IL-10-produced by human transitional B-cells down-regulates CD86 expression on B-cells leading to inhibition of CD4+T-cell responses.

Science.gov (United States)

Nova-Lamperti, Estefania; Fanelli, Giorgia; Becker, Pablo D; Chana, Prabhjoat; Elgueta, Raul; Dodd, Philippa C; Lord, Graham M; Lombardi, Giovanna; Hernandez-Fuentes, Maria P

2016-01-22

A novel subset of human regulatory B-cells has recently been described. They arise from within the transitional B-cell subpopulation and are characterised by the production of IL-10. They appear to be of significant importance in regulating T-cell immunity in vivo. Despite this important function, the molecular mechanisms by which they control T-cell activation are incompletely defined. Here we show that transitional B-cells produced more IL-10 and expressed higher levels of IL-10 receptor after CD40 engagement compared to other B-cell subsets. Furthermore, under this stimulatory condition, CD86 expressed by transitional B-cells was down regulated and T-cell proliferation was reduced. We provide evidence to demonstrate that the down-regulation of CD86 expression by transitional B-cells was due to the autocrine effect of IL-10, which in turn leads to decreased T-cell proliferation and TNF-α production. This analysis was further extended to peripheral B-cells in kidney transplant recipients. We observed that B-cells from patients tolerant to the graft maintained higher IL-10 production after CD40 ligation, which correlates with lower CD86 expression compared to patients with chronic rejection. Hence, the results obtained in this study shed light on a new alternative mechanism by which transitional B-cells inhibit T-cell proliferation and cytokine production.

IL-10-produced by human transitional B-cells down-regulates CD86 expression on B-cells leading to inhibition of CD4+T-cell responses

Science.gov (United States)

Nova-Lamperti, Estefania; Fanelli, Giorgia; Becker, Pablo D.; Chana, Prabhjoat; Elgueta, Raul; Dodd, Philippa C.; Lord, Graham M.; Lombardi, Giovanna; Hernandez-Fuentes, Maria P.

2016-01-01

A novel subset of human regulatory B-cells has recently been described. They arise from within the transitional B-cell subpopulation and are characterised by the production of IL-10. They appear to be of significant importance in regulating T-cell immunity in vivo. Despite this important function, the molecular mechanisms by which they control T-cell activation are incompletely defined. Here we show that transitional B-cells produced more IL-10 and expressed higher levels of IL-10 receptor after CD40 engagement compared to other B-cell subsets. Furthermore, under this stimulatory condition, CD86 expressed by transitional B-cells was down regulated and T-cell proliferation was reduced. We provide evidence to demonstrate that the down-regulation of CD86 expression by transitional B-cells was due to the autocrine effect of IL-10, which in turn leads to decreased T-cell proliferation and TNF-α production. This analysis was further extended to peripheral B-cells in kidney transplant recipients. We observed that B-cells from patients tolerant to the graft maintained higher IL-10 production after CD40 ligation, which correlates with lower CD86 expression compared to patients with chronic rejection. Hence, the results obtained in this study shed light on a new alternative mechanism by which transitional B-cells inhibit T-cell proliferation and cytokine production. PMID:26795594

Transcriptional regulation of miR-146b by C/EBPβ LAP2 in esophageal cancer cells

International Nuclear Information System (INIS)

Li, Junxia; Shan, Fabo; Xiong, Gang; Wang, Ju-Ming; Wang, Wen-Lin; Xu, Xueqing; Bai, Yun

2014-01-01

Highlights: • MiR-146b promotes esophageal cancer cell proliferation. • MiR-146b inhibits esophageal cancer cell apoptosis. • C/EBPβ directly binds to miR-146b promoter conserved region. • MiR-146b is up-regulated by C/EBPβ LAP2 transcriptional activation. - Abstract: Recent clinical study indicated that up-regulation of miR-146b was associated with poor overall survival of patients in esophageal squamous cell carcinoma. However, the underlying mechanism of miR-146b dysregulation remains to be explored. Here we report that miR-146b promotes cell proliferation and inhibits cell apoptosis in esophageal cancer cell lines. Mechanismly, two C/EBPβ binding motifs are located in the miR-146b promoter conserved region. Among the three isoforms of C/EBPβ, C/EBPβ LAP2 positively regulated miR-146b expression and increases miR-146b levels in a dose-dependent manner through transcription activation of miR-146b gene. Together, these results suggest a miR-146b regulatory mechanism involving C/EBPβ, which may contribute to the up-regulation of miR-146b in esophageal squamous cell carcinoma

The small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma and full-length FOXP1 exert similar oncogenic and transcriptional activity in human B cells.

Science.gov (United States)

van Keimpema, Martine; Grüneberg, Leonie J; Schilder-Tol, Esther J M; Oud, Monique E C M; Beuling, Esther A; Hensbergen, Paul J; de Jong, Johann; Pals, Steven T; Spaargaren, Marcel

2017-03-01

The forkhead transcription factor FOXP1 is generally regarded as an oncogene in activated B cell-like diffuse large B-cell lymphoma. Previous studies have suggested that a small isoform of FOXP1 rather than full-length FOXP1, may possess this oncogenic activity. Corroborating those studies, we herein show that activated B cell-like diffuse large B-cell lymphoma cell lines and primary activated B cell-like diffuse large B-cell lymphoma cells predominantly express a small FOXP1 isoform, and that the 5'-end of the Foxp1 gene is a common insertion site in murine lymphomas in leukemia virus- and transposon-mediated insertional mutagenesis screens. By combined mass spectrometry, (quantative) reverse transcription polymerase chain reaction/sequencing, and small interfering ribonucleic acid-mediated gene silencing, we determined that the small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma lacks the N-terminal 100 amino acids of full-length FOXP1. Aberrant overexpression of this FOXP1 isoform (ΔN100) in primary human B cells revealed its oncogenic capacity; it repressed apoptosis and plasma cell differentiation. However, no difference in potency was found between this small FOXP1 isoform and full-length FOXP1. Furthermore, overexpression of full-length FOXP1 or this small FOXP1 isoform in primary B cells and diffuse large B-cell lymphoma cell lines resulted in similar gene regulation. Taken together, our data indicate that this small FOXP1 isoform and full-length FOXP1 have comparable oncogenic and transcriptional activity in human B cells, suggesting that aberrant expression or overexpression of FOXP1, irrespective of the specific isoform, contributes to lymphomagenesis. These novel insights further enhance the value of FOXP1 for the diagnostics, prognostics, and treatment of diffuse large B-cell lymphoma patients. Copyright© Ferrata Storti Foundation.

In silico identification of NF-kappaB-regulated genes in pancreatic beta-cells

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Eizirik Decio L

2007-02-01

Full Text Available Abstract Background Pancreatic beta-cells are the target of an autoimmune attack in type 1 diabetes mellitus (T1DM. This is mediated in part by cytokines, such as interleukin (IL-1β and interferon (IFN-γ. These cytokines modify the expression of hundreds of genes, leading to beta-cell dysfunction and death by apoptosis. Several of these cytokine-induced genes are potentially regulated by the IL-1β-activated transcription factor (TF nuclear factor (NF-κB, and previous studies by our group have shown that cytokine-induced NF-κB activation is pro-apoptotic in beta-cells. To identify NF-κB-regulated gene networks in beta-cells we presently used a discriminant analysis-based approach to predict NF-κB responding genes on the basis of putative regulatory elements. Results The performance of linear and quadratic discriminant analysis (LDA, QDA in identifying NF-κB-responding genes was examined on a dataset of 240 positive and negative examples of NF-κB regulation, using stratified cross-validation with an internal leave-one-out cross-validation (LOOCV loop for automated feature selection and noise reduction. LDA performed slightly better than QDA, achieving 61% sensitivity, 91% specificity and 87% positive predictive value, and allowing the identification of 231, 251 and 580 NF-κB putative target genes in insulin-producing INS-1E cells, primary rat beta-cells and human pancreatic islets, respectively. Predicted NF-κB targets had a significant enrichment in genes regulated by cytokines (IL-1β or IL-1β + IFN-γ and double stranded RNA (dsRNA, as compared to genes not regulated by these NF-κB-dependent stimuli. We increased the confidence of the predictions by selecting only evolutionary stable genes, i.e. genes with homologs predicted as NF-κB targets in rat, mouse, human and chimpanzee. Conclusion The present in silico analysis allowed us to identify novel regulatory targets of NF-κB using a supervised classification method based on

KLF2 in Regulation of NF-κB-Mediated Immune Cell Function and Inflammation

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Prerana Jha

2017-11-01

Full Text Available KLF2 (Kruppel-like factor 2 is a member of the zinc finger transcription factor family, which critically regulates embryonic lung development, function of endothelial cells and maintenance of quiescence in T-cells and monocytes. It is expressed in naïve T-cells and monocytes, however its level of expression decreases during activation and differentiation. KLF2 also plays critical regulatory role in various inflammatory diseases and their pathogenesis. Nuclear factor-kappaB (NF-κB is an important inducer of inflammation and the inflammation is mediated through the transcription of several proinflammatory cytokines, chemokines and adhesion molecules. So, both transcriptional factors KLF2 and NF-κB are being associated with the similar cellular functions and their maintenance. It was shown that KLF2 regulates most of the NF-κB-mediated activities. In this review, we focused on emphasizing the involvement of KLF2 in health and disease states and how they interact with transcriptional master regulator NF-κB.

Regulation of development and function of different T cell subtypes by Rel/NF-{kappa}B family members

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Vallabhapurapu, S.

2004-09-01

This study reveals the requirement of distinct members of the Rel/NF-{kappa}B family in both hematopoietic and non-hematopoietic cells for the development of thymic NKT cells. Activation of NF-{kappa}B via the classical I{kappa}B{alpha}-regulated pathway is required within the NKT precursors for their efficient maturation from NK1.1{sup -} precursors to mature NK1.1{sup +} NKT cells. The Rel/NF-{kappa}B family member RelB, on the other hand, is required in thymic stromal cells for the generation of very early NK1.1{sup -} precursors. NF-{kappa}B-inducing kinase (NIK) has also been shown to be required in thymic stromal cells for NKT cell development and this study demonstrates that NIK specifically regulates both constitutive and signal-induced DNA binding of RelB, but not RelA. Moreover, NIK-induced DNA binding of RelB depends on the processing of inhibitory p100 to p52, revealing an alternate pathway of NF-{kappa}B induction. Thus, Rel/NF-{kappa}B complexes activated by the classical I{kappa}B{alpha}-regulated pathway in NKT precursors and an alternate NIK/p100/RelB pathway in thymic stromal cells regulate different stages of NKT cell development. (orig.)

Loss of PRDM1/BLIMP-1 function contributes to poor prognosis of activated B-cell-like diffuse large B-cell lymphoma

DEFF Research Database (Denmark)

Xia, Yi; Xu-Monette, Z Y; Tzankov, A

2017-01-01

PRDM1/BLIMP-1, a master regulator of plasma-cell differentiation, is frequently inactivated in activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) patients. Little is known about its genetic aberrations and relevant clinical implications. A large series of patients with de novo DLBC...

Regulation of development and function of different T cell subtypes by Rel/NF-κB family members

International Nuclear Information System (INIS)

Vallabhapurapu, S.

2004-09-01

This study reveals the requirement of distinct members of the Rel/NF-κB family in both hematopoietic and non-hematopoietic cells for the development of thymic NKT cells. Activation of NF-κB via the classical IκBα-regulated pathway is required within the NKT precursors for their efficient maturation from NK1.1 - precursors to mature NK1.1 + NKT cells. The Rel/NF-κB family member RelB, on the other hand, is required in thymic stromal cells for the generation of very early NK1.1 - precursors. NF-κB-inducing kinase (NIK) has also been shown to be required in thymic stromal cells for NKT cell development and this study demonstrates that NIK specifically regulates both constitutive and signal-induced DNA binding of RelB, but not RelA. Moreover, NIK-induced DNA binding of RelB depends on the processing of inhibitory p100 to p52, revealing an alternate pathway of NF-κB induction. Thus, Rel/NF-κB complexes activated by the classical IκBα-regulated pathway in NKT precursors and an alternate NIK/p100/RelB pathway in thymic stromal cells regulate different stages of NKT cell development. (orig.)

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

Science.gov (United States)

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

2015-01-01

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

Bi-directional activation between mesenchymal stem cells and CLL B-cells: implication for CLL disease progression.

Science.gov (United States)

Ding, Wei; Nowakowski, Grzegorz S; Knox, Traci R; Boysen, Justin C; Maas, Mary L; Schwager, Susan M; Wu, Wenting; Wellik, Linda E; Dietz, Allan B; Ghosh, Asish K; Secreto, Charla R; Medina, Kay L; Shanafelt, Tait D; Zent, Clive S; Call, Timothy G; Kay, Neil E

2009-11-01

It was hypothesized that contact between chronic lymphocytic leukaemia (CLL) B-cells and marrow stromal cells impact both cell types. To test this hypothesis, we utilized a long-term primary culture system from bone biopsies that reliably generates a mesenchymal stem cell (MSC). Co-culture of MSC with CLL B-cells protected the latter from both spontaneous apoptosis and drug-induced apoptosis. The CD38 expression in previously CD38 positive CLL B-cells was up-regulated with MSC co-culture. Upregulation of CD71, CD25, CD69 and CD70 in CLL B-cells was found in the co-culture. CD71 upregulation was more significantly associated with high-risk CLL, implicating CD71 regulation in the microenvironment predicting disease progression. In MSC, rapid ERK and AKT phosphorylation (within 30 min) were detected when CLL B-cells and MSC were separated by transwell; indicating that activation of MSC was mediated by soluble factors. These findings support a bi-directional activation between bone marrow stromal cells and CLL B-cells.

Cdc25A promotes cell survival by stimulating NF-κB activity through IκB-α phosphorylation and destabilization

International Nuclear Information System (INIS)

Hong, Hey-Young; Choi, Jiyeon; Cho, Young-Wook; Kim, Byung-Chul

2012-01-01

Highlights: ► We examine the antiapoptotic mechanisms of Cdc25A. ► Smad7 decreases the phosphorylation of IκB-alpha at Ser-32. ► Smad7 positively regulates NF-κB activity through IκB-alpha ubiquitination. -- Abstract: Cell division cycle 25A (Cdc25A), a dual specificity protein phosphatase, exhibits anti-apoptotic activity, but the underlying molecular mechanisms are poorly characterized. Here we report that Cdc25A inhibits cisplatin-induced apoptotic cell death by stimulating nuclear factor-kappa B (NF-κB) activity. In HEK-293 cells, Cdc25A decreased protein level of inhibitor subunit kappa B alpha (Iκ-Bα) in association with increased serine 32-phosphorylation, followed by stimulation of transcriptional activity of NF-κB. Inhibition of NF-κB activity by chemical inhibitor or overexpression of Iκ-Bα in Cdc25A-elevated cancer cells resistant to cisplatin improved their sensitivity to cisplatin-induced apoptosis. Our data show for the first time that Cdc25A has an important physiological role in NF-κB activity regulation and it may be an important survival mechanism of cancer cells.

Polycystin-1 promotes PKCα-mediated NF-κB activation in kidney cells

International Nuclear Information System (INIS)

Banzi, Manuela; Aguiari, Gianluca; Trimi, Viky; Mangolini, Alessandra; Pinton, Paolo; Witzgall, Ralph; Rizzuto, Rosario; Senno, Laura del

2006-01-01

Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-κB signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293 CTT ), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-κB nuclear levels and NF-κB-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-κB promoter activation was mediated by PKCα because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293 CTT cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-κB inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKCα-mediated NF-κB signalling and cell survival

All-trans retinoic acid negatively regulates cytotoxic activities of nature killer cell line 92

International Nuclear Information System (INIS)

Li Ang; He Meilan; Wang Hui; Qiao Bin; Chen Ping; Gu Hua; Zhang Mengjie; He Shengxiang

2007-01-01

NK cells are key components of innate immune systems and their activities are regulated by cytokines and hormones. All-trans retinoic acid (ATRA), as a metabolite of vitamin A and an immunomodulatory hormone, plays an important role in regulating immune responses. In the present study, we investigated the effect of ATRA on human NK cell line NK92. We found that ATRA dose-dependently suppressed cytotoxic activities of NK92 cells without affecting their proliferation. To explore the mechanisms underlying the ATRA influence on NK92 cells, we examined the production of cytokines (TNF-α, IFN-γ), gene expression of cytotoxic-associated molecules (perforin, granzyme B, nature killer receptors (NCRs), and NKG2D), and the activation of NF-κB pathways related with immune response. Our results demonstrated that ATRA suppressed NF-κB activity and prevented IκBα degradation in a dose-dependent way, inhibited IFN-γ production and gene expression of granzyme B and NKp46. Our findings suggest that ATRA is a negative regulator of NK92 cell activation and may act as a potential regulator of anti-inflammatory functions in vivo

PTP1B confers liver fibrosis by regulating the activation of hepatic stellate cells

Energy Technology Data Exchange (ETDEWEB)

Chen, Pei-Jie; Cai, Shuang-Peng; Yang, Yang; Li, Wan-Xia; Huang, Cheng; Meng, Xiao-Ming; Li, Jun, E-mail: lj@ahmu.edu.cn

2016-02-01

Liver fibrosis is a reversible wound-healing response to chronic hepatic injuries. Activation of hepatic stellate cells (HSCs) plays a pivotal role in the development of hepatic fibrosis. The currently accepted mechanism for the resolution of liver fibrosis is the apoptosis and inactivation of activated HSCs. Protein tyrosine phosphatase 1B (PTP1B), a prototype of non-receptor protein tyrosine phosphatase, is proved to be a vital modulator in cardiac fibrogenesis. However, the precise role of PTP1B on liver fibrosis and HSC activation is still unclear. Our study showed that the expression of PTP1B was elevated in fibrotic liver but reduced after spontaneous recovery. Moreover, stimulation of HSC-T6 cells with transforming growth factor-β1 (TGF-β1) resulted in a dose/time-dependent increase of PTP1B mRNA and protein. Co-incubation of HSC-T6 cells with PTP1B-siRNA inhibited the cell proliferation and activation induced by TGF-β1. Additionally, both mRNA and protein of PTP1B were dramatically decreased in inactivated HSCs after treated with adipogenic differentiation mixture (MDI). Over-expression of PTP1B hindered the inactivation of HSC-T6 cells induced by MDI. These observations revealed a regulatory role of PTP1B in liver fibrosis and implied PTP1B as a potential therapeutic target. - Highlights: • The expression of PTP1B in the fibrotic livers and recovery livers • The expression of PTP1B in activated and inactivated HSCs • Blockade of PTP1B inhibited the TGF-β1-induced proliferation and activation of HSCs. • Over-expression of PTP1B abolished the inactivation of HSCs induced by MDI.

PTP1B confers liver fibrosis by regulating the activation of hepatic stellate cells

International Nuclear Information System (INIS)

Chen, Pei-Jie; Cai, Shuang-Peng; Yang, Yang; Li, Wan-Xia; Huang, Cheng; Meng, Xiao-Ming; Li, Jun

2016-01-01

Liver fibrosis is a reversible wound-healing response to chronic hepatic injuries. Activation of hepatic stellate cells (HSCs) plays a pivotal role in the development of hepatic fibrosis. The currently accepted mechanism for the resolution of liver fibrosis is the apoptosis and inactivation of activated HSCs. Protein tyrosine phosphatase 1B (PTP1B), a prototype of non-receptor protein tyrosine phosphatase, is proved to be a vital modulator in cardiac fibrogenesis. However, the precise role of PTP1B on liver fibrosis and HSC activation is still unclear. Our study showed that the expression of PTP1B was elevated in fibrotic liver but reduced after spontaneous recovery. Moreover, stimulation of HSC-T6 cells with transforming growth factor-β1 (TGF-β1) resulted in a dose/time-dependent increase of PTP1B mRNA and protein. Co-incubation of HSC-T6 cells with PTP1B-siRNA inhibited the cell proliferation and activation induced by TGF-β1. Additionally, both mRNA and protein of PTP1B were dramatically decreased in inactivated HSCs after treated with adipogenic differentiation mixture (MDI). Over-expression of PTP1B hindered the inactivation of HSC-T6 cells induced by MDI. These observations revealed a regulatory role of PTP1B in liver fibrosis and implied PTP1B as a potential therapeutic target. - Highlights: • The expression of PTP1B in the fibrotic livers and recovery livers • The expression of PTP1B in activated and inactivated HSCs • Blockade of PTP1B inhibited the TGF-β1-induced proliferation and activation of HSCs. • Over-expression of PTP1B abolished the inactivation of HSCs induced by MDI.

NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

International Nuclear Information System (INIS)

Wang, Yi; Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing

2011-01-01

Highlights: → Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. → Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. → P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. → Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the regulation of foam

NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

Energy Technology Data Exchange (ETDEWEB)

Wang, Yi, E-mail: wangyi2004a@126.com [Department of Cardiology, Shanghai First People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080 (China); Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing [Department of Cardiology, Shanghai First People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080 (China)

2011-08-05

Highlights: {yields} Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. {yields} Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. {yields} P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. {yields} Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the

Dendritic branching of olfactory bulb mitral and tufted cells: regulation by TrkB.

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Fumiaki Imamura

2009-08-01

Full Text Available Projection neurons of mammalian olfactory bulb (OB, mitral and tufted cells, have dendrites whose morphologies are specifically differentiated for efficient odor information processing. The apical dendrite extends radially and arborizes in single glomerulus where it receives primary input from olfactory sensory neurons that express the same odor receptor. The lateral dendrites extend horizontally in the external plexiform layer and make reciprocal dendrodendritic synapses with granule cells, which moderate mitral/tufted cell activity. The molecular mechanisms regulating dendritic development of mitral/tufted cells is one of the unsolved important problems in the olfactory system. Here, we focused on TrkB receptors to test the hypothesis that neurotrophin-mediate mechanisms contributed to dendritic differentiation of OB mitral/tufted cells.With immunohistochemical analysis, we found that the TrkB neurotrophin receptor is expressed by both apical and lateral dendrites of mitral/tufted cells and that expression is evident during the early postnatal days when these dendrites exhibit their most robust growth and differentiation. To examine the effect of TrkB activation on mitral/tufted cell dendritic development, we cultured OB neurons. When BDNF or NT4 were introduced into the cultures, there was a significant increase in the number of primary neurites and branching points among the mitral/tufted cells. Moreover, BDNF facilitated filopodial extension along the neurites of mitral/tufted cells.In this report, we show for the first time that TrkB activation stimulates the dendritic branching of mitral/tufted cells in developing OB. This suggests that arborization of the apical dendrite in a glomerulus is under the tight regulation of TrkB activation.

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

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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.

Dopamine D1 receptor activation regulates the expression of the estrogen synthesis gene aromatase B in radial glial cell

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Lei eXing

2015-09-01

Full Text Available Radial glial cells (RGCs are abundant stem-like non-neuronal progenitors that are important for adult neurogenesis and brain repair, yet little is known about their regulation by neurotransmitters. Here we provide evidence for neuronal-glial interactions via a novel role for dopamine to stimulate RGC function. Goldfish were chosen as the model organism due to the abundance of RGCs and regenerative abilities of the adult central nervous system. A close anatomical relationship was observed between tyrosine hydroxylase-positive catecholaminergic cell bodies and axons and dopamine-D1 receptor expressing RGCs along the ventricular surface of telencephalon, a site of active neurogenesis. A primary cell culture model was established and immunofluorescence analysis indicates that in vitro RGCs from female goldfish retain their major characteristics in vivo, including expression of glial fibrillary acidic protein and brain lipid binding protein. The estrogen synthesis enzyme aromatase B is exclusively found in RGCs, but this is lost as cells differentiate to neurons and other glial types in adult teleost brain. Pharmacological experiments using the cultured RGCs established that specific activation of dopamine D1 receptors up-regulates aromatase B mRNA through a cyclic adenosine monophosphate-dependent molecular mechanism. These data indicate that dopamine enhances the steroidogenic function of this neuronal progenitor cell.

Food Additive Sodium Benzoate (NaB Activates NFκB and Induces Apoptosis in HCT116 Cells

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Betul Yilmaz

2018-03-01

Full Text Available NaB, the metabolite of cinnamon and sodium salt of benzoic acid is a commonly used food and beverage preservative. Various studies have investigated NaB for its effects on different cellular models. However, the effects of NaB on cancer cell viability signaling is substantially unknown. In this study, the effects of NaB on viability parameters and NFκB, one of the most important regulators in apoptosis, were examined in HCT116 colon cancer cells. Cell culture, light microscopy, spectrophotometry, flow cytometry, and western blot were used as methods to determine cell viability, caspase-3 activity, NFκB, Bcl-xl, Bim, and PARP proteins, respectively. NaB (6.25 mM–50 mM treatment inhibited cell viability by inducing apoptosis, which was evident with increased Annexin V-PE staining and caspase-3 activity. NFκB activation accompanied the induction of apoptosis in NaB treated cells. Inhibition of NFκB with BAY 11-7082 did not show a pronounced effect on cell viability but induced a more apoptotic profile, which was confirmed by increased PARP fragmentation and caspase-3 activity. This effect was mostly evident at 50 mM concentration of NaB. Bcl-xl levels were not affected by NaB or BAY 11-7082/NaB treatment; whereas, total Bim increased with NaB treatment. Inhibition of NFκB activity further increased Bim levels. Overall, these results suggest that NaB induces apoptosis and activates NFκB in HCT116 colon cancer cells. Activation of NFκB emerges as target in an attempt to protect cells against apoptosis.

Cdc25A promotes cell survival by stimulating NF-{kappa}B activity through I{kappa}B-{alpha} phosphorylation and destabilization

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Hong, Hey-Young; Choi, Jiyeon [Department of Biochemistry, College of Natural Sciences, Kangwon National University, 192-1 Hyoja-2-dong, Chuncheon 200-701 (Korea, Republic of); Cho, Young-Wook [Korea Basic Science Institute, Chuncheon Center, Gangwondaehak-gil 1, Chuncheon 200-701 (Korea, Republic of); Kim, Byung-Chul, E-mail: bckim@kangwon.ac.kr [Department of Biochemistry, College of Natural Sciences, Kangwon National University, 192-1 Hyoja-2-dong, Chuncheon 200-701 (Korea, Republic of)

2012-04-06

Highlights: Black-Right-Pointing-Pointer We examine the antiapoptotic mechanisms of Cdc25A. Black-Right-Pointing-Pointer Smad7 decreases the phosphorylation of I{kappa}B-alpha at Ser-32. Black-Right-Pointing-Pointer Smad7 positively regulates NF-{kappa}B activity through I{kappa}B-alpha ubiquitination. -- Abstract: Cell division cycle 25A (Cdc25A), a dual specificity protein phosphatase, exhibits anti-apoptotic activity, but the underlying molecular mechanisms are poorly characterized. Here we report that Cdc25A inhibits cisplatin-induced apoptotic cell death by stimulating nuclear factor-kappa B (NF-{kappa}B) activity. In HEK-293 cells, Cdc25A decreased protein level of inhibitor subunit kappa B alpha (I{kappa}-B{alpha}) in association with increased serine 32-phosphorylation, followed by stimulation of transcriptional activity of NF-{kappa}B. Inhibition of NF-{kappa}B activity by chemical inhibitor or overexpression of I{kappa}-B{alpha} in Cdc25A-elevated cancer cells resistant to cisplatin improved their sensitivity to cisplatin-induced apoptosis. Our data show for the first time that Cdc25A has an important physiological role in NF-{kappa}B activity regulation and it may be an important survival mechanism of cancer cells.

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

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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.

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.

Rictor positively regulates B cell receptor signaling by modulating actin reorganization via ezrin.

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Lu Huang

2017-08-01

Full Text Available As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2 has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell-specfic Rictor knockout (KO mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons tyrosine kinase (pBtk and phosphorylated SH2-containing inositol phosphatase (pSHIP, are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization.

Cirtical role for Salmonella effector SopB in regulating inflammasome activation.

Science.gov (United States)

Hu, Gui-Qiu; Song, Pei-Xuan; Chen, Wei; Qi, Shuai; Yu, Shui-Xing; Du, Chong-Tao; Deng, Xu-Ming; Ouyang, Hong-Sheng; Yang, Yong-Jun

2017-10-01

Salmonella is known to evolve many mechanisms to avoid or delay inflammasome activation which remain largely unknown. In this study, we investigated whether the SopB protein critical to bacteria virulence capacity was an effector that involved in the regulation of inflammasome activation. BMDMs from NLRC4-, NLRP3-, caspase-1/-11-, IFI16- and AIM2-deficient mice were pretreated with LPS, and subsequently stimulated with a series of SopB-related strains of Salmonella, inflammasome induced cell death, IL-1β secretion, cleaved caspase-1 production and ASC speckle formation were detected. We found that SopB could inhibit host IL-1β secretion, caspase-1 activation and inflammasome induced cell death using a series of SopB-related strains of Salmonella; however the reduction of IL-1β secretion was not dependent on sensor that contain PYD domain, such as NLRP3, AIM2 or IFI16, but dependent on NLRC4. Notably, SopB specifically prevented ASC oligomerization and the enzymatic activity of SopB was responsible for the inflammasome inhibition. Furthermore, inhibition of Akt signaling induced enhanced inflammasome activation. These results revealed a novel role in inhibition of NLRC4 inflammasome for Salmonella effector SopB. Copyright © 2017. Published by Elsevier Ltd.

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

OpenAIRE

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

2014-01-01

Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of li...

Lycopene inhibits ICAM-1 expression and NF-κB activation by Nrf2-regulated cell redox state in human retinal pigment epithelial cells.

Science.gov (United States)

Yang, Po-Min; Wu, Zhi-Zhen; Zhang, Yu-Qi; Wung, Being-Sun

2016-06-15

Age-related macular degeneration (AMD) is one of the most common diseases leading to blindness in elderly people. The progression of AMD may be prevented through anti-inflammation and antioxidation in retinal pigment epithelium (RPE) cells. Lycopene, a carotenoid, has been shown to possess both antioxidative and anti-inflammatory properties. This research was conducted to detail the mechanisms of these effects of lycopene-treated RPE cells. We exposed ARPE-19 cells to TNFα after pretreatment with lycopene, and measured monocyte adhesion, ICAM-1 expression, NF-κB nuclear translocation, and transcriptional activity. Cell viability was assayed with Alamar Blue. The cell redox state was tested by glutathione (GSH) and reactive oxygen species (ROS) levels. The importance of the Nrf2 pathway was tested in nuclear translocation, promoter reporter assay, and siRNA. Lycopene could reduce TNF-α-induced monocyte adhesion and H2O2- induced cell damage in RPE cells. Furthermore, lycopene inhibits ICAM-1 expression and abolishes NF-κB activation for up to 12h in TNFα-treated RPE cells. Lycopene upregulates Nrf2 levels in nuclear extracts and increases the transactivity of antioxidant response elements. The use of Nrf2 siRNA blocks the inhibitory effect of lycopene in TNF-α-induced ICAM-1 expression and NF-κB activation. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the de novo synthesis of GSH. We found that lycopene increases intracellular GSH levels and GCL expression. Following lycopene treatment, TNF-α-induced ROS production was abolished. The Nrf2-regulated antioxidant property plays a pivotal role in the anti-inflammatory mechanism underlying the inhibition of NF-κB activation in lycopene-treated ARPE-19 cells. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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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.

Quantitative regulation of B cell division destiny by signal strength.

Science.gov (United States)

Turner, Marian L; Hawkins, Edwin D; Hodgkin, Philip D

2008-07-01

Differentiation to Ab secreting and isotype-switched effector cells is tightly linked to cell division and therefore the degree of proliferation strongly influences the nature of the immune response. The maximum number of divisions reached, termed the population division destiny, is stochastically distributed in the population and is an important parameter in the quantitative outcome of lymphocyte responses. In this study, we further assessed the variables that regulate B cell division destiny in vitro in response to T cell- and TLR-dependent stimuli. Both the concentration and duration of stimulation were able to regulate the average maximum number of divisions undergone for each stimulus. Notably, a maximum division destiny was reached during provision of repeated saturating stimulation, revealing that an intrinsic limit to proliferation exists even under these conditions. This limit was linked directly to division number rather than time of exposure to stimulation and operated independently of the survival regulation of the cells. These results demonstrate that a B cell population's division destiny is regulable by the stimulatory conditions up to an inherent maximum value. Division destiny is a crucial parameter in regulating the extent of B cell responses and thereby also the nature of the immune response mounted.

No evidence for dualism in function and receptors: PD-L2/B7-DC is an inhibitory regulator of human T cell activation.

Science.gov (United States)

Pfistershammer, Katharina; Klauser, Christoph; Pickl, Winfried F; Stöckl, Johannes; Leitner, Judith; Zlabinger, Gerhard; Majdic, Otto; Steinberger, Peter

2006-05-01

The B7 family member programmed-death-1-ligand 2 (PD-L2/B7-DC) is a ligand for programmed-death-receptor 1 (PD-1), a receptor involved in negative regulation of T cell activation. Several independent studies have reported that PD-L2, however, can also potently costimulate murine T cells via an additional yet unidentified receptor. In this study, we evaluated the contribution of PD-L2 to the activation of human T cells using a novel system of engineered T cell stimulators that expresses membrane-bound anti-CD3 antibodies. Analyzing early activation markers, cytokine production and proliferation, we found PD-L2 to consistently inhibit T cell activation. PD-L2 inhibition affected CD4+ and CD8+ T cells and was not abrogated by costimulation via CD28. Blocking PD-1 reverted the inhibitory effect of PD-L2, demonstrating involvement of this pathway. In human T cells, we found no evidence for any of the costimulatory effects described for PD-L2 in murine systems. In line with our functional data that do not point to stimulatory PD-L2-ligands, we show that binding of PD-L2-immunoglobulin to activated human T cells is abrogated by PD-1 antibodies. Our results demonstrate that PD-L2 negatively regulates human T cell activation and thus might be a candidate molecule for immunotherapeutic approaches aimed to attenuate pathological immune responses.

The cell biology of T-dependent B cell activation

DEFF Research Database (Denmark)

Owens, T; Zeine, R

1989-01-01

The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been...... implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell...

ErbB2 regulates NHEJ repair pathway by affecting erbB1-triggered IR-induced Akt activity

International Nuclear Information System (INIS)

Toulany, Mahmoud; Peter Rodemann, H.

2009-01-01

We have already reported that erbBl-PI3K-AKT signaling is an important pathway in regulating radiation sensitivity and DNA double strand break repair of human tumor cells. In the present study using small interfering RNA and pharmacological inhibitors in non-small cell lung cancer cell lines we investigated the role of Aktl on radiation-induced DNA-PKcs activity and DNA-double strand break (DNA-DSB) repair. Likewise, the function of erbB2 as hetrodimerization partner of erbBl in radiation-induced Akt activity and regulation of DNA-dsb repair through DNA-PKcs was evaluated. In A549 and H460 transfected with AKTl-siRNA radiation-induced phosphorylation of DNA-PKcs the key enzyme regulating NHEJ repair pathway was markedly inhibited. In both cell lines downregulation of Aktl led to a significant enhancement of residual DNA-DSB, i.e. impaired DNA-DSB repair. Interestingly, in cells transfected with DNA-PKcs-siRNA a lack of effect of AKTl-siRNA on enhancement of residual DNA-DSBs was observed. This results indicate that Aktl regulates NHEJ repair in a DNA-PKcs dependent manner

Fucoidan Elevates MicroRNA-29b to Regulate DNMT3B-MTSS1 Axis and Inhibit EMT in Human Hepatocellular Carcinoma Cells

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Ming-De Yan

2015-09-01

Full Text Available Accumulating evidence has revealed that fucoidan exhibits anti-tumor activities by arresting cell cycle and inducing apoptosis in many types of cancer cells including hepatocellular carcinoma (HCC. Exploring its effect on microRNA expression, we found that fucoidan markedly upregulated miR-29b of human HCC cells. The induction of miR-29b was accompanied with suppression of its downstream target DNMT3B in a dose-dependent manner. The reduction of luciferase activity of DNMT3B 3′-UTR reporter by fucoidan was as markedly as that by miR-29b mimic, indicating that fucoidan induced miR-29b to suppress DNMT3B. Accordingly, the mRNA and protein levels of MTSS1 (metastasis suppressor 1, a target silenced by DNMT3B, were increased after fucoidan treatment. Furthermore, fucoidan also down-regulated TGF-β receptor and Smad signaling of HCC cells. All these effects leaded to the inhibition of EMT (increased E-cadherin and decreased N-cadherin and prevention of extracellular matrix degradation (increased TIMP-1 and decreased MMP2, 9, by which the invasion activity of HCC cells was diminished. Our results demonstrate the profound effect of fucoidan not only on the regulation of miR-29b-DNMT3B-MTSS1 axis but also on the inhibition of TGF-β signaling in HCC cells, suggesting the potential of using fucoidan as integrative therapeutics against invasion and metastasis of HCC.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation

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Cho, Seong-Jun; Kang, Hana [KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co, Seoul (Korea, Republic of); Kim, Min Young [Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan (Korea, Republic of); Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun [KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co, Seoul (Korea, Republic of); Pyo, Suhkneung [College of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do (Korea, Republic of); Yang, Kwang Hee, E-mail: kwangheey@khnp.co.kr [KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co, Seoul (Korea, Republic of)

2016-04-01

Purpose: To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Methods and Materials: Splenocytes and IM-9 cells were uniformly irradiated with various doses of a {sup 137}Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. Results: First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Conclusion: Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation.

Science.gov (United States)

Cho, Seong-Jun; Kang, Hana; Kim, Min Young; Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun; Pyo, Suhkneung; Yang, Kwang Hee

2016-04-01

To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Splenocytes and IM-9 cells were uniformly irradiated with various doses of a (137)Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast. Copyright © 2016 Elsevier Inc. All rights reserved.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation

International Nuclear Information System (INIS)

Cho, Seong-Jun; Kang, Hana; Kim, Min Young; Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun; Pyo, Suhkneung; Yang, Kwang Hee

2016-01-01

Purpose: To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Methods and Materials: Splenocytes and IM-9 cells were uniformly irradiated with various doses of a "1"3"7Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. Results: First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Conclusion: Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast.

Tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces cell proliferation in normal human bronchial epithelial cells through NFκB activation and cyclin D1 up-regulation

International Nuclear Information System (INIS)

Ho, Y.-S.; Chen, Chien-Ho; Wang, Y.-J.; Pestell, Richard G.; Albanese, Chris; Chen, R.-J.; Chang, M.-C.; Jeng, J.-H.; Lin, S.-Y.; Liang, Y.-C.; Tseng, H.; Lee, W.-S.; Lin, J.-K.; Chu, J.-S.; Chen, L.-C.; Lee, C.-H.; Tso, W.-L.; Lai, Y.-C.; Wu, C.-H.

2005-01-01

Cigarette smoke contains several carcinogens known to initiate and promote tumorigenesis as well as metastasis. Nicotine is one of the major components of the cigarette smoke and the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific carcinogen. Here, we demonstrated that NNK stimulated cell proliferation in normal human bronchial epithelial cells (NHBE) and small airway epithelial cells (SAEC). Cells exposed to NNK resulted in an increase in the level of cyclin D1 protein (as early as 3-6 h). Increased phosphorylation of the Rb Ser 795 was detected at 6-15 h after NNK treatment and thereby promoted cells entering into the S phase (at 15-21 h). The increased cyclin D1 protein level was induced through activation of the transcription factor, nuclear factor kB (NFκB), in the NHBE cells. Treatment of the NHBE cells with PD98059, an ERK1/2 (extracellular signal-regulated protein kinase)-specific inhibitor, specifically suppressed the NNK-induced IκBα phosphorylation at position 32 of the serine residue, suggesting that the ERK1/2 kinase was involved in the IκBα phosphorylation induced by NFκB activation. To determine whether the NNK-induced NFκB activation and cyclin D1 induction were also observed in vivo, A/J mice were treated with NNK (9.1 mg) for 20 weeks and the results showed a significant induction of cyclin D1 and NFκB translocation determined by immunoblotting analyses. We further demonstrated that the nicotine acetylcholine receptor (nAchR), which contains the α3-subunit, was the major target mediating NNK-induced cyclin D1 expression in the NHBE cells. In summary, our findings demonstrate for the first time that NNK could stimulate normal human bronchial cell proliferation through activation of the NFκB, which in turn up-regulated the cyclin D1 expression

Metabolic activity is necessary for activation of T suppressor cells by B cells

International Nuclear Information System (INIS)

Elkins, K.L.; Stashak, P.W.; Baker, P.J.

1990-01-01

Ag-primed B cells must express cell-surface IgM, but not IgD or Ia Ag, and must remain metabolically active, in order to activate suppressor T cells (Ts) specific for type III pneumococcal polysaccharide. Ag-primed B cells that were gamma-irradiated with 1000r, or less, retained the ability to activate Ts; however, Ag-primed B cells exposed to UV light were not able to do so. gamma-Irradiated and UV-treated Ag-primed B cells both expressed comparable levels of cell-surface IgM, and both localized to the spleen after in vivo transfer; neither could proliferate in vitro in response to mitogens. By contrast, gamma-irradiated primed B cells were still able to synthesize proteins, whereas UV-treated primed B cells could not. These findings suggest that in order for Ag-primed B cells to activate Ts, they must (a) express cell-associated IgM (sIgM) antibody bearing the idiotypic determinants of antibody specific for type III pneumococcal polysaccharide, and (b) be able to synthesize protein for either the continued expression of sIgM after cell transfer, or for the elaboration of another protein molecule that is also required for the activation of Ts; this molecule does not appear to be Ia Ag

Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

Energy Technology Data Exchange (ETDEWEB)

Berndt, Alexander, E-mail: alexander.berndt@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Büttner, Robert, E-mail: Robert-Buettner@gmx.net [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany); Gühne, Stefanie, E-mail: stefanie_guehne@gmx.net [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Gleinig, Anna, E-mail: annagleinig@yahoo.com [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Richter, Petra, E-mail: P.Richter@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Chen, Yuan, E-mail: Yuan.Chen@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Franz, Marcus, E-mail: Marcus.Franz@med.uni-jena.de [Clinic of Internal Medicine I, Jena University Hospital, 07740 Jena (Germany); Liebmann, Claus, E-mail: Claus.Liebmann@uni-jena.de [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany)

2014-04-01

Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM{sub TGF}, FCM{sub PDGF}) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM{sub B}). FCM{sub TGF} stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM{sub TGF}≫FCM{sub PDGF} induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM{sub TGF}>FCM{sub PDGF}) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin

Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

International Nuclear Information System (INIS)

Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

2014-01-01

Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM TGF , FCM PDGF ) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM B ). FCM TGF stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM TGF ≫FCM PDGF induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM TGF >FCM PDGF ) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin as sign of EMT. • Results qualify

Inhibition of Ubc13-mediated Ubiquitination by GPS2 Regulates Multiple Stages of B Cell Development.

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Lentucci, Claudia; Belkina, Anna C; Cederquist, Carly T; Chan, Michelle; Johnson, Holly E; Prasad, Sherry; Lopacinski, Amanda; Nikolajczyk, Barbara S; Monti, Stefano; Snyder-Cappione, Jennifer; Tanasa, Bogdan; Cardamone, M Dafne; Perissi, Valentina

2017-02-17

Non-proteolytic ubiquitin signaling mediated by Lys 63 ubiquitin chains plays a critical role in multiple pathways that are key to the development and activation of immune cells. Our previous work indicates that GPS2 (G-protein Pathway Suppressor 2) is a multifunctional protein regulating TNFα signaling and lipid metabolism in the adipose tissue through modulation of Lys 63 ubiquitination events. However, the full extent of GPS2-mediated regulation of ubiquitination and the underlying molecular mechanisms are unknown. Here, we report that GPS2 is required for restricting the activation of TLR and BCR signaling pathways and the AKT/FOXO1 pathway in immune cells based on direct inhibition of Ubc13 enzymatic activity. Relevance of this regulatory strategy is confirmed in vivo by B cell-targeted deletion of GPS2, resulting in developmental defects at multiple stages of B cell differentiation. Together, these findings reveal that GPS2 genomic and non-genomic functions are critical for the development and cellular homeostasis of B cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Early growth response gene-2 (Egr-2 regulates the development of B and T cells.

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Suling Li

2011-04-01

Full Text Available Understanding of how transcription factors are involved in lymphocyte development still remains a challenge. It has been shown that Egr-2 deficiency results in impaired NKT cell development and defective positive selection of T cells. Here we investigated the development of T, B and NKT cells in Egr-2 transgenic mice and the roles in the regulation of distinct stages of B and T cell development.The expression of Egr1, 2 and 3 were analysed at different stages of T and B cell development by RT-PCT and results showed that the expression was strictly regulated at different stages. Forced expression of Egr-2 in CD2(+ lymphocytes resulted in a severe reduction of CD4(+CD8(+ (DP cells in thymus and pro-B cells in bone marrow, which was associated with reduced expression of Notch1 in ISP thymocytes and Pax5 in pro-B cells, suggesting that retraction of Egr-2 at the ISP and pro-B cell stages is important for the activation of lineage differentiation programs. In contrast to reduction of DP and pro-B cells, Egr-2 enhanced the maturation of DP cells into single positive (SP T and NKT cells in thymus, and immature B cells into mature B cells in bone marrow.Our results demonstrate that Egr-2 expressed in restricted stages of lymphocyte development plays a dynamic, but similar role for the development of T, NKT and B cells.

Protein tyrosine phosphatase-1B (PTP1B) helps regulate EGF-induced stimulation of S-phase entry in human corneal endothelial cells

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Ishino, Yutaka; Zhu, Cheng; Harris, Deshea L.

2008-01-01

Purpose Human corneal endothelial cells (HCEC), particularly from older donors, only proliferate weakly in response to EGF. The protein tyrosine phosphatase, PTP1B, is known to negatively regulate EGF-induced signaling in several cell types by dephosphorylating the epidermal growth factor receptor (EGFR). The current studies were conducted to determine whether PTP1B plays a role in regulating cell cycle entry in HCEC in response to EGF stimulation. Methods Donor corneas were obtained from the National Disease Research Interchange and accepted for study based on established exclusion criteria. PTP1B was localized in the endothelium of ex vivo corneas and in cultured cells by immunocytochemistry. Western blot analysis verified PTP1B protein expression in HCEC and then compared the relative expression of EGFR and PTP1B in HCEC from young (60 years old). The effect of inhibiting the activity of PTP1B on S-phase entry was tested by comparing time-dependent BrdU incorporation in subconfluent HCEC incubated in the presence or absence of the PTP1B inhibitor, CinnGEL 2Me, before EGF stimulation. Results PTP1B was localized in a punctate pattern mainly within the cytoplasm of HCEC in ex vivo corneas and cultured cells. Western blots revealed the presence of three PTP1B-positive bands in HCEC and the control. Further western blot analysis showed no significant age-related difference in expression of EGFR (p=0.444>0.05); however, PTP1B expression was significantly higher in HCEC from older donors (p=0.024<0.05). Pre-incubation of HCEC with the PTP1B inhibitor significantly increased (p=0.019<0.05) the number of BrdU positive cells by 48 h after EGF stimulation. Conclusions Both immunolocalization and western blot studies confirmed that PTP1B is expressed in HCEC. Staining patterns strongly suggest that at least a subset of PTP1B is localized to the cytoplasm and most likely to the endoplasmic reticulum, the known site of EGFR/PTP1B interaction following EGF stimulation. PTP1B

Constitutively active ErbB2 regulates cisplatin-induced cell death in breast cancer cells via pro- and antiapoptotic mechanisms

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Sigurðsson, Haraldur H; Olesen, Christina Wilkens; Dybboe, Rie

2015-01-01

cancer cell death, and determine how NHE1 regulates this process. Cisplatin treatment elicited apoptosis, ATM phosphorylation, upregulation of p53, Noxa (PMAIP1), and PUMA (BBC3), and cleavage of caspase-9, -7, fodrin, and PARP-1 in MCF-7 cells. Inducible ΔNErbB2 expression strongly reduced cisplatin...

B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing.

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Dara K Mohammad

Full Text Available Protein kinase B (AKT phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206 dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins.

Classical NF-κB Metabolically Reprograms Sarcoma Cells Through Regulation of Hexokinase 2

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Priya Londhe

2018-04-01

Full Text Available BackgroundMetabolic reprogramming has emerged as a cancer hallmark, and one of the well-known cancer-associated metabolic alterations is the increase in the rate of glycolysis. Recent reports have shown that both the classical and alternative signaling pathways of nuclear factor κB (NF-κB play important roles in controlling the metabolic profiles of normal cells and cancer cells. However, how these signaling pathways affect the metabolism of sarcomas, specifically rhabdomyosarcoma (RMS and osteosarcoma (OS, has not been characterized.MethodsClassical NF-κB activity was inhibited through overexpression of the IκBα super repressor of NF-κB in RMS and OS cells. Global gene expression analysis was performed using Affymetrix GeneChip Human Transcriptome Array 2.0, and data were interpreted using gene set enrichment analysis. Seahorse Bioscience XFe24 was used to analyze oxygen consumption rate as a measure of aerobic respiration.ResultsInhibition of classical NF-κB activity in sarcoma cell lines restored alternative signaling as well as an increased oxidative respiratory metabolic phenotype in vitro. In addition, microarray analysis indicated that inhibition of NF-κB in sarcoma cells reduced glycolysis. We showed that a glycolytic gene, hexokinase (HK 2, is a direct NF-κB transcriptional target. Knockdown of HK2 shifted the metabolic profile in sarcoma cells away from aerobic glycolysis, and re-expression of HK2 rescued the metabolic shift induced by inhibition of NF-κB activity in OS cells.ConclusionThese findings suggest that classical signaling of NF-κB plays a crucial role in the metabolic profile of pediatric sarcomas potentially through the regulation of HK2.

Tight regulation between cell survival and programmed cell death in GBM stem-like cells by EGFR/GSK3b/PP2A signaling.

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Gürsel, Demirkan B; Banu, Matei A; Berry, Nicholas; Marongiu, Roberta; Burkhardt, Jan-Karl; Kobylarz, Keith; Kaplitt, Michael G; Rafii, Shahin; Boockvar, John A

2015-01-01

Malignant gliomas represent one of the most aggressive forms of cancer, displaying high mortality rates and limited treatment options. Specific subpopulations of cells residing in the tumor niche with stem-like characteristics have been postulated to initiate and maintain neoplasticity while resisting conventional therapies. The study presented here aims to define the role of glycogen synthase kinase 3 beta (GSK3b) in patient-derived glioblastoma (GBM) stem-like cell (GSC) proliferation, apoptosis and invasion. To evaluate the potential role of GSK3b in GBM, protein profiles from 68 GBM patients and 20 normal brain samples were analyzed for EGFR-mediated PI3kinase/Akt and GSK3b signaling molecules including protein phosphatase 2A (PP2A). To better understand the function of GSK3b in GBM, GSCs were isolated from GBM patient samples. Blocking GSK3b phosphorylation at Serine 9 attenuated cell proliferation while concomitantly stimulating apoptosis through activation of Caspase-3 in patient-derived GSCs. Increasing GSK3b protein content resulted in the inhibition of cell proliferation, colony formation and stimulated programmed cell death. Depleting GSK3b in GSCs down regulated PP2A. Furthermore, knocking down PP2A or blocking its activity by okadaic acid inactivated GSK3b by increasing GSK3b phosphorylation at Serine 9. Our data suggests that GSK3b may function as a regulator of apoptosis and tumorigenesis in GSCs. Therapeutic approaches targeting GSK3b in glioblastoma stem-like cells may be a useful addition to our current therapeutic armamentarium.

[Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

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Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

2004-01-01

To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P ras4B can enhance the expression of ERalpha and

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

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Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

2003-01-01

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

Caspase-10 Negatively Regulates Caspase-8-Mediated Cell Death, Switching the Response to CD95L in Favor of NF-κB Activation and Cell Survival

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Sebastian Horn

2017-04-01

Full Text Available Formation of the death-inducing signaling complex (DISC initiates extrinsic apoptosis. Caspase-8 and its regulator cFLIP control death signaling by binding to death-receptor-bound FADD. By elucidating the function of the caspase-8 homolog, caspase-10, we discover that caspase-10 negatively regulates caspase-8-mediated cell death. Significantly, we reveal that caspase-10 reduces DISC association and activation of caspase-8. Furthermore, we extend our co-operative/hierarchical binding model of caspase-8/cFLIP and show that caspase-10 does not compete with caspase-8 for binding to FADD. Utilizing caspase-8-knockout cells, we demonstrate that caspase-8 is required upstream of both cFLIP and caspase-10 and that DISC formation critically depends on the scaffold function of caspase-8. We establish that caspase-10 rewires DISC signaling to NF-κB activation/cell survival and demonstrate that the catalytic activity of caspase-10, and caspase-8, is redundant in gene induction. Thus, our data are consistent with a model in which both caspase-10 and cFLIP coordinately regulate CD95L-mediated signaling for death or survival.

Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis.

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Eeva, J; Nuutinen, U; Ropponen, A; Mättö, M; Eray, M; Pellinen, R; Wahlfors, J; Pelkonen, J

2009-12-01

During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

Up-regulation of granzyme B and perforin by staphylococcal enterotoxin C2 mutant induces enhanced cytotoxicity in Hepa1–6 cells

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Zhang, Guojun [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); University of Chinese Academy of Sciences, Beijing (China); Xu, Mingkai, E-mail: mkxu@iae.ac.cn [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); Zhang, Huiwen [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); Song, Yubo [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); University of Chinese Academy of Sciences, Beijing (China); Wang, Jian; Zhang, Chenggang [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China)

2016-12-15

Staphylococcal enterotoxin C2 (SEC2), a member of bacterial superantigen, is one of the most potent known activators of T lymphocytes. With this property, SEC2 has already been used in clinic as a tumor immunotherapy agent in China. To increase the antitumor activity, a SEC2 mutant named ST-4 (GKVTG102-106WWH) with amino acid substitutions in T cell receptor (TCR)-binding domain was generated by site-directed mutagenesis, and the molecular mechanism of the enhanced antitumor activity was investigated. Results showed that ST-4 could activate much more Vβ 8.2 and 8.3 T cells and NK cells compared with SEC2, and exhibited significantly enhanced immunocyte stimulation and antitumor activity in vitro. The synthetic peptide sequencing the residues of mutant TCR-binding domain could competitively inhibit the immunocyte stimulation activity of ST-4. Most importantly, ST-4 up-regulated granzyme B and perforin at both mRNA and protein levels. We also found that expression of proapoptotic proteins cytochrome c, BAX and activation of caspase-3, 9 was up-regulated, and antiapoptotic protein Bcl-xL was down-regulated in the treatment with either ST-4 or SEC2. When granzyme B inhibitor or perforin inhibitor is presented, tumor cell viability was significantly rescued. Taken together, we demonstrate that increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. These activated cells released up-regulated granzyme B and perforin, which induced the enhanced tumor cells apoptosis by mitochondrial apoptotic pathway, and ultimately led to enhanced tumor cell growth inhibition. ST-4 may be a promising candidate for antitumor clinic usage in future. - Highlights: • We obtained a SEC2 mutant ST-4 with enhanced superantigen and antitumor activity. • Increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. • Up-regulated GzmB and PRF1 in T cell by ST-4 induced enhanced tumor cells apoptosis. • Enhanced tumor cell apoptosis

Valproic acid inhibits neural progenitor cell death by activation of NF-κB signaling pathway and up-regulation of Bcl-XL

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Han Seol

2011-07-01

Full Text Available Abstract Background At the beginning of neurogenesis, massive brain cell death occurs and more than 50% of cells are eliminated by apoptosis along with neuronal differentiation. However, few studies were conducted so far regarding the regulation of neural progenitor cells (NPCs death during development. Because of the physiological role of cell death during development, aberration of normal apoptotic cell death is detrimental to normal organogenesis. Apoptosis occurs in not only neuron but also in NPCs and neuroblast. When growth and survival signals such as EGF or LIF are removed, apoptosis is activated as well as the induction of differentiation. To investigate the regulation of cell death during developmental stage, it is essential to investigate the regulation of apoptosis of NPCs. Methods Neural progenitor cells were cultured from E14 embryonic brains of Sprague-Dawley rats. For in vivo VPA animal model, pregnant rats were treated with VPA (400 mg/kg S.C. diluted with normal saline at E12. To analyze the cell death, we performed PI staining and PARP and caspase-3 cleavage assay. Expression level of proteins was investigated by Western blot and immunocytochemical assays. The level of mRNA expression was investigated by RT-PCR. Interaction of Bcl-XL gene promoter and NF-κB p65 was investigated by ChIP assay. Results In this study, FACS analysis, PI staining and PARP and caspase-3 cleavage assay showed that VPA protects cultured NPCs from cell death after growth factor withdrawal both in basal and staurosporine- or hydrogen peroxide-stimulated conditions. The protective effect of prenatally injected VPA was also observed in E16 embryonic brain. Treatment of VPA decreased the level of IκBα and increased the nuclear translocation of NF-κB, which subsequently enhanced expression of anti-apoptotic protein Bcl-XL. Conclusion To the best of our knowledge, this is the first report to indicate the reduced death of NPCs by VPA at developmentally

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

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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.

Immune Regulation of RAW264.7 Cells In Vitro by Flavonoids from Astragalus complanatus via Activating the NF-κB Signalling Pathway

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Yu Li

2018-01-01

Full Text Available The current study aimed at investigating the effects of flavonoids from Astragalus complanatus (FAC on the proliferation, the contents, and gene expression levels of cytokines, secretion of surface stimulating factors, cell cycle, and the expression level of the NF-κB signalling pathway in RAW264.7 cells. Our results revealed that compared with control group, the contents of IL-6, IL-1β, TNF-α, and NO and the mRNA expression levels of IL-6, IL-1β, TNF-α, and iNOS in FAC-treated groups significantly increased (p<0.01. Moreover, FAC induced macrophage activation to release the above-mentioned mediators partly involved in NF-κB/MAPK signalling pathways. Therefore, FAC regulates immune function in RAW264.7 cells via activating the NF-κB signalling pathway. FAC could be applicable for agriculture, drug research, and food industry as a potent immune-modulatory agent.

Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells.

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Jeong, Kwon; Kim, Kiyoon; Kim, Hunsung; Oh, Yoojung; Kim, Seong-Jin; Jo, Yunhee; Choe, Wonchae

2015-06-01

Hypoxia is an important form of physiological stress that induces cell death, due to the resulting endoplasmic reticulum (ER) stress, particularly in solid tumors. Although previous studies have indicated that cyclophilin B (CypB) plays a role in ER stress, there is currently no direct information supporting the mechanism of CypB involvement under hypoxic conditions. However, it has previously been demonstrated that ER stress positively regulates the expression of CypB. In the present study, it was demonstrated that CypB is transcriptionally regulated by hypoxia-mediated activation of transcription factor 6 (ATF6), an ER stress transcription factor. Subsequently, the effects of ATF6 on CypB promoter activity were investigated and an ATF6-responsive region in the promoter was identified. Hypoxia and ATF6 expression each increased CypB promoter activity. Collectively, these results demonstrate that ATF6 positively regulates the expression of CypB by binding to an ATF6-responsive region in the promoter, which may play an important role in the attenuation of apoptosis in the adaption to hypoxia. These results suggest that CypB may be a key molecule in the adaptation of cells to hypoxic conditions.

MiR-146a regulates PM1 -induced inflammation via NF-κB signaling pathway in BEAS-2B cells.

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Liu, Limin; Wan, Chong; Zhang, Wei; Guan, Longfei; Tian, Guoxiong; Zhang, Fang; Ding, Wenjun

2018-04-18

Exposure to particulate matter (PM) leads to kinds of cardiopulmonary diseases, such as asthma, COPD, arrhythmias, lung cancer, etc., which are related to PM-induced inflammation. We have found that PM 2.5 (aerodynamics diameter <2.5 µm) exposure induces inflammatory response both in vivo and in vitro. Since the toxicity of PM is tightly associated with its size and components, PM 1 (aerodynamics diameter <1.0 µm) is supposed to be more toxic than PM 2.5 . However, the mechanism of PM 1 -induced inflammation is not clear. Recently, emerging evidences prove that microRNAs play a vital role in regulating inflammation. Therefore, we studied the regulation of miR-146a in PM 1 -induced inflammation in human lung bronchial epithelial BEAS-2B cells. The results show that PM 1 induces the increase of IL-6 and IL-8 in BEAS-2B cells and up-regulates the miR-146a expression by activating NF-κB signaling pathway. Overexpressed miR-146a prevents the nuclear translocation of p65 through inhibiting the IRAK1/TRAF6 expression, and downregulates the expression of IL-6 and IL-8. Taken together, these results demonstrate that miR-146a can negatively feedback regulate PM 1 -induced inflammation via NF-κB signaling pathway in BEAS-2B cells. © 2018 Wiley Periodicals, Inc.

Mechanisms underlying regulation of cell cycle and apoptosis by hnRNP B1 in human lung adenocarcinoma A549 cells.

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Han, Juan; Tang, Feng-ming; Pu, Dan; Xu, Dan; Wang, Tao; Li, Weimin

2014-01-01

Overexpression of heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), a nuclear RNA binding protein, has been reported to occur in early-stage lung cancer and in premalignant lesions. DNA-dependent protein kinase (DNA-PK) is known to be involved in the repair of double-strand DNA breaks. Reduced capacity to repair DNA has been associated with the risk of lung cancer. We investigated a link between hnRNP B1 and DNA-PK and their effects on proliferation, cell cycle, and apoptosis in the human lung adenocarcinoma cell line A549. We found that hnRNP B1 and DNA-PK interact with each other in a complex fashion. Reducing hnRNP B1 expression in A549 cells with the use of RNAi led to upregulation of p53 activity through upregulation of DNA-PK activity but without inducing p53 expression. Further, suppression of hnRNP B1 in A549 cells slowed cell proliferation, promoted apoptosis, and induced cell cycle arrest at the G1 stage. The presence of NU7026 reduced the arrest of cells at the G1 stage and reduced the apoptosis rate while promoting cell growth. Taken together, our results demonstrate that by regulating DNA-PK activity, hnRNP B1 can affect p53-mediated cell cycle progression and apoptosis, resulting in greater cell survival and subsequent proliferation.

MicroRNA-193b represses cell proliferation and regulates cyclin D1 in melanoma.

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Chen, Jiamin; Feilotter, Harriet E; Paré, Geneviève C; Zhang, Xiao; Pemberton, Joshua G W; Garady, Cherif; Lai, Dulcie; Yang, Xiaolong; Tron, Victor A

2010-05-01

Cutaneous melanoma is an aggressive form of human skin cancer characterized by high metastatic potential and poor prognosis. To better understand the role of microRNAs (miRNAs) in melanoma, the expression of 470 miRNAs was profiled in tissue samples from benign nevi and metastatic melanomas. We identified 31 miRNAs that were differentially expressed (13 up-regulated and 18 down-regulated) in metastatic melanomas relative to benign nevi. Notably, miR-193b was significantly down-regulated in the melanoma tissues examined. To understand the role of miR-193b in melanoma, functional studies were undertaken. Overexpression of miR-193b in melanoma cell lines repressed cell proliferation. Gene expression profiling identified 314 genes down-regulated by overexpression of miR-193b in Malme-3M cells. Eighteen of these down-regulated genes, including cyclin D1 (CCND1), were also identified as putative miR-193b targets by TargetScan. Overexpression of miR-193b in Malme-3M cells down-regulated CCND1 mRNA and protein by > or = 50%. A luciferase reporter assay confirmed that miR-193b directly regulates CCND1 by binding to the 3'untranslated region of CCND1 mRNA. These studies indicate that miR-193b represses cell proliferation and regulates CCND1 expression and suggest that dysregulation of miR-193b may play an important role in melanoma development.

Novel Radiolytic Rotenone Derivative, Rotenoisin B with Potent Anti-Carcinogenic Activity in Hepatic Cancer Cells

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Srilatha Badaboina

2015-07-01

Full Text Available Rotenone, isolated from roots of derris plant, has been shown to possess various biological activities, which lead to attempting to develop a potent drug against several diseases. However, recent studies have demonstrated that rotenone has the potential to induce several adverse effects such as a neurodegenerative disease. Radiolytic transformation of the rotenone with gamma-irradiation created a new product, named rotenoisin B. The present work was designed to investigate the anticancer activity of rotenoisin B with low toxicity and its molecular mechanism in hepatic cancer cells compared to a parent compound, rotenone. Our results showed rotenoisin B inhibited hepatic cancer cells’ proliferation in a dose dependent manner and increased in apoptotic cells. Interestingly, rotenoisin B showed low toxic effects on normal cells compared to rotenone. Mitochondrial transmembrane potential has been decreased, which leads to cytochrome c release. Down regulation of anti-apoptotic Bcl-2 levels as well as the up regulation of proapoptotic Bax levels were observed. The cleaved PARP (poly ADP-ribose polymerase level increased as well. Moreover, phosphorylation of extracellular signal regulated kinase (ERK and p38 slightly up regulated and intracellular reactive oxygen species (ROS increased as well as cell cycle arrest predominantly at the G2/M phase observed. These results suggest that rotenoisin B might be a potent anticancer candidate similar to rotenone in hepatic cancer cells with low toxicity to normal cells even at high concentrations compared to rotenone.

LKB1 inhibition of NF-κB in B cells prevents T follicular helper cell differentiation and germinal center formation.

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Walsh, Nicole C; Waters, Lynnea R; Fowler, Jessica A; Lin, Mark; Cunningham, Cameron R; Brooks, David G; Rehg, Jerold E; Morse, Herbert C; Teitell, Michael A

2015-06-01

T-cell-dependent antigenic stimulation drives the differentiation of B cells into antibody-secreting plasma cells and memory B cells, but how B cells regulate this process is unclear. We show that LKB1 expression in B cells maintains B-cell quiescence and prevents the premature formation of germinal centers (GCs). Lkb1-deficient B cells (BKO) undergo spontaneous B-cell activation and secretion of multiple inflammatory cytokines, which leads to splenomegaly caused by an unexpected expansion of T cells. Within this cytokine response, increased IL-6 production results from heightened activation of NF-κB, which is suppressed by active LKB1. Secreted IL-6 drives T-cell activation and IL-21 production, promoting T follicular helper (TFH ) cell differentiation and expansion to support a ~100-fold increase in steady-state GC B cells. Blockade of IL-6 secretion by BKO B cells inhibits IL-21 expression, a known inducer of TFH -cell differentiation and expansion. Together, these data reveal cell intrinsic and surprising cell extrinsic roles for LKB1 in B cells that control TFH -cell differentiation and GC formation, and place LKB1 as a central regulator of T-cell-dependent humoral immunity. © 2015 The Authors.

Surface receptor Toso controls B cell-mediated regulation of T cell immunity.

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Yu, Jinbo; Duong, Vu Huy Hoang; Westphal, Katrin; Westphal, Andreas; Suwandi, Abdulhadi; Grassl, Guntram A; Brand, Korbinian; Chan, Andrew C; Föger, Niko; Lee, Kyeong-Hee

2018-05-01

The immune system is tightly controlled by regulatory processes that allow for the elimination of invading pathogens, while limiting immunopathological damage to the host. In the present study, we found that conditional deletion of the cell surface receptor Toso on B cells unexpectedly resulted in impaired proinflammatory T cell responses, which led to impaired immune protection in an acute viral infection model and was associated with reduced immunopathological tissue damage in a chronic inflammatory context. Toso exhibited its B cell-inherent immunoregulatory function by negatively controlling the pool of IL-10-competent B1 and B2 B cells, which were characterized by a high degree of self-reactivity and were shown to mediate immunosuppressive activity on inflammatory T cell responses in vivo. Our results indicate that Toso is involved in the differentiation/maintenance of regulatory B cells by fine-tuning B cell receptor activation thresholds. Furthermore, we showed that during influenza A-induced pulmonary inflammation, the application of Toso-specific antibodies selectively induced IL-10-competent B cells at the site of inflammation and resulted in decreased proinflammatory cytokine production by lung T cells. These findings suggest that Toso may serve as a novel therapeutic target to dampen pathogenic T cell responses via the modulation of IL-10-competent regulatory B cells.

Constitutive activation of extracellular signal-regulated kinase predisposes diffuse large B-cell lymphoma cell lines to CD40-mediated cell death

DEFF Research Database (Denmark)

Hollmann, C Annette; Owens, Trevor; Nalbantoglu, Josephine

2006-01-01

CD40 promotes survival, proliferation, and differentiation of normal B cells but can cause activation-induced cell death in malignant B lymphocytes. CD40 ligand and anti-CD40 antibodies have been used successfully to induce apoptosis in lymphoma lines both in vitro and in xenograft tumor models. ...

Up-regulation of OLR1 expression by TBC1D3 through activation of TNFα/NF-κB pathway promotes the migration of human breast cancer cells.

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Wang, Bei; Zhao, Huzi; Zhao, Lei; Zhang, Yongchen; Wan, Qing; Shen, Yong; Bu, Xiaodong; Wan, Meiling; Shen, Chuanlu

2017-11-01

Metastatic spread of cancer cells is the most life-threatening aspect of breast cancer and involves multiple steps including cell migration. We recently found that the TBC1D3 oncogene promotes the migration of breast cancer cells, and its interaction with CaM enhances the effects of TBC1D3. However, little is known regarding the mechanism by which TBC1D3 induces the migration of cancer cells. Here, we demonstrated that TBC1D3 stimulated the expression of oxidized low density lipoprotein receptor 1 (OLR1), a stimulator of cell migration, in breast cancer cells at the transcriptional level. Depletion of OLR1 by siRNAs or down-regulation of OLR1 expression using pomalidomide, a TNFα inhibitor, significantly decreased TBC1D3-induced migration of these cells. Notably, TBC1D3 overexpression activated NF-κB, a major effector of TNFα signaling, while inhibition of TNFα signaling suppressed the effects of TBC1D3. Consistent with this, NF-κB inhibition using its specific inhibitor caffeic acid phenethyl ester decreased both TBC1D3-induced OLR1 expression and cell migration, suggesting a critical role for TNFα/NF-κB signaling in TBC1D3-induced migration of breast cancer cells. Mechanistically, TBC1D3 induced activation of this signaling pathway on multiple levels, including by increasing the release of TNFα, elevating the transcription of TNFR1, TRAF1, TRAF5 and TRAF6, and decreasing the degradation of TNFR1. In summary, these studies identify the TBC1D3 oncogene as a novel regulator of TNFα/NF-κB signaling that mediates this oncogene-induced migration of human breast cancer cells by up-regulating OLR1. Copyright © 2017 Elsevier B.V. All rights reserved.

Aqueous extract of Tribulus terrestris Linn induces cell growth arrest and apoptosis by down-regulating NF-κB signaling in liver cancer cells.

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Kim, Hye Jin; Kim, Jin Chul; Min, Jung Sun; Kim, Mi-Jee; Kim, Ji Ae; Kor, Myung Ho; Yoo, Hwa Seung; Ahn, Jeong Keun

2011-06-14

A medicinal herb Tribulus terrestris Linn has been used to treat various diseases including hepatocellular carcinoma. The aim of the present study was to investigate the anticancer activity of Tribulus terrestris Linn (TT) in liver cancer cells. The antitumor activity of aqueous TT extract was analyzed by testing the cytotoxicity and the effect on clonogenecity in HepG2 cells. Apoptosis and cell cycle arrest induced by TT were dissected by flow cytometry and its inhibitory effect on NF-κB activity was determined by analyzing the expression levels of NF-κB/IκB subunit proteins. The suppression of NF-κB-regulated gene expression by TT was assessed by RT-PCR. TT extract repressed clonogenecity and proliferation, induced apoptosis, and enhanced accumulation in the G0/G1 phase of liver cancer cells. It also turned out that TT extract inhibited NF-κB-dependent reporter gene expression and NF-κB subunit p50 expression, while it enhanced the cellular level of IκBα by inhibiting the phosphorylation and degradation of IκBα. In addition, IKK activity was inhibited in a dose-dependent manner. Furthermore, TT extract suppressed the transcription of genes associated with cell cycle regulation, anti-apoptosis, and invasion. These data showed that TT extract blocks proliferation and induces apoptosis in human liver cancer cells through the inhibition of NF-κB signaling. Aqueous TT extract can be used as an anticancer drug for hepatocellular carcinoma patients. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

FOXP1 suppresses immune response signatures and MHC class II expression in activated B-cell-like diffuse large B-cell lymphomas

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Brown, P J; Wong, K K; Felce, S L

2016-01-01

The FOXP1 (forkhead box P1) transcription factor is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). Here microarray analysis of FOXP1-silenced DLBCL cell lines identified differential regulation of immune response signatures and major histocompatibility complex class II (MHC II......) genes as some of the most significant differences between germinal center B-cell (GCB)-like DLBCL with full-length FOXP1 protein expression versus activated B-cell (ABC)-like DLBCL expressing predominantly short FOXP1 isoforms. In an independent primary DLBCL microarray data set, multiple MHC II genes......, including human leukocyte antigen DR alpha chain (HLA-DRA), were inversely correlated with FOXP1 transcript expression (PABC-DLBCL cells led to increased cell-surface expression of HLA-DRA and CD74. In R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone...

TrkB-T1 regulates the RhoA signaling and actin cytoskeleton in glioma cells

International Nuclear Information System (INIS)

Ohira, Koji; Homma, Koichi J.; Hirai, Hirohisa; Nakamura, Shun; Hayashi, Motoharu

2006-01-01

Recently, the truncated TrkB receptor, T1, has been reported to be involved in the control of cell morphology via the regulation of Rho proteins, through which T1 binds Rho guanine nucleotide dissociation inhibitor (Rho GDI) 1 and dissociates it in a brain-derived neurotrophic factor (BDNF)-dependent manner. However, it is unclear whether T1 signaling regulates the downstream of Rho signaling and the actin cytoskeleton. In this study, we investigated this question using C6 rat glioma cells, which express T1 endogenously. Rho GDI1 was dissociated from T1 in a BDNF-dependent manner, which also causes decreases in the activities of Rho-signaling molecules such as RhoA, Rho-associated kinase, p21-activated kinase, and extracellular-signal regulated kinase1/2. Moreover, BDNF treatment resulted in the disappearance of stress fibers in the cells treated with lysophosphatidic acid, an activator of RhoA, and in morphological changes in cells. Furthermore, a competitive assay with cyan fluorescent protein fusion proteins of T1-specific sequences reduced the effects of BDNF. These results suggest that T1 regulates the Rho-signaling pathways and the actin cytoskeleton

Regulation of normal B-cell differentiation and malignant B-cell survival by OCT2.

Science.gov (United States)

Hodson, Daniel J; Shaffer, Arthur L; Xiao, Wenming; Wright, George W; Schmitz, Roland; Phelan, James D; Yang, Yandan; Webster, Daniel E; Rui, Lixin; Kohlhammer, Holger; Nakagawa, Masao; Waldmann, Thomas A; Staudt, Louis M

2016-04-05

The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3 Finally, by introducing mutations designed to disrupt the OCT2-OCA-B interface, we reveal a requirement for this protein-protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell-restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity.

PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

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Ana E. González Wusener

2016-01-01

Full Text Available Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO cells and PTP1B reconstituted (WT cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration.

PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

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González Wusener, Ana E.; González, Ángela; Nakamura, Fumihiko; Arregui, Carlos O.

2016-01-01

ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration. PMID:26700725

B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model.

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Da Rosa, Larissa C; Boldison, Joanne; De Leenheer, Evy; Davies, Joanne; Wen, Li; Wong, F Susan

2018-06-01

Type 1 diabetes is a T cell-mediated autoimmune disease characterised by the destruction of beta cells in the islets of Langerhans, resulting in deficient insulin production. B cell depletion therapy has proved successful in preventing diabetes and restoring euglycaemia in animal models of diabetes, as well as in preserving beta cell function in clinical trials in the short term. We aimed to report a full characterisation of B cell kinetics post B cell depletion, with a focus on pancreatic islets. Transgenic NOD mice with a human CD20 transgene expressed on B cells were injected with an anti-CD20 depleting antibody. B cells were analysed using multivariable flow cytometry. There was a 10 week delay in the onset of diabetes when comparing control and experimental groups, although the final difference in the diabetes incidence, following prolonged observation, was not statistically significant (p = 0.07). The co-stimulatory molecules CD80 and CD86 were reduced on stimulation of B cells during B cell depletion and repopulation. IL-10-producing regulatory B cells were not induced in repopulated B cells in the periphery, post anti-CD20 depletion. However, the early depletion of B cells had a marked effect on T cells in the local islet infiltrate. We demonstrated a lack of T cell activation, specifically with reduced CD44 expression and effector function, including IFN-γ production from both CD4 + and CD8 + T cells. These CD8 + T cells remained altered in the pancreatic islets long after B cell depletion and repopulation. Our findings suggest that B cell depletion can have an impact on T cell regulation, inducing a durable effect that is present long after repopulation. We suggest that this local effect of reducing autoimmune T cell activity contributes to delay in the onset of autoimmune diabetes.

TRPM4 expression is associated with activated B cell subtype and poor survival in diffuse large B cell lymphoma

DEFF Research Database (Denmark)

Loo, Suet K; Ch'ng, Ewe S; Md Salleh, Md Salzihan

2017-01-01

to investigate TRPM4 protein expression pattern in non-malignant tissues and DLBCL cases, and its association with clinico-demographic parameters and survival in DLBCL. METHODS AND RESULTS: Analysis of publicly available DLBCL microarray data sets showed that TRPM4 transcripts were up-regulated in DLBCL compared...... to normal germinal centre B (GCB) cells, were expressed more highly in the activated B cell-like DLBCL (ABC-DLBCL) subtype and higher TRPM4 transcripts conferred worse overall survival (OS) in R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-treated DLBCL cases (P ... immunohistochemical analysis showed that TRPM4 was expressed in various human tissues but not in normal B cells within lymphoid tissues (reactive tonsil, lymph node and appendix). TRPM4 protein was present in 26% (n = 49 of 189) of our cohort of R-CHOP-treated DLBCL cases and this was associated significantly...

Vitamin D Receptor Negatively Regulates Bacterial-Stimulated NF-κB Activity in Intestine

OpenAIRE

Wu, Shaoping; Liao, Anne P.; Xia, Yinglin; Li, Yan Chun; Li, Jian-Dong; Sartor, R. Balfour; Sun, Jun

2010-01-01

Vitamin D receptor (VDR) plays an essential role in gastrointestinal inflammation. Most investigations have focused on the immune response; however, how bacteria regulate VDR and how VDR modulates the nuclear factor (NF)-κB pathway in intestinal epithelial cells remain unexplored. This study investigated the effects of VDR ablation on NF-κB activation in intestinal epithelia and the role of enteric bacteria on VDR expression. We found that VDR−/− mice exhibited a pro-inflammatory bias. After ...

Transcriptional profiling of ErbB signalling in mammary luminal epithelial cells - interplay of ErbB and IGF1 signalling through IGFBP3 regulation

International Nuclear Information System (INIS)

Worthington, Jenny; Bertani, Mariana; Chan, Hong-Lin; Gerrits, Bertran; Timms, John F

2010-01-01

enhanced in ErbB2-overexpressing cells, whilst loss of ErbB2 expression by siRNA silencing reduced IGF1 signalling. Furthermore, IGFBP3 knockdown resulted in basal ERK and Akt activation in luminal epithelial cells and increased invasiveness and anchorage-independent colony formation in SKBR3 cells. These data show IGFBP3 as a negative regulator of transformation and that its down-regulation enhances IGF1-dependent signalling. They also show that ErbB2 can up-regulate IGF1-dependent signalling, possibly via the regulated expression of IGFBP3

B7h-expressing dendritic cells and plasma B cells mediate distinct outcomes of ICOS costimulation in T cell-dependent antibody responses

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Larimore Kevin

2012-06-01

Full Text Available Abstract Background The ICOS-B7h costimulatory receptor-ligand pair is required for germinal center formation, the production of isotype-switched antibodies, and antibody affinity maturation in response to T cell-dependent antigens. However, the potentially distinct roles of regulated B7h expression on B cells and dendritic cells in T cell-dependent antibody responses have not been defined. Results We generated transgenic mice with lineage-restricted B7h expression to assess the cell-type specific roles of B7h expression on B cells and dendritic cells in regulating T cell-dependent antibody responses. Our results show that endogenous B7h expression is reduced on B cells after activation in vitro and is also reduced in vivo on antibody-secreting plasma B cells in comparison to both naïve and germinal center B cells from which they are derived. Increasing the level of B7h expression on activated and plasma B cells in B-B7hTg mice led to an increase in the number of antibody-secreting plasma cells generated after immunization and a corresponding increase in the concentration of antigen-specific high affinity serum IgG antibodies of all isotypes, without affecting the number of responding germinal center B cells. In contrast, ICOS costimulation mediated by dendritic cells in DC-B7hTg mice contributed to germinal center formation and selectively increased IgG2a production without affecting the overall magnitude of antibody responses. Conclusions Using transgenic mice with lineage-restricted B7h expression, we have revealed distinct roles of ICOS costimulation mediated by dendritic cells and B cells in the regulation of T cell-dependent antibody responses.

Exposure to coplanar PCBs induces endothelial cell inflammation through epigenetic regulation of NF-κB subunit p65

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Liu, Dandan; Perkins, Jordan T. [Superfund Research Center, University of Kentucky, Lexington, KY 40536 (United States); Department of Animal and Food Sciences, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY 40536 (United States); Petriello, Michael C. [Superfund Research Center, University of Kentucky, Lexington, KY 40536 (United States); Graduate Center for Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536 (United States); Hennig, Bernhard, E-mail: bhennig@uky.edu [Superfund Research Center, University of Kentucky, Lexington, KY 40536 (United States); Department of Animal and Food Sciences, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY 40536 (United States)

2015-12-15

Epigenetic modifications of DNA and histones alter cellular phenotypes without changing genetic codes. Alterations of epigenetic marks can be induced by exposure to environmental pollutants and may contribute to associated disease risks. Here we test the hypothesis that endothelial cell dysfunction induced by exposure to polychlorinated biphenyls (PCBs) is mediated in part though histone modifications. In this study, human vascular endothelial cells were exposed to physiologically relevant concentrations of several PCBs congeners (e.g., PCBs 77, 118, 126 and 153) followed by quantification of inflammatory gene expression and changes of histone methylation. Only exposure to coplanar PCBs 77 and 126 induced the expression of histone H3K9 trimethyl demethylase jumonji domain-containing protein 2B (JMJD2B) and nuclear factor-kappa B (NF-κB) subunit p65, activated NF-κB signaling as evidenced by nuclear translocation of p65, and up-regulated p65 target inflammatory genes, such as interleukin (IL)-6, C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and IL-1α/β. The increased accumulation of JMJD2B in the p65 promoter led to a depletion of H3K9me3 repression mark, which accounts for the observed up-regulation of p65 and associated inflammatory genes. JMJD2B gene knockdown confirmed a critical role for this histone demethylase in mediating PCB-induced inflammation of the vascular endothelium. Finally, it was determined, via chemical inhibition, that PCB-induced up-regulation of JMJD2B was estrogen receptor-alpha (ER-α) dependent. These data suggest that coplanar PCBs may exert endothelial cell toxicity through changes in histone modifications. - Highlights: • Coplanar PCBs significantly induced histone demethylase JMJD2B expression. • Coplanar PCBs activated NF-κB through p65 up-regulation and nuclear translocation. • Histone H3K4 and K9 modifications were mediated by ER-α/JMJD2B/MLL2 complex. �

Protein Phosphotyrosine Phosphatase 1B (PTP1B) in Calpain-dependent Feedback Regulation of Vascular Endothelial Growth Factor Receptor (VEGFR2) in Endothelial Cells

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Zhang, Yixuan; Li, Qiang; Youn, Ji Youn; Cai, Hua

2017-01-01

The VEGF/VEGFR2/Akt/eNOS/NO pathway is essential to VEGF-induced angiogenesis. We have previously discovered a novel role of calpain in mediating VEGF-induced PI3K/AMPK/Akt/eNOS activation through Ezrin. Here, we sought to identify possible feedback regulation of VEGFR2 by calpain via its substrate protein phosphotyrosine phosphatase 1B (PTP1B), and the relevance of this pathway to VEGF-induced angiogenesis, especially in diabetic wound healing. Overexpression of PTP1B inhibited VEGF-induced VEGFR2 and Akt phosphorylation in bovine aortic endothelial cells, while PTP1B siRNA increased both, implicating negative regulation of VEGFR2 by PTP1B. Calpain inhibitor ALLN induced VEGFR2 activation, which can be completely blocked by PTP1B overexpression. Calpain activation induced by overexpression or Ca/A23187 resulted in PTP1B cleavage, which can be blocked by ALLN. Moreover, calpain activation inhibited VEGF-induced VEGFR2 phosphorylation, which can be restored by PTP1B siRNA. These data implicate calpain/PTP1B negative feedback regulation of VEGFR2, in addition to the primary signaling pathway of VEGF/VEGFR2/calpain/PI3K/AMPK/Akt/eNOS. We next examined a potential role of PTP1B in VEGF-induced angiogenesis. Endothelial cells transfected with PTP1B siRNA showed faster wound closure in response to VEGF. Aortic discs isolated from PTP1B siRNA-transfected mice also had augmented endothelial outgrowth. Importantly, PTP1B inhibition and/or calpain overexpression significantly accelerated wound healing in STZ-induced diabetic mice. In conclusion, our data for the first time demonstrate a calpain/PTP1B/VEGFR2 negative feedback loop in the regulation of VEGF-induced angiogenesis. Modulation of local PTP1B and/or calpain activities may prove beneficial in the treatment of impaired wound healing in diabetes. PMID:27872190

TRAF family member-associated NF-κB activator (TANK) induced by RANKL negatively regulates osteoclasts survival and function.

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Wu, Mengrui; Wang, Yiping; Deng, Lianfu; Chen, Wei; Li, Yi-Ping

2012-01-01

Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation, and confirmed by western blot and quantitative RT-PCR. Bone marrow macrophages (BMMs) transduced with lentivirus carrying tank-shRNA were induced to form osteoclast in the presence of RANKL and M-CSF. Tank expression was downregulated by 90% by Tank-shRNA, which is confirmed by western blot. Compared with wild-type (WT) cells, osteoclastogenesis of Tank-silenced BMMs was increased, according to tartrate-resistant acid phosphatase (TRAP) stain on day 5 and day 7. Number of bone resorption pits by Tank-silenced osteoclasts was increased by 176% compared with WT cells, as shown by wheat germ agglutinin (WGA) stain and scanning electronic microscope (SEM) analysis. Survival rate of Tank-silenced mature osteoclast is also increased. However, acid production of Tank-knockdown cells was not changed compared with control cells. IκBα phosphorylation is increased in tank-silenced cells, indicating that TANK may negatively regulate NF-κB activity in osteoclast. In conclusion, Tank, whose expression is increased during osteoclastogenesis, inhibits osteoclast formation, activity and survival, by regulating NF-κB activity and c-FLIP expression. Tank enrolls itself in a negative feedback loop in bone resorption. These results may provide means for therapeutic intervention in diseases of excessive bone resorption.

Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells.

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Sánchez-Martínez, Diego; Lanuza, Pilar M; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A; Pardo, Julián

2016-01-01

Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV ) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.

Cytotoxicity of diacetoxyscirpenol is associated with apoptosis by activation of caspase-8 and interruption of cell cycle progression by down-regulation of cdk4 and cyclin B1 in human Jurkat T cells

International Nuclear Information System (INIS)

Jun, Do Youn; Kim, Jun Seok; Park, Hae Sun; Song, Woo Sun; Bae, Young Seuk; Kim, Young Ho

2007-01-01

To understand the mechanism underlying T-cell toxicity of diacetoxyscirpenol (DAS) from Fusarium sambucinum, its apoptogenic as well as growth retardation activity was investigated in human Jurkat T cells. Exposure to DAS (0.01-0.15 μM) caused apoptotic DNA fragmentation along with caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3, and PARP degradation, without any alteration in the levels of Fas or FasL. Under these conditions, necrosis was not accompanied. The cytotoxicity of DAS was not blocked by the anti-Fas neutralizing antibody ZB-4. Although the DAS-induced apoptotic events were completely prevented by overexpression of Bcl-xL, the cells overexpressing Bcl-xL were unable to divide in the presence of DAS, resulting from the failure of cell cycle progression possibly due to down-regulation in the protein levels of cdk4 and cyclin B1. The DAS-mediated apoptosis and activation of caspase-8, -9, and -3 were abrogated by either pan-caspase inhibitor (z-VAD-fmk) or caspase-8 inhibitor (z-IETD-fmk). While the DAS-mediated apoptosis and activation of caspase-9 and caspase-3 were slightly suppressed by the mitochondrial permeability transition pore inhibitor (CsA), both caspase-8 activation and Bid cleavage were not affected by CsA. The activated normal peripheral T cells possessed a similar susceptibility to the cytotoxicity of DAS. These results demonstrate that the T-cell toxicity of DAS is attributable to not only apoptosis initiated by caspase-8 activation and subsequent mitochondrion-dependent or -independent activation of caspase cascades, which can be regulated by Bcl-xL, but also interruption of cell cycle progression caused by down-regulation of cdk4 and cyclin B1 proteins

Nogo-B (Reticulon-4B) functions as a negative regulator of the apoptotic pathway through the interaction with c-FLIP in colorectal cancer cells.

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Kawaguchi, Nao; Tashiro, Keitaro; Taniguchi, Kohei; Kawai, Masaru; Tanaka, Keitaro; Okuda, Junji; Hayashi, Michihiro; Uchiyama, Kazuhisa

2018-08-01

Nogo-B is a member of the Nogo/Reticulon-4 family and has been reported to be an inducer of apoptosis in certain types of cancer cells. However, the role of Nogo-B in human cancer remains less understood. Here, we demonstrated the functions of Nogo-B in colorectal cancer cells. In clinical colorectal cancer specimens, Nogo-B was obviously overexpressed, as determined by immunohistochemistry; and Western blot analysis showed its expression level to be significantly up-regulated. Furthermore, knockdown of Nogo-B in two colorectal cancer cell lines, SW480 and DLD-1, by transfection with si-RNA (siR) resulted in significantly reduced cell viability and a dramatic increase in apoptosis with insistent overexpression of cleaved caspase-8 and cleaved PARP. The transfection with Nogo-B plasmid cancelled that apoptosis induced by siRNogoB in SW480 cells. Besides, combinatory treatment with siR-Nogo-B/staurosporine (STS) or siR-Nogo-B/Fas ligand (FasL) synergistically reduced cell viability and increased the expression of apoptotic signaling proteins in colorectal cancer cells. These results strongly support our contention that Nogo-B most likely played an oncogenic role in colorectal cancer cells, mainly by negatively regulating the extrinsic apoptotic pathway in them. Finally, we revealed that suppression of Nogo-B caused down-regulation of c-FLIP, known as a major anti-apoptotic protein, and activation of caspase-8 in the death receptor pathway. Interaction between Nogo-B and c-FLIP was shown by immunoprecipitation and immunofluorescence studies. In conclusion, Nogo-B was shown to play an important negative role in apoptotic signaling through its interaction with c-FLIP in colorectal cancer cells, and may thus become a novel therapeutic target for colorectal cancer. Copyright © 2018 Elsevier B.V. All rights reserved.

IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

International Nuclear Information System (INIS)

Li, Yan; Ohms, Stephen J.; Shannon, Frances M.; Sun, Chao; Fan, Jun Y.

2012-01-01

Highlights: ► DNA methylation is dynamic and flexible and changes rapidly upon cell activation. ► DNA methylation controls the inducible gene expression in a given cell type. ► Some enzymes are involved in maintaining the methylation profile of immune cells. -- Abstract: DNA demethylation has been found to occur at the promoters of a number of actively expressed cytokines and is believed to play a critical role in transcriptional regulation. While many DNA demethylation studies have focused on T cell activation, proliferation and differentiation, changes in DNA methylation in other types of immune cells are less well studied. We found that the expression of two cytokines (IL-2 and GM-CSF) responded differently to activation in three types of immune cells: EL4, A20 and RAW264.7 cells. Using the McrBC and MeDIP approaches, we observed decreases in DNA methylation at a genome-wide level and at the promoters of the genes of these cytokines. The expression of several potential enzymes/co-enzymes involved in the DNA demethylation pathways seemed to be associated with immune cell activation.

PSMB4 promotes multiple myeloma cell growth by activating NF-κB-miR-21 signaling

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Zheng, Peihao; Guo, Honggang [Department of Hematology, Navy General Hospital, Beijing 100048 (China); Li, Guangchao [School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006 (China); Han, Siqi [Department of Medical Oncology, Jinling Hospital, Nanjing 210002 (China); Luo, Fei [Department of Stomatology, Jinling Hospital, Nanjing 210002 (China); Liu, Yi, E-mail: liuyi2033@163.com [Department of Hematology, Navy General Hospital, Beijing 100048 (China)

2015-03-06

Proteasomal subunit PSMB4, was recently identified as potential cancer driver genes in several tumors. However, the regulatory mechanism of PSMB4 on carcinogenesis process remains unclear. In this study, we investigated the expression and roles of PSMB4 in multiple myeloma (MM). We found a significant up-regulation of PSMB4 in MM plasma and cell lines. Ectopic overexpression of PSMB4 promoted cell growth and colony forming ability of MM cells, whereas inhibition of PSMB4 led to a decrease of such events. Furthermore, our results demonstrated the up-regulation of miR-21 and a positive correlation between the levels of miR-21 and PSMB4 in MM. Re-expression of miR-21 markedly rescued PSMB4 knockdown-mediated suppression of cell proliferation and clone-formation. Additionally, while enforced expression of PSMB4 profoundly increased NF-κB activity and the level of miR-21, PSMB4 knockdown or NF-κB inhibition suppressed miR-21 expression in MM cells. Taken together, our results demonstrated that PSMB4 regulated MM cell growth in part by activating NF-κB-miR-21 signaling, which may represent promising targets for novel specific therapies. - Highlights: • First reported upregulation of PSMB4 in MM plasma and cell lines. • PSMB4 promoted MM cell growth and colony forming ability. • Further found miR-21 was up-regulated by PSMB4 in MM plasma and cell lines. • PSMB4-induced miR-21 expression was modulated by NF-κB. • PSMB4-NF-κB-miR-21 axis may be potential therapeutic targets of MM.

CBX7 regulates stem cell-like properties of gastric cancer cells via p16 and AKT-NF-κB-miR-21 pathways.

Science.gov (United States)

Ni, Su-Jie; Zhao, Li-Qin; Wang, Xiao-Feng; Wu, Zhen-Hua; Hua, Rui-Xi; Wan, Chun-Hua; Zhang, Jie-Yun; Zhang, Xiao-Wei; Huang, Ming-Zhu; Gan, Lu; Sun, Hua-Lin; Dimri, Goberdhan P; Guo, Wei-Jian

2018-02-08

Chromobox protein homolog 7 (CBX7), a member of the polycomb group (PcG) family of proteins, is involved in the regulation of cell proliferation and cancer progression. PcG family members, such as BMI, Mel-18, and EZH2, are integral constituents of the polycomb repressive complexes (PRCs) and have been known to regulate cancer stem cell (CSC) phenotype. However, the role of other PRCs' constituents such as CBX7 in the regulation of CSC phenotype remains largely elusive. This study was to investigate the role of CBX7 in regulating stem cell-like properties of gastric cancer and the underlying mechanisms. Firstly, the role of CBX7 in regulating stem cell-like properties of gastric cancer was investigated using sphere formation, Western blot, and xenograft tumor assays. Next, RNA interference and ectopic CBX7 expression were employed to determine the impact of CBX7 on the expression of CSC marker proteins and CSC characteristics. The expression of CBX7, its downstream targets, and stem cell markers were analyzed in gastric stem cell spheres, common cancer cells, and gastric cancer tissues. Finally, the pathways by which CBX7 regulates stem cell-like properties of gastric cancer were explored. We found that CBX7, a constituent of the polycomb repressive complex 1 (PRC1), plays an important role in maintaining stem cell-like characteristics of gastric cancer cells via the activation of AKT pathway and the downregulation of p16. Spearman rank correlation analysis showed positive correlations among the expression of CBX7 and phospho-AKT (pAKT), stem cell markers OCT-4, and CD133 in gastric cancer tissues. In addition, CBX7 was found to upregulate microRNA-21 (miR-21) via the activation of AKT-NF-κB pathway, and miR-21 contributes to CBX7-mediated CSC characteristics. CBX7 positively regulates stem cell-like characteristics of gastric cancer cells by inhibiting p16 and activating AKT-NF-κB-miR-21 pathway.

Constitutive activation of alternative nuclear factor kappa B pathway in canine diffuse large B-cell lymphoma contributes to tumor cell survival and is a target of new adjuvant therapies.

Science.gov (United States)

Seelig, Davis M; Ito, Daisuke; Forster, Colleen L; Yoon, Una A; Breen, Matthew; Burns, Linda J; Bachanova, Veronika; Lindblad-Toh, Kerstin; O'Brien, Timothy D; Schmechel, Stephen C; Rizzardi, Anthony E; Modiano, Jaime F; Linden, Michael A

2017-07-01

Activation of the classical nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway is a common molecular event observed in both human and canine diffuse large B-cell lymphoma (DLBCL). Although the oncogenic potential of the alternative NFκB pathway (ANFκBP) has also been recently identified in DLBCL, its precise role in tumor pathogenesis and potential as a treatment target is understudied. We hypothesized that up-regulation of the ANFκBP plays an important role in the proliferation and survival of canine DLBCL cells, and we demonstrate that the ANFκBP is constitutively active in primary canine DLBCL samples and a cell line (CLBL1). We further demonstrate that a small interfering RNA inhibits the activation of the NFκB pathway and induces apoptosis in canine DLBCL cells. In conclusion, the ANFκBP facilitates survival of canine DLBCL cells, and thus, dogs with spontaneous DLBCL can provide a useful large animal model to study therapies targeting the ANFκBP.

Activation of ion transport systems during cell volume regulation

International Nuclear Information System (INIS)

Eveloff, J.L.; Warnock, D.G.

1987-01-01

This review discusses the activation of transport pathways during volume regulation, including their characteristics, the possible biochemical pathways that may mediate the activation of transport pathways, and the relations between volume regulation and transepithelial transport in renal cells. Many cells regulate their volume when exposed to an anisotonic medium. The changes in cell volume are caused by activation of ion transport pathways, plus the accompanying osmotically driven water movement such that cell volume returns toward normal levels. The swelling of hypertonically shrunken cells is termed regulatory volume increase (RVI) and involves an influx of NaCl into the cell via either activation of Na-Cl, Na-K-2Cl cotransport systems, or Na + -H + and Cl - -HCO 3 - exchangers. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease (RVD) and involves an efflux of KCl and water from the cell by activation of either separate K + and Cl - conductances, a K-Cl cotransport system, or parallel K + -H + and Cl - -HCO 3 - exchangers. The biochemical mechanisms involved in the activation of transport systems are largely unknown, however, the phosphoinositide pathway may be implicated in RVI; phorbol esters, cGMP, and Ca 2+ affect the process of volume regulation. Renal tubular cells, as well as the blood cells that transverse the medulla, are subjected to increasing osmotic gradients from the corticomedullary junction to the papillary tip, as well as changing interstitial and tubule fluid osmolarity, depending on the diuretic state of the animal. Medullary cells from the loop of Henle and the papilla can volume regulate by activating Na-K-2Cl cotransport or Na + -H + and Cl - -HCO 3 - exchange systems

Nontranscriptional regulation of SYK by the coactivator OCA-B is required at multiple stages of B cell development.

Science.gov (United States)

Siegel, Rachael; Kim, Unkyu; Patke, Alina; Yu, Xin; Ren, Xiaodi; Tarakhovsky, Alexander; Roeder, Robert G

2006-05-19

OCA-B was originally identified as a nuclear transcriptional coactivator that is essential for antigen-driven immune responses. The later identification of a membrane bound, myristoylated form of OCA-B suggested additional, unique functions in B cell signaling pathways. This study has shown that OCA-B also functions in the pre-B1-to-pre-B2 cell transition and, most surprisingly, that it directly interacts with SYK, a tyrosine kinase critical for pre-BCR and BCR signaling. This unprecedented type of interaction-a transcriptional coactivator with a signaling kinase-occurs in the cytoplasm and directly regulates SYK stability. This study indicates that OCA-B is required for pre-BCR and BCR signaling at multiple stages of B cell development through its nontranscriptional regulation of SYK. Combined with the deregulation of OCA-B target genes, this may help explain the multitude of defects observed in B cell development and immune responses of Oca-b-/- mice.

Porcine arterivirus activates the NF-κB pathway through IκB degradation

International Nuclear Information System (INIS)

Lee, Sang-Myeong; Kleiboeker, Steven B.

2005-01-01

Nuclear factor-kappaB (NF-κB) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-κB in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-κB activation was characterized by translocation of NF-κB from the cytoplasm to the nucleus, increased DNA binding activity, and NF-κB-regulated gene expression. NF-κB activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-κB activation. Degradation of IκB protein was detected late in PRRSV infection, and overexpression of the dominant negative form of IκBα (IκBαDN) significantly suppressed NF-κB activation induced by PRRSV. However, IκBαDN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-κB DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-κB was activated by PRRSV infection. Moreover, NF-κB-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-κB activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV

Activated allogeneic NK cells preferentially kill poor prognosis B-cell chronic lymphocytic leukemia cells

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Diego Sanchez-Martinez

2016-10-01

Full Text Available Mutational status of TP53 together with expression of wild type (wt IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL patients. Adoptive cell therapy using allogeneic HLA mismatched Natural Killer (NK cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs the most effective stimulus to activate NK cells. Here we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell activating receptors (NKG2D and NCRs and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.□

Bach2 regulates aberrant activation of B cell in systemic lupus erythematosus and can be negatively regulated by BCR-ABL/PI3K.

Science.gov (United States)

Zhu, Zhengwei; Yang, Chao; Wen, Leilei; Liu, Lu; Zuo, Xianbo; Zhou, Fusheng; Gao, Jinping; Zheng, Xiaodong; Shi, Yinjuan; Zhu, Caihong; Liang, Bo; Yin, Xianyong; Wang, Wenjun; Cheng, Hui; Shen, Songke; Tang, Xianfa; Tang, Huayang; Sun, Liangdan; Zhang, Anping; Yang, Sen; Cui, Yong; Zhang, Xuejun; Sheng, Yujun

2018-04-01

This study was aimed to explore the effect of Bach2 on B cells in systemic lupus erythematosus (SLE), as well as the underlying mechanisms. Expression of Bach2, phosphorylated-Bach2 (p-Bach2), Akt, p-Akt and BCR-ABL (p210) in B cells isolated from SLE patients and the healthy persons were assessed by Western blot. Immunofluorescence staining was performed to assess the localization of Bach2 in B cells. Enzyme-linked immunosorbent assay (ELISA) was employed to detect IgG produced by B cells. Cell counting kit-8 (CCK-8) and Annexin-V FITC/PI double staining assay were adopted to evaluate cell proliferation and apoptosis in B cells, respectively. Compared to the healthy controls, Bach2, p-Akt and p210 were significantly decreased, while nuclear translocation of Bach2, IgG, CD40 and CD86 obviously up-regulated in B cells from SLE patients. Bach2 significantly inhibited the proliferation, promoted apoptosis of B cells from SLE patients, whereas BCR-ABL dramatically reversed cell changes induced by Bach2. Besides, BCR-ABL also inhibited nuclear translocation of Bach2 in B cells from SLE patients. Further, LY294002 treatment had no effect on decreased expression of Bach2 induced by BCR-ABL, but significantly eliminated BCR-ABL-induced phosphorylation of Bach2 and restored reduced nuclear translocation of Bach2 induced by BCR-ABL in B cells from SLE. Bach2 may play a suppressive role in B cells from SLE, and BCR-ABL may inhibit the nuclear translocation of Bach2 via serine phosphorylation through the PI3K pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

Regulation of HTLV-1 Tax Stability, Cellular Trafficking and NF-κB Activation by the Ubiquitin-Proteasome Pathway

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Lavorgna, Alfonso; Harhaj, Edward William

2014-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%–5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-κB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-κB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-κB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis. PMID:25341660

Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function.

Science.gov (United States)

Geng, Jie; Altman, John D; Krishnakumar, Sujatha; Raghavan, Malini

2018-05-09

When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8 + T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8 + T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8 + T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8 + T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8 + T cell activation, mediated by the binding of empty HLA-I to CD8. © 2018, Geng et al.

The X protein of hepatitis B virus activates hepatoma cell proliferation through repressing melanoma inhibitory activity 2 gene

Energy Technology Data Exchange (ETDEWEB)

Xu, Yilin; Yang, Yang; Cai, Yanyan; Liu, Fang; Liu, Yingle; Zhu, Ying [State Key Laboratory of Virology, College of Life Sciences, and Chinese-French Liver Disease Research Institute at Zhongnan Hospital, Wuhan University, Wuhan 430072 (China); Wu, Jianguo, E-mail: jwu@whu.edu.cn [State Key Laboratory of Virology, College of Life Sciences, and Chinese-French Liver Disease Research Institute at Zhongnan Hospital, Wuhan University, Wuhan 430072 (China)

2011-12-16

Highlights: Black-Right-Pointing-Pointer We demonstrated that HBV represses MIA2 gene expression both invitro and in vivo. Black-Right-Pointing-Pointer The X protein of HBV plays a major role in such regulation. Black-Right-Pointing-Pointer Knock-down of MIA2 in HepG2 cells activates cell growth and proliferation. Black-Right-Pointing-Pointer HBx activates cell proliferation, over-expression of MIA2 impaired such regulation. Black-Right-Pointing-Pointer HBx activates hepatoma cell proliferation through repressing MIA2 expression. -- Abstract: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer deaths globally. Chronic hepatitis B virus (HBV) infection accounts for over 75% of all HCC cases; however, the molecular pathogenesis of HCC is not well understood. In this study, we found that the expression of the newly identified gene melanoma inhibitory activity 2 (MIA2) was reduced by HBV infection invitro and invivo, and that HBV X protein (HBx) plays a major role in this regulation. Recent studies have revealed that MIA2 is a potential tumor suppressor, and that, in most HCCs, MIA2 expression is down-regulated or lost. We found that the knock-down of MIA2 in HepG2 cells activated cell growth and proliferation, suggesting that MIA2 inhibits HCC cell growth and proliferation. In addition, the over-expression of HBx alone induced cell proliferation, whereas MIA2 over-expression impaired the HBx-mediated induction of proliferation. Taken together, our results suggest that HBx activates hepatoma cell growth and proliferation through repression of the potential tumor suppressor MIA2.

Natural IgM and TLR Agonists Switch Murine Splenic Pan-B to “Regulatory” Cells That Suppress Ischemia-Induced Innate Inflammation via Regulating NKT-1 Cells

Directory of Open Access Journals (Sweden)

Peter I. Lobo

2017-08-01

Full Text Available Natural IgM anti-leukocyte autoantibodies (IgM-ALAs inhibit inflammation by several mechanisms. Here, we show that pan-B cells and bone marrow-derived dendritic cells (BMDCs are switched to regulatory cells when pretreated ex vivo with IgM. B cells are also switched to regulatory cells when pretreated ex vivo with CpG but not with LPS. Pre-emptive infusion of such ex vivo induced regulatory cells protects C57BL/6 mice from ischemia-induced acute kidney injury (AKI via regulation of in vivo NKT-1 cells, which normally amplify the innate inflammatory response to DAMPS released after reperfusion of the ischemic kidney. Such ex vivo induced regulatory pan-B cells and BMDC express low CD1d and inhibit inflammation by regulating in vivo NKT-1 in the context of low-lipid antigen presentation and by a mechanism that requires costimulatory molecules, CD1d, PDL1/PD1, and IL10. Second, LPS and CpG have opposite effects on induction of regulatory activity in BMDC and B cells. LPS enhances regulatory activity of IgM-pretreated BMDC but negates the IgM-induced regulatory activity in B cells, while CpG, with or without IgM pretreatment, induces regulatory activity in B cells but not in BMDC. Differences in the response of pan-B and dendritic cells to LPS and CpG, especially in the presence of IgM-ALA, may have relevance during infections and inflammatory disorders where there is an increased IgM-ALA and release of TLRs 4 and 9 ligands. Ex vivo induced regulatory pan-B cells could have therapeutic relevance as these easily available cells can be pre-emptively infused to prevent AKI that can occur during open heart surgery or in transplant recipients receiving deceased donor organs.

Interferon-β-induced activation of c-Jun NH2-terminal kinase mediates apoptosis through up-regulation of CD95 in CH31 B lymphoma cells

International Nuclear Information System (INIS)

Takada, Eiko; Shimo, Kuniaki; Hata, Kikumi; Abiake, Maira; Mukai, Yasuo; Moriyama, Masami; Heasley, Lynn; Mizuguchi, Junichiro

2005-01-01

Type I interferon (IFN)-induced antitumor action is due in part to apoptosis, but the molecular mechanisms underlying IFN-induced apoptosis remain largely unresolved. In the present study, we demonstrate that IFN-β induced apoptosis and the loss of mitochondrial membrane potential (ΔΨm) in the murine CH31 B lymphoma cell line, and this was accompanied by the up-regulation of CD95, but not CD95-ligand (CD95-L), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL). Pretreatment with anti-CD95-L mAb partially prevented the IFN-β-induced loss of ΔΨm, suggesting that the interaction of IFN-β-up-regulated CD95 with CD95-L plays a crucial role in the induction of fratricide. IFN-β induced a sustained activation of c-Jun NH 2 -terminal kinase 1 (JNK1), but not extracellular signal-regulated kinases (ERKs). The IFN-β-induced apoptosis and loss of ΔΨm were substantially compromised in cells overexpressing a dominant-negative form of JNK1 (dnJNK1), and it was slightly enhanced in cells carrying a constitutively active JNK construct, MKK7-JNK1 fusion protein. The IFN-β-induced up-regulation of CD95 together with caspase-8 activation was also abrogated in the dnJNK1 cells while it was further enhanced in the MKK7-JNK1 cells. The levels of cellular FLIP (c-FLIP), competitively interacting with caspase-8, were down-regulated by stimulation with IFN-β but were reversed by the proteasome inhibitor lactacystin. Collectively, the IFN-β-induced sustained activation of JNK mediates apoptosis, at least in part, through up-regulation of CD95 protein in combination with down-regulation of c-FLIP protein

Time- and cell-resolved dynamics of redox-sensitive Nrf2, HIF and NF-κB activities in 3D spheroids enriched for cancer stem cells

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Anna P. Kipp

2017-08-01

Full Text Available Cancer cells have an altered redox status, with changes in intracellular signaling pathways. The knowledge of how such processes are regulated in 3D spheroids, being well-established tumor models, is limited. To approach this question we stably transfected HCT116 cells with a pTRAF reporter that enabled time- and cell-resolved activity monitoring of three redox-regulated transcription factors Nrf2, HIF and NF-κB in spheroids enriched for cancer stem cells. At the first day of spheroid formation, these transcription factors were activated and thereafter became repressed. After about a week, both HIF and Nrf2 were reactivated within the spheroid cores. Further amplifying HIF activation in spheroids by treatment with DMOG resulted in a dominant quiescent stem-cell-like phenotype, with high resistance to stress-inducing treatments. Auranofin, triggering oxidative stress and Nrf2 activation, had opposite effects with increased differentiation and proliferation. These novel high-resolution insights into spatiotemporal activation patterns demonstrate a striking coordination of redox regulated transcription factors within spheroids not occurring in conventional cell culture models. Keywords: Redox regulation, Cancer stem cells, Spheroids, Nrf2, HIF, NF-κB

[Regulatory B cells activated by CpG-ODN combined with anti-CD40 monoclonal antibody inhibit CD4(+)T cell proliferation].

Science.gov (United States)

Wang, Keng; Tao, Lei; Su, Jianbing; Zhang, Yueyang; Zou, Binhua; Wang, Yiyuan; Li, Xiaojuan

2016-09-01

Objective To observe the immunosuppressive function of regulatory B cells (Bregs) in vitro after activated by CpG oligodeoxynucleotide (CpG-ODN) and anti-CD40 mAb. Methods Mice splenic CD5(+)CD1d(high)B cells and CD5(-)CD1d(low)B cells were sorted by flow cytometry. These B cells were first stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours, and then co-cultured with purified CD4(+)T cells. The interleukin 10 (IL-10) expression in the activated Bregs and other B cell subset, as well as the proliferation and interferon γ (IFN-γ) expression in the CD4(+) T cells activated by anti-CD3 mAb plus anti-CD28 mAb were determined by flow cytometry. Results CD5(+)CD1d(high) B cells activated by CpG-ODN plus anti-CD40 mAb blocked the up-regulated CD4(+)T proliferation and significantly reduced the IFN-γ level. At the same time, activated CD5(-)CD1d(low)B cells showed no inhibitory effect on CD4(+)T cells. Further study revealed that IL-10 expression in the CD5(+)CD1d(high) B cells were much higher than that in the CD5(-)CD1d(low)B cells after stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours. Conclusion CD5(+)CD1d(high) B cells activated by CpG-ODN combined with anti-CD40 mAb have immune inhibitory effects on CD4(+)T cell activation in vitro , which possibly due to IL-10 secretion.

IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

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Li, Yan [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi 712100 (China); Department of Genome Biology, John Curtin School of Medical Research, The Australian National University, ACT 2601 (Australia); Ohms, Stephen J. [ACRF Biomolecular Resource Facility, John Curtin School of Medical Research, The Australian National University, ACT 2601 (Australia); Shannon, Frances M. [Department of Genome Biology, John Curtin School of Medical Research, The Australian National University, ACT 2601 (Australia); The University of Canberra, ACT 2602 (Australia); Sun, Chao, E-mail: sunchao2775@163.com [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi 712100 (China); Fan, Jun Y., E-mail: jun.fan@anu.edu.au [Department of Genome Biology, John Curtin School of Medical Research, The Australian National University, ACT 2601 (Australia)

2012-03-23

Highlights: Black-Right-Pointing-Pointer DNA methylation is dynamic and flexible and changes rapidly upon cell activation. Black-Right-Pointing-Pointer DNA methylation controls the inducible gene expression in a given cell type. Black-Right-Pointing-Pointer Some enzymes are involved in maintaining the methylation profile of immune cells. -- Abstract: DNA demethylation has been found to occur at the promoters of a number of actively expressed cytokines and is believed to play a critical role in transcriptional regulation. While many DNA demethylation studies have focused on T cell activation, proliferation and differentiation, changes in DNA methylation in other types of immune cells are less well studied. We found that the expression of two cytokines (IL-2 and GM-CSF) responded differently to activation in three types of immune cells: EL4, A20 and RAW264.7 cells. Using the McrBC and MeDIP approaches, we observed decreases in DNA methylation at a genome-wide level and at the promoters of the genes of these cytokines. The expression of several potential enzymes/co-enzymes involved in the DNA demethylation pathways seemed to be associated with immune cell activation.

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

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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.

Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10

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Matsunaga, Toshiyuki; Morikawa, Yoshifumi; Haga, Mariko; Endo, Satoshi; Soda, Midori; Yamamura, Keiko; El-Kabbani, Ossama; Tajima, Kazuo; Ikari, Akira; Hara, Akira

2014-01-01

Inhalation of 9,10-phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust, exerts fatal damage against a variety of cells involved in respiratory function. Here, we show that treatment with high concentrations of 9,10-PQ evokes apoptosis of lung cancer A549 cells through production of reactive oxygen species (ROS). In contrast, 9,10-PQ at its concentrations of 2 and 5 μM elevated the potentials for proliferation, invasion, metastasis and tumorigenesis, all of which were almost completely inhibited by addition of an antioxidant N-acetyl-L-cysteine, inferring a crucial role of ROS in the overgrowth and malignant progression of lung cancer cells. Comparison of mRNA expression levels of six aldo-keto reductases (AKRs) in the 9,10-PQ-treated cells advocated up-regulation of AKR1B10 as a major cause contributing to the lung cancer malignancy. In support of this, the elevation of invasive, metastatic and tumorigenic activities in the 9,10-PQ-treated cells was significantly abolished by the addition of a selective AKR1B10 inhibitor oleanolic acid. Intriguingly, zymographic and real-time PCR analyses revealed remarkable increases in secretion and expression, respectively, of matrix metalloproteinase 2 during the 9,10-PQ treatment, and suggested that the AKR1B10 up-regulation and resultant activation of mitogen-activated protein kinase cascade are predominant mechanisms underlying the metalloproteinase induction. In addition, HPLC analysis and cytochrome c reduction assay in in vitro 9,10-PQ reduction by AKR1B10 demonstrated that the enzyme catalyzes redox-cycling of this quinone, by which ROS are produced. Collectively, these results suggest that AKR1B10 is a key regulator involved in overgrowth and malignant progression of the lung cancer cells through ROS production due to 9,10-PQ redox-cycling. - Highlights: • 9,10-PQ promotes invasion, metastasis and tumorigenicity in lung cancer cells. • The 9,10-PQ-elicited promotion is possibly due to AKR1B10 up-regulation

Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10

Energy Technology Data Exchange (ETDEWEB)

Matsunaga, Toshiyuki, E-mail: matsunagat@gifu-pu.ac.jp [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Morikawa, Yoshifumi; Haga, Mariko; Endo, Satoshi [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Soda, Midori; Yamamura, Keiko [Laboratory of Clinical Pharmacy, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650 (Japan); El-Kabbani, Ossama [Monash Institute of Pharmaceutical Sciences, Monash University, Victoria 3052 (Australia); Tajima, Kazuo [Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181 (Japan); Ikari, Akira [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Hara, Akira [Faculty of Engineering, Gifu University, Gifu 501-1193 (Japan)

2014-07-15

Inhalation of 9,10-phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust, exerts fatal damage against a variety of cells involved in respiratory function. Here, we show that treatment with high concentrations of 9,10-PQ evokes apoptosis of lung cancer A549 cells through production of reactive oxygen species (ROS). In contrast, 9,10-PQ at its concentrations of 2 and 5 μM elevated the potentials for proliferation, invasion, metastasis and tumorigenesis, all of which were almost completely inhibited by addition of an antioxidant N-acetyl-L-cysteine, inferring a crucial role of ROS in the overgrowth and malignant progression of lung cancer cells. Comparison of mRNA expression levels of six aldo-keto reductases (AKRs) in the 9,10-PQ-treated cells advocated up-regulation of AKR1B10 as a major cause contributing to the lung cancer malignancy. In support of this, the elevation of invasive, metastatic and tumorigenic activities in the 9,10-PQ-treated cells was significantly abolished by the addition of a selective AKR1B10 inhibitor oleanolic acid. Intriguingly, zymographic and real-time PCR analyses revealed remarkable increases in secretion and expression, respectively, of matrix metalloproteinase 2 during the 9,10-PQ treatment, and suggested that the AKR1B10 up-regulation and resultant activation of mitogen-activated protein kinase cascade are predominant mechanisms underlying the metalloproteinase induction. In addition, HPLC analysis and cytochrome c reduction assay in in vitro 9,10-PQ reduction by AKR1B10 demonstrated that the enzyme catalyzes redox-cycling of this quinone, by which ROS are produced. Collectively, these results suggest that AKR1B10 is a key regulator involved in overgrowth and malignant progression of the lung cancer cells through ROS production due to 9,10-PQ redox-cycling. - Highlights: • 9,10-PQ promotes invasion, metastasis and tumorigenicity in lung cancer cells. • The 9,10-PQ-elicited promotion is possibly due to AKR1B10 up-regulation

The let-7/Lin28 axis regulates activation of hepatic stellate cells in alcoholic liver injury.

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McDaniel, Kelly; Huang, Li; Sato, Keisaku; Wu, Nan; Annable, Tami; Zhou, Tianhao; Ramos-Lorenzo, Sugeily; Wan, Ying; Huang, Qiaobing; Francis, Heather; Glaser, Shannon; Tsukamoto, Hidekazu; Alpini, Gianfranco; Meng, Fanyin

2017-07-07

The let-7/Lin28 axis is associated with the regulation of key cellular regulatory genes known as microRNAs in various human disorders and cancer development. This study evaluated the role of the let-7/Lin28 axis in regulating a mesenchymal phenotype of hepatic stellate cells in alcoholic liver injury. We identified that ethanol feeding significantly down-regulated several members of the let-7 family in mouse liver, including let-7a and let-7b. Similarly, the treatment of human hepatic stellate cells (HSCs) with lipopolysaccharide (LPS) and transforming growth factor-β (TGF-β) significantly decreased the expressions of let-7a and let-7b. Conversely, overexpression of let-7a and let-7b suppressed the myofibroblastic activation of cultured human HSCs induced by LPS and TGF-β, as evidenced by repressed ACTA2 (α-actin 2), COL1A1 (collagen 1A1), TIMP1 (TIMP metallopeptidase inhibitor 1), and FN1 (fibronectin 1); this supports the notion that HSC activation is controlled by let-7. A combination of bioinformatics, dual-luciferase reporter assay, and Western blot analysis revealed that Lin28B and high-mobility group AT-hook (HMGA2) were the direct targets of let-7a and let-7b. Furthermore, Lin28B deficiency increased the expression of let-7a/let-7b as well as reduced HSC activation and liver fibrosis in mice with alcoholic liver injury. This feedback regulation of let-7 by Lin28B is verified in hepatic stellate cells isolated by laser capture microdissection from the model. The identification of the let-7/Lin28 axis as an important regulator of HSC activation as well as its upstream modulators and down-stream targets will provide insights into the involvement of altered microRNA expression in contributing to the pathogenesis of alcoholic liver fibrosis and novel therapeutic approaches for human alcoholic liver diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mast cell activators as novel immune regulators.

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Johnson-Weaver, Brandi; Choi, Hae Woong; Abraham, Soman N; Staats, Herman F

2018-05-26

Mast cells are an important cell type of the innate immune system that when activated, play a crucial role in generating protective innate host responses after bacterial and viral infection. Additionally, activated mast cells influence lymph node composition to regulate the induction of adaptive immune responses. The recognition that mast cells play a beneficial role in host responses to microbial infection and induction of adaptive immunity has provided the rationale to evaluate mast cell activators for use as antimicrobials or vaccine adjuvants. This review summarizes the role of mast cell activators in antimicrobial responses while also discussing the use of different classes of mast cell activators as potent vaccine adjuvants that enhance the induction of protective immune responses. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

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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

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

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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

GCN5 regulates the activation of PI3K/Akt survival pathway in B cells exposed to oxidative stress via controlling gene expressions of Syk and Btk.

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Kikuchi, Hidehiko; Kuribayashi, Futoshi; Takami, Yasunari; Imajoh-Ohmi, Shinobu; Nakayama, Tatsuo

2011-02-25

Histone acetyltransferase(s) (HATs) are involved in the acetylation of core histones, which is an important event for transcription regulation through alterations in the chromatin structure in eukaryotes. General control non-depressible 5 (GCN5) was first identified as a global coactivator and transcription-related HAT. Here we report that GCN5 regulates the activation of phosphatidylinositol 3-kinase (PI3K)/acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) survival pathway in B cells exposed to oxidative stress via controlling gene expressions of spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk). The GCN5-deficiency remarkably caused apoptotic cell death by treatment with exogenous hydrogen peroxide (H(2)O(2)) in chicken DT40 cells. In GCN5-deficient DT40 cells, gene expressions of Syk and Btk, which are involved in activation of PI3K/Akt survival pathway in DT40 cells exposed to exogenous H(2)O(2), were remarkably decreased compared with those in wild type DT40 cells. In addition, phosphorylation of Akt in H(2)O(2)-treated GCN5-deficient cells was remarkably suppressed as compared to that of DT40. Chromatin immunoprecipitation assay revealed that GCN5 binds to proximal 5'-upstream regions of Syk and Btk genes in vivo. These results suggest that GCN5 takes part in transcriptional regulations of the Syk and Btk genes, and plays a key role in epigenetic regulation of PI3K/Akt survival pathway in B cells exposed to reactive oxygen species such as H(2)O(2). Copyright © 2011 Elsevier Inc. All rights reserved.

Induction of the nuclear IκB protein IκB-ζ upon stimulation of B cell antigen receptor

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Hijioka, Kuniaki; Matsuo, Susumu; Eto-Kimura, Akiko; Takeshige, Koichiro; Muta, Tatsushi

2007-01-01

The nuclear IκB protein IκB-ζ is barely detectable in resting cells and is induced in macrophages and fibroblasts following stimulation of innate immunity via Toll-like receptors. The induced IκB-ζ associates with nuclear factor (NF)-κB in the nucleus and plays crucial roles in its transcriptional regulation. Here, we examined the induction of IκB-ζ in B lymphocytes, one of the major players in adaptive immunity. Upon crosslinking of the surface immunoglobulin complex, IκB-ζ mRNA was robustly induced in murine B-lymphoma cell line A20 cells. While the crosslinking activated NF-κB and induced its target gene, IκB-α, co-crosslinking of Fcγ receptor IIB to the surface immunoglobulin complex inhibited NF-κB activation and the induction of IκB-ζ and IκB-α, suggesting critical roles for NF-κB in the induction. These results indicate that IκB-ζ is also induced by stimulation of B cell antigen receptor, suggesting that IκB-ζ is involved in the regulation of adaptive immune responses

Protein tyrosine phosphatase, PTP1B, expression and activity in rat corneal endothelial cells

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Harris, Deshea L.

2007-01-01

Purpose The current studies were conducted to determine whether the protein tyrosine phosphatase, PTP1B, plays a role in regulating epidermal growth factor receptor (EGFR) Tyr992 phosphorylation and cell cycle entry in rat corneal endothelial cells. Methods Corneas were obtained from male Sprague-Dawley rats. PTP1B mRNA and protein expression were compared in confluent and subconfluent cells by RT-PCR and western blots. Immunocytochemistry was used to determine the subcellular localization of both PTP1B and EGFR following epidermal growth factor (EGF) stimulation. Western blots were used to analyze the time-dependent effect of EGF on phosphorylation of EGFR Tyr992 plus or minus CinnGEL 2Me, an inhibitor of PTP1B activity. The effect of PTP1B inhibition on cell cycle entry was determined by calculating the percent of Ki67-positive cells following EGF treatment. Results PTP1B mRNA expression was similar in confluent and subconfluent cells, but PTP1B protein was expressed at 3 fold higher levels in subconfluent cells. Positive staining for PTP1B was localized in vesicular structures below the plasma membrane. EGFR staining was located at cell-cell borders in untreated endothelium, but was mainly cytoplasmic by 15 min after EGF treatment. In control cultures, phosphorylation of EGFR Tyr992 peaked by 5 min following EGF stimulation and rapidly decreased to basal levels by 30 min. In cultures pretreated with CinnGEL 2Me, Tyr992 phosphorylation peaked 2 min following EGF addition and was consistently sustained at a higher level than controls until 60 min after treatment. By 18 h following EGF treatment, cultures pretreated with CinnGEL 2Me exhibited a 1.7 fold increase in the number of Ki67-positive cells compared with control cultures. Conclusions Comparison of PTP1B mRNA and protein levels indicates that PTP1B expression is regulated mainly at the protein level and is higher in subconfluent cells. PTP1B was located in vesicles below the plasma membrane. The fact that

Altholactone Inhibits NF-κB and STAT3 Activation and Induces Reactive Oxygen Species-Mediated Apoptosis in Prostate Cancer DU145 Cells

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Chunwa Jiang

2017-02-01

Full Text Available Altholactone, a natural compound isolated from Goniothalamus spp., has demonstrated anti-inflammatory and anticancer activities, but its molecular mechanisms are still not fully defined. Nuclear factor kappa B (NF-κB and signal transducer and activator of transcription 3 (STAT3 play pivotal roles in the cell survival of many human tumors. The objective of this study was to elucidate the mechanism of action of altholactone against prostate cancer DU145 cells and to evaluate whether its effects are mediated by inhibition of NF-κB and STAT3 activity. Altholactone inhibited proliferation of DU145 cells and induced cell cycle arrest in S phase and triggered apoptosis. Reporter assays revealed that altholactone repressed p65- and TNF-α-enhanced NF-κB transcriptional activity and also inhibited both constitutive and IL-6-induced transcriptional activity of STAT3. Consistent with this, altholactone down-regulated phosphorylation of STAT3 and moreover, decreased constitutively active mutant of STAT3 (STAT3C-induced transcriptional activity. Altholactone treatment also results in down-regulation of STAT3 target genes such as survivin, and Bcl-2 followed by up regulation of pro-apoptotic Bax protein. However, pre-treatment with the antioxidant N-acetylcysteine (NAC significantly inhibited the activation of Bax and prevented down-regulation of STAT3 target genes. Collectively, our findings suggest that altholactone induces DU145 cells death through inhibition of NF-κB and STAT3 activity.

Involvement of mitogen-activated protein kinases and NFκB in LPS-induced CD40 expression on human monocytic cells

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Wu Weidong; Alexis, Neil E.; Chen Xian; Bromberg, Philip A.; Peden, David B.

2008-01-01

CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NFκB were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NFκB activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NFκB activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NFκB activation, and CD40 expression. Moreover, blockage of MAPK and NFκB activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NFκB

Fungal-induced cell cycle impairment, chromosome instability and apoptosis via differential activation of NF-κB.

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Mariem Ben-Abdallah

Full Text Available Microbial pathogens have developed efficient strategies to compromise host immune responses. Cryptococcus neoformans is a facultative intracellular pathogen, recognised as the most common cause of systemic fungal infections leading to severe meningoencephalitis, mainly in immunocompromised patients. This yeast is characterized by a polysaccharide capsule, which inhibits its phagocytosis. Whereas phagocytosis escape and macrophage intracellular survival have been intensively studied, extracellular survival of this yeast and restraint of host innate immune response are still poorly understood. In this study, we have investigated whether C. neoformans affected macrophage cell viability and whether NF-κB (nuclear factor-κB, a key regulator of cell growth, apoptosis and inflammation, was involved. Using wild-type (WT as well as mutant strains of C. neoformans for the pathogen side, and WT and mutant cell lines with altered NF-κB activity or signalling as well as primary macrophages for the host side, we show that C. neoformans manipulated NF-κB-mediated signalling in a unique way to regulate macrophage cell fate and viability. On the one hand, serotype A strains reduced macrophage proliferation in a capsule-independent fashion. This growth decrease, which required a critical dosage of NF-κB activity, was caused by cell cycle disruption and aneuploidy, relying on fungal-induced modification of expression of several cell cycle checkpoint regulators in S and G2/M phases. On the other hand, C. neoformans infection induced macrophage apoptosis in a capsule-dependent manner with a differential requirement of the classical and alternative NF-κB signalling pathways, the latter one being essential. Together, these findings shed new light on fungal strategies to subvert host response through uncoupling of NF-κB activity in pathogen-controlled apoptosis and impairment of cell cycle progression. They also provide the first demonstration of induction of

Nuclear protein IκB-ζ inhibits the activity of STAT3

International Nuclear Information System (INIS)

Wu, Zhihao; Zhang, Xiaoai; Yang, Juntao; Wu, Guangzhou; Zhang, Ying; Yuan, Yanzhi; Jin, Chaozhi; Chang, Zhijie; Wang, Jian; Yang, Xiaoming; He, Fuchu

2009-01-01

STAT3 (Signal transducer and activator of transcription 3) is a key transcription factor of the JAK-STAT (Janus kinase/signal transducer and activator of transcription) pathway that regulates cell proliferation and apoptosis. Activation of STAT3 is under tight regulation, and yet the different signaling pathways and the mechanisms that regulate its activity remain to be elucidated. Using a yeast two-hybrid screening, we have identified a nuclear protein IκB-ζ that interacts in a novel way with STAT3. This physical interaction was further confirmed by co-immunoprecipitation assays. The interaction regions were mapped to the coiled-coil domain of STAT3 and the C-terminal of IκB-ζ. Overexpression of IκB-ζ inhibited the transcriptional activity of STAT3. It also suppressed cell growth and induced cell apoptosis in SRC-simulated cells, which is partially mediated by down-regulation of expression of a known STAT3 target gene, MCL1. Our results suggest that IκB-ζ is a negative regulator of STAT3, and demonstrate a novel mechanism in which a component of the NF-κB signaling pathway inhibits the activation of STAT3.

Differential Role of Cathepsins S and B In Hepatic APC-Mediated NKT Cell Activation and Cytokine Secretion.

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de Mingo Pulido, Álvaro; de Gregorio, Estefanía; Chandra, Shilpi; Colell, Anna; Morales, Albert; Kronenberg, Mitchell; Marí, Montserrat

2018-01-01

Natural killer T (NKT) cells exhibit a specific tissue distribution, displaying the liver the highest NKT/conventional T cell ratio. Upon antigen stimulation, NKT cells secrete Th1 cytokines, including interferon γ (IFNγ), and Th2 cytokines, including IL-4 that recruit and activate other innate immune cells to exacerbate inflammatory responses in the liver. Cysteine cathepsins control hepatic inflammation by regulating κB-dependent gene expression. However, the contribution of cysteine cathepsins other than Cathepsin S to NKT cell activation has remained largely unexplored. Here we report that cysteine cathepsins, cathepsin B (CTSB) and cathepsin S (CTSS), regulate different aspects of NKT cell activation. Inhibition of CTSB or CTSS reduced hepatic NKT cell expansion in a mouse model after LPS challenge. By contrast, only CTSS inhibition reduced IFNγ and IL-4 secretion after in vivo α-GalCer administration. Accordingly, in vitro studies reveal that only CTSS was able to control α-GalCer-dependent loading in antigen-presenting cells (APCs), probably due to altered endolysosomal protein degradation. In summary, our study discloses the participation of cysteine cathepsins, CTSB and CTSS, in the activation of NKT cells in vivo and in vitro .

Differential Role of Cathepsins S and B In Hepatic APC-Mediated NKT Cell Activation and Cytokine Secretion

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Álvaro de Mingo Pulido

2018-02-01

Full Text Available Natural killer T (NKT cells exhibit a specific tissue distribution, displaying the liver the highest NKT/conventional T cell ratio. Upon antigen stimulation, NKT cells secrete Th1 cytokines, including interferon γ (IFNγ, and Th2 cytokines, including IL-4 that recruit and activate other innate immune cells to exacerbate inflammatory responses in the liver. Cysteine cathepsins control hepatic inflammation by regulating κB-dependent gene expression. However, the contribution of cysteine cathepsins other than Cathepsin S to NKT cell activation has remained largely unexplored. Here we report that cysteine cathepsins, cathepsin B (CTSB and cathepsin S (CTSS, regulate different aspects of NKT cell activation. Inhibition of CTSB or CTSS reduced hepatic NKT cell expansion in a mouse model after LPS challenge. By contrast, only CTSS inhibition reduced IFNγ and IL-4 secretion after in vivo α-GalCer administration. Accordingly, in vitro studies reveal that only CTSS was able to control α-GalCer-dependent loading in antigen-presenting cells (APCs, probably due to altered endolysosomal protein degradation. In summary, our study discloses the participation of cysteine cathepsins, CTSB and CTSS, in the activation of NKT cells in vivo and in vitro.

MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

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Hui Xu

2010-11-01

Full Text Available MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

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Xu, Hui; Yan, Yaping; Williams, Mark S; Carey, Gregory B; Yang, Jingxian; Li, Hongmei; Zhang, Guang-Xian; Rostami, Abdolmohamad

2010-11-01

MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK) and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

TGF-b and a specific TGF-b inhibitor regulate pericentrin B and MYH9 in glioma cell lines

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Óscar Álzate

2006-01-01

Full Text Available Malignant gliomas are heterogeneous, highly invasive vascular tumours. The multifunctional cytokine, transforming growth factor-beta (TGF-P, is expressed by grade III/IV gliomas and promotes tumour angiogenesis, invasión and immune escape. It has been shown previously that a small TGF-P receptor type I (TGF-(3-RI molecule inhibitor (SB-431542 blocks TGF-(3-mediated signal transduction, induction of angiogenic factor expression and cellular motility. As glioma cell lines display differential sensitivity to TGF-P, it was expected that they would also be differentially impacted by disruption of TGF-P signalling. Differential in gel expression (DIGE analysis and mass spectrometry was used in this work for determining protein regulation effects of both TGF-P and SB-431542 on human glioma cell lines. It was found that pericentrin B and non muscle myosin were differentially expressed in fragments which likely resulted from protease activation by the tumour growth mechanism. These results suggest that both pericentrin B and non-muscle myosin might be potential glioma biomarkers. Key words: DIGE, proteomics, glioma, TGF-P, mass spectrometry, non muscle myosin, pericentrin B.

The negative cell cycle regulator, Tob (transducer of ErbB-2), is involved in motor skill learning

International Nuclear Information System (INIS)

Wang Xinming; Gao Xiang; Zhang Xuehan; Tu Yanyang; Jin Meilei; Zhao Guoping; Yu Lei; Jing Naihe; Li Baoming

2006-01-01

Tob (transducer of ErbB-2) is a negative cell cycle regulator with anti-proliferative activity in peripheral tissues. Our previous study identified Tob as a protein involved in hippocampus-dependent memory consolidation (M.L. Jin, X.M. Wang, Y.Y. Tu, X.H. Zhang, X. Gao, N. Guo, Z.Q. Xie, G.P. Zhao, N.H. Jing, B.M. Li, Y.Yu, The negative cell cycle regulator, Tob (Transducer of ErbB-2), is a multifunctional protein involved in hippocampus-dependent learning and memory, Neuroscience 131 (2005) 647-659). Here, we provide evidence that Tob in the central nervous system is engaged in acquisition of motor skill. Tob has a relatively high expression in the cerebellum. Tob expression is up-regulated in the cerebellum after rats receive training on a rotarod-running task. Rats infused with Tob antisense oligonucleotides into the 4th ventricle exhibit a severe deficit in running on a rotating rod or walking across a horizontally elevated beam

Human B cells induce dendritic cell maturation and favour Th2 polarization by inducing OX-40 ligand

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Maddur, Mohan S.; Sharma, Meenu; Hegde, Pushpa; Stephen-Victor, Emmanuel; Pulendran, Bali; Kaveri, Srini V.; Bayry, Jagadeesh

2015-01-01

Dendritic cells (DCs) play a critical role in immune homeostasis by regulating the functions of various immune cells, including T and B cells. Notably, DCs also undergo education on reciprocal signalling by these immune cells and environmental factors. Various reports demonstrated that B cells have profound regulatory functions, although only few reports have explored the regulation of human DCs by B cells. Here we demonstrate that activated but not resting B cells induce maturation of DCs with distinct features to polarize Th2 cells that secrete interleukin (IL)-5, IL-4 and IL-13. B-cell-induced maturation of DCs is contact dependent and implicates signalling of B-cell activation molecules CD69, B-cell-activating factor receptor, and transmembrane activator and calcium-modulating cyclophilin ligand interactor. Mechanistically, differentiation of Th2 cells by B-cell-matured DCs is dependent on OX-40 ligand. Collectively, our results suggest that B cells have the ability to control their own effector functions by enhancing the ability of human DCs to mediate Th2 differentiation. PMID:24910129

Invasin of Yersinia pseudotuberculosis activates human peripheral B cells.

OpenAIRE

Lundgren, E; Carballeira, N; Vazquez, R; Dubinina, E; Bränden, H; Persson, H; Wolf-Watz, H

1996-01-01

The Yersinia pseudotuberculosis cell surface-located protein invasin was found to promote binding between the pathogen and resting peripheral B cells via beta 1 integrin receptors (CD29). B cells responded by expressing several activation markers and by growing, In contrast, T cells did not react, although these cells express CD29. An isogenic invA mutant failed to activate B cells. The mutation could be complemented by providing the invA+ gene in trans. Purified invasin alone did not activat...

Antitumor Activity of Tenacissoside H on Esophageal Cancer through Arresting Cell Cycle and Regulating PI3K/Akt-NF-κB Transduction Cascade

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Yong-sen Jia

2015-01-01

Full Text Available Objective. The purpose of the study was to elucidate the molecular mechanism of tenacissoside H (TDH inhibiting esophageal carcinoma infiltration and proliferation. Methods. In vitro, EC9706 cells were treated with TDH. Cells proliferation and cell cycle were assayed. PI3K and NF-κB mRNAs expression were determined by real time PCR. In vivo, model of nude mice with tumor was established. Mice were treated with TDH. Inhibition ratio of tumor volume was calculated. PCNA expression was examined. Protein expression in PI3K/Akt-NF-κB signaling pathway was determined. Results. In vitro, TDH significantly inhibited cells proliferation in a time-and-dose-dependent manner. TDH arrested the cell cycle in S phase and significantly inhibited PI3K and NF-κB mRNA expression, compared with blank controlled group (P<0.05. In vivo, TDH strongly inhibits tumor growth and volume. PCNA expression was significantly decreased after treatment of TDH. TDH downregulated proteins expression in PI3K/Akt-NF-κB transduction cascade (P<0.05. Conclusion. TDH inhibited esophageal carcinoma infiltration and proliferation both in vitro and in vivo. The anticancer activity has relation to arresting the cell cycle at the S phase, inhibited the PCNA expression of transplanted tumors in nude mice, and regulated the protein expression in the PI3K/Akt-NF-κB transduction cascade.

Iodine-131 treatment of thyroid cancer cells leads to suppression of cell proliferation followed by induction of cell apoptosis and cell cycle arrest by regulation of B-cell translocation gene 2-mediated JNK/NF-κB pathways

Energy Technology Data Exchange (ETDEWEB)

Zhao, L.M.; Pang, A.X., E-mail: zhaoliming515@126.com [Department of Nuclear Medicine, Linyi People' s Hospital, Linyi (China); Department of Urology, Linyi People' s Hospital, Linyi (China)

2017-10-01

Iodine-131 ({sup 131}I) is widely used for the treatment of thyroid-related diseases. This study aimed to investigate the expression of p53 and BTG2 genes following {sup 131}I therapy in thyroid cancer cell line SW579 and the possible underlying mechanism. SW579 human thyroid squamous carcinoma cells were cultured and treated with {sup 131}I. They were then assessed for {sup 131}I uptake, cell viability, apoptosis, cell cycle arrest, p53 expression, and BTG2 gene expression. SW579 cells were transfected with BTG2 siRNA, p53 siRNA and siNC and were then examined for the same aforementioned parameters. When treated with a JNK inhibitor of SP600125 and {sup 131}I or with a NF-kB inhibitor of BMS-345541 and {sup 131}I, non-transfected SW579 cells were assessed in JNK/NFkB pathways. It was observed that {sup 131}I significantly inhibited cell proliferation, promoted cell apoptosis and cell cycle arrest. Both BTG2 and p53 expression were enhanced in a dose-dependent manner. An increase in cell viability by up-regulation in Bcl2 gene, a decrease in apoptosis by enhanced CDK2 gene expression and a decrease in cell cycle arrest at G{sub 0}/G{sub 1} phase were also observed in SW579 cell lines transfected with silenced BTG2 gene. When treated with SP600125 and {sup 131}I, the non transfected SW579 cell lines significantly inhibited JNK pathway, NF-kB pathway and the expression of BTG2. However, when treated with BMS-345541 and {sup 131}I, only the NF-kB pathway was suppressed. {sup 131}I suppressed cell proliferation, induced cell apoptosis, and promoted cell cycle arrest of thyroid cancer cells by up-regulating B-cell translocation gene 2-mediated activation of JNK/NF--κB pathways. (author)

Down-regulation of LRP1B in colon cancer promoted the growth and migration of cancer cells.

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Wang, Zhiqiang; Sun, Peng; Gao, Chun; Chen, Ji; Li, Jun; Chen, Zhonghao; Xu, Ming; Shao, Jun; Zhang, Yunpeng; Xie, Jiang

2017-08-01

Aberrant activation of beta-catenin/TCF signaling is one of the hallmarks of colon cancer. It is of great interest to study the mechanism for the regulation of beta-catenin/TCF signaling. In this study, it was found that LRP1B was down-regulated in colon cancer tissues and inhibited the growth, migration and metastasis of colon cancer cells. The molecular mechanism study revealed that LRP1B interacted with DVL2, inhibited the interaction between DVL2 and Axin, and negatively regulated beta-catenin/TCF signaling. Taken together, our study demonstrated the suppressive roles of LRP1B in the progression of colon cancer, implicating that restoring the function of LRP1B would be a promising strategy for the treatment of colon cancer. Copyright © 2017. Published by Elsevier Inc.

NF-{kappa}B p50 promotes tumor cell invasion through negative regulation of invasion suppressor gene CRMP-1 in human lung adenocarcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Ming, Gao [Cancer Research Center, College of Medicine, National Taiwan University, Taipei, Taiwan (China); National Center of Excellence for Clinical Trial and Research, National Taiwan University, Hospital, Taipei, Taiwan (China); Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Yeh, P Y [Cancer Research Center, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Department of Oncology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 10016, Taiwan (China); Lu, Y -S [Cancer Research Center, College of Medicine, National Taiwan University, Taipei, Taiwan (China); National Center of Excellence for Clinical Trial and Research, National Taiwan University, Hospital, Taipei, Taiwan (China); Department of Oncology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 10016, Taiwan, ROC (China); Chang, W C [Cancer Research Center, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Kuo, M -L [Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Cheng, A -L [Cancer Research Center, College of Medicine, National Taiwan University, Taipei, Taiwan (China); National Center of Excellence for Clinical Trial and Research, National Taiwan University, Hospital, Taipei, Taiwan (China); Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Department of Oncology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 10016, Taiwan (China); Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 10016, Taiwan (China)], E-mail: alcheng@ntu.edu.tw

2008-11-14

Lung adenocarcinoma Cl1-5 cells were selected from parental Cl1-0 cells based on their high metastatic potential. In a previous study, CRMP-1, an invasion suppressor gene, was shown to be suppressed in Cl1-5 cells. However, the regulation of CRMP-1 expression has not been explored. In this study, we showed nuclear factor-{kappa}B controls CRMP-1 expression. The electromobility shift assay showed that while Cl1-0 cells exhibited low NF-{kappa}B activity in response to TNF-{alpha}, an abundance of basal and TNF-{alpha}-induced NF-{kappa}B-DNA complex was detected in Cl1-5 cells. Supershift-coupled EMSA and Western blotting of nuclear proteins, however, revealed p50 protein, but not classic p65/p50 heterodimer in the complex. ChIP and EMSA demonstrated that p50 binds to a {kappa}B site residing between -1753 and -1743 of the CRMP-1 promoter region. Transfection of antisense p50 gene into Cl1-5 cells increased the CRMP-1 protein level and decreased the invasive activity of Cl1-5 cells.

NF-κB p50 promotes tumor cell invasion through negative regulation of invasion suppressor gene CRMP-1 in human lung adenocarcinoma cells

International Nuclear Information System (INIS)

Gao Ming; Yeh, P.Y.; Lu, Y.-S.; Chang, W.C.; Kuo, M.-L.; Cheng, A.-L.

2008-01-01

Lung adenocarcinoma Cl1-5 cells were selected from parental Cl1-0 cells based on their high metastatic potential. In a previous study, CRMP-1, an invasion suppressor gene, was shown to be suppressed in Cl1-5 cells. However, the regulation of CRMP-1 expression has not been explored. In this study, we showed nuclear factor-κB controls CRMP-1 expression. The electromobility shift assay showed that while Cl1-0 cells exhibited low NF-κB activity in response to TNF-α, an abundance of basal and TNF-α-induced NF-κB-DNA complex was detected in Cl1-5 cells. Supershift-coupled EMSA and Western blotting of nuclear proteins, however, revealed p50 protein, but not classic p65/p50 heterodimer in the complex. ChIP and EMSA demonstrated that p50 binds to a κB site residing between -1753 and -1743 of the CRMP-1 promoter region. Transfection of antisense p50 gene into Cl1-5 cells increased the CRMP-1 protein level and decreased the invasive activity of Cl1-5 cells

MicroRNA expression profiling identifies activated B cell status in chronic lymphocytic leukemia cells.

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Shuqiang Li

2011-03-01

Full Text Available Chronic lymphocytic leukemia (CLL is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+ and IgV(H unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.

BolA inhibits cell elongation and regulates MreB expression levels.

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Freire, Patrick; Moreira, Ricardo Neves; Arraiano, Cecília Maria

2009-02-06

The morphogene bolA is a general stress response gene in Escherichia coli that induces a round morphology when overexpressed. Results presented in this report show that increased BolA levels can inhibit cell elongation mechanisms. MreB polymerization is crucial for the bacterial cell cytoskeleton, and this protein is essential for the maintenance of a cellular rod shape. In this report, we demonstrate that bolA overexpression affects the architecture of MreB filaments. An increase in BolA leads to a significant reduction in MreB protein levels and mreB transcripts. BolA affects the mreBCD operon in vivo at the level of transcription. Furthermore, our results show that BolA is a new transcriptional repressor of MreB. The alterations in cell morphology induced by bolA seem to be mediated by a complex pathway that integrates PBP5, PBP6, MreB, and probably other regulators of cell morphology/elongation.

The Regulation of NF-κB Subunits by Phosphorylation

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Frank Christian

2016-03-01

Full Text Available The NF-κB transcription factor is the master regulator of the inflammatory response and is essential for the homeostasis of the immune system. NF-κB regulates the transcription of genes that control inflammation, immune cell development, cell cycle, proliferation, and cell death. The fundamental role that NF-κB plays in key physiological processes makes it an important factor in determining health and disease. The importance of NF-κB in tissue homeostasis and immunity has frustrated therapeutic approaches aimed at inhibiting NF-κB activation. However, significant research efforts have revealed the crucial contribution of NF-κB phosphorylation to controlling NF-κB directed transactivation. Importantly, NF-κB phosphorylation controls transcription in a gene-specific manner, offering new opportunities to selectively target NF-κB for therapeutic benefit. This review will focus on the phosphorylation of the NF-κB subunits and the impact on NF-κB function.

Sustained Expression of Negative Regulators of Myelination Protects Schwann Cells from Dysmyelination in a Charcot-Marie-Tooth 1B Mouse Model.

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Florio, Francesca; Ferri, Cinzia; Scapin, Cristina; Feltri, M Laura; Wrabetz, Lawrence; D'Antonio, Maurizio

2018-05-02

Schwann cell differentiation and myelination in the PNS are the result of fine-tuning of positive and negative transcriptional regulators. As myelination starts, negative regulators are downregulated, whereas positive ones are upregulated. Fully differentiated Schwann cells maintain an extraordinary plasticity and can transdifferentiate into "repair" Schwann cells after nerve injury. Reactivation of negative regulators of myelination is essential to generate repair Schwann cells. Negative regulators have also been implicated in demyelinating neuropathies, although their role in disease remains elusive. Here, we used a mouse model of Charcot-Marie-Tooth neuropathy type 1B (CMT1B), the P0S63del mouse characterized by ER stress and the activation of the unfolded protein response, to show that adult Schwann cells are in a partial differentiation state because they overexpress transcription factors that are normally expressed only before myelination. We provide evidence that two of these factors, Sox2 and Id2, act as negative regulators of myelination in vivo However, their sustained expression in neuropathy is protective because ablation of Sox2 or/and Id2 from S63del mice of both sexes results in worsening of the dysmyelinating phenotype. This is accompanied by increased levels of mutant P0 expression and exacerbation of ER stress, suggesting that limited differentiation may represent a novel adaptive mechanism through which Schwann cells counter the toxic effect of a mutant terminal differentiation protein. SIGNIFICANCE STATEMENT In many neuropathies, Schwann cells express high levels of early differentiation genes, but the significance of these altered expression remained unclear. Because many of these factors may act as negative regulators of myelination, it was suggested that their misexpression could contribute to dysmyelination. Here, we show that the transcription factors Sox2 and Id2 act as negative regulators of myelination in vivo , but that their sustained

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

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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.

B16-BL6 melanoma cells release inhibitory factor(s) of active pump activity in isolated lymph vessels.

Science.gov (United States)

Nakaya, K; Mizuno, R; Ohhashi, T

2001-12-01

We investigated whether supernatant cultured with melanoma cell lines B16-BL6 and K1735 or the Lewis lung carcinoma cell line (LLC) can regulate lymphatic pump activity with bioassay preparations isolated from murine iliac lymph vessels. B16-BL6 and LLC supernatants caused significant dilation of lymph microvessels with cessation of pump activity. B16-BL6 supernatant produced dose-related cessation of lymphatic pump activity. There was no significant tachyphylaxis in the supernatant-mediated inhibitory response of lymphatic pump activity. Pretreatment with 3 x 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME) or 10(-7) M or 10(-6) M glibenclamide and 5 x 10(-4) M 5-hydroxydecanoic acid caused significant reduction of supernatant-mediated inhibitory responses. Simultaneous treatment with 10(-3) M L-arginine and 3 x 10(-5) M L-NAME significantly lessened L-NAME-induced inhibition of the supernatant-mediated response, suggesting that endogenous nitric oxide (NO) plays important roles in supernatant-mediated inhibitory responses. Chemical treatment dialyzed substances of B16-BL6 cells may release nonpeptide substance(s) of <1,000 MW, resulting in significant cessation of lymphatic pump activity via production and release of endogenous NO and activation of mitochondrial ATP-sensitive K(+) channels.

The potential impact of low dose ionizing γ-radiation on immune response activity up-regulated by Ikaros in IM-9 B lymphocytes

International Nuclear Information System (INIS)

Kim Sung Jn; Jang, Seon A; Yang, Kwang Hee; Kim, Ji Young; Kim, Cha Soon; Nam, Seon Young; Jeong, Mee Seon; Jin, Young Woo

2011-01-01

The biological effects of low dose ionizing radiation (LDIR) remain insufficiently understood. We examined for the scientific evidence to show the biological effects of LDIR using radiation-sensitive immune cells. We found that Ikaros protein was responded to low dose-dependent effects of gamma radiation in IM-9 B lymphocytes. Ikaros encodes zinc finger transcription factors that is important regulators of a hematopoietic stem cells (HSCs) progression to the B lymphoid lineage development, differentiation and proliferation. In this study, we observed that cell proliferation was enhanced from 10% to 20% by LDIR (0.05 Gy) in IM-9 B lymphocytes. The Ikaros protein was phosphorylated in its serine/threonine (S/T) region and decreased its DNA binding activity in the cells exposed to LDIR. We found that Ikaros phosphorylation was up-regulated by CK2/AKT pathway and the residues of ser-304 and ser-306 in Ikaros was phosphorylated by LDIR. We also observed that Ikaros protein was localized from the nucleus to the cytoplasm after LDIR and bound with Autotaxin (ENPP2, ATX) protein, stimulating proliferation, migration and survival of immune cells. In addition, we found that the lysoPLD activity of ATX was dependent on Ikaros-ATX binding activity. These results indicate that the Ikaros is an important regulator of immune activation. Therefore, we suggest that low dose ionizing radiation can be considered as a beneficial effects, stimulating the activation of immune cells.

Activated H-Ras regulates hematopoietic cell survival by modulating Survivin

International Nuclear Information System (INIS)

Fukuda, Seiji; Pelus, Louis M.

2004-01-01

Survivin expression and Ras activation are regulated by hematopoietic growth factors. We investigated whether activated Ras could circumvent growth factor-regulated Survivin expression and if a Ras/Survivin axis mediates growth factor independent survival and proliferation in hematopoietic cells. Survivin expression is up-regulated by IL-3 in Ba/F3 and CD34 + cells and inhibited by the Ras inhibitor, farnesylthiosalicylic acid. Over-expression of constitutively activated H-Ras (CA-Ras) in Ba/F3 cells blocked down-modulation of Survivin expression, G 0 /G 1 arrest, and apoptosis induced by IL-3 withdrawal, while dominant-negative (DN) H-Ras down-regulated Survivin. Survivin disruption by DN T34A Survivin blocked CA-Ras-induced IL-3-independent cell survival and proliferation; however, it did not affect CA-Ras-mediated enhancement of S-phase, indicating that the anti-apoptotic activity of CA-Ras is Survivin dependent while its S-phase enhancing effect is not. These results indicate that CA-Ras modulates Survivin expression independent of hematopoietic growth factors and that a CA-Ras/Survivin axis regulates survival and proliferation of transformed hematopoietic cells

Human telomerase activity regulation

OpenAIRE

Wojtyla, Aneta; Gladych, Marta; Rubis, Blazej

2010-01-01

Telomerase has been recognized as a relevant factor distinguishing cancer cells from normal cells. Thus, it has become a very promising target for anticancer therapy. The cell proliferative potential can be limited by replication end problem, due to telomeres shortening, which is overcome in cancer cells by telomerase activity or by alternative telomeres lengthening (ALT) mechanism. However, this multisubunit enzymatic complex can be regulated at various levels, including expression control b...

Type II NKT-TFH cells against Gaucher lipids regulate B-cell immunity and inflammation.

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Nair, Shiny; Boddupalli, Chandra Sekhar; Verma, Rakesh; Liu, Jun; Yang, Ruhua; Pastores, Gregory M; Mistry, Pramod K; Dhodapkar, Madhav V

2015-02-19

Chronic inflammation including B-cell activation is commonly observed in both inherited (Gaucher disease [GD]) and acquired disorders of lipid metabolism. However, the cellular mechanisms underlying B-cell activation in these settings remain to be elucidated. Here, we report that β-glucosylceramide 22:0 (βGL1-22) and glucosylsphingosine (LGL1), 2 major sphingolipids accumulated in GD, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT) cells. Human βGL1-22- and LGL1-reactive CD1d tetramer-positive T cells have a distinct T-cell receptor usage and genomic and cytokine profiles compared with the classical type I NKT cells. In contrast to type I NKT cells, βGL1-22- and LGL1-specific NKT cells constitutively express T-follicular helper (TFH) phenotype. Injection of these lipids leads to an increase in respective lipid-specific type II NKT cells in vivo and downstream induction of germinal center B cells, hypergammaglobulinemia, and production of antilipid antibodies. Human βGL1-22- and LGL1-specific NKT cells can provide efficient cognate help to B cells in vitro. Frequency of LGL1-specific T cells in GD mouse models and patients correlates with disease activity and therapeutic response. Our studies identify a novel type II NKT-mediated pathway for glucosphingolipid-mediated dysregulation of humoral immunity and increased risk of B-cell malignancy observed in metabolic lipid disorders. © 2015 by The American Society of Hematology.

Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane.

LENUS (Irish Health Repository)

Knodler, Leigh A

2009-11-01

The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella-containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono-ubiquitinated. Substitution of these six lysine residues with arginine, SopB-K(6)R, almost completely eliminated SopB ubiquitination. We found that ubiquitination does not affect SopB stability or membrane association, or SopB-dependent events in SCV biogenesis. However, two spatially and temporally distinct events are dependent on ubiquitination, downregulation of SopB activity at the plasma membrane and prolonged retention of SopB on the SCV. Activation of the mammalian pro-survival kinase Akt\\/PKB, a downstream target of SopB, was intensified and prolonged after infection with the SopB-K(6)R mutant. At later times, fewer SCV were decorated with SopB-K(6)R compared with SopB. Instead SopB-K(6)R was present as discrete vesicles spread diffusely throughout the cell. Altogether, our data show that ubiquitination of SopB is not related to its intracellular stability but rather regulates its enzymatic activity at the plasma membrane and intracellular localization.

Trib1 Is Overexpressed in Systemic Lupus Erythematosus, While It Regulates Immunoglobulin Production in Murine B Cells

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Léa Simoni

2018-03-01

Full Text Available Systemic lupus erythematosus (SLE is a severe and heterogeneous autoimmune disease with a complex genetic etiology, characterized by the production of various pathogenic autoantibodies, which participate in end-organ damages. The majority of human SLE occurs in adults as a polygenic disease, and clinical flares interspersed with silent phases of various lengths characterize the usual evolution of the disease in time. Trying to understand the mechanism of the different phenotypic traits of the disease, and considering the central role of B cells in SLE, we previously performed a detailed wide analysis of gene expression variation in B cells from quiescent SLE patients. This analysis pointed out an overexpression of TRIB1. TRIB1 is a pseudokinase that has been implicated in the development of leukemia and also metabolic disorders. It is hypothesized that Trib1 plays an adapter or scaffold function in signaling pathways, notably in MAPK pathways. Therefore, we planned to understand the functional significance of TRIB1 overexpression in B cells in SLE. We produced a new knock-in model with B-cell-specific overexpression of Trib1. We showed that overexpression of Trib1 specifically in B cells does not impact B cell development nor induce any development of SLE symptoms in the mice. By contrast, Trib1 has a negative regulatory function on the production of immunoglobulins, notably IgG1, but also on the production of autoantibodies in an induced model. We observed a decrease of Erk activation in BCR-stimulated Trib1 overexpressing B cells. Finally, we searched for Trib1 partners in B cells by proteomic analysis in order to explore the regulatory function of Trib1 in B cells. Interestingly, we find an interaction between Trib1 and CD72, a negative regulator of B cells whose deficiency in mice leads to the development of autoimmunity. In conclusion, the overexpression of Trib1 could be one of the molecular pathways implicated in the negative regulation

Proteomic analyses for profiling regulated proteins/enzymes by Fucus vesiculosus fucoidan in B16 melanoma cells: A combination of enzyme kinetics functional study.

Science.gov (United States)

Wang, Zhi-Jiang; Zheng, Li; Yang, Jun-Mo; Kang, Yani; Park, Yong-Doo

2018-06-01

Fucoidans are complex sulfated polysaccharides that have a wide range of biological activities. Previously, we reported the various effects of Fucus vesiculosus fucoidan on tyrosinase and B16 melanoma cells. In this study, to identify fucoidan-targeted proteins in B16 melanoma cells, we performed a proteomics study and integrated enzyme kinetics. We detected 19 candidate proteins dysregulated by fucoidan treatment. Among the probed proteins, the enzyme kinetics of two candidate enzymes, namely lactate dehydrogenase (LDH) as an upregulated protein and superoxide dismutase (SOD) as a downregulated enzyme, were determined. The enzyme kinetics results showed that Fucus vesiculosus fucoidan significantly inhibited LDH catalytic function while it did not affect SOD activity even at a high dose, while only slightly decreased activity (up to 10%) at a low dose. Based on our previous and present observations, fucoidan could inhibit B16 melanoma cells growth via regulating proteins/enzymes expression levels such as LDH and SOD known as cell survival biomarkers. Interestingly, both expression level and enzyme catalytic activity of LDH were regulated by fucoidan, which could directly induce the apoptotic effect on B16 melanoma cells along with SOD downregulation. This study highlights how combining proteomics with enzyme kinetics can yield valuable insights into fucoidan targets. Copyright © 2018 Elsevier B.V. All rights reserved.

The distribution of IL-13 receptor alpha1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4.

Science.gov (United States)

Graber, P; Gretener, D; Herren, S; Aubry, J P; Elson, G; Poudrier, J; Lecoanet-Henchoz, S; Alouani, S; Losberger, C; Bonnefoy, J Y; Kosco-Vilbois, M H; Gauchat, J F

1998-12-01

To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.

Mouse CD23 regulates monocyte activation through an interaction with the adhesion molecule CD11b/CD18.

Science.gov (United States)

Lecoanet-Henchoz, S; Plater-Zyberk, C; Graber, P; Gretener, D; Aubry, J P; Conrad, D H; Bonnefoy, J Y

1997-09-01

CD23 is expressed on a variety of hemopoietic cells. Recently, we have reported that blocking CD23 interactions in a murine model of arthritis resulted in a marked improvement of disease severity. Here, we demonstrate that CD11b, the alpha chain of the beta 2 integrin adhesion molecule complex CD11b/CD18 expressed on monocytes interacts with CD23. Using a recombinant fusion protein (ZZ-CD23), murine CD23 was shown to bind to peritoneal macrophages and peripheral blood cells isolated from mice as well as the murine macrophage cell line, RAW. The interactions between mouse ZZ-CD23 and CD11b/CD18-expressing cells were significantly inhibited by anti-CD11b monoclonal antibodies. A functional consequence was then demonstrated by inducing an up-regulation of interleukin-6 (IL-6) production following ZZ-CD23 incubation with monocytes. The addition of Fab fragments generated from the monoclonal antibody CD11b impaired this cytokine production by 50%. Interestingly, a positive autocrine loop was identified as IL-6 was shown to increase CD23 binding to macrophages. These results demonstrate that similar to findings using human cells, murine CD23 binds to the surface adhesion molecule, CD11b, and these interactions regulate biological activities of murine myeloid cells.

1,25-dihydroxyvitamin D3 impairs NF-κB activation in human naive B cells

International Nuclear Information System (INIS)

Geldmeyer-Hilt, Kerstin; Heine, Guido; Hartmann, Bjoern; Baumgrass, Ria; Radbruch, Andreas; Worm, Margitta

2011-01-01

Highlights: → In naive B cells, VDR activation by calcitriol results in reduced NF-κB p105 and p50 protein expression. → Ligating the VDR with calcitriol causes reduced nuclear translocation of NF-κB p65. → Reduced nuclear amount of p65 after calcitriol incubation results in reduced binding of p65 on the p105 promoter. → Thus, vitamin D receptor signaling may reduce or prevent activation of B cells and unwanted immune responses, e.g. in IgE dependent diseases such as allergic asthma. -- Abstract: 1α,25-dihydroxyvitamin D 3 (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-κB p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-κB mediated activation of human naive B cells. Naive B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human naive B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-κB activation by interference with NF-κB p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in naive B cells, namely by reducing CD40 signaling.

NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I

International Nuclear Information System (INIS)

Lacoste, J.; Cohen, L.; Hiscott, J.

1991-01-01

The effect of constitutive Tax expression on the interaction of NF-κ B with its recognition sequence and on NF-κ B-dependent gene expression was examined in T lymphoid Jurkat cell lines (19D and 9J) stably transformed with a Tax expression vector. Tax expressing T cell lines contained a constitutive level of NF-κ B binding activity, detectable by mobility shift assay and uv cross-linking using a palindromic NF-κ B probe homologous to the interferon beta PRDII site. In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85, 75, and 54 kDa, whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced. Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters. Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment. Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters

Clonorchis sinensis excretory-secretory products regulate migration and invasion in cholangiocarcinoma cells via extracellular signal-regulated kinase 1/2/nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

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Pak, Jhang Ho; Shin, Jimin; Song, In-Sung; Shim, Sungbo; Jang, Sung-Wuk

2017-01-01

Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory-secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory-secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory-secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory-secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory-secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory-secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

FK506 Binding Protein Mediates Glioma Cell Growth and Sensitivity to Rapamycin Treatment by Regulating NF-κB Signaling Pathway

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Wei Jiang

2008-03-01

Full Text Available FK506 binding protein 5 (FKBP5 belongs to a family of immunophilins named for their ability to bind immunosuppressive drugs, also known as peptidyl-prolyl cis-trans isomerases, and also with chaperones to help protein folding. Using glioma cDNA microarray analysis, we found that FKBP5 was overexpressed in glioma tumors. This finding was further validated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. The roles of FKBP5 in glioma cells were then examined. We found that cell growth was suppressed after FKBP5 expression was inhibited by short interfering RNA transfection and enhanced by FKBP5 overexpression. Electrophoretic mobility shift assay showed that nuclear factor-kappa B (NF-κB and DNA binding was enhanced by FKBP5 overexpression. The expression level of I-kappa B alpha and phosphorylated NF-κB was regulated by the expression of FKBP5. These data suggest that FKBP5 is involved in NF-κB pathway activation in glioma cells. In addition, FKBP5 overexpression in rapamycin-sensitive U87 cells blocked the cells' response to rapamycin treatment, whereas rapamycin-resistant glioma cells, both PTEN-positive and -negative, were synergistically sensitive to rapamycin after FKBP5 was knocked down, suggesting that the FKBP5 regulates glioma cell response to rapamycin treatment. In conclusion, our study demonstrates that FKBP5 plays an important role in glioma growth and chemoresistance through regulating signal transduction of the NF-κB pathway.

Thrombin selectively engages LIM kinase 1 and slingshot-1L phosphatase to regulate NF-κB activation and endothelial cell inflammation.

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Leonard, Antony; Marando, Catherine; Rahman, Arshad; Fazal, Fabeha

2013-11-01

Endothelial cell (EC) inflammation is a central event in the pathogenesis of many pulmonary diseases such as acute lung injury and its more severe form acute respiratory distress syndrome. Alterations in actin cytoskeleton are shown to be crucial for NF-κB regulation and EC inflammation. Previously, we have described a role of actin binding protein cofilin in mediating cytoskeletal alterations essential for NF-κB activation and EC inflammation. The present study describes a dynamic mechanism in which LIM kinase 1 (LIMK1), a cofilin kinase, and slingshot-1Long (SSH-1L), a cofilin phosphatase, are engaged by procoagulant and proinflammatory mediator thrombin to regulate these responses. Our data show that knockdown of LIMK1 destabilizes whereas knockdown of SSH-1L stabilizes the actin filaments through modulation of cofilin phosphorylation; however, in either case thrombin-induced NF-κB activity and expression of its target genes (ICAM-1 and VCAM-1) is inhibited. Further mechanistic analyses reveal that knockdown of LIMK1 or SSH-1L each attenuates nuclear translocation and thereby DNA binding of RelA/p65. In addition, LIMK1 or SSH-1L depletion inhibited RelA/p65 phosphorylation at Ser(536), a critical event conferring transcriptional competency to the bound NF-κB. However, unlike SSH-1L, LIMK1 knockdown also impairs the release of RelA/p65 by blocking IKKβ-dependent phosphorylation/degradation of IκBα. Interestingly, LIMK1 or SSH-1L depletion failed to inhibit TNF-α-induced RelA/p65 nuclear translocation and proinflammatory gene expression. Thus this study provides evidence for a novel role of LIMK1 and SSH-1L in selectively regulating EC inflammation associated with intravascular coagulation.

Eph receptor interclass cooperation is required for the regulation of cell proliferation

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Jurek, Aleksandra; Genander, Maria [Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Kundu, Parag [Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Lee Kong Chian School of Medicine, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Catchpole, Timothy [Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas TX 75390 (United States); He, Xiao; Strååt, Klas; Sabelström, Hanna [Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Xu, Nan-Jie [Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas TX 75390 (United States); Pettersson, Sven [Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Lee Kong Chian School of Medicine, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); The National Cancer Centre, Singapore General Hospital (Singapore); Henkemeyer, Mark [Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas TX 75390 (United States); Frisén, Jonas, E-mail: jonas.frisen@ki.se [Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm (Sweden)

2016-10-15

Cancer often arises by the constitutive activation of mitogenic pathways by mutations in stem cells. Eph receptors are unusual in that although they regulate the proliferation of stem/progenitor cells in many adult organs, they typically fail to transform cells. Multiple ephrins and Eph receptors are often co-expressed and are thought to be redundant, but we here describe an unexpected dichotomy with two homologous ligands, ephrin-B1 and ephrin-B2, regulating specifically migration or proliferation in the intestinal stem cell niche. We demonstrate that the combined activity of two different coexpressed Eph receptors of the A and B class assembled into common signaling clusters in response to ephrin-B2 is required for mitogenic signaling. The requirement of two different Eph receptors to convey mitogenic signals identifies a new type of cooperation within this receptor family and helps explain why constitutive activation of a single receptor fails to transform cells. - Highlights: • We demonstrate that ephrin-B1 and ephrin-B2 have largely non-overlapping functions in the intestinal stem cell niche. • Ephrin-B1 regulates cell positioning and ephrin-B2 regulates cell proliferation in the intestinal stem cell niche. • EphA4/B2 receptor cooperation in response to ephrin-B2 binding is obligatory to convey mitogenic signals in the intestine. • EphA4 facilitates EphB2 phosphorylation in response to ephrin-B2 in SW480 adenocarcinoma cells. • Ephrin-B1 and ephrin-B2 induce phosphorylation and degradation of the EphB2 receptor with different kinetics.

Eph receptor interclass cooperation is required for the regulation of cell proliferation

International Nuclear Information System (INIS)

Jurek, Aleksandra; Genander, Maria; Kundu, Parag; Catchpole, Timothy; He, Xiao; Strååt, Klas; Sabelström, Hanna; Xu, Nan-Jie; Pettersson, Sven; Henkemeyer, Mark; Frisén, Jonas

2016-01-01

Cancer often arises by the constitutive activation of mitogenic pathways by mutations in stem cells. Eph receptors are unusual in that although they regulate the proliferation of stem/progenitor cells in many adult organs, they typically fail to transform cells. Multiple ephrins and Eph receptors are often co-expressed and are thought to be redundant, but we here describe an unexpected dichotomy with two homologous ligands, ephrin-B1 and ephrin-B2, regulating specifically migration or proliferation in the intestinal stem cell niche. We demonstrate that the combined activity of two different coexpressed Eph receptors of the A and B class assembled into common signaling clusters in response to ephrin-B2 is required for mitogenic signaling. The requirement of two different Eph receptors to convey mitogenic signals identifies a new type of cooperation within this receptor family and helps explain why constitutive activation of a single receptor fails to transform cells. - Highlights: • We demonstrate that ephrin-B1 and ephrin-B2 have largely non-overlapping functions in the intestinal stem cell niche. • Ephrin-B1 regulates cell positioning and ephrin-B2 regulates cell proliferation in the intestinal stem cell niche. • EphA4/B2 receptor cooperation in response to ephrin-B2 binding is obligatory to convey mitogenic signals in the intestine. • EphA4 facilitates EphB2 phosphorylation in response to ephrin-B2 in SW480 adenocarcinoma cells. • Ephrin-B1 and ephrin-B2 induce phosphorylation and degradation of the EphB2 receptor with different kinetics.

Clusterin silencing sensitizes pancreatic cancer MIA-PaCa-2 cells to gmcitabine via regulation of NF-kB/Bcl-2 signaling.

Science.gov (United States)

Xu, Miao; Chen, Xiumei; Han, Yanling; Ma, Chunqing; Ma, Lin; Li, Shirong

2015-01-01

Clusterin (CLU) is known as a multifunctional protein involved in a variety of physiological processes including lipid transport, epithelial cell differentiation, tumorigenesis, and apoptosis. Our recent study has demonstrated that knockdown of clusterin sensitizes pancreatic cancer cell lines to gmcitabine treatment. However the details of this survival mechanism remain undefined. Of the various downstream targets of CLU, we examined activation of the NF-kB transcription factor and subsequent transcriptional regulation of BCL-2 gene in pancreatic cancer cell MIA-PaCa-2. The MIA-PaCa-2 cells were transfected with an antisense oligonucleotide (ASO) against clusterin, which led to a decreased protein level of the antiapoptotic gene BCL-2. Furthermore, inhibition of CLU decreased the function of NF-kB, which is capable of transcriptional regulation of the BCL-2 gene. Inhibiting this pathway increased the apoptotic effect of gmcitabine chemotherapy. Re-activated NF-kB resulted in attenuation of ASO-induced effects, followed by the bcl-2 upregulation, and bcl-2 re-inhibition resulted in attenuation of Re-activated NF-kB -induced effects. Animals injected with ASO CLU in MIA-PaCa-2 cells combined with gmcitabine treatment had fewer tumors than gmcitabine or ASO CLU alone. These findings suggest that knockdown of CLU sensitized MIA-PaCa-2 cells to gmcitabine chemotherapy through modulating NF-Kb/bcl-2 pathway.

Src inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity in Ramos Burkitt's lymphoma B cell line

International Nuclear Information System (INIS)

Hristov, K.; Mitev, V.; Knox, K.

2006-01-01

Reversible tyrosine phosphorylation, regulation of expression and proteolytic cleavage control tyrosine phosphatase contribution for the signalling pathways of B-cell antigen receptor (BCR), and CD40 during B cell selection. We used Ramos-BL B cell line to determine whether BCR and CD40 stimulation, or inhibition of the Src - tyrosine kinase, tyrosine phosphatase and caspase activity have an effect on the tyrosine phosphatase activities determined on in-gel phosphatase assay. The tyrosine phosphatase activities present in whole cell lysates of Ramos-BL B cells following treatment with 20 μg/ml anti-IgM, 1 μg/ml anti-CD40, 10 μM herbimycin A, 178 μM vanadate,100 μM phenylarsine oxide and 10 μM zVAD-fmk were detected with an in-gel phosphatase assay. Seven major tyrosine phosphatase activities with approximate molecular weight of 132.7, 63.9, 60.3, 54.2, 49.7, 44.6, and 39 kDa are present in whole cell lysates of Ramos-BL B cells. Treatment with Src-PTK inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity. We conclude that the catalytic activity of Src-PTK in Ramos-BL B cells is critical for the presence of this 132.7 kDa tyrosine phosphatase activity. (authors)

Bruton's tyrosine kinase mediates the synergistic signalling between TLR9 and the B cell receptor by regulating calcium and calmodulin.

Directory of Open Access Journals (Sweden)

Elaine F Kenny

Full Text Available B cells signal through both the B cell receptor (BCR which binds antigens and Toll-like receptors (TLRs including TLR9 which recognises CpG DNA. Activation of TLR9 synergises with BCR signalling when the BCR and TLR9 co-localise within an auto-phagosome-like compartment. Here we report that Bruton's tyrosine kinase (BTK is required for synergistic IL6 production and up-regulation of surface expression of MHC-class-II, CD69 and CD86 in primary murine and human B cells. We show that BTK is essential for co-localisation of the BCR and TLR9 within a potential auto-phagosome-like compartment in the Namalwa human B cell line. Downstream of BTK we find that calcium acting via calmodulin is required for this process. These data provide new insights into the role of BTK, an important target for autoimmune diseases, in B cell activation.

Morusin induces apoptosis and suppresses NF-κB activity in human colorectal cancer HT-29 cells

International Nuclear Information System (INIS)

Lee, J.-C.; Won, S.-J.; Chao, C.-L.; Wu, F.-L.; Liu, H.-S.; Ling Pin; Lin, C.-N.; Su, C.-L.

2008-01-01

Morusin is a pure compound isolated from root bark of Morusaustralis (Moraceae). In this study, we demonstrated that morusin significantly inhibited the growth and clonogenicity of human colorectal cancer HT-29 cells. Apoptosis induced by morusin was characterized by accumulation of cells at the sub-G 1 phase, fragmentation of DNA, and condensation of chromatin. Morusin also inhibited the phosphorylation of IKK-α, IKK-β and IκB-α, increased expression of IκB-α, and suppressed nuclear translocation of NF-κB and its DNA binding activity. Dephosphorylation of NF-κB upstream regulators PI3K, Akt and PDK1 was also displayed. In addition, activation of caspase-8, change of mitochondrial membrane potential, release of cytochrome c and Smac/DIABLO, and activation of caspase-9 and -3 were observed at the early time point. Downregulation in the expression of Ku70 and XIAP was exhibited afterward. Caspase-8 or wide-ranging caspase inhibitor suppressed morusin-induced apoptosis. Therefore, the antitumor mechanism of morusin in HT-29 cells may be via activation of caspases and inhibition of NF-κB

Chronic lymphocytic leukemia cells acquire regulatory B-cell properties in response to TLR9 and CD40 activation.

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Ringelstein-Harlev, Shimrit; Avivi, Irit; Fanadka, Mona; Horowitz, Netanel A; Katz, Tami

2018-02-15

Circulating chronic lymphocytic leukemia (CLL) cells share phenotypic features with certain subsets of regulatory B-cells (Bregs). The latter cells have been reported to negatively regulate immune cell responses, mostly by provision of IL-10. The purpose of the current study was to identify and delineate Breg properties of CLL cells. B-cells and T-cells were obtained from the peripheral blood of untreated CLL patients diagnosed according to the 2008 Guidelines of the International Workshop on Chronic Lymphocytic Leukemia. Co-culture assays were used to examine the ability of CLL cells to suppress autologous T-cell immune responses. IL-10 potency of CLL cells was assessed following stimulation with activators of the toll-like receptor 9 (TLR9) or CD40 and was correlated with the inhibitory activity of the cells. TLR9-activated CLL cells were found to increase the frequency of CD4 + CD25 hi FOXp3 + regulatory T-cells (Tregs) and to inhibit autologous CD4 + T-cell proliferation. This signaling cascade proved to control IL-10 generation in CLL cells, which in turn promoted the inhibition of T-cell proliferation by CLL cells. However, CD40 activation of CLL cells, while exhibiting a similar ability to augment Treg frequency, did not either affect IL-10 generation or T-cell proliferation. In conclusion, CLL cells demonstrate a unique clonal quality of adopting Breg properties which promote modulation of T-cell characteristics. TLR9 appears to be a potent activator of regulatory abilities in CLL cells, possibly contributing to preferential immune escape of TLR9-responsive cells.

MicroRNA-187, down-regulated in clear cell renal cell carcinoma and associated with lower survival, inhibits cell growth and migration though targeting B7-H3

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Zhao, Jun [Foshan Maternal and Child Health Care Hospital, Foshan (China); Lei, Ting [Zhongshan People’s Hospital, Zhongshan (China); Xu, Congjie [Department of Urology, Pepole’s Hospital of Hainan Province, Haikou (China); Li, Huan; Ma, Wenmin; Yang, Yunxia; Fan, Shuming [Foshan Maternal and Child Health Care Hospital, Foshan (China); Liu, Yuchen, E-mail: s_ycliu1@stu.edu.cn [Anhui Medical University, Hefei (China)

2013-08-23

Highlights: •miR-187 is down-regulated in clear cell renal cell carcinoma (ccRCC). •Down-regulation of miR-187 is associated with poor outcomes in patients with ccRCC. •miR-187 inhibits cell growth and migration though targeting B7-H3 in ccRCC. -- Abstract: Aberrantly expressed microRNAs (miRNAs) are frequently associated with the aggressive malignant behavior of human cancers, including clear cell renal cell carcinoma (ccRCC). Based on the preliminary deep sequencing data, we hypothesized that miR-187 may play an important role in ccRCC development. In this study, we found that miR-187 was down-regulated in both tumor tissue and plasma of ccRCC patients. Lower miR-187 expression levels were associated with higher tumor grade and stage. All patients with high miR-187 expression survived 5 years, while with low miR-187 expression, only 42% survived. Suppressed in vitro proliferation, inhibited in vivo tumor growth, and decreased motility were observed in cells treated with the miR-187 expression vector. Further studies showed that B7 homolog 3 (B7-H3) is a direct target of miR-187. Over-expression of miR-187 decreased B7-H3 mRNA level and repressed B7-H3-3′-UTR reporter activity. Knockdown of B7-H3 using siRNA resulted in similar phenotype changes as that observed for overexpression of miR-187. Our data suggest that miR-187 is emerging as a novel player in the disease state of ccRCC. miR-187 plays a tumor suppressor role in ccRCC.

MicroRNA-187, down-regulated in clear cell renal cell carcinoma and associated with lower survival, inhibits cell growth and migration though targeting B7-H3

International Nuclear Information System (INIS)

Zhao, Jun; Lei, Ting; Xu, Congjie; Li, Huan; Ma, Wenmin; Yang, Yunxia; Fan, Shuming; Liu, Yuchen

2013-01-01

Highlights: •miR-187 is down-regulated in clear cell renal cell carcinoma (ccRCC). •Down-regulation of miR-187 is associated with poor outcomes in patients with ccRCC. •miR-187 inhibits cell growth and migration though targeting B7-H3 in ccRCC. -- Abstract: Aberrantly expressed microRNAs (miRNAs) are frequently associated with the aggressive malignant behavior of human cancers, including clear cell renal cell carcinoma (ccRCC). Based on the preliminary deep sequencing data, we hypothesized that miR-187 may play an important role in ccRCC development. In this study, we found that miR-187 was down-regulated in both tumor tissue and plasma of ccRCC patients. Lower miR-187 expression levels were associated with higher tumor grade and stage. All patients with high miR-187 expression survived 5 years, while with low miR-187 expression, only 42% survived. Suppressed in vitro proliferation, inhibited in vivo tumor growth, and decreased motility were observed in cells treated with the miR-187 expression vector. Further studies showed that B7 homolog 3 (B7-H3) is a direct target of miR-187. Over-expression of miR-187 decreased B7-H3 mRNA level and repressed B7-H3-3′-UTR reporter activity. Knockdown of B7-H3 using siRNA resulted in similar phenotype changes as that observed for overexpression of miR-187. Our data suggest that miR-187 is emerging as a novel player in the disease state of ccRCC. miR-187 plays a tumor suppressor role in ccRCC

CD11b regulates antibody class switching via induction of AID.

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Park, Seohyun; Sim, Hyunsub; Kim, Hye-In; Jeong, Daecheol; Wu, Guang; Cho, Soo Young; Lee, Young Seek; Kwon, Hyung-Joo; Lee, Keunwook

2017-07-01

The integrin CD11b, which is encoded by the integrin subunit alpha M (ITGAM), is primarily expressed on the surface of innate immune cells. Genetic variations in ITGAM are among the strongest risk factors for systemic lupus erythematosus, an autoimmune disease characterized by the presence of autoantibodies. However, the regulatory function of CD11b in the antibody responses remains unclear. Here, we report the induction of CD11b in activated B2 B cells and define its unexpected role in immunoglobulin heavy chain class switch recombination (CSR). LPS-activated B cells lacking CD11b yielded fewer IgG subtypes such as IgG1 and IgG2a in vitro, and immunization-dependent CSR and affinity maturation of antibodies were severely impaired in CD11b-deficient mice. Notably, we observed the reduced expression of activation-induced cytidine deaminase (AID), an enzyme that initiates CSR and somatic hypermutation, and ectopic expression of AID was sufficient to rescue the defective CSR of CD11b-deficient B cells. LPS-induced phosphorylation of NF-κB p65 and IκBα was attenuated in CD11b-deficient B cells, and hyperactivation of IκB kinase 2 restored the defective AID expression and CSR, which implied that CD11b regulates the NF-κB-dependent induction of AID. Overall, our experimental evidence emphasized the function of CD11b in antibody responses and the role of CD11b as a vital regulator of CSR. Copyright © 2017 Elsevier Ltd. All rights reserved.

BRD4 regulates cellular senescence in gastric cancer cells via E2F/miR-106b/p21 axis.

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Dong, Xingchen; Hu, Xiangming; Chen, Jinjing; Hu, Dan; Chen, Lin-Feng

2018-02-12

Small molecules targeting bromodomains of BET proteins possess strong anti-tumor activities and have emerged as potential therapeutics for cancer. However, the underlying mechanisms for the anti-proliferative activity of these inhibitors are still not fully characterized. In this study, we demonstrated that BET inhibitor JQ1 suppressed the proliferation and invasiveness of gastric cancer cells by inducing cellular senescence. Depletion of BRD4, which was overexpressed in gastric cancer tissues, but not other BET proteins recapitulated JQ1-induced cellular senescence with increased cellular SA-β-Gal activity and elevated p21 levels. In addition, we showed that the levels of p21 were regulated at the post-transcriptional level by BRD4-dependent expression of miR-106b-5p, which targets the 3'-UTR of p21 mRNA. Overexpression of miR-106b-5p prevented JQ1-induced p21 expression and BRD4 inhibition-associated cellular senescence, whereas miR-106b-5p inhibitor up-regulated p21 and induced cellular senescence. Finally, we demonstrated that inhibition of E2F suppressed the binding of BRD4 to the promoter of miR-106b-5p and inhibited its transcription, leading to the increased p21 levels and cellular senescence in gastric cancer cells. Our results reveal a novel mechanism by which BRD4 regulates cancer cell proliferation by modulating the cellular senescence through E2F/miR-106b-5p/p21 axis and provide new insights into using BET inhibitors as potential anticancer drugs.

[6]-Gingerol Prevents Disassembly of Cell Junctions and Activities of MMPs in Invasive Human Pancreas Cancer Cells through ERK/NF-κB/Snail Signal Transduction Pathway

Directory of Open Access Journals (Sweden)

Sung Ok Kim

2013-01-01

Full Text Available To study the effects of [6]-gingerol, a ginger phytochemical, on tight junction (TJ molecules, we investigated TJ tightening and signal transduction pathways in human pancreatic duct cell-derived cancer cell line PANC-1. The following methods were utilized: MTT assay to determine cytotoxicity; zymography to examine matrix metalloproteinase (MMP activities; transepithelial electrical resistance (TER and paracellular flux for TJ measurement; RT-PCR and immunoblotting for proteins related to TJ and invasion; and EMSA for NF-κB activity in PANC-1 cells. Results revealed that TER significantly increased and claudin 4 and MMP-9 decreased compared to those of the control. TJ protein levels, including zonula occludens (ZO- 1, occludin, and E-cadherin, increased in [6]-gingerol-treated cells, which correlated with a decrease in paracellular flux and MMP activity. Furthermore, NF-κB/Snail nuclear translocation was suppressed via downregulation of the extracellular signal-regulated kinase (ERK pathway in response to [6]-gingerol treatment. Moreover, treatment with U0126, an ERK inhibitor, completely blocked NF-κB activity. In conclusion, these findings demonstrate that [6]-gingerol regulates TJ-related proteins and suppresses invasion and metastasis through NF-κB/Snail inhibition via inhibition of the ERK pathway. Therefore, [6]-gingerol may suppress the invasive activity of PANC-1 cells.

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.

miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis in osteosarcoma cells

Energy Technology Data Exchange (ETDEWEB)

Li, Zhi [Department of Human Anatomy and Histoembryology, College of Basic Medical Sciences, Jilin University (China); Li, Youjun, E-mail: liyoujunn@126.com [Department of Human Anatomy and Histoembryology, College of Basic Medical Sciences, Jilin University (China); Wang, Nan; Yang, Lifeng; Zhao, Wei; Zeng, Xiandong [Central Hospital Affiliated to Shenyang Medical College (China)

2016-03-18

miR-130b was significantly up-regulated in osteosarcoma (OS) cells. Naked cuticle homolog 2 (NKD2) inhibited tumor growth and metastasis in OS by suppressing Wnt signaling. We used three miRNA target analysis tools to identify potential targets of miR-130b, and found that NKD2 is a potential target of miR-130b. Based on these findings, we hypothesize that miR-130b might target NKD2 and regulate the Wnt signaling to promote OS growth. We detected the expression of miR-130b and NKD2 mRNA and protein by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found up-regulation of miR-130b and down-regulation of NKD2 mRNA and protein exist in OS cell lines. MTT and flow cytometry assays showed that miR-130b inhibitors inhibit proliferation and promote apoptosis in OS cells. Furthermore, we showed that NKD2 is a direct target of miR-130b, and miR-130b regulated proliferation and apoptosis of OS cells by targeting NKD2. We further investigated whether miR-130b and NKD2 regulate OS cell proliferation and apoptosis by inhibiting Wnt signaling, and the results confirmed our speculation that miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis of OS cells. These findings will offer new clues for OS development and progression, and novel potential therapeutic targets for OS. - Highlights: • miR-130b is up-regulated and NKD2 is down-regulated in osteosarcoma cell lines. • Down-regulation of miR-130b inhibits proliferation of osteosarcoma cells. • Down-regulation of miR-130b promotes apoptosis of osteosarcoma cells. • miR-130b directly targets NKD2. • NKD2 regulates OS cell proliferation and apoptosis by inhibiting the Wnt signaling.

miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis in osteosarcoma cells

International Nuclear Information System (INIS)

Li, Zhi; Li, Youjun; Wang, Nan; Yang, Lifeng; Zhao, Wei; Zeng, Xiandong

2016-01-01

miR-130b was significantly up-regulated in osteosarcoma (OS) cells. Naked cuticle homolog 2 (NKD2) inhibited tumor growth and metastasis in OS by suppressing Wnt signaling. We used three miRNA target analysis tools to identify potential targets of miR-130b, and found that NKD2 is a potential target of miR-130b. Based on these findings, we hypothesize that miR-130b might target NKD2 and regulate the Wnt signaling to promote OS growth. We detected the expression of miR-130b and NKD2 mRNA and protein by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found up-regulation of miR-130b and down-regulation of NKD2 mRNA and protein exist in OS cell lines. MTT and flow cytometry assays showed that miR-130b inhibitors inhibit proliferation and promote apoptosis in OS cells. Furthermore, we showed that NKD2 is a direct target of miR-130b, and miR-130b regulated proliferation and apoptosis of OS cells by targeting NKD2. We further investigated whether miR-130b and NKD2 regulate OS cell proliferation and apoptosis by inhibiting Wnt signaling, and the results confirmed our speculation that miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis of OS cells. These findings will offer new clues for OS development and progression, and novel potential therapeutic targets for OS. - Highlights: • miR-130b is up-regulated and NKD2 is down-regulated in osteosarcoma cell lines. • Down-regulation of miR-130b inhibits proliferation of osteosarcoma cells. • Down-regulation of miR-130b promotes apoptosis of osteosarcoma cells. • miR-130b directly targets NKD2. • NKD2 regulates OS cell proliferation and apoptosis by inhibiting the Wnt signaling.

Overexpression of Fc receptor-like 1 associated with B-cell activation during hepatitis B virus infection

Energy Technology Data Exchange (ETDEWEB)

Wang, Ke [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu Province (China); Pei, Hao [Wuxi Hospital of Infectious Disease, Wuxi, Jiangsu Province (China); Huang, Biao; Yang, Run-Lin [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu Province (China); Wu, Hang-Yuan [Wuxi Hospital of Infectious Disease, Wuxi, Jiangsu Province (China); Zhu, Xue; Zhu, Lan [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu Province (China)

2012-08-17

The role of B cells in the pathogenesis of hepatitis B virus (HBV) infection has not been explored in depth. In the present study, the activation status of B cells from peripheral blood of healthy controls (N = 20) and patients with acute hepatitis B (AHB, N = 15) or chronic hepatitis B (CHB, N = 30) was evaluated by measuring the expression levels of B-cell activation markers CD69 and CD86, using quantitative real-time PCR and flow cytometry. Moreover, the potential mechanism underlying B-cell activation during HBV infection was further investigated by analyzing the expression profile of FCRL1, an intrinsic activation molecule of B cells. An elevation in the levels of B-cell activation markers including CD69 and CD86 was observed in the AHB patients (44.31 ± 9.27, 27.64 ± 9.26%) compared to CHB patients (30.35 ± 11.27, 18.41 ± 6.56%, P < 0.05), which was still higher than healthy controls (12.23 ± 7.84, 8.22 ± 3.43%, P < 0.05). Furthermore, the expression of FCRL1 was found to be similar to B-cell activation markers, which was highest in AHB patients (70.15 ± 17.11%), lowest in healthy donors (36.32 ± 9.98%, P < 0.05) and half-way between these levels in patients with CHB (55.17 ± 12.03%, P < 0.05). The results were positively associated with aberrant B-cell activation. These data suggest that B cells can play a role in HBV infection, and therefore more effort should be devoted to exploring their functions.

HT-29 and Caco-2 Reporter Cell Lines for Functional Studies of Nuclear Factor Kappa B Activation

Directory of Open Access Journals (Sweden)

Giuliana Mastropietro

2015-01-01

Full Text Available The NF-κB is a transcription factor which plays a key role in regulating biological processes. In response to signals, NF-κB activation occurs via phosphorylation of its inhibitor, which dissociates from the NF-κB dimer allowing the translocation to the nucleus, inducing gene expression. NF-κB activation has direct screening applications for drug discovery for several therapeutic indications. Thus, pathway-specific reporter cell systems appear as useful tools to screen and unravel the mode of action of probiotics and natural and synthetic compounds. Here, we describe the generation, characterization, and validation of human epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory agents. Caco-2 and HT-29 cells were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. Three proinflammatory cytokines (TNF-α, IL-1β, and LPS were able to activate the reporter systems in a dose-response manner, which corresponds to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using lactic acid bacteria and a natural compound. We have established robust Caco-2-NF-κB-hrGFP and HT-29-NF-κB-hrGFP reporter cell lines which represent a valuable tool for primary screening and identification of bacterial strains and compounds with a potential therapeutic interest.

MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

Energy Technology Data Exchange (ETDEWEB)

Zhang, Yifei; Ghazwani, Mohammed; Li, Jiang [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Sun, Ming; Stolz, Donna B. [Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261 (United States); He, Fengtian [Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038 (China); Fan, Jie [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Xie, Wen [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Li, Song, E-mail: sol4@pitt.edu [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States)

2014-04-18

Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl{sub 4}-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.

MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

International Nuclear Information System (INIS)

Zhang, Yifei; Ghazwani, Mohammed; Li, Jiang; Sun, Ming; Stolz, Donna B.; He, Fengtian; Fan, Jie; Xie, Wen; Li, Song

2014-01-01

Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl 4 -treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation

The Hepatitis B Virus (HBV) HBx Protein Activates AKT To Simultaneously Regulate HBV Replication and Hepatocyte Survival

Science.gov (United States)

Rawat, Siddhartha

2014-01-01

ABSTRACT Chronic infection with hepatitis B virus (HBV) is a risk factor for developing liver diseases such as hepatocellular carcinoma (HCC). HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary hepatocytes and determined how this HBx activity affects HBV replication. We report that HBx activates AKT in primary hepatocytes and that the activation of AKT decreases HBV replication and HBV mRNA and core protein levels. We show that the transcription factor hepatocyte nuclear factor 4α (HNF4α) is a target of HBx-regulated AKT, and we link HNF4α to HBx-regulated AKT modulation of HBV transcription and replication. Although we and others have shown that HBx stimulates and is likely required for HBV replication, we now report that HBx also activates signals that can diminish the overall level of HBV replication. While this may seem counterintuitive, we show that an important effect of HBx activation of AKT is inhibition of apoptosis. Consequently, our studies suggest that HBx balances HBV replication and cell survival by stimulating signaling pathways that enhance hepatocyte survival at the expense of higher levels of HBV replication. IMPORTANCE Chronic hepatitis B virus (HBV) infection is a common cause of the development of liver cancer. Regulation of cell signaling pathways by the HBV HBx protein is thought to influence the development of HBV-associated liver cancer. HBx stimulates, and may be essential for, HBV replication. We show that HBx activates AKT in hepatocytes to reduce HBV replication. While this seems contradictory to an

Cloning of B cell-specific membrane tetraspanning molecule BTS possessing B cell proliferation-inhibitory function.

Science.gov (United States)

Suenaga, Tadahiro; Arase, Hisashi; Yamasaki, Sho; Kohno, Masayuki; Yokosuka, Tadashi; Takeuchi, Arata; Hattori, Takamichi; Saito, Takashi

2007-11-01

Lymphocyte proliferation is regulated by signals through antigen receptors, co-stimulatory receptors, and other positive and negative modulators. Several membrane tetraspanning molecules are also involved in the regulation of lymphocyte growth and death. We cloned a new B cell-specific tetraspanning (BTS) membrane molecule, which is similar to CD20 in terms of expression, structure and function. BTS is specifically expressed in the B cell line and its expression is increased after the pre-B cell stage. BTS is expressed in intracellular granules and on the cell surface. Overexpression of BTS in immature B cell lines induces growth retardation through inhibition of cell cycle progression and cell size increase without inducing apoptosis. This inhibitory function is mediated predominantly by the N terminus of BTS. The development of mature B cells is inhibited in transgenic mice expressing BTS, suggesting that BTS is involved in the in vivo regulation of B cells. These results indicate that BTS plays a role in the regulation of cell division and B cell growth.

Unirradiated cells rescue cells exposed to ionizing radiation: Activation of NF-κB pathway in irradiated cells

Energy Technology Data Exchange (ETDEWEB)

Lam, R.K.K. [Department of Physics and Materials Science, City University of Hong Kong (Hong Kong); Han, Wei [Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031 (China); Yu, K.N., E-mail: peter.yu@cityu.edu.hk [Department of Physics and Materials Science, City University of Hong Kong (Hong Kong); State Key Laboratory in Marine Pollution, City University of Hong Kong (Hong Kong)

2015-12-15

Highlights: • Rescue effect was observed in both irradiated and HeLa and NIH/3T3 cells. • Novel setup and procedures to separate the rescue signals and the bystander signals. • Confirmed activation of NF-κB pathway in rescue effect using activation inhibitor. • Confirmed activation of NF-κB pathway in rescue effect using anti-NF-κB p65 antibody. - Abstract: We studied the involvement of NF-κB pathway activation in the rescue effect in HeLa and NIH/3T3 cells irradiated by α particles. Firstly, upon irradiation by 5 cGy of α particles, for both cell lines, the numbers of 53BP1 foci/cell at 12 h post-irradiation were significantly smaller when only 2.5% of the cell population was irradiated as compared to 100% irradiation, which demonstrated the rescue effect. Secondly, we studied the effect of NF-κB on the rescue effect through the use of the NF-κB activation inhibitor BAY-11-7082. Novel experimental setup and procedures were designed to prepare the medium (CM) which had conditioned the bystander cells previously partnered with irradiated cells, to ensure physical separation between rescue and bystander signals. BAY-11-7082 itself did not inflict DNA damages in the cells or have effects on activation of the NF-κB response pathway in the irradiated cells through direct irradiation. The rescue effect was induced in both cell lines by the CM, which was abrogated if BAY-11-7082 was added to the CM. Thirdly, we studied the effect of NF-κB on the rescue effect through staining for phosphorylated NF-κB (p-NF-κB) expression using the anti-NF-κB p65 (phospho S536) antibody. When the fraction of irradiated cells dropped from 100% to 2.5%, the p-NF-κB expression in the cell nuclei of irradiated NIH/3T3 cells increased significantly, while that in the cell nuclei of irradiated HeLa cells also increased although not significantly. Moreover, the p-NF-κB expression in the cell nuclei of irradiated HeLa cells and NIH/3T3 cells treated with CM also increased

PSGL-1–mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

Science.gov (United States)

Foubert, Philippe; Silvestre, Jean-Sébastien; Souttou, Boussad; Barateau, Véronique; Martin, Coralie; Ebrahimian, Téni G.; Leré-Déan, Carole; Contreres, Jean Olivier; Sulpice, Eric; Levy, Bernard I.; Plouët, Jean; Tobelem, Gérard; Le Ricousse-Roussanne, Sophie

2007-01-01

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2–Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2–Fc–stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy. PMID:17510705

Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling*♦

Science.gov (United States)

Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu.; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I.; Nienhaus, G. Ulrich; Gierschik, Peter

2015-01-01

The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca2+. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca2+ and regulation of Ca2+-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca2+ release from intracellular stores; (iii) Ca2+ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca2+-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca2+ signaling. PMID:25903139

ErbB2-Driven Breast Cancer Cell Invasion Depends on a Complex Signaling Network Activating Myeloid Zinc Finger-1-Dependent Cathepsin B Expression

DEFF Research Database (Denmark)

Rafn, Bo; Nielsen, Christian Thomas Friberg; Andersen, Sofie Hagel

2012-01-01

Aberrant ErbB2 receptor tyrosine kinase activation in breast cancer is strongly linked to an invasive disease. The molecular basis of ErbB2-driven invasion is largely unknown. We show that cysteine cathepsins B and L are elevated in ErbB2 positive primary human breast cancer and function...... as effectors of ErbB2-induced invasion in vitro. We identify Cdc42-binding protein kinase beta, extracellular regulated kinase 2, p21-activated protein kinase 4, and protein kinase C alpha as essential mediators of ErbB2-induced cysteine cathepsin expression and breast cancer cell invasiveness. The identified...

1,25-dihydroxyvitamin D{sub 3} impairs NF-{kappa}B activation in human naive B cells

Energy Technology Data Exchange (ETDEWEB)

Geldmeyer-Hilt, Kerstin, E-mail: kerstin.hilt@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany); Heine, Guido, E-mail: guido.heine@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany); Deutsches Rheuma-Forschungszentrum Berlin, Chariteplatz 1, 10117 Berlin (Germany); Hartmann, Bjoern, E-mail: bjoern.hartmann@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany); Baumgrass, Ria, E-mail: baumgrass@drfz.de [Deutsches Rheuma-Forschungszentrum Berlin, Chariteplatz 1, 10117 Berlin (Germany); Radbruch, Andreas, E-mail: radbruch@drfz.de [Deutsches Rheuma-Forschungszentrum Berlin, Chariteplatz 1, 10117 Berlin (Germany); Worm, Margitta, E-mail: margitta.worm@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany)

2011-04-22

Highlights: {yields} In naive B cells, VDR activation by calcitriol results in reduced NF-{kappa}B p105 and p50 protein expression. {yields} Ligating the VDR with calcitriol causes reduced nuclear translocation of NF-{kappa}B p65. {yields} Reduced nuclear amount of p65 after calcitriol incubation results in reduced binding of p65 on the p105 promoter. {yields} Thus, vitamin D receptor signaling may reduce or prevent activation of B cells and unwanted immune responses, e.g. in IgE dependent diseases such as allergic asthma. -- Abstract: 1{alpha},25-dihydroxyvitamin D{sub 3} (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-{kappa}B p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-{kappa}B mediated activation of human naive B cells. Naive B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human naive B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-{kappa}B activation by interference with NF-{kappa}B p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in naive B cells, namely by reducing CD40 signaling.

Regulation of hepatitis B virus ENI enhancer activity by hepatocyte-enriched transcription factor HNF3.

Science.gov (United States)

Chen, M; Hieng, S; Qian, X; Costa, R; Ou, J H

1994-11-15

Hepatitis B virus (HBV) ENI enhancer can activate the expression of HBV and non-HBV genes in a liver-specific manner. By performing the electrophoretic mobility-shift assays, we demonstrated that the three related, liver-enriched, transcription factors, HNF3 alpha, HNF3 beta, and HNF3 gamma could all bind to the 2c site of HBV ENI enhancer. Mutations introduced in the 2c site to abolish the binding by HNF3 reduced the enhancer activity approximately 15-fold. Moreover, expression of HNF3 antisense sequences to suppress the expression of HNF3 in Huh-7 hepatoma cells led to reduction of the ENI enhancer activity. These results indicate that HNF3 positively regulates the ENI enhancer activity and this regulation is most likely mediated through the 2c site. The requirement of HNF3 for the ENI enhancer activity could explain the liver specificity of this enhancer element.

Activation of AMP-activated protein kinase by kainic acid mediates brain-derived neurotrophic factor expression through a NF-kappaB dependent mechanism in C6 glioma cells

International Nuclear Information System (INIS)

Yoon, Hana; Oh, Young Taek; Lee, Jung Yeon; Choi, Ji Hyun; Lee, Ju Hie; Baik, Hyung Hwan; Kim, Sung Soo; Choe, Wonchae; Yoon, Kyung-Sik; Ha, Joohun; Kang, Insug

2008-01-01

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. Kainic acid (KA), a prototype excitotoxin is known to induce brain-derived neurotrophic factor (BDNF) in brain. In this study, we examined the role of AMPK in KA-induced BDNF expression in C6 glioma cells. We showed that KA and KA receptor agonist induced activation of AMPK and KA-induced AMPK activation was blocked by inhibition of Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) β. We then showed that inhibition of AMPK by compound C, a selective inhibitor of AMPK, or small interfering RNA of AMPKα1 blocked KA-induced BDNF mRNA and protein expression. Inhibition of AMPK blocked KA-induced phosphorylation of CaMKII and I kappaB kinase (IKK) in C6 cells. Finally, we showed that inhibition of AMPK reduced DNA binding and transcriptional activation of nuclear factor-kappaB (NF-κB) in KA-treated cells. These results suggest that AMPK mediates KA-induced BDNF expression by regulating NF-κB activation

Tumor cell-released TLR4 ligands stimulate Gr-1+CD11b+F4/80+ cells to induce apoptosis of activated T cells.

Science.gov (United States)

Liu, Yan-Yan; Sun, Ling-Cong; Wei, Jing-Jing; Li, Dong; Yuan, Ye; Yan, Bin; Liang, Zhi-Hui; Zhu, Hui-Fen; Xu, Yong; Li, Bo; Song, Chuan-Wang; Liao, Sheng-Jun; Lei, Zhang; Zhang, Gui-Mei; Feng, Zuo-Hua

2010-09-01

Gr-1(+)CD11b(+)F4/80(+) cells play important roles in tumor development and have a negative effect on tumor immunotherapy. So far, the mechanisms underlying the regulation of their immunosuppressive phenotype by classical and alternative macrophage activation stimuli are not well elucidated. In this study, we found that molecules from necrotic tumor cells (NTC-Ms) stimulated Gr-1(+)CD11b(+)F4/80(+) cells to induce apoptosis of activated T cells but not nonstimulated T cells. The apoptosis-inducing capacity was determined by higher expression levels of arginase I and IL-10 relative to those of NO synthase 2 and IL-12 in Gr-1(+)CD11b(+)F4/80(+) cells, which were induced by NTC-Ms through TLR4 signaling. The apoptosis-inducing capacity of NTC-Ms-stimulated Gr-1(+)CD11b(+)F4/80(+) cells could be enhanced by IL-10. IFN-gamma may reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells only if their response to IFN-gamma was not attenuated. However, the potential of Gr-1(+)CD11b(+)F4/80(+) cells to express IL-12 in response to IFN-gamma could be attenuated by tumor, partially due to the existence of active STAT3 in Gr-1(+)CD11b(+)F4/80(+) cells and NTC-Ms from tumor. In this situation, IFN-gamma could not effectively reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells. Tumor immunotherapy with 4-1BBL/soluble programmed death-1 may significantly reduce, but not abolish the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells in local microenvironment. Blockade of TLR4 signaling could further reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and enhance the suppressive effect of 4-1BBL/soluble form of programmed death-1 on tumor growth. These findings indicate the relationship of distinct signaling pathways with apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and emphasize the importance of blocking TLR4 signaling to prevent the induction of T cell apoptosis by Gr-1(+)CD11b(+)F4/80(+) cells.

DHX15 is associated with poor prognosis in acute myeloid leukemia (AML) and regulates cell apoptosis via the NF-kB signaling pathway.

Science.gov (United States)

Pan, Lili; Li, Yang; Zhang, Hai-Ying; Zheng, Yi; Liu, Xiao-Li; Hu, Zheng; Wang, Yi; Wang, Jing; Cai, Yuan-Hua; Liu, Qiao; Chen, Wan-Ling; Guo, Ying; Huang, Yuan-Mao; Qian, Feng; Jin, Li; Wang, Jiucun; Wang, Shao-Yuan

2017-10-27

The role of DHX15 , a newly identified DEAH-box RNA helicase, in leukemogenesis remains elusive. Here, we identified a recurrent mutation in DHX15 (NM_001358:c.664C>G: p.(R222G)) in one familial AML patient and 4/240 sporadic AML patients. Additionally, DHX15 was commonly overexpressed in AML patients and associated with poor overall survival (OS) (P=0.019) and relapse-free survival (RFS) (P=0.032). In addition, we found a distinct expression pattern of DHX15 . DHX15 was highly expressed in hematopoietic stem cells and leukemia cells but was lowly expressed in mature blood cells. DHX15 was down-regulated when AML patients achieved disease remission or when leukemia cell lines were induced to differentiate. DHX15 silencing greatly inhibited leukemia cell proliferation and induced cell apoptosis and G1-phase arrest. In contrast, the restoration of DHX15 expression rescued cell viability and reduced cell apoptosis. In addition, we found that DHX15 was down-regulated when cell apoptosis was induced by ATO (arsenic trioxide); overexpression of DHX15 caused dramatic resistance to ATO-induced cell apoptosis, suggesting an important role for DHX15 in cell apoptosis. We further explored the mechanism of DHX15 in apoptosis and found that overexpression of DHX15 activated NF-kB transcription. Knockdown of DHX15 inhibited the nuclear translocation and activation of the NF-kB subunit P65 in leukemia cells. Several downstream targets of the NF-kB pathway were also down-regulated, and apoptosis-associated genes CASP3 and PARP were activated. In conclusion, this study represents the first demonstration that DHX15 plays an important role in leukemogenesis via the NF-kB signaling pathway and may serve as an independent prognostic marker for AML.

CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFκB signaling pathways.

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Wang, Chaoqun; Fok, Kin Lam; Cai, Zhiming; Chen, Hao; Chan, Hsiao Chang

2017-01-10

CD147 null mutant male mice are infertile with arrested spermatogenesis and increased apoptotic germ cells. Our previous studies have shown that CD147 prevents apoptosis in mouse spermatocytes but not spermatogonia. However, the underlying mechanism remains elusive. In the present study, we aim to determine the CD147-regulated apoptotic pathway in mouse spermatocytes. Our results showed that immunodepletion of CD147 triggered apoptosis through extrinsic apoptotic pathway in mouse testis and spermatocyte cell line (GC-2 cells), accompanied by activation of non-canonical NFκB signaling and suppression of canonical NFκB signaling. Furthermore, CD147 was found to interact with TRAF2, a factor known to regulate NFκB and extrinsic apoptotic signaling, and interfering CD147 led to the decrease of TRAF2. Consistently, depletion of CD147 by CRISPR/Cas9 technique in GC-2 cells down-regulated TRAF2 and resulted in cell death with suppressed canonical NFκB and activated non-canonical NFκB signaling. On the contrary, interfering of CD147 had no effect on NFκB signaling pathways as well as TRAF2 protein level in mouse spermatogonia cell line (GC-1 cells). Taken together, these results suggested that CD147 plays a key role in reducing extrinsic apoptosis in spermatocytes, but not spermatogonia, through modulating NFκB signaling pathway.

Bi-directional Activation between Mesenchymal Stem Cells and CLL B-Cells: Implication for CLL Disease Progression

OpenAIRE

Ding, Wei; Nowakowski, Grzegorz S.; Knox, Traci R.; Boysen, Justin C.; Maas, Mary L.; Schwager, Susan M.; Wu, Wenting; Wellik, Linda E.; Dietz, Allan B.; Ghosh, Asish K.; Secreto, Charla R.; Medina, Kay L.; Shanafelt, Tait D.; Zent, Clive S.; Call, Timothy G.

2009-01-01

It was hypothesized that contact between chronic lymphocytic leukemia (CLL) B-cells and marrow stromal cells impact both cell types. To test this hypothesis, we utilized a long-term primary culture system from bone biopsies that reliably generates a mesenchymal stem cell (MSC). Co-culture of MSC with CLL B-cells protected the latter from both spontaneous apoptosis and drug-induced apoptosis. The CD38 expression in previously CD38 positive CLL B-cells was up-regulated with MSC co-culture. Up-r...

TRAF Family Member-Associated NF-κB Activator (TANK) Induced by RANKL Negatively Regulates Osteoclasts Survival and Function

OpenAIRE

Mengrui Wu, Yiping Wang, Lianfu Deng, Wei Chen, Yi-Ping Li

2012-01-01

Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation...

TRAF Family Member-Associated NF-κB Activator (TANK) Induced by RANKL Negatively Regulates Osteoclasts Survival and Function

OpenAIRE

Wu, Mengrui; Wang, Yiping; Deng, Lianfu; Chen, Wei; Li, Yi-Ping

2012-01-01

Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation, and...

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

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Okazaki, Shogo [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nakatani, Fumi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Masuko, Kazue; Tsuchihashi, Kenji [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ueda, Shiho; Masuko, Takashi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Saya, Hideyuki [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nagano, Osamu, E-mail: osmna@sb3.so-net.ne.jp [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

2016-01-29

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

International Nuclear Information System (INIS)

Okazaki, Shogo; Nakatani, Fumi; Masuko, Kazue; Tsuchihashi, Kenji; Ueda, Shiho; Masuko, Takashi; Saya, Hideyuki; Nagano, Osamu

2016-01-01

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

Selective induction of DNA repair pathways in human B cells activated by CD4+ T cells.

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Xiaosheng Wu

Full Text Available Greater than 75% of all hematologic malignancies derive from germinal center (GC or post-GC B cells, suggesting that the GC reaction predisposes B cells to tumorigenesis. Because GC B cells acquire expression of the highly mutagenic enzyme activation-induced cytidine deaminase (AID, GC B cells may require additional DNA repair capacity. The goal of this study was to investigate whether normal human B cells acquire enhanced expression of DNA repair factors upon AID induction. We first demonstrated that several DNA mismatch repair, homologous recombination, base excision repair, and ATR signaling genes were overexpressed in GC B cells relative to naïve and memory B cells, reflecting activation of a process we have termed somatic hyperrepair (SHR. Using an in vitro system, we next characterized activation signals required to induce AID expression and SHR. Although AID expression was induced by a variety of polyclonal activators, SHR induction strictly required signals provided by contact with activated CD4+ T cells, and B cells activated in this manner displayed reduced levels of DNA damage-induced apoptosis. We further show the induction of SHR is independent of AID expression, as GC B cells from AID-/-mice retained heightened expression of SHR proteins. In consideration of the critical role that CD4+ T cells play in inducing the SHR process, our data suggest a novel role for CD4+ T cells in the tumor suppression of GC/post-GC B cells.

N-Cadherin Attenuates High Glucose-Induced Nucleus Pulposus Cell Senescence Through Regulation of the ROS/NF-κB Pathway.

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Hou, Gang; Zhao, Huiqing; Teng, Haijun; Li, Pei; Xu, Wenbin; Zhang, Junbin; Lv, Lulu; Guo, Zhiliang; Wei, Li; Yao, Hui; Xu, Yichun

2018-05-11

Diabetes mellitus (DM) is a potential etiology of disc degeneration. N-cadherin (N-CDH) helps maintain the cell viability, cell phenotype and matrix biosynthesis of nucleus pulposus (NP) cells. Here, we mainly aimed to investigate whether N-CDH can attenuate high glucose-induced NP cell senescence and its potential mechanism. Rat NP cells were cultured in a base culture medium and base culture medium with a 0.2 M glucose concentration. Recombinant lentiviral vectors were used to enhance N-CDH expression in NP cells. Senescence-associated β-galactosidase (SA-β-Gal) activity was measured by SA-β-Gal staining. NP cell proliferation was evaluated by CCK-8 assay. Telomerase activity and intracellular reactive oxygen species (ROS) content were tested by specific chemical kits according to the manufacturer's instructions. G0/G1 cell cycle arrest was evaluated by flow cytometry. Real-time PCR and Western blotting were used to analyze mRNA and protein expressions of senescence markers (p16 and p53) and matrix macromolecules (aggrecan and collagen II). Additionally, p-NF-κB expression was also analyzed by Western blotting to evaluate NF-κB pathway activity. High glucose significantly decreased N-CDH expression, increased ROS generation and NF-κB pathway activity, and promoted NP cell senescence, which was reflected in the increase in SA-β-Gal activity and senescence marker (p16 and p53) expression, compared to the control group. High glucose decreased telomerase activity and cell proliferation potency. However, N-CDH overexpression partially attenuated NP cell senescence, decreased ROS content and inhibited the activation of the NF-κB pathway under the high glucose condition. High glucose decreases N-CDH expression and promotes NP cell senescence. N-CDH overexpression can attenuate high glucose-induced NP cell senescence through the regulation of the ROS/ NF-κB pathway. This study suggests that N-CDH is a potential therapeutic target to slow DM-mediated disc NP

Inhibition of NFkappaB activation and IL-8 expression in human bronchial epithelial cells by acrolein.

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Valacchi, Giuseppe; Pagnin, Elisa; Phung, Anh; Nardini, Mirella; Schock, Bettina C; Cross, Carroll E; van der Vliet, Albert

2005-01-01

Lipid oxidation and environmental pollutants are major sources of alpha,beta-unsaturated aldehydes such as acrolein and 4-hydroxynonenal. Acrolein (2-propenal), a major product of organic combustion such as tobacco smoke, represents the most reactive alpha,beta-unsaturated aldehyde, with high reactivity toward nucleophilic targets such as sulfhydryl groups. To investigate how acrolein affects respiratory tract cell activation, we exposed either primary (NHBE) or immortalized human bronchial epithelial cells (HBE1) to 0-25 microM acrolein, and determined effects on basal and tumor necrosis factor-alpha (TNFalpha)-induced production of the chemokine interleukin (IL)-8. Cell exposure to acrolein dose-dependently suppressed IL-8 mRNA levels in HBE1 cells (26, 40, and 79% at 5, 10, and 25 microM acrolein concentrations, respectively) and resulted in corresponding decreases in IL-8 production. Studies of nuclear factor-kappaB (NFkappaB) activation, an essential event in IL-8 production, showed decreased TNFalpha-induced NFkappaB activation by acrolein, illustrated by inhibition of nuclear translocation of NFkappaB and reduced IkappaBalpha degradation. Immunochemical analysis of IkappaB kinase (IKK), a redox-sensitive regulator of NFkappaB activation, indicated direct modification of the IKK beta-subunit by acrolein, suggesting that acrolein may act directly on IKK. In summary, our results demonstrate that acrolein can suppress inflammatory processes in the airways by inhibiting epithelial IL-8 production through direct or indirect inhibitory effects on NFkappaB activation.

B Cell Help by CD1d-Rectricted NKT Cells

OpenAIRE

Livia Clerici; Giulia Casorati; Paolo Dellabona

2015-01-01

B cell activation and antibody production against foreign antigens is a central step of host defense. This is achieved via highly regulated multi-phase processes that involve a variety of cells of both innate and adaptive arms of the immune system. MHC class II-restricted CD4+ T cells specific for peptide antigens, which acquire professional follicular B cell helper functions, have been long recognized as key players in this process. Recent data, however, challenge this paradigm by showing th...

B cell activation by outer membrane vesicles--a novel virulence mechanism.

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Maria Laura A Perez Vidakovics

2010-01-01

Full Text Available Secretion of outer membrane vesicles (OMV is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR clustering and Ca(2+ mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19(+ IgD(+ lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86, whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.

Identification of a new gene regulatory circuit involving B cell receptor activated signaling using a combined analysis of experimental, clinical and global gene expression data

Science.gov (United States)

Schrader, Alexandra; Meyer, Katharina; Walther, Neele; Stolz, Ailine; Feist, Maren; Hand, Elisabeth; von Bonin, Frederike; Evers, Maurits; Kohler, Christian; Shirneshan, Katayoon; Vockerodt, Martina; Klapper, Wolfram; Szczepanowski, Monika; Murray, Paul G.; Bastians, Holger; Trümper, Lorenz; Spang, Rainer; Kube, Dieter

2016-01-01

To discover new regulatory pathways in B lymphoma cells, we performed a combined analysis of experimental, clinical and global gene expression data. We identified a specific cluster of genes that was coherently expressed in primary lymphoma samples and suppressed by activation of the B cell receptor (BCR) through αIgM treatment of lymphoma cells in vitro. This gene cluster, which we called BCR.1, includes numerous cell cycle regulators. A reduced expression of BCR.1 genes after BCR activation was observed in different cell lines and also in CD10+ germinal center B cells. We found that BCR activation led to a delayed entry to and progression of mitosis and defects in metaphase. Cytogenetic changes were detected upon long-term αIgM treatment. Furthermore, an inverse correlation of BCR.1 genes with c-Myc co-regulated genes in distinct groups of lymphoma patients was observed. Finally, we showed that the BCR.1 index discriminates activated B cell-like and germinal centre B cell-like diffuse large B cell lymphoma supporting the functional relevance of this new regulatory circuit and the power of guided clustering for biomarker discovery. PMID:27166259

Chronic Exposure to Malaria Is Associated with Inhibitory and Activation Markers on Atypical Memory B Cells and Marginal Zone-Like B Cells

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Itziar Ubillos

2017-08-01

Full Text Available In persistent infections that are accompanied by chronic immune activation, such as human immunodeficiency virus, hepatitis C virus, and malaria, there is an increased frequency of a phenotypically distinct subset of memory B cells lacking the classic memory marker CD27 and showing a reduced capacity to produce antibodies. However, critical knowledge gaps remain on specific B cell changes and immune adaptation in chronic infections. We hypothesized that expansion of atypical memory B cells (aMBCs and reduction of activated peripheral marginal zone (MZ-like B cells in constantly exposed individuals might be accompanied by phenotypic changes that would confer a tolerogenic profile, helping to establish tolerance to infections. To better understand malaria-associated phenotypic abnormalities on B cells, we analyzed peripheral blood mononuclear cells from 55 pregnant women living in a malaria-endemic area of Papua Nueva Guinea and 9 Spanish malaria-naïve individuals using four 11-color flow cytometry panels. We assessed the expression of markers of B cell specificity (IgG and IgM, activation (CD40, CD80, CD86, b220, TACI, and CD150, inhibition (PD1, CD95, and CD71, and migration (CCR3, CXCR3, and CD62l. We found higher frequencies of active and resting aMBC and marked reduction of MZ-like B cells, although changes in absolute cell counts could not be assessed. Highly exposed women had higher PD1+-, CD95+-, CD40+-, CD71+-, and CD80+-activated aMBC frequencies than non-exposed subjects. Malaria exposure increased frequencies of b220 and proapoptotic markers PD1 and CD95, and decreased expression of the activation marker TACI on MZ-like B cells. The increased frequencies of inhibitory and apoptotic markers on activated aMBCs and MZ-like B cells in malaria-exposed adults suggest an immune-homeostatic mechanism for maintaining B cell development and function while simultaneously downregulating hyperreactive B cells. This mechanism would keep the B cell

Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress.

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Kim, Kiyoon; Kim, Hunsung; Jeong, Kwon; Jung, Min Hyung; Hahn, Bum-Soo; Yoon, Kyung-Sik; Jin, Byung Kwan; Jahng, Geon-Ho; Kang, Insug; Ha, Joohun; Choe, Wonchae

2012-08-01

Cyclophilin, a cytosolic receptor for the immunosuppressive drug cyclosporin A, plays a role in diverse pathophysiologies along with its receptor, CD147. Although the interaction between cyclophilin A and CD147 is well established in inflammatory disease, that of cyclophilin B (CypB) with CD147 has not been fully explored, especially in cancer cell biology, and the exact molecular mechanism underlying such an association is poorly understood. In this study, we first identified high expression levels of CypB in 54 % of hepatocellular carcinoma patient tissues but in only 12.5 % of normal liver tissues. Then, we demonstrated that CypB overexpression protects human hepatoma cells against oxidative stress through its binding to CD147; this protective effect depends on the peptidyl prolyl isomerase activity of CypB. siRNA-mediated knockdown of CypB expression rendered hepatoma cells more vulnerable to ROS-mediated apoptosis. Furthermore, we also determined that a direct interaction between secreted CypB and CD147 regulates the extracellular signal-regulated kinase intracellular signaling pathway and is indispensible for the protective functions of CypB. For the first time, we demonstrated that CypB has an essential function in protecting hepatoma cells against oxidative stress through binding to CD147 and regulating the ERK pathway.

Akt-dependent NF-κB activation is required for bile acids to rescue colon cancer cells from stress-induced apoptosis

International Nuclear Information System (INIS)

Shant, Jasleen; Cheng, Kunrong; Marasa, Bernard S.; Wang Jianying; Raufman, Jean-Pierre

2009-01-01

Conjugated secondary bile acids promote human colon cancer cell proliferation by activating EGF receptors (EGFR). We hypothesized that bile acid-induced EGFR activation also mediates cell survival by downstream Akt-regulated activation of NF-κB. Deoxycholyltaurine (DCT) treatment attenuated TNF-α-induced colon cancer cell apoptosis, and stimulated rapid and sustained NF-κB nuclear translocation and transcriptional activity (detected by NF-κB binding to an oligonucleotide consensus sequence and by activation of luciferase reporter gene constructs). Both DCT-induced NF-κB nuclear translocation and attenuation of TNF-α-stimulated apoptosis were dependent on EGFR activation. Inhibitors of nuclear translocation, proteosome activity, and IκBα kinase attenuated NF-κB transcriptional activity. Cell transfection with adenoviral vectors encoding a non-degradable IκBα 'super-repressor' blocked the actions of DCT on both NF-κB activation and TNF-α-induced apoptosis. Likewise, transfection with mutant akt and treatment with a chemical inhibitor of Akt attenuated effects of DCT on NF-κB transcriptional activity and TNF-α-induced apoptosis. Chemical inhibitors of Akt and NF-κB activation also attenuated DCT-induced rescue of H508 cells from ultraviolet radiation-induced apoptosis. Collectively, these observations indicate that, downstream of EGFR, bile acid-induced colon cancer cell survival is mediated by Akt-dependent NF-κB activation. These findings provide a mechanism whereby bile acids increase resistance of colon cancer to chemotherapy and radiation

Comparative analysis of B7-1 and B7-2 expression in Langerhans cells: differential regulation by T helper type 1 and T helper type 2 cytokines.

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Kawamura, T; Furue, M

1995-07-01

Epidermal Langerhans cells (LC) are Ia-bearing potent antigen-presenting cells (APC) of dendritic cell lineage that play a crucial role in primary and secondary T cell-dependent immune responses. LC express several costimulatory molecules such as B7, which has been implicated as one of the important determinants of professional APC. Recently, B7 antigens have been shown to include three distinct molecules termed B7-1, B7-2, and B7-3, and the expression of B7-1 and B7-2 in LC has been already confirmed. However, little is known of the regulation of B7-1 and B7-2 expression in LC. We demonstrated that LC do not express B7-1 and B7-2 in situ; however, the expression of both molecules is rapidly induced during the first 3 days of culture, and high levels of expression are maintained at least until day 6. We show that the expression of B7-2 in LC is much higher than that of B7-1 in each experiment, and that B7-1 and B7-2 expression is reproducibly augmented by interleukin (IL)-4 in a dose-dependent manner; however, IL-2 affected expression very little. Finally, B7-1 expression is significantly and dose-dependently down-regulated by interferon (IFN)-gamma or IL-10, and B7-2 expression is consistently inhibited by IL-10, but not by IFN-gamma. The effects of these cytokines are active only in the induction phase (during first 3 days of culture) of B7 expression: the modulatory effects of cytokines are hardly detected in the plateau phase (days 4 to 6 of culture) of B7 expression in LC. These findings suggest that B7-1 and B7-2 expression are indeed selectively and differentially regulated by these T cell-derived cytokines, and that the cytokines may modulate the synthesis of B7 molecules rather than the degradation of already-expressed B7 molecules.

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

Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

International Nuclear Information System (INIS)

Nagata, Takayuki; Murata, Kazuko; Murata, Ryo; Sun, Shu-lan; Saito, Yutaro; Yamaga, Shuhei; Tanaka, Nobuyuki; Tamai, Keiichi; Moriya, Kunihiko; Kasai, Noriyuki; Sugamura, Kazuo; Ishii, Naoto

2014-01-01

Highlights: •ESCRT-0 protein regulates the development of peripheral B-cells. •BCR expression on cell surface should be controlled by the endosomal-sorting system. •Hrs plays important roles in responsiveness to Ag stimulation in B lymphocytes. -- Abstract: Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs flox/flox ;mb1 cre/+ :Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes

Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

Energy Technology Data Exchange (ETDEWEB)

Nagata, Takayuki [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Murata, Kazuko, E-mail: murata-k@iwakimu.ac.jp [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Murata, Ryo [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Sun, Shu-lan [Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Saito, Yutaro; Yamaga, Shuhei [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Tanaka, Nobuyuki; Tamai, Keiichi [Division of Immunology, Miyagi Cancer Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293 (Japan); Moriya, Kunihiko [Department of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Kasai, Noriyuki [Institute for Animal Experimentation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Sugamura, Kazuo [Division of Immunology, Miyagi Cancer Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293 (Japan); Ishii, Naoto [Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan)

2014-01-10

Highlights: •ESCRT-0 protein regulates the development of peripheral B-cells. •BCR expression on cell surface should be controlled by the endosomal-sorting system. •Hrs plays important roles in responsiveness to Ag stimulation in B lymphocytes. -- Abstract: Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs{sup flox/flox};mb1{sup cre/+}:Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

IL-15 inhibits pre-B cell proliferation by selectively expanding Mac-1+B220+ NK cells

International Nuclear Information System (INIS)

Nakajima, Shinsuke; Hida, Shigeaki; Taki, Shinsuke

2008-01-01

Natural killer (NK) cells are the cells critical for inhibition of repopulation of allogenic bone marrow cells. However, it is not well known if NK cells affect autologous lymphopoiesis. Here, we observed that NK cells could inhibit pre-B cell proliferation in vitro driven by interleukin (IL)-7 in a manner dependent on IL-15. Interestingly, the great majority of expanding NK cells were Mac-1 + B220 + , a recently identified potent interferon (IFN)-γ producer. Indeed, IFN-γ was produced in those cultures, and pre-B cells lacking IFN-γ receptors, but not those lacking type I IFN receptors, were resistant to such an inhibition. Furthermore, even NK cells from mice lacking β2-microglobulin, which were known to be functionally dampened, inhibited pre-B cell proliferation as well. Thus, activated NK cells, which were expanded selectively by IL-15, could potentially regulate B lymphopoiesis through IFN-γ beyond the selection imposed upon self-recognition

OCAM regulates embryonic spinal cord stem cell proliferation by modulating ErbB2 receptor.

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Loïc Deleyrolle

Full Text Available The proliferation and differentiation of neural stem cells are tightly controlled by intrinsic and extrinsic cues. Cell adhesion molecules are increasingly recognized as regulators of these processes. Here we report the expression of the olfactory cell adhesion molecule (OCAM/NCAM2/RNCAM during mouse spinal cord development and in neural stem cells cultured as neurospheres. OCAM is also weakly expressed in the dormant adult stem cell niche around the central canal and is overexpressed after spinal cord injury. Both transmembrane (TM and glycosylphosphatidylinositol (GPI-linked isoforms are present in neurospheres. Electron microscopy and internalisation experiments revealed a dynamic trafficking of OCAM between the membrane and intracellular compartments. After differentiation, OCAM remains in neurons and oligodendrocytes whereas no expression is detected in astrocytes. Using OCAM knockout (KO mice, we found that mutant spinal cord stem cells showed an increased proliferation and self-renewal rates although no effect on differentiation was observed. This effect was reversed by lentivirus-mediated re-introduction of OCAM. Mechanistically, we identified the ErbB2/Neu/HER2 protein as being implicated in the enhanced proliferation of mutant cells. ErbB2 protein expression and phosphorylation level were significantly increased in KO cells whereas no difference was observed at the mRNA level. Overexpression of ErbB2 in wild-type and mutant cells also increased their growth while reintroduction of OCAM in mutant cells reduced the level of phosphorylated ErbB2. These results indicate that OCAM exerts a posttranscriptional control on the ErbB2 signalling in spinal cord stem cells. This study adds further support for considering cell adhesion molecules as regulators of the ErbB signalling.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated disruption of the CD40 ligand-induced activation of primary human B cells

International Nuclear Information System (INIS)

Lu Haitian; Crawford, Robert B.; Kaplan, Barbara L.F.; Kaminski, Norbert E.

2011-01-01

Suppression of the primary antibody response is particularly sensitive to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice; however, surprisingly little is known concerning the effects of TCDD on humoral immunity or B cell function in humans. Results from a limited number of previous studies, primarily employing in vitro activation models, suggested that human B cell effector function is suppressed by TCDD. The present study sought to extend these findings by investigating, in primary human B cells, the effects of TCDD on several critical stages leading to antibody secretion including activation and plasmacytic differentiation using an in vitro CD40 ligand activation model. These studies revealed important differences in the response of human and mouse B cells to TCDD, the most striking being altered expression of plasmacytic differentiation regulators, B lymphocyte-induced maturation protein 1 and paired box protein 5, in mouse but not human B cells. The activation of human B cells was profoundly impaired by TCDD, as evidenced by decreased expression of activation markers CD80, CD86, and CD69. The impaired activation correlated with decreased cell viability, which prevented the progression of human B cells toward plasmacytic differentiation. TCDD treatment also attenuated the early activation of mitogen-activated protein kinases (MAPK) and Akt signaling in human B cells. Collectively, the present study provided experimental evidence for novel mechanisms by which TCDD impairs the effector function of primary human B cells. - Highlights: → In this study primary human and mouse B cell toxicity to TCDD was compared. → TCDD altered the expression of Blimp-1 and Pax5 in mouse but not human B cells. → TCDD markedly suppressed human B cell activation as characterized by CD80, CD86 and CD69 expression. → TCDD inhibited ERK, p38, and Akt phosphorylation in human B cells.

B cells promote inflammation in obesity and type 2 diabetes through regulation of T-cell function and an inflammatory cytokine profile.

Science.gov (United States)

DeFuria, Jason; Belkina, Anna C; Jagannathan-Bogdan, Madhumita; Snyder-Cappione, Jennifer; Carr, Jordan David; Nersesova, Yanina R; Markham, Douglas; Strissel, Katherine J; Watkins, Amanda A; Zhu, Min; Allen, Jessica; Bouchard, Jacqueline; Toraldo, Gianluca; Jasuja, Ravi; Obin, Martin S; McDonnell, Marie E; Apovian, Caroline; Denis, Gerald V; Nikolajczyk, Barbara S

2013-03-26

Patients with type 2 diabetes (T2D) have disease-associated changes in B-cell function, but the role these changes play in disease pathogenesis is not well established. Data herein show B cells from obese mice produce a proinflammatory cytokine profile compared with B cells from lean mice. Complementary in vivo studies show that obese B cell-null mice have decreased systemic inflammation, inflammatory B- and T-cell cytokines, adipose tissue inflammation, and insulin resistance (IR) compared with obese WT mice. Reduced inflammation in obese/insulin resistant B cell-null mice associates with an increased percentage of anti-inflammatory regulatory T cells (Tregs). This increase contrasts with the sharply decreased percentage of Tregs in obese compared with lean WT mice and suggests that B cells may be critical regulators of T-cell functions previously shown to play important roles in IR. We demonstrate that B cells from T2D (but not non-T2D) subjects support proinflammatory T-cell function in obesity/T2D through contact-dependent mechanisms. In contrast, human monocytes increase proinflammatory T-cell cytokines in both T2D and non-T2D analyses. These data support the conclusion that B cells are critical regulators of inflammation in T2D due to their direct ability to promote proinflammatory T-cell function and secrete a proinflammatory cytokine profile. Thus, B cells are potential therapeutic targets for T2D.

Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

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Tong, X; Kono, T; Evans-Molina, C

2015-06-18

The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

Regulation of Laminin γ2 Expression by CDX2 in Colonic Epithelial Cells Is Impaired During Active Inflammation

DEFF Research Database (Denmark)

Coskun, Mehmet; Soendergaard, Christoffer; Jørgensen, Steffen

2017-01-01

and to assess the influence of inflammation. Transcriptional regulation of LAMC2 was examined by reporter gene assays, overexpression, and shRNA-mediated knock-down of CDX2. CDX2-DNA interactions were assessed by chromatin immunoprecipitation on Caco-2 cells without or with TNF-α, as well as in purified colonic......The expression of Caudal-related homeobox transcription factor 2 (CDX2) is impaired by tumor necrosis factor-α (TNF-α)-mediated activation of nuclear factor-κB (NF-κB) in ulcerative colitis (UC). Laminin subunit γ2 (LAMC2) is an epithelial basement membrane protein implicated in cell migration......, proliferation, differentiation, as well as tumor invasion and intestinal inflammation, and its expression is enhanced by TNF-α in a NF-κB-dependent regulation of the recently identified LAMC2 enhancer. The aim was to determine whether CDX2 is involved in the basal regulation of LAMC2 in epithelial cells...

Roles of bHLH genes in neural stem cell differentiation

International Nuclear Information System (INIS)

Kageyama, Ryoichiro; Ohtsuka, Toshiyuki; Hatakeyama, Jun; Ohsawa, Ryosuke

2005-01-01

Neural stem cells change their characteristics over time during development: they initially proliferate only and then give rise to neurons first and glial cells later. In the absence of the repressor-type basic helix-loop-helix (bHLH) genes Hes1, Hes3 and Hes5, neural stem cells do not proliferate sufficiently but prematurely differentiate into neurons and become depleted without making the later born cell types such as astrocytes and ependymal cells. Thus, Hes genes are essential for maintenance of neural stem cells to make cells not only in correct numbers but also in full diversity. Hes genes antagonize the activator-type bHLH genes, which include Mash1, Math and Neurogenin. The activator-type bHLH genes promote the neuronal fate determination and induce expression of Notch ligands such as Delta. These ligands activate Notch signaling and upregulate Hes1 and Hes5 expression in neighboring cells, thereby maintaining these cells undifferentiated. Thus, the activator-type and repressor-type bHLH genes regulate each other, allowing only subsets of cells to undergo differentiation while keeping others to stay neural stem cells. This regulation is essential for generation of complex brain structures of appropriate size, shape and cell arrangement

Regulation of cancer stem cell properties by CD9 in human B-acute lymphoblastic leukemia

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Yamazaki, Hiroto [Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Wilson Xu, C. [Drug Development Program, Nevada Cancer Institute, Las Vegas, NV (United States); Naito, Motohiko [Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Nishida, Hiroko [Division of Hematology, Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Okamoto, Toshihiro; Ghani, Farhana Ishrat; Iwata, Satoshi [Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Inukai, Takeshi; Sugita, Kanji [Department of Pediatrics, School of Medicine, University of Yamanashi, Yamanashi (Japan); Morimoto, Chikao, E-mail: morimoto@ims.u-tokyo.ac.jp [Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Drug Development Program, Nevada Cancer Institute, Las Vegas, NV (United States)

2011-05-27

Highlights: {yields} We performed more detailed analysis of CD9 function for CSC properties in B-ALL. {yields} Leukemogenic fusion/Src family proteins were markedly regulated in the CD9{sup +} cells. {yields} Proliferation of B-ALL cells was inhibited by anti-CD9 monoclonal antibody. {yields} Knockdown of CD9 by RNAi remarkably reduced the leukemogenic potential. {yields} CD9-knockdown affected the expression and phosphorylation of Src family and USP22. -- Abstract: Although the prognosis of acute lymphoblastic leukemia (ALL) has improved considerably in recent years, some of the cases still exhibit therapy-resistant. We have previously reported that CD9 was expressed heterogeneously in B-ALL cell lines and CD9{sup +} cells exhibited an asymmetric cell division with greater tumorigenic potential than CD9{sup -} cells. CD9{sup +} cells were also serially transplantable in immunodeficient mice, indicating that CD9{sup +} cell possess self-renewal capacity. In the current study, we performed more detailed analysis of CD9 function for the cancer stem cell (CSC) properties. In patient sample, CD9 was expressed in the most cases of B-ALL cells with significant correlation of CD34-expression. Gene expression analysis revealed that leukemogenic fusion proteins and Src family proteins were significantly regulated in the CD9{sup +} population. Moreover, CD9{sup +} cells exhibited drug-resistance, but proliferation of bulk cells was inhibited by anti-CD9 monoclonal antibody. Knockdown of CD9 remarkably reduced the leukemogenic potential. Furthermore, gene ablation of CD9 affected the expression and tyrosine-phosphorylation of Src family proteins and reduced the expression of histone-deubiquitinase USP22. Taken together, our results suggest that CD9 links to several signaling pathways and epigenetic modification for regulating the CSC properties of B-ALL.

The VP7 Outer Capsid Protein of Rotavirus Induces Polyclonal B-Cell Activation

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Blutt, Sarah E.; Crawford, Sue E.; Warfield, Kelly L.; Lewis, Dorothy E.; Estes, Mary K.; Conner, Margaret E.

2004-01-01

The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered capsid and repetitively arranged antigens can induce activation of B cells, one or more of the capsid proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer capsid protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer capsid protein, VP7, initiate intestinal B-cell activation during rotavirus infection. PMID:15194774

Signal transduction by HLA-DR is mediated by tyrosine kinase(s) and regulated by CD45 in activated T cells

DEFF Research Database (Denmark)

Odum, Niels; Martin, P J; Schieven, G L

1991-01-01

Recently, it was shown that HLA class II molecules on B cells and activated human T cells can transmit signals involving tyrosine phosphorylation of specific proteins, activation of the inositol phospholipid pathway, and release of cytosolic free Ca2+(Ca2+)i. The regulation of class II induced si...

Lauroside B, a megastigmane glycoside from Laurus nobilis (bay laurel) leaves, induces apoptosis in human melanoma cell lines by inhibiting NF-κB activation.

Science.gov (United States)

Panza, Elisabetta; Tersigni, Mariaroberta; Iorizzi, Maria; Zollo, Franco; De Marino, Simona; Festa, Carmen; Napolitano, Maria; Castello, Giuseppe; Ialenti, Armando; Ianaro, Angela

2011-02-25

Malignant melanoma is a highly aggressive tumor that frequently resists chemotherapy, so the search for new agents for its treatment is of great importance. In the present study, the antiproliferative propensity against human melanoma cell lines of lauroside B (1), a megastigmane glycoside isolated from Laurus nobilis (bay laurel) leaves, was investigated. This compound suppressed the proliferation of three human melanoma cell lines, namely, A375, WM115, and SK-Mel-28. The 1-induced inhibition of human melanoma cell proliferation was due to the induction of apoptosis, as demonstrated by FACS analysis with annexin V/PI staining and confirmed by activation of caspase-3 and by the cleavage of poly(ADP-ribose) polymerase (PARP). Growing evidence implicates NF-κB as an important contributor to metastasis and increased chemoresistance of melanoma. Thus, it was hypothesized that 1-induced apoptosis could be associated with suppression of NF-κB activation. The results showed that exposure of human melanoma cells to 1 inhibited IκB-α degradation and constitutive NF-κB DNA-binding activity as well as the expression, regulated by NF-κB, of two antiapoptotic genes, XIAP and c-FLIP. Induction of apoptosis by 1 in human aggressive melanoma cell lines has a potential high biological value.

Pim-2 activates API-5 to inhibit the apoptosis of hepatocellular carcinoma cells through NF-kappaB pathway.

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Ren, Ke; Zhang, Wei; Shi, Yujun; Gong, Jianping

2010-06-01

Pim-2 is proved to be relevant to the tumorigenesis of hepatocellular carcinoma (HCC), but the mechanism is unclear. We studied the relationship among Pim-2, NF-kappaB and API-5. In our experiment, expression level of the three factors and phosphorylation level of API-5, as well as NF-kappaB activity, were detected in HCC tissues and the nontumorous controls. Then Pim-2 gene was transfected into nontumorous liver cells L02, and Pim-2 SiRNA was transfected into hepatoblastoma cell line HepG2. Parthenolide was added as NF-kappaB inhibitor. The same detections as above were repeated in the cells, along with the apoptosis analysis. We found the levels of Pim-2, NF-kappaB and API-5, as well as NF-kappaB activity, were significantly higher in HCC tissues. Pim-2 level was increased in L02 cells after the transfection of Pim-2 gene, but decreased in HepG2 cells after the transfection of Pim-2 SiRNA. The levels of NF-kappaB and API-5, as well as NF-kappaB activity and API-5 phosphorylation level, were in accordance with Pim-2 level, but could be reversed by Parthenolide. Cell apoptosis rates were negatively correlated with API-5 phosphorylation level. Therefore, we infer that Pim-2 could activate API-5 to inhibit the apoptosis of liver cells, and NF-kappaB is the key regulator.

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.

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