In vitro farnesoid X receptor ligand sensor assay using surface plasmon resonance and based on ligand-induced coactivator association

Ligand binding to nuclear receptors leads to a conformational change that increases the affinity of the receptors to coactivator proteins. We have developed a ligand sensor assay for farnesoid X receptor (FXR) in which the receptor–coactivator interaction can be directly monitored using surface plasmon resonance biosensor technology. A 25-mer peptide from coactivator SRC1 containing the LXXLL nuclear receptor interaction motif was immobilized on the surface of a BIAcore sensor chip. Injection of the FXR ligand binding domain (FXRLBD) with or without the most potent natural ligand, chenodeoxycholic acid (CDCA), over the surface of the chip resulted in a ligand- and LXXLL motif-dependent interaction. Kinetic analysis revealed that CDCA and its conjugates decreased the equilibrium dissociation constant (K_d) by 8–11-fold, indicating an increased affinity. Using this technique, we found that a synthetic bile acid sulfonate, 3,7-dihydroxy-5β-cholane-24-sulfonate, which was inactive in a FXR response element-driven luciferase assay using CV-1 cells, caused the most potent interaction, comparable to the reaction produced by CDCA. This method provides a rapid and reliable in vitro ligand assay for FXR. This kinetic analysis-featured technique may be applicable to mechanistic studies.     

Nuclear receptor ligands and cofactor recruitment: is there a coactivator "on deck"?

Farnesoid X receptor (FXR) is a bile acid sensor that regulates lipid homeostasis. New structural data suggest that, unlike other nuclear receptors, FXR contains a second coactivator binding site and binds bile acids with the steroid backbone flipped head to tail, both of which have important functional ramifications.     

Identification of intermediates in the bile acid synthetic pathway as ligands for the farnesoid X receptor

Bile acid synthesis from cholesterol is tightly regulated via a feedback mechanism mediated by the farnesoid X receptor (FXR), a nuclear receptor activated by bile acids. Synthesis via the classic pathway is initiated by a series of cholesterol ring modifications and followed by the side chain cleavage. Several intermediates accumulate or are excreted as end products of the pathway in diseases involving defective bile acid biosynthesis. In this study, we investigated the ability of these intermediates to activate human FXR. In a cell-based reporter assay and coactivator recruitment assays in vitro, early intermediates possessing an intact cholesterol side chain were inactive, whereas 26- or 25-hydroxylated bile alcohols and C27 bile acids were highly efficacious ligands for FXR at a level comparable to that of the most potent physiological ligand, chenodeoxycholic acid. Treatment of HepG2 cells with these precursors repressed the rate-limiting cholesterol 7alpha-hydroxylase mRNA level and induced the small heterodimer partner and the bile salt export pump mRNA, indicating the ability to regulate bile acid synthesis and excretion. Because 26-hydroxylated bile alcohols and C27 bile acids are known to be evolutionary precursors of bile acids in mammals, our findings suggest that human FXR may have retained affinity to these precursors during evolution.     

Polyunsaturated fatty acids are FXR ligands and differentially regulate expression of FXR targets

Polyunsaturated fatty acids (PUFAs) have been previously reported as agonists of peroxisome proliferatoractivated receptor and antagonists of the liver X receptor. The activities on these two nuclear receptors have been attributed to their beneficial effects such as improvement of dyslipidemia and insulin sensitivity and decrease of hepatic lipogenesis. Here we report that PUFAs are ligands of farnesoid X receptor (FXR), a nuclear receptor for bile acids. In a conventional FXR binding assay, arachidonic acid (AA, 20:4), docosahexaenoic acid (DA, 22:6), and linolenic acid (LA, 18:3) had an affinity of 2.6, 1.5, and 3.5 microM, respectively. In a cell-free coactivator association assay, AA, DA, and LA decreased FXR agonist-induced FXR activation with IC(50)s ranging from 0.9 to 4.7 microM. In HepG2 cells, PUFAs regulated the expression of two FXR targets, BSEP and kininogen, in an opposite fashion, although both genes were transactivated by FXR. All three PUFAs dose-dependently enhanced FXR agonist-induced BSEP expression but decreased FXR agonist-induced human kininogen mRNA. Saturated fatty acids such as stearic acid (SA, 18:0) and palmitic acid (PA, 16:0) did not bind to FXR and did not change BSEP or kininogen expression. The pattern of BSEP and kininogen regulation by PUFAs is closely similar to that of the guggulsterone, previously reported as a selective bile acid receptor modulator. Our results suggest that PUFAs may belong to the same class of FXR ligands as guggulsterone, and that the selective regulation of FXR targets may contribute to the beneficial effects of PUFAs in lipid metabolism.     

Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.

The major metabolic pathway for elimination of cholesterol is via conversion to bile acids. In addition to this metabolic function, bile acids also act as signaling molecules that negatively regulate their own biosynthesis. However, the precise nature of this signaling pathway has been elusive. We have isolated an endogenous biliary component (chenodeoxycholic acid) that selectively activates the orphan nuclear receptor, FXR. Structure-activity analysis defined a subset of related bile acid ligands that activate FXR and promote coactivator recruitment. Finally, we show that ligand-occupied FXR inhibits transactivation from the oxysterol receptor LXR alpha, a positive regulator of cholesterol degradation. We suggest that FXR (BAR) is the endogenous bile acid sensor and thus an important regulator of cholesterol homeostasis.     

5beta-Cholane activators of the farnesol X receptor

The farnesoid X receptor (FXR) is activated by bile acids, natural agonists for this nuclear receptor. FXR-target genes play important roles in cholesterol and lipid metabolism. We have found that a series of 5beta-cholanic acid derivatives, even though without a hydroxyl group or any other substituent on the steroidal rings, can activate FXR more potently than hydroxylated bile acids in a reporter gene assay. The most potent compound among these derivatives, N-methyl-5beta-glycocholanic acid (NMGCA), induces the formation of receptor/coactivator complex in a gel-shift assay and also increases the expression of FXR target genes in human hepatoma HepG2 cells. Furthermore, in rats, NMGCA causes hypolipidemic effects as well as induction of the FXR target genes in liver. Our results suggest that NMGCA and its derivatives are important FXR activators in the study of the physiological functions of FXR and are potentially useful as pharmaceutical agents for treatment of cholesterol and lipid-related diseases.     

The nuclear receptor for bile acids, FXR, transactivates human organic solute transporter-alpha and -beta genes

Bile acids are synthesized from cholesterol in the liver and are excreted into bile via the hepatocyte canalicular bile salt export pump. After their passage into the intestine, bile acids are reabsorbed in the ileum by sodium-dependent uptake across the apical membrane of enterocytes. At the basolateral domain of ileal enterocytes, bile acids are extruded into portal blood by the heterodimeric organic solute transporter OSTalpha/OSTbeta. Although the transport function of OSTalpha/OSTbeta has been characterized, little is known about the regulation of its expression. We show here that human OSTalpha/OSTbeta expression is induced by bile acids through ligand-dependent transactivation of both OST genes by the nuclear bile acid receptor/farnesoid X receptor (FXR). FXR agonists induced endogenous mRNA levels of OSTalpha and OSTbeta in cultured cells, an effect that was not discernible upon inhibition of FXR expression by small interfering RNAs. Furthermore, OST mRNAs were induced in human ileal biopsies exposed to the bile acid chenodeoxycholic acid. Reporter constructs containing OSTalpha or OSTbeta promoters were transactivated by FXR in the presence of its ligand. Two functional FXR binding motifs were identified in the OSTalpha gene and one in the OSTbeta gene. Targeted mutation of these elements led to reduced inducibility of both OST promoters by FXR. In conclusion, the genes encoding the human OSTalpha/OSTbeta complex are induced by bile acids and FXR. By coordinated control of OSTalpha/OSTbeta expression, bile acids may adjust the rate of their own efflux from enterocytes in response to changes in intracellular bile acid levels.     

Structure-activity relationship of bile acids and bile acid analogs in regard to FXR activation

The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that plays a major role in bile acid and cholesterol metabolism. To obtain an insight into the structure-activity relationships of FXR ligands, we investigated the functional roles of structural elements in the physiological ligands chenodeoxycholic acid [CDCA; (3alpha,7alpha)], cholic acid [CA; (3alpha,7alpha,12alpha)], deoxycholic acid [DCA; (3alpha,12alpha)], and lithocholic acid (3alpha) in regard to FXR activation in a cell-based FXR response element-driven luciferase assay and an in vitro coactivator association assay. Conversion of the carboxyl group of CDCA or CA to an alcohol did not greatly diminish their ability to activate FXR. In contrast, the 7beta-epimers of the alcohols were inactive, indicating that the bile alcohols retained the ligand properties of the original bile acids and that the 7beta-hydroxyl group diminished their FXR-activating effect. Similarly, hydroxyl epimers of DCA exhibited decreased activity compared with DCA, indicating a negative effect of 3beta- or 12beta-hydroxyl groups. Introduction of an alkyl group at the 7beta- or 3beta-position of CDCA resulted in diminished FXR activation in the following order of alkyl groups: 7-ethyl=7-propyl>3-methyl>7-methyl. These results indicate that bulky substituents, whether hydroxyl groups or alkyl residues, at the beta-position of cholanoids decrease their ability to activate FXR.     

Identification of farnesoid X receptor modulators by a fluorescence polarization-based interaction assay

Farnesoid X receptor (FXR) serves as a receptor for chenodeoxycholic acid (CDCA) and other bile acids, and it coordinates cholesterol and lipid metabolism. Because targeting the FXR-CDCA interaction might provide a way to regulate lipid homeostasis, we developed an FXR binding assay based on fluorescence polarization. Employing a fluorescently labeled CDCA (CDCA-F), we showed that CDCA-F selectively bound to the ligand binding domain of FXR (FXR-LBD) among nuclear receptors. The assay was then used for screening inhibitors against the FXR-CDCA interaction, thereby discovering four relatively potent inhibitors. The selected inhibitors were further studied for changes in intrinsic tryptophan fluorescence of FXR-LBD to gain structural insights into the interaction. Furthermore, transactivation effects of the inhibitors on the human bile salt excretory pump (BSEP) promoter were examined to reveal their cellular activities in the FXR-mediated pathway. Therefore, we demonstrated that the developed assay would offer an efficient primary screening tool for identifying FXR modulators.     

The farnesoid X receptor FXRalpha/NR1H4 acquired ligand specificity for bile salts late in vertebrate evolution

The nuclear receptor FXRalpha (NR1H4) plays a pivotal role in maintaining bile salt and lipid homeostasis by functioning as a bile salt sensor in mammals. In contrast, FXRbeta (NR1H5) from mouse is activated by lanosterol and does not share common ligands with FXRalpha. To further elucidate FXR ligand/receptor and structure/function relationships, we characterized a FXR gene from the marine skate, Leucoraja erinacea, representing a vertebrate lineage that diverged over 400 million years ago. Phylogenetic analysis of sequence data indicated that skate Fxr (sFxr) is a FXRbeta. There is an extra sequence in the middle of the sFxr ligand binding domain (LBD) compared with the LBD of FXRalpha. Luciferase reporter assays demonstrated that sFxr responds weakly to scymnol sulfate, bile salts, and synthetic FXRalpha ligands, in striking difference from human FXRalpha (hFXRalpha). Interestingly, all-trans retinoic acid was capable of transactivating both hFXRalpha and sFxr. When the extra amino acids in the sFxr LBD were deleted and replaced with the corresponding sequence from hFXRalpha, the mutant sFxr gained responsiveness to ursodeoxycholic acid, GW4064, and fexaramine. Surprisingly, chenodeoxycholic acid antagonized this activation. Together, these results indicate that FXR is an ancient nuclear receptor and suggest that FXRalpha may have acquired ligand specificity for bile acids later in evolution by deletion of a sequence from its LBD. Acquisition of this property may be an example of molecular exploitation, where an older molecule is recruited for a new functional role.     

Bile acids induce the expression of the human peroxisome proliferator-activated receptor alpha gene via activation of the farnesoid X receptor

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that controls lipid and glucose metabolism and exerts antiinflammatory activities. PPARalpha is also reported to influence bile acid formation and bile composition. Farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that mediates the effects of bile acids on gene expression and plays a major role in bile acid and possibly also in lipid metabolism. Thus, both PPARalpha and FXR appear to act on common metabolic pathways. To determine the existence of a molecular cross-talk between these two nuclear receptors, the regulation of PPARalpha expression by bile acids was investigated. Incubation of human hepatoma HepG2 cells with the natural FXR ligand chenodeoxycholic acid (CDCA) as well as with the nonsteroidal FXR agonist GW4064 resulted in a significant induction of PPARalpha mRNA levels. In addition, hPPARalpha gene expression was up-regulated by taurocholic acid in human primary hepatocytes. Cotransfection of FXR/retinoid X receptor in the presence of CDCA led to up to a 3-fold induction of human PPARalpha promoter activity in HepG2 cells. Mutation analysis identified a FXR response element in the human PPARalpha promoter (alpha-FXR response element (alphaFXRE)] that mediates bile acid regulation of this promoter. FXR bound the alphaFXRE site as demonstrated by gel shift analysis, and CDCA specifically increased the activity of a heterologous promoter driven by four copies of the alphaFXRE. In contrast, neither the murine PPARalpha promoter, in which the alphaFXRE is not conserved, nor a mouse alphaFXRE-driven heterologous reporter, were responsive to CDCA treatment. Moreover, PPARalpha expression was not regulated in taurocholic acid-fed mice. Finally, induction of hPPARalpha mRNA levels by CDCA resulted in an enhanced induction of the expression of the PPARalpha target gene carnitine palmitoyltransferase I by PPARalpha ligands. In concert, these results demonstrate that bile acids stimulate PPARalpha expression in a species-specific manner via a FXRE located within the human PPARalpha promoter. These results provide molecular evidence for a cross-talk between the FXR and PPARalpha pathways in humans.     

The amino acid residues asparagine 354 and isoleucine 372 of human farnesoid X receptor confer the receptor with high sensitivity to chenodeoxycholate

The critical steps in bile acid metabolism have remarkable differences between humans and mice. It is known that human cholesterol 7 alpha-hydroxylase, the enzyme catalyzing the rate-limiting step of bile acid synthesis, is more sensitive to bile acid suppression. In addition, hepatic bile acid export in humans is more dependent on the bile salt export pump (BSEP). To explore the molecular basis for these species differences, we analyzed the function of the ligand-binding domain (LBD) of human and murine farnesoid X receptor (FXR), a nuclear receptor for bile acids. We observed a strong interspecies difference in bile acid-mediated FXR function; in the coactivator association assay, chenodeoxycholate (CDCA) activated human FXR-LBD with 10-fold higher affinity and 3-fold higher maximum response than murine FXR-LBD. Consistently, in HepG2 cells human FXR-LBD increased reporter expression more robustly in the presence of CDCA. The basis for these differences was investigated by preparing chimeric receptors and by site-directed mutagenesis. Remarkably, the double replacements of Lys(366) and Val(384) in murine FXR (corresponding to Asn(354) and Ile(372) in human FXR) with Asn(366) and Ile(384) explained the difference in both potency and maximum activation; compared with the wild-type murine FXR-LBD, the double mutant gained 8-fold affinity and more than 250% maximum response to CDCA in vitro. This mutant also increased reporter expression to an extent comparable with that of human FXR-LBD in HepG2 cells. These results demonstrate that Asn(354) and Ile(372) are critically important for FXR function and that murine FXR can be "humanized" by substituting with the two corresponding residues of human FXR. Consistent with the difference in FXR-LBD transactivation, CDCA induced endogenous expression of human BSEP by 10-12-fold and murine BSEP by 2-3-fold in primary hepatocytes. This study not only provides the identification of critical residues for FXR function but may also explain the species difference in bile acids/cholesterol metabolism.