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Originally published In Press as doi:10.1074/jbc.M700897200 on June 1, 2007

J. Biol. Chem., Vol. 282, Issue 30, 21653-21661, July 27, 2007
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Regulation of the Sodium/Sulfate Co-transporter by Farnesoid X Receptor {alpha}*

Hans Lee{ddagger}, Melissa L. Hubbert{ddagger}§, Timothy F. Osborne, Katherine Woodford||, Noa Zerangue||, and Peter A. Edwards{ddagger}§**1

From the Departments of {ddagger}Biological Chemistry and §Medicine, David Geffen School of Medicine, and the **Molecular Biology Institute at UCLA, UCLA, Los Angeles, California 90095, the Department of Molecular Biology and Biochemistry at UCI, University of California, Irvine, California 92697, and ||XenoPort, Inc., Santa Clara, California 95051

Received for publication, January 31, 2007 , and in revised form, May 31, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fxr{alpha} is known to regulate a variety of metabolic processes, including bile acid, cholesterol, and carbohydrate metabolism. In this study, we show direct evidence that Fxr{alpha} is a key player in maintaining sulfate homeostasis. We identified and characterized the sodium/sulfate co-transporter (NaS-1; Slc13a1) as an Fxr{alpha} target gene expressed in the kidney and intestine. Electromobility shift assays, chromatin immunoprecipitation, and promoter reporter studies identified a single functional Fxr{alpha} response element in the second intron of the mouse Slc13a1 gene. Treatment of wild-type mice with GW4064, a synthetic Fxr{alpha} agonist, induced Slc13a1 mRNA in the intestine and kidney. Slc13a1 mRNA was also induced in the kidney and intestine of wild-type, but not Fxr{alpha}–/– mice, after treatment with the hepatotoxin {alpha}-naphthylisothiocyanate, which is known to result in elevated blood bile acid levels. Finally, we observed a decrease in Slc13a1 mRNA in the kidney and intestine of Fxr{alpha}–/– mice and a corresponding increase in urinary excretion of free sulfates as compared with wild-type mice. These results demonstrate that mouse Slc13a1 is a novel Fxr{alpha} target gene expressed in the kidney and intestine and that in the absence of Fxr{alpha}, mice waste sulfate into the urine. Thus, Fxr{alpha} is necessary for normal sulfate homeostasis in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Inorganic sulfate is one of the most abundant anions in mammalian blood and is essential for numerous physiological functions (1). Sulfation of endogenous and exogenous substances such as steroids, neurotransmitters, xenobiotics, and bile acids is a key process in their activation or detoxification of these compounds (2). Sulfation is also essential for the biosynthesis of a variety of structural molecules, such as glycosaminoglycan sulfate, cerebroside sulfate, and heparin sulfate (3).

In mammals, sulfate homeostasis is largely regulated by the kidney. The majority of filtered sulfate is reabsorbed in the proximal tubules, and only ~5–20% of the filtered load is excreted into the urine (4). Transcellular transport of sulfate from tubular lumen to blood depends on the sodium/sulfate co-transporter (NaS-1, Slc13a1) in the brush-border membrane (for sulfate entry into the cell) and a sulfate/anion exchanger (Sat-1, Slc26a1) in the basolateral membrane (for sulfate efflux into the blood) (4). A similar transcellular transport pathway is also used in the small intestine, mainly the distal ileum, for dietary sulfate absorption (1).

Slc13a1 was originally cloned from a rat renal cortex cDNA library using the Xenopus oocyte expression cloning system (5). This co-transporter is predominantly expressed in the renal cortex and ileum. Previous studies showed that thyroid hormone, glucocorticoids, and vitamin D can modulate serum sulfate levels, renal sulfate handling, and Slc13a1 expression (6).

Farnesoid X receptor {alpha} (Fxr{alpha}; NR1H4) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily (7). Fxr{alpha} binds to farnesoid X receptor response elements (FXREs)2 in the promoter or introns after heterodimerization with its obligate partner 9-cis-retinoic acid receptor {alpha} (RXR{alpha}) (7, 8). The optimal DNA binding sequence for the FXR{alpha}/RXR{alpha} heterodimer is an inverted repeat, composed of minor variants of two AGGTCA half-sites spaced by one nucleotide (IR-1) (9–11). However, FXR{alpha}/RXR{alpha} can bind to other sites, including everted and direct repeats (ER-8 and DR-1) to activate transcription (12, 13).

In 1995, Forman et al. (10) and Seol et al. (11) identified Fxr{alpha} transcripts in the liver, kidney, intestine, and the adrenal gland. In 1999, three groups independently identified bile acids as endogenous activators of Fxr{alpha}, providing critical insight into Fxr{alpha} function (14–16). Subsequent studies have shown tissue-specific expression of four distinct Fxr{alpha} isoforms (Fxr{alpha}1, Fxr{alpha}2, Fxr{alpha}3, and Fxr{alpha}4) that are derived from alternative promoters and splicing (17, 18). Along with their tissue-specific expression, certain target genes of Fxr{alpha} are regulated in an isoform-specific manner. Most of the Fxr{alpha} targets identified to date are regulated by all four Fxr{alpha} isoforms (18, 19). However, a few of the targets are highly regulated by the Fxr{alpha}2 and Fxr{alpha}4 isoforms and relatively refractory to Fxr{alpha}1 and Fxr{alpha}3. Such genes include I-BABP, FGF-19, CryAA, and SDC-1 (12, 18–21). The significance and mechanism of this isoform-specific gene activation has yet to be truly determined.

The identification of bile acids as ligands for Fxr{alpha} and the generation of potent synthetic (non-bile acid) agonists (22–25) have provided significant insights into the functional role of this nuclear receptor. Fxr{alpha} has been shown to have crucial roles in controlling bile acid homeostasis, lipoprotein and glucose metabolism, hepatic regeneration, intestinal bacterial growth, response to hepatotoxins, and resistance to hepatocarcinomas (19). These studies have identified important functions for Fxr{alpha} in both the liver and intestine. In contrast, the functional importance of Fxr{alpha} in the kidney and adrenal gland is poorly understood, despite the fact that these organs express high levels of Fxr{alpha}. Recently, we and others have shown that Fxr{alpha} regulates the organic solute transporters {alpha} and beta (Ost{alpha} and Ostbeta) in the intestine (21, 26–28) and kidney (21, 28). These two genes are also expressed in the adrenal gland (21). In the ileum, Ost{alpha} and Ostbeta function as a heteromeric basolateral bile acid transporter that functions in the enterohepatic circulation of bile acids (29, 30). Whether Ost{alpha} and Ostbeta also affect bile acid flux through the kidney and adrenal gland remains to be determined.

In the current study, we provide direct evidence that Fxr{alpha} is a key player in the regulation of sulfate homeostasis in mice. We identified the sodium/sulfate co-transporter, Slc13a1, as a gene regulated by Fxr{alpha} in the kidney and intestine. In vivo studies show that Fxr{alpha}–/– mice display increased sulfate wasting into the urine. The data presented herein indicate that Slc13a1 is a novel Fxr{alpha} target gene in the kidney and intestine and that Fxr{alpha} is necessary to maintain the basal expression of Slc13a1 and thus normal sulfate homeostasis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Expression constructs for the human FXR{alpha}1 and FXR{alpha}2 (pcDNA-hFXR{alpha}1 and pcDNA-hFXR{alpha}2) and human RXR{alpha} (pCMX-hRXR{alpha}) have been described previously (20). 3-(2,6-Dichlorophenyl)-4-(3'-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl-isoxazole (GW4064), a synthetic ligand for FXR{alpha}, was a gift from Drs. Tim Willson and Patrick Malloney (GlaxoSmithKline). {alpha}-Naphthylisothiocyanate (ANIT) was purchased from Sigma.

RNA Isolation and Real Time Quantitative PCR—RNA from tissues was isolated using TRIzol reagent (Invitrogen). Real time PCR was performed essentially as described (21). Briefly, 5 µg of DNase I-treated total RNA from tissues was reverse-transcribed with random hexamers using SuperScript II Reverse Transcriptase (Invitrogen). Each amplification mixture (20 µl) contained 50 ng of cDNA, 375 nM forward primer, 375 nM reverse primer, and 10 µl of SYBR Green Supermix (Bio-Rad). PCR thermocycling parameters were 95 °C for 2.5 min and 40 cycles of 95 °C for 10 s and 60 °C for 45 s. Real-time PCR was carried out using the Bio-Rad MyIQ single color real time PCR detection system. Each sample was assayed in duplicate and normalized to glyceraldehyde-3-phosphate dehydrogenase expression. Quantitative expression values were extrapolated from separate standard curves.

Identification of a Putative FXRE and EMSA—A putative FXRE in the second intron of Slc13a1 was identified using the NUBIScan computer algorithm (available on the World Wide Web). This approach is based on a weighted nucleotide distribution matrix compiled from published functional hexamer half-sites.

Electrophoretic mobility shift assays were performed essentially as described (21). Human FXR isoforms or human RXR{alpha} was synthesized in vitro using the TNT T7-coupled reticulocyte system (Promega). The sense strand sequence for wild-type and mutant Slc13a1 were 5'-cgagaggcccaggtcaatgtccggatggatgcc-3' (Slc13a1) and 5'-cgagaggcccagAAcaatgtTcggatggatgcc-3' (mutSlc13a1). The IR-1 is in boldface type, and mutations are capitalized.

Chromatin Immunoprecipitation—Wild-type mice were treated with either vehicle or GW4064 once a day for 5 days at 50 mg/kg and sacrificed, and kidneys were excised. The initial fixation of the kidneys, isolation of nuclear fraction, and sonication of chromatin were performed as described (31). Chromatin immunoprecipitation was performed using the Active Motif ChIP-IT kit according to the manufacturer's protocol with some modifications. Briefly, the sheared chromatin was precleared twice with goat IgG (20 µg) and Protein G beads, and an aliquot was stored to use subsequently as "input." Aliquots of the precleared sheared chromatin were then immunoprecipitated using goat IgG (20 µg) or anti-FXR{alpha} antibody (C-20X; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (20 µg). The resulting DNA was released from protein and used for real time quantitative PCR analysis. Quantitative PCR primers are listed in Table 2.


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  		TABLE 2
Primer sequences used for real time quantitative PCR and chromatin immunoprecipitation analysis

 
Reporter Gene and Transient Transfection Assays—The two-copy IR-1 reporter construct (pTK-2xIR-1 WT) was generated by annealing the oligonucleotides 5'-gatcccgagaggcccaggtcaatgtccggatggatgcccgagaggcccaggtcaatgtccggatggatgcca-3' and 5'-gatctggcatccatccggacattgacctgggcctctcgggcatccatccggacattgacctgggcctctcgg-3' before ligation into BamHI/BglII-digested TK-Luc. The two IR-1 sites are shown in boldface type. The two-copy mutant IR-1 reporter construct (pTK-2xIR-1mut) was generated using the same method and the oligonucleotides 5'-gatcccgagaggcccagAAcaatgtTcggatggatgcccgagaggcccagAAcaatgtTcggatggatgcca-3' and 5'-gatctggcatccatccgAacattgTTctgggcctctcgggcatccatccgAacattgTTctgggcctctcgg-3'. Mutations are capitalized.

The Slc13a1 proximal promoter (–1986 to +22 relative to the transcriptional start site) was amplified from mouse genomic DNA and cloned into KpnI/NheI sites of the pGL3 basic vector (Promega) to generate pGL3-Slc13a1. The Slc13a1 (–1986 to +22) construct was amplified using the 5' primer 5'-cccggtacctgggcattttggaattatgg-3' and the 3' primer 5'-cccgctagcgagcaggtgccttcaacagt-3'.

Transient transfections were performed on 48-well plates essentially as described (21). Briefly, 5 ng of pCMX-hRXR{alpha}, 100 ng of reporter construct, and 50 ng of pCMX-beta-galactosidase, together with 50 ng of pcDNA-hFXR{alpha}1 or pcDNA-hFXR{alpha}2, were cotransfected into HepG2 cells using the MBS mammalian transfection kit (Stratagene). The cells were treated with vehicle (Me2SO) or the indicated ligands in 10% superstripped fetal bovine serum (HyClone) for 24 h. Luciferase activity was assayed and normalized to beta-galactosidase activity. Each transfection was performed in triplicate, and experiments were repeated at least three times.

Animal Experiments—Eight to 10-week-old male and female Fxr{alpha}–/– mice (32) and their wild-type C57BL/6J littermates were housed in a pathogen-free barrier facility with a 12-h light/12-h dark cycle and fed a standard chow diet. Where indicated, wild-type mice (four mice/group) were gavaged twice a day for 4 days with either vehicle (2-hydroxypropyl-beta-cyclodextrin) or GW4064 (dissolved in vehicle) at 30 mg/kg. Animals were euthanized, and the tissues were excised and homogenized in TRIzol reagent (Invitrogen) for RNA extraction. In the initial screen (Table 1), RNA was isolated from the liver, kidney, and intestine of mice (two mice/group). RNA samples were subjected to real time quantitative PCR analysis in duplicate by Xenoport Inc. (Santa Clara, CA). Subsequent analyses were performed on RNA isolated from four individual mice treated as indicated. For the ANIT experiment, 8–10-week-old male and female Fxr{alpha}–/– mice and their wild-type C57BL/6J littermates were gavaged with a single dose of vehicle (olive oil) or ANIT dissolved in vehicle (75 mg/kg). Individual animals were placed in metabolic cages for urine collection and were provided a standard chow diet and water. Animals were euthanized at 48 h after the ANIT dose, and the indicated tissues were excised and homogenized in TRIzol reagent (Invitrogen) for RNA extraction. Blood was collected by cardiac puncture. Urine and serum were stored at –80 °C until further use.


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  		TABLE 1
Real time quantitative PCR screen of transporters and drug-metabolizing enzymes

Wild-type mice (two mice/group) were orally gavaged twice daily for 4 days with vehicle (2-hydroxypropyl-beta-cyclodextrin) or GW4064 (30 mg/kg). The resulting gene inductions in the tissues are listed.

 
Measurement of Serum and Urinary Sulfate—Urinary creatinine was measured using the Prameter Creatinine Kit (R & D Systems). Inorganic sulfate levels in urine or serum were assayed by a turbidimetric method as described (33). Briefly, urine was diluted 1:20 with 5% trichloroacetic acid. Serum samples were deproteinated by mixing with 10% trichloroacetic acid. The samples were placed on ice for 30 min and centrifuged, and the sulfate present in the supernatant was collected for the assay. Standards of Na2SO4 (0.5–2.0 mM for urine samples and 0.125–1.0 mM for serum samples) were prepared in 5% trichloroacetic acid. Fifty-µl samples, standards, or blanks were added to a 96-well plate in triplicate. Fifty µl of 30% glycerol was then added and covered, and the mixture was incubated for 10 min at room temperature. Twenty-five µl of 1% BaCl2 in 10% dextran was then added to all wells. After the contents in the wells were mixed, the absorbance was determined using a microplate reader set at a wavelength of 600 nm. The correlation coefficient of the standard curves was >0.99. Serum and urine sulfate levels were normalized to protein and creatinine, respectively.

Statistical Analysis—Mean values and S.E. were determined by the analysis of multiple independent samples, each assayed in duplicate or triplicate, as indicated in the figure legends. A two-tailed Student's t test was used to calculate p values.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Slc13a1 as a Putative Fxr{alpha} Target Gene—In an attempt to identify novel Fxr{alpha} target genes, we initially treated two wild-type mice with vehicle or the potent Fxr{alpha} agonist, GW4064, for 4 days. RNA was isolated from three (liver, kidney, and intestine) of the four tissues that express significant levels of Fxr{alpha}. The relative expression levels of 250 known transporters and drug-metabolizing enzymes were determined by real time quantitative PCR (see "Experimental Procedures"). Analysis of the data suggested that ~20 genes, including the sodium/sulfate co-transporter (NaS-1, Slc13a1) and sulfate anion transporter (Sat-1, Slc26a1), were induced greater than 1.5-fold in the kidney, intestine, or liver by GW4064 (Table 1).

In an attempt to confirm the data shown in Table 1 and to eliminate false positives, we treated additional wild-type mice (four mice/group) with either vehicle or GW4064 and isolated RNA from liver, kidney, and intestine. RNA levels of selective genes were quantified in individual mice using real time quantitative PCR using primer sets (Table 2) that differed from those used in the original screen. As indicated in Fig. 1, A and B, Slc13a1 message was significantly induced in the wild-type kidney and intestine in response to GW4064 (p < 0.05), whereas no discernable changes were observed with Slc26a1 mRNA expression. We conclude that the identification of Slc26a1 as a gene induced by Fxr{alpha} activation in the preliminary screen (Table 1) was a false positive.

In addition to Slc13a1, a sulfotransferase (Sult2a1) and a bile acid transporter (Mrp2), two previously identified Fxr{alpha} targets involved in bile acid detoxification in the liver (13, 34), were also induced in the kidney in response to GW4064 (Fig. 1A). These inductions were specific, since a xenobiotic sulfotransferase (Sult1a1) was not induced (Fig. 1A). Ost{alpha}, a previously identified Fxr{alpha} target gene expressed in the kidney and intestine, was induced and served as a positive control (Fig. 1, A and B). Moreover, quantitative PCR analysis revealed that Slc13a1 is not expressed in the liver (data not shown).


Figure 1
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  		FIGURE 1.
GW4064 treatment induces Slc13a1 mRNA in the kidney and intestine of wild-type mice. Wild-type mice (four mice/group) were orally gavaged twice daily for 4 days with vehicle or GW4064 (30 mg/kg). Total RNA was isolated from kidney (A) and intestine (B) and reverse transcribed for real time quantitative PCR analysis using the indicated gene-specific primers (Table 2). Samples from each mouse were assayed in duplicate and normalized to murine glyceraldehyde-3-phosphate dehydrogenase. Data are reported as mean ± S.E. (n = 4); *, p < 0.05.

 
The data in Fig. 1 suggest that activation of renal Fxr{alpha} following oral administration of GW4064 induces the mRNA expression of an apical sulfate transporter, Slc13a1, as well as genes involved in bile acid sulfation and transport. In contrast, the data demonstrated that the expression of the basolateral sulfate transporter, Slc26a1, was unaffected.

Identification of an FXRE within the Second Intron of the Mouse Slc13a1 Gene—Since Slc13a1 mRNA was significantly induced in both the kidney and intestine of GW4064-treated mice, we scanned the proximal promoter of the mouse Slc13a1 gene for putative FXREs using the NUBIScan computer algorithm (see "Experimental Procedures"). However, no putative IR-1 was identified in the region 10 kb upstream of the mouse Slc13a1 transcription start site. Further in silico analysis of the intronic regions of the gene identified a putative IR-1 within the second intron (Fig. 2A). We then used in vitro transcribed and translated RXR{alpha} and FXR{alpha}2 proteins in an electrophoretic mobility shift assay to determine whether the FXR{alpha}/RXR{alpha} heterodimer can bind to this putative FXRE. The data in Fig. 2B show that the FXR{alpha}2/RXR{alpha} heterodimer binds to radiolabeled probes containing the Slc13a1 IR-1 (Fig. 2B, lane 7). Probes containing a well characterized IR-1/FXRE from the PLTP proximal promoter (35) served as a positive control (Fig. 2B, lane 2). In addition, unlabeled wild-type Slc13a1 IR-1 effectively competed for the binding of FXR{alpha}2/RXR{alpha} heterodimers (Fig. 2B, lanes 3–5 and 8–10), whereas the formation of the shifted complex was relatively unaffected by the presence of the unlabeled DNA containing the mutated IR-1 sequences (mutSlc13a1) (Fig. 2B, lanes 11–13).


Figure 2
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  		FIGURE 2.
FXR{alpha}/RXR{alpha} heterodimers bind to an IR-1 element in the second intron of the murine Slc13a1 gene. A, scheme of the mouse Slc13a1 gene with the putative FXRE (IR-1) indicated. Exons are numbered and boxed. The arrow identifies the ATG in exon 1. B, FXR{alpha}/RXR{alpha} binds to Slc13a1 IR-1. Electrophoretic mobility shift assays were performed as described under "Experimental Procedures." FXR{alpha} was incubated with RXR{alpha} and the indicated radiolabeled probe in the absence or presence of unlabeled wild-type or mutant Slc13a1 IR-1 (50-, 100-, and 250-fold excess) competitor as indicated. The shifted DNA-protein complexes are identified by the arrow. PLTP, phospholipid transfer protein.

 
In order to confirm that Fxr{alpha} is associated with the putative FXRE identified in the second intron in vivo, we performed chromatin immunoprecipitation using DNA isolated from the kidneys of wild-type mice treated with GW4064. Chromatin was analyzed for Fxr{alpha} binding to the putative FXRE using an antibody to Fxr{alpha} and real time quantitative PCR primers specific for the relevant region of the second intron of the Slc13a1. A genomic region from the Slc13a1 gene, corresponding to exon 1, lacked any putative FXRE and served as a negative control (Fig. 3). The Slc13a1 second intron DNA was enriched over 3-fold relative to an equivalent sample, in which a control IgG was used in the immunoprecipitation. Fxr{alpha} binding to the negative control region of the Slc13a1 was negligible (Fig. 3). A DNA region containing a previously identified functional FXRE from a known Fxr{alpha} target gene, Ost{alpha}, was also enriched about 4-fold relative to a control IgG (Fig. 3). These data demonstrate that endogenous Fxr{alpha} is specifically recruited in vivo to the region in the second intron of Slc13a1 containing the putative FXRE.

Functional Identification of the FXRE within the Second Intron of the Mouse Slc13a1 Gene—The data in Figs. 2 and 3 suggest that the IR-1 located in intron 2 of the Slc13a1 gene is a functional FXRE. To confirm this hypothesis, we constructed luciferase reporter genes under the control of a minimal promoter and two copies of either the wild-type pTK-Slc13a1 2xIR-1 WT or mutant Slc13a1 IR-1 pTK-Slc13a1 2xIR-1 mut. The reporter constructs were transiently transfected into HepG2 cells in the presence or absence of plasmids encoding RXR{alpha} and FXR{alpha}1 or FXR{alpha}2, and the cells were then incubated for 24 h with an FXR agonist, GW4064 (Fig. 4, A and B). Transfection of pTK-2xIR-1 WT with plasmids encoding FXR{alpha}1 or -{alpha}2 and RXR{alpha} led to a robust increase in luciferase activity after the addition of GW4064 (Fig. 4A). Importantly, mutation of the IR-1 elements (pTK-2xIR-1 mut) completely abolished gene activation in response to the FXR{alpha} agonist (Fig. 4B). These data demonstrate that the single FXRE in the second intron of Slc13a1 is critical for the transcriptional activation in response to activated Fxr{alpha}.


Figure 3
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  		FIGURE 3.
Fxr{alpha} binds an FXRE in intron 2 of the Slc13a1 gene in vivo. Wild-type mice were orally gavaged once daily for 5 days with vehicle or GW4064 (50 mg/kg). Chromatin immunoprecipitation was performed as described under "Experimental Procedures." Chromatin was immunoprecipitated with either control goat IgG or anti-FXR{alpha} antibody, followed by real time quantitative PCR analysis of the precipitated DNA using primers for Slc13a1, Ost{alpha}, or a genomic region of Slc13a1 (exon 1) as negative control. *, p < 0.05.

 


Figure 4
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  		FIGURE 4.
FXR{alpha} activation of Slc13a1 IR-1 elements in a heterologous promoter. Triplicate dishes of HepG2 cells were transiently transfected with pTK-luciferase reporter construct with either wild-type (pTK-Slc13a1 2xIR-1 WT) (A) or mutant Slc13a1 FXREs (pTK-Slc13a1 2xIR-1 mut) (B) or pGL3-luciferase reporter construct under the control of the Slc13a1 proximal promoter (–1986 to +22 relative to the transcriptional start site; pGL3-Slc13a1-promoter) (C). The cells were also co-transfected with beta-galactosidase, RXR{alpha}, and either FXR{alpha}1 or FXR{alpha}2. Following treatment for 48 h with either vehicle (Me2SO; DMSO) or 1 µM GW4064, cells were lysed, and luciferase activity was determined, normalized to beta-galactosidase activity, and expressed as relative light units (RLU). All transfections were performed in triplicate, and the data are given as means ± S.E. The data shown are representative of at least two experiments. *, p < 0.05.

 
In order to rule out the possibility of the presence of a divergent but functional FXRE in the promoter of Slc13a1, we constructed a luciferase reporter gene under the control of 2 kb of the Slc13a1 proximal promoter (pGL3-Slc13a1-promoter). The reporter construct was transiently transfected into HepG2 cells in the presence or absence of plasmids encoding FXR{alpha}1 or FXR{alpha}2, and the cells were then incubated for 24 h with vehicle or GW4064. pGL3-Slc13a1-promoter was not induced following activation of FXR{alpha} (Fig. 4C), suggesting that the proximal promoter lacks a functional FXRE. The data demonstrate that the induction of the mouse Slc13a1 gene by FXR{alpha} activation is mediated by the FXRE identified in the second intron and not by an unidentified FXRE in the proximal promoter of the Slc13a1 gene.

Measurement of Urinary and Serum Sulfate Levels of Wild-type and Fxr{alpha}–/– Mice—Previous studies by Bolt et al. (36) demonstrated that vitamin D receptor null mice (Vdr–/–) exhibit a decrease in basal expression of Slc13a1 mRNA in the kidney and a corresponding decrease in renal reabsorption of free sulfate. In addition, Dawson et al. (37) observed that in addition to an increase in urinary sulfate excretion, Slc13a1–/– mice have increased levels of bile acids in the blood. The authors of this latter study concluded that decreased availability of free sulfates, as a result of Slc13a1 deletion, also affected bile acid homeostasis. However, the mechanism remains unknown.

Fxr{alpha}–/– mice also display increased bile acid levels in the blood (32). Therefore, in light of the finding that Fxr{alpha} activation also regulates Slc13a1 expression, we measured free sulfate levels in urine and serum of wild-type and Fxr{alpha}–/– mice. Mice were also treated with either vehicle or ANIT to further increase the overall bile acid levels in the blood. Previous work by our laboratory and others has shown that treatment of mice with ANIT increases the bile acid levels in the blood greater than 100-fold (21, 38), and this increase in bile acids results in activation of Fxr{alpha} target genes in the kidney and adrenal gland (21). ANIT treatment probably affects other regulatory mechanisms, in addition to bile acid activation of Fxr{alpha}. Nonetheless, the increase of bile acids in the blood in ANIT-treated mice serves as an alternative approach to the use of GW4064 to activate Fxr{alpha}.


Figure 5
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  		FIGURE 5.
Induction of Slc13a1 mRNA in the kidney and intestine from wild-type and Fxr{alpha}-deficient mice in response to ANIT. WT (hatched bars) and Fxr{alpha}–/– (black bars) mice were orally gavaged with a single dose of vehicle (olive oil) or ANIT (75 mg/kg) as indicated. The mice were euthanized after 48 h, and total RNA was isolated from kidney (A) and intestine (B) and reverse-transcribed for real time quantitative PCR analysis using the indicated gene-specific primers. Values are shown as means ± S.E. (n = 4 mice/group). *, p < 0.05. NS, not significant versus vehicle-treated wild-type mice.

 
With this caveat in mind, the data in Fig. 5 show that administration of ANIT to wild-type mice resulted in a significant (p < 0.05) increase in mRNA levels of Slc13a1 in both the kidney (Fig. 5A) and intestine (Fig. 5B). In contrast, kidney and intestinal Slc13a1 mRNA levels were unaffected after the administration of ANIT to Fxr{alpha}–/– mice (Fig. 5, A and B). ANIT treatment also increased Ost{alpha}, a previously identified Fxr{alpha} target gene (21), in the kidney and intestine of wild-type, but not Fxr{alpha}–/–, mice and served as a positive control (Fig. 5, A and B). Similarly, both the bile acid sulfotransferase (Sult2a1) and a bile acid transporter (Mrp2), two FXR targets involved in bile acid detoxification in the liver, were also induced in the kidney in response to ANIT treatment of wild-type mice. These changes were specific, since the xenobiotic sulfotransferase (Sult1a1) was not affected by ANIT treatment (Fig. 5A). Unexpectedly, the kidney mRNA levels of Slc26a1, the basolateral sulfate anion transporter, were significantly inhibited in response to ANIT treatment of wild-type mice or Fxr{alpha}–/– mice (Fig. 5A). We conclude that administration of ANIT increased plasma bile acids that activate Fxr{alpha} in the kidney and intestine, resulting in increased transcription of Fxr{alpha} target genes that include Slc13a1, Sult2a1, Mrp2, and Ost{alpha}.

Interestingly, the basal expression of Slc13a1 mRNA was significantly reduced in the Fxr{alpha}–/– as compared with wild-type mice, irrespective of the presence of ANIT (p < 0.05) (Fig. 5, A and B). In addition, the basal expression of Slc26a1 was also significantly inhibited in the intestine of Fxr{alpha}–/– mice (p < 0.05) (Fig. 5B). We also observed a slight reduction of Slc26a1 in the kidney from Fxr{alpha}–/– mice, although the decrease did not reach statistical significance (Fig. 5A).

In order to determine whether the decrease in basal expression of renal Slc13a1 mRNA resulted in a corresponding decrease in renal sulfate reabsorption, we analyzed both urine and serum for free sulfate levels. Similar to Vdr–/– mice (36), Fxr{alpha}–/– mice display an ~50% increase in sulfate levels in the urine (Fig. 6A), consistent with a defect in the ability of the renal tubular cells to reabsorb free sulfates. The data suggest that the deletion of Fxr{alpha} results in a decrease in the basal expression of Slc13a1 mRNA and a corresponding increase in renal excretion of sulfates into the urine. In addition, we observed a slight reduction in serum sulfate levels in the Fxr{alpha}–/– mice, although the decrease did not reach statistical significance (p < 0.08) (Fig. 6B). These data further suggest that the Fxr{alpha}–/– mice have adapted to the decrease in renal reabsorption of sulfates by an alternative mechanism to maintain sulfate homeostasis in the blood.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the original studies by Forman et al. (10), rodent Fxr{alpha} was shown to be expressed in the liver, intestine, adrenal cortex, and renal tubules. Subsequent studies established that Fxr{alpha} activates hepatic and intestinal target genes involved in the maintenance of bile acid, lipid, and glucose homeostasis (reviewed in Refs. 19, 39, and 40). This led to the proposal that Fxr{alpha} plays a major role in the enterohepatic circulation of bile acids. Recently, organic solute transporters {alpha} and beta (Ost{alpha} and Ostbeta) were identified as Fxr{alpha} target genes that are expressed in the adrenal gland and kidney as well as the small intestine (21, 26–28, 41). These data suggested a role for Fxr{alpha} in either bile acid or steroid transport in the kidney and adrenal gland. However, the physiological role and importance of Fxr{alpha} in the kidney and adrenal gland remain poorly understood, in part because very few Fxr{alpha} target genes have been identified in these two organs.


Figure 6
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  		FIGURE 6.
Fxr{alpha}-deficient mice have increased urinary sulfate excretion. Wild-type and Fxr{alpha}–/– mice were housed in metabolic cages and urine output was collected for 48 h. A, urinary sulfate concentration was normalized to urinary creatinine content. *, p < 0.01 versus wild-type (n = 4 mice/group). B, serum sulfate concentration in wild-type and Fxr{alpha}–/– mice. Serum sulfate concentration was normalized to serum protein content. NS, not significant versus wild-type mice (p < 0.08). Values are shown as means ± S.E. (n = 4 mice/group).

 
In order to identify novel Fxr{alpha} target genes, we treated wild-type mice with vehicle or GW4064 and compared the expression levels of ~250 transporters and drug-metabolizing enzymes that are expressed in the kidney, liver, and/or small intestine. The preliminary results suggested that only seven of the transporters, including previously identified Ost{alpha} and Ostbeta, were induced in the kidneys of GW4064-treated mice (Table 1). Analysis of additional mice treated with GW4064 confirmed that the sodium/sulfate co-transporter gene (NaS-1; Slc13a1) is induced in the kidney and intestine of mice in response to Fxr{alpha} activation (Fig. 1). The findings that Slc13a1 mRNA levels increase in an Fxr{alpha}-dependent manner in vivo and the identification of a functional FXRE in the second intron of Slc13a1 (Figs. 2–4), demonstrate that Slc13a1 is a direct Fxr{alpha} target gene. In addition, we show that Fxr{alpha} plays a major role in maintaining sulfate homeostasis, since Fxr{alpha}–/– mice display a decreased basal expression of Slc13a1 in the kidney (Fig. 5A) and increased sulfate wasting into the urine (Fig. 6).

All cells require inorganic sulfate for normal function. Sulfate is among the most important macronutrients in cells and is one of the most abundant anions in human plasma (1). Sulfate is essential for numerous physiological functions, including activation and detoxification of various endogenous and exogenous substances, including xenobiotics, steroids, neurotransmitters, and bile acids (2). Structural molecules, such as glycosaminoglycans, cerebrosides, and heparin, require sulfate conjugation for their biosynthesis (3).

A number of proteins have been identified to date that transport sulfate across the cell plasma membrane. The renal proximal tubular cells express two functionally and structurally distinct sulfate transporters: the sodium/sulfate co-transporter (NaS-1; Slc13a1) and the sulfate anion transporter 1 (Sat-1; Slc26a1) (4, 6, 42). These two transporters function to reabsorb filtered sulfate back into the blood. The most widely studied sulfate transporter, Slc13a1, is expressed mainly in the mammalian kidney and intestine. Slc13a1 is expressed on the brush border membrane of renal tubular cells and mediates the uptake of inorganic sulfate that has been filtered through the glomeruli. The inorganic sulfates that have been taken up by Slc13a1 are returned to the blood by Slc26a1 expressed on the basolateral membrane of renal tubular cells, thus maintaining sulfate homeostasis in the blood. Both Slc13a1 and Slc26a1 are also expressed in the intestine (43), where it has been proposed that these two proteins may function to absorb dietary sulfates.

The importance of Slc13a1 in sulfate homeostasis is well established. Mice deficient in Slc13a1 (Slc13a1–/–) display hyposulfatemia as a result of decreased renal sulfate reabsorption (37). In addition, these mice have major physiological defects, such as growth retardation, reduced fertility, and spontaneous seizures (37). Additional studies have shown that the Slc13a1 gene is regulated at the transcriptional level by the activation of the vitamin D receptor (Vdr) and thyroid hormone receptor (44), two members of the nuclear receptor family. The response elements in the promoter region of the mouse Slc13a1 gene that interact with these two nuclear receptors have been identified (44). In addition, the relative importance of Vdr in the maintenance of sulfate homeostasis has been established, since mice deficient in Vdr display increased sulfate excretion into the urine and a concurrent decrease in serum sulfate levels (36). The observed hyposulfatemic phenotype in the Vdr–/– mice is attributed to a decrease in the basal expression of Slc13a1 as compared with wild-type mice (36).

In the current study, we report changes in Slc13a1 mRNA expression and sulfate levels in the urine and serum of Fxr{alpha}–/– mice similar to those previously observed in Vdr–/– mice (36). We show that the Slc13a1 gene is regulated at the transcriptional level by the nuclear receptor Fxr{alpha}. Deficiency in Fxr{alpha} leads to a decrease in the basal expression of Slc13a1 mRNA (Fig. 5). More importantly, as a result of this decrease, there is an increase in the excretion of sulfates into the urine and a decrease in serum sulfate (Fig. 6), indicating a defect in sulfate reabsorption by renal tubular cells. The reduction of sulfate levels in the serum of Fxr{alpha}–/– mice, however, did not reach statistical significance (Fig. 6B). It is tempting to speculate that there are compensatory mechanisms that could induce metabolism of amino acids or sulfated macromolecules that allow for the maintenance of relatively normal sulfate levels in the blood (1). Taken together with previous studies, we conclude that Slc13a1 expression and sulfate homeostasis are controlled in vivo by both Vdr and Fxr{alpha}.

The importance of Fxr{alpha} activation, as opposed to loss of Fxr{alpha}, in maintaining sulfate homeostasis remains unclear. Although activation of Fxr{alpha} either by a synthetic agonist (Fig. 1) or by increased availability of bile acids (Fig. 5) can lead to the up-regulation of Slc13a1 mRNA, our data suggest that the increase in message has no significant effect on blood or urine sulfate levels (data not shown). The data in Fig. 5 suggest that increased bile acid levels in the renal tubular cells induce the expression of Slc13a1 by activation of Fxr{alpha}, thus increasing the availability of inorganic sulfates in the renal tubular cells. Since the accumulation of bile acids is toxic, the renal tubular cells may utilize the increased sulfate pool as a detoxification process to eliminate bile acids into the urine. Consistent with this hypothesis, we observed an increase in the expression of both a bile acid sulfotransferase (Sult2a1) and a bile acid transporter (Mrp2) in the kidney in response ANIT (Fig. 5A). In addition, we also observed a down-regulation of Slc26a1 mRNA, the basolateral sulfate transporter, in response to ANIT (Fig. 5A). In contrast to Slc13a1, the transcriptional regulation of Slc26a1 remains to be established. Nevertheless, such "contraregulation" of the two sulfate transporters (increase in Slc13a1 and decrease in Slc26a1) in response to bile acids may be required to retain sulfates in the renal tubular cells to be utilized for bile acid detoxification. Such sulfated and conjugated bile acids might then be excreted into the urine rather than being transported into the blood, where bile acids are already at supraphysiological levels as a result of the ANIT treatment. However, additional studies will be necessary to determine whether the repression of Slc26a1, observed in the kidneys of ANIT-treated mice, is a result of the toxic side effects of the drug.

Similar to the kidney, Slc13a1 mRNA levels are induced in the intestine of mice in response to Fxr{alpha} activation (Figs. 1B and 5B). Intestinal Slc13a1 is thought to mediate the absorption of dietary sulfates (43). Further analysis will be required to determine if reduced expression of intestinal Slc13a1 message in the Fxr{alpha}–/– mice is associated with changes in absorption of dietary sulfates.

In summary, the current study identifies the sodium/sulfate co-transporter, Slc13a1, which is expressed in the kidney and intestine, as a novel Fxr{alpha} target gene. The expression and regulation of Slc13a1 in the kidney is disrupted in Fxr{alpha}–/– mice. Such changes are associated with increased excretion of inorganic sulfates into the urine. The findings of this study implicate a novel role for Fxr{alpha} in sulfate homeostasis.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL-68445 and HL-30568 (to P. A. E.) and a grant from the Laubisch Fund (to P. A. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

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

2 The abbreviations and trivial name used are: FXRE, farnesoid X receptor response element; RXR{alpha}, 9-cis-retinoic acid receptor {alpha}; FXR{alpha}, farnesoid X receptor {alpha}; IR, inverted repeat; ANIT, {alpha}-naphthylisothiocyanate; GW4064, 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl-isoxazole; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Frank Gonzalez (National Institutes of Health) for initially providing Fxr{alpha}+/– mice.



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