非磷酸化肌球蛋白在血小板衍生生长因子诱导的血管平滑肌细胞迁移中的作用

为了阐明非磷酸化肌球蛋白在平滑肌细胞迁移中的作用,研究探讨了非磷酸化肌球蛋白是否介导了血小板衍生生长因子(PDGF)诱导豚鼠脑基底动脉平滑肌细胞(GbaSM-4)的迁移。研究结果显示,20ng/ml以下剂量的PDGF可诱导GbaSM-4细胞发生迁移,此时肌球蛋白轻链(MLC20)磷酸化水平无变化。该迁移作用可被肌球蛋白特异性抑制剂blebbistatin所拮抗。应用RNA干扰技术抑制肌球蛋白轻链激酶表达,经免疫印迹检测经果显示,MLC20的磷酸化水平发生了显著下降;但对PDGF诱导的迁移作用无影响;在RNA干扰后blebbistatin也可抑制其迁移作用。体外ATP酶活性测定结果显示,blebbistatin对从平滑肌中提取的非磷酸化肌球蛋白的ATP酶活性有明显的抑制作用,其主要作用位点位于肌球蛋白头的头部S1。上述结果提示,非磷酸化的肌球蛋白参与了PDGF诱导的平滑肌细胞迁移。

Effect of Myosin without Phosphorylation on the Migration of Vascular Smooth Muscle Cells induced by Platelet-derived Growth Factor

In order to demonstrate the effect of myosin without phosphorylation on the migration of smooth muscle cells, we investigated myosin without phosphorylation whether involved in the migration of GbaSM- 4 cells induced by platelet-derived growth factor (PDGF). It was found that lower 20 ng/ml PDGF was able to induce the migration of GbaSM-4 cells, in which myosin light chain (MLC20) with phosphorylation was not necessary. The migration can be depressed by blebbistatin, a specific inhibitor of myosin. The phosphorylation of MLC20 was downregulated in the GbaSM-4 cells by RNA interference (RNAi) targeting myosin light chain kinase (MLCK), examined by Western blot analysis. Moreover, PDGF still was able to induce the migration when RNAi was used, which could be blocked by blebbistatin. The ATPase activity assay for myosin in smooth muscle cells in vitro showed that blebbistatin was able to inhibit the ATPase activity of myosin without phosphorylation in smooth muscle cells. The target was mainly localized at the head of myosin S1. The data demonstrated that the non-phosphorylated myosin may involved in the migration of vascular smooth muscle cells induced by PDGF.