AMPK、p38 MAPK信号通路参与调控电刺激诱导骨骼肌细胞PGC-1α基因表达

AMPK、p38 MAPK Signaling Pathways Regulate Electrical Stimulation-mediated PGC-1α Gene Expression and Activity in Skeletal Muscle Cells

目的采用急性电刺激(acute electrical stimulation)C2C12成熟肌管细胞模型,结合使用相关信号激酶通路特异性抑制剂,探究急性电刺激所诱发的细胞内信号激酶对PGC-1α基因表达的调控作用。旨在从细胞水平揭示肌肉收缩刺激调控PGC-1α基因表达的分子机制。方法体外培养小鼠成肌细胞系C2C12细胞,采用脂质体法分别转染含小鼠PGC-1α启动子DNA全序列的质粒(p2533)及装载有能够与PGC-1α全长基因序列相结合GAL4DNA结合区域的p Bind载体,细胞在转染后分化5-6 d形成成熟肌管,5 Hz,10 V强度急性电刺激2 h后即刻收集各组细胞,进行总蛋白或双荧光素酶检测。抑制剂处理组分别采用40μM Compound C(CC,AMPK抑制剂)与10μM BIRB796(BIRB,p38抑制剂)处理细胞。结果与对照组(CON)相比,电刺激组(STIM)细胞PGC-1α启动子活性显著增高52%(P<0.05),PGC-1α辅激活转录活性显著增高35%(P<0.05),同时伴随PGC-1α蛋白由细胞质向细胞核迁移的现象。此外,与CON组相比,STIM组细胞AMPK、p38信号激酶通路磷酸化水平显著上调(P<0.05),在分别采用AMPK、p38抑制剂处理后,其磷酸化水平显著受到抑制(P<0.01)。结论急性电刺激可诱导骨骼肌细胞PGC-1α转录水平以及翻译后蛋白水平活性上调,这一作用部分是通过AMPK或p38信号通路实现的。

Objective In order to investigate the role of divergent contractile activity - induced intracellular signaling path- ways on the mouse PGC - 1αgene expression, mature C2C12 muscle cells were applied by acute electrical stimulation with or without some related protein kinase inhibitors treatment. Methods C2C12 murine skeletal muscle cells were cultured, C2C12 myoblasts were transiently transfected with 2 kb mouse PGC - 1α promoter plasmid or a chimeric gene encoding full - length PGC - 1 α fused to the DNA -binding domain （DBD） of GAlA of pBind vector, following 5 - 6 days of differentia- tion,C2C12 myotubes were subjected to 2 h of electrical stimulation -evoked contractile activity （CA） （5 Hz, 10 V ） , and protein or enzyme extracts were made immediately. Cells were treatment with or without 40 μM Compound C （ CC,AMPK in- hibitor） or 10 μM BIRB796 （BIRB,p38 inhibitor）. Results Compare to control group,PGC -1α promoter activity and the ability of PGC -1α coactivation were significantly enhanced by 52% and 35% respectively in response to acute contractile activity （ P 〈 0. 05 ） , associated with the translocation of PGC - 1 α protein from the cytosol to nuclear. Acute contractile activ- ity also resulted in a significant augmented the phosphorylation of AMPK and p38 （ P 〈 0. 05 ） , and the phosphorylation of AMPK and p38 were dramatically reduced with their inhibitors treatment. In addition, the contractile activity - induced in- creases in both PGC - 1αtranscription and activity were also inhibited by CC or BIRB （ P 〈 0. 01 ）. Conclusion The activa- tion of AMPK or p38 is necessary for the regulation of contractile activity - induced PGC - 1α gene expression at transcrip-tional and post- translational level.