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Androgens via the androgen receptor (AR) play crucial roles in prostate physiology and pathophysiology. These androgen actions can be either inhibited or potentiated by estrogens. The mechanisms of these seemingly opposing estrogen effects are unclear. We studied the effects of estrogens on the modulation of androgen induction of prostate specific antigen (PSA) gene expression and prostate tumor cell growth. Cotransfection analyses in CV-1, DU-145, and PC-3 cells showed that dihydrotestosterone (DHT)-induced PSA transcription activity was inhibited by 17β-estradiol, diethylstilbestrol, ICI182780, and 17α-estradiol, but not by tamoxifen via estrogen receptor α (ERα). In the presence of ERβ, 17β-estradiol and diethylstilbestrol had no significant effect, while 17α-estradiol inhibited and ICI182780 and tamoxifen potentiated DHT action. When both ERα and ERβ were present, all ER-ligands except tamoxifen inhibited DHT action. The inhibition of DHT action by 17β-estradiol via ERα was mainly dependent on the DNA binding domain, while the 17α-estradiol effect was mainly dependent on the ERα carboxyl terminus. Treatment with DHT in LAPC-4 prostate tumor cells that express a wild-type AR and both ERβ and ERα greatly increased the PSA gene expression and cell growth. These DHT effects were significantly attenuated by the addition of 17α-estradiol, 17β-estradiol, or cyproterone acetate in a dose-dependent manner. These results indicate that estrogens produce an ER-isoform– and ER-ligand–specific modulation of DHT induction of PSA gene expression and prostate tumor cell growth, providing a molecular basis for designing favorable agents for the prevention and control of prostate cancer.
Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer mortality in American and Western European males (Weir et al, 2003). Androgens play an important role in the pathogenesis of prostate cancer (Bosland, 2000). Androgen ablative therapy by castration or medication leads to prostate cell apoptosis and prostate atrophy and is a major therapeutic regimen in the treatment of advanced prostate cancer (Dreicer, 2000).
There are two natural potent androgens, testosterone and dihydrotestosterone (DHT), in humans and mammals (Zhu et al, 1998). Both androgens interact with the androgen receptor (AR) to regulate androgen-target gene expression. DHT, a potent androgen converted from testosterone by 5α-reductase isozymes, is the major intracellular androgen and the major mediator of androgen actions in the prostate (Anderson and Liao, 1968; Zhu et al, 2003). Clinical studies indicate that 46XY subjects with an inherited DHT deficiency attributable to a 5α-reductase-2 gene defect have a small undifferentiated prostate and an undetectable plasma prostate specific antigen (PSA) level. DHT replacement therapy results in an enlargement of the prostate and an elevation of plasma PSA levels in these subjects (Mendonca et al, 1996; Imperato-McGinley and Zhu, 2002; Zhu and Sung, 2005).
Like androgens, estrogens also display genomic actions via the estrogen receptors (ERs). Both AR and ER are members of the nuclear receptor superfamily and ligand-dependent nuclear transcription factors. Two ERs, ERα and ERβ, have been identified in humans and animals. The ERs share some common features but also possess differential estrogen actions (Kuiper et al, 1997; Paech et al, 1997). Both ERs are expressed in prostate cells along with the AR (Chang and Prins, 1999; Lau et al, 2000). Growing evidence indicates that estrogens can interact with androgens to modulate androgen actions in the prostate. Estrogens have been shown to either inhibit or potentiate androgen actions in the prostate, and theoretically they may either inhibit or promote the development of prostate diseases (Kumar et al, 1994; Suzuki et al, 1995; vom Saal et al, 1997; Bosland, 2000; Farrugia et al, 2000). The mechanisms of these apparently opposing estrogen effects are unclear.
In the present report, we analyzed the interaction between DHT, a natural potent androgen, and estrogens in the regulation of PSA gene expression and prostate tumor cell growth. The results obtained indicate that estrogen analogs possess receptor-isoform and ligand-specific modulation of DHT-induced PSA gene transcription and prostate tumor cell growth, which may be mediated via differential mechanisms.
A PSA promoter–directed reporter construct was generated by insertion of a 5.8-kb Hind III fragment (+12 approximately −5824) of human PSA promoter (Schuur et al, 1996) from pUPH6.0 (a gift of Dr A. Lundwall) into the Hind III site upstream of the chloramphenicol acetyltransferase (CAT) reporter gene (PSA5.8-CAT) of pBLCAT3 or the luciferase reporter gene (PSA5.8-pGL3) of pGL3-Basic (Promega, Madison, Wisc). Similar DHT induction of PSA transcription activity was observed using either PSA5.8-CAT or PSA5.8-pGL3 reporter construct. The 5.8-kb fragment of human PSA promoter contains the cis elements necessary for androgen induction and tissue-specific expression (Schuur et al, 1996). The full length of human ERβ cDNA obtained from Drs Mosselman and Jansen (N.V. Organon, The Netherlands) was subcloned into the pSG5 vector. A human AR expression vector (pSG5AR) was obtained from Dr S. Liao (The Ben May Institute, Chicago, Ill), the ERα mutants from Dr Chambon (IGBMC, France), and PRL-null from Promega. Other plasmids used in the experiments, ERE-tk-CAT, ERα, thyroid hormone receptor α1 (TRα1), vitamin D receptor, and RSV-β-galactosidase (β-gal), have been described previously (Zhu et al, 1996).
CV-1 and DU-145 prostate tumor cells (ATCC, Rockville, Md) were grown in Dulbecco modified Eagle medium (Sigma Chemical Co, St Louis, Mo), PC-3 cells (ATCC) in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gemini Bio-Products Inc, Calabasas, Calif), 2 mM l-glutamine, 50 U/mL of penicillin, and 50 μg/mL of streptomycin. All cells were maintained in a 5% CO2, 95% air-humidified atmosphere at 37°C and were cultured in phenol-red free medium with 5% stripped fetal bovine serum (Gemini Bio-Products Inc) 24 hours before experiments.
CV-1 cell cotransfections were performed using the calcium phosphate precipitation method (ProFection, Promega), as previously described (Zhu et al, 1996). Briefly, CV-1 cells were plated on 60-mm dishes or 6-well plates with an approximately 60% density and were cotransfected with 4 μg of a CAT-base (PSA5.8-CAT, MMTV-CAT, or ERE-tk-CAT) reporter plasmid, 2 μg β-gal, 1 μg receptor expression vector, and pBluescript-SK plasmid to a total of 15 μg DNA per 60-mm dish; or with 1.5 μg PSA5.8-pGL3 reporter plasmid, 1 μg PRL-null plasmid, 0.25 μg receptor expression vector, and pBluescript-SK plasmid to a total of 4 μg DNA per well, respectively. Sixteen hours later, the transfected cells were treated with various hormones for 48 hours, as indicated in the experiment. The CAT and luciferase activity was quantitated by phosphor-imager and Dual-Luciferase® Reporter Assay System (Promega), normalized to β-gal activity and Renilla luciferase activity, respectively, presented as PSA transcription activity, and expressed as folds of vehicle control or as a percentage of DHT-treated level.
Cotransfection of DU-145 and PC-3 cells was carried out in 6-well plates using SuperFect Transfection kit from Qiagen (Valencia, Calif), following the manufacturer’s instruction, and the luciferase activity was determined and presented as described above.
Total cellular RNA was extracted by using TriPURE reagents (Roche Diagnostic Inc, Indianapolis, Ind), and the concentrations of RNA were determined by ultraviolet absorbance at 260 nm.
Reverse transcription–polymerase chain reaction (RT-PCR) was carried out using the Titan 1-tube RT-PCR system (Roche Diagnostic) with 1 μg of total cellular RNA (Zhu et al, 2003). A pair of specific primers, 5′-ATGAGAGCTGCC AACCTTTG-3′ and 5′-AGAAATGTGTACACTCCAGAAT-3′, from human ERα gene, and a pair of primers, 5′-GATGAGGGGAA-ATGCGTAGA-3′ and 5′-CTTGTTACTCGCATGCCTGA-3′, from human ERβ gene, respectively, were used. The RT-PCR conditions were 50°C for 30 minutes, following 94°C for 2 minutes, and then 40 cycles of 94°C for 30 seconds, 62°C for 30 seconds for ERα, or 60°C for 30 for ERβ, and 68°C for 45 seconds, following a final cycle of 68°C for 7 minutes. The PCR products were fractionated in a 2% agarose gel and visualized by ethidium bromide staining. The expected sizes of specific PCR products for ERα and ERβ are 530 and 321 bp, respectively. Total cellular RNA from human ovary was used as a positive control, and yeast tRNA was used as a negative control of RT-PCR.
For analysis of cell growth, LAPC-4 prostate tumor cells (a gift from Dr C. Sawyer, University of California, Los Angeles) were cultured in Isove modified Eagle medium (IMEM) supplemented with 15% fetal bovine serum, 2 mM l-glutamine, 1 nM R1881, 50 U/mL of penicillin, and 50 μg/mL of streptomycin. R1881 was withdrawn 48 hours before cell passage for experiments. LAPC-4 cells were plated in 96-well plates (2.5 × 104 cells/well) in phenol-red free IMEM with 5% stripped fetal bovine serum, and 24 hours later, they were treated with various hormones for 72 hours, as indicated in the experiment. The number of viable cells was determined using the CellTiter One Solution Cell Proliferation Assay kit from Promega and was presented as a percentage of vehicle control in the same experiment.
The levels of total PSA in the culture medium were determined using an enzyme-linked immunosorbent assay kit from Diagnostic Systems Laboratories Inc (Wester, Tex) following the manufacturer’s instruction.
All chemicals and steroids were obtained from Sigma, except where indicated. ICI182780 (ICI), a pure estrogen antagonist, was kindly provided by Dr Wakeling at Zeneca Pharmaceuticals (United Kingdom). R1881 was purchased from Perkin Elmer (Shelton, Conn).
The data are presented as mean plus standard error of the mean (SEM). One-way analysis of variance (ANOVA) following Student Newman-Keuls test was used to analyze the dose-response effects and to determine the difference among multiple groups. A P value of less than .05 was accepted as the level of statistical significance.
Using cotransfection analysis of PSA promoter directed reporter constructs and AR in CV-1 cells that express neither endogenous AR nor ERs, DHT produced a dose-dependent induction of PSA transcription activity, as shown in Figure 1. This DHT induction of PSA transcription activity was AR-dependent, since DHT failed to increase PSA transcription activity in the absence of AR cotransfection.
Cotransfection of AR with ERα or ERβ in CV-1 cells noticeably, but not significantly, decreased the DHT-induced PSA transcription activity (Figure 2a). Concomitant treatment of the cotransfected cells with E2 (10 nM) greatly attenuated the DHT-induced PSA transcription activity via ERα, regardless of induction by either a low (0.5 nM) or a high dose (10 nM) of DHT (Figure 2b). However, in the presence of ERβ (Figure 2c), E2 had no significant effect, even at high doses (up to 1 μM) of E2, or with high levels (up to 4 μg) of ERβ cotransfection. The levels of ERβ cotransfected were detectable by Western blot analysis and were functionally sufficient to mediate E2 induction of estrogen-target gene expression using a consensus ERE-directed CAT reporter gene (ERE-tk-CAT) in the same system (data not shown).
The E2 inhibition of DHT action via ERα was not affected by the addition of ICI182780 (ICI), a pure estrogen antagonist (Figure 2d). Interestingly, ICI, like E2, also inhibited DHT-induced PSA transcription activity via ERα, as shown in Figure 2d. However, the combination of E2 (10 nM) and ICI (100 nM) did not display additive or synergistic effects at the doses tested.
The inhibition of DHT action by E2 via ERα was an ER and estrogen-specific event, since cotransfection of AR with either TRα1 or vitamin D receptor, treated with corresponding ligand triiodothyronine or 1,25-dihydroxyvitamin D, either had no significant effect or slightly potentiated DHT action at high-dose (1 μM) hormone treatment (data not shown).
A similar ER-isoform–specific inhibition of DHT (10 nM)–induced androgen-target gene transcription by E2 (10 nM) was observed by cotransfection of a MMTV-CAT reporter construct in CV-1 cells (data not shown), which has a CAT-reporter gene directed by the mouse mammary tumor virus (MMTV) promoter that contains a consensus androgen response element (Yeh et al, 1998).
By using DHT induction of PSA transcription activity in CV-1 cell cotransfection as a model system, the effects of various ER-ligands on the modulation of DHT action via ERα or ERβ were examined. As shown in Figure 3a and b, similar to E2, diethylstilbestrol, a synthetic potent estrogen agonist, inhibited DHT-induced PSA transcription activity via ERα in a dose-dependent manner, while it had no significant effect in the presence of ERβ. In contrast, tamoxifen, a partial estrogen agonist or a selective ER modulator, did not affect DHT-induced PSA transcription activity via ERα, while it significantly potentiated DHT action via ERβ. ICI, a pure estrogen antagonist, inhibited DHT action via ERα, while it might stimulate it via ERβ at a high concentration (1 μM).
Interestingly, 17α-estradiol (αE2), an isomer of E2 and a weaker estrogen agonist, produced a dose-dependent inhibition of DHT action via either ERα or ERβ (Figure 3a and b). The effects of all ER-ligands were dependent on ER, since they did not significantly alter DHT action in the absence of ER cotransfection.
When AR, ERα, and ERβ were cotransfected at a 1:1: 1 ratio, as shown in Figure 3c, treatment with E2, αE2, or diethylstilbestrol inhibited DHT action; tamoxifen potentiated DHT action; and ICI inhibited DHT action at a dose of 10 nM, while it had no significant effect at 100 nM.
Similar to CV-1 cotransfection analysis, estrogens produced an ER-isoform– and ER-ligand-specific modulation of DHT-induced PSA transcription activity in prostate tumor DU-145 and PC-3 cell cotransfection analyses. In DU-145 cells, both E2 and αE2 inhibited DHT action via ERα, while tamoxifen had no significant effect (Figure 4a). In the presence of ERβ, E2 had no significant effect, while αE2 significantly inhibited and tamoxifen potentiated DHT action (Figure 4b).
In PC-3 cells (Figure 4c and d), both E2 and αE2 significantly inhibited DHT action, while tamoxifen had no effect via ERα at a dose of 100 nM. In the presence of ERβ, tamoxifen potentiated DHT action, but αE2 and E2 failed to effect DHT action at the doses tested.
To analyze the functional significance of each ERα domain in this androgen-estrogen interaction, various ERα mutants, as illustrated in Figure 5a (Kumar et al, 1987), were studied by cotransfection in CV-1 cells. A deletion of ERα DNA binding domain (DBD, HE11) completely blocked the E2 inhibition of DHT action, while it only partially interfered with αE2 action (Figure 5b). A deletion of the carboxyl-terminal domain (F domain, HE13) completely blocked the αE2 inhibition of DHT action but only partially affected E2 action (Figure 5c). Deletion of ERα amino-terminal domain (HE19) had no significant effects on either E2 or αE2 inhibition of DHT action (Figure 5d) although this mutant greatly affects the E2-induced estrogen-target gene expression (Kumar et al, 1987). Both E2 and αE2 failed to inhibit DHT action in the presence of ERd-DBD alone (HE20, Figure 5e).
To determine the biological significance of androgen-estrogen interaction in prostate tumor cells, we first examined the levels of ER expression in LAPC-4 cells, an androgen-sensitive prostate tumor cell line containing a wild-type AR (Klein et al, 1997), using RT-PCR analysis. In LAPC-4 cells, ERβ was clearly expressed, while ERα expression was quite low or undetectable, as shown in Figure 6. Additionally, in MDA Pca-2b cells, an androgen-independent prostate cancer cell line (Zhao et al, 2000), both ERs were detected, but ERβ was predominant. As expected, both ERs were expressed in human ovarian tissues.
The biological significance of androgen-estrogen interaction is exemplified by studying the estrogen modulation of DHT-induced PSA expression in LAPC-4 cells, as shown in Figure 7a. Treatment with DHT at a dose of 10 nM for 72 hours produced an approximately fourfold induction of PSA levels in LAPC-4 cell culture medium. This DHT-induced PSA expression was significantly inhibited by concomitant treatment with E2, αE2, or cyproterone acetate (an AR antagonist) in a dose-dependent manner.
To further explore the biological significance of estrogen inhibition of DHT action, the effects of E2 and αE2 on DHT induction of LAPC-4 cell growth were examined. Treatment of LAPC-4 cells with DHT for 72 hours produced a dose-dependent induction of cell growth, as shown in Figure 7b. This DHT-induced LAPC-4 cell growth was significantly inhibited by the addition of E2, αE2, or cyproterone acetate in a dose-dependent manner (Figure 7c). However, treatment with E2 or αE2 alone for 72 hours had no significant effects on LAPC-4 cell growth.
PSA is a useful biomarker for screening prostate cancer and for evaluating prostate cancer progression (Kantoff and Talcott, 1994). The expression of PSA gene is induced by androgens via AR, and multiple AREs have been identified in the PSA gene promoter (Riegman et al, 1991; Cleutjens et al, 1996; Schuur et al, 1996). As a result of its tissue specificity and androgen inducibility, human PSA gene has been used as a model system in studying androgen action in prostate gene expression and in prostate cancer research.
Using DHT-induced PSA transcription in cotransfection assays as a model system, the effects of ER-isoforms and various ER-ligands on modulation of androgen action were analyzed. The observation that E2 inhibited DHT-induced PSA transcription activity and MMTV-CAT activity via ERα is in agreement with the results of previous studies (Kumar et al, 1994; Panet-Raymond et al, 2000). More importantly, we demonstrate the ER-isoform and ER-ligand specificity in this androgen-estrogen interaction. Based on these results, ER-ligands are classified into 4 different categories related to their effects on androgen action (the Table). This classification is different from the pharmacological classification based on estrogenic activity. E2, αE2, and ICI all inhibit DHT action via ERα, although they are pharmacologically classified as an estrogen agonist, a weaker agonist, and a pure antagonist, respectively.
The observed ER-isoform–specific actions are consistent with the demonstration that ERα and ERβ possess distinctive action in the regulation of gene expression. Paech et al (1997) have shown that ERα and ERβ signal in opposite ways in the presence of E2 from an AP1 site; with ERα, E2 activates transcription, whereas with ERβ, E2 inhibits transcription. Panet-Raymond et al (2000) observed that E2 inhibits androgen action via ERα, but not via ERβ, a result that is in agreement with our current results. Taken together, these data indicate that ERα and ERβ possess differential actions in mediating ligand-specific activity, although they share high homology and common functional modalities.
An ER-ligand–specific modulation of DHT action is observed via either ERα or ERβ. This ligand specificity is consistent with the concept that different ER-ligands cause differential conformational changes in the ERs (Egner et al, 2001; Nilsson et al, 2001; Margeat et al, 2003). The differential changes in ER conformation by various ligands may result in a recruitment of various transcriptional factors or coregulators (Shang and Brown, 2002; Margeat et al, 2003) and may process different downstream actions. This explains the differential ER-ligand effects, but it does not explain why various ER-ligands, including agonist, partial agonist, and pure antagonist, all lead to the inhibition of DHT action via ERα. The observation that both E2 and ICI inhibit DHT action is consistent with previous demonstrations that both E2 and ICI induce the same target gene transcription (Kim et al, 2003) and cause proteasome-dependent ERα degradation (Preisler-Mashek et al, 2002).
A stereo-specificity of αE2 and E2 in modulating DHT action is observed. Like E2, αE2 binds to ERs and processes estrogenic activity, although in a much weaker fashion (Edwards and McGuire, 1980). Unlike E2, which inhibits DHT-induced PSA transcription via ERα only in cotransfection assays, αE2 inhibits DHT action via either ERα or ERβ.
The importance of ERα functional domains in this estrogen-androgen interaction is analyzed using ERα mutants. The E2-inhibition of DHT action via ERα mainly involves the DBD. However, gel shift analysis shows that neither ERα nor ERβ directly binds to any of the 3 functional AREs in the human PSA promoter (our unpublished data), indicating that direct competition of ARE-binding between ligand-AR and ligand-ER is unlikely. On the other hand, αE2 inhibition of DHT action via ERα is completely eliminated when the ERα F-domain is deleted. The F-domain is highly variable in the nuclear receptor superfamily, and there is little homology between ERα and ERβ. The ERα F-domain potentially contains helix-13 and β-strand motifs (Kumar et al, 1987; Kim et al, 2003). The functional significance of the ERα F-domain is just emerging. It is required for the agonist/antagonist action of tamoxifen (Schwartz et al, 2002) and the E2 activation of ERα/SP1 pathway (Kim et al, 2003).
The biological significance of this androgen-estrogen interaction is exemplified by determining the effects of αE2 and E2 on the regulation of DHT induction of PSA gene expression and cell growth in LAPC-4 prostate tumor cells. Previous studies show that LAPC-4 cells express a wild-type AR and are sensitive to androgen stimulation (Klein et al, 1997). We here demonstrate that LAPC-4 cells also express ERs with predominantly ERβ and low level of ERα, which is consistent with previous demonstration in other prostate tumor cells (Lau et al, 2000). As expected, treatment with DHT in LAPC-4 cells increases PSA expression and cell growth (see Figure 7). These DHT effects are significantly inhibited by coadministration of E2, or αE2 in a dose-dependent manner. The inhibitory effects of these estrogen analogs are as potent as cyproterone acetate, an AR antagonist, although they act via different mechanisms.
The estrogen inhibition of DHT-induced PSA gene expression and cell growth in LAPC-4 cells may be mediated via ERs, as both ERα and ERβ are expressed in these cells. If this is the case, then the data obtained in LAPC-4 cells are consistent with the cotransfection studies, in which both E2 and αE2 inhibit DHT-induced PSA transcription activity when both ERα and ERβ are cotransfected in the cells. This hypothesis is further supported by our recent studies showing that the estrogen inhibition of AR action is mainly mediated via ERβ using RNA interference analysis (unpublished data). However, it should be noted that the receptor-isoform– and ligand-specific modulation of DHT action by estrogens needs to be further evaluated in prostate cells, and there is no direct linkage between DHT-induced PSA gene expression and prostate tumor cell growth. This notion is supported by comparing the dose-response data between estrogen inhibition of DHT-induced PSA expression and estrogen inhibition of DHT-induced cell growth in LAPC-4 cells (see Figure 7a and c), in which differential dose-response curves are observed. Although PSA has been shown to degrade insulin-like growth factor binding protein 3, thereby leading to a potentiation of insulin-like growth factor action on cell growth by in vitro analysis (Cohen et al, 1994), such PSA action has not been demonstrated in vivo, and the significance of PSA in prostate pathogenesis remains to be determined.
In vivo studies have shown that estrogens can either inhibit or enhance androgen effects, although the mechanisms of these apparent opposing estrogen actions are not explored. The present analyses using in vitro cotransfection assays provide a reasonable, potential explanation of these seemingly opposing estrogen actions. Since the outcome of androgen-estrogen interaction is cell-type, ER-isoform– and ligand-specific, the final result can be either inhibitory or stimulatory depending on the ER-isoforms expressed in the cells, as well as the ligands used in the experiment. Further study using ER knockout animals will provide valuable information for androgen-estrogen interaction in the prostate.
In summary, estrogens have been used in hormonal therapy of prostate cancer, presumably by inhibiting testosterone biosynthesis via the negative feedback of the hypothalamus-pituitary-gonadal axis (Huggins and Hodges, 1941). Our current study provides an additional mechanism for estrogens acting directly through ERs to antagonize androgen actions within the cells. The demonstration of ER-isoform and ligand specificity mediated via differential mechanisms in androgen-estrogen interaction explains the seemingly opposing estrogen action in the prostate and provides important information for the development of new estrogen analogs in the prevention and control of prostate cancer.
We are very grateful to Dr A. Lundwall (Lund University, Malmö, Sweden) for providing us with the pUPH6.0 plasmid; to Drs Mosselman and Jansen (N.V. Organon, The Netherlands) for an ERβ expression vector; to Dr Chambon (Strasbourg, France) for the ERα and ERα mutant expression vectors; to Dr Liao (The Ben May Institute, Chicago, Ill) for the AR expression vector; to Dr Sawyer (UCLA) for LAPC-4 cells; to Dr Wakeling (Zeneca Pharmaceuticals, United Kingdom) for ICI182780; and to Dr C. Chang (University of Rochester, NY) for valuable discussion.
Supported in part by grants from USAMRAA (DAMD17-02-1-0160) and NIH/NIDDK (DK061004) to Y.S.Z. Z-k.Z. was supported by a NIH training grant (T32 DK-07313-24) directed by J. Imperato-McGinley.