Ping; Zhu, Gang; Lin, Min; Zhang, Lei; Xu, Shangcheng; He, Mindi; Lu, Yonghui; Duan, Weixia; Pi, Huifeng; Cao, Zhengwang; Pei, Liping; Li, Min; Liu, Chuan; Zhang, Yanwen; Zhong, Min; Zhou, Zhou; Yu, Zhengping

    2016-01-01

    Exposure to extremely low-frequency electromagnetic fields (ELF-EMFs) can enhance hippocampal neurogenesis in adult mice. However, little is focused on the effects of ELF-EMFs on embryonic neurogenesis. Here, we studied the potential effects of ELF-EMFs on embryonic neural stem cells (eNSCs). We exposed eNSCs to ELF-EMF (50 Hz, 1 mT) for 1, 2, and 3 days with 4 hours per day. We found that eNSC proliferation and maintenance were significantly enhanced after ELF-EMF exposure in proliferation medium. ELF-EMF exposure increased the ratio of differentiated neurons and promoted the neurite outgrowth of eNSC-derived neurons without influencing astrocyes differentiation and the cell apoptosis. In addition, the expression of the proneural genes, NeuroD and Ngn1, which are crucial for neuronal differentiation and neurite outgrowth, was increased after ELF-EMF exposure. Moreover, the expression of transient receptor potential canonical 1 (TRPC1) was significantly up-regulated accompanied by increased the peak amplitude of intracellular calcium level induced by ELF-EMF. Furthermore, silencing TRPC1 expression eliminated the up-regulation of the proneural genes and the promotion of neuronal differentiation and neurite outgrowth induced by ELF-EMF. These results suggest that ELF-EMF exposure promotes the neuronal differentiation and neurite outgrowth of eNSCs via up-regulation the expression of TRPC1 and proneural genes (NeuroD and Ngn1). These findings also provide new insights in understanding the effects of ELF-EMF exposure on embryonic brain development. PMID:26950212

  17. Regulation of human mesenchymal stem cells differentiation into chondrocytes in extracellular matrix-based hydrogel scaffolds.

    Science.gov (United States)

    Du, Mingchun; Liang, Hui; Mou, Chenchen; Li, Xiaoran; Sun, Jie; Zhuang, Yan; Xiao, Zhifeng; Chen, Bing; Dai, Jianwu

    2014-02-01

    To induce human mesenchymal stem cells (hMSCs) to differentiate into chondrocytes in three-dimensional (3D) microenvironments, we developed porous hydrogel scaffolds using the cartilage extracellular matrix (ECM) components of chondroitin sulfate (CS) and collagen (COL). The turbidity and viscosity experiments indicated hydrogel could form through pH-triggered co-precipitation when pH=2-3. Enzyme-linked immunosorbent assay (ELISA) confirmed the hydrogel scaffolds could controllably release growth factors as envisaged. Transforming growth factor-β (TGF-β) was released to stimulate hMSCs differentiation into chondrocytes; and then collagen binding domain-basic fibroblast growth factor (CBD-bFGF) was released to improve the differentiation and preserve the chondrocyte phenotype. In in vitro cell culture experiments, the differentiation processes were compared in different microenvironments: 2D culture in culture plate as control, 3D culture in the fabricated scaffolds without growth factors (CC), the samples with CBD-bFGF (CC-C), the samples with TGF-β (CC-T), the samples with CBD-bFGF/TGF-β (CC-CT). Real-time polymerase chain reaction (RT-PCR) revealed the hMSC marker genes of CD44 and CD105 decreased; at the same time the chondrocyte marker genes of collagen type II and aggrecan increased, especially in the CC-CT sample. Immunostaining results further confirmed the hMSC marker protein of CD 44 disappeared and the chondrocyte marker protein of collagen type II emerged over time in the CC-CT sample. These results imply the ECM-based hydrogel scaffolds with growth factors can supply suitable 3D cell niches for hMSCs differentiation into chondrocytes and the differentiation process can be regulated by the controllably released growth factors. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. IL-15 expression on RA synovial fibroblasts promotes B cell survival.

    Directory of Open Access Journals (Sweden)

    Marta Benito-Miguel

    Full Text Available INTRODUCTION: The purpose of this study was to examine the role of RA Synovial Fibroblast (RASFib IL-15 expression on B cell survival. METHODS: Magnetically sorted peripheral blood memory B cells from 15 healthy subjects were cocultured with RASFib. RESULTS: RASFib constitutively expressed membrane IL-15. Survival of isolated B cells cultured for 6 days, below 5%, was extended in coculture with RASFib to 52+/-8% (p<0.001. IL-15 neutralizing agents but not isotype controls, reduced this rate to 31+/-6% (p<0.05. Interestingly, rhIL-15 had no effect on isolated B cells but significantly increased their survival in coculture with RASFib. In parallel, B cell IL-15R chains were upregulated in cocultures. BAFF and VCAM-1, that are expressed on RASFib, were tested as potential candidates involved in upregulating B cell IL-15R. Culture of B cells in the presence of rhBAFF or rhVCAM-1 resulted in significantly increased survival, together with upregulation of all three IL-15R chains; in parallel, rhIL-15 potentiated the anti-apoptotic effect of BAFF and VCAM-1. Both BAFF and VCAM-1 neutralizing agents downmodulated the effect of RASFib on B cell survival and IL-15R expression. In parallel, rhIL-15 had a lower effect on the survival of B cells cocultured with RASFib in the presence of BAFF or VCAM-1 neutralizing agents. Peripheral blood B cells from 15 early RA patients demonstrated an upregulated IL-15R and increased survival in cocultures. CONCLUSION: IL-15 expression on RASFib significantly contributes to the anti-apoptotic effect of RASFib on B cells. IL-15 action is facilitated by BAFF and VCAM-1 expressed on RASFib, through an upregulation of IL-15R chains.

  19. [PRODUCT OF THE BMI1--A KEY COMPONENT OF POLYCOMB--POSITIVELY REGULATES ADIPOCYTE DIFFERENTIATION OF MOUSE MESENCHYMAL STEM CELLS].

    Science.gov (United States)

    Petrov, N S; Vereschagina, N A; Sushilova, E N; Kropotov, A V; Miheeva, N F; Popov, B V

    2016-01-01

    Bmil is a key component of Polycomb (PcG), which in mammals controls the basic functions of mammalian somatic stem cells (SSC) such as self-renewal and differentiation. Bmi1 supports SSC via transcriptional suppression of genes associated with cell cycle and differentiation. The most studied target genes of Bmi1 are the genes of Ink4 locus, CdkI p16(Ink4a) and p1(Arf), suppression of which due to activating mutations of the BMI1 results in formation of cancer stem cells (CSC) and carcinomas in various tissues. In contrast, inactivation of BMI1 results in cell cycle arrest and cell senescence. Although clinical phenomena of hypo- and hyperactivation of BMI1 are well known, its targets and mechanisms of regulation of tissue specific SSC are still obscure. The goal of this study was to evaluate the regulatory role of BMI1 in adipocyte differentiation (AD) of mouse mesenchymal stem cells (MSC). Induction of AD in mouse MSC of the C3H10T1/2 cell line was associated with an increase in the expression levels of BMI1, the genes of pRb family (RB, p130) and demethylase UTX, but not methyltransferase EZH2, whose products regulate the methylation levels of H3K27. It was observed earlier that H3K27me3 may play the role of the epigenetic switch by promoting AD of human MSC via activating expression of the PPARγ2, the master gene of AD (Hemming et al., 2014). Here we show that inactivation of BMI1 using specific siRNA slows and decreases the levels of AD, but does not abolish it. This is associated with a complete inhibition of the expression of adipogenic marker genes--PPARγ2, ADIPOQ and a decrease in the expression of RB, p130, but not UTX. The results obtained give evidence that the epigenetic mechanism regulating AD differentiation in mouse and human MSC is different.

  20. Proteomic analysis of osteogenic differentiation of dental follicle precursor cells

    DEFF Research Database (Denmark)

    Morsczeck, Christian; Petersen, Jørgen; Völlner, Florian

    2009-01-01

    of differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B....... The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic...... proteins, plastin 3 T-isoform, beta-actin, superoxide dismutases, and transgelin were found to be highly up-regulated, whereas cofilin-1, pro-alpha 1 collagen, destrin, prolyl 4-hydrolase and dihydrolipoamide dehydrogenase were found to be highly down-regulated. The group of up-regulated proteins...

  1. Mechanical loading regulates human MSC differentiation in a multi-layer hydrogel for osteochondral tissue engineering.

    Science.gov (United States)

    Steinmetz, Neven J; Aisenbrey, Elizabeth A; Westbrook, Kristofer K; Qi, H Jerry; Bryant, Stephanie J

    2015-07-01

    A bioinspired multi-layer hydrogel was developed for the encapsulation of human mesenchymal stem cells (hMSCs) as a platform for osteochondral tissue engineering. The spatial presentation of biochemical cues, via incorporation of extracellular matrix analogs, and mechanical cues, via both hydrogel crosslink density and externally applied mechanical loads, were characterized in each layer. A simple sequential photopolymerization method was employed to form stable poly(ethylene glycol)-based hydrogels with a soft cartilage-like layer of chondroitin sulfate and low RGD concentrations, a stiff bone-like layer with high RGD concentrations, and an intermediate interfacial layer. Under a compressive load, the variation in hydrogel stiffness within each layer produced high strains in the soft cartilage-like layer, low strains in the stiff bone-like layer, and moderate strains in the interfacial layer. When hMSC-laden hydrogels were cultured statically in osteochondral differentiation media, the local biochemical and matrix stiffness cues were not sufficient to spatially guide hMSC differentiation after 21 days. However dynamic mechanical stimulation led to differentially high expression of collagens with collagen II in the cartilage-like layer, collagen X in the interfacial layer and collagen I in the bone-like layer and mineral deposits localized to the bone layer. Overall, these findings point to external mechanical stimulation as a potent regulator of hMSC differentiation toward osteochondral cellular phenotypes. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  2. Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-β Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation

    Directory of Open Access Journals (Sweden)

    Nagarajan Selvamurugan

    2017-01-01

    Full Text Available Pulsed electromagnetic fields (PEMFs have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs’ cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF-β signaling pathway and microRNA 21 (miR21 were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF-β signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-β signaling pathway, was found to be miR21-5p’s putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF-β signaling pathway and stimulation of expression of miR21-5p in hBMSCs.

  3. Time-resolved transcriptome and proteome landscape of human regulatory T cell (Treg) differentiation reveals novel regulators of FOXP3

    KAUST Repository

    Schmidt, Angelika

    2018-04-27

    BackgroundRegulatory T cells (Tregs) expressing the transcription factor FOXP3 are crucial mediators of self-tolerance, preventing autoimmune diseases but possibly hampering tumor rejection. Clinical manipulation of Tregs is of great interest, and first-in-man trials of Treg transfer have achieved promising outcomes. Yet, the mechanisms governing induced Treg (iTreg) differentiation and the regulation of FOXP3 are incompletely understood.ResultsTo gain a comprehensive and unbiased molecular understanding of FOXP3 induction, we performed time-series RNA sequencing (RNA-Seq) and proteomics profiling on the same samples during human iTreg differentiation. To enable the broad analysis of universal FOXP3-inducing pathways, we used five differentiation protocols in parallel. Integrative analysis of the transcriptome and proteome confirmed involvement of specific molecular processes, as well as overlap of a novel iTreg subnetwork with known Treg regulators and autoimmunity-associated genes. Importantly, we propose 37 novel molecules putatively involved in iTreg differentiation. Their relevance was validated by a targeted shRNA screen confirming a functional role in FOXP3 induction, discriminant analyses classifying iTregs accordingly, and comparable expression in an independent novel iTreg RNA-Seq dataset.ConclusionThe data generated by this novel approach facilitates understanding of the molecular mechanisms underlying iTreg generation as well as of the concomitant changes in the transcriptome and proteome. Our results provide a reference map exploitable for future discovery of markers and drug candidates governing control of Tregs, which has important implications for the treatment of cancer, autoimmune, and inflammatory diseases.

  4. Arginine Methylation Regulates MEIS2 Nuclear Localization to Promote Neuronal Differentiation of Adult SVZ Progenitors

    Directory of Open Access Journals (Sweden)

    Jasmine Kolb

    2018-04-01

    Full Text Available Summary: Adult neurogenesis is regulated by stem cell niche-derived extrinsic factors and cell-intrinsic regulators, yet the mechanisms by which niche signals impinge on the activity of intrinsic neurogenic transcription factors remain poorly defined. Here, we report that MEIS2, an essential regulator of adult SVZ neurogenesis, is subject to posttranslational regulation in the SVZ olfactory bulb neurogenic system. Nuclear accumulation of MEIS2 in adult SVZ-derived progenitor cells follows downregulation of EGFR signaling and is modulated by methylation of MEIS2 on a conserved arginine, which lies in close proximity to nested binding sites for the nuclear export receptor CRM1 and the MEIS dimerization partner PBX1. Methylation impairs interaction with CRM1 without affecting PBX1 dimerization and thereby allows MEIS2 nuclear accumulation, a prerequisite for neuronal differentiation. Our results describe a form of posttranscriptional modulation of adult SVZ neurogenesis whereby an extrinsic signal fine-tunes neurogenesis through posttranslational modification of a transcriptional regulator of cell fate. : A hallmark of adult neurogenesis is its strong dependence on physiological stimuli and environmental signals. Schulte and colleagues show that the nuclear localization and activity of a transcriptional regulator of adult neurogenesis is controlled by posttranslational modification. Their results link intrinsic control over neuron production to external signals and help to explain how adult neurogenesis can occur “on demand.” Keywords: subventricular zone, stem cell niche, posttranslational modification, controlled nuclear import, TALE-homdomain protein, MEIS2, PBX1, CRM1, neurogenesis, stem cell niche

  5. Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

    DEFF Research Database (Denmark)

    Farajzadeh, Leila; Hornshøj, Henrik; Momeni, Jamal

    2013-01-01

    , isoform, and transcription start site (TSS), and promoter level showed that several of the genes differed at all four levels. Interestingly, these genes were mainly annotated to the "electron transport chain" and neuronal differentiation, emphasizing that "tissue important" genes are regulated at several...

  6. Regulation of Silk Genes by Hox and Homeodomain Proteins in the Terminal Differentiated Silk Gland of the Silkworm Bombyx mori

    Science.gov (United States)

    Takiya, Shigeharu; Tsubota, Takuya; Kimoto, Mai

    2016-01-01

    The silk gland of the silkworm Bombyx mori is a long tubular organ that is divided into several subparts along its anteroposterior (AP) axis. As a trait of terminal differentiation of the silk gland, several silk protein genes are expressed with unique regional specificities. Most of the Hox and some of the homeobox genes are also expressed in the differentiated silk gland with regional specificities. The expression patterns of Hox genes in the silk gland roughly correspond to those in embryogenesis showing “colinearity”. The central Hox class protein Antennapedia (Antp) directly regulates the expression of several middle silk gland–specific silk genes, whereas the Lin-1/Isl-1/Mec3 (LIM)-homeodomain transcriptional factor Arrowhead (Awh) regulates the expression of posterior silk gland–specific genes for silk fiber proteins. We summarize our results and discuss the usefulness of the silk gland of Bombyx mori for analyzing the function of Hox genes. Further analyses of the regulatory mechanisms underlying the region-specific expression of silk genes will provide novel insights into the molecular bases for target-gene selection and regulation by Hox and homeodomain proteins. PMID:29615585

  7. Regulation of Silk Genes by Hox and Homeodomain Proteins in the Terminal Differentiated Silk Gland of the Silkworm Bombyx mori

    Directory of Open Access Journals (Sweden)

    Shigeharu Takiya

    2016-05-01

    Full Text Available The silk gland of the silkworm Bombyx mori is a long tubular organ that is divided into several subparts along its anteroposterior (AP axis. As a trait of terminal differentiation of the silk gland, several silk protein genes are expressed with unique regional specificities. Most of the Hox and some of the homeobox genes are also expressed in the differentiated silk gland with regional specificities. The expression patterns of Hox genes in the silk gland roughly correspond to those in embryogenesis showing “colinearity”. The central Hox class protein Antennapedia (Antp directly regulates the expression of several middle silk gland–specific silk genes, whereas the Lin-1/Isl-1/Mec3 (LIM-homeodomain transcriptional factor Arrowhead (Awh regulates the expression of posterior silk gland–specific genes for silk fiber proteins. We summarize our results and discuss the usefulness of the silk gland of Bombyx mori for analyzing the function of Hox genes. Further analyses of the regulatory mechanisms underlying the region-specific expression of silk genes will provide novel insights into the molecular bases for target-gene selection and regulation by Hox and homeodomain proteins.

  8. Dasatinib accelerates valproic acid-induced acute myeloid leukemia cell death by regulation of differentiation capacity.

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    Sook-Kyoung Heo

    Full Text Available Dasatinib is a compound developed for chronic myeloid leukemia as a multi-targeted kinase inhibitor against wild-type BCR-ABL and SRC family kinases. Valproic acid (VPA is an anti-epileptic drug that also acts as a class I histone deacetylase inhibitor. The aim of this research was to determine the anti-leukemic effects of dasatinib and VPA in combination and to identify their mechanism of action in acute myeloid leukemia (AML cells. Dasatinib was found to exert potent synergistic inhibitory effects on VPA-treated AML cells in association with G1 phase cell cycle arrest and apoptosis induction involving the cleavage of poly (ADP-ribose polymerase and caspase-3, -7 and -9. Dasatinib/VPA-induced cell death thus occurred via caspase-dependent apoptosis. Moreover, MEK/ERK and p38 MAPK inhibitors efficiently inhibited dasatinib/VPA-induced apoptosis. The combined effect of dasatinib and VPA on the differentiation capacity of AML cells was more powerful than the effect of each drug alone, being sufficiently strong to promote AML cell death through G1 cell cycle arrest and caspase-dependent apoptosis. MEK/ERK and p38 MAPK were found to control dasatinib/VPA-induced apoptosis as upstream regulators, and co-treatment with dasatinib and VPA to contribute to AML cell death through the regulation of differentiation capacity. Taken together, these results indicate that combined dasatinib and VPA treatment has a potential role in anti-leukemic therapy.

  9. Decreased Intracellular pH Induced by Cariporide Differentially Contributes to Human Umbilical Cord-Derived Mesenchymal Stem Cells Differentiation

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    Wei Gao

    2014-01-01

    Full Text Available Background/Aims: Na+/H+ exchanger 1 (NHE1 is an important regulator of intracellular pH (pHi. High pHi is required for cell proliferation and differentiation. Our previous study has proven that the pHi of mesenchymal stem cells is higher than that of normal differentiated cells and similar to tumor cells. NHE1 is highly expressed in both mesenchymal stem cells and tumor cells. Targeted inhibition of NHE1 could induce differentiation of K562 leukemia cells. In the present paper we explored whether inhibition of NHE1 could induce differentiation of mesenchymal stem cells. Methods: MSCs were obtained from human umbilical cord and both the surface phenotype and functional characteristics were analyzed. Selective NHE1 inhibitor cariporide was used to treat human umbilical cord-derived mesenchymal stem cells (hUC-MSCs. The pHi and the differentiation of hUC-MSCs were compared upon cariporide treatment. The putative signaling pathway involved was also explored. Results: The pHi of hUC-MSCs was decreased upon cariporide treatment. Cariporide up-regulated the osteogenic differentiation of hUC-MSCs while the adipogenic differentiation was not affected. For osteogenic differentiation, β-catenin expression was up-regulated upon cariporide treatment. Conclusion: Decreased pHi induced by cariporide differentially contributes to hUC-MSCs differentiation.

  10. Cholesterol negatively regulates IL-9-producing CD8+ T cell differentiation and antitumor activity.

    Science.gov (United States)

    Ma, Xingzhe; Bi, Enguang; Huang, Chunjian; Lu, Yong; Xue, Gang; Guo, Xing; Wang, Aibo; Yang, Maojie; Qian, Jianfei; Dong, Chen; Yi, Qing

    2018-05-09

    CD8 + T cells can be polarized into IL-9-secreting (Tc9) cells. We previously showed that adoptive therapy using tumor-specific Tc9 cells generated stronger antitumor responses in mouse melanoma than classical Tc1 cells. To understand why Tc9 cells exert stronger antitumor responses, we used gene profiling to compare Tc9 and Tc1 cells. Tc9 cells expressed different levels of cholesterol synthesis and efflux genes and possessed significantly lower cholesterol content than Tc1 cells. Unique to Tc9, but not other CD8 + or CD4 + T cell subsets, manipulating cholesterol content in polarizing Tc9 cells significantly affected IL-9 expression and Tc9 differentiation and antitumor response in vivo. Mechanistic studies showed that IL-9 was indispensable for Tc9 cell persistence and antitumor effects, and cholesterol or its derivatives inhibited IL-9 expression by activating liver X receptors (LXRs), leading to LXR Sumoylation and reduced p65 binding to Il9 promoter. Our study identifies cholesterol as a critical regulator of Tc9 cell differentiation and function. © 2018 Ma et al.

  11. LIF inhibits osteoblast differentiation at least in part by regulation of HAS2 and its product hyaluronan.

    Science.gov (United States)

    Falconi, Dominic; Aubin, Jane E

    2007-08-01

    LIF arrests osteogenesis in fetal rat calvaria cells in a differentiation stage-specific manner. Differential display identified HAS2 as a LIF-induced gene and its product, HA, modulated osteoblast differentiation similarly to LIF. Our data suggest that LIF arrests osteoblast differentiation by altering HA content of the extracellular matrix. Leukemia inhibitory factor (LIF) elicits both anabolic and catabolic effects on bone. We previously showed in the fetal rat calvaria (RC) cell system that LIF inhibits osteoblast differentiation at the late osteoprogenitor/early osteoblast stage. To uncover potential molecular mediators of this inhibitory activity, we used a positive-negative genome-wide differential display screen to identify LIF-induced changes in the developing osteoblast transcriptome. Although LIF signaling is active throughout the RC cell proliferation-differentiation sequence, only a relatively small number of genes, in several different functional clusters, are modulated by LIF specifically during the LIF-sensitive inhibitory time window. Based on their known and predicted functions, most of the LIF-regulated genes identified are plausible candidates to be involved in the LIF-induced arrest of osteoprogenitor differentiation. To test this hypothesis, we further analyzed the function of one of the genes identified, hyaluronan synthase 2 (HAS2), in the LIF-induced inhibition. Synthesis of hyaluronan (HA), the product of HAS enzymatic activity, was stimulated by LIF and mimicked the HAS2 expression profile, with highest expression in early/proliferative and late/maturing cultures and lowest levels in intermediate/late osteoprogenitor-early osteoblast cultures. Exogenously added high molecular weight HA, the product of HAS2, dose-dependently inhibited osteoblast differentiation, with pulse-treatment effective in the same differentiation stage-specific inhibitory window as seen with LIF. In addition, however, pulse treatment with HA in early cultures

  12. Serum cholinesterases are differentially regulated in normal and dystrophin-deficient mutant mice

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    Andrea R. Durrant

    2012-06-01

    Full Text Available The cholinesterases, acetylcholinesterase and butyrylcholinesterase (pseudocholinesterase, are abundant in the nervous system and in other tissues. The role of acetylcholinesterase in terminating transmitter action in the peripheral and central nervous system is well understood. However, both knowledge of the function(s of the cholinesterases in serum, and of their metabolic and endocrine regulation under normal and pathological conditions, is limited. This study investigates acetylcholinesterase and butyrylcholinesterase in sera of dystrophin-deficient mdx mutant mice, an animal model for the human Duchenne muscular dystrophy and in control healthy mice. The data show systematic and differential variations in the concentrations of both enzymes in the sera, and specific changes dictated by alteration of hormonal balance in both healthy and dystrophic mice. While acetylcholinesterase in mdx-sera is elevated, butyrylcholinesterase is markedly diminished, resulting in an overall cholinesterase decrease compared to sera of healthy controls. The androgen testosterone (T is a negative modulator of butyrylcholinesterase, but not of acetylcholinesterase, in male mouse sera. T-removal elevated both butyrylcholinesterase activity and the butyrylcholinesterase/acetylcholinesterase ratio in mdx male sera to values resembling those in healthy control male mice. Mechanisms of regulation of the circulating cholinesterases and their impairment in the dystrophic mice are suggested, and clinical implications for diagnosis and treatment are considered.

  13. Paralogous SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes differentially regulate leaf initiation and reproductive phase change in petunia.

    Science.gov (United States)

    Preston, Jill C; Jorgensen, Stacy A; Orozco, Rebecca; Hileman, Lena C

    2016-02-01

    Duplicated petunia clade-VI SPL genes differentially promote the timing of inflorescence and flower development, and leaf initiation rate. The timing of plant reproduction relative to favorable environmental conditions is a critical component of plant fitness, and is often associated with variation in plant architecture and habit. Recent studies have shown that overexpression of the microRNA miR156 in distantly related annual species results in plants with perennial characteristics, including late flowering, weak apical dominance, and abundant leaf production. These phenotypes are largely mediated through the negative regulation of a subset of genes belonging to the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family of transcription factors. In order to determine how and to what extent paralogous SPL genes have partitioned their roles in plant growth and development, we functionally characterized petunia clade-VI SPL genes under different environmental conditions. Our results demonstrate that PhSBP1and PhSBP2 differentially promote discrete stages of the reproductive transition, and that PhSBP1, and possibly PhCNR, accelerates leaf initiation rate. In contrast to the closest homologs in annual Arabidopsis thaliana and Mimulus guttatus, PhSBP1 and PhSBP2 transcription is not mediated by the gibberellic acid pathway, but is positively correlated with photoperiod and developmental age. The developmental functions of clade-VI SPL genes have, thus, evolved following both gene duplication and speciation within the core eudicots, likely through differential regulation and incomplete sub-functionalization.

  14. Neuron-NG2 Cell Synapses: Novel Functions for Regulating NG2 Cell Proliferation and Differentiation

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    Qian-Kun Yang

    2013-01-01

    Full Text Available NG2 cells are a population of CNS cells that are distinct from neurons, mature oligodendrocytes, astrocytes, and microglia. These cells can be identified by their NG2 proteoglycan expression. NG2 cells have a highly branched morphology, with abundant processes radiating from the cell body, and express a complex set of voltage-gated channels, AMPA/kainate, and GABA receptors. Neurons notably form classical and nonclassical synapses with NG2 cells, which have varied characteristics and functions. Neuron-NG2 cell synapses could fine-tune NG2 cell activities, including the NG2 cell cycle, differentiation, migration, and myelination, and may be a novel potential therapeutic target for NG2 cell-related diseases, such as hypoxia-ischemia injury and periventricular leukomalacia. Furthermore, neuron-NG2 cell synapses may be correlated with the plasticity of CNS in adulthood with the synaptic contacts passing onto their progenies during proliferation, and synaptic contacts decrease rapidly upon NG2 cell differentiation. In this review, we highlight the characteristics of classical and nonclassical neuron-NG2 cell synapses, the potential functions, and the fate of synaptic contacts during proliferation and differentiation, with the emphasis on the regulation of the NG2 cell cycle by neuron-NG2 cell synapses and their potential underlying mechanisms.

  15. Adaptor protein SH2-B linking receptor-tyrosine kinase and Akt promotes adipocyte differentiation by regulating peroxisome proliferator-activated receptor gamma messenger ribonucleic acid levels.

    Science.gov (United States)

    Yoshiga, Daigo; Sato, Naoichi; Torisu, Takehiro; Mori, Hiroyuki; Yoshida, Ryoko; Nakamura, Seiji; Takaesu, Giichi; Kobayashi, Takashi; Yoshimura, Akihiko

    2007-05-01

    Adipocyte differentiation is regulated by insulin and IGF-I, which transmit signals by activating their receptor tyrosine kinase. SH2-B is an adaptor protein containing pleckstrin homology and Src homology 2 (SH2) domains that have been implicated in insulin and IGF-I receptor signaling. In this study, we found a strong link between SH2-B levels and adipogenesis. The fat mass and expression of adipogenic genes including peroxisome proliferator-activated receptor gamma (PPARgamma) were reduced in white adipose tissue of SH2-B-/- mice. Reduced adipocyte differentiation of SH2-B-deficient mouse embryonic fibroblasts (MEFs) was observed in response to insulin and dexamethasone, whereas retroviral SH2-B overexpression enhanced differentiation of 3T3-L1 preadipocytes to adipocytes. SH2-B overexpression enhanced mRNA level of PPARgamma in 3T3-L1 cells, whereas PPARgamma levels were reduced in SH2-B-deficient MEFs in response to insulin. SH2-B-mediated up-regulation of PPARgamma mRNA was blocked by a phosphatidylinositol 3-kinase inhibitor, but not by a MAPK kinase inhibitor. Insulin-induced Akt activation and the phosphorylation of forkhead transcription factor (FKHR/Foxo1), a negative regulator of PPARgamma transcription, were up-regulated by SH2-B overexpression, but reduced in SH2-B-deficient MEFs. These data indicate that SH2-B is a key regulator of adipogenesis both in vivo and in vitro by regulating the insulin/IGF-I receptor-Akt-Foxo1-PPARgamma pathway.

  16. KCNK10, a Tandem Pore Domain Potassium Channel, Is a Regulator of Mitotic Clonal Expansion during the Early Stage of Adipocyte Differentiation

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    Makoto Nishizuka

    2014-12-01

    Full Text Available KCNK10, a member of tandem pore domain potassium channel family, gives rise to leak K+ currents. It plays important roles in stabilizing the negative resting membrane potential and in counterbalancing depolarization. We previously demonstrated that kcnk10 expression is quickly elevated during the early stage of adipogenesis of 3T3-L1 cells and that reduction of kcnk10 expression inhibits adipocyte differentiation. However, the molecular mechanism of KCNK10 in adipocyte differentiation remains unclear. Here we revealed that kcnk10 is induced by 3-isobutyl-1-methylxanthine, a cyclic nucleotide phosphodiesterase inhibitor and a potent inducer of adipogenesis, during the early stage of adipocyte differentiation. We also demonstrated that KCNK10 functions as a positive regulator of mitotic clonal expansion (MCE, a necessary process for terminal differentiation. The reduction of kcnk10 expression repressed the expression levels of CCAAT/enhancer-binding protein β (C/EBPβ and C/EBPδ as well as the phosphorylation level of Akt during the early phase of adipogenesis. In addition, knockdown of kcnk10 expression suppressed insulin-induced Akt phosphorylation. These results indicate that KCNK10 contributes to the regulation of MCE through the control of C/EBPβ and C/EBPδ expression and insulin signaling.

  17. Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Kotnik Halavaty, Katarina; Regan, Jennifer; Mehta, Kavi; Laimins, Laimonis, E-mail: l-laimins@northwestern.edu

    2014-03-15

    Human papillomaviruses (HPV) infect stratified epithelia and link their life cycles to epithelial differentiation. The HPV E5 protein plays a role in the productive phase of the HPV life cycle but its mechanism of action is still unclear. We identify a new binding partner of E5, A4, using a membrane-associated yeast-two hybrid system. The A4 protein co-localizes with HPV 31 E5 in perinuclear regions and forms complexes with E5 and Bap31. In normal keratinocytes, A4 is found primarily in basal cells while in HPV positive cells high levels of A4 are seen in both undifferentiated and differentiated cells. Reduction of A4 expression by shRNAs, enhanced HPV genome amplification and increased cell proliferation ability following differentiation but this was not seen in cells lacking E5. Our studies suggest that the A4 protein is an important E5 binding partner that plays a role in regulating cell proliferation ability upon differentiation. - Highlights: • A4 associates with HPV 31 E5 proteins. • A4 is localized to endoplasmic reticulum. • HPV proteins induce A4 expression in suprabasal layers of stratified epithelium. • E5 is important for proliferation ability of differentiating HPV positive cells.

  18. Delayed Mesoderm and Erythroid Differentiation of Murine Embryonic Stem Cells in the Absence of the Transcriptional Regulator FUBP1

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    Josephine Wesely

    2017-01-01

    Full Text Available The transcriptional regulator far upstream binding protein 1 (FUBP1 is essential for fetal and adult hematopoietic stem cell (HSC self-renewal, and the constitutive absence of FUBP1 activity during early development leads to embryonic lethality in homozygous mutant mice. To investigate the role of FUBP1 in murine embryonic stem cells (ESCs and in particular during differentiation into hematopoietic lineages, we generated Fubp1 knockout (KO ESC clones using CRISPR/Cas9 technology. Although FUBP1 is expressed in undifferentiated ESCs and during spontaneous differentiation following aggregation into embryoid bodies (EBs, absence of FUBP1 did not affect ESC maintenance. Interestingly, we observed a delayed differentiation of FUBP1-deficient ESCs into the mesoderm germ layer, as indicated by impaired expression of several mesoderm markers including Brachyury at an early time point of ESC differentiation upon aggregation to EBs. Coculture experiments with OP9 cells in the presence of erythropoietin revealed a diminished differentiation capacity of Fubp1 KO ESCs into the erythroid lineage. Our data showed that FUBP1 is important for the onset of mesoderm differentiation and maturation of hematopoietic progenitor cells into the erythroid lineage, a finding that is supported by the phenotype of FUBP1-deficient mice.

  19. MicroRNA-4739 regulates osteogenic and adipocytic differentiation of immortalized human bone marrow stromal cells via targeting LRP3

    DEFF Research Database (Denmark)

    Elsafadi, Mona; Manikandan, Muthurangan; Alajez, Nehad M

    2017-01-01

    3 (LRP3) in regulating the osteogenic and adipogenic differentiation of immortalized hBMSCs. Gene expression profiling revealed significantly higher LRP3 levels in the highly osteogenic hBMSC clone imCL1 than in the less osteogenic clone imCL2, as well as a significant upregulation of LRP3 during...

  20. CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes.

    Science.gov (United States)

    Singh, Randeep K; Dagnino, Lina

    2017-01-17

    The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation. Analysis of the structural determinants that govern these processes revealed a central role for S403 and T433. In particular, substitution of these two amino acid residues with non-phosphorylatable alanine (E2F1 ST/A) interferes with E2F1 nuclear export, K11- and K48-linked polyubiquitylation and degradation in differentiated keratinocytes. In contrast, replacement of S403 and T433 with phosphomimetic aspartic acid to generate a pseudophosphorylated E2F1 mutant protein (E2F1 ST/D) generates a protein that is regulated in a manner indistinguishable from that of wild type E2F1. Cdh1 is an activating cofactor that interacts with the anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, promoting proteasomal degradation of various substrates. We found that Cdh1 associates with E2F1 in keratinocytes. Inhibition or RNAi-mediated silencing of Cdh1 prevents E2F1 degradation in response to differentiation signals. Our results reveal novel regulatory mechanisms that jointly modulate post-translational modifications and downregulation of E2F1, which are necessary for proper epidermal keratinocyte differentiation.

  1. A single enhancer regulating the differential expression of duplicated red-sensitive opsin genes in zebrafish.

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    Taro Tsujimura

    2010-12-01

    Full Text Available A fundamental step in the evolution of the visual system is the gene duplication of visual opsins and differentiation between the duplicates in absorption spectra and expression pattern in the retina. However, our understanding of the mechanism of expression differentiation is far behind that of spectral tuning of opsins. Zebrafish (Danio rerio have two red-sensitive cone opsin genes, LWS-1 and LWS-2. These genes are arrayed in a tail-to-head manner, in this order, and are both expressed in the long member of double cones (LDCs in the retina. Expression of the longer-wave sensitive LWS-1 occurs later in development and is thus confined to the peripheral, especially ventral-nasal region of the adult retina, whereas expression of LWS-2 occurs earlier and is confined to the central region of the adult retina, shifted slightly to the dorsal-temporal region. In this study, we employed a transgenic reporter assay using fluorescent proteins and P1-artificial chromosome (PAC clones encompassing the two genes and identified a 0.6-kb "LWS-activating region" (LAR upstream of LWS-1, which regulates expression of both genes. Under the 2.6-kb flanking upstream region containing the LAR, the expression pattern of LWS-1 was recapitulated by the fluorescent reporter. On the other hand, when LAR was directly conjugated to the LWS-2 upstream region, the reporter was expressed in the LDCs but also across the entire outer nuclear layer. Deletion of LAR from the PAC clones drastically lowered the reporter expression of the two genes. These results suggest that LAR regulates both LWS-1 and LWS-2 by enhancing their expression and that interaction of LAR with the promoters is competitive between the two genes in a developmentally restricted manner. Sharing a regulatory region between duplicated genes could be a general way to facilitate the expression differentiation in duplicated visual opsins.

  2. Laser Capture and Deep Sequencing Reveals the Transcriptomic Programmes Regulating the Onset of Pancreas and Liver Differentiation in Human Embryos

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    Rachel E. Jennings

    2017-11-01

    Full Text Available To interrogate the alternative fates of pancreas and liver in the earliest stages of human organogenesis, we developed laser capture, RNA amplification, and computational analysis of deep sequencing. Pancreas-enriched gene expression was less conserved between human and mouse than for liver. The dorsal pancreatic bud was enriched for components of Notch, Wnt, BMP, and FGF signaling, almost all genes known to cause pancreatic agenesis or hypoplasia, and over 30 unexplored transcription factors. SOX9 and RORA were imputed as key regulators in pancreas compared with EP300, HNF4A, and FOXA family members in liver. Analyses implied that current in vitro human stem cell differentiation follows a dorsal rather than a ventral pancreatic program and pointed to additional factors for hepatic differentiation. In summary, we provide the transcriptional codes regulating the start of human liver and pancreas development to facilitate stem cell research and clinical interpretation without inter-species extrapolation.

  3. Energy Balance, Myostatin, and GILZ: Factors Regulating Adipocyte Differentiation in Belly and Bone

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    Xingming Shi

    2007-01-01

    Full Text Available Peroxisome proliferator-activated receptor gamma (PPAR-γ belongs to the nuclear hormone receptor subfamily of transcription factors. PPARs are expressed in key target tissues such as liver, fat, and muscle and thus they play a major role in the regulation of energy balance. Because of PPAR-γ's role in energy balance, signals originating from the gut (e.g., GIP, fat (e.g., leptin, muscle (e.g., myostatin, or bone (e.g., GILZ can in turn modulate PPAR expression and/or function. Of the two PPAR-γ isoforms, PPAR-γ2 is the key regulator of adipogenesis and also plays a role in bone development. Activation of this receptor favors adipocyte differentiation of mesenchymal stem cells, while inhibition of PPAR-γ2 expression shifts the commitment towards the osteoblastogenic pathway. Clinically, activation of this receptor by antidiabetic agents of the thiazolidinedione class results in lower bone mass and increased fracture rates. We propose that inhibition of PPAR-γ2 expression in mesenchymal stem cells by use of some of the hormones/factors mentioned above may be a useful therapeutic strategy to favor bone formation.

  4. Tight junction regulates epidermal calcium ion gradient and differentiation

    International Nuclear Information System (INIS)

    Kurasawa, Masumi; Maeda, Tetsuo; Oba, Ai; Yamamoto, Takuya; Sasaki, Hiroyuki

    2011-01-01

    Research highlights: → We disrupted epidermal tight junction barrier in reconstructed epidermis. → It altered Ca 2+ distribution and consequentially differentiation state as well. → Tight junction should affect epidermal homeostasis by maintaining Ca 2+ gradient. -- Abstract: It is well known that calcium ions (Ca 2+ ) induce keratinocyte differentiation. Ca 2+ distributes to form a vertical gradient that peaks at the stratum granulosum. It is thought that the stratum corneum (SC) forms the Ca 2+ gradient since it is considered the only permeability barrier in the skin. However, the epidermal tight junction (TJ) in the granulosum has recently been suggested to restrict molecular movement to assist the SC as a secondary barrier. The objective of this study was to clarify the contribution of the TJ to Ca 2+ gradient and epidermal differentiation in reconstructed human epidermis. When the epidermal TJ barrier was disrupted by sodium caprate treatment, Ca 2+ flux increased and the gradient changed in ion-capture cytochemistry images. Alterations of ultrastructures and proliferation/differentiation markers revealed that both hyperproliferation and precocious differentiation occurred regionally in the epidermis. These results suggest that the TJ plays a crucial role in maintaining epidermal homeostasis by controlling the Ca 2+ gradient.

  5. Differential regulation of glomerular and interstitial endothelial nitric oxide synthase expression in the kidney of hibernating ground squirrel.

    Science.gov (United States)

    Sandovici, Maria; Henning, Robert H; Hut, Roelof A; Strijkstra, Arjen M; Epema, Anne H; van Goor, Harry; Deelman, Leo E

    2004-09-01

    Hibernating animals transiently reduce renal function during their hypothermic periods (torpor), while completely restoring it during their periodical rewarming to euthermia (arousal). Moreover, structural integrity of the kidney is preserved throughout the hibernation. Nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) is a crucial vasodilatory mediator and a protective factor in the kidney. We investigated renal NOS expression in hibernating European ground squirrels after 1 day and 7 days of torpor (torpor short, TS, and torpor long, TL, respectively), at 1.5 and at 10 h of rewarming (arousal short, AS, and arousal long, AL, respectively), and in continuously euthermic animals after hibernation (EU). For that purpose, we performed NOS activity assay, immunohistochemistry and real-time PCR analysis. Immunohistochemistry revealed a decreased glomerular eNOS expression in hibernating animals (TS, TL, AS, and AL) compared to non-hibernating animals (EU, p EU. In all methods used, torpid and aroused squirrels did not differ. These results demonstrate differential regulation of eNOS in glomeruli and interstitium of hibernating animals, which is unaffected during arousal. The differential regulation of eNOS may serve to reduce ultrafiltration without jeopardizing tubular structures during hibernation.

  6. Signals of Ezh2, Src, and Akt Involve in Myostatin-Pax7 Pathways Regulating the Myogenic Fate Determination during the Sheep Myoblast Proliferation and Differentiation

    Science.gov (United States)

    Li, Li; Liu, Ruizao; Zhang, Li; Zhao, Fuping; Lu, Jian; Zhang, Xiaoning; Du, Lixin

    2015-01-01

    Myostatin and Pax7 have been well documented individually, however, the mechanism by which Myostatin regulates Pax7 is seldom reported. Here, based on muscle transcriptome analysis in Texel (Myostatin mutant) and Ujumqin (wild type) sheep across the five fetal stages, we constructed and examined the Myostatin-Pax7 pathways in muscle. Then we validated the signals by RNAi in the proliferating and differentiating sheep myoblasts in vitro at mRNA, protein, and cell morphological levels. We reveal that Myostatin signals to Pax7 at least through Ezh2, Src, and Akt during the sheep myoblast proliferation and differentiation. Other signals such as p38MAPK, mTOR, Erk1/2, Wnt, Bmp2, Smad, Tgfb1, and p21 are most probably involved in the Myostatin-affected myogenic events. Myostatin knockdown significantly reduces the counts of nucleus and myotube, but not the fusion index of myoblasts during cell differentiation. In addition, findings also indicate that Myostatin is required for normal myogenic differentiation of the sheep myoblasts, which is different from the C2C12 myoblasts. We expand the regulatory network of Myostatin-Pax7 pathways and first illustrate that Myostatin as a global regulator participates in the epigenetic events involved in myogenesis, which contributes to understand the molecular mechanism of Myostatin in regulation of myogenesis. PMID:25811841

  7. Differential regulation of renal Klotho and FGFR1 in normal and uremic rats.

    Science.gov (United States)

    Muñoz-Castañeda, Juan R; Herencia, Carmen; Pendón-Ruiz de Mier, Maria Victoria; Rodriguez-Ortiz, Maria Encarnación; Diaz-Tocados, Juan M; Vergara, Noemi; Martínez-Moreno, Julio M; Salmerón, Maria Dolores; Richards, William G; Felsenfeld, Arnold; Kuro-O, Makoto; Almadén, Yolanda; Rodríguez, Mariano

    2017-09-01

    In renal failure, hyperphosphatemia occurs despite a marked elevation in serum fibroblast growth factor (FGF)-23. Abnormal regulation of the FGFR1-Klotho receptor complex may cause a resistance to the phosphaturic action of FGF23. The purpose of the present study was to investigate the regulation of renal Klotho and FGF receptor (FEFR)-1 in healthy and uremic rats induced by 5/6 nephrectomy. In normal rats, the infusion of rat recombinant FGF23 enhanced phosphaturia and increased renal FGFR1 expression; however, Klotho expression was reduced. Uremic rats on a high-phosphate (HP) diet presented hyperphosphatemia with marked elevation of FGF23 and an increased fractional excretion of phosphate (P) that was associated with a marked reduction of Klotho expression and an increase in FGFR1. After neutralization of FGF23 by anti-FGF23 administration, phosphaturia was still abundant, Klotho expression remained low, and the FGFR1 level was reduced. These results suggest that the expression of renal Klotho is modulated by phosphaturia, whereas the FGFR1 expression is regulated by FGF23. Calcitriol (CTR) administration prevented a decrease in renal Klotho expression. In HEK293 cells HP produced nuclear translocation of β-catenin, together with a reduction in Klotho. Wnt/β-catenin inhibition with Dkk-1 prevented the P-induced down-regulation of Klotho. The addition of CTR to HP medium was able to recover Klotho expression. In summary, high FGF23 levels increase FGFR1, whereas phosphaturia decreases Klotho expression through the activation of Wnt/β-catenin pathway.-Muñoz-Castañeda, J. R., Herencia, C., Pendón-Ruiz de Mier, M. V., Rodriguez-Ortiz, M. E., Diaz-Tocados, J. M., Vergara, N., Martínez-Moreno, J. M., Salmerón, M. D., Richards, W. G., Felsenfeld, A., Kuro-O, M., Almadén, Y., Rodríguez, M. Differential regulation of renal Klotho and FGFR1 in normal and uremic rats. © FASEB.

  8. Identification of a Dynamic Core Transcriptional Network in t(8;21 AML that Regulates Differentiation Block and Self-Renewal

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    Anetta Ptasinska

    2014-09-01

    Full Text Available Oncogenic transcription factors such as RUNX1/ETO, which is generated by the chromosomal translocation t(8;21, subvert normal blood cell development by impairing differentiation and driving malignant self-renewal. Here, we use digital footprinting and chromatin immunoprecipitation sequencing (ChIP-seq to identify the core RUNX1/ETO-responsive transcriptional network of t(8;21 cells. We show that the transcriptional program underlying leukemic propagation is regulated by a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes, which bind to identical DNA sites in a mutually exclusive fashion. Perturbation of this equilibrium in t(8;21 cells by RUNX1/ETO depletion leads to a global redistribution of transcription factor complexes within preexisting open chromatin, resulting in the formation of a transcriptional network that drives myeloid differentiation. Our work demonstrates on a genome-wide level that the extent of impaired myeloid differentiation in t(8;21 is controlled by the dynamic balance between RUNX1/ETO and RUNX1 activities through the repression of transcription factors that drive differentiation.

  9. Hypoxia and hypoxia-inducible factors as regulators of T cell development, differentiation, and function

    Science.gov (United States)

    McNamee, Eóin N.; Johnson, Darlynn Korns; Homann, Dirk

    2014-01-01

    Oxygen is a molecule that is central to cellular respiration and viability, yet there are multiple physiologic and pathological contexts in which cells experience conditions of insufficient oxygen availability, a state known as hypoxia. Given the metabolic challenges of a low oxygen environment, hypoxia elicits a range of adaptive responses at the cellular, tissue, and systemic level to promote continued survival and function. Within this context, T lymphocytes are a highly migratory cell type of the adaptive immune system that frequently encounters a wide range of oxygen tensions in both health and disease. It is now clear that oxygen availability regulates T cell differentiation and function, a response orchestrated in large part by the hypoxia-inducible factor transcription factors. Here, we discuss the physiologic scope of hypoxia and hypoxic signaling, the contribution of these pathways in regulating T cell biology, and current gaps in our understanding. Finally, we discuss how emerging therapies that modulate the hypoxic response may offer new modalities to alter T cell function and the outcome of acute and chronic pathologies. PMID:22961658

  10. Differentiation, distribution and gammadelta T cell-driven regulation of IL-22-producing T cells in tuberculosis.

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    Shuyu Yao

    2010-02-01

    Full Text Available Differentiation, distribution and immune regulation of human IL-22-producing T cells in infections remain unknown. Here, we demonstrated in a nonhuman primate model that M. tuberculosis infection resulted in apparent increases in numbers of T cells capable of producing IL-22 de novo without in vitro Ag stimulation, and drove distribution of these cells more dramatically in lungs than in blood and lymphoid tissues. Consistently, IL-22-producing T cells were visualized in situ in lung tuberculosis (TB granulomas by confocal microscopy and immunohistochemistry, indicating that mature IL-22-producing T cells were present in TB granuloma. Surprisingly, phosphoantigen HMBPP activation of Vgamma2Vdelta2 T cells down-regulated the capability of T cells to produce IL-22 de novo in lymphocytes from blood, lung/BAL fluid, spleen and lymph node. Up-regulation of IFNgamma-producing Vgamma2Vdelta2 T effector cells after HMBPP stimulation coincided with the down-regulated capacity of these T cells to produce IL-22 de novo. Importantly, anti-IFNgamma neutralizing Ab treatment reversed the HMBPP-mediated down-regulation effect on IL-22-producing T cells, suggesting that Vgamma2Vdelta2 T-cell-driven IFNgamma-networking function was the mechanism underlying the HMBPP-mediated down-regulation of the capability of T cells to produce IL-22. These novel findings raise the possibility to ultimately investigate the function of IL-22 producing T cells and to target Vgamma2Vdelta2 T cells for balancing potentially hyper-activating IL-22-producing T cells in severe TB.

  11. NADPH oxidases differentially regulate ROS metabolism and nutrient uptake under cadmium toxicity.

    Science.gov (United States)

    Gupta, D K; Pena, L B; Romero-Puertas, M C; Hernández, A; Inouhe, M; Sandalio, L M

    2017-04-01

    The role of NADPH oxidases under cadmium (Cd) toxicity was studied using Arabidopsis thaliana mutants AtrbohC, AtrbohD and AtrbohF, which were grown under hydroponic conditions with 25 and 100 μM Cd for 1 and 5 days. Cadmium reduced the growth of leaves in WT, AtrbohC and D, but not in AtrbohF. A time-dependent increase in H 2 O 2 and lipid peroxidation was observed in all genotypes, with AtrbohC showing the smallest increase. An opposite behaviour was observed with NO accumulation. Cadmium increased catalase activity in WT plants and decreased it in Atrboh mutants, while glutathione reductase and glycolate oxidase activities increased in Atrboh mutants, and superoxide dismutases were down-regulated in AtrbohC. The GSH/GSSG and ASA/DHA couples were also affected by the treatment, principally in AtrbohC and AtrbohF, respectively. Cadmium translocation to the leaves was severely reduced in Atrboh mutants after 1 day of treatment and even after 5 days in AtrbohF. Similar results were observed for S, P, Ca, Zn and Fe accumulation, while an opposite trend was observed for K accumulation, except in AtrbohF. Thus, under Cd stress, RBOHs differentially regulate ROS metabolism, redox homeostasis and nutrient balance and could be of potential interest in biotechnology for the phytoremediation of polluted soils. © 2016 John Wiley & Sons Ltd.

  12. Differential microRNA Analysis of Glandular Trichomes and Young Leaves in Xanthium strumarium L. Reveals Their Putative Roles in Regulating Terpenoid Biosynthesis.

    Science.gov (United States)

    Fan, Rongyan; Li, Yuanjun; Li, Changfu; Zhang, Yansheng

    2015-01-01

    The medicinal plant Xanthium strumarium L. (X. strumarium) is covered with glandular trichomes, which are the sites for synthesizing pharmacologically active terpenoids such as xanthatin. MicroRNAs (miRNAs) are a class of 21-24 nucleotide (nt) non-coding RNAs, most of which are identified as regulators of plant growth development. Identification of miRNAs involved in the biosynthesis of plant secondary metabolites remains limited. In this study, high-throughput Illumina sequencing, combined with target gene prediction, was performed to discover novel and conserved miRNAs with potential roles in regulating terpenoid biosynthesis in X. strumarium glandular trichomes. Two small RNA libraries from leaves and glandular trichomes of X. strumarium were established. In total, 1,185 conserved miRNAs and 37 novel miRNAs were identified, with 494 conserved miRNAs and 18 novel miRNAs being differentially expressed between the two tissue sources. Based on the X. strumarium transcriptome data that we recently constructed, 3,307 annotated mRNA transcripts were identified as putative targets of the differentially expressed miRNAs. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that some of the differentially expressed miRNAs, including miR6435, miR5021 and miR1134, might be involved in terpenoid biosynthesis in the X. strumarium glandular trichomes. This study provides the first comprehensive analysis of miRNAs in X. strumarium, which forms the basis for further understanding of miRNA-based regulation on terpenoid biosynthesis.

  13. Differential microRNA Analysis of Glandular Trichomes and Young Leaves in Xanthium strumarium L. Reveals Their Putative Roles in Regulating Terpenoid Biosynthesis.

    Directory of Open Access Journals (Sweden)

    Rongyan Fan

    Full Text Available The medicinal plant Xanthium strumarium L. (X. strumarium is covered with glandular trichomes, which are the sites for synthesizing pharmacologically active terpenoids such as xanthatin. MicroRNAs (miRNAs are a class of 21-24 nucleotide (nt non-coding RNAs, most of which are identified as regulators of plant growth development. Identification of miRNAs involved in the biosynthesis of plant secondary metabolites remains limited. In this study, high-throughput Illumina sequencing, combined with target gene prediction, was performed to discover novel and conserved miRNAs with potential roles in regulating terpenoid biosynthesis in X. strumarium glandular trichomes. Two small RNA libraries from leaves and glandular trichomes of X. strumarium were established. In total, 1,185 conserved miRNAs and 37 novel miRNAs were identified, with 494 conserved miRNAs and 18 novel miRNAs being differentially expressed between the two tissue sources. Based on the X. strumarium transcriptome data that we recently constructed, 3,307 annotated mRNA transcripts were identified as putative targets of the differentially expressed miRNAs. KEGG (Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that some of the differentially expressed miRNAs, including miR6435, miR5021 and miR1134, might be involved in terpenoid biosynthesis in the X. strumarium glandular trichomes. This study provides the first comprehensive analysis of miRNAs in X. strumarium, which forms the basis for further understanding of miRNA-based regulation on terpenoid biosynthesis.

  14. Context-dependent Dynamic Processes in Attention Deficit/Hyperactivity Disorder : Differentiating Common and Unique Effects of State Regulation Deficits and Delay Aversion

    NARCIS (Netherlands)

    Sonuga-Barke, Edmund J. S.; Wiersema, Jan R.; van der Meere, Jacob J.; Roeyers, Herbert

    The ability to specify differential predictions is a mark of a scientific models' value. State regulation deficits (SRD) and delay aversion (DAv) have both been hypothesized as context-dependent dynamic dysfunctions in ADHD. However, to date there has been no systematic comparison of their common

  15. Chondroitin sulfate proteoglycans regulate the growth, differentiation and migration of multipotent neural precursor cells through the integrin signaling pathway

    Directory of Open Access Journals (Sweden)

    Lü He-Zuo

    2009-10-01

    Full Text Available Abstract Background Neural precursor cells (NPCs are defined by their ability to proliferate, self-renew, and retain the potential to differentiate into neurons and glia. Deciphering the factors that regulate their behaviors will greatly aid in their use as potential therapeutic agents or targets. Chondroitin sulfate proteoglycans (CSPGs are prominent components of the extracellular matrix (ECM in the central nervous system (CNS and are assumed to play important roles in controlling neuronal differentiation and development. Results In the present study, we demonstrated that CSPGs were constitutively expressed on the NPCs isolated from the E16 rat embryonic brain. When chondroitinase ABC was used to abolish the function of endogenous CSPGs on NPCs, it induced a series of biological responses including the proliferation, differentiation and migration of NPCs, indicating that CSPGs may play a critical role in NPC development and differentiation. Finally, we provided evidence suggesting that integrin signaling pathway may be involved in the effects of CSPGs on NPCs. Conclusion The present study investigating the influence and mechanisms of CSPGs on the differentiation and migration of NPCs should help us to understand the basic biology of NPCs during CNS development and provide new insights into developing new strategies for the treatment of the neurological disorders in the CNS.

  16. Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis

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    Abbas Jafari

    2017-02-01

    Full Text Available Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis.

  17. Platyphylloside Isolated From Betula platyphylla Inhibit Adipocyte Differentiation and Induce Lipolysis Via Regulating Adipokines Including PPARγ in 3T3-L1 Cells

    Science.gov (United States)

    Lee, Mina; Sung, Sang Hyun

    2016-01-01

    Background: Obesity causes or aggravates many health problems, both independently and in association with several pathological disorders, including Type II diabetes, hypertension, atherosclerosis, and cancer. Therefore, we screened small compounds isolated from natural products for the development of anti-obesity drugs. Objective: The purpose of this study was to investigate the anti-adipogenic activities of platyphylloside, diarylheptanoid isolated from Betula platyphylla, which was selected based on the screening using 3T3-L1 cells. Materials and Methods: To evaluate the inhibition of adipocyte differentiation and lipolysis, lipid contents of BPP on were measured using Oil Red O staining in 3T3-L1 cells. The mRNA and protein expression levels of various adipokines were measured by Quantitative real-time PCR and Western blotting analysis, respectively. Results: Platyphylloside showed significant inhibitory activity on adipocyte differentiation in 3T3-L1 cells and suppressed adipocyte differentiation even in the presence of troglitazone, a PPARγ agonist. Platyphylloside might suppress adipocyte differentiation through PPARγ, C/EBPα, and SREBP1-induced adipogenesis, which is synergistically associated with downstream adipocyte-specific gene promoters such as aP2, FAS, SCD-1, LPL, and Adiponectin. In addition, platyphylloside affected lipolysis by down-regulating perilipin and HSL and up-regulating TNFα. Conclusion: Taken together, the results reveal that platyphylloside has anti-adipogenic activity and highlight its potential in the prevention and treatment of obesity. SUMMARY The extract of B. platyphylla bark and its isolate, BPP, had anti-adipogenic activity in 3T3-L1 cells via suppression of adipocyte differentiation from preadipocytes.Treatment with BPP significantly down-regulated the expression of PPARγ, C/EBP, C/EBPβ, C/EBPδ, SREBP1c, SCD-1, FAS, aP2 and LPL.BPP induced a lipolytic response in mature adipocytes via up-regulation krof TNFá and down-regulation

  18. P27Kip1, regulated by glycogen synthase kinase-3β, results in HMBA-induced differentiation of human gastric cancer cells

    International Nuclear Information System (INIS)

    Wei, Min; Gu, Qinlong; Wang, Zhiwei; Yao, Hongliang; Yang, Zhongyin; Zhang, Qing; Liu, Bingya; Yu, Yingyan; Su, Liping; Zhu, Zhenggang

    2011-01-01

    Gastric cancer is the second most common cause of global cancer-related mortality. Although dedifferentiation predicts poor prognosis in gastric cancer, the molecular mechanism underlying dedifferentiation, which could provide fundamental insights into tumor development and progression, has yet to be elucidated. Furthermore, the molecular mechanism underlying the effects of hexamethylene bisacetamide (HMBA), a recently discovered differentiation inducer, requires investigation and there are no reported studies concerning the effect of HMBA on gastric cancer. Based on the results of FACS analysis, the levels of proteins involved in the cell cycle or apoptosis were determined using western blotting after single treatments and sequential combinations of HMBA and LiCl. GSK-3β and proton pump were investigated by western blotting after up-regulating Akt expression by Ad-Akt infection. To investigate the effects of HMBA on protein localization and the activities of GSK-3β, CDK2 and CDK4, kinase assays, immunoprecipitation and western blotting were performed. In addition, northern blotting and RNase protection assays were carried out to determine the functional concentration of HMBA. HMBA increased p27Kip1 expression and induced cell cycle arrest associated with gastric epithelial cell differentiation. In addition, treating gastric-derived cells with HMBA induced G0/G1 arrest and up-regulation of the proton pump, a marker of gastric cancer differentiation. Moreover, treatment with HMBA increased the expression and activity of GSK-3β in the nucleus but not the cytosol. HMBA decreased CDK2 activity and induced p27Kip1 expression, which could be rescued by inhibition of GSK-3β. Furthermore, HMBA increased p27Kip1 binding to CDK2, and this was abolished by GSK-3β inhibition. The results presented herein suggest that GSK-3β functions by regulating p27Kip1 assembly with CDK2, thereby playing a critical role in G0/G1 arrest associated with HMBA-induced gastric epithelial

  19. The homeobox gene Hex regulates hepatocyte differentiation from embryonic stem cell-derived endoderm.

    Science.gov (United States)

    Kubo, Atsushi; Kim, Yon Hui; Irion, Stefan; Kasuda, Shogo; Takeuchi, Mitsuaki; Ohashi, Kazuo; Iwano, Masayuki; Dohi, Yoshiko; Saito, Yoshihiko; Snodgrass, Ralph; Keller, Gordon

    2010-02-01

    We investigated the role of the hematopoietically expressed homeobox (Hex) in the differentiation and development of hepatocytes within embryonic stem cell (ESC)-derived embryoid bodies (EBs). Analyses of hepatic endoderm derived from Hex(-/-) EBs revealed a dramatic reduction in the levels of albumin (Alb) and alpha-fetoprotein (Afp) expression. In contrast, stage-specific forced expression of Hex in EBs from wild-type ESCs led to the up-regulation of Alb and Afp expression and secretion of Alb and transferrin. These inductive effects were restricted to c-kit(+) endoderm-enriched EB-derived populations, suggesting that Hex functions at the level of hepatic specification of endoderm in this model. Microarray analysis revealed that Hex regulated the expression of a broad spectrum of hepatocyte-related genes, including fibrinogens, apolipoproteins, and cytochromes. When added to the endoderm-induced EBs, bone morphogenetic protein 4 acted synergistically with Hex in the induction of expression of Alb, Afp, carbamoyl phosphate synthetase, transcription factor 1, and CCAAT/enhancer binding protein alpha. These findings indicate that Hex plays a pivotal role during induction of liver development from endoderm in this in vitro model and suggest that this strategy may provide important insight into the generation of functional hepatocytes from ESCs.

  20. Monocyte to macrophage differentiation-associated (MMD) positively regulates ERK and Akt activation and TNF-α and NO production in macrophages.

    Science.gov (United States)

    Liu, Qiang; Zheng, Jin; Yin, Dan-Dan; Xiang, Jie; He, Fei; Wang, Yao-Chun; Liang, Liang; Qin, Hong-Yan; Liu, Li; Liang, Ying-Min; Han, Hua

    2012-05-01

    Macrophage activation is modulated by both environmental cues and endogenous programs. In the present study, we investigated the role of a PAQR family protein, monocyte to macrophage differentiation-associated (MMD), in macrophage activation and unveiled its underlying molecular mechanism. Our results showed that while MMD expression could be detected in all tissues examined, its expression level is significantly up-regulated upon monocyte differentiation. Within cells, EGFP-MMD fusion protein could be co-localized to endoplasmic reticulum, mitochondria, Golgi apparatus, but not lysosomes and cytoplasm. MMD expression is up-regulated in macrophages after LPS stimulation, and this might be modulated by RBP-J, the critical transcription factor of Notch signaling. Overexpression of MMD in macrophages increased the production of TNF-α and NO upon LPS stimulation. We found that MMD overexpression enhanced ERK1/2 and Akt phosphorylation in macrophages after LPS stimulation. Blocking Erk or Akt by pharmacological agent reduced TNF-α or NO production in MMD-overexpressing macrophages, respectively. These results suggested that MMD modulates TNF-α and NO production in macrophages, and this process might involves Erk or Akt.

  1. A genome-wide RNAi screen reveals MAP kinase phosphatases as key ERK pathway regulators during embryonic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Shen-Hsi Yang

    Full Text Available Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Complex interlinked transcriptional and signaling networks control the fate of these cells towards maintenance of pluripotency or differentiation. In this study we have focused on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase and GSK3 signaling pathways play in this process. Through a genome-wide siRNA screen we have identified more than 400 genes involved in loss of pluripotency and promoting the onset of differentiation. These genes were functionally associated with the ERK and/or GSK3 pathways, providing an important resource for studying the roles of these pathways in controlling escape from the pluripotent ground state. More detailed analysis identified MAP kinase phosphatases as a focal point of regulation and demonstrated an important role for these enzymes in controlling ERK activation kinetics and subsequently determining early embryonic stem cell fate decisions.

  2. Pilocarpine-induced seizures trigger differential regulation of microRNA-stability related genes in rat hippocampal neurons.

    Science.gov (United States)

    Kinjo, Erika R; Higa, Guilherme S V; Santos, Bianca A; de Sousa, Erica; Damico, Marcio V; Walter, Lais T; Morya, Edgard; Valle, Angela C; Britto, Luiz R G; Kihara, Alexandre H

    2016-02-12

    Epileptogenesis in the temporal lobe elicits regulation of gene expression and protein translation, leading to reorganization of neuronal networks. In this process, miRNAs were described as being regulated in a cell-specific manner, although mechanistics of miRNAs activity are poorly understood. The specificity of miRNAs on their target genes depends on their intracellular concentration, reflecting the balance of biosynthesis and degradation. Herein, we confirmed that pilocarpine application promptly (PAPD4 gene expression in the hippocampus, two genes related to miRNA degradation and stability, respectively. Moreover, SE decreased the number of XRN2-positive cells in the hilus, while reduced the number of PAPD4-positive cells in CA1. XRN2 and PAPD4 levels did not change in calretinin- and CamKII-positive cells, although it was possible to determine that PAPD4, but not XRN2, was upregulated in parvalbumin-positive cells, revealing that SE induction unbalances the accumulation of these functional-opposed proteins in inhibitory interneurons that directly innervate distinct domains of pyramidal cells. Therefore, we were able to disclose a possible mechanism underlying the differential regulation of miRNAs in specific neurons during epileptogenesis.

  3. Spontaneous Vesicle Fusion Is Differentially Regulated at Cholinergic and GABAergic Synapses

    Directory of Open Access Journals (Sweden)

    Haowen Liu

    2018-02-01

    Full Text Available The locomotion of C. elegans is balanced by excitatory and inhibitory neurotransmitter release at neuromuscular junctions. However, the molecular mechanisms that maintain the balance of synaptic transmission remain enigmatic. Here, we investigated the function of voltage-gated Ca2+ channels in triggering spontaneous release at cholinergic and GABAergic synapses. Recordings of the miniature excitatory/inhibitory postsynaptic currents (mEPSCs and mIPSCs, respectively showed that UNC-2/CaV2 and EGL-19/CaV1 channels are the two major triggers for spontaneous release. Notably, however, Ca2+-independent spontaneous release was observed at GABAergic but not cholinergic synapses. Functional screening led to the identification of hypomorphic unc-64/Syntaxin-1A and snb-1/VAMP2 mutants in which mEPSCs are severely impaired, whereas mIPSCs remain unaltered, indicating differential regulation of these currents at cholinergic and GABAergic synapses. Moreover, Ca2+-independent spontaneous GABA release was nearly abolished in the hypomorphic unc-64 and snb-1 mutants, suggesting distinct mechanisms for Ca2+-dependent and Ca2+-independent spontaneous release.

  4. ERK2 protein regulates the proliferation of human mesenchymal stem cells without affecting their mobilization and differentiation potential

    International Nuclear Information System (INIS)

    Carcamo-Orive, Ivan; Tejados, Naiara; Delgado, Jesus; Gaztelumendi, Ainhoa; Otaegui, David; Lang, Valerie; Trigueros, Cesar

    2008-01-01

    Human Mesenchymal Stem Cells (hMSC), derived mainly from adult bone marrow, are valuable models for the study of processes involved in stem cell self-renewal and differentiation. As the Extracellular signal-Regulated Kinase (ERK) signalling pathway is a major contributor to cellular growth, differentiation and survival, we have studied the functions of this kinase in hMSC activity. Ablation of ERK2 gene expression (but not ERK1) by RNA interference significantly reduced proliferation of hMSC. This reduction was due to a defect in Cyclin D1 expression and subsequent arrest in the G0/G1 phase of the cell cycle. hMSC growth is enhanced through culture medium supplementation with growth factors (GFs) such as Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF). However, these supplements could not rescue the defect observed after ERK2 knockdown, suggesting a common signalling pathway used by these GFs for proliferation. In contrast, ERK1/2 may be dissociated from chemotactic signalling induced by the same GFs. Additionally, hMSCs were capable of differentiating into adipocytes even in the absence of either ERK1 or ERK2 proteins. Our data show that hMSCs do not require cell division to enter the adipogenic differentiation process, indicating that clonal amplification of these cells is not a critical step. However, cell-cell contact seems to be an essential requirement to be able to differentiate into mature adipocytes

  5. Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

    International Nuclear Information System (INIS)

    Yi, TacGhee; Kim, Hye-Jin; Cho, Je-Yoel; Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik; Baek, Jeong-Hwa

    2006-01-01

    Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity

  6. Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing.

    Science.gov (United States)

    Jain, Mukesh; Chevala, V V S Narayana; Garg, Rohini

    2014-11-01

    MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  7. The Regulation of Chemerin and CMKLR1 Genes Expression by TNF-α, Adiponectin, and Chemerin Analog in Bovine Differentiated Adipocytes

    Directory of Open Access Journals (Sweden)

    Y. Suzuki

    2012-09-01

    Full Text Available Adipokines, adipocyte-derived protein, have important roles in various kinds of physiology including energy homeostasis. Chemerin, one of adipocyte-derived adipokines, is highly expressed in differentiated adipocytes and is known to induce macrophage chemotaxis and glucose intolerance. The objective of the present study was to investigate the changes of chemerin and the chemokine-like-receptor 1 (CMKLR1 gene expression levels during differentiation of the bovine adipocyte and in differentiated adipocytes treated with tumor necrosis factor-α (TNF-α, adiponectin, leptin, and chemerin (peptide analog. The expression levels of the chemerin gene increased at d 6 and 12 of the differentiation period accompanied by increased cytoplasm lipid droplets. From d 6 onward, peroxisome proliferator-activated receptor-γ2 (PPAR-γ2 gene expression levels were significantly higher than that of d 0 and 3. In contrast, CMKLR1 expression levels decreased at the end of the differentiation period. In fully differentiated adipocytes (i.e. at d 12, the treatment of TNF-α and adiponectin upregulated both chemerin and CMKLR1 gene expression levels, although leptin did not show such effects. Moreover, chemerin analog treatment was shown to upregulate chemerin gene expression levels regardless of doses. These results suggest that the expression of chemerin in bovine adipocyte might be regulated by chemerin itself and other adipokines, which indicates its possible role in modulating the adipokine secretions in adipose tissues.

  8. Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis.

    Directory of Open Access Journals (Sweden)

    David Garciadiego-Cázares

    Full Text Available The Integrin β1 family is the major receptors of the Extracellular matrix (ECM, and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA. In this scenario, integrins modify their pattern expression and regulate chondrocyte differentiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β Superfamily, such as Growth differentiation factor 5 (Gdf-5 and Bone morphogenetic protein 7 (Bmp-7, play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedifferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressed αV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of the α5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh, Gdf-5 and α5 integrin to maintain articular cartilage and prevent

  9. Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

    International Nuclear Information System (INIS)

    Farajzadeh, Leila; Hornshøj, Henrik; Momeni, Jamal; Thomsen, Bo; Larsen, Knud; Hedegaard, Jakob; Bendixen, Christian; Madsen, Lone Bruhn

    2013-01-01

    Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i

  10. bHLH-O proteins balance the self-renewal and differentiation of Drosophila neural stem cells by regulating Earmuff expression.

    Science.gov (United States)

    Li, Xiaosu; Chen, Rui; Zhu, Sijun

    2017-11-15

    Balancing self-renewal and differentiation of stem cells requires differential expression of self-renewing factors in two daughter cells generated from the asymmetric division of the stem cells. In Drosophila type II neural stem cell (or neuroblast, NB) lineages, the expression of the basic helix-loop-helix-Orange (bHLH-O) family proteins, including Deadpan (Dpn) and E(spl) proteins, is required for maintaining the self-renewal and identity of type II NBs, whereas the absence of these self-renewing factors is essential for the differentiation of intermediate neural progenitors (INPs) generated from type II NBs. Here, we demonstrate that Dpn maintains type II NBs by suppressing the expression of Earmuff (Erm). We provide evidence that Dpn and E(spl) proteins suppress Erm by directly binding to C-sites and N-boxes in the cis-regulatory region of erm. Conversely, the absence of bHLH-O proteins in INPs allows activation of erm and Erm-mediated maturation of INPs. Our results further suggest that Pointed P1 (PntP1) mediates the dedifferentiation of INPs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins. Taken together, these findings reveal mechanisms underlying the regulation of the maintenance of type II NBs and differentiation of INPs through the differential expression of bHLH-O family proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.

    Science.gov (United States)

    Wang, Shuxin; Li, Zheyi; Shen, Hongyan; Zhang, Zhong; Yin, Yuxin; Wang, Qingsong; Zhao, Xuyang; Ji, Jianguo

    2016-08-01

    Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3β (Gsk3β). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101. © 2016 AlphaMed Press.

  12. Plasma Membrane Ca2+-Permeable Channels are Differentially Regulated by Ethylene and Hydrogen Peroxide to Generate Persistent Plumes of Elevated Cytosolic Ca2+ During Transfer Cell Trans-Differentiation.

    Science.gov (United States)

    Zhang, Hui-ming; van Helden, Dirk F; McCurdy, David W; Offler, Christina E; Patrick, John W

    2015-09-01

    The enhanced transport capability of transfer cells (TCs) arises from their ingrowth wall architecture comprised of a uniform wall on which wall ingrowths are deposited. The wall ingrowth papillae provide scaffolds to amplify plasma membranes that are enriched in nutrient transporters. Using Vicia faba cotyledons, whose adaxial epidermal cells spontaneously and rapidly (hours) undergo a synchronous TC trans-differentiation upon transfer to culture, has led to the discovery of a cascade of inductive signals orchestrating deposition of ingrowth wall papillae. Auxin-induced ethylene biosynthesis initiates the cascade. This in turn drives a burst in extracellular H2O2 production that triggers uniform wall deposition. Thereafter, a persistent and elevated cytosolic Ca(2+) concentration, resulting from Ca(2+) influx through plasma membrane Ca(2+)-permeable channels, generates a Ca(2+) signal that directs formation of wall ingrowth papillae to specific loci. We now report how these Ca(2+)-permeable channels are regulated using the proportionate responses in cytosolic Ca(2+) concentration as a proxy measure of their transport activity. Culturing cotyledons on various combinations of pharmacological agents allowed the regulatory influence of each upstream signal on Ca(2+) channel activity to be evaluated. The findings demonstrated that Ca(2+)-permeable channel activity was insensitive to auxin, but up-regulated by ethylene through two independent routes. In one route ethylene acts directly on Ca(2+)-permeable channel activity at the transcriptional and post-translational levels, through an ethylene receptor-dependent pathway. The other route is mediated by an ethylene-induced production of extracellular H2O2 which then acts translationally and post-translationally to up-regulate Ca(2+)-permeable channel activity. A model describing the differential regulation of Ca(2+)-permeable channel activity is presented. © The Author 2015. Published by Oxford University Press on

  13. REST represses a subset of the pancreatic endocrine differentiation program

    DEFF Research Database (Denmark)

    Martin, David; Kim, Yung-Hae; Sever, Dror

    2015-01-01

    in neurons and in endocrine cells, which is necessary for their normal function. During development, REST represses a subset of genes in the neuronal differentiation program and Rest is down-regulated as neurons differentiate. Here, we investigate the role of REST in the differentiation of pancreatic...... endocrine cells, which are molecularly close to neurons. We show that Rest is widely expressed in pancreas progenitors and that it is down-regulated in differentiated endocrine cells. Sustained expression of REST in Pdx1(+) progenitors impairs the differentiation of endocrine-committed Neurog3...

  14. DNA topoisomerase IIβ stimulates neurite outgrowth in neural differentiated human mesenchymal stem cells through regulation of Rho-GTPases (RhoA/Rock2 pathway) and Nurr1 expression.

    Science.gov (United States)

    Zaim, Merve; Isik, Sevim

    2018-04-25

    DNA topoisomerase IIβ (topo IIβ) is known to regulate neural differentiation by inducing the neuronal genes responsible for critical neural differentiation events such as neurite outgrowth and axon guidance. However, the pathways of axon growth controlled by topo IIβ have not been clarified yet. Microarray results of our previous study have shown that topo IIβ silencing in neural differentiated primary human mesenchymal stem cells (hMSCs) significantly alters the expression pattern of genes involved in neural polarity, axonal growth, and guidance, including Rho-GTPases. This study aims to further analyze the regulatory role of topo IIβ on the process of axon growth via regulation of Rho-GTPases. For this purpose, topo IIβ was silenced in neurally differentiated hMSCs. Cells lost their morphology because of topo IIβ deficiency, becoming enlarged and flattened. Additionally, a reduction in both neural differentiation efficiency and neurite length, upregulation in RhoA and Rock2, downregulation in Cdc42 gene expression were detected. On the other hand, cells were transfected with topo IIβ gene to elucidate the possible neuroprotective effect of topo IIβ overexpression on neural-induced hMSCs. Topo IIβ overexpression prompted all the cells to exhibit neural cell morphology as characterized by longer neurites. RhoA and Rock2 expressions were downregulated, whereas Cdc42 expression was upregulated. Nurr1 expression level correlated with topo IIβ in both topo IIβ-overexpressed and -silenced cells. Furthermore, differential translocation of Rho-GTPases was detected by immunostaining in response to topo IIβ. Our results suggest that topo IIβ deficiency could give rise to neurodegeneration through dysregulation of Rho-GTPases. However, further in-vivo research is needed to demonstrate if re-regulation of Rho GTPases by topo IIβ overexpression could be a neuroprotective treatment in the case of neurodegenerative diseases.

  15. Selective phosphorylation during early macrophage differentiation

    KAUST Repository

    Zhang, Huoming; Qian, Pei-Yuan; Ravasi, Timothy

    2015-01-01

    -regulated phosphoproteins in the early stages of differentiation. Further analysis of the PMA-regulated phosphoproteins revealed that transcriptional suppression, cytoskeletal reorganization and cell adhesion were among the most significantly activated pathways. Some key

  16. Bile acids deoxycholic acid and ursodeoxycholic acid differentially regulate human β-defensin-1 and -2 secretion by colonic epithelial cells.

    Science.gov (United States)

    Lajczak, Natalia K; Saint-Criq, Vinciane; O'Dwyer, Aoife M; Perino, Alessia; Adorini, Luciano; Schoonjans, Kristina; Keely, Stephen J

    2017-09-01

    Bile acids and epithelial-derived human β-defensins (HβDs) are known to be important factors in the regulation of colonic mucosal barrier function and inflammation. We hypothesized that bile acids regulate colonic HβD expression and aimed to test this by investigating the effects of deoxycholic acid (DCA) and ursodeoxycholic acid on the expression and release of HβD1 and HβD2 from colonic epithelial cells and mucosal tissues. DCA (10-150 µM) stimulated the release of both HβD1 and HβD2 from epithelial cell monolayers and human colonic mucosal tissue in vitro In contrast, ursodeoxycholic acid (50-200 µM) inhibited both basal and DCA-induced defensin release. Effects of DCA were mimicked by the Takeda GPCR 5 agonist, INT-777 (50 μM), but not by the farnesoid X receptor agonist, GW4064 (10 μM). INT-777 also stimulated colonic HβD1 and HβD2 release from wild-type, but not Takeda GPCR 5 -/- , mice. DCA stimulated phosphorylation of the p65 subunit of NF-κB, an effect that was attenuated by ursodeoxycholic acid, whereas an NF-κB inhibitor, BMS-345541 (25 μM), inhibited DCA-induced HβD2, but not HβD1, release. We conclude that bile acids can differentially regulate colonic epithelial HβD expression and secretion and discuss the implications of our findings for intestinal health and disease.-Lajczak, N. K., Saint-Criq, V., O'Dwyer, A. M., Perino, A., Adorini, L., Schoonjans, K., Keely, S. J. Bile acids deoxycholic acid and ursodeoxycholic acid differentially regulate human β-defensin-1 and -2 secretion by colonic epithelial cells. © FASEB.

  17. Proteomic Analysis of Fetal Ovaries Reveals That Primordial Follicle Formation and Transition Are Differentially Regulated

    Directory of Open Access Journals (Sweden)

    Mengmeng Xu

    2017-01-01

    Full Text Available Primordial follicle formation represents a critical phase of the initiation of embryonic reproductive organ development, while the primordial follicle transition into primary follicle determines whether oestrus or ovulation will occur in female animals. To identify molecular mechanism of new proteins which are involved in ovarian development, we employed 2D-DIGE to compare the protein expression profiles of primordial follicles and primary follicles of fetal ovaries in pigs. Fetal ovaries were collected at distinct time-points of the gestation cycle (g55 and g90. The identified proteins at the g55 time-point are mainly involved in the development of anatomical structures [reticulocalbin-1 (RCN1, reticulocalbin-3 (RCN3], cell differentiation (actin, and stress response [heterogeneous nuclear ribonucleoprotein K (HNRNPK]. Meanwhile, at the g90 stage, the isolated proteins with altered expression levels were mainly associated with cell proliferation [major vault protein (MVP] and stress response [heat shock-related 70 kDa protein 2 (HSPA2]. In conclusion, our work revealed that primordial follicle formation is regulated by RCN1, RCN3, actin, and HNRNPK, while the primordial follicle transformation to primary follicle is regulated by MVP and HSPA2. Therefore, our results provide further information for the prospective understanding of the molecular mechanism(s involved in the regulation of the ovarian follicle development.

  18. Pluripotency factors in embryonic stem cells regulate differentiation into germ layers.

    Science.gov (United States)

    Thomson, Matt; Liu, Siyuan John; Zou, Ling-Nan; Smith, Zack; Meissner, Alexander; Ramanathan, Sharad

    2011-06-10

    Cell fate decisions are fundamental for development, but we do not know how transcriptional networks reorganize during the transition from a pluripotent to a differentiated cell state. Here, we asked how mouse embryonic stem cells (ESCs) leave the pluripotent state and choose between germ layer fates. By analyzing the dynamics of the transcriptional circuit that maintains pluripotency, we found that Oct4 and Sox2, proteins that maintain ESC identity, also orchestrate germ layer fate selection. Oct4 suppresses neural ectodermal differentiation and promotes mesendodermal differentiation; Sox2 inhibits mesendodermal differentiation and promotes neural ectodermal differentiation. Differentiation signals continuously and asymmetrically modulate Oct4 and Sox2 protein levels, altering their binding pattern in the genome, and leading to cell fate choice. The same factors that maintain pluripotency thus also integrate external signals and control lineage selection. Our study provides a framework for understanding how complex transcription factor networks control cell fate decisions in progenitor cells. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. TGF-β1 is Involved in Vitamin D-Induced Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Regulating the ERK/JNK Pathway

    Directory of Open Access Journals (Sweden)

    Xiaorui Jiang

    2017-08-01

    Full Text Available Background/Aims: Osteoarthritis (OA is characterized by degradation of cartilage, sole cell type of which is chondrocytes. Bone marrow-derived mesenchymal stem cells (BMSCs possess multipotency and can be directionally differentiated into chondrocytes under stimulation. This study was aimed to explore the possible roles of vitamin D and transforming growth factor-β1 (TGF-β1 in the chondrogenic differentiation of BMSCs. Methods: BMSCs were isolated from femurs and tibias of rats and characterized by flow cytometry. After stimulation with vitamin D, BMSC proliferation and migration were measured by Cell Counting Kit-8 (CCK-8 and Transwell assays, respectively. Chondrogenic differentiation was estimated through expression levels of specific markers by qRT-PCR and Western blot analysis. After stable transfection, the effects of aberrantly expressed TGF-β1 on vitamin D-induced alterations, including BMSC viability, migration and chondrogenic differentiation, were all evaluated utilizing CCK-8 assay, Transwell assay, qRT-PCR and Western blot analysis. Finally, the phosphorylation levels of key kinases in the extracellular signal-regulated kinase (ERK and c-Jun N-terminal kinase (JNK pathways were determined by Western blot analysis. Results: Vitamin D remarkably promoted BMSC viability, migration and chondrogenic differentiation. These alterations of BMSCs induced by vitamin D were reinforced by TGF-β1 overexpression while were reversed by TGF-β1 silencing. Additionally, the phosphorylation levels of ERK, JNK and c-Jun were enhanced by TGF-β1 overexpression but were reduced by TGF-β1 knockdown. Conclusion: Vitamin D promoted BMSC proliferation, migration and chondrogenic differentiation. TGF-β1 might be implicated in the vitamin D-induced alterations of BMSCs through regulating ERK/JNK pathway.

  20. Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2.

    Science.gov (United States)

    Bylund, Jeffery B; Trinh, Linh T; Awgulewitsch, Cassandra P; Paik, David T; Jetter, Christopher; Jha, Rajneesh; Zhang, Jianhua; Nolan, Kristof; Xu, Chunhui; Thompson, Thomas B; Kamp, Timothy J; Hatzopoulos, Antonis K

    2017-05-01

    Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling.

  1. Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2

    Science.gov (United States)

    Bylund, Jeffery B.; Trinh, Linh T.; Awgulewitsch, Cassandra P.; Paik, David T.; Jetter, Christopher; Jha, Rajneesh; Zhang, Jianhua; Nolan, Kristof; Xu, Chunhui; Thompson, Thomas B.; Kamp, Timothy J.

    2017-01-01

    Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling. PMID:28125926

  2. Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress

    Directory of Open Access Journals (Sweden)

    Muhammed eJamsheer K

    2015-09-01

    Full Text Available Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1 signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response towards energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response.

  3. ADAMTS9-Regulated Pericellular Matrix Dynamics Governs Focal Adhesion-Dependent Smooth Muscle Differentiation

    Directory of Open Access Journals (Sweden)

    Timothy J. Mead

    2018-04-01

    Full Text Available Summary: Focal adhesions anchor cells to extracellular matrix (ECM and direct assembly of a pre-stressed actin cytoskeleton. They act as a cellular sensor and regulator, linking ECM to the nucleus. Here, we identify proteolytic turnover of the anti-adhesive proteoglycan versican as a requirement for maintenance of smooth muscle cell (SMC focal adhesions. Using conditional deletion in mice, we show that ADAMTS9, a secreted metalloprotease, is required for myometrial activation during late gestation and for parturition. Through knockdown of ADAMTS9 in uterine SMC, and manipulation of pericellular versican via knockdown or proteolysis, we demonstrate that regulated pericellular matrix dynamics is essential for focal adhesion maintenance. By influencing focal adhesion formation, pericellular versican acts upstream of cytoskeletal assembly and SMC differentiation. Thus, pericellular versican proteolysis by ADAMTS9 balances pro- and anti-adhesive forces to maintain an SMC phenotype, providing a concrete example of the dynamic reciprocity of cells and their ECM. : Mead et al. identify a proteolytic mechanism that actively maintains a pericellular microenvironment conducive to uterine smooth muscle activation prior to parturition. They show that pericellular matrix proteolysis by the secreted metalloprotease ADAMTS9 is crucial for maintenance of focal adhesions in uterine smooth muscle cells, and its absence impairs parturition. Keywords: metalloprotease, extracellular matrix, smooth muscle, proteoglycan, myometrium, parturition, uterus, focal adhesion, proteolysis, interference reflection microscopy

  4. Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells

    Science.gov (United States)

    Ali, Dalia; Hamam, Rimi; Alfayez, Musaed; Kassem, Moustapha; Aldahmash, Abdullah

    2016-01-01

    The epigenetic mechanisms promoting lineage-specific commitment of human skeletal (mesenchymal or stromal) stem cells (hMSCs) into adipocytes or osteoblasts are still not fully understood. Herein, we performed an epigenetic library functional screen and identified several novel compounds, including abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell proliferation and differentiation that were targeted by abexinostat. Concordantly, ChIP-quantitative polymerase chain reaction revealed marked increase in H3K9Ac epigenetic mark on the promoter region of AdipoQ, FABP4, PPARγ, KLF15, CEBPA, SP7, and ALPL in abexinostat-treated hMSCs. Pharmacological inhibition of focal adhesion kinase (PF-573228) or insulin-like growth factor-1R/insulin receptor (NVP-AEW51) signaling exhibited significant inhibition of abexinostat-mediated adipocytic differentiation, whereas inhibition of WNT (XAV939) or transforming growth factor-β (SB505124) signaling abrogated abexinostat-mediated osteogenic differentiation of hMSCs. Our findings provide insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways governing adipocyte and osteoblast differentiation. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies and tissue engineering. Significance This unbiased epigenetic library functional screen identified several novel compounds, including abexinostat, that promoted adipocytic and osteoblastic differentiation of human skeletal (mesenchymal or stromal) stem cells (hMSCs). These data provide new insight into the understanding of the relationship between the epigenetic effect of histone deacetylase

  5. NHE10, a novel osteoclast-specific member of the Na+/H+ exchanger family, regulates osteoclast differentiation and survival

    International Nuclear Information System (INIS)

    Lee, Seoung Hoon; Kim, Taesoo; Park, Eui-Soon; Yang, Sujeong; Jeong, Daewon; Choi, Yongwon; Rho, Jaerang

    2008-01-01

    Bone homeostasis is tightly regulated by the balanced actions of osteoblasts (OBs) and osteoclasts (OCs). We previously analyzed the gene expression profile of OC differentiation using a cDNA microarray, and identified a novel osteoclastogenic gene candidate, clone OCL-1-E7 [J. Rho, C.R. Altmann, N.D. Socci, L. Merkov, N. Kim, H. So, O. Lee, M. Takami, A.H. Brivanlou, Y. Choi, Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis, DNA Cell Biol. 21 (2002) 541-549]. In this study, we have isolated full-length cDNAs corresponding to this clone from mice and humans to determine the functional roles of this gene in osteoclastogenesis. The full-length cDNA of OCL-1-E7 encodes 12 membrane-spanning domains that are typical of isoforms of the Na + /H + exchangers (NHEs), indicating that this clone is a novel member of the NHE family (hereafter referred to as NHE10). Here, we show that NHE10 is highly expressed in OCs in response to receptor activator of nuclear factor-κB ligand signaling and is required for OC differentiation and survival

  6. Foxo1 and Foxp1 play opposing roles in regulating the differentiation and antitumor activity of TH9 cells programmed by IL-7

    Science.gov (United States)

    Bi, Enguang; Ma, Xingzhe; Lu, Yong; Yang, Maojie; Wang, Qiang; Xue, Gang; Qian, Jianfei; Wang, Siqing; Yi, Qing

    2018-01-01

    Tumor-specific CD4+ T helper 9 (TH9) cells, so-called because of their production of the cytokine interleukin-9 (IL-9), are a powerful effector T cell subset for cancer immunotherapy. We found that pretreatment of naïve CD4+ T cells with IL-7 further enhanced their differentiation into TH9 cells and augmented their antitumor activity. IL-7 markedly increased the abundance of the histone acetyltransferase p300 by activating the STAT5 and PI3K-AKT-mTOR signaling pathways and promoting the acetylation of histones at the Il9 promoter. As a result, the transcriptional regulator Foxo1 was dephosphorylated and translocated to the nucleus, bound to the Il9 promoter, and induced the production of IL-9 protein. In contrast, Foxp1, which bound to the Il9 promoter in naïve CD4+ T cells and inhibited Il9 expression, was outcompeted for binding to the Il9 promoter by Foxo1 and translocated to the cytoplasm. Furthermore, forced expression of Foxo1 or a deficiency in Foxp1 in CD4+ T cells markedly increased the production of IL-9, whereas a deficiency in Foxo1 inhibited the ability of IL-7 to enhance the differentiation and antitumor activity of TH9 cells. Thus, we identified the roles of Foxo1 as a positive regulator and Foxp1 as a negative regulator of TH9 cell differentiation and antitumor activity, which may provide potential targets for cancer immunotherapy. PMID:29018172

  7. Sex Reversal and Comparative Data Undermine the W Chromosome and Support Z-linked DMRT1 as the Regulator of Gonadal Sex Differentiation in Birds.

    Science.gov (United States)

    Hirst, Claire E; Major, Andrew T; Ayers, Katie L; Brown, Rosie J; Mariette, Mylene; Sackton, Timothy B; Smith, Craig A

    2017-09-01

    The exact genetic mechanism regulating avian gonadal sex differentiation has not been completely resolved. The most likely scenario involves a dosage mechanism, whereby the Z-linked DMRT1 gene triggers testis development. However, the possibility still exists that the female-specific W chromosome may harbor an ovarian determining factor. In this study, we provide evidence that the universal gene regulating gonadal sex differentiation in birds is Z-linked DMRT1 and not a W-linked (ovarian) factor. Three candidate W-linked ovarian determinants are HINTW, female-expressed transcript 1 (FET1), and female-associated factor (FAF). To test the association of these genes with ovarian differentiation in the chicken, we examined their expression following experimentally induced female-to-male sex reversal using the aromatase inhibitor fadrozole (FAD). Administration of FAD on day 3 of embryogenesis induced a significant loss of aromatase enzyme activity in female gonads and masculinization. However, expression levels of HINTW, FAF, and FET1 were unaltered after experimental masculinization. Furthermore, comparative analysis showed that FAF and FET1 expression could not be detected in zebra finch gonads. Additionally, an antibody raised against the predicted HINTW protein failed to detect it endogenously. These data do not support a universal role for these genes or for the W sex chromosome in ovarian development in birds. We found that DMRT1 (but not the recently identified Z-linked HEMGN gene) is male upregulated in embryonic zebra finch and emu gonads, as in the chicken. As chicken, zebra finch, and emu exemplify the major evolutionary clades of birds, we propose that Z-linked DMRT1, and not the W sex chromosome, regulates gonadal sex differentiation in birds. Copyright © 2017 Endocrine Society.

  8. Interferon-γ regulates the proliferation and differentiation of mesenchymal stem cells via activation of indoleamine 2,3 dioxygenase (IDO.

    Directory of Open Access Journals (Sweden)

    Juliana Croitoru-Lamoury

    Full Text Available The kynurenine pathway (KP of tryptophan metabolism is linked to antimicrobial activity and modulation of immune responses but its role in stem cell biology is unknown. We show that human and mouse mesenchymal and neural stem cells (MSCs and NSCs express the complete KP, including indoleamine 2,3 dioxygenase 1 (IDO and IDO2, that it is highly regulated by type I (IFN-β and II interferons (IFN-γ, and that its transcriptional modulation depends on the type of interferon, cell type and species. IFN-γ inhibited proliferation and altered human and mouse MSC neural, adipocytic and osteocytic differentiation via the activation of IDO. A functional KP present in MSCs, NSCs and perhaps other stem cell types offers novel therapeutic opportunities for optimisation of stem cell proliferation and differentiation.

  9. Differential regulation of kiss1 expression by melatonin and gonadal hormones in male and female Syrian hamsters

    DEFF Research Database (Denmark)

    Ansel, L; Bolborea, M; Bentsen, A H

    2010-01-01

    In seasonal breeders, reproduction is synchronized to seasons by day length via the pineal hormone melatonin. Recently, we have demonstrated that Kiss1, a key activator of the reproductive function, is down-regulated in sexually inactive hamsters maintained in inhibitory short days (SDs). In rode......In seasonal breeders, reproduction is synchronized to seasons by day length via the pineal hormone melatonin. Recently, we have demonstrated that Kiss1, a key activator of the reproductive function, is down-regulated in sexually inactive hamsters maintained in inhibitory short days (SDs......). In rodents, Kiss1 is expressed in the anteroventral periventricular nucleus (AVPV) and in the arcuate nucleus (ARC). Because both the duration of the nocturnal peak of melatonin and circulating sex steroid levels vary with photoperiod, the aim of this study was to determine whether melatonin and sex steroids...... differentially regulate Kiss1 expression in the ARC and the AVPV. Kiss1 expression was examined by in situ hybridization in both male and female hamsters kept in various experimental conditions, and we observed that 1) SD exposure markedly reduced Kiss1 expression in the ARC and AVPV of male and female hamsters...

  10. Transgelin is a TGFβ-inducible gene that regulates osteoblastic and adipogenic differentiation of human skeletal stem cells through actin cytoskeleston organization

    DEFF Research Database (Denmark)

    Elsafadi, E; Manikandan, M; Dawud, R. A.

    2016-01-01

    Regenerative medicine is a novel approach for treating conditions in which enhanced bone regeneration is required. We identified transgelin (TAGLN), a transforming growth factor beta (TGFβ)-inducible gene, as an upregulated gene during in vitro osteoblastic and adipocytic differentiation of human......MSC by regulating cytoskeleton organization. Targeting TAGLN is a plausible approach to enrich for committed hMSC cells needed for regenerative medicine application....... bone marrow-derived stromal (skeletal) stem cells (hMSC). siRNA-mediated gene silencing of TAGLN impaired lineage differentiation into osteoblasts and adipocytes but enhanced cell proliferation. Additional functional studies revealed that TAGLN deficiency impaired hMSC cell motility and in vitro...... transwell cell migration. On the other hand, TAGLN overexpression reduced hMSC cell proliferation, but enhanced cell migration, osteoblastic and adipocytic differentiation, and in vivo bone formation. In addition, deficiency or overexpression of TAGLN in hMSC was associated with significant changes...

  11. Obligatory Role for B Cells in the Development of Angiotensin II-Dependent Hypertension.

    Science.gov (United States)

    Chan, Christopher T; Sobey, Christopher G; Lieu, Maggie; Ferens, Dorota; Kett, Michelle M; Diep, Henry; Kim, Hyun Ah; Krishnan, Shalini M; Lewis, Caitlin V; Salimova, Ekaterina; Tipping, Peter; Vinh, Antony; Samuel, Chrishan S; Peter, Karlheinz; Guzik, Tomasz J; Kyaw, Tin S; Toh, Ban-Hock; Bobik, Alexander; Drummond, Grant R

    2015-11-01

    Clinical hypertension is associated with raised serum IgG antibodies. However, whether antibodies are causative agents in hypertension remains unknown. We investigated whether hypertension in mice is associated with B-cell activation and IgG production and moreover whether B-cell/IgG deficiency affords protection against hypertension and vascular remodeling. Angiotensin II (Ang II) infusion (0.7 mg/kg per day; 28 days) was associated with (1) a 25% increase in the proportion of splenic B cells expressing the activation marker CD86, (2) an 80% increase in splenic plasma cell numbers, (3) a 500% increase in circulating IgG, and (4) marked IgG accumulation in the aortic adventitia. In B-cell-activating factor receptor-deficient (BAFF-R(-/-)) mice, which lack mature B cells, there was no evidence of Ang II-induced increases in serum IgG. Furthermore, the hypertensive response to Ang II was attenuated in BAFF-R(-/-) (Δ30±4 mm Hg) relative to wild-type (Δ41±5 mm Hg) mice, and this response was rescued by B-cell transfer. BAFF-R(-/-) mice displayed reduced IgG accumulation in the aorta, which was associated with 80% fewer aortic macrophages and a 70% reduction in transforming growth factor-β expression. BAFF-R(-/-) mice were also protected from Ang II-induced collagen deposition and aortic stiffening (assessed by pulse wave velocity analysis). Finally, like BAFF-R deficiency, pharmacological depletion of B cells with an anti-CD20 antibody attenuated Ang II-induced hypertension by ≈35%. Hence, these studies demonstrate that B cells/IgGs are crucial for the development of Ang II-induced hypertension and vessel remodeling in mice. Thus, B-cell-targeted therapies-currently used for autoimmune diseases-may hold promise as future treatments for hypertension. © 2015 American Heart Association, Inc.

  12. Differential regulation of HIF-1α and HIF-2α in neuroblastoma: Estrogen-related receptor alpha (ERRα) regulates HIF2A transcription and correlates to poor outcome

    International Nuclear Information System (INIS)

    Hamidian, Arash; Stedingk, Kristoffer von; Munksgaard Thorén, Matilda; Mohlin, Sofie; Påhlman, Sven

    2015-01-01

    Hypoxia-inducible factors (HIFs) are differentially regulated in tumor cells. While the current paradigm supports post-translational regulation of the HIF-α subunits, we recently showed that hypoxic HIF-2α is also transcriptionally regulated via insulin-like growth factor (IGF)-II in the childhood tumor neuroblastoma. Here, we demonstrate that transcriptional regulation of HIF-2α seems to be restricted to neural cell-derived tumors, while HIF-1α is canonically regulated at the post-translational level uniformly across different tumor forms. Enhanced expression of HIF2A mRNA at hypoxia is due to de novo transcription rather than increased mRNA stability, and chemical stabilization of the HIF-α proteins at oxygen-rich conditions unexpectedly leads to increased HIF2A transcription. The enhanced HIF2A levels do not seem to be dependent on active HIF-1. Using a transcriptome array approach, we identified members of the Peroxisome proliferator-activated receptor gamma coactivator (PGC)/Estrogen-related receptor (ERR) complex families as potential regulators of HIF2A. Knockdown or inhibition of one of the members, ERRα, leads to decreased expression of HIF2A, and high expression of the ERRα gene ESRRA correlates with poor overall and progression-free survival in a clinical neuroblastoma material consisting of 88 tumors. Thus, targeting of ERRα and pathways regulating transcriptional HIF-2α are promising therapeutic avenues in neuroblastoma. - Highlights: • Transcriptional control of HIF-2α is restricted to neural cell-derived tumors. • Enhanced transcription of HIF2A is not due to increased mRNA stability. • Chemical stabilization of the HIF-α subunits leads to increased HIF2A transcription. • ERRα regulates HIF2A mRNA expression in neuroblastoma. • High expression of ESRRA correlates to poor outcome in neuroblastoma

  13. Differential regulation of HIF-1α and HIF-2α in neuroblastoma: Estrogen-related receptor alpha (ERRα) regulates HIF2A transcription and correlates to poor outcome

    Energy Technology Data Exchange (ETDEWEB)

    Hamidian, Arash; Stedingk, Kristoffer von; Munksgaard Thorén, Matilda; Mohlin, Sofie; Påhlman, Sven, E-mail: sven.pahlman@med.lu.se

    2015-06-05

    Hypoxia-inducible factors (HIFs) are differentially regulated in tumor cells. While the current paradigm supports post-translational regulation of the HIF-α subunits, we recently showed that hypoxic HIF-2α is also transcriptionally regulated via insulin-like growth factor (IGF)-II in the childhood tumor neuroblastoma. Here, we demonstrate that transcriptional regulation of HIF-2α seems to be restricted to neural cell-derived tumors, while HIF-1α is canonically regulated at the post-translational level uniformly across different tumor forms. Enhanced expression of HIF2A mRNA at hypoxia is due to de novo transcription rather than increased mRNA stability, and chemical stabilization of the HIF-α proteins at oxygen-rich conditions unexpectedly leads to increased HIF2A transcription. The enhanced HIF2A levels do not seem to be dependent on active HIF-1. Using a transcriptome array approach, we identified members of the Peroxisome proliferator-activated receptor gamma coactivator (PGC)/Estrogen-related receptor (ERR) complex families as potential regulators of HIF2A. Knockdown or inhibition of one of the members, ERRα, leads to decreased expression of HIF2A, and high expression of the ERRα gene ESRRA correlates with poor overall and progression-free survival in a clinical neuroblastoma material consisting of 88 tumors. Thus, targeting of ERRα and pathways regulating transcriptional HIF-2α are promising therapeutic avenues in neuroblastoma. - Highlights: • Transcriptional control of HIF-2α is restricted to neural cell-derived tumors. • Enhanced transcription of HIF2A is not due to increased mRNA stability. • Chemical stabilization of the HIF-α subunits leads to increased HIF2A transcription. • ERRα regulates HIF2A mRNA expression in neuroblastoma. • High expression of ESRRA correlates to poor outcome in neuroblastoma.

  14. Let7a involves in neural stem cell differentiation relating with TLX level

    Energy Technology Data Exchange (ETDEWEB)

    Song, Juhyun [Department of Anatomy, Yonsei University College of Medicine, Seoul (Korea, Republic of); Cho, Kyoung Joo; Oh, Yumi [Department of Anatomy, Yonsei University College of Medicine, Seoul (Korea, Republic of); BK21 Plus Project for Medical Sciences, and Brain Research Institute, Yonsei University College of Medicine, Seoul (Korea, Republic of); Lee, Jong Eun, E-mail: jelee@yuhs.ac [Department of Anatomy, Yonsei University College of Medicine, Seoul (Korea, Republic of); BK21 Plus Project for Medical Sciences, and Brain Research Institute, Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2015-07-10

    Neural stem cells (NSCs) have the potential for differentiation into neurons known as a groundbreaking therapeutic solution for central nervous system (CNS) diseases. To resolve the therapeutic efficiency of NSCs, recent researchers have focused on the study on microRNA's role in CNS. Some micro RNAs have been reported significant functions in NSC self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. MicroRNA-Let7a (Let7a) has known as the regulator of diverse cellular mechanisms including cell differentiation and proliferation. In present study, we investigated whether Let7a regulates NSC differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal, proliferation and differentiation. We performed the following experiments: western blot analysis, TaqMan assay, RT-PCR, and immunocytochemistry to confirm the alteration of NSCs. Our data showed that let7a play important roles in controlling NSC fate determination. Thus, manipulating Let-7A and TLX could be a novel strategy to enhance the efficiency of NSC's neuronal differentiation for CNS disorders. - Highlights: • Let7a influences on NSC differentiation and proliferation. • Let7a involves in mainly NSC differentiation rather than proliferation. • Let7a positively regulates the TLX expression.

  15. Let7a involves in neural stem cell differentiation relating with TLX level

    International Nuclear Information System (INIS)

    Song, Juhyun; Cho, Kyoung Joo; Oh, Yumi; Lee, Jong Eun

    2015-01-01

    Neural stem cells (NSCs) have the potential for differentiation into neurons known as a groundbreaking therapeutic solution for central nervous system (CNS) diseases. To resolve the therapeutic efficiency of NSCs, recent researchers have focused on the study on microRNA's role in CNS. Some micro RNAs have been reported significant functions in NSC self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. MicroRNA-Let7a (Let7a) has known as the regulator of diverse cellular mechanisms including cell differentiation and proliferation. In present study, we investigated whether Let7a regulates NSC differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal, proliferation and differentiation. We performed the following experiments: western blot analysis, TaqMan assay, RT-PCR, and immunocytochemistry to confirm the alteration of NSCs. Our data showed that let7a play important roles in controlling NSC fate determination. Thus, manipulating Let-7A and TLX could be a novel strategy to enhance the efficiency of NSC's neuronal differentiation for CNS disorders. - Highlights: • Let7a influences on NSC differentiation and proliferation. • Let7a involves in mainly NSC differentiation rather than proliferation. • Let7a positively regulates the TLX expression

  16. 5-Azacytidine delivered by mesoporous silica nanoparticles regulates the differentiation of P19 cells into cardiomyocytes

    Science.gov (United States)

    Cheng, Jin; Ding, Qian; Wang, Jia; Deng, Lin; Yang, Lu; Tao, Lei; Lei, Haihong; Lu, Shaoping

    2016-01-01

    Heart disease is one of the deadliest diseases causing mortality due to the limited regenerative capability of highly differentiated cardiomyocytes. Stem cell-based therapy in tissue engineering is one of the most exciting and rapidly growing areas and raises promising prospects for cardiac repair. In this study, we have synthesized FITC-mesoporous silica nanoparticles (FMSNs) based on a sol-gel method (known as Stöber's method) as a drug delivery platform to transport 5-azacytidine in P19 embryonic carcinoma stem cells. The surfactant CTAB is utilized as a liquid crystal template to self-aggregate into micelles, resulting in the synthesis of MSNs. Based on the cell viability assay, treatment with FMSNs + 5-azacytidine resulted in much more significant inhibition of the proliferation than 5-azacytidine alone. To study the mechanism, we have tested the differentiation genes and cardiac marker genes in P19 cells and found that these genes have been up-regulated in P19 embryonic carcinoma stem cells treated with FMSNs + 5-azacytidine + poly(allylamine hydrochloride) (PAH), with the changes of histone modifications on the regulatory region. In conclusion, with FMSNs as drug delivery platforms, 5-azacytidine can be more efficiently delivered into stem cells and can be used to monitor and track the transfection process in situ to clarify their effects on stem cell functions and the differentiation process, which can serve as a promising tool in tissue engineering and other biomedical fields.

  17. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

    International Nuclear Information System (INIS)

    Robinson, Claire; Kolb, Andreas F.

    2009-01-01

    The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A β-galactosidase reporter gene was inserted in place of the β-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the β-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal β-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the β-casein gene

  18. An empirical investigation of spatial differentiation and price floor regulations in retail markets for gasoline

    Science.gov (United States)

    Houde, Jean-Francois

    In the first essay of this dissertation, I study an empirical model of spatial competition. The main feature of my approach is to formally specify commuting paths as the "locations" of consumers in a Hotelling-type model of spatial competition. The main consequence of this location assumption is that the substitution patterns between stations depend in an intuitive way on the structure of the road network and the direction of traffic flows. The demand-side of the model is estimated by combining a model of traffic allocation with econometric techniques used to estimate models of demand for differentiated products (Berry, Levinsohn and Pakes (1995)). The estimated parameters are then used to evaluate the importance of commuting patterns in explaining the distribution of gasoline sales, and compare the economic predictions of the model with the standard home-location model. In the second and third essays, I examine empirically the effect of a price floor regulation on the dynamic and static equilibrium outcomes of the gasoline retail industry. In particular, in the second essay I study empirically the dynamic entry and exit decisions of gasoline stations, and measure the impact of a price floor on the continuation values of staying in the industry. In the third essay, I develop and estimate a static model of quantity competition subject to a price floor regulation. Both models are estimated using a rich panel dataset on the Quebec gasoline retail market before and after the implementation of a price floor regulation.

  19. Insulin/IGF-I regulation of necdin and brown adipocyte differentiation via CREB- and FoxO1-associated pathways

    DEFF Research Database (Denmark)

    Cypess, Aaron M; Zhang, Hongbin; Schulz, Tim J

    2011-01-01

    is regulated by the phosphoinositide 3 kinase-Akt pathway, increased necdin promoter activity. Based on reporter gene assays using truncations of the necdin promoter and chromatin immunoprecipitation studies, we demonstrated that CREB and FoxO1 are recruited to the necdin promoter, likely interacting......Brown adipose tissue plays an important role in obesity, insulin resistance, and diabetes. We have previously shown that the transition from brown preadipocytes to mature adipocytes is mediated in part by insulin receptor substrate (IRS)-1 and the cell cycle regulator protein necdin. In this study...... with specific consensus sequences in the proximal region. Based on these results, we propose that insulin/IGF-I act through IRS-1 phosphorylation to stimulate differentiation of brown preadipocytes via two complementary pathways: 1) the Ras-ERK1/2 pathway to activate CREB and 2) the phosphoinositide 3 kinase-Akt...

  20. Differential Dopamine Regulation of Ca2+ Signaling and Its Timing Dependence in the Nucleus Accumbens

    Directory of Open Access Journals (Sweden)

    Immani Swapna

    2016-04-01

    Full Text Available Dopamine action in the nucleus accumbens (NAc is thought to drive appetitive behavior and Pavlovian reward learning. However, it remains controversial how dopamine achieves these behavioral effects by regulating medium spiny projection neurons (MSNs of the NAc, especially on a behaviorally relevant timescale. Metabotropic glutamate receptor (mGluR-induced Ca2+ signaling dependent on the Ca2+- releasing messenger inositol 1,4,5-triphosphate (IP3 plays a critical role in controlling neuronal excitability and synaptic plasticity. Here, we show that transient dopamine application facilitates mGluR/IP3-induced Ca2+ signals within a time window of ∼2–10 s in a subpopulation of MSNs in the NAc core. Dopamine facilitation of IP3-induced Ca2+ signaling is mediated by D1 dopamine receptors. In dopamine-insensitive MSNs, activation of A2A adenosine receptors causes enhancement of IP3-evoked Ca2+ signals, which is reversed by D2 dopamine receptor activation. These results show that dopamine differentially regulates Ca2+ signaling on the order of seconds in two distinct MSN subpopulations.

  1. STUDY OF INTRA TESTICULAR REGULATIONS OF SPERMATOGENESIS DIFFERENTIATION BY EX-VIVO APPROACH

    Directory of Open Access Journals (Sweden)

    A. Adaika

    2010-12-01

    Full Text Available The aim of this work is to study the regulation of intratesticular during spermatogenesis ex vivo. To highlight the progress of spermatogenesis ex vivo, we developed two cell culture systems of seminiferous tubules to study the role of local factors that control the proliferation and differentiation of male germ cells. Our studies are based on two main techniques: RT-PCR and RNA extraction to examine changes in the expression of some growth factors in the culture of seminiferous tubules as the SCF, c- Kit and TGFß. The results show, using RT-PCR, that expression of SCF, c-Kit and TGFb is probably not involved in the alterations of spermatogenesis ex vivo. Indeed, their expressions are not modified during three weeks of culture, and their expressions depend on the proportion of cells where they are expressed. Our results also show that clusterin is a marker of Sertoli cells in the culture of seminiferous tubules and its expression is not altered by the presence of germ cells.

  2. PRELP (proline/arginine-rich end leucine-rich repeat protein) promotes osteoblastic differentiation of preosteoblastic MC3T3-E1 cells by regulating the β-catenin pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Haiying; Cui, Yazhou; Luan, Jing [School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Ji' nan, Shandong (China); Key Laboratory for Rare Disease Research of Shandong Province, Key Laboratory for Biotech Drugs of the Ministry of Health, Shandong Medical Biotechnological Center, Shandong Academy of Medical Sciences, Ji' nan, Shandong (China); Zhang, Xiumei [School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Ji' nan, Shandong (China); Li, Chengzhi; Zhou, Xiaoyan; Shi, Liang [School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Ji' nan, Shandong (China); Key Laboratory for Rare Disease Research of Shandong Province, Key Laboratory for Biotech Drugs of the Ministry of Health, Shandong Medical Biotechnological Center, Shandong Academy of Medical Sciences, Ji' nan, Shandong (China); Wang, Huaxin [Shandong University of Traditional Chinese Medicine, Ji' an, Shandong (China); Han, Jinxiang, E-mail: jxhan9888@aliyun.com [School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Ji' nan, Shandong (China); Key Laboratory for Rare Disease Research of Shandong Province, Key Laboratory for Biotech Drugs of the Ministry of Health, Shandong Medical Biotechnological Center, Shandong Academy of Medical Sciences, Ji' nan, Shandong (China)

    2016-02-12

    Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that β-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit the level of connexin43, a key regulator of osteoblast differentiation by affecting β-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating β-catenin/connexin43 pathway and osteoblast differentiation.

  3. The critical role of myostatin in differentiation of sheep myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chenxi [College of Life Science and Technology, Xinjiang University, Urumqi (China); Xinjiang Laboratory of Animal Biotechnology, Urumqi (China); Li, Wenrong; Zhang, Xuemei; Zhang, Ning; He, Sangang; Huang, Juncheng [Xinjiang Laboratory of Animal Biotechnology, Urumqi (China); Laboratory of Grass-fed Animal Genetics, Breeding and Reproduction of Ministry of Agriculture, Urumqi (China); Animal Biotechnological Research Center, Xinjiang Academy of Animal Science, Urumqi (China); Ge, Yubin [The State Engineering Laboratory of AIDS Vaccine, College of Life Science, Jilin University, Changchun (China); Liu, Mingjun, E-mail: xjlmj2004@yahoo.com.cn [Xinjiang Laboratory of Animal Biotechnology, Urumqi (China); Laboratory of Grass-fed Animal Genetics, Breeding and Reproduction of Ministry of Agriculture, Urumqi (China); Animal Biotechnological Research Center, Xinjiang Academy of Animal Science, Urumqi (China)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Identification of the effective and specific shRNA to knockdown MSTN. Black-Right-Pointing-Pointer Overexpression of MSTN reversibly suppressed myogenic differentiation. Black-Right-Pointing-Pointer shRNA knockdown of endogenous MSTN promoted ovine myoblast differentiation. Black-Right-Pointing-Pointer MSTN inhibits myogenic differentiation through down-regulation of MyoD and Myogenin and up-regulation of Smad3. Black-Right-Pointing-Pointer Provides a promise for the generation of transgenic sheep to improve meat productivity. -- Abstract: Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.

  4. The critical role of myostatin in differentiation of sheep myoblasts

    International Nuclear Information System (INIS)

    Liu, Chenxi; Li, Wenrong; Zhang, Xuemei; Zhang, Ning; He, Sangang; Huang, Juncheng; Ge, Yubin; Liu, Mingjun

    2012-01-01

    Highlights: ► Identification of the effective and specific shRNA to knockdown MSTN. ► Overexpression of MSTN reversibly suppressed myogenic differentiation. ► shRNA knockdown of endogenous MSTN promoted ovine myoblast differentiation. ► MSTN inhibits myogenic differentiation through down-regulation of MyoD and Myogenin and up-regulation of Smad3. ► Provides a promise for the generation of transgenic sheep to improve meat productivity. -- Abstract: Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.

  5. The excretory-secretory products of Echinococcus granulosus protoscoleces directly regulate the differentiation of B10, B17 and Th17 cells.

    Science.gov (United States)

    Pan, Wei; Hao, Wen-Ting; Shen, Yu-Juan; Li, Xiang-Yang; Wang, Yan-Juan; Sun, Fen-Fen; Yin, Jian-Hai; Zhang, Jing; Tang, Ren-Xian; Cao, Jian-Ping; Zheng, Kui-Yang

    2017-07-21

    Excretory-secretory products (ESPs) released by helminths are well-known to regulate T cell responses in the host. However, their direct influence in the differentiation of naïve T cells, and especially B cells, remains largely unknown. This study investigated the effects of Echinococcus granulosus protoscoleces ESPs (EgPSC-ESPs) on the differentiation of IL-10-producing B cells (B10), IL-17A-producing B cells (B17) and Th17 cells. BALB/c mice injected with EgPSC were used to evaluate the in vivo profiles of B10, B17 and Th17 cells. In vitro purified CD19 + B and naïve CD4 + T cells were cultured in the presence of native, heat-inactivated or periodate-treated EgPSC-ESPs, and the differentiation of these cell subsets were compared. In contrast to the control group, infected mice showed higher frequencies of B10, B17 and Th17 cells, and higher levels of IL-10 and IL-17A in the sera. Interestingly, B17 cells were first identified to express CD19 + CD1d high . In vitro, B cells cultured with native ESPs exhibited a higher percentage of B10 cells but lower percentage of B17 and Th17 cells compared to the PBS group. Moreover, the relative expression of IL-10 and IL-17A mRNA were consistent with the altered frequencies. However, ESPs subjected to heat-inactivation or periodate treatment exhibited an inverse effect on the induction of these cell subsets. Our findings indicate that ESPs released by EgPSC can directly regulate the differentiation of B10, B17 and Th17 cells, which appear to be heat-labile and carbohydrate-dependent.

  6. Network-based differential gene expression analysis suggests cell cycle related genes regulated by E2F1 underlie the molecular difference between smoker and non-smoker lung adenocarcinoma

    Science.gov (United States)

    2013-01-01

    Background Differential gene expression (DGE) analysis is commonly used to reveal the deregulated molecular mechanisms of complex diseases. However, traditional DGE analysis (e.g., the t test or the rank sum test) tests each gene independently without considering interactions between them. Top-ranked differentially regulated genes prioritized by the analysis may not directly relate to the coherent molecular changes underlying complex diseases. Joint analyses of co-expression and DGE have been applied to reveal the deregulated molecular modules underlying complex diseases. Most of these methods consist of separate steps: first to identify gene-gene relationships under the studied phenotype then to integrate them with gene expression changes for prioritizing signature genes, or vice versa. It is warrant a method that can simultaneously consider gene-gene co-expression strength and corresponding expression level changes so that both types of information can be leveraged optimally. Results In this paper, we develop a gene module based method for differential gene expression analysis, named network-based differential gene expression (nDGE) analysis, a one-step integrative process for prioritizing deregulated genes and grouping them into gene modules. We demonstrate that nDGE outperforms existing methods in prioritizing deregulated genes and discovering deregulated gene modules using simulated data sets. When tested on a series of smoker and non-smoker lung adenocarcinoma data sets, we show that top differentially regulated genes identified by the rank sum test in different sets are not consistent while top ranked genes defined by nDGE in different data sets significantly overlap. nDGE results suggest that a differentially regulated gene module, which is enriched for cell cycle related genes and E2F1 targeted genes, plays a role in the molecular differences between smoker and non-smoker lung adenocarcinoma. Conclusions In this paper, we develop nDGE to prioritize

  7. 5 CFR 591.204 - Who can receive COLAs and post differentials?

    Science.gov (United States)

    2010-01-01

    ... REGULATIONS ALLOWANCES AND DIFFERENTIALS Cost-of-Living Allowance and Post Differential-Nonforeign Areas Cost-Of-Living Allowances and Post Differentials § 591.204 Who can receive COLAs and post differentials...

  8. 5 CFR 591.203 - Why does the Government pay post differentials?

    Science.gov (United States)

    2010-01-01

    ... REGULATIONS ALLOWANCES AND DIFFERENTIALS Cost-of-Living Allowance and Post Differential-Nonforeign Areas Cost-Of-Living Allowances and Post Differentials § 591.203 Why does the Government pay post differentials...

  9. Fluctuations in Blood Marginal Zone B-Cell Frequencies May Reflect Migratory Patterns Associated with HIV-1 Disease Progression Status.

    Science.gov (United States)

    Gauvin, Julie; Chagnon-Choquet, Josiane; Poudrier, Johanne; Roger, Michel

    2016-01-01

    We have previously shown that overexpression of BLyS/BAFF was associated with increased relative frequencies of innate "precursor" marginal zone (MZ)-like B-cells in the blood of HIV-1-infected rapid and classic progressors. However, along with relatively normal BLyS/BAFF expression levels, these cells remain unaltered in elite-controllers (EC), rather, percentages of more mature MZ-like B-cells are decreased in the blood of these individuals. Fluctuations in frequencies of blood MZ-like B-cell populations may reflect migratory patterns associated with disease progression status, suggesting an important role for these cells in HIV-1 pathogenesis. We have therefore longitudinally measured plasma levels of B-tropic chemokines by ELISA-based technology as well as their ligands by flow-cytometry on blood B-cell populations of HIV-1-infected individuals with different rates of disease progression and uninfected controls. Migration potential of B-cell populations from these individuals were determined by chemotaxis assays. We found important modulations of CXCL13-CXCR5, CXCL12-CXCR4/CXCR7, CCL20-CCR6 and CCL25-CCR9 chemokine-axes and increased cell migration patterns in HIV progressors. Interestingly, frequencies of CCR6 expressing cells were significantly elevated within the precursor MZ-like population, consistent with increased migration in response to CCL20. Although we found little modulation of chemokine-axes in EC, cell migration was greater than that observed for uninfected controls, especially for MZ-like B-cells. Overall the immune response against HIV-1 may involve recruitment of MZ-like B-cells to peripheral sites. Moreover, our findings suggest that "regulated" attraction of these cells in a preserved BLyS/BAFF non-inflammatory environment, such as encountered in EC could be beneficial to the battle and even control of HIV.

  10. Profile of atacicept and its potential in the treatment of systemic lupus erythematosus

    Directory of Open Access Journals (Sweden)

    Cogollo E

    2015-03-01

    Full Text Available Estafania Cogollo,1,* Marta Amaral Silva,2,* David Isenberg3 1Department of Internal Medicine, Hospital Principe de Asturias, Alcala de Henares, Madrid, Spain; 2Department of Internal Medicine, Hospital Distrital da Figueira da Foz, Coimbra, Portugal; 3Centre for Rheumatology, Department of Medicine, University College London, London, UK *These authors are regarded as equal first authors Abstract: The importance of B cell activating factors in the generation of autoantibodies in patients with systemic lupus erythematosus (SLE is now recognized. The two key factors, known as BAFF and APRIL, produced by a variety of cells including monocytes, dendritic cells and T cells, also help to regulate B cell maturation, function and survival. Biologic agents that block these factors have now been developed and tried out in large scale clinical trials in SLE patients. Benlysta which blocks BAFF has met some of its end points in clinical trials and is approved for use in patients with skin and joint disease who have failed conventional drugs. In contrast, clinical trials using atacicept which blocks both BAFF and APRIL have been more challenging to interpret. An early study in lupus nephritis was, mistakenly, abandoned due to serious infections thought to be linked to the biologic when in fact the dramatic fall in the immunoglobulin levels took place when the patients were given mycophenolate, prior to the introduction of the atacicept. Likewise the higher dose arm (150 mgm of a flare prevention study was terminated prematurely when 2 deaths occurred. However, the mortality rate in this study was identical to that seen in the Benlysta studies and a post hoc analysis found a highly significant benefit for the 150mgm arm compared to the lower dose (75 mgm and placebo arms. Other trials with both Benlysta and atacicept are on-going. Keywords: cytokines, lupus nephritis, BLyS, APRIL

  11. Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

    Science.gov (United States)

    Qing, Hua; Aono, Jun; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

    2016-06-01

    Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. © 2015 Wiley Periodicals, Inc.

  12. Differential arousal regulation by prokineticin 2 signaling in the nocturnal mouse and the diurnal monkey.

    Science.gov (United States)

    Zhou, Qun-Yong; Burton, Katherine J; Neal, Matthew L; Qiao, Yu; Kanthasamy, Anumantha G; Sun, Yanjun; Xu, Xiangmin; Ma, Yuanye; Li, Xiaohan

    2016-08-18

    The temporal organization of activity/rest or sleep/wake rhythms for mammals is regulated by the interaction of light/dark cycle and circadian clocks. The neural and molecular mechanisms that confine the active phase to either day or night period for the diurnal and the nocturnal mammals are unclear. Here we report that prokineticin 2, previously shown as a circadian clock output molecule, is expressed in the intrinsically photosensitive retinal ganglion cells, and the expression of prokineticin 2 in the intrinsically photosensitive retinal ganglion cells is oscillatory in a clock-dependent manner. We further show that the prokineticin 2 signaling is required for the activity and arousal suppression by light in the mouse. Between the nocturnal mouse and the diurnal monkey, a signaling receptor for prokineticin 2 is differentially expressed in the retinorecipient suprachiasmatic nucleus and the superior colliculus, brain projection targets of the intrinsically photosensitive retinal ganglion cells. Blockade with a selective antagonist reveals the respectively inhibitory and stimulatory effect of prokineticin 2 signaling on the arousal levels for the nocturnal mouse and the diurnal monkey. Thus, the mammalian diurnality or nocturnality is likely determined by the differential signaling of prokineticin 2 from the intrinsically photosensitive retinal ganglion cells onto their retinorecipient brain targets.

  13. NRF2 Signaling Negatively Regulates Phorbol-12-Myristate-13-Acetate (PMA-Induced Differentiation of Human Monocytic U937 Cells into Pro-Inflammatory Macrophages.

    Directory of Open Access Journals (Sweden)

    Min-Gu Song

    Full Text Available Blood monocytes are recruited to injured tissue sites and differentiate into macrophages, which protect against pathogens and repair damaged tissues. Reactive oxygen species (ROS are known to be an important contributor to monocytes' differentiation and macrophages' function. NF-E2-related factor 2 (NRF2, a transcription factor regulating cellular redox homeostasis, is known to be a critical modulator of inflammatory responses. We herein investigated the role of NRF2 in macrophage differentiation using the human monocytic U937 cell line and phorbol-12-myristate-13-acetate (PMA. In U937 cells with NRF2 silencing, PMA-stimulated cell adherence was significantly facilitated when compared to control U937 cells. Both transcript and protein levels for pro-inflammatory cytokines, including interleukine-1β (IL-1β, IL-6, and tumor necrosis factor-α (TNFα were highly elevated in PMA-stimulated NRF2-silenced U937 compared to the control. In addition, PMA-inducible secretion of monocyte chemotactic protein 1 (MCP-1 was significantly high in NRF2-silenced U937. As an underlying mechanism, we showed that NRF2-knockdown U937 retained high levels of cellular ROS and endoplasmic reticulum (ER stress markers expression; and subsequently, PMA-stimulated levels of Ca2+ and PKCα were greater in NRF2-knockdown U937 cells, which caused enhanced nuclear accumulation of nuclear factor-ҡB (NFҡB p50 and extracellular signal-regulated kinase (ERK-1/2 phosphorylation. Whereas the treatment of NRF2-silenced U937 cells with pharmacological inhibitors of NFҡB or ERK1/2 largely blocked PMA-induced IL-1β and IL-6 expression, indicating that these pathways are associated with cell differentiation. Taken together, our results suggest that the NRF2 system functions to suppress PMA-stimulated U937 cell differentiation into pro-inflammatory macrophages and provide evidence that the ROS-PKCα-ERK-NFҡB axis is involved in PMA-facilitated differentiation of NRF2-silenced U937

  14. Proliferation and osteo/odontogenic differentiation of stem cells from apical papilla regulated by Zinc fingers and homeoboxes 2: An in vitro study.

    Science.gov (United States)

    Wan, Fang; Gao, Lifen; Lu, Yating; Ma, Hongxin; Wang, Hongxing; Liang, Xiaohong; Wang, Yan; Ma, Chunhong

    2016-01-15

    In the process of tooth root development, stem cells from the apical papilla (SCAPs) can differentiate into odontoblasts and form root dentin, however, molecules regulating SCAPs differentiation have not been elucidated. Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. It is reported to modulate the development of nerve cells, liver cells, B cells, red blood cells, and so on. However, the role of ZHX2 in tooth root development remains unclear. In this study, we explored the potential role of ZHX2 in the process of SCAPs differentiation. The results showed that overexpression of ZHX2 upregulated the expression of osteo/odontogenic related genes and ALP activity, inhibited the proliferation of SCAPs. Consistently, ZHX2 knockdown reduced SCAPs mineralization and promoted SCAPs proliferation. These results indicated that ZHX2 plays a critical role in the proliferation and osteo/odontogenic differentiation of SCAPs. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Classical and alternative NF-κB signaling cooperate in regulating adipocyte differentiation and function

    DEFF Research Database (Denmark)

    Weidemann, A.; Lovas, A.; Rauch, A.

    2016-01-01

    Background and objective:Inflammation of adipose tissue (AT) is a central mediator of insulin resistance. However, the molecular mechanisms triggered by inflammatory cells are not fully understood. The aim of this study was to analyze the metabolic functions of lymphotoxin-β-receptor (LTβ...... to adipocytes. The molecular mechanism was elucidated by chromatin immunoprecipitation and combinatorial treatment with α-LTβR and tumor necrosis factor (TNF).Results:RelB FatKO mice showed improved insulin sensitivity despite increased adiposity and adipocyte hypertrophy. LTβR-induced activation of p52-Rel.......Conclusions:Our data describe an anti-adipogenic action of LTβR signaling and a novel synergism of alternative and classical NF-κB signaling in the regulation of adipocytes. In conclusion, this strong synergism between the two NF-κB pathways shows a method to inhibit adipocyte differentiation and to improve insulin...

  16. MicroRNA-130b targets Fmr1 and regulates embryonic neural progenitor cell proliferation and differentiation

    International Nuclear Information System (INIS)

    Gong, Xi; Zhang, Kunshan; Wang, Yanlu; Wang, Junbang; Cui, Yaru; Li, Siguang; Luo, Yuping

    2013-01-01

    Highlights: •We found that the 3′ UTR of the Fmr1 mRNA is a target of miR-130b. •MiR-130b suppresses the expression of Fmr1 in mouse embryonic stem cell. •MiR-130b alters the proliferation of mouse embryonic stem cell. •MiR-130b alters fate specification of mouse embryonic stem cell. -- Abstract: Fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by expansion of the CGG repeat in the 5′-untranslated region of the X-linked Fmr1 gene, which results in transcriptional silencing and loss of expression of its encoded protein FMRP. The loss of FMRP increases proliferation and alters fate specification in adult neural progenitor cells (aNPCs). However, little is known about Fmr1 mRNA regulation at the transcriptional and post-transcriptional levels. In the present study, we report that miR-130b regulated Fmr1 expression by directly targeting its 3′-untranslated region (3′ UTR). Up-regulation of miR-130b in mouse embryonic neural progenitor cells (eNPCs) decreased Fmr1 expression, markedly increased eNPC proliferation and altered the differentiation tendency of eNPCs, suggesting that antagonizing miR-130b may be a new therapeutic entry point for treating Fragile X syndrome

  17. MicroRNA-130b targets Fmr1 and regulates embryonic neural progenitor cell proliferation and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Xi [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China); Zhang, Kunshan [Department of Regenerative Medicine, Stem Cell Center, Tongji University School of Medicine, Shanghai 200092 (China); Wang, Yanlu; Wang, Junbang; Cui, Yaru [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China); Li, Siguang, E-mail: siguangli@163.com [Department of Regenerative Medicine, Stem Cell Center, Tongji University School of Medicine, Shanghai 200092 (China); Luo, Yuping, E-mail: luoyuping@163.com [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China)

    2013-10-04

    Highlights: •We found that the 3′ UTR of the Fmr1 mRNA is a target of miR-130b. •MiR-130b suppresses the expression of Fmr1 in mouse embryonic stem cell. •MiR-130b alters the proliferation of mouse embryonic stem cell. •MiR-130b alters fate specification of mouse embryonic stem cell. -- Abstract: Fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by expansion of the CGG repeat in the 5′-untranslated region of the X-linked Fmr1 gene, which results in transcriptional silencing and loss of expression of its encoded protein FMRP. The loss of FMRP increases proliferation and alters fate specification in adult neural progenitor cells (aNPCs). However, little is known about Fmr1 mRNA regulation at the transcriptional and post-transcriptional levels. In the present study, we report that miR-130b regulated Fmr1 expression by directly targeting its 3′-untranslated region (3′ UTR). Up-regulation of miR-130b in mouse embryonic neural progenitor cells (eNPCs) decreased Fmr1 expression, markedly increased eNPC proliferation and altered the differentiation tendency of eNPCs, suggesting that antagonizing miR-130b may be a new therapeutic entry point for treating Fragile X syndrome.

  18. Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

    Science.gov (United States)

    Gogos, J A; Thompson, R; Lowry, W; Sloane, B F; Weintraub, H; Horwitz, M

    1996-08-01

    To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

  19. Regulation of a TGF-β1-CD147 self-sustaining network in the differentiation plasticity of hepatocellular carcinoma cells.

    Science.gov (United States)

    Wu, J; Lu, M; Li, Y; Shang, Y-K; Wang, S-J; Meng, Y; Wang, Z; Li, Z-S; Chen, H; Chen, Z-N; Bian, H

    2016-10-20

    Cellular plasticity has an important role in the progression of hepatocellular carcinoma (HCC). In this study, the involvement of a TGF-β1-CD147 self-sustaining network in the regulation of the dedifferentiation progress was fully explored in HCC cell lines, hepatocyte-specific basigin/CD147-knockout mice and human HCC tissues. We demonstrated that TGF-β1 stimulation upregulated CD147 expression and mediated the dedifferentiation of HCC cells, whereas all-trans-retinoic acid induced the downregulation of CD147 and promoted differentiation in HCC cells. Overexpression of CD147 induced the dedifferentiation and enhanced the malignancy of HCC cells, and increased the transcriptional expression of TGF-β1 by activating β-catenin. CD147-induced matrix metalloproteinase (MMP) production activated pro-TGF-β1. The activated TGF-β1 signaling subsequently repressed the HNF4α expression via Smad-Snail1 signaling and enhanced the dedifferentiation progress. Hepatocyte-specific basigin/CD147-knockout mice decreased the susceptibility to N-nitrosodiethylamine-induced tumorigenesis by suppressing TGF-β1-CD147 signaling and inhibiting dedifferentiation in hepatocytes during tumor progression. CD147 was positively correlated with TGF-β1 and negatively correlated with HNF4α in human HCC tissues. Positive CD147 staining and lower HNF4α levels in tumor tissues were significantly associated with poor survival of patients with HCC. The overexpression of HNF4α and Smad7 and the deletion of CD147 by lentiviral vectors jointly reprogrammed the expression profile of hepatocyte markers and attenuated malignant properties including proliferation, cell survival and tumor growth of HCC cells. Our results highlight the important role of the TGF-β1-CD147 self-sustaining network in driving HCC development by regulating differentiation plasticity, which provides a strong basis for further investigations of the differentiation therapy of HCC targeting TGF-β1 and CD147.

  20. Estrogen-Induced Maldevelopment of the Penis Involves Down-Regulation of Myosin Heavy Chain 11 (MYH11) Expression, a Biomarker for Smooth Muscle Cell Differentiation1

    Science.gov (United States)

    Okumu, L.A.; Bruinton, Sequoia; Braden, Tim D.; Simon, Liz; Goyal, Hari O.

    2012-01-01

    ABSTRACT Cavernous smooth muscle cells are essential components in penile erection. In this study, we investigated effects of estrogen exposure on biomarkers for smooth muscle cell differentiation in the penis. Neonatal rats received diethylstilbestrol (DES), with or without the estrogen receptor (ESR) antagonist ICI 182,780 (ICI) or the androgen receptor (AR) agonist dihydrotestosterone (DHT), from Postnatal Days 1 to 6. Tissues were collected at 7, 10, or 21 days of age. The smooth muscle cell biomarker MYH11 was studied in depth because microarray data showed it was significantly down-regulated, along with other biomarkers, in DES treatment. Quantitative real time-PCR and Western blot analyses showed 50%–80% reduction (P ≤ 0.05) in Myh11 expression in DES-treated rats compared to that in controls; and ICI and DHT coadministration mitigated the decrease. Temporally, from 7 to 21 days of age, Myh11 expression was onefold increased (P ≥ 0.05) in DES-treated rats versus threefold increased (P ≤ 0.001) in controls, implying the long-lasting inhibitory effect of DES on smooth muscle cell differentiation. Immunohistochemical localization of smooth muscle alpha actin, another biomarker for smooth muscle cell differentiation, showed fewer cavernous smooth muscle cells in DES-treated animals than in controls. Additionally, DES treatment significantly up-regulated Esr1 mRNA expression and suppressed the neonatal testosterone surge by 90%, which was mitigated by ICI coadministration but not by DHT coadministration. Collectively, results provided evidence that DES treatment in neonatal rats inhibited cavernous smooth muscle cell differentiation, as shown by down-regulation of MYH11 expression at the mRNA and protein levels and by reduced immunohistochemical staining of smooth muscle alpha actin. Both the ESR and the AR pathways probably mediate this effect. PMID:22976277

  1. Growth/differentiation factor 15 promotes EGFR signalling, and regulates proliferation and migration in the hippocampus of neonatal and young adult mice.

    Science.gov (United States)

    Carrillo-García, Carmen; Prochnow, Sebastian; Simeonova, Ina K; Strelau, Jens; Hölzl-Wenig, Gabriele; Mandl, Claudia; Unsicker, Klaus; von Bohlen Und Halbach, Oliver; Ciccolini, Francesca

    2014-02-01

    The activation of epidermal growth factor receptor (EGFR) affects multiple aspects of neural precursor behaviour, including proliferation and migration. Telencephalic precursors acquire EGF responsiveness and upregulate EGFR expression at late stages of development. The events regulating this process and its significance are still unclear. We here show that in the developing and postnatal hippocampus (HP), growth/differentiation factor (GDF) 15 and EGFR are co-expressed in primitive precursors as well as in more differentiated cells. We also provide evidence that GDF15 promotes responsiveness to EGF and EGFR expression in hippocampal precursors through a mechanism that requires active CXC chemokine receptor (CXCR) 4. Besides EGFR expression, GDF15 ablation also leads to decreased proliferation and migration. In particular, lack of GDF15 impairs both processes in the cornu ammonis (CA) 1 and only proliferation in the dentate gyrus (DG). Importantly, migration and proliferation in the mutant HP were altered only perinatally, when EGFR expression was also affected. These data suggest that GDF15 regulates migration and proliferation by promoting EGFR signalling in the perinatal HP and represent a first description of a functional role for GDF15 in the developing telencephalon.

  2. Dioxinodehydroeckol Enhances the Differentiation of Osteoblasts by Regulating the Expression of Phospho-Smad1/5/8

    Directory of Open Access Journals (Sweden)

    Byul-Nim Ahn

    2016-09-01

    Full Text Available Lack of bone formation-related health problems are a major problem for the aging population in the modern world. As a part of the ongoing trend of developing natural substances that attenuate osteoporotic bone loss conditions, dioxinodehydroeckol (DHE from edible brown alga Ecklonia cava was tested for its effects on osteoblastogenic differentiation in MC3T3-E1 pre-osteoblasts. DHE was observed to successfully enhance osteoblast differentiation, as indicated by elevated cell proliferation, alkaline phosphatase activity, intracellular cell mineralization, along with raised levels of osteoblastogenesis indicators at the concentration of 20 μM. Results suggested a possible intervening of DHE on the bone morphogenetic protein (BMP signaling pathway, according to elevated protein levels of BMP-2, collagen-I, and Smads. In addition, the presence of DHE was also able to raise the phosphorylated extracellular signal–regulated kinase (ERK and c-Jun N-terminal kinase (JNK levels which are also activated by the BMP signaling pathway. In conclusion, DHE is suggested to be a potential bioactive compound against bone loss that could enhance osteoblastogenesis with a suggested BMP pathway interaction.

  3. The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation.

    Science.gov (United States)

    Linares, Anthony J; Lin, Chia-Ho; Damianov, Andrey; Adams, Katrina L; Novitch, Bennett G; Black, Douglas L

    2015-12-24

    The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development.

  4. Angiotensin II Regulates Th1 T Cell Differentiation Through Angiotensin II Type 1 Receptor-PKA-Mediated Activation of Proteasome.

    Science.gov (United States)

    Qin, Xian-Yun; Zhang, Yun-Long; Chi, Ya-Fei; Yan, Bo; Zeng, Xiang-Jun; Li, Hui-Hua; Liu, Ying

    2018-01-01

    Naive CD4+ T cells differentiate into T helper cells (Th1 and Th2) that play an essential role in the cardiovascular diseases. However, the molecular mechanism by which angiotensin II (Ang II) promotes Th1 differentiation remains unclear. The aim of this study was to determine whether the Ang II-induced Th1 differentiation regulated by ubiquitin-proteasome system (UPS). Jurkat cells were treated with Ang II (100 nM) in the presence or absence of different inhibitors. The gene mRNA levels were detected by real-time quantitative PCR analysis. The protein levels were measured by ELISA assay or Western blot analysis, respectively. Ang II treatment significantly induced a shift from Th0 to Th1 cell differentiation, which was markedly blocked by angiotensin II type 1 receptor (AT1R) inhibitor Losartan (LST). Moreover, Ang II significantly increased the activities and the expression of proteasome catalytic subunits (β1, β1i, β2i and β5i) in a dose- and time-dependent manner. However, Ang II-induced proteasome activities were remarkably abrogated by LST and PKA inhibitor H-89. Mechanistically, Ang II-induced Th1 differentiation was at least in part through proteasome-mediated degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB. This study for the first time demonstrates that Ang II activates AT1R-PKA-proteasome pathway, which promotes degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB thereby leading to Th1 differentiation. Thus, inhibition of proteasome activation might be a potential therapeutic target for Th1-mediated diseases. © 2018 The Author(s). Published by S. Karger AG, Basel.

  5. Current perspectives in Set7 mediated stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Nazanin Karimnia

    2016-12-01

    Full Text Available Set7 is a key regulatory enzyme involved in the methylation of lysine residues of histone and non-histone proteins. This lysine methyltransferase is induced during stem cell differentiation and regulates lineage specific gene transcription and cell fate. In this article we discuss recent experimental evidence identifying regulatory targets under the control of Set7 as well as emerging evidence of regulation in stem cell differentiation. Furthermore, we discuss the function of non-coding RNAs regulated by Set7 implicated in cell plasticity.

  6. Differential regulation of GluA1 expression by ketamine and memantine.

    Science.gov (United States)

    Zhang, Ke; Yamaki, Vitor Nagai; Wei, Zhisheng; Zheng, Yu; Cai, Xiang

    2017-01-01

    Evidence from preclinical and clinical studies shows that ketamine, a noncompetitive NMDA receptor antagonist, exerts rapid and sustained antidepressant responses. However, ketamine's psychotomimetic side effects and abuse liability limit the clinical use of the compound. Interestingly, memantine, another NMDA receptor channel blocker, processes no defined antidepressant property but is much safer and clinical tolerated. Understanding why ketamine but not memantine exhibits rapid antidepressant responses is important to elucidate the cellular signaling underlying the fast antidepressant actions of ketamine and to design a new safer generation of fast-acting antidepressants. Here we show that ketamine but memantine caused a rapid and sustained antidepressant-like responses in forced swim test (FST). Both drugs enhanced GluA1 S845 phosphorylation and potentiated Schaffer collateral-CA1 synaptic transmission. However, ketamine but not memantine elevated the expression of GluA1. Incubating acutely prepared hippocampal slices with ketamine but not memantine enhanced mTOR phosphorylation in a time course parallel to the time course of GluA1 elevation. Our results suggest that distinct properties in regulation of mTOR phosphorylation and synaptic protein expression may underlie the differential effectiveness of ketamine and memantine in their antidepressant responses. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Bone morphogenetic protein signaling and olig1/2 interact to regulate the differentiation and maturation of adult oligodendrocyte precursor cells.

    Science.gov (United States)

    Cheng, Xiaoxin; Wang, Yaping; He, Qian; Qiu, Mengsheng; Whittemore, Scott R; Cao, Qilin

    2007-12-01

    Promotion of remyelination is an important therapeutic strategy for the treatment of the demyelinating neurological disorders. Adult oligodendrocyte precursor cells (OPCs), which normally reside quiescently in the adult central nervous system (CNS), become activated and proliferative after demyelinating lesions. However, the extent of endogenous remyelination is limited because of the failure of adult OPCs to mature into myelinating oligodendrocytes (OLs) in the demyelinated CNS. Understanding the molecular mechanisms that regulate the differentiation of adult OPCs could lead to new therapeutic strategies to treat these disorders. In this study, we established a stable culture of adult spinal cord OPCs and developed a reliable in vitro protocol to induce their sequential differentiation. Adult OPCs expressed bone morphogenetic protein (BMP) type Ia, Ib, and II receptor subunits, which are required for BMP signal transduction. BMP2 and 4 promoted dose-dependent astrocyte differentiation of adult OPCs with concurrent suppression of OL differentiation. Treatment of OPCs with BMP2 and 4 increased ID4 expression and decreased the expression of olig1 and olig2. Overexpression of olig1 or olig2 blocked the astrocyte differentiation of adult OPCs induced by BMP2 and 4. Furthermore, overexpression of both olig1 and olig2, but not olig1 or olig2 alone, rescued OL differentiation from inhibition by BMP2 and 4. Our results demonstrated that downregulation of olig1 and olig2 is an important mechanism by which BMP2 and 4 inhibit OL differentiation of adult OPCs. These data suggest that blocking BMP signaling combined with olig1/2 overexpression could be a useful therapeutic strategy to enhance endogenous remyelination and facilitate functional recovery in CNS demyelinated disorders. Disclosure of potential conflicts of interest is found at the end of this article.

  8. Differential feedback regulation of ethylene biosynthesis in pulp and peel tissues of banana fruit.

    Science.gov (United States)

    Inaba, Akitsugu; Liu, Xuejun; Yokotani, Naoki; Yamane, Miki; Lu, Wang-Jin; Nakano, Ryohei; Kubo, Yasutaka

    2007-01-01

    The feedback regulation of ethylene biosynthesis in banana [Musa sp. (AAA group, Cavendish subgroup) cv. Grand Nain] fruit was investigated in an attempt to clarify the opposite effect of 1-methylcyclopropene (1-MCP), an ethylene action inhibitor, before and after the onset of ripening. 1-MCP pre-treatment completely prevented the ripening-induced effect of propylene in pre-climacteric banana fruit, whereas treatment after the onset of ripening stimulated ethylene production. In pre-climacteric fruit, higher concentrations of propylene suppressed ethylene production more strongly, despite their earlier ethylene-inducing effect. Exposure of the fruit ripened by propylene to 1-MCP increased ethylene production concomitantly with an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC content, and prevented a transient decrease in MA-ACS1 transcripts in the pulp tissues. In contrast, in the peel of ripening fruit, 1-MCP prevented the increase in ethylene production and subsequently the ripening process by reduction of the increase in MA-ACS1 and MA-ACO1 transcripts and of ACC synthase and ACC oxidase activities. These results suggest that ethylene biosynthesis in ripening banana fruit may be controlled negatively in the pulp tissue and positively in the peel tissue. This differential regulation by ethylene in pulp and peel tissues was also observed for MA-PL, MA-Exp, and MA-MADS genes.

  9. Rho GTPases in ameloblast differentiation

    Directory of Open Access Journals (Sweden)

    Keishi Otsu

    2016-05-01

    Full Text Available During tooth development, ameloblasts differentiate from inner enamel epithelial cells to enamel-forming cells by modulating the signal pathways mediating epithelial–mesenchymal interaction and a cell-autonomous gene network. The differentiation process of epithelial cells is characterized by marked changes in their morphology and polarity, accompanied by dynamic cytoskeletal reorganization and changes in cell–cell and cell–matrix adhesion over time. Functional ameloblasts are tall, columnar, polarized cells that synthesize and secrete enamel-specific proteins. After deposition of the full thickness of enamel matrix, ameloblasts become smaller and regulate enamel maturation. Recent significant advances in the fields of molecular biology and genetics have improved our understanding of the regulatory mechanism of the ameloblast cell life cycle, mediated by the Rho family of small GTPases. They act as intracellular molecular switch that transduce signals from extracellular stimuli to the actin cytoskeleton and the nucleus. In our review, we summarize studies that provide current evidence for Rho GTPases and their involvement in ameloblast differentiation. In addition to the Rho GTPases themselves, their downstream effectors and upstream regulators have also been implicated in ameloblast differentiation.

  10. Effect of auxin on xylem tracheids differentiation in decapitated stems of Pinus silvestris L. and its interaction with some vitamins and growth regulators

    Directory of Open Access Journals (Sweden)

    T. J. Wodzicki

    2015-01-01

    Full Text Available The effects of several vitamins and substances known as important agents in regulation of cell metabolism upon secondary xylem differentiation were studied in interaction with auxin (IAA as applied in lanoline to decapitated stems of 5-year-old Pinus silvestris trees in early and late-summer. Tested substances were: gibberellic acid, kinetin, nicotinic acid, thiamine, pyridoxine, calcium panthotenate, choline chloride, riboflavin, inositol, ascorbic acid, vitamin, A (alcohol, vitamin A (ester, saponin. None of the effects of these substances appeared significant enough to indicate the involvement in the seasonal variation of the response of cambium or differentiating tracheids to auxin. However, several effects, especially those of inositol, vitamin A and pyridoxine upon cambial xylem production and further stages of tracheid differentiation were observed. Auxin (IAA affected cambial activity and subsequent differentiation of tracheids during the earliest stages of cell ontogenesis. At these stages auxin treatment induced quantitative expression of the developmental processes involving radial growth and secondary wall formation by tracheids. In this respect, auxin did not affect cells advanced in differentiation, however, it proved to be an essential factor in the completion of the full cycle of tracheid ontogenesis.

  11. Proliferation and osteo/odontogenic differentiation of stem cells from apical papilla regulated by Zinc fingers and homeoboxes 2: An in vitro study

    International Nuclear Information System (INIS)

    Wan, Fang; Gao, Lifen; Lu, Yating; Ma, Hongxin; Wang, Hongxing; Liang, Xiaohong; Wang, Yan; Ma, Chunhong

    2016-01-01

    In the process of tooth root development, stem cells from the apical papilla (SCAPs) can differentiate into odontoblasts and form root dentin, however, molecules regulating SCAPs differentiation have not been elucidated. Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. It is reported to modulate the development of nerve cells, liver cells, B cells, red blood cells, and so on. However, the role of ZHX2 in tooth root development remains unclear. In this study, we explored the potential role of ZHX2 in the process of SCAPs differentiation. The results showed that overexpression of ZHX2 upregulated the expression of osteo/odontogenic related genes and ALP activity, inhibited the proliferation of SCAPs. Consistently, ZHX2 knockdown reduced SCAPs mineralization and promoted SCAPs proliferation. These results indicated that ZHX2 plays a critical role in the proliferation and osteo/odontogenic differentiation of SCAPs. - Highlights: • Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. • we found another new biological function of ZHX2 for the first time. • ZHX2 inhibit SCAPs proliferation. • ZHX2 promote the osteo/odontogenic differentiation of SCAPs.

  12. Proliferation and osteo/odontogenic differentiation of stem cells from apical papilla regulated by Zinc fingers and homeoboxes 2: An in vitro study

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Fang [Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); VIP Center, Shandong Provincial Key Laboratory of Oral Biomedicine, School and Hospital of Stomatology, Shandong University, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); Gao, Lifen [Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); Lu, Yating [VIP Center, Shandong Provincial Key Laboratory of Oral Biomedicine, School and Hospital of Stomatology, Shandong University, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); Ma, Hongxin; Wang, Hongxing; Liang, Xiaohong [Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); Wang, Yan, E-mail: wangyan1965@sdu.edu.cn [VIP Center, Shandong Provincial Key Laboratory of Oral Biomedicine, School and Hospital of Stomatology, Shandong University, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); Ma, Chunhong, E-mail: machunhong@sdu.edu.cn [Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China)

    2016-01-15

    In the process of tooth root development, stem cells from the apical papilla (SCAPs) can differentiate into odontoblasts and form root dentin, however, molecules regulating SCAPs differentiation have not been elucidated. Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. It is reported to modulate the development of nerve cells, liver cells, B cells, red blood cells, and so on. However, the role of ZHX2 in tooth root development remains unclear. In this study, we explored the potential role of ZHX2 in the process of SCAPs differentiation. The results showed that overexpression of ZHX2 upregulated the expression of osteo/odontogenic related genes and ALP activity, inhibited the proliferation of SCAPs. Consistently, ZHX2 knockdown reduced SCAPs mineralization and promoted SCAPs proliferation. These results indicated that ZHX2 plays a critical role in the proliferation and osteo/odontogenic differentiation of SCAPs. - Highlights: • Zinc fingers and homeoboxes 2 (ZHX2) is a novel transcriptional inhibitor. • we found another new biological function of ZHX2 for the first time. • ZHX2 inhibit SCAPs proliferation. • ZHX2 promote the osteo/odontogenic differentiation of SCAPs.

  13. Regulation of insulin-like growth factor (IGF) I receptor expression during muscle cell differentiation. Potential autocrine role of IGF-II.

    OpenAIRE

    Rosenthal, S M; Brunetti, A; Brown, E J; Mamula, P W; Goldfine, I D

    1991-01-01

    Muscle is an important target tissue for insulin-like growth factor (IGF) action. The presence of specific, high affinity IGF receptors, as well as the expression of IGF peptides and binding proteins by muscle suggest that a significant component of IGF action in this tissue is mediated through autocrine and/or paracrine mechanisms. To explore autocrine/paracrine action of IGFs in muscle, we studied the regulation of the IGF-I receptor and the expression of IGF peptides during differentiation...

  14. Cancer stem cells and differentiation therapy.

    Science.gov (United States)

    Jin, Xiong; Jin, Xun; Kim, Hyunggee

    2017-10-01

    Cancer stem cells can generate tumors from only a small number of cells, whereas differentiated cancer cells cannot. The prominent feature of cancer stem cells is its ability to self-renew and differentiate into multiple types of cancer cells. Cancer stem cells have several distinct tumorigenic abilities, including stem cell signal transduction, tumorigenicity, metastasis, and resistance to anticancer drugs, which are regulated by genetic or epigenetic changes. Like normal adult stem cells involved in various developmental processes and tissue homeostasis, cancer stem cells maintain their self-renewal capacity by activating multiple stem cell signaling pathways and inhibiting differentiation signaling pathways during cancer initiation and progression. Recently, many studies have focused on targeting cancer stem cells to eradicate malignancies by regulating stem cell signaling pathways, and products of some of these strategies are in preclinical and clinical trials. In this review, we describe the crucial features of cancer stem cells related to tumor relapse and drug resistance, as well as the new therapeutic strategy to target cancer stem cells named "differentiation therapy."

  15. Matrix Metalloproteinases Are Differentially Regulated and Responsive to Compression Therapy in a Red Duroc Model of Hypertrophic Scar.

    Science.gov (United States)

    Travis, Taryn E; Ghassemi, Pejhman; Prindeze, Nicholas J; Moffatt, Lauren T; Carney, Bonnie C; Alkhalil, Abdulnaser; Ramella-Roman, Jessica C; Shupp, Jeffrey W

    2018-01-01

    Objective: Proteins of the matrix metalloproteinases family play a vital role in extracellular matrix maintenance and basic physiological processes in tissue homeostasis. The function and activities of matrix metalloproteinases in response to compression therapies have yet to be defined. Here, a swine model of hypertrophic scar was used to profile the transcription of all known 26 matrix metalloproteinases in scars treated with a precise compression dose. Methods: Full-thickness excisional wounds were created. Wounds underwent healing and scar formation. A subset of scars underwent 2 weeks of compression therapy. Biopsy specimens were preserved, and microarrays, reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry were performed to characterize the transcription and expression of various matrix metalloproteinase family members. Results: Microarray results showed that 13 of the known 26 matrix metalloproteinases were differentially transcribed in wounds relative to the preinjury skin. The predominant upregulation of these matrix metalloproteinases during early wound-healing stages declined gradually in later stages of wound healing. The use of compression therapy reduced this decline in 10 of the 13 differentially regulated matrix metalloproteinases. Further investigation of MMP7 using reverse transcription-polymerase chain reaction confirmed the effect of compression on transcript levels. Assessment of MMP7 at the protein level using Western blotting and immunohistochemistry was concordant. Conclusions: In a swine model of hypertrophic scar, the application of compression to hypertrophic scar attenuated a trend of decreasing levels of matrix metalloproteinases during the process of hypertrophic wound healing, including MMP7, whose enzyme regulation was confirmed at the protein level.

  16. GSK3 is a regulator of RAR-mediated differentiation

    Science.gov (United States)

    Gupta, K; Gulen, F; Sun, L; Aguilera, R; Chakrabarti, A; Kiselar, J; Agarwal, MK; Wald, DN

    2015-01-01

    Acute myeloid leukemia (AML) is the most common form of leukemia in adults. Unfortunately, the standard therapeutic agents used for this disease have high toxicities and poor efficacy. The one exception to these poor outcomes is the use of the retinoid, all-trans retinoic acid (ATRA), for a rare subtype of AML (APL). The use of the differentiation agent, ATRA, in combination with low-dose chemotherapy leads to the long-term survival and presumed cure of 75–85% of patients. Unfortunately ATRA has not been clinically useful for other subtypes of AML. Though many non-APL leukemic cells respond to ATRA, they require significantly higher concentrations of ATRA for effective differentiation. Here we show that the combination of ATRA with glycogen synthase kinase 3 (GSK3) inhibition significantly enhances ATRA-mediated AML differentiation and growth inhibition. These studies have revealed that ATRA's receptor, the retinoic acid receptor (RAR), is a novel target of GSK3 phosphorylation and that GSK3 can impact the expression and transcriptional activity of the RAR. Overall, our studies suggest the clinical potential of ATRA and GSK3 inhibition for AML and provide a mechanistic framework to explain the promising activity of this combination regimen. PMID:22222598

  17. Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

    Science.gov (United States)

    Imamura, Katsuyuki; Maeda, Shingo; Kawamura, Ichiro; Matsuyama, Kanehiro; Shinohara, Naohiro; Yahiro, Yuhei; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

    2014-04-04

    Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

  18. Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

    Science.gov (United States)

    Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

    2012-01-01

    Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

  19. Biglycan deficiency increases osteoclast differentiation and activity due to defective osteoblasts

    DEFF Research Database (Denmark)

    Bi, Yanming; Nielsen, Karina L; Kilts, Tina M

    2006-01-01

    to be independent of the differential production of soluble RANKL and OPG and, instead, due to a decrease in osteoblast maturation accompanied by increase in osteoblastic proliferation. In addition to the imbalance between differentiation and proliferation, there was a differential decrease in secretory leukocyte......Bone mass is maintained by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. Although osteoblasts and osteoclasts have different developmental origins, it is generally believed that the differentiation, function, and survival of osteoclasts are regulated...... by osteogenic cells. We have previously shown that the extracellular matrix protein, biglycan (Bgn), plays an important role in the differentiation of osteoblast precursors. In this paper, we showed that Bgn is involved in regulating osteoclast differentiation through its effect on osteoblasts...

  20. Opposing regulation of PROX1 by interleukin-3 receptor and NOTCH directs differential host cell fate reprogramming by Kaposi sarcoma herpes virus.

    Directory of Open Access Journals (Sweden)

    Jaehyuk Yoo

    Full Text Available Lymphatic endothelial cells (LECs are differentiated from blood vascular endothelial cells (BECs during embryogenesis and this physiological cell fate specification is controlled by PROX1, the master regulator for lymphatic development. When Kaposi sarcoma herpes virus (KSHV infects host cells, it activates the otherwise silenced embryonic endothelial differentiation program and reprograms their cell fates. Interestingly, previous studies demonstrated that KSHV drives BECs to acquire a partial lymphatic phenotype by upregulating PROX1 (forward reprogramming, but stimulates LECs to regain some BEC-signature genes by downregulating PROX1 (reverse reprogramming. Despite the significance of this KSHV-induced bidirectional cell fate reprogramming in KS pathogenesis, its underlying molecular mechanism remains undefined. Here, we report that IL3 receptor alpha (IL3Rα and NOTCH play integral roles in the host cell type-specific regulation of PROX1 by KSHV. In BECs, KSHV upregulates IL3Rα and phosphorylates STAT5, which binds and activates the PROX1 promoter. In LECs, however, PROX1 was rather downregulated by KSHV-induced NOTCH signal via HEY1, which binds and represses the PROX1 promoter. Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1. Upon co-activation of IL3Rα and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs. Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV.

  1. Keratinocyte proliferation, differentiation, and apoptosis-Differential mechanisms of regulation by curcumin, EGCG and apigenin

    International Nuclear Information System (INIS)

    Balasubramanian, Sivaprakasam; Eckert, Richard L.

    2007-01-01

    We have proposed that it is important to examine the impact of chemopreventive agents on the function of normal human epidermal keratinocytes since these cells comprise the barrier that protects the body from a range of environmental insults. In this context, it is widely appreciated that cancer may be retarded by consumption or topical application of naturally occurring food-derived chemopreventive agents. Our studies show that (-)-epigallocatechin-3-gallate (EGCG), a green tea-derived polyphenol, acts to enhance the differentiation of normal human keratinocytes as evidenced by its ability to increase involucrin (hINV), transglutaminase type 1 (TG1) and caspase-14 gene expression. EGCG also stimulates keratinocyte morphological differentiation. These actions of EGCG are mediated via activation of a nPKC, Ras, MEKK1, MEK3, p38δ-ERK1/2 signaling cascade which leads to increased activator protein 1 (AP1) and CAATT enhancer binding protein (C/EBP) transcription factor expression, increased binding of these factors to DNA, and increased gene transcription. In contrast, apigenin, a dietary flavonoid derived from plants and vegetables, and curcumin, an agent derived from turmeric, inhibit differentiation by suppressing MAPK signal transduction and reducing API transcription factor level. Curcumin also acts to enhance apoptosis, although EGCG and apigenin do not stimulate apoptosis. In addition, all of these agents inhibit keratinocyte proliferation. These findings indicate that each of these diet-derived chemopreventive agents has a profound impact on normal human keratinocyte function and that they operate via distinct and sometimes opposing mechanisms. However, all are expected to act as chemopreventive agents

  2. Role of Alternative Polyadenylation during Adipogenic Differentiation: An In Silico Approach

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    Spangenberg, Lucía; Correa, Alejandro; Dallagiovanna, Bruno; Naya, Hugo

    2013-01-01

    Post-transcriptional regulation of stem cell differentiation is far from being completely understood. Changes in protein levels are not fully correlated with corresponding changes in mRNAs; the observed differences might be partially explained by post-transcriptional regulation mechanisms, such as alternative polyadenylation. This would involve changes in protein binding, transcript usage, miRNAs and other non-coding RNAs. In the present work we analyzed the distribution of alternative transcripts during adipogenic differentiation and the potential role of miRNAs in post-transcriptional regulation. Our in silico analysis suggests a modest, consistent, bias in 3′UTR lengths during differentiation enabling a fine-tuned transcript regulation via small non-coding RNAs. Including these effects in the analyses partially accounts for the observed discrepancies in relative abundance of protein and mRNA. PMID:24143171

  3. Repression of TSC1/TSC2 mediated by MeCP2 regulates human embryo lung fibroblast cell differentiation and proliferation.

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    Wang, Yuanyuan; Chen, Chen; Deng, Ziyu; Bian, Erbao; Huang, Cheng; Lei, Ting; Lv, Xiongwen; Liu, Liping; Li, Jun

    2017-03-01

    Pulmonary fibrosis (PF) is a severe inflammatory disease with limited effective treatments. It is known that the transdifferentiation of human embryo lung fibroblast (HELF) cells from pulmonary fibroblasts into myofibroblasts, contributes to the progression of pulmonary fibrogenesis. The tuberous sclerosis proteins TSC1 and TSC2 are two key signaling factors which can suppress cell growth and proliferation. However, the roles of TSC1 and TSC2 in lung fibroblast are unclear. Here, we developed a PF model with bleomycin (BLM) in mice and conducted several simulation experiments in HELF cells. Our study shows that the expression of TSC1 and TSC2 in fibrotic mice lung was reduced and stimulation of HELF cells with TGF-β1 resulted in a down-regulation of TSC1 and TSC2. In addition, overexpression of TSC1 or TSC2 decreased cell proliferation and differentiation. Furthermore, we found that reduced expression of TSC1 and TSC2 caused by TGF-β1 is associated with the promoter methylation status of TSC1 and TSC2. MeCP2, controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis. We found that expression of TSC1 and TSC2 can be repressed by MeCP2, which regulates HELF cell differentiation and proliferation as myofibroblasts and lead to PF ultimately. Copyright © 2016. Published by Elsevier B.V.

  4. Human mesenchymal stem cell proliferation is regulated by PGE2 through differential activation of cAMP-dependent protein kinase isoforms

    International Nuclear Information System (INIS)

    Kleiveland, Charlotte Ramstad; Kassem, Moustapha; Lea, Tor

    2008-01-01

    The conditions used for in vitro differentiation of hMSCs contain substances that affect the activity and expression of cyclooxygenase enzymes (COX1/COX2) and thereby the synthesis of prostanoids. hMSC constitutively produce PGE2 when cultivated in vitro. In this study we have investigated effects of PGE2 on proliferation of hMSC. We here demonstrate that one of the main control molecules in the Wnt pathway, GSK-3β, is phosphorylated at the negative regulatory site ser-9 after treating the cells with PGE2. This phosphorylation is mediated by elevation of cAMP and subsequent activation of PKA. Furthermore, PGE2 treatment leads to enhanced nuclear translocation of β-catenin, thus influencing cell proliferation. The presence of two PKA isoforms, types I and II, prompted us to investigate their individual contribution in PGE2-mediated regulation of proliferation. Specific activation of PKA type II with synthetic cAMP analogues, resulted in enhancement of proliferation. On the other side, we found that treatment of hMSC with high concentrations of PGE2 inhibited cell proliferation by arresting the cells in G 0 /G 1 phase, an effect we found to be mediated by PKA I. Hence, the two different PKA isoforms seem to have opposing functions in the regulation of proliferation and differentiation in these cells

  5. Transcriptomic Analysis of Flower Bud Differentiation in Magnolia sinostellata

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    Lijie Fan

    2018-04-01

    Full Text Available Magnolias are widely cultivated for their beautiful flowers, but despite their popularity, the molecular mechanisms regulating flower bud differentiation have not been elucidated. Here, we used paraffin sections and RNA-seq to study the process of flower bud differentiation in Magnolia sinostellata. Flower bud development occurred between 28 April and 30 May 2017 and was divided into five stages: undifferentiated, early flower bud differentiation, petal primordium differentiation, stamen primordium differentiation, and pistil primordium differentiation. A total of 52,441 expressed genes were identified, of which 11,592 were significantly differentially expressed in the five bud development stages. Of these, 82 genes were involved in the flowering. In addition, MADS-box and AP2 family genes play critical roles in the formation of flower organs and 20 differentially expressed genes associated with flower bud differentiation were identified in M. sinostellata. A qRT-PCR analysis verified that the MADS-box and AP2 family genes were expressed at high levels during flower bud differentiation. Consequently, this study provides a theoretical basis for the genetic regulation of flowering in M. sinostellata, which lays a foundation for further research into flowering genes and may facilitate the development of new cultivars.

  6. Differential Regulation of Receptor Activation and Agonist Selectivity by Highly Conserved Tryptophans in the Nicotinic Acetylcholine Receptor Binding Site

    OpenAIRE

    Williams, Dustin K.; Stokes, Clare; Horenstein, Nicole A.; Papke, Roger L.

    2009-01-01

    We have shown previously that a highly conserved Tyr in the nicotinic acetylcholine receptor (nAChR) ligand-binding domain (LBD) (α7 Tyr188 or α4 Tyr195) differentially regulates the activity of acetylcholine (ACh) and the α7-selective agonist 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) in α4β2 and α7 nAChR. In this study, we mutated two highly conserved LBD Trp residues in human α7 and α4β2 and expressed the receptors in Xenopus laevis oocytes. α7 Re...

  7. Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation

    International Nuclear Information System (INIS)

    Vezeridis, Peter S.; Semeins, Cornelis M.; Chen Qian; Klein-Nulend, Jenneke

    2006-01-01

    Osteocytes are thought to orchestrate bone remodeling, but it is unclear exactly how osteocytes influence neighboring bone cells. Here, we tested whether osteocytes, osteoblasts, and periosteal fibroblasts subjected to pulsating fluid flow (PFF) produce soluble factors that modulate the proliferation and differentiation of cultured osteoblasts and periosteal fibroblasts. We found that osteocyte PFF conditioned medium (CM) inhibited bone cell proliferation, and osteocytes produced the strongest inhibition of proliferation compared to osteoblasts and periosteal fibroblasts. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the inhibitory effects of osteocyte PFF CM, suggesting that a change in NO release is at least partially responsible for the inhibitory effects of osteocyte PFF CM. Furthermore, osteocyte PFF CM stimulated osteoblast differentiation measured as increased alkaline phosphatase activity, and L-NAME decreased the stimulatory effects of osteocyte PFF CM on osteoblast differentiation. We conclude that osteocytes subjected to PFF inhibit proliferation but stimulate differentiation of osteoblasts in vitro via soluble factors and that the release of these soluble factors was at least partially dependent on the activation of a NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast with respect to the production of soluble signaling molecules affecting osteoblast proliferation and differentiation

  8. Dopamine receptors D3 and D5 regulate CD4(+)T-cell activation and differentiation by modulating ERK activation and cAMP production.

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    Franz, Dafne; Contreras, Francisco; González, Hugo; Prado, Carolina; Elgueta, Daniela; Figueroa, Claudio; Pacheco, Rodrigo

    2015-07-15

    Dopamine receptors have been described in T-cells, however their signalling pathways coupled remain unknown. Since cAMP and ERKs play key roles regulating T-cell physiology, we aim to determine whether cAMP and ERK1/2-phosphorylation are modulated by dopamine receptor 3 (D3R) and D5R, and how this modulation affects CD4(+) T-cell activation and differentiation. Our pharmacologic and genetic evidence shows that D3R-stimulation reduced cAMP levels and ERK2-phosphorylation, consequently increasing CD4(+) T-cell activation and Th1-differentiation, respectively. Moreover, D5R expression reinforced TCR-triggered ERK1/2-phosphorylation and T-cell activation. In conclusion, these findings demonstrate how D3R and D5R modulate key signalling pathways affecting CD4(+) T-cell activation and Th1-differentiation. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Targeting sarcoma tumor-initiating cells through differentiation therapy

    Directory of Open Access Journals (Sweden)

    Dan Han

    2017-05-01

    Full Text Available Human leukocyte antigen class I (HLA-I down-regulation has been reported in many human cancers to be associated with poor clinical outcome. However, its connection to tumor-initiating cells (TICs remains unknown. In this study, we report that HLA-I is down-regulated in a subpopulation of cells that have high tumor initiating capacity in different types of human sarcomas. Detailed characterization revealed their distinct molecular profiles regarding proliferation, apoptosis and stemness programs. Notably, these TICs can be induced to differentiate along distinct mesenchymal lineages, including the osteogenic pathway. The retinoic acid receptor signaling pathway is overexpressed in HLA-1 negative TICs. All-trans retinoic acid treatment successfully induced osteogenic differentiation of this subpopulation, in vitro and in vivo, resulting in significantly decreased tumor formation. Thus, our findings indicate down-regulated HLA-I is a shared feature of TICs in a variety of human sarcomas, and differentiation therapy strategies may specifically target undifferentiated TICs and inhibit tumor formation.

  10. Hematopoietic stem/progenitor cell proliferation and differentiation is differentially regulated by high-density and low-density lipoproteins in mice.

    Directory of Open Access Journals (Sweden)

    Yingmei Feng

    Full Text Available RATIONALE: Hematopoietic stem/progenitor cells (HSPC are responsible for maintaining the blood system as a result of their self-renewal and multilineage differentiation capacity. Recently, studies have suggested that HDL cholesterol may inhibit and impaired cholesterol efflux may increase HSPC proliferation and differentiation. OBJECTIVES: We hypothesized that LDL may enhance HSPC proliferation and differentiation while HDL might have the opposing effect which might influence the size of the pool of inflammatory cells. METHODS AND RESULTS: HSPC number and function were studied in hypercholesterolemic LDL receptor knockout (LDLr(-/- mice on high fat diet. Hypercholesterolemia was associated with increased frequency of HSPC, monocytes and granulocytes in the peripheral blood (PB. In addition, an increased proportion of BM HSPC was in G(2M of the cell cycle, and the percentage of HSPC and granulocyte-macrophage progenitors (GMP increased in BM of LDLr(-/- mice. When BM Lin-Sca-1+cKit+ (i.e. "LSK" cells were cultured in the presence of LDL in vitro we also found enhanced differentiation towards monocytes and granulocytes. Furthermore, LDL promoted lineage negative (Lin- cells motility. The modulation by LDL on HSPC differentiation into granulocytes and motility was inhibited by inhibiting ERK phosphorylation. By contrast, when mice were infused with human apoA-I (the major apolipoprotein of HDL or reconstituted HDL (rHDL, the frequency and proliferation of HSPC was reduced in BM in vivo. HDL also reversed the LDL-induced monocyte and granulocyte differentiation in vitro. CONCLUSION: Our data suggest that LDL and HDL have opposing effects on HSPC proliferation and differentiation. It will be of interest to determine if breakdown of HSPC homeostasis by hypercholesterolemia contributes to inflammation and atherosclerosis progression.

  11. Factors that regulate embryonic gustatory development

    Directory of Open Access Journals (Sweden)

    Krimm Robin F

    2007-09-01

    Full Text Available Abstract Numerous molecular factors orchestrate the development of the peripheral taste system. The unique anatomy/function of the taste system makes this system ideal for understanding the mechanisms by which these factors function; yet the taste system is underutilized for this role. This review focuses on some of the many factors that are known to regulate gustatory development, and discusses a few topics where more work is needed. Some attention is given to factors that regulate epibranchial placode formation, since gustatory neurons are thought to be primarily derived from this region. Epibranchial placodes appear to arise from a pan-placodal region and a number of regulatory factors control the differentiation of individual placodes. Gustatory neuron differentiation is regulated by a series of transcription factors and perhaps bone morphongenic proteins (BMP. As neurons differentiate, they also proliferate such that their numbers exceed those in the adult, and this is followed by developmental death. Some of these cell-cycling events are regulated by neurotrophins. After gustatory neurons become post-mitotic, axon outgrowth occurs. Axons are guided by multiple chemoattractive and chemorepulsive factors, including semaphorins, to the tongue epithelium. Brain derived neurotrophic factor (BDNF, functions as a targeting factor in the final stages of axon guidance and is required for gustatory axons to find and innervate taste epithelium. Numerous factors are involved in the development of gustatory papillae including Sox-2, Sonic hedge hog and Wnt-β-catenin signaling. It is likely that just as many factors regulate taste bud differentiation; however, these factors have not yet been identified. Studies examining the molecular factors that regulate terminal field formation in the nucleus of the solitary tract are also lacking. However, it is possible that some of the factors that regulate geniculate ganglion development, outgrowth, guidance and

  12. The retinoblastoma protein regulates hypoxia-inducible genetic programs, tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells

    Science.gov (United States)

    Labrecque, Mark P.; Takhar, Mandeep K.; Nason, Rebecca; Santacruz, Stephanie; Tam, Kevin J.; Massah, Shabnam; Haegert, Anne; Bell, Robert H.; Altamirano-Dimas, Manuel; Collins, Colin C.; Lee, Frank J.S.; Prefontaine, Gratien G.; Cox, Michael E.; Beischlag, Timothy V.

    2016-01-01

    Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies. PMID:27015368

  13. Metabolic reprogramming during neuronal differentiation.

    Science.gov (United States)

    Agostini, M; Romeo, F; Inoue, S; Niklison-Chirou, M V; Elia, A J; Dinsdale, D; Morone, N; Knight, R A; Mak, T W; Melino, G

    2016-09-01

    Newly generated neurons pass through a series of well-defined developmental stages, which allow them to integrate into existing neuronal circuits. After exit from the cell cycle, postmitotic neurons undergo neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. Lack of a global metabolic analysis during early cortical neuronal development led us to explore the role of cellular metabolism and mitochondrial biology during ex vivo differentiation of primary cortical neurons. Unexpectedly, we observed a huge increase in mitochondrial biogenesis. Changes in mitochondrial mass, morphology and function were correlated with the upregulation of the master regulators of mitochondrial biogenesis, TFAM and PGC-1α. Concomitant with mitochondrial biogenesis, we observed an increase in glucose metabolism during neuronal differentiation, which was linked to an increase in glucose uptake and enhanced GLUT3 mRNA expression and platelet isoform of phosphofructokinase 1 (PFKp) protein expression. In addition, glutamate-glutamine metabolism was also increased during the differentiation of cortical neurons. We identified PI3K-Akt-mTOR signalling as a critical regulator role of energy metabolism in neurons. Selective pharmacological inhibition of these metabolic pathways indicate existence of metabolic checkpoint that need to be satisfied in order to allow neuronal differentiation.

  14. Methylation of miR-145a-5p promoter mediates adipocytes differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Du, Jingjing; Cheng, Xiao; Shen, Linyuan; Tan, Zhendong; Luo, Jia; Wu, Xiaoqian; Liu, Chendong [College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130 (China); Yang, Qiong [Department of Animal Husbandry and Veterinary Medicine, Chengdu Agricultural College, Chengdu 611100, Sichuan (China); Jiang, Yanzhi [College of Life and Science, Sichuan Agricultural University, Chengdu 611130 (China); Tang, Guoqing; Li, Xuewei [College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130 (China); Zhang, Shunhua, E-mail: zhangsh1919@163.com [College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130 (China); Zhu, Li, E-mail: zhuli7508@163.com [College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130 (China)

    2016-06-17

    MicroRNAs (miRNAs, miR) play important roles in adipocyte development. Recent studies showed that the expression of several miRNAs is closely related with promoter methylation. However, it is not known whether miRNA mediates adipocytes differentiation by means of DNA methylation. Here, we showed that miR-145a-5p was poorly expressed in adipose tissue from mice fed a high fat diet (HFD). Overexpression or inhibition of miR-145a-5p was unfavorable or beneficial, respectively, for adipogenesis, and these effects were achieved by regulating adipocyte-specific genes involved in lipogenic transcription, fatty acid synthesis, and fatty acid transportation. Particularly, we first suggested that miR-145a-5p mimics or inhibitors promoted or repressed adipocytes proliferation by regulating p53 and p21, which act as cell cycle regulating factors. Surprisingly, the miR-145a-5p-repressed adipocyte differentiation was enhanced or rescued when cells treated with 5-Aza-dC were transfected with miR-145a-5p mimics or inhibitors, respectively. These data indicated that, as a new mean to positively regulate adipocyte proliferation, the process of miR-145a-5p-inhibited adipogenesis may be regulated by DNA methylation. -- Highlights: •MiR-145a-5p promotes adipocytes proliferation. •MiR-145a-5p is negatively correlated with obesity. •MiR-145a-5p mediates adipocytes differentiation via regulating pathway related adipocytes differentiation. MiR-145a-5p mediating adipocytes differentiation was regulated by DNA methylation.

  15. Let7a involves in neural stem cell differentiation relating with TLX level.

    Science.gov (United States)

    Song, Juhyun; Cho, Kyoung Joo; Oh, Yumi; Lee, Jong Eun

    2015-07-10

    Neural stem cells (NSCs) have the potential for differentiation into neurons known as a groundbreaking therapeutic solution for central nervous system (CNS) diseases. To resolve the therapeutic efficiency of NSCs, recent researchers have focused on the study on microRNA's role in CNS. Some micro RNAs have been reported significant functions in NSC self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. MicroRNA-Let7a (Let7a) has known as the regulator of diverse cellular mechanisms including cell differentiation and proliferation. In present study, we investigated whether Let7a regulates NSC differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal, proliferation and differentiation. We performed the following experiments: western blot analysis, TaqMan assay, RT-PCR, and immunocytochemistry to confirm the alteration of NSCs. Our data showed that let7a play important roles in controlling NSC fate determination. Thus, manipulating Let-7A and TLX could be a novel strategy to enhance the efficiency of NSC's neuronal differentiation for CNS disorders. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Review of Signaling Pathways Governing MSC Osteogenic and Adipogenic Differentiation

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    Aaron W. James

    2013-01-01

    Full Text Available Mesenchymal stem cells (MSC are multipotent cells, functioning as precursors to a variety of cell types including adipocytes, osteoblasts, and chondrocytes. Between osteogenic and adipogenic lineage commitment and differentiation, a theoretical inverse relationship exists, such that differentiation towards an osteoblast phenotype occurs at the expense of an adipocytic phenotype. This balance is regulated by numerous, intersecting signaling pathways that converge on the regulation of two main transcription factors: peroxisome proliferator-activated receptor-γ (PPARγ and Runt-related transcription factor 2 (Runx2. These two transcription factors, PPARγ and Runx2, are generally regarded as the master regulators of adipogenesis and osteogenesis. This review will summarize signaling pathways that govern MSC fate towards osteogenic or adipocytic differentiation. A number of signaling pathways follow the inverse balance between osteogenic and adipogenic differentiation and are generally proosteogenic/antiadipogenic stimuli. These include β-catenin dependent Wnt signaling, Hedgehog signaling, and NELL-1 signaling. However, other signaling pathways exhibit more context-dependent effects on adipogenic and osteogenic differentiation. These include bone morphogenic protein (BMP signaling and insulin growth factor (IGF signaling, which display both proosteogenic and proadipogenic effects. In summary, understanding those factors that govern osteogenic versus adipogenic MSC differentiation has significant implications in diverse areas of human health, from obesity to osteoporosis to regenerative medicine.

  17. Social defeat during adolescence and adulthood differentially induce BDNF-regulated immediate early genes

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    Caroline M. Coppens

    2011-11-01

    Full Text Available Stressful life events generally enhance the vulnerability for the development of human psychopathologies such as anxiety disorders and depression. The incidence rates of adult mental disorders steeply rises during adolescence in parallel with a structural and functional reorganization of the neural circuitry underlying stress reactivity. However, the mechanisms underlying susceptibility to stress and manifestation of mental disorders during adolescence are little understood. We hypothesized that heightened sensitivity to stress during adolescence reflects age-dependent differences in the expression of activity-dependent genes involved in synaptic plasticity. Therefore, we compared the effect of social stress during adolescence with social stress in adulthood on the expression of a panel of genes linked to induction of long-term potentiation (LTP and brain-derived neurotrophic factor (BDNF signaling. We show that social defeat during adolescence and adulthood differentially regulates expression of the immediate early genes BDNF, Arc, Carp, and Tieg1, as measured by qPCR in tissue lysates from prefrontal cortex, nucleus accumbens, and hippocampus. In the hippocampus, mRNA levels for all four genes were robustly elevated following social defeat in adolescence, whereas none were induced by defeat in adulthood. The relationship to coping style was also examined using adult reactive and proactive coping rats. Gene expression levels of reactive and proactive animals were similar in the prefrontal cortex and hippocampus. However, a trend toward a differential expression of BDNF and Arc mRNA in the nucleus accumbens was detected. BDNF mRNA was increased in the nucleus accumbens of proactive defeated animals, whereas the expression level in reactive defeated animals was comparable to control animals. The results demonstrate striking differences in immediate early gene expression in response to social defeat in adolescent and adult rats.

  18. Retinoic acid-loaded polymeric nanoparticles enhance vascular regulation of neural stem cell survival and differentiation after ischaemia

    Science.gov (United States)

    Ferreira, R.; Fonseca, M. C.; Santos, T.; Sargento-Freitas, J.; Tjeng, R.; Paiva, F.; Castelo-Branco, M.; Ferreira, L. S.; Bernardino, L.

    2016-04-01

    Stroke is one of the leading causes of death and disability worldwide. However, current therapies only reach a small percentage of patients and may cause serious side effects. We propose the therapeutic use of retinoic acid-loaded nanoparticles (RA-NP) to safely and efficiently repair the ischaemic brain by creating a favourable pro-angiogenic environment that enhances neurogenesis and neuronal restitution. Our data showed that RA-NP enhanced endothelial cell proliferation and tubule network formation and protected against ischaemia-induced death. To evaluate the effect of RA-NP on vascular regulation of neural stem cell (NSC) survival and differentiation, endothelial cell-conditioned media (EC-CM) were collected. EC-CM from healthy RA-NP-treated cells reduced NSC death and promoted proliferation while EC-CM from ischaemic RA-NP-treated cells decreased cell death, increased proliferation and neuronal differentiation. In parallel, human endothelial progenitor cells (hEPC), which are part of the endogenous repair response to vascular injury, were collected from ischaemic stroke patients. hEPC treated with RA-NP had significantly higher proliferation, which further highlights the therapeutic potential of this formulation. To conclude, RA-NP protected endothelial cells from ischaemic death and stimulated the release of pro-survival, proliferation-stimulating factors and differentiation cues for NSC. RA-NP were shown to be up to 83-fold more efficient than free RA and to enhance hEPC proliferation. These data serve as a stepping stone to use RA-NP as vasculotrophic and neurogenic agents for vascular disorders and neurodegenerative diseases with compromised vasculature.

  19. Gene function in early mouse embryonic stem cell differentiation

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    Campbell Pearl A

    2007-03-01

    Full Text Available Abstract Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5 undergoing undirected differentiation into embryoid bodies (EBs over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1, our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2 that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of m

  20. Nanomaterials modulate stem cell differentiation: biological interaction and underlying mechanisms.

    Science.gov (United States)

    Wei, Min; Li, Song; Le, Weidong

    2017-10-25

    Stem cells are unspecialized cells that have the potential for self-renewal and differentiation into more specialized cell types. The chemical and physical properties of surrounding microenvironment contribute to the growth and differentiation of stem cells and consequently play crucial roles in the regulation of stem cells' fate. Nanomaterials hold great promise in biological and biomedical fields owing to their unique properties, such as controllable particle size, facile synthesis, large surface-to-volume ratio, tunable surface chemistry, and biocompatibility. Over the recent years, accumulating evidence has shown that nanomaterials can facilitate stem cell proliferation and differentiation, and great effort is undertaken to explore their possible modulating manners and mechanisms on stem cell differentiation. In present review, we summarize recent progress in the regulating potential of various nanomaterials on stem cell differentiation and discuss the possible cell uptake, biological interaction and underlying mechanisms.

  1. Up-regulated microRNA-143 in cancer stem cells differentiation promotes prostate cancer cells metastasis by modulating FNDC3B expression

    International Nuclear Information System (INIS)

    Fan, Xinlan; Chen, Xu; Deng, Weixi; Zhong, Guangzheng; Cai, Qingqing; Lin, Tianxin

    2013-01-01

    Metastatic prostate cancer is a leading cause of cancer-related death in men. Cancer stem cells (CSCs) are involved in tumor progression and metastasis, including in prostate cancer. There is an obvious and urgent need for effective cancer stem cells specific therapies in metastatic prostate cancer. MicroRNAs (miRNAs) are an important class of pervasive genes that are involved in a variety of biological functions, especially in cancer. The goal of this study was to identify miRNAs involved in prostate cancer metastasis and cancer stem cells. A microarray and qRT-PCR were performed to investigate the miRNA expression profiles in PC-3 sphere cells and adherent cells. A transwell assay was used to evaluate the migration of PC-3 sphere cells and adherent cells. MiR-143 was silenced with antisense oligonucleotides in PC-3, PC-3-M and LNCaP cells. The role of miR-143 in prostate cancer metastasis was measured by wound-healing and transwell assays in vitro and bioluminescence imaging in vivo. Bioinformatics and luciferase report assays were used to identify the target of miR-143. The expression of miR-143 and the migration capability were reduced in PC-3 sphere cells and progressively increased during sphere re-adherent culture. Moreover, the down-regulation of miR-143 suppressed prostate cancer cells migration and invasion in vitro and systemically inhibited metastasis in vivo. Fibronectin type III domain containing 3B (FNDC3B), which regulates cell motility, was identified as a target of miR-143. The inhibition of miR-143 increased the expression of FNDC3B protein but not FNDC3B mRNA in vitro and vivo. These data demonstrate for the first time that miR-143 was up-regulated during the differentiation of prostate cancer stem cells and promoted prostate cancer metastasis by repressing FNDC3B expression. This sheds a new insight into the post-transcriptional regulation of cancer stem cells differentiation by miRNAs, a potential approach for the treatment of prostate cancer

  2. Zirconium ions up-regulate the BMP/SMAD signaling pathway and promote the proliferation and differentiation of human osteoblasts.

    Directory of Open Access Journals (Sweden)

    Yongjuan Chen

    Full Text Available Zirconium (Zr is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2 or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV oxynitrate (ZrO(NO32 at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.

  3. Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

    Science.gov (United States)

    Chen, Yongjuan; Roohani-Esfahani, Seyed-Iman; Lu, ZuFu; Zreiqat, Hala; Dunstan, Colin R.

    2015-01-01

    Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV) oxynitrate (ZrO(NO3)2) at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling. PMID:25602473

  4. Integrating and differentiating aspects of self-regulation: effortful control, executive functioning, and links to negative affectivity.

    Science.gov (United States)

    Bridgett, David J; Oddi, Kate B; Laake, Lauren M; Murdock, Kyle W; Bachmann, Melissa N

    2013-02-01

    Subdisciplines within psychology frequently examine self-regulation from different frameworks despite conceptually similar definitions of constructs. In the current study, similarities and differences between effortful control, based on the psychobiological model of temperament (Rothbart, Derryberry, & Posner, 1994), and executive functioning are examined and empirically tested in three studies (n = 509). Structural equation modeling indicated that effortful control and executive functioning are strongly associated and overlapping constructs (Study 1). Additionally, results indicated that effortful control is related to the executive function of updating/monitoring information in working memory, but not inhibition (Studies 2 and 3). Study 3 also demonstrates that better updating/monitoring information in working memory and better effortful control were uniquely linked to lower dispositional negative affect, whereas the executive function of low/poor inhibition was uniquely associated with an increased tendency to express negative affect. Furthermore, dispositional negative affect mediated the links between effortful control and, separately, the executive function of updating/monitoring information in working memory and the tendency to express negative affect. The theoretical implications of these findings are discussed, and a potential framework for guiding future work directed at integrating and differentiating aspects of self-regulation is suggested. PsycINFO Database Record (c) 2013 APA, all rights reserved.

  5. Positional mapping and candidate gene analysis of the mouse Ccs3 locus that regulates differential susceptibility to carcinogen-induced colorectal cancer.

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    Charles Meunier

    Full Text Available The Ccs3 locus on mouse chromosome 3 regulates differential susceptibility of A/J (A, susceptible and C57BL/6J (B6, resistant mouse strains to chemically-induced colorectal cancer (CRC. Here, we report the high-resolution positional mapping of the gene underlying the Ccs3 effect. Using phenotype/genotype correlation in a series of 33 AcB/BcA recombinant congenic mouse strains, as well as in groups of backcross populations bearing unique recombinant chromosomes for the interval, and in subcongenic strains, we have delineated the maximum size of the Ccs3 physical interval to a ∼2.15 Mb segment. This interval contains 12 annotated transcripts. Sequencing of positional candidates in A and B6 identified many either low-priority coding changes or non-protein coding variants. We found a unique copy number variant (CNV in intron 15 of the Nfkb1 gene. The CNV consists of two copies of a 54 bp sequence immediately adjacent to the exon 15 splice site, while only one copy is found in CRC-susceptible A. The Nfkb1 protein (p105/p50 expression is much reduced in A tumors compared to normal A colonic epithelium as analyzed by immunohistochemistry. Studies in primary macrophages from A and B6 mice demonstrate a marked differential activation of the NfκB pathway by lipopolysaccharide (kinetics of stimulation and maximum levels of phosphorylated IκBα, with a more robust activation being associated with resistance to CRC. NfκB has been previously implicated in regulating homeostasis and inflammatory response in the intestinal mucosa. The interval contains another positional candidate Slc39a8 that is differentially expressed in A vs B6 colons, and that has recently been associated in CRC tumor aggressiveness in humans.

  6. Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation

    Science.gov (United States)

    Marconett, Crystal N.; Zhou, Beiyun; Rieger, Megan E.; Selamat, Suhaida A.; Dubourd, Mickael; Fang, Xiaohui; Lynch, Sean K.; Stueve, Theresa Ryan; Siegmund, Kimberly D.; Berman, Benjamin P.

    2013-01-01

    Elucidation of the epigenetic basis for cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how epigenetic changes are integrated with transcriptional activation to determine cell phenotype during differentiation. We performed epigenomic profiling in conjunction with transcriptomic profiling using in vitro differentiation of human primary alveolar epithelial cells (AEC). This model recapitulates an in vivo process in which AEC transition from one differentiated cell type to another during regeneration following lung injury. Interrogation of histone marks over time revealed enrichment of specific transcription factor binding motifs within regions of changing chromatin structure. Cross-referencing of these motifs with pathways showing transcriptional changes revealed known regulatory pathways of distal alveolar differentiation, such as the WNT and transforming growth factor beta (TGFB) pathways, and putative novel regulators of adult AEC differentiation including hepatocyte nuclear factor 4 alpha (HNF4A), and the retinoid X receptor (RXR) signaling pathways. Inhibition of the RXR pathway confirmed its functional relevance for alveolar differentiation. Our incorporation of epigenetic data allowed specific identification of transcription factors that are potential direct upstream regulators of the differentiation process, demonstrating the power of this approach. Integration of epigenomic data with transcriptomic profiling has broad application for the identification of regulatory pathways in other models of differentiation. PMID:23818859

  7. The JAK-STAT transcriptional regulator, STAT-5, activates the ATM DNA damage pathway to induce HPV 31 genome amplification upon epithelial differentiation.

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    Shiyuan Hong

    Full Text Available High-risk human papillomavirus (HPV must evade innate immune surveillance to establish persistent infections and to amplify viral genomes upon differentiation. Members of the JAK-STAT family are important regulators of the innate immune response and HPV proteins downregulate expression of STAT-1 to allow for stable maintenance of viral episomes. STAT-5 is another member of this pathway that modulates the inflammatory response and plays an important role in controlling cell cycle progression in response to cytokines and growth factors. Our studies show that HPV E7 activates STAT-5 phosphorylation without altering total protein levels. Inhibition of STAT-5 phosphorylation by the drug pimozide abolishes viral genome amplification and late gene expression in differentiating keratinocytes. In contrast, treatment of undifferentiated cells that stably maintain episomes has no effect on viral replication. Knockdown studies show that the STAT-5β isoform is mainly responsible for this activity and that this is mediated through the ATM DNA damage response. A downstream target of STAT-5, the peroxisome proliferator-activated receptor γ (PPARγ contributes to the effects on members of the ATM pathway. Overall, these findings identify an important new regulatory mechanism by which the innate immune regulator, STAT-5, promotes HPV viral replication through activation of the ATM DNA damage response.

  8. Effects of let-7b and TLX on the proliferation and differentiation of retinal progenitor cells in vitro.

    Science.gov (United States)

    Ni, Ni; Zhang, Dandan; Xie, Qing; Chen, Junzhao; Wang, Zi; Deng, Yuan; Wen, Xuyang; Zhu, Mengyu; Ji, Jing; Fan, Xianqun; Luo, Min; Gu, Ping

    2014-10-20

    MicroRNAs manifest significant functions in brain neural stem cell (NSC) self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. Let-7b is expressed in the mammalian brain and regulates NSC proliferation and differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal. Whether let-7b and TLX act as important regulators in retinal progenitor cell (RPC) proliferation and differentiation remains unknown. Here, our data show that let-7b and TLX play important roles in controlling RPC fate determination in vitro. Let-7b suppresses TLX expression to negatively regulate RPC proliferation and accelerate the neuronal and glial differentiation of RPCs. The overexpression of let-7b downregulates TLX levels in RPCs, leading to reduced RPC proliferation and increased neuronal and glial differentiation, whereas antisense knockdown of let-7b produces robust TLX expression,enhanced RPC proliferation and decreased differentiation. Moreover, the inhibition of endogenous TLX by small interfering RNA suppresses RPC proliferation and promotes RPC differentiation. Furthermore, overexpression of TLX rescues let-7b-induced proliferation deficiency and weakens the RPC differentiation enhancement caused by let-7b alone. These results suggest that let-7b, by forming a negative feedback loop with TLX, provides a novel model to regulate the proliferation and differentiation of retinal progenitors in vitro.

  9. PTEN drives Th17 cell differentiation by preventing IL-2 production.

    Science.gov (United States)

    Kim, Hyeong Su; Jang, Sung Woong; Lee, Wonyong; Kim, Kiwan; Sohn, Hyogon; Hwang, Soo Seok; Lee, Gap Ryol

    2017-11-06

    T helper 17 (Th17) cells are a CD4 + T cell subset that produces IL-17A to mediate inflammation and autoimmunity. IL-2 inhibits Th17 cell differentiation. However, the mechanism by which IL-2 is suppressed during Th17 cell differentiation remains unclear. Here, we show that phosphatase and tensin homologue (PTEN) is a key factor that regulates Th17 cell differentiation by suppressing IL-2 production. Th17-specific Pten deletion ( Pten fl/fl Il17a cre ) impairs Th17 cell differentiation in vitro and ameliorated symptoms of experimental autoimmune encephalomyelitis (EAE), a model of Th17-mediated autoimmune disease. Mechanistically, Pten deficiency up-regulates IL-2 and phosphorylation of STAT5, but reduces STAT3 phosphorylation, thereby inhibiting Th17 cell differentiation. PTEN inhibitors block Th17 cell differentiation in vitro and in the EAE model. Thus, PTEN plays a key role in Th17 cell differentiation by blocking IL-2 expression. © 2017 Kim et al.

  10. Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-α with BCAR1 and Traf6

    International Nuclear Information System (INIS)

    Robinson, Lisa J.; Yaroslavskiy, Beatrice B.; Griswold, Reed D.; Zadorozny, Eva V.; Guo, Lida; Tourkova, Irina L.; Blair, Harry C.

    2009-01-01

    The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at ∼ 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-β-estradiol. Estrogen receptor-α (ERα) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ERα. However, ERα was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ERα in the presence of estrogen, was abundant. Immunoprecipitation showed rapid (∼ 5 min) estrogen-dependent formation of ERα-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-κB activity, precipitated with this complex. Reduction of NF-κB nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of IκB in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ERα.

  11. Estrogen inhibits RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of estrogen receptor-{alpha} with BCAR1 and Traf6

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, Lisa J., E-mail: robinsonlj@msx.upmc.edu [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Yaroslavskiy, Beatrice B.; Griswold, Reed D.; Zadorozny, Eva V.; Guo, Lida; Tourkova, Irina L. [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Blair, Harry C. [Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Veteran' s Affairs Medical Center, Pittsburgh, PA 15243 (United States)

    2009-04-15

    The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at {approx} 1 nM reduced RANKL-dependent osteoclast differentiation by 40-50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-{beta}-estradiol. Estrogen receptor-{alpha} (ER{alpha}) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ER{alpha}. However, ER{alpha} was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ER{alpha} in the presence of estrogen, was abundant. Immunoprecipitation showed rapid ({approx} 5 min) estrogen-dependent formation of ER{alpha}-BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-{kappa}B activity, precipitated with this complex. Reduction of NF-{kappa}B nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of I{kappa}B in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ER{alpha}.

  12. Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture.

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    Boon Siang Nicholas Tan

    Full Text Available Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL cells from mouse embryonic stem (mES cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation.

  13. Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

    Science.gov (United States)

    Tan, Boon Siang Nicholas; Kwek, Joly; Wong, Chong Kum Edwin; Saner, Nicholas J.; Yap, Charlotte; Felquer, Fernando; Morris, Michael B.; Gardner, David K.; Rathjen, Peter D.; Rathjen, Joy

    2016-01-01

    Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. PMID:27723793

  14. Differentially expressed miRNAs after GnRH treatment and their potential roles in FSH regulation in porcine anterior pituitary cell.

    Directory of Open Access Journals (Sweden)

    Rui-Song Ye

    Full Text Available Hypothalamic gonadotropin-releasing hormone (GnRH is a major regulator of follicle-stimulating hormone (FSH secretion in gonadotrope cell in the anterior pituitary gland. microRNAs (miRNAs are small RNA molecules that control gene expression by imperfect binding to the 3'-untranslated region (3'-UTR of mRNA at the post-transcriptional level. It has been proven that miRNAs play an important role in hormone response and/or regulation. However, little is known about miRNAs in the regulation of FSH secretion. In this study, primary anterior pituitary cells were treated with 100 nM GnRH. The supernatant of pituitary cell was collected for FSH determination by enzyme-linked immunosorbent assay (ELISA at 3 hours and 6 hours post GnRH treatment respectively. Results revealed that GnRH significantly promoted FSH secretion at 3 h and 6 h post-treatment by 1.40-fold and 1.80-fold, respectively. FSHβ mRNA at 6 h post GnRH treatment significantly increased by 1.60-fold. At 6 hours, cells were collected for miRNA expression profile analysis using MiRCURY LNA Array and quantitative PCR (qPCR. Consequently, 21 up-regulated and 10 down-regulated miRNAs were identified, and qPCR verification of 10 randomly selected miRNAs showed a strong correlation with microarray results. Chromosome location analysis indicated that 8 miRNAs were mapped to chromosome 12 and 4 miRNAs to chromosome X. Target and pathway analysis showed that some miRNAs may be associated with GnRH regulation pathways. In addition, In-depth analysis indicated that 10 up-regulated and 3 down-regulated miRNAs probably target FSHβ mRNA 3'-UTR directly, including miR-361-3p, a highly conserved X-linked miRNA. Most importantly, functional experimental results showed that miR-361-3p was involved in FSH secretion regulation, and up-regulated miR-361-3p expression inhibited FSH secretion, while down-regulated miR-361-3p expression promoted FSH secretion in pig pituitary cell model. These differentially

  15. Proteomic analysis of PC12 cell differentiation induced by ionizing radiation

    International Nuclear Information System (INIS)

    Zhang Junquan; Gao Ronglian; Chen Xiaohua; Wang Zhidong; Dong Bo; Rao Yalan; Hou Lili; Zhang Hao; Mao Bingzhi

    2005-01-01

    Objective: To explore the molecular mechanism of PC12 cell differentiation induced by ionizing radiation and screen the molecular target of nervous system injured by irradiation. Methods: PC12 cells were irradiated with 16 Gy 60 Co γ ray. Total proteins of normal and irradiated cells were prepared 48 hours after irradiation and separated with two dimensional gel electrophoresis. Some differential expressed proteins were characterized with mass spectrometry. Results: 876 differential expressed proteins were observed. Up-regulated expression of ubiquitin carboxyl-terminal hydratase L1 was found. Down-regulated expression of new protein similar to HP1α was found. Conclusion: The characterization of some differential expressed proteins through proteomic analysis would benefit the research of molecular mechanism of PC12 cell differentiation induced by ionizing radiation. (authors)

  16. Decoding cell signalling and regulation of oligodendrocyte differentiation.

    Science.gov (United States)

    Santos, A K; Vieira, M S; Vasconcellos, R; Goulart, V A M; Kihara, A H; Resende, R R

    2018-05-22

    Oligodendrocytes are fundamental for the functioning of the nervous system; they participate in several cellular processes, including axonal myelination and metabolic maintenance for astrocytes and neurons. In the mammalian nervous system, they are produced through waves of proliferation and differentiation, which occur during embryogenesis. However, oligodendrocytes and their precursors continue to be generated during adulthood from specific niches of stem cells that were not recruited during development. Deficiencies in the formation and maturation of these cells can generate pathologies mainly related to myelination. Understanding the mechanisms involved in oligodendrocyte development, from the precursor to mature cell level, will allow inferring therapies and treatments for associated pathologies and disorders. Such mechanisms include cell signalling pathways that involve many growth factors, small metabolic molecules, non-coding RNAs, and transcription factors, as well as specific elements of the extracellular matrix, which act in a coordinated temporal and spatial manner according to a given stimulus. Deciphering those aspects will allow researchers to replicate them in vitro in a controlled environment and thus mimic oligodendrocyte maturation to understand the role of oligodendrocytes in myelination in pathologies and normal conditions. In this study, we review these aspects, based on the most recent in vivo and in vitro data on oligodendrocyte generation and differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Functions of Heterogeneous Nuclear Ribonucleoproteins in Stem Cell Potency and Differentiation

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    Qishan Chen

    2013-01-01

    Full Text Available Stem cells possess huge importance in developmental biology, disease modelling, cell replacement therapy, and tissue engineering in regenerative medicine because they have the remarkable potential for self-renewal and to differentiate into almost all the cell types in the human body. Elucidation of molecular mechanisms regulating stem cell potency and differentiation is essential and critical for extensive application. Heterogeneous nuclear ribonucleoproteins (hnRNPs are modular proteins consisting of RNA-binding motifs and auxiliary domains characterized by extensive and divergent functions in nucleic acid metabolism. Multiple roles of hnRNPs in transcriptional and posttranscriptional regulation enable them to be effective gene expression regulators. More recent findings show that hnRNP proteins are crucial factors implicated in maintenance of stem cell self-renewal and pluripotency and cell differentiation. The hnRNPs interact with certain sequences in target gene promoter regions to initiate transcription. In addition, they recognize 3′UTR or 5′UTR of specific gene mRNA forming mRNP complex to regulate mRNA stability and translation. Both of these regulatory pathways lead to modulation of gene expression that is associated with stem cell proliferation, cell cycle control, pluripotency, and committed differentiation.

  18. Glimpse into Hox and tale regulation of cell differentiation and reprogramming.

    Science.gov (United States)

    Cerdá-Esteban, Nuria; Spagnoli, Francesca M

    2014-01-01

    During embryonic development, cells become gradually restricted in their developmental potential and start elaborating lineage-specific transcriptional networks to ultimately acquire a unique differentiated state. Hox genes play a central role in specifying regional identities, thereby providing the cell with critical information on positional value along its differentiation path. The exquisite DNA-binding specificity of the Hox proteins is frequently dependent upon their interaction with members of the TALE family of homeodomain proteins. In addition to their function as Hox-cofactors, TALE homeoproteins control multiple crucial developmental processes through Hox-independent mechanisms. Here, we will review recent findings on the function of both Hox and TALE proteins in cell differentiation, referring mostly to vertebrate species. In addition, we will discuss the direct implications of this knowledge on cell plasticity and cell reprogramming. Copyright © 2013 Wiley Periodicals, Inc.

  19. CDDO and ATRA Instigate Differentiation of IMR32 Human Neuroblastoma Cells

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    Namrata Chaudhari

    2017-09-01

    Full Text Available Neuroblastoma is the most common solid extra cranial tumor in infants. Improving the clinical outcome of children with aggressive tumors undergoing one of the multiple treatment options has been a major concern. Differentiating neuroblastoma cells holds promise in inducing tumor growth arrest and treating minimal residual disease. In this study, we investigated the effect of partial PPARγ agonist 2-cyano-3,12-dioxooleana-1,9(11-dien-28-oic acid (CDDO on human neuroblastoma IMR32 cells. Our results demonstrate that treatment with low concentration of CDDO and particularly in combination with all trans retinoic acid (ATRA induced neurite outgrowth, increased the percentage of more than two neurites bearing cells, and decreased viability in IMR32 cells. These morphological changes were associated with an increase in expression of bonafide differentiation markers like β3-tubulin and Neuron Specific Enolase (NSE. The differentiation was accompanied by a decrease in the expression of MYCN whose amplification is known to contribute to the pathogenesis of neuroblastoma. MYCN is known to negatively regulate NMYC downstream-regulated gene 1 (NDRG1 in neuroblastomas. MYCN down-regulation induced by CDDO correlated with increased expression of NDRG1. CDDO decreased Anaplastic Lymphoma Kinase (ALK mRNA expression without affecting its protein level, while ATRA significantly down-regulated ALK. Antagonism of PPARγ receptor by T0070907 meddled with differentiation inducing effects of CDDO as observed by stunted neurite growth, increased viability and decreased expression of differentiation markers. Our findings indicate that IMR32 differentiation induced by CDDO in combination with ATRA enhances, differentiation followed by cell death via cAMP-response-element binding protein (CREB independent and PPARγ dependent signaling mechanisms.

  20. Mechanisms involved in regulation of osteoclastic differentiation by mechanical stress-loaded osteoblasts

    International Nuclear Information System (INIS)

    Kaneuji, Takeshi; Ariyoshi, Wataru; Okinaga, Toshinori; Toshinaga, Akihiro; Takahashi, Tetsu; Nishihara, Tatsuji

    2011-01-01

    Highlights: → Effect of compressive force on osteoblasts were examined. → Compressive force induced OPG expression and suppressed osteoclastogenesis. → This enhancement of OPG is dependent on Wnt/Ca2+ signal pathway. -- Abstract: Mechanical stress is known to be important for regulation of bone turnover, though the detailed mechanisms are not fully understood. In the present study, we examined the effect of mechanical stress on osteoblasts using a novel compression model. Mouse osteoblastic MC3T3-E1 cells were embedded in three-dimensional (3D) gels and cultured with continuous compressive force (0-10.0 g/cm 2 ) for 48 h, and the conditioned medium were collected. RAW264.7 cells were then incubated with the conditioned medium for various times in the presence of receptor activator of nuclear factor-κB ligand (RANKL). Conditioned medium was found to inhibit the differentiation of RAW264.7 cells into osteoclasts induced by RANKL via down-regulation of the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of IκBα, and nuclear translocation of p50 and p65. Interestingly, the conditioned medium also had a high level of binding activity to RANKL and blocked the binding of RANK to RANKL. Furthermore, the binding activity of conditioned medium to RANKL was reduced when the 3D gel was supplemented with KN-93, an inhibitor of non-canonical Wnt/Ca 2+ pathway. In addition, expression level of osteoprotegerin (OPG) mRNA was increased in time- and force-dependent manners, and remarkably suppressed by KN-93. These results indicate that osteoblastic cells subjected to mechanical stress produce OPG, which binds to RANKL. Furthermore, this binding activity strongly inhibited osteoclastogenesis through suppression of TRAF6 and the nuclear factor-kappa B (NF-κB) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt/Ca 2+ pathway.