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Endocrinology Vol. 140, No. 5 2241-2251
Copyright © 1999 by The Endocrine Society

ARTICLES

The Role of Protein Kinases A and C Pathways in the Regulation of Mitogen-Activated Protein Kinase Activation in Response to Gonadotropin-Releasing Hormone Receptor Activation

Xin-bing Han and P. Michael Conn

Oregon Regional Primate Research Center (X.-B.H., P.M.C.), Oregon Health Sciences University, Beaverton, Oregon 97006; and the Department of Physiology and Pharmacology, Oregon Health Sciences University (P.M.C.), Portland, Oregon 97201

Address all correspondence and requests for reprints to: Dr. P. Michael Conn, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: connm{at}OHSU.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is convincing evidence that mitogen-activated protein kinase (MAPK) activation is coupled to both receptor tyrosine kinase and G protein-coupled receptors. The presence of the epidermal growth factor (EGF) receptor and the GnRH receptor on the surface of GGH31' cells makes this cell line a good model for the assessment of MAPK activation by receptor tyrosine kinases and G protein-coupled receptors. In this study, to assess the activated and total (i.e. activated plus inactivated) MAPK, the phosphorylation state of p44 and p42 MAPKs was examined using antisera that distinguish phospho-p44/42 MAPK (Thr202/Tyr204) from p44/42 MAPK (phosphorylation state independent). The data show that both EGF (200 ng/ml) and Buserelin (a GnRH agonist; 10 ng/ml) provoke rapid activation of MAPK (within 5 and 15 min, respectively) after binding to their receptors. The role of protein kinase A (PKA) and protein kinase C (PKC) signal transduction pathways in mediating MAPK activation was also assessed. Both phorbol ester (phorbol 12-myristate 13-acetate; 10 ng/ml) and (Bu)2cAMP (1 mM) trigger the phosphorylation of MAPK, suggesting potential roles for PKC and PKA signaling events in MAPK activation in GGH31' cells. Treatment of PKC-depleted cells with Buserelin activated MAPK, suggesting involvement of PKC-independent signal transduction pathways in MAPK activation in response to GnRH. Similarly, treatment of PKC-depleted cells with forskolin (50 µM) or cholera toxin (100 ng/ml) stimulated MAPK activation, whereas pertussis toxin (100 ng/ml) had no measurable effect. To further assess the role of PKA in response to EGF and Buserelin, cells were treated with EGF (200 ng/ml) for 3 min or with Buserelin (10 ng/ml) for 10 min after pretreatment with 3-isobutyl-1-methylxanthine (0.5 mM), forskolin (50 µM), or (Bu)2cAMP (1 mM) for 15 min. The results show that MAPK can be activated in a PKA-dependent manner in GGH31' cells. Consistent with previous reports, the current data support the view that MAPK activation can be achieved via both PKC- and PKA-dependent signaling pathways triggered by the GnRH receptor that couples to Gq/11 and Gs {alpha}-subunit proteins. In contrast, Gi/o{alpha} does not appear to participate in MAPK activation in GGH31' cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MITOGEN-ACTIVATED protein (MAP) kinases (MAPKs) are a group of protein serine/threonine kinases that is activated in response to a variety of extracellular stimuli and mediates signal transduction from the cell surface to the nucleus (1). Upon activation, MAPK translocates to the nucleus and phosphorylates transcription factors (2, 3). The MAPK cascade is involved in processes regulating cell growth, division, and differentiation. The most widely studied cascades are the extracellular signal-regulated kinase-1 (ERK1; p44 MAPK) and ERK2 (p42 MAPK). The activation of MAPK results from two distinct classes of cell surface receptors: receptor tyrosine kinases [RTK; such as the epidermal growth factor (EGF) receptor (EGFR)] and G protein-coupled receptors [GPCR; such as GnRH receptor (GnRH-R)].

As both the EGFR (4) and GPCRs, such as GnRH-R and TRH receptor (5), are expressed on the surface of GGH31' cells, these cells are a potentially useful model to investigate differential regulation of MAPK activation mediated by EGFR and GnRH-R. It was reported that TRH stimulates MAPK activity in GH3 cells in both protein kinase C (PKC)-dependent and -independent pathways (6). However, MAPK activation triggered by EGFR and GnRH-R has not been investigated in such cells.

It is well known that RTKs regulate MAPK in a multistep process that involves Ras and a limited number of well understood molecules (2, 3). In contrast, MAPK activation by GPCRs is not fully understood (7). Heterotrimeric G proteins consist of three heterologous subunits ({alpha}, ß, and {gamma}). G protein {alpha}-subunits are classified into four major classes based on their function: Gs, Gi/o, Gq/11, and G12. Gi/o, Gq/11, as well as Gs{alpha} can activate MAPK in cells, Gi-coupled receptors (such as the {alpha}2A-adrenergic receptor) mediate Ras-dependent MAPK activation by G protein ß{gamma}-subunits (Gß{gamma}) (8, 9), Gq/11-coupled receptors (such as M1 acetylcholine receptor) activate MAPK via a pathway that is Ras independent but requires the activity of PKC (10), and Go-coupled receptors (e.g. M1 acetylcholine receptor) activate MAPK via a novel PKC-dependent mechanism (3, 11).

The role of Gs in 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 (12, 13, 14). 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 (15). Similarly, cAMP stimulates MAPK activity in PC12 cells (16). More recently, analysis of the Gs/MAPK pathway in mutant S49 cells provided molecular genetic evidence that Gs{alpha} is responsible for transducing the ß-adrenergic receptor signal to MAPK in the protein kinase A (PKA)-dependent pathway involving Rap1 and Raf (but not Ras) molecules (17). The MAPK cascade is activated by GnRH in a PKC-, Ca2+-, and protein tyrosine kinase-dependent fashion (18, 19, 20). However, the roles of Gi/o and Gs in MAPK activation in response to GnRH are still controversial even in the same cell line ({alpha}T3 cell line) (20, 21). The PKA signal transduction pathway appears to be involved in GnRH action in GGH31' cells (22, 23, 24). Considering the fact that the GnRH-R couples to several G protein subunits, Western blotting by phospho-p44/42 MAPK antibody and p44/42 MAPK antibody was performed in this study to assess the role of PKC as well as that of PKA in MAPK activation in GGH31' cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The GnRH agonist Buserelin (D-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH) was a gift from Hoechst-Roussel Pharmaceuticals (Somerville, NJ). Cholera toxin (CTX) and pertussis toxin (PTX; List Biological Laboratories, Campbell, CA), (Bu)2cAMP, forskolin, phorbol esters [phorbol 12-myristate 13-acetate (PMA)], 3-isobutyl-1-methylxanthine (MIX; Sigma Chemical Co., St. Louis, MO), DMEM (Irvine Scientific, Santa Ana, CA), EGF (receptor grade, Collaborative Biomedical Products, Becton Dickinson and Co., Franklin Lakes, NJ), and GF109203X (Calbiochem, La Jolla, CA) were purchased from the indicated vendors. The PhosphoPlus p44/42 MAP kinase antibody kit was purchased from New England Biolabs, Inc. (Beverly, MA).

Cell culture
GH3 cells stably transfected with the rat GnRH-R complementary DNA (GGH31') (25) were maintained in growth medium [DMEM containing 10% FCS (HyClone Laboratories, Inc., Logan, UT) and 20 µg/ml gentamicin (Gemini Bioproducts, Calabasas, CA)] in a humid atmosphere (37 C) with 5% CO2. Cells were grown to confluence in 162-cm2 T-flask (Costar, Cambridge, MA), then scraped and plated at a density of 5 x 105 cells/well in six-well culture plates. Approximately 24 h after plating, cells were washed once, and the medium was replaced for another 24 h in growth medium. Approximately 48 h after seeding, the cells were washed twice with DMEM-0.1% BSA-20 µg/ml gentamicin and incubated for another 4 h before treatment with the agents at the indicated concentrations for the indicated lengths of time. The cells were washed with cold PBS, then lysed by the addition of SDS sample buffer.

Western blotting
SDS-polyacylamide gels (10%) were run and transferred to nitrocellulose membranes by standard methods (26). Briefly, cell extracts were subjected to SDS-PAGE gels, then electrotransferred to nitrocellulose membranes (Hoefer Scientific, San Francisco, CA). Western blotting was performed according to the instructions in the PhosphoPlus p44/42 MAP kinase antibody kit, except that a syringe with a 25-gauge 5/8th-in. hypodermic needle was used to shear DNA and reduce sample viscosity before loading 100-µl samples onto SDS-PAGE gel (15 x 15 cm). The membrane was probed with a rabbit polyclonal primary antibody for phospho-p44/42 MAPK (Thr202/Tyr204), which detects p42 and p44 MAPK only when phosphorylated at Thr202 and Tyr204, and alternatively probed with a rabbit polyclonal antibody for p44/42 MAPK, which detects total MAPK (phosphorylation state independent) levels. After washing the membrane, the signals were visualized using horseradish peroxidase-conjugated second antibody and the enhanced chemiluminescence method provided by the kit. Phosphorylated and nonphosphorylated MAPK were used as controls, and biotinylated protein markers were used to determine mol wt: maltose binding protein ß-galactosidase (165K), glutamic dehydrogenase (57K), maltose binding protein 2 (46.5K), lactate dehydrogenase M (28K), tyrosine inhibitor (20.5K), lysozyme (14.5K), and aprotinin (6.5K). The bands on the x-ray film were scanned, and the intensities of the bands corresponding to p42 MAPK were quantitated by an imaging densitometer.

The data shown are band densities in arbitrary optical density units. Experiments were repeated two or three times each in duplicate or triplicate wells, and the results shown are from a representative experiment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EGF stimulation of MAPK in GGH31' cells
Treatment of the GGH31' cells with EGF (200 ng/ml) stimulated a rapid increase in MAPK activity. Activation of MAPK reached its peak within 5 min after a challenge with EGF, then decreased rapidly to basal levels (upper panel and Fig. 1aGo). In contrast, the total MAPK level in GGH31' cells remained constant for 1 h despite the continued presence of EGF (lower panel).



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  		Figure 1. a, Time course of EGF-stimulated MAPK activation in GGH31' cells. GGH31' cells were grown in DMEM with 10% FCS and 20 µg/ml gentamicin. Plated cells were incubated in DMEM-0.1% BSA for 4 h before treatment with EGF (200 ng/ml). The cells were stimulated with EGF for 0, 1, 3, 5, 10, 20, 30, 40, or 60 min. After the indicated time, cells were washed with cold PBS. The cells were lysed in 100 µl SDS sample buffer and centrifuged at 10,000 x g for 10 min. Each supernatant was immunoblotted with antibody for phospho-p44/42 to determine the phosphorylated p44/42 MAPK as described in Materials and Methods. Alternatively, the same samples were immunoblotted with antibody for p44/42 MAPK to determine total p42 MAPK as a control. The upper panel shows bands for activated MAPK (phosphorylated p44/42 MAPK); the lower panel shows bands for total MAPK. The intensity of the bands corresponding to p42 MAPK was quantitated with an imaging densitometer. The results show one of three similar experiments. b, Time course of Buserelin-stimulated MAPK activation in GGH31' cells. GGH31' cells were incubated, and the supernatant was prepared from treated cells as described in a. The cells were stimulated with Buserelin (10 ng/ml) for 0, 3, 5, 7.5, 10, 20, 30, 40, or 60 min, then washed and lysed as described above. Phosphorylated p44/42 MAPK and total p42 MAPK were analyzed with Western immunoblotting as described in Materials and Methods. The upper panel shows bands for phosphorylated p44/42 MAPK. The lower panel shows bands for total MAPK in response to Buserelin. The intensities of the bands corresponding to p42 MAPK in the upper panel were quantitated with an imaging densitometer (b). The results show one of three similar experiments.

 
Buserelin stimulation of MAPK in GGH31' cells
The GnRH agonist, Buserelin, stimulated rapid activation of MAPK in GGH31' cells (Fig. 1bGo). Maximum stimulation by Buserelin (10 ng/ml) was observed within 15 min. Similar to the EGF response, the activated MAPK level decreased rapidly to basal levels after MAPK activation reached a peak in GGH31' cells (upper panel), whereas the total MAPK activity in GGH31' cells remained unchanged for 1 h (lower panel).

Effect of Buserelin on MAPK activation in GGH31' cells
To investigate stimulation of MAPK activation in response to 10-11–10-7 M Buserelin, the cells were treated for 10 min. The data show that treatment with Buserelin stimulates MAPK activation in a dose-dependent manner, with maximum stimulation obtained at 10-9–10-8 M (Fig. 2Go).



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  		Figure 2. The effect of Buserelin with different concentrations on MAPK activation in GGH31' cells. GGH31' cells were incubated, and the supernatant was prepared as described in Materials and Methods. The cells were stimulated with Buserelin with concentrations between 10-11–10-7 M for 10 min. Then, activated MAPK was analyzed by Western blotting with the antibody recognizing phosphorylated p44/42 MAPK. The intensities of the bands were quantitated as described in Fig. 1aGo. The results show one of three similar experiments.

 
Effect of PMA on MAPK activation in GGH31' cells
To assess whether the PKC signal transduction pathway is involved in MAPK activation in GGH31' cells, the cells were treated with 10 ng/ml PMA. The time course of PMA showed that treatment with PMA led to a maximum level of activated MAPK after 30 min, followed by a decline of activated MAPK in GGH31' cells (Fig. 3Go). Of note, the time course for the maximum activation of MAPK in response to PMA was different from that evoked by EGF and Buserelin, suggesting that distinct signal transduction pathways might be involved in MAPK activation in GGH31' cells, apart from the PKC signaling pathway.



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  		Figure 3. Time course of PMA-stimulated MAPK activation in GGH31' cells. GGH31' cells were incubated, and the supernatant was prepared as described in Fig. 1Go. The cells were stimulated with 10 ng/ml PMA for 0, 1, 3, 5, 10, 20, 30, 40, or 60 min. The cells were washed and lysed as described in Fig. 1aGo. Phosphorylated p44/42 MAPK in the upper panel and total MAPK in the lower panel were analyzed as described in Fig. 1Go. The densities of bands corresponding to phospho-p42 MAPK were quantitated using an imaging densitometer. The results show one of three similar experiments.

 
Effect of PKC inhibitor GF109203X on MAPK activation in GGH31' cells
To assess the role of PKC signal transduction pathways in MAPK activation in GGH31' cells, the cells were pretreated with the PKC inhibitor GF109203X (25 µM for 10 min), followed by stimulation with EGF (200 ng/ml), Buserelin (10 ng/ml), or PMA (10 ng/ml) for 3, 10, or 30 min, respectively. The time required for maximum activation of MAPK in response to EGF, Buserelin, and PMA is shown in Fig. 1Go, a and b, and Fig. 3Go. As shown in Fig. 4Go, GF109203X failed to influence the basic level of phosphorylated MAPK in response to EGF, whereas it caused complete inhibition of MAPK activation in response to Buserelin or PMA.



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  		Figure 4. The effect of GF109203X on MAPK activation in GGH31' cells. GGH31' cells were incubated, and the supernatant was prepared as described in Fig. 1Go. The cells were treated with EGF (200 ng/ml), Buserelin (10 ng/ml), or PMA (10 ng/ml) for 3, 10, and 30 min, respectively. Alternatively, the cells were incubated with GF109203X (25 µM) or DMEM-0.1% BSA alone (control) for 3, 10, and 30 min, respectively. Alternatively, the cells were pretreated with GF109203X (25 µM) for 10 min before stimulation with Buserelin (10 ng/ml), EGF (200 ng/ml), or PMA (10 ng/ml) for 3, 10, and 30 min, respectively. The amount of phosphorylated MAPK was determined by an imaging densitometer as described in Materials and Methods. The results show one of three similar experiments.

 
Effect of PKC depletion on MAPK activation in response to EGF, Buserelin, and PMA
To investigate whether other distinct signaling pathways might be involved in the activation of MAPK in GGH31' cells, the cells were pretreated with PMA (100 ng/ml) for 18 h to deplete PKC or were pretreated with medium alone, then challenged with EGF (200 ng/ml), Buserelin (10 ng/ml), or PMA (10 ng/ml) for 3, 10, and 30 min, respectively. The results are shown in Fig. 5aGo. The data indicate that treatment with both EGF and Buserelin provoked MAPK activation in PKC-depleted cells despite the attenuation of stimulation in response to EGF and Buserelin in such cells. On the other hand, although treatment with PMA alone led to an increase in the phosphorylated MAPK level, PMA failed to stimulate activation of MAPK in GGH31' cells that were pretreated with PMA (100 ng/ml) for 18 h to deplete intracellular PKC.



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  		Figure 5. a, The effect of PKC depletion on MAPK activation in response to EGF, Buserelin, and PMA. GGH31' cells were incubated, and the lysate for Western blotting was prepared from treated cells as described in Fig. 1Go. The cells were pretreated with or without PMA (100 ng/ml) for 18 h to deplete intracellular PKC. The cells were washed three times with DMEM-0.1% BSA before stimulation with EGF (200 ng/ml), Buserelin (100 ng/ml), or PMA (10 ng/ml) for 3, 10, and 30 min, respectively. The control cells were incubated with medium alone for 3 min. The amount of activated MAPK (phosphorylated MAPK) was determined by an imaging densitometer as described inMaterials and Methods. The results show one of three similar experiments. b, The effect of PKC depletion on MAPK activation in response to PKA signal transduction pathway. GGH31' cells were incubated, and the lysate for Western blotting was prepared from treated cells as described in Fig. 1Go. The cells were pretreated with or without PMA (100 ng/ml) for 18 h to deplete intracellular PKC. The cells were washed three times with DMEM-0.1% BSA before treatment with or without forskolin (50 µM), PTX (100 ng/ml), or CTX (100 ng/ml) for 30 min. The amount of activated MAPK (phosphorylated MAPK) was determined by an imaging densitometer as described inMaterials and Methods. The results show one of three similar experiments.

 
Effect of PKC depletion on MAPK activation in response to the PKA signal transduction pathway
To investigate the role of PKA in MAPK activation in GGH31' cells, forskolin (50 µM), PTX (100 ng/ml), or CTX (100 ng/ml) was used to treat PKC-depleted or cells with the normal PKC complement. The data in Fig. 5bGo indicate that treatment with forskolin or CTX results in activation of MAPK independent of PKC depletion, suggesting that PKA can stimulate activation of MAPK in a PKC-independent manner. Although treatment with PTX alone seemed to have a modest effect on MAPK activation, PTX failed to have any effect in PKC-depleted cells, suggesting that there is little or no effect of the Gi/o subunit on MAPK activation in GGH31' cells (Fig. 5bGo).

Role of PKA on MAPK activation in response to EGF in GGH31' cells
The cells were pretreated for 15 min with medium alone (control) or with medium containing MIX (0.5 mM), forskolin (50 µM), (Bu)2cAMP (1 mM), MIX (0.5 mM) plus forskolin (50 µM), or MIX (0.5 mM) plus (Bu)2cAMP (1 mM) to increase cAMP levels, followed by stimulation with medium in the presence (200 ng/ml) or absence of EGF for 3 min. The data show that pretreatment with MIX, forskolin, (Bu)2cAMP, MIX plus forskolin, or MIX plus (Bu)2cAMP stimulated activation of MAPK (Fig. 6aGo). The results of MAPK activation resulting from an increased cAMP level provide further evidence of involvement of Gs in MAPK activation in GGH31' cells. MAPK activation in response to EGF was not affected by the level of intracellular cAMP, suggesting that the PKA signal transduction pathway appeared not to play an important role in MAPK activation by EGF in GGH31' cells.



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  		Figure 6. a, Role of PKA in MAPK activation induced by EGF in GGH31' cells. GGH31' cells were incubated, and the lysate for Western blotting was prepared from treated cells as described in Fig. 1Go. Cells were treated with DMEM-0.1% BSA alone (control) or with DMEM-0.1% BSA containing MIX (0.5 mM), forskolin (50 µM; Forsk), (Bu)2cAMP (1 mM) (cAMP), forskolin (50 µM) plus MIX (0.5 mM), or (Bu)2cAMP (1 mM) plus MIX (0.5 mM) for 15 min. The cells were then stimulated with medium in the presence or absence of EGF (200 ng/ml) for another 3 min. The amount of activated MAPK (phosphorylated MAPK) was determined by an imaging densitometer as described in Materials and Methods. The results show one of three similar experiments. b, Role of PKA in MAPK activation in response to Buserelin in GGH31' cells. GGH31' cells were incubated, and the lysate for Western blotting was prepared from treated cells as described in Fig. 1Go. After pretreatment of the cells with MIX (0.5 mM), forskolin (50 µM; Forsk), (Bu)2cAMP (1 mM; cAMP), forskolin (50 µM) plus MIX (0.5 mM), (Bu)2cAMP (1 mM) plus MIX (0.5 mM), or DMEM-0.1% BSA alone for 15 min, the cells were stimulated with or without Buserelin (10 ng/ml) for another 10 min. The amount of activated MAPK (phosphorylated MAPK) was determined by an imaging densitometer as described in Materials and Methods. The results show one of three similar experiments.

 
Role of PKA on MAPK activation in response to Buserelin in GGH31' cells
After pretreatment with MIX (0.5 mM), forskolin (50 µM), (Bu)2cAMP (1 mM), MIX (0.5 mM) plus forskolin (50 µM), or MIX (0.5 mM) plus (Bu)2cAMP (1 mM) for 15 min, the cells were then challenged with Buserelin (10 ng/ml) for an additional 10 min. The results indicate that the level of intracellular cAMP is associated with MAPK activation by Buserelin in GGH31' cells (Fig. 6bGo).

Role of (Bu)2cAMP in MAPK activation in GGH31' cells (time course)
Direct evidence of involvement of the Gs {alpha}-subunit in MAPK activation was provided by the results of a time course of (Bu)2cAMP treatment (1 mM) in GGH31' cells. Treatment with (Bu)2cAMP provoked MAPK activation in GGH31' cells after 1 h of incubation (Fig. 7Go). The maximum stimulation of MAPK activation was observed after the cells were treated for 30 and 40 min, then decreased rapidly.



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  		Figure 7. Time course of (Bu)2cAMP-stimulated MAPK activation in GGH31' cells. GGH31' cells were incubated, and the supernatant was prepared as described in Fig. 1Go. The cells were stimulated with (Bu)2cAMP (1 mM) for 0, 1, 3, 5, 10, 20, 30, 40, or 60 min. The cells were washed and lysed as described in Fig. 1Go. Phosphorylated MAPK in the upper panel and total MAPK in the lower panel were analyzed as described in Fig. 1Go. The densities of bands corresponding to phospho-p42 MAPK in the upper panel were quantitated with an imaging densitometer. The results show one of three similar experiments.

 
Roles of PTX and CTX in MAPK activation in response to EGF, Buserelin, and PMA
To determine the roles of Gs and Gi/o in MAPK activation of GGH31' cells, we investigated the effect of PTX and CTX on the activation of MAPK in response to EGF (200 ng/ml), Buserelin (10 ng/ml), or PMA (10 ng/ml). Pretreatment of the cells with PTX (100 ng/ml) or CTX (100 ng/ml for 15 min) had no influence on MAPK activation in response to EGF (Fig. 8aGo), whereas pretreatment with CTX (100 ng/ml) significantly enhanced the phosphorylation of MAPK in response to Buserelin (Fig. 8bGo). The results demonstrate that the Gs subunit coupled to the GnRH-R leads to MAPK activation in GGH31' cells. In turn, PTX pretreatment had almost no measurable effect on MAPK activation in response to Buserelin, indicating that Gi/o did not have a major role in mediation of MAPK activation. On the other hand, stimulation with PMA significantly enhanced MAPK activation in GGH31' cells after pretreatment with CTX (Fig. 8cGo). The additive action of CTX and PMA in MAPK activation further supports the hypothesis that Gs and Gq/11 mediate the activation of MAPK in response to GnRH-R action. In contrast, pretreatment with PTX for 15 min did not alter the level of phosphorylated MAPK (activated MAPK) in response to PMA in GGH31' cells. The data described above show that Gq/11 and Gs, but not Gi/o, play significant roles in MAPK activation in GGH31' cells.



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  		Figure 8. a, Roles of PTX and CTX in MAPK activation in response to EGF. GGH31' cells were incubated, and the lysate for Western blotting was prepared from treated cells as described in Fig. 1Go. The cells were incubated with DMEM-0.1% BSA alone (control) or with DMEM-0.1% BSA in the presence of PTX (100 ng/ml) or CTX (100 ng/ml) for 15 min, followed by stimulation with or without EGF (200 ng/ml) for another 3 min. The amount of activated MAPK (phosphorylated MAPK) was determined by an imaging densitometer as described in Materials and Methods. The results show one of three similar experiments. b, Roles of PTX and CTX in MAPK activation in response to Buserelin. GGH31' cells were incubated, and the lysate for Western blotting was prepared from treated cells as described in Fig. 1Go. The cells were incubated with DMEM-0.1% BSA alone (control) or with DMEM-0.1% BSA in the presence of PTX (100 ng/ml) or CTX (100 ng/ml) for 15 min, followed by stimulation with or without Buserelin (10 ng/ml) for another 10 min. The amount of activated MAPK (phosphorylated MAPK) was determined by an imaging densitometer as described in Materials and Methods. The results show one of three similar experiments. c, Roles of PTX and CTX in MAPK activation in response to PMA. GGH31' cells were incubated, and the lysate for Western blotting was prepared from treated cells as described in Fig. 1Go. The cells were incubated with DMEM-0.1% BSA alone (control) or with DMEM-0.1% BSA in the presence of PTX (100 ng/ml) or CTX (100 ng/ml) for 15 min, followed by stimulation with or without PMA (10 ng/ml) for another 30 min. The amount of activated MAPK (phosphorylated MAPK) was determined by an imaging densitometer as described in Materials and Methods. The results show one of three similar experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we investigated the roles of PKC and PKA in MAPK activation in response to EGF and GnRH. The data indicate that treatment with either EGF or Buserelin stimulated MAPK activation in GGH31' cells. Treatment of the cells with PMA led to MAPK activation; treatment of the cells with MIX, forskolin, (Bu)2cAMP, or CTX appeared to activate MAPK in PKC-depleted cells, and PTX was without any measurable effect. The data implicate both PKC and PKA signal transduction pathways in MAPK activation in GGH31' cells.

The GnRH-R is a GPCR and mediates signaling events, including stimulation of calcium influx and activation of phospholipase C, leading to increased protein kinase activity (23, 27). There is convincing evidence that agonist occupancy of the GnRH-R leads to MAPK activation. The PKC/MAPK pathway activated by GnRH preferentially stimulates {alpha}-subunit gene transcription by phosphorylation of the Ets family transcription factors (28), whereas induction of the LHß gene is dependent on calcium influx (29). Stimulation of MAPK reached its peak within 5 min in response to EGF and within 15 min in response to Buserelin followed by an abrupt decrease in phosphorylated MAPK in GGH31' cells, whereas the total MAPK (inactivated and activated MAPK) level remained nearly unchanged for 1 h. Haisenleder et al. showed that a pulsatile GnRH signal is required to maintain MAPK activity for periods longer than 2 h; additionally, MAPK appears to play a central role in the induction of gonadotropin {alpha}, FSHß, and GnRH-R messenger RNA (mRNA) responses to pulsatile GnRH (30).

Several G protein {alpha}-subunits, such as Gi/o, Gq/11, and Gs, are involved in MAPK activation (8, 9, 10). MAPK activation in response to GnRH is associated with the PKC signal transduction pathway and the Ca2+ signal (18, 19, 20), which is apparently involved in the regulation of Gq/11 subunit protein in GnRH-R signaling (31, 32). The mechanism by which PKC activates the MAPK cascade remains unclear; however, there is evidence that the stimulation of MAPK activity by Gq/11-coupled receptor is mediated by both p21 Ras-dependent (33) and p21 independent pathways (34). The results of MAPK activation in response to Buserelin in PKC-depleted cells suggest that Buserelin activates MAPK by both PKC-dependent and PKC-independent pathways in GGH31' cells.

Treatment with GF109203X (25 µM) failed to block MAPK activation in response to EGF, but completely inhibited MAPK activation in response to Buserelin or PMA. A cell-permeable PKC inhibitor (Ki = 10 nM), GF109203X, is structurally similar to staurosporine and shows high selectivity for PKC{alpha}, -ß1, -ß11, -{gamma}, -{delta}, and -{epsilon} isozymes. Interestingly, there is evidence that H7, another specific inhibitor of PKC, failed to block the activation of MAPK resulting from GnRH action (35). This discrepancy may result from different specificity toward the various PKC isoforms that are present in the cells, including {alpha}T3 (36) and COS-7 cells (37). Previous reports showed that MAPK activation by GnRH was inhibited by GF109203X with an IC50 value of 1.8 ± 0.2 µM (20, 21). However, GF109203X may inhibit PKA at much higher concentrations (Ki = 2 µM). Although GF109203X (25 µM) inhibits MAPK activation mediated by GnRH-R, the possibility that the PKA signal transduction pathway is involved in MAPK activation by GnRH could not be excluded. The difference between MAPK activation by EGFR and that by GnRH-R may reflect the fact that GPCRs and Raf-1 protein kinase-dependent RTKs mediate distinct cell responses. Similarly, results from the PKC-depleted cells and from cells pretreated with MIX, forskolin, or (Bu)2cAMP suggest a distinct mechanism by which MAP kinases were activated via EGFR and GnRH-R in GGH31' cells.

In gonadotrope-derived cell lines ({alpha}T3–1 cells) and in lactotrope-derived cell lines stably expressing the GnRH-R (GGH3 cells), Gq/11{alpha} couples to the GnRH-R (31, 32). Although the PKA pathway appears not to be involved in stimulated hormone release from {alpha}T3–1 cells or gonadotrope cells, this pathway may have an effect on increasing the biosynthesis of LH (38), regulating the gonadotropin subunit mRNA (39) or GnRH-R mRNA (40) or the stimulation of proliferation in {alpha}T3–1 cells (41). Recently, Garrel et al. (42) reported that catalytic and regulatory subunits of PKA are subject to both hormonal and receptor-independent regulation in {alpha}T3–1 cells, whereas the effects of PKA activation probably involve proteolytic degradation of the dissociated PKA holoenzyme, providing direct evidence for cross-talk between PKA and PKC pathways in {alpha}T3–1 cells (42).

Apart from Gq/11{alpha}, the GnRH-R is also coupled to Gs{alpha}, which activates adenylate cyclase in GGH31' cells, resulting in the production of cAMP (22, 23, 24). Treatment of GGH3 cells with Buserelin stimulates cAMP production (5) and evokes dose- and time-dependent PRL synthesis in GGH3 cells via a cAMP-dependent pathway (43). Similar effects were reported for transiently expressed GnRH-R and constitutively activated Gs{alpha} in COS-7 cells (44). More recently, the results of palmitoylation of G protein subunits provided more direct evidence for activation of Gq/11{alpha}, Gs{alpha}, and Gi{alpha} by the GnRH-R in both GGH3 cells and primary pituitary cells (45). Evidence from loop fragment transfection also supports the idea that the third intracellular loop of the rat GnRH-R couples to the Gs- and Gq/11-mediated signaling pathway in GGH3 cells (46). Taken together, the PKA signaling pathway participates in GnRH action in pituitary cells and in GGH3 cells. The present data demonstrate that the PKA signal transduction pathway is involved in the activation of MAPK, consistent with the previous findings that the GnRH-R couples to Gs{alpha} to activate adenylate cyclase and subsequent production of cAMP. Thus, MAPK activation by GnRH appeared to involve both PKC- and PKA-dependent signal transduction pathways in GGH3 cells. There is evidence that a sustained high level of cAMP causes degradation of the catalytic subunit of PKA, thereby diminishing its activity in several kinds of cells, including GH3 cells (47), {alpha}T3 cells (42), and rat hepatocytes (48). It is reasonable to believe that a similar phenomenon could be responsible for the lack of the additive action in MAPK activation in response to either forskolin-MIX or (Bu)2cAMP-MIX. The mechanistic details of the precise relation between the PKA signaling pathway and the MAPK cascade remain elusive.

In {alpha}T-3 cells, MAPK was activated by the GnRH-R through a mechanism involving PKC (20, 21). There is evidence that the GnRH-R is coupled to Gi{alpha} in reproductive tract tumor (49). On the other hand, the GnRH-R expressed in insect cells activates adenylyl cyclase (50). Increasing evidence that multiple G proteins mediate the effects of GnRH-R raises the possibility that the same kind of GPCR exerts regulation via differential signal transduction pathways in distinct tissue or cells, further determining the specific function mediated by the receptor in these cells.

It was noteworthy that treatment of GGH31' cells with either PMA (10 ng/ml) or (Bu)2cAMP (1 mM) produced significant stimulation of MAPK after 30 min, then declined, while Buserelin provoked the rapid activation of MAPK. The results implied the involvement of the distinct signal transduction pathways in mediating MAPK activation by the GnRH-R apart from the PKA- and PKC-dependent pathways. There is convincing evidence that signaling from GPCR to MAPK involves ß{gamma}-subunits of heterotrimeric G proteins acting on a Ras-dependent pathway (7, 8, 9, 44). Thus, it is reasonable to conclude that both GPCR and RTK induce p21 Ras activation via convergent signaling pathways, leading to the rapid activation of MAPK in response to Buserelin. RTKs and GPCRs, two important families of membrane receptors, mediate MAPK activation after binding to their specific ligands. Therefore, the convergence of signaling events induced by RTKs and GPCRs also implied the significance of MAPK in mediating cell functions. Although the signaling events of RTKs and GPCRs await further investigation, the data presented here not only establish the importance of PKA and PKC function in MAPK activation in GGH31' cells, but further identify the cross-talk between GPCR and RTK signals, thereby providing important insights into the signaling network involving two major classes of cell surfaces in GGH31' cells.

The data presented here provide evidence that both EGFR and GnRH-R mediate MAPK activation in GGH31' cells. Treatment with Buserelin provoked MAPK activation in a PKA- and PKC-dependent manner, suggesting that both Gs and Gq/11 are involved in the action mediated by the GnRH-R in GGH31' cells.


   Acknowledgments
 
We thank Jo Ann Janovick, Dziennis Suzan, and Guadalupe Maya-Nunez for commenting on the manuscript.

Received October 14, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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