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Sample records for bmp signaling induces

  1. BMP signaling in rats with TNBS-induced colitis following BMP7 therapy.

    Science.gov (United States)

    Maric, Ivana; Kucic, Natalia; Turk Wensveen, Tamara; Smoljan, Ivana; Grahovac, Blazenka; Zoricic Cvek, Sanja; Celic, Tanja; Bobinac, Dragica; Vukicevic, Slobodan

    2012-05-15

    Beyond stimulating bone formation, bone morphogenetic proteins (BMPs) are important in development, inflammation, and malignancy of the gut. We have previously shown that BMP7 has a regenerative, anti-inflammatory, and antiproliferative effect on experimental inflammatory bowel disease (IBD) in rats. To further investigate the BMP signaling pathway we monitored the effect of BMP7 therapy on the BMP signaling components in the rat colon during different stages of experimentally induced colitis by 2,4,6-trinitrobenzene sulfonic acid (TNBS). The results showed a significantly decreased BMP7 expression in the acute phase, followed by a significantly increased BMP2 and decreased BMP6 expression during the chronic phase of colitis. BMP7 therapy influenced the expression of several BMPs with the most prominent effect on downregulation of BMP2 and upregulation of BMP4 in the chronic phase of colitis. Importantly, connective tissue growth factor and noggin expression were elevated in the acute stage and significantly decreased upon BMP7 therapy. BMP receptor I expression was unchanged, whereas BMP receptor II was decreased at day 2 and increased at days 14 and 30 of TNBS inflammation. However, an opposite pattern of expression following BMP7 therapy has been observed. BMP7 increased the expression of BR-Smad including Smad3 and Smad4. Inhibitory Smads were increased in colitis and significantly decreased following BMP7 therapy at later stages of the disease. We suggest that BMP signaling was altered during TNBS-induced colitis and was recovered with BMP7 administration, suggesting that IBD is a reversible process.

  2. Arginine methylation initiates BMP-induced Smad signaling

    Science.gov (United States)

    Xu, Jian; Wang, A. Hongjun; Oses-Prieto, Juan; Makhijani, Kalpana; Katsuno, Yoko; Pei, Ming; Yan, Leilei; Zheng, Y. George; Burlingame, Alma; Brückner, Katja; Derynck, Rik

    2014-01-01

    Summary Kinase activation and substrate phosphorylation commonly form the backbone of signaling cascades. Bone morphogenetic proteins (BMPs), a subclass of TGF-β family ligands, induce activation of their signaling effectors, the Smads, through C-terminal phosphorylation by transmembrane receptor kinases. However, the slow kinetics of Smad activation in response to BMP suggests a preceding step in the initiation of BMP signaling. We now show that arginine methylation, which is known to regulate gene expression, yet also modifies some signaling mediators, initiates BMP-induced Smad signaling. BMP-induced receptor complex formation promotes interaction of the methyltransferase PRMT1 with the inhibitory Smad6, resulting in Smad6 methylation and relocalization at the receptor, leading to activation of effector Smads through phosphorylation. PRMT1 is required for BMP-induced biological responses across species, as evidenced by the role of its ortholog Dart1 in BMP signaling during Drosophila wing development. Activation of signaling by arginine methylation may also apply to other signaling pathways. PMID:23747011

  3. BMP signaling induces digit regeneration in neonatal mice.

    Science.gov (United States)

    Yu, Ling; Han, Manjong; Yan, Mingquan; Lee, Eun-Chee; Lee, Jangwoo; Muneoka, Ken

    2010-02-01

    The regenerating digit tip of mice is a novel epimorphic response in mammals that is similar to fingertip regeneration in humans. Both display restricted regenerative capabilities that are amputation-level dependent. Using this endogenous regeneration model in neonatal mice, we have found that noggin treatment inhibits regeneration, thus suggesting a bone morphogenetic protein (BMP) requirement. Using non-regenerating amputation wounds, we show that BMP7 or BMP2 can induce a regenerative response. BMP-induced regeneration involves the formation of a mammalian digit blastema. Unlike the endogenous regeneration response that involves redifferentiation by direct ossification (evolved regeneration), the BMP-induced response involves endochondral ossification (redevelopment). Our evidence suggests that BMP treatment triggers a reprogramming event that re-initiates digit tip development at the amputation wound. These studies demonstrate for the first time that the postnatal mammalian digit has latent regenerative capabilities that can be induced by growth factor treatment.

  4. Endocytosis contributes to BMP2-induced Smad signalling and neuronal growth.

    Science.gov (United States)

    Hegarty, Shane V; Sullivan, Aideen M; O'Keeffe, Gerard W

    2017-02-08

    Bone morphogenetic protein 2 (BMP2) is a neurotrophic factor which induces the growth of midbrain dopaminergic (DA) neurons in vitro and in vivo, and its neurotrophic effects have been shown to be dependent on activation of BMP receptors (BMPRs) and Smad 1/5/8 signalling. However, the precise intracellular cascades that regulate BMP2-BMPR-Smad-signalling-induced neurite growth remain unknown. Endocytosis has been shown to regulate Smad 1/5/8 signalling and differentiation induced by BMPs. However, these studies were carried out in non-neural cells. Indeed, there are scant reports regarding the role of endocytosis in BMP-Smad signalling in neurons. To address this, and to further characterise the mechanisms regulating the neurotrophic effects of BMP2, the present study examined the role of dynamin-dependent endocytosis in BMP2-induced Smad signalling and neurite growth in the SH-SY5Y neuronal cell line. The activation, temporal kinetics and magnitude of Smad 1/5/8 signalling induced by BMP2 were significantly attenuated by dynasore-mediated inhibition of endocytosis in SH-SY5Y cells. Furthermore, BMP2-induced increases in neurite length and neurite branching in SH-SY5Y cells were significantly reduced following inhibition of dynamin-dependent endocytosis using dynasore. This study demonstrates that BMP2-induced Smad signalling and neurite growth is regulated by dynamin-dependent endocytosis in a model of human midbrain dopaminergic neurons.

  5. Regulation of Notch signaling genes during BMP2-induced differentiation of osteoblast precursor cells.

    NARCIS (Netherlands)

    Jong, D.S. de; Steegenga, W.T.; Hendriks, J.M.; Zoelen, E.J.J. van; Olijve, W.; Dechering, K.J.

    2004-01-01

    The bone morphogenetic protein (BMP)-induced Smad signal transduction pathway is an important positive regulator of osteoblast differentiation. BMP and other members of the transforming growth factor-beta (TGF-beta) family have distinct effects on osteoblast differentiation, depending on cell type a

  6. Regulation of Notch signaling genes during BMP2-induced differentiation of osteoblast precursor cells

    NARCIS (Netherlands)

    Jong, de D.S.; Steegenga, W.T.; Hendriks, J.M.A.; Zoelen, van E.J.J.; Olijve, W.; Dechering, K.J.

    2004-01-01

    The bone morphogenetic protein (BMP)-induced Smad signal transduction pathway is an important positive regulator of osteoblast differentiation. BMP and other members of the transforming growth factor-beta (TGF-beta) family have distinct effects on osteoblast differentiation, depending on cell type a

  7. BMP signaling mediates stem/progenitor cell-induced retina regeneration.

    Science.gov (United States)

    Haynes, Tracy; Gutierrez, Christian; Aycinena, Juan-Carlos; Tsonis, Panagiotis A; Del Rio-Tsonis, Katia

    2007-12-18

    We identified a mechanism whereby retina regeneration in the embryonic chick can be induced by the contribution of stem/progenitor cells. We show that bone morphogenetic protein (BMP) signaling is sufficient and necessary to induce retina regeneration and that its action can be divided into two phases. By 3 days after postretinectomy (d PR), the BMP pathway directs proliferation and regeneration through the activation of Smad (canonical BMP pathway) and the up-regulation of FGF signaling by the MAPK pathway. By 7d PR, it induces apoptosis by activating p38 (a noncanonical BMP pathway) and down-regulating FGF signaling (by both MAPK and AKT pathways). Apoptosis at this later stage can be prevented, and BMP-induced regeneration can be further induced by inhibition of p38. These results unravel a mechanism for stem/progenitor cell-mediated retina regeneration, where BMP activation establishes a cross-talk with the FGF pathway and selectively activates the canonical and noncanonical BMP pathways. Retina stem/progenitor cells exist in other species, including humans. Thus, our findings provide insights on how retinal stem cells can be activated for possible regenerative therapies.

  8. Fgf signaling induces posterior neuroectoderm independently of Bmp signaling inhibition.

    NARCIS (Netherlands)

    Rentzsch, F.; Bakkers, J.; Kramer, C.; Hammerschmidt, M.

    2004-01-01

    Whereas according to the neural default model, neural specification is induced by extracellular inhibitors of bone morphogenetic proteins (Bmps), the role of fibroblast growth factors (Fgfs) during neural induction is heavily debated. Here, we show that, in zebrafish embryos, Bmps and Fgfs play diff

  9. Kaempferol induces chondrogenesis in ATDC5 cells through activation of ERK/BMP-2 signaling pathway.

    Science.gov (United States)

    Nepal, Manoj; Li, Liang; Cho, Hyoung Kwon; Park, Jong Kun; Soh, Yunjo

    2013-12-01

    Endochondral bone formation occurs when mesenchymal cells condense to differentiate into chondrocytes, the primary cell types of cartilage. The aim of the present study was to identify novel factors regulating chondrogenesis. We investigated whether kaempferol induces chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Kaempferol treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. Kaempferol-treated ATDC5 cells stained more intensely with alcian blue staining than control cells, suggesting greater synthesis of matrix proteoglycans in the kaempferol-treated cells. Similarly, kaempferol induced greater activation of alkaline phosphatase activity than control cells, and it enhanced the expression of chondrogenic marker genes, such as collagen type I, collagen type X, OCN, Runx2, and Sox9. Kaempferol induced an acute activation of extracellular signal-regulated kinase (ERK) but not c-jun N-terminal kinase or p38 MAP kinase. PD98059, an inhibitor of MAPK/ERK, decreased in stained cells treated with kaempferol. Furthermore, kaempferol greatly expressed the protein and mRNA levels of BMP-2, suggesting chondrogenesis was stimulated via a BMP-2 pathway. Taken together, our results suggest that kaempferol has chondromodulating effects via an ERK/BMP-2 signaling pathway and could potentially be used as a therapeutic agent for bone growth disorders.

  10. FGFs and BMP4 induce both Msx1-independent and Msx1-dependent signaling pathways in early tooth development.

    Science.gov (United States)

    Bei, M; Maas, R

    1998-11-01

    During early tooth development, multiple signaling molecules are expressed in the dental lamina epithelium and induce the dental mesenchyme. One signal, BMP4, has been shown to induce morphologic changes in dental mesenchyme and mesenchymal gene expression via Msx1, but BMP4 cannot substitute for all the inductive functions of the dental epithelium. To investigate the role of FGFs during early tooth development, we examined the expression of epithelial and mesenchymal Fgfs in wild-type and Msx1 mutant tooth germs and tested the ability of FGFs to induce Fgf3 and Bmp4 expression in wild-type and Msx1 mutant dental mesenchymal explants. Fgf8 expression is preserved in Msx1 mutant epithelium while that of Fgf3 is not detected in Msx1 mutant dental mesenchyme. Moreover, dental epithelium as well as beads soaked in FGF1, FGF2 or FGF8 induce Fgf3 expression in dental mesenchyme in an Msx1-dependent manner. These results indicate that, like BMP4, FGF8 constitutes an epithelial inductive signal capable of inducing the expression of downstream signaling molecules in dental mesenchyme via Msx1. However, the BMP4 and FGF8 signaling pathways are distinct. BMP4 cannot induce Fgf3 nor can FGFs induce Bmp4 expression in dental mesenchyme, even though both signaling molecules can induce Msx1 and Msx1 is necessary for Fgf3 and Bmp4 expression in dental mesenchyme. In addition, we have investigated the effects of FGFs and BMP4 on the distal-less homeobox genes Dlx1 and Dlx2 and we have clarified the relationship between Msx and Dlx gene function in the developing tooth. Dlx1,Dlx2 double mutants exhibit a lamina stage arrest in maxillary molar tooth development (Thomas B. L., Tucker A. S., Qiu M. , Ferguson C. A., Hardcastle Z., Rubenstein J. L. R. and Sharpe P. T. (1997) Development 124, 4811-4818). Although the maintenance of molar mesenchymal Dlx2 expression at the bud stage is Msx1-dependent, both the maintenance of Dlx1 expression and the initial activation of mesenchymal Dlx1

  11. Delta-like 1/fetal antigen 1(DLK1/FA1) inhibits BMP2 induced osteoblast differentiation through modulation of NFκB signaling pathway

    DEFF Research Database (Denmark)

    Qiu, Weimin; Abdallah, Basem; Kassem, Moustapha

    as assessed by reduced Alp activity and osteogenic gene expression including Alp, Col1a1, Runx2 and Bglap. In addition, DLK1/FA1 inhibited BMP signaling as demonstrated by reduced gene expression of BMP-responsive genes: Junb and Id1, reduced BMP2 induced luciferase activity in C2C12 BMP luciferase reporter....... Besides, we observed that DLK1/FA1 induced strong NFκB activity evidenced by NFκB responsive luciferase reporter assay and real-time RT-PCR analysis of NFκB target genes. The inhibitory effect of NFκB signaling on BMP signaling was confirmed by luciferase assay in C2C12 BMP luciferase reporter cells...

  12. 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.

  13. Arsenite suppression of BMP signaling in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    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

  14. β3 integrin-mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner.

    Science.gov (United States)

    Fourel, Laure; Valat, Anne; Faurobert, Eva; Guillot, Raphael; Bourrin-Reynard, Ingrid; Ren, Kefeng; Lafanechère, Laurence; Planus, Emmanuelle; Picart, Catherine; Albiges-Rizo, Corinne

    2016-03-14

    Understanding how cells integrate multiple signaling pathways to achieve specific cell differentiation is a challenging question in cell biology. We have explored the physiological presentation of BMP-2 by using a biomaterial that harbors tunable mechanical properties to promote localized BMP-2 signaling. We show that matrix-bound BMP-2 is sufficient to induce β3 integrin-dependent C2C12 cell spreading by overriding the soft signal of the biomaterial and impacting actin organization and adhesion site dynamics. In turn, αvβ3 integrin is required to mediate BMP-2-induced Smad signaling through a Cdc42-Src-FAK-ILK pathway. β3 integrin regulates a multistep process to control first BMP-2 receptor activity and second the inhibitory role of GSK3 on Smad signaling. Overall, our results show that BMP receptors and β3 integrin work together to control Smad signaling and tensional homeostasis, thereby coupling cell adhesion and fate commitment, two fundamental aspects of developmental biology and regenerative medicine.

  15. β3 integrin–mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner

    Science.gov (United States)

    Fourel, Laure; Valat, Anne; Faurobert, Eva; Guillot, Raphael; Bourrin-Reynard, Ingrid; Ren, Kefeng; Lafanechère, Laurence; Planus, Emmanuelle; Albiges-Rizo, Corinne

    2016-01-01

    Understanding how cells integrate multiple signaling pathways to achieve specific cell differentiation is a challenging question in cell biology. We have explored the physiological presentation of BMP-2 by using a biomaterial that harbors tunable mechanical properties to promote localized BMP-2 signaling. We show that matrix-bound BMP-2 is sufficient to induce β3 integrin–dependent C2C12 cell spreading by overriding the soft signal of the biomaterial and impacting actin organization and adhesion site dynamics. In turn, αvβ3 integrin is required to mediate BMP-2–induced Smad signaling through a Cdc42–Src–FAK–ILK pathway. β3 integrin regulates a multistep process to control first BMP-2 receptor activity and second the inhibitory role of GSK3 on Smad signaling. Overall, our results show that BMP receptors and β3 integrin work together to control Smad signaling and tensional homeostasis, thereby coupling cell adhesion and fate commitment, two fundamental aspects of developmental biology and regenerative medicine. PMID:26953352

  16. Insights into the osteoblast precursor differentiation towards mature osteoblasts induced by continuous BMP-2 signaling

    Directory of Open Access Journals (Sweden)

    Omar F. Zouani

    2013-07-01

    Mature osteoblasts are the cells responsible for bone formation and are derived from precursor osteoblasts. However, the mechanisms that control this differentiation are poorly understood. In fact, unlike the majority of organs in the body, which are composed of “soft” tissue from which cells can easily be isolated and studied, the “hard” mineralized tissue of bone has made it difficult to study the function of bone cells. Here, we established an in vitro model that mimics this differentiation under physiological conditions. We obtained mature osteoblasts and characterized them on the basis of the following parameters: the strong expression of osteoblastic markers, such as Runx2 and Col-I; the achievement of specific dimensions (the cell volume increases 26-fold compared to the osteoblast precursors; and the production of an abundant extracellular matrix also called osteoid. We demonstrated that the differentiation of osteoblast precursors into mature osteoblasts requires the continuous activation of Bone Morphogenetic Protein (BMP receptors, which we established with the immobilization of a BMP-2mimetic peptide on a synthetic matrix mimicking in vivo microenvironment. Importantly, we demonstrated that the organization of the F-actin network and acetylated microtubules of the cells were modified during the differentiation process. We showed that the perturbation of the F-actin cytoskeleton organization abolished the differentiation process. In addition, we demonstrated that expression of the Runx2 gene is required for this differentiation. These findings demonstrate the retro-regulation of cytoplasmic and genic components due to the continuous induction of BMP-2 and also provide more detailed insights into the correct signaling of BMPs for cell differentiation in bone tissue.

  17. Activation of Bmp2-Smad1 signal and its regulation by coordinated alteration of H3K27 trimethylation in Ras-induced senescence.

    Directory of Open Access Journals (Sweden)

    Atsushi Kaneda

    2011-11-01

    Full Text Available Cellular senescence involves epigenetic alteration, e.g. loss of H3K27me3 in Ink4a-Arf locus. Using mouse embryonic fibroblast (MEF, we here analyzed transcription and epigenetic alteration during Ras-induced senescence on genome-wide scale by chromatin immunoprecipitation (ChIP-sequencing and microarray. Bmp2 was the most activated secreted factor with H3K4me3 gain and H3K27me3 loss, whereas H3K4me3 loss and de novo formation of H3K27me3 occurred inversely in repression of nine genes, including two BMP-SMAD inhibitors Smad6 and Noggin. DNA methylation alteration unlikely occurred. Ras-activated cells senesced with nuclear accumulation of phosphorylated SMAD1/5/8. Senescence was bypassed in Ras-activated cells when Bmp2/Smad1 signal was blocked by Bmp2 knockdown, Smad6 induction, or Noggin induction. Senescence was induced when recombinant BMP2 protein was added to Bmp2-knocked-down Ras-activated cells. Downstream Bmp2-Smad1 target genes were then analyzed genome-wide by ChIP-sequencing using anti-Smad1 antibody in MEF that was exposed to BMP2. Smad1 target sites were enriched nearby transcription start sites of genes, which significantly correlated to upregulation by BMP2 stimulation. While Smad6 was one of Smad1 target genes to be upregulated by BMP2 exposure, Smad6 repression in Ras-activated cells with increased enrichment of Ezh2 and gain of H3K27me3 suggested epigenetic disruption of negative feedback by Polycomb. Among Smad1 target genes that were upregulated in Ras-activated cells without increased repressive mark, Parvb was found to contribute to growth inhibition as Parvb knockdown lead to escape from senescence. It was revealed through genome-wide analyses in this study that Bmp2-Smad1 signal and its regulation by harmonized epigenomic alteration play an important role in Ras-induced senescence.

  18. Dynamics of BMP signaling in limb bud mesenchyme and polydactyly.

    Science.gov (United States)

    Norrie, Jacqueline L; Lewandowski, Jordan P; Bouldin, Cortney M; Amarnath, Smita; Li, Qiang; Vokes, Martha S; Ehrlich, Lauren I R; Harfe, Brian D; Vokes, Steven A

    2014-09-15

    Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.

  19. Brown-fat paucity due to impaired BMP signalling induces compensatory browning of white fat.

    Science.gov (United States)

    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

    2013-03-21

    Maintenance of body temperature is essential for the survival of homeotherms. Brown adipose tissue (BAT) is a specialized fat tissue that is dedicated to thermoregulation. Owing to its remarkable capacity to dissipate stored energy and its demonstrated presence in adult humans, BAT holds great promise for the treatment of obesity and metabolic syndrome. Rodent data suggest the existence of two types of brown fat cells: constitutive BAT (cBAT), which is of embryonic origin and anatomically located in the interscapular region of mice; and recruitable BAT (rBAT), which resides within white adipose tissue (WAT) and skeletal muscle, and has alternatively been called beige, brite or inducible BAT. Bone morphogenetic proteins (BMPs) regulate the formation and thermogenic activity of BAT. Here we use mouse models to provide evidence for a systemically active regulatory mechanism that controls whole-body BAT activity for thermoregulation and energy homeostasis. Genetic ablation of the type 1A BMP receptor (Bmpr1a) in brown adipogenic progenitor cells leads to a severe paucity of cBAT. This in turn increases sympathetic input to WAT, thereby promoting the formation of rBAT within white fat depots. This previously unknown compensatory mechanism, aimed at restoring total brown-fat-mediated thermogenic capacity in the body, is sufficient to maintain normal temperature homeostasis and resistance to diet-induced obesity. These data suggest an important physiological cross-talk between constitutive and recruitable brown fat cells. This sophisticated regulatory mechanism of body temperature may participate in the control of energy balance and metabolic disease.

  20. Improving the osteogenic efficacy of BMP2 with mechano growth factor by regulating the signaling events in BMP pathway.

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    Deng, Moyuan; Liu, Peng; Xiao, Hualiang; Zhang, Yuanyuan; Wang, Yuanliang; Zhao, Jianhua; Xu, Jianzhong

    2015-09-01

    Local application of bone morphogenetic protein 2 (BMP2) is known to promote large bone defect healing and BMP2-initiated bone regeneration could be enhanced by an additional mechanical stimulation. The C-terminal 24-a.a. peptide of mechano growth factor (MGF24E), a mechanical-sensitive molecule, has been demonstrated to promote bone healing. Here, we propose a hypothesis that MGF24E could also improve the osteogenic efficacy of BMP2 by regulating the signaling events in the BMP pathway. To confirm the hypothesis, the potentials of MGF24E, BMP2 and BMP2/MGF24E combination treatments on the phosphorylation of Smad 1/5/8, the downstream osteogenesis-related gene expression and osteoblasts mineralization, are investigated with or without the blocking of Smad 5 siRNA. Furthermore, 15-mm rabbit radial bone defects were healed with the cytokine treatments and then evaluated by radiographic examination, histological assessment and immunohistochemical analysis. MGF24E could enhance the BMP2-induced Smad signaling pathway by upregulating the p-Smad protein expression and the downstream osteogenic gene expression. An amount of 5 nM BMP2 in a sub-25 nM concentration of MGF24E medium achieved a higher expression for ALP mRNA and a greater calcium mineral content compared with BMP2 alone. Nevertheless, the inhibition of the MGF24E-regulated BMP pathway could block osteogenesis induced by the dual treatment. In vivo, MGF24E treatment upregulated the endogenous BMP2 expression and the addition of MGF24E into the BMP2 treatment remarkably enhanced the bone mineral density (BMD), the radiographic scores and the histological restoration of the regenerated tissue against BMP2 treatment, suggesting a new strategy for BMP2 in bone defect healing.

  1. Regulation of BMP2-induced intracellular calcium increases in osteoblasts.

    Science.gov (United States)

    Xu, Wenfeng; Liu, Bo; Liu, Xue; Chiang, Martin Y M; Li, Bo; Xu, Zichen; Liao, Xiaoling

    2016-10-01

    Although bone morphogenetic protein-2 (BMP2) is a well-characterized regulator that stimulates osteoblast differentiation, little is known about how it regulates intracellular Ca(2+) signaling. In this study, intracellular Ca(2+) concentration ([Ca(2+) ]i ) upon BMP2 application, focal adhesion kinase (FAK) and Src activities were measured in the MC3T3-E1 osteoblast cell line using fluorescence resonance energy transfer-based biosensors. Increase in [Ca(2+) ]i , FAK, and Src activities were observed during BMP2 stimulation. The removal of extracellular calcium, the application of membrane channel inhibitors streptomycin or nifedipine, the FAK inhibitor PF-573228 (PF228), and the alkaline phosphatase (ALP) siRNA all blocked the BMP2-stimulated [Ca(2+) ]i increase, while the Src inhibitor PP1 did not. In contrast, a gentle decrease of endoplasmic reticulum calcium concentration was found after BMP2 stimulation, which could be blocked by both streptomycin and PP1. Further experiments revealed that BMP2-induced FAK activation could not be inhibited by PP1, ALP siRNA or the calcium channel inhibitor nifedipine. PF228, but not PP1 or calcium channel inhibitors, suppressed ALP elevation resulting from BMP2 stimulation. Therefore, our results suggest that BMP2 can increase [Ca(2+) ]i through extracellular calcium influx regulated by FAK and ALP and can deplete ER calcium through Src signaling simultaneously. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1725-1733, 2016.

  2. BMP type I receptor ALK2 is required for angiotensin II-induced cardiac hypertrophy.

    Science.gov (United States)

    Shahid, Mohd; Spagnolli, Ester; Ernande, Laura; Thoonen, Robrecht; Kolodziej, Starsha A; Leyton, Patricio A; Cheng, Juan; Tainsh, Robert E T; Mayeur, Claire; Rhee, David K; Wu, Mei X; Scherrer-Crosbie, Marielle; Buys, Emmanuel S; Zapol, Warren M; Bloch, Kenneth D; Bloch, Donald B

    2016-04-15

    Bone morphogenetic protein (BMP) signaling contributes to the development of cardiac hypertrophy. However, the identity of the BMP type I receptor involved in cardiac hypertrophy and the underlying molecular mechanisms are poorly understood. By using quantitative PCR and immunoblotting, we demonstrated that BMP signaling increased during phenylephrine-induced hypertrophy in cultured neonatal rat cardiomyocytes (NRCs), as evidenced by increased phosphorylation of Smads 1 and 5 and induction of Id1 gene expression. Inhibition of BMP signaling with LDN193189 or noggin, and silencing of Smad 1 or 4 using small interfering RNA diminished the ability of phenylephrine to induce hypertrophy in NRCs. Conversely, activation of BMP signaling with BMP2 or BMP4 induced hypertrophy in NRCs. Luciferase reporter assay further showed that BMP2 or BMP4 treatment of NRCs repressed atrogin-1 gene expression concomitant with an increase in calcineurin protein levels and enhanced activity of nuclear factor of activated T cells, providing a mechanism by which BMP signaling contributes to cardiac hypertrophy. In a model of cardiac hypertrophy, C57BL/6 mice treated with angiotensin II (A2) had increased BMP signaling in the left ventricle. Treatment with LDN193189 attenuated A2-induced cardiac hypertrophy and collagen deposition in left ventricles. Cardiomyocyte-specific deletion of BMP type I receptor ALK2 (activin-like kinase 2), but not ALK1 or ALK3, inhibited BMP signaling and mitigated A2-induced cardiac hypertrophy and left ventricular fibrosis in mice. The results suggest that BMP signaling upregulates the calcineurin/nuclear factor of activated T cell pathway via BMP type I receptor ALK2, contributing to cardiac hypertrophy and fibrosis.

  3. Sox9 potentiates BMP2-induced chondrogenic differentiation and inhibits BMP2-induced osteogenic differentiation.

    Science.gov (United States)

    Liao, Junyi; Hu, Ning; Zhou, Nian; Lin, Liangbo; Zhao, Chen; Yi, Shixiong; Fan, Tingxu; Bao, Wei; Liang, Xi; Chen, Hong; Xu, Wei; Chen, Cheng; Cheng, Qiang; Zeng, Yongming; Si, Weike; Yang, Zhong; Huang, Wei

    2014-01-01

    Bone morphogenetic protein 2 (BMP2) is one of the key chondrogenic growth factors involved in the cartilage regeneration. However, it also exhibits osteogenic abilities and triggers endochondral ossification. Effective chondrogenesis and inhibition of BMP2-induced osteogenesis and endochondral ossification can be achieved by directing the mesenchymal stem cells (MSCs) towards chondrocyte lineage with chodrogenic factors, such as Sox9. Here we investigated the effects of Sox9 on BMP2-induced chondrogenic and osteogenic differentiation of MSCs. We found exogenous overexpression of Sox9 enhanced the BMP2-induced chondrogenic differentiation of MSCs in vitro. Also, it inhibited early and late osteogenic differentiation of MSCs in vitro. Subcutaneous stem cell implantation demonstrated Sox9 potentiated BMP2-induced cartilage formation and inhibited endochondral ossification. Mouse limb cultures indicated that BMP2 and Sox9 acted synergistically to stimulate chondrocytes proliferation, and Sox9 inhibited BMP2-induced chondrocytes hypertrophy and ossification. This study strongly suggests that Sox9 potentiates BMP2-induced MSCs chondrogenic differentiation and cartilage formation, and inhibits BMP2-induced MSCs osteogenic differentiation and endochondral ossification. Thus, exogenous overexpression of Sox9 in BMP2-induced mesenchymal stem cells differentiation may be a new strategy for cartilage tissue engineering.

  4. A new class of small molecule inhibitor of BMP signaling.

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    Caroline E Sanvitale

    Full Text Available Growth factor signaling pathways are tightly regulated by phosphorylation and include many important kinase targets of interest for drug discovery. Small molecule inhibitors of the bone morphogenetic protein (BMP receptor kinase ALK2 (ACVR1 are needed urgently to treat the progressively debilitating musculoskeletal disease fibrodysplasia ossificans progressiva (FOP. Dorsomorphin analogues, first identified in zebrafish, remain the only BMP inhibitor chemotype reported to date. By screening an assay panel of 250 recombinant human kinases we identified a highly selective 2-aminopyridine-based inhibitor K02288 with in vitro activity against ALK2 at low nanomolar concentrations similar to the current lead compound LDN-193189. K02288 specifically inhibited the BMP-induced Smad pathway without affecting TGF-β signaling and induced dorsalization of zebrafish embryos. Comparison of the crystal structures of ALK2 with K02288 and LDN-193189 revealed additional contacts in the K02288 complex affording improved shape complementarity and identified the exposed phenol group for further optimization of pharmacokinetics. The discovery of a new chemical series provides an independent pharmacological tool to investigate BMP signaling and offers multiple opportunities for pre-clinical development.

  5. Bmp and Shh signaling mediate the expression of satb2 in the pharyngeal arches.

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    Sheehan-Rooney, Kelly; Swartz, Mary E; Lovely, C Ben; Dixon, Michael J; Eberhart, Johann K

    2013-01-01

    In human, mutation of the transcription factor SATB2 causes severe defects to the palate and jaw. The expression and sequence of SATB2 is highly conserved across vertebrate species, including zebrafish. We sought to understand the regulation of satb2 using the zebrafish model system. Due to the normal expression domains of satb2, we analyzed satb2 expression in mutants with disrupted Hh signaling or defective ventral patterning. While satb2 expression appears independent of Edn1 signaling, appropriate expression requires Shha, Smo, Smad5 and Hand2 function. Transplantation experiments show that neural crest cells receive both Bmp and Hh signaling to induce satb2 expression. Dorsomorphin- and cyclopamine-mediated inhibition of Bmp and Hh signaling, respectively, suggests that proper satb2 expression requires a relatively earlier Bmp signal and a later Hh signal. We propose that Bmp signaling establishes competence for the neural crest to respond to Hh signaling, thus inducing satb2 expression.

  6. Targeting osteoblastic casein kinase-2 interacting protein-1 to enhance Smad-dependent BMP signaling and reverse bone formation reduction in glucocorticoid-induced osteoporosis

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    Liu, Jin; Lu, Changwei; Wu, Xiaohao; Zhang, Zongkang; Li, Jie; Guo, Baosheng; Li, Defang; Liang, Chao; Dang, Lei; Pan, Xiaohua; Peng, Songlin; Lu, Aiping; Zhang, Baoting; Zhang, Ge

    2017-01-01

    The underlying mechanism of the reduced bone formation during the development of glucocorticoid-induced osteoporosis (GIO) remains unclear. Here, we found that the highly expressed CKIP-1 together with lowly expressed total and phosphorylated Smad1/5 in bone samples was accompanied by either the reduced serum bone formation markers in GIO patients or the decreased bone formation in GIO mice. In vitro studies showed that the highly expressed CKIP-1 could promote Smad1 ubiquitination to suppress the Smad-dependent BMP signaling and inhibit osteogenic differentiation and mineral deposition in MC3T3-E1 cells during glucocorticoid treatment. Further, the reduced bone formation in GIO mice could not only be prevented by osteoblasts-specific Ckip-1 ablation, but also be attenuated after osteoblasts-specific Smad1 overexpression. Moreover, osteoblasts-targeting CKIP-1 siRNA treatment also attenuated the bone formation reduction in GIO mice. These study suggest that the highly expressed CKIP-1 in osteoblasts could suppress the Smad-dependent BMP signaling and contribute to the bone formation reduction in GIO. Targeting osteoblastic CKIP-1 would be a novel bone anabolic strategy for GIO patients. PMID:28128304

  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.

  8. TGF-b/BMP signaling and other molecular events:regulation of osteoblastogenesis and bone formation

    Institute of Scientific and Technical Information of China (English)

    Md Shaifur Rahman; Naznin Akhtar; Hossen Mohammad Jamil; Rajat Suvra Banik; Sikder M Asaduzzaman

    2015-01-01

    Transforming growth factor-beta (TGF-b)/bone morphogenetic protein (BMP) plays a fundamental role in the regulation of bone organogenesis through the activation of receptor serine/threonine kinases. Perturbations of TGF-b/BMP activity are almost invariably linked to a wide variety of clinical outcomes, i.e., skeletal, extra skeletal anomalies, autoimmune, cancer, and cardiovascular diseases. Phosphorylation of TGF-b (I/II) or BMP receptors activates intracellular downstream Smads, the transducer of TGF-b/BMP signals. This signaling is modulated by various factors and pathways, including transcription factor Runx2. The signaling network in skeletal development and bone formation is overwhelmingly complex and highly time and space specific. Additive, positive, negative, or synergistic effects are observed when TGF-b/BMP interacts with the pathways of MAPK, Wnt, Hedgehog (Hh), Notch, Akt/mTOR, and miRNA to regulate the effects of BMP-induced signaling in bone dynamics. Accumulating evidence indicates that Runx2 is the key integrator, whereas Hh is a possible modulator, miRNAs are regulators, and b-catenin is a mediator/regulator within the extensive intracellular network. This review focuses on the activation of BMP signaling and interaction with other regulatory components and pathways highlighting the molecular mechanisms regarding TGF-b/BMP function and regulation that could allow understanding the complexity of bone tissue dynamics.

  9. Direct BMP2/4 signaling through BMP receptor IA regulates fetal thymocyte progenitor homeostasis and differentiation to CD4+CD8+ double-positive cell.

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    Hager-Theodorides, Ariadne L; Ross, Susan E; Sahni, Hemant; Mishina, Yuji; Furmanski, Anna L; Crompton, Tessa

    2014-01-01

    BMP2/4 signaling is required for embryogenesis and involved in thymus morphogenesis and T-lineage differentiation. In vitro experiments have shown that treatment of thymus explants with exogenous BMP4 negatively regulated differentiation of early thymocyte progenitors and the transition from CD4-CD8- (DN) to CD4+CD8+ (DP). Here we show that in vivo BMP2/4 signaling is required for fetal thymocyte progenitor homeostasis and expansion, but negatively regulates differentiation from DN to DP cell. Unexpectedly, conditional deletion of BMPRIA from fetal thymocytes (using the Cre-loxP system and directing excision to hematopoietic lineage cells with the Vav promoter) demonstrated that physiological levels of BMP2/4 signaling directly to thymocytes through BMPRIA are required for normal differentiation and expansion of early fetal DN thymocytes. In contrast, the arrest in early thymocyte progenitor differentiation caused by exogenous BMP4 treatment of thymus explants is induced in part by direct signaling to thymocytes through BMPRIA, and in part by indirect signaling through non-hematopoietic cells. Analysis of the transition from fetal DN to DP cell, both by ex vivo analysis of conditional BMPRIA-deficient thymocytes and by treatment of thymus explants with the BMP4-inhibitor Noggin demonstrated that BMP2/4 signaling is a negative regulator at this stage. We showed that at this stage of fetal T-cell development BMP2/4 signals directly to thymocytes through BMPRIA.

  10. BMP2 induces osteoblast apoptosis in a maturation state and noggin-dependent manner.

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    Hyzy, Sharon L; Olivares-Navarrete, Rene; Schwartz, Zvi; Boyan, Barbara D

    2012-10-01

    Large doses of bone morphogenetic protein 2 (BMP2) are used clinically to induce bone formation in challenging bone defects. However, complications after treatment include swelling, ectopic bone formation, and adjacent bone resorption. While BMP2 can be effective, it is important to characterize the mechanism of the deleterious effects to optimize its use. The aim of this study was to determine the effect of BMP2 on apoptosis in osteoblast lineage cells and to determine the role of the BMP inhibitor Noggin in this process. Human mesenchymal stem cells (MSCs), immature osteoblast-like MG63 cells, and mature normal human osteoblasts (NHOst) were treated with BMP2. A model system of increased endogenous BMP signaling was created by silencing Noggin (shNOG-MG63). Finally, the BMP pathway regulating apoptosis in NHOst was examined using BMP signaling inhibitors (5Z-7-oxozeaenol, dorsomorphin, H-8). Apoptosis was characterized by caspase-3, BAX/BCL2, p53, and DNA fragmentation. BMP2 induced apoptosis in a cell-type dependent manner. While the effect was minor in MSCs, MG63 cells had modest increases and NHOst cells had robust increases apoptosis after BMP2 treatment. Apoptosis was significantly higher in shNOG-MG63 than MG63 cells. 5Z-7-oxozeaenol and dorsomorphin eliminated the BMP2-induced increase in DNA fragmentation in NHOst, suggesting roles for TAB/TAK1 and Smad signaling. These results indicate that the apoptotic effect of BMP2 is dependent on cell maturation state, inducing apoptosis in committed osteoblasts through Smad and TAB/TAK1 signaling, and is regulated by Noggin. Dose and delivery must be optimized in therapeutic applications of BMP2 to minimize complications.

  11. BMP2-SMAD signaling represses the proliferation of embryonic neural stem cells through YAP.

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    Yao, Minghui; Wang, Yadong; Zhang, Peng; Chen, Hong; Xu, Zhiheng; Jiao, Jianwei; Yuan, Zengqiang

    2014-09-03

    Previous studies have shown that the Hippo pathway effector yes-associated protein (YAP) plays an important role in maintaining stem cell proliferation. However, the precise molecular mechanism of YAP in regulating murine embryonic neural stem cells (NSCs) remains largely unknown. Here, we show that bone morphogenetic protein-2 (BMP2) treatment inhibited the proliferation of mouse embryonic NSCs, that YAP was critical for mouse NSC proliferation, and that BMP2 treatment-induced inhibition of mouse NSC proliferation was abrogated by YAP knockdown, indicating that the YAP protein mediates the inhibitory effect of BMP2 signaling. Additionally, we found that BMP2 treatment reduced YAP nuclear translocation, YAP-TEAD interaction, and YAP-mediated transactivation. BMP2 treatment inhibited YAP/TEAD-mediated Cyclin D1 (ccnd1) expression, and knockdown of ccnd1 abrogated the BMP2-mediated inhibition of mouse NSC proliferation. Mechanistically, we found that Smad1/4, effectors of BMP2 signaling, competed with YAP for the interaction with TAED1 and inhibited YAP's cotranscriptional activity. Our data reveal mechanistic cross talk between BMP2 signaling and the Hippo-YAP pathway in murine NSC proliferation, which may be exploited as a therapeutic target in neurodegenerative diseases and aging.

  12. BMP4 signaling is involved in the generation of inner ear sensory epithelia

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    Wang Yucheng

    2005-08-01

    Full Text Available 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 the development of inner ear cell types and show that hair cell and supporting cell generation is remarkably reduced when BMP signaling is blocked, either with its antagonist noggin or by using soluble BMP receptors. Conversely, we observed an increase in the number of hair cells when cultured otocysts were treated with exogenous BMP4. BMP4 treatment additionally prompted down-regulation of Pax-2 protein in proliferating sensory epithelial progenitors, leading to reduced progenitor cell proliferation. Conclusion Our results implicate BMP4 in two events during chicken inner ear sensory epithelium formation: first, in inducing the switch from proliferative sensory epithelium progenitors to differentiating epithelial cells and secondly, in promoting the differentiation of hair cells within the developing sensory epithelia.

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

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    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.

  14. Spatial segregation of BMP/Smad signaling affects osteoblast differentiation in C2C12 cells.

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    Eva Heining

    Full Text Available BACKGROUND: Bone morphogenetic proteins (BMPs are involved in a plethora of cellular processes in embryonic development and adult tissue homeostasis. Signaling specificity is achieved by dynamic processes involving BMP receptor oligomerization and endocytosis. This allows for spatiotemporal control of Smad dependent and non-Smad pathways. In this study, we investigate the spatiotemporal regulation within the BMP-induced Smad transcriptional pathway. METHODOLOGY/PRINCIPAL FINDINGS: Here we discriminate between Smad signaling events that are dynamin-dependent (i.e., require an intact endocytic pathway and dynamin-independent. Inhibition of dynamin-dependent endocytosis in fluorescence microscopy and fractionation studies revealed a delay in Smad1/5/8 phosphorylation and nuclear translocation after BMP-2 stimulation of C2C12 cells. Using whole genome microarray and qPCR analysis, we identified two classes of BMP-2 induced genes that are differentially affected by inhibition of endocytosis. Thus, BMP-2 induced gene expression of Id1, Id3, Dlx2 and Hey1 is endocytosis-dependent, whereas BMP-2 induced expression of Id2, Dlx3, Zbtb2 and Krt16 is endocytosis-independent. Furthermore, we demonstrate that short term inhibition of endocytosis interferes with osteoblast differentiation as measured by alkaline phosphatase (ALP production and qPCR analysis of osteoblast marker gene expression. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that dynamin-dependent endocytosis is crucial for the concise spatial activation of the BMP-2 induced signaling cascade. Inhibition of endocytic processes during BMP-2 stimulation leads to altered Smad1/5/8 signaling kinetics and results in differential target gene expression. We show that interfering with the BMP-2 induced transcriptional network by endocytosis inhibition results in an attenuation of osteoblast differentiation. This implies that selective sensitivity of gene expression to endocytosis provides an

  15. Specification of osteoblast cell fate by canonical Wnt signaling requires Bmp2.

    Science.gov (United States)

    Salazar, Valerie S; Ohte, Satoshi; Capelo, Luciane P; Gamer, Laura; Rosen, Vicki

    2016-12-01

    Enhanced BMP or canonical Wnt (cWnt) signaling are therapeutic strategies employed to enhance bone formation and fracture repair, but the mechanisms each pathway utilizes to specify cell fate of bone-forming osteoblasts remain poorly understood. Among all BMPs expressed in bone, we find that singular deficiency of Bmp2 blocks the ability of cWnt signaling to specify osteoblasts from limb bud or bone marrow progenitors. When exposed to cWnts, Bmp2-deficient cells fail to progress through the Runx2/Osx1 checkpoint and thus do not upregulate multiple genes controlling mineral metabolism in osteoblasts. Cells lacking Bmp2 after induction of Osx1 differentiate normally in response to cWnts, suggesting that pre-Osx1(+) osteoprogenitors are an essential source and a target of BMP2. Our analysis furthermore reveals Grainyhead-like 3 (Grhl3) as a transcription factor in the osteoblast gene regulatory network induced during bone development and bone repair, which acts upstream of Osx1 in a BMP2-dependent manner. The Runx2/Osx1 transition therefore receives crucial regulatory inputs from BMP2 that are not compensated for by cWnt signaling, and this is mediated at least in part by induction and activation of Grhl3.

  16. Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells.

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    Redhai, Siamak; Hellberg, Josephine E E U; Wainwright, Mark; Perera, Sumeth W; Castellanos, Felix; Kroeger, Benjamin; Gandy, Carina; Leiblich, Aaron; Corrigan, Laura; Hilton, Thomas; Patel, Benjamin; Fan, Shih-Jung; Hamdy, Freddie; Goberdhan, Deborah C I; Wilson, Clive

    2016-10-01

    Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion.

  17. 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.

  18. Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling

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    Fitzpatrick, Vincent; Fourel, Laure; Destaing, Olivier; Gilde, Flora; Albigès-Rizo, Corinne; Picart, Catherine; Boudou, Thomas

    2017-01-01

    In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

  19. Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling

    Science.gov (United States)

    Fitzpatrick, Vincent; Fourel, Laure; Destaing, Olivier; Gilde, Flora; Albigès-Rizo, Corinne; Picart, Catherine; Boudou, Thomas

    2017-01-01

    In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro. PMID:28134270

  20. BMP9-Induced Survival Effect in Liver Tumor Cells Requires p38MAPK Activation

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    María García-Álvaro

    2015-08-01

    Full Text Available The study of bone morphogenetic proteins (BMPs role in tumorigenic processes, and specifically in the liver, has gathered importance in the last few years. Previous studies have shown that BMP9 is overexpressed in about 40% of hepatocellular carcinoma (HCC patients. In vitro data have also shown evidence that BMP9 has a pro-tumorigenic action, not only by inducing epithelial to mesenchymal transition (EMT and migration, but also by promoting proliferation and survival in liver cancer cells. However, the precise mechanisms driving these effects have not yet been established. In the present work, we deepened our studies into the intracellular mechanisms implicated in the BMP9 proliferative and pro-survival effect on liver tumor cells. In HepG2 cells, BMP9 induces both Smad and non-Smad signaling cascades, specifically PI3K/AKT and p38MAPK. However, only the p38MAPK pathway contributes to the BMP9 growth-promoting effect on these cells. Using genetic and pharmacological approaches, we demonstrate that p38MAPK activation, although dispensable for the BMP9 proliferative activity, is required for the BMP9 protective effect on serum withdrawal-induced apoptosis. These findings contribute to a better understanding of the signaling pathways involved in the BMP9 pro-tumorigenic role in liver tumor cells.

  1. Osteogenesis on nanoparticulate mineralized collagen scaffolds via autogenous activation of the canonical BMP receptor signaling pathway.

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    Ren, Xiaoyan; Bischoff, David; Weisgerber, Daniel W; Lewis, Michael S; Tu, Victor; Yamaguchi, Dean T; Miller, Timothy A; Harley, Brendan A C; Lee, Justine C

    2015-05-01

    Skeletal regenerative medicine frequently incorporates deliverable growth factors to stimulate osteogenesis. However, the cost and side effects secondary to supraphysiologic dosages of growth factors warrant investigation of alternative methods of stimulating osteogenesis for clinical utilization. In this work, we describe growth factor independent osteogenic induction of human mesenchymal stem cells (hMSCs) on a novel nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG). hMSCs demonstrated elevated osteogenic gene expression and mineralization on MC-GAG with minimal to no effect upon addition of BMP-2 when compared to non-mineralized scaffolds (Col-GAG). To investigate the intracellular pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Constitutive Smad1/5 phosphorylation with nuclear translocation occurred on MC-GAG independent of BMP-2, whereas Smad1/5 phosphorylation depended on BMP-2 stimulation on Col-GAG. When non-canonical BMPR signaling molecules were examined, ERK1/2 phosphorylation was found to be decreased in MC-GAG but elevated in Col-GAG. No differences in Smad2/3 or p38 activation were detected. Collectively, these results demonstrated that MC-GAG scaffolds induce osteogenesis without exogenous BMP-2 addition via endogenous activation of the canonical BMP receptor signaling pathway.

  2. Neural retina identity is specified by lens-derived BMP signals.

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    Pandit, Tanushree; Jidigam, Vijay K; Patthey, Cedric; Gunhaga, Lena

    2015-05-15

    The eye has served as a classical model to study cell specification and tissue induction for over a century. Nevertheless, the molecular mechanisms that regulate the induction and maintenance of eye-field cells, and the specification of neural retina cells are poorly understood. Moreover, within the developing anterior forebrain, how prospective eye and telencephalic cells are differentially specified is not well defined. In the present study, we have analyzed these issues by manipulating signaling pathways in intact chick embryo and explant assays. Our results provide evidence that at blastula stages, BMP signals inhibit the acquisition of eye-field character, but from neural tube/optic vesicle stages, BMP signals from the lens are crucial for the maintenance of eye-field character, inhibition of dorsal telencephalic cell identity and specification of neural retina cells. Subsequently, our results provide evidence that a Rax2-positive eye-field state is not sufficient for the progress to a neural retina identity, but requires BMP signals. In addition, our results argue against any essential role of Wnt or FGF signals during the specification of neural retina cells, but provide evidence that Wnt signals together with BMP activity are sufficient to induce cells of retinal pigment epithelial character. We conclude that BMP activity emanating from the lens ectoderm maintains eye-field identity, inhibits telencephalic character and induces neural retina cells. Our findings link the requirement of the lens ectoderm for neural retina specification with the molecular mechanism by which cells in the forebrain become specified as neural retina by BMP activity.

  3. BMP4 Cross-talks With Estrogen/ERα Signaling to Regulate Adiposity and Glucose Metabolism in Females

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    Shu-wen Qian

    2016-09-01

    Full Text Available Similar to estrogens, bone morphogenetic protein 4 (BMP4 promotes the accumulation of more metabolically active subcutaneous fat and reduction of visceral fat. However, whether there is a cross-talk between BMP4 and estrogen signaling remained unknown. Herein, we found that BMP4 deficiency in white adipose tissue (WAT increased the estrogen receptor α (ERα level and its signaling, which prevented adult female mice from developing high fat diet (HFD-induced obesity and insulin resistance; estrogens depletion up regulated BMP4 expression to overcome overt adiposity and impaired insulin sensitivity with aging, and failure of BMP4 regulation due to genetic knockout led to more fat gain in aged female mice. This mutual regulation between BMP4 and estrogen/ERα signaling may also happen in adipose tissue of women, since the BMP4 level significantly increased after menopause, and was inversely correlated with body mass index (BMI. These findings suggest a counterbalance between BMP4 and estrogen/ERα signaling in the regulation of adiposity and relative metabolism in females.

  4. Fstl1 antagonizes BMP signaling and regulates ureter development.

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    Jingyue Xu

    Full Text Available Bone morphogenetic protein (BMP signaling pathway plays important roles in urinary tract development although the detailed regulation of its activity in this process remains unclear. Here we report that follistatin-like 1 (Fstl1, encoding a secreted extracellular glycoprotein, is expressed in developing ureter and antagonizes BMP signaling activity. Mouse embryos carrying disrupted Fstl1 gene displayed prominent hydroureter arising from proximal segment and ureterovesical junction defects. These defects were associated with significant reduction in ureteric epithelial cell proliferation at E15.5 and E16.5 as well as absence of subepithelial ureteral mesenchymal cells in the urinary tract at E16.5 and E18.5. At the molecular level, increased BMP signaling was found in Fstl1 deficient ureters, indicated by elevated pSmad1/5/8 activity. In vitro study also indicated that Fstl1 can directly bind to ALK6 which is specifically expressed in ureteric epithelial cells in developing ureter. Furthermore, Sonic hedgehog (SHH signaling, which is crucial for differentiation of ureteral subepithelial cell proliferation, was also impaired in Fstl1(-/- ureter. Altogether, our data suggest that Fstl1 is essential in maintaining normal ureter development by antagonizing BMP signaling.

  5. Differential requirements of BMP and Wnt signalling during gastrulation and neurulation define two steps in neural crest induction.

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    Steventon, Ben; Araya, Claudio; Linker, Claudia; Kuriyama, Sei; Mayor, Roberto

    2009-03-01

    The neural crest is induced by a combination of secreted signals. Although previous models of neural crest induction have proposed a step-wise activation of these signals, the actual spatial and temporal requirement has not been analysed. Through analysing the role of the mesoderm we show for the first time that specification of neural crest requires two temporally and chemically different steps: first, an induction at the gastrula stage dependent on signals arising from the dorsolateral mesoderm; and second, a maintenance step at the neurula stage dependent on signals from tissues adjacent to the neural crest. By performing tissue recombination experiments and using specific inhibitors of different inductive signals, we show that the first inductive step requires Wnt activation and BMP inhibition, whereas the later maintenance step requires activation of both pathways. This change in BMP necessity from BMP inhibition at gastrula to BMP activation at neurula stages is further supported by the dynamic expression of BMP4 and its antagonists, and is confirmed by direct measurements of BMP activity in the neural crest cells. The differential requirements of BMP activity allow us to propose an explanation for apparently discrepant results between chick and frog experiments. The demonstration that Wnt signals are required for neural crest induction by mesoderm solves an additional long-standing controversy. Finally, our results emphasise the importance of considering the order of exposure to signals during an inductive event.

  6. Reactive oxygen species are involved in BMP-induced dendritic growth in cultured rat sympathetic neurons.

    Science.gov (United States)

    Chandrasekaran, Vidya; Lea, Charlotte; Sosa, Jose Carlo; Higgins, Dennis; Lein, Pamela J

    2015-07-01

    Previous studies have shown that bone morphogenetic proteins (BMPs) promote dendritic growth in sympathetic neurons; however, the downstream signaling molecules that mediate the dendrite promoting activity of BMPs are not well characterized. Here we test the hypothesis that reactive oxygen species (ROS)-mediated signaling links BMP receptor activation to dendritic growth. In cultured rat sympathetic neurons, exposure to any of the three mechanistically distinct antioxidants, diphenylene iodinium (DPI), nordihydroguaiaretic acid (NGA) or desferroxamine (DFO), blocked de novo BMP-induced dendritic growth. Addition of DPI to cultures previously induced with BMP to extend dendrites caused dendritic retraction while DFO and NGA prevented further growth of dendrites. The inhibition of the dendrite promoting activity of BMPs by antioxidants was concentration-dependent and occurred without altering axonal growth or neuronal cell survival. Antioxidant treatment did not block BMP activation of SMAD 1,5 as determined by nuclear localization of these SMADs. While BMP treatment did not cause a detectable increase in intracellular ROS in cultured sympathetic neurons as assessed using fluorescent indicator dyes, BMP treatment increased the oxygen consumption rate in cultured sympathetic neurons as determined using the Seahorse XF24 Analyzer, suggesting increased mitochondrial activity. In addition, BMPs upregulated expression of NADPH oxidase 2 (NOX2) and either pharmacological inhibition or siRNA knockdown of NOX2 significantly decreased BMP-7 induced dendritic growth. Collectively, these data support the hypothesis that ROS are involved in the downstream signaling events that mediate BMP7-induced dendritic growth in sympathetic neurons, and suggest that ROS-mediated signaling positively modulates dendritic complexity in peripheral neurons.

  7. Designer Nodal/BMP2 Chimeras Mimic Nodal Signaling, Promote Chondrogenesis, and Reveal a BMP2-like Structure

    Science.gov (United States)

    Esquivies, Luis; Blackler, Alissa; Peran, Macarena; Rodriguez-Esteban, Concepcion; Izpisua Belmonte, Juan Carlos; Booker, Evan; Gray, Peter C.; Ahn, Chihoon; Kwiatkowski, Witek; Choe, Senyon

    2014-01-01

    Nodal, a member of the TGF-β superfamily, plays an important role in vertebrate and invertebrate early development. The biochemical study of Nodal and its signaling pathway has been a challenge, mainly because of difficulties in producing the protein in sufficient quantities. We have developed a library of stable, chemically refoldable Nodal/BMP2 chimeric ligands (NB2 library). Three chimeras, named NB250, NB260, and NB264, show Nodal-like signaling properties including dependence on the co-receptor Cripto and activation of the Smad2 pathway. NB250, like Nodal, alters heart looping during the establishment of embryonic left-right asymmetry, and both NB250 and NB260, as well as Nodal, induce chondrogenic differentiation of human adipose-derived stem cells. This Nodal-induced differentiation is shown to be more efficient than BPM2-induced differentiation. Interestingly, the crystal structure of NB250 shows a backbone scaffold similar to that of BMP2. Our results show that these chimeric ligands may have therapeutic implications in cartilage injuries. PMID:24311780

  8. Shh Signaling is Involved in Regulating BMP9-induced Osteogenic Differentiation of Mesenchymal Stem Cells%Shh信号参与调控BMP9诱导的间充质干细胞成骨分化

    Institute of Scientific and Technical Information of China (English)

    李丽; 蒙秋蓉; 郭琦; 王岚; 商蕾; 欧欣颖; 罗进勇

    2014-01-01

    目的:观察sonic hedgehog (Shh)信号通路在骨形态发生蛋白9(BMP9)诱导的小鼠间充质干细胞(MSCs) C3H10T1/2和C2C12成骨分化中的作用,并初步探讨其作用机制.方法:Shh信号通路抑制剂Cyclopamine和激活剂Purmorphamine以及过表达Shh腺病毒分别作用于BMP9处理的C3H10T1/2和C2C12细胞,碱性磷酸酶(ALP)检测早期成骨指标ALP,茜素红S染色检测晚期成骨指标钙盐沉积,RT-PCR检测Shh信号相关基因以及成骨关键转录因子的表达,Westernblot检测Shh的表达,荧光素酶报告基因检测Smad1/5/8的转录调控活性.结果:BMP9促进Shh信号相关基因的表达,激活Shh信号可增强BMP9诱导的C3H10T1/2和C2C12细胞早晚期成骨分化并促进了BMP9诱导的Smad荧光素酶活性,抑制Shh信号后作用相反.结论:激活Shh信号通路可促进BMP9诱导的小鼠MSCs成骨分化,抑制其活性后作用相反.

  9. 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.

  10. Age-Associated Increase in BMP Signaling Inhibits Hippocampal Neurogenesis.

    Science.gov (United States)

    Yousef, Hanadie; Morgenthaler, Adam; Schlesinger, Christina; Bugaj, Lukasz; Conboy, Irina M; Schaffer, David V

    2015-05-01

    Hippocampal neurogenesis, the product of resident neural stem cell proliferation and differentiation, persists into adulthood but decreases with organismal aging, which may contribute to the age-related decline in cognitive function. The mechanisms that underlie this decrease in neurogenesis are not well understood, although evidence in general indicates that extrinsic changes in an aged stem cell niche can contribute to functional decline in old stem cells. Bone morphogenetic protein (BMP) family members are intercellular signaling proteins that regulate stem and progenitor cell quiescence, proliferation, and differentiation in various tissues and are likewise critical regulators of neurogenesis in young adults. Here, we establish that BMP signaling increases significantly in old murine hippocampi and inhibits neural progenitor cell proliferation. Furthermore, direct in vivo attenuation of BMP signaling via genetic and transgenic perturbations in aged mice led to elevated neural stem cell proliferation, and subsequent neurogenesis, in old hippocampi. Such advances in our understanding of mechanisms underlying decreased hippocampal neurogenesis with age may offer targets for the treatment of age-related cognitive decline.

  11. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

  14. Tsukushi functions as an organizer inducer by inhibition of BMP activity in cooperation with chordin.

    Science.gov (United States)

    Ohta, Kunimasa; Lupo, Giuseppe; Kuriyama, Sei; Keynes, Roger; Holt, Christine E; Harris, William A; Tanaka, Hideaki; Ohnuma, Shin-ichi

    2004-09-01

    During chick gastrulation, inhibition of BMP signaling is required for primitive streak formation and induction of Hensen's node. We have identified a unique secreted protein, Tsukushi (TSK), which belongs to the Small Leucine-Rich Proteoglycan (SLRP) family and is expressed in the primitive streak and Hensen's node. Grafts of cells expressing TSK in combination with the middle primitive streak induce an ectopic Hensen's node, while electroporation of TSK siRNA inhibits induction of the node. In Xenopus embryos, TSK can block BMP function and induce a secondary dorsal axis, while it can dorsalize ventral mesoderm and induce neural tissue in embryonic explants. Biochemical analysis shows that TSK binds directly to both BMP and chordin and forms a ternary complex with them. These observations indicate that TSK is an essential dorsalizing factor involved in the induction of Hensen's node.

  15. Autotaxin-mediated lipid signaling intersects with LIF and BMP signaling to promote the naive pluripotency transcription factor program

    Science.gov (United States)

    Kime, Cody; Sakaki-Yumoto, Masayo; Goodrich, Leeanne; Hayashi, Yohei; Sami, Salma; Derynck, Rik; Asahi, Michio; Panning, Barbara; Yamanaka, Shinya; Tomoda, Kiichiro

    2016-01-01

    Developmental signaling molecules are used for cell fate determination, and understanding how their combinatorial effects produce the variety of cell types in multicellular organisms is a key problem in biology. Here, we demonstrate that the combination of leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), lysophosphatidic acid (LPA), and ascorbic acid (AA) efficiently converts mouse primed pluripotent stem cells (PSCs) into naive PSCs. Signaling by the lipid LPA through its receptor LPAR1 and downstream effector Rho-associated protein kinase (ROCK) cooperated with LIF signaling to promote this conversion. BMP4, which also stimulates conversion to naive pluripotency, bypassed the need for exogenous LPA by increasing the activity of the extracellular LPA-producing enzyme autotaxin (ATX). We found that LIF and LPA-LPAR1 signaling affect the abundance of signal transducer and activator of transcription 3 (STAT3), which induces a previously unappreciated Kruppel-like factor (KLF)2-KLF4-PR domain 14 (PRDM14) transcription factor circuit key to establish naive pluripotency. AA also affects this transcription factor circuit by controlling PRDM14 expression. Thus, our study reveals that ATX-mediated autocrine lipid signaling promotes naive pluripotency by intersecting with LIF and BMP4 signaling. PMID:27738243

  16. BMP signaling negatively regulates bone mass through sclerostin by inhibiting the canonical Wnt pathway.

    Science.gov (United States)

    Kamiya, Nobuhiro; Ye, Ling; Kobayashi, Tatsuya; Mochida, Yoshiyuki; Yamauchi, Mitsuo; Kronenberg, Henry M; Feng, Jian Q; Mishina, Yuji

    2008-11-01

    Bone morphogenetic proteins (BMPs) are known to induce ectopic bone. However, it is largely unknown how BMP signaling in osteoblasts directly regulates endogenous bone. This study investigated the mechanism by which BMP signaling through the type IA receptor (BMPR1A) regulates endogenous bone mass using an inducible Cre-loxP system. When BMPR1A in osteoblasts was conditionally disrupted during embryonic bone development, bone mass surprisingly was increased with upregulation of canonical Wnt signaling. Although levels of bone formation markers were modestly reduced, levels of resorption markers representing osteoclastogenesis were severely reduced, resulting in a net increase in bone mass. The reduction of osteoclastogenesis was primarily caused by Bmpr1a-deficiency in osteoblasts, at least through the RANKL-OPG pathway. Sclerostin (Sost) expression was downregulated by about 90% and SOST protein was undetectable in osteoblasts and osteocytes, whereas the Wnt signaling was upregulated. Treatment of Bmpr1a-deficient calvariae with sclerostin repressed the Wnt signaling and restored normal bone morphology. By gain of Smad-dependent BMPR1A signaling in mice, Sost expression was upregulated and osteoclastogenesis was increased. Finally, the Bmpr1a-deficient bone phenotype was rescued by enhancing BMPR1A signaling, with restoration of osteoclastogenesis. These findings demonstrate that BMPR1A signaling in osteoblasts restrain endogenous bone mass directly by upregulating osteoclastogenesis through the RANKL-OPG pathway, or indirectly by downregulating canonical Wnt signaling through sclerostin, a Wnt inhibitor and a bone mass mediator.

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

    Directory of Open Access Journals (Sweden)

    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

  18. Bmp signaling is at the heart of vertebrate left-right asymmetry

    NARCIS (Netherlands)

    Verhoeven, M.C.

    2009-01-01

    Bone morphogenetic protein (Bmp) signaling is vitally important in many aspects of cardiac development. These include cardiac induction and differentiation and establishing the L/R axis. In this thesis, we focus on the role of Bmp signaling in securing proper cardiac asymmetry, by (1) establishing c

  19. 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

  20. Efficient differentiation of embryonic stem cells into mesodermal precursors by BMP, retinoic acid and Notch signalling.

    Directory of Open Access Journals (Sweden)

    Josema Torres

    Full Text Available The ability to direct differentiation of mouse embryonic stem (ES cells into specific lineages not only provides new insights into the pathways that regulate lineage selection but also has translational applications, for example in drug discovery. We set out to develop a method of differentiating ES cells into mesodermal cells at high efficiency without first having to induce embryoid body formation. ES cells were plated on a feeder layer of PA6 cells, which have membrane-associated stromal-derived inducing activity (SDIA, the molecular basis of which is currently unknown. Stimulation of ES/PA6 co-cultures with Bone Morphogenetic Protein 4 (BMP4 both favoured self-renewal of ES cells and induced differentiation into a Desmin and Nestin double positive cell population. Combined stimulation with BMP4 and all-trans Retinoic Acid (RA inhibited self-renewal and resulted in 90% of cells expressing Desmin and Nestin. Quantitative reverse transcription-polymerase chain reaction (qPCR analysis confirmed that the cells were of mesodermal origin and expressed markers of mesenchymal and smooth muscle cells. BMP4 activation of a MAD-homolog (Smad-dependent reporter in undifferentiated ES cells was attenuated by co-stimulation with RA and co-culture with PA6 cells. The Notch ligand Jag1 was expressed in PA6 cells and inhibition of Notch signalling blocked the differentiation inducing activity of PA6 cells. Our data suggest that mesodermal differentiation is regulated by the level of Smad activity as a result of inputs from BMP4, RA and the Notch pathway.

  1. BMP signaling and microtubule organization regulate synaptic strength.

    Science.gov (United States)

    Ball, R W; Peled, E S; Guerrero, G; Isacoff, E Y

    2015-04-16

    The strength of synaptic transmission between a neuron and multiple postsynaptic partners can vary considerably. We have studied synaptic heterogeneity using the glutamatergic Drosophila neuromuscular junction (NMJ), which contains multiple synaptic connections of varying strengths between a motor axon and muscle fiber. In larval NMJs, there is a gradient of synaptic transmission from weak proximal to strong distal boutons. We imaged synaptic transmission with the postsynaptically targeted fluorescent calcium sensor SynapCam, to investigate the molecular pathways that determine synaptic strength and set up this gradient. We discovered that mutations in the Bone Morphogenetic Protein (BMP) signaling pathway disrupt production of strong distal boutons. We find that strong connections contain unbundled microtubules in the boutons, suggesting a role for microtubule organization in transmission strength. The spastin mutation, which disorganizes microtubules, disrupted the transmission gradient, supporting this interpretation. We propose that the BMP pathway, shown previously to function in the homeostatic regulation of synaptic growth, also boosts synaptic transmission in a spatially selective manner that depends on the microtubule system.

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

    OpenAIRE

    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...

  3. 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.

  4. Alcohol Activates TGF-Beta but Inhibits BMP Receptor-Mediated Smad Signaling and Smad4 Binding to Hepcidin Promoter in the Liver

    Directory of Open Access Journals (Sweden)

    Lisa Nicole Gerjevic

    2012-01-01

    Full Text Available Hepcidin, a key regulator of iron metabolism, is activated by bone morphogenetic proteins (BMPs. Mice pair-fed with regular and ethanol-containing L. De Carli diets were employed to study the effect of alcohol on BMP signaling and hepcidin transcription in the liver. Alcohol induced steatosis and TGF-beta expression. Liver BMP2, but not BMP4 or BMP6, expression was significantly elevated. Despite increased BMP expression, the BMP receptor, and transcription factors, Smad1 and Smad5, were not activated. In contrast, alcohol stimulated Smad2 phosphorylation. However, Smad4 DNA-binding activity and the binding of Smad4 to hepcidin promoter were attenuated. In summary, alcohol stimulates TGF-beta and BMP2 expression, and Smad2 phosphorylation but inhibits BMP receptor, and Smad1 and Smad5 activation. Smad signaling pathway in the liver may therefore be involved in the regulation of hepcidin transcription and iron metabolism by alcohol. These findings may help to further understand the mechanisms of alcohol and iron-induced liver injury.

  5. Analysis of BMP4 and BMP7 signaling in breast cancer cells unveils time-dependent transcription patterns and highlights a common synexpression group of genes

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    Rodriguez-Martinez Alejandra

    2011-11-01

    Full Text Available Abstract Background Bone morphogenetic proteins (BMPs are members of the TGF-beta superfamily of growth factors. They are known for their roles in regulation of osteogenesis and developmental processes and, in recent years, evidence has accumulated of their crucial functions in tumor biology. BMP4 and BMP7, in particular, have been implicated in breast cancer. However, little is known about BMP target genes in the context of tumor. We explored the effects of BMP4 and BMP7 treatment on global gene transcription in seven breast cancer cell lines during a 6-point time series, using a whole-genome oligo microarray. Data analysis included hierarchical clustering of differentially expressed genes, gene ontology enrichment analyses and model based clustering of temporal data. Results Both ligands had a strong effect on gene expression, although the response to BMP4 treatment was more pronounced. The cellular functions most strongly affected by BMP signaling were regulation of transcription and development. The observed transcriptional response, as well as its functional outcome, followed a temporal sequence, with regulation of gene expression and signal transduction leading to changes in metabolism and cell proliferation. Hierarchical clustering revealed distinct differences in the response of individual cell lines to BMPs, but also highlighted a synexpression group of genes for both ligands. Interestingly, the majority of the genes within these synexpression groups were shared by the two ligands, probably representing the core molecular responses common to BMP4 and BMP7 signaling pathways. Conclusions All in all, we show that BMP signaling has a remarkable effect on gene transcription in breast cancer cells and that the functions affected follow a logical temporal pattern. Our results also uncover components of the common cellular transcriptional response to BMP4 and BMP7. Most importantly, this study provides a list of potential novel BMP target

  6. Disruption of BMP Signaling in Osteoblasts Through Type IA Receptor (BMPRIA) Increases Bone Mass*

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    Kamiya, Nobuhiro; Ye, Ling; Kobayashi, Tatsuya; Lucas, Donald J; Mochida, Yoshiyuki; Yamauchi, Mitsuo; Kronenberg, Henry M; Feng, Jian Q; Mishina, Yuji

    2008-01-01

    Bone morphogenetic proteins (BMPs) are known as ectopic bone inducers. The FDA approved BMPs (BMP2 and BMP7) for clinical use. However, direct effects of BMPs on endogenous bone metabolism are not yet well known. We conditionally disrupted BMP receptor type IA (BMPRIA) in osteoblasts during weanling and adult stages to show the impact of BMP signaling on endogenous bone modeling and remodeling. Cre recombination was detected in immature osteoblasts in the periosteum, osteoblasts, and osteocytes but not in chondrocytes and osteoclasts after tamoxifen administration. Bmpr1a conditional knockout mice (cKO) showed increased bone mass primarily in trabecular bone at P21 and 22 wk as determined by H&E staining. Vertebrae, tails, and ribs showed increased radiodensity at 22 wk, consistent with a significant increase in BMD. Both μCT and histomorphometry showed an increase in trabecular BV/TV and thickness of cKO adult bones, whereas osteoclast number, bone formation rate, and mineral apposition rate were decreased. Expression levels of bone formation markers (Runx2 and Bsp), resorption markers (Mmp9, Ctsk, and Tracp), and Rankl were decreased, and Opg was increased in adult bones, resulting in a reduction in the ratio of Rankl to osteoprotegerin (Opg). The reduction in osteoclastogenesis through the RANKL–OPG pathway was also observed in weanling stages and reproduced in newborn calvaria culture. These results suggest that Bmpr1a cKO increased endogenous bone mass primarily in trabecular bone with decreased osteoclastogenesis through the RANKL–OPG pathway. We conclude that BMPRIA signaling in osteoblasts affects both bone formation and resorption to reduce endogenous bone mass in vivo. PMID:18684091

  7. Disruption of BMP signaling in osteoblasts through type IA receptor (BMPRIA) increases bone mass.

    Science.gov (United States)

    Kamiya, Nobuhiro; Ye, Ling; Kobayashi, Tatsuya; Lucas, Donald J; Mochida, Yoshiyuki; Yamauchi, Mitsuo; Kronenberg, Henry M; Feng, Jian Q; Mishina, Yuji

    2008-12-01

    Bone morphogenetic proteins (BMPs) are known as ectopic bone inducers. The FDA approved BMPs (BMP2 and BMP7) for clinical use. However, direct effects of BMPs on endogenous bone metabolism are not yet well known. We conditionally disrupted BMP receptor type IA (BMPRIA) in osteoblasts during weanling and adult stages to show the impact of BMP signaling on endogenous bone modeling and remodeling. Cre recombination was detected in immature osteoblasts in the periosteum, osteoblasts, and osteocytes but not in chondrocytes and osteoclasts after tamoxifen administration. Bmpr1a conditional knockout mice (cKO) showed increased bone mass primarily in trabecular bone at P21 and 22 wk as determined by H&E staining. Vertebrae, tails, and ribs showed increased radiodensity at 22 wk, consistent with a significant increase in BMD. Both muCT and histomorphometry showed an increase in trabecular BV/TV and thickness of cKO adult bones, whereas osteoclast number, bone formation rate, and mineral apposition rate were decreased. Expression levels of bone formation markers (Runx2 and Bsp), resorption markers (Mmp9, Ctsk, and Tracp), and Rankl were decreased, and Opg was increased in adult bones, resulting in a reduction in the ratio of Rankl to osteoprotegerin (Opg). The reduction in osteoclastogenesis through the RANKL-OPG pathway was also observed in weanling stages and reproduced in newborn calvaria culture. These results suggest that Bmpr1a cKO increased endogenous bone mass primarily in trabecular bone with decreased osteoclastogenesis through the RANKL-OPG pathway. We conclude that BMPRIA signaling in osteoblasts affects both bone formation and resorption to reduce endogenous bone mass in vivo.

  8. Angiocrine Bmp2 signaling in murine liver controls normal iron homeostasis.

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    Koch, Philipp-Sebastian; Olsavszky, Victor; Ulbrich, Friederike; Sticht, Carsten; Demory, Alexandra; Leibing, Thomas; Henzler, Thomas; Meyer, Mathias; Zierow, Johanna; Schneider, Sven; Breitkopf-Heinlein, Katja; Gaitantzi, Haristi; Spencer-Dene, Bradley; Arnold, Bernd; Klapproth, Kay; Schledzewski, Kai; Goerdt, Sergij; Géraud, Cyrill

    2017-01-26

    Microvascular endothelial cells (ECs) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific ECs control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal endothelial cells (LSECs) are uniquely differentiated to fulfill important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSECs. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSECs using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2(fl/fl) (Bmp2(LSECKO)) mice caused massive iron overload in the liver and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is nonredundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ but also for the homeostasis of the whole organism.

  9. 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.

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

    Science.gov (United States)

    Chen, Yongjuan; Roohani-Esfahani, Seyed-Iman; Lu, ZuFu; Zreiqat, Hala; Dunstan, Colin R

    2015-01-01

    Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV) oxynitrate (ZrO(NO3)2) at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.

  11. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Shimada, Koichi, E-mail: shimada-ki@dent.nihon-u.ac.jp [Department of Periodontology, Nihon University School of Dentistry, Tokyo (Japan); Division of Advanced Dental Treatment, Dental Research Center, Nihon University School of Dentistry, Tokyo (Japan); Ikeda, Kyoko [Department of Periodontology, Nihon University School of Dentistry, Tokyo (Japan); Ito, Koichi [Department of Periodontology, Nihon University School of Dentistry, Tokyo (Japan); Division of Advanced Dental Treatment, Dental Research Center, Nihon University School of Dentistry, Tokyo (Japan)

    2009-12-18

    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-{alpha} stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-{alpha}-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-{alpha} and BMP signaling pathways.

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

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    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.

  14. BRITER: a BMP responsive osteoblast reporter cell line.

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    Prem Swaroop Yadav

    Full Text Available BACKGROUND: BMP signaling pathway is critical for vertebrate development and tissue homeostasis. High-throughput molecular genetic screening may reveal novel players regulating BMP signaling response while chemical genetic screening of BMP signaling modifiers may have clinical significance. It is therefore important to generate a cell-based tool to execute such screens. METHODOLOGY/PRINCIPAL FINDINGS: We have established a BMP responsive reporter cell line by stably integrating a BMP responsive dual luciferase reporter construct in the immortalized calvarial osteoblast cells isolated from tamoxifen inducible Bmp2; Bmp4 double conditional knockout mouse strain. This cell line, named BRITER (BMP Responsive Immortalized Reporter cell line, responds robustly, promptly and specifically to exogenously added BMP2 protein. The sensitivity to added BMP may be further increased by depleting the endogenous BMP2 and BMP4 proteins. CONCLUSION: As the dynamic range of the assay (for BMP responsiveness is very high for BRITER and as it responds specifically and promptly to exogenously added BMP2 protein, BRITER may be used effectively for chemical or molecular genetic screening for BMP signaling modifiers. Identification of novel molecular players capable of influencing BMP signaling pathway may have clinical significance.

  15. BMP signaling protects telencephalic fate by repressing eye identity and its Cxcr4-dependent morphogenesis.

    Science.gov (United States)

    Bielen, Holger; Houart, Corinne

    2012-10-16

    Depletion of Wnt signaling is a major requirement for the induction of the anterior prosencephalon. However, the molecular events driving the differential regionalization of this area into eye-field and telencephalon fates are still unknown. Here we show that the BMP pathway is active in the anterior neural ectoderm during late blastula to early gastrula stage in zebrafish. Bmp2b mutants and mosaic loss-of-function experiments reveal that BMP acts as a repressor of eye-field fate through inhibition of its key transcription factor Rx3, thereby protecting the future telencephalon from acquiring eye identity. This BMP-driven mechanism initiates the establishment of the telencephalon prior to the involvement of Wnt antagonists from the anterior neural border. Furthermore, we demonstrate that Rx3 and BMP are respectively required to maintain and restrict the chemokine receptor cxcr4a, which in turn contributes to the morphogenetic separation of eye-field and telencephalic cells during early neurulation.

  16. BMP-Smad4 signaling is required for precartilaginous mesenchymal condensation independent of Sox9 in the mouse.

    Science.gov (United States)

    Lim, Joohyun; Tu, Xiaolin; Choi, Kyunghee; Akiyama, Haruhiko; Mishina, Yuji; Long, Fanxin

    2015-04-01

    Bone morphogenetic proteins (BMPs) regulate multiple aspects of skeletal development in vertebrates. Although exogenously applied BMPs can induce chondrogenesis de novo, the role and mechanism of physiologic BMP signaling during precartilaginous mesenchymal condensation is not well understood. By deleting the type I BMP receptors or the transcription factor Smad4 in the limb bud mesenchyme, we find that loss of BMP-Smad signaling abolishes skeletal development due to a failure in mesenchymal condensation. In the absence of Smad4, expression of Sox9, an essential transcription factor for chondrogenesis, initiates normally in the proximal mesenchyme of the limb bud, but fails to maintain its level or expand to the more distal territory at the later stages. However, forced-expression of Sox9 does not restore cartilage formation in the Smad4-deficeint embryo. In vitro micromass cultures show that the Smad4-deficient cells fail to condense in a cell-autonomous manner, even though they express several cell adhesion molecules either normally or even at a higher level. Thus, BMP-Smad signaling critically controls mesenchymal condensation to initiate skeletal development likely through a Sox9-independent mechanism.

  17. 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.

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

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    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.

  19. WNT4 acts downstream of BMP2 and functions via β-catenin signaling pathway to regulate human endometrial stromal cell differentiation.

    Science.gov (United States)

    Li, Quanxi; Kannan, Athilakshmi; Das, Amrita; Demayo, Franco J; Hornsby, Peter J; Young, Steven L; Taylor, Robert N; Bagchi, Milan K; Bagchi, Indrani C

    2013-01-01

    Differentiation of endometrial stromal cells into decidual cells is a prerequisite for successful embryo implantation. Our previous studies in the mouse have shown that bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFβ superfamily, is essential for this differentiation process. BMP2 is markedly induced in human primary endometrial stromal cells (HESCs) as they undergo differentiation in response to steroid hormones and cAMP. The present study was undertaken to identify the BMP2-mediated molecular pathways in primary cultures of HESCs during decidualization. Using gene expression profiling, we identified wingless-related murine mammary tumor virus integration site 4 (WNT4) as a target of BMP2 regulation during decidualization. Attenuation of WNT4 expression in HESCs by small interfering RNA administration greatly reduced BMP2-induced stromal differentiation. Additionally, adenovirus-mediated overexpression of WNT4 in HESCs markedly advanced the differentiation program, indicating that it is a key regulator of decidualization. The stimulatory effect of WNT4 was accompanied by the accumulation of active β-catenin in the nuclei of decidualizing stromal cells, indicating the involvement of the canonical WNT signaling pathway. Functional inhibition of WNT4/β-catenin pathway by Dickkopf-1, an inhibitor of the canonical WNT signaling, or small interfering RNA-mediated silencing of β-catenin expression, greatly reduced the BMP2- and WNT4-induced decidualization. Gene expression profiling revealed that Forkhead box protein O1, a forkhead family transcription factor and previously reported regulator of HESC differentiation, is a common downstream mediator of both BMP2 and WNT4 signaling. Taken together, these studies uncovered a linear pathway involving BMP2, WNT4/β-catenin, and Forkhead box protein O1 that operates in human endometrium to critically control decidualization.

  20. A protein kinase A (PKA)/β-catenin pathway sustains the BMP2/DLX3-induced osteogenic differentiation in dental follicle cells (DFCs).

    Science.gov (United States)

    Viale-Bouroncle, S; Klingelhöffer, C; Ettl, T; Reichert, T E; Morsczeck, C

    2015-03-01

    The directed expression of osteogenic transcription factors via a balanced activation of signaling pathways is an important prerequisite for the development of mineralized tissues. A positive-feedback loop of the BMP2-dependent SMAD signaling pathway and the DLX3 transcription factor (BMP2/DLX3 pathway) directs the osteogenic differentiation of periodontal precursor cells from the dental follicle (DFCs). However, little is known how this BMP2/DLX3 pathway interacts with other crucial signaling pathways such as the WNT/β-catenin signaling pathway. This study investigated the interaction between the BMP2/DLX3 pathway and the WNT pathway during the osteogenic differentiation of DFCs. BMP2 induced the WNT/β-catenin pathway in DFCs and phosphorylates β-catenin via protein kinase A (PKA). Moreover, only BMP2 facilitated the binding of LEF1/SMAD4/β-catenin complex to the DLX3 promoter, while an inducer of the canonical WNT pathway, WNT3A, act as an inhibitor. Although WNT3A inhibits the osteogenic differentiation of DFCs the expression of β-catenin was crucial for both the expression of DLX3 and for the osteogenic differentiation. In conclusion, while the activation of the canonical WNT pathway inhibits the osteogenic differentiation of DFCs, β-catenin sustains the BMP2/DLX3-mediated osteogenic differentiation via the activation of PKA.

  1. Gallic acid inhibits vascular calcification through the blockade of BMP2-Smad1/5/8 signaling pathway.

    Science.gov (United States)

    Kee, Hae Jin; Cho, Soo-Na; Kim, Gwi Ran; Choi, Sin Young; Ryu, Yuhee; Kim, In Kyeom; Hong, Young Joon; Park, Hyung Wook; Ahn, Youngkeun; Cho, Jeong Gwan; Park, Jong Chun; Jeong, Myung Ho

    2014-11-01

    Vascular calcification is associated with increased risk of morbidity and mortality in patients with cardiovascular diseases, chronic kidney diseases, and diabetes. Gallic acid, a natural compound found in gallnut and green tea, is known to be antifungal, antioxidant, and anticancer. Here we investigated the effect of gallic acid on vascular smooth muscle cell (VSMC) calcification and the underlying mechanism. Gallic acid inhibited inorganic phosphate-induced osteoblast differentiation markers as well as calcification phenotypes (as determined by calcium deposition, Alizarin Red, and Von Kossa staining). Knockdown of BMP2 or Noggin blocked phosphate-induced calcification. Gallic acid suppressed phosphorylation of Smad1/5/8 protein induced by inorganic phosphate. Taken together, we suggest that gallic acid acts as a novel therapeutic agent of vascular calcification by mediating BMP2-Smad1/5/8 signaling pathway.

  2. Long-term exposure to bisphenol A or benzo(a)pyrene alters the fate of human mammary epithelial stem cells in response to BMP2 and BMP4, by pre-activating BMP signaling

    Science.gov (United States)

    Clément, Flora; Xu, Xinyi; Donini, Caterina F; Clément, Alice; Omarjee, Soleilmane; Delay, Emmanuel; Treilleux, Isabelle; Fervers, Béatrice; Le Romancer, Muriel; Cohen, Pascale A; Maguer-Satta, Véronique

    2017-01-01

    Bone morphogenetic protein 2 (BMP2) and BMP4 are key regulators of the fate and differentiation of human mammary epithelial stem cells (SCs), as well as of their niches, and are involved in breast cancer development. We established that MCF10A immature mammary epithelial cells reliably reproduce the BMP response that we previously identified in human primary epithelial SCs. In this model, we observed that BMP2 promotes luminal progenitor commitment and expansion, whereas BMP4 prevents lineage differentiation. Environmental pollutants are known to promote cancer development, possibly by providing cells with stem-like features and by modifying their niches. Bisphenols, in particular, were shown to increase the risk of developing breast cancer. Here, we demonstrate that chronic exposure to low doses of bisphenol A (BPA) or benzo(a)pyrene (B(a)P) alone has little effect on SCs properties of MCF10A cells. Conversely, we show that this exposure affects the response of immature epithelial cells to BMP2 and BMP4. Furthermore, the modifications triggered in MCF10A cells on exposure to pollutants appeared to be predominantly mediated by altering the expression and localization of type-1 receptors and by pre-activating BMP signaling, through the phosphorylation of small mothers against decapentaplegic 1/5/8 (SMAD1/5/8). By analyzing stem and progenitor properties, we reveal that BPA prevents the maintenance of SC features prompted by BMP4, whereas promoting cell differentiation towards a myoepithelial phenotype. Inversely, B(a)P prevents BMP2-mediated luminal progenitor commitment and expansion, leading to the retention of stem-like properties. Overall, our data indicate that BPA and B(a)P distinctly alter the fate and differentiation potential of mammary epithelial SCs by modulating BMP signaling. PMID:27740625

  3. Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation

    Science.gov (United States)

    Liu, Jia; Saito, Kan; Maruya, Yuriko; Nakamura, Takashi; Yamada, Aya; Fukumoto, Emiko; Ishikawa, Momoko; Iwamoto, Tsutomu; Miyazaki, Kanako; Yoshizaki, Keigo; Ge, Lihong; Fukumoto, Satoshi

    2016-01-01

    Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling. PMID:27030100

  4. Expression Products of Chimeric BMP2 and BMP7 Induce Osteoblast Differentiation%BMP2与BMP7嵌合表达产物可诱导成骨细胞分化

    Institute of Scientific and Technical Information of China (English)

    胡丽玲; 李晓霞; 张镜宇; 王宝利

    2009-01-01

    目的:构建骨形态发生蛋白(BMP)2与BMP7嵌合表达的分泌型基因载体pcDNA3-BMP2/7,检测表达产物的成骨诱导活性.方法:聚合酶链反应(PCR)扩增BMP2与BMP7的成熟肽编码基因,利用重叠延伸PCR以柔性肽(Gly_4Ser)_3编码序列使两者嵌合并克隆到质粒pcDNA3/sec上,转染CHO-K1细胞筛选得到稳定克隆,以其条件培养基处理鼠胚胎成纤维细胞C3H10T1/2,通过RT-PCR研究BMP2/7嵌合表达产物的活性.结果:BMP2/7嵌合表达产物可以明显提高C3H10T1/2细胞碱性磷酸酶(Alkaline phosphatase,ALP)、骨钙素(Osteocalcin,Oc)成骨细胞表型基因以及特异性转录因子Runx2 (runt-related transcription factor 2)mRNA的表达(P < 0.01).结论:制备的BMP2/7嵌合表达产物能够形成异源二聚体,诱导非骨源性细胞向成骨细胞分化.%Objective: To study the osteoinductive activity of chimeric molecule of bone morphogenetic protein(BMP)2 and BMP7 expressed in mammalian cells. Methods: Sequences encoding mature peptides of BMP2 and BMP7 were separately amplified by PCR and then linked by overlap-extension PCR with a DNA sequence encoding a flexible peptide (Gly_4Ser)_3 between them. The chimeric DNA sequence was cloned into secretory expression plasmid pcDNA3/sec and then the recombinant plasmid pcDNA3 -BMP2/7 was transfected into CHO-K1 cells. In the presence of G418,cells that stably expressed BMP2/7 were screened out. Thereafter, the conditioned culture medium of the transfected cells was collected and used to treat C3H10T1/2 cells. RT-PCR was employed to study the activity of the recombinant product in inducing osteoblast differentiation. Results: The expression products of chimeric BMP2/7 significantly enhanced the mRNA expression levels of osteoblast phenotype genes, such as alkaline phosphatase, osteocalcin and osteoblast specific transcription factor runt-related transcription factor 2 in C3H10T1/2 cells(P < 0.01). Conclusion: The chimeric expression products of BMP2

  5. Mouse bone marrow stromal cells differentiate to neuron-like cells upon inhibition of BMP signaling.

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    Saxena, Monika; Prashar, Paritosh; Yadav, Prem Swaroop; Sen, Jonaki

    2016-01-01

    Bone marrow stromal cells (BMSCs) are a source of autologous stem cells that have the potential for undergoing differentiation into multiple cell types including neurons. Although the neuronal differentiation of mesenchymal stem cells has been studied for a long time, the molecular players involved are still not defined. Here we report that the genetic deletion of two members of the bone morphogenetic protein (Bmp) family, Bmp2 and Bmp4 in mouse BMSCs causes their differentiation into cells with neuron-like morphology. Surprisingly these cells expressed certain markers characteristic of both neuronal and glial cells. Based on this observation, we inhibited BMP signaling in mouse BMSCs through a brief exposure to Noggin protein which also led to their differentiation into cells expressing both neuronal and glial markers. Such cells seem to have the potential for further differentiation into subtypes of neuronal and glial cells and thus could be utilized for cell-based therapeutic applications.

  6. The response of early neural genes to FGF signaling or inhibition of BMP indicate the absence of a conserved neural induction module

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    Rogers Crystal D

    2011-12-01

    Full Text Available Abstract Background The molecular mechanism that initiates the formation of the vertebrate central nervous system has long been debated. Studies in Xenopus and mouse demonstrate that inhibition of BMP signaling is sufficient to induce neural tissue in explants or ES cells respectively, whereas studies in chick argue that instructive FGF signaling is also required for the expression of neural genes. Although additional signals may be involved in neural induction and patterning, here we focus on the roles of BMP inhibition and FGF8a. Results To address the question of necessity and sufficiency of BMP inhibition and FGF signaling, we compared the temporal expression of the five earliest genes expressed in the neuroectoderm and determined their requirements for induction at the onset of neural plate formation in Xenopus. Our results demonstrate that the onset and peak of expression of the genes vary and that they have different regulatory requirements and are therefore unlikely to share a conserved neural induction regulatory module. Even though all require inhibition of BMP for expression, some also require FGF signaling; expression of the early-onset pan-neural genes sox2 and foxd5α requires FGF signaling while other early genes, sox3, geminin and zicr1 are induced by BMP inhibition alone. Conclusions We demonstrate that BMP inhibition and FGF signaling induce neural genes independently of each other. Together our data indicate that although the spatiotemporal expression patterns of early neural genes are similar, the mechanisms involved in their expression are distinct and there are different signaling requirements for the expression of each gene.

  7. BMP2 induces chondrogenic differentiation, osteogenic differentiation and endochondral ossification in stem cells.

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    Zhou, Nian; Li, Qi; Lin, Xin; Hu, Ning; Liao, Jun-Yi; Lin, Liang-Bo; Zhao, Chen; Hu, Zhen-Ming; Liang, Xi; Xu, Wei; Chen, Hong; Huang, Wei

    2016-10-01

    Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) super-family, is one of the main chondrogenic growth factors involved in cartilage regeneration. BMP2 is known to induce chondrogenic differentiation in various types of stem cells in vitro. However, BMP2 also induces osteogenic differentiation and endochondral ossification in mesenchymal stem cells (MSCs). Although information regarding BMP2-induced chondrogenic and osteogenic differentiation within the same system might be essential for cartilage tissue engineering, few studies concerning these issues have been conducted. In this study, BMP2 was identified as a regulator of chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. BMP2 was used to regulate chondrogenic and osteogenic differentiation in stem cells within the same culture system in vitro and in vivo. Any changes in the differentiation markers were assessed. BMP2 was found to induce chondrogenesis and osteogenesis in vitro via the expression of Sox9, Runx2 and its downstream markers. According to the results of the subcutaneous stem cell implantation studies, BMP2 not only induced cartilage formation but also promoted endochondral ossification during ectopic bone/cartilage formation. In fetal limb cultures, BMP2 promoted chondrocyte hypertrophy and endochondral ossification. Our data reveal that BMP2 can spontaneously induce chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. Thus, BMP2 can be used in cartilage tissue engineering to regulate cartilage formation but has to be properly regulated for cartilage tissue engineering in order to retain the cartilage phenotype.

  8. Magnesium modification up-regulates the bioactivity of bone morphogenetic protein-2 upon calcium phosphate cement via enhanced BMP receptor recognition and Smad signaling pathway.

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    Ding, Sai; Zhang, Jing; Tian, Yu; Huang, Baolin; Yuan, Yuan; Liu, Changsheng

    2016-09-01

    Efficient presentation of growth factors is one of the great challenges in tissue engineering. In living systems, bioactive factors exist in soluble as well as in matrix-bound forms, both of which play an integral role in regulating cell behaviors. Herein, effect of magnesium on osteogenic bioactivity of recombinant human bone morphogenetic protein-2 (rhBMP-2) was investigated systematically with a series of Mg modified calcium phosphate cements (xMCPCs, x means the content of magnesium phosphate cement wt%) as matrix model. The results indicated that the MCPC, especially 5MCPC, could promote the rhBMP-2-induced in vitro osteogenic differentiation via Smad signaling of C2C12 cells. Further studies demonstrated that all MCPC substrates exhibited similar rhBMP-2 release rate and preserved comparable conformation and biological activity of the released rhBMP-2. Also, the ionic extracts of MCPC made little difference to the bioactivity of rhBMP-2, either in soluble or in matrix-bound forms. However, with the quartz crystal microbalance (QCM), we observed a noticeable enhancement of rhBMP-2 mass-uptake on 5MCPC as well as a better recognition of the bound rhBMP-2 to BMPR IA and BMPR II. In vivo results demonstrated a better bone regeneration capacity of 5MCPC/rhBMP-2. From the above, our results demonstrated that it was the Mg anchored on the underlying substrates that tailored the way of rhBMP-2 bound on MCPC, and thus facilitated the recognition of BMPRs to stimulate osteogenic differentiation. The study will guide the development of Mg-doped bioactive bone implants for tissue regeneration.

  9. BMP9-Induced Osteogenetic Differentiation and Bone Formation of Muscle-Derived Stem Cells

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    Li Xiang

    2012-01-01

    Full Text Available Efficient osteogenetic differentiation and bone formation from muscle-derived stem cells (MDSCs should have potential clinical applications in treating nonunion fracture healing or bone defects. Here, we investigate osteogenetic differentiation ability of MDSCs induced by bone morphogenetic protein 9 (BMP9 in vitro and bone formation ability in rabbit radius defects repairing model. Rabbit's MDSCs were extracted by type I collagenase and trypsin methods, and BMP9 was introduced into MDSCs by infection with recombinant adenovirus. Effects of BMP9-induced osteogenetic differentiation of MDSCs were identified with alkaline phosphatase (ALP activity and expression of later marker. In stem-cell implantation assay, MDSCs have also shown valuable potential bone formation ability induced by BMP9 in rabbit radius defects repairing test. Taken together, our findings suggest that MDSCs are potentiated osteogenetic stem cells which can be induced by BMP9 to treat large segmental bone defects, nonunion fracture, and/or osteoporotic fracture.

  10. Akt1 signaling coordinates BMP signaling and β-catenin activity to regulate second heart field progenitor development.

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    Luo, Wen; Zhao, Xia; Jin, Hengwei; Tao, Lichan; Zhu, Jingai; Wang, Huijuan; Hemmings, Brian A; Yang, Zhongzhou

    2015-02-15

    Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/β-catenin signaling. PTEN-Akt signaling regulates the stem cell/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract. Cell proliferation of cardiac progenitors was enhanced, whereas cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding the BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of β-catenin, thus enhancing its activity. Reducing β-catenin levels could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and β-catenin activity.

  11. Embryonic hair follicle fate change by augmented beta-catenin through Shh and Bmp signaling.

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    Suzuki, Kentaro; Yamaguchi, Yuji; Villacorte, Mylah; Mihara, Kenichiro; Akiyama, Masashi; Shimizu, Hiroshi; Taketo, Makoto M; Nakagata, Naomi; Tsukiyama, Tadasuke; Yamaguchi, Terry P; Birchmeier, Walter; Kato, Shigeaki; Yamada, Gen

    2009-02-01

    beta-catenin signaling is one of the key factors regulating the fate of hair follicles (HFs). To elucidate the regulatory mechanism of embryonic HF fate determination during epidermal development/differentiation, we analyzed conditional mutant mice with keratinocytes expressing constitutively active beta-catenin (K5-Cre Catnb(ex3)fl/+). The mutant mice developed scaly skin with a thickened epidermis and showed impaired epidermal stratification. The hair shaft keratins were broadly expressed in the epidermis but there was no expression of the terminal differentiation markers K1 and loricrin. Hair placode markers (Bmp2 and Shh) and follicular dermal condensate markers (noggin, patched 1 and Pdgfra) were expressed throughout the epidermis and the upper dermis, respectively. These results indicate that the embryonic epidermal keratinocytes have switched extensively to the HF fate. A series of genetic studies demonstrated that the epidermal switching to HF fate was suppressed by introducing the conditional mutation K5-Cre Catnb(ex3)fl/+Shhfl/- (with additional mutation of Shh signaling) or K5-Cre Catnb(ex3)fl/+BmprIAfl/fl (with additional mutation of Bmp signaling). These results demonstrate that Wnt/beta-catenin signaling relayed through Shh and Bmp signals is the principal regulatory mechanism underlying the HF cell fate change. Assessment of Bmp2 promoter activities suggested a putative regulation by beta-catenin signaling relayed by Shh signaling towards Bmp2. We also found that Shh protein expression was increased and expanded in the epidermis of K5-Cre Catnb(ex3)fl/+BmprIAfl/fl mice. These results indicate the presence of growth factor signal cross-talk involving beta-catenin signaling, which regulates the HF fate.

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

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

    2016-05-01

    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

  13. 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

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

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    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.

  15. 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

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

    Science.gov (United States)

    Sengle, Gerhard; Carlberg, Valerie; Tufa, Sara F; Charbonneau, Noe L; Smaldone, Silvia; Carlson, Eric J; Ramirez, Francesco; Keene, Douglas R; Sakai, Lynn Y

    2015-06-01

    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 fibrillin-2 can

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

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    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

  18. PELA microspheres with encapsulated arginine-chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation.

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    Xu, Xiaolong; Qiu, Sujun; Zhang, Yuxian; Yin, Jie; Min, Shaoxiong

    2017-03-01

    Repair of the bone injury remains a challenge in clinical practices. Recent progress in tissue engineering and therapeutic gene delivery systems have led to promising new strategies for successful acceleration of bone repair process. The aim of this study was to create a controlled-release system to slowly release the arginine-chitosan/plasmid DNA nanoparticles encoding BMP-2 gene (Arg-CS/pBMP-2 NPs), efficiently transfect osteoblastic progenitor cells, secrete functional BMP-2 protein, and promote osteogenic differentiation. In this study, chitosan was conjugated with arginine to generate arginine-chitosan polymer (Arg-CS) for gene delivery. Mix the Arg-CS with pBMP-2 to condense pBMP-2 into nano-sized particles. In vitro transfection assays demonstrated that the transfection efficiency of Arg-CS/pBMP-2 nanoparticles and the expression level of BMP-2 was obviously exceed control groups. Further, PELA microspheres as the controlled-release carrier for the nanoparticles were used to encapsulate Arg-CS/pBMP-2 NPs. We demonstrated that the Arg-CS/pBMP-2 NPs could slowly release from the PELA microspheres at least for 42 d. During the co-culture with the PELA microspheres, the content of BMP-2 protein secreted by MC3T3-E1 reached the peak at 7 d. After 21d, the secretion of BMP-2 protein still maintain a higher level. The alkaline phosphatase activity, alizarin red staining, and osteogenesis-related gene expression by real-time quantitative PCR analysis all showed the PELA microspheres entrapping with Arg-CS/pBMP-2 NPs can obviously induce the osteogenic differentiation. The results indicated that the Arg-CS is a suitable gene vector which can promote the gene transfection. And the novel PELA microspheres-nanoparticle controlled-release system has potential clinical application in the future after further research.

  19. 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.

  20. 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.

  1. 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.

  2. Uptake of nickel from 316L stainless steel into contacting osteoblastic cells and metal ion interference with BMP-2-induced alkaline phosphatase.

    Science.gov (United States)

    Mölders, Martina; Felix, Joachim; Bingmann, Dieter; Hirner, Alfred; Wiemann, Martin

    2007-11-01

    Bone cells contacting nickel (Ni)-containing implant materials may be affected by Ni species via disturbed signaling pathways involved in bone cell development. Here we analyze effects of the Ni-containing steel 316L and major metal constituents thereof on bone morphogenetic protein-2 (BMP-2)-induced alkaline phosphatase (ALP) of MC3T3-E1 cells. While cells grew normally on 316L, cellular Ni content increased 10-fold vs. control within 4 days. With respect to the major components of 316L, Ni2+ (3-50 microM) was most inhibitory to BMP-2-induced ALP, whereas even 50 microM Fe3+, Cr3+, Mo5+, or Mn2+ had no such effect. In line with this, BMP-2-induced ALP was significantly reduced in cells on 316L. This effect was not prevented by the metal ion chelator diethylenetriaminepentaacetic acid (DTPA). Instead, DTPA abolished the stimulatory effect of BMP-2 on ALP, pointing to chelatable metal ions involved. Zn2+, as one possible candidate, antagonized the Ni2+ inhibition of BMP-2-induced ALP in both MC3T3-E1 and human bone marrow stromal cells. Results show that cells contacting 316L steel are exposed to increased concentrations of Ni which suffice to impair BMP-2-induced ALP activity. Zn2+, as a competitor of this inhibition, may help to restore normal osteoblastic function and bone development under these conditions.

  3. BMP2-induced inflammation can be suppressed by the osteoinductive growth factor NELL-1.

    Science.gov (United States)

    Shen, Jia; James, Aaron W; Zara, Janette N; Asatrian, Greg; Khadarian, Kevork; Zhang, James B; Ho, Stephanie; Kim, Hyun Ju; Ting, Kang; Soo, Chia

    2013-11-01

    Bone-morphogenetic protein 2 (BMP2) is currently the only Food and Drug Administration-approved osteoinductive growth factor used in clinical settings for bone regeneration and repair. However, the use of BMP2 is encumbered by numerous clinical complications, including postoperative inflammation and life-threatening cervical swelling. Thus, methods to prevent BMP2-induced inflammation would have far-reaching clinical implications toward improving current BMP2-based methods for bone regeneration. For the first time, we investigate the potential role of the growth factor Nel-like molecule-1 (NELL-1) in inhibiting BMP2-induced inflammation. Adult rats underwent a femoral bone onlay procedure, treated with either BMP2 protein (4 mg/mL), NELL-1 protein (4 mg/mL), or both proteins combined. Animals were evaluated at 3, 7, and 14 days postoperatively by histology, histomorphometry, immunohistochemistry, and real-time PCR for markers of inflammation (TNFα, IL6). The relative levels of TNFα and IL6 in serum were also detected by ELISA. The mechanism for NELL-1's anti-inflammatory effect was further assessed through examining inflammatory markers and generation of reactive oxygen species (ROS) in the mouse embryonic fibroblast NIH3T3 cells. BMP2 significantly induced local inflammation, including an early and pronounced polymorphonuclear cell infiltration accompanied by increased expression of TNFα and IL6. Treatment with NELL-1 alone elicited no significant inflammatory response. However, NELL-1 significantly attenuated BMP2-induced inflammation by all markers and at all timepoints. These local findings were also confirmed using systemic serum inflammatory biomarkers (TNFα, IL6). In each case, NELL-1 fully reversed BMP2-induced systemic inflammation. Lastly, our findings were recapitulated in vitro, where NELL-1 suppressed BMP2 induced expression of inflammatory markers, as well as NF-κB transcriptional activity and generation of ROS. BMP2-induced inflammation is a

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

    Science.gov (United States)

    Hakim, Md Abdul; Jiang, Wenbin; Luo, Lei; Li, Bowen; Yang, Shilong; Song, Yuzhu; Lai, Ren

    2015-09-14

    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.

  5. BMP receptor signaling is required for postnatal maintenance of articular cartilage.

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    Ryan B Rountree

    2004-11-01

    Full Text Available Articular cartilage plays an essential role in health and mobility, but is frequently damaged or lost in millions of people that develop arthritis. The molecular mechanisms that create and maintain this thin layer of cartilage that covers the surface of bones in joint regions are poorly understood, in part because tools to manipulate gene expression specifically in this tissue have not been available. Here we use regulatory information from the mouse Gdf5 gene (a bone morphogenetic protein [BMP] family member to develop new mouse lines that can be used to either activate or inactivate genes specifically in developing joints. Expression of Cre recombinase from Gdf5 bacterial artificial chromosome clones leads to specific activation or inactivation of floxed target genes in developing joints, including early joint interzones, adult articular cartilage, and the joint capsule. We have used this system to test the role of BMP receptor signaling in joint development. Mice with null mutations in Bmpr1a are known to die early in embryogenesis with multiple defects. However, combining a floxed Bmpr1a allele with the Gdf5-Cre driver bypasses this embryonic lethality, and leads to birth and postnatal development of mice missing the Bmpr1a gene in articular regions. Most joints in the body form normally in the absence of Bmpr1a receptor function. However, articular cartilage within the joints gradually wears away in receptor-deficient mice after birth in a process resembling human osteoarthritis. Gdf5-Cre mice provide a general system that can be used to test the role of genes in articular regions. BMP receptor signaling is required not only for early development and creation of multiple tissues, but also for ongoing maintenance of articular cartilage after birth. Genetic variation in the strength of BMP receptor signaling may be an important risk factor in human osteoarthritis, and treatments that mimic or augment BMP receptor signaling should be

  6. 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

  7. Protein palmitoylation regulates osteoblast differentiation through BMP-induced osterix expression.

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    Wai Fook Leong

    Full Text Available Osteoporosis is one of the most common diseases and can be treated by either anti-resorption drugs, anabolic drugs, or both. To search for anabolic drug targets for osteoporosis therapy, it is crucial to understand the biology of bone forming cells, osteoblasts, in terms of their proliferation, differentiation, and function. Here we found that protein palmitoylation participates in signaling pathways that control osterix expression and osteoblast differentiation. Mouse calvarial osteoblasts express most of the 24 palmitoyl transferases, with some being up-regulated during differentiation. Inhibition of protein palmitoylation, with a substrate-analog inhibitor, diminished osteoblast differentiation and mineralization, but not proliferation or survival. The decrease in differentiation capacity is associated with a reduction in osterix, but not Runx2 or Atf4. Inhibition of palmitoyl transferases had little effect in p53(-/- osteoblasts that show accelerated differentiation due to overexpression of osterix, suggesting that osterix, at least partially, mediated the effect of inhibition of palmitoyl transferases on osteoblast differentiation. BMPs are the major driving force of osteoblast differentiation in the differentiation assays. We found that inhibition of palmitoyl transferases also compromised BMP2-induced osteoblast differentiation through down-regulating osterix induction. However, palmitoyl transferases inhibitor did not inhibit Smad1/5/8 activation. Instead, it compromised the activation of p38 MAPK, which are known positive regulators of osterix expression and differentiation. These results indicate that protein palmitoylation plays an important role in BMP-induced MAPK activation, osterix expression, and osteoblast differentiation.

  8. Disrupted dorsal neural tube BMP signaling in the cilia mutant Arl13b hnn stems from abnormal Shh signaling.

    Science.gov (United States)

    Horner, Vanessa L; Caspary, Tamara

    2011-07-01

    In the embryonic neural tube, multiple signaling pathways work in concert to create functional neuronal circuits in the adult spinal cord. In the ventral neural tube, Sonic hedgehog (Shh) acts as a graded morphogen to specify neurons necessary for movement. In the dorsal neural tube, bone morphogenetic protein (BMP) and Wnt signals cooperate to specify neurons involved in sensation. Several signaling pathways, including Shh, rely on primary cilia in vertebrates. In this study, we used a mouse mutant with abnormal cilia, Arl13b(hnn), to study the relationship between cilia, cell signaling, and neural tube patterning. Arl13b(hnn) mutants have abnormal ventral neural tube patterning due to disrupted Shh signaling; in addition, dorsal patterning defects occur, but the cause of these is unknown. Here we show that the Arl13b(hnn) dorsal patterning defects result from abnormal BMP signaling. In addition, we find that Wnt ligands are abnormally expressed in Arl13b(hnn) mutants; surprisingly, however, downstream Wnt signaling is normal. We demonstrate that Arl13b is required non-autonomously for BMP signaling and Wnt ligand expression, indicating that the abnormal Shh signaling environment in Arl13b(hnn) embryos indirectly causes dorsal defects.

  9. BMP-7 Signaling and its Critical Roles in Kidney Development, the Responses to Renal Injury, and Chronic Kidney Disease.

    Science.gov (United States)

    Manson, Scott R; Austin, Paul F; Guo, Qiusha; Moore, Katelynn H

    2015-01-01

    Chronic kidney disease (CKD) is a significant health problem that most commonly results from congenital abnormalities in children and chronic renal injury in adults. The therapeutic potential of BMP-7 was first recognized nearly two decades ago with studies demonstrating its requirement for kidney development and ability to inhibit the pathogenesis of renal injury in models of CKD. Since this time, our understanding of CKD has advanced considerably and treatment strategies have evolved with the identification of many additional signaling pathways, cell types, and pathologic processes that contribute to disease progression. The purpose of this review is to revisit the seminal studies that initially established the importance of BMP-7, highlight recent advances in BMP-7 research, and then integrate this knowledge with current research paradigms. We will provide an overview of the evolutionarily conserved roles of BMP proteins and the features that allow BMP signaling pathways to function as critical signaling nodes for controlling biological processes, including those related to CKD. We will discuss the multifaceted functions of BMP-7 during kidney development and the potential for alterations in BMP-7 signaling to result in congenital abnormalities and pediatric kidney disease. We will summarize the renal protective effects of recombinant BMP-7 in experimental models of CKD and then propose a model to describe the potential physiological role of endogenous BMP-7 in the innate repair mechanisms of the kidneys that respond to renal injury. Finally, we will highlight emerging clinical approaches for applying our knowledge of BMP-7 toward improving the treatment of patients with CKD.

  10. BMP7 activates brown adipose tissue and reduces diet-induced obesity only at subthermoneutrality.

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    Mariëtte R Boon

    Full Text Available 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

  11. Endothelial follistatin-like 1 regulates the maturation of the pulmonary vasculature by modulating BMP/SMAD signaling

    NARCIS (Netherlands)

    Tania, Navessa Padma; Maarsingh, Harm; Bos, Sophie T.; Mattiotti, Andrea; Prakash, Stuti; Timens, Wim; Schmidt, Martina; Van Den Hoff, Maurice; Gosens, Reinoud

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a progressive disease that is characterized by vascular remodeling and sustained vasoconstriction which consequently lead to high blood pressure in the pulmonary vasculature and right ventricle remodeling. Altered bone morphogenetic protein (BMP) signaling ha

  12. Spatially Resolved Genome-wide Transcriptional Profiling Identifies BMP Signaling as Essential Regulator of Zebrafish Cardiomyocyte Regeneration.

    Science.gov (United States)

    Wu, Chi-Chung; Kruse, Fabian; Vasudevarao, Mohankrishna Dalvoy; Junker, Jan Philipp; Zebrowski, David C; Fischer, Kristin; Noël, Emily S; Grün, Dominic; Berezikov, Eugene; Engel, Felix B; van Oudenaarden, Alexander; Weidinger, Gilbert; Bakkers, Jeroen

    2016-01-11

    In contrast to mammals, zebrafish regenerate heart injuries via proliferation of cardiomyocytes located near the wound border. To identify regulators of cardiomyocyte proliferation, we used spatially resolved RNA sequencing (tomo-seq) and generated a high-resolution genome-wide atlas of gene expression in the regenerating zebrafish heart. Interestingly, we identified two wound border zones with distinct expression profiles, including the re-expression of embryonic cardiac genes and targets of bone morphogenetic protein (BMP) signaling. Endogenous BMP signaling has been reported to be detrimental to mammalian cardiac repair. In contrast, we find that genetic or chemical inhibition of BMP signaling in zebrafish reduces cardiomyocyte dedifferentiation and proliferation, ultimately compromising myocardial regeneration, while bmp2b overexpression is sufficient to enhance it. Our results provide a resource for further studies on the molecular regulation of cardiac regeneration and reveal intriguing differential cellular responses of cardiomyocytes to a conserved signaling pathway in regenerative versus non-regenerative hearts.

  13. crm-1 facilitates BMP signaling to control body size in Caenorhabditis elegans.

    Science.gov (United States)

    Fung, Wong Yan; Fat, Ko Frankie Chi; Eng, Cheah Kathryn Song; Lau, Chow King

    2007-11-01

    We have identified in Caenorhabditis elegans a homologue of the vertebrate Crim1, crm-1, which encodes a putative transmembrane protein with multiple cysteine-rich (CR) domains known to have bone morphogenetic proteins (BMPs) binding activity. Using the body morphology of C. elegans as an indicator, we showed that attenuation of crm-1 activity leads to a small body phenotype reminiscent of that of BMP pathway mutants. We showed that the crm-1 loss-of-function phenotype can be rescued by constitutive supply of sma-4 activity. crm-1 can enhance BMP signaling and this activity is dependent on the presence of the DBL-1 ligand and its receptors. crm-1 is expressed in neurons at the ventral nerve cord, where the DBL-1 ligand is produced. However, ectopic expression experiments reveal that crm-1 gene products act outside the DBL-1 producing cells and function non-autonomously to facilitate dbl/sma pathway signaling to control body size.

  14. Proteolytic Processing as a Regulator of BMP-type Signaling in Drosophila Development

    OpenAIRE

    2013-01-01

    A small set of highly conserved signaling molecules performs a great number of tasks in different animals and developmental contexts. Among them, the bone morphogenetic proteins (BMPs) constitute a group of growth and differentiation factors that are involved in numerous developmental processes affecting cell proliferation, apoptosis and differentiation. In the fruit fly, Drosophila melanogaster, three BMP type proteins have been identified, each of which has a homolog in mammals. Decapentapl...

  15. BMP6 reverses TGF-β1-induced changes in HK-2 cells:implications for the treatment of renal fibrosis

    Institute of Scientific and Technical Information of China (English)

    Ji-dong YAN; Shuang YANG; Jie ZHANG; Tian-hui ZHU

    2009-01-01

    Aim:The aim of the study was to investigate the potential role of BMP6 in TGF-β1-mediated changes in HK-2 cells.Methods:BMP6 was purified via heparin affinity and reverse phase liquid chromatography.The purity,specificity,and bioactivity of BMP6 were determined by SDS-PAGE,Western blot assays,and the induction of alkaline phosphatase (ALP) activity,respectively.Cell proliferation,morphology,and expression levels of α-SMA and E-cadherin were assessed by cell viability,microscopy,and Western blot assays,respectively.In addition,cell adhesion abilities were determined by countingthe number of attached cells.The expression of fibronectin,collagen IV,matrix metalloproteinases 2 (MMP-2),and tissue inhibitors of matrix metalloproteinases 2 (TIMP-2) were analyzed using RT-PCR.MMP-2 activity was analyzed by zymography,whereas the activation of the MAPKs and Smad signaling were analyzed using Western blot assays and a reporter gene assay,respectively.Results:Our results indicated that recombinant BMP6 induced ALP activity in a dose-dependent and time-course-dependent manner.Treatment with TGF-β1 reduced both the cell proliferation and the expression of E-cadherin,induced a morphological transformation,decreased the expression and activity of MMP-2,and increased the expression levels of a-SMA,fibronectin,and TIMP-2 in HK-2 cells,All of these effects were inhibited when cells were treated with TGF-β1 in combination with rhBMP6,whereas rhBMP6 alone demonstrated no such effect.Treatment with TGF-β1,rhBMP6,or a combination of both had no effect on the expression of collagen Ⅳ.In addition,the administration of rhBMP6 prevented the enhanced adhesion behavior triggered by TGF-β1.Furthermore,the addition of rhBMP6 abrogated the JNK and Smad2/3 signaling that was activated by TGF-β1.Conclusion:BMP6 ameliorated the TGF-β1-induced changes in HK-2 cells.The suppression of TGF-β1-mediated JNK and Smad2/3 signaling activation were implicated in these effects.

  16. Stiffness-dependent cellular internalization of matrix-bound BMP-2 and its relation to Smad and non-Smad signaling.

    Science.gov (United States)

    Gilde, Flora; Fourel, Laure; Guillot, Raphael; Pignot-Paintrand, Isabelle; Okada, Takaharu; Fitzpatrick, Vincent; Boudou, Thomas; Albiges-Rizo, Corinne; Picart, Catherine

    2016-12-01

    Surface coatings delivering BMP are a promising approach to render biomaterials osteoinductive. In contrast to soluble BMPs which can interact with their receptors at the dorsal side of the cell, BMPs presented as an insoluble cue physically bound to a biomimetic matrix, called here matrix-bound (bBMP-2), are presented to cells by their ventral side. To date, BMP-2 internalization and signaling studies in cell biology have always been performed by adding soluble (sBMP-2) to cells adhered on cell culture plates or glass slides, which will be considered here as a "reference" condition. However, whether and how matrix-bound BMP-2 can be internalized by cells and its relation to canonical (SMAD) and non-canonical signaling (ALP) remain open questions. In this study, we investigated the uptake and processing of BMP-2 by C2C12 myoblasts. This BMP-2 was presented either embedded in polyelectrolyte multilayer films (matrix-bound presentation) or as soluble form. Using fluorescently labeled BMP-2, we showed that the amount of matrix-bound BMP-2 internalized is dependent on the level of crosslinking of the polyelectrolyte films. Cav-1-mediated internalization is related to both SMAD and ALP signaling, while clathrin-mediated is only related to ALP signaling. BMP-2 internalization was independent of the presentation mode (sBMP-2 versus bBMP-2) for low crosslinked films (soft, EDC10) in striking contrast with high crosslinked (stiff, EDC70) films where internalization was much lower and slower for bBMP-2. As anticipated, internalization of sBMP-2 barely depended on the underlying matrix. Taken together, these results indicate that BMP-2 internalization can be tuned by the underlying matrix and activates downstream BMP-2 signaling, which is key for the effective formation of bone tissue.

  17. Compound C prevents the unfolded protein response during glucose deprivation through a mechanism independent of AMPK and BMP signaling.

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    Sakae Saito

    Full Text Available Inhibiting the unfolded protein response (UPR can be a therapeutic approach, especially for targeting the tumor microenvironment. Here, we show that compound C (also known as dorsomorphin, a small-molecule inhibitor of AMP-activated protein kinase (AMPK and bone morphogenetic protein (BMP signaling, inhibit the UPR-induced transcription program depending on the glucose deprivation conditions. We found that compound C prevented UPR marker glucose-regulated protein 78 (GRP78 accumulation and exerted enhanced cytotoxicity during glucose deprivation. Gene expression profiling, together with biochemical analysis, revealed that compound C had a unique mode of action to suppress the transcriptional activation of UPR-targeted genes, as compared with the classic UPR inhibitors versipelostatin and biguanides. Surprisingly, the UPR-inhibiting activity of compound C was not associated with either AMPK or BMP signaling inhibition. We further found that combination treatments of compound C and the classic UPR inhibitors resulted in synergistic cell death with UPR suppression during glucose deprivation. Our findings demonstrate that compound C could be a unique tool for developing a UPR-targeted antitumor therapy.

  18. Decreased BMP2 signal in GIT1 knockout mice slows bone healing.

    Science.gov (United States)

    Sheu, T J; Zhou, Wei; Fan, Jin; Zhou, Hao; Zuscik, Michael J; Xie, Chao; Yin, Guoyong; Berk, Bradford C

    2014-12-01

    Endochondral ossification, an important stage of fracture healing, is regulated by a variety of signaling pathways. Transforming growth factor β (TGFβ) superfamily plays important roles and comprises TGFβs, bone morphogenetic proteins (BMPs), and growth differentiation factors. TGFβs primarily regulate cartilage formation and endochondral ossification. BMP2 shows diverse efficacy, from the formation of skeleton and extraskeletal organs to the osteogenesis and remodeling of bone. G-protein-coupled receptor kinase 2-interacting protein-1 (GIT1), a shuttle protein in osteoblasts, facilitates fracture healing by promoting bone formation and increasing the secretion of vascular endothelial growth factor. Our study examined whether GIT1 regulates fracture healing through the BMP2 signaling pathway and/or through the TGFβ signaling pathway. GIT1 knockout (KO) mice exhibited delayed fracture healing, chondrocyte accumulation in the fracture area, and reduced staining intensity of phosphorylated Smad1/5/8 (pSmad1/5/8) and Runx2. Endochondral mineralization diminished while the staining intensity of phosphorylated Smad2/3 (pSmad2/3) showed no significant change. Bone marrow mesenchymal stem cells extracted from GIT1 KO mice showed a decline of pSmad1/5/8 levels and of pSmad1/5/8 translocated into the cell nucleus after BMP2 stimulus. We detected no significant change in the pSmad2/3 level after TGFβ1 stimulus. Data obtained from reporter gene analysis of C3H10T1/2 cells cultured in vitro confirmed these findings. GIT1-siRNA inhibited transcription in the cell nucleus via pSmad1/5/8 after BMP2 stimulus but had no significant effect on transcription via pSmad2/3 after TGFβ1 stimulus. Our results indicate that GIT1 regulates Smad1/5/8 phosphorylation and mediates BMP2 regulation of Runx2 expression, thus affecting endochondral ossification at the fracture site.

  19. 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

    2016-01-01

    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

  20. The Functions of BMP3 in Rabbit Articular Cartilage Repair

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    Zhe Zhang

    2015-10-01

    Full Text Available Bone morphogenetic proteins (BMPs play important roles in skeletal development and repair. Previously, we found fibroblast growth factor 2 (FGF2 induced up-regulation of BMP2, 3, 4 in the process of rabbit articular cartilage repair, which resulted in satisfactory repair effects. As BMP2/4 show a clearly positive effect for cartilage repair, we investigated the functions of BMP3 in rabbit articular cartilage repair. In this paper, we find that BMP3 inhibits the repair of partial-thickness defect of articular cartilage in rabbit by inducing the degradation of extracellular matrix, interfering with the survival of chondrocytes surrounding the defect, and directly inhibiting the expression of BMP2 and BMP4. Meanwhile BMP3 suppress the repair of full-thickness cartilage defect by destroying the subchondral bone through modulating the proliferation and differentiation of bone marrow stem cells (BMSCs, and directly increasing the expression of BMP4. Although BMP3 has different functions in the repair of partial and full-thickness defects of articular cartilage in rabbit, the regulation of BMP expression is involved in both of them. Together with our previous findings, we suggest the regulation of the BMP signaling pathway by BMP3 is essential in articular cartilage repair.

  1. The Notch intracellular domain integrates signals from Wnt, Hedgehog, TGFβ/BMP and hypoxia pathways.

    Science.gov (United States)

    Borggrefe, Tilman; Lauth, Matthias; Zwijsen, An; Huylebroeck, Danny; Oswald, Franz; Giaimo, Benedetto Daniele

    2016-02-01

    Notch signaling is a highly conserved signal transduction pathway that regulates stem cell maintenance and differentiation in several organ systems. Upon activation, the Notch receptor is proteolytically processed, its intracellular domain (NICD) translocates into the nucleus and activates expression of target genes. Output, strength and duration of the signal are tightly regulated by post-translational modifications. Here we review the intracellular post-translational regulation of Notch that fine-tunes the outcome of the Notch response. We also describe how crosstalk with other conserved signaling pathways like the Wnt, Hedgehog, hypoxia and TGFβ/BMP pathways can affect Notch signaling output. This regulation can happen by regulation of ligand, receptor or transcription factor expression, regulation of protein stability of intracellular key components, usage of the same cofactors or coregulation of the same key target genes. Since carcinogenesis is often dependent on at least two of these pathways, a better understanding of their molecular crosstalk is pivotal.

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

    Directory of Open Access Journals (Sweden)

    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

  3. 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.

  4. 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.

  5. L51P - A BMP2 variant with osteoinductive activity via inhibition of Noggin.

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    Albers, Christoph E; Hofstetter, Wilhelm; Sebald, Hans-Jörg; Sebald, Walter; Siebenrock, Klaus A; Klenke, Frank M

    2012-09-01

    Bone morphogenetic proteins (BMP) have to be applied at high concentrations to stimulate bone healing. The limited therapeutic efficacy may be due to the local presence of BMP antagonists such as Noggin. Thus, inhibiting BMP antagonists is an attractive therapeutic option. We hypothesized that the engineered BMP2 variant L51P stimulates osteoinduction by antagonizing Noggin-mediated inhibition of BMP2. Primary murine osteoblasts (OB) were treated with L51P, BMP2, and Noggin. OB proliferation and differentiation were quantified with XTT and alkaline phosphatase (ALP) assays. BMP receptor dependent intracellular signaling in OB was evaluated with Smad and p38 MAPK phosphorylation assays. BMP2, Noggin, BMP receptor Ia/Ib/II, osteocalcin, and ALP mRNA expressions were analyzed with real-time PCR. L51P stimulated OB differentiation by blocking Noggin mediated inhibition of BMP2. L51P did not induce OB differentiation directly and did not activate BMP receptor dependent intracellular signaling via the Smad pathway. Treatment of OB cultures with BMP2 but not with L51P resulted in an increased expression of ALP, BMP2, and Noggin mRNA. By inhibiting the BMP antagonist Noggin, L51P enhances BMP2 activity and stimulates osteoinduction without exhibiting direct osteoinductive function. Indirect osteoinduction with L51P seems to be advantageous to osteoinduction with BMP2 as BMP2 stimulates the expression of Noggin thereby self-limiting its own osteoinductive activity. Treatment with L51P is the first protein-based approach available to augment BMP2 induced bone regeneration through inhibition of BMP antagonists. The described strategy may help to decrease the amounts of exogenous BMPs currently required to stimulate bone healing.

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

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    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.

  7. TGF-β Prevents Phosphate-Induced Osteogenesis through Inhibition of BMP and Wnt/β-Catenin Pathways

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    Almadén, Yolanda; Martínez-Moreno, Julio M.; Montes de Oca, Addy; Rodriguez-Ortiz, María Encarnación; Diaz-Tocados, Juan M.; Canalejo, Antonio; Florio, Mónica; López, Ignacio; Richards, William G.; Rodriguez, Mariano; Aguilera-Tejero, Escolástico; Muñoz-Castañeda, Juan R.

    2014-01-01

    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. PMID:24586576

  8. Biological Silicon Stimulates Collagen Type 1 and Osteocalcin Synthesis in Human Osteoblast-Like Cells Through the BMP-2/Smad/RUNX2 Signaling Pathway.

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    Dong, Meng; Jiao, Guangjun; Liu, Haichun; Wu, Wenliang; Li, Shangzhi; Wang, Qingshi; Xu, Daxia; Li, Xiaofeng; Liu, Huan; Chen, Yunzhen

    2016-10-01

    Silicon is essential for bone formation. A low-silicon diet leads to bone defects, and numerous animal models have demonstrated that silicon supplementation increases bone mineral density (BMD) and reduces bone fragility. However, the exact mechanism of this action has not been characterized. In this study, we aimed to determine the role of biological silicon in the induction of osteoblast differentiation and the possible underlying mechanism. We examined whether orthosilicic acid promotes collagen type 1 (COL-1) and osteocalcin synthesis through the bone morphogenetic protein-2 (BMP-2)/Smad1/5/runt-related transcription factor 2 (RUNX2) signaling pathway by investigating its effect in vitro at several concentrations on COL-1 and osteocalcin synthesis in human osteosarcoma cell lines (MG-63 and U2-OS). The expression of relevant proteins was detected by Western blotting following exposure to noggin, an inhibitor of BMP-2. In MG-63 cells, immunofluorescence methods were applied to detect changes in the expression of BMP-2, phosphorylated Smad1/5 (P-Smad1/5), and RUNX2. Furthermore, rat bone mesenchymal stem cells (BMSCs) were used to determine the effect of orthosilicic acid on osteogenic differentiation. Exposure to 10 μM orthosilicic acid markedly increased the expression of BMP-2, P-Smad1/5, RUNX2, COL-1, and osteocalcin in osteosarcoma cell lines. Enhanced ALP activity and the formation of mineralized nodules were also observed under these conditions. Furthermore, preconditioning with noggin inhibited the silicon-induced upregulation of P-Smad1/5, RUNX2, and COL-1 expression. In conclusion, the BMP-2/Smad1/5/RUNX2 signaling pathway participates in the silicon-mediated induction of COL-1 and osteocalcin synthesis, and orthosilicic acid promotes the osteogenic differentiation of rat BMSCs.

  9. Fucoidan promotes osteoblast differentiation via JNK- and ERK-dependent BMP2-Smad 1/5/8 signaling in human mesenchymal stem cells.

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    Kim, Beom Su; Kang, Hyo-Jin; Park, Ji-Yun; Lee, Jun

    2015-01-09

    Fucoidan has attracted attention as a potential drug because of its biological activities, which include osteogenesis. However, the molecular mechanisms involved in the osteogenic activity of fucoidan in human alveolar bone marrow-derived mesenchymal stem cells (hABM-MSCs) remain largely unknown. We investigated the action of fucoidan on osteoblast differentiation in hABM-MSCs and its impact on signaling pathways. Its effect on proliferation was determined using the crystal violet staining assay. Osteoblast differentiation was evaluated based on alkaline phosphatase (ALP) activity and the mRNA expression of multiple osteoblast markers. Calcium accumulation was determined by Alizarin red S staining. We found that fucoidan induced hABM-MSC proliferation. It also significantly increased ALP activity, calcium accumulation and the expression of osteoblast-specific genes, such as ALP, runt-related transcription factor 2, type I collagen-α 1 and osteocalcin. Moreover, fucoidan induced the expression of bone morphogenetic protein 2 (BMP2) and stimulated the activation of extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase by increasing phosphorylation. However, the effect of fucoidan on osteogenic differentiation was inhibited by specific inhibitors of ERK (PD98059) and JNK (SP600125) but not p38 (SB203580). Fucoidan enhanced BMP2 expression and Smad 1/5/8, ERK and JNK phosphorylation. Moreover, the effect of fucoidan on osteoblast differentiation was diminished by BMP2 knockdown. These results indicate that fucoidan induces osteoblast differentiation through BMP2-Smad 1/5/8 signaling by activating ERK and JNK, elucidating the molecular basis of the osteogenic effects of fucoidan in hABM-MSCs.

  10. Regulation of FSHβ induction in LβT2 cells by BMP2 and an Activin A/BMP2 chimera, AB215.

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    Jung, Jae Woo; Ahn, Chihoon; Shim, Sun Young; Gray, Peter C; Kwiatkowski, Witek; Choe, Senyon

    2014-10-01

    Activins and bone morphogenetic proteins (BMPs) share activin type 2 signaling receptors but utilize different type 1 receptors and Smads. We designed AB215, a potent BMP2-like Activin A/BMP2 chimera incorporating the high-affinity type 2 receptor-binding epitope of Activin A. In this study, we compare the signaling properties of AB215 and BMP2 in HEK293T cells and gonadotroph LβT2 cells in which Activin A and BMP2 synergistically induce FSHβ. In HEK293T cells, AB215 is more potent than BMP2 and competitively blocks Activin A signaling, while BMP2 has a partial blocking activity. Activin A signaling is insensitive to BMP pathway antagonism in HEK293T cells but is strongly inhibited by constitutively active (CA) BMP type 1 receptors. By contrast, the potencies of AB215 and BMP2 are indistinguishable in LβT2 cells and although AB215 blocks Activin A signaling, BMP2 has no inhibitory effect. Unlike HEK293T, Activin A signaling is strongly inhibited by BMP pathway antagonism in LβT2 cells but is largely unaffected by CA BMP type 1 receptors. BMP2 increases phospho-Smad3 levels in LβT2 cells, in both the absence and the presence of Activin A treatment, and augments Activin A-induced FSHβ. AB215 has the opposite effect and sharply decreases basal phospho-Smad3 levels and blocks Smad2 phosphorylation and FSHβ induction resulting from Activin A treatment. These findings together demonstrate that while AB215 activates the BMP pathway, it has opposing effects to those of BMP2 on FSHβ induction in LβT2 cells apparently due to its ability to block Activin A signaling.

  11. 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.

  12. BMP-SHH signaling network controls epithelial stem cell fate via regulation of its niche in the developing tooth.

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    Li, Jingyuan; Feng, Jifan; Liu, Yang; Ho, Thach-Vu; Grimes, Weston; Ho, Hoang Anh; Park, Shery; Wang, Songlin; Chai, Yang

    2015-04-20

    During embryogenesis, ectodermal stem cells adopt different fates and form diverse ectodermal organs, such as teeth, hair follicles, mammary glands, and salivary glands. Interestingly, these ectodermal organs differ in their tissue homeostasis, which leads to differential abilities for continuous growth postnatally. Mouse molars lose the ability to grow continuously, whereas incisors retain this ability. In this study, we found that a BMP-Smad4-SHH-Gli1 signaling network may provide a niche supporting transient Sox2+ dental epithelial stem cells in mouse molars. This mechanism also plays a role in continuously growing mouse incisors. The differential fate of epithelial stem cells in mouse molars and incisors is controlled by this BMP/SHH signaling network, which partially accounts for the different postnatal growth potential of molars and incisors. Collectively, our study highlights the importance of crosstalk between two signaling pathways, BMP and SHH, in regulating the fate of epithelial stem cells during organogenesis.

  13. Twisted gastrulation, a BMP antagonist, exacerbates podocyte injury.

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    Sachiko Yamada

    Full Text Available Podocyte injury is the first step in the progression of glomerulosclerosis. Previous studies have demonstrated the beneficial effect of bone morphogenetic protein 7 (Bmp7 in podocyte injury and the existence of native Bmp signaling in podocytes. Local activity of Bmp7 is controlled by cell-type specific Bmp antagonists, which inhibit the binding of Bmp7 to its receptors. Here we show that the product of Twisted gastrulation (Twsg1, a Bmp antagonist, is the central negative regulator of Bmp function in podocytes and that Twsg1 null mice are resistant to podocyte injury. Twsg1 was the most abundant Bmp antagonist in murine cultured podocytes. The administration of Bmp induced podocyte differentiation through Smad signaling, whereas the simultaneous administration of Twsg1 antagonized the effect. The administration of Bmp also inhibited podocyte proliferation, whereas simultaneous administration of Twsg1 antagonized the effect. Twsg1 was expressed in the glomerular parietal cells (PECs and distal nephron of the healthy kidney, and additionally in damaged glomerular cells in a murine model of podocyte injury. Twsg1 null mice exhibited milder hypoalbuminemia and hyperlipidemia, and milder histological changes while maintaining the expression of podocyte markers during podocyte injury model. Taken together, our results show that Twsg1 plays a critical role in the modulation of protective action of Bmp7 on podocytes, and that inhibition of Twsg1 is a promising means of development of novel treatment for podocyte injury.

  14. Reciprocal repression between Fgf8 and miR-133 regulates cardiac induction through Bmp2 signaling

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    Carmen Lopez-Sanchez

    2015-12-01

    Full Text Available This data article contains complementary figures and results related to the research article entitled “Negative Fgf8-Bmp2 feed-back is controlled by miR-130 during early cardiac specification” [15], which reveals what specific role miR-130 plays during the cardiac induction process. This study evidenced miR-130 a putative microRNA that targets Erk1/2 (Mapk1 3′UTR- as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2. Thus, miR-130 regulates a negative Fgf8-Bmp2 feed-back loop responsible to achieve early cardiac specification. A significant aspect supporting our conclusions is given by the expression pattern of miR-130 during early cardiac specification, as well as by those results obtained after the designed experimental procedures. The data presented here reveal that miR-133 is also expressed within the precardiac areas during early cardiogenesis, pattern which is comparable to that of FGFR1, receptor involved in the Fgf8/ERK signaling pathway. Interestingly, our miR-133 overexpression experiments resulted in a decrease of Fgf8 expression, whereas we observed an increase of Bmp2 and subsequently of cardiac specific markers Nkx-2.5 and Gata4. Additionally, our loss-of-function experiments -through Fgf8 siRNA electroporation- showed an increase of miR-133 expression. Finally, after our Bmp2 experiments, we observed that miR-133 is upstream-regulated by Bmp2. All those results suggest that miR-133 also constitutes a crucial linkage in the crosstalk between Fgf8 and Bmp2 signaling by regulating the Fgf8/ERK pathway during cardiac induction.

  15. Reciprocal repression between Fgf8 and miR-133 regulates cardiac induction through Bmp2 signaling.

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    Lopez-Sanchez, Carmen; Franco, Diego; Bonet, Fernando; Garcia-Lopez, Virginio; Aranega, Amelia; Garcia-Martinez, Virginio

    2015-12-01

    This data article contains complementary figures and results related to the research article entitled "Negative Fgf8-Bmp2 feed-back is controlled by miR-130 during early cardiac specification" [15], which reveals what specific role miR-130 plays during the cardiac induction process. This study evidenced miR-130 a putative microRNA that targets Erk1/2 (Mapk1) 3'UTR- as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2. Thus, miR-130 regulates a negative Fgf8-Bmp2 feed-back loop responsible to achieve early cardiac specification. A significant aspect supporting our conclusions is given by the expression pattern of miR-130 during early cardiac specification, as well as by those results obtained after the designed experimental procedures. The data presented here reveal that miR-133 is also expressed within the precardiac areas during early cardiogenesis, pattern which is comparable to that of FGFR1, receptor involved in the Fgf8/ERK signaling pathway. Interestingly, our miR-133 overexpression experiments resulted in a decrease of Fgf8 expression, whereas we observed an increase of Bmp2 and subsequently of cardiac specific markers Nkx-2.5 and Gata4. Additionally, our loss-of-function experiments -through Fgf8 siRNA electroporation- showed an increase of miR-133 expression. Finally, after our Bmp2 experiments, we observed that miR-133 is upstream-regulated by Bmp2. All those results suggest that miR-133 also constitutes a crucial linkage in the crosstalk between Fgf8 and Bmp2 signaling by regulating the Fgf8/ERK pathway during cardiac induction.

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

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    Si, Lina; Shi, Jin; Gao, Wenqun; Zheng, Min; Liu, Lingjuan; Zhu, Jing; Tian, Jie

    2014-07-18

    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 GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.

  17. Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.

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    Robbie D Schultz

    Full Text Available In mammals, Bone Morphogenetic Protein (BMP pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.

  18. 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

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

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    Lapraz, François; Besnardeau, Lydia; Lepage, Thierry

    2009-11-01

    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, we show that

  20. BMP-2 up-regulates PTEN expression and induces apoptosis of pulmonary artery smooth muscle cells under hypoxia.

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    Weifeng Pi

    Full Text Available AIM: To investigate the role of bone morphogenetic protein 2 (BMP-2 in regulation of phosphatase and tensin homologue deleted on chromosome ten (PTEN and apoptosis of pulmonary artery smooth muscle cells (PASMCs under hypoxia. METHODS: Normal human PASMCs were cultured in growth medium (GM and treated with BMP-2 from 5-80 ng/ml under hypoxia (5% CO(2+94% N(2+1% O(2 for 72 hours. Gene expression of PTEN, AKT-1 and AKT-2 were determined by quantitative RT-PCR (QRT-PCR. Protein expression levels of PTEN, AKT and phosph-AKT (pAKT were determined. Apoptosis of PASMCs were determined by measuring activities of caspases-3, -8 and -9. siRNA-smad-4, bpV(HOpic (PTEN inhibitor and GW9662 (PPARγ antagonist were used to determine the signalling pathways. RESULTS: Proliferation of PASMCs showed dose dependence of BMP-2, the lowest proliferation rate was achieved at 60 ng/ml concentration under hypoxia (82.2±2.8%. BMP-2 increased PTEN gene expression level, while AKT-1 and AKT-2 did not change. Consistently, the PTEN protein expression also showed dose dependence of BMP-2. AKT activity significantly reduced in BMP-2 treated PASMCs. Increased activities of caspase-3, -8 and -9 of PASMCs were found after cultured with BMP-2. PTEN expression remained unchanged when Smad-4 expression was inhibited by siRNA-Smad-4. bpV(HOpic and GW9662 (PPARγ inhibitor inhibited PTEN protein expression and recovered PASMCs proliferation rate. CONCLUSION: BMP-2 increased PTEN expression under hypoxia in a dose dependent pattern. BMP-2 reduced AKT activity and increased caspase activity of PASMCs under hypoxia. The increased PTEN expression may be mediated through PPARγ signalling pathway, instead of BMP/Smad signalling pathway.

  1. Antagonistic signals between BMP4 and FGF8 define the expression of Pitx1 and Pitx2 in mouse tooth-forming anlage.

    Science.gov (United States)

    St Amand, T R; Zhang, Y; Semina, E V; Zhao, X; Hu, Y; Nguyen, L; Murray, J C; Chen, Y

    2000-01-15

    Members of the Pitx/RIEG family of homeodomain-containing transcription factors have been implicated in vertebrate organogenesis. In this study, we examined the expression and regulation of Pitx1 and Pitx2 during mouse tooth development. Pitx1 expression is detected in early development in a widespread pattern, in both epithelium and mesenchyme, covering the tooth-forming region in the mandible, and is then maintained in the dental epithelium from the bud stage to the late bell stage. Pitx2 expression, on the other hand, is restricted to the dental epithelium throughout odontogenesis. Interestingly, from E9.5 to E10.5, the expression domains of Pitx1 and Pitx2, in the developing mandible, overlap with that of Fgf8 but are exclusive to the zone of Bmp4 expression. Bead implantation experiments demonstrate that ectopic expression of Fgf8 can induce/maintain the expression of both Pitx1 and Pitx2 at E9.5. In contrast, Bmp4-expressing tissues and BMP4-soaked beads were able to repress Pitx1 expression in mandibular mesenchyme and Pitx2 expression in the presumptive dental epithelium, respectively. However, the effects of FGF8 and BMP4 are transient. It thus appears that the early expression patterns of Pitx1 and Pitx2 in the developing mandible are regulated by the antagonistic effects of FGF8 and BMP4 such that the Pitx1 and Pitx2 expression patterns are defined. These results indicate that the epithelial-derived signaling molecules are responsible not only for restricting specific gene expression in the dental mesenchyme, but also for defining gene expression in the dental epithelium.

  2. TGF-β and iron differently alter HBV replication in human hepatocytes through TGF-β/BMP signaling and cellular microRNA expression.

    Directory of Open Access Journals (Sweden)

    Sun O Park

    Full Text Available The nature of host-virus interactions in hepatitis B virus infection is incompletely understood. Since soluble factors, e.g., cytokines and metals, may exacerbate liver injury in chronic hepatitis, we considered that defining the effects of receptor-mediated signaling upon viral replication will be significant. Consequently, we studied effects of iron or TGF-β-induced TGF-β/BMP signaling in the HepG2 2.2.15 cell model of hepatitis B virus replication. We found iron and TGF-β increased hepcidin mRNA expression or TGF-β receptor kinase activity, respectively, which indicated that 2.2.15 cells responded appropriately to these substances. However, iron increased but TGF-β decreased hepatitis B virus mRNA and DNA expression. TGF-β induced expression at the mRNA level of multiple TGF-β/BMP pathway genes. This change was not observed in iron-treated cells. On the other hand, presence of SMAD proteins in iron or TGF-β-treated cells, including of SMAD4, did confirm convergence of TGF-β/BMP signaling pathways under these conditions. Since transcription factors in TGF-β/BMP signaling pathways could not have directly targeted hepatitis B virus itself, we studied whether iron or TGF-β exerted their effects through alternative mechanisms, such as by involvement of antiviral cellular microRNAs. We discovered cellular microRNA expression profiles were significantly different in iron or TGF-β-treated cells compared with untreated control cells. In many cases, exposure to iron or TGF-β changed microRNA expression in opposite directions. Introduction in cells of sequences representing such differentially expressed microRNAs, e.g., hsa-miR-125a-5p and -151-5p, even reproduced effects on virus replication of iron- or TGF-β. We surmised that TGF-β/BMP pathway members, i.e., SMADs, likely governed iron or TGF-β-induced microRNA expression. Iron may have mediated Drosha/DGCR8/heme-mediated processing of microRNAs. In turn, cellular microRNAs regulated

  3. BST2 Mediates Osteoblast Differentiation via the BMP2 Signaling Pathway in Human Alveolar-Derived Bone Marrow Stromal Cells.

    Science.gov (United States)

    Yoo, Su-Hyang; Kim, Jae Goo; Kim, Beom-Su; Lee, Jun; Pi, Sung-Hee; Lim, Hyun-Dae; Shin, Hong-In; Cho, Eui-Sic; You, Hyung-Keun

    2016-01-01

    The molecular mechanisms controlling the differentiation of bone marrow stromal stem cells into osteoblasts remain largely unknown. In this study, we investigated whether bone marrow stromal antigen 2 (BST2) influences differentiation toward the osteoblasts lineage. BST2 mRNA expression in human alveolar-derived bone marrow stromal cells (hAD-BMSCs) increased during differentiation into osteoblasts. hAD-BMSCs differentiation into osteoblasts and the mRNA expression of the bone-specific markers alkaline phosphatase, collagen type α 1, bone sialoprotein, osteocalcin, and osterix were reduced by BST2 knockdown using siRNA. Furthermore, BST2 knockdown in hAD-BMSCs resulted in decreased RUNX2 mRNA and protein expression. We hypothesized that BST2 is involved in differentiation of into osteoblasts via the BMP2 signaling pathway. Accordingly, we evaluated the mRNA expression levels of BMP2, BMP receptors (BMPR1 and 2), and the downstream signaling molecules SMAD1, SMAD4, and p-SMAD1/5/8 in BST2 knockdown cells. BMP2 expression following the induction of differentiation was significantly lower in BST2 knockdown cells than in cells treated with a non-targeting control siRNA. Similar results were found for the knockdown of the BMP2 receptor- BMPR1A. We also identified significantly lower expression of SMAD1, SMAD4, and p-SMAD1/5/8 in the BST2 knockdown cells than control cells. Our data provide the first evidence that BST2 is involved in the osteogenic differentiation of bone marrow stromal cells via the regulation of the BMP2 signaling pathway.

  4. Inducible chondrocyte-specific overexpression of BMP2 in young mice results in severe aggravation of osteophyte formation in experimental OA without altering cartilage damage

    NARCIS (Netherlands)

    Davidson, E.N.; Vitters, E.L.; Bennink, M.B.; Lent, P.L. van; Caam, A.P. van; Blom, A.B.; Berg, W.B. van den; Loo, F.A.J. van de; Kraan, P.M. van der

    2015-01-01

    OBJECTIVES: In osteoarthritis (OA) chondrocytes surrounding lesions express elevated bone morphogenetic protein 2 (BMP2) levels. To investigate the functional consequence of chondrocyte-specific BMP2 expression, we made a collagen type II dependent, doxycycline (dox)-inducible BMP2 transgenic mouse

  5. Metallofullerene nanoparticles promote osteogenic differentiation of bone marrow stromal cells through BMP signaling pathway

    Science.gov (United States)

    Yang, Kangning; Cao, Weipeng; Hao, Xiaohong; Xue, Xue; Zhao, Jing; Liu, Juan; Zhao, Yuliang; Meng, Jie; Sun, Baoyun; Zhang, Jinchao; Liang, Xing-Jie

    2013-01-01

    Although endohedral metallofullerenol [Gd@C82(OH)22]n nanoparticles have anti-tumor efficiency and mostly deposit in the bones of mice, how these nanoparticles act in bone marrow stromal cells (MSCs) remains largely unknown. Herein, we observed that [Gd@C82(OH)22]n nanoparticles facilitated the differentiation of MSCs toward osteoblasts, as evidenced by the enhancement of alkaline phosphatase (ALP) activity and mineralized nodule formation upon [Gd@C82(OH)22]n nanoparticle treatment. Mechanistically, the effect of [Gd@C82(OH)22]n nanoparticles on ALP activity was inhibited by the addition of noggin as an inhibitor of the BMP signaling pathway. Moreover, the in vivo results of the ovariectomized rats further indicated that [Gd@C82(OH)22]n nanoparticles effectively improved bone density and prevented osteoporosis.Although endohedral metallofullerenol [Gd@C82(OH)22]n nanoparticles have anti-tumor efficiency and mostly deposit in the bones of mice, how these nanoparticles act in bone marrow stromal cells (MSCs) remains largely unknown. Herein, we observed that [Gd@C82(OH)22]n nanoparticles facilitated the differentiation of MSCs toward osteoblasts, as evidenced by the enhancement of alkaline phosphatase (ALP) activity and mineralized nodule formation upon [Gd@C82(OH)22]n nanoparticle treatment. Mechanistically, the effect of [Gd@C82(OH)22]n nanoparticles on ALP activity was inhibited by the addition of noggin as an inhibitor of the BMP signaling pathway. Moreover, the in vivo results of the ovariectomized rats further indicated that [Gd@C82(OH)22]n nanoparticles effectively improved bone density and prevented osteoporosis. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr33575a

  6. Smurf-mediated differential proteolysis generates dynamic BMP signaling in germline stem cells during Drosophila testis development.

    Science.gov (United States)

    Chang, Yi-Jie; Pi, Haiwei; Hsieh, Chang-Che; Fuller, Margaret T

    2013-11-01

    Germline stem cells (GSCs) produce gametes throughout the reproductive life of many animals, and intensive studies have revealed critical roles of BMP signaling to maintain GSC self-renewal in Drospophila adult gonads. Here, we show that BMP signaling is downregulated as testes develop and this regulation controls testis growth, stem cell number, and the number of spermatogonia divisions. Phosphorylated Mad (pMad), the activated Drosophila Smad in germ cells, was restricted from anterior germ cells to GSCs and hub-proximal cells during early larval development. pMad levels in GSCs were then dramatically downregulated from early third larval instar (L3) to late L3, and maintained at low levels in pupal and adult GSCs. The spatial restriction and temporal down-regulation of pMad, reflecting the germ cell response to BMP signaling activity, required action in germ cells of E3 ligase activity of HECT domain protein Smurf. Analyses of Smurf mutant testes and dosage-dependent genetic interaction between Smurf and mad indicated that pMad downregulation was required for both the normal decrease in stem cell number during testis maturation in the pupal stage, and for normal limit of four rounds of spermatogonia cell division for control of germ cell numbers and testis size. Smurf protein was expressed at a constant low level in GSCs and spermatogonia during development. Rescue experiments showed that expression of exogenous Smurf protein in early germ cells promoted pMad downregulation in GSCs in a stage-dependent but concentration-independent manner, suggesting that the competence of Smurf to attenuate response to BMP signaling may be regulated during development. Taken together, our work reveals a critical role for differential attenuation of the response to BMP signaling in GSCs and early germ cells for control of germ cell number and gonad growth during development.

  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. Bone morphogenetic protein 2 (BMP2) induces growth suppression and enhances chemosensitivity of human colon cancer cells

    DEFF Research Database (Denmark)

    Vishnubalaji, Radhakrishnan; Yue, Shijun; Alfayez, Musaad

    2016-01-01

    datasets revealed significant downregulation of BMP2 in metastatic recurrent compared to non-metastatic cancer (p = 0.02). Global gene expression analysis in CRC cells over-expressing BMP2 revealed multiple dysregulated pathways mostly affecting cell cycle and DNA damage response. Concordantly, lentiviral......BACKGROUND: Molecular profiling of colorectal cancer (CRC) based on global gene expression has revealed multiple dysregulated signalling pathways associated with drug resistance and poor prognosis. However, the role of BMP2 signaling in CRC is not fully characterised. METHODS: Bioinformatics data...... expression were assessed using qRT-PCR. AlamarBlue assay was used to assess cell viability in vitro. In vivo experiments were conducted using SCID mice. RESULTS: Our data revealed frequent downregulation of BMP2 in primary CRC tissues. Additionally, interrogation of publically available gene expression...

  9. Effect of grafting BMP2-derived peptide to nanoparticles on osteogenic and vasculogenic expression of stromal cells.

    Science.gov (United States)

    Mercado, Angel E; Yang, Xiaoming; He, Xuezhong; Jabbari, Esmaiel

    2014-01-01

    Bone morphogenetic protein-2 (BMP2) plays a major role in initiating the cascade of osteogenesis. However, high doses of exogenous BMP2 coupled with diffusion away from the intended site cause adverse side-effects. An alternative is to use biodegradable polymeric nanoparticles (NPs) grafted with peptides of the active domains of BMP2. NPs present a multivalent form of the peptide for stronger interaction with cell surface receptors, leading to a stronger activation of osteogenic signalling pathways. The objective of this work was to compare osteogenic activity of the BMP2 peptide (BMP2Pe), corresponding to residues 73-92 of BMP2 protein (BMP2Pr), grafted to biodegradable NPs with that of BMP2 protein (BMP2Pr). BMP2Pe was functionalized with a cysteine residue and grafted to poly(lactide fumarate) and poly(lactide-co-ethylene oxide fumarate) (PLAF/PLEOF) NPs via a thioether link. The calcium content of bone marrow stromal (BMS) cells cultured in osteogenic medium supplemented with BMP2 peptide/protein-grafted NPs (BMP2Pe-gNP and BMP2Pr-gNP) was slightly higher than other BMP2-treated groups, but all osteogenic groups showed similar levels of mineralization after 21 days. The expression pattern of master transcription factors Dlx5 and Runx2 indicated that BMP2 protein induced faster osteogenic signalling than the BMP peptide. The expression level of Osteopontin (OP), Osteocalcin (OC) and PECAM-1 in the NP-grafted BMP2 groups was significantly higher than those of ungrafted BMP2Pr and BMP2Pe groups, which may be due to a more effective presentation of the peptide/protein to cell surface receptors, thus leading to a stronger interaction of the peptide/protein with clustered cell surface receptors.

  10. Robust Generation of Cardiomyocytes from Human iPS Cells Requires Precise Modulation of BMP and WNT Signaling.

    Science.gov (United States)

    Kadari, Asifiqbal; Mekala, SubbaRao; Wagner, Nicole; Malan, Daniela; Köth, Jessica; Doll, Katharina; Stappert, Laura; Eckert, Daniela; Peitz, Michael; Matthes, Jan; Sasse, Philipp; Herzig, Stefan; Brüstle, Oliver; Ergün, Süleyman; Edenhofer, Frank

    2015-08-01

    Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

  11. Nanoparticulate Mineralized Collagen Scaffolds and BMP-9 Induce a Long-Term Bone Cartilage Construct in Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Ren, Xiaoyan; Weisgerber, Daniel W; Bischoff, David; Lewis, Michael S; Reid, Russell R; He, Tong-Chuan; Yamaguchi, Dean T; Miller, Timothy A; Harley, Brendan A C; Lee, Justine C

    2016-07-01

    Engineering the osteochondral junction requires fabrication of a microenvironment that supports both osteogenesis and chondrogenesis. Multiphasic scaffold strategies utilizing a combination of soluble factors and extracellular matrix components are ideally suited for such applications. In this work, the contribution of an osteogenic nanoparticulate mineralized glycosaminoglycan scaffold (MC-GAG) and a dually chondrogenic and osteogenic growth factor, BMP-9, in the differentiation of primary human mesenchymal stem cells (hMSCs) is evaluated. Although 2D cultures demonstrate alkaline phosphatase activity and mineralization of hMSCs induced by BMP-9, MC-GAG scaffolds do not demonstrate significant differences in the collagen I expression, osteopontin expression, or mineralization. Instead, BMP-9 increases expression of collagen II, Sox9, aggrecan (ACAN), and cartilage oligomeric protein. However, the hypertrophic chondrocyte marker, collagen X, is not elevated with BMP-9 treatment. In addition, histologic analyses demonstrate that while BMP-9 does not increase mineralization, BMP-9 treatment results in an increase of sulfated glycosaminoglycans. Thus, the combination of BMP-9 and MC-GAG stimulates chondrocytic and osteogenic differentiation of hMSCs.

  12. 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.

  13. BMP-Smad 1/5/8 signalling in the development of the nervous system.

    Science.gov (United States)

    Hegarty, Shane V; O'Keeffe, Gerard W; Sullivan, Aideen M

    2013-10-01

    The transcription factors, Smad1, Smad5 and Smad8, are the pivotal intracellular effectors of the bone morphogenetic protein (BMP) family of proteins. BMPs and their receptors are expressed in the nervous system (NS) throughout its development. This review focuses on the actions of Smad 1/5/8 in the developing NS. The mechanisms by which these Smad proteins regulate the induction of the neuroectoderm, the central nervous system (CNS) primordium, and finally the neural crest, which gives rise to the peripheral nervous system (PNS), are reviewed herein. We describe how, following neural tube closure, the most dorsal aspect of the tube becomes a signalling centre for BMPs, which directs the pattern of the development of the dorsal spinal cord (SC), through the action of Smad1, Smad5 and Smad8. The direct effects of Smad 1/5/8 signalling on the development of neuronal and non-neuronal cells from various neural progenitor cell populations are then described. Finally, this review discusses the neurodevelopmental abnormalities associated with the knockdown of Smad 1/5/8.

  14. BMP4 and FGF strongly induce differentiation of mouse ES cells into oral ectoderm

    Directory of Open Access Journals (Sweden)

    Hiroshi Ochiai

    2015-09-01

    Full Text Available During embryonic development, oral ectoderm differentiates into the adenohypophysis, dental epithelia, salivary glands, and nasal pit. Few reports exist concerning the induction of oral ectoderm from embryonic stem (ES cells. Generally, any lot differences in fetal bovine serum (FBS and serum replacer may affect the induction of ES cell-differentiation. Using a previously established culture strategy for differentiation, the proportion of cell aggregates containing Pitx1+ oral ectoderm varied widely between 9–36% when several different lots of FBS or serum replacer were used. We therefore tried to enhance the differentiation method. We found that bone morphogenetic protein (BMP 4 and fibroblast growth factor (FGF treatments improved oral ectoderm induction. Such treatment also improved the differentiation of oral ectoderm into the adenohypophysis. Furthermore, increased BMP4 treatment induced dental epithelium and mesenchyme. Such differentiation suggests that the Pitx1+ layer displays similar properties to oral ectoderm, as found in vivo. Differentiation of ES cells into oral ectoderm using different lots of FBS and serum replacer increased 78–90% after treatment with BMP4 and FGF. In summary, we have established a robust strategy for the induction of oral ectoderm differentiation from mouse ES cells.

  15. Chondrocyte BMP2 signaling plays an essential role in bone fracture healing.

    Science.gov (United States)

    Mi, Meng; Jin, Hongting; Wang, Baoli; Yukata, Kiminori; Sheu, Tzong-Jen; Ke, Qiao Han; Tong, Peijian; Im, Hee-Jeong; Xiao, Guozhi; Chen, Di

    2013-01-10

    The specific role of endogenous Bmp2 gene in chondrocytes and in osteoblasts in fracture healing was investigated by generation and analysis of chondrocyte- and osteoblast-specific Bmp2 conditional knockout (cKO) mice. The unilateral open transverse tibial fractures were created in these Bmp2 cKO mice. Bone fracture callus samples were collected and analyzed by X-ray, micro-CT, histology analyses, biomechanical testing and gene expression assays. The results demonstrated that the lack of Bmp2 expression in chondrocytes leads to a prolonged cartilage callus formation and a delayed osteogenesis initiation and progression into mineralization phase with lower biomechanical properties. In contrast, when the Bmp2 gene was deleted in osteoblasts, the mice showed no significant difference in the fracture healing process compared to control mice. These findings suggest that endogenous BMP2 expression in chondrocytes may play an essential role in cartilage callus maturation at an early stage of fracture healing. Our studies may provide important information for clinical application of BMP2.

  16. LRP4 association to bone properties and fracture and interaction with genes in the Wnt- and BMP signaling pathways.

    Science.gov (United States)

    Kumar, Jitender; Swanberg, Maria; McGuigan, Fiona; Callreus, Mattias; Gerdhem, Paul; Akesson, Kristina

    2011-09-01

    Osteoporosis is a common complex disorder in postmenopausal women leading to changes in the micro-architecture of bone and increased risk of fracture. Members of the low-density lipoprotein receptor-related protein (LRP) gene family regulates the development and physiology of bone through the Wnt/β-catenin (Wnt) pathway that in turn cross-talks with the bone morphogenetic protein (BMP) pathway. In two cohorts of Swedish women: OPRA (n=1002; age 75 years) and PEAK-25 (n=1005; age 25 years), eleven single nucleotide polymorphisms (SNPs) from Wnt pathway genes (LRP4; LRP5; G protein-coupled receptor 177, GPR177) were analyzed for association with Bone Mineral Density (BMD), rate of bone loss, hip geometry, quantitative ultrasound and fracture. Additionally, interaction of LRP4 with LRP5, GPR177 and BMP2 were analyzed. LRP4 (rs6485702) was associated with higher total body (TB) and lumbar spine (LS) BMD in the PEAK-25 cohort (p=0.006 and 0.005 respectively), and interaction was observed with LRP5 (p=0.007) and BMP2 (p=0.004) for TB BMD. LRP4 also showed significant interaction with LRP5 for femoral neck (FN) and LS BMD in this cohort. In the OPRA cohort, LRP4 polymorphisms were associated with significantly lower fracture incidence overall (p=0.008-0.001) and fewer hip fractures (rs3816614, p=0.006). Significant interaction in the OPRA cohort was observed for LRP4 with BMP2 and GPR177 for FN BMD as well as for rate of bone loss at TB and FN (p=0.007-0.0001). In conclusion, LRP4 and interaction between LRP4 and genes in the Wnt and BMP signaling pathways modulate bone phenotypes including peak bone mass and fracture, the clinical endpoint of osteoporosis.

  17. Intestinal Mucosal Barrier Is Injured by BMP2/4 via Activation of NF-κB Signals after Ischemic Reperfusion

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    Kang Chen

    2014-01-01

    Full Text Available Intestinal ischemic reperfusion (I/R can cause dysfunction of the intestinal mucosal barrier; however, the mechanism of the intestinal mucosal barrier dysfunction caused by I/R remains unclear. In this study, using intestinal epithelial cells under anaerobic cultivation and an in vivo rat intestinal I/R model, we found that hypoxia and I/R increased the expression of BMP2/4 and upregulated BMP type Ia receptor and BMP type II receptor expression. We also found that exogenous BMP2/4 can activate the ERK and AKT signaling pathways in rat small intestine (IEC-6 cells, thereby activating NF-κB signaling, which leads to increased levels of inflammatory factors, such as TNF-α and IL-6. Furthermore, recombinant BMP2/4 decreased the expression of the tight junction protein occludin via the activation of the NF-κB pathway; these effects were abolished by treatment with the BMP-specific antagonist noggin or the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC. All these factors can destroy the intestinal mucosal barrier, thereby leading to weaker barrier function. On the basis of these data, we conclude that BMP2/4 may act as the pathogenic basis for intestinal mucosal barrier dysfunction when the intestines suffer an I/R injury. Our results provide background for the development pharmacologic interventions in the management of I/R injury.

  18. The BMP Pathway Participates in Human Naive CD4+ T Cell Activation and Homeostasis.

    Directory of Open Access Journals (Sweden)

    Víctor G Martínez

    Full Text Available Bone Morphogenetic Proteins (BMPs form a group of secreted factors that belongs to the TGF-β superfamily. Among different roles in a number of immune cell types, BMPs are known to regulate T cell development within the thymus, although the role of BMP signaling in human mature T cells remains elusive. In this study, we demonstrate that canonical BMP signaling is necessary during two critical events that regulate the size and function of human naive CD4+ T cell population: activation and homeostasis. Upon stimulation via TCR, naive CD4+ T cells upregulate the expression of BMP ligands triggering canonical BMP signaling in CD25+ cells. Blockade of BMP signaling severely impairs CD4+ T cell proliferation after activation mainly through regulation of IL-2, since the addition of this cytokine recuperates normal T cell expansion after inhibition of BMP signaling. Similarly, activation of canonical BMP pathway is required for both the maintenance of cell survival and the homeostatic proliferation induced by IL-7, a key factor for T cell homeostasis. Moreover, upregulation of two critical receptors for T cell homeostasis, CXCR4 and CCR9, triggered by IL-7 is also abrogated in the absence of BMP signaling. Collectively, we describe important roles of the canonical BMP signaling in human naive CD4+ T cell activation and homeostasis that could be valuable for clinical application.

  19. Ror2 signaling is required for local upregulation of GDF6 and activation of BMP signaling at the neural plate border.

    Science.gov (United States)

    Schille, Carolin; Bayerlová, Michaela; Bleckmann, Annalen; Schambony, Alexandra

    2016-09-01

    The receptor tyrosine kinase Ror2 is a major Wnt receptor that activates β-catenin-independent signaling and plays a conserved role in the regulation of convergent extension movements and planar cell polarity in vertebrates. Mutations in the ROR2 gene cause recessive Robinow syndrome in humans, a short-limbed dwarfism associated with craniofacial malformations. Here, we show that Ror2 is required for local upregulation of gdf6 at the neural plate border in Xenopus embryos. Ror2 morphant embryos fail to upregulate neural plate border genes and show defects in the induction of neural crest cell fate. These embryos lack the spatially restricted activation of BMP signaling at the neural plate border at early neurula stages, which is required for neural crest induction. Ror2-dependent planar cell polarity signaling is required in the dorsolateral marginal zone during gastrulation indirectly to upregulate the BMP ligand Gdf6 at the neural plate border and Gdf6 is sufficient to rescue neural plate border specification in Ror2 morphant embryos. Thereby, Ror2 links Wnt/planar cell polarity signaling to BMP signaling in neural plate border specification and neural crest induction.

  20. Local delivery of siRNA using a biodegradable polymer application to enhance BMP-induced bone formation.

    Science.gov (United States)

    Manaka, Tomoya; Suzuki, Akinobu; Takayama, Kazushi; Imai, Yuuki; Nakamura, Hiroaki; Takaoka, Kunio

    2011-12-01

    Small interfering RNA (siRNA) is useful tool for specific and efficient knockdown of disease-related genes. However, in vivo applications of siRNA are limited due to difficulty in its efficient delivery to target cells. In this study, we investigated the efficacy of a biodegradable hydrogel, poly-d,l-lactic acid-p-dioxanone-polyethylene glycol block co-polymer (PLA-DX-PEG), as a siRNA carrier. PLA-DX-PEG pellets with or without fluorescein-labeled dsRNA were implanted into mouse dosal muscle pouches. The cellular uptake of dsRNA surround the polymer was confirmed by fluorescent microscopy. The fluorescence intensity was dose-dependent of the dsRNA, and exhibited a time-dependent decrease. To investigate its biological efficiency, noggin (antagonoist to BMPs) gene-silencing with siRNA (siRNA/Noggin) was examined by the amount of suppression of BMP-2-induced noggin expression and the level of performance of BMP, indicated by ectopic bone formation. Noggin gene expression induced by BMP-2 was suppressed by addition of siRNA/Noggin to the implant, and the ectopic bone formation induced by implants with both BMP-2 and siRNA/Noggin was significantly greater than those induced by implants with BMP-2 alone. These results indicate the efficacy of local delivery of siRNAs by PLA-DX-PEG polymer, which intensified bone-inducing effects of BMP and promoted new bone formation by suppressing gene expression of Noggin.

  1. Augmented BMPRIA-mediated BMP signaling in cranial neural crest lineage leads to cleft palate formation and delayed tooth differentiation.

    Directory of Open Access Journals (Sweden)

    Lu Li

    Full Text Available The importance of BMP receptor Ia (BMPRIa mediated signaling in the development of craniofacial organs, including the tooth and palate, has been well illuminated in several mouse models of loss of function, and by its mutations associated with juvenile polyposis syndrome and facial defects in humans. In this study, we took a gain-of-function approach to further address the role of BMPR-IA-mediated signaling in the mesenchymal compartment during tooth and palate development. We generated transgenic mice expressing a constitutively active form of BmprIa (caBmprIa in cranial neural crest (CNC cells that contributes to the dental and palatal mesenchyme. Mice bearing enhanced BMPRIa-mediated signaling in CNC cells exhibit complete cleft palate and delayed odontogenic differentiation. We showed that the cleft palate defect in the transgenic animals is attributed to an altered cell proliferation rate in the anterior palatal mesenchyme and to the delayed palatal elevation in the posterior portion associated with ectopic cartilage formation. Despite enhanced activity of BMP signaling in the dental mesenchyme, tooth development and patterning in transgenic mice appeared normal except delayed odontogenic differentiation. These data support the hypothesis that a finely tuned level of BMPRIa-mediated signaling is essential for normal palate and tooth development.

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

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    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

  3. Retinal and choroidal expression of BMP-2 in lens-induced myopia and recovery from myopia in guinea pigs.

    Science.gov (United States)

    Li, Honghui; Wu, Juan; Cui, Dongmei; Zeng, Junwen

    2016-03-01

    The present study investigated the retinal and choroidal expression of bone morphogenetic protein-2 (BMP-2) in myopia and in myopia recovery in a guinea pig model. For this investigation, two groups of guinea pigs, lens‑induced myopia and recovery from myopia, were used, and defocused myopia was induced the guinea pigs wearing ‑4.00 D lenses on the right eyes for 3 weeks, with the left eyes serving as the contralateral. In the following week, the lenses of the guinea pigs in the recovery group were removed, and the refractive power and axial length were measured. The expression of BMP‑2 in the eyeballs was observed using immunohistochemistry and analyzed using Western blot analysis. After 3 weeks, the eyes acquired relative myopia and longer axial lengths in the two groups of guinea pigs. After 1 week without lenses in the recovery group, the myopia and axial lengths regressed. Immunofluorescence staining showed that BMP‑2 was expressed in the posterior retina, RPE, choroid and sclera. The expression of BMP‑2 decreased in the myopic retina of the guinea pigs. Following the regression of myopia in the recovery group, no difference in the expression of BMP‑2 was observed between the recovered treated eyes and the contralateral eyes. The choroidal expression level of BMP‑2 in the treated eyes showed no significant changes in either group. Therefore, BMP‑2 may be involved in the development of myopia, however, it does not have a primary role in the retinal and choroidal signals regulating scleral remodeling.

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

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

    2012-11-13

    How dermal papilla (DP) niche cells regulate hair follicle progenitors to control hair growth remains unclear. Using Tbx18(Cre) 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 speed of differentiating hair shaft progenitors. Transcriptional profiling of Sox2 null DPs reveals increased Bmp6 and decreased BMP inhibitor Sostdc1, a direct Sox2 transcriptional target. Subsequently, we identify upregulated BMP signaling in knockout hair shaft progenitors and demonstrate that Bmp6 inhibits cell migration, an effect that can be attenuated by Sostdc1. A shorter and Sox2-negative hair type lacks Sostdc1 in the DP and shows reduced migration and increased BMP activity of hair shaft progenitors. Collectively, our data identify Sox2 as a key regulator of hair growth that controls progenitor migration by fine-tuning BMP-mediated mesenchymal-epithelial crosstalk.

  5. Extensive sequence turnover of the signal peptides of members of the GDF/BMP family: exploring their evolutionary landscape

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    Veitia Reiner A

    2009-07-01

    Full Text Available Abstract We show that the predicted signal peptide (SP sequences of the secreted factors GDF9, BMP15 and AMH are well conserved in mammals but dramatic divergence is noticed for more distant orthologs. Interestingly, bioinformatic predictions show that the divergent protein segments do encode SPs. Thus, such SPs have undergone extensive sequence turnover with full preservation of functionality. This can be explained by a pervasive accumulation of neutral and compensatory mutations. An exploration of the potential evolutionary landscape of some SPs is presented. Some of these signal sequences highlight an apparent paradox: they are encoded, by definition, by orthologous DNA segments but they are, given their striking divergence, examples of what can be called functional convergence. Reviewers: This article was reviewed by Fyodor Kondrashov and Eugene V. Koonin.

  6. BMP pathway regulation of and by macrophages.

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    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.

  7. Acute BMP2 upregulation following induction of ischemic osteonecrosis in immature femoral head.

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    Kamiya, Nobuhiro; Shafer, Sasha; Oxendine, Ila; Mortlock, Douglas P; Chandler, Ronald L; Oxburgh, Leif; Kim, Harry K W

    2013-03-01

    Juvenile ischemic osteonecrosis of the femoral head (IOFH) is one of the most serious hip conditions causing the femoral head deformity. Little is known about BMP signaling following ischemic osteonecrosis. In this study, we found acute BMP2 upregulation in the femoral head cartilage 24h after ischemic induction using our immature pig IOFH model. Similarly, in our ischemic osteonecrosis mouse model, BMP2 expression and BMP signaling were enhanced in the articular cartilage surrounding the necrotic bone. BMP2 was increased in cartilage explants and primary chondrocytes under hypoxia (1% O(2)) compared with normoxia (21% O(2)). Addition of the hypoxia inducible factor 1 (HIF1) activator DFO significantly increased BMP2 while HIF1 silencing (siHIF1) only partially reduced BMP2, suggesting other mechanisms of BMP2 upregulation being present. Hypoxia is known to induce the production of free oxygen radicals, which are converted to hydrogen peroxide (H(2)O(2)) by superoxide dismutase 2 (SOD2). As an alternative mechanism, we investigated the effect of H(2)O(2)/SOD2 production on BMP2 upregulation. Chondrocytes produced more H(2)O(2) under hypoxia than normoxia. H(2)O(2) addition to the chondrocyte culture also significantly increased BMP2 expression. SOD2 was also dramatically increased in the ischemic pig cartilage at 24h following surgery and in primary chondrocytes/cartilage explants culture under hypoxia. SOD2 protein addition to the chondrocyte culture significantly increased BMP2. Moreover, DFO significantly increased SOD2 while HIF1 silencing only partially reduced SOD2. These results suggest that the acute BMP2 response of chondrocytes to ischemic osteonecrosis is more dominantly through the H(2)O(2) production and only partly through the HIF1 pathway.

  8. Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists.

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    Alborzinia, Hamed; Schmidt-Glenewinkel, Hannah; Ilkavets, Iryna; Breitkopf-Heinlein, Katja; Cheng, Xinlai; Hortschansky, Peter; Dooley, Steven; Wölfl, Stefan

    2013-01-01

    Bone morphogenetic proteins (BMPs) are members of the TGFβ family of signaling proteins and play an important role during development and in tissue formation. BMP signaling is a well-studied process, which is initiated through binding of cognate receptors and processed through activation of Smad downstream mediators. A hallmark of BMP signaling is its modulation at the extracellular level through specific antagonists. Although it had been shown that BMP and TGFβ receptors are internalized following activation, little is known about the fate of BMP ligands. We prepared biologically active fluorescently labeled BMP2 and quantitatively analyzed its binding and uptake in cells using flow cytometry and confocal microscopy. Exogenous BMP2 was rapidly bound to the cell surface and subsequently internalized in a time-dependent manner and accumulated in the cell center. Although binding to the cell surface was limited by binding sites at the beginning, internalization continously increased with time, after a short delay. Using different inhibitors we found that internalization of BMP2 through endosomal particles occurred in a clathrin-dependent pathway. Furthermore, uptake of BMP2 was modulated in strikingly different ways by BMP2 antagonists. Although Noggin and Gremlin increased BMP2 uptake, Chordin blocked BMP2 uptake, which was concentration dependent in both cases. In conclusion, our findings present interesting mechanisms for the modulation of BMP signaling by concentration gradients of BMP ligands and antagonists in a dose- and time-dependent manner, which can provide an explanation of some properties of the BMP regulatory network.

  9. The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster.

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    Justin Schleede

    2015-10-01

    Full Text Available The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog. However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues.

  10. AcvR1-mediated BMP signaling in second heart field is required for arterial pole development: implications for myocardial differentiation and regional identity.

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    Thomas, Penny S; Rajderkar, Sudha; Lane, Jamie; Mishina, Yuji; Kaartinen, Vesa

    2014-06-15

    BMP signaling plays an essential role in second heart field-derived heart and arterial trunk development, including myocardial differentiation, right ventricular growth, and interventricular, outflow tract and aortico-pulmonary septation. It is mediated by a number of different BMP ligands, and receptors, many of which are present simultaneously. The mechanisms by which they regulate morphogenetic events and degree of redundancy amongst them have still to be elucidated. We therefore assessed the role of BMP Type I receptor AcvR1 in anterior second heart field-derived cell development, and compared it with that of BmpR1a. By removing Acvr1 using the driver Mef2c[AHF]-Cre, we show that AcvR1 plays an essential role in arterial pole morphogenesis, identifying defects in outflow tract wall and cushion morphology that preceded a spectrum of septation defects from double outlet right ventricle to common arterial trunk in mutants. Its absence caused dysregulation in gene expression important for myocardial differentiation (Isl1, Fgf8) and regional identity (Tbx2, Tbx3, Tbx20, Tgfb2). Although these defects resemble to some degree those in the equivalent Bmpr1a mutant, a novel gene knock-in model in which Bmpr1a was expressed in the Acvr1 locus only partially restored septation in Acvr1 mutants. These data show that both BmpR1a and AcvR1 are needed for normal heart development, in which they play some non-redundant roles, and refine our understanding of the genetic and morphogenetic processes underlying Bmp-mediated heart development important in human congenital heart disease.

  11. Potential Roles of Bone Morphogenetic Protein (BMP-9 in Human Liver Diseases

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    Blanca Herrera

    2014-03-01

    Full Text Available Bone morphogenetic proteins (BMP-2 to BMP-15 belong to the Transforming Growth Factor (TGF-β superfamily and, besides their well-documented roles during embryogenesis and bone formation, some of them have recently been described to be involved in the pathogenesis of different organs, including the liver. The role of BMPs in liver damage responses including hepatocellular carcinoma (HCC development has only begun to be addressed and strong evidence supports the concept of a pro-tumorigenic role of BMP signaling in HCC cells. BMP-9 (also termed Growth and Differentiation Factor (GDF-2 represents the most recently discovered member of the BMP family. We have previously demonstrated that in HCC patient samples BMP-9 expression was positively associated with the tumor seize (“T stage” and that it enhanced cell migration and induced epithelial to mesenchymal transition (EMT in HCC cells in vitro. In another study we recently found that BMP-9 promotes growth in HCC cells, but not in non-transformed hepatocytes. Published as well as unpublished results obtained with primary hepatocytes support the concept of a dual function of BMP-9 in the liver: while in primary, non-malignant cells BMP-9 stabilizes the epithelial phenotype and inhibits proliferation, in HCC cells it induces cell growth and the acquisition of a migratory phenotype. In this review article we summarize current knowledge about BMPs in liver diseases, with special focus on the role of BMP-9 in HCC development and progression, that may provide new clues for a better understanding of the contribution of BMP-signaling to chronic liver diseases.

  12. Potential Roles of Bone Morphogenetic Protein (BMP)-9 in Human Liver Diseases

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    Herrera, Blanca; Dooley, Steven; Breitkopf-Heinlein, Katja

    2014-01-01

    Bone morphogenetic proteins (BMP-2 to BMP-15) belong to the Transforming Growth Factor (TGF)-β superfamily and, besides their well-documented roles during embryogenesis and bone formation, some of them have recently been described to be involved in the pathogenesis of different organs, including the liver. The role of BMPs in liver damage responses including hepatocellular carcinoma (HCC) development has only begun to be addressed and strong evidence supports the concept of a pro-tumorigenic role of BMP signaling in HCC cells. BMP-9 (also termed Growth and Differentiation Factor (GDF)-2) represents the most recently discovered member of the BMP family. We have previously demonstrated that in HCC patient samples BMP-9 expression was positively associated with the tumor seize (“T stage”) and that it enhanced cell migration and induced epithelial to mesenchymal transition (EMT) in HCC cells in vitro. In another study we recently found that BMP-9 promotes growth in HCC cells, but not in non-transformed hepatocytes. Published as well as unpublished results obtained with primary hepatocytes support the concept of a dual function of BMP-9 in the liver: while in primary, non-malignant cells BMP-9 stabilizes the epithelial phenotype and inhibits proliferation, in HCC cells it induces cell growth and the acquisition of a migratory phenotype. In this review article we summarize current knowledge about BMPs in liver diseases, with special focus on the role of BMP-9 in HCC development and progression, that may provide new clues for a better understanding of the contribution of BMP-signaling to chronic liver diseases. PMID:24670474

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

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    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.

  14. The BMP2 antagonist inhibitor L51P enhances the osteogenic potential of BMP2 by simultaneous and delayed synergism.

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    Khattab, Hany Mohamed; Ono, Mitsuaki; Sonoyama, Wataru; Oida, Yasutaka; Shinkawa, Shigehiko; Yoshioka, Yuya; Maekawa, Kenji; Tabata, Yasuhiko; Sugama, Kazushige; Sebald, Walter; Kuboki, Takuo

    2014-12-01

    Bone morphogenetic protein 2 (BMP2) is a potent osteoinductive cytokine that plays crucial roles in bone repair. However, large amounts of BMP2 are required to induce sufficient bone formation in humans possibly due to a feedback response of BMP antagonists. The engineered BMP2 variant L51P is deficient in BMP receptor type I activation but maintains affinity for BMP antagonists and can allow for the inactivation of BMP antagonists, and eventually enhance BMP2 action. As hypothesized, simultaneous addition of L51P enhanced the BMP2-induced osteogenesis. To test the ability of L51P to competitively inactivate BMP antagonists, cell binding affinity of BMP2 ligands was investigated in the presence or absence of L51P. Because the BMP antagonists were highly expressed 3 days after exogenous BMP2 stimulation, we collected supernatants from 3-day stimulated cell cultures and used as condition culture media (CM). The results showed a significant decrease in the cell binding of BMP2 ligands when cells were incubated with exogenous BMP2 and CM, whereas L51P addition competitively rescued the suppression of BMP2-to-cell binding induced by CM incubation. In a delayed experimental model, L51P was applied 3 days after exogenous BMP2 stimulation and we could observe a striking enhancement of the BMP2-induced SMAD-1/5/8 phosphorylation and luciferase activity of the Id1 promoter compared to the simultaneous addition of the two factors. These findings provide a deeper insight into the cellular and molecular mechanisms involved in the effect of L51P in suppressing the BMP antagonists and enhancing BMP activity. Additionally, these results demonstrate that L51P is a promising down regulator of BMP-induced negative feedback, which could have a significant impact in future applications of BMP2 in research and clinical settings.

  15. Shh signaling, negatively regulated by BMP signaling, inhibits the osteo/dentinogenic differentiation potentials of mesenchymal stem cells from apical papilla.

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    Jiang, Qingsong; Du, Juan; Yin, Xiaonan; Shan, Zhaochen; Ma, Yushi; Ma, Ping; Du, Juan; Fan, Zhipeng

    2013-11-01

    Mesenchymal stem cells (MSCs) derived from dental tissues show promise for use in tooth-related tissue regeneration, but the molecular mechanisms underlying their directed differentiation remain unclear, limiting their usefulness. Sonic Hedgehog (Shh) signaling is a major signaling pathway that regulates cell differentiation and osteogenesis. We found that when Shh signaling was activated by human recombinant SHH-N protein or by overexpression of active mutant M2-Smoothened (SMO) in stem cells from apical papilla (SCAPs), GLI1, a key downstream transcription factor and a marker of Shh signaling, was upregulated. Subsequently, in vitro osteo/dentinogenic differentiation and in vivo osteogenesis were inhibited in SCAPs. Moreover, the expression of GLI1 and SMO were downregulated by BMP signaling while osteo/dentinogenic differentiation in SCAPs was upregulated. These results provide insights into the role of Shh signaling in the directed differentiation of MSCs derived from dental tissues and suggest possible target genes for optimizing the use of stem cells of dental origin for tissue regeneration applications.

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

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    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 (pBMP2 at doses of 50ng/ml, 100ng/ml and 200ng/ml. A significant upregulation of ALP gene in BMP2 treated cells was seen compared to HUMSCs treated in osteogenic medium (pBMP2 dose of

  17. 民猪BMP2基因的冷诱导研究%Expression of BMP2 in Min Pig during Cold Induced

    Institute of Scientific and Technical Information of China (English)

    张冬杰

    2010-01-01

    以75日龄民猪为试验材料,将骨形成蛋白2(BMP2)基因作为影响民猪抗寒特性的候选基因,对其在低温冷诱导后在民猪肌肉组织内的表达变化情况进行了分析.结果表明:BMP2基因在民猪冷诱导前后表达水平没有显著变化.

  18. YAP stabilizes SMAD1 and promotes BMP2-induced neocortical astrocytic differentiation.

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    Huang, Zhihui; Hu, Jinxia; Pan, Jinxiu; Wang, Ying; Hu, Guoqing; Zhou, Jiliang; Mei, Lin; Xiong, Wen-Cheng

    2016-07-01

    ‪YAP (yes-associated protein), a key transcriptional co-factor that is negatively regulated by the Hippo pathway, is crucial for the development and size control of multiple organs, including the liver. However, its role in the brain remains unclear. Here, we provide evidence for YAP regulation of mouse neocortical astrocytic differentiation and proliferation. YAP was undetectable in neurons, but selectively expressed in neural stem cells (NSCs) and astrocytes. YAP in NSCs was required for neocortical astrocytic differentiation, with no apparent role in self-renewal or neural differentiation. However, YAP in astrocytes was necessary for astrocytic proliferation. Yap (Yap1) knockout, Yap(nestin) conditional knockout and Yap(GFAP) conditional knockout mice displayed fewer neocortical astrocytes and impaired astrocytic proliferation and, consequently, death of neocortical neurons. Mechanistically, YAP was activated by BMP2, and the active/nuclear YAP was crucial for BMP2 induction and stabilization of SMAD1 and astrocytic differentiation. Expression of SMAD1 in YAP-deficient NSCs partially rescued the astrocytic differentiation deficit in response to BMP2. Taken together, these results identify a novel function of YAP in neocortical astrocytic differentiation and proliferation, and reveal a BMP2-YAP-SMAD1 pathway underlying astrocytic differentiation in the developing mouse neocortex.

  19. Bmp4 from the optic vesicle specifies murine retina formation.

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    Huang, Jie; Liu, Ying; Oltean, Alina; Beebe, David C

    2015-06-01

    Previous studies of mouse embryos concluded that after the optic vesicle evaginates from the ventral forebrain and contacts the surface ectoderm, signals from the ectoderm specify the distal region of the optic vesicle to become retina and signals from the optic vesicle induce the lens. Germline deletion of Bmp4 resulted in failure of lens formation. We performed conditional deletion of Bmp4 from the optic vesicle to test the function of Bmp4 in murine eye development. The optic vesicle evaginated normally and contacted the surface ectoderm. Lens induction did not occur. The optic cup failed to form and the expression of retina-specific genes decreased markedly in the distal optic vesicle. Instead, cells in the prospective retina expressed genes characteristic of the retinal pigmented epithelium. We conclude that Bmp4 is required for retina specification in mice. In the absence of Bmp4, formation of the retinal pigmented epithelium is the default differentiation pathway of the optic vesicle. Differences in the signaling pathways required for specification of the retina and retinal pigmented epithelium in chicken and mouse embryos suggest major changes in signaling during the evolution of the vertebrate eye.

  20. Endocardial to myocardial notch-wnt-bmp axis regulates early heart valve development.

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    Yidong Wang

    Full Text Available Endocardial to mesenchymal transformation (EMT is a fundamental cellular process required for heart valve formation. Notch, Wnt and Bmp pathways are known to regulate this process. To further address how these pathways coordinate in the process, we specifically disrupted Notch1 or Jagged1 in the endocardium of mouse embryonic hearts and showed that Jagged1-Notch1 signaling in the endocardium is essential for EMT and early valvular cushion formation. qPCR and RNA in situ hybridization assays reveal that endocardial Jagged1-Notch1 signaling regulates Wnt4 expression in the atrioventricular canal (AVC endocardium and Bmp2 in the AVC myocardium. Whole embryo cultures treated with Wnt4 or Wnt inhibitory factor 1 (Wif1 show that Bmp2 expression in the AVC myocardium is dependent on Wnt activity; Wnt4 also reinstates Bmp2 expression in the AVC myocardium of endocardial Notch1 null embryos. Furthermore, while both Wnt4 and Bmp2 rescue the defective EMT resulting from Notch inhibition, Wnt4 requires Bmp for its action. These results demonstrate that Jagged1-Notch1 signaling in endocardial cells induces the expression of Wnt4, which subsequently acts as a paracrine factor to upregulate Bmp2 expression in the adjacent AVC myocardium to signal EMT.

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

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    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.

  2. Manipulation of Fgf and Bmp signaling in teleost fishes suggests potential pathways for the evolutionary origin of multicuspid teeth.

    Science.gov (United States)

    Jackman, William R; Davies, Shelby H; Lyons, David B; Stauder, Caitlin K; Denton-Schneider, Benjamin R; Jowdry, Andrea; Aigler, Sharon R; Vogel, Scott A; Stock, David W

    2013-01-01

    Teeth with two or more cusps have arisen independently from an ancestral unicuspid condition in a variety of vertebrate lineages, including sharks, teleost fishes, amphibians, lizards, and mammals. One potential explanation for the repeated origins of multicuspid teeth is the existence of multiple adaptive pathways leading to them, as suggested by their different uses in these lineages. Another is that the addition of cusps required only minor changes in genetic pathways regulating tooth development. Here we provide support for the latter hypothesis by demonstrating that manipulation of the levels of Fibroblast growth factor (Fgf) or Bone morphogenetic protein (Bmp) signaling produces bicuspid teeth in the zebrafish (Danio rerio), a species lacking multicuspid teeth in its ancestry. The generality of these results for teleosts is suggested by the conversion of unicuspid pharyngeal teeth into bicuspid teeth by similar manipulations of the Mexican Tetra (Astyanax mexicanus). That these manipulations also produced supernumerary teeth in both species supports previous suggestions of similarities in the molecular control of tooth and cusp number. We conclude that despite their apparent complexity, the evolutionary origin of multicuspid teeth is positively constrained, likely requiring only slight modifications of a pre-existing mechanism for patterning the number and spacing of individual teeth.

  3. Bone morphogenetic protein signaling protects against cerulein-induced pancreatic fibrosis.

    Directory of Open Access Journals (Sweden)

    Xuxia Gao

    Full Text Available Bone morphogenetic proteins (BMPs have an anti-fibrogenic function in the kidney, lung, and liver. However, their role in chronic pancreatitis (CP is unknown. The aim of this study was to define the anti-fibrogenic role of BMP signaling in the pancreas in vivo under CP induction. Mice with a deletion of BMP type II receptor (BMPR2(+/- were used in this study in comparison with wild-type mice. CP was induced by repetitive cerulein injection intraperitoneally for 4 weeks, and the severity of CP was evaluated. Pancreatic stellate cells (PSCs were isolated from the mice and treated with BMP2 and TGF-β in vitro, and extracellular matrix protein (ECM production was measured. Smad and mitogen-activated protein kinase (MAPK signaling was also evaluated. BMPR2(+/- mice revealed a greater pancreatic fibrosis, PSC activation and leukocyte infiltration after CP induction compared to wild-type mice (P<0.05. Under CP induction, phospho (pSmad1/5/8 was elevated in wild-type mice and this effect was abolished in BMPR2(+/- mice; pSmad2 and pp38(MAPK were further enhanced in BMPR2(+/- mice compared to wild-type mice (P<0.05. In vitro, BMP2 inhibited TGF-β-induced ECM protein fibronectin production in wild-type PSCs; this effect was abolished in BMPR2(+/- PSCs (P<0.05. In BMPR2(+/- PSCs, pSmad1/5/8 level was barely detectable upon BMP2 stimulation, while pSmad2 level was further enhanced by TGF-β stimulation, compared to wild-type PSCs (P<0.05. BMPR2/Smad1/5/8 signaling plays a protective role against cerulein-induced pancreatic fibrosis by inhibiting Smad2 and p38(MAPK signaling pathways.

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

    Science.gov (United States)

    Hsieh, Wen-Hui; Chen, Yi-Jyun; Hung, Chi-Jr; Huang, Tsui-Hsien; Kao, Chia-Tze; Shie, Ming-You

    2014-11-01

    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.

  5. Noggin and Wnt3a enable BMP4-dependent differentiation of telencephalic stem cells into GluR-agonist responsive neurons

    DEFF Research Database (Denmark)

    Andersson, Therese; Duckworth, Joshua K; Fritz, Nicolas;

    2011-01-01

    of multipotent telencephalic neural stem cells (NSCs) with BMP4 induces differentiation primarily into astrocytic and mesenchymal cells. However, BMP4-mediated mesenchymal differentiation is inhibited at certain culture conditions of NSCs, corresponding to in vivo developmental contexts. These inhibitory......Early telencephalic development is dependent on the spatially and temporally coordinated regulation by essential signaling factors. For example, members of the Bone Morphogenetic Protein (BMP) family, such as BMP4, are crucial for proper development of dorsal telencephalic structures. Stimulation...... to be essential for the development of neurons derived from the dorsal telencephalon, and co-stimulation of NSCs with BMP4+Wnt3a resulted in a synergistic effect yielding significantly increased number of mature neurons compared to stimulation with each factor alone. Thus whereas only a subset of BMP4-induced...

  6. Tfap2a promotes specification and maturation of neurons in the inner ear through modulation of Bmp, Fgf and notch signaling.

    Science.gov (United States)

    Kantarci, Husniye; Edlund, Renee K; Groves, Andrew K; Riley, Bruce B

    2015-03-01

    Neurons of the statoacoustic ganglion (SAG) transmit auditory and vestibular information from the inner ear to the hindbrain. SAG neuroblasts originate in the floor of the otic vesicle. New neuroblasts soon delaminate and migrate towards the hindbrain while continuing to proliferate, a phase known as transit amplification. SAG cells eventually come to rest between the ear and hindbrain before terminally differentiating. Regulation of these events is only partially understood. Fgf initiates neuroblast specification within the ear. Subsequently, Fgf secreted by mature SAG neurons exceeds a maximum threshold, serving to terminate specification and delay maturation of transit-amplifying cells. Notch signaling also limits SAG development, but how it is coordinated with Fgf is unknown. Here we show that transcription factor Tfap2a coordinates multiple signaling pathways to promote neurogenesis in the zebrafish inner ear. In both zebrafish and chick, Tfap2a is expressed in a ventrolateral domain of the otic vesicle that includes neurogenic precursors. Functional studies were conducted in zebrafish. Loss of Tfap2a elevated Fgf and Notch signaling, thereby inhibiting SAG specification and slowing maturation of transit-amplifying cells. Conversely, overexpression of Tfap2a inhibited Fgf and Notch signaling, leading to excess and accelerated SAG production. However, most SAG neurons produced by Tfap2a overexpression died soon after maturation. Directly blocking either Fgf or Notch caused less dramatic acceleration of SAG development without neuronal death, whereas blocking both pathways mimicked all observed effects of Tfap2a overexpression, including apoptosis of mature neurons. Analysis of genetic mosaics showed that Tfap2a acts non-autonomously to inhibit Fgf. This led to the discovery that Tfap2a activates expression of Bmp7a, which in turn inhibits both Fgf and Notch signaling. Blocking Bmp signaling reversed the effects of overexpressing Tfap2a. Together, these data

  7. Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B

    DEFF Research Database (Denmark)

    Olsen, Oddrun Elise; Wader, Karin Fahl; Hella, Hanne;

    2015-01-01

    BACKGROUND: Activins are members of the TGF-β family of ligands that have multiple biological functions in embryonic stem cells as well as in differentiated tissue. Serum levels of activin A were found to be elevated in pathological conditions such as cachexia, osteoporosis and cancer. Signaling...

  8. 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.

  9. Modulation of Bmp4 signalling in the epithelial-mesenchymal interactions that take place in early thymus and parathyroid development in avian embryos.

    Science.gov (United States)

    Neves, Hélia; Dupin, Elisabeth; Parreira, Leonor; Le Douarin, Nicole M

    2012-01-15

    Epithelial-mesenchymal interactions are crucial for the development of the endoderm of the pharyngeal pouches into the epithelia of thymus and parathyroid glands. Here we investigated the dynamics of epithelial-mesenchymal interactions that take place at the earliest stages of thymic and parathyroid organogenesis using the quail-chick model together with a co-culture system capable of reproducing these early events in vitro. The presumptive territories of thymus and parathyroid epithelia were identified in three-dimensionally preserved pharyngeal endoderm of embryonic day 4.5 chick embryos on the basis of the expression of Foxn1 and Gcm2, respectively: the thymic rudiment is located in the dorsal domain of the third and fourth pouches, while the parathyroid rudiment occupies a more medial/anterior pouch domain. Using in vitro quail-chick tissue associations combined with in ovo transplantations, we show that the somatopleural but not the limb bud mesenchyme, can mimic the role of neural crest-derived pharyngeal mesenchyme to sustain development of these glands up to terminal differentiation. Furthermore, mesenchymal-derived Bmp4 appears to be essential to promote early stages of endoderm development during a short window of time, irrespective of the mesenchymal source. In vivo studies using the quail-chick system and implantation of growth factor soaked-beads further showed that expression of Bmp4 by the mesenchyme is necessary during a 24 h-period of time. After this period however, Bmp4 is no longer required and another signalling factor produced by the mesenchyme, Fgf10, influences later differentiation of the pouch endoderm. These results show that morphological development and cell differentiation of thymus and parathyroid epithelia require a succession of signals emanating from the associated mesenchyme, among which Bmp4 plays a pivotal role for triggering thymic epithelium specification.

  10. Shh/Bmp4信号在直肠末端的表达与先天性肛门直肠畸形发生的关系%Down-regulation of Shh/Bmp4 signaling pathway in congenital ano- rectal malformations

    Institute of Scientific and Technical Information of China (English)

    张娟; 张志波; 高红; 王维林

    2008-01-01

    Objective To assess the relationship between the expression of Shh/Bmp4 signaling pathway and congenital anorectal malformations (CAM). Methods The posterior wall of terminal rectum of 40 anorectal malformation patients (15 high and 25 low) and 10 normal controls were sampled. The Shh、Gli2 and Bmp4 mRNA were extracted, assayed by RT-PCR and the difference between the two groups was compared. Results The expression levels of Shh、 Gli2 and Bmp4 mRNA were significantly lower in high CAM than those in low CAM and in control group. There was no significant difference between the low CAM and control group except the Gli2 gene. Conclusions Down-regulation of Shh/Bmp4 signaling pathway may be one of the factors contributing to the pathogenesis of CAM. There may be other mechanisms involved in low CAM.%目的 研究先天性肛门直肠畸形(CAM)直肠末端Shh/Bmp4信号表达水平,探讨其与畸形发生的关系.方法 采用RT-PCR方法检测15例高位CAM、25例低位CAM和10例正常对照组直肠后壁末端Shh、Gli2和Bmp4表达水平,比较正常组与CAM组、CAM组不同类型之间表达水平的差异.结果 高位CAM的Shh、Gli2和Bmp4表达水平明显低于正常及低位CAM(P0.05).结论 高位CAM的发生可能与Shh/Bmp4信号表达下调有关,低位CAM可能涉及其他机制.

  11. Smad7 dependent expression signature highlights BMP2 and HK2 signaling in HSC transdifferentiation

    Institute of Scientific and Technical Information of China (English)

    Bernd; Denecke; Lucia; Wickert; Loredana; Ciuclan; Steven; Dooley; Nadja; M; Meindl-Beinker

    2010-01-01

    AIM: To analyse the influence of Smad7, antagonist of transforming growth factor (TGF)-β canonical signaling pathways on hepatic stellate cell (HSC) transdifferentia-tion in detail. METHODS: We systematically analysed genes regulated by TGF-β/Smad7 in activated HSCs by microarray analy-sis and validated the results using real time polymerase chain reaction and Western blotting analysis. RESULTS: We identif ied 100 known and unknown tar-gets underlying the regulation of Smad7 expression and delineated 8 gene...

  12. BMP2诱导C3H10细胞成骨分化的TGF-βⅠ型受体的体外分析%Analyses of TypeⅠTGF-β Receptors in BMP2 Induced Osteogenic Differentiation of C3H10 Cell in Vitro

    Institute of Scientific and Technical Information of China (English)

    张燕; 翁亚光; 文巍; 冯涛; 罗进勇

    2009-01-01

    筛选和分析与BMP2诱导间充质干细胞C3H10成骨分化有关的TGF-βⅠ型受体.利用显性负性突变型TGF-βⅠ型受体竞争抑制配体功能的特性,运用碱性磷酸酶定量测定、Real time PCR等方法,初步筛选出可能与BMP2诱导间充质干细胞C3H10成骨分化有关的的TGF-βⅠ型受体,随后运用RNA干扰的方法抑制相应TGF-βⅠ型受体的表达,进一步证实相关TGF-βⅠ型受体与BMP2发挥诱导成骨活性的关系.结果证实,显性负性突变的ALK3和ALK6能够抑制BMP2诱导的C3H10细胞成骨分化;RNA干扰抑制ALK3或(和)ALK6表达后,BMP2诱导C3H10细胞向成骨分化的趋势受到抑制.因此,ALK3和ALK6是与BMP2诱导C3H10细胞成骨分化有关的TGF-βⅠ型受体.%It was to analysis the typeⅠ TGF-β receptors in BMP2 induced osteogenic differentiation. The typeⅠ TGF-β receptors involved in BMP2 induced osteogenic differentiation of C3H10 cell was identified by using ALP quantitative assay and Real time PCR. The expression of BMP2 related typeⅠ TGF-β receptors was knock down by RNA interference, to confirm the relationship between osteogenic activity of BMP2 and typeⅠ TGF-β receptors. Results showed that dominant negative forms of ALK3 and ALK6 can inhibit BMP2 induced osteogenic differentiation of C3H10 cell,and ALK3/ALK6 knock-down by RNAi can inhibit BMP2 induced osteogenic differentiation of C3H10 cell. It can prove that ALK3 and ALK6 are the typeⅠ TGF-β receptors that can involve in BMP2 induced osteogenic differentiation of C3H10 cell.

  13. Differential expression of Bmp2, Bmp4 and Bmp3 in embryonic development of mouse anterior and posterior palate

    Institute of Scientific and Technical Information of China (English)

    NIE Xu-guang

    2005-01-01

    Background The palate is differently regulated and developed along the anterior-posterior axis. The Bmp signal pathway plays a crucial role in palatogenesis. Conditioned-inactivation of Bmp type I receptor Alk2 or Alk3 in the neural crest or craniofacial region leads to palatal cleft in mice. However, how different Bmp members are involved in palatogenesis remains to be elucidated. In the present study, mRNA expression patterns of Bmp2, Bmp3 and Bmp4 in the developing anterior and posterior palates were examined and compared, focusing on the fusion stage. Methods To detect the expression of Bmp mRNA, antisense riboprobes were synthesized by in vitro transcription. Radioactive in situ hybridization was performed on sagital and coronal sections of mice head from E13 to E18. Results The expression of these Bmps were developmentally regulated in the anterior and posterior palates prior to, during and after palatal fusion. During palatal fusion, Bmp4 expression shifted from the anterior to the posterior palate, Bmp2 was highly expressed in both the anterior and posterior palates in this process, whereas Bmp3 was only localized in the posterior palate. They showed generally non-overlapping pattern in their expression domains. Thereafter, their expression was detected in both the anterior and posterior palates regulating osteogenesis and myogenesis respectively. Conclusions Bmp signalling is involved in palatogenesis in multiple stages and has multiple roles in regulating anterior and posterior palatal development. Disturbances of Bmp signalling during palatogenesis might be a possible mechanism of cleft palate.

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

    Science.gov (United States)

    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

    2013-02-26

    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 BMP4 transgene in white adipocytes of mice gives rise to reduced WAT mass and white adipocyte size along with an increased number of a white adipocyte cell types with brown adipocyte characteristics comparable to those of beige or brite adipocytes. These changes correlate closely with increased energy expenditure, improved insulin sensitivity, and protection against diet-induced obesity and diabetes. Conversely, BMP4-deficient mice exhibit enlarged white adipocyte morphology and impaired insulin sensitivity. We identify peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α) as the target of BMP signaling required for these brown fat-like changes in WAT. This effect of BMP4 on WAT appears to extend to human adipose tissue, because the level of expression of BMP4 in WAT correlates inversely with body mass index. These findings provide a genetic and metabolic basis for BMP4's role in altering insulin sensitivity by affecting WAT development.

  15. BMP Sustains Embryonic Stem Cell Self-Renewal through Distinct Functions of Different Krüppel-like Factors

    Directory of Open Access Journals (Sweden)

    Masato Morikawa

    2016-01-01

    Full Text Available Bone morphogenetic protein (BMP signaling exerts paradoxical roles in pluripotent stem cells (PSCs; it sustains self-renewal of mouse embryonic stem cells (ESCs, while it induces differentiation in other PSCs, including human ESCs. Here, we revisit the roles of BMP-4 using mouse ESCs (mESCs in naive and primed states. SMAD1 and SMAD5, which transduce BMP signals, recognize enhancer regions together with KLF4 and KLF5 in naive mESCs. KLF4 physically interacts with SMAD1 and suppresses its activity. Consistently, a subpopulation of cells with active BMP-SMAD can be ablated without disturbing the naive state of the culture. Moreover, Smad1/5 double-knockout mESCs stay in the naive state, indicating that the BMP-SMAD pathway is dispensable for it. In contrast, the MEK5-ERK5 pathway mediates BMP-4-induced self-renewal of mESCs by inducing Klf2, a critical factor for the ground state pluripotency. Our study illustrates that BMP exerts its self-renewing effect through distinct functions of different Krüppel-like factors.

  16. Identification of bone morphogenetic protein 7 (BMP7) as an instructive factor for human epidermal Langerhans cell differentiation.

    Science.gov (United States)

    Yasmin, Nighat; Bauer, Thomas; Modak, Madhura; Wagner, Karin; Schuster, Christopher; Köffel, Rene; Seyerl, Maria; Stöckl, Johannes; Elbe-Bürger, Adelheid; Graf, Daniel; Strobl, Herbert

    2013-11-18

    Human Langerhans cell (LC) precursors populate the epidermis early during prenatal development and thereafter undergo massive proliferation. The prototypic antiproliferative cytokine TGF-β1 is required for LC differentiation from human CD34(+) hematopoietic progenitor cells and blood monocytes in vitro. Similarly, TGF-β1 deficiency results in LC loss in vivo. However, immunohistology studies revealed that human LC niches in early prenatal epidermis and adult basal (germinal) keratinocyte layers lack detectable TGF-β1. Here we demonstrated that these LC niches express high levels of bone morphogenetic protein 7 (BMP7) and that Bmp7-deficient mice exhibit substantially diminished LC numbers, with the remaining cells appearing less dendritic. BMP7 induces LC differentiation and proliferation by activating the BMP type-I receptor ALK3 in the absence of canonical TGF-β1-ALK5 signaling. Conversely, TGF-β1-induced in vitro LC differentiation is mediated via ALK3; however, co-induction of ALK5 diminished TGF-β1-driven LC generation. Therefore, selective ALK3 signaling by BMP7 promotes high LC yields. Within epidermis, BMP7 shows an inverse expression pattern relative to TGF-β1, the latter induced in suprabasal layers and up-regulated in outer layers. We observed that TGF-β1 inhibits microbial activation of BMP7-generated LCs. Therefore, TGF-β1 in suprabasal/outer epidermal layers might inhibit LC activation, resulting in LC network maintenance.

  17. Oxygen-glucose deprivation preconditioning protects neurons against oxygen-glucose deprivation/reperfusion induced injury via bone morphogenetic protein-7 mediated ERK, p38 and Smad signalling pathways.

    Science.gov (United States)

    Guan, Junhong; Du, Shaonan; Lv, Tao; Qu, Shengtao; Fu, Qiang; Yuan, Ye

    2016-01-01

    Bone morphogenetic protein (BMP)-7 mediated neuroprotective effect of cerebral ischemic preconditioning (IPC) has been studied in an ischemic animal model, but the underlying cellular mechanisms have not been clearly clarified. In this study, primary cortical neurons and the SH-SY5Y cell line were used to investigate the role of BMP-7 and its downstream signals in the neuroprotective effects of oxygen-glucose deprivation preconditioning (OGDPC). Immunocytochemistry was used to detect the expression of neurofilament in neurons. MTT and lactate dehydrogenase activity assays were used to measure the cytotoxicity. Western blot was used to detect the protein expression of BMP-7 and downstream signals. BMP inhibitor, mitogen-activated protein kinase inhibitors, Smad inhibitor and siRNA of Smad 1 were used to investigate the role of corresponding signalling pathways in the OGDPC. Results showed that OGDPC-induced overexpression of BMP-7 in primary cortical neurons and SH-SY5Y cells. Both of endogenous and exogenous BMP-7 could replicate the neuroprotective effects seen in OGDPC pretreatment. In addition, extracellular regulated protein kinases, p38 and Smad signalling pathway were found to be involved in the neuroprotective effects mediated by OGDPC via BMP-7. This study primarily reveals the cellular mechanisms of the neuroprotection mediated by OGDPC, and provides evidence for better understanding of this intrinsic factor against ischemia.

  18. Rat adipose-derived stromal cells expressing BMP4 induce ectopic bone formation in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Lin LIN; Xin FU; Xin ZHANG; Lian-xu CHEN; Ji-ying ZHANG; Chang-long YU; Kang-tao MA; Chun-yan ZHOU

    2006-01-01

    Aim: Bone morphogenetic protein 4 (BMP4) is one of the main local contributing factors in callus formation in the early phase of fracture healing. Adipose-derived stromal cells (ADSC) are multipotent cells. The present study was conducted to investigate the osteogenic potential of ADSC when exposed to adenovirus containing BMP4 cDNA (Ad-BMP4). Methods: ADSC were harvested from Sprague-Dawley rats. After exposure to Ad-BMP4, ADSC were assessed by alkaline phos-phatase activity (ALP) assay, RT-PCR and von Kossa staining. BMP4 expression was assessed by RT-PCR, immunofluorescence and Western blot analysis. ADSC transduced with Ad-BMP4 were directly injected into the hind limb muscles of athymic mice. ADSC Ad-EGFP(enhanced green fluorescence protein) served as controls. All animals were examined by X-ray film and histological analysis. Results: The expression of BMP4 was confirmed at both mRNA and protein levels. The expression of the osteoblastic gene, ALP activity and von Kossa staining confirmed that ADSC transduced with Ad-BMP4 underwent rapid and marked osteoblast differentiation, whereas ADSC transduced with Ad-EGFP and cells left alone displayed no osteogenic differentiation. X-ray and histological examination confirmed new bone formation in athymic mice transplanted with ADSC transduced with Ad-BMP4. Conclusion: Our data demonstrated successful osteogenic differentiation of ADSC transduced with Ad-BMP4 in vitro and in vivo. ADSC may be an ideal source of mesenchyme lineage stem cells for gene therapy and tissue engineering.

  19. Local BMP-SMAD1 Signaling Increases LIF Receptor-Dependent STAT3 Responsiveness and Primed-to-Naive Mouse Pluripotent Stem Cell Conversion Frequency

    Directory of Open Access Journals (Sweden)

    Kento Onishi

    2014-07-01

    Full Text Available Conversion of EpiSCs to naive ESCs is a rare event that is driven by the reestablishment of the naive transcription factor network. In mice, STAT3 activation is sufficient to drive conversion of EpiSCs to the naive pluripotent stem cell (PSC state. However, the lack of responsiveness of EpiSCs to LIF presents a bottleneck in this conversion process. Here, we demonstrate that local accumulation of BMP-SMAD1 signaling, in cooperation with GP130 ligands, enhances the recovery of LIF responsiveness by directly controlling transcription of the LIF receptor (Lif-r. Addition of BMP and LIF to EpiSCs increases both LIF responsiveness and conversion frequencies to naive PSCs. Mechanistically, we show that the transcriptional cofactor P300 plays a critical role by mediating complex formation between STAT3 and SMAD1. This demonstration of how the local microenvironment or stem cell niche reactivates dormant signaling responsiveness and developmental potential may be applicable to other stem cell niche-containing systems.

  20. MAB21L2, a vertebrate member of the Male-abnormal 21 family, modulates BMP signaling and interacts with SMAD1

    Directory of Open Access Journals (Sweden)

    Longhi Renato

    2004-12-01

    Full Text Available Abstract Background Through in vivo loss-of-function studies, vertebrate members of the Male abnormal 21 (mab-21 gene family have been implicated in gastrulation, neural tube formation and eye morphogenesis. Despite mounting evidence of their considerable importance in development, the biochemical properties and nature of MAB-21 proteins have remained strikingly elusive. In addition, genetic studies conducted in C. elegans have established that in double mutants mab-21 is epistatic to genes encoding various members of a Transforming Growth Factor beta (TGF-beta signaling pathway involved in the formation of male-specific sensory organs. Results Through a gain-of-function approach, we analyze the interaction of Mab21l2 with a TGF-beta signaling pathway in early vertebrate development. We show that the vertebrate mab-21 homolog Mab21l2 antagonizes the effects of Bone Morphogenetic Protein 4 (BMP4 overexpression in vivo, rescuing the dorsal axis and restoring wild-type distribution of Chordin and Xvent2 transcripts in Xenopus gastrulae. We show that MAB21L2 immunoprecipitates in vivo with the BMP4 effector SMAD1, whilst in vitro it binds SMAD1 and the SMAD1-SMAD4 complex. Finally, when targeted to an heterologous promoter, MAB21L2 acts as a transcriptional repressor. Conclusions Our results provide the first biochemical and cellular foundation for future functional studies of mab-21 genes in normal neural development and its pathological disturbances.

  1. Calcium phosphate nanoparticles carrying BMP-7 plasmid DNA induce an osteogenic response in MC3T3-E1 pre-osteoblasts.

    Science.gov (United States)

    Hadjicharalambous, Chrystalleni; Kozlova, Diana; Sokolova, Viktoriya; Epple, Matthias; Chatzinikolaidou, Maria

    2015-12-01

    Functionalized calcium phosphate nanoparticles with osteogenic activity were prepared. Polyethyleneimine-stabilized calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization with thiol groups. Between the calcium phosphate surface and the outer silica shell, plasmid DNA which encoded either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein was incorporated as cargo. The plasmid DNA-loaded calcium phosphate nanoparticles were used for the transfection of the pre-osteoblastic MC3T3-E1 cells. The cationic nanoparticles showed high transfection efficiency together with a low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring the alkaline phosphatase (ALP) activity and calcium deposition with alizarin red staining. The expression of the osteogenic markers Alp, Runx2, ColIa1 and Bsp was investigated by means of real-time quantitative polymerase chain reaction. It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically ALP activity and calcium accumulation in the extracellular matrix. Future strategies to stimulate bone regeneration focus into enhancing transfection efficiency and achieving higher levels of BMP-7 produced by the transfected cells.

  2. TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants and arrests downstream differentiation at an early stage of hypertrophy.

    Directory of Open Access Journals (Sweden)

    Nahoko Shintani

    Full Text Available BACKGROUND: Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2 induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2 and transforming growth factor beta 1 (TGF-ß1 were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml for 4 (or 6 weeks. FGF-2 (10 ng/ml or TGF-ß1 (10 ng/ml was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2, but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume. CONCLUSIONS/SIGNIFICANCE: TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of

  3. A Crucial Role of Bone Morphogenetic Protein Signaling in the Wound Healing Response in Acute Liver Injury Induced by Carbon Tetrachloride

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    Nao Oumi

    2012-01-01

    Full Text Available Background. Acute liver injury induced by administration of carbon tetrachloride (CCl4 has used a model of wound repair in the rat liver. Previously, we reported transient expression of bone morphogenetic protein (Bmp 2 or Bmp4 at 6–24 h after CCl4 treatment, suggesting a role of BMP signaling in the wound healing response in the injured liver. In the present study, we investigated the biological meaning of the transient Bmp expression in liver injury. Methods. Using conditional knockout mice carrying a floxed exon in the BMP receptor 1A gene, we determined the hepatic gene expressions and proliferative activity following CCl4-treated liver. Results. We observed retardation of the healing response in the knockout mice treated with CCl4, including aggravated histological feature and reduced expressions of the albumin and Tdo2 genes, and a particular decrease in the proliferative activity shown by Ki-67 immunohistochemistry. Conclusion. Our findings suggest a crucial role of BMP signaling in the amelioration of acute liver injury.

  4. 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.

  5. Acoustically-Induced Electrical Signals

    Science.gov (United States)

    Brown, S. R.

    2014-12-01

    We have observed electrical signals excited by and moving along with an acoustic pulse propagating in a sandstone sample. Using resonance we are now studying the characteristics of this acousto-electric signal and determining its origin and the controlling physical parameters. Four rock samples with a range of porosities, permeabilities, and mineralogies were chosen: Berea, Boise, and Colton sandstones and Austin Chalk. Pore water salinity was varied from deionized water to sea water. Ag-AgCl electrodes were attached to the sample and were interfaced to a 4-wire electrical resistivity system. Under computer control, the acoustic signals were excited and the electrical response was recorded. We see strong acoustically-induced electrical signals in all samples, with the magnitude of the effect for each rock getting stronger as we move from the 1st to the 3rd harmonics in resonance. Given a particular fluid salinity, each rock has its own distinct sensitivity in the induced electrical effect. For example at the 2nd harmonic, Berea Sandstone produces the largest electrical signal per acoustic power input even though Austin Chalk and Boise Sandstone tend to resonate with much larger amplitudes at the same harmonic. Two effects are potentially responsible for this acoustically-induced electrical response: one the co-seismic seismo-electric effect and the other a strain-induced resistivity change known as the acousto-electric effect. We have designed experimental tests to separate these mechanisms. The tests show that the seismo-electric effect is dominant in our studies. We note that these experiments are in a fluid viscosity dominated seismo-electric regime, leading to a simple interpretation of the signals where the electric potential developed is proportional to the local acceleration of the rock. Toward a test of this theory we have measured the local time-varying acoustic strain in our samples using a laser vibrometer.

  6. 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.

  7. BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence

    Science.gov (United States)

    Hayashi, Yohei; Hsiao, Edward C.; Sami, Salma; Lancero, Mariselle; Schlieve, Christopher R.; Nguyen, Trieu; Yano, Koyori; Nagahashi, Ayako; Ikeya, Makoto; Matsumoto, Yoshihisa; Nishimura, Ken; Fukuda, Aya; Hisatake, Koji; Tomoda, Kiichiro; Asaka, Isao; Toguchida, Junya; Conklin, Bruce R.; Yamanaka, Shinya

    2016-01-01

    Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes. PMID:27794120

  8. A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells

    DEFF Research Database (Denmark)

    Hansson, Mattias; Olesen, Dorthe R; Peterslund, Janny M L

    2009-01-01

    Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous....... Notably, activin induction of Gsc-GFP(+) cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17(+) cells by activin while BMP4-induced T expression...... requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro....

  9. A novel therapeutic approach with Caviunin-based isoflavonoid that en routes bone marrow cells to bone formation via BMP2/Wnt-β-catenin signaling.

    Science.gov (United States)

    Kushwaha, P; Khedgikar, V; Gautam, J; Dixit, P; Chillara, R; Verma, A; Thakur, R; Mishra, D P; Singh, D; Maurya, R; Chattopadhyay, N; Mishra, P R; Trivedi, R

    2014-09-18

    Recently, we reported that extract of Dalbergia sissoo made from leaves and pods have antiresorptive and bone-forming effects. The positive skeletal effect attributed because of active molecules present in the extract of Dalbergia sissoo. Caviunin 7-O-[β-D-apiofuranosyl-(1-6)-β-D-glucopyranoside] (CAFG), a novel isoflavonoid show higher percentage present in the extract. Here, we show the osteogenic potential of CAFG as an alternative for anabolic therapy for the treatment of osteoporosis by stimulating bone morphogenetic protein 2 (BMP2) and Wnt/β-catenin mechanism. CAFG supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur and decreased bone turnover markers better than genistein. Oral administration of CAFG to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased the expression of osteogenic genes in femur and show new bone formation without uterine hyperplasia. CAFG increased mRNA expression of osteoprotegerin in bone and inhibited osteoclast activation by inhibiting the expression of skeletal osteoclastogenic genes. CAFG is also an effective accelerant for chondrogenesis and has stimulatory effect on the repair of cortical bone after drill-hole injury at the tissue, cell and gene level in mouse femur. At cellular levels, CAFG stimulated osteoblast proliferation, survival and differentiation. Signal transduction inhibitors in osteoblast demonstrated involvement of p-38 mitogen-activated protein kinase pathway stimulated by BMP2 to initiate Wnt/β-catenin signaling to reduce phosphorylation of GSK3-β and subsequent nuclear accumulation of β-catenin. Osteogenic effects were abrogated by Dkk1, Wnt-receptor blocker and FH535, inhibitor of TCF-complex by reduction in β-catenin levels. CAFG modulated MSC responsiveness to BMP2, which promoted osteoblast differentiation via Wnt/β-catenin mechanism. CAFG at 1 mg/kg(/)day dose in

  10. Sequential Treatment with SDF-1 and BMP-2 Potentiates Bone Formation in Calvarial Defects.

    Science.gov (United States)

    Hwang, Hee-Don; Lee, Jung-Tae; Koh, Jeong-Tae; Jung, Hong-Moon; Lee, Heon-Jin; Kwon, Tae-Geon

    2015-07-01

    Stromal cell-derived factor-1 (SDF-1) protein and its receptor, CXCR-4, play an important role in tissue repair and regeneration in various organs, including the bone. SDF-1 is indispensable for bone morphogenetic protein-2 (BMP-2)-induced osteogenic differentiation. However, SDF-1 is not needed after the osteogenic induction has been activated. Since the precise condition for the additive effects of combined DF-1 and BMP-2 in bone healing had not been fully investigated, we aimed to determine the optimal conditions for SDF-1- and BMP-2-mediated bone regeneration. We examined the in vitro osteoblastic differentiation and cell migration after sequential treatments with SDF-1 and BMP-2. Based on the in vitro additive effects of SDF-1 and BMP-2, the critical size defects of mice calvaria were treated with these cytokines in various sequences. Phosphate buffered saline (PBS)-, SDF-1-, or BMP-2-soaked collagen scaffolds were implanted into the calvarial defects (n=36). Periodic percutaneous injections of PBS or the cytokine SDF-1 and BMP-2 into the implanted scaffolds were performed on days 3 and 6, postoperatively. Six experimental groups were used according to the types and sequences of the cytokine treatments. After 28 days, the mice were euthanized and bone formation was evaluated with microcomputed tomography and histology. The molecular mechanism of the additive effect of SDF-1 and BMP-2 was evaluated by analyzing intracellular signal transduction through Smad and Erk phosphorylation. The in vitro experiments revealed that, among all the treatments, the treatment with BMP-2 after SDF-1 showed the strongest osteoblastic differentiation and enhanced cell migration. Similarly, in the animal model, the treatment with SDF-1 followed by BMP-2 treatment showed the highest degree of new bone regeneration than any other groups, including the one with continuous BMP-2 treatment. This new bone formation can be partially explained by the activation of Smad and Erk pathways

  11. The hedgehog-signaling pathway is repressed during the osteogenic differentiation of dental follicle cells

    DEFF Research Database (Denmark)

    Morsczeck, Christian; Reck, A; Beck, H C

    2017-01-01

    differentiation by BMP2 remains elusive. We investigated therefore the phosphoproteome of DFCs after the induction of the osteogenic differentiation with BMP2. In this study, phosphoproteins of the hedgehog "off" state were differentially expressed. Further analyses revealed that BMP2 induced the expression...... of repressors of the hedgehog-signaling pathway such as Patched 1 (PTCH1), Suppressor of Fused (SUFU), and Parathyroid Hormone-Related Peptide (PTHrP). Previous studies suggested that hedgehog proteins induce the osteogenic differentiation of mesenchymal stem cells via a paracrine pathway. Indian hedgehog (IHH....... In conclusion, our results suggest that BMP2 inhibits the hedgehog signaling after the induction of the osteogenic differentiation in DFCs....

  12. Bone morphogenetic protein 2 promotes osteogenesis of bone marrow stromal cells in type 2 diabetic rats via the Wnt signaling pathway.

    Science.gov (United States)

    Qian, Chao; Zhu, Chenyuan; Yu, Weiqiang; Jiang, Xinquan; Zhang, Fuqiang; Sun, Jian

    2016-11-01

    Type 2 diabetes mellitus impairs osteogenesis in bone marrow stromal cells (BMSCs). Bone morphogenetic protein 2 (BMP2) has been extensively applied for bone defect restoration and has been shown to activate the Wnt signaling pathway. The objective of this study was to investigate the effects of BMP2 on the cell proliferation and osteogenesis of type 2 diabetic BMSCs in rats and explore whether BMP2 induced osteogenesis via the stimulation of Wnt signaling pathway. The cell experiments were divided into DM (diabetic BMSCs), BMP25 (induced with 25ng/ml BMP2), BMP100 (induced with 100ng/ml BMP2) and BMP25 +XAV groups. All cells with or without the different concentrations of BMP2 were cultured under the same experimental conditions. The in vitro results indicated that BMP2 enhanced cell proliferation by 130%-157% and osteogenic differentiation by approximately two-fold in type 2 diabetic BMSCs. The expression levels of β-catenin, cyclin D1, Runx2 and c-myc related to the Wnt signaling pathway were also upregulated from 180% to 212% in BMP2-induced type 2 diabetic rat BMSCs, while the level of GSK3β decreased to 43%. In BMP2-induced type 2 diabetic BMSCs with calcium phosphate cement (CPC) scaffolds for osteoblast study in vivo, the appearance of newly formed bone dramatically increased to 175% compared with type 2 diabetic BMSCs. These data demonstrated that BMP2 enhanced bone regeneration in diabetic BMSCs by stimulating the Wnt signaling pathway with the accumulation of β-catenin and the depressed expression of GSK3β. Diabetic BMSCs associated with BMP2 might be a potential tissue-engineered construct for bone defects in type 2 diabetes mellitus.

  13. Periosteal BMP2 activity drives bone graft healing.

    Science.gov (United States)

    Chappuis, Vivianne; Gamer, Laura; Cox, Karen; Lowery, Jonathan W; Bosshardt, Dieter D; Rosen, Vicki

    2012-10-01

    Bone graft incorporation depends on the orchestrated activation of numerous growth factors and cytokines in both the host and the graft. Prominent in this signaling cascade is BMP2. Although BMP2 is dispensable for bone formation, it is required for the initiation of bone repair; thus understanding the cellular mechanisms underlying bone regeneration driven by BMP2 is essential for improving bone graft therapies. In the present study, we assessed the role of Bmp2 in bone graft incorporation using mice in which Bmp2 has been removed from the limb prior to skeletal formation (Bmp2(cKO)). When autograft transplantations were performed in Bmp2cKO mice, callus formation and bone healing were absent. Transplantation of either a vital wild type (WT) bone graft into a Bmp2(cKO) host or a vital Bmp2(cKO) graft into a WT host also resulted in the inhibition of bone graft incorporation. Histological analyses of these transplants show that in the absence of BMP2, periosteal progenitors remain quiescent and healing is not initiated. When we analyzed the expression of Sox9, a marker of chondrogenesis, on the graft surface, we found it significantly reduced when BMP2 was absent in either the graft itself or the host, suggesting that local BMP2 levels drive periosteal cell condensation and subsequent callus cell differentiation. The lack of integrated healing in the absence of BMP2 was not due to the inability of periosteal cells to respond to BMP2. Healing was achieved when grafts were pre-soaked in rhBMP2 protein, indicating that periosteal progenitors remain responsive in the absence of BMP2. In contrast to the requirement for BMP2 in periosteal progenitor activation in vital bone grafts, we found that bone matrix-derived BMP2 does not significantly enhance bone graft incorporation. Taken together, our data show that BMP2 signaling is not essential for the maintenance of periosteal progenitors, but is required for the activation of these progenitors and their subsequent

  14. Neurotrophin-3 Induces BMP-2 and VEGF Activities and Promotes the Bony Repair of Injured Growth Plate Cartilage and Bone in Rats.

    Science.gov (United States)

    Su, Yu-Wen; Chung, Rosa; Ruan, Chun-Sheng; Chim, Shek Man; Kuek, Vincent; Dwivedi, Prem P; Hassanshahi, Mohammadhossein; Chen, Ke-Ming; Xie, Yangli; Chen, Lin; Foster, Bruce K; Rosen, Vicki; Zhou, Xin-Fu; Xu, Jiake; Xian, Cory J

    2016-06-01

    Injured growth plate is often repaired by bony tissue causing bone growth defects, for which the mechanisms remain unclear. Because neurotrophins have been implicated in bone fracture repair, here we investigated their potential roles in growth plate bony repair in rats. After a drill-hole injury was made in the tibial growth plate and bone, increased injury site mRNA expression was observed for neurotrophins NGF, BDNF, NT-3, and NT-4 and their Trk receptors. NT-3 and its receptor TrkC showed the highest induction. NT-3 was localized to repairing cells, whereas TrkC was observed in stromal cells, osteoblasts, and blood vessel cells at the injury site. Moreover, systemic NT-3 immunoneutralization reduced bone volume at injury sites and also reduced vascularization at the injured growth plate, whereas recombinant NT-3 treatment promoted bony repair with elevated levels of mRNA for osteogenic markers and bone morphogenetic protein (BMP-2) and increased vascularization and mRNA for vascular endothelial growth factor (VEGF) and endothelial cell marker CD31 at the injured growth plate. When examined in vitro, NT-3 promoted osteogenesis in rat bone marrow stromal cells, induced Erk1/2 and Akt phosphorylation, and enhanced expression of BMPs (particularly BMP-2) and VEGF in the mineralizing cells. It also induced CD31 and VEGF mRNA in rat primary endothelial cell culture. BMP activity appears critical for NT-3 osteogenic effect in vitro because it can be almost completely abrogated by co-addition of the BMP inhibitor noggin. Consistent with its angiogenic effect in vivo, NT-3 promoted angiogenesis in metatarsal bone explants, an effect abolished by co-treatment with anti-VEGF. This study suggests that NT-3 may be an osteogenic and angiogenic factor upstream of BMP-2 and VEGF in bony repair, and further studies are required to investigate whether NT-3 may be a potential target for preventing growth plate faulty bony repair or for promoting bone fracture healing. © 2016

  15. Simulated Microgravity Regulates Osteoblast BMP2-Smad Signaling via Actin Microfilament%模拟微重力效应通过微丝骨架调控成骨细胞BMP2-Smad信号

    Institute of Scientific and Technical Information of China (English)

    徐洪杰; 戴钟铨; 吴峰; 张洪玉; 杨超; 商澎; 李莹辉

    2014-01-01

    细胞微丝骨架在力信号传导和基因表达调控中起重要作用.为了研究微丝骨架在模拟微重力效应调控成骨细胞BMP2-Smad信号中的作用,作者通过构建反映Smad活性的报告基因载体转染MC3T3-E1细胞,并通过报告基因活性分析、Western blot等方法检测了微丝骨架解聚剂和回转模拟微重力效应对BMP2诱导Samd磷酸化、核质分布和转录活性的作用.结果显示,构建的报告基因载体在成骨细胞中正确表达并响应BMP2;破坏微丝骨架会抑制BMP2诱导的Smad1/5/8蛋白磷酸化、入核及转录活性;回转抑制Smad1/5/8磷酸化、入核及其转录活性,而微丝骨架稳定剂可对抗回转的抑制作用.因此,认为回转模拟微重力效应可通过解聚微丝骨架抑制BMP2-Smad信号传导.

  16. Negative and positive auto-regulation of BMP expression in early eye development.

    Science.gov (United States)

    Huang, Jie; Liu, Ying; Filas, Benjamen; Gunhaga, Lena; Beebe, David C

    2015-11-15

    Previous results have shown that Bone Morphogenetic Protein (BMP) signaling is essential for lens specification and differentiation. How BMP signals are regulated in the prospective lens ectoderm is not well defined. To address this issue we have modulated BMP activity in a chicken embryo pre-lens ectoderm explant assay, and also studied transgenic mice, in which the type I BMP receptors, Bmpr1a and Acvr1, are deleted from the prospective lens ectoderm. Our results show that chicken embryo pre-lens ectoderm cells express BMPs and require BMP signaling for lens specification in vitro, and that in vivo inhibition of BMP signals in the mouse prospective lens ectoderm interrupts lens placode formation and prevents lens invagination. Furthermore, our results provide evidence that BMP expression is negatively auto-regulated in the lens-forming ectoderm, decreasing when the tissue is exposed to exogenous BMPs and increasing when BMP signaling is prevented. In addition, eyes lacking BMP receptors in the prospective lens placode develop coloboma in the adjacent wild type optic cup. In these eyes, Bmp7 expression increases in the ventral optic cup and the normal dorsal-ventral gradient of BMP signaling in the optic cup is disrupted. Pax2 becomes undetectable and expression of Sfrp2 increases in the ventral optic cup, suggesting that increased BMP signaling alter their expression, resulting in failure to close the optic fissure. In summary, our results suggest that negative and positive auto-regulation of BMP expression is important to regulate early eye development.

  17. The hedgehog signal induced modulation of bone morphogenetic protein signaling: an essential signaling relay for urinary tract morphogenesis.

    Directory of Open Access Journals (Sweden)

    Ryuma Haraguchi

    Full Text Available BACKGROUND: Congenital diseases of the urinary tract are frequently observed in infants. Such diseases present a number of developmental anomalies such as hydroureter and hydronephrosis. Although some genetically-modified mouse models of growth factor signaling genes reproduce urinary phenotypes, the pathogenic mechanisms remain obscure. Previous studies suggest that a portion of the cells in the external genitalia and bladder are derived from peri-cloacal mesenchymal cells that receive Hedgehog (Hh signaling in the early developmental stages. We hypothesized that defects in such progenitor cells, which give rise to urinary tract tissues, may be a cause of such diseases. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the pathogenic mechanisms of upper urinary tract malformations, we analyzed a series of Sonic hedgehog (Shh deficient mice. Shh(-/- displayed hydroureter and hydronephrosis phenotypes and reduced expression of several developmental markers. In addition, we suggested that Shh modulation at an early embryonic stage is responsible for such phenotypes by analyzing the Shh conditional mutants. Tissue contribution assays of Hh-responsive cells revealed that peri-cloacal mesenchymal cells, which received Hh signal secreted from cloacal epithelium, could contribute to the ureteral mesenchyme. Gain- and loss-of-functional mutants for Hh signaling revealed a correlation between Hh signaling and Bone morphogenetic protein (Bmp signaling. Finally, a conditional ablation of Bmp receptor type IA (BmprIA gene was examined in Hh-responsive cell lineages. This system thus made it possible to analyze the primary functions of the growth factor signaling relay. The defective Hh-to-Bmp signaling relay resulted in severe urinary tract phenotypes with a decrease in the number of Hh-responsive cells. CONCLUSIONS/SIGNIFICANCE: This study identified the essential embryonic stages for the pathogenesis of urinary tract phenotypes. These results suggested that Hh

  18. BMP2 Mediates Decorin-induced Vascular Smooth Muscle Cell Calcification%BMP2参与Decorin诱导的血管平滑肌细胞钙化

    Institute of Scientific and Technical Information of China (English)

    颜建云; 周芹; 陈燕玲; 宋艳; 陆立鹤

    2014-01-01

    [目的]阐明BMP2是否参与了Decorin诱导的血管平滑肌细胞(VSMC)钙化.[方法]本研究采用人血管平滑肌细胞钙化体外模型,用腺病毒载体过表达Decorin,检测骨相关蛋白BMP2、Msx2、Osterix的表达和细胞钙化程度.观察抑制或增加BMP2信号对Decorin诱导的血管平滑肌细胞钙化和Msx2、Osterix表达的影响.[结果]Decorin能促进人血管平滑肌细胞钙化,同时上调BMP2、Msx2、Osterix的表达水平.抑制BMP2能减轻Decorin诱导的细胞钙化,而增加BMP2加速Decorin诱导的细胞钙化.[结论]Decofin通过调节BMP2促进人血管平滑肌细胞钙化.

  19. Bone morphogenetic protein 4 (BMP4) induces buffalo (Bubalus bubalis) embryonic stem cell differentiation into germ cells.

    Science.gov (United States)

    Shah, Syed Mohmad; Saini, Neha; Ashraf, Syma; Singh, Manoj Kumar; Manik, Radhey Sham; Singla, Suresh Kumar; Palta, Prabhat; Chauhan, Manmohan Singh

    2015-12-01

    The aim of the present study was to investigate the effect of Bone morphogenetic protein 4 (BMP4) stimulation on differentiation of buffalo embryonic stem (ES) cells into germ lineage cells. ES cells were subjected to in vitro differentiation in floating and adherent cultures, under different BMP4 concentrations (20, 50 and 100 ngml(-1)) for different culture intervals (4, 8 and 14 days). qPCR analysis revealed that BMP4 at a concentration of 50-100 ngml(-1) for a culture period of 14 days led to maximum induction of germ lineage genes like DAZL, VASA, PLZF (PGC-specific); SYCP3, MLH1, TNP1/2 and PRM2 (Meiotic genes); BOULE and TEKT1 (Spermatocyte markers); GDF9, ZP2 and 3 (Oocyte markers). The expression levels of all the genes were significantly higher under BMP4 differentiation as compared to BMP4 + NOGGIN and spontaneously differentiated cultures. Immunocytochemical analysis of embryoid bodies (EBs) and monolayer adherent cultures revealed expression of PGC- (c-KIT, DAZL and VASA); Meiotic- (SYCP3, MLH1 and PROTAMINE1); Spermatocyte- (ACROSIN and HAPRIN); and Oocyte- markers (GDF9 and ZP4). Western blotting was positive for VASA, GDF9 and ZP4. Oocyte-like structures (OLS) obtained in monolayer differentiated cultures harbored a big nucleus and a ZP4 coat. They showed embryonic development and progressed through 2-cell, 4-cell, 8-cell and blastocyst-like structures. Global DNA methylation analysis showed significantly (p < 0.05) decreased levels of 5-methyl-2-deoxycytidine in EBs obtained in optimum differentiation medium. The expression of meiotic markers coupled with expression of spermatocyte and oocyte markers is an indication of post-meiotic progression into spermatogenesis and oogenesis, respectively.

  20. 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.

  1. The regulation of osteogenic differentiation by BMP2 and the canonical Wnt signaling pathway and the crosstalk between them%BMP2信号通路与经典 Wnt 信号通路及相互交联对成骨分化的调控

    Institute of Scientific and Technical Information of China (English)

    刘光源; 田发明; 张柳; 刘家寅

    2014-01-01

    The osteogenic differentiation process in mesenchymal stem cells and other lineages that are capable of differentiating into osteoblasts, is regulated by several signal transduction pathways and the dynamic regulatory networks consisted of them.In this process, more and more attention has been paid to the role of BMP2 signaling pathway and canonical Wnt signaling pathway. Previous studies have shown that BMP2 and canonical Wnt signaling pathway can regulate the osteogenic differentiation process in target cells, during which the expression level and functional activity of key molecules in one signaling pathway are regulated by the other.Moreover, they also exhibit cooperative or antagonize interaction on the regulation of osteogenic differentiation biomarkers. This paper briefly summarizes the role of BMP2 and canonical Wnt signaling pathway in regulating osteogenic differentiation and the crosstalk between the two signaling pathways in this process.%在以间充质干细胞为代表的具有成骨分化潜能的细胞中,多条信号传导通路及其构成的动态调控网络参与调控其成骨分化过程。其中BMP2和经典Wnt信号通路在此过程中的作用日益受到关注。国内外研究表明,BMP2和经典Wnt信号通路在调控靶细胞成骨分化过程中伴有彼此通路中的关键分子的表达水平和功能活性的改变,并在一定条件下表现出对成骨分化特异性标志物的协同和拮抗调控。成骨增殖分化过程是一个动态过程,在成骨分化的不同阶段,分子通路的作用不同,相互之间的调节也动态变化的,研究BMP2和经典Wnt信号通路在成骨分化过程中的相互调节作用,对于指导治疗骨质疏松和骨折有重要的作用,本文就BMP2和经典Wnt信号通路对靶细胞成骨分化的调控作用以及在此过程中两条信号通路之间的交联作一简要综述。

  2. TGF-β1刺激下损伤的前交叉韧带和内侧副韧带中BMP-1基因的表达%Differential BMP-1 expression in injured anterior cruciate ligament and medial collateral ligament fibroblasts induced by TGF-β1

    Institute of Scientific and Technical Information of China (English)

    尹琳; 谢静; 蒋稼欢; 王春莉; 张艳君; 许春明; KL Paul Sung

    2012-01-01

    Objective To investigate the differential expression of bone morphogenetic protein-1 (BMP-1) in injured anterior cruciate ligament ( ACL) and medial collateral ligament ( MCL) fibroblasts induced by TGF-p,, and to find out the differences between the poorly self-healing ACL and well functionally self-healing MCL fibroblasts. Methods Fibroblasts were primarily cultured from clinical ACL and MCL samples, and then given 12% mechanical stretch injury and treated by 1, 5 and 50 ng/ml TGF-β1 for 2 h, or by 5 ng/ml TGF-β,1 for 2, 6, 12 and 24 h respectively at the same time. The expression of BMP-1 in the above treated and untreated fibroblasts were detected by reversed-transcript PCR and real-time quantitative PCR. Western blotting was used to detect the expression of BMP-1 in the injured fibroblasts induced by 5 ng/ml TGF-p, for 48 h. Results TGF-p, treatment resulted in an increased mRNA expression of BMP-1 in the injured fibroblasts in a dose-depended manner, especially in the cells from MCL than in those from ACL (about 1 times higher, P <0. 05). Compared with the fibroblasts without TGF-p, treatment, 5 ng/ml TGF-p, treatment for 24 h made the expression of BMP-1 reach the summit in ACL (6.1 folds higher, P <0.05) and in MCL (9.84 folds higher, P <0.05). Compared with the control, the protein expression of BMP-1 were elevated to 2. 32 and 3. 84 folds higher in ACL and MCL respectively after 5 ng/ml TGF-p, treatment for 48 h (P <0.01). Conclusion TGF-p, affects the expression of BMP-1 in the injured fibroblasts, and then directly affects activities of lysyl oxiadse in the extracellular matrix, implying its potential significant value and clinical usage in repair of injured ACL.%目的 观察在转化生长因子-β1(transforming growth factor betal,TGF-β1)作用下,损伤的前交叉韧带(anterior cruciate ligament,ACL)和内侧副韧带(medial collateral ligament,MCL)中骨形态发生蛋白-1(bone morphogenetic protein-1,BMP-1)基因的表达,找出TGF-31、BMP

  3. Experimental study of the synergistic effect of Wnt11 and BMP-2 on inducing the differentiation of rat bone marrow mesenchymal stem cells into myocardium-like cells under%心肌微环境中Wnt11与BMP2协同作用促大鼠BM-MSCs分化为心肌样细胞的实验研究

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    马志峰; 董红燕; 张志峰; 袁红花; 张中明

    2011-01-01

    Objective To investigate the synergetic effect of Wnt11 and BMP2 on inducing differentiation of BM -MSCs into cardiomyocyte - like cells under the paracrine effect of myocardial micro - environment. Methods The Transwell co - culture model of BM - MSCs cells was establishment, with cardiac myocytes (CMs) together with Wnt11 plasmid -transfected 3T3 cells laid on the upper layer and BM - MSCs underneath. For the first three days, cells were cultured with 50 μg/L noggin ( BMP - inhibitor), followed by replacement with 0.5 μg/L BMP2 protein till the 14th day. According to the different inducing conditions, cells were divided into seven groups: positive control (heart group), myocardial -induction ( CM group), BMP2 - induction in myocardial micro - environment ( CM + BMP2 group), Wnt11 - induction in myocardial micro- environment (CM + Wnt11 group), Wnt11 and BMP2 co- induction (Wnt11 + BMP2 group), Wnt11 and BMP2 co- induction in myocardial micro- environment (CM + Wnt11 + BMP2 group), and negative control (BM -MSCs group). At the end of 14 days of induction culture, RT - PCR was employed to detect the expression levels of nucleic acid of the myocardium- specific transcription factors (Nkx2.5, GATA4 and Mef2c) and mature CM -specific genes (cTrtl, ANP, α - MHC and β - MHC) to evaluate the biological effect of diversified induction on the differentiation of BM -MSCs into cardiomyocyte - like cells. Results RT - PCR results revealed that in the myocardial micro - environment of paracrine the expression levels of myocardium - specific transcription factors and mature CM - specific genes in CM + Wnt11 + BMP2 group was significantly higher than the BM - MSCs group and the other co - culture groups but lower than the heart group ( P < 0. 05 ). Conclusions In cardiac paracrine micro - environment, the synergistic effect of Wnt11 and BMP - 2 can improve the efficiency of the differentiation from BM - MSCs into cardiomyocyte - like cells.%目的 探讨在心肌微

  4. BMP-7 enhances cell migration and αvβ3 integrin expression via a c-Src-dependent pathway in human chondrosarcoma cells.

    Directory of Open Access Journals (Sweden)

    Jui-Chieh Chen

    Full Text Available Bone morphogenic protein (BMP-7 is a member of the transforming growth factor (TGF-beta superfamily, which is originally identified based on its ability to induce cartilage and bone formation. In recent years, BMP-7 is also defined as a potent promoter of cell motility, invasion, and metastasis. However, there is little knowledge of the role of BMP-7 and its cellular function in chondrosarcoma cells. In the present study, we investigated the biological impact of BMP-7 on cell motility using transwell assay. In addition, the intracellular signaling pathways were also investigated by pharmacological and genetic approaches. Our results demonstrated that treatment with exogenous BMP-7 markedly increased cell migration by activating c-Src/PI3K/Akt/IKK/NF-κB signaling pathway, resulting in the transactivation of αvβ3 integrin expression. Indeed, abrogation of signaling activation, by chemical inhibition or expression of a kinase dead form of the protein attenuated BMP-7-induced expression of integrin αvβ3 and cell migration. These findings may provide a useful tool for diagnostic/prognostic purposes and even therapeutically in late-stage chondrosarcoma as an anti-metastatic agent.

  5. Crosstalk of FGF-2 and BMP-2 in Osteoblastic Differentiation of Cranial Suture Cells%FGF-2与BMP-2在颅缝细胞成骨分化中的相互作用

    Institute of Scientific and Technical Information of China (English)

    姜陶然; 曹德君

    2015-01-01

    expression of FGF2- and BMP2-induced OC and BSP by 1.26-fold and 1.20-fold respectively. Conclusion BMP2 is a downstream target of FGF-2, and BMP-2 signals are required for FGF-2-dependent induction of later-stage osteoblast differentiation in cranial suture cells.

  6. Turning Bone Morphogenetic Protein 2 (BMP2) on and off in Mesenchymal Cells.

    Science.gov (United States)

    Rogers, Melissa B; Shah, Tapan A; Shaikh, Nadia N

    2015-10-01

    The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels cause congenital anomalies and diseases involving the mesenchymal cells that differentiate into muscle, fat, cartilage, and bone. The molecules and conditions that influence BMP2 synthesis are diverse. Understandably, complex mechanisms control Bmp2 gene expression. This review includes a compilation of agents and conditions that can induce Bmp2. The currently known trans-regulatory factors and cis-regulatory elements that modulate Bmp2 expression are summarized and discussed. Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell behavior. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence the allocation of cells to muscle, fat, cartilage, and bone, the mechanisms that regulate the Bmp2 gene are crucial. Key early mesodermal events that require precise Bmp2 regulation include heart specification and morphogenesis. Originally named for its osteoinductive properties, healing fractures requires BMP2. The human Bmp2 gene also has been linked to osteoporosis and osteoarthritis. In addition, all forms of pathological calcification in the vasculature and in cardiac valves involve the pro-osteogenic BMP2. The diverse tissues, mechanisms, and diseases influenced by BMP2 are too numerous to list here (see OMIM: 112261). However, in all BMP2-influenced pathologies, changes in the behavior and differentiation of pluripotent mesenchymal cells are a recurring theme. Consequently, much effort has been devoted to identifying the molecules and conditions that influence BMP2 synthesis and the complex mechanisms that control Bmp2 gene expression. This review begins with an

  7. High-concentration of BMP2 reduces cell proliferation and increases apoptosis via DKK1 and SOST in human primary periosteal cells.

    Science.gov (United States)

    Kim, Harry K W; Oxendine, Ila; Kamiya, Nobuhiro

    2013-05-01

    BMP2, a well-known osteoinductive agent approved by FDA, is currently being used for various off-label orthopedic applications. Recently, concerns about its efficacy for off-label use, concentration, and complications have emerged. Interestingly, there is an extremely large discrepancy in BMP2 concentration between clinical use (i.e. 1.5mg/ml) and in vitro studies (50-300 ng/ml). The purpose of this study was to determine the effects of a relatively high-concentration of BMP2 on cell proliferation and apoptosis using human primary periosteal cells as BMP2 is generally applied around the periosteum in orthopedic surgeries. We isolated periosteal cells from three independent patients. The cell proliferation assessed by MTT activity was significantly reduced by a high-concentration of BMP2 (~2000 ng/ml), while such a reduction was not observed by using a low-concentration of BMP2 (~200 ng/ml). The cell apoptosis assessed by caspase activity was significantly increased by high-concentration BMP2, while such an increase was not observed by low-concentration BMP2. We found that Wnt signaling activity was significantly reduced by high-concentration BMP2 along with a dramatic increase in DKK1 and SOST, key inhibitors of Wnt signaling in bone. The addition of DKK1 or SOST protein to the primary periosteal cells reduced MTT activity and significantly increased caspase activity. Silencing the DKK1 or SOST expression using the siRNA technique normalized cell proliferation and apoptosis in the periosteum-derived cells when exposed to a high-concentration BMP2. Taken together, these results suggest that a high-concentration BMP2 decreases human periosteal cell proliferation and induces apoptosis via the activation of Wnt inhibitors DKK1 and SOST. This study provides new insights to the effects of high BMP2 concentration on human periosteal cells and brings out the possibility of multiple effects of current BMP2 therapy on various skeletal tissues.

  8. Induced disease resistance signaling in plants

    NARCIS (Netherlands)

    Verhagen, B.W.M.; Loon, L.C. van; Pieterse, C.M.J.

    2006-01-01

    To protect themselves from disease, plants have evolved sophisticated inducible defense mechanisms in which the signal molecules salicylic acid, jasmonic acid and ethylene often play crucial roles. Elucidation of signaling pathways controlling induced disease resistance is a major objective in resea

  9. Metastatic function of BMP-2 in gastric cancer cells: The role of PI3K/AKT, MAPK, the NF-{kappa}B pathway, and MMP-9 expression

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Myoung Hee [Graduate School of Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Oh, Sang Cheul [Division of Oncology/Hematology, Department of Internal Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Lee, Hyun Joo [Department of Pathology, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Kang, Han Na; Kim, Jung Lim [Graduate School of Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Kim, Jun Suk [Division of Oncology/Hematology, Department of Internal Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Yoo, Young A., E-mail: ydanbi@korea.ac.kr [Brain Korea 21 Program for Biomedical Science, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of)

    2011-07-15

    Bone morphogenetic proteins (BMPs) have been implicated in tumorigenesis and metastatic progression in various types of cancer cells, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of phosphoinositide 3-kinase (PI3K)/AKT, extracellular signal-regulated protein kinase (ERK), nuclear factor (NF)-{kappa}B, and matrix metalloproteinase (MMP) expression in BMP-2-mediated metastatic function in gastric cancer. We found that stimulation of BMP-2 in gastric cancer cells enhanced the phosphorylation of AKT and ERK. Accompanying activation of AKT and ERK kinase, BMP-2 also enhanced phosphorylation/degradation of I{kappa}B{alpha} and the nuclear translocation/activation of NF-{kappa}B. Interestingly, blockade of PI3K/AKT and ERK signaling using LY294002 and PD98059, respectively, significantly inhibited BMP-2-induced motility and invasiveness in association with the activation of NF-{kappa}B. Furthermore, BMP-2-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with PI3K/AKT, ERK, or NF-{kappa}B inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of BMP-2 and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the BMP-2 signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the PI3K/AKT or MAPK pathway followed by the induction of NF-{kappa}B and MMP-9 activity, indicating that BMP-2 has the potential to be a therapeutic molecular target to decrease metastasis.

  10. Dynamics and cellular localization of Bmp2, Bmp4, and Noggin transcription in the postnatal mouse skeleton.

    Science.gov (United States)

    Pregizer, Steven K; Mortlock, Douglas P

    2015-01-01

    Transcription of BMPs and their antagonists in precise spatiotemporal patterns is essential for proper skeletal development, maturation, maintenance, and repair. Nevertheless, transcriptional activity of these molecules in skeletal tissues beyond embryogenesis has not been well characterized. In this study, we used several transgenic reporter mouse lines to define the transcriptional activity of two potent BMP ligands, Bmp2 and Bmp4, and their antagonist, Noggin, in the postnatal skeleton. At 3 to 4 weeks of age, Bmp4 and Noggin reporter activity was readily apparent in most cells of the osteogenic or chondrogenic lineages, respectively, whereas Bmp2 reporter activity was strongest in terminally differentiated cells of both lineages. By 5 to 6 months, activity of the reporters had generally abated; however, the Noggin and Bmp2 reporters remained remarkably active in articular chondrocytes and persisted there indefinitely. We further found that endogenous Bmp2, Bmp4, and Noggin transcript levels in postnatal bone and cartilage mirrored the activity of their respective reporters in these tissues. Finally, we found that the activity of the Bmp2, Bmp4, and Noggin reporters in bone and cartilage at 3 to 4 weeks could be recapitulated in both osteogenic and chondrogenic culture models. These results reveal that Bmp2, Bmp4, and Noggin transcription persists to varying degrees in skeletal tissues postnatally, with each gene exhibiting its own cell type-specific pattern of activity. Illuminating these patterns and their dynamics will guide future studies aimed at elucidating both the causes and consequences of aberrant BMP signaling in the postnatal skeleton.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. BMP9 inhibits proliferation and metastasis of HER2-positive SK-BR-3 breast cancer cells through ERK1/2 and PI3K/AKT pathways.

    Directory of Open Access Journals (Sweden)

    Wei Ren

    Full Text Available Bone morphogenetic protein 9 (BMP9, a member of TGF-β superfamily, is reported to inhibit the growth and migration of prostate cancer, osteosarcoma and triple-negative MDA-MB-231 breast cancer cells. However, little is known about the effect of on the biological behaviors of HER2-positive SK-BR-3 breast cancer cells and the underlying mechanisms. This study aimed to investigate the effects of BMP9 on the proliferation and metastasis of SK-BR-3 cells with BMP9 over-expression or BMP9 down-regulated expression. Results indicated that exogenously expressed BMP9 inhibited the proliferation and metastasis of SK-BR-3 cells while decreased endogenous BMP9 expression in SK-BR-3 cells promoted the proliferation and migration of breast cancer cells in vitro and in vivo. In SK-BR-3 cells with BMP9 over-expression, the phosphorylation of HER2, ERK1/2 and AKT was markedly suppressed and the HER2 expression decreased at both mRNA and protein levels, while opposite results were observed in SK-BR-3 cells with BMP9 knock down. When the phosphorylation of ERK1/2 and PI3K/AKT was inhibited by PD98059 and LY294002, respectively, the decreased proliferation and invasion induced by BMP9 knock down were eliminated. These findings suggest that BMP9 can inhibit the proliferation and metastasis of SK-BR-3 cells via inactivating ERK1/2 and PI3K/AKT signaling pathways. Thus, BMP9 may serve as a useful agent in the treatment of HER-2 positive breast cancer.

  13. BMP9 inhibits proliferation and metastasis of HER2-positive SK-BR-3 breast cancer cells through ERK1/2 and PI3K/AKT pathways.

    Science.gov (United States)

    Ren, Wei; Liu, Yuehong; Wan, Shaoheng; Fei, Chang; Wang, Wei; Chen, Yingying; Zhang, Zhihui; Wang, Ting; Wang, Jinshu; Zhou, Lan; Weng, Yaguang; He, Tongchuan; Zhang, Yan

    2014-01-01

    Bone morphogenetic protein 9 (BMP9), a member of TGF-β superfamily, is reported to inhibit the growth and migration of prostate cancer, osteosarcoma and triple-negative MDA-MB-231 breast cancer cells. However, little is known about the effect of on the biological behaviors of HER2-positive SK-BR-3 breast cancer cells and the underlying mechanisms. This study aimed to investigate the effects of BMP9 on the proliferation and metastasis of SK-BR-3 cells with BMP9 over-expression or BMP9 down-regulated expression. Results indicated that exogenously expressed BMP9 inhibited the proliferation and metastasis of SK-BR-3 cells while decreased endogenous BMP9 expression in SK-BR-3 cells promoted the proliferation and migration of breast cancer cells in vitro and in vivo. In SK-BR-3 cells with BMP9 over-expression, the phosphorylation of HER2, ERK1/2 and AKT was markedly suppressed and the HER2 expression decreased at both mRNA and protein levels, while opposite results were observed in SK-BR-3 cells with BMP9 knock down. When the phosphorylation of ERK1/2 and PI3K/AKT was inhibited by PD98059 and LY294002, respectively, the decreased proliferation and invasion induced by BMP9 knock down were eliminated. These findings suggest that BMP9 can inhibit the proliferation and metastasis of SK-BR-3 cells via inactivating ERK1/2 and PI3K/AKT signaling pathways. Thus, BMP9 may serve as a useful agent in the treatment of HER-2 positive breast cancer.

  14. Gremlin-2 is a BMP antagonist that is regulated by the circadian clock

    Science.gov (United States)

    Yeung, Ching-Yan Chloé; Gossan, Nicole; Lu, Yinhui; Hughes, Alun; Hensman, James J.; Bayer, Monika L.; Kjær, Michael; Kadler, Karl E.; Meng, Qing-Jun

    2014-01-01

    Tendons are prominent members of the family of fibrous connective tissues (FCTs), which collectively are the most abundant tissues in vertebrates and have crucial roles in transmitting mechanical force and linking organs. Tendon diseases are among the most common arthropathy disorders; thus knowledge of tendon gene regulation is essential for a complete understanding of FCT biology. Here we show autonomous circadian rhythms in mouse tendon and primary human tenocytes, controlled by an intrinsic molecular circadian clock. Time-series microarrays identified the first circadian transcriptome of murine tendon, revealing that 4.6% of the transcripts (745 genes) are expressed in a circadian manner. One of these genes was Grem2, which oscillated in antiphase to BMP signaling. Moreover, recombinant human Gremlin-2 blocked BMP2-induced phosphorylation of Smad1/5 and osteogenic differentiation of human tenocytes in vitro. We observed dampened Grem2 expression, deregulated BMP signaling, and spontaneously calcifying tendons in young CLOCKΔ19 arrhythmic mice and aged wild-type mice. Thus, disruption of circadian control, through mutations or aging, of Grem2/BMP signaling becomes a new focus for the study of calcific tendinopathy, which affects 1-in-5 people over the age of 50 years. PMID:24897937

  15. Platelet-released supernatant induces osteoblastic differentiation of human mesenchymal stem cells: potential role of BMP-2

    Directory of Open Access Journals (Sweden)

    M Alini

    2010-12-01

    Full Text Available Platelet-rich preparations have recently gained popularity in maxillofacial and dental surgery, but their beneficial effect is still under debate. Furthermore, very little is known about the effect of platelet preparations at the cellular level, and the underlying mechanisms. In this study, we tested the effect of platelet-released supernatant (PRS on human mesenchymal stem cell (MSC differentiation towards an osteoblastic phenotype in vitro. Cultures of MSC were supplemented with PRS and typical osteoblastic markers were assessed at up to 28 days post-confluence. PRS showed an osteoinductive effect on MSC, as shown by an increased expression of typical osteoblastic marker genes such as collagen Ialpha1, bone sialoprotein II, BMP-2 and MMP-13, as well as by increased 45Ca2+ incorporation. Our results suggest that the effect of PRS on human MSC could be at least partially mediated by BMP-2.Activated autologous PRS could therefore provide an alternative to agents like recombinant bone growth factors by increasing osteoblastic differentiation of bone precursor cells at bone repair sites, although further studies are needed to fully support our observations.

  16. 抑制Runx2的表达增强BMP2诱导的干细胞成软骨分化%Suppression of Runx2 Potentiates BMP2-induced Chondrogenic Differentiation

    Institute of Scientific and Technical Information of China (English)

    孙泽绪; 赵辰; 廖军义; 王琦; 徐伟; 陈诚; 黄伟

    2016-01-01

    目的:利用腺病毒介导RNA干扰抑制成骨分化关键转录调控因子Runx2(Runt-related transcription factor 2)的表达,研究其对骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)诱导间充质干细胞(mesenchymal stem cells,MSCs)成软骨分化的影响,并初步探讨相关机制.方法:利用腺病毒Ad-GFP、Ad-BMP2、Ad-SiRunx2感染C3H10T1/2细胞;共分4组:GFP组、BMP2组、BMP2+ SiRunx2组和SiRunx2组.Alcian blue染色检测各组软骨细胞基质糖胺聚糖分泌;RT-PCR检测Runx2、早期成软骨标志物(Col2a1)、蛋白聚糖(Aggrecan)及晚期成软骨标志物(Col10a1) mRNA表达水平;Western blot检测目的蛋白BMP2、Ⅱ型胶原(Col2a1)及X型胶原(Col10a1)的蛋白表达.结果:在BMP2诱导C3H10T1/2细胞成软骨分化过程中,下调Runx2的表达可以增强Col2a1、Aggrecan及软骨细胞外基质糖胺聚糖的合成,抑制Col10a1的合成.结论:下调Runx2表达可以增强BMP2诱导间充质干细胞成软骨分化能力,并抑制软骨细胞的成熟和肥大.

  17. Bmp indicator mice reveal dynamic regulation of transcriptional response.

    Directory of Open Access Journals (Sweden)

    Anna L Javier

    Full Text Available Cellular responses to Bmp ligands are regulated at multiple levels, both extracellularly and intracellularly. Therefore, the presence of these growth factors is not an accurate indicator of Bmp signaling activity. While a common approach to detect Bmp signaling activity is to determine the presence of phosphorylated forms of Smad1, 5 and 8 by immunostaining, this approach is time consuming and not quantitative. In order to provide a simpler readout system to examine the presence of Bmp signaling in developing animals, we developed BRE-gal mouse embryonic stem cells and a transgenic mouse line that specifically respond to Bmp ligand stimulation. Our reporter identifies specific transcriptional responses that are mediated by Smad1 and Smad4 with the Schnurri transcription factor complex binding to a conserved Bmp-Responsive Element (BRE, originally identified among Drosophila, Xenopus and human Bmp targets. Our BRE-gal mES cells specifically respond to Bmp ligands at concentrations as low as 5 ng/ml; and BRE-gal reporter mice, derived from the BRE-gal mES cells, show dynamic activity in many cellular sites, including extraembryonic structures and mammary glands, thereby making this a useful scientific tool.

  18. BMP-7 PROTEIN EXPRESSION IS DOWNREGULATED IN HUMAN DIABETIC NEPHROPATHY.

    Science.gov (United States)

    Ivanac-Janković, Renata; Ćorić, Marijana; Furić-Čunko, Vesna; Lovičić, Vesna; Bašić-Jukić, Nikolina; Kes, Petar

    2015-06-01

    Bone morphogenetic protein-7 (BMP-7) is expressed in all parts of the normal kidney parenchyma, being highest in the epithelium of proximal tubules. It protects kidney against acute and chronic injury, inflammation and fibrosis. Diabetic nephropathy is the leading cause of chronic kidney disease, and is characterized by decreased expression of BMP-7. The aim of our study was to analyze whether the expression of BMP-7 is significantly changed in advanced stages of human diabetic nephropathy. Immunohistochemical analysis of the expression of BMP-7 was performed on archival material of 30 patients that underwent renal biopsy and had confirmed diagnosis of diabetic nephropathy. Results showed that BMP-7 was differently expressed in the cytoplasm of epithelial cells of proximal tubules and podocytes among all stages of diabetic nephropathy. At early stages of diabetic nephropathy, BMP-7 was strongly positive in proximal tubules and podocytes, while low expression was recorded in the majority of samples at advanced stages. In conclusion, increased expression of BMP-7 at initial stages of diabetic nephropathy with subsequent decrease at advanced stage highlights the role of BMP-7 in the protection of kidney structure and function. Further investigations should be focused on disturbances of BMP-7 receptors and signaling pathways in patients with diabetic nephropathy.

  19. A dynamic Shh expression pattern, regulated by SHH and BMP signaling, coordinates fusion of primordia in the amniote face.

    Science.gov (United States)

    Hu, Diane; Young, Nathan M; Li, Xin; Xu, Yanhua; Hallgrímsson, Benedikt; Marcucio, Ralph S

    2015-02-01

    The mechanisms of morphogenesis are not well understood, yet shaping structures during development is essential for establishing correct organismal form and function. Here, we examine mechanisms that help to shape the developing face during the crucial period of facial primordia fusion. This period of development is a time when the faces of amniote embryos exhibit the greatest degree of similarity, and it probably results from the necessity for fusion to occur to establish the primary palate. Our results show that hierarchical induction mechanisms, consisting of iterative signaling by Sonic hedgehog (SHH) followed by Bone morphogenetic proteins (BMPs), regulate a dynamic expression pattern of Shh in the ectoderm covering the frontonasal (FNP) and maxillary (MxP) processes. Furthermore, this Shh expression domain contributes to the morphogenetic processes that drive the directional growth of the globular process of the FNP toward the lateral nasal process and MxP, in part by regulating cell proliferation in the facial mesenchyme. The nature of the induction mechanism that we discovered suggests that the process of fusion of the facial primordia is intrinsically buffered against producing maladaptive morphologies, such as clefts of the primary palate, because there appears to be little opportunity for variation to occur during expansion of the Shh expression domain in the ectoderm of the facial primordia. Ultimately, these results might explain why this period of development constitutes a phylotypic stage of facial development among amniotes.

  20. Cell signalling pathways underlying induced pluripotent stem cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Kate; Hawkins; Shona; Joy; Tristan; Mc; Kay

    2014-01-01

    Induced pluripotent stem(i PS) cells, somatic cells reprogrammed to the pluripotent state by forced expression of defined factors, represent a uniquely valuable resource for research and regenerative medicine. However, this methodology remains inefficient due to incomplete mechanistic understanding of the reprogramming process. In recent years, various groups have endeavoured to interrogate the cell signalling that governs the reprogramming process, including LIF/STAT3, BMP, PI3 K, FGF2, Wnt, TGFβ and MAPK pathways, with the aim of increasing our understanding and identifying new mechanisms of improving safety, reproducibility and efficiency. This has led to a unified model of reprogramming that consists of 3 stages: initiation, maturation and stabilisation. Initiation of reprogramming occurs in almost all cells that receive the reprogramming transgenes; most commonly Oct4, Sox2, Klf4 and c Myc, and involves a phenotypic mesenchymal-to-epithelial transition. The initiation stage is also characterised by increased proliferation and a metabolic switch from oxidative phosphorylation to glycolysis. The maturation stage is considered the major bottleneck within the process, resulting in very few "stabilisation competent" cells progressing to the final stabilisation phase. To reach this stage in both mouse and human cells, pre-i PS cells must activate endogenous expression of the core circuitry of pluripotency, comprising Oct4, Sox2, and Nanog, and thus reach a state of transgene independence. By the stabilisation stage, i PS cells generally use the same signalling networks that govern pluripotency in embryonic stem cells. These pathways differ between mouse and human cells although recent work has demonstrated that this is context dependent. As i PS cell generation technologies move forward, tools are being developed to interrogate the process in more detail, thus allowing a greater understanding of this intriguing biological phenomenon.

  1. 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.

  2. Dopaminergic neuronal differentiation from the forebrain-derived human neural stem cells induced in cultures by using a combination of BMP-7 and pramipexole with growth factors

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    Hongna eYang

    2016-04-01

    Full Text Available Transplantation of dopaminergic (DA neurons is considered to be the most promising therapeutic strategy for replacing degenerated dopamine cells in the midbrain of Parkinson’s disease (PD, thereby restoring normal neural circuit function and slow clinical progression of the disease. Human neural stem cells (hNSCs derived from fetal forebrain are thought to be the important cell sources for producing DA neurons because of their multipotency for differentiation and long-term expansion property in cultures. However, low DA differentiation of the forebrain-derived hNSCs limited their therapeutic potential in PD. In the current study, we explored a combined application of Pramipexole (PRX, bone morphogenetic proteins 7 (BMP-7, and growth factors, including acidic fibroblast factor (aFGF, forskolin, and phorbol-12-myristae-13-acetate (TPA, to induce differentiation of forebrain-derived hNSCs towards DA neurons in cultures. We found that DA neuron-associated genes, including Nurr1, Neurogenin2 (Ngn2, and tyrosine hydroxylase (TH were significantly increased after 24h of differentiation by RT-PCR analysis (p<0.01. Fluorescent examination showed that about 25% of cells became TH-positive neurons at 24h, about 5% of cells became VMAT2 (vascular monoamine transporter 2-positive neurons, and less than 5% of cells became DAT (dopamine transporter-positive neurons at 72h following differentiation in cultures. Importantly, these TH-, VMAT2- and DAT-expressing neurons were able to release dopamine into cultures under both of the basal and evoked conditions. Dopamine levels released by DA neurons produced using our protocol were significantly higher compared to the control groups (P<0.01, as examined by ELISA. Our results demonstrated that the combination of PRX, BMP-7, and growth factors was able to greatly promote differentiation of the forebrain-derived hNSCs into DA-releasing neurons.

  3. A BMP regulatory network controls ectodermal cell fate decisions at the neural plate border.

    Science.gov (United States)

    Reichert, Sabine; Randall, Rebecca A; Hill, Caroline S

    2013-11-01

    During ectodermal patterning the neural crest and preplacodal ectoderm are specified in adjacent domains at the neural plate border. BMP signalling is required for specification of both tissues, but how it is spatially and temporally regulated to achieve this is not understood. Here, using a transgenic zebrafish BMP reporter line in conjunction with double-fluorescent in situ hybridisation, we show that, at the beginning of neurulation, the ventral-to-dorsal gradient of BMP activity evolves into two distinct domains at the neural plate border: one coinciding with the neural crest and the other abutting the epidermis. In between is a region devoid of BMP activity, which is specified as the preplacodal ectoderm. We identify the ligands required for these domains of BMP activity. We show that the BMP-interacting protein Crossveinless 2 is expressed in the BMP activity domains and is under the control of BMP signalling. We establish that Crossveinless 2 functions at this time in a positive-feedback loop to locally enhance BMP activity, and show that it is required for neural crest fate. We further demonstrate that the Distal-less transcription factors Dlx3b and Dlx4b, which are expressed in the preplacodal ectoderm, are required for the expression of a cell-autonomous BMP inhibitor, Bambi-b, which can explain the specific absence of BMP activity in the preplacodal ectoderm. Taken together, our data define a BMP regulatory network that controls cell fate decisions at the neural plate border.

  4. Regulatory role of BMP-9 in steroidogenesis by rat ovarian granulosa cells.

    Science.gov (United States)

    Hosoya, Takeshi; Otsuka, Fumio; Nakamura, Eri; Terasaka, Tomohiro; Inagaki, Kenichi; Tsukamoto-Yamauchi, Naoko; Hara, Takayuki; Toma, Kishio; Komatsubara, Motoshi; Makino, Hirofumi

    2015-03-01

    BMPs expressed in the ovary differentially regulate steroidogenesis by granulosa cells. BMP-9, a circulating BMP, is associated with cell proliferation, apoptosis and differentiation in various tissues. However, the effects of BMP-9 on ovarian function have yet to be elucidated. Here we investigated BMP-9 actions on steroidogenesis using rat primary granulosa cells. BMP-9 potently suppressed FSH-induced progesterone production, whereas it did not affect FSH-induced estradiol production by granulosa cells. The effects of BMP-9 on FSH-induced steroidogenesis were not influenced by the presence of oocytes. FSH-induced cAMP synthesis and FSH-induced mRNA expression of steroidogenic factors, including StAR, P450scc, 3βHSD2 and FSHR, were suppressed by treatment with BMP-9. BMP-9 mRNA expression was detected in granulosa cells but not in oocytes. BMP-9 readily activated Smad1/5/8 phosphorylation and Id-1 transcription in granulosa cells. Analysis using ALK inhibitors indicated that BMP-9 actions were mediated via type-I receptors other than ALK-2, -3 and -6. Furthermore, experiments using extracellular domains (ECDs) for BMP type-I and -II receptor constructs revealed that the effects of BMP-9 were reversed by ECDs for ALK-1 and BMPRII. Thus, the functional receptors for BMP-9 in granulosa cells were most likely to be the complex of ALK-1 and BMPRII. Collectively, the results of the present study showed that BMP-9 can affect luteinization and that there are two possible sources of BMP-9, serum and granulosa cells in the ovary.

  5. Bone Morphogenetic Protein-7 Antagonizes Myocardial Fibrosis Induced by Atrial Fibrillation by Restraining Transforming Growth Factor-β (TGF-β)/Smads Signaling

    Science.gov (United States)

    Chen, Xinjun; Xu, Jing; Jiang, Baozhou; Liu, Danping

    2016-01-01

    Background This aim of this study was to investigate the expression of BMP-7 in atrial fibrillation and illuminate the role of BMP-7 and TGF-β/Smads signaling in myocardial fibrosis. Material/Methods Fibrosis of myocardial fibroblasts was induced by TGF-β1 and the optimal condition was determined by the MTT assay. Cells with TGF-β1 treatment were sub-divided into 4 groups: TGF-β1 group, TGF-β1 + Smad3 siRNA group, TGF-β1 + BMP-7 group, and TGF-β1 + BMP-7 + Smad1/5 siRNA group. Cells were then analyzed by detecting the expression of epithelial cadherin (E-cadherin), collagen I, alpha smooth muscle cell actin (α-SMA), and activated Smads using Western blot. Mice were injected daily with Ach-CaCl2 with or without the addition of BMP-7 and Smad1/5 siRNA over a period of 4 weeks. Cardiac functions were tested by echocardiogram assay and fibrosis was diagnosed by histopathological examination. Finally, molecule biomarkers were detected using standard procedures. Results TGF-β1 treatment significantly down-regulated E-cadherin expression and up-regulated expressions of Collagen I, α-SMA, and pSmad3 (P<0.05). The effects of TGF-β1 treatment can be significantly suppressed by Smad3 siRNA (P<0.05). Cells in the BMP-7 group exhibited significantly higher expression levels of E-cadherin and pSmad1/5 together with lower expression levels of pSmad3, collagen I, and α-SMA (P<0.05). Moreover, Smad1/5 siRNA can substantially repress the effects of BMP-7 (P<0.05) and results from the mice model coincided with those in myocardial fibroblasts. Conclusions BMP-7 can regulate TGF-β1/Smad3 by targeting Smad1/5 to antagonize fibrosis in myocardial fibroblasts resulting from atrial fibrillation. PMID:27677228

  6. Negative Fgf8-Bmp2 feed-back is regulated by miR-130 during early cardiac specification.

    Science.gov (United States)

    Lopez-Sanchez, Carmen; Franco, Diego; Bonet, Fernando; Garcia-Lopez, Virginio; Aranega, Amelia; Garcia-Martinez, Virginio

    2015-10-01

    It is known that secreted proteins from the anterior lateral endoderm, FGF8 and BMP2, are involved in mesodermal cardiac differentiation, which determines the first cardiac field, defined by the expression of the earliest specific cardiac markers Nkx-2.5 and Gata4. However, the molecular mechanisms responsible for early cardiac development still remain unclear. At present, microRNAs represent a novel layer of complexity in the regulatory networks controlling gene expression during cardiovascular development. This paper aims to study the role of miR130 during early cardiac specification. Our model is focused on developing chick at gastrula stages. In order to identify those regulatory factors which are involved in cardiac specification, we conducted gain- and loss-of-function experiments in precardiac cells by administration of Fgf8, Bmp2 and miR130, through in vitro electroporation technique and soaked beads application. Embryos were subjected to in situ hybridization, immunohistochemistry and qPCR procedures. Our results reveal that Fgf8 suppresses, while Bmp2 induces, the expression of Nkx-2.5 and Gata4. They also show that Fgf8 suppresses Bmp2, and vice versa. Additionally, we observed that Bmp2 regulates miR-130 -a putative microRNA that targets Erk1/2 (Mapk1) 3'UTR, recognizing its expression in precardiac cells which overlap with Erk1/2 pattern. Finally, we evidence that miR-130 is capable to inhibit Erk1/2 and Fgf8, resulting in an increase of Bmp2, Nkx-2.5 and Gata4. Our data present miR-130 as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2, establishing a negative feed-back loop responsible to achieve early cardiac specification.

  7. Laminins, via heparan sulfate proteoglycans, participate in zebrafish myotome morphogenesis by modulating the pattern of Bmp responsiveness.

    Science.gov (United States)

    Dolez, Morgane; Nicolas, Jean-François; Hirsinger, Estelle

    2011-01-01

    In zebrafish, Hedgehog-induced Engrailed expression defines a muscle fibre population that includes both slow and fast fibre types and exhibits an organisational role on myotome and surrounding tissues, such as motoneurons and lateral line. This Engrailed-positive population is restricted in the myotome to a central domain. To understand how this population is established, we have analysed the phenotype of the sly/lamc1 mutation in the Laminin γ1 chain that was shown to specifically affect Engrailed expression in pioneers. We find that the sly mutation affects Engrailed expression in the entire central domain and that Hedgehog signalling does not mediate this effect. We show that Bmp-responding cells are excluded from the central domain and that this pattern is modulated by laminins, but not by Hedgehog signalling. Knockdown of Bmp signalling rescues Engrailed expression in the sly mutant and ectopically activates Engrailed expression in slow and fast lineages in wild-type embryos. Last, extracellular matrix-associated heparan sulfate proteoglycans are absent in sly and their enzymatic removal mimics the sly phenotype. Our results therefore show that laminins, via heparan sulfate proteoglycans, are instrumental in patterning Bmp responsiveness and that Bmp signalling restricts Engrailed expression to the central domain. This study underlines the importance of extracellular cues for the precise spatial modulation of cell response to morphogens.

  8. Induced signals in resistive plate chambers

    CERN Document Server

    Riegler, W

    2002-01-01

    We derive theorems for induced signals on electrodes embedded in a medium with a position and frequency dependent permittivity $\\vep(\\vx,s)$ and conductivity $\\sigma(\\vx,s)$ that are connected with arbitrary discrete elements. The problem is treated using the quasi-static approximation of Maxwell's equations for weakly conducting media \\cite{melcher}\\cite{quasi}. The induced signals can be derived by time dependent weighting fields and potentials and the result is the same as the one given in \\cite{gatti}. We also show how these time dependent weighting fields can be derived from electrostatic solutions. Finally we will apply the results to Resistive Plate Chambers (RPCs) where we discuss the effects of the resistive plates and thin resistive layers on the signals induced on plane electrodes and strips.

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

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    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

  10. HIF-1α potentiates BMP2-induced chondrogenic differentiation but inhibits osteogenic differentiation in stem cells%低氧诱导因子-1α对骨形态发生蛋白2诱导的干细胞成软骨、成骨分化的影响

    Institute of Scientific and Technical Information of China (English)

    周年; 黄伟; 廖军义; 胡宁; 陈筱蓉; 梁熙; 司维柯; 杨忠; 易世雄

    2014-01-01

    目的 探讨低氧通路中关键转录调控因子低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)对骨形态发生蛋白2(bone morpho-genetic protein 2,BMP2)诱导干细胞骨、软骨分化的影响,阐明HIF-1 α在干细胞成骨、软骨分化中的作用.方法 构建相应腺病毒AdBMP2、AdHIF-1 α、AdGFP,单独或共同感染干细胞,Western blot法检测成软骨、成骨分化关键转录调控因子Sox9、Runx2的表达,Real-time PCR法检测成软骨、成骨分化标志物COL2A1、aggrecan、COL1 A1和ALP mRNA表达,Alcian blue、ALP及Alizarin red S染色检测软骨细胞外基质及骨基质钙盐沉积情况.进行干细胞裸鼠皮下移植,观察不同处理组形成骨块的组织结构情况,探讨HIF-1α对BMP2诱导干细胞成骨、软骨分化的影响.结果 诱导分化后第1、3天,BMP2+ HIF-1α组Sox9蛋白表达明显高于BMP2单独处理组,而BMP2+ HIF-1α组Runx2蛋白表达明显低于BMP2单独处理组.诱导分化后第7、9天,BMP2+ HIF-1α组COL2A1、aggrecan mRNA相对表达明显高于BMP2单独处理组(P<0.05),而BMP2+ HIF-1α组COL1 A1、ALP mRNA相对表达明显低于BMP2单独处理组(P<0.05).Alcianblue染色发现BMP2+ HIF-1α组软骨细胞外基质分泌多于BMP2单独处理组,染色更深;ALP染色发现BMP2+ HIF-1 α组ALP的活性弱于BMP2单独处理组;茜素红染色发现BMP2+ HIF-1α组较BMP2单独处理组骨基质钙盐沉积更少;体内试验组织学观察见BMP2+ HIF-1α组软骨成分更多,骨化不明显,BMP2单独处理组软骨成分少,软骨内骨化更明显.结论 HIF-1α明显增强了BMP2诱导的干细胞成软骨分化,抑制了成骨分化及软骨内骨化,维持了软骨分化表型.

  11. 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.

  12. Tgfβ2 and 3 are coexpressed with their extracellular regulator Ltbp1 in the early limb bud and modulate mesodermal outgrowth and BMP signaling in chicken embryos

    Directory of Open Access Journals (Sweden)

    Garcia-Porrero Juan A

    2010-06-01

    Full Text Available Abstract Background Transforming growth factor β proteins (Tgfβs are secreted cytokines with well-defined functions in the differentiation of the musculoskeletal system of the developing limb. Here we have studied in chicken embryos, whether these cytokines are implicated in the development of the embryonic limb bud at stages preceding tissue differentiation. Results Immunohistochemical detection of phosphorylated Smad2 and Smad3 indicates that signaling by this pathway is active in the undifferentiated mesoderm and AER. Gene expression analysis shows that transcripts of tgfβ2 and tgfβ3 but not tgfβ1 are abundant in the growing undifferentiated limb mesoderm. Transcripts of tgfβ2 are also found in the AER, which is the signaling center responsible for limb outgrowth. Furthermore, we show that Latent Tgfβ Binding protein 1 (LTBP1, which is a key extracellular modulator of Tgfβ ligand bioavailability, is coexpressed with Tgfβs in the early limb bud. Administration of exogenous Tgfβs to limb buds growing in explant cultures provides evidence of these cytokines playing a role in the regulation of mesodermal limb proliferation. In addition, analysis of gene regulation in these experiments revealed that Tgfβ signaling has no effect on the expression of master genes of musculoskeletal tissue differentiation but negatively regulates the expression of the BMP-antagonist Gremlin. Conclusion We propose the occurrence of an interplay between Tgfβ and BMP signaling functionally associated with the regulation of early limb outgrowth by modulating limb mesenchymal cell proliferation.

  13. 骨钙素在BMP2/7异源二聚体诱导种植体周围骨缺损再生中的表达%Expression of osteocalcin using rhBMP2/7 heterodimer in peri-implant bone defects compared to BMP2 homodimer and BMP7 homodimer

    Institute of Scientific and Technical Information of China (English)

    孙平; 王利民; 冯剑颖

    2011-01-01

    目的:研究骨钙素在使用BMP2/7异源二聚体促进种植体周骨缺损再生中的表达.方法:建立小型猪种植体周骨缺损模型,并使用BMP2/7异源二聚体及BMP2、BMP7同源二聚体促进骨再生,采用免疫组化方法分别在2、3、6周时检测新骨中骨钙素表达.结果:在各实验组中,骨钙素3周时表达达到最高值.在2、3、6周,BMP2/7组骨钙素表达均强于BMP2、BMP7同源二聚体组及对照组.结论:在以同样较低浓度(30ng/ml)作用于小型猪种植体周骨缺损诱导新骨形成过程中,BMP2/7异源二聚体较BMP、BMP7同源二聚体有效促进骨钙素的表达.%To delineate expression of osteocalcin induced by recombinant human bone morphogenetic protein (BMP)2/7 heterodimer in peri-implant bone defects in comparison to BMP2 and BMP7 homodimer.Methods: Identical peri-implant bone defects were created on the frontal skull of minipig. Collagen sponges with the same dose BMP2/7 heterodimer、 BMP2 homodimer 、 BMP7 homodimer 、 or BMPs were adopted to treat the freshly created implant bed. Titanium implants were centrally implanted with 4mm-flxture within bone defects. Immunohistochemical method was applied to evaluate the expression of osteocalcin (OCN) after 2, 3, and 6 weeks of implantation. Results: The OCN proteins displayed intensity peaks at 3 weeks (P<0.05) in all the groups. Immunoreactive expression of OCN of BMP2/7 group was more intensity than BMP2、 BMP7 and control group at 2, 3, and 6 weeks post-operation (P<0.05). Conclusions: Purified recombinant human BMP2/7 heterodimer can enhance the expression of OCN with significant differences over BMP2 homodimers or BMP7 homodimers with the same low-dose (30ng/ml).

  14. Distinct steps of neural induction revealed by Asterix, Obelix and TrkC, genes induced by different signals from the organizer.

    Directory of Open Access Journals (Sweden)

    Sonia Pinho

    Full Text Available The amniote organizer (Hensen's node can induce a complete nervous system when grafted into a peripheral region of a host embryo. Although BMP inhibition has been implicated in neural induction, non-neural cells cannot respond to BMP antagonists unless previously exposed to a node graft for at least 5 hours before BMP inhibitors. To define signals and responses during the first 5 hours of node signals, a differential screen was conducted. Here we describe three early response genes: two of them, Asterix and Obelix, encode previously undescribed proteins of unknown function but Obelix appears to be a nuclear RNA-binding protein. The third is TrkC, a neurotrophin receptor. All three genes are induced by a node graft within 4-5 hours but they differ in the extent to which they are inducible by FGF: FGF is both necessary and sufficient to induce Asterix, sufficient but not necessary to induce Obelix and neither sufficient nor necessary for induction of TrkC. These genes are also not induced by retinoic acid, Noggin, Chordin, Dkk1, Cerberus, HGF/SF, Somatostatin or ionomycin-mediated Calcium entry. Comparison of the expression and regulation of these genes with other early neural markers reveals three distinct "epochs", or temporal waves, of gene expression accompanying neural induction by a grafted organizer, which are mirrored by specific stages of normal neural plate development. The results are consistent with neural induction being a cascade of responses elicited by different signals, culminating in the formation of a patterned nervous system.

  15. Pax9 regulates a molecular network involving Bmp4, Fgf10, Shh signaling and the Osr2 transcription factor to control palate morphogenesis.

    Science.gov (United States)

    Zhou, Jing; Gao, Yang; Lan, Yu; Jia, Shihai; Jiang, Rulang

    2013-12-01

    Cleft palate is one of the most common birth defects in humans. Whereas gene knockout studies in mice have shown that both the Osr2 and Pax9 transcription factors are essential regulators of palatogenesis, little is known about the molecular mechanisms involving these transcription factors in palate development. We report here that Pax9 plays a crucial role in patterning the anterior-posterior axis and outgrowth of the developing palatal shelves. We found that tissue-specific deletion of Pax9 in the palatal mesenchyme affected Shh expression in palatal epithelial cells, indicating that Pax9 plays a crucial role in the mesenchyme-epithelium interactions during palate development. We found that expression of the Bmp4, Fgf10, Msx1 and Osr2 genes is significantly downregulated in the developing palatal mesenchyme in Pax9 mutant embryos. Remarkably, restoration of Osr2 expression in the early palatal mesenchyme through a Pax9(Osr2KI) allele rescued posterior palate morphogenesis in the absence of Pax9 protein function. Our data indicate that Pax9 regulates a molecular network involving the Bmp4, Fgf10, Shh and Osr2 pathways to control palatal shelf patterning and morphogenesis.

  16. Kaempferol inhibits vascular smooth muscle cell migration by modulating BMP-mediated miR-21 expression.

    Science.gov (United States)

    Kim, Kwangho; Kim, Sunghwan; Moh, Sang Hyun; Kang, Hara

    2015-09-01

    Bioflavonoids are known to induce cardioprotective effects by inhibiting vascular smooth muscle cell (VSMC) proliferation and migration. Kaempferol has been shown to inhibit VSMC proliferation. However, little is known about the effect of kaempferol on VSMC migration and the underlying molecular mechanisms. Our studies provide the first evidence that kaempferol inhibits VSMC migration by modulating the BMP4 signaling pathway and microRNA expression levels. Kaempferol activates the BMP signaling pathway, induces miR-21 expression and downregulates DOCK4, 5, and 7, leading to inhibition of cell migration. Moreover, kaempferol antagonizes the PDGF-mediated pro-migratory effect. Therefore, our study uncovers a novel regulatory mechanism of VSMC migration by kaempferol and suggests that miRNA modulation by kaempferol is a potential therapy for cardiovascular diseases.

  17. 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.

  18. Mechanisms of UV-induced signal transduction

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    Kulms, D.; Schwarz, T. [Univ. Muenster, Muenster (Germany). Ludwing Boltzmann Inst. for Cell Biology and Immunobiology of the Skin

    2002-04-01

    Ultraviolet radiation (UV) causes a variety of biological effects that can be either beneficial or harmful for human health. To exert these effects on a cellular basis, UV uses a variety of signaling pathways. DNA is the major chromophore for UVB. Thus, nuclear DNA damage has been detected to be a major mediator of numerous UVB effects, and experimental reduction of DNA damage is associated with a loss of these effects. On the other hand, UV has been found to utilize molecular components within the cytoplasm or at the cell membrane for signaling. UV can directly activate cell surface receptors, kinases, and transcription factors. The nuclear and extranuclear signaling pathways are generated independently and have been recently recognized to be not mutually exclusive but to contribute to various UV effects in an independent and additive way. Further knowledge of how these signaling pathways relate to each other will certainly increase our understanding of how UV acts as a pathogen. The following review will briefly discuss current aspects of the mechanisms involved in UV-induced signal transduction. (author)

  19. Genetic analysis reveals an unexpected role of BMP7 in initiation of ureteric bud outgrowth in mouse embryos.

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    Alexandre Gonçalves

    Full Text Available BACKGROUND: Genetic analysis in the mouse revealed that GREMLIN1 (GREM1-mediated antagonism of BMP4 is essential for ureteric epithelial branching as the disruption of ureteric bud outgrowth and renal agenesis in Grem1-deficient embryos is restored by additional inactivation of one Bmp4 allele. Another BMP ligand, BMP7, was shown to control the proliferative expansion of nephrogenic progenitors and its requirement for nephrogenesis can be genetically substituted by Bmp4. Therefore, we investigated whether BMP7 in turn also participates in inhibiting ureteric bud outgrowth during the initiation of metanephric kidney development. METHODOLOGY/PRINCIPAL FINDINGS: Genetic inactivation of one Bmp7 allele in Grem1-deficient mouse embryos does not alleviate the bilateral renal agenesis, while complete inactivation of Bmp7 restores ureteric bud outgrowth and branching. In mouse embryos lacking both Grem1 and Bmp7, GDNF/WNT11 feedback signaling and the expression of the Etv4 target gene, which regulates formation of the invading ureteric bud tip, are restored. In contrast to the restoration of ureteric bud outgrowth and branching, nephrogenesis remains aberrant as revealed by the premature loss of Six2 expressing nephrogenic progenitor cells. Therefore, very few nephrons develop in kidneys lacking both Grem1 and Bmp7 and the resulting dysplastic phenotype is indistinguishable from the one of Bmp7-deficient mouse embryos. CONCLUSIONS/SIGNIFICANCE: Our study reveals an unexpected inhibitory role of BMP7 during the onset of ureteric bud outgrowth. As BMP4, BMP7 and GREM1 are expressed in distinct mesenchymal and epithelial domains, the localized antagonistic interactions of GREM1 with BMPs could restrict and guide ureteric bud outgrowth and branching. The robustness and likely significant redundancy of the underlying signaling system is evidenced by the fact that global reduction of Bmp4 or inactivation of Bmp7 are both able to restore ureteric bud outgrowth

  20. Bone morphogenetic protein-5 (BMP-5 promotes dendritic growth in cultured sympathetic neurons

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    Higgins Dennis

    2001-09-01

    Full Text Available Abstract Background BMP-5 is expressed in the nervous system throughout development and into adulthood. However its effects on neural tissues are not well defined. BMP-5 is a member of the 60A subgroup of BMPs, other members of which have been shown to stimulate dendritic growth in central and peripheral neurons. We therefore examined the possibility that BMP-5 similarly enhances dendritic growth in cultured sympathetic neurons. Results Sympathetic neurons cultured in the absence of serum or glial cells do not form dendrites; however, addition of BMP-5 causes these neurons to extend multiple dendritic processes, which is preceded by an increase in phosphorylation of the Smad-1 transcription factor. The dendrite-promoting activity of BMP-5 is significantly inhibited by the BMP antagonists noggin and follistatin and by a BMPR-IA-Fc chimeric protein. RT-PCR and immunocytochemical analyses indicate that BMP-5 mRNA and protein are expressed in the superior cervical ganglia (SCG during times of initial growth and rapid expansion of the dendritic arbor. Conclusions These data suggest a role for BMP-5 in regulating dendritic growth in sympathetic neurons. The signaling pathway that mediates the dendrite-promoting activity of BMP-5 may involve binding to BMPR-IA and activation of Smad-1, and relative levels of BMP antagonists such as noggin and follistatin may modulate BMP-5 signaling. Since BMP-5 is expressed at relatively high levels not only in the developing but also the adult nervous system, these findings suggest the possibility that BMP-5 regulates dendritic morphology not only in the developing, but also the adult nervous system.

  1. Dkk1 haploinsufficiency requires expression of Bmp2 for bone anabolic activity.

    Science.gov (United States)

    Intini, Giuseppe; Nyman, Jeffry S

    2015-06-01

    Bone fractures remain a serious health burden and prevention and enhanced healing of fractures have been obtained by augmenting either BMP or Wnt signaling. However, whether BMP and Wnt signaling are both required or are self-sufficient for anabolic and fracture healing activities has never been fully elucidated. Mice haploinsufficient for Dkk1 (Dkk1(+/-)) exhibit a high bone mass phenotype due to an up-regulation of canonical Wnt signaling while mice lacking Bmp2 expression in the limbs (Bmp2(c/c);Prx1::cre) succumb to spontaneous fracture and are unable to initiate fracture healing; combined, these mice offer an opportunity to examine the requirement for activated BMP signaling on the anabolic and fracture healing activity of Wnts. When Dkk1(+/-) mice were crossed with Bmp2(c/c);Prx1::cre mice, the offspring bearing both genetic alterations were unable to increase bone mass and heal fractures, indicating that increased canonical Wnt signaling is unable to exploit its activity in absence of Bmp2. Thus, our data suggest that BMP signaling is required for Wnt-mediated anabolic activity and that therapies aimed at preventing fractures and fostering fracture repair may need to target both pathways for maximal efficacy.

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

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    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.

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    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.

  4. 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

  5. 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

    2016-05-05

    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.

  6. Hedgehog target genes: mechanisms of carcinogenesis induced by aberrant hedgehog signaling activation.

    Science.gov (United States)

    Katoh, Y; Katoh, M

    2009-09-01

    Hedgehog signaling is aberrantly activated in glioma, medulloblastoma, basal cell carcinoma, lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, and other tumors. Hedgehog signals activate GLI family members via Smoothened. RTK signaling potentiates GLI activity through PI3K-AKT-mediated GSK3 inactivation or RAS-STIL1-mediated SUFU inactivation, while GPCR signaling to Gs represses GLI activity through adenylate cyclase-mediated PKA activation. GLI activators bind to GACCACCCA motif to regulate transcription of GLI1, PTCH1, PTCH2, HHIP1, MYCN, CCND1, CCND2, BCL2, CFLAR, FOXF1, FOXL1, PRDM1 (BLIMP1), JAG2, GREM1, and Follistatin. Hedgehog signals are fine-tuned based on positive feedback loop via GLI1 and negative feedback loop via PTCH1, PTCH2, and HHIP1. Excessive positive feedback or collapsed negative feedback of Hedgehog signaling due to epigenetic or genetic alterations leads to carcinogenesis. Hedgehog signals induce cellular proliferation through upregulation of N-Myc, Cyclin D/E, and FOXM1. Hedgehog signals directly upregulate JAG2, indirectly upregulate mesenchymal BMP4 via FOXF1 or FOXL1, and also upregulate WNT2B and WNT5A. Hedgehog signals induce stem cell markers BMI1, LGR5, CD44 and CD133 based on cross-talk with WNT and/or other signals. Hedgehog signals upregulate BCL2 and CFLAR to promote cellular survival, SNAI1 (Snail), SNAI2 (Slug), ZEB1, ZEB2 (SIP1), TWIST2, and FOXC2 to promote epithelial-to-mesenchymal transition, and PTHLH (PTHrP) to promote osteolytic bone metastasis. KAAD-cyclopamine, Mu-SSKYQ-cyclopamine, IPI-269609, SANT1, SANT2, CUR61414 and HhAntag are small-molecule inhibitors targeted to Smoothened, GANT58, GANT61 to GLI1 and GLI2, and Robot-nikinin to SHH. Hedgehog signaling inhibitors should be used in combination with RTK inhibitors, GPCR modulators, and/or irradiation for cancer therapy.

  7. Mouse Oocytes Enable LH-Induced Maturation of the Cumulus-Oocyte Complex via Promoting EGF Receptor-Dependent Signaling

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    Su, You-Qiang; Sugiura, Koji; Li, Qinglei; Wigglesworth, Karen; Matzuk, Martin M.; Eppig, John J.

    2010-01-01

    LH triggers the maturation of the cumulus-oocyte complex (COC), which is followed by ovulation. These ovarian follicular responses to LH are mediated by epidermal growth factor (EGF)-like growth factors produced by granulosa cells and require the participation of oocyte-derived paracrine factors. However, it is not clear how oocytes coordinate with the EGF receptor (EGFR) signaling to achieve COC maturation. The aim of the present study was to test the hypothesis that oocytes promote the expression of EGFR by cumulus cells, thus enabling them to respond to the LH-induced EGF-like peptides. Egfr mRNA and protein expression were dramatically reduced in cumulus cells of mutant mice deficient in the production of the oocyte-derived paracrine factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15). Moreover, microsurgical removal of oocytes from wild-type COCs dramatically reduced expression of Egfr mRNA and protein, and these levels were restored by either coculture with oocytes or treatment with recombinant GDF9 or GDF9 plus recombinant BMP15. Blocking Sma- and Mad-related protein (SMAD)2/3 phosphorylation in vitro inhibited Egfr expression in wild-type COCs and in GDF9-treated wild-type cumulus cells, and conditional deletion of Smad2 and Smad3 genes in granulosa cells in vivo resulted in the reduction of Egfr mRNA in cumulus cells. These results indicate that oocytes promote expression of Egfr in cumulus cells, and a SMAD2/3-dependent pathway is involved in this process. At least two oocyte-derived growth factors, GDF9 and BMP15, are required for EGFR expression by cumulus cells. PMID:20382892

  8. A phenotypic screen in zebrafish identifies a novel small-molecule inducer of ectopic tail formation suggestive of alterations in non-canonical Wnt/PCP signaling.

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    Evelien Gebruers

    Full Text Available Zebrafish have recently emerged as an attractive model for the in vivo bioassay-guided isolation and characterization of pharmacologically active small molecules of natural origin. We carried out a zebrafish-based phenotypic screen of over 3000 plant-derived secondary metabolite extracts with the goal of identifying novel small-molecule modulators of the BMP and Wnt signaling pathways. One of the bioactive plant extracts identified in this screen - Jasminum gilgianum, an Oleaceae species native to Papua New Guinea - induced ectopic tails during zebrafish embryonic development. As ectopic tail formation occurs when BMP or non-canonical Wnt signaling is inhibited during the tail protrusion process, we suspected a constituent of this extract to act as a modulator of these pathways. A bioassay-guided isolation was carried out on the basis of this zebrafish phenotype, identifying para-coumaric acid methyl ester (pCAME as the active compound. We then performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos, including a tissue-specific marker analysis of the secondary tails. We found pCAME to synergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails, and causing convergence-extension defects in compound-treated embryos. These results indicate that pCAME may interfere with non-canonical Wnt signaling. Inhibition of Jnk, a downstream target of Wnt/PCP signaling (via morpholino antisense knockdown and pharmacological inhibition with the kinase inhibitor SP600125 phenocopied pCAME-treated embryos. However, immunoblotting experiments revealed pCAME to not directly inhibit Jnk-mediated phosphorylation of c-Jun, suggesting additional targets of SP600125, and/or other pathways, as possibly being involved in the ectopic tail formation activity of pCAME. Further investigation of pCAME's mechanism of action will help determine this compound's pharmacological utility.

  9. Zygotic LvBMP5-8 is required for skeletal patterning and for left-right but not dorsal-ventral specification in the sea urchin embryo.

    Science.gov (United States)

    Piacentino, Michael L; Chung, Oliver; Ramachandran, Janani; Zuch, Daniel T; Yu, Jia; Conaway, Evan A; Reyna, Arlene E; Bradham, Cynthia A

    2016-04-01

    Skeletal patterning in the sea urchin embryo requires coordinated signaling between the pattern-dictating ectoderm and the skeletogenic primary mesenchyme cells (PMCs); recent studies have begun to uncover the molecular basis for this process. Using an unbiased RNA-Seq-based screen, we have previously identified the TGF-ß superfamily ligand, LvBMP5-8, as a skeletal patterning gene in Lytechinus variegatus embryos. This result is surprising, since both BMP5-8 and BMP2/4 ligands have been implicated in sea urchin dorsal-ventral (DV) and left-right (LR) axis specification. Here, we demonstrate that zygotic LvBMP5-8 is required for normal skeletal patterning on the left side, as well as for normal PMC positioning during gastrulation. Zygotic LvBMP5-8 is required for expression of the left-side marker soxE, suggesting that LvBMP5-8 is required for left-side specification. Interestingly, we also find that LvBMP5-8 knockdown suppresses serotonergic neurogenesis on the left side. While LvBMP5-8 overexpression is sufficient to dorsalize embryos, we find that zygotic LvBMP5-8 is not required for normal DV specification or development. In addition, ectopic LvBMP5-8 does not dorsalize LvBMP2/4 morphant embryos, indicating that, in the absence of BMP2/4, BMP5-8 is insufficient to specify dorsal. Taken together, our data demonstrate that zygotic LvBMP5-8 signaling is essential for left-side specification, and for normal left-side skeletal and neural patterning, but not for DV specification. Thus, while both BMP2/4 and BMP5-8 regulate LR axis specification, BMP2/4 but not zygotic BMP5-8 regulates DV axis specification in sea urchin embryos.

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

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    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.

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

    Science.gov (United States)

    Horasawa, Noriko; Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki

    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.

  12. Effects of Qizhi Jiangtang Capsule on BMP-7 and TGF-β1/Smads Signaling Pathway of Kidney in Diabetic Nephropathy Rats%芪蛭降糖胶囊对糖尿病肾病大鼠肾组织BMP-7及TGF-β1/smads信号传导通路的影响

    Institute of Scientific and Technical Information of China (English)

    武帅; 郭兆安; 于春江; 李悦; 姜蓓蓓; 肖荣; 李敏; 刘玉臻; 董雄飞

    2014-01-01

    目的:观察芪蛭降糖胶囊对糖尿病肾病(diabetic nephropathy,DN)大鼠肾组织骨形成蛋白-7(bone morpho-genetic protein-7,BMP-7)及转化生长因子(transforming growth factor,TGF)-β1/Smads信号传导通路的影响。方法:48只清洁级雄性Wistar大鼠按体重随机抽取40只,采用切除右肾加腹腔注射链脲菌素( streptozotocin,STZ)的方法制备DN模型,另外8只大鼠行右肾假切术。造模成功后按尿微量白蛋白( microAlbumin,mAlb)高低,两头随机抽取分为模型组、缬沙坦对照组、芪蛭降糖胶囊低剂量组、芪蛭降糖胶囊高剂量组。成模2 d起各组给予相应浓度和剂量的药物,给药12周时观察DN大鼠尿mAlb、α1微球蛋白(α1-MG)和血清肌酐( Scr)、尿素氮( BUN)。然后,处死所有动物,肾组织行HE染色、PAS染色和Masson染色,观察肾组织病理变化并半定量计算肾小管损伤指数(tubulointerstitial injury index,TII),免疫组化法检测BMP-7、TGF-β1、Smad2、Smad7在肾组织的表达。结果:给药12周后,模型组大鼠尿 mAlb、α1-MG较假手术组显著增多( P 0.05),但2个中药治疗组较模型组明显降低(P0. 05). ocompared with the control group, these indicators of 2 groups of QJC were obviously decreased(P<0. 05~0. 01). HE staining showed that renal pathological damage of 3 treated groups rats were lessened. Renal pathological of 2 groups of QJC was improved more obvious than that of control group. PAS staining showed that TII of 3 treated groups was significantly lower than that of the model group,and TII of 2 groups of QJC was more lower than the control group. Tubulointerstitial was improved by Masson stain in the 2 groups of QJC. Compared with the model group, the protein expression of TGF-β1 ,Smad2 were down-regulated in the renal tissue of 3 treated groups,while the protein expression were further down-regulated by QJC. At the same time,the protein expression of BMP-7、Smad7 were up-regulated in the 3

  13. Effect of BMP-2 and BMP-7 homodimers and a mixture of BMP-2/BMP-7 homodimers on osteoblast adhesion and growth following culture on a collagen scaffold

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    Laflamme, Claude; Rouabhia, Mahmoud [Groupe de recherche en ecologie buccale, Faculte de medecine dentaire, Universite Laval, Quebec City, Quebec G1K 7P4 (Canada)], E-mail: Mahmoud.rouabhia@fmd.ulaval.ca

    2008-03-01

    In the present study, we studied the involvement of BMP-2 and BMP-7 as homodimers and as a mixture of homodimers in bone regeneration using an engineered bone model. The engineered bone model consisted of a collagen scaffold populated with osteoblasts that acted as a carrier for the BMPs. BMP-2, BMP-7 and a mixture of BMP-2/BMP-7 were used at final concentrations of 10 and 100 ng ml{sup -1}. Osteoblasts seeded onto a collagen scaffold were cultured for 24 h before being stimulated with the BMPs. Four days later, osteoblast adhesion to and growth on the scaffold were assessed. Osteocalcin, IL-6, metalloproteinase (MMP-2 and MMP-9) and protease inhibitor (TIMP-1 and TIMP-2) mRNA and protein levels were measured. Our results showed that the BMP-2, BMP-7 and a mixture of BMP-2/BMP-7 all promoted osteoblast growth on the collagen scaffold, with the mixture of BMP-2/BMP-7 enhancing the most growth. BMP-2 and the mixture of BMP-2/BMP-7 enhanced osteocalcin (an osteoblast differentiation marker) mRNA expression and protein secretion, likely via the IL-6 pathway given that IL-6 secretion was upregulated by BMP-7 and a mixture of BMP-2/BMP-7. BMPs promote extracellular matrix production by inhibiting MMP-2 mRNA and increasing TIMP-1 and TIMP-2 mRNA expressions and protein secretions. BMP-2, BMP-7 and the mixture of BMP-2/BMP-7 could promote bone regeneration via different mechanisms involving IL-6 and MMP inhibitors.

  14. BMP2/BMPR1A is linked to tumour progression in dedifferentiated liposarcomas.

    Science.gov (United States)

    O'Neill, Hannah L; Cassidy, Amy P; Harris, Olivia B; Cassidy, John W

    2016-01-01

    Bone Morphogenic Protein 2 (BMP2) is a multipurpose cytokine, important in the development of bone and cartilage, and with a role in tumour initiation and progression. BMP2 signal transduction is dependent on two distinct classes of serine/threonine kinase known as the type I and type II receptors. Although the type I receptors (BMPR1A and BMPR1B) are largely thought to have overlapping functions, we find tissue and cellular compartment specific patterns of expression, suggesting potential for distinct BMP2 signalling outcomes dependent on tissue type. Herein, we utilise large publicly available datasets from The Cancer Genome Atlas (TCGA) and Protein Atlas to define a novel role for BMP2 in the progression of dedifferentiated liposarcomas. Using disease free survival as our primary endpoint, we find that BMP2 confers poor prognosis only within the context of high BMPR1A expression. Through further annotation of the TCGA sarcoma dataset, we localise this effect to dedifferentiated liposarcomas but find overall BMP2/BMP receptor expression is equal across subsets. Finally, through gene set enrichment analysis we link the BMP2/BMPR1A axis to increased transcriptional activity of the matrisome and general extracellular matrix remodelling. Our study highlights the importance of continued research into the tumorigenic properties of BMP2 and the potential disadvantages of recombinant human BMP2 (rhBMP2) use in orthopaedic surgery. For the first time, we identify high BMP2 expression within the context of high BMPR1A expression as a biomarker of disease relapse in dedifferentiated liposarcomas.

  15. BMP-2 gene-fibronectin-apatite composite layer enhances bone formation

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    Sogo Yu

    2011-08-01

    Full Text Available Abstract Background Safe and efficient gene transfer systems are needed for tissue engineering. We have developed an apatite composite layer including the bone morphogenetic protein-2 (BMP-2 gene and fibronectin (FB, and we evaluated its ability to induce bone formation. Methods An apatite composite layer was evaluated to determine the efficiency of gene transfer to cells cultured on it. Cells were cultured on a composite layer including the BMP-2 gene and FB, and BMP-2 gene expression, BMP-2 protein concentrations, alkaline phosphatase (ALP activity, and osteocalcin (OC concentrations were measured. A bone defect on the cranium of rats was treated with hydroxyapatite (HAP-coated ceramic buttons with the apatite composite layer including the BMP-2 gene and FB (HAP-BMP-FB. The tissue concentration of BMP-2, bone formation, and the expression levels of the BMP-2, ALP, and OC genes were all quantified. Results The apatite composite layer provided more efficient gene transfer for the cultured cells than an apatite composite layer without FB. The BMP-2 concentration was approximately 100~600 pg/mL in the cell-culture medium. Culturing the cells on the apatite composite layer for 27 days increased ALP activity and OC concentrations. In animal experiments, the tissue concentrations of BMP-2 were over 100 pg/mg in the HAP-BMP-FB group and approximately 50 pg/mg in the control groups. Eight weeks later, bone formation was more enhanced in the HAP-BMP-FB group than in the control groups. In the tissues surrounding the HAP button, the gene expression levels of ALP and OC increased. Conclusion The BMP-2 gene-FB-apatite composite layer might be useful for bone engineering.

  16. The effect of Smad6 RNA interference on BMP-2 induced osteogenic differentiation of mesenchymal stem cells%Smad6信号干扰对MSCs骨向分化的影响

    Institute of Scientific and Technical Information of China (English)

    刘猛; 董伟; 冯晓洁; 邓久鹏; 戚孟春; 李金源

    2011-01-01

    Objective To investigate the effect of Smad6 mRNA interference on bone morphogenetic protein 2 (BMP-2) induced osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). Methods The bone marrow MSCs of mice were cultured and underwent BMP-2 induced osteogenic differentiation. The cells were divided into 3 groups: the cells in group A were transfected with recombinant Smad6 RNA interference vector,which was labeled with green fluorescent protein (GFP) , and the cells in group B were transfected with control vector,and the cells in group C served as controls. The activity of alkaline phosphonate (ALP) and levels of osteocalcin were detected at five days after transfection by ALP staining and radioimmunoassay, respectively. The formation of mineralization nodus was also examined by alizarin red staining. Results The GFP was obviously expressed in MSCs after viral transfection, and viral transfection efficiency reached 98. 5%. As compared with group B,Smad6 RNA interference increased significantly ALP activity and osteocalcin levels in group A ( P < 0.01) .however,both ALP activity and osteocalcin levels in group C were significantly lower than those in the other two groups( P <0.01). The results of alizarin red staining showed that the counts of mineralization nodus in group A were significantly more than those in group B ( P <0.05) ,but no mineralization nodus was found in group C. Conclusion Smad6 mRNA interference. Can promote effectively BMP-2 induced osteogenic differentiation of MSCs,which may be a valuable method for bone regeneration for bone defect in bone tissue engineering.%目的 研究Smad6信号干扰对骨形态发生蛋白2(BMP-2)诱导的骨髓间充质干细胞(MSCs)骨向分化的促进效应.方法 培养小鼠MSCs,用BMP-2诱导骨向分化.细胞分为3组:A组细胞用携带绿色荧光蛋白(GFP)的Smad6重组RNA干扰载体转染;B组细胞用空白载体转染;C组细胞作为对照.结果 病毒转染后GFP在MSCs中有

  17. Bmp2 and Bmp4 accelerate alveolar bone development.

    Science.gov (United States)

    Ou, Mingming; Zhao, Yibing; Zhang, Fangming; Huang, Xiaofeng

    2015-06-01

    Alveolar bone remodeling is a continuous process that takes place during development and in response to various physiological and pathological stimuli. However, detailed knowledge regarding the underlying mechanisms involved in alveolar bone development is still lacking. This study aims at improving our understanding of alveolar bone formation and the role of bone morphogenetic proteins (Bmps) in this process. Mice at embryonic (E) day 13.5 to postnatal (PN) day 15.5 were selected to observe the process of alveolar bone development. Alveolar bone development was found to be morphologically observable at E14.5. Molar teeth isolated from mice at PN7.5 were pretreated with Bmp2, Bmp4, Noggin, or BSA, and grafted subcutaneously into mice. The subcutaneously implanted tooth germs formed alveolar bone indicating the role of the dental follicle in alveolar bone development. Alveolar bone formation was increased after pretreatment with Bmp2 and Bmp4, but not with Noggin. Gene expression levels in dental follicle cells from murine molars were also determined by real-time RT-PCR. The expression levels of Runx2, Bsp, and Ocn were significantly higher in dental follicle cells cultured with Bmp2 or Bmp4, and significantly lower in those cultured with Noggin when compared with that of the BSA controls. Our results suggest that the dental follicle participates in alveolar bone formation and Bmp2/4 appears to accelerate alveolar bone development.

  18. 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.

  19. Bone morphogenetic protein 2-induced human dental pulp cell differentiation involves p38 mitogen-activated protein kinase-activated canonical WNT pathway

    Institute of Scientific and Technical Information of China (English)

    Jing Yang; Ling Ye; Tian-Qian Hui; Dong-Mei Yang; Ding-Ming Huang; Xue-Dong Zhou; Jeremy J Mao; Cheng-Lin Wang

    2015-01-01

    Both bone morphogenetic protein 2 (BMP2) and the wingless-type MMTV integration site (WNT)/b-catenin signalling pathway play important roles in odontoblast differentiation and dentinogenesis. Cross-talk between BMP2 and WNT/b-catenin in osteoblast differentiation and bone formation has been identified. However, the roles and mechanisms of the canonical WNT pathway in the regulation of BMP2 in dental pulp injury and repair remain largely unknown. Here, we demonstrate that BMP2 promotes the differentiation of human dental pulp cells (HDPCs) by activating WNT/b-catenin signalling, which is further mediated by p38 mitogen-activated protein kinase (MAPK) in vitro. BMP2 stimulation upregulated the expression of b-catenin in HDPCs, which was abolished by SB203580 but not by Noggin or LDN193189. Furthermore, BMP2 enhanced cell differentiation, which was not fully inhibited by Noggin or LDN193189. Instead, SB203580 partially blocked BMP2-induced b-catenin expression and cell differentiation. Taken together, these data suggest a possible mechanism by which the elevation of b-catenin resulting from BMP2 stimulation is mediated by the p38 MAPK pathway, which sheds light on the molecular mechanisms of BMP2-mediated pulp reparative dentin formation.

  20. Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

    Science.gov (United States)

    Guo, Feng; Feng, Junsheng; Wang, Feng; Li, Wentong; Gao, Qingping; Chen, Zhuo; Shoff, Lisa; Donly, Kevin J; Gluhak-Heinrich, Jelica; Chun, Yong Hee Patricia; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

    2015-08-01

    Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo.

  1. BMP2/BMP4 colorectal cancer susceptibility loci in northern and southern European populations.

    Science.gov (United States)

    Fernandez-Rozadilla, Ceres; Palles, Claire; Carvajal-Carmona, Luis; Peterlongo, Paolo; Nici, Carmela; Veneroni, Silvia; Pinheiro, Manuela; Teixeira, Manuel R; Moreno, Victor; Lamas, Maria-Jesus; Baiget, Montserrat; Lopez-Fernandez, L A; Gonzalez, Dolors; Brea-Fernandez, Alejandro; Clofent, Juan; Bujanda, Luis; Bessa, Xavier; Andreu, Montserrat; Xicola, Rosa; Llor, Xavier; Jover, Rodrigo; Castells, Antoni; Castellvi-Bel, Sergi; Carracedo, Angel; Tomlinson, Ian; Ruiz-Ponte, Clara

    2013-02-01

    Genome-wide association studies have successfully identified 20 colorectal cancer susceptibility loci. Amongst these, four of the signals are defined by tagging single nucleotide polymorphisms (SNPs) on regions 14q22.2 (rs4444235 and rs1957636) and 20p12.3 (rs961253 and rs4813802). These markers are located close to two of the genes involved in bone morphogenetic protein (BMP) signaling (BMP4 and BMP2, respectively). By investigating these four SNPs in an initial cohort of Spanish origin, we found substantial evidence that minor allele frequencies (MAFs) may be different in northern and southern European populations. Therefore, we genotyped three additional southern European cohorts comprising a total of 2028 cases and 4273 controls. The meta-analysis results show that only one of the association signals (rs961253) is effectively replicated in the southern European populations, despite adequate power to detect all four. The other three SNPs (rs4444235, rs1957636 and rs4813802) presented discordant results in MAFs and linkage disequilibrium patterns between northern and southern European cohorts. We hypothesize that this lack of replication could be the result of differential tagging of the functional variant in both sets of populations. Were this true, it would have complex consequences in both our ability to understand the nature of the real causative variants, as well as for further study designs.

  2. Expression and functional study of extracellular BMP antagonists during the morphogenesis of the digits and their associated connective tissues.

    Directory of Open Access Journals (Sweden)

    Carlos I Lorda-Diez

    Full Text Available The purpose of this study is to gain insight into the role of BMP signaling in the diversification of the embryonic limb mesodermal progenitors destined to form cartilage, joints, and tendons. Given the importance of extracellular BMP modulators in in vivo systems, we performed a systematic search of those expressed in the developing autopod during the formation of the digits. Here, we monitored the expression of extracellular BMP modulators including: Noggin, Chordin, Chordin-like 1, Chordin-like 2, Twisted gastrulation, Dan, BMPER, Sost, Sostdc1, Follistatin, Follistatin-like 1, Follistatin-like 5 and Tolloid. These factors show differential expression domains in cartilage, joints and tendons. Furthermore, they are induced in specific temporal patterns during the formation of an ectopic extra digit, preceding the appearance of changes that are identifiable by conventional histology. The analysis of gene regulation, cell proliferation and cell death that are induced by these factors in high density cultures of digit progenitors provides evidence of functional specialization in the control of mesodermal differentiation but not in cell proliferation or apoptosis. We further show that the expression of these factors is differentially controlled by the distinct signaling pathways acting in the developing limb at the stages covered by this study. In addition, our results provide evidence suggesting that TWISTED GASTRULATION cooperates with CHORDINS, BMPER, and NOGGIN in the establishment of tendons or cartilage in a fashion that is dependent on the presence or absence of TOLLOID.

  3. Expression and Functional Study of Extracellular BMP Antagonists during the Morphogenesis of the Digits and Their Associated Connective Tissues

    Science.gov (United States)

    Lorda-Diez, Carlos I.; Montero, Juan A.; Rodriguez-Leon, Joaquin; Garcia-Porrero, Juan A.; Hurle, Juan M.

    2013-01-01

    The purpose of this study is to gain insight into the role of BMP signaling in the diversification of the embryonic limb mesodermal progenitors destined to form cartilage, joints, and tendons. Given the importance of extracellular BMP modulators in in vivo systems, we performed a systematic search of those expressed in the developing autopod during the formation of the digits. Here, we monitored the expression of extracellular BMP modulators including: Noggin, Chordin, Chordin-like 1, Chordin-like 2, Twisted gastrulation, Dan, BMPER, Sost, Sostdc1, Follistatin, Follistatin-like 1, Follistatin-like 5 and Tolloid. These factors show differential expression domains in cartilage, joints and tendons. Furthermore, they are induced in specific temporal patterns during the formation of an ectopic extra digit, preceding the appearance of changes that are identifiable by conventional histology. The analysis of gene regulation, cell proliferation and cell death that are induced by these factors in high density cultures of digit progenitors provides evidence of functional specialization in the control of mesodermal differentiation but not in cell proliferation or apoptosis. We further show that the expression of these factors is differentially controlled by the distinct signaling pathways acting in the developing limb at the stages covered by this study. In addition, our results provide evidence suggesting that TWISTED GASTRULATION cooperates with CHORDINS, BMPER, and NOGGIN in the establishment of tendons or cartilage in a fashion that is dependent on the presence or absence of TOLLOID. PMID:23573253

  4. 3-OST-7 regulates BMP-dependent cardiac contraction.

    Directory of Open Access Journals (Sweden)

    Shiela C Samson

    2013-12-01

    Full Text Available The 3-O-sulfotransferase (3-OST family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing tropomyosin4 (tpm4 expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin tpm4 but not by troponin tnnt2, indicating that tpm4 serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of bmp4. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, kcnh2, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, tnnt2, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function.

  5. Construction of pcDNA3-BMP2 and Induced Expression in E. coli DH-5α%BMP2转基因载体的构建及在大肠杆菌DH-5α中的诱导表达

    Institute of Scientific and Technical Information of China (English)

    王国贤; 崔大祥; 刘丹平; 刘贺亮; 陈勇; 郭晏海; 刘晓虹

    2003-01-01

    BMP2基因来源于pSP6-BMP2质粒,用pcDNA3作载体,构建BMP2转基因载体并在大肠杆菌DH-5α中的诱导表达.用HindⅢ与XbaI双酶切pSP6-BMP2与pcDNA3质粒,回收BMP2基因片段与pcDNA3载体,用连接酶连接后转化JM109,提质粒后酶切鉴定;把构建好的载体转化DH-5α细菌,并诱导表达,用SDS-PAGE电泳鉴定有无表达,用WesternBlot鉴定表达蛋白.pcDNA3-BMP2载体酶切鉴定与预期片段相符,SDS-PAGE显示有BMP2蛋白表达;Western Blot证明表达蛋白有免疫原活性.成功地构建了表明pcDNA3-BMP2转基因载体并在大肠杆菌DH-5α中诱导表达了BMP蛋白.

  6. Low-dose rhBMP2/7 heterodimer to reconstruct peri-implant bone defects: a micro-CT evaluation

    NARCIS (Netherlands)

    Wang, J.; Zheng, Y.; Zhao, J.; Liu, T.; Gao, L.; Gu, Z.; Wu, G.

    2012-01-01

    Objectives To delineate the dynamic micro-architectures of bone induced by low-dose bone morphogenetic protein (BMP)-2/7 heterodimer in peri-implant bone defects compared to BMP2 and BMP7 homodimer. Material and Methods Peri-implant bone defects (8 mm in diameter, 4 mm in depth) were created surroun

  7. Wnt3a enhances bone morphogenetic protein 9-induced osteogenic differentiation of C3H10T1/2 cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiao; LIN Liang-bo; XU Dao-jing; CHEN Rong-fu; TAN Ji-xiang; LIANG Xi; HU Ning

    2013-01-01

    Background Bone morphogenetic protein 9 (BMP9) and Wnt/β-catenin signaling pathways are able to induce osteogenic differentiation of mesenchymal stem cells (MSCs),but the role of Wnt/β-catenin signaling pathway in BMP9-induced osteogenic differentiation is not well understood.Thus,our experiment was undertaken to investigate the interaction between BMP9 and Wnt/β-catenin pathway in inducing osteogenic differentiation of MSCs.Methods C3H10T1/2 cells were infected with recombinant adenovirus expressing BMP9,Wnt3a,and BMP9+Wnt3a.ALP,the early osteogenic marker,was detected by quantitative and staining assay.Later osteogenic marker,mineral calcium deposition,was determined by Alizarin Red S staining.The expression of osteopotin (OPN),osteocalcin (OC),and Runx2 was analyzed by Real time PCR and Western blotting.In vivo animal experiment was carried out to further confirm the role of Wnt3a in ectopic bone formation induced by BMP9.Results The results showed that Wnt3a enhanced the ALP activity induced by BMP9 and increased the expressions of OC and OPN,with increase of mineral calcium deposition in vitro and ectopic bone formation in vivo.Furthermore,we also found that Wnt3a increased the level of Runx2,an important nuclear transcription factor of BMP9.Conclusion Canonical Wnt/β-catenin signal pathway may play an important role in BMP9-induced osteogenic differentiation of MSCs,and Runx2 may be a linkage between the two signal pathways.

  8. Bmp7 functions via a polarity mechanism to promote cloacal septation.

    Directory of Open Access Journals (Sweden)

    Kun Xu

    Full Text Available BACKGROUND: During normal development in human and other placental mammals, the embryonic cloacal cavity separates along the axial longitudinal plane to give rise to the urethral system, ventrally, and the rectum, dorsally. Defects in cloacal development are very common and present clinically as a rectourethral fistula in about 1 in 5,000 live human births. Yet, the cellular mechanisms of cloacal septation remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We previously detected Bone morphogenetic protein 7 (Bmp7 expression in the urorectal mesenchyme (URM, and have shown that loss of Bmp7 function results in the arrest of cloacal septation. Here, we present evidence that cloacal partitioning is driven by Bmp7 signaling in the cloacal endoderm. We performed TUNEL and immunofluorescent analysis on cloacal sections from Bmp7 null and control littermate embryos. We found that loss of Bmp7 results in a dramatic decrease in the endoderm survival and a delay in differentiation. We used immunological methods to show that Bmp7 functions by activating the c-Jun N-terminal kinase (JNK pathway. We carried out confocal and 3D imaging analysis of mitotic chromosome bundles to show that during normal septation cells in the cloacal endoderm divide predominantly in the apical-basal direction. Loss of Bmp7/JNK signaling results in randomization of mitotic angles in the cloacal endoderm. We also conducted immunohistochemical analysis of human fetal sections to show that BMP/phospho-SMAD and JNK pathways function in the human cloacal region similar as in the mouse. CONCLUSION/SIGNIFICANCE: Our results strongly indicate that Bmp7/JNK signaling regulates remodeling of the cloacal endoderm resulting in a topological separation of the urinary and digestive systems. Our study points to the importance of Bmp and JNK signaling in cloacal development and rectourethral malformations.

  9. Regulation of BMP4 on the proliferation and differentiation in SVZa neural stem cells

    Institute of Scientific and Technical Information of China (English)

    LIU Shiyong; ZHANG Zhiyuan; SONG Yechun; QIU Kejun; ZHANG Kecheng; AN Ning; ZHOU Zheng; CAI Wenqin; YANG Hui

    2004-01-01

    The neural stem cells in the anterior subventricular zone (SVZa) mainly generate the progenitors that will differentiate into neurons, and along a highly circumscribed migratory access--Rostral migratory stream (RMS), they migrate to the olfactory bulbs (OB). To understand the effects of BMPs on SVZa neural stem cells, in this study BMP4 at various concentrations was used to induce SVZa neural stem cells, and the living cell labeling using BMP4 promotor conjugated with red fluorescence protein showed the expression of BMP4 dynamically. The results demonstrated that low BMP4 doses (1-5 ng/mL)promoted while high doses (10-100 ng/mL) inhibited the proliferation of SVZa neural stem cells, and BMP4 promoted neuron differentiation in the early stage (1-3 d), howeverm,it inhibited the neuron commitment after 4 d. Noggin, the antagonist of BMP4, blocked the physiological effects of BMP4. In OB, BMP4 is mainly to accelerate the progenitors to withdraw from the cell cycle and trigger the differentiation, and in RMS, it promotes the proliferation of committed progenitors and not differentiation, further in SVZa, BMP4 enhances astrocyte commitment.

  10. Wnt/BMP信号通路激活高效诱导人多能干细胞分化为血管平滑肌细胞%Generation of vascular smooth muscle cells from human pluripotent stem cells via the activation of Wnt and BMP signaling

    Institute of Scientific and Technical Information of China (English)

    李艳辉; 罗丹; 高颖

    2016-01-01

    cells were sorted by flow cytometry at day 9,and then directly differentiated into vascular smooth muscle cells with PDGF-BB.The differentiated cells were characterized by immunofluorescent staining and real-time quantitative PCR.Results Flow cytometric analyses demonstrated the percentage of CD31 +,CD34 + cells sorted from the differentiated cell population in A group is significantly higher than that in B group (38.8%-51.4% vs.11.7%-22.5%.P < 0.05).After in vitro direct differentiation,all differentiated cells stained positively and had gene expressions of myogenic marker,such as SM22-α,SMA-α and Calponin.Conclusion Simultaneous activation of the BMP signaling and Wnt signaling can rapidly and efficiently induced the hPSCs differentiation into vascular smooth muscle cells.It holds a promise to provide a population of clinical-grade vascular smooth muscle cells for cell therapy.

  11. Post-hypoxic and ischemic neuroprotection of BMP-7 in the cerebral cortex and caudate-putamen tissue of rat.

    Science.gov (United States)

    Luan, Liju; Yang, Xiaomei; Zhou, Changman; Wang, Ke; Qin, Lihua

    2015-03-01

    Previous reports have indicated that exogenous bone morphogenetic protein-7 (BMP-7) has a neuroprotective effect after cerebral ischemia injury and promotes motor function recovery, but the appropriate BMP-7 concentration and time course are unclear. Here, we assessed endogenous BMP-7 expression in hypoxia and ischemia-damaged brain tissues and investigated the effects of different BMP-7 concentrations in pre- and post-hypoxic primary rat neurons. The results showed that BMP-7 expression was significantly higher in the ischemic hemisphere. The expressions of BMP-7 and caspase-3 were localized in the cytoplasm of the primary cerebral cortical and caudate-putamen neurons 24h after hypoxia/reoxygenation. After BMP-7 treatment, the number of caspase-3 positive neurons began to decrease with increasing BMP-7 concentrations up to 80ng/ml, but not beyond. Although the numbers of caspase-3-positive neurons between pre- and post-hypoxia/reoxygenation were not significantly different, more dendrites were observed in the groups treated prior to hypoxia/reoxygenation. These results suggest that increased BMP-7 expression can be induced in the cerebral cortex and caudate-putamen both in vivo and in vitro in hypoxic-ischemic states. The neuroprotective mechanism of BMP-7 may include apoptosis suppression, and its effect was enhanced from 40 to 80ng/ml. Pre-hypoxic BMP-7 treatment may be useful to stimulate dendrite sprouting in non-injured neurons.

  12. Soluble VEGFR1 reverses BMP2 inhibition of intramembranous ossification during healing of cortical bone defects.

    Science.gov (United States)

    Hu, Kai; Besschetnova, Tatiana Y; Olsen, Bjorn R

    2016-09-07

    BMP2 is widely used for promotion of bone repair and regeneration. However, bone formation induced by BMP2 is quite variable. Bone forming progenitor cells in different locations appear to respond to BMP2 in different ways, and repair outcomes can vary as a consequence of modulating effects by other factors. In this study, we have examined the effects of VEGF on BMP2-induced repair of a cortical bone defect, a 1 mm diameter drill hole, in the proximal tibia of mice. Treatment of the defect with either a bolus of PBS or soluble VEGFR1 (sVEGFR1), a decoy receptor for VEGF, had the same effects on bone formation via intramembranous ossification in the defect and cartilage formation and injured periosteum, during the healing process. In contrast, treatment with BMP2 inhibited intramembranous bone formation in the defect while it promoted cartilage and endochondral bone formation in the injured periosteum compared with mice treated with PBS or sVEGFR1. The inhibitory effect of BMP2 on bone formation was unlikely due to increased osteoclast activity and decreased invasion of blood vessels in the defect. Most importantly, co-delivery of BMP2 and sVEGFR1 reversed the inhibition of intramembranous bone formation by BMP2. Furthermore, the decreased accumulation of collagen and production of bone matrix proteins in the defect of groups with BMP2 treatment could also be prevented by co-delivery of BMP2 and sVEGFR1. Our data indicate that introducing a VEGF-binding protein, such as sVEGFR1, to reduce levels of extracellular VEGF, may enhance the effects of BMP2 on intramembranous bone formation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

  13. A selection fit mechanism in BMP receptor IA as a possible source for BMP ligand-receptor promiscuity.

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    Stefan Harth

    Full Text Available BACKGROUND: Members of the TGF-β superfamily are characterized by a highly promiscuous ligand-receptor interaction as is readily apparent from the numeral discrepancy of only seven type I and five type II receptors available for more than 40 ligands. Structural and functional studies have been used to address the question of how specific signals can be deduced from a limited number of receptor combinations and to unravel the molecular mechanisms underlying the protein-protein recognition that allow such limited specificity. PRINCIPAL FINDINGS: In this study we have investigated how an antigen binding antibody fragment (Fab raised against the extracellular domain of the BMP receptor type IA (BMPR-IA recognizes the receptor's BMP-2 binding epitope and thereby neutralizes BMP-2 receptor activation. The crystal structure of the complex of the BMPR-IA ectodomain bound to the Fab AbD1556 revealed that the contact surface of BMPR-IA overlaps extensively with the contact surface for BMP-2 interaction. Although the structural epitopes of BMPR-IA to both binding partners coincides, the structures of BMPR-IA in the two complexes differ significantly. In contrast to the structural differences, alanine-scanning mutagenesis of BMPR-IA showed that the functional determinants for binding to the antibody and BMP-2 are almost identical. CONCLUSIONS: Comparing the structures of BMPR-IA bound to BMP-2 or bound to the Fab AbD1556 with the structure of unbound BMPR-IA shows that binding of BMPR-IA to its interaction partners follows a selection fit mechanism, possibly indicating that the ligand promiscuity of BMPR-IA is inherently encoded by structural adaptability. The functional and structural analysis of the BMPR-IA binding antibody AbD1556 mimicking the BMP-2 binding epitope may thus pave the way for the design of low-molecular weight synthetic receptor binders/inhibitors.

  14. Bone Morphogenetic Protein (BMP-4 and BMP-7 regulate differentially Transforming Growth Factor (TGF-β1 in normal human lung fibroblasts (NHLF

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    Lloyd Clare M

    2010-06-01

    Full Text Available Abstract Background Airway remodelling is thought to be under the control of a complex group of molecules belonging to the Transforming Growth Factor (TGF-superfamily. The Bone Morphogenetic Proteins (BMPs belong to this family and have been shown to regulate fibrosis in kidney and liver diseases. However, the role of BMPs in lung remodelling remains unclear. BMPs may regulate tissue remodelling in asthma by controlling TGF-β-induced profibrotic functions in lung fibroblasts. Methods Cell cultures were exposed to TGF-β1 alone or in the presence of BMP-4 or BMP-7; control cultures were exposed to medium only. Cell proliferation was assessed by quantification of the incorporation of [3H]-thymidine. The expression of the mRNA encoding collagen type I and IV, tenascin C and fibronectin in normal human lung fibroblasts (NHLF was determined by real-time quantitative PCR and the main results were confirmed by ELISA. Cell differentiation was determined by the analysis of the expression of α-smooth muscle actin (α-SMA by western blot and immunohistochemistry. The effect on matrix metalloproteinase (MMP activity was assessed by zymography. Results We have demonstrated TGF-β1 induced upregulation of mRNAs encoding the extracellular matrix proteins, tenascin C, fibronectin and collagen type I and IV when compared to unstimulated NHLF, and confirmed these results at the protein level. BMP-4, but not BMP-7, reduced TGF-β1-induced extracellular matrix protein production. TGF-β1 induced an increase in the activity of the pro-form of MMP-2 which was inhibited by BMP-7 but not BMP-4. Both BMP-4 and BMP-7 downregulated TGF-β1-induced MMP-13 release compared to untreated and TGF-β1-treated cells. TGF-β1 also induced a myofibroblast-like transformation which was partially inhibited by BMP-7 but not BMP-4. Conclusions Our study suggests that some regulatory properties of BMP-7 may be tissue or cell type specific and unveil a potential regulatory role for

  15. 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.

  16. Bone morphogenetic protein 2 signaling negatively modulates lymphatic development in vertebrate embryos

    DEFF Research Database (Denmark)

    Dunworth, William P; Cardona-Costa, Jose; Bozkulak, Esra Cagavi

    2014-01-01

    signaling in zebrafish embryos and mouse embryonic stem cell-derived embryoid bodies substantially decrease the emergence of LECs. Mechanistically, BMP2 signaling induces expression of miR-31 and miR-181a in a SMAD-dependent mechanism, which in turn results in attenuated expression of prospero homeobox...

  17. A Functional Link between AMPK and Orexin Mediates the Effect of BMP8B on Energy Balance

    Directory of Open Access Journals (Sweden)

    Luís Martins

    2016-08-01

    Full Text Available AMP-activated protein kinase (AMPK in the ventromedial nucleus of the hypothalamus (VMH and orexin (OX in the lateral hypothalamic area (LHA modulate brown adipose tissue (BAT thermogenesis. However, whether these two molecular mechanisms act jointly or independently is unclear. Here, we show that the thermogenic effect of bone morphogenetic protein 8B (BMP8B is mediated by the inhibition of AMPK in the VMH and the subsequent increase in OX signaling via the OX receptor 1 (OX1R. Accordingly, the thermogenic effect of BMP8B is totally absent in ox-null mice. BMP8B also induces browning of white adipose tissue (WAT, its thermogenic effect is sexually dimorphic (only observed in females, and its impact on OX expression and thermogenesis is abolished by the knockdown of glutamate vesicular transporter 2 (VGLUT2, implicating glutamatergic signaling. Overall, our data uncover a central network controlling energy homeostasis that may be of considerable relevance for obesity and metabolic disorders.

  18. Interactions between BMP-7 and USAG-1 (uterine sensitization-associated gene-1 regulate supernumerary organ formations.

    Directory of Open Access Journals (Sweden)

    Honoka Kiso

    Full Text Available Bone morphogenetic proteins (BMPs are highly conserved signaling molecules that are part of the transforming growth factor (TGF-beta superfamily, and function in the patterning and morphogenesis of many organs including development of the dentition. The functions of the BMPs are controlled by certain classes of molecules that are recognized as BMP antagonists that inhibit BMP binding to their cognate receptors. In this study we tested the hypothesis that USAG-1 (uterine sensitization-associated gene-1 suppresses deciduous incisors by inhibition of BMP-7 function. We learned that USAG-1 and BMP-7 were expressed within odontogenic epithelium as well as mesenchyme during the late bud and early cap stages of tooth development. USAG-1 is a BMP antagonist, and also modulates Wnt signaling. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Using explant culture and subsequent subrenal capsule transplantation of E15 USAG-1 mutant maxillary incisor tooth primordia supplemented with BMP-7 demonstrated in USAG-1+/- as well as USAG-1-/- rescue and supernumerary tooth development. Based upon these results, we conclude that USAG-1 functions as an antagonist of BMP-7 in this model system. These results further suggest that the phenotypes of USAG-1 and BMP-7 mutant mice reported provide opportunities for regenerative medicine and dentistry.

  19. Structure of neuroblastoma suppressor of tumorigenicity 1 (NBL1): insights for the functional variability across bone morphogenetic protein (BMP) antagonists.

    Science.gov (United States)

    Nolan, Kristof; Kattamuri, Chandramohan; Luedeke, David M; Angerman, Elizabeth B; Rankin, Scott A; Stevens, Mariana L; Zorn, Aaron M; Thompson, Thomas B

    2015-02-20

    Bone morphogenetic proteins (BMPs) are antagonized through the action of numerous extracellular protein antagonists, including members from the differential screening-selected gene aberrative in neuroblastoma (DAN) family. In vivo, misregulation of the balance between BMP signaling and DAN inhibition can lead to numerous disease states, including cancer, kidney nephropathy, and pulmonary arterial hypertension. Despite this importance, very little information is available describing how DAN family proteins effectively inhibit BMP ligands. Furthermore, our understanding for how differences in individual DAN family members arise, including affinity and specificity, remains underdeveloped. Here, we present the structure of the founding member of the DAN family, neuroblastoma suppressor of tumorigenicity 1 (NBL1). Comparing NBL1 to the structure of protein related to Dan and Cerberus (PRDC), a more potent BMP antagonist within the DAN family, a number of differences were identified. Through a mutagenesis-based approach, we were able to correlate the BMP binding epitope in NBL1 with that in PRDC, where introduction of specific PRDC amino acids in NBL1 (A58F and S67Y) correlated with a gain-of-function inhibition toward BMP2 and BMP7, but not GDF5. Although NBL1(S67Y) was able to antagonize BMP7 as effectively as PRDC, NBL1(S67Y) was still 32-fold weaker than PRDC against BMP2. Taken together, this data suggests that alterations in the BMP binding epitope can partially account for differences in the potency of BMP inhibition within the DAN family.

  20. Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis

    Science.gov (United States)

    Garciadiego-Cázares, David; Aguirre-Sánchez, Hilda I.; Abarca-Buis, René F.; Kouri, Juan B.; Velasquillo, Cristina; Ibarra, Clemente

    2015-01-01

    The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy

  1. Expression of BMP2/Smad1/Rnnx2 Signal Pathway in Renal Artery of Rat with Vascular Calcification%血管钙化大鼠肾动脉BMP2/Smad1/Runx2信号通路的表达

    Institute of Scientific and Technical Information of China (English)

    覃春美; 魏晓; 龚财判; 吴蔚桦; 刘建; 樊均明; 欧三桃

    2016-01-01

    目的 观察血管钙化大鼠模型肾动脉上骨形态蛋白2 (BMP2)/Smad1/Runt相关转录因子2(Runx2)信号通路的表达及其变化规律,探讨BMP2/Smad1/Runx2信号通路激活在肾动脉钙化中的作用.方法 将24只雄性SD大鼠随机分为对照组和钙化组,钙化组建立维生素D3和尼古丁诱导的大鼠血管钙化模型,对照组给予生理盐水和花生油.6周后采用Von Kossa染色检测肾动脉钙化程度,钙离子试剂盒测定大鼠肾动脉钙含量,实时荧光定量PCR检测肾动脉组织中BMP2、Smad1、Runx2 mRNA水平,免疫组化法观察BMP2、Smad1、Runx2及α-平滑肌肌动蛋白(α-SMA)的表达.结果 Von Kossa染色见钙化组大鼠肾动脉有大量黑色颗粒沉淀.钙化组血管钙含量高于对照组(P<0.05).与对照组相比,钙化组大鼠肾动脉组织BMP2、Smad1、Runx2 mRNA的水平升高(P<0.05),BMP2/Smad1/Runx2信号通路蛋白在肾动脉的表达亦同步上调,而α-SMA的表达较对照组下降(P<0.05).相关分析表明,大鼠肾血管钙含量与肾动脉组织BMP2 mRNA(r=0.655,P<0.05)、Smad1 mRNA (r=0.735,P<0.05)、Runx2 mRNA (r=0.734,P<0.05)均呈正相关.结论 BMP2/Smad1/Runx2通路的表达变化与肾动脉钙化的严重程度相关,提示BMP2/Smad1/Runx2信号通路参与了肾动脉钙化的发生发展.

  2. 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...... enhanced in cultures enriched with either BMP-2 or Dex. Cell proliferation was only increased significantly by Dex treatment. In conclusion, the model described produces cells with an osteoblastic phenotype, and both Dex and BMP-2 can be used as osteoblast inducers. However, the two treatments produce...

  3. Decreased body fat, elevated plasma transforming growth factor-β levels, and impaired BMP4-like signaling in biglycan-deficient mice.

    Science.gov (United States)

    Tang, Tao; Thompson, Joel C; Wilson, Patricia G; Nelson, Christina; Williams, Kevin Jon; Tannock, Lisa R

    2013-01-01

    Biglycan (BGN), a small leucine-rich proteoglycan, binds the pro-fibrotic cytokine transforming growth factor β (TGFβ) and inhibits its bioactivity in vitro. Nevertheless, it is controversial whether BGN plays an inhibitory role in vivo. Therefore, the purpose of this study was to evaluate the effect of BGN deficiency on TGFβ activity in vivo by studying 1-year-old Bgn null and wild-type (WT) mice on an Ldlr-null background. Phenotypic and metabolic characterization showed that the Bgn null mice had lower body weight, shorter body length, and shorter femur length (all p kidney histology. Overall, we propose that this unexpected phenotype arises from the effects of BGN deficiency in vivo to elevate TGFβ levels while decreasing bone morphogenetic protein 4-like signaling.

  4. 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.

  5. Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries

    Directory of Open Access Journals (Sweden)

    Adelina A. Hernandez-Hurtado

    2016-01-01

    Full Text Available Adipose-derived mesenchymal stem cells (ADMSCs are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs. This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology.

  6. Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries

    Science.gov (United States)

    Hernandez-Hurtado, Adelina A.; Lara-Arias, Jorge; Romero-Diaz, Viktor J.; Abrego-Guerra, Adalberto; Vilchez-Cavazos, Jose F.; Elizondo-Riojas, Guillermo; Martinez-Rodriguez, Herminia G.; Espinoza-Juarez, Marcela A.; Mendoza Lemus, Oscar F.

    2016-01-01

    Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology. PMID:27818692

  7. Role of Smad7 in Sox9-potentiated and BMP2-induced differentiation of mouse mesenchymal stem cells into chondrocytes%Smad7在Sox9增强BMP2成软骨效应中的作用

    Institute of Scientific and Technical Information of China (English)

    赵辰; 黄伟; 梁熙; 廖军义; 周年; 胡宁; 赵智; 简长春

    2015-01-01

    目的 探讨Smad7在Sox9增强BMP2成软骨效应中的作用和机制.方法 将小鼠骨髓间充质干细胞(C3 H10T1/2)作为种子细胞,重组腺病毒Ad-BMP2和/或Ad-Sox9感染细胞,Ad-GFP感染细胞为对照.采用Real-time PCR、免疫细胞化学和Western blot分别检测感染后各组Smad7 mRNA表达水平和蛋白表达水平.采用Real-time PCR检测与Smad7相关因子MMP13与OPN mRNA的表达.结果 BMP2+Sox9组感染细胞7、11d时,Smad7 mRNA和蛋白表达水平均明显低于BMP2组(P<0.05);免疫细胞化学染色结果显示,BMP2+Sox9组Smad7染色明显弱于BMP2组;同时BMP2+Sox9组中与软骨最终成熟因子OPN与MMP13的表达均低于BMP2组(P<0.05).结论 在BMP2诱导间充质干细胞成软骨分化中,高表达的Smad7可被Sox9抑制,并抑制Smad7相关因子MMP13与OPN表达,从而解除了Smad7对BMP2成软骨的抑制作用,阻止了软骨细胞最终成熟骨化,有利于保持软骨发育与正常状态.

  8. 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

  9. 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

  10. Restoring effect of rhBMP-4m on bone marrow hematopoietic injury induced by irradiation in mice%重组人骨形成蛋白成熟肽-4对辐射小鼠骨髓造血损伤的修复作用

    Institute of Scientific and Technical Information of China (English)

    刘绍凡; 陈愉; 万锐杰; 徐敏敏

    2013-01-01

    目的探讨小鼠受到辐射后,重组人骨形成蛋白成熟肽-4(rhBMP-4m)对辐射引起的骨髓造血损伤的修复作用。方法通过60 Coγ射线照射,建立小鼠骨髓造血损伤模型,经 rhBMP-4m 连续治疗后,抽血检测小鼠外周血白细胞数,并进行骨髓单个核细胞计数,对脾结节进行计数并通过流式细胞仪检测骨髓单个核细胞中 CD34+细胞比例,比较辐射组和实验组之间的差别。结果经 rhBMP-4m 治疗的小鼠,白细胞数、单个核细胞数、脾结节数和 CD34+细胞比例均明显高于辐射组(P<0.05)。结论对于辐射引起的小鼠骨髓造血损伤,rhBMP-4m 能够加快骨髓造血系统的重建。%Objective To explore the restoring effect of recombinant human bone morphogenetic protein-4m (rhBMP-4m) on bone marrow hematopoietic injury induced by irradiation in mice .Methods Animal models of bone marrow hematopoietic injury were established by radiating with 60 Coγ-ray .After continuous treatment with rhBMP-4m ,the number of white blood cells (WBC) in peripheral blood ,and total and CD34 + mononuclear cells in bone mar-row were detected .Results The number of WBC ,mononuclear cells and the proportion of CD34 + cells in experiment group were higher than radiation group (P< 0 .05) .Conclusion rhBMP-4m could accelerate the restoring process of hematopoietic function in mice with bone marrow hematopoietic injury induced by irradiation .

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

    Directory of Open Access Journals (Sweden)

    Tim N. Beck

    2016-07-01

    Full Text Available 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.

  12. Hedgehog signaling pathway and gastrointestinal stem cell signaling network (review).

    Science.gov (United States)

    Katoh, Yuriko; Katoh, Masaru

    2006-12-01

    Hedgehog, BMP/TGFbeta, FGF, WNT and Notch signaling pathways constitute the stem cell signaling network, which plays a key role in a variety of processes, such as embryogenesis, maintenance of adult tissue homeostasis, tissue repair during chronic persistent inflammation, and carcinogenesis. Sonic hedgehog (SHH), Indian hedgehog (IHH) and Desert hedgehog (DHH) bind to PTCH1/PTCH or PTCH2 receptor to release Smoothened (SMO) signal transducer from Patched-dependent suppression. SMO then activates STK36 serine/threonine kinase to stabilize GLI family members and to phosphorylate SUFU for nuclear accumulation of GLI. Hedgehog signaling activation leads to GLI-dependent transcriptional activation of target genes, such as GLI1, PTCH1, CCND2, FOXL1, JAG2 and SFRP1. GLI1-dependent positive feedback loop combined with PTCH1-dependent negative feedback loop gives rise to transient proliferation of Hedgehog target cells. Iguana homologs (DZIP1 and DZIP1L) and Costal-2 homologs (KIF7 and KIF27) are identified by comparative integromics. SHH-dependent parietal cell proliferation is implicated in gastric mucosal repair during chronic Helicobacter pylori infection. BMP-RUNX3 signaling induces IHH expression in surface differentiated epithelial cells of stomach and intestine. Hedgehog signals from epithelial cells then induces FOXL1-mediated BMP4 upregulation in mesenchymal cells. Hedgehog signaling is frequently activated in esophageal cancer, gastric cancer and pancreatic cancer due to transcriptional upregulation of Hedgehog ligands and epigenetic silencing of HHIP1/HHIP gene, encoding the Hedgehog inhibitor. However, Hedgehog signaling is rarely activated in colorectal cancer due to negative regulation by the canonical WNT signaling pathway. Hedgehog signaling molecules or targets, such as SHH, IHH, HHIP1, PTCH1 and GLI1, are applied as biomarkers for cancer diagnostics, prognostics and therapeutics. Small-molecule inhibitors for SMO or STK36 are suitable to be used for

  13. Mapping the follicle-stimulating hormone-induced signalling networks

    Directory of Open Access Journals (Sweden)

    Pauline eGloaguen

    2011-10-01

    Full Text Available Follicle-stimulating hormone (FSH is a central regulator of male and female reproductive function. Over the last decade, there has been a growing perception of the complexity associated with FSH-induced cellular signalling. It is now clear that the canonical Gs/cAMP/PKA pathway is not the sole mechanism that must be considered in FSH biological actions. In parallel, consistent with the emerging concept of biased agonism, several examples of ligand-mediated selective signalling pathway activation by gonadotropin receptors have been reported. In this context, it is important to gain an integrative view of the signalling pathways induced by FSH and how they interconnect to form a network. In this review, we propose a first attempt at building topological maps of various pathways known to be involved in the FSH-induced signalling network. We discuss the multiple facets of FSH-induced signalling and how they converge to the hormone integrated biological response. Despite of their incompleteness, these maps of the FSH-induced signalling network represent a first step towards gaining a system-level comprehension of this hormone’s actions, which may ultimately facilitate the discovery of novel regulatory processes and therapeutic strategies for infertilities and non-steroidal contraception.

  14. Human BMP sequences can confer normal dorsal-ventral patterning in the Drosophila embryo.

    Science.gov (United States)

    Padgett, R W; Wozney, J M; Gelbart, W M

    1993-04-01

    The type beta transforming growth factor family is composed of a series of processed, secreted growth factors, several of which have been implicated in important regulatory roles in cell determination, inductive interactions, and tissue differentiation. Among these factors, the sequence of the DPP protein from Drosophila is most similar to two of the vertebrate bone morphogenetic proteins, BMP2 and BMP4. Here we report that the human BMP4 ligand sequences can function in lieu of DPP in Drosophila embryos. We introduced the ligand region from human BMP4 into a genomic fragment of the dpp gene in place of the Drosophila ligand sequences and recovered transgenic flies by P-element transformation. We find that this chimeric dpp-BMP4 transgene can completely rescue the embryonic dorsal-ventral patterning defect of null dpp mutant genotypes. We infer that the chimeric DPP-BMP4 protein can be processed properly and, by analogy with the action of other family members, can activate the endogenous DPP receptor to carry out the events necessary for dorsal-ventral patterning. Our evidence suggests that the DPP-BMP4 signal transduction pathway has been functionally conserved for at least 600 million years.

  15. Signaling by bone morphogenetic proteins directs formation of an ectodermal signaling center that regulates craniofacial development.

    Science.gov (United States)

    Foppiano, Silvia; Hu, Diane; Marcucio, Ralph S

    2007-12-01

    We previously described a signaling center, the Frontonasal Ectodermal Zone (FEZ) that regulates growth and patterning of the frontonasal process (FNP). The FEZ is comprised of FNP ectoderm flanking a boundary between Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8) expression domains. Our objective was to examine BMP signaling during formation of the FEZ. We blocked BMP signaling throughout the FNP prior to FEZ formation by infecting chick embryos at stage 10 (HH10) with a replication-competent avian retrovirus encoding the BMP antagonist Noggin. We assessed gene expression patterns in the FNP 72 h after infection (approximately HH22) and observed that Shh expression was reduced or absent. In the mesenchyme, we observed that Bmp2 transcripts were absent while the Bmp4 expression domain was expanded proximally. In addition to the molecular changes, infected embryos also exhibited facial malformations at 72 and 96 h after infection suggesting that the FEZ did not form. Our data indicate that reduced cell proliferation, but not apoptosis, in the mesenchyme contributed to the phenotype that we observed. Additionally, adding exogenous SHH into the mesenchyme of RCAS-Noggin-infected embryos did not restore Bmp2 and Bmp4 to a normal pattern of expression. These data indicate that BMP signaling mediates interactions between tissues in the FNP that regulate FEZ formation; and that the correct pattern of Bmp2 and Bmp4, but not Bmp7, expression in the FNP mesenchyme requires signaling by the BMP pathway.

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

    Science.gov (United States)

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

    2014-12-01

    Clinical trials using bone morphogenetic protein-2 (BMP2) for bone reconstruction have shown promising results. However, the relatively high concentration needed to be effective raises concerns for efficacy and safety. The aim of this study was to investigate the osteogenic effect of an alternative 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 osteogenic medium. The effects of preconditioning were analyzed with regard to proliferation and expression of osteogenic markers at both gene and protein level. The results were compared to those from cultures with continuous BMP2 stimulation. A significant increase in proliferation was seen with both precondition and continuous stimulation with BMP2, with no difference between the treatments. Preconditioning with BMP2 significantly increased gene expression of RUNX2, COLI, ALP, and OC, and protein levels of COLI and ALP. This was not found with continuous stimulation. The role of preconditioning with BMP2 in osteogenesis was validated by findings of increased gene expression of SMAD1 and an increase in dual phosphorylation of ser 463 and ser 465 in the SMAD 1/5/8 pathway. We concluded that preconditioning hMSCs with BMP2 stimulates osteogenesis: proliferation with matrix secretion and matrix 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 be avoided.

  17. 转化生长因子β1(TGF-β1)和骨形成蛋白2(BMP2)体外联合诱导成牙本质细胞样细胞分化%Transforming Growth Factor β1 (TGFβ1) and Bone Morphogenetic Protein 2 (BMP2) Induce Odontoblast-like Cell Differentiation in vitro

    Institute of Scientific and Technical Information of China (English)

    朱奇; 樊明文; 张旗; 陈智; 边专

    2005-01-01

    目的:观察转化生长因子β1(transforming growth factor β1, TGF-β1)和骨形成蛋白2(bone morphogenetic protein 2,BMP2)联合应用对体外培养的鼠牙乳头成牙本质细胞分化的影响.方法:取17 d胎龄小鼠下颌第一磨牙牙胚,胰蛋白酶消化分离牙乳头,置半固态培养基培养6 d,半固态培养基中单独加入重组TGFβ1、BMP2,或分别与肝素联合,或TGFβ1和BMP2联合,组织学观察牙乳头的形态学变化.结果:TGF-β1或BMP2 单独加入时未见细胞极化, 但基质分泌增加.TGF-β1或BMP2 加肝素可诱导牙乳头周边细胞发生极化,并分泌胞外基质.半固态培养基中同时加入 TGF-β1和BMP2的牙乳头培养6 d后,牙乳头周边细胞出现极化和功能性分化,牙乳头周边胞外基质沉积明显,且可见牙乳头尖形态的维持及从牙乳头尖至牙乳头基底部成牙本质细胞分化梯度的维持.结论:本研究结果证实,在没有内釉上皮和完整的基底膜存在的情况下,TGF-β1和BMP2加肝素可诱导培养的牙乳头出现成牙本质细胞分化,并促进胞外基质的分泌.TGF-β1和BMP2 均能诱导成牙本质细胞的细胞学分化和分泌功能,二者联合应用可协同发挥作用增强诱导效果.

  18. Bone Morphogenetic Protein (BMP-7 expression is decreased in human hypertensive nephrosclerosis

    Directory of Open Access Journals (Sweden)

    Cohen Clemens D

    2010-11-01

    Full Text Available Abstract Background Bone Morphogenetic Protein (BMP-7 is protective in different animal models of acute and chronic kidney disease. Its role in human kidneys, and in particular hypertensive nephrosclerosis, has thus far not been described. Methods BMP-7 mRNA was quantified using real-time PCR and localised by immunostaining in tissue samples from normal and nephrosclerotic human kidneys. The impact of angiotensin (AT-II and the AT-II receptor antagonist telmisartan on BMP-7 mRNA levels and phosphorylated Smad 1/5/8 (pSmad 1/5/8 expression was quantified in proximal tubular cells (HK-2. Functional characteristics of BMP-7 were evaluated by testing its influence on TGF-β induced epithelial-to-mesenchymal transition (EMT, expression of TGF-β receptor type I (TGF-βRI and phosphorylated Smad 2 (pSmad 2 as well as on TNF-α induced apoptosis of proximal tubular cells. Results BMP-7 was predominantly found in the epithelia of the distal tubule and the collecting duct and was less abundant in proximal tubular cells. In sclerotic kidneys, BMP-7 was significantly decreased as demonstrated by real-time PCR and immunostaining. AT-II stimulation in HK-2 cells led to a significant decrease of BMP-7 and pSmad 1/5/8, which was partially ameliorated upon co-incubation with telmisartan. Only high concentrations of BMP-7 (100 ng/ml were able to reverse TNF-α-induced apoptosis and TGF-β-induced EMT in human proximal tubule cells possibly due to a decreased expression of TGF-βRI. In addition, BMP-7 was able to reverse TGF-β-induced phosphorylation of Smad 2. Conclusions The findings suggest a protective role for BMP-7 by counteracting the TGF-β and TNF-α-induced negative effects. The reduced expression of BMP-7 in patients with hypertensive nephrosclerosis may imply loss of protection and regenerative potential necessary to counter the disease.

  19. BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects.

    Science.gov (United States)

    Xiong, Long; Zeng, Jianhua; Yao, Aihua; Tu, Qiquan; Li, Jingtang; Yan, Liang; Tang, Zhiming

    2015-01-01

    The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2), a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 μm) with a core (60±18 μm) and a mesoporous shell (180±42 m(2)/g surface area) were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 μg/mg). There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option for bone graft substitutes in bone regeneration.

  20. Identification of BMP2 as an epigenetically silenced growth inhibitor in rhabdomyosarcoma.

    Science.gov (United States)

    Wolf, Sebastian; Hagl, Beate; Kappler, Roland

    2014-05-01

    Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of infancy and although therapy has improved over the years, mortality is still fairly high. The establishment of new treatments has been hampered by the limited knowledge of the molecular mechanisms driving development of RMS. One characteristic of cancer cells is aberrant DNA methylation, which could lead to silencing of tumor suppressor genes. However, only a few epigenetically silenced genes have been described in RMS so far. We performed an expression profiling analysis of three RMS cell lines that were treated with the demethylating agent 5'-aza-2'-deoxycytidine (5-Aza‑dC) facilitating re-expression of epigenetically silenced genes. This treatment induced the gene BMP2 (bone morphogenetic protein 2) throughout all cell lines. Detailed methylation analysis of CpG sites in the BMP2 promoter region by bisulfite sequencing and methylation-specific PCR revealed that a high degree of DNA methylation is causatively associated with the suppression of BMP2 in RMS cells. Consequently, treatment of the RMS cell lines with 5-Aza-dC resulted in DNA demethylation of the BMP2 promoter, most prominently in alveolar RMS. Supplementation of recombinant human BMP2 (rhBMP2) led to a reduced viability of RMS cells. Altogether, these findings suggest that suppression of BMP2 by epigenetic silencing may play a critical role in the genesis of RMS, thereby providing a rationale for the development of a new treatment strategy for RMS.

  1. 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.

  2. 促红细胞生成素通过 BMP/SMAD 通路对肾性贫血大鼠肾脏的保护作用及其机制%Renoprotective action by erythropoietin via BMP/SMAD signaling pathway in renal anemia in rats and its mechanism

    Institute of Scientific and Technical Information of China (English)

    张妍; 李银岚; 陈孟华

    2015-01-01

    目的:探讨促红细胞生成素(EPO)对肾性贫血大鼠肾脏的保护作用及其可能的作用机制。方法将实验大鼠随机分为对照组(NC 组,n=18)和实验组(n=72),采用腺嘌呤注射建立肾性贫血大鼠模型,再随机分为单纯贫血组(SA 组)、铁剂+促红素组(IR+EPO 组)、促红素组(EPO 组)、铁剂组(IR 组)。药物干预4周后,处死大鼠(心脏取血),自动化血液分析仪检测血清肌酐(Scr)、尿素氮(Bun)、血红蛋白(Hb)、血细胞比容(Hct);ELISA 法检测铁调素(Hepcidin)。免疫组织化学染色法和 Western blot 法分别检测大鼠肾脏骨形态蛋白-6(BMP-6)、丝/苏氨酸激酶受体-1(SMAD-1)和 SMAD-4的表达。结果①一般指标的比较:与 SA 组相比较,IR+EPO 组和 EPO 组 BUN、Scr 显著降低(P <0.01);IR+EPO 组和 EPO 组 Hb、Hct 显著升高(P <0.01)。②ELISA 法检测示 IR+EPO 组和 EPO 组铁调素显著降低(P <0.01)。③病理学观察提示 EPO 可以减轻大鼠肾脏病理及功能改变。④免疫组织化学染色示各组大鼠肾小球细胞胞质和肾小管上皮细胞内均表达 SMAD-4、SMAD-1、BMP-6。⑤Western blot 进一步提示,与 SA组相比,IR+EPO 组和 EPO 组 SMAD-4蛋白表达量显著下调(P <0.01),IR+EPO 组和 EPO 组 SMAD-1蛋白表达量下调(P <0.05),IR+EPO 组和 EPO 组 BMP-6蛋白表达量显著上调(P <0.01)。结论EPO 通过 BMP/SMAD 传导通路,抑制铁调素的表达及分泌,纠正贫血,发挥肾脏保护作用。%Objective To investigate the protective effect of erythropoietin (EPO)on the kidney of renal anemia rats and further explore the renoprotective action and possible mechanisms.Methods A total of 90 SD rats were randomly divided into 5 groups:normal control rats (NC),simple anemia group (SA),iron + EPO group (IR+EPO),erythropoietin group (EPO),and iron group (IR)with 18 in each.The rats were sacrificed after 4 weeks’ treatment with intragastrically-injected adenine.Serum creatinine (Scr

  3. Jasmonic acid signaling modulates ozone-induced hypersensitive cell death.

    Science.gov (United States)

    Rao, M V; Lee, H; Creelman, R A; Mullet, J E; Davis, K R

    2000-09-01

    Recent studies suggest that cross-talk between salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O(3)) exposure activates a hypersensitive response (HR)-like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O(3)-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O(3)-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O(3)-induced H(2)O(2) content and SA concentrations and completely abolished O(3)-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O(3) exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O(3) of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O(3)-induced HR-like cell death.

  4. 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.

  5. Immune-Signaling Molecules and Obesity-Induced Inflammation.

    Science.gov (United States)

    Yu, Rina

    2015-01-01

    Obesity-induced inflammation is closely associated with the development of metabolic complications such as insulin resistance and type 2 diabetes. Several immune-signaling receptors and their counterpart ligands are known to be crucial for crosstalk between the adaptive and innate immune system, and they are implicated in various inflammatory pathologies. In this mini-review, I will discuss the involvement of the immune costimulatory molecule 4-1BB and its ligand in obesity-induced inflammation and metabolic complications.

  6. BMP-mediated induction of GATA4/5/6 blocks somitic responsiveness to SHH.

    Science.gov (United States)

    Daoud, Georges; Kempf, Hervé; Kumar, Deepak; Kozhemyakina, Elena; Holowacz, Tamara; Kim, Dae-Won; Ionescu, Andreia; Lassar, Andrew B

    2014-10-01

    The relative timing of SHH and BMP signals controls whether presomitic mesoderm (PSM) cells will adopt either a chondrogenic or lateral plate mesoderm fate. Here we document that SHH-mediated induction of Nkx3.2 maintains the competence of somitic cells to initiate chondrogenesis in response to subsequent BMP signals by repressing BMP-dependent induction of GATA genes. Conversely, administration of BMP signals to PSM or forced expression of GATA family members in chick PSM explants blocks induction of hedgehog-dependent gene expression. We demonstrate that GATA factors can interact with Gli factors and can recruit the transcriptional co-factor FOG1 (ZFPM1) to the regulatory region of the mouse Gli1 gene, repressing the induction of Gli1 by SHH by binding to both GATA and Gli binding sites. Knockdown of FOG1 reverses the ability of GATA factors to repress Gli1 expression. Our findings uncover a novel role for GATA transcription factors as repressors of hedgehog signaling, and document that NKX3.2 maintains the ability of sclerotomal cells to express SHH transcriptional targets in the presence of BMP signals by repressing the induction of Gata4/5/6.

  7. BMP-2 regulates the formation of oral sulcus in mouse tongue by altering the balance between TIMP-1 and MMP-13.

    Science.gov (United States)

    Fukui, Tadayoshi; Suga, Takeo; Iida, Ryo-Hei; Morito, Mitsuhiko; Luan, Xianghong; Diekwisch, Thomas G H; Nakamura, Yoshiki; Yamane, Akira

    2010-08-01

    The aim of this study is to investigate whether BMP-2 regulates the oral sulcus formation of mouse embryonic tongue by modifying the expression of TIMP and MMP. The BMP-2 siRNA induced a 180% increase in the depth of oral sulcus cavity (P sulcus into the mesenchymal tissues consisting of tongue floor, whereas the recombinant BMP-2 suppressed the process in the organ culture system of mouse embryonic tongue. The BMP-2 siRNA induced a 60% decrease in the expression of TIMP-1 mRNA (P sulcus in the BMP-2 siRNA treated mandibles. The recombinant BMP-2 induced a 220% increases in the expression of TIMP-1 mRNA and the area of the immunostaining for TIMP-1 around the oral sulcus was larger in the mandibles treated with the recombinant BMP-2 than the vehicle. The BMP-2 siRNA induced a 60% increase in the expression of MMP-13 protein and a marked increase in the staining intensity for MMP-13 was observed in the epithelial region of the BMP-2 siRNA treated mandibles. The recombinant BMP-2 induced a 70% decrease in the expression of MMP-13 mRNA and the decrease was mainly observed in the tissues around oral sulcus. The expressions of BMP-2, TIMP-1, and MMP-13 were verified in the tissues around in vivo developing oral sulcus at E11, 12, and 13 by immunohistochemistry. These results suggest that BMP-2 regulates the formation of oral sulcus by altering the balance between TIMP-1 and MMP-13.

  8. The Role of Hedgehog Signaling in Tumor Induced Bone Disease

    Directory of Open Access Journals (Sweden)

    Shellese A. Cannonier

    2015-08-01

    Full Text Available Despite significant progress in cancer treatments, tumor induced bone disease continues to cause significant morbidities. While tumors show distinct mutations and clinical characteristics, they behave similarly once they establish in bone. Tumors can metastasize to bone from distant sites (breast, prostate, lung, directly invade into bone (head and neck or originate from the bone (melanoma, chondrosarcoma where they cause pain, fractures, hypercalcemia, and ultimately, poor prognoses and outcomes. Tumors in bone secrete factors (interleukins and parathyroid hormone-related protein that induce RANKL expression from osteoblasts, causing an increase in osteoclast mediated bone resorption. While the mechanisms involved varies slightly between tumor types, many tumors display an increase in Hedgehog signaling components that lead to increased tumor growth, therapy failure, and metastasis. The work of multiple laboratories has detailed Hh signaling in several tumor types and revealed that tumor establishment in bone can be controlled by both canonical and non-canonical Hh signaling in a cell type specific manner. This review will explore the role of Hh signaling in the modulation of tumor induced bone disease, and will shed insight into possible therapeutic interventions for blocking Hh signaling in these tumors.

  9. The Role of Hedgehog Signaling in Tumor Induced Bone Disease

    Energy Technology Data Exchange (ETDEWEB)

    Cannonier, Shellese A.; Sterling, Julie A., E-mail: Julie.sterling@vanderbilt.edu [Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN 37235 (United States); Vanderbilt Center for Bone Biology, Department of Medicine, Division of Clinical Pharmacology Vanderbilt University, Nashville, TN 372335 (United States); Department of Cancer Biology, Vanderbilt University, Nashville, TN 37235 (United States)

    2015-08-26

    Despite significant progress in cancer treatments, tumor induced bone disease continues to cause significant morbidities. While tumors show distinct mutations and clinical characteristics, they behave similarly once they establish in bone. Tumors can metastasize to bone from distant sites (breast, prostate, lung), directly invade into bone (head and neck) or originate from the bone (melanoma, chondrosarcoma) where they cause pain, fractures, hypercalcemia, and ultimately, poor prognoses and outcomes. Tumors in bone secrete factors (interleukins and parathyroid hormone-related protein) that induce RANKL expression from osteoblasts, causing an increase in osteoclast mediated bone resorption. While the mechanisms involved varies slightly between tumor types, many tumors display an increase in Hedgehog signaling components that lead to increased tumor growth, therapy failure, and metastasis. The work of multiple laboratories has detailed Hh signaling in several tumor types and revealed that tumor establishment in bone can be controlled by both canonical and non-canonical Hh signaling in a cell type specific manner. This review will explore the role of Hh signaling in the modulation of tumor induced bone disease, and will shed insight into possible therapeutic interventions for blocking Hh signaling in these tumors.

  10. Mastoparan-Induced Cell Death Signalling in Chlamydomonas Reinhardtii

    NARCIS (Netherlands)

    Yordanova, Z.P.; Kapchina-Toteva, V.M.; Woltering, E.J.; Cristescu, S.M.; Harren, F.J.M.; Yakimova, E.T.

    2009-01-01

    The present study was focused on the elucidation of stress-induced cell death signaling events in the unicellular alga Chlamydomonas reinhardtii exposed to treatment with wasp venom mastoparan. By applying pharmacological approach with specific inhibitors, we have investigated the involvement of eth

  11. 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.

  12. Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics.

    Science.gov (United States)

    Tang, Zhurong; Wang, Zhe; Qing, Fangzhu; Ni, Yilu; Fan, Yujiang; Tan, Yanfei; Zhang, Xingdong

    2015-03-01

    Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway.

  13. A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology.

    Directory of Open Access Journals (Sweden)

    Courtney M Tate

    Full Text Available Bone morphogenetic proteins (BMPs, members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.

  14. A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology

    Science.gov (United States)

    Pallini, Roberto; Vakana, Eliza; Wyss, Lisa; Blosser, Wayne; Ricci-Vitiani, Lucia; D’Alessandris, Quintino Giorgio; Morgante, Liliana; Giannetti, Stefano; Maria Larocca, Luigi; Todaro, Matilde; Benfante, Antonina; Colorito, Maria Luisa; Stassi, Giorgio; De Maria, Ruggero; Rowlinson, Scott; Stancato, Louis

    2015-01-01

    Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis. PMID:25919028

  15. Phenotype characterization and sequence analysis of BMP2 and BMP4 variants in two Mexican families with oligodontia.

    Science.gov (United States)

    Mu, Y; Xu, Z; Contreras, C I; McDaniel, J S; Donly, K J; Chen, S

    2012-11-28

    Both BMP2 and BMP4 are involved in tooth development. We examined phenotypes and BMP2 and BMP4 gene variations in two Mexican oligodontia families. Physical and oral examinations and panoramic radiographs were performed on affected and unaffected members in these two families. The affected members lacked six or more teeth. DNA sequencing was performed to detect BMP2 and BMP4 gene variations. Three single nucleotide polymorphisms (SNPs) in BMP2 and BMP4 genes were identified in the two families, including one synonymous and two missense SNPs: BMP2 c261A>G, pS87S, BMP2 c570A>T, pR190S, and BMP4 c455T>C, pV152A. Among the six affected patients, 67% carried "GG" or "AG" genotype in BMP2 c261A>G and four were "TT" or "AT" genotype in BMP2 c570A>T (pR190S). Polymorphism of BMP4 c455T>C resulted in amino acid changes of Val/Ala (pV152A). BMP2 c261A>G and BMP4 c455T>C affect mRNA stability. This was the first time that BMP2 and BMP4 SNPs were observed in Mexican oligodontia families.

  16. Reconstruction and expression of recombinant adenovirus co-expressing BMP2 and BMP9%双表达骨形态发生蛋白2、9重组腺病毒载体的构建和表达

    Institute of Scientific and Technical Information of China (English)

    林春阳; 陈亮; 罗进勇; 邓忠良

    2011-01-01

    Objective To reconstruct and identify the recombinant adenovirus co-expressing BMP2 and BMP9.Method The genes of BMP2 and BMP9 were amplified from AdEasy vector by PCR and sub-cloned into pASG2 vector.The co-expression shuttle plasmid pASG2-BMP2,9 was confirmed by restriction endonuclease digestion, PCR and gene sequencing, then pASG2-BMP2,9 was electro-transducted into competent AdEasier cells to acquire recombinant adenovirus plasmid.Then, the recombinant vector was transfected into HEK293 cells and high-titer recombinant adenovirus (AdBMP2,9) was gained after rounds of amplification.The expression and bone induction capacity of AdBMP2,9 was observed in C3H10 cells.Result AdBMP2,9 was constructed successfully and the virus titer was 1010 IU/mL after amplification.AdBMP2,9 could express and induce alkaline phosphatase activity in C3H10 cells.Conclusion The recombinant adenovirus co-expressing BMP2 and BMP9 was constructed successfully, which provides a useful tool for bone tissue engineering.%目的 构建双表达骨形态发生蛋白(Bone morphogenic protein,BMP)2、9腺病毒重组体并进行鉴定.方法 自单一表达的BMP2或BMP9 AdEasy质粒上扩增BMP2和BMP9片段,先后亚克隆至穿梭质粒pASG2,获得双表达穿梭质粒pASG2-BMP2、9.酶切及PCR鉴定确认、测序正确后同源重组获得双表达BMP2、BMP9腺病毒质粒,转染至HEK-293细胞中包装和扩增得到高滴度双表达BMP2、BMP9腺病毒,体外感染C3H10细胞,RT-PCR鉴定并观察其早期诱导成骨情况.结果 成功构建双表达BMP2、BMP9的腺病毒,滴度约为1010IU/mL,RT-PCR证实双表达腺病毒在C3H10细胞中表达,其感染的C3H10细胞早期碱性磷酸酶含量较单一表达的BMP2或BMP9腺病毒组增加.结论 成功构建双表达BMP2、9的重组腺病毒载体,为进一步研究BMP2和BMP9的协同成骨作用和制备高效的组织工程人工骨提供了有利的工具.

  17. Overcoming low-alignment signal contrast induced alignment failure by alignment signal enhancement

    Science.gov (United States)

    Lee, Byeong Soo; Kim, Young Ha; Hwang, Hyunwoo; Lee, Jeongjin; Kong, Jeong Heung; Kang, Young Seog; Paarhuis, Bart; Kok, Haico; de Graaf, Roelof; Weichselbaum, Stefan; Droste, Richard; Mason, Christopher; Aarts, Igor; de Boeij, Wim P.

    2016-03-01

    Overlay is one of the key factors which enables optical lithography extension to 1X node DRAM manufacturing. It is natural that accurate wafer alignment is a prerequisite for good device overlay. However, alignment failures or misalignments are commonly observed in a fab. There are many factors which could induce alignment problems. Low alignment signal contrast is one of the main issues. Alignment signal contrast can be degraded by opaque stack materials or by alignment mark degradation due to processes like CMP. This issue can be compounded by mark sub-segmentation from design rules in combination with double or quadruple spacer process. Alignment signal contrast can be improved by applying new material or process optimization, which sometimes lead to the addition of another process-step with higher costs. If we can amplify the signal components containing the position information and reduce other unwanted signal and background contributions then we can improve alignment performance without process change. In this paper we use ASML's new alignment sensor (as was introduced and released on the NXT:1980Di) and sample wafers with special stacks which can induce poor alignment signal to demonstrate alignment and overlay improvement.

  18. 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

  19. Wnt signaling induces epithelial differentiation during cutaneous wound healing

    Directory of Open Access Journals (Sweden)

    Hocking Anne

    2006-01-01

    Full Text Available Abstract Background Cutaneous wound repair in adult mammals does not regenerate the original epithelial architecture and results in altered skin function. We propose that lack of regeneration may be due to the absence of appropriate molecular signals to promote regeneration. In this study, we investigated the regulation of Wnt signaling during cutaneous wound healing and the consequence of activating either the beta-catenin-dependent or beta-catenin-independent Wnt signaling on epidermal architecture during wound repair. Results We determined that the expression of Wnt ligands that typically signal via the beta-catenin-independent pathway is up-regulated in the wound while the beta-catenin-dependent Wnt signaling is activated in the hair follicles adjacent to the wound edge. Ectopic activation of beta-catenin-dependent Wnt signaling with lithium chloride in the wound resulted in epithelial cysts and occasional rudimentary hair follicle structures within the epidermis. In contrast, forced expression of Wnt-5a in the deeper wound induced changes in the interfollicular epithelium mimicking regeneration, including formation of epithelia-lined cysts in the wound dermis, rudimentary hair follicles and sebaceous glands, without formation of tumors. Conclusion These findings suggest that adult interfollicular epithelium is capable of responding to Wnt morphogenic signals necessary for restoring epithelial tissue patterning in the skin during wound repair.

  20. Bone Regeneration using an Alpha 2 Beta 1 Integrin-Specific Hydrogel as a BMP-2 Delivery Vehicle

    Science.gov (United States)

    Shekaran, Asha; García, José R.; Clark, Amy Y.; Kavanaugh, Taylor E.; Lin, Angela S.; Guldberg, Robert E.; García, Andrés J.

    2014-01-01

    Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential. PMID:24726536

  1. Bacillus thuringiensis metalloproteinase Bmp1 functions as a nematicidal virulence factor.

    Science.gov (United States)

    Luo, Xiaoxia; Chen, Ling; Huang, Qiong; Zheng, Jinshui; Zhou, Wei; Peng, Donghai; Ruan, Lifang; Sun, Ming

    2013-01-01

    Some Bacillus thuringiensis strains have high toxicity to nematodes. Nematicidal activity has been found in several families of crystal proteins, such as Cry5, Cry6, and Cry55. The B. thuringiensis strain YBT-1518 has three cry genes that have high nematicidal activity. The whole genome sequence of this strain contains multiple potential virulence factors. To evaluate the pathogenic potential of virulence factors, we focused on a metalloproteinase called Bmp1. It encompasses a consecutive N-terminal signal peptide, an FTP superfamily domain, an M4 neutral protease GluZincin superfamily, two Big-3 superfamily motifs, and a Gram-positive anchor superfamily motif as a C-terminal domain. Here, we showed that purified Bmp1 protein showed metalloproteinase activity and toxicity against Caenorhabditis elegans (the 50% lethal concentration is 610 ± 9.37 μg/ml). In addition, mixing Cry5Ba with Bmp1 protein enhanced the toxicity 7.9-fold (the expected toxicity of the two proteins calculated from their separate toxicities) against C. elegans. Confocal microscopic observation revealed that Bmp1 protein was detected from around the mouth and esophagus to the intestine. Striking microscopic images revealed that Bmp1 degrades intestine tissues, and the Cry5Ba causes intestinal shrinkage from the body wall. Thus, the B. thuringiensis Bmp1 metalloproteinase is a nematicidal virulence factor. These findings give a new insight into the relationship between B. thuringiensis and its host nematodes.

  2. Effect of BMP-2 Delivery Mode on Osteogenic Differentiation of Stem Cells

    Science.gov (United States)

    Kim, Yong-Jin; Kim, Ki-Suk; Jang, Hyon-Seok; Chung, Hyung-Min

    2017-01-01

    Differentiation of stem cells is an important strategy for regeneration of defective tissue in stem cell therapy. Bone morphogenetic protein-2 (BMP-2) is a well-known osteogenic differentiation factor that stimulates stem cell signaling pathways by activating transmembrane type I and type II receptors. However, BMPs have a very short half-life and may rapidly lose their bioactivity. Thus, a BMP delivery system is required to take advantage of an osteoinductive effect for osteogenic differentiation. Previously, BMP delivery has been designed and evaluated for osteogenic differentiation, focusing on carriers and sustained release system for delivery of BMPs. The effect of the delivery mode in cell culture plate on osteogenic differentiation potential was not evaluated. Herein, to investigate the effect of delivery mode on osteogenic differentiation of BM-MSCs in this study, we fabricated bottom-up release and top-down release systems for culture plate delivery of BMP-2. And also, we selected Arg-Gly-Asp- (RGD-) conjugated alginate hydrogel for BMP-2 delivery because alginate is able to release BMP-2 in a sustained manner and it is a biocompatible material. After 7 days of culture, the bottom-up release system in culture plate significantly stimulated alkaline phosphate activity of human bone marrow-mesenchymal stem cells. The present study highlights the potential value of the tool in stem cell therapy. PMID:28197209

  3. 核心结合因子α1在BMP-2调控细胞外基质蛋白表达中的作用%Study of cbfα1 on the expression of extracellular matrix proteins in dental papilla cells induced by BMP-2 in vitro

    Institute of Scientific and Technical Information of China (English)

    余擎; 朱庆林; 孙汉堂; 田宇; 何文喜; 肖明振

    2008-01-01

    目的:探讨核心结合因子α1(cbfa1)在BMP-2调控体外培养的牙乳头细胞表达细胞外基质蛋白中的作用.方法:采用反义核酸技术,体外阻断培养的牙乳头细胞中cbfα1的表达,分别用RT-PCR、Western印迹等方法观察200ng/mL BMP-2作用6h后细胞中相关基质蛋白,碱性磷酸酶(ALP)、骨钙素(OC)、骨连蛋白(ON)、骨桥素(OPN)、骨涎蛋白(BSP)、牙本质基质蛋白1(DMP-1)以及牙本质涎磷蛋白(DSPP)的表达,采用SPSS 11.0软件包对数据进行方差分析.结果:外源性BMP-2能明显上调牙乳头细胞中ALP、OC含量以及OPN、BSP和ON的表达,当反义阻断cbfα1的表达时,ALP、OC、OPN和BSP的表达显著降低(P<0.01).结论:cbfα1参与了BMP-2调控体外培养的牙乳头细胞表达细胞外基质蛋白的信号转导过程.

  4. BMP7 gene transfer via gold nanoparticles into stroma inhibits corneal fibrosis in vivo.

    Directory of Open Access Journals (Sweden)

    Ashish Tandon

    Full Text Available This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA, F-actin and fibronectin], immune reaction (CD11b and F4/80, keratocyte apoptosis (TUNEL, calcification (alizarin red, vonKossa and osteocalcin, and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF and in vitro experiments were used to characterize the molecular mechanism mediating BMP7's anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×10(4 gene copies/ug DNA. Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p88%; p<0.0001, and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001 and Smad6 (53%, p<0.001, and decreased αSMA (78%; p<0.001 protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling.

  5. Morphine dependence and withdrawal induced changes in cholinergic signaling

    Science.gov (United States)

    Neugebauer, Nichole M.; Einstein, Emily B.; Lopez, Maria B.; McClure-Begley, Tristan D.; Mineur, Yann S.; Picciotto, Marina R.

    2013-01-01

    Cholinergic signaling is thought to be involved in morphine dependence and withdrawal, but the specific mechanisms involved remain unclear. The current study aimed to identify alterations in the cholinergic system that may contribute to the development of morphine dependence and withdrawal. Acetylcholinesterase (AChE) activity and [3H]-epibatidine binding were evaluated in order to determine if morphine dependence and withdrawal induces alterations in cholinergic signaling or expression of high affinity nicotinic acetylcholine receptors (nAChRs) in the midbrain (MB), medial habenula (MHb) and interpeduncular nucleus (IPN). The effect of cholinergic signaling through nAChRs on morphine-withdrawal induced jumping behavior was then determined. Lastly, the contribution of β4-containing nAChRs receptors in the MHb to morphine-withdrawal induced jumping behavior and neuronal activity as indicated by c-fos expression was assessed. Chronic morphine administration decreased AChE activity in MB and MHb, an effect that was no longer present following precipitated withdrawal. Morphine dependent mice showed increased nicotinic acetylcholine receptor (nAChR) levels in MB. Further, nicotine (0.4 mg/kg) and lobeline (3 mg/kg) decreased jumping behavior while mecamylamine (1 mg/kg) had no effect. Knock-down of β4 subunit-containing nAChRs in the MHb attenuated c-fos activation, but did not decrease morphine withdrawal-induced jumping. Thus, morphine withdrawal induces cholinergic signaling in the MHb, but this does not appear to be responsible for the effects of cholinergic drugs on somatic signs of opiate withdrawal, as measured by jumping behavior. PMID:23651795

  6. Improving Osteogenesis Activity on BMP-2-Immobilized PCL Fibers Modified by the γ-Ray Irradiation Technique

    Directory of Open Access Journals (Sweden)

    Young-Pil Yun

    2015-01-01

    Full Text Available The purpose of this study was to demonstrate the ability of BMP-2-immobilized polycaprolactone (PCL fibers modified using the γ-ray irradiation technique to induce the osteogenic differentiation of MG-63 cells. Poly acrylic acid (AAc was grafted onto the PCL fibers by the γ-ray irradiation technique. BMP-2 was then subsequently immobilized onto the AAc-PCL fibers (BMP-2/AAc-PCL. PCL and surface-modified PCL fibers was characterized by evaluation with a scanning electron microscope (SEM, X-ray photoelectron spectroscopy (XPS, and contact angle. The biological activity of the PCL and surface-modified PCL fibers were characterized by alkaline phosphatase (ALP activity, calcium deposition, and the mRNA expression of osteocalcin and osteopontin in MG-63 cells. Successfully grafted AAc and PCL fibers with immobilized BMP-2 were confirmed by XPS results. The results of the contact angle showed that BMP-2/AAc-PCL fibers have more hydrophilic properties in comparison to PCL fibers. The ALP activity, calcium deposition, and gene expressions of MG-63 cells grown on BMP-2/AAc-PCL fibers showed greatly induced osteogenic differentiation in comparison to the PCL fibers. In conclusion, these results demonstrated that BMP-2/AAc-PCL fibers have the potential to effectively induce the osteogenic differentiation of MG-63 cells.

  7. 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

  8. Roxarsone induces angiogenesis via PI3K/Akt signaling

    OpenAIRE

    2016-01-01

    Background 3-Nitro-4-hydroxy phenyl arsenic acid, roxarsone, is widely used as an organic arsenic feed additive for livestock and poultry, which may increase the level of arsenic in the environment and the risk of exposure to arsenic in human. Little information is focused on the angiogenesis roxarsone-induced and its mechanism at present. This paper aims to study the role of PI3K/Akt signaling in roxarsone-induced angiogenesis in rat vascular endothelial cells and a mouse B16–F10 melanoma xe...

  9. PI3K signaling supports amphetamine-induced dopamine efflux.

    Science.gov (United States)

    Lute, Brandon J; Khoshbouei, Habibeh; Saunders, Christine; Sen, Namita; Lin, Richard Z; Javitch, Jonathan A; Galli, Aurelio

    2008-08-01

    The dopamine (DA) transporter (DAT) is a major molecular target of the psychostimulant amphetamine (AMPH). AMPH, as a result of its ability to reverse DAT-mediated inward transport of DA, induces DA efflux thereby increasing extracellular DA levels. This increase is thought to underlie the behavioral effects of AMPH. We have demonstrated previously that insulin, through phosphatidylinositol 3-kinase (PI3K) signaling, regulates DA clearance by fine-tuning DAT plasma membrane expression. PI3K signaling may represent a novel mechanism for regulating DA efflux evoked by AMPH, since only active DAT at the plasma membrane can efflux DA. Here, we show in both a heterologous expression system and DA neurons that inhibition of PI3K decreases DAT cell surface expression and, as a consequence, AMPH-induced DA efflux.

  10. Electrical signaling in Aloe vera induced by localized thermal stress.

    Science.gov (United States)

    Volkov, Alexander G; Lang, Ryan D; Volkova-Gugeshashvili, Maia I

    2007-11-01

    Action potentials in higher plants are theorized as the information carriers in intercellular and intracellular communication in the presence of environmental stressors. Among the most common stressors is heat shock. Under stressful conditions, the response reactions of plant tissues and organs can be local or transmitted over long distances. In this article, the speeds of propagation of thermally induced action potentials in green plants are discussed, and their speeds were found to be comparable to those occurring in various mammalian species. These rapid action potentials in green plants were recorded in real time using modern data acquisition techniques. According to our measurements, a single application of localized heat stress induces fast action potentials in Aloe vera (67 m/s). Electrical signals propagated along all leaves of the A. vera plants were studied. Possible pathways for electrical signal propagation in vascular plants are also discussed.

  11. Different activation signals induce distinct mast cell degranulation strategies

    Science.gov (United States)

    Sibilano, Riccardo; Marichal, Thomas; Reber, Laurent L.; Cenac, Nicolas; McNeil, Benjamin D.; Dong, Xinzhong; Hernandez, Joseph D.; Sagi-Eisenberg, Ronit; Hammel, Ilan; Roers, Axel; Valitutti, Salvatore; Tsai, Mindy

    2016-01-01

    Mast cells (MCs) influence intercellular communication during inflammation by secreting cytoplasmic granules that contain diverse mediators. Here, we have demonstrated that MCs decode different activation stimuli into spatially and temporally distinct patterns of granule secretion. Certain signals, including substance P, the complement anaphylatoxins C3a and C5a, and endothelin 1, induced human MCs rapidly to secrete small and relatively spherical granule structures, a pattern consistent with the secretion of individual granules. Conversely, activating MCs with anti-IgE increased the time partition between signaling and secretion, which was associated with a period of sustained elevation of intracellular calcium and formation of larger and more heterogeneously shaped granule structures that underwent prolonged exteriorization. Pharmacological inhibition of IKK-β during IgE-dependent stimulation strongly reduced the time partition between signaling and secretion, inhibited SNAP23/STX4 complex formation, and switched the degranulation pattern into one that resembled degranulation induced by substance P. IgE-dependent and substance P–dependent activation in vivo also induced different patterns of mouse MC degranulation that were associated with distinct local and systemic pathophysiological responses. These findings show that cytoplasmic granule secretion from MCs that occurs in response to different activating stimuli can exhibit distinct dynamics and features that are associated with distinct patterns of MC-dependent inflammation. PMID:27643442

  12. Biologic effect and immunoisolating behavior of BMP-2 gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules.

    Science.gov (United States)

    Ding, H F; Liu, R; Li, B G; Lou, J R; Dai, K R; Tang, T T

    2007-11-03

    We investigated the encapsulation of BMP-2 gene-modified mesenchymal stem cells (MSCs) in alginate-poly-L-lysine (APA) microcapsules for the persistent delivery of bone morphogenic protein-2 (BMP-2) to induce bone formation. An electrostatic droplet generator was employed to produce APA microcapsules containing encapsulated beta-gal or BMP-2 gene-transfected bone marrow-derived MSCs. We found that X-gal staining was still positive 28 days after encapsulation. Encapsulated BMP-2 gene-transfected cells were capable of constitutive delivery of BMP-2 proteins for at least 30 days. The encapsulated BMP-2 gene-transfected MSCs or the encapsulated non-gene transfer MSCs (control group) were cocultured with the undifferentiated MSCs. The gene products from the encapsulated BMP-2 cells could induce the undifferentiated MSCs to become osteoblasts that had higher alkaline phosphatase (ALP) activity than those in the control group (pAPA microcapsules could inhibit the permeation of fluorescein isothiocyanate-conjuncted immunoglobulin G. Mixed lymphocyte reaction also indicates that the APA microcapsules could prevent the encapsulated BMP-2 gene-transfected MSCs from initiating the cellular immune response. These results demonstrated that the nonautologous BMP-2 gene-transfected stem cells are of potential utility for enhancement of bone repair and bone regeneration in vivo.

  13. Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice.

    Science.gov (United States)

    McBride-Gagyi, Sarah Howe; McKenzie, Jennifer A; Buettmann, Evan G; Gardner, Michael J; Silva, Matthew J

    2015-12-01

    Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10-24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2.

  14. Overexpression of constitutively active BMP-receptor-IB in mouse skin causes an ichthyosis-vulgaris-like disease.

    Science.gov (United States)

    Yu, Xueyan; Espinoza-Lewis, Ramón A; Sun, Cheng; Lin, Lisong; He, Fenglei; Xiong, Wei; Yang, Jing; Wang, Alun; Chen, Yiping

    2010-12-01

    The skin is the outer layer of protection against the environment. The development and formation of the skin is regulated by several genetic cascades including the bone morphogenetic protein (BMP) signaling pathway, which has been suggested to play an important role during embryonic organ development. Several skin defects and diseases are caused by genetic mutations or disorders. Ichthyosis is a common genetic skin disorder characterized by dry scaly skin. Loss-of-function mutations in the filaggrin (FLG) gene have been identified as the cause of the ichthyosis vulgaris (IV) phenotype; however, the direct regulation of filaggrin expression in vivo is unknown. We present evidence that BMP signaling regulates filaggrin expression in the epidermis. Mice expressing a constitutively active form of BMP-receptor-IB in the developing epidermis exhibit a phenotype resembling IV in humans, including dry flaky skin, compact hyperkeratosis, and an attenuated granular layer associated with a significantly downregulated expression of filaggrin. Regulation of filaggrin expression by BMP signaling has been further confirmed by the application of exogenous BMP2 in skin explants and by a transgenic model overexpressing Noggin in the epidermis. Our results demonstrate that aberrant BMP signaling in the epidermis causes overproliferation and hyperkeratinization, leading to an IV-like skin disease.

  15. Activation of p38 mitogen-activated protein kinase contribute to BMP4-induced alkaline phosphatase expression in MC3T3-E1 preosteoblast

    Institute of Scientific and Technical Information of China (English)

    YUAN Ye; Wu Zhi-jun; YAO Hui-yu; YU Xiao-dan; GUO Zi-kuan; CHEN Xiao-san; TANG Pei-xian; MAO Ning

    2006-01-01

    @@ Bone morphogenetic proteins (BMPs) induce ectopic bone formation and promote osteoblast differentiation.1 It has been documented that Smad transcriptional factors function as primary mediators of BMPs activity. Receptor-regulated Smad (Smad1, 5, 8) could be phosphorylated by activated BMPR-I and form complex with Smad4. The Smad complex translocates to the nucleus and regulate target gene transcription.2

  16. 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...

  17. A carboxy terminal BMP/TGF-β binding site in secreted phosphoprotein 24 kD independently affects BMP-2 activity.

    Science.gov (United States)

    Tian, Haijun; Li, Chen-Shuang; Zhao, Ke-Wei; Wang, Jeffrey C; Duarte, M Eugenia L; David, Cynthia L; Phan, Kevin; Atti, Elisa; Brochmann, Elsa J; Murray, Samuel S

    2015-04-01

    Secreted phosphoprotein 24 kD (spp24) is a bone matrix protein isolated during attempts to identify osteogenic proteins. It is not osteogenic but performs other important roles in the regulation of bone metabolism, at least in part, by binding to and affecting the activity of members of the BMP/TGF-β family of cytokines. Spp24 exists in a number of forms that preserve the N-terminus and are truncated at the C-terminus. The hypothesized cytokine binding domain is present within the cystatin domain which is preserved in all of the N-terminal products. In this report, we describe a C-terminal fragment that is distinct from the cystatin domain and which independently binds to BMP-2 and TGF-β. This fragment inhibited BMP-2 activity in an ectopic bone forming assay. A shorter C-terminal product did not inhibit BMP-2 activity but improved bone quality induced by BMP-2 and produced increased calcium deposition outside of bone. Spp24 has been used to develop several potential therapeutic proteins. These results provide more information on the function of spp24 and provide other materials that can be exploited for clinical interventions.

  18. Molecular characterization, expression and methylation status analysis of BMP4 gene in skin tissue of Liaoning cashmere goat during hair follicle cycle.

    Science.gov (United States)

    Bai, Wen L; Dang, Yun L; Wang, Jiao J; Yin, Rong H; Wang, Ze Y; Zhu, Yu B; Cong, Yu Y; Xue, Hui L; Deng, Liang; Guo, Dan; Wang, Shi Q; Yang, Shu H

    2016-08-01

    Bone morphogenetic protein 4 (BMP4) is a member of the bone morphogenetic protein family (BMPs). It is involved in the development and cycle of hair follicle, as well as, is thought to be a potential candidate gene for cashmere traits in goats. In the present study, we isolated and characterized a full-length open reading frame (ORF) of BMP4 cDNA from the skin tissue of Liaoning cashmere goat, and investigated the transcriptional pattern and methylation status of BMP4 gene in skin tissue of this breed during different stages of hair follicle cycle. The sequence analysis indicated that the isolated cDNA was 1264-bp in length containing a complete ORF of 1230-bp. It encoded a precursor peptide of 409 amino acids with a signal peptide of 19 amino acids. The structural analysis indicated that goat BMP4 contains typical TGF-β propeptide and TGF-β domains. In skin tissue, BMP4 is generally transcribed in an ascendant pattern from anagen to telogen. The methylation level of 5' flanking regulatory region of BMP4 gene might be involved in its mRNA expression in skin tissue: a higher BMP4 methylation level in skin coincides with a lower expression of BMP4 mRNA. These results from the present work provided a foundation for further insight into the functional and regulatory characteristics of BMP4 in the development and cycle of hair follicle in Liaoning Cashmere goat.

  19. Cultured Human Periosteum-Derived Cells Can Differentiate into Osteoblasts in a Perioxisome Proliferator-Activated Receptor Gamma-Mediated Fashion via Bone Morphogenetic Protein signaling.

    Science.gov (United States)

    Chung, Jin-Eun; Park, Jin-Ho; Yun, Jeong-Won; Kang, Young-Hoon; Park, Bong-Wook; Hwang, Sun-Chul; Cho, Yeong-Cheol; Sung, Iel-Yong; Woo, Dong Kyun; Byun, June-Ho

    2016-01-01

    The differentiation of mesenchymal stem cells towards an osteoblastic fate depends on numerous signaling pathways, including activation of bone morphogenetic protein (BMP) signaling components. Commitment to osteogenesis is associated with activation of osteoblast-related signal transduction, whereas inactivation of this signal transduction favors adipogenesis. BMP signaling also has a critical role in the processes by which mesenchymal stem cells undergo commitment to the adipocyte lineage. In our previous study, we demonstrated that an agonist of the perioxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipocyte differentiation, stimulates osteoblastic differentiation of cultured human periosteum-derived cells. In this study, we used dorsomorphin, a selective small molecule inhibitor of BMP signaling, to investigate whether BMP signaling is involved in the positive effects of PPARγ agonists on osteogenic phenotypes of cultured human periosteum-derived cells. Both histochemical detection and bioactivity of ALP were clearly increased in the periosteum-derived cells treated with the PPARγ agonist at day 10 of culture. Treatment with the PPARγ agonist also caused an increase in alizarin red S staining and calcium content in the periosteum-derived osteoblasts at 2 and 3 weeks of culture. In contrast, dorsomorphin markedly decreased ALP activity, alizarin red S staining and calcium content in both the cells treated with PPARγ agonist and the cells cultured in osteogenic induction media without PPARγ agonist during the culture period. In addition, the PPARγ agonist clearly increased osteogenic differentiation medium-induced BMP-2 upregulation in the periosteum-derived osteoblastic cells at 2 weeks of culture as determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), immunoblotting, and immunocytochemical analyses. Although further study will be needed to clarify the mechanisms of PPARγ-regulated osteogenesis

  20. Leptin-Induced JAK/STAT Signaling and Cancer Growth

    Directory of Open Access Journals (Sweden)

    McKay Mullen

    2016-07-01

    Full Text Available Growth factor and cytokine signaling can influence the development of several cancer types. One of the key players in the development of cancer is the Janus kinas (JAK signal transducer of activators of transcription (STAT signaling pathway. The majority of growth factors and cytokine interactions with their membrane-bound receptors trigger JAK-STAT activation. The influential relationship between obesity and cancer is a fact. However, there is a complex sequence of events contributing to the regulation of this mechanism to promote tumor growth, yet to be fully elucidated. The JAK-STAT pathway is influenced by obesity-associated changes that have been shown to impact cancer growth and progression. This intricate process is highly regulated by a vast array of adipokines and cytokines that exert their pleiotropic effects on cancer cells to enhance metastasis to distant target sites. Leptin is a cytokine, or more precise, an adipokine secreted mainly by adipose tissue that requires JAK-STAT activation to exert its biological functions. Leptin is the central regulator of energy balance and appetite. Leptin binding to its receptor OB-R in turn activates JAK-STAT, which induces proliferation, angiogenesis, and anti-apoptotic events in normal cells and malignant cells expressing the receptor. Leptin also induces crosstalk with Notch and IL-1 (NILCO, which involves other angiogenic factors promoting tumor growth. Therefore, the existence of multiple novel classes of therapeutics that target the JAK/STAT pathway has significant clinical implications. Then, the identification of the signaling networks and factors that regulate the obesity-cancer link to which potential pharmacologic interventions can be implemented to inhibit tumor growth and metastasis. In this review, we will discuss the specific relationship between leptin-JAK-STAT signaling and cancer.

  1. Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis.

    Science.gov (United States)

    Nemoto, Eiji; Ebe, Yukari; Kanaya, Sousuke; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi

    2012-06-15

    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.

  2. Silymarin Suppresses Cellular Inflammation By Inducing Reparative Stress Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Lovelace, Erica S.; Wagoner, Jessica; MacDonald, James; Bammler, Theo; Bruckner, Jacob; Brownell, Jessica; Beyer, Richard; Zink, Erika M.; Kim, Young-Mo; Kyle, Jennifer E.; Webb-Robertson, Bobbie-Jo M.; Waters, Katrina M.; Metz, Thomas O.; Farin, Federico; Oberlies, Nicholas H.; Polyak, Steve

    2015-08-28

    Silymarin (SM), a natural product, is touted as a liver protectant and preventer of both chronic inflammation and diseases. To define how SM elicits these effects at a systems level, we performed transcriptional profiling, metabolomics, and signaling studies in human liver and T cell lines. Multiple pathways associated with cellular stress and metabolism were modulated by SM treatment within 0.5 to four hours: activation of Activating Transcription Factor 4 (ATF-4) and adenosine monophosphate protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) signaling, the latter being associated with induction of DNA-damage-inducible transcript 4 (DDIT4). Metabolomics analyses revealed suppression of glycolytic, TCA cycle, and amino acid metabolism by SM treatment. Antiinflammatory effects arose with prolonged (i.e. 24 hours) SM exposure, with suppression of multiple proinflammatory mRNAs and nuclear factor kappa B (NF-κB) and forkhead box O (FOXO) signaling. Studies with murine knock out cells revealed that SM inhibition of both mTOR and NF-κB was partially AMPK dependent, while SM inhibition of the mTOR pathway in part required DDIT4. Thus, SM activates stress and repair responses that culminate in an anti-inflammatory phenotype. Other natural products induced similar stress responses, which correlated with their ability to suppress inflammation. Therefore, natural products like SM may be useful as tools to define how metabolic, stress, and repair pathways regulate cellular inflammation.

  3. Epidermal growth factor signaling induces behavioral quiescence in Caenorhabditis elegans.

    Science.gov (United States)

    Van Buskirk, Cheryl; Sternberg, Paul W

    2007-10-01

    The epidermal growth factor receptor (EGFR)/ErbB receptor tyrosine kinases regulate several aspects of development, including the development of the mammalian nervous system. ErbB signaling also has physiological effects on neuronal function, with influences on synaptic plasticity and daily cycles of activity. However, little is known about the effectors of EGFR activation in neurons. Here we show that EGF signaling has a nondevelopmental effect on behavior in Caenorhabditis elegans. Ectopic expression of the EGF-like ligand LIN-3 at any stage induces a reversible cessation of feeding and locomotion. These effects are mediated by neuronal EGFR (also called LET-23) and phospholipase C-gamma (PLC-gamma), diacylglycerol-binding proteins, and regulators of synaptic vesicle release. Activation of EGFR within a single neuron, ALA, is sufficient to induce a quiescent state. This pathway modulates the cessation of pharyngeal pumping and locomotion that normally occurs during the lethargus period that precedes larval molting. Our results reveal an evolutionarily conserved role for EGF signaling in the regulation of behavioral quiescence.

  4. Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

    Energy Technology Data Exchange (ETDEWEB)

    Yonezawa, Takayuki [Department of Nutriproteomics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Lee, Ji-Won [Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Hibino, Ayaka; Asai, Midori [Department of Biological Chemistry, College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Hojo, Hironori [Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Cha, Byung-Yoon [Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Teruya, Toshiaki [Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Faculty of Education, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213 (Japan); Nagai, Kazuo [Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Department of Biological Chemistry, College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto, Kasugai, Aichi 487-8501 (Japan); Chung, Ung-Il [Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Yagasaki, Kazumi [Department of Nutriproteomics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Division of Applied Biological Chemistry, Institute of Agriculture, Tokyo Noko University, 3-5-8 Saiwai, Fuchu, Tokyo 183-8509 (Japan); and others

    2011-06-03

    Highlights: {yields} Harmine promotes the activity and mRNA expression of ALP. {yields} Harmine enhances the expressions of osteocalcin mRNA and protein. {yields} Harmine induces osteoblastic mineralization. {yields} Harmine upregulates the mRNA expressions of BMPs, Runx2 and Osterix. {yields} 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 {beta}-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 {beta}-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

  5. Adenosine signaling and the energetic costs of induced immunity.

    Directory of Open Access Journals (Sweden)

    Brian P Lazzaro

    2015-04-01

    Full Text Available Life history theory predicts that trait evolution should be constrained by competing physiological demands on an organism. Immune defense provides a classic example in which immune responses are presumed to be costly and therefore come at the expense of other traits related to fitness. One strategy for mitigating the costs of expensive traits is to render them inducible, such that the cost is paid only when the trait is utilized. In the current issue of PLOS Biology, Bajgar and colleagues elegantly demonstrate the energetic and life history cost of the immune response that Drosophila melanogaster larvae induce after infection by the parasitoid wasp Leptopilina boulardi. These authors show that infection-induced proliferation of defensive blood cells commands a diversion of dietary carbon away from somatic growth and development, with simple sugars instead being shunted to the hematopoetic organ for rapid conversion into the raw energy required for cell proliferation. This metabolic shift results in a 15% delay in the development of the infected larva and is mediated by adenosine signaling between the hematopoietic organ and the central metabolic control organ of the host fly. The adenosine signal thus allows D. melanogaster to rapidly marshal the energy needed for effective defense and to pay the cost of immunity only when infected.

  6. Effects of BMP2 and VEGF165 on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Lin, Zhaowei; Wang, Jiang-Sheng; Lin, Lijun; Zhang, Jingwen; Liu, Yunlong; Shuai, Ming; Li, Qi

    2014-03-01

    Bone marrow-derived mesenchymal stem cells (MSCs) are dominant seed cell sources for bone regeneration. Bone morphogenetic proteins (BMPs) initiate cartilage and bone formation in a sequential cascade. Vascular endothelial growth factor (VEGF) is an essential coordinator of extracellular matrix remodeling, angiogenesis and bone formation. In the present study, the effects of the vascular endothelial growth factor 165 (VEGF165) and bone morphogenetic protein 2 (BMP2) genes on bone regeneration were investigated by the lentivirus-mediated cotransfection of the two genes into rat bone marrow-derived MSCs. The successful co-expression of the two genes in the MSCs was confirmed using quantitative polymerase chain reaction (qPCR) and western blot analysis. The results of alizarin red and alkaline phosphatase (ALP) staining at 14 days subsequent to transfection showed that the area of staining in cells transfected with BMP2 alone was higher than that in cells transfected with BMP2 and VEGF165 or untransfected control cells, while the BMP2 + VEGF165 group showed significantly more staining than the untransfected control. This indicated that BMP2 alone exhibited a stronger effect in bone regeneration than BMP2 in combination with VEGF165. Similarly, in inducing culture medium, the ALP activity of the BMP2 + VEGF165 group was notably suppressed compared with that of the BMP2 group. The overexpression of VEGF165 inhibited BMP2-induced MSC differentiation and osteogenesis in vitro. Whether or not local VEGF gene therapy is likely to affect bone regeneration in vivo requires further investigation.

  7. 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...

  8. Tbx2 regulates anterior neural specification by repressing FGF signaling pathway.

    Science.gov (United States)

    Cho, Gun-Sik; Park, Dong-Seok; Choi, Sun-Cheol; Han, Jin-Kwan

    2017-01-15

    During early embryogenesis, FGF signals regulate the antero-posterior (AP) patterning of the neural plate by promoting posterior cell fates. In particular, BMP signal-mediated attenuation of FGF pathway plays a critical role in the determination of the anterior neural region. Here we show that Tbx2, a T-box transcriptional repressor regulates anterior neural specification by suppressing FGF8 signaling pathway in Xenopus embryo. Tbx2 is expressed in the anterior edge of the neural plate in early neurulae. Overexpression and knockdown of Tbx2 induce expansion and reduction in the expression of anterior neural markers, respectively. It also suppresses FGF8-induced ERK phosphorylation and neural caudalization. Tbx2, which is a target gene of BMP signal, down-regulates FGF8 signaling by inhibiting the expression of Flrt3, a positive regulator of this pathway. We found that Tbx2 binds directly to the T-box element located in the promoter region of Flrt3 gene, thereby interfering with the activity of the promoter. Consistently, Tbx2 augmentation of anterior neural formation is inhibited by co-expression of Flrt3. Furthermore, disruption of the anterior-most structures such as eyes in Tbx2-depleted embryos can be rescued by inhibition of Flrt3 function or FGF signaling. Taken together, our results suggest that Tbx2 mediates BMP signal to down-regulate FGF signaling pathway by repressing Flrt3 expression for anterior tissue formation.

  9. Secreted Stress-Induced Phosphoprotein 1 Activates the ALK2-SMAD Signaling Pathways and Promotes Cell Proliferation of Ovarian Cancer Cells

    Directory of Open Access Journals (Sweden)

    Chia-Lung Tsai

    2012-08-01

    Full Text Available Stress-induced phosphoprotein 1 (STIP1, a cochaperone that organizes other chaperones, heat shock proteins (HSPs, was recently shown to be secreted by human ovarian cancer cells. In neuronal tissues, binding to prion protein was required for STIP1 to activate the ERK (extracellular-regulated MAP kinase signaling pathways. However, we report that STIP1 binding to a bone morphogenetic protein (BMP receptor, ALK2 (activin A receptor, type II-like kinase 2, was necessary and sufficient to stimulate proliferation of ovarian cancer cells. The binding of STIP1 to ALK2 activated the SMAD signaling pathway, leading to transcriptional activation of ID3 (inhibitor of DNA binding 3, promoting cell proliferation. In conclusion, ovarian-cancer-tissue-secreted STIP1 stimulates cancer cell proliferation by binding to ALK2 and activating the SMAD-ID3 signaling pathways. Although animal studies are needed to confirm these mechanisms in vivo, our results may pave the way for developing novel therapeutic strategies for ovarian cancer.

  10. 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;

    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 osteogenesis was validated by findings of increased gene expression of SMAD1 and an increase in dual phosphorylation of ser 463 and ser 465 in the SMAD 1/5/8 pathway. We concluded that preconditioning hMSCs with BMP2 stimulates osteogenesis: proliferation with matrix secretion and matrix maturation of h......MSCs. 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 be avoided....

  11. 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.

  12. Accelerated bone growth in vitro by the conjugation of BMP2 peptide with hydroxyapatite on titanium alloy.

    Science.gov (United States)

    Cai, Yanli; Wang, Xiaoyan; Poh, Chye Khoon; Tan, Hark Chuan; Soe, Min Tun; Zhang, Sam; Wang, Wilson

    2014-04-01

    Titanium alloys have been widely used in orthopedic practice due to their inherent bioactivity, however it is still insufficient to truly and reliably incorporate into living bone. In this work, polydopamine film was employed to induce the growth of hydroxyapatite (HA) on titanium alloy to enhance its osteoconductivity. Bone morphogenetic protein-2 (BMP2) peptide was absorbed into the HA particles for osteoinductivity. The precipitation of HA and the existence of BMP2 peptide were examined by X-ray diffraction, X-ray photoelectron spectroscopy and fluorescence microscopy. The dissolution of HA and the release of BMP2 peptide were monitored by measuring the concentrations of calcium ions and BMP2 peptide in phosphate buffered saline solution, respectively. The effect of BMP2 peptide incorporated into HA coating on bone growth was evaluated in vitro by cell culture tests, including cell attachment, alkaline phosphatase (ALP) activity, and gene expression. The results show that the HA particles grown on the substrate are mediated by the polydopamine film. The BMP2 peptide is distributed uniformly on HA-coated substrate and released in a sustained manner. Moreover, the conjunction of HA and BMP2 peptide increases cell adhesion, ALP activity and gene expression of osteogenic markers, which are potentially useful in the development of enhanced orthopedic medical devices.

  13. In anemia of multiple myeloma, hepcidin is induced by increased bone morphogenetic protein 2

    Science.gov (United States)

    Maes, Ken; Nemeth, Elizabeta; Roodman, G. David; Huston, Alissa; Esteve, Flavia; Freytes, Cesar; Callander, Natalie; Katodritou, Eirini; Tussing-Humphreys, Lisa; Rivera, Seth; Vanderkerken, Karin; Lichtenstein, Alan

    2010-01-01

    Hepcidin is the principal iron-regulatory hormone and a pathogenic factor in anemia of inflammation. Patients with multiple myeloma (MM) frequently present with anemia. We showed that MM patients had increased serum hepcidin, which inversely correlated with hemoglobin, suggesting that hepcidin contributes to MM-related anemia. Searching for hepcidin-inducing cytokines in MM, we quantified the stimulation of hepcidin promoter-luciferase activity in HuH7 cells by MM sera. MM sera activated the hepcidin promoter significantly more than did normal sera. We then examined the role of bone morphogenetic proteins (BMPs) and interleukin-6 (IL-6), the major transcriptional regulators of hepcidin. Mutations in both BMP-responsive elements abrogated the activation dramatically, while mutations in the IL-6–responsive signal transducer and activator of transcription 3-binding site (STAT3-BS) had only a minor effect. Cotreatment with anti–BMP-2/4 or noggin-Fc blocked the promoter induction with all MM sera, anti–IL-6 blocked it with a minority of sera, whereas anti–BMP-4, -6, or -9 antibodies had no effect. BMP-2–immunodepleted MM sera had decreased promoter stimulatory capacity, and BMP-2 concentrations in MM sera were significantly higher than in normal sera. Our results demonstrate that BMP-2 is a major mediator of the hepcidin stimulatory activity of MM sera. PMID:20679527

  14. Exercise-induced endocannabinoid signaling is modulated by intensity.

    Science.gov (United States)

    Raichlen, David A; Foster, Adam D; Seillier, Alexandre; Giuffrida, Andrea; Gerdeman, Gregory L

    2013-04-01

    Endocannabinoids (eCB) are endogenous ligands for cannabinoid receptors that are densely expressed in brain networks responsible for reward. Recent work shows that exercise activates the eCB system in humans and other mammals, suggesting eCBs are partly responsible for the reported improvements in mood and affect following aerobic exercise in humans. However, exercise-induced psychological changes reported by runners are known to be dependent on exercise intensity, suggesting that any underlying molecular mechanism should also change with varying levels of exercise intensity. Here, we examine circulating levels of eCBs following aerobic exercise (treadmill running) in recreationally fit human runners at four different intensities. We show that eCB signaling is indeed intensity dependent, with significant changes in circulating eCBs observed following moderate intensities only (very high and very low intensity exercises do not significantly alter circulating eCB levels). Our results are consistent with intensity-dependent psychological state changes with exercise and therefore support the hypothesis that eCB activity is related to neurobiological effects of exercise. Thus, future studies examining the role of exercise-induced eCB signaling on neurobiology or physiology must take exercise intensity into account.

  15. A signaling network in phenylephrine-induced benign prostatic hyperplasia.

    Science.gov (United States)

    Kim, Jayoung; Yanagihara, Yutaka; Kikugawa, Tadahiko; Ji, Mihee; Tanji, Nozomu; Masayoshi, Yokoyama; Freeman, Michael R

    2009-08-01

    Benign prostatic hyperplasia (BPH) is an age-related disease of unknown etiology characterized by prostatic enlargement and coinciding with distinctive alterations in tissue histomorphology. To identify the molecular mechanisms underlying the development of BPH, we conducted a DNA microarray study using a previously described animal model in which chronic alpha(1)-adrenergic stimulation by repeated administration of phenylephrine evokes histomorphological changes in the rat prostate that resemble human BPH. Bioinformatic tools were applied to microarray data obtained from prostate tissue to construct a network model of potentially relevant signal transduction pathways. Significant involvement of inflammatory pathways was demonstrable, including evidence for activation of a TGF-beta signaling cascade. The heterodimeric protein clusterin (apolipoprotein J) was also identified as a prominent node in the network. Responsiveness of TGF-beta signaling and clusterin gene and protein expression were confirmed independently of the microarray data, verifying some components of the model. This is the first attempt to develop a comprehensive molecular network for histological BPH induced by adrenergic activation. The study also implicated clusterin as a novel biochemical target for therapy.

  16. Multiple Signaling Pathways Control Tbx6 Expression during Xenopus Myogenesis

    Institute of Scientific and Technical Information of China (English)

    Pan-Feng FANG; Rui-Ying HU; Xing-Yue HE; Xiao-Yan DING

    2004-01-01

    Tbx6 is critical for somite specification and myogenesis initiation.It has been shown that Activin/Nodal,VegT/Nodal,FGF,and BMP signaling pathways are involved early in specifying mesoderm or later in patterning mesoderm,and Xnot plays roles in setting up the boundary between notochord and paraxial mesoderm.In this study,we introduce the dominant negative form of above genes into embryos to evaluate if they are responsible for regulating Tbx6 expression.The results show that: (1)Activin/Nodal and VegT/Nodal signals are necessary for both initiation and maintenance of Tbx6 expression,and Nodal is sufficient to induce ectopic Tbx6 expression;(2) FGF signal is necessary for the initiation and maintenance of Tbx6,but it is not sufficient to induce Tbx6 expression;(3) BMP is also necessary for the expression of Tbx6,and the induction of Tbx6 expression by BMP is dose dependent;(4) Xnot has no effect on the expression of Tbx6.Our results suggest that several signaling pathways are involved in regulating Tbx6expression,and pave the route to reveal the molecular mechanism of initiating myogenesis.

  17. Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

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    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

  18. 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.

  19. Klotho Ameliorates Chemically Induced Endoplasmic Reticulum (ER Stress Signaling

    Directory of Open Access Journals (Sweden)

    Srijita Banerjee

    2013-05-01

    Full Text Available Background: Both endoplasmic reticulum (ER stress, a fundamental cell response associated with stress-initiated unfolded protein response (UPR, and loss of Klotho, an anti-aging hormone linked to NF-κB-induced inflammation, occur in chronic metabolic diseases such as obesity and type 2 diabetes. We investigated if the loss of Klotho is causally linked to increased ER stress. Methods: We treated human renal epithelial HK-2, alveolar epithelial A549, HEK293, and SH-SH-SY5Y neuroblastoma cells with ER stress-inducing agents, thapsigargin and/or tunicamycin. Effects of overexpression or siRNA-mediated knockdown of Klotho on UPR signaling was investigated by immunoblotting and Real-time PCR. Results: Elevated Klotho levels in HK-2 cells decreased expression of ER stress markers phospho-IRE1, XBP-1s, BiP, CHOP, pJNK, and phospho-p38, all of which were elevated in response to tunicamycin and/or thapsigargin. Similar results were observed using A549 cells for XBP-1s, BiP, and CHOP in response to thapsigargin. Conversely, knockdown of Klotho in HEK 293 cells using siRNA caused further thapsigargin-induced increases in pIRE-1, XBP-1s, and BiP. Klotho overexpression in A549 cells blocked thapsigargin-induced caspase and PARP cleavage and improved cell viability. Conclusion: Our data indicate that Klotho has an important role in regulating ER stress and that loss of Klotho is causally linked to ER stress-induced apoptosis.

  20. Coleusin factor, a novel anticancer diterpenoid, inhibits osteosarcoma growth by inducing bone morphogenetic protein-2-dependent differentiation.

    Science.gov (United States)

    Geng, Shuo; Sun, Bo; Lu, Ran; Wang, Jingze

    2014-06-01

    Coleusin factor is a diterpenoid compound isolated from the root of a tropical plant, Coleus forskohlii. Although Coleusin factor has been reported to suppress proliferation of and induce apoptosis in several types of cancer cells, the effects of Coleusin factor on osteosarcoma and the underlying mechanism are still not fully understood. In this study, we show that Coleusin factor treatment potently inhibits the growth of osteosarcoma cells associated with G(1) cell-cycle arrest. Interestingly, apoptosis and cell death are not induced. Instead, Coleusin factor causes osteosarcoma cells to exhibit typical properties of differentiated osteoblasts, including a morphologic alteration resembling osteoblasts, the expression of osteoblast differentiation markers, elevated alkaline phosphatase activity, and increased cellular mineralization. Coleusin factor treatment significantly increases the expression of bone morphogenetic protein-2 (BMP-2), a crucial osteogenic regulator, and runt-related transcription factor 2 (RUNX2), one of the key transcription factors of the BMP pathway. When BMP-2 signaling is blocked, Coleusin factor fails to inhibit cell proliferation and to induce osteoblast differentiation. Thus, upregulation of BMP-2 autocrine is critical for Coleusin factor to induce osteoblast differentiation and exert its anticancer effects on osteosarcoma. Importantly, administration of Coleusin factor inhibits the growth of osteosarcoma xenografted in nude mice without systemic or immunologic toxicity. Osteosarcoma is a highly aggressive cancer marked by the loss of normal differentiation. Coleusin factor represents a new type of BMP-2 inducer that restores differentiation in osteosarcoma cells. It may provide a promising therapeutic strategy against osteosarcoma with minimal side effects.

  1. β-Catenin-dependent transcription is central to Bmp-mediated formation of venous vessels.

    Science.gov (United States)

    Kashiwada, Takeru; Fukuhara, Shigetomo; Terai, Kenta; Tanaka, Toru; Wakayama, Yuki; Ando, Koji; Nakajima, Hiroyuki; Fukui, Hajime; Yuge, Shinya; Saito, Yoshinobu; Gemma, Akihiko; Mochizuki, Naoki

    2015-02-01

    β-catenin regulates the transcription of genes involved in diverse biological processes, including embryogenesis, tissue homeostasis and regeneration. Endothelial cell (EC)-specific gene-targeting analyses in mice have revealed that β-catenin is required for vascular development. However, the precise function of β-catenin-mediated gene regulation in vascular development is not well understood, since β-catenin regulates not only gene expression but also the formation of cell-cell junctions. To address this question, we have developed a novel transgenic zebrafish line that allows the visualization of β-catenin transcriptional activity specifically in ECs and discovered that β-catenin-dependent transcription is central to the bone morphogenetic protein (Bmp)-mediated formation of venous vessels. During caudal vein (CV) formation, Bmp induces the expression of aggf1, a putative causative gene for Klippel-Trenaunay syndrome, which is characterized by venous malformation and hypertrophy of bones and soft tissues. Subsequently, Aggf1 potentiates β-catenin transcriptional activity by acting as a transcriptional co-factor, suggesting that Bmp evokes β-catenin-mediated gene expression through Aggf1 expression. Bmp-mediated activation of β-catenin induces the expression of Nr2f2 (also known as Coup-TFII), a member of the nuclear receptor superfamily, to promote the differentiation of venous ECs, thereby contributing to CV formation. Furthermore, β-catenin stimulated by Bmp promotes the survival of venous ECs, but not that of arterial ECs. Collectively, these results indicate that Bmp-induced activation of β-catenin through Aggf1 regulates CV development by promoting the Nr2f2-dependent differentiation of venous ECs and their survival. This study demonstrates, for the first time, a crucial role of β-catenin-mediated gene expression in the development of venous vessels.

  2. The influence of Aloe vera and xenograft XCB toward of bone morpho protein 2 BMP2 expression and amount of osteoblast of alveolar bone induced into tooth extraction sockets Cavia cobaya

    Directory of Open Access Journals (Sweden)

    Utari Kresnoadi

    2014-12-01

    Full Text Available Tooth extraction can cause inflammation leading to alveolar ridge resorption. In addition, prominent ridge has crucial role for making denture su-ccessfully. Thus, socket preservation is needed to prevent greater alveolar ridge resorption. An innovative material, a combination of Aloe vera and xe-nograft (XCB, is then considered as a biogenic stimulator that can reduce inflammation, as a result, the growth of alveolar bone is expected to be impro-ved. This research is aimed to prove whether the mixture of Aloe vera and xenograft can stimulate BMP2 and increase osteoblasts. Forty-eight Cavia co-baya animals were divided into eight groups each of which consisted of six animals. The mandibular incisors of those Cavia cobaya animals were then extracted and filled with PEG as Group Control, XCB as Group XCB, Aloe vera as Group Aloe vera, and a combination of Aloe vera +XCB as Group Aloe vera +XCB. Next, the first four groups were sacrificed seven days after extraction, and the second four groups were sacrificed 30 days after extrac-tion. And then, immunohistochemical and histopathology examinations were conducted to examine BMP2 expression and osteoblasts. Based on the re-sult known that the mixture of Aloe vera and xenograft can increase BMP2 expression and amount of osteoblasts. It can be concluded that the mixture of Aloe vera and xenograft can increase BMP2 expression and amount of osteoblast cel . It can be used as an alternative material to increase the growth of alveolar bone after extraction.

  3. Smad4-Shh-Nfic signaling cascade-mediated epithelial-mesenchymal interaction is crucial in regulating tooth root development.

    Science.gov (United States)

    Huang, Xiaofeng; Xu, Xun; Bringas, Pablo; Hung, Yee Ping; Chai, Yang

    2010-05-01

    Transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) signaling is crucial for regulating epithelial-mesenchymal interaction during organogenesis, and the canonical Smad pathway-mediated TGF-beta/BMP signaling plays important roles during development and disease. During tooth development, dental epithelial cells, known as Hertwig's epithelial root sheath (HERS), participate in root formation following crown development. However, the functional significance of HERS in regulating root development remains unknown. In this study we investigated the signaling mechanism of Smad4, the common Smad for TGF-beta/BMP signaling, in HERS in regulating root development. Tissue-specific inactivation of Smad4 in HERS results in abnormal enamel and dentin formation in K14-Cre;Smad4(fl/fl) mice. HERS enlarges but cannot elongate to guide root development without Smad4. At the molecular level, Smad4-mediated TGF-beta/BMP signaling is required for Shh expression in HERS and Nfic (nuclear factor Ic) expression in the cranial neural crest (CNC)-derived dental mesenchyme. Nfic is crucial for root development, and loss of Nfic results in a CNC-derived dentin defect similar to the one of K14-Cre;Smad4(fl/fl) mice. Significantly, we show that ectopic Shh induces Nfic expression in dental mesenchyme and partially rescues root development in K14-Cre;Smad4(fl/fl) mice. Taken together, our study has revealed an important signaling mechanism in which TGF-beta/BMP signaling relies on a Smad-dependent mechanism in regulating Nfic expression via Shh signaling to control root development. The interaction between HERS and the CNC-derived dental mesenchyme may guide the size, shape, and number of tooth roots.

  4. Combined delivery of PDGF-BB and BMP-6 for enhanced osteoblastic differentiation.

    Science.gov (United States)

    Demirtaş, T Tolga; Göz, Eda; Karakeçili, Ayşe; Gümüşderelioğlu, Menemşe

    2016-01-01

    Natural microenvironment during bone tissue regeneration involves integration of multiple biological growth factors which regulate mitogenic activities and differentiation to induce bone repair. Among them platelet derived growth factor (PDGF-BB) and bone morphogenic protein-6 (BMP-6) are known to play a prominent role. The aim of this study was to investigate the benefits of combined delivery of PDGF-BB and BMP-6 on proliferation and osteoblastic differentiation of MC3T3-E1 preosteoblastic cells. PDGF-BB and BMP-6 were loaded in gelatin and poly (3-hydroxybutyric acid-co-3-hydroxyvaleric acid) particles, respectively. The carrier particles were then loaded into 3D chitosan matrix fabricated by freeze drying. The fast release of PDGF-BB during 7 days was accompanied by slower and prolonged release of BMP-6. The premising release of mitogenic factor PDGF-BB resulted in an increased MC3T3-E1 cell population seeded on chitosan scaffolds. Osteogenic markers of RunX2, Col 1, OPN were higher on chitosan scaffolds loaded with growth factors either individually or in combination. However, OCN expression and bone mineral formation were prominent on chitosan scaffolds incorporating PDGF-BB and BMP-6 as a combination.

  5. BMP2 rescues deficient cell migration in Tgfbr3(-/-) epicardial cells and requires Src kinase.

    Science.gov (United States)

    Allison, Patrick; Espiritu, Daniella; Camenisch, Todd D

    2016-05-03

    During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types which contribute to the coronary vessels. The type III transforming growth factor-β receptor (TGFβR3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Bone Morphogenic Protein-2 (BMP2) is a driver of epicardial cell migration. Utilizing a primary epicardial cell line derived from Tgfbr3(+/+) and Tgfbr3(-/-) mouse embryos, we show that Tgfbr3(-/-) epicardial cells are deficient in BMP2 mRNA expression. Tgfbr3(-/-) epicardial cells are deficient in 2-dimensional migration relative to Tgfbr3(+/+) cells; BMP2 induces cellular migration to Tgfbr3(+/+) levels without affecting proliferation. We further demonstrate that Src kinase activity is required for BMP2 driven Tgfbr3(-/-) migration. BMP2 also requires Src for filamentous actin polymerization in Tgfbr3(-/-) epicardial cells. Taken together, our data identifies a novel pathway in epicardial cell migration required for development of the coronary vessels.

  6. 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

  7. Secreted phosphoprotein 24 kD (Spp24) inhibits growth of human pancreatic cancer cells caused by BMP-2.

    Science.gov (United States)

    Li, Chen-Shuang; Tian, Haijun; Zou, Min; Zhao, Ke-Wei; Li, Yawei; Lao, Lifeng; Brochmann, Elsa J; Duarte, M Eugenia L; Daubs, Michael D; Zhou, Yan-Heng; Murray, Samuel S; Wang, Jeffrey C

    2015-10-16

    The emerging role of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers has drawn great attention in cancer research. In this study, we report that BMP-2 can promote the proliferation of the pancreatic tumor cell line, PANC-1. Secreted phosphoprotein 24 kD (Spp24), a BMP binding protein, did not affect the proliferation of the cells but promoted the apoptosis of the cells in vitro. In a xeneograft tumor model using PANC-1 cells, BMP-2 dramatically promoted tumor growth, while Spp24 not only abolished the effect of BMP-2, but also dramatically induced tumor shrinking when used alone. Activation of Smad1/5/8 participated in this process as demonstrated by immunohistochemical staining of phosphorylated Smad 1/5/8. We conclude that Spp24 can be developed into a therapeutic agent that could be employed in clinical situations where the inhibition of BMPs and related proteins is advantageous.

  8. 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.

  9. Enhancement of endocannabinoid signaling protects against cocaine-induced neurotoxicity.

    Science.gov (United States)

    Vilela, Luciano R; Gobira, Pedro H; Viana, Thercia G; Medeiros, Daniel C; Ferreira-Vieira, Talita H; Doria, Juliana G; Rodrigues, Flávia; Aguiar, Daniele C; Pereira, Grace S; Massessini, André R; Ribeiro, Fabíola M; de Oliveira, Antonio Carlos P; Moraes, Marcio F D; Moreira, Fabricio A

    2015-08-01

    Cocaine is an addictive substance with a potential to cause deleterious effects in the brain. The strategies for treating its neurotoxicity, however, are limited. Evidence suggests that the endocannabinoid system exerts neuroprotective functions against various stimuli. Thus, we hypothesized that inhibition of fatty acid amide hydrolase (FAAH), the main enzyme responsible for terminating the actions of the endocannabinoid anandamide, reduces seizures and cell death in the hippocampus in a model of cocaine intoxication. Male Swiss mice received injections of endocannabinoid-related compounds followed by the lowest dose of cocaine that induces seizures, electroencephalographic activity and cell death in the hippocampus. The molecular mechanisms were studied in primary cell culture of this structure. The FAAH inhibitor, URB597, reduced cocaine-induced seizures and epileptiform electroencephalographic activity. The cannabinoid CB1 receptor selective agonist, ACEA, mimicked these effects, whereas the antagonist, AM251, prevented them. URB597 also inhibited cocaine-induced activation and death of hippocampal neurons, both in animals and in primary cell culture. Finally, we investigated if the PI3K/Akt/ERK intracellular pathway, a cell surviving mechanism coupled to CB1 receptor, mediated these neuroprotective effects. Accordingly, URB597 injection increased ERK and Akt phosphorylation in the hippocampus. Moreover, the neuroprotective effect of this compound was reversed by the PI3K inhibitor, LY294002. In conclusion, the pharmacological facilitation of the anandamide/CB1/PI3K signaling protects the brain against cocaine intoxication in experimental models. This strategy may be further explored in the development of treatments for drug-induced neurotoxicity.

  10. Enhancement of endocannabinoid signaling protects against cocaine-induced neurotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Vilela, Luciano R. [Graduate Program in Neuroscience, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil); Gobira, Pedro H.; Viana, Thercia G. [Department of Pharmacology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil); Medeiros, Daniel C.; Ferreira-Vieira, Talita H. [Department of Physiology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil); Doria, Juliana G. [Graduate Program in Neuroscience, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil); Rodrigues, Flávia [Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil); Aguiar, Daniele C. [Department of Pharmacology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil); Pereira, Grace S.; Massessini, André R. [Department of Physiology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil); Ribeiro, Fabíola M. [Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil); Oliveira, Antonio Carlos P. de [Department of Pharmacology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil); Moraes, Marcio F.D., E-mail: mfdm@icb.ufmg.br [Department of Physiology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil); Moreira, Fabricio A., E-mail: fabriciomoreira@icb.ufmg.br [Department of Pharmacology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil)

    2015-08-01

    Cocaine is an addictive substance with a potential to cause deleterious effects in the brain. The strategies for treating its neurotoxicity, however, are limited. Evidence suggests that the endocannabinoid system exerts neuroprotective functions against various stimuli. Thus, we hypothesized that inhibition of fatty acid amide hydrolase (FAAH), the main enzyme responsible for terminating the actions of the endocannabinoid anandamide, reduces seizures and cell death in the hippocampus in a model of cocaine intoxication. Male Swiss mice received injections of endocannabinoid-related compounds followed by the lowest dose of cocaine that induces seizures, electroencephalographic activity and cell death in the hippocampus. The molecular mechanisms were studied in primary cell culture of this structure. The FAAH inhibitor, URB597, reduced cocaine-induced seizures and epileptiform electroencephalographic activity. The cannabinoid CB{sub 1} receptor selective agonist, ACEA, mimicked these effects, whereas the antagonist, AM251, prevented them. URB597 also inhibited cocaine-induced activation and death of hippocampal neurons, both in animals and in primary cell culture. Finally, we investigated if the PI3K/Akt/ERK intracellular pathway, a cell surviving mechanism coupled to CB{sub 1} receptor, mediated these neuroprotective effects. Accordingly, URB597 injection increased ERK and Akt phosphorylation in the hippocampus. Moreover, the neuroprotective effect of this compound was reversed by the PI3K inhibitor, LY294002. In conclusion, the pharmacological facilitation of the anandamide/CB1/PI3K signaling protects the brain against cocaine intoxication in experimental models. This strategy may be further explored in the development of treatments for drug-induced neurotoxicity. - Highlights: • Cocaine toxicity is characterized by seizures and hippocampal cell death. • The endocannabinoid anandamide acts as a brain protective mechanism. • Inhibition of anandamide hydrolysis

  11. Identification of a BMP inhibitor-responsive promoter module required for expression of the early neural gene zic1.

    Science.gov (United States)

    Tropepe, Vincent; Li, Shuhong; Dickinson, Amanda; Gamse, Joshua T; Sive, Hazel L

    2006-01-15

    Expression of the transcription factor zic1 at the onset of gastrulation is one of the earliest molecular indicators of neural fate determination in Xenopus. Inhibition of bone morphogenetic protein (BMP) signaling is critical for activation of zic1 expression and fundamental for establishing neural identity in both vertebrates and invertebrates. The mechanism by which interruption of BMP signaling activates neural-specific gene expression is not understood. Here, we report identification of a 215 bp genomic module that is both necessary and sufficient to activate Xenopus zic1 transcription upon interruption of BMP signaling. Transgenic analyses demonstrate that this BMP inhibitory response module (BIRM) is required for expression in the whole embryo. Multiple consensus binding sites for specific transcription factor families within the BIRM are required for its activity and some of these regions are phylogenetically conserved between orthologous vertebrate zic1 genes. These data suggest that interruption of BMP signaling facilitates neural determination via a complex mechanism, involving multiple regulatory factors that cooperate to control zic1 expression.

  12. Nitric oxide-induced signalling in rat lacrimal acinar cells

    DEFF Research Database (Denmark)

    Looms, Dagnia Karen; Tritsaris, K.; Dissing, S.

    2002-01-01

    The aim of the present study was to investigate the physiological role of nitric oxide (NO) in mediating secretory processes in rat lacrimal acinar cells. In addition, we wanted to determine whether the acinar cells possess endogenous nitric oxide synthase (NOS) activity by measuring NO productio...... not by itself causing fast transient increases in [Ca2+]i. In addition, we suggest that endogenously produced NO activated by ß-adrenergic receptor stimulation, plays an important role in signalling to the surrounding tissue.......The aim of the present study was to investigate the physiological role of nitric oxide (NO) in mediating secretory processes in rat lacrimal acinar cells. In addition, we wanted to determine whether the acinar cells possess endogenous nitric oxide synthase (NOS) activity by measuring NO production......-adrenergic stimulation and not by a rise in [Ca2+]i alone.   We show that in rat lacrimal acinar cells, NO and cGMP induce Ca2+ release from intracellular stores via G kinase activation. However, the changes in [Ca2+]i are relatively small, suggesting that this pathway plays a modulatory role in Ca2+ signalling, thus...

  13. Signaling pathways and tissue interactions in neural plate border formation.

    Science.gov (United States)

    Schille, Carolin; Schambony, Alexandra

    2017-01-01

    The neural crest is a transient cell population that gives rise to various cell types of multiple tissues and organs in the vertebrate embryo. Neural crest cells arise from the neural plate border, a region localized at the lateral borders of the prospective neural plate. Temporally and spatially coordinated interaction with the adjacent tissues, the non-neural ectoderm, the neural plate and the prospective dorsolateral mesoderm, is required for neural plate border specification. Signaling molecules, namely BMP, Wnt and FGF ligands and corresponding antagonists are derived from these tissues and interact to induce the expression of neural plate border specific genes. The present mini-review focuses on the current understanding of how the NPB territory is formed and accentuates the need for coordinated interaction of BMP and Wnt signaling pathways and precise tissue communication that are required for the definition of the prospective NC in the competent ectoderm.

  14. 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.

  15. Disequilibrium of BMP2 levels in the breast stem cell niche launches epithelial transformation by overamplifying BMPR1B cell response.

    Science.gov (United States)

    Chapellier, Marion; Bachelard-Cascales, Elodie; Schmidt, Xenia; Clément, Flora; Treilleux, Isabelle; Delay, Emmanuel; Jammot, Alexandre; Ménétrier-Caux, Christine; Pochon, Gaëtan; Besançon, Roger; Voeltzel, Thibault; Caron de Fromentel, Claude; Caux, Christophe; Blay, Jean-Yves; Iggo, Richard; Maguer-Satta, Véronique

    2015-02-10

    Understanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer.

  16. Disequilibrium of BMP2 Levels in the Breast Stem Cell Niche Launches Epithelial Transformation by Overamplifying BMPR1B Cell Response

    Directory of Open Access Journals (Sweden)

    Marion Chapellier

    2015-02-01

    Full Text Available Understanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer.

  17. Disequilibrium of BMP2 Levels in the Breast Stem Cell Niche Launches Epithelial Transformation by Overamplifying BMPR1B Cell Response

    Science.gov (United States)

    Chapellier, Marion; Bachelard-Cascales, Elodie; Schmidt, Xenia; Clément, Flora; Treilleux, Isabelle; Delay, Emmanuel; Jammot, Alexandre; Ménétrier-Caux, Christine; Pochon, Gaëtan; Besançon, Roger; Voeltzel, Thibault; Caron de Fromentel, Claude; Caux, Christophe; Blay, Jean-Yves; Iggo, Richard; Maguer-Satta, Véronique

    2015-01-01

    Summary Understanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer. PMID:25601208

  18. Serotonergic signaling inhibits hyperalgesia induced by spinal cord damage.

    Science.gov (United States)

    Horiuchi, Hideki; Ogata, Tadanori; Morino, Tadao; Takeba, Jun; Yamamoto, Haruyasu

    2003-02-14

    Although dysesthesia is one of the most serious problems in patients with spinal cord injury, most of them being unresponsive to conventional treatments. In this study, we established a rat thoracic spinal cord mild-compression model that revealed thermal hyperalgesia in the hind limb. The thoracic spinal cord was compressed gently, using a 20 g weight for 20 min. The withdrawal latency of the thermal stimulation of the bilateral hind-limb was monitored using Hargreaves' Plantar test apparatus. In this model, thermal-hyperalgesia was observed for 1 week after the injury. The spinal cord injury-induced thermal-hyperalgesia was mimicked by the intrathecal application of metergoline, a non-selective 5-HT antagonist, 1-(2-methoxyphenyl)-4-[4-(2-phthalimido) butyl]-piperazine hydrobromide (NAN190), a selective 5-HT1 antagonist, and 3-tropanyl-3,5-dichlorobenzoate (MDL72222), a selective 5-HT3 antagonist. Intraperitoneal application of fluvoxamine maleate, a selective serotonin reuptake inhibitor, reduced the intensity of hyperalgesia induced by spinal cord injury. The inhibitory effect of fluvoxamine maleate on thermal hyperalgesia was prevented by the application of the aforementioned nonselective or selective 5-HT receptor antagonists. Intrathecal application of fluvoxamine maleate and selective 5-HT receptor agonists, i.e., 8-hydroxy-2-(di-n-proplyamino)-tetralin hydrobromide (8-OH-DPAT: 5HT-1 selective) and 2-methyl-5-hydroxytryptamine maleate (2-m-5-HT: 5HT-3 selective), inhibited the spinal cord injury-induced hyperalgesia. These results suggest that the change in the descending serotonergic signal plays an important role in hyperalgesia after the spinal cord injury, and that the application of selective serotonin reuptake inhibitors will be one of the candidates for new therapeutic methods against post-spinal cord injury dysesthesia.

  19. Distinct and overlapping gene regulatory networks in BMP- and HDAC-controlled cell fate determination in the embryonic forebrain

    Directory of Open Access Journals (Sweden)

    Scholl Catharina

    2012-07-01

    Full Text Available Abstract Background Both bone morphogenetic proteins (BMPs and histone deacetylases (HDACs have previously been established to play a role in the development of the three major cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. We have previously established a connection between these two protein families, showing that HDACs suppress BMP-promoted astrogliogenesis in the embryonic striatum. Since HDACs act in the nucleus to effect changes in transcription, an unbiased analysis of their transcriptional targets could shed light on their downstream effects on BMP-signaling. Results Using neurospheres from the embryonic striatum as an in vitro system to analyze this phenomenon, we have performed microarray expression profiling on BMP2- and TSA-treated cultures, followed by validation of the findings with quantitative RT-PCR and protein analysis. In BMP-treated cultures we first observed an upregulation of genes involved in cell-cell communication and developmental processes such as members of BMP and canonical Wnt signaling pathways. In contrast, in TSA-treated cultures we first observed an upregulation of genes involved in chromatin modification and transcription. Interestingly, we could not record direct changes in the protein levels of canonical members of BMP2 signaling, but we did observe an upregulation of both the transcription factor STAT3 and its active isoform phospho-STAT3 at the protein level. Conclusions STAT3 and SMAD1/5/8 interact synergistically to promote astrogliogenesis, and thus we show for the first time that HDACs act to suppress BMP-promoted astrogliogenesis by suppression of the crucial partner STAT3.

  20. Msx1 and Tbx2 antagonistically regulate Bmp4 expression during the bud-to-cap stage transition in tooth development.

    Science.gov (United States)

    Saadi, Irfan; Das, Pragnya; Zhao, Minglian; Raj, Lakshmi; Ruspita, Intan; Xia, Yan; Papaioannou, Virginia E; Bei, Marianna

    2013-07-01

    Bmp4 expression is tightly regulated during embryonic tooth development, with early expression in the dental epithelial placode leading to later expression in the dental mesenchyme. Msx1 is among several transcription factors that are induced by epithelial Bmp4 and that, in turn, are necessary for the induction and maintenance of dental mesenchymal Bmp4 expression. Thus, Msx1(-/-) teeth arrest at early bud stage and show loss of Bmp4 expression in the mesenchyme. Ectopic expression of Bmp4 rescues this bud stage arrest. We have identified Tbx2 expression in the dental mesenchyme at bud stage and show that this can be induced by epithelial Bmp4. We also show that endogenous Tbx2 and Msx1 can physically interact in mouse C3H10T1/2 cells. In order to ascertain a functional relationship between Msx1 and Tbx2 in tooth development, we crossed Tbx2 and Msx1 mutant mice. Our data show that the bud stage tooth arrest in Msx1(-/-) mice is partially rescued in Msx1(-/-);Tbx2(+/-) compound mutants. This rescue is accompanied by formation of the enamel knot (EK) and by restoration of mesenchymal Bmp4 expression. Finally, knockdown of Tbx2 in C3H10T1/2 cells results in an increase in Bmp4 expression. Together, these data identify a novel role for Tbx2 in tooth development and suggest that, following their induction by epithelial Bmp4, Msx1 and Tbx2 in turn antagonistically regulate odontogenic activity that leads to EK formation and to mesenchymal Bmp4 expression at the key bud-to-cap stage transition.

  1. Integration of signals along orthogonal axes of the vertebrate neural tube controls progenitor competence and increases cell diversity.

    Science.gov (United States)

    Sasai, Noriaki; Kutejova, Eva; Briscoe, James

    2014-07-01

    A relatively small number of signals are responsible for the variety and pattern of cell types generated in developing embryos. In part this is achieved by exploiting differences in the concentration or duration of signaling to increase cellular diversity. In addition, however, changes in cellular competence-temporal shifts in the response of cells to a signal-contribute to the array of cell types generated. Here we investigate how these two mechanisms are combined in the vertebrate neural tube to increase the range of cell types and deliver spatial control over their location. We provide evidence that FGF signaling emanating from the posterior of the embryo controls a change in competence of neural progenitors to Shh and BMP, the two morphogens that are responsible for patterning the ventral and dorsal regions of the neural tube, respectively. Newly generated neural progenitors are exposed to FGF signaling, and this maintains the expression of the Nk1-class transcription factor Nkx1.2. Ventrally, this acts in combination with the Shh-induced transcription factor FoxA2 to specify floor plate cells and dorsally in combination with BMP signaling to induce neural crest cells. As development progresses, the intersection of FGF with BMP and Shh signals is interrupted by axis elongation, resulting in the loss of Nkx1.2 expression and allowing the induction of ventral and dorsal interneuron progenitors by Shh and BMP signaling to supervene. Hence a similar mechanism increases cell type diversity at both dorsal and ventral poles of the neural tube. Together these data reveal that tissue morphogenesis produces changes in the coincidence of signals acting along orthogonal axes of the neural tube and this is used to define spatial and temporal transitions in the competence of cells to interpret morphogen signaling.

  2. Integration of signals along orthogonal axes of the vertebrate neural tube controls progenitor competence and increases cell diversity.

    Directory of Open Access Journals (Sweden)

    Noriaki Sasai

    2014-07-01

    Full Text Available A relatively small number of signals are responsible for the variety and pattern of cell types generated in developing embryos. In part this is achieved by exploiting differences in the concentration or duration of signaling to increase cellular diversity. In addition, however, changes in cellular competence-temporal shifts in the response of cells to a signal-contribute to the array of cell types generated. Here we investigate how these two mechanisms are combined in the vertebrate neural tube to increase the range of cell types and deliver spatial control over their location. We provide evidence that FGF signaling emanating from the posterior of the embryo controls a change in competence of neural progenitors to Shh and BMP, the two morphogens that are responsible for patterning the ventral and dorsal regions of the neural tube, respectively. Newly generated neural progenitors are exposed to FGF signaling, and this maintains the expression of the Nk1-class transcription factor Nkx1.2. Ventrally, this acts in combination with the Shh-induced transcription factor FoxA2 to specify floor plate cells and dorsally in combination with BMP signaling to induce neural crest cells. As development progresses, the intersection of FGF with BMP and Shh signals is interrupted by axis elongation, resulting in the loss of Nkx1.2 expression and allowing the induction of ventral and dorsal interneuron progenitors by Shh and BMP signaling to supervene. Hence a similar mechanism increases cell type diversity at both dorsal and ventral poles of the neural tube. Together these data reveal that tissue morphogenesis produces changes in the coincidence of signals acting along orthogonal axes of the neural tube and this is used to define spatial and temporal transitions in the competence of cells to interpret morphogen signaling.

  3. 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.

  4. Discovery of a Small-Molecule BMP Sensitizer for Human Embryonic Stem Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Lingling Feng

    2016-05-01

    Full Text Available Sorely missing from the “toolkit” for directed differentiation of stem/progenitor cells are agonists of the BMP-signaling pathway. Using a high-throughput chemical screen, we discovered that PD407824, a checkpoint kinase 1 (CHK1 inhibitor, increases the sensitivity of cells to sub-threshold amounts of BMP4. We show utility of the compound in the directed differentiation of human embryonic stem cells toward mesoderm or cytotrophoblast stem cells. Blocking CHK1 activity using pharmacological compounds or CHK1 knockout using single guide RNA (sgRNA confirmed that CHK1 inhibition increases the sensitivity to BMP4 treatment. Additional mechanistic studies indicate that CHK1 inhibition depletes p21 levels, thereby activating CDK8/9, which then phosphorylates the SMAD2/3 linker region, leading to decreased levels of SMAD2/3 protein and enhanced levels of nuclear SMAD1. This study provides insight into mechanisms controlling the BMP/transforming growth factor beta (TGF-β signaling pathways and a useful pharmacological reagent for directed differentiation of stem cells.

  5. 前列腺癌骨转移灶中BMP-2、BMP-4、BMP-7的表达及临床意义%Expression of BMP-2, BMP-4 and BMP-7 in human metastatic prostate cancer and their clinical significance

    Institute of Scientific and Technical Information of China (English)

    宫丽华; 孙晓淇; 刘宝岳; 黄啸原

    2012-01-01

    目的 检测BMP-2、BMP-4、BMP-7在前列腺癌骨转移灶中的表达,探讨其在前列腺癌成骨性转移中的作用.方法 采用免疫组化EnVision法检测28例前列腺癌骨转移病例及17例良性前列腺增生(benign prostate hyperplasia,BPH)病例中BMP-2、BMP-4、BMP-7的表达并对其进行对比分析.结果 BMP-2在所有前列腺癌骨转移灶及BPH病例中均表达,二者中其阳性率及表达强度无明显差异(P>0.05).BMP-4在前列腺癌骨转移灶及BPH中的阳性率无明显差异(P>0.05),但在前者中BMP-4的表达强度明显高于后者(P<0.05).BMP-7在前列腺癌骨转移灶中的阳性率及表达强度均明显高于BPH (P<0.05).在BPH的阳性表达病例中,BMP-2、BMP-4、BMP-7细胞质与细胞核同时阳性的表达率分别为13.3%、7.1%和11.1%,在前列腺癌骨转移灶的阳性表达病例中,BMP-2、BMP-4、BMP-7细胞质与细胞核的同时阳性的表达率均为100%,且细胞核的表达强度明显高于细胞质.结论 BMP-4、BMP-7在前列腺癌骨转移灶中高表达,提示其在前列腺癌的成骨性转移中可能起重要作用.%Purpose To analyze the expression of BMP-2, BMP-4, BMP-7 in prostate cancer with bone metastasis. Methods Iininu-nohistochemical technique was used to detect the expression of BMP-2, BMP-4 and BMP-7 in the bone osteobastic metastastic lesions of prostate cancer and the benign prostate hyperplasia ( BPH ). Results The expression of BMP-2 was detected in all samples and there was no difference between prostate cancer and BPH. The intensity of positivity of BMP-4 was higher in prostate cancer than that in BPH ( P < 0. 05 ), but the difference of positive rate between these two groups showed no significance. The intensity of positivity and the positive rate of BMP-7 were higher in prostate cancer than that in BPH ( P < 0. 05 ). The positive rate of staining involving both cyto-plasm and nuclear in BMP-2, BMP-4 and BMP-7-positive samples in BPH was respectively 13. 3

  6. ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Xinhua [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China); Wang, Xiaoyuan [Department of Nephrology, Xi An Honghui Hospital, Xi an (China); Hu, Xiongke; Chen, Yong; Zeng, Kefeng [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China); Zhang, Hongqi, E-mail: zhq9699@126.com [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China)

    2015-07-01

    Although 17β-estradial (E2) is known to stimulate bone formation, the underlying mechanisms are not fully understood. Recent studies have implicated the Wnt/β-catenin pathway as a major signaling cascade in bone biology. The interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. In this study, E2 significantly increased the expression of β-catenin by inducing phosphorylations of GSK3β at serine 9. ERβ siRNAs were transfected into MC3T3-E1 cells and revealed that ERβ involved E2-induced osteoblasts proliferation and differentiation via Wnt/β-catenin signaling. The osteoblast differentiation genes (BGP, ALP and OPN) and proliferation related gene (cyclin D1) expression were significantly induced by E2-mediated ERβ. Furthermore immunofluorescence and immunoprecipitation analysis demonstrated that E2 induced the accumulation of β-catenin protein in the nucleus which leads to interaction with T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors. Taken together, these findings suggest that E2 promotes osteoblastic proliferation and differentiation by inducing proliferation-related and differentiation-related gene expression via ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. Our findings provide novel insights into the mechanisms of action of E2 in osteoblastogenesis. - Highlights: • 17β-estradial (E2) promotes GSK3-β phosphorylation. • E2 activates the Wnt/β-catenin signaling pathway. • The Wnt/β-catenin signaling pathway interacts with estrogen signaling pathways. • E2-mediated ER induced osteoblast differentiation and proliferation related genes expression.

  7. Skeletal trauma generates systemic BMP2 activation that is temporally related to the mobilization of CD73+ cells.

    Science.gov (United States)

    Marsell, Richard; Steen, Brandon; Bais, Manish V; Mortlock, Douglas P; Einhorn, Thomas A; Gerstenfeld, Louis C

    2014-01-01

    The relationship between BMP2 expression and the recruitment of skeletogenic stem cells was assessed following bone marrow reaming. BMP2 expression was examined using transgenic mice in which β-galactosidase had been inserted into the coding region of BMP