The type I TGFβ receptor TGFβRI (encoded by Tgfbr1) was ablated in cartilage. The resulting Tgfbr1^Col2 mice exhibited lethal chondrodysplasia. Similar defects were not seen in mice lacking the type II TGFβ receptor or SMADs 2 and 3, the intracellular mediators of canonical TGFβ signaling. However, we detected elevated BMP activity in Tgfbr1^Col2 mice. As previous studies showed that TGFβRI can physically interact with ACVRL1, a type I BMP receptor, we generated cartilage-specific Acvrl1 (Acvrl1^Col2 ) and Acvrl1/Tgfbr1 (Acvrl1/Tgfbr1^Col2) knockouts. Loss of ACVRL1 alone had no effect, but Acvrl1/Tgfbr1^Col2 mice exhibited a striking reversal of the chondrodysplasia seen in Tgfbr1^Col2 mice. Loss of TGFβRI led to a redistribution of the type II receptor ACTRIIB into ACVRL1/ACTRIIB complexes, which have high affinity for BMP9. Although BMP9 is not produced in cartilage, we detected BMP9 in the growth plate, most likely derived from the circulation. These findings demonstrate that the major function of TGFβRI in cartilage is not to transduce TGFβ signaling, but rather to antagonize BMP signaling mediated by ACVRL1.

Background and Aims: It's reported that bone morphogenetic protein 9 (BMP9) played an important role in lipid and glucose metabolism, but the role of BMP9 in nonalcoholic fatty liver disease (NAFLD) is unclear. Here, we evaluated the therapeutic efficacy of recombined BMP9 in NAFLD mice and investigated the potential mechanism. Methods: The effects of recombinant BMP9 on NAFLD were assessed in HFD-induced NAFLD mice. C57BL/6 mice were administrated with high-fat diet (HFD) for 12 weeks. In the last 4 weeks, mice were treated with PBS or recombined BMP9 once daily. Insulin sensitivity was evaluated by glucose tolerance test (GTT) and insulin tolerance test (ITT) at the end of the 12th week. Then NAFLD related indicators were assessed by a variety of biological methods, including histology, western blotting, real-time PCR, RNA-seq and assay for transposase-accessible chromatin using sequencing (ATAC-seq) analyses. Results: BMP9 reduced obesity, improved glucose metabolism, alleviated hepatic steatosis and decreased liver macrophages infiltration in HFD mice. RNA-seq showed that Cers6, Cidea, Fabp4 involved in lipid and glucose metabolism and Fos, Ccl2, Tlr1 involved in inflammatory response downregulated significantly after BMP9 treatment in HFD mouse liver. ATAC-seq showed that chromatin accessibility on promoters of Cers6, Fabp4, Ccl2 and Fos decreased after BMP9 treatment in HFD mouse liver. KEGG pathway analysis of dysregulated genes in RNA-seq and integration of RNA-seq and ATAC-seq showed that TNF signaling pathway and Toll-like receptor signaling pathway decreased in BMP9 treated HFD mouse liver. Conclusion: Our data revealed that BMP9 might alleviate NAFLD via improving glucose and lipid metabolism, decreasing inflammatory response and reshaping chromatin accessibility in HFD mouse liver. BMP9 downregulate genes related to lipid metabolism, glucose metabolism and inflammation expression, at least partially via decreasing promoter chromatin accessibility of Cers6, Fabp4, Fos and Tlr1. BMP9 may also reduce the expression of liver Ccl2, thereby changing the number or composition of liver macrophages, and ultimately reducing liver inflammation. The effect of BMP9 on NAFLD might be all-round, and not limit to lipid and glucose metabolism. Therefore, the underlying mechanism needs to be studied in detail further.

Keywords: ATAC-seq; BMP9; NAFLD; RNA-seq; glucose metabolism; hepatic steatosis; inflammatory response.

Obesity drives the development of nonalcoholic fatty liver disease (NAFLD) characterized by hepatic steatosis. Several bone morphogenetic proteins (BMPs) except BMP9 were reported related to metabolic syndrome. This study demonstrates that liver cytokine BMP9 is decreased in the liver and serum of NAFLD model mice and patients. BMP9 knockdown induces lipid accumulation in Hepa 1-6 cells. BMP9-knockout mice exhibit hepatosteatosis due to down-regulated peroxisome proliferator-activated receptor α (PPARα) expression and reduced fatty acid oxidation. In vitro, recombinant BMP9 treatment attenuates triglyceride accumulation by enhancing PPARα promoter activity via the activation of p-smad. PPARα-specific antagonist GW6471 abolishes the effect of BMP9 knockdown. Furthermore, adeno-associated virus-mediated BMP9 overexpression in mouse liver markedly relieves liver steatosis and obesity-related metabolic syndrome. These findings indicate that BMP9 plays a critical role in regulating hepatic lipid metabolism in a PPARα-dependent manner and may provide a previously unknown insight into NAFLD therapeutic approaches.

The aim of the present work was to address the role of BMP9 in different genetic backgrounds (C57BL/6, BALB/c, and 129/Ola) of mice deleted for Bmp9. We found that Bmp9 deletion led to premature mortality only in the 129/Ola strain. We have previously shown that Bmp9 deletion led to liver sinusoidal endothelial cells (LSEC) capillarization and liver fibrosis in the 129/Ola background. Here, we showed that this is not the case in the C57BL/6 background. Analysis of LSEC from Wild-type (WT) versus Bmp9-KO mice in the C57BL/6 background showed no difference in LSEC fenestration and in the expression of differentiation markers. Comparison of the mRNA expression of LSEC differentiation markers between WT C57BL/6 and 129/Ola mice showed a significant decrease in Stabilin2, Plvap, and CD209b, suggesting a more capillary-like phenotype in WT C57BL/6 LSECs. C57BL/6 mice also had lower BMP9 circulating concentrations and hepatic Vegfr2 mRNA levels, compared to the 129/Ola mice. Taken together, our observations support a role for BMP9 in liver endothelial cell fenestration and prevention of fibrosis that is dependent on genetic background. It also suggests that 129/Ola mice are a more suitable model than C57BL/6 for the study of liver fibrosis subsequent to LSEC capillarization.

Bone morphogenetic protein 9 (BMP9) is a recently discovered cytokine mainly secreted by the liver and is a member of the transforming growth factor β (TGF-β) superfamily. In recent years, an increasing number of studies have shown that BMP9 is associated with liver diseases, including nonalcoholic fatty liver disease (NAFLD), liver fibrosis and hepatocellular carcinoma (HCC), and BMP9 signaling may play dual roles in liver diseases. In this review, we mainly summarized and discussed the roles and potential mechanisms of BMP9 signaling in NAFLD, liver fibrosis and HCC. Specifically, this article will provide a better understanding of BMP9 signaling and new clues for the treatment of liver diseases.

Keywords: Bone morphogenetic protein 9 (BMP9); Hepatocellular carcinoma (HCC); Liver fibrosis; Nonalcoholic fatty liver disease (NAFLD); Signaling pathway.

Bone morphogenetic protein 9 (BMP9) has previously been characterized as the strongest osteoinductive growth factor among the BMP family. The aim of the present study was to evaluate the possibility of combining rhBMP9 with an injectable biphasic calcium phosphate (I-BCP, maxresorb inject®), since I-BCP is an easy to handle biomaterial with ideal properties for bone augmentation procedures. The adsorption potential of rhBMP9 as well as the cell behavior of bone stromal ST2 cells were investigated on cell viability, adhesion, proliferation and osteogenic differentiation for I-BCP combined with/without rhBMP9 in vitro. I-BCP demonstrated excellent adsorption/retention potential of rhBMP9 with a slow and steady release over a 10 day period by ELISA. Cell attachment at 8 hours and cell proliferation at 1, 3 and 5 days was decreased on I-BCP with/without rhBMP9 when compared to control tissue-culture plastic. While I-BCP had little influence on osteoblast differentiation, its combination with rhBMP9 significantly increased ALP activity at 7 days and mRNA levels of osteoblast differentiation markers including ALP and osteocalcin at 14 days. I-BCP served as an excellent carrier for rhBMP9 clearly demonstrating its osteoinductive potential. We therefore confirm the great potential of rhBMP9 to serve as a future regenerative growth factor for bone applications.

Heritable forms of pulmonary arterial hypertension (PAH) and pulmonary veno-occlusive disease/pulmonary capillary haemangiomatosis (PVOD/PCH) diverge by lung histopathological lesions, clinical and para-clinical presentation, their responsible genes, and mode of transmission. Since the identification of the BMPR2 gene in families affected by PAH, mutations in several other genes have been discovered for both forms. The mutation landscape in these new genes is not yet well known.

We set up a next-generation sequencing-based targeted sequencing gene panel allowing known genes for PAH and PVOD/PCH to be analysed simultaneously. Genetic analysis was prospectively performed on 263 PAH and PVOD/PCH patients (adult and paediatric cases).

Pathogenic mutations were identified in 19.5% of sporadic PAH patients (n=180), 54.5% of familial PAH patients and 13.3% of PVOD/PCH patients. BMPR2 was the most frequently mutated gene, followed by TBX4 in both paediatric and adult PAH. BMP9 mutations were identified in 1.2% of adult PAH cases. EIF2AK4 biallelic mutations were restricted to PVOD/PCH. A truncating mutation and a predicted loss-of-function variant were also identified in BMP10 in two severely affected sporadic PAH female patients.

Our results confirm that mutations are found in genes beyond BMPR2 in heritable PAH, emphasise the role of TBX4 and BMP9, and designate BMP10 as a new PAH gene.

The diagnosis of hereditary hemorrhagic telangiectasia (HHT) is based on the Curaçao criteria: epistaxis, telangiectases, arteriovenous malformations in internal organs, and family history. Genetically speaking, more than 90% of HHT patients show mutations in ENG or ACVRL1/ALK1 genes, both belonging to the TGF-β/BMP9 signaling pathway. Despite clear knowledge of the symptoms and genes of the disease, we still lack a definite cure for HHT, having just palliative measures and pharmacological trials. Among the former, two strategies are: intervention at "ground zero" to minimize by iron and blood transfusions in order to counteract anemia. Among the later, along the last 15 years, three different strategies have been tested: (1) To favor coagulation with antifibrinolytic agents (tranexamic acid); (2) to increase transcription of ENG and ALK1 with specific estrogen-receptor modulators (bazedoxifene or raloxifene), antioxidants (N-acetylcysteine, resveratrol), or immunosuppressants (tacrolimus); and (3) to impair the abnormal angiogenic process with antibodies (bevacizumab) or blocking drugs like etamsylate, and propranolol. This manuscript reviews the main strategies and sums up the clinical trials developed with drugs alleviating HHT.

Keywords: ALK1; FK506; HHT; N-acetylcysteine; bazedoxifene; endoglin; etamsylate; propranolol; raloxifene; tranexamic acid.

Osteoarthritis (OS) is a common disease in orthopedics. Although OS is known as an inflammation mediated by inflammatory cytokines; however, the mechanism is poorly understood. In the present study, the role of bone morphogenetic protein-9 (BMP9) was investigated in chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADMSCs). ADMSCs were transfected with BMP9. BMP9 mRNA expression was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Type II collagen and aggrecan expression was detected by western blotting and RT-qPCR. Mouse models of knee OS were established. Hematoxylin-eosin staining and toluidine blue staining were performed to observe changes in the OS-affected knee joint. After intra-articular injection of ADMSCs transfected with BMP9, intra-articular expression of type II collagen and aggrecan was detected by western blot analysis and RT-qPCR. After the Notch signaling pathway was inhibited in ADMSCs, ADMSCs were injected into the articular cavity. The expression of Notch signaling pathway-related proteins Notch1 and Jagged1 was detected by western blot analysis and RT-qPCR. BMP9 promoted chondrogenic differentiation of ADMSCs. After injection of BMP9 overexpressing ADMSCs into the articular space, type II collagen and aggrecan expression was increased. When the Notch signaling pathway of ADMSCs was inhibited, the ability of BMP9 overexpressing ADMSCs to repair the cartilage in the OS-affected knee joint was attenuated. These results demonstrate that upregulating BMP9 protein expression may promote the chondrogenic differentiation of ADMSCs. Intra-articular injection of ADMSCs contributes to cartilage repair in OS-affected knee joints through the Notch1/Jagged1 signaling pathway.

Fracture healing involves the coordinated actions of multiple cytokines. Bone morphogenetic protein 9 (BMP9) is an important factor in bone formation. The present study aimed to investigate the osteogenic potential of bone marrow stem cells (BMSCs) in response to adenoviral (Ad)BMP9, and the early fracture repair properties of AdBMP9 in surgically‑created fractures in osteoporotic rats. Alkaline phosphatase (ALP) activity was assayed and matrix mineralization was examined by Alizarin Red S staining. mRNA and protein expression levels of BMP9, runt‑related transcription factor 2 (RUNX2) and type 1 collagen (COL‑1) were detected in vitro and in vivo. Femoral bone mineral density was assessed for osteoporosis in ovariectomized rats. An open femora fracture was subsequently created, and gelatin sponges containing AdBMP9 were implanted. The femora were harvested for radiographical, micro‑computed tomography, biomechanical and histological analysis 4 weeks later. BMP9 successfully increased ALP activity and induced mineralized nodule formation in BMSCs. BMP9 in gelatin sponges demonstrated marked effects on microstructural parameters and the biomechanical strength of bone callus. In addition, it upregulated the expression levels of RUNX2 and COL‑1. AdBMP9 in gelatin sponges significantly mediated callus formation, and increased bone mass and strength in osteoporotic rats with femora fractures. The results of the present study suggested that BMP9 enhanced callus formation and maintained early mechanical stability during fracture healing in osteoporotic rats, implicating it as a potential novel therapeutic target for fracture healing.

Background: Disrupted endothelial BMP9/10 signaling may contribute to the pathophysiology of both hereditary hemorrhagic telangiectasia (HHT) and pulmonary arterial hypertension (PAH), yet loss of circulating BMP9 has not been confirmed in individuals with ultra-rare homozygous GDF2 (BMP9 gene) nonsense mutations. We studied two pediatric patients homozygous for GDF2 (BMP9 gene) nonsense mutations: one with PAH (c.[76C>T];[76C>T] or p.[Gln26Ter];[Gln26Ter] and a new individual with pulmonary arteriovenous malformations (PAVMs; c.[835G>T];[835G>T] or p.[Glu279Ter];[Glu279Ter]); both with facial telangiectases.

Methods: Plasma samples were assayed for BMP9 and BMP10 by ELISA. In parallel, serum BMP activity was assayed using an endothelial BRE-luciferase reporter cell line (HMEC1-BRE). Proteins were expressed for assessment of secretion and processing.

Results: Plasma levels of both BMP9 and BMP10 were undetectable in the two homozygous index cases and this corresponded to low serum-derived endothelial BMP activity in the patients. Measured BMP9 and BMP10 levels were reduced in the asymptomatic heterozygous p.[Glu279Ter] parents, but serum activity was normal. Although expression studies suggested alternate translation can be initiated at Met57 in the p.[Gln26Ter] mutant, this does not result in secretion of functional BMP9.

Conclusion: Collectively, these data show that homozygous GDF2 mutations, leading to a loss of circulating BMP9 and BMP10, can cause either pediatric PAH and/or "HHT-like" telangiectases and PAVMs. Although patients reported to date have manifestations that overlap with those of HHT, none meet the Curaçao criteria for HHT and seem distinct from HHT in terms of the location and appearance of telangiectases, and a tendency for tiny, diffuse PAVMs.

Keywords: bone morphogenetic protein; hereditary hemorrhagic telangiectasia; pulmonary arterial hypertension; pulmonary arteriovenous malformations.

Aims: BMP9 and BMP10 mutations were recently identified in patients with pulmonary arterial hypertension (PAH), but their specific roles in the pathogenesis of the disease are still unclear. We aimed to study the roles of BMP9 and BMP10 in cardiovascular homeostasis and pulmonary hypertension using transgenic mouse models deficient in Bmp9 and/or Bmp10.

Methods and results: Single- and double-knockout mice for Bmp9 (constitutive) and/or Bmp10 (tamoxifen inducible) were generated. Single-KO mice developed no obvious age-dependent phenotype when compared with their wild-type littermates. However, combined deficiency in Bmp9 and Bmp10 led to vascular defects resulting in a decrease in peripheral vascular resistance and blood pressure and the progressive development of high-output heart failure (HOHF) and pulmonary hemosiderosis. RNAseq analysis of the lungs of the double-KO mice revealed differential expression of genes involved in inflammation and vascular homeostasis. We next challenged these mice to chronic hypoxia. After three weeks of hypoxic exposure, Bmp10-cKO mice showed an enlarged heart. However, although genetic deletion of Bmp9 in the single and double-KO mice attenuated the muscularization of pulmonary arterioles induced by chronic hypoxia, we observed no differences in Bmp10-cKO mice. Consistent with these results, endothelin-1 levels were significantly reduced in Bmp9 deficient mice but not Bmp10-cKO mice. Furthermore, the effects of BMP9 on vasoconstriction were inhibited by bosentan, an endothelin receptor antagonist, in a chick chorioallantoic membrane assay.

Conclusions: Our data show redundant roles for BMP9 and BMP10 in cardiovascular homeostasis under normoxic conditions (only combined deletion of both Bmp9 and Bmp10 was associated with severe defects) but highlight specific roles under chronic hypoxic conditions. We obtained evidence that BMP9 contributes to chronic hypoxia-induced pulmonary vascular remodeling, whereas BMP10 plays a role in hypoxia-induced cardiac remodeling in mice.

Keywords: bone morphogenetic proteins; high-output heart failure; pulmonary hypertension; pulmonary vascular remodeling; vascular anomalies.

Objective: Pulmonary arterial hypertension is a disease of proliferative vascular occlusion that is strongly linked to mutations in BMPR2-the gene encoding the BMPR-II (BMP [bone morphogenetic protein] type II receptor). The endothelial-selective BMPR-II ligand, BMP9, reverses disease in animal models of pulmonary arterial hypertension and suppresses the proliferation of healthy endothelial cells. However, the impact of BMPR2 loss on the antiproliferative actions of BMP9 has yet to be assessed. Approach and Results: BMP9 suppressed proliferation in blood outgrowth endothelial cells from healthy control subjects but increased proliferation in blood outgrowth endothelial cells from pulmonary arterial hypertension patients with BMPR2 mutations. This shift from growth suppression to enhanced proliferation was recapitulated in control human pulmonary artery endothelial cells following siRNA-mediated BMPR2 silencing, as well as in mouse pulmonary endothelial cells isolated from endothelial-conditional Bmpr2 knockout mice (Bmpr2^EC-/-). BMP9-induced proliferation was not attributable to altered metabolic activity or elevated TGFβ (transforming growth factor beta) signaling but was linked to the prolonged induction of the canonical BMP target ID1 in the context of BMPR2 loss. In vivo, daily BMP9 administration to neonatal mice impaired both retinal and lung vascular patterning in control mice (Bmpr2^EC+/+) but had no measurable effect on mice bearing a heterozygous endothelial Bmpr2 deletion (Bmpr2^EC+/-) and caused excessive angiogenesis in both vascular beds for Bmpr2^EC-/- mice.

Conclusions: BMPR2 loss reverses the endothelial response to BMP9, causing enhanced proliferation. This finding has potential implications for the proposed translation of BMP9 as a treatment for pulmonary arterial hypertension and suggests the need for focused patient selection in clinical trials.

Keywords: endothelial cells; hypertension, pulmonary; lung; mice, knockout; signal transduction.

Bone morphogenic proteins (BMPs) and blood flow regulate vascular remodeling and homeostasis. In this issue, Baeyens et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201603106) show that blood flow sensitizes endothelial cells to BMP9 signaling by triggering Alk1/ENG complexing to suppress cell proliferation and to recruit mural cells, thereby establishing endothelial quiescence.

Rationale: Pulmonary hypertension (PH) related to pulmonary fibrosis (PF) belongs to the WHO Group III, one of the most common subgroups which lacks effective treatment options. In this study, we aim to uncover the underlying mechanism of action, especially the BMP9/BMPR2/SMAD signaling pathway in this subtype of PH.

Methods: Male Sprague Dawley rats were used to establish a bleomycin (BLM)-induced PF and PH animal model, and primarily cultured rat pulmonary microvascular endothelial cells (PMVECs) were applied as the cell model.

Results: Typical PH characteristics were observed at early stage after BLM treatment. First, significantly increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) occurred after 14-days of BLM treatment, while the excessive pulmonary arterial (PA) thickening and severe loss of PA endothelium were observed as early as 7-days after BLM treatment. Then, markedly downregulation of the BMP9/BMPR2/SMAD signaling pathway were determined in both the lung tissues from BLM-treated rats (throughout the 7- to 35-days treatment period) and BLM-treated rat PMVECs, in correlation with the aggravated cell apoptosis and loss of PA endothelium. Notably, treatment of recombinant human bone morphogenetic protein 9 (rhBMP9) significantly attenuated the BLM-induced PH characteristics by restoring the disrupted BMP9/BMPR2/SMAD signaling pathway.

Conclusion: In BLM-treated rats, an early-onset and persistent suppression of the BMP9/BMPR2/SMAD signaling pathway might be a trigger to induce the severe loss of PA endothelium and subsequent PA vascular remodeling, contributing to the development of PH. Therapeutic approaches reinforcing the BMP9/BMPR2/SMAD signaling might be ideal strategies for this subtype of PH.

Keywords: BMP9; BMPR2; Smad1/5/9; bleomycin; endothelial cell; pulmonary hypertension.

ActRIIB (activin receptor type-2B) is an activin receptor subtype constitutively expressed in the whole body, playing a role in cellular proliferation, differentiation, and metabolism. For its various physiological activities, ActRIIB interacts with activin and multiple other ligands including myostatin (MSTN), growth differentiation factor 11 (GDF11), and bone morphogenetic protein 9 (BMP9). Notably, the protein-protein interaction (PPI) between ActRIIB and MSTN negatively controls muscular development. Therefore, this PPI has been targeted for effective treatment of muscle degenerative diseases such as muscular dystrophy and sarcopenia. Here, we report the identification of ligand-selective peptidic ActRIIB-antagonists by phage display technology. Our peptides bound to the extracellular domain of ActRIIB, inhibited PPIs between ActRIIB expressed on the cell surface and its ligands, and subsequently suppressed activation of Smad that serves as the downstream signal of the ActRIIB pathway. Interestingly, these peptidic antagonists displayed different ligand selectivities; the AR2mini peptide inhibited multiple ligands (activin A, MSTN, GDF11, and BMP9), AR9 inhibited MSTN and GDF11, while AR8 selectively inhibited MSTN. This is the first report of artificial peptidic ActRIIB-antagonists possessing ligand-selectivity.

Although mutations in the RASA1 gene in vein of Galen aneurysmal malformation (VGAM) and an endoglin gene mutation in a VGAM patient with a family history of hereditary hemorrhagic telangiectasia (HHT) have been identified, most VGAM cases have no mutation in these genes. We sought to detect mutations in other genes related to HHT. We screened for mutations in RASA1 and three genes (endoglin, activin receptor-like kinase 1 (ACVRL1), encoding ALK1, and SMAD4) related to HHT in four VGAM patients. One variant (c.652 C>T p.R218W) in ACVRL1 was identified. Immunoblotting revealed that the ALK1-R218W protein could not promote SMAD1/5/8 phosphorylation by BMP9 stimulation. On the other hand, wild-type ALK1 could enhance the phosphorylation as expected. Furthermore, the transcriptional activation of ALK1-R218W was less efficient than that of wild-type ALK1. We identified 1 variant in ACVRL1 in a VGAM patient. These findings suggest that the ACVRL1 variant-R218W may be associated with the pathogenesis of VGAM.

Osteopontin (OPN) is an osteoblast-derived secretory protein that plays a role in bone remodeling, osteoblast responsiveness, and inflammation. We recently found that osteoblast differentiation is type-specific, with conditions of JNK inactivation inducing osteoblasts that preferentially express OPN (OPN-type). Since OPN-type osteoblasts highly express osteogenesis-inhibiting proteins and Rankl, an important inducer of osteoclastogenesis, an increased appearance of OPN-type osteoblasts may be associated with inefficient and poor-quality bone regeneration. However, whether specific osteogenic inducers can modulate OPN-type osteoblast differentiation is completely unknown. Here, we demonstrate that bone morphogenic protein 9 (BMP9) prevents induction of OPN-type osteoblast differentiation under conditions of JNK inhibition. Although JNK inactivation suppressed both BMP2- and BMP9-induced matrix mineralization and osteocalcin expression, the expression of Rankl and specific cytokines such as Gpha2, Esm1, and Sfrp1 under similar conditions was increased in all cells except those treated with BMP9. Increased expression of Id4, a critical transcriptional regulator of OPN-type osteoblast differentiation, was similarly prevented only in BMP9-treated cells. We also found that BMP9 specifically induces the expression of Hey1, a bHLH transcriptional repressor, and that Id4 inhibits the suppressive effects of Hey1 on Opn promoter activity by forming Id4-Hey1 complexes in osteoblasts. Using site-direct mutagenesis, ChIP, and immunoprecipitation, we elucidated that BMP9-induced overexpression of Hey1 can overcome the effects of Id4 and suppress OPN expression. We further found that p38 activation and JNK inactivation are involved in BMP9-induced Hey1 expression. Collectively, these data suggest that BMP9 is a unique osteogenic inducer that regulates OPN-type osteoblast differentiation.

Bone morphogenetic protein 9 (BMP9), a member of the TGF-β superfamily, is considered a regulator of glucose homeostasis as well as a neuronal differentiation factor. BMP9 induces phosphorylation of Smad1/5 through activin receptor-like kinase 1 and 2 (ALK1 and ALK2). Recently, many studies have shown that BMP9 contributes to tumorigenesis, and aberrant ALK2 expression is involved in many diseases. To investigate the role of BMP9-ALK2 signaling in cancer cells, we used TF-1 cells that require granulocyte-macrophage colony-stimulating factor (GM-CSF) for cell proliferation. BMP9 promoted the proliferation of TF-1 cells in media lacking GM-CSF. TF-1 cells overexpressing ALK2 resulted in the autophosphorylation of Smad1/5, leading to consequent increase in cell growth. Through high-throughput screening (HTS), we found two ALK2-specific inhibitors, KRC203 and KRC360, with IC50 values of 0.9 nM and 0.3 nM. These compounds were more potent and specific for the inhibition of ALK2 when compared to LDN193189. In cell-based assays, these compounds effectively inhibited the proliferation and migration of cancer cells induced by ALK2 and BMP9. Therefore, we propose that our compounds are promising candidates for the treatment of cancer or diseases with abnormal ALK2 or BMP9 signaling.

OBJECTIVE

To investigate the roles of miR-149 in the progression of human osteosarcoma (OS).

RESULTS

miR-149 level was upregulated in tissues from OS patients more than in normal subjects. Cell proliferation and apoptosis assays revealed that miR-149 increased cell proliferation and inhibited cell apoptosis in OS cell line (MG63). An increase of Bcl-2 gene expression and a decrease of cleaved-caspase-3, and cleaved-PARP expression were observed in MG63 cells with transfection of miR-149. Additionally, bone morphogenetic protein 9 (BMP9) was identified as a target of miR-149 in MG63 cells, and BMP9 expression was negatively correlated with miR149 level in OS clinical samples. Co-overexpression of BMP9 with miR-149 in MG63 cells prohibited miR-149-mediated promotive effects on OS progression. Importantly, overexpression of miR-149 conferred chemoresistance in MG63 cells.

CONCLUSIONS

miR-149 promotes OS progression via targeting BMP9.

COX-2 specific inhibitor, which has been widely used, can delay bone fracture healing and reduce osteogenic potential of bone marrow stromal cells. However, it remains unknown how to prevent these side-effects of COX-2 inhibitor. In this study, we introduced BMP9-induced osteogenic differentiation as model to evaluate whether all-trans retinoic acid (ATRA) could ameliorate these adverse effects of COX-2 specific inhibitor on bone metabolism with in vitro and in vivo experiments, and uncover the possible mechanism underlying this process. Results showed that ATRA enhanced the potential of BMP9 to induce the osteogenic markers, such as alkaline phosphates (ALP) and mineralization; but retinoic acid receptor a (RARa) inhibitor showed the reversal effects. COX-2 specific inhibitor (NS398) reduced the osteogenic markers induced by BMP9, and ATRA almost eliminated the inhibitory effect of NS398. BMP9 up-regulated the protein level of β-catenin and promoted it translocate to nucleus, and both were reduced by NS398. On the contrary, ATRA notablely attenuated the inhibitory effect of NS398 on BMP9-increased β-catenin. Exogenous RXRa obviously ameliorated the inhibitory effect of silencing COX-2 on ectopic bone formation induced by BMP9. NS398 reduced the level of phosphorylated CREB, which was almost reversed by ATRA. Besides, RXRa interacted with phosphorylated CREB directly and both were recruited at β-catenin promoter region. Thus, we demonstrated that ATRA may reverse the side-effects of COX-2 inhibitor on bone metabolism through increasing the activation of Wnt/β-catenin pathway partly.