Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells
Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells
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DOI：
10.1002/jcb.21411
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发表时间：
2008-02-01
影响因子：
4
通讯作者：
Xiao, Guozhi
中科院分区：
文献类型：
--
作者：
Singha, Ujjal K.;Jiang, Yu;Xiao, Guozhi
虽然哺乳动物雷帕霉素靶蛋白（mTOR）信号转导在调节细胞生长、增殖和存活中的作用已在各种细胞类型中得到充分证实，但其在成骨细胞中的作用知之甚少。在这项研究中，我们确定了雷帕霉素，mTOR的特异性抑制剂，对成骨细胞增殖和分化的影响，使用MC 3 T3-E1前成骨细胞（MC-4）和原代小鼠骨髓基质细胞（BMSCs）。雷帕霉素在低至0.1 nM的浓度下显著抑制MC-4细胞和BMSC的增殖。Western印迹分析表明，雷帕霉素治疗显着降低细胞周期蛋白A和D1蛋白在两种细胞类型的水平。在分化成骨细胞，雷帕霉素显着降低成骨细胞特异性骨钙素（OCN），骨唾液蛋白（BSP），和osterix（Osx）mRNA的表达，ALP活性，矿化能力。然而，当细胞完全分化时，药物处理对成骨细胞分化参数没有影响。重要的是，雷帕霉素显着降低Runx 2蛋白在增殖和分化，但不分化成骨细胞的水平。最后，在COS-7细胞中过表达S6 K显著增加Runx 2蛋白水平和Runx 2活性。总之，我们的研究表明，mTOR信号通过靶向成骨细胞增殖和成骨细胞分化的早期阶段影响成骨细胞功能。
While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation.