Bone Morphogenetic Protein Type IA Research Articles

Novel bone Morphogenetic Protein (BMP)-2/4 Consensus Peptide (BCP) for the Osteogenic Differentiation of C2C12 Cells.

Despite the promising clinical potential of bone morphogenetic protein (BMP)-related therapies for bone formation, their side effects warrant the need for alternative therapeutic peptides. BMP family members can aid in bone repair; however, peptides derived from BMP2/ 4 have not yet been investigated. In this study, three candidates BMP2/4 consensus peptide (BCP) 1, 2, and 3 were identified and their ability to induce osteogenesis in C2C12 cells was analyzed. First, an alkaline phosphatase (ALP) staining assay was performed to evaluate the osteogenic effects of BCPs. Next, the effects of BCPs on RNA expression levels and protein abundances of osteogenic markers were explored. Furthermore, the transcriptional activity of ALP by BCP1 and in silico molecular docking model on BMP type IA receptor (BRIA) were performed. BCP1-3 induced higher RUNX2 expression than BMP2. Interestingly, among them, BCP1 significantly promoted osteoblast differentiation more than BMP2 in ALP staining with no cytotoxicity. BCP1 significantly induced the osteoblast markers, and the highest RUNX2 expression was observed at 100 ng/mL compared to other concentrations. In transfection experiments, BCP1 stimulated osteoblast differentiation via RUNX2 activation and the Smad signaling pathway. Finally, in silico molecular docking suggested the possible binding sites of BCP1 on BRIA. These results show that BCP1 promotes osteogenicity in C2C12 cells. This study suggests that BCP1 is the most promising candidate peptide to replace BMP2 for osteoblast differentiation.

Controversy of physiological vs. pharmacological effects of BMP signaling: Constitutive activation of BMP type IA receptor-dependent signaling in osteoblast lineage enhances bone formation and resorption, not affecting net bone mass.

Bone morphogenetic proteins (BMPs) were first described over 50years ago as potent inducers of ectopic bone formation when administrated subcutaneously. Preclinical studies have extensively examined the osteoinductive properties of BMPs in vitro and new bone formation in vivo. BMPs (BMP-2, BMP-7) have been used in orthopedics over 15years. While osteogenic function of BMPs has been widely accepted, our previous studies demonstrated that loss-of-function of BMP receptor type IA (BMPR1A), a potent receptor for BMP-2, increased net bone mass by significantly inhibiting bone resorption in mice, indicating a positive role of BMP signaling in bone resorption. The physiological role of BMPs (i.e. osteogenic vs. osteoclastogenic) is still largely unknown. The purpose of this study was to investigate the physiological role of BMP signaling in endogenous long bones during adult stages. For this purpose, we conditionally and constitutively activated the Smad-dependent canonical BMP signaling thorough BMPR1A in osteoblast lineage cells using the mutant mice (Col1CreER™:caBmpr1a). Because trabecular bones were largely increased in the loss-of-function mouse study for BMPR1A, we hypothesized that the augmented BMP signaling would affect endogenous trabecular bones. In the mutant bones, the Smad phosphorylation was enhanced within physiological level three-fold while the resulting gross morphology, bodyweights, bone mass/shape/length, serum calcium/phosphorus levels, collagen cross-link patterns, and healing capability were all unchanged. Interestingly, we found; 1) increased expressions of both bone formation and resorption markers in femoral bones, 2) increased osteoblast and osteoclast numbers together with dynamic bone formation parameters by trabecular bone histomorphometry, 3) modest bone architectural phenotype with reduced bone quality (i.e. reduced trabecular bone connectivity, larger diametric size but reduced cortical bone thickness, and reduced bone mechanical strength), and 4) increased expression of SOST, a downstream target of the Smad-dependent BMPR1A signaling, in the mutant bones. This study is clinically insightful because gain-of-function of BMP signaling within a physiological window does not increase bone mass while it alters molecular and cellular aspects of osteoblast and osteoclast functions as predicted. These findings help explain the high-doses of BMPs (i.e. pharmacological level) in clinical settings required to substantially induce a bone formation, concurrent with potential unexpected side effects (i.e. bone resorption, inflammation) presumably due to a broader population of cell-types exposed to the high-dose BMPs rather than osteoblastic lineage cells.

Disruption of bone morphogenetic protein type IA receptor in osteoblasts impairs bone quality and bone strength in mice.

In recent years, several studies have found that the disruption of type IA receptor of bone morphogenetic proteins (BMPR1A) could increase bone mass. However, whether disruption of BMPR1A could have an effect on bone quality and bone strength is currently unknown. Osteoblast-targeted conditional knockout (cKO) of BMPRIA by crossing 3.2-kb Col1-CreER™ mice with BMPR1A fx +/+ mice was conducted. Then, in vitro and in vivo studies were employed to examine the effect of BMPR1A knockout on bone quality and bone strength. It was found that the ultimate force and stiffness of the femora decreased significantly in cKO mice when compared to control mice. The content of collagen and mineralization level decreased as the structure of the collagen became disorganized. The morphology of osteocytes in cKO mice was abnormal as well. The expression level of osteocalcin, a marker for the terminal differentiation of osteoblasts, decreased in cKO mice. This data indicated that the differentiation of osteoblasts in cKO mice was impaired. Immunohistochemistry examination revealed deregulated expression of dickkopf 1(DKK1) in osteocytes in cKO mice. Adding DKK1 to the culture medium reversed these effects. In conclusion, even though disruption of BMPR1A could increase bone mass, it also impairs bone quality and bone strength.

Bone Morphogenetic Protein Signaling Is Required for Aortic Valve Calcification.

Calcific aortic valve disease (CAVD) is the most prevalent type of heart valve disease, affecting ≈2% of the US population. CAVD is characterized by the presence of calcific nodules, resulting in aortic valve (AoV) stenosis; however, the underlying mechanisms driving disease remain unknown. Studies of human diseased AoV provide initial evidence that bone morphogenetic protein (BMP) signaling, essential for normal bone formation, is activated during CAVD. Mice deficient in Klotho, an FGF23 transmembrane coreceptor, exhibit premature aging and develop AoV calcific nodules as occurs in human CAVD. The role of BMP signaling in the development of CAVD was examined in porcine aortic valve interstitial cells (VICs) and Klotho(-/-) mice. We show that activation of BMP signaling, as indicated by pSmad1/5/8 expression, precedes and later localizes with AoV calcification in Klotho(-/-) mice. In addition, cellular and extracellular matrix changes resembling features of normal bone formation are accompanied by increased osteochondrogenic gene induction in calcified Klotho(-/-) AoV. Likewise, osteogenic media treatment of porcine VICs results in BMP pathway activation, increased osteochondrogenic gene induction, and formation of calcific nodules in vitro. We demonstrate that genetic inactivation of the BMP type IA receptor in Klotho(-/-) aortic VICs, as well as BMP pathway inhibition of osteogenic media-treated aortic VICs in vitro, results in the inhibition of AoV calcification. BMP signaling and osteochondrogenic gene induction are active in calcified Klotho(-/-) AoV in vivo and calcified porcine aortic VICs in vitro. Importantly, BMP signaling is required for the development of AoV calcification in vitro and in vivo.

Bone morphogenetic protein signaling promotes tumorigenesis in a murine model of high-grade glioma.

Improved therapies for high-grade glioma (HGG) are urgently needed as the median survival for grade IV gliomas is only 15 months. Bone morphogenetic protein (BMP) signaling plays critical and complex roles in many types of cancer, including glioma, with most of the recently published work focusing on BMP-mediated regulation of glioma stem cells (GSCs). We hypothesized that BMP signaling may be an important modulator of tumorigenic properties in glioma cells outside of the GSC compartment. We used a human HGG tissue microarray and performed immunohistochemistry for phospho-Smads1,5,8. To examine the role of BMP signaling in tumorigenic astrocytes, transgenic mice were used to delete the BMP type IA receptor (Bmpr1a) and generate astrocytes transformed with oncogenic Ras and homozygous deletion of p53. The cells were transplanted orthotopically into immunocompetent adult host mice. First we established that BMP signaling is active within the vast majority of HGG tumor cells. Mice implanted with BMPR1a-knockout transformed astrocytes showed an increase in median survival compared with mice that received BMPR1a-intact transformed astrocytes (52.5 vs 16 days). In vitro analysis showed that deletion of BMPR1a in oncogenic astrocytes resulted in decreased proliferation, decreased invasion, decreased migration, and increased expression of stemness markers. In addition, inhibition of BMP signaling in murine cells and astrocytoma cells with a small molecule BMP receptor kinase inhibitor resulted in similar tumor suppressive effects in vitro. BMP inhibition may represent a viable therapeutic approach in adult HGG.

Bone Morphogenetic Protein-4 (BMP4): A Paracrine Regulator of Human Adrenal C19 Steroid Synthesis.

Bone morphogenetic proteins (BMPs) comprise one of the largest subgroups in the TGF-β ligand superfamily. We have identified a functional BMP system equipped with the ligand (BMP4), receptors (BMP type II receptor, BMP type IA receptor, also called ALK3) and the signaling proteins, namely the mothers against decapentaplegic homologs 1, 4, and 5 in the human adrenal gland and the human adrenocortical cell line H295R. Microarray, quantitative RT-PCR, and immunohistochemistry confirmed that BMP4 expression was highest in the adrenal zona glomerulosa followed by the zona fasciculata and zona reticularis. Treatment of H295R cells with BMP4 caused phosphorylation of the mothers against decapentaplegic and a profound decrease in synthesis of the C19 steroids dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione. Administration of BMP4 to cultures of H295R cells also caused a profound decrease in the mRNA and protein levels of 17α-hydroxylase/17,20-lyase (CYP17A1 and P450c17, respectively) but no significant effect on the mRNA levels of cholesterol side-chain cleavage cytochrome P450 (CYP11A1) or type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). Furthermore, Noggin (a BMP inhibitor) was able to reverse the negative effects of BMP4 with respect to both CYP17A1 transcription and DHEA secretion in the H295R cell line. Collectively the present data suggest that BMP4 is an autocrine/paracrine negative regulator of C19 steroid synthesis in the human adrenal and works by suppressing P450c17.

Influence of bone morphogenetic protein type IA receptor conditional knockout in lens on expression of bone morphogenetic protein 4 in lens.

To investigate the influence of bone morphogenetic protein type IA receptor [BMPR-IA (ALK3)] conditional knockout in lens on expression of bone morphogenetic protein 4 (BMP4) in lens during the development of the vertebrate eye. Cre-positive mice were mated with Cre-negative mice to generate 50% Cre-positive (conditional knockout, CKO) 4 embryos, 8 eyes and 50% Cre-negative offspring (wild type, WT) 4 embryos, 8 eyes. The embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 µm. Removal of paraffin wax and dehydrating for sections, and then the procedure of in situ hybridization was processed, BMP4 MK1784-m (BOSTER) was used, and observed the expression of BMP4 in the lens in experimental group and control group. We selected SPSS11.0 software for statistical analysis, P<0.05 showed that the difference was statistically significant. Four embryos of each genotype were examined, totally we had 8 embryos, 16 eyes. We got the uniform outcomes in all the embryos. We found ALK3 was required during lens growing, but was not essential for the formation of lens. We observed that the expression of Bmp4 in the lens was significantly reduced in all 8 ALK3 CKO lens, BMP4 expression was normal in all the 8 WT lens, P<0.01. This phenomenon became increasingly visible in accordance with embryo development. The most apparent alteration was present at stage E15.5. ALK3 is essential for lens growth. The influence of ALK3 on the expression of BMP4 is present during the development of mice lens.

Augmented BMP signaling in the neural crest inhibits nasal cartilage morphogenesis by inducing p53-mediated apoptosis.

Bone morphogenetic protein (BMP) signaling plays many roles in skull morphogenesis. We have previously reported that enhanced BMP signaling through the BMP type IA receptor (BMPR1A) in cranial neural crest cells causes craniosynostosis during postnatal development. Additionally, we observed that 55% of Bmpr1a mutant mice show neonatal lethality characterized by a distended gastrointestinal tract. Here, we show that severely affected mutants exhibit defective nasal cartilage, failure of fusion between the nasal septum and the secondary palate, and higher levels of phosphorylated SMAD1 and SMAD5 in the nasal tissue. TUNEL demonstrated an increase in apoptosis in both condensing mesenchymal tissues and cartilage of the nasal region in mutants. The levels of p53 (TRP53) tumor suppressor protein were also increased in the same tissue. Injection of pifithrin-α, a chemical inhibitor of p53, into pregnant mice prevented neonatal lethality while concomitantly reducing apoptosis in nasal cartilage primordia, suggesting that enhanced BMP signaling induces p53-mediated apoptosis in the nasal cartilage. The expression of Bax and caspase 3, downstream targets of p53, was increased in the mutants; however, the p53 expression level was unchanged. It has been reported that MDM2 interacts with p53 to promote degradation. We found that the amount of MDM2-p53 complex was decreased in all mutants, and the most severely affected mutants had the largest decrease. Our previous finding that the BMP signaling component SMAD1 prevents MDM2-mediated p53 degradation coupled with our new data indicate that augmented BMP signaling induces p53-mediated apoptosis by prevention of p53 degradation in developing nasal cartilage. Thus, an appropriate level of BMP signaling is required for proper craniofacial morphogenesis.

CS-13 * BONE MORPHOGENETIC PROTEIN SIGNALING PROMOTES TUMORIGENESIS IN A TRANSGENIC MOUSE MODEL OF GLIOMA

Improved therapies for high grade glioma (HGG) are imperative, as the median survival after diagnosis with grade IV glioma is 15 months. Recently pathways regulating neural development, such as the bone morphogenetic protein (BMP) pathway, have been investigated as potential therapeutic targets in HGG. In xenograft transplant models, BMP signaling has been shown to have a tumor suppressive effect on the subpopulation of cells known as glioma-initiating or glioma stem cells. However, the degree to which BMP signaling plays a role in the bulk tumor cells or the more differentiated component in HGG is unknown. To investigate these questions we used both human samples of HGG and a novel murine model of human HGG established in our laboratory. We determined that BMP signaling is present and active in human HGG using a human HGG tissue-microarray. Our analysis of phospho-smad1/5/8 staining suggests that BMP signaling is present and active in most cells in many HGG gliomas, suggesting that BMP signaling is not limited to the glioma stem cell compartment. To examine the role of BMP signaling in differentiated, tumorigenic astrocytes, we deleted the BMP type IA receptor gene (Bmpr1a) in transformed astrocytes, effectively abrogating canonical BMP signaling in these cells. The cells were then transplanted orthotopically into immunocompetent adult host mice. Preliminary data suggest that, while BMP signaling may be tumor suppressive in stem-like cells, it acts as a tumor promoter in differentiated tumorigenic astrocytes. Compared to controls receiving cells with intact Bmpr1a, mice receiving Bmpr1a-knockout cells showed a significant increase in survival time upon orthotopic injection (21 vs. 52 days, p = 0.001). In vitro, deletion of Bmpr1a in oncogenic astrocytes resulted in decreased proliferation and increased Olig2 expression. Studies to further investigate the role of BMPs in HGG are underway.

Augmentation of smad-dependent BMP signaling in neural crest cells causes craniosynostosis in mice

Craniosynostosis describes conditions in which one or more sutures of the infant skull are prematurely fused, resulting in facial deformity and delayed brain development. Approximately 20% of human craniosynostoses are thought to result from gene mutations altering growth factor signaling; however, the molecular mechanisms by which these mutations cause craniosynostosis are incompletely characterized, and the causative genes for diverse types of syndromic craniosynostosis have yet to be identified. Here, we show that enhanced bone morphogenetic protein (BMP) signaling through the BMP type IA receptor (BMPR1A) in cranial neural crest cells, but not in osteoblasts, causes premature suture fusion in mice. In support of a requirement for precisely regulated BMP signaling, this defect was rescued on a Bmpr1a haploinsufficient background, with corresponding normalization of Smad phosphorylation. Moreover, in vivo treatment with LDN-193189, a selective chemical inhibitor of BMP type I receptor kinases, resulted in partial rescue of craniosynostosis. Enhanced signaling of the fibroblast growth factor (FGF) pathway, which has been implicated in craniosynostosis, was observed in both mutant and rescued mice, suggesting that augmentation of FGF signaling is not the sole cause of premature fusion found in this model. The finding that relatively modest augmentation of Smad-dependent BMP signaling leads to premature cranial suture fusion suggests an important contribution of dysregulated BMP signaling to syndromic craniosynostoses and potential strategies for early intervention.

Patchy deletion of Bmpr1a potentiates proximal pulmonary artery remodeling in mice exposed to chronic hypoxia

Reduced vascular expression of bone morphogenetic protein type IA receptor (Bmpr1a) has been found in patients with pulmonary arterial hypertension. Our previous studies in mice with patchy deletion of Bmpr1a in vascular smooth muscle cells and cardiac myocytes showed decreased distal vascular remodeling despite a similar severity of hypoxic pulmonary hypertension (HPH). We speculate increased stiffness from ectopic deposition of collagen in proximal pulmonary arteries might account for HPH. Pulsatile pressure-flow relationships were measured in isolated, ventilated, perfused lungs of SM22α;TRE-Cre;R26R;Bmpr1a(flox/flox) (KO) mice and wild-type littermates, following 21 days (hypoxia) and 0 days (control) of chronic hypoxia. Pulmonary vascular impedance, which yields insight into proximal and distal arterial remodeling, was calculated. Reduced Bmpr1a expression had no effect on input impedance Z(0) (P = 0.52) or characteristic impedance Z(C) (P = 0.18) under control conditions; it also had no effect on the decrease in Z(0) via acute rho kinase inhibition. However, following chronic hypoxia, reduced Bmpr1a expression increased Z(C) (P < 0.001) without affecting Z(0) (P = 0.72). These results demonstrate that Bmpr1a deficiency does not significantly alter the hemodynamic function of the distal vasculature or its response to chronic hypoxia but larger, more proximal arteries are affected. In particular, reduced Bmpr1a expression likely decreased dilatation and increased stiffening in response to hypoxia, probably by collagen accumulation. Increased PA stiffness can have a significant impact on right ventricular function. This study illustrates for the first time how proximal pulmonary artery changes in the absence of distal pulmonary artery changes contribute to pulmonary arterial hypertension.

Bone Morphogenetic Protein 2 Acts via Inhibitor of DNA Binding Proteins to Synergistically Regulate Follicle-Stimulating Hormone β Transcription with Activin A

We recently reported that bone morphogenetic proteins (BMPs) 2 and 4 can stimulate FSHbeta-subunit (Fshb) transcription alone and in synergy with activins. We further showed that BMP2 signals via the BMP type IA receptor (or activin receptor-like kinase 3) to mediate its effects. However, the intracellular mechanisms through which BMP2 regulates Fshb are unknown. In the current study, we used cDNA microarray analyses (and validation by real-time quantitative RT-PCR) to identify BMP2 target genes in the murine gonadotrope cell line, LbetaT2. Short-interfering RNA-mediated knockdown, overexpression, and coimmunoprecipitation experiments were used to examine the potential functional roles of selected gene products. Quantitative RT-PCR analysis largely confirmed the results of the array analyses, and inhibitors of DNA binding 1, 2, and 3 (Id1, Id2, and Id3) were selected for functional analyses. Knockdown of endogenous Id2 or Id3, but not Id1, diminished the synergistic effects of BMP2 and activin A on Fshb transcription. Overexpression of Id1, Id2, or Id3 alone had no effect, but all three potentiated activin A or mothers against decapentaplegic homolog (SMAD)3 induction of Fshb transcription. Though the precise mechanism through which Ids produce their effects are not yet known, we observed physical interactions between Id1, Id2, or Id3 and SMAD3. Collectively, the data suggest that BMP2 synergistically regulates Fshb transcription with activins, at least in part, through the combined actions of Ids 2 or 3 and SMAD3.

Altered Bone Morphogenetic Protein and Transforming Growth Factor-β Signaling in Rat Models of Pulmonary Hypertension

Recent genetic studies have highlighted the role of the bone morphogenetic protein (BMP)/transforming growth factor (TGF)-beta signaling pathways in the pathogenesis of familial pulmonary arterial hypertension (PAH). It remains unclear whether alterations in these pathways contribute to other forms of pulmonary hypertension and to what extent these changes can be exploited for therapeutic intervention. We studied BMP/TGF-beta signaling in 2 rat models of PAH due to chronic hypoxia and monocrotaline. In both models, there was a significant reduction in lung BMP type IA receptor and BMP type II receptor mRNA expression, although these changes were more pronounced in the monocrotaline model. This was accompanied by a reduction in lung levels of phospho-Smad1/5 and Id (inhibitor of DNA binding) gene expression in the monocrotaline model. In contrast, we observed increased TGF-beta activity, again more marked in the monocrotaline model, as evidenced by increased phospho-Smad2/3 and increased expression of TGF-beta-regulated genes. Immunohistochemistry revealed increased TGF-beta(1) expression in pulmonary artery smooth muscle cells and macrophages surrounding remodeled pulmonary arteries in monocrotaline rats. Inhibition of activin receptor-like kinase-5 signaling in vivo with the selective small-molecule inhibitor IN-1233 prevented PAH, right ventricular hypertrophy, and vascular remodeling after monocrotaline injection and inhibited the progression of established PAH in this model. No significant effect was observed in hypoxic PAH. In vitro studies confirmed that TGF-beta stimulated migration of distal rat pulmonary artery smooth muscle cells and that this effect was inhibited by IN-1233. Disruption of BMP/TGF-beta signaling is more pronounced in the monocrotaline model of PAH than in the chronic hypoxia model. Increased TGF-beta activity is associated with greater macrophage recruitment with monocrotaline treatment. Inhibition of TGF-beta signaling via activin receptor-like kinase-5 prevents development and progression of PAH in the monocrotaline model and may involve inhibition of pulmonary artery smooth muscle cell migration.

BMP signaling through BMPRIA in astrocytes is essential for proper cerebral angiogenesis and formation of the blood–brain-barrier

Bone morphogenetic protein (BMP) signaling is involved in differentiation of neural precursor cells into astrocytes, but its contribution to angiogenesis is not well characterized. This study examines the role of BMP signaling through BMP type IA receptor (BMPRIA) in early neural development using a conditional knockout mouse model, in which Bmpr1a is selectively disrupted in telencephalic neural stem cells. The conditional mutant mice show a significant increase in the number of cerebral blood vessels and the level of vascular endothelial growth factor (VEGF) is significantly upregulated in the mutant astrocytes. The mutant mice also show leakage of immunoglobulin around cerebral microvessels in neonatal mice, suggesting a defect in formation of the blood–brain-barrier. In addition, astrocytic endfeet fail to encircle cortical blood vessels in the mutant mice. These results suggest that BMPRIA signaling in astrocytes regulates the expression of VEGF for proper cerebrovascular angiogenesis and has a role on in the formation of the blood–brain-barrier.

Bone Morphogenetic Protein Signaling Inhibits Hair Follicle Anagen Induction by Restricting Epithelial Stem/Progenitor Cell Activation and Expansion

Epithelial stem cells (EP-SCs) located in the bulge region of a hair follicle (HF) have the potential to give rise to hair follicle stem/progenitor cells that migrate down to regenerate HFs. Bone morphogenetic protein (BMP) signaling has been shown to regulate the HF cycle by inhibiting anagen induction. Here we show that active BMP signaling functions to prevent EP-SC activation and expansion. Dynamic expression of Noggin, a BMP antagonist, releases EP-SCs from BMP-mediated restriction, leading to EP-SC activation and initiation of the anagen phase. Experimentally induced conditional inactivation of the BMP type IA receptor (Bmpr1a) in EP-SCs leads to overproduction of HF stem/progenitor cells and the eventual formation of matricomas. This genetic manipulation of the BMP signaling pathway also reveals unexpected activation of beta-catenin, a major mediator of Wnt signaling. We propose that BMP activity controls the HF cycle by antagonizing Wnt/beta-catenin activity. This is at least partially achieved by BMP-mediated enhancement of transforming growth factor-beta-regulated epithelial cell-specific phosphatase (PTEN) function. Subsequently, PTEN, through phosphatidyl inositol 3-kinase-Akt, inhibits the activity of beta-catenin, the convergence point of the BMP and Wnt signaling pathways.

BMP signaling in the epiblast is required for proper recruitment of the prospective paraxial mesoderm and development of the somites

Bmpr1a encodes the BMP type IA receptor for bone morphogenetic proteins (BMPs), including 2 and 4. Here, we use mosaic inactivation of Bmpr1a in the epiblast of the mouse embryo (Bmpr-MORE embryos) to assess functions of this gene in mesoderm development. Unlike Bmpr1a-null embryos, which fail to gastrulate, Bmpr-MORE embryos initiate gastrulation, but the recruitment of prospective paraxial mesoderm cells to the primitive streak is delayed. This delay causes a more proximal distribution of cells with paraxial mesoderm character within the primitive streak, resulting in a lateral expansion of somitic mesoderm to form multiple columns. Inhibition of FGF signaling restores the normal timing of recruitment of prospective paraxial mesoderm and partially rescues the development of somites. This suggests that BMP and FGF signaling function antagonistically during paraxial mesoderm development.

Heparin Potentiates the in Vivo Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists.

Mutual Regulation of Follicle-Stimulating Hormone Signaling and Bone Morphogenetic Protein System in Human Granulosa Cells1

Bone morphogenetic proteins (BMPs) play critical roles in folliculogenesis by modulating the actions of follicle-stimulating hormone (FSH) in the ovary. However, the effects of FSH on the BMP system remain unknown. Here, we have investigated the effects of FSH on BMP signaling using the human granulosa-like tumor cell line KGN. KGN cells express BMP type I and type II receptors and the BMP signaling molecules SMADs. FSH administration upregulated BMP type IA (BMPR1A) and IB (BMPR1B) receptors, activin type II receptor (ACVR2), and BMP type II receptor (BMPR2). FSH also augmented SMAD1 and SMAD5 expression, and conversely, FSH suppressed the expression of the inhibitory SMADs, SMAD6 and SMAD7. Bioassays revealed that FSH enhances BMP-induced SMAD1/5/8 phosphorylation and cellular DNA synthesis induced by BMP6 and BMP7. Since overexpression of BMPR1A and BMPR1B, but not SMADs, significantly enhanced the BMP responses, these type I receptors were revealed to be limiting factors for BMP signaling in KGN cells. BMPs significantly suppressed progesterone synthesis induced by forskolin and dibutyryl-cAMP (BtcAMP) but had no effect on estradiol induced by the same factors. KGN cAMP levels induced by forskolin were not altered by BMPs, suggesting that BMPs regulate steroidogenesis at a level downstream of cAMP synthesis in KGN cells. In this regard, BMPs specifically reduced the STAR transcription, whereas the levels of CYP11A, HSD3B2, and CYP19 stimulated by forskolin as well as BtcAMP were not altered. Collectively, the two major factors, FSH-cAMP pathway and BMP system, are reciprocally and functionally linked. Given that BMPs downregulate FSH receptors in KGN cells, this interaction may contribute to fine-tuning of the mutual sensitivity toward BMP ligands and FSH.

Bone morphogenetic proteins: from structure to clinical use

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor ss superfamily. Family members are expressed during limb development, endochondral ossification, early fracture, and cartilage repair. The activity of BMPs was first identified in the 1960s but the proteins responsible for bone induction were unknown until the purification and cloning of human BMPs in the 1980s. To date, about 15 BMP family members have been identified and characterized. The signal triggered by BMPs is transduced through serine/threonine kinase receptors, type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, namely: type IA and IB BMP receptors and type IA activin receptors. BMPs seem to be involved in the regulation of cell proliferation, survival, differentiation and apoptosis, but their hallmark is their ability to induce bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. This suggests that, in the future, they may play a major role in the treatment of bone diseases. Several animal studies have illustrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. Difficulties in producing and purifying BMPs from bone tissue have prompted the attempts made by several laboratories, including ours, to express these proteins in the recombinant form in heterologous systems. This review focuses on BMP structure, molecular mechanisms of action and significance and potential applications in medical, dental and veterinary practice for the treatment of cartilage and bone-related diseases.

Bone Morphogenetic Proteins

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor beta (TGFbeta) superfamily. The roles of BMPs in embryonic development and cellular functions in postnatal and adult animals have been extensively studied in recent years. Signal transduction studies have revealed that Smad1, 5 and 8 are the immediate downstream molecules of BMP receptors and play a central role in BMP signal transduction. Studies from transgenic and knockout mice and from animals and humans with naturally occurring mutations in BMPs and related genes have shown that BMP signaling plays critical roles in heart, neural and cartilage development. BMPs also play an important role in postnatal bone formation. BMP activities are regulated at different molecular levels. Preclinical and clinical studies have shown that BMP-2 can be utilized in various therapeutic interventions such as bone defects, non-union fractures, spinal fusion, osteoporosis and root canal surgery. Tissue-specific knockout of a specific BMP ligand, a subtype of BMP receptors or a specific signaling molecule is required to further determine the specific role of a BMP ligand, receptor or signaling molecule in a particular tissue. BMPs are members of the TGFbeta superfamily. The activity of BMPs was first identified in the 1960s (Urist, M.R. (1965) "Bone formation by autoinduction", Science 150, 893-899), but the proteins responsible for bone induction remained unknown until the purification and sequence of bovine BMP-3 (osteogenin) and cloning of human BMP-2 and 4 in the late 1980s (Wozney, J.M. et al. (1988) "Novel regulators of bone formation: molecular clones and activities", Science 242, 1528-1534; Luyten, F.P. et al. (1989) "Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation", J. Biol. Chem. 264, 13377-13380; Wozney, J.M. (1992) "The bone morphogenetic protein family and osteogenesis", Mol. Reprod. Dev. 32, 160-167). To date, around 20 BMP family members have been identified and characterized. BMPs signal through serine/threonine kinase receptors, composed of type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, type IA and IB BMP receptors (BMPR-IA or ALK-3 and BMPR-IB or ALK-6) and type IA activin receptor (ActR-IA or ALK-2) (Koenig, B.B. et al. (1994) "Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells", Mol. Cell. Biol. 14, 5961-5974; ten Dijke, P. et al. (1994) "Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4", J. Biol. Chem. 269, 16985-16988; Macias-Silva, M. et al. (1998) "Specific activation of Smad1 signaling pathways by the BMP7 type I receptor, ALK2", J. Biol. Chem. 273, 25628-25636). Three type II receptors for BMPs have also been identified and they are type II BMP receptor (BMPR-II) and type II and IIB activin receptors (ActR-II and ActR-IIB) (Yamashita, H. et al. (1995) "Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects", J. Cell. Biol. 130, 217-226; Rosenzweig, B.L. et al. (1995) "Cloning and characterization of a human type II receptor for bone morphogenetic proteins", Proc. Natl Acad. Sci. USA 92, 7632-7636; Kawabata, M. et al. (1995) "Cloning of a novel type II serine/threonine kinase receptor through interaction with the type I transforming growth factor-beta receptor", J. Biol. Chem. 270, 5625-5630). Whereas BMPR-IA, IB and II are specific to BMPs, ActR-IA, II and IIB are also signaling receptors for activins. These receptors are expressed differentially in various tissues. Type I and II BMP receptors are both indispensable for signal transduction. After ligand binding they form a heterotetrameric-activated receptor complex consisting of two pairs of a type I and II receptor complex (Moustakas, A. and C.H. Heldi (2002) "From mono- to oligo-Smads: the heart of the matter in TGFbeta signal transduction" Genes Dev. 16, 67-871). The type I BMP receptor substrates include a protein family, the Smad proteins, that play a central role in relaying the BMP signal from the receptor to target genes in the nucleus. Smad1, 5 and 8 are phosphorylated by BMP receptors in a ligand-dependent manner (Hoodless, P.A. et al. (1996) "MADR1, a MAD-related protein that functions in BMP2 signaling pathways", Cell 85, 489-500; Chen Y. et al. (1997) "Smad8 mediates the signaling of the receptor serine kinase", Proc. Natl Acad. Sci. USA 94, 12938-12943; Nishimura R. et al. (1998) "Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12", J. Biol. Chem. 273, 1872-1879). After release from the receptor, the phosphorylated Smad proteins associate with the related protein Smad4, which acts as a shared partner. This complex translocates into the nucleus and participates in gene transcription with other transcription factors (). A significant advancement about the understanding of in vivo functions of BMP ligands, receptors and signaling molecules has been achieved in recent years.