Am J Physiol Gastrointest Liver Physiol 290: G912-G922, 2006.
First published December 15, 2005; doi:10.1152/ajpgi.00479.2005
0193-1857/06 $8.00
LIVER AND BILIARY TRACT
Regulation of the mouse organic solute transporter 
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, Ost-Ost, by bile acids
Tamara Frankenberg,1
Anuradha Rao,2
Frank Chen,1
Jamie Haywood,2
Benjamin L. Shneider,1 and
Paul A. Dawson2
1Department of Pediatrics, Mount Sinai School of Medicine, New York, New York; and 2Department of Internal Medicine and Center for Lipid Sciences, Wake Forest University School of Medicine, Winston-Salem, North Carolina
Submitted 11 October 2005
; accepted in final form 9 December 2005
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ABSTRACT
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The mechanisms responsible for bile acid regulation of mouse intestinal organic solute transporter 
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(Ost-Ost) expression were investigated. Expression of Ost-Ost mRNA was increased in cecum and proximal colon of cholic acid-fed mice and in chenodeoxycholate-treated mouse CT26 colon adenocarcinoma cells. Sequence analysis revealed potential cis-acting elements for farnesoid X receptor (FXR) and liver receptor homolog-1 (LRH-1) in the mouse Ost and Ost promoters and reporter constructs containing Ost and Ost 5'-flanking sequences were positively regulated by bile acids. Expression of a dominant-negative FXR, reduction of FXR with interfering small RNA (siRNA), or mutation of the potential FXR elements decreased Ost and Ost promoter activity and abolished the induction by chenodeoxycolic acid. Negative regulation of the Ost and Ost promoters by bile acids was mediated through LRH-1 elements. Ost and Ost promoter activities were increased by coexpression of LRH-1 and decreased by coexpression of SHP. Mutation of the potential LRH-1 elements and siRNA-mediated reduction of LRH-1 expression decreased basal promoter activity. As predicted from the promoter analyses, ileal Ost and Ost mRNA expressions were increased in wild-type mice administered the FXR agonist GW4064 and decreased in FXR-null mice. Immunoblotting analysis revealed that Ost and Ost intestinal protein expressions correlated with mRNA expression. The mouse Ost and Ost promoters are unusual in that they contain functional FXR and LRH elements, which mediate, respectively, positive and negative feedback regulation by bile acids. Although the positive regulatory pathway appears to be dominant, this arrangement provides a mechanism to finely titrate Ost-Ost expression to the bile acid flux.
intestine; ileum; cecum; farnesoid X receptor; liver receptor homolog-1; small heterodimer partner; dynamic regulation
BILE ACIDS ARE SYNTHESIZED from cholesterol in the liver and secreted into the small intestine where they facilitate the absorption of dietary lipids and fat-soluble vitamins. Most bile acids are reabsorbed from the intestine, returned to the liver via the portal venous circulation, and resecreted into bile (29). The major mechanism for intestinal absorption is active transport in the distal ileum by the well-characterized apical sodium bile acid cotransporter (Asbt, gene name: Slc10a2) (3, 4). Loss-of-function mutations in the human Asbt gene are associated with intestinal bile acid malabsorption (24), and targeted deletion of the Abst gene eliminates enterohepatic cycling of bile acids in mice (8). The regulation of Abst expression by intestinal bile acid flux has been studied in a variety of animal models, including guinea pigs, rabbits, rats, and mice. Previous studies in C57BL/6 mice and New Zealand White rabbits have shown that Abst expression is negatively regulated by bile acids (6, 18). In these species, the Abst promoter harbors a liver receptor homolog-1 (LRH-1) element and negative regulation by bile acids is mediated through the farnesoid X receptor (FXR)-SHP-LRH-1 pathway.
In contrast to apical transport, there is limited information regarding the regulation of ileal basolateral bile acid transport. Recently, the organic solute transporter - (Ost-Ost) was identified as a potential ileal basolateral bile acid transporter based in part on their hypothesized regulation by bile acids (9, 28). Ost-Ost is a heteromeric solute transporter that includes a 340-amino acid seven-potential transmembrane domain protein (Ost) and a 128-amino acid single transmembrane ancillary polypeptide (Ost) (9, 28, 33). Although the functions of the individual subunits have not yet been elucidated, coexpressions of both Ost and Ost are essential for delivery of the individual proteins to the plasma membrane (9). Ost and Ost mRNA expressions closely parallel one another in mouse tissues, and Ost and Ost mRNAs are expressed at particularly high levels in terminal ileum. Beside their tissue distribution, little is known about the regulation of Ost and Ost expression. In Asbt-null mice that exhibit significant intestinal bile acid malabsorption, expression of Ost and Ost mRNA was decreased in ileum and induced in cecum, presumably in response to changes in the bile acid flux through those tissues (9). The present study was designed to elucidate potential mechanisms responsible for the regulation of mouse Ost-Ost by bile acids. The results demonstrate that Ost-Ost expression is both positively and negatively regulated by bile acids. The mouse Ost and Ost promoters harbor both FXR and LRH-1 elements that mediate, respectively, this positive and negative feedback regulation. Whereas the negative regulatory pathway appears to be subordinated to the positive feedback by bile acids, the presence of both positive and negative elements provides a mechanism for dynamic fine regulation of Ost-Ost expression.
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MATERIALS AND METHODS
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Animals and diets.
The mice were housed in our American Association for the Accreditation of Laboratory Animal Care-approved animal facility. The Wake Forest University Institutional Animal Care and Use Committee approved all animal procedures. Male C57BL/6J mice were obtained from the Jackson Laboratory, fed ad libitum, and group housed in plastic colony cages in a temperature-controlled room (22°C) with 12:12-h light-dark cycling. The animals were maintained on a basal diet containing 16% fat, 20% protein, 64% carbohydrate (% of calories), and 0.017 mg/calorie of cholesterol (0.006%) (8). For the diet studies, male mice (12 wk of age) were fed the basal diet supplemented with 0.2% (wt/wt) cholic acid (CA) (Sigma, St. Louis, MO) for 14 days.
Cloning of the mouse Ost and Ost promoters and preparation of promoter constructs.
Oligonucleotide primers based on the mouse Ost and Ost cDNA sequences were used to screen a mouse (strain 129S6/SvEvTac) PAC library (RPCI-21; Invitrogen). Three genomic clones each for Ost and Ost were identified and mapped by Southern blotting and partial DNA sequencing. Mouse Ost and Ost gene sequences were obtained from GenBank. The putative transcription start sites were assigned with the sequences of the most 5' expressed sequence tag (EST) clones for mouse Ost (BY075854
[GenBank]
.1) and Ost (BY704432
[GenBank]
.1). For the Ost transcription studies, a 1.7-kb XbaI-HindIII fragment encompassing nucleotides –1564 and +183 (relative to the Ost putative transcription start site) was subcloned from the PAC genomic clone into pBluescript II KS and sequenced using an Applied Biosystems model 3100 instrument. The pBluescript clone was then used as template for PCR. The forward primer 5'-GCGCGCCTCGAGTCTAGATGTGGAGCCTTGATG-3' inserted an XhoI site next to the 5' XbaI site. The reverse primer 5'-GCGCGCAAGCTTTTTATCTCAGAGTTCACCCCT-3' utilized the existing 3' HindIII site. This 1.7-kb Ost promoter fragment was then subcloned into the XhoI and HindIII sites of pGL3 basic (Promega, Madison, WI). This construct was designated as pGL3–1.7Ost (see Fig. 3A). A second Ost 1.2-kb promoter construct encompassing nucleotides –1081 to +183 was constructed by digestion of pGL3–1.7Ost with SmaI and HindIII and subcloning the promoter fragment into pGL3 basic. This construct was designated as pGL3–1.2Ost (see Fig. 3A).
For the Ost transcription studies, a 3.0-kb BamHI/ApaI fragment encompassing nucleotides –2998 to +65 was PCR-amplified with the PAC genomic clone as template. The forward and reverse primers are 5'-TGCAGAGGATCCAGGTTCTGTTCC-3' and 5'-ACC AGCAGGGGCCCATTTGG-3', respectively. A similar strategy was also used to PCR-amplify a 0.9-kb fragment that encompassed SacI sites at positions –824 and +81 (relative to the Ost putative transcription start site); the following forward and reverse primers were used: 5'-CTTGGGGATCGAGCTCCTGT-3' and 5'-ATCCACCTACAGAGCTCACACC-3'. The PCR products were digested with the indicated restriction enzymes and subcloned into pBluescript II KS. After the inserts were sequenced, the 3.0-kb and 0.9-kb fragments were subcloned into the SmaI and SacI sites of pGL3 basic, respectively. These constructs are designated as pGL3–3.0Ost and pGL3–0.9Ost (see Fig. 3B). Point mutations in the FXR element (FXRE) and LRH-1 sequences of the 1.7-kb Ost and the 0.9-kb Ost promoter fragments were generated with the Quick-Change mutagenesis kit (Strategene) and verified by DNA sequencing. The constructs are denoted as follows: pGL3–1.7Ost µFXRE has mutations in the potential FXRE at nucleotide position –1221. The forward and reverse primers used to mutate this site are 5'-GGTGTGGCTGAGATCACTGCCCTTCAGCAG-3' and 5'-GCTGCTGAAGGGCAGTGATCTCAGCCACACCCCA-3', respectively. The pGL3–1.7Ost µLRH construct has mutations in the potential LRH site at nucleotide position +55. The following forward and reverse primers were used to generate this mutant: 5'-GGGTAGTTCAGCTAAAAGGTGGTGATCAACCGC-3' and 5'-CCCATCAAGTCGATTTTCCACCACTAGTTGGCG-3'. pGL3–0.9Ost µFXRE has mutations in the potential FXRE at nucleotide position –5, and the following forward and reverse primers were used to generate this mutant: 5'-GGTCATGCATTCGTGGGAACATTCTTCCTGGACTGGCAGC-3 and 5'-GCTGCCAGTCCAGGAAGAATGTTCCCACGAATGCATGACC-3'. pGL3–0.9Ost µLRH-240 has mutations in the potential LRH site at nucleotide position –240, and the following forward and reverse mutagenic primers were used to generate this mutant: 5'-GATCGCACAATCTCCAGCTTCAATCGTCTGACTTTATGGTTCC-3' and 5'-GGAACCATAAAGTCAGACGATTGAAGCTGGAGATTGTGCGATC-3'. pGL3–0.9Ost µLRH-393 has mutations in the potential LRH site at nucleotide position –393, and the following forward and reverse primers were used to generate the mutant: 5'-CCACTTTTTAAGTCAAAGAGGACGATTGACTTGGCACTGACCC-3' and 5'-GGGTCAGTGCCAAGTCAATCGTCCTCTTTGACTTAAAAAGTGG-3'. pGL3–0.9Ost doubleµLRH has mutations in both potential LRH sites at nucleotide positions –240 and –393. Nucleotides that were mutated are underlined. The oligonucleotide primers were all obtained from Integrated DNA Technologies.
Cell culture, transient transfections, and luciferase assays.
The mouse colon carcinoma cell line CT26 (CRL-2638; American Type Culture Collection, Rockville, MD) was grown and maintained in RPMI 1640 medium, which was supplemented with 10% FCS. To minimize interference from serum bile acids, the cell medium was changed to RPMI 1640 plus 0.5% charcoal-treated FCS when cells were treated with exogenous chenodeoxycolic acid (CDCA) (Sigma). For Northern blot analysis of endogenous RNA expression, CT26 cells were treated for 40 h with vehicle control (DMSO), pRNA-siFIC1, or 100 µM sodium CDCA. Transient transfection of the CT26 cells was carried out as described previously (6). Briefly, confluent cells (5 x 106) were harvested and resuspended in 700 µl of PBS containing 4 µg of mouse Ost- or Ost-luciferase reporter constructs and 0.1 µg of a control plasmid (pRL-TK) encoding the Renilla luciferase gene under the control of the thymidine kinase promoter (Promega). The cells were transfected by electroporation at 0.22 kV and 0.95 µF x 1,000 (Bio-Rad). After electroporation, the cells were resuspended in the medium and cultured for 40 h. Luciferase activities were determined by the dual luciferase reporter assay system (Promega) according to manufacturer’s instructions, using a Turner 20/20n Luminometer (Turner BioSystems) with a 10-s counting window. All transfections were performed in triplicate and repeated in two separate sets of experiments (i.e., 6 individual data points for each experimental transient transfection). After transfection, cells were cultured for 40 h at 37°C and in 5% CO2 before they were harvested for reporter gene assays.
To study the Ost and Ost promoter activities in the presence of exogenous FXR, SHP, or LRH, the Ost promoter constructs were cotransfected with expression plasmid constructs pCMX-rFXR (10), pCMX-mSHP (20) (gift from Dr. David Mangelsdorf, University of Texas, Southwestern Medical Center, Dallas, TX), or pcDNA3.1/myc-LRH (21) (gift from Dr. Alan R. Tall, Columbia University, New York, NY). Additional cotransfections were performed with an FXR-dominant negative plasmid, pCMX-hFXR-w469A (FXRdn), which encodes a mutant FXR protein that binds to the FXR cis-element but does not activate transcription (2). To knock down the endogenous expression of SHP, FIC1, LRH, and FXR, cells were transfected with the following interfering small RNA (siRNA) expression constructs: pRNA-siSHP, 5'-GAUUCUGCUGGAGGAGCCC-3' (23) (Orbigen, San Diego, CA); pRNA-siFIC1, 5'-GUGAGGUUGUUCGUGGUAC-3' (5); pRNA-siLRH, 5'-GCCAAUGGACUUAAGCUGGAA-3'; and pRNA-siFXR, 5'-UAGAUGCCAGGAGAAUACCAG-3' (GenScript). Scrambled RNA antisense constructs were used as controls. One microgram of each siRNA construct was cotransfected along with pRL-TK and the respective Ost reporter construct.
Analysis of RNA expression.
Total RNA was extracted from cells or frozen tissue from individual mice using TRIzol Reagent (Invitrogen) as suggested by the manufacturer. For Northern blot analysis, total RNA from cells (20 µg) or mouse tissues (10 µg) was fractionated on 1.2% (wt/vol) agarose gels containing 2.2 M formaldehyde and transferred to Nytran (0.45 µm; Schleicher & Schuell). The CT26 cell RNA blots were hybridized with 32P-labeled DNA probes labeled by nick translation, and the tissue RNA blots were hybridized with 32P-labeled random hexamer-primed DNA probes. Expression levels were quantified with a Typhoon 8600 PhosphorImager (Molecular Dynamics). The mouse probes for Ost, Ost, Asbt, Ibabp, SHP, Fic1, and -actin have been described previously (8, 9). The 28S rRNA (3) and -actin RNA levels were used as load controls for CT26 and tissue Northern blots, respectively. The PhosphorImager signal for the 28S rRNA loading is linear over more than a 100-fold range, and the RNA measurements were performed in the linear range for this assay (data not shown).
Real-time PCR analysis was carried out with intestinal RNA isolated from wild-type, FXR-null (31), and SHP-null mice (15). The mice were 3–4 mo of age and maintained on mouse chow (Teklad 7001). These studies included ileal RNA from male mixed-strain (C57BL/6–129/OlaHsd) mice treated by daily oral gavage of vehicle (1% Tween 80, 1% methylcellulose) or the FXR agonist GW-4064 (100 mg/kg body wt) for 5 days, ileal RNA from male mixed-strain (C57BL/6–129/OlaHsd) SHP-null mice and wild-type litter-mate controls (a kind gift of Dr. Steve Kliewer, Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX), and RNA from the distal third of the small intestine of male mixed-strain (C57BL/6–129/SVJ-FVB) FXR-null mice and wild-type litter-mate controls (a kind gift from Dr. Joyce Repa, Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX). Real-time PCR analysis of Ost and Ost expression was performed as described previously (9). The following primer sequences were also used for the real-time PCR analysis: SHP, 5'-CAGCGCTGCCTGGAGTCT-3' and 5'-AGGATCGTGCCCTTCAGGTA-3'; Ibabp, 5'-CAAGGCTACCGTGAAGATGGA-3' and 5'-CCCACGACCTCCGAAGTCT-3'; cyclophilin, 5'-TGGAGAGCACCAAGACAGACA-3' and 5'-TGCCGGAGTCGACAATGAT-3'. The SHP and Ibabp primers were used at a concentration of 500 nM, whereas cyclophilin primers were used at 700 nM.
Analysis of Ost protein expression.
For mouse Ost, a synthetic peptide corresponding to amino acids 315–329, MYYRRKDDKVGYEAC, was synthesized, coupled via the COOH-terminal cysteine residue to keyhole limpet hemocyanin using sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (1), and used to immunize New Zealand White rabbits (AnaSpec). The cDNA coding for the COOH-terminal 28 amino acids of mouse Ost was PCR amplified, subcloned into pGEX 3x (Amersham Biosciences), and sequenced. The glutathione S-transferase (GST)-Ost fusion protein was purified from E. coli (BL21) cytosol by glutathione affinity chromatography and coupled to agarose beads (Amino-Link immobilization kit; Pierce) according to the manufacturer’s instructions. The anti-Ost (rabbits 2729 and 2730) antibody was partially purified by ammonium sulfate precipitation and then isolated by affinity chromatography using the GST-Ost-coupled agarose. The affinity-purified antibodies were stored at –70°C in PBS containing 1 mg/ml of BSA (IgG-free, protease-free; Jackson ImmunoResearch) and subjected to only one freeze-thaw cycle. For mouse Ost, cDNA coding for Ost amino acids 54–128 was PCR amplified, subcloned into pGEX 3x (Amersham Biosciences), and sequenced. The GST-Ost fusion protein was purified from E. coli (BL21) cytosol by glutathione affinity chromatography. Two New Zealand White rabbits (rabbits 4053 and 4054) were immunized with 500 µg of the GST-Ost fusion protein in Freund’s complete adjuvant (Lampire Biological Laboratories, Pipersville, PA). The production bleeds were used without further purification.
For immunoblotting analysis of tissue extracts, intestinal segments were homogenized on ice in 25 mM Tris·HCl (pH 7.4), 300 mM NaCl, and 1% Triton X-100 in the presence of a protease inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, 10 µg/ml aprotonin, 10 µg/ml leupeptin, 10 mM EDTA) with a Polytron (model PT 1200C; Kinematia). After homogenization, samples were centrifuged twice at 10,000 g for 10 min each at 4°C, and the supernatant was aliquoted and stored at –70°C. Samples were diluted into laemmli sample buffer containing 50 mM Tris·HCl (pH 6.8), 4% SDS, 10 mM EDTA, 10% glycerol, 100 mM DTT, and 0.004% bromophenol blue, heated at 37°C for 5 min, and subjected to SDS-PAGE on 10% or 12% polyacrylamide gels. After transfer to nitrocellulose membranes, blots were blocked and incubated with antibody as described previously (9). The following dilutions of antibodies were used: anti-Ost or anti-Ost, 1:1,000; anti--actin, 1:10,000; horseradish peroxidase-conjugated anti-rabbit antibody (GE Healthcare), 1:5,000; and horseradish peroxidase-conjugated anti-mouse antibody (GE Healthcare), 1:10,000. Antibody binding was detected with an enhanced chemiluminescence technique (SuperSignal West Pico; Pierce). Pilot immunoblotting experiments were performed to determine the amount of ileal protein extract that falls in the linear range for quantitating Ost and Ost protein expression (see Fig. 1 in supplemental material) (supplemental data for this article may be found at http://ajpgi.physiology.org/cgi/content/full/00479.2005/DC1). The blots were also probed with mouse anti--actin antibody (Sigma; catalog number A5441) as a control for protein loading. Ost and Ost protein expression was quantified by scanning the X-ray films with an Alpha Innotec (San Leandro, CA) 5500 imaging system, and the expression data were normalized to the levels of -actin loading control.
Statistical analysis.
For the transfection and CT26 cell RNA studies, we performed statistical analyses with In-Stat software (Graph Pad Software, San Diego, CA). Unless otherwise stated, means were compared with the Turkey-Kramer multiple comparisons test. All values are means ± SD, and a value of P < 0.05 was considered statistically significant. For the mouse studies, data were evaluated for statistically significant differences with the two-tailed Student’s t-test assuming equal variance. The levels of significance for identified differences were determined according to the formulas provided with the statistical software (Statview, Mountain View, CA). Mean values ± SE are indicated. Differences were considered statistically significant at P < 0.05.
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RESULTS
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Bile acid response in the mouse and CT26 murine colon adenocarcinoma cells.
Male C57BL/6J mice were fed a synthetic diet containing 0.2% cholic acid for 14 days, and the expression of Ost and Ost mRNA was examined by Northern blotting. Bile acid feeding did not alter the overall gradient of expression for Ost and Ost along the cephalocaudal axis, and both mRNAs were expressed at highest levels in ileum (see Fig. 2 in supplemental material) (supplemental data for this article may be found at http://ajpgi.physiology.org/cgi/content/full/00479.2005/DC1). Because Ost-Ost expression was not significantly changed in the proximal small intestine and there was little expression of Ost mRNA in distal colon, subsequent studies focused on Ost-Ost expression in ileum (segment 5), cecum, and proximal colon. Analysis of RNA from individual animals by Northern blotting showed that Ost and Ost expression was not significantly increased in ileum but showed modest increases in cecum and proximal colon of the cholic acid-fed mice (Fig. 1). Across all mice in this study fed the control or cholic acid-containing diets, there was a significant correlation between Ost and Ost mRNA expression (r = 0.93) in ileum, cecum, and colon, underscoring the importance of coexpression of the heteromeric transporter’s subunits for activity (see Fig. 3 in supplemental material) (supplemental data for this article may be found at http://ajpgi.physiology.org/cgi/content/full/00479.2005/DC1).
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Fig. 2. Expression of Ost, Ost, SHP, and Asbt mRNA in CT26 mouse colon adenocarcinoma cells. A: total RNA was isolated from untreated CT26 cells, CT26 cells incubated with 100 µM chenodeoxycolic acid (CDCA), or CT26 cells transfected with an interfering small RNA to decrease expression of endogenous Fic1 (siFIC1). Ost, Ost, SHP, and Asbt mRNA expression levels were measured in triplicate using total RNA (20 µg) for Northern blot analysis. The level of 28S rRNA was used as a loading control. B: quantitation of Ost, Ost, SHP, and Asbt mRNA levels in CT26 cells. mRNA expression levels were quantitated using a PhosphorImager. CDCA treatment resulted in a statistically significant increase (P < 0.001) in Ost, Ost, and SHP mRNA expression and a significant decrease (P < 0.01) in Asbt mRNA expression compared with mRNA from untreated cells. Treatment with siFIC1 significantly decreased Ost, Ost, and SHP mRNA expression (P < 0.01) and significantly increased Asbt mRNA expression (P < 0.001) compared with mRNA from untreated cells. *Statistically significant differences from untreated CT26 cells. 28S rRNA expression was similar in all samples.
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Ibabp mRNA expression is upregulated by bile acids via FXR (12, 13, 19, 20) and was used as a positive control for the cholic acid feeding. Ibabp expression increased in the ileum, cecum, and proximal colon of cholic acid-fed mice (Fig. 1). The expression of Asbt mRNA is negatively regulated by bile acids (6, 18) and was used as an additional control for this study. Asbt mRNA expression significantly decreased in the ileum and proximal colon of cholic acid-fed mice (Fig. 1) but was only barely detectable in cecum by Northern blot analysis (data not shown). The expression of Asbt mRNA was also measured in ileum and cecum by use of the more sensitive method of real-time PCR, and results similar to the Northern blotting data were obtained with the RNA samples from the individual mice (normalized threshold values, means ± SE, n = 5 mice per group): Asbt, ileum: control 264 ± 9 vs. CA 138 ± 11; Asbt, cecum: control 53 ± 7 vs. CA 48 ± 8.
The CT26 murine colon adenocarcinoma cell line endogenously expresses Ost and Ost mRNA (Fig. 2). Treatment of these cells with 100 µM CDCA increased Ost, Ost, and SHP mRNA expression by 
300%, whereas Asbt mRNA expression decreased 65% (Fig. 2). These results are consistent with the hypothesis that the mRNA expression of mouse Ost-Ost is regulated by bile acids, potentially through the nuclear receptor FXR. The progressive familial intrahepatic cholestasis type 1-associated gene product, Fic1, is hypothesized to activate FXR by promoting posttranslational modifications and subsequent FXR nuclear translocation (5, 30). Therefore, siRNA-mediated reduction of mouse Fic1 (siFIC1) was also used to support the involvement of FXR in regulating Ost-Ost expression. Control experiments showed that transfection of the Fic1 siRNA, but not a scrambled Fic1 sequence siRNA, reduced endogenous Fic1 mRNA expression by more than 80% (data not shown). Transfection of the CT26 cells with the Fic1 siRNA resulted in an 70% decrease of Ost, Ost, and SHP mRNA levels and a 300% increase in Asbt mRNA expression.
Analysis of the mouse Ost promoter and identification of a functional FXRE.
The increases in Ost and Ost mRNA expression in vivo after bile acid feeding and in vitro after incubation with CDCA suggests that both genes are direct targets of FXR activation. Analysis of the mouse Ost and Ost promoters using the Web-based program MatInspector (http://www.genomatrix.de/) revealed several potential FXR as well as LRH-1 cis-elements (Fig. 3). Analysis of the Ost promoter region revealed potential FXRE sequences located 1221, 1285, and 1419 bp upstream of the putative transcription start site. Of these potential sites, only the FXRE at position –1221 was identical to the FXR consensus sequence 5'-AGGCAnTGACCT-3' site (Fig. 3C). In addition to the FXR binding sites, a potential LRH-1 cis-element is located at position +55 (Fig. 3A).
To determine the role of these elements in the regulation of Ost expression, the indicated promoter fragments were cloned into the luciferase reporter vector pGL3 basic. After transient transfection of pGL3–1.7Ost into CT26 cells and incubation with 100 µM CDCA for 40 h, expression of the luciferase reporter was increased 500% compared with basal activity in vehicle-treated cells (Table 1). Deletion of distal promoter sequences (–1081 to –1564; pGL3–1.2Ost), including the putative FXRE sequences, reduced the basal promoter activity by 70%, and incubation with CDCA further reduced expression of the luciferase reporter by 75% (Table 2). These results suggest that the bile acid induction of Ost promoter activity was mediated by the upstream FXR cis-acting elements and also that the potential LRH-1 element at +55 may be mediating a negative regulation by bile acids. To further investigate the role of the FXRE sequences in the Ost promoter, CT26 cells were cotransfected with pGL3–1.7Ost and an FXR expression plasmid, pCMX-rFXR. Overexpression of FXR did not affect Ost basal promoter activity but markedly enhanced the promoter activity by almost 20-fold when incubated in the presence of CDCA. Furthermore, cotransfection with an expression plasmid encoding a dominant-negative FXR mutant that blocks FXR activity (dnFXR) (2), siRNA-mediated reduction of endogenous FXR expression, or siRNA-mediated reduction of mouse Fic1 all decreased basal and CDCA-stimulated activity of the pGL3–1.7Ost promoter construct (Table 1). Control experiments showed that transient transfection of siFXR or siFic1 expression constructs, but not scrambled FXR or Fic1 sequence expression constructs, were effective in reducing endogenous levels of FXR protein or Fic1 mRNA in the CT26 cells (data not shown). The decreased promoter activity was not because of siRNA-mediated nonspecific effects because transfection of a scrambled FIC1 siRNA (scrFIC1) did not affect the pGL3–1.7Ost basal promoter activity or its induction in CDCA-treated CT26 cells (Table 1). Similar control experiments also showed that the scrambled siRNAs (scrFXR, scrLRH, and scrSHP) did not affect Ost promoter activity: (luciferase activity relative to Ost alone; means ± SD, n = 3) 1.7Ost alone, 100 ± 10%; 1.7Ost plus scrFXR, 112 ± 7%; 1.7Ost plus scrSHP, 115 ± 3%; and 1.7Ost plus scrLRH, 117 ± 5%.
The potential FXRE sequence at position –1221 in the mouse Ost promoter is identical to the FXR consensus, 5'-AGGCAnTGACCT-3' (Fig. 3C). To determine whether this FXRE is responsible for mediating the bile acid induction of mouse Ost expression, the element was mutated to 5'-AGaTCAcTGcCAT-3' in pGL3–1.7Ost µFXRE. Mutation of the FXRE at position –1221 reduced the mouse Ost basal promoter activity and abolished the CDCA induction (Table 1). Interestingly, addition of CDCA further decreased luciferase activity of the pGL3–1.7Ost µFXRE construct. This is potentially because of the loss of bile acid induction via the FXRE at –1221 while retaining an intact LRH-1 element at +55. In support of this hypothesis, cotransfection with an FXR expression plasmid, pCMX-rFXR, did not increase basal luciferase activity of pGL3–1.7Ost µFXRE, whereas addition of CDCA and expression of exogenous FXR decreased luciferase activity to 11% of the pGL3–1.7Ost level (Table 1).
Analysis of the mouse Ost promoter and identification of a functional LRH-1 element.
The next experiments were performed to study the role of the potential LRH-1 element at position +55 in the Ost promoter. This element, ACAAGGTTG, is a 78% match to the LRH-1 consensus sequence YCAAGGYCR (Fig. 3D). Cotransfection of pGL3–1.7Ost and a SHP expression plasmid, pCMX-mSHP, decreased the promoter activity to 28% of basal activity (Table 2). Furthermore, the siRNA-mediated decrease in SHP expression (siSHP) increased the Ost promoter activity by 400% under basal conditions and almost 20-fold when incubated with CDCA. This dramatic increase in Ost promoter activity is presumably due to prevention of SHP from repressing the constitutive positive transcriptional activity of LRH-1 while permitting induction by FXR. Cotransfection with an expression plasmid encoding LRH-1 and pGL3–1.7Ost induced Ost promoter activity. In contrast, siRNA-mediated reduction of endogenous LRH-1 expression decreased basal promoter activity by 67%. Mutation of the putative LRH-1 element at +55 to 5'-aaAAGGTgG-3'in pGL3–1.7OstµLRH reduced the Ost basal promoter activity but did not alter its induction by CDCA (Table 2). Cotransfection of pGL3–1.7OstµLRH and pCMX-mSHP decreased Ost promoter activity; however, siRNA-mediated reduction of endogenous SHP expression did not further increase pGL3–1.7Ost µLRH basal activity. Reduction of endogenous SHP expression, plus incubation with CDCA, increased pGL3–1.7Ost µLRH promoter activity as expected because of the presence of the FXRE at position –1221. Expression of exogenous LRH-1 did not increase the promoter activity of pGL3–1.7-Ost µLRH above basal levels (Table 2).
The Ost promoter construct, pGL3–1.2Ost, lacks an FXRE but retains the LRH-1 element at +55. Addition of CDCA to cells transfected with this promoter construct decreases promoter activity by 75%, presumably due to an induction of endogenous SHP expression and repression of the constitutive LRH-1 activity. Expression of exogenous SHP reduces pGL3–1.2Ost activity to similar levels, whereas siRNA-mediated reduction of endogenous SHP expression removes the effects of this negative regulator and dramatically increases pGL3–1.2Ost activity. The siRNA-mediated reduction of endogenous SHP (siSHP) together with CDCA does not further increase pGL3–1.2Ost activity due to lack of the FXRE. The promoter activity of pGL3–1.2Ost was increased by coexpression of exogenous LRH-1 and decreased by siRNA-mediated reduction of endogenous LRH-1 expression (Table 2).
Analysis of the mouse Ost promoter and identification of functional FXR and LRH-1 elements.
Analysis of the 5'-flanking region of the mouse Ost promoter revealed a cluster of three potential FXREs located close to the putative transcription start site (Fig. 3B). Further examination of these sites revealed a region beginning at position –5 that most closely matched the FXRE consensus sequence (Fig. 3C). This region 5'-gGGTCAtTcACCc-3' shares 75% sequence identity with the consensus FXRE (capitalized bases indicate identity with the consensus). These sites are present in the reporter constructs pGL3–0.9Ost and pGL3–3.0Ost (Fig. 3B). Two additional potential FXREs are located further upstream at positions –1693 and –2175 and are present only in pGL3–3.0Ost. Examination of the mouse Ost 5'-flanking sequence revealed five potential LRH-1 elements; those at positions –240 and –393 are present in both reporter constructs, whereas the remaining three potential LRH-1 elements at positions –2464, –2054, and –2014 are present only in pGL3–3.0Ost (Fig. 3B). Comparison of the promoter constructs showed that the basal activity of pGL3–0.9Ost was 62% that of pGL3–3.0Ost (data not shown). However, the percent induction of promoter activity upon treatment with CDCA was similar (greater than 500%) for both the pGL3–0.9Ost and pGL3–3.0Ost constructs (Table 1 of supplemental material) (supplemental data for this article may be found at http://ajpgi.physiology.org/cgi/content/full/00479.2005/DC1). Therefore, although the distal potential FXRE sequences may play some role, we chose to focus on the 0.9-kb Ost construct as this promoter region appears to contain all the elements required for bile acid regulation. Parallel experiments with were conducted with pGL3–3.0Ost and are presented as supplementary material (Tables 1 and 2 of supplementary material) (supplemental data for this article may be found at http://ajpgi.physiology.org/cgi/content/full/00479.2005/DC1).
Coexpression of exogenous FXR did not increase pGL3–0.9Ost activity. However, exogenous FXR together with CDCA treatment increased promoter activity by 20-fold (Table 3). Surprisingly, cotransfection with an expression plasmid encoding a dominant-negative FXR mutant that blocks FXR activity, siRNA-mediated reduction of endogenous FXR expression, and siRNA-mediated reduction of Fic1 all increased the promoter basal activity by approximately three- for fourfold (Table 3). Inhibition of endogenous FXR activity or expression may decrease endogenous SHP expression, thereby removing its inhibitory effects on LRH-1. This putative of decreased SHP expression may be amplified by the presence of multiple LRH-1 elements in the Ost promoter. Addition of CDCA to the dnFXR, siFXR, or siFIC-treated cells did not further affect promoter activity because of loss of FXR activity. As a control, cells were treated with a scrambled sequence siRNA for Fic1 (scrFIC1). The scrFIC1 had no effect on the basal or CDCA-stimulated promoter activity of the Ost promoter constructs (Table 3 and Table 1 in supplemental material) (supplemental data for this article may be found at http://ajpgi.physiology.org/cgi/content/full/00479.2005/DC1). Similar control experiments also showed that the scrambled siRNAs scrFXR, scrLRH, and scrSHP did not affect Ost promoter activity: (luciferase activity relative to Ost alone; mean ± SD, n = 3) 0.9Ost alone, 100 ± 10%; 0.9Ost plus scrFXR, 110 ± 9%; 0.9Ost plus scrSHP, 98 ± 2%; and 0.9Ost plus scrLRH, 93 ± 3%. Inhibiting both FXR and LRH activity with siLRH and siFXR decreased promoter activity to just 13% of basal levels (Table 3 and Table 1 in supplemental material) (supplemental data for this article may be found at http://ajpgi.physiology.org/cgi/content/full/00479.2005/DC1), demonstrating that both FXR and LRH are involved in positive regulation of the Ost promoter. To determine whether the putative FXRE at position –5 in the Ost promoter is functional, the sequences were mutated to 5'-gGaaCAtTcttCc-3'. The plasmid pGL3–0.9Ost µFXRE has 30% the basal activity of pGL3–0.9Ost, and addition of CDCA further decreases activity. Expression of exogenous FXR alone and expression of exogenous FXR plus the addition of CDCA further decreased promoter activity. This decrease is presumably due to increased FXR expression or activity leading to decreased SHP expression, coupled with lack of a functional positively regulated FXRE and the presence of two intact negatively regulated LRH-1 elements (Table 3).
Coexpression of exogenous SHP and pGL3–0.9Ost decreased promoter activity by 60%, whereas siRNA-mediated reduction of endogenous SHP (siRNA) increased promoter activity by 448% that of basal levels (Table 4). The siRNA-mediated reduction of SHP plus addition of CDCA further increased luciferase levels. Coexpression of exogenous LRH-1 caused a 496% increase in activity over basal levels, whereas siRNA-mediated reduction of LRH-1 (siLRH) decreased luciferase expression to 62% of basal levels. To determine whether the potential LRH-1 elements at positions –240 and –393 in the Ost promoter are responsible for this activation by LRH-1, the sequences were mutated slightly singly (pGL30.9Ost µLRH-240, pGL30.9Ost µLRH-393) and in combination (pGL3 0.9Ost doubleµLRH). The pGL30.9Ost µLRH-240 and pGL30.9Ost µLRH-393 plasmids both exhibited 70% of the basal promoter activity for pGL3–0.9Ost. Coexpression of exogenous LRH-1 increased basal activity by 10%, because of the remaining intact LRH-1 element in each promoter construct. However, the pGL3–0.9Ost doubleµLRH plasmid exhibited only 31% of the basal promoter activity for pGL3–0.9Ost, and coexpression of LRH-1 did not significantly increase basal activity (Table 4). These results demonstrate that both LRH-1 elements in the Ost promoter are functional.
Real-time PCR analysis of Ost and Ost RNA expression in FXR- and SHP-null mice.
To determine whether FXR regulates Ost-Ost mRNA expression in vivo, real-time PCR analysis was performed with RNA isolated from the ileum of mice treated with the FXR agonist GW4064. As shown in Fig. 4A, the FXR agonist induced ileal Ost and Ost expression 1.5-fold. The expressions of SHP and Ibabp mRNAs were also increased 1.5-fold and 4-fold, respectively, whereas ASBT mRNA expression was not significantly altered by the FXR agonist (1,390 ± 394 vs. 1,297 ± 275; normalized threshold values, means ± SE, n = 3–5 mice per group). We hypothesized that the positive regulation of Ost and Ost mRNA expression is mediated by FXR, and as such the expression Ost-Ost expression would therefore decrease in FXR-null mice. The results depicted in Fig. 4B demonstrate that there is approximately a 60% decrease in Ost and Ost mRNA levels in the distal small intestine of FXR-null mice compared with wild-type control littermates. Expression of SHP and Ibabp mRNA, both positively regulated by FXR, also decrease 60% and 90%, respectively. ASBT mRNA expression was also measured in the FXR-null mice and decreased from 4,426 ± 682 to 2,255 ± 526 (normalized threshold values, means ± SE, n = 5 mice per group). We examined Ost and Ost expression in SHP-null mice and wild-type littermates (Fig. 4C). In contrast to the FXR-null mice, there was little change in the expression of Ost and Ost mRNA in the SHP-null mice, suggesting that the positive regulatory pathway is dominant. Ibabp levels were also not significantly altered in the SHP-null mice (Fig. 5C), whereas ASBT levels were slightly decreased (1,390 ± 394 vs. 917 ± 57; normalized threshold values, means ± SE, n = 4 or 5 mice per group).
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Fig. 4. Analysis of Ost and Ost mRNA expression in wild-type, FXR-null, and SHP-null mice. A: RNA was isolated from the ileum of wild-type male mice administered vehicle or the FXR agonist GW4064 (100 mg/kg) by gavage for 5 days and used for real-time PCR to measure Ost, Ost, SHP, and Ibabp mRNA expression. B: RNA was isolated from the distal third of the small intestine from male FXR-null mice and wild-type littermates fed a chow diet and used for real-time PCR to measure Ost, Ost, SHP, and Ibabp mRNA expression. C: RNA was isolated from the ileum of male SHP-null mice and wild-type littermates fed a chow diet and used for real-time PCR to measure Ost, Ost, and Ibabp mRNA expression. For each condition or genotype, expression levels in individual mice were determined in triplicate and normalized to cyclophilin expression. The normalized threshold values are plotted as percentage of the mRNA expression in the control wild-type mice (mean ± SE; n = 4 or 5 mice per group). *P < 0.05 vs. wild-type levels.
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Fig. 5. Expression of Ost and Ost protein in ileum, cecum, and proximal colon in wild-type mice fed control and cholic acid-containing diets. Male C57BL/6J mice (3 mo of age) were fed a control synthetic diet or the synthetic diet containing 0.2% cholic acid for 14 days. Ileum (A), cecum (B), and proximal colon (C) were used to isolate protein extracts. Ost, Ost, and -actin protein expression levels were then measured by immunoblotting, using the following amounts of protein extract from individual animals: ileum, Ost (50 µg), Ost (25 µg); cecum, Ost, (150 µg), Ost (25 µg); proximal colon, Ost (50 µg), Ost (25 µg). D–F: protein expression levels were quantitated by densitometry. Values for Ost and Ost protein expression were normalized to -actin expression and are expressed relative to the control diet (means ± SE; n = 5 mice/group). *P < 0.05 vs. control levels.
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Analysis of Ost and Ost protein expression.
To determine whether the levels of Ost and Ost protein were regulated in parallel with their RNA, immunoblotting analysis was performed using tissue extracts from ileum, cecum, and proximal colon of mice fed control and 0.2% cholic acid-containing diets. As shown previously, Ost migrated as an 40-kDa protein (9) (Fig. 1 with supplemental material) (supplemental data for this article may be found at http://ajpgi.physiology.org/cgi/content/full/00479.2005/DC1). The anti-Ost antibody detected a closely spaced doublet of 22 and 24 kDa, and both bands were efficiently competed with the GST-Ost fusion protein but not GST protein alone (Fig. 1 in supplemental material) (supplemental data for this article may be found at http://ajpgi.physiology.org/cgi/content/full/00479.2005/DC1). In previous studies of Ost-transfected cells, the Ost protein migrated primarily as a monomer of 24 kDa (9), and the modification responsible for the doublet in the tissue extracts has not yet been identified. For these studies, the upper and lower bands were both included in the Ost protein mass quantitation. There is a small increase in the expression of Ost and Ost protein in proximal colon (Fig. 5F) but not in ileum following cholic acid feeding (Fig. 5D). Similar to their mRNA, Ost and Ost protein expression was greater in cecum following cholic acid feeding (Fig. 5, B and E). Ost and Ost protein levels generally paralleled their mRNA expression in ileum, proximal colon, and to a lesser extent in cecum (Figs. 1 and 5) in the cholic acid-fed mice. In cecum, Ost mRNA and protein expressions were increased 2.0- and 2.4-fold, respectively, whereas Ost mRNA and protein increased 4.8 and 8.3-fold, respectively. The larger apparent increase in Ost protein vs. mRNA may reflect posttranscriptional regulation or, more likely, difficulty in quantifying the lower levels of Ost (and Ost) protein expression in cecum. The expression of Ost and Ost protein in cecum is 30% and 10%, respectively, of ileum in mice fed the control diet (data not shown).
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DISCUSSION
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The major finding of this study is that expression of mouse Ost and Ost mRNA is positively and negatively regulated by bile acids. The promoters for these genes are unusual in that they harbor both FXR and LRH-1 elements that mediate, respectively, this positive and negative feedback regulation. Such a dual or push-pull mechanism of dynamic regulation enables more precise regulation of expression with lower intrinsic noise (25). In Asbt-null mice, Ost and Ost mRNA expression was decreased in ileum and increased in the cecum and colon, suggesting that these genes are positively regulated by bile acids (9). In the present study where mice were fed a 0.2% cholic acid-containing diet for 2 wk, the mRNA expression for Ost and Ost showed little changed in ileum, was modestly increased in proximal colon, and significantly increased in cecum. These tissue and experimental model expression differences can be explained by differences in the concentration of bile acids to which these tissues are normally exposed, as well as differences in the mechanism of bile acid uptake. In mice fed a control diet, intestinal bile acids are efficiently taken up by the ileum before reaching the cecum and colon. However, in mice fed the cholic acid-containing diet, ileal bile acid uptake is partially limited by a decrease in Asbt expression (Fig. 1), thereby blunting the increase in ileal Ost-Ost expression. In contrast to cholic acid, the FXR agonist GW4064 is not dependent on the Asbt for uptake by the ileum, and Ost-Ost mRNA expression was increased in the GW4064-treated mice (Fig. 4A). In the cholic acid-fed mice, the combination of an increased bile acid load and decreased ileal Asbt expression results in an increased passage of bile acids into the cecum and colon. The cecum and colon can passively absorb bile acids after their 7-dehydroxylation by the endogenous bacterial flora, so that changes in cecal or colonic Asbt expression do not significantly limit their uptake by those tissues. This is evident by the similar increases in Ost-Ost mRNA expression in cecum and colon for the cholic acid-fed wild-type mice in this study and our previous study using Asbt-null mice (9).
Functional analysis of the promoters for mouse Ost and Ost revealed that the bile acid-dependent upregulation is mediated by FXR. Ost-Ost has an atypical push-pull mechanism of dynamic regulation that is secondary to the presence of both FXR and LRH-1 cis-elements. This type of dual regulation has been previously described for SHP, which also contains both FXR and LRH-1 elements (20). The expression differences between genes that are regulated strictly in a positive fashion by FXR, such as Ibabp, and genes regulated in a more complex fashion are nicely illustrated in the FXR-null mouse (Fig. 4B). Whereas Ost, Ost, and SHP mRNA expression is decreased by 50% in the FXR-null mice, Ibabp mRNA expression is decreased by 95%. In contrast to the FXR-null mice, Ost and Ost expression was not significantly changed in the SHP-null mice (Fig. 4C), suggesting that this negative regulatory pathway is subordinate under basal conditions in the ileum. The potential positive and negative regulation of mouse Ost-Ost by bile acids is summarized in Fig. 6. These findings raise the question as to why Ost-Ost would require more complex regulation than induction via the FXR-bile acid complex. One possibility is that this push-pull regulatory system is acting to closely titrate Ost-Ost expression to match the bile acid flux through the cell. There is good evidence that Ost-Ost can function as a bi-directional facilitative carrier and transport other solutes besides bile acids (9, 28). As such, it may be deleterious to regulate Ost-Ost expression only in a positive fashion because an excess basolateral Ost-Ost transport capacity above the bile acid load could allow unintended ileal transport of other solutes to or from the blood.
These studies focused on the mouse Ost and Ost genes; however, inspection of the human OST and OST genes revealed that the FXR and LRH-1 cis-elements identified in the mouse genes are also conserved in their human orthologues (data not shown), suggesting that human OST-OST may be under similar regulation. These new findings could have interesting clinical implications. Familial intrahepatic cholestasis type 1 (Fic1 disease, originally described as Byler disease) is a specific form of intrahepatic cholestasis that causes intense pruritus, fat malabsorption, variable degrees of diarrhea, and frequent progression to cirrhosis (7, 30). The disease results from defects in the gene FIC1 (ATP8B1), a P-type ATPase, that may function as an aminophospholipid flippase facilitating the transfer of phosphatidylserine and phosphatidylethanolamine from the outer to the inner hemi-leaflet of cellular membranes (32). Recent studies suggest that Fic1 activates the FXR gene product, presumably via posttranslational modifications that lead to its nuclear translocation. In the absence of Fic1, FXR activity and expression were found to be reduced (5). This reduction in FXR activity is predicted to significantly affect the expression of bile acid transporters. The siRNA-mediated reduction of endogenous Fic1 expression in CT26 mouse cells resulted in an increase in Asbt mRNA expression and a significant decrease in Ost-Ost mRNA expression. A similar decrease in Ost and Ost mRNA expression was observed in the ileum of FXR-null mice (Fig. 4B). From these results, it is predicted that a decrease in FXR activity as a consequence of Fic1 deficiency would have deleterious consequences for the enterocyte. Continued Asbt-mediated bile acid uptake combined with downregulation of OST-OST would create the potential for ileal enterocyte toxicity and possibly explain the diarrhea seen in Fic1 disease. Interestingly, severe and incapacitating diarrhea has been described after liver transplantation in patients with Fic1 disease (11, 22), potentially reflecting ileal enterocyte toxicity in the face of a normal hepatic capacity for bile secretion.
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GRANTS
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This work was supported by the National Institutes of Health Grants DK-47987 (to P. A. Dawson) and DK-54165 and DK-069942 (to B. L. Shneider).
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ACKNOWLEDGMENTS
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We thank Drs. Joyce Repa and Steve Kliewer (University of Texas Southwestern Medical Center, Dallas, TX) for providing intestinal mRNA samples from FXR- and SHP-null mice for this study.
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FOOTNOTES
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Address for reprint requests and other correspondence: P. A. Dawson, Dept. of Internal Medicine, Section of Gastroenterology, Wake Forest Univ. School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157 (e-mail: pdawson{at}wfubmc.edu)
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.
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ABSTRACT
MATERIALS AND METHODS
