Download Activators of the farnesoid X receptor negatively regulate androgen

Biochem. J. (2008) 410, 245–253 (Printed in Great Britain)
245
doi:10.1042/BJ20071136
Activators of the farnesoid X receptor negatively regulate androgen
glucuronidation in human prostate cancer LNCAP cells
Jenny KAEDING*†, Emmanuel BOUCHAERT‡, Julie BÉLANGER*†, Patrick CARON*†, Sarah CHOUINARD*§, Mélanie
VERREAULT*†, Olivier LAROUCHE*§, Georges PELLETIER*§, Bart STAELS‡, Alain BÉLANGER*§ and Olivier BARBIER*†1
*Molecular Endocrinology and Oncology Research Center, CHUL Research Center, Quebec, QC, Canada G1V 4G2, †Faculty of Pharmacy, Laval University, QC, Canada G1K 7P4, ‡Unité
545 INSERM, Lille, Département d’athérosclérose, Institut Pasteur de Lille and Faculté de Pharmacie, Université Lille II, Lille F-59019, France, and §Faculty of Medicine, Laval
University, QC, Canada G1K 7P4
Androgens are major regulators of prostate cell growth and
physiology. In the human prostate, androgens are inactivated
in the form of hydrophilic glucuronide conjugates. These
metabolites are formed by the two human UGT2B15 [UGT
(UDP-glucuronosyltransferase) 2B15] and UGT2B17 enzymes.
The FXR (farnesoid X receptor) is a bile acid sensor controlling hepatic and/or intestinal cholesterol, lipid and glucose
metabolism. In the present study, we report the expression of
FXR in normal and cancer prostate epithelial cells, and we
demonstrate that its activation by chenodeoxycholic acid or
GW4064 negatively interferes with the levels of UGT2B15 and
UGT2B17 mRNA and protein in prostate cancer LNCaP cells.
FXR activation also causes a drastic reduction of androgen
glucuronidation in these cells. These results point out activators
of FXR as negative regulators of androgen-conjugating UGT
expression in the prostate. Finally, the androgen metabolite
androsterone, which is also an activator of FXR, dose-dependently
reduces the glucuronidation of androgens catalysed by UGT2B15
and UGT2B17 in an FXR-dependent manner in LNCaP cells.
In conclusion, the present study identifies for the first time the
activators of FXR as important regulators of androgen metabolism
in human prostate cancer cells.
INTRODUCTION
ive reduced metabolites ADT and 3α-DIOL; however, since these
metabolic reactions can be reversed, complete androgen inactivation is only ensured through glucuronidation [2]. The importance
of glucuronidation for androgen metabolism in the human prostate
has further been emphasized by the observation that polymorphisms within androgen-glucuronidating genes are associated
with an increased risk for prostate cancer [8–11].
Among the 18 functional UGT (UDP-glucuronosyltransferase)
enzymes identified in humans, UGT2B7, UGT2B15 and
UGT2B17 have a remarkable capacity to conjugate androgens
[2,12]. However, only UGT2B15 and UGT2B17 are expressed
at detectable levels in the human prostate [5,13]. The sequence
homology between these two UGTs is very high (95 %), but
they possess a distinct substrate specificity towards 5α-reduced
androgens. UGT2B17 glucuronidates the 3-hydroxy position of
ADT and the 17-hydroxy group of 3α-DIOL or DHT and has
been identified as the major glucuronidating enzyme for ADT
in the prostate. The conjugating activity of UGT2B15 is limited
to the 17-hydroxy position of 3α-DIOL and DHT [13,14].
The FXR (farnesoid X receptor; NR1H4) is a bile acid
sensor that belongs to the family of ligand-activated transcription
factors [15]. The regulatory activity of FXR is primarily associated with the control of genes involved in hepatic bile acid homoeostasis [16]. However, recent data suggest that FXR plays
a broader role in controlling numerous genes involved in
glucose, lipid, cholesterol and steroid metabolism [17,18]. Various
observations suggest that FXR may also be involved in androgen
activation and activity: (i) FXR is a receptor for certain androgen
metabolites such as ADT and etiocholanolone [19]; (ii) FXR
The glucuronidation reaction, which corresponds to the transfer
of the polar glucuronosyl group from UDP-GA (UDP-glucuronic
acid) to an acceptor substrate, is an essential metabolic pathway
for a wide variety of endogenous and exogenous molecules
[1]. Among glucuronidated endobiotics, androgens occupy an
important place. Indeed, there is much clinical and in vivo
evidence that glucuronidation constitutes the major elimination
route for the active androgen DHT (dihydrotestosterone) and
its 5α-reduced metabolites 3α-DIOL (androstane-3α,17β-diol)
and ADT (androsterone) [2]. Likewise, ADT-3G (androsterone-3glucuronide) and 3α-DIOL-17G (3α-DIOL-17-glucuronide) are
the predominant androgen metabolites found in circulation in men
[2]. Whereas glucuronidation is generally considered as a hepatic
detoxification mechanism, extrahepatic glucuronidation is now
established as an efficient way to locally inactivate endogenous
bioactive molecules [2,3]. This is particularly true for androgens,
which are efficiently glucuronidated within their target tissues
such as the human prostate [2,4,5].
The prostate’s growth and function are tightly regulated by
androgens, and changes in the synthesis and inactivation of the
active hormone can lead to excessive androgen influence and
thereby initiate or support the progression of prostate cancer
[6]. Androgenic precursors secreted from adrenals or testis are
converted in the prostate into DHT [2], the active androgen that
activates the AR (androgen receptor; NR3C4) and thereby initiates
the transcription of genes involved in prostate cell proliferation
and differentiation [7]. DHT is locally converted into the inact-
Key words: androgen metabolism, farnesoid X receptor (FXR),
gene expression, glucuronidation, prostate, UDP-glucuronosyltransferase (UGT) 2B15 (UGT2B15) and UGT2B17.
Abbreviations used: ADT, androsterone; ADT-3G, androsterone-3-glucuronide; AP-1, activator protein-1; AR, androgen receptor; CDCA,
chendeoxycholic acid; DHT, dihydrotestosterone; 3α-DIOL, androstane-3α,17β-diol; 3α-DIOL-17G, 3α-DIOL-17-glucuronide; DTT, dithiothreitol; FBS, fetal
bovine serum; FXR, farnesoid X receptor; HSD, hydroxysteroid dehydrogenase; LC, liquid chromatography; LC–MS/MS, LC–tandem MS; PrEC, prostate
epithelial cells; UDP-GA, UDP-glucuronic acid; UGT, UDP-glucuronosyltransferase.
1
To whom correspondence should be addressed, at Laboratory of Transcriptomics and Molecular Pharmacology, CHUL Research Center (CHUQ),
2705, Blvd Laurier, Quebec, QC, Canada G1V 4G2 (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
246
J. Kaeding and others
activation results in the modulation of genes encoding androgen
precursor-synthesizing enzymes, namely dehydroepiandrosterone
sulfotransferase (SULT2A1), 5α-reductase and 3β-HSD (3βhydroxysteroid dehydrogenase) in the liver [20,21]; (iii) FXR
was recently identified as a negative modulator of the androgen–
oestrogen-converting aromatase enzyme in human breast carcinoma MDA-MB-468 cells [18]; and (iv) FXR is expressed at
very high levels in the adrenal gland, where androgen precursor
synthesis occurs, and at detectable levels in androgen-sensitive
tissues, such as the prostate [22].
On the other hand, several UGT enzymes have already been
shown to be regulated by FXR. In rat hepatocytes treated with the
specific FXR agonist GW4064, a significant increase in UGT2B1
and UGT1A7 mRNA levels has been reported [20]. Similarly,
in human hepatocytes, FXR activation resulted in increased
UGT2B4 expression [23], whereas Lu et al. [24] reported
that ligand-activated FXR inhibits the UGT2B7 expression in
intestinal Caco-2 cells.
Based on these observations, we hypothesized that FXR
may also control the expression and activity of the androgenconjugating UGT2B15 and UGT2B17 enzymes in the prostate. To
address this hypothesis, we have investigated the presence of FXR
transcript and proteins in human prostate tissue, epithelial cells
and/or in the androgen-sensitive prostate cancer LNCaP cell line.
Furthermore, the effect of endogenous as well as synthetic FXR
activators on UGT2B15 and UGT2B17 expression and activity
was analysed in LNCaP cells.
MATERIALS AND METHODS
Materials
Prostate tissue slides were purchased from US Biomax
(Toronto, ON, Canada) [25]. UDP-GA, poly(L-lysine) and
steroid substrates (3α-DIOL, ADT and DHT) were obtained
from Sigma (St. Louis, MO, U.S.A.). CDCA (chendeoxycholic
acid), 3α-DIOL-17G, ADT-3G and DHT-17G were purchased
from Steraloids (Newport, RI, U.S.A.). Protein assay reagents
were obtained from Bio-Rad Laboratories (Marnes-la-Coquette,
France). Cell culture materials and Lipofectin® transfection
reagent were obtained from Invitrogen (Burlington, ON,
Canada). SYBR Green PCR Mastermix was purchased from
Applied Biosystems (Foster City, CA, U.S.A.). Casodex (bicalutamide) was provided by Endorecherche (Quebec, QC, Canada).
R1881 was from PerkinElmer (Woodbridge, ON, Canada) and
GW4064 was as described previously [23]. The anti-human
UGT2B15 and UGT2B17 antibodies have been described
previously [5,12,26]; the rabbit polyclonal anti-human FXR
antibodies were from Abcam (ab28676; Cambridge, MA, U.S.A.)
and Santa Cruz Biotechnology (sc-13063; Santa Cruz, CA,
U.S.A.) respectively, the rabbit polyclonal anti-human actin
antibody was purchased from Sigma (A5060) and the secondary
anti-rabbit IgG (RPN4301) was obtained from Amersham
(Oakville, ON, Canada).
Immunohistochemistry
Immunohistochemical staining was conducted using standard
techniques as previously described [25,27]. Briefly, immunostaining was performed by using anti-human FXR antibodies diluted
in Tris saline (1:20 for sc-13063 and 1:200 for ab28676) (pH 7.6)
for 1 h at room temperature (22 ◦C). Sections were subsequently
washed with PBS and incubated with a biotin-labelled goat
anti-rabbit γ -globulin for 10 min. After incubation, the sections
were treated with streptavidin coupled with peroxidase, and
diaminobenzidine was used as the chromogen to visualize the
c The Authors Journal compilation c 2008 Biochemical Society
Table 1
Primers and conditions used in PCR analyses
FWD, forward; REV, reverse.
Gene
Primers
Final concn. (nM)
28S
FWD: 5 -AAACTCTGGTGGAGGTCCGT-3
REV: 5 -CTTACCAAAGTGGCCCACTA-3
200
UGT2B15
FWD: 5 -GTGTTGGGAATATTATGACTACAGTAAC-3
REV: 5 -GGGTATGTTAAATAGTTCAGCCAGT-3
125
UGT2B17
FWD: 5 -TGACTTTTGGTTTCAAGC-3
REV: 5 -TTCCATTTCCTTAGGCAA-3
300
biotin–streptavidin peroxidase complex (Vectastain ABC kit;
Vector Laboratories). The sections were then counterstained with
haematoxylin. For negative control, the antibodies were replaced
by a rabbit pre-immune serum.
Cell culture
Human PrEC (prostate epithelial cells; Cambrex Walkersville,
MD, U.S.A.) and human prostate cancer LNCaP cells (A.T.C.C.,
Rockville, MD, U.S.A.) were cultured as described in [25]. For all
experiments, LNCaP cells were used between passages 21 and 33
and in culture plates pretreated with poly(L-lysine) overnight prior
to cell seeding. CDCA, GW4064, DHT, Casodex and androgens
were dissolved in ethanol and the final concentration of the
solvent never exceeded 0.1 % (v/v) in cell culture experiments.
For RNA, protein and cell viability analyses, 3 × 105 LNCaP
cells per well were seeded on to 6-well plates and cultured
in RPMI 1640 medium supplemented with 0.2 % FBS (fetal
bovine serum) for 48 h to allow cell attachment. Cells were
then treated with vehicle [0.1 % (v/v) ethanol], CDCA, GW4064,
DHT or ADT alone and/or in combination with Casodex at the
indicated concentrations in RPMI 1640 medium supplemented
with 0.2 % FBS for different time periods. For glucuronidation
assays, 2 × 106 cells were seeded on to 10 cm culture dishes and
cultured in RPMI 1640 medium, supplemented with 0.2 % FBS,
for 48 h. Cells were then treated with vehicle (ethanol), CDCA
(50 µM), GW4064 (5 µM) or ADT (10 µM) for 24 or 72 h in
the same medium, which was changed after 48 h during the
72 h treatments. Finally, cells were harvested and resuspended
in PBS (pH 7.4; 50 mM) with DTT (dithiothreitol; 0.5 mM) and
homogenized by pipetting.
RNA analysis
Total RNA from human hepatocytes was obtained as previously
described [28]. Extraction of total RNA from PrEC or LNCaP cells
and reverse transcription of 1 µg of RNA were performed using
respectively the TRI Reagent acid phenol protocol (Molecular
Research Center, Cincinnati, OH, U.S.A.) and 200 units of
SuperScript II reverse transcriptase (Invitrogen) as previously
reported [23,25].
UGT2B15 and UGT2B17 mRNA levels were determined by
real-time PCR with SYBR Green by using the ABI Prism 7500
detection system from Applied Biosystems. For each reaction, the
final volume of 20 µl comprised 10 µl of SYBR Green PCR mix,
2 µl of each primer (Table 1) and 6 µl of a reverse transcription
product (dilution 1:500). The cycle parameters for real-time PCR
were as follows: initial 95 ◦C for 10 min, followed by 40 cycles
of 95 ◦C for 15 s and 56 ◦C for 1 min. The specific amplification of
each UGT mRNA was ensured by testing PCR strategies with all
human UGT2B cDNAs (1.0 ng) and by direct sequencing of PCR
products. Threshold cycle (Ct ) values were analysed using the
comparative Ct (Ct ) method as described by the manufacturer
Farnesoid X receptor reduces androgen glucuronidation in LNCaP cells
(Applied Biosystems). The amount of target gene (2−Ct ) was
obtained by normalizing to the endogenous reference (28S) and
was expressed relatively to vehicle-treated control as the baseline.
For each gene, the amplification efficiency and the accuracies
of Ct of target genes compared with 28S were tested using
2–5 log of concentrations of cDNA produced from LNCaP-cell
purified mRNA.
The absolute quantification of FXR cDNA in reversetranscribed mRNA (1 µg) from human hepatocytes, PrEC and
LNCaP cells was performed using the absolute standard curve
method as recommended by the manufacturer’s instructions
(Applied Biosystems). Briefly, FXR cDNA was quantified in both
reverse-transcribed mRNA and in aqueous solutions containing
increasing amounts (from 1 fg to 1 ng) of FXR cDNA by using
the TaqMan® gene expression assay Hs00231968_m1 (Applied
Biosystems). Threshold cycle (Ct ) values from the standard curve
were interpolated to calculate the FXR mRNA concentration in
each reverse-transcribed total RNA. In these experiments, the
cycle parameters were: initially 95 ◦C for 10 min, followed by
40 cycles of 95 ◦C for 15 s and 60 ◦C for 1 min.
Cell viability
After treatment, LNCaP cells were trypsinized, centrifuged and
resuspended in PBS containing propidium iodide (20 µg/ml).
After 15 min of incubation on ice, the reaction was stopped by
adding 25 % ethanol in PBS. Cells were then stained with Hoechst
33 342 (112 µg/ml) for 10 min. Analysis by flow cytometry was
performed on an Epics Elite ESP instrument (Beckman Coulter)
by using an argon laser (488 nm) and a helium–cadmium laser
(325 nm). For each condition, 100 000 cells were evaluated.
Protein analyses
Total proteins from LNCaP cells were purified according to the
TRI Reagent acid phenol protocol. Human liver tissue samples as
previously described [28] were homogenized in PBS (pH 7.4;
50 mM) with DTT (0.5 mM). Nuclear extracts of confluent
LNCaP cells (approx. 20 million) were prepared by the protocol
of Liu et al. [30]. For Western-blot experiments, 1 µg of human
liver microsomes (BD Biosciences Discovery Labware, Woburn,
MA, U.S.A.) and 10 µg of total proteins were size-separated by
SDS/10 %-(w/v)-PAGE and immunoblotted with anti-UGT2B15
or -UGT2B17 antibodies as previously reported [5,12]. The
same amount of protein was also immunoblotted with an antiactin antibody as a loading control. Relative protein levels were
quantified by using BioImage Visage 110s (Genomic Solution,
Ann Arbor, MI, U.S.A.).
Glucuronidation assays and steroid quantification
Glucuronidation assays were performed for 1 h at 37 ◦C by
incubating 3 µg of total protein from homogenized LNCaP cells in
the presence of 1 mM UDP-GA, 200 µM ADT, DHT or 3α-DIOL
in a glucuronidation assay buffer as previously described [13,25].
Assays were ended by adding 100 µl of methanol with 0.02 %
BHT (butylated hydroxytoluene) followed by centrifugation at
14 000 g for 10 min. The formation of glucuronide conjugates
was resolved by LC (liquid chromatography) coupled with MS
[LC–MS/MS (LC–tandem MS)] by using a previously established analytical method [12,25]. The LC–MS/MS system was
composed of an Alliance 2690 apparatus (Waters, Milford, MA,
U.S.A.) coupled with an API-3200 mass spectrometer (Applied
Biosystems, Concord, ON, Canada). Non-conjugated DHT was
determined by GC–MS (gas chromatography coupled with MS)
on an HP5973 quadrupole mass spectrometer equipped with a
chemical ionization source as previously reported [31].
247
Statistical analyses
All results are presented as means +
− S.D. Comparisons between
two groups were performed using a two-tailed Student’s t test.
Comparisons between three or more groups were made using
ANOVA followed by the Dunnett’s procedure for multiple
comparisons as the post hoc analysis (SAS V9.1.3 software; SAS
Institute, Cary, NC, U.S.A.). In all cases, P 0.05 was considered
statistically significant.
RESULTS
FXR is expressed in LNCaP cells
Previous studies reported the presence of FXR transcripts in the
human prostate [22]; however, the receptor expression in normal
or cancer prostate cells has never been investigated. To address
this question, we quantified FXR mRNA levels in total RNA from
PrEC in primary culture, prostate cancer LNCaP cells and human
hepatocytes (positive control) (Figure 1A). PrEC and LNCaP cells
contained similar concentrations of FXR mRNA (54 and 42 fg/µg
of total RNA respectively). As expected, FXR mRNA was approx.
30-fold more abundant in human hepatocytes when compared
with prostate cells (Figure 1A). It is of interest that such a variation
is close to the 56-fold difference in FXR mRNA levels reported
between human liver and prostate tissues [22].
Since the presence of the FXR protein has never been
demonstrated in the human prostate tissue and in prostate cancer
cell lines, Western-blot and immunohistochemistry analyses were
performed with nuclear extract from LNCaP cells and slices from
the human prostate respectively (Figures 1B and 1C). Westernblot analyses performed with the anti-FXR antibody (ab28676;
Abcam) revealed the presence of two bands in both LNCaP cell
nuclear extracts and human liver homogenate (positive control)
(Figure 1B). These 54 and 56 kDa bands present similar migration
properties as the previously reported functional isoforms of FXRα
identified in humans: 1–2 and 3–4 [17,32,33]. Interestingly,
LNCaP nuclear extracts are enriched in the FXRα3/4 isoforms,
which contain an extended N-terminus and are fully active to
regulate gene transcription [32,33]. The expression of the FXR
proteins in normal prostate tissue has also been investigated
through immunohistochemistry, by using two different sources of
anti-FXR antibodies (sc-13063; Santa Cruz Biotechnology; and
Ab28676; Abcam). Both antibodies yielded an intense staining of
nuclei from both androgens-responsive luminal and androgensproducing basal cells of the prostate epithelium (Figure 1C). The
nuclear localization of the FXR protein is consistent with labelling
reported with anti-FXR antibodies in cell nuclei from other human
tissues [34,35].
Overall, these observations demonstrate that FXR mRNA and
protein are expressed at significant levels in androgen-sensitive
prostate cells.
CDCA represses the expression of androgen-glucuronidating UGT
enzymes in LNCaP cells
To investigate whether the FXR activator CDCA affects
UGT2B15 and UGT2B17 gene expressions, LNCaP cells were
treated with 50 µM CDCA for the indicated time periods
(Figures 2A and 2B). UGT2B15 and UGT2B17 mRNA levels
were time-dependently reduced, with maximal inhibition after 72 h
of treatment (Figures 2A and 2B). Likewise, treatment of LNCaP
cells with increasing concentrations of CDCA (5–50 µM) for
48 h resulted in a dose-dependent decrease in both UGT2B15 and
UGT2B17 transcript levels in the range of 15–50 µM (Figures 2C
and 2D). A 5 µM portion of CDCA slightly induced UGT2B15
c The Authors Journal compilation c 2008 Biochemical Society
248
J. Kaeding and others
not significantly altered after 24 h of CDCA treatment (50 µM;
Figure 3A), longer exposure (72 h) resulted in 69 and 78 %
reduction in UGT2B15 and UGT2B17 respectively (Figure 3B).
Subsequently, enzymatic assays were performed using
homogenized LNCaP cells incubated in the presence of ethanol
(vehicle) or 50 µM CDCA for 24 or 72 h (Figures 3C and 3D).
Apart from DHT, its two major metabolites 3α-DIOL and ADT
were used as substrates. After 24 h of exposure to CDCA, no
significant changes in the formation of 3α-DIOL-17G, DHT-17G
and ADT-3G were observed (Figure 3C). However, after 72 h of
treatment, the formation of 3α-DIOL-17G, DHT-17G and ADT3G was 55, 52 and 54 % reduced respectively when compared
with vehicle-treated cells (Figure 3D).
Bile acids are natural detergents that exert pro-apoptotic and
pro-necrotic effects at high concentrations [36]. Thus it was
possible that the reduction of UGT2B only reflected non-specific
toxicity of CDCA. To exclude this possibility, we performed a
flow cytometric cell viability/toxicity analysis using combined
staining with propidium iodide and Hoechst 33342 (Figure 4).
Treatment with 50 µM CDCA caused significant reduction of
cell viability only after 72 h (31 % reduction; P < 0.05; Figure 4),
whereas viability of cells treated for 24 and 48 h was not affected
by CDCA. In contrast, exposure to CDCA for 96 h resulted in
50 % induced cell mortality, which was not statistically significant
due to high variability. We also analysed the effect of the selective
FXR agonist GW4064 (5 µM) on cell viability as a negative control (Figure 4). This synthetic activator of FXR did not show any
significant effect on cell viability, even after treatment for 96 h.
Taken together, these observations demonstrate that CDCA
specifically reduces the expression and androgen-conjugating
activity of the UGT2B15 and UGT2B17 enzymes in prostate
cancer LNCaP cells.
FXR mediates the CDCA-dependent inhibition of androgen
glucuronidation in LNCaP cells
Figure 1 The FXR receptor is expressed in human prostate epithelial and
LNCaP cells
(A) FXR mRNA levels were quantified in PrEC in primary culture, in LNCaP cells as well as human
hepatocytes (HH) by using the absolute standard curve method of real-time PCR as described in
the Materials and methods section. (B) LNCaP nuclear extracts (100 µg) were immunoblotted
with an anti-human FXR antibody (Abcam; 1:500) using human liver homogenate (100 µg) as
a positive control. (C) Normal prostate tissues (n = 4) were subjected to immunohistochemical
analyses using two different anti-human FXR antibodies: from Abcam (Ab28676; 1:200; 1, 3,
5) and Santa Cruz Biotechnology (sc-13063; 1:20; 7). A rabbit pre-immune serum was used as
the negative control (2, 4, 6, 8). Magnifications are × 400 (1–6) and × 200 (7, 8). Arrows
indicate luminal cells and arrowheads indicate basal cells.
expression; however, this effect was not statistically significant
and lower doses were inefficient in affecting UGT expression in
LNCaP cells (results not shown).
CDCA decreases UGT2B15 and UGT2B17 protein levels and
androgen-glucuronidation activity in LNCaP cells without affecting
cell viability
Analysis of UGT2B15 and UGT2B17 protein levels in CDCAtreated LNCaP cells further confirmed the time-dependent
reduction of UGT2B15 and UGT2B17 enzymes (Figures 3A
and 3B). Whereas UGT2B15 and UGT2B17 protein levels were
c The Authors Journal compilation c 2008 Biochemical Society
Because CDCA may also exert FXR-independent effects, the
influence of GW4064 on UGT2B15 and UGT2B17 gene expressions was also investigated (Figure 5). We observed that GW4064
(5 µM) reduced UGT2B15 and UGT2B17 mRNA (Figure 5A)
and protein (Figure 5B) levels to a similar order of magnitude
as CDCA (Figures 2 and 3). However, in contrast with CDCA,
GW4064 already yielded a clear reduction in UGT protein
levels as early as 24 h when compared with vehicle-treated
cells (Figure 5B). Consistent with the reduced concentration of
UGT2B15 and UGT2B17 proteins, we also observed that treatment with GW4064 for 72 h drastically reduced the glucuronide
conjugation of DHT, 3α-DIOL and ADT (78, 77 and 73 %
reduction respectively) (Figure 5C).
Overall, these results confirm that FXR activation reduces
UGT2B gene expression and activity in prostate cancer LNCaP
cells.
ADT also affects androgen glucuronidation in
an FXR-dependent manner
The 5α-reduced androgen metabolite ADT was recently identified
as an activator of FXR [37]. Since ADT is a substrate for
UGT2B17 in the prostate, it was of interest to determine whether
this androgen derivative could also reduce its own glucuronidation
by activating FXR. To verify this hypothesis, LNCaP cells were
treated with increasing concentrations of ADT, and levels of
UGT2B15 and UGT2B17 mRNA, protein and activity were
determined (Figure 6). No significant reduction in UGT2B15
and UGT2B17 mRNA levels was observed in the presence of
10 nM ADT (Figure 6A). However, a significant dose-dependent
Farnesoid X receptor reduces androgen glucuronidation in LNCaP cells
Figure 2
249
CDCA represses androgen-glucuronidating UGT2B15 and UGT2B17 mRNA expression in human prostate cancer cells
(A, B) LNCaP cells were treated with ethanol (vehicle) or CDCA at 50 µM in RPMI 1640 medium supplemented with 0.2 % FBS for the indicated time periods. (C, D) LNCaP cells were treated
with ethanol (vehicle) or increasing concentrations of CDCA in RPMI 1640 medium supplemented with 0.2 % FBS for 48 h. UGT2B15 (A, C) and UGT2B17 (B, D) mRNA levels were measured by
real-time PCR and normalized to 28S expression. Values are expressed as means +
− S.D. (n = 3) and statistically significant differences between control- and CDCA-treated samples are indicated
by asterisks [(A, B) Student’s t test; (C, D) one-way ANOVA; n.s., not significant; ∗ P < 0.05; ∗∗ P < 0.005; ∗∗∗ P < 0.001].
reduction in UGT mRNA levels was observed in the presence
of 100 nM to 10 µM ADT (Figure 6A). Furthermore, UGT2B
protein levels also decreased significantly after 24 h of treatment
with ADT (10 µM) and were further reduced after exposure for
72 h (Figure 6B). Likewise, ADT (10 µM) treatment for 72 h
resulted in a significant reduction in 3α-DIOL-17G, DHT-17G
and ADT-3G formation (Figure 6C).
DHT is also a potent reducer of UGT2B15 and UGT2B17
expression in LNCaP cells [25]. Because 3α-HSD enzymes,
which can re-convert ADT into DHT, are expressed in LNCaP
cells [38], it remained plausible that the reduction in UGT
expression observed in the presence of ADT only reflected an
effect of the activated AR after re-conversion of ADT into DHT.
To test this hypothesis, we determined the formation of DHT in
treated LNCaP cells (Table 2) and compared the response to ADT
and DHT treatment in the presence or absence of Casodex, a
specific AR antagonist (Figures 6D and 6E). Casodex treatment
alone did not result in DHT formation at a detectable level in
LNCaP cells (Table 2). However, the addition of ADT (10 µM) led
to the synthesis of approx. 3 nM DHT in the presence of Casodex
(Table 2). When LNCaP cells were treated with the corresponding
concentration of DHT (3 nM), UGT2B15 and UGT2B17 levels
were reduced by 50 and 35 % respectively (Figures 6D and 6E).
This effect was significantly weaker than treatment with 10 µM
ADT, which resulted in 85 and 75 % reduction of UGT2B15 and
UGT2B17 mRNA expression respectively (Figures 6D and 6E).
In addition, co-treatment with Casodex resulted in a complete
loss of the DHT-dependent inhibition of the expression of both
UGT enzymes, but failed to significantly alter the effect of ADT
(Figures 6D and 6E).
These results demonstrate that ADT inhibits androgen glucuronidation in LNCaP cells in an AR-independent manner.
DISCUSSION
The present study demonstrates the presence of FXR in epithelial
cells of the human prostate and establishes its activators as
important regulators of androgen metabolism in prostate cancer
LNCaP cells. FXR is classically considered as a bile acid
sensor that regulates hepatic and intestinal bile acid synthesis,
metabolism and transport [16]. However, this receptor also
exerts important functions in the control of lipid and glucose
homoeostasis [17]. The results presented here further reinforce
the idea that FXR plays different physiological roles in various
extrahepatic tissues.
FXR activation caused a drastic reduction of UGT2B15 and
UGT2B17 gene expression, resulting in a decreased glucuronide
conjugation of the active androgen DHT and its reduced
metabolites ADT and 3α-DIOL. The observation that FXR affects
UGT2B15 and UGT2B17 gene expressions in an identical manner
is consistent with the very high homology of their proximal
promoter regions (more than 90 % nucleotide homology within
the proximal 1600 bp sequences of the UGT2B15 and UGT2B17
genes) [39,40] and supports the idea that both genes share an
identical FXR response element within this region. In human
c The Authors Journal compilation c 2008 Biochemical Society
250
Figure 3
J. Kaeding and others
CDCA decreases UGT2B15 and UGT2B17 protein levels and glucuronidation of androgen metabolites in LNCaP cells
(A, B) LNCaP cells were treated with ethanol (vehicle) or CDCA (50 µM) in RPMI 1640 medium supplemented with 0.2 % FBS for the indicated time periods. Then, 10 µg of total protein of each
sample was immunoblotted with the anti-UGT2B15 antibody (1:1500; top panels) or the anti-UGT2B17 antibody (1:2000; middle panels). Human liver microsomes (2 µg) were used as a positive
control. The equal loading of each lane was ensured by hybridizing with an anti-human actin antibody (1:1000; bottom panels). Immunoreactions with anti-UGT antibodies were quantified by
densitometry and are expressed as relative quantity of proteins compared with vehicle-treated cells (set as 1). (C, D) LNCaP cells were treated with ethanol (vehicle) or CDCA (50 µM) in RPMI 1640
medium supplemented with 0.2 % FBS, for 24 h (C) or 72 h (D), and glucuronidation assays were performed with 3α-DIOL, ADT and DHT as substrates (200 µM) using 3 µg of cell homogenates.
Glucuronide formation was quantified by LC–MS/MS analyses and corrected according to the amount of protein. Values (means +
− S.D.; n = 4) are expressed as µg of glucuronide formation/µg of
protein per hour, and statistically significant differences between control- and CDCA-treated samples are indicated by asterisks (Student’s t test; n.s., not significant; ∗ P < 0.05; ∗∗∗ P < 0.001).
Figure 4 Treatment with FXR agonists does not significantly interfere with
LNCaP cell viability
LNCaP cells were treated with ethanol (vehicle) or CDCA at 50 µM in RPMI 1640 medium
supplemented with 0.2 % FBS for the indicated time periods. Cell viability was determined by flow
cytometry after staining with propidium iodide and Hoechst 33342 as described in the Materials
and methods section. Significant differences between control- and CDCA-/GW4064-treated
samples are indicated by asterisks (Student’s t test; ∗ P < 0.05).
prostate epithelium, the UGT2B15 protein is found in luminal
cells, whereas UGT2B17 is expressed in the basal layer [5,25].
Thus the presence of FXR protein in the two cell types is also
consistent with its regulatory role in the control of the respective UGT enzyme expression. Nevertheless, the exact mechanism
by which FXR negatively regulates UGT2B15 and UGT2B17
genes remains to be determined. Negative FXR response elements
have been reported in bile acid-down-regulated genes [24,41],
including the human UGT2B7 gene [24]. However, bioinformatics analysis of the UGT2B15 and UGT2B17 promoter se
c The Authors Journal compilation c 2008 Biochemical Society
quences failed to identify such a responsive sequence (results not
shown). While we cannot exclude the presence of other negative
FXR elements within UGT promoters, this last observation
does not support a direct down-regulation of the UGT2B15 and
UGT2B17 genes by FXR. On the other hand, FXR also negatively
regulates gene transcription by interfering with other regulatory
pathways. Indeed, FXR was suggested as a mediator of the bile
acid-dependent activation of the AP-1 (activator protein-1) transcription pathway in colon cancer cells [2]. Considering the
importance of AP-1 factors in the pathogenesis of prostate cancer
[8,42], it would be possible that the FXR-dependent regulatory
pathway of UGT2B genes in LNCaP cells involves AP-1 factors.
Such a hypothesis is supported by the presence of at least four
conserved AP-1-binding sites within the highly homologous
1.6 kb proximal promoter sequences of both UGT2B15 and
UGT2B17 genes [39,40].
The FXR-dependent inhibition of androgen-glucuronidating
UGT2B enzymes in LNCaP cells was unexpected, since previous
studies reported that CDCA fails to modulate UGT2B15
mRNA levels in human hepatocytes [23]. However, the present
observation suggests that FXR regulates UGT2B15 expression
in a tissue-specific manner. Similar observations were reported
for the UGT2B7 gene, which was inhibited by bile acid-activated
FXR in colon carcinoma Caco-2 cells, while remaining unaffected
in hepatocytes [23,24]. It is generally accepted that such a differential regulation may be associated with the tissue-specific
balance between nuclear receptor co-activators and co-repressors
[18]. We also observed that Ugt2b expression seems to increase
in the prostate from FXR-null mice, compared with wild-type
Farnesoid X receptor reduces androgen glucuronidation in LNCaP cells
251
Figure 5 GW4064 also reduces UGT2B15 and UGT2B17 expression and
androgen glucuronidation activity in LNCaP cells
(A) LNCaP cells were treated with ethanol (vehicle) or GW4064 (5 µM) in RPMI 1640 medium
supplemented with 0.2 % FBS for 24 h. UGT2B15 and UGT2B17 mRNA levels were measured
by real-time PCR and normalized to 28S expression. Values are expressed as means +
− S.D.
(n = 3) and statistically significant differences between control- and GW4064-treated samples
are indicated by asterisks (Student’s t test: ∗∗∗ P < 0.001). (B) LNCaP cells were treated with
ethanol (vehicle) or GW4064 (5 µM) in RPMI 1640 medium supplemented with 0.2 % FBS for
24 h. Then, 10 µg of total protein of each sample was immunoblotted with the anti-UGT2B15
antibody (1:1500; top panel) or the anti-UGT2B17 antibody (1:2000; middle panel). Human
liver microsomes (HL) (2 µg) were used as a positive control. The equal loading of each
lane was ensured by hybridizing with an anti-human actin antibody (1:1000; lower panel).
Immunoreactions with anti-UGT antibodies were quantified by densitometry and are expressed
as relative quantity of protein content compared with vehicle-treated cells (set as 1). (C) LNCaP
cells were treated with ethanol (vehicle) or GW4064 (5 µM) in RPMI 1640 medium supplemented
with 0.2 % FBS, for 72 h, and glucuronidation assays were performed with 3α-DIOL, ADT
and DHT (200 µM) by using 3 µg of cell homogenate as described in the Materials and
methods section. Glucuronide formation was quantified by LC–MS/MS analysis and corrected
according to the amount of protein. Statistically significant differences between control- and
GW4064-treated samples are indicated by asterisks (Student’s t test; n.s., not significant;
∗∗
P < 0.005; ∗∗∗ P < 0.001).
animals (see Supplementary Figure 1 at http://www.BiochemJ.
org/bj/410/bj4100245add.htm). While being consistent with the
idea that FXR down-regulates UGT2B enzymes in the prostate,
the physiological implications of this observation are unclear
since androgen glucuronidation is almost absent in the rodent
prostate [2,43,44].
While being structurally different, the bile acid CDCA and
the 5α-reduced androgen metabolite ADT are two ultimate chol-
Figure 6 Elevated concentrations of ADT also affect androgen-conjugating
UGT expression and activity in an FXR-dependent manner
(A) LNCaP cells were treated with ethanol (vehicle) or ADT at indicated concentrations in RPMI
1640 medium supplemented with 0.2 % FBS for 48 h. UGT2B15 and UGT2B17 mRNA levels
were measured by real-time PCR and normalized to 28S. Values are expressed as means +
− S.D.
(n = 3) and statistically significant differences between control- and ADT-treated samples
are indicated by asterisks (one-way ANOVA; n.s., not significant; ∗ P < 0.05; ∗∗∗ P < 0.001).
(B) LNCaP cells were treated with ethanol (vehicle) or ADT (10 µM) in RPMI 1640 medium
supplemented with 0.2 % FBS for 24 or 72 h. For each sample, 10 µg of cell homogenate
was immunoblotted with the anti-UGT2B15 antibody (1:1500; top panel) or the anti-UGT2B17
antibody (1:2000; middle panel). The equal loading of each lane was ensured by hybridizing
with an anti-human actin antibody (1:1000; bottom panel). Immunoreactions with anti-UGT
antibodies were quantified by densitometry and are expressed as relative quantity of protein
content compared with vehicle-treated cells (set as 1). (C) LNCaP cells were treated with ethanol
(vehicle) or ADT (10 µM) in RPMI 1640 medium supplemented with 0.2 % FBS, for 72 h,
and glucuronidation assays were performed with 200 µM of 3α-DIOL, ADT or DHT by using
3 µg of cell homogenate. Glucuronide formation was quantified by LC–MS/MS analysis and
corrected for the amount of protein. Values (means +
− S.D.; n = 4) are expressed as µg of
glucuronide formation/µg of protein per hour, and statistically significant differences between
control- and GW4064-treated samples are indicated by asterisks (Student’s t test; ∗∗ P < 0.005;
∗∗∗
P < 0.001). (D) LNCaP cells were treated with ethanol (vehicle), ADT (10 µM) and DHT
(3 nM) in the presence or absence of Casodex (10 µM) in RPMI 1640 medium supplemented
with 0.2 % FBS for 24 h. UGT2B15 and UGT2B17 mRNA levels were measured by real-time PCR
and normalized to 28S. Values are expressed as means +
− S.D. (n = 3). Statistically significant
differences between control and ADT/DHT treatments, as well as their effects in the presence or
absence of casodex, were determined by two-way ANOVA and are indicated by asterisks (n.s.,
not significant; ∗ P < 0.05; ∗∗ P < 0.005; ∗∗∗ P < 0.001).
c The Authors Journal compilation c 2008 Biochemical Society
252
Table 2
J. Kaeding and others
DHT formation in LNCaP cells in the presence of Casodex
BLQ, below limit of quantification.
Treatment
[DHT] (nM)
Casodex (10 µM)
ADT (10 µM) + Casodex (10 µM)
BLQ
3.04 +
− 0.45
esterol catabolites, which have been identified as FXR activators
[37,45]. Since the exact mechanism by which FXR negatively
regulates UGT2B15 and UGT2B17 genes remains to be determined in the prostate, it was of interest to investigate whether
ADT can affect its own glucuronidation. We observed that ADT
modulates UGT2B15 and UGT2B17 expression and activity
at concentrations above 100 nM. While this concentration is
drastically elevated when compared with levels of unconjugated
ADT found in human circulation (i.e. approx. 1–3 nM) [46,47], it
is more similar to the concentration range of circulating ADT3G (i.e. 35–190 nM) [2,47–50]. Because ADT is efficiently
glucuronidated and constitutes the most abundant androgen
metabolite found in humans, this observation suggests that
intratissular concentrations of ADT may be much higher than
expected from plasma quantification. Thus it is likely that ADTdependent activation of FXR is of physiological importance and
is able to result in decreased UGT in the human prostate tissue.
Furthermore, ADT activates FXR at a concentration close to
the K m value of its glucuronidation by UGT2B17: 400–600 nM
[14,51]. From this point of view, ADT glucuronidation may
be considered as a relevant inactivating pathway, which can
compete with the activation of FXR by ADT, as was previously
demonstrated for CDCA [28]. The reduction of UGT2B17
expression in the presence of ADT may therefore be interpreted
as a feed-forward mechanism by which this androgen metabolite
is able to sustain its FXR-mediated biological activity through
reduction of its inactivation. Therefore our results provide the
first evidence for a likely physiological role of ADT as an FXR
ligand in the human prostate. However, it is also possible that FXR
is activated by a yet unidentified endogenous metabolite present
at physiologically relevant levels in the human prostate.
The present study constitutes the first evidence that FXR plays
an important role in the control of androgen metabolism in
prostate cells. However, activation of the FXR regulatory pathway
also negatively interferes with the PMA-dependent induction of
aromatase gene expression in human breast cancer cells [18].
Aromatase catalyses the conversion of androgens into oestrogens.
Together with the present study, this observation suggests that in
hormone-sensitive tissues, such as the mammary and prostate
glands, FXR activation may result in accumulation of androgens.
In prostate cancer cells, FXR activation and subsequent
reduction of androgen glucuronidation may correspond to a procarcinogenic mechanism. Indeed, it has already been reported
that stimuli that reduce androgen glucuronidation also stimulate
prostate cancer cell proliferation [2,26]. However, Choi et al.
[52] reported that CDCA was ineffective in modulating prostate
carcinoma PC-3 cell growth, which does not support the abovestated hypothesis. However, and in contrast with LNCaP, PC-3
cells do not express the androgen-conjugating UGT2B enzymes
and grow in an androgen-independent manner [53]. Consequently,
the effects we report here would have not been observed in PC-3
cells.
Our results clearly show that FXR is expressed in PrEC, where
its activation reduces the glucuronide conjugation of androgens.
Interestingly, the development of cholestasis has been reported in
c The Authors Journal compilation c 2008 Biochemical Society
various patients with prostate cancer [54–57]. During cholestasis,
plasma levels of bile acids are drastically increased [16], and
based on the present study, it is tempting to speculate that, in
such patients, glucuronidation of androgen may be reduced in the
prostate tissue. On the other hand, GW4064 is considered as a
promising treatment for cholestasis [16], and based on the present
study, it would be of importance to monitor circulating markers
of prostate cancer in GW4064-treated patients.
In conclusion, the present study shows for the first time a regulatory function of the nuclear receptor FXR in androgen metabolism in prostate cancer LNCaP cells. In concert with other
recent reports, this suggests that FXR may be an important, yet
not well studied, regulator of androgen homoeostasis.
This work was supported by the CIHR (Canadian Institute of Health Research)
(MOP118446 to O. B. and MOP64273 to A. B.), FRSQ (Fonds de la Recherche en Santé
du Québec) and CFI (Canada Foundation for Innovation) (to O. B.) and the Faculty
of Pharmacy, Laval University. S. C. is supported by ‘Fonds pour la Recherche et
l’Enseignement’ (to R. V. and S. K.) from the CIHR and O. B. and J. K. by the Health
Research Foundation of Rx&D-CIHR. We thank Endorecherche for providing Casodex
and Professor F. Gonzalez (National Institutes of Health, Bethesda, MD, U.S.A.) for
providing the FXR-null animals. We are grateful to Valérie Jomphe from the Department
of Statistics, Laval University, for her assistance with statistical analysis. We also thank
Dr Virginie Bocher for a critical reading of this paper and Dr Chantal Guillemette for helpful
discussions.
REFERENCES
1 Mackenzie, P., Walter Bock, K., Burchell, B., Guillemette, C., Ikushiro, S., Iyanagi, T.,
Miners, J., Owens, I. S. and Nebert, D. W. (2005) Nomenclature update for the
mammalian UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenet. Genomics
15, 677–685
2 Bélanger, A., Pelletier, G., Labrie, F., Barbier, O. and Chouinard, S. (2003)
Inactivation of androgens by UDP-glucuronosyltransferase enzymes in humans.
Trends Endocrinol. Metab. 14, 473–479
3 Guillemette, C., Bélanger, A. and Lépine, J. (2004) Metabolic inactivation of estrogens in
breast tissue by UDP-glucuronosyltransferase enzymes: an overview. Breast Cancer Res.
6, 246–254
4 Barbier, O. and Bélanger, A. (2003) The cynomolgus monkey (Macaca fascicularis ) is the
best animal model for the study of steroid glucuronidation. J. Steroid Biochem. Mol. Biol.
85, 235–245
5 Barbier, O., Lapointe, H., El Alfy, M., Hum, D. W. and Bélanger, A. (2000) Cellular
localization of uridine diphosphoglucuronosyltransferase 2B enzymes in the human
prostate by in situ hybridization and immunohistochemistry. J. Clin. Endocrinol. Metab.
85, 4819–4826
6 Soronen, P., Laiti, M., Torn, S., Harkonen, P., Patrikainen, L., Li, Y., Pulkka, A., Kurkela, R.,
Herrala, A. and Kaija, H. (2004) Sex steroid hormone metabolism and prostate cancer.
J. Steroid Biochem. Mol. Biol. 92, 281–286
7 Platz, E. A. and Giovannucci, E. (2004) The epidemiology of sex steroid hormones and
their signaling and metabolic pathways in the etiology of prostate cancer. J. Steroid
Biochem. Mol. Biol. 92, 237–253
8 Park, J., Chen, L., Ratnashinge, L., Sellers, T. A., Tanner, J.-P., Lee, J.-H., Dossett, N.,
Lang, N., Kadlubar, F. F., Ambrosone, C. B. et al. (2006) Deletion polymorphism of
UDP-glucuronosyltransferase 2B17 and risk of prostate cancer in African American and
Caucasian men. Cancer Epidemiol. Biomarkers Prev. 15, 1473–1478
9 Park, J., Chen, L., Shade, K., Lazarus, P., Seigne, J., Patterson, S., Helal, M. and
Pow-Sang, J. (2004) Asp85tyr polymorphism in the UDP-glucuronosyltransferase (UGT)
2B15 gene and the risk of prostate cancer. J. Urol. 171, 2484–2488
10 Hajdinjak, T. and Zagradisnik, B. (2004) Prostate cancer and polymorphism D85Y in gene
for dihydrotestosterone degrading enzyme UGT2B15: frequency of DD homozygotes
increases with Gleason score. Prostate 59, 436–439
11 MacLeod, S. L., Nowell, S., Plaxco, J. and Lang, N. P. (2000) An allele-specific
polymerase chain reaction method for the determination of the D85Y polymorphism in the
human UDP-glucuronosyltransferase 2B15 gene in a case-control study of prostate
cancer. Ann. Surg. Oncol. 7, 777–782
12 Chouinard, S., Pelletier, G., Bélanger, A. and Barbier, O. (2004) Cellular specific
expression of the androgen-conjugating enzymes UGT2B15 and UGT2B17 in the human
prostate epithelium. Endocr. Res. 30, 717–725
13 Turgeon, D., Carrier, J.-S., Lévesque, E., Hum, D. W. and Bélanger, A. (2001) Relative
enzymatic activity, protein stability, and tissue distribution of human steroid-metabolizing
UGT2B subfamily members. Endocrinology 142, 778–787
Farnesoid X receptor reduces androgen glucuronidation in LNCaP cells
14 Dubois, S., Beaulieu, M., Lévesque, E., Hum, D. W. and Bélanger, A. (1999) Alteration of
human UDP-glucuronosyltransferase UGT2B17 regio-specificity by a single amino acid
substitution. J. Mol. Biol. 289, 29–39
15 Laffitte, B. A., Kast, H. R., Nguyen, C. M., Zavacki, A. M., Moore, D. D. and Edwards, P. A.
(2000) Identification of the DNA binding specificity and potential target genes for the
farnesoid X-activated receptor. J. Biol. Chem. 275, 10638–10647
16 Trottier, J., Milkiewicz, P., Kaeding, J., Verreault, M. and Barbier, O. (2006) Coordinate
regulation of hepatic bile acid oxidation and conjugation by nuclear receptors.
Mol. Pharm. 3, 212–222
17 Lee, F. Y., Lee, H., Hubbert, M. L., Edwards, P. A. and Zhang, Y. (2006) FXR, a
multipurpose nuclear receptor. Trends Biochem. Sci. 31, 572–580
18 Swales, K. E., Korbonits, M., Carpenter, R., Walsh, D. T., Warner, T. D. and
Bishop-Bailey, D. (2006) The farnesoid X receptor is expressed in breast cancer and
regulates apoptosis and aromatase expression. Cancer Res. 66, 10120–10126
19 Howard, W. R., Pospisil, J. A., Njolito, E. and Noonan, D. J. (2000) Catabolites of
cholesterol synthesis pathways and forskolin as activators of the farnesoid X-activated
nuclear receptor. Toxicol. Appl. Pharmacol. 163, 195–202
20 Pircher, P. C., Kitto, J. L., Petrowski, M. L., Tangirala, R. K., Bischoff, E. D., Schulman,
I. G. and Westin, S. K. (2003) Farnesoid X receptor regulates bile acid–amino acid
conjugation. J. Biol. Chem. 278, 27703–27711
21 Miyata, M., Matsuda, Y., Tsuchiya, H., Kitada, H., Akase, T., Shimada, M., Nagata, K.,
Gonzalez, F. J. and Yamazoe, Y. (2006) Chenodeoxycholic acid-mediated activation
of the farnesoid X receptor negatively regulates hydroxysteroid sulfotransferase.
Drug Metab. Pharmacokinet. 21, 315–323
22 Nishimura, M., Naito, S. and Yoko, T. (2004) Tissue-specific mRNA expression profiles of
human nuclear receptor subfamilies. Drug Metab. Pharmacokinet. 19, 135–149
23 Barbier, O., Torra, I. P., Sirvent, A., Claudel, T., Blanquart, C., Duran-Sandoval, D.,
Kuipers, F., Kosykh, V., Fruchart, J. C. and Staels, B. (2003) FXR induces the UGT2B4
enzyme in hepatocytes: a potential mechanism of negative feedback control of FXR
activity. Gastroenterology 124, 1926–1940
24 Lu, Y., Heydel, J.-M., Li, X., Bratton, S., Lindblom, T. and Radominska-Pandya, A. (2005)
Lithocholic acid decreases expression of UGT2B7 in Caco-2 cells: a potential role for a
negative farnesoid X receptor response element. Drug Metab. Dispos. 33, 937–946
25 Chouinard, S., Pelletier, G., Bélanger, A. and Barbier, O. (2006) Isoform-specific
regulation of UDP-glucuronosyltransferase (UGT)2B enzymes in the human prostate:
differential consequences for androgen and bioactive lipid inactivation. Endocrinology
147, 5431–5442
26 Guillemette, C., Lévesque, E., Beaulieu, M., Turgeon, D., Hum, D. W. and Bélanger, A.
(1997) Differential regulation of two uridine diphospho-glucuronosyltransferases,
UGT2B15 and UGT2B17, in human prostate LNCaP cells. Endocrinology 138,
2998–3005
27 Barbier, O., Lapointe, H., El Alfy, M., Hum, D. W. and Bélanger, A. (2000) Cellular
localization of uridine diphosphoglucuronosyltransferase 2B enzymes in the human
prostate by in situ hybridization and immunohistochemistry. J. Clin. Endocrinol. Metab.
85, 4819–4826
28 Trottier, J., Verreault, M., Grepper, S., Monté, D., Bélanger, J., Kaeding, J., Caron, P.,
Inaba, T. T. and Barbier, O. (2006) Human UDP-glucuronosyltransferase (UGT)1A3
enzyme conjugates chenodeoxycholic acid in the liver. Hepatology 44, 1158–1170
29 Reference deleted
30 Liu, Y.-Y., Nguyen, C. and Peleg, S. (2000) Regulation of ligand-induced
heterodimerization and coactivator interaction by the activation function-2 domain
of the vitamin D receptor. Mol. Endocrinol. 14, 1776–1787
31 Labrie, F., Bélanger, A., Bélanger, P., Bérubé, R., Martel, C., Cusan, L., Gomez, J.,
Candas, B., Chaussade, V., Castiel, I. et al. (2007) Metabolism of DHEA in
postmenopausal women following percutaneous administration. J. Steroid Biochem.
Mol. Biol. 103, 178–188
32 Zhang, Y., Kast-Woelbern, H. R. and Edwards, P. A. (2003) Natural structural variants of
the nuclear receptor farnesoid X receptor affect transcriptional activation. J. Biol. Chem.
278, 104–110
33 Huber, R. M., Murphy, K., Miao, B., Link, J. R., Cunningham, M. R., Rupar, M. J.,
Gunyuzlu, P. L., Haws, T. F., Kassam, A., Powell, F. et al. (2002) Generation of multiple
farnesoid-X-receptor isoforms through the use of alternative promoters. Gene 290, 35–43
34 De Gottardi, A., Dumonceau, J. M., Bruttin, F., Vonlaufen, A., Morard, I., Spahr, L.,
Rubbia-Brandt, L., Frossard, J. L., Dinjens, W. N., Rabinovitch, P. S. et al. (2006)
Expression of the bile acid receptor FXR in Barrett’s esophagus and enhancement of
apoptosis by guggulsterone in vitro . Mol. Cancer 5, 48
253
35 Bishop-Bailey, D., Walsh, D. T. and Warner, T. D. (2004) Expression and activation of the
farnesoid X receptor in the vasculature. Proc. Natl. Acad. Sci. U.S.A. 101, 3668–3673
36 Trauner, M. and Boyer, J. L. (2003) Bile salt transporters: molecular characterization,
function, and regulation. Physiol. Rev. 83, 633–671
37 Wang, S., Lai, K., Moy, F. J., Bhat, A., Hartman, H. B. and Evans, M. J. (2006) The nuclear
hormone receptor farnesoid X receptor (FXR) is activated by androsterone. Endocrinology
147, 4025–4033
38 Simard, J., Ricketts, M. L., Gingras, S., Soucy, P., Feltus, F. A. and Melner, M. H. (2005)
Molecular biology of the 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase
gene family. Endocr. Rev. 26, 525–582
39 Beaulieu, M., Lévesque, É., Tchernof, A., Beatty, B. G., Bélanger, A. and Hum, D. W.
(1997) Chromosomal localization, structure, and regulation of the UGT2B17 gene,
encoding a C19 steroid metabolizing enzyme. DNA Cell Biol. 16, 1143–1154
40 Turgeon, D., Carrier, J. S., Lévesque, É., Beatty, B. G., Bélanger, A. and Hum, D. W. (2000)
Isolation and characterization of the human UGT2B15 gene, localized within a cluster of
UGT2B genes and pseudogenes on chromosome 4. J. Mol. Biol. 295, 489–504
41 Claudel, T., Sturm, E., Duez, H., Torra, I. P., Sirvent, A., Kosykh, V., Fruchart, J. C.,
Dallongeville, J., Hum, D. W., Kuipers, F. et al. (2002) Bile acid-activated nuclear receptor
FXR suppresses apolipoprotein A-I transcription via a negative FXR response element.
J. Clin. Invest. 109, 961–971
42 Jochum, W., Passegue, E. and Wagner, E. F. (2001) AP-1 in mouse development and
tumorigenesis. Oncogene 20, 2401–2412
43 Barbier, O. and Bélanger, A. (2003) The cynomolgus monkey (Macaca fascicularis ) is the
best animal model for the study of steroid glucuronidation. J. Steroid Biochem. Mol. Biol.
85, 235–245
44 Guillemette, C., Hum, D. W. and Bélanger, A. (1996) Levels of plasma C19 steroids and 5
alpha-reduced C19 steroid glucuronides in primates, rodents, and domestic animals.
Am. J. Physiol. Endocrinol. Metab. 271, E348–E353
45 Parks, D. J., Blanchard, S. G., Bledsoe, R. K., Chandra, G., Consler, T. G., Kliewer, S. A.,
Stimmel, J. B., Willson, T. M., Zavacki, A. M., Moore, D. D. et al. (1999) Bile acids:
natural ligands for an orphan nuclear receptor. Science 284, 1365–1368
46 Williams, R. H., Larsen, P. R., Kronenberg, H. M., Melmed, S., Polonsky, K. S., Wilson,
J. D. and Foster, D. W. (2002) Williams Text Book of Endocrinology, Saunders,
Philadelphia, PA, U.S.A.
47 Bélanger, A., Candas, B., Dupont, A., Cusan, L., Diamond, P., Gomez, J. and Labrie, F.
(1994) Changes in serum concentrations of conjugated and unconjugated steroids in
40–80-year-old men. J. Clin. Endocrinol. Metab. 79, 1086–1090
48 Hong, Y., Gagnon, J., Rice, T., Perusse, L., Leon, A., Skinner, J., Wilmore, J.,
Bouchard, C. and Rao, D. (2001) Familial resemblance for free androgens and
androgen glucuronides in sedentary black and white individuals: the HERITAGE family
study. Health, risk factors, exercise training and genetics. J. Endocrinol. 170,
485–492
49 Guillemette, C., Hum, D. W. and Bélanger, A. (1996) Levels of plasma C19 steroids and 5
alpha-reduced C19 steroid glucuronides in primates, rodents, and domestic animals.
Am. J. Physiol. Endocrinol. Metab. 271, E348–E353
50 Wu, A. H., Whittemore, A. S., Kolonel, L. N., Stanczyk, F. Z., John, E. M., Gallagher, R. P.
and West, D. W. (2001) Lifestyle determinants of 5{alpha}-reductase metabolites in older
African-American, white, and Asian-American men. Cancer Epidemiol. Biomarkers Prev.
10, 533–538
51 Beaulieu, M., Lévesque, E., Hum, D. W. and Bélanger, A. (1996) Isolation and
characterization of a novel cDNA encoding a human UDP-glucuronosyltransferase active
on C19 steroids. J. Biol. Chem. 271, 22855-22862
52 Choi, Y. H., Im, E. O., Suh, H., Jin, Y., Yoo, Y. H. and Kim, N. D. (2003) Apoptosis and
modulation of cell cycle control by synthetic derivatives of ursodeoxycholic acid and
chenodeoxycholic acid in human prostate cancer cells. Cancer Lett. 199, 157–167
53 Koh, E., Kanaya, J. and Namiki, M. (2001) Adrenal steroids in human prostatic cancer cell
lines. Arch. Androl. 46, 117–125
54 Ben-Ishay, D., Slavin, S., Levij, I. S. and Eliakim, M. (1975) Obstructive jaundice
associated with carcinoma of the prostate. Isr. J. Med. Sci. 11, 838–844
55 Bloch, W. E. and Block, N. L. (1992) Metastatic prostate cancer presenting as obstructive
jaundice. Urology 40, 456–457
56 Koruk, M., Buyukberber, M., Savas, C. and Kadayifci, A. (2004) Paraneoplastic
cholestasis associated with prostate carcinoma. Turk. J. Gastroenterol. 15, 53–55
57 Karakolios, A., Kasapis, C., Kallinikidis, T., Kalpidis, P. and Grigoriadis, N. (2003)
Cholestatic jaundice as a paraneoplastic manifestation of prostate adenocarcinoma.
Clin. Gastroenterol. Hepatol. 1, 480–483
Received 17 August 2007/26 October 2007; accepted 7 November 2007
Published as BJ Immediate Publication 7 November 2007, doi:10.1042/BJ20071136
c The Authors Journal compilation c 2008 Biochemical Society