Li Rui, Zhang Rui, Li Yang, Zhu Bing, Chen Wei, Zhang Yan, Chen Guoxun
School of Public Health, Wuhan University, Wuhan, Hubei, 430071, P. R. China; Department of Nutrition, University of Tennessee at Knoxville, Knoxville, TN 37996, USA.
Department of Nutrition, University of Tennessee at Knoxville, Knoxville, TN 37996, USA.
J Nutr Biochem. 2014 Sep;25(9):964-76. doi: 10.1016/j.jnutbio.2014.04.009. Epub 2014 May 22.
The expression of hepatic glucokinase gene (Gck) is regulated by hormonal and nutritional signals. How these signals integrate to regulate the hepatic Gck expression is unclear. We have shown that the hepatic Gck expression is affected by Vitamin A status and synergistically induced by insulin and retinoids in primary rat hepatocytes. We hypothesized that this is mediated by a retinoic acid responsive element (RARE) in the hepatic Gck promoter. Here, we identified the RARE in the hepatic Gck promoter using standard molecular biology techniques. The single nucleotide mutations affecting the promoter activation by retinoic acid (RA) were also determined for detail analysis of protein and DNA interactions. We have optimized experimental conditions for performing electrophoresis mobility shift assay and demonstrated the interactions of the retinoic acid receptor α (RARα), retinoid X receptor α (RXRα), hepatocyte nuclear factor 4α (HNF4α) and chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) in the rat nuclear extract with this RARE, suggesting their roles in the regulation of Gck expression. Chromatin immunoprecipitation assays demonstrated that recombinant adenovirus-mediated overexpression of RARα, HNF4α and COUP-TFII, but not RXRα, significantly increased their occupancy in the hepatic Gck promoter in primary rat hepatocytes. Overexpression of RARα, HNF4α and COUP-TFII, but not RXRα, also affected the RA- and insulin-mediated Gck expression in primary rat hepatocytes. In summary, this hepatic Gck promoter RARE interacts with RARα, HNF4α and COUP-TFII to integrate Vitamin A and insulin signals.
肝葡萄糖激酶基因（Gck）的表达受激素和营养信号调控。目前尚不清楚这些信号如何整合以调节肝脏Gck的表达。我们已经表明，肝脏Gck的表达受维生素A状态的影响，并在原代大鼠肝细胞中由胰岛素和类视黄醇协同诱导。我们推测这是由肝脏Gck启动子中的视黄酸反应元件（RARE）介导的。在此，我们使用标准分子生物学技术鉴定了肝脏Gck启动子中的RARE。还确定了影响视黄酸（RA）对启动子激活的单核苷酸突变，以详细分析蛋白质与DNA的相互作用。我们优化了进行电泳迁移率变动分析的实验条件，并证明了大鼠核提取物中的视黄酸受体α（RARα）、视黄醇X受体α（RXRα）、肝细胞核因子4α（HNF4α）和鸡卵清蛋白上游启动子转录因子II（COUP-TFII）与该RARE相互作用，表明它们在Gck表达调控中的作用。染色质免疫沉淀分析表明，重组腺病毒介导的RARα、HNF4α和COUP-TFII过表达，但不是RXRα，显著增加了它们在原代大鼠肝细胞肝脏Gck启动子中的占有率。RARα、HNF4α和COUP-TFII的过表达，但不是RXRα，也影响原代大鼠肝细胞中RA和胰岛素介导的Gck表达。总之，这种肝脏Gck启动子RARE与RARα、HNF4α和COUP-TFII相互作用，以整合维生素A和胰岛素信号。
