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  1. Constitutive activation of BMP signalling abrogates experimental metastasis of OVCA429 cells via reduced cell adhesion

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    Shepherd Trevor G

    2010-02-01

    Full Text Available Abstract Background Activation of bone morphogenetic protein (BMP4 signalling in human ovarian cancer cells induces a number of phenotypic changes in vitro, including altered cell morphology, adhesion, motility and invasion, relative to normal human ovarian surface epithelial cells. From these in vitro analyses, we had hypothesized that active BMP signalling promotes the metastatic potential of ovarian cancer. Methods To test this directly, we engineered OVCA429 human ovarian cancer cells possessing doxycycline-inducible expression of a constitutively-active mutant BMP receptor, ALK3QD, and administered these cells to immunocompromised mice. Further characterization was performed in vitro to address the role of activated BMP signalling on the EOC phenotype, with particular emphasis on epithelial-mesenchymal transition (EMT and cell adhesion. Results Unexpectedly, doxycycline-induced ALK3QD expression in OVCA429 cells reduced tumour implantation on peritoneal surfaces and ascites formation when xenografted into immunocompromised mice by intraperitoneal injection. To determine the potential mechanisms controlling this in vivo observation, we followed with several cell culture experiments. Doxycycline-induced ALK3QD expression enhanced the refractile, spindle-shaped morphology of cultured OVCA429 cells eliciting an EMT-like response. Using in vitro wound healing assays, we observed that ALK3QD-expressing cells migrated with long, cytoplasmic projections extending into the wound space. The phenotypic alterations of ALK3QD-expressing cells correlated with changes in specific gene expression patterns of EMT, including increased Snail and Slug and reduced E-cadherin mRNA expression. In addition, ALK3QD signalling reduced β1- and β3-integrin expression, critical molecules involved in ovarian cancer cell adhesion. The combination of reduced E-cadherin and β-integrin expression correlates directly with the reduced EOC cell cohesion in spheroids and

  2. Abrogation of epithelial BMP2 and BMP4 causes Amelogenesis Imperfecta by reducing MMP20 and KLK4 expression

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    Xie, Xiaohua; Liu, Chao; Zhang, Hua; Jani, Priyam H.; Lu, Yongbo; Wang, Xiaofang; Zhang, Bin; Qin, Chunlin

    2016-01-01

    Amelogenesis Imperfecta (AI) can be caused by the deficiencies of enamel matrix proteins, molecules responsible for the transportation and secretion of enamel matrix components, and proteases processing enamel matrix proteins. In the present study, we discovered the double deletion of bone morphogenetic protein 2 (Bmp2) and bone morphogenetic protein 4 (Bmp4) in the dental epithelium by K14-cre resulted in hypoplastic enamel and reduced density in X-ray radiography as well as shortened enamel rods under scanning electron microscopy. Such enamel phenotype was consistent with the diagnosis of hypoplastic amelogenesis imperfecta. Histological and molecular analyses revealed that the removal of matrix proteins in the mutant enamel was drastically delayed, which was coincided with the greatly reduced expression of matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4). Although the expression of multiple enamel matrix proteins was down-regulated in the mutant ameloblasts, the cleavage of ameloblastin was drastically impaired. Therefore, we attributed the AI primarily to the reduction of MMP20 and KLK4. Further investigation found that BMP/Smad4 signaling pathway was down-regulated in the K14-cre;Bmp2f/f;Bmp4f/fameloblasts, suggesting that the reduced MMP20 and KLK4 expression may be due to the attenuated epithelial BMP/Smad4 signaling. PMID:27146352

  3. Abrogation of epithelial BMP2 and BMP4 causes Amelogenesis Imperfecta by reducing MMP20 and KLK4 expression.

    Science.gov (United States)

    Xie, Xiaohua; Liu, Chao; Zhang, Hua; Jani, Priyam H; Lu, Yongbo; Wang, Xiaofang; Zhang, Bin; Qin, Chunlin

    2016-01-01

    Amelogenesis Imperfecta (AI) can be caused by the deficiencies of enamel matrix proteins, molecules responsible for the transportation and secretion of enamel matrix components, and proteases processing enamel matrix proteins. In the present study, we discovered the double deletion of bone morphogenetic protein 2 (Bmp2) and bone morphogenetic protein 4 (Bmp4) in the dental epithelium by K14-cre resulted in hypoplastic enamel and reduced density in X-ray radiography as well as shortened enamel rods under scanning electron microscopy. Such enamel phenotype was consistent with the diagnosis of hypoplastic amelogenesis imperfecta. Histological and molecular analyses revealed that the removal of matrix proteins in the mutant enamel was drastically delayed, which was coincided with the greatly reduced expression of matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4). Although the expression of multiple enamel matrix proteins was down-regulated in the mutant ameloblasts, the cleavage of ameloblastin was drastically impaired. Therefore, we attributed the AI primarily to the reduction of MMP20 and KLK4. Further investigation found that BMP/Smad4 signaling pathway was down-regulated in the K14-cre;Bmp2(f/f);Bmp4(f/f)ameloblasts, suggesting that the reduced MMP20 and KLK4 expression may be due to the attenuated epithelial BMP/Smad4 signaling. PMID:27146352

  4. Enhanced BMP signaling results in supernumerary tooth formation in USAG-1 deficient mouse

    International Nuclear Information System (INIS)

    Uterine sensitization associated gene-1 (USAG-1) is a BMP antagonist, and also modulates Wnt signaling. We previously reported that USAG-1 deficient mice have supernumerary teeth. The supernumerary maxillary incisor appears to form as a result of the successive development of the rudimentary upper incisor. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. We confirmed that BMPs were expressed in both the epithelium and mesenchyme of the rudimentary incisor at E14 and E15. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Wnt signaling as demonstrated by the nuclear localization of β-catenin was also up-regulated. Inhibition of BMP signaling rescues supernumerary tooth formation in E15 incisor explant culture. Based upon these results, we conclude that enhanced BMP signaling results in supernumerary teeth and BMP signaling was modulated by Wnt signaling in the USAG-1 deficient mouse model

  5. Smad1 and its target gene Wif1 coordinate BMP and Wnt signaling activities to regulate fetal lung development

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    Xu, Bing; Chen, Cheng; Chen, Hui; Zheng, Song-Guo; Bringas, Pablo; Xu, Min; Zhou, Xianghong; Chen, Di; Umans, Lieve; Zwijsen, An; SHI, Wei

    2011-01-01

    Bone morphogenetic protein 4 (Bmp4) is essential for lung development. To define the intracellular signaling mechanisms by which Bmp4 regulates lung development, BMP-specific Smad1 or Smad5 was selectively knocked out in fetal mouse lung epithelial cells. Abrogation of lung epithelial-specific Smad1, but not Smad5, resulted in retardation of lung branching morphogenesis and reduced sacculation, accompanied by altered distal lung epithelial cell proliferation and differentiation and, consequen...

  6. BMP signaling in the nephron progenitor niche

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    Oxburgh, Leif; Brown, Aaron C.; Fetting, Jennifer; Hill, Beth

    2011-01-01

    Bone morphogenic proteins (BMPs) play diverse roles in embryonic kidney development, regulating essential aspects of both ureteric bud and nephron development. In this review, we provide an overview of reported expression patterns and functions of BMP signaling components within the nephrogenic zone or nephron progenitor niche of the developing kidney. Reported in situ hybridization results are relatively challenging to interpret and sometimes conflicting. Comparing these with high-resolution...

  7. BMP2 Transfer to Neighboring Cells and Activation of Signaling.

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    Alborzinia, Hamed; Shaikhkarami, Marjan; Hortschansky, Peter; Wölfl, Stefan

    2016-09-01

    Morphogen gradients and concentration are critical features during early embryonic development and cellular differentiation. Previously we reported the preparation of biologically active, fluorescently labeled BMP2 and quantitatively analyzed their binding to the cell surface and followed BMP2 endocytosis over time on the level of single endosomes. Here we show that this internalized BMP2 can be transferred to neighboring cells and, moreover, also activates downstream BMP signaling in adjacent cells, indicated by Smad1/5/8 phosphorylation and activation of the downstream target gene id1. Using a 3D matrix to modulate cell-cell contacts in culture we could show that direct cell-cell contact significantly increased BMP2 transfer. Using inhibitors of vesicular transport, transfer was strongly inhibited. Interestingly, cotreatment with the physiological BMP inhibitor Noggin increased BMP2 uptake and transfer, albeit activation of Smad signaling in neighboring cells was completely suppressed. Our findings present a novel and interesting mechanism by which morphogens such as BMP2 can be transferred between cells and how this is modulated by BMP antagonists such as Noggin, and how this influences activation of Smad signaling by BMP2 in neighboring cells. PMID:27306974

  8. Arsenite suppression of BMP signaling in human keratinocytes

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    Phillips, Marjorie A.; Qin, Qin [Department of Environmental Toxicology, University of California, Davis, CA 95616-8588 (United States); Hu, Qin; Zhao, Bin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Rice, Robert H., E-mail: rhrice@ucdavis.edu [Department of Environmental Toxicology, University of California, Davis, CA 95616-8588 (United States)

    2013-06-15

    Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction, BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression. - Highlights: • BMP induces FOXN1 transcription. • BMP induces DUSP2 and DUSP14, suppressing ERK activation. • Arsenite suppresses levels of phosphorylated Smad1/5 and FOXN1 and DUSP mRNA. • These actions rationalize arsenite suppression of keratinocyte

  9. Identification of small molecule activators of BMP signaling.

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    Karen Vrijens

    Full Text Available Bone Morphogenetic Proteins (BMPs are morphogens that play a major role in regulating development and homeostasis. Although BMPs are used for the treatment of bone and kidney disorders, their clinical use is limited due to the supra-physiological doses required for therapeutic efficacy causing severe side effects. Because recombinant BMPs are expensive to produce, small molecule activators of BMP signaling would be a cost-effective alternative with the added benefit of being potentially more easily deliverable. Here, we report our efforts to identify small molecule activators of BMP signaling. We have developed a cell-based assay to monitor BMP signaling by stably transfecting a BMP-responsive human cervical carcinoma cell line (C33A with a reporter construct in which the expression of luciferase is driven by a multimerized BMP-responsive element from the Id1 promoter. A BMP-responsive clone C33A-2D2 was used to screen a bioactive library containing ∼5,600 small molecules. We identified four small molecules of the family of flavonoids all of which induced luciferase activity in a dose-dependent manner and ventralized zebrafish embryos. Two of the identified compounds induced Smad1, 5 phosphorylation (P-Smad, Id1 and Id2 expression in a dose-dependent manner demonstrating that our assays identified small molecule activators of BMP signaling.

  10. IGF1 potentiates BMP9-induced osteogenic differentiation in mesenchymal stem cells through the enhancement of BMP/Smad signaling

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    Chen, Liang; Zou, Xiang; Zhang, Ran-Xi; Pi, Chang-Jun; Wu, Nian; Yin, Liang-Jun; Deng, Zhong-Liang

    2016-01-01

    Engineered bone tissue is thought to be the ideal alternative for bone grafts in the treatment of related bone diseases. BMP9 has been demonstrated as one of the most osteogenic factors, and enhancement of BMP9-induced osteogenesis will greatly accelerate the development of bone tissue engineering. Here, we investigated the effect of insulin-like growth factor 1 (IGF1) on BMP9-induced osteogenic differentiation, and unveiled a possible molecular mechanism underling this process. We found that IGF1 and BMP9 are both detectable in mesenchymal stem cells (MSCs). Exogenous expression of IGF1 potentiates BMP9-induced alkaline phosphatase (ALP), matrix mineralization, and ectopic bone formation. Similarly, IGF1 enhances BMP9-induced endochondral ossification. Mechanistically, we found that IGF1 increases BMP9-induced activation of BMP/Smad signaling in MSCs. Our findings demonstrate that IGF1 can enhance BMP9-induced osteogenic differentiation in MSCs, and that this effect may be mediated by the enhancement of the BMP/Smad signaling transduction triggered by BMP9. [BMB Reports 2016; 49(2): 122-127] PMID:26645636

  11. Canonical Wnt activity regulates trunk neural crest delamination linking BMP/noggin signaling with G1/S transition.

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    Burstyn-Cohen, Tal; Stanleigh, Jonathan; Sela-Donenfeld, Dalit; Kalcheim, Chaya

    2004-11-01

    Delamination of premigratory neural crest cells depends on a balance between BMP/noggin and on successful G1/S transition. Here, we report that BMP regulates G1/S transition and consequent crest delamination through canonical Wnt signaling. Noggin overexpression inhibits G1/S transition and blocking G1/S abrogates BMP-induced delamination; moreover, transcription of Wnt1 is stimulated by BMP and by the developing somites, which concomitantly inhibit noggin production. Interfering with beta-catenin and LEF/TCF inhibits G1/S transition, neural crest delamination and transcription of various BMP-dependent genes, which include Cad6B, Pax3 and Msx1, but not that of Slug, Sox9 or FoxD3. Hence, we propose that developing somites inhibit noggin transcription in the dorsal tube, resulting in activation of BMP and consequent Wnt1 production. Canonical Wnt signaling in turn stimulates G1/S transition and generation of neural crest cell motility independently of its proposed role in earlier neural crest specification. PMID:15456730

  12. Hepcidin regulation by BMP signaling in macrophages is lipopolysaccharide dependent.

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    Xinggang Wu

    Full Text Available Hepcidin is an antimicrobial peptide, which also negatively regulates iron in circulation by controlling iron absorption from dietary sources and iron release from macrophages. Hepcidin is synthesized mainly in the liver, where hepcidin is regulated by iron loading, inflammation and hypoxia. Recently, we have demonstrated that bone morphogenetic protein (BMP-hemojuvelin (HJV-SMAD signaling is central for hepcidin regulation in hepatocytes. Hepcidin is also expressed by macrophages. Studies have shown that hepcidin expression by macrophages increases following bacterial infection, and that hepcidin decreases iron release from macrophages in an autocrine and/or paracrine manner. Although previous studies have shown that lipopolysaccharide (LPS can induce hepcidin expression in macrophages, whether hepcidin is also regulated by BMPs in macrophages is still unknown. Therefore, we examined the effects of BMP signaling on hepcidin expression in RAW 264.7 and J774 macrophage cell lines, and in primary peritoneal macrophages. We found that BMP4 or BMP6 alone did not have any effect on hepcidin expression in macrophages although they stimulated Smad1/5/8 phosphorylation and Id1 expression. In the presence of LPS, however, BMP4 and BMP6 were able to stimulate hepcidin expression in macrophages, and this stimulation was abolished by the NF-κB inhibitor Ro1069920. These results suggest that hepcidin expression is regulated differently in macrophages than in hepatocytes, and that BMPs regulate hepcidin expression in macrophages in a LPS-NF-κB dependent manner.

  13. Bmp signaling mediates endoderm pouch morphogenesis by regulating Fgf signaling in zebrafish.

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    Lovely, C Ben; Swartz, Mary E; McCarthy, Neil; Norrie, Jacqueline L; Eberhart, Johann K

    2016-06-01

    The endodermal pouches are a series of reiterated structures that segment the pharyngeal arches and help pattern the vertebrate face. Multiple pathways regulate the complex process of endodermal development, including the Bone morphogenetic protein (Bmp) pathway. However, the role of Bmp signaling in pouch morphogenesis is poorly understood. Using genetic and chemical inhibitor approaches, we show that pouch morphogenesis requires Bmp signaling from 10-18 h post-fertilization, immediately following gastrulation. Blocking Bmp signaling during this window results in morphological defects to the pouches and craniofacial skeleton. Using genetic chimeras we show that Bmp signals directly to the endoderm for proper morphogenesis. Time-lapse imaging and analysis of reporter transgenics show that Bmp signaling is necessary for pouch outpocketing via the Fibroblast growth factor (Fgf) pathway. Double loss-of-function analyses demonstrate that Bmp and Fgf signaling interact synergistically in craniofacial development. Collectively, our analyses shed light on the tissue and signaling interactions that regulate development of the vertebrate face. PMID:27122171

  14. Hepcidin Regulation by BMP Signaling in Macrophages Is Lipopolysaccharide Dependent

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    Wu, Xinggang; Yung, Lai-Ming; Cheng, Wai-Hang; Yu, Paul B.; Babitt, Jodie L.; Lin, Herbert Yih-Fuu; Xia, Yin

    2012-01-01

    Hepcidin is an antimicrobial peptide, which also negatively regulates iron in circulation by controlling iron absorption from dietary sources and iron release from macrophages. Hepcidin is synthesized mainly in the liver, where hepcidin is regulated by iron loading, inflammation and hypoxia. Recently, we have demonstrated that bone morphogenetic protein (BMP)-hemojuvelin (HJV)-SMAD signaling is central for hepcidin regulation in hepatocytes. Hepcidin is also expressed by macrophages. Studies ...

  15. Regulation of the BMP Signaling-Responsive Transcriptional Network in the Drosophila Embryo.

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    Deignan, Lisa; Pinheiro, Marco T; Sutcliffe, Catherine; Saunders, Abbie; Wilcockson, Scott G; Zeef, Leo A H; Donaldson, Ian J; Ashe, Hilary L

    2016-07-01

    The BMP signaling pathway has a conserved role in dorsal-ventral axis patterning during embryonic development. In Drosophila, graded BMP signaling is transduced by the Mad transcription factor and opposed by the Brinker repressor. In this study, using the Drosophila embryo as a model, we combine RNA-seq with Mad and Brinker ChIP-seq to decipher the BMP-responsive transcriptional network underpinning differentiation of the dorsal ectoderm during dorsal-ventral axis patterning. We identify multiple new BMP target genes, including positive and negative regulators of EGF signaling. Manipulation of EGF signaling levels by loss- and gain-of-function studies reveals that EGF signaling negatively regulates embryonic BMP-responsive transcription. Therefore, the BMP gene network has a self-regulating property in that it establishes a balance between its activity and that of the antagonistic EGF signaling pathway to facilitate correct patterning. In terms of BMP-dependent transcription, we identify key roles for the Zelda and Zerknüllt transcription factors in establishing the resulting expression domain, and find widespread binding of insulator proteins to the Mad and Brinker-bound genomic regions. Analysis of embryos lacking the BEAF-32 insulator protein shows reduced transcription of a peak BMP target gene and a reduction in the number of amnioserosa cells, the fate specified by peak BMP signaling. We incorporate our findings into a model for Mad-dependent activation, and discuss its relevance to BMP signal interpretation in vertebrates. PMID:27379389

  16. BMP4 Signaling is Involved in the Generation of Inner Ear Sensory Epithelia

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    Wang Yucheng; Zhao Yanling; Wang Zhengmin; Corrales Carleton E; Li Huawei; Liu Hong; Heller Stefan

    2005-01-01

    Abstract Background The robust expression of BMP4 in the incipient sensory organs of the inner ear suggests possible roles for this signaling protein during induction and development of auditory and vestibular sensory epithelia. Homozygous BMP4-/- animals die before the inner ear's sensory organs develop, which precludes determining the role of BMP4 in these organs with simple gene knockout experiments. Results Here we use a chicken otocyst culture system to perform quantitative studies on th...

  17. TGF-βand BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease

    Institute of Scientific and Technical Information of China (English)

    Mengrui Wu; Guiqian Chen; and Yi-Ping Li

    2016-01-01

    Transforming growth factor-beta (TGF-β) and bone morphogenic protein (BMP) signaling has fundamental roles in both embryonic skeletal development and postnatal bone homeostasis. TGF-βs and BMPs, acting on a tetrameric receptor complex, transduce signals to both the canonical Smad-dependent signaling pathway (that is, TGF-β/BMP ligands, receptors, and Smads) and the non-canonical-Smad-independent signaling pathway (that is, p38 mitogen-activated protein kinase/p38 MAPK) to regulate mesenchymal stem cell differentiation during skeletal development, bone formation and bone homeostasis. Both the Smad and p38 MAPK signaling pathways converge at transcription factors, for example, Runx2 to promote osteoblast differentiation and chondrocyte differentiation from mesenchymal precursor cells. TGF-βand BMP signaling is controlled by multiple factors, including the ubiquitin–proteasome system, epigenetic factors, and microRNA. Dysregulated TGF-βand BMP signaling result in a number of bone disorders in humans. Knockout or mutation of TGF-βand BMP signaling-related genes in mice leads to bone abnormalities of varying severity, which enable a better understanding of TGF-β/BMP signaling in bone and the signaling networks underlying osteoblast differentiation and bone formation. There is also crosstalk between TGF-β/BMP signaling and several critical cytokines’ signaling pathways (for example, Wnt, Hedgehog, Notch, PTHrP, and FGF) to coordinate osteogenesis, skeletal development, and bone homeostasis. This review summarizes the recent advances in our understanding of TGF-β/BMP signaling in osteoblast differentiation, chondrocyte differentiation, skeletal development, cartilage formation, bone formation, bone homeostasis, and related human bone diseases caused by the disruption of TGF-β/BMP signaling.

  18. Signaling Crosstalk between PPARγ and BMP2 in Mesenchymal Stem Cells

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    Ichiro Takada

    2012-01-01

    Full Text Available Recent studies have revealed that PPARγ’s transactivation function is regulated by extracellular signals. In particular, cytokines and Wnt family proteins suppress the ligand-inducible transactivation function of PPARγ and attenuate adipogenesis/osteoblastogenesis switching in mesenchymal stem cells (MSCs. For example, Wnt5a suppresses PPARγ transcriptional activity through the NLK/SETDB1/CHD7 pathway. Among these factors, BMP2 strongly induces bone formation, but the effect of BMP2 on PPARγ function remains unclear. We examined the effect of BMP2 and PPARγ in ST2 cells and found that PPARγ activation affected BMP2’s signaling pathway through epigenetic regulation. Although BMP2 did not interfere with PPARγ-mediated adipogenesis, BMP2 increased mRNA expression levels of PPARγ target genes (such as Fabp4 and Nr1h3 when cells were first treated with troglitazone (TRO. Moreover, PPARγ activation affected BMP2 through enhancement of histone activation markers (acetylated histone H3 and trimethylated Lys4 of histone H3 on the Runx2 promoter. After TRO treatment for three hours, BMP2 enhanced the levels of active histone marks on the promoter of a PPARγ target gene. These results suggest that the order of treatment with BMP2 and a PPARγ ligand is critical for adipogenesis and osteoblastogenesis switching in MSCs.

  19. BMP signaling mediates effects of exercise on hippocampal neurogenesis and cognition in mice.

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    Kevin T Gobeske

    Full Text Available Exposure to exercise or to environmental enrichment increases the generation of new neurons in the adult hippocampus and promotes certain kinds of learning and memory. While the precise role of neurogenesis in cognition has been debated intensely, comparatively few studies have addressed the mechanisms linking environmental exposures to cellular and behavioral outcomes. Here we show that bone morphogenetic protein (BMP signaling mediates the effects of exercise on neurogenesis and cognition in the adult hippocampus. Elective exercise reduces levels of hippocampal BMP signaling before and during its promotion of neurogenesis and learning. Transgenic mice with decreased BMP signaling or wild type mice infused with a BMP inhibitor both exhibit remarkable gains in hippocampal cognitive performance and neurogenesis, mirroring the effects of exercise. Conversely, transgenic mice with increased BMP signaling have diminished hippocampal neurogenesis and impaired cognition. Exercise exposure does not rescue these deficits, suggesting that reduced BMP signaling is required for environmental effects on neurogenesis and learning. Together, these observations show that BMP signaling is a fundamental mechanism linking environmental exposure with changes in cognitive function and cellular properties in the hippocampus.

  20. Inhibition of Bmp signaling affects growth and differentiation in the anagen hair follicle

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    Kulessa, Holger; Turk, Gail; Hogan, Brigid L. M.

    2000-01-01

    Growth and differentiation of postnatal hair follicles are controlled by reciprocal interactions between the dermal papilla and the surrounding epidermal hair precursors. The molecular nature of these interactions is largely unknown, but they are likely to involve several families of signaling molecules, including Fgfs, Wnts and Bmps. To analyze the function of Bmp signaling in postnatal hair development, we have generated transgenic mice expressing the Bmp inhibitor, Noggin, under the contro...

  1. BMP signaling turns up in fragile X syndrome: FMRP represses BMPR2.

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    Broihier, Heather T

    2016-01-01

    Fragile X syndrome is the most common inherited form of intellectual disability and results from a loss of function of the translational repressor FMRP. In this issue of Science Signaling, Kashima et al find that FMRP binds to and represses a specific isoform of BMPR2, a type II bone morphogenetic protein (BMP) receptor. Reducing signaling through this BMP pathway reverses neuroanatomical defects observed in fragile X models. PMID:27273094

  2. Transforming growth factor β1 inhibits bone morphogenic protein (BMP-2 and BMP-7 signaling via upregulation of Ski-related novel protein N (SnoN: possible mechanism for the failure of BMP therapy?

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    Ehnert Sabrina

    2012-09-01

    Full Text Available Abstract Background Bone morphogenic proteins (BMPs play a key role in bone formation. Consequently, it was expected that topical application of recombinant human (rhBMP-2 and rhBMP-7 would improve the healing of complex fractures. However, up to 36% of fracture patients do not respond to this therapy. There are hints that a systemic increase in transforming growth factor β1 (TGFβ1 interferes with beneficial BMP effects. Therefore, in the present work we investigated the influence of rhTGFβ1 on rhBMP signaling in primary human osteoblasts, with the aim of more specifically delineating the underlying regulatory mechanisms. Methods BMP signaling was detected by adenoviral Smad-binding-element-reporter assays. Gene expression was determined by reverse transcription polymerase chain reaction (RT-PCR and confirmed at the protein level by western blot. Histone deacetylase (HDAC activity was determined using a test kit. Data sets were compared by one-way analysis of variance. Results Our findings showed that Smad1/5/8-mediated rhBMP-2 and rhBMP-7 signaling is completely blocked by rhTGFβ1. We then investigated expression levels of genes involved in BMP signaling and regulation (for example, Smad1/5/8, TGFβ receptors type I and II, noggin, sclerostin, BMP and activin receptor membrane bound inhibitor (BAMBI, v-ski sarcoma viral oncogene homolog (Ski, Ski-related novel protein N (SnoN and Smad ubiquitination regulatory factors (Smurfs and confirmed the expression of regulated genes at the protein level. Smad7 and SnoN were significantly induced by rhTGFβ1 treatment while expression of Smad1, Smad6, TGFβRII and activin receptor-like kinase 1 (Alk1 was reduced. Elevated SnoN expression was accompanied by increased HDAC activity. Addition of an HDAC inhibitor, namely valproic acid, fully abolished the inhibitory effect of rhTGFβ1 on rhBMP-2 and rhBMP-7 signaling. Conclusions rhTGFβ1 effectively blocks rhBMP signaling in osteoblasts. As possible

  3. BMP-2 Overexpression Augments Vascular Smooth Muscle Cell Motility by Upregulating Myosin Va via Erk Signaling

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    Ming Zhang

    2014-01-01

    Full Text Available Background. The disruption of physiologic vascular smooth muscle cell (VSMC migration initiates atherosclerosis development. The biochemical mechanisms leading to dysfunctional VSMC motility remain unknown. Recently, cytokine BMP-2 has been implicated in various vascular physiologic and pathologic processes. However, whether BMP-2 has any effect upon VSMC motility, or by what manner, has never been investigated. Methods. VSMCs were adenovirally transfected to genetically overexpress BMP-2. VSMC motility was detected by modified Boyden chamber assay, confocal time-lapse video assay, and a colony wounding assay. Gene chip array and RT-PCR were employed to identify genes potentially regulated by BMP-2. Western blot and real-time PCR detected the expression of myosin Va and the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2. Immunofluorescence analysis revealed myosin Va expression locale. Intracellular Ca2+ oscillations were recorded. Results. VSMC migration was augmented in VSMCs overexpressing BMP-2 in a dose-dependent manner. siRNA-mediated knockdown of myosin Va inhibited VSMC motility. Both myosin Va mRNA and protein expression significantly increased after BMP-2 administration and were inhibited by Erk1/2 inhibitor U0126. BMP-2 induced Ca2+ oscillations, generated largely by a “cytosolic oscillator”. Conclusion. BMP-2 significantly increased VSMCs migration and myosin Va expression, via the Erk signaling pathway and intracellular Ca2+ oscillations. We provide additional insight into the pathophysiology of atherosclerosis, and inhibition of BMP-2-induced myosin Va expression may represent a potential therapeutic strategy.

  4. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration.

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    Plikus, Maksim V; Mayer, Julie Ann; de la Cruz, Damon; Baker, Ruth E; Maini, Philip K; Maxson, Robert; Chuong, Cheng-Ming

    2008-01-17

    In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge are regulated by the surrounding microenvironment, or niche. The activation of such stem cells is cyclic, involving periodic beta-catenin activity. In the adult mouse, regeneration occurs in waves in a follicle population, implying coordination among adjacent follicles and the extrafollicular environment. Here we show that unexpected periodic expression of bone morphogenetic protein 2 (Bmp2) and Bmp4 in the dermis regulates this process. This BMP cycle is out of phase with the WNT/beta-catenin cycle, thus dividing the conventional telogen into new functional phases: one refractory and the other competent for hair regeneration, characterized by high and low BMP signalling, respectively. Overexpression of noggin, a BMP antagonist, in mouse skin resulted in a markedly shortened refractory phase and faster propagation of the regenerative wave. Transplantation of skin from this mutant onto a wild-type host showed that follicles in donor and host can affect their cycling behaviours mutually, with the outcome depending on the equilibrium of BMP activity in the dermis. Administration of BMP4 protein caused the competent region to become refractory. These results show that BMPs may be the long-sought 'chalone' inhibitors of hair growth postulated by classical experiments. Taken together, results presented in this study provide an example of hierarchical regulation of local organ stem cell homeostasis by the inter-organ macroenvironment. The expression of Bmp2 in subcutaneous adipocytes indicates physiological integration between these two thermo-regulatory organs. Our findings have practical importance for studies using mouse skin as a model for carcinogenesis, intra-cutaneous drug

  5. Matrix-immobilized BMP-2 on microcontact printed fibronectin as in vitro tool to study BMP-mediated signaling and cell migration

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    Kristin eHauff

    2015-05-01

    Full Text Available During development, bone morphogenetic proteins (BMPs exert important functions in several tissues by regulating signaling for cell differentiation and migration. In vivo the extracellular matrix (ECM not only provides a support for adherent cells, but also presents a reservoir of growth factors (GFs. Several constituents of the ECM provide adhesive cues, which serve as binding sites for cell transmembrane receptors, such as integrins, which convey adhesion-mediated signaling to the intracellular compartment. Integrins do not function alone but rather crosstalk and cooperate with other receptors, such as GF receptors, in regulating cell responses to extracellular signals. To this, we present here the immobilization of BMP-2 onto cellular fibronectin (cFN, a key protein of the ECM, to investigate their impact on GF-mediated signaling and migration.Following biotinylation, BMP-2 was linked to biotinylated cFN using NeutrAvidin (NA as cross-linker. Characterization with QCM-D and ELISA confirmed the efficient immobilization of BMP-2 on cFN over a period of 24 h.To validate the bioactivity of matrix-immobilized BMP-2 (iBMP-2 we investigated short- and long-term responses of C2C12 myoblasts in comparison to soluble BMP-2 (sBMP-2 or in absence of GFs. Similarly to sBMP-2, iBMP-2 triggered Smad 1/5 phosphorylation and translocation into the nucleus corresponding to the activation of BMP-mediated Smad-dependent pathway. Additionally, successful suppression of myotube formation was observed after six days.We next implemented this approach to fabricate cFN micro patterned stripes by soft lithography. These stripes only allowed cell-surface interaction on the pattern due to passivation of the surface in between, thus serving as platform for studies on directed cell migration. During a 10 h-period, cells showed an increased migratory activity upon BMP-2 exposure.Thus, this versatile tool retains the GF's bioactivity and allows the presentation of ECM

  6. Circadian period integrates network information through activation of the BMP signaling pathway.

    Directory of Open Access Journals (Sweden)

    Esteban J Beckwith

    2013-12-01

    Full Text Available Living organisms use biological clocks to maintain their internal temporal order and anticipate daily environmental changes. In Drosophila, circadian regulation of locomotor behavior is controlled by ∼150 neurons; among them, neurons expressing the PIGMENT DISPERSING FACTOR (PDF set the period of locomotor behavior under free-running conditions. To date, it remains unclear how individual circadian clusters integrate their activity to assemble a distinctive behavioral output. Here we show that the BONE MORPHOGENETIC PROTEIN (BMP signaling pathway plays a crucial role in setting the circadian period in PDF neurons in the adult brain. Acute deregulation of BMP signaling causes period lengthening through regulation of dClock transcription, providing evidence for a novel function of this pathway in the adult brain. We propose that coherence in the circadian network arises from integration in PDF neurons of both the pace of the cell-autonomous molecular clock and information derived from circadian-relevant neurons through release of BMP ligands.

  7. Circadian Period Integrates Network Information Through Activation of the BMP Signaling Pathway

    Science.gov (United States)

    Beckwith, Esteban J.; Gorostiza, E. Axel; Berni, Jimena; Rezával, Carolina; Pérez-Santángelo, Agustín; Nadra, Alejandro D.; Ceriani, María Fernanda

    2013-01-01

    Living organisms use biological clocks to maintain their internal temporal order and anticipate daily environmental changes. In Drosophila, circadian regulation of locomotor behavior is controlled by ∼150 neurons; among them, neurons expressing the PIGMENT DISPERSING FACTOR (PDF) set the period of locomotor behavior under free-running conditions. To date, it remains unclear how individual circadian clusters integrate their activity to assemble a distinctive behavioral output. Here we show that the BONE MORPHOGENETIC PROTEIN (BMP) signaling pathway plays a crucial role in setting the circadian period in PDF neurons in the adult brain. Acute deregulation of BMP signaling causes period lengthening through regulation of dClock transcription, providing evidence for a novel function of this pathway in the adult brain. We propose that coherence in the circadian network arises from integration in PDF neurons of both the pace of the cell-autonomous molecular clock and information derived from circadian-relevant neurons through release of BMP ligands. PMID:24339749

  8. An FGF3-BMP Signaling Axis Regulates Caudal Neural Tube Closure, Neural Crest Specification and Anterior-Posterior Axis Extension

    Science.gov (United States)

    Anderson, Matthew J.; Schimmang, Thomas; Lewandoski, Mark

    2016-01-01

    During vertebrate axis extension, adjacent tissue layers undergo profound morphological changes: within the neuroepithelium, neural tube closure and neural crest formation are occurring, while within the paraxial mesoderm somites are segmenting from the presomitic mesoderm (PSM). Little is known about the signals between these tissues that regulate their coordinated morphogenesis. Here, we analyze the posterior axis truncation of mouse Fgf3 null homozygotes and demonstrate that the earliest role of PSM-derived FGF3 is to regulate BMP signals in the adjacent neuroepithelium. FGF3 loss causes elevated BMP signals leading to increased neuroepithelium proliferation, delay in neural tube closure and premature neural crest specification. We demonstrate that elevated BMP4 depletes PSM progenitors in vitro, phenocopying the Fgf3 mutant, suggesting that excessive BMP signals cause the Fgf3 axis defect. To test this in vivo we increased BMP signaling in Fgf3 mutants by removing one copy of Noggin, which encodes a BMP antagonist. In such mutants, all parameters of the Fgf3 phenotype were exacerbated: neural tube closure delay, premature neural crest specification, and premature axis termination. Conversely, genetically decreasing BMP signaling in Fgf3 mutants, via loss of BMP receptor activity, alleviates morphological defects. Aberrant apoptosis is observed in the Fgf3 mutant tailbud. However, we demonstrate that cell death does not cause the Fgf3 phenotype: blocking apoptosis via deletion of pro-apoptotic genes surprisingly increases all Fgf3 defects including causing spina bifida. We demonstrate that this counterintuitive consequence of blocking apoptosis is caused by the increased survival of BMP-producing cells in the neuroepithelium. Thus, we show that FGF3 in the caudal vertebrate embryo regulates BMP signaling in the neuroepithelium, which in turn regulates neural tube closure, neural crest specification and axis termination. Uncovering this FGF3-BMP signaling axis is

  9. An FGF3-BMP Signaling Axis Regulates Caudal Neural Tube Closure, Neural Crest Specification and Anterior-Posterior Axis Extension.

    Directory of Open Access Journals (Sweden)

    Matthew J Anderson

    2016-05-01

    Full Text Available During vertebrate axis extension, adjacent tissue layers undergo profound morphological changes: within the neuroepithelium, neural tube closure and neural crest formation are occurring, while within the paraxial mesoderm somites are segmenting from the presomitic mesoderm (PSM. Little is known about the signals between these tissues that regulate their coordinated morphogenesis. Here, we analyze the posterior axis truncation of mouse Fgf3 null homozygotes and demonstrate that the earliest role of PSM-derived FGF3 is to regulate BMP signals in the adjacent neuroepithelium. FGF3 loss causes elevated BMP signals leading to increased neuroepithelium proliferation, delay in neural tube closure and premature neural crest specification. We demonstrate that elevated BMP4 depletes PSM progenitors in vitro, phenocopying the Fgf3 mutant, suggesting that excessive BMP signals cause the Fgf3 axis defect. To test this in vivo we increased BMP signaling in Fgf3 mutants by removing one copy of Noggin, which encodes a BMP antagonist. In such mutants, all parameters of the Fgf3 phenotype were exacerbated: neural tube closure delay, premature neural crest specification, and premature axis termination. Conversely, genetically decreasing BMP signaling in Fgf3 mutants, via loss of BMP receptor activity, alleviates morphological defects. Aberrant apoptosis is observed in the Fgf3 mutant tailbud. However, we demonstrate that cell death does not cause the Fgf3 phenotype: blocking apoptosis via deletion of pro-apoptotic genes surprisingly increases all Fgf3 defects including causing spina bifida. We demonstrate that this counterintuitive consequence of blocking apoptosis is caused by the increased survival of BMP-producing cells in the neuroepithelium. Thus, we show that FGF3 in the caudal vertebrate embryo regulates BMP signaling in the neuroepithelium, which in turn regulates neural tube closure, neural crest specification and axis termination. Uncovering this FGF3

  10. Bmp signaling in colonic mesenchyme regulates stromal microenvironment and protects from polyposis initiation.

    Science.gov (United States)

    Allaire, Joannie M; Roy, Sébastien A B; Ouellet, Camille; Lemieux, Étienne; Jones, Christine; Paquet, Marilène; Boudreau, Francois; Perreault, Nathalie

    2016-06-01

    In the colon, myofibroblasts are primary contributors in the establishment of the microenvironment involved in tissue homeostasis. Alterations in myofibroblast functions lead to changes resulting in a toxic microenvironment nurturing tumorigenesis. Bone morphogenetic proteins (Bmps) are morphogens known to play key roles in adult gut homeostasis. Studies in genetically-modified mice have shown that Bmp disruption in all cell layers leads to the development of gut polyposis. In contrast, our studies showed that loss of Bmp exclusively in the gastrointestinal epithelium resulted in increased epithelial proliferation without polyposis initiation, thus suggesting a key role for mesenchymal Bmp signaling in polyposis initiation. In order to identify the role of mesenchymal Bmp signaling on the microenvironment and its impact on colonic mucosa, a mouse model was generated with suppression of Bmp signaling exclusively in myofibroblasts (Bmpr1aΔMES). Bmpr1aΔMES mice exhibited increased subepithelial proliferation with changes in cellular composition leading to the development of a primed stroma with modulation of extracellular matrix proteins, immune cells and cytokines as early as 90 days of age. This microenvironmental deregulation was associated with increased polyposis initiation at one year of age. These results are the first to demonstrate that mesenchymal Bmpr1a inactivation alone is sufficient to prompt an expansion of myofibroblasts leading to the development of a reactive mesenchyme that contributes to polyposis initiation in the colon. These findings support the novel concept that inhibition of Bmp signaling in mesenchymal cells surrounding the normal epithelium leads to important changes instructing a toxic microenvironment sufficient to induce colonic polyposis. PMID:26773796

  11. BMP signaling is essential in neonatal surfactant production during respiratory adaptation.

    Science.gov (United States)

    Luo, Yongfeng; Chen, Hui; Ren, Siying; Li, Nan; Mishina, Yuji; Shi, Wei

    2016-07-01

    Deficiency in pulmonary surfactant results in neonatal respiratory distress, and the known genetic mutations in key components of surfactant only account for a small number of cases. Therefore, determining the regulatory mechanisms of surfactant production and secretion, particularly during the transition from prenatal to neonatal stages, is essential for better understanding of the pathogenesis of human neonatal respiratory distress. We have observed significant increase of bone morphogenetic protein (BMP) signaling in neonatal mouse lungs immediately after birth. Using genetically manipulated mice, we then studied the relationship between BMP signaling and surfactant production in neonates. Blockade of endogenous BMP signaling by deleting Bmpr1a (Alk3) or Smad1 in embryonic day 18.5 in perinatal lung epithelial cells resulted in severe neonatal respiratory distress and death, accompanied by atelectasis in histopathology and significant reductions of surfactant protein B and C, as well as Abca3, whereas prenatal lung development was not significantly affected. We then identified a new BMP-Smad1 downstream target, Nfatc3, which is known as an important transcription activator for surfactant proteins and Abca3. Furthermore, activation of BMP signaling in cultured lung epithelial cells was able to promote endogenous Nfatc3 expression and also stimulate the activity of an Nfatc3 promoter that contains a Smad1-binding site. Therefore, our study suggests that the BMP-Alk3-Smad1-Nfatc3 regulatory loop plays an important role in enhancing surfactant production in neonates, possibly helping neonatal respiratory adaptation from prenatal amniotic fluid environment to neonatal air breathing. PMID:27190064

  12. Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice.

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    Gerhard Sengle

    2015-06-01

    Full Text Available Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that

  13. Signaling cross-talk between TGF-β/BMP and other path-ways

    Institute of Scientific and Technical Information of China (English)

    Xing Guo; Xiao-Fan Wang

    2009-01-01

    Transforming growth factor-beta(TGF-β)/bone morphogenic protein(BMP)signaling is involved in the vast majority of cellular processes and is fundamentally important during the entire life of alI metazoans.Deregulation of TGF-β/BMP activity almost invariably leads to developmental defects and/or diseases.including cancer.The proper functioning of the TGF-β/BMP pathway depends on its constitutive and extensive communication with other signaling pathways,leading to synergistic or antagonistic effects and eventually desirable biological outcomes.The nature of such signaling cross-talk iS overwhelmingly complex and highly context-dependent.Here we review the difierent modes of cross-talk between TGF-β/BMP and the signaling pathways of Mitogen-activated protein kinase,phosphatidyIinositoI-3 kinase/Akt,Wnt,Hedgehog,Notch,and the interleukin/interferon-gamma/tumor necrosis factor-alpha cytokines,with an emphasis on the underlying molecular mechanisms.

  14. Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells

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    Kimberly A. Wong

    2015-03-01

    Full Text Available Retina formation requires the correct spatiotemporal patterning of key regulatory factors. While it is known that repression of several signaling pathways lead to specification of retinal fates, addition of only Noggin, a known BMP antagonist, can convert pluripotent Xenopus laevis animal cap cells to functional retinal cells. The aim of this study is to determine the intracellular molecular events that occur during this conversion. Surprisingly, blocking BMP signaling alone failed to mimic Noggin treatment. Overexpressing Noggin in pluripotent cells resulted in a concentration-dependent suppression of both Smad1 and Smad2 phosphorylation, which act downstream of BMP and Activin signaling, respectively. This caused a decrease in downstream targets: endothelial marker, xk81, and mesodermal marker, xbra. We treated pluripotent cells with dominant-negative receptors or the chemical inhibitors, dorsomorphin and SB431542, which each target either the BMP or Activin signaling pathway. We determined the effect of these treatments on retina formation using the Animal Cap Transplant (ACT assay; in which treated pluripotent cells were transplanted into the eye field of host embryos. We found that inhibition of Activin signaling, in the presence of BMP signaling inhibition, promotes efficient retinal specification in Xenopus tissue, mimicking the affect of adding Noggin alone. In whole embryos, we found that the eye field marker, rax, expanded when adding both dominant-negative Smad1 and Smad2, as did treating the cells with both dorsomorphin and SB431542. Future studies could translate these findings to a mammalian culture assay, in order to more efficiently produce retinal cells in culture.

  15. Reduced BMP signaling results in hindlimb fusion with lethal pelvic/urogenital organ aplasia: a new mouse model of sirenomelia.

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    Kentaro Suzuki

    Full Text Available Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4(flox/flox and the Isl1 (Islet1-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4(flox/flox conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.

  16. Histone deacetylases control neurogenesis in embryonic brain by inhibition of BMP2/4 signaling.

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    Maya Shakèd

    Full Text Available BACKGROUND: Histone-modifying enzymes are essential for a wide variety of cellular processes dependent upon changes in gene expression. Histone deacetylases (HDACs lead to the compaction of chromatin and subsequent silencing of gene transcription, and they have recently been implicated in a diversity of functions and dysfunctions in the postnatal and adult brain including ocular dominance plasticity, memory consolidation, drug addiction, and depression. Here we investigate the role of HDACs in the generation of neurons and astrocytes in the embryonic brain. PRINCIPAL FINDINGS: As a variety of HDACs are expressed in differentiating neural progenitor cells, we have taken a pharmacological approach to inhibit multiple family members. Inhibition of class I and II HDACs in developing mouse embryos with trichostatin A resulted in a dramatic reduction in neurogenesis in the ganglionic eminences and a modest increase in neurogenesis in the cortex. An identical effect was observed upon pharmacological inhibition of HDACs in in vitro-differentiating neural precursors derived from the same brain regions. A reduction in neurogenesis in ganglionic eminence-derived neural precursors was accompanied by an increase in the production of immature astrocytes. We show that HDACs control neurogenesis by inhibition of the bone morphogenetic protein BMP2/4 signaling pathway in radial glial cells. HDACs function at the transcriptional level by inhibiting and promoting, respectively, the expression of Bmp2 and Smad7, an intracellular inhibitor of BMP signaling. Inhibition of the BMP2/4 signaling pathway restored normal levels of neurogenesis and astrogliogenesis to both ganglionic eminence- and cortex-derived cultures in which HDACs were inhibited. CONCLUSIONS: Our results demonstrate a transcriptionally-based regulation of BMP2/4 signaling by HDACs both in vivo and in vitro that is critical for neurogenesis in the ganglionic eminences and that modulates cortical

  17. The BMP signaling pathway at the Drosophila neuromuscular junction and its links to neurodegenerative diseases

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    Bayat, Vafa; Jaiswal, Manish; Bellen, Hugo J

    2010-01-01

    The Drosophila neuromuscular junction (NMJ) has recently provided new insights into the roles of various proteins in neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy (SMA), Multiple Sclerosis (MS) Hereditary Spastic Paraplegia (HSP), and Huntington’s Disease (HD). Several developmental signaling pathways including WNT, MAPK and BMP/TGF-β signaling play important roles in the formation and growth of the Drosophila NMJ. Studies of the fly homolog...

  18. miR-143 interferes with ERK5 signaling, and abrogates prostate cancer progression in mice.

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    Cyrielle Clapé

    Full Text Available BACKGROUND: Micro RNAs are small, non-coding, single-stranded RNAs that negatively regulate gene expression at the post-transcriptional level. Since miR-143 was found to be down-regulated in prostate cancer cells, we wanted to analyze its expression in human prostate cancer, and test the ability of miR-43 to arrest prostate cancer cell growth in vitro and in vivo. RESULTS: Expression of miR-143 was analyzed in human prostate cancers by quantitative PCR, and by in situ hybridization. miR-143 was introduced in cancer cells in vivo by electroporation. Bioinformatics analysis and luciferase-based assays were used to determine miR-143 targets. We show in this study that miR-143 levels are inversely correlated with advanced stages of prostate cancer. Rescue of miR-143 expression in cancer cells results in the arrest of cell proliferation and the abrogation of tumor growth in mice. Furthermore, we show that the effects of miR-143 are mediated, at least in part by the inhibition of extracellular signal-regulated kinase-5 (ERK5 activity. We show here that ERK5 is a miR-143 target in prostate cancer. CONCLUSIONS: miR-143 is as a new target for prostate cancer treatment.

  19. PARM-1 promotes cardiomyogenic differentiation through regulating the BMP/Smad signaling pathway

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    Nakanishi, Naohiko [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan); Takahashi, Tomosaburo, E-mail: ttaka@koto.kpu-m.ac.jp [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan); Ogata, Takehiro; Adachi, Atsuo; Imoto-Tsubakimoto, Hiroko [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan); Ueyama, Tomomi, E-mail: toueyama-circ@umin.ac.jp [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan); Matsubara, Hiroaki [Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566 (Japan)

    2012-11-30

    Highlights: Black-Right-Pointing-Pointer PARM-1 expression is induced during cardiomyogenesis. Black-Right-Pointing-Pointer PARM-1 expression precedes Nkx2.5 and Tbx5 during cardiomyogenesis. Black-Right-Pointing-Pointer PARM-1 activates BMP/Smad signaling. Black-Right-Pointing-Pointer PARM-1 enhances cardiac specification, resulting in promoted cardiomyogenesis. -- Abstract: PARM-1, prostatic androgen repressed message-1, is an endoplasmic reticulum (ER) molecule that is involved in ER stress-induced apoptosis in cardiomyocytes. In this study, we assessed whether PARM-1 plays a role in the differentiation of stem cells into cardiomyocytes. While PARM-1 was not expressed in undifferentiated P19CL6 embryonic carcinoma cells, PARM-1 expression was induced during cardiomyogenic differentiation. This expression followed expression of mesodermal markers, and preceded expression of cardiac transcription factors. PARM-1 overexpression did not alter the expression of undifferentiated markers and the proliferative property in undifferentiated P19CL6 cells. Expression of cardiac transcription factors during cardiomyogenesis was markedly enhanced by overexpression of PARM-1, while expression of mesodermal markers was not altered, suggesting that PARM-1 is involved in the differentiation from the mesodermal lineage to cardiomyocytes. Furthermore, overexpression of PARM-1 induced BMP2 mRNA expression in undifferentiated P19CL6 cells and enhanced both BMP2 and BMP4 mRNA expression in the early phase of cardiomyogenesis. PARM-1 overexpression also enhanced phosphorylation of Smads1/5/8. Thus, PARM-1 plays an important role in the cardiomyogenic differentiation of P19CL6 cells through regulating BMP/Smad signaling pathways, demonstrating a novel role of PARM-1 in the cardiomyogenic differentiation of stem cells.

  20. Apc bridges Wnt/{beta}-catenin and BMP signaling during osteoblast differentiation of KS483 cells

    Energy Technology Data Exchange (ETDEWEB)

    Miclea, Razvan L., E-mail: R.L.Miclea@lumc.nl [Department of Pediatrics, Leiden University Medical Centre (LUMC), Leiden (Netherlands); Horst, Geertje van der, E-mail: G.van_der_Horst@lumc.nl [Department of Urology, LUMC, Leiden (Netherlands); Robanus-Maandag, Els C., E-mail: E.C.Robanus@lumc.nl [Department of Human Genetics, LUMC, Leiden (Netherlands); Loewik, Clemens W.G.M., E-mail: C.W.G.M.Lowik@lumc.nl [Department of Endocrinology and Metabolic Diseases, LUMC, Leiden (Netherlands); Oostdijk, Wilma, E-mail: W.Oostdijk@lumc.nl [Department of Pediatrics, Leiden University Medical Centre (LUMC), Leiden (Netherlands); Wit, Jan M., E-mail: J.M.Wit@lumc.nl [Department of Pediatrics, Leiden University Medical Centre (LUMC), Leiden (Netherlands); Karperien, Marcel, E-mail: H.B.J.Karperien@tnw.utwente.nl [MIRA Institute for Biomedical Technology and Technical Medicine, Department of Tissue Regeneration, University of Twente, Zuidhorst Room ZH 144, Drienerlolaan 5, 7522 NB Enschede (Netherlands)

    2011-06-10

    The canonical Wnt signaling pathway influences the differentiation of mesenchymal cell lineages in a quantitative and qualitative fashion depending on the dose of {beta}-catenin signaling. Adenomatous polyposis coli (Apc) is the critical intracellular regulator of {beta}-catenin turnover. To better understand the molecular mechanisms underlying the role of Apc in regulating the differentiation capacity of skeletal progenitor cells, we have knocked down Apc in the murine mesenchymal stem cell-like KS483 cells by stable expression of Apc-specific small interfering RNA. In routine culture, KSFrt-Apc{sub si} cells displayed a mesenchymal-like spindle shape morphology, exhibited markedly decreased proliferation and increased apoptosis. Apc knockdown resulted in upregulation of the Wnt/{beta}-catenin and the BMP/Smad signaling pathways, but osteogenic differentiation was completely inhibited. This effect could be rescued by adding high concentrations of BMP-7 to the differentiation medium. Furthermore, KSFrt-Apc{sub si} cells showed no potential to differentiate into chondrocytes or adipocytes. These results demonstrate that Apc is essential for the proliferation, survival and differentiation of KS483 cells. Apc knockdown blocks the osteogenic differentiation of skeletal progenitor cells, a process that can be overruled by high BMP signaling.

  1. Apc bridges Wnt/β-catenin and BMP signaling during osteoblast differentiation of KS483 cells

    International Nuclear Information System (INIS)

    The canonical Wnt signaling pathway influences the differentiation of mesenchymal cell lineages in a quantitative and qualitative fashion depending on the dose of β-catenin signaling. Adenomatous polyposis coli (Apc) is the critical intracellular regulator of β-catenin turnover. To better understand the molecular mechanisms underlying the role of Apc in regulating the differentiation capacity of skeletal progenitor cells, we have knocked down Apc in the murine mesenchymal stem cell-like KS483 cells by stable expression of Apc-specific small interfering RNA. In routine culture, KSFrt-Apcsi cells displayed a mesenchymal-like spindle shape morphology, exhibited markedly decreased proliferation and increased apoptosis. Apc knockdown resulted in upregulation of the Wnt/β-catenin and the BMP/Smad signaling pathways, but osteogenic differentiation was completely inhibited. This effect could be rescued by adding high concentrations of BMP-7 to the differentiation medium. Furthermore, KSFrt-Apcsi cells showed no potential to differentiate into chondrocytes or adipocytes. These results demonstrate that Apc is essential for the proliferation, survival and differentiation of KS483 cells. Apc knockdown blocks the osteogenic differentiation of skeletal progenitor cells, a process that can be overruled by high BMP signaling.

  2. The Wnt and BMP Families of Signaling Morphogens at the Vertebrate Neuromuscular Junction

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    Juan P. Henríquez

    2011-12-01

    Full Text Available The neuromuscular junction has been extensively employed in order to identify crucial determinants of synaptogenesis. At the vertebrate neuromuscular synapse, extracellular matrix and signaling proteins play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in invertebrate species have revealed crucial functions of early morphogens during the assembly and maturation of the neuromuscular junction. Here, we discuss growing evidence addressing the function of Wnt and Bone morphogenetic protein (BMP signaling pathways at the vertebrate neuromuscular synapse. We focus on the emerging role of Wnt proteins as positive and negative regulators of postsynaptic differentiation. We also address the possible involvement of BMP pathways on motor neuron behavior for the assembly and/or regeneration of the neuromuscular junction.

  3. mTOR signaling promotes stem cell activation via counterbalancing BMP-mediated suppression during hair regeneration.

    Science.gov (United States)

    Deng, Zhili; Lei, Xiaohua; Zhang, Xudong; Zhang, Huishan; Liu, Shuang; Chen, Qi; Hu, Huimin; Wang, Xinyue; Ning, Lina; Cao, Yujing; Zhao, Tongbiao; Zhou, Jiaxi; Chen, Ting; Duan, Enkui

    2015-02-01

    Hair follicles (HFs) undergo cycles of degeneration (catagen), rest (telogen), and regeneration (anagen) phases. Anagen begins when the hair follicle stem cells (HFSCs) obtain sufficient activation cues to overcome suppressive signals, mainly the BMP pathway, from their niche cells. Here, we unveil that mTOR complex 1 (mTORC1) signaling is activated in HFSCs, which coincides with the HFSC activation at the telogen-to-anagen transition. By using both an inducible conditional gene targeting strategy and a pharmacological inhibition method to ablate or inhibit mTOR signaling in adult skin epithelium before anagen initiation, we demonstrate that HFs that cannot respond to mTOR signaling display significantly delayed HFSC activation and extended telogen. Unexpectedly, BMP signaling activity is dramatically prolonged in mTOR signaling-deficient HFs. Through both gain- and loss-of-function studies in vitro, we show that mTORC1 signaling negatively affects BMP signaling, which serves as a main mechanism whereby mTORC1 signaling facilitates HFSC activation. Indeed, in vivo suppression of BMP by its antagonist Noggin rescues the HFSC activation defect in mTORC1-null skin. Our findings reveal a critical role for mTOR signaling in regulating stem cell activation through counterbalancing BMP-mediated repression during hair regeneration. PMID:25609845

  4. Tsukushi modulates Xnr2, FGF and BMP signaling: regulation of Xenopus germ layer formation.

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    Samantha A Morris

    Full Text Available BACKGROUND: Cell-cell communication is essential in tissue patterning. In early amphibian development, mesoderm is formed in the blastula-stage embryo through inductive interactions in which vegetal cells act on overlying equatorial cells. Members of the TGF-beta family such as activin B, Vg1, derrière and Xenopus nodal-related proteins (Xnrs are candidate mesoderm inducing factors, with further activity to induce endoderm of the vegetal region. TGF-beta-like ligands, including BMP, are also responsible for patterning of germ layers. In addition, FGF signaling is essential for mesoderm formation whereas FGF signal inhibition has been implicated in endoderm induction. Clearly, several signaling pathways are coordinated to produce an appropriate developmental output; although intracellular crosstalk is known to integrate multiple pathways, relatively little is known about extracellular coordination. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that Xenopus Tsukushi (X-TSK, a member of the secreted small leucine rich repeat proteoglycan (SLRP family, is expressed in ectoderm, endoderm, and the organizer during early development. We have previously reported that X-TSK binds to and inhibits BMP signaling in cooperation with chordin. We now demonstrate two novel interactions: X-TSK binds to and inhibits signaling by FGF8b, in addition to binding to and enhancement of Xnr2 signaling. This signal integration by X-TSK at the extracellular level has an important role in germ layer formation and patterning. Vegetally localized X-TSK potentiates endoderm formation through coordination of BMP, FGF and Xnr2 signaling. In contrast, X-TSK inhibition of FGF-MAPK signaling blocks ventrolateral mesoderm formation, while BMP inhibition enhances organizer formation. These actions of X-TSK are reliant upon its expression in endoderm and dorsal mesoderm, with relative exclusion from ventrolateral mesoderm, in a pattern shaped by FGF signals. CONCLUSIONS

  5. Repressed BMP signaling reactivates NKL homeobox gene MSX1 in a T-ALL subset.

    Science.gov (United States)

    Nagel, Stefan; Ehrentraut, Stefan; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G; MacLeod, Roderick A F

    2015-02-01

    In T-cell acute lymphoblastic leukemia (T-ALL), several members of the NK-like (NKL) homeobox genes are aberrantly expressed. Here, we have analyzed the activity of NKL homeobox gene MSX1 using pediatric T-ALL in silico data, detecting overexpression in 11% of patients. Quantification of MSX1 transcripts in a panel of 24 T-ALL cell lines demonstrated overexpression in two examples. Comparative expression profiling indicated inhibition of the bone morphogenetic protein (BMP) signaling pathway, which was shown to inhibit MSX1 transcription. In the LOUCY cell line we identified conspicuous expression of CHRDL1 encoding a BMP inhibitor which mediated activation of MSX1. Promoter analyses demonstrated activation of CHRDL1 by oncogenic PITX1. Furthermore, knockdown and overexpression studies of hematopoietic transcription factors demonstrated that GATA2 and FOXC1 mediate activation and GATA3, LEF1, TAL1 and TOX repression of MSX1 transcription. Collectively, our findings suggest that MSX1 is physiologically restricted to lymphoid progenitors. The identification of deregulated BMP signaling may provide novel therapeutic options for the treatment of T-ALL. PMID:24844359

  6. Transdifferentiation from cornea to lens in Xenopus laevis depends on BMP signalling and involves upregulation of Wnt signalling

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    Day Robert C

    2011-09-01

    Full Text Available Abstract Background Surgical removal of the lens from larval Xenopus laevis results in a rapid transdifferention of central corneal cells to form a new lens. The trigger for this process is understood to be an induction event arising from the unprecedented exposure of the cornea to the vitreous humour that occurs following lens removal. The molecular identity of this trigger is unknown. Results Here, we have used a functional transgenic approach to show that BMP signalling is required for lens regeneration and a microarray approach to identify genes that are upregulated specifically during this process. Analysis of the array data strongly implicates Wnt signalling and the Pitx family of transcription factors in the process of cornea to lens transdifferentiation. Our analysis also captured several genes associated with congenital cataract in humans. Pluripotency genes, in contrast, were not upregulated, supporting the idea that corneal cells transdifferentiate without returning to a stem cell state. Several genes from the array were expressed in the forming lens during embryogenesis. One of these, Nipsnap1, is a known direct target of BMP signalling. Conclusions Our results strongly implicate the developmental Wnt and BMP signalling pathways in the process of cornea to lens transdifferentiation (CLT in Xenopus, and suggest direct transdifferentiation between these two anterior eye tissues.

  7. BMP Signaling Modulates Hepcidin Expression in Zebrafish Embryos Independent of Hemojuvelin

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    Gibert, Yann; Lattanzi, Victoria J.; Zhen, Aileen W.; Vedder, Lea; Brunet, Frédéric; Faasse, Sarah A.; Babitt, Jodie L.; Lin, Herbert Y.; Hammerschmidt, Matthias; Fraenkel, Paula G.

    2011-01-01

    Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv. PMID:21283739

  8. BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs

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    Finnson Kenneth

    2010-02-01

    Full Text Available Abstract Background Axolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration. Results The expression of BMP-2 and SOX-9 in developing and regenerating axolotl limbs corresponded to the first digits forming in the anterior portion of the autopods. The inhibition of sonic hedgehog signaling with cyclopamine caused hypomorphic limbs (during development and regeneration but did not affect the expression of BMP-2 and SOX-9. Overexpression of BMP-2 in regenerating limbs caused a loss of digits. Overexpression of Noggin (BMP inhibitor in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to BMP-2 overexpression was the result of increased cell condensation and apoptosis while the loss caused by Noggin was due to a decrease in cell division. Conclusion The expression of BMP-2 and its target SOX-9 was independent of sonic hedgehog signaling in developing and regenerating limbs. Their expression correlated with chondrogenesis and the appearance of skeletal elements has

  9. A transient wave of BMP signaling in the retina is necessary for Müller glial differentiation.

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    Ueki, Yumi; Wilken, Matthew S; Cox, Kristen E; Chipman, Laura B; Bermingham-McDonogh, Olivia; Reh, Thomas A

    2015-02-01

    The primary glial cells in the retina, the Müller glia, differentiate from retinal progenitors in the first postnatal week. CNTF/LIF/STAT3 signaling has been shown to promote their differentiation; however, another key glial differentiation signal, BMP, has not been examined during this period of Müller glial differentiation. In the course of our analysis of the BMP signaling pathway, we observed a transient wave of Smad1/5/8 signaling in the inner nuclear layer at the end of the first postnatal week, from postnatal day (P) 5 to P9, after the end of neurogenesis. To determine the function of this transient wave, we blocked BMP signaling during this period in vitro or in vivo, using either a BMP receptor antagonist or noggin (Nog). Either treatment leads to a reduction in expression of the Müller glia-specific genes Rlbp1 and Glul, and the failure of many of the Müller glia to repress the bipolar/photoreceptor gene Otx2. These changes in normal Müller glial differentiation result in permanent disruption of the retina, including defects in the outer limiting membrane, rosette formation and a reduction in functional acuity. Our results thus show that Müller glia require a transient BMP signal at the end of neurogenesis to fully repress the neural gene expression program and to promote glial gene expression. PMID:25605781

  10. The signalling mucin Msb2 regulates surface sensing and host penetration via BMP1 MAP kinase signalling in Botrytis cinerea.

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    Leroch, Michaela; Mueller, Nathalie; Hinsenkamp, Isabel; Hahn, Matthias

    2015-10-01

    Botrytis cinerea is a necrotrophic fungus that infects a wide range of fruit, vegetable and flower crops. Penetration of the host cuticle occurs via infection structures that are formed in response to appropriate plant surface signals. The differentiation of these structures requires a highly conserved mitogen-activated protein (MAP) kinase cascade including the MAP kinase BMP1. In yeast and several plant-pathogenic fungi, the signalling mucin Msb2 has been shown to be involved in surface recognition and MAP kinase activation. In this study, a B. cinerea msb2 mutant was generated and characterized. The mutant showed normal growth, sporulation, sclerotia formation and stress resistance. In the absence of nutrients, abnormal germination with multiple germ tubes was observed. In the presence of sugars, normal germination occurred, but msb2 germlings were almost unable to form appressoria or infection cushions on hard surfaces. Nevertheless, the msb2 mutant showed only a moderate delay in lesion formation on different host plants, and formed expanding lesions similar to the wild-type. Although the wild-type showed increasing BMP1 phosphorylation during the first hours of germination on hard surfaces, the phosphorylation levels in the msb2 mutant were strongly reduced. Several genes encoding secreted proteins were found to be co-regulated by BMP1 and Msb2 during germination. Taken together, B. cinerea Msb2 is likely to represent a hard surface sensor of germlings and hyphae that triggers infection structure formation via the activation of the BMP1 MAP kinase pathway. PMID:25582910

  11. Human carcinoma-associated mesenchymal stem cells promote ovarian cancer chemotherapy resistance via a BMP4/HH signaling loop

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    Coffman, Lan G.; Choi, Yun-Jung; McLean, Karen; Allen, Benjamin L.; di Magliano, Marina Pasca; Buckanovich, Ronald J.

    2016-01-01

    The tumor microenvironment is critical to cancer growth and therapy resistance. We previously characterized human ovarian carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are multi-potent cells that can differentiate into tumor microenvironment components including fibroblasts, myofibroblasts and adipocytes. We previously reported CA-MSCs, compared to normal MSCs, express high levels of BMP proteins and promote tumor growth by increasing numbers of cancer stem-like cells (CSCs). We demonstrate here that ovarian tumor cell-secreted Hedgehog (HH) induces CA-MSC BMP4 expression. CA-MSC-derived BMP4 reciprocally increases ovarian tumor cell HH expression indicating a positive feedback loop. Interruption of this loop with a HH pathway inhibitor or BMP4 blocking antibody decreases CA-MSC-derived BMP4 and tumor-derived HH preventing enrichment of CSCs and reversing chemotherapy resistance. The impact of HH inhibition was only seen in CA-MSC-containing tumors, indicating the importance of a humanized stroma. These results are reciprocal to findings in pancreatic and bladder cancer, suggesting HH signaling effects are tumor tissue specific warranting careful investigation in each tumor type. Collectively, we define a critical positive feedback loop between CA-MSC-derived BMP4 and ovarian tumor cell-secreted HH and present evidence for the further investigation of HH as a clinical target in ovarian cancer. PMID:26755648

  12. Limiting hepatic Bmp-Smad signaling by matriptase-2 is required for erythropoietin-mediated hepcidin suppression in mice.

    Science.gov (United States)

    Nai, Antonella; Rubio, Aude; Campanella, Alessandro; Gourbeyre, Ophélie; Artuso, Irene; Bordini, Jessica; Gineste, Aurélie; Latour, Chloé; Besson-Fournier, Céline; Lin, Herbert Y; Coppin, Hélène; Roth, Marie-Paule; Camaschella, Clara; Silvestri, Laura; Meynard, Delphine

    2016-05-12

    Hepcidin, the main regulator of iron homeostasis, is repressed when erythropoiesis is acutely stimulated by erythropoietin (EPO) to favor iron supply to maturing erythroblasts. Erythroferrone (ERFE) has been identified as the erythroid regulator that inhibits hepcidin in stress erythropoiesis. A powerful hepcidin inhibitor is the serine protease matriptase-2, encoded by TMPRSS6, whose mutations cause iron refractory iron deficiency anemia. Because this condition has inappropriately elevated hepcidin in the presence of high EPO levels, a role is suggested for matriptase-2 in EPO-mediated hepcidin repression. To investigate the relationship between EPO/ERFE and matriptase-2, we show that EPO injection induces Erfe messenger RNA expression but does not suppress hepcidin in Tmprss6 knockout (KO) mice. Similarly, wild-type (WT) animals, in which the bone morphogenetic protein-mothers against decapentaplegic homolog (Bmp-Smad) pathway is upregulated by iron treatment, fail to suppress hepcidin in response to EPO. To further investigate whether the high level of Bmp-Smad signaling of Tmprss6 KO mice counteracts hepcidin suppression by EPO, we generated double KO Bmp6-Tmprss6 KO mice. Despite having Bmp-Smad signaling and hepcidin levels that are similar to WT mice under basal conditions, double KO mice do not suppress hepcidin in response to EPO. However, pharmacologic downstream inhibition of the Bmp-Smad pathway by dorsomorphin, which targets the BMP receptors, improves the hepcidin responsiveness to EPO in Tmprss6 KO mice. We concluded that the function of matriptase-2 is dominant over that of ERFE and is essential in facilitating hepcidin suppression by attenuating the BMP-SMAD signaling. PMID:26755707

  13. Patterning of the dorsal-ventral axis in echinoderms: insights into the evolution of the BMP-chordin signaling network.

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    François Lapraz

    2009-11-01

    Full Text Available Formation of the dorsal-ventral axis of the sea urchin embryo relies on cell interactions initiated by the TGFbeta Nodal. Intriguingly, although nodal expression is restricted to the ventral side of the embryo, Nodal function is required for specification of both the ventral and the dorsal territories and is able to restore both ventral and dorsal regions in nodal morpholino injected embryos. The molecular basis for the long-range organizing activity of Nodal is not understood. In this paper, we provide evidence that the long-range organizing activity of Nodal is assured by a relay molecule synthesized in the ventral ectoderm, then translocated to the opposite side of the embryo. We identified this relay molecule as BMP2/4 based on the following arguments. First, blocking BMP2/4 function eliminated the long-range organizing activity of an activated Nodal receptor in an axis rescue assay. Second, we demonstrate that BMP2/4 and the corresponding type I receptor Alk3/6 functions are both essential for specification of the dorsal region of the embryo. Third, using anti-phospho-Smad1/5/8 immunostaining, we show that, despite its ventral transcription, the BMP2/4 ligand triggers receptor mediated signaling exclusively on the dorsal side of the embryo, one of the most extreme cases of BMP translocation described so far. We further report that the pattern of pSmad1/5/8 is graded along the dorsal-ventral axis and that two BMP2/4 target genes are expressed in nested patterns centered on the region with highest levels of pSmad1/5/8, strongly suggesting that BMP2/4 is acting as a morphogen. We also describe the very unusual ventral co-expression of chordin and bmp2/4 downstream of Nodal and demonstrate that Chordin is largely responsible for the spatial restriction of BMP2/4 signaling to the dorsal side. Thus, unlike in most organisms, in the sea urchin, a single ventral signaling centre is responsible for induction of ventral and dorsal cell fates. Finally

  14. Transferrin receptor facilitates TGF-β and BMP signaling activation to control craniofacial morphogenesis.

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    Lei, R; Zhang, K; Liu, K; Shao, X; Ding, Z; Wang, F; Hong, Y; Zhu, M; Li, H; Li, H

    2016-01-01

    The Pierre Robin Sequence (PRS), consisting of cleft palate, glossoptosis and micrognathia, is a common human birth defect. However, how this abnormality occurs remains largely unknown. Here we report that neural crest cell (NCC)-specific knockout of transferrin receptor (Tfrc), a well known transferrin transporter protein, caused micrognathia, cleft palate, severe respiratory distress and inability to suckle in mice, which highly resemble human PRS. Histological and anatomical analysis revealed that the cleft palate is due to the failure of palatal shelves elevation that resulted from a retarded extension of Meckel's cartilage. Interestingly, Tfrc deletion dramatically suppressed both transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling in cranial NCCs-derived mandibular tissues, suggesting that Tfrc may act as a facilitator of these two signaling pathways during craniofacial morphogenesis. Together, our study uncovers an unknown function of Tfrc in craniofacial development and provides novel insight into the etiology of PRS. PMID:27362800

  15. Zirconium ions up-regulate the BMP/SMAD signaling pathway and promote the proliferation and differentiation of human osteoblasts.

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    Yongjuan Chen

    Full Text Available Zirconium (Zr is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2 or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV oxynitrate (ZrO(NO32 at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.

  16. Sprouty2 regulates endochondral bone formation by modulation of RTK and BMP signaling.

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    Joo, Adriane; Long, Roger; Cheng, Zhiqiang; Alexander, Courtney; Chang, Wenhan; Klein, Ophir D

    2016-07-01

    Skeletal development is regulated by the coordinated activity of signaling molecules that are both produced locally by cartilage and bone cells and also circulate systemically. During embryonic development and postnatal bone remodeling, receptor tyrosine kinase (RTK) superfamily members play critical roles in the proliferation, survival, and differentiation of chondrocytes, osteoblasts, osteoclasts, and other bone cells. Recently, several molecules that regulate RTK signaling have been identified, including the four members of the Sprouty (Spry) family (Spry1-4). We report that Spry2 plays an important role in regulation of endochondral bone formation. Mice in which the Spry2 gene has been deleted have defective chondrogenesis and endochondral bone formation, with a postnatal decrease in skeletal size and trabecular bone mass. In these constitutive Spry2 mutants, both chondrocytes and osteoblasts undergo increased cell proliferation and impaired terminal differentiation. Tissue-specific Spry2 deletion by either osteoblast- (Col1-Cre) or chondrocyte- (Col2-Cre) specific drivers led to decreased relative bone mass, demonstrating the critical role of Spry2 in both cell types. Molecular analyses of signaling pathways in Spry2(-/-) mice revealed an unexpected upregulation of BMP signaling and decrease in RTK signaling. These results identify Spry2 as a critical regulator of endochondral bone formation that modulates signaling in both osteoblast and chondrocyte lineages. PMID:27130872

  17. Traf2 interacts with Smad4 and regulates BMP signaling pathway in MC3T3-E1 osteoblasts

    International Nuclear Information System (INIS)

    Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate in the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from bone marrow, to indentify the proteins interacting with Smad4. cDNA clones for Tumor necrosis factor (TNF) receptor-associated factor 2 (Traf2) were identified, and the interaction between the endogenous proteins was confirmed in the mouse osteoblast cell line MC3T3-E1. To investigate the function of Traf2, we silenced it with siRNA. The level of BMP-2 protein in the medium, the expression levels of the Bmp2 gene and BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Sp7, and the phosphorylated-Smad1 protein level were increased in cells transfected with Traf2 siRNA. The nuclear accumulation of Smad1 increased with TNF-α stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-α-stimulated nuclear accumulation of Smad1 may be dependent on Traf2. Thus, the interaction between Traf2 and Smad4 may play a role in the cross-talk between TNF-α and BMP signaling pathways.

  18. Integration of a Notch-dependent mesenchymal gene program and Bmp2-driven cell invasiveness regulates murine cardiac valve formation.

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    Luna-Zurita, Luis; Prados, Belén; Grego-Bessa, Joaquim; Luxán, Guillermo; del Monte, Gonzalo; Benguría, Alberto; Adams, Ralf H; Pérez-Pomares, José María; de la Pompa, José Luis

    2010-10-01

    Cardiac valve formation is crucial for embryonic and adult heart function. Valve malformations constitute the most common congenital cardiac defect, but little is known about the molecular mechanisms regulating valve formation and homeostasis. Here, we show that endocardial Notch1 and myocardial Bmp2 signal integration establish a valve-forming field between 2 chamber developmental domains. Patterning occurs through the activation of endocardial epithelial-to-mesenchymal transition (EMT) exclusively in prospective valve territories. Mice with constitutive endocardial Notch1 activity ectopically express Hey1 and Heyl. They also display an activated mesenchymal gene program in ventricles and a partial (noninvasive) EMT in vitro that becomes invasive upon BMP2 treatment. Snail1, TGF-β2, or Notch1 inhibition reduces BMP2-induced ventricular transformation and invasion, whereas BMP2 treatment inhibits endothelial Gsk3β, stabilizing Snail1 and promoting invasiveness. Integration of Notch and Bmp2 signals is consistent with Notch1 signaling being attenuated after myocardial Bmp2 deletion. Notch1 activation in myocardium extends Hey1 expression to nonchamber myocardium, represses Bmp2, and impairs EMT. In contrast, Notch deletion abrogates endocardial Hey gene transcription and extends Bmp2 expression to the ventricular endocardium. This embryonic Notch1-Bmp2-Snail1 relationship may be relevant in adult valve disease, in which decreased NOTCH signaling causes valve mesenchyme cell formation, fibrosis, and calcification. PMID:20890042

  19. Calycosin-7-O-β-d-glucopyranoside stimulates osteoblast differentiation through regulating the BMP/WNT signaling pathways

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    Jing Jian

    2015-09-01

    Full Text Available The isoflavone calycosin-7-O-β-d-glucopyranoside (CG is a principal constituent of Astragalus membranaceus (AR and has been reported to inhibit osteoclast development in vitro and bone loss in vivo. The aim of this study was to investigate the osteogenic effects of CG and its underlying mechanism in ST2 cells. The results show that exposure of cells to CG in osteogenic differentiation medium increases ALP activity, osteocalcin (Ocal mRNA expression and the osteoblastic mineralization process. Mechanistically, CG treatment increased the expression of bone morphogenetic protein 2 (BMP-2, p-Smad 1/5/8, β-catenin and Runx2, all of which are regulators of the BMP- or wingless-type MMTV integration site family (WNT/β-catenin-signaling pathways. Moreover, the osteogenic effects of CG were inhibited by Noggin and DKK-1 which are classical inhibitors of the BMP and WNT/β-catenin-signaling pathways, respectively. Taken together, the results indicate that CG promotes the osteoblastic differentiation of ST2 cells through regulating the BMP/WNT signaling pathways. On this basis, CG may be a useful lead compound for improving the treatment of bone-decreasing diseases and enhancing bone regeneration.

  20. Abrogation of prostaglandin E-EP4 signaling in osteoblasts prevents the bone destruction induced by human prostate cancer metastases.

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    Watanabe, Kenta; Tominari, Tsukasa; Hirata, Michiko; Matsumoto, Chiho; Maruyama, Takayuki; Murphy, Gillian; Nagase, Hideaki; Miyaura, Chisato; Inada, Masaki

    2016-09-01

    The metastasis of tumors to bone is known to be promoted by prostaglandin E2 (PGE2) produced by the tumor host stromal tissue. Although bone metastases frequently occur in prostate cancer patients, the significance of PGE2 in stromal responses to the tumor is not known. In this study, we report that PGE2 and its receptor EP4 play a pivotal role in bone destruction and metastasis in an experimental metastasis model of prostate cancer in nude mice. Using human prostate cancer PC-3 cells that are stably transfected with luciferase, we showed that the development of bone metastasis was accompanied by increased osteoclastic bone resorption in the bone metastasis microenvironment, and could be abrogated by an EP4 receptor antagonist. The growth of PC-3 cells in vitro was not influenced by PGE2 or by the EP4 receptor. However, cell-cell interactions between fixed PC-3 cells and host osteoblasts induced PGE2 production and RANKL expression in the osteoblasts. Addition of an EP4 antagonist suppressed both PGE2 and RANKL expression induced by the PC3-osteoblast interaction, which would have consequent effects on osteoclast activation and osteolysis. These results indicate that the blockage of PGE2-EP4 signaling prevents the bone destruction required for prostate cancer metastases, and that this is, in part due to the abrogation of bone cell responses. The study provides further evidence that an EP4 antagonist is a candidate for the treatment of prostate cancer in the blockade of bone metastasis. PMID:27450806

  1. ERK5 signalling rescues intestinal epithelial turnover and tumour cell proliferation upon ERK1/2 abrogation

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    de Jong, Petrus R.; Taniguchi, Koji; Harris, Alexandra R.; Bertin, Samuel; Takahashi, Naoki; Duong, Jen; Campos, Alejandro D.; Powis, Garth; Corr, Maripat; Karin, Michael; Raz, Eyal

    2016-01-01

    The ERK1/2 MAPK signalling module integrates extracellular cues that induce proliferation and differentiation of epithelial lineages, and is an established oncogenic driver, particularly in the intestine. However, the interrelation of the ERK1/2 module relative to other signalling pathways in intestinal epithelial cells and colorectal cancer (CRC) is unclear. Here we show that loss of Erk1/2 in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration and secretory cell differentiation. However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion of Erk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines. These results suggest that ERK5 provides a common bypass route in intestinal epithelial cells, which rescues cell proliferation upon abrogation of ERK1/2 signalling, with therapeutic implications in CRC. PMID:27187615

  2. The Balance of Cell Surface and Soluble Type III TGF-β Receptor Regulates BMP Signaling in Normal and Cancerous Mammary Epithelial Cells

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    Catherine E. Gatza

    2014-06-01

    Full Text Available Bone morphogenetic proteins (BMPs are members of the TGF-β superfamily that are over-expressed in breast cancer, with context dependent effects on breast cancer pathogenesis. The type III TGF-β receptor (TβRIII mediates BMP signaling. While TβRIII expression is lost during breast cancer progression, the role of TβRIII in regulating BMP signaling in normal mammary epithelium and breast cancer cells has not been examined. Restoring TβRIII expression in a 4T1 murine syngeneic model of breast cancer suppressed Smad1/5/8 phosphorylation and inhibited the expression of the BMP transcriptional targets, Id1 and Smad6, in vivo. Similarly, restoring TβRIII expression in human breast cancer cell lines or treatment with sTβRIII inhibited BMP-induced Smad1/5/8 phosphorylation and BMP-stimulated migration and invasion. In normal mammary epithelial cells, shRNA-mediated silencing of TβRIII, TβRIII over-expression, or treatment with sTβRIII inhibited BMP-mediated phosphorylation of Smad1/5/8 and BMP induced migration. Inhibition of TβRIII shedding through treatment with TAPI-2 or expression of a non-shedding TβRIII mutant rescued TβRIII mediated inhibition of BMP induced Smad1/5/8 phosphorylation and BMP induced migration and/or invasion in both in normal mammary epithelial cells and breast cancer cells. Conversely, expression of a TβRIII mutant, which exhibited increased shedding, significantly reduced BMP-mediated Smad1/5/8 phosphorylation, migration, and invasion. These data demonstrate that TβRIII regulates BMP-mediated signaling and biological effects, primarily through the ligand sequestration effects of sTβRIII in normal and cancerous mammary epithelial cells and suggest that the ratio of membrane bound versus sTβRIII plays an important role in mediating these effects.

  3. Specific induction of cranial placode cells from Xenopus ectoderm by modulating the levels of BMP, Wnt, and FGF signaling.

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    Watanabe, Tomoko; Kanai, Yuna; Matsukawa, Shinya; Michiue, Tatsuo

    2015-10-01

    The neural-epidermal boundary tissues include the neural crest and preplacodal ectoderm (PPE) as primordial constituents. The PPE region is essential for the development of various sensory and endocrine organs, such as the anterior lobe of the pituitary, olfactory epithelium, lens, trigeminal ganglion, and otic vesicles. During gastrulation, a neural region is induced in ectodermal cells that interacts with mesendodermal tissue and responds to several secreted factors. Among them, inhibition of bone morphogenetic protein (BMP) in the presumptive neuroectoderm is essential for the induction of neural regions, and formation of a Wnt and fibroblast growth factor (FGF) signaling gradient along the midline determines anterior-posterior patterning. In this study, we attempted to specifically induce PPE cells from undifferentiated Xenopus cells by regulating BMP, Wnt, and FGF signaling. We showed that the proper level of BMP inhibition with an injection of truncated BMP receptor or treatment with a chemical antagonist triggered the expression of PPE genes. In addition, by varying the amount of injected chordin, we optimized specific expression of the PPE genes. PPE gene expression is increased by adding an appropriate dose of an FGF receptor antagonist. Furthermore, co-injection with either wnt8 or the Wnt inhibitor dkk-1 altered the expression levels of several region-specific genes according to the injected dose. We specifically induced PPE cell differentiation in animal cap cells from early-stage Xenopus embryos by modulating BMP, Wnt, and FGF signaling. This is not the first research on placode induction, but our simple method could potentially be applied to mammalian stem cell systems. PMID:26249012

  4. Wnt signaling interacts with bmp and edn1 to regulate dorsal-ventral patterning and growth of the craniofacial skeleton.

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    Courtney Alexander

    2014-07-01

    Full Text Available Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D and ventral (V elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3, (Tg(hsp70I:tcf3-GFP, or the canonical Wnt inhibitor dickkopf1 (dkk1, (Tg(hsp70i:dkk1-GFP after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe, and ventral cartilage differentiation (e.g. lower jaws. These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1 protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.

  5. Embryonic Ethanol Exposure Dysregulates BMP and Notch Signaling, Leading to Persistent Atrio-Ventricular Valve Defects in Zebrafish.

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    Sarmah, Swapnalee; Muralidharan, Pooja; Marrs, James A

    2016-01-01

    Fetal alcohol spectrum disorder (FASD), birth defects associated with ethanol exposure in utero, includes a wide spectrum of congenital heart defects (CHDs), the most prevalent of which are septal and conotruncal defects. Zebrafish FASD model was used to dissect the mechanisms underlying FASD-associated CHDs. Embryonic ethanol exposure (3-24 hours post fertilization) led to defects in atrio-ventricular (AV) valvulogenesis beginning around 37 hpf, a morphogenetic event that arises long after ethanol withdrawal. Valve leaflets of the control embryos comprised two layers of cells confined at the compact atrio-ventricular canal (AVC). Ethanol treated embryos had extended AVC and valve forming cells were found either as rows of cells spanning the AVC or as unorganized clusters near the AV boundary. Ethanol exposure reduced valve precursors at the AVC, but some ventricular cells in ethanol treated embryos exhibited few characteristics of valve precursors. Late staged larvae and juvenile fish exposed to ethanol during embryonic development had faulty AV valves. Examination of AVC morphogenesis regulatory networks revealed that early ethanol exposure disrupted the Bmp signaling gradient in the heart during valve formation. Bmp signaling was prominent at the AVC in controls, but ethanol-exposed embryos displayed active Bmp signaling throughout the ventricle. Ethanol exposure also led to mislocalization of Notch signaling cells in endocardium during AV valve formation. Normally, highly active Notch signaling cells were organized at the AVC. In ethanol-exposed embryos, highly active Notch signaling cells were dispersed throughout the ventricle. At later stages, ethanol-exposed embryos exhibited reduced Wnt/β-catenin activity at the AVC. We conclude that early embryonic ethanol exposure alters Bmp, Notch and other signaling activities during AVC differentiation leading to faulty valve morphogenesis and valve defects persist in juvenile fish. PMID:27556898

  6. A Targeted Glycan-Related Gene Screen Reveals Heparan Sulfate Proteoglycan Sulfation Regulates WNT and BMP Trans-Synaptic Signaling

    Science.gov (United States)

    Dani, Neil; Nahm, Minyeop; Lee, Seungbok; Broadie, Kendal

    2012-01-01

    A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS) 6-O-sulfotransferase (hs6st) and sulfatase (sulf1), which bidirectionally control HS proteoglycan (HSPG) sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st) and increased (sulf1) neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg) and BMP (Glass Bottom Boat; Gbb) ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects. PMID:23144627

  7. A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling.

    Directory of Open Access Journals (Sweden)

    Neil Dani

    Full Text Available A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS 6-O-sulfotransferase (hs6st and sulfatase (sulf1, which bidirectionally control HS proteoglycan (HSPG sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st and increased (sulf1 neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg and BMP (Glass Bottom Boat; Gbb ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects.

  8. Augmented noncanonical BMP type II receptor signaling mediates the synaptic abnormality of fragile X syndrome.

    Science.gov (United States)

    Kashima, Risa; Roy, Sougata; Ascano, Manuel; Martinez-Cerdeno, Veronica; Ariza-Torres, Jeanelle; Kim, Sunghwan; Louie, Justin; Lu, Yao; Leyton, Patricio; Bloch, Kenneth D; Kornberg, Thomas B; Hagerman, Paul J; Hagerman, Randi; Lagna, Giorgio; Hata, Akiko

    2016-01-01

    Epigenetic silencing of fragile X mental retardation 1 (FMR1) causes fragile X syndrome (FXS), a common inherited form of intellectual disability and autism. FXS correlates with abnormal synapse and dendritic spine development, but the molecular link between the absence of the FMR1 product FMRP, an RNA binding protein, and the neuropathology is unclear. We found that the messenger RNA encoding bone morphogenetic protein type II receptor (BMPR2) is a target of FMRP. Depletion of FMRP increased BMPR2 abundance, especially that of the full-length isoform that bound and activated LIM domain kinase 1 (LIMK1), a component of the noncanonical BMP signal transduction pathway that stimulates actin reorganization to promote neurite outgrowth and synapse formation. Heterozygosity for BMPR2 rescued the morphological abnormalities in neurons both in Drosophila and in mouse models of FXS, as did the postnatal pharmacological inhibition of LIMK1 activity. Compared with postmortem prefrontal cortex tissue from healthy subjects, the amount of full-length BMPR2 and of a marker of LIMK1 activity was increased in this brain region from FXS patients. These findings suggest that increased BMPR2 signal transduction is linked to FXS and that the BMPR2-LIMK1 pathway is a putative therapeutic target in patients with FXS and possibly other forms of autism. PMID:27273096

  9. Increased iron loading induces Bmp6 expression in the non-parenchymal cells of the liver independent of the BMP-signaling pathway.

    Directory of Open Access Journals (Sweden)

    Caroline A Enns

    Full Text Available Bone morphogenetic protein 6 (BMP6 is an essential cytokine for the expression of hepcidin, an iron regulatory hormone secreted predominantly by hepatocytes. Bmp6 expression is upregulated by increased iron-levels in the liver. Both hepatocytes and non-parenchymal liver cells have detectable Bmp6 mRNA. Here we showed that induction of hepcidin expression in hepatocytes by dietary iron is associated with an elevation of Bmp6 mRNA in the non-parenchymal cells of the liver. Consistently, incubation with iron-saturated transferrin induces Bmp6 mRNA expression in isolated hepatic stellate cells, but not in hepatocytes. These observations suggest an important role of the non-parenchymal liver cells in regulating iron-homeostasis by acting as a source of Bmp6.

  10. Combined inhibition of p38 and Akt signaling pathways abrogates cyclosporine A-mediated pathogenesis of aggressive skin SCCs

    International Nuclear Information System (INIS)

    Highlights: ► p38 and Akt are the crucial molecular targets in the pathogenesis of SCCs in OTRs. ► Combined inhibition of these targets diminished tumor growth by 90%. ► Inhibition of these targets act through downregulating mTOR signaling pathway. -- Abstract: Non-melanoma skin cancers (NMSCs) are the most common neoplasm in organ transplant recipients (OTRs). These cancers are more invasive and metastatic as compared to those developed in normal cohorts. Previously, we have shown that immunosuppressive drug, cyclosporine A (CsA) directly alters tumor phenotype of cutaneous squamous cell carcinomas (SCCs) by activating TGF-β and TAK1/TAB1 signaling pathways. Here, we identified novel molecular targets for the therapeutic intervention of these SCCs. We observed that combined blockade of Akt and p38 kinases-dependent signaling pathways in CsA-promoted human epidermoid carcinoma A431 xenograft tumors abrogated their growth by more than 90%. This diminution in tumor growth was accompanied by a significant decrease in proliferation and an increase in apoptosis. The residual tumors following the combined treatment with Akt inhibitor triciribine and p38 inhibitors SB-203580 showed significantly diminished expression of phosphorylated Akt and p38 and these tumors were less invasive and highly differentiated. Diminished tumor invasiveness was associated with the reduced epithelial–mesenchymal transition as ascertained by the enhanced E-cadherin and reduced vimentin and N-cadherin expression. Consistently, these tumors also manifested reduced MMP-2/9. The decreased p-Akt expression was accompanied by a significant reduction in p-mTOR. These data provide first important combinatorial pharmacological approach to block the pathogenesis of CsA-induced highly aggressive cutaneous neoplasm in OTRs.

  11. Combined inhibition of p38 and Akt signaling pathways abrogates cyclosporine A-mediated pathogenesis of aggressive skin SCCs

    Energy Technology Data Exchange (ETDEWEB)

    Arumugam, Aadithya; Walsh, Stephanie B.; Xu, Jianmin; Afaq, Farrukh [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Elmets, Craig A. [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer p38 and Akt are the crucial molecular targets in the pathogenesis of SCCs in OTRs. Black-Right-Pointing-Pointer Combined inhibition of these targets diminished tumor growth by 90%. Black-Right-Pointing-Pointer Inhibition of these targets act through downregulating mTOR signaling pathway. -- Abstract: Non-melanoma skin cancers (NMSCs) are the most common neoplasm in organ transplant recipients (OTRs). These cancers are more invasive and metastatic as compared to those developed in normal cohorts. Previously, we have shown that immunosuppressive drug, cyclosporine A (CsA) directly alters tumor phenotype of cutaneous squamous cell carcinomas (SCCs) by activating TGF-{beta} and TAK1/TAB1 signaling pathways. Here, we identified novel molecular targets for the therapeutic intervention of these SCCs. We observed that combined blockade of Akt and p38 kinases-dependent signaling pathways in CsA-promoted human epidermoid carcinoma A431 xenograft tumors abrogated their growth by more than 90%. This diminution in tumor growth was accompanied by a significant decrease in proliferation and an increase in apoptosis. The residual tumors following the combined treatment with Akt inhibitor triciribine and p38 inhibitors SB-203580 showed significantly diminished expression of phosphorylated Akt and p38 and these tumors were less invasive and highly differentiated. Diminished tumor invasiveness was associated with the reduced epithelial-mesenchymal transition as ascertained by the enhanced E-cadherin and reduced vimentin and N-cadherin expression. Consistently, these tumors also manifested reduced MMP-2/9. The decreased p-Akt expression was accompanied by a significant reduction in p-mTOR. These data provide first important combinatorial pharmacological approach to block the pathogenesis of CsA-induced highly aggressive cutaneous neoplasm in OTRs.

  12. Quercetin abrogates IL-6/STAT3 signaling and inhibits glioblastoma cell line growth and migration

    Energy Technology Data Exchange (ETDEWEB)

    Michaud-Levesque, Jonathan; Bousquet-Gagnon, Nathalie; Beliveau, Richard, E-mail: oncomol@nobel.si.uqam.ca

    2012-05-01

    Evidence has suggested that STAT3 functions as an oncogene in gliomagenesis. As a consequence, changes in the inflammatory microenvironment are thought to promote tumor development. Regardless of its origin, cancer-related inflammation has many tumor-promoting effects, such as the promotion of cell cycle progression, cell proliferation, cell migration and cell survival. Given that IL-6, a major cancer-related inflammatory cytokine, regulates STAT3 activation and is upregulated in glioblastoma, we sought to investigate the inhibitory effects of the chemopreventive flavonoid quercetin on glioblastoma cell proliferation and migration triggered by IL-6, and to determine the underlying mechanisms of action. In this study, we show that quercetin is a potent inhibitor of the IL-6-induced STAT3 signaling pathway in T98G and U87 glioblastoma cells. Exposure to quercetin resulted in the reduction of GP130, JAK1 and STAT3 activation by IL-6, as well as a marked decrease of the proliferative and migratory properties of glioblastoma cells induced by IL-6. Interestingly, quercetin also modulated the expression of two target genes regulated by STAT3, i.e. cyclin D1 and matrix metalloproteinase-2 (MMP-2). Moreover, quercetin reduced the recruitment of STAT3 at the cyclin D1 promoter and inhibited Rb phosphorylation in the presence of IL-6. Overall, these results provide new insight into the role of quercetin as a blocker of the STAT3 activation pathway stimulated by IL-6, with a potential role in the prevention and treatment of glioblastoma.

  13. Quercetin abrogates IL-6/STAT3 signaling and inhibits glioblastoma cell line growth and migration

    International Nuclear Information System (INIS)

    Evidence has suggested that STAT3 functions as an oncogene in gliomagenesis. As a consequence, changes in the inflammatory microenvironment are thought to promote tumor development. Regardless of its origin, cancer-related inflammation has many tumor-promoting effects, such as the promotion of cell cycle progression, cell proliferation, cell migration and cell survival. Given that IL-6, a major cancer-related inflammatory cytokine, regulates STAT3 activation and is upregulated in glioblastoma, we sought to investigate the inhibitory effects of the chemopreventive flavonoid quercetin on glioblastoma cell proliferation and migration triggered by IL-6, and to determine the underlying mechanisms of action. In this study, we show that quercetin is a potent inhibitor of the IL-6-induced STAT3 signaling pathway in T98G and U87 glioblastoma cells. Exposure to quercetin resulted in the reduction of GP130, JAK1 and STAT3 activation by IL-6, as well as a marked decrease of the proliferative and migratory properties of glioblastoma cells induced by IL-6. Interestingly, quercetin also modulated the expression of two target genes regulated by STAT3, i.e. cyclin D1 and matrix metalloproteinase-2 (MMP-2). Moreover, quercetin reduced the recruitment of STAT3 at the cyclin D1 promoter and inhibited Rb phosphorylation in the presence of IL-6. Overall, these results provide new insight into the role of quercetin as a blocker of the STAT3 activation pathway stimulated by IL-6, with a potential role in the prevention and treatment of glioblastoma.

  14. Naringin abrogated radiation induced oxidative stress through modulation of redox regulated cellular signaling system

    International Nuclear Information System (INIS)

    Ionizing radiation is widely used as major diagnostic and therapeutic applications. However, the deleterious effects of ionizing radiation are due to generation of reactive oxygen species. The amounts of ionizing radiation that can be given to treat malignant tumours are often limited by toxicity in the surrounding normal tissues and organs. The aim of this study was to investigate the role of Naringin (NG), a natural flavonoid, present in many plant parts against radiation induced oxidative stress with an evidence based exploration of the mechanism involved. Isolated murine splenocyte were irradiated with γ radiation (6 Gy) along with/without different concentrations of NG (50 and 100 μM). Biochemical, immunoblot, flow cytometry and immunofluorescence study was subject to be performed to observe its molecular mechanisms of action. Pretreatment with NG significantly prevented the radiation induced intracellular ROS generation, therefore prevented cellular TBARS formation and development of cellular nitrite. NG showed the significant reduction in nuclear DNA damage with respect to irradiated splenocyte through inhibition of DNA-PKcs and p-γH2AX. It recovered radiation induced reduced cell viability through modulation of redox regulated cell signaling system. It resulted in significant inhibition of radiation induced G1/S phase cell cycle arrest mediated by modulation of p53 dependent p21/WAF1 expression followed by Cyclin E and CDK2 expression. NG was involved in blocking radiation induced p38 function; reversed radiation mediated differential stress response through inhibition of NF-κB pathway. It prevented p-IKKα/β, p-IκBα, p-p65, COX2 expression. It also reversed the radiation induced p38/NF-κB guided inflammatory development. Thus it down regulated radiation induced CRP, MCP-1, and iNOS2 gene expression. This novel role of naringin provides a basis for therapeutic applications in future against radiation induced molecular and cellular functional

  15. Essential role of Bmp signaling and its positive feedback loop in the early cell fate evolution of chordates

    Czech Academy of Sciences Publication Activity Database

    Kozmiková, Iryna; Candiani, S.; Fabian, Peter; Gurská, Daniela; Kozmik, Zbyněk

    2013-01-01

    Roč. 382, č. 2 (2013), s. 538-554. ISSN 0012-1606 R&D Projects: GA ČR GCP305/10/J064; GA MŠk EE2.3.30.0027 Institutional support: RVO:68378050 Keywords : Bmp signaling * axial patterning * cell fate * chordates * evolution Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.637, year: 2013

  16. BMP signalling regulates the pre-implantation development of extra-embryonic cell lineages in the mouse embryo

    OpenAIRE

    Graham, Sarah J. L.; Wicher, Krzysztof B.; Jedrusik, Agnieszka; Guo, Guoji; Herath, Wishva; Robson, Paul; Zernicka-Goetz, Magdalena

    2014-01-01

    Pre-implantation development requires the specification and organization of embryonic and extra-embryonic lineages. The separation of these lineages takes place when asymmetric divisions generate inside and outside cells that differ in polarity, position and fate. Here we assess the global transcriptional identities of these precursor cells to gain insight into the molecular mechanisms regulating lineage segregation,. Unexpectedly, this reveals that complementary components of the BMP signall...

  17. Integration of nodal and BMP signals in the heart requires FoxH1 to create left-right differences in cell migration rates that direct cardiac asymmetry.

    Directory of Open Access Journals (Sweden)

    Kari F Lenhart

    Full Text Available Failure to properly establish the left-right (L/R axis is a major cause of congenital heart defects in humans, but how L/R patterning of the embryo leads to asymmetric cardiac morphogenesis is still unclear. We find that asymmetric Nodal signaling on the left and Bmp signaling act in parallel to establish zebrafish cardiac laterality by modulating cell migration velocities across the L/R axis. Moreover, we demonstrate that Nodal plays the crucial role in generating asymmetry in the heart and that Bmp signaling via Bmp4 is dispensable in the presence of asymmetric Nodal signaling. In addition, we identify a previously unappreciated role for the Nodal-transcription factor FoxH1 in mediating cell responsiveness to Bmp, further linking the control of these two pathways in the heart. The interplay between these TGFβ pathways is complex, with Nodal signaling potentially acting to limit the response to Bmp pathway activation and the dosage of Bmp signals being critical to limit migration rates. These findings have implications for understanding the complex genetic interactions that lead to congenital heart disease in humans.

  18. Angelman Syndrome Protein Ube3a Regulates Synaptic Growth and Endocytosis by Inhibiting BMP Signaling in Drosophila.

    Science.gov (United States)

    Li, Wenhua; Yao, Aiyu; Zhi, Hui; Kaur, Kuldeep; Zhu, Yong-Chuan; Jia, Mingyue; Zhao, Hui; Wang, Qifu; Jin, Shan; Zhao, Guoli; Xiong, Zhi-Qi; Zhang, Yong Q

    2016-05-01

    Altered expression of the E3 ubiquitin ligase UBE3A, which is involved in protein degradation through the proteasome-mediated pathway, is associated with neurodevelopmental and behavioral defects observed in Angelman syndrome (AS) and autism. However, little is known about the neuronal function of UBE3A and the pathogenesis of UBE3A-associated disorders. To understand the in vivo function of UBE3A in the nervous system, we generated multiple mutations of ube3a, the Drosophila ortholog of UBE3A. We found a significantly increased number of total boutons and satellite boutons in conjunction with compromised endocytosis in the neuromuscular junctions (NMJs) of ube3a mutants compared to the wild type. Genetic and biochemical analysis showed upregulation of bone morphogenetic protein (BMP) signaling in the nervous system of ube3a mutants. An immunochemical study revealed a specific increase in the protein level of Thickveins (Tkv), a type I BMP receptor, but not other BMP receptors Wishful thinking (Wit) and Saxophone (Sax), in ube3a mutants. Ube3a was associated with and specifically ubiquitinated lysine 227 within the cytoplasmic tail of Tkv, and promoted its proteasomal degradation in Schneider 2 cells. Negative regulation of Tkv by Ube3a was conserved in mammalian cells. These results reveal a critical role for Ube3a in regulating NMJ synapse development by repressing BMP signaling. This study sheds new light onto the neuronal functions of UBE3A and provides novel perspectives for understanding the pathogenesis of UBE3A-associated disorders. PMID:27232889

  19. Cooperative inputs of Bmp and Fgf signaling induce tail regeneration in urodele amphibians.

    Science.gov (United States)

    Makanae, Aki; Mitogawa, Kazumasa; Satoh, Akira

    2016-02-01

    Urodele amphibians have remarkable organ regeneration ability. They can regenerate not only limbs but also a tail throughout their life. It has been demonstrated that the regeneration of some organs are governed by the presence of neural tissues. For instance, limb regeneration cannot be induced without nerves. Thus, identifying the nerve factors has been the primary focus in amphibian organ regeneration research. Recently, substitute molecules for nerves in limb regeneration, Bmp and Fgfs, were identified. Cooperative inputs of Bmp and Fgfs can induce limb regeneration in the absence of nerves. In the present study, we investigated whether similar or same regeneration mechanisms control another neural tissue governed organ regeneration, i.e., tail regeneration, in Ambystoma mexicanum. Neural tissues in a tail, which is the spinal cord, could transform wound healing responses into organ regeneration responses, similar to nerves in limb regeneration. Furthermore, the identified regeneration inducer Fgf2+Fgf8+Bmp7 showed similar inductive effects. However, further analysis revealed that the blastema cells induced by Fgf2+Fgf8+Bmp7 could participate in the regeneration of several tissues, but could not organize a patterned tail. Regeneration inductive ability of Fgf2+Fgf8+Bmp7 was confirmed in another urodele, Pleurodeles waltl. These results suggest that the organ regeneration ability in urodele amphibians is controlled by a common mechanism. PMID:26703427

  20. The level of BMP4 signaling is critical for the regulation of distinct T-box gene expression domains and growth along the dorso-ventral axis of the optic cup

    Directory of Open Access Journals (Sweden)

    Sowden Jane C

    2006-12-01

    Full Text Available Abstract Background Polarised gene expression is thought to lead to the graded distribution of signaling molecules providing a patterning mechanism across the embryonic eye. Bone morphogenetic protein 4 (Bmp4 is expressed in the dorsal optic vesicle as it transforms into the optic cup. Bmp4 deletions in human and mouse result in failure of eye development, but little attempt has been made to investigate mammalian targets of BMP4 signaling. In chick, retroviral gene overexpression studies indicate that Bmp4 activates the dorsally expressed Tbx5 gene, which represses ventrally expressed cVax. It is not known whether the Tbx5 related genes, Tbx2 and Tbx3, are BMP4 targets in the mammalian retina and whether BMP4 acts at a distance from its site of expression. Although it is established that Drosophila Dpp (homologue of vertebrate Bmp4 acts as a morphogen, there is little evidence that BMP4 gradients are interpreted to create domains of BMP4 target gene expression in the mouse. Results Our data show that the level of BMP4 signaling is critical for the regulation of distinct Tbx2, Tbx3, Tbx5 and Vax2 gene expression domains along the dorso-ventral axis of the mouse optic cup. BMP4 signaling gradients were manipulated in whole mouse embryo cultures during optic cup development, by implantation of beads soaked in BMP4, or the BMP antagonist Noggin, to provide a local signaling source. Tbx2, Tbx3 and Tbx5, showed a differential response to alterations in the level of BMP4 along the entire dorso-ventral axis of the optic cup, suggesting that BMP4 acts across a distance. Increased levels of BMP4 caused expansion of Tbx2 and Tbx3, but not Tbx5, into the ventral retina and repression of the ventral marker Vax2. Conversely, Noggin abolished Tbx5 expression but only shifted Tbx2 expression dorsally. Increased levels of BMP4 signaling caused decreased proliferation, reduced retinal volume and altered the shape of the optic cup. Conclusion Our findings suggest

  1. Laminin and integrin expression in the ventral ectodermal ridge of the mouse embryo: implications for regulation of BMP signalling

    Science.gov (United States)

    Lopez-Escobar, Beatriz; de Felipe, Beatriz; Sanchez-Alcazar, Jose Antonio; Sasaki, Takako; Copp, Andrew J.; Ybot-Gonzalez, Patricia

    2013-01-01

    Background The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail-bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear. Results We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co-expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell-matrix interactions regulating BMP signalling at this site of caudal morphogenesis. Conclusions Laminin532 could interact with α6-containing integrin to direct differentiation of the specialised VER cells from surface ectoderm. PMID:22911573

  2. BMP and TGFbeta pathways in human central chondrosarcoma: enhanced endoglin and Smad 1 signaling in high grade tumors

    Directory of Open Access Journals (Sweden)

    Boeuf Stephane

    2012-10-01

    Full Text Available Abstract Background As major regulators of normal chondrogenesis, the bone morphogenic protein (BMP and transforming growth factor β (TGFB signaling pathways may be involved in the development and progression of central chondrosarcoma. In order to uncover their possible implication, the aim of this study was to perform a systematic quantitative study of the expression of BMPs, TGFBs and their receptors and to assess activity of the corresponding pathways in central chondrosarcoma. Methods Gene expression analysis was performed by quantitative RT-PCR in 26 central chondrosarcoma and 6 healthy articular cartilage samples. Expression of endoglin and nuclear localization of phosphorylated Smad1/5/8 and Smad2 was assessed by immunohistochemical analysis. Results The expression of TGFB3 and of the activin receptor-like kinase ALK2 was found to be significantly higher in grade III compared to grade I chondrosarcoma. Nuclear phosphorylated Smad1/5/8 and Smad2 were found in all tumors analyzed and the activity of both signaling pathways was confirmed by functional reporter assays in 2 chondrosarcoma cell lines. Immunohistochemical analysis furthermore revealed that phosphorylated Smad1/5/8 and endoglin expression were significantly higher in high-grade compared to low-grade chondrosarcoma and correlated to each other. Conclusions The BMP and TGFβ signaling pathways were found to be active in central chondrosarcoma cells. The correlation of Smad1/5/8 activity to endoglin expression suggests that, as described in other cell types, endoglin could enhance Smad1/5/8 signaling in high-grade chondrosarcoma cells. Endoglin expression coupled to Smad1/5/8 activation could thus represent a functionally important signaling axis for the progression of chondrosarcoma and a regulator of the undifferentiated phenotype of high-grade tumor cells.

  3. BMP and TGFbeta pathways in human central chondrosarcoma: enhanced endoglin and Smad 1 signaling in high grade tumors

    International Nuclear Information System (INIS)

    As major regulators of normal chondrogenesis, the bone morphogenic protein (BMP) and transforming growth factor β (TGFB) signaling pathways may be involved in the development and progression of central chondrosarcoma. In order to uncover their possible implication, the aim of this study was to perform a systematic quantitative study of the expression of BMPs, TGFBs and their receptors and to assess activity of the corresponding pathways in central chondrosarcoma. Gene expression analysis was performed by quantitative RT-PCR in 26 central chondrosarcoma and 6 healthy articular cartilage samples. Expression of endoglin and nuclear localization of phosphorylated Smad1/5/8 and Smad2 was assessed by immunohistochemical analysis. The expression of TGFB3 and of the activin receptor-like kinase ALK2 was found to be significantly higher in grade III compared to grade I chondrosarcoma. Nuclear phosphorylated Smad1/5/8 and Smad2 were found in all tumors analyzed and the activity of both signaling pathways was confirmed by functional reporter assays in 2 chondrosarcoma cell lines. Immunohistochemical analysis furthermore revealed that phosphorylated Smad1/5/8 and endoglin expression were significantly higher in high-grade compared to low-grade chondrosarcoma and correlated to each other. The BMP and TGFβ signaling pathways were found to be active in central chondrosarcoma cells. The correlation of Smad1/5/8 activity to endoglin expression suggests that, as described in other cell types, endoglin could enhance Smad1/5/8 signaling in high-grade chondrosarcoma cells. Endoglin expression coupled to Smad1/5/8 activation could thus represent a functionally important signaling axis for the progression of chondrosarcoma and a regulator of the undifferentiated phenotype of high-grade tumor cells

  4. Abrogation of donor T-cell IL-21 signaling leads to tissue-specific modulation of immunity and separation of GVHD from GVL

    OpenAIRE

    Hanash, Alan M.; Kappel, Lucy W.; Yim, Nury L.; Nejat, Rebecca A.; Goldberg, Gabrielle L.; Smith, Odette M.; Rao, Uttam K.; Dykstra, Lindsay; Na, Il-Kang; Holland, Amanda M.; Dudakov, Jarrod A.; Liu, Chen; Murphy, George F.; Leonard, Warren J.; Heller, Glenn

    2011-01-01

    IL-21 is a proinflammatory cytokine produced by Th17 cells. Abrogation of IL-21 signaling has recently been shown to reduce GVHD while retaining graft-versus-leukemia/lymphoma (GVL) responses. However, the mechanisms by which IL-21 may lead to a separation of GVHD and GVL remain incompletely understood. In a murine MHC-mismatched BM transplantation model, we observed that IL-21 receptor knockout (IL-21R KO) donor T cells mediate decreased systemic and gastrointestinal GVHD in recipients of a ...

  5. Syncrip/hnRNP Q influences synaptic transmission and regulates BMP signaling at the Drosophila neuromuscular synapse

    Directory of Open Access Journals (Sweden)

    James M. Halstead

    2014-08-01

    Full Text Available Synaptic plasticity involves the modulation of synaptic connections in response to neuronal activity via multiple pathways. One mechanism modulates synaptic transmission by retrograde signals from the post-synapse that influence the probability of vesicle release in the pre-synapse. Despite its importance, very few factors required for the expression of retrograde signals, and proper synaptic transmission, have been identified. Here, we identify the conserved RNA binding protein Syncrip as a new factor that modulates the efficiency of vesicle release from the motoneuron and is required for correct synapse structure. We show that syncrip is required genetically and its protein product is detected only in the muscle and not in the motoneuron itself. This unexpected non-autonomy is at least partly explained by the fact that Syncrip modulates retrograde BMP signals from the muscle back to the motoneuron. We show that Syncrip influences the levels of the Bone Morphogenic Protein ligand Glass Bottom Boat from the post-synapse and regulates the pre-synapse. Our results highlight the RNA-binding protein Syncrip as a novel regulator of synaptic output. Given its known role in regulating translation, we propose that Syncrip is important for maintaining a balance between the strength of presynaptic vesicle release and postsynaptic translation.

  6. Canonical BMP Signaling Pathway And Mammalian Lung Development%经典BMP信号通路与哺乳动物肺器官发育

    Institute of Scientific and Technical Information of China (English)

    肖爱平; 滕鸿琦; 李小兵; 张明凤

    2012-01-01

      肺器官发育是上皮和间充质相互作用的过程,由多条信号通路共同调控。已知经典BMP信号通路调控了细胞的增殖、凋亡及分化过程,对哺乳动物肺器官形态发生极为重要。在小鼠等模式生物上研究发现,它参与了哺乳动物肺器官发育的调控过程。本文综合了近年来经典BMP信号通路成员在哺乳动物肺器官发育过程中的表达变化、作用功能及表达异常可能诱发的肺部疾病,以期为研究经典BMP信号通路调控人类肺器官发育的分子机制及相关肺部疾病的诊治奠定基础。%  Lung development is a consequence of interaction between epithelium and mesenchyme,which is regulated by several sig-naling pathways. It is well known that the canonical BMP signal pathway plays an important role in the regulation of cell proliferation, apoptosis and differentiation. They are also found to play pivotal roles in morphogenesis of mammalian lung. Recent studies on model organisms,such as mouse and rat,verified that the canonical BMP signal pathway participated in the regulation of mammalian lung development. This review has summarized the main recent research findings on the role of canonical BMP signaling components in the regulation of mammalian lung development. The gene expression and function in all members of canonical BMP signaling pathway, and the possible lung diseases induced by abnormal expression of canonical BMP signaling components during the mammalian lung development were emphasized to state. It’s anticipated to provide valuable information for further illustration of the molecular mecha-nisms of BMP signals in the regulation of human lung development,as well as will be in favor of finding the way to diagnose and cure human lung diseases.

  7. A New Subtype of Multiple Synostoses Syndrome Is Caused by a Mutation in GDF6 That Decreases Its Sensitivity to Noggin and Enhances Its Potency as a BMP Signal.

    Science.gov (United States)

    Wang, Jian; Yu, Tingting; Wang, Zhigang; Ohte, Satoshi; Yao, Ru-En; Zheng, Zhaojing; Geng, Juan; Cai, Haiqing; Ge, Yihua; Li, Yuchan; Xu, Yunlan; Zhang, Qinghua; Gusella, James F; Fu, Qihua; Pregizer, Steven; Rosen, Vicki; Shen, Yiping

    2016-04-01

    Growth and differentiation factors (GDFs) are secreted signaling molecules within the BMP family that have critical roles in joint morphogenesis during skeletal development in mice and humans. Using genetic data obtained from a six-generation Chinese family, we identified a missense variant in GDF6 (NP_001001557.1; p.Y444N) that fully segregates with a novel autosomal dominant synostoses (SYNS) phenotype, which we designate as SYNS4. Affected individuals display bilateral wrist and ankle deformities at birth and progressive conductive deafness after age 40 years. We find that the Y444N variant affects a highly conserved residue of GDF6 in a region critical for binding of GDF6 to its receptor(s) and to the BMP antagonist NOG, and show that this mutant GDF6 is a more potent stimulator of the canonical BMP signaling pathway compared with wild-type GDF6. Further, we determine that the enhanced BMP activity exhibited by mutant GDF6 is attributable to resistance to NOG-mediated antagonism. Collectively, our findings indicate that increased BMP signaling owing to a GDF6 gain-of-function mutation is responsible for loss of joint formation and profound functional impairment in patients with SYNS4. More broadly, our study highlights the delicate balance of BMP signaling required for proper joint morphogenesis and reinforces the critical role of BMP signaling in skeletal development. PMID:26643732

  8. Expressional and functional analyses of transcription factors activated by BMP-4s signaling in early xenopus embryo; BMP-4 shigunaru dentatsu kiko to sono hyoteki kakunai tensha inshi ni kansuru kenkyu

    Energy Technology Data Exchange (ETDEWEB)

    Maeno, Mitsugu [Niigata University, Niigata (Japan). Faculty of Science

    1998-12-16

    The expression and physiological function of two transcription factors, GATA-2 and Xmsx-1, in amphibian embryos has been analyzed. The expression of these mRNAs in embryonic cells were firmly regulated by the BMP-4 signaling, that plays a central role in the formation of ventral tissues. The microinjection studies of GATA-2 RNA into embryonic cells suggested that this factor functions in two adjacent germ layers, mesoderm and ectoderm, to participate in blood cell formation in ventral area of embryo. Embryos injected with Xmsx-1 RNA, but not with GATA-2, in dorsal blastomeres exhibited a ventralized phenotype, with microcephaly and swollen abdomen. Thus, Xmsx-1 is a ventralizing agent. However, on the basis of molecular marker analyses, Xmsx-1 did not promote erythropoietic differentiation, but promoted muscle tissue formation. It has been concluded that Xmsx-1 si a target transcription factor of the BMP-4 signaling, but possesses a distinct activity on dorso-ventral patterning of mesodermal tissues. (author)

  9. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Si, Lina; Shi, Jin; Gao, Wenqun [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Zheng, Min [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Liu, Lingjuan; Zhu, Jing [Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Tian, Jie, E-mail: jietian@cqmu.edu.cn [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China)

    2014-07-18

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  10. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    International Nuclear Information System (INIS)

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  11. Id4 functions downstream of Bmp signaling to restrict TCF function in endocardial cells during atrioventricular valve development.

    Science.gov (United States)

    Ahuja, Suchit; Dogra, Deepika; Stainier, Didier Y R; Reischauer, Sven

    2016-04-01

    The atrioventricular canal (AVC) connects the atrial and ventricular chambers of the heart and its formation is critical for the development of the cardiac valves, chamber septation and formation of the cardiac conduction system. Consequently, problems in AVC formation can lead to congenital defects ranging from cardiac arrhythmia to incomplete cardiac septation. While our knowledge about early heart tube formation is relatively comprehensive, much remains to be investigated about the genes that regulate AVC formation. Here we identify a new role for the basic helix-loop-helix factor Id4 in zebrafish AVC valve development and function. id4 is first expressed in the AVC endocardium and later becomes more highly expressed in the atrial chamber. TALEN induced inactivation of id4 causes retrograde blood flow at the AV canal under heat induced stress conditions, indicating defects in AV valve function. At the molecular level, we found that id4 inactivation causes misexpression of several genes important for AVC and AV valve formation including bmp4 and spp1. We further show that id4 appears to control the number of endocardial cells that contribute to the AV valves by regulating Wnt signaling in the developing AVC endocardium. PMID:26892463

  12. The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways.

    Directory of Open Access Journals (Sweden)

    Toshiyuki Fukada

    Full Text Available BACKGROUND: Zinc (Zn is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS. The Slc39a13 knockout (Slc39a13-KO mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP and TGF-beta signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice. CONCLUSIONS/SIGNIFICANCE: Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-beta signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-beta signaling and connective tissue dysfunction.

  13. Abrogation of STAT3 signaling cascade by zerumbone inhibits proliferation and induces apoptosis in renal cell carcinoma xenograft mouse model.

    Science.gov (United States)

    Shanmugam, Muthu K; Rajendran, Peramaiyan; Li, Feng; Kim, Chulwon; Sikka, Sakshi; Siveen, Kodappully Sivaraman; Kumar, Alan Prem; Ahn, Kwang Seok; Sethi, Gautam

    2015-10-01

    Persistent activation of signal transducer and activator of transcription 3 (STAT3) is one of the characteristic features of renal cell carcinoma (RCC) and often linked to its deregulated proliferation, survival, and angiogenesis. In the present report, we investigated whether zerumbone, a sesquiterpene, exerts its anticancer effect through modulation of STAT3 activation pathway. The pharmacological effect of zerumbone on STAT3 activation, associated protein kinases and phosphatase, and apoptosis was investigated using both RCC cell lines and xenograft mouse model. We observed that zerumbone suppressed STAT3 activation in a dose- and time-dependent manner in RCC cells. The suppression was mediated through the inhibition of activation of upstream kinases c-Src, Janus-activated kinase 1, and Janus-activated kinase 2. Pervanadate treatment reversed zerumbone-induced downregulation of STAT3, suggesting the involvement of a tyrosine phosphatase. Indeed, we found that zerumbone induced the expression of tyrosine phosphatase SHP-1 that correlated with its ability to inhibit STAT3 activation. Interestingly, deletion of SHP-1 gene by siRNA abolished the ability of zerumbone to inhibit STAT3 activation. The inhibition of STAT3 activation by zerumbone also caused the suppression of the gene products involved in proliferation, survival, and angiogenesis. Finally, when administered i.p., zerumbone inhibited STAT3 activation in tumor tissues and the growth of human RCC xenograft tumors in athymic nu/nu mice without any side effects. Overall, our results suggest for the first time that zerumbone is a novel blocker of STAT3 signaling cascade and thus has an enormous potential for the treatment of RCC and other solid tumors. PMID:24797723

  14. P38 MAPK信号通路参与BMP-13诱导C3H10T1/2细胞向心肌样细胞分化%P38 MAPK signaling pathway is involved in BMP-13-induced cardiomyocyte-like differentiation from C3H10T1/2 cells

    Institute of Scientific and Technical Information of China (English)

    孙文静; 陈沅; 张芬; 陈露; 陈妙月; 耿雪静; 朱高慧

    2013-01-01

    interference group and C3H10 blank group. The t-P38 MAPK was detected by Western blot. 3) The influence of BMP-13 induced differentiation after Ad-si-P38 blocking P38 MAPK signal pathway;si-P38 + Ad-BMP-13 transfec-tion group,si-NC + Ad-BMP-13 transfection group,si-NC + Ad-GFP transfection group and C3H10 blank group. cTnT and Cx43 were detected by Western blot and the GATA-4 and MEF-2C were detected by fluorescent quantitative PCR. 4)The influence of BMP-13 induced differentiation after SB203580 blocking P38 MAPK signal pathway; DMSO + Ad-BMP-13 transfection group, SB203580 (2,5 and 10 μmol/L) + Ad-BMP-13 transfection group. The GATA-4 and MEF-2C were detected by by fluorescent quantitative PCR. Results BMP-13 promoted P38 MAPK phosphorylation. Ad-si-P38 effectively inhibited the P38 MAPK expression. Ad-si-P38 which blocked P38 MAPK signal pathway significantly inhibited the BMP-13-induced expression of cTnT, Cx43 (P 〈 0. 05) and GATA4, MEF-2C(P〈0.05). With the increased concentration of P38 MAPK specific inhibitor SB203580, expression of GATA-4 ,MEF-2C was significantly reduced. Conclusion P38 MAPK signal pathway can be activated by Ad-BMP-13 to promote cardiomyocyte-like cells differentiation from C3H10T1/2 cells.

  15. Galectin-9 Signaling through TIM-3 Is Involved in Neutrophil-Mediated Gram-Negative Bacterial Killing: An Effect Abrogated within the Cystic Fibrosis Lung

    Science.gov (United States)

    Vega-Carrascal, Isabel; Bergin, David A.; McElvaney, Oliver J.; McCarthy, Cormac; Banville, Nessa; Pohl, Kerstin; Hirashima, Mitsuomi; Kuchroo, Vijay K.; Reeves, Emer P.; McElvaney, Noel G.

    2016-01-01

    The T cell Ig and mucin domain–containing molecule (TIM) family of receptors have emerged as potential therapeutic targets to correct abnormal immune function in chronic inflammatory conditions. TIM-3 serves as a functional receptor in structural cells of the airways and via the ligand galectin-9 (Gal-9) can modulate the inflammatory response. The aim of this study was to investigate TIM-3 expression and function in neutrophils, focusing on its potential role in cystic fibrosis (CF) lung disease. Results revealed that TIM-3 mRNA and protein expression values of circulating neutrophils were equal between healthy controls (n = 20) and people with CF (n = 26). TIM-3 was detected on resting neutrophil membranes by FACS analysis, and expression levels significantly increased post IL-8 or TNF-α exposure (p < 0.05). Our data suggest a novel role for TIM-3/Gal-9 signaling involving modulation of cytosolic calcium levels. Via TIM-3 interaction, Gal-9 induced neutrophil degranulation and primed the cell for enhanced NADPH oxidase activity. Killing of Pseudomonas aeruginosa was significantly increased upon bacterial opsonization with Gal-9 (p < 0.05), an effect abrogated by blockade of TIM-3 receptors. This mechanism appeared to be Gram-negative bacteria specific and mediated via Gal-9/ LPS binding. Additionally, we have demonstrated that neutrophil TIM-3/Gal-9 signaling is perturbed in the CF airways due to proteolytic degradation of the receptor. In conclusion, results suggest a novel neutrophil defect potentially contributing to the defective bacterial clearance observed in the CF airways and suggest that manipulation of the TIM-3 signaling pathway may be of therapeutic value in CF, preferably in conjunction with antiprotease treatment. PMID:24477913

  16. Abnormalities in the Enamel in Bmp2-Deficient Mice

    OpenAIRE

    Feng, Junsheng; Yang, Guobin; Yuan, Guohua; GLUHAK-HEINRICH, JELICA; Yang, Wuchen; Wang, Lynn; Chen, Zhi; Schulze McDaniel, Jennifer; DONLY, KEVIN J; Harris, Stephen E.; Macdougall, Mary; Chen, Shuo

    2011-01-01

    Tooth development is regulated by epithelial-mesenchymal interactions and their reciprocal molecular signaling. Bone morphogenetic protein 2 (Bmp2) is essential for tooth formation. However, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in the regulation of postnatal enamel formation was investigated via the conditional ablation of Bmp2 in enamel using the (Osx-Cre) mouse. Bmp2 gene ablation was confirmed by PCR analysis in Osx-Cre, Bmp2flox...

  17. N-cadherin mediated distribution of beta-catenin alters MAP kinase and BMP-2 signaling on chondrogenesis-related gene expression.

    Science.gov (United States)

    Modarresi, Rozbeh; Lafond, Toulouse; Roman-Blas, Jorge A; Danielson, Keith G; Tuan, Rocky S; Seghatoleslami, M Reza

    2005-05-01

    We have examined the effect of calcium-dependent adhesion, mediated by N-cadherin, on cell signaling during chondrogenesis of multipotential embryonic mouse C3H10T1/2 cells. The activity of chondrogenic genes, type II collagen, aggrecan, and Sox9 were examined in monolayer (non-chondrogenic), and micromass (chondrogenic) cultures of parental C3H10T1/2 cells and altered C3H10T1/2 cell lines that express a dominant negative form of N-cadherin (delta390-T1/2) or overexpress normal N-cadherin (MNCD2-T1/2). Our findings show that missexpression or inhibition of N-cadherin in C3H10T1/2 cells results in temporal and spatial changes in expression of the chondrogenic genes Sox9, aggrecan, and collagen type II. We have also analyzed activity of the serum response factor (SRF), a nuclear target of MAP kinase signaling implicated in chondrogenesis. In semi-confluent monolayer cultures (minimum cell-cell contact) of C3H10T1/2, MNCD2-T1/2, or delta390-T1/2 cells, there was no significant change in the pattern of MAP kinase or bone morphogenetic protein-2 (BMP-2) regulation of SRF. However, in micromass cultures, the effect of MAP kinase and BMP-2 on SRF activity was proportional to the nuclear localization of beta-catenin, a Wnt stabilized cytoplasmic factor that can associate with lymphoid enhancer-binding factor (LEF) to serve as a transcription factor. Our findings suggest that the extent of adherens junction formation mediated by N-cadherin can modulate the potential Wnt-induced nuclear activity of beta-catenin. PMID:15723280

  18. BMP-13 Emerges as a Potential Inhibitor of Bone Formation

    Directory of Open Access Journals (Sweden)

    Bojiang Shen, Divya Bhargav, Aiqun Wei, Lisa A Williams, Helen Tao, David D F Ma, Ashish D Diwan

    2009-01-01

    Full Text Available Bone morphogenetic protein-13 (BMP-13 plays an important role in skeletal development. In the light of a recent report that mutations in the BMP-13 gene are associated with spine vertebral fusion in Klippel-Feil syndrome, we hypothesized that BMP-13 signaling is crucial for regulating embryonic endochondral ossification. In this study, we found that BMP-13 inhibited the osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs in vitro. The endogenous BMP-13 gene expression in MSCs was examined under expansion conditions. The MSCs were then induced to differentiate into osteoblasts in osteo-inductive medium containing exogenous BMP-13. Gene expression was analysed by real-time PCR. Alkaline phosphatase (ALP expression and activity, proteoglycan (PG synthesis and matrix mineralization were assessed by cytological staining or ALP assay. Results showed that endogenous BMP-13 mRNA expression was higher than BMP-2 or -7 during MSC growth. BMP-13 supplementation strongly inhibited matrix mineralization and ALP activity of osteogenic differentiated MSCs, yet increased PG synthesis under the same conditions. In conclusion, BMP-13 inhibited osteogenic differentiation of MSCs, implying that functional mutations or deficiency of BMP-13 may allow excess bone formation. Our finding provides an insight into the molecular mechanisms and the therapeutic potential of BMP-13 in restricting pathological bone formation.

  19. Osteogenesis differentiation of human periodontal ligament cells by CO2 laser-treatment stimulating macrophages via BMP2 signalling pathway

    International Nuclear Information System (INIS)

    Immune reactions play an important role in determining the biostimulation of bone formation, either in new bone formation or inflammatory fibrous tissue encapsulation. Macrophage cell, the important effector cells in the immune reaction, which are indispensable for osteogenesis and their heterogeneity and plasticity, render macrophages a primer target for immune system modulation. However, there are very few studies about the effects of macrophage cells on laser treatment-regulated osteogenesis. In this study, we used CO2 laser as a model biostimulation to investigate the role of macrophage cells on the CO2 laser stimulated osteogenesis. Bone morphogenetic protein 2 (BMP2) was also significantly up regulated by the CO2 laser stimulation, indicating that macrophage may participate in the CO2 laser stimulated osteogenesis. Interestingly, when laser treatment macrophage-conditioned medium were applied to human periodontal ligament cells (hPDLs), the osteogenesis differentiation of hPDLs was significantly enhanced, indicating the important role of macrophages in CO2 laser-induced osteogenesis. These findings provided valuable insights into the mechanism of CO2 laser-stimulated osteogenic differentiation, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of laser treatment. (paper)

  20. Mechanical Loading Synergistically Increases Trabecular Bone Volume and Improves Mechanical Properties in the Mouse when BMP Signaling Is Specifically Ablated in Osteoblasts.

    Directory of Open Access Journals (Sweden)

    Ayaka Iura

    Full Text Available Bone homeostasis is affected by several factors, particularly mechanical loading and growth factor signaling pathways. There is overwhelming evidence to validate the importance of these signaling pathways, however, whether these signals work synergistically or independently to contribute to proper bone maintenance is poorly understood. Weight-bearing exercise increases mechanical load on the skeletal system and can improves bone quality. We previously reported that conditional knockout (cKO of Bmpr1a, which encodes one of the type 1 receptors for Bone Morphogenetic Proteins (BMPs, in an osteoblast-specific manner increased trabecular bone mass by suppressing osteoclastogenesis. The cKO bones also showed increased cortical porosity, which is expected to impair bone mechanical properties. Here, we evaluated the impact of weight-bearing exercise on the cKO bone phenotype to understand interactions between mechanical loading and BMP signaling through BMPR1A. Male mice with disruption of Bmpr1a induced at 9 weeks of age, exercised 5 days per week on a motor-driven treadmill from 11 to 16 weeks of age. Trabecular bone volume in cKO tibia was further increased by exercise, whereas exercise did not affect the trabecular bone in the control genotype group. This finding was supported by decreased levels of osteoclasts in the cKO tibiae. The cortical porosity in the cKO bones showed a marginally significant decrease with exercise and approached normal levels. Exercise increased ductility and toughness in the cKO bones. Taken together, reduction in BMPR1A signaling may sensitize osteoblasts for mechanical loading to improve bone mechanical properties.

  1. Emerging roles of BMP9 and BMP10 in hereditary hemorrhagic telangiectasia

    Directory of Open Access Journals (Sweden)

    Emmanuelle eTillet

    2015-01-01

    Full Text Available Rendu-Osler-Weber syndrome, also known as hereditary hemorrhagic telangiectasia (HHT, is an autosomal dominant vascular disorder. Three genes are causally related to HHT: the ENG gene encoding endoglin, a co-receptor of the TGFß family (HHT1, the ACVRL1 gene encoding ALK1 (activin receptor-like kinase 1, a type I receptor of the TGFß family (HHT2, and the SMAD4 gene, encoding a transcription factor critical for this signaling pathway. Bone morphogenetic proteins (BMPs are growth factors of the TGFß family. Among them, BMP9 and BMP10 have been shown to bind directly with high affinity to ALK1 and endoglin, and BMP9 mutations have recently been linked to a vascular-anomaly syndrome that has phenotypic overlap with HHT. BMP9 and BMP10 are both circulating cytokines in blood, and the current working model is that BMP9 and BMP10 maintain a quiescent endothelial state that is dependent on the level of ALK1/endoglin activation on endothelial cells. In accordance with this model, to explain the etiology of HHT we hypothesize that a deficient BMP9/BMP10/ALK1/endoglin pathway may lead to re-activation of angiogenesis or a greater sensitivity to an angiogenic stimulus. Resulting endothelial hyperproliferation and hypermigration may lead to vasodilatation and formation of arteriovenous malformation (AVM. HHT would thus result from a defect in the angiogenic balance. This review will focus on the emerging role played by BMP9 and BMP10 in the development of this disease and the therapeutic approaches that this opens.

  2. BMP signaling pathway is required for commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage

    OpenAIRE

    Huang, Haiyan; Song, Tan-Jing; Li, Xi; Hu, Lingling; He, Qun; Liu, Mei; Lane, M. Daniel; Tang, Qi-Qun

    2009-01-01

    Obesity is accompanied by an increase in both adipocyte number and size. The increase in adipocyte number is the result of recruitment to the adipocyte lineage of pluripotent stem cells present in the vascular stroma of adipose tissue. These pluripotent cells have the potential to undergo commitment and then differentiate into adipocytes, as well as myocytes, osteocytes, and chondrocytes. In this article, we show that both bone morphogenetic protein (BMP)2 and BMP4 can induce commitment of C3...

  3. Sox2 in the dermal papilla niche controls hair growth by fine-tuning Bmp signaling in differentiating hair shaft progenitors

    OpenAIRE

    Clavel, Carlos; Grisanti, Laura; Zemla, Roland; Rezza, Amelie; Barros, Rita; Sennett, Rachel; Mazloom, Amin; Chung, Chi-Yeh; Cai, Xiaoqiang; Cai, Chen-Leng; Pevny, Larysa; Nicolis, Silvia; Ma’ayan, Avi; Rendl, Michael

    2012-01-01

    How dermal papilla (DP) niche cells regulate hair follicle progenitors to control hair growth remains unclear. Using Tbx18Cre to target embryonic DP precursors, we ablate the transcription factor Sox2 early and efficiently, resulting in diminished hair shaft outgrowth. We find that DP niche expression of Sox2 controls the migration rate of differentiating hair shaft progenitors. Transcriptional profiling of Sox2 null DPs reveals increased Bmp6 and decreased Bmp inhibitor Sostdc1, a direct Sox...

  4. MMP-2 suppression abrogates irradiation-induced microtubule formation in endothelial cells by inhibiting αvβ3-mediated SDF-1/CXCR4 signaling

    OpenAIRE

    Maddirela, Dilip Rajasekhar; Kesanakurti, Divya; Gujrati, Meena; Rao, Jasti S.

    2013-01-01

    The majority of glioblastoma multiforme (GBM) tumors recur after radiation (IR) treatment due to increased angiogenesis and IR-induced signaling events in endothelial cells (ECs) that are involved in tumor neovascularization; however, these signaling events have yet to be well characterized. In the present study, we observed that IR (8 Gy) significantly elevated MMP-2 expression and gelatinolytic activity in 4910 and 5310 human GBM xenograft cells. In addition, ECs treated with tumor-conditio...

  5. Phosphorylation of Sox9 is required for neural crest delamination and is regulated downstream of BMP and canonical Wnt signaling.

    Science.gov (United States)

    Liu, Jessica A J; Wu, Ming-Hoi; Yan, Carol H; Chau, Bolton K H; So, Henry; Ng, Alvis; Chan, Alan; Cheah, Kathryn S E; Briscoe, James; Cheung, Martin

    2013-02-19

    Coordination of neural crest cell (NCC) induction and delamination is orchestrated by several transcription factors. Among these, Sry-related HMG box-9 (Sox9) and Snail2 have been implicated in both the induction of NCC identity and, together with phoshorylation, NCC delamination. How phosphorylation effects this function has not been clear. Here we show, in the developing chick neural tube, that phosphorylation of Sox9 on S64 and S181 facilitates its SUMOylation, and the phosphorylated forms of Sox9 are essential for trunk neural crest delamination. Both phosphorylation and to a lesser extent SUMOylation, of Sox9 are required to cooperate with Snail2 to promote delamination. Moreover, bone morphogenetic protein and canonical Wnt signaling induce phosphorylation of Sox9, thereby connecting extracellular signals with the delamination of NCCs. Together the data suggest a model in which extracellular signals initiate phosphorylation of Sox9 and its cooperation with Snail2 to induce NCC delamination. PMID:23382206

  6. Target genes of Dpp/BMP signaling pathway revealed by transcriptome profiling in the early D.melanogaster embryo.

    Science.gov (United States)

    Dominguez, Calixto; Zuñiga, Alejandro; Hanna, Patricia; Hodar, Christian; Gonzalez, Mauricio; Cambiazo, Verónica

    2016-10-10

    In the early Drosophila melanogaster embryo, the gene regulatory network controlled by Dpp signaling is involved in the subdivision of dorsal ectoderm into the presumptive dorsal epidermis and amnioserosa. In this work, we aimed to identify new Dpp downstream targets involved in dorsal ectoderm patterning. We used oligonucleotide D. melanogaster microarrays to identify the set of genes that are differential expressed between wild type embryos and embryos that overexpress Dpp (nos-Gal4>UAS-dpp) during early stages of embryo development. By using this approach, we identified 358 genes whose relative abundance significantly increased in response to Dpp overexpression. Among them, we found the entire set of known Dpp target genes that function in dorsal ectoderm patterning (zen, doc, hnt, pnr, ush, tup, and others) in addition to several up-regulated genes of unknown functions. Spatial expression pattern of up-regulated genes in response to Dpp overexpression as well as their opposing transcriptional responses to Dpp loss- and gain-of-function indicated that they are new candidate target genes of Dpp signaling pathway. We further analyse one of the candidate genes, CG13653, which is expressed at the dorsal-most cells of the embryo during a restricted period of time. CG13653 orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa. We characterized the enhancer region of CG13653 and revealed that CG13653 is directly regulated by Dpp signaling pathway. PMID:27397649

  7. Drosophila motor neuron retraction during metamorphosis is mediated by inputs from TGF-β/BMP signaling and orphan nuclear receptors.

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    Ana Boulanger

    Full Text Available Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. In this study, we describe the motor neuron retraction as opposed to degeneration based on the early disappearance of β-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, we show that phagocytes have a key role in this process. Importantly, we show the presence of peripheral glial cells close to the neuro-muscular junction that retracts before the motor neuron. We show also that in muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor triggers axon retraction. We propose that a signal from a TGF-β family ligand is produced by the dismantling muscle (postsynapse compartment and received by the motor neuron (presynaptic compartment resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation.

  8. Astaxanthin Inhibits JAK/STAT-3 Signaling to Abrogate Cell Proliferation, Invasion and Angiogenesis in a Hamster Model of Oral Cancer

    OpenAIRE

    Kowshik, J.; Baba, Abdul Basit; Giri, Hemant; Deepak Reddy, G.; Dixit, Madhulika; Nagini, Siddavaram

    2014-01-01

    Identifying agents that inhibit STAT-3, a cytosolic transcription factor involved in the activation of various genes implicated in tumour progression is a promising strategy for cancer chemoprevention. In the present study, we investigated the effect of dietary astaxanthin on JAK-2/STAT-3 signaling in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model by examining the mRNA and protein expression of JAK/STAT-3 and its target genes. Quantitative RT...

  9. Single-molecule imaging of BMP4 dimerization on human periodontal ligament cells.

    Science.gov (United States)

    Mi, H-W; Lee, M-C; Chiang, Y-C; Chow, L-P; Lin, C-P

    2011-11-01

    We expressed bone morphogenetic protein 4 (BMP4) fused with enhanced green fluorescent protein (BMP4-EGFP) in the secretory pathways of producer cells. Fluorescent EGFP was acquired only after we interrupted the transport of BMP4-EGFP by culturing cells at a lower temperature (20°C), and the dynamics of BMP4-EGFP could be monitored by single-molecule microscopy. Western blotting analysis confirmed that exposure to low temperature helped the integrated formation of BMP4-EGFP fusion proteins. In this study, for the first time, we could image the fluorescently labeled BMP4 molecules localized on the plasma membrane of living hPDL cells. The one-step photobleaching with EGFP and the "blinking" behavior of quantum dots suggest that the fluorescent spots represent the events of single BMP4 molecules. Single-molecule tracking showed that the BMP receptors (BMPR) dimerize after BMP4 stimulation, or that a complex of one BMP4 molecule and a pre-formed BMPR dimer develops first, followed by the binding of the second BMP4 molecule. Furthermore, BMP4-EGFP enhanced the osteogenic differentiation of hPDL cells via signal transduction involving BMP receptors. This single-molecule imaging technique might be a valuable tool for the future development of BMP4 gene therapy and regenerative medicine mediated by hPDLs. PMID:21841042

  10. Function of BMP-7/Smad6 signaling pathway in the rat model of alcoholic liver disease%BMP -7/Smad6信号通路在酒精性肝损伤大鼠模型中的调节作用

    Institute of Scientific and Technical Information of China (English)

    何培元; 王明娟; 张聪; 马立新; 侯志平; 李炳庆

    2014-01-01

    目的:探讨 BMP -7/ Smad6信号传导通路在酒精性肝病发病机制中的作用并检测其在酒精性肝病大鼠模型中的基因水平和蛋白水平。方法以自由摄取白酒加高脂饲料诱导酒精性肝纤维化大鼠模型,并比较普通饲料与高脂饲料对酒精性肝病的影响。实验结束后取各实验组肝组织观察病理学变化,用生化检测仪检测大鼠血浆 ALT、AST变化。用实时 RCR 检测肝的骨形成蛋白7(BMP -7)和 Smad6 mRNA 水平变化,并用 Western Blot 检测蛋白水平变化。结果肝脏组织形态学改变显示正常饲料组肝组织出现脂肪肝表现,高脂饲料组脂肪变性程度加重并出现了肝炎及胶原纤维增生,而对照组大鼠肝脏组织结构正常。生化分析仪检测 ALT、AST 结果显示:至实验结束时,实验组比对照组转氨酶活性明显升高( P ﹤0.05)。BMP 和 Smad6这两个基因的蛋白丰度与它们各自的 mRNA 表达水平呈正相关,与对照组比较两个实验组 BMP -7表达水平降低,并与肝脏病理损伤程度呈负相关,两个实验组 Smad6表达水平较对照组升高,但与肝脏病理变化严重程度无相关性。结论饮用白酒水溶液结合高脂饲料的方法可以成功建立酒精性肝损伤的大鼠模型,BMP -7/ Smad6信号通路的失活促进了肝纤维化的形成。%Objective To investigate the function of BMP - 7 / Smad6 signaling pathway in alcoholic liver disease rat model and detect the two genes in both mRNA and protein levels. Methods Rats were fed with alcohol and high fat diet to establish alcoholic liver disease model. In the end of the experiment,liver of rats were dissected for histological studies whereas plasma obtained from rats was taken for biochemical testing to detect the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST). mRNA was extracted from the liver to perform real- time PCR in order to detect the mRNA level of

  11. A Bmp/Admp regulatory circuit controls maintenance and regeneration of dorsal-ventral polarity in planarians

    OpenAIRE

    Gaviño, Michael A; Reddien, Peter W.

    2011-01-01

    Animal embryos have diverse anatomy and vary greatly in size. It is therefore remarkable that a common signaling pathway – BMP signaling – controls development of the dorsoventral (DV) axis throughout the Bilateria [1-8]. In vertebrates, spatially opposed expression of the BMP-family signaling proteins Bmp4 and Admp (anti-dorsalizing morphogenetic protein) can promote restoration of DV pattern following tissue removal [9-11]. bmp4 orthologs have been identified in all three groups of the Bila...

  12. n-3 polyunsaturated fatty acids abrogate mTORC1/2 signaling and inhibit adrenocortical carcinoma growth in vitro and in vivo.

    Science.gov (United States)

    Liu, Jun; Xu, Meinian; Zhao, Yongbin; Ao, Chunping; Wu, Yukun; Chen, Zhenguo; Wang, Bangqi; Bai, Xiaochun; Li, Ming; Hu, Weilie

    2016-06-01

    n-3 polyunsaturated fatty acids (PUFAs) are essential for human health and have been reported to reduce the risk of cancer, inhibit the growth of various types of tumors both in vitro and in vivo, and affect adrenal function. However, their effects on adrenocortical carcinoma (ACC) are not known. In the present study, we demonstrated that docosahexenoic acid (DHA) inhibited ACC cell proliferation, colony formation and cell cycle progression, and promoted apoptosis. In addition, ectopic expression of fat-1, a desaturase that converts n-6 to n-3 PUFAs endogenously, also inhibited ACC cell proliferation. Moreover, supplementing n-3 PUFAs in the diet efficiently prevented ACC cell growth in xenograft models. Notably, implanted ACC cells were unable to grow in fat-1 transgenic severe combined immune deficiency mice. Further study revealed that exogenous and endogenous n-3 PUFAs efficiently suppressed both mTOR complex 1 (mTORC1) and mTORC2 signaling in ACC in vitro and in vivo. Taken together, our findings provide comprehensive preclinical evidence that n-3 PUFAs efficiently prevent ACC growth by inhibiting mTORC1/2, which may have important implications in the treatment of ACC. PMID:27035283

  13. Combination of spices and herbal extract restores macrophage foam cell migration and abrogates the athero-inflammatory signalling cascade of atherogenesis.

    Science.gov (United States)

    Nimgulkar, Chetan; Ghosh, Sudip; Sankar, Anand B; Uday, Kumar P; Surekha, M V; Madhusudhanachary, P; Annapurna, B R; Raghu, P; Bharatraj, Dinesh Kumar

    2015-09-01

    The trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step in atherogenesis. However, information about possible treatments for this condition is lacking. Here, we hypothesized that combining the polyphenol-rich fractions (PHC) of commonly consumed spices (Allium sativum L (Liliaceae), Zingiber officinale R (Zingiberaceae), Curcuma longa L (Zingiberaceae)) and herbs (Terminalia arjuna (R) W & A (Combretaceae) and Cyperus rotundus L (Cyperaceae)) prevents foam cell formation and atherogenesis. Using an in vitro foam cell formation assay, we found that PHC significantly inhibited lipid-laden macrophage foam cell formation compared to the depleted polyphenol fraction of PHC (F-PHC). We further observed that PHC attenuated the LDL and LPS induced CD36, p-FAK and PPAR-γ protein expression in macrophages and increased their migration. NK-κB-DNA interaction, TNF-α, ROS generation, and MMP9 and MMP2 protein expression were suppressed in PHC-treated macrophages. The anti-atherosclerotic activity of PHC was investigated in a high fat- and cholesterol-fed rabbit model. The inhibition of foam cell deposition within the aortic intima and atheroma formation confirmed the atheroprotective activity of PHC. Therefore, we conclude that the armoury of polyphenols in PHC attenuates the CD36 signalling cascade-mediated foam cell formation, enhances the migration of these cells and prevents atherogenesis. PMID:25869517

  14. BMP pathway regulation of and by macrophages.

    Directory of Open Access Journals (Sweden)

    Megha Talati

    Full Text Available Pulmonary arterial hypertension (PAH is a disease of progressively increasing pulmonary vascular resistance, associated with mutations of the type 2 receptor for the BMP pathway, BMPR2. The canonical signaling pathway for BMPR2 is through the SMAD family of transcription factors. BMPR2 is expressed in every cell type, but the impact of BMPR2 mutations affecting SMAD signaling, such as Bmpr2delx4+, had only previously been investigated in smooth muscle and endothelium. In the present study, we created a mouse with universal doxycycline-inducible expression of Bmpr2delx4+ in order to determine if broader expression had an impact relevant to the development of PAH. We found that the most obvious phenotype was a dramatic, but patchy, increase in pulmonary inflammation. We crossed these double transgenic mice onto an NF-κB reporter strain, and by luciferase assays on live mice, individual organs and isolated macrophages, we narrowed down the origin of the inflammatory phenotype to constitutive activation of tissue macrophages. Study of bone marrow-derived macrophages from mutant and wild-type mice suggested a baseline difference in differentiation state in Bmpr2 mutants. When activated with LPS, both mutant and wild-type macrophages secrete BMP pathway inhibitors sufficient to suppress BMP pathway activity in smooth muscle cells (SMC treated with conditioned media. Functionally, co-culture with macrophages results in a BMP signaling-dependent increase in scratch closure in cultured SMC. We conclude that SMAD signaling through BMP is responsible, in part, for preventing macrophage activation in both live animals and in cells in culture, and that activated macrophages secrete BMP inhibitors in sufficient quantity to cause paracrine effect on vascular smooth muscle.

  15. Inhibition of KIT-glycosylation by 2-deoxyglucose abrogates KIT-signaling and combination with ABT-263 synergistically induces apoptosis in gastrointestinal stromal tumor.

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    Thomas Mühlenberg

    Full Text Available Positron emission tomography (PET with 18F-fluorodeoxyglucose (FDG is frequently used for visualizing gastrointestinal stromal tumors (GIST, which are highly glucose-avid tumors. Dramatic metabolic responses following imatinib treatment indicate a high, KIT-dependent glucose turnover which has been particularly helpful for predicting tumor response to imatinib. The glucose analogue 2-deoxyglucose (2DG inhibits glucose metabolism in cancer cells that depend on aerobic glycolysis for ATP production. We show that 2DG inhibits proliferation in both imatinib-sensitive and imatinib-resistant GIST cell lines at levels that can be achieved clinically. KIT-negative GIST48B have 3-14-fold higher IC50 levels than KIT-positive GIST cells indicating that oncogenic KIT may sensitize cells to 2DG. GIST sensitivity to 2DG is increased in low-glucose media (110 mg/dl. 2DG leads to dose- and glucose dependent inhibition of KIT glycosylation with resultant reduction of membrane-bound KIT, inhibition of KIT-phosphorylation and inactivation of KIT-dependent signaling intermediates. In contrast to imatinib, 2DG caused ER-stress and elicited the unfolded protein response (UPR. Mannose but not pyruvate rescued GIST cells from 2DG-induced growth arrest, suggesting that loss of KIT integrity is the predominant effect of 2DG in GIST. Additive anti-tumoral effects were seen with imatinib and BH3-mimetics. Our data provide the first evidence that modulation of the glucose-metabolism by 2DG may have a disease-specific effect and may be therapeutically useful in GIST.

  16. BMP10 inhibited the growth and migration of gastric cancer cells.

    Science.gov (United States)

    Lei, Haiming; Wang, Jian; Lu, Peihua; Si, Xinghua; Han, Koulan; Ruan, Tingyan; Lu, Junjie

    2016-03-01

    Bone morphogenetic protein 10 (BMP10), a novel member of BMP family, has been identified as an important regulator for angiogenesis. Dysregulation of BMP has been observed in several cancer types. However, its roles in gastric cancer (GC) remain unknown. In this study, the expression of BMP10 was found to be down-regulated in GC samples. Forced expression of BMP10 in GC cells inhibited its growth and migration, while knocking down the expression of BMP10 in GC cells promoted cell growth, migration, and metastasis. BMP10 was shown to negatively regulated beta-catenin/TCF signaling by up-regulating Axin protein level. Taken together, the present study revealed the suppressive function of BMP10 in gastric cancer. PMID:26419594

  17. BMP is an important regulator of proepicardial identity in the chick embryo.

    Science.gov (United States)

    Schlueter, Jan; Männer, Jörg; Brand, Thomas

    2006-07-15

    The proepicardium (PE) is a transient structure formed by pericardial coelomic mesothelium at the venous pole of the embryonic heart and gives rise to several cell types of the mature heart. In order to study PE development in chick embryos, we have analyzed the expression pattern of the marker genes Tbx18, Wt1, and Cfc. During PE induction, the three marker genes displayed a left-right asymmetric expression pattern. In each case, expression on the right side was stronger than on the left side. The left-right asymmetric gene expression observed here is in accord with the asymmetric formation of the proepicardium in the chick embryo. While initially the marker genes were expressed in the primitive sinus horn, subsequently, expression became confined to the PE mesothelium. In order to search for signaling factors involved in PE development, we studied Bmp2 and Bmp4 expression. Bmp2 was bilaterally expressed in the sinus venosus. In contrast, Bmp4 expression was initially expressed unilaterally in the right sinus horn and subsequently in the PE. In order to assess its functional role, BMP signaling was experimentally modulated by supplying exogenous BMP2 and by inhibiting endogenous BMP signaling through the addition of Noggin. Both supplying BMP and blocking BMP signaling resulted in a loss of PE marker gene expression. Surprisingly, both experimental situations lead to cardiac myocyte formation in the PE cultures. Careful titration experiments with exogenously added BMP2 or Noggin revealed that PE-specific marker gene expression depends on a low level of BMP signaling. Implantation of BMP2-secreting cells or beads filled with Noggin protein into the right sinus horn of HH stage 11 embryos resulted in downregulation of Tbx18 expression, corresponding to the results of the explant assay. Thus, a distinct level of BMP signaling is required for PE formation in the chick embryo. PMID:16677627

  18. Bu-Shen-Ning-Xin decoction: inhibition of osteoclastogenesis by abrogation of the RANKL-induced NFATc1 and NF-κB signaling pathways via selective estrogen receptor α

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    Wang L

    2015-07-01

    in osteoclast precursor cells; the inhibitory effect was abolished by methyl-piperidino-pyrazole but not the estrogen receptor β antagonist or androgen receptor antagonist.Conclusion: These results collectively suggest that administration of BSNXD presents inhibitory effects on osteoclast differentiation by abrogating the RANKL-induced nuclear factor of activated T-cells, cytoplasmic 1 and NF-κB signaling pathways downstream of estrogen receptor α, thereby contributing to the inhibitory effect on bone resorption.Keywords: herbal formula, osteoclastogenesis, estrogen receptor α, NF-κB, NFATc1

  19. Targeted disruption of BMP signaling through type IA receptor (BMPR1A) in osteocyte suppresses SOST and RANKL, leading to dramatic increase in bone mass, bone mineral density and mechanical strength.

    Science.gov (United States)

    Kamiya, Nobuhiro; Shuxian, Lin; Yamaguchi, Ryosuke; Phipps, Matthew; Aruwajoye, Olumide; Adapala, Naga Suresh; Yuan, Hui; Kim, Harry K W; Feng, Jian Q

    2016-10-01

    Recent studies suggest a critical role of osteocytes in controlling skeletal development and bone remodeling although the molecular mechanism is largely unknown. This study investigated BMP signaling in osteocytes by disrupting Bmpr1a under the Dmp1-promoter. The conditional knockout (cKO) mice displayed a striking osteosclerotic phenotype with increased trabecular bone volume, thickness, number, and mineral density as assessed by X-ray and micro-CT. The bone histomorphometry, H&E, and TRAP staining revealed a dramatic increase in trabecular and cortical bone masses but a sharp reduction in osteoclast number. Moreover, there was an increase in BrdU positive osteocytes (2-5-fold) and osteoid volume (~4-fold) but a decrease in the bone formation rate (~85%) in the cKO bones, indicating a defective mineralization. The SEM analysis revealed poorly formed osteocytes: a sharp increase in cell numbers, a great reduction in cell dendrites, and a remarkable change in the cell distribution pattern. Molecular studies demonstrated a significant decrease in the Sost mRNA levels in bone (>95%), and the SOST protein levels in serum (~85%) and bone matrices. There was a significant increase in the β-catenin (>3-fold) mRNA levels as well as its target genes Tcf1 (>6-fold) and Tcf3 (~2-fold) in the cKO bones. We also showed a significant decrease in the RANKL levels of serum proteins (~65%) and bone mRNA (~57%), and a significant increase in the Opg mRNA levels (>20-fold) together with a significant reduction in the Rankl/Opg ratio (>95%), which are responsible for a sharp reduction in the cKO osteoclasts. The values of mechanical strength were higher in cKO femora (i.e. max force, displacement, and work failure). These results suggest that loss of BMP signaling specifically in osteocytes dramatically increases bone mass presumably through simultaneous inhibition of RANKL and SOST, leading to osteoclast inhibition and Wnt activation together. Finally, a working hypothesis is

  20. DMH1, a small molecule inhibitor of BMP type i receptors, suppresses growth and invasion of lung cancer.

    Directory of Open Access Journals (Sweden)

    Jijun Hao

    Full Text Available The bone morphogenetic protein (BMP signaling cascade is aberrantly activated in human non-small cell lung cancer (NSCLC but not in normal lung epithelial cells, suggesting that blocking BMP signaling may be an effective therapeutic approach for lung cancer. Previous studies demonstrated that some BMP antagonists, which bind to extracellular BMP ligands and prevent their association with BMP receptors, dramatically reduced lung tumor growth. However, clinical application of protein-based BMP antagonists is limited by short half-lives, poor intra-tumor delivery as well as resistance caused by potential gain-of-function mutations in the downstream of the BMP pathway. Small molecule BMP inhibitors which target the intracellular BMP cascades would be ideal for anticancer drug development. In a zebrafish embryo-based structure and activity study, we previously identified a group of highly selective small molecule inhibitors specifically antagonizing the intracellular kinase domain of BMP type I receptors. In the present study, we demonstrated that DMH1, one of such inhibitors, potently reduced lung cell proliferation, promoted cell death, and decreased cell migration and invasion in NSCLC cells by blocking BMP signaling, as indicated by suppression of Smad 1/5/8 phosphorylation and gene expression of Id1, Id2 and Id3. Additionally, DMH1 treatment significantly reduced the tumor growth in human lung cancer xenograft model. In conclusion, our study indicates that small molecule inhibitors of BMP type I receptors may offer a promising novel strategy for lung cancer treatment.

  1. BMP8B Increases Brown Adipose Tissue Thermogenesis through Both Central and Peripheral Actions

    OpenAIRE

    Whittle AJ; Carobbio S; Martins L; Slawik M; Hondares E; V\\xe1zquez MJ; Morgan D; Csikasz RI; Gallego R; Rodriguez-Cuenca S; Dale M.; Virtue S; Villarroya F; Cannon B; Rahmouni K

    2012-01-01

    Summary Thermogenesis in brown adipose tissue (BAT) is fundamental to energy balance and is also relevant for humans. Bone morphogenetic proteins (BMPs) regulate adipogenesis, and, here, we describe a role for BMP8B in the direct regulation of thermogenesis. BMP8B is induced by nutritional and thermogenic factors in mature BAT, increasing the response to noradrenaline through enhanced p38MAPK/CREB signaling and increased lipase activity. Bmp8b−/− mice exhibit impaired thermogenesis and reduce...

  2. Mutations in GDF5 reveal a key residue mediating BMP inhibition by NOGGIN.

    Directory of Open Access Journals (Sweden)

    Petra Seemann

    2009-11-01

    Full Text Available Signaling output of bone morphogenetic proteins (BMPs is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T in patients with multiple synostosis syndrome (SYM1 in the BMP-related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP-inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.

  3. A Bmp/Admp regulatory circuit controls maintenance and regeneration of dorsal-ventral polarity in planarians.

    Science.gov (United States)

    Gaviño, Michael A; Reddien, Peter W

    2011-02-22

    Animal embryos have diverse anatomy and vary greatly in size. It is therefore remarkable that a common signaling pathway, BMP signaling, controls development of the dorsoventral (DV) axis throughout the Bilateria. In vertebrates, spatially opposed expression of the BMP family proteins Bmp4 and Admp (antidorsalizing morphogenetic protein) can promote restoration of DV pattern following tissue removal. bmp4 orthologs have been identified in all three groups of the Bilateria (deuterostomes, ecdysozoans, and lophotrochozoans). By contrast, the absence of admp orthologs in ecdysozoans such as Drosophila and C. elegans has suggested that a regulatory circuit of oppositely expressed bmp4 and admp genes represents a deuterostome-specific innovation. Here we describe the existence of spatially opposed bmp and admp expression in a protostome. An admp ortholog (Smed-admp) is expressed ventrally and laterally in adult Schmidtea mediterranea planarians, opposing the dorsal-pole expression of Smed-bmp4. Smed-admp is required for regeneration following parasagittal amputation. Furthermore, Smed-admp promotes Smed-bmp4 expression and Smed-bmp4 inhibits Smed-admp expression, generating a regulatory circuit that buffers against perturbations of Bmp signaling. These results suggest that a Bmp/Admp regulatory circuit is a central feature of the Bilateria, used broadly for the establishment, maintenance, and regeneration of the DV axis. PMID:21295483

  4. Dual Inhibition of Activin/Nodal/TGF-β and BMP Signaling Pathways by SB431542 and Dorsomorphin Induces Neuronal Differentiation of Human Adipose Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Vedavathi Madhu

    2016-01-01

    Full Text Available Damage to the nervous system can cause devastating diseases or musculoskeletal dysfunctions and transplantation of progenitor stem cells can be an excellent treatment option in this regard. Preclinical studies demonstrate that untreated stem cells, unlike stem cells activated to differentiate into neuronal lineage, do not survive in the neuronal tissues. Conventional methods of inducing neuronal differentiation of stem cells are complex and expensive. We therefore sought to determine if a simple, one-step, and cost effective method, previously reported to induce neuronal differentiation of embryonic stem cells and induced-pluripotent stem cells, can be applied to adult stem cells. Indeed, dual inhibition of activin/nodal/TGF-β and BMP pathways using SB431542 and dorsomorphin, respectively, induced neuronal differentiation of human adipose derived stem cells (hADSCs as evidenced by formation of neurite extensions, protein expression of neuron-specific gamma enolase, and mRNA expression of neuron-specific transcription factors Sox1 and Pax6 and matured neuronal marker NF200. This process correlated with enhanced phosphorylation of p38, Erk1/2, PI3K, and Akt1/3. Additionally, in vitro subcutaneous implants of SB431542 and dorsomorphin treated hADSCs displayed significantly higher expression of active-axonal-growth-specific marker GAP43. Our data offers novel insights into cell-based therapies for the nervous system repair.

  5. High-performance scaffolds on titanium surfaces: Osteoblast differentiation and mineralization promoted by a globular fibrinogen layer through cell-autonomous BMP signaling

    Energy Technology Data Exchange (ETDEWEB)

    Horasawa, Noriko, E-mail: horasawa@po.mdu.ac.jp [Department of Dental Materials, Matsumoto Dental University, 1780 Hiro-oka Gobara, Shiojiri, Nagano 399-0781 (Japan); Yamashita, Teruhito [Institute for Oral Science, Matsumoto Dental University, 1780 Hiro-oka Gobara, Shiojiri, Nagano 399-0781 (Japan); Uehara, Shunsuke; Udagawa, Nobuyuki [Department of Biochemistry, Matsumoto Dental University, 1780 Hiro-oka Gobara, Shiojiri, Nagano 399-0781 (Japan)

    2015-01-01

    Titanium has been widely used as a dental implant material. However, it takes several months for the implant body to bind with the jawbone. To develop new bioactive modification on titanium surfaces to achieve full osseointegration expeditiously, we used fibrinogen and fibronectin as bioactive scaffolds on the titanium plate, which are common extracellular matrix (ECM) proteins. We analyzed the features of the surface of ECM-modified titanium plates by atomic force microscopy and Fourier transform infrared spectrophotometry. We also evaluated the effect of ECM modification on promoting the differentiation and mineralization of osteoblasts on these surfaces. Fibrinogen had excellent adsorption on titanium surfaces even at low concentrations, due to the binding ability of fibrinogen via its RGD motif. The surface was composed of a fibrinogen monolayer, in which the ratio of β-sheets was decreased. Osteoblast proliferation on ECM-modified titanium surface was significantly promoted compared with titanium alone. Calcification on the modified surface was also accelerated. These ECM-promoting effects correlated with increased expression of bone morphogenetic proteins (BMPs) by the osteoblasts themselves and were inhibited by Noggin, a BMP inhibitor. These results suggest that the fibrinogen monolayer-modified titanium surface is recognized as bioactive scaffolds and promotes bone formation, resulting in the acceleration of osseointegration. - Highlights: • Fibrinogen had an excellent adsorption on titanium at low concentrations. • Fibrinogen on titanium formed composite layer with a decrease in β-sheet structure. • Osteoblast proliferation and calcification on the ECM-modified titanium plates were significant. • These effects of fibrinogen were increased of BMPs by osteoblasts themselves. • The scaffolds of fibrinogen on titanium might accelerate osseointegration.

  6. High-performance scaffolds on titanium surfaces: Osteoblast differentiation and mineralization promoted by a globular fibrinogen layer through cell-autonomous BMP signaling

    International Nuclear Information System (INIS)

    Titanium has been widely used as a dental implant material. However, it takes several months for the implant body to bind with the jawbone. To develop new bioactive modification on titanium surfaces to achieve full osseointegration expeditiously, we used fibrinogen and fibronectin as bioactive scaffolds on the titanium plate, which are common extracellular matrix (ECM) proteins. We analyzed the features of the surface of ECM-modified titanium plates by atomic force microscopy and Fourier transform infrared spectrophotometry. We also evaluated the effect of ECM modification on promoting the differentiation and mineralization of osteoblasts on these surfaces. Fibrinogen had excellent adsorption on titanium surfaces even at low concentrations, due to the binding ability of fibrinogen via its RGD motif. The surface was composed of a fibrinogen monolayer, in which the ratio of β-sheets was decreased. Osteoblast proliferation on ECM-modified titanium surface was significantly promoted compared with titanium alone. Calcification on the modified surface was also accelerated. These ECM-promoting effects correlated with increased expression of bone morphogenetic proteins (BMPs) by the osteoblasts themselves and were inhibited by Noggin, a BMP inhibitor. These results suggest that the fibrinogen monolayer-modified titanium surface is recognized as bioactive scaffolds and promotes bone formation, resulting in the acceleration of osseointegration. - Highlights: • Fibrinogen had an excellent adsorption on titanium at low concentrations. • Fibrinogen on titanium formed composite layer with a decrease in β-sheet structure. • Osteoblast proliferation and calcification on the ECM-modified titanium plates were significant. • These effects of fibrinogen were increased of BMPs by osteoblasts themselves. • The scaffolds of fibrinogen on titanium might accelerate osseointegration

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

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

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

  8. The etiology of cleft palate formation in BMP7-deficient mice.

    Science.gov (United States)

    Kouskoura, Thaleia; Kozlova, Anastasiia; Alexiou, Maria; Blumer, Susanne; Zouvelou, Vasiliki; Katsaros, Christos; Chiquet, Matthias; Mitsiadis, Thimios A; Graf, Daniel

    2013-01-01

    Palatogenesis is a complex process implying growth, elevation and fusion of the two lateral palatal shelves during embryogenesis. This process is tightly controlled by genetic and mechanistic cues that also coordinate the growth of other orofacial structures. Failure at any of these steps can result in cleft palate, which is a frequent craniofacial malformation in humans. To understand the etiology of cleft palate linked to the BMP signaling pathway, we studied palatogenesis in Bmp7-deficient mouse embryos. Bmp7 expression was found in several orofacial structures including the edges of the palatal shelves prior and during their fusion. Bmp7 deletion resulted in a general alteration of oral cavity morphology, unpaired palatal shelf elevation, delayed shelf approximation, and subsequent lack of fusion. Cell proliferation and expression of specific genes involved in palatogenesis were not altered in Bmp7-deficient embryos. Conditional ablation of Bmp7 with Keratin14-Cre or Wnt1-Cre revealed that neither epithelial nor neural crest-specific loss of Bmp7 alone could recapitulate the cleft palate phenotype. Palatal shelves from mutant embryos were able to fuse when cultured in vitro as isolated shelves in proximity, but not when cultured as whole upper jaw explants. Thus, deformations in the oral cavity of Bmp7-deficient embryos such as the shorter and wider mandible were not solely responsible for cleft palate formation. These findings indicate a requirement for Bmp7 for the coordination of both developmental and mechanistic aspects of palatogenesis. PMID:23516636

  9. The p38/MK2/Hsp25 pathway is required for BMP-2-induced cell migration.

    Directory of Open Access Journals (Sweden)

    Cristina Gamell

    Full Text Available BACKGROUND: Bone morphogenetic proteins (BMPs have been shown to participate in the patterning and specification of several tissues and organs during development and to regulate cell growth, differentiation and migration in different cell types. BMP-mediated cell migration requires activation of the small GTPase Cdc42 and LIMK1 activities. In our earlier report we showed that activation of LIMK1 also requires the activation of PAKs through Cdc42 and PI3K. However, the requirement of additional signaling is not clearly known. METHODOLOGY/PRINCIPAL FINDINGS: Activation of p38 MAPK has been shown to be relevant for a number of BMP-2's physiological effects. We report here that BMP-2 regulation of cell migration and actin cytoskeleton remodelling are dependent on p38 activity. BMP-2 treatment of mesenchymal cells results in activation of the p38/MK2/Hsp25 signaling pathway downstream from the BMP receptors. Moreover, chemical inhibition of p38 signaling or genetic ablation of either p38α or MK2 blocks the ability to activate the downstream effectors of the pathway and abolishes BMP-2-induction of cell migration. These signaling effects on p38/MK2/Hsp25 do not require the activity of either Cdc42 or PAK, whereas p38/MK2 activities do not significantly modify the BMP-2-dependent activation of LIMK1, measured by either kinase activity or with an antibody raised against phospho-threonine 508 at its activation loop. Finally, phosphorylated Hsp25 colocalizes with the BMP receptor complexes in lamellipodia and overexpression of a phosphorylation mutant form of Hsp25 is able to abolish the migration of cells in response to BMP-2. CONCLUSIONS: These results indicate that Cdc42/PAK/LIMK1 and p38/MK2/Hsp25 pathways, acting in parallel and modulating specific actin regulatory proteins, play a critical role in integrating responses during BMP-induced actin reorganization and cell migration.

  10. "Abrogation of Rulings” Methodology: A Critique

    OpenAIRE

    Gasser Auda

    2004-01-01

    Surveying the subject of abrogation (naskh) in the Qur’ān, ḥādīth and Islamic literature, it is clear that most abrogation cases were introduced after the Prophetic era in order to interpret certain Qur’ānic verses and Prophetic narrations (aḥādīth) that some scholars perceived as “conflicting.” Two striking examples are “The Verse of the Sword” (āyat al-saif) and “The Verse of the Barrier” (āyat al-ḥijāb). The Qur’ānic verses and aḥādīth,...

  11. GDF11/BMP11 activates both smad1/5/8 and smad2/3 signals but shows no significant effect on proliferation and migration of human umbilical vein endothelial cells

    Science.gov (United States)

    Zhang, Yong-Hui; Cheng, Feng; Du, Xue-Ting; Gao, Jin-Lai; Xiao, Xiao-Lin; Li, Na; Li, Shan-Liang; Dong, De-Li

    2016-01-01

    GDF11/BMP11, a member of TGF-β superfamily, was reported to rejuvenate heart, skeletal muscle and blood vessel architecture in aged mice. However, the rejuvenative effects of GDF11 were questioned recently. Here, we investigated the effects of GDF11 on smad and non-smad signals in human umbilical vein endothelial cells (HUVECs) and the effects of GDF11 on proliferation and migration of HUVECs and primary rat aortic endothelial cells (RAECs). GDF11 factor purchased from two different companies (PeproTech and R&D Systems) was comparatively studied. Western blot was used to detect the protein expressions. The cell viability and migration were examined by using MTT and wound healing assays. Results showed that GDF11 activated both smad1/5/8 and smad2/3 signals in HUVECs. GDF11 increased protein expression of NADPH oxidase 4(NOX4) in HUVECs. GDF11 showed no significant effect on the protein level of p38, p-p38, ERK, p-ERK, Akt, p-Akt (Ser473) and p-Akt(Thr308), but increased the protein level of p-JNK and p-AMPK in HUVECs, and these increases were inhibited by antioxidant mitoTEMPO treatment. GDF11 slightly increased cell viability after short-term treatment and slightly decreased cell viability after long-term treatment. GDF11 showed no significant effect on cell proliferation and migration. These data indicated that the notion of GDF11 as a rejuvenation-related factor for endothelial cells needs to be cautious. PMID:26919250

  12. Advanced BMP Gene Therapies for Temporal and Spatial Control of Bone Regeneration

    OpenAIRE

    Wilson, C.G.; Martín-Saavedra, F.M.; Vilaboa, N.; Franceschi, R.T.

    2013-01-01

    Spatial and temporal patterns of bone morphogenetic protein (BMP) signaling are crucial to the assembly of appropriately positioned and shaped bones of the face and head. This review advances the hypothesis that reconstitution of such patterns with cutting-edge gene therapies will transform the clinical management of craniofacial bone defects attributed to trauma, disease, or surgical resection. Gradients in BMP signaling within developing limbs and orofacial primordia regulate proliferation ...

  13. BMP antagonists enhance myogenic differentiation and ameliorate the dystrophic phenotype in a DMD mouse model.

    Science.gov (United States)

    Shi, SongTing; Hoogaars, Willem M H; de Gorter, David J J; van Heiningen, Sandra H; Lin, Herbert Y; Hong, Charles C; Kemaladewi, Dwi U; Aartsma-Rus, Annemieke; ten Dijke, Peter; 't Hoen, Peter A C

    2011-02-01

    Duchenne Muscular Dystrophy (DMD) is an X-linked lethal muscle wasting disease characterized by muscle fiber degeneration and necrosis. The progressive pathology of DMD can be explained by an insufficient regenerative response resulting in fibrosis and adipose tissue formation. BMPs are known to inhibit myogenic differentiation and in a previous study we found an increased expression of a BMP family member BMP4 in DMD myoblasts. The aim of the current study was therefore to investigate whether inhibition of BMP signaling could be beneficial for myoblast differentiation and muscle regeneration processes in a DMD context. All tested BMP inhibitors, Noggin, dorsomorphin and LDN-193189, were able to accelerate and enhance myogenic differentiation. However, dorsomorphin repressed both BMP and TGFβ signaling and was found to be toxic to primary myoblast cell cultures. In contrast, Noggin was found to be a potent and selective BMP inhibitor and was therefore tested in vivo in a DMD mouse model. Local adenoviral-mediated overexpression of Noggin in muscle resulted in an increased expression of the myogenic regulatory genes Myog and Myod1 and improved muscle histology. In conclusion, our results suggest that repression of BMP signaling may constitute an attractive adjunctive therapy for DMD patients. PMID:20940052

  14. Activation of JNKs is essential for BMP9-induced osteogenic differentiation of mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yan-fang Zhao

    2013-08-01

    Full Text Available Although BMP9 is highly capable of promoting osteogenicdifferentiation of mesenchymal stem cell (MSCs, the molecularmechanism involved remains to be fully elucidated. Here, weexplore the possible involvement and detail role of JNKs (c-JunN-terminal kinases in BMP9-induced osteogenic differentiationof MSCs. It was found that BMP9 stimulated the activation ofJNKs in MSCs. BMP9-induced osteogenic differentiation ofMSCs was dramatically inhibited by JNKs inhibitor SP600125.Moreover, BMP9-activated Smads signaling was decreased bySP600125 treatment in MSCs. The effects of inhibitor arereproduced with adenoviruses expressing siRNA targeted JNKs.Taken together, our results revealed that JNKs was activated inBMP9-induced osteogenic differentiation of MSCs. What ismost noteworthy, however, is that inhibition of JNKs activityresulted in reduction of BMP9-induced osteogenic differentiationof MSCs, implying that activation of JNKs is essential forBMP9 osteoinductive activity. [BMB Reports 2013; 46(8:422-427

  15. Delayed BMP4 exposure increases germ cell differentiation in mouse embryonic stem cells.

    Science.gov (United States)

    Talaei-Khozani, Tahereh; Zarei Fard, Nehleh; Bahmanpour, Soghra; Jaberipour, Mansoureh; Hosseini, Ahmah; Esmaeilpour, Tahereh

    2014-01-01

    Fate mapping studies have revealed that bone morphogenetic protein 4 (BMP4) signaling has a key role in segregation of primordial germ cells from proximal epiblast. Adding BMP4 to the culture media of embryonic stem (ES) cells could induce expression of germ cell markers; however, to provide a desired number of germ cells has remained a challenge. In the current study, we intended to establish an in vitro system to obtain reliable germ cells derived from ES cells. Differentiation was induced in ES cells via embryoid body (EB) and monolayer culture system. Cells were cultured with BMP4 from the beginning (++BMP4) or after 48 hours (+BMP4) of culturing for five days. The cultures were assessed for alkaline phosphatase (ALP) activity, expression of Oct4, Mvh and c-kit. In EB culture protocol, the expression of Mvh, Oct4 and ALP activity significantly increased in +BMP4 culture condition, but a significant down-regulation in the expression of germ cell markers was shown in ++BMP4 condition compared with the control group. Parallel differentiation experiments using monolayer culture system indicated the number of putative germ cells did not change. In the current study, we compared two differentiation methods (EB and monolayer) to achieve an optimal germ cell production. The EBs with a short exposure time period to BMP4, showing typical characteristics of germ cells. Therefore, our approach provides a strategy for the production of germline cells from ES cells. PMID:24969978

  16. Delivery of dexamethasone from bioactive nanofiber matrices stimulates odontogenesis of human dental pulp cells through integrin/BMP/mTOR signaling pathways.

    Science.gov (United States)

    Lim, Hyun-Chang; Nam, Ok Hyung; Kim, Mi-Joo; El-Fiqi, Ahmed; Yun, Hyung-Mun; Lee, Yoo-Mi; Jin, Guang-Zhen; Lee, Hae-Hyoung; Kim, Hae-Won; Kim, Eun-Cheol

    2016-01-01

    Therapeutically relevant design of scaffolds is of special importance in the repair and regeneration of tissues including dentin and pulp. Here we exploit nanofiber matrices that incorporate bioactive glass nanoparticles (BGNs) and deliver the odontogenic drug dexamethasone (DEX) to stimulate the odontogenic differentiation of human dental pulp cells (HDPCs). DEX molecules were first loaded onto the BGN, and then the DEX-BGN complex was incorporated within the biopolymer nanofiber matrix through electrospinning. The release of DEX continued over a month, showing a slow releasing profile. HDPCs cultured on the DEX-releasing BGN matrices were viable, proliferating well up to 14 days. The odontogenic differentiation, as assessed by alkaline phosphatase activity, mRNA expression of genes, and mineralization, was significantly stimulated on the matrices incorporating BGN and further on those releasing DEX. The DEX-releasing BGN matrices highly upregulated the expression of the integrin subsets α1, α5, and β3 as well as integrin downstream signaling molecules, including focal adhesion kinase (FAK), Paxillin, and RhoA, and activated bone morphogenetic protein mRNA and phosphorylation of Smad1/5/8. Furthermore, the DEX-releasing BGN-matrices stimulated Akt and mammalian target of rapamycin (mTOR), which was proven by the inhibition study. Collectively, the designed therapeutic nanofiber matrices that incorporate BGN and deliver DEX were demonstrated to promote odontogenesis of HDPCs, and the integrins, bone morphogenetic protein, and mTOR signaling pathways are proposed to be the possible molecular mechanisms. While further in vivo studies are still needed, the DEX-releasing bioactive scaffolds are considered as a potential therapeutic nanomatrix for regenerative endodontics and tissue engineering. PMID:27354790

  17. Delivery of dexamethasone from bioactive nanofiber matrices stimulates odontogenesis of human dental pulp cells through integrin/BMP/mTOR signaling pathways

    Science.gov (United States)

    Lim, Hyun-Chang; Nam, Ok Hyung; Kim, Mi-joo; El-Fiqi, Ahmed; Yun, Hyung-Mun; Lee, Yoo-Mi; Jin, Guang-Zhen; Lee, Hae-Hyoung; Kim, Hae-Won; Kim, Eun-Cheol

    2016-01-01

    Therapeutically relevant design of scaffolds is of special importance in the repair and regeneration of tissues including dentin and pulp. Here we exploit nanofiber matrices that incorporate bioactive glass nanoparticles (BGNs) and deliver the odontogenic drug dexamethasone (DEX) to stimulate the odontogenic differentiation of human dental pulp cells (HDPCs). DEX molecules were first loaded onto the BGN, and then the DEX-BGN complex was incorporated within the biopolymer nanofiber matrix through electrospinning. The release of DEX continued over a month, showing a slow releasing profile. HDPCs cultured on the DEX-releasing BGN matrices were viable, proliferating well up to 14 days. The odontogenic differentiation, as assessed by alkaline phosphatase activity, mRNA expression of genes, and mineralization, was significantly stimulated on the matrices incorporating BGN and further on those releasing DEX. The DEX-releasing BGN matrices highly upregulated the expression of the integrin subsets α1, α5, and β3 as well as integrin downstream signaling molecules, including focal adhesion kinase (FAK), Paxillin, and RhoA, and activated bone morphogenetic protein mRNA and phosphorylation of Smad1/5/8. Furthermore, the DEX-releasing BGN-matrices stimulated Akt and mammalian target of rapamycin (mTOR), which was proven by the inhibition study. Collectively, the designed therapeutic nanofiber matrices that incorporate BGN and deliver DEX were demonstrated to promote odontogenesis of HDPCs, and the integrins, bone morphogenetic protein, and mTOR signaling pathways are proposed to be the possible molecular mechanisms. While further in vivo studies are still needed, the DEX-releasing bioactive scaffolds are considered as a potential therapeutic nanomatrix for regenerative endodontics and tissue engineering. PMID:27354790

  18. Abrogation of CD40–CD154 Signaling Impedes the Homeostasis of Thymic Resident Regulatory T Cells by Altering the Levels of IL-2, but Does Not Affect Regulatory T Cell Development

    OpenAIRE

    Cuss, Steven M.; Green, E. Allison

    2012-01-01

    Identification of costimulatory signals required for murine regulatory T (Treg) cell development relies on measuring the frequency of total thymic Treg cells. However, the thymus contains both resident and newly developed Treg cells; whether such signals target both populations is unknown. In this study, we show that CD40–CD154 blockade specifically targeted thymic resident Treg cells, but not, as was previously believed, newly developed Treg cells. Unlike CD28–CD80/CD86 signals, CD40–CD154 s...

  19. Delivery of dexamethasone from bioactive nanofiber matrices stimulates odontogenesis of human dental pulp cells through integrin/BMP/mTOR signaling pathways

    Directory of Open Access Journals (Sweden)

    Lim HC

    2016-06-01

    Full Text Available Hyun-Chang Lim,1,* Ok Hyung Nam,2,* Mi-joo Kim,3 Ahmed El-Fiqi,4,5 Hyung-Mun Yun,3 Yoo-Mi Lee,3 Guang-Zhen Jin,4,5 Hae-Hyoung Lee,5,6 Hae-Won Kim,4–6 Eun-Cheol Kim3 1Department of Periodontology, 2Department of Pediatric Dentistry, 3Department of Oral and Maxillofacial Pathology, Research Center for Tooth and Periodontal Regeneration (MRC, School of Dentistry, Kyung Hee University, Seoul, 4Department of Nanobiomedical Science, BK21 PLUS NBM Global Research Center for Regenerative Medicine, 5Institute of Tissue Regeneration Engineering, 6Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan, Republic of Korea *These authors contributed equally to this work as first authors Abstract: Therapeutically relevant design of scaffolds is of special importance in the repair and regeneration of tissues including dentin and pulp. Here we exploit nanofiber matrices that incorporate bioactive glass nanoparticles (BGNs and deliver the odontogenic drug dexamethasone (DEX to stimulate the odontogenic differentiation of human dental pulp cells (HDPCs. DEX molecules were first loaded onto the BGN, and then the DEX-BGN complex was incorporated within the biopolymer nanofiber matrix through electrospinning. The release of DEX continued over a month, showing a slow releasing profile. HDPCs cultured on the DEX-releasing BGN matrices were viable, proliferating well up to 14 days. The odontogenic differentiation, as assessed by alkaline phosphatase activity, mRNA expression of genes, and mineralization, was significantly stimulated on the matrices incorporating BGN and further on those releasing DEX. The DEX-releasing BGN matrices highly upregulated the expression of the integrin subsets α1, α5, and β3 as well as integrin downstream signaling molecules, including focal adhesion kinase (FAK, Paxillin, and RhoA, and activated bone morphogenetic protein mRNA and phosphorylation of Smad1/5/8. Furthermore, the DEX-releasing BGN

  20. Nutritional abrogation of photoimmunosuppression: in vivo investigations.

    Science.gov (United States)

    Pilkington, Suzanne M; Gibbs, Neil K; Friedmann, Peter S; Rhodes, Lesley E

    2014-01-01

    Skin cancer is a major public health concern, and the primary aetiological factor in the majority of skin cancers is ultraviolet radiation (UVR) exposure. UVR not only induces potentially mutagenic DNA damage but also suppresses cell-mediated immunity (CMI), allowing cancerous cells to escape destruction and progress to tumours. A considerable proportion of an individual's annual sun exposure is obtained outside the vacation period when topical and physical measures for photoprotection are irregularly used. Certain nutrients could provide an adjunctive protective role, and evidence is accruing from experimental studies to support their use in abrogation of photoimmunosuppression. Moreover, developments in clinical research methods to evaluate impact of solar-simulated radiation on cutaneous CMI allow the immune protective potential of nutritional agents to be examined in humans in vivo. This article summarises the mediation of CMI and its suppression by UVR, evaluates the methodology for quantitative assessment in vivo, reviews the human studies reported on nutritional abrogation of photoimmunosuppression including recent randomized controlled trials and discusses the mechanisms of photoprotection by the nutrients. This includes, in addition to antioxidants, novel studies of omega-3 polyunsaturated fatty acids and nicotinamide. PMID:24283330

  1. Fibrin Hydrogel Based Bone Substitute Tethered with BMP-2 and BMP-2/7 Heterodimers

    Directory of Open Access Journals (Sweden)

    Lindsay S. Karfeld-Sulzer

    2015-03-01

    Full Text Available Current clinically used delivery methods for bone morphogenetic proteins (BMPs are collagen based and require large concentrations that can lead to dangerous side effects. Fibrin hydrogels can serve as osteoinductive bone substitute materials in non-load bearing bone defects in combination with BMPs. Two strategies to even further optimize such a fibrin based system include employing more potent BMP heterodimers and engineering growth factors that can be covalently tethered to and slowly released from a fibrin matrix. Here we present an engineered BMP-2/BMP-7 heterodimer where an N-terminal transglutaminase substrate domain in the BMP-2 portion provides covalent attachment to fibrin together with a central plasmin substrate domain, a cleavage site for local release of the attached BMP-2/BMP-7 heterodimer under the influence of cell-activated plasmin. In vitro and in vivo results revealed that the engineered BMP-2/BMP-7 heterodimer induces significantly more alkaline phosphatase activity in pluripotent cells and bone formation in a rat calvarial model than the engineered BMP-2 homodimer. Therefore, the engineered BMP-2/BMP-7 heterodimer could be used to reduce the amount of BMP needed for clinical effect.

  2. Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis.

    Science.gov (United States)

    Wu, Lian; Wang, Feng; Donly, Kevin J; Wan, Chunyan; Luo, Daoshu; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

    2015-11-01

    Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages. PMID:26037045

  3. A feed-forward loop coupling extracellular BMP transport and morphogenesis in Drosophila wing.

    Directory of Open Access Journals (Sweden)

    Shinya Matsuda

    2013-03-01

    Full Text Available A variety of extracellular factors regulate morphogenesis during development. However, coordination between extracellular signaling and dynamic morphogenesis is largely unexplored. We address the fundamental question by studying posterior crossvein (PCV development in Drosophila as a model, in which long-range BMP transport from the longitudinal veins plays a critical role during the pupal stages. Here, we show that RhoGAP Crossveinless-C (Cv-C is induced at the PCV primordial cells by BMP signaling and mediates PCV morphogenesis cell-autonomously by inactivating members of the Rho-type small GTPases. Intriguingly, we find that Cv-C is also required non-cell-autonomously for BMP transport into the PCV region, while a long-range BMP transport is guided toward ectopic wing vein regions by loss of the Rho-type small GTPases. We present evidence that low level of ß-integrin accumulation at the basal side of PCV epithelial cells regulated by Cv-C provides an optimal extracellular environment for guiding BMP transport. These data suggest that BMP transport and PCV morphogenesis are tightly coupled. Our study reveals a feed-forward mechanism that coordinates the spatial distribution of extracellular instructive cues and morphogenesis. The coupling mechanism may be widely utilized to achieve precise morphogenesis during development and homeostasis.

  4. Foxc1 Expression in Early Osteogenic Differentiation Is Regulated by BMP4-SMAD Activity.

    Science.gov (United States)

    Hopkins, Alexander; Mirzayans, Freda; Berry, Fred

    2016-07-01

    FOXC1 is an important regulator of the initial steps in intramembranous and endochondral ossification processes. As BMP signalling is a key initiator of these processes, we sought to determine whether Foxc1 expression is regulated by such signalling factors. BMP4 treatment of C2C12 cells resulted in an induction in Foxc1 mRNA levels. Chromatin immunoprecipitation assays demonstrated that SMAD proteins interacted with the mouse Foxc1 promoter approximately 300 bp upstream of the transcription start site. This ChIP positive region was cloned into a luciferase reporter and demonstrated to be responsive to BMP4 stimulation. Reduction of Foxc1 levels in C2C12 cells though siRNA impaired BMP4 osteogenic differentiation. In contrast, BMP4 treatment repressed Foxc1 expression in 10T1/2 or D1-ORL mesenchymal cells and MC3T3 preosteoblasts. Finally, siRNA knock-down of Foxc1 in MC3T3 cells resulted in an induction of markers of osteoblast differentiation and an accelerated mineralization. These data indicate that Foxc1 expression is regulated by BMP4 and FOXC1 functions in the commitment of progenitor cells to the osteoblast fate and its expression is reduced when differentiation proceeds. J. Cell. Biochem. 117: 1707-1717, 2016. © 2015 Wiley Periodicals, Inc. PMID:26666591

  5. Repressive BMP2 gene regulatory elements near the BMP2 promoter

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Shan [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States); Chandler, Ronald L. [Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University School of Medicine, Nashville, TN (United States); Fritz, David T. [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States); Mortlock, Douglas P. [Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University School of Medicine, Nashville, TN (United States); Rogers, Melissa B., E-mail: rogersmb@umdnj.edu [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States)

    2010-02-05

    The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.

  6. Repressive BMP2 gene regulatory elements near the BMP2 promoter

    International Nuclear Information System (INIS)

    The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.

  7. BMP4 Is a Peripherally-Derived Factor for Motor Neurons and Attenuates Glutamate-Induced Excitotoxicity In Vitro

    Science.gov (United States)

    Chou, Hui-Ju; Lai, Dar-Ming; Huang, Cheng-Wen; McLennan, Ian S.; Wang, Horng-Dar; Wang, Pei-Yu

    2013-01-01

    Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-β) superfamily, have been shown to play important roles in the nervous system, including neuronal survival and synaptogenesis. However, the physiological functions of BMP signaling in the mammalian neuromuscular system are not well understood. In this study, we found that proteins of the type II bone morphogenetic receptors (BMPRII) were detected at the neuromuscular junction (NMJ), and one of its ligands, BMP4, was expressed by Schwann cells and skeletal muscle fibers. In double-ligated nerves, BMP4 proteins accumulated at the proximal and distal portions of the axons, suggesting that Schwann cell- and muscle fiber-derived BMP4 proteins were anterogradely and retrogradely transported by motor neurons. Furthermore, BMP4 mRNA was down-regulated in nerves but up-regulated in skeletal muscles following nerve ligation. The motor neuron-muscle interactions were also demonstrated using differentiated C2C12 muscle cells and NG108-15 neurons in vitro. BMP4 mRNA and immunoreactivity were significantly up-regulated in differentiated C2C12 muscle cells when the motor neuron-derived factor, agrin, was present in the culture. Peripherally-derived BMP4, on the other hand, promotes embryonic motor neuron survival and protects NG108-15 neurons from glutamate-induced excitotoxicity. Together, these data suggest that BMP4 is a peripherally-derived factor that may regulate the survival of motor neurons. PMID:23472198

  8. The impact of bone morphogenetic protein 4 (BMP4) on breast cancer metastasis in a mouse xenograft model.

    Science.gov (United States)

    Ampuja, M; Alarmo, E L; Owens, P; Havunen, R; Gorska, A E; Moses, H L; Kallioniemi, A

    2016-06-01

    Bone morphogenetic protein 4 (BMP4) is a key regulator of cell proliferation and differentiation. In breast cancer cells, BMP4 has been shown to reduce proliferation in vitro and interestingly, in some cases, also to induce migration and invasion. Here we investigated whether BMP4 influences breast cancer metastasis formation by using a xenograft mouse model. MDA-MB-231 breast cancer cells were injected intracardially into mice and metastasis formation was monitored using bioluminescence imaging. Mice treated with BMP4 developed metastases slightly earlier as compared to control animals but the overall number of metastases was similar in both groups (13 in the BMP4 group vs. 12 in controls). In BMP4-treated mice, bone metastases were more common (10 vs. 7) but adrenal gland metastases were less frequent (1 vs. 5) than in controls. Immunostaining revealed no differences in signaling activation, proliferation rate, blood vessel formation, EMT markers or the number of cancer-associated fibroblasts between the treatment groups. In conclusion, BMP4 caused a trend towards accelerated metastasis formation, especially in bone. More work is needed to uncover the long-term effects of BMP4 and the clinical relevance of these findings. PMID:26970275

  9. Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis

    Science.gov (United States)

    Wu, Lian; Wang, Feng; Donly, Kevin J.; Wan, Chunyan; Luo, Daoshu; Harris, Stephen E.; Macdougall, Mary; Chen, Shuo

    2016-01-01

    Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2ko/ko dp) cell line by introducing Cre fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2fx/fx dp) cells. iBmp2ko/ko dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2ko/ko dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmpko/ko cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages. PMID:26037045

  10. Bmp4 is essential for the formation of the vestibular apparatus that detects angular head movements.

    Directory of Open Access Journals (Sweden)

    Weise Chang

    2008-04-01

    Full Text Available Angular head movements in vertebrates are detected by the three semicircular canals of the inner ear and their associated sensory tissues, the cristae. Bone morphogenetic protein 4 (Bmp4, a member of the Transforming growth factor family (TGF-beta, is conservatively expressed in the developing cristae in several species, including zebrafish, frog, chicken, and mouse. Using mouse models in which Bmp4 is conditionally deleted within the inner ear, as well as chicken models in which Bmp signaling is knocked down specifically in the cristae, we show that Bmp4 is essential for the formation of all three cristae and their associated canals. Our results indicate that Bmp4 does not mediate the formation of sensory hair and supporting cells within the cristae by directly regulating genes required for prosensory development in the inner ear such as Serrate1 (Jagged1 in mouse, Fgf10, and Sox2. Instead, Bmp4 most likely mediates crista formation by regulating Lmo4 and Msx1 in the sensory region and Gata3, p75Ngfr, and Lmo4 in the non-sensory region of the crista, the septum cruciatum. In the canals, Bmp2 and Dlx5 are regulated by Bmp4, either directly or indirectly. Mechanisms involved in the formation of sensory organs of the vertebrate inner ear are thought to be analogous to those regulating sensory bristle formation in Drosophila. Our results suggest that, in comparison to sensory bristles, crista formation within the inner ear requires an additional step of sensory and non-sensory fate specification.

  11. Smad6 determines BMP-regulated invasive behaviour of breast cancer cells in a zebrafish xenograft model

    Science.gov (United States)

    de Boeck, Miriam; Cui, Chao; Mulder, Aat A; Jost, Carolina R; Ikeno, Souichi; ten Dijke, Peter

    2016-01-01

    The transforming growth factor-β (TGF-β) family is known to play critical roles in cancer progression. While the dual role of TGF-β is well described, the function of bone morphogenetic proteins (BMPs) is unclear. In this study, we established the involvement of Smad6, a BMP-specific inhibitory Smad, in breast cancer cell invasion. We show that stable overexpression of Smad6 in breast cancer MCF10A M2 cells inhibits BMP signalling, thereby mitigating BMP6-induced suppression of mesenchymal marker expression. Using a zebrafish xenograft model, we demonstrate that overexpression of Smad6 potentiates invasion of MCF10A M2 cells and enhances the aggressiveness of breast cancer MDA-MB-231 cells in vivo, whereas a reversed phenotype is observed after Smad6 knockdown. Interestingly, BMP6 pre-treatment of MDA-MB-231 cells induced cluster formation at the invasive site in the zebrafish. BMP6 also stimulated cluster formation of MDA-MB-231 cells co-cultured on Human Microvascular Endothelial Cells (HMEC)-1 in vitro. Electron microscopy illustrated an induction of cell-cell contact by BMP6. The clinical relevance of our findings is highlighted by a correlation of high Smad6 expression with poor distant metastasis free survival in ER-negative cancer patients. Collectively, our data strongly indicates the involvement of Smad6 and BMP signalling in breast cancer cell invasion in vivo. PMID:27113436

  12. Smad6 determines BMP-regulated invasive behaviour of breast cancer cells in a zebrafish xenograft model.

    Science.gov (United States)

    de Boeck, Miriam; Cui, Chao; Mulder, Aat A; Jost, Carolina R; Ikeno, Souichi; Ten Dijke, Peter

    2016-01-01

    The transforming growth factor-β (TGF-β) family is known to play critical roles in cancer progression. While the dual role of TGF-β is well described, the function of bone morphogenetic proteins (BMPs) is unclear. In this study, we established the involvement of Smad6, a BMP-specific inhibitory Smad, in breast cancer cell invasion. We show that stable overexpression of Smad6 in breast cancer MCF10A M2 cells inhibits BMP signalling, thereby mitigating BMP6-induced suppression of mesenchymal marker expression. Using a zebrafish xenograft model, we demonstrate that overexpression of Smad6 potentiates invasion of MCF10A M2 cells and enhances the aggressiveness of breast cancer MDA-MB-231 cells in vivo, whereas a reversed phenotype is observed after Smad6 knockdown. Interestingly, BMP6 pre-treatment of MDA-MB-231 cells induced cluster formation at the invasive site in the zebrafish. BMP6 also stimulated cluster formation of MDA-MB-231 cells co-cultured on Human Microvascular Endothelial Cells (HMEC)-1 in vitro. Electron microscopy illustrated an induction of cell-cell contact by BMP6. The clinical relevance of our findings is highlighted by a correlation of high Smad6 expression with poor distant metastasis free survival in ER-negative cancer patients. Collectively, our data strongly indicates the involvement of Smad6 and BMP signalling in breast cancer cell invasion in vivo. PMID:27113436

  13. A secreted BMP antagonist, Cer1, fine tunes the spatial organization of the ureteric bud tree during mouse kidney development.

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    Lijun Chi

    Full Text Available The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1 fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching.

  14. Induction of chronic pancreatitis by pancreatic duct ligation activates BMP2, apelin, and PTHrP expression in mice.

    Science.gov (United States)

    Rastellini, Cristiana; Han, Song; Bhatia, Vandanajay; Cao, Yanna; Liu, Ka; Gao, Xuxia; Ko, Tien C; Greeley, George H; Falzon, Miriam

    2015-10-01

    Chronic pancreatitis (CP) is a devastating disease with no treatments. Experimental models have been developed to reproduce the parenchyma and inflammatory responses typical of human CP. For the present study, one objective was to assess and compare the effects of pancreatic duct ligation (PDL) to those of repetitive cerulein (Cer)-induced CP in mice on pancreatic production of bone morphogenetic protein-2 (BMP2), apelin, and parathyroid hormone-related protein (PTHrP). A second objective was to determine the extent of cross talk among pancreatic BMP2, apelin, and PTHrP signaling systems. We focused on BMP2, apelin, and PTHrP since these factors regulate the inflammation-fibrosis cascade during pancreatitis. Findings showed that PDL- and Cer-induced CP resulted in significant elevations in expression and peptide/protein levels of pancreatic BMP2, apelin, and PTHrP. In vivo mouse and in vitro pancreatic cell culture experiments demonstrated that BMP2 stimulated pancreatic apelin expression whereas apelin expression was inhibited by PTHrP exposure. Apelin or BMP2 exposure inhibited PTHrP expression, and PTHrP stimulated upregulation of gremlin, an endogenous inhibitor of BMP2 activity. Transforming growth factor-β (TGF-β) stimulated PTHrP expression. Together, findings demonstrated that PDL- and Cer-induced CP resulted in increased production of the pancreatic BMP2, apelin, and PTHrP signaling systems and that significant cross talk occurred among pancreatic BMP2, apelin, and PTHrP. These results together with previous findings imply that these factors interact via a pancreatic network to regulate the inflammation-fibrosis cascade during CP. More importantly, this network communicated with TGF-β, a key effector of pancreatic pathophysiology. This novel network may be amenable to pharmacologic manipulations during CP in humans. PMID:26229008

  15. Simultaneous gene transfer of bone morphogenetic protein (BMP -2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression

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    Miyazaki Jun-ichi

    2006-08-01

    Full Text Available Abstract Background Transcutaneous in vivo electroporation is expected to be an effective gene-transfer method for promoting bone regeneration using the BMP-2 plasmid vector. To promote enhanced osteoinduction using this method, we simultaneously transferred cDNAs for BMP-2 and BMP-7, as inserts in the non-viral vector pCAGGS. Methods First, an in vitro study was carried out to confirm the expression of BMP-2 and BMP-7 following the double-gene transfer. Next, the individual BMP-2 and BMP-7 plasmids or both together were injected into rat calf muscles, and transcutaneous electroporation was applied 8 times at 100 V, 50 msec. Results In the culture system, the simultaneous transfer of the BMP-2 and BMP-7 genes led to a much higher ALP activity in C2C12 cells than did the transfer of either gene alone. In vivo, ten days after the treatment, soft X-ray analysis showed that muscles that received both pCAGGS-BMP-2 and pCAGGS-BMP-7 had better-defined opacities than those receiving a single gene. Histological examination showed advanced ossification in calf muscles that received the double-gene transfer. BMP-4 mRNA was also expressed, and RT-PCR showed that its level increased for 3 days in a time-dependent manner in the double-gene transfer group. Immunohistochemistry confirmed that BMP-4-expressing cells resided in the matrix between muscle fibers. Conclusion The simultaneous transfer of BMP-2 and BMP-7 genes using in vivo electroporation induces more rapid bone formation than the transfer of either gene alone, and the increased expression of endogenous BMP-4 suggests that the rapid ossification is related to the induction of BMP-4.

  16. Endoplasmic reticulum (ER stress inducible factor cysteine-rich with EGF-like domains 2 (Creld2 is an important mediator of BMP9-regulated osteogenic differentiation of mesenchymal stem cells.

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    Jiye Zhang

    Full Text Available Mesenchymal stem cells (MSCs are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2 in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.

  17. Heterozygous Mutations in BMP6 Pro-peptide Lead to Inappropriate Hepcidin Synthesis and Moderate Iron Overload in Humans

    OpenAIRE

    Daher, Raed; Kannengiesser, Caroline; Houamel, Dounia; Lefebvre, Thibaud; Bardou-Jacquet, Edouard; Ducrot, Nicolas; Kerguenec, Caroline,; Jouanolle, Anne-Marie; Robreau, Anne-Marie; Oudin, Claire; Le Gac, Gerald; Moulouel, Boualem; Loustaud-Ratti, Véronique; Bedossa, Pierre; Valla, Dominique

    2015-01-01

    Background & Aims Hereditary hemochromatosis is a heterogeneous group of genetic disorders characterized by parenchymal iron overload. It is caused by defective expression of liver hepcidin, the main regulator of iron homeostasis. Iron stimulates the gene encoding (HAMP) hepcidin via the BMP6 signaling to SMAD. Although several genetic factors have been found to cause late-onset hemochromatosis, many patients have unexplained signs of iron overload. We investigated BMP6 function in these indi...

  18. Biochemicalmethane potential (BMP) of solid organic substrates

    DEFF Research Database (Denmark)

    Raposo, F.; Fernández-Cegrí, V.; de la Rubia, M.A.;

    2011-01-01

    BACKGROUND: This paper describes results obtained for different participating research groups in an interlaboratory study related to biochemical methane potential (BMP). In this research work, all experimental conditions influencing the test such as inoculum, substrate characteristics and experim...

  19. Glucocorticoid regulation of human BMP-6 transcription.

    Science.gov (United States)

    Liu, Yunshan; Titus, Louisa; Barghouthi, Mejd; Viggeswarapu, Manjula; Hair, Gregory; Boden, Scott D

    2004-09-01

    Addition of dexamethasone (Dex) to human mesenchymal stem cells (hMSCs) resulted in a 16-fold increase in human bone morphogenetic protein-6 (hBMP-6) mRNA levels 24 h after treatment. Evaluation of luciferase expression after transfection of HeLa cells with hBMP-6 promoter/luciferase reporter constructs indicated that the hBMP-6 promoter activity was contained in a 268-bp region (-1051 to -784 where +1 is the translation start site) over 600 bases 5' to that previously published. It further showed that the promoter activity is regulated by glucocorticoid treatment. Analysis of RNA from hMSCs and HeLa cells by primer extension, RNase protection, and 5' RACE further narrowed the location of the transcription start site to an 84-bp region (-940 to -857). To determine whether this start site was regulated in hMSCs, hBMP-6 mRNA levels in control and Dex-treated cells were quantitated by RT-PCR using one primer set in the translated region of the gene and one located just 3' of the 84-bp region. Both primer sets showed hBMP-6 mRNA levels approximately 16- to 22-fold higher in the Dex-treated cells, demonstrating that hBMP-6 transcription is being regulated by glucocorticoids in the pluripotent hMSCs at the upstream transcription start site. PMID:15336603

  20. BMP3 expression in the adult rat CNS.

    Science.gov (United States)

    Yamashita, Kanna; Mikawa, Sumiko; Sato, Kohji

    2016-07-15

    Bone morphogenetic protein-3 (BMP3) is a very unique member of the TGF-β superfamily, because it functions as an antagonist to both the canonical BMP and activin pathways and plays important roles in multiple biological events. Although BMP3 expression has been described in the early development of the kidney, intestine and bone, little information is available for BMP3 expression in the central nervous system (CNS). We, thus, investigated BMP3 expression in the adult rat CNS using immunohistochemistry. BMP3 was intensely expressed in most neurons and their axons. Furthermore, we found that astrocytes and ependymal cells also express BMP3 protein. These data indicate that BMP3 is widely expressed throughout the adult CNS, and its abundant expression in the adult brain strongly supports the idea that BMP3 plays important roles in the adult brain. PMID:27130896

  1. Osthole-mediated cell differentiation through bone morphogenetic protein-2/p38 and extracellular signal-regulated kinase 1/2 pathway in human osteoblast cells.

    Science.gov (United States)

    Kuo, Po-Lin; Hsu, Ya-Ling; Chang, Cheng-Hsiung; Chang, Jiunn-Kae

    2005-09-01

    The survival of osteoblast cells is one of the determinants of the development of osteoporosis in patients. Osthole (7-methoxy-8-isopentenoxycoumarin) is a coumarin derivative present in many medicinal plants. By means of alkaline phosphatase (ALP) activity, osteocalcin, osteopontin, and type I collagen, enzyme-linked immunosorbent assay, we have shown that osthole exhibits a significant induction of differentiation in two human osteoblast-like cell lines, MG-63 and hFOB. Induction of differentiation by osthole was associated with increased bone morphogenetic protein (BMP)-2 production and the activations of SMAD1/5/8 and p38 and extracellular signal-regulated kinase (ERK) 1/2 kinases. Addition of purified BMP-2 protein did not increase the up-regulation of ALP activity and osteocalcin by osthole, whereas the BMP-2 antagonist noggin blocked both osthole and BMP-2-mediated ALP activity enhancement, indicating that BMP-2 production is required in osthole-mediated osteoblast maturation. Pretreatment of osteoblast cells with noggin abrogated p38 activation but only partially decreased ERK1/2 activation, suggesting that BMP-2 signaling is required in p38 activation and is partially involved in ERK1/2 activation in osthole-treated osteoblast cells. Cotreatment of p38 inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole] or p38 small interfering RNA (siRNA) expression inhibited osthole-mediated activation of ALP but only slightly affected osteocalcin production. In contrast, the production of osteocalcin induced by osthole was inhibited by the mitogen-activated protein kinase kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) or by expression of an ERK2 siRNA. These data suggest that BMP-2/p38 pathway links to the early phase, whereas ERK1/2 pathway is associated with the later phase in osthole-mediated differentiation of osteoblast cells. In this study, we demonstrate that osthole is a promising agent for treating osteoporosis

  2. Simultaneous gene transfer of bone morphogenetic protein (BMP) -2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression

    OpenAIRE

    Kawai, Mariko; Bessho, Kazuhisa; Maruyama, Hiroki; Miyazaki, Jun-ichi; Yamamoto, Toshio

    2006-01-01

    Background: Transcutaneous in vivo electroporation is expected to be an effective gene-transfer method for promoting bone regeneration using the BMP-2 plasmid vector. To promote enhanced osteoinduction using this method, we simultaneously transferred cDNAs for BMP-2 and BMP-7, as inserts in the non-viral vector pCAGGS.

  3. The BMP pathway is essential for re-specification and maintenance of the dorsoventral axis in regenerating and intact planarians.

    Science.gov (United States)

    Molina, M Dolores; Saló, Emili; Cebrià, Francesc

    2007-11-01

    The bone morphogenetic protein (BMP) pathway has been shown to play an important role in the establishment of the dorsoventral axis during development in both vertebrate and invertebrate species. In an attempt to unravel the role of BMPs in pattern formation during planarian regeneration, we studied this signaling pathway in Schmidtea mediterranea. Here, we functionally characterize planarian homologues of two key elements of the pathway: Smed-BMP and Smed-Smad1. Whole-mount in situ hybridization showed that Smed-BMP is expressed at the planarian dorsal midline, suggesting a role in dorsoventral patterning, while Smed-Smad1 is widely expressed throughout the mesenchyme and in the central nervous system. RNA interference (RNAi) knockdowns of Smed-BMP or Smed-Smad1 led to the disappearance of dorsal markers along with the ectopic expression of ventral markers on the dorsal side of the treated animals. In almost all cases, a duplicated central nervous system differentiated dorsally after Smed-BMP or Smed-Smad1 RNAi. These defects were observed not only during regeneration but also in intact non-regenerating animals. Our results suggest that the BMP signaling pathway is conserved in planarians and that it plays a key role in the regeneration and maintenance of the dorsoventral axis. PMID:17905225

  4. Cross talk between insulin and bone morphogenetic protein signaling systems in brown adipogenesis

    DEFF Research Database (Denmark)

    Zhang, Hongbin; Schulz, Tim J; Espinoza, Daniel O;

    2010-01-01

    Both insulin and bone morphogenetic protein (BMP) signaling systems are important for adipocyte differentiation. Analysis of gene expression in BMP7-treated fibroblasts revealed a coordinated change in insulin signaling components by BMP7. To further investigate the cross talk between insulin and...... BMP7's suppressive effect on pref-1 transcription. Together, these data suggest cross talk between the insulin and BMP signaling systems by which BMP7 can rescue brown adipogenesis in cells with insulin resistance.......Both insulin and bone morphogenetic protein (BMP) signaling systems are important for adipocyte differentiation. Analysis of gene expression in BMP7-treated fibroblasts revealed a coordinated change in insulin signaling components by BMP7. To further investigate the cross talk between insulin and...... BMP signaling systems in brown adipogenesis, we examined the effect of BMP7 in insulin receptor substrate 1 (IRS-1)-deficient brown preadipocytes, which exhibit a severe defect in differentiation. Treatment of these cells with BMP7 for 3 days prior to adipogenic induction restored differentiation and...

  5. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Chieri [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp [Division of Pharmacotherapy, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

  6. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    International Nuclear Information System (INIS)

    Highlights: ► We investigated the role of S1P signaling for osteoblast differentiation. ► Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. ► S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. ► MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P receptor-mediated signaling plays a crucial role for osteoblast differentiation.

  7. Functional cardiomyocytes derived from Isl1 cardiac progenitors via Bmp4 stimulation.

    Directory of Open Access Journals (Sweden)

    Esra Cagavi

    Full Text Available As heart failure due to myocardial infarction remains a leading cause of morbidity worldwide, cell-based cardiac regenerative therapy using cardiac progenitor cells (CPCs could provide a potential treatment for the repair of injured myocardium. As adult CPCs may have limitations regarding tissue accessibility and proliferative ability, CPCs derived from embryonic stem cells (ESCs could serve as an unlimited source of cells with high proliferative ability. As one of the CPCs that can be derived from embryonic stem cells, Isl1 expressing cardiac progenitor cells (Isl1-CPCs may serve as a valuable source of cells for cardiac repair due to their high cardiac differentiation potential and authentic cardiac origin. In order to generate an unlimited number of Isl1-CPCs, we used a previously established an ESC line that allows for isolation of Isl1-CPCs by green fluorescent protein (GFP expression that is directed by the mef2c gene, specifically expressed in the Isl1 domain of the anterior heart field. To improve the efficiency of cardiac differentiation of Isl1-CPCs, we studied the role of Bmp4 in cardiogenesis of Isl1-CPCs. We show an inductive role of Bmp directly on cardiac progenitors and its enhancement on early cardiac differentiation of CPCs. Upon induction of Bmp4 to Isl1-CPCs during differentiation, the cTnT+ cardiomyocyte population was enhanced 2.8±0.4 fold for Bmp4 treated CPC cultures compared to that detected for vehicle treated cultures. Both Bmp4 treated and untreated cardiomyocytes exhibit proper electrophysiological and calcium signaling properties. In addition, we observed a significant increase in Tbx5 and Tbx20 expression in differentiation cultures treated with Bmp4 compared to the untreated control, suggesting a link between Bmp4 and Tbx genes which may contribute to the enhanced cardiac differentiation in Bmp4 treated cultures. Collectively these findings suggest a cardiomyogenic role for Bmp4 directly on a pure population of

  8. BMP-2-enhanced chondrogenesis involves p38 MAPK-mediated down-regulation of Wnt-7a pathway.

    Science.gov (United States)

    Jin, Eun-Jung; Lee, Sun-Young; Choi, Young-Ae; Jung, Jae-Chang; Bang, Ok-Sun; Kang, Shin-Sung

    2006-12-31

    The bone morphogenetic protein (BMP) family has been implicated in control of cartilage development. Here, we demonstrate that BMP-2 promotes chondrogenesis by activating p38 mitogen-activated protein kinase (MAPK), which in turn downregulates Wnt-7a/b-catenin signaling responsible for proteasomal degradation of Sox9. Exposure of mesenchymal cells to BMP-2 resulted in upregulation of Sox9 protein and a concomitant decrease in the level of b-catenin protein and Wnt-7a signaling. In agreement with this, the interaction of Sox9 with b-catenin was inhibited in the presence of BMP-2. Inhibition of the p38 MAPK pathway using a dominant negative mutant led to sustained Wnt-7a signaling and decreased Sox9 expression, with consequent inhibition of precartilage condensation and chondrogenic differentiation. Moreover, overexpression of b-catenin caused degradation of Sox9 via the ubiquitin/26S proteasome pathway. Our results collectively indicate that the increase in Sox9 protein resulting from downregulation of b-catenin/Wnt-7a signaling is mediated by p38 MAPK during BMP-2 induced chondrogenesis in chick wing bud mesenchymal cells. PMID:17202865

  9. A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction

    OpenAIRE

    Sulkowski, Mikolaj J.; Tae Hee Han; Carolyn Ott; Qi Wang; Verheyen, Esther M.; Jennifer Lippincott-Schwartz; Mihaela Serpe

    2016-01-01

    Author Summary Synaptic activity and synapse development are intimately linked, but our understanding of the coupling mechanisms remains limited. Anterograde and retrograde signals together with trans-synaptic complexes enable intercellular communications. How synapse activity status is monitored and relayed across the synaptic cleft remains poorly understood. The Drosophila NMJ is a very powerful genetic system to study synapse development. BMP signaling modulates NMJ growth via a canonical,...

  10. Identification of bone morphogenetic protein 9 (BMP9) as a novel profibrotic factor in vitro.

    Science.gov (United States)

    Muñoz-Félix, José M; Cuesta, Cristina; Perretta-Tejedor, Nuria; Subileau, Mariela; López-Hernández, Francisco J; López-Novoa, José M; Martínez-Salgado, Carlos

    2016-09-01

    Upregulated synthesis of extracellular matrix (ECM) proteins by myofibroblasts is a common phenomenon in the development of fibrosis. Although the role of TGF-β in fibrosis development has been extensively studied, the involvement of other members of this superfamily of cytokines, the bone morphogenetic proteins (BMPs) in organ fibrosis has given contradictory results. BMP9 is the main ligand for activin receptor-like kinase-1 (ALK1) TGF-β1 type I receptor and its effect on fibrosis development is unknown. Our purpose was to study the effect of BMP9 in ECM protein synthesis in fibroblasts, as well as the involved receptors and signaling pathways. In cultured mice fibroblasts, BMP9 induces an increase in collagen, fibronectin and connective tissue growth factor expression, associated with Smad1/5/8, Smad2/3 and Erk1/2 activation. ALK5 inhibition with SB431542 or ALK1/2/3/6 with dorsomorphin-1, inhibition of Smad3 activation with SIS3, and inhibition of the MAPK/Erk1/2 with U0126, demonstrates the involvement of these pathways in BMP9-induced ECM synthesis in MEFs. Whereas BMP9 induced Smad1/5/8 phosphorylation through ALK1, it also induces Smad2/3 phosphorylation through ALK5 but only in the presence of ALK1. Summarizing, this is the first study that accurately identifies BMP9 as a profibrotic factor in fibroblasts that promotes ECM protein expression through ALK1 and ALK5 receptors. PMID:27208502

  11. Deficiency of retinaldehyde dehydrogenase 1 induces BMP2 and increases bone mass in vivo.

    Directory of Open Access Journals (Sweden)

    Shriram Nallamshetty

    Full Text Available The effects of retinoids, the structural derivatives of vitamin A (retinol, on post-natal peak bone density acquisition and skeletal remodeling are complex and compartment specific. Emerging data indicates that retinoids, such as all trans retinoic acid (ATRA and its precursor all trans retinaldehyde (Rald, exhibit distinct and divergent transcriptional effects in metabolism. Despite these observations, the role of enzymes that control retinoid metabolism in bone remains undefined. In this study, we examined the skeletal phenotype of mice deficient in retinaldehyde dehydrogenase 1 (Aldh1a1, the enzyme responsible for converting Rald to ATRA in adult animals. Bone densitometry and micro-computed tomography (µCT demonstrated that Aldh1a1-deficient (Aldh1a1(-/- female mice had higher trabecular and cortical bone mass compared to age and sex-matched control C57Bl/6 wild type (WT mice at multiple time points. Histomorphometry confirmed increased cortical bone thickness and demonstrated significantly higher bone marrow adiposity in Aldh1a1(-/- mice. In serum assays, Aldh1a1(-/- mice also had higher serum IGF-1 levels. In vitro, primary Aldh1a1(-/- mesenchymal stem cells (MSCs expressed significantly higher levels of bone morphogenetic protein 2 (BMP2 and demonstrated enhanced osteoblastogenesis and adipogenesis versus WT MSCs. BMP2 was also expressed at higher levels in the femurs and tibias of Aldh1a1(-/- mice with accompanying induction of BMP2-regulated responses, including expression of Runx2 and alkaline phosphatase, and Smad phosphorylation. In vitro, Rald, which accumulates in Aldh1a1(-/- mice, potently induced BMP2 in WT MSCs in a retinoic acid receptor (RAR-dependent manner, suggesting that Rald is involved in the BMP2 increases seen in Aldh1a1 deficiency in vivo. Collectively, these data implicate Aldh1a1 as a novel determinant of cortical bone density and marrow adiposity in the skeleton in vivo through modulation of BMP signaling.

  12. BMP and Hedgehog Regulate Distinct AGM Hematopoietic Stem Cells Ex Vivo.

    Science.gov (United States)

    Crisan, Mihaela; Solaimani Kartalaei, Parham; Neagu, Alex; Karkanpouna, Sofia; Yamada-Inagawa, Tomoko; Purini, Caterina; Vink, Chris S; van der Linden, Reinier; van Ijcken, Wilfred; Chuva de Sousa Lopes, Susana M; Monteiro, Rui; Mummery, Christine; Dzierzak, Elaine

    2016-03-01

    Hematopoietic stem cells (HSC), the self-renewing cells of the adult blood differentiation hierarchy, are generated during embryonic stages. The first HSCs are produced in the aorta-gonad-mesonephros (AGM) region of the embryo through endothelial to a hematopoietic transition. BMP4 and Hedgehog affect their production and expansion, but it is unknown whether they act to affect the same HSCs. In this study using the BRE GFP reporter mouse strain that identifies BMP/Smad-activated cells, we find that the AGM harbors two types of adult-repopulating HSCs upon explant culture: One type is BMP-activated and the other is a non-BMP-activated HSC type that is indirectly controlled by Hedgehog signaling through the VEGF pathway. Transcriptomic analyses demonstrate that the two HSC types express distinct but overlapping genetic programs. These results revealing the bifurcation in HSC types at early embryonic stages in the AGM explant model suggest that their development is dependent upon the signaling molecules in the microenvironment. PMID:26923823

  13. BMP and Hedgehog Regulate Distinct AGM Hematopoietic Stem Cells Ex Vivo

    Directory of Open Access Journals (Sweden)

    Mihaela Crisan

    2016-03-01

    Full Text Available Hematopoietic stem cells (HSC, the self-renewing cells of the adult blood differentiation hierarchy, are generated during embryonic stages. The first HSCs are produced in the aorta-gonad-mesonephros (AGM region of the embryo through endothelial to a hematopoietic transition. BMP4 and Hedgehog affect their production and expansion, but it is unknown whether they act to affect the same HSCs. In this study using the BRE GFP reporter mouse strain that identifies BMP/Smad-activated cells, we find that the AGM harbors two types of adult-repopulating HSCs upon explant culture: One type is BMP-activated and the other is a non-BMP-activated HSC type that is indirectly controlled by Hedgehog signaling through the VEGF pathway. Transcriptomic analyses demonstrate that the two HSC types express distinct but overlapping genetic programs. These results revealing the bifurcation in HSC types at early embryonic stages in the AGM explant model suggest that their development is dependent upon the signaling molecules in the microenvironment.

  14. TNIK inhibition abrogates colorectal cancer stemness

    Science.gov (United States)

    Masuda, Mari; Uno, Yuko; Ohbayashi, Naomi; Ohata, Hirokazu; Mimata, Ayako; Kukimoto-Niino, Mutsuko; Moriyama, Hideki; Kashimoto, Shigeki; Inoue, Tomoko; Goto, Naoko; Okamoto, Koji; Shirouzu, Mikako; Sawa, Masaaki; Yamada, Tesshi

    2016-01-01

    Canonical Wnt/β-catenin signalling is essential for maintaining intestinal stem cells, and its constitutive activation has been implicated in colorectal carcinogenesis. We and others have previously identified Traf2- and Nck-interacting kinase (TNIK) as an essential regulatory component of the T-cell factor-4 and β-catenin transcriptional complex. Consistent with this, Tnik-deficient mice are resistant to azoxymethane-induced colon tumorigenesis, and Tnik−/−/Apcmin/+ mutant mice develop significantly fewer intestinal tumours. Here we report the first orally available small-molecule TNIK inhibitor, NCB-0846, having anti-Wnt activity. X-ray co-crystal structure analysis reveals that NCB-0846 binds to TNIK in an inactive conformation, and this binding mode seems to be essential for Wnt inhibition. NCB-0846 suppresses Wnt-driven intestinal tumorigenesis in Apcmin/+ mice and the sphere- and tumour-forming activities of colorectal cancer cells. TNIK is required for the tumour-initiating function of colorectal cancer stem cells. Its inhibition is a promising therapeutic approach. PMID:27562646

  15. Effects of Cadmium on BMP Induced Bone Formation

    Institute of Scientific and Technical Information of China (English)

    陈秋生; 徐顺清

    2003-01-01

    To demonstrate the direct effects of cadmium on activities of bone morphogenetic protein (BMP), a complex containing BMP and cadmium chloride (CdCl2) was implanted beneath the abdominal skin of young male Wistar rats. The activity of BMP was studied by observing the histological changes, and measuring the activity of alkaline phosphatase (ALP) and acid phosphatase (ACP) and calcium content of the implants at different time points. Our results showed that during bone formation induced by BMP, cadmium inhibited the activities of osteoblasts and osteoclasts, and slowed the deposition of calcium. It is concluded that cadmium can directly affect biological activities of BMP directly.

  16. Vergleich von BMP-4 versus BMP-2 für die osteogene Differenzierung von Periostzellen

    OpenAIRE

    Klumpp, Florian (Alexander Stephan)

    2010-01-01

    Es ist heute bekannt, dass humane periostale mesenchymale Stammzellen (PMSCs) eine aussichtsreiche Grundlage für ein erfolgreiches Knochen Tissue Engineering darstellen. Dennoch ist die osteogene Differenzierung noch nicht vollständig be-schrieben. Da BMP-2 und BMP-4 nachweislich Regulatoren der Osteogenese sind, bestand die Aufgabe der vorliegenden Arbeit darin, die Wirkung derer auf die osteo-gene Differenzierung humaner PMSCs zu untersuchen. Isolierte humane PMSCs wurden mit Hilfe von o...

  17. Glypican-3 modulates BMP- and FGF-mediated effects during renal branching morphogenesis.

    Science.gov (United States)

    Grisaru, S; Cano-Gauci, D; Tee, J; Filmus, J; Rosenblum, N D

    2001-03-01

    The kidney of the Gpc3-/ mouse, a novel model of human renal dysplasia, is characterized by selective degeneration of medullary collecting ducts preceded by enhanced cell proliferation and overgrowth during branching morphogenesis. Here, we identify cellular and molecular mechanisms underlying this renal dysplasia. Glypican-3 (GPC3) deficiency was associated with abnormal and contrasting rates of proliferation and apoptosis in cortical (CCD) and medullary collecting duct (MCD) cells. In CCD, cell proliferation was increased threefold. In MCD, apoptosis was increased 16-fold. Expression of Gpc3 mRNA in ureteric bud and collecting duct cells suggested that GPC3 can exert direct effects in these cells. Indeed, GPC3 deficiency abrogated the inhibitory activity of BMP2 on branch formation in embryonic kidney explants, converted BMP7-dependent inhibition to stimulation, and enhanced the stimulatory effects of KGF. Similar comparative differences were found in collecting duct cell lines derived from GPC3-deficient and wild type mice and induced to form tubular progenitors in vitro, suggesting that GPC3 directly controls collecting duct cell responses. We propose that GPC3 modulates the actions of stimulatory and inhibitory growth factors during branching morphogenesis. PMID:11180950

  18. Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

    Directory of Open Access Journals (Sweden)

    DeCarlo Arthur A

    2012-09-01

    Full Text Available Abstract Background Many growth factors, such as bone morphogenetic protein (BMP-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS glycosaminoglycans (GAGs, which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS, regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. Results Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1 expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™. Conclusions A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid

  19. MRI of transforaminal lumbar interbody fusion: imaging appearance with and without the use of human recombinant bone morphogenetic protein-2 (rhBMP-2)

    Energy Technology Data Exchange (ETDEWEB)

    Fox, Michael G.; Goldberg, Judd M.; Gaskin, Cree M.; Barr, Michelle S.; Alford, Bennett [University of Virginia, Department of Radiology and Medical Imaging, Charlottesville, VA (United States); Patrie, James T. [University of Virginia, Department of Public Health Sciences, Charlottesville, VA (United States); Shen, Francis H. [University of Virginia, Department of Orthopedic Surgery, Charlottesville, VA (United States)

    2014-09-15

    To describe the vertebral endplate and intervertebral disc space MRI appearance following TLIF, with and without the use of rhBMP-2, and to determine if the appearance is concerning for discitis/osteomyelitis. After institutional review board approval, 116 TLIF assessments performed on 75 patients with rhBMP-2 were retrospectively and independently reviewed by five radiologists and compared to 73 TLIF assessments performed on 45 patients without rhBMP-2. MRIs were evaluated for endplate signal, disc space enhancement, disc space fluid, and abnormal paraspinal soft tissue. Endplate edema-like signal was reported when T1-weighted hypointensity, T2-weighted hyperintensity, and endplate enhancement were present. Subjective concern for discitis/osteomyelitis on MRI was graded on a five-point scale. Generalized estimating equation binomial regression model analysis was performed with findings correlated with rhBMP-2 use, TLIF level, graft type, and days between TLIF and MRI. The rhBMP-2 group demonstrated endplate edema-like signal (OR 5.66; 95 % CI [1.58, 20.24], p = 0.008) and disc space enhancement (OR 2.40; 95 % CI [1.20, 4.80], p = 0.013) more often after adjusting for the TLIF level, graft type, and the number of days following TLIF. Both groups had a similar temporal distribution for endplate edema-like signal but disc space enhancement peaked earlier in the rhBMP-2 group. Disc space fluid was only present in the rhBMP-2 group. Neither group demonstrated abnormal paraspinal soft tissue and discitis/osteomyelitis was not considered likely in any patient. Endplate edema-like signal and disc space enhancement were significantly more frequent and disc space enhancement developed more rapidly following TLIF when rhBMP-2 was utilized. The concern for discitis/osteomyelitis was similar and minimal in both groups. (orig.)

  20. MRI of transforaminal lumbar interbody fusion: imaging appearance with and without the use of human recombinant bone morphogenetic protein-2 (rhBMP-2)

    International Nuclear Information System (INIS)

    To describe the vertebral endplate and intervertebral disc space MRI appearance following TLIF, with and without the use of rhBMP-2, and to determine if the appearance is concerning for discitis/osteomyelitis. After institutional review board approval, 116 TLIF assessments performed on 75 patients with rhBMP-2 were retrospectively and independently reviewed by five radiologists and compared to 73 TLIF assessments performed on 45 patients without rhBMP-2. MRIs were evaluated for endplate signal, disc space enhancement, disc space fluid, and abnormal paraspinal soft tissue. Endplate edema-like signal was reported when T1-weighted hypointensity, T2-weighted hyperintensity, and endplate enhancement were present. Subjective concern for discitis/osteomyelitis on MRI was graded on a five-point scale. Generalized estimating equation binomial regression model analysis was performed with findings correlated with rhBMP-2 use, TLIF level, graft type, and days between TLIF and MRI. The rhBMP-2 group demonstrated endplate edema-like signal (OR 5.66; 95 % CI [1.58, 20.24], p = 0.008) and disc space enhancement (OR 2.40; 95 % CI [1.20, 4.80], p = 0.013) more often after adjusting for the TLIF level, graft type, and the number of days following TLIF. Both groups had a similar temporal distribution for endplate edema-like signal but disc space enhancement peaked earlier in the rhBMP-2 group. Disc space fluid was only present in the rhBMP-2 group. Neither group demonstrated abnormal paraspinal soft tissue and discitis/osteomyelitis was not considered likely in any patient. Endplate edema-like signal and disc space enhancement were significantly more frequent and disc space enhancement developed more rapidly following TLIF when rhBMP-2 was utilized. The concern for discitis/osteomyelitis was similar and minimal in both groups. (orig.)

  1. Dickkopf-1 is involved in BMP9-induced osteoblast differentiation of C3H10T1/2 mesenchymal stem cells

    Science.gov (United States)

    Lin, Liangbo; Qiu, Quanhe; Zhou, Nian; Dong, Wen; Shen, Jieliang; Jiang, Wei; Fang, Ji; Hao, Jie; Hu, Zhenming

    2016-01-01

    Bone morphogenetic protein 9 (BMP9) is a potent inducer of osteogenic differentiation of mesenchymal stem cells. The Wnt antagonist Dickkopf-1 (Dkk1) is involved in skeletal development and bone remodeling. Here, we investigated the role of Dkk1 in BMP9-induced osteogenic differentiation of MSCs. We found that overexpression of BMP9 induced Dkk1 expression in a dose-dependent manner, which was reduced by the P38 inhibitor SB203580 but not the ERK inhibitor PD98059. Moreover, Dkk1 dramatically decreased not only BMP9-induced alkaline phosphatase (ALP) activity but also the expression of osteocalcin (OCN) and osteopontin (OPN) and matrix mineralization of C3H10T1/2 cells. Furthermore, exogenous Dkk1 expression inhibited Wnt/β-catenin signaling induced by BMP9. Our findings indicate that Dkk1 negatively regulates BMP9-induced osteogenic differentiation through inhibition of the Wnt/β-catenin pathway and it could be used to optimize the therapeutic use of BMP9 and for bone tissue engineering. [BMB Reports 2016; 49(3): 179-184] PMID:26674341

  2. Antibody-mediated neutralization of virus is abrogated by mycoplasma.

    OpenAIRE

    Dickson, C; Elkington, J; Hales, A.; Weiss, R.

    1980-01-01

    The ability of a mouse mammary tumor cell line to abrogate antibody neutralization of vesicular stomatitis virus was shown to be due to the presence of mycoplasma. The mycoplasma was isolated from the cell line and typed as Mycoplasma orale. Colonies of this mycoplasma were used to deliberately infect cell cultures which then gained the capacity to reactivate antibody-neutralized virus. The extent of the reactivation depended on the source of neutralizing antiserum. Other species of mycoplasm...

  3. Platelet-derived growth factor (PDGF)-induced activation of Erk5 MAP-kinase is dependent on Mekk2, Mek1/2, PKC and PI3-kinase, and affects BMP signaling.

    Science.gov (United States)

    Tsioumpekou, Maria; Papadopoulos, Natalia; Burovic, Fatima; Heldin, Carl-Henrik; Lennartsson, Johan

    2016-09-01

    Platelet-derived growth factor-BB (PDGF-BB) binds to its tyrosine kinase receptors (PDGFRs) and stimulates mitogenicity and survival of cells of mesenchymal origin. Activation of PDGFRs initiates a number of downstream signaling pathways, including phosphatidyl 3'-inositol kinase (PI3-kinase), phospholipase Cγ and MAP kinase pathways. In this report, we show that Erk5 MAP kinase is activated in response to PDGF-BB in the smooth muscle cell line MOVAS in a manner dependent on Mekk2, Mek1/2, Mek5, PI3-kinase and protein kinase C (PKC). The co-operation of Mek1/2 and Mekk2 in the activation of Erk5, suggests a close co-regulation between the Erk1/2 and Erk5 MAP kinase pathways. Furthermore, we found that classical PKCs are important for Erk5 activation. In addition, we found that PKCζ interacts with Erk5 and may exert a negative feed-back effect. We observed no nuclear accumulation of Erk5 in response to PDGF-BB stimulation, however, we identified a mechanism by which cytoplasmic Erk5 influences gene expression; Erk5 was essential for PDGF-BB-mediated Smad1/5/8 signaling by stimulating release and/or activation of bone morphogenetic protein(s) (BMPs). Thus, PDGF-BB-induced Erk5 activation involves parallel stimulatory and inhibitory pathways and promotes Smad1/5/8 signaling. PMID:27339033

  4. Chronic inflammation and angiogenic signaling axis impairs differentiation of dental-pulp stem cells.

    Directory of Open Access Journals (Sweden)

    Michael Boyle

    Full Text Available Dental-pulp tissue is often exposed to inflammatory injury. Sequested growth factors or angiogenic signaling proteins that are released following inflammatory injury play a pivotal role in the formation of reparative dentin. While limited or moderate angiogenesis may be helpful for dental pulp maintenance, the induction of significant level of angiogenesis is probably highly detrimental. Hitherto, several studies have addressed the effects of proinflammatory stimuli on the survival and differentiation of dental-pulp stem cells (DPSC, in vitro. However, the mechanisms communal to the inflammatory and angiogenic signaling involved in DPSC survival and differentiation remain unknown. Our studies observed that short-term exposure to TNF-α (6 and 12 hours [hrs] induced apoptosis with an upregulation of VEGF expression and NF-κB signaling. However, long-term (chronic exposure (14 days to TNF-α resulted in an increased proliferation with a concomitant shortening of the telomere length. Interestingly, DPSC pretreated with Nemo binding domain (NBD peptide (a cell permeable NF-κB inhibitor significantly ameliorated TNF-α- and/or VEGF-induced proliferation and the shortening of telomere length. NBD peptide pretreatment significantly improved TNF-α-induced downregulation of proteins essential for differentiation, such as bone morphogenic proteins (BMP-1 & 2, BMP receptor isoforms-1&2, trasnforming growth factor (TGF, osteoactivin and osteocalcin. Additionally, inhibition of NF-κB signaling markedly increased the mineralization potential, a process abrogated by chronic exposure to TNF-α. Thus, our studies demonstrated that chronic inflammation mediates telomere shortening via NF-κB signaling in human DPSC. Resultant chromosomal instability leads to an emergence of increased proliferation of DPSC, while negatively regulating the differentiation of DPSC, in vitro.

  5. Bone Enhancement with BMP-2 for Safe Clinical Translation

    OpenAIRE

    Kisiel, Marta

    2013-01-01

    Bone morphogenetic protein-2 (BMP-2) is considered a promising adjuvant for the treatment of bone regeneration. However, BMP-2 delivery in a conventional collagen scaffold needs a high dose to achieve an effective outcome. Moreover, such dosage may lead to serious side effects. The aim of the following thesis was to find clinically acceptable strategies reducing the required dose of BMP-2 by improving the delivery and optimizing the preclinical testing of the new approaches. In all the studie...

  6. Unveiling the Bmp13 Enigma: Redundant Morphogen or Crucial Regulator?

    Directory of Open Access Journals (Sweden)

    Lisa A Williams, Divya Bhargav, Ashish D Diwan

    2008-01-01

    Full Text Available Bone morphogenetic proteins are a diverse group of morphogens with influences not only on bone tissue, as the nomenclature suggests, but on multiple tissues in the body and often at crucial and influential periods in development. The purpose of this review is to identify and discuss current knowledge of one vertebrate BMP, Bone Morphogenetic Protein 13 (BMP13, from a variety of research fields, in order to clarify BMP13's functional contribution to developing and maintaining healthy tissues, and to identify potential future research directions for this intriguing morphogen. BMP13 is highly evolutionarily conserved (active domain >95% across diverse species from Zebrafish to humans, suggesting a crucial function. In addition, mutations in BMP13 have recently been associated with Klippel-Feil Syndrome, causative of numerous skeletal and developmental defects including spinal disc fusion. The specific nature of BMP13's crucial function is, however, not yet known. The literature for BMP13 is focused largely on its activity in the healing of tendon-like tissues, or in comparisons with other BMP family molecules for whom a clear function in embryo development or osteogenic differentiation has been identified. There is a paucity of detailed information regarding BMP13 protein activity, structure or protein processing. Whilst some activity in the stimulation of osteogenic or cartilaginous gene expression has been reported, and BMP13 expression is found in post natal cartilage and tendon tissues, there appears to be a redundancy of function in the BMP family, with several members capable of stimulating similar tissue responses. This review aims to summarise the known or potential role(s for BMP13 in a variety of biological systems.

  7. BMP4 and LGL1 are Down Regulated in an Ovine Model of Congenital Diaphragmatic Hernia

    Directory of Open Access Journals (Sweden)

    Heather eEmmerton-Coughlin

    2014-11-01

    Full Text Available Background/Purpose: The molecular pathophysiology of lung hypoplasia in congenital diaphragmatic hernia (CDH remains poorly understood. The Wnt signaling pathway and downstream targets, such as bone morphogenetic proteins (BMP 4 and other factors such as late gestation lung protein 1 (LGL1, are essential to normal lung development. Nitrofen-induced hypoplastic CDH rodent lungs demonstrate down regulation of the Wnt pathway including BMP4 and reduced LGL1 expression. The aim of the current study was to examine the molecular pathophysiology associated with a surgically induced CDH in an ovine model. Methods: Left thoracotomy was performed at 80 days in 14 fetal sheep; CDH was created in 7 experimental animals. Lungs were harvested at 136 days (term=145d. Lung weight and mean terminal bronchiole density (MTBD were measured to determine the degree of pulmonary hypoplasia. Quantitative real time PCR was undertaken to analyze Wnt2, Wnt7b, BMP4 and LGL1 mRNA expression. Results: Total lung weight was decreased while MTBD was increased in the CDH group (p<0.05, confirming pulmonary hypoplasia. BMP4 and LGL1 mRNA was significantly reduced in CDH lungs (p<0.05. Wnt2 mRNA was decreased, although not significantly (p<0.06. Conclusions: For the first time, down regulation of BMP4 and Lgl1 are reported in an ovine CDH model. In contrast to other animal models, these changes are persistent to near term. These findings suggest that mechanical compression from herniated viscera may play a more important role in causing pulmonary hypoplasia in CDH, rather than a primary defect in lung organogenesis.

  8. Bmp2 Is Required for Odontoblast Differentiation and Pulp Vasculogenesis

    OpenAIRE

    Yang, W; Harris, M.A.; Y. Cui; Mishina, Y; Harris, S.E.; Gluhak-Heinrich, J.

    2012-01-01

    Using the Bmp2 floxed/3.6Col1a1-Cre (Bmp2-cKOod) mouse model, we have observed severe defects in odontogenesis and dentin formation with the removal of the Bmp2 gene in early-polarizing odontoblasts. The odontoblasts in the Bmp2-cKOod do not mature properly and fail to form proper dentin with normal dentinal tubules and activate terminal differentiation, as reflected by decreased Osterix, Col1a1, and Dspp expression. There is less dentin, and the dentin is hypomineralized and patchy. We also ...

  9. NOX4 mediates BMP4-induced upregulation of TRPC1 and 6 protein expressions in distal pulmonary arterial smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Qian Jiang

    Full Text Available RATIONALE: Our previous studies demonstrated that bone morphogenetic protein 4 (BMP4 mediated, elevated expression of canonical transient receptor potential (TRPC largely accounts for the enhanced proliferation in pulmonary arterial smooth muscle cells (PASMCs. In the present study, we sought to determine the signaling pathway through which BMP4 up-regulates TRPC expression. METHODS: We employed recombinant human BMP4 (rhBMP4 to determine the effects of BMP4 on NADPH oxidase 4 (NOX4 and reactive oxygen species (ROS production in rat distal PASMCs. We also designed small interfering RNA targeting NOX4 (siNOX4 and detected whether NOX4 knockdown affects rhBMP4-induced ROS, TRPC1 and 6 expression, cell proliferation and intracellular Ca2+ determination in PASMCs. RESULTS: In rhBMP4 treated rat distal PASMCs, NOX4 expression was (226.73±11.13 %, and the mean ROS level was (123.65±1.62 % of that in untreated control cell. siNOX4 transfection significantly reduced rhBMP4-induced elevation of the mean ROS level in PASMCs. Moreover, siNOX4 transfection markedly reduced rhBMP4-induced elevation of TRPC1 and 6 proteins, basal [Ca2+]i and SOCE. Furthermore, compared with control group (0.21±0.001, the proliferation of rhBMP4 treated cells was significantly enhanced (0.41±0.001 (P<0.01. However, such increase was attenuated by knockdown of NOX4. Moreover, external ROS (H2O2 100 µM, 24 h rescued the effects of NOX4 knockdown, which included the declining of TRPC1 and 6 expression, basal intracellular calcium concentration ([Ca2+]i and store-operated calcium entry (SOCE, suggesting that NOX4 plays as an important mediator in BMP4-induced proliferation and intracellular calcium homeostasis. CONCLUSION: These results suggest that BMP4 may increase ROS level, enhance TRPC1 and 6 expression and proliferation by up-regulating NOX4 expression in PASMCs.

  10. KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1{alpha} survival pathways

    Energy Technology Data Exchange (ETDEWEB)

    Oommen, Deepu, E-mail: oommen1978@gmail.com [Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Belfast, Northern Ireland (United Kingdom); Prise, Kevin M. [Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Belfast, Northern Ireland (United Kingdom)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer KNK437, a benzylidene lactam compound, is a novel radiosensitizer. Black-Right-Pointing-Pointer KNK437 inhibits AKT signaling and abrogates the accumulation of HIF-1{alpha} under hypoxia. Black-Right-Pointing-Pointer KNK437 abrogates hypoxia induced resistance to radiation. -- Abstract: KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1{alpha} (HIF-1{alpha}). HIF-1{alpha} is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1{alpha}. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1{alpha} in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1{alpha} levels in KNK437-treated cells. This suggested that the absence of HIF-1{alpha} in hypoxic cells was not due to the enhanced protein degradation. HIF-1{alpha} is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1{alpha} mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1{alpha} levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

  11. KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1α survival pathways

    International Nuclear Information System (INIS)

    Highlights: ► KNK437, a benzylidene lactam compound, is a novel radiosensitizer. ► KNK437 inhibits AKT signaling and abrogates the accumulation of HIF-1α under hypoxia. ► KNK437 abrogates hypoxia induced resistance to radiation. -- Abstract: KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1α. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1α in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1α levels in KNK437-treated cells. This suggested that the absence of HIF-1α in hypoxic cells was not due to the enhanced protein degradation. HIF-1α is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1α mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1α levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

  12. BMP7 and EREG Contribute to the Inductive Potential of Dental Mesenchyme.

    Science.gov (United States)

    Gao, Bo; Zhou, Xin; Zhou, Xuedong; Pi, Caixia; Xu, Ruoshi; Wan, Mian; Yang, Jing; Zhou, Yue; Liu, Chengcheng; Sun, Jianxun; Zhang, Yan; Zheng, Liwei

    2015-01-01

    Odontogenesis is accomplished by reciprocal signaling between the epithelial and mesenchymal compartments. It is generally accepted that the inductive mesenchyme is capable of inducing the odontogenic commitment of both dental and non-dental epithelial cells. However, the duration of this signal in the developing dental mesenchyme and whether adult dental pulp tissue maintains its inductive capability remain unclear. This study investigated the contribution of growth factors to regulating the inductive potential of the dental mesenchyme. Human oral epithelial cells (OEs) were co-cultured with either human dental mesenchymal/papilla cells (FDPCs) or human dental pulp cells (ADPCs) under 2-dimensional or 3-dimensional conditions. Odontogenic-associated genes and proteins were detected by qPCR and immunofluorescence, respectively, and significant differences were observed between the two co-culture systems. The BMP7 and EREG expression levels in FDPCs were significantly higher than in ADPCs, as indicated by human growth factor PCR arrays and immunofluorescence analyses. OEs co-cultured with ADPCs supplemented with BMP7 and EREG expressed ameloblastic differentiation genes. Our study suggests that BMP7 and EREG expression in late bell-stage human dental papilla contributes to the inductive potential of dental mesenchyme. Furthermore, adult dental pulp cells supplemented with these two growth factors re-established the inductive potential of postnatal dental pulp tissue. PMID:25952286

  13. BMP type II receptor regulates positioning of outflow tract and remodeling of atrioventricular cushion during cardiogenesis.

    Science.gov (United States)

    Beppu, Hideyuki; Malhotra, Rajeev; Beppu, Yuko; Lepore, John J; Parmacek, Michael S; Bloch, Kenneth D

    2009-07-15

    Signaling of bone morphogenetic protein (BMP) via type I and type II receptors is involved in multiple processes contributing to cardiogenesis. To investigate the role of the BMP type II receptor (BMPRII) in heart development, the BMPRII gene was deleted throughout the embryo during gastrulation using a Mox2-Cre transgene. BMPRII(flox/-);Mox2-Cre mice exhibited cardiac defects including double-outlet right ventricle, ventricular septal defect (VSD), atrioventricular (AV) cushion defects, and thickened valve leaflets. To characterize the tissue-specific functions of BMPRII in cardiogenesis, a series of Cre transgenes (alphaMHC-, Tie2-, Wnt1-, and SM22alpha-Cre) was employed. Interestingly, myocardial development was normal when the BMPRII gene was deleted in myocardial cells using Mox2-Cre, alphaMHC-Cre, or SM22alpha-Cre transgenes, suggesting that signaling by other BMP type II receptors may compensate for the absence of BMPRII in the myocardial cells. AV cushion defects including atrial septal defect, membranous VSD, and thickened valve leaflets were found in BMPRII(flox/-);Tie2-Cre mice. Abnormal positioning of the aorta was observed in BMPRII(flox/-);Wnt1-Cre and BMPRII(flox/-);SM22alpha-Cre mice. Taken together, these results demonstrate that endocardial BMPRII expression is required for septal formation and valvulogenesis. Moreover, mesenchymal BMPRII expression in the outflow tract cushion is required for proper positioning of the aorta. PMID:19409885

  14. EMBRYO DEVELOPMENT. BMP gradients: A paradigm for morphogen-mediated developmental patterning.

    Science.gov (United States)

    Bier, Ethan; De Robertis, Edward M

    2015-06-26

    Bone morphogenetic proteins (BMPs) act in dose-dependent fashion to regulate cell fate choices in a myriad of developmental contexts. In early vertebrate and invertebrate embryos, BMPs and their antagonists establish epidermal versus central nervous system domains. In this highly conserved system, BMP antagonists mediate the neural-inductive activities proposed by Hans Spemann and Hilde Mangold nearly a century ago. BMPs distributed in gradients subsequently function as morphogens to subdivide the three germ layers into distinct territories and act to organize body axes, regulate growth, maintain stem cell niches, or signal inductively across germ layers. In this Review, we summarize the variety of mechanisms that contribute to generating reliable developmental responses to BMP gradients and other morphogen systems. PMID:26113727

  15. BMP-2 and titanium particles synergistically activate osteoclast formation

    Energy Technology Data Exchange (ETDEWEB)

    Sun, S.X. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedics, Yinchuan, Ningxia Hui Autonomous Region, China, Department of Orthopedics, Affiliated Hospital of Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region (China); Guo, H.H. [Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region, China, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region (China); Zhang, J. [Institute of Pathology, Xi' an Jiaotong University, Xi' an Shaanxi, China, Institute of Pathology, Xi' an Jiaotong University, Xi' an Shaanxi (China); Yu, B. [Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region, China, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region (China); Sun, K.N.; Jin, Q.H. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedics, Yinchuan, Ningxia Hui Autonomous Region, China, Department of Orthopedics, Affiliated Hospital of Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region (China)

    2014-05-09

    A previous study showed that BMP-2 (bone morphogenetic protein-2) and wear debris can separately support osteoclast formation induced by the receptor activator of NF-κB ligand (RANKL). However, the effect of BMP-2 on wear debris-induced osteoclast formation is unclear. In this study, we show that neither titanium particles nor BMP-2 can induce osteoclast formation in RAW 264.7 mouse leukemic monocyte macrophage cells but that BMP-2 synergizes with titanium particles to enhance osteoclast formation in the presence of RANKL, and that at a low concentration, BMP-2 has an optimal effect to stimulate the size and number of multinuclear osteoclasts, expression of osteoclast genes, and resorption area. Our data also clarify that the effects caused by the increase in BMP-2 on phosphorylated SMAD levels such as c-Fos expression increased throughout the early stages of osteoclastogenesis. BMP-2 and titanium particles stimulate the expression of p-JNK, p-P38, p-IkB, and P50 compared with the titanium group. These data suggested that BMP-2 may be a crucial factor in titanium particle-mediated osteoclast formation.

  16. BMP-2 Is Involved in Scleral Remodeling in Myopia Development.

    Directory of Open Access Journals (Sweden)

    Honghui Li

    Full Text Available The development of myopia is associated with scleral remodeling, but it is unclear which factors regulate this process. This study investigated bone morphogenetic protein-2 (BMP-2 expression in the sclera of guinea pigs with lens-induced myopia (LIM and after recovery from myopia and evaluated the effect of BMP-2 on extracellular matrix (ECM synthesis in human scleral fibroblasts (HSFs cultured in vitro. Lens-induced myopia was brought about in two groups of guinea pigs (the lens-induced myopia and myopia recovery groups by placing -4.00 D lenses on the right eye for three weeks. The left eye served as a contralateral control. In the recovery group, the lenses were removed after one week. The refractive power and axial length of the eyes were measured, and the BMP-2 expression levels in the sclera were measured. After three weeks, the lens-induced eyes acquired relative myopia in both groups of guinea pigs. Immunostaining of the eyeballs revealed significantly decreased BMP-2 expression in the posterior sclera of the myopic eyes compared to the contralateral eyes. One week after lens removal, BMP-2 expression recovered, and no differences were observed between the experimental and contralateral eyes in the recovery group. HSFs were cultured with BMP-2 or transforming growth factor-β1 (TGF-β1. Type I and type III collagen synthesis was significantly up-regulated following BMP-2 treatment in culture after one and two weeks, but the ratio of type III to type I collagen mRNA was not increased. Biosynthesis of glycosaminoglycan (GAG and aggrecan was increased in HSFs treated with BMP-2. Some chondrogenesis-associated genes expression increased in HSFs treated with BMP-2. From this study, we concluded that BMP-2 is involved in scleral remodeling in the development and recovery of lens-induced myopia.

  17. BMP-2 Is Involved in Scleral Remodeling in Myopia Development

    Science.gov (United States)

    Li, Honghui; Cui, Dongmei; Zhao, Feng; Huo, Lijun; Hu, Jianmin; Zeng, Junwen

    2015-01-01

    The development of myopia is associated with scleral remodeling, but it is unclear which factors regulate this process. This study investigated bone morphogenetic protein-2 (BMP-2) expression in the sclera of guinea pigs with lens-induced myopia (LIM) and after recovery from myopia and evaluated the effect of BMP-2 on extracellular matrix (ECM) synthesis in human scleral fibroblasts (HSFs) cultured in vitro. Lens-induced myopia was brought about in two groups of guinea pigs (the lens-induced myopia and myopia recovery groups) by placing -4.00 D lenses on the right eye for three weeks. The left eye served as a contralateral control. In the recovery group, the lenses were removed after one week. The refractive power and axial length of the eyes were measured, and the BMP-2 expression levels in the sclera were measured. After three weeks, the lens-induced eyes acquired relative myopia in both groups of guinea pigs. Immunostaining of the eyeballs revealed significantly decreased BMP-2 expression in the posterior sclera of the myopic eyes compared to the contralateral eyes. One week after lens removal, BMP-2 expression recovered, and no differences were observed between the experimental and contralateral eyes in the recovery group. HSFs were cultured with BMP-2 or transforming growth factor-β1 (TGF-β1). Type I and type III collagen synthesis was significantly up-regulated following BMP-2 treatment in culture after one and two weeks, but the ratio of type III to type I collagen mRNA was not increased. Biosynthesis of glycosaminoglycan (GAG) and aggrecan was increased in HSFs treated with BMP-2. Some chondrogenesis-associated genes expression increased in HSFs treated with BMP-2. From this study, we concluded that BMP-2 is involved in scleral remodeling in the development and recovery of lens-induced myopia. PMID:25965995

  18. BMP-2 and ALP gene expression induced by a BMP-2 gene-fibronectin-apatite composite layer

    Energy Technology Data Exchange (ETDEWEB)

    Wang Xiupeng; Sogo, Yu; Li Xia; Ito, Atsuo [Human Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Oyane, Ayako [Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan); Tsurushima, Hideo, E-mail: xp-wang@aist.go.jp, E-mail: xiupengw@hotmail.com [Department of Neurosurgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575 (Japan)

    2011-08-15

    The bone morphogenetic protein 2 (BMP-2) gene delivery system with a gene-fibronectin (Fn)-apatite composite layer was fabricated on the surface of a hydroxyapatite ceramic scaffold. The BMP-2 gene-Fn-apatite composite layer was coated on the scaffold using a supersaturated calcium phosphate solution supplemented with BMP-2 DNA and Fn. The scaffolds were ectopically implanted into the dorsal subcutaneous tissue of rats. Four weeks after the implantation, the hydroxyapatite scaffold coated with the BMP-2 gene-Fn-apatite composite layer showed improved gene expressions of BMP-2 and alkaline phosphatase as compared with the scaffold coated with the apatite layer. Although these results suggest the possibility of ectopic bone formation induced by the present gene delivery system, further study is necessary to prove this.

  19. Expression of genes for bone morphogenetic proteins BMP-2, BMP-4 and BMP-6 in various parts of the human skeleton

    Directory of Open Access Journals (Sweden)

    Włodarski Krzysztof

    2007-12-01

    Full Text Available Abstract Background Differences in duration of bone healing in various parts of the human skeleton are common experience for orthopaedic surgeons. The reason for these differences is not obvious and not clear. Methods In this paper we decided to measure by the use of real-time RT-PCR technique the level of expression of genes for some isoforms of bone morphogenetic proteins (BMPs, whose role is proven in bone formation, bone induction and bone turnover. Seven bone samples recovered from various parts of skeletons from six cadavers of young healthy men who died in traffic accidents were collected. Activity of genes for BMP-2, -4 and -6 was measured by the use of fluorescent SYBR Green I. Results It was found that expression of m-RNA for BMP-2 and BMP-4 is higher in trabecular bone in epiphyses of long bones, cranial flat bones and corpus mandibulae then in the compact bone of diaphyses of long bones. In all samples examined the expression of m-RNA for BMP-4 was higher than for BMP-2. Conclusion It was shown that m-RNA for BMP-6 is not expressed in the collected samples at all. It is postulated that differences in the level of activation of genes for BMPs is one of the important factors which determine the differences in duration of bone healing of various parts of the human skeleton.

  20. Effects of bone morphogenic protein 4 (BMP4 and its inhibitor, Noggin, on in vitro maturation and culture of bovine preimplantation embryos

    Directory of Open Access Journals (Sweden)

    Fernandez-Martin Rafael

    2011-02-01

    , our findings demonstrate, for the first time, that a correct balance of BMP signaling is needed for proper pre-implantation development of bovine embryos.

  1. Dominant negative Bmp5 mutation reveals key role of BMPs in skeletal response to mechanical stimulation

    Directory of Open Access Journals (Sweden)

    Kingsley David M

    2008-04-01

    Full Text Available Abstract Background Over a hundred years ago, Wolff originally observed that bone growth and remodeling are exquisitely sensitive to mechanical forces acting on the skeleton. Clinical studies have noted that the size and the strength of bone increase with weight bearing and muscular activity and decrease with bed rest and disuse. Although the processes of mechanotransduction and functional response of bone to mechanical strain have been extensively studied, the molecular signaling mechanisms that mediate the response of bone cells to mechanical stimulation remain unclear. Results Here, we identify a novel germline mutation at the mouse Bone morphogenetic protein 5 (Bmp5 locus. Genetic analysis shows that the mutation occurs at a site encoding the proteolytic processing sequence of the BMP5 protein and blocks proper processing of BMP5. Anatomic studies reveal that this mutation affects the formation of multiple skeletal features including several muscle-induced skeletal sites in vivo. Biomechanical studies of osteoblasts from these anatomic sites show that the mutation inhibits the proper response of bone cells to mechanical stimulation. Conclusion The results from these genetic, biochemical, and biomechanical studies suggest that BMPs are required not only for skeletal patterning during embryonic development, but also for bone response and remodeling to mechanical stimulation at specific anatomic sites in the skeleton.

  2. Shaping skeletal growth by modular regulatory elements in the Bmp5 gene.

    Directory of Open Access Journals (Sweden)

    Catherine Guenther

    2008-12-01

    Full Text Available Cartilage and bone are formed into a remarkable range of shapes and sizes that underlie many anatomical adaptations to different lifestyles in vertebrates. Although the morphological blueprints for individual cartilage and bony structures must somehow be encoded in the genome, we currently know little about the detailed genomic mechanisms that direct precise growth patterns for particular bones. We have carried out large-scale enhancer surveys to identify the regulatory architecture controlling developmental expression of the mouse Bmp5 gene, which encodes a secreted signaling molecule required for normal morphology of specific skeletal features. Although Bmp5 is expressed in many skeletal precursors, different enhancers control expression in individual bones. Remarkably, we show here that different enhancers also exist for highly restricted spatial subdomains along the surface of individual skeletal structures, including ribs and nasal cartilages. Transgenic, null, and regulatory mutations confirm that these anatomy-specific sequences are sufficient to trigger local changes in skeletal morphology and are required for establishing normal growth rates on separate bone surfaces. Our findings suggest that individual bones are composite structures whose detailed growth patterns are built from many smaller lineage and gene expression domains. Individual enhancers in BMP genes provide a genomic mechanism for controlling precise growth domains in particular cartilages and bones, making it possible to separately regulate skeletal anatomy at highly specific locations in the body.

  3. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation.

    Science.gov (United States)

    Watanabe, Yukihide; Papoutsoglou, Panagiotis; Maturi, Varun; Tsubakihara, Yutaro; Hottiger, Michael O; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-06-10

    We previously established a mechanism of negative regulation of transforming growth factor β signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation. PMID:27129221

  4. BMP-2 Is Involved in Scleral Remodeling in Myopia Development

    OpenAIRE

    Honghui Li; Dongmei Cui; Feng Zhao; Lijun Huo; Jianmin Hu; Junwen Zeng

    2015-01-01

    The development of myopia is associated with scleral remodeling, but it is unclear which factors regulate this process. This study investigated bone morphogenetic protein-2 (BMP-2) expression in the sclera of guinea pigs with lens-induced myopia (LIM) and after recovery from myopia and evaluated the effect of BMP-2 on extracellular matrix (ECM) synthesis in human scleral fibroblasts (HSFs) cultured in vitro. Lens-induced myopia was brought about in two groups of guinea pigs (the lens-induced ...

  5. Expression of human bone morphogenetic protein (BMP-2 and BMP-4 genes in transgenic bovine fibroblasts Expressão dos genes bone morphogenetic protein (BMP-2 e BMP-4 em fibroblastos bovinos transgênicos

    Directory of Open Access Journals (Sweden)

    C. Oleskovicz

    2004-08-01

    Full Text Available cDNAs dos genes bone morphogenetic protein-2 (BMP-2 e bone morphogenetic protein-4 (BMP-4 foram sintetizados a partir de RNA total extraído de tecidos ósseos de pacientes que apresentavam trauma facial (fraturas do maxilar entre o 7º e o 10º dia pós-trauma e clonados num vetor para expressão em células mamíferas, sob controle do promotor de citomegalovírus (CMV. Os vetores contendo os genes BMP-2 e o BMP-4 foram utilizados para a transfecção de fibroblastos bovinos. mRNAs foram indiretamente detectados por RT-PCR nas células transfectadas. As proteínas BMP-2 e BMP-4 foram detectadas mediante análises de Western blot. Os resultados demonstram a possibilidade de produção desses fatores de crescimento celular em fibroblastos bovinos. Essas células poderão ser utilizadas como fontes doadoras de material genético para a técnica de transferência nuclear na geração de animais transgênicos.

  6. Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on colonization and bone formation in vivo.

    Science.gov (United States)

    Schofer, Markus D; Tünnermann, Lisa; Kaiser, Hendric; Roessler, Philip P; Theisen, Christina; Heverhagen, Johannes T; Hering, Jacqueline; Voelker, Maximilian; Agarwal, Seema; Efe, Turgay; Fuchs-Winkelmann, Susanne; Paletta, Jürgen R J

    2012-09-01

    The reconstruction of large bone defects after injury or tumor resection often requires the use of bone substitution. Artificial scaffolds based on synthetic biomaterials can overcome disadvantages of autologous bone grafts, like limited availability and donor side morbidity. Among them, scaffolds based on nanofibers offer great advantages. They mimic the extracellular matrix, can be used as a carrier for growth factors and allow the differentiation of human mesenchymal stem cells. Differentiation is triggered by a series of signaling processes, including integrin and bone morphogenetic protein (BMP), which act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in artificial poly-(l)-lactide acid (PLLA) based nanofiber scaffolds in vivo. Electrospun matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were implanted in calvarial critical size defects in rats. Cranial CT-scans were taken 4, 8 and 12 weeks after implantation. Specimens obtained after euthanasia were processed for histology and immunostainings on osteocalcin, BMP-2 and Smad5. After implantation the scaffolds were inhomogeneously colonized and cells were only present in wrinkle- or channel-like structures. Ossification was detected only in focal areas of the scaffold. This was independent of whether BMP-2 was incorporated in the scaffold. However, cells that migrated into the scaffold showed an increased ratio of osteocalcin and Smad5 positive cells compared to empty defects. Furthermore, in case of BMP-2 incorporated PLLA-collagen type I scaffolds, 4 weeks after implantation approximately 40 % of the cells stained positive for BMP-2 indicating an autocrine process of the ingrown cells. These findings indicate that a cooperative effect between BMP-2 and collagen type I can be transferred to PLLA nanofibers and furthermore, that this effect is active in vivo. However, this had no effect on bone formation. The reason for

  7. The origin of bmp16, a novel Bmp2/4 relative, retained in teleost fish genomes

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    Meyer Axel

    2009-12-01

    Full Text Available Abstract Background Whole genome sequences have allowed us to have an overview of the evolution of gene repertoires. The target of the present study, the TGFβ superfamily, contains many genes involved in vertebrate development, and provides an ideal system to explore the relationships between evolution of gene repertoires and that of developmental programs. Results As a result of a bioinformatic survey of sequenced vertebrate genomes, we identified an uncharacterized member of the TGFβ superfamily, designated bmp16, which is confined to teleost fish species. Our molecular phylogenetic study revealed a high affinity of bmp16 to the Bmp2/4 subfamily. Importantly, further analyses based on the maximum-likelihood method unambiguously ruled out the possibility that this teleost-specific gene is a product of teleost-specific genome duplication. This suggests that the absence of a bmp16 ortholog in tetrapods is due to a secondary loss. In situ hybridization showed embryonic expression of the zebrafish bmp16 in the developing swim bladder, heart, tail bud, and ectoderm of pectoral and median fin folds in pharyngula stages, as well as gut-associated expression in 5-day embryos. Conclusion Comparisons of expression patterns revealed (1 the redundancy of bmp16 expression with its homologs in presumably plesiomorphic expression domains, such as the fin fold, heart, and tail bud, which might have permitted its loss in the tetrapod lineage, and (2 the loss of craniofacial expression and gain of swim bladder expression of bmp16 after the gene duplication between Bmp2, -4 and -16. Our findings highlight the importance of documenting secondary changes of gene repertoires and expression patterns in other gene families.

  8. Relaxin requires the angiotensin II type 2 receptor to abrogate renal interstitial fibrosis.

    Science.gov (United States)

    Chow, Bryna S Man; Kocan, Martina; Bosnyak, Sanja; Sarwar, Mohsin; Wigg, Belinda; Jones, Emma S; Widdop, Robert E; Summers, Roger J; Bathgate, Ross A D; Hewitson, Tim D; Samuel, Chrishan S

    2014-07-01

    Fibrosis is a hallmark of chronic kidney disease, for which there is currently no effective cure. The hormone relaxin is emerging as an effective antifibrotic therapy; however, its mechanism of action is poorly understood. Recent studies have shown that relaxin disrupts the profibrotic actions of transforming growth factor-β1 (TGF-β1) by its cognate receptor, relaxin family peptide receptor 1 (RXFP1), extracellular signal-regulated kinase phosphorylation, and a neuronal nitric oxide synthase-dependent pathway to abrogate Smad2 phosphorylation. Since angiotensin II also inhibits TGF-β1 activity through its AT2 receptor (AT2R), we investigated the extent to which relaxin interacts with the AT2R. The effects of the AT2R antagonist, PD123319, on relaxin activity were examined in primary rat kidney myofibroblasts, and in kidney tissue from relaxin-treated male wild-type and AT2R-knockout mice subjected to unilateral ureteric obstruction. Relaxin's antifibrotic actions were significantly blocked by PD123319 in vitro and in vivo, or when relaxin was administered to AT2R-knockout mice. While heterodimer complexes were formed between RXFP1 and AT2Rs independent of ligand binding, relaxin did not directly bind to AT2Rs but signaled through RXFP1-AT2R heterodimers to induce its antifibrotic actions. These findings highlight a hitherto unrecognized interaction that may be targeted to control fibrosis progression. PMID:24429402

  9. JAK Kinase Inhibition Abrogates STAT3 Activation and Head and Neck Squamous Cell Carcinoma Tumor Growth

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    Malabika Sen

    2015-03-01

    Full Text Available Aberrant activation of the Janus kinase (JAK/signal transducer and activator of transcription (STAT 3 has been implicated in cell proliferation and survival of many cancers including head and neck squamous cell carcinoma (HNSCC. AZD1480, an orally active pharmacologic inhibitor of JAK1/JAK2, has been tested in several cancer models. In the present study, the in vitro and in vivo effects of AZD1480 were evaluated in HNSCC preclinical models to test the potential use of JAK kinase inhibition for HNSCC therapy. AZD1480 treatment decreased HNSCC proliferation in HNSCC cell lines with half maximal effective concentration (EC50 values ranging from 0.9 to 4 μM in conjunction with reduction of pSTAT3Tyr705 expression. In vivo antitumor efficacy of AZD1480 was demonstrated in patient-derived xenograft (PDX models derived from two independent HNSCC tumors. Oral administration of AZD1480 reduced tumor growth in conjunction with decreased pSTAT3Tyr705 expression that was observed in both PDX models. These findings suggest that the JAK1/2 inhibitors abrogate STAT3 signaling and may be effective in HNSCC treatment approaches.

  10. Curcumin and folic acid abrogated methotrexate induced vascular endothelial dysfunction.

    Science.gov (United States)

    Sankrityayan, Himanshu; Majumdar, Anuradha S

    2016-01-01

    Methotrexate, an antifolate drug widely used in rheumatoid arthritis, psoriasis, and cancer, is known to cause vascular endothelial dysfunction by causing hyperhomocysteinemia, direct injury to endothelium or by increasing the oxidative stress (raising levels of 7,8-dihydrobiopterin). Curcumin is a naturally occurring polyphenol with strong antioxidant and anti-inflammatory action and therapeutic spectra similar to that of methotrexate. This study was performed to evaluate the effects of curcumin on methotrexate induced vascular endothelial dysfunction and also compare its effect with that produced by folic acid (0.072 μg·g(-1)·day(-1), p.o., 2 weeks) per se and in combination. Male Wistar rats were exposed to methotrexate (0.35 mg·kg(-1)·day(-1), i.p.) for 2 weeks to induce endothelial dysfunction. Methotrexate exposure led to shedding of endothelium, decreased vascular reactivity, increased oxidative stress, decreased serum nitrite levels, and increase in aortic collagen deposition. Curcumin (200 mg·kg(-1)·day(-1) and 400 mg·kg(-1)·day(-1), p.o.) for 4 weeks prevented the increase in oxidative stress, decrease in serum nitrite, aortic collagen deposition, and also vascular reactivity. The effects were comparable with those produced by folic acid therapy. The study shows that curcumin, when concomitantly administered with methotrexate, abrogated its vascular side effects by preventing an increase in oxidative stress and abating any reduction in physiological nitric oxide levels. PMID:26571019

  11. Defining BMP functions in the hair follicle by conditional ablation of BMP receptor IA

    OpenAIRE

    Kobielak, Krzysztof; Pasolli, H. Amalia; Alonso, Laura; Polak, Lisa; Fuchs, Elaine

    2003-01-01

    Using conditional gene targeting in mice, we show that BMP receptor IA is essential for the differentiation of progenitor cells of the inner root sheath and hair shaft. Without BMPRIA activation, GATA-3 is down-regulated and its regulated control of IRS differentiation is compromised. In contrast, Lef1 is up-regulated, but its regulated control of hair differentiation is still blocked, and BMPRIA-null follicles fail to activate Lef1/β-catenin–regulated genes, including keratin genes. Wnt-medi...

  12. Crimpy enables discrimination of pre and postsynaptic pools of a BMP at the Drosophila NMJ

    Science.gov (United States)

    James, Rebecca E.; Hoover, Kendall M.; Bulgari, Dinara; McLaughlin, Colleen N.; Wilson, Christopher G.; Wharton, Kristi A.; Levitan, Edwin S.; Broihier, Heather T.

    2014-01-01

    Summary Distinct pools of the BMP Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, while muscle-derived Gbb regulates NMJ growth. Yet how cells differentiate between these ligand pools is not known. Here we present evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbb's pro-neurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, we propose that Gbb/Cmpy co-release from presynaptic terminals defines a neuronal pro-transmission signal. PMID:25453556

  13. Threshold-dependent BMP-mediated repression: a model for a conserved mechanism that patterns the neuroectoderm.

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    Claudia Mieko Mizutani

    2006-10-01

    Full Text Available Subdivision of the neuroectoderm into three rows of cells along the dorsal-ventral axis by neural identity genes is a highly conserved developmental process. While neural identity genes are expressed in remarkably similar patterns in vertebrates and invertebrates, previous work suggests that these patterns may be regulated by distinct upstream genetic pathways. Here we ask whether a potential conserved source of positional information provided by the BMP signaling contributes to patterning the neuroectoderm. We have addressed this question in two ways: First, we asked whether BMPs can act as bona fide morphogens to pattern the Drosophila neuroectoderm in a dose-dependent fashion, and second, we examined whether BMPs might act in a similar fashion in patterning the vertebrate neuroectoderm. In this study, we show that graded BMP signaling participates in organizing the neural axis in Drosophila by repressing expression of neural identity genes in a threshold-dependent fashion. We also provide evidence for a similar organizing activity of BMP signaling in chick neural plate explants, which may operate by the same double negative mechanism that acts earlier during neural induction. We propose that BMPs played an ancestral role in patterning the metazoan neuroectoderm by threshold-dependent repression of neural identity genes.

  14. Titanium With Nanotopography Induces Osteoblast Differentiation by Regulating Endogenous Bone Morphogenetic Protein Expression and Signaling Pathway.

    Science.gov (United States)

    M S Castro-Raucci, Larissa; S Francischini, Marcelo; N Teixeira, Lucas; P Ferraz, Emanuela; B Lopes, Helena; T de Oliveira, Paulo; Hassan, Mohammad Q; Losa, Adalberto L; Beloti, Marcio M

    2016-07-01

    We aimed at evaluating the effect of titanium (Ti) with nanotopography (Nano) on the endogenous expression of BMP-2 and BMP-4 and the relevance of this process to the nanotopography-induced osteoblast differentiation. MC3T3-E1 cells were grown on Nano and machined (Machined) Ti surfaces and the endogenous BMP-2/4 expression and the effect of BMP receptor BMPR1A silencing in both osteoblast differentiation and expression of genes related to TGF-β/BMP signaling were evaluated. Nano supported higher BMP-2 gene and protein expression and upregulated the osteoblast differentiation compared with Machined Ti surface. The BMPR1A silencing inhibited the osteogenic potential induced by Nano Ti surface as indicated by reduced alkaline phosphatase (ALP), osteocalcin and RUNX2 gene expression, RUNX2 protein expression and ALP activity. In addition, the expression of genes related to TGF-β/BMP signaling was deeply affected by BMPR1A-silenced cells grown on Nano Ti surface. In conclusion, we have demonstrated for the first time that nanotopography induces osteoblast differentiation, at least in part, by upregulating the endogenous production of BMP-2 and modulating BMP signaling pathway. J. Cell. Biochem. 117: 1718-1726, 2016. © 2015 Wiley Periodicals, Inc. PMID:26681207

  15. Comparison of osteogenic potentials of human rat BMP4 and BMP6 gene therapy using [E1-] and [E1-,E2b-] adenoviral vectors

    Directory of Open Access Journals (Sweden)

    Hongwei Li, Jin Zhong Li, Debra D. Pittman, Andy Amalfitano, Gerald R. Hankins, Gregory A. Helm

    2006-01-01

    Full Text Available Osteogenic potentials of some recombinant human bone morphogenetic protein (BMP first-generation adenoviral vectors (ADhBMPs are significantly limited in immunocompetent animals. It is unclear what role expression of viral proteins and foreign proteins transduced by adenoviral vectors play in the host immune response and in ectopic bone formation. In this study two sets of experiments were designed and performed. First, rat BMP6 cDNA were amplified, sequenced, and recombined in first-generation adenoviral vector (ADrBMP6. A comparison of human and rat BMP6 adenoviral vectors demonstrated identical osteogenic activities in both immunodeficient and immunocompetent rats. Second, the activities of recombinant human BMP6 in E1- (ADhBMP6 and [E1-,E2b-] ( [E1-,E2b-]ADGFP&hBMP6, and [E1-,E2b-]ADhBMP6 adenoviral vectors were compared in both in vitro and in vivo models. Similar activities of these two generations of BMP adenoviral vectors were found in all models. These results indicate that the amount of viral gene expression and the source of the BMP cDNA are not major factors in the interruption of osteogenic potentials of recombinant BMP6 adenoviral vectors in immunocompetent animals.

  16. Extraembryonic signals under the control of MGA, Max, and Smad4 are required for dorsoventral patterning.

    Science.gov (United States)

    Sun, Yuhua; Tseng, Wei-Chia; Fan, Xiang; Ball, Rebecca; Dougan, Scott T

    2014-02-10

    In vertebrates, extraembryonic tissues can act as signaling centers that impose a reproducible pattern of cell types upon the embryo. Here, we show that the zebrafish yolk syncytial layer (YSL) secretes a ventralizing signal during gastrulation. This activity is mediated by Bmp2b/Swirl (Swr) expressed under the control of Max's giant associated protein (MGA) and its binding partners, Max and Smad4. MGA coimmunoprecipitates with both Max and Smad4 in embryo extracts, and the three proteins form a complex in vitro. Furthermore, all three proteins bind to a DNA fragment upstream of the bmp2b transcription start site. Targeted depletion of MGA, its binding partners, or Bmp2b/Swr from the YSL reduces BMP signaling throughout the embryo, resulting in a mildly dorsalized phenotype. We conclude that MGA, Max, and Smad4 act in the extraembryonic YSL to initiate a positive feedback loop of Bmp signaling within the embryo. PMID:24525188

  17. Phenotype discovery by gene expression profiling: mapping of biological processes linked to BMP-2-mediated osteoblast differentiation.

    Science.gov (United States)

    Balint, Eva; Lapointe, David; Drissi, Hicham; van der Meijden, Caroline; Young, Daniel W; van Wijnen, Andre J; Stein, Janet L; Stein, Gary S; Lian, Jane B

    2003-05-15

    Understanding physiological control of osteoblast differentiation necessitates characterization of the regulatory signals that initiate the events directing a cell to lineage commitment and establishing competency for bone formation. The bone morphogenetic protein, BMP-2, a member of the TGFbeta superfamily, induces osteoblast differentiation and functions through the Smad signal transduction pathway during in vivo bone formation. However, the molecular targets of BMP-mediated gene transcription during the process of osteoblast differentiation have not been comprehensively identified. In the present study, BMP-2 responsive factors involved in the early stages of commitment and differentiation to the osteoblast phenotype were analyzed by microarray gene expression profiling in samples ranging from 1 to 24 h following BMP-2 dependent differentiation of C2C12 premyoblasts into the osteogenic lineage. A total of 1,800 genes were responsive to BMP-2 and expression was modulated from 3- to 14-fold for less than 100 genes during the time course. Approximately 50% of these 100 genes are either up- or downregulated. Major events associated with phenotypic changes towards the osteogenic lineage were identified from hierarchical and functional clustering analyses. BMP-2 immediately responsive genes (1-4 h), which exhibited either transient or sustained expression, reflect activation and repression of non-osseous BMP-2 developmental systems. This initial response was followed by waves of expression of nuclear proteins and developmental regulatory factors including inhibitors of DNA binding, Runx2, C/EBP, Zn finger binding proteins, forkhead, and numerous homeobox proteins (e.g., CDP/cut, paired, distaless, Hox) which are expressed at characterized stages during osteoblast differentiation. A sequential profile of genes mediating changes in cell morphology, cell growth, and basement membrane formation is observed as a secondary transient early response (2-8 h). Commitment to the

  18. Inhibitory Smads and bone morphogenetic protein (BMP) modulate anterior photoreceptor cell number during planarian eye regeneration.

    Science.gov (United States)

    González-Sastre, Alejandro; Molina, Ma Dolores; Saló, Emili

    2012-01-01

    Planarians represent an excellent model to study the processes of body axis and organ re-specification during regeneration. Previous studies have revealed a conserved role for the bone morphogenetic protein (BMP) pathway and its intracellular mediators Smad1/5/8 and Smad4 in planarian dorsoventral (DV) axis re-establishment. In an attempt to gain further insight into the role of this signalling pathway in planarians, we have isolated and functionally characte-rized the inhibitory Smads (I-Smads) in Schmidtea mediterranea. Two I-Smad homologues have been identified: Smed-smad6/7-1 and Smed-smad6/7-2. Expression of smad6/7-1 was detected in the parenchyma, while smad6/7-2 was found to be ex-pressed in the central nervous system and the eyes. Neither single smad6/7-1 and smad6/7-2 nor double smad6/7-1,-2 silencing gave rise to any apparent disruption of the DV axis. However, both regenerating and intact smad6/7-2 (RNAi) planarians showed defects in eye morphogenesis and displayed small, rounded eyes that lacked the anterior subpopulation of photoreceptor cells. The number of pigment cells was also reduced in these animals at later stages of regeneration. In contrast, after low doses of Smed-bmp(RNAi), planarians regenerated larger eyes in which the anterior subpopulation of photoreceptor cells was expanded. Our results suggest that Smed-smad6/7-2 and Smed-bmp control the re-specification and maintenance of anterior photoreceptor cell number in S. mediterranea. PMID:22451003

  19. Nanotopography Directs Mesenchymal Stem Cells to Osteoblast Lineage through Regulation of microRNA-SMAD-BMP-2 Circuit

    Science.gov (United States)

    KATO, ROGERIO B.; ROY, BHASKAR; DE OLIVEIRA, FABIOLA S.; FERRAZ, EMANUELA P.; DE OLIVEIRA, PAULO T.; KEMPER, AUSTIN G.; HASSAN, MOHAMMAD Q.; ROSA, ADALBERTO L.; BELOTI, MARCIO M.

    2016-01-01

    The aim of this study was to investigate if chemically produced nanotopography on titanium (Ti) surface induces osteoblast differentiation of cultured human bone marrow mesenchymal stem cells (hMSCs) by regulating the expression of microRNAs (miRs). It was demonstrated that Ti with nanotopography induces osteoblast differentiation of hMSCs as evidenced by upregulation of osteoblast specific markers compared with untreated (control) Ti at day 4. At this time-point, miR-sequencing analysis revealed that 20 miRs were upregulated (>2 fold) while 20 miRs were downregulated (>3 fold) in hMSCs grown on Ti with nanotopography compared with control Ti. Three miRs, namely miR-4448, -4708 and -4773, which were significantly downregulated (>5 fold) by Ti with nanotopography affect osteoblast differentiation of hMSCs. These miRs that directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (BMP-2) osteogenic signal, were upregulated by Ti with nanotopography. Overexpression of miR-4448, -4708 and 4773 in MC3T3-E1 pre-osteoblasts noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and therefore repressed the gene expression of key bone markers. Additionally, it was observed that the treatment with BMP-2 displayed a higher osteogenic effect on MC3T3-E1 cells grown on Ti with nanotopography compared with control Ti, suggesting that the BMP-2 signaling pathway was more effective on this surface. Taken together, these results indicate that a complex regulatory network involving a miR-SMAD-BMP-2 circuit governs the osteoblast differentiation induced by Ti with nanotopography. PMID:24619927

  20. Targeting ILK and β4 integrin abrogates the invasive potential of ovarian cancer

    International Nuclear Information System (INIS)

    Highlights: ► The potential of targeting ILK and integrins for highly aggressive ovarian cancer. ► Unanticipated synergistic effect for the combination of ILK/β4 integrin. ► Combination of ILK/β4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade. ► Targeting of β4 integrin/ILK had potent inhibitory effects in ovarian cancer. -- Abstract: Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of β1 and β4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expression of β1 and β4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of β4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of β4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting β4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.

  1. Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid--an experimental study in rats.

    Science.gov (United States)

    Schliephake, Henning; Weich, Herbert A; Dullin, Christian; Gruber, Rudolf; Frahse, Sarah

    2008-01-01

    The aim of the present study was to test the hypothesis that human recombinant bone morphogenic protein 2 (rhBMP-2) implanted in a slow release carrier of polylactic acid (PLA) can repair a non-healing defect in the rat mandible and maintain the thickness of an augmented volume. p-DL-lactic acid discs were produced and loaded with 48 and 96 microg rhBMP-2 and inserted into non-healing defects of the mandible of 45 Wistar rats. Fifteen rats received implants with 96 microg rhBMP-2 (Group 2), 48 microg rhBMP-2 (Group 1) and blank implants without BMP (Group 0) each on one side of the mandible. Unfilled defects of the same size on the contralateral sides of the mandibles served as empty controls. After 6, 13 and 26 weeks, implants of each group were retrieved from five animals each and submitted to flat panel detector computed tomography. Bone formation and thickness of augmentation was assessed by computer-assisted histomorphometry. In Group 2 significantly more bone was produced than in Group 1. Implants of Group 1 induced significantly more bone than the blank controls only after 6 weeks, whereas the difference was not significant after 13 and 26 weeks. Differences between Group 2 and Group 1 were clearly significant after 26 weeks. The thickness of bone tissue was maintained in Group 2 whereas it decreased in Group 1 and was negligible in Group 0. It is concluded that the PLA implants with 96 microg rhBMP-2 were able to bridge a non-healing defect in the rat mandible and maintained the thickness of an augmented volume. However, continuous supply of osteogenic signals appears to be required to compensate for adverse effects during polymer degradation. PMID:17936352

  2. Glycosylation of Twisted gastrulation is required for BMP binding and activity during craniofacial development

    Directory of Open Access Journals (Sweden)

    CharlesJ.Billington

    2011-09-01

    Full Text Available Twisted Gastrulation (TWSG1 is a conserved, secreted glycoprotein that modulates signaling of bone morphogenetic proteins (BMPs in the extracellular space. Deletion of exon 4 of mouse Twsg1 (mTwsg1 is associated with significant craniofacial defects. However, little is understood about the biochemical properties of the corresponding region of the protein. We have uncovered a significant role for exon 4 sequences as encoding the only two glycosylation sites of the mTWSG1 protein. Deletion of the entire exon 4 or mutation of both glycosylation sites within exon 4 abolishes glycosylation of mTWSG1. Importantly, we find that constructs with mutated glycosylation sites have significantly reduced BMP binding activity. We further show that glycosylation and activity of TWSG1 recombinant proteins vary markedly by cellular source. Non-glycosylated mTWSG1 made in E. coli has both reduced affinity for BMPs, as shown by surface plasmon resonance analysis, and reduced BMP inhibitory activity in a mandibular explant culture system compared to glycosylated proteins made in insect cells or murine myeloma cells. This study highlights an essential role for glycosylation in Twisted gastrulation action.

  3. OPTIMIZATION OF DECENTRALIZED BMP CONTROLS IN URBAN AREAS

    Science.gov (United States)

    This paper will present an overview of a recently completed project for the US EPA entitled, Optimization of Urban Wet-weather Flow Control Systems. The focus of this effort is on techniques that are suitable for evaluating decentralized BMP controls. The four major components ...

  4. Biological activity of a genetically modified BMP-2 variant with inhibitory activity

    Directory of Open Access Journals (Sweden)

    Kübler Alexander C

    2009-02-01

    Full Text Available Abstract Background Alterations of the binding epitopes of bone morphogenetic protein-2 (BMP-2 lead to a modified interaction with the ectodomains of BMP receptors. In the present study the biological effect of a BMP-2 double mutant with antagonistic activity was evaluated in vivo. Methods Equine-derived collagenous carriers were loaded with recombinant human BMP-2 (rhBMP-2 in a well-known dose to provide an osteoinductive stimulus. The study was performed in a split animal design: carriers only coupled with rhBMP-2 (control were implanted into prepared cavities of lower limb muscle of rats, specimens coupled with rhBMP-2 as well as BMP-2 double mutant were placed into the opposite limb in the same way. After 28 days the carriers were explanted, measured radiographically and characterized histologically. Results As expected, the BMP-2 loaded implants showed a typical heterotopic bone formation. The specimens coupled with both proteins showed a significant decreased bone formation in a dose dependent manner. Conclusion The antagonistic effect of a specific BMP-2 double mutant could be demonstrated in vivo. The dose dependent influence on heterotopic bone formation by preventing rhBMP-2 induced osteoinduction suggests a competitive receptor antagonism.

  5. BMP analysis system for watershed-based stormwater management.

    Science.gov (United States)

    Zhen, Jenny; Shoemaker, Leslie; Riverson, John; Alvi, Khalid; Cheng, Mow-Soung

    2006-01-01

    Best Management Practices (BMPs) are measures for mitigating nonpoint source (NPS) pollution caused mainly by stormwater runoff. Established urban and newly developing areas must develop cost effective means for restoring or minimizing impacts, and planning future growth. Prince George's County in Maryland, USA, a fast-growing region in the Washington, DC metropolitan area, has developed a number of tools to support analysis and decision making for stormwater management planning and design at the watershed level. These tools support watershed analysis, innovative BMPs, and optimization. Application of these tools can help achieve environmental goals and lead to significant cost savings. This project includes software development that utilizes GIS information and technology, integrates BMP processes simulation models, and applies system optimization techniques for BMP planning and selection. The system employs the ESRI ArcGIS as the platform, and provides GIS-based visualization and support for developing networks including sequences of land uses, BMPs, and stream reaches. The system also provides interfaces for BMP placement, BMP attribute data input, and decision optimization management. The system includes a stand-alone BMP simulation and evaluation module, which complements both research and regulatory nonpoint source control assessment efforts, and allows flexibility in the examining various BMP design alternatives. Process based simulation of BMPs provides a technique that is sensitive to local climate and rainfall patterns. The system incorporates a meta-heuristic optimization technique to find the most cost-effective BMP placement and implementation plan given a control target, or a fixed cost. A case study is presented to demonstrate the application of the Prince George's County system. The case study involves a highly urbanized area in the Anacostia River (a tributary to Potomac River) watershed southeast of Washington, DC. An innovative system of

  6. New insights into BMP-7 mediated osteoblastic differentiation of primary human mesenchymal stem cells.

    Science.gov (United States)

    Lavery, Karen; Hawley, Sara; Swain, Pamela; Rooney, Robert; Falb, Dean; Alaoui-Ismaili, Moulay Hicham

    2009-07-01

    Bone Morphogenetic Proteins (BMPs) are members of the TGF-beta superfamily of growth factors. Several BMPs exhibit osteoinductive bioactivities, and are critical for bone formation in both developing and mature skeletal systems. BMP-7 (OP-1) is currently used clinically in revision of posterolateral spine fusions and long bone non-unions. The current study characterizes BMP-7 induced gene expression during early osteoblastic differentiation of human mesenchymal stem cells (hMSC). Primary hMSC were treated with BMP-7 for 24 or 120 h and gene expression across the entire human genome was evaluated using Affymetrix HG-U133 Plus 2.0 Arrays. 955 probe sets representing 655 genes and 95 ESTs were identified as differentially expressed and were organized into three major expression profiles (Profiles A, B and C) by hierarchical clustering. Genes from each profile were classified according to biochemical pathway analyses. Profile A, representing genes upregulated by BMP-7, revealed strong enrichment for established osteogenic marker genes, as well as several genes with undefined roles in osteoblast function, including MFI2, HAS3, ADAMTS9, HEY1, DIO2 and FGFR3. A functional screen using siRNA suggested roles for MFI2, HEY1 and DIO2 in osteoblastic differentiation of hMSC. Profile B contained genes transiently downregulated by BMP-7, including numerous genes associated with cell cycle regulation. Follow-up studies confirmed that BMP-7 attenuates cell cycle progression and cell proliferation during early osteoblastic differentiation. Profile C, comprised of genes continuously downregulated by BMP-7, exhibited strong enrichment for genes associated with chemokine/cytokine activity. Inhibitory effects of BMP-7 on cytokine secretion were verified by analysis of enriched culture media. Potent downregulation of CHI3L1, a potential biomarker for numerous joint diseases, was also observed in Profile C. A focused evaluation of BMP, GDF and BMP inhibitor expression elucidated feedback

  7. Complexation and sequestration of BMP-2 from an ECM mimetic hyaluronan gel for improved bone formation.

    Directory of Open Access Journals (Sweden)

    Marta Kisiel

    Full Text Available Bone morphogenetic protein-2 (BMP-2 is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an efficacious outcome. To lower its effective dose, we precomplexed BMP-2 with the glycosaminoglycans (GAGs dermatan sulfate (DS or heparin (HP, prior to loading it into a hyaluronic acid (HA hydrogel. In vitro release studies showed that BMP-2 precomplexed with DS or HP had a prolonged delivery compared to without GAG. BMP-2-DS complexes achieved a slightly faster release in the first 24 h than HP; however, both delivered BMP-2 for an equal duration. Analysis of the kinetic interaction between BMP-2 and DS or HP showed that HP had approximately 10 times higher affinity for BMP-2 than DS, yet it equally stabilized the protein, as determined by alkaline phosphatase activity. Ectopic bone formation assays at subcutaneous sites in rats demonstrated that HA hydrogel-delivered BMP-2 precomplexed with GAG induced twice the volume of bone compared with BMP-2 delivered uncomplexed to GAG.

  8. BMP-2 induces versican and hyaluronan that contribute to post-EMT AV cushion cell migration.

    Directory of Open Access Journals (Sweden)

    Kei Inai

    Full Text Available Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM components, versican and hyaluronan (HA, and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions.

  9. The culture of referendum in Albania: Technical and theoritecal reflections on the abrogative referendum

    OpenAIRE

    Valbona Pajo Bala

    2014-01-01

    The aim of this paper is to analyse the Albanian constitutional and legal framework on referenda, in general, focusing special attention to the abrogative referenda of a law or part thereof. Given the absence of any concrete case of an abrogative referenda held in Albania, which does not creates very much room for discussion in that regard, the paper, through a comparative approach on the referenda culture in other european states, aims at offering to the reader a more complete view on the me...

  10. Biochemical methane potential (BMP) of solid organic materials

    DEFF Research Database (Denmark)

    Raposo, Francisco; Fernández-Cegrí, V.; De la Rubia, M.A.;

    2010-01-01

    experimental conditions were reported. The study was performed using 4 samples: 3 reference substrates (starch, cellulose and gelatine), and 1 raw material (mung bean). The BMP of mung bean was carried out at two inoculum to substrate ratios (ISR), specifically 2 and 1. The methane yields of reference......This paper describes the results obtained for different participating research groups in an interlaboratory study related to the biochemical methane potential (BMP). In this research work, the full experimental conditions influencing the test such as inoculum, substrate characteristics and...... substrates for starch, cellulose and gelatine were 352±33, 353±29 and 382±42 mL/g VSadded, respectively. The percentages of biotransformation of these substrates into methane were 85±8, 85±7 and 88±10%, respectively. On the other hand, the values of methane yields and biodegradability for MB were 373±35 m...

  11. Fibronectin induction abrogates the BRAF inhibitor response of BRAF V600E/PTEN-null melanoma cells.

    Science.gov (United States)

    Fedorenko, I V; Abel, E V; Koomen, J M; Fang, B; Wood, E R; Chen, Y A; Fisher, K J; Iyengar, S; Dahlman, K B; Wargo, J A; Flaherty, K T; Sosman, J A; Sondak, V K; Messina, J L; Gibney, G T; Smalley, K S M

    2016-03-10

    The mechanisms by which some melanoma cells adapt to Serine/threonine-protein kinase B-Raf (BRAF) inhibitor therapy are incompletely understood. In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF mutant and PTEN null. Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN dependent. These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs. Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment. Analysis of a melanoma tissue microarray showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis. Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5β1 integrin. This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1. The protection conveyed by the induction of FN expression could be overcome through combined treatment with a BRAF and PI3K inhibitor. PMID:26073081

  12. The role of BMP inhibitors on craniofacial development

    Czech Academy of Sciences Publication Activity Database

    Buchtová, Marcela; Richman, J. M.

    Hradec Králové: Czech Anatomical Society, 2012. 37-37. [Morphology 2012 International Congress on Anatomy /47./ and Lojda Symposium on Histochemistry /49./. 09.09.2012-12.09.2012, Hradec Králové] R&D Projects: GA ČR GA304/09/0725 Institutional research plan: CEZ:AV0Z50450515 Keywords : BMP inhibitors Subject RIV: EA - Cell Biology

  13. BMP2 induced osteogenic differentiation of human umbilical cord stem cells in a peptide-based hydrogel scaffold

    Science.gov (United States)

    Lakshmana, Shruthi M.

    Craniofacial tissue loss due to traumatic injuries and congenital defects is a major clinical problem around the world. Cleft palate is the second most common congenital malformation in the United States occurring with an incidence of 1 in 700. Some of the problems associated with this defect are feeding difficulties, speech abnormalities and dentofacial anomalies. Current treatment protocol offers repeated surgeries with extended healing time. Our long-term goal is to regenerate bone in the palatal region using tissue-engineering approaches. Bone tissue engineering utilizes osteogenic cells, osteoconductive scaffolds and osteoinductive signals. Mesenchymal stem cells derived from human umbilical cord (HUMSCs) are highly proliferative with the ability to differentiate into osteogenic precursor cells. The primary objective of the study was to characterize HUMSCs and culture them in a 3D hydrogel scaffold and investigate their osteogenic potential. PuraMatrix(TM) is an injectable 3D nanofiber scaffold capable of self-assembly when exposed to physiologic conditions. Our second objective was to investigate the effect of Bone Morphogenic Protein 2 (BMP2) in enhancing the osteogenic differentiation of HUMSCs encapsulated in PuraMatrix(TM). We isolated cells isolated from Wharton's Jelly region of the umbilical cord obtained from NDRI (New York, NY). Isolated cells satisfied the minimal criteria for mesenchymal stem cells (MSCs) as defined by International Society of Cell Therapy in terms of plastic adherence, fibroblastic phenotype, surface marker expression and osteogenic differentiation. Flow Cytometry analysis showed that cells were positive for CD73, CD90 and CD105 while negative for hematopoietic marker CD34. Alkaline phosphatase activity (ALP) of HUMSCs showed peak activity at 2 weeks (p<0.05). Cells were encapsulated in 0.2% PuraMatrix(TM) at cell densities of 10x104, 20x104, 40x10 4 and 80x104. Cell viability with WST and proliferation with Live-Dead cell assays

  14. Bone regeneration by implantation of adipose-derived stromal cells expressing BMP-2

    International Nuclear Information System (INIS)

    In this study, we reported that the adipose-derived stromal cells (ADSCs) genetically modified by bone morphogenetic protein 2 (BMP-2) healed critical-sized canine ulnar bone defects. First, the osteogenic and adipogenic differentiation potential of the ADSCs derived from canine adipose tissue were demonstrated. And then the cells were modified by the BMP-2 gene and the expression and bone-induction ability of BMP-2 were identified. Finally, the cells modified by BMP-2 gene were applied to a β-tricalcium phosphate (TCP) carrier and implanted into ulnar bone defects in the canine model. After 16 weeks, radiographic, histological, and histomorphometry analysis showed that ADSCs modified by BMP-2 gene produced a significant increase of newly formed bone area and healed or partly healed all of the bone defects. We conclude that ADSCs modified by the BMP-2 gene can enhance the repair of critical-sized bone defects in large animals

  15. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    International Nuclear Information System (INIS)

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  16. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Li-An [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi-an (China); Yuan, Guohua; Yang, Guobin [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Key Laboratory of Oral Biomedical Engineering Ministry of Education, Wuhan (China); Ortiz-Gonzalez, Iris [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Yang, Wuchen; Cui, Yong [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); MacDougall, Mary [Department of Oral/Maxillofacial Surgery, University of Alabama, Birmingham, AL (United States); Donly, Kevin J. [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Harris, Stephen [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); Chen, Shuo, E-mail: chens0@uthscsa.edu [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States)

    2009-08-14

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  17. Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

    International Nuclear Information System (INIS)

    Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression

  18. In Vitro and In Vivo Studies of BMP-2-Loaded PCL–Gelatin–BCP Electrospun Scaffolds

    OpenAIRE

    Kim, Bo-Ram; Nguyen, Thuy Ba Linh; Min, Young-Ki; Lee, Byong-Taek

    2014-01-01

    To confirm the effect of recombinant human bone morphogenetic protein-2 (BMP-2) for bone regeneration, BMP-2-loaded polycaprolactone (PCL)–gelatin (Gel)–biphasic calcium phosphate (BCP) fibrous scaffolds were fabricated using the electrospinning method. The electrospinning process to incorporate BCP nanoparticles into the PCL–Gel scaffolds yielded an extracellular matrix-like microstructure that was a hybrid system composed of nano- and micro-sized fibers. BMP-2 was homogeneously loaded on th...

  19. Multifunctional Thin Film Biomatrice Biosensor in a Degradable Scaffold Containing Bone Morphogenetic Protein-2 (BMP-2) for Controlled Release in Skeletal Tissue Engineering

    Science.gov (United States)

    McDaniel, Harvey; Lomax, Linda

    2001-03-01

    Bone morphonogenetic proteins (BMP-2) have been under investigation for three decades. Deminerialized bone and extracts of deminerialized bone are o steoinductive with a temporal sequence of bone induction. Native and recombi nant BMP's have shown the ability, thru growth and differentiative factors t o induce de novo bone formation both invitro and invivo. Their principle fun ction is to induce transformation of undifferentiated mesenchymal cells into osteoblasts. Native and recombinant BMP's, when purified and used without carrier disp erse after implantation and exert no effect on bone induction. The delivery system provides the missing component to successsfully applying osteogenic p roteins for clinical need. Biological and physio-chemical properties are str ictly adhered tofor a successful delivery system. The BMP delivery system ca rrier for osteo inductive payload provided; 1)non tumorgenic genecity, 2) no n immunogenecity, 3) water insoluble, 4) biosorbability with predictable enz ymatic degradation, and 5) an optimized surface for compatibility, cell migr ation and attachment with a negative surface change that encouraged target c ell attachment. Being a controlled Release System, it binded the proteins wi th predictible BMP released kinetics. Porosity with interconnecting voids pr otected the BMP from noon specific proteolysis and promoted rapid vascular a nd mesenchymal invasion. Far wide ranging clinical applications of mechanica l and biofunctional requirements were met with the BMP delivery system. Cohe sion and malleability were reqiured forcontour augmentation, and reconstruct ion of the discontinuity defects, prevented dislocation and retained the sha pe and bone replaced the system. Biological systems have elastic activity associated with them. The activi ty was current associated with a time dependant biological/biochemical react ion (enzymic activity). Bioelectric phoenomena associated with charged molec ules in a biologic structure caused

  20. Conditional Deletion of BMP7 from the Limb Skeleton Does Not Affect Bone Formation or Fracture Repair

    OpenAIRE

    Tsuji, Kunikazu; Cox, Karen; Gamer, Laura; Graf, Daniel; Economides, Aris; Rosen, Vicki

    2010-01-01

    While the osteoinductive activity of recombinant bone morphogenetic protein 7 (BMP7) is well established, evaluation of the role of endogenous BMP7 in bone formation and fracture healing has been hampered by perinatal lethality in BMP7 knockout mice. Here we employ conditional deletion of BMP7 from the embryonic limb prior to the onset of skeletogenesis to create limb bones lacking BMP7. We find that the absence of locally produced BMP7 has no effect on postnatal limb growth, articular cartil...

  1. New Roles and Mechanism of Action of BMP4 in Postnatal Tooth Cytodifferentiation

    OpenAIRE

    Gluhak-Heinrich, J.; Guo, D; Yang, W; Harris, MA; Lichtler, A; Kream, B; Zhang, J; Feng, JQ; Harris, SE

    2010-01-01

    During the phase of overt tooth cytodifferentiation that occurs after birth in the mouse and using the 3.6Collagen1a-Cre, the BMP4 floxed and BMP4 knock-out mice, the BMP4 gene was deleted in early collagen producing odontoblasts around postnatal day 1. BMP4 expression was reduced over 90% in alveolar osteoblasts and odontoblasts. There was decreased rate of predentin to dentin formation and decreased mature odontoblast differentiation reflected in reduced DMP1 expression and proper dentinal ...

  2. Study on Z-H/BMP Toughened Compound Artificial Bone and Its Osteogenesis

    Institute of Scientific and Technical Information of China (English)

    XU Wei-guo; CHEN An-min; SUN Shu-zhen

    2003-01-01

    The purpose of this study was to find a kind of new artificial bone for anterior spinal fusion.ZrO2 stabilized by Y2O3 ( Y- PSZ), porous hydroxyapatite ( HA ) and bone morphogenetic protein (BMP) were used to make artificial compound bone ( Y2O3 ) ZrO2 -HA/ BMP( Z-H/ BMP ) , whose function was tested, microstructure and mineralogic composition constitution were analysised by SEM and XRD , and the corresponding animal tests were porformed. Osteogenesis of the material was observed by eyes, histology and SEM. Experimental results show that the component and ossific activity of Z-H/BMP were satisfactory.

  3. BMP7 retards peripheral myelination by activating p38 MAPK in Schwann cells.

    Science.gov (United States)

    Liu, Xiaoyu; Zhao, Yahong; Peng, Su; Zhang, Shuqiang; Wang, Meihong; Chen, Yeyue; Zhang, Shan; Yang, Yumin; Sun, Cheng

    2016-01-01

    Schwann cell (SC) myelination is pivotal for the proper physiological functioning of the nervous system, but the underlying molecular mechanism remains less well understood. Here, we showed that the expression of bone morphogenetic protein 7 (BMP7) inversely correlates with myelin gene expression during peripheral myelination, which suggests that BMP7 is likely a negative regulator for myelin gene expression. Our experiments further showed that the application of BMP7 attenuates the cAMP induced myelin gene expression in SCs. Downstream pathway analysis suggested that both p38 MAPK and SMAD are activated by exogenous BMP7 in SCs. The pharmacological intervention and gene silence studies revealed that p38 MAPK, not SMAD, is responsible for BMP7-mediated suppression of myelin gene expression. In addition, c-Jun, a potential negative regulator for peripheral myelination, was up-regulated by BMP7. In vivo experiments showed that BMP7 treatment greatly impaired peripheral myelination in newborn rats. Together, our results established that BMP7 is a negative regulator for peripheral myelin gene expression and that p38 MAPK/c-Jun axis might be the main downstream target of BMP7 in this process. PMID:27491681

  4. Factors Affecting Pollutant Load Reduction with Uncertainty Analysis in Urban Stormwater BMP Systems

    Science.gov (United States)

    Park, D.

    2015-12-01

    This study incorporates uncertainty analysis into a model of the performance of stormwater best management practices (BMPs) to characterize the uncertainty in stormwater BMP effluent load that results from uncertainty in the BMP performance modeling in an urban stormwater system. Detention basins are used as BMPs in the urban stormwater systems, and the total suspended solids (TSS) are used as an urban nonpoint source pollutant in Los Angeles, CA. The k-C* model, which incorporates uncertainty analysis, is applied to the uncertainty of the stormwater effluent concentration in urban stormwater systems. This study presents a frequency analysis of the runoff volume and BMP overflows to characterize the uncertainty of BMP effluent loads, and the load frequency curve (LFC) is simulated with and without BMP conditions and verified using the observed TSS load. Finally, the effects of imperviousness, BMP volume, and BMP surface area are investigated using a reliability analysis. The results of this study can be used to determine the appropriate BMP size to achieve a specific watershed runoff pollutant load. The result of this evaluation method can support the adequate sizing of a BMP to meet the defined nonpoint source pollutant regulations. Acknowlegments This research was supported by a grant (14AWMP-B082564-01) from Advanced Water Management Research Program funded by Ministry of Land, Infrastructure and Transport of Korean government.

  5. TGF-β prevents phosphate-induced osteogenesis through inhibition of BMP and Wnt/β-catenin pathways.

    Directory of Open Access Journals (Sweden)

    Fátima Guerrero

    Full Text Available BACKGROUND: Transforming growth factor-β (TGF-β is a key cytokine during differentiation of mesenchymal stem cells (MSC into vascular smooth muscle cells (VSMC. High phosphate induces a phenotypic transformation of vascular smooth muscle cells (VSMC into osteogenic-like cells. This study was aimed to evaluate signaling pathways involved during VSMC differentiation of MSC in presence or not of high phosphate. RESULTS: Our results showed that TGF-β induced nuclear translocation of Smad3 as well as the expression of vascular smooth muscle markers, such as smooth muscle alpha actin, SM22α, myocardin, and smooth muscle-myosin heavy chain. The addition of high phosphate to MSC promoted nuclear translocation of Smad1/5/8 and the activation of canonical Wnt/β-catenin in addition to an increase in BMP-2 expression, calcium deposition and alkaline phosphatase activity. The administration of TGF-β to MSC treated with high phosphate abolished all these effects by inhibiting canonical Wnt, BMP and TGF-β pathways. A similar outcome was observed in high phosphate-treated cells after the inhibition of canonical Wnt signaling with Dkk-1. Conversely, addition of both Wnt/β-catenin activators CHIR98014 and lithium chloride enhanced the effect of high phosphate on BMP-2, calcium deposition and alkaline phosphatase activity. CONCLUSIONS: Full VSMC differentiation induced by TGF-β may not be achieved when extracellular phosphate levels are high. Moreover, TGF-β prevents high phosphate-induced osteogenesis by decreasing the nuclear translocation of Smad 1/5/8 and avoiding the activation of Wnt/β-catenin pathway.

  6. Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels.

    Science.gov (United States)

    Bosman, Erika A; Lawson, Kirstie A; Debruyn, Joke; Beek, Lisette; Francis, Annick; Schoonjans, Luc; Huylebroeck, Danny; Zwijsen, An

    2006-09-01

    Smad5 is an intracellular mediator of bone morphogenetic protein (Bmp) signalling. It is essential for primordial germ cell (PGC) development, for the development of the allantois and for amnion closure, as demonstrated by loss of Bmp signalling. By contrast, the appearance of ectopic PGC-like cells and regionalized ectopic vasculogenesis and haematopoiesis in thickened Smad5(m1/m1) amnion are amnion defects that have not been associated with loss of Bmp signalling components. We show that defects in amnion and allantois can already be detected at embryonic day (E) 7.5 in Smad5 mutant mice. However, ectopic Oct4-positive (Oct4(+)) and alkaline phosphatase-positive (AP(+)) cells appear suddenly in thickened amnion at E8.5, and at a remote distance from the allantois and posterior primitive streak, suggesting a change of fate in situ. These ectopic Oct4(+), AP(+) cells appear to be Stella negative and hence cannot be called bona fide PGCs. We demonstrate a robust upregulation of Bmp2 and Bmp4 expression, as well as of Erk and Smad activity, in the Smad5 mutant amnion. The ectopic expression of several Bmp target genes in different domains and the regionalized presence of cells of several Bmp-sensitive lineages in the mutant amnion suggest that different levels of Bmp signalling may determine cell fate. Injection of rBMP4 in the exocoelom of wild-type embryos can induce thickening of amnion, mimicking the early amnion phenotype in Smad5 mutants. These results support a model in which loss of Smad5 results paradoxically in gain of Bmp function defects in the amnion. PMID:16887830

  7. Risk variants in BMP4 promoters for nonsyndromic cleft lip/palate in a Chilean population

    Directory of Open Access Journals (Sweden)

    Suazo José

    2011-12-01

    Full Text Available Abstract Background Bone morphogenetic protein 4 gene (BMP4 plays a key role during maxillofacial development, since orofacial clefts are observed in animals when this gene is conditionally inactivated. We recently reported the existence of association between nonsyndromic cleft lip/palate (NSCLP and BMP4 polymorphisms by detecting transmission deviations for haplotypes that include a region containing a BMP4 promoter in case-parent trios. The aim of the present study was to search for possible causal mutations within BMP4 promoters (BMP4.1 and BMP4.2. Methods We analyzed the sequence of BMP4.1 and BMP4.2 in 167 Chilean NSCLP cases and 336 controls. Results We detected three novel variants in BMP4.1 (c.-5514G > A, c.-5365C > T and c.-5049C > T which could be considered as cleft risk factors due to their absence in controls. Additionally, rs2855530 G allele (BMP4.2 carriers showed an increased risk for NSCLP restricted to males (OR = 1.52; 95% C.I. = 1.07-2.15; p = 0.019. For this same SNP the dominant genotype model showed a higher frequency of G/G+G/C and a lower frequency of C/C in cases than controls in the total sample (p = 0.03 and in the male sample (p = 0.003. Bioinformatic prediction analysis showed that all the risk variants detected in this study could create new transcription factor binding motifs. Conclusions The sex-dependent association between rs2855530 and NSCLP could indirectly be related to the differential gene expression observed between sexes in animal models. We concluded that risk variants detected herein could potentially alter BMP4 promoter activity in NSCLP. Further functional and developmental studies are necessary to support this hypothesis.

  8. Cardiogenic induction of pluripotent stem cells streamlined through a conserved SDF-1/VEGF/BMP2 integrated network.

    Directory of Open Access Journals (Sweden)

    Anca Chiriac

    Full Text Available BACKGROUND: Pluripotent stem cells produce tissue-specific lineages through programmed acquisition of sequential gene expression patterns that function as a blueprint for organ formation. As embryonic stem cells respond concomitantly to diverse signaling pathways during differentiation, extraction of a pro-cardiogenic network would offer a roadmap to streamline cardiac progenitor output. METHODS AND RESULTS: To resolve gene ontology priorities within precursor transcriptomes, cardiogenic subpopulations were here generated according to either growth factor guidance or stage-specific biomarker sorting. Innate expression profiles were independently delineated through unbiased systems biology mapping, and cross-referenced to filter transcriptional noise unmasking a conserved progenitor motif (55 up- and 233 down-regulated genes. The streamlined pool of 288 genes organized into a core biological network that prioritized the "Cardiovascular Development" function. Recursive in silico deconvolution of the cardiogenic neighborhood and associated canonical signaling pathways identified a combination of integrated axes, CXCR4/SDF-1, Flk-1/VEGF and BMP2r/BMP2, predicted to synchronize cardiac specification. In vitro targeting of the resolved triad in embryoid bodies accelerated expression of Nkx2.5, Mef2C and cardiac-MHC, enhanced beating activity, and augmented cardiogenic yield. CONCLUSIONS: Transcriptome-wide dissection of a conserved progenitor profile thus revealed functional highways that coordinate cardiogenic maturation from a pluripotent ground state. Validating the bioinformatics algorithm established a strategy to rationally modulate cell fate, and optimize stem cell-derived cardiogenesis.

  9. Development of Regional Excel-Based Stormwater/Nutrient BMP Optimization Tool (Opti-Tool)

    Science.gov (United States)

    During 2014, EPA Region 1 contracted with Tetra Tech, Inc. to work with a regional technical Advisory Committee to develop an Excel-based stormwater/nutrient BMP optimization tool (Opti-Tool) using regional precipitation data and regionally calibrated BMP performance data from UN...

  10. SHH/BMP4 in the development of enteric nervous system in rats with anorectal malformation%SHH/BMP4信号在肛门直肠畸形肠神经系统发育中的作用研究

    Institute of Scientific and Technical Information of China (English)

    任红霞; 陈新新; 陈兰萍; 吴晓霞; 赵宝红

    2012-01-01

    目的 研究不同类型先天性肛门直肠畸形(ARM)胎鼠直肠末端肠神经系统(ENS)发育程度,探讨SHH/BMP4在其发育过程中的作用.方法 利用全反式维甲酸(ATRA)诱导大鼠产生肛门直肠畸形胚胎,孕20d剖宫取胎,应用免疫组织化学方法检测对照组和高、低位ARM组直肠末端神经元特异性烯醇化酶(NSE)及骨形态发生蛋白4(BMP4)的表达;反转录-聚合酶链反应(RT-PCR)方法检测各组直肠末端SHH/BMP4 mRNA的表达差异.结果 对照组肠壁肌间及黏膜下神经丛可见NSE和BMP4抗体染色阳性细胞,ARM高位组与ARM低位组、对照组比较,阳性细胞的平均光密度(MOD)值明显降低,差异均有统计学意义(P<0.05);ARM低位组和对照组相比,阳性细胞的MOD值略降低,差异有统计学意义(P<0.05).在ARM直肠末端SHH和BMP4基因表达呈正相关(P<0.01,r=0.884),对照组与ARM组相比较,BMP4和SHH mRNA的表达水平明显高于ARM组,差异均有统计学意义(P<0.05),不同ARM组之间比较,高位ARM组的表达水平显著减弱低于低位ARM组,差异也有统计学意义(P<0.05).结论 ①不同类型ARM胎鼠直肠末端ENS发育程度存在差异,不仅与闭锁的位置密切相关,还可能与BMP4基因的表达水平有关.②过量的ATRA可能抑制了SHH/BMP4信号表达,干扰了直肠ENS的正常发育.%Objective To study the development of enteric nervous system (ENS) in the rectal terminal of rat embryos with different types of congenital anorectal malformation (ARM) and to assess the role that SHH/Bmp4 signaling pathway played in the development of ENS. Methods The rat embryos with all-trans retinoic acid-induced ARM were harvested via caesarean section at week 20 during gestation. This entailed measurement of neuron specific enolase (NSE) and bone morphogenetic protein-4 (BMP4) expression in the rectal terminal in control group and rats with high-and low-ARM via immunohistochemistry assay. The difference in Shh/BMP

  11. BMP2 Regulation of CXCL12 Cellular, Temporal, and Spatial Expression is Essential During Fracture Repair

    Science.gov (United States)

    Myers, Timothy J; Longobardi, Lara; Willcockson, Helen; Temple, Joseph D; Tagliafierro, Lidia; Ye, Ping; Li, Tieshi; Esposito, Alessandra; Moats-Staats, Billie M; Spagnoli, Anna

    2016-01-01

    The cellular and humoral responses that orchestrate fracture healing are still elusive. Here we report that bone morphogenic protein 2 (BMP2)-dependent fracture healing occurs through a tight control of chemokine C-X-C motif-ligand-12 (CXCL12) cellular, spatial, and temporal expression. We found that the fracture repair process elicited an early site-specific response of CXCL12+-BMP2+ endosteal cells and osteocytes that was not present in unfractured bones and gradually decreased as healing progressed. Absence of a full complement of BMP2 in mesenchyme osteoprogenitors (BMP2cKO/+) prevented healing and led to a dysregulated temporal and cellular upregulation of CXCL12 expression associated with a deranged angiogenic response. Healing was rescued when BMP2cKO/+ mice were systemically treated with AMD3100, an antagonist of CXCR4 and agonist for CXCR7 both receptors for CXCL12. We further found that mesenchymal stromal cells (MSCs), capable of delivering BMP2 at the endosteal site, restored fracture healing when transplanted into BMP2cKO/+ mice by rectifying the CXCL12 expression pattern. Our in vitro studies showed that in isolated endosteal cells, BMP2, while inducing osteoblastic differentiation, stimulated expression of pericyte markers that was coupled with a decrease in CXCL12. Furthermore, in isolated BMP2cKO/cKO endosteal cells, high expression levels of CXCL12 inhibited osteoblastic differentiation that was restored by AMD3100 treatment or coculture with BMP2-expressing MSCs that led to an upregulation of pericyte markers while decreasing platelet endothelial cell adhesion molecule (PECAM). Taken together, our studies show that following fracture, a CXCL12+-BMP2+ perivascular cell population is recruited along the endosteum, then a timely increase of BMP2 leads to downregulation of CXCL12 that is essential to determine the fate of the CXCL12+-BMP2+ to osteogenesis while departing their supportive role to angiogenesis. Our findings have far

  12. BMP2 Regulation of CXCL12 Cellular, Temporal, and Spatial Expression is Essential During Fracture Repair.

    Science.gov (United States)

    Myers, Timothy J; Longobardi, Lara; Willcockson, Helen; Temple, Joseph D; Tagliafierro, Lidia; Ye, Ping; Li, Tieshi; Esposito, Alessandra; Moats-Staats, Billie M; Spagnoli, Anna

    2015-11-01

    The cellular and humoral responses that orchestrate fracture healing are still elusive. Here we report that bone morphogenic protein 2 (BMP2)-dependent fracture healing occurs through a tight control of chemokine C-X-C motif-ligand-12 (CXCL12) cellular, spatial, and temporal expression. We found that the fracture repair process elicited an early site-specific response of CXCL12(+)-BMP2(+) endosteal cells and osteocytes that was not present in unfractured bones and gradually decreased as healing progressed. Absence of a full complement of BMP2 in mesenchyme osteoprogenitors (BMP2(cKO/+)) prevented healing and led to a dysregulated temporal and cellular upregulation of CXCL12 expression associated with a deranged angiogenic response. Healing was rescued when BMP2(cKO/+) mice were systemically treated with AMD3100, an antagonist of CXCR4 and agonist for CXCR7 both receptors for CXCL12. We further found that mesenchymal stromal cells (MSCs), capable of delivering BMP2 at the endosteal site, restored fracture healing when transplanted into BMP2(cKO/+) mice by rectifying the CXCL12 expression pattern. Our in vitro studies showed that in isolated endosteal cells, BMP2, while inducing osteoblastic differentiation, stimulated expression of pericyte markers that was coupled with a decrease in CXCL12. Furthermore, in isolated BMP2(cKO/cKO) endosteal cells, high expression levels of CXCL12 inhibited osteoblastic differentiation that was restored by AMD3100 treatment or coculture with BMP2-expressing MSCs that led to an upregulation of pericyte markers while decreasing platelet endothelial cell adhesion molecule (PECAM). Taken together, our studies show that following fracture, a CXCL12(+)-BMP2(+) perivascular cell population is recruited along the endosteum, then a timely increase of BMP2 leads to downregulation of CXCL12 that is essential to determine the fate of the CXCL12(+)-BMP2(+) to osteogenesis while departing their supportive role to angiogenesis. Our findings have far

  13. Expression of active hBMP2 in transgenic tobacco plants.

    Science.gov (United States)

    Suo, Guangli; Chen, Bing; Zhang, Jingyu; Gao, Yuan; Wang, Xia; He, Zhengquan; Dai, Jianwu

    2006-12-01

    Bone morphogenetic protein 2 (BMP2) is important for bone tissue repair. The goal of this research is to construct a high level human BMP2 (hBMP2) expression system using transgenic tobacco plants as a bioreactor. Cauliflower mosaic virus (CaMV) 35S promoter, alfalfa mosaic virus (AMV) enhancer, tobacco mosaic virus (TMV) enhancer, matrix attachment regions (MARs) sequence, and "Kozak" sequence were used to construct recombinant expression vectors and the high-expression vectors were screened out through GUS-fusions assay. The promoter is the most important factor; double-CaMV 35S promoter is more effective than single promoter. The AMV or TMV enhancer is able to promote the foreign protein expression. After four-step purification, the activated hBMP2 (0.02% total soluble protein) was obtained. Our results suggested that the transgenic tobacco has great potential to be used as a bioreactor to produce hBMP2. PMID:16819603

  14. TLR4 initiated and cAMP mediated abrogation of bacterial invasion of the bladder

    OpenAIRE

    Song, Jeongmin; Bishop, Brian L.; Li, Guojie; Duncan, Matthew J.; Abraham, Soman N

    2007-01-01

    The remarkable resistance of the urinary tract to infection has been attributed to its physical properties and the innate immune responses triggered by pattern recognition receptors lining the tract. We report a distinct TLR4 mediated mechanism in bladder epithelial cells (BECs) that abrogates bacterial invasion, a necessary step for successful infection. Compared to controls, uropathogenic type 1 fimbriated Escherichia coli and Klebsiella pneumoniae invaded BECs of TLR4 mutant mice in 10-fol...

  15. BMP2, 4 and 6 and BMPR1B are altered from early stages of bovine cystic ovarian disease development.

    Science.gov (United States)

    Díaz, Pablo U; Hein, Gustavo J; Belotti, Eduardo M; Rodríguez, Fernanda M; Rey, Florencia; Amweg, Ayelén N; Matiller, Valentina; Baravalle, María E; Ortega, Hugo H; Salvetti, Natalia R

    2016-10-01

    Cystic ovarian disease (COD) is an important cause of subfertility in dairy cattle. Bone morphogenetic proteins (BMPs), mainly BMP2, BMP4 and BMP6, play a key role in female fertility. In this study, we hypothesized that an altered BMP system is associated with ovarian alterations contributing to COD pathogenesis. Therefore, we examined the expression of BMP2, BMP4 and BMP6 and BMP receptor 1B (BMPR1B) in the ovaries of animals with spontaneous or ACTH-induced COD, as well as during the development of the disease, in a model of follicular persistence induced by low doses of progesterone (at 5, 10 and 15 days of follicular persistence). Results showed changes in BMP2, BMP4 and BMP6 expression during folliculogenesis, in granulosa and theca cells in the COD groups, as well as at different stages of follicular persistence. Results also showed changes in BMPR1B expression in developing follicles in animals with COD, and at the initial stages of follicular persistence (P5). Comparison between groups showed significant differences, mainly in BMP4 and BMP6 expression, in granulosa and theca cells of different follicular categories. The expression of these BMPs also increased in cystic and persistent follicles, in relation to antral follicles of the control group. BMPR1B showed high expression in cystic follicles. Together, these results may indicate an alteration in BMPs, especially in BMP4 and BMP6, as well as in BMPR1B, which occurs early in folliculogenesis and incipiently during the development of COD, which could be a major cause of recurrence of this disease in cattle.Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/early/2016/08/01/REP-15-0315/suppl/DC1. PMID:27486268

  16. Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint

    Energy Technology Data Exchange (ETDEWEB)

    Nakadate, Yusuke [Shien-Lab, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Department of Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585 (Japan); Kodera, Yasuo; Kitamura, Yuka [Shien-Lab, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Tachibana, Taro [Department of Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585 (Japan); Tamura, Tomohide [Division of Thoracic Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Koizumi, Fumiaki, E-mail: fkoizumi@ncc.go.jp [Division of Thoracic Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2013-11-29

    Highlights: •Radiosensitization by PARG silencing was observed in multiple lung cancer cells. •PAR accumulation was enhanced by PARG silencing after DNA damage. •Radiation-induced G2/M arrest and checkpoint activation were impaired by PARG siRNA. -- Abstract: Poly(ADP-ribose) glycohydrolase (PARG) is a major enzyme that plays a role in the degradation of poly(ADP-ribose) (PAR). PARG deficiency reportedly sensitizes cells to the effects of radiation. In lung cancer, however, it has not been fully elucidated. Here, we investigated whether PARG siRNA contributes to an increased radiosensitivity using 8 lung cancer cell lines. Among them, the silencing of PARG induced a radiosensitizing effect in 5 cell lines. Radiation-induced G2/M arrest was largely suppressed by PARG siRNA in PC-14 and A427 cells, which exhibited significantly enhanced radiosensitivity in response to PARG knockdown. On the other hand, a similar effect was not observed in H520 cells, which did not exhibit a radiosensitizing effect. Consistent with a cell cycle analysis, radiation-induced checkpoint signals were not well activated in the PC-14 and A427 cells when treated with PARG siRNA. These results suggest that the increased sensitivity to radiation induced by PARG knockdown occurs through the abrogation of radiation-induced G2/M arrest and checkpoint activation in lung cancer cells. Our findings indicate that PARG could be a potential target for lung cancer treatments when used in combination with radiotherapy.

  17. Effect of rhBMP-2 Immobilized Anorganic Bovine Bone Matrix on Bone Regeneration

    Directory of Open Access Journals (Sweden)

    Jung-Bo Huh

    2015-07-01

    Full Text Available Anorganic bovine bone matrix (Bio-Oss® has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2 has been suggested to overcome this limitation of Bio-Oss®. In the present study, heparin-mediated rhBMP-2 was combined with Bio-Oss® in animal experiments to investigate bone formation performance; heparin was used to control rhBMP-2 release. Two calvarial defects (8 mm diameter were formed in a white rabbit model and then implanted or not (controls with Bio-Oss® or BMP-2/Bio-Oss®. The Bio-Oss® and BMP-2/Bio-Oss® groups had significantly greater new bone areas (expressed as percentages of augmented areas than the non-implanted controls at four and eight weeks after surgery, and the BMP-2/Bio-Oss® group (16.50 ± 2.87 (n = 6 had significantly greater new bone areas than the Bio-Oss® group (9.43 ± 3.73 (n = 6 at four weeks. These findings suggest that rhBMP-2 treated heparinized Bio-Oss® markedly enhances bone regeneration.

  18. Effect of rhBMP-2 Immobilized Anorganic Bovine Bone Matrix on Bone Regeneration.

    Science.gov (United States)

    Huh, Jung-Bo; Yang, June-Jip; Choi, Kyung-Hee; Bae, Ji Hyeon; Lee, Jeong-Yeol; Kim, Sung-Eun; Shin, Sang-Wan

    2015-01-01

    Anorganic bovine bone matrix (Bio-Oss®) has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been suggested to overcome this limitation of Bio-Oss®. In the present study, heparin-mediated rhBMP-2 was combined with Bio-Oss® in animal experiments to investigate bone formation performance; heparin was used to control rhBMP-2 release. Two calvarial defects (8 mm diameter) were formed in a white rabbit model and then implanted or not (controls) with Bio-Oss® or BMP-2/Bio-Oss®. The Bio-Oss® and BMP-2/Bio-Oss® groups had significantly greater new bone areas (expressed as percentages of augmented areas) than the non-implanted controls at four and eight weeks after surgery, and the BMP-2/Bio-Oss® group (16.50 ± 2.87 (n = 6)) had significantly greater new bone areas than the Bio-Oss® group (9.43 ± 3.73 (n = 6)) at four weeks. These findings suggest that rhBMP-2 treated heparinized Bio-Oss® markedly enhances bone regeneration. PMID:26184187

  19. Rac1 modulates the formation of primordial follicles by facilitating STAT3-directed Jagged1, GDF9 and BMP15 transcription in mice.

    Science.gov (United States)

    Zhao, Lihua; Du, Xinhua; Huang, Kun; Zhang, Tuo; Teng, Zhen; Niu, Wanbao; Wang, Chao; Xia, Guoliang

    2016-01-01

    The size of the primordial follicle pool determines the reproductive potential of mammalian females, and establishment of the pool is highly dependent on specific genes expression. However, the molecular mechanisms by which the essential genes are regulated coordinately to ensure primordial follicle assembly remain a mystery. Here, we show that the small GTPase Rac1 plays an indispensable role in controlling the formation of primordial follicles in mouse ovary. Employing fetal mouse ovary organ culture system, we demonstrate that disruption of Rac1 retarded the breakdown of germline cell cysts while Rac1 overexpression accelerated the formation of primordial follicles. In addition, in vivo inhibitor injection resulted in the formation of multi-oocyte follicles. Subsequent investigation showed that Rac1 induced nuclear import of STAT3 by physical binding. In turn, nuclear STAT3 directly activated the transcription of essential oocyte-specific genes, including Jagged1, GDF9, BMP15 and Nobox. Further, GDF9 and BMP15 regulated the translation of Notch2 via mTORC1 activation in pregranulosa cells. Overexression or addition of Jagged1, GDF9 and BMP15 not only reversed the effect of Rac1 disruption, but also accelerated primordial follicle formation via Notch2 signaling activation. Collectively, these results indicate that Rac1 plays important roles as a key regulator in follicular assembly. PMID:27050391

  20. Low dose BMP-2 treatment for bone repair using a PEGylated fibrinogen hydrogel matrix.

    Science.gov (United States)

    Ben-David, Dror; Srouji, Samer; Shapira-Schweitzer, Keren; Kossover, Olga; Ivanir, Eran; Kuhn, Gisela; Müller, Ralph; Seliktar, Dror; Livne, Erella

    2013-04-01

    Bone repair strategies utilizing resorbable biomaterial implants aim to stimulate endogenous cells in order to gradually replace the implant with functional repair tissue. These biomaterials should therefore be biodegradable, osteoconductive, osteoinductive, and maintain their integrity until the newly formed host tissue can contribute proper function. In recent years there has been impressive clinical outcomes for this strategy when using osteoconductive hydrogel biomaterials in combination with osteoinductive growth factors such as human recombinant bone morphogenic protein (hrBMP-2). However, the success of hrBMP-2 treatments is not without risks if the factor is delivered too rapidly and at very high doses because of a suboptimal biomaterial. Therefore, the aim of this study was to evaluate the use of a PEGylated fibrinogen (PF) provisional matrix as a delivery system for low-dose hrBMP-2 treatment in a critical size maxillofacial bone defect model. PF is a semi-synthetic hydrogel material that can regulate the release of physiological doses of hrBMP-2 based on its controllable physical properties and biodegradation. hrBMP-2 release from the PF material and hrBMP-2 bioactivity were validated using in vitro assays and a subcutaneous implantation model in rats. Critical size calvarial defects in mice were treated orthotopically with PF containing 8 μg/ml hrBMP-2 to demonstrate the capacity of these bioactive implants to induce enhanced bone formation in as little as 6 weeks. Control defects treated with PF alone or left empty resulted in far less bone formation when compared to the PF/hrBMP-2 treated defects. These results demonstrate the feasibility of using a semi-synthetic biomaterial containing small doses of osteoinductive hrBMP-2 as an effective treatment for maxillofacial bone defects. PMID:23375953

  1. Extracellular Ca2+ Promotes Odontoblastic Differentiation of Dental Pulp Stem Cells via BMP2-Mediated Smad1/5/8 and Erk1/2 Pathways.

    Science.gov (United States)

    Li, Shiting; Hu, Jing; Zhang, Gang; Qi, Wei; Zhang, Ping; Li, Pengfei; Zeng, Yong; Zhao, Wenfeng; Tan, Yinghui

    2015-09-01

    Ca(2+) is the main element of many pulp capping materials that are used to promote the regeneration of tertiary dentin, but the underlying molecular mechanism is not clear. In this study, we found that Ca(2+) increased the expression of the odontoblastic differentiation marker gene DSPP and promoted odontoblastic differentiation and mineralization of DPSCs, but inhibited ALP activity. Ca(2+) increases the expression of endogenous BMP2, which activates the Smad1/5/8 pathway and promotes the Smad1-Runx2 and Runx2-DSPP interaction in DPSCs. Inhibition of Smad1/5/8 with dorsomorphin partially blocked Runx2 activity; however, inhibition of the BMP2 receptor with Noggin nearly fully suppressed Runx2 activity. These results indicate that Ca(2+) promotes cell differentiation mainly via BMP2-mediated Smad-dependent and Smad-independent pathways. We then determined that the phosphorylation level of Erk1/2, but not JNK or p38, was significantly increased as a result of Ca(2+) stimulation. Blockage of Erk1/2 was found to inhibit Runx2 activity, indicating that Ca(2+) triggers the Erk1/2 pathway, which subsequently regulates Runx2 activity. In addition, inhibition of Erk1/2 differentially attenuated the phosphorylation levels of Smad1/5/8 and Smad2/3. Collectively, this study demonstrates that Ca(2+) activates the BMP2-mediated Smad1/5/8 and Erk1/2 pathways in DPSCs and that Smad1/5/8 and Erk1/2 signaling converge at Runx2 to control the odontoblastic differentiation of DPSCs. PMID:25656933

  2. Effect of rhBMP-2 Immobilized Anorganic Bovine Bone Matrix on Bone Regeneration

    OpenAIRE

    Jung-Bo Huh; June-Jip Yang; Kyung-Hee Choi; Ji Hyeon Bae; Jeong-Yeol Lee; Sung-Eun Kim; Sang-Wan Shin

    2015-01-01

    Anorganic bovine bone matrix (Bio-Oss®) has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been suggested to overcome this limitation of Bio-Oss®. In the present study, heparin-mediated rhBMP-2 was combined with Bio-Oss® in animal experiments to investigate bone formation performance; heparin was used to control rhBMP-2 release. Two calvarial defects (8 mm diameter) were fo...

  3. BMP7 Activates Brown Adipose Tissue and Reduces Diet-Induced Obesity Only at Subthermoneutrality

    Science.gov (United States)

    Boon, Mariëtte R.; van den Berg, Sjoerd A. A.; Wang, Yanan; van den Bossche, Jan; Karkampouna, Sofia; Bauwens, Matthias; De Saint-Hubert, Marijke; van der Horst, Geertje; Vukicevic, Slobodan; de Winther, Menno P. J.; Havekes, Louis M.; Jukema, J. Wouter; Tamsma, Jouke T.; van der Pluijm, Gabri; van Dijk, Ko Willems; Rensen, Patrick C. N.

    2013-01-01

    Background/Aims Brown adipose tissue (BAT) dissipates energy stored in triglycerides as heat via the uncoupling protein UCP-1 and is a promising target to combat hyperlipidemia and obesity. BAT is densely innervated by the sympathetic nervous system, which increases BAT differentiation and activity upon cold exposure. Recently, Bone Morphogenetic Protein 7 (BMP7) was identified as an inducer of BAT differentiation. We aimed to elucidate the role of sympathetic activation in the effect of BMP7 on BAT by treating mice with BMP7 at varying ambient temperature, and assessed the therapeutic potential of BMP7 in combating obesity. Methods and Results High-fat diet fed lean C57Bl6/J mice were treated with BMP7 via subcutaneous osmotic minipumps for 4 weeks at 21°C or 28°C, the latter being a thermoneutral temperature in which sympathetic activation of BAT is largely diminished. At 21°C, BMP7 increased BAT weight, increased the expression of Ucp1, Cd36 and hormone-sensitive lipase in BAT, and increased total energy expenditure. BMP7 treatment markedly increased food intake without affecting physical activity. Despite that, BMP7 diminished white adipose tissue (WAT) mass, accompanied by increased expression of genes related to intracellular lipolysis in WAT. All these effects were blunted at 28°C. Additionally, BMP7 resulted in extensive ‘browning’ of WAT, as evidenced by increased expression of BAT markers and the appearance of whole clusters of brown adipocytes via immunohistochemistry, independent of environmental temperature. Treatment of diet-induced obese C57Bl6/J mice with BMP7 led to an improved metabolic phenotype, consisting of a decreased fat mass and liver lipids as well as attenuated dyslipidemia and hyperglycemia. Conclusion Together, these data show that BMP7-mediated recruitment and activation of BAT only occurs at subthermoneutral temperature, and is thus likely dependent on sympathetic activation of BAT, and that BMP7 may be a promising tool to

  4. Abrogation of the Transactivation Activity of p53 by BCCIP Down-regulation*

    OpenAIRE

    Meng, Xiangbing; Yue, Jingyin; Liu, Zhihe; Shen, Zhiyuan

    2006-01-01

    The tumor suppression function of p53 is mostly conferred by its transactivation activity, which is inactivated by p53 mutations in ~50% of human cancers. In cancers harboring wild type p53, the p53 transactivation activity may be compromised by other mechanisms. Identifying the mechanisms by which wild type p53 transactivation activity can be abrogated may provide insights into the molecular etiology of cancers harboring wild type p53. In this report, we show that BCCIP, a BRCA2 and CDKN1A-i...

  5. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Henriksen, Z; Sørensen, O H;

    2004-01-01

    D), 100 nM Dex, and/or 100 ng/ml BMP-2. The osteoblast phenotype was assessed as alkaline phosphatase (AP) activity/staining, production of osteocalcin and procollagen type 1 (P1NP), parathyroid hormone (PTH)-induced cyclic adenosine mono-phosphate (cAMP) production, and in vitro mineralization. AP...... activity was increased by Dex, but not by BMP-2 treatment. P1NP production was decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with vitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels, but in...... osteoblastic cells with different phenotypic characteristics, and a selective activation of some of the most important genes and functions of the mature osteoblast can thus be performed in vitro....

  6. Crystallization of BMP receptor type IA bound to the antibody Fab fragment AbD1556

    International Nuclear Information System (INIS)

    The crystallization of BMP receptor type IA bound to the neutralizing antibody Fab fragment AbD1556 obtained by phage-display selection is reported. An antibody Fab fragment, AbD1556, was selected against the extracellular domain of BMP receptor type IA, which blocks the binding of BMP-2 to BMPR-IA and thereby neutralizes BMP-2 activity. To study the mechanism by which BMPR-IA is recognized and bound by the Fab fragment, the complex of AbD1556 bound to BMPR-IA was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group P21, with unit-cell parameters a = 89.32, b = 129.25, c = 100.24 Å, β = 92.27°

  7. Bone induction at physiological doses of BMP through localization by clay nanoparticle gels.

    Science.gov (United States)

    Gibbs, D M R; Black, C R M; Hulsart-Billstrom, G; Shi, P; Scarpa, E; Oreffo, R O C; Dawson, J I

    2016-08-01

    Bone Morphogenic Protein 2 (BMP2) can induce ectopic bone. This ability, which first motivated the widespread application of BMP2 in fracture healing and spinal arthrodesis has, more recently, been indicated as one of several serious adverse effects associated with the supra-physiological doses of BMP2 relied upon for clinical efficacy. Key to harnessing BMPs and other agents safely and effectively will be the ability to localize activity at a target site at substantially reduced doses. Clay (Laponite) nanoparticles can self assemble into gels under physiological conditions and bind growth factors for enhanced and localized efficacy. Here we show the ability to localize and enhance the activity of BMP2 to achieve ectopic bone formation at doses within the sub-microgram per ml range of concentrations sufficient to induce differentiation of responsive cell populations in vitro and at approximately 3000 fold lower than those employed in clinical practice. PMID:27209259

  8. Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer

    Directory of Open Access Journals (Sweden)

    E Ferreira

    2012-07-01

    Full Text Available Transplantation of mesenchymal stem cells (MSCs with electrotransferred bone morphogenetic protein-2 (BMP-2 transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications.

  9. Evolution, gene regulation and functional analysis of BMP2 in fish

    OpenAIRE

    Marques, Cátia Andreia Lourenço

    2013-01-01

    Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor β (TGFβ) superfamily with a central role in bone formation and mineralization. BMP2, a founding member of this family, has demonstrated remarkable osteogenic properties and is clinically used to promote bone repair and fracture healing. Lack of basic data on factors regulating BMP2 expression and activity have hampered a better understanding of its role in bone formation and bone-...

  10. BMP7 Activates Brown Adipose Tissue and Reduces Diet-Induced Obesity Only at Subthermoneutrality

    OpenAIRE

    Boon, M.R.; Berg, S.A.A. van den; Wang, Y; Bossche, J. van den; Karkampouna, S.; Bauwens, M.; Saint-Hubert, M. de; Horst, G; Vukicevic, S.; de Winther, M. P. J.; Havekes, L M; Jukema, J.W.; Tamsma, J. T.; Pluijm, G. van der; van Dijk, K W

    2013-01-01

    Background/Aims:Brown adipose tissue (BAT) dissipates energy stored in triglycerides as heat via the uncoupling protein UCP-1 and is a promising target to combat hyperlipidemia and obesity. BAT is densely innervated by the sympathetic nervous system, which increases BAT differentiation and activity upon cold exposure. Recently, Bone Morphogenetic Protein 7 (BMP7) was identified as an inducer of BAT differentiation. We aimed to elucidate the role of sympathetic activation in the effect of BMP7...

  11. Ion Mobility and Tandem Mass Spectrometry of Phosphatidylglycerol and Bis(monoacylglycerol)phosphate (BMP)

    OpenAIRE

    Hankin, Joseph A.; Murphy, Robert C.; Barkley, Robert M.; Gijón, Miguel A.

    2015-01-01

    The tandem mass spectrometry, ion mobility, and normal phase HPLC of isomeric phosphatidylglycerol (PG) and bis(monoacylglycerol)phosphate (BMP) have been investigated in this study with the objective of differentiating these unique classes of lipids. Measurement of ion mobility using the traveling wave method for negative molecular and product ions from isomeric PG and BMP yielded identical results, but different ion mobilities were observed for positive product ions arising from collision-i...

  12. 人骨形态发生蛋白-2基因的真核表达载体构建%Construction of the Eukaryotic Expression Vector for hBMP-gene

    Institute of Scientific and Technical Information of China (English)

    沈伟; 郭善一; 鲍秋野; 梁东春; 张镜宇

    2001-01-01

    Objective:To construct the recombinant bacmid DNA of human bone morpbogenetic protein-2 (hBMP-2) gene and ex press hBMP-2 by transfecting the insect cell line (Sf 9). Methods:The full-length coding region (1188 bp) for hBMP-2 in cluding signal peptide,inter pro-peptide and mature peptide was inserted to the multi-cloning sites of baculovirus vector pFastBacl which was controlled by the promoter pPolh, the recombinant plasmid (pFBBMP) was constructed, and pF- BBMP was transformed into E. coli DH 10 Bac. After transposition, the positive colonies were selected and the bacmid DNA was extracted. Confirmed by PCR,Sf 9 was transfected with recombinant bacmid DNA by liposome-mediated gene transfer method. Results: The recombinant bacmid DNA which can directly transfect Sf 9 was produced,and the expression produc tion (hBMP-2) was of 35.6% of total insect cell protein. Conclusion: The recombinant bacmid DNA is successfully con structed by baculovirus vector,and hBMP-2 highly expresses in insect cells.%目的:利用杆状病毒载体,构建可直接转染昆虫细胞Sf 9 的含人骨形态发生蛋白-2(hBMP-2)基因的重 组穿梭载体(bacmid DNA),最终获得重组蛋白。方法:将编码包含hBMP-2 N-端信号肽,中间前肽以及C-端成熟肽 共1188bp的cDNA片断,插入真核表达载体pFastBacl的多克隆位点,受控于pPolh启动子,构建成pFastBacl/ hBMP-2重组转移载体。重组子转化大肠杆菌DH10Bac。转座后,挑选阳性菌落,提取bacmid DNA,经PCR鉴定 后,以重组bacmid DNA转染Sf9细胞。收集重组病毒,扩大转染Sf9,表达产物行SDS-PAGE初步鉴定。结果:获 得了含hBMP-2全长cDNA片段的重组bacmid DNA和重组病毒,经SDS-PAGE证实在昆虫细胞中表达了分泌型 hBMP-2蛋白,经激光密度扫描仪扫描显示分泌性蛋白表达量占细胞蛋白总量的35.6%。结论:利用杆状病毒载 体成功构建了可用于直接转染昆虫细胞而

  13. Sustained release of BMP-2 in bioprinted alginate for osteogenicity in mice and rats.

    Directory of Open Access Journals (Sweden)

    Michelle T Poldervaart

    Full Text Available The design of bioactive three-dimensional (3D scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2 influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.

  14. Effect of a Novel Nonviral Gene Delivery of BMP-2 on Bone Healing

    Directory of Open Access Journals (Sweden)

    P. Schwabe

    2012-01-01

    Full Text Available Background. Gene therapeutic drug delivery approaches have been introduced to improve the efficiency of growth factors at the site of interest. This study investigated the efficacy and safety of a new nonviral copolymer-protected gene vector (COPROG for the stimulation of bone healing. Methods. In vitro, rat osteoblasts were transfected with COPROG + luciferase plasmid or COPROG + hBMP-2 plasmid. In vivo, rat tibial fractures were intramedullary stabilized with uncoated versus COPROG+hBMP-2-plasmid-coated titanium K-wires. The tibiae were prepared for biomechanical and histological analyses at days 28 and 42 and for transfection/safety study at days 2, 4, 7, 28, and 42. Results. In vitro results showed luciferase expression until day 21, and hBMP-2-protein was measured from day 2 – day 10. In vivo, the local application of hBMP-2-plasmid showed a significantly higher maximum load after 42 days compared to that in the control. The histomorphometric analysis revealed a significantly less mineralized periosteal callus area in the BMP-2 group compared to the control at day 28. The rt-PCR showed no systemic biodistribution of luciferase RNA. Conclusion. A positive effect on fracture healing by nonviral BMP-2 plasmid application from COPROG-coated implants could be shown in this study; however, the effect of the vector may be improved with higher plasmid concentrations. Transfection showed no biodistribution to distant organs and was considered to be safe.

  15. Scorpion Toxin, BmP01, Induces Pain by Targeting TRPV1 Channel

    Directory of Open Access Journals (Sweden)

    Md Abdul Hakim

    2015-09-01

    Full Text Available The intense pain induced by scorpion sting is a frequent clinical manifestation. To date, there is no established protocol with significant efficacy to alleviate the pain induced by scorpion envenomation. One of the important reasons is that, little information on pain-inducing compound from scorpion venoms is available. Here, a pain-inducing peptide (BmP01 has been identified and characterized from the venoms of scorpion (Mesobuthus martensii. In an animal model, intraplantar injection of BmP01 in mouse hind paw showed significant acute pain in wild type (WT mice but not in TRPV1 knock-out (TRPV1 KO mice during 30 min recording. BmP01 evoked currents in WT dorsal root ganglion (DRG neurons but had no effect on DRG neurons of TRPV1 KO mice. Furthermore, OPEN ACCESS Toxins 2015, 7 3672 BmP01 evoked currents on TRPV1-expressed HEK293T cells, but not on HEK293T cells without TRPV1. These results suggest that (1 BmP01 is one of the pain-inducing agents in scorpion venoms; and (2 BmP01 induces pain by acting on TRPV1. To our knowledge, this is the first report about a scorpion toxin that produces pain by targeting TRPV1. Identification of a pain-inducing compound may facilitate treating pain induced by scorpion envenomation.

  16. Osteogenesis Capacity of a Novel BMP/α-TCP Bioactive Composite Bone Cement

    Institute of Scientific and Technical Information of China (English)

    YANG Wei-zhong; ZHOU Da-li; YIN Shao-ya; YIN Guang-fu; GAO Li-da; ZHANG Yun

    2004-01-01

    To improve the osteogenesis ability of α-tricalcium phosphate (α-TCP) bone cement,a novel BMP/α-TCP composite bone cement was prepared.By measuring the setting time and compressive strength,the hydration characteristic of bone cement was evaluated.Animal experiments including histological observation,radiographic investigation as well as digital image analyses reveal the difference of osteogenesis ability among BMP,α-TCP bone cement and BMP/α-TCP composite bone cement.Results show that α-TCP bone cement possesses excellent hydration and setting properties as well as high mechanical property.Comparison experiments show that BMP/α-TCP composite bone cement has a stronger osteogenesis ability.The gross observation of the implant site does not exhibit any inflammation or necrosis.Histological analyses reveal that the material has good osteointegration with host bone,and new bone formation is detected within the materials,which are degrading.Strong osteogenesis ability of the composite is due to not only the excellent osteoconductive potential but also the osteoinductive potential contributed by active BMP releasing and the material degradation.Large skull defect could be well-healed by filling BMP/α-TCP composite bone cement.This novel material proves itself to be an absorbable and bioactive bone cement with an osteogenesis ability.

  17. BMP-2 and BMP-2/7 Heterodimers Conjugated to a Fibrin/Hyaluronic Acid Hydrogel in a Large Animal Model of Mild Intervertebral Disc Degeneration.

    Science.gov (United States)

    Peeters, Mirte; Detiger, Suzanne E L; Karfeld-Sulzer, Lindsay S; Smit, Theo H; Yayon, Avner; Weber, Franz E; Helder, Marco N

    2015-01-01

    Intervertebral disc (IVD) degeneration is etiologically associated with low back pain and is currently only treated in severe cases with spinal fusion. Regenerative medicine attempts to restore degenerated tissue by means of cells, hydrogels, and/or growth factors and can therefore be used to slow, halt, or reverse the degeneration of the IVD in a minimally invasive manner. Previously, the growth factors bone morphogenetic proteins 2 and 7 (BMP-2, -7) were shown to enhance disc regeneration, in vitro and in vivo. Since BMPs have only a short in vivo half-life, and to prevent heterotopic ossification, we evaluated the use of a slow release system for BMP-2 homodimers and BMP-2/7 heterodimers for IVD regeneration. BMP growth factors were conjugated to a fibrin/hyaluronic acid (FB/HA) hydrogel and intradiscally injected in a goat model of mild IVD degeneration to study safety and efficacy. Mild degeneration was induced in five lumbar discs of seven adult Dutch milk goats, by injections with the enzyme chondroitinase ABC. After 12 weeks, discs were treated with either FB/HA-hydrogel only or supplemented with 1 or 5 μg/mL of BMP-2 or BMP-2/7. BMPs were linked to the FB/HA hydrogels using a transglutaminase moiety, to be released through an incorporated plasmin cleavage site. After another 12 weeks, goats were sacrificed and discs were assessed using radiography, MRI T2* mapping, and biochemical and histological analyses. All animals maintained weight throughout the study and no heterotopic bone formation or other adverse effects were noted during follow-up. Radiographs showed significant disc height loss upon induction of mild degeneration. MRI T2* mapping showed strong and significant correlations with biochemistry and histology as shown before. Surprisingly, no differences could be demonstrated in any parameter between intervention groups. To our knowledge, this is the first large animal study evaluating BMPs conjugated to an FB/HA-hydrogel for the treatment of

  18. The toxic effects of Tris-(2,3-dibromopropyl)isocyanurate(TBC) on genes expression of bmp2b and bmp4 of zebrafish embryos

    Institute of Scientific and Technical Information of China (English)

    JIA Wan-jun

    2016-01-01

    We exposed zebrafish embryos to Tris-(2,3-dibromopropyl)isocyanurate(TBC)at the concentration of 20ppb, 100ppb, 400ppb, 1000ppb for 120h and 0.1%DMSO was set as the control group. Bmp2b and bmp4 were chosen perform RT-PCR to determine their genes expression level. The results showed that, TBC influenced their genes expression level in some extent and it significantly raised the genes expression level at the concentration of 20ppb.

  19. Differential expression of a BMP4 reporter allele in anterior fungiform versus posterior circumvallate taste buds of mice

    Directory of Open Access Journals (Sweden)

    Barlow Linda A

    2010-10-01

    Full Text Available Abstract Background Bone Morphogenetic Protein 4 (BMP4 is a diffusible factor which regulates embryonic taste organ development. However, the role of BMP4 in taste buds of adult mice is unknown. We utilized transgenic mice with LacZ under the control of the BMP4 promoter to reveal the expression of BMP4 in the tongues of adult mice. Further we evaluate the pattern of BMP4 expression with that of markers of specific taste bud cell types and cell proliferation to define and compare the cell populations expressing BMP4 in anterior (fungiform papillae and posterior (circumvallate papilla tongue. Results BMP4 is expressed in adult fungiform and circumvallate papillae, i.e., lingual structures composed of non-taste epithelium and taste buds. Unexpectedly, we find both differences and similarities with respect to expression of BMP4-driven ß-galactosidase. In circumvallate papillae, many fusiform cells within taste buds are BMP4-ß-gal positive. Further, a low percentage of BMP4-expressing cells within circumvallate taste buds is immunopositive for markers of each of the three differentiated taste cell types (I, II and III. BMP4-positive intragemmal cells also expressed a putative marker of immature taste cells, Sox2, and consistent with this finding, intragemmal cells expressed BMP4-ß-gal within 24 hours after their final mitosis, as determined by BrdU birthdating. By contrast, in fungiform papillae, BMP4-ß-gal positive cells are never encountered within taste buds. However, in both circumvallate and fungiform papillae, BMP4-ß-gal expressing cells are located in the perigemmal region, comprising basal and edge epithelial cells adjacent to taste buds proper. This region houses the proliferative cell population that gives rise to adult taste cells. However, perigemmal BMP4-ß-gal cells appear mitotically silent in both fungiform and circumvallate taste papillae, as we do not find evidence of their active proliferation using cell cycle immunomarkers

  20. BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis.

    Science.gov (United States)

    Shea, Colleen M; Edgar, Cory M; Einhorn, Thomas A; Gerstenfeld, Louis C

    2003-12-15

    The molecular mechanisms by which bone morphogenetic proteins (BMPs) promote skeletal cell differentiation were investigated in the murine mesenchymal stem cell line C3H10T1/2. Both BMP-7 and BMP-2 induced C3H10T1/2 cells to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media. Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes. Subsequent experiments using BMP-7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells. Continuous treatment with BMP-7 increased the expression of Sox9, Msx 2, and c-fos during the periods of chondrogenic differentiation after which point their expression decreased. In contrast, Dlx 5 expression was induced by BMP-7 treatment and remained elevated throughout the time-course of skeletal cell differentiation. Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP-7. The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP-7-regulated expression of other morphogenetic factors. An examination of the expression of Wnt, transforming growth factor-beta (TGF-beta), and the hedgehog family of morphogens showed that Wnt 5b, Wnt 11, BMP-4, growth and differentiation factor-1 (GDF-1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells. Wnt 11, BMP-4, and GDF-1 expression were inhibited by BMP-7 treatment in a dose-dependent manner while Wnt 5b and Shh were selectively induced by BMP-7 during the period of chondrogenic

  1. A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells.

    Science.gov (United States)

    Yigit, Burcu; Halibozek, Peter J; Chen, Shih-Shih; O'Keeffe, Michael S; Arnason, Jon; Avigan, David; Gattei, Valter; Bhan, Atul; Cen, Osman; Longnecker, Richard; Chiorazzi, Nicholas; Wang, Ninghai; Engel, Pablo; Terhorst, Cox

    2016-05-01

    The signaling lymphocyte activation molecule family [SLAMF] of cell surface receptors partakes in both the development of several immunocyte lineages and innate and adaptive immune responses in humans and mice. For instance, the homophilic molecule SLAMF6 (CD352) is in part involved in natural killer T cell development, but also modulates T follicular helper cell and germinal B cell interactions. Here we report that upon transplantation of a well-defined aggressive murine B220+CD5+ Chronic Lymphocytic Leukemia (CLL) cell clone, TCL1-192, into SCID mice one injection of a monoclonal antibody directed against SLAMF6 (αSlamf6) abrogates tumor progression in the spleen, bone marrow and blood. Similarly, progression of a murine B cell lymphoma, LMP2A/λMyc, was also eliminated by αSlamf6. But, surprisingly, αSLAMF6 neither eliminated TCL1-192 nor LMP2A/λMyc cells, which resided in the peritoneal cavity or omentum. This appeared to be dependent upon the tumor environment, which affected the frequency of sub-populations of the TCL1-192 clone or the inability of peritoneal macrophages to induce Antibody Dependent Cellular Cytotoxicity (ADCC). However, co-administering αSlamf6 with the Bruton tyrosine kinase (Btk) inhibitor, ibrutinib, synergized to efficiently eliminate the tumor cells in the spleen, bone marrow, liver and the peritoneal cavity. Because an anti-human SLAMF6 mAb efficiently killed human CLL cells in vitro and in vivo, we propose that a combination of αSlamf6 with ibrutinib should be considered as a novel therapeutic approach for CLL and other B cell tumors. PMID:27029059

  2. Combination therapy with BMP-2 and a systemic RANKL inhibitor enhances bone healing in a mouse critical-sized femoral defect.

    Science.gov (United States)

    Bougioukli, Sofia; Jain, Ashish; Sugiyama, Osamu; Tinsley, Brian A; Tang, Amy H; Tan, Matthew H; Adams, Douglas J; Kostenuik, Paul J; Lieberman, Jay R

    2016-03-01

    Recombinant human BMP-2 (rhBMP-2) is a potent osteoinductive agent, but has been associated not only with bone formation, but also osteoclastogenesis and bone resorption. Osteoprotegerin (OPG) is a RANKL inhibitor that blocks differentiation and function of osteoclasts. We hypothesized that the combination of local BMP-2 (recombinant protein or a product of gene therapy) plus systemic OPG-Fc is more effective than BMP-2 alone in promoting bone repair. To test this hypothesis we used a mouse critical-sized femoral defect model. Col2.3eGFP (osteoblastic marker) male mice were treated with rhBMP-2 (group I), rhBMP-2 and systemic OPG (group II), rhBMP-2 and delayed administration of OPG (group III), mouse BM cells transduced with a lentiviral vector containing the BMP-2 gene (LV-BMP-2; group IV), LV-BMP-2 and systemic OPG (group V), a carrier alone (group VI) and administration of OPG alone (group VII). All bone defects treated with BMP-2 (alone or combined with OPG) healed, whereas minimal bone formation was noted in animals treated with the carrier alone or OPG alone. MicroCT analysis showed that bone volume (BV) in rhBMP-2+OPG and LV-BMP-2+OPG groups was significantly higher compared to rhBMP-2 alone (p<0.01) and LV-BMP-2 alone (p<0.001). Similar results were observed in histomorphometry, with rhBMP-2 alone defects exhibiting significantly lower bone area (B.Ar) compared to rhBMP-2+OPG defects (p<0.005) and LV-BMP-2 defects having a significantly lower B.Ar compared to all BMP-2+OPG treated groups (p≤0.01). TRAP staining demonstrated a major osteoclast response in the groups that did not receive OPG (rhBMP-2, LV-BMP-2 and sponge alone) beginning as early as 7days post-operatively. In conclusion, we demonstrated that locally delivered BMP-2 (recombinant protein or gene therapy) in combination with systemically administered OPG improved bone healing compared to BMP-2 alone in a mouse critical-sized bone defect. These data indicate that osteoclasts can diminish

  3. Preconditioning Human Mesenchymal Stem Cells with a Low Concentration of BMP2 Stimulates Proliferation and Osteogenic Differentiation In Vitro

    DEFF Research Database (Denmark)

    Lysdahl, Helle; Baatrup, Anette; Foldager, Casper Bindzus; Bünger, Cody

    2014-01-01

    treatment strategy in which human bone marrow-derived mesenchymal stem cells (hMSCs) are preconditioned with low concentrations of BMP2 for a short time in vitro. hMSCs in suspension were stimulated for 15 min with 10 and 20 ng/mL of BMP2. After the BMP2 was removed, the cells were seeded and cultured in...... maturation of hMSCs. This implies that preconditioning with BMP2 might be more effective at inducing proliferation and osteogenic differentiation of hMSCs than continuous stimulation. Preconditioning with BMP2 could benefit the clinical application of BMP2 since side effects from high-dose treatments could...

  4. Influence of p53 abrogation on penclomedine and radiation-induced cell kill

    International Nuclear Information System (INIS)

    Purpose: Penclomedine [(3.5-dichloro-2,4-dimethoxy-6-trichloromethyl)pyridine], a synthetic pyridine derivative, has documented activity against human and murine tumor cells and is currently being investigated in Phase I clinical trials. Cells exposed to Penclomedine reveal abundant chromosomal aberrations suggesting that its mechanism of action may be through DNA alkylation. Since p53 is considered to be a DNA damage responsive element, we investigated the influence of p53 abrogation on Penclomedine sensitivity, on sensitivity to ionizing radiation and to both modalities combined. Material and Methods: Three malignant cell lines, derived from a human colorectal carcinoma, were used in this study: RKO cells which contain wild-type p53 alleles, RKO cells transfected with an over expressed mutant p53 transgene (p53.13) and RKO cells transfected with an over expressed HPV-16 E6 gene (RC 10.03) resulting in abrogation of normal p53 function. Cells were exposed to increasing concentrations of Penclomedine (0-120 μg/ml) for variable periods of time (4, 6, 8, 12, 24 hours) as well as to graded doses of ionizing radiation (0-8 Gy), either alone or following 24 hour exposure to Penclomedine. Cell viability was monitored by the colony-forming assay and Trypan blue exclusion. Cell numbers were counted using a hemocytometer and Coulter counter. Both asynchronous and synchronized cell populations were studied with synchrony attained by mitotic shake off. S phase cell determination was by anti-BrdU immuno labeling. Results: Each cell line exhibited a dose and time-dependent reduction in cell viability following Penclomedine treatment. However, RC 10.03 cells showed a dramatic enhancement in cell kill compared to the parental RKO cells (P<0.05). No significant difference in radiation sensitivity amongst the 3 cell lines was observed when cells were exposed either as an asynchronous population or in early G1. Conclusion: The results of this study suggest that radiation

  5. Anti-Müllerian Hormone Signaling Regulates Epithelial Plasticity and Chemoresistance in Lung Cancer.

    Science.gov (United States)

    Beck, Tim N; Korobeynikov, Vladislav A; Kudinov, Alexander E; Georgopoulos, Rachel; Solanki, Nehal R; Andrews-Hoke, Magda; Kistner, Timothy M; Pépin, David; Donahoe, Patricia K; Nicolas, Emmanuelle; Einarson, Margret B; Zhou, Yan; Boumber, Yanis; Proia, David A; Serebriiskii, Ilya G; Golemis, Erica A

    2016-07-19

    Anti-Müllerian hormone (AMH) and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-β)/bone morphogenetic protein (BMP) signaling and epithelial-mesenchymal transition (EMT) in lung cancer. AMH is a TGF-β/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3) also used by the type II receptor for BMP (BMPR2). AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB) and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC). AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90) inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-β/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT. PMID:27396341

  6. BMP4-mediated brown fat-like changes in white adipose tissue alter glucose and energy homeostasis

    OpenAIRE

    Qian, Shu-Wen; Tang, Yan; Li, Xi; Liu, Yuan; Zhang, You-you; Huang, Hai-yan; Xue, Rui-Dan; Yu, Hao-Yong; Guo, Liang; Gao, Hui-Di; Liu, Yan; Sun, Xia; Li, Yi-ming; Jia, Wei-Ping; Tang, Qi-Qun

    2013-01-01

    Expression of bone morphogenetic protein 4 (BMP4) in adipocytes of white adipose tissue (WAT) produces “white adipocytes” with characteristics of brown fat and leads to a reduction of adiposity and its metabolic complications. Although BMP4 is known to induce commitment of pluripotent stem cells to the adipocyte lineage by producing cells that possess the characteristics of preadipocytes, its effects on the mature white adipocyte phenotype and function were unknown. Forced expression of a BMP...

  7. Non-stringent tissue-source requirements for BMP ligand expression in regulation of body size in Caenorhabditis elegans

    OpenAIRE

    Savage-Dunn, Cathy; Yu, Ling; Gill, Kwesi; Awan, Muhammad; Fernando, Thilini

    2011-01-01

    In Caenorhabditis elegans, the Bone Morphogenetic Protein (BMP)-related ligand Dpp- and BMP-like-1 (DBL-1) regulates body size by promoting the larval and adult growth of the large epidermal syncytium hyp7 without affecting cell division. This system provides an excellent model for dissecting the growth-promoting activities of BMP ligands, since in this context the growth and differentiation functions of DBL-1 are naturally uncoupled. dbl-1 is expressed primarily in neurons and the DBL-1 liga...

  8. Sustained presentation of BMP-2 enhances osteogenic differentiation of human adipose-derived stem cells in gelatin hydrogels.

    Science.gov (United States)

    Samorezov, Julia E; Headley, Emma B; Everett, Christopher R; Alsberg, Eben

    2016-06-01

    Human adipose-derived stem cells (hASCs) show great potential for healing bone defects. Bone morphogenetic protein-2 (BMP-2) has been reported to stimulate their osteogenic differentiation both in vitro and in vivo. Here, methacrylated gelatin (GelMA) hydrogels were evaluated as a system to deliver BMP-2 to encapsulated hASCs from two different donors, and BMP-2 delivered from the hydrogels was compared to BMP-2 presented exogenously in culture media. GelMA hydrogels were shown to provide sustained, localized presentation of BMP-2 due to electrostatic interactions between the growth factor and biomaterial after an initial burst release. Both donors exhibited similar responses to the loaded and exogenous growth factor; BMP-2 from the hydrogels had a statistically significant effect on hASC osteogenic differentiation compared to exogenous BMP-2. Expression of alkaline phosphatase was accelerated, and cells in hydrogels with loaded BMP-2 deposited more calcium at one, two, and four weeks than cells without BMP-2 or with the growth factor presented in the media. There were no statistically significant differences in calcium content between groups with 25, 50, or 100 µg/mL loaded BMP-2, suggesting that using a lower growth factor dose may be as effective as a higher loading amount in this system. Taken together, these findings suggest that controlled delivery of BMP-2 from the GelMA enhances its osteogenic bioactivity compared to free growth factor presented in the media. Thus, the GelMA system is a promising biomaterial for BMP-2-mediated hASC osteogenesis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1387-1397, 2016. PMID:26822338

  9. Polymorphism of BMP2 Gene Associated with Growth Traits in Guizhou Semi-Fine Wool Sheep

    OpenAIRE

    Xiao Yun Shen; Xin Wang; Wen Ting Li; Yong Jun Li; Li Juan Li

    2012-01-01

    Bone Morphogenetic Protein 2 (BMP2) plays a crucial role in bone growth. The objective of this study was to investigate variations in sheep BMP2 gene and their associations with growth traits in 320 Guizhou Semi-Fine Wool sheep. Five fragments of BMP-2 gene were investigated only exon2 region of BMP-2 gene showed polymorphism after PCR-SSCP and DNA sequencing methods. There was one G>A (g. 273 G>A) mutation located in nucleotide position of GenBank Accession No. EU854586 which constructed thr...

  10. Abrogation of Early Apoptosis Does Not Alter Late Inhibition of Hippocampal Neurogenesis After Irradiation

    International Nuclear Information System (INIS)

    Purpose: Irradiation of the adult brain results in acute apoptosis of neural progenitors and vascular endothelial cells, as well as late dysfunction of neural progenitors and inhibition of neurogenesis. We sought to determine whether the early apoptotic response has a causative role in late inhibition of neurogenesis after cranial irradiation. Methods and Materials: Using a genetic approach with p53 and smpd1 transgenic mice and a pharmacologic approach with basic fibroblast growth factor (bFGF) to abrogate the early apoptotic response, we evaluated the late inhibition of neurogenesis in the hippocampal dentate gyrus after cranial irradiation. Results: In dentate gyrus, subgranular neural progenitors underwent p53-dependent apoptosis within 24 h after irradiation. Despite a near abrogation of neural progenitor apoptosis in p53-/- mice, the reduction in newborn neurons in dentate gyrus at 9 weeks after irradiation in p53-/- mice was not different from that observed in wildtype controls. Endothelial cell apoptosis after radiation is mediated by membrane damage initiated by activation of acid sphingomyelinase (ASMase). Deletion of the smpd1 gene (which encodes ASMase) attenuated the apoptotic response of endothelial cells. At 9 weeks after irradiation, the inhibition of hippocampal neurogenesis was not rescued by ASMase deficiency. Intravenous administration of bFGF protected both endothelial cells and neural progenitors against radiation-induced apoptosis. There was no protection against inhibition of neurogenesis at 9 weeks after irradiation in bFGF-treated mice. Conclusion: Early apoptotic death of neural progenitors, endothelial cells, or both does not have a causative association with late inhibition of neurogenesis after irradiation.

  11. HCV NS5A abrogates p53 protein function by interfering with p53-DNA binding

    Institute of Scientific and Technical Information of China (English)

    Guo-Zhong Gong; Yong-Fang Jiang; Yan He; Li-Ying Lai; Ying-Hua Zhu; Xian-Shi Su

    2004-01-01

    AIM: To evaluate the inhibition effect of HCV NS5A on p53 transactivation on p21 promoter and explore its possible mechanism for influencing p53 function.METHODS: p53 function of transactivation on p21 promoter was studied with a luciferase reporter system in which the luciferase gene is driven by p21 promoter, and the p53-DNA binding ability was observed with the use of electrophoretic mobility-shift assay (EMSA). Lipofectin mediated p53 or HCV NS5A expression vectors were used to transfect hepatoma cell lines to observe whether HCV NS5A could abrogate the binding ability of p53 to its specific DNA sequence and p53 transactivation on p21 promoter.Western blot experiment was used for detection of HCV NS5A and p53 proteins expression.RESULTS: Relative luciferase activity driven by p21 promoter increased significantly in the presence of endogenous p53 protein. Compared to the control group, exogenous p53 protein also stimulated p21 promoter driven luciferase gene expression in a dose-dependent way. HCV NS5A protein gradually inhibited both endogenous and exogenous p53 transactivation on p21 promoter with increase of the dose of HCV NS5A expression plasmid. By the experiment of EMSA, we could find p53 binding to its specific DNA sequence and, when co-transfected with increased dose of HCV NS5A expression vector, the p53 binding affinity to its DNA gradually decreased and finally disappeared. Between the Huh 7 cells transfected with p53 expression vector alone or co-transfected with HCV NS5A expression vector, there was no difference in the p53 protein expression.CONCLUSION: HCV NS5A inhibits p53 transactivation on p21 promoter through abrogating p53 binding affinity to its specific DNA sequence. It does not affect p53 protein expression.

  12. Local Application of BMP-2 Specific Plasmids in Fibrin Glue does not Promote Implant Fixation

    Directory of Open Access Journals (Sweden)

    Plank Christan

    2011-07-01

    Full Text Available Abstract Background BMP-2 is known to accelerate fracture healing and might also enhance osseointegration and implant fixation. Application of recombinant BMP-2 has a time-limited effect. Therefore, a gene transfer approach with a steady production of BMP-2 appears to be attractive. The aim of this study was to examine the effect of locally applied BMP-2 plasmids on the bone-implant integration in a non-weight bearing rabbit tibia model using a comparatively new non-viral copolymer-protected gene vector (COPROG. Methods Sixty rabbits were divided into 4 groups. All of them received nailing of both tibiae. The verum group had the nails inserted with the COPROG vector and BMP-2 plasmids using fibrin glue as a carrier. Controls were a group with fibrin glue only and a blank group. After 28 and 56 days, these three groups were sacrificed and one tibia was randomly chosen for biomechanical testing, while the other tibia underwent histomorphometrical examination. In a fourth group, a reporter-gene was incorporated in the fibrin glue instead of the BMP-2 formula to prove that transfection was successful. Results Implant fixation strength was significantly lower after 28 and 56 days in the verum group. Histomorphometry supported the findings after 28 days, showing less bone-implant contact. In the fourth group, successful transfection could be confirmed by detection of the reporter-gene in 20 of 22 tibiae. But, also systemic reporter-gene expression was found in heterotopic locations, showing an undesired spreading of the locally applied gene formula. Conclusion Our results underline the transfecting capability of this vector and support the idea that BMP-2 might diminish osseointegration. Further studies are necessary to specify the exact mechanisms and the systemic effects.

  13. Regulating the osteogenic function of rhBMP 2 by different titanium surface properties.

    Science.gov (United States)

    Xiao, Ming; Biao, Meina; Chen, Yangmei; Xie, Meiju; Yang, Bangcheng

    2016-08-01

    Bone morphogenetic protein 2 (BMP-2) is important for regulating the osteogenic differentiation of mesenchymal stem cells and the response of bone tissue. It adsorbs on the surface of biomedical implants immediately and plays a role of mediator between the materials surfaces and the host cells. Studies usually connect the material surface properties and the new bone formation directly. However, interaction between the adsorbed BMP-2 on the implant surface and the cells in the tissue is the key to explaining the osteogenic properties of the material. So, in this article, we investigated the conformational and functional changes induced by the surface modified titanium metals. We found that the α-helix and β-sheet structure of rhBMP-2 can be well maintained on the anodic oxidation treated titanium surface. The osteogenic function of rhBMP-2 can sustain for a relatively long time even though there is less amount adhere to the surface compared with that on the acid alkali treated titanium. Surface properties, especially the morphology enable a larger amount of rhBMP-2 to adsorb to the surface of the acid alkali treated titanium, but the conformation of the protein is severely influenced. The percentage of α-helix structure is also significantly decreased so that the efficacy of rhBMP-2 is only maintained in the early time. This study indicated that different surface modification of the surface could regulate the structure of rhBMP-2 and then further influence its osteogenic function. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1882-1893, 2016. PMID:26991341

  14. Osseointegration of titanium implants by addition of recombinant bone morphogenetic protein 2 (rhBMP-2)

    Energy Technology Data Exchange (ETDEWEB)

    Lichtinger, T.K.; Mueller, R.T.; Schuermann, N.; Oldenburg, M. [Essen Univ. (Germany). Dept. of Orthopaedic Surgery; Wiemann, M. [Inst. of Physiology, Univ. of Essen (Germany); Chatzinikolaidou, M.; Jennissen, H.P. [Inst. of Physiological Chemistry, Univ. of Essen (Germany); Rumpf, H.M.

    2001-12-01

    The osseointegration of long-term implants is often incomplete such that gaps remain between the implant surface and the surrounding hard tissue. This study examines the effect of soluble recombinant human bone morphogenic protein 2 (rhBMP-2) on gap healing and osseous integration. The effect of a single, intraoperative application of soluble rhBMP-2 on the formation of new bone around titanium implants was studied. A total of 8 titanium-alloy cylinders (Ti-6Al-4V) with a plasma spray coating (TPS; 400 {mu}m thickness) were implanted into femoral condyles of mature sheep: rhBMP-2 solution (1 {mu}g) was pipetted into the 1 mm wide cleft around 4 implants; 4 further implants served as rhBMP-2-free controls. Two of these controls exhibited an additional calciumphosphate-coating. The cleft around the implants served as testing zone to study the formation of new bone by microradiographical and histological analyses. The follow-up periods were 4 and 9 weeks, respectively. A significant amount of new bone contacting the implants' surface was detected where rhBMP-2-solution had been used: In 50% a circumferential osseoinduction occurred within 4 weeks and a nearly complete osseointegration was observed after 9 weeks. In all cases bone formation was exaggerated and filled the spongiosa with compact bone. Time matched TPS-controls and controls with calciumphosphate coating showed no notable formation of new bone. The results suggest that a single administration of soluble rhBMP-2 into a bone cavity can augment bone formation and also osseointegration of titanium implants. Further investigations based on these findings are necessary to develop long-term implants (e.g. joint replacements) with rhBMP-2-biocoating for humans. (orig.)

  15. Bone morphogenetic protein-9 suppresses growth of myeloma cells by signaling through ALK2 but is inhibited by endoglin

    International Nuclear Information System (INIS)

    Multiple myeloma is a malignancy of plasma cells predominantly located in the bone marrow. A number of bone morphogenetic proteins (BMPs) induce apoptosis in myeloma cells in vitro, and with this study we add BMP-9 to the list. BMP-9 has been found in human serum at concentrations that inhibit cancer cell growth in vitro. We here show that the level of BMP-9 in serum was elevated in myeloma patients (median 176 pg/ml, range 8–809) compared with healthy controls (median 110 pg/ml, range 8–359). BMP-9 was also present in the bone marrow and was able to induce apoptosis in 4 out of 11 primary myeloma cell samples by signaling through ALK2. BMP-9-induced apoptosis in myeloma cells was associated with c-MYC downregulation. The effects of BMP-9 were counteracted by membrane-bound (CD105) or soluble endoglin present in the bone marrow microenvironment, suggesting a mechanism for how myeloma cells can evade the tumor suppressing activity of BMP-9 in multiple myeloma

  16. Convergence of bone morphogenetic protein and laminin-1 signaling pathways promotes proliferation and colony formation by fetal mouse pancreatic cells

    International Nuclear Information System (INIS)

    We previously reported that bone morphogenetic proteins (BMPs), members of the transforming growth factor superfamily, together with the basement membrane glycoprotein laminin-1 (Ln-1), promote proliferation of fetal pancreatic cells and formation of colonies containing peripheral insulin-positive cells. Here, we further investigate the cross-talk between BMP and Ln-1 signals. By RT-PCR, receptors for BMP (BMPR) (excepting BMPR-1B) and Ln-1 were expressed in the fetal pancreas between E13.5 and E17.5. Specific blocking antibodies to BMP-4 and -6 and selective BMP antagonists partially inhibited colony formation by fetal pancreas cells. Colony formation induced by BMP-6 and Ln-1 was completely abolished in a dose-dependent manner by blocking Ln-1 binding to its α6 integrin and α-dystroglycan receptors or by blocking the Ln-1 signaling molecules, phosphatidyl-inositol-3-kinase (P13K) and MAP kinase kinase-1. These results demonstrate a convergence of BMP and Ln-1 signaling through P13K and MAP kinase pathways to induce proliferation and colony formation in E15.5 fetal mouse pancreatic cells

  17. Vasohibin-1 Expression Is Regulated by Transforming Growth Factor-β/Bone Morphogenic Protein Signaling Pathway Between Tumor-Associated Macrophages and Pancreatic Cancer Cells

    Science.gov (United States)

    Seppänen, Hanna; Kauttu, Tuuli; Vainionpää, Sanna; Ye, Yingjiang; Mustonen, Harri

    2013-01-01

    Vasohibin-1 has been detected in endothelial cells as an intrinsic angiogenesis inhibitor. Both tumor-associated macrophages (TAMs) and transforming growth factor-β (TGF-β)/bone morphogenic protein (BMP) signaling have been reported to promote angiogenesis in cancer. However, whether vasohibin-1 expression is regulated by TGF-β/BMP signaling between TAMs and cancer cells remains unclear. The expression of TGF-β1, TGF-β2, BMP-4, and BMP-7 in TAMs and the expression of vasohibin-1, vascular endothelial growth factor-A (VEGF-A), and VEGF-C in two pancreatic cancer cell lines (a nonmetastatic cell line Panc-1 and a distant metastatic cell line HPAF-II) were measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). The TGF-β receptor 1 and BMP receptor 1 were inhibited by the inhibitor SB-431542 and LDN193189, respectively. Thereafter, vasohibin-1, VEGF-A, and VEGF-C expression was detected by real-time RT-PCR. We found that the expression of TGF-β1, TGF-β2, BMP-4, and BMP-7 was upregulated in TAMs cocultured with pancreatic cancer cells. Vasohibin-1, VEGF-A, and VEGF-C mRNA expression in pancreatic cancer cells was upregulated by TAMs. Vasohibin-1 expression in pancreatic cancer cells cocultured with TAMs was upregulated significantly when TGF-β receptors or BMP receptors were inhibited, but VEGF-C expression was downregulated. Therefore, Vasohibin-1 expression is regulated by the TGF-β/BMP signaling between TAMs and pancreatic cancer cells. These results might shed a new light on the antiangiogenesis therapy in the pancreatic cancer. PMID:23651239

  18. No advantage to rhBMP-2 in addition to autogenous graft for fracture nonunion.

    Science.gov (United States)

    Takemoto, Richelle; Forman, Jordanna; Taormina, David P; Egol, Kenneth A

    2014-06-01

    Bone morphogenetic proteins are a necessary component of the fracture healing cascade. Few studies have delineated the efficacy of iliac crest bone graft and recombinant human bone morphogenetic protein 2 (rhBMP-2), especially, in comparison with the gold standard treatment of nonunion, which is autogenous bone graft alone. This study compared the outcome of patients with fracture nonunion treated with autogenous bone graft plus rhBMP-2 adjuvant vs patients treated with autogenous bone graft alone. A total of 118 consecutive patients who were to undergo long bone nonunion surgery with autogenous bone graft (50) or autogenous bone graft plus rhBMP-2 (68) were identified. Surgical intervention included either harvested iliac autogenous bone graft or autogenous bone graft plus 1.5 mg/mL of rhBMP-2 placed in and around the site of nonunion. No differences were found in the distribution of nonunion sites included within each group. Twelve-month follow-up was obtained on 100 of 118 patients (84.7%). Analyses of demographic characteristics (including tobacco), medical comorbidities, previous surgeries, and nonunion type (atrophic vs hypertrophic) did not differ. Postoperative complication rates did not differ. The percentage of patients who progressed to union did not differ. Mean time to union in the autogenous bone graft plus rhBMP-2 group was 6.6 months (±3.9) vs 5.4 (±2.7) months in the autogenous bone graft-only group (P=.06). Rates of revision (16.2% for rhBMP-2 plus autogenous bone graft vs 8% for autogenous bone graft) did not differ statistically (P=.19), nor did 12-month scores of pain and functional assessment. Although rhBMP-2 is a safe adjuvant, there was no benefit seen when rhBMP-2 was added to autogenous bone graft in the treatment of long bone nonunion. Given its high cost, rhBMP-2 should be reconsidered as an aid to autogenous bone graft in the treatment of nonunion. PMID:24972432

  19. A late role for bmp2b in the morphogenesis of semicircular canal ducts in the zebrafish inner ear.

    Directory of Open Access Journals (Sweden)

    Katherine L Hammond

    Full Text Available The Bone Morphogenetic Protein (BMP genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear.We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b(-/- embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal.Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.

  20. BMP6-Engineered MSCs Induce Vertebral Bone Repair in a Pig Model: A Pilot Study.

    Science.gov (United States)

    Pelled, Gadi; Sheyn, Dmitriy; Tawackoli, Wafa; Jun, Deuk Soo; Koh, Youngdo; Su, Susan; Cohn Yakubovich, Doron; Kallai, Ilan; Antebi, Ben; Da, Xiaoyu; Gazit, Zulma; Bae, Hyun; Gazit, Dan

    2016-01-01

    Osteoporotic patients, incapacitated due to vertebral compression fractures (VCF), suffer grave financial and clinical burden. Current clinical treatments focus on symptoms' management but do not combat the issue at the source. In this pilot study, allogeneic, porcine mesenchymal stem cells, overexpressing the BMP6 gene (MSC-BMP6), were suspended in fibrin gel and implanted into a vertebral defect to investigate their effect on bone regeneration in a clinically relevant, large animal pig model. To check the effect of the BMP6-modified cells on bone regeneration, a fibrin gel only construct was used for comparison. Bone healing was evaluated in vivo at 6 and 12 weeks and ex vivo at 6 months. In vivo CT showed bone regeneration within 6 weeks of implantation in the MSC-BMP6 group while only minor bone formation was seen in the defect site of the control group. After 6 months, ex vivo analysis demonstrated enhanced bone regeneration in the BMP6-MSC group, as compared to control. This preclinical study presents an innovative, potentially minimally invasive, technique that can be used to induce bone regeneration using allogeneic gene modified MSCs and therefore revolutionize current treatment of challenging conditions, such as osteoporosis-related VCFs. PMID:26770211

  1. Osteogenic effect of controlled released rhBMP-2 in 3D printed porous hydroxyapatite scaffold.

    Science.gov (United States)

    Wang, Hai; Wu, Gui; Zhang, Jing; Zhou, Kui; Yin, Bo; Su, Xinlin; Qiu, Guixing; Yang, Guang; Zhang, Xianglin; Zhou, Gang; Wu, Zhihong

    2016-05-01

    Recently, 3D printing as effective technology has been highlighted in the biomedical field. Previously, a porous hydroxyapatite (HA) scaffold with the biocompatibility and osteoconductivity has been developed by this method. However, its osteoinductivity is limited. The main purpose of this study was to improve it by the introduction of recombinant human bone morphogenetic protein-2 (rhBMP-2). This scaffold was developed by coating rhBMP-2-delivery microspheres with collagen. These synthesized scaffolds were characterized by Scanning Electron Microscopy (SEM), a delivery test in vitro, cell culture, and the experiments in vivo by a Micro-computed tomography (μCT) scan and histological evaluation of VanGieson staining. SEM results indicated the surface of scaffolds were more fit for the adhesion of hMSCs to coat collagen/rhBMP-2 microspheres. Biphasic release of rhBMP-2 could continue for more than 21 days, and keep its osteoinductivity to induce osteogenic differentiation of hMSCs in vitro. In addition, the experiments in vivo showed that the scaffold had a good bone regeneration capacity. These findings demonstrate that the HA/Collagen/Chitosan Microspheres system can simultaneously achieve localized long-term controlled release of rhBMP-2 and bone regeneration, which provides a promising route for improving the treatment of bone defects. PMID:26896655

  2. A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro.

    Science.gov (United States)

    Lee, Yura; Bae, Kyoung Jun; Chon, Hae Jung; Kim, Seong Hwan; Kim, Soon Ae; Kim, Jiyeon

    2016-05-31

    Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders. PMID:27025387

  3. BMP13 Prevents the Effects of Annular Injury in an Ovine Model

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    Aiqun Wei, Lisa A Williams, Divya Bhargav, Bojiang Shen, Thomas Kishen, Neil Duffy, Ashish D Diwan

    2009-01-01

    Full Text Available Chronic back pain is a global health problem affecting millions of people worldwide and carries significant economic and social morbidities. Intervertebral disc damage and degeneration is a major cause of back pain, characterised by histological and biochemical changes that have been well documented in animal models. Recently there has been intense interest in early intervention in disc degeneration using growth factors or stem cell transplantation, to replenish the diseased tissues. Bone Morphogenetic Proteins (BMPs have been approved for clinical use in augmenting spinal fusions, and may represent candidate molecules for intervertebral disc regeneration. BMP13 has an important role in embryonic development and recent genetic evidence shows a role in the development of the human spine. This study explores the effect of BMP13 on a damaged intervertebral disc in an ovine model of discal degeneration. We found that, when injected at the time of injury, BMP13 reversed or arrested histological changes that occurred in the control discs such as loss of extracellular matrix proteins. In addition, BMP13 injected discs retained greater hydration after 4months, and possessed more cells in the NP. Taken together, BMP13 may be a potent clinical therapeutic agent when used early in the degeneration cascade to promote healthy disc tissue.

  4. Effects of codon modification on human BMP2 gene expression in tobacco plants.

    Science.gov (United States)

    Suo, Guangli; Chen, Bing; Zhang, Jingyu; Duan, Ziyuan; He, Zhengquan; Yao, Wei; Yue, Chaoyin; Dai, Jianwu

    2006-07-01

    Bone morphogenetic protein 2 (BMP2) has great potential in therapeutic applications. We are working on generating transgenic plants as a bioreactor to produce BMP2. We have studied the effects of codon optimization on the expression of human BMP2 (hBMP2) in tobacco plants. Three modified hBMP2 genes were transformed into tobacco under the control of either cauliflower mosaic virus 35S (CaMV35S) promoter or double-CaMV35S promoter plus alfalfa mosaic virus (AMV) enhancer. The fused beta-glucuronidase (GUS) reporter gene was used to facilitate the assay of protein expression. The results indicated that codon optimization could increase the protein expression level obviously under CaMV35S promoter. However, under relatively stronger initiation condition (double-CaMV35S promoter plus AMV enhancer), only the gene with the lowest degree of codon optimization could increase the protein expression level. Our findings suggest that the action of codon optimization may be influenced by the factors of promoter strength and A+T content in tobacco plants. PMID:16491379

  5. Tracheal cartilage regeneration and new bone formation by slow release of bone morphogenetic protein (BMP)-2.

    Science.gov (United States)

    Igai, Hitoshi; Chang, Sung Soo; Gotoh, Masashi; Yamamoto, Yasumichi; Yamamoto, Masaya; Tabata, Yasuhiko; Yokomise, Hiroyasu

    2008-01-01

    We investigated the efficiency of bone morphogenetic protein (BMP)-2 released slowly from gelatin sponge for tracheal cartilage regeneration. A 1-cm gap was made in the mid-ventral portion of each of 10 consecutive tracheal cartilages. In the control group (n = 4), the resulting gap was left untreated. In the gelatin group (n = 4), plain gelatin was implanted in the gap. In the BMP-2 group (n = 4), gelatin containing 100 microg BMP-2 was implanted. We euthanatized all dogs in each group at 1, 3, 6, and 12 months after the implantation, respectively, and then examined the implant site macro- and microscopically. In the BMP-2 group, regenerated fibrous cartilage and newly formed bone were observed at 1 and 12 months. Regenerated cartilage was observed at the ends of the host cartilage stumps, with newly formed bone in the middle portion. The gaps were filled with regenerated cartilage and newly formed bone. At 3 and 6 months, regenerated cartilage, but not newly formed bone, was evident. The regenerated cartilage was covered with perichondrium and showed continuity with the host cartilage. We succeeded in inducing cartilage regeneration and new bone formation in canine trachea by slow release of 100 microg BMP-2 from gelatin. PMID:18204324

  6. Regulation of Axolotl (Ambystoma mexicanum Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

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    Jeffrey Lehrberg

    Full Text Available We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

  7. Cerium oxide nanoparticles promote neurogenesis and abrogate hypoxia-induced memory impairment through AMPK–PKC–CBP signaling cascade

    Directory of Open Access Journals (Sweden)

    Arya A

    2016-03-01

    Full Text Available Aditya Arya,1 Anamika Gangwar,1 Sushil Kumar Singh,2 Manas Roy,3,4 Mainak Das,3 Niroj Kumar Sethy,1 Kalpana Bhargava1 1Peptide and Proteomics Division, Defense Institute of Physiology and Allied Sciences, 2Functional Materials Division, Solid State Physics Laboratory, Defense Research and Development Organization, Timarpur, Delhi, 3Biological Science and Bioengineering, Indian Institute of Technology, Kanpur, 4Department of Chemistry, Indian Institute of Engineering Science and Technology, Howrah, India Abstract: Structural and functional integrity of the brain is adversely affected by reduced oxygen saturation, especially during chronic hypoxia exposure and often encountered by altitude travelers or dwellers. Hypoxia-induced generation of reactive nitrogen and oxygen species reportedly affects the cortex and hippocampus regions of the brain, promoting memory impairment and cognitive dysfunction. Cerium oxide nanoparticles (CNPs, also known as nanoceria, switch between +3 and +4 oxidation states and reportedly scavenge superoxide anions, hydrogen peroxide, and peroxynitrite in vivo. In the present study, we evaluated the neuroprotective as well as the cognition-enhancing activities of nanoceria during hypobaric hypoxia. Using polyethylene glycol-coated 3 nm nanoceria (PEG-CNPs, we have demonstrated efficient localization of PEG-CNPs in rodent brain. This resulted in significant reduction of oxidative stress and associated damage during hypoxia exposure. Morris water maze-based memory function tests revealed that PEG-CNPs ameliorated hypoxia-induced memory impairment. Using microscopic, flow cytometric, and histological studies, we also provide evidences that PEG-CNPs augmented hippocampus neuronal survival and promoted neurogenesis. Molecular studies revealed that PEG-CNPs promoted neurogenesis through the 5'-adenine monophosphate-activated protein kinase–protein kinase C–cyclic adenosine monophosphate response element-binding protein binding (AMPK-PKC-CBP protein pathway. Our present study results suggest that nanoceria can be translated as promising therapeutic molecules for neurodegenerative diseases. Keywords: cerium oxide nanoparticles, oxidative stress, memory, hypoxia, neuroprotection

  8. Resveratrol abrogates lipopolysaccharide-induced depressive-like behavior, neuroinflammatory response, and CREB/BDNF signaling in mice.

    Science.gov (United States)

    Ge, Li; Liu, Liwei; Liu, Hansen; Liu, Song; Xue, Hao; Wang, Xueer; Yuan, Lin; Wang, Zhen; Liu, Dexiang

    2015-12-01

    Current evidence supports that depression is accompanied by the activation of the inflammatory-response system, and overproduction of pro-inflammatory cytokines may play a role in the pathophysiology of depressive disorders. Resveratrol has anti-inflammatory, antioxidant and anti-depressant-like properties. Using an animal model of depression induced by a single administration of lipopolysaccharide (LPS), the present study investigated the effects of resveratrol on LPS-induced depressive-like behavior and inflammatory-response in adult mice. Our results showed that pretreatment with resveratrol (80mg/kg, i.p.) for 7 consecutive days reversed LPS-increased the immobility time in the forced swimming test and tail suspension test, and LPS-reduced sucrose preference test. Moreover, the antidepressant action of resveratrol was paralleled by significantly reducing the expression levels of pro-inflammatory cytokines, and up-regulating phosphorylated cAMP response-element-binding protein (pCREB)/brain-derived neurotrophic factor (BDNF) expression in prefrontal cortex (PFC) and hippocampus. In addition, resveratrol ameliorated LPS-induced NF-κB activation in the PFC and hippocampus. The results demonstrate that resveratrol may be an effective therapeutic agent for LPS-induced depressive-like behavior, partially due to its anti-inflammatory aptitude and by modulating pCREB and BDNF expression in the brain region of mice. PMID:26485503

  9. Abrogation of Nrf2 impairs antioxidant signaling and promotes atrial hypertrophy in response to high-intensity exercise stress

    OpenAIRE

    Kumar, Radhakrishnan Rajesh; Narasimhan, Madhusudhanan; Shanmugam, Gobinath; Hong, Jennifer; Devarajan, Asokan; Palaniappan, Sethu; Zhang, Jianhua; Halade, Ganesh V; Darley-Usmar, Victor M.; Hoidal, John R.; Rajasekaran, Namakkal S.

    2016-01-01

    Background Anomalies in myocardial structure involving myocyte growth, hypertrophy, differentiation, apoptosis, necrosis etc. affects its function and render cardiac tissue more vulnerable to the development of heart failure. Although oxidative stress has a well-established role in cardiac remodeling and dysfunction, the mechanisms linking redox state to atrial cardiomyocyte hypertrophic changes are poorly understood. Here, we investigated the role of nuclear erythroid-2 like factor-2 (Nrf2),...

  10. The BMP ligand Gdf6 prevents differentiation of coronal suture mesenchyme in early cranial development.

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    Dawn E Clendenning

    Full Text Available Growth Differentiation Factor-6 (Gdf6 is a member of the Bone Morphogenetic Protein (BMP family of secreted signaling molecules. Previous studies have shown that Gdf6 plays a role in formation of a diverse subset of skeletal joints. In mice, loss of Gdf6 results in fusion of the coronal suture, the intramembranous joint that separates the frontal and parietal bones. Although the role of GDFs in the development of cartilaginous limb joints has been studied, limb joints are developmentally quite distinct from cranial sutures and how Gdf6 controls suture formation has remained unclear. In this study we show that coronal suture fusion in the Gdf6-/- mouse is due to accelerated differentiation of suture mesenchyme, prior to the onset of calvarial ossification. Gdf6 is expressed in the mouse frontal bone primordia from embryonic day (E 10.5 through 12.5. In the Gdf6-/- embryo, the coronal suture fuses prematurely and concurrently with the initiation of osteogenesis in the cranial bones. Alkaline phosphatase (ALP activity and Runx2 expression assays both showed that the suture width is reduced in Gdf6+/- embryos and is completely absent in Gdf6-/- embryos by E12.5. ALP activity is also increased in the suture mesenchyme of Gdf6+/- embryos compared to wild-type. This suggests Gdf6 delays differentiation of the mesenchyme occupying the suture, prior to the onset of ossification. Therefore, although BMPs are known to promote bone formation, Gdf6 plays an inhibitory role to prevent the osteogenic differentiation of the coronal suture mesenchyme.

  11. Does Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Use in Adult Spinal Deformity (ASD) Increase Complications and Are Complications Associated With Location of rhBMP-2 Use?: A Prospective, Multicenter Study of 279 Consecutive Patients.

    Science.gov (United States)

    Bess, Shay; Line, Breton G; Lafarge, Virginie; Schwab, Frank; Shaffrey, Christopher I; Hart, Robert A; Boachie-Adjei, Oheneba; Akbarnia, Behrooz A; Ames, Christopher P; Burton, Douglas C; Deverin, Vedat; Fu, Kai-Ming G; Gupta, Munish; Hostin, Richard; Kebaish, Khaled; Klineberg, Eric; Mundis, Gregory; O'Brien, Michael; Shelokov, Alexis; Smith, Justin S

    2013-11-18

    Study Design. Multi-center, prospective analysis of consecutive ASD patients.Objective. Evaluate complications associated with rhBMP-2 use in ASDSummary of Background Data. Off-label rhBMP-2 use is common, however under-reporting of rhBMP-2 associated complications has been recently scrutinized.Methods. ASD patients consecutively enrolled into a prospective, multicenter database, were evaluated for type and timing of acute perioperative complications. Inclusion criteria: age ≥ 18 years, ASD, spinal arthrodesis >4 levels, and ≥3 months follow-up. Patients divided into those receiving rhBMP-2 (BMP) or no rhBMP-2 (NOBMP). BMP divided into location of use: posterior (PBMP), interbody (IBMP), and interbody + posterior spine (I+PBMP). Correlations between acute perioperative complications and rhBMP-2 use including total dose, dose/level and location of use were evaluated.Results. 279 patients (mean age 57 years, mean spinal levels fused 12.0, mean follow-up 28.8 months) met inclusion criteria. BMP (n = 172; average posterior dose = 2.5 mg/level, average interbody dose = 5 mg/level) had similar age, smoking history, previous spine surgery, total spinal levels fused, estimated blood loss, and duration of hospital stay as NOBMP (n = 107; p>0.05). BMP had greater Charlson Comorbidity Index (1.9 vs. 1.2), greater scoliosis (43° vs. 38°), longer operative time (488.2 vs. 414.6 minutes), more osteotomies/patient (4.0 vs. 1.6) and greater percentage of anteroposterior fusion (APSF; 20.9% vs. 8.4%) than NOBMP, respectively (p0.05). Multivariate analysis demonstrated small to non-existent correlations between rhBMP-2 use and complications.Conclusions. RhBMP-2 use and location of rhBMP-2 use in ASD surgery, at reported doses, does not increase acute major, neurological or wound complications. Research is needed for higher rhBMP-2 dosing and long-term follow-up. PMID:24253783

  12. Improving the osteogenic potential of BMP-2 with hyaluronic acid hydrogel modified with integrin-specific fibronectin fragment

    NARCIS (Netherlands)

    Kisiel, M.; Martino, M.M.; Ventura, M.; Hubbell, J.A.; Hilborn, J.; Ossipov, D.A.

    2013-01-01

    While human bone morphogenetic protein-2 (rhBMP-2) is a promising growth factor for bone regeneration, its clinical efficacy has recently shown to be below expectation. In order to improve the clinical translation of rhBMP-2, there exists strong motivation to engineer better delivery systems. Hyalur

  13. Herpesvirus telomerase RNA (vTR with a mutated template sequence abrogates herpesvirus-induced lymphomagenesis.

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    Benedikt B Kaufer

    2011-10-01

    Full Text Available Telomerase reverse transcriptase (TERT and telomerase RNA (TR represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5 by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1 that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2 that this strategy could be used to generate novel vaccine candidates

  14. Tetrandrine: A Potent Abrogator of G2 Checkpoint Function in Tumor Cells and Its Mechanism

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To assess the ability of tetrandrine (Tet) to enhance the sensitivity to irradiation and its mechanism in cell lines of human breast cancer p53-mutant MCF-7/ADR, p53-wild-type MCF-7 and human colon carcinoma p53-mutant HT-29 as well as in C26 colorectal carcinoma-bearing BALB/c mice. Methods MCF-7/ADR, HT-29 and MCF-7 cells were exposed to irradiation in the absence or presence of tetrandrine. The effect of Tet on the cytotoxicity of X-irradiation in these three cells was determined and the effect of tetrandrine on cell cycle arrest induced by irradiation in its absence or presence was studied by flow cytometry, Moreover, mitotic index measurement determined mitosis of cells to enter mitosis. Western blotting was employed to detect cyclin B1 and Cdc2 proteins in extracts from irradiated or non-irradiated cells of MCF-7/ADR, HT-29 and MCF-7 treated with tetrandrine at various concentrations. Tumor growth delay assay was conducted to determine the radio-sensitization of tetrandrine in vivo. Results Clonogenic assay showed that tetrandrine markedly enhanced the lethal effect of X-rays on p53-mutant MCF-7/ADR and HT-29 cells and the sensitization enhancement ratio (SER) of tetrandrine was 1.51 and 1.63, but its SER was only 1.1 in p53-wt MCF-7 cells. Irradiated p53-mutant MCF-7/ADR and HT-29 cells were only arrested in G2/M phase while MCF-7 cells were arrested in G1 and G2/M phases. Radiation-induced G2 phase arrests were abrogated by tetrandrine in a concentration-dependent manner in MCF-7/ADR and HT-29 cells,whereas redistribution within MCF-7 cell cycle changed slightly. The proportion of cells in M phase increased from 1.3% to 14.7% in MCF-7/ADR cells, and from 1.5% to 13.2% in HT-29 cells, but 2.4% to 7.1% in MCF-7 cells. Furthermore, the levels of cyclin B 1 and Cdc2 expression decreased after X-irradiation in MCF-7/ADR and HT-29 cells, and the mitotic index was also lower. Tet could reverse the decrease and induce the irradiated cells to enter mitosis

  15. Effect of heat treatment at 600 degree C for 10 hours on human BMP

    International Nuclear Information System (INIS)

    Viral infection are an extremely serious in allogeneic bone transplantations. While it is essential to kill viruses such as HIV in allogeneic bone graft, the osteoinductive activity must be preserved. Heat treatment of allogeneic bone graft at 60 degree C for 10 hours is effective in killing viruses such as HIV, but it is unclear to what extent the activity of human bone morphogenetic protein (hBMP) is preserved. In this experiment crude hBMP was extracted from both heated and non-heated human bones which were decalcified by the Urist method. Gelatin capsules containing 5mg of crude hBMP were transplanted into the thigh muscles of 5 week old mice. Human bone samples heated in a water bath at 60 degree C for 10 hours and non-heated samples were each transplanted into 5 mice. At 20 days after transplantation, the heterotopic bone formation was compared by evaluation of X-ray and histologicic analysis. X-rays showed heterotopic bone formation in both heated and non-heated samples. Further, histologic analysis showed that peripheral osteoid tissue had developed into laminar bone formation and interlaminar bone marrow was observed. Heterotopic bone formation was induced by crude hBMP from heated bones in a similar way to crude hBMP from non-heated bones observed in X-ray. There was no significant difference in histologic analysis. The crude hBMP, extracted from bones which were heat-treated at 60 degree C for 10 hours induced heterotopic bone formation similar to that in non-heated bone observed by X-ray and histologic analysis at 20 days after transplantation. This demostrates that the heat-treated bone preserved osteoinduction

  16. BMP7 transfection induces in-vitro osteogenic differentiation of dental pulp mesenchymal stem cells

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    Ka Po John Yau

    2013-01-01

    Full Text Available Objective: To assess whether in-vitro osteogenic differentiation of human dental pulp mesenchymal stem cells can be induced by transient transfection with the gene encoding human bone morphogenic protein 7 (BMP7. Materials and Methods: A mesenchymal stem cell population was isolated from the dental pulp of two extracted permanent premolars, expanded and characterized. The human BMP7 gene, as a recombinant pcDNA3.1/V5-His-TOPO-BMP7 plasmid, was transfected into the cells. Three negative controls were used: No plasmid, empty vector, and an unrelated vector encoding green fluorescent protein. After the interval of 24 and 48 h, mRNA levels of alkaline phosphatase and osteocalcin as markers of in-vitro osteogenic differentiation were measured by real-time polymerase chain reaction and standardized against β-actin mRNA levels. Results: The level of alkaline phosphatase mRNA was significantly higher for the BMP7 group than for all three negative controls 48 h after transfection (706.9 vs. 11.24 for untransfected cells, 78.05 for empty vector, and 73.10 for green fluorescent protein vector. The level of osteocalcin mRNA was significantly higher for the BMP7 group than for all three negative controls 24 h after transfection (1.0, however, decreased after another 24 h. Conclusions: In-vitro osteoblastic differentiation of human dental pulp mesenchymal stem cells, as indicated by expression of alkaline phosphatase and osteocalcin, can be induced by transient transfection with the BMP7 gene.

  17. Wnt/β-catenin regulates the activity of Epiprofin/Sp6, SHH, FGF and BMP to coordinate the stages of odontogenesis

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    Maitane Aurrekoetxea

    2016-03-01

    Full Text Available Background: We used an in vitro tooth development model to investigate the effects of overactivation of the Wnt/β-catenin pathway during odontogenesis by bromoindirubin oxime reagent (BIO, a specific inhibitor of GSK-3 activity. Results: Overactivatingthe Wnt/β-catenin pathway at tooth initiation upregulated and ectopically expressed the epithelial markers Sonic Hedgehog (Shh, Epiprofin (Epfn and Fibroblast growth factor8 (Fgf8, which are involved in the delimitation of odontogenic fields in the oral ectoderm. This result indicated an ectopic extension of the odontogenic potential. During tooth morphogenesis, Fibroblast growth factor4 (Fgf4, Fibroblast growth factor10 (Fgf10, Muscle segment homeobox 1 (Msx-1, Bone Morphogenetic protein 4 (Bmp4 and Dickkopf WNT signaling pathway inhibitor 1 (Dkk-1 were overexpressed in first molars cultured with BIO. Conversely, the expression levels of Wingless integration site 10b (Wnt-10b and Shh were reduced. Additionally, the odontoblast differentiation markers Nestin and Epfn showed ectopic overexpression in the dental mesenchyme of BIO-treated molars. Moreover, alkaline phosphatase activity increased in the dental mesenchyme, again suggesting aberrant, ectopic mesenchymal cell differentiation. Finally, Bmp4 downregulated Epfn expression during dental morphogenesis. Conclusions: We suggest the presence of a positive feedback loop wherein Epfn and β-catenin activate each other. The balance of the expression of these two molecules is essential for proper tooth development. We propose a possible link between Wnt, Bmp and Epfn that would critically determine the correct patterning of dental cusps and the differentiation of odontoblasts and ameloblasts.

  18. Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

    Science.gov (United States)

    Wu, Li-An; Wang, Feng; Donly, Kevin J; Baker, Andrew; Wan, Chunyan; Luo, Daoshu; MacDougall, Mary; Chen, Shuo

    2016-06-01

    Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko) ) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages. J. Cell. Physiol. 231: 1189-1198, 2016. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:26595646

  19. Characterisation of the biochemical methane potential (BMP) of individual material fractions in Danish source-separated organic household waste

    DEFF Research Database (Denmark)

    Naroznova, Irina; Møller, Jacob; Scheutz, Charlotte

    2016-01-01

    composition in Denmark (untreated) was calculated, and the BMP contribution of the individual material fractions was then evaluated. Material fractions of the two general waste types, defined as "food waste" and "fibre-rich waste," were found to be anaerobically degradable with considerable BMP. Material......This study is dedicated to characterising the chemical composition and biochemical methane potential (BMP) of individual material fractions in untreated Danish source-separated organic household waste (SSOHW). First, data on SSOHW in different countries, available in the literature, were evaluated......) and material degradability (BMP from laboratory incubation tests divided by TBMP) were expressed. Moreover, the degradability of lignocellulose biofibres (the share of volatile lignocellulose biofibre solids degraded in laboratory incubation tests) was calculated. Finally, BMP for average SSOHW...

  20. Testing the Biomethane Yield of Degradable Wastes of Meat Industry by BMP Test

    OpenAIRE

    Imamović, I.; Goletić, Š.

    2014-01-01

    The optimal mix of solid waste from the meat industry (MI) for anaerobic digestion (AD) treatment can be selected by defining the biomethane potential (BMP test) of the waste in relation to the unit value of chemical oxygen demand (COD). In this paper, the BMP test of biodegradable wastes from MI has been performed. For the purposes of the experiment, two types of input substrates have been defined: manure (manure from cattle depots and transport vehicles for cattle transport) labeled as O1 a...

  1. EXPRESSION OF rhBMP-7 GENE IN TRANSDUCED BONE MARROW DERIVED STROMAL CELLS

    Institute of Scientific and Technical Information of China (English)

    段德宇; 杜靖远; 王洪; 刘勇; 郭晓东

    2002-01-01

    Objective. To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells(BMSCs). Methods. The marker gene , pbLacZ, was transferred into cultured BMSCs and the expression of transduced gene by X-gal staining was examined. Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. Results. The exogenous gene could be expressed efficiently in transduced BMSCs. Conculsion. The present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.

  2. Function and Regulation of Bone Morphogenetic Protein 7 (BMP7) in Cerebral Cortex Development

    OpenAIRE

    Ortega Cano, Juan Alberto

    2011-01-01

    [eng] Brain derived neurotrophic factor (BDNF) is a chemokine which levels are regulated by neuronal activity and could act as a sensor in front of distinct physiologic stimulus, activating the transcription of specific group of genes. In this work we show that BDNF induces the expression of BMP7 in neurons through TrkB receptor and MAPK/ERK pathways, an induction mechanism that is mediated in part by the release of the transcriptional repression exerted by p53 family proteins. BMP member...

  3. Arsenic induces apoptosis in mouse liver is mitochondria dependent and is abrogated by N-acetylcysteine

    International Nuclear Information System (INIS)

    Arsenicosis, caused by arsenic contamination of drinking water supplies, is a major public health problem in India and Bangladesh. Chronic liver disease, often with portal hypertension occurs in chronic arsenicosis, contributes to the morbidity and mortality. The early cellular events that initiate liver cell injury due to arsenicosis have not been studied. Our aim was to identify the possible mechanisms related to arsenic-induced liver injury in mice. Liver injury was induced in mice by arsenic treatment. The liver was used for mitochondrial oxidative stress, mitochondrial permeability transition (MPT). Evidence of apoptosis was sought by TUNEL test, caspase assay and histology. Pretreatment with N-acetyl-L-cysteine (NAC) was done to modulate hepatic GSH level. Arsenic treatment in mice caused liver injury associated with increased oxidative stress in liver mitochondria and alteration of MPT. Altered MPT facilitated cytochrome c release in the cytosol, activation of caspase 9 and caspase 3 activities and apoptotic cell death. Pretreatment of NAC to arsenic-treated mice abrogated all these alteration suggesting a glutathione (GSH)-dependent mechanism. Oxidative stress in mitochondria and inappropriate MPT are important in the pathogenesis of arsenic induced apoptotic liver cell injury. The phenomenon is GSH dependent and supplementation of NAC might have beneficial effects

  4. Beclin1-induced autophagy abrogates radioresistance of lung cancer cells by suppressing osteopontin

    International Nuclear Information System (INIS)

    Osteopontin (OPN) serves as an indicator of resistance to radiotherapy. However, the role of OPN in the development of acquired radioresistance in human lung cancer cells has not yet been fully elucidated. Therefore, the potential importance of OPN as a marker of lung cancer with a potential significant role in the development of radioresistance against repeated radiotherapy has prompted us to define the pathways by which OPN regulates lung cancer cell growth. In addition, autophagy has been reported to play a key role in the radiosensitization of cancer cells. Here, we report that increased OPN expression through induction of nuclear p53 following irradiation was inhibited by exogenous beclin-1 (BECN1). Our results clearly show that BECN1 gene expression led to induction of autophagy and inhibition of cancer cell growth and angiogenesis. Our results suggest that the induction of autophagy abrogated the radioresistance of the cancer cells. Interestingly, we showed that knockdown of OPN by lentivirus-mediated shRNA induced the autophagy of human lung cancer cell. Taken together, these results suggest that OPN and BECN1 can be molecular targets for overcoming radioresistance by controlling autophagy. (author)

  5. Abrogation of plasminogen activator inhibitor-1-vitronectin interaction ameliorates acute kidney injury in murine endotoxemia.

    Directory of Open Access Journals (Sweden)

    Kamlesh K Gupta

    Full Text Available Sepsis-induced acute kidney injury (AKI contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1 and vitronectin (Vn are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1-/- or expressing a PAI-1-mutant (PAI-1R101A/Q123K in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1-/- and PAI-1R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT mice after LPS challenge. Also, PAI-1-/- mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity.

  6. Abrogation of Plasminogen Activator Inhibitor-1-Vitronectin Interaction Ameliorates Acute Kidney Injury in Murine Endotoxemia

    Science.gov (United States)

    Gupta, Kamlesh K.; Donahue, Deborah L.; Sandoval-Cooper, Mayra J.; Castellino, Francis J.; Ploplis, Victoria A.

    2015-01-01

    Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1−/−) or expressing a PAI-1-mutant (PAI-1R101A/Q123K) in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1−/− and PAI-1R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT) mice after LPS challenge. Also, PAI-1−/− mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC) in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity. PMID:25799354

  7. Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B

    DEFF Research Database (Denmark)

    Olsen, Oddrun Elise; Wader, Karin Fahl; Hella, Hanne; Mylin, Anne Kærsgaard; Turesson, Ingemar; Nesthus, Ingerid; Waage, Anders; Sundan, Anders; Holien, Toril

    2015-01-01

    activin A through canonical ALK4-ACVR2 receptor complexes activates the transcription factors SMAD2 and SMAD3. Activin A has a strong affinity to type 2 receptors, a feature that they share with some of the bone morphogenetic proteins (BMPs). Activin A is also elevated in myeloma patients with advanced...

  8. Klumpfuss controls FMRFamide expression by enabling BMP signaling within the NB5-6 lineage

    OpenAIRE

    Losada-Perez, Maria; Gabilondo, Hugo; Molina, Isabel; Turiegano, Enrique; Torroja, Laura; Thor, Stefan; Benito-Sipos, Jonathan

    2013-01-01

    A number of transcription factors that are expressed within most, if not all, embryonic neuroblast (NB) lineages participate in neural subtype specification. Some have been extensively studied in several NB lineages (e.g. components of the temporal gene cascade) whereas others only within specific NB lineages. To what extent they function in other lineages remains unknown. Klumpfuss (Klu), the Drosophila ortholog of the mammalian Wilms tumor 1 (WT1) protein, is one such transcription factor. ...

  9. rBMP Represses Wnt Signaling and Influences Skeletal Progenitor Cell Fate Specification During Bone Repair

    OpenAIRE

    Minear, Steve; Leucht, Philipp; Miller, Samara; Helms, Jill A.

    2010-01-01

    Bone morphogenetic proteins (BMPs) participate in multiple stages of the fetal skeletogenic program from promoting cell condensation to regulating chondrogenesis and bone formation through endochondral ossification. Here, we show that these pleiotropic functions are recapitulated when recombinant BMPs are used to augment skeletal tissue repair. In addition to their well-documented ability to stimulate chondrogenesis in a skeletal injury, we show that recombinant BMPs (rBMPs) simultaneously su...

  10. The adipocyte clock controls brown adipogenesis through the TGF-Beta and BMP signaling pathways

    Science.gov (United States)

    The molecular clock is intimately linked to metabolic regulation, and brown adipose tissue plays a key role in energy homeostasis. However, whether the cell-intrinsic clock machinery participates in brown adipocyte development is unknown. Here, we show that Bmal1 (also known as ARNTL), the essential...

  11. Cyclic dermal BMP signaling regulates stem cell activation during hair regeneration

    OpenAIRE

    Plikus, Maksim V; Mayer, Julie; de la Cruz, Damon; Baker, Ruth E.; Maini, Philip K.; Maxson, Robert; Chuong, Cheng-ming

    2008-01-01

    In the age of stem cell engineering, it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life,1 and are an important model for organ regeneration. Hair stem cells located in the follicle bulge2 are regulated by the surrounding micro-environment, or niche3. The activation of such stem cells is cyclic, involving periodic β-catenin activity4–7. In adult mouse, regeneration occurs in waves in...

  12. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration

    OpenAIRE

    Plikus, M. V.; Mayer, J. A.; de La Cruz, D.; Baker, Ruth E.; Maini, P.K.; Maxson, R.; Chuong, C M

    2008-01-01

    In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life1, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge2 are regulated by the surrounding microenvironment, or niche3. The activation of such stem cells is cyclic, involving periodic -catenin activity4, 5, 6, 7. In the adult mouse, regeneration occurs in ...

  13. Brown Fat Paucity Due to Impaired BMP Signaling Induces Compensatory Browning of White Fat

    OpenAIRE

    Schulz, Tim J.; Huang, Ping; Huang, Tian Lian; Xue, Ruidan; McDougall, Lindsay E.; Townsend, Kristy L.; Cypess, Aaron M.; Mishina, Yuji; Gussoni, Emanuela; Tseng, Yu-Hua

    2013-01-01

    Summary Maintenance of body temperature is essential for survival of homeotherms. Brown adipose tissue (BAT) is a specialized fat tissue that is dedicated to thermoregulation 1 . Due to its remarkable capacity to dissipate stored energy and its demonstrated presence in adult humans 2-5 , BAT holds great promise for the treatment of obesity and metabolic syndrome 1 . Rodent data suggest the existence of two types of brown fat cells: the constitutive BAT (cBAT), which is of embryonic origin and...

  14. Brown Fat Paucity Due to Impaired BMP Signaling Induces Compensatory Browning of White Fat

    OpenAIRE

    Schulz, Tim J.; Huang, Ping; Huang, Tian Lian; Xue, Ruidan; McDougall, Lindsay E.; Townsend, Kristy L.; Cypess, Aaron M.; Mishina, Yuji; Gussoni, Emanuela; Tseng, Yu-Hua

    2013-01-01

    Summary Maintenance of body temperature is essential for survival of homeotherms. Brown adipose tissue (BAT) is a specialized fat tissue that is dedicated to thermoregulation1. Due to its remarkable capacity to dissipate stored energy and its demonstrated presence in adult humans2-5, BAT holds great promise for the treatment of obesity and metabolic syndrome1. Rodent data suggest the existence of two types of brown fat cells: the constitutive BAT (cBAT), which is of embryonic origin and anato...

  15. Autocrine Bone Morphogenetic Protein-9 signals via Activin Receptor Like Kinase-2/Smad1/Smad4 to promote ovarian cancer cell proliferation

    OpenAIRE

    Herrera, Blanca; van Dinther, Maarten; ten Dijke, Peter; Inman, Gareth J.

    2009-01-01

    Bone morphogenetic proteins (BMPs) act as central regulators of ovarian physiology and may be involved in ovarian cancer development. In an effort to understand these processes we characterized TGFβ/BMP receptor and Smad expression in immortalised ovarian surface epithelial cells (IOSE) and a panel of ovarian cancer cell lines. These studies prompted us to evaluate the potential role of BMP9 signalling in ovarian cancer. Using siRNA, ligand trap, inhibitor and ligand stimulation approaches we...

  16. Characteristics and Stimulation Potential with BMP-2 and BMP-7 of Tenocyte-Like Cells Isolated from the Rotator Cuff of Female Donors

    Science.gov (United States)

    Klatte-Schulz, Franka; Pauly, Stephan; Scheibel, Markus; Greiner, Stefan; Gerhardt, Christian; Hartwig, Jelka; Schmidmaier, Gerhard; Wildemann, Britt

    2013-01-01

    Tendon bone healing of the rotator cuff is often associated with non-healing or recurrent defects, which seems to be influenced by the patient’s age and sex. The present study aims to examine cellular biological characteristics of tenocyte-like cells that may contribute to this impaired rotator cuff healing. Moreover, a therapeutic approach using growth factors could possibly stimulate tendon bone healing. Therefore, our second aim was to identify patient groups who would particularly benefit from growth factor stimulation. Tenocyte-like cells isolated from supraspinatus tendons of female donors younger and older than 65 years of age were characterized with respect to different cellular biological parameters, such as cell density, cell count, marker expression, collagen-I protein synthesis, and stem cell potential. Furthermore, cells of the donor groups were stimulated with BMP-2 and BMP-7 (200 and 1000 ng/ml) in 3D-culture and analyzed for cell count, marker expression and collagen-I protein synthesis. Female donors older than 65 years of age showed significantly decreased cell count and collagen-I protein synthesis compared to cells from donors younger than 65 years. Cellular biological parameters including cell count, collagen-I and –III expression, and collagen-I protein synthesis of cells from both donor groups were stimulated with BMP-2 and BMP-7. The cells from donors older than 65 years revealed a decreased stimulation potential for cell count compared to the younger group. Cells from female donors older than 65 years of age showed inferior cellular biological characteristics. This may be one reason for a weaker healing potential observed in older female patients and should be taken into consideration for tendon bone healing of the rotator cuff. PMID:23825642

  17. Characteristics and stimulation potential with BMP-2 and BMP-7 of tenocyte-like cells isolated from the rotator cuff of female donors.

    Directory of Open Access Journals (Sweden)

    Franka Klatte-Schulz

    Full Text Available Tendon bone healing of the rotator cuff is often associated with non-healing or recurrent defects, which seems to be influenced by the patient's age and sex. The present study aims to examine cellular biological characteristics of tenocyte-like cells that may contribute to this impaired rotator cuff healing. Moreover, a therapeutic approach using growth factors could possibly stimulate tendon bone healing. Therefore, our second aim was to identify patient groups who would particularly benefit from growth factor stimulation. Tenocyte-like cells isolated from supraspinatus tendons of female donors younger and older than 65 years of age were characterized with respect to different cellular biological parameters, such as cell density, cell count, marker expression, collagen-I protein synthesis, and stem cell potential. Furthermore, cells of the donor groups were stimulated with BMP-2 and BMP-7 (200 and 1000 ng/ml in 3D-culture and analyzed for cell count, marker expression and collagen-I protein synthesis. Female donors older than 65 years of age showed significantly decreased cell count and collagen-I protein synthesis compared to cells from donors younger than 65 years. Cellular biological parameters including cell count, collagen-I and -III expression, and collagen-I protein synthesis of cells from both donor groups were stimulated with BMP-2 and BMP-7. The cells from donors older than 65 years revealed a decreased stimulation potential for cell count compared to the younger group. Cells from female donors older than 65 years of age showed inferior cellular biological characteristics. This may be one reason for a weaker healing potential observed in older female patients and should be taken into consideration for tendon bone healing of the rotator cuff.

  18. Wnt signaling and the activation of myogenesis in mammals.

    OpenAIRE

    Cossu, G.; Borello, U

    1999-01-01

    In the amniote embryos, specification of skeletal myoblasts occurs in the paraxial mesoderm in response to a number of signaling molecules produced by neighboring tissues such as neural tube, notochord and dorsal ectoderm. Candidate molecules for this complex signaling activity include Sonic hedgehog, Wnts and Noggin as positive activators and BMP4 as a possible inhibitor. Recently, the receptors and the post-receptor pathways for Sonic hedgehog and Wnts have been characterized, and this has ...

  19. Genetic predisposition to fracture non-union: a case control study of a preliminary single nucleotide polymorphisms analysis of the BMP pathway

    Directory of Open Access Journals (Sweden)

    Giannoudis Peter V

    2011-02-01

    Full Text Available Abstract Background Despite the known multi-factorial nature of atrophic fracture non-unions, a possible genetic predisposition for the development of this complication after long bone fractures remains unknown. This pilot study aimed to address this issue by performing a preliminary SNP analysis of specific genes known to regulate fracture healing. Methods A total of fifteen SNPs within four genes of the Bone Morphogenetic Protein (BMP pathway (BMP-2, BMP-7, NOGGIN and SMAD6 were examined, in 109 randomly selected patients with long bone fractures as a result of motor vehicle accident, fall or direct blow. There were sixty-two patients with atrophic non-union and forty-seven patients (54 fractures with uneventful fracture union. Overall SNPs frequencies were computed with respect to patient's age, gender, smoking habits, fracture-associated parameters and the use of nonsteroidal anti-inflammatory drugs (NSAIDs, and tested for their association to the impaired bone healing process, using binary logistic regression (STATA 11.1; StataCorp, Texas USA. Results Statistical analysis revealed age to be an important covariate in the development of atrophic non-union (p = 0.01, OR 1.05 [per year], and two specific genotypes (G/G genotype of the rs1372857 SNP, located on NOGGIN and T/T genotype of the rs2053423 SNP, located on SMAD6 to be associated with a greater risk of fracture non-union (p = 0.02, OR 4.56 and p = 0.04, OR 10.27, respectively, after adjustment for age. Conclusions This is the first clinical study to investigate the potential existence of genetic susceptibility to fracture non-union. Even though no concrete conclusions can be obtained from this pilot study, our results indicate the existence of a potential genetically predetermined impairment within the BMP signalling cascade, initiated after a fracture and when combined with other risk factors could synergistically increase the susceptibility of a patient to develop non-union. Further

  20. Possible Involvement of Smad Signaling Pathways in Induction of Odontoblastic Properties in KN-3 Cells by Bone Morphogenetic Protein-2: A Growth Factor to Induce Dentin Regeneration

    Directory of Open Access Journals (Sweden)

    Ayako Washio

    2012-01-01

    Full Text Available We examined the effects of bone morphogenetic protein-2 (BMP-2 on growth, differentiation, and intracellular signaling pathways of odontoblast-like cells, KN-3 cells, to clarify molecular mechanisms of odontoblast differentiation during pulp regeneration process. After treatment with BMP-2, the cell morphology, growth, alkaline phosphatase (ALP activity, and the activation and expression of BMP-induced intracellular signaling molecules, such as Smad1/5/8 and Smad6/7, as well as activities of dentin sialoprotein (DSP and dentin matrix protein 1 (DMP1, were examined. BMP-2 had no effects on the morphology, growth, or ALP activity of KN-3 cells, whereas it induced the phosphorylation of Smad1/5/8 and expression of Smad6/7. BMP-2 also induced the expressions of DSP and DMP-1. Our results suggest that KN-3 cells may express an odontoblastic phenotype with the addition of BMP-2 through the activation of Smad signaling pathways.

  1. Fabrication of an rhBMP-2 loaded porous β-TCP microsphere-hyaluronic acid-based powder gel composite and evaluation of implant osseointegration

    OpenAIRE

    Lee, Jae Hyup; Kim, Jungju; Baek, Hae-Ri; Lee, Kyung Mee; Seo, Jun-Hyuk; Lee, Hyun-Kyung; Lee, A-Young; Zheng, Guang Bin; Chang, Bong-Soon; Lee, Choon-Ki

    2014-01-01

    Methods to improve osseointegration that include implantation of rhBMP-2 with various kinds of carriers are currently of considerable interest. The present study was conducted to evaluate if the rhBMP-2 loaded β-TCP microsphere-hyaluronic acid-based powder-like hydrogel composite (powder gel) can act as an effective rhBMP-2 carrier for implantation in host bone with a bone defect or poor bone quality. The release pattern for rhBMP-2 was then evaluated against an rhBMP-2-loaded collagen sponge...

  2. Low-power GaAlAs laser irradiation promotes the proliferation and osteogenic differentiation of stem cells via IGF1 and BMP2.

    Directory of Open Access Journals (Sweden)

    Jyun-Yi Wu

    Full Text Available Low-power laser irradiation (LPLI has been found to induce various biological effects and cellular processes. Also, LPLI has been shown to promote fracture repair. Until now, it has been unclear how LPLI promotes bone formation and fracture healing. The aim of this study was to investigate the potential mechanism of LPLI-mediated enhancement of bone formation using mouse bone marrow mesenchymal stem cells (D1 cells. D1 cells were irradiated daily with a gallium-aluminum-arsenide (GaAlAs laser at dose of 0, 1, 2, or 4 J/cm(2. The lactate dehydrogenase (LDH assay showed no cytotoxic effects of LPLI on D1 cells, and instead, LPLI at 4 J/cm(2 significantly promoted D1 cell proliferation. LPLI also enhanced osteogenic differentiation in a dose-dependent manner and moderately increased expression of osteogenic markers. The neutralization experiments indicated that LPLI regulated insulin-like growth factor 1 (IGF1 and bone morphogenetic protein 2 (BMP2 signaling to promote cell proliferation and/or osteogenic differentiation. In conclusion, our study suggests that LPLI may induce IGF1 expression to promote both the proliferation and osteogenic differentiation of D1 cells, whereas it may induce BMP2 expression primarily to enhance osteogenic differentiation.

  3. CLONING AND SEQUENCING OF MATURE FRAGMENT OF HUMAN BMP4 GENE

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To study the cloning and sequencing of mature fragment of human bone morphogenetic protein-4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT- PCR method, the cDNA coding for the mature fragment of BMP-4 was amplified, cloned into the vector pUC19, and sequenced by Sanger Dideoxy-mediated Chain Termination method. Results The mature fragment of BMP4 cDNA was obtained by RT-PCR and determined by sequencing. Through the computer search on Genebank, the analysis showed that the homology of nucleotides and amino acids between cDNA of rhBMP4 mature fragment of this study and the published sequence was 99%. Sequence analysis showed that there were two differences, one was at base 1154 (201): G→C, which had no influence on the corresponding amino acids (Val). Another was at basel222 (269):C→T, the mutation at the base 1222 had the change of Ala to Val. Conclusion The mature fragment of BMP4 gene has been cloned. The results will be of great significance in treatment of skeletal injuries and diseases.

  4. Biodegradable Chitosan Nanoparticle Coatings on Titanium for the Delivery of BMP-2

    Directory of Open Access Journals (Sweden)

    Nils Poth

    2015-01-01

    Full Text Available A simple method for the functionalization of a common implant material (Ti6Al4V with biodegradable, drug loaded chitosan-tripolyphosphate (CS-TPP nanoparticles is developed in order to enhance the osseointegration of endoprostheses after revision operations. The chitosan used has a tailored degree of acetylation which allows for a fast biodegradation by lysozyme. The degradability of chitosan is proven via viscometry. Characteristics and degradation of nanoparticles formed with TPP are analyzed using dynamic light scattering. The particle degradation via lysozyme displays a decrease in particle diameter of 40% after 4 days. Drug loading and release is investigated for the nanoparticles with bone morphogenetic protein 2 (BMP-2, using ELISA and the BRE luciferase test for quantification and bioactivity evaluation. Furthermore, nanoparticle coatings on titanium substrates are created via spray-coating and analyzed by ellipsometry, scanning electron microscopy and X-ray photoelectron spectroscopy. Drug loaded nanoparticle coatings with biologically active BMP-2 are obtained in vitro within this work. Additionally, an in vivo study in mice indicates the dose dependent induction of ectopic bone growth through CS-TPP-BMP-2 nanoparticles. These results show that biodegradable CS-TPP coatings can be utilized to present biologically active BMP-2 on common implant materials like Ti6Al4V.

  5. Chrysin, an anti-inflammatory molecule, abrogates renal dysfunction in type 2 diabetic rats

    International Nuclear Information System (INIS)

    Diabetic nepropathy (DN) is considered as the leading cause of end-stage renal disease (ESRD) worldwide, but the current available treatments are limited. Recent experimental evidences support the role of chronic microinflammation in the development of DN. Therefore, the tumor necrosis factor-alpha (TNF-α) pathway has emerged as a new therapeutic target for the treatment of DN. We investigated the nephroprotective effects of chrysin (5, 7-dihydroxyflavone) in a high fat diet/streptozotocin (HFD/STZ)-induced type 2 diabetic Wistar albino rat model. Chrysin is a potent anti-inflammatory compound that is abundantly found in plant extracts, honey and bee propolis. The treatment with chrysin for 16 weeks post induction of diabetes significantly abrogated renal dysfunction and oxidative stress. Chrysin treatment considerably reduced renal TNF-α expression and inhibited the nuclear transcription factor-kappa B (NF-kB) activation. Furthermore, chrysin treatment improved renal pathology and suppressed transforming growth factor-beta (TGF-β), fibronectin and collagen-IV protein expressions in renal tissues. Chrysin also significantly reduced the serum levels of pro-inflammatory cytokines, interleukin-1beta (IL-1β) and IL-6. Moreover, there were no appreciable differences in fasting blood glucose and serum insulin levels between the chrysin treated groups compared to the HFD/STZ-treated group. Hence, our results suggest that chrysin prevents the development of DN in HFD/STZ-induced type 2 diabetic rats through anti-inflammatory effects in the kidney by specifically targeting the TNF-α pathway. - Highlights: • Chrysin reduced renal oxidative stress and inflammation in diabetic rats. • Chrysin reduced serum levels of pro-inflammatory in diabetic rats. • Chrysin exhibited renal protective effect by suppressing the TNF-α pathway

  6. Selenium deficiency abrogates inflammation-dependent plasma cell tumors in mice.

    Science.gov (United States)

    Felix, Klaus; Gerstmeier, Simone; Kyriakopoulos, Antonios; Howard, O M Zack; Dong, Hui-Fang; Eckhaus, Michael; Behne, Dietrich; Bornkamm, Georg W; Janz, Siegfried

    2004-04-15

    The role of the micronutrient, selenium, in human cancers associated with chronic inflammations and persistent infections is poorly understood. Peritoneal plasmacytomas (PCTs) in strain BALB/c (C), the premier experimental model of inflammation-dependent plasma cell transformation in mice, may afford an opportunity to gain additional insights into the significance of selenium in neoplastic development. Here, we report that selenium-depleted C mice (n = 32) maintained on a torula-based low-selenium diet (5-8 micro g of selenium/kg) were totally refractory to pristane induction of PCT. In contrast, 11 of 26 (42.3%) control mice maintained on a selenium adequate torula diet (300 micro g of selenium/kg) and 15 of 40 (37.5%) control mice fed standard Purina chow (440 micro g of selenium/kg) developed PCT by 275 days postpristane. Abrogation of PCT was caused in part by the striking inhibition of the formation of the inflammatory tissue in which PCT develop (pristane granuloma). This was associated with the reduced responsiveness of selenium-deficient inflammatory cells (monocytes and neutrophils) to chemoattractants, such as thioredoxin and chemokines. Selenium-deficient C mice exhibited little evidence of disturbed redox homeostasis and increased mutant frequency of a transgenic lacZ reporter gene in vivo. These findings implicate selenium, via the selenoproteins, in the promotion of inflammation-induced PCT and suggest that small drug inhibitors of selenoproteins might be useful for preventing human cancers linked with chronic inflammations and persistent infections. PMID:15087411

  7. Chrysin, an anti-inflammatory molecule, abrogates renal dysfunction in type 2 diabetic rats

    Energy Technology Data Exchange (ETDEWEB)

    Ahad, Amjid [Lipid Metabolism Laboratory, Department of Biochemistry, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi 110062 (India); Ganai, Ajaz Ahmad [Department of Biotechnology, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi 110062 (India); Mujeeb, Mohd [Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062 (India); Siddiqui, Waseem Ahmad, E-mail: was.sid121@gmail.com [Lipid Metabolism Laboratory, Department of Biochemistry, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi 110062 (India)

    2014-08-15

    Diabetic nepropathy (DN) is considered as the leading cause of end-stage renal disease (ESRD) worldwide, but the current available treatments are limited. Recent experimental evidences support the role of chronic microinflammation in the development of DN. Therefore, the tumor necrosis factor-alpha (TNF-α) pathway has emerged as a new therapeutic target for the treatment of DN. We investigated the nephroprotective effects of chrysin (5, 7-dihydroxyflavone) in a high fat diet/streptozotocin (HFD/STZ)-induced type 2 diabetic Wistar albino rat model. Chrysin is a potent anti-inflammatory compound that is abundantly found in plant extracts, honey and bee propolis. The treatment with chrysin for 16 weeks post induction of diabetes significantly abrogated renal dysfunction and oxidative stress. Chrysin treatment considerably reduced renal TNF-α expression and inhibited the nuclear transcription factor-kappa B (NF-kB) activation. Furthermore, chrysin treatment improved renal pathology and suppressed transforming growth factor-beta (TGF-β), fibronectin and collagen-IV protein expressions in renal tissues. Chrysin also significantly reduced the serum levels of pro-inflammatory cytokines, interleukin-1beta (IL-1β) and IL-6. Moreover, there were no appreciable differences in fasting blood glucose and serum insulin levels between the chrysin treated groups compared to the HFD/STZ-treated group. Hence, our results suggest that chrysin prevents the development of DN in HFD/STZ-induced type 2 diabetic rats through anti-inflammatory effects in the kidney by specifically targeting the TNF-α pathway. - Highlights: • Chrysin reduced renal oxidative stress and inflammation in diabetic rats. • Chrysin reduced serum levels of pro-inflammatory in diabetic rats. • Chrysin exhibited renal protective effect by suppressing the TNF-α pathway.

  8. Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

    Directory of Open Access Journals (Sweden)

    Grassi Rici Rose

    2012-02-01

    Full Text Available Abstract Background The bone morphogenetic proteins (BMPs belong to a unique group of proteins that includes the growth factor TGF-β. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs and belong to the University of São Paulo, College of Veterinary Medicine (FMVZ-USP stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p

  9. Enhanced reconstruction of long bone architecture by a growth factor mutant combining positive features of GDF-5 and BMP-2.

    Science.gov (United States)

    Kleinschmidt, Kerstin; Ploeger, Frank; Nickel, Joachim; Glockenmeier, Julia; Kunz, Pierre; Richter, Wiltrud

    2013-08-01

    Non healing bone defects remain a worldwide health problem and still only few osteoinductive growth factors are available for clinical use in bone regeneration. By introducing BMP-2 residues into growth and differentiation factor (GDF)-5 we recently produced a mutant GDF-5 protein BB-1 which enhanced heterotopic bone formation in mice. Designed to combine positive features of GDF-5 and BMP-2, we suspected that this new growth factor variant may improve long bone healing compared to the parent molecules and intended to unravel functional mechanisms behind its action. BB-1 acquired an increased binding affinity to the BMP-IA receptor, mediated enhanced osteogenic induction of human mesenchymal stem cells versus GDF-5 and higher VEGF secretion than BMP-2 in vitro. Rabbit radius defects treated with a BB-1-coated collagen carrier healed earlier and with increased bone volume compared to BMP-2 and GDF-5 according to in vivo micro-CT follow-up. While BMP-2 callus often remained spongy, BB-1 supported earlier corticalis and marrow cavity formation, showing no pseudojoint persistence like with GDF-5. Thus, by combining positive angiogenic and osteogenic features of GDF-5 and BMP-2, only BB-1 restored a natural bone architecture within 12 weeks, rendering this promising growth factor variant especially promising for long bone regeneration. PMID:23680368

  10. [Expression of BMP-4 in stromal cells in vitro derived from human aorta-gonad-mesonephros region].

    Science.gov (United States)

    Chen, Hui-Qin; Zhang, Xu-Chao; Wu, Yan-Feng; Wu, Bei-Yan; Huang, Shao-Liang

    2007-08-01

    The objective of this study was to investigate the expression of BMP-4 in stromal cells in vitro derived from human aorta-gonad-mesonephros (AGM) region. Stromal cells derived from human AGM region (hAGM S1-S5) and fibroblasts derived from human fetal trunk (hFT) were cultured in vitro. RT-PCR was used to analyze the expression of BMP-4 in hAGM S1-S5 stromal cells at mRNA level. And BMP-4 level was detected in the supernatant of hAGM S1-S5 stromal cells by ELISA assay. hFT cells were used as control group. The results showed that the heterogenous hAGM S1-S5 stromal cells displyed shapes of fibroblast-like and endothelial-like cells. hAGM S1-S5 stromal cells expressed BMP-4 mRNA, but fetal trunk fibroblasts (hFT) did not express BMP-4 mRNA. In the supernatant of hAGM S1-S5 cells, BMP-4 could be detected by ELISA assay ana its levels were statistically higher than that in hFT group (p 0.05). It is concluded that human AGM-derived stromal cells in vitro express BMP-4, and the establishment of a new culture system based on the feeder cells of AGM stroma would promote the differention of embryonic stem cells into hematopoietic stem cells at a high proportion. PMID:17708801

  11. Study of the association between the BMP4 gene and congenital anomalies of the kidney and urinary tract

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    Geisilaine Soares dos Reis

    2014-01-01

    Full Text Available OBJECTIVE: To determine the frequency of different phenotypes for congenital anomalies of the kidney and urinary tract (CAKUT in a Brazilian sample, and to evaluate the association between the CAKUT phenotypes and the BMP4 gene. METHODS: In this study, 457 Brazilian individuals were analyzed in an attempt to establish the association between the BMP4 gene and the CAKUT diagnosis. A case-control sample was genotyped for three BMP4 gene polymorphisms. RESULTS: Association data was established with CAKUT sample as a whole and with the three most important CAKUT phenotypes: multicystic dysplastic kidney disease (MDK, ureteropelvic junction obstruction (UPJO and vesicoureteral reflux (VUR. When the sample was segregated in these three phenotypes, associations between the BMP4 gene were observed with UPJO and with MDK. Conversely, VUR was not associated to the polymorphisms of the BMP4 gene. CONCLUSIONS: The present data suggest that Brazilian individuals with polymorphisms of the BMP4 gene have a higher risk to develop CAKUT, especially the malformations related to nephrogenesis and initial branching such as MDK and UPJO. Conversely, VUR appeared not to be related to BMP4 gene.

  12. Lung remodeling in a mouse model of asthma involves a balance between TGF-β1 and BMP-7.

    Directory of Open Access Journals (Sweden)

    Camila Leindecker Stumm

    Full Text Available A key event in chronic allergic asthma is the TGF-β-induced activation of fibroblasts into α-SMA-positive myofibroblasts which synthesize type-I collagen. In the present study we investigated the effect of the anti-fibrotic molecule BMP-7 in asthma. Balb/c mice were immunized i.p. with ovalbumin in alum and challenged every 2 days with ovalbumin aerosol (two or six challenges for acute and chronic protocols, respectively. The lung was evaluated for: α-SMA and type-I collagen by immunohistochemistry; BMP-7 and TGF- β1 gene expression by qRT-PCR; type-I collagen and Smads 2 and 3 by immunoblotting; mucus by PSA staining. Type-I collagen around bronchi, α-SMA, mucus secretion, TGF- β1 and BMP-7 gene expression were all increased in asthma. The TGF- β1/BMP-7 ratio was higher in the chronic group and correlated with higher levels of collagen. Fibroblasts isolated from asthmatic and healthy lungs produced type-I collagen upon stimulation with TGF- β1 via phosphorylation of Smad-2, Smad-3. Pre-treatment of the fibroblasts with BMP-7 reduced collagen production and Smads phosphorylation. Intranasal treatment of asthmatic mice with recombinant BMP-7 during the immunization protocol reduced lung inflammation and type I collagen deposition. These results suggest a protective role for BMP-7 in lung allergic inflammation, opposing the pro-fibrotic effects of TGF- β1.

  13. The human leukemia oncogene bcr-abl abrogates the anchorage requirement but not the growth factor requirement for proliferation.

    OpenAIRE

    Renshaw, M W; McWhirter, J R; Wang, J Y

    1995-01-01

    Proliferation of normal cells in a multicellular organism requires not only growth factors but also the proper attachment to the extracellular matrix. A hallmark of neoplastic transformation is the loss of anchorage dependence which usually accompanies the loss of growth factor requirement. The Bcr-Abl tyrosine kinase of human leukemias is shown here to abrogate only the anchorage, not the growth factor, requirement. Bcr-Abl-transformed cells grow in soft agar but do not proliferate in serum-...

  14. Are we ready to abrogate compulsory vaccinations for children?: The opinion of apulian family pediatricians; Italy, 2013

    OpenAIRE

    Martinelli, Domenico; Tafuri, Silvio; Fortunato, Francesca; Cozza, Vanessa; Germinario, Cinzia A; Prato, Rosa

    2014-01-01

    In Italy, vaccination against diphtheria, tetanus, polio and hepatitis B is compulsory for infants countrywide, except in Veneto region where since 2007 Health Authorities have experimented the suspension of mandatory vaccination. In light of the recent discussion on the potential abrogation in other regions, we explored the opinion of family pediatricians who play a crucial role in promoting immunization programmes in Italy. In November 2009, we interviewed by phone the family pediatricians ...

  15. Effect of growth factors (BMP-4/7 & bFGF on proliferation & osteogenic differentiation of bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Shaohui Yuan

    2013-01-01

    Full Text Available Background & objectives: BMP (bone morphogenetic protein-4/7 and bFGF (basic fibroblast growth factor significantly promote the osteogenic activity and the proliferation of rabbit BMSCs (bone marrow stromal cells, respectively. However, their synergistic effects on the proliferation and the differentiation of BMSCs remain unclear. In the present study, the effects of bFGF and BMP-4/7 were investigated on the proliferation and the differentiation of rat BMSCs in vitro. Methods: BMSCs were isolated from New Zealand white rabbits and cultured to the third passage. The samples were divided into five groups according to the material implanted: (A 80 ng/ml BMP-4/7; (B 80 ng/ml bFGF; (C 30 ng/ml BMP-4/7 and 30 ng/ml bFGF; (D 50 ng/ml BMP-4/7 and 50 ng/ml bFGF; and (E 80 ng/ml BMP-4/7 and 80 ng/ml bFGF. Cell proliferation was analyzed using methyl thiazolyl tetrazolium (MTT assay. Alkaline phosphatase activity and osteocalcin (OC dynamics were also measured. Results: BMP-4/7 alone significantly (P<0.05 promoted the proliferation of BMSCs. At the same time, it also promoted or inhibited the osteogenic differentiation of BMSCs. The synergistic effects of BMP-4/7 and bFGF significantly promoted both the proliferation and the osteogenic differentiation of BMSCs. The treatment of the synergistic effects was dose and time dependent. Interpretation & conclusions: A rational combination of BMP-4/7 and bFGF can promote the proliferation and the osteogenic differentiation of BMSCs. In addition, the synergistic functions are effective.

  16. hBMP-7 induces the differentiation of adipose-derived mesenchymal stem cells into osteoblast-like cells.

    Science.gov (United States)

    Ren, Y; Han, C; Wang, J; Jia, Y; Kong, L; Eerdun, T; Wu, L; Jiang, D

    2016-01-01

    The aim of this study was to investigate the differentiation potential of adipose-derived mesenchymal stem cells (ADMSCs) into osteoblasts by human bone morphogenetic protein-7 (hBMP-7) induction. ADMSCs were isolated from the subcutaneous adipose tissue of a rabbit, and then transfected with the pcDNA3.1 vector alone and pcDNA3.1-hBMP-7 (hBMP-7), respectively. Untransfected ADMSCs were used as the control group. After transfection, the morphology and green fluorescent protein (GFP) fluorescence intensity of ADMSCs were observed by fluorescent microscopy. The 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay was performed to detect the growth of ADMSCs at 1, 3, and 5 days, respectively. Transmission electron microscopy was performed to observe the ultrastructural morphology of ADMSCs. In addition, ADMSCs were stained with quinalizarin and toluidine blue to reflect the content of osteoblasts and chondrocytes, respectively. Finally, the expression of collagen I and osteocalcin in ADMSCs was detected by western blot. ADMSCs were successfully isolated. Obvious GFP fluorescence and high expression of hBMP-7 demonstrated the successful transfection of hBMP-7. Specific morphological characters with a metabolically active ultrastructure were exhibited on the ADMSCs transfected with hBMP- 7. In addition, the growth rate of ADMSCs transfected with hBMP-7 was significantly higher than that of the cells in the vector and control groups. Successfully induced osteoblast-like cells were identified by an obvious erythrine area and high expression of collagen I and osteocalcin in ADMSCs transfected with hBMP-7. Thus, ADMSCs can be successfully differentiated into osteoblast-like cells by hBMP-7 induction in vitro. PMID:27525862

  17. The efficacy of rhBMP-2 versus autograft for posterolateral lumbar spine fusion in elderly patients

    OpenAIRE

    Lee, Kwang-Bok; Taghavi, Cyrus E.; Hsu, Margaret S.; Song, Kyung-Jin; Yoo, Jeong Hyun; Keorochana, Gun; Ngo, Stephanie S.; Wang, Jeffrey C.

    2009-01-01

    Few studies have specifically examined the outcomes following rhBMP-2 usage in patients 65 years and older. The purpose of this retrospective study is to evaluate the efficacy of rhBMP-2 with allograft versus autograft for posterolateral lumbar fusion in patients 65 years and older. One hundred twenty-seven patients were divided into three groups based on fusion material and age. Subjects in group A (n = 34) consisted of patients 65 years and older who received rhBMP-2 and allograft. Group B ...

  18. Gene gun transferring-bone morphogenetic protein 2 (BMP-2) gene enhanced bone fracture healing in rabbits

    OpenAIRE

    Li, Wenju; Wei, Haifeng; Xia, Chunmei; Zhu, Xiaomeng; Hou, Guozhu; Xu, Feng; Xinghua SONG; Zhan, Yulin

    2015-01-01

    Purpose: Transferring the bone morphogenetic protein 2 (BMP-2) genes into the tissues or cells can improve the bone healing of the fracture has been widely accepted. We evaluated the efficiency of using gene gun to transfer the BMP-2 gene thereby affected the healing of a fractured bone. Methods: The vector coding for BMP-2 was constructed by a non-replicating encephalo-myocarditis virus (ECMV)-based vector. The segmental bone defect (1.5 cm) model was created by a wire-saw at the middle part...

  19. Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

    International Nuclear Information System (INIS)

    Highlights: → Harmine promotes the activity and mRNA expression of ALP. → Harmine enhances the expressions of osteocalcin mRNA and protein. → Harmine induces osteoblastic mineralization. → Harmine upregulates the mRNA expressions of BMPs, Runx2 and Osterix. → BMP signaling pathways are involved in the actions of harmine. -- Abstract: Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a β-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure-activity relationship studies using harmine-related β-carboline alkaloids revealed that the C3-C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and Runx2

  20. Y-tocotrienol inhibits angiogenesis-dependent growth of human hepatocellular carcinoma through abrogation of AKT/mTOR pathway in an orthotopic mouse model.

    Science.gov (United States)

    Siveen, Kodappully Sivaraman; Ahn, Kwang Seok; Ong, Tina H; Shanmugam, Muthu K; Li, Feng; Yap, Wei Ney; Kumar, Alan Prem; Fong, Chee Wai; Tergaonkar, Vinay; Hui, Kam M; Sethi, Gautam

    2014-04-15

    Angiogenesis is one of the key hallmarks of cancer. In this study, we investigated whether γ-tocotrienol can abrogate angiogenesis-mediated tumor growth in hepatocellular carcinoma (HCC) and if so, through what molecular mechanisms. We observed that γ-tocotrienol inhibited vascular endothelial growth factor (VEGF)-induced migration, invasion, tube formation and viability of HUVECs in vitro. Moreover, γ-tocotrienol reduced the number of capillary sprouts from matrigel embedded rat thoracic aortic ring in a dose-dependent manner. Also, in chick chorioallantoic membrane assay, γ-tocotrienol significantly reduced the blood vessels formation. We further noticed that γ-tocotrienol blocked angiogenesis in an in vivo matrigel plug assay. Furthermore, γ-tocotrienol inhibited VEGF-induced autophosphorylation of VEGFR2 in HUVECs and also suppressed the constitutive activation of AKT/mammalian target of rapamycin (mTOR) signal transduction cascades in HUVECs as well as in HCC cells. Interestingly, γ-tocotrienol was also found to significantly reduce the tumor growth in an orthotopic HCC mouse model and inhibit tumor-induced angiogenesis in HCC patient xenografts through the suppression of various biomarkers of proliferation and angiogenesis. Taken together, our findings strongly suggest that γ-tocotrienol might be a promising anti-angiogenic drug with significant antitumor activity in HCC. PMID:24722367

  1. Knockdown of platinum-induced growth differentiation factor 15 abrogates p27-mediated tumor growth delay in the chemoresistant ovarian cancer model A2780cis

    International Nuclear Information System (INIS)

    Molecular mechanisms underlying the development of resistance to platinum-based treatment in patients with ovarian cancer remain poorly understood. This is mainly due to the lack of appropriate in vivo models allowing the identification of resistance-related factors. In this study, we used human whole-genome microarrays and linear model analysis to identify potential resistance-related genes by comparing the expression profiles of the parental human ovarian cancer model A2780 and its platinum-resistant variant A2780cis before and after carboplatin treatment in vivo. Growth differentiation factor 15 (GDF15) was identified as one of five potential resistance-related genes in the A2780cis tumor model. Although A2780-bearing mice showed a strong carboplatin-induced increase of GDF15 plasma levels, the basal higher GDF15 plasma levels of A2780cis-bearing mice showed no further increase after short-term or long-term carboplatin treatment. This correlated with a decreased DNA damage response, enhanced AKT survival signaling and abrogated cell cycle arrest in the carboplatin-treated A2780cis tumors. Furthermore, knockdown of GDF15 in A2780cis cells did not alter cell proliferation but enhanced cell migration and colony size in vitro. Interestingly, in vivo knockdown of GDF15 in the A2780cis model led to a basal-enhanced tumor growth, but increased sensitivity to carboplatin treatment as compared to the control-transduced A2780cis tumors. This was associated with larger necrotic areas, a lobular tumor structure and increased p53 and p16 expression of the carboplatin-treated shGDF15-A2780cis tumors. Furthermore, shRNA-mediated GDF15 knockdown abrogated p27 expression as compared to control-transduced A2780cis tumors. In conclusion, these data show that GDF15 may contribute to carboplatin resistance by suppressing tumor growth through p27. These data show that GDF15 might serve as a novel treatment target in women with platinum-resistant ovarian cancer

  2. Genistein abrogates G2 arrest induced by curcumin in p53 deficient T47D cells

    Science.gov (United States)

    2012-01-01

    Background The high cost and low level of cancer survival urge the finding of new drugs having better mechanisms. There is a high trend of patients to be “back to nature” and use natural products as an alternative way to cure cancer. The fact is that some of available anticancer drugs are originated from plants, such as taxane, vincristine, vinblastine, pacitaxel. Curcumin (diferuloylmethane), a dietary pigment present in Curcuma longa rizhome is reported to induce cell cycle arrest in some cell lines. Other study reported that genistein isolated from Glycine max seed inhibited phosphorylation of cdk1, gene involved during G2/M transition and thus could function as G2 checkpoint abrogator. The inhibition of cdk1 phosphorylation is one of alternative strategy which could selectively kill cancer cells and potentially be combined with DNA damaging agent such as curcumin. Methods T47D cell line was treated with different concentrations of curcumin and genistein, alone or in combination; added together or with interval time. Flow Cytometry and MTT assay were used to evaluate cell cycle distribution and viability, respectively. The presence of apoptotic cells was determined using acridine orange-ethidium bromide staining. Results In this study curcumin induced G2 arrest on p53 deficient T47D cells at the concentration of 10 μM. Increasing concentration up to 30 μM increased the number of cell death. Whilst genistein alone at low concentration (≤10 μM) induced cell proliferation, addition of genistein (20 μM) 16 h after curcumin resulted in more cell death (89%), 34% higher than that administered at the same time (56%). The combination treatment resulted in apoptotic cell death. Combining curcumin with high dose of genistein (50 μM) induced necrotic cells. Conclusions Genistein increased the death of curcumin treated T47D cells. Appropriate timing of administration and concentration of genistein determine the outcome of treatment and this method

  3. Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Nemoto, Eiji, E-mail: e-nemoto@dent.tohoku.ac.jp [Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Ebe, Yukari; Kanaya, Sousuke [Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Tsuchiya, Masahiro [Department of Aging and Geriatric Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Nakamura, Takashi [Department of Pediatric Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Tamura, Masato [Department of Biochemistry and Molecular Biology, Hokkaido University Graduate School of Dentistry, Sapporo 060-8586 (Japan); Shimauchi, Hidetoshi [Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Wnt5a is identified in osteoblasts in tibial growth plate and bone marrow. Black-Right-Pointing-Pointer Osteoblastic differentiation is associated with increased expression of Wnt5a/Ror2. Black-Right-Pointing-Pointer Wnt5a/Ror2 signaling is important for BMP-2-mediated osteoblastic differentiation. Black-Right-Pointing-Pointer Wnt5a/Ror2 operates independently of BMP-Smad pathway. -- Abstract: Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through {beta}-catenin-dependent canonical and {beta}-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent

  4. Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

    International Nuclear Information System (INIS)

    Highlights: ► Wnt5a is identified in osteoblasts in tibial growth plate and bone marrow. ► Osteoblastic differentiation is associated with increased expression of Wnt5a/Ror2. ► Wnt5a/Ror2 signaling is important for BMP-2-mediated osteoblastic differentiation. ► Wnt5a/Ror2 operates independently of BMP-Smad pathway. -- Abstract: Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.

  5. Sirenomelia phenotype in bmp7;shh compound mutants: a novel experimental model for studies of caudal body malformations.

    Directory of Open Access Journals (Sweden)

    Carlos Garrido-Allepuz

    Full Text Available Sirenomelia is a severe congenital malformation of the lower body characterized by the fusion of the legs into a single lower limb. This striking external phenotype consistently associates severe visceral abnormalities, most commonly of the kidneys, intestine, and genitalia that generally make the condition lethal. Although the causes of sirenomelia remain unknown, clinical studies have yielded two major hypotheses: i a primary defect in the generation of caudal mesoderm, ii a primary vascular defect that leaves the caudal part of the embryo hypoperfused. Interestingly, Sirenomelia has been shown to have a genetic basis in mice, and although it has been considered a sporadic condition in humans, recently some possible familial cases have been reported. Here, we report that the removal of one or both functional alleles of Shh from the Bmp7-null background leads to a sirenomelia phenotype that faithfully replicates the constellation of external and internal malformations, typical of the human condition. These mutants represent an invaluable model in which we have analyzed the pathogenesis of sirenomelia. We show that the signaling defect predominantly impacts the morphogenesis of the hindgut and the development of the caudal end of the dorsal aortas. The deficient formation of ventral midline structures, including the interlimb mesoderm caudal to the umbilicus, leads to the approximation and merging of the hindlimb fields. Our study provides new insights for the understanding of the mechanisms resulting in caudal body malformations, including sirenomelia.

  6. BMP-15 m-RNA expression of mouse oocytes in vitro maturation in different droplet medium volume

    Institute of Scientific and Technical Information of China (English)

    Sri Rahayu; Nashi Widodo; Yumi Hoshino; Eimei Sato

    2015-01-01

    Objective:To investigate droplet medium volume effect on the BMP-15 mRNA expression. Methods:Oocytes are collected from mice ovaries by puncturing with a sterile 26-G needle. The droplet medium volumes are using 50 µL, 100 µL and 200 µL. The BMP-15 mRNA expression is determined in each group.Results:The results indicated that BMP-15 mRNA expression did not significantly differ when oocyte were cultured in 50 and 100 µL/droplet medium volume, but significant difference (P < 0.05) was found when oocytes were cultured in 200 µL/droplet medium volume.Conclusions:The highest BMP-15 m-RNA expression occur when oocytes are cultured in 200 µL/droplet medium volume.

  7. E. coli-Produced BMP-2 as a Chemopreventive Strategy for Colon Cancer: A Proof-of-Concept Study

    Directory of Open Access Journals (Sweden)

    Saravanan Yuvaraj

    2012-01-01

    Full Text Available Colon cancer is a serious health problem, and novel preventive and therapeutical avenues are urgently called for. Delivery of proteins with anticancer activity through genetically modified bacteria provides an interesting, potentially specific, economic and effective approach here. Interestingly, bone morphogenetic protein 2 (BMP-2 is an important and powerful tumour suppressor in the colon and is thus an attractive candidate protein for delivery through genetically modified bacteria. It has not been shown, however, that BMP production in the bacterial context is effective on colon cancer cells. Here we demonstrate that transforming E. coli with a cDNA encoding an ileal-derived mature human BMP-2 induces effective apoptosis in an in vitro model system for colorectal cancer, whereas the maternal organism was not effective in this respect. Furthermore, these effects were sensitive to cotreatment with the BMP inhibitor Noggin. We propose that prevention and treatment of colorectal cancer using transgenic bacteria is feasible.

  8. Small Molecules Dorsomorphin and LDN-193189 Inhibit Myostatin/GDF8 Signaling and Promote Functional Myoblast Differentiation*

    OpenAIRE

    Horbelt, Daniel; Boergermann, Jan H.; Chaikuad, Apirat; Alfano, Ivan; Williams, Eleanor; Lukonin, Ilya; Timmel, Tobias; Bullock, Alex N.; Knaus, Petra

    2014-01-01

    GDF8, or myostatin, is a member of the TGF-β superfamily of secreted polypeptide growth factors. GDF8 is a potent negative regulator of myogenesis both in vivo and in vitro. We found that GDF8 signaling was inhibited by the small molecule ATP competitive inhibitors dorsomorphin and LDN-193189. These compounds were previously shown to be potent inhibitors of BMP signaling by binding to the BMP type I receptors ALK1/2/3/6. We present the crystal structure of the type II receptor ActRIIA with do...

  9. Delivery of RANKL-Binding Peptide OP3-4 Promotes BMP-2-Induced Maxillary Bone Regeneration.

    Science.gov (United States)

    Uehara, T; Mise-Omata, S; Matsui, M; Tabata, Y; Murali, R; Miyashin, M; Aoki, K

    2016-06-01

    Although bone morphogenetic protein 2 (BMP-2) is known to stimulate osteogenesis, there is evidence that high doses of BMP-2 can lead to side effects, including inflammation and carcinogenesis. The supplementation of other bone-augmenting agents is considered helpful in preventing such side effects by reducing the amount of BMP-2 required to obtain a sufficient amount of bone. We recently showed that a receptor activator of nuclear factor κB ligand (RANKL)-binding peptide promotes osteoblast differentiation. In the present study, we aimed to investigate whether OP3-4, a RANKL-binding peptide, promotes BMP-2-induced bone formation in the murine maxilla using an injectable gelatin hydrogel (GH) carrier. A GH carrier containing OP3-4 with BMP-2 was subperiosteally injected into the murine maxillary right diastema between the incisor and the first molar. The mice were sacrificed 28 d after the injections. The local bone formation in the OP3-4-BMP-2-injected group was analyzed in comparison to the carrier-injected, BMP-2-injected, and control-peptide-BMP-2-injected groups. The GH carrier containing OP3-4 with BMP-2 enlarged the radio-opaque area and increased the bone mineral content and density in the radiological analyses in comparison to the other experimental groups. Interestingly, fluorescence-based histological analyses revealed that the mineralization had started from the outside, then proceeded inward, suggesting that the size of the newly formed bone had already been set before calcification started and that the effects of OP3-4 might be involved in accelerating the early steps of osteogenesis. Actually, OP3-4 enhanced the BMP-2-induced 5-bromo-2'-deoxyuridine (BrdU)-positive cell numbers at the injected site on day 7 and the expression of Runx2 and Col1a1, which are early osteogenic cell markers, on day 10 after the subperiosteal injections. In summary, we demonstrated, for the first time, that the application of OP3-4 by subperiosteal injection promoted BMP

  10. Inhibitory effect of BMP-2 gene transfection mediated by nanoparticles on proliferation of graft vascular inner membrane

    International Nuclear Information System (INIS)

    Objective: To investigate the inhibitory effect of bone morphogenetic protein 2 (BMP-2) gene transfection mediated by nanoparticles (NP) on proliferation of vascular smooth muscle cells (VSMC) in rat vein grafting model, and to provide a new method to prevent restenosis. Methods: The VSMC were transduced in vitro with NP BMP-2 DNA complex prepared with PLGA and used as BMP-2-PLGA group, meanwhile simple PLGA group and control group were set up. The cell proliferation was determined by flow cytometry. Autogenous vein graft models were established in 72 rabbits by transplanting internal branch of jugular vein to carotid artery, then divided into BMP-2 group, empty vector group and simple graft control group. The grafted veins were harvested at 3, 7, 14 and 28 d respectively after operation. The thickness of vascular inner membrane was detected with Verhoeff staining. The exogenous BMP-2 protein expression in veins was determined by Western blotting. The expressions of proliferating nuclear antigen (PCNA) and BMP-2 were detected by immunohistochemistry. Results: Compared with control group, the inhibitory rate of cell proliferation and apoptotic rate in BMP-2-PLGA group were increased obviously (P<0.05) and the cell cycle was arrested in G1 phase (P<0.052). Compared with empty vector group and simple graft control group, the thickness of vascular inner membrane in BMP-2 group was decreased (P<0.01), but there was no significant difference between empty vector group and simple graft control group. Compared with empty vector group and graft simple graft control group, the BMP-2 protein expressions in rabbit vein graft tissue in BMP-2 group 3, 7, 14 d after operation were significantly increased (P<0.05); the PCNA expressions were significantly decreased 7-28 d after operation (P<0.05); but there was no significant difference between empty vector group and simple graft control group. Conclusion: Expression of BMP-2 gene can prevent intimal hyperplasia (IH) and VSMC

  11. Osteo-/odontogenic differentiation of BMP2 and VEGF gene-co-transfected human stem cells from apical papilla.

    Science.gov (United States)

    Zhang, Wen; Zhang, Xiaolei; Ling, Junqi; Wei, Xi; Jian, Yutao

    2016-05-01

    Stem cells from apical papilla (SCAP) possess clear osteo‑/odontogenic differentiation capabilities, and are regarded as the major cellular source for root dentin development. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) serve pivotal roles in the modulation of tooth development and dentin formation. However, the synergistic effects of BMP2 and VEGF on osteo‑/odontogenic differentiation of SCAP remain unclear. The current study aimed to investigate the proliferative and osteo‑/odontogenic differentiating capabilities of BMP2 and VEGF gene-co-transfected SCAP (SCAP-BMP2-VEGF) in vitro. The basic characteristics of the isolated SCAP were identified by the induction of multipotent differentiation and by flow cytometry. Lentiviral vector‑mediated gene transfection was conducted with SCAP in order to construct blank vector‑transfected SCAP (SCAP-green fluorescent protein), BMP2 gene-transfected SCAP (SCAP-BMP2), VEGF gene‑transfected SCAP (SCAP‑VEGF) and SCAP-BMP2-VEGF. The Cell Counting Kit 8 assay was used to analyze the proliferative capacities of the four groups of cells. The expression of osteo-/odontogenic genes and proteins in the cells were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. The mineralized nodules formed by the four group cells were visualized by alkaline phosphatase (ALP) staining. Among the four groups of cells, SCAP‑VEGF was demonstrated to exhibit increased proliferation, and SCAP‑BMP2‑VEGF exhibited reduced proliferation during eight days observation. SCAP‑BMP2‑VEGF exhibited significantly increased expression levels of ALP, osteocalcin, dentin sialophosphoprotein, dentin matrix acidic phosphoprotein gene 1 and dentin sialoprotein than the other three groups at the majority of the time points. Furthermore, the SCAP‑BMP2‑VEGF group exhibited a significantly greater number of ALP‑positive mineralized nodules than the other

  12. Characterisation of the biochemical methane potential (BMP) of individual material fractions in Danish source-separated organic household waste.

    Science.gov (United States)

    Naroznova, Irina; Møller, Jacob; Scheutz, Charlotte

    2016-04-01

    This study is dedicated to characterising the chemical composition and biochemical methane potential (BMP) of individual material fractions in untreated Danish source-separated organic household waste (SSOHW). First, data on SSOHW in different countries, available in the literature, were evaluated and then, secondly, laboratory analyses for eight organic material fractions comprising Danish SSOHW were conducted. No data were found in the literature that fully covered the objectives of the present study. Based on laboratory analyses, all fractions were assigned according to their specific properties in relation to BMP, protein content, lipids, lignocellulose biofibres and easily degradable carbohydrates (carbohydrates other than lignocellulose biofibres). The three components in lignocellulose biofibres, i.e. lignin, cellulose and hemicellulose, were differentiated, and theoretical BMP (TBMP) and material degradability (BMP from laboratory incubation tests divided by TBMP) were expressed. Moreover, the degradability of lignocellulose biofibres (the share of volatile lignocellulose biofibre solids degraded in laboratory incubation tests) was calculated. Finally, BMP for average SSOHW composition in Denmark (untreated) was calculated, and the BMP contribution of the individual material fractions was then evaluated. Material fractions of the two general waste types, defined as "food waste" and "fibre-rich waste," were found to be anaerobically degradable with considerable BMP. Material degradability of material fractions such as vegetation waste, moulded fibres, animal straw, dirty paper and dirty cardboard, however, was constrained by lignin content. BMP for overall SSOHW (untreated) was 404mL CH4 per g VS, which might increase if the relative content of material fractions, such as animal and vegetable food waste, kitchen tissue and dirty paper in the waste, becomes larger. PMID:26878771

  13. From fruitflies to mammals: mechanisms of signalling via the Sonic hedgehog pathway in lung development

    OpenAIRE

    Tuyl, Minke; Post, Martin

    2000-01-01

    The hedgehog signalling pathway has been implicated in many different processes in fly and vertebrate development. It is now known that the hedgehog cascade is crucial for the patterning of the early respiratory system. Hedgehog signalling in the lung involves Gli transcription proteins, but their potential downstream target genes have yet to be identified. Bmp4 and Fgf10 have been shown to regulate lung branching morphogenesis but seem not to be targets of hedgehog signalling.

  14. Korean Red Ginseng Extract Enhances the Anticancer Effects of Imatinib Mesylate Through Abrogation p38 and STAT5 Activation in KBM-5 Cells.

    Science.gov (United States)

    Jung, Sang Yoon; Kim, Chulwon; Kim, Wan-Seok; Lee, Seok-Geun; Lee, Jun-Hee; Shim, Bum Sang; Kim, Sung-Hoon; Ahn, Kyoo Seok; Ahn, Kwang Seok

    2015-07-01

    Although imatinib mesylate (IM) in the treatment of chronic myelogenous leukemia (CML) remains the best example of successful targeted therapy, majority of patients with CML suffer its toxicity profile and develop chemoresistance to existing therapeutic agents. Thus, there is a need to develop novel alternative therapies for the treatment of CML. Here, we investigated whether Korean red ginseng extract (KRGE) could suppress the proliferation and induce chemosensitization in human CML cells. Also, we used a human phospho-antibody array containing 46 antibodies against signaling molecules to examine a subset of phosphorylation events after treatment. Korean red ginseng extract broadly suppressed the proliferation of five different cell lines, but KRGE was found to be the most potent inducer of apoptosis against KBM-5 cells. It also abrogated the expression of Bcl-2 (B-cell lymphoma 2), Bcl-xL (B-cell lymphoma-extra large), survivin, inhibitors of apoptosis protein 1/2, COX-2 (Cyclooxygenase-2), cyclin D1, matrix metalloproteinase-9, and VEGF (Vascular endothelial growth factor), as well as upregulated the expression of pro-apoptotic gene products. Interestingly, KRGE also enhanced the cytotoxic and apoptotic effect of IM in KBM-5 cells. The combination treatment of KRGE and IM caused pronounced suppression of p38 and signal transducer and activator of transcription 5 phosphorylation and induced phosphorylation of p53 compared with the individual treatment. Our results demonstrate that KRGE can enhance the anticancer activity of IM and may have a substantial potential in the treatment of CML. PMID:25857479

  15. Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Xiao Caiwen; Zhou Huifang; Fu Yao; Gu Ping; Fan Xianqun [Department of Ophthalmology, Shanghai Ninth People' s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China); Liu Guangpeng [Key Laboratory of Tissue Engineering, Shanghai Ninth People' s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China); Zhang Peng [Center for Translational Medicine Research and Development, Shenzhen Institute of Advanced Technology, Chinese Academy of Science (China); Hou Hongliang; Tang Tingting, E-mail: drfanxianqun@126.com [Department of Orthopedics, Shanghai Ninth People' s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China)

    2011-02-15

    Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

  16. An experimental study on application of implant to irradiated bone. Effect of combination with rhBMP-2

    International Nuclear Information System (INIS)

    The purpose of this study was to investigate the effects of rhBMP-2 on wound healing around implants placed in irradiated bone. Fifty-four male Wistar rats were used. A single dose of 30 Gy irradiation from a Linac source was delivered to the right lower leg of all rats. The left leg was kept as a non-irradiated site. A pure titanium screw with a block of Poly D, L-lactic-co-glycolic acid and gelatin sponge (PGS) containing 100 ng rhBMP-2 was installed to the bilateral tibial proximal metaphysis three months after irradiation. The rats in which the screw and PGS without rhBMP-2 were implanted and those in which only the screw was implanted served as controls. The rats were sacrificed one, two, and eight weeks after the placement. Non-decalcified specimens stained with toluidine blue were used for histological analyses. The bone volume in the medullary cavity and bone-implant contact ratio was also quantified with a contact microradiogram. Administration of rhBMP-2 promoted bone formation around the implant of the irradiated group. Administration of rhBMP-2 improved the bone-implant contact of the irradiated group in the early time period. The results indicate that simultaneous administration of rhBMP-2 is effective in implant placement into irradiated bone. (author)

  17. Optimization of entrapping conditions to improve the release of BMP-2 from PELA carriers by response surface methodology

    International Nuclear Information System (INIS)

    A microcapsule prepared from triblock copolymer poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PLA, PELA) was investigated as a controlled release carrier for recombinant human bone morphogenetic protein-2 (rhBMP-2). The rhBMP-2/PELA microspheres were prepared using the water-in-oil-in-water (W/O/W) solvent evaporation method. This work was conducted to optimize the entrapping conditions of the rhBMP-2 loaded PELA copolymer. The effects on encapsulation efficiency (EE) of different molecular weights (MW) of PEG in the copolymer, the amount of PELA, the amount of rhBMP-2, the span-20 concentration, the polyvinyl alcohol (PVA) concentration and stirring time were tested. On the basis of single-factor experiments, the optimum parameters were achieved using response surface methodology (RSM). The results showed that the highest EE of BMP-2 was achieved with a span-20 concentration of 0.5%, PEG MW 4000 Da, a stirring time of 30 min at 800 rpm min−1, 282.3 mg of PELA, 1 μg of rhBMP-2 and PVA concentration 0.79%. Under these optimal conditions, it was predicted that the highest EE to be achieved would be 76.5%; the actual EE achieved was 75%. (paper)

  18. Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration

    International Nuclear Information System (INIS)

    Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

  19. RhBMP-2 microspheres-loaded chitosan/collagen scaffold enhanced osseointegration: an experiment in dog.

    Science.gov (United States)

    Shi, Shanshan; Cheng, Xiangrong; Wang, Jiawei; Zhang, Wei; Peng, Lin; Zhang, Yufeng

    2009-01-01

    The purpose of this study is to develop a novel recombinant human bone morphogenetic protein-2 (rhBMP-2) sustained release scaffold for dental implant osseointegration, and to evaluate the effect of this scaffold on promoting bone formation. RhBMP-2 was encapsulated in the poly-D,L-lactide-co-glycolide (PLGA) biodegradable microspheres, which were subsequently dispersed in a chitosan/collagen composite scaffold. This rhBMP-2 microspheres-loaded scaffold (S-MB) was compared with a chitosan/collagen scaffold without microspheres that directly encapsulated rhBMP-2 (S-B) in vitro and in vivo. The microstructure of the new scaffold was examined with scanning electron microscopy. The release profile of rhBMP-2 in vitro was measured at interval periods. The effect of rhBMP-2 encapsulated scaffolds on enhancing bone formation through implantation in dogs' mandibles was identified by histological examination of the regenerated bone after 4 weeks of implantation. Due to PLGA microspheres being loaded, the S-MB exhibited lower values at porosity and swelling rate, as well as a higher effective release dose than that of the S-B. Bone density, bone-implant contact, and bone-fill values measured from dog experiments demonstrated that the S-MB induced bone regeneration more quickly and was timely substituted by new bone. It was concluded that this sustained carrier scaffold based on microspheres was more effective to induce implant osseointegration. PMID:18667455

  20. Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling.

    Directory of Open Access Journals (Sweden)

    Poulomi Ray

    Full Text Available Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF, Bone Morphogenetic Protein (BMP and Transforming Growth Factor beta (TGF-β signaling pathways. Rho Kinase (ROCK-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis.

  1. BMP-9 enhances fibroblast growth factor 21 expression and suppresses obesity.

    Science.gov (United States)

    Kim, Sooho; Choe, Senyon; Lee, Dong Kun

    2016-07-01

    Although BMP-9 has been reported to induce browning of white adipose tissues (WATs) and suppress high fat diet-induced obesity, detailed molecular mechanism needs to be further elucidated. We report here that administration of MB109, a recombinant derivative of human BMP-9, into obese mice enhanced gene expression of fibroblast growth factor 21 (FGF21), a metabolic regulator, and alleviates a spectrum of pathological symptoms due to high fat diet-induced obesity. In addition, periodical injection of MB109 (500μg/kg/week) reduced an amount of lipid droplets in the liver, serum levels of alanine aminotransferase (ALT), and total cholesterol. These results indicate that MB109 is also effective to treat obesity-mediated non-alcoholic fatty liver disease (NAFLD). PMID:27085971

  2. BMP-6 promotes E-cadherin expression through repressing δEF1 in breast cancer cells

    International Nuclear Information System (INIS)

    Bone morphogenetic protein-6 (BMP-6) is critically involved in many developmental processes. Recent studies indicate that BMP-6 is closely related to tumor differentiation and metastasis. Quantitative RT-PCR was used to determine the expression of BMP-6, E-cadherin, and δEF1 at the mRNA level in MCF-7 and MDA-MB-231 breast cancer cells, as well as in 16 breast cancer specimens. Immunoblot analysis was used to measure the expression of δEF1 at the protein level in δEF1-overexpressing and δEF1-interfered MDA-MB-231 cells. Luciferase assay was used to determine the rhBMP-6 or δEF1 driven transcriptional activity of the E-cadherin promoter in MDA-MB-231 cells. Quantitative CHIP assay was used to detect the direct association of δEF1 with the E-cadherin proximal promoter in MDA-MB-231 cells. MCF-7 breast cancer cells, an ER+ cell line that expressed high levels of BMP-6 and E-cadherin exhibited very low levels of δEF1 transcript. In contrast, MDA-MB-231 cells, an ER- cell line had significantly reduced BMP-6 and E-cadherin mRNA levels, suggesting an inverse correlation between BMP-6/E-cadherin and δEF1. To determine if the same relationship exists in human tumors, we examined tissue samples of breast cancer from human subjects. In 16 breast cancer specimens, the inverse correlation between BMP-6/E-cadherin and δEF1 was observed in both ER+ cases (4 of 8 cases) and ER- cases (7 of 8 cases). Further, we found that BMP-6 inhibited δEF1 transcription, resulting in an up-regulation of E-cadherin mRNA expression. This is consistent with our analysis of the E-cadherin promoter demonstrating that BMP-6 was a potent transcriptional activator. Interestingly, ectopic expression of δEF1 was able to block BMP-6-induced transactivation of E-cadherin, whereas RNA interference-mediated down-regulation of endogenous δEF1 in breast cancer cells abolished E-cadherin transactivation by BMP-6. In addition to down-regulating the expression of δEF1, BMP-6 also physically

  3. The chemical NMP as a potent BMP enhancer for bone tissue regeneration

    OpenAIRE

    San Miguel, B; Ghayor, C; Ehrbar, M.; Jung, R.E.; Zwahlen, R A; Hortschansky, P; Schmökel, H G; Weber, F. E.

    2009-01-01

    In medicine N-methylpyrrolidone (NMP) has a long track record as constituent in FDA approved medical devices and thus can be considered as safe and biological inactive small chemical. In the present study we report on the newly discovered pharmaceutical properties of NMP as it enhances bone regeneration in a rabbit calvarial defect model in vivo. At the cellular level, the pharmaceutical effect of NMP was confirmed, in particular, in combination with BMP-2, as NMP increased early and late mar...

  4. Scorpion Toxin, BmP01, Induces Pain by Targeting TRPV1 Channel

    OpenAIRE

    Md Abdul Hakim; Wenbin Jiang; Lei Luo; Bowen Li; Shilong Yang; Yuzhu Song; Ren Lai

    2015-01-01

    The intense pain induced by scorpion sting is a frequent clinical manifestation. To date, there is no established protocol with significant efficacy to alleviate the pain induced by scorpion envenomation. One of the important reasons is that, little information on pain-inducing compound from scorpion venoms is available. Here, a pain-inducing peptide (BmP01) has been identified and characterized from the venoms of scorpion (Mesobuthus martensii). In an animal model, intraplantar injection of ...

  5. Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

    International Nuclear Information System (INIS)

    It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown

  6. BMP-2 in der Therapie der Pseudarthrose langer Röhrenknochen

    OpenAIRE

    Hellriegel, Tom

    2010-01-01

    Effective therapy for long bone non-unions is still a challenge in trauma and orthopedic surgery and treatment is time and cost-intensive. Complications can lead to ensuing health-related problems for the patient and their ability to work can be restricted. An innovative approach to stimulate bone regeneration is the application of growth factors. Bone morphogenetic protein-2 (BMP-2) has a high osteoinductive capacity and might stimulate human non-union healing. The purpose of this stud...

  7. Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hye-Lim; Park, Hyun-Jung; Kwon, Arang [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Baek, Kyunghwa [Department of Pharmacology, College of Dentistry and Research Institute of Oral Science, Gangneung-Wonju National University, Gangneung 210-702, Gangwondo (Korea, Republic of); Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Baek, Jeong-Hwa, E-mail: baekjh@snu.ac.kr [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of)

    2014-05-01

    It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown.

  8. 伯氏疏螺旋体膜蛋白BmpA研究进展%Progresses on Borrelia burgdorferi Membrance Protein A (BmpA)

    Institute of Scientific and Technical Information of China (English)

    宝福凯; 赖名耀; 张云波; 董坚; 赵桂萍; 陈明清; 柳爱华

    2012-01-01

    Lyme disease, a global health concern, is a zoonosis, which has been a serious threat to human. The spiroehete Borrelia burgdorferi that is transmitted by the bite of hard tick (Ixodidae) is the pathogen of Lyme disease. Borrelia burgdorferi contains many membrane proteins with immungenicity and pathogenicity. Recent researches show that BmpA is an dominant immune protein of Borrelia burgdorferi, a laminin-binding protein, and an arthritogennic factor. Research progresses of BmpA protein in biological function, Lyme arthritis pathogenesis and diagnosis of lyme disease are reviewed.%莱姆病是一种人兽共患病,已严重威胁人类健康,成全球公共卫生问题,引起全球关注.伯氏疏螺旋体是莱姆病病原体,通过蜱叮咬传播而引起莱姆病,其表面存在的膜蛋白具有免疫性和致病性.BmpA (Borreli burgdorferi membrance protein A)是伯氏疏螺旋体的主要抗原之一,为层粘连蛋白结合蛋白,是莱姆关节炎的重要致病因子,对蛋白功能、诊断应用和莱姆关节炎致病机理三方面的研究进展进行概述.

  9. Lineage tracking of mesenchymal and endothelial progenitors in BMP-induced bone formation.

    Science.gov (United States)

    Kolind, Mille; Bobyn, Justin D; Matthews, Brya G; Mikulec, Kathy; Aiken, Alastair; Little, David G; Kalajzic, Ivo; Schindeler, Aaron

    2015-12-01

    To better understand the relative contributions of mesenchymal and endothelial progenitor cells to rhBMP-2 induced bone formation, we examined the distribution of lineage-labeled cells in Tie2-Cre:Ai9 and αSMA-creERT2:Col2.3-GFP:Ai9 reporter mice. Established orthopedic models of ectopic bone formation in the hind limb and spine fusion were employed. Tie2-lineage cells were found extensively in the ectopic bone and spine fusion masses, but co-staining was only seen with tartrate-resistant acid phosphatase (TRAP) activity (osteoclasts) and CD31 immunohistochemistry (vascular endothelial cells), and not alkaline phosphatase (AP) activity (osteoblasts). To further confirm the lack of a functional contribution of Tie2-lineage cells to BMP-induced bone, we developed conditional knockout mice where Tie2-lineage cells are rendered null for key bone transcription factor osterix (Tie2-cre:Osx(fx/fx) mice). Conditional knockout mice showed no difference in BMP-induced bone formation compared to littermate controls. Pulse labeling of mesenchymal cells with Tamoxifen in mice undergoing spine fusion revealed that αSMA-lineage cells contributed to the osteoblastic lineage (Col2.3-GFP), but not to endothelial cells or osteoclast populations. These data indicate that the αSMA+ and Tie2+ progenitor lineages make distinct cellular contributions to bone formation, angiogenesis, and resorption/remodeling. PMID:26141839

  10. BMP2 gene delivery to bone mesenchymal stem cell by chitosan-g-PEI nonviral vector

    Science.gov (United States)

    Yue, Jianhui; Wu, Jun; Liu, Di; Zhao, Xiaoli; Lu, William W.

    2015-04-01

    Nanotechnology has made a significant impact on the development of nanomedicine. Nonviral vectors have been attracting more attention for the advantage of biosafety in gene delivery. Polyethylenimine (PEI)-conjugated chitosan (chitosan-g-PEI) emerged as a promising nonviral vector and has been demonstrated in many tumor cells. However, there is a lack of study focused on the behavior of this vector in stem cells which hold great potential in regenerative medicine. Therefore, in this study, in vitro gene delivering effect of chitosan-g-PEI was investigated in bone marrow stem cells. pIRES2-ZsGreen1-hBMP2 dual expression plasmid containing both the ZsGreen1 GFP reporter gene and the BMP2 functional gene was constructed for monitoring the transgene expression level. Chitosan-g-PEI-mediated gene transfer showed 17.2% of transfection efficiency and more than 80% of cell viability in stem cells. These values were higher than that of PEI. The expression of the delivered BMP2 gene in stem cells enhanced the osteogenic differentiation. These results demonstrated that chitosan-g-PEI is capable of applying in delivering gene to stem cells and providing potential applications in stem cell-based gene therapy.

  11. Safety and efficacy of rhBMP2 in posterior cervical spinal fusion for subaxial degenerative spine disease: Analysis of outcomes in 204 patients

    OpenAIRE

    Xu, Risheng; Bydon, Mohamad; Sciubba, Daniel M.; Witham, Timothy F.; Wolinsky, Jean-Paul; Gokaslan, Ziya L; Bydon, Ali

    2011-01-01

    Background: Many studies offer excellent demonstration of the ability of bone morphogenic protein (BMP) to enhance fusion rates in anterior as well as posterior lumbar surgery. Recently, BMP has also been shown to increase arthrodesis rates in anterior cervical surgery, albeit with concomitant increases in complication rates. To date, however, few studies have investigated the safety and efficacy of BMP in cervical surgeries approached posteriorly. Methods: We retrospectively reviewed 204 con...

  12. Effects of bone morphogenic protein 4 (BMP4) and its inhibitor, Noggin, on in vitro maturation and culture of bovine preimplantation embryos

    OpenAIRE

    Fernandez-Martin Rafael; Pereira Michele M; Camargo Luiz SA; La Rosa Isabel; Paz Dante A; Salamone Daniel F

    2011-01-01

    Abstract Background BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments) and embryos in vitro development (c experiments) of bovine. Methods For m experiments, COCs were collected from slaughterhouse ovaries and in vitro matured in...

  13. Serum Heme Oxygenase-1 and BMP-7 Are Potential Biomarkers for Bone Metabolism in Patients with Rheumatoid Arthritis and Ankylosing Spondylitis

    Science.gov (United States)

    Yuan, Tong-ling; Chen, Jin; Tong, Yan-li; Zhang, Yan; Liu, Yuan-yuan; Wei, James Cheng-Chung; Liu, Yi; Herrmann, Martin

    2016-01-01

    Backgrounds. Heme oxygenase-1 (HO-1) has been reported to play a regulatory role in osteoclastogenesis. Bone morphogenetic protein (BMP) pathways induce osteoblastic differentiation and bone remodeling. Aims. To identify serum levels of HO-1, BMP-7, and Runt related-transcription factor 2 (Runx2) in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS) and to investigate the relationships between HO-1, BMP-7, Runx2, and other common biomarkers for bone metabolism. Results. Serum levels of HO-1 and BMP-7 were revealed to be significantly higher in patients with RA or AS than in healthy controls (p < 0.01). In RA group, HO-1 was positively correlated with BMP-7, Runx2, and tartrate-resistant acid phosphatase-5b (TRAP-5b) (p < 0.05, resp.), BMP-7 was positively correlated with Runx2 and TRAP-5b (p < 0.05, resp.), and Runx2 was negatively correlated with N-terminal midfragment of osteocalcin (NMID) (p < 0.05). In AS group, we observed identical correlation between HO-1 and BMP-7, but opposite correlations between BMP-7 and TRAP-5b and between Runx2 and NMID, when comparing with the RA cohort. Conclusion. Our findings suggest that HO-1 and BMP-7 are potential biomarkers for bone metabolism in patients with RA and AS. The different correlations between the bone markers point to distinct differences in bone remodeling pathways in the two types of arthritis. PMID:27314037

  14. Osteoinductivity Assessment of BMP-2 Loaded Composite Chitosan-Nano-Hydroxyapatite Scaffolds in a Rat Muscle Pouch

    Directory of Open Access Journals (Sweden)

    Warren O. Haggard

    2011-08-01

    Full Text Available The objective of this study was to evaluate the osteoinductivity of composite chitosan-nano-hydroxyapatite scaffolds in a rat muscle pouch model. Previous in vitro characterization demonstrated the ability of the scaffolds to promote bone regeneration and as a carrier for local delivery of BMP-2. Composite microspheres were prepared using a co-precipitation method, and scaffolds were fabricated using an acid wash to adhere beads together. To determine the in vivo osteoinductivity of the scaffolds, the following groups (n = 6 were implanted into muscle pouches created in the latissimus dorsi of Sprague Dawley rats: (A lyophilized scaffolds without rhBMP-2, (B lyophilized scaffolds with rhBMP-2, (C non-lyophilized scaffolds with rhBMP-2, and (D absorbable collagen sponge with rhBMP-2 (control. Groups B, C, and D were loaded with 4 mL of a 9.0 μg/mL solution of rhBMP-2 for 48 h. The rats were sacrificed after one month and samples were analyzed for amount of residual implant material, new bone, and osteoid. Although the experimental groups displayed minimal degradation after one month, all of the scaffolds contained small amounts of woven bone and considerable amounts of osteoid. Approximately thirty percent of the open space available for tissue ingrowth in the scaffolds contained new bone or osteoid in the process of mineralization. The ability of the composite scaffolds (with and without BMP-2 to promote ectopic bone growth in vivo was demonstrated.

  15. Abrogation of ospAB constitutively activates the Rrp2-RpoN-RpoS pathway (sigmaN-sigmaS cascade) in Borrelia burgdorferi.

    Science.gov (United States)

    He, Ming; Oman, Tara; Xu, Haijun; Blevins, Jon; Norgard, Michael V; Yang, X Frank

    2008-12-01

    Molecular mechanisms underlying the reciprocal regulation of the two major surface lipoproteins and virulence factors of Borrelia burgdorferi, OspA and OspC, are not fully understood. Herein, we report that inactivation of the ospAB operon resulted in overproduction of OspC and many other lipoproteins via the constitutive activation of the Rrp2-RpoN-RpoS pathway. Complementing the ospAB mutant with a wild-type copy of ospA, but not an ospA variant that lacks the lipoprotein signal sequence, restored normal regulation of the Rrp2-RpoN-RpoS pathway; these results indicate that the phenotype was not caused by spurious mutations. Interestingly, while most of the ospAB mutant clones displayed a constitutive ospC expression phenotype, some ospAB mutant clones showed little or no ospC expression. Further analyses revealed that this OspC-negative phenotype was independent of abrogation of ospAB. While activation of the Rrp2-RpoN-RpoS pathway was recently shown to downregulate ospA, our findings suggest that reduction of OspA can also activate this pathway. We postulate that the activation of the Rrp2-RpoN-RpoS pathway and downregulation of OspA form a positive feedback loop that allows spirochaetes to produce and maintain a constant high level of OspC and other lipoproteins during tick feeding, a strategy that is critical for spirochaetal transmission and mammalian infection. PMID:19019147

  16. Transplanted astrocytes derived from BMP- or CNTF-treated glial-restricted precursors have opposite effects on recovery and allodynia after spinal cord injury

    Directory of Open Access Journals (Sweden)

    Davies Jeannette E

    2008-09-01

    Full Text Available Abstract Background Two critical challenges in developing cell-transplantation therapies for injured or diseased tissues are to identify optimal cells and harmful side effects. This is of particular concern in the case of spinal cord injury, where recent studies have shown that transplanted neuroepithelial stem cells can generate pain syndromes. Results We have previously shown that astrocytes derived from glial-restricted precursor cells (GRPs treated with bone morphogenetic protein-4 (BMP-4 can promote robust axon regeneration and functional recovery when transplanted into rat spinal cord injuries. In contrast, we now show that transplantation of GRP-derived astrocytes (GDAs generated by exposure to the gp130 agonist ciliary neurotrophic factor (GDAsCNTF, the other major signaling pathway involved in astrogenesis, results in failure of axon regeneration and functional recovery. Moreover, transplantation of GDACNTF cells promoted the onset of mechanical allodynia and thermal hyperalgesia at 2 weeks after injury, an effect that persisted through 5 weeks post-injury. Delayed onset of similar neuropathic pain was also caused by transplantation of undifferentiated GRPs. In contrast, rats transplanted with GDAsBMP did not exhibit pain syndromes. Conclusion Our results show that not all astrocytes derived from embryonic precursors are equally beneficial for spinal cord repair and they provide the first identification of a differentiated neural cell type that can cause pain syndromes on transplantation into the damaged spinal cord, emphasizing the importance of evaluating the capacity of candidate cells to cause allodynia before initiating clinical trials. They also confirm the particular promise of GDAs treated with bone morphogenetic protein for spinal cord injury repair.

  17. The effects of 3D bioactive glass scaffolds and BMP-2 on bone formation in rat femoral critical size defects and adjacent bones

    International Nuclear Information System (INIS)

    Reconstruction of critical size defects in the load-bearing area has long been a challenge in orthopaedics. In the past, we have demonstrated the feasibility of using a biodegradable load-sharing scaffold fabricated from poly(propylene fumarate)/tricalcium phosphate (PPF/TCP) loaded with bone morphogenetic protein-2 (BMP-2) to successfully induce healing in those defects. However, there is limited osteoconduction observed with the PPF/TCP scaffold itself. For this reason, 13-93 bioactive glass scaffolds with local BMP-2 delivery were investigated in this study for inducing segmental defect repairs in a load-bearing region. Furthermore, a recent review on BMP-2 revealed greater risks in radiculitis, ectopic bone formation, osteolysis and poor global outcome in association with the use of BMP-2 for spinal fusion. We also evaluated the potential side effects of locally delivered BMP-2 on the structures of adjacent bones. Therefore, cylindrical 13-93 glass scaffolds were fabricated by indirect selective laser sintering with side holes on the cylinder filled with dicalcium phosphate dehydrate as a BMP-2 carrier. The scaffolds were implanted into critical size defects created in rat femurs with and without 10 μg of BMP-2. The x-ray and micro-CT results showed that a bridging callus was found as soon as three weeks and progressed gradually in the BMP group while minimal bone formation was observed in the control group. Degradation of the scaffolds was noted in both groups. Stiffness, peak load and energy to break of the BMP group were all higher than the control group. There was no statistical difference in bone mineral density, bone area and bone mineral content in the tibiae and contralateral femurs of the control and BMP groups. In conclusion, a 13-93 bioactive glass scaffold with local BMP-2 delivery has been demonstrated for its potential application in treating large bone defects. (paper)

  18. Influence of BMP-2 on early follicular development and mRNA expression of oocyte specific genes in bovine preantral follicles cultured in vitro.

    Science.gov (United States)

    Rossi, Rodrigo O D S; da Cunha, Ellen V; Portela, Antonia M L R; Passos, José R S; Costa, José J N; Silva, Anderson W B; Saraiva, Márcia V A; Peixoto, Christina A; Donato, Mariana A M; van den Hurk, Robert; Silva, José R V

    2016-03-01

    This study evaluates the effect of different concentrations (0, 10, 50 and 100ng/mL) of bone morphogenetic protein-2 (BMP-2) on primordial and secondary follicle development. It also investigates the effects of FSH and BMP-2 on the growth, morphology, ultrastructure and expression of mRNA for GDF9, NLRP5 and NPM2 genes in secondary follicles cultured for 18 days. The presence of BMP-2 at all tested concentrations increased the development of primordial follicles in vitro, but the highest concentration of BMP-2 (100 ng/mL) reduced the percentage of normal follicles when compared with tissues cultured with 10 ng/mL BMP-2. During culture of secondary follicles, in contrast to higher concentrations (50 or 100 ng/mL), 10 ng/mL BMP-2 kept the morphology of follicles during initial stages of in vitro culture. This concentration of BMP-2 also benefits maintenance of the ultrastructure of 18-day cultured follicles. The presence of both BMP-2 and FSH in culture medium resulted in a significant (PFSH and BMP-2 reduced follicular mRNA expression of GDF9 and NLRP5 when compared to follicles cultured in media containing only FSH. In combination with FSH, BMP-2 reduced the mRNA levels of NPM2, when compared to follicles cultured in control medium. It is concluded from these data that 10 ng/mL BMP-2 promotes the growth of primordial in vitro and it helps to maintain the ultrastructure of secondary follicles, while FSH is more important for better expression of follicular markers like GDF9 and NLRP5. PMID:26435174

  19. Reduction of Adipose Tissue Formation by the Controlled Release of BMP-2 Using a Hydroxyapatite-Coated Collagen Carrier System for Sinus-Augmentation/Extraction-Socket Grafting

    Directory of Open Access Journals (Sweden)

    Jung-Seok Lee

    2015-11-01

    Full Text Available The effects of hydroxyapatite (HA-coating onto collagen carriers for application of recombinant human bone morphogenetic protein 2 (rhBMP-2 on cell differentiation in vitro, and on in vivo healing patterns after sinus-augmentation and alveolar socket-grafting were evaluated. In vitro induction of osteogenic/adipogenic differentiation was compared between the culture media with rhBMP-2 solution and with the released rhBMP-2 from the control collagen and from the HA-coated collagen. Demineralized bovine bone and collagen/HA-coated collagen were grafted with/without rhBMP-2 in sinus-augmentation and tooth-extraction-socket models. Adipogenic induction by rhBMP-2 released from HA-coated collagen was significantly reduced compared to collagen. In the sinus-augmentation model, sites that received rhBMP-2 exhibited large amounts of vascular tissue formation at two weeks and increased adipose tissue formation at eight weeks; this could be significantly reduced by using HA-coated collagen as a carrier for rhBMP-2. In extraction-socket grafting, dimensional reduction of alveolar ridge was significantly decreased at sites received rhBMP-2 compared to control sites, but adipose tissue was increased within the regenerated socket area. In conclusion, HA-coated collagen carrier for Escherichia coli-derived rhBMP-2 (ErhBMP-2 may reduce in vitro induction of adipogenic differentiation and in vivo adipose bone marrow tissue formation in bone tissue engineering by ErhBMP-2.

  20. BMP7 enhances the effect of BMSCs on extracellular matrix remodeling in a rabbit model of intervertebral disc degeneration.

    Science.gov (United States)

    Xu, Jun; E, Xiao-Qiang; Wang, Nan-Xiang; Wang, Mo-Nan; Xie, Huan-Xin; Cao, Yan-Hui; Sun, Li-Hua; Tian, Jun; Chen, Hua-Jiang; Yan, Jing-Long

    2016-05-01

    Intervertebral discs (IVDs) provide stability and flexibility to the spinal column; however, IVDs, and in particular the nucleus pulposus (NP), undergo a degenerative process characterized by changes in the disc extracellular matrix (ECM), decreased cell viability, and reduced synthesis of proteoglycan and type II collagen. Here, we investigated the efficacy and feasibility of stem cell therapy using bone marrow mesenchymal stem cells (BMSCs) over-expressing bone morphogenetic protein 7 (BMP7) to promote ECM remodeling of degenerated IVDs. Lentivirus-mediated BMP7 over-expression induced differentiation of BMSCs into an NP phenotype, as indicated by expression of the NP markers collagen type II, aggrecan, SOX9 and keratins 8 and 19, increased the content of glycosaminoglycan, and up-regulated β-1,3-glucuronosyl transferase 1, a regulator of chondroitin sulfate synthesis in NP cells. These effects were suppressed by Smad1 silencing, indicating that the effect of BMP7 on ECM remodeling was mediated by the Smad pathway. In vivo analysis in a rabbit model of disc degeneration showed that implantation of BMSCs over-expressing BMP7 promoted cell differentiation and proliferation in the NP, as well as their own survival, and these effects were mediated by the Smad pathway. The results of the present study indicate the beneficial effects of BMP7 on restoring ECM homeostasis in NP cells, and suggest potential strategies for improving cell therapy for the treatment of disc diseases. PMID:26929154

  1. Segmental bone regeneration using rhBMP-2-loaded collagen/chitosan microspheres composite scaffold in a rabbit model

    International Nuclear Information System (INIS)

    The reconstruction of segmental bone defects remains an urgent problem in the orthopaedic field, and bone morphogenetic protein-2 (BMP-2) is known for its potent osteoinductive properties in bone regeneration. In this study, chitosan microspheres (CMs) were prepared and combined with absorbable collagen sponge to maintain controlled-release recombinant human bone morphogenetic protein-2 (rhBMP-2). The rhBMP-2-loaded composite scaffolds were implanted into 15 mm radius defects of rabbits and the bone-repair ability was evaluated systematically. CMs were spherical in shape and had a polyporous surface, according to SEM images. The complex scaffold exhibited an ideal releasing profile in vitro. The micro-computed tomographic analysis revealed that the rhBMP-2-loaded composite scaffold not only bridged the defects as early as 4 weeks, but also healed the defects and presented recanalization of the bone-marrow cavity at 12 weeks. These results were confirmed by x-ray. When compared with other control groups, the composite scaffold group remarkably enhanced new bone formation and mechanical properties, as evidenced by bone mineral content evaluation, histological observations and biomechanical testing. Moreover, the biocompatibility and appropriate degradation of the composite scaffold could be obtained. All of these results clearly demonstrated that the composite scaffold is a promising carrier of BMP-2 for the treatment of segmental bone defects. (paper)

  2. A composited PEG-silk hydrogel combining with polymeric particles delivering rhBMP-2 for bone regeneration.

    Science.gov (United States)

    Ma, Dakun; An, Gang; Liang, Min; Liu, Yugang; Zhang, Bin; Wang, Yansong

    2016-08-01

    Given the fabulous potential of promoting bone regeneration, BMP-2 has been investigated widely in the bone tissue engineering field. A sophisticated biomaterial loaded with BMP-2, which could avoid the required supraphysiological dose leading to high medical costs and risks of complications, has been considered as a promising strategy to treat non-healing bone defects. In this study, we developed a simple approach to engineer a composited hydrogel consisting polymeric particles (PLA/PLGA) used as a BMP-2 delivery vehicle. Compared with other groups, the introduction of PLA into PEG-silk gels endowed the hydrogel new physicochemical characteristics especially hydrophobicity which inhibited the burst release of BMP-2 and enhanced gel's structural stability. Moreover, such composited gels could stabilize entrapped proteins and maintain their bioactivity fully in vitro. In vivo, the bio-degradability experiment demonstrated this system was biocompatible and the reinforced hydrophobicity significantly decreased degradation rate, and in rat critical-sized cranial defects model, the gel containing PLA promoted the most bone formation. These findings demonstrated the introduction of PLA changed physicochemical features of gels more suitable as a BMP-2 carrier indicated by inducing bone regeneration efficiently in large bone defects at low delivered dose and this system may own translational potential. PMID:27157747

  3. Attenuation of bone morphogenetic protein signaling during amphibian limb development results in the generation of stage-specific defects.

    Science.gov (United States)

    Jones, Tamsin E M; Day, Robert C; Beck, Caroline W

    2013-11-01

    The vertebrate limb is one of the most intensively studied organs in the field of developmental biology. Limb development in tetrapod vertebrates is highly conserved and dependent on the interaction of several important molecular pathways. The bone morphogenetic protein (BMP) signaling cascade is one of these pathways and has been shown to be crucial for several aspects of limb development. Here, we have used a Xenopus laevis transgenic line, in which expression of the inhibitor Noggin is under the control of the heat-shock promoter hsp70 to examine the effects of attenuation of BMP signaling at different stages of limb development. Remarkably different phenotypes were produced at different stages, illustrating the varied roles of BMP in development of the limb. Very early limb buds appeared to be refractory to the effects of BMP attenuation, developing normally in most cases. Ectopic limbs were produced by overexpression of Noggin corresponding to a brief window of limb development at about stage 49/50, as recently described by Christen et al. (2012). Attenuation of BMP signaling in stage 51 or 52 tadpoles lead to a reduction in the number of digits formed, resulting in hypodactyly or ectrodactyly, as well as occasional defects in the more proximal tibia-fibula. Finally, inhibition at stage 54 (paddle stage) led to the formation of dramatically shortened digits resulting from loss of distal phalanges. Transcriptome analysis has revealed the possibility that more Noggin-sensitive members of the BMP family could be involved in limb development than previously suspected. Our analysis demonstrates the usefulness of heat-shock-driven gene expression as an effective method for inhibiting a developmental pathway at different times during limb development. PMID:23981117

  4. Investigation of Serum BMP-7 and Insulin Resistance Levels in Pregnant Women%肥胖孕妇血清BMP-7及胰岛素抵抗水平的研究

    Institute of Scientific and Technical Information of China (English)

    汪小娟; 郝艳华; 李建梅; 刘莉莉; 刘香萍; 李小永

    2012-01-01

    Objective To investigate the changes and significances of serum BMP-7 and insulin resistance levels in obese pregnant women. Methods A number of 22 obese pregnant women were presented as case group, and 122 normal pregnant women were presented as control group. Fasting serum BMP-7,fasting glucose and fasting insulin (FINS) were examined to evaluate the insulin resistance index (HOMA-IR). Results The serum level of BMP-7 was significantly higher in obese pregnant women than that in normal pregnant women (P<0. 05) ,with an average of 1 619. 61i633. 23 ng/L and 1 472. 05 + 1 196. 18 ng/L,respectively. The HOMA-IR in obese pregnant women and normal pregnant women was 2. 30±l. 21 and 1. 80± 1. 41, respectively. Significant difference was obtained between these two groups (P<0. 05). Statistical Results showed that fasting serum BMP-7 level had a positive correlation with FINS (P<0. 05). Conclusion The rising level of serum BMP-7 in obese pregnant women may be involved in pathological insulin resistance during pregnancy.%目的 探讨肥胖孕妇血清BMP-7(人骨成形蛋白7)和胰岛素抵抗水平的变化及意义.方法 选取妊娠期肥胖孕妇22例,正常妊娠孕妇122例,测定空腹血清BMP-7、空腹血糖、空腹胰岛素(FINS),计算胰岛素抵抗指数(HOMA-IR).结果 妊娠期肥胖组血清BMP-7含量平均值1 619.61±633.23 ng/L,正常妊娠组血清BMP-7含量平均值1 472.05±1196.18 ng/L;妊娠期肥胖组血清BMP-7水平明显高于正常妊娠组(P<0.05);妊娠期肥胖组胰岛素抵抗指数平均值2.30±1.21,正常妊娠组胰岛素抵抗指数平均值1.80±1.41,两组之间的差异有统计学意义(P<0.05);分析结果显示,空腹血清BMP-7水平与FINS呈正相关(P<0.05).结论 妊娠期肥胖组孕妇血清BMP-7水平的升高,可能参与了妊娠期糖尿病病理性胰岛素抵抗的发生.

  5. Abrogating endocrine resistance by targeting ER alpha and PI3K in breast cancer

    Directory of Open Access Journals (Sweden)

    ToddWMiller

    2012-10-01

    Full Text Available Antiestrogen therapies targeting estrogen receptor alpha (ER signaling are a mainstay for patients with ER+ breast cancer. While many cancers exhibit resistance to antiestrogen therapies, a large body of clinical and experimental evidence indicates that hyperactivation of the phosphatidylinositol 3-kinase (PI3K pathway promotes antiestrogen resistance. In addition, continued ligand-independent ER signaling in the setting of estrogen deprivation may contribute to resistance to endocrine therapy. PI3K activates several proteins which promote cell cycle progression and survival. In ER+ breast cancer cells, PI3K promotes ligand-dependent and -independent ER transcriptional activity. Models of antiestrogen-resistant breast cancer often remain sensitive to estrogen stimulation and PI3K inhibition, suggesting that clinical trials with combinations of drugs targeting both the PI3K and ER pathways are warranted. Herein, we review recent findings on the roles of PI3K and ER in antiestrogen resistance, and clinical trials testing drug combinations which target both pathways. We also discuss the need for clinical investigation of ER downregulators in combination with PI3K inhibitors.

  6. Intestinal microbiota shifts towards elevated commensal Escherichia coli loads abrogate colonization resistance against Campylobacter jejuni in mice.

    Directory of Open Access Journals (Sweden)

    Lea-Maxie Haag

    Full Text Available BACKGROUND: The zoonotic pathogen Campylobacter jejuni is a leading cause of bacterial foodborne enterocolitis in humans worldwide. The understanding of immunopathology underlying human campylobacteriosis is hampered by the fact that mice display strong colonization resistance against the pathogen due to their host specific gut microbiota composition. METHODOLOGY/PRINCIPAL FINDINGS: Since the microbiota composition changes significantly during intestinal inflammation we dissected factors contributing to colonization resistance against C. jejuni in murine ileitis, colitis and in infant mice. In contrast to healthy animals C. jejuni could stably colonize mice suffering from intestinal inflammation. Strikingly, in mice with Toxoplasma gondii-induced acute ileitis, C. jejuni disseminated to mesenteric lymphnodes, spleen, liver, kidney, and blood. In infant mice C. jejuni infection induced enterocolitis. Mice suffering from intestinal inflammation and C. jejuni susceptible infant mice displayed characteristical microbiota shifts dominated by increased numbers of commensal Escherichia coli. To further dissect the pivotal role of those distinct microbiota shifts in abrogating colonization resistance, we investigated C. jejuni infection in healthy adult mice in which the microbiota was artificially modified by feeding live commensal E. coli. Strikingly, in animals harboring supra-physiological intestinal E. coli loads, colonization resistance was significantly diminished and C. jejuni infection induced enterocolitis mimicking key features of human campylobacteriosis. CONCLUSION/SIGNIFICANCE: Murine colonization resistance against C. jejuni is abrogated by changes in the microbiota composition towards elevated E. coli loads during intestinal inflammation as well as in infant mice. Intestinal inflammation and microbiota shifts thus represent potential risk factors for C. jejuni infection. Corresponding interplays between C. jejuni and microbiota might

  7. Abrogation of Age-Induced MicroRNA-195 Rejuvenates the Senescent Mesenchymal Stem Cells by Reactivating Telomerase.

    Science.gov (United States)

    Okada, Motoi; Kim, Ha Won; Matsu-ura, Kaoru; Wang, Yi-Gang; Xu, Meifeng; Ashraf, Muhammad

    2016-01-01

    Previously, we reported that a novel subpopulation of young mesenchymal stem cells (YMSCs) existed in old bone marrow, which possessed high antiaging properties as well as excellent efficacy for cardiac repair. MicroRNAs (miRNAs) have emerged as key regulators in post-transcriptional gene expression programs, and however, it is unknown whether miRNAs directly control stem cell senescence. Here we present the first evidence that miR-195 overexpressed in old MSCs (OMSCs) induces stem cell senescence deteriorating their regenerative ability by directly deactivating telomerase reverse transcriptase (Tert), and abrogation of miR-195 can reverse stem cell aging. MiRNAs profiling analysis in YMSCs and OMSCs by microarray showed that miR-140, miR-146a/b, and miR-195 were significantly upregulated in OMSCs, which led us to hypothesize that these are age-induced miRNAs involved in stem cell senescence. Of these miRNAs, we found miR-195 directly targeted 3'-untranslated region of Tert gene by computational target prediction analysis and luciferase assay, and knockdown of miR-195 significantly increased Tert expression in OMSCs. Strikingly, miR-195 inhibition significantly induced telomere relengthening in OMSCs along with reduced expression of senescence-associated β-galactosidase. Moreover, silencing miR-195 in OMSCs by transfection of miR-195 inhibitor significantly restored antiaging factors expression including Tert and Sirt1 as well as phosphorylation of Akt and FOXO1. Notably, abrogation of miR-195 markedly restored proliferative abilities in OMSCs. Transplantation of OMSCs with knocked out miR-195 reduced infarction size and improved LV function. In conclusion, rejuvenation of aged stem cells by miR-195 inhibition would be a promising autologous therapeutic strategy for cardiac repair in the elderly patients. PMID:26390028

  8. BMP2基因修饰犬脂肪源性基质细胞修复自体大段骨缺损%Repairing canine segmental bone defects using BMP2 gene modified adipose-derived stromal cells

    Institute of Scientific and Technical Information of China (English)

    李慧武; 戴尅戎; 汤亭亭; 张晓玲; 唐坚; 孙晓江; 张双燕; 楼觉人

    2008-01-01

    Objective To evaluate osteogenetic effectiveness of porous β-tricalcium phosphate(β-TCP) ceramic mixed with human bone morphogenetic protein2 gene(Adv-hBMP2)modified adipose derived stromal cells (ADSCs) in the repair of critical-sized bone defects..Methods The ADSCs taken from the back of beagle dogs were modified by the BMP2 gene.The expression and bone-induction ability of BMP2 was identified by ELISA and ectopic bone formation in nude mice.The cells were applied to a β-tricalcium phosphate (TGP)carrier and implanted into ulnar bone defects in the canine model.18 ulnar bone defects were divided into three groups randomly and filled with granular TCP alone,granular TCP and ADSCs,or TCP and ADSCs transduced with Adv-hBMP2 respectively.All dogs were followed clinically and roentgenographically for 16 weeks and then sacrificed.Results ELISA and ectopic bone formation in nude mice showed the recombinant ADSCs could express BMP2 highly and stably.No bone defects healed after implanting granular TCP alone or granular TCP and ADSCs.In the TCP and ADSCs transduced with AdvhBMP2 group,two defects healed,four partly healed.Histological examination showed woven bone at the both end of the cortices but entirelv fibrous tissue in the middle in which defects filled with TCP alone or TCP and ADSCs.Defects filled with TCP and transduced ADSCs showed substatial new bone formation.Histomorphometry showed TCP combined with ADSCs did not significantly increase new bone area compared with TCP alone.TCP and recombinant ADSCs produced a significant increase in newly formed bone area.Conclusion ADSCs tansduced with BMP2 gene in a TCP carrier can enhance bone regeneratmn to repair the critically-sized bone defect.%目的 评价BMP2基因修饰的犬脂肪源性基质细胞(ADSCs)与β-磷酸三钙(β-TCP)复合修复自体大段骨缺损的疗效.方法 从比格犬背部脂肪组织中提取基质细胞,转染腺病毒介导的人BMP2基因(Adv-hBMP2),通过ELISA和裸鼠体内异位成骨实验鉴定BMP

  9. Induction of osteoconductivity by BMP-2 gene modification of mesenchymal stem cells combined with plasma-sprayed hydroxyapatite coating

    Science.gov (United States)

    Wu, Jiang; Guo, Ying-qiang; Yin, Guang-fu; Chen, Huai-qing; Kang, Yunqing

    2008-11-01

    Success in bone implant depends greatly on the composition and surface features of the implant. The surface-modification measures not only favor the implant's osteoconductivity, but also promote both bone anchoring and biomechanical stability. This paper reports an approach to combine a hydroxyapatite (HA) coated substrate with a cellular vehicle for the delivery of bone morphogenetic protein-2 (BMP-2) synergistically enhancing the osteoconductivity of implant surfaces. We examined the attachment, growth and osteoinductive activity of transfected BMP-producing bone marrow mesenchymal stem cells (BMSCs) on a plasma-sprayed HA coated substrate. It was found that the HA coated substrate could allow the attachment and growth of BMP-2 gene modified BMSCs, and this combined application synergistically enhanced osteconductivity of the substrate surface. This synergistic method may be of osseointegration value in orthopedic and dental implant surgery.

  10. Identification of Bmpr-1b and Bmp15 gene mutations in fat tail sheep

    OpenAIRE

    Maskur .; Chairussyuhur Arman

    2012-01-01

    Fat tail sheep (FTS) is regarded as highly prolific local sheep and have been well adapted under tropical climate of Lombok island. BMPR-1B and BMP15 genes that controll reproductive traits such as ovulation rate and litter size in different type of sheep will be studied as candidate genes for prolific traits in FTS. These genes  have been reported by various investigators have different prolificacy mechanism between several breeds of sheep, and it is very likely will occur in FTS. This study...

  11. Evaluation and modeling of biochemical methane potential (BMP) of landfilled solid waste: a pilot scale study

    DEFF Research Database (Denmark)

    Bilgili, M Sinan; Demir, Ahmet; Varank, Gamze

    2009-01-01

    The main goal of this study was to present a comparison of landfill performance with respect to solids decomposition. Biochemical methane potential (BMP) test was used to determine the initial and the remaining CH(4) potentials of solid wastes during 27 months of landfilling operation in two pilot...... scale landfill reactors. The initial methane potential of solid wastes filled to the reactors was around 0.347 L/CH(4)/g dry waste, which decreased with operational time of landfill reactors to values of 0.117 and 0.154 L/CH(4)/g dry waste for leachate recirculated (R1) and non-recirculated (R2...

  12. Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration.

    Science.gov (United States)

    Bouyer, Michael; Guillot, Raphael; Lavaud, Jonathan; Plettinx, Cedric; Olivier, Cécile; Curry, Véronique; Boutonnat, Jean; Coll, Jean-Luc; Peyrin, Françoise; Josserand, Véronique; Bettega, Georges; Picart, Catherine

    2016-10-01

    The rapid and effective bone regeneration of large non-healing defects remains challenging. Bioactive proteins, such as bone morphogenetic protein (BMP)-2, are proved their osteoinductivity, but their clinical use is currently limited to collagen as biomaterial. Being able to deliver BMP-2 from any other biomaterial would broaden its clinical use. This work presents a novel means for repairing a critical size volumetric bone femoral defect in the rat by combining a osteoinductive surface coating (2D) to a polymeric scaffold (3D hollow tube) made of commercially-available PLGA. Using a polyelectrolyte film as BMP-2 carrier, we tune the amount of BMP-2 loaded in and released from the polyelectrolyte film coating over a large extent by controlling the film crosslinking level and initial concentration of BMP-2 in solution. Using microcomputed tomography and quantitative analysis of the regenerated bone growth kinetics, we show that the amount of newly formed bone and kinetics can be modulated: an effective and fast repair was obtained in 1-2 weeks in the best conditions, including complete defect bridging, formation of vascularized and mineralized bone tissue. Histological staining and high-resolution computed tomography revealed the presence of bone regeneration inside and around the tube with spatially distinct organization for trabecular-like and cortical bones. The amount of cortical bone and its thickness increased with the BMP-2 dose. In view of the recent developments in additive manufacturing techniques, this surface-coating technology may be applied in combination with various types of polymeric or metallic scaffolds to offer new perspectives of bone regeneration in personalized medicine. PMID:27454063

  13. BMP2 genetically engineered MSCs and EPCs promote vascularized bone regeneration in rat critical-sized calvarial bone defects.

    Directory of Open Access Journals (Sweden)

    Xiaoning He

    Full Text Available Current clinical therapies for critical-sized bone defects (CSBDs remain far from ideal. Previous studies have demonstrated that engineering bone tissue using mesenchymal stem cells (MSCs is feasible. However, this approach is not effective for CSBDs due to inadequate vascularization. In our previous study, we have developed an injectable and porous nano calcium sulfate/alginate (nCS/A scaffold and demonstrated that nCS/A composition is biocompatible and has proper biodegradability for bone regeneration. Here, we hypothesized that the combination of an injectable and porous nCS/A with bone morphogenetic protein 2 (BMP2 gene-modified MSCs and endothelial progenitor cells (EPCs could significantly enhance vascularized bone regeneration. Our results demonstrated that delivery of MSCs and EPCs with the injectable nCS/A scaffold did not affect cell viability. Moreover, co-culture of BMP2 gene-modified MSCs and EPCs dramatically increased osteoblast differentiation of MSCs and endothelial differentiation of EPCs in vitro. We further tested the multifunctional bone reconstruction system consisting of an injectable and porous nCS/A scaffold (mimicking the nano-calcium matrix of bone and BMP2 genetically-engineered MSCs and EPCs in a rat critical-sized (8 mm caviarial bone defect model. Our in vivo results showed that, compared to the groups of nCS/A, nCS/A+MSCs, nCS/A+MSCs+EPCs and nCS/A+BMP2 gene-modified MSCs, the combination of BMP2 gene -modified MSCs and EPCs in nCS/A dramatically increased the new bone and vascular formation. These results demonstrated that EPCs increase new vascular growth, and that BMP2 gene modification for MSCs and EPCs dramatically promotes bone regeneration. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for CSBDs.

  14. BMP2 Genetically Engineered MSCs and EPCs Promote Vascularized Bone Regeneration in Rat Critical-Sized Calvarial Bone Defects

    Science.gov (United States)

    He, Xiaoning; Dziak, Rosemary; Yuan, Xue; Mao, Keya; Genco, Robert; Swihart, Mark; Sarkar, Debanjan; Li, Chunyi; Wang, Changdong; Lu, Li; Andreadis, Stelios; Yang, Shuying

    2013-01-01

    Current clinical therapies for critical-sized bone defects (CSBDs) remain far from ideal. Previous studies have demonstrated that engineering bone tissue using mesenchymal stem cells (MSCs) is feasible. However, this approach is not effective for CSBDs due to inadequate vascularization. In our previous study, we have developed an injectable and porous nano calcium sulfate/alginate (nCS/A) scaffold and demonstrated that nCS/A composition is biocompatible and has proper biodegradability for bone regeneration. Here, we hypothesized that the combination of an injectable and porous nCS/A with bone morphogenetic protein 2 (BMP2) gene-modified MSCs and endothelial progenitor cells (EPCs) could significantly enhance vascularized bone regeneration. Our results demonstrated that delivery of MSCs and EPCs with the injectable nCS/A scaffold did not affect cell viability. Moreover, co-culture of BMP2 gene-modified MSCs and EPCs dramatically increased osteoblast differentiation of MSCs and endothelial differentiation of EPCs in vitro. We further tested the multifunctional bone reconstruction system consisting of an injectable and porous nCS/A scaffold (mimicking the nano-calcium matrix of bone) and BMP2 genetically-engineered MSCs and EPCs in a rat critical-sized (8 mm) caviarial bone defect model. Our in vivo results showed that, compared to the groups of nCS/A, nCS/A+MSCs, nCS/A+MSCs+EPCs and nCS/A+BMP2 gene-modified MSCs, the combination of BMP2 gene -modified MSCs and EPCs in nCS/A dramatically increased the new bone and vascular formation. These results demonstrated that EPCs increase new vascular growth, and that BMP2 gene modification for MSCs and EPCs dramatically promotes bone regeneration. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for CSBDs. PMID:23565253

  15. Enhancement of the Regenerative Potential of Anorganic Bovine Bone Graft Utilizing a Polyglutamate-Modified BMP2 Peptide with Improved Binding to Calcium-Containing Materials.

    Science.gov (United States)

    Bain, Jennifer L; Bonvallet, Paul P; Abou-Arraj, Ramzi V; Schupbach, Peter; Reddy, Michael S; Bellis, Susan L

    2015-09-01

    Autogenous bone is the gold standard material for bone grafting in craniofacial and orthopedic regenerative medicine. However, due to complications associated with harvesting donor bone, clinicians often use commercial graft materials that may lose their osteoinductivity due to processing. This study was aimed to functionalize one of these materials, anorganic bovine bone (ABB), with osteoinductive peptides to enhance regenerative capacity. Two peptides known to induce osteoblastic differentiation of mesenchymal stem cells were evaluated: (1) DGEA, an amino acid motif within collagen I and (2) a biomimetic peptide derived from bone morphogenic protein 2 (BMP2pep). To achieve directed coupling of the peptides to the graft surface, the peptides were engineered with a heptaglutamate domain (E7), which confers specific binding to calcium moieties within bone mineral. Peptides with the E7 domain exhibited greater anchoring to ABB than unmodified peptides, and E7 peptides were retained on ABB for at least 8 weeks in vivo. To assess the osteoinductive potential of the peptide-conjugated ABB, ectopic bone formation was evaluated utilizing a rat subcutaneous pouch model. ABB conjugated with full-length recombinant BMP2 (rBMP2) was also implanted as a model for current clinical treatments utilizing rBMP2 passively adsorbed to carriers. These studies showed that E7BMP2pep/ABB samples induced more new bone formation than all other peptides, and an equivalent amount of new bone as compared with rBMP2/ABB. A mandibular defect model was also used to examine intrabony healing of peptide-conjugated ABB. Bone healing was monitored at varying time points by positron emission tomography imaging with (18)F-NaF, and it was found that the E7BMP2pep/ABB group had greater bone metabolic activity than all other groups, including rBMP2/ABB. Importantly, animals implanted with rBMP2/ABB exhibited complications, including inflammation and formation of cataract-like lesions in the eye, whereas

  16. Betulinic acid synergically enhances BMP2-induced bone formation via stimulating Smad 1/5/8 and p38 pathways

    OpenAIRE

    Choi, Hyuck; Jeong, Byung-Chul; Kook, Min-Suk; Koh, Jeong-Tae

    2016-01-01

    Background Healing of bone defects is a dynamic and orchestrated process that relies on multiple growth factors and cell types. Bone morphogenetic protein 2 (BMP2) is a key growth factor for bone healing, which stimulates mesenchymal stem cells to differentiate into osteoblasts. Betulinic acid (BetA) is a natural pentacyclic triterpenoid from plants. This study aimed to examine combinatory effects of BetA and BMP2 on ectopic bone generation in mice. Results In MC3T3-E1 preosteoblast culture, ...

  17. Repair of Cranial Bone Defects Using rhBMP2 and Submicron Particle of Biphasic Calcium Phosphate Ceramics with Through-Hole

    Directory of Open Access Journals (Sweden)

    Byung-Chul Jeong

    2015-01-01

    Full Text Available Recently a submicron particle of biphasic calcium phosphate ceramic (BCP with through-hole (donut-shaped BCP (d-BCP was developed for improving the osteoconductivity. This study was performed to examine the usefulness of d-BCP for the delivery of osteoinductive rhBMP2 and the effectiveness on cranial bone regeneration. The d-BCP was soaked in rhBMP2 solution and then freeze-dried. Scanning electron microscope (SEM, energy dispersive spectroscopy (EDS, and Raman spectroscopy analyses confirmed that rhBMP2 was well delivered onto the d-BCP surface and the through-hole. The bioactivity of the rhBMP2/d-BCP composite was validated in MC3T3-E1 cells as an in vitro model and in critical-sized cranial defects in C57BL/6 mice. When freeze-dried d-BCPs with rhBMP2 were placed in transwell inserts and suspended above MC3T3-E1, alkaline phosphatase activity and osteoblast-specific gene expression were increased compared to non-rhBMP2-containing d-BCPs. For evaluating in vivo effectiveness, freeze-dried d-BCPs with or without rhBMP2 were implanted into critical-sized cranial defects. Microcomputed tomography and histologic analysis showed that rhBMP2-containing d-BCPs significantly enhanced cranial bone regeneration compared to non-rhBMP2-containing control. These results suggest that a combination of d-BCP and rhBMP2 can accelerate bone regeneration, and this could be used to develop therapeutic strategies in hard tissue healing.

  18. Potential Roles of Stevia rebaudiana Bertoni in Abrogating Insulin Resistance and Diabetes: A Review

    Directory of Open Access Journals (Sweden)

    Nabilatul Hani Mohd-Radzman

    2013-01-01

    Full Text Available Insulin resistance is a key factor in metabolic disorders like hyperglycemia and hyperinsulinemia, which are promoted by obesity and may later lead to Type II diabetes mellitus. In recent years, researchers have identified links between insulin resistance and many noncommunicable illnesses other than diabetes. Hence, studying insulin resistance is of particular importance in unravelling the pathways employed by such diseases. In this review, mechanisms involving free fatty acids, adipocytokines such as TNFα and PPARγ and serine kinases like JNK and IKKβ, asserted to be responsible in the development of insulin resistance, will be discussed. Suggested mechanisms for actions in normal and disrupted states were also visualised in several manually constructed diagrams to capture an overall view of the insulin-signalling pathway and its related components. The underlying constituents of medicinal significance found in the Stevia rebaudiana Bertoni plant (among other plants that potentiate antihyperglycemic activities were explored in further depth. Understanding these factors and their mechanisms may be essential for comprehending the progression of insulin resistance towards the development of diabetes mellitus.

  19. Abrogation of junctional adhesion molecule-A expression induces cell apoptosis and reduces breast cancer progression.

    Directory of Open Access Journals (Sweden)

    Masato Murakami

    Full Text Available Intercellular junctions promote homotypic cell to cell adhesion and transfer intracellular signals which control cell growth and apoptosis. Junctional adhesion molecule-A (JAM-A is a transmembrane immunoglobulin located at tight junctions of normal epithelial cells of mammary ducts and glands. In the present paper we show that JAM-A acts as a survival factor for mammary carcinoma cells. JAM-A null mice expressing Polyoma Middle T under MMTV promoter develop significantly smaller mammary tumors than JAM-A positive mice. Angiogenesis and inflammatory or immune infiltrate were not statistically modified in absence of JAM-A but tumor cell apoptosis was significantly increased. Tumor cells isolated from JAM-A null mice or 4T1 cells incubated with JAM-A blocking antibodies showed reduced growth and increased apoptosis which paralleled altered junctional architecture and adhesive function. In a breast cancer clinical data set, tissue microarray data show that JAM-A expression correlates with poor prognosis. Gene expression analysis of mouse tumor samples showed a correlation between genes enriched in human G3 tumors and genes over expressed in JAM-A +/+ mammary tumors. Conversely, genes enriched in G1 human tumors correlate with genes overexpressed in JAM-A-/- tumors. We conclude that down regulation of JAM-A reduces tumor aggressive behavior by increasing cell susceptibility to apoptosis. JAM-A may be considered a negative prognostic factor and a potential therapeutic target.

  20. Autophagy-related gene 5 and Wnt5 signaling pathway requires differentiation of embryonic stem cells into odontoblast-like cells.

    Science.gov (United States)

    Ozeki, Nobuaki; Mogi, Makio; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kawai, Rie; Kondo, Ayami; Matsumoto, Toru; Nakata, Kazuhiko

    2016-02-01

    We previously confirmed a unique and unanticipated role for an α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and matrix metalloproteinase (MMP)-3-mediated signaling cascade, in driving the odontoblast-like differentiation of mouse embryonic stem (ES) cells in a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). To explore the early signaling cascade for odontoblastic differentiation, we examined the upregulation of autophagy-related gene (Atg) and Wnt signaling by CS/BMP-4 mediated odontoblast differentiation. In a screening experiment, CS/BMP-4 increased the mRNA and protein levels of Atg5, Lrp5/Fzd9 (an Atg5 receptor), and Wnt5, but not microtubule-associated protein 1 light chain (LC3; a mammalian homolog of yeast Atg8), TFE3, Beclin1, and Atg12, together with the amount of autophagosomes and autophagy fluxes. Treatment with siRNAs against Atg5 and Wnt5 individually suppressed the CS/BMP-4-induced increase in odontoblast differentiation. The odontoblastic phenotype, involving dentin matrix protein-1 and dentin sialophosphoprotein expression, decreased when autophagy was inhibited by chloroquine, but increased after treatment with rapamycin (an autophagy enhancer). Taken together with our previous findings, we have revealed a unique sequential cascade involving Atg5, Wnt5a, α2 integrin, Emmprin, and MMP-3. This cascade results in a potent increase in odontoblastic cell differentiation, indicating the unique involvement of Atg5, autophagy and Wnt5 signaling in CS/BMP-4-induced differentiation of ES cells into odontoblast-like cells, at a relatively early stage. PMID:26806855

  1. Evaluation of nanostructure and microstructure of bone regenerated by BMP-2-porous scaffolds.

    Science.gov (United States)

    Del Rosario, Carlos; Rodríguez-Evora, Maria; Reyes, Ricardo; González-Orive, Alejandro; Hernández-Creus, Alberto; Shakesheff, Kevin M; White, Lisa J; Delgado, Araceli; Evora, Carmen

    2015-09-01

    In this study, three systems containing BMP-2 were fabricated, including two electrospun sandwich-like-systems of PLGA 75:25 and PLGA 50:50 and a microsphere system of PLGA 50:50 to be implanted in a critical size defect in rat calvaria. The in vivo BMP-2 release profiles of the three systems were similar. The total dose was released during the first two weeks. To evaluate the nano and microstructure of the regenerated bone a multi-technique analysis was used, including stereo microscope, X-Ray; AFM, micro-CT, and histological analyses. The progression of bone regeneration was followed at 4, 8, and 12 weeks after the microsphere system implantation whereas the two electrospun systems were evaluated at fixed 12 weeks. All the techniques applied showed high bone regeneration. The average values of bone volume density, bone mineral density, Young's modulus, and the percent of bone repair were ∼70% of the values of the native bone. Besides, SEM-EDX analysis indicated that the main chemical elements in the new bone were oxygen, calcium, and phosphorus in a ratio similar to that of native bone. In comparison, the micro-CT may provide an alternative to histology for the evaluation of bone formation at the defect size. PMID:25689580

  2. Abrogation of Chk1-mediated S/G2 checkpoint by UCN-01 enhances ara-C-induced cytotoxicity in human colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    Rong-guang SHAO; Chun-Xia CAO; Yves POMMIER

    2004-01-01

    AIM: To investigate whether 7-hydroxystaurosporine (UCN-01) affects cell cycle progression in arabinosylcytosine (ara-C) treated human colon carcinoma HT-29 cells. METHODS: Cytotoxicity, DNA synthesis, cell cycle distribution,protein level, and kinase activity were determined by clonogenic assay, flow cytometry, DNA synthesis assay,immunoblotting, and kinase assays, respectively. RESULTS: UCN-01 abrogated an S/G2-phase checkpoint in HT29 cells treated with ara-C. When UCN-01 was added after treatment with ara-C, the rate of recovery of DNA synthesis was enhanced and colony-forming ability diminished. Thus, premature recovery of DNA synthesis was associated with increased cytotoxicity. Measurements of cyclin A and B protein levels, Cdk2 and Cdc2 kinase activities, Cdc25C phosphorylation, and Chkl kinase activity were consistent with UCN-01-induced abrogation of the S/G2-phase checkpoint in ara-C treated cells. CONCLUSION: The abrogation of the S/G2 checkpoint may be due to inhibition of Chkl kinase by UCN-01. The enhanced cytotoxicity produced when UCN-01 was combined with ara-C suggested a rationale for the use of this drug combination for tumors that might be susceptible to cell cycle checkpoint abrogation.

  3. 脊柱融合术患者BMP-2基因突变的检测及其意义%Mutational analysis of BMP-2 gene in the patients of spinal fusion

    Institute of Scientific and Technical Information of China (English)

    周传利; 陈晓亮

    2007-01-01

    目的 检测脊柱融合术患者的骨形态发生蛋白-2(BMP-2)基因突变状况.方法 从80例行脊柱融合术患者的术前空腹静脉血中提取DNA,采用聚合酶链反应-单链构象多态性分析(PCR-SSCP)及测序技术,检测其BMP-2基因部分编码区及其侧翼序列的突变.结果 脊柱融合术患者的外周静脉血中BMP-2基因有突变:TCG→GCG,TCA→TCG,并引起相应多肽的结构改变.结论 脊柱融合术患者中存在BMP-2基因突变及多态性分布,并有可能影响植骨融合效果.

  4. Odontoblast β-catenin signaling regulates fenestration of mouse Hertwig's epithelial root sheath.

    Science.gov (United States)

    Zhang, Ran; Teng, Yan; Zhu, Liang; Lin, JingTing; Yang, Xiao; Yang, Guan; Li, TieJun

    2015-09-01

    The interaction between Hertwig's epithelial root sheath (HERS) and the adjacent mesenchyme is vitally important in mouse tooth root development. We previously generated odontoblast-specific Ctnnb1 (encoding β-catenin) deletion mice, and demonstrated that odontoblast β-catenin signaling regulates odontoblast proliferation and differentiation. However, the role of odontoblast β-catenin signaling in regulation of HERS behavior has not been fully investigated. Here, using the same odontoblast- specific Ctnnb1 deletion mice, we found that ablation of β-catenin signaling in odontoblasts led to aberrant HERS formation. Mechanistically, odontoblast-specific Ctnnb1 deletion resulted in elevated bone morphogenetic protein 7 (Bmp7) expression and reduced expression of noggin and follistatin, both of which encode extracellular inhibitors of BMPs. Furthermore, the levels of phosphorylated Smad1/5/8 were increased in HERS cells. In vitro tissue culture confirmed that BMP7 treatment disrupted the HERS structure. Taken together, we demonstrated that odontoblast β-catenin signaling may act through regulation of BMP signaling to maintain the integrity of HERS cells. PMID:26208822

  5. 3D bioprinting of BMSC-laden methacrylamide gelatin scaffolds with CBD-BMP2-collagen microfibers.

    Science.gov (United States)

    Du, Mingchun; Chen, Bing; Meng, Qingyuan; Liu, Sumei; Zheng, Xiongfei; Zhang, Cheng; Wang, Heran; Li, Hongyi; Wang, Nuo; Dai, Jianwu

    2015-12-01

    Three-dimensional (3D) bioprinting combines biomaterials, cells and functional components into complex living tissues. Herein, we assembled function-control modules into cell-laden scaffolds using 3D bioprinting. A customized 3D printer was able to tune the microstructure of printed bone mesenchymal stem cell (BMSC)-laden methacrylamide gelatin scaffolds at the micrometer scale. For example, the pore size was adjusted to 282 ± 32 μm and 363 ± 60 μm. To match the requirements of the printing nozzle, collagen microfibers with a length of 22 ± 13 μm were prepared with a high-speed crusher. Collagen microfibers bound bone morphogenetic protein 2 (BMP2) with a collagen binding domain (CBD) as differentiation-control module, from which BMP2 was able to be controllably released. The differentiation behaviors of BMSCs in the printed scaffolds were compared in three microenvironments: samples without CBD-BMP2-collagen microfibers in the growth medium, samples without microfibers in the osteogenic medium and samples with microfibers in the growth medium. The results indicated that BMSCs showed high cell viability (>90%) during printing; CBD-BMP2-collagen microfibers induced BMSC differentiation into osteocytes within 14 days more efficiently than the osteogenic medium. Our studies suggest that these function-control modules are attractive biomaterials and have potential applications in 3D bioprinting. PMID:26684899

  6. Vitapex can promote the expression of BMP-2 during the bone regeneration of periapical lesions in rats

    Directory of Open Access Journals (Sweden)

    Xianyin Xia

    2013-01-01

    Full Text Available Purpose: To investigate the effect of Vitapex on the healing of periapical lesions and the expression of bone morphogenetic protein (BMP-2 during the periapical bone regeneration. Materials and Methods: Periapical lesions were induced in Sprague-Dawley (S-D rats by an occlusal pulp exposure in the mandibular first molars and were verified by X-ray. Total of 36 rats were randomly divided into three groups, and they were obturated with Zinc Oxide Eugenol (ZOE, or with Vitapex, or non-treated as negative control group. The rats of three groups were randomly killed at week 0, 2, 4, and 8 after root canal therapy, and then the mandibles were processed for histological examination and immunohistochemistry analysis. Results: At week 0, only a few BMP-2 positive cells could be observed in all rats. While the expression of BMP-2 was dramatically increased in case of Vitapex group at week 2 and week 4, and then climaxed at week 8. However, no apparent changes were observed in ZOE group and negative group at week 2, 4, and 8. Conclusion: These observations suggested that Vitapex has a greater ability in inducing bone regeneration than ZOE by the expression of BMP-2 induction in the treatment of rats experimental periapical lesions.

  7. [BMP-2 gene carried by biodegradable scaffold and fibrinous gel for repairing segmental radial defect in rabbit].

    Science.gov (United States)

    Li, Jianjun; Wang, Enbo; Sun, Hongbin; Han, Dong; Wang, Huan; Bai, Lunhao; Li, Lei; Liu, Xueyong; Xu, Xinxiang

    2007-04-01

    Adenovirus carrying BMP-2 gene, after being mixed with fibrinous gel, was siphoned off on biodegradable scaffolds (PLA/PCL). The composite was used to repair 1.5 cm long radius defect in rabbits. Four methods were in use in the experiments: Ad-BMP-2 plus fibrinous gel and PLA/PCL (Group A), reconstructed hBMP-2 plus fibrinous gel and PLA/PCL (Group B), Ad-Lacz plus fibrinous gel and PLA/PCL (Group C), and fibrinous gel and PLA/PCL (Group D). Results showed that the defects treated in Group A were repaired with much more new bone regenerated, bridged earlier and stronger than those in Group B 12 weeks after operation. The defects treated in the other two groups could not attain osseous tissue healing. BMP-2 gene carried by biodegradable scaffold and fibrinous gel is easy to conduct and has very strong osteoinduction ability. It is really a good method to repair segmental bone defects. PMID:17591257

  8. In vitro and in vivo evaluation of bone morphogenetic protein-2 (BMP-2) immobilized collagen-coated polyetheretherketone (PEEK)

    Science.gov (United States)

    Du, Ya-Wei; Zhang, Li-Nan; Ye, Xin; Nie, He-Min; Hou, Zeng-Tao; Zeng, Teng-Hui; Yan, Guo-Ping; Shang, Peng

    2015-03-01

    Polyetheretherketone (PEEK) is regarded as one of the most potential candidates of biomaterials in spinal implant applications. However, as a bioinert material, PEEK plays a limited role in osteoconduction and osseointegration. In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) was immobilized onto the surface of collagen-coated PEEK in order to prepare a multi-functional material. After adsorbed onto the PEEK surface by hydrophobic interaction, collagen was cross-linked with N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). EDC/NHS system also contributed to the immobilization of rhBMP-2. Water contact angle tests, XPS and SEM clearly demonstrated the surface changes. ELISA tests quantified the amount of rhBMP-2 immobilized and the release over a period of 30 d. In vitro evaluation proved that the osteogenesis differentiation rate was higher when cells were cultured on modified PEEK discs than on regular ones. In vivo tests were conducted and positive changes of major parameters were presented. This report demonstrates that the rhBMP-2 immobilized method for PEEK modification increase bioactivity in vitro and in vivo, suggesting its practicability in orthopedic and spinal clinical applications.

  9. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    F Wegman

    2011-03-01

    Full Text Available Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2, which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix.

  10. Kinetics and thermodynamics studies on the BMP-2 adsorption onto hydroxyapatite surface with different multi-morphological features

    International Nuclear Information System (INIS)

    The effect of the surface topography on protein adsorption process is of great significance for designing hydroxyapatite (HA) ceramic material surfaces. In this work, three different topographies of HA materials HA-sheet, HA-rod, and HA-whisker were synthesized and testified by X-ray diffraction (XRD), Fourier transform infrared (FT-IR), Brunauer–Emmett–Teller (BET) and a field emission scanning electron microscopy (FE-SEM). We have systematically investigated the adsorption kinetics and thermodynamics of bone morphogenetic proteins (BMP-2) on the three different topography surfaces of HA, respectively. The results showed that the maximum adsorption capacities of HA-sheet, HA-rod and HA-whisker were (219.96 ± 10.18), (247.13 ± 12.35), and (354.67 ± 17.73) μg · g−1, respectively. Kinetic parameters, rate constants, equilibrium adsorption capacities and related correlation coefficients, for each kinetic model were calculated as well as discussed. It demonstrated that the adsorption of BMP-2 onto HA could be described by the pseudo second-order equation. Adsorption of BMP-2 onto HA followed the Langmuir isotherm. It confirmed that compared with other samples HA-whisker had more adsorption sites for its high specific surface area which could provide more opportunities for protein molecules. The adsorption processes were endothermic (ΔH > 0), spontaneous (ΔG < 0) and entropy increasing (ΔS > 0). A possible adsorption mechanism has been proposed. In addition, the BMP-2 could be adsorbed to the surface which existed slight conformational changes by FT-IR. - Highlights: • A novel protein adsorption studies based on sheet, rod and whisker of HA were designed. • Kinetic and thermodynamics parameters of BMP-2 and HA bonded materials were evaluated. • Surface topographies of the HA effect BMP-2 adsorption • The HA-whisker material had excellent adsorption performance for protein enrichment. • The electrostatic interaction is responsible for the BMP-2

  11. Kinetics and thermodynamics studies on the BMP-2 adsorption onto hydroxyapatite surface with different multi-morphological features

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Zhiwei; Huangfu, Changxin; Wang, Yanying; Ge, Hongwei; Yao, Yao; Zou, Ping; Wang, Guangtu [College of Science, Sichuan Agricultural University, Ya' an 625014 (China); He, Hua [Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Wenjiang, Sichuan 611130 (China); Rao, Hanbing, E-mail: rhbscu@gmail.com [College of Science, Sichuan Agricultural University, Ya' an 625014 (China)

    2015-07-01

    The effect of the surface topography on protein adsorption process is of great significance for designing hydroxyapatite (HA) ceramic material surfaces. In this work, three different topographies of HA materials HA-sheet, HA-rod, and HA-whisker were synthesized and testified by X-ray diffraction (XRD), Fourier transform infrared (FT-IR), Brunauer–Emmett–Teller (BET) and a field emission scanning electron microscopy (FE-SEM). We have systematically investigated the adsorption kinetics and thermodynamics of bone morphogenetic proteins (BMP-2) on the three different topography surfaces of HA, respectively. The results showed that the maximum adsorption capacities of HA-sheet, HA-rod and HA-whisker were (219.96 ± 10.18), (247.13 ± 12.35), and (354.67 ± 17.73) μg · g{sup −1}, respectively. Kinetic parameters, rate constants, equilibrium adsorption capacities and related correlation coefficients, for each kinetic model were calculated as well as discussed. It demonstrated that the adsorption of BMP-2 onto HA could be described by the pseudo second-order equation. Adsorption of BMP-2 onto HA followed the Langmuir isotherm. It confirmed that compared with other samples HA-whisker had more adsorption sites for its high specific surface area which could provide more opportunities for protein molecules. The adsorption processes were endothermic (ΔH > 0), spontaneous (ΔG < 0) and entropy increasing (ΔS > 0). A possible adsorption mechanism has been proposed. In addition, the BMP-2 could be adsorbed to the surface which existed slight conformational changes by FT-IR. - Highlights: • A novel protein adsorption studies based on sheet, rod and whisker of HA were designed. • Kinetic and thermodynamics parameters of BMP-2 and HA bonded materials were evaluated. • Surface topographies of the HA effect BMP-2 adsorption • The HA-whisker material had excellent adsorption performance for protein enrichment. • The electrostatic interaction is responsible for the

  12. Fabrication of Core-Shell PEI/pBMP2-PLGA Electrospun Scaffold for Gene Delivery to Periodontal Ligament Stem Cells

    Directory of Open Access Journals (Sweden)

    Qiao Xie

    2016-01-01

    Full Text Available Bone tissue engineering is the most promising technology for enhancing bone regeneration. Scaffolds loaded with osteogenic factors improve the therapeutic effect. In this study, the bioactive PEI (polyethylenimine/pBMP2- (bone morphogenetic protein-2 plasmid- PLGA (poly(D, L-lactic-co-glycolic acid core-shell scaffolds were prepared using coaxial electrospinning for a controlled gene delivery to hPDLSCs (human periodontal ligament stem cells. The pBMP2 was encapsulated in the PEI phase as a core and PLGA was employed to control pBMP2 release as a shell. First, the scaffold characterization and mechanical properties were evaluated. Then the gene release behavior was analyzed. Our results showed that pBMP2 was released at high levels in the first few days, with a continuous release behavior in the next 28 days. At the same time, PEI/pBMP2 showed high transfection efficiency. Moreover, the core-shell electrospun scaffold showed BMP2 expression for a much longer time (more than 28 days compared with the single axial electrospun scaffold, as evaluated by qRT-PCR and western blot after culturing with hPDLSCs. These results suggested that the core-shell PEI/pBMP2-PLGA scaffold fabricated by coaxial electrospinning had a good gene release behavior and showed a prolonged expression time with a high transfection efficiency.

  13. NS1-binding protein abrogates the elevation of cell viability by the influenza A virus NS1 protein in association with CRKL

    International Nuclear Information System (INIS)

    Highlights: •NS1 induced excessive phosphorylation of ERK and elevated cell viability. •NS1-BP expression and CRKL knockdown abolished survival effect of NS1. •NS1-BP and NS1 formed the complex through the interaction with CRKL-SH3(N). -- Abstract: The influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor consisting of an RNA binding domain and several Src-homology (SH) 2 and SH3 binding motifs, which promotes virus replication in the host cell and helps to evade antiviral immunity. NS1 modulates general host cell physiology in association with various cellular molecules including NS1-binding protein (NS1-BP) and signaling adapter protein CRK-like (CRKL), while the physiological role of NS1-BP during influenza A virus infection especially in association with NS1 remains unclear. In this study, we analyzed the intracellular association of NS1-BP, NS1 and CRKL to elucidate the physiological roles of these molecules in the host cell. In HEK293T cells, enforced expression of NS1 of A/Beijing (H1N1) and A/Indonesia (H5N1) significantly induced excessive phosphorylation of ERK and elevated cell viability, while the over-expression of NS1-BP and the abrogation of CRKL using siRNA abolished such survival effect of NS1. The pull-down assay using GST-fusion CRKL revealed the formation of intracellular complexes of NS1-BP, NS1 and CRKL. In addition, we identified that the N-terminus SH3 domain of CRKL was essential for binding to NS1-BP using GST-fusion CRKL-truncate mutants. This is the first report to elucidate the novel function of NS1-BP collaborating with viral protein NS1 in modulation of host cell physiology. In addition, an alternative role of adaptor protein CRKL in association with NS1 and NS1-BP during influenza A virus infection is demonstrated

  14. NS1-binding protein abrogates the elevation of cell viability by the influenza A virus NS1 protein in association with CRKL

    Energy Technology Data Exchange (ETDEWEB)

    Miyazaki, Masaya [Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638 (Japan); Nishihara, Hiroshi, E-mail: hnishihara@med.hokudai.ac.jp [Department of Translational Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638 (Japan); Hasegawa, Hideki [Department of Pathology, National Institute of Infectious Diseases, Sinjuku-ku, Tokyo (Japan); Tashiro, Masato [Influenza Virus Research Center, National Institute of Infectious Diseases, Sinjuku-ku, Tokyo (Japan); Wang, Lei [Department of Translational Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638 (Japan); Kimura, Taichi; Tanino, Mishie; Tsuda, Masumi [Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638 (Japan); Tanaka, Shinya [Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638 (Japan); Department of Translational Pathology, Hokkaido University Graduate School of Medicine, N15W7, Kita-ku, Sapporo 060-8638 (Japan)

    2013-11-29

    Highlights: •NS1 induced excessive phosphorylation of ERK and elevated cell viability. •NS1-BP expression and CRKL knockdown abolished survival effect of NS1. •NS1-BP and NS1 formed the complex through the interaction with CRKL-SH3(N). -- Abstract: The influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor consisting of an RNA binding domain and several Src-homology (SH) 2 and SH3 binding motifs, which promotes virus replication in the host cell and helps to evade antiviral immunity. NS1 modulates general host cell physiology in association with various cellular molecules including NS1-binding protein (NS1-BP) and signaling adapter protein CRK-like (CRKL), while the physiological role of NS1-BP during influenza A virus infection especially in association with NS1 remains unclear. In this study, we analyzed the intracellular association of NS1-BP, NS1 and CRKL to elucidate the physiological roles of these molecules in the host cell. In HEK293T cells, enforced expression of NS1 of A/Beijing (H1N1) and A/Indonesia (H5N1) significantly induced excessive phosphorylation of ERK and elevated cell viability, while the over-expression of NS1-BP and the abrogation of CRKL using siRNA abolished such survival effect of NS1. The pull-down assay using GST-fusion CRKL revealed the formation of intracellular complexes of NS1-BP, NS1 and CRKL. In addition, we identified that the N-terminus SH3 domain of CRKL was essential for binding to NS1-BP using GST-fusion CRKL-truncate mutants. This is the first report to elucidate the novel function of NS1-BP collaborating with viral protein NS1 in modulation of host cell physiology. In addition, an alternative role of adaptor protein CRKL in association with NS1 and NS1-BP during influenza A virus infection is demonstrated.

  15. Injectable perlecan domain 1-hyaluronan microgels potentiate the cartilage repair effect of BMP2 in a murine model of early osteoarthritis

    International Nuclear Information System (INIS)

    The goal of this study was to use bioengineered injectable microgels to enhance the action of bone morphogenetic protein 2 (BMP2) and stimulate cartilage matrix repair in a reversible animal model of osteoarthritis (OA). A module of perlecan (PlnD1) bearing heparan sulfate (HS) chains was covalently immobilized to hyaluronic acid (HA) microgels for the controlled release of BMP2 in vivo. Articular cartilage damage was induced in mice using a reversible model of experimental OA and was treated by intra-articular injection of PlnD1-HA particles with BMP2 bound to HS. Control injections consisted of BMP2-free PlnD1-HA particles, HA particles, free BMP2 or saline. Knees dissected following these injections were analyzed using histological, immunostaining and gene expression approaches. Our results show that knees treated with PlnD1-HA/BMP2 had lesser OA-like damage compared to control knees. In addition, the PlnD1-HA/BMP2-treated knees had higher mRNA levels encoding for type II collagen, proteoglycans and xylosyltransferase 1, a rate-limiting anabolic enzyme involved in the biosynthesis of glycosaminoglycan chains, relative to control knees (PlnD1-HA). This finding was paralleled by enhanced levels of aggrecan in the articular cartilage of PlnD1-HA/BMP2-treated knees. Additionally, decreases in the mRNA levels encoding for cartilage-degrading enzymes and type X collagen were seen relative to controls. In conclusion, PlnD1-HA microgels constitute a formulation improvement compared to HA for efficient in vivo delivery and stimulation of proteoglycan and cartilage matrix synthesis in mouse articular cartilage. Ultimately, PlnD1-HA/BMP2 may serve as an injectable therapeutic agent for slowing or inhibiting the onset of OA after knee injury.

  16. Effect of adipose-derived stromal cells and BMP12 on intrasynovial tendon repair: A biomechanical, biochemical, and proteomics study.

    Science.gov (United States)

    Gelberman, Richard H; Shen, Hua; Kormpakis, Ioannis; Rothrauff, Benjamin; Yang, Guang; Tuan, Rocky S; Xia, Younan; Sakiyama-Elbert, Shelly; Silva, Matthew J; Thomopoulos, Stavros

    2016-04-01

    The outcomes of flexor tendon repair are highly variable. As recent efforts to improve healing have demonstrated promise for growth factor- and cell-based therapies, the objective of the current study was to enhance repair via application of autologous adipose derived stromal cells (ASCs) and the tenogenic growth factor bone morphogenetic protein (BMP) 12. Controlled delivery of cells and growth factor was achieved in a clinically relevant canine model using a nanofiber/fibrin-based scaffold. Control groups consisted of repair-only (no scaffold) and acellular scaffold. Repairs were evaluated after 28 days of healing using biomechanical, biochemical, and proteomics analyses. Range of motion was reduced in the groups that received scaffolds compared to normal. There was no effect of ASC + BMP12 treatment for range of motion or tensile properties outcomes versus repair-only. Biochemical assays demonstrated increased DNA, glycosaminoglycans, and crosslink concentration in all repair groups compared to normal, but no effect of ASC + BMP12. Total collagen was significantly decreased in the acellular scaffold group compared to normal and significantly increased in the ASC + BMP12 group compared to the acellular scaffold group. Proteomics analysis comparing healing tendons to uninjured tendons revealed significant increases in proteins associated with inflammation, stress response, and matrix degradation. Treatment with ASC + BMP12 amplified these unfavorable changes. In summary, the treatment approach used in this study induced a negative inflammatory reaction at the repair site leading to poor healing. Future approaches should consider cell and growth factor delivery methods that do not incite negative local reactions. PMID:26445383

  17. Non-virally engineered human adipose mesenchymal stem cells produce BMP4, target brain tumors, and extend survival.

    Science.gov (United States)

    Mangraviti, Antonella; Tzeng, Stephany Y; Gullotti, David; Kozielski, Kristen L; Kim, Jennifer E; Seng, Michael; Abbadi, Sara; Schiapparelli, Paula; Sarabia-Estrada, Rachel; Vescovi, Angelo; Brem, Henry; Olivi, Alessandro; Tyler, Betty; Green, Jordan J; Quinones-Hinojosa, Alfredo

    2016-09-01

    There is a need for enabling non-viral nanobiotechnology to allow safe and effective gene therapy and cell therapy, which can be utilized to treat devastating diseases such as brain cancer. Human adipose-derived mesenchymal stem cells (hAMSCs) display high anti-glioma tropism and represent a promising delivery vehicle for targeted brain tumor therapy. In this study, we demonstrate that non-viral, biodegradable polymeric nanoparticles (NPs) can be used to engineer hAMSCs with higher efficacy (75% of cells) than leading commercially available reagents and high cell viability. To accomplish this, we engineered a poly(beta-amino ester) (PBAE) polymer structure to transfect hAMSCs with significantly higher efficacy than Lipofectamine™ 2000. We then assessed the ability of NP-engineered hAMSCs to deliver bone morphogenetic protein 4 (BMP4), which has been shown to have a novel therapeutic effect by targeting human brain tumor initiating cells (BTIC), a source of cancer recurrence, in a human primary malignant glioma model. We demonstrated that hAMSCs genetically engineered with polymeric nanoparticles containing BMP4 plasmid DNA (BMP4/NP-hAMSCs) secrete BMP4 growth factor while maintaining their multipotency and preserving their migration and invasion capacities. We also showed that this approach can overcome a central challenge for brain therapeutics, overcoming the blood brain barrier, by demonstrating that NP-engineered hAMSCs can migrate to the brain and penetrate the brain tumor after both intranasal and systemic intravenous administration. Critically, athymic rats bearing human primary BTIC-derived tumors and treated intranasally with BMP4/NP-hAMSCs showed significantly improved survival compared to those treated with control GFP/NP-hAMCSs. This study demonstrates that synthetic polymeric nanoparticles are a safe and effective approach for stem cell-based cancer-targeting therapies. PMID:27240162

  18. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Zhang Yingang; Guo Xiong; Liu Zheng; Wang Shijie

    2007-01-01

    Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42.8±6.1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

  19. A boost of BMP4 accelerates the commitment of human embryonic stem cells to the endothelial lineage.

    Science.gov (United States)

    Goldman, Orit; Feraud, Olivier; Boyer-Di Ponio, Julie; Driancourt, Catherine; Clay, Denis; Le Bousse-Kerdiles, Marie-Caroline; Bennaceur-Griscelli, Annelise; Uzan, Georges

    2009-08-01

    Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133(+)KDR(+) population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144(+)KDR(+) population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144(+)KDR(+) peak was shifted to day 7. Most of these cells, however, were CD31(-), becoming CD31(+) in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144(+)KDR(+)CD31(-) population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12. PMID:19544443

  20. In silico Mechano-Chemical Model of Bone Healing for the Regeneration of Critical Defects: The Effect of BMP-2.

    Directory of Open Access Journals (Sweden)

    Frederico O Ribeiro

    Full Text Available The healing of bone defects is a challenge for both tissue engineering and modern orthopaedics. This problem has been addressed through the study of scaffold constructs combined with mechanoregulatory theories, disregarding the influence of chemical factors and their respective delivery devices. Of the chemical factors involved in the bone healing process, bone morphogenetic protein-2 (BMP-2 has been identified as one of the most powerful osteoinductive proteins. The aim of this work is to develop and validate a mechano-chemical regulatory model to study the effect of BMP-2 on the healing of large bone defects in silico. We first collected a range of quantitative experimental data from the literature concerning the effects of BMP-2 on cellular activity, specifically proliferation, migration, differentiation, maturation and extracellular matrix production. These data were then used to define a model governed by mechano-chemical stimuli to simulate the healing of large bone defects under the following conditions: natural healing, an empty hydrogel implanted in the defect and a hydrogel soaked with BMP-2 implanted in the defect. For the latter condition, successful defect healing was predicted, in agreement with previous in vivo experiments. Further in vivo comparisons showed the potential of the model, which accurately predicted bone tissue formation during healing, bone tissue distribution across the defect and the quantity of bone inside the defect. The proposed mechano-chemical model also estimated the effect of BMP-2 on cells and the evolution of healing in large bone defects. This novel in silico tool provides valuable insight for bone tissue regeneration strategies.

  1. Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

    Energy Technology Data Exchange (ETDEWEB)

    Prins, Kathleen C.; Delpeut, Sebastien; Leung, Daisy W.; Reynard, Olivier; Volchkova, Valentina A.; Reid, St. Patrick; Ramanan, Parameshwaran; Cárdenas, Washington B.; Amarasinghe, Gaya K.; Volchkov, Viktor E.; Basler, Christopher F. (CNRS-INSERM); (Mount Sinai Hospital); (LB-Ecuador); (Iowa State)

    2010-10-11

    Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-{alpha}/{beta} responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-{alpha}/{beta} production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.

  2. A Molecular Switch Abrogates Glycoprotein 100 (gp100) T-cell Receptor (TCR) Targeting of a Human Melanoma Antigen*

    Science.gov (United States)

    Bianchi, Valentina; Bulek, Anna; Fuller, Anna; Lloyd, Angharad; Attaf, Meriem; Rizkallah, Pierre J.; Dolton, Garry; Sewell, Andrew K.; Cole, David K.

    2016-01-01

    Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280–288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3 → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination. PMID:26917722

  3. Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence.

    Science.gov (United States)

    Ananth, Abhirami A; Tai, Lee-Hwa; Lansdell, Casey; Alkayyal, Almohanad A; Baxter, Katherine E; Angka, Leonard; Zhang, Jiqing; Tanese de Souza, Christiano; Stephenson, Kyle B; Parato, Kelley; Bramson, Jonathan L; Bell, John C; Lichty, Brian D; Auer, Rebecca C

    2016-01-01

    Anti-tumor CD8+ T cells are a key determinant for overall survival in patients following surgical resection for solid malignancies. Using a mouse model of cancer vaccination (adenovirus expressing melanoma tumor-associated antigen (TAA)-dopachrome tautomerase (AdDCT) and resection resulting in major surgical stress (abdominal nephrectomy), we demonstrate that surgical stress results in a reduction in the number of CD8+ T cell that produce cytokines (IFNγ, TNFα, Granzyme B) in response to TAA. This effect is secondary to both reduced proliferation and impaired T cell function following antigen binding. In a prophylactic model, surgical stress completely abrogates tumor protection conferred by vaccination in the immediate postoperative period. In a clinically relevant surgical resection model, vaccinated mice undergoing a positive margin resection with surgical stress had decreased survival compared to mice with positive margin resection alone. Preoperative immunotherapy with IFNα significantly extends survival in surgically stressed mice. Importantly, myeloid derived suppressor cell (MDSC) population numbers and functional impairment of TAA-specific CD8+ T cell were altered in surgically stressed mice. Our observations suggest that cancer progression may result from surgery-induced suppression of tumor-specific CD8+ T cells. Preoperative immunotherapies aimed at targeting the prometastatic effects of cancer surgery will reduce recurrence and improve survival in cancer surgery patients. PMID:27196057

  4. An exon 53 frameshift mutation in CUBN abrogates cubam function and causes Imerslund-Gräsbeck syndrome in dogs.

    Science.gov (United States)

    Fyfe, John C; Hemker, Shelby L; Venta, Patrick J; Fitzgerald, Caitlin A; Outerbridge, Catherine A; Myers, Sherry L; Giger, Urs

    2013-08-01

    Cobalamin malabsorption accompanied by selective proteinuria is an autosomal recessive disorder known as Imerslund-Gräsbeck syndrome in humans and was previously described in dogs due to amnionless (AMN) mutations. The resultant vitamin B12 deficiency causes dyshematopoiesis, lethargy, failure to thrive, and life-threatening metabolic disruption in the juvenile period. We studied 3 kindreds of border collies with cobalamin malabsorption and mapped the disease locus in affected dogs to a 2.9Mb region of homozygosity on canine chromosome 2. The region included CUBN, the locus encoding cubilin, a peripheral membrane protein that in concert with AMN forms the functional intrinsic factor-cobalamin receptor expressed in ileum and a multi-ligand receptor in renal proximal tubules. Cobalamin malabsorption and proteinuria comprising CUBN ligands were demonstrated by radiolabeled cobalamin uptake studies and SDS-PAGE, respectively. CUBN mRNA and protein expression were reduced ~10 fold and ~20 fold, respectively, in both ileum and kidney of affected dogs. DNA sequencing demonstrated a single base deletion in exon 53 predicting a translational frameshift and early termination codon likely triggering nonsense mediated mRNA decay. The mutant allele segregated with the disease in the border collie kindred. The border collie disorder indicates that a CUBN mutation far C-terminal from the intrinsic factor-cobalamin binding site can abrogate receptor expression and cause Imerslund-Gräsbeck syndrome. PMID:23746554

  5. Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

    Directory of Open Access Journals (Sweden)

    Bell Graham

    2005-12-01

    Full Text Available Abstract Background Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages. Results We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages. Conclusion Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.

  6. Three-Dimensional Printing of rhBMP-2-Loaded Scaffolds with Long-Term Delivery for Enhanced Bone Regeneration in a Rabbit Diaphyseal Defect

    OpenAIRE

    Shim, Jin-Hyung; Kim, Se Eun; Park, Ju Young; Kundu, Joydip; Kim, Sung Won; Kang, Seong Soo; Cho, Dong-Woo

    2014-01-01

    In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) delivery system with slow mode was successfully developed in three-dimensional (3D) printing-based polycaprolactone (PCL)/poly(lactic-co-glycolic acid) (PLGA) scaffolds for bone formation of critical-sized rabbit segmental diaphyseal defect. To control the delivery of the rhBMP-2, collagen (for long-term delivery up to 28 days) and gelatin (for shor-term delivery within a week) solutions encapsulating rhBMP-2 were dispens...

  7. Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid--an experimental study in rats.

    OpenAIRE

    Schliephake, Henning; Weich, Herbert A.; Dullin, Christian; Gruber, Rudolf; Frahse, Sarah

    2008-01-01

    The aim of the present study was to test the hypothesis that human recombinant bone morphogenic protein 2 (rhBMP-2) implanted in a slow release carrier of polylactic acid (PLA) can repair a non-healing defect in the rat mandible and maintain the thickness of an augmented volume. p-DL-lactic acid discs were produced and loaded with 48 and 96 microg rhBMP-2 and inserted into non-healing defects of the mandible of 45 Wistar rats. Fifteen rats received implants with 96 microg rhBMP-2 (Group 2), 4...

  8. Effects of rhBMP-2 on Sandblasted and Acid Etched Titanium Implant Surfaces on Bone Regeneration and Osseointegration: Spilt-Mouth Designed Pilot Study

    OpenAIRE

    Nam-Ho Kim; So-Hyoun Lee; Jae-Jun Ryu; Kyung-Hee Choi; Jung-Bo Huh

    2015-01-01

    This study was conducted to evaluate effects of rhBMP-2 applied at different concentrations to sandblasted and acid etched (SLA) implants on osseointegration and bone regeneration in a bone defect of beagle dogs as pilot study using split-mouth design. Methods. For experimental groups, SLA implants were coated with different concentrations of rhBMP-2 (0.1, 0.5, and 1 mg/mL). After assessment of surface characteristics and rhBMP-2 releasing profile, the experimental groups and untreated contro...

  9. Akt Signaling Accelerates Tumor Recurrence Following Ras Inhibition in the Context of Ink4a/Arf Loss

    OpenAIRE

    Robinson, Gemma L.; Robinson, James P.; Lastwika, Kristin J.; Holmen, Sheri L.; VanBrocklin, Matthew W.

    2013-01-01

    Aberrant activation of the RAS signaling pathway contributes to nearly all human cancers, including gliomas. To determine the dependence of high-grade gliomas on this signaling pathway, we developed a doxycycline-regulated KRas glioma mouse model. Using this model we previously demonstrated that inhibition of KRas expression in gliomas induced by activated KRas and Akt results in complete tumor regression. We have also shown that, in the context of Ink4a/Arf loss, abrogation of KRas signaling...

  10. Porcine pluripotency cell signaling develops from the inner cell mass to the epiblast during early development

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane; Christensen, Josef; Gao, Yu;

    2009-01-01

    (LIF, LIFR, GP130), FGF pathway (bFGF, FGFR1, FGFR2), BMP pathway (BMP4), and downstream-activated genes (STAT3, c-Myc, c-Fos, and SMAD4). We discovered two different expression profiles exist in the developing porcine embryo. The D6 porcine blastocyst (inner cell mass stage) is devoid in the......  The signaling mechanisms regulating pluripotency in porcine embryonic stem cells and embryos are unknown. In this study, we characterize cell signaling in the in-vivo porcine inner cell mass and later-stage epiblast. We evaluate expression of OCT4, NANOG, SOX2, genes within the JAK/STAT pathway...... pluripotency in human embryonic stem cells is detectable in the porcine epiblast, but not in the inner cell mass. Copyright (c) 2009 Wiley-Liss, Inc....

  11. Dithiothreitol abrogates the effect of arsenic trioxide on normal rat liver mitochondria and human hepatocellular carcinoma cells

    International Nuclear Information System (INIS)

    Arsenic trioxide (ATO) is a known environmental toxicant and a potent chemotherapeutic agent. Significant correlation has been reported between consumption of arsenic-contaminated water and occurrence of liver cancer; moreover, ATO-treated leukemia patients also suffers from liver toxicity. Hence, modulation of ATO action may help to prevent populations suffering from arsenic toxicity as well as help reduce the drug-related side effects. Dithiothreitol (DTT) is a well-known dithiol agent reported to modulate the action of ATO. Controversial reports exist regarding the effect of DTT on ATO-induced apoptosis in leukemia cells. To the best of our knowledge, no report illustrates the modulatory effect of DTT on ATO-induced liver toxicity, the prime target for arsenic. Mitochondria serve as the doorway to apoptosis and have been implicated in ATO-induced cell death. Hence, we attempted to study the modulatory effect of DTT on ATO-induced dysfunction of mammalian liver mitochondria and human hepatocellular carcinoma cell line (Hep3B). We, for the first time, report that ATO produces complex I-mediated electron transfer inhibition, reactive oxygen species (ROS) generation, respiration inhibition, and ATO-induced ROS-mediated mitochondrial permeability transition (MPT) opening. DTT at low concentration (100 μM and less) prevents the effect of ATO-induced complex I-malfunctions. DTT protects mitochondria from ATO-mediated opening of MPT and membrane potential depolarization. DTT also prevented ATO-induced Hep3B cell death. Thus, at low concentrations DTT abrogates the effect of ATO on rat liver mitochondria and Hep3B cell line. Therefore, the present result suggests, that use of low concentration of dithiols as food supplement may prevent arsenic toxicity in affected population

  12. Mutations that abrogate transactivational activity of the feline leukemia virus long terminal repeat do not affect virus replication

    International Nuclear Information System (INIS)

    The U3 region of the LTR of oncogenic Moloney murine leukemia virus (Mo-MuLV) and feline leukemia viruses (FeLV) have been previously reported to activate expression of specific cellular genes in trans, such as MHC class I, collagenase IV, and MCP-1, in an integration-independent manner. It has been suggested that transactivation of these specific cellular genes by leukemia virus U3-LTR may contribute to the multistage process of leukemogenesis. The U3-LTR region, necessary for gene transactivational activity, also contains multiple transcription factor-binding sites that are essential for normal virus replication. To dissect the promoter activity and the gene transactivational activity of the U3-LTR, we conducted mutational analysis of the U3-LTR region of FeLV-A molecular clone 61E. We identified minimal nucleotide substitution mutants on the U3 LTR that did not disturb transcription factor-binding sites but abrogated its ability to transactivate the collagenase gene promoter. To determine if these mutations actually have altered any uncharacterized important transcription factor-binding site, we introduced these U3-LTR mutations into the full-length infectious molecular clone 61E. We demonstrate that the mutant virus was replication competent but could not transactivate cellular gene expression. These results thus suggest that the gene transactivational activity is a distinct property of the LTR and possibly not related to its promoter activity. The cellular gene transactivational activity-deficient mutant FeLV generated in this study may also serve as a valuable reagent for testing the biological significance of LTR-mediated cellular gene activation in the tumorigenesis caused by leukemia viruses

  13. Contact-dependent abrogation of bone marrow-derived plasmacytoid dendritic cell differentiation by murine mesenchymal stem cells.

    Science.gov (United States)

    Hackstein, Holger; Tschipakow, Inna; Bein, Gregor; Nold, Philipp; Brendel, Cornelia; Baal, Nelli

    2016-07-15

    Plasmacytoid dendritic cells (pDCs) are rare central regulators of antiviral immunity and unsurpassed producers of interferon-α (IFN-α). Despite their crucial role as a link between innate and adaptive immunity, little is known about the modulation of pDC differentiation by other bone marrow (BM) cells. In this study, we investigated the modulation of pDC differentiation in Flt-3 ligand (Flt3L)-supplemented BM cultures, using highly purified mesenchymal stem cells (MSCs) that were FACS-isolated from murine BM based on surface marker expression and used after in vitro expansion. Initial analysis revealed an almost complete inhibition of BM-derived pDC expansion in the presence of >2% MSC. This inhibition was cell contact-dependent and soluble factor-independent, as indicated by trans-well experiments. The abrogation of functional pDC development by MSCs was confirmed after TLR9 stimulation, revealing a complete, contact-dependent suppression of the IFN-a producing capacity of pDCs in Flt3L MSC BM co-cultures. MSC selectively inhibited pDC development in contrast to myeloid DC development, as indicated by the significantly increased numbers of myeloid DC in Flt3L-supplemented BM cultures. The absence of significant MSC-mediated inhibitory effects on myeloid DC differentiation was confirmed by additional experiments in GM-CSF/IL-4-supplemented BM cultures. In summary, we describe a novel contact-dependent immunomodulatory mechanism of MSC that targets the BM-derived expansion of functional pDCs. PMID:27233615

  14. BMP and retinoic acid regulate anterior–posterior patterning of the non-axial mesoderm across the dorsal–ventral axis

    Science.gov (United States)

    Naylor, Richard W.; Skvarca, Lauren Brilli; Thisse, Christine; Thisse, Bernard; Hukriede, Neil A.; Davidson, Alan J.

    2016-01-01

    Despite the fundamental importance of patterning along the dorsal–ventral (DV) and anterior–posterior (AP) axes during embryogenesis, uncertainty exists in the orientation of these axes for the mesoderm. Here we examine the origin and formation of the zebrafish kidney, a ventrolateral mesoderm derivative, and show that AP patterning of the non-axial mesoderm occurs across the classic gastrula stage DV axis while DV patterning aligns along the animal–vegetal pole. We find that BMP signalling acts early to establish broad anterior and posterior territories in the non-axial mesoderm while retinoic acid (RA) functions later, but also across the classic DV axis. Our data support a model in which RA on the dorsal side of the embryo induces anterior kidney fates while posterior kidney progenitors are protected ventrally by the RA-catabolizing enzyme Cyp26a1. This work clarifies our understanding of vertebrate axis orientation and establishes a new paradigm for how the kidney and other mesodermal derivatives arise during embryogenesis. PMID:27406002

  15. Disruption of PPARγ/β-catenin–mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival

    Science.gov (United States)

    Alastalo, Tero-Pekka; Li, Molong; de Jesus Perez, Vinicio; Pham, David; Sawada, Hirofumi; Wang, Jordon K.; Koskenvuo, Minna; Wang, Lingli; Freeman, Bruce A.; Chang, Howard Y.; Rabinovitch, Marlene

    2011-01-01

    Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with pulmonary arterial hypertension (PAH) can impair pulmonary arterial EC (PAEC) function. This can adversely affect EC survival and promote SMC proliferation. We hypothesized that interventions to normalize expression of genes that are targets of BMPR2 signaling could restore PAEC function and prevent or reverse PAH. Here we have characterized, in human PAECs, a BMPR2-mediated transcriptional complex between PPARγ and β-catenin and shown that disruption of this complex impaired BMP-mediated PAEC survival. Using whole genome-wide ChIP-Chip promoter analysis and gene expression microarrays, we delineated PPARγ/β-catenin–dependent transcription of target genes including APLN, which encodes apelin. We documented reduced PAEC expression of apelin in PAH patients versus controls. In cell culture experiments, we showed that apelin-deficient PAECs were prone to apoptosis and promoted pulmonary arterial SMC (PASMC) proliferation. Conversely, we established that apelin, like BMPR2 ligands, suppressed proliferation and induced apoptosis of PASMCs. Consistent with these functions, administration of apelin reversed PAH in mice with reduced production of apelin resulting from deletion of PPARγ in ECs. Taken together, our findings suggest that apelin could be effective in treating PAH by rescuing BMPR2 and PAEC dysfunction. PMID:21821917

  16. Phosphorylation of Sox9 is required for neural crest delamination and is regulated downstream of BMP and canonical Wnt signaling

    OpenAIRE

    Liu, Jessica A. J.; Wu, Ming-Hoi; Yan, Carol H.; Chau, Bolton K H; So,Henry; Ng, Alvis; CHAN, Alan; Cheah, Kathryn S.E.; Briscoe, James; Cheung, Martin

    2013-01-01

    Coordination of neural crest cell (NCC) induction and delamination is orchestrated by several transcription factors. Among these, Sry-related HMG box-9 (Sox9) and Snail2 have been implicated in both the induction of NCC identity and, together with phoshorylation, NCC delamination. How phosphorylation effects this function has not been clear. Here we show, in the developing chick neural tube, that phosphorylation of Sox9 on S64 and S181 facilitates its SUMOylation, and the phosphorylated forms...

  17. Hox5 Genes Regulate the Wnt2/2b-Bmp4-Signaling Axis during Lung Development

    OpenAIRE

    Steven M. Hrycaj; Briana R. Dye; Nicholas C. Baker; Brian M. Larsen; Ann C. Burke; Jason R. Spence; Deneen M. Wellik

    2015-01-01

    Hox genes are required for proper anteroposterior axial patterning and the development of several organ systems. Here, we show that all three Hox5 paralogous genes play redundant roles in the developing lung. Hoxa5;Hoxb5;Hoxc5 triple-mutant embryos develop severely hypoplastic lungs with reduced branching and proximal-distal patterning defects. Hox5 genes are exclusively expressed in the lung mesoderm; however, defects are observed in both lung mesenchyme and endodermally derived epithelium, ...

  18. Reduced BMP Signaling Results in Hindlimb Fusion with Lethal Pelvic/Urogenital Organ Aplasia: A New Mouse Model of Sirenomelia

    OpenAIRE

    Suzuki, Kentaro; Adachi, Yasuha; Numata, Tomokazu; Nakada, Shoko; Yanagita, Motoko; Nakagata, Naomi; Evans, Sylvia M.; Graf, Daniel; Economides, Aris; Haraguchi, Ryuma; Moon, Anne M.; Yamada, Gen

    2012-01-01

    Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of pheno...

  19. Essential role of Bmp signaling and its positive feedback loop in the early cell fate evolution of chordates

    OpenAIRE

    Kozmiková, I. (Iryna); S Candiani; P. Fabian; Gurská, D. (Daniela); Kozmik, Z

    2013-01-01

    In chordates, early separation of cell fate domains occurs prior to the final specification of ectoderm to neural and non-neural as well as mesoderm to dorsal and ventral during development. Maintaining such division with the establishment of an exact border between the domains is required for the formation of highly differentiated structures such as neural tube and notochord. We hypothesized that the key condition for efficient cell fate separation in a chordate embryo is the presence of a p...

  20. TGF-β1 induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells is enhanced by IL-1β but not abrogated by corticosteroids

    OpenAIRE

    Doerner, Astrid M; Zuraw, Bruce L

    2009-01-01

    Background Chronic persistent asthma is characterized by ongoing airway inflammation and airway remodeling. The processes leading to airway remodeling are poorly understood, and there is increasing evidence that even aggressive anti-inflammatory therapy does not completely prevent this process. We sought to investigate whether TGFβ1 stimulates bronchial epithelial cells to undergo transition to a mesenchymal phenotype, and whether this transition can be abrogated by corticosteroid treatment o...

  1. The effect of SDF-1α on low dose BMP-2 mediated bone regeneration by release from heparinized mineralized collagen type I matrix scaffolds in a murine critical size bone defect model.

    Science.gov (United States)

    Zwingenberger, Stefan; Langanke, Robert; Vater, Corina; Lee, Geoffrey; Niederlohmann, Eik; Sensenschmidt, Markus; Jacobi, Angela; Bernhardt, Ricardo; Muders, Michael; Rammelt, Stefan; Knaack, Sven; Gelinsky, Michael; Günther, Klaus-Peter; Goodman, Stuart B; Stiehler, Maik

    2016-09-01

    The treatment of critical size bone defects represents a challenge. The growth factor bone morphogenetic protein 2 (BMP-2) is clinically established but has potentially adverse effects when used at high doses. The aim of this study was to evaluate if stromal derived factor-1 alpha (SDF-1α) and BMP-2 released from heparinized mineralized collagen type I matrix (MCM) scaffolds have a cumulative effect on bone regeneration. MCM scaffolds were functionalized with heparin, loaded with BMP-2 and/or SDF-1α and implanted into a murine critical size femoral bone defect (control group, low dose BMP-2 group, low dose BMP-2 + SDF-1α group, and high dose BMP-2 group). After 6 weeks, both the low dose BMP-2 + SDF-1α group (5.8 ± 0.6 mm³, p = 0.0479) and the high dose BMP-2 group (6.5 ± 0.7 mm³, p = 0.008) had a significantly increased regenerated bone volume compared to the control group (4.2 ± 0.5 mm³). There was a higher healing score in the low dose BMP-2 + SDF-1α group (median grade 8; Q1-Q3 7-9; p = 0.0357) than in the low dose BMP-2 group (7; Q1-Q3 5-9) histologically. This study showed that release of BMP-2 and SDF-1α from heparinized MCM scaffolds allows for the reduction of the applied BMP-2 concentration since SDF-1α seems to enhance the osteoinductive potential of BMP-2. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2126-2134, 2016. PMID:27060915

  2. Follistatin、Activin A与BMP-4在大鼠脑发育过程中的表达及意义%Expression and significance of follistatin, activin A and BMP-4 during the development of rat brain

    Institute of Scientific and Technical Information of China (English)

    卢彦春; 张蕾; 穆长征; 李伟伟; 金辉

    2012-01-01

    目的 观察卵泡抑素(FS)、激活素(Activin)A与骨形态发生蛋白(BMP)-4在大鼠脑发育过程中的表达变化规律.方法 将同期受孕30只SD大鼠按照胎鼠发育时间随机分为胚胎8.5 d(E8.5组)、13 d(E13组)、18d(E18组)及出生后3 d(P3组)、7d(P7组)、30 d(P30组)各5只,采用免疫组化ABC法检测各组脑皮质、纹状体、海马齿状回、嗅球组织中FS、Activin A、BMP-4表达情况.结果 FS与Activin A在大鼠脑内广泛分布,二者在E8.5组表达最高,并以E13组表