ALK1 is an endothelial-specific type I receptor of the TGFbeta receptor family whose heterozygous mutations cause hereditary hemorrhagic telangiectasia type 2. Although TGFbeta1 and TGFbeta3 have been shown to bind ALK1 under specific experimental conditions, they may not represent the physiological ligands for this receptor. In the present study, we demonstrate that BMP9 induces the phosphorylation of Smad1/5/8 in microvascular endothelial cells, and this phosphorylation lasts over a period of 24 hours. BMP9 also activates the ID1 promoter-derived BMP response element (BRE) in a dose-dependent manner (EC50 = 45 +/- 27 pg/mL), and this activation is abolished by silencing ALK1 expression or addition of ALK1 extracellular domain. Overexpression of endoglin increases the BMP9 response, whereas silencing of both BMPRII and ActRIIA expressions completely abolishes it. BMP10, which is structurally close to BMP9, is also a potent ALK1 ligand. Finally, we demonstrate that BMP9 and BMP10 potently inhibit endothelial cell migration and growth, and stimulate endothelial expression of a panel of genes that was previously reported to be activated by the constitutively active form of ALK1. Taken together, our results suggest that BMP9 and BMP10 are two specific ALK1 ligands that may physiologically trigger the effects of ALK1 on angiogenesis.

Expression of hepcidin, a key regulator of intestinal iron absorption, can be induced in vitro by several bone morphogenetic proteins (BMPs), including BMP2, BMP4 and BMP9 (refs. 1,2). However, in contrast to BMP6, expression of other BMPs is not regulated at the mRNA level by iron in vivo, and their relevance to iron homeostasis is unclear. We show here that targeted disruption of Bmp6 in mice causes a rapid and massive accumulation of iron in the liver, the acinar cells of the exocrine pancreas, the heart and the renal convoluted tubules. Despite their severe iron overload, the livers of Bmp6-deficient mice have low levels of phosphorylated Smad1, Smad5 and Smad8, and these Smads are not significantly translocated to the nucleus. In addition, hepcidin synthesis is markedly reduced. This indicates that Bmp6 is critical for iron homeostasis and that it is functionally nonredundant with other members of the Bmp subfamily. Notably, Bmp6-deficient mice retain their capacity to induce hepcidin in response to inflammation. The iron burden in Bmp6 mutant mice is significantly greater than that in mice deficient in the gene associated with classical hemochromatosis (Hfe), suggesting that mutations in BMP6 might cause iron overload in humans with severe juvenile hemochromatosis for which the genetic basis has not yet been characterized.

Efficacious bone regeneration could revolutionize the clinical management of many bone and musculoskeletal disorders. Bone morphogenetic proteins (BMPs) can regulate the differentiation of mesenchymal stem cells into cartilage, bone, tendon/ligament, and fat lineages. Early data documented the osteogenic potential of rhBMP2 and rhBMP7/OP-1. However, prior to this work that summarized several of our recent studies, no comprehensive analysis had been undertaken to characterize relative osteogenic activity of all BMPs. Using recombinant adenoviruses expressing 14 BMPs, we have demonstrated that, besides BMP2 and BMP7, BMP6 and BMP9 exhibit the highest osteogenic activity both in vitro and in vivo. We further demonstrated that several BMPs may exert synergistic effect on osteogenic differentiation, and that osteogenic BMPs produce a distinct set of molecular fingerprints during osteogenic differentiation. The reported work should expand our current understanding of BMP functions during osteogenic differentiation. It is conceivable that osteogenic BMPs (i.e., BMP2, 4, 6, 7, and 9) may be used to formulate synergistic pairs among themselves and/or with other less osteogenic BMPs for efficacious bone regeneration in clinical settings.

Activin receptor-like kinase 1 (ALK1) is an endothelial-specific member of the TGF-β/BMP receptor family that is inactivated in patients with hereditary hemorrhagic telangiectasia (HHT). How ALK1 signaling regulates angiogenesis remains incompletely understood. Here we show that ALK1 inhibits angiogenesis by cooperating with the Notch pathway. Blocking Alk1 signaling during postnatal development in mice leads to retinal hypervascularization and the appearance of arteriovenous malformations (AVMs). Combined blockade of Alk1 and Notch signaling further exacerbates hypervascularization, whereas activation of Alk1 by its high-affinity ligand BMP9 rescues hypersprouting induced by Notch inhibition. Mechanistically, ALK1-dependent SMAD signaling synergizes with activated Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2, thereby repressing VEGF signaling, tip cell formation, and endothelial sprouting. Taken together, these results uncover a direct link between ALK1 and Notch signaling during vascular morphogenesis that may be relevant to the pathogenesis of HHT vascular lesions.

Angiogenesis is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals. ALK1 (activin receptor like-kinase 1) is an endothelial-specific type 1 receptor of the transforming growth factor-beta receptor family. Heterozygotes with mutations in the ALK1 gene develop hereditary hemorrhagic telangiectasia type 2 (HHT2). Recently, we reported that bone morphogenetic protein (BMP)9 and BMP10 are specific ligands for ALK1 that potently inhibit microvascular endothelial cell migration and growth. These data lead us to suggest that these factors may play a role in the control of vascular quiescence. To test this hypothesis, we checked their presence in human serum. We found that human serum induced Smad1/5 phosphorylation. To identify the active factor, we tested neutralizing antibodies against BMP members and found that only the anti-BMP9 inhibited serum-induced Smad1/5 phosphorylation. The concentration of circulating BMP9 was found to vary between 2 and 12 ng/mL in sera and plasma from healthy humans, a value well above its EC(50) (50 pg/mL). These data indicated that BMP9 is circulating at a biologically active concentration. We then tested the effects of BMP9 in 2 in vivo angiogenic assays. We found that BMP9 strongly inhibited sprouting angiogenesis in the mouse sponge angiogenesis assay and that BMP9 could inhibit blood circulation in the chicken chorioallantoic membrane assay. Taken together, our results demonstrate that BMP9, circulating under a biologically active form, is a potent antiangiogenic factor that is likely to play a physiological role in the control of adult blood vessel quiescence.

Recently, it has been suggested that hepcidin, a peptide involved in iron homeostasis, is regulated by bone morphogenetic proteins (BMPs), apparently by binding to hemojuvelin (Hjv) as a coreceptor and signaling through Smad4. We investigate the role of Hfe, Tfr2 (transferrin receptor 2), and IL-6 in BMP2-, BMP4-, and BMP9-stimulated up-regulation of murine hepcidin, because these molecules, like Hjv, are known to be involved in hepcidin signaling. We show that the BMP signaling pathway acts independently of Hfe, Tfr2, and IL-6: The response to BMP2, BMP4, and BMP9 is similar in isolated hepatocytes of wild-type, Hfe(-/-), IL-6(-/-), and Tfr2(m) mutant mice. The potency of different human BMPs in stimulating hepcidin transcription by murine primary hepatocytes is BMP9>> BMP4>> BMP2. However, in human HepG2 cells, BMP4 and BMP9 are equally potent, whereas BMP2 requires a higher dose to become an effective hepcidin activator. Moreover, all of the tested BMPs are more potent regulators of hepcidin than IL-6 and thus are the most potent known stimulators of hepcidin transcription.

The peptide hormone hepcidin is the principal regulator of systemic iron homeostasis. We examined the pathway by which iron stimulates the production of hepcidin. In humans who ingested 65 mg of iron, the increase in transferrin saturation preceded by hours the increase in urinary hepcidin excretion. Increases in urinary hepcidin concentrations were proportional to the increment in transferrin saturation. Paradoxically, in previous studies in primary hepatocytes and cell lines, hepcidin response to iron or iron transferrin was not observed. We now report that freshly isolated murine primary hepatocytes responded to holotransferrin but not apotransferrin by increasing hepcidin mRNA. Hepcidin increase was not due to contamination of the transferrin preparations by endotoxin, a potent pathologic stimulus of hepcidin synthesis. Using this culture system, we showed that holotransferrin concentrations regulate hepcidin mRNA concentrations through a hemojuvelin/BMP2/4-dependent pathway. Although BMP9 is known to be expressed in the liver and potently increased the basal concentrations of hepcidin mRNA, it did not interact with hemojuvelin, and interference with its signaling pathway did not affect iron regulation. Fresh primary hepatocytes constitute a sufficient system for the regulation of hepcidin by physiologic iron stimuli and will greatly facilitate studies of major disorders of iron homeostasis.

Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor beta (TGFbeta) superfamily. Recent observations clearly emphasize the emerging role of BMPs in angiogenesis: (i) two genetic vascular diseases (hereditary hemorrhagic telangiectasia (HHT) and pulmonary arterial hypertension (PAH)) are caused by mutations in genes encoding components of the BMP signalling pathway (endoglin, ALK1 and BMPRII). (ii) BMP9 has been identified as the physiological ligand of the endothelial receptor ALK1 in association with BMPRII. This review will focus on the diverse functions of BMPs in angiogenesis. We will propose a model that distinguishes the BMP2, BMP7 and GDF5 subgroups from the BMP9 subgroup on the basis of their functional implication in the two phases of angiogenesis (activation and maturation).

ALK1 is a type I receptor of the TGF-β family that is involved in angiogenesis. Circulating BMP9 was identified as a specific ligand for ALK1 inducing vascular quiescence. In this work, we found that blocking BMP9 with a neutralizing antibody in newborn mice significantly increased retinal vascular density. Surprisingly, Bmp9-KO mice did not show any defect in retinal vascularization. However, injection of the extracellular domain of ALK1 impaired retinal vascularization in Bmp9-KO mice, implicating another ligand for ALK1. Interestingly, we detected a high level of circulating BMP10 in WT and Bmp9-KO pups. Further, we found that injection of a neutralizing anti-BMP10 antibody to Bmp9-KO pups reduced retinal vascular expansion and increased vascular density, whereas injection of this antibody to WT pups did not affect the retinal vasculature. These data suggested that BMP9 and BMP10 are important in postnatal vascular remodeling of the retina and that BMP10 can substitute for BMP9. In vitro stimulation of endothelial cells by BMP9 and BMP10 increased the expression of genes involved in the Notch signaling pathway (Jagged1, Dll4, Hey1, Hey2, Hes1) and decreased apelin expression, suggesting a possible cross-talk between these pathways and the BMP pathway.

Genetic evidence implicates the loss of bone morphogenetic protein type II receptor (BMPR-II) signaling in the endothelium as an initiating factor in pulmonary arterial hypertension (PAH). However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy. Here, we identify BMP9 as the preferred ligand for preventing apoptosis and enhancing monolayer integrity in both pulmonary arterial endothelial cells and blood outgrowth endothelial cells from subjects with PAH who bear mutations in the gene encoding BMPR-II, BMPR2. Mice bearing a heterozygous knock-in allele of a human BMPR2 mutation, R899X, which we generated as an animal model of PAH caused by BMPR-II deficiency, spontaneously developed PAH. Administration of BMP9 reversed established PAH in these mice, as well as in two other experimental PAH models, in which PAH develops in response to either monocrotaline or VEGF receptor inhibition combined with chronic hypoxia. These results demonstrate the promise of direct enhancement of endothelial BMP signaling as a new therapeutic strategy for PAH.

Endoglin (CD105), a transmembrane protein of the transforming growth factor β superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. The BMP9/BMP10 binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26-359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.

Mutations in transforming growth factor-beta (TGF-beta) receptor superfamily members underlie conditions characterized by vascular dysplasia. Mutations in endoglin and activin-like kinase receptor 1 (ALK1) cause hereditary hemorrhagic telangiectasia, whereas bone morphogenetic protein type II receptor (BMPR-II) mutations underlie familial pulmonary arterial hypertension. To understand the functional roles of these receptors, we examined their relative contributions to BMP signaling in human pulmonary artery endothelial cells (HPAECs). BMP9 potently and selectively induced Smad1/5 phosphorylation and Id gene expression in HPAECs. Contrary to expectations, BMP9 also stimulated Smad2 activation. Furthermore, BMP9 induced the expression of interleukin 8 and E-selectin. Using small interfering RNA, we demonstrate that the type I receptor, ALK1, is essential for these responses. However, small interfering RNA and inhibitor studies showed no involvement of ALK5 or endoglin. We further demonstrate that, of the candidate type II receptors, BMPR-II predominantly mediated IL-8 and E-selectin induction and mitogenic inhibition by BMP9. Conversely, activin receptor type II (ActR-II) contributed more to BMP9-mediated Smad2 activation. Only abolition of both type II receptors significantly reduced the Smad1/5 and Id responses. Both ALK1 and BMPR-II contributed to growth inhibition of HPAECs, whereas ActR-II was not involved. Taken together, our findings demonstrate the critical role of type II receptors in balancing BMP9 signaling via ALK1 and emphasize the essential role for BMPR-II in a subset of BMP9 responses (interleukin 8, E-selectin, and proliferation). This differential signaling may contribute to the contrasting pathologies of hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension.

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We previously demonstrated that bone morphogenetic protein (BMP) 9 is one of the most potent and yet least characterized BMPs that are able to induce osteogenic differentiation of MSCs both in vitro and in vivo. Here, we conducted gene expression-profiling analysis and identified that Hey1 of the hairy/Enhancer of split-related repressor protein basic helix-loop-helix family was among the most significantly up-regulated early targets in BMP9-stimulated MSCs. We demonstrated that Hey1 expression was up-regulated at the immediate early stage of BMP9-induced osteogenic differentiation. Chromatin immunoprecipitation analysis indicated that Hey1 may be a direct target of the BMP9-induced Smad signaling pathway. Silencing Hey1 expression diminished BMP9-induced osteogenic differentiation both in vitro and in vivo and led to chondrogenic differentiation. Likewise, constitutive Hey1 expression augmented BMP9-mediated bone matrix mineralization. Hey1 and Runx2 were shown to act synergistically in BMP9-induced osteogenic differentiation, and Runx2 expression significantly decreased in the absence of Hey1, suggesting that Runx2 may function downstream of Hey1. Accordingly, the defective osteogenic differentiation caused by Hey1 knockdown was rescued by exogenous Runx2 expression. Thus, our findings suggest that Hey1, through its interplay with Runx2, may play an important role in regulating BMP9-induced osteoblast lineage differentiation of MSCs.

Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs.

Members of the transforming growth factor beta (TGF-beta) family have been genetically linked to vascular formation during embryogenesis. However, contradictory studies about the role of TGF-beta and other family members with reported vascular functions, such as bone morphogenetic protein (BMP) 9, in physiological and pathological angiogenesis make the need for mechanistic studies apparent. We demonstrate, by genetic and pharmacological means, that the TGF-beta and BMP9 receptor activin receptor-like kinase (ALK) 1 represents a new therapeutic target for tumor angiogenesis. Diminution of ALK1 gene dosage or systemic treatment with the ALK1-Fc fusion protein RAP-041 retarded tumor growth and progression by inhibition of angiogenesis in a transgenic mouse model of multistep tumorigenesis. Furthermore, RAP-041 significantly impaired the in vitro and in vivo angiogenic response toward vascular endothelial growth factor A and basic fibroblast growth factor. In seeking the mechanism for the observed effects, we uncovered an unexpected signaling synergy between TGF-beta and BMP9, through which the combined action of the two factors augmented the endothelial cell response to angiogenic stimuli. We delineate a decisive role for signaling by TGF-beta family members in tumor angiogenesis and offer mechanistic insight for the forthcoming clinical development of drugs blocking ALK1 in oncology.

Hereditary hemorrhagic telangiectasia (HHT), the most common inherited vascular disorder, is caused by mutations in genes involved in the transforming growth factor beta (TGF-β) signaling pathway (ENG, ACVRL1, and SMAD4). Yet, approximately 15% of individuals with clinical features of HHT do not have mutations in these genes, suggesting that there are undiscovered mutations in other genes for HHT and possibly vascular disorders with overlapping phenotypes. The genetic etiology for 191 unrelated individuals clinically suspected to have HHT was investigated with the use of exome and Sanger sequencing; these individuals had no mutations in ENG, ACVRL1, and SMAD4. Mutations in BMP9 (also known as GDF2) were identified in three unrelated probands. These three individuals had epistaxis and dermal lesions that were described as telangiectases but whose location and appearance resembled lesions described in some individuals with RASA1-related disorders (capillary malformation-arteriovenous malformation syndrome). Analyses of the variant proteins suggested that mutations negatively affect protein processing and/or function, and a bmp9-deficient zebrafish model demonstrated that BMP9 is involved in angiogenesis. These data confirm a genetic cause of a vascular-anomaly syndrome that has phenotypic overlap with HHT.

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and play a critical role in skeletal development, bone formation and stem cell differentiation. Disruptions in BMP signaling result in a variety of skeletal and extraskeletal anomalies. BMP9 is a poorly characterized member of the BMP family and is among the most osteogenic BMPs, promoting osteoblastic differentiation of mesenchymal stem cells (MSCs) both in vitro and in vivo. Recent findings from various in vivo and molecular studies strongly suggest that the mechanisms governing BMP9-mediated osteoinduction differ from other osteogenic BMPs. Many signaling pathways with diverse functions have been found to play a role in BMP9-mediated osteogenesis. Several of these pathways are also critical in the differentiation of other cell lineages, including adipocytes and chondrocytes. While BMP9 is known to be a potent osteogenic factor, it also influences several other pathways including cancer development, angiogenesis and myogenesis. Although BMP9 has been demonstrated as one of the most osteogenic BMPs, relatively little is known about the specific mechanisms responsible for these effects. BMP9 has demonstrated efficacy in promoting spinal fusion and bony non-union repair in animal models, demonstrating great translational promise. This review aims to summarize our current knowledge of BMP9-mediated osteogenesis by presenting recently completed work which may help us to further elucidate these pathways.

BACKGROUND

Bone morphogenetic proteins (BMPs) are pleiotropic members of the TGF-beta superfamily which regulate many biological processes during development and adult tissue homeostasis and are implicated in the pathogenesis of a number of human diseases. Their involvement in both normal and aberrant physiology creates a need for rapid, sensitive and methodologically simple assays to evaluate their activity from a variety of biological samples. Previously alkaline phosphatase based assays, ELISA and luciferase based bioassays have been developed to evaluate either individual or total BMP activity. In this paper, we describe a highly sensitive, rapid and specific cell based assay for the simultaneous quantification of total and isoform specific BMP activity from biological samples.

RESULTS

A C2C12 cell line stably transfected with a reporter plasmid consisting of the BMP response element (BRE) from the Id1 promoter fused to a luciferase reporter gene was generated. Exposure of this cell line to human recombinant BMP2, BMP4, BMP6, BMP7, BMP9 and BMP10 induced the expression of luciferase which was quantified using a luminometer. This assay was specific for BMP activity as the other TGF-beta superfamily members TGF-beta 1, Nodal and Mullerian Inhibiting Substance (MIS) did not induce the reporter. Pretreatment of samples with isoform specific BMP blocking antibodies coupled with isoform specific titration analysis allowed the simultaneous identification and quantification of BMP4, BMP6 and BMP9 in serum samples.

CONCLUSIONS

The assay is rapid (<48 hours) and can be used to simultaneously measure isoform specific and total BMP activity in complex solutions.

Activin receptor-like kinase-1 (ALK1) is a type I, endothelial cell-specific member of the transforming growth factor-beta superfamily of receptors known to play an essential role in modulating angiogenesis and vessel maintenance. In the present study, we sought to examine the angiogenic and tumorigenic effects mediated upon the inhibition of ALK1 signaling using a soluble chimeric protein (ALK1-Fc). Of 29 transforming growth factor-beta-related ligands screened by surface plasmon resonance, only bone morphogenetic protein (BMP9) and BMP10 displayed high-affinity binding to ALK1-Fc. In cell-based assays, ALK1-Fc inhibited BMP9-mediated Id-1 expression in human umbilical vein endothelial cells and inhibited cord formation by these cells on a Matrigel substrate. In a chick chorioallantoic membrane assay, ALK1-Fc reduced vascular endothelial growth factor-, fibroblast growth factor-, and BMP10-mediated vessel formation. The growth of B16 melanoma explants was also inhibited significantly by ALK1-Fc in this assay. Finally, ALK1-Fc treatment reduced tumor burden in mice receiving orthotopic grafts of MCF7 mammary adenocarcinoma cells. These data show the efficacy of chimeric ALK1-Fc proteins in mitigating vessel formation and support the view that ALK1-Fc is a powerful antiangiogenic agent capable of blocking vascularization.

Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-β superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-β family ligands BMP9 and BMP10 as well as the three type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.

BACKGROUND

As one of the least studied bone morphogenetic proteins (BMPs), BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA) signaling is known to play an important role in development, differentiation and bone metabolism. In this study, we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs).

RESULTS

Both primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9, RARalpha and RXRalpha into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as osteopontin (OPN) and osteocalcin (OC). BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants, we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRalpha or RARalpha significantly increases trabecular bone and osteoid matrix formation.

CONCLUSIONS

Our results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore, a combination of BMP9 and retinoic acid (or its agonists) may be explored as effective bone regeneration therapeutics to treat large segmental bony defects, non-union fracture, and/or osteoporotic fracture.

Mesenchymal stromal progenitor cells (MSCs) are multipotent progenitors that can be isolated from numerous tissues. MSCs can undergo osteogenic differentiation under proper stimuli. We have recently demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most osteogenic BMPs. As one of the least studied BMPs, BMP9 has been shown to regulate angiogenesis in endothelial cells. However, it is unclear whether BMP9-regulated angiogenic signaling plays any important role in the BMP9-initiated osteogenic pathway in MSCs. Here, we investigate the functional role of hypoxia-inducible factor 1α (HIF1α)-mediated angiogenic signaling in BMP9-regulated osteogenic differentiation of MSCs. We find that BMP9 induces HIF1α expression in MSCs through Smad1/5/8 signaling. Exogenous expression of HIF1α potentiates BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo. siRNA-mediated silencing of HIF1α or HIF1α inhibitor CAY10585 profoundly blunts BMP9-induced osteogenic signaling in MSCs. HIF1α expression regulated by cobalt-induced hypoxia also recapitulates the synergistic effect between HIF1α and BMP9 in osteogenic differentiation. Mechanistically, HIF1α is shown to exert its synergistic effect with BMP9 by inducing both angiogenic signaling and osteogenic signaling in MSCs. Thus, our findings should not only expand our understanding of the molecular basis behind BMP9-regulated osteoblastic lineage-specific differentiation, but also provide an opportunity to harness the BMP9-induced synergy between osteogenic and angiogenic signaling pathways in regenerative medicine.

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and have diverse functions during development and organogenesis. BMPs play a major role in skeletal development and bone formation, and disruptions in BMP signaling cause a variety of skeletal and extraskeletal anomalies. Several knockout models have provided insight into the mechanisms responsible for these phenotypes. Proper bone formation requires the differentiation of osteoblasts from mesenchymal stem cell (MSC) precursors, a process mediated in part by BMP signaling. Multiple BMPs, including BMP2, BMP6, BMP7 and BMP9, promote osteoblastic differentiation of MSCs both in vitro and in vivo. BMP9 is one of the most osteogenic BMPs yet is a poorly characterized member of the BMP family. Several studies demonstrate that the mechanisms controlling BMP9-mediated osteogenesis differ from other osteogenic BMPs, but little is known about these specific mechanisms. Several pathways critical to BMP9-mediated osteogenesis are also important in the differentiation of other cell lineages, including adipocytes and chondrocytes. BMP9 has also demonstrated translational promise in spinal fusion and bone fracture repair. This review will summarize our current knowledge of BMP-mediated osteogenesis, with a focus on BMP9, by presenting recently completed work which may help us to further elucidate these pathways.

Growth hormone (GH) is usually released by somatotrophs in the anterior pituitary in response to the GH-releasing hormone and plays an important role in skeleton development and postnatal growth. However, it is unclear if extrapituitary GH exerts any effect on murine multilineage cells (MMCs). MMCs are multipotent progenitors that give rise to several lineages, including bone, cartilage, and fat. We have identified bone morphogenic protein 9 (BMP9) as one of the most osteogenic BMPs in MMCs by regulating a distinct set of downstream mediators. In this study, we find that GH is one of the most significantly upregulated genes by BMP9 in mouse MMCs through expression-profiling analysis. We confirm that GH is a direct early target of and upregulated by BMP9 signaling. Exogenous GH synergizes with BMP9 on inducing early and late osteogenic markers in MMCs. Furthermore, BMP9 and GH costimulation leads to a significant expansion of growth plate in cultured limb explants. Although GH alone does not induce de novo bone formation in an ectopic bone formation model, BMP9 and GH costimulated MMCs form more mature bone, which can be inhibited by silencing GH expression. The synergistic osteogenic activity between BMP9 and GH can be significantly blunted by JAK/STAT inhibitors, leading to a decrease in GH-regulated insulin-like growth factor 1 (IGF1) expression in MMCs. Our results strongly suggest that BMP9 may effectively regulate extrapituitary GH expression in MMCs. Thus, it is conceivable that the BMP9-GH-IGF axis may be exploited as an innovative strategy to enhance osteogenesis in regenerative medicine.