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Endocrinology Vol. 142, No. 2 887-895
Copyright © 2001 by The Endocrine Society

ARTICLES

Induction of c-fos and c-junMessenger Ribonucleic Acid Expression by Prostaglandin F2{alpha} Is Mediated by a Protein Kinase C-Dependent Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Pathway in Bovine Luteal Cells1

Dongbao Chen2, Hon Wa Fong and John S. Davis

The Women’s Research Institute (D.C., H.W.F., J.S.D.), Departments of Obstetrics and Gynecology, and Internal Medicine, University of Kansas School of Medicine-Wichita, and Research Service of the Department of Veterans Affairs (J.S.D.), 1010 North Kansas, Wichita, Kansas 67214

Address all correspondence and requests for reprints to: John S. Davis, Ph.D., The Women’s Research Institute, 1010 North Kansas, Wichita, Kansas 67214-3199. E-mail: jdavis3{at}kumc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PGF2{alpha} triggers the demise of the corpus luteum whereby progesterone synthesis is inhibited, the luteal structure regresses, and the estrus cycle resumes. Upon binding to its heterotrimeric G-protein-coupled receptors, PGF2{alpha} initiates the phospholipase C/diacylglycerol and inositol-1,4,5-trisphosphate/Ca2+-protein kinase C (PKC) signaling pathway. More recently, we have demonstrated that PGF2{alpha} activates extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase signaling through a Raf-dependent mechanism in bovine luteal cells. However, the relationship between PKC and ERK activation in PGF2{alpha} signaling has not been clearly defined. Moreover, the signaling pathway that PGF2{alpha} uses to regulate gene expression is unknown. In this report, primary cultures of bovine luteal cells were used to address the role of PKC in ERK activation and the signaling pathway for induction of c-fos and c-jun messenger RNA (mRNA) expression in response to PGF2{alpha}. By using a PKC inhibitor and a PKC-deficient luteal cell model, we observed that phorbol ester-responsive isoforms of PKC were required for ERK phosphorylation and activation by PGF2{alpha} (1 µM) or phorbol 12-myristate 13-acetate (PMA) (20 nM). In PGF2{alpha}- and PMA-treated cells, active ERK MAP kinase was localized in the nucleus. PGF2{alpha}-induced ERK phosphorylation was dose-dependently inhibited by the MEK1 inhibitor PD098059 (1–50 µM). The expression of c-fos and c-jun mRNA in luteal cells was markedly increased by treatment with PGF2{alpha} (1 µM) or PMA (20 nM) for 30 min. We also observed that activation of ERK MAP kinase was required for the expression of c-fos and c-jun mRNA in response to PGF2{alpha} and PMA because it was abrogated by blocking the ERK pathway with PD098059. In addition, PGF2{alpha} and PMA-induced c-fos and c-jun mRNA expression was abolished in the PKC-deficient cells. Taken together, our data demonstrate that a PKC-dependent ERK MAP kinase pathway mediates the expression of c-fos and c-jun mRNA in PGF2{alpha}-treated bovine luteal cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PGF2{alpha} triggers the demise of the corpus luteum (luteolysis) in domestic farm animals whereby progesterone synthesis is inhibited, the luteal structure regresses, and the estrous cycle resumes (reviewed in 1, 2). Despite extensive studies demonstrating the physiological role of PGF2{alpha} in the corpus luteum, the cellular and molecular mechanisms of PGF2{alpha}-induced luteolysis remain poorly understood. It is well-known that, in bovine luteal cells, PGF2{alpha} binds to specific G-protein-coupled receptors (3) and activates phospholipase C (PLC), which leads to the generation of two second messengers, i.e. diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (InsP3) (4). Accumulation of InsP3 elevates intracellular Ca2+ levels (4, 5). PGF2{alpha} stimulates this signaling pathway presumptively to activate the Ca2+- and phospholipid-dependent protein kinase C (PKC). Indeed, PGF2{alpha} has been demonstrated to increase PKC activity and PKC translocation in luteal cells (6, 7, 8, 9). Activation of PKC is thought to, at least in part, mediate PGF2{alpha}-induced luteolysis in vivo (10).

Ligands that signal via G-protein-coupled receptors have been shown to activate the mitogen-activated protein (MAP) kinase signaling cascade. This cytoplasmic protein kinase cascade transduces signals initiated at the plasma membrane to the nucleus, where it regulates the expression of specific target genes (11, 12). In vertebrates, multiple isoforms of MAP kinase have been identified and categorized into three subfamilies, i.e. the extracellular signal-regulated kinases (ERKs), p38mapk, and the Jun N-terminal kinases (JNKs) or stress-activated protein kinases. The classical ERKs, ERK2 or p42mapk and ERK1 or p44mapk, are positioned downstream of Raf-1 and MEK1, and together comprise an orderly signaling cascade (13). The MAP kinase signaling pathways have been implicated in control of different, and even opposite, cellular responses (including proliferation, differentiation, and cell death). Such actions are elicited, at least in part, through translocation of activated MAP kinase into the nucleus (14), where it phosphorylates and thereby activates nuclear transcription factors, including Elk-1 (15), Sap1 (16), c-Jun (17), ATF2 (15, 17), and others. These transcription factors stimulate the expression of the immediate-early response oncogenes, i.e. c-fos and c-jun (15, 17). Fos and Jun proteins are constituents of the activator protein-1 (AP-1) transcription factors that, in turn, regulate the transcription of numerous genes possessing promoter AP-1 binding sites (18). In bovine luteal cells, we have recently reported that PGF2{alpha} activates each component of the Raf/MEK1/ERK signaling cascade (19), increases levels of c-fos and c-jun messenger RNA (mRNA), and activates the AP-1 transcription factors (20). It seems likely that the Raf/MEK1/ERK signaling cascade constitutes a pivotal intracellular network directly regulating the expression and activation of the transcription factor AP-1 that may play an important role in programming gene expression during PGF2{alpha}-induced luteolysis. However, the mechanisms regulating the expression of AP-1 transcription factors in the corpus luteum are currently unknown.

The mechanism by which PGF2{alpha} regulates the activation of the Raf/MEK1/ERK signaling cascade is also unclear. Watanabe et al. (21) suggested that the mitogenic actions of PGF2{alpha} in NIH3T3 cells are mediated via activation of p21Ras, Raf, and subsequent activation of MEK1 and ERK2. Also of interest in this study were findings demonstrating that ERK activation by PGF2{alpha} was independent of phorbol ester-responsive isoforms of PKC (21). G-protein-ß{gamma}-subunit-mediated tyrosine phosphorylation of Shc and Sos has been recently implied in PGF2{alpha}-induced Raf-1 activation in myometrial cells (22). Additionally, Hakeda et al. (23) reported that PGF2{alpha} activates the ERK MAP kinase pathway in osteoblastic MC3T3-E1 cells via PKC-dependent activation of Raf-1. Our recent data suggests that PKC is involved in PGF2{alpha}-induced activation of the Raf/MEK/ERK signaling cascade in luteal cells, because the response to PGF2{alpha} was mimicked by the PKC activator phorbol 12-myristate 13-acetate (PMA) (19). In the present study, experiments were performed to determine: 1) the role of PKC in regulating the activation of the ERK MAP kinase; and 2) the role of PKC and ERK MAP kinase in the expression of c-fos and c-jun by PGF2{alpha} in bovine luteal cells. Our results indicate that activation of the c-fos and c-jun protooncogenes by PGF2{alpha} in luteal cells is mediated by a PKC-dependent ERK MAP kinase signaling pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell isolation and culture
Bovine ovaries from early pregnancy (fetal crown rump length <15 cm) were collected from a local slaughterhouse and transported to the laboratory in cold M-199. The luteal tissue was dissociated with collagenase as described (19). Cell viability was determined by the trypan blue exclusion test. Only those cell preparations with viability more than 90% were used, and they were plated in standard tissue culture plasticware in M-199 with 5% FCS, for 18 h at 37 C, in a humidified atmosphere of 5% CO2 in air. The cells were serum-starved, for 48 h before experiments, by replacing medium with serum-free M-199 supplemented with 0.1% BSA (Fraction V) and 5 µg/ml bovine insulin.

The PKC-depleted luteal cells were obtained by chronic treatment with high levels of PMA. After serum starvation for 24 h, luteal cells were treated for an additional 24 h with 2.5 µM PMA. Total cell extracts were prepared as described below. Western blot analysis with specific anti-PKC-{alpha}, -ßII, and -{epsilon} antibodies was used to verify depletion of phorbol ester-responsive isoforms of PKC.

Chemicals, cell treatments, and total cell extracts
All chemicals, unless noted, were purchased from Sigma (St. Louis, MO). The MEK-1 inhibitor PD098059 and the PKC inhibitor bisindoylmaleimide (GF109203x) were purchased from Calbiochem (La Jolla, CA). PGF2{alpha} was dissolved in absolute ethanol at 2 mM. PMA, PD098059, and GF109203x were prepared in dimethylsulfoxide at concentrations of 5 mM, 20 mM, and 10 mM, respectively. Cell treatments were performed with serum-free M-199 plus 0.1% BSA after 1-h equilibration at 37 C. Experiments were stopped by rapidly rinsing twice with cold PBS. The cells were lysed with nondenaturing lysis buffer A [10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-100, 0.5% NP-40, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin-A] on ice, with continuous shaking for 30 min. The total cell extracts were collected using a disposable cell scraper, vortexed vigorously, and clarified by centrifugation (15,000 x g, 5 min). The protein content of the samples was measured by a Bio-Rad Laboratories, Inc. procedure using BSA as the standard. Aliquots of the extracts were subjected to immunoprecipitation or frozen at -80 C until Western blot analysis could be performed.

SDS-PAGE and Western blotting
The cell extracts were heat-denatured in Laemmili buffer and subjected to discontinuous SDS-PAGE with 5% and 10% polyacrylamide in the stacking and resolving gels, respectively. Proteins in the gel were electrophoretically (100 V, 1.5 h) transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). The membrane was preincubated in TBST [50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 0.05% Tween-20] containing 5% fat-free milk, overnight at 4 C, and then incubated with appropriate amounts of primary antibodies in TBST-3% BSA (for monoclonal antibodies) or in TBST-5% fat-free milk (for polyclonal antibodies) at room temperature for 1 h. After four washes with 15 ml TBST, the membrane was incubated with corresponding antimouse (1:2000) or antirabbit (1:4000) peroxidase-conjugated IgG for 1 h, respectively. The membrane was washed again with TBST, and bound antibodies were detected by the Amersham Pharmacia Biotech enhanced chemiluminescence reagents.

Immunoprecipitation
Clarified cell extracts were precleared by coincubation with 1 µl normal goat serum and 20 µl protein-A conjugated agarose beads for 1 h at 4 C. After centrifugation (15,000 x g, 4 C, 5 min), the precleared cell extracts were transferred to new tubes. Equal amounts of protein were immunoprecipitated at 4 C, either with the rabbit polyclonal anti-ERK2 or anti-ERK1 conjugated agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in 1 ml of lysis buffer A, with continuous rotation for 2 h. The beads (immunocomplex) were collected by centrifugation. The immunoprecipitates were washed three times with buffer A, three times with LiCl solution (0.5 M LiCl, 100 mM Tris-HCl, pH 8.0), and two times with kinase assay buffer B [50 mM ß-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM dithiothreitol, and 0.09% Brig35]. The beads were resuspended in 40 µl kinase buffer B. Aliquots of the immunoprecipitates were prepared in Laemmili buffer for subsequent Western blot analysis to verify that the same amounts of immunoprecipitated MAP kinase were subjected to the kinase assay (data not shown).

Immunocomplex kinase assay
The transcription activation domain of human Elk-1 (amino acids 307–428) was obtained by nucleotide sequence analysis (24). The Elk-1 trans-activation domain was cloned into pGEX3 to produce a GST-Elk-1 fusion gene. GST-Elk-1 fusion protein was expressed in bacteria and partially purified as previously described (25) and used as the substrate for ERK kinase assay as reported (19). The kinase activity of ERK2 and ERK1 was measured by in vitro phosphorylation of GST-Elk-1 as described (19, 24). To perform these kinase assays: briefly, 20 µl of immunoprecipitates were coincubated with 0.5 µg GST-Elk-1 in the kinase cocktail [30 µM ATP, 20 mM Tris-HCl (pH 7.4), 0.5 mM MnCl2, 5 mM MgCl2]. The reaction was initiated by the addition of 10 µCi [{gamma}-32P]-ATP, incubated at 30 C for 30 min, and stopped by the addition of Laemmili buffer. The reaction mixtures were resolved on 10% SDS-PAGE, after which the gels were stained. Autoradiography was performed on dried gels to visualize phosphorylated GST-Elk-1. The corresponding bands of GST-Elk-1 were cut out and quantified by liquid scintillation counting.

Fluorescence immunocytochemistry
Luteal cells were cultured in 8-well Lab-Tek glass chamber slides as described above. After treatment with PGF2{alpha} (1 µM) and PMA (20 nM) for 5 min, the cells were washed with PBS, and fixed with cold methanol for 10 min at -20 C. The cells were air-dried and washed with 0.1% BSA-TBS [10 mM Tris-HCl (pH7.4), 150 mM NaCl], and permeabilized with 0.02% Triton-100 in 0.1% BSA-TBS for 10 min. Nonspecific binding was blocked by incubation with 3% BSA-TBS for 30 min at room temperature, after which the phospho-MAP kinase antibody (5 µg/ml; Promega Corp., Madison, WI) or polyclonal rabbit anti-3ß-hydroxysteroid dehydrogenase (anti-3ß-HSD) antibody (1:100) diluted in 0.1% BSA-TBS was applied and incubated overnight at 4 C. After four washes (10 min each) with 0.1% BSA-TBS, the samples were incubated with Cy3-conjugated antirabbit IgG (1:100) for 1 h at room temperature. After four washes (5 min each) with 0.1% BSA-TBS, the samples were examined by confocal microscopy (Bio-Rad Laboratories, Inc. MRC-1024, Laser Scanning Confocal Imaging System using an argon/krypton lamp). Digital imaging was captured by the Bio-Rad Laboratories, Inc. laser-shop software. For negative controls, the same procedure described above was performed either without primary antibody or without secondary antibody (data not shown).

RNA extraction and Northern blot analysis
After cell stimulation for 30 min, the medium was discarded. Total RNA was isolated by the single-step guanidine-thiocyanate-phenol-chloroform procedure (26) and quantified based upon the absorbance value at 260 nM by UV spectrophotometer. RNA samples (15 µg/lane) were electrophoresed on a denaturing gel of 1% agarose and 1.5% formaldehyde, transferred and UV cross-linked to a zeta probe blotting membrane (Bio-Rad Laboratories, Inc., Richmond, CA). The membrane was prehybridized in 500 mM NaHPO4 (pH 7.2), 7% SDS, and 1 mM EDTA, at 60 C for 4 h. Hybridization was performed in the same buffer containing denatured [32P]-labeled bovine c-fos (GenBank accession number AF069575) and c-jun (GenBank accession number 06951) complementary DNA (cDNA) probes (approximately 2.5 x 106 cpm/ml), overnight at 60 C. The 1.8-kb human ß-actin cDNA probe was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA) and served as a control for sample loading. The cDNAs were radiolabeled with [{gamma}-32P] deoxycycidine triphosphate by using a random-primed labeling kit (Roche Molecular Biochemicals, Indianapolis, IN). After four washes (20 min each) in prewarmed (60 C) buffer [40 mM NaHPO4 (pH 7.2), 1% SDS, 1 mM EDTA (pH 8.0)] at room temperature, the membranes were subjected to autoradiography.

Statistical analysis
Data are presented as mean ± SEM. Each experiment was performed at least three times. Data were analyzed using the InStat program. One-way ANOVA was followed by Dunn’s t test. P < 0.05 values were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunoblotting of PKC isoforms in bovine luteal cells
Direct evidence of PKC activation upon PGF2{alpha} exposure is limited and largely based on comparison with the response to phorbol esters. In the corpus luteum, this comparison has been applied to evaluate the possible role of phorbol ester-responsive isoforms of PKC in mediating the luteolytic effects of PGF2{alpha} (8, 9, 10). Additionally, we have recently demonstrated that PMA mimicked the stimulatory effects of PGF2{alpha} on each component of the Raf-1/MEK1/ERK signaling cascade in bovine luteal cells (19). Therefore, the PKC isoforms involved in PGF2{alpha} action in the corpus luteum are presumptively the PMA-sensitive conventional and novel isoforms of PKC. To test this possibility, a PKC-deficient bovine luteal cell model was established based on the fact that chronic treatment with PMA degrades phorbol ester-responsive isoforms of PKC (27, 28). Luteal cells were rendered PKC-deficient by pretreatment with 2.5 µM PMA for 24 h (Fig. 1Go). Western blotting experiments, with subtype-specific antipeptide antibodies raised against different isoforms of PKC, were performed to confirm the depletion of PKC isoforms. A monoclonal antibody (Transduction Laboratories, Inc., Lexington, KY) against a C-terminal domain epitope (270–427) of human PKC-{alpha} recognized a single band with an apparent molecular mass of 84 kDa in bovine luteal cells. The specific PKC-ßII antibody (SC-210, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), raised against the C-terminal amino acids 657–673 of human PKC-ßII, recognized the predicted 84-kDa protein but also cross-reacted with an unknown protein of approximately 95 kDa. The specific PKC-{epsilon} antibody (Transduction Laboratories, Inc.), raised against the NH2-terminal amino acids 1–175 of human PKC-{epsilon}, recognized a single protein with the predicted molecular mass of 96 kDa. It is noteworthy that, compared with controls (Fig. 1Go, lanes 1–3), the levels of these PKC isoforms were greatly reduced after exposure to 2.5 µM PMA for 24 h (Fig. 1Go, lanes 4–6). With the same samples, Western blotting experiments were also performed with a specific antibody raised against human 3ß-HSD, the enzyme that converts progenolene to progesterone (kindly provided by Dr. Ian Mason, University of Texas Southwestern Medical Center). High levels of the 40-kDa 3ß-HSD protein were observed in all samples and were not altered after exposure to 2.5 µM PMA for 24 h (Fig. 1Go). Additionally, acute treatment with PGF2{alpha} and PMA for 5 min did not alter the cellular levels of PKC isoforms or 3ß-HSD (Fig. 1Go).



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  		Figure 1. Immunoblot analysis of PKC isoforms in bovine luteal cells. After attachment, bovine luteal cells were cultured in serum-free media for 24 h and pretreated with control media (lanes 1–3) or with 2.5 µM PMA (lanes 4–6) for 24 h. The cells were then treated with PGF2{alpha} (1 µM) and PMA (20 nM) for 5 min and lysed in a nondenaturing buffer as described in Materials and Methods. Total cell lysates (30 µg/lane) were size-fractionated on 8% SDS-PAGE and transferred to a PVDF membrane. The membrane was analyzed by Western blotting with monoclonal antibodies against PKC{alpha} (1:500) and PKC-{epsilon} (1:500) followed by antimouse peroxidase-conjugated IgG (1:2000) or with polyclonal rabbit antibodies against PKC-ßII (1:250) and 3ß-HSD (1:1000) followed by antirabbit peroxidase-conjugated IgG (1:4000). Bound antibodies were visualized with the ECL reagents according to the manufacturer’s instructions. Results from one of four independent experiments are shown.

 
PKC is required for ERK activation by PGF2{alpha} and PMA in bovine luteal cells
Activation of phosphatidylinositol-specific PLC and subsequent DAG-activation of PKC upon PGF2{alpha} exposure takes place within seconds in ovarian cells (4, 5). Acute treatment with PGF2{alpha} and PMA also results in a rapid activation of ERK MAP kinase in a number of cell types (21, 22, 23), including ERK2 and ERK1 in primary cultures of bovine luteal cells (19). To investigate whether PKC is required for activation of ERK2 and ERK1 by PGF2{alpha} in bovine luteal cells, we first assessed whether phosphorylation and activation of ERK2 and ERK1 by PGF2{alpha} and PMA was mediated by phorbol ester-responsive isoforms of PKC. For this purpose, a comparison of phosphorylation and activation of ERK2 and ERK1 by PGF2{alpha} and PMA was made by using cells with or without chronic PMA pretreatment (Fig. 2Go). Immunoblotting experiments with monoclonal {alpha}PanERK antibody (Transduction Laboratories, Inc.) showed that the cellular levels of ERK2 and ERK1 were not altered by chronic pretreatment with 2.5 µM PMA (lanes 4–6) or by acute treatment with 1 µM PGF2{alpha} (lanes 2 and 5) and 20 nM PMA (lanes 3 and 6). In keeping with our previous report (19), acute treatment with PGF2{alpha} and PMA caused the gel-shift phosphorylation of ERK2 and ERK1 (lanes 2 and 3). Notably, the PGF2{alpha} and PMA-induced gel shifts (lanes 2 and 3) were not apparent in PMA-pretreated cells (lanes 5 and 6). Western blot analysis of these samples was also performed using a phospho-MAP kinase antibody. Figure 3Go demonstrates that treatment with PGF2{alpha} (1 µM) and PMA (20 nM) for 5 min markedly stimulated the phosphorylation (Fig. 3AGo) of ERK2 and ERK1 in bovine luteal cells. Compared with untreated cells (lanes 1–3), chronic PMA pretreatment (lanes 4–6) did not alter the basal phosphorylation status of ERK2 and ERK1 (Fig. 3aGo). When cells were pretreated with 2.5 µM PMA for 24 h, the phosphorylation (Fig. 3AGo) of ERK2 and ERK1 by PGF2{alpha} and PMA was completely attenuated. These data indicate an obligatory role of phorbol ester-responsive isoforms of PKC in mediating ERK phosphorylation in response to both PGF2{alpha} and PMA in bovine luteal cells. When the cells were pretreated with a PKC inhibitor GF102903x (100 nM, 1 h), which possesses high selectivity for PKC-{alpha}, -ß, -{gamma}, -{delta}, and -{epsilon} isozymes, the phosphorylation of ERK2 and ERK1 by PGF2{alpha} was also completely blocked (Fig. 3BGo).



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  		Figure 2. Immunoblotting of ERK2 and ERK1 in bovine luteal cells. After attachment, bovine luteal cells were cultured in serum-free media for 24 h and pretreated with control media (lanes 1–3) or with 2.5 µM PMA (lanes 4–6) for 24 h. The cells were then treated with PGF2{alpha} (1 µM) or PMA (20 nM) for 5 min and lysed in a nondenaturing buffer. Total cell lysates (20 µg/lane) were size-fractionated by 10% SDS-PAGE, electrically transferred onto a PVDF membrane, and analyzed by Western blotting with the {alpha}PanERK antibody (1:500). A gel-shift on SDS-PAGE, demonstrating in ERK1 and ERK2 activation, is shown in the upper panel (A) with longer exposure (45 sec) and ERK2 activation shown in the lower panel (B) with shorter exposure (15 sec), respectively. Results from one of three independent experiments are shown.

 


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  		Figure 3. Phosphorylation of ERK2 and ERK1 in response to PGF2{alpha} and PMA requires PKC. A, Luteal cells were treated exactly as described in Fig. 2Go. After attachment, bovine luteal cells were cultured in serum-free media for 24 h and pretreated with control media (lanes 1–3) or with 2.5 µM PMA (lanes 4–6) for 24 h. The cells were then treated with PGF2{alpha} (1 µM) and PMA (20 nM) for 5 min. B, Luteal cells were cultured as described in Fig. 2Go. Cultures were pretreated with or without the PKC inhibitor GF109203x (100 nM) for 1 h, then challenged with PGF2{alpha} or PMA for 5 min. Total cell lysates were prepared with a nondenaturing buffer. Proteins (20 µg/lane) were size-fractionated with 10% SDS-PAGE and electrically transferred to a PVDF membrane. Phosphorylation of ERK2 and ERK1 was analyzed with an active MAP kinase antibody (1:1000) that recognizes the phosphorylated forms of ERK2 and ERK1. The results represent one of four similar independent experiments.

 
Immunocomplex protein kinase assays (Fig. 4Go) were performed to demonstrate that the phosphorylation of ERK1 and ERK2 was associated with an increase in the activity of the enzymes. Treatment with PGF2{alpha} (1 µM) or PMA (20 nM) for 5 min increased the activities of both ERK2 (Fig. 4AGo) and ERK1 (Fig. 4BGo). Treatment with PMA (2.5 µM) for 24 h had little effect on basal activities of ERK1 and ERK2 but inhibited the acute stimulatory actions of both PGF2{alpha} and PMA. This result further supports a pivotal role of PKC in mediating ERK MAP kinase activation by PGF2{alpha} and PMA in bovine luteal cells.



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  		Figure 4. Activation of ERK2 and ERK1 in response to PGF2{alpha} and PMA. After attachment, bovine luteal cells were cultured in serum-free media for 24 h and pretreated with control media (lanes 1–3) or with 2.5 µM PMA (lanes 4–6) for 24 h. Total cell lysates (200 µg protein) were subjected to ERK2 and ERK1 immunoprecipitation protein kinase assays as described in Materials and Methods. The top and bottom panels summarize activation of ERK2 and ERK1, respectively, in response to PGF2{alpha} and PMA. A representative autoradiogram, showing phosphorylation of GST-Elk-1 by activated ERK2 and ERK1, is included in their corresponding panels. The results are expressed as means ± SEM from three independent experiments. (*, P < 0.05 vs. control).

 
Active ERK MAP kinases are found in the nucleus upon PGF2{alpha} and PMA stimulation in bovine luteal cells
Upon stimulation, activated MAP kinase rapidly translocates into the nucleus (14), where it phosphorylates and thereby activates nuclear substrates to regulate gene transcription (11, 12, 13). To test whether activated ERK MAP kinases are found in the nucleus upon PGF2{alpha} and PMA stimulation in bovine luteal cells, we localized the activated ERK by indirect fluorescence immunocytochemistry using a specific phospho-MAP kinase antibody (Fig. 5Go). The active MAP kinase signal observed either in the cytoplasm or the nucleus of untreated control cells was weak (A). After treatment with PGF2{alpha} (C) and PMA (D) for 5 min, fluorescence immunocytochemistry revealed a strong nuclear labeling of activated MAP kinase, whereas the cytoplasmic signals of activated MAP kinase labeling remained weak. In parallel, 3ß-HSD immunolabeling ( B) was primarily present in the cytoplasm of untreated control cells. The majority of the cells were 3ß-HSD positive cells, demonstrating their steroidogenic nature. In keeping with the evidence of various gene expression upon PGF2{alpha} stimulation in bovine corpus luteum (29), nuclear location of activated MAP kinase in response to PGF2{alpha} may be a key step that transmits the initial PGF2{alpha}-induced PLC/InsP3-DAG/Ca2+-PKC signaling pathway into the nucleus and further regulates the transcription of PGF2{alpha}-inducible genes in luteal cells.



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  		Figure 5. Nuclear translocation of active ERK in response to PGF2{alpha} and PMA in bovine luteal cells. Fluorescence immunocytochemistry: Bovine luteal cells were cultured on 8-well Lab-Tek glass chamber slides. The cells were treated with PGF2{alpha} (1 µM) and PMA (20 nM) for 5 min, washed twice with cold-PBS, and fixed, and immunocytochemistry was performed as described in the Materials and Methods. The cells were examined by confocal microscopy (400x). The labeling patterns of active MAP kinase in untreated cells, PGF2{alpha}, and PMA-treated cells are shown in A, C, and D, respectively. B shows cells labeled with anti-3ß-HSD antibodies.

 
Blocking ERK activation by PD098059 attenuates PGF2{alpha} and PMA-induced c-fos/c-jun mRNA expression
Elk-1, a member of the Ets family of nuclear transcription factors, is a well-documented substrate for activated MAP kinase, including members of all three MAP kinase subfamilies (30, 31). Elk-1 phosphorylation stimulates the transcription of the immediate early response gene c-fos by facilitating the formation of a ternary complex with serum response factor (SRF) and c-fos promoter serum response element (SRE) (15). Therefore, the question that arises is whether ERK is responsible for the induction of c-fos and c-jun mRNA expression by PGF2{alpha} and PMA. To test this, we first examined the effects of the specific MEK1 inhibitor PD098059 on phosphorylation of ERK2 and ERK1 by PGF2{alpha} in bovine luteal cells (Fig. 6AGo). Western blot analysis with the active MAP kinase antibody showed a marked increase of ERK2 and ERK1 phosphorylation with treatment of 1 µM PGF2{alpha} in bovine luteal cells. Pretreatment with increasing concentrations of PD098059 for 1 h resulted in a concentration-dependent inhibition of PGF2{alpha}-induced ERK2 and ERK1 phosphorylation (Fig. 6AGo). Nearly complete inhibition of ERK phosphorylation was observed with 50 µM PD098059, a concentration without cytotoxic effects in a number of cell types (32). Treatment with PD098059 had no effect on levels of ERK1 or ERK2 protein (data not shown). Therefore, 50 µM PD098059 was chosen to examine the role of ERK activation in PGF2{alpha} and PMA-induced c-fos and c-jun mRNA expression.



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  		Figure 6. ERK activation is required for increased c-fos and c-jun mRNA expression in response to PGF2{alpha} and PMA in bovine luteal cells. A, PD098059 blocks ERK phosphorylation in response to PGF2{alpha} and PMA. Bovine luteal cell cultures were pretreated with 1–50 µM PD098059 for 1 h as indicated in the figure, then treated with PGF2{alpha} (1 µM) and PMA (20 µM) for 5 min. Phosphorylation of ERK2 and ERK1 was determined by immunoblotting with active MAP kinase antibody as described in Fig. 3Go. The results represent one of three similar independent experiments. B, PD098059 blocks increased c-fos and c-jun mRNA expression in response to PGF2{alpha} and PMA. Bovine luteal cells were pretreated with 50 µM PD098095 for 1 h, followed by treatment with PGF2{alpha} (1 µM) or PMA (20 nM) for 30 min. Total RNA was isolated. The RNA samples (15 µg/lane) were size-fractionated on formaldehyde-1% agrose gel, and Northern blot analysis was performed with [{alpha}-32P] deoxycycidine triphosphate labeled bovine specific c-fos and c-jun cDNA probes as described in Materials and Methods. The membranes were stripped and reprobed with human ß-actin cDNA probe as an internal control. The results represent one of three similar independent experiments.

 
In keeping with our observation that treatment with PGF2{alpha} (1 µM) and PMA (20 nM) for 30 min induced the maximal expression of c-fos and c-jun mRNA in bovine luteal cells (20), experiments were performed to determine whether blocking ERK activation attenuates the induction of c-fos and c-jun mRNA expression by PGF2{alpha} and PMA (Fig. 6BGo). As expected, treatment with PGF2{alpha} and PMA for 30 min significantly stimulated both c-fos and c-jun mRNA expression (lanes 3 and 5, respectively). In comparison with cells without PD098059 pretreatment (lane 3), the expression of c-fos and c-jun mRNA induced by PGF2{alpha} was completely blocked in cells pretreated with PD098059 (lane 4). This result indicates that ERK activation is required for PGF2{alpha}-induced transcription of c-fos and c-jun protooncogenes. Additionally, blocking ERK activation with PD098059 also inhibited PMA-stimulated c-fos and c-jun mRNA expression (lane 6). This result suggests that ERK MAP kinase is also involved in c-fos and c-jun mRNA expression upon activation of phorbol ester-responsive isoforms of PKC by PMA.

Stimulation of c-fos and c-jun mRNA by PGF2{alpha} and PMA was blocked by chronic pretreatment with PMA
Having demonstrated that (1) PGF2{alpha} and PMA activate a PKC-dependent ERK signaling pathway, and (2) ERK2 and ERK1 mediate PGF2{alpha} and PMA stimulation of c-fos and c-jun mRNA expression, we further tested whether PKC was required for c-fos and c-jun expression in response to PGF2{alpha} and PMA. For this purpose, the PKC-deficient cell model was applied. As shown in Fig. 7Go, pretreatment with PMA (2.5 µM) for 24 h did not alter the basal levels of c-fos and c-jun mRNA expression. However, PGF2{alpha} and PMA failed to stimulate c-fos and c-jun mRNA expression in PKC-deficient cells (lanes 4–6), in comparison with cells without chronic PMA pretreatment (lanes 1–3). These data suggest that phorbol ester-responsive isoforms of PKC play an obligatory role in PGF2{alpha}-induced c-fos and c-jun mRNA expression.



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  		Figure 7. Increased c-fos and c-jun mRNA expression in response to PGF2{alpha} and PMA requires PKC. After attachment, bovine luteal cells were cultured in serum-free media for 24 h, and pretreated with control media (lanes 1–3) or with 2.5 µM PMA (lanes 4–6) for 24 h to deplete phorbol ester-responsive isoforms of PKC. The cells were washed, then challenged with PGF2{alpha} (1 µM) or PMA (20 nM) for 30 min. Total RNA was isolated, size-fractionated on formaldehyde-1% agrose gel, transferred, and cross-linked on a zeta-probe membrane. Northern blot analysis was performed to determine mRNA levels of c-fos and c-jun as described in Fig. 6Go. The results represent one of three similar independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Ligand-G-protein-coupled receptor activation of PLC, resulting in InsP3-induced Ca2+ mobilization and DAG activation of PKC, is believed to be the initial transmembrane signaling mechanism by which PGF2{alpha} exerts its luteolytic effects (4, 5, 33). However, a gap exists in our knowledge, between this initial signaling event and the physiological sequelae observed in response to PGF2{alpha}. In a recent report, we demonstrated that PGF2{alpha} activates the Raf-1/MEK1/ERK signaling cascade in bovine luteal cells (19). We now extend these observations by showing that in bovine luteal cells: 1) inhibition of PKC by a PKC inhibitor or by depletion of phorbol ester-responsive PKC isoforms abrogated phosphorylation and activation of ERK2 and ERK1 in response to both PGF2{alpha} and PMA; 2) activated ERKs were present in the nucleus; 3) blocking the ERK MAP kinase pathway by the specific MEK1 inhibitor PD098059 attenuated ERK activation and expression of c-fos and c-jun mRNA in response to PGF2{alpha} and PMA; and 4) depletion of phorbol ester-responsive isoforms of PKC blocked PGF2{alpha} and PMA-induced expression of c-fos and c-jun mRNA. These data indicate that a PKC-dependent ERK MAP kinase pathway mediates the expression of c-fos and c-jun transcription factors in response to PGF2{alpha}. This novel evidence supports the idea that a PKC-dependent ERK MAP kinase pathway is, perhaps, the predominant cytoplasmic protein kinase cascade that links the plasma membrane receptor-initiated signaling events to transcription factor regulation of the expression of PGF2{alpha}-responsive genes.

Activation of the ERK MAP kinases involves a signaling cascade comprising Raf, MEK1, and ERK2/ERK1 (11, 12, 13). Multiple mechanisms exist for regulating Raf-1 activation, e.g. membrane association upon Ras-binding (34, 35), tyrosine phosphorylation by Src family of tyrosine kinases (36), interaction with 14–3-3 proteins (37), and direct serine phosphorylation by PKC (38, 39). Immediate upstream activators other than Raf are also identified for MEK1, which represent a Raf-independent mechanism for ERK activation, possibly involving PKC-{zeta} (40). It is clear, therefore, that the ERK MAP kinases can be activated through different mechanisms by different cell surface receptors. Likewise, PGF2{alpha} seems to be able to activate ERK differentially, based on the cell types and coupling to specific G protein subunits. PGF2{alpha} is generally thought to activate PLC by coupling to G{alpha}q. PGF2{alpha}, possibly via G{alpha}q subunits, stimulated the formation of p21Ras-GTP complexes and the subsequent activation of Raf in NIH3T3 cells, suggesting a Ras-dependent pathway for ERK activation by PGF2{alpha} (21). In NIH3T3 cells, PGF2{alpha}-induced ERK2 activation was also shown to be independent of PMA-sensitive PKC isoforms (21). Additionally, in rat myometrial cells, G-protein-ß{gamma}-subunit-mediated tyrosine phosphorylation of Shc and Sos is thought to mediate PGF2{alpha}-induced Raf-1 activation of ERKs (22). In contrast, activation of the ERK MAP kinase signaling cascade by PGF2{alpha} in osteoblastic MC3T3-E1 cells seems to be mediated by PKC-dependent activation of Raf-1 (23). Likewise, PKC has been implicated in the activation of the Raf/MEK1/ERK signaling cascade by PGF2{alpha} in luteal cells, because a similar activation pattern was observed in response to the PKC activator PMA (19). Our present data further implicate PKC in the activation of ERK MAP kinase signaling. Moreover, preliminary results suggest that p21ras activation is not required for PGF2{alpha}-induced ERK activation in bovine luteal cells (Davis and Obholz, unpublished observation). It seems, therefore, that PGF2{alpha} may use cell-type-specific signaling pathways to activate ERKs in various tissues and cell lines.

Multiple isoforms of PKC (41) are present in the corpus luteum (33), including members of the conventional class (cPKC-{alpha}, -ßI, and -ßII), a novel class (nPKC-{epsilon} and -{theta}), and atypical class (aPKC-{zeta} and -{iota}/{lambda}). Although PGF2{alpha} activates PKC in luteal cells (6, 7, 8, 9, 10), little is known about the specific PKC isoform(s) and the physiological substrates lying immediately downstream of PKC activation. The present study demonstrates that PKC is required for phosphorylation and activation of ERK2 and ERK1 by PGF2{alpha} and PMA in luteal cells. This is supported by studies showing that a specific PKC inhibitor and depletion of phorbol ester-responsive PKC isoforms completely blocked ERK phosphorylation and activation in response to both PGF2{alpha} and PMA. The isoforms of PKC involved in ERK activation and in response to PGF2{alpha} are most likely the phorbol ester-sensitive isoforms of cPKCs and nPKCs. Whereas the phorbol ester-unresponsive atypical PKC-{zeta} and {iota}/{lambda} isoforms are present in bovine luteal cells (33), it seems unlikely that these PKC isoforms mediate PGF2{alpha}-induced ERK activation. Further investigation is required to characterize the mechanisms by which PKC mediates PGF2{alpha}-induced ERK activation. It is tempting to speculate that specific cPKCs or nPKCs serve as direct upstream kinases for Raf-1 activation (38, 39).

Transcription of the prototypic immediate-early response genes, i.e. c-fos and c-jun, is rapidly induced in cells on exposure to a wide variety of extracellular stimuli. The data presented herein demonstrate that treatment with PGF2{alpha} and PMA stimulated c-fos and c-jun mRNA expression in primary cultures of bovine luteal cells. Our results in vitro are in keeping with observations that treatment with PGF2{alpha} in vivo stimulated c-jun mRNA expression in bovine (42) and rat (43) corpora lutea. Therefore, our present data underline the relevance of expression of c-fos and c-jun protooncogenes as a physiological criterion for PGF2{alpha} action in the corpus luteum.

The transcription of c-fos and c-jun is controlled by the cis-acting elements in their promoter regions, including the SRE and the 12-O-tetradecanoylphorbol 13-acetate response element. The best-characterized SRE is that of the c-fos gene (15, 44). Full-activation of the c-fos SRE requires association with the ubiquitous transcription factor SRF and formation of a ternary complex with ternary complex factors, including the Ets family of transcription factors Elk-1 and Sap1 (44, 45). The DNA binding domain at the N terminus of Elk-1 and Sap1 facilitates ternary complex formation, whereas the trans-activation domain at the C-terminus of Elk-1 and Sap1 contains several conserved MAP kinase phosphorylation sites. Upon cell stimulation, MAP kinase translocates into the nucleus (14), where it phosphorylates the Elk-1 C-terminus, which then cooperates with the C-terminus of the SRF activation domain to initiate c-fos transcription (46). Our data suggest that phosphorylation of Elk-1 by the ERK pathway is involved in the activation of the c-fos gene by PGF2{alpha} in bovine luteal cells. This notion is supported by the following evidence. First, ERK2 and ERK1 were phosphorylated and activated upon PGF2{alpha} and PMA stimulation. Second, PGF2{alpha}-activated ERK2 and ERK1 were able to phosphorylate Elk-1 in vitro. Third, preincubation of cells with the specific MEK1 inhibitor PD098059 attenuated the phosphorylation of ERK2 and ERK1 by PGF2{alpha} and PMA. Fourth, active ERK was observed in the nucleus of cells treated with PGF2{alpha} and PMA. Last, preincubation of cells with PD098059 blocked the stimulatory action of PGF2{alpha} on the expression of c-fos mRNA.

The regulatory mechanism for induction of the c-jun gene seems more complicated and much less understood. Early studies suggested that a putative AP-1 element in the c-jun promoter mediates the positive autoinduction of c-jun by mitogens (47). However, several consensus binding sites for different transcription factors, including AP-1, MEF2, Sp-1, and CTF are present in the c-jun promoter (17). Recent studies demonstrated that the MEF2 site and the AP-1 site, but not the Sp-1 and CTF sites, are required for induction of the c-jun promoter in response to activators of epidermal growth factor (EGF) receptors (48) and G-protein-coupled m1 acetylcholine receptors (49). Therefore, the AP-1 transcription factors seem to be important regulators of c-jun expression and may be involved in PGF2{alpha} regulation of c-jun mRNA expression in luteal cells.

Current evidence suggests that c-Jun activity can be regulated by posttranslational modifications of preexisting c-Jun protein. Phosphorylation of serine and threonine residues within the N-terminal trans-activation domain (50, 51) results in enhanced trans-activation, DNA-binding, and stability of the c-Jun protein (51, 52). The direct upstream kinases that phosphorylate the residues within c-Jun N terminus include members of the two MAP kinase subfamilies, i.e. the ERKs, including ERK2 and ERK1 (48, 53), and JNK (48, 54, 55). Similar to the ERK pathway, JNK activation involves a parallel protein kinase cascade comprising MEKK1, SEK, and JNKs (54, 55). The small G proteins Rac1, cdc42, and Ras (56) have been identified as upstream activators of MEKK1. In Hela cells, transfection of active forms of Ras, RacI, cdc42Hs, and MEKK1 increased, whereas transfection of dominant-negative mutants of Ras, RacI, and MEKK1 inhibited the expression of a c-jun promoter-driven luciferase reporter gene in response to EGF (48). This result suggested that the Rac-MEKK and JNK pathway was involved in the activation of the c-jun promoter by EGF. However, in NIH3T3 cells expressing G-protein-coupled m1 acetylcholine receptors, the JNK pathway did not seem to be involved in the activation of the c-jun promoter in response to carbacol, because coexpression of MEKK1 effectively increased JNK activity but only with limited c-jun promoter activity (49). This suggests that G-protein-coupled receptors can use mechanisms other than JNK to regulate c-jun expression. In this regard, recent evidence points to a role of ERK MAP kinase in regulating the nucleosomal response associated with the induction of immediate-early response genes (57, 58). In the present report, we demonstrate that PGF2{alpha} and PMA rapidly stimulated the expression of c-jun mRNA in bovine luteal cells. Moreover, enhanced c-jun mRNA expression by PGF2{alpha} was attenuated by blocking the ERK pathway with the specific MEK1 inhibitor PD098059. Although the exact mechanism(s) remain to be established, our data suggest that ERK2 and ERK1 are involved in the activation of the c-jun gene by PGF2{alpha} in bovine luteal cells.

Lastly, we showed that depletion of PMA-responsive PKC isoforms by chronic pretreatment with PMA blocked the expression of c-fos and c-jun mRNA in response to PGF2{alpha} and PMA. These results indicate that the phorbol ester-responsive isoforms of PKC play a pivotal role in mediating the expression of the AP-1 transcription factors by PGF2{alpha} in bovine luteal cells. Because depletion of phorbol ester-responsive isoforms of PKC completely inhibited ERK activation by PGF2{alpha} and PMA, and ERK activation was required for the enhanced expression of c-fos and c-jun mRNA by PGF2{alpha} and PMA, these data allow us to conclude that induction of c-fos and c-jun mRNA expression by PGF2{alpha} in bovine luteal cells is mediated by a PKC-dependent ERK MAP kinase pathway.


   Acknowledgments
 
The authors thank Arleene Moore for help in the preparation of the manuscript. The authors are grateful to Dr. Mark S. Roberson (Cornell University) for providing the glutathione-S-transferase Elk-1, and Dr. Ian Mason for providing the polyclonal rabbit antihuman 3ß-HSD antibody. We also thank Dr. John W. Schmidt (Wichita State University) for his assistance in confocal microscopy.


   Footnotes
 
1 The present study was supported, in part, by the Lalor Foundation, the Research Service of the Department of Veterans Affairs, the Women’s Research Institute, and the Wesley Medical Research Institute. The data was partially presented at the XI Ovarian Workshop as Abstract 7, July 26–30, 1996, London, Ontario, Canada. Back

2 Current address: Perinatal Research Laboratories, Department of Obstetrics and Gynecology, University of Wisconsin Medical School, 7E Meriter Hospital/Park, 202 South Park Street, Madison, Wisconsin 53715. Back

Received September 8, 2000.


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 Materials and Methods
 Results
 Discussion
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