TNF-α-NF-κB对成骨细胞分化过程中BMP-2-Smad1信号通路的影响

The effects of TNF-α and NF-κB on BMP-2-Smad1 signaling pathway during osteoblast differentiation

目的探讨肿瘤坏死因子-α(TNF-α)-NF-κB信号通路通过骨形态发生蛋白-2(BMP-2)-Smad1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Smad1与核转录因子(NF-kB)及其抑制剂(IκBα),研究TNF-α和NF-κB对BMP-2-Smad1信号通路的影响。结果 TNF-α能够将Smad1(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-κB过表达则将Smad1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Smad1的含量,而RT-PCR研究发现其并不能改变Smad1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-kB而降低BMP-2信号通路中磷酸化的Smad1,进而抑制BMP-2-Smad1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。

Objective To investigate the molecular mechanisms of the effects of TNF-α and NF-κB on BMP-2-Smadl 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 effeetors. 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- Smadl signaling pathway. Results TNF-α decreased the activity of its downstream molecule Smadl from 2.04 times in the control to 0. 63 times. Overexpression of NF-κB decreased the activity of Smadl from 2.04 times to 0. 39 times. Western blotting data showed that TNF-α significantly reduced the expression of phospho-Smadl 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.