Tenascin-C表达降低抑制成骨细胞分化并促进骨质疏松的机制

Tenascin-C knockdown suppresses osteoblast differentiation and promotes osteoporosis in mice by inhibiting Wnt signaling

目的探讨Tenascin-C在成骨细胞分化中的作用及机制,并分析其表达变化对骨质疏松的调控。方法以免疫印迹法检测去卵巢致骨质疏松的小鼠股骨松质骨的Tenascin-C表达变化。体外诱导成骨前体细胞MC3T3-E1分化,检测瞬时转染Tenascin-C的si RNA后其碱性磷酸酶的活性。以蛋白质相互作用预测软件STRING筛选与Tenascin-C可能存在相互作用的蛋白质。在上述候选蛋白中,以免疫沉淀法验证了Tenascin-C与Wnt通路内源性抑制蛋白Dickkopf-1(Dkk-1)的相互作用,并以免疫印迹检测Tenascin-C对DKK-1的调控。此后进一步采用荧光素酶报告基因系统检测了Tenascin-C对Wnt信号通路转录活性的影响。结果去卵巢骨质疏松的小鼠股骨松质中Tenascin-C蛋白表达急剧下降。降低Tenascin-C的表达能够阻遏MC3T3-E1向成熟成骨细胞分化,反之,在此基础上过表达β-Catenin可逆转前者导致的成骨细胞分化阻滞。Tenascin-C结合于DKK-1,敲低Tenascin-C可上调DKK-1的表达并降低Wnt通路的转录活性。结论 Tenascin-C的下调可能通过减弱对DKK-1的负调控从而抑制Wnt通路的转录活性,进而抑制成骨细胞的分化并介导骨质疏松。

Objective To understand the mechanism by which tenascin-C regulates osteoblast differentiation and the role of tenascin-C in osteoporosis. Methods Tenascin-C protein expression in femoral spongy bone of mice with or without osteoporosis was analyzed using Western blotting. In MC3T3-E1 osteoblasts with or without tenascin-C depletion by a specific si RNA targeting tenascin-C, alkaline phosphatase activity and Dickkopf-1(DKK-1) expression were determined using quantitative RT-PCR and Western blotting, and the transcriptional activity of Wnt signaling pathway was analyzed using a luciferase reporter assay. The possible interaction of tenascin-C with DKK-1 predicted by STRING software was verified by immunoprecipitation. Results Tenascin-C was markedly down-regulated in hemoral spongy bone of mice with osteoporosis as compared with the control mice. Osteoblastic differentiation was markedly suppressed in MC3T3-E1 osteoblast after tenascinC depletion, and was significantly reversed by simultaneous β-catenin over-expression. si RNA-mediated knockdown of tenascin-C, which bound DKK-1, up-regulated the expression of DKK-1 and consequently lowered the transcriptional activity of Wnt pathway. Conclusion Tenascin-C knockdown attenuates its negative control on DKK-1 to suppress the transcriptional activity of Wnt pathway, which in turn suppresses osteoblastic differentiation and promotes osteoporosis.