Our aim was to examine whether the dysregulated sumoylation could regulate the ethanol-induced CYP2E1 expression in ALD and elucidate the molecular mechanism(s). Methods: Studies were done using in vivo binge ethanol-fed mice, primary mouse hepatocytes in matrigel. 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. Results: We found Ubc9 mRNA level is increased in the livers of binge mice and ethanol-treated hepatocytes whereas CYP2E1 mRNA levels increased minimally. In contrast, protein levels of Ubc9 and CYP2E1 are both induced. Ubc9 knockdown reduced CYP2E1 mRNA level and CYP2E1 promoter activity. BMN673 Chip assay showed that Ubc9 is required for NF-κB and C/EBPp, two positive regulators of CYP2E1 promoter activity known to be sumoylated, to interact with CYP2E1 promoter in hepatocytes. In addition, silencing Ubc9 prevented ethanol-mediated induction in CYP2E1 protein level when de novo mRNA synthesis is blocked by actinomycin D, suggesting that most of the increase in CYP2E1 protein level
was at the translational level and this required Ubc9. Finally, we found that CYP2E1 is sumoylated in vitro and in vivo in binge mice livers and increased following ethanol treatment. Conclusions: Ethanol-mediated Urease sumoylation Selleckchem Napabucasin increased CYP2E1 expression in livers of binge mice and primary hepatocytes. Sumoylation positively regulates CYP2E1 promoter activity but the primary role of sumoylation
is to stabilize CYP2E1 protein after ethanol treatment. To our knowledge, this is the first report of ethanol-mediated up-regulation of CYP2E1 via sumoylation induction, leading to increased CYP2E1 protein stability. Disclosures: The following people have nothing to disclose: Maria Lauda Tomasi, Minjung Ryoo, Diana Arsene Background. Vinyl chloride (VC) is a ubiquitous environmental contaminant and ranks 4th on the ATSDR Hazardous Substances Priority List. We have previously reported increased hepatocellular necrosis in a highly exposed occupational cohort and in vitro models. A major paradigm shift in environmental research is to assess the impact of underlying disorders that may modify risk. Arguably, the most ubiquitous underlying disorder in the developed world is obesity. The impact of obesity and obesity-induced liver damage (i.e., NAFLD) on hepatic injury caused by VC is not known. The purpose of the current study was therefore to investigate hepatic injury caused by chloroethanol (ClEtOH; VC metabolite) in an experimental model of high-fat diet (HFD) induced obesity. Methods. Mice were administered a bolus dose of chloroethanol (or vehicle) 10 wks after being fed an HFD (42% milk fat)-fed or low fat control diet (LFD; 13% milk fat).