固醇调节元件结合蛋白1c抑制骨骼肌胰岛素受体底物1基因表达的分子机制

The molecular mechanism of sterol regulatory element binding protein-lc suppressing insulin receptor substrate-1 in skeletal muscle

目的探讨固醇调节元件结合蛋白lc（SREBP—lc）抑制骨骼肌细胞胰岛素受体底物1（IRS-1）基因转录的具体分子机制。方法将SREBP-1c表达质粒、IRS-1启动子荧光素酶报告质粒及海肾质粒胸腺嘧啶核苷激酶启动子{pRL-TK）采用Lipofectamin2000共转染L6细胞，以pCDNA3．1空质粒为对照，36h后裂解细胞按双荧光素酶报告基因检测试剂盒说明书检测荧光素酶。将表达SREBP-1c的腺病毒感染L6肌管细胞，以表达绿色荧光蛋白（GFP）的腺病毒作对照，感染48h后收取细胞抽提核蛋白，进行凝胶迁移滞后实验（EMSA）和染色质免疫共沉淀（CHIP）实验，分析SREBP-1c转录因子与IRS-1启动子区域的相互作用。组间比较采用双因素方差分析。结果荧光素酶截断实验显示IRS．1启动子区域-450～-210bp为SREBP-1c的作用范围。序列分析显示IRS-1启动子-302～-292bp处存在SREBP-1c潜在的结合序列GCCTCCCGAG，该序列突变为TGTYAAATTA，荧光素酶实验显示SREBP．1c抑制野生型IRS-1启动子活性，而对突变型IRS-1启动子无抑制作用（分别为7．03±1．28比19．09±2．45、3．55±1．68比3．96±1．09，F=114．437、0．251，均P〉0．05）；EMSA结果显示SREBP-lc蛋白与野生型探针直接结合，而不被突变探针所竞争。CHIP结果显示SREBP-1c可直接结合到IRS-1的启动子区域，定量分析显示PA组细胞SREBP-1c结合到IRS．1启动子区域的量为对照组的2倍，SREBP-1c过表达组为GFP组的5倍（分别为2．15±0．03比1．07＋±0．24，5．48±1．28比0．86±0．19，t=6．8775、5．495，均P〈0．05）。结论SREBP-1c通过直接结合到IRS．1启动子区域的非典型固醇反应元件序列抑制IRS-1基因转录表达，继而影响胰岛素信号通路的转导。

Objective To investigate the molecular mechanism of sterol regulatory element binding protein-lc （SREBP-lc） suppressing insulin receptor substrate-I（IRS-1） in skeletal muscle cells. Methods Luciferase plasmid for rat IRS- 1 promoter, expression plasmid of SREBP- lc and pRL-TK renilla plasmid were cotransfected into L6 cells using Lipofectamine 2000 （Invitrogen）. After transfection for 36 h, luciferase activity was measured using the dual-luciferase reporter assay system following the manufacturer＇s instructions. L6 myotubes were infected with adenoviral vectors expressing SREBP-lc. Adenovirus expressing green fluorescent protein （GFP） was used as control. The cells were harvested for nucleus protein after infection for 48 h. Electrophoretic mobility shift assay （EMSA） was performed using a Light Shift Chemiluminescent EMSA Kit. Chromatin immunoprecipitation assay was performed by CHIP Kit. The interaction between the transcription factor SREBP-lc and the promoter region of IRS-1 was assessed by above experiments. Differences among the groups were determined using two-way ANOVA. Results The luciferase deletion studies suggested the region from -450 to -210 bp on the IRS-I promoter was a potential target region for SREBP-lc. Sequence analysis showed that the target region of the rat IRS-1 promoter gene contained a potential binding site （GCCTCCCGAG）, which was located between- 302 and- 292 bp. PCR site-directed mutagenesis （TGTTAAATTA） was generated and analyzed using a luciferase assay. The transcriptional activity of wild-type IRS-1 promoter was dramatically down-regulated by SREBP-lc, whereas SREBP-lc had no effect on the IRS-1 promoter bearing mutation of SREBP-lc binding site （7.03 ± 1.28 vs 19.09±2.45,3.55±1.68 vs 3.96±1,09, F=114.437,0.251, all P〉0.05）. The EMSA results showed that the labeled wild-type probe successfully formed a complex with nuclear proteins. But the unlabelled mutant probe did not interfere the complex. The results from CHIP assay showed that SREBP-lc could bind directly to the IRS- 1 promoter region. The quantity of SREBP- le protein binding to the IRS- 1 promoter was two times of control under PA conditions in L6 cells or five times of control when SREBP-lc was over-expressed （2.15±0.03 vs 1.07±0.24,5.48±1.28 vs 0.86±0.19,t=6.877,5.495,all P〈0.05）. Conclusion SREBP-lc could directly bind to the atypical SRE sequence in the promoter region of IRS- 1, suppressing IRS- 1 expression and the subsequent insulin signaling pathway.