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Endocrinology Vol. 145, No. 5 2141-2147
Copyright © 2004 by The Endocrine Society

Synergistic Induction of the Nicotinamide Adenine Dinucleotide-Linked 15-Hydroxyprostaglandin Dehydrogenase by an Androgen and Interleukin-6 or Forskolin in Human Prostate Cancer Cells

Min Tong and Hsin-Hsiung Tai

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082

Address all correspondence and requests for reprints to: Hsin-Hsiung Tai, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082.E-mail: htai1{at}uky.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The nicotinamide adenine dinucleotide-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of 15 (S)-hydroxyl group of prostaglandins and lipoxins and participates along with cyclooxygenases and lipoxygenases in controlling the cellular levels of prostaglandins and lipoxins. 15-PGDH could be induced by IL-6 and forskolin in addition to androgens in a time- and dose-dependent manner but not by other cytokines and growth factors in LNCaP cells. Concurrent addition of IL-6 and forskolin showed additive effect in the induction of 15-PGDH activity. However, combined addition of dihydrotestosterone (DHT) and IL-6 or DHT plus forskolin exhibited synergistic induction of 15-PGDH activity. The increase in enzyme activity was correlated with the expression of the enzyme protein as shown by Western blot analysis. The induction by DHT or IL-6 or forskolin or their combinations was inhibited by antiandrogen, casodex, in a dose-dependent manner, indicating that a functional androgen receptor was required for the action of any of these three agents. The induction by forskolin plus DHT or by either agent or by IL-6 alone was greatly inhibited by H-89, indicating the involvement of protein kinase A in the actions of forskolin, DHT, and IL-6. The induction of 15-PGDH by IL-6 was also blocked by some other protein kinase inhibitors, indicating the participation of MAPK, MAPK/ERK kinase, and STAT3 in the signaling pathway of IL-6. These results indicate that the induction of 15-PGDH by DHT, IL-6, and forskolin in LNCaP cells may involve a functional androgen receptor and phosphorylation-dependent multiple signaling pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDINS ARE DERIVED from arachidonic acid through the cyclooxygenase (COX) pathway. Two isoforms of COX have been recognized. COX-1 is expressed constitutively, whereas COX-2 is induced by growth factors, tumor promoters, and proinflammatory cytokines (1). COX catalyzes oxidative cyclization of arachidonic acid to prostaglandin endoperoxide, which is the immediate precursor of prostaglandins, thromoboxane, and prostacyclin (2). These biologically potent autocoids are rapidly metabolized in vivo. The first step of metabolism of prostaglandins is catalyzed by the nicotinamide adenine dinucleotide (NAD+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which produces 15-keto-prostaglandins of greatly reduced biological activities (3). Therefore, this enzyme has been considered the key enzyme responsible for biological inactivation of prostaglandins. Furthermore, this enzyme coupled with COX may regulate the levels of biologically active prostaglandins. In addition to regulating prostaglandin actions and bio-half-life, 15-PGDH also regulates additional eicosanoids that act within immune responses that can impact tumor progression such as lipoxins and aspirin-triggered lipoxins (4, 5). Its role in autocoid metabolism and actions may be broader than previously recognized. The involvement of COX in prostate cancer has been suggested in a number of reports in which a marked overexpression of COX-2 but not COX-1 in tumor tissues was described (6, 7, 8). Furthermore, COX inhibitors such as nonsteroidal antiinflammatory drugs have been shown to exhibit promising chemopreventive and therapeutic effects against prostate cancer in animal models (9) and humans (10). However, the participation of 15-PGDH in prostate tumorigenesis remains to be determined.

Androgens are known to play a critical role in the tumorigenesis and progression of prostate cancer with androgen-regulated gene expression being mediated by the ligand-activated androgen receptor (11). Prostate cancer initially occurs as an androgen-dependent tumor, which can be treated with androgen ablation therapy. However, the cancer eventually recurs as an androgen-independent tumor and becomes resistant to treatment. Thus, the progression from androgen dependence to androgen independence is a critical step in prostate cancer development. The molecular mechanism underlying this critical progression is not clear. One attractive hypothesis is that aberrant activation of the androgen receptor through the signaling pathways independent of androgen may be responsible for the progression of prostate tumors to the rapidly proliferating androgen-independent state. In fact, recruitment of these nonsteroid receptor signaling pathways to activate androgen receptor has been reported to contribute to prostate cancer progression (12, 13). Among these nonsteroid receptor signaling pathways, IL-6 and cAMP have been shown to activate the androgen receptor through Janus kinase (JAK)/signal transducer and activator of transcription (STAT)3 and protein kinase A (PKA) pathways, respectively (14, 15). IL-6 is a pleiotropic cytokine that regulates not only immune and inflammatory responses but also the growth of many tumor cells. The expression of IL-6 and its receptor has been consistently demonstrated not only in human prostate carcinoma but also in benign prostate hyperplasia. Furthermore, serum levels of IL-6 have been shown to elevate in patients with metastatic prostatic carcinoma (16).

Previously, we showed that androgens are able to induce significantly the expression of 15-PGDH in human hormone-responsive LNCaP cells but not in hormone refractory PC-3 cells (17). The induction of 15-PGDH expression can be also achieved partially by progesterone and 17ß-estradiol. In this report, we extend our findings to two other stimulants, IL-6 and forskolin, which also induce the expression of 15-PGDH in LNCaP cells. Furthermore, we discovered that simultaneous addition of an androgen and IL-6 or forskolin stimulated synergistically the induction of 15-PGDH expression. The present regulation by an androgen and IL-6 or forskolin of the 15-PGDH expression may affect the functions and actions of prostaglandins and lipoxins in vivo as they relate to the tumor growth in prostate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Dihydrotestosterone (DHT), dithiothreitol (DTT), bovine liver glutamate dehydrogenase, dexamethasone, forskolin, benzamidine, genistein, leupeptin, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, epidermal growth factor, tyrphostin AG 490, H-89, NAD+, and RPMI 1640 were obtained form Sigma Chemical Co. (St. Louis, MO). GF-109203X, PD-98059, U-0126, and wortmannin were purchased from Alexis Biochemical (San Diego, CA). IL-1ß, IL-4, IL-6, IL-10, basic fibroblast growth factor, nerve growth factor, TGF-1ß, TNF-{alpha}, TNF-ß, and IGF-1 were obtained from Intergen (Burlington, MA). PGE2 was supplied by Cayman Chemical Co. (Ann Arbor, MI). Casodex (bicalutamide) was a kind gift of Dr. David Feldman (Stanford University, Palo Alto, CA). Biotinylated goat antirabbit IgG and streptavidin-horseradish peroxidase conjugate was supplied by the Jackson Immunolaboratory (West Grove, PA). ECL+ plus Western blotting detection system RPN 2132 was obtained form Amersham Pharmacia Biotech (Piscataway, NH). Rabbit antiserum against human placental 15-PGDH was generated as described previously (18). 15(S)-[15-3H]-PGE2 was prepared according to a previously published procedure (19). LNCaP and PC3 cells were obtained from the American Type Culture Collection (Manassas, VA). Other reagents were obtained from the best commercial sources.

Cell culture
LNCaP and PC3 cells were cultured in RPMI 1640 medium containing 10% fetal calf serum, 100 U penicillin per milliliter, and 100 µg streptomycin per milliliter at 37 C in a humidified atmosphere of 5% CO2. The cells were plated in a six-well plate (2 ml per well) at about 2 x 105 cells/ml in duplicate and grown for 36 h before treatment.

Treatment of prostate cancer cells
After LNCaP or PC3 cells were grown for 36 h, cells in each well received treatment with IL-6, DHT, forskolin, or other compounds either alone or in different combinations with other reagents as described in the figure legends.

Preparation of cell homogenate
Prostate cancer cells from the above culture were scraped and spun down at maximal speed in a microfuge for 2 min and washed once with saline. Approximately 1 x 106 cells were suspended in 1 ml of 0.05 M Tris-HCl buffer (pH 7.5) containing 1 mM DTT and sonicated in an ice bath for 3 x 10 sec by an ultrasonic sonicator set at 4. The crude homogenate was used as an enzyme preparation.

Enzyme assay
15-PGDH was routinely assayed by measuring the transfer of tritium from 15(S)-[15-3H]-PGE2 to glutamate by coupling 15-PGDH with glutamate dehydrogenase as described previously (19). Briefly, the reaction mixture contained NH4Cl (5 µmol), {alpha}-oxo-glutarate (1 µmol), NAD+ (1 µmol), 15(S)-[15-3H]-PGE2 (1 nmol, 20,000 cpm), glutamate dehydrogenase (100 µg), DTT (1 µmol), and 15-PGDH enzyme preparation in a final volume of 1 ml of 0.05 M Tris-HCl (pH 7.5). The reaction was allowed to continue for 10 min at 37 C and was terminated by the addition of 0.3 ml of 10% aqueous charcoal suspension. After incubation for 5 min, the mixture was centrifuged at 2000 x g for 5 min. The radioactivity in the supernatant was determined by liquid scintillation counting. Calculation of the amount of PGE2 oxidized was based on the assumption that no kinetic isotope effect was involved in the oxidation of 15(S)-hydroxyl group of 15(S)-[15-3H]-PGE2 as a substrate. Enzyme activity of each sample was always assayed in duplicate.

SDS-PAGE and immunoblot analysis
LNCaP cells either treated or incubated with different stimuli were homogenized in PBS containing 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 50 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, and 1 mM benzamidine by sonication in an ice bath for 3 x 10 sec. Approximately 50–150 µg cellular extract were resolved by SDS-PAGE (10%) gel according to the method of Laemmli (20). Electrophoretic transfer of proteins from the gel to polyvinyl difluoride membrane was performed, and the membrane was blocked with 5% nonfat dry milk in 0.02 M Tris-HCl (pH 7.6) containing 0.8% NaCl and 0.1% Tween 20 (TBST) followed by incubation with a rabbit antiserum against human placental 15-PGDH (1 to 10,000 dilution in TBST with 5% nonfat milk) at room temperature for 1 h. After washing with TBST three times, the membrane was incubated with biotinylated goat antirabbit IgG (1 to 10,000 dilution in TBST with 5% nonfat milk) at room temperature for 1 h. After extensive washing with TBST, the membrane was incubated with streptavidin-horseradish peroxidase conjugate (1 to 2000 dilution) for 1 h. After extensive washing with TBST, the immunoreactive bands were detected with ECL+ Plus Western blotting detection system.

Statistical analysis
Each enzyme sample was performed in duplicate. The data were expressed as the mean ± SE. Statistical significance was assessed by Student’s t test using P < 0.05. Each figure is a representative of two to four replications.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Previously, we reported that DHT, testosterone, and to a lesser extent progesterone and 17ß-estradiol stimulated the induction of 15-PGDH expression in LNCaP cells but not PC-3 cells (17). In search of other nonsteroidal stimulators, we found that IL-6 and forskolin also increased the induction of 15-PGDH expression in LNCaP cells but not in PC-3 cells, whereas other growth factors and cytokines tested were inactive (Fig. 1Go). These observations including the effect of DHT described below were made in medium containing 10% fetal calf serum. Identical findings were made using medium containing 10% charcoal stripped serum, indicating that the fetal calf serum used contained minimal concentrations of androgens, cytokines, and other growth factors. IL-6 stimulated the induction of 15-PGDH activity in a time and dose-dependent manner (Fig. 2Go, A and B)]. The time course of induction by IL-6 indicated that the expression began to show at 5 h after the addition of IL-6 and continued to increase up to 36 h, although it appeared to begin reaching plateau at 24 h. Subsequent experiments were therefore carried out for 24 h of induction. IL-6 at 10 ng/ml stimulated 15-PGDH activity near the maximal level. Similarly, forskolin stimulated the induction of 15-PGDH activity in a dose-dependent manner. Forskolin at 25 µM stimulated 15-PGDH activity near the maximal level (Fig. 3Go). Concurrent addition of IL-6 and forskolin exhibited additive effect in the induction of 15-PGDH activity.



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  		FIG. 1. Effect of forskolin and various growth factors on the induction of 15-PGDH activity in LNCaP cells. LNCaP cells were treated with forskolin (25 µM) or various growth factors or cytokines (10 ng/ml) for 24 h. 15-PGDH activity was assayed as described in Materials and Methods.

 


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  		FIG. 2. Time- and dose-dependent induction of 15-PGDH activity by IL-6. A, LNCaP cells were treated with IL-6 (10 ng/ml) for the indicated amount of time. 15-PGDH activity was assayed as described in Materials and Methods. B, LNCaP cells were treated with IL-6 at the indicated concentrations for 24 h. 15-PGDH activity was assayed as described in Materials and Methods.

 


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  		FIG. 3. Dose-dependent induction of 15-PGDH activity by forskolin in the absence and presence of IL-6. LNCaP cells were treated with forskolin at the indicated concentrations in the absence ({diamondsuit}) or presence ({blacksquare}) of IL-6 (10 ng/ml) for 24 h. 15-PGDH activity was assayed as described in Materials and Methods.

 
DHT (0.1 µM) was shown earlier to stimulate 15-PGDH activity. Further addition of either forskolin (up to 25 µM) or IL-6 (up to 10 ng/ml) exhibited synergistic induction of 15-PGDH activity (Fig. 4AGo). The increases in 15-PGDH activity by DHT (0.1 µM) and IL-6 (10 ng/ml) were correlated with the expression of 15-PGDH enzyme protein as shown by Western blot analysis (Fig. 4BGo). However, there is no increase in immunoreactivity of 15-PGDH by forskolin (data not shown). The induction by DHT was inhibited by a specific antagonist of the classical androgen receptor, casodex (21), in a dose-dependent manner (Fig. 5Go). Casodex at 10 µM inhibited 50–60% of the induction. Similarly, induction by IL-6 alone or in combination with DHT was also inhibited by casodex at 10 µM (Fig. 6Go). The induction by forskolin (25 µM) alone or in combination with DHT was significantly inhibited by casodex at 10 µM (Fig. 7Go). Similar inhibition was observed by using another antagonist, flutamide, at 10 µM (data not shown). Furthermore, the induction by forskolin plus DHT or by either agent alone was greatly inhibited by PKA inhibitor, H-89, at 25 µM. Even the induction by IL-6 alone was also inhibited by H-89.



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  		FIG. 4. Synergistic induction of 15-PGDH expression by DHT and forskolin or DHT and IL-6. A, LNCaP cells were treated with DHT (0.1 µM) in the presence of increasing concentrations of forskolin (upper panel) or IL-6 (lower panel) for 24 h. 15-PGDH activity was assayed as described in Materials and Methods. B, LNCaP cells were treated with IL-6 (10 ng/ml), DHT (0.1 µM), or IL-6 plus DHT for 24 h. Immunoreactivity of 15-PGDH was determined by Western blot as described in Materials and Methods.

 


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  		FIG. 5. Dose-dependent inhibition of DHT-induced 15-PGDH activity by casodex. LNCaP cells were treated with DHT (0.1 µM) in the presence of increasing concentrations of casodex for 24 h. 15-PGDH activity was assayed as described in Materials and Methods.

 


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  		FIG. 6. Effect of antiandrogen casodex on the induction of 15-PGDH activity by IL-6, DHT, and IL-6 plus DHT. LNCaP cells were treated with IL-6 (10 ng/ml), DHT (0.1 µM), or IL-6 plus DHT in the absence or presence of casodex (10 µM) for 24 h. 15-PGDH activity was assayed as described in Materials and Methods.

 


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  		FIG. 7. Effect of PKA inhibitor H-89 on the induction of 15-PGDH activity by forskolin, DHT, and forskolin plus DHT. LNCaP cells were treated with forskolin (FK, 25 µM), DHT (0.1 µM), IL-6 (10 ng/ml), or forskolin plus DHT in the absence or presence of H-89 (25 µM) or casodex (10 µM) for 24 h. 15-PGDH activity was assayed as described in Materials and Methods.

 
To study the signal transduction pathway of IL-6, various protein kinase inhibitors were examined if they affected IL-6-induced 15-PGDH activity. Figure 8Go shows that MAPK kinase (MEK) inhibitor U-0126 at 10 µM, MAPK/ERK inhibitor PD 98059 at 30 µM, JAK inhibitor tyrphostin AG 490 at 30 µM, and tyrosine kinase inhibitor genistein at 50 µM all inhibited almost completely the IL-6-induced 15-PGDH activity. However, protein kinase C inhibitor GF-109203X at 1 µM and phosphatidylinositol 3-kinase inhibitor wortmannin at 100 nM did not significantly inhibit the induction by IL-6. The concentrations selected for these inhibitors are based on their optimal inhibition of the enzyme systems tested in other cell lines.



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  		FIG. 8. Effect of various kinase inhibitors on IL-6-induced 15-PGDH activity. LNCaP cells were incubated with IL-6 (10 ng/ml) in the presence or absence (control) of wortmannin (100 nM), U-0126 (10 µM), AG490 (30 µM), genistein (50 µM), GF-109203X (1 µM), or PD-98059 (30 µM). 15-PGDH activity was assayed as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that both IL-6 and forskolin are capable of stimulating the induction of 15-PGDH expression in a dose- and time-dependent manner in androgen receptor-positive LNCaP cells but not in androgen receptor-negative PC-3 cells. The induction by either agent was blocked by the addition of antagonists of the classical androgen receptor, casodex or flutamide, indicating that the induction was mediated by the classical androgen receptor. IL-6 is known to act as a growth factor for both normal prostatic epithelial cells and LNCaP prostate cancer cells (22). Its action is believed to signal through JAK and the downstream signaling components, STAT3, MEK, and MAPK, in LNCaP cells (14, 23, 24). Both MAPK and STAT3 have been reported to mediate signaling cross-talk between steroid receptor and other signaling pathways. The fact that casodex was able to block IL-6-induced 15-PGDH expression suggests that cross-talk between the androgen receptor and other signaling pathways is hampered by the association of casodex and the androgen receptor. Our finding that the induction of 15-PGDH expression by IL-6 was antagonized by JAK inhibitor, AG 490, and MEK inhibitor, U-0126, is consistent with this signal transduction mechanism. Furthermore, the induction by IL-6 was also blocked by a PKA inhibitor, H-89, indicating that PKA may be involved in the induction of 15-PGDH expression by IL-6. Our finding is in good agreement with other studies in which IL-6 signaling was thought to include the PKA pathway (25). The mechanism of IL-6-induced activation of PKA pathway is not clear. There has been no report showing the direct stimulation of cAMP synthesis by IL-6. However, IL-6 could stimulate cAMP synthesis indirectly through increased synthesis of PGE2 because IL-6 has been shown to induce COX-2 expression in human esophageal cancer cells (26). It is likely that IL-6 may induce PGE2 synthesis through induction of COX-2 expression in LNCaP cells and cause the elevation of cAMP, which activates PKA. IL-6 alone is able to induce 15-PGDH expression and this induction is blocked by casodex indicating that androgen receptor can be activated to mediate IL-6-induced 15-PGDH expression in a ligand-independent manner. Similar conclusion was made with IL-6-induced expression of prostate-specific antigen in LNCaP cells (27).

The induction of 15-PGDH expression by IL-6 was inhibited by inhibitors of MAPK, PKA, and JAK. The interrelationship among MAPK, PKA, and JAK pathways in IL-6-induced 15-PGDH expression remains to be elucidated. However, the interrelationship between these pathways in the IL-6-induced prostate-specific antigen gene expression has been proposed (24). IL-6 appears to induce ligand-independent activation of the androgen receptor through the N-terminal domain (24, 25). Activation of the androgen receptor by IL-6 was found to be mediated by the PKA pathway (15, 25, 27) as well as the MAPK and JAK/STAT3 pathways (23, 24). Activation of the IL-6 receptor leads to the stimulation of JAK, which catalyzes the phosphorylation of STAT3 at Tyr-706. Concurrently, activation of the IL-6 receptor also induces stimulation of MEK and subsequently MAPK, which catalyzes the phosphorylation of STAT3 at Ser-727. Activation of PKA may also lead to the phosphorylation of MEK, which in turn activates MAPK. Both fully activated STAT3 and MAPK can turn on the androgen receptor, which binds to the regulatory site of prostate-specific antigen gene and induces the expression of prostate-specific antigen. Our findings that these kinase inhibitors inhibit the induction of 15-PGDH by IL-6 indicate that these kinase pathways also can be involved in the IL-6-induced 15-PGDH expression. Further experimental support of this mechanistic model using 15-PGDH promoter-luciferase construct as an exploratory system is currently in progress.

Forskolin, an activator of adenylate cyclase, is known to elevate intracellular level of cAMP and activate PKA (28). The induction by forskolin was totally inhibited by H-89, supporting the contention that PKA mediates the induction by forskolin. Forskolin-induced 15-PGDH activity was also shown to be fully blocked by casodex, suggesting that the induction by forskolin was dependent on a functional androgen receptor. The induction by forskolin is very similar to that by IL-6. The situation is analogous to the induction of prostate-specific antigen by either agent in LNCaP cells (27). In this study, forskolin was shown to activate androgen receptor in the absence of androgen when androgen receptor is cotransfected into PC-3 cells with androgen-responsive reporters (27). Western blotting revealed no significant change in receptor level after forskolin treatment, suggesting that the enhanced activity was due to activation of the receptor, presumably by PKA-mediated phosphorylation of the receptor or its associated proteins. Our finding that H-89 and casodex were able to block forskolin-induced 15-PGDH expression is consistent with the hypothesis that forskolin-induced phosphorylation of the receptor or its associated proteins is responsible for the activation of the receptor.

The molecular mechanisms for the synergistic stimulation of 15-PGDH expression by IL-6 and DHT and by forskolin and DHT are not clear. It has been recognized that glucocorticoids synergize the IL-6 response (29, 30). Examination of the synergistic relationship between glucocorticoids and IL-6 signaling identified a novel function for STAT3. Zhang et al. (31) have shown that IL-6 activated STAT3 interacts with ligand bound glucocorticoid receptor to augment glucocorticoid signaling by acting as a potent coactivator of the glucocorticoid receptor. Recently several groups have also shown that STAT3 can also mediate IL-6-induced activation of the androgen receptor by functioning as a coactivator of the receptor even in the absence of the receptor (14, 23, 32). Furthermore, IL-6 was shown to induce the phosphorylation of both MAPK and STAT3 (24). It is possible that MAPK and STAT3 may cross-talk productively to facilitate the role of STAT3 as a coactivator. The ligand-independent activation of the androgen receptor by IL-6 was found to be inhibited by antiandrogens as shown in this study as well as in other studies (24). The androgen-dependent activation of the androgen receptor was also shown to be sensitive to antiandrogens as indicated in this study. Previous failure to demonstrate the inhibition of the induction by antiandrogens is due to the relatively low levels of the antagonists used (17). Apparently receptor activations by an androgen and by IL-6 or forskolin are separate events, but the signaling molecules derived from the separate pathways may interact productively to induce the expression of 15-PGDH. Previously studies have shown that IL-6 induces activation of STAT3 (24, 25, 27). Activation of STAT3 may augment potently the androgen bound receptor signaling and contribute to synergistic induction of 15-PGDH expression by combined treatments with IL-6 and DHT. Furthermore, IL-6 was shown to induce androgen receptor expression in addition to increasing androgen receptor activity (33). Increased androgen receptor expression by IL-6 may in part account for the synergism in 15-PGDH expression by IL-6 and DHT.

Similarly, forskolin and DHT were also found to act synergistically in inducing the expression of 15-PGDH. Induction by forskolin was sensitive to casodex indicating that the ligand-independent and forskolin-stimulated pathway is an androgen receptor-mediated event. Forskolin was shown to induce the phosphorylation of MAPK (24) by which phosphorylation of the androgen receptor or its associated proteins may occur. In the presence of an androgen, phosphorylated receptor, or its associated proteins may be more active leading to the synergistic induction of 15-PGDH expression. Synergistic induction of the expression of prostate-specific antigen by forskolin and an androgen was also observed in LNCaP cells (23). Furthermore, synergistic stimulation of the expression of prostate-specific antigen by forskolin and IL-6 was also demonstrated in the same cells (24). However, an additive induction of 15-PGDH expression by forskolin and IL-6 was found in the current study. The difference in synergism of the expression between 15-PGDH and prostate-specific antigen by forskolin and IL-6 is not clear.

The implications of our findings for alternate mechanisms in activating androgen receptor and enhancing the expression of 15-PGDH may be important in understanding the progression of prostate cancer. The activation of androgen receptor by factors other than androgens has been well documented (34). Although forskolin and a variety of cytokines and growth factors have been shown to activate the androgen receptor signaling pathway in the absence of androgens, IL-6 is the only cytokine that we discovered so far capable of activating the androgen receptor and stimulating the expression of 15-PGDH in LNCaP cells. This cytokine coupled with an androgen can synergistically stimulate the expression of 15-PGDH. Overexpression of COX-2 has been observed in prostate tumors (6, 7, 8). Increased level of PGE2 may stimulate production of IL-6 and enhance soluble IL-6 receptor release, gp130 dimerization, and STAT3 phosphorylation, leading to the cell proliferation (35, 36). Increased expression of 15-PGDH may function to facilitate the metabolism and inactivation of prostaglandins that are overly produced by tumor cells as a defensive regulatory mechanism. When the induction of the expression of 15-PGDH is impaired, overproduction of prostaglandins, particularly PGE2, may lead to the unconstrained growth of the tumor cells. Prostate cancer cells that overexpress COX-2 and are unresponsive to androgens or IL-6 or other growth factors in inducing 15-PGDH expression may therefore lead to the promotion and growth of the tumors. Alternatively, increased expression of 15-PGDH may also function to inactivate lipoxins and aspirin-triggered 15-epi-lipoxins because these lipoxins are excellent substrates of 15-PGDH (5, 37). These lipoxins have also been shown to be potent inhibitors of cell proliferation (38). The consequence is that facilitated catabolism of these antiproliferative eicosanoids may enhance tumorigenesis.


   Footnotes
 
This work was supported by National Institutes of Heath Grant HL-46296.

Abbreviations: COX, Cyclooxygenase; DHT, dihydrotestosterone; DTT, dithiothreitol; EGF, epidermal growth factor; JAK, Janus kinase; MEK, MAPK/ERK kinase; NAD+, nicotinamide adenine dinucleotide; 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; PKA, protein kinase A; STAT, signal transducers and activators of transcription; TBST, Tris-HCl containing NaCl and 0.1% Tween 20.

Received September 22, 2003.

Accepted for publication January 14, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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