For Farnesoid X Receptor Research Articles

3D-QSAR and Molecular Dynamics Study of Isoxazole Derivatives to Identify the Structural Requirements for Farnesoid X Receptor (FXR) Agonists.

The farnesoid X receptor (FXR) has been recognized as a potential drug target for the treatment of non-alcoholic fatty liver disease (NAFLD). FXR agonists benefit NAFLD by modulating bile acid synthesis and transport, lipid metabolism, inflammation, and fibrosis pathways. However, there are still great challenges involved in developing safe and effective FXR agonists. To investigate the critical factors contributing to their activity on the FXR, 3D-QSAR molecular modeling was applied to a series of isoxazole derivatives, using comparative molecular field analysis (CoMFA (q2 = 0.664, r2 = 0.960, r2pred = 0.872)) and comparative molecular similarity indices analysis (CoMSIA (q2 = 0.706, r2 = 0.969, r2pred = 0.866)) models, which demonstrated strong predictive ability in our study. The contour maps generated from molecular modeling showed that the presence of hydrophobicity at the R2 group and electronegativity group at the R3 group in these compounds is crucial to their agonistic activity. A molecular dynamics (MD) simulation was carried out to further understand the binding modes and interactions between the FXR and its agonists in preclinical or clinical studies. The conformational motions of loops L: H1/H2 and L: H5/H6 in FXR-ligand binding domain (LBD) were crucial to the protein stability and agonistic activity of ligands. Hydrophobic interactions were formed between residues (such as LEU287, MET290, ALA291, HIS294, and VAL297) in helix H3 and ligands. In particular, our study found that residue ARG331 participated in salt bridges, and HIS447 participated in salt bridges and hydrogen bonds with ligands; these interactions were significant to protein-ligand binding. Eight new potent FXR agonists were designed according to our results, and their activities were predicted to be better than that of the first synthetic FXR agonist, GW4064.

Hepatic farnesoid X receptor is necessary to facilitate ductular reaction and expression of heme biosynthetic genes.

Bile, which contains bile acids, the natural ligands for farnesoid x receptor (FXR), moves from the liver to the intestine through bile ducts. Ductular reaction often occurs during biliary obstruction. A subset of patients with erythropoietic protoporphyria, an inherited genetic mutation in heme biosynthetic enzyme ferrochelatase, accumulate porphyrin-containing bile plugs, leading to cholestasis. Here, we examined the link between FXR, bile plug formation, and how heme biosynthesis relates to this connection. We treated female and male wild-type and global and tissue-specific Fxr knockout mice with a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine, an inhibitor of ferrochelatase, and examined the expression of heme biosynthetic genes. We mined FXR mouse ChIP-Seq data, performed biochemical and histological analysis, and tested HepG2 and primary human hepatocytes after treatment with obeticholic acid, an FXR agonist. We observed that hepatic but not intestinal Fxr loss resulted in reduced bile plugs and ductular reaction in the liver. Then, we examined if FXR plays a regulatory role in heme biosynthesis and found significantly lower porphyrin accumulation in 3,5-diethoxycarbonyl-1, 4-dihydrocollidine-fed Fxr knockout mice. Gene expression and FXR mouse ChIP-Seq atlas analysis revealed that FXR orchestrates the expression of multiple heme biosynthetic enzymes. Finally, human HepG2 cells and primary human hepatocytes treated with obeticholic acid, showed increased expression of several heme biosynthetic genes. Overall, our data show that hepatic Fxr is necessary to maintain ductular reaction and accumulation of bile plugs. FXR can direct the expression of multiple heme biosynthetic genes. Thus, modulating FXR activity in EPP patients may help alleviate its associated liver disease.

Development and optimization of a cell screening system for farnesoid X receptor agonist

This study aims to develop an improved cell screening system for farnesoid X receptor (FXR) agonists based on a dual luciferase reporter gene system. FXR response element (FXRE) fragments from FXR target genes were cloned and inserted into upstream of firefly luciferase (Luc) gene in the plasmid pGL4-luc2P-Hygro. In combination with the internal reference plasmid containing renilla luciferase, a dual luciferase reporter gene system was developed and used for high throughput screening of FXR agonists. After studying the effects of over-expression of RXR, mouse or human FXR, various FXRE fragments, and different ratio of FXR plasmid amount to reporter gene plasmid, induction efficiency of the screening system was optimized by the known FXR agonist GW4064, and Z factor for the system reached 0.83 under optimized conditions. In summary, an improved cell screening system based on double luciferase reporter gene detection system was developed to facilitate the discovery of FXR agonists, where a new enhanced FXRE element was formed by a superposition of multiple FXRE fragments from FXR target genes, instead of a superposition of traditional IR-1 (inverted repeats-1) fragments.

N1-Substituted benzimidazole scaffold for farnesoid X receptor (FXR) agonists accompanying prominent selectivity against vitamin D receptor (VDR)

As a cellular bile acid sensor, farnesoid X receptor (FXR) participates in regulation of bile acid, lipid and glucose homeostasis, and liver protection. With respect to the bone metabolism, FXR positively regulates bone metabolism through both bone formation and resorption of the bone remodeling pathways. Some of FXR agonists possessing isoxazole moiety are undergoing clinical trials for the treatment of non-alcoholic steatohepatitis. To date, therefore, the activation of FXR leads to considerable interest in FXR as potential therapeutic targets. We have identified a series of nonsteroidal FXR agonists bearing N1-methyl benzimidazole and isoxazole moieties that are bridged with aromatic derivatives. They showed affinity to FXR, but also weak affinity toward the vitamin D receptor (VDR) that involves regulation of calcium and phosphate homeostasis and is activated by bile acids. The deployment of FXR agonists without activity against VDR as off-target is therefore crucial in the development of FXR ligands. Our efforts focusing on increasing the agonist properties towards FXR led to the discovery of 19, which activates FXR at and below nanomolar levels (EC50 = 26.5 ± 10.5 nM TR-FRET and 0.8 ± 0.2 nM luciferase, respectively) and functions as a FXR agonist: the affinity toward FXR over eight nuclear receptors, including VDR [IC50 (VDR) / EC50 (FXR) > 5000] and TGR5, effects FXR target genes, and activates bone morphogenetic protein-2-induced differentiation of mouse bone marrow-derived mesenchymal stem cell-like ST2 cells into osteoblast.

Repositioning an Immunomodulatory Drug Vidofludimus as a Farnesoid X Receptor Modulator With Therapeutic Effects on NAFLD.

Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disorder, and yet with no pharmacological treatment approved worldwide. The repositioning of old drugs provides a safe approach for drug development. Vidofludimus, an inhibitor for dihydroorotate dehydrogenase (DHODH) for the treatment of autoimmune disorders, is herein uncovered as a novel modulator for farnesoid X receptor (FXR) by biochemical and crystallographic analysis. We further revealed that vidofludimus exerts in vivo therapeutic effects on dextran sodium sulfate (DSS)-induced colitis in an FXR-dependent manner. Notably, vidofludimus also possesses remarkable beneficial effects in reducing NAFLD by targeting FXR, which may represent a unique approach in developing the treatment for NAFLD. Our findings not only reveal a promising template for the design of novel FXR ligands in treating autoimmune disorders, but also uncover a novel therapeutic effect for vidofludimus on NAFLD based on the newly established relationships among drugs, targets, and diseases.

Anthranilic acid derivatives as novel ligands for farnesoid X receptor (FXR)

Nuclear farnesoid X receptor (FXR) has important physiological roles in various metabolic pathways including bile acid, cholesterol and glucose homeostasis. The clinical use of known synthetic non-steroidal FXR ligands is restricted due to toxicity or poor bioavailability. Here we report the development, synthesis, in vitro activity and structure–activity relationship (SAR) of anthranilic acid derivatives as novel FXR ligands. Starting from a virtual screening hit we optimized the scaffold to a series of potent partial FXR agonists with appealing drug-like properties. The most potent derivative exhibited an EC50 value of 1.5±0.2μM and 37±2% maximum relative FXR activation. We investigated its SAR regarding polar interactions with the receptor by generating derivatives and computational docking.

The retinoid X receptor in a marine invertebrate chordate: Evolutionary insights from urochordates

Retinoid X receptors (RXRs) are highly conserved members of the nuclear hormone receptor family that mediate various physiological processes in vertebrates and invertebrates. We examined the expression patterns of RXR in the ascidian Halocynthia roretzi across a wide range of tissues and stages of embryo development, as well as the regulation of gene transcription by the ascidian RXR. H. roretzi RXR cDNA (HrRXR) was cloned from 64-cell stage embryos. The overall amino acid sequence of HrRXR showed high sequence identity with a urochordate Ciona intestinalis RXR (58%), but the ligand-binding domain of HrRXR was more similar to vertebrate orthologs than to those of invertebrate RXRs. Based on a phylogenetic analysis, HrRXR belongs to a group of urochordates that are separate from vertebrate RXRs, showing a clear evolutionary history. Real-time quantitative polymerase chain reaction and whole-mount in situ hybridization analyses revealed that the HrRXR mRNA is of maternal origin during embryogenesis, and in the examined adult tissues it is expressed in the muscles, gills, gonads, and the hepatopancreas. Immunofluorescence and immunohistochemical staining demonstrated that HrRXR is localized to the nucleus and highly expressed in the gills and hepatopancreas. Unlike human RXRα, HrRXR did not show 9-cis retinoic acid- and bexarotene (LGD1069)-dependent transactivation. While a synthetic ligand for farnesoid X receptor (FXR), GW4064, did not increase the transcriptional activation in HrRXR- or HrRXR/HrFXR-transfected HEK-293 cells, the ligand showed weak but significant activity for a single amino acid mutant of HrRXR (Phe231Cys) and HrFXR cotransfected cells. The present study suggests that the marine invertebrate chordate RXR may possess endogenous ligands that are different than vertebrate RXR ligands and which function during early embryonic stages.

Inhibition of Apolipoprotein(a) Synthesis by Farnesoid X Receptor and Fibroblast Growth Factor 15/19

Lipoprotein(a), or Lp(a), is a plasma lipoprotein that accumulates in concentrations ranging from undetectable to as high as 200 mg/dL, mostly depending on the synthesis rate of its unique protein, apo(a), which consists of multiple repetitions of kringle IV, type 2 of plasminogen.1 Additionally, plasma levels of Lp(a) are inversely proportional to the size of the apo(a) isoform, which is genetically determined.2 Considering that cholesterol represents about one fifth of the particle, Lp(a) can be equivalent to an extra 40 mg/dL of low-density lipoprotein (LDL) cholesterol in some individuals. The complex is the result of a covalent association between apoB100 and the function-free apo(a). The size of this lipoprotein ranges from that of a large high-density lipoprotein (HDL) to that of a small remnant depending on its triglyceride content. It is an apoB-containing lipoprotein and therefore viewed as an atherogenic particle. Because apo(a) does not form Lp(a) in murine plasma, development of a transgenic mouse model has required the concomitant expression of human apo(a) and apoB100.3 Apo(a) binds via a single disulfide bridge to the carboxyl-terminus of apoB100 and thus cannot associate with chylomicrons, which only contain apoB48.4 Also, apo(a) preferentially binds apoB100 of LDL-sized particles, although the linkage between the 2 proteins is likely to take place on the surface of hepatocytes, which produce exclusively VLDL-sized particles.5 On the clearance front, Lp(a) does not bind to the LDL receptor, and only modest increases in Lp(a) levels are seen in patients with familial hypercholesterolemia.6 This means that cholesterol-lowering agents acting more or less directly via upregulation of the LDL receptor (statins, resins, and cholesterol absorption inhibitors) have modest to no effects or may even raise plasma Lp(a) levels.7–9 Other agents, such as fibrates and niacins, share similar lipid-modulating effects (decreasing triglycerides, …

Sodium taurocholate inhibits intestinal adenoma formation in APCMin/+ mice, potentially through activation of the farnesoid X receptor

In light of clinical and biological evidence that bile constituents exert preventive effects against colorectal cancer, we evaluated the influence of oral bilirubin and sodium taurocholate (NaTC) on intestinal tumor formation in APC(Min/+) mice. Mice received bilirubin and/or bovine serum albumin (BSA) and NaTC in the drinking water for 8 weeks, after which the number, size and location of intestinal adenomas were determined. Tissue specimens were analyzed by light microscopy, TUNEL staining, immunohistochemistry for beta-catenin and Ki-67 and quantitative polymerase chain reaction for farnesoid X receptor (FXR)-dependent gene expression. Colon tumor formation also was assessed in azoxymethane (AOM)-treated hyperbilirubinemic Gunn (j/j) and wild-type (+/+) rats. Compared with untreated APC(Min/+) mice, the mean number of intestinal adenomas was markedly lower in both bilirubin (10.5 +/- 0.9 versus 37.0 +/- 5.2; +/-SEM; P < 0.001) and NaTC plus BSA (14.3 +/- 5.4; P = 0.01)-treated animals. Both treatment groups exhibited reduced levels of cellular proliferation in the ileum (by Ki-67 staining), but no differences in TUNEL staining or the percentage of beta-catenin-positive crypts. Bilirubin feeding reduced intestinal inducible nitric oxide synthase expression, but did not alter adenoma multiplicity in APC(Min/+) mice or in AOM-treated j/j versus +/+ rats. Mice receiving NaTC manifested increased intestinal expression of the FXR-regulated genes, Shp, FGF15 and IBABP, and a concomitant decrease in cyclin D1 message. Administering NaTC to APC(Min/+) mice causes a marked reduction in intestinal adenomas. We postulate that this effect is mediated through activation of FXR, leading to increased Shp expression and consequent downregulation of cyclin D1.

Back Door Modulation of the Farnesoid X Receptor: Design, Synthesis, and Biological Evaluation of a Series of Side Chain Modified Chenodeoxycholic Acid Derivatives

Carbamate derivatives of bile acids were synthesized with the aim of systematically exploring the potential for farnesoid X receptor (FXR) modulation endowed with occupancy of the receptor's back door, localized between loops H1-H2 and H4-H5. Since it was previously shown that bile acids bind to FXR by projecting the carboxylic tail opposite the transactivation function 2 (AF-2, helix 12), functionalization of the side chain is not expected to interfere directly with the orientation of H12 but can result in a more indirect way of receptor modulation. The newly synthesized compounds were extensively characterized for their ability to modulate FXR function in a variety of assays, including the cell-free fluorescence resonance energy transfer (FRET) assay and the cell-based luciferase transactivation assay, and displayed a broad range of activity from full agonism to partial antagonism. Docking studies clearly indicate that the side chain of the new derivatives fits in a so far unexploited receptor cavity localized near the "back door" of FXR. We thus demonstrate the possibility of achieving a broad FXR modulation without directly affecting the H12 orientation.

Regulation of the mouse organic solute transporter α-β, Ostα-Ostβ, by bile acids

The mechanisms responsible for bile acid regulation of mouse intestinal organic solute transporter alpha-beta (Ostalpha-Ostbeta) expression were investigated. Expression of Ostalpha-Ostbeta mRNA was increased in cecum and proximal colon of cholic acid-fed mice and in chenodeoxycholate-treated mouse CT26 colon adenocarcinoma cells. Sequence analysis revealed potential cis-acting elements for farnesoid X receptor (FXR) and liver receptor homolog-1 (LRH-1) in the mouse Ostalpha and Ostbeta promoters and reporter constructs containing Ostalpha and Ostbeta 5'-flanking sequences were positively regulated by bile acids. Expression of a dominant-negative FXR, reduction of FXR with interfering small RNA (siRNA), or mutation of the potential FXR elements decreased Ostalpha and Ostbeta promoter activity and abolished the induction by chenodeoxycolic acid. Negative regulation of the Ostalpha and Ostbeta promoters by bile acids was mediated through LRH-1 elements. Ostalpha and Ostbeta promoter activities were increased by coexpression of LRH-1 and decreased by coexpression of SHP. Mutation of the potential LRH-1 elements and siRNA-mediated reduction of LRH-1 expression decreased basal promoter activity. As predicted from the promoter analyses, ileal Ostalpha and Ostbeta mRNA expressions were increased in wild-type mice administered the FXR agonist GW4064 and decreased in FXR-null mice. Immunoblotting analysis revealed that Ostalpha and Ostbeta intestinal protein expressions correlated with mRNA expression. The mouse Ostalpha and Ostbeta promoters are unusual in that they contain functional FXR and LRH elements, which mediate, respectively, positive and negative feedback regulation by bile acids. Although the positive regulatory pathway appears to be dominant, this arrangement provides a mechanism to finely titrate Ostalpha-Ostbeta expression to the bile acid flux.

Role of FXR in Regulating Bile Acid Homeostasis and Relevance for Human Diseases

Recent studies reveal that bile acids are signalling molecules that activate several nuclear receptors and regulate many physiological pathways and processes to maintain bile acid and cholesterol homeostasis. Analysis of orphan receptor expression patterns in enterohepatic tissues identified bile acids as ligands for farnesoid X receptor (FXR). The primary bile acid chenodeoxycholic acid (CDCA) was shown to be the most potent FXR ligand in vitro at an EC50 of 10-50 microM. FXR can also be activated by the secondary bile acids lithocholic acid (LCA) and deoxycholic acid (DCA). Upon activation FXR heterodimerises with 9-cis retinoic X receptor (RXR) and regulates a cohort of genes involved in cholesterol catabolism and bile acids biosynthesis. Thus bile acid-activated FXR directly induces expression of Small Heterodimer Partner (SHP), a nuclear receptor that suppresses bile acid biosynthesis down-regulates the Na+ taurocholate cotransport peptide (NTCP), a pump depicted to transport bile acids from the lumen into hepatocyte, and induces expression of bile salt export pump (BSEP), the principal bile acid efflux transporter in the liver. As demonstrated by the Fxr null mice, FXR defends the liver against cholestasis. The 6-ethyl derivative of CDCA (6-ECDCA) is approximately 100 fold more potent than CDCA in activating FXR in vitro. In vivo administration of 6-ECDCA protects against cholestasis induced by estrogen and LCA in rats providing evidence that development of potent FXR agonists might represent a new approach for the treatment of cholestastic disorders.

Disrupted coordinate regulation of farnesoid X receptor target genes in a patient with cerebrotendinous xanthomatosis

Cerebrotendinous xanthomatosis (CTX), sterol 27-hydroxylase (CYP27A1) deficiency, is associated with markedly reduced chenodeoxycholic acid (CDCA), the most powerful activating ligand for farnesoid X receptor (FXR). We investigated the effects of reduced CDCA on FXR target genes in humans. Liver specimens from an untreated CTX patient and 10 control subjects were studied. In the patient, hepatic CDCA concentration was markedly reduced but the bile alcohol level exceeded CDCA levels in control subjects (73.5 vs. 37.8 +/- 6.2 nmol/g liver). Cholesterol 7alpha-hydroxylase (CYP7A1) and Na+/taurocholate-cotransporting polypeptide (NTCP) were upregulated 84- and 8-fold, respectively. However, small heterodimer partner (SHP) and bile salt export pump were normally expressed. Marked CYP7A1 induction with normal SHP expression was not explained by the regulation of liver X receptor alpha (LXRalpha) or pregnane X receptor. However, another nuclear receptor, hepatocyte nuclear factor 4alpha (HNF4alpha), was induced 2.9-fold in CTX, which was associated with enhanced mRNA levels of HNF4alpha target genes, CYP7A1, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase, CYP27A1, and NTCP. In conclusion, the coordinate regulation of FXR target genes was lost in CTX. The mechanism of the disruption may be explained by a normally stimulated FXR pathway attributable to markedly increased bile alcohols with activation of HNF4alpha caused by reduced bile acids in CTX liver.

Multiple mechanisms of ontogenic regulation of nuclear receptors during rat liver development.

Nuclear receptors (NRs) play pivotal roles in the regulation of genes contributing to hepatobiliary cholesterol and bile acid homeostasis. We have previously shown that transporters involved in bile formation are developmentally regulated and are poorly developed during the fetal stage, but their expression reached gradual maturity during the postnatal period. To define the molecular mechanisms underlying this regulation and the role that class II NRs and associated members [liver receptor homolog-1 (LRH-1) and short heterodimer partner (SHP)] play, we have analyzed the ontogeny of NR expression during liver development. Real-time PCR analysis of hepatic NR expression from fetal day 17 through adult revealed that steady-state mRNA levels for all NRs were very low during the embryonic period. However, mRNA levels peaked close to that of adult rats (>6 wk-old rats) by 4 wk of age for farnesoid X receptor (FXR), pregnane X receptor (PXR), liver X receptor-alpha (LXRalpha), peroxisome proliferator-activated receptor-alpha (PPARalpha), retinoid acid receptor-alpha (RARalpha), LRH-1, and SHP, whereas RXRalpha mRNA lagged behind. FXR, PXR, LXRalpha, RARalpha, and PPARalpha functional activity in liver nuclear extracts assayed by gel EMSA demonstrated that the activity attained adult levels by 4 wk of age, exhibiting a strict correlation with mRNA levels. Surprisingly, PPARalpha activity was delayed as seen by EMSA assay. Protein levels for NRs also corresponded to the mRNA and functional activity except for RXRalpha. RXRalpha protein levels were higher than message levels, suggesting increased protein stability. We conclude that expression of NRs during rat liver development is primarily regulated by transcriptional mechanisms, which in turn, control the regulation of bile acid and cholesterol metabolic pathways.

Bile acid-induced negative feedback regulation of the human ileal bile acid transporter.

Ileal expression of the apical sodium-dependent bile acid transporter (ASBT) in the rat is unaffected by bile salts, yet in the mouse it is under negative-feedback regulation. The bile acid responsiveness of human ASBT is unknown. The human ASBT promoter linked to a luciferase reporter was studied in Caco-2 cells treated with chenodeoxycholic acid (CDCA) and transfected with expression plasmids for farnesoid X-receptor (FXR), short heterodimer partner (SHP), and retinoic acid receptor/retinoid X receptor (RAR/RXR). CDCA treatment of Caco-2 cells led to a 75% reduction in steady-state ASBT messenger RNA levels and a 78% reduction in human ASBT promoter activity. A dominant negative FXR abrogated the response to CDCA. Site-directed mutagenesis of an RAR/RXR cis element in the human ASBT promoter reduced its activity by 50% and eliminated the bile acid response. Retinoic acid activated the human ASBT promoter fourfold. SHP repressed the activity of the ASBT promoter and reduced activation by retinoic acid. Antisense mediated knock-down of SHP in Caco-2 cells partially offset the bile acid mediated repression of ASBT promoter activity. In conclusion, the human ASBT is positively regulated by retinoic acid. Bile acids induce a negative feedback regulation of human ASBT via an FXR-mediated, SHP-dependent effect upon RAR/RXR activation of ASBT.

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, 3α,7α-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.

Guggulsterone Is a Farnesoid X Receptor Antagonist in Coactivator Association Assays but Acts to Enhance Transcription of Bile Salt Export Pump

Guggulipid is an extract of the guggul tree Commiphora mukul and has been widely used to treat hyperlipidemia in humans. The plant sterol guggulsterone (GS) is the active agent in this extract. Recent studies have shown that GS can act as an antagonist ligand for farnesoid X receptor (FXR) and decrease expression of bile acid-activated genes. Here we show that GS, although an FXR antagonist in coactivator association assays, enhances FXR agonist-induced transcription of bile salt export pump (BSEP), a major hepatic bile acid transporter. In HepG2 cells, in the presence of an FXR agonist such as chenodeoxycholate or GW4064, GS enhanced endogenous BSEP expression with a maximum induction of 400-500% that induced by an FXR agonist alone. This enhancement was also readily observed in FXR-dependent BSEP promoter activation using a luciferase reporter construct. In addition, GS alone slightly increased BSEP promoter activation in the absence of an FXR agonist. Consistent with the results in HepG2, guggulipid treatment in Fisher rats increased BSEP mRNA. Interestingly, in these animals expression of the orphan nuclear receptor SHP (small heterodimer partner), a known FXR target, was also significantly increased, whereas expression of other FXR targets including cholesterol 7alpha-hydroxylase (Cyp 7a1), sterol 12alpha-hydroxylase (Cyp 8b1), and the intestinal bile acid-binding protein (I-BABP), remained unchanged. Thus, we propose that GS is a selective bile acid receptor modulator that regulates expression of a subset of FXR targets. Guggulipid treatment in rats lowered serum triglyceride and raised serum high density lipoprotein levels. Taken together, these data suggest that guggulsterone defines a novel class of FXR ligands characterized by antagonist activities in coactivator association assays but with the ability to enhance the action of agonists on BSEP expression in vivo.

Down-regulation of Cholesterol 7α-Hydroxylase (CYP7A1) Gene Expression by Bile Acids in Primary Rat Hepatocytes Is Mediated by the c-Jun N-terminal Kinase Pathway

Cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in the neutral pathway of bile acid biosynthesis, is feedback-inhibited at the transcriptional level by hydrophobic bile acids. Recent studies show that bile acids are physiological ligands for farnesoid X receptor (FXR). Activated FXR indirectly represses CYP7A1 transcription through induction of small heterodimer protein (SHP-1). In this study, we provide evidence that bile acids rapidly down-regulate CYP7A1 transcription via activation of the JNK/c-Jun pathway. Furthermore, we demonstrate that SHP-1 is also a direct target of activated c-Jun. In primary rat hepatocyte cultures, taurocholate (TCA) strongly activated JNK in a time- and concentration-dependent manner. Tumor necrosis factor-alpha, a potent activator of JNK, also rapidly activated JNK and down-regulated CYP7A1 mRNA levels. Overexpression of dominant-negative JNK1 or a transactivating domain mutant of c-Jun significantly blocked the ability of TCA to down-regulate CYP7A1 mRNA. In contrast, overexpression of wild-type c-Jun (c-Jun(wt)) enhanced the repression of CYP7A1 by TCA. Moreover, overexpression of c-Jun(wt) resulted in increased SHP-1 promoter activity. Mutation of a putative AP-1 (c-Jun) element suppressed c-Jun-mediated activation of the SHP-1 promoter construct. These results indicate that the bile acid-activated JNK pathway plays a pivotal role in regulating CYP7A1 levels in primary rat hepatocytes.