Irrcher Isabella, Ljubicic Vladimir, Kirwan Angie F, Hood David A
Department of Biology, York University, Toronto, Ontario, Canada.
PLoS One. 2008;3(10):e3614. doi: 10.1371/journal.pone.0003614. Epub 2008 Oct 31.
The mechanisms by which PGC-1alpha gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1alpha using AICAR, an activator of AMPK, that is known to increase PGC-1alpha expression. A 2.2 kb fragment of the human PGC-1alpha promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-kappaB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1alpha promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at -495 within the PGC-1alpha promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1alpha promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1alpha promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1alpha promoter activity. The USF-1-mediated increase in PGC-1alpha promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1alpha gene expression. This could represent a potential therapeutic target to control PGC-1alpha expression in skeletal muscle.
在骨骼肌中，PGC-1α基因表达的调控机制在很大程度上仍不明确。因此，我们试图利用AICAR（一种已知可增加PGC-1α表达的AMPK激活剂）来研究PGC-1α的转录调控。克隆了人PGC-1α启动子的一个2.2 kb片段，序列分析表明，这个无TATA的序列包含推定的共有位点，包括一个GC盒、一个CRE、几个IRS、一个SRE、GATA、MEF2、p53、NF-κB和E盒结合蛋白的结合位点。AMPK激活24小时可增加PGC-1α启动子活性，并伴随mRNA表达增加。基于凝胶迁移分析显示GATA/E盒DNA结合增加，AICAR对转录激活的作用是由PGC-1α启动子内-495处的一个重叠GATA/E盒结合位点介导的。启动子中GATA/E盒结合位点内的E盒突变降低了基础启动子活性，并完全消除了AICAR的作用。超迁移分析确定USF-1是一种可能参与调节PGC-1α启动子活性的DNA结合转录因子，这在体内通过染色质免疫沉淀得到证实。单独过表达GATA-4或USF-1分别使p851 PGC-1α启动子活性增加1.7倍和2.0倍，而GATA-4和USF-1共表达导致PGC-1α启动子活性的累加增加。USF-1介导的PGC-1α启动子激活增加在mRNA水平上也有类似的增加。我们的数据确定了一种新的AMPK介导的调节途径，该途径调节PGC-1α基因表达。这可能代表了一个控制骨骼肌中PGC-1α表达的潜在治疗靶点。
