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The human prostate cancer cell line LNCaP bears functional membrane testosterone receptors that increase PSA secretion and modify actin cytoskeleton¹

MARILENA KAMPA^*, EVANGELIA A. PAPAKONSTANTI, ANASTASSIA HATZOGLOU^*, EFSTATHIOS N. STATHOPOULOS, CHRISTOS STOURNARAS and ELIAS CASTANAS^*2

Laboratories of
^* Experimental Endocrinology,
Biochemistry and
Pathology, University of Crete, School of Medicine, Heraklion, GR-71110, Greece

²Correspondence: University of Crete, School of Medicine, Laboratory of Experimental Endocrinology, P.O. Box 1393, Heraklion, GR-71110, Greece. E-mail: castanas{at}med.uoc.gr

SPECIFIC AIM

Different steroids enter the cell and exert their action after binding to intracellular proteins, the respective cognitive hormone receptors, sharing characteristics of nuclear transcription factors. In recent years, however, some reports have indicated that these hormones may also act through membrane binding sites by a mechanism not involving nuclear effects. In the present work, we report the identification of a membrane receptor for testosterone in the human hormone receptor-positive cell line LNCaP. We show that this binding site is different from the classical androgen receptor and that its activation results, within minutes, in actin polymerization, redistribution of actin cytoskeleton, and an increase of PSA secretion in the culture medium.

PRINCIPAL FINDINGS

1. Testosterone induces PSA secretion by LNCaP cells through membrane sites, different from classical androgen receptors
In LNCaP cells, testosterone and its biologically active metabolite dihydrotestosterone (DHT) increased, in a dose-dependent manner, the synthesis and secretion of PSA after 24 h incubation. This effect involves intracellular androgen receptors and modulation of androgen-dependent PSA gene through induction of an androgen-responsive element. We further applied testosterone for brief incubation times (1 min-1 h) and measured its effect on PSA secretion. DHT increased significantly (by 20%) PSA secretion after 10 min. The same effect was found when testosterone was replaced by BSA-testosterone conjugate, which cannot penetrate the cell. The same action was observed when the nonsteroidal anti-androgen flutamide was applied alone or in addition to testosterone-BSA, indicating a membrane-mediated effect of testosterone. The above result indicates that testosterone might bind to membrane sites on LNCaP cells, different from the classical androgen receptor, as androgen and anti-androgen show the same effect; an anti-androgen receptor primary antibody, identifying cytosolic and nuclear-translocated androgen receptors produced no membrane staining of LNCaP cells in the absence or in the presence of testosterone.

2. Identification of membrane binding of testosterone in LNCaP cells
To further investigate the presence of membrane testosterone binding sites, we used a testosterone-BSA-FITC conjugate that cannot translocate in the cell. FITC-related fluorescence was followed by flow cytometry and identified by confocal laser microscopy. After 1 min of incubation, 50% of the cells were labeled by the testosterone-BSA-FITC conjugate. With time, an increase in specific fluorescence intensity was observed. Fluorescence intensity reached a maximum after 30 min incubation and was reduced when LNCaP cells where incubated with testosterone-BSA-FITC in the presence of 1000-fold excess of DHT. Confocal laser microscopy revealed a specific membrane staining by the testosterone-BSA-FITC conjugate. The specific fluorescence intensity was increased from 1 to 30 min.

To further identify specific membrane testosterone binding sites, we prepared membranes form LNCaP cells cultured in the absence of serum, in order to eliminate the presence of sex hormone binding globulins and performed saturation binding of radiolabeled testosterone. As shown in Fig. 1 A, [³H]testosterone ranging from 1 to 50 nM induces a specific saturable binding. Scatchard analysis of the results (Fig. 1A , insert) revealed a high binding affinity for testosterone (K_D 10.9 nM) and binding sites of 144.3 fmol/mg protein corresponding to 13,340 sites/cell. This membrane binding component was androgen selective, as shown in Fig. 1B . DHT produced a displacement of radiolabeled testosterone whereas estradiol and progesterone displaced radiolabeled testosterone with a significant lower affinity (10⁴- and 10²-fold, respectively).

View larger version (27K): [in this window] [in a new window]   Figure 1. Saturation (A) and displacement binding (B) of [3H]testosterone on membranes of LNCaP cells. A) Saturation binding. Cell membranes were incubated at 4°C with 6 concentrations of [3H]testosterone, varying from 2 to 50 nM, in the absence or presence of a thousand-fold excess of unlabeled androgen (DHT). The insert presents analysis of the data in Scatchard coordinates. Results of a typical experiment performed in triplicate. B) Displacement binding. Cell membranes (200 µg) were incubated with 5 nM of [3H] Testosterone alone (Bo) or in the presence of the indicated concentrations of unlabeled steroids (DHT, estradiol, progesterone) ranging from 10-12 to 10-6 M. Nonspecific binding was assayed by introducing 5 µM DHT. The figure (means of three different experiments performed in duplicate) presents the ratio of specific binding in the presence of the indicated concentrations of DHT (Bs) to the specific binding in the absence of DHT (Bo), Bs/Bo.

3. Brief incubation with DHT redistributes the actin cytoskeleton
As indicated above, testosterone binding sites activation resulted in an increased secretion of PSA. PSA, a serine-protease, is stored in intracellular secretion vesicles. To verify whether cytoskeletal modifications were involved, we analyzed the effects of cytochalasin B, an actin cytoskeleton-disrupting agent, on testosterone-induced PSA secretion. The secretion of PSA was significantly decreased in control cells that had been incubated with 100 µg/ml cytochalasin B, whereas incubation of cells with 10^-7 M DHT or BSA-testosterone for 30 min led to a significant increase of PSA release. Preincubation of cells with cytochalasin B before the application of DHT resulted in a complete reversal of DHT effects on PSA release. This effect was comparable to the action of cytochalasin B on control cells, suggesting the involvement of actin cytoskeleton in the effect of membrane testosterone action.

To further analyze the involvement of membrane testosterone binding sites on the cytoskeleton, we measured modification of the actin polymerization dynamics in LNCaP cells after brief testosterone incubation. The ratio of Triton-soluble (monomeric) to Triton-insoluble (filamentous) actin ratio decreased significantly by 36% and 47% 10 min after DHT or testosterone-BSA application, respectively, indicating an increase in the proportion of filamentous actin. These quantitative data were confirmed by confocal laser microscopic analysis of the actin cytoskeleton in LNCaP cells incubated with DHT or testosterone-BSA. A complete submembrane redistribution of the actin polymers became evident, as shown in Fig. 2 .

View larger version (20K): [in this window] [in a new window]   Figure 2. Confocal laser image of the actin cytoskeleton after brief incubation with DHT. LNCaP cells cultured in coverslips were fixed and stained for filamentous actin. They were examined in a confocal laser microscope at a 100x magnification. Sections of 8 microns are presented. Upper lane: control (untreated) cells; lower lane: cells treated with DHT for 10 min. Similar results were obtained when DHT was replaced testosterone-BSA.

CONCLUSIONS

Prostate cancer is the second most common neoplasia in men. It is a hormone-regulated cancer, and its treatment at initial stages involves anti-androgen adjuvant therapy. Steroid hormones, including androgen, are small molecules that move freely through the cell membrane and exert their actions after binding to intracellular receptors. The complex androgen-androgen receptor dimerizes, translocates to the nucleus, binds to specific responsive elements of DNA, and affects the transcription of androgen-responsive genes (Fig. 3 ). This pathway is the classical model of action for all steroids and is exerted after relative long periods of steroid application, since it involves mRNA and protein synthesis (genomic action). In recent years, however, it was reported that besides this genomic effect, steroid hormones could also exert effects that are not mediated via the classical receptors. These effects are mediated through membrane steroid binding sites, explaining the very rapid effect of steroids in nonclassical steroid receptor-bearing cells and cell lines. The first evidence of a nongenomic membrane testosterone action (hepatic glycogen phosphorylase activity) was reported in 1984. Membrane testosterone binding sites have been identified in chicken osteoblasts and Sertoli cells, rat T cells (both CD4 and CD8 positive), macrophages, rat epididymis, and rat ventral prostate. In all cases, membrane testosterone binding induced intracellular Ca²⁺ fluxes.

View larger version (23K): [in this window] [in a new window]   Figure 3. Schematic representation of testosterone genomic and nongenomic actions in LNCaP cells.

The results of the present study show for the first time a functional androgen binding element on membranes of the human prostate cancer cell line LNCaP. This component is different immunologically and functionally from classical intracellular androgen receptors and presents all the characteristics of a true binding site, being saturable (Fig. 1A ), and selective for androgen (Fig. 1B ). Our results indicate that the activation of membrane testosterone receptors induces the secretion of PSA from LNCaP cells in a dose- and time-related manner. PSA synthesis and secretion has been described as a genomic testosterone effect that occurs after relatively long incubation periods. To exclude such a genomic action of testosterone, we repeated the experiments using a nonpermeable testosterone-BSA conjugate that had exactly the same effects as on PSA secretion, indicating a membrane effect of testosterone on PSA secretion by LNCaP cells. Flutamide, a nonsteroid anti-androgen, exhibited the same stimulatory effects on PSA secretion.

Actin cytoskeleton changes play a central role in early cellular responses induced by a variety of stimuli. Several cellular functions such as endo- and exocytosis, secretion, membrane trafficking, and membrane transport involve changes in the polymerization state of actin. Our results show that this was also the case in our study. Indeed, PSA secretion under DHT is completely reversed by the blocker of actin polymerization cytochalasin B, whereas short-term actions of testosterone and testosterone-BSA modify profoundly the redistribution of monomeric and polymeric actin and remodel completely the actin cytoskeleton (Fig. 2) , redistributing it under the cell membrane. Preliminary results from our laboratory indicate that testosterone membrane signaling triggers profoundly actin-regulating proteins, indicating a direct implication of these membrane receptors on cytoskeletal reorganization, which may regulate trafficking and secretion of molecules, including PSA (nongenomic effects, Fig. 3 ).

The results of the present study identify for the first time a membrane functional androgen receptor, different from the classical intracellular androgen binding molecules, in prostate cancer LNCaP cells. We have shown that this membrane site is involved in early androgen-related PSA secretion and in the remodeling of the submembranous actin cytoskeleton structures. It would be of great interest to investigate the possible expression of this site in normal, adenomatous, and neoplastic lesions of the prostate. Implication of this site to the reorganization of the cellular cytoskeleton and activation of actin signaling molecules could possibly provide useful additional hints for prostate cancer chemotherapy, in which cytoskeleton-modifying agents are used with promising results.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0131fje; to cite this article, use FASEB J. (July 18, 2002) 10.1096/fj.02-0131fje

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