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Endocrinology Vol. 140, No. 3 1310-1318
Copyright © 1999 by The Endocrine Society

ARTICLES

Activation of the p38 Mitogen-Activated Protein Kinase Pathway by Gonadotropin-Releasing Hormone1

Mark S. Roberson, Tong Zhang, Hui Ling Li and Jennifer M. Mulvaney

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

Address all correspondence and requests for reprints to: Dr. Mark S. Roberson, Department of Biomedical Sciences, T6–008a Veterinary Research Tower, Cornell University, Ithaca, New York 14853. E-mail: msr14{at}cornell.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that interaction of GnRH with its serpentine, G protein-coupled receptor results in activation of the extracellular signal regulated protein kinase (ERK) and the Jun N-terminal protein kinase (JNK) pathways in pituitary gonadotropes. In the present study, we examined GnRH-stimulated activation of an additional member of the mitogen-activated protein kinase (MAPK) superfamily, p38 MAPK. GnRH treatment of {alpha}T3–1 cells resulted in tyrosine phosphorylation of several intracellular proteins. Separation of phosphorylated proteins by ion exchange chromatography suggested that GnRH receptor stimulation can activate the p38 MAPK pathway. Immunoprecipitation studies using a phospho-tyrosine antibody resulted in increased amounts of immunoprecipitable p38 MAPK from {alpha}T3–1 cells treated with GnRH. Immunoblot analysis of whole cell lysates using a phospho-specific antibody directed against dual phosphorylated p38 kinase revealed that GnRH-induced phosphorylation of p38 kinase was dose and time dependent and was correlated with increased p38 kinase activity in vitro. Activation of p38 kinase was blocked by chronic phorbol ester treatment, which depletes protein kinase C isozymes {alpha} and {epsilon}. Overexpression of p38 MAPK and an activated form of MAPK kinase 6 resulted in activation of c-jun and c-fos reporter genes, but did not alter the expression of the glycoprotein hormone {alpha}-subunit reporter. Inhibition of p38 activity with SB203580 resulted in attenuation of GnRH-induced c-fos reporter gene expression, but was not sufficient to reduce GnRH-induced c-jun or glycoprotein hormone {alpha}-subunit promoter activity. These studies provide evidence that the GnRH signaling pathway in {alpha}T3–1 cells includes protein kinase C-dependent activation of the p38 MAPK pathway. GnRH integration of c-fos promoter activity may include regulation by p38 MAPK.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH IS REQUIRED for normal function of the hypothalamo-pituitary-gonadal axis in mammals. The GnRH receptor is a serpentine, G protein-coupled receptor. Binding of GnRH to its receptor results in G{alpha}q/11-mediated activation of phospholipase C, leading to the production of inositol 1,4,5-trisphosphate and diacylglycerol, increased intracellular calcium, and activation of protein kinase C (PKC) (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). The consequences of GnRH receptor occupancy include increased synthesis and secretion of the gonadotropic hormones, LH and FSH. Ablation of GnRH stimulation of the anterior pituitary results in decreased glycoprotein hormone subunit messenger RNA concentrations and diminished secretion of the gonadotropic hormones (13, 14). In addition to the glycoprotein hormone subunit genes, GnRH stimulates increased c-fos (15, 16), c-jun (15), mitogen-activated protein kinase phosphatase 2 messenger RNA and transcriptional activation of the ternary complex factor Elk-1 (17).

GnRH-dependent intracellular signaling events downstream of PKC have only recently been characterized. Several groups have demonstrated that GnRH receptor occupancy results in activation of extracellular signal regulated protein kinases (ERK) (17, 18, 19, 20) and c-jun N-terminal protein kinase (21) (Mulvaney, J. M., T. Zhang, and M. S. Roberson, in preparation), members of the mitogen-activated protein kinase (MAPK) superfamily. Acute phorbol ester administration can mimic GnRH action on ERKs (17, 18, 19). Chronic phorbol ester treatment to deplete PKC isozymes reduces GnRH-induced ERK activity, suggesting a requirement for PKC in GnRH activation of the ERK pathway (18, 19). GnRH activation of the JNK cascade is dependent on the low mol wt GTP-binding protein, Cdc42 (21), and appears to be activated independently of diacylglycerol-dependent PKC isozymes, which differentiates GnRH modulation of the ERK and JNK cascades (Mulvaney, J. M., T. Zhang, and M. S. Roberson, in preparation).

In addition to the ERK and JNK cascades, the MAPK superfamily includes the p38 kinase pathway (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). Studies of other G{alpha}q-coupled receptors have shown that ligand association results in activation of all three MAPK pathways (36, 37). Activation of the p38 signaling pathway has been linked to activation of Elk-1 ternary complex factor, which binds to the c-fos promoter (38), and myocyte enhancer family (MEF-2)-dependent c-jun gene expression (39), suggesting a potential role for p38 MAPK in the modulation of immediate early gene expression in pituitary gonadotropes. The present studies investigated the possibility that GnRH induces activation of p38 MAPK in the {alpha}T3–1 gonadotrope cell line. We report here that GnRH stimulates activation of the p38 kinase pathway. Activation of the p38 kinase by GnRH requires PKC. Our studies suggest that GnRH-induced p38 MAPK activation may selectively contribute to the regulation of c-fos protooncogene expression, but not c-jun or the glycoprotein hormone {alpha}-subunit gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression vectors and plasmids
The coding sequence for p38 kinase was obtained by PCR and verified by nucleotide sequence analysis (GenBank accession no. U91847) (40). The p38 coding sequence was fused in-frame with the AU1 epitope (peptide sequence DTYRYI) and cloned downstream of the cytomegalovirus promoter in pcDNA3 expression vector. The glycoprotein hormone {alpha}-subunit promoter linked to luciferase has been described previously (17). Expression vector for Gal4-Elk-1 was prepared as previously described (17). Expression vector for MAPK kinase-6 (MKK-6) Glu was a gift from Dr. R. Davis (University of Massachusetts, Boston, MA). Bacterial expression vector for glutathione-S-transferase (GST)-activating transcription factor-2 (ATF-2) was a gift from Dr. M. Green (University of Massachusetts). Bacterial expression and partial purification of GST fusion proteins were accomplished as previously described (41). Expression vector for c-jun-luciferase reporter was a gift from Dr. Ron Prywes (Columbia University, New York, NY) and has been described previously (42). All plasmid DNA was prepared by two cycles of centrifugation through cesium chloride.

Cell culture and transfection
{alpha}T3–1 cells (provided by Dr. P. Mellon, University of California-San Diego) (17, 43, 44) were maintained in monolayer culture in DMEM supplemented with 5% FBS and 5% horse serum (Life Technologies, Grand Island, NY). For immunoblotting and transient transfection studies, cells were grown to approximately 60% confluence before transfection. For immunoblot studies, cells were serum deprived for 2 h before hormone treatments. Agonists included the GnRH analog buserelin ([D-Ser(tBu)6,Pro9-ethylamide]GnRH; this analog is referred to as GnRHa), phorbol myristate acetate (PMA), or sorbitol (0.3 M) in DMEM. In some experiments, the GnRH antagonist ([N-Ac-D-Nal(2)1-pCl-D-Phe2-D-Pal(3)3-Lys(Nic)5-D-Lys(Nic)6-Lys(iPr)8-D-Ala10]GnRH; antide, Bachem, Torrance, CA) was added to the medium 30 min before and during treatment with buserelin. For transient transfection studies, cells were transfected by electroporation using a single electrical pulse at 220 V and 950 µF, as described previously (17). In transfection studies, {alpha}T3–1 cells were treated with buserelin at a concentration of 10 nM for 16 h before collection (18 h after electroporation) to allow for accumulation of luciferase activity. Some transfected cells were given 20 µM SB203580 (Calbiochem, La Jolla, CA) beginning 2 h before buserelin administration. SB203580 remained on transfected cells for the duration of hormone treatment. After cell collection, lysates were prepared by three freeze-thaw cycles and clarified by centrifugation, and luciferase activity was determined in equal amounts for cellular protein (45).

Mono Q chromatography
For preparation of large scale {alpha}T3–1 cell lysate for fractionation by Mono Q chromatography, cells were cultured in 150-mm dishes to approximately 50% confluence. Cells were serum starved for 2 h, then treated (where indicated) with control solution or the GnRH analog buserelin (10 nM) for 15 min. The dishes were then placed on ice, washed three times with ice-cold buffer containing 0.15 M NaCl and 10 mM HEPES (pH 7.5), and lysed with gentle agitation for 15 min at 4 C. The lysis buffer contained 70 mM ß-glycerophosphate (pH 7.2), 2 mM sodium vanadate, 2 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 2.5 µg leupeptin/ml, 2.5 µg pepstatin/ml, 0.2 mM phenylmethylsulfonylfluoride, 5 mM benzamidine, and 1 mM dithiothreitol (buffer A). Cell debris was removed by centrifugation, and clarified supernatants (equal amounts of total protein, 1.0–1.5 mg) were loaded onto a Mono Q ion exchange chromatography column (Pharmacia Biotech, Piscataway, NJ) maintained at 4 C. During all procedures, the column was maintained at a flow rate of 0.3 ml/min, and fractions were collected in 1.0-ml volumes. After loading of the cell lysate, the column was washed with 5 ml buffer A and eluted in a NaCl gradient (buffer A plus 1.0 M NaCl) from 0–0.4 M NaCl delivered over a 25-ml column elution. For experiments with cells treated without or with buserelin, control cell lysates were processed first, followed immediately (column was washed and reequilibrated between lysate preparations) by lysates from cells receiving buserelin treatment. Fractions (50 µl) were then subjected to immunoblot analysis as described above.

Antibodies, immunoprecipitation, immunoblotting, and kinase assays
Monoclonal antibodies directed against phosphotyrosine (4G10; 3.9 mg/ml) were provided by Dr. B. Drucker (Oregon Health Sciences University, Portand, OR). An antibody dilution of 1:12,500 was used for all phosphotyrosine blots, and 5% BSA was used as a blocking agent. AU1 monoclonal antibody was purchased from BAbCO (Berkeley, CA). Specific polyclonal antibodies directed against ERKs and p38 kinase, horseradish peroxidase-coupled secondary antibodies (antimouse and antirabbit), and protein A/G-agarose were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and used according to the manufacturer’s instruction. Phospho-specific antibodies for ERK and p38 kinase were purchased from New England Biolabs, Inc. (Beverly, MA), and used according to the manufacturer’s instructions. The antibodies for PKC isozymes, {alpha}, {epsilon}, and {zeta} were obtained from Life Technologies. For immunoprecipitations (IP) and immunoblotting, {alpha}T3–1 cells were treated for the specified time periods, then washed in ice-cold buffer containing 0.15 M NaCl and 10 mM HEPES (pH 7.5). The cells were lysed in a buffer containing 25 mM HEPES (pH 7.5), 3 mM ß-glycerophosphate, 3 mM EDTA, 3 mM EGTA, 250 mM NaCl, 2 mM sodium vanadate, 1% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, and 5 mM benzamidine (referred to as lysis buffer) at 4 C for 10 min with gentle rocking. The cell lysates were scraped from the dishes and clarified by centrifugation. Methods for IP and subsequent washes have been described previously (17). For whole cell lysates (40–80 µg total protein) or IPs, proteins were resolved on denaturing polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membrane by electroblotting. After immunostaining, specific proteins or phosphotyrosine accumulation was visualized with enhanced chemiluminescence reagents using protocols described by the supplier (New England Nuclear-DuPont, Boston, MA). Stripping of PVDF membranes was accomplished by placing the membrane in a solution containing 62.5 mM Tris (pH 6.8), 2% SDS, and 100 mM 2-mercaptoethanol and heating to 55 C for 30 min. The membrane was then washed three times (10 min each) in Tris-buffered saline (pH 7.5) containing 0.1% Tween-20. For p38 kinase assays, IP complexes were washed four times in 1 ml lysis buffer followed by one wash in a kinase buffer containing 20 mM HEPES (pH 7.5), 20 mM MgCl2, 25 mM ß-glycerol phosphate, 100 µM sodium vanadate, 50 mM ATP, and 2 mM dithiothreitol. The IP agarose beads were resuspended in kinase buffer, and [{gamma}-32P]ATP (5 µCi) and a specific p38 substrate (recombinant GST-ATF-2) were added last. Samples were subjected to kinase reaction for 30 min at 30 C. The reaction was stopped by the addition of SDS loading buffer. Samples were then boiled for 2 min and resolved by SDS-PAGE. Kinase activity was visualized by autoradiography and analyzed by scanning densitometry.

Statistical analysis
Transfection data were subjected to ANOVA, and treatment differences were determined by either pairwise t test or Tukey’s Studentized range test. Differences were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRH stimulation of {alpha}T3–1 cells results in induction of tyrosine-phosphorylated proteins that include p38 kinase
To examine signaling molecules associated with GnRH action, phosphotyrosine immunoblot analysis was performed on whole cell lysates from {alpha}T3–1 cells (Fig. 1Go). {alpha}T3–1 cells are a clonal cell line derived from targeted oncogenesis of the gonadotrope cell lineage of the anterior pituitary (43, 44). {alpha}T3–1 cells were treated with control solution or the GnRH agonist buserelin (GnRHa; Fig. 1Go). Administration of GnRHa for 15–30 min resulted in phosphotyrosine accumulation of at least three proteins in the approximate molecular mass range of 43 and 29 kDa. We have previously demonstrated that GnRH receptor occupancy activates the ERK signaling pathway (17), and p44/p42 ERKs probably accounted for two of the phosphotyrosine-containing proteins. The presence of multiple phosphotyrosine-containing bands suggested the possibility that GnRH also induced phosphorylation of other members of the MAPK family.



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  		Figure 1. GnRH stimulation of {alpha}T3–1 cells results in tyrosine phosphorylation of intracellular proteins. {alpha}T3–1 cells were cultured in the absence of serum for 2 h, followed by administration of the GnRH analog buserelin (GnRHa; 10 nM) for the indicated times. Cells were lysed, and intracellular proteins were resolved on denaturing gels and then transferred to PVDF membrane. Immunoblots were probed with antiphosphotyrosine antibodies. Arrows indicate bands of phosphotyrosine accumulation with GnRHa. The molecular size standard (MW) is indicated at the left of the figure.

 
Biochemical fractionation of GnRH-induced MAPK activity in {alpha}T3–1 cells was used to identify other MAPK family members that may play a role in GnRH action. Cell extracts from GnRHa-treated {alpha}T3–1 cells were separated by ion exchange chromatography on a Mono Q column, fractions were further resolved by denaturing PAGE, and phosphotyrosine-containing proteins were identified by immunoblotting (Fig. 2AGo). The results of these studies revealed that phosphotyrosine-containing proteins in the 43–29 kDa range could be resolved into two groups of fractions (fractions 38–44 and fractions 48–50). Analysis with antibodies for specific members of the MAPK family indicated that ERK-1 and ERK-2 elute with fractions 38–44 (Fig. 2BGo), whereas p38 elutes in fractions 48–50 (Fig. 2CGo). In a second study, {alpha}T3–1 cells were treated with control solution (Fig. 2DGo) or GnRHa (Fig. 2EGo) for 15 min, cells were lysed, and lysates were subjected to Mono Q fractionation. Fractions expected to contain p38 immunoactivity were then assayed by phosphotyrosine immunoblotting. A marked accumulation of a phosphotyrosine-containing band with the appropriate size and elution profile for p38 kinase was observed after GnRH treatment. These studies suggest that GnRH treatment may activate the p38 MAPK.



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  		Figure 2. Separation of ERK and p38 kinase immunoactivity by ion exchange chromatography. Cell lysates from GnRHa-treated {alpha}T3–1 cells were resolved on a Mono Q column, and fractions were eluted with increasing ionic strength. A portion of the denoted fractions (fraction number) were resolved on denaturing gels and transferred to the PVDF membrane. The immunoblot was probed with antiphosphotyrosine antiserum (A), stripped, and reprobed with ERK antibody (B), then restripped and reprobed with p38 antibody (C). Molecular size standards are denoted on the left axis. A second experiment was conducted to determine the effects of GnRH administration on accumulation of phosphotyrosine in fractions containing p38 kinase. Cell lysates from {alpha}T3–1 cells treated with either control solution (D) or for 15 min with buserelin (GnRHa; 10 nM; E) were fractionated by Mono Q chromatography and resolved on denaturing gels. Proteins were transferred to PVDF membrane and immunoblotted with antiphosphotyrosine antibodies. The arrows denote changes in phosphotyrosine observed after GnRH treatment.

 
Biochemical studies predicted that immunoprecipitation (IP) of tyrosine-phosphorylated proteins from GnRHa-treated {alpha}T3–1 cells would result in IP of increased amounts of the p38 MAPK. {alpha}T3–1 cells were treated with GnRHa for 15 min, and whole cell lysates were prepared. An antiphosphotyrosine antibody was used to IP tyrosine-phosphorylated proteins induced by GnRHa. The proteins present in the IP were resolved by SDS-PAGE and transferred to PVDF membrane. Immunoblot analysis was performed using antibodies directed against p38 kinase (Fig. 3Go). Consistent with our prediction, a greater amount of p38 kinase was present in the IP from {alpha}T3–1 cells treated with GnRHa compared with control cells. This study is consistent with observations from Mono Q chromatography and provides additional evidence that mechanisms of GnRH signaling include phosphorylation of p38 MAPK.



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  		Figure 3. GnRHa induces tyrosine phosphorylation of p38 kinase. {alpha}T3–1 cells were serum starved for 2 h, then treated with buserelin (GnRHa; 10 nM) for 15 min. The cells were lysed, and phosphotyrosine-containing proteins were immunoprecipitated (IP) with a phosphotyrosine antibody and protein A agarose. The products of the IP were resolved with SDS-PAGE and transferred to the PVDF membrane. The blot were probed with a p38 antibody. Thearrows indicate IgG present in the IP and p38 kinase (p38).

 
GnRH induces p38 kinase activity
We used two approaches to directly examine the effects of GnRH on activation of the p38 kinase. Recently, phospho-specific p38 kinase antibodies became commercially available (New England Biolabs, Inc., Beverly, MA). Phospho-specific p38 antibodies recognize the p38 MAPK when phosphorylated on threonine 180 and tyrosine 182. Activation of the p38 MAPK depends upon dual phosphorylation of these two residues (28); thus, the phospho-specific p38 kinase antibody putatively recognizes activated p38 kinase. Initial studies examined the effects of GnRHa for a 15-min period in a dose-response study (Fig. 4AGo). GnRHa induced activation of p38 MAPK at doses as low as 0.1 nM (4.3-fold) and appeared to have a modest peak in activation at 10 nM GnRHa (5.5-fold). Time-course studies revealed that increased phospho-specific staining of the p38 MAPK was evident after 15 min (5.2-fold) of GnRHa treatment, began to decline at 30 min (4.4-fold), and had essentially returned to baseline 2 h after hormone administration (Fig. 4AGo). Similar amounts of p38 kinase (independent of the phosphorylation state) were present in all lanes. The second approach to assay p38 kinase activation was an immune complex kinase assay using GST-ATF-2 as a substrate (Fig. 4BGo). GnRHa administration for 15 or 30 min stimulated an increase (2.5- and 3.5-fold for 15 and 30 min, respectively) in p38 kinase activity consistent with results obtained with phospho-specific p38 antiserum. Sorbitol was administered in these studies to induce osmotic shock, and p38 kinase activity was used as a positive control (40, 46). Concurrent administration of the specific GnRH receptor antagonist, antide, at a 10-fold molar excess blocked GnRHa-induced p38 MAPK activation (data not shown). These studies provide direct evidence that GnRH receptor occupancy results in activation of p38 kinase in {alpha}T3–1 cells.



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  		Figure 4. GnRHa induces p38 kinase activity. Immunoblot analysis with phospho-specific antiserum and in vitro kinase assay were used to examine the activation state of p38 kinase after treatment of {alpha}T3–1 cells with buserelin (GnRHa). {alpha}T3–1 cells were serum starved for 2 h, then were treated for 15 min with increasing doses (0, 0.1, 1, 10, or 100 nM) of GnRHa (A). In a second study, serum-starved {alpha}T3–1 cells were administered GnRHa (10 nM) for 0, 15, 30, 60, 120, and 240 min (A). Lysates were prepared and resolved by SDS-PAGE and probed with a phospho-specific p38 kinase antibody (phospho-p38). The blots were then stripped and reprobed with an antibody to p38 kinase to demonstrate equal protein amounts in each lane (p38). In a third study, {alpha}T3–1 cells were administered GnRHa for 0, 15, or 30 min or were subjected to osmotic shock by administration of sorbitol (300 mM) as a positive control. Cell lysates were prepared, and p38 kinase activity was isolated by immunoprecipition (IP) with specific p38 kinase antiserum (B). The IP complexes were washed, and GST-ATF-2 was added to a kinase reaction in the presence of Mg and 32P-labeled ATP. Labeled substrate (GST-ATF-2) was then resolved by SDS-PAGE, and bands were visualized by autoradiography.

 
PKC-depletion blocks GnRH-mediated phosphorylation of p38 kinase
PKC isozymes are required for GnRH-mediated activation of ERKs in {alpha}T3–1 cells (18, 19). Further, phorbol ester treatment has been shown to activate the p38 kinase pathway in some cell types (47). We tested the hypothesis that GnRH-induced activation of p38 kinase was PKC dependent. Chronic treatment of {alpha}T3–1 cells with PMA (100 nM for 20 h) was used to deplete PKC isozymes. After PKC depletion, {alpha}T3–1 cells were treated with GnRHa for the specified times (Fig. 5AGo). Phospho-specific p38 and phospho-specific ERK immunoblots were conducted on {alpha}T3–1 whole cell lysates to determine the activation state of MAPKs. In the absence of chronic PMA treatment, GnRHa induced activation of p38 kinase and ERKs (Fig. 5AGo). PKC depletion by chronic PMA administration resulted in a marked attenuation of GnRHa-induced phosphorylation of p38 kinase and ERKs. PKC isozyme depletion in this paradigm did not appreciably alter the total amounts of p38 or ERKs present in {alpha}T3–1 cells (Fig. 5AGo). To confirm the efficacy of our PKC depletion studies, amounts of PKC isozymes present before and after chronic administration of PMA were investigated by immunoblot analysis (Fig. 5BGo). {alpha}T3–1 cells express at least three different isozymes of the PKC family ({alpha}, {epsilon}, and {zeta}) (48). Chronic phorbol ester administration at 100 nM PMA for 20 h resulted in a near-complete loss of the {alpha} and {epsilon} isozymes (Fig. 5BGo). Additional immunoblot analysis for the PKC{zeta} isozyme revealed that this isozyme remained unchanged after chronic phorbol ester treatment in {alpha}T3–1 cells (data not shown). These findings suggest a requirement for diacylglycerol-dependent PKC isozymes in GnRH-mediated p38 kinase activation similar to that demonstrated for GnRH-induced activation of ERKs (18, 19).



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  		Figure 5. PKC depletion by chronic phorbol ester treatment blocks GnRH-mediated phosphorylation of p38 kinase. {alpha}T3–1 cells were administered DMSO or PMA (100 nM) for 20 h before agonist treatment to depleted PKC isozymes. Cells were then administered GnRHa (10 nM) for the indicated times. Cell lysates were prepared and subjected to immunoblot analysis. Initially, blots were probed with phospho-specific p38 antibodies (phospho-p38; A), stripped, and reprobed with p38 antibodies (p38) to determine the amounts of p38 present in each lane. The immunoblot was then stripped again and probed with antibodies directed against tyrosine-phosphorylated p42/p44 ERKs (phospho-ERK; A). The blot was then stripped again and reprobed with ERK antibodies (ERK) to determine the amount of ERK protein present in each lane. B depicts immunoblot analysis of {alpha}T3–1 whole cell lysates derived from control cells or cells treated for 20 h with phorbol ester (PMA; 100 nM). Blots were probed with antibodies specific for PKC{alpha} and PKC{epsilon} isozymes.

 
Activation of the p38 MAPK by constitutively active MKK-6 activates the c-jun and c-fos promoters in {alpha}T3–1 cells
GnRH receptor occupancy results in increased immediate early gene activation of the protooncogenes, c-fos and c-jun, and cell-specific activation of the glycoprotein hormone {alpha}-subunit promoter (15, 16, 17, 38). The {alpha}-subunit promoter is regulated by the ERK pathway (17, 18, 19); however, regulation by other MAPK pathways has not been examined. Studies examined whether p38 activation was sufficient and/or required for the regulation of protooncogene expression or the {alpha}-subunit promoter. Initial investigation focused on isolating the activation of the p38 signaling cascade in the absence of ERK or JNK activation in {alpha}T3–1 cells to determine whether activation of the p38 pathway alone was sufficient to activate the various reporter genes. The MKK-6 has been reported as a specific activator of p38 kinase (47, 49). Substitution mutations at Ser207 and Thr211 with Glu results in partial activation of MKK-6 (referred to as MKK-6 Glu). Cotransfection of p38 and MKK-6 Glu expression vectors resulted in a dramatic increase in p38 kinase activity in COS-1 cells (47). Based upon this approach, {alpha}T3–1 cells were transfected with expression vector for epitope-tagged (AU1) p38 kinase alone or in combination with MKK-6 (Glu). p38 kinase activity was then isolated by IP using the AU1 monoclonal antibody and subjected to kinase assay using GST-ATF-2 as substrate. AU1-p38 kinase protein levels were similar in the absence or presence of MKK-6 Glu expression vector in transfected {alpha}T3–1 cells (Fig. 6AGo). Transfection of MKK-6 Glu expression alone was not sufficient to activate endogenous p38 kinase (not shown). Cotransfection of AU1-p38 with MKK-6 Glu expression vector resulted in marked activation of p38 kinase. MKK-6 has been shown to be a specific activator of p38 kinase, but not ERKs or JNK, and our results were consistent with previous studies with activation of p38 kinase by overexpression of MKK-6 Glu in COS-1 cells (47). This approach provided an overexpression paradigm to specifically activate the p38 kinase without activation of parallel MAPK (ERK and JNK) pathways. Based upon these experimental conditions, we examine the effects of specific p38 kinase activation on expression of the {alpha}-subunit, c-jun, and c-fos reporter genes (Fig. 6BGo). Specific activation of p38 kinase did not alter expression of the glycoprotein hormone {alpha}-subunit reporter gene activity. Overexpression and activation of p38 kinase was sufficient to activate the c-jun and c-fos reporter genes. Replacement of the p38 expression vector with expression vectors for wild-type ERK-2 or JNK in additional transfection studies did not result in activation of the c-jun or c-fos reporter constructs (data not shown), suggesting that the effects of overexpression of MKK-6 Glu and p38 were specific.



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  		Figure 6. Overexpression and activation of p38 kinase by MKK-6 results in increased expression of c-jun and c-fos reporter genes. {alpha}T3–1 cells were transfected by electroporation with expression vectors for epitope (AU1)-tagged p38 kinase (5 µg) in the absence or presence of a constitutively activated form of MKK-6 (MKK-6 Glu; 5 µg; A). Eighteen to 20 h after transfection, cells were lysed. A portion of the lysate was resolved by SDS-PAGE and transferred to PVDF for immunoblot analysis with the AU1 antibody (AU 1-p38), demonstrating approximately equal overexpression of p38 kinase. The remainder of the lysates were subjected to IP with an AU1 monoclonal antibody. The products of the IP were subjected to p38 kinase assay (as described in Materials and Methods) using GST-ATF-2 as a substrate. The products of the kinase reaction were resolved by SDS-PAGE and visualized by autoradiography. In a separate study (B), {alpha}T3–1 cells were transfected with either pcDNA3 (control; 10 µg) or expression vectors for MKK-6 Glu and AU1-p38 (MKK-6 and p38; 5 µg each). In addition to expression vectors, cells were transfected with one of three reporter genes, the glycoprotein hormone {alpha}-subunit or c-jun or c-fos luciferase fusion genes (1 µg each). Eighteen to 20 h after transfection, cells were harvested by scraping, and lysates were prepared by three freeze-thaw cycles. Luciferase activity was then determined. Data are reported as mean luciferase activity ± SEM. For the {alpha}-subunit and c-fos reporters, relative luciferase activity is expressed as light units x 10-4. For the c-jun reporter, relative luciferase activity is expressed as light units x 10-5. The study was conducted on three separate occasions (in triplicate) all with similar results. The asterisk denotes a difference from the control value (P < 0.05).

 
Studies next examined whether GnRH-induction of the p38 cascade was required for {alpha}-subunit or protooncogene expression. In these studies, we used the specific p38 kinase inhibitor, SB203580 (50). SB203580 is a pyridinyl imidazole that specifically inhibits the p38 MAPK in a dose-dependent manner, but does not inhibit ERK or JNK activity (50). {alpha}T3–1 cells were transfected with the various reporter genes and cultured in the absence or presence of 20 µM SB203580 for 2 h. Some cells then received GnRHa for approximately 16 h. All cells were collected approximately 18 h after electroporation and assayed for luciferase activity. Administration of SB203580 resulted in a reduction of basal levels of {alpha}-subunit reporter activity. GnRHa administration induced the {alpha}-subunit reporter approximately 6-fold, and administration of SB203580 did not alter this effect (Fig. 7Go). Thus, the reduction in the absolute magnitude of GnRHa-induced {alpha}-subunit reporter activity with SB203580 pretreatment was attributable to changes in basal level of {alpha}-subunit reporter activity. SB203580 administration did not alter basal or GnRH-induced c-jun reporter gene expression levels (Fig. 7Go). In contrast, pretreating transfected cells with SB203580 resulted in a partial loss (~50%) of GnRHa-induced activation of the c-fos protooncogene (Fig. 7Go). Similar partial inhibition was obtained in experiments using 10 µM SB203580 (not shown), suggesting that 20 µM SB203580 was saturating. Collectively, these studies provide evidence that GnRH induction of the p38 MAPK cascade is probably not required for the activation of the {alpha}-subunit or c-jun promoters, but can selectively contribute to GnRH-induced transcriptional activation of the c-fos protooncogene.



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  		Figure 7. Inhibition of p38 kinase selectively attenuates GnRHa-induced c-fos reporter gene activity. {alpha}T3–1 cells were transiently transfected by electroporation with reporter genes for the glycoprotein hormone {alpha}-subunit ({alpha}), c-jun (jun), or c-fos (fos) coupled to luciferase (1 µg each). After electroporation, transfected cells were cultured in the absence or presence of 20 µM SB203580. Two hour after electroporation, some cells were treated with GnRHa (10 nM) for 16 h. All cells were then collected by scraping and lysed by three freeze-thaw cycles. Luciferase activity was then determined. Data are reported as the mean luciferase activity or fold induction (Fold) ± SEM. The study was conducted on three separate occasions (in triplicate), all with similar results. The asterisk denotes a difference from the control value (P < 0.05).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies support the conclusion that GnRH receptor occupancy results in activation of multiple MAPK pathways. Our laboratory and others have demonstrated that GnRH receptor occupancy results in activation of the ERK and JNK cascades (17, 18, 19, 20, 21) (Mulvaney, J. M., T. Zhang, and M. S. Roberson, in preparation). In the present studies, GnRH agonist administration to {alpha}T3–1 cells resulted in an accumulation of tyrosine-phosphorylated p38 kinase within 15 min of treatment. Tyrosine phosphorylation of p38 kinase was correlated with increased enzyme activity in vitro. The time course of p38 activation was consistent with activation of the ERK cascade, with peak activity at 15 min and a return to baseline by 1–2 h after hormone administration (Mulvaney, J. M., T. Zhang, and M. S. Roberson, unpublished observations). In contrast, GnRH induction of the JNK cascade is slightly delayed, with peak activity occurring at 30 min after hormone treatment (21) (Mulvaney, J. M., T. Zhang, and M. S. Roberson, unpublished observations). GnRH receptor occupancy resulting in activation of all three MAPK pathways is consistent with other serpentine receptors that couple to G{alpha}q/11. Endothelin (ET) interaction with the ET-B receptor results in ERK, JNK, and p38 activation (51). In studies of the ET-B receptor, the carboxyl-terminal 146 amino acids of the cytoplasmic tail were required to couple to multiple MAPK pathways (37). The GnRH receptor has only two amino acids associated with the carboxyl-terminal cytoplasmic tail (for review, see Ref. 52). Recent studies (53) have shown that overexpression of the third intracellular loop from the GnRH receptor appears to interfere with inositol phosphate production in GH3 cells stably transfected with the GnRH receptor complementary DNA; however, the role of intracellular loops within the GnRH receptor in modulating multiple MAPK signaling has not yet been determined. Thus, the actual mechanism(s) of coupling of serpentine receptors to multiple MAPK pathways may vary with structural differences among receptor subtypes.

The p38 MAPK is a mammalian homolog of the yeast protein HOG-1. HOG-1 was originally identified as an intracellular mediator of osmotic stress (28, 40, 46). The p38 kinase has been shown to be activated by osmotic manipulation, UV irradiation, endotoxins, and cytokines (28, 46, 47, 54, 55). The present studies demonstrate that the effects of osmotic stress (induced by sorbitol treatment) on p38 kinase activity were greater than activation induced by GnRHa in {alpha}T3–1 cells. Osmotic stress-induced p38 kinase activity also does not appear to require PKC (Roberson, M. S., unpublished observation), demonstrating a possible divergence in upstream regulators of the p38 kinase. The reasons for variable magnitude in p38 kinase activity induced by stress or GnRH are not presently known. However, it is possible that in response to adverse environmental conditions, high level or prolonged activation of p38 kinase (combined perhaps with other MAPK pathways) may be required to regulate critical mechanisms of cell survival (or cell death). Activation of p38 kinase by GnRH appears to support cellular mechanisms that contribute to immediate early gene expression.

Previous studies have demonstrated a requirement for PKC isozymes on GnRH-mediated ERK activation (18). The present studies extend these observations, demonstrating that PKC isozyme depletion can block GnRH-induced p38 activation. Our studies and those of others also using {alpha}T3–1 cells (56) have shown that the cellular content of both PKC{alpha} and PKC{epsilon} are depleted by chronic phorbol treatment. Interestingly, recent studies (56) failed to detect redistribution of PKC{alpha} to the membrane after GnRH stimulation of {alpha}T3–1 cells, raising the possibility that this isozyme is not involved in GnRH signaling. However, as with other cell systems (57), more extensive time-course studies will be required to confirm these results and investigate the possibility the PKC{alpha} may have translocated to the membrane more rapidly than the reported time course. Thus, it may be premature to discount a role for PKC{alpha} in mediating the effects of GnRH on activation of ERKs or p38 MAPK. In contrast to the {alpha} isozyme, PKC{epsilon} was found to redistribute to the membrane after GnRH administration (56) consistent with a role for the {epsilon} isozyme in mediating responses to GnRH, including activation of the ERK and p38 cascades. More definitive studies of the role of specific PKC isozymes in mediating GnRH effects will require the development of reagents that can selectively block the action of specific PKC isozymes.

To date, several downstream targets of p38 kinase have been identified. Inflammatory cytokines and heat stress result in p38 kinase-dependent activation of MAPK-activated protein kinase-2 (MAPKAP-2) and MAPKAP-3, leading to the phosphorylation of the small heat shock protein, Hsp27 (58, 59, 60, 61). Two additional targets of p38 kinase are transcription factors that are presumably activated by p38 kinase-dependent phosphorylation, including the ternary complex factor, Elk-1 (38, 47) and MEF-2 (39). In the present studies, both MKK-6 Glu/p38 overexpression and studies using SB203580 demonstrated that p38 activation was sufficient and at least partially required for GnRH signaling to the c-fos reporter. These observations are consistent with studies of p38 activation of the c-fos serum response element and activation of the ternary complex factor Elk-1 (38, 61). Our present studies suggest that while p38 may contribute to c-fos gene expression, it is probably not the primary mechanism for GnRH action on this immediately early gene. This is based on two observations. First, overexpression studies using MKK-6 Glu and p38 did increase c-fos promoter activity. We used this overexpression paradigm simply to isolate p38 kinase activity in the absence of other related MAPK family members. The caveat to these studies is that the level of p38 kinase activity induced by MKK-6 overexpression far exceeded that induced by GnRH. This may suggest that high levels of p38 activity are sufficient to activate the c-fos reporter. The possibility exists that lower levels of p38 activity (such as that induced by GnRH) may contribute less to c-fos expression probably relative to more robust contributions of GnRH-induced ERK and JNK activities. Second, only partial attenuation of GnRH-induced c-fos reporter gene activity by SB203580 was observed. In 3T3 cells, 10 µM SB203580 was sufficient to block 95% of p38 kinase activity without significant alteration of ERK or JNK activity (50). This suggests that the conditions used in the present study (20 µM SB203580) were sufficient to block more than 95% of GnRH-induced p38 enzyme activity. Probably other signaling mechanisms, such as GnRH-induced ERK or JNK activation, may play a primary role in up-regulation of the c-fos protooncogene, whereas GnRH-induced p38 activity may represent a contributory, but secondary, mechanism.

The p38 MAPK has been linked to transcriptional regulation of c-jun via an MEF-2-binding site present on the c-jun promoter (39). In those studies, MEF-2C was shown to be a specific target of the p38 MAPK pathway. Specific activation of p38 kinase by overexpression of MKK-6 Glu/p38 MAPK in the present studies did result in a 2-fold activation of the c-jun promoter in {alpha}T3–1 cells. However, administration of SB203580 was not sufficient to block GnRH-induced activation of the c-jun reporter, suggesting that p38 kinase activation was not required for GnRH action on c-jun. These data support the conclusion that overexpression of activated p38 MAPK at high levels was sufficient to activate the c-jun reporter probably in a manner not consistent with GnRH action. Lower levels of p38 MAPK activity induced by GnRH are not apparently required for GnRH-induced c-jun promoter activity in {alpha}T3–1 cells.

The studies presented have identified the p38 MAPK signaling pathway as a target of GnRH hormone action. Activation of p38 kinase by GnRH requires PKC isozymes. One possible consequence of GnRH action on p38 kinase appears to be contribution to the transcriptional regulation of the c-fos protooncogene. These studies also suggest that GnRH action on the tissue-specific promoter for the glycoprotein hormone {alpha}-subunit gene does not require activation of p38 kinase. Activation of the ERK, JNK, and p38 MAPK pathways by GnRH receptor activation is consistent with other serpentine receptors and provides a mechanism to modulate the expression and activity of multiple transcriptional activators.


   Acknowledgments
 
The authors thank Drs. R. Davis, M. Green, R. Prywes, and B. Drucker for generously supplying valuable reagents. We thank Drs. Richard Cerione and Joanne Fortune for critical reading of the manuscript, and Dr. Ron Prywes for helpful discussions. We are indebted to Dr. Brett White for aid with statistical analysis. The authors are most grateful to the guidance and generosity provided by Dr. Richard A. Maurer at Oregon Health Sciences University.


   Footnotes
 
1 This work was supported by a grant from the NIH (HD-34722; to M.S.R.). Back

Received September 8, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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