Objective To investigate the molecular mechanisms of the effects of TNF-αt and NF-κB on BMP-2-Smad1 signaling pathways during osteoblast differentiation.Methods Mouse myoblast C2C12 cells treated with BMP-2 were used as model of osteoblast differentiation.Transient transfection was performed to express the downstream effectors.Luciferase reporter activity assay and RT-PCR were performed to measure the expressions of NF-κB and its inhibitor IκBα, for studying the effect of TNF-α and NF-κB on BMP-2-Smad1 signaling pathway.Results TNF-α decreased the activity of its downstream molecule Smad1 from 2.04 times in the control to 0.63 times.Overexpression of NF-κB decreased the activity of Smad1 from 2.04 times to 0.39 times.Western blotting data showed that TNF-α significantly reduced the expression of phospho-Smad1 induced by BMP2 in C2C12 cells.However, RT-PCR data indicated that TNF-α did not change the mRNA expression of Smadl.Conclusion TNF-α inhibited osteoblast differentiation through the activation of NF-κB by TNF-α, causing a decrease of phosphorylated Smadl in BMP-2 signaling pathway which further inhibited the BMP-2-Smadl signaling pathway.This suggested that persistent inflammation could directly inhibit osteoblast differentiation and bone formation.%目的 探讨肿瘤坏死因子-α(TNF-α)-NF-кB信号通路通过骨形态发生蛋白-2(BMP-2)-Smadl信号通路对成骨细胞分化影响的分子机制.方法 应用BMP-2体外诱导鼠肌源细胞 C2C12 向成骨细胞分化模型,瞬时转染表达其下游效应物,通过实时RT-PCR 以及蛋白电泳和免疫印迹技术(Western-blot),检测其下游效应物Smadl与核转录因子(NF-кB)及其抑制剂(IкBα),研究 TNF-α和NF-кB对BMP-2-Smadl信号通路的影响.结果 TNF-α能够将Smadl(BMP-2的下游效应物)的活性从对照组的2.04倍降低至0.63倍.NF-кB过表达则将Smadl活性从2.04倍降低至0.39倍.蛋白质印迹实验可见,TNF-α能明显降低BMP2活化的C2C12细胞中磷酸化Smadl的含量,而RT-PCR研究发现其并不能改变Smadl的mRNA丰度.结论 TNF-α抑制成骨细胞分化其分子机制是通过TNF-α激活NF-кB而降低BMP-2信号通路中磷酸化的Smadl,进而抑制BMP-2-Smadl通路活性而发挥作用.提示持续性的炎症反应能直接抑制成骨细胞的分化和骨形成.
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