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Endocrinology Vol. 139, No. 5 2375-2381
Copyright © 1998 by The Endocrine Society

ARTICLES

Effect of Interleukins on UGT2B15 and UGT2B17 Steroid Uridine Diphosphate-Glucuronosyltransferase Expression and Activity in the LNCaP Cell Line1

Éric Lévesque2,3, Martin Beaulieu3, Chantal Guillemette3, Dean W. Hum and Alain Bélanger

Medical Research Council Group in Molecular Endocrinology, CHUL Research Center and Laval University, Québec, Canada G1V 4G2

Address all correspondence and requests for reprints to: Dr. Alain Bélanger or Dr. Dean Hum, Laboratory of Molecular Endocrinology CHUL Research Center, 2705 Laurier Boulevard, Québec, Canada G1V 4G2. E-mail: alain.belanger@crchul.ulaval.ca; or Dean.Hum{at}crchul.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cytokines are known to modulate the level of both phase 1 and phase 2 drug-metabolizing enzymes in hepatocytes. Although the effects of cytokines on cytochrome P450 (CYP450) enzymes are well understood, there is limited knowledge on how cytokines may affect steroid UDP-glucuronosyltransferase (UGT) phase 2 enzyme activity and expression in different cell types, including hepatocytes and steroid target cells. LNCaP cells, which is a human prostate cancer cell line, is a good model to study the effect of cytokines in steroid target cells because it is known to express steroidogenic enzymes, including UGT2B15 and UGT2B17, which are widely expressed steroid UGT enzymes known to conjugate androgens. In this study, we examined the possible interaction among interleukin-1{alpha} (IL-1{alpha}), IL-4, IL-6, and steroid UGT enzymes (UGT2B15 and UGT2B17). Treatment of LNCaP cells with IL-1{alpha} led to a dose-dependent inhibition of dihydrotestosterone (DHT) glucuronidation. IL-1{alpha} decreased both UGT activity and LNCaP cell proliferation in the absence and presence of DHT (0.5 nM); a maximal inhibition of 70% was observed. IL-6 inhibited LNCaP cell proliferation as well as the DHT-induced proliferation of these cells. However, neither IL-4 nor IL-6 significantly affected the formation of DHT glucuronide. Ribonuclease protection and Western blot analyses demonstrated a specific reduction of UGT2B17 transcript and protein levels in IL-1{alpha}-treated LNCaP cells. The level of UGT2B15 was not affected by cytokine treatments, indicating a differential regulation between these two UGT enzymes. Transfection experiments performed with the UGT2B17 gene promoter region indicates that the regulation occurs at the transcription level via putative cis-acting elements. This study indicates that cell proliferation and UGT expression in steroid-responsive cancer cells are differentially regulated depending on the cytokines present in the cell microenvironment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IMPAIRMENT of drug metabolism during inflammation and infection is associated with a decrease in hepatic activity and expression of phase 1 and phase 2 metabolic enzymes (1, 2, 3, 4, 5, 6). Cytochrome P-450 monooxygenases, a superfamily of phase 1 enzymes, are suppressed in hepatocytes by proinflammatory cytokines, leading to a decrease in drug metabolism (1, 2, 4, 5, 7). Recent studies have demonstrated that IL-1 and IL-6, two proinflammatory cytokines, are involved in the down-regulation of CYP450 enzyme activities and expression (1, 2, 8, 9). These modulations of CYP450 enzymes by cytokines were reported to occur at different levels, including specific regulation of transcription (1, 7, 10, 11). The phase 2 metabolizing UDP-glucuronosyltransferase (UGT) enzymes are also affected by cytokines, as UGT activities on morphine, 1-naphtol, and paracetamol were decreased in pig hepatocytes after IL-1 and IL-6 treatments (2). Although regulation of CYP450 enzymes has been extensively described, there is limited knowledge about how cytokines affect UGT2B enzymes.

UGT enzymes represent a family of microsomal enzymes that catalyze the transfer of glucuronic acid from uridine 5'-diphosphoglucuronic acid to endogenous and exogenous molecules with functional groups of oxygen, nitrogen, sulfur, or carbon (12). Glucuronidation of aglycone substrates renders the molecules more polar, generally water soluble, less toxic, and more easily excreted from the body (12, 13, 14, 15). Based on evolutionary divergence, UGT enzymes have been classified into two families, UGT1 and UGT2; the latter was further subdivided into subfamilies 2A and 2B (16). The UGT2B subfamily is implicated in the conjugation of bile acids, phenolic drugs, carcinogens, and particularly steroid hormones (16). Glucuronidation of a steroid molecule is believed to prevent its interaction with its nuclear receptor and favor its elimination from the steroid target cells (17, 18). In humans, high levels of glucuronidated dihydrotestosterone (DHT-G) metabolites, including androsterone glucuronide and androstane-3{alpha},17ß-diol glucuronide, are present in the circulation (19, 20). As UGT2B enzymes are widely expressed in extrahepatic tissues (18, 21, 22, 23), it has been proposed that glucuronidation inactivates steroid hormones in steroid target tissues, including breast and prostate.

Androgens are known to have important effects on the prostate; however, recent findings also indicate that complex paracrine interactions between epithelial and stromal cells regulate normal and pathological development of the prostate (24, 25, 26). The prostate produces factors, including peptide growth factors and interleukins (ILs), that are capable of enhancing or inhibiting cellular proliferation of the prostate and altering its function (25, 27, 28). ILs are also potent regulators of steroidogenic enzymes. A series of cytokines, such as interleukin-1{alpha} (IL-1{alpha}), IL-2, IL-4, IL-6, and IL-13, has been shown to modulate 17ß-hydroxysteroid dehydrogenase, 3ß-hydroxysteroid dehydrogenase, and CYP450 aromatase activities (29, 30, 31). It is also well known that steroids can modulate IL secretion (32, 33), indicating close interactions between the endocrine and immune systems. A wide range of cancer cell lines, including human prostate cancer LNCaP cells, secrete cytokines and express cytokine receptors (26, 34, 35, 36, 37), suggesting that these physiological regulators may alter the proliferation of different cancer cells (26, 34, 38, 39).

We have demonstrated that androgens and growth factors induce a marked decrease in the glucuronidation of 5{alpha}-reduced C19 steroids in LNCaP cells, which appear to occur via their respective receptors (17, 40, 41). Human prostate and LNCaP cells express both UGT2B15 and UGT2B17, which are two widely distributed UGT isoforms implicated in 5{alpha}-reduced C19 steroid glucuronidation (18, 22, 23). However, it was demonstrated that only UGT2B17 transcript and protein levels are down-regulated by these factors, correlating with the decrease in androgen conjugation (17, 41). Transfection experiments using the 5'-flanking regulatory region of the UGT2B17 gene revealed that the regulation of UGT2B17 expression by these factors occurs at least partially at the transcriptional level, as observed with several CYP450 genes (1, 11, 42).

The purpose of the present study was to investigate the effects of IL-1{alpha}, IL-4, and IL-6 on steroid UGT2B enzyme activity and expression in the LNCaP cell line. We demonstrate that both IL-1{alpha} and IL-6 inhibit cell proliferation of LNCaP cells. However, only IL-1{alpha} was able to reduce the formation of DHT-G by specifically decreasing UGT2B17 transcript and protein levels. Results obtained with the UGT2B17 gene promoter demonstrate that this regulation occurs at the transcriptional level and that cis-acting elements are implicated in suppression of transcription. The regulation of steroid UGT expression by cytokines is potentially an important pathway by which these factors can alter the level of DHT in the prostate and thus influence the growth of hormone-dependent cancers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
All media and supplements for cell culture, IL-1{alpha}, and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma Chemical Co. (St. Louis, MO). FBS was purchased from Immunocorp (Quebec, Canada). IL-4 was obtained from Schering-Plough (Kenilworth, NJ), and IL-6 was purchased from Boehringer Mannheim (Quebec, Canada). IL-1 receptor antagonist (IL-1ra) was obtained from R&D Systems (Minneapolis, MN). [1,2-3H]DHT (47 Ci/mmol) and {alpha}-[32P]deoxy-UTP (3000 Ci/mmol) were purchased from Amersham (Ontario, Canada). Unlabeled DHT was obtained from Steraloids (Wilton, NH). Protein assay reagents were purchased from Bio-Rad (Richmond, CA). Restriction enzymes and other molecular biology reagents were obtained from Pharmacia LKB Biotechnology (Milwaukee, WI), Life Technologies (Ontario, Canada), Stratagene (La Jolla, CA), Boehringer Mannheim (Indianapolis, IN), and Promega (Madison, WI). Lipofectin and Opti-MEM medium were obtained from Life Technologies (Ontario, Canada). Luciferase reagents were purchased from Promega. The anti-UGT2B17 antibodies and all UGT2B17 gene promoter constructs were obtained as previously described (17, 42).

Cell experiments
The LNCaP cell line was obtained from American Type Culture Collection (Rockville, MD) at passage 21 and was used between passages 22–29. The cells were routinely maintained as previously described (43). Fresh medium containing the indicated concentrations of ILs was added to cells every 2 days in presence or absence of 1 µM IL-1ra. At the end of the experiment, cells were washed with fresh medium, and labeled DHT (10 nM), previously dissolved in the medium, was added for 3 h. The medium was removed, and the measurement of glucuronide formation was performed as previously described (17, 43). Methanol was added to the cells, and plates were left to dry at room temperature in the absence of light. DNA content was quantitated by fluorometric assay with 3,5-diaminobenzoic acid (44). Steroid glucuronide analysis, dose-response curves, IC50 determinations, and analysis of statistical significance were performed as previously described (43, 45, 46).

Northern blot analysis
Total RNA was isolated by the Tri-Reagent acid-phenol method (Molecular Research Center, Inc., Cincinnati, OH). Ten micrograms of total RNA were separated on a 1% agarose gel and transferred to a Nylon-N membrane (Amersham, Ontario, Canada) using 10 x SSC (standard saline citrate). Northern blot conditions were previously described (17). A full-length UGT2B15 complementary DNA (cDNA), radiolabeled by the random hexamer primer technique in the presence of [{alpha}-32P]deoxy-CTP, was used as probe (17).

Ribonuclease (RNase) protection assays
To generate a probe specific for UGT2B17, the pBK-CMV-UGT2B17 construct was linearized by EcoRI digestion, and a radiolabeled complementary RNA (cRNA) probe of 318 bases, from nucleotides 1394–1629, including 83 bases from the vector, was generated using the T7 RNA polymerase and [{alpha}-32P]UTP as described in the instructions provided with the MAXIscript kit (Ambion, Austin, TX). The probe specific for UGT2B15 was generated as previously described (21). For all RNase protection assays, 25 µg total RNA were hybridized with 200,000 cpm of the appropriate cRNA probe for 16 h at 42 C. cRNA-RNA hybrids were digested with 0.5 U RNase A and 20.0 U RNase T1 for 30 min at 37 C, and the protected products were analyzed on a 7 M urea-6% polyacrylamide gel. The amount of protected probe corresponding to the bands on the gel was quantitated by phosphorimaging (Molecular Dynamics, Sunnyvale, CA). The quantity of RNA was normalized using a 137-bp 18S probe and the protected fragments of 110 bp in each RNA preparation.

Immunoblot analysis
Microsomes were purified as previously described (17, 23, 47). Microsomal proteins isolated from human liver, prostate, and untreated and IL-1{alpha} treated LNCaP cells were separated on a 12% SDS-PAGE gel. The gel was transferred onto a nitrocellulose filter and probed with a 1:2000 dilution of rabbit antiserum EL-95, a polyclonal antibody directed against the recombinant UGT2B17 fusion protein (17). Antirabbit IgG horseradish peroxidase conjugates (Amersham) were used as secondary antibodies, and the recognized proteins were visualized using enhanced chemiluminescence (Renaissance, Quebec, Canada) and exposed on Hyperfilm for 30 min (Eastman Kodak, Rochester, NY).

Transfection experiments using the UGT2B17 gene promoter
All cells were grown in six-well plates for 5 days before transfection in 5% charcoal-absorbed FBS in RPMI 1640 medium (40, 42). Transfection experiments were carried out with 3 nmol plasmid DNA, using Lipofectin according to the manufacturer’s instructions (Life Technologies, Ontario, Canada). After transfections, cells were treated for 48 h with 10 ng/ml IL-1{alpha} or 10 nM PMA. Cells were washed with 1 ml Tris-buffered saline (48) and harvested in 200 µl of a cell lysis buffer (42). Fifteen microliters of cell lysate were used for luciferase assays according to the manufacturer’s instructions (Promega), using a luminometer (Lumat LB 9501, Berthold). One hundred nanograms of cytomegalovirus-ß-galactosidase plasmid were used to normalize transfection efficiency as previously reported (42). All final values are the means of three independent experiments performed in duplicate using two different preparations of plasmid DNA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of ILs on UGT activity and cell growth
To determine whether ILs can affect cell proliferation and UGT activity in steroid target cells, we incubated LNCaP cells with IL-1{alpha}, IL-4, and IL-6. Increasing concentrations of IL-1{alpha} inhibited the proliferation of LNCaP cells in a dose-dependent manner in both the presence and absence of DHT. However, the ability of DHT to stimulate cell proliferation was retained at the highest concentration of IL-1{alpha} used (Fig. 1AGo). Treatment of LNCaP cells with IL-1{alpha} inhibited the glucuronidation of DHT with an IC50 of 0,57 nM (Fig. 1BGo). As previously shown, DHT inhibited glucuronidation in these cells; however, the addition of IL-1{alpha} caused a further inhibition. To demonstrate the specificity of the IL-1{alpha} interaction with its receptor, we incubated LNCaP cells with increasing amounts of IL-1{alpha} (0.1–10 ng/ml) in the presence of IL-1ra (1 µM). IL-1ra reversed the effect of IL-1{alpha} on cell growth and steroid glucuronidation. The antagonist inhibited the effect of IL-1{alpha}, and a higher concentration of the cytokine was required to obtain the same inhibition of androgen glucuronidation in LNCaP cells (Fig. 2Go). Treatment of LNCaP cells with IL-4 had no effect on cell proliferation or UGT activity in the presence or absence of DHT (Fig. 3Go, A and C). IL-6 inhibited cell growth and led to a decrease in DHT-induced cell proliferation (Fig. 3BGo). However, IL-6 did not affect the formation of DHT-G (Fig. 3DGo).



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  		Figure 1. Effects of IL-1{alpha} on LNCaP cell proliferation and DHT glucuronidation. The proliferation (A) and UGT activity (B) of human prostatic cancer LNCaP cells were determined after continuous exposure (6 days) to increasing concentrations of IL-1{alpha} (1–10 ng/ml) in the presence and absence of DHT (0.5 nM). Cells were initially plated at a density of 45,000 cells/well. DNA content was measured after determination of DHT-G formation, as described in Materials and Methods. The data are the mean ± SEM from three separate experiments, each consisting of triplicate determinations. **, P <= 0.01; *, P <= 0.001 (treatments vs. control without IL-1{alpha}).

 


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  		Figure 2. Effect of IL-1ra on IL-1{alpha}-treated LNCaP cells. The glucuronidation of DHT in LNCaP cells was determined after continuous exposure (6 days) to increasing concentrations of IL-1{alpha} (0.1–10 ng/ml) in the presence or absence of IL-1ra (1 µM). Cells were initially plated at a density of 45,000 cells/well. Values represent the mean of two independent experiments performed in triplicate.

 


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  		Figure 3. Effects of IL-4 and IL-6 on LNCaP cell proliferation and UGT activity. The proliferation (A) and level of DHT glucuronidation (B) in human prostatic cancer LNCaP cells were determined after continuous exposure (6 days) to increasing concentrations of IL-4 (0.5–25 ng/ml; A and C) or IL-6 (10–150 ng/ml; B and D). Cells were initially plated at a density of 45,000 cells/well, and triplicate wells were used per treatment. DNA content was measured after determination of DHT-G formation, as described in Materials and Methods. The data represent the mean ± SEM from two separate experiments, each performed in triplicate. *, P <= 0.001, treatments vs. control without ILs. Empty and bold columns represent results obtained in the absence or presence of DHT (0.5 nM), respectively.

 
Effect of IL-1{alpha} on the expression of UGT2B15 and UGT2B17
Having shown that IL-1{alpha} can inhibit the glucuronidation of DHT in LNCaP cells, we determined whether this effect is mediated by changes in the level of UGT transcripts. Northern blot analysis of LNCaP cell messenger RNA probed with the UGT2B15 cDNA showed that the level of UGT2B transcript was decreased after IL-1{alpha} treatment (5 ng/ml; Fig. 4Go). Due to the high homology among the different UGT2B cDNA clones that have been characterized to date, it is probable that the UGT2B15 cDNA probe can hybridize to several UGT2B transcripts, including UGT2B15 and UGT2B17, on a Northern blot. Knowing that the nucleotide sequences of UGT2B15 and UGT2B17 are 97% identical and that both proteins can conjugate DHT in LNCaP cells (17, 18, 21), we performed RNase protection analyses to distinguish between the two transcripts and to specifically measure any changes in expression level. The level of UGT2B15 transcript was not affected by IL treatment (data not shown); however, the level of UGT2B17 transcript was decreased by 83% in cells treated with IL-1{alpha} (10 ng/ml; Fig. 5Go). To determine whether the decrease in UGT2B17 transcript in response to IL-1{alpha} is reflected by the level of protein expression, microsomes from untreated and IL-1{alpha}-treated LNCaP cells were isolated, and immunoblot analyses were performed using the EL-95 polyclonal anti-UGT2B17 antibody (17). IL-1{alpha} decreased the level of UGT2B17 protein by 75%, as shown in Fig. 6Go.



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  		Figure 4. Northern blot analysis of UGT2B transcripts in LNCaP cells treated with IL-1{alpha}. Ten micrograms of total RNA were isolated from control (untreated) or IL-1{alpha}-treated LNCaP cells and separated on a 1% agarose gel. The blot was hybridized with a full-length UGT2B15 cDNA probe. Cells were treated for 6 days with IL-1{alpha} (5 ng/ml). The quantity of RNA was normalized with a GAPDH cDNA as probe (bottom).

 


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  		Figure 5. RNase protection analyses of UGT2B17 transcript in LNCaP cells treated with IL-1{alpha}, IL-4, and IL-6. Twenty-five micrograms of total RNA isolated from untreated and treated LNCaP cells were hybridized to a UGT2B17 cRNA probe. The UGT2B17 probe of 318 bp protected a fragment of appropriately 224 bp. The integrity of the RNA samples was assessed using a 137-bp 18S cRNA probe and protected a fragment of 110 bp in each RNA preparation. The sizes of the probe and protected fragments are indicated on the left. The sequence shown on the right was used to confirm the sizes of the cRNA probes and protected fragments. All samples were separated on a denaturing 6% polyacrylamide gel.

 


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  		Figure 6. Immunoblot analysis of UGT2B17 protein after treatment of LNCaP cells with IL-1{alpha}. Microsomal proteins from human liver (2 µg), prostate (10 µg), untreated LNCaP cells, and IL-1{alpha}-treated LNCaP cells (10 µg) were separated on a 12% SDS-PAGE gel and transferred to a nitrocellulose filter for analysis with the anti-UGT2B17 antibody. Cells were treated as described in Materials and Methods.

 
Effect of IL-1{alpha} on the UGT2B17 gene promoter
As IL-1{alpha} diminished the level of UGT2B17 transcript and protein, the ability of IL-1{alpha} to affect expression of the UGT2B17 gene conferred by the 5'-flanking region was assessed. Treatment of transfected LNCaP cells with IL-1{alpha} (10 ng/ml) significantly decreased the luciferase activity of the -2942B17/Luc reporter construct. Progressive 5'-deletions of the 5'-flanking region of the UGT2B17 gene did not show a loss of inhibition in response to IL-1{alpha} (Fig. 7Go). As IL-1{alpha} has been proposed to regulated gene expression via activation of the Raf-1 kinase [mitogen-activated protein (MAP) kinase pathway], transfected cells were also treated with phorbol ester (PMA), which activates the protein kinase C pathway and, subsequently, Raf-1. PMA (10 nM) also inhibited transcription conferred by the 5'-flanking region of the UGT2B17 gene. Treatment of the longest construct (-2942/B17Luc) with IL-1{alpha} or PMA conferred maximal inhibitions of 85% and 90% on transcriptional activity. Treatment of the shortest construct (-267/B17Luc) with IL-1{alpha} decreased the luciferase activity by 70%, whereas no significant effect was observed with PMA. Incubation of transfected LNCaP cells with IL-4 and IL-6 (-2942/B17Luc) did not affect the basal transcriptional activity of the reporter constructs (data not shown).



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  		Figure 7. Effect of IL-1{alpha} and PMA on UGT2B17 gene promoter activity. Equimolar amounts of each reporter construct were transiently transfected into LNCaP cells in the presence or absence of IL-1{alpha} (10 ng/ml) and PMA (10 nM). After 48 h of incubation, the cells were harvested, and luciferase activity was determined. Each value represents the mean ± SEM of three independent experiments, each performed in duplicate. All experiments were normalized with cotransfection of the cytomegalovirus-ß-galactosidase vector.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To establish whether specific steroid UGT enzymes can be modulated in response to cytokines, as is the case for CYP450 enzymes, we treated LNCaP cells with IL-1{alpha}, IL-4, and IL-6. The proinflammatory cytokines IL-1{alpha} and IL-6 were able to inhibit the proliferation of LNCaP cells, in contrast to treatment with IL-4, an antiinflammatory cytokine, which had no effect on cell proliferation. These results are in agreement with previous observations indicating that IL-1{alpha} and IL-6 can inhibit LNCaP cell proliferation (26, 35, 49). Treatment of LNCaP cells with IL-1{alpha} in the presence of DHT increased the dose at which the inhibitory effect of IL-1{alpha} on glucuronidation activity is observed (2-fold increase in IC50 value). However, the maximal inhibition of glucuronidation is the same in the presence or absence of DHT. The variation in the IC50 value may be explained by the possibility that the effects of IL-1{alpha} and DHT involve a common factor. As androgens can induce I{kappa}B expression in LNCaP cells, and IL-1{alpha} can activate the nuclear factor-{kappa}B (NF-{kappa}B) pathway, it is possible that one pathway may interfere with the other, thus increasing the IC50 of the effect of IL-1{alpha} on androgen glucuronidation activity (50).

The inhibition of DHT glucuronide formation by IL-1{alpha} was specifically mediated by the IL-1 receptor, as the antagonist IL-1ra, which is known to compete with IL-1{alpha} for receptor binding (51), prevents the inhibitory effect of IL-1{alpha} on glucuronide formation in LNCaP cells. However, no inhibition of androgen glucuronidation activity was observed when LNCaP cells were treated with IL-4 and IL-6, which was surprising because IL-6 is known to modulate CYP450 enzyme activities and expression as well as decrease UGT activity on morphine, paracetamol, and 1-naphtol in pig hepatocytes (1, 2, 52). IL-6, however, was able to modulate LNCaP cell proliferation; therefore, the results obtained with IL-6 clearly indicate that the modulation of UGT activity can be dissociated from and is not dependent on the modulation of LNCaP cell proliferation. The absence of an effect on androgen glucuronidation by IL-4, which acts via the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway (53), correlates with the absence of potential STAT-responsive consensus sequences in the UGT2B17 gene promoter.

Treatment of LNCaP cells with IL-1{alpha} decreases the level of UGT2B transcript. UGT2B15 and UGT2B17 are both expressed in LNCaP cells and are able to conjugate androgens; however, only the level of UGT2B17 was inhibited by IL-1{alpha}. As expected, the effect of IL-1{alpha} was observed at the protein level, where a 75% decrease of UGT2B17 was observed. Transfections of LNCaP cells with reporter constructs under the control of the 5'-flanking region of the UGT2B17 gene (42) revealed that IL-1{alpha} represses transcriptional activity. Maximal inhibition (90%) was obtained with the -2942/B17Luc construct, whereas a 70% inhibition was still present with the shortest -267/B17Luc construct. This inhibitory effect of IL-1{alpha} observed with the minimal promoter -267/B17Luc suggests that active cis-acting elements are found in the proximal promoter region of the UGT2B17 gene from nucleotides -267 to -1, as previously observed for the CYP450 2C11 gene (1). The response to IL-1{alpha} may be conferred by several of the putative cis-acting elements found in the 5'-flanking region of the UGT2B17 gene, which contain five potential activating protein-1 (AP-1)-like binding sites, five Rel/NF-{kappa}B-like binding sites, and seven C/EBP-like binding sites, all of which can be activated by the IL-1{alpha} transduction pathway (54). Transfection of 5'-deletion constructs of the UGT2B17 gene promoter showed a progressive loss of inhibition in response to PMA, and no significant effect was observed with the -267B17/Luc construct. The response to PMA may be conferred by the potential cis-acting elements found in the UGT2B17 gene, as it is known to activate protein kinase C, the MAP kinase pathway, and NF-{kappa}B (55, 56, 57). The absence of inhibition by PMA of the shortest construct suggests that different cis-acting elements and trans-acting factors are implicated in the proximal promoter region of the UGT2B17 gene in response to IL-1{alpha} and PMA.

Growth factors acting through an intrinsic tyrosine kinase receptor, such as the epidermal growth factor and the fibroblast growth factor, down-regulate UGT2B17 gene transcription, probably via putative AP-1-binding sites (17, 41, 42). Activation of MAP kinases by PMA or IL-1{alpha} can activate c-fos and c-jun (54, 55). AP-1 may bind to negative regulatory elements in the UGT2B17 gene to decrease its expression, as has been shown to be the case for the c-myc gene (58). As AP-1, C/EBP, and NF-{kappa}B transcription factors are known to interact with each other (59, 60, 61, 62, 63), further studies are required to identify trans-acting factors involved and to determine the mechanism by which the UGT2B17 gene is regulated by growth factors, cytokines, androgens, and phorbol esters.

The ILs investigated in the present study exert their biological effects through specific receptors, signal transducers, and transcription factors, which may explain their differential effects on cell proliferation and UGT expression in LNCaP cells (53, 54, 55). This in vitro study did not establish whether the effects of IL-1{alpha} on LNCaP cell proliferation are direct or indirect, considering that IL-1{alpha} induces the synthesis of other cytokines, including IL-6, which, in turn, may affect cell proliferation. However, IL-1{alpha} was demonstrated to be responsible for the repression of the UGT2B17 gene promoter.

In conclusion, an important finding from the present study is that UGT2B17 enzyme activity and expression can be regulated at the level of transcription by cytokines and phorbol esters. It is clear that several factors involved in the regulation of inflammation, infection, and tumor proliferation can regulate UGT enzymes. UGT2B17 is widely distributed in human tissues, where the modification of cytokine secretion or the expression of cytokine receptors will affect steroid conjugation by UGT2B17. Our data suggest that androgen metabolism in steroid target tissues is controlled by several factors, including cytokines.


   Acknowledgments
 
We thank Dr. Pei min Rong for technical assistance with immunoblot analysis.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada, the Fonds de la Recherche en Santé du Québec, and Endorecherche. Back

2 Recipient of a scholarship from the Medical Research Council of Canada. Back

3 These authors contributed equally. Back

Received October 8, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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