THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 35, Issue of August 29, pp. 32852–32860, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Peroxisome Proliferator-activated Receptor ␣ Induces Hepatic Expression of the Human Bile Acid Glucuronidating UDP-glucuronosyltransferase 2B4 Enzyme*

Received for publication, May 22, 2003 Published, JBC Papers in Press, June 16, 2003, DOI 10.1074/jbc.M305361200

Olivier Barbier‡, Daniel Duran-Sandoval‡, Ine´s Pineda-Torra‡, Vladimir Kosykh§, Jean-Charles Fruchart‡, and Bart Staels‡¶ From the ‡Unite´de Recherche 545, Institut National de la Sante´et de la Recherche Me´dicale (INSERM), De´partement d’Athe´roscle´rose, Institut Pasteur de Lille and the Faculte´de Pharmacie, Universite´de Lille II, 59019 Lille, France and §Institute of Experimental Cardiology, Russian Cardiology Complex, Moscow 121552, Russia

Glucuronidation, a major metabolic pathway for a pollutants (1). This reaction consists in the transfer of the large variety of endobiotics and xenobiotics, is cata- glucuronosyl group from UDP-glucuronic acid to the acceptor lyzed by enzymes belonging to the UDP-glucuronosyl- molecule (1). The addition of the glucuronosyl group on a com- transferase (UGT) family. Among UGT enzymes, pound results in a more water-soluble molecule, which can be

UGT2B4 conjugates a large variety of endogenous and excreted into bile or urine. Glucuronidation is catalyzed by Downloaded from exogenous molecules and is considered to be the major enzymes belonging to the UDP-glucuronosyltransferase (UGT) bile acid conjugating UGT enzyme in human liver. In the family, and based on primary structure homology, UGT pro- present study, we identify UGT2B4 as a novel target teins have been divided into two major subfamilies, UGT1A gene of the nuclear receptor peroxisome proliferator- and UGT2B (2). In humans, seven members of the UGT2B ␣ ␣ activated receptor (PPAR ), which mediates the hypo- subfamily have been characterized: UGT2B4, UGT2B7, lipidemic action of fibrates. Incubation of human hepa- UGT2B10, UGT2B11, UGT2B15, UGT2B17, and UGT2B28 (3, http://www.jbc.org/ tocytes or hepatoblastoma HepG2 and Huh7 cells with 4). synthetic PPAR␣ agonists, fenofibric acid, or Wy 14643 Among the UGT2B enzymes, UGT2B4 catalyzes the glucu- resulted in an increase of UGT2B4 mRNA levels. Fur- ␣ thermore, treatment of HepG2 cells with Wy 14643 in- ronide conjugation of various molecules, including BAs, 5 - duced the glucuronidation of hyodeoxycholic acid, a reduced androgens, catecholesterogens, and phenolic and specific bile acid UGT2B4 substrate. Analysis of UGT2B monoterpenoid compounds (4–7). A certain degree of overlap- mRNA and protein levels in PPAR␣ wild type and null ping substrate specificity exists among the UGT2Bs, and these by guest on December 24, 2015 mice revealed that PPAR␣ regulates both basal and fi- compounds are also conjugated by other UGT2B isoforms. brate-induced expression of these enzymes in rodents However, various studies established the crucial role that also. Finally, a PPAR response element was identified in UGT2B4 plays in hepatic BA glucuronide conjugation. Pillot et the UGT2B4 promoter by site-directed mutagenesis and al. (7) carried out immunoprecipitation studies to demonstrate electromobility shift assays. These results demonstrate the strict substrate specificity of UGT2B4 for the 6␣-hydroxy- that PPAR␣ agonists may control the catabolism of cy- lated BA hyodeoxycholic acid (HDCA) in human liver. Further- totoxic bile acids and reinforce recent data indicating more, no or low glucuronidation activity of HDCA was observed that PPAR␣, which has been largely implicated in the in colon where UGT2B4 is not expressed (8, 9). Finally, a recent control of lipid and cholesterol metabolism, is also an study revealed that UGT2B4 expression is positively regulated important modulator of the metabolism of endobiotics by the BA sensor farnesoid X-receptor (FXR) and suggested and xenobiotics in human hepatocytes. that UGT2B4 induction by BAs may be part of a negative feedback mechanism by which BAs limit their biological activ- ity and control their intracellular levels to avoid a pathophys- Glucuronide conjugation is a major metabolic pathway for iological accumulation (10). numerous endogenous and exogenous compounds, including An important consequence of BA glucuronidation is the in- 1 bile acids (BA), bilirubin, steroids, drugs, and environmental troduction of an additional negative charge in the molecule that allows their transport by conjugate transporters such as the multidrug-resistance related proteins, MRP2 (ABCC2) and * This work was supported by grants from the Fondation Lefoulon- MRP3 (ABCC3), which are present in liver (11, 12), and favors Delalande, Institut de France (to O. B.), the European community (ERBFMBICT983214) (to I. P.-T.), the ministerio de Hacienda del Go- their excretion in urine. Whereas BAs are biological detergents bierno de Chile (to D. D.-S.), the Fonds Europe´ens de De´veloppement with numerous important functions, these compounds are in- Re´gional, Conseil Re´gional Re´gion Nord/Pas-de-Calais (Genopole Pro- herently cytotoxic and perturbations in their normal synthesis, ject Grant 01360124), and the Leducq Foundation (to B. S. and J.-C. F.). transport, or secretion can result in a variety of pathophysio- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked logical conditions including intrahepatic cholestasis (13). Dur- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to ing their enterohepatic circulation, BAs undergo several met- indicate this fact. abolic alterations, including glucuronide conjugation at ring ¶ To whom correspondence should be addressed: Unite´INSERM 545, Institut Pasteur de Lille, 1, rue du Pr Calmette, BP 245, 59019 Lille, France. Tel.: 33-3-20-87-73-87; Fax: 33-3-20-87-71-98; E-mail: bart. acid; LCA, lithocholic acid; PPAR, peroxisome proliferator-activated [email protected]. receptor ␣; RXR, retinoid X-receptor; PPRE, PPAR response elements; 1 The abbreviations used are: BA, bile acids; UGT, UDP-glucurono- RT, reverse transcription; UDPGA, UDP-glucuronic acid; TLC, thin syltransferase; HDCA, hyodeoxycholic acid; FXR, farnesoid X-receptor; layer chromatography; ANOVA, analysis of variance; PXR, pregnane EMSA, electrophoretic mobility shift assays; CDCA, chenodeoxycholic X-receptor; CMV, cytomegalovirus; CYP, cytochrome P450.

32852 This paper is available on line at http://www.jbc.org PPAR␣ Induces Hepatic Expression of UGT2B4 Enzyme 32853 hydroxyl groups (7, 14). The most abundant glucuronide con- HepG2 and Huh7 cells. The induction of UGT2B4 gene expres- jugate reported in human plasma is the primary BA chenode- sion is accompanied by an increased glucuronidation activity of oxycholic acid (CDCA) glucuronide followed by the secondary HDCA. This positive regulation occurs at the transcriptional lithocholic acid (LCA) glucuronide (7, 15). In the urine of cho- level via binding of PPAR␣ to a DR1 response element located lestatic patients, the proportion of BA glucuronide metabolites at Ϫ1193 bp in the promoter region of the UGT2B4 gene. increases to up to 35% of total BAs (16, 17) and HDCA is EXPERIMENTAL PROCEDURES exclusively found as a glucuronide derivative (18). Animal Studies—Animal studies were performed in compliance with Peroxisome proliferator-activated receptors (PPARs) belong European Union specifications regarding the use of laboratory animals. to the family of nuclear receptors that are ligand-activated Details of experimental conditions have been described previously (33). transcription factors. Three distinct types of PPARs have been Male Sv/129 homozygous wild type (ϩ/ϩ)(n ϭ 6) and PPAR␣ null (Ϫ/Ϫ) identified as PPAR␣, PPAR␦ (or PPAR␤), and PPAR␥. Each (n ϭ 6) mice (34) (a kind gift of Dr. F. Gonzalez, National Cancer isotype is encoded by a distinct gene and shows different dis- Institute, National Institutes of Health, Bethesda, MD) were fed for 17 days with a standard mouse chow diet containing 0.2% (wt/wt) fenofi- tribution patterns (19, 20). Upon ligand activation, PPARs brate or not. At the end of the treatment period, the animals were fasted regulate gene transcription by dimerizing with the retinoid for 4 h and sacrificed and livers were removed immediately, weighed, X-receptor (RXR) and binding to PPAR response elements rinsed in 0.9% (w/v) NaCl, frozen in liquid nitrogen, and stored at (PPREs) within the regulatory regions of target genes (19). Ϫ80 °C until total RNA or microsome preparation. These PPREs usually consist of a direct repeat of the hex- Materials—UDP-glucuronic acid (UDPGA), leupeptin, pepstatin, phosphatidylcholine, and BAs were obtained from Sigma. Human hepa- anucleotide AGGTCA sequence separated by one or two nucle- toblastoma HepG2 cells were from the American Type Culture Collec- otides (DR1 or DR2) (19). Furthermore, PPARs can also nega- tion (Manassas, VA). Restriction enzymes and other molecular biology tively interfere with pro-inflammatory transcription factor reagents were from New England Biolabs (distributed by Ozyme, Saint- ␣ Quentin, France), Stratagene (La Jolla, CA), Promega, and Roche Ap- pathways by a mechanism termed transrepression (21). PPAR Downloaded from is highly expressed in various tissues such as liver, muscle, plied Science. Protein assay reagents were obtained from Bio-Rad. [␣-32P]dCTP, [␥-32P]ATP, and [14C]UDPGA (180mCi/mmol) were pur- kidney, and heart where it stimulates the ␤-oxidative degrada- chased from PerkinElmer Life Sciences. Cell culture reagents were tion of fatty acids (22). Natural eicosanoids derived from from Invitrogen. ExGen 500 was from Euromedex (Souffelweyersheim, arachidonic acid via the lipoxygenase pathway, such as 8-hy- France). The anti-UGT2B antibody was kindly provided by Dr. A. droxytetraenoic acid, 15-hydroxytetraenoic acid, and leukotri- Be´langer (Laval University, Quebec, Canada), and the secondary anti- ene B4 as well as oxidized phospholipids, activate PPAR␣ (23– body against rabbit IgG was purchased from Sigma. Real-time PCR kits http://www.jbc.org/ were purchased from Stratagene. 25). The hypolipidemic fibrates (gemfibrozil, bezafibrate, Cell Culture—Human primary hepatocytes were isolated as de- ciprofibrate, and fenofibrate) are synthetic PPAR␣ ligands scribed previously (35) and incubated for the indicated times in used in the treatment of dyslipidemia (23). William’s E medium containing fenofibric acid (250 ␮M). Human hepa- Recent findings indicate that PPAR␣ also regulates BA syn- toma HepG2 and Huh7 cells were grown as described previously (36, 6 thesis and transport. In cultured rat hepatocytes, PPAR␣ ago- 37). For RNA analyses, 10 HepG2 or Huh7 cells were treated with Wy

14643 at the indicated concentrations in the presence or absence of 75 by guest on December 24, 2015 nists decrease bile acid synthesis and suppress the expression ␮M CDCA for 24 h. In all of the experiments, controls were incubated of two key BA-synthesizing enzymes, the cytochrome P450 with an identical volume of Me2SO (vehicle). cholesterol 7␣-hydroxylase (CYP)7A1 and the sterol 27-hydrox- RNA Analysis—Total RNA was isolated from mice liver, primary ylase (CYP27), which is paralleled by a similar reduction of human hepatocytes, HepG2, and Huh7 cells using TRIzol (Invitrogen). their respective activities (26). By contrast, ligand-activated Northern blot analyses were performed as described previously (38) ␣ using human UGT2B4 and 36B4 cDNAs as probes. For quantitative PPAR stimulates the expression and activity of the murine RT-PCR analyses of UGT2B4 gene expression, RNA was reverse-tran- sterol 12␣-hydroxylase enzyme (CYP8B1), a hepatic microso- scribed using random hexamer primers and 200 units of Moloney mu- mal enzyme that acts as a branch point in the bile acid syn- rine leukemia virus reverse transcriptase (Invitrogen). Reverse-tran- thetic pathway, determining the ratio of cholic acid/CDCA (27). scribed UGT2B4 and 28 S cDNAs were quantified by real-time PCR on In human hepatoma HepG2 cells, the PPAR␣ ligand Wy 14643 a MX4000 apparatus (Stratagene) using specific primers for UGT2B4 and 28 S as described previously (36, 39). PCR amplifications were suppresses CYP7A1 gene promoter activity (28). Furthermore, performed in a volume of 25 ␮l containing 100 nM of each primer, 4 mM in mouse liver, treatment with the PPAR␣ agonist, ciprofi- MgCl2, the Brilliant Quantitative PCR Core reagent kit mixture (Strat- brate, results in a decreased expression of the bile salt trans- agene), and SYBR Green 0.33X (Sigma). The conditions were 95 °C for porters, such as Naϩ-taurocholate co-transporting polypeptide 10 min followed by 40 cycles of 30 s at 95 °C,30sat60°C, and 30 s at 1, Naϩ-independent organic anion-transporting polypeptide 72 °C. UGT2B4 mRNA levels were normalized to 28 S mRNA (36). Plasmid Cloning and Site-directed Mutagenesis—The B4p-2400- (Oatp1), and the bile salt export pump (29). By contrast, cipro- pGL3 construct was obtained as described previously (10). The B4p- fibrate activation of PPAR␣ induces the promoter activity of 2084, B4p-1214, B4p-1149, and B4p-524 reporter constructs were gen- human apical sodium-dependent bile salt transporter (ASBT) erated by PCR amplification with Pfu Turbo polymerase (Stratagene) gene in human colon carcinoma Caco2 cells (30). Overall, these and 100 pmol of the sense oligonucleotides: B4-2084, 5Ј-CATCAGAGT- Ј Ј data suggest that PPAR␣ activation may result in a decreased AGTGACTGCTAGTAGTTG-3 ; B4-1214, 5 -TTTAAGTTATTATCTAT- AGAACAG-3Ј; B4-1149, 5Ј-TATTAGGAAGCGAGTCAGAGAG-3Ј; and BAs synthesis and secretion into bile. Furthermore, several B4-524, 5Ј-CATTTCTGAAATATATTACATGAG-3Ј, respectively. The studies in both humans and animals reported that treatment reverse primer was pGL3-512, (5Ј-TATGCAGTTGCTCTCCAGCGGTT- with PPAR␣ activators results in enhanced glucuronidation CCATCTTCC-3Ј) from the pGL3 basic plasmid. PCR products were activity and UGT expression (31, 32). Indeed, clofibrate induces subsequently digested with NcoI, gel-purified, and cloned into a SmaI the bilirubin-conjugating UGT1A1 protein in microsomes from plus NcoI-digested pGL3 basic plasmid. Mutations were introduced in the PPRE using the QuikChange site-directed mutagenesis kit (Strat- rat liver (32). agene) and the oligonucleotide B4-PPREmt (5Ј-AGTTAAGATAAAAT- Since PPAR␣ is an important regulator of BAs synthesis and TTAATCTGTA-3Ј) (nucleotide in boldface indicate the mutated bases). transport and considering the major role that UGT2B4 plays in The B4-PPREwtx6-TKpGL3 plasmid was obtained by cloning six copies hepatic glucuronidation of BAs, we investigated in the present of the corresponding dimerized oligonucleotides in the thymidine kinase study whether hepatic UGT2B4 expression and activity are promoter-driven luciferase reporter (TKpGL3) vector. ϫ 3 ␣ ␣ Transient Transfection Assays—60 10 HepG2 or Huh7 cells were regulated by PPAR . Our results demonstrate that PPAR transfected with 100 ng of the indicated luciferase reporter plasmids, 50 activation results in the induction of UGT2B4 gene expression ng of the pCMV-␤-galactosidase expression vector, and with or without in human primary hepatocytes and human hepatoblastoma 30 ng of the pSG5-PPAR␣ plasmid. All of the samples were comple- 32854 PPAR␣ Induces Hepatic Expression of UGT2B4 Enzyme

FIG.1.PPAR␣ activation increases UGT2B4 mRNA levels in primary human hepatocytes. a, primary human hepatocytes were treated ␮ for 24 h with Me2SO (Vehicle) or fenofibric acid (250 M), and UGT2B mRNA levels were analyzed by Northern blot. 36B4 RNA was measured as ␮ a control. b, primary human hepatocytes were treated for 6, 12, 24, and 48 h with Me2SO (Vehicle) or fenofibric acid (250 M), and UGT2B4 transcripts were quantified using real-time RT-PCR analyses. Values are expressed as means Ϯ S.D. (n ϭ 6) relative to the control set as 1. Statistically significant differences between vehicle- and fenofibric acid-treated cells are indicated by asterisks (Mann-Whitney test: ***, p Ͻ 0.001). Downloaded from http://www.jbc.org/ by guest on December 24, 2015

FIG.2.PPAR␣ agonists dose-dependently induce the UGT2B4 gene expression in human hepatoblastoma HepG2 and Huh7 cells. ␮ HepG2 (a) or Huh7 (b) cells were incubated with increasing concentrations of Wy 14643 (25, 50, 75, and 100 M) or vehicle (Me2SO) for 24 h. UGT2B4 mRNA levels were measured by real-time RT-PCR and expressed relative to control set as 1. Values are means Ϯ S.D. (n ϭ 6). Statistically significant differences between vehicle- and Wy 14643-treated cells are indicated by asterisks (Mann-Whitney test, *, p Ͻ 0.05; ***, p Ͻ 0.001).

mented with pBS-SKϩ plasmid (Stratagene) to an identical amount of plexes were visualized using the Western blot Chemiluminescence Re- 500 ng/well. Cells were transfected with ExGen reagent (Euromedex) agent Plus as specified by the manufacturer (PerkinElmer Life for6hat37°C and subsequently incubated overnight with Dulbecco’s Sciences). modified Eagle’s medium, 0.2% fetal bovine serum and then treated for Glucuronidation Assay—HepG2 cells were resuspended in Tris-buff- ␮ 24 h with either Me2SO (vehicle) or Wy 14643 (50 M) as indicated. ered saline containing 0.5 mM dithiothreitol and homogenized using a Electrophoretic Mobility Shift Assays (EMSA)—EMSA using in vitro Brinkman Polytron. Enzyme assays were performed as described pre- produced PPAR␣ and RXR were performed as described previously (40) viously (5). 100 ␮g of cell homogenate were incubated with 25 ␮M using the radiolabeled probes B4-PPREwt, 5Ј-TAAGATGAACTTTAA- [14C]UDP-glucuronic acid, 2 mM unlabeled UDPGA, and 200 ␮M HDCA TCTTGTAAC-3Ј; B4-PPREmt5Ј,5Ј-TAAGATAAAATTTAATCTTGTA- in a final volume of 100 ␮l of glucuronidation assay buffer for 8 h (5). AC-3Ј; and B4-PPREmt3Ј,5Ј-TAAGATGAACTTTAAAATTGTAAC-3Ј Assays were terminated by adding 100 ␮l of methanol, and the samples (where underlined nucleotides represent response element half-sites were centrifuged at 14,000 rpm for 2 min to remove the precipitated and bases in boldface are mutated). For supershift experiments, 0.2 ␮g proteins. 100 ␮l of glucuronidation assays were applied onto a thin layer of the anti-PPAR␣ antibody (Santa-Cruz Biotechnology) was preincu- chromatography (TLC) plate (Merck) and migrated using a toluene: bated for 20 min in the binding buffer before the addition of PPAR␣ and methanol:acetic acid (7:3:1) mixture. The extent of HDCA glucuronida- RXR proteins. For competition experiments, the unlabeled oligonucleo- tion was analyzed and quantified by PhosphorImager analysis. tides were included in the binding reaction at the indicated excess Statistical Analyses—A nonparametric Mann-Whitney test was used concentrations over the probe just before adding the labeled to analyze for significant difference between the experimental groups. oligonucleotide. Analyses of variance (ANOVA) and Tukey post-hoc tests were used for Microsome Purification and Western Blot Analysis—Microsomal pro- analysis of the effects of simultaneous treatment with FXR and PPAR␣ teins were purified from wild type or PPAR␣-null mouse livers as agonists. previously described (41). Microsome pellets were resuspended in 300 ␮l of homogenization buffer, and the protein content was determined RESULTS using Bradford reagent (Bio-Rad) and bovine serum albumin for stand- PPAR␣ Activators Induce UGT2B4 Expression ard curves. Samples were aliquoted and kept at Ϫ 80 °C until Western blot analysis or glucuronidation assays. For Western blot, 25 ␮gof in Human Hepatocytes microsomal proteins were separated on a 10% SDS-polyacrylamide gel. Primary human hepatocytes were treated with fenofibric The gel was transferred onto a nitrocellulose membrane, which was ␮ then hybridized with the anti-UGT2B antibody (dilution, 1/2000). An acid (250 M) for 24 h, and UGT2B mRNA levels were deter- anti-rabbit IgG antibody conjugated with peroxidase was used as sec- mined by Northern blot analysis. A significant increase in ondary antibody (dilution, 1/10000), and the resulting immunocom- UGT2B mRNA was observed in fenofibric acid-treated cells PPAR␣ Induces Hepatic Expression of UGT2B4 Enzyme 32855

␣ FIG.3.PPAR activation increases bile acid glucuronidation activity in HepG2 cells. HepG2 cells were treated with vehicle (Me2SO) or Wy 14643 (75 ␮M) for 36 h. Cell homogenates (100 ␮g) were incubated with [14C]UDPGA (25 ␮M), unlabeled UDPGA (2 mM), and HDCA (200 ␮M)for8hat37°C. a, radiolabeled HDCA-glucuronide was subsequently analyzed by TLC. Conjugated HDCA migrates at the top of TLC, whereas the free UDPGA is detected at the bottom of the chromatogram. b, formation of HDCA-glucuronide was quantified by PhosphorImager analysis. Values represent means Ϯ S.D. (n ϭ 3). Downloaded from

FIG.4.PPAR␣ is required for the fenofibrate-dependent induction of UGT2B mRNA and protein levels in mouse liver. Wild type ϩ ϩ ␣ Ϫ Ϫ ( / ) and PPAR -null ( / ) mice were fed for 17 days with standard mouse chow diet containing fenofibrate 0.2% (wt/wt) or not. a, UGT2B mRNA http://www.jbc.org/ levels were analyzed by Northern blot using the human UGT2B4 cDNA as radiolabeled probe. 36B4 RNA was measured as a control. b, microsomal proteins were extracted from livers and immunoblotted using an anti-UGT2B antibody (1/2000). compared with vehicle-treated cells (Fig. 1a). With the cDNA the groups. These data indicate that PPAR␣ is a crucial regu- sequences of the different human UGT2B isoforms being Ͼ85% lator of basal and fibrate-activated murine UGT2B gene homologous, a UGT2B4-specific real time RT-PCR method was expression. by guest on December 24, 2015 used to specifically quantify UGT2B4 mRNA levels in hepato- To determine whether PPAR␣ also regulates murine UGT2B cytes treated for 6, 12, 24, or 48 h with fenofibric acid (250 ␮M) protein levels, liver microsomes from wild type and PPAR␣- (Fig. 1b). Fenofibric acid rapidly induced UGT2B4 expression, null mice were subjected to Western blot analysis using an because a maximal 10-fold increase in the concentration of anti-UGT2B antibody. In wild type mice, a pronounced in- UGT2B4 transcripts was observed within 12 h (Fig. 1b). crease in UGT2B protein levels was observed in fenofibrate- To further characterize the PPAR␣-dependent regulation of treated compared with vehicle-treated animals (Fig. 4b). As for UGT2B4 expression, human hepatoma HepG2 and Huh7 cells their mRNAs, UGT2B protein levels were almost undetectable were incubated in the presence of increasing concentrations of in liver microsomes from PPAR␣-null mice and treatment with the PPAR␣ ligand Wy 14643 (Fig. 2, a and b). UGT2B4 mRNA fenofibrate failed to increase UGT2B protein concentration levels were induced in a dose-dependent manner to a maximum (Fig. 4b). In fact, longer exposure of the Western blot revealed of 2.7- and 2.3-fold activation in HepG2 and Huh7 cells, respec- the presence of low amounts of UGT2Bs in PPAR␣-null mice, tively (Fig. 2, a and b). which were not affected by fenofibrate treatment (data not shown). These results clearly demonstrate that, similar to hu- PPAR␣ Activators Induce UGT2B4 Activity man UGT2B4, murine UGT2B enzymes are positively regu- in HepG2 Cells lated PPAR␣ target genes. ␣ To determine whether PPAR activation of UGT2B4 expres- ␣ sion modifies its activity, HepG2 cells were treated with Wy PPAR Activates the UGT2B4 Gene Promoter 14643 (75 ␮M) for 36 h and their glucuronidation activity was To decipher the molecular mechanisms of human UGT2B4 analyzed using the UGT2B4-specific substrate HDCA (Fig. 3a). induction by PPAR␣ activators, a 2.4-kb fragment of the Treatment with Wy 14643 provoked a 3-fold increase in HDCA UGT2B4 gene promoter cloned in front of the pGL3-luciferase glucuronidation (Fig. 3b), thus demonstrating that PPAR␣ ago- reporter gene was transfected into HepG2 cells in the presence nists induce UGT2B4 activity. or absence of a PPAR␣ expression plasmid. Transfected cells were subsequently treated with the PPAR␣ ligand Wy 14643 PPAR␣ Gene Disruption Abolishes Fibrate Induction of (Fig. 5). Wy 14643 alone slightly induced UGT2B4 promoter UGT2B mRNA and Protein Levels in Mouse Liver activity, whereas co-transfection of PPAR␣ significantly en- The PPAR␣-dependent induction of UGT2B expression was hanced Wy 14643-induced promoter activity to ϳ3-fold in measured in male Sv/129 homozygous wild type (ϩ/ϩ) and HepG2 cells (Fig. 5). To localize the region within the UGT2B4 PPAR␣-null (Ϫ/Ϫ) mice by Northern blotting (Fig. 4a). In wild promoter that confers transcriptional responsiveness to PPAR␣ type mice, fenofibrate treatment resulted in an ϳ5-fold in- ligands, serial deletions from Ϫ2084 to Ϫ524 bp of the UGT2B4 crease of UGT2B mRNA levels compared with vehicle-treated promoter were also co-transfected with or without the expres- animals. Interestingly, UGT2B transcripts were undetectable sion vector for PPAR␣ (Fig. 5). A marked increase in reporter by this method in both fenofibrate- and vehicle-treated PPAR␣- activities of the two larger fragments (Ϫ2084 and Ϫ1214 bp) null mice. As control, 36B4 mRNA levels were similar in all of was observed in HepG2 cells treated with Wy 14643, and co- 32856 PPAR␣ Induces Hepatic Expression of UGT2B4 Enzyme

FIG.5.Identification of a functional PPRE in the human UGT2B4 promoter. HepG2 cells were transfected with the indicated human UGT2B4 promoter-driven luciferase (Luc) reporter plasmids (100 ng) in the absence or presence of pSG5-PPAR␣ (30 ng), and a CMV-driven ␤ ␤ ␮ -galactosidase expression plasmid (pCMV- -galactosidase, 50 ng). Cells were subsequently treated with Wy 14643 (50 M) or vehicle (Me2SO) for 24 h. Values are expressed as fold induction of the controls (pGL3) set at 1 normalized to internal ␤-galactosidase activity as described under Downloaded from “Experimental Procedures.” Values represent the means Ϯ S.D. http://www.jbc.org/ by guest on December 24, 2015

FIG.6. PPAR␣ activates the DR1-1193 response element. a, HepG2 cells were transfected with the wild type (B4p-2400) or mutated ␣ ␤ (B4p-2400mt) reporter plasmids (100 ng) in the absence or presence of pSG5-PPAR (30 ng) and a CMV-driven -galactosidase expression plasmid ␤ ␮ (pCMV- -galactosidase, 50 ng). Cells were subsequently treated with Wy 14643 (50 M) or vehicle (Me2SO) for 24 h. b, six copies of the wild type DR1-1193 response element were cloned upstream of the thymidine kinase minimal promoter-driven luciferase reporter TKpGL3. The resulting constructs (100 ng) were co-transfected with the pCMV-␤-galactosidase plasmid (50 ng) in HepG2 cells in the presence or absence of pSG5-hPPAR␣ (30 ng). Cells were subsequently treated or not with Wy 14643 (50 ␮M) for 24 h. Values are expressed as fold induction of the controls (pGL3) set at 1 normalized to internal ␤-galactosidase activity as described under “Experimental Procedures.” Values represent the means Ϯ S.D. transfection of PPAR␣ further increased the activities of the revealed the presence of a degenerated DR1 sequence, two constructs (Fig. 5). Further 5Ј deletion (Ϫ1149 and Ϫ524 TGAACTTTAATCT, at positions from Ϫ1193 to Ϫ1180. To test bp) constructs were no longer induced by Wy 14643-activated whether this site mediates the induction by PPAR␣, mutations PPAR␣, indicating that the region between Ϫ1214 and Ϫ1149 were introduced in this site in the context of the Ϫ2400 bp bp mediates the effect of PPAR␣ ligands on the UGT2B4 pro- UGT2B4 promoter constructs (Fig. 6a). Mutation of this site moter. Identical results were obtained when these reported abolished the induction of UGT2B4 promoter activity by Wy constructs were co-transfected with or without PPAR␣ in Huh7 14643-activated PPAR␣. Furthermore, the UGT2B4 DR1 site cells (data not shown). was cloned in multiple copies upstream of the luciferase re- porter gene driven by the heterologous thymidine kinase pro- Identification of a PPRE within moter TKpGL3 and subsequently transfected in the presence the UGT2B4 Gene Promoter or absence of the pSG5-PPAR␣ in HepG2 cells, which were Consensus DR1 sites have been previously reported to bind treated or not with Wy 14643 (Fig. 6b). Reporter activity was the PPAR␣/RXR heterodimer (19). A computer-assisted analy- increased upon co-transfection of constructs containing six cop- sis (42) of the Ϫ1214/Ϫ1149 region of the UGT2B4 promoter ies of the wild type DR1 with the PPAR␣ plasmid. This activity PPAR␣ Induces Hepatic Expression of UGT2B4 Enzyme 32857 Downloaded from http://www.jbc.org/ by guest on December 24, 2015

FIG.7.PPAR␣ binds to the PPRE in the UGT2B4 promoter. a and b, EMSAs were performed with end-labeled wild type or mutated B4-PPRE probes as indicated in the presence of unprogrammed reticulocyte lysate, RXR, PPAR␣, or both RXR and PPAR␣ as indicated. Supershift experiments were carried out using the anti-PPAR␣ antibody (0.2 ␮g). c, competition EMSAs on radiolabeled B4-PPRE probe were performed by adding 1-, 10-, or 50-fold molar excess of the indicated cold consensus DR1 (DR1cons), B4-PPREwt, or B4-PPREmt5Ј oligonucleotides in EMSA with unprogrammed reticulocyte lysate, RXR, and/or PPAR␣.

was further enhanced by the addition of the PPAR␣ ligand (Fig. (Fig. 7c). By contrast, the mutated B4-PPREmt3Ј did not com- 6b). By contrast, no change in activity was observed when pete for PPAR␣ binding to the DR1. Taken together, these data either the empty TKpGL3 vector (Fig. 6b) or the TKpGL3 demonstrate that PPAR␣ binds to the PPRE site at nucleotides vector containing three copies of the mutated DR1 (data not from Ϫ1193 to Ϫ1180 in the UGT2B4 gene promoter. shown) was transfected. These results indicate that the Ϫ1193 ␣ to Ϫ1180 site in the UGT2B4 promoter is a positive PPRE. PPAR and FXR Activators Additively To demonstrate that PPAR␣ binds to the PPRE identified in Induce UGT2B4 Expression the UGT2B4 gene promoter, EMSAs were performed using a We previously reported that CDCA-activated FXR positively probe spanning nucleotides from Ϫ1199 to Ϫ1175 (B4- regulates the expression of UGT2B4 in human hepatocytes and PPREwt) in the presence of in vitro translated PPAR␣ and RXR HepG2 cells (10). To test whether ligand-activated FXR and proteins (Fig. 7). As expected, neither RXR nor PPAR␣ alone PPAR␣ can cooperate to regulate UGT2B4 expression, HepG2 bound the probe (Fig. 7a, lanes 2 and 3). By contrast, a clear cells were treated for 24 h with Wy 14643, CDCA, or both Wy shift was observed when this oligonucleotide was incubated in 14643 and CDCA together. As expected, UGT2B4 mRNA levels the presence of both RXR and PPAR␣ (Fig. 7a, lane 4). Fur- were induced 2.6-fold by Wy 14643, whereas CDCA-induced thermore, this complex was supershifted by the anti-PPAR␣ UGT2B4 gene expression was ϳ10-fold (Fig. 8). Interestingly, antibody (lane 5), thus demonstrating that the PPAR␣/RXR cells treated with both PPAR␣ and FXR activators contained heterodimer specifically binds the Ϫ1193 DR1 site. By contrast, 14-fold higher concentrations of UGT2B4 transcripts, indicat- no protein-DNA complex was observed when mutated probes in ing that the two receptors coordinately regulate UGT2B4 gene the 5Ј and 3Ј half-sites (B4-PPREmt5Ј and B4-PPREmt3Ј, re- expression. spectively) were tested (Fig. 7b, lanes 5–12). For competition experiments, increasing amounts (1-, 10-, and 50-fold excess) of DISCUSSION unlabeled oligonucleotides encompassing either a consensus In this study, we identify the human UGT2B4 enzyme as a DR1 site (DR1cons), the B4-PPREwt, or the B4-PPREmt5Ј positively regulated PPAR␣ target gene. UGT2B4 induction by were added to binding reactions containing PPAR␣ in the pres- fibrates occurs via PPAR␣ binding to a PPRE in the UGT2B4 ence of RXR (Fig. 7c). PPAR␣ binding to the B4-PPREwt was promoter. Furthermore, we show that fenofibrate induces he- competed by the DR1 consensus site and by the B4-PPREwt patic UGT2B mRNA and protein levels only in Sv/129 wild type 32858 PPAR␣ Induces Hepatic Expression of UGT2B4 Enzyme mice, whereas a drastically lowered expression of UGT2Bs is PPAR␣ activation may affect BA glucuronidation in HepG2 observed in livers from PPAR␣-null mice treated or not with cells. Indeed, we observed that Wy 14643-dependent PPAR␣ fenofibrate. This observation demonstrates that PPAR␣ is a activation provoked a 2-fold increase of HDCA-glucuronidation crucial regulator of both human UGT2B4 and murine UGT2B activity. HDCA is a 6␣-hydroxylated metabolite of LCA, which enzyme expression. Interestingly, PPAR␣ gene disruption also is primarily excreted as a glucuronide derivative in urine (18, critically reduced the basal expression of mitochondrial fatty 44). Because of its high degree of lipophilicity, LCA is a potent acid-metabolizing enzymes such as very long chain acyl-CoA cholestatic agent and possesses an elevated cytotoxicity (45, dehydrogenase, long chain acyl-CoA dehydrogenase, and long 46). However, conjugation of LCA with sulfate, a conjugation chain acyl-CoA synthetase enzymes (43). Thus, the present reaction catalyzed by the dehydroepiandrosterone sulfotrans- findings demonstrate that in mice, PPAR␣ plays a crucial role ferase (SULT2A1) enzyme, allows an increased hydrosolubility in the constitutive expression of not only mitochondrial fatty of LCA and facilitates its biliary excretion (47–49). In addition acid-metabolizing enzymes but also microsomal UGT2B to sulfation, LCA is efficiently 6␣-hydroxylated into HDCA by enzymes. the hepatic CYP3A4 enzyme, and this modification facilitates Considering the major role that UGT2B4 plays in BA glucu- its glucuronidation by UGT2B4 at the 6␣-hydroxy position ronidation, we hypothesized that UGT2B4 induction following prior to renal excretion (50). Thus, glucuronidation of HDCA has been proposed as an alternative mechanism for reducing the hepatic toxicity of monohydroxylated LCA (44, 50). Recent studies indicate that the BA sensors pregnane X-receptor (PXR) and FXR play important roles in LCA detoxification. As such, activation of PXR induces both SULT2A1 and CYP3A4 expression, whereas BA-activated FXR stimulates SULT2A1 and UGT2B4 expression (10, 44, 49, 51). Results from the Downloaded from present study prove that PPAR␣ also participates in the control of LCA detoxification in addition to PXR and FXR (Fig. 9). Furthermore, PPAR␣, FXR, and PXR inhibit CYP7A1 expres- sion (26, 52, 53), thus suggesting that the three receptors may cooperate to control BA homeostasis and detoxification by both reducing BA synthesis and inducing their metabolism (Fig. 9). http://www.jbc.org/ Recently, PPAR␣ was identified as a FXR target gene, thus providing molecular evidence for a cross-talk between the FXR and PPAR␣ transcriptional pathways in humans (54). Consid- ering that UGT2B4 expression is also up-regulated upon CDCA activation of FXR (10), we investigated whether this cross-talk between FXR and PPAR␣ can affect UGT2B4 expression in by guest on December 24, 2015 FIG.8.PPAR␣ and FXR induce UGT2B4 gene expression in an HepG2 cells. We observed that upon ligand activation, PPAR␣ additive manner. HepG2 cells were treated for 24 h with Wy 14643 and FXR act in concert to stimulate BA glucuronidation. Over- (75 ␮M), CDCA (75 ␮M), or both Wy 14 643 and CDCA. UGT2B4 mRNA all, these results demonstrate that FXR and PPAR␣ control not levels were measured by real-time RT-PCR and expressed relative to control set as 1. Values are means Ϯ S.D. (n ϭ 6). Values followed by only the same BA-metabolizing enzyme but also share cooper- different letters are statistically significantly different from each other ative activity to induce BA glucuronidation catalyzed by (ANOVA followed by Mann-Whitney test, p Ͻ 0.01). UGT2B4. It would be interesting to determine whether a sim-

FIG.9.PPAR␣ participates with PXR and FXR in the control of bile acid homeostasis and detoxification. By inhibiting CYP7A1 expression and inducing CYP3A4, SULT2A1, and UGT2B4 enzyme expression, PXR, FXR, and PPAR␣ form a cluster of ligand-activated transcription factors that control bile acid homeostasis and reduce bile acid toxicity. HDCA-G, HDCA-glucuronide; LCA-S: LCA-sulfate; SULT2A1, sulfotransferase 2A1. PPAR␣ Induces Hepatic Expression of UGT2B4 Enzyme 32859 ilar cross-talk also exists between PXR and PPAR␣ and/or R. H., and Manns, M. P. (2000) J. Biol. Chem. 275, 36164–36171 10. Barbier, O., Pineda Torra, I., Sirvent, A., Claudel, T., Blanquart, C., Duran- FXR. Sandoval, D., Kuipers, F., Kosykh, V., Fruchart, J. C., and Staels, B. (2003) UGT2B4 is considered to be the specific BA-conjugating UGT Gastroenterology 124, 1926–1940 enzyme in human liver, although it also participates to the 11. Kuipers, F., Radominska, A., Zimniak, P., Little, J. M., Havinga, R., Vonk, R. 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2991–2998 by guest on December 24, 2015 in rodents. The role of different nuclear receptors, such as PXR 34. Lee, S. S., Pineau, T., Drago, J., Lee, E. J., Owens, J. W., Kroetz, D. L., and constitutive androstane receptor, in the control of xenobi- Fernandez-Salguero, P. M., Westphal, H., and Gonzalez, F. J. (1995) Mol. otic metabolizing enzyme expression has been fully character- Cell. Biol. 15, 3012–3022 35. Bode, B. P., Kaminski, D. L., Souba, W. W., and Li, A. P. (1995) Hepatology 21, ized, whereas PPAR␣ received less attention regarding xeno- 511–520 biotic metabolizing enzyme regulation. Nevertheless, the 36. Claudel, T., Sturm, E., Duez, H., Torra, I. P., Sirvent, A., Kosykh, V., Fruchart, J. C., Dallongeville, J., Hum, D. W., Kuipers, F., and Staels, B. (2002) present findings added to previous reports indicate that J. Clin. Invest. 109, 961–971 PPAR␣ is also an important xenobiotic sensor that regulates 37. 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Olivier Barbier, Daniel Duran-Sandoval, Inés Pineda-Torra, Vladimir Kosykh, Jean-Charles Fruchart and Bart Staels J. Biol. Chem. 2003, 278:32852-32860.

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