UNC-Chapel Hill ENVR 442 - The Orphan Human Pregnane X Receptor Mediates the Transcriptional Activation of CYP3A4

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The Orphan Human Pregnane X Receptor Mediates theTranscriptional Activation of CYP3A4 by Rifampicin through aDistal Enhancer ModuleBRYAN GOODWIN, ECUSHLA HODGSON, and CHRISTOPHER LIDDLEDepartment of Clinical Pharmacology and Storr Liver Unit, University of Sydney at Westmead Hospital, Westmead, AustraliaReceived July 6, 1999; accepted August 26, 1999 This paper is available online at http://www.molpharm.orgABSTRACTCytochrome P-450 3A4 (CYP3A4), the predominant cyto-chrome P-450 expressed in adult human liver, is subject totranscriptional induction by a variety of structurally unrelatedxenobiotics, including the antibiotic rifampicin. The molecularmechanisms underlying this phenomenon are poorly under-stood. We transfected a human liver-derived cell line (HepG2)with various CYP3A4-luciferase reporter gene constructs con-taining a nested set of 59-deletions of the CYP3A4 59-flankingregion. Rifampicin-inducible transcription of the reporter genewas observed only with the longest construct, which encom-passed bases 213000 to 153 of CYP3A4 (3-fold induction).The responsive region was functional regardless of its positionor orientation relative to the proximal promoter of CYP3A4 andwas capable of conferring rifampicin-inducible expression on aheterologous promoter. Further deletion mutants localized theinduction to bases 27836 to 27607. In vitro DNase I footprintanalysis of this region revealed four protected sites (FP1, FP2,FP3, and FP4). Two of these sites, FP3 (bases 27738 to27715) and FP4 (bases 27698 to 27682), overlapped bindingmotifs for the orphan human pregnane X receptor (hPXR). Co-transfection of responsive constructs with a hPXR expressionvector substantially increased the rifampicin-inducibility to;50-fold. In addition, the rifampicin-responsive constructswere strongly activated by a range of CYP3A inducers. Finally,we demonstrate cooperativity between elements within thedistal enhancer region and cis-acting elements in the proximalpromoter of CYP3A4. Our results provide evidence for theexistence of a potent enhancer module, 8 kb distal to thetranscription start point, which mediates the transcriptional in-duction of CYP3A4 by activators of hPXR.Cytochromes P-450 (P-450) are a superfamily of hemopro-teins that play a pivotal role in the oxidative metabolism ofnumerous endogenous and exogenous compounds (Nelson etal., 1996). The human P-450 3A subfamily contains threefunctional members: CYP3A4, CYP3A5, and CYP3A7.CYP3A4 is the predominant isoform expressed in adult hu-man liver and is reported to be responsible for the metabo-lism of more than 60% of therapeutic drugs (Li et al., 1995).In addition, this enzyme is the primary catalyst of steroid6b-hydroxylation (Waxman et al., 1991) and therefore has acentral role in steroid hormone homeostasis. CYP3A4 is alsoinvolved in the bioactivation of anticancer drugs and envi-ronmental procarcinogens, including benzo(a)pyrene andother dihydrodiol derivatives of polycyclic aromatic hydrocar-bons and carcinogenic mycotoxins (Li et al., 1995). Thus,CYP3A4 is considered a key enzyme in chemical carcinogen-esis in both the liver and extrahepatic tissues.CYP3A4 is transcriptionally regulated by a variety of hor-mones, including glucocorticoids, growth hormone, and tri-iodothyronine (Schuetz et al., 1993; Liddle et al., 1998), andxenobiotics such as phenobarbital, clotrimazole, mifepristone(RU486), and rifampicin (Daujat et al., 1991; Schuetz et al.,1993; Kocarek et al., 1995). Rifampicin, a macrocyclic anti-biotic, is known to be one of the most potent inducers ofCYP3A4 expression both in vivo and in cultured hepatocytes(Schuetz et al., 1993; Kocarek et al., 1995; Michalets, 1998).This work was supported by a grant from the National Health and MedicalResearch Council (Australia) and the Robert W. Storr bequest to the MedicalFoundation, University of Sydney. B.G. was a recipient of the National Healthand Medical Research Council (Australia) Dora Lush Biomedical Scholarship.1Three independent studies have implicated an orphan nuclear receptor inCYP3A regulation. The human PXR (hPXR; Lehmann et al., 1998) and humanpregnane activated receptor (hPAR; Bertilsson et al., 1998) are identical in thederived amino acid sequence. The human steroid and xenobiotic receptor(hSXR; Blumberg et al., 1998) contains a single base-pair insertion at position1225 and a single base deletion at 1279, relative to hPXR, which results in ashift in the reading frame for amino acid residues 215–233. However, hPXR,hPAR, and hSXR almost certainly represent products of the same gene. In thisstudy, we used the “hPXR” nomenclature.ABBREVIATIONS: P-450, cytochrome P-450; PXR, pregnane X receptor; RXRa,9-cis retinoic acid receptor-a; PXRE, pregnane X receptorresponse element; prPXRE, proximal pregnane X receptor response element; HNF, hepatocyte nuclear factor; EMSA, electrophoretic mobility shiftassay; RORa1, retinoic acid receptor-related receptora-1; COUP-TF, chicken ovalbumin upstream promoter-transcription factor; PCR, polymer-ase chain reaction; PCN, pregnenolone 16a-carbonitrile; tk, herpes simplex virus thymidine kinase; hGR, human glucocorticoid receptor; GRE,glucocorticoid-responsive element; XREM, xenobiotic-responsive enhancer module; DMSO, dimethyl sulfoxide.0026-895X/99/061329-11$3.00/0Copyright © The American Society for Pharmacology and Experimental TherapeuticsAll rights of reproduction in any form reserved.MOLECULAR PHARMACOLOGY, 56:1329–1339 (1999).1329 at Univ Of NC Acq Srvcs on September 4, 2009 molpharm.aspetjournals.orgDownloaded fromInduction of CYP3A4 expression by rifampicin and otherxenobiotics underlies many reported drug interactions and isof considerable importance for patients subject to combina-tion drug therapy such as for HIV/AIDS (Michalets, 1998).Although the nucleotide sequence of the proximal 59-flank-ing region of CYP3A4 has been reported (Hashimoto et al.,1993), the molecular mechanisms underlying the transcrip-tional regulation of this gene have yet to be elucidated. Theproximal promoter of the CYP3A4 gene (bases 2172 to 2149)contains two copies of an AG(G/T)TCA hexamer, the recog-nition sequence for the nuclear receptor family of transcrip-tion factors (Mangelsdorf et al., 1995). Barwick et al. (1996)demonstrated that these half-sites, organized as an ER-6(everted repeat separated by six nucleotides), conferred ri-fampicin-responsiveness on heterologous reporter gene con-structs when transfected into rabbit but not rat


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UNC-Chapel Hill ENVR 442 - The Orphan Human Pregnane X Receptor Mediates the Transcriptional Activation of CYP3A4

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