FAK和ILK介导骨桥蛋白诱导的血管平滑肌细胞黏附和迁移

FAK and ILK Regulate Adhesion and Migration of Vascular Smooth Muscle Cells Induced by Osteopontin

黏着斑激酶(FAK)和整合素偶联激酶(ILK)是整合素信号途径中的重要信号转导分子,为阐明两者在血管平滑肌细胞(VSMC)黏附和迁移中的作用,以骨桥蛋白(OPN)作为VSMC黏附和迁移的诱导剂,检测其对FAK和ILK磷酸化以及对两者之间结合的影响.在此基础上,用FAK磷酸化特异性抑制剂黏着斑相关非激酶(FRNK)或ILK反义RNA分别阻断FAK磷酸化或ILK表达,进一步探讨两者在VSMC黏附和迁移中所起的作用.结果显示,OPN诱导可促进FAK磷酸化,诱导10min后FAK磷酸化水平升高到对照组的2·4倍;与此同时,ILK的磷酸化受到抑制,30min降至对照细胞的44·6%.OPN诱导FAK磷酸化的同时使FAK与ILK的结合减少.外源性FRNK在VSMC中的过表达显著降低FAK的磷酸化水平,促进ILK磷酸化和FAK与ILK之间的结合,抑制VSMC的黏附和迁移.用ILK反义RNA抑制ILK表达使VSMC在OPN上的黏附增加1·8倍,迁移细胞数降低45·5%.结果提示,FAK和ILK介导OPN诱导的VSMC黏附和迁移过程,两者通过对同一刺激信号产生不同的磷酸化变化而对VSMC的黏附和迁移产生不同的影响.

Focal adhesion kinase （FAK） and integrin-linked kinase （ILK） have been emphatically implicated in integrin signaling. To investigate their roles in the adhesion and migration of vascular smooth muscle ceils （VSMCs） induced by osteopontin （OPN）, the phosphorylation of FAK and ILK, and their interaction were detected by immunoprecipitation and co-immunoprecipitation. Then the adhesion and migration of VSMCs were evaluated, which were transfected by the recombinants of FRNK （an endogenous inhibitor of FAK activation） or the vector expressing ILK antisense RNA. The results showed that there was increase in the level of phosphorylated FAK, and it was increased about 2.4 times after VSMCs were treated with OPN for 10 min, compared with that of control. At the same time, there was decrease in the level of phosphorylated ILK, which was reduced to 44.6 % of control in VSMCs stimulated by OPN for 30 min. OPN stimulation also inhibited the association of FAK and ILK. Overexpressing FRNK suppressed obviously the phosphorylation of FAK and the adhesion and migration of VSMCs, but stimulated the phosphorylation of ILK and the association of FAK and ILK induced by OPN. The adhesion activity of VSMCs transfected by ILK antisense RNA was increased to 1.8 folds, compared with that of untransfected VSMCs, but the migration activity was reduced to 54.5 % of untransfectants. These results suggest that FAK and ILK are involved in regulating the adhesion and migration activities of VSMCs induced by OPN, and OPN exerts different effect on the adhesion and migration of VSMCs through regulating differently the phosphorylation of FAK and ILK.