1. Endogenous Bile Acids Are Ligands for the Nuclear Receptor FXR/BAR
Haibo Wang, Jasmine Chen, Kevin Hollister, Lawrence C Sowers, Barry M Forman 
Molecular Cell 
Volume 3, Issue 5, Pages (May 1999)
DOI: /S (00)

2. Figure 1 A Bile Extract Specifically Activates FXR–RXR Heterodimers
CV-1 cells were transiently transfected with the indicated plasmids and treated with a methanol extract of porcine bile (100 μg/ml). Reporter activity was normalized to the internal control and the data plotted as fold activation relative to untreated cells. The following reporter/receptor pairs (see Experimental Procedures) were used: EcRE × 6 / FXR + RXR; βRE2 × 3 / CARβ; PPRE × 3 / PPARα,δ, TR2-11; LXRE × 3 / LXRα; DR0 × 2 / GCNF; SF1 × 4 / SF1; UASG × 4 / GAL-RORα, GAL-Nurr1, GAL-DAX, GAL-ERR2. All transfections contained CMX-β-gal as an internal control.
Molecular Cell 1999 3, DOI: ( /S (00) )

3. Figure 2
The Activity in the Bile Extract Is Mediated by the FXR Subunit
(A) RXRm responds to ligand with a 10-fold decrease in potency relative to the wild-type receptor. CV-1 cells were transiently transfected with CMX-β-gal, UASG × 4, and GAL-L-RXR or the GAL-L-RXRm LBD mutant. After transfection, cells were treated with the indicated concentrations of the RXR-specific ligand LG268 (6-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl]nicotinic acid).
(B) RXRm recruits coactivators with a 100-fold decrease in potency. Coactivator recruitment assays were performed as described (Experimental Procedures) using 1.2 μl of in vitro translated RXR (top panel) or RXRm (lower panel), 5 μg of purified GST-GRIP1, a 32P-labeled DR1 probe, and the concentrations of LG268 ranging from 0 to 1000 nM. CoA refers to a GST fusion containing the three receptor interaction domains from the coactivator GRIP1.
(C) Quantitation of (B) above. The amount of material in the RXR–CoA complex was determined by phosphorimager analysis and plotted as a function of the LG268 concentration.
(D) The bile extract activates FXR but not RXR. CV-1 cells were transfected with the indicated plasmids and treated with either 100 nM LG268 (left panel) or a methanol extract of porcine bile (200 μg/ml, right panel). Note that FXR–RXRm responds to bile acids with about 50% of the maximal fold activation observed with the wild-type heterodimer (data not shown and Figure 4A).
Molecular Cell 1999 3, DOI: ( /S (00) )

4. Figure 4
Structure–Activity and Dose–Response Profiles for FXR and Bile Acids
(A) Efficacy of various bile acids in activating FXR. CV-1 cells were transfected with CMX-β-gal, EcRE × 6, and FXR + RXR (left panel) or FXR + RXRm (right panel) and treated with the indicated bile acids (100 μM), juvenile hormone III (JH III, 50 μM), or LG268 (100 nM). Bile acids are denoted as follows: CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; UDCA, ursodeoxycholic acid.
(B) Bile acid transport is required for activation of FXR by cholic acid and conjugated bile acids. CV-1 cells were transfected with CMX-β-gal, EcRE × 6, and FXR + RXR alone (left panel) or FXR + RXR and the liver bile acid transporter (right panel). After transfection, cells were treated with 100 μM concentrations of the indicated bile acid.
(C) Potency of various bile acids in activating FXR. CV-1 cells were transfected as above and treated with the indicated concentrations of each bile acid.
(D) Comparison of the chemical structure of key bile acids and their efficacy as FXR activators. ++, >200-fold activation; +, 100- to 150-fold activation of FXR–RXR heterodimers.
Molecular Cell 1999 3, DOI: ( /S (00) )

5. Figure 5 Bile Acids Act as Ligands for FXR
(A) Bile acids activate transcription through the ligand-binding domains of FXR–RXR heterodimers. CV-1 cells were transfected with CMX-β-gal, UASG × 4, and constructs containing the ligand-binding domain of RXR (L-RXR) and/or the GAL4 DNA-binding domain fused to the ligand-binding domains of either FXR (GAL-L-FXR) or RXR (VP-GAL-RXR). After transfection, cells were treated with 100 μM CDCA or LCA.
(B) Bile acids induce coactivator recruitment in a mammalian two-hybrid assay. CV-1 cells were transfected with CMX-β-gal, UASG × 4, and GAL-CoA (a GAL4 fusion containing the three receptor interaction domains of SRC-1). Where indicated, cells were also cotransfected with constructs containing the ligand-binding domain of RXR (L-RXR) and/or the VP16 transactivation domain fused to the ligand-binding domains of FXR (VP-L-FXR). After transfection, cells were treated with 100 μM CDCA or LCA.
(C) Bile acids induce coactivator recruitment in vitro. Coactivator recruitment assays were performed by incubating 0.6 μl each of in vitro translated FXR + RXR (left panel) or FXR + RXRm (right panel), 5 μg of purified GST-GRIP1, and a 32P-labeled hsp27 EcRE probe. LCA (300 μM), glycolithocholic acid (300 μM), or LG268 (100 nM) was added as indicated. The resulting complexes were separated by electrophoresis through a nondenaturing gel as described (Experimental Procedures).
Molecular Cell 1999 3, DOI: ( /S (00) )

6. Figure 3 Chenodeoxycholic Acid Is the Active Component in Bile
(A) Fractionation of bile by preparative thin-layer chromatography (PTLC). CV-1 cells were transfected with the indicated plasmids and treated with each of the five PTLC fractions at 25 μg/ml.
(B) HPLC fractionation of bile extract. PTLC fraction B was fractionated by HPLC on a C18 column and the absorbance monitored at 200 nm.
(C) HPLC fraction Z contains the FXR activator. CV-1 cells were transfected as in (A) above and treated with the indicated HPLC fractions at concentrations of 25 μg/ml.
(D) Gas chromatogram of HPLC peak Z. The material in peak Z was methylated and analyzed by gas chromatography/mass spectrometry. The chromatogram demonstrates that fraction Z has one major peak and has been purified to near homogeneity. Compound Z has a retention time of min and is indistinguishable from a chenodeoxycholic acid standard.
(E) Mass spectrum of peak Z. The material eluting at min has a mass spectrum identical to that of chenodeoxycholic acid.
(F) 13C NMR of peak Z. Chemical shifts determined by 13C NMR (CD3OD) are as follows: C1 (36.80), C2 (31.61), C3 (73.09), C4 (40.77), C5 (43.45), C6 (36.14), C7 (69.27), C8 (41.08), C9 (34.33), C10 (36.42), C11 (21.99), C12 (41.28), C13 (43.92), C14 (51.74), C15 (24.77), C16 (29.29), C17 (57.63), C18 (12.55), C19 (23.51), C20 (36.86), C21 (19.01), C22 (32.58), C23 (32.32), and C24 (178.25). In separate experiments (data not shown), 1H NMR (CD3OD) revealed the following chemical shifts: H1α (1.84), H1β (0.96), H2α (1.55), H2β (1.65), H3β (3.36), H4α (2.25), H4β (1.64), H5β (1.38), H6α (1.55), H6β (1.99), H7β (3.81), H8β (1.50), H9α (1.88), H11α (1.53), H11β (1.27), H12α (1.20), H12β (2.02), H14α (1.51), H15α (1.12), H15β (1.75), H16α (1.92), H16β (1.32), H17α (1.20), H18 (0.71, CH3), H19 (0.92, CH3), H20 (1.48), H21 (0.97, CH3), H22α (1.78), H22β (1.29), H23α (2.36), and H23β (2.16). These data further confirm that material in peak Z is CDCA (structure shown).
Molecular Cell 1999 3, DOI: ( /S (00) )

7. Figure 6 Ligand-Occupied FXR Inhibits LXRα
(A) Activation by LXRα and T3R in the presence of FXR. CV-1 cells were transfected with CMX-β-gal, LXRE × 3 (left panel), or T3RE × 3 (right panel), and the indicated expression vectors. All transfections also contain an expression vector for FXR. For LXRα, (left panel), reporter activity was measured in the absence of added ligand. For T3R, activity was measured in the absence or presence of T3 (L-triiodothyronine, 100 nM).
(B) Ligand-occupied FXR inhibits transcription by LXRα but not T3R. CV-1 cells were transfected as in (A) above but this time in the presence or absence of an FXR expression vector. For the LXR transfection (left panel), cells were treated in the absence or presence of 100 μM concentrations of LCA or CDCA. Fold repression represents inhibition of the constitutive activity of LXR by these bile acids. In the case of the T3R transfection (right panel), cells were treated with 100 nM T3 alone or in the presence of 100 μM LCA or CDCA. Fold repression represents inhibition of T3-stimulated activity.
Molecular Cell 1999 3, DOI: ( /S (00) )

8. Figure 7
A Model for Coordinate Regulation of Cholesterol Homeostasis by FXR and LXRα
The major pathway for the degradation of cholesterol to bile acids is via the classical pathway of bile acid synthesis (Chiang 1998) in the liver. The rate-limiting step for this pathway is mediated by Cyp7a (cholesterol 7α-hydroxylase). It is known that transcription of this enzyme is induced by cholesterol (Jelinek et al. 1990), presumably by its conversion to an oxysterol ligand that binds to and activates LXRα (Peet et al. 1998). In contrast, bile acids inhibit transcription of Cyp7a and decrease the conversion of cholesterol to bile acids (Chiang 1998). Our data indicate that FXR acts as a bile acid sensor that inhibits LXRα in response to physiologically relevant bile acids. In addition to the liver, FXR and LXRα are coexpressed in the intestine and kidney, suggesting that these receptors may also serve opposing functions in these tissues.
Molecular Cell 1999 3, DOI: ( /S (00) )