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

大鼠肝脏中大鼠脂肪酸合成酶(Fas)基因的转录可以通过喂食高碳水化合物饮食来调节。位于大鼠Fas基因启动子上的一个碳水化合物反应元件(Chore)已被鉴定。我们克隆了一个碱性螺旋-环-螺旋蛋白，裂解和毛发相关蛋白的增强子-2(Sharp-2)，作为Chore结合蛋白的cdna。Northern印迹分析显示，当给正常大鼠喂食高碳水化合物食物或给糖尿病大鼠注射胰岛素时，Sharp-2mRNA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-2mRNA的积累，并被Wortmannin和LY294002阻断。此外，核连续实验显示大鼠Sharp-2基因的转录是由胰岛素诱导的。因此，我们得出结论，胰岛素通过磷脂酰肌醇3-激酶途径诱导大鼠夏普-2基因的转录。接下来，我们研究了Sharp-2mrna的表达是否调节…的问题。在其他胰岛素反应细胞中，如3T3-L1脂肪细胞和L6肌管，更多地受到胰岛素的影响。我们发现Sharp-2在这些细胞中也是一种胰岛素诱导的转录因子，环状AMP也以相加的方式增加了两种细胞中Sharp-2的mRNA水平。然后，检测了Sharp-2转录抑制因子对大鼠磷酸烯醇式丙酮酸羧酸激酶(PEPCK)基因表达的影响。腺病毒介导的SHARP-2在H4IIE细胞和原代培养的大鼠肝细胞中过表达，导致PEPCK mRNA水平下降。最后，当将SHARP-2表达载体瞬时导入MH1C1细胞时，PEPCK报告载体的启动子活性明显降低。基于这些发现，我们得出结论，SHARP-2是一个潜在的PEPCK基因表达的抑制因子。我们还报道了促性腺激素对大鼠SHARP-2基因表达的调控，并对该基因的转录调控机制进行了分子克隆和鉴定。较少

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