Caveolae/caveolin-1/ERK_(1/2)信号通路在降钙素基因相关肽抑制血管平滑肌细胞增殖中的作用(英文)  

Inhibitory Effects of CGRP on Vascular Smooth Muscle Cell Proliferation: Role of Caveolae/caveolin-1/ERK_(1/2) Signal Pathway

作　　者：谌赟[1] 戴忠[2] 刘彦梅[1] 田海红[1] 邓水秀[1] 陈临溪[1] Wang H Donna 秦旭平[1] 

机构地区：[1]南华大学药物药理研究所,动脉硬化学湖南省重点实验室,衡阳421001 [2]广东医学院药理教研室,东莞523808 [3]Deportment of Medicine,Michigan State University,East Lansing,MI 48824,USA

出　　处：《生物化学与生物物理进展》2013年第5期445-453,共9页Progress In Biochemistry and Biophysics

基　　金：supported by grants from The National Natural Science Foundation of China(30572192,81173060);Aid program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province~~

摘　　要：业已证明,Caveolae及其蛋白caveolin-1参与了细胞膜的胆固醇转运和细胞膜的信号转导.我们前期工作发现降钙素基因相关肽(CGRP)抑制血管平滑肌细胞(VSMC)增殖的信号通路与抑制ERK1/2活性和上调caveolin-1表达有关.本文研究Caveolae及caveolin-1在CGRP抑制VSMC增殖中的作用,进一步研究caveolin-1表达增加是否有直接抑制ERK1/2信号激酶活性的作用.采用大鼠主动脉贴块法培养VSMC,取3～10代VSMC用于实验,10%小牛血清(FBS)用于刺激VSMC增殖,用β-环糊精(cyclodextrin)或菲律宾菌素(filipin)剥夺胆固醇破坏Caveolae结构;MTT法和流式细胞仪用于检测细胞增殖;蛋白质印迹和免疫共沉淀法分别用于检测目的蛋白的表达或蛋白质间相互作用.结果显示,CGRP呈时间和浓度依赖性显著抑制10%FBS诱导的VSMC增殖.细胞Caveolae结构的破坏能降低CGRP抑制VSMC增殖作用,同时也增加了ERK1/2的磷酸化;β-环糊精孵育细胞能降低caveolin-1的表达.免疫共沉淀发现10%FBS和/或CGRP共同孵育细胞对非磷酸化ERK1/2与caveolin-1的结合无差别,但10%FBS能降低磷酸化ERK1/2与caveolin-1的结合,CGRP预孵育细胞能增加这两者的相互作用.结果揭示,Caveolae及caveolin-1可以正调控CGRP抑制VSMC增殖作用,其机制可能与CGRP增加caveolin-1与p-ERK1/2在Caveolae的结合,并抑制p-ERK1/2核转位作用有关.Caveolae and caveolin-1 participate in the transportation of cholesterol and cell signal transduction. Our previous studies showed that the inhibitory effect of calcitonin gene-related peptide （CGRP） on the vascular smooth muscle cells （VSMCs） was related to decreasing the activity of extracellular signal-regulated kinase （ERK）1/2 and increasing the expression of caveolin-1. In the present study, we investigated the role of caveolae and caveolin-1 in proliferation of VSMCs and whether there are interaction between the caveolin-1 and ERK1/2 in the inhibitory effect of CGRP signal pathway. VSMCs were prepared from thoracic aorta of male Sprague-Dawley rat by the classic explants method, the passage 3 - 10 VSMCs were used for the present study. 10% fetal bovine serum （FBS） was employed as a stimulus for the proliferation of VSMCs. β-Cyclodextrin or filipin was used to deplete cholesterol in the caveolae. Proliferation of VSMCs was estimated by methylthiazoletrazoliurn （MTT） assay and Flow Cytometry. Western blotting and co-immunoprecipitation were used to determine interaction of p-ERKI/2 or caveolin-1. Results showed that CGRP significantly inhibited VSMC proliferation and down-regulated phosphorylation of ERKla. Incubation of VSMCs with β-cyclodextrin or filipin promoted cells proliferation, up-regulated phosphorylation of ERKxa, attenuated the inhibitory action of CGRP on VSMC proliferation and decreased caveolin-1 expression. Pretreatment with CGRP increased the direct binding of cavolin-1 with phosphorylated（p-） ERK1/2 but not non-phosphorylated ERK1/2 in the presence of 10%FBS. Our results revealed that caveolae and caveolin-1 may contribute to the inhibitory effect of CGRP on the VSMC proliferation, and the mechanism may be related to the deceased nuclei translocation of p-ERK1/2 because of the increased binding of caveolin-1 with p-ERK1/2.

关 键 词：CAVEOLAE CAVEOLIN-1 降钙素基因相关肽 细胞外信号调节酶 血管平滑肌细胞 增殖 

分 类 号：R322.12[医药卫生—人体解剖和组织胚胎学]