The farnesoid X receptor (FXR) functions as a bile acid (BA) sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. However, BAs are poor reagents for characterizing FXR functions due to multiple receptor independent properties. Accordingly, using combinatorial chemistry we evolved a small molecule agonist termed fexaramine with 100-fold increased affinity relative to natural compounds. Gene-profiling experiments conducted in hepatocytes with FXR-specific fexaramine versus the primary BA chenodeoxycholic acid (CDCA) produced remarkably distinct genomic targets. Highly diffracting cocrystals (1.78 A) of fexaramine bound to the ligand binding domain of FXR revealed the agonist sequestered in a 726 A(3) hydrophobic cavity and suggest a mechanistic basis for the initial step in the BA signaling pathway. The discovery of fexaramine will allow us to unravel the FXR genetic network from the BA network and selectively manipulate components of the cholesterol pathway that may be useful in treating cholesterol-related human diseases.

The Farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily that has been shown to play an important role in bile acid and cholesterol homeostasis. Here we identify four murine FXR transcripts, derived from a single gene, that encode four isoforms, FXR␣1, FXR␣2, FXR␤1, and FXR␤2. FXR␣ and FXR␤ differ at their amino terminus, and FXR␣1 and FXR␤1 have a four-amino acid residue insertion in the hinge region immediately adjacent to the DNA binding domain. Real time PCR and 5-rapid amplification of cDNA ends followed by Southern blotting reveal that these four transcripts are expressed differentially in liver, intestine, kidney, adrenals, stomach, fat, and heart. Electrophoretic mobility shift assays demonstrate that FXR␣2 and FXR␤2 bind to FXR response elements with a higher affinity as compared with FXR␣1 and FXR␤1, suggesting that the four-amino acid insert may affect FXR function. Consistent with this idea, the results of transient transfection experiments demonstrate that the four FXR isoforms differentially transactivated a number of promoter-reporter genes; activation of an ileal bile acid-binding protein promoter-reporter gene varied 20-fold depending on the FXR isoform; the rank order of activation was FXR␤2 > FXR␣2 > > FXR␣1 ‫؍‬ FXR␤1. In contrast, SHP reporter or BSEP reporter genes were activated to similar degrees by each of the FXR isoforms. Finally, NIH3T3 cells were stably infected with individual murine FXR isoforms, and the cells were treated with FXR ligands. The endogenous ileal bile acid-binding protein gene was activated by the four FXR isoforms with the same rank order as seen in transfections. This effect was gene-specific, since induction of bile salt export pump mRNA was independent of the FXR isoform. These observations suggest that there are four distinct murine FXR isoforms that differentially regulate gene expression in numerous tissues in vivo.

Insulin-induced gene 1 (INSIG-1) is a key regulator in the processing of the sterol regulatory element-binding proteins (SREBPs). We demonstrated that

Syndecan-1 (SDC1), a transmembrane heparan sulfate proteoglycan that participates in the binding and internalization of extracellular ligands, was identified in a screen designed to isolate genes that are regulated by the farnesoid X-receptor (FXR, NR1H4). Treatment of human hepatocytes with either naturally occurring (chenodeoxycholic acid) or synthetic (GW4064) FXR ligands resulted in both induction of SDC1 mRNA and enhanced binding, internalization, and degradation of low density lipoprotein. Transient transfection assays, using wild-type and mutant SDC1 promoter-luciferase genes, led to the identification of a nuclear hormone receptor-binding hexad arranged as a direct repeat separated by one nucleotide (DR-1) in the proximal promoter that was necessary and sufficient for activation by FXR. The wild-type, but not a mutated DR-1 element, conferred FXR responsiveness to a heterologous thymidine kinase promoter-reporter gene. Four murine FXR isoforms have been identified recently that differ either at their amino terminus and/or by the presence or absence of four amino acids in the hinge region. Interestingly, the activities of the human SDC1 promoter-reporter constructs were highly induced by the two FXR isoforms that do not contain the four-amino acid insert and were unresponsive to the isoforms containing the four amino acids. Thus, current studies demonstrate that hepatic SDC1 is induced in an FXR isoform-specific manner. Increased expression of SDC1 may account in part for the hypotriglyceridemic effect that can result from the administration of chenodeoxycholic acid to humans.

Affymetrix microarray data and Northern blot assays demonstrated that phospholipid transfer protein (PLTP) was induced 6-fold when either murine or human macrophages were incubated in the presence of ligands for the liver X receptor (LXR) and the retinoid X receptor. Two functional LXR response elements (LXREs) were identified and characterized in the proximal promoter of the human PLTP gene. One LXRE corresponds to a traditional direct repeat separated by 4 bp. However, the second LXRE is novel in that it corresponds to an inverted repeat separated by 1 bp, and is identical to the farnesoid X receptor response element. These studies demonstrate that PLTP is a direct target for activated LXR and farnesoid X receptor (FXR). In addition, apolipoprotein E (apoE), a known LXR target gene in macrophages, was shown to be activated in liver cells by FXR ligands.Taken together, the current data suggest that a small number of genes that currently include PLTP, apoE, and apoC-II, are induced in macrophages by activated LXR and in liver by activated FXR. Phospholipid transfer protein (PLTP) is a member of the lipid transfer/lipopolysaccharide binding protein gene family that includes CETP, the lipopolysaccharide binding protein, and the bactericidal/permeability increasing protein (1). PLTP has broad substrate specificity and functions to transfer phospholipids from triglyceride rich particles (VLDL and chylomicrons) to HDL during lipoprotein lipolysis (2). PLTP mRNA is expressed in a number of tissues, including liver, ovary, thymus, and placenta (3). Recent studies have demonstrated that PLTP is a target gene for activated farnesoid X receptor (FXR) (4, 5). This conclusion was based on the findings that i ) FXR ligands induced PLTP mRNA levels in cultured human hepatocytes (4) and in the livers of wild type but not FXR null mice (6), and ii ) the proximal promoter of the human gene contains a functional FXR response element (FXRE) (4, 5).In the current report, we utilized murine and human macrophages to demonstrate that PLTP is also an liver X receptor (LXR) target gene. Since macrophages do not express FXR, the results demonstrate that PLTP can be regulated independently by either receptor. Apolipoprotein E (apoE) is a known LXR target gene in macrophages (7,8). We now demonstrate that the hepatic expression of apoE is induced by FXR ligands. Taken together, these studies provide evidence that a subset of genes, which currently includes PLTP, apoE, and apoC-II (4-8), are induced following activation of LXR or FXR and control lipid metabolism. EXPERIMENTAL PROCEDURES Reagents, RNA isolation, Northern blot analysis, transient transfections, and reporter gene assaysExpression plasmids, sources of all reagents, and the indicated procedures have been described (6, 7, 9, 10). T0901317, chenodeoxycholate (CDCA), LG100153 (LG), and GW4064 (GW) were obtained from Cayman Chemical (Ann Arbor, MI), Sigma (St. Louis, MO), Dr. R. Heyman (Ligand Pharmaceuticals, La Jolla, CA), and Dr. T. Willson (GlaxoSmithKline), respectivel...