14-3-3参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径研究(英文)  

14-3-3 Is Involved in ERK1/2 Signaling Pathway of Rat Vascular Smooth Muscle Cells Proliferation Induced by Apelin-13

作　　者：潘伟男[1,2] 李峰[1,3] 毛小环[1,4] 秦旭平[1] 邓水秀[1,2] 封芬[1] 陈锋[1] 李兰芳[1] 廖端芳[3] 陈临溪[1] 

机构地区：[1]南华大学药物药理研究所,湖南省高等学校药物蛋白质组学重点实验室,湖南省动脉硬化学重点实验室,衡阳421001 [2]湖南省食品药品职业学院,长沙410001 [3]湖南中医药大学,长沙410001 [4]湖南省环境生物职业技术学院,衡阳421001

出　　处：《生物化学与生物物理进展》2011年第12期1153-1161,共9页Progress In Biochemistry and Biophysics

基　　金：supported by grants from The National Natural Scientific Foundation of China(30901577);The Hengyang Technological Bureau of China(2009KJ14);Ministry of Education of Returned Overseas Students to Start Research and Fund Projects(20091590);Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province~~

摘　　要：本室以前已经报道了G蛋白偶联受体APJ的内源性配体多肽,apelin-13,通过激活ERK1/2促进大鼠血管平滑肌细胞增殖.本文研究14-3-3信号蛋白是否参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径,探讨apelin/APJ系统的细胞信号转导机制.组织贴块法培养大鼠胸主动脉VSMCs;Western blotting方法检测14-3-3、pRaf-1、Raf-1、pERK1/2、ERK1/2、cyclinD1、cyclinE的表达;MTT方法观察14-3-3抑制剂Difopein对VSMCs的增殖作用;免疫共沉淀方法检测14-3-3和Raf-1蛋白复合物的形成.Western blotting方法结果显示,apelin-13(0、0.5、1、2、4μmol/L)浓度依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,以2μmol/L最为明显;2μmol/L apelin-13时间依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,在4 h增加最为显著;14-3-3蛋白抑制剂Difopein明显抑制apelin-13诱导的Raf-1磷酸化、ERK1/2磷酸化、cyclinD1及cyclinE表达;免疫共沉淀方法发现apelin-13诱导14-3-3与Raf-1结合增加,而Difopein明显抑制两者结合;MTT法显示Difopein明显抑制apelin-13诱导的血管平滑肌细胞增殖.上述结果表明,Apelin-13通过14-3-3/Raf-1复合物-ERK1/2信号转导通路促进大鼠血管平滑肌细胞增殖.Previously,we found that G protein-coupled receptor APJ endogenous ligand apelin-13 stimulates vascular smooth muscle cells（VSMC） proliferation mediated in part by PKC-PI3K-ERK1/2-cyclinD1 signaling cascades.In this study,Raf-1-14-3-3 signaling in rat VSMCs proliferation stimulated by apelin-13 was further investigated.Cell proliferation was measured with MTT assay.Expression of PI3K,phospho-PI3K,Raf-1,phospho-Raf-1,ERK1/2,phospho-ERK1/2,cyclinD1 and cyclinE were detected byWestern blotting.14-3-3 protein combining with Raf-1 was detected by immunoprecipitation.Here,we demonstrated that apelin-13 increased the expression of 14-3-3,Raf-1 phosphorylation and ERK1/2 phosphorylation in a concentration-dependent and time-dependent manner at 0～4 μmol/L and 0～48 h.14-3-3 inhibitor Difopein decreased the apelin-13-induced Raf-1 phosphorylation,ERK1/2 phosphorylation,expression of cyclinD1 and cyclinE.Furthermore,apelin-13 promoted the combination of 14-3-3 protein and Raf-1,Difopein significantly inhibited the combination of 14-3-3 and Raf-1 stimulated by apelin-13.Similarly,Difopein significantly inhibited the VSMCs proliferation stimulated by apelin-13.Our results revealed that Raf-1 ＋14-3-3-ERK1/2 signaling cascades mediated the effect of apelin-13 on rat VSMCs proliferation.

关 键 词：APELIN APJ 14-3-3 血管平滑肌细胞 ERK1/2 RAF-1 

分 类 号：R966[医药卫生—药理学]