阻断ERK1／2激酶可增强骨形态发生蛋白9诱导的间充质干细胞成骨分化

Inhibition of ERK1/2 Kinase Enhances BMP9-induced Osteogenic Differentiation of Mesenchymal Stem Cells

骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)具有很强的诱导间充质干细胞定向成骨分化的能力.但对于其所涉及的相关分子机理了解并不深入.利用BMP9重组腺病毒感染间充质干细胞,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,或以RNA干扰抑制ERK1/2表达,通过体外细胞实验和体内动物实验,初步分析和揭示ERK1/2对于BMP9诱导的间充质干细胞成骨分化的调控作用及其可能机制.结果发现:BMP9可以促进ERK1/2激酶的磷酸化,ERK1/2抑制剂PD98059可增强由BMP9诱导的碱性磷酸酶(alkaline phosphatase,ALP)活性、骨桥蛋白(osteopontin,OPN)表达和钙盐沉积,并促进由BMP9诱导的Runx2基因的表达和转录活性,以及Smad经典途径的活化;而RNA干扰导致ERK1/2基因沉默同样也可进一步促进BMP9诱导的ALP活性和钙盐沉积,并促进BMP9诱导的间充质干细胞在裸鼠皮下异位成骨.因此,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诱导的成骨分化,ERK1/2极可能对于BMP9诱导的间充质干细胞成骨分化起着负向调控作用.

In our previous reports, BMP9 has shown potent function to induce osteogenic differentiation of mesenchymal stem cells, however, the underlying molecular mechanism of BMP9-induced osteogenesis is needed to be deep explored. In this study, BMP9 was introduced into mesenchymal stem cells by recombinant adenoviruses protocol, then, in vitro and in vivo assays were conducted to evidence whether BMP9 can regulate osteogenic differentiation of mesenchymal stem cells through ERK1/2 kinase pathway. The results showed that BMP9 can activate ERK1/2 kinase through increase the phosphorylated form of ERK1/2 kinase. ERK1/2 kinase inhibitor PD98059 can increase the ALP activity, OPN expression and calcium deposition of C3H10T1/2 cells induced by BMP9. Furthermore, PD98059 also led to enhancement of Runx2 activity and activation of canonical Smad pathway stimulated by BMP9. When ERK1/2 kinase was silenced by RNA interference, BMP9-activated Smad pathway was further enhanced. Moreover, ALP activity, calcium deposition and in invo ectopic bone formation were accordingly increased along with knockdown of ERK1/2 kinase. Taken together, those results intensively suggested that BMP9 can activate ERK1/2 kinase, and inhibition of ERK1/2 kinase can enhance BMP9-induced osteogenic differentiation of mesenchymal stem cells. ERK1/2 are highly capable of negatively regulate BMP9-induced osteoblastic differentiation of mesenchymal stem cells.