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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.
Apelin-13, an endogenous ligand polypeptide of GJ-coupled receptor APJ, has been reported previously in our laboratory to promote the proliferation of rat vascular smooth muscle cells by activating ERK1 / 2.We investigated whether 14-3-3 signaling protein is involved in apelin -13 to promote ERK1 / 2 signal pathway in rat aortic smooth muscle cells to explore the cell signal transduction mechanism of apelin / APJ system.Methods Rat VSMCs were cultured by tissue patch method.The levels of 14-3-3 and pRaf 1, Raf-1, pERK1 / 2, ERK1 / 2, cyclinD1 and cyclinE were detected by MTT assay. The proliferative effects of 14-3-3 inhibitor Difopein on VSMCs were observed by MTT assay. Immunoprecipitation assay was used to detect the expression of 14-3-3 and Raf -1 protein complex.Western blotting results showed that apelin-13 (0,0.5,1,2,4μmol / L) stimulated the expression of 14-3-3 in rat VSMCs in a concentration-dependent manner.Raf-1 and ERK1 / 2 phosphorylation was the most obvious at 2μmol / L, and the expression of 14-3-3 in rat VSMCs was stimulated by 2μmol / L apelin-13 in a time-dependent manner. The phosphorylation of Raf-1 and ERK1 / 2 increased most significantly at 4 h. Difopein, a 3-3 protein inhibitor, significantly inhibited apelin-13-induced phosphorylation of Raf-1, phosphorylation of ERK1 / 2, cyclinD1 and cyclinE; co-immunoprecipitation Apelin-13 induced the increase of 14-3-3 binding to Raf-1, while Difopein significantly inhibited the binding of the two.The MTT assay showed that Difopein significantly inhibited apelin-13-induced proliferation of vascular smooth muscle cells.The above results show that Apelin-13 through 14 -3-3 / Raf-1 complex-ERK1 / 2 signal transduction pathway promotes the proliferation of rat vascular smooth muscle cells.