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Endocrinology, doi:10.1210/en.2006-0229
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Endocrinology Vol. 147, No. 11 5431-5442
Copyright © 2006 by The Endocrine Society

Isoform-Specific Regulation of Uridine Diphosphate-Glucuronosyltransferase 2B Enzymes in the Human Prostate: Differential Consequences for Androgen and Bioactive Lipid Inactivation

Sarah Chouinard, Georges Pelletier, Alain Bélanger and Olivier Barbier

Oncology and Molecular Endocrinology Research Center (S.C., G.P., A.B., O.B.), Centre Hospitalier de l’Université Laval Research Center, and Faculties of Medicine (S.C., G.P., A.B.) and Pharmacy (O.B.), Laval University, Québec, Canada G1V 4G2

Address all correspondence and requests for reprints to: Olivier Barbier or Alain Bélanger, Oncology and Molecular Endocrinology Research Center, CHUL Research Center, 2705 Laurier Boulevard Bloc T3-67, Québec, Canada G1V 4G2. E-mail: olivier.barbier{at}pha.ulaval.ca or alain.belanger{at}ap.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens as well as monohydroxy-fatty acids are implicated in the pathogenesis of prostate cancer. Like a huge variety of endo- and xenobiotics, they are eliminated as glucuronide conjugates formed by uridine diphosphate-glucuronosyltransferase (UGT) enzymes. In the present study, we observe that treatment of the prostate cancer cells LNCaP with natural and synthetic androgens, IL-1{alpha}, or epidermal growth factor (EGF) differently modulates the glucuronidation of androgen and bioactive lipid metabolites. Indeed, glucuronidation of 5{alpha}-androstane-3{alpha},17ß-diol and 13-hydroxyoctadecadienoic acid was drastically reduced, whereas 12-hydroxyeicosatetraenoic acid conjugation by UGT was increased after androgen treatment. These effects reflected the reduction of UGT2B10, -B15, and -B17 enzyme expression, and the activation of the UGT2B11 gene. In human prostate epithelial cells, only UGT2B11 and -B15 mRNAs are detected and are regulated by androgens in a similar manner as in LNCaP cells. In LNCaP cells, IL-1{alpha} and EGF also regulate UGT2B expression in an isoform-specific manner; IL-1{alpha} induced UGT2B10 and reduced UGT2B17, while having no effects on UGT2B11 mRNA levels. EGF treatment resulted in a decreased UGT2B17 expression, whereas UGT2B10 and -B11 mRNA remained at their basal levels. Overall, these results demonstrate that in the human prostate, androgens do not only affect their own inactivation but also influence the levels of monohydroxy-fatty acids by regulating the expression of UGT2B enzymes in an isoform-specific manner. These differential effects of androgens, IL-1{alpha}, and EGF on lipid metabolism likely constitute an additional mechanism by which these endogenous factors promote prostate cancer development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAJOR ROLE that androgens play in the physiology of normal prostate as well as in the initiation and development of prostate cancer has been widely documented (for review see Ref. 1). More recently, evidence suggested that hydroxy-fatty acids, such as linoleic acid (LA) and arachidonic acid (AA) metabolites, are also implicated in the pathogenesis of prostate cancer. Indeed, overexpression of the 12-lipoxygenase (LOX) enzyme, which converts AA into 12-hydroxyeicosatetraenoic acid (12-HETE) has been observed in prostate cancers and tends to correlate with the aggressive tumor behavior (2, 3, 4). Consistently, urinary levels of 12-LOX metabolites are increased in patients with prostatic diseases (5). On the other hand, expression of the 15-LOX type 1 enzyme, which catalyzes the transformation of LA into 13-hydroxyoctadecadienoic acid (HODE), is also correlated with a high grade of prostate cancer (6, 7). These recent observations suggest that such monohydroxy-fatty acids promote prostate cancer development by activating cellular proliferation and angiogenesis (8). Interestingly, recent findings also indicate that androgens and monohydroxy-fatty acids share an inactivating metabolic pathway called glucuronidation (9, 10, 11).

Uridine diphosphate-glucuronosyltransferase (UGT) enzymes represent a family of microsomal enzymes that catalyze the transfer of glucuronic acid from uridine 5'-diphosphoglucuronic acid (UDPGA) to endogenous and exogenous molecules with oxygen, nitrogen, and sulfur functional groups (12). Glucuronidation renders aglycon substrates water soluble and more easily excretable from the body (12, 13). Based on evolutionary divergences, human UGT enzymes have been classified into two subfamilies, UGT1 and UGT2; the latter was further subdivided into UGT2A and -2B (12). The seven UGT2B enzymes isolated to date, UGT2B4, -B7, -B10, -B11, -B15, -B17, and -B28, are encoded by different genes clustered on chromosome 4 (4q13–4q21.1) (12). UGT2B4 and -B7 conjugate bile acids, whereas the latter also glucuronidates androgen, estrogen, mineralocorticoid, and glucocorticoid hormones (14, 15, 16, 17, 18). The only substrates identified to date for UGT2B10 and -B11 are the metabolites of LA and AA, namely 13-HODE, 12-HETE, and 15-HETE (10). In addition to UGT2B7, the group of androgen-conjugating enzymes also comprises UGT2B15, -B17, and -B28; and UGT2B7, -B15, and -B17 are less specific for hydroxy-fatty acids but also conjugate some metabolites of LA and AA (9, 10, 14).

Expression of the seven human UGT2B enzymes has been detected in various androgen target tissues such as prostate and skin where the glucuronidation of dihydrotestosterone (DHT), and its metabolites 5{alpha}-androstane-3{alpha},17ß-diol (3{alpha}-Diol) and androsterone has been reported (9, 17, 19). These observations suggest that androgens are inactivated locally before being released into the circulation. Indeed, high levels of androgen glucuronides were measured in human prostate (20), whereas the proportion of unconjugated vs. glucuronidated 3{alpha}-Diol and androsterone is relatively low in blood (21).

The local inactivation of biologically active endobiotics constitutes an efficient mechanism to control their effects (9, 22, 23, 24). Recent observations indicate that expression of UGT2B15 and -B17 enzymes in LNCaP cells, an androgen-dependent prostate cancer cell line, is modulated by endogenous factors such as androgens, cytokines, and growth factors (25, 26, 27). Furthermore, the down-regulation of UGT2B15 and -B17 expression and activity in LNCaP cells cultured in the presence of androgens has been correlated to increased concentrations of DHT in the cell media, to induced cell growth, and to elevated prostate-specific antigen secretion (21, 25). Sun et al. (28) also reported that isoflavone treatment of LNCaP cells resulted in an increased inactivation of DHT by glucuronidation, which caused lower prostate-specific antigen production.

In addition to the androgen-conjugating enzymes UGT2B15 and -B17, UGT2B11 is also expressed in the human prostate (17). Regulation of UGT2B15 and -B17 expression and activity has been previously studied, whereas the relative limited number of substrates for UGT2B11 reduced the interest of investigators for this isoform. However, Turgeon et al. (10) recently reported that UGT2B10 and -B11 catalyze the glucuronidation of HODE and HETE substrates. Considering the emerging role that such LA and AA metabolites play in the development of prostate cancer, the present study was aimed at analyzing the global effect of androgens, IL-1{alpha}, and epidermal growth factor (EGF) on the expression and androgen- and hydroxy-fatty-acid-conjugating activity of UGT2B enzymes in the classical prostate cell model, LNCaP and in normal human prostate epithelial cells (PrEC).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
R1881 was purchased from DuPont NEN Life Science Products (Boston, MA). Protein assay reagents were obtained from Bio-Rad Laboratories Inc. (Marnes-la-Coquette, France). IL-1{alpha} and EGF were purchased from Sigma (Oakville, Ontario, Canada). UDPGA was obtained from Sigma Chemical Co. (St. Louis, MO). 13-HODE, 12-HETE, and 5-HETE were purchased from Cayman Chemicals (Ann Arbor, MI). 3{alpha}-Diol was purchased from Steraloids (Newport, RI). Standards of 3{alpha}-Diol-glucuronides and Casodex were provided by Endorecherche (Medicinal Chemistry Division of our laboratory). The positional isomers of 3{alpha}-Diol and the regioselectivity of the glucuronidated metabolites were ascertain using proton-nuclear magnetic resonance analysis. Ammonium formate was from Aldrich Chemical Co. (Milwaukee, WI), and HPLC-grade methanol was provided by VWR Canlab (Montreal, Quebec, Canada). Human embryonic kidney (HEK293) and human prostate cancer (LNCaP) cells were obtained from the American Type Culture Collection (Rockville, MD). PrEC and related culture medium were obtained from Cambrex (Walkersville, MD), and human liver microsomes were obtained from the Human Cell Culture Center (Laurel, MD). SyBr Green PCR Mix 2x was obtained from Applied Biosystems (Foster City, CA). The slide of prostate tissue was obtained from US Biomax Inc. (Toronto, Ontario, Canada).

Cell culture
HEK293 and LNCaP cells were grown as previously described (10, 26). PrEC were grown in PrEC basal medium as recommended by the supplier (Cambrex, Walkersville, MD). HEK293 cell lines stably expressing UGT2B enzymes were obtained as previously reported (10). For treatments with androgen receptor (AR) agonists, Casodex (5 µM), IL-1{alpha} (2 ng/ml), EGF (10 ng/ml), cycloheximide (20 µg/ml), and actinomycin D (1 µg/ml), LNCaP cells were precultured for 24 h in serum-free media, and all treatments were performed in the absence of serum for the indicated duration and at the indicated concentrations. For cycloheximide treatment, LNCaP cells were incubated for 24 h in the presence or absence of R1881 (10 nM), and for actinomycin D, LNCaP cells were pretreated with actinomycin D for 2 h and then media were changed to novel media containing or not R1881 (10 nM) for 24 h. AR agonists and Casodex were dissolved in ethanol, and the volume added to media never exceeded 0.1% ethanol (vol/vol). For longer treatments, fresh medium was added every 48 h.

Production of the anti-UGT2B10-B11 antibody
A 34.8-kDa fragment of the UGT2B11 protein (amino acids 60–140) fused to the glutathione S-transferase protein has been constructed as previously described (26, 29). Production and purification of the fusion protein and immunization procedures were performed as fully described elsewhere (30). Briefly, three rabbits were injected at multiple sites with 300 µg purified fusion protein, in the presence of 300 µl complete Freund’s adjuvant. Subsequently, two booster injections were given at 4-wk intervals with the same quantity of protein in the presence of incomplete Freund’s adjuvant. The production of antibodies was checked 14 d after each injection on blood collected by ear puncture. Rabbit sera were tested for the presence of antibodies against UGT2B by Western blot using microsomal extracts from human liver and HEK293 cells stably expressing each human UGT2B protein. Whereas all three sera were reactive with UGT2B proteins, serum 1845 recognized only UGT2B10 and -B11.

Western blot experiments
Microsomal preparations of UGT2B-HEK293 and LNCaP cells were obtained by differential centrifugation, as previously described (10), and were resuspended in homogenization buffer at 20 µg/µl and stored at –80 C. Total PrEC and LNCaP proteins from R1881 treated or control cells were purified according to the Tri-Reagent acid phenol protocol as specified by the supplier (Molecular Research Center, Cincinnati, OH). For Western blot experiments, 10 µg microsomal proteins or 30 µg total proteins were separated by 10% SDS-polyacrylamide gel. The gel was transferred onto nitrocellulose membrane and probed with the 1845 anti-UGT2B10-B11 (1:6000 dilution), 1849 anti-UGT2B15 (1:1500 dilution), or anti-calnexin (1:5000 dilution) antibodies (Stressgen, Victoria, British Columbia, Canada). An antirabbit IgG horse antibody conjugated with peroxidase (Amersham, Oakville, Ontario, Canada) was used as the second antibody, and the resulting immunocomplexes were visualized using a chemiluminescence kit (ECL) (Renaissance, Quebec, Canada) and exposed on hyperfilm (Kodak Corp., Rochester, NY) for 15–60 sec.

Immunohistochemical analyses
Immunohistochemical analyses were performed as previously described (20, 29). Briefly, immunostaining was performed by using anti-UGT2B10-B11 (1845) antisera diluted 1:500 in Tris saline (pH 7.6) for 1 h at room temperature. Sections were subsequently washed with PBS and incubated with a peroxidase-labeled goat antirabbit {gamma}-globulin diluted 1:200 for 10 min (Hyclone Laboratories, Inc., Logan, UT). After incubation, the peroxidase complex was revealed with diaminobenzidine after exposure for 2 min. The intensity of the staining was controlled under the microscope. The sections were then counterstained with hematoxylin. Control experiments were performed on consecutive sections using preimmune rabbit serum (1:500) or antisera anti-UGT2B10-B11 preincubated with the corresponding preparation of stably transfected HK293 cells (20 µM).

Glucuronidation assay
Enzymatic assays were conducted using 30–40 µg microsomal proteins or 30–80 µg proteins from cells homogenized in Tris-buffered saline (pH 7.4; 50 mM) with dithiothreitol (0.5 mM) in the presence of 1 mM UDPGA, 10 mM MgCl2, 100 µg/ml phosphatidylcholine, 8.5 mM saccharolactone, and 200 µM of the different aglycons in a glucuronidation assay buffer (10). Assays were incubated at 37 C for 2 h and terminated by adding 100 µl methanol with 0.02% butylated Hydroxytoluene followed by centrifugation at 14,000 x g for 10 min, as previously described (30). The formation of glucuronide conjugates was measured by using liquid chromatography coupled with mass spectrometry (LC-MS/MS) as previously described (10).

RNA isolation, reverse transcription, and real-time PCR
Total RNA was isolated from LNCaP cells or PrEC according to the Tri-Reagent acid phenol protocol as specified by the supplier (Molecular Research Center). The RT reaction was performed using 200 U Superscript II (Invitrogen, Burlington, Ontario, Canada) with 1 µg total RNA and 7.5 ng random hexamers (Roche, Laval, Quebec, Canada) at 42 C for 50 min. The real-time PCR were performed using an ABI Prism 7000 instrument from Applied Biosystems (Foster City, CA). For each gene, the amplification efficiency was tested using 2–5 log of concentrations of cDNA produced from LNCaP-cell purified mRNA. The conditions for real-time PCR are described in Table 1Go. For each reaction, the final volume of 20 µl comprised 10 µl SyBr Green PCR mix, 2 µl of each primer (Table 1Go), and 6 µl of a RT product (1/100 dilution for LNCaP cells and 1/20 for PrEC). Conditions for real-time PCR were 95 C for 10 min, 95 C for 15 sec, and 56 C (36B4, AR, UGT2B10, UGT2B15, UGT2B28 and 28S) or 62 C (36B4, UGT2B4, UGT2B7, UGT2B11, and 28S) for 60 sec for 40 cycles. Cycle threshold (Ct) values were calculated by normalizing UGT2B mRNA expression with 36B4 or with 28S. Treatment with actinomycin D and cycloheximide affected the Ct values of the housekeeping gene 36B4; therefore, the UGT expression was normalized with the unaffected 28S for these analyses.


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  		TABLE 1. Primers and conditions used in real-time PCR analyses

 
Statistical analyses
A nonparametric Student’s t test was used to analyze for significant differences between the experimental groups, by using the JMP version 5.0.2 software (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LC-MS/MS analyses of glucuronides formed from 3{alpha}-Diol, 5-HETE, 12-HETE, and 13-HODE in LNCaP cells
Although the formation of 3{alpha}-Diol-glucuronide has been previously reported (31, 32), the position of the glucuronide group on the steroid molecule remains to be clarified. In the present study, the formation of 3{alpha}-Diol-3-glucuronide and 3{alpha}-Diol-17-glucuronide by microsomal proteins extracted from LNCaP cells was investigated by LC-MS/MS using synthetic standards. Furthermore, because the ability of LNCaP to form glucuronide conjugates of 12-HETE, 5-HETE, and 13-HODE has never been investigated, similar experiments were performed using these fatty acids as substrates. As shown in Fig. 1Go, the major proportion of glucuronides detected by LC-MS/MS corresponded to 3{alpha}-Diol-17-glucuronide, a metabolite specifically formed by microsomal preparations of UGT2B15 and -B17 proteins (Fig. 1Go). By contrast, 3{alpha}-Diol-3-glucuronide formation was minor in the presence of microsomes from LNCaP cells, while being the major glucuronidation product of 3{alpha}-Diol formed by the UGT2B7 isoform (Fig. 1Go). Interestingly, the production of these two 3{alpha}-Diol-glucuronide conjugates correlates to the previously reported low levels of UGT2B7 and high levels of UGT2B15 and -B17 expressions in LNCaP cells (30, 32). In the presence of LNCaP cell extracts, 12-HETE and 13-HODE were converted to polar metabolites that were detected by LC-MS/MS at m/z and retention times corresponding to their glucuronide derivatives (data not shown). However, the formation of 5-HETE-glucuronide was below the limit of detection.


Figure 1
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  		FIG. 1. Gene specificity and regiospecificity of 3{alpha}-Diol glucuronidation. Shown is a representation of the regiospecificity of glucuronidation on 3{alpha}-Diol by LNCaP cells and stably UGT2B7-, -B15-, and -B17-transfected HEK293 cells. Chromatographic separation was performed by HPLC, and glucuronidated metabolites were detected in the multiple reaction monitoring mode of the mass spectrometer. G, Glucuronide.

 
R1881 treatment reduces 3{alpha}-Diol and 13-HODE glucuronidation but increases 12-HETE-glucuronide formation in LNCaP cells
The effects of androgens on the metabolism of 3{alpha}-Diol and hydroxy-fatty acids were investigated by treating LNCaP cells with R1881. Previous studies demonstrated that in contrast to DHT, R1881 is not a substrate for human UGTs and is less susceptible to other metabolic pathways (33). As expected (25, 26, 27), we observe that this treatment resulted in a 2-fold lower formation of 3{alpha}-Diol-17-glucuronide compared with control cells (Fig. 2AGo). Interestingly, we also observed that the glucuronidation of 13-HODE was decreased by 3-fold (28 to 8 pmol/min/mg proteins) in R1881-treated compared with control cells (Fig. 2BGo), whereas a 2-fold increase of 12-HETE conjugation was measured (Fig. 2CGo). These results indicate that R1881 differentially regulates the expression of the UGT enzymes involved in the glucuronidation of androgens and monohydroxy-fatty acids.


Figure 2
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  		FIG. 2. R1881 differently affects the glucuronidation of endogenous molecules in LNCaP cells. LNCaP cells were incubated in the presence or absence of R1881 (10 nM) for 48 h, and glucuronidation assays were performed with 3{alpha}-Diol (A), 13-HODE (B), and 12-HETE (C) and homogenized cells as described in Materials and Methods. Relative activities were calculated with 3{alpha}-Diol-17-glucuronide as standard or with the peak area of glucuronide metabolite for the other aglycons and corrected according to the time of incubation and quantity of proteins. Values are means ± SEM (n = 6; two experiments in triplicate). Statistically significant differences between vehicle- and R1881-treated cells are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test).

 
Treatment of LNCaP cells with R1881 reduces UGT2B10, -B15, and -B17 mRNA levels but stimulates UGT2B11 gene expression
Whereas the expression and regulation of the UGT2B15 and -B17 genes in LNCaP cells are well documented (25, 26), the presence or absence of other UGT2B members as well as the effects of AR agonists on these enzymes were not investigated. To address this point, LNCaP cells were cultured in the presence or absence of R1881 (10 nM) for 24 h, and the mRNA levels of all human UGT2B enzymes were quantified by using real-time PCR. UGT2B4, -B7, and -B28 mRNAs were extremely low in both control and R1881-treated cells (see Table 2Go), indicating that these genes are minimally expressed in this cell line. Similarly to the drastic reduction in both UGT2B15 and -B17 transcript concentrations (78 and 62% reduction, respectively) (Fig. 3Go, A and B), UGT2B10 mRNA levels were significantly reduced (56%) by the treatment with R1881 (Fig. 3CGo). By contrast, we found that treatment with the synthetic AR agonist resulted in 4-fold higher UGT2B11 mRNA levels compared with control cells (Fig. 3DGo). Incidentally, LNCaP cells treated with the natural AR activator DHT also exhibited a down-regulation of UGT2B10, -B15, and -B17 genes and a similar induction of the UGT2B11 gene expression (data not shown). Because previous studies investigated the negative effects that androgens exert on the expression of androgen-conjugating UGT (25, 26, 33, 34), the present study was aimed at characterizing the positive regulation of UGT2B11, which was then compared with the reduction in UGT2B15 expression.


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  		TABLE 2. Expression levels of UGT2B enzymes in LNCaP cells and PrEC

 

Figure 3
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  		FIG. 3. R1881 treatment reduces UGT2B15 (A), -B17 (B), and -B10 (C) mRNA levels but strongly induces UGT2B11 (D) expression in LNCaP cells. LNCaP cells were incubated in the presence or absence of R1881 at 10 nM for 24 h, and the UGT2B mRNA content was quantified using real-time PCR as described in Materials and Methods. UGT2B mRNA levels were normalized with the 36B4 mRNA concentration. Values are means ± SEM (n = 6; two experiments in triplicate). Statistically significant differences between vehicle- and R1881-treated cells are indicated: **, P < 0.01 (Student’s t test).

 
Time-course and dose-response experiments were performed by incubating LNCaP cells in the presence of R1881 (10 nM) for 6, 12, 18, 24, 48, and 72 h or in the presence of R1881 at different concentrations (0.1, 0.5, 1, 2, 5, and 10 nM) for 24 h (Fig. 4Go). These experiments reveal that the induction of UGT2B11 mRNA levels occurred as early as 6 h of treatment to reach a maximal activation (>150-fold) after 72 h (Fig. 4AGo). On the other hand, inhibition of UGT2B15 expression started only after 12 h of treatment and reached undetectable levels at 72 h of R1881 treatment (Fig. 4BGo). In addition, the induction of the UGT2B11 mRNA level occurred at 0.5 nM R1881 to reach a maximal activation at 2 nM (Fig. 4CGo). For UGT2B15, the inhibition was detected in the presence of a very low concentration of R1881 (0.1 nM), with a maximal effect at 0.5 nM (Fig. 4DGo).


Figure 4
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  		FIG. 4. R1881 treatment induces UGT2B11 mRNA levels (A and C) but reduces the UGT2B15 gene expression (B and D) in a time-dependent (A and B) and dose-dependent (C and D) manner. LNCaP cells were incubated either in the presence or absence of R1881 (10 nM) for different durations (6, 12, 18, 24, 48, and 72 h) (A and B) or in the presence or absence of increasing amounts of R1881 (0.1, 0.5, 1, 2, 5, and 10 nM) for 48 h (C and D) and mRNA content was quantified using real-time PCR as described in Materials and Methods. UGT2B mRNA levels were normalized with the 36B4 mRNA concentration. Values are means ± SEM (n = 6; two experiments in triplicate). Statistically significant differences between vehicle- and R1881-treated cells are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test).

 
R1881 also affects UGT2B11 and -B15 protein levels in LNCaP cells
The effect of the R1881 treatment on UGT2B11 and -B15 protein expression was investigated by Western blot analyses, using specific antibodies. Although the anti-UGT2B15 antiserum was previously characterized (29), the specificity of the antibody raised against the UGT2B11 peptide remained to be established. For this purpose, 10 µg microsomal proteins of HEK293 cells stably expressing human UGT2B4, -B7, -B10, -B11, -B15, or B17 proteins were size separated by SDS-PAGE and immunoblotted with the anti-UGT2B15 or -2B11 antisera (Fig. 5Go, B and C). As expected, the anti-UGT2B15 specifically bound the corresponding protein (26, 29). By contrast, the anti-UGT2B11 antiserum recognized both UGT2B10 and -B11 proteins but remained specific for these isoforms because no binding was detected with other UGT2B enzymes (Fig. 5BGo). Equal loading of each lane was ensured by immunoblotting the same membrane with the anti-calnexin antibody as a control (Fig. 5AGo). Subsequently, microsomal proteins from LNCaP cells treated with R1881 for 48 h were probed with these antibodies. The expression of calnexin remained unchanged within treated cells, whereas the protein levels of UGT2B15 were drastically decreased (Fig. 5Go, A, and C). By contrast, the anti-UGT2B10-B11 antibody revealed an accumulation of proteins in microsomal fractions from treated compared with control cells (Fig. 5BGo, treatments 1 and 2). Considering that R1881 treatment resulted in a 50% reduction of UGT2B10 mRNA levels after 24 h (Fig. 3Go), this protein accumulation likely reflects higher concentrations of the UGT2B11 protein. These analyses demonstrate that activation of AR with the synthetic agonist differently affects UGT2B expression at both mRNA and protein levels.


Figure 5
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  		FIG. 5. R1881 increases UGT2B11 protein levels while reducing other UGT2B protein contents in LNCaP cells. LNCaP cells were incubated in the presence or absence of R1881 at 10 nM for 48 h, and microsomal proteins were subsequently purified and immunoblotted using anti-calnexin (A, internal control), anti-UGT2B10-B11 (B), and anti-UGT2B15 (C) antibodies. Specificities of the antibodies were assessed by using microsomal proteins from HEK293 cells stably expressing UGT2B4, -B7, -B10, -B11, -B15, and -B17 proteins.

 
UGT2B11 expression is found in epithelial cells of the human prostate and R1881 dose-dependently regulates UGT2B11 and -B15 gene expression in PrEC
Previous analyses showed that UGT proteins are expressed in a cell-type-specific manner in the human prostate epithelium, with UGT2B17 found in basal and UGT2B15 in luminal cells (29). Therefore, immunohistochemical analyses were performed with the specific antibody against UGT2B10-B11 to determine in which cells of the human prostate UGT2B11 is expressed. Considering that the UGT2B10 enzyme is not found in the human prostate (17), the results demonstrate that UGT2B11 is localized in both luminal and basal epithelial cells of the human prostate (Fig. 6AGo). The absence of signal when the anti-UGT2B10-B11 was preincubated with the corresponding protein demonstrates the specificity of the binding.


Figure 6
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  		FIG. 6. UGT2B11 protein is localized in luminal and basal cells of the human prostate (A), and R1881 dose-dependently regulates UGT2B11 (B) and -B15 (C) mRNA levels as well as quantity of UGT2B11 and -B15 proteins (D) in PrEC in the same manner as in LNCaP cells. A, Immunohistochemistry analysis (x20 magnification) using the preimmune serum, the specific anti-UGT2B10-B11 antibody, or antisera anti-UGT2B10-B11 preincubated with the corresponding microsomal preparation of stably transfected HEK293 cells performed on a slide of human prostate revealed that the UGT2B11 is expressed in both basal (arrowhead) and luminal (arrow) cells of the prostate epithelium. B and C, PrEC were incubated either in the absence or presence of increasing amounts of R1881 (0.1, 1, and 10 nM) for 48 h, and total RNA was extracted and analyzed by real-time PCR for UGT2B11 (B) and -B15 (C) expression. UGT2B mRNA levels were normalized with 36B4. Values are means ± SEM (n = 6; two experiments in triplicate). Statistically significant differences are indicated: **, P < 0.01; ***, P < 0.001 (Student’s t test). D, Total proteins (30 µg) from PrEC treated or not with R1881 (1 nM) for 48 h were immunoblotted with the anti-calnexin (a, internal control), anti-UGT2B10-B11 (b), or anti-UGT2B15 (c) antibodies.

 
Subsequently and to determine whether the androgen-dependent regulation of UGT2B11 and -B15 expression also occurs in noncarcinogenic cells, we analyzed the effects of R1881 (0.1, 1, and 10 nM) on UGT2B expression in human primary PrEC (Fig. 6Go). As shown in Table 2Go, of the seven enzymes, only UGT2B11 and -B15 were found to be expressed in this preparation of prostate cells. Furthermore, a significant expression of the androgen receptor was detected in these cells (Table 2Go). We also observed that R1881 dose-dependently increased the concentration of UGT2B11 transcripts, whereas the UGT2B15 gene expression was inhibited (Fig. 6Go, B and C). In addition, as in LNCaP cells, the concentration of UGT2B11 protein was induced in R1881-treated PrEC (1 nM for 48 h), whereas the same treatment resulted in a drastic reduction of the UGT2B15 protein contents (Fig. 6DGo). These observations demonstrate that androgens act as regulators of UGT2B11 and -B15 genes in both proliferative LNCaP cells and noncancer PrEC.

AR controls UGT2B11 and -B15 genes expression
Upon ligand-dependent activation, AR positively regulates gene expression through direct binding to DNA (35). We ascertained the involvement of AR in the R1881-dependent regulation of UGT2B enzymes by using the pure AR antagonist Casodex, and we investigated whether R1881-activated AR modulates UGT2B mRNA levels by acting directly on the corresponding gene by using inhibitors of gene transcription (actinomycin D) or of protein synthesis (cycloheximide) (Fig. 7Go). As expected, incubation of LNCaP in the presence of R1881 resulted in reduced UGT2B15 and increased UGT2B11 mRNA levels (Fig. 7Go, A and B). However, coincubation with Casodex significantly affected the response of both UGT2B15 (Fig. 7AGo) and UGT2B11 (Fig. 7BGo) genes to R1881, indicating that AR mediates the differential regulation of these genes in the presence of natural or synthetic androgens. Furthermore, we also observed that preincubation with actinomycin D also abolished the R1881-dependent regulation of UGT2B15 and UGT2B11 genes expression (Fig. 7Go, C and D). By contrast, in the presence of cycloheximide, R1881 retained its inducing effect on the UGT2B11 gene expression (Fig. 7DGo) but failed to significantly reduce UGT2B15 mRNA levels (Fig. 7CGo). Together these observations indicate that R1881 regulates UGT2B11 and UGT2B15 genes in a transcriptional manner that involves AR. Despite that this regulatory effect is direct in the case of UGT2B11, it requires de novo protein synthesis in the case of UGT2B15.


Figure 7
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  		FIG. 7. AR agonist differently modulates UGT2B gene expression in LNCaP cells via a direct or indirect transcriptional mechanism. A and B, LNCaP cells were treated for 48 h with ethanol (vehicle) or R1881 (1 nM) in the presence or absence of Casodex (5 µM). C and D, LNCaP cells were incubated for 24 h with ethanol (vehicle) or R1881 (10 nM) in the absence or presence of cycloheximide (20 µg/ml) or after a pretreatment (2 h) with actinomycin D (1 µg/ml). mRNA was extracted and analyzed by real-time PCR using specific oligonucleotides for UGT2B15 (A and C) and -B11 (B and D). UGT mRNA levels were normalized with 36B4 (A and B) or 28S (C and D). Values are means ± SEM (n = 6; two experiments in triplicate). In A and B, values followed by different letters are statistically significantly different at P < 0.05 (Student’s t test). In C and D, statistically significant differences are indicated by asterisks: *, P < 0.05; **, P < 0.01 (Student’s t test).

 
Effects of IL-1{alpha} and EGF on UGT2B11 expression in LNCaP cells
It was previously shown that IL-1{alpha} and EGF exert opposite effects on LNCaP cell proliferation; a stimulation is observed with EGF treatment, whereas IL-1{alpha} inhibits the growth of these prostate cancer cells (26, 27). Both factors induce, however, a marked down-regulation in UGT2B17 expression without affecting the level of UGT2B15 transcripts (26, 27), suggesting that the modulation of the two androgen-conjugating enzymes is dissociated from the modulation in cell proliferation. In the present study, the effects of IL-1{alpha} and EGF treatments were investigated on UGT2B10, -B11, and -B17 (positive control) gene expression. As expected (26, 27), IL-1{alpha} and EGF treatments diminished the levels of UGT2B17 transcripts (Fig. 8AGo). However, the UGT2B11 transcripts concentration remained unaffected (Fig. 8CGo), and UGT2B10 expression was significantly stimulated by IL-1{alpha} treatment while being not affected in the presence of EGF (Fig. 8BGo). These observations also indicate dissociation between modulation of cell proliferation and action of IL-1{alpha} and EGF on UGT2B transcripts in LNCaP cells.


Figure 8
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  		FIG. 8. IL-1{alpha} and EGF differently affect UGT2B expression in LNCaP cells. LNCaP cells were incubated in the presence or absence of IL-1{alpha} (2 ng/ml) or EGF (10 ng/ml) for 24 h. mRNA was extracted and analyzed by real-time PCR using specific oligonucleotides for UGT2B17 (A), -B10 (B), and -B11 (C). mRNA expression was normalized with 36B4. Values are means ± SEM (n = 6; two experiments in triplicate). Statistically significant differences are indicated: ***, P < 0.001 (Student’s t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we report that activation of LNCaP cancer cells with synthetic or natural androgens affects the metabolism of not only 5{alpha}-reduced C19 steroids but also of monohydroxy-fatty acids. Treatment of LNCaP cells with androgens resulted in a drastic reduction of 3{alpha}-Diol and 13-HODE glucuronidation but in an enhanced formation of 12-HETE-glucuronide. Numerous studies established the importance of androgen glucuronidation as a major metabolic pathway of androgen inactivation in normal and cancerous prostate tissues, thus suggesting glucuronidation as a protective mechanism against prostate cancer (9, 26, 34, 36, 37, 38, 39, 40). By contrast, the role of LA and AA glucuronidation in this tissue is poorly understood. In fact, only recent studies revealed the presence in prostate cells of 12- and 15-LOX-1 enzymes capable of converting LA and AA into biologically active hydroxylated metabolites (41). In this tissue, the locally produced 13-HODE displays mitogenic effects, whereas 12-HETE induces angiogenesis (7, 42, 43, 44). The present study indicates that these active monohydroxy-fatty acids that are formed in the human prostate are also inactivated as glucuronide derivatives in a prostate cancer cell line.

Among the UGT enzymes expressed in LNCaP cells (Table 2Go), only UGT2B10, -B11, and -B17 catalyze the glucuronide conjugation of 13-HODE (10). Thus, the decrease in glucuronidation of this substrate by R1881-treated LNCaP cells suggests that this molecule is mainly conjugated by UGT2B10 and -B17, whereas UGT2B11 (the expression of which is strongly induced upon androgen treatment) is less efficient for glucuronidating this substrate. By contrast, the conjugation of 12-HETE, which is a substrate for UGT2B10, -B11, and -B15, was increased in treated compared with control cells, which may reflect the accumulation of the UGT2B11 protein. Overall, these observations indicate that the inhibitory effect on 13-HODE glucuronidation is a result of the reduction in UGT2B10 and -B17 activity, whereas the relatively small stimulatory effect observed for 12-HETE reflects both the reduction of UGT2B10 and -B15 expression and the induction of the UGT2B11 enzyme. Furthermore, it is interesting to note that UGT2B10 expression, like that of -B15 and -B17, is drastically reduced in the presence of androgens (25). Considering the high nucleotide sequence homology between coding regions of the UGT2B10 and -B11 genes (45) and their similar substrate specificity, which is restricted to hydroxy-fatty acids (10), it could have been anticipated that both enzymes would be regulated in a similar manner, as observed with UGT2B15 and -B17, two highly homologous proteins (30). Therefore, the differential regulation of UGT2B10 and -B11 by androgens indicates that the high sequence homology of these genes may not be extended to the 5'-flanking regions or that minor nucleotide changes in these regions may alter their responding property to androgens. Nevertheless, the results of the present study clearly demonstrate that androgens regulate differently the expression of the four UGT2B genes expressed in LNCaP cells. In addition, the data indicate that UGT2B11 is modulated by a direct action of the androgen receptor in its gene, whereas regulation of UGT2B15 transcripts by androgen necessitated the synthesis of intermediary protein. However, the implication of AR in the regulation of both genes is consistent with their cellular localization into luminal secretory cells of the prostate epithelium, where AR is expressed (20, 46).

Previous studies established that androgens reduce the expression and activity of androgen-conjugating UGT enzymes, i.e. UGT2B15 and -B17, in prostate carcinoma cells (25, 26, 27, 33, 34). Those negative regulatory effects were closely associated with an increased growth and proliferation of prostate cancer LNCaP cells (25). Here, we observe that the androgen-dependent regulation of UGT2B gene expression resulted in a reduced glucuronidation of 13-HODE and in increased production of 12-HETE-glucuronide. Kelavkar et al. (44) recently demonstrated that the 15-LOX-1 metabolite of LA, 13-HODE, is a strong stimulator of prostate cancer cell proliferation. It is therefore tempting to speculate that the reduced glucuronidation of this compound may increase the cellular concentration of unconjugated 13-HODE, thus further enhancing its proliferative activity. In addition, these observations also suggest that reduction of 13-HODE glucuronidation in the presence of AR agonists is part of the complex manner in which androgens stimulate prostate cancer cell proliferation and tumor growth. Interestingly, treatment of LNCaP cells with EGF, which stimulates LNCaP cell proliferation, has no effect on the expression of conjugating UGT2B10 and -B11 enzymes, whereas UGT2B17 expression is markedly inhibited, thus suggesting a diminution in 13-HODE glucuronidation. It is therefore possible that the increase in endogenous 13-HODE levels caused by the EGF treatment may also facilitate the action of EGF on MAPK activity, on peroxisome proliferator-activated receptor-{gamma} phosphorylation, and finally, on cell proliferation as recently demonstrated by Hsi et al. (47). By contrast, the slight increase in UGT2B10 expression observed in the presence of IL-1{alpha} may contribute to the previously reported inhibitory effect of the cytokine on LNCaP cell proliferation. We also observe that R1881-treated LNCaP cells produce higher concentrations of 12-HETE glucuronide. Although this effect was less marked than the reduction of 13-HODE glucuronidation, this would also mean that androgens, by stimulating 12-HETE glucuronidation, reduce the angiogenic and metastatic effects of 12-LOX metabolite (48). IL-1{alpha}, which stimulates the level of UGT2B10 transcript, may also exert the same effect on 12-HETE levels. Whereas the pathophysiological consequences of such findings remain to be fully understood, the present study is the first reporting that the metabolism of hydroxy-fatty acids is under the control of androgens in prostate cells. However, additional analyses on the role of UGT2B enzymes in the control of the biological activity of AA and LA metabolites are required to fully understand the consequences of the androgen-, cytokine-, and growth-factor-dependent regulation of UGTs for the carcinogenicity of these hydroxy-fatty acids.

In summary, the present data establish that treatments of normal and prostate cancer cells with androgens influence the expression of UGT2B enzymes in an isoform-specific manner. These enzymes are implicated in the local inactivation of endogenous substrates, namely androgens and monohydroxy-fatty acids, and this may contribute to alter their intracellular concentrations and actions. Finally, this study reveals for the first time a physiological link between androgens and hydroxy-fatty acids in prostate cells during carcinogenesis.


   Acknowledgments
 
We thank Patrick Caron and David Poisson-Paré for technical assistance with liquid chromatography and immunostaining analysis, respectively. We also thank Dr. Virginie Bocher for the critical reading of this manuscript.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research (CIHR; MOP-64273) and the Fonds de la Recherche en Santé du Québec. S.C. is holder of a scholarship from the Fonds de la Recherche en Santé du Québec. O.B. is granted by the Health Research Foundation of Rx&D-CIHR.

Disclosure statement: The authors have nothing to disclose.

First Published Online August 3, 2006

Abbreviations: AA, Arachidonic acid; AR, androgen receptor; Ct, cycle threshold; DHT, dihydrotestosterone; 3{alpha}-Diol, 5{alpha}-androstane-3{alpha},17ß-diol; EGF, epidermal growth factor; HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid; LA, linoleic acid; LC-MS/MS, liquid chromatography coupled with mass spectrometry; LOX, lipoxygenase; PrEC, prostate epithelial cells; UDPGA, uridine 5'-diphosphoglucuronic acid; UGT, uridine diphosphate-glucuronosyltransferase.

Received February 23, 2006.

Accepted for publication July 17, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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