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Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5; Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University (K.-C.C.), Ithaca, New York 14853; Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University (S.K.K.), Seoul, Korea 151-742; and Taipei Medical College Hospital (C.-J.T.), 110 Taipei, Taiwan
Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: . peleung{at}interchange.ubc.ca
Abstract
To investigate the role of FSH in ovarian cancer development, the present study examined the expression of FSH receptor (FSH-R) and the effect of FSH on proliferation of normal, preneoplastic, and neoplastic ovarian surface epithelium (OSE) cells. Recently, immortalized OSE (IOSE) cell lines, including IOSE-29 (preneoplastic) and IOSE-29EC (neoplastic), were used. Our results indicated that FSH-R mRNA was expressed and that FSH exerted a growth stimulatory effect in normal, preneoplastic, and neoplastic OSE cells. To investigate the mechanism of the growth stimulatory effect, the activation of MAPKs by FSH was examined in preneoplastic and neoplastic OSE cells. Treatment with FSH resulted in MAPK activation of IOSE-29 and IOSE-29EC cells, whereas the stimulatory effect of FSH on cellular proliferation and MAPK activation was completely abolished in the presence of PD98059, a MAPK kinase inhibitor, suggesting that the growth stimulatory effect of FSH is mediated through MAPK activation in these OSE cells. In a time-dependent study, FSH significantly increased MAPK activity at 5–10 min in IOSE-29 cells. The activated MAPK declined to the control level after 20 min in these cells. Similarly, treatment with FSH significantly induced MAPK activation after 5 min and sustained it for 60 min in IOSE-29EC cells. In addition, treatment with FSH resulted in substantial phosphorylation of Elk-1, confirming that FSH action is mediated via activation of MAPK. In conclusion, we have demonstrated that FSH-R was expressed, and FSH induced growth stimulation in normal, preneoplastic, and neoplastic OSE cells. Furthermore, treatment with FSH stimulated activation of the MAPK cascade and phosphorylated Elk-1 in neoplastic OSE cells. These results suggest that the MAPK cascade may be involved in cellular functions such as growth stimulation in response to FSH in preneoplastic and neoplastic OSE cells.
MOST OVARIAN TUMORS appear to arise from the ovarian surface epithelium (OSE), which is a simple squamous to cuboidal mesothelium covering the ovary based on histopathological observations, even though the exact mechanism of ovarian tumorigenesis has not been elucidated (1). Repeated ovulation and the process of healing ruptured OSE have been suggested to contribute to neoplastic transformation of OSE (2, 3). It has been suggested that endocrine factors such as gonadotropins and steroid hormones may influence the occurrence of ovarian tumors (2, 3, 4, 5, 6, 7). In addition to its well established function in reproductive physiology, FSH has been implicated in ovarian cancer development (8, 9). Epidemiological studies demonstrated an increased occurrence of ovarian cancer with exposure to high levels of gonadotropins during postmenopause or infertility therapy (2, 10, 11). Expression of FSH receptor (FSH-R), G protein-coupled receptor, has been demonstrated in normal OSE (12), ovarian inclusions, and epithelial tumors (9), implicating a possible role of FSH in these cells. In addition, treatment with FSH resulted in growth stimulation of rabbit OSE (13) and ovarian cancer cells (9, 14) in a dose- and time-dependent manner in vitro. Despite these findings the precise molecular mechanism of FSH in terms of growth stimulation and intracellular signaling in ovarian cancer remains unknown.
MAPKs are a group of serine/threonine kinases that are activated in response to a diverse array of extracellular stimuli and mediate signal transduction from the cell surface to the nucleus (15, 16). These MAPKs are divided into three subgroups, ERKs, Jun N-terminal kinases/stress-activated protein kinases, and p38. It is well known that the MAPK cascade is activated by two distinct classes of cell surface receptors, receptor tyrosine kinases and G protein-coupled receptors (16, 17, 18, 19, 20). The signals transmitted through this cascade lead to activation of a set of molecules that regulate cell growth, division, and differentiation. The most extensively studied members of the cascade are ERK1 (p44 MAPK) and ERK2 (p42 MAPK). In addition, it has been demonstrated that MAPK is regulated by cisplatin (21), paclitaxel (22), endothelin-1 (23), and GnRH (24) in ovarian cancer cells. However, the role of FSH in the MAPK cascade in neoplastic OSE cells has not been reported.
Recently, immortalized OSE (IOSE) cell lines, IOSE-29 (preneoplastic) and IOSE-29EC (neoplastic and tumorigenic), were generated from normal OSE directly by transfection with simian virus 40-large T antigen and subsequent E- cadherin (25, 26). The IOSE-29EC was found to be anchorage independent and formed transplantable, invasive sc and ip adenocarcinomas in SCID (severe combined immune deficiency) mice (27). Little is known about the molecular events that mediate FSH actions in normal, preneoplastic, and neoplastic ovarian cells. Considering that FSH might play a role in these cells (8, 9, 10, 11, 12, 13, 14), we sought to investigate the effect of FSH in normal OSE and IOSE cell lines. In the present study experiments were designed to investigate 1) the expression of FSH-R at the mRNA in normal OSE and IOSE cell lines, 2) a proliferative effect by FSH in these cells, and 3) the effect of FSH on ERK1/2 activation.
Materials and Methods
Materials
Human recombinant FSH was a gift from Dr. A. F. Parlow (National Hormone and Pituitary Program, Harbor-University of California- Los Angeles Medical Center, Torrance, CA). PD98059 [2-(2-amino-3- methoxyphenyl)-4H-1-benzopyran-4-one], a MAPK/ERK kinase (MEK) inhibitor, and 3-isobutyl-1-methylxanthine were purchased from New England Biolabs, Inc. (Beverly, MA), and Sigma-Aldrich Corp. (St. Louis, MO), respectively.
Cell culture
Normal human OSE cells were scraped from the ovarian surface during laparoscopies from premenopausal women for nonmalignant disorders and cultured as previously described (28) in medium 199/MCDB 105 (Sigma-Aldrich Corp.) containing 10% FBS (HyClone Laboratories, Inc., Logan, UT), 100 U/ml penicillin G, and 100 µg/ml streptomycin (Life Technologies, Inc., Gaithersburg, MD) in a humidified atmosphere of 5% CO2/95% air and passaged with 0.06% trypsin (1:250)/0.01% EDTA in Mg2+/Ca2+-free HBSS when confluent.
The nontumorigenic simian virus 40-Tag IOSE-derived line (IOSE-29), its tumorigenic derivatives (IOSE-29EC), and the cell lines derived from IOSE-29EC-inoculated SCID mice (IOSE-29EC/T4 and IOSE-29EC/T5) were cultured as previously described (25, 26, 27). For monolayer culture, the cell lines were maintained on tissue culture dishes (Falcon, Becton Dickinson and Co., Franklin Lakes, NJ) in a 1:1 mixture of medium 199/MCDB 105 medium supplemented with 10% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin. OVCAR-3 and SKOV-3 cells, which are ovarian carcinoma cancer cell lines, were cultured in the above-mentioned culture conditions and used for the following experiments.
RNA extraction, RT-PCR procedure, and Southern blot analysis
Total RNA was prepared from cultured cells using the RNaid kit (Bio/Can Scientific, Mississauga, Canada) according to the manufacturer’s suggested procedure. RNA integrity was confirmed using agarose gel electrophoresis and ethidium bromide staining. The total RNA concentration was determined from spectrophotometric analysis at A260/280. cDNA was synthesized from 2.5 µg total RNA by RT at 37 C for 2 h using a first-strand cDNA synthesis kit (Amersham Pharmacia Biotech., Québec, Canada). The synthesized cDNA was used as template for PCR amplification. A semiquantitative PCR amplification was carried out with denaturing for 1 min at 94 C, annealing for 35 sec at 55 C, extension for 90 sec at 72 C, and a final extension for 15 min at 72 C using a thermal cycler (DNA Thermal Cycler, Perkin-Elmer Corp., Norwalk, CT). The primers were designed to amplify FSH-R mRNA based on the published sequences of human FSH-R (12). In addition, amplification of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed using specific primers (29) to rule out the possibility of RNA degradation and was used to control the variation in mRNA amount in PCR reaction. The primers of FSH-R are composed of: sense, (5'-GAGAG CAAGG TGACA GAGAT TCC-3 (exon 1, nucleotides 97–120); and antisense, 5'-CCTTT TGGAG AGAAT GAATC TT-3' (exon 5, nucleotides 439–417). The sequences of GAPDH amplification are: sense, 5'-ATGTT CGTCA TGGGT GTGAA CCA-3'; and antisense, 5'-TGGCA GGTTT TTCTA GACGG CAG-3'. The PCR reactions were performed in 25 µl PCR mixture containing 1x PCR buffer, 0.2 mM of each dNTP, 1.6 mM MgCl2, 50 pmol specific primers, each cDNA template, and 0.25 U Taq polymerase. Twelve microliters of PCR products were analyzed by agarose (1%) gel electrophoresis and visualized by ethidium bromide staining, and the sizes were estimated by comparison to DNA mol wt markers. After electrophoresis, Southern blot analysis was performed to detect a specific signal with digoxigenin-labeled probes for FSH-R or GAPDH as previously described (30). In addition, PCR products isolated from the gel were cloned into pCRII vector using the TA cloning kit (Invitrogen, San Diego, CA) and were sequenced by the dideoxynucleotide chain termination method using the T7 DNA polymerase sequencing kit (Amersham Pharmacia Biotech.).
Northern blot analysis
Total RNAs (50 µg) were denatured in 50% formamide/2.2 M formaldehyde, incubated at 60 C for 15 min, and electrophoresed on 1% denaturing agarose gel (20 mM MOPS, 2.2 M formaldehyde, 8 mM sodium acetate, and 1 mM EDTA, pH 8.0) at a constant 70 V for 3–4 h. A capillary transfer of RNA to nylon membrane was performed overnight in 10x SSC, as described for Southern blot analysis. The membrane was then irradiated by UV light for 5 min to cross-link the RNA to the membrane. A radioactively labeled cDNA probe of FSH-R (provided by Dr. T. Minegishi, Department of Obstetrics and Gynecology, Gunma University School of Medicine, Gunma, Japan) (31) was prepared using a Random Labeling Kit (Life Technologies, Inc.) according to the manufacturer’s suggested procedure. DNA templates (100 ng) were denatured at 100 C for 5 min and placed on ice. Two microliters of each dNTP (dCTP, dGTP, and dTTP), 15 µl random priming buffer, 50 µCi [
-32P]dATP (3000 Ci/mmol; Amersham Pharmacia Biotech), and 3 U Klenow fragment were mixed. After a 3-h incubation at 25 C, the reaction was stopped by adding 5 µl Stop Buffer (0.2 mM Na2EDTA, pH 7.5). The labeled DNA was purified on a Sephadex G-50 column (Amersham Pharmacia Biotech). The membrane was prehybridized in standard hybridization solution (50% formamide, 5x SSPE, 5x Denhardt’s solution, 0.5% SDS, and 100 µg/ml denatured herring sperm DNA) for 3 h at 42 C. The membrane was washed once with 2x SSC/0.1% SDS at room temperature and twice with 0.1x SSC/0.1% SDS at 65 C for 15 min. After washing, the membrane was exposed to Kodak OMAT x-ray film (Eastman Kodak Co., Rochester, NY).
[3H]Thymidine incorporation assay
A [3H]thymidine incorporation assay was performed to analyze the effect of FSH on the proliferative index in normal, preneoplastic, and neoplastic OSE cells. The cells were plated in 24-well plates at 2 x 104 cells/well in 0.5 ml medium 199/MCDB105 supplemented with 10% FBS and antibiotics and incubated for 48 h. Before treatment with FSH, the cells were starved in serum-free medium for 4 h. After starvation, the cells were incubated with increasing concentrations (10, 100, and 1000 ng/ml) of FSH and 1 µCi [3H]thymidine (5.0 Ci/mmol; Amersham Pharmacia Biotech) in serum-free medium for 24 h. At the end of the incubation period, the culture medium was removed, and cells were washed three times with PBS, followed by precipitation with 0.5 ml 10% trichloroacetic acid for 20 min at 4 C. The precipitate was washed in methanol twice and solubilized in 0.5 ml 0.1 N sodium hydroxide, and the incorporated radioactivity was measured in a 1217 Rackbeta liquid scintillation counter (LKB Wallac, Inc., Turku, Finland) as previously described (30). In addition, the effect of FSH in the presence or absence of PD98059 was examined to elucidate whether MAPK activation by FSH is related to growth stimulation.
Immunoblot assay
The cells were seeded at a density of 2 x 105 cells in 35-mm culture dishes and cultured in a humidified atmosphere of 5% CO2-95% air at 37 C. Cells were washed once with medium and serum-starved for 4 h before treatments with FSH in the presence or absence of PD98059 (50 µM) in a time- and/or dose-dependent manner. After treatments, the cells were washed twice with ice-cold PBS and lysed in ice-cold RIPA buffer [150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris (pH 7.5), 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 100 µg/ml aprotinin]. The extracts were placed on ice for 15 min and centrifuged to remove cellular debris. The amount of protein in the supernatants was determined using a Bradford assay (Bio-Rad Laboratories, Inc., Richmond, CA). Thirty micrograms of total protein were run on 10% SDS-PAGE gels and electrotransferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Oakville, Canada). The membrane was immunoblotted using a mouse monoclonal antibody specific to the phosphorylated p44/p42 MAPK (P-MAPK; Thr202/Tyr204, New England Biolabs, Inc.) (32). Alternatively, a membrane was probed with a rabbit polyclonal antibody for p44/42 MAPK (New England Biolabs, Inc.), which detects total MAPK (T-MAPK; phosphorylation-state independent) levels. After washing, the signals were detected with horseradish peroxidase-conjugated secondary antibody and visualized using the ECL chemiluminescent system (Amersham Pharmacia Biotech). P-MAPK levels (p44 and p42) were quantified using a computerized visual light densitometer (model 620, Bio-Rad Laboratories, Inc.) and normalized against the levels of T-MAPK per sample.
In vitro MAPK assay
IOSE-29 and IOSE-29EC cells were serum-starved for 4 h. The cells were then treated with FSH (100 ng/ml) in the presence or absence of PD98059 for 10 and 30 min, respectively, washed twice with ice-cold PBS, and lysed in 1 x lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerol phosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonylfluoride]. The extracts were placed on ice for 15 min and centrifuged to remove cellular debris, and the amount of protein in the supernatants was determined. Cellular protein (200 µg) was immunoprecipitated with immobilized phospho-p44/42 MAPK monoclonal antibody. In vitro MAPK assays were performed using the Elk-1 fusion protein as a substrate for activated MAPK according to the manufacturer’s suggested procedure (New England Biolabs, Inc.).
RIA for intracellular cAMP
To measure intracellular cAMP, IOSE cell lines and human granulosa luteal cells (2 x 105 cells) were plated onto 35-mm culture dishes and cultured for 4 d. The cells were then preincubated in serum-free medium containing 0.1% BSA and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich Corp.) for 30 min and treated with FSH for 0, 5, 10, 20, or 60 min. Intracellular cAMP levels were measured using a [3H]cAMP assay system (Amersham Pharmacia Biotech), according to the manufacturer’s suggested procedure.
Data analysis
Data are shown as the mean ± SD of two or three individual experiments with triplicate samples. In the [3H]thymidine incorporation assay, values are expressed as the percentage of growth compared with the control value and are the mean ± SD of two individual experiments with triplicate samples. Data were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test or Dunnett’s test. P < 0.05 was considered statistically significant.
Results
Expression of FSH-R mRNA
The mRNA expression of FSH-R in OSE cells was investigated by RT-PCR and Southern blot analysis. A predicted PCR product of FSH-R was obtained as 369 bp and was confirmed by Southern blot analysis using digoxigenin- labeled probes (Fig. 1A
) and sequence analysis (data not shown). The human granulosa luteal cells (hGLCs) were used for a positive control. As demonstrated in Fig. 1A, FSH-R mRNAs are expressed in normal OSE and IOSE cell lines (IOSE-29, IOSE-29EC, IOSE-29EC/T4, and IOSE-29EC/T5). To confirm FSH-R mRNA expression by RT-PCR and Southern blot, Northern blot analysis was performed in IOSE-29, IOSE-29EC, and ovarian cancer cell lines, including OVCAR-3 and SKOV-3 cells (Fig. 1B). As shown in Fig. 1B, the low levels of two transcripts (4.1 and 2.4 kb) of FSH-R mRNA were demonstrated in IOSE cell lines (IOSE-29 and IOSE-29EC) and two ovarian cancer cell lines (OVCAR-3 and SKOV-3). The high level of FSH-R mRNA transcripts was observed in human granulosa-luteal cells as a positive control.
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To evaluate the role of FSH in normal and immortalized OSE cell lines, the cells were treated with increasing concentrations (10, 100, and 1000 ng/ml) of human recombinant FSH for 24 h, and a [3H]thymidine incorporation assay was performed as previously described (30). Treatments with increasing doses of FSH (10, 100, or 1000 ng/ml) resulted in a significant growth stimulation in normal OSE (Fig. 2A; 100.0 ± 8.33% vs. 135.1 ± 7.49%, 137.1 ± 9.06%, or 135.4 ± 13.90%) and OVCAR-3 cells (Fig. 2B; 100.0 ± 9.38% vs. 128.4 ± 7.21% or 128.9 ± 9.60%). In addition, treatments with the same concentrations of FSH induced a significant growth stimulation of IOSE-29 (Fig. 2C; 100.0 ± 9.08% vs. 126.9 ± 10.12%, 130.6 ± 11.23%, or 129.9 ± 7.25%) and IOSE-29EC cells (Fig. 2D; 100.0 ± 11.05% vs.137.5 ± 8.21%, 147.6 ± 11.65%, or 154.6 ± 12.45%). This result indicates that normal, preneoplastic, and neoplastic OSE cells are responsive to FSH treatments, resulting in growth stimulation of these cells.
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To investigate the role of FSH on MAPK activation, the cells were treated with increasing doses of FSH (10, 100, or 1000 ng/ml) for 10 min in the presence or absence of 50 µM PD98059, a MEK inhibitor. As shown in Fig. 3, A and B, treatments with FSH induced a significant increase in MAPK activation in IOSE-29 cells (100.0 ± 9.94% vs.147.8 ± 9.31%, 151.9 ± 10.62%, or 156.7 ± 9.83%). The stimulatory effect of FSH was completely reversed by pretreatment with PD98059 (157.3 ± 11.21% vs. 56.3 ± 5.18%). Similarly, treatments with FSH resulted in a significant increase in MAPK activation in IOSE-29EC cells (Fig. 3, C and D; 100.0 ± 7.54% vs.182.9 ± 11.84%, 183.4 ± 9.52%, or 179.2 ± 9.00%). This stimulatory effect of FSH was completely blocked by pretreatment with PD98059 (179.3 ± 12.04% vs. 65.7 ± 4.98%). Treatment with PD98059 alone resulted in a significant decrease in basal P-MAPK in both IOSE-29 and IOSE-29EC cells (Fig. 3, B and D).
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, A and B, treatment with FSH induced a significant increase in P-MAPK at 5–10 min in IOSE-29 cells (100.0 ± 9.15% vs.132.6 ± 7.07% or 163.3 ± 9.22%). The activated MAPK declined to the control level after 20 min in these cells. In contrast, treatment with FSH significantly activated MAPK after 5 min and sustained it for 60 min in IOSE-29EC cells (Fig. 4, C and D; 100.0 ± 9.35% vs. 195.0 ± 10.70%, 184.6 ± 14.47%, or 190.8 ± 14.26%). FSH-stimulated MAPK activation was completely abolished by pretreatment with PD98059 in both cell lines. In addition, treatment with PD98059 alone significantly decreased MAPK activity in both IOSE-29 and IOSE-29EC cells (Fig. 4, B and D).
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To examine the role of FSH on MAPKs in OVCAR-3 and SKOV-3 cells, the cells were pretreated with 50 µM PD98059 for 30 min, followed by treatment with 100 ng/ml FSH for 10 min. As shown in Fig. 5A, treatment with FSH appeared to induce a significant increase in MAPK activation in OVCAR-3 cells. In contrast, no difference was observed in SKOV-3 cells after FSH treatment (Fig. 5B). Pretreatment with PD98059 resulted in a decrease in FSH-induced MAPK activation in OVCAR-3 cells (Fig. 5A), whereas pretreatment with PD98059 did not affect MAPK activity in SKOV-3 cells (Fig. 5B).
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The Ets family transcription factor, Elk-1, is a physiological substrate for p42 MAPK and p44 MAPK (33, 34). To investigate whether the FSH-induced activation of MAPK leads to phosphorylation of Elk-1 in vitro, the cells were treated with FSH (100 ng/ml) for 10 min and/or PD98059 (50 µM) for 30 min. As shown in Fig. 6, treatment with FSH resulted in a significant increase in Elk-1 phosphorylation, whereas pretreatment with PD98059 significantly inhibited FSH-induced Elk-1 phosphorylation in both IOSE-29 (100.0 ± 9.66% vs. 173.5 ± 14.59% or 52.0 ± 6.27%) and IOSE-29EC cells (100.0 ± 10.46% vs. 196.3 ± 13.76% or 56.1 ± 7.70%).
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To evaluate the effect of MAPK inhibitor on FSH-stimulated cell growth, IOSE-29 and IOSE-29EC cells were pretreated with PD98059 (50 µM) for 30 min and then treated with FSH (100 ng/ml) for 24 h. [3H]Thymidine incorporation assay was performed as previously described in Materials and Methods. Treatment with FSH (100 ng/ml) resulted in a significant growth stimulation in these cells as expected (Fig. 7). In addition, as shown in Fig. 7, pretreatment with PD98059 completely attenuated FSH-stimulated cell growth in both IOSE-29 and IOSE-29EC cells.
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To investigate whether FSH modulates intracellular cAMP levels, the cells were treated with FSH (100 ng/ml), and intracellular cAMP levels were measured. Treatment with FSH did not affect basal intracellular cAMP levels in IOSE-29 and IOSE-29EC cells (Fig. 8), whereas FSH treatment induced a significant increase in intracellular cAMP in hGLCs.
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In addition to its well documented role in ovarian physiology, FSH, one of pituitary glycoprotein hormones, has been suggested to play a role in ovarian cancer development. An increased occurrence of ovarian cancer with exposure to high levels of gonadotropins during postmenopause or infertility therapy has been suggested by epidemiological studies (2, 10, 11). Even though it has been suggested that FSH may play a role in ovarian tumorigenesis (8, 9), little information is available regarding the expression of FSH-R and the exact role of FSH in normal and neoplastic OSE cells. FSH-R, a G protein-coupled receptor, is expressed in normal OSE (12), ovarian inclusions, and epithelial tumors (9). In addition, treatment with FSH resulted in a growth stimulation of rabbit OSE (13) and ovarian cancer cells (9, 14) in a dose- and time-dependent manner in vitro. In agreement with these previous reports the present study demonstrated that FSH-R mRNA was expressed in normal, preneoplastic, and neoplastic OSE cells; in addition, treatment with FSH (10–1000 ng/ml) induced the growth stimulation of these cells. Recent studies demonstrated that treatment with FSH is potent in increasing cell growth, and FSH-R mRNA is expressed in normal and malignant human OSE cells (34A ). The circulating level of FSH in premenopausal women varies between 10–25 mIU/ml; however, in postmenopausal women, FSH is elevated up to 66 mIU/ml (34A ). It has been shown that FSH-R expression was decreased with increasing tumor grade in epithelial inclusions, cystadenomas, borderline tumors, and carcinomas, suggesting that constitutive expression of FSH-R may represent a cellular differentiation marker for epithelial ovarian tumors (9). However, in the present study no difference was observed in the expression level of FSH-R in normal, preneoplastic (IOSE-29), neoplastic (IOSE-29EC), and late tumorigenic OSE (IOSE-29EC/T4 and/T5) cells. By Northern blot analysis, low levels of two transcripts (4.1 and 2.4 kb) of FSH-R mRNA were demonstrated in IOSE-29, IOSE-29EC, OVCAR-3, and SKOV-3 to confirm mRNA expression by RT-PCR and Southern blot analysis. The predominant transcript was 4.1 kb in size, as previously described (31). A recent report demonstrated that elevated level of gonadotropins stimulated the growth of ovarian carcinoma by induction of tumor angiogenesis, and the FSH effect was connected with the expression of vascular endothelial growth factor, which is an angiogenic factor presumably involved in ovarian tumorigenesis (35), implying that gonadotropins may facilitate the growth of existing microtumors by enhancing the blood supply.
Protein phosphorylation is a critical regulatory response to cellular stimulation and differentiation. The MAPK cascade is known to regulate acute cellular responses and to control transcriptional events through phosphorylation of target enzymes and transcriptional factors (15, 16, 17). Activation of ERK is induced by phosphorylation of both threonine and tyrosine residues of the enzymes as a result of stimulation of Ras, ERK kinase kinase, MEK kinase, and MEK (15, 16, 36). The MAPKs have been shown to mediate a diverse range of regulatory molecules, such as FSH (37), PGF2 (38), TGF (39), and epidermal growth factor (EGF) (40) in ovarian cells. In addition, treatment with GnRH analog resulted in an increase in ERK up to 24 h, and suppression of ERK activation by PD98059, which binds MEK, blocked GnRH analog- induced growth inhibition as well as hypophosphorylation of pRB in CaOV-3 cells (24). Considering that the mechanism of FSH action in ovarian tumors is unclear, we investigated the molecular mechanism of FSH-induced MAPK activation and its role in neoplastic OSE cells. In the present study FSH stimulated MAPK activation in both IOSE-29 and IOSE-29EC cells, whereas the stimulatory effect of FSH was completely reversed by pretreatment with PD98059, a MEK inhibitor, in both FSH-induced MAPK activation and FSH-stimulated proliferative index, suggesting that the growth stimulatory effect of FSH may be related to MAPK activation in neoplastic OSE cells. In addition, treatment with PD98059 alone resulted in a significant decrease in MAPK activation in both cell lines. In the previous reports EGF activated ERK1/2 and increased and sustained levels of c-Jun mRNA, but had no effect on Jun N-terminal kinase 1 activation in IOSE-29 cells (40). Similarly, EGF has been demonstrated to induce activation of ERK, and cellular proliferation was partially inhibited by PD98059 in a prostate cancer cell line (41). Additionally, it has been shown that EGF-induced cell proliferation, MMP-9 induction, and invasion through reconstituted basement membrane were significantly reduced when breast epithelial cells were exposed to MEK inhibitor (PD 98059) or MAPK inhibitors (apigenin or MAPK antisense phosphorothioate oligodeoxynucleotides). These results suggest that interference with MAPK activity may affect the growth and invasiveness of tumors in which the signaling cascade is activated (42).
In a time-dependent study, treatment with FSH induced a significant increase in MAPK activation at 5–10 min in IOSE-29 cells. The activated MAPK declined to control levels after 20 min in these cells. Treatment with FSH significantly stimulated MAPK activation after 5 min and sustained it for 60 min in IOSE-29EC cells. It appears that cellular responses to MAPK may be influenced by the duration of its activation. Sustained activation of MAPK is associated with cellular differentiation by nerve growth factor in PC12 cells, whereas transient activation of MAPK by EGF leads to cellular proliferation (43, 44). Thus, a rapid activation of MAPK by FSH in IOSE-29 and IOSE-29EC cells is related to growth stimulation in the present study. However, the cause of the sustained response after FSH treatment in IOSE-29EC cells has yet to be elucidated. EGF stimulated an early rise in ERK activity at 4 min, followed by a rapid decline in normal breast epithelium, whereas sustained ERK activity (1 h) was observed in neoplastic breast cells (45), suggesting that the time course of ERK activity may be different in normal and neoplastic cells. In addition, PD98059 inhibited EGF-stimulated proliferation and ERK activity in a parallel and dose-dependent manner, indicating that ERK activation is permissive for the proliferative response to EGF (45).
FSH-R belongs to a superfamily of G protein-coupled receptors that interact with intracellular signaling system through seven-transmembrane domains (46). Transient increases in c-Fos and c-Myc expression and MAPK activation were demonstrated in granulosa cell cultures in response to FSH (37, 47, 48); these actions were mediated by either a cAMP-dependent or -independent pathway. MAPK pathway has been shown to mediate cAMP-independent FSH activation in growth promotion (49). As demonstrated in this study, FSH did not stimulate the basal cAMP level, suggesting that the PKA pathway is not involved in FSH-induced MAPK activation in neoplastic OSE cells. This is in contrast to human granulosa-luteal cells, where FSH stimulates cAMP accumulation, and MAPK activation occurs via a PKA-dependent pathway. It is tempting to investigate further which pathway, PKA or PKC, is associated with FSH-induced MAPK activation in neoplastic OSE cells for further studies. In the other ovarian cancer cell lines examined, it appears that FSH induced a significant increase in MAPK activation in OVCAR-3 cells, but not in SKOV-3 cells. Pretreatment of PD98059 resulted in a decrease in FSH-induced MAPK activation in OVCAR-3 cells, whereas it appears that pretreatment with PD98059 did not affect MAPK activity in SKOV-3 cells. Several studies have shown that MAPKs phosphorylate ternary complex factor proteins such as Elk-1 and serum response factor accessary protein-1 (33, 34, 50). The activated ternary complex factor protein regulates the expression of c-fos and other coregulated genes through their actions on the serum response element. Therefore, the ability of FSH to activate a downstream effector of the MAPK pathway was examined using the immunoprecipitation method. The present study demonstrated that treatment with FSH resulted in substantial phosphorylation of Elk-1 fusion protein in vitro. These results confirmed that FSH action is mediated by the activation of MAPK, as treatment with PD98059 completely reversed the effect of FSH on Elk-1 phosphorylation. Taken together, these results suggest that FSH-stimulated MAPK activation resulted in a phosphorylation of Elk-1, the Ets family transcription factor, which possibly mediates cellular functions in response to FSH in neoplastic OSE cells.
In conclusion, we demonstrated that FSH-R was expressed, and FSH induced growth stimulation in normal, preneoplastic, and neoplastic OSE cells. In addition, treatment with FSH resulted in activation of the MAPK cascade and activated MAPK-phosphorylated Elk-1 in IOSE-29 and IOSE-29EC cells. These results suggest that the MAPK cascade may be involved in cellular function such as growth stimulation in response to FSH in preneoplastic and neoplastic OSE cells.
Acknowledgments
We are very grateful to Ms. Clara Salamanca for providing human normal OSE cells. We also thank Dr. A. F. Parlow, National Hormone and Pituitary Program of Harbor-University of California-Los Angeles Medical Center for recombinant human FSH and Dr. T. Minegishi for human FSH-R cDNA clone, respectively.
Footnotes
This work was supported by grants from the Canadian Institutes of Health Research and the National Cancer Institute of Canada, and a career investigatorship (to P.C.K.L.) and a studentship award (to S.K.K.) from the British Columbia Research Institute of Children’s and Women’s Health.
Abbreviations: EGF, Epidermal growth factor; FSH-R, FSH receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hGLC, human granulosa luteal cell; IOSE, immortalized ovarian surface epithelial cells; MEK, MAPK/ERK kinase; OSE, ovarian surface epithelium; P-MAPK, phosphorylated p44/p42 MAPK; T-MAPK, total MAPK.
Received June 19, 2001.
Accepted February 12, 2002.
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  H.-M. Wu, J.-C. Cheng, H.-S. Wang, H.-Y. Huang, C. D. MacCalman, and P. C.K. Leung Gonadotropin-Releasing Hormone Type II Induces Apoptosis of Human Endometrial Cancer Cells by Activating GADD45{alpha} Cancer Res., May 15, 2009; 69(10): 4202 - 4208. [Abstract] [Full Text] [PDF]
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 F. C.-K. CHEN, G. OSKAY-OZCELIK, K. J. BUHLING, U. KOPSTEIN, M. MENTZE, W. LICHTENEGGER, and J. SEHOULI Prognostic Value of Serum and Ascites Levels of Estradiol, FSH, LH and Prolactin in Ovarian Cancer Anticancer Res, May 1, 2009; 29(5): 1575 - 1578. [Abstract] [Full Text] [PDF]
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 J.-H. Choi, C.-L. Chen, S. L. Poon, H.-S. Wang, and P. C K Leung Gonadotropin-stimulated epidermal growth factor receptor expression in human ovarian surface epithelial cells: involvement of cyclic AMP-dependent exchange protein activated by cAMP pathway Endocr. Relat. Cancer, March 1, 2009; 16(1): 179 - 188. [Abstract] [Full Text] [PDF]
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 S. S. Deepa and L. Q. Dong APPL1: role in adiponectin signaling and beyond Am J Physiol Endocrinol Metab, January 1, 2009; 296(1): E22 - E36. [Abstract] [Full Text] [PDF]
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 C. N. Landen Jr, M. J. Birrer, and A. K. Sood Early Events in the Pathogenesis of Epithelial Ovarian Cancer J. Clin. Oncol., February 20, 2008; 26(6): 995 - 1005. [Abstract] [Full Text] [PDF]
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 J.-H. Choi, A. S. T. Wong, H.-F. Huang, and P. C. K. Leung Gonadotropins and Ovarian Cancer Endocr. Rev., June 1, 2007; 28(4): 440 - 461. [Abstract] [Full Text] [PDF]
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 P. C.K. Leung and J.-H. Choi Endocrine signaling in ovarian surface epithelium and cancer Hum. Reprod. Update, March 1, 2007; 13(2): 143 - 162. [Abstract] [Full Text] [PDF]
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J.-H. Choi, K.-C. Choi, N. Auersperg, and P. C K Leung Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid by gonadotropins in human immortalized ovarian surface epithelium and ovarian cancer cells. Endocr. Relat. Cancer, June 1, 2006; 13(2): 641 - 651. [Abstract] [Full Text] [PDF]
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J.-H. Choi, K.-C. Choi, N. Auersperg, and P. C.K. Leung Gonadotropins Activate Proteolysis and Increase Invasion through Protein Kinase A and Phosphatidylinositol 3-Kinase Pathways in Human Epithelial Ovarian Cancer Cells. Cancer Res., April 1, 2006; 66(7): 3912 - 3920. [Abstract] [Full Text] [PDF]
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J.-H. Choi, K.-C. Choi, N. Auersperg, and P. C K Leung Gonadotropins upregulate the epidermal growth factor receptor through activation of mitogen-activated protein kinases and phosphatidyl-inositol-3-kinase in human ovarian surface epithelial cells Endocr. Relat. Cancer, June 1, 2005; 12(2): 407 - 421. [Abstract] [Full Text] [PDF]
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 K.-Y. Kim, K.-C. Choi, S.-H. Park, N. Auersperg, and P. C. K. Leung Extracellular Signal-Regulated Protein Kinase, But Not c-Jun N-Terminal Kinase, Is Activated by Type II Gonadotropin-Releasing Hormone Involved in the Inhibition of Ovarian Cancer Cell Proliferation J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1670 - 1677. [Abstract] [Full Text] [PDF]
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 A. Lukanova and R. Kaaks Endogenous Hormones and Ovarian Cancer: Epidemiology and Current Hypotheses Cancer Epidemiol. Biomarkers Prev., January 1, 2005; 14(1): 98 - 107. [Abstract] [Full Text] [PDF]
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J.-H. Choi, K.-C. Choi, N. Auersperg, and P. C. K. Leung Overexpression of Follicle-Stimulating Hormone Receptor Activates Oncogenic Pathways in Preneoplastic Ovarian Surface Epithelial Cells J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5508 - 5516. [Abstract] [Full Text] [PDF]
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 C. A. Nechamen, R. M. Thomas, B. D. Cohen, G. Acevedo, P. I. Poulikakos, J. R. Testa, and J. A. Dias Human Follicle-Stimulating Hormone (FSH) Receptor Interacts with the Adaptor Protein APPL1 in HEK 293 Cells: Potential Involvement of the PI3K Pathway in FSH Signaling Biol Reprod, August 1, 2004; 71(2): 629 - 636. [Abstract] [Full Text] [PDF]
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K.-Y. Kim, K.-C. Choi, S.-H. Park, C.-S. Chou, N. Auersperg, and P. C. K. Leung Type II Gonadotropin-Releasing Hormone Stimulates p38 Mitogen-Activated Protein Kinase and Apoptosis in Ovarian Cancer Cells J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 3020 - 3026. [Abstract] [Full Text] [PDF]
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 P. P. Kayampilly and K. M. J. Menon Inhibition of Extracellular Signal-Regulated Protein Kinase-2 Phosphorylation by Dihydrotestosterone Reduces Follicle-Stimulating Hormone-Mediated Cyclin D2 Messenger Ribonucleic Acid Expression in Rat Granulosa Cells Endocrinology, April 1, 2004; 145(4): 1786 - 1793. [Abstract] [Full Text] [PDF]
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