The transcription factor E4bp4 regulates the expression and activity of Cyp3a11 in mice

Abstract

Human CYP3A4 (Cyp3a11 in mice) is one of the most important enzymes for drug metabolism and detoxification. Here, we aimed to investigate a potential role for E4bp4 in regulation of Cyp3a11 expression and activity. The regulatory effects of E4bp4 on Cyp3a11 enzyme were assessed using E4bp4−/− mice and Hepa-1c1c7 cells. The mRNA and protein levels were quantified using qPCR and Western blotting, respectively. In vitro microsomal Cyp3a11 activity was probed using its specific substrates midazolam and testosterone. Pharmacokinetic studies were performed with wild-type and E4bp4−/− mice after midazolam administration. Global deletion of E4bp4 led to significant upregulation of Cyp3a11 mRNA and protein in major metabolic organs (i.e., the liver, kidney and small intestine). E4bp4 ablation also caused an increased microsomal Cyp3a11 activity consistent with the enzyme’s expression change. Overexpression of E4bp4 in Hepa-1c1c7 cells resulted in reduced levels of Cyp3a11 mRNA and protein, whereas E4bp4 knockdown caused upregulation of Cyp3a11 expression. In addition, the systemic exposure of midazolam was lowered in E4bp4−/− mice compared with wild-type mice. This was accompanied by enhanced formation of its metabolite 1′-hydroxymidazolam. Furthermore, luciferase reporter and mobility shift assays revealed that E4bp4 repressed Cyp3a11 transcription via direct binding to C-site (−1539/−1529 bp) in the promoter region. In conclusion, E4bp4 negatively regulates Cyp3a11 expression, thereby impacting drug metabolism and pharmacokinetics.