Post-transcriptional regulation of sterol regulatory element-binding protein-1 by ethanol induces class I alcohol dehydrogenase in rat liver

Members of the sterol regulatory element-binding protein ( SREBP) family of transcription factors control the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids. Continuous intragastric infusion of ethanol-containing diets as part of total enteral nutrition generates well defined 6-day cycles ( pulses) of urine ethanol concentrations (UECs) in rats. Pulsatile UECs are the result of cyclical expression and activity of the principal alcohol-metabolizing enzyme, hepatic Class I alcohol dehydrogenase (ADH), and this mechanism involves regulated CCAAT/enhancer-binding protein-beta expression and binding to the ADH promoter. In this study, we further explore the molecular mechanism for ethanol-induced ADH expression during the UEC pulse in adult male rats fed ethanol by total enteral nutrition for 21 - 30 days. In hypophysectomized rats, in which the ADH protein increased by similar to6-fold, the nuclear form of SREBP-1 decreased by similar to7-fold. Because the ADH promoter contains two canonical sterol response element (SRE) sites ( - 63 to - 53 and - 52 to - 40 relative to the transcription start site), electrophoretic mobility shift assays were conducted using an ADH- specific SRE site. Hepatic nuclear protein binding decreased by 2.4-fold on the ascending limbs and by 3.6-fold on the descending limbs of UEC pulses ( p < 0.05). The specificity of nuclear protein binding to the ADH- SRE site was confirmed using antibody and UV cross-link assays. The in vivo binding status of SREBP-1 to ADH- SRE sites, as measured by the chromatin immunoprecipitation assay, had a pattern very similar to the electrophoretic mobility shift assay results. Functional analysis of the ADH- SREs demonstrated these sites to be essential for ADH transcription. In vitro transcription assays demonstrated that depletion of the SREBP-1 protein from nuclear extracts increased transcription activity by similar to 5-fold and that the liver X receptor agonist T0901317 ( a known activator of SREBP-1c transcription) reduced in vitro expression of ADH mRNA by 2-fold. We conclude that SREBP-1 is a negative regulator of the ADH gene and may work in concert with the CCAAT/enhancer-binding proteins to mediate ethanol induction of ADH in vivo.