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Molecular Endocrinology, doi:10.1210/me.2007-0025
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Molecular Endocrinology 21 (8): 1769-1780
Copyright © 2007 by The Endocrine Society

A Common Polymorphism in the Bile Acid Receptor Farnesoid X Receptor Is Associated with Decreased Hepatic Target Gene Expression

Catia Marzolini, Rommel G. Tirona, Guillermo Gervasini, Balasubramanian Poonkuzhali, Mahfoud Assem, Wooin Lee, Brenda F. Leake, John D. Schuetz, Erin G. Schuetz and Richard B. Kim

Division of Clinical Pharmacology, Department of Medicine (C.M., B.F.L.) and Division of Hematology/Oncology, Department of Medicine (W.L.), Vanderbilt University Medical Center, Nashville, Tennessee 37232; Division of Clinical Pharmacology, Department of Medicine (R.G.T., R.B.K.) and Department of Physiology and Pharmacology (R.G.T., R.B.K.), The University of Western Ontario, London, Ontario, Canada N6A 5A5; Department of Pharmacology and Psychiatry (G.G.), Medical School, University of Extremadura, 06071 Badajoz, Spain; and Department of Pharmaceutical Sciences (B.P., M.A., J.D.S., E.G.S.), St. Jude Children’s Research Hospital, Memphis, Tennessee 38105

Address all correspondence and requests for reprints to: Dr. Richard B. Kim, Division of Clinical Pharmacology, Department of Medicine, The University of Western Ontario, 339 Windermere Road, Room ALL-152, London Health Sciences Centre, University Hospital, London, Ontario, Canada N6A 5A5. E-mail: richard.kim{at}lhsc.on.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The farnesoid X receptor (FXR or NR1H4) is an important bile-acid-activated, transcriptional regulator of genes involved in bile acid, lipid, and glucose homeostasis. Accordingly, interindividual variations in FXR expression and function could manifest as variable susceptibility to conditions such as cholesterol gallstone disease, atherosclerosis, and diabetes. We performed an FXR polymorphism discovery analysis of European-, African-, Chinese-, and Hispanic-Americans and identified two rare gain-of-function variants and a common single nucleotide polymorphism resulting in a G-1T substitution in the nucleotide adjacent to the translation initiation site (FXR*1B) with population allelic frequencies ranging from 2.5 to 12%. In cell-based transactivation assays, FXR*1B (-1T) activity was reduced compared with FXR*1A (-1G). This reduced activity for FXR*1B resulted from neither decreased translational efficiency nor the potential formation of a truncated translational variant. To further define the relevance of this polymorphism, gene expression was examined in a human liver bank to reveal that levels of the FXR target genes small heterodimer partner and organic anion transporting polypeptide 1B3 were significantly reduced in livers harboring an FXR*1B allele. These findings are the first to identify the presence of a common genetic variant in FXR with functional consequences that could contribute to disease risk or therapeutic outcomes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE FARNESOID X RECEPTOR (FXR or NR1H4) is an intracellular, bile-acid-activated nuclear receptor (NR) that regulates the genes involved in bile acid (1, 2, 3), lipid (1), and glucose homeostasis (4). In humans as in other species, the FXR gene, also called FXR{alpha} (5), encodes four isoforms (FXR{alpha}1, FXR{alpha}2, FXR{alpha}3, and FXR{alpha}4). FXR{alpha}1 and FXR{alpha}2 are derived from a common promoter but differ in that FXR{alpha}1 contains an additional four amino acid (MYTG) insertion within the hinge region of the receptor as a result of differential splicing (6). FXR{alpha}3 and FXR{alpha}4 are produced from a separate promoter, creating isoforms with a larger N-terminal activation function (AF-1) region than FXR{alpha}1 and FXR{alpha}2 but differ from each other because of the MYTG insertion. FXR isoforms may differentially regulate the expression of target genes, but the physiological relevance of these findings is uncertain at present (7). FXR{alpha}1 and FXR{alpha}2 are highly expressed in the adrenals, liver, small intestine, and kidney, whereas FXR{alpha}3 and FXR{alpha}4 are expressed at lower levels and with differing tissue distribution. For example, FXR{alpha}3 and FXR{alpha}4 are not well expressed in liver and adrenals, tissues that highly express FXR{alpha}1 and FXR{alpha}2 (6).

The physiological importance of FXR has primarily been revealed in FXR-deficient mice (1, 8), which are shown to develop a constellation of disorders, including dyslipidemia (1), cholesterol gallstone disease (9), cholestasis (10), hepatic steatosis, blunted hepatic regenerative capacity (11), hyperglycemia (12, 13), peripheral insulin resistance (14), and loss of intestinal bacterial defense (15). Hence, there is considerable preclinical data to suggest potential for treatments of human diseases such as atherosclerosis, diabetes, cholestatic liver, and gallstones by pharmacological modulation of FXR activity (16). With appreciation that FXR plays a central role in metabolism, it is conceivable that functional genetic polymorphisms in FXR, if they exist, as well as variation in its expression would impact multiple pathways governing lipid, bile acid, and glucose homeostasis particularly in organs such as the liver. This possibility is not without precedence because mutations and polymorphisms in several members of the NR superfamily such as vitamin D receptor (17), peroxisome proliferator activated receptor {alpha} (18), peroxisome proliferator activated receptor {gamma} (19), liver X receptor (20), small heterodimer partner (SHP) (21), and hepatocyte nuclear factor 1{alpha} (22) and hepatocyte nuclear factor 4{alpha} (23) are associated with various metabolic disorders. However, to date, identification and functional characterization of FXR polymorphisms have not been described.

Herein, we screened DNA from European-, African-, Chinese-, and Hispanic-Americans and identified several single nucleotide polymorphisms (SNPs) in the FXR gene and assessed their functional consequences. In addition to identifying two rare nonsynonymous SNPs associated with an apparent gain in activity, a common FXR polymorphism was found to have reduced functional activity in vitro and was associated with decreased target gene expression in liver. We therefore propose that this functional FXR polymorphism may be linked to diseases involving bile, lipid, and glucose metabolism.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genetic Polymorphisms in FXR
Given the predominant expression of FXR{alpha}1 and FXR{alpha}2, as opposed to FXR{alpha}3 and FXR{alpha}4, in liver and intestine, organs responsible for bile acid, cholesterol, and glucose homeostasis, we examined genetic variability in the chromosomal regions encoding these specific isoforms. The FXR gene encoding the major isoforms is found on chromosome 12 and is composed of 11 exons, with the translation initiation site located within exon 3 (Fig. 1Go). Genetic screening of the coding exons (exons 3–11) from 142 European-American and 95 DNA samples for each African-, Chinese-, and Hispanic-American ethnic groups was performed by PCR and temperature-dependent capillary electrophoresis. On comparison with the FXR reference sequence (GenBank accession no. NM_005123; defined here as FXR*1A), several polymorphisms were identified: G-1T, C643T (H215Y), and G646T (A216S), which we define as FXR*1B, FXR*2, and FXR*3, respectively. Two synonymous polymorphisms (C783T, 261N; 1341T, 447H) were also found, and their locations predict amino acid differences within the hinge region of FXR (Fig. 1Go). Most coding region variants were rare with low genotypic frequencies (<1%) (Table 1Go). However, a polymorphism immediately 5' to the initiation AUG codon (G-1T, FXR*1B) (Fig. 1Go) was relatively common among Chinese (12.1%)-, Hispanic (5.8%)-, African (3.2%)-, or European (2.5%)-Americans. The C643T variation was observed only in European-Americans (0.7%), whereas G646T and C787T were only seen in Chinese-Americans (0.5%) and C1341T was detected only in Hispanic-Americans (0.5%). After comparison of FXR cDNA sequences among various species, it is seen that the nonsynonymous polymorphisms (FXR*2 and FXR*3) predict amino acid changes in relatively conserved residues (Fig. 1Go).


Figure 1
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  		Fig. 1. Polymorphisms in the Human FXR Gene

The major FXR isoforms, FXR{alpha}1 and FXR{alpha}2, are encoded by a gene on chromosome 12 containing 11 exons with the translation initiation site located in exon 3. Polymorphisms in FXR were identified in exon 3 (G-1T, FXR*1B) and exon 6 (C643T, FXR*2; G646T, FXR*3). The structure domains of FXR are defined by the N-terminal ligand-independent transcriptional activation AF-1 domain, the DNA-binding C domain, the hinge region, and the C-terminal ligand-binding domain containing the ligand-dependent AF-2 activation domain. FXR*2 and FXR*3 polymorphisms encode changes in amino acids within the hinge region that are relatively conserved among species.

 

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  		Table 1. Allellic Frequencies of SNPs in FXR among Various Ethnic Populations

 
Functional Analysis of FXR Variants
A luciferase-based reporter driven by the FXR-sensitive, bile salt export pump (BSEP) promoter was transfected into human hepatocellular carcinoma cells (HepG2) along with expression plasmids containing FXR*1A, FXR*1B, FXR*2, and FXR*3 or vector control. This reporter construct is driven by the proximal promoter region of the human BSEP gene (–1540 to +75 relative to the transcription start site). We previously identified a functional FXR response element (IR-1) in BSEP at the +50 position (24). Luciferase activity was measured after treatment of cells with the FXR ligand chenodeoxycholic acid (CDCA), at a physiological concentration of 20 µM. FXR*1A strongly activated the BSEP promoter in a bile-acid-dependent manner as evidenced by a 24-fold increase in reporter signal (Fig. 2AGo). For FXR*1B, both basal and bile-acid activated reporter activities were lower (52 and 76% of FXT *1A, respectively) than those observed for FXR*1A. For FXR*2 and FXR*3, basal (162 and 125% of FXR*1A, respectively) and CDCA-treated (114 and 129% of FXR*1A, respectively) reporter activities were higher than FXR*1A (Fig. 2AGo). Over a wide concentration range (0.1–25 µM), bile-acid activation of BSEP promoter continued to show that FXR*1B activity is reduced (Fig. 2BGo). Furthermore, FXR*1B activity remained lower than FXR*1A when the reporter assay was performed in other human cell lines, including Caco-2 (colorectal carcinoma) and HeLa (cervical carcinoma) (data not shown). Because the G-1T polymorphism in FXR*1B is not located within the predicted coding sequence, lower activity of this variant in vitro compared with FXR*1A may have resulted from differences in transcription and translation efficiency.


Figure 2
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  		Fig. 2. Cell-Based Transactivation by FXR Variants in Response to Bile-Acid Treatment

HepG2 cells were transfected with BSEP promoter-luciferase (firefly luciferase), pRL-CMV (Renilla luciferase to control transfection efficiency), and either vector control or FXR*1A, FXR*1B, FXR*2, or FXR*3. After 24 h of treatment with (filled bars) or without (open bars) CDCA (20 µM) (A) or increasing concentrations of CDCA (B), luciferase activities were determined. Bars represent mean ± SD of firefly luciferase/Renilla luciferase activities (relative luciferase activity). In A, statistically significant differences (P < 0.05) between the FXR variants and FXR*1A in basal (*) or bile-acid-treated activities (#) were calculated with the Student’s t test. Statistical significance in differences of bile-acid-mediated luciferase activities between FXR variants and FXR*1A at the same CDCA concentration (*) was determined with Student’s t test (B).

 
Transcription and Translation Efficiency of the G-1T Polymorphism (FXR*1B)
To assess whether decreased in vitro activity of FXR*1B compared with FXR*1A was attributable to lower steady-state FXR mRNA levels, quantitative real-time PCR analysis was performed. Recombinant FXR originating from the expression plasmids was detected by PCR using one primer located at the 3' end of the FXR open reading frame and another primer located in the artificial 3' untranslated region coded by the expression plasmid (pEF-V5/His; Invitrogen, Carlsbad, CA) (supplemental Table 2, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). There was a lack of amplicon detected in HepG2 cells transfected with blank (mock) plasmid, whereas a robust signal was detected in cells transfected with FXR*1A or FXR*1B (Fig. 3AGo), indicating that the observed amplification was specific to ectopic FXR rather than endogenous FXR expression. Levels of ectopic FXR*1A and FXR*1B expression in transfected cells did not appear different (Fig. 3AGo). This finding suggests that the G-1T polymorphism did not decrease FXR transcription to result in decreased FXR activity. Because the G-1T polymorphism lies within the sequence spanned by the Kozak consensus motif (25), the rate of FXR protein synthesis derived from FXR*1A and FXR*1B expression plasmids was evaluated in a cell-free, rabbit reticulocyte lysate transcription/translation system. In this assay, we used C-terminus tagged (V5) FXR cDNAs because the rabbit polyclonal antibody (C-20; Santa Cruz Biotechnology, Santa Cruz, CA) used to detect high-level FXR expression in human livers (see below) reacted nonspecifically with elements of the rabbit reticulocyte lysate in Western blot analyses. With the V5 tag, we were able to use mouse monoclonal antibodies to clearly detect the recombinant protein synthesized. Although C-terminal modification around the AF-2 region is known to modulate ligand-dependent transactivation of NRs, we have used the V5 tags only in experiments examining protein expression and not in functional activity assays. There remains the possibility, however, that the V5 tags alter protein stability. Given these considerations, time-dependent FXR protein (~55 kDa) expression was observed in both transcription/translation reactions containing FXR*1A or FXR*1B plasmids, which was apparent after 15 min and reached a plateau by 45 min (Fig. 3AGo). The rates for FXR protein synthesis with FXR*1A and FXR*1B were not markedly different. To further examine transcription/translation in an intact cell system, FXR*1A and FXR*1B expression plasmids were transfected into HeLa cells using a vaccinia virus system (26). This expression system differs from the method used in the reporter assays in that FXR cDNA-transfected HeLa cells are transduced with vaccinia virus harboring recombinant T7 RNA polymerase to enhance cellular expression. With this method, we can readily detect FXR protein levels by Western blot, whereas in the case of transiently transfected HepG2 cells, FXR protein levels remain too low to be observed. Again, with overexpression in this cell-based system, there were no discernable differences in FXR protein expression in cells transfected with FXR*1A and FXR*1B.


Figure 3
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  		Fig. 3. Effect of the G-1T (FXR*1B) Polymorphism on the Efficiency of the Transcription/Translation

A, HepG2 cells were transiently transfected with FXR*1A, FXR*1B, or blank vector control, and ectopic FXR expression was determined by quantitative real-time PCR analysis. B, At various intervals, 2 µl of the in vitro transcription/translation reaction (rabbit reticulocyte lysate system) with FXR*1A or FXR*1B cDNA were separated by SDS-PAGE and electroblotted. Blots were probed with anti-V5 antibody, which detects the C-terminus tag. A main band of approximately 55 kDa is present in both -1G and -1T plasmids, which was quantified by densitometry. C, HeLa cells were transiently transfected with vector control, FXR*1A, or FXR*1B using a recombinant vaccinia virus (VTF-7 virus) expression system. Whole-cell lysates (2 µg) were analyzed by immunoblot using V5 antibody.

 
Analysis of a Potential Translational Variant Arising from FXR*1B
Because the rates of transcription/translation did not appear be different between FXR*1A and FXR*1B, we examined whether reduced function of the G-1T polymorphism resulted from synthesis of a low-activity protein variant synthesized through translational initiation at a downstream start codon located four amino acids after the first AUG site (Fig. 4AGo). This was considered a possibility because FXR in several species such as dog and rat lack the four N-terminal amino acids found for the human ortholog (Fig. 4AGo). Hence, an expression plasmid encoding a four-amino acid, N-terminus, truncated version of human FXR ({Delta}hFXR) (Fig. 4BGo) was created and assessed in the cell-based transactivation assay. When compared with wild-type FXR (hFXR WT), {Delta}hFXR possessed both higher basal (482%) and bile-acid-inducible (204%) activity (Fig. 4CGo). In the converse scenario, when the four amino acids found in the hFXR N terminus were inserted into rat FXR (rFXR) to produce the expression plasmid 4A.A. + rPXR, the modification did not have a significant effect on basal or ligand-activated reporter expression (Fig. 4CGo). Together, these findings indicate that reduced transactivation activity of FXR*1B relative to FXR*1A was not attributable to differences in transcription/translation or the formation of a translational variant lacking four N-terminal amino acids.


Figure 4
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  		Fig. 4. Effect of a Potential N-Terminus Translational Variant on FXR Activity

A, Alignment of N-terminal FXR amino acid sequences among various species. B, N-terminal amino acid sequence from mutated FXR expression plasmids; hFXR (FXR*1A), a four amino acid truncated human FXR ({Delta}hFXR), rFXR, and a four amino acid N-terminus addition to rFXR (rFXR + 4A.A.). C, Cell-based transactivation by FXR mutants in response to bile-acid treatment. HepG2 cells were transfected with BSEP promoter-luciferase and vector control or hFXR, {Delta}hFXR, rFXR, or rFXR + 4A.A. After 24 h of treatment with (filled bars) or without (open bars) CDCA (20 µM), luciferase activities were determined. Bars represent mean ± SD. Statistically significant differences (P < 0.05) between the FXR mutants and hFXR in basal (*) or bile-acid-treated (#) activities were calculated with the Student’s t test.

 
Variability in Hepatic FXR Expression
The abundance of FXR in tissues likely influences the degree of regulation of its target genes. Therefore, interindividual variability in FXR expression was assessed by quantitative analysis of mRNA and protein in a bank of 21 human livers from Caucasian-American donors (Nashville Regional Organ Procurement Agency, Nashville, TN). FXR mRNA varied 7-fold in these livers with relative copy numbers of 14–100 per 106 copies of 18S ribosomal RNA (Fig. 5BGo). Similarly, there was an approximately 10-fold variation in immunodectectable FXR protein among livers (Fig. 5AGo). A significant correlation exists between FXR mRNA and FXR protein content in the 21 livers (r2 = 0.29; P = 0.01) as illustrated in Fig. 5BGo. Genotypic analysis revealed that each liver examined in this bank was homozygous for FXR*1A. These findings demonstrate the presence of relatively high interindividual variability in hepatic FXR expression.


Figure 5
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  		Fig. 5. Relationship between Hepatic FXR mRNA and Protein

A, FXR protein expression in 21 human livers (Vanderbilt University). Total cell lysate proteins were analyzed by immunoblot with anti-FXR antibody (C-20; Santa Cruz Biotechnology) and then stripped and probed with anti-calnexin (Stressgen Biotechnologies) antibody. Bands were analyzed by densitometric analysis. Human liver 140 (HL140) was used as a control in each blot. B, Correlation between FXR mRNA and FXR protein in human livers. FXR mRNA was determined by quantitative real-time PCR and FXR protein in each liver was divided by the corresponding expression of calnexin to obtain the normalized protein expression. All livers in this liver bank were FXR*1A/*1A genotype.

 
FXR*1B Genotype Is Associated with Reduced Hepatic FXR Target Gene Expression
Another liver bank (n = 165) at St. Jude Children’s Research Hospital contained eight donors who were heterozygous for FXR*1A/*1B. Gene expression analysis of these livers showed that the presence of an FXR*1B allele did not have an impact on FXR mRNA levels (Fig. 6AGo). Similarly, there was a lack of differences in mRNA expression of the FXR target gene BSEP (24) between livers homozygous for FXR*1A and those heterozygous for FXR*1B (Fig. 6BGo). Furthermore, whereas the bile-acid receptor is an indirect negative regulator of the hepatic basolateral sodium-dependent taurocholate transporting polypeptide (NTCP) (27), FXR genotype was without influence on the expression of this transporter (Fig. 6DGo). However, livers carrying one FXR*1B allele had significantly lower mRNA levels of the FXR target genes SHP (2, 3) and organic anion transporting polypeptide (OATP) 1B3 (28) than those homozygous for FXR*1A (Fig. 6Go, C and F). Evidently, the expression of the sodium-independent hepatic bile-acid transporter OATP1B1 was similarly associated with FXR genotype (Fig. 6EGo) despite that it has not been considered a direct target of FXR.


Figure 6
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  		Fig. 6. Relationship between FXR Genotype and Hepatic Gene Expression

A human liver bank (St. Jude Children’s Research Hospital) was genotyped for FXR*1B and mRNA gene expression for FXR (A), BSEP (B), SHP (C), NTCP (D), OATP1B1 (E), and OATP1B3 (F) was determined by quantitative real-time PCR analysis. In the left column, each vertical bar represents the gene expression in a single liver whose identification number is indicated on the horizontal axis. The FXR genotype of each liver is shown as a gray square (WT, FXR*1A/*1A) or as an orange square (heterozygous, FXR*1B/*1A). In the right column, gene expression in livers is shown in relation to FXR genotype. Significant differences (P < 0.05) were observed in SHP, OATP1B1, and OATP1B3 expression between livers genotyped with FXR*1A/*1A (WT) and those with FXR*1A/*1B (HET).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
To date, the extent or functional relevance of genetic polymorphisms in FXR has not been reported despite increasing evidence of a critical role of this NR in bile acid, lipid, and glucose homeostasis. In this report, we describe the identification of five polymorphisms in the coding region of FXR, including two nonsynonymous polymorphisms, in populations of European-, African-, Chinese-, and Hispanic-Americans. Genotypic analysis revealed that G-1T polymorphism (FXR*1B) located in the sequence flanking the start codon was relatively common among populations (2.5- 12%). The nonsynonymous polymorphisms FXR*2 and FXR*3 that alter amino acids localized within the FXR hinge region separating the DNA binding and ligand binding domains were uncommon.

Given the relative prevalence of FXR*1B, we focused our attention on its potential functional relevance. Although FXR*1B had reduced transactivational activity in vitro compared with FXR*1A and that this SNP is located in the Kozak consensus motif (25), we could not confirm that this effect was a result of decreased transcription/translation or the formation of a low-activity, translational variant. A lack of influence of transcription on the reduced activity of FXR*1B is also evidenced by similar FXR mRNA expression in livers that were FXR*1A/*1A or FXR*1A/*1B. Although in the current study we were unable to show in vitro differences in the transcription or translational efficiency FXR*1A vs. *1B, it is of interest to note that the SNP that defines the FXR*1B appears in a position that could influence the affinity for the AUG start codon. In dogs and rats, FXR is encoded by genes that use an AUG start codon that creates a protein four amino acids smaller at the N terminus than that for humans, chimpanzees, and chickens (Fig. 4AGo). Similarly, N-terminal translational isoforms of the glucocorticoid receptor have been described whose abundance differs in tissues to cause differential effects on target gene expression (29). In vivo, as evidenced by human liver target gene expression data, it would seem FXR*1B is clearly associated with reduced target gene expression, fully consistent with our in vitro luciferase-based reporter activity data (Fig. 2Go). It should be noted that sequences flanking the initiating AUG codon are capable of modulating the ability of the 40S ribosomal subunits to stop and initiate translation in vitro, especially those at positions –3 and +4. However, although a C at position –1 is optimal, this identity of this nucleotide is not considered to strongly impact translation (30). Despite this, polymorphisms or mutations at the –1 position of the {alpha}-tocopherol transfer protein (31), annexin V genes (32), and CD40 (33) are associated with increased risk of ataxia, myocardial infarction, and Grave’s disease, respectively.

Consistent with a reduced in vitro functional activity, the hepatic expression of the FXR target genes SHP and OATP1B3 are significantly lower in livers harboring FXR*1B than those homozygous for FXR*1A. Although it remains uncertain how decreased SHP expression in individuals with FXR*1B genotype would manifest, studies in SHP-null mice demonstrate that SHP deficiency causes increased hepatic insulin sensitivity (34). Interestingly, the hepatic expression of the FXR target gene BSEP was not associated with FXR genotype. Hence, the impact of FXR polymorphisms appears to be target gene dependent, suggesting that the overall influence of FXR on the regulated expression of individual genes within the larger network regulating hepatic bile acid, lipid, and glucose metabolism may be complex.

The relatively unannotated nature of the liver samples examined in this study must be considered in evaluating the impact of the FXR genotype-phenotype relationships. In this study, we identified 8 of 186 archived livers with the FXR*1B genotype. Given the allelic frequency of the FXR*1B polymorphism (~2–12%), a larger cohort of livers needs to be examined to draw better conclusions regarding the hepatic effects of FXR genotype. Furthermore, interindividual differences in intrahepatic bile-acid exposure and concentrations could impact on the relative magnitude of FXR activation and target gene expression. Because diurnal variation in NR expression is known to occur (35), the time of tissue acquisition needs to be considered in the evaluation of FXR target gene expression. Clearly, population-based studies in metabolic diseases are required to understand the clinical relevance of the current findings.

Overall, there is a remarkable paucity of nonsynonymous SNPs in FXR, suggesting that this is an important and evolutionary conserved gene critical to the maintenance of organ function and cellular homeostasis. The common, noncoding polymorphism FXR*1B is intriguing in that both in vitro and phenotypic analyses are consistent with an overall reduced function. We therefore propose that FXR*1B may be linked to disease and risk factor phenotypes or therapeutic outcomes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Identification of SNPs in FXR and Their Allelic Frequencies
Genomic DNA was isolated from blood samples obtained from healthy volunteers (47 European-Americans) using the QiAmp system (Qiagen, Valencia, CA). The protocol was approved by the Vanderbilt University Institutional Review Board, and an informed consent was obtained for every sample. In addition, genomic DNA isolated from peripheral blood lymphocytes of healthy European-, African-, Chinese-, or Hispanic-American volunteers was purchased from Coriell Cell Repositories (Camden, NJ) (n = 95) (Camden, NJ). Primers pairs (found in supplemental information, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org) used in the amplification of the FXR gene were designed to span all coding exons and at least 100 bp of the flanking intronic region. PCR followed by temperature-dependent capillary electrophoresis (Reveal; SpectruMedix, State College, PA) was used to identify the presence of FXR allelic variants. Samples presenting differences in thermodynamic stability and mobility of heteroduplexes from homoduplexes were sequenced (ABI 3700 DNA Analyzer; Applied Biosystems, Foster City, CA). FXR genotyping of a liver bank (St. Jude Children’s Research Hospital) containing donors from 100 Caucasian-Americans, 32 Hispanic-Americans, and 33 African-Americans was done by PCR and direct sequencing.

Plasmid Constructs
The human FXR cDNA (including four bases upstream to the start codon) was obtained by PCR using the primers 5'-GAGGATGGGATCAAAAATGAATCTCATTG-3' and 5'- TCACTGCACGTCCCAGATTTCACAGAG-3', followed by cloning into pEF6/V5-His vector (Invitrogen). pEF-hFXR was sequenced using an ABI 3700 DNA analyzer (Applied Biosystems) and found to match the published reference sequence (GenBank accession no. NM_005123; FXR*1A). This clone corresponds to the FXR{alpha}1 isoform and was used as a template for the creation of FXR variants. Allelic variants G-1T (noncoding, FXR*1B), C643T (H215Y, FXR*2), and G646T (A216S, FXR*3) were created by site-directed mutagenesis (Stratagene, La Jolla, CA). {Delta}hFXR expression plasmid was obtained by PCR using the primers 5'- CAAAAATGAATCTCATTGAACATTCC-3' and 5'-TCACTGCACGTCCCAGATTTCACAGAG-3'. rFXR cDNA was amplified by PCR with primer pairs 5'-ATGAATCTGATTGGGCCCTCCC-3' and 5'-TCACTGCACATGCCAGATCTCA-3' and cloned into pEF6/V5-His. A four-amino acid insertion (MGSK) at the N terminus of rFXR was created by PCR methods with primer pairs 5'-ATGGGATCAAAAATGAATCTGATTGGG-3' and 5'-TCACTGCACATGCCAGATCTCA-3'. The BSEP luciferase promoter construct was prepared by PCR from the proximal 1.6-kb fragment of the BSEP promoter (–1540/+75) using the primers 5'-GATCTTCATGGCACCAGA-3' and 5'-GGAAATAATGGACTCCACTGTG-3'. This fragment was subcloned into pGL3 Basic vector (Promega, Madison, WI). Plasmid DNA was quantified using the Picogreen assay (Invitrogen).

Transient Transfection Assay
Human hepatoma cells (HepG2; American Type Culture Collection, Manassas, VA) were grown in 24-well plates and transfected in triplicate as described previously (36) with 250 ng of the human BSEP promoter-firefly luciferase reporter, 250 ng of the FXR expression plasmids, as well as control Renilla luciferase reporter (pRL-CMV) plasmid (2.5 ng) driven by the cytomegalovirus (CMV) promoter to normalize transfection efficiency with Lipofectin (Invitrogen). After treatment with CDCA (Sigma, St. Louis, MO) for 24 h, dual-luciferase activities were determined using Dual-Luciferase Reporter reagents (Promega). Data are expressed as the ratio of firefly to Renilla luciferase activities (i.e. relative luciferase activity).

Cell-Free Transcription/Translation and Cellular Expression
V5-tagged FXR expression plasmids were created by removing the termination codons from the plasmids by mutagenesis and for further use in reactions and transfections. In transactivation assays, tagged proteins were functionally similar to untagged FXR. Transcription/translation assay was performed using the TNT T7 quick coupled transcription/translation system (Promega). Expression of FXR variants in HeLa cells was achieved by a transfection and recombinant vaccinia virus method described previously (26). FXR protein was determined by Western blot using anti-V5 antibodies (Invitrogen).

Gene Expression
RNA was extracted from transfected HepG2 cells with RNeasy kit (Qiagen) or Trizol (Invitrogen) and from human liver samples (Nashville Regional Organ Procurement Agency) obtained from Caucasian donors without any history of liver pathology or previous medications using Trizol reagent. First-strand cDNA was obtained using SuperScript II Reverse Transcriptase (Invitrogen) and random hexamers. Quantitative real-time PCR analysis using SYBR green detection (Bio-Rad, Hercules, CA) was performed with analysis by relative or absolute quantitation. Primer pairs are detailed in supplemental data (published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Protein was extracted from the human liver samples and analyzed by Western blot using anti-FXR (C-20; Santa Cruz Biotechnology) and anti-calnexin (Stressgen Biotechnologies, San Diego, CA) antibodies.

Statistical Analysis
For in vitro functional analyses, determination of the statistical differences between various groups was performed using Student’s t test. Statistics for hepatic gene expression differences were calculated using Kruskal-Wallis test. A P value of <0.05 was considered significant.


   ACKNOWLEDGMENTS
 
We are indebted to Kathy Lee, Rachel Hammers, and Chris Lemke for excellent technical assistance. We also thank Dr. F. Peter Guengerich (Vanderbilt University) for the human liver samples.


   FOOTNOTES
 
This work was supported by United States Public Health Service Grants GM60346 (to E.G.S.), GM61393 (to E.G.S.), GM31304 (to R.B.K.), and GM54724 (to R.B.K.). C.M. was supported by Swiss National Research Foundation Grant 81LA-69443.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 22, 2007

Abbreviations: AF, Activation function; BSEP, bile salt export pump; CDCA, chenodeoxycholic acid; CMV, cytomegalovirus; FXR, farnesoid X receptor; h, human; {Delta}hFXR, truncated form of the human farnesoid X receptor; NR, nuclear receptor; NTCP, sodium-dependent taurocholate transporting polypeptide; OATP, organic anion transporting polypeptide; r, rat; SHP, small heterodimer partner; SNP, single nucleotide polymorphism; WT, wild type.

Received for publication January 16, 2007. Accepted for publication May 18, 2007.


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 MATERIALS AND METHODS
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