CYR61 Regulates BMP-2-dependent Osteoblast Differentiation through the αvβ3 Integrin/Integrin-linked Kinase/ERK Pathway

Osteoporosis is one of the most common bone pathologies. A number of novel molecules have been reported to increase bone formation including cysteine-rich protein 61 (CYR61), a ligand of integrin receptor, but mechanisms remain unclear. It is known that bone morphogenetic proteins (BMPs), especially BMP-2, are crucial regulators of osteogenesis. However, the interaction between CYR61 and BMP-2 is unclear. We found that CYR61 significantly increases proliferation and osteoblastic differentiation in MC3T3-E1 osteoblasts and primary cultured osteoblasts. CYR61 enhances mRNA and protein expression of BMP-2 in a time- and dose-dependent manner. Moreover, CYR61-mediated proliferation and osteoblastic differentiation are significantly decreased by knockdown of BMP-2 expression or inhibition of BMP-2 activity. In this study we found integrin α_vβ₃ is critical for CYR61-mediated BMP-2 expression and osteoblastic differentiation. We also found that integrin-linked kinase, which is downstream of the α_vβ₃ receptor, is involved in CYR61-induced BMP-2 expression and subsequent osteoblastic differentiation through an ERK-dependent pathway. Taken together, our results show that CYR61 up-regulates BMP-2 mRNA and protein expression, resulting in enhanced cell proliferation and osteoblastic differentiation through activation of the α_vβ₃ integrin/integrin-linked kinase/ERK signaling pathway.     

洛伐他汀促进成骨细胞增殖、BMP-2表达和矿化的实验研究

目的研究洛伐他汀对体外培养大鼠颅骨成骨细胞生物学功能的影响,探讨其促进骨形成的作用机制.方法洛伐他汀作用于体外培养大鼠颅骨成骨细胞,化学染色观察对成骨细胞矿化结节形成的影响;用免疫细胞化学单标计数测定成骨细胞增殖率及染色吸光度测定BMP-2的表达的变化;BMP-2和BrdU免疫双标染色吸光度测定新生成骨细胞BMP-2的表达情况.结果实验组成骨细胞矿化结节的数量和面积、细胞增殖率及BMP-2的表达明显高于空白对照组(P<0.05);实验组新生成骨细胞BMP-2的表达显著高于对照组(P<0.01).结论洛伐他汀可促进成骨细胞的增殖、分化、BMP-2的表达和矿化结节的形成,从而发挥促进骨形成的作用.     

Bone morphogenetic protein-3b (BMP-3b) gene expression is correlated with differentiation in rat calvarial osteoblasts

BMP-3b (also called GDF-10) is a novel BMP-3-related protein recently discovered in rat femur tissue. Gene expression of BMP-3b in osteoblastic cells and its regulation by prolonged culture, BMP-2 and transforming growth factor beta1 (TGF-beta1) were examined. The BMP-3b gene was highly expressed in rat osteoblasts obtained from calvarial bones but not in the osteoblastic cell lines (MC3T3-E1 and U2-OS). BMP-3b mRNA increased during osteoblastic differentiation in prolonged culture and was associated with increased alkaline phosphatase (ALPase) activity. When BMP-2, an enhancer of ALPase activity, was added to the primary osteoblast culture, BMP-3b mRNA increased 6.9-fold after 24 h. In contrast, TGF-beta1 treatment, which suppresses ALPase activity, rapidly and completely inhibited gene expression of BMP-3b. The regulation of BMP-3 mRNA differed from that of BMP-3b, even though both proteins share 81% identity. These findings indicate that BMP-3b gene expression is regulated by osteoblastic differentiation and BMP-3b functions in highly differentiated osteoblasts.     

Effect of TAK1 on osteogenic differentiation of mesenchymal stem cells by regulating BMP-2 via Wnt/β-catenin and MAPK pathway

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteoblasts and chondrocytes. In vitro osteogenic differentiation is critical but the molecular mechanism has yet to be further clarified. The role of TGF-β activated kinase 1 (TAK1) in MSCs osteogenesis differentiation has not been reported. By adding si-TAK1 and rhTAK1, the osteogenic differentiation of MSCs was measured. Expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs were checked. As well as molecules involved in BMP and Wnt/β-catenin signaling pathways. The phosphorylation of p38 and JNK was also checked. TAK1 is essential for mineralization of MSCs at low concentration, but excessive rhTAK1 inhibits mineralization of MSCs. It up regulates the expression levels of bone sialoprotein (BSP), osteocalcin (OSC), Alkaline phosphatase (ALP), and RUNX2 during osteogenic differentiation of MSCs. It can also promote TGF-β/BMP-2 gene expression and β-catenin expression, and down regulate GSK-3β expression. Meanwhile, TAK1 promotes the phosphorylation of p38 and JNK. Additionally, TAK1 up regulates the expression of BMP-2 at all concentration under the inhibition of p38 and JNK. Our results suggested that TAK1 is essential in MSCs osteogenesis differentiation, and functions as a double-edged sword, probably through regulation of β-catenin and p38/JNK.     

CCN3 increases BMP-4 expression and bone mineralization in osteoblasts

The nephroblastoma overexpressed (NOV) gene, also called CCN3, regulates differentiation of skeletal mesenchymal cells. Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and bone formation, but the effects of CCN3 on BMP expression and bone formation in cultured osteoblasts are largely unknown. Here we found that CCN3 increased BMP-4 expression and bone nodule formation in cultured osteoblast. Monoclonal antibodies for α5β1 and αvβ5 integrins, and inhibitors of integrin-linked kinase (ILK), p38, and JNK, all inhibited CCN3-induced bone nodule formation and BMP-4 up-regulation of osteoblasts. CCN3 stimulation increased the kinase activity of ILK and phosphorylation of p38 and JNK. Inhibitors of activator protein-1 (AP-1) also suppressed bone nodule formation and BMP-4 expression enhanced by CCN3. Moreover, CCN3-induced c-Jun translocation into the nucleus, and the binding of c-Jun to the AP-1 element on the BMP-4 promoter were both inhibited by specific inhibitors of the ILK, p38, and JNK cascades. Taken together, our results provide evidence that CCN3 enhances BMP-4 expression and bone nodule formation in osteoblasts, and that the integrin receptor, ILK, p38, JNK, and AP-1 signaling pathways may be involved.     

M-Ras is activated by bone morphogenetic protein-2 and participates in osteoblastic determination, differentiation, and transdifferentiation

The small GTPase M-Ras is highly expressed in the central nervous system and plays essential roles in neuronal differentiation. However, its other cellular and physiological functions remain to be elucidated. Here, we clarify the novel functions of M-Ras in osteogenesis. M-Ras was prominently expressed in developing mouse bones particularly in osteoblasts and hypertrophic chondrocytes. Its expression was elevated in C3H/10T1/2 (10T1/2) mesenchymal cells and in MC3T3-E1 preosteoblasts during differentiation into osteoblasts. Treatment of C2C12 skeletal muscle myoblasts with bone morphogenetic protein-2 (BMP-2) to bring about transdifferentiation into osteoblasts also induced M-Ras mRNA and protein expression. Moreover, the BMP-2 treatment activated the M-Ras protein. Stable expression of the constitutively active M-Ras(G22V) in 10T1/2 cells facilitated osteoblast differentiation. M-Ras(G22V) also induced transdifferentiation of C2C12 cells into osteoblasts. In contrast, knockdown of endogenous M-Ras by RNAi interfered with osteoblast differentiation in 10T1/2 and MC3T3-E1 cells. Osteoblast differentiation in M-Ras(G22V)-expressing C2C12 cells was inhibited by treatment with inhibitors of p38 MAP kinase (MAPK) and c-Jun N-terminal kinase (JNK) but not by inhibitors of MAPK and ERK kinase (MEK) or phosphatidylinositol 3-kinase. These results imply that M-Ras, induced and activated by BMP-2 signaling, participates in the osteoblastic determination, differentiation, and transdifferentiation under p38 MAPK and JNK regulation.     

BMP-13 Emerges as a Potential Inhibitor of Bone Formation

Bone morphogenetic protein-13 (BMP-13) plays an important role in skeletal development. In the light of a recent report that mutations in the BMP-13 gene are associated with spine vertebral fusion in Klippel-Feil syndrome, we hypothesized that BMP-13 signaling is crucial for regulating embryonic endochondral ossification. In this study, we found that BMP-13 inhibited the osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. The endogenous BMP-13 gene expression in MSCs was examined under expansion conditions. The MSCs were then induced to differentiate into osteoblasts in osteo-inductive medium containing exogenous BMP-13. Gene expression was analysed by real-time PCR. Alkaline phosphatase (ALP) expression and activity, proteoglycan (PG) synthesis and matrix mineralization were assessed by cytological staining or ALP assay. Results showed that endogenous BMP-13 mRNA expression was higher than BMP-2 or -7 during MSC growth. BMP-13 supplementation strongly inhibited matrix mineralization and ALP activity of osteogenic differentiated MSCs, yet increased PG synthesis under the same conditions. In conclusion, BMP-13 inhibited osteogenic differentiation of MSCs, implying that functional mutations or deficiency of BMP-13 may allow excess bone formation. Our finding provides an insight into the molecular mechanisms and the therapeutic potential of BMP-13 in restricting pathological bone formation.     

Protein related to DAN and cerberus (PRDC) inhibits osteoblastic differentiation and its suppression promotes osteogenesis in vitro

Protein related to DAN and cerberus (PRDC) is a secreted protein characterized by a cysteine knot structure, which binds bone morphogenetic proteins (BMPs) and thereby inhibits their binding to BMP receptors. As an extracellular BMP antagonist, PRDC may play critical roles in osteogenesis; however, its expression and function in osteoblastic differentiation have not been determined. Here, we investigated whether PRDC is expressed in osteoblasts and whether it regulates osteogenesis in vitro. PRDC mRNA was found to be expressed in the pre-osteoblasts of embryonic day 18.5 (E18.5) mouse calvariae. PRDC mRNA expression was elevated by treatment with BMP-2 in osteoblastic cells isolated from E18.5 calvariae (pOB cells). Forced expression of PRDC using adenovirus did not affect cell numbers, whereas it suppressed exogenous BMP activity and endogenous levels of phosphorylated Smad1/5/8 protein. Furthermore, PRDC inhibited the expression of bone marker genes and bone-like mineralized matrix deposition in pOB cells. In contrast, the reduction of PRDC expression by siRNA elevated alkaline phosphatase activity, increased endogenous levels of phosphorylated Smad1/5/8 protein, and promoted bone-like mineralized matrix deposition in pOB cells. These results suggest that PRDC expression in osteoblasts suppresses differentiation and that reduction of PRDC expression promotes osteogenesis in vitro. PRDC is accordingly identified as a potential novel therapeutic target for the regulation of bone formation.     

Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.     

Osteoblastic differentiation of monkey embryonic stem cells in vitro

Monkey embryonic stem (ES) cell is a useful tool for preclinical studies of regenerative medicine. In this paper, we investigated whether monkey ES cells can be differentiated into osteoblasts in vitro using factors known to promote osteogenesis. We prepared embryoid bodies (EB) in the presence of retinoic acid (RA) and subsequently differentiated in the medium containing either dexamethasone (DEX) or bone morphogenetic protein (BMP)-2 in addition to osteogenic supplements (OS), specifically ascorbic acid and beta-glycerophosphate. RA treatment during EB formation induced osteoblastic marker genes, such as collagen type 1, osteopontin, and Cbfa1. For the expression of osteocalcin, however, cultivation with medium containing either DEX or BMP-2 in addition to OS was required. These results showed that osteoblasts could be derived from monkey ES cells in vitro and BMP-2 + OS was effective to induce calcification.