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.     

Effects of farnesoid X receptor on the expression of the fatty acid synthetase and hepatic lipase

The farnesoid X receptor (FXR) is a nuclear receptor that regulates gene expression in response to bile acids (BAs). FXR plays an important role in the homeostasis of bile acid, cholesterol, lipoprotein and triglyceride. In this report, we identified fatty acid synthase (FAS) and hepatic lipase (HL) genes as novel target genes of FXR. Human hepatoma HepG2 cells were treated with chenodeoxycholic acid, the natural FXR ligand, and the messenger RNA and protein levels of FAS and HL were determined by RT-PCR and Western blot analysis, respectively. Chenodeoxycholic acid (CDCA) down-regulated the expression of FAS and HL genes in a dose and time-dependent manner in human hepatoma HepG2 cells. In addition, treatment of mice with CDCA significantly decreased the expression of FAS and HL in mouse liver and the activity of HL. These results demonstrated that FAS and HL might be FXR-regulated genes in liver cells. In view of the role of FAS and HL in lipogenesis and plasma lipoprotein metabolism, our results further support the central role of FXR in the homeostasis of fatty acid and lipid.     

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.     

Dehydroepiandrosterone sulfotransferase gene induction by bile acid activated farnesoid X receptor

Dehydroepiandrosterone sulfotransferase (STD) is a hydroxysteroid sulfo-conjugating enzyme with preferential substrate specificity for C-19 androgenic steroids and C-24 bile acids. STD is primarily expressed in the liver, intestine and adrenal cortex. Earlier studies have shown that androgens inhibit the rat Std promoter function through a negative androgen response region located between -235 and -310 base pair positions (Song, C. S., Jung, M. H., Kim, S. C., Hassan, T., Roy, A. K., and Chatterjee, B. (1998) J. Biol. Chem. 273, 21856-21866). Here we report that the primary bile acid chenodeoxycholic acid (CDCA) also acts as an important regulator of the Std gene promoter. CDCA is a potent inducer of the Std gene, and its inducing effect is mediated through the bile acid-activated farnesoid X receptor (FXR), a recently characterized member of the nuclear receptor superfamily. The ligand-activated FXR acts as a heterodimer with the 9-cis-retinoic acid receptor (RXR) and regulates the Std gene by binding to an upstream region at base pair positions -169 to -193. This specific binding region was initially identified by bile acid responsiveness of the progressively deleted forms of the Std promoter in transfected HepG2 hepatoma and enterocyte-like Caco-2 cells. Subsequently, the precise RXR/FXR binding position was established by protein-DNA interaction using in vitro footprinting and electrophoretic mobility shift analyses. Unlike all other previously characterized FXR target genes, which contain an inverted repeat (IR) of the consensus hexanucleotide half-site (A/G)G(G/T)TCA with a single nucleotide spacer (IR-1), the bile acid response element of the Std promoter does not contain any spacer between the two hexanucleotide repeats (IR-0). A promoter-reporter construct carrying three tandem copies of the IR-0 containing -169/-193 element, linked to a minimal thymidine kinase promoter, can be stimulated more than 70-fold in transfected Caco-2 cells upon CDCA treatment. Autoregulation of the STD gene by its bile acid substrate may provide an important contributing role in the enterohepatic bile acid metabolism and 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.