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J. Biol. Chem., Vol. 277, Issue 34, 30559-30566, August 23, 2002

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Human Apical Sodium-dependent Bile Salt Transporter Gene (SLC10A2) Is Regulated by the Peroxisome Proliferator-activated Receptor ^*

Diana Jung, Michael Fried, and Gerd A. Kullak-Ublick

From the Laboratory of Molecular Gastroenterology and Hepatology and the Division of Clinical Pharmacology and Toxicology, University Hospital, CH-8091 Zurich, Switzerland

Received for publication, April 11, 2002, and in revised form, May 28, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The apical sodium-dependent bile salt transporter (ASBT/SLC10A2), also called the ileal bile acid transporter, mediates the intestinal absorption of bile salts. The efficiency of this transport process is a determinant of hepatic bile salt synthesis from cholesterol and of serum triglyceride levels. Our aim was to characterize the human ASBT gene promoter with respect to regulatory mechanisms that coordinately affect ASBT expression and hepatic lipid and bile salt metabolism. The minimal construct that confers full promoter activity contains three functional hepatocyte nuclear factor 1 (HNF1) recognition sites, explaining the dependence of ASBT gene expression upon HNF1. A nuclear receptor binding site arranged as a direct hexanucleotide repeat (DR1 motif) is localized ~1.6 kb upstream of the transcription initiation site. Constructs containing this element were transactivated by WY14643 and ciprofibrate, ligands of the peroxisome proliferator-activated receptor  (PPAR), in Caco2 cells. The DR1 element was shown to bind the PPAR/9-cis-retinoic acid receptor heterodimer, and targeted mutagenesis of the DR1 motif abolished PPAR responsiveness. Ciprofibrate treatment of SK-ChA cholangiocytes increased ASBT mRNA levels, suggesting a physiologic role for PPAR-mediated ASBT gene regulation. This study identifies PPAR as a novel link between ileal bile salt absorption and hepatic lipid metabolism.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bile salts undergo extensive enterohepatic circulation through the coordinated action of several transport systems in the intestine and the liver. In man, the bile salt pool circulates 6-10 times/24 h, resulting in a daily hepatic bile salt excretion of 20-40 g. Only about 0.5 g of bile salts escape intestinal absorption and are lost through fecal excretion. This loss is compensated for by de novo hepatic synthesis. The intrinsic link between intestinal bile salt absorption and hepatic synthesis has become apparent from studies showing that hydrophobic bile salts can transcriptionally induce the ileal bile acid-binding protein (I-BABP)¹ and repress hepatic cholesterol 7-hydroxylase (CYP7A1) through the action of the nuclear bile salt receptor, farnesoid X receptor (FXR) (1, 2). By this mechanism, bile salts can regulate their own enterohepatic circulation.

The chief intestinal bile salt uptake system is the apical sodium-dependent bile salt transporter (ASBT/SLC10A2), also called the ileal bile acid transporter. The human ASBT protein consists of 348 amino acids and is encoded by a major 4.0-kb transcript that has been detected in the ileum, cecum, and kidney by Northern blot analyses (3). Expression on the apical surface of ileal enterocytes, renal proximal tubular cells, and large cholangiocytes has been shown in rat studies (4, 5). Human ASBT is an efficient transport system for conjugated and unconjugated bile salts, with a higher affinity for the dihydroxy bile salts chenodeoxycholate (CDCA) and deoxycholate than for the trihydroxy bile salt, taurocholate (3). Intestinal ASBT expression is a critical determinant of the bile salt pool size and activity of the bile salt biosynthetic enzymes CYP7A1 (classic or neutral synthetic pathway) and sterol 27-hydroxylase (alternative or acidic pathway) in the liver (6). The ASBT gene is localized on chromosome 13q33 (7).

Several lines of evidence indicate that ASBT gene expression is tightly regulated at the transcriptional level. First, mice with null mutations in the gene coding for hepatocyte nuclear factor 1 (HNF1) (encoded by Tcf1) have no expression of ASBT in intestine and kidneys, indicating that ASBT gene expression is dependent upon HNF1 (8). Second, the rat ASBT gene promoter contains an AP-1 element that binds c-Jun and c-Fos and coexpression of c-Jun enhances promoter activity (9). Third, intestinal inflammation in rabbits decreases ileal ASBT mRNA levels, whereas glucocorticoid administration in rats leads to up-regulation (10, 11). Whether or not bile salts regulate the ASBT gene remains controversial; a direct involvement of FXR seems unlikely because FXR^/ mice have no obvious alteration in ASBT expression level (12).

Intestinal bile salt absorption rates have been shown to correlate inversely with serum triglyceride levels, suggesting a connection between ASBT function and lipid metabolism (13, 14). The pharmacologic inhibition of ASBT function and expression could, therefore, have a major impact not only on the amount of potentially carcinogenic bile salts that enter the colon but also on hepatic VLDL synthesis (15), serum triglyceride levels and hepatic breakdown of cholesterol to bile salts (6). The primary aim of this study was to characterize the human ASBT gene promoter and to investigate whether a regulatory pathway exists that could account for the observed correlation between ileal bile salt absorption and hepatic lipid and bile salt metabolism. Specifically, we studied whether the human ASBT gene is regulated by a member of the nuclear receptor superfamily. Prototypic "lipid sensors" within the nuclear receptor superfamily include FXR, which regulates CYP7A1 and bile salt transporter genes (1, 2, 16, 17), and the liver X receptor (LXR), which also regulates CYP7A1 as well as cholesterol and bile salt transport systems including I-BABP (18, 19). The results indicate an important new mechanism of ASBT gene regulation and identify the peroxisome proliferator-activated receptor  (PPAR) as a novel link between ileal bile salt absorption and hepatic lipid metabolism.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [-³²P]Adenosine triphosphate (3000 Ci/mmol) was purchased from Amersham Biosciences. Restriction enzymes were from Roche Molecular Biochemicals, PfuTurbo DNA polymerase from Invitrogen, and T4 polynucleotide kinase from Stratagene. Polyacrylamide was obtained from BioRad. Unless stated otherwise, all chemicals were purchased from Sigma-Aldrich.

Plasmid Construction-- Three fragments of the 5'-region of the human ASBT gene were PCR-amplified using human genomic DNA as a template, upstream primers ASBT/1688for or ASBT/+26for, downstream primers ASBT/+21rev or ASBT/+526rev (Table I), and PfuTurbo DNA polymerase. The upstream primers contained an internal SacI restriction site and the downstream primer an internal XhoI site. The resulting PCR products were digested with SacI and ligated into the luciferase reporter gene vector, pGL3-Basic (Promega Catalys AG, Wallisellen, Switzerland), which had been predigested with SacI and SmaI, yielding the following promoter constructs: 1688-Luc, 1688/UTR-Luc, UTR-Luc. The 1492/UTR-Luc construct was generated by excising a StuI/SacI fragment from 1688/UTR-Luc. Additional deletional constructs of the 5'-untranslated region (5'-UTR) of the ASBT gene were constructed by PCR amplification of the 5'-UTR using upstream primers ASBT/+324for or ASBT/+292for and downstream primer ASBT/+539rev (Table I). The upstream primers contained an internal SacI restriction site and the downstream primer a BglII site. The resulting PCR products were digested with SacI and BglII and were ligated into the pGL3-Basic vector predigested with SacI and BglII, resulting in the constructs UTR292-539-Luc and UTR324-539-Luc. UTR26-251-Luc was generated by excising a XhoI fragment from UTR-Luc. The DR1-TK-Luc and mutDR1-TK-Luc plasmids were constructed by ligating a dimerized oligonucleotide (DR1 and mutDR1, respectively, in Table I) containing the DR1 element of the ASBT gene and 5'- HindIII and 3'-BamHI overhangs into TK-Luc plasmid predigested with HindIII and BamHI. Sequence identity of all constructs with the ASBT gene was verified by sequence analysis. Plasmid DNA was prepared using the Qiagen system (Basel, Switzerland).

Site-directed Mutagenesis-- A 1688/UTR-Luc derived construct containing staggered nicks was generated by PCR using two complementary oligonucleotides mutated in the DR1 binding site (sequence sdmut-DR1 in Table I) and PfuTurbo DNA polymerase. The product was digested with DpnI to remove the parental DNA template and select for DNA containing the mutation. The mutated plasmid was termed mutDR1-1688/UTR-Luc.

Culture and Transfection of Human Cell Lines-- Caco2 and HEK293 cell lines were purchased from ATCC (Manassas, VA). SK-ChA cells were kindly provided by Dr. M. Strazzabosco (Division of Gastroenterology, Ospedali Riuniti di Bergamo, Bergamo, Italy). SK-ChA cells were maintained in minimum essential medium (-MEM, Invitrogen), Caco2 and HEK293 in Dulbecco's modified Eagle's medium (Sigma). All media were supplemented with 10% fetal calf serum (20% in the case of Caco2 cells), 100 units/ml penicillin, and 100 µg/ml streptomycin. For transactivation assays, Caco2 cells were grown for 3 days in medium containing 20% charcoal-stripped bovine calf serum and then seeded at 90-95% density in 48-well plates. Caco2 cells were transfected with LipofectAMINE 2000 reagent (Invitrogen) and SK-ChA and HEK293 cells with FuGENE 6 (Roche Molecular Biochemicals). Plasmid DNA for reporter assays comprised 350 ng of luciferase promoter construct, 50 ng of pSV--galactosidase plasmid, and 50 ng each of pCMX-hRXR (kindly provided by Dr. D. J. Mangelsdorf, Howard Hughes Medical Institute and Dept. of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX) and pSG5-hPPAR (kindly provided by Dr. B. Staels, Département d'Athérosclerose, Institut Pasteur de Lille, Lille, France) expression plasmid or 100 ng of HNF1/HNF1 expression plasmids (pRSVhumHNF1-correct and pRSV-vHNF1Ahuman, kindly provided by Dr. M. Yaniv, Unité des Virus Oncogènes, Institut Pasteur, Paris, France). To ensure that the total DNA amount transfected remained constant, pBluescript vector (pB-SKII, CLONTECH, Basel, Switzerland) was used as carrier DNA as required. 18 h after transfection, cells were treated with 1 µM 9-cis-retinoic acid (9cRA) and either 10 µM WY14643 (Calbiochem) or 30 µM ciprofibrate (Sigma) as indicated for 24 h. Controls were treated with dimethyl sulfoxide (Me₂SO) alone.

Culture and Transfection of LMH Cells-- LMH cells were obtained from ATCC and were grown in William's medium E (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 2 mM glutamine, 1× nonessential amino acids, 100 units/ml penicillin, and 100 µg/ml streptomycin on gelatin-coated dishes (Sigma). 48 h before transfection, cells were seeded on to gelatin-coated 24-well plates in medium supplemented with 10% charcoal-stripped bovine calf serum (Sigma) at 75-80% density. Transfections were performed with 1.5 µl of FuGENE 6 transfection reagent (Roche Molecular Biochemicals) and 500 ng plasmid DNA, the latter comprising 450 ng of luciferase construct and 50 ng of pSV--galactosidase plasmid (Promega Catalys AG). Six hours after transfection, cells were stimulated with 30 µM ciprofibrate (Sigma), 50 µM CDCA (Sigma), 10 µM 22(R)-hydroxycholesterol (22-HC, Sigma), or Me₂SO for 24 h.

Luciferase and -Galactosidase Reporter Assays-- Cells were lysed with passive lysis buffer (Promega Catalys AG) 24 h after transfection (in the absence of ligand) or after treatment with ligand. Luciferase activity was quantified using the luciferase assay system (Promega Catalys AG) in a Lumat LB 9507-2 luminometer (Berthold, Bad Wildbad, Germany). -Galactosidase activity was quantified with a high sensitivity assay (Stratagene, Amsterdam, Netherlands) in a UVmax kinetic microplate reader (Molecular Devices, Sunnyvale, CA) at 595 nm.

Electrophoretic Mobility Shift Assays-- Double-stranded oligonucleotide probes were obtained by hybridizing single-stranded complementary oligonucleotides (Microsynth, Balgach, Switzerland). Dimers with the DR1 sequence corresponding to the sequence found in the ASBT gene promoter (Table I) were labeled with [-³²P]ATP using T4 polynucleotide kinase (Stratagene, Amsterdam, Netherlands). In vitro translation was performed with the TnT Quick-coupled transcription/translation system (Promega Catalys AG).

For gel mobility shift assays, 5 µl of in vitro translated PPAR or 9-cis-retinoic acid receptor (RXR) protein were incubated on ice for 20 min with 2-5 fmol of [³²P]-end-labeled dimerized oligonucleotide and 1 µg of poly(dI)poly(dC) (Amersham Biosciences) in 20 mM HEPES-KOH, pH 7.9, 20% glycerol, 100 mM KCl, 2 mM MgCl₂, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. For competition assays a 500-fold excess of unlabeled dimerized oligonucleotides was added. Sequence mutDR1 (Table I) corresponded to the wild-type ASBT sequence mutated within the DR1 site. For supershift experiments 2 µl of antibody against PPAR (N-19, sc-1985X, Santa Cruz Biotechnology Inc., Heidelberg, Germany) or RXR (D-20, sc-553X, Santa Cruz Biotechnology) was added to the reaction mix. Reactions were analyzed by electrophoresis through 3.4% polyacrylamide gels in 0.25× TBE buffer (Tris borate/EDTA) at 120 V for 2 h.

Quantitation of ASBT Gene Expression-- 1 µg of total RNA isolated from SK-ChA cholangiocytes was reversed-transcribed (Reverse Transcription System, Promega Catalys AG). Real-time PCR was performed with the ABI PRISM 7700 sequence detection system using one-sixth of the reverse transcription reaction and was analyzed with Applied Biosystems 1.7 software (Rotkreuz, Switzerland). Amplification of the endogenous control was performed with the ribosomal 18 S TaqMan PCR master system (Applied Biosystems). ASBT was amplified with the primers ASBTquant-for and ASBTquant-rev (Table I) using cyber green incorporation (SYBR Green PCR-Master Mix, Qiagen). Because validation experiments showed that the amplification efficiencies of the target and the reference were approximately equal, quantitation was performed using the comparative C_T method.

Statistical Analysis-- Reporter gene activities are expressed as the mean ± 1 standard error of the mean (S.E.) of 6-10 individual transfection experiments. All data were reproduced at least once using two different preparations of plasmid DNA.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Analysis of Basal ASBT (SLC10A2) Gene Promoter Activity in Human Cell Lines-- As shown in previous studies on the structure of the human ASBT gene (3, 7), the major transcription initiation site (designated +1 in Fig. 1) is separated by a 598-bp untranslated region from the translation start site on exon 1. Because previous studies had indicated the presence of regulatory elements within the 5'-UTR of the ASBT gene (8, 9) we constructed reporter gene vectors containing the major part of the 5'-UTR and up to 1688 nucleotides of 5'-flanking sequence (plasmids 1688/UTR-Luc and 1492/UTR-Luc in Fig. 1). Additional constructs contained only the 5'-UTR (UTR-Luc) or only 5'-flanking sequence (1688-Luc). These constructs were used to characterize the basal promoter function of the human ASBT gene.

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Analysis of ASBT promoter function in cell lines. Human colon-derived Caco2, embryonic kidney HEK293, and cholangiocyte SK-ChA cell lines were transiently transfected with four chimeric promoter constructs. The ASBT (SLC10A2) gene sequence is derived from GenBankTM accession number Z54350, and nucleotide numbering is relative to the transcription initiation site (nt +1). The 5'-UTR consists of 598 nucleotides and contains a region from nt +252 to +408 that is highly conserved among mammalian species. Constructs, designed as shown, contained up to 1688 nucleotides of 5'-flanking sequence. Promoter fragments were inserted into the pGL3-Basic luciferase vector; the position of the luciferase gene is symbolized by the curved arrow. Transfection efficiency was normalized by cotransfection of pSV--galactosidase. Promoter activity was measured as relative units of firefly luciferase per unit of -galactosidase. Promoter activity is shown as the factor of induction of luciferase over background activity measured in cells transfected with pGL3-Basic alone. Results are expressed as the mean ± 1 S.E. of 6-10 transfections.

For reporter gene assays, cell lines derived from human intestine (Caco2), embryonic kidney (HEK293), and bile duct epithelium (SK-ChA), tissues that have been shown to express the ASBT mRNA (3, 5), were employed. Transfection of cells with plasmids UTR-Luc, 1492/UTR-Luc, and 1688/UTR-Luc produced significant luciferase activity compared with the promoterless pGL3-Basic vector. The degree of activation was 8-12-fold in Caco2 cells, 2-3-fold in HEK293 cells, and up to 1.7-fold in SK-ChA cells (Fig. 1). Thus ASBT promoter activity was strongest in Caco2 cells, in accordance with real-time PCR experiments that showed the highest endogenous ASBT mRNA levels in Caco2 cells (data not shown).

The 1688-Luc construct, that contained only the 5'-flanking sequence without the 5'-UTR, produced no luciferase activity, indicating that factors that bind within the 5'-UTR are essential for minimal promoter function.

HNF1 Binds within the 5'-UTR and Is a Potent Transactivator of the ASBT Promoter-- The importance of HNF1 for expression of the ASBT gene became evident in Tcf1^/ (HNF1^/) mice with a null mutation in the HNF1 gene (8). These mice show no expression of the ASBT (Slc10a2) mRNA in the terminal ileum and kidney and a 6-fold elevation of fecal bile salt concentrations. A single HNF1 binding site, corresponding to nt +253 to +267 in the 5'-UTR of the human ASBT gene (Fig. 2, Table II) was described by Shih et al. (8). This site conferred inducibility by coexpressed HNF1 in HIT-T15 cells and bound HNF1 in gel shift mobility assays. Although mutagenesis of the site decreased transactivation by HNF1, the mutated construct was still activated ~12-fold by coexpressed HNF1 (8). This suggested additional HNF1 binding sites in the human ASBT gene.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Functional analysis of the HNF1 binding sites in the 5'-UTR of the ASBT gene. Caco2 cells were transfected with the indicated ASBT luciferase constructs together with either an HNF1 (HNF1, black bars) or HNF1 (HNF1, gray bars) expression plasmid or with pBluescript vector as a control (Carrier, white bars). Coexpression of HNF1 led to a 3.8-fold (UTR26-251-Luc) to 13-fold (UTR-Luc) activation of constructs that contained an HNF1 binding site. No significant induction was observed with the UTR324-539-Luc construct that lacked an HNF1 site. Coexpression of HNF1 had no effect on any of the constructs. The data indicate that all three HNF1 binding sites in the 5'-UTR of the ASBT gene are activated by HNF1.

Sequence analysis of the human ASBT promoter from nt 1688 to +598 using the program Mat Inspector (Genomatix Software, Munich, Germany) identified two additional HNF1 binding sites at nt +125 to +139 and +304 to +318, both also localized in the 5'-UTR (Fig. 2, Table II). The site at nt +125 to +139 is human-specific compared with the rodent ASBT gene sequences (9). To assess the role of each of the three HNF1 binding sites in activating the ASBT gene, deletional reporter constructs were characterized in cotransfection assays with an HNF1 expression plasmid. The UTR-Luc construct, which contained all three HNF1 binding sites, was induced 13-fold by cotransfection of an HNF1 expression vector compared with carrier DNA in Caco2 cells (Fig. 2). Cotransfection of HNF1, which binds to the identical sequence as HNF1 and is expressed in the kidney, liver, pancreas, and intestine (20), had no effect on ASBT promoter activity. A 5'-UTR construct containing only the site at nt 304-318 still conferred a 4.4-fold induction by HNF1 (UTR292-539-Luc in Fig. 2), indicating that this HNF1 element is also functional. A minimal construct containing only nt 324-539 of the 5'-UTR but lacking an HNF1 binding site exhibited residual luciferase activity (~1.8-fold induction compared with the promoterless pGL3-Basic vector) but was not inducible by HNF1 (UTR324-539-Luc in Fig. 2). Finally, a construct extending from nt 26-251, which contained only the human-specific site at nt 125-139, exhibited no basal luciferase activity compared with the pGL3-Basic vector but was induced 3.8-fold by coexpression of HNF1 (UTR26-251-Luc in Fig. 2). The data thus indicate that all three HNF1 sites in the 5'-UTR of the human ASBT gene are functional response elements that act synergistically to induce gene transcription. This may explain why the complete 5'-UTR is required for full ASBT promoter activity.

Identification of a PPAR Response Element in the ASBT Promoter-- Because the main objective of this study was to investigate whether a transcriptional mechanism exists that coordinately regulates ASBT and genes involved in hepatic bile salt and lipid metabolism, the ASBT promoter sequence was analyzed for potential binding sites of nuclear receptors. Nuclear receptors have also been termed lipid sensors; important candidates known to regulate other bile salt and lipid transporters include FXR, LXR, and PPAR (21). To study whether the ASBT gene promoter is regulated by one of these nuclear receptors, we initially transfected LMH chicken hepatoma cells with the 1688/UTR-Luc and UTR-Luc constructs and with the control vector pGL3-Basic. LMH cells have been used extensively as a model for ligand-dependent activation of endogenously expressed nuclear receptors (22, 23). ASBT promoter activity in LMH cells was assayed in the presence or absence of the PPAR ligand ciprofibrate, the LXR ligand, 22(R)-hydroxycholesterol, and the FXR ligand, chenodeoxycholic acid. A 1.75-fold activation of the ASBT promoter construct was observed in the presence of ciprofibrate (Fig. 3A), suggesting a possible involvement of PPAR. The UTR-Luc construct, lacking the 5'-flanking region of the ASBT gene, was not inducible by ciprofibrate, indicating that the response element is located in the untranscribed 5'-flanking region of the ASBT gene.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Activation of the human ASBT promoter by PPAR. A, LMH cells were transiently transfected with two ASBT reporter gene constructs containing up to 1688 nucleotides of 5'-flanking sequence (1688/UTR-Luc) or only the untranslated region (UTR-Luc) and with the promoterless control vector pGL3-Basic. 6 h after transfection, cells were treated with ligands for PPAR (Ciprofibrate, 30 µM), LXR (22-HC, 10 µM), or FXR (CDCA, 50 µM). Ciprofibrate led to a 1.75-fold induction of the 1688/UTR-Luc construct. Promoter activity of each construct is expressed in relation to values obtained in dimethyl sulfoxide Me2SO (DMSO)-treated controls. B, Caco2 cells transfected with ASBT reporter constructs were additionally cotransfected with PPAR/RXR expression plasmids where indicated (Co-). 12 h after transfection, cells were treated with the PPAR ligands WY14643 (10 µM) or ciprofibrate (30 µM) and the RXR ligand 9cRA (1 µM) or with dimethyl sulfoxide alone. Promoter activity is shown as the ratio of luciferase to -galactosidase activities.

Nuclear receptor DNA recognition sites contain consensus hexameric repeat motifs (AGAACA or AGGTCA) that can be organized as direct (DR), everted (ER), or inverted (IR) repeats and are spaced by a defined number of nucleotides (24, 25). Using a weighted matrix-based computational approach (26), no obvious binding sites for FXR or LXR could be identified. However, a highly conserved binding site arranged as a direct hexanucleotide repeat separated by a single base (DR1 motif, AGGCCAgAGGTCA) was found at position 1565 to 1577 of the human ASBT promoter sequence (Fig. 3). No comparable binding site was detected in the rat ASBT promoter (9). The DR1 motif has been shown to bind the PPAR/RXR heterodimer (27-29). Typical ligands for PPAR include fatty acids, eicosanoids, hypolipidemic fibrate drugs, and a broad range of synthetic ligands such as WY14643 (21, 30, 31).

To investigate whether the DR1 motif in the ASBT gene promoter is a functional PPAR response element, we transfected Caco2 cells with three ASBT luciferase constructs and with the promoterless pGL3-Basic vector. ASBT promoter activity was assayed in the presence or absence of the PPAR ligands, WY14643 and ciprofibrate, and the RXR ligand, 9cRA. Because Caco2 cells have low endogenous PPAR and RXR expression levels, the presence of 10 µM WY14643 and 1 µM 9cRA had no effect on ASBT promoter activity compared with Me₂SO-treated controls (Fig. 3B). Cotransfection of human PPAR and RXR expression plasmids increased luciferase activity of the 1688/UTR-Luc construct 2.5-fold. This effect was enhanced by the addition of PPAR ligands. Treatment with 10 µM WY14643/1 µM 9cRA led to a further 1.9-fold induction and treatment with 30 µM ciprofibrate/1 µM 9cRA to a 2.2-fold induction over Me₂SO-treated controls. No comparable effect was observed with the 1492/UTR-Luc, UTR-Luc, or pGL3-Basic vectors, suggesting a functional role of the DR1 motif at nt 1565 to 1577 as a PPAR response element.

The DR1 Motif Confers Activation by PPAR Ligands-- To further confirm the role of the DR1 motif in activation by PPAR ligands, the DR1 element was cloned in front of the thymidine kinase (TK) promoter of the luciferase reporter gene vector, TK-Luc (Fig. 4). The resulting reporter plasmid, DR1-TK-Luc, contained a single copy of the DR1 binding site as present in the sequence of the human ASBT gene. The mutDR1-TK-Luc reporter plasmid contained targeted mutations within the DR1 motif. In PPAR/RXR cotransfected Caco2 cells, all TK plasmids had similar basal luciferase activities attributable to the TK promoter (Fig. 4). Treatment with 9cRA and WY14643 (10 µM) or ciprofibrate (30 µM) resulted in a ~1.5-fold induction of DR1-TK-Luc, whereas native TK-Luc and mutDR1-TK-Luc showed decreased activity in the presence of ligands. These data confirmed the functional role of the DR1 motif as a PPAR response element.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Role of the DR1 binding site in PPAR activation in a heterologous promoter context. Caco2 cells were transfected with expression plasmids for PPAR and RXR and either the TK-Luc plasmid, the DR1-TK-Luc plasmid containing the wild-type DR1 element in front of the TK promoter, or the mutDR1-TK-Luc plasmid containing targeted mutations within the DR1 element (bold letters). Treatment with 1 µM 9cRA and WY14643 (10 µM) or ciprofibrate (30 µM) resulted in a 1.4-1.55-fold induction of DR1-TK-Luc, whereas TK-Luc-associated luciferase activity was reduced. Mutation of the DR1 site in the mutDR1-TK-Luc construct resulted in a loss of inducibility by PPAR ligands. Promoter activity of each construct is expressed in relation to values obtained in dimethyl sulfoxide Me2SO (DMSO)-treated controls.

To assess the importance of the DR1 element for PPAR-dependent activation of the ASBT promoter, targeted mutations were introduced into the DR1 sequence of the 1688/UTR-Luc construct by site-directed mutagenesis, resulting in the reporter plasmid mutDR1-1688/UTR-Luc. Whereas activation of the wild-type 1688/UTR-Luc construct and even of the 1688-Luc construct in transfected Caco2 cells was 2-fold in the presence of 9cRA and WY14643 or ciprofibrate, inducibility of the ASBT promoter was completely abolished in mutDR1-1688/UTR-Luc transfected cells (Fig. 5). Furthermore, luciferase activity in the absence of ligand was also significantly reduced (30%) compared with the wild-type 1688/UTR-Luc construct, suggesting that the DR1 element is operative in basal ASBT promoter function in Caco2 cells.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Mutagenesis of the DR1 element results in a loss of ASBT promoter activation by PPAR ligands. Caco2 cells were transfected with PPAR and RXR expression plasmids and with the indicated ASBT reporter gene constructs. 1688-Luc and 1688/UTR-Luc contained the wild-type ASBT gene sequence, whereas the mutDR1-1688/UTR-Luc construct contained targeted mutations within the DR1 element. Mutation of the DR1 site resulted in reduced overall promoter activity and a loss of inducibility by WY14643 (10 µM) or ciprofibrate (30 µM).

The DR1 Element in the ASBT Gene Binds the PPAR/RXR Heterodimer-- To determine whether the PPAR/RXR heterodimer indeed binds to the DR1 element in the ASBT gene promoter, electrophoretic mobility shift assays were performed. A ³²P-labeled dimerized oligonucleotide corresponding to the DR1 element of the ASBT gene (DR1 in Table I) was incubated with in vitro translated HNF1 (control), monomeric PPAR or RXR, or with the PPAR/RXR heterodimer. Whereas neither HNF1 nor monomeric PPAR and RXR protein were able to bind to the DR1 element, incubation with heterodimeric PPAR/RXR resulted in the formation of a DNA-protein complex (lower arrow in Fig. 6). The addition of specific antibodies against RXR or PPAR in the presence of the PPAR and RXR proteins produced single supershifted complexes (top and middle arrow, respectively, in Fig. 6). Furthermore, DNA-protein complex formation was inhibited by the addition of an excess of unlabeled DR1 oligonucleotide but was unaffected by the presence of excess mutDR1 oligonucleotide (Table I) with a mutated DR1 element (Fig. 6). These results confirmed the specificity of binding of heterodimeric PPAR/RXR to the DR1 motif in the human ASBT gene promoter.

View larger version (47K): [in this window] [in a new window]   Fig. 6.   The DR1 element in the ASBT promoter binds PPAR/RXR. Electrophoretic mobility shift assays were performed using a 32P-labeled DR1 oligonucleotide (Table I) that contained the wild-type DR1 binding site. The addition of in vitro translated HNF1 (as a negative control), RXR, or PPAR did not produce a visible complex. The addition of PPAR and RXR in combination resulted in the binding of heterodimeric PPAR/RXR (lower arrow). The addition of antibodies targeted against RXR or PPAR resulted in a supershift (top and middle arrow, respectively) and disappearance of the lower DNA-protein complex. Competition experiments using a 500-fold excess of unlabeled DR1 oligonucleotide (wt) inhibited binding of PPAR/RXR to the 32P-labeled DR1 oligonucleotide, whereas a 500-fold excess of mutDR1 oligonucleotide (mut) with a mutated DR1 recognition site (Table I) had no effect.

Quantitation of Endogenous ASBT Expression in SK-ChA Cholangiocytes-- Having established that the ASBT promoter binds PPAR/RXR and is transactivated by PPAR ligands, we tested whether endogenous ASBT expression is induced in response to ciprofibrate in a human cell line. SK-ChA cholangiocytes in passage 77-79 were treated with Me₂SO or 30 µM ciprofibrate/1 µM 9cRA. Subsequently, endogenous ASBT mRNA levels were quantitated by real-time PCR. Because a validation experiment showed that amplification efficiencies of target (ASBT) and reference (18 S RNA) were approximately equal, relative quantitation was performed with the comparative C_T method (32, 33). Ciprofibrate treatment of SK-ChA cholangiocytes resulted in a marked induction of the ASBT mRNA, whereas Me₂SO-treated control cells did not exhibit detectable ASBT mRNA (Table III). These data showed that binding of PPAR/RXR to the ASBT promoter is physiologically relevant and confers induction of endogenous ASBT mRNA levels in human cholangiocytes by the PPAR ligand ciprofibrate.

View this table: [in this window] [in a new window]   Table III Induction of ASBT expression in SK-ChA cholangiocytes by ciprofibrate SK-ChA cells in passage 77-79 were treated with Me2SO/EtOH as a control, or with 30 µM ciprofibrate and 1 µM 9cRA, for 24 h. 1 µg of total RNA from either Me2SO/EtOH- or ciprofibrate-treated cells was reverse-transcribed and used for relative quantitation of gene expression. Amplification of endogenous 18 S ribosomal RNA was performed to standardize the amount of ASBT mRNA. For relative quantitation, the comparative CT method was used. CT is the threshold cycle for target amplification; CT represents the difference in threshold cycles for target and reference. The amount of target (ASBT) mRNA, normalized for the endogenous reference and measured in relation to the calibrator (Me2SO/EtOH-treated cells), was calculated as 2CT.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study reports the characterization of the promoter region of the human ASBT (SLC10A2) gene and the identification of three binding sites for HNF1 and a binding site for the peroxisome proliferator-activated receptor . The region from nt 1565 to 1577 relative to the transcription initiation site contains a direct hexanucleotide repeat, a so-called DR1 element, with hexameric repeat motifs characteristic of the binding sites for nuclear receptors (Fig. 3) (24, 25). The DR1 motif has been shown to bind the nuclear receptor PPAR (27-29). This study is the first to show that PPAR, which has been established as a regulator of fatty acid catabolism, hepatic bile salt synthesis, and inflammation control (21, 31), also regulates a bile salt transporter gene.

Initial transfection experiments using ASBT reporter gene constructs indicated that promoter activity was strongest in Caco2 cells that endogenously express the ASBT mRNA (Fig. 1). Significant luciferase activity was conferred by constructs that extended downstream to nt +526 of the 5'-untranslated region, whereas constructs containing only 5'-flanking sequence upstream of nt +1 lacked relevant reporter activity (Fig. 1). Sequence analysis revealed the presence of three HNF1 binding sites within the 5'-UTR (Fig. 2, Table II). The site spanning nt 253-267 has previously been shown to bind and mediate responsiveness to HNF1 (8). The current study extends the previous findings and demonstrates the functional importance of the other two HNF1 sites in cotransfection assays. The data shown in Fig. 2 explain the reported observations that ASBT promoter activity is strongly activated by HNF1 and that ASBT gene expression is absent in Tcf1^/ (HNF1^/) mice (8). ASBT thus represents the third bile salt transporter gene, next to the liver-specific Na⁺-taurocholate cotransporting polypeptide (Ntcp/Slc10a1) and OATP-C (SLC21A6) genes, which are critically dependent upon HNF1 for basal promoter function (8, 34, 35). A potential clinical implication of ASBT activation by HNF1 concerns the reduction in ASBT expression that occurs during intestinal inflammation (10). In the liver, cytokines have been shown to decrease HNF1 expression (36, 37). If HNF1 expression in the intestine is similarly affected by cytokines in inflammatory bowel disorders, decreased nuclear HNF1 levels could be the cause of reduced ASBT expression.

To study whether the ASBT gene is regulated by nuclear receptors involved in bile salt and lipid metabolism, chicken hepatoma LMH cells, known to endogenously express nuclear receptors at sufficient levels to confer induction of target genes by the appropriate ligands (22, 23), were transfected with chimeric ASBT promoter constructs. Significant activation of the ASBT promoter was conferred by ciprofibrate (Fig. 3A), suggesting that the ASBT gene could be regulated by PPAR. We concluded that the response element was localized within the 5'-flanking region of the ASBT gene, because a construct that contained only the 5'-untranslated region was not responsive to ciprofibrate. We next investigated whether the DR1 motif at nt 1565 to 1577 is the response element that confers inducibility by PPAR ligands. Caco2 cells transfected with ASBT promoter constructs were cotransfected with PPAR and RXR expression plasmids and exposed to specific PPAR ligands. The ASBT promoter construct that contained the DR1 motif was induced ~2.4-fold by coexpression of PPAR/RXR and up to 5.5-fold by the addition of the PPAR ligands WY14643 or ciprofibrate and the RXR ligand 9cRA (Fig. 3B). In a heterologous promoter context the DR1 motif enhanced reporter gene activity from a thymidine kinase promoter luciferase construct in the presence of PPAR ligands, an effect that was abolished by mutagenesis of the DR1 motif (Fig. 4). Accordingly, in the full-length ASBT promoter construct, the induction of luciferase activity by WY14643 and ciprofibrate in combination with 9cRA was abolished by targeted mutagenesis of the DR1 motif (Fig. 5). Direct binding of PPAR/RXR to the DR1 element was confirmed in electrophoretic mobility shift assays (Fig. 6). Finally, induction of endogenous ASBT gene expression by ciprofibrate was shown in cultured SK-ChA cholangiocytes by real-time PCR (Table III), suggesting a physiological role of the PPAR pathway for ASBT gene regulation. Collectively, the data provide conclusive evidence that the human ASBT gene binds and is transactivated by PPAR.

The implications of ASBT activation by PPAR, which is expressed in the liver, renal proximal tubular cells, small and large intestine, and heart and skeletal muscle (30, 38, 39), should be considered in the light of recent experimental data that have revealed a central role for PPAR not only in fatty acid but also in bile salt metabolism. First, two key enzymes in bile salt biosynthesis, CYP7A1 and sterol 27-hydroxylase, are down-regulated transcriptionally and post-transcriptionally, respectively, by PPAR (40, 41). In vivo studies in human subjects show reduced 7-hydroxylation rates during treatment with fibrates (42). Second, the murine and rat sterol 12-hydroxylase gene, a branch-point enzyme in the bile salt biosynthetic pathway that determines the ratio of cholic acid to CDCA, is transcriptionally activated through direct binding of PPAR to an imperfect DR1 sequence in the promoter region. Accordingly, treatment of wild-type mice with WY14643 for 1 week resulted in an increased relative amount of CA, an effect that was abolished in PPAR null mice (43). Third, the inducing effects of PPAR ligands on target genes encoding enzymes of fatty acid -oxidation appear to be antagonized in mice by concomitant feeding of a bile salt-enriched diet, suggesting an influence of bile salts on PPAR-dependent gene regulation (44). The current study now adds the first bile salt transporter gene to the spectrum of target genes involved in bile salt homeostasis that are regulated by PPAR. A coordinate regulation of bile salt synthetic and transporter genes has so far been shown only for FXR-controlled genes including CYP7A1, the hepatocellular bile salt efflux pump, BSEP, I-BABP, and the hepatocellular organic anion uptake system, OATP8 (1, 2, 16, 17). This study introduces PPAR as a novel signaling mechanism in the coordinate regulation of bile salt biosynthetic and transport pathways.

A key function of PPAR is the regulation of genes involved in various steps of fatty acid metabolism (30). An important class of PPAR ligands are fibrate drugs, such as ciprofibrate, that effectively lower serum triglyceride levels by up-regulating genes involved in cellular uptake and -oxidation of fatty acids. It is thus of particular interest in the context of this study that a major determinant of serum triglyceride levels appears to be the rate of ileal bile salt absorption. Thus, patients with type IV hypertriglyceridemia exhibit decreased intestinal bile salt absorption compared with healthy controls, and ileal expression of ASBT mRNA and protein in these patients is reduced by 32 and 53%, respectively (13, 45). Although the cause-and-effect relationship of this association has not been clarified, the results of the current study open the possibility that decreased activity of PPAR could be the cause of both hypertriglyceridemia and decreased ileal bile salt absorption. The flux of bile salts through the enterohepatic circulation appears to have a direct effect on hepatic VLDL synthesis because patients administered CDCA exhibit decreased serum triglyceride concentrations (46). Conversely, interruption of the enterohepatic circulation of bile salts with cholestyramine transiently increases serum VLDL-triglyceride levels (47-49). One study investigated whether sequence polymorphisms of the ASBT gene are more prevalent in hypertriglyceridemic patients than in healthy controls, but this could not be confirmed (15). The authors postulated that the combined phenotype of hypertriglyceridemia and decreased ileal bile salt absorption results not from ASBT gene mutations but from a defect in a single regulatory factor influencing intestinal bile salt absorption, hepatic bile salt synthesis, and hepatic triglyceride metabolism. This factor was proposed to be FXR. The results of this study now support a role for PPAR as a global regulator of these metabolic cascades.

The suppression of hepatic bile salt synthesis is the presumed mechanism of fibrate induced decreased biliary bile salt excretion and consequently increased biliary cholesterol saturation with the risk of cholesterol gallstone formation (41). Because the adverse changes in biliary lipid composition have been measured in gallbladder or T-tube bile, it is conceivable that not only hepatocellular bile formation but also cholangiocellular modification of primary hepatic bile is affected by fibrate drugs. ASBT is expressed at the luminal surface of cholangiocytes, where it mediates reabsorption of bile salts from primary hepatic bile. Increased expression and function of ASBT secondary to fibrate-induced PPAR activation could increase cholangiocellular bile salt absorption, thereby contributing to the decreased biliary excretion of bile salts.

One final implication of PPAR-mediated ASBT induction deserves consideration. PPARs play an important role in inflammation control due to their activation by lipid-derived inflammatory mediators (31). This feature has led to the evaluation of PPAR ligands such as the thiazolidinedione derivatives as a treatment option in inflammatory bowel disease (IBD) (50). Patients with IBD have increased fecal excretion of bile salts and decreased absorption of the orally ingested radiolabeled bile acid ⁷⁵Se-homotaurocholic acid (51). Simultaneous activation of PPAR by novel therapeutic PPAR ligands in IBD could correct the bile salt absorptive defect by inducing ASBT expression. Reduced spillover of bile salts into the colon would not only ameliorate the chologenic component of diarrhea in these patients but could also reduce the carcinogenic risk conferred by increased exposure of the colonic mucosa to bile salts (52).

In summary, this study shows that the human ASBT gene is critically dependent upon HNF1 for base-line promoter function and that promoter activity is induced by the PPAR ligands WY14643 and ciprofibrate through direct binding of the PPAR/RXR heterodimer to a DR1 motif. Regulation of the major human intestinal bile salt uptake system by PPAR has major implications for the interplay between bile salt homeostasis, lipid metabolism, and inflammation control. The central role of PPAR in linking these cascades should be considered in the development of treatment strategies for lipid and inflammatory disorders associated with alterations in ASBT function and expression.

	ACKNOWLEDGEMENT

The technical assistance of Claudia Seitz is gratefully acknowledged.

	FOOTNOTES

^* This work was supported by Grants 32-59155.99 and 632-062773 from the Swiss National Science Foundation, Bern, Switzerland.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.

To whom correspondence should be addressed: Division of Gastroenterology and Hepatology, University Hospital, CH-8091 Zurich, Switzerland. Tel.: 41-1-255-4097; Fax: 41-1-255-4598; E-mail: gerd.kullak@dim.usz.ch.

Published, JBC Papers in Press, June 7, 2002, DOI 10.1074/jbc.M203511200

	ABBREVIATIONS

The abbreviations used are: I-BABP, ileal bile acid-binding protein; CYP7A1, cholesterol 7-hydroxylase; FXR, farnesoid X receptor; ASBT, apical sodium-dependent bile salt transporter; Slc/SLC, rodent/human solute carrier gene family; CDCA, chenodeoxycholate; HNF, hepatocyte nuclear factor; VLDL, very low density lipoprotein; LXR, liver X receptor; PPAR, peroxisome proliferator-activated receptor; UTR, untranslated region; Caco2 cells, colon carcinoma-derived cells; HEK293 cells, human embryonic kidney-derived cells; SK-ChA cells, human cholangiocyte-derived cells; 9cRA, 9-cis-retinoic acid; WY14643, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid; LMH cells, chicken hepatoma cells; RXR, 9-cis-retinoic acid receptor; TK, thymidine kinase; OATP, organic anion transporting polypeptide; nt, nucleotide(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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