Abstract
Bile acids are essential for the solubilization and transport of dietary lipids and are the major products of cholesterol catabolism. Results presented here show that bile acids are physiological ligands for the farnesoid X receptor (FXR), an orphan nuclear receptor. When bound to bile acids, FXR repressed transcription of the gene encoding cholesterol 7α-hydroxylase, which is the rate-limiting enzyme in bile acid synthesis, and activated the gene encoding intestinal bile acid–binding protein, which is a candidate bile acid transporter. These results demonstrate a mechanism by which bile acids transcriptionally regulate their biosynthesis and enterohepatic transport.
The enzymatic conversion of cholesterol to bile acids is regulated through feed-forward activation by oxysterols and feedback repression by bile acids (1,2). Because the feed-forward pathway is mediated by the liver X receptor (LXRα), a nuclear receptor that binds oxysterols (3), we speculated that the feedback repression pathway may also be regulated by a nuclear receptor that can bind to bile acids. The orphan receptor FXR (also called RIP14) is an ideal candidate for the bile acid receptor, because it is specifically expressed in tissues where bile acids function (such as the liver, intestine, and kidney); it is evolutionarily related to LXRα; and, like other class II nuclear receptors, it functions as a heterodimer with the retinoid X receptor (RXR) (4–6). In addition, several isoprenoid lipids can weakly activate FXR at supraphysiological concentrations; however, these compounds do not activate all species of FXR and do not bind as ligands (4, 7). Thus, the identity and physiologic function of FXR ligands have remained unknown.
To test the hypothesis that FXR is the bile acid receptor, murine or human FXR expression plasmids were transfected into monkey kidney CV-1 cells or human hepatoma HepG2 cells. Cells were then treated with a series of bile acid metabolites and screened for the expression of a luciferase reporter gene. The reporter construct contained multiple copies of an inverted repeat response element (IR-1) that binds to FXR-RXR heterodimers (4). FXR was strongly activated by bile acids that can regulate gene expression in vivo (Fig. 1A) (8, 9). The activation of FXR was specific and limited to the primary bile acid chenodeoxycholic acid (CDCA) and to a much lesser extent to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) (Fig. 1, A through C). Other closely related compounds such as cholesterol, oxysterols, steroid hormones, and other bile acid metabolites were inactive in this assay at concentrations up to 100 μM (Fig. 1) (10). The most potent activator, CDCA, had a half-maximal effective concentration (EC50) of 50 μM and 10 μM on murine and human FXR, respectively (Fig. 1B). These concentrations are well within the physiologic intracellular range reported in vivo (11). In addition, FXR was the only protein activated by bile acids (Fig. 1C). These results suggest that bile acids are the physiologic ligands of FXR.
Bile acids are physiologic ligands for FXR. (A) Specificity of bile acids that transactivate FXR. CV-1 cells were cotransfected with a rat FXR expression plasmid and a luciferase reporter gene, treated with 50 μM concentrations of the indicated compounds, and assayed as described (23). CDCA, chenodeoxycholic acid; CA, cholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; DHCA, dehydrocholic acid; UDCA, ursodeoxycholic acid; α- and β-MCA; α– and β–muricholic acid; GCDCA, glycochenodeoxycholic acid; GCA, glycocholic acid; TCDCA, taurochenodeoxycholic acid; TCA, taurocholic acid; TDCA, taurodeoxycholic acid. The inset shows the structure of CDCA. (B) Bile acid dose response for activation of a reporter gene by rat FXR in CV-1 cells (left panel) or human FXR in HepG2 cells (right panel) (23). RLU, relative light units. (C) CDCA (50 μM) specifically activates FXR but not other nuclear receptors (HNF4, hepatocyte nuclear factor 4). HepG2 cells were cotransfected as in (B) with Gal4-receptor chimeras in which the DNA binding domain of yeast Gal4 was fused in frame to the ligand binding domain of the indicated nuclear receptor (23). (D) Bile acids promote association of FXR and SRC-1 in vivo (24). (Eand F) Bile acids directly bind to FXR in vitro as analyzed by two coactivator/receptor interaction assays (25). 24,25-epoxychol., 24(S),25-epoxycholesterol. In (E), FRET measured the ligand-induced binding of europium-labeled FXR to an APC-labeled fragment of SRC-1. In (F), an ELISA measured ligand-induced association of FXR with a biotin-labeled LXXLL peptide.
After binding to ligand, nuclear receptors undergo a conformational change that increases their affinity for coactivator proteins such as SRC-1, a key step in the assembly of an active transcription complex (12). To further demonstrate that bile acids are physiologic ligands for FXR, a mammalian two-hybrid assay (13) was used to probe for a functional interaction in vivo between the FXR ligand-binding domain (LBD) and SRC-1. In transfected HEK-293 kidney cells, CDCA treatment promoted the association of FXR LBD and SRC-1 as expected (Fig. 1D). The EC50 of this response (20 μM) is similar to that for CDCA-induced transactivation of FXR (Fig. 1B).
The receptor-interacting domain of the coactivator SRC-1 has been mapped to a short motif with the amino acid sequence LXXLL, where L is leucine and X is any amino acid. Fragments of SRC-1 or short synthetic peptides containing one LXXLL motif or more bind nuclear receptors in a ligand-dependent manner (14). Hence, the association of LXXLL-containing peptides with nuclear receptors can be used to obtain a quantitative measurement of ligand-receptor binding in a simplified biochemical assay. Two experiments were performed to demonstrate that CDCA directly interacts with FXR. In the first experiment, a fluorescence resonance energy transfer (FRET) assay (15) measured interactions between the FXR LBD and a fragment of SRC-1 (amino acids 595 through 822) containing three LXXLL motifs (Fig. 1E). In the second experiment, we used an enzyme-linked immunosorbent assay (ELISA) (16) to measure the interaction between the FXR LBD and a biotin-labeled peptide containing an LXXLL motif (Fig. 1F). CDCA stimulated binding in both in vitro assays, yielding EC50's of 10 to 20 μM that correlated well with results from the mammalian two-hybrid (Fig. 1D) and transactivation (Fig. 1B) assays. Derivatives of CDCA conjugated with glycine (GCDCA) or taurine (TCDCA) were also active in both biochemical assays (Fig. 1, E and F) but were inactive in the cell-based assays (Fig. 1, A, B, and D). The ability of conjugated bile acids to bind FXR in vitro but not to transactivate FXR in cells is presumably due to the absence in these cells of the ileal bile acid transporter (I-BAT), which is required for conjugated bile acid uptake (17). The accompanying paper by Parks et al. (18) confirms this notion by demonstrating that conjugated bile acids become efficient FXR activators in cells expressing I-BAT. Neither CDCA nor other tested bile acids induced interaction of SRC-1 or LXXLL peptides with other nuclear receptors (such as LXRα, peroxisome proliferator-activated receptor-γ, or RXRα), and ligands for these other receptors did not promote the association of FXR with SRC-1 or LXXLL peptides (19). Together, these data provide convincing evidence that bile acids bind to FXR as ligands.
The return of bile acids to the liver is thought to be facilitated by specific binding proteins that transport bile acids across intestinal enterocytes. One of these proteins is the cytosolic intestinal bile acid–binding protein (I-BABP) (8). Expression of the gene encoding I-BABP increases in response to bile acids (20). To determine whether this response is induced by FXR acting as a bile acid receptor, approximately 1 kb of the 5′-flanking promoter region of the mouse I-BABP gene was linked to a luciferase reporter gene (pIBABP1031-Luc) and tested for FXR-specific activation in a cotransfection assay. This portion of the I-BABP promoter is sufficient for proper in vivo expression of I-BABP (21) and contains an inverted repeat sequence that may function as an FXR-specific bile acid response element (Fig. 2A). CDCA induced expression of the I-BABP–Luc reporter gene 12-fold in the presence of FXR and 29-fold when FXR was coexpressed with an increased amount of its heterodimeric partner RXR. Truncation of the I-BABP promoter to within 500 base pairs (bp) of the start site (pIBABP496-Luc) resulted in a similar CDCA-induced response (Fig. 2C). The rank order of potency of the various bile acids for FXR-specific induction of I-BABP reporter gene expression (Fig. 2D) was identical to that demonstrated in vivo (8). Finally, mutation of the IR-1 in the I-BABP promoter completely eliminated activation by CDCA (Fig. 2E). This IR-1 sequence alone drove CDCA- and FXR-dependent activation (10).
The I-BABP promoter is activated by FXR and bile acids (26). (A) Schematic map of the luciferase reporter gene containing the mouse I-BABP promoter used in these studies. The IR-1 sequence that functions as a bile acid response element (BARE) is shown. Cotransfection assays in CV-1 cells demonstrate that the FXR-RXR heterodimer is required for bile acid transactivation of a luciferase reporter containing either 1000 bp (B) or 500 bp (C) of upstream I-BABP promoter sequence. (D) Specificity of bile acids that activate the I-BABP promoter. CV-1 cells were cotransfected with a FXR and RXR expression plasmids and the pIBABP496-Luc reporter and treated with 100 μM concentrations of the indicated bile acids. (E) Mutation of the IR-1 motif in the I-BABP promoter eliminates activation by FXR and bile acid. The nucleotides in the IR-1 that are mutated are underlined in (A). Etoh, ethanol solvent control.
In addition to regulating their own transport, bile acids also regulate their synthesis by feedback that represses the expression of cholesterol 7α-hydroxylase (Cyp7a), the rate-limiting enzyme (2). In keeping with the notion that this regulation is also mediated by FXR, the rank order of bile acids that repress endogenous expression of human Cyp7a protein (Fig. 3A) and mRNA (Fig. 3B) in a hepatocyte-derived cell line was identical to that for binding and activating FXR. In addition, significant repression of a luciferase reporter containing the Cyp7a gene promoter occurred only when FXR, but not other nuclear receptors, was expressed (Fig. 3C). FXR-mediated repression of the Cyp7a promoter by CDCA was dose-dependent and had an EC50 of 20 μM (Fig. 3D). Feedback regulation of bile acid synthesis is conserved between human and murine species, and CDCA-mediated repression of the rat Cyp7a promoter also required FXR (10).
The Cyp7a promoter is repressed by bile acids and FXR. (A) Immunoblot analysis and (B) quantitative RT-PCR demonstrate that endogenous human Cyp7a protein and mRNA synthesis are repressed by bile acids. Immunoblots using an antibody to Cyp7a were performed on lysates from human HepG2 cells after treatment with bile acids. RT-PCR was performed as described (27) from cells treated with 50 μM bile acids. Results are normalized to control levels of glyceraldehyde phosphate dehydrogenase mRNA. (C) CDCA-mediated suppression of human Cyp7a requires FXR. HepG2 cells were cotransfected with expression plasmids for the indicated nuclear receptors and a luciferase reporter plasmid containing nucleotides –716 to +14 of the human Cyp7a promoter (pGL3-CYP7A). Cells were then treated with 10 μM CDCA and analyzed for luciferase activity relative to methanol solvent control (cont.). (D) Dose-dependent effects of bile acids on FXR suppression of Cyp7a. Cells were transfected as in (C) and treated with various concentrations of bile acids.
The above results show that bile acid regulation of I-BABP and Cyp7a gene expression depends on the presence of FXR in a manner consistent with FXR's role as a bile acid receptor. That FXR functions as a bile acid receptor suggests an expanded model for the regulation of cholesterol homeostasis by nuclear receptors (Fig. 4). The accumulation of dietary cholesterol in the liver results in the synthesis of oxysterols that bind to LXRα. LXRα activation in turn enhances the catabolic clearance of cholesterol by increasing bile acid synthesis and excretion. As demonstrated in this work, elevated bile acids regulate their synthesis and transport through their nuclear receptor FXR. Thus, bile acids are able to efficiently repress their further synthesis in the liver and increase the synthesis of transport proteins in the intestine. The discovery of oxysterol and bile acid signaling pathways mediated by nuclear receptors complements the extensive pioneering studies that have defined the intricate feedback and feed-forward control of sterol metabolism (22). Furthermore, the observation that LXRα and FXR function as key regulators of cholesterol and bile acid homeostasis has important therapeutic implications for the discovery of drugs targeted against these receptors.
Model of the transcriptional control of cholesterol and bile acid homeostasis by nuclear receptors.
