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Endocrinology, doi:10.1210/en.2007-1318
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Endocrinology Vol. 149, No. 2 565-573
Copyright © 2008 by The Endocrine Society

Estrogens Promote Human Testicular Germ Cell Cancer through a Membrane-Mediated Activation of Extracellular Regulated Kinase and Protein Kinase A

Adil Bouskine, Marielle Nebout, Baharia Mograbi, Françoise Brücker-Davis, Cyril Roger and Patrick Fenichel

Unité Mixte de Recherche Institut National de la Santé et de la Recherche Médicale Unité 670 (A.B., M.N., B.M., F.B.-D., C.R., P.F.), University of Nice-Sophia-Antipolis (UNSA), 06100 Nice, France; and Department of Reproductive Endocrinology (F.B.-D., P.F.) University Hospital of Nice (CHUN), 06200 Nice, France

Address all correspondence and requests for reprints to: Professor Patrick Fénichel, Unité Mixte de Recherche Institut National de la Santé et de la Recherche Médicale Unité 670 Faculty of Medicine of Nice, Avenue de Vallombrose, 06102 Nice cedex 02, France. E-mail: fenichel.p{at}chu-nice.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Clinical and experimental studies have suggested that estrogens, the archetype of female hormones, participate in the control of male germ cell proliferation and that fetal exposure to environmental estrogens may contribute to hypofertility and/or to testicular germ cell cancer. However, the underlying mechanisms remain to be elucidated. 17β-Estradiol (E2) conjugated to BSA was able to stimulate human testicular seminoma cell proliferation by triggering a rapid, nongenomic, membrane-mediated activation of ERK1/2 and cAMP-dependent protein kinase A (PKA). Both ERK1/2 and PKA participated in this promoting effect. This activation was associated with phosphorylation of the transcription factor cAMP response element-binding protein and the nuclear factor retinoblastoma protein. Enhanced proliferation together with ERK activation could be reversed by pertussis toxin, a G protein inhibitor. Estrogen receptors (ERs) in JKT-1 were characterized by immunofluorescence, subcellular fractioning, and Western blot. JKT-1 cells did not express ER{alpha} but ERβ, which localized to the mitochondria and the nucleus but not to the membrane. Moreover, neither ICI-182,780, a classical ER antagonist, nor tamoxifen, a selective ER modulator, could reverse the 17β-estradiol-BSA-induced promoting effect. Estrogens contribute to human testicular germ cell cancer proliferation by rapid activation of ERK1/2 and PKA through a membrane nonclassical ER. This nongenomic effect represents a new basis for understanding the estrogenic control of spermatogenesis and evaluating the role of fetal exposure to xenoestrogens during malignant transformation of testicular germ stem cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS NOW WELL established that estrogens, the archetype of female hormones, participate in the control of normal spermatogenesis (1). Indeed, 17β-estradiol (E2) is present at high concentrations in the adult testis where it is produced after testosterone conversion by aromatase (2). Human germ cells (3) express both aromatase and estrogen receptor (ER)-β. Strikingly, invalidation of the aromatase gene in mice results in male infertility with aging (4), and consistently, men with loss-of-function mutations in this gene display abnormal spermograms (5). However, the physiological role of estrogens during spermatogenesis and the molecular mechanisms by which they may regulate germ cell proliferation remain to be elucidated. Identifying these mechanisms is of particular interest because fetal exposure to environmental estrogens has been blamed for the increasing incidence of male infertility and testicular cancer (6), which stem, respectively, from impaired or excessive germ cell proliferation.

Spermatogenesis includes two steps of proliferation that concern first the fetal germ cells, the gonocytes, and second the adult germ stem cells, the spermatogonia. Testicular germ cell cancer, the most frequent cancer of young men, is considered to be raised from transformed gonocytes or undifferentiated spermatogonia (6). Interestingly, estrogens are able to stimulate proliferation of rat neonatal gonocytes in vitro (7), to induce spermatogenesis in the hypogonadal mouse (8), and to prevent apoptosis of human adult postmeiotic germ cells (9) cultivated in preserved seminiferous tubules. Conversely, we have also shown that E2 is able to inhibit human seminoma cell proliferation in vitro through an ERβ-dependent mechanism (10) suggesting that ERβ acts on germ cells as a tumor suppressor according to the observations made in ERβ knockout mice by Delbes et al. (11) on neonatal gonocytes. All these reports suggest that estrogens may either stimulate or inhibit germ cell proliferation via different opposite mechanisms. Indeed, there is now convincing evidence that estrogens, in addition to the classical ER-mediated nuclear regulation of estrogen-responsive genes, are able to trigger rapid membrane activation of a variety of second messenger-mediated signal transduction pathways (12) with possible implications on cell proliferation, apoptosis, or survival (13). However, the nature of these membrane ERs, related or unrelated to the classical ERs, and the precise signaling pathways that are activated remain to be clarified (14, 15). Thus, we investigated in this paper the hypothesis that estrogens, in addition to the abovementioned ERβ-dependent suppressive effect (10), could stimulate seminoma cell proliferation through such a nongenomic effect. We were able to show a rapid activation of MAPK and protein kinase A (PKA) signaling pathways induced by estrogens through a membrane, nonclassical ER, which requires G protein activation, illustrating that estrogens and xenoestrogens, suspected as etiological factors in breast or prostate cancers, could also represent in testicular germ cell cancer, through this nongenomic pathway, possible promoting agents.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and cell proliferation assay
The JKT-1 cell line was established from a human pure testicular seminoma developed from the testis of a 40-yr-old man (16). JKT-1 cells express placental alkaline phosphatase (17), a specific seminoma marker. JKT-1 cells were maintained in DMEM (Life Technologies, Inc., BRL, Rockville, MD) supplemented with penicillin-streptomycin (1%), sodium pyruvate (2%), and 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. Cells were seeded in six-well plates (0.6 x 106 cells per well). After 48 h, the cells were washed and estrogen starved overnight in fresh phenol red-free DMEM (Sigma Chemical Co., St. Louis, MO) supplemented with 1% charcoal-stripped fetal bovine serum, before adding E2 (Sigma), 17{alpha}-estradiol (Sigma), E2-BSA devoid of free E2, testosterone-BSA (Sigma), ICI-182,780 (Falsodex; Astra-Zeneca, Birmingham, UK), or tamoxifen (Sigma) at different concentrations or ethanol as vehicle control.

Cells were harvested by trypsin and counted using a Malassez hemocytometer. Results are expressed as percentages of variation as compared with the control.

RT-PCR analysis
Total RNAs were prepared from normal human testis and JKT-1 and MCF-7 cells. RT-PCR analysis was performed as described previously (10).

Western blot analysis
JKT-1 cells were grown in 10-cm dishes at a density of 4.9 x 106 cells per dish. After 48 h, the cells were washed with PBS and incubated overnight in phenol red-free DMEM/0.1% BSA and exposed to ligands for different times. Anisomycin and forskolin were purchased from Sigma, and PP2, PD98059, and H89 were obtained from VWR Calbiochem (La Jolla, CA). After washing with PBS, cell pellets were lysed in ice-cold lysis buffer Brij/Nonidet P-40 [50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 1% Brij 96 (Fluka, St. Quentin Fallavier, France), 1 mM Na3VO4, 10 mM β-glycerophosphate, 10 mM NaF, 2 mM EDTA, and protease inhibitors (Complete; Roche Diagnostics, Indianapolis, IN)]. Lysates were sonicated 7 sec on ice twice and then centrifuged for 15 min at 14,000 rpm. Equal amounts of whole protein extract were resolved on a 9% SDS-polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane (Immobilon P), probed with the antibodies against p44/42 MAPK, phospho-cAMP response element-binding protein (phospho-CREB), caspase 3, heat-shock protein 60 (Cell Signaling), ER{alpha}, ERβ, and lamin B (Santa Cruz Biotechnology, Santa Cruz, CA). After the blots were stripped, equal loading of proteins was verified by reprobing the same blots with anti-ERK antibody (Cell Signaling).

Immunocytochemical procedures
Cells were fixed in cold methanol at –20 C, washed twice in PBS, and then saturated in PBS/0.1% Triton for 20 min. ER{alpha} and ERβ were detected using a rabbit anti-ER{alpha} (Santa Cruz) and a goat anti-ERβ (Santa Cruz). After three washes in PBS, the antibodies were detected using an antirabbit-Texas Red antibody 1/30 (Amersham, Piscataway, NJ) and a fluorescein isothiocyanate-labeled antigoat (Dako, Carpinteria, CA) 1/50 in PBS with 5% goat serum. The nucleus was stained with Hoechst 33253 (blue). Sections were examined with a confocal laser scanning microscope (Leica TCS SP).

Subcellular fractioning
JKT-1 cells were grown in 15-cm dishes at a density of 4.9 x 106 cells per dish. After 48 h, the cells were washed twice with 1x PBS and then resuspended in isotonic buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.5 mM dithiothreitol] and a cocktail of proteases (1 mM Na3VO4, 10 mM β-glycerophosphate, 10 mM NaF, 2 mM EDTA) and phosphatase inhibitors. After incubation on ice for 5 min, the suspension was centrifuged to remove unbroken cells and nuclei (1000 x g, 10 min). The mitochondria were then pelleted by centrifugation at 15,000 x g for 10 min. The supernatant was further centrifuged at 100,000 x g for 1 h. The resultant supernatant was designated as cytosol and the pellet as the membrane fraction. The nuclear pellet was resuspended in 10 mM Tris-HCl (pH 7.5), 2.5 mM KCl, 2.5 mM MgCl2 and isolated after centrifugation at 90,000 x g for 30 min through 2.1 M sucrose, containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2. All steps were performed on ice or at 4 C.

Purity of cytosolic, mitochondrial, and nuclear fractions was then tested using compartment-specific antibodies directed, respectively, against caspase 3, heat-shock protein 60 and lamin B.

Statistical analysis
Results of cell count or densitometric analysis are all expressed as percentages of variation as compared with the control. A nonparametric Mann-Whitney U test was used for statistical analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E2-BSA stimulates JKT-1 cell proliferation by activating ERK1/2
E2 at 10–9 M, a physiological intratesticular concentration, induced after 24 h exposition a significant decrease of cell proliferation (Fig. 1AGo) in agreement with our previous report (10), which was not reproduced by 17{alpha}-estradiol (Fig. 1AGo). Conversely, E2-BSA, an impermeable E2 conjugate, was able in the same conditions to stimulate JKT-1 cell proliferation (Fig. 1AGo) with a maximal effect around 10–9/10–12 M. This stimulation, not reproduced with testosterone-BSA (Fig. 1AGo) was still observed after 4 d of culture (Fig. 1BGo). This promoting effect was MAPK dependent because it was prevented (Fig. 1CGo) by pretreatment with PD98059, a MAPK kinase-specific inhibitor, whereas the suppressive effect triggered by E2 alone was MAPK independent (Fig. 1CGo).


Figure 1
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  		FIG. 1. E2-BSA stimulates JKT-1 cell proliferation by activating ERK1/2. JKT-1 cells were incubated with steroid-free medium or medium containing E2, 17{alpha}-estradiol (17{alpha}-E2), E2-BSA, or testosterone-BSA (T-BSA) for 24 h (A) or several days (B). C, Effect of pretreatment for 90 min with PD98059 (1 nM), an inhibitor of MEK, was evaluated after 24 h stimulation with E2 or E2-BSA. Results are given as means + SEM of three independent experiments and represent the percentage of variation of cell numbers compared with control (steroid-free medium with ethanol). *, P < 0.05.

 
To assess MAPK activation by estrogens, Western blot analysis of JKT-1 cellular extracts was performed using specific antibodies recognizing the phosphorylated forms of p42/p44 ERK (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). At 10–9 M E2-BSA, activation of ERK1/2 was observed as early as 5 min, with a maximum at 15 min (Fig. 2AGo) and a return to basal levels within a few hours (data not shown). This MAPK activation was ERK specific because neither stress kinase p38 nor JNK were significantly activated by E2-BSA (Fig. 2BGo). The same concentration of E2 activated ERK1/2 (Fig. 2CGo) with a quite similar time course as for E2-BSA (data not shown). This estrogen-induced ERK phosphorylation was abolished by PP2, an inhibitor of the Src tyrosine kinase family (Fig. 2CGo).


Figure 2
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  		FIG. 2. E2-BSA induced activation of ERK1/2. A, Western blot analysis of phospho-ERK1/2 and total ERK during activation by E2-BSA after pretreatment or not with PD98059 for 90 min. B, Western blot analysis of phospho-p38 and phospho-JNK during activation with E2-BSA; activation by anisomycin (A) is shown as positive control. C, Western blot analysis of phospho-ERK1/2 and total ERK after 15 min activation by E2-BSA or E2 after pretreatment or not with PP2 for 90 min (the blot shown is representative of three independent experiments).

 
E2-BSA activates PKA and CREB in JKT-1 cells
Activation of PKA was also necessary for E2-BSA to promote JKT-1 cell proliferation because H89, a specific PKA inhibitor, totally prevented cell proliferation increase (Fig. 3AGo). Extracellular stimuli elicit changes in gene expression in target cells by activating intracellular protein kinase cascades that phosphorylate transcription factors within the nucleus. CREB is one of these factors that activates gene transcription after phosphorylation of serine 133 induced by a variety of protein kinases including PKA and ERK (18). Using an anti-phospho-CREB antibody that recognizes phosphorylated serine 133, we were able to observe a very rapid (5 min) E2-BSA-induced activation of CREB in JKT-1 cells with a maximum at 15 min (Fig. 3BGo). This activation was PKA dependent because H89 was able to abolish completely CREB phosphorylation (Fig. 3BGo). It was partially ERK dependent because pretreatment with PD98059 reduced only moderately CREB phosphorylation (Fig. 3BGo). However, ERK activation was not PKA dependent because H89 did not prevent E2-BSA-induced ERK phosphorylation (Fig. 3CGo), demonstrating that ERK and PKA activations were independently necessary to promote E2-BSA-induced JKT-1 cell proliferation and that at the same time they were both contributing to CREB phosphorylation.


Figure 3
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  		FIG. 3. E2-BSA activates PKA and CREB in JKT-1 cells. A, JKT-1 cells were incubated for 24 h with steroid-free medium containing E2-BSA (1 nM) after pretreatment or not with H89 (5 µM) for 90 min. Results are given as means + SEM of three independent experiments and represent the percentages of variation of cell number compared with control (steroid-free medium with ethanol). B, Time-dependent phosphorylation of CREB. Cells were incubated with E2-BSA (1 nM) for the indicated time in serum-free DMEM. Cells were pretreated with 5 µM H89 or with 1 nM PD98059 for 90 min. Top, Western blot representative of three independent experiments. Forskolin (25 µM) was used as positive control for CREB activation. Bottom, Bands from three experiments were quantified by densitometry. Results were normalized to total ERK1/2 expression in each sample and were plotted with SEM. For each time, CREB phosphorylation was compared with or without pretreatment with kinase inhibitors PD98059 (PD) or H89. *, P < 0.05. C, Time-dependent phosphorylation of ERK1/2 analyzed by Western blot. Cells were incubated with E2-BSA (1 nM) for the indicated time in serum-free DMEM after pretreatment or not with 5 µM H89 or with 1 nM PD98059 (PD) for 90 min. EGF (10 ng/ml) was used as positive control for ERK1/2 activation (the blot shown is representative of three independent experiments).

 
Activation of a Gi protein is required for E2-BSA-induced JKT-1 promotion
G protein-coupled receptors have been proposed to be involved in triggering membrane action of steroids (19), including estrogens (20, 21). For this reason, we studied the effect of pertussis toxin, an inhibitor of Gi proteins, in E2-BSA-induced activation of JKT-1 cells. This toxin was able to prevent completely any increase of cell proliferation induced by E2-BSA (Fig. 4AGo) associated with an inhibition of ERK1/2 activation (Fig. 4BGo).


Figure 4
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  		FIG. 4. Effect of pertussis toxin on E2-BSA-induced cell proliferation. Serum-deprived JKT-1 cells were treated with vehicle or 100 ng/ml pertussis toxin (PTX) for 3 h, followed by incubation with E2-BSA (1 nM). A, Effect of PTX on JKT-1 cell proliferation after E2-BSA activation for 24 h. Results are given as means + SEM of three independent experiments and represent the percentages of variation of cell number compared with control (steroid-free medium with ethanol). *, P < 0.05. B, Western blot analysis of ERK1/2 phosphorylation. Anisomycin (A) (10 pg/ml) was used as positive control Top, The blot shown is representative of three independent experiments); bottom, bands from three experiments were quantified by densitometry and compared with control (steroid-free medium with ethanol). *, P < 0.05.

 
E2-BSA stimulates cell cycle progression by phosphorylating retinoblastoma protein (Rb) in JKT-1 cells
Rb is a nuclear factor that participates to the regulation of the cell cycle, interfering with cyclin action when nonphosphorylated. This suppressive effect is prevented through phosphorylation of Rb. In JKT-1 cells, E2-BSA induced a rapid (2 h) and intensive phosphorylation of Rb (Fig. 5Go), leading to phospho-Rb inactivation during estrogenic stimulation.


Figure 5
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  		FIG. 5. Rb phosphorylation during E2-BSA activation. Top, Western blot analysis of Rb phosphorylation during activation with E2-BSA (1 nM) for the indicated time; bottom, bands from three experiments were quantified by densitometry and compared with control (steroid-free medium with ethanol). *, P < 0.05.

 
Expression of ERs in JKT-1 cells
As we recently reported (10), ER{alpha} was not expressed at all in JKT-1 cells as verified by RT-PCR, Western blot, or immunofluorescence (Fig. 6AGo). MCF-7 breast cancer cells were used as positive control (Fig. 6AGo). Concerning ERβ, it appeared by immunofluorescence to have an intracytoplasmic and nuclear localization (Fig. 6BGo). Western blot after subcellular fractioning allowed localizing this receptor in the mitochondria and in the nucleus without any membrane localization (Fig. 6CGo). To verify whether classical ERβ was involved in this membrane-induced MAPK-mediated activation, we tested also the effect of ICI-182,780, a pure ER antagonist, and tamoxifen, a selective ER modulator. ICI-182,780 and tamoxifen were both able to counteract completely the suppressive effect of E2 on JKT-1 cell proliferation (Fig. 7Go), suggesting an ERβ-dependent mechanism. However, neither ICI-182,780 nor tamoxifen could prevent the promoting effect of E2-BSA, supporting that the rapid effect induced by E2-BSA was likely not dependent on a classical ER (Fig 7Go).


Figure 6
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  		FIG. 6. Expression of ERs in JKT-1 cells. A, Analysis of ER{alpha} expression by RT-PCR, Western blot, and immunofluorescence. J, JKT-1; M, MCF-7; T, human testis. B, ERβ expression by immunofluorescence in JKT-1 cells. The nucleus was stained by Hoechst 32533. C, Western blot analysis after subcellular fractioning; specific markers were us for each compartment: Cyt, cytoplasm; Mb, membrane; Mit, mitochondria; N, nucleus; Wcl, whole cell.

 

Figure 7
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  		FIG. 7. Effect of ERs antagonists on estrogen-induced cell proliferation. JKT-1 cells were incubated for 24 h with E2 (1 nM) or E2-BSA (1 nM) with or without ICI-182,780 or tamoxifen. Results are given as means + SEM of three independent experiments and represent the percentages of variation of cell number compared with control (steroid-free medium with ethanol). *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show here for the first time that estrogens were able to stimulate in vitro proliferation of human malignant testicular germ cells by inducing a rapid, nongenomic, membrane-initiated activation of ERK1/2 and PKA. This promoting effect was triggered by a nonclassical membrane ER and required activation of a Gi protein. The effect of low and physiological concentrations suggests a stimulatory role for E2 in the control of germ cell proliferation and supports estrogenic dependency of testicular cancer.

Estrogens classically mediate their action after binding to nuclear receptors that modulate as transcription factors the activity of target genes by interacting with several DNA response elements. In addition to their ability to mediate gene transcription, estrogens also elicit rapid, nontranscriptional effects by membrane-mediated signaling pathways leading to calcium influx (22), cAMP (23) or nitric oxide production (24), phospholipase C activation, inositol phosphate generation (25), or MAPK ERK1/2 activation (26, 27, 28). Interestingly, the two isoforms p42/p44 MAPK play a critical role in the control of cell proliferation, survival, and apoptosis (29).

In JKT-1 cells, rapidity of E2-induced activation of ERK1/2, around a few minutes with a peak at 15 min and return to basal after a few hours, fits well with the known implication of MAPK in growth factor receptor signaling pathways and rules out a transcriptional effect. The fact that BSA-conjugated E2, a membrane-impermeable steroid, without free E2, was able to activate ERK1/2 in the same way, illustrates the membrane impact of this activation and allows us to further discriminate between the ER-mediated suppressive effect of E2 crossing the membrane, described by our team in 2005 (10), and the membrane-mediated promoting effect described in this paper with E2-BSA. Inhibition by an antagonist of the Src tyrosine kinase family supported the participation of Src kinases in mediating the ERK1/2 activation by estrogens, as described in several models such as adipocytes or cerebellar neurons (26, 30). Both ERK phosphorylation and the induced promoting effect required activation of a Gi protein.

In addition to ERK1/2 activation, the participation of PKA in triggering the promoting effect of estrogens was illustrated by the complete reversion obtained after pretreatment with a specific PKA inhibitor on cell proliferation. PKA may act through CREB phosphorylation, allowing CRE-associated cell cycle gene regulation, because H89 inhibited both CREB phosphorylation and enhanced cell proliferation. ERK activation participated also in CREB phosphorylation as described in several models (26, 31), as illustrated by the moderate inhibition observed after PD98059 pretreatment. However ERK-mediated cell proliferation implies other transcription factors because PD98059 prevented completely the E2-BSA promoting effect. Moreover, activation of ERK1/2 is necessary per se and not mediated by PKA activation (26) because H89 did not inhibit E2-BSA-induced ERK activation. These results support in fact that in JKT-1 cells, both ERK1/2 and PKA activation are necessary to promote cell proliferation as represented in Fig. 8Go. Phosphorylation of Rb was observed during E2-BSA-induced JKT-1 activation, illustrating the stimulating effect of estrogens on cell cycle regulation.


Figure 8
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  		FIG. 8. Proposed model for signal transduction pathways triggered by E2-BSA in JKT-1 cells. E2-BSA stimulates JKT-1 cell proliferation through a nonclassical membrane ER. This effect involves Gi protein activation and triggers PKA and ERK1/2 signaling pathways; both PKA and ERK activate CREB and are individually able to control proliferation-regulating gene expression.

 
One important question remains regarding the nature of the membrane ER that triggers this promoting effect in JKT-1 cells. Different studies of nonclassical estrogen signaling in a variety of target cells, such as endothelial, neuronal, and pituitary cells (31, 32), have strongly suggested that nuclear classical ER or ER-like proteins are candidates for the membrane ERs. Transfection of naive CHO cells (33) with cDNA encoding for rat or human ERs, either ER{alpha} or ERβ, have shown that the majority of the ER pool localizes into the nucleus, but about 3% of the transfected ER{alpha} is present in the plasma membrane, and that both receptors are able to mediate such a membrane-initiated transduction pathway with a possible inhibition for ER{alpha} by a pure ER antagonist, ICI-182,780 (33). Moreover, interactions with signaling proteins that migrate to the plasma membrane such as the raft protein caveolin-1 (34) have been shown in human vascular smooth muscle or breast cancer cells (MCF-7) to help association of ERs with the cell surface in discrete plasma membrane regions rich in sphingolipid and cholesterol called caveolae. This has been shown to occur in several E2 target cells, for the membrane ER{alpha} bound to its ligand or for ERβ, although less frequently (35). We have clearly shown (10) and confirmed by RT-PCR and Western blot that JKT-1 cells do not express at all ER{alpha}. Concerning ERβ that we have identified by means of two different monoclonal antibodies (10), our subfractioning and Western blot studies clearly demonstrate that ERβ is localized in JKT-1 cells in the nucleus and in the mitochondria but not at the membrane. Moreover, classical ER antagonists, such as ICI-182780 or tamoxifen, did not inhibit the promoting effect, which makes unlikely the role of ERβ as the starter of the signaling cascade leading to ERK1/2 activation, although the participation of an ERβ isoform unrecognized by the antibodies we have used cannot be excluded.

Another possibility is the participation of a completely different and nonclassical steroid receptor as described for estrogens and progestogens (19, 36). Filardo (37) has shown that estrogen-induced ERK activation may occur in human breast cancer cells that do not express either ER{alpha} or ERβ. Consistently, in several cell types, evidence has been provided for the involvement of membrane ERs associated with G proteins, in nongenomic estrogenic actions (38, 39). GPR30, an orphan G protein-coupled receptor, has been proposed as a nonclassical ER mediating rapid cell signaling in breast, endometrial, or ovarian cancer cells (37, 39, 40, 41). Thus, GPR30 may represent a pertinent candidate for our nonclassical membrane ER expressed by JKT-1 cells, able to activate both PKA and ERK pathways. Its expression and involvement in triggering an estrogenic promoting effect in human malignant testicular germ cells are now under verification in our laboratory.

Our results showing a promoting effect of E2-BSA on human malignant testicular germ cells via a nongenomic membrane-mediated activation of ERK1/2 and PKA complement our recent report describing a suppressive effect obtained with E2 via ERβ (10). They strongly suggest, as for breast, ovarian, endometrial, or prostatic cancers, estrogen dependency of testicular cancer.

The resulting impact of estrogens on normal or malignant germ cells may depend on the relative expression of both receptors (ERβ and nonclassical membrane ER) and on the respective binding affinity of the estrogenic compound. Human gonocytes do not express the active ERβ1 isoform until the prenatal period (42). They should then during fetal life be exclusively submitted to the nongenomic membrane-mediated promoting effect. Most testicular germ cell cancers share biomarkers with and are considered to be issued from gonocytes or undifferentiated spermatogonia (6). Our results with JKT-1 cells issued from a pure human testicular seminoma and expressing placental alkaline phosphatase (10), a gonocyte biomarker, suggest therefore that excessive fetal exposure of gonocytes to xenoestrogens with high affinity for nonclassical membrane ERs, as shown for example for ortho, para-dichlorodiphenyl-dichloroethylene in pancreatic β-cells (36), may contribute to the malignant germ stem cell transformation, leading at birth, as proposed by Skakkebaek et al. (6), to carcinoma in situ and then after puberty to testicular cancer.


   Footnotes
 
A.B. was supported in part by a grant from Fond d’Aide à la Recherche Organon and in part by the French Society of Endocrinology.

Disclosure Statement: The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

First Published Online November 26, 2007

Abbreviations: CREB, cAMP response element-binding protein; E2, 17β-estradiol; ER, estrogen receptor; JNK, c-Jun N-terminal kinase; PKA, protein kinase A; Rb, retinoblastoma protein.

Received September 24, 2007.

Accepted for publication November 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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R. Sirianni, A. Chimento, C. Ruggiero, A. De Luca, R. Lappano, S. Ando, M. Maggiolini, and V. Pezzi
The Novel Estrogen Receptor, G Protein-Coupled Receptor 30, Mediates the Proliferative Effects Induced by 17{beta}-Estradiol on Mouse Spermatogonial GC-1 Cell Line
Endocrinology, October 1, 2008; 149(10): 5043 - 5051.
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