| Objectives:Migration and proliferation of VSMCs from the medium to the intima layer of the vessel wall is considered to play crucial roles in the pathogenesis of many cardiovascular diseases, such as hypertension, restenosis after coronary angioplasty and atherosclerosis. Platelet-derived growth factor (PDGF) which is secreted by platelets, is a strong mitogen and has a chemotactic activity, its target cell is mainly VSMCs .It critically involves in the procession of vascular remodeling response to injury. PDGF is regarded as the strangest chemotactic agent in vitro by now, which can mediate the migration and proliferation of VSMCs. There are many signaling pathways in which impact the VSMCs migration and proliferation. In the Rho/ROCK pathway, ROCK can phosphorylate the downstream protein, regulate the expression of cell cycle proteins. There are two isoforms of ROCK, ROCKⅠand ROCKⅡ, which have 65% amino acid sequence homology but kinase domain homology as high as 92%. It is believed that they have same functions in the cells. With further researchs show that in some cells the isoforms have different substrates and functions. MAPK family members are involved in cell proliferation and migration via transducting extracellular signal into the cell. MAPK family members include JNK, ERK1/2, p38 and ERK5 in eukaryotic cells. As reported, p38 is involved in the procession of restenosis after vascular injury in vivo, meanwhile ERK1/2 pathway are closely related with cell migration and proliferation, thus p38 and ERK1/2 have a synergistic effect in restenosis. At present, it is not clear that whether ROCK isforms have the same function in VSMCs migration and proliferation. This study is aimed to clarify the functions and molecular mechanism of ROCKⅠand ROCKⅡin cell migration and proliferation.Methods: (1) Genes of ROCKⅠand ROCKⅡwere down-regulated by siRNA transfection. (2)The phosphorylation of MAPK (ERK1/2 and p38) and the expressions of cell cycle related proteins (CyclinD1, CDK4 and PCNA) were detected by western blot. (3)The effects of downregulation of ROCKⅠ/Ⅱgene expression and Y27632 on PDGF stimulated cell migration were detected by Boyden chamber. (4)Cell proliferation was detected by MTT. (5)PDGF-induced cell cycle was dermined by Flowcytometry analysis.Results: (1) The protein expressions of ROCKⅠand ROCKⅡwere decreased by transfecting corresponding siRNA, and PDGF can not influence the efficiency of siRNA. (2) ROCKⅠsiRNA transfection and Y27632 decreased the migration of A7r5 cells, but ROCKⅡsiRNA transfection had no significant effects at the same conditions. (3) In cytosol, down regulation of ROCKⅠand ROCKⅡhad no influence on the phosphorylation of ERK1/2 induced by PDGF, as well as Y27632; only when ROCKⅡwas knockdown leading the expression of p-p38 increased. (4) In nucleus, the level of p-p38 was decreased in ROCKⅠand ROCKⅡsiRNA and inhibitors groups. (5)When ROCKⅠand ROCKⅡwere down regulated, cell proliferation was inhibited. (6)Down regulation of ROCKⅠand ROCKⅡcan alter G1/S phase of cell cycle. (7) Cell cycle-related proteins were inhibited after down regulating ROCK isoforms.Conclusions: (1) ROCKⅠplays a major role in VSMCs migration. (2) ROCKⅠregulates PDGF-induced translocation of p-ERK into nucleus, whereas ROCKⅡcan up regulate p-p38 in cytoplasm, thus ROCK maybe serve as a upstream regulator for MAPK pathway. (3) PDGF-induced cell proliferation and cell cycle alteration can also be regulated by ROCK isoforms. |
PDF Full Text Request