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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 8 2535-2541
Copyright © 1997 by The Endocrine Society

Reproductive Endocrinology

Interplay between Sex Steroids and Melatonin in Regulation of Human Benign Prostate Epithelial Cell Growth

Eli Gilad, Haim Matzkin and Nava Zisapel

Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences (E.G., N.Z.), and the Department of Urology, Tel Aviv Medical Center and Sackler Faculty of Medicine (H.M.), Tel Aviv University, Tel Aviv 69978, Israel

Address all correspondence and requests for reprints to: Prof. Nava Zisapel, Ph.D., Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. E-mail: navazis{at}ccsg.tau.ac.il


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human benign prostatic epithelial cells contain functional melatonin receptors that can suppress cell growth and viability. The development of benign prostatic hyperplasia in men is assumed to result from androgen-estrogen imbalance. The impact of sex steroids on melatonin receptors in human benign prostate epithelial cells was investigated. The suppression by melatonin of [3H]thymidine incorporation and cGMP, and the enhancement of cAMP levels in the cells were used as markers of melatonin responses.

Dihydrotestosterone (DHT) and 17ß-estradiol (E2) separately increased [3H]thymidine incorporation into the cells, but suppressed it when combined. In cells grown with DHT, melatonin responses were extenuated. E2 greatly reduced the apparent affinity of [125I]melatonin binding in these cells without affecting binding site density. In parallel, the ability of melatonin to suppress [3H]thymidine incorporation into the cells was ablated within 1 h after the addition of E2. The melatonin-mediated increase in cAMP and decrease in cGMP concentrations were also ablated by E2.

Preincubation of the cells with bis-indolylmaleimide (GF 102903X), a specific inhibitor of protein kinase C, prevented the E2-mediated inactivation of melatonin binding and the inhibitory action on [3H]thymidine incorporation. Prolonged (18-h) incubation of the cells with phorbol 12-myristate 13-acetate to down regulate protein kinase activity, partially restored [125I]melatonin binding and responsiveness in the E2-treated cells.

These data indicate that 1) DHT and E2 enhance prostate epithelial cells growth, but reduce cell growth when combined; 2) DHT extenuates the inhibitory effects of melatonin on epithelial cell growth; and 3) E2 acts to inactivate melatonin receptors and consequently responses in human epithelial benign prostatic hyperplasia cells. This process is probably mediated by protein kinase C.

Together, these results show an interplay between melatonin and sex steroids in the regulation of benign prostatic epithelial cell growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BENIGN PROSTATE hyperplasia (BPH) is a disease of men characterized by symptoms of urinary obstruction caused by abnormal, nonmalignant, prostate growth. The prevalence of BPH increases sharply in men over 50 yr of age (1). Age-related changes in circulating concentrations of testosterone and estrogen, their balance, or their receptors in the prostate have been postulated to play a role in the development of BPH in man (2, 3, 4). In dogs, induction of BPH required administration of both dihydrotestosterone (DHT) and 17ß-estradiol (E2) (5). The presence of DHT and E2 receptors was confirmed in BPH tissues (6), although most of the E2 receptors were located in stromal cells (7, 8).

Melatonin, produced nocturnally by the pineal gland, plays a major role in the coordination of seasonal reproduction and pubertal development in mammals (9). The production of melatonin declines with age (9). We recently found by in vitro autoradiography, specific binding sites for 125I-labeled melatonin ([125I]melatonin) in human benign prostate tissue (10). Equilibrium binding and competition experiments revealed reversible, saturable, and specific binding of [125I]melatonin to sites primarily associated with the microsome-enriched fraction of prostate epithelial cells. The binding was inhibited by GTP analogs. In culture, human benign prostate epithelial cells were found to bind [125I]melatonin with high affinity (Kd = 68 pmol/L) (11). Melatonin at physiological concentrations inhibited DNA and protein synthesis in these cells and reduced their viability (11).

In a number of studies, melatonin-binding sites in the brain as well as melatonin responses have been shown to depend on the presence of sex steroids. In the female rat hypothalamus and medulla-pons, ovariectomy produced a large estradiol-reversible decrease in low affinity [125I]melatonin-binding sites (12). In parallel, the inhibitory effects of melatonin on dopamine release from the hypothalamus diminished in ovariectomized female rats and was reinstated by estradiol supplementation (13). In male hamsters maintained in a long day environment, castration produced a large testosterone-reversible decrease in low affinity [125I]melatonin-binding sites in the hypothalamus, medulla-pons, and hippocampus (14). On the other hand, castration has been shown to increase the density of high affinity [125I]melatonin-binding sites in the male rat anterior pituitary (15). Castration also affected [125I]melatonin-binding sites in the Harderian gland of the golden hamster and blind mole rat (16, 17). Thus, we explored the modulation by sex steroids of melatonin receptors and responses in human benign prostate epithelial cells.

Besides inhibition of [3H]thymidine incorporation (11), we found (unpublished) that melatonin increased cAMP and decreased cGMP in human benign prostate epithelial cells. Hence, we used the melatonin-mediated inhibition of thymidine incorporation, suppression of cGMP, and increase in cAMP levels as markers for melatonin responses in prostate epithelial cells.

Our results indicate E2-mediated inactivation of melatonin receptor binding and responses in prostate epithelial cells. We recently observed protein kinase C (PKC)-induced inactivation of melatonin receptors in these cells (unpublished). Nongenomic activation of PKC by E2 has been reported in human uterine endometrium, rat ovary, and rat pituitary (18, 19, 20, 21). Activation of PKC by androgens has also been reported in some studies (22), but not in others (23, 24). Human BPH tissue has been reported to contain phospholipid-dependent, phorbol 12-myristate 13-acetate (TPA)-activated, PKC isoforms (25). Hence, in the present study we investigated the possibility that the effects of E2 on melatonin receptors and responses in prostate epithelial cells were mediated by PKC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Melatonin, BSA, phenylmethylsulfonylfluoride, DHT, E2, and TPA were obtained from Sigma Chemical Co. (St. Louis, MO). Bis-indolylmaleimide (GF 102903X) was obtained from Calbiochem (La Jolla, CA). [Methyl-3H]thymidine was obtained from Rotem Industries (Beer-Sheva, Israel). The cAMP assay kit was obtained from DuPont (Wilmington, DE). Na125I and the cGMP assay kit were obtained from Amersham (Arlington Heights, IL). [125I]Melatonin was prepared as previously described (26).

RPMI 1640 medium, RPMI 1640 medium without phenol red supplemented with L-glutamine (RPMI-P), newborn calf serum (NBS), charcoal-stripped NBS (NBSC), 0.05% insulin, transferrin, selenium, and antibiotics were obtained from Biological Industries (Beit Haemek, Israel).

Epithelial cell cultures

Human prostate tissue samples were obtained from patients undergoing elective transabdominal prostatectomies for BPH. All patients (aged 55–80 yr; n = 21) were otherwise healthy. Approval for the use of the tissue was obtained from the local ethical committee. Epithelial cells were cultured as previously described (11) and grown at 37 C in growth medium (RPMI containing 10% NBS, 10 ng/mL epidermal growth factor, 5 ng/mL insulin, 5 ng/mL transferrin, 5 ng/mL selenium, 50 U/mL penicillin, 50 µg/mL streptomycin, 250 ng/mL amphotericin B, and 10 ng/mL DHT) in a humidified atmosphere with 5% CO2. Before each experiment, cells were harvested by trypsin and adjusted to a density of 106 cells/mL in culture medium (RPMI containing 10% NBSC, 10 ng/mL epidermal growth factor, 5 ng/mL insulin, 5 ng/mL transferrin, 5 ng/mL selenium, 50 U/mL penicillin, 50 µg/mL streptomycin, and 10 ng/mL DHT) and replated in 24-well multiplates. After 24 h, the culture medium was replaced with fresh culture medium containing 5% NBSC and used in the experiments. Protein content was determined in each plate (27). As shown below (see Table 2Go) the ratio of protein per cell was constant under the experimental procedures employed. Hence, data from all experiments were normalized to protein content.


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  		Table 2. Effects of E2 and melatonin on prostate epithelial cell count and protein content

 
Sex steroid treatment

Cells attached to the plates were incubated with DHT, E2, or both (10 nmol/L unless otherwise stated) for 1 h to 4 days at 37 C. Melatonin responses and [125I]melatonin binding were then assessed. In some experiments, cells grown in the presence of DHT (10 nmol/L) were incubated with GF 102903X (500 nmol/L, 30 min at 37 C) or TPA (100 ng/mL, 18 h) before the addition of E2 (10 nmol/L), and incubation was resumed for an additional 1-h period.

[125I]Melatonin binding

Equilibrium binding studies were carried out as previously described (11). Briefly, cells were incubated for 60 min at 37 C with 10 pmol/L and 1 nmol/L [125I]melatonin (2000 Ci/mmol) in RPMI-P medium in the absence (total binding) or presence (nonspecific binding) of 2 µmol/L melatonin. The media were then removed, and cells were immediately washed (twice, 2 mL each time) with ice-cold phosphate-buffered saline (PBS) and dissolved in 0.1 mol/L NaOH. Aliquots were collected for determination of protein content. The amount of radioactivity associated with the cells was determined in a {gamma}-counter.

The equilibrium binding data at the concentration range used were compatible with a single type of binding site (Scatchard analysis). The equilibrium binding parameters were obtained by nonlinear regression analysis of the binding data (Sigmaplot, Jandel Scientific, San Rafael, CA).

Thymidine incorporation

[3H]Thymidine incorporation was assessed as previously described (11). Briefly, cells attached to the plates were incubated with buffer or melatonin for 1 h at 37 C. [3H]Thymidine (60 Ci/mmol; 1 µCi/well) was then added, and incubation was resumed for 1 h. Media were then discarded, and the cells were washed (twice, 2 mL each time) with ice-cold PBS and harvested by trypsin. Aliquots were retained for protein determination. Trichloroacetic acid was added, and insoluble materials were collected by filtration on GF/C glass fiber filters (Whatman, Clifton, NJ). The amount of radioactivity was determined by scintillation spectrometry.

Determination of cAMP and cGMP in cells

Cells were washed twice with PBS, harvested mechanically using a rubber policeman, and suspended in RPMI-P medium containing 200 µmol/L phenylmethylsulfonylfluoride. Aliquots of the cell suspension were incubated for 10 min at 37 C with the phosphodiesterase inhibitor isobutylmethaxine (10–6 mol/L). Melatonin (10-10–10-6 mol/L) or the same volume of vehicle (0.01% ethanol in PBS) was then added, and incubation resumed for 10 min. The reaction mixture was then boiled for 4 min, frozen, thawed, sonicated, and centrifuged for 10 min (10,000 x g). The supernatant was collected. The cGMP and cAMP contents in samples were determined by RIAs. Data were normalized to protein content.

Statistical analyses

Results were compared by ANOVA followed by Student’s t tests for paired comparisons; significance was set at P < 0.050 (28).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of E2 on [125I]melatonin binding

The effects of E2 treatment on [125I]melatonin-binding sites in benign prostate epithelial cells in culture are shown in Fig. 1Go. Cells cultured with DHT (10 nmol/L) exhibited high affinity [125I]melatonin-binding sites (Kd = 80 ± 5 pmol/L; Fig. 1aGo). E2 treatment (10-8 mol/L for 60 min and for 4 days) reduced the apparent affinities of the [125I]melatonin-binding sites in the cells (Kd = 425 ± 78 and 1973 ± 390 pmol/L, respectively), whereas the apparent density of binding sites was not affected (Fig. 1Go, b and c, and Table 1Go).



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  		Figure 1. Specific [125I]melatonin binding to cultured epithelial cells from human benign prostate as a function of [125I]melatonin concentrations. Cells were incubated with a) vehicle (30 min), b) E2 (10-8 mol/L, 4 days), c) E2 (10-8 mol/L, 60 min), d) GF 102903X (30 min) and then GF plus E2 (60 min), and e) TPA (18 h) and then E2 (60 min). Specific binding (mean ± SEM; n = 12) was determined at equilibrium and calculated from the difference between the amount of [125I]melatonin bound in the absence and presence of 2 µmol/L melatonin. The solid lines are theoretical curves reconstructed from the mean Kd and apparent density of binding sites (Bmax) depicted in Table 1Go. Insets are Scatchard plots of the specific [125I]melatonin binding data.

 

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  		Table 1. Apparent Kd and Bmax values calculated from the regression analyses of the specific [125I]melatonin binding data (see Fig. 1Go)

 
Preincubation of the cells with GF 102903X (30 min) before treatment with E2 (10 nmol/L, 60 min) prevented the E2-mediated decrease in the apparent affinity of [125I]melatonin binding, but the apparent density of the sites significantly decreased (Fig. 1dGo and Table 1Go). Prolonged incubation (18 h) of the cells with TPA before E2 treatment (10 nmol/L, 1 h) resulted in a decreased affinity of the [125I]melatonin-binding sites (Kd = 277 ± 100 pmol/L; Table 1Go) compared to the control value, but a higher affinity than that in cells treated with E2 alone. Here, too, the apparent binding site density was not affected (Fig. 1eGo and Table 1Go).

Effects of sex steroids and melatonin on [3H]thymidine incorporation

The effects of various concentrations of DHT and E2 or their equimolar combinations on [3H]thymidine incorporation by prostate epithelial cells are shown in Fig. 2Go. Treatment of cells maintained in charcoal-stripped medium with DHT (10-12–10-6 mol/L for 4 days) resulted in a concentration-dependent increase in [3H]thymidine incorporation (Fig. 2Go, a and b). This response was maximal between 10-10–10-8 mol/L and was attenuated at higher concentrations. Melatonin (1 nmol/L, 1 h) inhibited [3H]thymidine incorporation significantly in the charcoal-stripped medium (Fig. 2aGo), and its effects were extenuated in the presence of DHT (Fig. 2bGo).



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  		Figure 2. Effects of sex steroids and melatonin on [3H]thymidine incorporation into human prostate epithelial cells. Cells were incubated with vehicle or various concentrations of a) vehicle (control), b) DHT, c) E2, or d) E2 plus DHT for 4 days and then incubated with melatonin (10-8 mol/L; solid) or the same volume of vehicle (open) for 1 h. The incorporation of [3H]thymidine was then assessed. Results (mean ± SEM of three independent studies run in quintuplicate) are expressed as a percentage of the incorporation in control cells incubated with vehicle. *, P < 0.01 within pairs; +, P < 0.05 within pairs; **, at concentrations equal to and above 10 nmol/L, P < 0.05 compared to control.

 
Similar treatment of the cells with E2 (10-12–10-6 mol/L for 4 days) enhanced [3H]thymidine incorporation into the cells compared to that in charcoal-stripped medium (Fig. 2cGo). The maximal increase was observed with 10-9–10-8 mol/L, and the effect was attenuated at higher E2 concentrations. However, the inhibition by melatonin of [3H]thymidine incorporation was significant only with 10-9 mol/L E2 and was ablated at higher (10-8–10-6 mol/L) E2 concentrations (Fig. 2cGo).

Treatment of the cells with equimolar concentrations of DHT and E2 paradoxically reduced [3H]thymidine incorporation by the cells in a concentration-dependent manner. No further suppression of [3H]thymidine incorporation by melatonin was evident under these conditions (Fig. 2dGo).

The incorporation of [3H]thymidine into cells cultured with DHT (10-8 mol/L) at various times after the addition of E2 (10-8 mol/L) was examined (Fig. 3Go). E2 significantly suppressed [3H]thymidine incorporation in the cells within 1 h of addition and ablated the inhibitory response to melatonin (1 nmol/L). [3H]Thymidine incorporation by the cells remained low during the 4 days of E2 treatment, and no further response to melatonin was observed (Fig. 3Go). The effects of E2 treatment on cell count and protein content are depicted in Table 2Go. The decrease in [3H]thymidine incorporation by the cells was followed by decreases in cell count and, concomitantly, protein content of the plates. A protein to cell count ratio of 4.7 µg/10,000 cells was found in cells grown without DHT. This value did not significantly change in cells grown with DHT or in the course of E2 treatment.



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  		Figure 3. Effects of E2 and melatonin on [3H]thymidine incorporation into prostate epithelial cells. Cells cultured with DHT (10 nmol/L) were treated with vehicle (control) or E2 (10-8 mol/L) for the specified period of time and then incubated with melatonin (10-8 mol/L; black bars) or the same volume of vehicle (dotted bars) for 1 h. The incorporation of [3H]thymidine was then assessed. Results (mean ± SEM of three independent studies run in quintuplicate) are expressed as a percentage of the incorporation into the control cells treated with vehicle. *, P < 0.05 within pairs.

 
The effects of the PKC inhibitor GF 102903X (500 nmol/L, 30 min) or the PKC activator TPA (100 ng/mL, 18 h) on the responsiveness of control and E2-treated cells to melatonin were studied (Fig. 4Go). In the control cells grown with DHT (10 nmol/L), melatonin (1 h) inhibited [3H]thymidine incorporation by up to 50% in a concentration-dependent manner (half-maximal effect at 1 nmol/L; Fig. 4Go). Treatment of the cells with E2 (10 nmol/L, 1 h) abolished this effect of melatonin. Pretreatment of the cells with GF 102903X (30 min) before E2 treatment (10 nmol/L, 1 h) enhanced [3H]thymidine incorporation (i.e. by 150% compared to that in E2-treated cells and by 60% compared to that in control cells; Fig 4Go). In parallel, pretreatment with GF 102903X prevented the E2-mediated loss of the melatonin response, and the melatonin-mediated inhibition of [3H]thymidine incorporation was extenuated (Fig. 4Go).



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  		Figure 4. Effects of melatonin on [3H]thymidine incorporation into prostate epithelial cells. Cells cultured with DHT (10-8 mol/L) were treated with vehicle (60 min; {blacktriangledown}), E2 (10-8 mol/L, 60 min; {square}), GF 102903X (30 min) and then GF and E2 (60 min; •), or TPA (18 h) and then E2 (60 min; {circ}). The treated cells were incubated in the absence (cont) and presence of various concentrations of melatonin for 1 h. The incorporation of [3H]thymidine was then assessed. Results (mean ± SEM of three independent studies run in quintuplicate) are expressed as a percentage of the incorporation in control cells treated with vehicle.

 
Prolonged (18-h) incubation of the cells with TPA before E2 treatment (10 nmol/L, 1 h) led to diminution of [3H]thymidine incorporation by the cells. Under these conditions, a small, but significant, melatonin-mediated reduction of [3H]thymidine incorporation was observed (Fig. 4Go).

Effects of E2 and melatonin on cAMP and cGMP concentrations

The impact of E2 on the melatonin-mediated effects on cAMP and cGMP were investigated (Fig. 5Go). In the control cells cultured with DHT (10 nmol/L), melatonin increased cAMP and reduced cGMP contents. These effects were concentration dependent with the 50% effect (IC50) at 0.2 and 2 nmol/L melatonin for cAMP and cGMP levels, respectively (Fig. 5aGo). Treatment of the cells with E2 (10 nmol/L, 1 h) also enhanced cAMP and suppressed cGMP levels (Fig. 5bGo). In the presence of E2, the effects of melatonin on cAMP and cGMP levels in the cells were abolished (Fig. 5bGo).



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  		Figure 5. Effects of melatonin on cAMP and cGMP concentrations in human benign prostate epithelial cells. Cell were incubated with a) DHT (10-8 mol/L) or b) DHT and E2 (10-8 mol/L each) for 4 days, harvested, and incubated with various melatonin concentrations or the same volume of vehicle for 10 min. The amounts of cells cAMP ({triangledown}) and cGMP ({blacksquare}) were determined by RIAs. Results (mean ± SEM of three independent studies run in triplicate) are expressed as a percentage of the respective cyclic nucleotide content in control cells treated with vehicle.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented here reveal an interplay among DHT, estradiol, and melatonin in the regulation of benign prostate epithelial cell growth. DHT and E2 enhance prostate epithelial cell growth, but reduce cell growth when combined. DHT attenuates the inhibitory effects of melatonin on epithelial cell growth, whereas E2 acts to inactivate melatonin receptors and, consequently, responses in human epithelial BPH cells.

Of particular interest is the relationship between the ef-fects of the sex steroid combinations on [3H]thymidine incorporation and their effects on cAMP and cGMP levels in the cells. In the presence of equimolar concentrations of E2 and DHT, [3H]thymidine incorporation was suppressed, whereas cAMP was greatly increased, and cGMP was decreased. An increase in cAMP is frequently associated with E2 activity in various tissues, including human and dog prostate (29). Elevation of cAMP frequently causes cell growth arrest (30). Hence, the inhibition of [3H]thymidine incorporation by E2 (as also by melatonin) in cells grown with DHT may be due to the increase in cellular cAMP levels.

On the other hand, the effects of E2 on cGMP seem to be tissue specific. In rat uterus and human coronary arteries, E2 increased cGMP content (31, 32), whereas in human granulosa cells, cGMP production may be reduced or increased, depending on E2 concentrations (33). PKC has been shown to inhibit the activity of guanylyl cyclase in NIH-3T3 cells and in the human renal cell line SK-NEP-1 (34, 35). Hence, the decrease in cGMP may result from the E2-mediated activation of PKC in human benign prostate epithelial cells.

Notably, E2 rapidly enhanced [3H]thymidine incorporation in the presence of DHT in GF 102903X-pretreated cells, while suppressing the incorporation in GF 102903X-untreated cells. These data point to the possibility that E2 may act to enhance prostate cell growth by some undefined mechanism, whereas a concomitant E2-mediated activation of PKC prevents this action. The effect of E2 on benign prostate epithelial cells has not been explored. However, in organ cultures of the rat ventral prostate, pharmacological E2 concentrations have been found to block the androgen-mediated activation of [3H]thymidine incorporation into DNA in the epithelium, whereas E2 alone increased the volume density of epithelium (36). These data are in agreement with our interpretation.

E2 rapidly (within 1 h) inactivates melatonin binding and desensitizes prostate epithelial cells to melatonin. The desensitization encompassed three different melatonin responses in these cells, namely the inhibition of [3H]thymidine incorporation, the suppression of cellular cGMP, and the enhancement of cAMP. The E2 effect might be due to inactivation of melatonin receptors, as indicated by the marked decrease in apparent ligand binding affinity.

The ability of the PKC inhibitor GF 102903X to prevent the E2-mediated desensitization of melatonin receptors implies the involvement of PKC in this effect. This explanation is compatible with previous reports on the activation of PKC by E2 in a number of tissues at the transcriptional and posttranscription levels (18, 19, 20, 21). E2 has recently been found to stimulate a rapid Ca2+ influx in LNCaP human prostate cancer cells (37). Preliminary data obtained in our laboratory (unpublished) indicate that E2 induced a rapid and large increase in 45Ca influx into human benign prostate epithelial cells. Hence, E2 may activate PKC activity in these cells by enhancing intracellular Ca2+. It should be noted that human BPH tissue reportedly contains TPA- but not Ca2+-activated PKC isoforms (25). Hence, in prostate epithelial cells, the effect of E2 on melatonin receptors may be predominantly mediated by a PKC isoform(s) that is not directly regulated by Ca2+.

The mechanism by which PKC mediates melatonin receptor desensitization is unknown. Uncoupling of receptor-G protein complexes has been shown to decrease binding affinity, but not site density, in some G protein-coupled receptors (38, 39). It is thus reasonable to assume that a PKC phosphorylation site is located at or close to a site that participates in melatonin binding or in coupling of the receptor to a G protein.

Prolonged incubations (>4 h) with TPA have been repeatedly shown to cause down-regulation of PKC (40, 41, 42). However, the long term (18-h) pretreatment with TPA partially preserved the binding and responsiveness to melatonin in the E2-treated (1 h) cells. Hence, the effects of E2 might be partially mediated via a PKC isoform that becomes down-regulated by TPA. These conclusions should be regarded with caution due to the diminished (20% of the control value) [3H]thymidine incorporation by the cells after the long term TPA treatment.

On the other hand, the responsiveness of prostate cells to melatonin (as evidenced by melatonin’s effects on [3H]thymidine incorporation, cAMP, and cGMP) did not resume upon prolonged incubation (4 days) with E2. Moreover, the apparent affinity of the binding sites was further reduced. These data are compatible with a role for a nondown-regulated PKC isoform in the E2-mediated inactivation of melatonin receptors. On the other hand, it is possible that besides activating PKC, E2 also acts via genomic pathways to increase PKC expression. Future experiments will be aimed at exploring E2-sensitive PKC isoforms in the prostate epithelial cells.

As shown here, pretreatment of the cells with GF 102903X prevented the E2-mediated decrease in apparent binding affinity in the cells, but significantly reduced the density of the binding sites. A similar phenomenon was noted in our recent studies in cells maintained in charcoal-stripped medium. In the latter, pretreatment with GF 102903X prevented the decrease in apparent binding affinity provoked by short term (30-min) TPA treatment, but significantly reduced binding site density. In both experiments, the reduction of binding site density did not suppress melatonin efficacy. These data imply that, as with other hormone receptors, there are spare receptors for melatonin in the cells.

Taken together, the data presented here show that melatonin responses in human prostate epithelial cells depend on the prevailing hormonal state. Namely, DHT supports, whereas E2 inactivates, melatonin receptor binding and responses. These data demonstrate a modulatory role for melatonin in the androgen-estrogen interplay in human prostate epithelium.

Received February 12, 1997.

Revised April 14, 1997.

Accepted April 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Guess HA. 1992 benign prostatic hyperplasia antecedents and natural history. Epidemiol Rev. 14:131–153.[Free Full Text]
  2. Gann PH, Hennekens CH, Longcope C, Verhoek-Oftedahl W, Gordestien F, Stamfer MJ. 1995 A prospective study of plasma hormonal levels, nonhormonal factors, and development of benign prostate hyperplasia. Prostate. 26:40–49.[Medline]
  3. Partin AW, Oesterling JE, Epstein JI, Horton R, Walsh PC. 1991 Influence of age and endocrine factors on volume of benign prostate hyperplasia. J Urol. 145:504–409.
  4. Kumar VL, Wadhwa SN, Kumar V, Farooq A. 1990 Androgen, estrogen, and progesterone receptor contents and serum hormone profiles in patients with benign hypertrophy and carcinoma of the prostate. J Surg Oncol. 44:122–128.[Medline]
  5. Winter ML, Liehr JG. 1996 Possible mechanism of induction of benign prostatic hyperplasia by estradiol and dihydrotestosterone in dogs. Toxicol Appl Pharmacol. 136:211–219.[CrossRef][Medline]
  6. Mobbs BG, Johnson IE, Liu Y. 1990 Quantitation of cytosolic and nuclear estrogen and progesterone receptor in benign, untreated, and treated malignant human prostatic tissue by radioligand binding and enzyme-immunoassays. Prostate. 16:235–244.[Medline]
  7. Ehara H, Koji T, Deguchi T, Yoshii A, Nakano M, Nakane PK, Kawada Y. 1995 Expression of estrogen receptor in diseased human prostate assessed by non-radioactive in situ hybridization and immunohistochemistry. Prostate. 27:304–313.[Medline]
  8. Schulze H, Claus S. 1990 Histological localization of estrogen receptors in normal and diseased human prostates by immunohistochemistry. Prostate. 16:331–343.[Medline]
  9. Reiter RJ. 1991 Pineal melatonin: cell biology of its synthesis and of its physiological interaction. Endocr Rev. 12:151–180.[Medline]
  10. Laudon M, Gilad E, Matzkin H, Braf Z, Zisapel N. 1996 Putative melatonin receptors in benign human prostate tissue J Clin Endocrinol Metab. 81:1336–1342.[Abstract]
  11. Gilad E, Laudon M, Matzkin H, et al. 1996 Functional melatonin receptors in human prostate epithelial cells. Endocrinology 137:1412–1417.
  12. Laudon M, Zisapel N. 1986 Impact of circulating estradiol on melatonin binding sites in discrete areas of the female rat brain. Brain Res. 402:146–150.[CrossRef]
  13. Zisapel N, Shaharabani M, Laudon M. 1987 Regulation of melatonin’s activity in the female rat brain by estradiol: effects on neurotransmitter release and on iodomelatonin binding sites. Neuroendocrinology. 46:207–217.[Medline]
  14. Anis Y, Zisapel N. 1991 Castration affects brain iodomelatonin binding in hamsters maintained in long but not short days. Mol Cell Endocrinol. 76:23–34.[CrossRef][Medline]
  15. Vanecek J, Kosar E, Vorlicek J. 1990 Daily changes in melatonin binding sites and the effect of castration. Mol Cell Endocrinol. 73:165–170.[CrossRef][Medline]
  16. Menendez-Pelaez A, Lopez-Gonzalez MA, Guerrero JM. 1993 Melatonin binding sites in the Harderian gland of Syrian hamsters: sexual differences and effect of castration. J Pineal Res. 14:34–38.[Medline]
  17. Gilad E, Shanas U, Terkel J, Zisapel N. 1997 Putative melatonin receptors in harderian gland of the blind mole rat. J Exp Zool. 277:435–441.[CrossRef][Medline]
  18. Fujimoto J, Hori M, Ichigo S, Morishita S, Tamaya T. 1996 Estrogen induced expression of c-fos and c-jun via activation of protein kinase C in an endometrial cancer cell line and fibroblasts derived from human uterine endometrium. Gynecol Endocrinol. 10:109–111.[Medline]
  19. Cutler RE, Maizels ET, Hunzicker-Dunn M. 1994 Delta protein kinase C in rat ovary: estrogen regulation and localization. Endocrinology. 135:1669–1678.[Abstract]
  20. Maeda T, Lloyd RV. 1993 Protein kinase C activity and messenger RNA modulation estrogen in normal and neoplastic rat pituitary tissue. Lab Invest. 68:472–480.[Medline]
  21. Levy L, Alvaro V, Dubray C, Joubert D. 1994 Ca(2+)-dependent protein kinase C isoforms in rat pituitary hyperplasia: effect of in vivo treatment with quinagolide. Eur J Pharmacol. 268:327–334.[CrossRef][Medline]
  22. Wetsel WC, Negro-Vilar A. 1989 Testosterone selectively influences protein kinase-C-coupled secretion of proluteinizing hormone-releasing hormone-derived peptides. Endocrinology. 125:538–547.[Abstract]
  23. Lacasa D, Agli B, Mur B, Dieudonne MN, Giudicelli Y. 1993 Protein kinase in rat adipocytes: influence of androgenic status and regional fat distribution. J Endocrinol. 138:493–501.[Abstract]
  24. Galdieri M, Caporale C, Adamo S. 1986 Calcium-, phospholipid-dependent protein kinase activity of cultured rat Sertoli cells and its modifications by vitamin A. Mol Cell Endocrinol. 48:213–220.[CrossRef][Medline]
  25. Garcia-Paramino P, Carmena MJ, Roman F, Colas B, Prieto JC. 1993 Characterization of protein kinase C in rat and human prostates. Biosci Rep. 13:313–323.[CrossRef][Medline]
  26. Vakkuri O, Leppaluoto J, Vuolteenaho O. 1984 Development and validation of a melatonin radioimmunoassay using radioiodinated melatonin tracer. Acta Endocrinol (Copenh). 106:152–157.[Medline]
  27. Markwell MAK, Haas SM, Bieber LC, Talbert NE. 1978 A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 87:206–210.[CrossRef][Medline]
  28. Sokal RR, Rholf FJ. 1969 Biometry. San Francisco: Freeman.
  29. Nakhla AM, Ding VD, Khan MS, et al. 1995 5{alpha}-Androstan-3{alpha},17ß-diol is a hormone: stimulation of cAMP accumulation in human and dog prostate. J Clin Endocrinol Metab. 80:2259–2262.[Abstract]
  30. Steiner MS, Wand GS, Barrack ER. 1994 Effects of transforming growth factor beta-1 on the adenylyl cyclase-cAMP pathway in prostate cancer. Growth Factors. 11:283–290.[Medline]
  31. Galand P, Rooryck J. 1996 Mediation by epidermal growth factor of the estradiol-induced increased in cyclic guanosine 3',5'-monophosphate content in rat uterus. Endocrinology. 137:1932–1937.[Abstract]
  32. White RE, Darkow DJ, Lang JL. 1995 Estrogen relaxes coronary arteries by opening BKCa channels through a cGMP-dependent mechanism. Circ Res. 77:936–942.[Abstract/Free Full Text]
  33. Sirotkin AV, Mlyncek J, Lavrincik J, Bulla J, Hetenyi L. 1995 The ability of steroid hormones to control cAMP and cGMP production by human granulosa cells in culture. Cell Signal. 7:61–65.[CrossRef][Medline]
  34. Potter LR, Garbers DL. 1994 Protein kinase C-dependent desensitization of atrial natriuretic peptide receptor is mediated by dephosphorylation. J Biol Chem. 269:14636–14642.[Abstract/Free Full Text]
  35. Shigematsu Y, Vaughan J, Frohlich ED, Cole FE. 1994 Adenosine 5'-triphosphate, phorbol ester, and pertussis toxin effects on atrial natriuretic peptide stimulation of guanylate cyclase in a human renal cell line. Life Sci. 54:213–221.[CrossRef][Medline]
  36. Martikainen PM, Makela SI, Santti RS, Harkonen PL, Suominen JJ. 1987 Interaction of male and female sex hormones in cultured rat prostate. Prostate. 11:291–303.[Medline]
  37. Audt MC, Vacher P, Dufy B. 1996 17ß estradiol stimulated a rapid Ca2+ influx in LNCaP human prostate cancer cells. Eur J Endocrinol. 135:367–373.[Abstract]
  38. Uchida MK, Mita M, Oishi K, Hishinuma S. 1994 Desensitization of the m3-muscarinic acetylcholine-receptor in the smooth muscle and the intracellular signal transduction. Nippon Yakurigaku Zasshi. 104:251–262.[Medline]
  39. Nilsson L, Bergstrom L, Meyersson G, Pahlman S, Winblad B, Folkesson R. 1995 Somatostatinergic phenotype markers in the human neuroblastoma cell-line LA-N-2. FEBS Lett. 372:88–92.[CrossRef][Medline]
  40. Nagata KI, Okano Y, Nozawa. 1996 Protein kinase C isozymes in human megakaryoblastic leukemia cell line, MEG-01: possible involvement of the isozymes in the differentiation process of MEG-01 cells. Br J Haematol. 93:762–771.[CrossRef][Medline]
  41. Oka M, Ogita K, Ando H, Kikkawa U, Ichihashi M. 1995 Differential down-regulation of protein kinase C subspecies in normal human melanocytes: possible involvement of the zeta subspecies in growth regulation. J Invest Dermatol. 105:567–567.[CrossRef][Medline]
  42. Chen CC, Cheng CS, Chang J, Huang HC. 1995 Differential correlation between translocation and down-regulation of conventional and new protein kinase C isozymes in C6 glioma cells. J Neurochem. 64:818–824.[Medline]



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A. GARCÍA RATO, J. GARCÍA PEDRERO, M. A. MARTÍNEZ, B. DEL RIO, P. S. LAZO, and S. RAMOS
Melatonin blocks the activation of estrogen receptor for DNA binding
FASEB J, May 1, 1999; 13(8): 857 - 868.
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