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Control of Steroid, Heme, and Carcinogen Metabolism by Nuclear Pregnane X Receptor and Constitutive Androstane Receptor

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Control of Steroid, Heme, and Carcinogen Metabolism by Nuclear Pregnane X Receptor and Constitutive Androstane Receptor Control of steroid, heme, and carcinogen metabolism by nuclear pregnane X receptor and constitutive androstane receptor Wen Xie*†‡, Mei-Fei Yeuh‡§, Anna Radominska-Pandya¶, Simrat P. S. Saini*, Yoichi Negishi*, Bobbie Sue Bottroff†, Geraldine Y. Cabrera†, Robert H. Tukey§, and Ronald M. Evans†ʈ *Center for Pharmacogenetics and Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15213; †Howard Hughes Medical Institute, Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; §Laboratory of Environmental Toxicology, Departments of Chemistry, and Biochemistry and Pharmacology, University of California at San Diego, La Jolla, CA 92093; and ¶Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 Contributed by Ronald M. Evans, December 31, 2002 Through a multiplex promoter spanning 218 kb, the phase II UDP- ogens, estrogen, and thyroxin metabolism. Moreover, activation of glucuronosyltransferase 1A (UGT1) gene encodes at least eight dif- PXR in transgenic mice is sufficient to increase levels of UGT ferently regulated mRNAs whose protein products function as the expression and activity, as well as bilirubin and steroid clearance. principal means to eliminate a vast array of steroids, heme metabo- lites, environmental toxins, and drugs. The orphan nuclear receptors Materials and Methods pregnane X receptor (PXR) and constitutive androstane receptor Animals. The generation of VP-hPXR, hPXR (previously known as (CAR) were originally identified as sensors able to respond to numer- VPSXR and SXR) transgenic mice and PXR null mice has been ous environmentally derived foreign compounds (xenobiotics) to described (8). promote detoxification by phase I cytochrome P450 genes. In this report, we show that both receptors can induce specific UGT1A UGT1A1 Promoter Cloning and Site-Directed Mutagenesis. The hu- isoforms including those involved in estrogen, thyroxin, bilirubin, and man UGT1A1 promoter was amplified by using a BAC clone carcinogen metabolism. Transgenic mice expressing a constitutively containing the entire UGT1 locus template (2). All primers were active form of human PXR show markedly increased UGT activity designed based on the sequence encoding the human UGT1A locus toward steroid, heme, and carcinogens, enhanced bilirubin clearance, published in the National Center for Biotechnology Information as well as massively increased steroid clearance. The ability of PXR and GenBank (accession no. AF297093). Site-directed mutagenesis was constitutive androstane receptor and their ligands to transduce both performed by PCR and confirmed by DNA sequencing. the phase I and phase II adaptive hepatic response defines a unique transcriptional interface that bridges the ingestion and metabolism of DNA-Binding Analysis. Electrophoretic mobility-shift assays environmental compounds to body physiology. (EMSA) were performed by using in vitro-transcribed and -trans- lated protein (TNT, Promega) as described (14). Oligonucleotides Ј he metabolism of steroid hormones, other endogenous com- were: UGT1A1, 5 -CTAACGGTTCATAAAGGGTATTAGGT- 3Ј; UGT1A6, 5Ј-CGAGTAGGTCATAAAGGTCACA-3Ј; and Tpounds, and xenobiotics occurs in the hepatogastrointestinal Ј Ј tract. This catabolic process is mediated by phase I enzymes, such UGT1A6m, 5 -CGAGTAGAACATAAAGAACACA-3 . as the monooxygenase CYP enzymes (1), as well as the phase II ͞ conjugating enzymes, such as UDP-glucuronosyltransferase Plasmid Construction and Transfection. The tk-UGT1A6 DR3-Luc (UGTs), sulfotransferases, and GSTs (2). The UGT 1A (UGT1A) and its mutant variants were generated by insertion of correspond- locus is controlled by 13 promoters (A1–A12), spanning Ͼ200 kb ing annealed oligonucleotides into the tk-Luc vector. The expres- of upstream sequence encoding overlapping but distinct mRNAs sion vectors for hPXR, VP-hPXR, and VP-CAR have been (3). Using UDP-glucuronic acid as a cosubstrate, UGT enzymes described (14). CV-1 cell transfection using N-[1-(2,3-dioleoy- convert a diverse set of lipophilic substances to water-soluble loxy)propyl]-N,N,N-trimethylammonium methylsulfate (Roche glucuronides and function as the principal means to eliminate Biochemicals) and HepG2 cell transfections using Lipofectamine steroids, heme metabolites, environmental toxins, and drugs from Plus (Invitrogen) were carried out as before (14, 15). the body (2). Crigler–Najjar syndrome type I results in a lethal Northern and Western Blot Analysis. accumulation of bilirubin due to a defect in the 1A1 promoter, Total RNA was prepared from liver tissues by using TRIzol reagent (Invitrogen). Northern hy- whereas Gilbert’s disease (7% of the population) is a more mild 1A1 bridization was carried out as described (8). Liver microsomes were promoter defect typified by increased sensitivity to certain drugs prepared and analyzed for UGT1A expression. Western blot pro- like Tylenol (2). files were conducted by using an anti-UGT1A antibody, which Nuclear receptor (NR) pregnane X receptor (PXR, also known recognizes only the UGT1A family of proteins, and an anti- as the steroid and xenobiotic receptor or SXR) and the constitutive UGT1A1 antibody that is specific for UGT1A1. androstane receptor (CAR) were originally shown to act through NR response elements localized in the promoters of the target UGT Analysis. The UGT analysis was similar to that described before CYP3A and CYP2B genes (4–10). Pharmaceutical compounds (16). In brief, mouse liver microsomes were prepared as described such as phenobarbital and rifampicin (RIF) have been reported as (16). The [14C]UDP-GlcUA was used as the sugar donor, and TLC UGT inducers, although the molecular basis remains to be defined was used to separate glucuronidation products. The products were (11, 12). Both phenobarbital and RIF have been empirically used to treat hyperbilirubinemia, a clinical accumulation of serum bili- rubin due to insufficient glucuronidation (11, 13). These com- Abbreviations: CAR, constitutive androstane receptor; PXR, pregnane X receptor; RIF, pounds are activators of PXR and CAR, suggesting a plausible rifampicin; TCPOBOP, 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene; UGT, UDP-glucurono- signaling pathway for UGT transcription. syltransferase; NR, nuclear receptor; EMSA, electrophoretic mobility-shift assay. In this report, we show that both receptors can induce specific ‡W.X. and M.-F.Y. contributed equally to this work. UGT1A isoforms including those specialized for bilirubin, carcin- ʈTo whom correspondence should be addressed. E-mail: [email protected]. 4150–4155 ͉ PNAS ͉ April 1, 2003 ͉ vol. 100 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0438010100 Downloaded by guest on September 26, 2021 MEDICAL SCIENCES Fig. 1. Induction of UGT1A expression and glucuronidation by PXR activation. (A) Liver microsomes were prepared from WT and VP-hPXR transgenic mice and subject to Western blot profiling by using a pan anti-UGT1A antibody and a specific anti-UGT1A1 antibody. (B) Mouse liver total RNAs were subjected to Northern blot analysis. The membranes were probed for UGT1A6, UGT1As, CYP3A11, and GAPDH as a loading control. (C) Induction of UGT1A mRNA by RIF in hPXR transgenic mice. WT or transgenic males were gavaged with a single dose of solvent or RIF (50 mg͞kg) 24 h before death. (D) The parental HepG2 cells or the HepG2-hPXR stable cells were treated with DMSO or 25 ␮M RIF for 24 h and subsequently harvested for Northern blot analysis to detect UGT1A1 mRNA. (E) Glucuronidation activity toward ␤-estradiol. The result is shown as the autoradiograph of a TLC plate. (F) Glucuronidation toward thyroid hormones (rT3 and T4) and xenobiotics (4-nitrophenol and 4-OH-PhIP). Results are presented as fold increase in glucuronidation activity over WTs and represent the mean and standard error. visualized with a Molecular Dynamics Storm 820 PhosphorImager. at 4 p.m. by retroorbital eye bleeding within 1 min of initial Alternatively, silica gel in zones corresponding to the glucuronide disturbance. The 24-h urine was collected by using mouse metabolic bands were visualized by autoradiography, or corresponding areas cages (Nalge). Corticosterone levels in plasma or urine were from control lanes were scraped into scintillation vials, and radio- measured with a Corticosterone [125I]RIA kit from ICN. The activity was measured by liquid scintillation counting. statistical analysis was performed with INSTAT 2.03 software. Bilirubin Clearance. Adult males were given a single does of bilirubin Results (10 mg͞kg body weight). Blood samples were collected in untreated tubes1hafterinjection.Serumwasprepared by centrifugation at Activation of PXR Induces UGT Expression and Glucuronidation in 4,000 rpm for 10 min. Total and conjugated bilirubin levels were Transgenic Mice. The regulatory locus of the UGT1A gene is highly measured by Antech Diagnostic (Lake Success, NY). The statistical complex. It harbors 12 promoters that control the independent analysis was performed with INSTAT 2.03 software. expression of a specific UGT isoform with distinctive substrate specificity (2). The isoforms vary at the amino terminus and Blood and Urine Collection and Hormone Analysis. Mouse blood share a common body coded by exons 2–5. For example, the 1A1 samples were collected in EDTA-coated tubes (Becton Dickinson) isoform metabolizes estrogens but not androgens or other Xie et al. PNAS ͉ April 1, 2003 ͉ vol. 100 ͉ no. 7 ͉ 4151 Downloaded by guest on September 26, 2021 Fig. 2. Cloning of the UGT1A1
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