Blocking c-Met signaling enhances bone morphogenetic protein-2-induced osteoblast differentiation.
Blocking c-Met signaling enhances bone morphogenetic protein-2-induced osteoblast differentiation.
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阻断 c-Met 信号传导可增强骨形态发生蛋白 2 诱导的成骨细胞分化。
DOI：
10.1016/j.fob.2015.04.008
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发表时间：
2015
期刊：
影响因子：
2.6
通讯作者：
Iwasaki, Tsuyoshi
中科院分区：
文献类型：
--
作者：
Shibasaki, Seiji;Kitano, Sachie;Karasaki, Miki;Tsunemi, Sachi;Sano, Hajime;Iwasaki, Tsuyoshi
探讨c-Met信号在成骨细胞分化中的作用。通过观察C2C12和MC3T3-E1细胞产生的ALP和骨钙素来判断成骨细胞的分化情况。c-Met信号负向调控成骨细胞分化。阻断c-Met信号传导可能作为类风湿关节炎的治疗策略。我们之前证明，阻断肝细胞生长因子（HGF）受体/c-Met信号可以抑制类风湿关节炎（RA）小鼠模型中的关节炎和关节骨破坏。在本研究中，我们利用小鼠卫星细胞衍生的C2C12成肌细胞系和MC3T3-E1小鼠成骨前细胞系，研究了c-Met信号在成骨细胞分化中的作用。在HGF拮抗剂（NK4）或c-Met抑制剂（SU11274）存在或不存在的情况下，骨形态发生蛋白(BMP)-2或成骨诱导试剂可诱导成骨细胞分化。通过Runx2的表达、碱性磷酸酶（ALP）和骨钙素的产生证实成骨细胞分化。酶联免疫吸附试验证实ALP、骨钙素和HGF的产生。逆转录- pcr分析证实Runx2表达。Western blot检测ERK1/2、AKT和Smads的磷酸化状态。在bmp -2刺激的C2C12细胞中，NK4和SU11274均增强Runx2的表达、ALP和骨钙素的产生，但抑制HGF的产生。SU11274还能促进成骨诱导剂刺激的MC3T3-E1细胞中ALP和骨钙素的产生。在hgf刺激的C2C12细胞中，SU11274抑制ERK1/2和AKT磷酸化。这一结果表明，ERK和AKT在c-Met信号通路的下游发挥作用。然而，在bmp -2刺激的C2C12细胞中，丝裂原活化蛋白激酶/ERK激酶（MEK）和磷脂酰肌醇3-激酶（PI3K）抑制剂均抑制骨钙素和HGF的产生。此外，SU11274、MEK和PI3K抑制剂抑制bmp -2刺激的C2C12细胞中Smad的磷酸化。这些结果表明，尽管c-Met-MEK-ERK-Smad和c-Met-PI3K-AKT-Smad信号通路正向调节成骨细胞分化，但c-Met信号通路负向调节成骨细胞分化，独立于MEK-ERK-Smad和PI3K-AKT-Smad信号通路。因此，阻断c-Met信号传导可能是修复RA患者受损骨的一种治疗策略。
Role of c-Met signaling in osteoblast differentiation was investigated. Osteoblast differentiation was determined by ALP and osteocalcin production by C2C12 and MC3T3-E1 cells. c-Met signaling negatively regulates osteoblast differentiation. Blocking c-Met signaling might serve as a therapeutic strategy for rheumatoid arthritis. We previously demonstrated that blocking hepatocyte growth factor (HGF) receptor/c-Met signaling inhibited arthritis and articular bone destruction in mouse models of rheumatoid arthritis (RA). In the present study, we investigated the role of c-Met signaling in osteoblast differentiation using the C2C12 myoblast cell line derived from murine satellite cells and the MC3T3-E1 murine pre-osteoblast cell line. Osteoblast differentiation was induced by treatment with bone morphogenetic protein (BMP)-2 or osteoblast-inducer reagent in the presence or absence of either HGF antagonist (NK4) or c-Met inhibitor (SU11274). Osteoblast differentiation was confirmed by Runx2 expression, and alkaline phosphatase (ALP) and osteocalcin production by the cells. Production of ALP, osteocalcin and HGF was verified by enzyme-linked immunosorbent assay. Runx2 expression was confirmed by reverse transcription-PCR analysis. The phosphorylation status of ERK1/2, AKT, and Smads was determined by Western blot analysis. Both NK4 and SU11274 enhanced Runx2 expression, and ALP and osteocalcin production but suppressed HGF production in BMP-2-stimulated C2C12 cells. SU11274 also enhanced ALP and osteocalcin production in osteoblast-inducer reagent-stimulated MC3T3-E1 cells. SU11274 inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated C2C12 cells. This result suggested that ERK and AKT were functional downstream of the c-Met signaling pathway. However, both mitogen-activated protein kinase/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitor suppressed osteocalcin and HGF production in BMP-2-stimulated C2C12 cells. Furthermore, SU11274, MEK, and PI3K inhibitor suppressed Smad phosphorylation in BMP-2-stimulated C2C12 cells. These results indicate that although the c-Met-MEK-ERK-Smad and c-Met-PI3K-AKT-Smad signaling pathways positively regulate osteoblast differentiation, c-Met signaling negatively regulates osteoblast differentiation, independent of the MEK-ERK-Smad and PI3K-AKT-Smad pathways. Therefore, blocking c-Met signaling might serve as a therapeutic strategy for the repair of destructed bone in patients with RA.
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影响因子：
4.1
作者：
Chen, Ketian;Perez-Stable, Carlos;Howard, Guy A.
通讯作者：
Howard, Guy A.
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