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Endocrinology Vol. 147, No. 9 4025-4033
Copyright © 2006 by The Endocrine Society

The Nuclear Hormone Receptor Farnesoid X Receptor (FXR) Is Activated by Androsterone

Shuguang Wang, KehDih Lai, Franklin J. Moy, Anitha Bhat, Helen B. Hartman and Mark J. Evans

Cardiovascular and Metabolic Disease Research (S.W., K.L., A.B., H.B.B., M.J.E.), Wyeth Research, Collegeville, Pennsylvania 19426; and Chemical and Screening Sciences (F.J.M.), Wyeth Research, Cambridge, Massachusetts 02140

Address all correspondence and requests for reprints to: Mark Evans, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426. E-mail: Evansm{at}wyeth.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Farnesoid X receptor (FXR) uses bile acids as endogenous ligands. Here, we demonstrate that androsterone, a metabolic product of testosterone, is also an FXR ligand. Treatment of castrated male mice with androsterone induced expression of the FXR target gene small heterodimer partner (SHP). In mouse AML-12 hepatocytes, chenodeoxycholic acid (CDCA) or androsterone induced SHP expression with a similar kinetic pattern. The FXR antagonist guggulsterone blocked the induction of SHP by androsterone in AML-12 cells. Nuclear magnetic resonance spectroscopy demonstrated the direct binding of androsterone to purified human FXR (hFXR) ligand-binding domain (LBD) protein, resulting in the recruitment of steroid receptor coactivator protein-1 (SRC-1) coactivator peptide. In HEK293 cells, androsterone activated gal4-mouse FXR-LBD and gal4-hFXR-LBD fusion proteins, although in contrast to CDCA, androsterone activation was significantly greater for the mouse FXR-LBD than for the hFXR-LBD. Site-directed mutagenesis of the hFXR-LBD defined amino acids Asn354 and Ser345 as critical for differential species sensitivity to CDCA and androsterone, respectively. Crystal structure studies suggest that the orientation of the steroid nucleus of bile acids within the binding pocket of FXR is reversed from all other nuclear hormone receptors. In support of this model, we show here that mutations M265I or R331H, residues predicted by crystal structure to interact with the carboxylic acid tail of CDCA but not with androsterone, altered CDCA activation but had no effect on androsterone activation. Activation of FXR by androsterone may provide an additional means for physiological or pharmacological modulation of FXR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FARNESOID X RECEPTOR (FXR), originally identified as an orphan nuclear hormone receptor (1, 2), is now known to bind bile acids as physiologically relevant ligands (3, 4, 5) and to play a critical role in bile acid metabolism through regulation of two critical pathways. First, when FXR binds to bile acids within the hepatocytes, activated FXR in turn induces expression of another orphan nuclear hormone receptor, small heterodimer partner (SHP). Elevated levels of SHP then function to inhibit expression of cholesterol 7{alpha}-hydroxylase (CYP7A1) and sterol 12{alpha}-hydroxylase (CYP8B1), two enzymes within the bile acid synthetic pathway that play critical roles in regulating the synthesis and composition of the bile acid pool (6, 7). Second, FXR activation by bile acids in the ileum activates expression of fibroblast growth factor 15 in mice or fibroblast growth factor 19 in humans, which then further represses expression of CYP7A1 within hepatocytes (8, 9). Activation of both pathways is essential for regulation of CYP7A1 and CYP8B1 expression. Conversion of cholesterol into bile acids is a major contributor to determination of plasma cholesterol levels, with polymorphisms within the human CYP7A1 promoter demonstrated to be a major determinant for variability in plasma low-density lipoprotein concentrations (10). Loss of FXR activity has detrimental effects on plasma cholesterol levels and development of atherosclerosis in mice (11, 12, 13). Furthermore, activation of FXR has now been shown to reduce plasma triglyceride levels, with multiple mechanisms suggested, including elevation of apolipoprotein CII (apoCII) expression, repression of apoCIII expression, and modulation of sterol regulatory element binding protein 1 expression (14, 15, 16). FXR activity thus plays a significant role in modulation of the overall lipid profile.

Sex hormones also play a significant role in modulating the lipid profile. Estrogens have well characterized effects including reduction in low-density lipoprotein cholesterol, elevation of high-density lipoprotein cholesterol, and elevation of triglyceride levels (17). The effects of androgens on plasma lipids are less consistent, with the results dependent upon the study population as well as the specific androgen and route of administration used (reviewed in Ref. 18). However, accumulating evidence suggests that testosterone may have net overall beneficial effects for inhibiting development of atherosclerosis both in humans and in experimental animals (19, 20, 21). Testosterone itself is a poor activator of nuclear hormone receptor activity. Rather, testosterone is converted into dihydrotestosterone (DHT), a potent activator of the androgen receptor. In turn, DHT is converted into 5{alpha}-androstan-17-one and then into androsterone, two steroids thought to lack activity toward nuclear hormone receptors.

Previously, we have found that estrogens induce expression of SHP via activation of SHP promoter activity at an estrogen receptor (ER) binding site that overlaps with the FXR response element within the SHP promoter (22). These results raised the question of whether male sex hormones might also regulate SHP expression. Androsterone increases the activity of a transfected synthetic FXR response element reporter plasmid when this plasmid is cotransfected with a rat FXR expression plasmid (23). However, it is not known whether this is a direct effect upon FXR or whether endogenous levels of FXR can respond to activation by androsterone to regulate gene expression either in cultured cells or in animals. Furthermore, there are significant species differences between FXR in regard to activation by bile acids (24), and it is unknown whether androsterone activation is limited to rat FXR. Here we demonstrate that androsterone can function as a direct activator of the mouse (m) and human (h) FXR ligand-binding domain (LBD) in a manner similar to bile acids, with androsterone having significantly greater activity on mFXR than on human FXR (hFXR).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Castrated male mice 8–10 wk of age (Taconic, Lexington, KY) were fed a casein diet (no. 8117; Test Diet, Richmond, IN) for 2 wk before the start of the study. The mice received daily sc injections of 0.1 ml 90% corn oil/10% ethanol vehicle, 10 mg/ml androsterone (Steraloids, Newport, RI) in vehicle, or 1 mg/ml 4-propyl-1,3,5-Tris(4-hydroxy-phenyl) pyrazole (PPT) (25) in vehicle. Animals were killed 2 h after the fifth daily treatment, approximately 4 h after commencement of the light cycle. Total RNA was prepared, and mRNA levels for specific genes were determined by real-time PCR as previously described (22). All treatments were in accord with accepted standards of care as specified by the Wyeth animal care committee.

Plasmid constructs
The LBDs of human or murine FXR were amplified by PCR and cloned into the BamHI/MluI sites of the vector pM (CLONTECH, Mountain View, CA), containing the Gal4 DNA-binding domain. Epitope-tagged fusion proteins were generated by introducing a FLAG tag (DYKDDDDK) between the carboxy terminus of the Gal4 DNA binding domain and the amino terminus of the FXR-LBD. hFXR-LBD mutants were constructed by overlapping PCR. Correct sequence of all constructs was verified by complete sequencing of both strands of all DNA regions generated by PCR.

Cell culture
AML-12 cells (26) were obtained from the American Type Culture Collection (Manassas, VA) and cultured in a 1:1 mix of DMEM/Ham’s F12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Life Technologies, Inc., Rockville, MD), 1% insulin-transferrin-selenium mix (Life Technologies), 40 ng/ml dexamethasone (Sigma Chemical Co., St. Louis, MO), 100 U/ml penicillin, and 100 µg/ml streptomycin. For gene regulation studies, AML-12 cells were plated in growth medium at 4 x 105 cells per well in six-well plates. The following day, treatments including chenodeoxycholic acid (CDCA), androsterone, DHT, 5{alpha}-androstan-17-one, or guggulsterone (all obtained from Steraloids) or dimethylsulfoxide (DMSO) vehicle were added to the medium. At various times after treatment addition, total RNA was prepared by the RNeasy method (QIAGEN, Valencia, CA). mRNA levels for individual genes were determined by real-time PCR as previously described (27).

For transfections, AML-12 cells were plated at 3 x 106 cells per 100-mm dish in 10 ml growth medium on d 1. On d 2, AML-12 cells were washed with OPTI-MEM (Invitrogen) and cotransfected with 1 µg pCDNA3.1(+) and 16 µg of either pGL3 hSHP/-1383 or pGL3 hSHP LS-291/-286 in 10 ml OPTI-MEM per dish using the Lipofectamine 2000 (Invitrogen) transfection procedure. Growth medium was replaced 6 h after transfection. On d 3, the medium was changed to growth medium supplemented with 800 µg/ml geneticin, and the cells were replated by limiting dilution into 96-well plates. After 12 d, single colonies were isolated and expanded. For determination of compound activities, the resulting stable AML-12 cell lines containing either pGL3 hSHP/-1383 or pGL3 hSHP LS-291/-286 were treated for 24 h with either 0.1% DMSO vehicle, 30 µM CDCA, or 30 µM androsterone. Cell lysates were prepared and assayed for luciferase by kit (Promega, Madison, WI).

HEK293 cells (American Type Culture Collection) were maintained in high-glucose DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 1% glutamax, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin. HEK293 cells were seeded in growth medium into 24-well plates at a density of 1.5 x 105 cells per well the day before transfection. Each well was transfected using 1 µg pFR-Luc luciferase reporter plasmid (Stratagene, La Jolla, CA), 0.05 µg ß-galactosidase expression plasmid pEF1 V5-His LacZ (Invitrogen), and 0.1 µg various pM-FXR-LBD fusion plasmids using Lipofectamine 2000 (Invitrogen). Twenty hours after transfection, fresh medium containing either DMSO vehicle or various concentrations of compounds (30 µM CDCA, 30 µM androsterone, or 1 µM GW4064) was added as indicated. Twenty-four hours after treatment addition, cell lysates from triplicate wells were prepared in lysis buffer (Tropix, Bedford, MA) and were analyzed for luciferase activity (Promega) and ß-galactosidase activity (Tropix). Luciferase activities were normalized to ß-galactosidase activities individually for each well.

Gal4-FXR fusion protein expression analysis
HEK293 cells were seeded into 60-mm dishes at a density of 1.5 x 106 cells per dish the day before transfection. Each transfection contained 8 µg pM-FLAG-hFXR-LBD wt or mutant as indicated using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, nuclear extracts were prepared using a nuclear and cytoplasmic extraction kit (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions. Seventeen micrograms of total nuclear protein were separated by polyacrylamide gel electrophoresis under reducing conditions using a 4–15% gradient SDS-denaturing gel (Bio-Rad, Hercules, CA). Western blotting was carried out using monoclonal anti-FLAG M2 primary antibody (Sigma) at a 1:200 dilution, goat antimouse IgG-horseradish peroxidase (HRP) secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:5000 dilution, and ECL plus Western blotting detection system (Amersham Biosciences, Arlington Heights, IL).

Nuclear magnetic resonance (NMR) spectroscopy
Escherichia coli strain BL21(DE3) expressing an N-terminal 8xHis and thrombin-tagged hFXR-LBD (amino acids 244–472) were expanded in a Biostat C-10 bioreactor (Sartorius BBI Systems, Inc., Bethlehem, PA) and induced with 1.0 mM isopropyl-ß-D-thiogalactopyranoside for 3 h at 25C. Cell pellets were quick frozen in liquid nitrogen and stored at –80 C until purification. The cells were resuspended in 20 mM Tris (pH 8), 500 mm NaCl, 5 mM 2-mercaptoethanol, 1 mm EDTA, and 5 mM imidazole. After disruption with a microfluidizer and centrifugation, the supernatant was applied to a Ni-NTA Superflow column (QIAGEN). The hFXR-LBD was eluted with a gradient of 5–150 mm imidazole. After digestion with thrombin to remove the N-terminal His-tag, hFXR-LBD was loaded onto a TSK gel G3000sw column (Tosoh Bioscience, South San Francisco, CA) equilibrated with a buffer of 20 mM Tris (pH 8), 50 mm NaCl, 5 mM 2-mercaptoethanol, and 1 mm EDTA. Fractions containing hFXR-LBD were applied onto a Q-Sepharose column and eluted with a 50–550 mM NaCl gradient. The yield of purified hFXR-LBD was 7 mg per 10 g of cell pellet.

One-dimensional (1D) 1H and saturation transfer difference (STD) (28) spectra were collected on a Bruker Avance 600-MHz spectrometer equipped with a triple-resonance 5-mm inverse cryoprobe. All samples were in a buffer containing 20 mM Tris, 1 mm dithiothreitol, 50 mM NaCl (pH 8.0), and 2.5% DMSO in D2O. All spectra were measured at 25 C. For the STD experiment, the concentration of FXR and androsterone was 5 and 50 µM, respectively. Nearly complete saturation of the protein was achieved with FXR irradiation at 0.78 ppm for 3 sec, using a train of 60 G4 Gaussian cascade shape pulses (29) of 50 msec length, each separated by a 1-msec delay. Off-resonance irradiation was applied at –4 ppm, void of any protein resonances. The subtraction of STD spectra was performed internally with on/off resonance protein excitation using phase cycling (30). Two negative control STD experiments were also performed with FXR alone and androsterone alone showing no signal at the androsterone resonance positions. All STD experiments were collected with 400 scans with a total time of 35 min along with a 1D 1H reference spectrum with 200 scans.

For the 1D line-broadening experiments (31), the spectra were recorded using a 10-msec spin lock pulse to reduce the protein background resonances to facilitate analysis. For the determination of agonist activity of androsterone, hFXR-LBD and androsterone were incubated together for 10 min followed by addition of steroid receptor coactivator protein-1 (SRC-1) peptide (biotin-Ser-His-Ser-Ser-Leu-Thr-Glu-Arg-His-Lys-Ile-Leu-His-Arg-Leu-Leu-Gln-Glu-Gly-Ser) and additional incubation for 15 min. The 1D 1H spectra were then collected with 768 scans and total time of 50 min. To demonstrate the antagonist effect of guggulsterone, a fresh sample of FXR, SRC-1 peptide, and androsterone was made as above followed by an incubation of guggulsterone for 15 min with subsequent 1D 1H NMR spectrum collection. Agonist and antagonist properties were monitored by a decrease or increase of SRC-1 peptide resonances, respectively. Concentrations used in the 1D 1H line-broadening experiments were 10 µM SRC-1, 10 µM hFXR-LBD, 50 µM androsterone, and 50 µM guggulsterone.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Androsterone activates mFXR
Induction of SHP expression in the liver is considered as one of the canonical features of FXR activity in vivo. To first determine whether androsterone could function as an FXR activator in vivo, castrated male mice were treated with either androsterone or the synthetic ER{alpha} ligand PPT as a control. Both treatments resulted in a significant induction of SHP (Fig. 1Go), suggesting androsterone may activate endogenous FXR.


Figure 1
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  		FIG. 1. Androsterone induces expression of SHP in mouse liver. Castrated male C57BL/6 mice fed a casein-based diet were administered a daily sc injection of corn oil/ethanol vehicle, 50 mg/kg androsterone, or 5 mg/kg PPT for 5 d. Two hours after the final treatment, livers were removed and the mRNA levels for SHP and glyceraldehyde-3-phosphate dehydrogenase in each individual animal was quantified by real-time PCR. The SHP expression levels were normalized for expression of glyceraldehyde-3-phosphate dehydrogenase and are presented as the mean ± SE, with six mice per group. The mean expression level of SHP in mice receiving vehicle treatment was defined as 1.0. *, P < 0.05.

 
To demonstrate that androsterone directly activated SHP expression through FXR activation in hepatocytes, AML-12 cells, a differentiated nontransformed mouse hepatocyte cell line (26), were treated with androsterone or the natural FXR ligand CDCA. Both treatments (Fig. 2AGo) as well as the synthetic FXR agonist GW4064 (not shown) rapidly induced SHP expression. To further verify that these inductions occurred through activation of FXR, AML-12 cells were treated with either CDCA or androsterone in the absence or presence of the FXR antagonist guggulsterone (32, 33, 34). Both CDCA and androsterone induction of SHP was completely blocked by guggulsterone (Fig. 2BGo). DHT and 5{alpha}-androstan-17-one, the intermediate in the conversion of DHT to androsterone, were both inactive for induction of SHP expression (Fig. 2BGo), indicating the lack of involvement of androgen receptor in SHP induction.


Figure 2
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  		FIG. 2. Androsterone induction of SHP in AML-12 cells is dependent upon FXR. A, AML-12 cells were cultured for the indicated times in medium containing vehicle ({circ}), 30 µM CDCA (bullet), or 100 µM androsterone ({square}). Total RNA was prepared, and the expression levels for SHP and glyceraldehyde-3-phosphate dehydrogenase were determined by real-time PCR. Expression levels for SHP were normalized for glyceraldehyde-3-phosphate dehydrogenase expression and are presented as the mean ± SE. The level of expression for SHP in cells before any treatment was defined as 1.0. B, AML-12 hepatocytes were cultured for 24 h in the presence of vehicle, 30 µM CDCA, 30 µM CDCA plus 30 µM guggulsterone, 100 µM androsterone, 100 µM androsterone plus 30 µM guggulsterone, 100 µM DHT, or 100 µM 5{alpha}-androstan-17-one (Adiol) as indicated. SHP mRNA was subsequently quantified by real-time PCR. Values were normalized for glyceraldehyde-3-phosphate dehydrogenase expression and are presented as the mean ± SE. The level of SHP expression in vehicle-treated cells was defined as 1.0. *, P < 0.05 vs. vehicle; **, P < 0.05 for guggulsterone inhibition.

 
To further verify that the FXR-LBD was directly activated by androsterone, we compared the ability of GW4064, androsterone, and CDCA to activate gal4-mFXR-LBD and gal4-hFXR-LBD fusion proteins in HEK293 cells (Fig. 3AGo). GW4064 activated both hFXR-LBD and mFXR-LBD to nearly the same extent, and CDCA activated the hFXR-LBD to a greater extent than the mFXR-LBD, as previously noted (24). Androsterone activated both the mFXR-LBD and the hFXR-LBD, although the reciprocal pattern of species sensitivity was observed, with androsterone activating the mFXR-LBD more strongly than the hFXR-LBD. Furthermore, cotransfection studies with gal4-hFXR-LBD and SRC-1/VP16 expression plasmids demonstrated that androsterone potentiated the interaction of SRC-1 with the hFXR-LBD (Fig. 3BGo).


Figure 3
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  		FIG. 3. Mouse and human Gal4/FXR-LBD fusion proteins are activated by androsterone. A, HEK293 cells plated at a density of 1.5 x 105 cells per well in 24-well plates were cotransfected with 1 µg pFR-Luc luciferase reporter plasmid, 0.05 µg ß-galactosidase expression plasmid pEF1 V5-His LacZ, and 0.1 µg of either pM-hFXR-LBD, expressing Gal4-hFXR-LBD, or pM-mFXR-LBD, expressing Gal4-mFXR-LBD. Twenty-four hours after transfection, the cells were treated with either DMSO vehicle or various concentrations of CDCA, androsterone, or GW4064. Cell lysates were prepared 24 h after addition of compounds and were assayed for luciferase and ß-galactosidase activities. Luciferase activities were normalized for ß-galactosidase determinations, with the normalized luciferase activity in cells treated with vehicle defined as 1.0. Values shown are mean ± SE. B, HEK293 cells were cotransfected with 1 µg pFR-Luc luciferase reporter plasmid and 0.05 µg ß-galactosidase expression plasmid pEF1 V5-His LacZ together with 0.1 µg pM-hFXR-LBD or pSRC-1/VP16 expression plasmid as indicated. Twenty-four hours after transfection, the cells were treated with vehicle or androsterone for 24 h as indicated and assayed for luciferase and ß-galactosidase activities.

 
Direct binding of androsterone to the hFXR-LBD was tested by STD spectroscopy (28) using purified bacterially synthesized hFXR-LBD. Comparison of the 1D spectrum of androsterone alone (Fig. 4AGo, tracing 3) with the 1D spectrum of androsterone plus hFXR LBD (Fig. 4AGo, tracing 1) demonstrated a significant STD signal (Fig. 4AGo, tracing 2). No signal was detected by STD for androsterone alone (Fig. 4AGo, tracing 4) whereas the STD spectrum of hFXR-LBD alone (Fig. 4AGo, tracing 5) indicated that the positive STD signal obtained with androsterone plus hFXR-LBD was not because of protein resonances. Together these results indicate a direct interaction of androsterone with the hFXR-LBD. To determine whether androsterone binding of hFXR-LBD could recruit coactivator, we compared the 1D NMR spectra of SRC-1 peptide alone (Fig. 4BGo, tracing 1), SRC-1 peptide plus hFXR-LBD (Fig. 4BGo, tracing 2), and SRC-1 peptide plus hFXR-LBD and 50 µM androsterone. A weak interaction between the SRC-1 peptide and hFXR-LBD was noted through the minor line broadening of SRC-1 peaks at 7.70 ppm (a), 6.95 ppm (b), and 6.90 ppm (c) in the presence of hFXR-LBD. In the presence of 50 µM androsterone, these resonances were strongly reduced (Fig. 4BGo, tracing 3), indicating tight binding of the SRC-1 peptide to the hFXR-LBD. In the presence of androsterone plus guggulsterone (Fig. 4BGo, tracing 4), these resonance peaks returned to a pattern similar to that observed in the absence of any ligand. Line-broadened guggulsterone resonances at 5.9 ppm (d) and 5.75 ppm (e) reflect the binding of guggulsterone to the hFXR-LBD. These results confirm that androsterone can directly bind to the hFXR-LBD in a manner that can be competed by guggulsterone and that binding of androsterone results in recruitment of an SRC-1 coactivator peptide to the hFXR-LBD.


Figure 4
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  		FIG. 4. Androsterone binds to hFXR-LBD and induces recruitment of SRC-1 coactivator peptide. A, For STD NMR spectroscopy, saturation of purified hFXR-LBD protein was achieved by selectively irradiating FXR resonances at 0.78 ppm for 3 sec with detection of the saturation transferred to bound androsterone by intermolecular spin diffusion. The presence of a positive STD spectrum (defined as the difference between the 1D spectrum with and without protein saturation) indicates ligand binding. Tracing 1, 1D spectrum of 50 µM androsterone and 5 µM FXR; 2, STD spectrum of 50 µM androsterone plus 5 µM FXR; 3, 1D spectrum of 50 µM androsterone; 4, STD spectrum of 50 µM androsterone; 5, STD spectrum of 5 µM FXR. B, NMR 1D spectrum measurement of androsterone recruitment of SRC-1 peptide to the hFXR-LBD. Tracing 1, 10 µM SRC-1 peptide; 2, 10 µM SRC-1 peptide and 10 µM hFXR-LBD; 3, 10 µM SRC-1 peptide, 10 µM hFXR-LBD, and 50 µM androsterone; 4, 10 µM SRC-1 peptide, 10 µM hFXR-LBD, 50 µM androsterone, and 50 µM guggulsterone; 5, 10 µM hFXR-LBD; 6, 50 µM guggulsterone. a–c, SRC-1 peptide resonances altered by binding to hFXR-LBD; d and e, guggulsterone line-broadened resonances indicative of guggulsterone binding to hFXR-LBD. Arrows delineate the decrease in peaks a– c observed in the presence of hFXR-LBD plus androsterone but not in the presence of the androsterone plus guggulsterone.

 
Differential sensitivity of mFXR and hFXR to CDCA and androsterone activation is determined primarily by two amino acids
Within the LBD, human and mFXR differ at 12 amino acid positions (Fig. 5Go). To determine which amino acids might confer the differential sensitivity to CDCA and androsterone induction, a series of Gal4-hFXR-LBD fusion proteins were constructed with the corresponding murine amino acids introduced at various positions (Fig. 6Go). As expected from the similar GW4064 induction curves for human and mFXR-LBD, no human-to-murine amino acid change had a significant effect upon induction by GW4064. The N354K conversion strongly diminished activation of the hFXR-LBD by CDCA but had no effect on activation by androsterone. Additionally, three additional conversions, N293S, S342A, and I372V all partially reduced activation by CDCA but had no effect on activation by androsterone. In contrast, activation of hFXR-LBD by androsterone was significantly enhanced by the S345A conversion. This amino acid change had no effect on activation by CDCA. The difference in sensitivity to androsterone and CDCA between human and mFXR is thus largely controlled by two amino acid differences, with serine or alanine at position 345 determining androsterone sensitivity and asparagine or lysine at position 354 determining CDCA sensitivity.


Figure 5
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  		FIG. 5. Mutations of the hFXR-LBD. The sequence of the hFXR-LBD used for Gal4 fusions is shown, with amino acids differing in the mFXR-LBD shown below the human sequence. Underlined amino acids have been suggested by crystallography to be important for binding of 6-ECDCA to rat FXR (35 ) or in binding of the synthetic ligand fexaramine to hFXR (36 ). Numbers designate positions of amino acid mutations created here in the hFXR-LBD.

 

Figure 6
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  		FIG. 6. Amino acid differences between human and mFXR-LBD determine relative sensitivity to CDCA and androsterone activation. A, HEK293 cells plated at a density of 1.5 x 105 cells per well in 24-well plates were cotransfected with 1 µg pFR-Luc luciferase reporter plasmid, 0.05 µg ß-galactosidase expression plasmid pEF1 V5-His LacZ, and 0.1 µg of either pMF-hFXR-LBD, expressing Flag-tagged Gal4-hFXR-LBD, or 0.1 µg plasmids expressing Flag-tagged Gal4-hFXR-LBD with the indicated amino acids converted to the corresponding mouse amino acid. Twenty-four hours after transfection, the cells were treated with DMSO vehicle, 30 µM androsterone (black bars), 30 µM CDCA (blue bars), or 1 µM GW4064 (red bars). Cell lysates were prepared 24 h after addition of compounds and were assayed for luciferase and ß-galactosidase activities. Luciferase activities were normalized for ß-galactosidase activity. The magnitude of inductions ± SE for each mutant is expressed as a percentage of the magnitude of induction for the native hFXR-LBD, defined as 100% for each compound. The absolute fold induction for hFXR was 6.3 ± 0.7 with androsterone, 192 ± 18 with CDCA, and 787 ± 88 with GW4064, based upon 11 determinations. B, HEK293 cells were transfected with either pMF-hFXR-LBD, expressing Flag-tagged Gal4-hFXR-LBD, or plasmids expressing Flag-tagged Gal4-hFXR-LBD with the indicated amino acid changes. Forty-eight hours after transfection, nuclear extracts were prepared and 17 µg of total nuclear protein was subjected to SDS-PAGE on a 4–15% gel. Western blotting was carried out using the monoclonal anti-FLAG M2 antibody as the primary antibody, goat antimouse IgG-HRP as the secondary antibody, and enhanced chemiluminescence as the detection system.

 
Although single amino acid differences between the human and mFXR-LBD can alter the relative sensitivity to CDCA or androsterone activation, these amino acids have not been reported to interact with ligand in the crystal structures of rat FXR bound with 6{alpha}-ethyl-CDCA (6-ECDCA) (35) or hFXR bound with fexaramine (36) (Fig. 5Go). Furthermore, the crystal structure of rat FXR indicates that bile acids occupy the FXR binding pocket with the steroid nucleus positioned in the reverse orientation compared with all other steroid receptors (35). Androsterone and CDCA share significant structural similarities, suggesting that androsterone might also interact with the FXR-LBD in a reverse orientation. If this were the case, then androsterone and CDCA should share significant similarities in sensitivity to the introduction of amino acid changes at ligand contact sites within the FXR ligand-binding pocket. To address this question, a series of mutations was created in which single amino acids of the hFXR-LBD were converted to the corresponding amino acid present in murine FXRß, which is not activated by CDCA (37). Several amino acid changes (M328I, I352S, I357H, F366S, L451Y, L465F, and Y369Q) strongly diminished the level of gal4-hFXR-LBD protein expression as measured by Western blotting (data not shown) and were not further characterized. The remaining mutations all had similar levels of expression and were tested for activation by androsterone, CDCA, or GW4064 (Fig. 7Go). For several mutations, results were similar for all three ligands. Thus A291S, L348A, and W469F strongly reduced activation by all three ligands, and E350V had no effect on activation by any ligand. The basis for the disruption by the A291S mutation may reflect disruption of the general hydrophobic character of the FXR ligand-binding pocket, whereas the mutation W469F may have weakened the {pi}-cation interaction between W469 and H444 critical for helix 12 interaction with ligand. The basis for the global disruption by L348A is less clear, because this has been reported as a contact site for fexaramine but not 6-ECDCA. Several amino acid changes including H294S, S332V, R351H, and Y361F significantly reduced both androsterone and CDCA activation while having much less influence on GW4064 activation. These results suggest the two steroid ligands androsterone and CDCA make several similar interactions with the FXR-LBD and that androsterone may thus likely also be in a reverse orientation within the ligand-binding pocket.


Figure 7
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  		FIG. 7. Amino acid differences between human and mFXR-LBD determine relative sensitivity to CDCA and androsterone activation. HEK293 cells plated at a density of 1.5 x 105 cells per well in 24-well plates were cotransfected with 1 µg pFR-Luc luciferase reporter plasmid, 0.05 µg ß-galactosidase expression plasmid pEF1 V5-His LacZ, and 0.1 µg of either pMF-hFXR-LBD, expressing Flag-tagged Gal4-hFXR-LBD, or 0.1 µg plasmids expressing Flag-tagged Gal4-hFXR-LBD with the indicated amino acid changes. One microgram of plasmids expressing the mutants M290V and A291S was used to compensate for low expression levels. Twenty-four hours after transfection, the cells were treated with DMSO vehicle, 100 µM androsterone (black bars), 30 µM CDCA (blue bars), or 1 µM GW4064 (red bars). Cell lysates were analyzed as described in Fig. 6Go. B, HEK293 cells were transfected with either pMF-hFXR-LBD, expressing Flag-tagged Gal4-hFXR-LBD, or plasmids expressing Flag-tagged Gal4-hFXR-LBD with the indicated amino acid changes. Forty-eight hours after transfection, nuclear extracts were prepared and 17 µg of total nuclear protein was subjected to SDS-PAGE on a 4–15% gel. Western blotting was carried out using the monoclonal anti-FLAG M2 antibody as the primary antibody, goat antimouse IgG-HRP as the secondary antibody, and enhanced chemiluminescence as the detection system.

 
There are two major structural distinctions between CDCA and androsterone. First is the presence of a 7{alpha}-hydroxyl group in CDCA, absent from androsterone, which would be predicted to interact with Y369 of the hFXR based upon the crystal structure of rat FXR with 6-ECDCA. The effect of mutagenesis of Y369 could not be determined because of poor expression levels. A second major structural distinction between CDCA and androsterone is the presence of a five-carbon carboxylic acid tail at position C17 of CDCA that would be predicted to make a hydrogen bond with arginine at position 331 and a hydrophobic interaction with methionine at position 262 of the hFXR-LBD. These interactions are expected to be absent for androsterone, which has a carbonyl group at position C17. In agreement with this model, the mutation R331H completely abolished CDCA activation of hFXR-LBD but had no effect on androsterone activation. Similarly, the substitution M265I enhanced activation by CDCA but had no effect on androsterone activation.

Finally, although androsterone is significantly smaller than CDCA, androsterone appears to make contacts with FXR that are not made by CDCA. For example, the mutation L340T nearly abolished activation by androsterone but had no effect on CDCA activation. Together, these mutation results indicate that androsterone and CDCA share many similarities in their interaction with the hFXR-LBD, yet may place the FXR-LBD into structurally distinct conformations.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
FXR has been established to play a critical role in the regulation of bile acid synthesis in the liver, predominantly by induction of expression of SHP (6, 7). Additionally, FXR regulates the enterohepatic recirculation of bile acids both through hepatic effects such as modulation of bile acid transporter expression (38, 39) and through effects within the intestine such as regulation of ileal bile acid-binding protein expression (40). FXR expression is high in the liver and intestine, consistent with this role. However, FXR is also expressed abundantly in the kidney and adrenal gland as well as numerous other organs and in vascular smooth muscle cells (2, 41). These other sites of FXR expression are not usually considered as targets for bile acid regulation, suggesting that other potential natural ligands may exist for FXR. Here we demonstrate by NMR spectroscopy that the steroid androsterone directly binds to the FXR-LBD and induces recruitment of coactivators such as SRC-1. Furthermore, androsterone can function as an activator of FXR both in a transfection system using gal4-FXR fusion protein induction of reporter genes and in cultured cells using endogenous FXR/retinoid X receptor heterodimer activation of gene expression. Finally, androsterone treatment of castrated male mice induced expression of SHP, suggesting that androsterone may also regulate gene expression in vivo via the FXR. It will be of interest to determine whether androsterone can regulate gene expression through FXR in other organs such as the adrenal gland.

Structurally diverse synthetic FXR ligands such as GW4064 and fexaramine have very different effects on the global pattern of gene regulation in hepatocytes (36). This has given rise to the concept that different ligands may place the FXR-LBD into distinct conformations that are responsible for different patterns of gene regulation. This model would be analogous to the situation for ER, where it has been demonstrated that ligands such as genistein can induce a conformation of the ER-LBD that still exerts the antiinflammatory gene regulations of estrogens yet is unable to induce expression of classical estrogen-response-element-driven gene transcription (42, 43). Limited profiling of gene regulation by CDCA and androsterone in mouse AML-12 cells identified at least one difference in gene regulation. Both CDCA and androsterone induced SHP, whereas in contrast, CDCA but not androsterone significantly increased apoCII expression (data not shown). Thus, just as synthetic FXR ligands can drive different patterns of gene activation, different endogenous FXR ligands may also have distinct patterns of gene regulation.

The crystal structure of FXR is unique among the known structures of nuclear hormone receptors. First, the FXR crystal structure indicates two coactivator binding sties. Second, bile acids are suggested to bind FXR with the steroid core in the reverse orientation relative to all other steroid hormones binding to their receptors. The structural similarity of the steroid nucleus between CDCA and androsterone suggested a method to address whether this unique feature of the crystal structure was reflective of the FXR-LBD structure within cells. If so, then mutations that altered amino acids predicted to interact with the carboxylic acid tail of CDCA would not be expected to alter androsterone activity. In agreement with this prediction, two amino acids that interact with the carboxylic acid tail of CDCA, M265 and R331, regulated activation by CDCA but had no effect on activation by androsterone. Several mutations (M290V, H294S, S332V, R351H, and Y361F) strongly inhibited both androsterone and CDCA activation but had little effect upon GW4064 activation, potentially indicative of amino acids interacting with the shared steroid core of CDCA and androsterone. Finally, at least one mutation, L340T, strongly inhibited androsterone activation but had no effect on CDCA activation, suggesting that androsterone may make contacts with the FXR-LBD distinct from those made by CDCA. Confirmation of this model awaits crystallization of the hFXR-LBD bound with CDCA or androsterone.

Activation of FXR by the bile acid CDCA is highly dependent upon the species examined, with CDCA having both a greater potency and magnitude of induction of hFXR than of mFXR (24). Conversely, androsterone had a higher activation capability for mFXR than for hFXR. A significant component for this species difference resides at amino acid position 354, asparagine in the human and hamster FXR and lysine in the mouse and rat FXR. Previously, it has been shown that introduction of an asparagine residue at position 366 in the mFXR-LBD (corresponding to position 354 in the hFXR-LBD) strongly increased activation by CDCA (24). Here, we demonstrate the converse introduction of a lysine residue at position 354 of hFXR-LBD decreased CDCA activation but had no effect on androsterone activation. Similarly, differences in species sensitivity to androsterone activation of the FXR-LBD can be largely ascribed to position 345, an alanine in hamster, mouse, and rat FXR but a serine in hFXR. Introduction of an alanine in this position in the hFXR-LBD was sufficient to significantly increase androsterone activation. The molecular basis for the control of FXR activation by either androsterone or CDCA through amino acid positions 345 or 354, respectively, is not clear, because neither position was identified as a ligand contact site in the hFXR-LBD bound with fexaramine or in the rat FXR-LBD bound with 6-ECDCA.

The levels of androsterone, androsterone-glucuronide, and androsterone-sulfate in male human serum are approximately 5 nM, 0.1 µM, and 1 µM, respectively (44), approaching levels required for FXR-LBD activation by androsterone. The concentration of androsterone or conjugates within organs expressing FXR such as liver or adrenal is unknown. Furthermore, additional androstanes such as etiocholanolone are able to activate FXR to a degree comparable to that seen for androsterone (data not shown), suggesting the total plasma androstane content is the most relevant variable. Intriguingly, the plasma level of androsterone in the rat is 150-fold lower than in the human (45). A potential physiological basis for the diminished sensitivity of hFXR toward androsterone may thus be to reduce cross-talk between bile acid signaling and androgen signaling in humans, where the level of circulating androsterone is far greater than in rodents.

The effects of testosterone on the development of cardiovascular disease are highly complex and may act through multiple mechanisms that exert both beneficial and negative effects. In general, testosterone levels in men are inversely proportional to atherosclerotic risk (19, 20, 21), suggesting a net overall protective effect of testosterone (or testosterone metabolites). Whether androgen receptor is directly responsible for the myriad activities of testosterone remains to be demonstrated. Some of the protective activities of testosterone on the development of atherosclerosis in mice are mediated by conversion of testosterone to estrogens, followed by activation of ER (46). The activation of FXR by androsterone suggests consideration of other nuclear hormone receptors as candidates to mediate in vivo testosterone activity should not be limited to ER, but rather the potential involvement of FXR signaling needs to also be considered in evaluating possible mechanisms of androgen activity on the cardiovascular system.

Finally, there is great interest in development of FXR agonists because of the beneficial modulation of triglyceride levels. Furthermore, it has recently been demonstrated that the synthetic bile acid 6-ECDCA can protect rats against liver fibrosis through an FXR-SHP regulatory cascade (47). Whether androstanes or synthetically modified androstanes will have beneficial activities on lipid parameters or for the treatment of liver fibrosis awaits future studies.


   Acknowledgments
 
We thank Janet Paulsen, Karl Malakian, and Wah-Tung Hum for construction, production, and purification of the hFXR-LBD protein.


   Footnotes
 
Disclosure summary: F.J.M. and H.B.H. are employed by Wyeth. S.W., K.L, and M.J.E. are employed by and have equity interests in Wyeth. A.B. was previously employed by Wyeth.

First Published Online May 4, 2006

Abbreviations: apoCII, Apolipoprotein CII; CDCA, chenodeoxycholic acid; CYP7A1, cholesterol 7{alpha}-hydroxylase; CYP8B1, sterol 12{alpha}-hydroxylase; 1D, one-dimensional; DHT, dihydrotestosterone; DMSO, dimethylsulfoxide; 6-ECDCA, 6{alpha}-ethyl-CDCA; ER, estrogen receptor; FXR, farnesoid X receptor; hFXR human FXR; HRP, horseradish peroxidase; LBD, ligand-binding domain; NMR, nuclear magnetic resonance; PPT, 4-propyl-1,3,5-Tris(4-hydroxy-phenyl) pyrazole; SHP, small heterodimer partner; SRC-1, steroid receptor coactivator protein-1; STD, saturation transfer difference.

Received November 22, 2005.

Accepted for publication April 24, 2006.


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 Results
 Discussion
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