平滑肌细胞迁移的肌球蛋白轻链非磷酸化途径

为了阐明平滑肌细胞迁移存在肌球蛋白轻链非磷酸化调节途径,研究花生四烯酸(arachidonicacid,AA)对肌球蛋白轻链非磷酸化状态下平滑肌细胞迁移的影响及其相关的信号传导途径.经Boyden小室跨膜迁移实验发现,AA对培养的兔血管平滑肌SM3细胞具有明显的诱导迁移作用.然而,当预先用10μmolL肌球蛋白轻链激酶(myosinlightchainkinase,MLCK)特异性抑制剂ML7作用SM3细胞后,发现AA对SM3细胞仍然具有明显的诱导迁移作用,并呈剂量依赖性,这种诱导作用可被细胞外信号调节激酶12(ERK12)的特异性抑制剂PD98059或磷脂酶C(PLC)的特异性抑制剂U73122所拮抗.此外,Ⅱ型肌球蛋白抑制剂blebbistatin(BLB)可部分抑制“非磷酸化”状态下AA的诱导迁移作用.经Western印迹检测显示,10μmolLML7可完全抑制SM3细胞中20kD肌球蛋白轻链(MLC20)磷酸化,并且加入AA后MLC20仍为非磷酸化状态.应用免疫荧光染色法观察肌动蛋白在SM3细胞中分布的变化,发现在AA作用下肌动蛋白呈细胞边缘聚集现象,有伪足形成,细胞形态表现为迁移状态.预先用ML7作用后再加入AA,肌动蛋白的分布与上述结果相同.研究结果初步表明,在平滑肌细胞迁移的作用途径中,在MLC磷酸化调节途径受到抑制时,AA可诱导MLC非磷酸化的平滑肌细胞发生迁移,其分子机理可能与ERK12和PLC信号传导途径有关,非磷酸化的肌球蛋白直接参与了该迁移过程.

Signal Pathway of Migration of Smooth Muscle Cells in Myosin Light Chain Without Phosphorylaion

The purpose is to explore the effect of myosin light chain (MLC20) without phosphorylation on migration of smooth muscle cells and its signaling pathway. SM3 cells,a cell line of rabbit aorta,were treated with arachidonic acid (AA) and ML-7,a specific inhibitor of myosin light chain kinase (MLCK). The migration of the cells was measured by Boyden's chamber assay. The MLC20 without phosphorylation was examined by Western blot analysis. Moreover,the distribution of actin was observed in SM3 cells by immunofluorescence staining. To reveal the mechanism,the signaling pathways were investigated by using inhibitors of extracellular signal-regulated kinase 1/2(ERK1/2) and phospholipase C(PLC). The results shown that 20 μmol/L AA was able to promote the migration after the phosphorylation of MLC20 was completely controlled in SM3 cells by 10 μmol/L ML-7. In a dose dependent manner,however,the MLC20 without phosphorylation was not changed in the presence of AA. In morphology,actin was congregated along the brim of the cells and the pseudopod appeared,suggesting that the cells were in a manner of migration. Interestingly,the affect was significantly abrogated by PD98059,a specific inhibitor of ERK1/2, and U73122, a specific inhibitor of PLC. The same result was observed in the presence of ML-7. In conclusion, our finding demonstrated that in the pathways of migration for smooth muscle cells, it is avaliable to migration in the presence of AA when MLC20 phosphorylation was completely controlled,ERK1/2 and PLC maybe involved in the signaling pathways.