阻断ERK1/2蛋白激酶可促进BMP9诱导的C_3H10T1/2细胞成骨分化

Inhibition of ERK1/2 kinase increases BMP9-induced osteogenic differentiation of C_3H10T1/2 mesenchymal stem cells

目的:初步分析丝裂原活化蛋白激酶家族成员ERK1/2在BMP9诱导间充质干细胞成骨分化中的作用及可能机制。方法:BMP9重组腺病毒感染C3H10T1/2细胞,Western blot检测ERK1/2激酶的磷酸化。ERK1/2的特异性抑制剂PD98059阻断ERK1/2活性,细胞化学染色和活性定量检测碱性磷酸酶(Alkaline Phosphatase,ALP)的表达情况;Western blot检测骨桥蛋白(Osteopontin,OPN)表达,茜素红染色检测钙盐沉积情况;荧光素酶报告基因实验检测Smad经典途径的变化。RNA干扰抑制ERK1/2表达,分析其对于BMP9诱导的C3H10T1/2细胞ALP活性和钙盐沉积的影响。结果:BMP9可以促进ERK1/2激酶的磷酸化;ERK1/2抑制剂PD98059可增强由BMP9诱导的C3H10T1/2细胞早期和晚期成骨分化,并促进由BMP9诱导的Smad经典途径的活化;RNA干扰导致ERK1/2基因沉默同样也可促进BMP9诱导的C3H10T1/2细胞成骨分化。结论:BMP9可以促进ERK1/2蛋白激酶的活化,而阻断ERK1/2蛋白激酶可进一步增强BMP9诱导的C3H10T1/2细胞成骨分化。

Objective：To analysis the functional role of ERK1/2 kinase in BMP9 - induced osteogenic differentiation of C3H10T 1/2 mesenchymal stem cells. Methods： CsHIOT 1/2 cells were infected by recombinant adenovirus expressing BMPg, phosphorylated/activated form of ERK1/2 kinase were determined by Western blot. Mter treating C3H10T 1/2 cells with ERK1/2 specific inhibitor PD98059, the early osteogenic marker ALP activity was detected by quantitative and cell chemistry assay, OPN expression was determined by western blot, calcium deposition was detected by Alizarin Red S staining, the change of Smad pathway was ass^sed by hiciferase reporter assay. Knockdown of ERK1/2 expression was achieved by RNAi, and then the effects of ERK1/2 gene silence on BM1x） - indcued osteogenic differentiation was analyzed in vitro. Results： BMP9 promoted the phosphorylated form of ERK1/2 kinase in GsH10T1/2 ceils. ERK1/2 specific inhibitor PD98059 dose - dependently accelerated BMP9 - indcued ALP activity, enhanced OPN expression, and increased calcium deposition of CsH10T 1/2 cells. Gene silence of ERK1/2 by RNA interference also led to an increase of ALP activity and calcium deposing. Conclusion： BMP9 was able to activate ERK1/2 kinase and inhibition of ERKI/2 could effectively promote BMP9 - incued osteogenic differentiation of CsH10TI/2 mesenchymal stem cells.