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BMP Signaling (bmp + signaling)
Selected AbstractsSustained BMP Signaling in Osteoblasts Stimulates Bone Formation by Promoting Angiogenesis and Osteoblast Differentiation,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2009Fengjie Zhang Abstract Angiogenesis and bone formation are tightly coupled during the formation of the skeleton. Bone morphogenetic protein (BMP) signaling is required for both bone development and angiogenesis. We recently identified endosome-associated FYVE-domain protein (endofin) as a Smad anchor for BMP receptor activation. Endofin contains a protein-phosphatase pp1c binding domain, which negatively modulates BMP signals through dephosphorylation of the BMP type I receptor. A single point mutation of endofin (F872A) disrupts interaction between the catalytic subunit pp1c and sensitizes BMP signaling in vitro. To study the functional impact of this mutation in vivo, we targeted expression of an endofin (F872A) transgene to osteoblasts. Mice expressing this mutant transgene had increased levels of phosphorylated Smad1 in osteoblasts and showed increased bone formation. Trabecular bone volume was significantly increased in the transgenic mice compared with the wildtype littermates with corresponding increases in trabecular bone thickness and number. Interestingly, the transgenic mice also had a pronounced increase in the density of the bone vasculature measured using contrast-enhanced ,CT imaging of Microfil-perfused bones. The vessel surface and volume were both increased in association with elevated levels of vascular endothelial growth factor (VEGF) in osteoblasts. Endothelial sprouting from the endofin (F872A) mutant embryonic metatarsals cultured ex vivo was increased compared with controls and was abolished by an addition of a VEGF neutralizing antibody. In conclusion, osteoblast targeted expression of a mutant endofin protein lacking the pp1c binding activity results in sustained signaling of the BMP type I receptor, which increases bone formation and skeletal angiogenesis. [source] Dysregulated BMP Signaling and Enhanced Osteogenic Differentiation of Connective Tissue Progenitor Cells From Patients With Fibrodysplasia Ossificans Progressiva (FOP),JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2008Paul C Billings Abstract The study of FOP, a disabling genetic disorder of progressive heterotopic ossification, is hampered by the lack of readily available connective tissue progenitor cells. We isolated such cells from discarded primary teeth of patients with FOP and controls and discovered dysregulation of BMP signaling and rapid osteoblast differentiation in FOP cells compared with control cells. Introduction: Fibrodysplasia ossificans progressiva (FOP), the most disabling condition of progressive heterotopic ossification in humans, is caused by a recurrent heterozygous missense mutation in activin receptor IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor, in all classically affected individuals. A comprehensive understanding of FOP has been limited, in part, by a lack of readily available connective tissue progenitor cells in which to study the molecular pathology of this disorder. Materials and Methods: We derived connective tissue progenitor cells from discarded primary teeth (SHED cells) of patients with FOP and controls and examined BMP signaling and osteogenic differentiation in these cells. Results: SHED cells transmitted BMP signals through both the SMAD and p38 mitogen-activated protein kinase (MAPK) pathways and responded to BMP4 treatment by inducing BMP responsive genes. FOP cells showed ligand-independent BMP signaling and ligand-dependent hyper-responsiveness to BMP stimulation. Furthermore, FOP cells showed more rapid differentiation to an osteogenic phenotype than control cells. Conclusions: This is the first study of BMP signaling and osteogenic differentiation in connective tissue progenitor cells from patients with FOP. Our data strongly support both basal and ligand-stimulated dysregulation of BMP signaling consistent with in silico studies of the mutant ACVR1 receptor in this condition. This study substantially extends our understanding of dysregulated BMP signaling in a progenitor cell population relevant to the pathogenesis of this catastrophic disorder of progressive ectopic ossification. [source] Smad3-Deficient Chondrocytes Have Enhanced BMP Signaling and Accelerated Differentiation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2006Tian-Fang Li Abstract Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-, signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3,/, chondrocytes. BMP signaling is increased in TGF-, deficiency and is required for accelerated chondrocyte maturation. Introduction: Disruption of TGF-, signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures. Materials and Methods: Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3,/, mice. Expressions of maturational markers, as well as genes involved in TGF-, and BMP signaling were examined. Chondrocytes were treated with TGF-, and BMP-2, and effects on maturation-related genes and BMP/TGF-, responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures. Results: Expression of colX and other maturational markers was markedly increased in Smad3,/, chondrocytes. Smad3,/, chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF - ,1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-, toward BMP signaling. In Smad3,/, chondrocytes, alternative TGF-, signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-, pathways reduced colX expression and alkaline phosphatase activity in TGF-,-treated Smad3,/, cultures, but only partially. In contrast, Smad3,/, chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor. Conclusions: These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-, signaling with important implications for osteoarthritis and cartilage diseases. [source] Dan is required for normal morphogenesis and patterning in the developing chick inner earDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2007Takahiro Yamanishi During vertebrate inner ear development, compartmentalization of the auditory and vestibular apparatuses along two axes depends on the patterning of transcription factors expressed in a region-specific manner. Although most of the patterning is regulated by extrinsic signals, it is not known how Nkx5.1 and Msx1 are patterned. We focus on Dan, the founding member of the Cerberus/Dan gene family that encodes BMP antagonists, and describe its function in morphogenesis and patterning. First, we confirmed that Dan is expressed in the dorso-medial region of the otic vesicle that corresponds to the presumptive endolymphatic duct and sac (ed/es). Second, we used siRNA knockdown to demonstrate that depletion of Dan induced both a severe reduction in the size of the ed/es and moderate deformities of the semicircular canals and cochlear duct. Depletion of Dan also caused suppression of Nkx5.1 in the dorso-lateral region, suppression of Msx1 in the dorso-medial region, and ectopic induction of Nkx5.1 and Msx1 in the ventro-medial region. Most of these phenotypes also appeared following misexpression of the constitutively active form of BMP receptor type Ib. Thus, Dan is required for the normal morphogenesis of the inner ear and, by inhibiting BMP signaling, for the patterning of the transcription factors Nkx5.1 and Msx1. [source] Patterning the embryonic kidney: BMP signaling mediates the differentiation of the pronephric tubules and duct in Xenopus laevisDEVELOPMENTAL DYNAMICS, Issue 1 2008Christina M. Bracken Abstract The Bone morphogenetic proteins (BMPs) mediate a wide range of diverse cellular behaviors throughout development. Previous studies implicated an important role for BMP signaling during the differentiation of the definitive mammalian kidney, the metanephros. In order to examine whether BMP signaling also plays an important role during the patterning of earlier renal systems, we examined the development of the earliest nephric system, the pronephros. Using the amphibian model system Xenopus laevis, in combination with reagents designed to inhibit BMP signaling during specific stages of nephric development, we revealed an evolutionarily conserved role for this signaling pathway during renal morphogenesis. Our results demonstrate that conditional BMP inhibition after specification of the pronephric anlagen is completed, but prior to the onset of morphogenesis and differentiation of renal tissues, results in the severe malformation of both the pronephric duct and tubules. Importantly, the effects of BMP signaling on the developing nephron during this developmental window are specific, only affecting the developing duct and tubules, but not the glomus. These data, combined with previous studies examining metanephric development in mice, provide further support that BMP functions to mediate morphogenesis of the specified renal field during vertebrate embryogenesis. Specifically, BMP signaling is required for the differentiation of two types of nephric structures, the pronephric tubules and duct. Developmental Dynamics 237:132,144, 2008. © 2007 Wiley-Liss, Inc. [source] Temporal and spatial regulation of bone morphogenetic protein signaling in late lung developmentDEVELOPMENTAL DYNAMICS, Issue 10 2007Miguel A. Alejandre-Alcázar Abstract Bone morphogenetic proteins (BMPs) play important roles in early lung development. No study to date has addressed a role for BMP signaling in late lung development. We describe changes in the expression and localization of BMP receptors (Bmpr1a, Bmpr1b, and Bmpr2) and Smad (Smad1, Smad4, Smad5, and Smad8) intracellular signaling proteins during the saccular and alveolarization stages of late lung development. BMP signaling, assessed by Smad1/5 phosphorylation, nuclear translocation, and induction of id1, id2, and id3 gene expression, was evident throughout late lung development. Our data indicate that BMP signaling is active during late lung development, and points to roles for the BMP system in septal and vascular development, and in the homeostasis of the epithelial layer of large conducting airways in the mature lung. Developmental Dynamics 236:2825,2835, 2007. © 2007 Wiley-Liss, Inc. [source] Restricted expression of Fgf16 within the developing chick inner earDEVELOPMENTAL DYNAMICS, Issue 8 2006Susan C. Chapman Abstract Fibroblast growth factor (FGF) signaling is required for otic placode induction and patterning of the developing inner ear. We have cloned the chick ortholog of Fgf16 and analyzed its expression pattern in the early chick embryo. Expression is restricted to the otic placode and developing inner ear through all the stages examined. By the closed otocyst stage, expression has resolved to anterior and posterior domains that partially overlap with those of bone morphogenetic protein 4 (Bmp4), a marker of the developing sensory patches, the cristae of the anterior and posterior semicircular canals. Platelet-derived growth factor alpha (PDGFA), another growth factor with restricted otic expression, also overlaps with Fgf16 expression. The restricted expression pattern of Fgf16 suggests a role for FGF signaling in the patterning of the sensory cristae, together with BMP signaling. Developmental Dynamics 235:2276,2281, 2006. © 2006 Wiley-Liss, Inc. [source] Xnr2 and Xnr5 unprocessed proteins inhibit Wnt signaling upstream of dishevelledDEVELOPMENTAL DYNAMICS, Issue 4 2005Yasuko Onuma Abstract Nodal and Nodal-related proteins activate the Activin-like signal pathway and play a key role in the formation of mesoderm and endoderm in vertebrate development. Recent studies have shown additional activities of Nodal-related proteins apart from the canonical Activin-like signal pathway. Here we report a novel function of Nodal-related proteins using cleavage mutants of Xenopus nodal-related genes (cmXnr2 and cmXnr5), which are known to be dominant-negative inhibitors of nodal family signaling. cmXnr2 and cmXnr5 inhibited both BMP signaling and Wnt signaling without activating the Activin-like signal in animal cap assays. Pro region construct of Xnr2 and Xnr5 did not inhibit Xwnt8, and pro/mature region chimera mutant cmActivin - Xnr2 and cmActivin- Xnr5 also did not inhibit Xwnt8 activity. These results indicate that the pro domains of Xnr2 and Xnr5 are necessary, but not sufficient, for Wnt inhibition, by Xnr family proteins. In addition, Western blot analysis and immunohistochemistry analysis revealed that the unprocessed Xnr5 protein is stably produced and secreted as effectively as mature Xnr5 protein, and that the unprocessed Xnr5 protein diffused in the extracellular space. These results suggest that unprocessed Xnr2 and Xnr5 proteins may be involved in inhibiting both BMP and Wnt signaling and are able to be secreted to act on somewhat distant target cells, if these are highly produced. Developmental Dynamics 234:900,910, 2005. © 2005 Wiley-Liss, Inc. [source] Activity-dependent regulation of synaptic size in Drosophila neuromuscular junctionsDEVELOPMENTAL NEUROBIOLOGY, Issue 9 2006Hiroaki Nakayama Abstract One of the fundamental questions in neural development is how neurons form synapses of the appropriate size for the efficient transfer of information across neural circuits. Here we investigated the mechanisms that bring about the size correlation between synapses and postsynaptic cells during development of Drosophila neuromuscular junctions (NMJs). To do this, we made use of a unique system in which two neighboring muscles (M6 and M7) are innervated by the same neurons. In mature NMJs, synaptic size on M6 is normally larger than that on M7, in accordance with the difference in muscle volume; this ensures the same extent of contraction of both muscles, and we refer to this correspondence as "matching". We found that matching was apparent in larvae 8 h after hatching, but not in newly hatched larvae despite the difference in muscle volume. When sensory inputs were suppressed by the expression of tetanus toxin in sensory neurons, matching did not occur, although synapses were able to grow. Matching was also suppressed by the inhibition of motoneuronal activity. These results suggest that matching is induced by regulating the rate of synaptic growth on M6 and M7 in an experience- and activity-dependent manner. It seems most likely that retrograde signals from the postsynaptic to the presynaptic cell convey the information about muscle cell size. We thus examined whether a candidate of retrograde signaling in NMJs, BMP signaling, is involved inmatching. However, there was no effect on matching inBMP type II receptor gene mutants, suggesting thatother experience-driven mechanisms besides BMP signaling are involved in the proper development of synapses. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2,GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 4 2006Tomokazu Fukuda Abstract BMP signaling plays pleiotropic roles in various tissues. Transgenic mouse lines that overexpress BMP signaling in a tissue-specific manner would be beneficial; however, production of each tissue-specific transgenic mouse line is labor-intensive. Here, using a Cre-loxP system, we generated a conditionally overexpressing mouse line for BMP signaling through the type I receptor ALK2 (alternatively known as AVCRI, ActRI, or ActRIA). By mating this line with Cre-expression mouse lines, Cre-mediated recombination removes an intervening floxed lacZ expression cassette and thereby permits the expression of a constitutively active form of Alk2 (caAlk2) driven by a ubiquitous promoter, CAG. Tissue specificity of Cre recombination was monitored by a bicistronically expressed EGFP following Alk2 cDNA. Increased BMP signaling was confirmed by ectopic phosphorylation of SMAD1/5/8 in the areas where Cre recombination had occurred. The conditional overexpression system described here provides versatility in investigating gene functions in a tissue-specific manner without having to generate independent tissue-specific transgenic lines. genesis 44:159,167, 2006. Published 2006 Wiley-Liss, Inc. [source] Dysregulated BMP Signaling and Enhanced Osteogenic Differentiation of Connective Tissue Progenitor Cells From Patients With Fibrodysplasia Ossificans Progressiva (FOP),JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2008Paul C Billings Abstract The study of FOP, a disabling genetic disorder of progressive heterotopic ossification, is hampered by the lack of readily available connective tissue progenitor cells. We isolated such cells from discarded primary teeth of patients with FOP and controls and discovered dysregulation of BMP signaling and rapid osteoblast differentiation in FOP cells compared with control cells. Introduction: Fibrodysplasia ossificans progressiva (FOP), the most disabling condition of progressive heterotopic ossification in humans, is caused by a recurrent heterozygous missense mutation in activin receptor IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor, in all classically affected individuals. A comprehensive understanding of FOP has been limited, in part, by a lack of readily available connective tissue progenitor cells in which to study the molecular pathology of this disorder. Materials and Methods: We derived connective tissue progenitor cells from discarded primary teeth (SHED cells) of patients with FOP and controls and examined BMP signaling and osteogenic differentiation in these cells. Results: SHED cells transmitted BMP signals through both the SMAD and p38 mitogen-activated protein kinase (MAPK) pathways and responded to BMP4 treatment by inducing BMP responsive genes. FOP cells showed ligand-independent BMP signaling and ligand-dependent hyper-responsiveness to BMP stimulation. Furthermore, FOP cells showed more rapid differentiation to an osteogenic phenotype than control cells. Conclusions: This is the first study of BMP signaling and osteogenic differentiation in connective tissue progenitor cells from patients with FOP. Our data strongly support both basal and ligand-stimulated dysregulation of BMP signaling consistent with in silico studies of the mutant ACVR1 receptor in this condition. This study substantially extends our understanding of dysregulated BMP signaling in a progenitor cell population relevant to the pathogenesis of this catastrophic disorder of progressive ectopic ossification. [source] Dysregulation of the BMP-p38 MAPK Signaling Pathway in Cells From Patients With Fibrodysplasia Ossificans Progressiva (FOP),,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2006Jennifer L Fiori Abstract FOP is a disabling disorder in which skeletal muscle is progressively replaced with bone. Lymphocytes, our model system for examining BMP signaling, cannot signal through the canonical Smad pathway unless exogenous Smad1 is supplied, providing a unique cell type in which the BMP,p38 MAPK pathway can be examined. FOP lymphocytes exhibit defects in the BMP,p38 MAPK pathway, suggesting that altered BMP signaling underlies ectopic bone formation in this disease. Introduction: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by progressive heterotopic ossification of connective tissues. Whereas the primary genetic defect in this condition is unknown, BMP4 mRNA and protein and BMP receptor type IA (BMPRIA) protein are overexpressed in cultured lymphocytes from FOP patients, supporting that altered BMP signaling is involved in this disease. In this study, we examined downstream signaling targets to study the BMP,Smad and BMP,p38 mitogen-activated protein kinase (MAPK) pathways in FOP. Materials and Methods: Protein phosphorylation was assayed by immunoblots, and p38 MAPK activity was measured by kinase assays. To examine BMP target genes, the mRNA expression of ID1, ID3, and MSX2 was determined by quantitative real-time PCR. Statistical analysis was performed using Student's t -test or ANOVA. Results: FOP lymphocytes exhibited increased levels of p38 phosphorylation and p38 MAPK activity in response to BMP4 stimulation. Furthermore, in response to BMP4, FOP cells overexpressed the downstream signaling targets ID1 by 5-fold and ID3 by 3-fold compared with controls. ID1 and ID3 mRNA induction was specifically blocked with a p38 MAPK inhibitor, but not extracellular signal-related kinase (ERK) or c-Jun N-terminal kinase (JNK) inhibitors. MSX2, a known Smad pathway target gene, is not upregulated in control or FOP cells in response to BMP, suggesting that lymphocytes do not use this limb of the BMP pathway. However, introduction of Smad1 into lymphocytes made the cells competent to regulate MSX2 mRNA after BMP4 treatment. Conclusions: Lymphocytes are a cell system that signals primarily through the BMP,p38 MAPK pathway rather than the BMP,Smad pathway in response to BMP4. The p38 MAPK pathway is dysregulated in FOP lymphocytes, which may play a role in the pathogenesis of FOP. [source] Smad3-Deficient Chondrocytes Have Enhanced BMP Signaling and Accelerated Differentiation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2006Tian-Fang Li Abstract Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-, signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3,/, chondrocytes. BMP signaling is increased in TGF-, deficiency and is required for accelerated chondrocyte maturation. Introduction: Disruption of TGF-, signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures. Materials and Methods: Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3,/, mice. Expressions of maturational markers, as well as genes involved in TGF-, and BMP signaling were examined. Chondrocytes were treated with TGF-, and BMP-2, and effects on maturation-related genes and BMP/TGF-, responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures. Results: Expression of colX and other maturational markers was markedly increased in Smad3,/, chondrocytes. Smad3,/, chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF - ,1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-, toward BMP signaling. In Smad3,/, chondrocytes, alternative TGF-, signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-, pathways reduced colX expression and alkaline phosphatase activity in TGF-,-treated Smad3,/, cultures, but only partially. In contrast, Smad3,/, chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor. Conclusions: These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-, signaling with important implications for osteoarthritis and cartilage diseases. [source] Hepatocyte Growth Factor Contributes to Fracture Repair by Upregulating the Expression of BMP Receptors,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2005Yuuki Imai MD Abstract Hepatocyte growth factor (HGF) is activated and the expression of BMP receptors (BMPRs) is induced around the fracture site during the early phase of fracture repair. HGF facilitates the expression of BMPRs in mesenchymal cells. This study suggests that HGF contributes to fracture repair by inducing the expression of BMPRs. Introduction: The precise mechanisms that control the upregulation of BMP, BMPRs, and other molecules involved in bone repair are not completely understood. In this study, we hypothesized that HGF, activated through the action of thrombin on the HGF activator, may enhance BMP action through the local induction of BMP or BMPRs. Materials and Methods: Callus samples from tibial fractures in mice were harvested for immunohistochemical analysis of HGF and phosphorylated c-Met, for in situ hybridization of BMPRs, and for real-time RT-PCR analysis for the expression of HGF, c-Met, and BMPRs. To study the changes in gene expression of BMPRs in response to HGF, C3H10T1/2 cells were cultured with or without HGF and harvested for real-time RT-PCR and for Western blot analysis. To evaluate the contribution of HGF to the biological action of BMP2, C3H10T1/2 cells and primary muscle-derived mesenchymal cells were precultured with HGF and cultured with BMP2. In addition, the expression of the luciferase gene linked to the Id1 promoter containing the BMP responsive element and alkaline phosphatase (ALP) activity were assayed. Results: Positive immunostaining of HGF and phosphorylated c-Met was detected around the fracture site at 1 day after the fracture was made. mRNA expression of BMPRs was increased 1 day after fracture and localized in mesenchymal cells at the fracture site. From an in vitro study, the expression of mRNA for BMPRs was elevated by treatment with HGF, but the expression of BMP4 did not change. Western blot analysis also showed the upregulation of BMPR2 by HGF treatment. The results from the luciferase and ALP assays indicated increased responsiveness to BMPs by treating with HGF. Conclusions: This study indicates that HGF is activated and expressed at the fracture site and that HGF induces the upregulation of BMPRs in mesenchymal cells. Furthermore, HGF may facilitate BMP signaling without altering the expression of BMP molecules. [source] Fibrodysplasia Ossificans Progressiva (FOP), a Disorder of Ectopic Osteogenesis, Misregulates Cell Surface Expression and Trafficking of BMPRIA,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2005Lourdes Serrano de la Peña Abstract FOP is a disorder in which skeletal muscle is progressively replaced with bone. FOP lymphocytes, a model system for exploring the BMP pathway in these patients, exhibit a defect in BMPRIA internalization and increased activation of downstream signaling, suggesting that altered BMP receptor trafficking underlies ectopic bone formation in this disease. Introduction: Fibrodysplasia ossificans progressiva (FOP) is a severely disabling disorder characterized by progressive heterotopic ossification of connective tissues. Whereas the genetic defect and pathophysiology of this condition remain enigmatic, BMP4 mRNA and protein are overexpressed, and mRNAs for a subset of secreted BMP antagonists are not synthesized at appropriate levels in cultured lymphocytes from FOP patients. These data suggest involvement of altered BMP signaling in the disease. In this study, we investigate whether the abnormality is associated with defective BMP receptor function in lymphocytes. Materials and Methods: Cell surface proteins were quantified by fluorescence-activated cell sorting (FACS). Protein phosphorylation was assayed by immunoprecipitation and immunoblotting. Protein synthesis and degradation were examined by [35S]methionine labeling and pulse-chase assays. mRNA was detected by RT-PCR. Results: FOP lymphocytes expressed 6-fold higher levels of BMP receptor type IA (BMPRIA) on the cell surface compared with control cells and displayed a marked reduction in ligand-stimulated internalization and degradation of BMPRIA. Moreover, in control cells, BMP4 treatment increased BMPRIA phosphorylation, whereas BMPRIA showed ligand-insensitive constitutive phosphorylation in FOP cells. Our data additionally support that the p38 mitogen-activated protein kinase (MAPK) signaling pathway is a major BMP signaling pathway in these cell lines and that expression of inhibitor of DNA binding and differentiation 1 (ID-1), a transcriptional target of BMP signaling, is enhanced in FOP cells. Conclusions: These data extend our previous observations of misregulated BMP4 signaling in FOP lymphocytes and show that cell surface overabundance and constitutive phosphorylation of BMPRIA are associated with a defect in receptor internalization. Altered BMP receptor trafficking may play a significant role in FOP pathogenesis. [source] CREB Cooperates with BMP-stimulated Smad signaling to enhance transcription of the Smad6 promoterJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Andreia M. Ionescu Growth plate chondrocytes integrate a multitude of growth factor signals during maturation. PTHrP inhibits maturation through stimulation of PKA/CREB signaling while the bone morphogenetic proteins (BMPs) stimulate maturation through Smad mediated signaling. In this manuscript, we show that interactions between CREB and the BMP associated Smads are promoter specific, and demonstrate for the first time the requirement of CREB signaling for Smad mediated activation of a BMP responsive region of the Smad6 promoter. The 28 base pairs (bp) BMP responsive element of the Smad6 promoter contains an 11 bp Smad binding region and an adjacent 17 bp region in which we characterize a putative CRE site. PKA/CREB gain of function enhanced BMP stimulation of this reporter, while loss of CREB function diminished transcriptional activity. In contrast, ATF-2 and AP-1 transcription factors had minimal effects. Electrophoretic mobility shift assay (EMSA) confirmed CREB binding to the Smad6 promoter element. Mutations eliminating binding resulted in loss of transcriptional activity, while mutations that maintained CREB binding had continued reporter activation by CREB and BMP-2. The Smad6 gene was similarly regulated by CREB. Dominant negative CREB reduced BMP-2 stimulated Smad6 gene transcription by 50%, but markedly increased BMP-2 mediated stimulation of colX and Ihh expression. In contrast, PTHrP which activates CREB signaling, blocked the stimulatory effect of BMP-2 on colX and Ihh, but minimally inhibited the stimulatory effect of BMP on Smad6. These findings are the first to demonstrate a cooperative association between CREB and BMP regulated Smads in cells from vertebrates and demonstrate that promoter-specific rather than generalized interactions between PKA/CREB and BMP signaling regulate gene expression in chondrocytes. J. Cell. Physiol. 198: 428,440, 2004© 2003 Wiley-Liss, Inc. [source] Prostaglandin E2 inhibits BMP signaling and delays chondrocyte maturationJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2009Christine A. Clark Abstract While cyclooxygenases are important in endochondral bone formation during fracture healing, mechanisms involved in prostaglandin E2 (PGE2) regulation of chondrocyte maturation are incompletely understood. The present study was undertaken to determine if PGE2 effects on chondrocyte differentiation are related to modulation of the bone morphogenetic protein (BMP) signaling pathway. In primary murine sternal chondrocytes, PGE2 differentially regulated genes involved in differentiation. PGE2 induced type II collagen and MMP-13, had minimal effects on alkaline phosphatase, and inhibited the expression of the maturational marker, type X collagen. In BMP-2,treated cultures, PGE2 blocked the induction of type X collagen. All four EP receptors were expressed in chondrocytes and tended to be inhibited by BMP-2 treatment. RCJ3.1C5.18 chondrocytes transfected with the protein kinase A (PKA) responsive reporter, CRE-luciferase, showed luciferase induction following exposure to PGE2, consistent with activation of PKA signaling and the presence of the EP2 and EP4 receptors. Both PGE2 and the PKA agonist, dibutyryl cAMP, blocked the induction of the BMP-responsive reporter, 12XSBE, by BMP-2 in RCJ3.1C5.18 chondrocytes. In contrast, PGE2 increased the ability of TGF-, to activate the TGF-,-responsive reporter, 4XSBE. Finally, PGE2 down-regulated BMP-mediated phosphorylation of Smads 1, 5, and 8 in RCJ3.1C5.18 cells and in primary murine sternal chondrocytes. Altogether, the findings show that PGE2 regulates chondrocyte maturation in part by targeting BMP/Smad signaling and suggest an important role for PGE2 in endochondral bone formation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 785,792, 2009 [source] Mouse models of gastric tumors: Wnt activation and PGE2 inductionPATHOLOGY INTERNATIONAL, Issue 9 2010Hiroko Oshima Accumulating evidence has suggested that cooperation of oncogenic activation and the host responses is important for cancer development. In gastric cancer, activation of Wnt signaling appears to be a major oncogenic pathway that causes tumorigenesis. In the chronic gastritis caused by Helicobacter pylori infection, cyclooxigenase-2 induces prostaglandin E2 (PGE2) biosythesis, which plays an important role in tumorigenesis. We constructed a series of mouse models and investigated the role of each pathway in the gastric tumorigenesis. Wnt activation in gastric epithelial cells suppresses differentiation, and induces development of preneoplastic lesions. On the other hand, induction of the PGE2 pathway in gastric mucosa induces development of spasmolytic polypeptide-expressing metaplasia (SPEM), which is a possible preneoplastic metaplasia. Importantly, simultaneous activation of Wnt and PGE2 pathways leads to dysplastic gastric tumor development. Moreover, induction of the PGE2 pathway also promotes gastric hamartoma development when bone morphogenetic protein (BMP) signaling is suppressed. These results indicate that alteration in the Wnt or BMP signaling impairs epithelial differentiation, and the PGE2 pathway accelerates tumor formation regardless of the types of oncogenic pathways. We review the phenotypes and gene expression profiles of the respective models, and discuss the cooperation of oncogenic pathways and host responses in gastric tumorigenesis. [source] Roles of bone morphogenetic protein signaling and its antagonism in holoprosencephaly,AMERICAN JOURNAL OF MEDICAL GENETICS, Issue 1 2010John Klingensmith Abstract Holoprosencephaly (HPE) is the most common malformation of the forebrain, resulting from a failure to completely septate the left and right hemispheres at the rostral end of the neural tube. Because of the tissue interactions that drive head development, these forebrain defects are typically accompanied by midline deficiencies of craniofacial structures. Early events in setting up tissue precursors of the head, as well as later interactions between these tissues, are critical for normal head formation. Defects in either process can result in HPE. Signaling by bone morphogenetic proteins (BMPs), a family of secreted cytokines, generally plays negative roles in early stages of head formation, and thus must be attenuated in multiple contexts to ensure proper forebrain and craniofacial development. Chordin and Noggin are endogenous, extracellular antagonists of BMP signaling that promote the normal organization of the forebrain and face. Mouse mutants with reduced levels of both factors display mutant phenotypes remarkably analogous to the range of malformations seen in human HPE sequence. Chordin and Noggin function in part by antagonizing the inhibitory effects of BMP signaling on the Sonic hedgehog and Nodal pathways, genetic lesions in each being associated with human HPE. Study of Chordin;Noggin mutant mice is helping us to understand the molecular, cellular, and genetic pathogenesis of HPE and associated malformations. © 2010 Wiley-Liss, Inc. [source] Overexpression of the transcription factor Msx1 is insufficient to drive complete regeneration of refractory stage Xenopus laevis hindlimbsDEVELOPMENTAL DYNAMICS, Issue 6 2009Donna M. Barker Abstract Xenopus laevis tadpoles are capable of hindlimb regeneration, although this ability declines with age. Bmp signaling is one pathway known to be necessary for successful regeneration to occur. Using an inducible transgenic line containing an activated version of the Bmp target Msx1, we assessed the ability of this transcription factor to enhance regeneration in older limbs. Despite considerable evidence correlating msx1 expression with regenerative success in vertebrate regeneration models, we show that induction of msx1 during hindlimb regeneration fails to induce complete regeneration. However, we did observe some improvement in regenerative outcome, linked to morphological changes in the early wound epithelium and a corresponding increase in proliferation in the underlying distal mesenchyme, neither of which are maintained later. Additionally, we show that Msx1 is not able to rescue limb regeneration in a Bmp signalling-deficient background, indicating that additional Bmp targets are required for regeneration in anuran limbs. Developmental Dynamics 238:1366,1378, 2009. © 2009 Wiley-Liss, Inc. [source] | |||||||||||||