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Endocrinology Vol. 142, No. 2 671-679
Copyright © 2001 by The Endocrine Society

ARTICLES

Stimulation of Mitogen-Activated Protein Kinase by Gonadotropin-Releasing Hormone in Human Granulosa-Luteal Cells1

Sung Keun Kang2, Chen-Jei Tai, Parimal S. Nathwani, Kyung-Chul Choi and Peter C. K. Leung3

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5

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
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study investigated the activation of mitogen-activated protein kinases (MAPKs) by a GnRH agonist (GnRHa) in human granulosa-luteal cells (hGLCs). The phosphorylation state of p44 and p42 MAPK was examined using antibodies that distinguish phospho-p44/42 MAPK (Thr202/Tyr204) from total p44/42 MAPK (activated plus inactivated). Activation of MAPK by GnRHa was observed within 5 min and was sustained for 60 min after treatment. GnRHa stimulated MAPK activation in a dose-dependent manner, with maximum stimulation (6.7-fold over basal levels) at 10-7 M. Pretreatment with a protein kinase C (PKC) inhibitor, GF109203X, completely blocked GnRHa-induced MAPK activation. In addition, pretreatment with a PKC activator, phorbol-12-myristate 13-acetate, potentiated GnRH-induced MAPK activation. These results indicate that GnRHa stimulates MAPK activation through a PKC-dependent pathway in hGLCs, possibly coupled to Gq{alpha} protein. MAPK activation was also observed in response to 8-bromo-cAMP or cholera toxin, but not pertussis toxin. Forskolin (50 µM) substantially stimulated a rapid cAMP accumulation, whereas GnRHa (10-7 M) or pertussis toxin (100 mg/ml) did not affect basal intracellular cAMP levels. Cotreatment of GnRHa (10-7 M) did not attenuate forskolin- or hCG-stimulated cAMP accumulation. These results suggest that the GnRH receptor is probably not coupled to Gs{alpha} or Gi{alpha} in hGLCs. Finally, GnRHa (10-7 M) stimulated a significant increase in Elk-1 phosphorylation and c-fos messenger RNA expression, as revealed by an in vitro kinase assay and Northern blot analysis, respectively. These results clearly demonstrate that GnRH activates the MAPK cascade through a PKC-dependent pathway in the human ovary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MITOGEN-ACTIVATED protein kinases (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 (1). Two distinct classes of cell surface receptors, receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), are known to activate the MAPK cascade (2, 3). The signals transmitted through this cascade lead to activation of a set of molecules that regulate cell growth, division, and differentiation. The most widely studied members of the cascade are the extracellular signal-regulated kinase-1 (ERK1; p44 MAPK) and kinase-2 (ERK2; p42 MAPK).

GnRH regulates the synthesis and secretion of the pituitary gonadotropins. The GnRH receptor (GnRHR) is a G protein-coupled receptor and is hypothesized to couple to multiple G proteins (4, 5, 6, 7, 8, 9). GnRHR couples to Gq{alpha} on gonadotroph cells, culminating in the activation of multiple signaling pathways, including phosphoinositol turnover, release of intracellular calcium, influx of extracellular calcium, and activation of protein kinase C (PKC) (4, 5). In addition, coupling of the GnRHR to the Gs{alpha} subunit has been suggested to increase the biosynthesis of LH (6), regulating the gonadotropin subunit messenger RNA (mRNA) (7) or GnRHR mRNA in {alpha}T3–1 cells (8). On the other hand, there is evidence to suggest that the GnRHR is coupled to Gi{alpha} in reproductive tract tumors (9). As the same receptor couples to multiple G proteins, it is suggested that the GnRH activates different intracellular signaling pathways and exerts a cell- or tissue-specific function.

Diverse mechanisms were proposed for MAPK activation by ligands operating via G protein-coupled receptors (10, 11, 12, 13, 14, 15). For example, thrombin and lysophosphatidic acid stimulate MAPK activation via a Gi{alpha} in a pertussis toxin (PTX)-sensitive (10, 11), PKC-independent pathway. In contrast, endothelin-1 acts in a PTX-sensitive and PKC-dependent mechanism (12). Recently, it has been demonstrated that GnRH is capable of activating the MAPK cascade in pituitary organ cultures (16, 17), in the pituitary in vivo (18), and the {alpha}T3–1 gonadotroph cell line (17) through a PKC dependent pathway. Functionally, activation of MAPK is involved in the transcriptional regulation of the glycoprotein {alpha}-subunit gene.

In addition to its well established function in the pituitary, GnRH is thought to be an autocrine/paracrine regulator in the gonads. This concept is based on the detection of GnRH and its receptor gene transcripts, synthesis of GnRH, and a multitude of effects attributed to GnRH receptor-mediated signaling in extrapituitary tissues (19, 20, 21, 22, 23, 24, 25). In the ovary, GnRH modulates basal and gonadotropin-stimulated steroidogenesis (22, 23) and induces transcription of several genes involved in follicular maturation and ovulation (24, 25). However, little is known about the molecular events that mediate GnRH actions in the extrapituitary tissues. Considering that differential actions of GnRH may be mediated via different signaling pathways in extrapituitary tissues (26), we investigated the GnRH-induced activation of MAPK and its intracellular signaling pathway in the human ovary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and treatment
The use of human granulosa-luteal cells (hGLCs) was approved by the clinical screening committee for research and other studies involving human subjects of the University of British Columbia. Follicular aspirates were collected during oocyte retrieval from women undergoing in vitro fertilization. hGLCs were prepared as previously described (22) and cultured in DMEM (Life Technologies, Inc., Burlington, Canada) supplemented with 10% FBS (Life Technologies, Inc.), 100 U/ml penicillin G, and 100 µg/ml streptomycin (Life Technologies, Inc.) for 4 days before treatment. Cells were seeded at a density of 5 x 105 cells in 35-mm culture dishes and cultured in a humidified atmosphere of 5% CO2-95% air at 37 C. Cell cultures were washed once with medium and serum starved for 4 h before treatment with a GnRH agonist, (D-Ala6)-GnRH (GnRHa), phorbol 12-myristate 13-acetate (PMA), 8-bromo-cAMP (8-Br-cAMP), or PTX in a time- and/or dose-dependent manner. All agents were purchased from Sigma-Aldrich Corp. (Oakville, Canada). Before GnRHa treatment, the appropriate cultures were pretreated with the GnRH antagonist, antide (10-7 M; Sigma-Aldrich Corp.) and a MAPK/ERK kinase (MEK) inhibitor, PD98059 (10 µM; New England Biolabs, Inc., Beverly, MA) for 10 and 60 min, respectively. To block PKC activation, cells were pretreated with a specific PKC inhibitor, GF109203X (2 µM; Calbiochem, San Diego, CA) for 15 min, followed by treatment with 10-7 M GnRHa and PMA for 20 and 30 min, respectively. To further determine the role of the PKC pathway in GnRH-induced MAPK activation, the cells were cultured and pretreated with PMA (10-7 M) for 5 min, followed by stimulation with GnRHa (10-7 M) for 20 min. To investigate the involvement of the Gs{alpha} and Gi{alpha} in GnRH-induced MAPK activation, cells were pretreated with 100 ng/ml PTX and cholera toxin (CTX; Sigma-Aldrich Corp.) for 15 min before GnRHa treatment for 20 min. After the indicated treatments, 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 protein content of the supernatants was determined using a Bradford assay (Bio-Rad Laboratories, Inc., Richmond, CA).

Immunoblot assay
Phospho-specific MAPK antibody, which detects p44 and p42 MAPK only when phosphorylated at Thr202 and Tyr204, was used to measure MAPK activation by Western blot analysis (New England Biolabs, Inc.) (18). Forty micrograms of total protein were run on 10% SDS-PAGE gels and electrotransferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Oakville, Canada) (27). The membrane was immunoblotted using a mouse monoclonal antibody specific to the phosphorylated p44/p42 MAPK (P-MAPK, Thr202/Tyr204). Alternatively, the membrane was stripped and probed with a rabbit polyclonal antibody for p44/42 MAPK, 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, Piscataway, NJ), followed by autoradiography. P-MAPK levels were quantitated by densitometry (NIH Image ß 3), and standardized against the levels of T-MAPK per sample.

RIA for intracellular cAMP
To measure intracellular cAMP, hGLCs (2 x 105 cells) were plated onto 35-mm culture dishes and cultured for 4 days. 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 50 µM forskolin (Sigma-Aldrich Corp.), 10-7 M GnRHa for 0, 5, 20, 60, 120, or 240 min. To determine whether GnRH modulates forskolin- or hCG-induced cAMP accumulation, cells were cotreated with GnRHa (10-7 M) and forskolin (50 µM) or hCG (1 IU; Sigma-Aldrich Corp.) for 20 min. Cells were also treated with PTX (100 ng/ml) for 20 min. Control cells were treated with vehicle. Intracellular cAMP levels were measured using a [3H]cAMP assay system (Amersham Pharmacia Biotech), according to the manufacturer’s suggested procedure.

In vitro MAPK assay
hGLCs were serum starved for 4 h and pretreated with vehicle and PD98059 for 1 h. The cells were then treated with 10-7 M GnRHa for 20 min, washed twice with ice-cold PBS, and lysed in 1x 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-glycerophosphate, 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 protein content of the supernatants was determined. Cellular protein (300 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 the MAPKs, according to the manufacturer’s suggested procedure (New England Biolabs, Inc.).

Northern blot analysis for c-fos
hGLCs (1.0 x 106 cells) were plated onto 60-mm culture dishes and cultured for 4 days. The cells were then serum starved for 6 h and treated with 10-7 M GnRHa for 0, 10, 20, 30, or 60 min. Total RNA was prepared using the RNaid kit (Bio/Can Scientific, Mississauga, Canada) according to the manufacturer’s suggested procedure, and the concentration of RNA was determined by absorbance at 260 nm. Total RNA (25 µg) was resolved by formaldehyde denaturing agarose gel electrophoresis and prepared for Northern blot analysis. Radioactive labeled c-fos probe (28) was prepared from the Random Labeling Kit (Life Technologies, Inc.). The membrane was prehybridized and hybridized in standard hybridization solution (50% formamide, 5 x SSPE, 5x Denhardt’s solution, 0.5% SDS, and 100 µg/ml denatured herring sperm DNA) at 42 C for 16 h, followed by high stringency washes (0.1 x SSPE and 0.1% SDS at 65 C for 10 min) and exposed to Kodak O-Mat x-ray film (Eastman Kodak Co., Rochester, NY). The membrane was stripped and rehybridized with radioactive labeled probe for glyceraldehyde-3-phosphate dehydrogenase (23).

Data analysis
The data were analyzed by ANOVA followed by Tukey’s multiple comparison test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRH-induced MAPK activation
The GnRH agonist, (D-Ala6)-GnRH, stimulated a rapid and sustained activation of MAPK in primary cultures of hGLCs cells at a concentration of 10-7 M (Fig. 1AGo). Stimulation of MAPK activity was observed within 5 min and was sustained for 60 min after treatment. Maximal activity (6.1-fold over basal levels) was observed within 20 min. Dose-response studies with various concentration of GnRHa for 20 min indicated that GnRHa stimulated MAPK activation in a dose-dependent manner, with maximum stimulation (6.7-fold over basal levels) at 10-7 M (Fig. 1BGo).



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  		Figure 1. The effects of GnRH on MAPK activation in hGLCs. hGLCs were cultured and treated with 10-7 M GnRHa in a time dependent manner (A). The cells were also treated with various concentrations of (D-Ala6)-GnRH for 20 min (B). Control cultures were treated with vehicle. Total (T-MAPK) and activated MAPK (P-MAPK) levels were analyzed by immunoblot assay. P-MAPK levels were quantitated by densitometry (NIH Image ß 3) and standardized against the levels of T-MAPK per sample. MAPK levels are expressed as the relative fold change compared with basal levels. The data were analyzed by ANOVA, followed by Tukey’s multiple comparison test. Values are the mean ± SD of three individual experiments. a, P < 0.05 vs. control.

 
Effects of antide and PD98059 on GnRH-induced MAPK activation
To confirm the specificity of the GnRH-induced activation of MAPK, the cells were pretreated with 10-7 M antide for 10 min, followed by treatment with 10-7 M GnRHa for 20 min. As shown in Fig. 2AGo, the GnRHa induced a significant increase in MAPK activation. This stimulatory effect was completely reversed by pretreatment with antide, demonstrating that the activation of the MAPK signaling pathway is a receptor-mediated event. Treatment with antide alone had no effect on MAPK activation. To investigate whether GnRH-induced MAPK activation is mediated through activation of MEK, cells were pretreated with 10 µM PD98059 before GnRHa treatment for 20 min. As shown in Fig. 2BGo, a 68% decrease in GnRH-induced MAPK activation was observed in hGLCs pretreated with PD98059, demonstrating that GnRH-induced MAPK activation is mediated through the activation of MEK. Treatment with PD98059 alone had no effect on MAPK activation.



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  		Figure 2. The effects of antide and PD98059 on GnRH-induced MAPK activation in hGLCs. hGLCs were cultured and pretreated with a GnRH antagonist, antide (10-7 M) for 10 min (A) and with a specific inhibitor for MEK, PD98059 (10 µM), for 1 h (B). The cells were then treated with 10-7 M GnRHa for 20 min. Control cultures were treated with vehicle. Total (T-MAPK) and activated MAPK (P-MAPK) levels were analyzed by immunoblot assay. P-MAPK levels were quantitated by densitometry (NIH Image ß 3) and standardized against the levels of T-MAPK per sample. MAPK levels are expressed as the relative fold change compared with basal levels. The data were analyzed by ANOVA, followed by Tukey’s multiple comparison test. Values are the mean ± SD of three individual experiments. a, P < 0.05 vs. control; b, P < 0.05 vs. antide plus GnRHa; c, P < 0.05 vs. PD98059 plus GnRHa.

 
Effects of PMA, GnRH, and PKC inhibitor GF109203X on MAPK activation
To assess whether the PKC signal transduction pathway is involved in MAPK activation in hGLCs, the cells were treated with a PKC activator, PMA (10-7 M) in a time- dependent manner. The results showed that PMA stimulated a rapid MAPK activation, with maximum stimulation (9.8-fold over basal levels) within 30 min (Fig. 3AGo). To determine whether PKC is necessary for MAPK activation by GnRH and PMA, hGLCs were pretreated with the PKC inhibitor GF109203X (2 µM) for 10 min, followed by stimulation with 10-7 M GnRHa and PMA for 20 and 30 min, respectively. As shown in Fig. 3BGo, GF109203X completely blocked the GnRHa-induced MAPK activation and decreased PMA- induced MAPK activity to 33% of the control value. GF109203X alone had no effect on the MAPK activity. To further determine the role of the PKC pathway in GnRH-induced MAPK activation, the cells were pretreated with PMA (10-7 M) for 5 min, followed by stimulation with GnRHa (10-7 M) for 20 min. As shown in Fig. 3CGo, the GnRHa or PMA treatment induced a substantial increase in MAPK activation. Pretreatment of the cells with PMA significantly potentiated MAPK activity in response to GnRHa, further indicating that GnRH stimulates MAPK activation through a PKC-dependent pathway.



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  		Figure 3. The effects of PMA, GnRH, and PKC inhibitor GF109203X on MAPK activation. hGLCs were cultured and treated with a PKC activator, PMA (100 ng/ml), in a time-dependent manner (A). The cells were pretreated with the PKC inhibitor, GF109203X (GF; 2 µM) for 10 min, followed by stimulation with 10-7 M GnRHa and PMA (100 ng/ml) for 20 or 30 min, respectively (B). Cell cultures were pretreated with PMA (100 ng/ml) for 5 min, followed by stimulation with 10-7 M GnRHa for 20 min (C). Control cultures were treated with vehicle. Total (T-MAPK) and activated MAPK (P-MAPK) levels were analyzed by immunoblot assay. P-MAPK levels were quantitated by densitometry (NIH Image ß 3) and standardized against the levels of T-MAPK per sample. MAPK levels are expressed as the relative fold change compared with basal levels. The data were analyzed by ANOVA, followed by Tukey’s multiple comparison test. Values are the mean ± SD of three individual experiments. a, P < 0.05 vs. control; b, P < 0.05 vs. GF plus PMA; c, P < 0.05 vs. GF plus GnRHa; d, P < 0.05 vs. GnRHa; e, P < 0.05 vs. PMA.

 
Effects of 8-Br-cAMP and PTX on the MAPK activity
hGLCs were cultured and treated with 8-Br-cAMP (1 mM) and various doses of PTX. As shown in Fig. 4AGo, stimulation of MAPK activity was observed within 5 min and reached a maximum level within 20 min after treatment. No activation of MAPK was observed in response to various doses of PTX in hGLCs (Fig. 4BGo).



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  		Figure 4. The effects of 8-Br-cAMP and PTX on the MAPK activation. hGLCs were cultured and treated with 8-Br-cAMP (1 mM) in a time-dependent manner (A) and with PTX in a dose-dependent manner (B). Control cultures were treated with vehicle. Total (T-MAPK) and activated MAPK (P-MAPK) levels were analyzed by immunoblot assay. P-MAPK levels were quantitated by densitometry (NIH Image ß 3) and standardized against the levels of T-MAPK per sample. MAPK levels are expressed as the relative fold change compared with basal levels. The data were analyzed by ANOVA, followed by Tukey’s multiple comparison test. Values are the mean ± SD of three individual experiments. a, P < 0.05 vs. control.

 
Effects of CTX and PTX on GnRH-induced MAPK activation
To further determine the roles of the Gs{alpha} and Gi{alpha} in MAPK activation, hGLCs were pretreated with CTX (100 ng/ml) and PTX (100 ng/ml) for 15 min, followed by stimulation with 10-7 M GnRHa for 20 min. The preliminary experiments were performed to obtain the maximum concentration of PTX and time of CTX treatment. The maximal effect of CTX was observed in hGLCs treated with 100 ng/ml CTX for 15 min (data not shown). As shown in Fig. 5AGo, GnRHa and CTX treatment stimulated MAPK activation. An additive action of CTX and GnRHa, rather than potentiative, was observed in hGLCs cells, suggesting that activation of MAPK by CTX and GnRHa is independent. Pretreatment with PTX (100 ng/ml) for 15 min (Fig. 5BGo) or longer (1, 6, or 24 h; data not shown) did not affect either basal or GnRH-induced MAPK levels (Fig. 5BGo). In contrast, the pretreatment with PTX (100 ng/ml, 15 min) significantly attenuated lysophosphatidic acid- induced MAPK activation in hGLCs (data not shown).



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  		Figure 5. The effects of CTX and PTX pretreatment on GnRH-induced MAPK activation. hGLCs were pretreated with 100 ng/ml CTX (A) or 100 ng/ml PTX (B) for 15 min, followed by stimulation with 10-7 M GnRHa for 20 min. Control cultures were treated with vehicle. Total (T-MAPK) and activated MAPK (P-MAPK) levels were analyzed by immunoblot assay. P-MAPK levels were quantitated by densitometry (NIH Image ß 3) and standardized against the levels of T-MAPK per sample. MAPK levels are expressed as the relative fold change compared with basal levels. The data were analyzed by ANOVA, followed by Tukey’s multiple comparison test. Values are the mean ± SD of three individual experiments. a, P < 0.05 vs. control; b, P < 0.05 vs. GnRHa; c, P < 0.05 vs. CTX; d, P < 0.05 vs. PTX.

 
Effects of forskolin, hCG, and GnRHa on intracellular cAMP accumulation
To investigate whether GnRH modulates intracellular cAMP levels, hGLCs were treated with forskolin (50 µM) or GnRHa (10-7 M), and intracellular cAMP levels were measured. Forskolin substantially stimulated a rapid cAMP accumulation within 5 min, and a maximum increase was observed within 20 min (Fig. 6AGo). In contrast, GnRHa did not affect basal intracellular cAMP levels (Fig. 6BGo), suggesting that GnRHR may not be coupled to the Gs{alpha} in hGLCs. Activation of Gi{alpha} subunit protein is negatively correlated with cAMP production. To explore the possibility that GnRHR may be coupled to Gi{alpha} and affect cAMP production in hGLCs, cells were cotreated with GnRHa (10-7 M) and forskolin (50 µM) or hCG (1 IU) for 20 min. As shown in Fig. 6CGo, cotreatment of cells with GnRHa (10-7 M) did not attenuate forskolin- or hCG-stimulated cAMP production. PTX alone did not affect basal intracellular cAMP levels.



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  		Figure 6. The effects of forskolin, hCG, and GnRHa on intracellular cAMP accumulation. hGLCs were preincubated with serum-free medium containing 0.1% BSA and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) for 30 min, and then treated with 50 µM forskolin (A) or 10-7 M GnRHa (B). Intracellular cAMP levels were measured by an established RIA. The cell cultures were treated with 50 µM forskolin or 1 IU hCG plus 10-7 M GnRHa, and intracellular cAMP levels were measured (C). Control cultures were treated with vehicle. The amount of cAMP was calculated from standard curve. The data were analyzed by ANOVA, followed by Tukey’s multiple comparison test. Values are the mean ± SD of five individual experiments from five different patients. a, P < 0.05 vs. control.

 
Effects of GnRH and PD98059 on Elk-1 phosphorylation
The Ets family transcription factor, Elk-1, is a physiological substrate for p42 MAPK and p44 MAPK (29, 30, 31). To investigate whether the GnRH-induced activation of MAPK leads to phosphorylation of Elk-1 in vitro, hGLCs were pretreated with 10 µM PD98059 for 1 h before treatment with GnRHa (10-7 M). As shown in Fig. 7Go, GnRHa (10-7 M) stimulated a significant increase in Elk-1 phosphorylation. Pretreatment with 10 µM PD98089 for 1 h completely blocked the GnRH-induced phosphorylation of Elk-1. As expected, active p42 MAPK (ERK-2) substantially stimulated Elk-1 phosphorylation.



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  		Figure 7. The effects of GnRH and PD98059 on Elk-1 phosphorylation. hGLCs were cultured and pretreated with 10 µM PD98059 for 1 h, followed by stimulation with 10-7 M GnRHa for 20 min. Control cultures were treated with vehicle. The activated MAPK in the cell lysate was immunoprecipitated with immobilized phospho-p44/42 MAPK antibody at 4 C overnight. In vitro MAPK assays were performed using the Elk-1 fusion protein as a substrate for the MAPKs. Active p42 MAPK (ERK-2) was included as a positive control. The phosphorylation state of Elk-1 was analyzed by immunoblot assay using a specific antibody for phospho-Elk-1. The levels of Elk-1 phosphorylation were quantitated by densitometry (NIH Image ß 3) and are expressed as the relative fold change compared with basal levels. The data were analyzed by ANOVA, followed by Tukey’s multiple comparison test. Values are the mean ± SD of three individual experiments. a, P < 0.05 vs. control; b, P < 0.05 vs. PD98059 plus GnRHa.

 
Effect of GnRH on c-fos mRNA levels
Several studies have shown that Elk-1 phosphorylation stimulates transcription of the immediate early response gene, c-fos, by facilitating the formation of a ternary complex with serum response element and the serum response factor (29, 30, 31). To investigate whether GnRH stimulates the transcription of the c-fos gene, hGLCs were treated with 10-7 M GnRHa in a time-dependent manner. As shown in Fig. 8Go, GnRHa stimulated a significant increase in c-fos mRNA expression. Maximal stimulation (4.1-fold over basal levels) was observed within 30 min.



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  		Figure 8. The effect of GnRH on c-fos mRNA levels. hGLCs were cultured and treated with 10-7 M GnRHa in a time-dependent manner. Control cultures were treated with vehicle. Total RNA (25 µg) was prepared and resolved by formaldehyde-denaturing agarose gel electrophoresis. Northern blot analysis was performed using a radioactively labeled c-fos probe. The levels of c-fos mRNA were expressed as the percent change from the control value and standardized against the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA level. Data are the means of three individual experiments and are presented as the mean ± SD. The data were analyzed by ANOVA, followed by Tukey’s multiple comparison test. a, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The MAPKs have been shown to mediate a diverse range of regulatory molecules, such as FSH (32), PGF2{alpha} (33), transforming growth factor-{alpha} (34), epidermal growth factor (35), and GnRH (36), in the ovarian cell, including hGLCs. Considering that the mechanism of action of GnRH in the human ovary is unclear, we investigated the mechanism of GnRH-induced MAPK activation and its role in hGLCs. We demonstrated that GnRH stimulates rapid and sustained MAPK activation via a PKC-dependent pathway. The activated MAPK phosphorylates Elk-1 transcription factor and may result in transcription of the c-fos gene.

Interestingly, MAPK activation in the present study was rapid and sustained. It is hypothesized that cellular responses to MAPK may be influenced by the duration of its activation. Sustained activation of MAPK is associated with cell differentiation by nerve growth factor in PC12 cells, whereas transient activation of MAPK by epidermal growth factor leads to cell proliferation (37, 38). GnRHa was capable of inducing a sustained MAPK signal (up to 60 min) in the gonadotroph {alpha}T3–1 cell line (16) and GGH31' (39), whereas EGF stimulated rapid MAPK activation and reached its peak within 5 min (39). GnRH-induced MAPK activation was involved in differentiated cellular functions such as gonadotropin secretion and synthesis (17, 40). Thus, sustained activation of MAPK by GnRHa in hGLCs may be associated with differentiated cellular functions, such as steroidogenesis.

GnRHR is thought to couple to multiple G protein subunits (Gq{alpha}, Gs{alpha}, and Gi{alpha}) and activate multiple signaling pathways (4, 5, 41). Increasing evidence that multiple G proteins mediate the effects of GnRHR raised the possibility that the same kind of GPCR exerts regulation via differential signal transduction pathways in distinct tissues or cells, thereby determining the specific function mediated by the receptor in these cells. The PKC pathway has been well studied in response to GnRH stimulation (42, 43), including in hGLCs (44). GnRH induces translocation of PKC activity from the cytosol to the plasma membrane and stimulates enzyme activity. Our data indicate that the activation of MAPK by GnRH is mediated via PKC, as a specific PKC inhibitor GF109203X completely blocked the MAPK stimulation. Furthermore, pretreatment of the cells with PMA significantly enhanced GnRH-induced MAPK activity. These results indicate that the GnRHR activation stimulates MAPK activation via a PKC-dependent pathway, possibly coupled to Gq{alpha} protein.

There is evidence to suggest that the GnRHR is coupled to Gi{alpha} in reproductive tract tumors (9, 45). On the other hand, in insect cells expressing GnRHR and in the stable cell line GGH31' cells, GnRHR coupled to Gs{alpha}, which activates adenylate cyclase and results in the production of cAMP (46, 47). Although the PKA pathway appears not to be involved in stimulated hormone release from gonadotroph {alpha}T3–1 cells, this pathway may have an effect on increasing the biosynthesis of LH (6), regulating the gonadotropin subunit (7) and GnRHR mRNA (8) or the stimulation of proliferation in {alpha}T3–1 cells (8). The role of Gs{alpha} in the regulation of MAPK activation is poorly understood, but apparently cell specific. In some cells, such as fibroblasts, rat adipocytes, human arterial smooth muscle cells, and NIH-3T3 cells, increased cAMP attenuates activation of MAPK (48, 49, 50), resulting in reduced responsiveness to mitogenic stimuli (50). Conversely, elevation of intracellular cAMP is a potent mitogenic signal for a number of cell types, including Swiss 3T3 cells, thyroid epithelial cells, and the somatotrope cells of the anterior pituitary (51). The roles of Gi{alpha} and Gs{alpha} in MAPK activation in response to GnRH are still controversial in {alpha}T3–1 cells (52, 53). In this study, the possible involvement of the PKA pathway in GnRH-induced MAPK activation was investigated. Our results suggest that the PKA pathway is not involved in the GnRH-induced activation of MAPK. This conclusion is based on the following observations. First, in contrast to forskolin, GnRHa did not affect basal intracellular cAMP levels. Although 8-Br-cAMP and CTX activate MAPK in hGLCs, it is unlikely that the effect of GnRHa in stimulation of MAPK involves GnRHR/Gs{alpha} coupling. Second, activation of the GnRHR by its ligand did not attenuate forskolin- or hCG-induced cAMP accumulation. These data together with the effect of each with PTX on basal or GnRHa-induced MAPK activation make it unlikely that the effect of GnRHa in hGLCs involves Gi{alpha} coupling to GnRHR.

We examined the ability of GnRH to activate a downstream effector of the MAPK pathway. Several studies have shown that MAPK phosphorylates ternary complex factor proteins such as Elk-1 and SAP-1 (29, 30, 31). The activated ternary complex factor protein regulates the expression of c-fos and other coregulated genes through their actions on the serum response element. Our studies demonstrate that treatment of hGLCs with GnRHa resulted in substantial phosphorylation of Elk-1 fusion protein in vitro. This effect appears to be mediated by the activation of MAPK, as treatment of hGLCs with PD98059 completely reversed the effect of the GnRHa on Elk-1 phosphorylation. Furthermore, GnRHa stimulates the expression of c-fos mRNA in hGLCs. Our results are consistent with the finding that GnRH stimulates transcriptional activation of Elk-1 (40) and increased immediate-early response gene mRNA such as c-fos and c-jun in gonadotroph-derived {alpha}T3–1 cells (54). Taken together, these results suggest that activated Ets family transcription factors may regulate the expression of immediate early genes or other coregulated genes, which possibly mediate cellular functions in response to GnRH in hGLCs.

The physiological implications of MAPK activation by GnRH in hGLCs remain to be determined. The activation of MAPK cascades in response to diverse stimuli is known to regulate cell growth and differentiation. Considering that GnRH modulates differentiated cellular function, such as steroidogenesis (22, 23), and also regulates cell growth, such as follicle development (55), in the ovary, it is possible that GnRH-induced MAPK activation in hGLCs may be involved in these cellular functions. This idea was supported by the recent finding that GnRH-induced MAPK activation was involved in inhibitory effect of GnRH in progesterone secretion from hGLCs (36).

In summary, we have demonstrated that GnRH stimulates activation of the MAPK cascade through a PKC-dependent pathway. This activation stimulates transcription of the c-fos gene and may be involved in cellular functions, such as modulation of steroidogenesis in response to GnRH in hGLCs.


   Acknowledgments
 
We express our gratitude to Dr. Margo Fluker and the Genesis Fertility Center (Vancouver, Canada) for providing the follicular aspirates for hGLC isolation. We also thank Dr. Takashi Nagaya (Nagoya University, Nagoya, Japan) for providing the complementary DNA for c-fos gene.


   Footnotes
 
1 This work was supported grants from the Medical Research Council of Canada. Back

2 Recipient of a studentship award from the British Columbia Research Institute of Children’s and Women’s Health. Back

3 Recipient of a career investigatorship from the British Columbia Research Institute of Children’s and Women’s Health. Back

Received August 29, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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