title: Aryl hydrocarbon receptor and NF-E2-related factor 2 are key regulators of human MRP4 expression.
abstract: Multidrug resistance protein 4 (MRP4; ABCC4) is an ATP binding cassette transporter that facilitates the excretion of bile salt conjugates and other conjugated steroids in hepatocytes and renal proximal tubule epithelium. MRP4/Mrp4 undergoes adaptive upregulation in response to oxidative and cholestatic liver injury in human and animal models of cholestasis. However, the molecular mechanism of this regulation remains to be determined. The aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) play important roles in protecting cells from oxidative stress. Here we examine the role of these two nuclear factors in the regulation of the expression of human MRP4. HepG2 cells and human hepatocytes were treated with the AhR and Nrf2 activators, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC), or oltipraz and other nuclear receptor agonists. TCDD, 3-MC, and oltipraz significantly increased MRP4 expression at mRNA and protein levels. Computer program analysis revealed three Xenobiotic response element (XRE) and one Maf response element sites within the first 500 bp of the MRP4 proximal promoter. Luciferase reporter assay detected strong promoter activity (53-fold higher than vector control) in this region. TCDD and 3-MC also induced promoter activity in the reporter assays. Mutation of any of these XRE sites significantly decreased MRP4 promoter activity in reporter assays, although XRE2 demonstrated the strongest effects on both basal and TCDD-inducible activity. EMSA and chromatin immunoprecipitation assays further confirmed that both AhR and Nrf2 bind to the proximal promoter of MRP4. Our findings indicate that AhR and Nrf2 play important roles in regulating MRP4 expression and suggest that agents that activate their activity may be of therapeutic benefit for cholestasis.
authors.affiliation: Department of Medicine and Liver Center, Yale University School of Medicine, New Haven, CT 06520-8019, USA.
authors.forename: Shuhua
authors.initials: S
authors.lastname: Xu
authors.forename: Jittima
authors.initials: J
authors.lastname: Weerachayaphorn
authors.forename: Shi-Ying
authors.initials: SY
authors.lastname: Cai
authors.forename: Carol J
authors.initials: CJ
authors.lastname: Soroka
authors.forename: James L
authors.initials: JL
authors.lastname: Boyer
mesh_terms: D024506:5' Flanking Region; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D002082:Butylamines / Q000494:pharmacology; D047369:Chromatin Immunoprecipitation; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008748:Methylcholanthrene / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011719:Pyrazines / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D013871:Thiones; D013876:Thiophenes; D013997:Time Factors; D014162:Transfection
chemical_list: D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D002082:Butylamines; D008748:Methylcholanthrene; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D051267:NF-E2-Related Factor 2; D000072317:Polychlorinated Dibenzodioxins; D011719:Pyrazines; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D013871:Thiones; D013876:Thiophenes; C121358:4-phenylbutylamine; C073492:ABCC4 protein, human; C111235:AHR protein, human; C497396:ARNT protein, human; C495635:NFE2L2 protein, human; C495636:Nfe2l2 protein, mouse; C026209:oltipraz
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