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.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
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~~
