科研之友
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摘要
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 alpha (RAR alpha), retinoid X receptor alpha (RXR alpha), hepatocyte nuclear factor 4 alpha (HNF4 alpha) 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 RAR alpha, HNF4 alpha and COUP-TFII, but not RXR alpha, significantly increased their occupancy in the hepatic Gck promoter in primary rat hepatocytes. Overexpression of RAR alpha, HNF4 alpha and COUP-TFII, but not RXR alpha, also affected the RA- and insulin-mediated Gck expression in primary rat hepatocytes. In summary, this hepatic Gck promoter RARE interacts with RAR alpha, HNF4 alpha and COUP-TFII to integrate Vitamin A and insulin signals.
