FXR-activating ligands inhibit rabbit ASBT expression via FXR-SHP-FTF cascade

Hai Li,1 Frank Chen,3 Quan Shang,1 Luxing Pan,2 Benjamin L. Shneider,3 John Y. L. Chiang,4 Barry M. Forman,5 M. Ananthanarayanan,3 G. Stephen Tint,1,2 Gerald Salen,1 and Guorong Xu1,2

1Department of Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark; 2Medical Service, Veterans Affairs Medical Center, East Orange, New Jersey; 3Department of Pediatrics, Mount Sinai School of Medicine, New York, New York; 4Department of Biochemistry and Molecular Pathology, Northeastern Ohio University College of Medicine, Rootstown, Ohio; and 5Division of Molecular Medicine, The Beckman Research Institute, the City of Hope National Medical Center, Duarte, California

Submitted 17 April 2004 ; accepted in final form 17 August 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The regulation of the rabbit apical sodium-dependent bile acid transporter (ASBT) was studied both in vivo and in vitro. New Zealand White rabbits were fed 0.5% deoxycholic acid (DCA) or SC-435, a competitive ASBT inhibitor, for 1 wk. In DCA-fed rabbits, ASBT expression was repressed, associated with activated FXR, and evidenced by increased ileal short heterodimer partner (SHP) mRNA. Feeding SC-435 to the rabbits blocked bile acid absorption, decreased SHP mRNA, and increased ASBT expression. A 1.9-kb rabbit ASBT 5'-flanking region (promoter) was cloned, and a cis-acting element for {alpha}-fetoprotein transcription factor (FTF) was identified (–1166/ –1158). The effects of transcriptional factors and different bile acids on the rabbit ASBT promoter were studied in Caco-2 cells. FTF stimulated the rabbit ASBT promoter activity fourfold but not after the FTF binding site was deleted from the promoter. Increasing the SHP protein notably inhibited FTF-dependent trans-activation of rabbit ASBT. Adding hydrophobic bile acids deoxycholic acid, chenodeoxycholic acid, and cholic acid, activating ligands for FXR, inhibited rabbit ASBT promoter activity in Caco-2 cells, but this inhibitory effect was abolished after the FTF binding site was deleted. Ursodeoxycholic acid and ursocholic acid, nonactivating ligands for FXR, did not repress ASBT promoter activity. Thus the rabbit ASBT promoter is negative-feedback regulated by bile acids via a functional FTF binding site. Only FXR-activating ligands can downregulate rabbit ASBT expression through the regulatory cascade FXR-SHP-FTF.


THE APICAL SODIUM-DEPENDANT bile acid cotransporter (ASBT/SLC10A2) is the primary bile salt uptake protein in the intestine. It is mainly located on the apical surface of the terminal ileal enterocytes and is also expressed on renal proximal tubular cells and large cholangiocytes (12, 19). ASBT is an efficient transporter for conjugated and unconjugated bile salts. Bile salt reabsorption by ASBT in the ileum is sodium dependent and can be saturated (7). ASBT has been cloned from the human (26), rabbit (11), rat (19), mouse (17), and hamster (25).

Regulation of ASBT expression by intestinal bile acid flux has been studied in guinea pigs (13), rats (2, 8, 10, 18, 20), and mice (21). However, whether ASBT expression is positively or negatively regulated by increasing bile acid flux remains controversial. Observations in guinea pigs (13) and mice (21) showed that ASBT was negatively regulated by the intestinal bile acid flux, whereas in rats, ASBT was positively regulated by bile acids (8, 10, 18, 20). Nevertheless, the results reported by Arrese et al. (2) showed that in rats, no regulatory response to changes in the intestinal bile acid flux occurred. Recently, Chen et al. (5) identified a physiologically functional liver receptor homolog-1 (LRH-1; also called FTF, {alpha}-fetoprotein transcription factor in other species) transcriptional binding site in the mouse ASBT promoter that was not present in the rat. As a result, chenodeoxycholic acid (CDCA), an activating ligand of nuclear receptor farnesoid X receptor (FXR), repressed mouse ASBT expression through the FXR/short heterodimer partner (SHP)/LRH-1 [{alpha}-fetoprotein transcription factor (FTF)] cascade. This negative feedback regulation did not occur in rat ASBT because of lack of the FTF binding site. It has been proposed that bile acids downregulate CYP7A1, the rate-limiting enzyme for bile acid synthesis, by activating the FXR/SHP/FTF cascade (9, 14). Our recent study (27) showed that in rabbits, hydrophobic deoxycholic acid (DCA), but not hydrophilic ursocholic acid (UCA), activated FXR and then repressed CYP7A1 expression through the FXR/SHP/FTF cascade.

This study was designed to determine whether in rabbits, ASBT expression is negatively regulated by bile acids, in particular, FXR-activating ligands; whether there is a functional FTF binding site in rabbit ASBT promoter region, and rabbit ASBT is also regulated by bile acids via FXR/SHP/FTF cascade; and whether rabbit ASBT should be downregulated only by FXR-activating ligands.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Studies

Male New Zealand White (NZW) (n = 18) rabbits weighing 2.5–2.75 kg (Convance, Denver, PA) were used in this study. Six rabbits were fed regular rabbit chow, six rabbits were fed regular chow containing 0.5% DCA (Purina Mills, St. Louis, MO), and six rabbits were fed a competitive inhibitor of ASBT, SC-435 (24) (125 mg·kg–1·day–1), with regular chow for 7 days. Feces were collected during the last 2 days of the feeding experiment for measurement of fecal bile acid outputs. One-half of the rabbits in each study group was killed to collect distal one-third ileal mucosa specimens, which were immediately frozen for measurement of mRNA levels of ASBT and SHP. Bile fistulas were constructed with the remaining half of the rabbits (3/6 in each group). The bile drainage continued for 5 days to recover the DCA pool to calculate the total bile acid pool as previously described (28).

Animal protocol was approved by the Institutional Animal Care and Use Committee at Veterans Affairs Medical Center, East Orange, NJ and the Institutional Animal Care and Use Committee at University of Medicine and Dentistry-New Jersey Medical School, Newark, NJ.

Cell Culture

Human Caco-2 colon epithelial cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM, containing 10% fetal calf serum. Plasmid-transfected cells were cultured for 40 h in DMEM containing 0.5% charcoal-treated fetal calf serum before harvesting for reporter gene assays. The change in medium was designed to minimize the effect of bile salts found in fetal calf serum, as previously reported (5).

Cloning and Construction of Rabbit ASBT Promoter

The rabbit ASBT5'-flanking 3.4-kb fragment was amplified by PCR using the following primers: sense: 5'-gaatatcatgctgccttttacc-3' and antisense: 5'-agccaccgtagagaagcaata-3' according to GeneBank accession no. AJ002005. Then its fragments were subcloned into a mammalian expression vector pGL3-Basic (Promega, Madison, WI) upstream of a firefly luciferase gene to form rabbit pGL3-ASBT5'/3.4 kb. With the use of this 3.4-kb ASBT5' sequence as a template, another two fragments were PCR synthesized and subcloned into pGL3 basic vector forming the rabbit ASBT5' construct pGL3-ASBT5'/1.9 kb (P2) and pGL3-ASBT5'/1.1 kb (P1). P1 corresponds to pGL3-ASBT5'/–1082/+79, and P2 corresponds to pGL3-ASBT5'/–1816/+79.

DNA Oligonucleotides and Site-Directed Mutagenesis

To prepare ASBT5', FTF binding site-deleted mutant construct, complimentary strands of DNA oligonucleotides containing the appropriate sequences and the desired nine nucleotides-deleted mutation for whole FTF element were synthesized (Integrated DNA Technologies, Coralville, IA): 5'-gccttgttagaaaaaaaTCAAAGCCTactccggggttcca-3';5'-tggaaccccggaGTAGGCTTTgatttttttctaacaaggc-3'; and 5'-gccttgttagaaaaaaatccggggttcca-3'; and 5'-tggaaccccggatttttttctaacaaggc-3'. The site-directed point mutagenesis of rabbit ASBT5' sequence was performed by a Quick Change site-directed mutagenesis kit (Stratagene, La Jolla, CA) targeted to the potential FTF cis-acting element (positioned from –1166 to –1158). The resulting construct P2m was the FTF binding site-deleted P2. The mutation (deletion of FTF cis-acting element) was confirmed by DNA sequencing.

Nuclear Protein Extraction and Gel-Shifting

Nuclear proteins were extracted from frozen rabbit ileal mucosa using NE-PER nuclear extraction reagents (Pierce, Rockford, IL). The protein concentration of nuclear extracts was determined by the protein assay kits (Bio-Rad, Hercules, CA), with bovine serum albumin used as a standard. The DNA protein binding reaction was carried out as described previously (27), with 5 µg of nuclear protein extract and 5,000 counts/min of end-labeling DNA oligos. Competition experiments were performed using unlabeled cis-element oligo probes and unlabeled mutant cis-element oligo probes. Samples were analyzed by 7% native polyacrylamide gel. The cis-element probe, used as a rabbit FTF-specific probe, was a double-stranded oligonucleotide containing the sequence sense (strand): 5'-gcttgttgttagaaaaaaatcaaagcctactccggg-3', and antisense: 5'-cccggagtaggctttgatttttttctaacaagc-3'. The sequence of the FTF mutant probes were5'-gcttgttagaaaaaaatcttagcagactccgg-3' and 5'-cccggagtctgctaagatttttttctaacaagc-3'.

Transient Transfection and Firefly Luciferase Assay

Confluent Caco-2 cells (~5 million cells/plate) were harvested and resuspended in 700 µl of phosphate-buffered saline containing 20 µg of rabbit ASBT5'/luciferase hybrid plasmid construct and 0.1 µg of control plasmid containing a thymidine kinase promoter-driven Renilla luciferase gene (Promega). Transfection was accomplished by electroporation (6) at 0.22 kV and 0.95 µF x 1,000 (Bio-Rad). After eletroporation, the cells were cultured for an additional 40 h before performing dual luciferase assays (Promega) as described by manufacturer.

Expression plasmid constructs

pCDNA3-mLRH-1 (a generous gift from Dr. Alan R. Tall, Columbia University, New York, New York) directs expression of the mouse LRH-1 gene, which is a homolog of the orphan nuclear receptor fushi tarazu F1 from Drosophila and is called {alpha}-fetoprotein transcription factor (FTF) in other species. This mouse LRH-1 plasmid construct was used to express FTF in the present study. pCMX-mutant SHP, pCDNA3-human FXR (hFXR), and pCMX-human retinoid X receptor (hRXR; generous gift from Dr. David Mangelsdorf, University of Texas Southern, Dallas, TX) harbor a copy of the mouse SHP, hFXR, and human RXR genes, respectively. pCMX-hFXR-W469A (from Dr. David Mangelsdorf), a mutated hFXR, contains a point mutation within the AF-2 domain of the gene (1). The resulting gene product contains bile acid and DNA binding domains but lacks a functional trans-activation motif. The effect of the orphan nuclear receptors on basal activity and bile acid-mediated responses was assessed by cotransfection of these constructs with pGL3-rabbit ASBT5' (the cloned rabbit ASBT promoter) in luciferase-transfected Caco-2 cells.

Real-Time PCR Assay

In this study, mRNA levels were also quantitatively measured by real-time PCR, and the data shown in the text are relative fold. Total RNA was isolated and treated with DNase I by Absolutely RNA RT-PCR mini-prep kit (Stratagene). One microgram of DNase I of treated total RNA was reverse transcripted by Omniscript Reverse Transcriptase (Qiagen, Valencia, CA) using the oligo-dT primer. Real-time PCR was performed with the ABI PRISM 7700 sequence detection system using one fiftieth of the reverse transcription (RT) reaction, and was analyzed with the 1.7 software (Applied Biosystems, Forest City, CA). Rabbit SHP, ASBT, cyclophilin primers, minor groove binder (MGB) probes, human SHP, FTF, cyclophilin primers, and MGB probes were synthesized by Applied Biosystems with Assays-by-Design Service (Applied Biosystems, Forest City, CA). PCR was carried out in a 50-µl reaction volume containing 1 x TaqMan Universal PCR Master Mix (Applied Biosystems), 20-ng cDNA templates, 0.9 µM of each forward and reverse primer, and a 0.25 µM MGB probe. Cyclophilin was set as loading control. Because validation experiments showed that amplification efficiency of the target and the cyclophilin were approximately equal, accumulation was performed using the comparative {Delta}{Delta}Ct method (4, 22). The detecting primers and probes were rabbit ASBT (sense: 5'-cgttatgccctttatgctacaccaa-3'; antisense: 5'-aagagcaaccaaggaagtacctatc-3'; probe: tggtcccagagtcc), rabbit SHP (sense: 5'-tggcccaagacatggtgac-3'; antisense: 5'-gctcctccagcaggatcttct-3'; probe: ctgaagccccggtgc), and rabbit cyclophilin (sense: 5'-ccttcgagctatttgcagacaa-3'; antisense: 5'-gaacccttataaccgaatcctttct-3'; probe: cagcagaaacttc). The primers and probe assay ID of human SHP, FTF, and cyclophillin in Assays-by-Demand (for detecting Caco-2 cell's SHP, and FTF mRNA expression after P2 cotransfection and DCA treatment) were Hs00222677 m1, Hs00155029 m1, Hs00187067 m1, and Hs99999904 m1, respectively (Applied Biosystems).

Bile Acid Measurement

Biliary bile acid analysis. Bile acids in the bile were measured by the capillary gas-liquid chromatograph (GLC) method as previously described (28).

Fecal bile acid analysis. Internal standard (nor-cholic acid, 20 µg) in 200 µl n-butanol (10–15 mg) was added to the freeze-dried feces. Concentrated hydrochloric acid (20 µl) was then added, and the suspension was heated at 55°C for 4 h. The solvents were evaporated, and the residue are subjected to trimethylsilylation, taken in 200 µl n-hexane, and an aliquot was subjected to GLC (3).

Statistical methods. Data are shown as means ± SD and were compared statistically by ANOVA followed by the Bonferroni multiple comparisons test. BMDP statistical software (BMDP Statistical Software, Los Angeles, CA) was used for statistical evaluations.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In Vivo Studies in Rabbits Fed 0.5% DCA or the Ileal ASBT Inhibitor SC-435

In NZW rabbits fed 0.5% DCA for 1 wk, the circulating bile acid pool size, which consisted of 89% DCA and 10% cholic acid (CA), increased greater than twofold from 274 ± 24 to 587 ± 68 mg (P < 0.01). Ileal mucosa ASBT mRNA expression decreased 40% (1.03 ± 0.07 vs. 0.62 ± 0.02-fold; P < 0.05), whereas SHP mRNA levels rose 50% (1.00 ± 0.09 vs. 1.54 ± 0.26-fold; P < 0.05) compared with baseline values measured by real-time PCR (Fig. 1, A and B).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1. Expression of apical sodium-dependent bile acid transporter (ASBT) and short heterodimer partner (SHP) in the ileum of rabbits fed deoxycholic acid (DCA) and SC-435. A: ileal ASBT mRNA expression in rabbits fed DCA or ileal ASBT inhibitor SC-435. New Zealand White (NZW) rabbits were fed 0.5% DCA or SC-435 (an ASBT competitive inhibitor) for 1 wk. ASBT mRNA expression decreased in rabbits fed DCA (P < 0.05) but increased (P < 0.01) in the SC-435-treated group compared with that in control. ASBT mRNA levels were measured by real-time PCR. B: ileal SHP mRNA expression in rabbits fed DCA and ileal ASBT inhibitor SC-435. SHP mRNA levels, which reflected farnesoid X receptor (FXR) activity, were increased (P < 0.05) in rabbits fed 0.5% DCA but decreased in the SC-435-treated rabbits compared with that in controls. SHP mRNA levels were measured by real-time PCR.

 
In NZW rabbits treated with SC-435 (125 mg·kg–1·day–1), a competitive inhibitor of ASBT (19) for 1 wk, the reabsorbed bile acid flux containing 85% DCA and 14% CA through the ileum decreased as the fecal bile acid outputs increased ninefold (1.9 ± 1.5 to 17.0 ± 10.1 mg/day; P < 0.001). Ileal mucosa ASBT mRNA expression rose 50% (1.53 ± 0.24-fold; P < 0.05), whereas SHP mRNA declined 34% (0.66 ± 0.13-fold; P > 0.05; Fig. 1, A and B).

Identification of FTF Element Involved in Rabbit ASBT Expression

Sequence analysis of 5'-flanking regions of rat (19), human (26), rabbit (11), and mouse (17) ASBT genes revealed several basic transcription factor elements, hepatic nuclear factor {alpha}-1, u-AP1, and dAP1, which are located at the proximal region of the rabbit ASBT promoter. At 5'-flanking region –1166/ –1158, a nine-nucleotide putative FTF binding element (TCAAAGCCT) was identified (Fig. 2). It was highly conserved to the FTF binding site in the human and rat CYP7A1 promoter. Two rabbit ASBT 5'-flanking region-pGL3 luciferase reporter vectors were constructed: a 1.9-kb 5'-flanking region-pGL3 (P2), which contained the putative FTF binding site, and a 1.1-kb ASBT 5'-flanking region-pGL3 (P1), which did not contain the FTF binding site. We found both constructs expressed luciferase activities in Caco-2 cells. The activity of P2 was twofold (P < 0.001) stronger than P1 (4,013 ± 267 vs. 1,670 ± 89 U; Fig. 3).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. {alpha}-Fetoprotein transcription (FTF) binding site in the cloned 1.9-kb ASBT promoter (P2). Only part of the nucleotide sequence (from –1086 to –1286) in the cloned rabbit ASBT promoter (P2) is shown. The suggested FTF binding site is located at 5'-flanking region –1166/–1158, a 9-nt sequence (tcaaagcct) that is highly conserved to the FTF [called liver receptor homolog (LRH)-1 in the mouse] binding site in the rat and human CYP7A1 promoter region.

 


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 3. Measurement of baseline activity of the 1.1 (P1)- and 1.9-kb cloned ASBT promoter (P2). A dual-luciferase reporter assay system (Promega) was employed to examine transcriptional activities of the rabbit ASBT promoter. All activities were normalized by thymidine kinase promoter-driven Renilla luciferase. The results showed that in Caco-2 cells transfected with P2, which contained the FTF binding site, ASBT expression indicated by luciferase activity was 2-fold higher (P < 0.001) than that with P1 without the FTF binding site. The high expression level in the Caco-2 cells transfected with a known positive promoter SV40 demonstrated good performance of this experimental system.

 
To further determine whether the FTF cis-element in the rabbit ASBT promoter was functional, a pair of oligonucleotide probes was synthesized according to the putative FTF cis-element sequence in the rabbit ASBT promoter region. Protein binding for FTF was seen after using EMSA using nuclear extracts from NZW rabbit ileal mucosa. FTF binding could be competitively blocked by unlabeled (cold) cis-element oligonucleotide probe (Fig. 4A, lanes 3 and 4), but was not competitively blocked by a cold mutant oligonucleotide probe (Fig. 4A, lanes 7 and 8). A shift of the binding band of FTF protein/FTF cis-element was seen after adding the FTF antibody (goat polyclonal antibody from Santa Cruz Biotechnology; Fig. 4A, lane 9). However, the shift did not occur when a control antibody (anti-histone H1 antibody) was applied instead (Fig. 4A, lane 10).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4. Identification of FTF cis-element in the rabbit ASBT promoter. A: binding of ileal mucosal nuclear protein extract to FTF cis-element. Gel-shift assays were carried out with 5 µg of nuclear protein extract from NZW rabbit ileal mucosa and 25 pM of 32P-labeled FTF binding site sequence oligo probe (F). DNA binding protein was detected (lane 2). The binding band can be competed by a 20 (competitor F+)- and 100-fold (competitor F++) cold probe (lanes 3 and 4) but cannot be competed by either a 20 (Fm+)- or 100-fold (Fm++) cold-mutated FTF oligos (lanes 7 and 8). This FTF probe/protein binding can be shifted by addition of anti-FTF antibody Fa (goat polyclonal antibody, Santa Cruz Biotechnology) with a final concentration of 20 µg/ml (lane 9). Anti-histone H1 antibody (H) was used as control antibody and, as expected, did not shift the binding (lane 10). Mutated FTF oligos (Fm) did not bind the extracted nuclear protein (lane 6). B: FTF protein stimulates ASBT promoter activity through FTF cis-element. Luciferase activity of P2, which contains FTF binding site, was increased 4-fold (P < 0.001) after adding 4 µg of FTF expression construct. P2m, where FTF binding site was deleted, had only 47% (P < 0.01) luciferase activity of P2. After FTF expression construct was added, P2m luciferase activity did not increase.

 
To determine the relationship between FTF cis-element and its binding protein, P2 with the deleted FTF binding site (P2m) was constructed. After cotransfection into Caco-2 cells, P2m had less than one-half the activity (1,620 ± 81 U; P < 0.01) than P2 with the intact FTF binding site (3,451 ± 191 U). FTF protein increased the activity fourfold (14,263 ± 950 U; P ≤ 0.001) in P2 with the intact FTF binding site but did not stimulate the activity of P2m (1,809 ± 108 U; Fig. 4B).

SHP is a Potent Repressor of Rabbit ASBT by Deactivating FTF

The effect of SHP on the potential transcriptional activity of the rabbit ASBT-luciferase reporter gene was determined using transfection assays in Caco-2 cells. Cotransfection with mouse SHP expression plasmid resulted in a 58% decrease (1,443 ± 32 vs. 3,451 ± 191 U, P < 0.001) in the activity of rabbit P2. This inhibitory effect of SHP disappeared in rabbit P2m, where the binding element was deleted (Fig. 5). In addition, increasing the concentration of the cotransfected SHP expression plasmid resulted in a dose-dependant decrease in the FTF-induced transactivation of rabbit P2 (Fig. 5). The data showed when the ratio of SHP to FTF reached 1:1, FTF-induced P2 activity was totally inhibited.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5. SHP represses FTF dependent rabbit ASBT promoter expression. After P2 and 4 µg of mouse SHP plasmid were cotransfected, P2 expression indicated that luciferase activity decreased 58% (P < 0.001). The change of P2 luciferase activity was examined after cotransfection of 2 µg mouse LRH-1 (FTF) plasmid and increasing cotransfected SHP plasmid (20 ng, 200 ng, and 2 µg). When the ratio of FTF to SHP (FTF:SHP) transfected into Caco-2 cells reached 1:1, FTF-induced P2 activity was totally abolished. Ctrl, control.

 
FXR and DCA Repressed Rabbit ASBT Promoter Activity

Adding 50 µM DCA, an activating ligand for FXR, to Caco-2 cells cotransfected with P2 decreased the promoter activity by 76% (969 ± 39 U; P < 0.001) compared with the baseline value of P2 (4,013 ± 267 U). When human FXR (hFXR) and human RXR (hRXR) expression vectors were cotransfected into the cells, rabbit P2 activity was repressed by 46% (2,175 ± 126 U; P < 0.001). Adding 50 µM DCA to Caco-2 cells cotransfected with hFXR and hRXR expression vectors inhibited rabbit P2 activity by 79% (840 ± 60 U; P < 0.001). After being cotransfected with RXR and mutated FXR W469A (FXRm), which could bind bile acids (ligands) but cannot transactivate promoter of its target genes (SHP), P2 activity (7,382 ± 153 U) was 84% (P < 0.001) higher than baseline. After 50 µM DCA was added to the Caco-2 cells cotransfected with FXRm and RXR, P2 activity still remained 34% (P < 0.01) higher than the baseline level (Fig. 6).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6. The downregulation of rabbit ASBT promoter (P2) by bile acids required functioning FXR. Caco-2 cells were cotransfected with 4 µg of expression plasmids for human FXR or FXR mutant (FXRm) and retinoid X receptor (RXR) in combination with P2. Some cells were incubated with 50 µM DCA. FXR/RXR and its ligand DCA inhibited P2 activity. When FXR was replaced by FXRm, P2 luciferase activity increased and remained higher than baseline value even after adding DCA.

 
Dose-Dependant Inhibition of Rabbit ASBT Promoter by FXR-Activating Ligands

Rabbit P2 hybrid construct transfected Caco-2 cells were treated with different concentrations of DCA (0.25, 1, 4, 20, and 50 µM). Rabbit P2 promoter activity, represented by luciferase activity, showed an inverse relationship with increasing concentrations of DCA in the medium (Fig. 7). Because there were no gross changes in cell viability and no effect on Renilla luciferase activity (as a control construct under the TK promoter), the dose-dependent reduction of P2 activity in this experiment was not due to a nonspecific effect of cellular toxicity but to a biological effect of DCA.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 7. Dose-dependent inhibition of rabbit ASBT promoter by DCA. The Caco-2 cells were cotransfected with P2, which was connected to a reporter gene, luciferase. The cells were treated with DCA for 40 h at different doses as indicated in the figure. P2 luciferase activity decreased, whereas the concentration of DCA was gradually increased in the medium.

 
FXR-Activating Ligands Repressed ASBT Promoter Activity via FTF

Caco-2 cells were treated with 10 µM hydrophobic bile acids DCA, CDCA, and CA, which activate FXR, and 10 µM UCA and UDCA, which almost do not activate FXR, for 40 h. Rabbit P2 activity decreased in Caco-2 cells treated with DCA (–47%; P < 0.001), CDCA (–42%; P < 0.001), and CA (–36%; P < 0.01; Fig. 8A). However, the inhibitory effects of the DCA, CDCA, and CA were abolished in P2m, where the FTF binding site was deleted (Fig. 8B). Neither UCA nor UDCA significantly reduced luciferase activity in P2 with intact FTF binding site (Fig. 8A).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 8. FXR-activating ligands repress ASBT promoter activity via FTF. A: Caco-2 cells were cotransfected with P2 and incubated with either 10 µM DCA, chenodeoxycholic acid (CDCA), cholic acid (CA), ursocholic acid (UCA), or ursochenodeoxycholic acid (UDCA) each for 40 h. Activating FXR ligands (DCA, CDCA, and CA) inhibited P2 luciferase activity, but nonactivating FXR ligands (UCA and UDCA) did not. B: Caco-2 cells were cotransfected with Pm, which is FTF binding site-deleted P2. Pm luciferase activity was not affected by DCA, CDCA, or CA treatment.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, after rabbits were fed 0.5% DCA, an FXR-activating ligand, for 1 wk, the influx of bile acid increased through the ileal enterocytes. This was demonstrated by a twofold enlargement of the circulating bile acid pool size, whereas ileal ASBT mRNA decreased by 40%. This in vivo study demonstrated feedback regulation of ASBT expression in rabbits by ileal bile acid influx. To confirm this observation, NZW rabbits were treated with SC-435, a competitive inhibitor of ASBT, for 1 wk. The influx of bile acids into the ileal apical epithelium was reduced, as evidenced by a ninefold increase in fecal bile acid outputs, whereas ASBT mRNA expression increased 50%. These results further demonstrated the negative feedback regulation of ASBT expression by the FXR-activating ligand influx into ileal epithelium in the in vivo rabbit studies.

We know that FXR played a crucial role in the feedback regulation of ASBT in mice via the FXR/SHP/LRH-1 (FTF homolog in mice) cascade (5). CA inhibited ASBT expression in wild-type (FXR+/+) mice but not in mice in which FXR was knockout. Furthermore, the feedback regulation did not occur in rats because there was no functional FTF binding element in the rat ASBT promoter. To clarify the mechanisms involved in the feedback regulation of rabbit ASBT expression by bile acids, we cloned a 1.1 (P1)- and a 1.9-kb (P2) rabbit ASBT 5'-flanking region-PGL3 construct. We identified that P2 contained a putative FTF binding site at 5'-flanking region –1166/–1158 (TCAAAGCCT). Although both P2 and P1 showed baseline expression indicated by luciferase activities in Caco-2 cells, the activity of P2 was twice as strong as P1 (Fig. 4). With the use of this putative rabbit FTF binding site (TCAAAGCCT) as a probe, gel shift assays showed that the binding of FTF protein with this probe could be competed by nonlabeled cold probe but not the mutant probe and was shifted by adding the FTF antibody. Thus this putative FTF binding site can specifically bind to rabbit FTF protein extracted from rabbit ileal mucosa (Fig. 5A). Furthermore, FTF protein increased ASBT activity fourfold in the Caco-2 cells cotransfected with P2 but not in those cells with P2m, where FTF binding site was deleted (Fig. 5B). These results confirmed that the proposed element in P2 was a functional rabbit FTF binding site.

The present study demonstrated that the negative regulation of ASBT expression by bile acids in the rabbit is also through the FXR/SHP/FTF cascade. The data in Fig. 6 showed that in Caco-2 cells, SHP inhibited rabbit ASBT promoter (P2) baseline expression, whereas FTF induced the expression. This inhibitory effect of SHP was absent in P2m, where the FTF binding site was deleted. These results indicate that SHP has an inhibitory effect against the stimulatory effect of FTF on rabbit ASBT expression, and this inhibitory effect requires the presence of the FTF binding site in the ASBT promoter. Furthermore, the studies in Caco-2 cells showed that adding DCA, an FXR-activating ligand, resulted in a decrease of ASBT expression by 76%. However, when FXR in the Caco-2 cells was replaced by FXRm, which binds bile acids (ligands) but could not transactivate the promoter of its target genes, including SHP, not only the inhibitory effect of FXR was absent but the expression of ASBT was even higher than baseline value regardless of whether DCA was added (Fig. 7). We hypothesize that ASBT expression level at baseline (control in Fig. 7) resulted from the inhibitory effect of bile acid/FXR endogenously present in the Caco-2 cells. After FXRm was added, W469A FXR, which competitively replaced FXR, ASBT was released from the inhibitory effect of the DCA/FXR/SHP/FTF cascade such that ASBT expression was higher than baseline value. Without functioning FXR, DCA could not repress ASBT expression. These results demonstrated that the negative feedback regulation of rabbit ASBT by bile acids is FXR dependent.

In addition, not all bile acids activate the FXR protein (15, 16, 23). Our previous in vivo study in rabbits showed that DCA, but not UCA, activated FXR (27). In the present study, we found that FXR-activating ligands CDCA, DCA, and CA but not FXR nonactivating ligands, UDCA and UCA, repressed FTF-dependant transactivation of rabbit ASBT in Caco-2 cells. The inhibitory effects of FXR-activating ligands were absent when the FTF binding site was deleted. These data further demonstrated that in rabbits, downregulation of ASBT expression by bile acids is through the activation of FXR and is FTF dependent. More importantly, because only FXR-activating ligands downregulate ASBT expression, the composition of activating FXR ligands in the bile acid pool as well as the pool size will be crucial in the regulation of ASBT expression in vivo. This opinion is supported by the results observed in the in vivo studies (Figs. 1 and 2). ASBT mRNA decreased with increased SHP expression in rabbits fed DCA, where circulating bile acid pool with 89% DCA enlarged twofold. ASBT expression increased with reduced SHP mRNA in the rabbits treated with ASBT inhibitor SC-435. In those rabbits, the reabsorbed bile acid flux, containing 85% DCA, decreased significantly.

It should be emphasized that the expression of ASBT is not only regulated by the FXR-SHP-FTF cascade, because there are other binding elements for transcriptional factors in the rabbit promoter region. In this article, we only demonstrate the effect of bile acids on the regulation of ASBT through the FXR/SHP/FTF cascade using Caco-2 cells as the experimental model.

In summary, we identified a FTF functional binding element in the rabbit ASBT promoter. A functional FTF binding site as well as functioning FXR are required for the negative feedback regulation of rabbit ASBT by bile acids. Only FXR-activating ligands can downregulate rabbit ASBT expression via the regulatory cascade FXR-SHP-FTF.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by grants from the Department of Veterans Affairs Research Service, Washington, D.C. and National Institutes of Health Grants DK-56830, HL-18094, DK-54165, DK-57636, DK-26756, and HD-20632.


    ACKNOWLEDGMENTS
 
Present address for H. Li: Shanghai Institute of Digestive Disease; GI Division, Department of Medicine, Ren-Ji Hospital, Shanghai Second Medical University, Shanghai, 200001 Peoples Republic of China.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. Xu, GI Lab (15A), Veterans Affairs Medical Center, 385 Tremont Ave. East Orange, NJ 07018–1095 (E-mail: xugu{at}umdnj.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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ananthanarayanan M, Balasubramanian N, Makishima M, Mangelsdorf DJ, and Suchy FJ. Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptor. J Biol Chem 276: 28857–28865, 2001.[Abstract/Free Full Text]
  2. Arrese M, Trauner M, Sacchiero RJ, Crossman MW, and Shneider BL. Neither intestinal sequestration of bile acids nor common bile duct ligation modulate the expression and function of the rat ileal bile acid transporter. Hepatology 28: 1081–1087, 1998.[CrossRef][ISI][Medline]
  3. Batta AK, Salen G, Rapole KR, Batta M, Batta P, Alberts D, and Earnest D. Highly simplified method for gas-liquid chromatographic quantization of bile acids and sterols in human stool. J Lipid Res 40: 1148–1154, 1999.[Abstract/Free Full Text]
  4. Bustin SA. Absolute quantification of m RNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25: 169–193, 2000.[Abstract/Free Full Text]
  5. Chen F, Ma L, Dawson PA, Sinal CJ, Sehayek E, Gonzalez FJ, Breslow J, Ananthanarayanan M, and Shneider BL. Liver receptor homologue-1 mediates species- and cell line-specific bile acid-dependent negative feedback regulation of the apical sodium-dependent bile acid transporter. J Biol Chem 278: 19909–19916, 2003.[Abstract/Free Full Text]
  6. Chu G, Hayakawa H, and Berg P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res 15: 1311–1326, 1987. 6.[Abstract]
  7. Craddock AL, Love MW, Daniel RW, Kirby LC, Walters HC, Wong MH, and Dawson PA. Expression and transport properties of the human ileal and renal sodium-dependent bile acid transporter. Am J Physiol Gastrointest Liver Physiol 274: G157–G169, 1998.[Abstract/Free Full Text]
  8. Dumaswala R, Berkowitz D, and Heubi JE. Adaptive response of the enterohepatic circulation of bile acids to extrahepatic cholestasis. Hepatology 23: 623–629, 1996.[ISI][Medline]
  9. Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, Galardi C, Wilson JG, Lewis MC, Roth ME, Maloney PR, Willson TM, and Kliewer SA. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 6: 517–526, 2000.[ISI][Medline]
  10. Higgins JV, Paul JM, Dumaswala R, and Heubi JE. Downregulation of taurocholate transport by ileal BBM and liver BLM in biliary-diverted rats. Am J Physiol Gastrointest Liver Physiol 267: G501–G507, 1994.[Abstract/Free Full Text]
  11. Kramer W, Stengelin S, Baringhaus KH, Enhsen A, Heuer H, Becker W, Corsiero D, Girbig F, Noll R, and Weyland C. Substrate specificity of the ileal and the hepatic Na(+)/bile acid cotransporters of the rabbit. I. Transport studies with membrane vesicles and cell lines expressing the cloned transporters. J Lipid Res 40: 1604–1617, 1999.[Abstract/Free Full Text]
  12. Lazaridis KN, Pham L, Tietz P, Marinelli RA, deGroen PC, Levine S, Dawson PA, and LaRusso NF. Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter. J Clin Invest 100: 2714–2721, 1997.[Abstract/Free Full Text]
  13. Lillienau J, Crombie DL, Munoz J, Longmire-Cook SJ, Hagey LR, and Hofmann AF. Negative feedback regulation of the ileal bile acid transport system in rodents. Gastroenterology 104: 38–46, 1993.[ISI][Medline]
  14. Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J, and Mangelsdorf DJ. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol Cell 6: 507–515, 2000.[ISI][Medline]
  15. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, Hull MV, Lustig KD, Mangelsdorf DJ, and Shan B. Identification of a nuclear receptor for bile acids. Science 284: 1362–1365, 1999.[Abstract/Free Full Text]
  16. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, Stimmel JB, Willson TM, Zavacki AM, Moore DD, and Lehmann JM. Bile acids: natural ligands for an orphan nuclear receptor. Science 284: 1365–1368, 1999.[Abstract/Free Full Text]
  17. Saeki T, Matoba K, Furukawa H, Kirifuji K, Kanamoto R, and Iwami K. Characterization, cDNA cloning, and functional expression of mouse ileal sodium-dependent bile acid transporter. J Biol Chem 125: 846–851, 1999.
  18. Sauer P, Stiehl A, Fitscher BA, Riedel HD, Benz C, Kloters-Plachky P, Stengelin S, Stremmel W, and Kramer W. Downregulation of ileal bile acid absorption in bile-duct-ligated rats. J Hepatol 33: 2–8, 2000.[CrossRef][ISI][Medline]
  19. Shneider BL, Dawson PA, Christie DM, Hardikar W, Wong MH, and Suchy FJ. Cloning and molecular characterization of the ontogeny of a rat ileal sodium-dependent bile acid transporter. J Clin Invest 95: 745–754, 1995.[ISI][Medline]
  20. Stravitz RT, Sanyal AJ, Pandak WM, Vlahcevic ZR, Beets JW, and Dawson PA. Induction of sodium-dependent bile acid transporter messenger RNA, protein, and activity in rat ileum by cholic acid. Gastroenterology 113: 1599–1608, 1997.[ISI][Medline]
  21. Torchia EC, Cheema SK, and Agellon LB. Coordinate regulation of bile acid biosynthetic and recovery pathways. Biochem Biophys Res Commun 225: 128–133, 1996.[CrossRef][ISI][Medline]
  22. Walker NJ. Real time and quantitative PCR: applications to mechanism base toxicology. J Biochem Mol Toxocol 15: 121–127, 2001.[CrossRef]
  23. Wang H, Chen J, Hollister K, Sowers LC, and Forman BM. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol Cell 3: 543–553, 1999.[ISI][Medline]
  24. West KL, Ramjiganesh T, Roy S, Keller BT, and Fernandez ML. 1-[4-[4[(4R,5R)-3,3-dibutyl-7-(dimethylamino)-2,3,4,5-tetrahydro-4-hydroxy-1,1-dioxido-1-benzothiepin-5yl] phenoxy] butyl]-4-aza-1-azoniabicyclo[2.22]octane methanesulfonate (SC-435), an ileal apical sodium-codependent bile acid transporter inhibitor alters hepatic cholesterol metabolism and lowers plasma low-density lipoprotein-cholesterol concentrations in guinea pigs. J Pharmacol Exp Ther 303: 293–299, 2002.[Abstract/Free Full Text]
  25. Wong MH, Oelkers P, Craddock AL, and Dawson PA. Expression cloning and characterization of the hamster ileal sodium-dependent bile acid transporter. J Biol Chem 269: 1340–1347, 1994.[Abstract/Free Full Text]
  26. Wong MH, Oelkers P, and Dawson PA. Identification of a mutation in the ileal sodium-dependent bile acid transporter gene that abolishes transport activity. J Biol Chem 270: 27228–27234, 1995.[Abstract/Free Full Text]
  27. Xu G, Pan LX, Li H, Forman BM, Erickson SK, Shefer S, Bollineni J, Batta AK, Christie J, Wang TH, Michel J, Yang S, Tsai R, Lai L, Shimada K, Tint GS, and Salen G. Regulation of the farnesoid X receptor (FXR) by bile acid flux in rabbits. J Biol Chem 277: 50491–50496, 2002.[Abstract/Free Full Text]
  28. Xu G, Salen G, Shefer S, Tint GS, Kren BT, Nguyen LB, Steer CJ, Chen TS, Salen L, and Greenblatt D. Increased bile acid pool inhibits cholesterol 7{alpha}-hydroxylase in cholesterol-fed rabbits. Gastroenterology 113: 1958–1965, 1997.[ISI][Medline]