title: Post-transcriptional regulation of sterol regulatory element-binding protein-1 by ethanol induces class I alcohol dehydrogenase in rat liver.
abstract: 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 approximately 6-fold, the nuclear form of SREBP-1 decreased by approximately 7-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 approximately 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.
authors.affiliation: Arkansas Children's Nutrition Center, University of Arkansas for Medical Sciences, 1120 Marshall Street, Little Rock, AR 72202, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: He
authors.forename: Frank A
authors.initials: FA
authors.lastname: Simmen
authors.forename: Martin J J
authors.initials: MJ
authors.lastname: Ronis
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Badger
mesh_terms: D000426:Alcohol Dehydrogenase / Q000096:biosynthesis*; D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002492:Central Nervous System Depressants / Q000494:pharmacology; D002790:Cholesterol 7-alpha-Hydroxylase / Q000096:biosynthesis; D002843:Chromatin / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology*; D064429:Fatty Acid Synthases / Q000096:biosynthesis; D006845:Hydrocarbons, Fluorinated; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D000071518:Liver X Receptors; D008297:Male; D008957:Models, Genetic; D057093:Orphan Nuclear Receptors; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000378:metabolism; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D013449:Sulfonamides; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D005466:Fluorocarbons; D000096926:Benzenesulfonamides
chemical_list: D000426:Alcohol Dehydrogenase; D000924:Anticholesteremic Agents; D022762:CCAAT-Enhancer-Binding Proteins; D002492:Central Nervous System Depressants; D002790:Cholesterol 7-alpha-Hydroxylase; D002843:Chromatin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D000431:Ethanol; D064429:Fatty Acid Synthases; D006845:Hydrocarbons, Fluorinated; D000071518:Liver X Receptors; D057093:Orphan Nuclear Receptors; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D012260:Ribonucleases; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D013449:Sulfonamides; D014157:Transcription Factors; C497389:Srebf1 protein, rat; C423915:T0901317; D005466:Fluorocarbons; D000096926:Benzenesulfonamides