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

胰岛素诱导转录因子的基因表达调控和功能分析

基本信息

批准号：
15590933
负责人：
YAMADA Kazuya
金额：
$ 2.3万
依托单位：
University of Fukui (2004)福井医科大学 (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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

高碳水化合物饮食可调节大鼠肝脏脂肪酸合成酶（FAS）基因的转录。在大鼠FAS基因启动子上发现了一个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，即分裂和毛发相关蛋白-2 （SHARP-2）的增强子，作为chore结合蛋白的cDNA。Northern blot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注射胰岛素时，夏普-2 mRNA水平升高。在原代培养的大鼠肝细胞中，胰岛素可迅速诱导SHARP-2 mRNA的积累，而这种积累可被wortmannin和LY294002阻断。此外，核运行试验显示胰岛素诱导大鼠SHARP-2基因的转录。因此，我们得出结论，胰岛素通过磷酸肌苷3激酶途径诱导大鼠夏普-2基因的转录。接下来，我们研究了其他胰岛素应答细胞（如3T3-L1脂肪细胞和L6肌管）中夏普-2 mRNA的表达是否受胰岛素调控。我们发现在这些细胞中夏普-2也是胰岛素诱导的转录因子，并且环状AMP也以加性的方式增加了两种细胞中夏普-2 mRNA的水平。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H4IIE细胞和原代培养的大鼠肝细胞中的过表达导致PEPCK mRNA水平降低。最后，当SHARP-2表达质粒与各种报告质粒短暂转染到MH1C1细胞时，PEPCK报告质粒的启动子活性特异性降低。基于这些发现，我们得出结论，SHARP-2是PEPCK基因表达的潜在抑制因子。我们还报道了促性腺激素对大鼠夏普-2基因表达的调控，以及该基因的分子克隆和转录调控机制的表征。少