A RARE of hepatic Gck promoter interacts with RARα, HNF4α and COUP-TFII that affect retinoic acid- and insulin-induced Gck expression.

Abstract

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.