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Endocrinology Vol. 138, No. 4 1673-1682
Copyright © 1997 by The Endocrine Society


ARTICLES

Mechanism of Mitogen-Activated Protein Kinase Activation by Gonadotropin-Releasing Hormone in the Pituitary {alpha}T3–1 Cell Line: Differential Roles of Calcium and Protein Kinase C1

Nachum Reiss, Linet N. Llevi, Sharon Shacham, Dagan Harris, Rony Seger2 and Zvi Naor

Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University (N.R., S.S., D.H., Z.N.), Ramat Aviv 69978; and the Department of Membrane Research and Biophysics, The Weizmann Institute of Science (L.N.-L., R.S.), Rehovot 76100, Israel

Address all correspondence and requests for reprints to: Zvi Naor, Ph.D., Department of Biochemistry, Tel Aviv University, Tel-Aviv 69978, Israel. E-mail: NAORZVI{at}CCSG.TAU.AC.IL


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mechanism of mitogen-activated protein kinase (MAPK, ERK) stimulation by the GnRH analog [D-Trp6]GnRH (GnRH-a) was investigated in the gonadotroph-derived {alpha}T3–1 cell line. GnRH-a as well as the protein kinase C (PKC) activator 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated a sustained response of MAPK activity, whereas epidermal growth factor (EGF) stimulated a transient response. MAPK kinase (MEK) is also activated by GnRH-a, but in a transient manner. GnRH-a and TPA apparently activated mainly the MAPK isoform ERK1, as revealed by Mono-Q fast protein liquid chromatography followed by Western blotting as well as by gel kinase assay. GnRH-a and TPA stimulated the tyrosine phosphorylation of several proteins, and this effect as well as the stimulation of MAPK activity were inhibited by the PKC inhibitor GF 109203X. Similarly, down-regulation of TPA-sensitive PKC subspecies nearly abolished the effect of GnRH-a and TPA on MAPK activity. Furthermore, the protein tyrosine kinase (PTK) inhibitor genistein inhibited protein tyrosine phosphorylation and reduced GnRH-a-stimulated MAPK activity by 50%, suggesting the participation of genistein-sensitive and insensitive pathways in GnRH-a action. Although Ca2+ ionophores have only a marginal stimulatory effect, the removal of Ca2+ markedly reduced MAPK activation by GnRH-a and TPA, but had no effect on GnRH-a and TPA stimulation of protein tyrosine phosphorylation. Interestingly, the removal of Ca2+ also partly inhibited the activation of MAPK by EGF and vanadate/H2O2. Thus, a calcium-dependent component(s) downstream of PKC and PTK might also participate in MAPK activation. Elevation of cAMP by forskolin exerted partial inhibition on EGF, but not on TPA or GnRH-a action, suggesting that MEK activators other than Raf-1 might be involved in GnRH action. We conclude that Ca2+, PTK, and PKC participate in the activation of MAPK by GnRH-a, with Ca2+ being necessary downstream to PKC and PTK.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH REGULATES the synthesis and release of the pituitary gonadotropins LH and FSH. The signaling events triggered by GnRH include interaction of the GnRH receptor (GnRHR) with Gq, enhanced phosphoinositide turnover, Ca2+ mobilization and influx, translocation and activation of protein kinase C (PKC), activation of phospholipase D and A2, and formation of bioactive lipoxygenase products, culminating in gonadotropin (LH and FSH) release and gonadotropin subunit gene expression (see Refs. 1–3 for reviews). However, little is known about the molecular events downstream of PKC activation during GnRH action.

Signal transduction elicited by hormones and growth factors has been found to involve sequential activation of several cytosolic protein kinases collectively known as the mitogen-activated protein kinase (MAPK) cascade (4, 5, 6, 7, 8, 9, 10). The MAPK gene family converges signals from G protein-coupled receptors (GPCRs) or growth factor receptor tyrosine kinases (RTKs), leading to cellular responses such as differentiation and proliferation. The mechanism of MAPK activation seems to differ among various stimuli. RTKs signal via an adaptor molecule, GRB2, and a guanine nucleotide exchange factor, mSOS, to activate the small GTP-binding protein (G protein), Ras, followed by activation of the MAPK cascade: Raf-1, MAPK kinase (MAPKK; MEK), and MAPK. On the other hand, GPCRs are thought to act via diverse mechanisms, including PKC-dependent and independent pathways, to activate the above cascade (6, 7, 8, 9, 10).

The signals transmitted through the MAPK cascade lead to activation of a set of regulatory molecules that eventually initiates cellular responses such as growth and differentiation (4, 5, 6, 7, 8, 9, 10). Recently, it has been shown that GnRH is capable of activating MAPK in pituitary organ culture (11) and the {alpha}T3–1 gonadotroph cell line (12, 13) via PKC, and that MAPK is involved in regulation of gene expression of the gonadotropin {alpha}-subunit (12, 13). We decided to follow the above reports and study further the mechanism of MAPK (ERK1 and ERK2) activation by GnRH. We found that GnRHR signaling results in stimulation of mainly the ERK1 isoform and involves a Ca2+-dependent pathway that does not include Gi and Go and seems to require MEK activators other than Raf1 as well as a protein tyrosine kinase (PTK) upstream to the MAPK cascade. Furthermore, we show that Ca2+ is required, but not sufficient, and its site of action is downstream to PKC and PTK signaling.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Materials
The GnRH analog [D-Trp6]GnRH (GnRH-a) was a gift from Dr. R. Millar (Capetown, South Africa). The epidermal growth factor (EGF) receptor peptide [EGF receptor-(662–681)] was used as a selective MAPK substrate (14) and was synthesized by the Peptide Synthesis Facility at the Weizmann Institute of Science (Rehovot, Israel). All media, sera, and antibiotics for cell culture were obtained from Biological Industries (Kibbutz Beit Ha’Emek, Israel). [{gamma}-32P]ATP was purchased from Rotem Industries (Beersheba, Israel). Genistein, phenylmethylsulfonylfluoride, EGTA, 12-O-tetradecanoyl phorbol-13-acetate (TPA), EGF (recombinant), forskolin, and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant ERK2 used in the MAPKK assay was prepared as previously described (15). Anti-P-tyrosine antibodies were obtained from Zymed (San Francisco, CA). Anti-MAPK serum was generously donated by Dr. Y. Granot, Ben-Gurion University (Beer-Sheva, Israel). The selective PKC inhibitor bisindolylmaleimide (GF 109203X) (16) was purchased from Calbiochem (Laufelfingen, Switzerland).

Buffers
Detergent lysis buffer consisted of 50 mM Tris-HCl (pH 6.8), 2 mM EGTA, 1 mM sodium orthovanadate, 20 mM NaCl, 10 mM ß-glycerophosphate, 28 mM ß-mercaptoethanol, 1 mM phenylmethylsulfonylfluoride, 20% glycerol, 50 nM each of okadaic acid and calyculin A, and 0.5% Nonidet P-40.

Buffer A consisted of 50 mM ß-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM sodium orthovanadate.

Buffer H consisted of 50 mM ß-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM sodium orthovanadate, 1 mM benzamidine, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 2 mg/ml pepstatin A.

Cell culture
{alpha}T3–1 cells were grown in DMEM supplemented with 5% FCS, 5% horse serum, penicillin (100 U/ml), and streptomycin (0.1 mg/ml). At 70–80% confluence, the cells were serum-starved for 18 h in DMEM, and 0.25% each of FCS and horse serum and stimulants were added in DMEM at the indicated concentration for the given length of time.

Fractionation and assay of MAPK and MAPKK
For determination of MAPK activity in cell extracts, the cells were rinsed twice with ice-cold PBS and lysed for 10 min in detergent lysis buffer. Samples of 5 µl (~2 µg protein) were subjected to MAPK assay at 22 C for 20 min. The reaction mixture (25 µl) contained 50 mM HEPES (pH 7.8), 50 µM [{gamma}-32P]ATP (1–2 cpm/fmol), 2 mM EGTA, 28 mM ß-mercaptoethanol, 2 mM MnCl2, 10 mM MgCl2, 0.8 mM sodium orthovanadate, and EGF receptor peptide (1 mg/ml). Reactions were quenched by spotting 10 µl of the reaction mixture on phosphocellulose paper squares (P81, Whatman, Clifton, NJ), which were washed in 150 mM phosphoric acid. Phosphate incorporation was measured by the Cerenkov method.

An in-gel kinase assay was performed as previously described (17), using myelin basic protein (MBP; 0.5 mg/ml gel) as a substrate.

For determination of fractionated MAPK and MAPKK activities (15), cells were harvested in buffer H and disrupted by two 7-sec sonication (50 watts) on ice, followed by centrifugation at 20,000 x g for 15 min at 4 C.

For DE-52 cellulose fractionation, the cytosolic extracts (0.5 ml) were applied to minicolumns (0.35 ml). The flow-through and wash in 0.02 M NaCl in buffer A were collected and measured as previously described (18) for MAPKK activity in a double couple assay in the presence or absence of recombinant ERK2. The eluate of 0.22 M NaCl in buffer A (0.75 ml) contained more than 85% MAPK activity, measured toward MBP as recently described (15).

For fast protein liquid chromatography fractionation, the cytosolic extracts (6 mg protein from 2 x 108 cells) were loaded on a 1 ml Mono-Q column and fractionated using a 0–0.4 M linear NaCl gradient in buffer A (1 ml/min; 1-ml fractions) as described by Ahn et al. (19). Even fractions were assayed for MAPKK and MAPK activities as described below.

Immunoblotting
Proteins in the cell extracts were separated on 7.5–18% PAGE-SDS gels (ratio of acrylamide to bisacrylamide, 30:0.5) and electrotransferred to nitrocellulose papers at 4 C and 100 V in 50 mM glycine and 50 mM Tris-HCl, pH 8.8. For detection of phosphotyrosine-containing proteins, the papers were blocked for 60 min in 1% BSA and 0.5% Tween-20 in TBS (20 mM Tris, pH 8.3, and 150 mM NaCl) and treated overnight with 0.1 µg/ml affinity-purified rabbit anti-P-tyrosine antibodies (Zymed). After intensive washing, the signals were visualized using horseradish peroxidase-conjugated goat antirabbit IgG and the enhanced chemiluminescence method.

Experiments, in duplicate or triplicate, were repeated two or three times, and the results shown are from a representative experiment.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sustained activation of MAPK by GnRH-a and TPA
Treatment of {alpha}T3–1 cells with GnRH-a or TPA resulted in a rapid activation of MAPK (within 1 min), as revealed by phosphorylation of EGF receptor peptide or MBP (Fig. 1Go). Maximal activity (4- to 5-fold over basal levels) was noticed around 10 min of incubation and persisted for the entire period examined here (60 min). The stimulatory effect of GnRH-a and TPA on MAPK activity declined to near-basal levels only after 2–4 h of incubation (not shown). On the other hand, EGF stimulated a transient response of MAPK activation that peaked 5 min after treatment and declined to basal levels within 20 min (Fig. 1Go, inset). The similarity of the GnRH-a and TPA time course was consistent with PKC being involved in GnRH-a stimulation of MAPK, as recently shown by Sundaresan et al. (13), whereas EGF might operate via a different mechanism (4, 5, 6, 7, 8, 9, 10).



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Figure 1. Time course of MAPK activation by GnRH-a, TPA, and EGF. Cells were stimulated with GnRH-a (10 nM; solid squares and circles), TPA (160 nM; open squares), or EGF (100 ng/ml; triangles, inset) for the time indicated. MAPK activity was determined in cell extracts (using 1 mg/ml EGF receptor peptide; left scale for open and solid squares and triangles) or after fractionation on diethylaminoethyl cellulose minicolumns using MBP (0.3 mg/ml) as a substrate (right scale for dark circles). Results represent one of three similar experiments.

 
Both GnRH-a and TPA showed a dose-related increase in MAPK activity with ED50 values of about 1 and 30 nM, respectively (Fig. 2Go). On the other hand, the Ca2+ ionophores, ionomycin and A23187 (not shown), had marginal or no stimulatory effect on MAPK activity (Fig. 2Go) even when combined with TPA (data not shown).



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Figure 2. Dose response of the effects of GnRH-a, TPA, and ionomycin on MAPK activity. Cells were stimulated with GnRH-a (closed circles), TPA (open circles), or ionomycin (squares) for 15 min at the indicated concentration. MAPK activity was determined in cell extracts, as described in Materials and Methods. Results represent one of three similar experiments.

 
Transient activation of MAPKK (MEK) by GnRH-a
Activation of MAPK by GnRH-a followed activation of the physiological activator of MAPK, namely MAPKK (Fig. 3Go). No MAPKK activity was observed in nonstimulated cells (not shown). However, unlike that of MAPK, activation of MAPKK by GnRH-a was of short duration, peaked at 5 min of stimulation, and declined to near-basal levels at 10 min, suggesting different regulatory termination mechanisms at the two levels of the MAPK signaling cascade.



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Figure 3. Time course of MAPKK (MEK) activation by GnRH-a. {alpha}T3–1 cells were stimulated with GnRH-a (100 nM) for the time indicated. Cytosolic extracts were fractionated on diethylaminoethyl cellulose minicolumns, and MAPKK activity was determined in the presence and absence of recombinant ERK2 as described in Materials and Methods. Results represent one of three similar experiments.

 
GnRH-a and TPA stimulate mainly the ERK1 isoform of MAPK
To further characterize the protein kinases activated by GnRH-a and TPA, cell extracts were fractionated on a Mono-Q column to detect stimulation of MBP-phosphorylating activities, including the MAPK isoforms ERK1 and ERK2, which under the conditions used here are eluted specifically in two peaks between 0.1–0.15 M NaCl (19). MAPKK activity eluted in the flow-through fractions of the Mono-Q column (18) was markedly enhanced by GnRH-a treatment of the cells, whereas nonstimulated cells showed no activity (Fig. 4AGo and data not shown). As MAPKK is highly specific toward its MAPK substrates (18), its activation generally results in MAPK stimulation. Indeed, as shown in Fig. 4BGo, two peaks of MBP phosphorylating activities are markedly activated by GnRH-a. Their elution in peak fractions 36 and 46 corresponds to the elution of ERK2 and ERK1 as previously described (15) and verified here by Western immunoblotting using anti-MAPK serum (Fig. 4BGo, inset, lanes 2 and 3), which recognizes both MAPK isoforms to the same extent (data not shown). An in-gel kinase renaturation assay of crude {alpha}T3–1 cell lysate also revealed the enhanced activation of ERK1 by GnRH-a (not shown). Similar to GnRH-a, TPA caused preferential activation of ERK1 (not shown). As the ratio of ERK2 to ERK1 in {alpha}T3–1 cells is about 3:1 (Fig. 4BGo, inset, lane 1), the data suggest that in pituitary gonadotrophs, GnRH-a as well as TPA stimulate mainly the ERK1 isoform of MAPK. This is in contrast to most other cells and ligands that seem to activate mainly the ERK2 isoform (9). Thus, ERK1 might be involved in regulation of differentiated cellular functions of GnRH.



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Figure 4. Mono-Q fractionation of GnRH-a-activated MAPKK and MAPK. Cytosolic extracts of untreated (B, basal, open circles) and stimulated (100 nM GnRH-a for 10 min; A and B, dark circles) {alpha}T3–1 cells were Mono-Q fast protein liquid chromatography fractionated using a linear NaCl gradient (0–0.4 M; right scale). Even fractions were assayed for MAPKK activity (A) as well as MBP kinase activity (B) as described in Materials and Methods. Under these conditions, MAPKK is eluted in the flow-through, whereas activated ERK2 and ERK1 are eluted by the salt gradient (peaking at fractions 36 and 46, respectively). The inset shows Western immunoblot analysis of the MAPK isoforms in the total {alpha}T3–1 cell extract (lane 1) and in peak MBP kinase fractions 36 and 46 (lanes 2 and 3, respectively). The anti-MAPK antibodies used recognize ERK1 and ERK2 to the same extent. Fraction 36 also contains nonactive ERK1, whereas the bulk of nonactive ERK2 is present in fractions 30–34 (not shown). In A, open circles represent the activity obtained in the absence of added recombinant ERK2. Results represent one of five similar experiments.

 
Effect of pertussis toxin (PTX) on activation of MAPK
PTX has been shown (20, 21, 22, 23) to inhibit MAPK activation by thrombin, lysophosphatidic acid, GnRH, and endothelin-1, which share some common mechanisms with GnRH in pituitary gonadotrophs (24, 25). Hence, we examined the effect of PTX on GnRH-a-, TPA-, and EGF-induced MAPK activation. In contrast to a previous report (23), prolonged preincubation of {alpha}T3–1 cells with PTX (100 ng/ml; 18 h) had no significant effect on stimulation of MAPK activity by GnRH-a, TPA, and EGF (Fig. 5Go). Thus, the effects of GnRH-a, TPA, and EGF on MAPK activity in {alpha}T3–1 cells do not involve PTX-sensitive G proteins such as Gi or Go. On the other hand, PTX inhibited (by 47%) serum (10% FCS) stimulation of MAPK activity (Fig. 5Go).



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Figure 5. Effect of PTX on GnRH-a, TPA, serum, and EGF stimulation of MAPK activity. {alpha}T3–1 cells were pretreated with PTX (100 ng/ml) for 18 h. Cells were then washed in DMEM and stimulated for 15 min with GnRH-a (10 nM) or TPA (160 nM) or serum (10%) or for 5 min with EGF (100 ng/ml). MAPK activity was determined in cell extracts as described in Materials and Methods. Results (mean ± SE) are from a representative experiment.

 
Stimulation of protein tyrosine phosphorylation by GnRH-a
Treatment of {alpha}T3–1 cells with GnRH-a resulted in the elevation of tyrosine phosphorylation of several proteins of 42, 46, 85, 95, 100, 125, and 170 kDa (Fig. 6Go and data not shown). The magnitude of the response ranged from 2- to 4-fold over basal levels, and the ED50 ranged from 1 nM, similar to that observed for MAPK activation (Fig. 2Go). A similar protein phosphorylation pattern was noticed with TPA (not shown).



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Figure 6. Effect of GnRH-a on protein tyrosine phosphorylation. {alpha}T3–1 cells were stimulated with GnRH-a at the indicated concentrations for 15 min. Cell extracts were run on SDS gel, transferred to nitrocellulose paper, and treated with anti-P-tyrosine antibodies. The bands were visualized with ECL reagents (A) and quantified by densitometric scanning (B) as described in Materials and Methods. Lanes 1–7 represent GnRH-a at 0, 0.1, 0.5, 1, 2, 10, and 100 nM, respectively. Arrows indicate the positions of the stimulated protein tyrosine phosphorylation.

 
Consistent with a previous report (11), tyrosine phosphorylation stimulated by GnRH-a was inhibited by the PTK inhibitor genistein (200 µM; not shown). However, in contrast to that report (11), genistein reduced by 50% the activation of MAPK by GnRH-a (Fig. 7Go), suggesting the participation of PTK-dependent and independent pathways in MAPK activation by GnRH-a. Genistein also inhibited the stimulation of MAPK by EGF by 90% (not shown).



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Figure 7. Effect of genistein on MAPK activation by GnRH-a. {alpha}T3–1 cells were pretreated with 200 µM genistein for 30 min. Cells were then stimulated for 5 min with GnRH-a (10 nM). MAPK activity was determined in cell extracts as described in Materials and Methods. Results (mean ± SE; n = 3) represent one of three similar experiments, each carried out in triplicates.

 
Role of PKC in activation of MAPK and protein tyrosine phosphorylation
To further explore the role of PKC in activation of MAPK and protein tyrosine phosphorylation, inhibition and depletion of the enzyme were employed. Using the selective PKC inhibitor GF 109203X (16), we found inhibition of GnRH-a- and TPA-induced MAPK activation both with an IC50 value of 1.8 µM (Fig. 8AGo).



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Figure 8. Effect of the PKC inhibitor GF 109203X on GnRH-a and TPA activation of MAPK (A) and protein tyrosine phosphorylation (B). {alpha}T3–1 cells were preincubated with increasing doses of GF 109203X for 10 min. A, Cells were then further incubated with GnRH-a (10 nM; closed circles) or TPA (160 nM; open circles) for 15 min. MAPK activity was determined in cell extracts as described in Materials and Methods. B, Cell extracts were run on SDS gels, transfered to nitrocellulose papers, and treated with anti-P-tyrosine antibodies. The bands were visualized with enhanced chemiluminescent reagents and quantified by densitometric scanning as described in Materials and Methods. GnRH-a- and TPA-stimulated phoshoproteins are represented by closed and open symbols, respectively (circles, pp42; triangles, pp46; squares, pp125). Inset: Lane 1, control; lane 2, GnRH-a (10 nM; 15 min); lane 3, GF109203X (10 µM) plus GnRH-a (10 nM; 15 min).

 
Similar to MAPK activity (Fig. 8AGo), protein tyrosine phosphorylation stimulated by GnRH-a and TPA was also inhibited by GF 109203X, with an IC50 ranging from 0.4–0.8 µM, with the exception of pp125 phosphorylation by GnRH-a (IC50, 30 µM; Fig. 8BGo and data not shown). The data strongly suggest that PKC mediates the activation of MAPK and PTKs by GnRH-a. The drug alone (up to 30 µM) had no effect on MAPK activity. In addition, the drug showed no inhibition of MAPK activity in vitro, of the PTK activity of EGF receptor (from A431 cells), or of the total PTK activity of {alpha}T3–1 cell lysate (not shown).

We also examined the activation of MAPK in PKC down-regulated cells. Prolonged pretreatment of the cells with TPA (100 ng/ml; 16 h) reduced endogenous PKC activity by about 90% (26, 27). GnRH-a and TPA stimulation of MAPK activity was abolished in the PKC down-regulated cells (Fig. 9Go). Further examination of GnRH-a stimulation of MAPK in control and PKC down-regulated cells at different time points revealed loss of responsiveness at all time points examined (Fig. 9Go, inset). The data rules out a possible PKC-independent transient response elicited by GnRH-a.



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Figure 9. Effect of PKC depletion on GnRH-a and TPA activation of MAPK. {alpha}T3–1 cells were pretreated with or without TPA (160 nM) for 16 h. Cells were then washed and stimulated with GnRH-a (10 nM) or TPA (100 nM) for 15 min. MAPK activity was determined in cell extracts as described in Materials and Methods. The inset shows the time course of MAPK activation by GnRH-a in control and TPA-induced down-regulated cells; cytosolic extracts were fractionated on diethylaminoethyl cellulose minicolumns, and MAPK activity in the 0.22 M NaCl fractions was determined toward MBP as substrate as described in Materials and Methods.

 
Effect of cAMP on activation of MAPK
Several reports have recently demonstrated that elevation of cAMP results in inhibition of Raf-1-MAPK signaling stimulated by growth factors, ligands for GPCR, and TPA (17, 28, 29; see Ref. 30 for review). We, therefore, examined the effect of forskolin, a diterpene that directly activates adenylyl cyclase, on GnRH-a, TPA, and EGF stimulation of MAPK (Fig. 10Go). Pretreatment of the cells with forskolin (50 µM; 15 min) stimulated basal MAPK activity (50%; P < 0.05). The EGF response (5-min treatment) was partially inhibited (31%; P < 0.02), but no significant effect on GnRH-a and TPA stimulation of MAPK activity was exerted by forskolin at 15 min.



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Figure 10. Effect of forskolin on GnRH-a, TPA, and EGF stimulation of MAPK activity. {alpha}T3–1 cells were pretreated with forskolin (50 µM) for 15 min and further stimulated for 15 min with GnRH-a (10 nM) or TPA (160 nM), or for 5 min with EGF (100 ng/ml). MAPK activity was determined in cell extracts as described in Materials and Methods. Forskolin alone elevated basal MAPK by 50% (P < 0.05). Results (mean ± SE) are from seven experiments. P < 0.02 for EGF plus forskolin vs. EGF, by two-way ANOVA.

 
Role of Ca2+ in MAPK activation
As mentioned above, the Ca2+ ionophores ionomycin and A23187 showed marginal or no stimulatory effect on MAPK activity (Fig. 2Go). On the other hand, transfer of the cells to Ca2+-free medium containing EGTA (250 µM) markedly reduced the stimulatory effect of both GnRH-a and TPA on MAPK activity (Fig. 11Go), but not on protein tyrosine phosphorylation (Fuchs, T., M. Liskovitch, and Z. Naor, in preparation). Thus, Ca2+ is necessary, but not sufficient, for mediating the effect of GnRH-a on MAPK activity. Surprisingly, removal of Ca2+ resulted in partial inhibition (50%) of EGF or vanadate/H2O2 stimulation of MAPK activity, but had no effect on PTK activity stimulated by vanadate/H2O2 (Fig. 11Go and data not shown). The data suggest the participation of a Ca2+-dependent component distinct from PKC in the activation mechanism of MAPK.



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Figure 11. Effect of Ca2+ removal on GnRH-a, TPA, and EGF stimulation of MAPK activity. {alpha}T3–1 cells in DMEM or in Ca2+-free DMEM containing 0.25 mM EGTA were treated with GnRH-a (10 nM) or TPA (160 nM) for 15 min or with EGF (100 ng/ml) for 5 min. MAPK activity was determined in cell extracts as described in Materials and Methods. Results shown are from a representative experiment.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The signaling events involved in GnRH-stimulated gonadotropin release and synthesis emphasize the role of Ca2+ and PKC (1, 2, 3, 13, 26, 27). Nevertheless, the sequence of events downstream from PKC activation is not known. As convergence of signaling originating from RTKs and some GPCRs is manifested at the MAPK level (4, 5, 6, 7, 8, 9, 10), we examined further the activation of MAPK by GnRH-a (11, 12, 13) and the relative roles of PKC and Ca2+. We observed here a prolonged activation of MAPK by GnRH-a and TPA that lasted at least 60 min, whereas activation by EGF was shorter in duration and returned to basal levels within 20 min. The similarity of the GnRH-a and TPA time response suggested that PKC mediates the activation process as recently shown by Sundaresan et al. (13), and that different regulatory termination signals operate during GnRH-a and TPA vs. EGF actions.

Transient activation of MAPK is thought to be associated with cellular growth and proliferation, whereas prolonged activation is involved in differentiation (31, 32). GnRH-a, which triggers differentiated cellular functions such as gonadotropin secretion and synthesis, is also found capable of inducing a sustained MAPK signal in the gonadotroph {alpha}T3–1 cell line. Thus, prolonged activation of MAPK might be associated with alternative cellular functions. Furthermore, GnRH-a is shown here to apparently activate mainly the ERK1 isoform, whereas other ligands were reported to activate mainly ERK2 (4, 5, 6, 7, 8, 9, 10). The significance of the differential activation of ERK1 vs. ERK2 is under investigation. Interestingly, unlike the prolonged MAPK activation, stimulation of MAPKK (MEK) by GnRH-a was transient, suggesting that distinct termination signals and protein phosphatases operate on these two members of the signaling cascade during GnRH-a action.

Different mechanisms were proposed for MAPK activation by ligands operating via GPCRs. Thrombin and lysophosphatidic acid stimulate MAPK via Gi in a PTX-sensitive, PKC-independent pathway (20, 21), whereas endothelin-1 acts in a PTX-sensitive and PKC-dependent mechanism (22). The agents acting via muscarinic receptors m1 and m2 stimulate MAPK apparently via the ß{gamma}-subunits of G proteins (Gi and Gq) in a Ras-dependent, PKC-independent process (33). Other Gq-linked receptors, such as that for bombesin, are thought to mediate activation of MAPK almost entirely via PKC (34), or in the case of TRH, activation is mediated partly by PKC and also by a Ras-dependent pathway (35). The results presented here for GnRH-a represent another aspect of signaling, emphasizing the differential role of PKC and Ca2+ in MAPK activation. In contrast to a previous report (23), our results indicate that Gi and Go are not involved in GnRH-a activation of MAPK by demonstrating the lack of inhibition of the GnRH-a response by pertussis toxin. The reason for the discrepancy is not known. Thus, GnRH-a stimulation of MAPK activity is most likely mediated by Gq via activation of phospholipase C (36, 37).

Activation of MAPK by RTKs, GPCRs, and phorbol esters was inhibited by elevation of cAMP (17, 28, 29; see Ref. 30 for review). However, in the PC12 cell line, forskolin seems to slightly elevate basal MAPK activity without affecting its maximal stimulated activity (17). The reason for this discrepancy is probably due to the pattern of MEK activator expression in PC12 cells (17). Indeed, the stimulation of basal MAPK activity by forskolin and the lack of inhibition of the GnRH-a and TPA responses observed here resemble the effect of forskolin in PC12 cells. However, the partial inhibition of EGF-stimulated activity suggests that more than one signaling mechanism leads to MAPK activation in {alpha}T3–1 cells. Thus, EGF stimulates MAPK activity by both cAMP-sensitive and insensitive pathways, whereas GnRH-a and TPA act by a cAMP-insensitive mechanism. One possible explanation for these distinct pathways is that GnRH-a and TPA signals might be transmitted by MEK activators that are distinct from the cAMP-sensitive Raf-1 (30, 38), whereas EGF signals use both Raf-1 and the other MEK activators.

Elevation of Ca2+ by ionomycin had marginal effect on MAPK activity in {alpha}T3–1 cells. The results differ from those reported in other cell systems, where elevated Ca2+ activated MAPK (39, 40). Furthermore, raising the levels of extracellular Ca2+ inhibited EGF-induced ERK2 activity in human primary keratinocytes (41). On the contrary, in our system, removal of Ca2+ markedly reduced the GnRH-a and EGF responses. Thus, Ca2+ is necessary, but not sufficient, for mediating GnRH-a elevation of MAPK activity. On the other hand, TPA elevates MAPK activity, whereas PKC inhibition or its depletion resulted in inhibition of the GnRH-a response. Thus, PKC is necessary and sufficient for mediating the GnRH-a effect. As activation of phosphoinositide turnover by GnRH is Ca2+ independent (36), it is possible that the Ca2+-requiring step involves the activation of a Ca2+-dependent PKC subtype, consistent with the report that PKC{alpha} activates Raf-1 by direct phosphorylation (42). However, the inhibitory effect of Ca2+ removal on the action of GnRH-a as well as that of EGF or vanadate/H2O2 on MAPK activity, but not on protein tyrosine phosphorylation or PTK activity, suggests that another Ca2+-binding protein might participate in the activation process downstream from PKC and PTK.

One of the targets of MAPK is the cytosolic phospholipase A2, which is phosphorylated and activated by MAPK (43). As the release of arachidonic acid and formation of its lipoxygenase products play a role in GnRH-induced gonadotropin release and gonadotropin subunit gene expression (44, 45, 46, 47, 48, 49), it is likely that activation of MAPK by GnRH is required to enable stimulation of the arachidonate cascade. In addition, some potential MAPK substrates, such as c-fos, Elk1, Ets2, and TAL-1 (6, 7, 8, 9, 10), might be required for GnRH actions as regulators of nuclear transcriptional activity.

Consistent with the reported effect of a general PKC activator such as TPA on protein tyrosine phosphorylation (50, 51), GnRH-a, stimulated the tyrosine phosphorylation of proteins of 42, 46, 85, 95, 100, 125, and 170 kDa, which might represent p42/p44 MAPKs, focal adhesion kinase (p125FAK) (21), and others. Interestingly, the phosphotyrosyl proteins induced by GnRH-a were inhibited by the selective PKC inhibitor GF 109203X, with an IC50 of about 1 µM, with the exception of pp125, which required higher doses of the drug (IC50, 30 µM). As inhibition of GnRH-a- and TPA-induced MAPK activation by GF 109203X was observed at an IC50 of 1.8 µM, some of the tyrosine-phosphorylated proteins, but not pp125, might be linked to MAPK in a manner yet to be determined. This is further supported by the inhibitory effect of the PTK inhibitor genistein on GnRH-a stimulation of MAPK activity. Furthermore, as the GnRH effect on protein tyrosine phosphorylation and MAPK activation is fully dependent on PKC, whereas genistein only partly inhibits (50%) the activation of MAPK by GnRH, it seems likely that at least two PKC subspecies that specialize in PTK-dependent and -independent signaling participate in the action of GnRH on MAPK. Nevertheless, PKC-sensitive mechanisms are involved in GnRH-a-induced protein tyrosine phosphorylation, which apparently are manifested upstream of MAPK activation. Based on the data presented here, we propose a model for GnRH-induced MAPK activation (Fig. 12Go). The present study opens a new vista in elucidating the mechanism of action of the neurohormone GnRH, which is the first key hormone of reproduction, and sheds further light on the pathways involved in RTKs vs. GPCRs leading to activation of MAPK.



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Figure 12. Proposed model for the mechanism of MAPK activation by GnRH. Gq is activated by the GnRHR and causes enhanced phospholipase C (PLC) activity and elevation of the second messengers inositol trisphosphate, which elevates intracellular Ca2+, and diacylglycerol (DAG), which activates various PKC isoforms. GnRH stimulates in a PKC-dependent manner the tyrosine phosphorylation of several proteins whose role in MEK and MAPK activation is still to be determined. PKCs cause the activation apparently of MEK activators other than Raf-1 or B-Raf, leading to MEK and ERK activation in a manner that is Ca2+ dependent.

 


    Acknowledgments
 
We thank Dr. P. Mellon for the {alpha}T3–1 cells, and Ms. T. Fuchs and Drs. U. Zor, Y. Granot, and M. Liscovitch for their interest and help during the studies. We also thank Mrs. Angela Cohen for editorial assistance.


    Footnotes
 
1 This work was supported by research grants from the United States-Israel Binational Science Foundation, the Israel Academy of Sciences and Humanities, the Eisne Foundation, the Tel Aviv University Special Fund (to Z.N.), the Paul Godfrey Foundation in Children’s Diseases (to R.S.), and ICRF Fellowship (to L.N.L.) Back

2 Incumbent of the Samuel and Isabela Friedman Career Development Chair. Back

Received October 3, 1996.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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