Sumoylation regulates Cytochrome P450 2E1 (CYP2E1) expression in alcoholic liver disease

Abstract

BackgroundEthanol‐inducible CYP2E1 contributes to increased oxidative stress and steatosis in alcoholic liver disease (ALD). Ubiquitin‐conjugation enzyme 9 (Ubc9), the sole E2‐conjugating enzyme for sumoylation, is induced in the livers of ethanol‐fed mice. Our aim was to examine whether the dysregulated sumoylation could regulate the ethanol‐induced CYP2E1 expression in ALD and elucidate the molecular mechanism(s).MethodsStudies were done using in vivo binge ethanol‐fed mice and primary mouse hepatocytes. Gene and protein expression were measured by real‐time PCR and Western blot analyses, respectively. Promoter activity, Chromatin Immunoprecipitation (Chip) assay and in vitro protein sumoylation were analyzed using commercial kits.ResultsWe found Ubc9 mRNA level is increased in the livers of binge mice and ethanol‐treated hepatocytes whereas CYP2E1 mRNA level increased minimally. In contrast, protein levels of Ubc9 and CYP2E1 are both induced. Ubc9 knockdown reduced CYP2E1 mRNA level and CYP2E1 promoter activity. Chip assay showed that Ubc9 is required for NF‐kB and C/EBPβ, two positive regulators of CYP2E1 promoter activity known to be sumoylated, to interact with CYP2E1 promoter in hepatocytes. Silencing of Ubc9 prevented ethanol‐mediated induction in CYP2E1 protein level when de novo mRNA synthesis is blocked by actinomycin D. Finally, we found that CYP2E1 is sumoylated in vitro and in vivo in binge mice livers and increased following ethanol treatment.ConclusionsEthanol‐mediated sumoylation increased CYP2E1 expression in livers of binge mice and primary hepatocytes regulating CYP2E1 promoter activity but the primary role of sumoylation is to stabilize CYP2E1 protein after ethanol treatment.