title: Upregulation of UGT2B4 Expression by 3'-Phosphoadenosine-5'-Phosphosulfate Synthase Knockdown: Implications for Coordinated Control of Bile Acid Conjugation.
abstract: During cholestasis, the bile acid-conjugating enzymes, SULT2A1 and UGT2B4, work in concert to prevent the accumulation of toxic bile acids. To understand the impact of sulfotransferase deficiency on human hepatic gene expression, we knocked down 3'-phosphoadenosine-5'-phosphosulfate synthases (PAPSS) 1 and 2, which catalyze synthesis of the obligate sulfotransferase cofactor, in HepG2 cells. PAPSS knockdown caused no change in SULT2A1 expression; however, UGT2B4 expression increased markedly (∼41-fold increase in UGT2B4 mRNA content). Knockdown of SULT2A1 in HepG2 cells also increased UGT2B4 expression. To investigate the underlying mechanism, we transfected PAPSS-deficient HepG2 cells with a luciferase reporter plasmid containing ∼2 Kb of the UGT2B4 5'-flanking region, which included a response element for the bile acid-sensing nuclear receptor, farnesoid X receptor (FXR). FXR activation or overexpression increased UGT2B4 promoter activity; however, knocking down FXR or mutating or deleting the FXR response element did not significantly decrease UGT2B4 promoter activity. Further evaluation of the UGT2B4 5'-flanking region indicated the presence of distal regulatory elements between nucleotides -10090 and -10037 that negatively and positively regulated UGT2B4 transcription. Pulse-chase analysis showed that increased UGT2B4 expression in PAPSS-deficient cells was attributable to both increased mRNA synthesis and stability. Transfection analysis demonstrated that the UGT2B4 3'-untranslated region decreased luciferase reporter expression less in PAPSS-deficient cells than in control cells. These data indicate that knocking down PAPSS increases UGT2B4 transcription and mRNA stability as a compensatory response to the loss of SULT2A1 activity, presumably to maintain bile acid-conjugating activity.
authors.affiliation: Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan (K.G.B., H.F., T.A.K., M.R.-M.); and Department of Pediatrics, Wayne State University, Detroit, Michigan (D.C., A.A.D.).
authors.forename: Kathleen G
authors.initials: KG
authors.lastname: Barrett
authors.affiliation: Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan (K.G.B., H.F., T.A.K., M.R.-M.); and Department of Pediatrics, Wayne State University, Detroit, Michigan (D.C., A.A.D.).
authors.forename: Hailin
authors.initials: H
authors.lastname: Fang
authors.affiliation: Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan (K.G.B., H.F., T.A.K., M.R.-M.); and Department of Pediatrics, Wayne State University, Detroit, Michigan (D.C., A.A.D.).
authors.forename: Daniela
authors.initials: D
authors.lastname: Cukovic
authors.affiliation: Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan (K.G.B., H.F., T.A.K., M.R.-M.); and Department of Pediatrics, Wayne State University, Detroit, Michigan (D.C., A.A.D.).
authors.forename: Alan A
authors.initials: AA
authors.lastname: Dombkowski
authors.affiliation: Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan (K.G.B., H.F., T.A.K., M.R.-M.); and Department of Pediatrics, Wayne State University, Detroit, Michigan (D.C., A.A.D.).
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kocarek
authors.affiliation: Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan (K.G.B., H.F., T.A.K., M.R.-M.); and Department of Pediatrics, Wayne State University, Detroit, Michigan (D.C., A.A.D.) m.runge-morris@wayne.edu.
authors.forename: Melissa
authors.initials: M
authors.lastname: Runge-Morris
mesh_terms: D024506:5' Flanking Region / Q000235:genetics; D001647:Bile Acids and Salts / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D055785:Gene Knockdown Techniques; D014453:Glucuronosyltransferase / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D009097:Multienzyme Complexes / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D013430:Sulfate Adenylyltransferase / Q000235:genetics*; D015238:Sulfotransferases / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D015854:Up-Regulation / Q000235:genetics; D000099150:Receptor, Farnesoid X-Activated
chemical_list: D001647:Bile Acids and Salts; D014453:Glucuronosyltransferase; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D013430:Sulfate Adenylyltransferase; D015238:Sulfotransferases; D000099150:Receptor, Farnesoid X-Activated; C486404:UGT2B4 protein, human; C022322:PAPS synthetase; C028152:alcohol sulfotransferase