BMP-2/4 and BMP-6/7 differentially utilize cell surface receptors to induce osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily of growth factors and are used clinically to induce new bone formation. The purpose of this study was to evaluate receptor utilization by BMP-2, BMP-4, BMP-6, and BMP-7 in primary human mesenchymal stem cells (hMSC), a physiologically relevant cell type that probably mediates the in vivo effects of BMPs. RNA interference-mediated gene knockdown revealed that osteoinductive BMP activities in hMSC are elicited through the type I receptors ACVR1A and BMPR1A and the type II receptors ACVR2A and BMPR2. BMPR1B and ACVR2B were expressed at low levels and were not found to play a significant role in signaling by any of the BMPs evaluated in this study. Type II receptor utilization differed significantly between BMP-2/4 and BMP-6/7. A greater reliance on BMPR2 was observed for BMP-2/4 relative to BMP-6/7, whereas ACVR2A was more critical to signaling by BMP-6/7 than BMP-2/4. Significant differences were also observed for the type I receptors. Although BMP-2/4 used predominantly BMPR1A for signaling, ACVR1A was the preferred type I receptor for BMP-6/7. Signaling by both BMP-2/4 and BMP-6/7 was mediated by homodimers of ACVR1A or BMPR1A. A portion of BMP-2/4 signaling also required concurrent BMPR1A and ACVR1A expression, suggesting that BMP-2/4 signal in part through ACVR1A/BMPR1A heterodimers. The capacity of ACVR1A and BMPR1A to form homodimers and heterodimers was confirmed by bioluminescence resonance energy transfer analyses. These results suggest different mechanisms for BMP-2/4- and BMP-6/7-induced osteoblastic differentiation in primary hMSC.     

Effects of BMP-2 compound with fibrin on osteoporotic vertebral fracture healing in rats

Objectives:To investigate the effects of bone morphogenetic protein-2 (BMP-2) compound with fibrin on osteoporotic vertebral fracture healing in rats.Methods:For the present study 160 Specific-Pathogen Free 32-week-old female Sprague-Dawley rats were used. 120 rats were randomly divided in three groups (experimental, model and sham operation group- n=40 per group) and were ovariectomized to establish the osteoporosis model. 40 rats served as a control group without treatment. The expression of BMP-2 in the fracture zone at the 4^th, 6^th, 8^th, and 12^th weeks was detected by qRT-PCR. The expression of BALP and CTX-I in serum at the 12^th week was detected by Elisa.Results:At week 8, the morphology of the sham operation group was the same and the fracture healing occurred more slowly than in the other groups. At week 12, the expression of BMP-2 in the model group was significantly higher than that in the other three groups (p<0.05). At week 12, the maximum load, maximum strain, and elastic modulus of model group were significantly lower than those of the other three groups.Conclusions:BMP-2 compound with fibrin can enhance the timing and quality of bone fracture healing in rats.     

BMP-2/6 Heterodimer Is More Effective than BMP-2 or BMP-6 Homodimers as Inductor of Differentiation of Human Embryonic Stem Cells

Background

Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types, and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells.

Methodology/Principal Findings

We have analyzed the ability for inducing differentiation of the heterodimer BMP-2/BMP-6 (BMP-2/6) compared to the homodimers BMP-2 or BMP-6, using human embryonic stem (hES) cells H9 as model system. When incubated in a medium with high concentration of basic fibroblastic growth factor (FGF2), 100 ng/ml of human recombinant BMPs induced morphological changes and differentiation of hES cells in 24 to 48 hours. After 5 days, expression of differentiation markers was induced and quantified by quantitative PCR (qPCR) and flow cytometry. BMP-2/6 exhibited stronger activity for the induction of the expression of trophectodermal (CDX2) and endodermal (SOX17, GATA4, AFP) markers than BMP-2 or BMP-6 homodimers. BMP-2/6 also induced the expression of BMPR2 gene more effectively than BMP-2 or BMP-6 when used at the same concentration and time. Moreover, the percentage of cells expressing the surface endodermal marker CXCR4 was also increased for the heterodimer when compared to both homodimers. BMP-2/6 was a more potent activator of Smad-dependent (SMAD1/5) and Smad-independent signaling (mitogen-activated protein kinases ERK and p38) than BMP-2 and BMP-6, and the activation of these pathways might play a role in its increased potency for inducing hES cell differentiation.

Conclusions/Significance

Therefore, we conclude that BMP-2/6 is more potent than BMP-2 or BMP-6 for inducing differentiation of hES cells, and it can be used as a more powerful substitute of these BMPs in in vitro differentiation guidance.     

Osteogenesis versus chondrogenesis by BMP-2 and BMP-7 in adipose stem cells

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain chondrogenesis and osteogenesis. We hypothesize that BMP-2 induces an osteogenic, and BMP-7 a chondrogenic phenotype in adipose tissue-derived mesenchymal stem cells (AT-MSCs). We compared the effects of a short 15min BMP-2 or BMP-7 (10ng/ml) treatment on osteogenic and chondrogenic differentiation of AT-MSCs. Gene expression was studied 4 and 14 days after BMP-treatment. At day 4 BMP-2, but not BMP-7, stimulated runx-2 and osteopontin gene expression, and at day 14 BMP-7 down-regulated expression of these genes. At day 4 BMP-2 and BMP-7 stimulated biglycan gene expression, which was down-regulated by BMP-7 at day 14. BMP-7 stimulated aggrecan gene expression at day 14. Our data indicate that BMP-2 treatment for 15min induces osteogenic differentiation, whereas BMP-7 stimulates a chondrogenic phenotype of AT-MSCs. Therefore, AT-MSCs triggered for only 15min with BMP-2 or BMP-7 provide a feasible tool for bone and cartilage tissue engineering.     

The prodomain of BMP-7 targets the BMP-7 complex to the extracellular matrix

Biochemical and biophysical methods are used to show that BMP-7 is secreted as a stable complex consisting of the processed growth factor dimer noncovalently associated with its two prodomain propeptide chains and that the BMP-7 complex is structurally similar to the small transforming growth factor beta (TGFbeta) complex. Because the prodomain of TGFbeta interacts with latent TGFbeta-binding proteins, a family of molecules homologous to the fibrillins, the prodomain of BMP-7 was tested for binding to fibrillin-1 or to LTBP-1. The BMP-7 prodomain and BMP-7 complex, but not the separated growth factor dimer, interact with N-terminal regions of fibrillin-1. This interaction may target the BMP-7 complex to fibrillin microfibrils in the extracellular matrix. Immunolocalization of BMP-7 in tissues like the kidney capsule and skin reveals co-localization with fibrillin. However, BMP-7 immunolocalization in other tissues known to be active sites for BMP-7 signaling is not apparent, suggesting that immunolocalization of BMP-7 in certain tissues represents specific extracellular storage sites. These studies suggest that the prodomains of TGFbeta-like growth factors are important for positioning and concentrating growth factors in the extracellular matrix. In addition, they raise the possibility that prodomains of other TGFbeta-like growth factors interact with fibrillins and/or LTBPs and are also targeted to the extracellular matrix.     

Heparin inhibits BMP-2 osteogenic bioactivity by binding to both BMP-2 and BMP receptor

Heparin demonstrates several kinds of biological activities by binding to various extracellular molecules and plays pivotal roles in bone metabolism. However, the role of heparin in the biological activity of bone morphogenetic protein (BMP) remains unclear. In the present study, we examined whether heparin has the effects on osteoblast differentiation induced by BMP-2 in vitro and also elucidated the precise mechanism by which heparin regulates bone metabolism induced by this molecule. Our results showed that heparin inhibited alkaline phosphatase (ALP) activity and mineralization in osteoblastic cells cultured with BMP-2. Heparin was found to suppress the mRNA expressions of osterix, Runx2, ALP and osteocalcin, as well as phosphorylation of Smad1/5/8 and p38 MAPK. Further, heparin bound to both BMP-2 and BMP receptor (BMPR). These results suggest that heparin suppresses BMP-2-BMPR binding, and inhibits BMP-2 osteogenic activity in vitro.     

BMP-3 and BMP-6 structures illuminate the nature of binding specificity with receptors

Bone morphogenetic proteins (BMPs) are extracellular messenger ligands involved in controlling a wide array of developmental and intercellular signaling processes. To initiate their specific intracellular signaling pathways, the ligands recognize and bind two structurally related serine/threonine kinase receptors, termed type I and type II, on the cell surface. Here, we present the crystal structures of BMP-3 and BMP-6, of which BMP-3 has remained poorly understood with respect to its receptor identity, affinity, and specificity. Using surface plasmon resonance (BIAcore) we show that BMP-3 binds Activin Receptor type II (ActRII) with Kd approximately 1.8 microM but ActRIIb with 30-fold higher affinity at Kd approximately 53 nM. This low affinity for ActRII may involve Ser-28 and Asp-33 of BMP-3, which are found only in BMP-3's type II receptor-binding interfaces. Point mutations of either residue to alanine results in up to 20-fold higher affinity to either receptor. We further demonstrate by Smad-based whole cell luciferase assays that the increased affinity of BMP-3S28A to ActRII enables the ligand's signaling ability to a level comparable to that of BMP-6. Focusing on BMP-3's preference for ActRIIb, we find that Lys-76 of ActRII and the structurally equivalent Glu-76 of ActRIIb are distinct between the two receptors. We demonstrate that ActRIIbE76K and ActRII bind BMP-3 with similar affinity, indicating BMP-3 receptor specificity is controlled by the interaction of Lys-30 of BMP-3 with Glu-76 of ActRIIb. These studies illustrate how a single amino acid can regulate the specificity of ligand-receptor binding and potentially alter biological signaling and function in vivo.     

Atherogenic phospholipids attenuate osteogenic signaling by BMP-2 and parathyroid hormone in osteoblasts

Cardiovascular disease, such as atherosclerosis, has been associated with reduced bone mineral density and fracture risk. A major etiologic factor in atherogenesis is believed to be oxidized phospholipids. We previously found that these phospholipids inhibit spontaneous osteogenic differentiation of marrow stromal cells, suggesting that they may account for the clinical link between atherosclerosis and osteoporosis. Currently, anabolic agents that promote bone formation are increasingly used as a new treatment for osteoporosis. It is not known, however, whether atherogenic phospholipids alter the effects of bone anabolic agents, such as bone morphogenetic protein (BMP)-2 and parathyroid hormone (PTH). Therefore we investigated the effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) on osteogenic signaling induced by BMP-2 and PTH in MC3T3-E1 cells. Results showed that ox-PAPC attenuated BMP-2 induction of osteogenic markers alkaline phosphatase and osteocalcin. Ox-PAPC also inhibited both spontaneous and BMP-induced expression of PTH receptor. Consistently, pretreatment of cells with ox-PAPC inhibited PTH-induced cAMP production and expression of immediate early genes Nurr1 and IL-6. Results from immunofluorescence and Western blot analyses showed that inhibitory effects of ox-PAPC on BMP-2 signaling were associated with inhibition of SMAD 1/5/8 but not p38-MAPK activation. These effects appear to be due to ox-PAPC activation of the ERK pathway, as the ERK inhibitor PD98059 reversed ox-PAPC inhibitory effects on BMP-2-induced alkaline phosphatase activity, osteocalcin expression, and SMAD activation. These results suggest that atherogenic lipids inhibit osteogenic signaling induced by BMP-2 and PTH, raising the possibility that hyperlipidemia and atherogenic phospholipids may interfere with anabolic therapy.     

BMP-3 is a novel inhibitor of both activin and BMP-4 signaling in Xenopus embryos

In Xenopus, the biological effects of BMP-3 oppose those of ventralizing BMPs, but the mechanism for this antagonism remains unclear. Here, we demonstrate that BMP-3 is a dorso-anteriorizing factor in Xenopus embryos that interferes with both activin and BMP signaling. BMP-3 acts by binding to ActRIIB, the common type II receptor for these proteins. Once BMP-3 binds to ActRIIB, it cannot be competed off by excess ligand making a receptor complex that is unable to activate R-Smads and transduce signal. Consistent with a model where BMP-3 interferes with activin and BMPs through a shared receptor, we show that overexpression of BMP-3 can only be rescued by co-injection of xActRIIB. Our results identify BMP-3 as a novel antagonist of both activin and BMPs and uncover how some of the diverse developmental processes that are regulated by both activin and BMP signaling can be modulated during embryogenesis.     

BMP-2 induction and TGF-beta 1 modulation of rat periosteal cell chondrogenesis

Periosteum contains osteochondral progenitor cells that can differentiate into osteoblasts and chondrocytes during normal bone growth and fracture healing. TGF-beta 1 and BMP-2 have been implicated in the regulation of the chondrogenic differentiation of these cells, but their roles are not fully defined. This study was undertaken to investigate the chondrogenic effects of TGF-beta 1 and BMP-2 on rat periosteum-derived cells during in vitro chondrogenesis in a three-dimensional aggregate culture. RT-PCR analyses for gene expression of cartilage-specific matrix proteins revealed that treatment with BMP-2 alone and combined treatment with TGF-beta 1 and BMP-2 induced time-dependent mRNA expression of aggrecan core protein and type II collagen. At later times in culture, the aggregates treated with BMP-2 exhibited expression of type X collagen and osteocalcin mRNA, which are markers of chondrocyte hypertrophy. Aggregates incubated with both TGF-beta 1 and BMP-2 showed no such expression. Treatment with TGF-beta 1 alone did not lead to the expression of type II or X collagen mRNA, indicating that this factor itself did not independently induce chondrogenesis in rat periosteal cells. These data were consistent with histological and immunohistochemical results. After 14 days in culture, BMP-2-treated aggregates consisted of many hypertrophic chondrocytes within a metachromatic matrix, which was immunoreactive with anti-type II and type X collagen antibodies. In contrast, at 14 days, TGF-beta 1 + BMP-2-treated aggregates did not contain any morphologically identifiable hypertrophic chondrocytes and their abundant extracellular matrix was not immunoreactive to the anti-type X collagen antibody. Expression of BMPR-IA, TGF-beta RI, and TGF-beta RII receptors was detected at all times in each culture condition, indicating that the distinct responses of aggregates to BMP-2, TGF-beta 1 and TGF-beta 1 + BMP-2 were not due to overt differences in receptor expression. Collectively, our results suggest that BMP-2 induces neochondrogenesis of rat periosteum-derived cells, and that TGF-beta 1 modulates the terminal differentiation in BMP-2 induced chondrogenesis.     

Tuning cellular responses to BMP-2 with material surfaces

Bone morphogenetic protein 2 (BMP-2) has been known for decades as a strong osteoinductive factor and for clinical applications is combined solely with collagen as carrier material. The growing concerns regarding side effects and the importance of BMP-2 in several developmental and physiological processes have raised the need to improve the design of materials by controlling BMP-2 presentation. Inspired by the natural cell environment, new material surfaces have been engineered and tailored to provide both physical and chemical cues that regulate BMP-2 activity. Here we describe surfaces designed to present BMP-2 to cells in a spatially and temporally controlled manner. This is achieved by trapping BMP-2 using physicochemical interactions, either covalently grafted or combined with other extracellular matrix components. In the near future, we anticipate that material science and biology will integrate and further develop tools for in vitro studies and potentially bring some of them toward in vivo applications.     

Bone-inducing activity of mature BMP-2b produced from a hybrid BMP-2a/2b precursor

The human osteoinductive proteins BMP-2a and BMP-2b have been cloned and expressed in mammalian cells. In order to improve expression levels we examined the role of the proregion in assembly and export. Use of the BMP-2a proregion combined with the mature region of BMP-2b leads to dramatically improved expression of mature BMP-2b. Mature BMP-2b has been purified to near homogeneity from the BMP-2a/2b hybrid, and its structural properties and biological activity determined. Recombinant mature BMP-2b homodimer elicits bone formation in vivo.     

Wnt signaling during BMP-2 stimulation of mesenchymal chondrogenesis

Members of both the Wnt and bone morphogenetic protein (BMP) families of signaling molecules have been implicated in the regulation of cartilage development. A key component of the Wnt signaling pathway is the cytosolic protein, beta-catenin. We have recently shown that the chondrogenic activity of BMP-2 in vitro involves the action of the cell-cell adhesion protein, N-cadherin, which functionally complexes with beta-catenin. The aim of this study is to test the hypothesis that Wnts may be involved in BMP-2 induced chondrogenesis, using an in vitro model of high-density micromass cultures of the murine multipotent mesenchymal cell line, C3H10T1/2. Expression of a number of Wnt members was detected in these cultures, including Wnt-3A and Wnt-7A, whose levels were up- and downregulated, respectively, by BMP-2. To assess the functional involvement of Wnt signaling in BMP-2 induced chondrogenesis, cultures were treated with lithium chloride, a Wnt-7A mimetic that acts by inhibiting the serine/threonine phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta). Lithium treatment significantly inhibited BMP-2 stimulation of chondrogenesis as well as GSK-3beta enzymatic activity, and decreased the levels of N-cadherin protein and mRNA. Furthermore, lithium decreased BMP-2 upregulation of total and nuclear levels of LEF-1 and beta-catenin as well as their interaction during later chondrogenesis; similarly, the interaction of beta-catenin with N-cadherin was also decreased. Interestingly, lithium treatment did not affect the ability of BMP-2 to decrease ubiquitination of beta-catenin, although it did reduce the interaction of beta-catenin with GSK-3beta during late chondrogenesis (days 9-13). We suggest that the chondro-inhibitory effect of lithium on BMP-2 induced chondrogenesis indicates antagonism between lithium-like Wnts and BMP-2 during mesenchymal condensation.     

Expression of BMP-2 and Ets1 in BMP-2-stimulated mouse pre-osteoblast differentiation is regulated by microRNA-370

MicroRNAs (miRs) regulate several biological functions such as cell growth, cell differentiation, and carcinogenesis, by binding to the 3'-untranslated regions (3'-UTR) of specific target genes, in order to repress translation or promote degradation of the transcribed mRNAs. In the present study, using microRNA array and in silico analyses, we found that miR-370 regulates the expression of bone morphogenetic protein-2 (BMP-2) and V-ets Erythroblastosis Virus E26 Oncogene Homolog 1 (Ets1) in BMP-2-stimulated murine pre-osteoblast MC3T3-E1 cell differentiation. The enforced expression of mature miR-370 in MC3T3-E1 cells or primary osteoblast cells remarkably attenuated BMP-2-induced pre-osteoblast differentiation. To ascertain the mechanisms underlying the regulation of osteoblast differentiation by miR-370, we hypothesized a BMP-2-Ets1-PTHrP feed-forward loop regulatory mechanism.