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Originally published In Press as doi:10.1074/jbc.M109326200 on November 12, 2001

J. Biol. Chem., Vol. 277, Issue 4, 2908-2915, January 25, 2002
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Regulation of Multidrug Resistance-associated Protein 2 (ABCC2) by the Nuclear Receptors Pregnane X Receptor, Farnesoid X-activated Receptor, and Constitutive Androstane Receptor*

Heidi R. KastDagger §, Bryan Goodwin§, Paul T. TarrDagger , Stacey A. Jones, Andrew M. AnisfeldDagger , Catherine M. Stoltz, Peter Tontonoz||, Steve Kliewer, Timothy M. Willson, and Peter A. EdwardsDagger **DaggerDagger

From the Dagger  Department of Biological Chemistry and Medicine, the || Department of Pathology and Laboratory Medicine and Howard Hughes Medical Institute, and the ** Molecular Biology Institute, UCLA, Los Angeles, California 90095 and  GlaxoSmithKline, Nuclear Receptor Discovery Research, Research Triangle Park, North Carolina 27709

Received for publication, September 26, 2001, and in revised form, November 9, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The multidrug resistance-associated protein 2 (MRP2, ABCC2), mediates the efflux of several conjugated compounds across the apical membrane of the hepatocyte into the bile canaliculi. We identified MRP2 in a screen designed to isolate genes that are regulated by the farnesoid X-activated receptor (FXR, NR1H4). MRP2 mRNA levels were induced following treatment of human or rat hepatocytes with either naturally occurring (chenodeoxycholic acid) or synthetic (GW4064) FXR ligands. In addition, we have shown that MRP2 expression is regulated by the pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3). Thus, treatment of rodent hepatocytes with PXR or CAR agonists results in a robust induction of MRP2 mRNA levels. The dexamethasone- and pregnenolone 16alpha -carbonitrile-dependent induction of MRP2 expression was not evident in hepatocytes derived from PXR null mice. In contrast, induction of MRP2 by phenobarbital, an activator of CAR, was comparable in wild-type and PXR null mice. An unusual 26-bp sequence was identified 440 bp upstream of the MRP2 transcription initiation site that contains an everted repeat of the AGTTCA hexad separated by 8 nucleotides (ER-8). PXR, CAR, and FXR bound with high affinity to this element as heterodimers with the retinoid X receptor alpha  (RXRalpha , NR2B1). Luciferase reporter gene constructs containing 1 kb of the rat MRP2 promoter were prepared and transiently transfected into HepG2 cells. Luciferase activity was induced in a PXR-, CAR-, or FXR-dependent manner. Furthermore, the isolated ER-8 element was capable of conferring PXR, CAR, and FXR responsiveness on a heterologous thymidine kinase promoter. Mutation of the ER-8 element abolished the nuclear receptor response. These studies demonstrate that MRP2 is regulated by three distinct nuclear receptor signaling pathways that converge on a common response element in the 5'-flanking region of this gene.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Members of the nuclear receptor superfamily of ligand-activated transcription factors have critical roles in many aspects of development and adult physiology, including cholesterol homeostasis, bile acid biosynthesis and transport, and xenobiotic metabolism. Recently, two orphan nuclear receptors, the farnesoid X-activated receptor (FXR,1 NRIH4) and the pregnane X receptor (PXR, NR1I2) were shown to be activated by an overlapping spectrum of bile acids (1-5). These results indicate that bile acids function as hormonal ligands, in addition to their well established roles in the solubilization and absorption of lipids and fat-soluble vitamins from the intestinal lumen.

The primary bile acids chenodeoxycholic acid (CDCA) and cholic acid are synthesized in the liver from either cholesterol or oxysterols via the neutral or acidic pathways before being transported across the basement membrane of the hepatocyte into the bile canaliculi and stored in the gall bladder (reviewed in Refs. 6 and 7). Following their excretion into the intestinal lumen, bile acids can be further metabolized by bacteria into secondary (deoxycholic acid (DCA) or lithocholic acid (LCA)) or tertiary bile acids prior to their resorption in the distal ileum. Defects in this cycle are associated with various diseases. For example, impaired bile flow (cholestasis) can result in the hepatic accumulation of supraphysiological levels of both toxic bile acids (e.g. LCA, 3-keto-LCA) and toxins that would normally be excreted in the bile (8).

FXR is activated by bile acids with the rank order of potency CDCA > DCA = LCA > cholic acid (3). In vitro studies have shown that FXR binds as a heterodimer with the retinoid X receptor (RXRalpha ; NR2B1) to repeats of the AGGTCA hexad. These elements can be either inverted repeats with a single nucleotide spacer (IR-1) and direct repeats separated by 3 or 4 bases (DR-3 or DR-4, respectively) (9, 10). However, to date, all known FXR target genes including the small heterodimer partner (SHP, NR0B2) (11, 12), ileal bile acid-binding protein (I-BABP) (5, 13), bile salt export pump (BSEP, ABCC11) (14, 15), phospholipid transfer protein (10, 16), and apolipoprotein C-II (apoC-II) (17) contain a functional IR-1 element in either the proximal promoter or distal enhancer elements. Activation of FXR in vivo is associated with a reduction in plasma triglyceride levels (14, 17, 18), inhibition of hepatic bile acid biosynthesis (11, 12), and increased transport of bile acids from the intestinal lumen into the enterocytes and back to the liver (5, 13, 14). Thus, FXR appears to play important roles in the regulation of bile acid biosynthesis, bile acid transport, and lipoprotein metabolism.

Recent studies have demonstrated that PXR is also activated by bile acids, although the rank order of potency (3-keto-LCA > LCA > DCA = CA) differs from that of FXR (1, 4). Unlike FXR, PXR is also highly activated by a number of diverse, structurally unrelated, foreign compounds (referred to as xenobiotics) (19, 20) that include pregnenolone 16alpha -carbonitrile (PCN) and dexamethasone (21). Activation of PXR results in increased expression of a number of cytochrome P450 (CYP) genes including Cyp3a11, CYP3A4, and various CYP2B subfamily members that are involved in the metabolism of a wide array of xenobiotics and endogenous substrates prior to their excretion into the bile (19, 20, 22-27).

Hepatotoxic bile acids, such as LCA and its metabolite 3-keto-LCA, activate PXR and result in increased hepatic expression of the genes encoding Cyp3a and Oatp2, the organic anion-transporting polypeptide (1, 4). Rat OATP2 is involved in the transport of bile acids and organic anions from the blood into hepatocytes (28, 29), while CYP3A superfamily members are active in the hepatic metabolism of these compounds, prior to their excretion into the bile (4, 30, 31). Defects in these pathways result in hepatic accumulation of numerous compounds and subsequent hepatotoxicity. Activation of PXR also results in decreased expression of CYP7A1, which encodes the regulatory enzyme of bile acid biosynthesis (1) by an as yet undefined mechanism. These data suggest that PXR regulates genes that control hepatic uptake and metabolism of many compounds, including bile acids.

The constitutive androstane receptor (CAR, NR1I4), like FXR and PXR, binds DNA as a heterodimer with RXRalpha (27, 32-34). CAR exhibits a high degree of constitutive transcriptional activity, which can be inhibited by the androstane metabolites androstenol and androstanol (35). Unlike PXR, CAR is located in the cytoplasm and translocates to the nucleus upon exposure of the cell to a variety of structurally diverse compounds, including the barbiturate phenobarbital (PB) (25, 36-38). PXR and CAR bind to common response elements in the promoters of CYP2B and CYP3A subfamily members that conform to the DR-3, DR-4, or ER-6 (everted repeat with 6-bp spacer) architecture (19-26, 39). The convergence of the PXR and CAR signaling pathways on common response elements suggests that interplay between these receptors is likely to be a significant factor in the regulation of these xenobiotic metabolizing enzymes.

Multidrug resistance-associated protein 2 (MRP2, ABCC2) is a member of the ATP-binding cassette family of transporter proteins. Formerly known as the canalicular multispecific organic anion transporter, MRP2 is a 190-kDa phosphoglycoprotein localized in the canalicular (apical) membrane of hepatocytes and is involved in the transport of organic anions, including sulfated and glucuronidated bile salts, as well as glutathione, a major driving force in the bile salt-independent bile flow. Notably, MRP2 is active in the transport of xenobiotics (including the anti-cancer drugs cisplatin, anthacyclines, vinca alkaloids, and methotrexate) and various glutathione, glucuronate, and sulfate conjugates (40-45). Natural mutations in the MRP2 gene have been linked to Dubin-Johnson syndrome/hyperbilirubinemia II, a disorder in which patients exhibit impaired transfer of anionic conjugates into the bile (reviewed in Ref. 46). Mutations in the MRP2 gene in Wistar (TR-) or Eisai hyperbilirubinemic (EBHR) rats result in impaired secretion of conjugates of xenobiotics and endogenous compounds, such as bilirubin, into the bile (41, 42). Although MRP2 expression is highest in the canalicular membrane of the liver, it is also expressed in the kidney, jejunum, and ileum, where it may also be involved in the excretion of toxic compounds from the body (45).

Recent studies have demonstrated that rifampicin, a PXR ligand, induces MRP2 mRNA expression in the liver of rhesus monkeys, small intestine of humans, and primary cultures of human hepatocytes (47-49). In addition, earlier studies had shown that MRP2 mRNA and protein levels were induced when rats were treated with dexamethasone, by a glucocorticoid receptor-independent manner (50, 51). Furthermore, the up-regulation of MRP2 gene expression upon treatment of rats with the anti-glucocorticoid/anti-progestin RU486 and the anti-fungal clotrimazole, known PXR ligands, has been observed (50). Since rifampicin, dexamethasone, PCN, RU486, and clotrimazole have been shown to activate PXR (36, 52), it seemed likely that the MRP2 gene was a target for activated PXR.

In the current study, we demonstrate that treatment of human, rat, or murine hepatocytes with ligands for FXR, PXR, or CAR results in increased expression of MRP2 mRNA. In addition, we identify and characterize a novel binding site in the proximal promoter of the rat MRP2 gene that is bound by PXR/RXR, CAR/RXR, or FXR/RXR heterodimers. This site functions as a highly responsive FXR, PXR, and CAR response element. Finally, we demonstrate that ligands for FXR, PXR, or CAR can activate expression of reporter genes controlled either by the rat MRP2 proximal promoter or by two copies of the novel hormone response element identified in the MRP2 promoter. These studies suggest that ligands for FXR, PXR, and CAR activate transcription of the MRP2 gene in order to promote excretion of conjugated toxic agents from the hepatocyte into the bile.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Materials-- The FXR-specific agonist GW4064 was a gift from Dr. Patrick Maloney (GlaxoSmithKline) (18). LG100153 was kindly provided by Dr. Richard Heyman (Ligand Pharmaceuticals) (53). The retroviral vector MSCV-IRES-neo plasmid was a gift from Dr. Owen Witte (UCLA). Mammalian expression vectors for rat FXR (pCMX-rFXR), and human RXRalpha (pCMX-hRXRalpha ), were gifts from Dr. Ron Evans (Salk Institute, La Jolla, CA). The sources of other reagents and plasmids have been noted elsewhere (17). PCN, dexamethasone, rifampicin, and sodium PB were purchased from Sigma.

Cell Culture and Stable Cell Lines-- The generation and maintenance of wild-type and stably infected HepG2 cells has been described (10). The FAO cells were maintained in Dulbecco's modified Eagle's medium plus 10% calf serum. Primary cultures of rat and mouse hepatocytes, prepared as described elsewhere (54), were maintained in Williams' medium E (Invitrogen, Rockville, MD) supplemented with 100 nM dexamethasone, 100 units/ml penicillin G, 100 µg/ml streptomycin, and insulin/transferrin/selenium (Invitrogen). Human hepatocytes were obtained from Dr. Stephen Strom (University of Pittsburgh). Twenty-four h after isolation, hepatocytes were treated with either GW4064 (1 or 10 µM), PCN (10 µM), PB (1 mM), or rifampicin (0.1-10 µM), which were added to the culture medium as 1000× stocks in Me2SO (PB was directly dissolved into the medium). Control cultures received vehicle (Me2SO) alone. Cells were cultured for 48 h prior to harvest, and total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions.

Supression-subtractive Hybridization-- Total RNA was isolated from HepG2-vector cells treated with Me2SO (driver RNA) and from HepG2-FXR cells treated for 24 h with 50 µM CDCA (tester RNA). The driver and tester RNA were used in suppression-subtractive hybridization (55) using the PCR-Select Subtraction Kit (CLONTECH Laboratories, Palo Alto, CA) according to the manufacturer's instructions.

RNA Isolation and Northern Blot Hybridization-- Unless otherwise indicated, HepG2-derived cell lines were cultured in medium containing superstripped FBS for 24 h before the addition of ligand or Me2SO (vehicle) for an additional 24-48 h. Total RNA was isolated using TRIzol reagent and was resolved (10 µg/lane) on a 1% agarose, 2.2 M formaldehyde gel, transferred to a nylon membrane (Hybond N+; Amersham Biosciences, Inc.), and cross-linked to the membrane with UV light. cDNA probes were radiolabeled with [alpha -32P]dCTP using the rediprimeTM II labeling kit (Amersham Biosciences, Inc.). Membranes were hybridized using the QuikHyb hybridization solution (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Blots were normalized for variations of RNA loading by hybridization to a control probe, either glyceraldehyde-3-phosphate dehydrogenase, rat 18 S ribosomal cDNA, or the ribosomal protein 36B4. The RNA levels were quantitated using a PhosphorImager (ImageQuant software; Molecular Dynamics, Inc., Sunnyvale, CA).

Electrophoretic Mobility Shift Assays (EMSAs)-- EMSAs were performed essentially as described elsewhere (23). rPXR and rCAR, hFXR, and hRXRalpha were synthesized from the pSG5-rPXR, pSG5-rCAR, pSG5-hFXR, and pSG5-hRXRalpha expression vectors, respectively, using the TNT T7 Coupled Reticulocyte System (Promega, Madison, WI). Unprogrammed lysate was prepared using the pSG5 expression vector (Stratagene). Binding reactions contained 10 mM HEPES, pH 7.8, 60 mM KCl, 0.2% Nonidet P-40, 6% glycerol, 2 mM dithiothreitol, 2 µg of poly(dI-dC)·poly(dI-dC), and 1-2 µl each of synthesized receptor protein. Control incubations received unprogrammed lysate alone. Reactions were preincubated on ice for 10 min prior to the addition of 32P-labeled double-stranded oligonucleotide probe (0.2 pmol). Competitor oligonucleotides were added to the preincubation at a 100- or 500-fold molar excess. Samples were held on ice for a further 20 min, and the protein-DNA complexes were resolved on a pre-electrophoresed 5% polyacrylamide gel in 0.5× TBE (45 mM Tris borate, 1 mM EDTA) at room temperature. Gels were dried and autoradiographed at -70 °C for 1-2 h. Oligonucleotide probes used in EMSAs are shown in Fig. 5A.

Reporter Genes-- The rat MRP2 proximal promoter (-1034 to-15, relative to the translation start site) (56) was amplified from rat genomic DNA using primers 5'-ggggtaccgcaaaagaacaagttgtttaa-3' and 5'-ggggctagcgcttctgttacgaagactcttc-3' (reverse primer 1) and cloned into KpnI/NheI sites of the pGL3 basic vector (Promega), generating pGL3-MRP2-1. The two-copy ER-8 (pTk-2xER-8) was generated by annealing the oligonucleotides 5'-gatctaacatctctgtgaactcttaaccaagttcaaactgaatgatgtaacatctctgtgaactcttaaccaagttcaaactgaa-3' and 5'-gatcttcagtttgaacttggttaagagttcacagagatgttacatcattcagtttgaacttggttaagagttcacagagatgtta-3' before ligation into BamHI/BglII-digested Tk-Luc. The two ER-8 sites are in boldface type. The two-copy mutant ER-8 (pTk-Mut2xER-8) was generated using the same method and the primers 5'-gatctaacatctctgtgTTctcttaaccaagAAcaaactgaatgatgtaacatctctgtgTTctcttaaccaagAAcaaactgaa-3' and 5'-gatcttcagttttgTTcttggttaagagAAcacagagatgttacatcattcagtttgTTctggttaagagAAcacagagatgtta-3'. Mutations are capitalized.

Transient Transfections and Reporter Gene Assays-- HepG2 cells were transiently transfected using the MBS mammalian transfection kit (Stratagene), with minor modifications. HepG2 cells in 48-well plates were transiently transfected with reporter plasmid (100 ng) and either 50 ng of pSG5-rCAR, 50 ng of pSG5-rPXR, or 25 ng of pCMX-rFXR, together with 5 ng of pCMX-hRXRalpha and 50 ng of pCMV-beta -galactosidase as indicated in the specific figure legends. Each well received a total of 200 ng of DNA. After 3.5 h, the cells were treated with 10% superstripped FBS and one of the following ligands: PCN, LG100153 (synthetic RXR-agonist), 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl-isoxazole (GW4064), or CDCA. After 24-48 h, the cells were lysed and assayed for luciferase and beta -galactosidase activity (10).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Induction of MRP2 by Bile Acids and the Synthetic FXR Ligand GW4064-- In an effort to identify target genes that are regulated by the bile acid receptor FXR, we infected human HepG2 cells with retroviral vectors that expressed either FXR and the neomycin-resistant gene or the neomycin-resistant gene alone (17). G418-resistant cells were isolated, and pooled cell populations were propagated that harbored either the vector alone (HepG2-vector) or overexpressed FXR (HepG2-FXR). Total RNA was isolated from HepG2-vector or HepG2-FXR cells that had been treated for 24 h with either vehicle (Me2SO) or the FXR ligand CDCA (50 µM), respectively. Suppression-subtractive hybridization was then used to identify mRNAs (cDNAs) that were induced when the HepG2-FXR cells were treated with 50 µM CDCA (see "Experimental Procedures"). The isolated cDNAs that encode putative FXR-regulated genes were sequenced and shown to correspond to a number of known genes, including APOC-II (17). Here we report on MRP2, a second FXR target gene identified using this approach.

In order to confirm that MRP2 mRNA levels were induced in response to CDCA, we performed Northern blot assays (Fig. 1). The addition of CDCA to HepG2 cells resulted in a dose- and time-dependent increase in MRP2 mRNA; induction was observed within 12 h, continued for 48 h (Fig. 1A), and was maximal at 200 µM CDCA (Fig. 1, B and C). In parallel, two previously reported FXR-target genes, that encode SHP (11, 12) and APOC-II (17), were induced by CDCA (Fig. 1, B and C). In addition, the synthetic FXR ligand GW4064 (18) increased both MRP2 and SHP expression, further suggesting that this induction is FXR-dependent (Fig. 1C). Maximal induction of MRP2 was observed with 200 µM CDCA, which distinguishes it from other FXR target genes including APOC-II (17) and SHP (Fig. 1, B and C). The induction of these latter two mRNAs is maximal at 100 µM CDCA, and the induction is largely attenuated at 200 µM CDCA (Fig. 1, B and C).


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  		Fig. 1.   Induction of MRP2 mRNA by bile acids. A, MRP2 mRNA levels are induced by CDCA. HepG2-FXR cells were incubated with 100 µM CDCA for the indicated times. Total RNA was isolated, separated on a 1% agarose/formaldehyde gel, transferred to a nylon membrane, and sequentially hybridized to radiolabeled cDNA probes for MRP2, SHP, apo-CII, and 18 S ribosomal RNA, as described under "Experimental Procedures." B, different concentrations of CDCA are required for maximum induction of MRP2, apoC-II and SHP mRNA levels. HepG2-FXR cells were treated for 24 h with increasing concentrations of CDCA (0, 50, 100, 150, 200, and 250 µM). RNA was isolated, and Northern analysis was performed as described above. C, MRP2 is regulated by a synthetic FXR-specific agonist. HepG2 cells were treated with the indicated concentrations of the FXR synthetic agonist GW4064 or CDCA for 24 h. Total RNA was isolated, and Northern analysis was performed as described above. The results shown in A-C are representative of two or three experiments. The relative mRNA levels are shown in B and C.

Induction of MRP2 mRNA Levels Following Activation of PXR or CAR-- The dose-dependent induction of MRP2 expression by CDCA (maximal at >= 200 µM) suggested that the human MRP2 gene is regulated by PXR, which is known to be activated by pathophysiological concentrations of bile acids. To test the hypothesis that MRP2 mRNA levels are induced by a PXR-dependent mechanism, primary human hepatocytes were treated for 48 h with the macrocyclic antibiotic rifampicin or the putative anti-depressant hyperforin, two drugs known to activate human PXR (21, 57) (Fig. 2). The data of Fig. 2 show that low levels (1 µM) of both rifampicin and hyperforin result in a parallel increase in the levels of the mRNAs encoding MRP2 and CYP3A4. These results provide support for the hypothesis that MRP2 mRNA levels are induced following activation of PXR. In contrast, SHP mRNA levels were unaffected by either drug (Fig. 2).


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  		Fig. 2.   Induction of MRP2 mRNA in human hepatocytes by PXR ligands. Human primary hepatocytes were treated for 48 h with vehicle (Me2SO), the indicated concentrations of rifampicin (Rif), or hyperforin (1 µM). RNA was isolated and analyzed as described in the legend to Fig. 1.

The data shown in Figs. 1 and 2 demonstrate that MRP2 mRNA levels are induced when human liver cells are incubated with ligands for either FXR or PXR. To determine whether MRP2 expression responded in a similar fashion in rodents, we incubated primary rat hepatocytes with ligands known to activate different nuclear receptors. MRP2 mRNA levels increased significantly when the hepatocytes were incubated in the presence of PXR ligands (PCN or dexamethasone), an FXR ligand (GW4064), or a CAR activator (PB) (Fig. 3A). As expected, PCN, dexamethasone, and PB also increased the expression of CYP3A23, a known PXR/CAR target gene (19, 25) (Fig. 3A). Additionally, GW4064 treatment resulted in a modest increase in CYP3A23 expression. In contrast, SHP mRNA levels were induced only when cells were incubated with GW4064 but were unaffected by PCN, dexamethasone, or PB (Fig. 3A). Interestingly, expression of BSEP was significantly induced by GW4064, PCN, and dexamethasone and, to a lesser extent, by PB (Fig. 3A). Although BSEP expression is regulated by FXR in both rodents and humans (14, 15, 58) (Fig. 3A), this is the first report to demonstrate regulation of this gene by PXR agonists. Taken together, these data suggest that the expression of both BSEP and MRP2 mRNAs are induced by ligands known to activate FXR, PXR, or CAR.


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  		Fig. 3.   Ligands for FXR, PXR, and CAR induce MRP2 mRNA expression in rat hepatocytes. A, MRP2 mRNA levels are induced in primary rat hepatocytes. Primary rat hepatocytes were incubated with 10 µM dexamethasone, PCN, GW4064, 1 mM PB, or vehicle alone (0.1% Me2SO) as described under "Experimental Procedures." RNA was isolated and Northern blots were performed as described in the legend to Fig. 1. The same membrane was hybridized sequentially with the indicated radiolabeled probes. B, induction of MRP2 mRNA levels in FAO cells. FAO cells were treated with CDCA (100 or 200 µM), GW4064 (1 µM), PCN (10 µM), 3-keto-LCA (100 µM), or Me2SO (0.1%) for 24 h. Total RNA was isolated, and Northern analysis was performed as described in the legend to Fig. 1.

In order to extend our results, we treated a rat hepatoma cell line (FAO) with several ligands that activate FXR, PXR, or CAR. MRP2 mRNA levels are significantly induced when FAO cells are treated with CDCA, GW4064, PCN, or 3-keto-LCA (Fig. 3B). As expected, SHP expression was highly activated by the FXR ligands CDCA and GW4064, weakly activated by 3-keto-LCA, and unaffected by the PXR ligand PCN (Fig. 3B). Taken together, the data of Figs. 1-3 suggest that MRP2 mRNA levels are induced in both human and rodent cells following activation of either FXR, PXR, or CAR.

To further establish the role of PXR in the induction of MRP2, we incubated primary hepatocytes, derived from PXR wild-type or PXR null mice, with ligands for PXR or CAR (Fig. 4). As expected, Cyp3a11 expression in the wild-type cells was induced by PXR ligands (PCN and dexamethasone) and/or a CAR activator (PB) (Fig. 4) (36). In line with an earlier report (1), CYP3A11 mRNA levels were not induced when the PXR ligands (PCN or dexamethasone) were added to the PXR null cells (Fig. 4). In contrast to PCN and dexamethasone, PB slightly induced expression of Cyp3a11 in the PXR null cells (Fig. 4), consistent with a PXR-independent mechanism of induction by this drug in rodent hepatocytes. As expected from the data shown in Figs. 1-3, MRP2 mRNA levels were induced when primary hepatocytes, derived from wild-type mice, were incubated with PCN, PB, or dexamethasone (Fig. 4). However, a different pattern emerged when the PXR null hepatocytes were treated with the same drugs; first, we noted that the basal levels of MRP2 mRNA were increased 2-fold in the PXR null cells, indicating that PXR may function to repress MRP2 expression under basal, nonactivated conditions. In addition, in the absence of PXR, the MRP2 promoter may become more accessible to other transcription factors with a higher basal activity. Such factors include CAR, which we demonstrate binds to the same response element as PXR. Second, we noted that MRP2 mRNA levels were no longer induced by PCN or dexamethasone, consistent with the proposal that PXR is required for activation of MRP2 by these two drugs. Third, we noted that in response to PB the MRP2 mRNA levels were still induced in the PXR null cells (Fig. 4), consistent with this drug activating MRP2 by a PXR-independent mechanism. Since PB is known to induce nuclear translocation of CAR in rodents (36), this observation further suggests that Mrp2 is activated by this third member of the nuclear receptor superfamily.


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  		Fig. 4.   Induction of MRP2 mRNA in primary cultures of hepatocytes from wild-type and PXR null mice. Primary hepatocytes from wild-type (+/+) and PXR (-/-) mice were treated for 48 h with PCN (10 µM), dexamethasone (Dex; 10 µM), PB (1 mM), or PCN and PB prior to isolation and preparation of RNA. Northern analysis of MRP2 and CYP3A11 was performed as described in the legend to Fig. 1.

The MRP2 Promoter Contains a Novel Element That Is Bound by Either FXR/RXR, PXR/RXR, or CAR/RXR-- Analysis of the published nucleotide sequence 5' of the transcriptional start site of the rat MRP2 gene identified a 26-bp sequence (-401 to -376, relative to the translation start site) that contains two copies of the AGTTCA hexad organized as an ER-8 (everted repeat with 8-bp spacer) (Fig. 5A). Although neither FXR, PXR, nor CAR have been shown to bind to an ER-8, we performed EMSAs with an oligonucleotide corresponding to this site, designated rMRP2 ER-8, (Fig. 5, B and C). The results shown in Fig. 5B demonstrate that PXR, CAR, and FXR were incapable of binding the rMRP2 ER-8 probe in the absence of RXRalpha (Fig. 5B). However, the addition of recombinant RXRalpha resulted in the appearance of a robust protein-DNA complex. The ability of PXR/CAR/FXR to interact with this element was further examined by competition EMSAs. A 100-fold molar excess of unlabeled rMRP2 ER-8 efficiently competed with a previously reported PXR- and CAR-binding motif, the ER-6 element from the CYP3A4 promoter (20, 22, 23, 27), for binding to PXR-RXRalpha and CAR-RXRalpha heterodimers (Fig. 5C, left panel). Mutation of either half-site (rMRP2 ER-8mut1 and rMRP2 ER-8mut2 probes) abolished the direct binding of PXR/CAR-RXRalpha heterodimers (data not shown) and the ability of the probe to compete for PXR/CAR-RXRalpha binding with the CYP3A4 ER-6 (Fig. 5C, left panel). Similarly, the rMRP2 ER-8 oligonucleotide was capable of effectively competing with an FXR response element from the human I-BABP gene (hI-BABP IR-1; Fig. 5C, right panel). In contrast, no competition was observed when the competitor DNA contained mutations of either half-site (rMRP2 ER-8mut1 and rMRP2 ER-8mut2 probes) (Fig. 5C, lower panel). These results indicate that the rMRP2 ER-8 site is a high affinity PXR, CAR, and FXR binding motif. Moreover, the observation that mutation of either of the AGTTCA half-sites results in complete loss of nuclear receptor binding function demonstrates that PXR/CAR/FXR-RXRalpha heterodimers are directly interacting with the ER-8 and not cryptic binding motifs embedded within this site.


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  		Fig. 5.   The PXR/RXR, CAR/RXR, and FXR/RXR heterodimers bind to the ER-8 in the proximal promoter of the MRP2 gene. A, the nucleotide sequence corresponding to the putative PXR/RXR, CAR/RXR, and FXR/RXR binding site at -401 to -376 from the rat MPR2 promoter is shown (rMRP2 ER-8). Mutations within the ER-8 are shown in lowercase type. Hexameric consensus sites are in boldface type. In addition, the sequences of the ER-6 located in the CYP3A4 promoter and the IR-1 from the human I-BABP gene are shown. B, PXR, CAR, and FXR bind to the rMRP2 ER-8. In vitro translated PXR, CAR or FXR were incubated with RXR and the 26-bp rat MRP2 oligonucleotide from the proximal promoter (rMRP2 ER-8), as described under "Experimental Procedures." The shifted DNA-protein complexes were identified by autoradiography. C, PXR/RXR, CAR/RXR, and FXR/RXR heterodimers bind to the rMRP2 ER-8 with high affinity. Either PXR, CAR, or FXR was incubated with RXR and the indicated radiolabeled probes in the presence of unlabeled competitor DNA (100- or 500-fold molar excess) as indicated and described under "Experimental Procedures."

The ER-8 in the MRP2 Proximal Promoter Is Required for Transcriptional Activation-- To investigate the mechanism involved in transcriptional regulation of the MRP2 gene, we cloned the rat MRP2 proximal promoter (-1034 to -15, relative to the translation start site) into a luciferase reporter construct. The reporter plasmid pGL3-MRP2-1 was transiently transfected into HepG2 cells in the presence or absence of plasmids encoding PXR, CAR, or FXR and RXR, and the cells were subsequently treated with receptor-specific ligands. In the absence of co-transfected PXR, the MRP2 promoter reporter gene was not activated by PCN (Fig. 6A). Co-transfection of a rat PXR expression vector resulted in ~2-fold induction in reporter gene activity following the addition of PCN. In addition, co-transfection of the pGL3-MRP2-1 construct and a CAR expression vector resulted in a 2-fold increase in reporter gene activity. No further induction was observed when PCN was added to the cell medium, consistent with a previous report that demonstrated that CAR is constitutively active when expressed in HepG2 cells (33). The MPR2 promoter-reporter construct was also activated 2-fold in the presence of the FXR synthetic compound GW4064, the RXR ligand LG100153, and FXR (Fig. 6B).


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  		Fig. 6.   Transactivation of the rat MRP2 proximal promoter by PXR, CAR, and FXR. A, HepG2 cells were co-transfected with either rat PXR or rat CAR and the pGL3 reporter gene under the control of the rat proximal promoter (-1034 to -15, relative to the translation start site, pGL3-MRP2-1). Cells were treated with vehicle (Me2SO) or PCN (10 µM) for 48 h following the transfection. Relative light units for the reporter gene are shown after normalization for minor changes in transfection efficiencies. B, HepG2 cells were transiently transfected with rat FXR and the pGL3-MRP2-1 reporter construct. Cells were treated with vehicle (Me2SO) or GW4064 (1 µM) and LG100153 (100 nM) for 48 h. Relative light units (RLUs) are shown following normalization with beta -galactosidase. C, HepG2 cells were transfected with a reporter gene construct containing either two copies of the ER-8 from the rat MRP2 promoter linked to a luciferase reporter gene (pTk-2xER-8), the Tk-luciferase vector under the control of two copies of the mutant ER-8 (pTk-Mut2xER-8), or three copies of the PXR response element from the CYP3A23 promoter (3xCyp3A23). Each reporter was cotransfected with either rat PXR, rat CAR, or rat FXR. Following the transfection, cells were treated with Me2SO, PCN (10 µM), or GW4064 (1 µM) for 24 h. RLUs were determined after normalization for changes in transfection efficiencies. All transfections were performed in triplicate, and the results varied less than 10%. The data shown in A-C are representative of two or three experiments.

To investigate the effects of multiple ligands and multiple nuclear receptors on the activity of the ER-8 in the MRP2 promoter, we constructed a luciferase reporter gene under the control of either two copies of the wild-type ER-8 (pTk-2xER-8) or the mutant ER-8 (pTk-Mut2xER-8). The expression of the pTk-luciferase control vector was not affected by PXR, CAR, FXR, or ligands for PXR or FXR (data not shown). However, the pTk-2xER-8-Luc reporter was activated over 100-fold in a PXR- and PCN-dependent manner (Fig. 6C). The reporter gene was activated ~150-fold by CAR, and this induction was independent of PCN or GW4064 (Fig. 6C), which is consistent with the constitutive activation of CAR in HepG2 cells. The Tk-2xER-8 reporter gene was also activated ~60-fold by a process that required FXR co-expression and the addition of the FXR ligand GW4064 (Fig. 6C). In contrast, the activity of the mutant ER-8 reporter gene was low and was unaffected by coexpression of PXR, CAR, or FXR and their ligands (Fig. 6C). As expected, a luciferase reporter gene driven by three copies of the PXR response element derived from the CYP3A23 gene was specifically activated in a PXR- and PCN-dependent manner (Fig. 6C). These data demonstrate that the ER-8 from the rat MRP2 proximal promoter acts as a functional response element and mediates the transcriptional activation of the MRP2 gene by ligand-activated PXR or FXR or the constitutively active nuclear receptor CAR.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The current study identifies MRP2 as a gene that is activated by FXR and bile acids. MRP2 is a member of the ATP-binding cassette superfamily of transporter proteins and is predicted to contain multiple transmembrane domains and two ATP binding cassettes (64). In addition, we demonstrate that hepatic MRP2 expression is induced by CAR or ligand-activated PXR. Since hepatic MRP2 is localized to the canalicular membrane and functions to transport a variety of conjugated compounds from the hepatocyte into bile, our data suggest that activation of FXR, PXR, or CAR is likely to enhance the excretion of these compounds from the body (Fig. 7).


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  		Fig. 7.   A model of transporter proteins in the rodent liver. Activation of the hepatic FXR/RXR heterodimer stimulates the export of bile salts (BS) and organic anions (OA) from the hepatocyte into the bile by increasing the expression of BSEP and MRP2. FXR and its target genes are shown in red. Activation of the PXR/RXR heterodimer increases the uptake of organic anions from the blood across the basal membrane by increasing the expression levels of the organic anion-transporting polypeptide (OATP2). PXR and its target genes are shown in blue. Within the hepatocyte, organic anions and bile salts are further metabolized by CYP3A, which is itself regulated by PXR and RXR. These metabolites are then transported from the hepatocyte into the bile canaliculi via MRP2. MRP2 expression is induced by activated PXR/RXR or CAR/RXR (fuschia) in addition to FXR/RXR. PXR/RXR also increase the expression of MDR1, which transports amphipathic compounds across the canalicular membrane in an ATP-dependent manner. Sodium taurocholate-cotransporting polypeptide (NTCP) facilitates the uptake of bile acids from the blood.

Surprisingly, all three nuclear receptors were shown to activate MRP2 gene expression via a novel hormone response element (ER-8) in the proximal promoter of the gene. Further studies will be necessary to determine whether activation of the MRP2 gene by different ligands is dependent upon competition between FXR/RXR, PXR/RXR, and CAR/RXR for this ER-8 element under normal physiological conditions.

The DNA sequences that function as hormone response elements for FXR, PXR, or CAR were once thought to be fairly specific. However, even with the identification of relatively few target genes for each nuclear receptor, it has become apparent that such DNA sequences can be quite varied. For example, IR-1 (5, 10, 11, 13, 14, 16, 17) and ER-8 (current study) have now been shown to function as FXR response elements in a number of genes. DR-3, DR-4, ER-6, and ER-8 (current study) elements have been shown to function as PXR and CAR binding motifs (reviewed in Refs. 39, 60, and 61). It is not known how each nuclear receptor heterodimer can form a transcriptionally active complex with such diverse DNA sequences. Presumably, the two DNA binding motifs present in the heterodimeric complex have considerable flexibility so as to allow stable interaction with the two hexads (AGGTCA or variants thereof) that may be separated by 0-8 nucleotides.

In addition to MRP2, a number of other transporter proteins, including BSEP, MDR1, and MDR3 are localized to the canalicular membrane of the hepatocyte (Fig. 7). These proteins facilitate the export of bile salts (BSEP), phospholipids (human MDR3/murine MDR2), and hydrophobic compounds/drugs (MDR1) into the bile. BSEP expression has recently been shown to be stimulated by bile acids in an FXR-dependent manner (14, 15). Likewise, MDR1 has been shown to be a direct PXR target (62). These findings provide an additional link between multiple nuclear receptors and hepatic excretion of various compounds.

As illustrated in Fig. 7, PXR induces transcription of the rodent OATP2 gene, which results in the uptake of organic anions and bile salts across the basal membrane (1). Hepatic uptake of bile acids is facilitated by a number of transporters including the sodium taurocholate-cotransporting polypeptide (63). PXR also mediates the transcriptional induction of hepatic CYP3A4, which, in turn, metabolizes a large number of endogenous and exogenous compounds (21, 23). The current study demonstrates that FXR, PXR, and CAR activate the expression of MRP2, which is involved in the subsequent transport of many of these compounds into the bile. This coordinate regulation results in a net clearance of compounds from the blood (OATP2), detoxification of these compounds within the hepatocyte (CYP3A), and efflux of these compounds into the bile (MRP2), whereupon they can ultimately be excreted into the intestine and from the body.

    ACKNOWLEDGEMENTS

We thank Drs. R. Heyman, O. Witte, P. Maloney, and R. Evans for providing plasmids and reagents.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL30568 and HL68445 (to P. A. E.), American Heart Association Grant 0150381N (to P. A. E.), a grant from the Laubisch Fund (to P. A. E.), and United States Department of Education Grant P200A80113 (to H. R. K. and A. M. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These two authors contributed equally to this work.

Dagger Dagger To whom correspondence should be addressed: Dept. of Biological Chemistry, CHS 33-257, UCLA, 10833 Le Conte Ave., Los Angeles, CA 90095. Tel.: 310-206-3717; Fax: 310-794-7345; E-mail: pedwards@mednet.ucla.edu.

Published, JBC Papers in Press, November 12, 2001, DOI 10.1074/jbc.M109326200

    ABBREVIATIONS

The abbreviations used are: FXR, farnesoid X-activated receptor; apoC-II, apolipoprotein C-II; BSEP, bile salt export pump; CAR, constitutive androstane receptor; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; DR-n, direct repeat with n-bp spacer; EMSA, electrophoretic mobility shift assay; ER-n, everted repeat with n-bp spacer; GW4064, 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl-isoxazole; I-BABP, ileal bile acid-binding protein; IR-n, inverted repeat with n-bp spacer; MRP1, -2, and -3, multidrug resistance-associated protein 1, 2, and 3, respectively; PB, phenobarbital; PCN, pregnenolone 16alpha -carbonitrile; PXR, pregnane X receptor; rCAR, rat CAR; rPXR, rat PXR; rFXR, rat FXR; RXR, retinoid X receptor; hRXRalpha , human retinoid X receptor alpha ; SHP, small heterodimer partner; LCA, lithocholic acid.

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ABSTRACT
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