Changes in bone morphogenetic protein receptor-IB localisation regulate osteogenic responses of human bone cells to bone morphogenetic protein-2

Cell responses to bone morphogenetic proteins (BMP) depend on the expression and surface localisation of transmembrane receptors BMPR-IA, -IB and -II. The present study shows that all three antigens are readily detected in human bone cells. However, only BMPR-II was found primarily at the plasma membrane, whereas BMPR-IA was expressed equally in the cytoplasm and at the cell surface. Notably, BMPR-IB was mainly intracellular, where it was associated with a number of cytoplasmic structures and possibly the nucleus. Treatment with transforming growth factor β1 (TGF-β1) caused rapid translocation of BMPR-IB to the cell surface, mediated via the p38 mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) pathways. The TGF-β1-induced increase in surface BMPR-IB resulted in significantly elevated BMP-2 binding and Smad1/5/8 phosphorylation, although the receptor was subsequently internalised and the functional response to BMP-2 consequently down-regulated. The results show, for the first time, that BMPR-IB is localised primarily in intracellular compartments in bone cells and that TGF-β1 induces rapid surface translocation from the cytoplasm to the cell surface, resulting in increased sensitivity of the cells to BMP-2.     

Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2.

Matrix GLA protein (MGP) has been identified as a calcification inhibitor in cartilage and vasculature. Part of this effect may be attributed to its influence on osteoinductive activity of bone morphogenetic protein-2 (BMP-2). To detect binding between MGP and BMP-2, we performed immunoprecipitation using MGP and BMP-2 tagged with FLAG and c-Myc. The results showed co-precipitation of BMP-2 with MGP. To quantify the effect of MGP on BMP-2 activity, we assayed for alkaline phosphatase activity and showed a dose-dependent effect. Low levels of MGP relative to BMP-2 (<1-fold excess) resulted in mild enhancement of osteoinduction, whereas intermediate levels (1-15-fold excess) resulted in strong inhibition. High levels of MGP (>15-fold excess), however, resulted in pronounced enhancement of the osteoinductive effect of BMP-2. Cross-linking studies showed that inhibitory levels of MGP abolished BMP-2 receptor binding. Immunoblotting showed a corresponding decrease in activation of Smad1, part of the BMP signaling system. Enhancing levels of MGP resulted in increased Smad1 activation. To determine the cellular localization of BMP-2 in the presence of MGP, binding assays were performed on whole cells and cell-synthesized matrix. Inhibitory levels of MGP yielded increased matrix binding of BMP-2, suggesting that MGP inhibits BMP-2 in part via matrix association. These results suggest that MGP is a BMP-2 regulatory protein.     

Post-translational modification of bone morphogenetic protein-1 is required for secretion and stability of the protein

Bone morphogenetic protein (BMP)-1 is a glycosylated metalloproteinase that is fundamental to the synthesis of a normal extracellular matrix because it cleaves type I procollagen, as well as other precursor proteins. Sequence analysis suggests that BMP-1 has six potential N-linked glycosylation sites (i.e. NXS/T) namely: Asn(91) (prodomain), Asn(142) (metalloproteinase domain), Asn(332) and Asn(363) (CUB1 domain), Asn(599) (CUB3 domain), and Asn(726) in the C-terminal-specific domain. In this study we showed that all these sites are N-glycosylated with complex-type oligosaccharides containing sialic acid, except Asn(726) presumably because proline occurs immediately C-terminal of threonine in the consensus sequence. Recombinant BMP-1 molecules lacking all glycosylation sites or the three CUB-specific sites were not secreted. BMP-1 lacking CUB glycosylation was translocated to the proteasome for degradation. BMP-1 molecules lacking individual glycosylation sites were efficiently secreted and exhibited full procollagen C-proteinase activity, but N332Q and N599Q exhibited a slower rate of cleavage. BMP-1 molecules lacking any one of the CUB-specific glycosylation sites were sensitive to thermal denaturation. The study showed that the glycosylation sites in the CUB domains of BMP-1 are important for secretion and stability of the molecule.     

Regulation of apoAI processing by procollagen C-proteinase enhancer-2 and bone morphogenetic protein-1

Given the increased prevalence of cardiovascular disease in the world, the search for genetic variations controlling the levels of risk factors associated with the development of the disease continues. Multiple genetic association studies suggest the involvement of procollagen C-proteinase enhancer-2 (PCPE2) in modulating HDL-C levels. Therefore biochemical and mechanistic studies were undertaken to determine whether there might be a basis for a role of PCPE2 in HDL biogenesis. Our studies indicate that PCPE2 accelerates the proteolytic processing of pro-apolipoprotein (apo) AI by enhancing the cleavage of the hexapeptide extension present at the N terminus of apoAI. Surface Plasmon Resonance and immunoprecipitation studies indicate that PCPE2 interacts with BMP-1 and pro-apoAI to form a ternary pro-apoAI/BMP-1/PCPE2 complex. The most favorable interaction among these proteins begins with the association of BMP-1 to pro-apoAI followed by the binding of PCPE2 which further stabilizes the complex. PCPE2 resides, along with apoAI, on the HDL fraction of lipoproteins in human plasma supporting a relationship between HDL and PCPE2. Taken together, the findings from our studies identify a new player in the regulation of apoAI post-translational processing and open a new avenue to the study of mechanisms involved in the regulation of apoAI synthesis, HDL levels, and potentially, cardiovascular disease.     

Lysyl oxidase-like protein from bovine aorta. Isolation and maturation to an active form by bone morphogenetic protein-1.

Recently several cDNAs have been described encoding lysyl oxidase-like proteins. Their deduced amino acid sequences are characterized by a strong similarity in the C-terminal region, corresponding to the lysyl oxidase family catalytic domain, and by marked differences in the N-terminal regions. Different biological functions have been described for lysyl oxidases in addition to their traditionally assumed cross-linking role. To answer the question of whether these different functions are carried out by different lysyl oxidases, purified and active forms of these enzymes are required. At present only the classical form of lysyl oxidase has been purified and characterized. The purpose of this study was to isolate and characterize the lysyl oxidase-like protein. In view of the strong sequence homology with the C-terminal domain of other lysyl oxidases, we chose to purify the protein from bovine aorta using antibodies specific to the N-terminal domain of the proenzyme. We have isolated a 56-kDa protein identified by amino acid sequencing as the bovine lysyl oxidase-like precursor, which is cleaved at the Arg-Arg-Arg sequence at positions 89-91 by a furin-like activity, as revealed after deblocking of the N-terminal residue. The immunopurified protein was largely inactive, but further processing in vitro by bone morphogenetic protein-1 led to an enzyme that was active on elastin and collagen substrates.     

Bone morphogenetic protein-1 processes insulin-like growth factor-binding protein 3

The bone morphogenetic protein-1 (BMP1)-like metalloproteinases play key roles in extracellular matrix formation, by converting precursors into mature functional proteins involved in forming the extracellular matrix. The BMP1-like proteinases also play roles in activating growth factors, such as BMP2/4, myostatin, growth differentiation factor 11, and transforming growth factor β1, by cleaving extracellular antagonists. The extracellular insulin-like growth factor-binding proteins (IGFBPs) are involved in regulating the effects of insulin-like growth factors (IGFs) on growth, development, and metabolism. Of the six IGFBPs, IGFBP3 has the greatest interaction with the large pool of circulating IGFs. It is also produced locally in tissues and is itself regulated by proteolytic processing. Here, we show that BMP1 cleaves human and mouse IGFBP3 at a single conserved site, resulting in markedly reduced ability of cleaved IGFBP3 to bind IGF-I or to block IGF-I-induced cell signaling. In contrast, such cleavage is shown to result in enhanced IGF-I-independent ability of cleaved IGFBP3 to block FGF-induced proliferation and to induce Smad phosphorylation. Consistent with in vivo roles for such cleavage, it is shown that, whereas wild type mouse embryo fibroblasts (MEFs) produce cleaved IGFBP3, MEFs doubly null for the Bmp1 gene and for the Tll1 gene, which encodes the related metalloproteinase mammalian Tolloid-like 1 (mTLL1), produce only unprocessed IGFBP3, thus demonstrating endogenous BMP1-related proteinases to be responsible for IGFBP3-processing activity in MEFs. Similarly, in zebrafish embryos, overexpression of Bmp1a is shown to reverse an Igfbp3-induced phenotype, consistent with the ability of BMP1-like proteinases to cleave IGFBP3 in vivo.     

Regulation of bone morphogenetic protein-4 by matrix GLA protein in vascular endothelial cells involves activin-like kinase receptor 1

Matrix GLA protein (MGP) has previously been shown to enhance expression of vascular endothelial growth factor (VEGF) through the activin-like kinase receptor 1 (ALK1) in bovine aortic endothelial cells. MGP has also been identified as an inhibitor of bone morphogenetic protein-2 (BMP-2). This study showed that the effect of MGP on ALK1 signaling and VEGF expression in bovine aortic endothelial cells was dose-dependent, that a progressive increase of MGP levels ceased to be stimulatory and instead turned inhibitory. We identified a new regulatory pathway involving BMP that may explain this response. BMP-2 and BMP-4 induced expression of ALK1 in a dose-dependent fashion as determined by real-time PCR and immunoblotting. Activation of ALK1 signaling induced expression of MGP in addition to that of VEGF, allowing for negative feedback regulation of BMP by MGP. MGP inhibited BMP-4 activity similarly to that of BMP-2 and interacted with BMP-4 on a protein level as determined by co-immunoprecipitation. The dose-dependent effect on ALK1 expression and the stimulation of MGP and VEGF expression were dependent on signaling by transforming growth factor-beta (TGF-beta) and ALK1. Inhibition of TGF-beta by neutralizing antibodies abolished the inhibitory effect of high BMP-4 levels on ALK1 expression and the induction of MGP and VEGF. Depletion of ALK1 by small interfering RNA abolished the induction of MGP and VEGF. MGP promoter activity was also stimulated by BMP-4 in a TGF-beta-dependent fashion. The results suggest that the effects of BMP on endothelial cells occur in part through induction of ALK1, an effect that may be limited by ALK1-induced MGP.     

Bone morphogenetic protein 1 prodomain specifically binds and regulates signaling by bone morphogenetic proteins 2 and 4

Highly purified fractions of bone extracts capable of inducing ectopic bone formation have been reported to contain peptides corresponding to the mature active regions of the TGF-beta-like bone morphogenetic proteins (BMPs) 2-7, and to the prodomain region of the metalloproteinase BMP1. Co-purification of BMPs 2-7 with BMP1 prodomain sequences through the multiple biochemical steps used in these previous reports has suggested the possibility of interactions between the BMP1 prodomain and BMPs 2-7. Here we demonstrate that the BMP1 prodomain binds BMPs 2 and 4 with high specificity and with a KD of approximately 11 nM, in the physiological range. It is further demonstrated that the BMP1 prodomain is capable of modulating signaling by BMPs 2 and 4 in vitro and in vivo, and that endogenous BMP1 prodomain-BMP4 complexes exist in cell culture media and in tissues.     

Opposing effects by glucocorticoid and bone morphogenetic protein-2 in fetal rat bone cell cultures

Glucocorticoid in excess produces bone loss in vivo. Consistent with this, it reduces the stimulatory effect of transforming growth factor β (TGF-β) on collagen synthesis in osteoblast-enriched cultures in vitro, where it also suppresses TGF-β binding to its type I receptors. Analogous studies with bone morphogenetic protein-2 (BMP-2) show directly opposite results. These findings prompted us to assess the effect of glucocorticoid on BMP-2 activity in cultured bone cells, and whether either agent had a dominant influence on TGF-β binding or function. BMP-2 activity was retained in part in osteoblast-enriched cultures pre-treated or co-treated with cortisol, and was fully evident when glucocorticoid exposure followed BMP-2 treatment. In addition, BMP-2 suppressed the effects of cortisol on TGF-β activity, on TGF-β binding, and on gene promoter activity directed by a glucocorticoid sensitive transfection construct. While BMP-2 also alters the function of less-differentiated bone cells, it only minimally prevented cortisol activity in these cultures. Our studies indicate that BMP-2 can oppose certain effects by cortisol on differentiated osteoblasts, and may reveal useful ways to diminish glucocorticoid-dependent bone wasting. J. Cell. Biochem. 67:528–540, 1997. © 1997 Wiley-Liss, Inc.     

Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer

Introduction  

The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures.     

Involvement of p70 S6 kinase in bone morphogenetic protein signaling: vascular endothelial growth factor synthesis by bone morphogenetic protein-4 in osteoblasts

In the present study, we investigated the effect of bone morphogenetic protein (BMP)-4 on the synthesis of vascular endothelial growth factor (VEGF) in osteoblast-like MC3T3-E1 cells. BMP-4 significantly stimulated VEGF synthesis time-dependently up to 48 h. The stimulatory effect was dose-dependent in the range between 1 and 100 ng/ml. BMP-4 time-dependently phosphorylated p70 S6 kinase. Rapamycin, an inhibitor of p70 S6 kinase, suppressed the BMP-4-stimulated VEGF synthesis as well as the phosphorylation of p70 S6 kinase. The VEGF synthesis by BMP-4 was suppressed by wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase. Both wortmannin and LY294002 inhibited the BMP-4-stimulated phosphorylation of p70 S6 kinase. BMP-4 did not affect the phosphorylation of Akt/protein kinase B. Taken together, our results strongly suggest that p70 S6 kinase takes part in BMP-4-stimulated VEGF synthesis as a positive regulator in osteoblasts and that phosphatidylinositol 3-kinase acts at a point upstream from p70 S6 kinase.     

大肠杆菌表达的重组人骨形成蛋白-7的复性

带有pBV221-hBMP-7的E．coli表达得到的rhBMP-7以不溶的包涵体形式存在,用高浓度的变性利溶解后,经过DEAE-FF纯化,得到高纯度的目的蛋白,达95％以上。分别用尿素浓度梯度降低法、添加促复性剂及人工分子伴侣法对蛋白质进行复性,并通过不同方法对复性结果进行比较。Western blot中辉度扫描结果显示,GSH／GSSG法样品二聚体／单体比例为79．5／20．5,尿素浓度梯度降低法二聚体／单体比例为73．6／26．4,表明GSH／GSSG法复性样品溶液上清中含较高比例的蛋白质二聚体。根据不同复性样品对NIH3T3细胞ALP活性影响大小的比较结果,氧化还原剂最有助于二聚体的形成,蛋白质活性最高。     

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.     

Matrix GLA protein modulates differentiation induced by bone morphogenetic protein-2 in C3H10T1/2 cells

Matrix GLA protein (MGP) is ubiquitously expressed with high accumulation in bone and cartilage, where it was found to associate with bone morphogenetic proteins (BMP) during protein purification. To test whether MGP affects BMP-induced differentiation, three sets of experiments were performed. First, pluripotent C3H10T1/2 cells transfected with human MPG (hMGP) or antisense to hMGP (AS-hMGP) were treated with BMP-2. In cells overexpressing hMGP, osteogenic and chondrogenic differentiation was inhibited indicating decreased BMP-2 activity. Conversely, in cells overexpressing AS-hMGP, BMP-2 activity was enhanced. Second, cells were prepared from homozygous and heterozygous MPG-deficient mice aortas. When treated with BMP-2, these cells underwent chondrogenic and osteogenic differentiation, respectively, whereas controls did not. Third, FLAG-tagged hMGP with the same biological effect as native hMGP inhibited BMP-induced differentiation, when exogenously added to culture media. Together, these results suggest that MGP modulates BMP activity. To test whether hMGP fragments would retain the effect of full-length hMGP, three subdomains were overexpressed in C3H10T1/2 cells. In cells expressing the mid-region, alone (amino acids (aa) 35-54) or in combination with the N terminus (aa 1-54) but not the C terminus (aa 35-84), osteogenic differentiation was enhanced and occurred even without added BMP-2. Thus, two subdomains had the opposite effect of full-length hMGP, possibly due to different expression levels or domain characteristics.     

Bone morphogenetic protein-1 processes probiglycan

Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays important roles in regulating the deposition of fibrous extracellular matrix in vertebrates, including provision of the procollagen C-proteinase activity that processes the major fibrillar collagens I-III. Biglycan, a small leucine-rich proteoglycan, is a nonfibrillar extracellular matrix component with functions that include the positive regulation of bone formation. Biglycan is synthesized as a precursor with an NH(2)-terminal propeptide that is cleaved to yield the mature form found in vertebrate tissues. Here, we show that BMP-1 cleaves probiglycan at a single site, removing the propeptide and producing a biglycan molecule with an NH(2) terminus identical to that of the mature form found in tissues. BMP-1-related proteases mammalian Tolloid and mammalian Tolloid-like 1 (mTLL-1) are shown to have low but detectable levels of probiglycan-cleaving activity. Comparison shows that wild type mouse embryo fibroblasts (MEFs) produce only fully processed biglycan, whereas MEFs derived from embryos homozygous null for the Bmp1 gene, which encodes both BMP-1 and mammalian Tolloid, produce predominantly unprocessed probiglycan, and MEFs homozygous null for both the Bmp1 gene and the mTLL-1 gene Tll1 produce only unprocessed probiglycan. Thus, all detectable probiglycan-processing activity in MEFs is accounted for by the products of these two genes.