Bone Morphogenetic Protein 2 Action Research Articles

Association Between BMP2 Functional Polymorphisms and Sheep Tail Type.

Simple SummaryWe previously detected selection markers of sheep with different tail types and found that bone morphogenetic protein 2 (BMP2) was strongly selected. We therefore predicted that BMP2 might be involved in fat deposition in sheep tails or related to sheep tail type. However, no studies have investigated the molecular mechanism of BMP2 in the formation of sheep tails. In this study, we performed flight mass spectrometry genotyping on the BMP2 gene of 533 sheep with different tail types and functionally verified our results at the cellular level. This aimed to provide basic research data for the regulation of fat deposition in sheep tails and to identify genetic markers for molecular breeding.Bone morphogenetic protein 2 (BMP2) is strongly selected in both fat-tailed and thin-tailed sheep and may be a candidate gene for sheep tail type selection. However, the mechanism of action of BMP2 in sheep tail fat deposition remains unclear. This study investigated genetic variation and haplotype combinations of the BMP2 gene in sheep with different tail types, aiming to reveal the molecular mechanism of BMP2 in sheep tail fat deposition. We detected a total of three single nucleotide polymorphisms (SNPs) (g.48401619 T > A, g.48401272 C > A, and g.48401136 C > T) among 533 sheep. The alleles and genotype frequencies of these SNPs were in Hardy–Weinberg equilibrium and showed significant correlations with tail length. Linkage disequilibrium existed between the g.48401272 C > A and g.48401136 C > T sites, where CACT was the predominant genotype. At the cellular level, the expression levels of peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL) were upregulated after BMP2 overexpression; there were significantly higher levels of PPARγ than controls at 0 d and 1 d, and of LPL than controls at 1 d and 7 d. These results indicate that the BMP2 gene may participate in sheep tail fat deposition and could be used for molecular-marker-assisted selection of sheep tail type.

Icariin Promotes the Osteogenic Action of BMP2 by Activating the cAMP Signaling Pathway.

Icariin (ICA) is the main active flavonoid glucoside from herbs of the genus Epimedium; in traditional Chinese medicine, these herbs have long been prescribed for the treatment of bone fractures and osteoporosis. Several studies have shown that treatment with ICA can increase osteogenic differentiation and reduce bone loss in vivo and in vitro. However, the definite signaling pathway of this osteogenic effect remains unclear. In this study, we selected bone morphogenetic protein 2 (BMP2)-induced osteoblastic differentiation of multipotent mesenchymal progenitor C2C12 cells as a model of osteoblast differentiation. We investigated the effects of ICA on C2C12 cells osteogenic differentiation and the underlying molecular mechanisms. We found that ICA could enhance BMP2-mediated osteoblastic differentiation of C2C12 cells in a dose-dependent manner. Treatment with ICA activated the cAMP/PKA/CREB signaling axis in a time-dependent manner. Blocking cAMP signaling using the PKA selective inhibitor H89 significantly inhibited the stimulatory effect of ICA on osteogenesis. Therefore, the osteoinductive potential and the low cost of ICA indicate that it is a promising alternative treatment or promoter for enhancing the therapeutic effects of BMP2.

Effects of Bone Morphogenetic Protein-2 on Neovascularization During Large Bone Defect Regeneration.

Insufficient blood vessel supply is a primary limiting factor for regenerative approaches to large bone defect repair. Recombinant bone morphogenetic protein-2 (BMP-2) delivery induces robust bone formation and has been observed to enhance neovascularization, but whether the angiogenic effects of BMP-2 are due to direct endothelial cell stimulation or due to indirect paracrine signaling remain unclear. In this study, we evaluated the effects of BMP-2 delivery on vascularized bone regeneration and tested whether BMP-2 induces neovascularization directly or indirectly. We found that delivery of BMP-2 (5 μg) enhanced both bone formation and neovascularization in critically sized (8 mm) rat femoral bone defects; however, BMP-2 did not directly stimulate angiogenesis in vitro. In contrast, conditioned medium from both mesenchymal progenitor cells and osteoblasts induced endothelial cell migration in vitro, suggesting a paracrine mechanism of BMP-2 action. Consistent with this inference, codelivery of BMP-2 with endothelial colony forming cells to a heterotopic site, distant from the skeletal stem cell-rich bone marrow niche, induced ossification but had no effect on neovascularization. Taken together, these data suggest that paracrine activation of osteoprogenitor cells is an important contributor to neovascularization during BMP-2-mediated bone regeneration. Impact Statement In this study, we show that bone morphogenetic protein-2 (BMP-2) robustly induces neovascularization during tissue-engineered large bone defect regeneration, and we found that BMP-2 induced angiogenesis, in part, through paracrine signaling from osteoprogenitor cells.

Stimulation of primordial follicle assembly by estradiol-17\u03b2 requires the action of bone morphogenetic protein-2 (BMP2)

Primordial follicle (PF) pool determines the availability of follicles for ovulation in all mammals. Premature depletion of the PF reserve leads to subfertility or infertility. Bone morphogenetic protein 2 (BMP2) promotes PF formation by facilitating oocyte and granulosa cell development. Estradiol-17β (E2) upregulates PF formation in developing hamster ovaries. However, if BMP2 mediates E2 effect is not known. We hypothesize that E2 facilitates the effect of BMP2 on somatic to granulosa cell transition. BMP2 and E2 together significantly upregulated the percentage of PFs in hamster fetal ovaries in vitro compared with either of the treatments alone. E2 also promoted BMP2 expression in vivo. Inhibition of BMP2 receptors suppressed E2-stimulation of PF formation while knockdown of BMP2 in vitro significantly suppressed the E2 effect. In contrast, estrogen receptor blocker did not affect BMP2 action. Inhibition of the activity of E2 or BMP2 receptors, either alone or combined during the last two days of the culture (C6-C8) resulted in a significant decrease in PF formation by C8, suggesting that both BMP2 and E2 action is essential for somatic cell differentiation for PF formation. Together, the results suggest that E2 activates BMP2-BMPR system leading to the formation of primordial follicles.

Mollugin enhances the osteogenic action of BMP-2 via the p38-Smad signaling pathway.

Bone morphogenetic protein 2 (BMP-2) has been used clinically to encourage bone regeneration; although, there can be major side effects with larger doses. Therefore, there is a need to identify new small molecules to potentiate the osteogenic action of BMP-2. In this study, we investigated the effect of mollugin on bone formation in murine bi-potential mesenchymal progenitor C2C12 cells by combination with BMP-2. We found mollugin could enhance the BMP-2-mediated osteoblast differentiation of C2C12 cells. This was accompanied by the induction of other osteogenic BMPs. We also found the enhancing potential of mollugin may involve activation of the p38-Smad1/5/8 signaling axis. Furthermore, mollugin promoted skeletal development in zebrafish. The combination of BMP-2 with small molecules, including mollugin, could minimize its clinical limitations, and these molecules might lead to the development of effective stem cell stimulants for bone regeneration and fracture healing.

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

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 3days 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 3days 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.

BMP2 Protein Regulates Osteocalcin Expression via Runx2-mediated Atf6 Gene Transcription

Bone morphogenetic protein 2 (BMP2) activates unfolded protein response (UPR) transducers, such as PERK and OASIS, in osteoblast cells. ATF6, a bZIP transcription factor, is also a UPR transducer. However, the involvement of ATF6 in BMP2-induced osteoblast differentiation has not yet been elucidated. In the present study, BMP2 treatment was shown to markedly induce the expression and activation of ATF6 with an increase in alkaline phosphatase (ALP) and OC expression in MC3T3E1 cells. In contrast, ATF6 activation by BMP2 was not observed in the Runx2(-/-) primary calvarial osteoblasts, and Runx2 overexpression recovered BMP2 action. BMP2 stimulated ATF6 transcription by enhancing the direct binding of Runx2 to the osteoblast-specific cis-acting element 2 (OSE2, ACCACA, -205 to -200 bp) motif of the Atf6 promoter region. In addition, the overexpression of ATF6 increased the Oc promoter activity by enhancing the direct binding to a putative ATF6 binding motif (TGACGT, -1126 to -1121 bp). The inhibition of ATF6 function with the dominant negative form of ATF6 (DN-ATF6) blocked BMP2- or Runx2-induced OC expression. Interestingly, OASIS, which is structurally similar to ATF6, did not induce Oc expression. ALP and Alizarin red staining results confirmed that BMP2-induced matrix mineralization was also dependent on ATF6 in vitro. Overall, these results suggest that BMP2 induces osteoblast differentiation through Runx2-dependent ATF6 expression, which directly regulates Oc transcription.

Phospho-Smad1 modulation by nedd4 e3 ligase in BMP/TGF-β signaling

A considerable number of studies have focused on the regulation of mothers against decapentaplegic homologue (Smad)-dependent or -independent pathways in the signaling by each transforming growth factor β (TGF-β) superfamily member in diverse biologic contexts. The sophisticated regulation of the actions of these molecules and the underlying molecular mechanisms still remain elusive. Here we show new mechanisms of ambilateral R (receptor-regulated)-Smad regulation of bone morphogenetic protein 2 (BMP-2)/TGF-β1 signals. In a specific context, both signals regulate the nonclassic Smads pathway reciprocally, BMP-2 to Smad2/3 and TGF-β1 to Smad1/5/8, as well as their own classic linear Smad pathway. Interestingly, in this study, we found that C-terminal phosphorylated forms of each pathway Smad degraded rapidly 3 hours after stimulation of nonclassic signals but are dramatically restored by treatment with via proteasomal inhibition. Furthermore, an E3 ligase, neural precursor cell expressed, developmentally down-regulated 4 (Nedd4), also was found as one of the important modulators of the p-Smad1 in both BMP-2 and TGF-β1 action. Overexpressed Nedd4 suppressed the BMP-induced osteoblast transdifferentiation process of premyoblast C2C12 cells or alkaline phosphatase (ALP) level of human osteosarcoma cells and promoted TGF-β1-induced degradation of p-Smad1 via physical interaction and polyubiquitination. Conversely, siNedd4 potentiated BMP signals through upregulation of p-Smad1 and ALP activity, the effect of which led to an increased the rate of P(i) -induced calcification of human vascular smooth muscle cells. These new insights about proteasomal degradation-mediated phosphorylated nonclassic Smad regulation of BMP-2/TGF-β1 could, in part, help to unravel the complex mechanisms of abnormal nonosseous calcification by the aberrant activity of BMP/TGF-β/Smads.

Role of PI3K on the regulation of BMP2-induced β-Catenin activation in human bone marrow stem cells

Bone morphogenetic protein 2 (BMP2), a very potent bone-inducing agent, promotes the differentiation of bone marrow stem cells (BMSCs) to osteoblasts. However, the potency of BMP2 action is variable and its perturbed dynamic signaling pathways in human BMSCs has not been fully elucidated. In this study, we used a combination of stable isotope labeling by amino acids during cell culture (SILAC) and liquid-chromatography electrospray ionization mass spectrometry (LC-ESI-MS/MS) technology to reveal the BMP2 action in BMSC. In this quantitative proteomic analysis, 414 of 449 proteins were successfully quantified with 79.2% peptide quantification efficiency. Interestingly, β-Catenin was identified in BMP2-stimulated heavy isotope-labeled cells, and further analysis confirmed that BMP2 increased β-Catenin mRNA and protein levels. The increment effects of BMP2 on the β-Catenin expression levels and its translocation to nucleus were diminished by blocking the PI3K signal pathway. In addition, BMP2-induced β-Catenin activity and ALP activity were blocked by PI3K inhibition. Thus, our quantitative proteomics analysis and further biochemical investigations showed that BMP2 modulates β-Catenin signaling via PI3K pathway and that this pathway plays roles in BMP2-induced osteoblast differentiation of hBMSCs.

Effect of IL-1α on the expression of cartilage matrix proteins in human chondrosarcoma cell line OUMS-27

We examined the effect of the inflammatory mediator interleukin-1α (IL-1α) on cell proliferation, alkaline phosphatase (ALPase) activity, and the expressions of cartilage matrix proteins, bone morphogenetic protein-2 (BMP-2), and BMP-2 receptors in human chondrosarcoma cell line OUMS-27 (chondrocytes). The cells were cultured with Dulbecco's modified Eagle's medium containing 15% fetal bovine serum with 0, 1, 10, or 100 units/ml of IL-1α for up to 14 days. The expressions of cartilage matrix proteins, BMP-2, and BMP-2 receptors were estimated by determining mRNA levels using semiquantitative or real-time PCR and/or by determining protein levels using Enzyme-linked immunosorbent assay. Cell proliferation was decreased after 5 days in culture with IL-1α. The ALPase activity was decreased significantly in the presence of IL-1α until day 10 of culture. The expression of type II collagen was significantly decreased after 7 days in culture with IL-1α. The expressions of aggrecan and link protein were significantly decreased through day 14 of culture with IL-1α. The expression of BMP-2 was increased at days 3, 7, and 14 of culture with IL-1α, while the expression of type II receptor for BMP-2 was significantly decreased in the samples. These results suggest that IL-1α suppresses the expression of cartilage matrix proteins through a suppression of the autocrine action of BMP-2, brought about by the decrease in BMP-2 receptor expression in chondrocytes.

Effect of carnosine on runt-related transcription factor-2/core binding factor alpha-1 and Sox9 expressions of human periodontal ligament cells.

Previous studies have reported that beta-alanyl-l-histidine (carnosine) enhanced the process of wound healing by stimulating effusion at the initial stage of inflammation, and also enhanced runt-related transcription factor-2/core binding factor alpha-1 (RUNX2/Cbfa1), Sox9, bone morphogenetic protein-2 (BMP-2) and BMP-7 expressions of human periodontal ligament cells. In order to clarify the relationship between RUNX2/Cbfa1 or Sox9 expressions and BMP-2 or BMP-7 expressions enhanced by carnosine, we determined the effect of carnosine on the expression of BMP receptors and activation of their downstream signaling molecules in human periodontal ligament cells. Human periodontal ligament cells were cultured with alpha-minimum essential medium containing 10% fetal bovine serum with or without 10(-4) or 10(-5) m carnosine for up to 10 days. The gene expression of BMP receptors, RUNX2/Cbfa1 and Sox9 was measured using semiquantitative reverse transcription-polymerase chain reaction. Phosphorylation of Smad1 was determined using a western blot analysis. Alkaline phosphatase activity increased in cultures with carnosine. Among the BMP receptors, expression of ActR-I and BMPR-II increased in cultures with carnosine, whereas expression of BMPR-IA, BMPR-IB, ActR-IIA and ActR-IIB was not affected. Culture with carnosine increased phosphorylation of Smad1, a signal-transducing molecule for BMP-2 and BMP-7. Noggin reduced carnosine-induced up-regulation of RUNX2/Cbfa1 and Sox9 mRNA, suggesting that BMPs were responsible for up-regulating RUNX2/Cbfa1 gene expression in human periodontal ligament cells. These results suggest that carnosine enhance RUNX2/Cbfa1 and Sox9 expression of human periodontal ligament cells via the autocrine action of BMP-2 or BMP-7 produced by the cells.

BMP-2 modulates the proliferation and differentiation of normal and cancerous gastric cells

Bone morphogenetic protein 2 (BMP-2), a member of the transforming growth factor β super-family, has been shown to act as an antiproliferative agent for a variety of cell lines by activating signaling cascades that cause cell cycle arrest. However, the biological effect and mechanism of action of BMP-2 on gastric cells remain unknown. In the present study, we showed that recombinant human BMP-2 dose-dependently inhibited the growth of OUMS37 rat gastric cells and MKN74 human gastric cancer cells. The antiproliferation seems to be due to cell cycle arrest in the G 1-phase, which was revealed by flow cytometric assays. BMP-2 increased the level of p21/WAF1/CIP1, suggesting that BMP-2-mediated inhibition of cell proliferation may be induced through p21/WAF1/CIP1. In addition, BMP-2 increased the expression of pepsinogen II, a differentiation marker of the stomach, in MKN74 cells. These results indicate that BMP-2 plays important roles in modulating the proliferation and differentiation of gastric epithelial cells.

Bone Morphogenetic Protein Receptor IB Signaling Mediates Apoptosis Independently of Differentiation in Osteoblastic Cells

Bone morphogenetic protein-2 (BMP-2) is an important regulator of osteoblast differentiation. However, the regulation of osteoblast apoptosis by BMP signaling remains poorly understood. Here we examined the role of type I BMP receptor (BMP-RI) in osteoblast apoptosis promoted by BMP-2. Despite undetectable BMP-RIB expression in OHS4 cells, BMP-2 or BMP-2 overexpression increased osteoblast differentiation similarly as in SaOS2 cells which express BMP-RIB, as shown by alkaline phosphatase and CBFA1/RUNX2 expression. In contrast to SaOS2 cells, however, BMP-2 or BMP-2 overexpression did not increase caspase-9 and caspases-3, -6, and -7 activity and DNA fragmentation in OHS4 cells. Consistently, BMP-2 increased protein kinase C (PKC) activity, and PKC inhibition suppressed BMP-2-induced caspase activity in SaOS2 but not in OHS4 cells that lack BMP-RIB. A dominant negative BMP-RIB inhibited BMP-2-induced caspase activity, whereas wild-type BMP-RIB promoted caspase activity induced by BMP-2 in SaOS2 and MC3T3-E1 cells. Wild-type BMP-RIB rescued the apoptotic response to BMP-2, and a constitutively active BMP-RIB restored the apoptotic signal in OHS4 cells, supporting an essential role for BMP-RIB in osteoblast apoptosis. We also assessed whether BMP-2-induced apoptosis occurred independently of osteoblast differentiation. General inhibition of caspases did not abolish BMP-2-induced alkaline phosphatase and CBFA1/RUNX2 expression in SaOS2 cells. Furthermore, broad caspases inhibition increased matrix mineralization but did not reverse the BMP-2 effect on mineralization in MC3T3-E1 cells. These results indicate that BMP-2-induced apoptosis was mediated by BMP-RIB in osteoblasts and occurred independently of BMP-2-induced osteoblast differentiation, which provides additional insights into the dual mechanism of BMP-2 action on osteoblast fate.

Brief Bone Morphogenetic Protein 2 Treatment of Glucocorticoid-inhibited MC3T3-E1 Osteoblasts Rescues Commitment-associated Cell Cycle and Mineralization without Alteration of Runx2

Glucocorticoids (GCs) inhibit bone formation in vivo. In MC3T3-E1 osteoblasts, chronic administration of 1 microm dexamethasone (DEX) starting at confluency results in >98% inhibition of bone morphogenetic protein 2 (BMP-2) expression and apatite mineral deposition. Here, it is shown that brief exposure to recombinant human BMP-2 (rhBMP-2), as short as 6 h, is sufficient to induce irreversible commitment to mineralization in DEX-treated cultures. RhBMP-2 dose dependently rescued mineralization but not collagen accumulation in DEX-inhibited cultures. The selective restoration of mineralization was evident in the higher mineral to matrix ratios of DEX/rhBMP-2 co-treated cultures compared with control. We tested the involvement of the runt-related transcription factor 2 (Runx2) in the DEX inhibition and rhBMP-2 rescue of mineralization. Surprisingly, DEX did not decrease Runx2 DNA binding activity, transactivation, or association with the endogenous osteocalcin gene promoter. Furthermore, the rhBMP-2 rescue did not involve Runx2 stimulation, suggesting an important role for factors other than Runx2 in BMP-2 action. Finally, we studied the differentiation-related cell cycle, which persists during commitment to mineralization in untreated cultures, but is inhibited along with mineralization in DEX-treated cultures. Although both rhBMP-2 alone and DEX alone were antimitogenic, rhBMP-2 stimulated this cell cycle in DEX-inhibited cultures. In conclusion, brief rhBMP-2 treatment restores mineralization in DEX-inhibited MC3T3-E1 osteoblasts via a mechanism different from Runx2 stimulation. This restoration may be functionally related to the accompanying rescue of the differentiation-related cell cycle. The efficacy of short term BMP-2 treatment supports the potential of short-lived BMP vectors for skeletal therapy in both traditional and gene therapeutic approaches.

Autocrine action of BMP2 regulates expression of GDNF-mRNA in sciatic Schwann cells

Schwann cell is a cell type that forms myelin sheath and provides trophic supports for neuronal cells by producing neurotrophic factors in both normal and traumatic situations. It was recently reported that after lesion of sciatic nerve, mRNA for glial cell line-derived neurotrophic factor (GDNF) is induced in nonneuronal cells in the nerve. However, the mechanism regulating GDNF-mRNA has remained largely unknown. In the present study, we searched for factors regulating the GDNF-mRNA expression in Schwann cells. First, we found that after transfer into explant culture as an in vitro lesion model, sciatic nerve segments began to express mRNA for bone morphogenetic protein-2 (BMP2) concomitantly with the induction of GDNF-mRNA. Treatment of the Schwann cells isolated from the sciatic nerve with combination of BMP2 and retinoic acid (RA) dramatically induced GDNF-mRNA, while BMP2 or RA alone had no effect. Furthermore, ionomycin, a calcium ionophore, which had even stronger activity on the induction of GDNF-mRNA also induced also BMP2-mRNA in cultured Schwann cells. Effects of inhibitors of intracellular signaling pathways such as protein kinase C inhibitor and MAPKK inhibitor suggested that the molecular mechanism of the induction of GDNF-mRNA is distinct from that of BMP2-mRNA. These results suggest that the Schwann cell-produced BMP2 plays an important role in the induction of GDNF after nerve injury in an autocrine fashion.

Parathyroid hormone-related peptide interacts with bone morphogenetic protein 2 to increase osteoblastogenesis and decrease adipogenesis in pluripotent C3H10T 1/2 mesenchymal cells.

We examined the effect of PTH-related peptide (PTHrP) on modulating adipogenesis and osteoblastogenesis in the pluripotent mesenchymal cell line C3H10T(1/2). These cells express the type 1 PTH/PTHrP receptor, thereby allowing PTHrP to inhibit bone morphogenetic protein 2 (BMP2) from enhancing gene expression of peroxisome proliferator-activated receptor gamma and the adipocyte-specific protein aP2 and from augmenting the accumulation of lipid. In the presence of BMP2, PTHrP or a protein kinase C (PKC) stimulator (phorbol ester) increased the expression of indexes of the osteoblast phenotype, including alkaline phosphatase, type I collagen, and osteocalcin, whereas a PKC inhibitor (chelerythrin chloride) inhibited PTHrP action. PTHrP and a phorbol ester increased gene expression of the BMP IA receptor, and both enhanced BMP2-dependent increases in promoter activity of the signaling molecule SMAD6. Overexpression of the BMP IA receptor facilitated the capacity of BMP2 to increase osteoblastogenesis in the absence of PTHrP and a dominant negative BMP IA receptor variant inhibited this effect of BMP2. These results demonstrate that PTHrP can direct osteoblastic, rather then adipogenic, commitment of mesenchymal cells, implicate PKC signaling in this activity, and show that PTHrP action involves enhanced gene expression of the BMP IA receptor, which facilitates BMP2 action in enhancing osteoblastogenesis in pluripotent mesenchymal cells.

BMP-2 regulates cardiomyocyte contractility in a phosphatidylinositol 3 kinase-dependent manner

Bone morphogenetic protein-2 (BMP-2) regulates development of heart during vertebrate embryogenesis. In vitro BMP-2 induces differentiation of precardiac cells into mature cardiomyocytes by inducing the expression of cardiac-specific genes. However, the role of BMP-2 and its signaling in other cardiac functions have not been studied. We examined the action of phosphatidylinositol (PI) 3 kinase in isolated adult rat cardiomyocytes. Incubation of rat ventricular cardiomyocytes with BMP-2 increased the PI 3 kinase activity. Ly294002, a pharmacological inhibitor of PI 3 kinase, blocked BMP-2-induced PI 3 kinase activity completely. To investigate the contractility of isolated cardiomyocytes, fractional shortening was examined. BMP-2 significantly increased the percent fractional shortening of the cardiomyocytes. Inhibition of PI 3 kinase activity completely abolished this action of BMP-2. These data indicate that PI 3 kinase regulates BMP-2-induced myocyte contractility. To further confirm this observation, we used adenovirus-mediated gene transfer to express a constitutively active myristoylated catalytic subunit of PI 3 kinase in rat cardiomyocytes. Infection of cardiomyocytes with the adenovirus vector increased the expression of constitutively active PI 3 kinase within 24 h. Expression of constitutively active PI 3 kinase significantly increased cardiomyocyte contractility. Together, these data show for the first time that the growth and differentiation factor, BMP-2, stimulates cardiomyocyte contractility. Also we provide the first evidence that BMP-2-induced PI 3 kinase activity regulates this cardiomyocyte function.

Identification of a BMP-responsive element in Id1, the gene for inhibition of myogenesis.

Bone morphogenetic protein-2 (BMP-2) stimulates osteoblast differentiation, but inhibits myogenic differentiation in C2C12 myoblasts. BMP-2 induces transcription of Id1, an inhibitor for myogenesis, within 1 h in the cells. To examine the molecular mechanism of the action of BMP-2, we analysed a BMP-2-responsive element (BRE) in the 5' flanking region of the human Id1 gene. A GC-rich region between -985 bp and -957 bp of the human Id1 gene was identified as a BRE. The BRE containing promoter activity was stimulated by BMP-2 or by constitutively active BMP receptors (BMPR-IA and BMPR-IB). The stimulation was blocked by co-transfecting with dominant negative BMPR-IA or Smad7. A unique DNA-protein complex was induced in response to BMP-2 on the BRE. The complex induced by BMP-2 contained Smad1 and Smad4, possibly as a complex of both Smads. BMP-2 failed to stimulate the expression of Id1 mRNA in Smad4-deficient cells. Over-expression of Smad4, but not Smad1, stimulated the Id1 reporter activity and the expression of endogenous Id1 mRNA in Smad4-deficient cells. Signalling of BMP-2 to stimulate the expression of Id1 would be transduced by BMPR-IA and mediated by Smad1 and Smad4, both of which form a complex on the 29 bp GC-rich element.

Effect of prostaglandin E2 on recombinant human bone morphogenetic protein-2-stimulated osteoblastic differentiation in human periodontal ligament cells.

Recombinant human (rh) bone morphogenetic protein-2 (BMP-2) stimulates osteoblastic differentiation in cells isolated from human periodontal ligament (HPLC), and this action of rhBMP-2 may be modulated by prostaglandins (PGs), which are local regulatory factors in the bone metabolism. In the present study, we investigated the effect of prostaglandin E2 (PGE2) on rhBMP-2-stimulated osteoblastic differentiation in cultured HPLC. rhBMP-2 (500 ng/ml)-stimulated alkaline phosphatase (ALPase) activity was enhanced by simultaneous treatment with low concentrations (10(-10)-10(-8) M) of PGE2, whereas a high concentration (10(-6) M) of PGE2 suppressed it. rhBMP-2 did not induce cyclo-oxygenase-2 (COX-2) mRNA expression or subsequent PGE2 production, whereas it remarkably suppressed rhIL-1 beta-induced COX-2 mRNA expression and PGE2 production. The rhBMP-2 action on osteoblastic differentiation in HPLC was also enhanced by co-treatment with 0.25 to 25 ng/ml of rh interleukin-1 beta (IL-1 beta). The ALPase activity stimulated by simultaneous treatment with rhBMP-2 and rhIL-1 beta was partially inhibited by addition of 10(-6) M of indomethacin, which completely inhibited rhIL-1 beta-induced PGE2 production. These results reveal that PGE2 at different concentrations exerts a biphasic effect on BMP-2-stimulated osteoblastic differentiation in HPLC, BMP-2 inhibits IL-1 beta-induced PGE2 production through suppressing COX-2 expression, and the BMP-2-stimulated osteoblastic differentiation may be enhanced by the endogenous PGE2 induced by BMP-2 and IL-1 beta. These suggest that BMP-2 action on osteoblastic differentiation in HPLC may be modulated by PGE2 in autocrine and paracrine fashions.

Bone morphogenetic protein 2 transiently enhances expression of a gene, Id (inhibitor of differentiation), encoding a helix-loop-helix molecule in osteoblast-like cells.

Bone morphogenetic protein 2 (BMP-2) is a potent inducer of differentiation of osteoblasts both in vivo and in vitro. We examined the action of BMP-2 on expression of a helix-loop-helix-type transcription factor, Id (inhibitor of differentiation), in osteoblast-like cells, as well as in osteoblast-enriched cells and possible precursor cells. To our surprise, BMP-2 enhanced Id gene expression in the cell types of osteoblastic lineage we examined. The maximal BMP-2 enhancement was observed within 24 hr in early proliferating cultures and the enhancement lasted up to 96 hr. The BMP-2 effect was not blocked by actinomycin D, while it was blocked by cycloheximide, suggesting that BMP-2 regulates Id gene expression at least in part via posttranscriptional events, which require protein synthesis. Other experiments indicated that BMP-2 did not further enhance Id mRNA levels promoted by dexamethasone, while BMP-2 did not resume the Id mRNA levels suppressed by 1,25-dihydroxyvitamin D3. Similar BMP-2 enhancement of Id message expression was also observed in osteoblast-enriched fetal rat calvaria cells as well as C3H10T1/2 cells. These results indicate that BMP-2 enhances expression of Id in early cultures of osteoblastic cells and suggest that enhancement of Id expression may somehow be involved in the promotion of differentiation by this cytokine in these osteoblastic cells and in their precursor cells.