FXR Induces the UGT2B4 Enzyme in Hepatocytes: A PotentialMechanism of Negative Feedback Control of FXR Activity
OLIVIER BARBIER,* INES PINEDA TORRA,* AUDREY SIRVENT,‡ THIERRY CLAUDEL,*CHRISTOPHE BLANQUART,* DANIEL DURAN–SANDOVAL,* FOLKERT KUIPERS,§ VLADIMIR KOSYKH,�
JEAN–CHARLES FRUCHART,* and BART STAELS**U545 INSERM, Department of Atherosclerosis, Lille Pasteur Institute and Faculty of Pharmacie, University of Lille II, Lille, France; ‡GenfitSA, Parc Eurasante, Loos, France; §Center for Liver, Digestive and Metabolic Diseases, Laboratory of Pediatrics, University Hospital,Groningen, The Netherlands; and �Institute of Experimental Cardiology, Russian Cardiology Complex, Moscow, Russia
Background & Aims: Bile acids are essential for bileformation and intestinal absorption of lipids and fat-soluble vitamins. However, the intrinsic toxicity ofhydrophobic bile acids demands a tight control oftheir intracellular concentrations. Bile acids are li-gands for the farnesoid X receptor (FXR) that regu-lates the expression of genes controlling bile acidsynthesis and transport. The human uridine 5�-diphos-phate–glucuronosyltransferase 2B4 (UGT2B4) con-verts hydrophobic bile acids into more hydrophilicglucuronide derivatives. In this study, we identifyUGT2B4 as an FXR target gene. Methods: Humanhepatocytes or hepatoblastoma HepG2 cells weretreated with chenodeoxycholic acid or the syntheticFXR agonist GW4064, and the levels of UGT2B4 mes-senger RNA, protein, and activity were determined byusing real-time polymerase chain reaction, Westernblot, and glucuronidation assays. Results: Treatmentof hepatocytes and HepG2 cells with FXR agonistsresulted in an increase of UGT2B4 messenger RNA,protein, and activity. A bile acid response element inthe UGT2B4 promoter (B4-BARE) to which FXR, butnot retinoid X receptor, binds, was identified by site-directed mutagenesis, electromobility shift, and chro-matin immunoprecipitation assays. Retinoid X recep-tor activation abolished the induction of UGT2B4expression and inhibited binding of FXR to the B4-BARE, suggesting that retinoid X receptor modulatesFXR target gene activation. Overexpression of UGT2B4in HepG2 cells resulted in the attenuation of bile acidinduction of the FXR target gene small heterodi-meric partner. Conclusions: These data suggest thatUGT2B4 gene induction by bile acids contributes to afeed-forward reduction of bile acid toxicity and a de-crease of the activity of these biological FXR activa-tors.
Bile acids (BAs) are biological detergents that sub-serve a number of important functions, including
the uptake of cholesterol, fat-soluble vitamins, and
other lipids in the intestine1 and the hepatic genera-tion of bile flow.2 Furthermore, the biosynthesis ofBAs from cholesterol is the most important pathwayfor the elimination of cholesterol from the body. How-ever, because of their detergent properties, BAs areinherently cytotoxic, and perturbations in their nor-mal synthesis, transport, or secretion can result in avariety of pathophysiological conditions, includingintrahepatic cholestasis.3
The farnesoid X receptor (FXR; NR1H4) is a nu-clear receptor that is activated by the primary BAchenodeoxycholic acid (CDCA) and the secondary BAsdeoxycholic acid and lithocholic acid (LCA), as well astheir tauro- and glycoconjugates.4 In addition to BAs,synthetic FXR agonists have been identified.5,6 BA-activated FXR regulates the expression of target genesthrough binding as a heterodimer with the retinoid Xreceptor (RXR; NR2B1) to FXR response elements intheir promoter. Most of the FXR response elementscorrespond to an inverted repeat of the AGGTCAhalf-site spaced by 1 nucleotide (IR-1).7 However, theheterodimer FXR/RXR can also bind to direct repeatsof the AGGTCA hexamer separated by 3 or 4 nucle-otides.7 Moreover, FXR also binds as a monomer to anegative regulatory site in the apolipoprotein AI genepromoter.8
FXR negatively regulates BA synthesis by inhibitingtranscription of the cytochrome P450 7A1 gene, which
Abbreviations used in this paper: BA, bile acid; BARE, bile acid responseelement; CDCA, chenodeoxycholic acid; EMSA, electrophoretic mobilityshift assay; FXR, farnesoid X receptor; HDCA, hyodeoxycholic acid; IR-1,inverted repeat of the AGGTCA half-site spaced by 1 nucleotide; LCA,lithocholic acid; MRP, multidrug-resistance related protein; PCR, polymer-ase chain reaction; RT, reverse transcription; RXR, retinoid X receptor;SHP, small heterodimeric partner; UDPGA, uridine 5�-diphosphate–glucu-ronic acid; UGT, uridine 5�-diphosphate glucuronosyltransferase.
encodes a rate-limiting enzyme in BA synthesis.9,10 FXRalso regulates the expression of various proteins involvedin the uptake, intracellular transport, and export of BAs,such as the intestinal BA-binding protein,11 the bile saltexport pump,12 and the Na�-taurocholate co-transport-ing polypeptide (NTCP).13 BA-activated FXR also up-regulates the expression of SULT2A1, a sulfotransferaseenzyme involved in BA conjugation.14 Overall, BA ac-tivation of FXR in enterocytes or hepatocytes is thoughtto confer protection on the cells from their detergenteffects.
During their enterohepatic circulation, BAs undergoseveral metabolic alterations, including glucuronide con-jugation at ring hydroxyl groups.15,16 The glucu-ronidated BAs represent 7%–8% of the BA pool in theplasma of cholestatic patients, whereas in urine the pro-portion of these metabolites increases to up to 35% oftotal BAs.17,18 The most abundant glucuronide conjugatereported in human plasma is CDCA glucuronide, fol-lowed by LCA glucuronide.16,17,19
Glucuronidation is a major metabolic pathway fornumerous endogenous and exogenous compounds.20 Thisreaction consists of the transfer of the glucuronosyl groupfrom uridine 5�-diphosphate–glucuronic acid (UDPGA)to the acceptor molecule and is catalyzed by enzymesbelonging to the uridine 5�-diphosphate–glucuronosyl-transferase (UGT) family.21 Glucuronidated products aregenerally less biologically active, more hydrophilic, andmore easily excreted into bile or urine.21 On the basis ofamino acid sequence homology, UGT enzymes have beendivided into 2 major subfamilies: UGT1A andUGT2B.22 The human UGT1A isoforms are producedfrom a single gene, whereas UGT2B proteins are encodedby different genes clustered on chromosome 4q13–4q21.1.23–25 Whereas the UGT1A gene is conservedamong mammalian species, the UGT2B enzymes are notconserved between rodents and humans, thus precludingthe use of nonprimate animal models for the study ofUGT2B gene regulation. In humans, 7 different UGT2Bproteins have been characterized: UGT2B4, UGT2B7,UGT2B10, UGT2B11, UGT2B15, UGT2B17, andUGT2B28.20,26 UGT2B4 has been shown to glucu-ronidate 6�-hydroxylated BAs, such as hyodeoxycholicacid (HDCA).16,27 An important consequence of BAglucuronidation is the introduction of an additional neg-ative charge to the molecule, which allows their trans-port by conjugate transporters such as multidrug resis-tance associated 2 (adenosine triphosphate–bindingcassette C2) and multidrug resistance associated 3 (aden-osine triphosphate–binding cassette C3), which arepresent in liver and intestine.28,29 CDCA-activated FXR
induces the expression of these hepatocytic organic aniontransport proteins.30,31
The tight control of intracellular and circulating BAlevels is partially achieved through the regulation of BAsynthesis and transport. However, the metabolic path-ways involved in their inactivation and excretion mustalso be tightly regulated, in particular under conditionsof disturbed bile formation, i.e., cholestasis. Recently, Liet al.32 suggested that, in rats, BAs may modulate theirown metabolism via regulating UGT enzyme expression.However, regulation of human UGT enzymes by BAshas not yet been analyzed. Because both FXR andUGT2B4 proteins are expressed in liver,33,34 the mostimportant organ in BA metabolism, we studied whetherhuman UGT2B expression is regulated by BAs. Here wereport that UGT2B4 is a target gene for FXR and thatits induction results in increased BA glucuronidation inhuman hepatoma HepG2 cells. Furthermore, FXR bindsas a monomer to a BA response element (BARE) in theUGT2B4 gene promoter. In addition, activation ofRXR, the heterodimer partner of FXR, repressesUGT2B4 gene expression induced by CDCA. Finally,overexpression of UGT2B4 in HepG2 cells results inreduced FXR-mediated induction of the small het-erodimeric partner (SHP). Because the induction ofUGT2B4 by FXR agonists seems delayed compared withSHP, our data suggest that UGT2B4 may be part of asignal terminator mechanism of the FXR transcriptionpathway.
Materials and MethodsMaterials
UDPGA, leupeptin, pepstatin, phosphatidylcholine,and BAs were obtained from Sigma (Saint-Quentin, France).Human hepatoblastoma HepG2 cells were from the AmericanType Culture Collection (Rockville, MD). Restriction enzymesand other molecular biology reagents were from New EnglandBiolabs (Beverly, MA; distributed by Ozyme, Saint-Quentin,France), Stratagene (La Jolla, CA), Promega Corp. (Charbon-nieres, France), and Roche (Mannheim, Germany). Proteinassay reagents were obtained from Bio-Rad Laboratories Inc.(Marnes-la-Coquette, France). [�-32P]Deoxycytidine triphos-phate and [14C]UDPGA (180 mCi/mmol) were purchasedfrom NEN-Life Sciences (Paris, France). Cell culture reagentsand G418 were from Life Technologies (Cergy-Pontoise,France). ExGen 500 was from Euromedex (Souffelweyersheim,France). The anti-UGT2B antibody was provided by Dr. A.Belanger (Laval University, Quebec, Canada), and the second-ary antibody against rabbit immunoglobulin G was purchasedfrom Sigma. GW4064 was synthesized in the chemistry de-partment of Genfit SA (Loos, France), according to Maloney etal.5 CD3640 was a gift of U. Reichert (Le Cird-Galderma,
Human primary hepatocytes were isolated as de-scribed35 and incubated for 24 hours with CDCA (30 �mol/L).Immortalized human hepatocytes were cultured as reportedpreviously.36 Human hepatoblastoma HepG2 cells were grownas described.8 For RNA analyses, 106 HepG2 cells were treatedwith CDCA, GW4064, or CD3640 in the absence or presenceof actinomycin D or cycloheximide at the indicated concen-trations for 24 hours. For time-course experiments, cells wereincubated in the presence of CDCA (50 �mol/L) for theindicated periods of time. In all experiments, controls wereincubated with an identical volume of ethanol (vehicle).HepG2 cells stably overexpressing UGT2B4 or UGT1A6 wereobtained by transfection in OptiMEM medium (InvitrogenCorp., Carlsbad, CA) with 2 �g of expression plasmid by usingExGen reagent according to the manufacturer’s instruction(Euromedex). Stable transfectants were selected by using 800�g/mL of G418 (Invitrogen Corp.) for 60 days. Stably express-ing cells were pooled and cultured in Dulbecco’s modifiedEagle medium containing 400 �g/mL of G418. Treatmentswere performed in the absence of G418 for 24 hours, asdescribed previously.
RNA Analysis
RNA was isolated from primary human hepatocytes orHepG2 cells by using Trizol (Life Technologies). Northernblot analyses were performed as previously described37 byusing human UGT2B4 and 36B4 complementary DNAs asprobes. For reverse-transcription (RT)-PCR analyses ofUGT2B gene expression, RNA was reverse transcribed withrandom hexamer primers and 200 U of Moloney murineleukemia virus RT (Life Technologies) and subsequently am-plified by PCR with sense and antisense primers as de-scribed.26,34,38–40 For quantitative PCR, reverse-transcribedUGT2B4, UGT1A6, SHP, and 28S complementary DNAswere quantified by real-time PCR on an MX 4000 apparatus(Stratagene) by using specific primers as described forUGT2B4, UGT1A6, SHP, and 28S.8,41 PCR amplificationswere performed in a volume of 25 �L containing 100 nmol/Lof each primer, 4 mmol/L of MgCl2 , the Brilliant QuantitativePCR Core Reagent Kit mix (Stratagene), and SYBR Green0.33� (Sigma-Aldrich, St. Louis, MO). The conditions were95°C for 10 minutes, followed by 40 cycles of 30 seconds at95°C, 30 seconds at 55°C (SHP and 28S) or 60°C (UGT2B4,UGT1A6, and 28S), and 30 seconds at 72°C. Messenger RNA(mRNA) levels were normalized to 28S mRNA.8
Plasmid Cloning and Site-DirectedMutagenesis
A 2.4-kilobase (kb) fragment of the human UGT2B4promoter was amplified by PCR from human genomic DNAby using Herculase polymerase (Stratagene) and 100 pmol ofsense (5�-GGGGTACCTCATATTAAGTGATTTTGCTAA-
3�) and antisense (5�-CGGGATCCTGATGCAAATGCAAT-3�) primers based on the UGT2B4 gene sequence.24 KpnI andBamHI restriction sites were inserted in the sense and antisenseprimers, respectively. The PCR product was cloned in theKpnI/BglII restriction sites of the pGL3 luciferase vector. Mu-tations were introduced in the �1193 BARE by using theQuick Change Site Directed Mutagenesis Kit (Stratagene) andthe oligonucleotide BAREmt (5�-AGTTAAGATAAAATT-TAATCTGTA) (bold nucleotides indicate the mutated bases).The IR-1-TKpGL3, B4-BAREwt-TKpGL3, and B4-BAREmt-TKpGL3 plasmids were obtained by cloning 3 copies of thecorresponding dimerized oligonucleotides in the thymidine ki-nase promoter-driven luciferase reporter (TKpGL3) vector.
Transient Transfection Assays
A total of 60 � 103 HepG2 cells were transfected with100 ng of the indicated luciferase reporter plasmids and 50 ngof the pCMV-�galactosidase expression vector, with or with-out 30 ng of the indicated expression plasmids. All sampleswere complemented with pBS-SK� plasmid (Stratagene) to anidentical amount of 500 ng per well. HepG2 cells weretransfected with ExGen reagent (Euromedex) for 6 hours at37°C, subsequently incubated overnight with Dulbecco’smodified Eagle medium 0.2% fetal bovine serum, and thentreated for 24 hours with either ethanol (vehicle) or CDCA (50�mol/L), as indicated.
Electrophoretic Mobility Shift Assays
Electrophoretic mobility shift assays (EMSAs) using invitro produced FXR and RXR were performed as described8
by using radiolabeled probes (IR-1: 5�-GATCTCAAGAGGT-CATTGACCTTTTTG-3�; B4-BAREwt: 5�-TAAGATGAA-CTTTAATCTTGTAAC-3�; and B4-BAREmt: 5�-TAAGA-TAAAATTTAATCTTGTAAC-3�, where underlinednucleotides represent response element half-sites and basesin bold are mutated). For supershift experiments, the anti-FXR antibody (sc1204; Santa Cruz Biochemicals, SantaCruz, CA) was preincubated for 20 minutes in the bindingbuffer before the addition of FXR and/or RXR proteins. Forcompetition experiments, the unlabeled oligonucleotideswere included in the binding reaction at the indicatedexcess concentrations over the probe just before the labeledoligonucleotides were added. In experiments using immor-talized human hepatocyte nuclear proteins, extracts werepurified as previously described,42 and EMSA with 2.5 �gof nuclear extracts was performed as described previously,except that incubations were performed at 4°C.
Chromatin Immunoprecipitation Assays
Chromatin immunoprecipitation assays were per-formed according to the method of Shang et al.,43 as modifiedby Giraud et al.44 Briefly, 200 � 106 HepG2 cells were grownto 60% confluence and serum-starved for 16 hours. Cells weresubsequently treated with ethanol (vehicle), CDCA (50 �mol/L), CD3640 (2.5 �mol/L), or both CDCA and CD3640 for 6hours. Cell lysates were sonicated on ice 15 times for 15
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seconds separated by 45 seconds. A volume of lysate equivalentof 20 � 106 cells was immunoprecipitated with 4 �g ofanti-FXR (sc13063; Santa Cruz), anti-RXR� (sc553; SantaCruz) antibodies, or 4 �g of the anti-UGT2B antibody asnegative control. The same lysate volume was kept withoutimmunoprecipitation for subsequent purification of inputgenomic DNA. One tenth of the immunoprecipitated DNAwas PCR amplified for 35 cycles (30 seconds at 95°C, 30 secondsat 58°C, and 30 seconds at 72°C) by using the following primers:B4-2402 sense, 5�-TACCTCATATTAAGTGATTTTGCTAA;B4-2169 antisense, 5�-GGCTGAGATGAGTGTTACTAC-3�;B4-1344 sense, 5�-CTTGAGAGCAGCTGGCATCAGG-3�;B4-1104 antisense, 5�-AACCAAGCCTGTAGTACATTCA-3�;SHP-438 sense, 5�-TGCATTCAAGGCCCACCCAGCTCT-3�;SHP-203 antisense, 5�-CACGTGGCACTGATATCACCTCA-3�; �-actin forward, 5�- CGAGCCATAAAAGGCAACTTTCG-3�; and �-actin reverse, 5�-AGGAAGAGGAGGAGG-GAGAGTTT-3�. An equal volume of nonprecipitated (input)genomic DNA was amplified as a positive control. One in 15(input) or 1 in 5 (precipitated DNA) PCR products wereseparated on an ethidium bromide–stained 2% agarose gel.
Microsome Purification and Western BlotAnalysis
Microsomal protein purification was performed as de-scribed.40 Microsome pellets were resuspended in 100 �L ofhomogenization buffer, and the protein content was deter-mined by using Bradford’s reagent (Bio-Rad) and bovine se-rum albumin for standard curves (Bio-Rad). Samples werealiquoted and kept at �80°C until Western blot analysis.Thirty micrograms of microsomal proteins from treated orcontrol HepG2 cells was separated on a 10% sodium dodecylsulfate polyacrylamide gel. The gel was transferred onto anitrocellulose membrane that was stained with Ponceau Ssolution to ensure equal protein loading on each lane. Mem-branes were then hybridized with the anti-UGT2B antibody(dilution, 1/1000). An anti-rabbit immunoglobulin G anti-body conjugated with peroxidase was used as secondary anti-body (dilution, 1/10,000), and resulting immunocomplexeswere visualized with Western blot Chemiluminescence ReagentPlus as specified by the manufacturer (NEN-Life Sciences).
Glucuronidation Assay
HepG2 cells were resuspended in Tris-buffered salinecontaining 0.5 mmol/L of dithiothreitol and homogenizedwith a Brinkman polytron. Enzyme assays were performed aspreviously described.38 Briefly, 100 �g of cell homogenate wasincubated with 15 �mol/L of [14C]UDPGA, 485 �mol/L ofunlabeled UDPGA, and 200 �mol/L of HDCA in a finalvolume of 100 �L of glucuronidation assay buffer38 for 8 hoursat 37°C. Assays were terminated by adding 100 �L of meth-anol, and the samples were centrifuged at 14,000 rpm for 2minutes to remove the precipitated proteins. One hundredmicroliters of glucuronidation assay was applied onto a thin-layer chromatography plate (Merck, Darmstadt, Germany) andchromatographed in a solvent of toluene, methanol, and acetic
acid (7:3:1). The extent of HDCA glucuronidation was ana-lyzed and quantified by PhosphorImager (Bio-Rad Laborato-ries) analysis.
Statistical Analyses
A nonparametric Mann–Whitney test was used toanalyze for significant differences between the experimentalgroups. Analyses of variance and Tukey post hoc tests wereused for analysis of the effects of cotreatment with FXR andRXR agonists.
ResultsFXR Agonists Increase UGT2B4 Expressionin Human Liver Cells
Human hepatocytes were treated with CDCA (30�mol/L) for 24 hours, and UGT2B mRNA levels weredetermined by Northern blot analysis. A significant in-crease in UGT2B mRNA levels was observed in CDCA-treated compared with vehicle-treated cells (Figure 1A ).Nucleic acid sequences of the different human UGT2Bcomplementary DNAs are more than 85% homologous;therefore, an isoform-specific RT-PCR approach was de-signed to determine which UGT2B gene is regulated byCDCA. Neither UGT2B17 nor UGT2B28 transcriptswere detected in these cells (not shown). Only UGT2B4mRNA increased after treatment, whereas none of theother UGT2B isoforms was regulated by CDCA (Figure1B). To quantify the increase of UGT2B4 mRNA afterCDCA treatment, real-time RT-PCR analysis was per-formed. Treatment with CDCA resulted in a 5-foldinduction of UGT2B4 mRNA levels (Figure 1C ).
Furthermore, CDCA induced UGT2B4 expression ina dose- and time-dependent manner in HepG2 cells(Figure 2A and B), indicating that this human hepatomacell line is a suitable cell model to investigate the mo-lecular and cellular mechanisms by which BAs regulateUGT2B4 expression. Preincubation with actinomycin D(1 �g/mL) abolished the induction of UGT2B4 mRNAby CDCA completely, whereas cycloheximide treatmentdid not have any influence (Figure 2C ). These resultsindicate that BAs induce expression of the UGT2B4gene at the transcriptional level without requiring denovo protein synthesis. The role of FXR in the CDCA-induced expression of UGT2B4 was ascertained by usingthe specific synthetic FXR agonist GW40648,45 (5�mol/L) for 24 hours. GW4064 treatment led to a3.2-fold increase of UGT2B4 transcripts when comparedwith vehicle (Figure 2D).
To determine whether UGT2B protein levels are in-duced by BAs, HepG2 cells were treated with CDCA for36 hours, and UGT2B protein content was analyzed byWestern blotting. A significant increase in UGT2B pro-
June 2003 FXR REGULATION OF UGT2B4 1929
tein levels was observed in CDCA-treated compared withvehicle-treated cells (Figure 3A ). Furthermore, this in-duction of UGT2B protein levels was accompanied by a2-fold increase of HDCA-glucuronide formation (Fig-ure 3B).
FXR Activates the UGT2B4 Promoter
To further decipher the molecular mechanisms ofUGT2B4 up-regulation by BAs, a 2.4-kb fragment ofthe human UGT2B4 gene promoter24 was cloned in thepGL3-luciferase reporter plasmid and transfected inHepG2 cells in the presence or absence of FXR, CDCA,or both. FXR slightly increased UGT2B4 promoter ac-tivity, an effect that was significantly enhanced byCDCA (Figure 4A ). Analysis of the UGT2B4 promotershowed the presence of a TGAACT sequence at nucleo-tides �1193 to �1187, which resembles the consensusnuclear receptor binding half-site TGACCT. To testwhether this site mediates the induction by FXR, mu-tations were introduced in this site in the context of the�2400 base pair (bp) UGT2B4 promoter (Figure 4A ).Mutation of the �1193 B4-BARE site abolished theinduction of UGT2B4 promoter activity by CDCA-activated FXR. Furthermore, CDCA-activated FXR in-creased the activity of a heterologous promoter driven by3 copies of the wild-type but not the mutated B4-BARE(Figure 4B), thus confirming that the B4-BARE is apositive FXR response element.
FXR Binds the Bile Acid Response Elementof the UGT2B4 Promoter
EMSAs were performed by using the B4-BARE asradiolabeled probe and the consensus IR-1 site as positivecontrol. In the absence of FXR, RXR did not bind anyof the probes (Figure 5A, lanes 2 and 7). FXR bound theIR-1 site both in the presence and absence of RXR (lanes3 and 4), and this complex was supershifted by an FXRantibody (lane 5). In contrast, FXR bound in the absenceof RXR to the B4-BARE probe, whereas no complexcorresponding to the FXR/RXR heterodimer was formed(Figure 5A, lanes 8 and 9). The FXR complex wassupershifted by the FXR antibody (lane 11). In thepresence of RXR, FXR binding was greatly reduced(Figure 5A, lane 9), and the addition of the FXR anti-body caused a complete inhibition of FXR binding (Fig-ure 5A, lane 10). By contrast, whereas FXR bound to thewild-type B4-BARE (Figure 5B, lanes 3 and 4), noprotein/DNA complex was observed on the mutatedB4-BAREmt oligonucleotide (lanes 7 and 8), which wasfunctionally inactive, as shown previously. Binding ofFXR on the B4-BAREwt site was competed to a similarextent by the consensus IR-1 and B4-BAREwt (Figure5C, lanes 4–10), whereas the B4-BAREmt oligonucleo-tide did not compete for binding (lanes 11–14).
Occupancy of the UGT2B4 promoter by FXR andRXR in living cells was analyzed by using chromatin
Figure 1. Bile acids increase UGT2B4 mRNA levels in primary human hepatocytes. Primary human hepatocytes were treated for 24 hours withethanol (vehicle) or chenodeoxycholic acid (CDCA; 30 �mol/L). (A) Northern blot analysis of human UGT2B mRNA (36B4 RNA was measured ascontrol). (B) Semiquantitative RT-PCR analyses of the human UGT2B isoforms (28S RNA was measured as control). C, RT-PCR negative control.(C) Real-time RT-PCR analysis of UGT2B4 RNA. Values are expressed as means � SD (n 6) relative to the control set as 1. Statisticallysignificant differences between vehicle- and CDCA-treated cells are indicated by asterisks (Mann–Whitney test: ***P 0.001).
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immunoprecipitation assays performed on DNA fromHepG2 cells treated with either vehicle or CDCA, withanti-FXR or anti-RXR� antibodies (Figure 5D). TheDNA encompassing the B4-BARE was precipitated bythe anti-FXR antibody in CDCA-treated cells (Figure5D, lane 6, panel B), whereas no amplification wasobserved in untreated cells. PCR amplification of DNAfrom control and treated cells precipitated with theanti-RXR antibody failed to amplify the 240-bp frag-ment containing the B4-BARE (lanes 5 and 6, panel C).Moreover, when the same DNA samples were PCR-amplified with primers covering a region 900 bp up-stream of the BARE, no signal was observed (lanes 3 and4). In contrast, both antibodies precipitated the IR-1 ofthe SHP gene in CDCA-treated, but not in control, cells
(lanes 1 and 2, panels B and C). Finally, PCR amplifi-cation with oligonucleotides for �-actin, as a negativecontrol for the immunoprecipitation, did not result inany signal (lanes 7 and 8). Taken together, these resultsindicate that in presence of CDCA, the B4-BARE isimmunoprecipitated by the anti-FXR, but not by theanti-RXR, antibody.
To investigate whether endogenous FXR binds theB4-BARE site in the absence of any partner, EMSAswere performed with nuclear extracts from immortal-ized human hepatocytes, and the migration of thecomplex was compared with FXR (Figure 5E ). Asnoted previously, in vitro–produced FXR bound theB4-BARE probe, whereas no FXR/RXR heterodimercomplexes were observed (lanes 1 and 2). That incu-
Figure 2. Bile acid and synthetic FXR agonists induce UGT2B4 gene expression in human hepatoblastoma HepG2 cells in a time- anddose-dependent manner via a direct transcriptional mechanism. (A) HepG2 cells were incubated with increasing concentrations of CDCA (10, 25,and 50 �mol/L) or vehicle (ethanol) for 24 hours. (B) HepG2 cells were incubated with CDCA (50 �mol/L) or vehicle (ethanol) for 6, 12, 24, and36 hours. (C) HepG2 cells were incubated for 24 hours with ethanol (vehicle) or CDCA (50 �mol/L) in the absence and presence of cycloheximide(CHX; 20 �g/mL) or actinomycin D (Act. D; 1 �g/mL). (D) Cells were incubated for 24 hours with vehicle (ethanol), CDCA (50 �mol/L), or GW4064(5 �mol/L). RNA levels were measured by real-time (bars) or semiquantitative (ethidium bromide–stained gels) RT-PCR. Values are means � SD(n 6). Statistically significant differences between vehicle- and FXR agonist–treated cells are indicated by asterisks (Mann–Whitney test: *P 0.05; ***P 0.001; ns, not significant).
June 2003 FXR REGULATION OF UGT2B4 1931
bation of the nuclear extract with the radiolabeledprobe resulted in the formation of a protein/DNAcomplex that co-migrated with the FXR/B4-BAREcomplex (lane 4). Furthermore, the anti-FXR anti-
body, which supershifted the FXR/B4-BARE complex(lane 3), resulted in an inhibition of the shift observedwith nuclear extract (lane 5), indicating that the pro-tein bound to the probe corresponded to FXR. These
Figure 3. Bile acids increase UGT2B protein levels and activity in HepG2 cells. (A) HepG2 cells were incubated with vehicle (ethanol) or CDCA(50 �mol/L) for 36 hours. Microsomal proteins were subsequently purified and immunoblotted with an anti-UGT2B antibody (upper panel). Equalloading of protein in each lane was assessed by Ponceau S staining (lower panel). (B) HepG2 cells were incubated for 36 hours with CDCA (50�mol/L) or vehicle (ethanol). Cell homogenates (100 �g) were incubated with [14C]UDPGA (15 �mol/L), unlabeled UDPGA (485 �mol/L), andhyodeoxycholic acid (HDCA; 200 �mol/L) for 8 hours at 37°C. Radiolabeled HDCA/glucuronide was subsequently analyzed by thin-layerchromatography and quantified by PhosphorImager analysis. Values represent means � SD (n 3).
Figure 4. Identification of abile acid response element(B4-BARE) in the humanUGT2B4 promoter. (A) HepG2cells were transfected with theindicated human UGT2B4 pro-moter-driven luciferase (Luc) re-porter plasmids (100 ng) in theabsence or presence ofpcDNA3-hFXR (30 ng) and a cy-tomegalovirus-driven �-galacto-sidase expression plasmid(pCMV-�-gal; 50 ng). Cells weresubsequently treated withCDCA (50 �mol/L) or vehiclefor 24 hours. (B) HepG2 cellswere transfected with the indi-cated plasmids (100 ng) con-taining 3 copies of the wild-typeor mutated B4-BARE upstreamof the thymidine kinase (TK)minimal promoter-driven lucif-erase reporter (TKpGL3) andpCMV-�-gal (50 ng) and in theabsence or presence of hFXR(30 ng). Cells were subse-quently treated with CDCA (50�mol/L) or vehicle for 24hours. Values are expressedas -fold induction of controls(pGL3 or TKpGL3) set at 1, nor-malized to internal �-galactosi-dase activity as described inMaterials and Methods. Valuesrepresent the means � SD.
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Figure 5. FXR binds the B4-BARE as a monomer. (A) Electromobility shift assays (EMSA) were performed with end-labeled consensus IR-1 orB4-BAREwt oligonucleotides in the presence of RXR, FXR, both RXR and FXR, or unprogrammed reticulocyte lysate, as indicated. Supershiftexperiments were performed with anti-FXR antibody (0.2 �g). (B) EMSA assays were performed on radiolabeled B4-BAREwt and B4-BAREmtoligonucleotides by using in vitro transcribed/translated RXR (lanes 2 and 6), FXR (lanes 3 and 7 ), both RXR and FXR (lanes 4 and 8 ), orunprogrammed reticulocyte lysate (lanes 1 and 5 ). (C) Competition EMSAs on B4-BAREwt as radiolabeled probe were performed by adding 1-,10-, 50-, or 100-fold molar excess of the indicated cold IR-1, B4-BAREwt, or B4-BAREmt oligonucleotides in EMSA and FXR, RXR, FXR and RXR,or unprogrammed reticulocyte lysate. (D) Soluble chromatin was prepared from HepG2 cells treated with vehicle (ethanol) or CDCA (50 �mol/L)for 6 hours and immunoprecipitated with antibodies directed against FXR and RXR or with anti-UGT2B antibody as a negative control. The finalDNA extractions were amplified by using pairs of primers covering the B4-BARE, a distal region of the UGT2B4 gene promoter, or the �-actin geneas negative control. As a positive control for FXR/RXR binding, a 235-bp DNA fragment encompassing the IR-1 of the SHP gene promoter wasamplified. (E) EMSAs were performed on end-labeled B4-BAREwt oligonucleotides by using in vitro transcribed/translated FXR (lane 1 ), both RXRand FXR (lanes 2 and 3 ), or 2.5 �g of nuclear extract from immortalized human hepatocytes (IHH-NE) (lanes 4 and 5 ). Supershift analyses wereperformed by adding the anti-FXR antibody (lanes 3 and 5 ).
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data show that endogenous FXR binds the B4-BAREwithout RXR.
RXR Activation Inhibits the Induction ofUGT2B4 by CDCATo show that the RXR-dependent inhibition of
FXR binding to the B4-BARE was due to formationof inactive FXR/RXR heterodimer complexes, EMSAswere performed with an RXR dimerization-incompe-tent FXR mutant protein,8 FXRLeu433Arg (Figure6A ). As shown previously, binding of wild-type FXRto the B4-BARE was reduced in the presence of RXR(Figure 6A, lanes 3 and 4). In contrast, RXR did not
affect the DNA binding activity of FXRLeu433Arg(lanes 5 and 6), indicating that FXR/RXR het-erodimer formation impedes binding of FXR to theB4-BARE.
To assess whether RXR activation prevents FXRbinding to the UGT2B4 promoter in cells, chromatinimmunoprecipitation experiments were performedwith FXR or RXR antibodies on HepG2 cells treatedwith vehicle, CDCA, the RXR agonist CD3640, orboth CDCA and CD3640. The anti-FXR antibodyimmunoprecipitated genomic DNA encompassing theB4-BARE in CDCA-treated cells (Figure 6B, lane 7,
Figure 6. RXR activation reduces the bile acid–induced up-regulation of UGT2B4 in HepG2 cells. (A) EMSAs were performed with the radiolabeledB4-BAREwt oligonucleotide in the presence of RXR (lane 2 ), FXR (lane 3 ), FXR and RXR (lane 4 ), FXRLeu433Arg (lane 5 ), FXRLeu433Arg and RXR(lane 6 ), or unprogrammed reticulocyte lysate (lane 1 ). (B) Chromatin immunoprecipitation experiments on DNA from HepG2 cells treated withCDCA (50 �mol/L), CD4064 (2.5 �mol/L), both CDCA and CD4064, or vehicle (ethanol) for 6 hours. Soluble chromatin was prepared asdescribed in Materials and Methods and immunoprecipitated with anti-FXR, anti-RXR, or anti-UGT2B antibodies. The extracted DNA was amplifiedwith pairs of primers that cover B4-BARE, a distal region of the UGT2B4 gene promoter, or the �-actin gene as a negative control. As a positivecontrol, a 235-bp DNA fragment encompassing the IR-1 of the SHP gene promoter was amplified. (C) HepG2 cells were transfected with theTK-pGL3 reporter plasmid or the TK-pGL3 plasmid containing 3 copies of the B4-BARE and pCMV-�-gal in the absence or presence of FXR,FXRLeu433Arg, and/or RXR. Cells were subsequently treated with CDCA (50 �mol/L) or vehicle (ethanol) for 24 hours. (D and E) HepG2 cellswere treated with vehicle (ethanol), CDCA (50 �mol/L), CD3640 (2.5 �mol/L), or both CDCA and CD3640 for 24 hours. Total RNA was extracted,and UGT2B4 (D) and SHP (E) mRNA levels were measured by real-time PCR. Values are means � SD (n 4). Values followed by different lettersare statistically significantly different (analysis of variance followed by the Mann–Whitney test; P 0.05).
1934 BARBIER ET AL. GASTROENTEROLOGY Vol. 124, No. 7
panel B), whereas the anti-RXR antibody did not(Figure 6B, lanes 5– 8, panel C). CD3640 treatmentprevented FXR binding to the B4-BARE, as shown bythe absence of PCR products in anti-FXR–precipi-tated DNA from CDCA-/CD3640-treated cells (Fig-ure 6B, lane 8, panel B). By contrast, both FXR andRXR bound to the IR-1 of the SHP gene in CD3640-,CDCA-, and CDCA-/CD3640-treated cells (lanes 10 –12, panels B and C). These results clearly show thatactivation of RXR abolishes CDCA-dependent bind-ing of FXR to the B4-BARE.
Functional consequences of inhibition of FXR bind-ing to the B4-BARE by RXR was tested by transfect-ing HepG2 cells with the TKpGL3 vector driven by 3copies of the B4-BARE in the presence of FXR aloneor in combination with RXR (Figure 6C ). As control,the activity of the empty TK-pGL3 reporter gene wasnot influenced at any of the conditions tested. Activityof the B4-BARE-TKpGL3 vector increased in thepresence of CDCA-activated FXR, whereas RXR in-hibited this induction (Figure 6C ). In contrast, RXRfailed to inhibit the activity of the B4-BARE-TK-pGL3 vector induced by FXRLeu433Arg. These re-sults indicate that RXR inhibits CDCA-induced ac-tivation of the B4-BARE by FXR.
The physiological relevance of this potential com-petitive activity of RXR was studied by analyzing theinfluence of CD3640 on CDCA-induced UGT2B4 ex-pression in HepG2 cells. CDCA treatment resulted ina clear induction of UGT2B4 mRNA, whereasCD3640 did not affect it (Figure 6D). UGT2B4 in-duction was lower when cells were treated with bothCDCA and CD3640. As control, the expression ofSHP, whose induction occurs via FXR/RXR het-erodimer formation, was increased in cells treated withCDCA either with or without CD3640 (Figure 6E ).These results indicate that RXR reduces CDCA-in-duced UGT2B4 expression by inhibiting FXR bind-ing to the B4-BARE and transcriptional activation ofthe UGT2B4 promoter.
Overexpression of UGT2B4 in HepG2 CellsNegatively Interferes With the Ability of BileAcids to Activate FXR
To start assessing the physiological conse-quence of UGT2B4 induction by FXR on the glucu-ronidation of BAs, HepG2 cells stably transfectedwith UGT2B4 were generated. As negative control, aHepG2 cell line overexpressing UGT1A6, a UGTenzyme that is not involved in BA glucuronidation,20
was also generated. Transfected and untransfected cellswere treated with vehicle or CDCA for 24 hours, and
SHP, UGT2B4, or UGT1A6 mRNA levels were ana-lyzed by semiquantitative and real-time RT-PCR(Figure 7A and B). In both untransfected andUGT1A6-transfected HepG2 cells, CDCA treatmentsignificantly increased SHP mRNA (Figure 7A andB). In contrast, CDCA treatment did not induce SHPmRNA in cells overexpressing UGT2B4. BecauseCDCA-induced SHP expression is dependent onFXR,46 these data suggest that overexpression ofUGT2B4 interferes negatively with the induction ofSHP by FXR.
Bile Acid Treatment of HepG2 Cells InducesSmall Heterodimeric Partner More RapidlyThan UGT2B4
To compare the kinetics of UGT2B4 induction byCDCA-activated FXR with other FXR target genes,SHP and UGT2B4 mRNA levels were measured inHepG2 cells incubated with CDCA (50 �mol/L) for 6,12, and 24 hours. CDCA treatment rapidly induced SHPgene expression, which decreased progressively at longertimes of treatment (Figure 8). By contrast, UGT2B4mRNA levels started to increase only after 6 hours, toprogressively reach a maximum after 24 hours (Figure 8).Overall, these data indicate that the induction ofUGT2B4 expression by CDCA-activated FXR is delayedcompared with SHP.
DiscussionIn this study, we identify human UGT2B4 as a
target gene of BA-activated FXR. UGT2B4 inductionby CDCA occurs via FXR binding to a novel atypicalresponse element in the UGT2B4 promoter that con-sists of a hexamer half-site to which FXR binds, mostlikely as a monomer. Most previously characterizedFXR target genes contain FXR response elementsconsisting of an IR-1 to which FXR binds as a het-erodimer with RXR.7 FXR binds, also as a monomer,to the C site of the apolipoprotein AI promoter,resulting in a negative regulation of apolipoprotein AIexpression.8 To the best of our knowledge, this is thefirst demonstration of an RXR-independent, posi-tively regulated FXR target gene. The factors thatdetermine whether FXR acts as an activator or inhib-itor of transcription on monomeric sites are currentlyunknown and require further study. The promotercontext and nature of the response element and/orsurrounding transcription factor complexes may bepossible discriminating factors. Remarkably, RXR ac-tivation inhibits the binding of FXR to the B4-BAREin EMSA and chromatin immunoprecipitation exper-
June 2003 FXR REGULATION OF UGT2B4 1935
iments. This inhibition of FXR binding results in adecrease of FXR-induced UGT2B4 mRNA expres-sion. Thus, in contrast to target genes to which FXRbinds as a heterodimer, with RXR leading to a per-missive activation of both receptors by their respectiveligands, RXR activity may limit FXR induction oftarget genes driven by response elements similar tothe B4-BARE. Thus, SHP expression is induced byboth RXR and FXR ligands, whereas RXR activation
reduces FXR induction of UGT2B4. Recently, Kas-sam et al.47 reported that RXR agonists antagonizeFXR induction of bile salt export pump expression bydecreasing the binding of FXR/RXR heterodimers tothe bile salt export pump/BARE and by the inabilityof RXR agonists to recruit coactivators to the FXR/RXR heterodimer complex. However, because RXRdoes not bind to the B4-BARE, inhibition of FXR/B4-BARE complex formation by RXR may be due tothe squelching of FXR by RXR to form FXR/RXRheterodimers, which then bind to target genes drivenby heterodimer-binding response elements such asSHP. Altogether, these data clearly indicate a role forRXR ligands in BA homeostasis. Although the effectsof RXR ligands on the FXR-dependent down-regula-tion of the apolipoprotein AI gene were not investi-gated, it will be of interest to determine whetherretinoids, which have been shown to increase apoli-poprotein AI transcription via RXR/RXR, RXR/RAR, or RXR/PPAR� complexes,48 –50 also inhibitBA-dependent repression of apolipoprotein AI. Reti-noids may up-regulate apolipoprotein AI expressionvia simultaneous activation of heterodimeric com-plexes and inhibition of FXR-mediated repression viathe monomeric FXR-binding site.
Two human UGT2B enzymes catalyze the glucu-ronidation of BAs, i.e., UGT2B4 and UGT2B7.16,51,52
Whereas both isoforms glucuronidate HDCA at the6�-hydroxyl group, the predominant role of UGT2B4was clearly established by immunoprecipitation stud-
Figure 7. UGT2B4 overexpression in HepG2 cells interferes nega-tively with the bile acid induction of SHP gene expression. Stableoverexpressing UGT2B4- and UGT1A6-HepG2 cells were obtained asdescribed in Materials and Methods. Cells were treated with vehicle(ethanol) or CDCA (50 �mol/L) for 24 hours. Levels of SHP, UGT2B4,and UGT1A6 mRNAs were analyzed by semiquantitative RT-PR (A) andquantified with real-time RT-PCR (B). Statistically significant differ-ences of SHP mRNA levels between vehicle- and FXR agonist–treatedcells are indicated by asterisks (Mann–Whitney test; ***P 0.001;ns, not significant).
Figure 8. CDCA-induced UGT2B4 expression is delayed comparedwith SHP in HepG2 cells. HepG2 cells were incubated with CDCA orethanol for 6, 12, and 24 hours. RNA levels were measured byreal-time RT-PCR. Values are means � SD (n 4). Statisticallysignificant differences between UGT2B4 and SHP inductions are in-dicated by asterisks (Mann–Whitney test; **P 0.005; ***P 0.001).
1936 BARBIER ET AL. GASTROENTEROLOGY Vol. 124, No. 7
ies.16 Furthermore, no or low glucuronidation activityof HDCA was observed in colon, where UGT2B7 butnot UGT2B4 is expressed,53,54 thus indicating thatCDCA-activated FXR specifically induces the princi-pal BA-conjugating UGT2B isoform, UGT2B4.
BA activation of FXR regulates a number of genesinvolved in control of intracellular BA levels, such ascytochrome P450 7A1, bile salt export pump, orSULT2A1.10,12,14 Induction of UGT2B4 by FXR consti-tutes an additional mechanism via which BA metabolismmay be controlled. CDCA-activated FXR also regulatesthe expression of the hepatocytic organic anion transportMRP2 protein.31 MRP2 and MRP3 have been shown totransport glucuronidated and sulfated BAs.28,55–57 MRP2is localized at the canalicular (apical) pole of hepatocytes,and MRP3 is localized at the basolateral membranes.Up-regulation of MRP3 under conditions of experimen-tal cholestasis may constitute a mechanism to facilitatesecretion of BA metabolites into blood, followed byenhanced urinary excretion.58–62 An increase of circulat-ing levels of BA glucuronides in patients with chronicintrahepatic cholestasis has been reported.18,63,64 BA-dependent FXR activation increased HDCA/glucuronideformation 2-fold in HepG2 cells. HDCA is a 6�-hy-droxylated metabolite of LCA, which is primarily ex-creted as a glucuronide derivative in urine.63 Glucu-ronidation of HDCA has been proposed as an alternativemechanism for reducing hepatic toxicity of monohy-droxylated LCA.65 Thus, LCA conjugates are rapidlyexcreted in bile via MRP2 when there is no cholestasis orare converted to HDCA and excreted as glucuronide inplasma via MRP3 to be eliminated in urine when bileformation is compromised. Overall, these data suggestthat BA-induced UGT2B4 expression may be involvedin a switch between biliary and urinary BA eliminationduring cholestasis. However, a clear understanding of thephysiological consequences of FXR-dependent inductionof UGT2B4 under normal and cholestatic conditionsrequires additional in vivo experiments. Unfortunately,assessment of the relevance of UGT2B4 regulation undercholestatic conditions in established rodent models ishampered by the fact that, in contrast to humans, addi-tional hydroxylation of bile salts, rather than glucu-ronidation, is a major response during cholestasis in ratsand mice.65 This renders generally accepted animal mod-els of cholestasis unsuitable to evaluate our concept. Inaddition, because rodent and human UGT2B enzymesare not conserved, analysis of the regulation of rodentUGT2B enzymes does not provide any information withrespect to the human UGT2B4 gene. Thus, carefullydesigned human studies are required to further under-
stand the pathophysiological role of UGT2B4 regulationby BAs.
In addition to BA metabolism, UGT2B4 is in-volved in the inactivation of various endobiotics andxenobiotics, such as catecholestrogens, C19-steroids,and phenolic and monoterpenoid compounds.16,20,66
Because MRPs have a broad substrate specificity thatencompasses glucuronide, sulfate, and glutathioneconjugates of a variety of endo- and xenobiotics, FXRseems to be a key factor for the elimination of manyendogenous and exogenous molecules from the liver.Using a synthetic steroidal FXR activator, Pelliciari etal.6 reported that FXR activation induces protectingpathways challenged with toxic xenobiotics. Inductionof UGT2B4-dependent glucuronidation of these sub-stances may concourse to this hepatocellular protect-ing effect of FXR activation.
UGT2B4 overexpression in HepG2 cells interferednegatively with CDCA induction of the FXR targetgene SHP. Furthermore, the observation that activa-tion of UGT2B4 is delayed compared with SHP in-dicates that glucuronidation may constitute a meta-bolic pathway that leads to the termination ofFXR-mediated transcriptional activity. A similarmechanism of signal termination by glucuronidationis observed for odorant signals in which aerial mole-cules are conjugated with glucuronic acid in the ol-factory epithelium.67 To the best of our knowledge,this is the first demonstration that endogenous nuclearreceptor ligands induce their own inactivationthrough glucuronidation in a human model. Physio-logical relevance of such signal termination is plausi-ble, because the Michaelis constant values for CDCAglucuronidation by UGT enzymes are in the samerange as their median effective concentration values ofFXR activation (respectively 82 and 50 �mol inrats).10,15 Thus, BAs bind UGT enzymes and FXRwith a similar affinity, and the preferential pathwaywill be determined mainly by the relative amounts ofthe respective proteins. However, the contribution ofother mechanisms, such as the loss of expression ofspecific cofactors and other transcription factors, to thenegative control of FXR activation by BAs inUGT2B4-HepG2 cells cannot be excluded.
In conclusion, this study identifies UGT2B4 as a novelFXR target gene. UGT2B4 induction by BAs may bepart of a negative feedback mechanism by which BAslimit their biological activity and control their intracel-lular level to avoid a pathophysiological accumulation.Furthermore, our results indicate that BA-activated FXRdirectly induces human UGT2B4 transcription through
June 2003 FXR REGULATION OF UGT2B4 1937
binding to a novel type of FXR response element in itspromoter.
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June 2003 FXR REGULATION OF UGT2B4 1939
Received November 20, 2002. Accepted March 13, 2003.Address requests for reprints to: Bart Staels, Ph.D., Unite INSERM
545, Institut Pasteur de Lille, 1 Rue du Pr Calmette, BP 245, 59019,Lille, France. e-mail: [email protected]; fax: (33) 3-20-87-71-98.Supported by grants from the Ligue Nationale Contre le Cancer
(France) (to O.B.); the Region Nord-Pas-de-Calais (France) (to C. B.); theEuropean Community (ERBFMBICT983214) (to I.P.T.); the Ministerio
de Hacienda del Gabierno de Chile (to D.D.-S.); the Fonds Europeensde Developpement Regional, Conseil Regional Region Nord/Pas-de-Calais (Genopole Project 01360124); and the Leducq Foundation (toJ.-C.F.).
A. Belanger (Centre Hospitalier de l’Universite Laval Research Cen-ter, Canada), K. Bertrand (Genfit SA, France), and U. Reichert (Cird-Galderma, Nice, France) are thanked for providing the anti-UGT2Bantibody, GW4064, and CD3640, respectively.
1940 BARBIER ET AL. GASTROENTEROLOGY Vol. 124, No. 7
