摘要
为阐明整合素 β3 粘着斑激酶 (FAK)信号途径在骨桥蛋白 (OPN)诱导血管平滑肌细胞(VSMC)迁移中的作用 ,用FAK磷酸化特异性抑制剂粘着斑相关非激酶 (FRNK)选择性阻断FAK磷酸化 ,观察对OPN 整合素 β3 相互作用所激活的FAK信号通路的影响及其与OPN诱导VSMC迁移之间的关系 .外源性FRNK在VSMC中的过表达可显著抑制OPN诱导的VSMC迁移 ,使跨膜迁移细胞数下降 5 0 5 8% (P <0 0 5 ) .OPN刺激不但明显诱导FAK表达 ,而且还促进其磷酸化 .外源性FRNK对OPN诱导的FAK磷酸化具有显著抑制作用 ,使磷酸化型FAK水平比相应对照细胞下降5 9 1% ,但其对FAK表达不产生明显的影响 .FRNK还具有下调整合素 β3 表达的作用 ,免疫荧光细胞化学分析结果显示 ,在转染FRNK的VSMC中 ,粘着斑蛋白的磷酸化水平降低 ,粘着斑数量明显减少 .结果提示 ,整合素 β3 FAK是介导VSMC迁移的重要信号途径 ,外源性FRNK通过下调 β3 表达、抑制FAK磷酸化和减少粘着斑蛋白磷酸化及粘着斑形成等机制 ,减弱OPN刺激信号的跨膜转导及沿胞内途径传递 ,发挥抑制OPN促VSMC迁移的效应 .
To determine whether integrin β-3-focal adhesion kinase (FAK) signal pathway was involved in osteopontin (OPN)-induced migration of vascular smooth muscle cells (VSMC), the recombinants of FAK related non-kinase (FRNK), which is an endogenous inhibitor of FAK activation, were transfected into VSMC. The phosphorylation of FAK and the migration activity of VSMC were detected by immunoprecipitation and transmembrane assay, respectively, following OPN stimulating. The results showed that there were increase in the expression and the phosphorylation level of FAK in VSMC stimulated by OPN. VSMC overexpressing FRNK after transfected by pGZ21δxZ-FRNK suppressed the OPN-induced activation of FAK, the level of phosphorylated FAK in transfected VSMC was reduced by 59.1%, compared with the untransfected VSMC. The decline in FAK activity was positively associated with the decrease in the number of transmembrane cells. In addition, FRNK could downregulate expression of integrin β-3. Immunofluorescence revealed lower phosphorylation of focal adhesion proteins and fewer numbers of focal adhesions in VSMC transfected by FRNK. These results suggested that integrin β-3-FAK was the main signal pathway mediated VSMC migration, and overexpression of FRNK might inhibit the signal transduction triggered by OPN-integrin β-3 interaction via the depression of the phosphorylation of FAK and the formation of focal adhesions, and in turn, result in the decrease in migration of VSMC.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2004年第1期131-137,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金资助项目 (No .90 2 0 80 14 )
国家科技攻关计划 (No .2 0 0 2BA75 5C)
北省自然科学基金资助项目 (No .3 0 3 45 5 )~~
关键词
骨桥蛋白
粘着斑激酶
粘着斑相关非激酶
血管平滑肌细胞
迁移
osteopontin, focal adhesion kinase(FAK), FAK related non-kinase, vascular smooth muscle cell, migration
