Sclerostin inhibition of Wnt-3a-induced C3H10T1/2 cell differentiation is indirect and mediated by bone morphogenetic proteins

High bone mass diseases are caused both by activating mutations in the Wnt pathway and by loss of SOST, a bone morphogenetic protein (BMP) antagonist, leading to the activation of BMP signaling. Given the phenotypic similarity between mutations that activate these signaling pathways, it seems likely that BMPs and Wnts operate in parallel or represent components of the same pathway, modulating osteoblast differentiation. In this study, we show that in C3H10T1/2 cells, Wnt-3A and BMP-6 proteins were inducers of osteoblast differentiation, as measured by alkaline phosphatase (ALP) induction. Surprisingly, sclerostin, noggin, and human BMP receptor 1A (BMPR1A)-FC fusion proteins blocked Wnt-3A-induced ALP as well as BMP-6-induced ALP activity. Dkk-1, a Wnt inhibitor, blocked Wnt-induced ALP activity but not BMP-induced ALP activity. Early Wnt-3A signaling as measured by beta-catenin accumulation was not affected by the BMP antagonists but was blocked by Dkk-1. Wnt-3A induced the appearance of BMP-4 mRNA 12 h prior to that of ALP in C3H10T1/2 cells. We propose that sclerostin and other BMP antagonists do not block Wnt signaling directly. Sclerostin blocks Wnt-induced ALP activity by blocking the activity of BMP proteins produced by Wnt treatment. The expression of BMP proteins in this autocrine loop is essential for Wnt-3A-induced osteoblast differentiation.     

Tanshinone IIA enhances BMP-2-stimulated commitment of C2C12 cells into osteoblasts via p38 activation

In this study, we demonstrate a stimulatory effect of tanshinone IIA isolated from the root of Salvia miltiorrhiza on the commitment of bi-potential mesenchymal precursor C2C12 cells into osteoblasts in the presence of bone morphogenetic protein (BMP)-2. At low concentrations, tanshinone IIA enhanced BMP-2-stimulated induction of alkaline phosphatase (ALP), an early phase biomarker of osteoblast differentiation, and mRNA expression of BMPs. ALP induction was inhibited by the BMP antagonist noggin, suggesting that tanshinone IIA enhances the osteogenic activity of BMP signaling. Furthermore, considering the tanshinone IIA-mediated enhancement of BMP-2-stimulated Smad-Runx2 activities, tanshinone IIA could enhance the osteogenic activity of BMP-2 via acceleration of Smad-Runx2 activation. Additionally, pharmacologic inhibition studies suggest the possible involvement of p38 in the action of tanshinone IIA. The p38 inhibitor SB202190 strongly and dose-dependently inhibited tanshinone IIA-enhanced ALP induction. SB202190 also dose-dependently inhibited the tanshinone IIA-induced p38 activation and combined tanshinone IIA-BMP-2-induced Smad activation. In conclusion, tanshinone IIA enhances the commitment of C2C12 cells into osteoblasts and their differentiation through synergistic cross talk between tanshinone IIA-induced p38 activation and BMP-2-induced Smad activation. These activations could subsequently induce the activation of Runx2, which induces osteogenesis via regulation of the osteogenic factors BMP and ALP expression.     

Bone morphogenetic protein receptor IB signaling mediates apoptosis independently of differentiation in osteoblastic cells

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

Bone morphogenetic protein-7 (OP1) and transforming growth factor-beta1 modulate 1,25(OH)2-vitamin D3-induced differentiation of human osteoblasts

Bone morphogenetic proteins (BMPs) and transforming growth factor-beta (TGFbeta) are potent regulators of osteoblast differentiation and proliferation, processes that are crucial in bone remodeling. BMPs and TGFbeta act in concert with other local factors and hormones, among them 1,25(OH)2-vitamin D3 and insulin. Here we show that BMP7 inhibits 1,25(OH)2-vitamin D3-induced differentiation of human osteoblasts, whereas TGFbeta1 stimulates it, as assessed by assays for alkaline phosphatase (ALP) induction, matrix mineralization, and morphology changes. BMP7 or TGFbeta1 alone affects the differentiation of human osteoblasts. Similar results were obtained in assays for ALP induction using conditionally immortalized human osteoblasts (hFOB) and primary osteoblasts obtained from trabecular bone of the femoral head after hip replacement surgery. BMP7 stimulation led to a decrease of 1,25(OH)2-vitamin D3-induced binding of nuclear proteins to a vitamin D response element, as shown by electrophoretic mobility shift assay. Our results suggest that 1,25(OH)2-vitamin D3 modulates in opposite ways the effects of BMP7 and TGFbeta1 on osteoblast differentiation.     

AMP-activated protein kinase (AMPK) positively regulates osteoblast differentiation via induction of Dlx5-dependent Runx2 expression in MC3T3E1 cells

This study examined the role of AMPK activation in osteoblast differentiation and the underlining mechanism. An AMPK activator (AICAR or metformin) stimulated osteoblast differentiation with increases in ALP and OC protein production as well as the induction of AMPK phosphorylation in MC3T3E1 cells. In addition, metformin induced the phosphorylation of Smad1/5/8 and expression of Dlx5 and Runx2, whereas compound C or dominant negative AMPK inhibited these effects. Transient transfection studies also showed that metformin increased the BRE-Luc and Runx2-Luc activities, which were inhibited by DN-AMPK or compound C. Down-regulation of Dlx5 expression by siRNA suppressed metformin-induced Runx2 expression. These results suggest that the activation of AMPK stimulates osteoblast differentiation via the regulation of Smad1/5/8-Dlx5-Runx2 signaling pathway.     

RUNX2对BMP9诱导的间充质干细胞C3H10T1/2成骨分化的影响

目的:研究和确认RUNX2在骨形态发生蛋白9(BMP9)诱导的间充质干细胞C3H10T1/2成骨分化中的作用。方法:通过Western blot、RT-PCR、荧光素酶活性分析检测BMP9对RUNX2表达的影响;分别在过表达RUNX2和RNA干扰抑制RUNX2表达的情况下,利用碱性磷酸酶(ALP)活性测定和染色、钙盐沉积实验,免疫细胞化学和裸鼠皮下异位成骨实验分析RUNX2对于BMP9诱导的间充质干细胞成骨分化的影响。结果:BMP9可以促进RUNX2的表达;RUNX2体外可促进BMP9诱导的C3H10T1/2的ALP活性和钙盐沉积,却抑制了OCN表达,RUNX2还可促进BMP9诱导的裸鼠皮下异位成骨;而在降低RUNX2表达后,BMP9诱导的C3H10T1/2细胞的ALP活性、钙盐沉积、OCN表达和裸鼠皮下异位成骨均受到抑制。结论:RUNX2可以促进BMP9诱导的间充质干细胞C3H10T1/2细胞成骨分化。     

The protein kinase 2 inhibitor CX-4945 regulates osteoclast and osteoblast differentiation In vitro

Drug repositioning can identify new therapeutic applications for existing drugs, thus mitigating high R&D costs. The Protein kinase 2 (CK2) inhibitor CX-4945 regulates human cancer cell survival and angiogenesis. Here we found that CX-4945 significantly inhibited the RANKL-induced osteoclast differentiation, but enhanced the BMP2-induced osteoblast differentiation in a cell culture model. CX-4945 inhibited the RANKL-induced activation of TRAP and NFATc1 expression accompanied with suppression of Akt phosphorylation, but, in contrast, it enhanced the BMP2-mediated ALP induction and MAPK ERK1/2 phosphorylation. CX-4945 is thus a novel drug candidate for bone-related disorders such as osteoporosis.     

CCN3/NOV inhibits BMP-2-induced osteoblast differentiation by interacting with BMP and Notch signaling pathways

We elucidate the role of CCN3/NOV, a member of the CCN family proteins, in osteoblast differentiation using MC3T3-E1 osteoblastic cells. Transduction with CCN3 adenovirus (AdCCN3) alone induced no apparent changes in the expression of osteoblast-related markers, whereas cotransduction with BMP-2 adenovirus (AdBMP-2) and AdCCN3 significantly inhibited the AdBMP-2-induced mRNA expression of Runx2, osterix, ALP, and osteocalcin. Immunoprecipitation-western analysis revealed that CCN3 associated with BMP-2. Compared to transduction with AdBMP-2 alone, cotransduction with AdBMP-2 and AdCCN3 attenuated the expression of phosphorylated Smad1/5/8 and the mRNA for Id1, Id2, and Id3. Transduction with AdCCN3 stimulated the expression of cleaved Notch1, the mRNA expression of Hes1 and Hey1/Hesr1, and the promoter activities of Hes1 and Hey1. The inhibitory effects of CCN3 on the expression of BMP-2-induced osteoblast-related markers were nullified in Hey1-deficient osteoblastic cells. These results indicate that CCN3 exerts inhibitory effects on BMP-2-induced osteoblast differentiation by its involvement of the BMP and Notch signaling pathways.     

BMP7 can promote osteogenic differentiation of human periosteal cells in vitro

To study and evaluate BMP7s functions in osteogenic differentiation of human periosteal cells in vitro. Human periosteal cells from adult tibia were collected and cultured as experimental samples. BMP7 was used to induce periosteal cells in the experiment group with common osteogenic medium. The proliferative activity of periosteal cells was detected by CCK-8. The potentials of osteogenic differentiation were demonstrated as follows: (1) realtime-PCR and ELISA to confirm the expression of the OC, ALP and OPN, (2) Colorimetry, ALP staining and Von Kossa staining were performed to identify ALP activity, ALP expression and calcium nodules, respectively. Based on the significant different expression of OC, ALP and OPN, BMP7 ability of osteogenic differentiation can be identified. ALP activity detection, calcium nodules staining and toluidine staining also provide the power evidence to support BMP7 can promote osteogenic differentiation of human periosteal cells in vitro. To human periosteal cells, BMP7 is a good inducer for osteogenic differentiation. Therefore, it's maybe a potential tool for clinical application.