Regulation of gene expression and functional analysis of an insulin-inducible transcription factor

大鼠脂肪酸合成酶（FAS）基因在大鼠肝脏中的转录可以通过喂养高碳水化合物饮食来调节。已鉴定出位于大鼠FAS基因启动子上的碳水化合物反应元件（ChoRE）。我们克隆了一个基本的螺旋-环-螺旋蛋白，分裂和毛相关蛋白增强子-2（SHARP-2），作为ChoRE结合蛋白的cDNA。北方印迹分析显示，当给正常大鼠喂食高碳水化合物饮食或给糖尿病大鼠施用胰岛素时，SHARP-2 mRNA的水平增加。在原代培养的大鼠肝细胞中，胰岛素迅速诱导SHARP-2 mRNA的积累，并通过渥曼青霉素和LY 294002阻断该增加。此外，核运行分析显示，大鼠SHARP-2基因的转录被胰岛素诱导。因此，我们得出结论，胰岛素诱导大鼠SHARP-2基因的转录通过磷酸肌醇3-激酶途径。接下来，我们研究了SHARP-2 mRNA的表达是否是调节性的。 ...更多信息 在其他胰岛素应答细胞中，如3 T3-L1脂肪细胞和L 6肌管中，胰岛素可使其活化。我们发现SHARP-2也是一种胰岛素诱导的转录因子，环腺苷酸（cAMP）也以相加的方式增加SHARP-2 mRNA在两种细胞中的表达，然后检测SHARP-2转录抑制因子对大鼠磷酸烯醇丙酮酸羧激酶（PEPCK）基因表达的影响。腺病毒介导的SHARP-2在H4 IIE细胞和原代培养的大鼠肝细胞中的过表达导致PEPCK mRNA水平的降低。最后，当SHARP-2表达质粒与各种报告质粒一起瞬时转染到MH 1C 1细胞中时，PEPCK报告质粒的启动子活性特异性降低。基于以上结果，我们认为SHARP-2是PEPCK基因表达的潜在抑制因子，并报道了促性腺激素对大鼠SHARP-2基因表达的调控以及该基因转录调控机制的分子克隆和鉴定。少

Transcription of the rat fatty acid synthase (FAS) gene in the rat liver can be regulated by feeding a high-carbohydrate diet. A carbohydrate response element (ChoRE) located on the rat FAS gene promoter has been identified. We cloned a basic helix-loop-helix protein, enhancer of split-and hairy-related protein-2 (SHARP-2), as the cDNA of ChoRE-binding proteins. Northern blot analysis revealed that the levels of SHARP-2 mRNA increase, when a high-carbohydrate diet is fed to normal rats or when insulin is administered to diabetic rats. In primary cultured rat hepatocytes, insulin rapidly induced an accumulation of SHARP-2 mRNA and the increase was blocked by wortmannin and LY294002. In addition, nuclear run-on assay revealed that transcription of the rat SHARP-2 gene was induced by insulin. Thus, we conclude that insulin induces the transcription of the rat SHARP-2 gene via a phosphoinositide 3-kinase pathway. Next, we examined the issue of whether the expression of SHARP-2 mRNA is regu … More lated by insulin in other insulin-responsive cells, such as 3T3-L1 adipocytes and L6 myotubes. We found that SHARP-2 is also an insulin-inducible transcription factor in these cells and cyclic AMP also increased the level of SHARP-2 mRNA in both cells in an additive manner.Then, the influence of the SHARP-2 transcriptional repressor on the expression of rat phosphoenolpyruvate carboxykinase (PEPCK) gene was examined. The adenovirus-mediated overexpression of SHARP-2 in H4IIE cells and primary cultured rat hepatocytes led to a decrease in the levels of PEPCK mRNA. Finally, when a SHARP-2 expression plasmid was transiently transfected with various reporter plasmids into MH1C1 cells, the promoter activity of a PEPCK reporter plasmid was specifically decreased. Based on these findings, we concluded that SHARP-2 is a potential repressor of PEPCK gene expression.We also reported regulation of expression of the rat SHARP-2 gene by gonadotropins and molecular cloning and characterization of transcriptional regulatory mechanism of the gene. Less