G蛋白耦联受体激酶结合蛋白1通过调节细胞外调节激酶1/2的活性促进成骨细胞迁移

GIT1 promotes osteoblast migration by regulating ERK1/2 activity in focal adhesions

目的探索G蛋白耦联受体激酶结合蛋白1(GITI)在成骨细胞迁移中的作用,并分析其机理。方法通过Western blot方法检测GIT1蛋白在鼠的成骨细胞内的表达；用免疫荧光染色方法确定：在血小板衍生生长因子(PDGF)不刺激和刺激的条件下,GIT1和细胞外调节激酶1/2(ERK1/2)在成骨细胞内的位置；用共同免疫沉淀的方法测定GIT1和ERK1/2相互结合,并且用免疫荧光双染的方法确定这两种蛋白相互结合的位置；用包含GIT1-RNA发夹结构的腺病毒感染成骨细胞后,用免疫荧光染色方法确定磷酸化ERK1/2(pERK1/2)在成骨细胞内的位置,用划痕愈合法检测在PDGF刺激下的迁移能力。结果在成骨细胞内,PDGF刺激导致了GIT1和ERK1/2的相互结合,并且这种结合发生在成骨细胞的局部粘附内。包含GIT1-RNA发夹结构的腺病毒明显抑制了pERK1/2招募至成骨细胞局部粘附内以及PDGF所刺激的成骨细胞的迁移。结论在PDGF刺激下,GIT1招募pERK1/2至成骨细胞的局部粘附内,从而促进成骨细胞的迁移。

Objective To study the function and mechanism of GIT1 （G protein coupled receptor kinase interacting protein 1） in osteoblast migration. Methods GIT1 and ERK1/2 （Extracellular Signal-regulated kinase 1/2） were detected in mice primary osteoblasts. The localizations of GIT1 and ERK1/2 were determined by immunofluorescence stain with or without PDGF （platelet-derived growth factor） stimulation. The association of GIT1 and ERK1/2 was analyzed by co-immunoprecipitation and western blot. After stimulation, the co-localization of GIT1 and pERK1/2 in osteoblasts was detected by double-immunofluorescence stain. The pERK1/2 localization was detected by immunofluorescence stain after GIT1 RNAh adenovirns infection of osteoblasts. The role of this association was determined by wound healing assay. Results The co-immunoprecipitation results showed that GIT1 interacted with ERK1/2 in osteoblasts induced by PDGF and this association occurred in focal adhesions. GIT1 RNAh adenovirus significantly inhibited the pERK1/2 translocation to focal adhesions and osteoblast migration induced by PDGF. Conclusion GIT1 associates with ERK1/2 in osteoblasts, which is required for pERK1/2 translocation to focal adhesions and osteoblast migration.