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Activation of MAPK Cascades by GnRH: ERK and Jun N-Terminal Kinase Are Involved in Basal and GnRH-Stimulated Activity of the Glycoprotein Hormone LHß-Subunit Promoter

Department of Biochemistry (D.H., D.B., D.C., Z.N.), The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel; and Department of Biological Regulation (S.K., R.S.), The Weizmann Institute of Science, Rehovot 76100, Israel

Address all correspondence and requests for reprints to: Dr. Zvi Naor, Department of Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel. E-mail: . Naorzvi{at}post.tau.ac.il

	Abstract

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The role of ERK and Jun N-terminal kinase (JNK) in basal- and GnRH-stimulated LHß-promoter activity was examined in the gonadotroph cell line LßT-2. GnRH agonist (GnRH-A) stimulates the MAPK cascades ERK, JNK, and p38MAPK, with a peak at 7 min for ERK and at 60 min for JNK and p38MAPK. The rat glycoprotein hormone LHß-subunit promoter, linked to the chloramphenicol acetyl transferase (CAT) reporter gene, was used to follow its activation. Addition of GnRH-A (10 nM) to LßT-2 cells resulted in a 6-fold increase in LHß-CAT activity at 8 h, which was markedly reduced by a GnRH antagonist. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca²⁺ ionophore ionomycin, stimulated LHß-CAT activity. Addition of GnRH-A and TPA together did not produce an additive response. Down-regulation of PKC, but not removal of Ca²⁺, abolished the GnRH-A and the TPA response. Cotransfection of the LHß-promoter and the constitutively active form of Raf-1 stimulated basal and GnRH-A-induced LHß-CAT activity. The dominant negative forms of the ERK cascade members Ras, Raf-1, and MAPK/ERK kinase (MEK) markedly reduced basal and GnRH-A-induced LHß-CAT activity, Similar results were obtained with the MEK inhibitor PD 098059. Cotransfection of the LHß-promoter and the constitutively active CDC42 stimulated basal and GnRH-A-induced LHß-CAT activity. The dominant negative forms of the JNK cascade members Rac, CDC42, and SEK markedly diminished basal and GnRH-A-induced LHß-CAT activity. Interestingly, the constitutively active form of c-Src stimulated the basal and the GnRH-A response, whereas the dominant negative form of c-Src, or the c-Src inhibitor PP1 diminished basal and the GnRH-A response. We conclude that ERK and JNK are involved in basal and GnRH-A stimulation of LHß-CAT activity. c-Src participates also in LHß-promoter activation by a mechanism which might be linked to ERK and JNK activation.

	Introduction

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THE HYPOTHALAMIC NEUROHORMONE GnRH stimulates the synthesis and release of LH and FSH from pituitary gonadotrophs. Common to the pituitary heterodimeric glycoprotein hormones LH, FSH, and TSH is the -subunit, which binds noncovalently to a specific ß-subunit (1, 2, 3). We and others have demonstrated that GnRH elicits a rapid and sequential activation of the phospholipases C, D, and A₂, followed by Ca²⁺ mobilization and influx, activation of PKC, and stimulation of the MAPK cascades ERK, Jun N-terminal kinase (JNK), and p38MAPK (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). Although much is known about mechanisms involved in gonadotropin release, relatively little is known about signaling of the GnRH receptor (GnRHR) during activation of gonadotropin subunit gene expression (see Refs. 2 and 3 for reviews).

The MAPK cascades consist of up to six tiers of protein kinases, which sequentially activate each other by phosphorylation (see Refs. 8 and 18 for reviews). Four distinct MAPK cascades have been identified in mammals: the ERK cascade, also known as p42, and p44 MAPKs, JNK, p38MAPK, and the big MAPK (BMK, ERK5) (see Refs. 8 and 18 for reviews). A small GTP-binding protein of the Ras family often initiates the activation of a MAPK cascade, followed by activation of MAP3K (MEK kinase; MEKK), MEK, and MAPK. In the ERK cascade, Raf kinase (MAP3K, MEKK) activates MEK and ERK 1, 2. In the JNK cascade, several MEKKs, including MEKK1–4, activate MKK4, 7, and JNK1–3. The translocation to the nucleus and activation of various transcription factors, which are a hallmark of MAPK signaling, make this group of kinases important regulators of the transcriptional machinery.

The role of MAPKs in the regulation of gonadotropin gene expression is not clear. It was reported that ERK is involved in basal as well as GnRH-stimulated transcription of the -subunit (10, 19). On the other hand, others have reported that ERK is mainly involved in the regulation of the basal, but not in the GnRH-stimulated, -subunit promoter activity (11). Controversy also exists concerning the role of ERK in GnRH regulation of LHß gene expression, with some investigators reporting a positive role for ERK (20), whereas others have found no role for ERK in the transcriptional regulation of LHß by GnRH (19, 21).

Activation of JNK by GnRH (13, 17, 21) could be important in common -subunit gene expression, because Jun and ATF2, which are known substrates of JNK, have been shown to contribute to -subunit gene transcription via binding to the CRE domain of the promoter (22). Nevertheless, we have recently found that ERK, but not JNK, is partially involved in basal and in GnRH-induced -subunit gene regulation (Harris, D., O. Benard, D. Chuderland, R. Seger, and Z. Naor, manuscript submitted). Others have recently reported that JNK, but not ERK, is involved in GnRH-induced LHß gene expression (21). We report here that ERK and JNK are involved in regulation of basal and GnRH stimulation of LHß transcription.

	Materials and Methods

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Materials
The stable GnRH agonist, [D-Trp⁶]-GnRH (GnRH-A), ionomycin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) were obtained from Sigma (St. Louis, MO); PD 098059 and PP1, from Calbiochem (La Jolla, CA). The GnRH antagonist (antide) was a kind gift from Dr. D. Coy (Tulane University, New Orleans, LA). Mouse monoclonal antiactive (doubly phosphorylated) MAPKs (ERK, JNK, p38MAPK) and polyclonal antibodies to general MAPKs were from Sigma (Rehovot, Israel). The plasmids for the various ERK and JNK cascade members used here were recently described (13, 24). The rat LHß-subunit promoter, linked to chloramphenicol acetyl transferase (CAT), was kindly provided by Dr. W. Chin (Boston, MA).

Activation of MAPK cascades
LßT-2 cells were grown in 6-well plates and serum starved (0.5% FCS) for 16 h. After stimulation with the various ligands, the cells were washed twice with ice-cold PBS and once with ice-cold buffer A (50 mM ß-glycerophosphate, 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 1 mM sodium orthovanadate). Cells were harvested in 0.3 ml buffer H: buffer A containing 1 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride, followed by sonication (2 x 7 sec, 40 W) and centrifugation (15,000 x g, 15 min, 4 C). The supernatants, which contained cytosolic proteins, were collected, and aliquots from each sample (20 µg) were separated on 10% SDS-PAGE, followed by Western blotting with mouse monoclonal antiactive MAPKs (ERK, JNK, p38MAPK), as recently described (24). Total MAPKs were detected, with polyclonal antibodies for the various MAPKs as a control. The blots were developed with alkaline phosphatase or horseradish peroxidase-conjugated antimouse or antirabbit Fab antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) (24).

Cell culture, transfection, and CAT assay
Subconfluent LßT-2 cells were cotransfected, for 8 h, with 5 µg LHßCAT and 10 µg of the plasmids described in the legends, using the calcium phosphate method. The total amount of plasmid was adjusted to 20 µg with vector DNA in control experiments. The transfection efficiency was 10–30%, as determined by transfection with a plasmid that contained RSV-LUC; 24–36 h after transfection, the cells were serum starved for 16 h and incubated with GnRH-A or other stimulants or inhibitors as described in the legends. Cells were then harvested, and the cell extracts were analyzed for CAT activities (25), which were normalized to the level of RSV-LUC activity. Results were then calculated as fold increase relative to expression in control wells, and the data are shown as the mean ± SEM. The t test was used to determine statistically different groups, and significance was set at P < 0.05.

	Results

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Effect of GnRH-A on MAPKs activity
LßT-2 cells were incubated with GnRH-A for the time indicated, and cell lysates were subjected to Western blot analysis using antibodies which specifically recognize the doubly phosphorylated, active forms of the MAPKs (24). ERK, JNK, and p38MAPK were activated, to various extents, upon GnRH-A stimulation (Fig. 1A). The peak of activation was observed at 7 min for ERK and at 60 min for JNK and p38MAPK. The selective MEK inhibitor, PD 098059 (50 µM), abolished GnRH-A-stimulated ERK but not JNK activation (Fig. 1B).

View larger version (51K): [in this window] [in a new window]   Figure 1. Effect of GnRH-A on MAPK activity in LßT2 cells. Subconfluent LßT2 cells were treated with GnRH-A (100 nM), in the presence (B) or the absence (A) of PD 098059 (50 µM), for the indicated times. MAPK [ERK, JNK, and p38MAPK (p38)] activity was determined by Western blotting with type-specific antibodies to the phosphorylated (hence, activated) forms of the enzymes. A representative experiment is shown.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effect of GnRH-A on LHß-subunit gene expression. Subconfluent LßT2 cells were transfected with LHßCAT for 8 h, washed several times, and incubated for 24 h. GnRH-A was then added for 8 h (A), or for the indicated times (B, GnRH-A, 10 nM), or with a GnRH antagonist (antide, 100 nM) for 8 h (C). LHßCAT activity was determined as described in Materials and Methods. Results are expressed as fold increase, relative to untreated controls, and represent the average of three experiments + SEM.

View larger version (16K): [in this window] [in a new window]   Figure 3. Role of PKC in GnRH-A stimulation of LHß-subunit gene expression. Subconfluent LßT2 cells were transfected with LHßCAT for 8 h, washed several times, and incubated for 24 h. A, The cells were incubated with TPA (100 ng/ml) for 8 h. B, To remove endogenous PKC activity, cells were down-regulated by including TPA (100 ng/ml) during the 24-h incubation (D-reg) or were left untreated. GnRH-A (10 nM) and TPA (100 ng/ml) were then added to untreated or down-regulated cells for 8 h. LHßCAT activity was determined as described in Materials and Methods. Results are expressed as fold increase, relative to untreated controls, and represent the average of three experiments + SEM.

View larger version (17K): [in this window] [in a new window]   Figure 4. Role of Ca2+ in GnRH-A stimulation of LHß-subunit gene expression. Subconfluent LßT2 cells were transfected with LHßCAT for 8 h, washed several times, and incubated for 24 h. A. The cells were incubated with the Ca2+ ionophore ionomycin (1 µM) for 8 h. B. The transfected cells were transferred to normal DMEM (+), or to Ca2+-free DMEM (-), or to Ca2+-free DMEM also containing EGTA (0.25 M) and incubated with or without GnRH-A (10 nM) for 8 h. C. The transfected cells were incubated with GnRH-A (10 nM), TPA (100 ng/ml), ionomycin (1 µM), or with the indicated combinations, for 8 h. LHßCAT activity was determined as described in Materials and Methods. Results are expressed as fold increase, relative to untreated controls, and represent the average of three experiments + SEM.

View larger version (19K): [in this window] [in a new window]   Figure 5. Role of ERK in GnRH-A stimulation of LHß-subunit gene expression. A, Effect of constitutively active Raf1. Subconfluent LßT2 cells were transfected with LHßCAT, with or without the constitutively active form of Raf1, for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h, and LHßCAT activity was determined as described in Materials and Methods. Results are expressed as fold increase, relative to untreated controls, and represent the average of three experiments + SEM. B, Effect of dominant negative Ras, Raf1 and MEK. Subconfluent LßT2 cells were transfected with LHßCAT, with or without the dominant negative plasmids of Ras, Raf1, or MEK, for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h, and LHßCAT activity was determined as described in Materials and Methods. Results are expressed as fold increase, relative to untreated controls (cont.), and represent the average of three experiments. The relative fold increase is shown in the inset (1, GnRH-A; 2, GnRH-A+ Ras D. N; 3, GnRH-A+ Raf D. N; 4, GnRH-A+ MEK D. N). C. Effect of PD 098059. Subconfluent LßT2 cells were transfected with LHßCAT for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added, for 8 h, in the presence and absence of the MEK inhibitor PD 098059 (50 µM); and LHßCAT activity was determined as described in Materials and Methods. Results are expressed as fold increase, relative to untreated controls, and represent the average of three experiments. The relative fold increase is shown in the inset (1, GnRH-A; 2, GnRH-A+ PD).

View larger version (20K): [in this window] [in a new window]   Figure 6. Role of JNK in GnRH-A stimulation of LHß-subunit gene expression. A, Effect of constitutively active CDC42. Subconfluent LßT2cells were transfected with LHßCAT, with or without the constitutively active form of CDC42, for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h, and LHßCAT activity was determined as described in Materials and Methods. Results are expressed as fold increase, relative to untreated controls, and represent the average of three experiments. B, Effect of dominant negative Rac, CDC42, and SEK. Subconfluent LßT2 cells were transfected with LHßCAT, with or without the dominant negative plasmids of Rac, CDC42, and SEK, for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h, and LHßCAT activity was determined as described in Materials and Methods. Results are expressed as fold increase, relative to untreated controls, and represent the average of three experiments. The relative fold increase is shown in the inset (1, GnRH-A; 2, GnRH-A+Rac D. N; 3, GnRH-A+ CDC42 D. N; 4, GnRH-A+ SEK D. N).

View larger version (18K): [in this window] [in a new window]   Figure 7. Role of cSrc in GnRH-A stimulation of LHß-subunit gene expression. A, Effect of constitutively active cSrc. Subconfluent LßT2 cells were transfected with LHßCAT, with or without the constitutively active form of cSrc, for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h, and LHßCAT activity was determined as described in Materials and Methods. Results are expressed as fold increase, relative to untreated controls, and represent the average of three experiments. B, Effect of dominant negative cSrc and a cSrc inhibitor. Subconfluent LßT2 cells were transfected with LHßCAT, with or without the dominant negative plasmids of cSrc (CSK), for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h alone, or in CSK-pretreated cells, or in cells treated with the cSrc inhibitor PP1 (2 µM). LHßCAT activity was determined as described in Materials and Methods. Results are expressed as fold increase, relative to untreated controls, and represent the average of three experiments. The relative fold increase is shown in the inset (1, GnRH-A; 2, GnRH-A+ PP1; 3, GnRH-A+ CSK).

	Discussion

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The role of MAPK cascades in GnRH actions is controversial. Weck et al. (19) reported that PKC and ERK are involved in GnRH regulation of common -, but not that of LHß subunit, which is mediated by elevation of Ca²⁺. Saunders et al. (28) reported opposite results, namely that Ca²⁺ mediates the GnRH effect on -subunit expression and PKC mediates the effect on LHß. Call and Wolf (20) found that PKC and ERK, but not Ca²⁺, are involved in GnRH regulation of LHß gene expression. On the other hand, Yokoi et al. (21) found a role for JNK, but not for PKC and ERK, in regulation of LHß gene expression by GnRH. Still, we report here a role for ERK and JNK in GnRH regulation of the LHß gene. The opposing results can be explained by different cell types, promoters, and species used by the various authors. For example, whereas Yokoi et al. (21) are using a -156- to +7-bp rat LHß-promoter, we are using a -797- to +45-bp rat LHß-promoter, which might explain the different results.

The Ets family of transcription factors, such as Elk1, was implicated in mediating transcriptional responses to activation of the ERK cascade (10). Because common - and LHß-promoters contain Ets or Ets-like family binding sites, they might mediate ERK involvement in common - and LHß gene transcription. It was recently shown that expression of the LHß gene is modulated by the orphan nuclear receptor steroidogenic factor-1 (SF-1) and the early growth response (Egr) protein-1, both acting in a synergistic manner (29). Whereas SF-1 regulates both basal and GnRH-stimulated activities, Egr-1 participates only in the GnRH response. Indeed, Egr-1 expression is increased by GnRH in a PKC-dependent manner and is known to be modulated by MAPK (20, 29). Whether GnRH modulates SF-1 is not clear at the present time (3, 29, 30). Egr-1 might, therefore, be involved in the PKC/ERK pathway leading to LHß gene transcription. Kaiser et al. (30, 31) have identified two regions in the rat LHß-promoter, at -490 to -352 and -207 to -82 bp, that mediate the GnRH response and bind the transcription factor Sp-1, which might also be modulated by PKC (3, 30, 31). In addition, the JNK and ERK cascades might operate via the activation of Jun and Fos, respectively, acting on AP-1-like sites within the promoter. Indeed, c-Jun was recently implicated in GnRH activation of the LHß-promoter (21). Although both - and LHß-promoters contain CREs that are usually activated by cAMP (28), it is unlikely that GnRH uses this signaling pathway as we and others have demonstrated (4, 5, 6). Interestingly, Jun and ATF-2, which are known substrates of JNK, were shown to bind to the CRE domain of the -promoter (22). Nevertheless, unlike LHß, we could not find a role for JNK in basal or GnRH-stimulated -subunit gene expression (Harris, D., O. Benard, D. Chuderland, R. Seger, and Z. Naor, manuscript submitted). It is therefore interesting to determine whether Jun and ATF-2 mediate JNK involvement in LHß-promoter activity by binding to the CRE domain.

Addition of ionomycin or removal of Ca²⁺ had no effect on GnRH-induced LHß-promoter activity. On the other hand, removal of Ca²⁺ abolished GnRH stimulation of common -subunit gene regulation (Harris, D., O. Benard, D. Chuderland, R. Seger, and Z. Naor, manuscript submitted). Hence, Ca²⁺ participates in mediating differential activation of gonadotropin subunit gene expression. Removal of Ca²⁺ will reduce the exocytotic response to GnRH (see Refs. 4, 5, 6 for reviews). The interesting observation that LHß-promoter activity is enhanced by GnRH in a Ca²⁺-independent manner indicates dissociation between release and synthesis of LH, and that exocytosis is not a prerequisite for LHß gene expression as observed also for common -subunit (32).

Stimulation of PKC by TPA enhanced LHß but not common -subunit promoter activity; and down-regulation of endogenous PKC abolished GnRH-stimulated LHß but not -promoter activity (present results and Harris, D., O. Benard, D. Chuderland, R. Seger, and Z. Naor, manuscript submitted). Also, TPA enhanced GnRH-induced -, but not LHß subunit promoter activity. We therefore suggest that PKC plays a supportive and a central role in common - and LHß-subunit promoter activities, respectively. Furthermore, as with Ca²⁺, PKC also participates in mediating the differential regulation of gonadotropin subunit gene expression by GnRH in pituitary gonadotrophs.

In this study we described a role for c-Src in GnRH-stimulated LHß gene regulation. Previously we found that c-Src is involved in ERK and JNK activation by GnRH (13, 24). It is therefore likely that c-Src participates in GnRH-stimulated LHß-subunit promoter activity in a JNK- and ERK-dependent mechanism.

Differential regulation of gonadotropin subunit genes can be achieved by various mechanisms. Some researches have proposed that alterations in the frequency of GnRH pulses will dictate specificity of the signal (33). Others have suggested that the density of GnRHRs might be involved in signal specificity (34). The present results and others (19, 20, 21, 23, 28, 32) emphasize that GnRH might use different signaling cascades to regulate selective gonadotropin subunit genes. We therefore propose that ERK be involved in regulating basal and GnRH-stimulated - and LHß genes, whereas JNK mediates basal and GnRH-stimulated LHß-gene expression. Thus, by altering the relative activation of MAPK cascades, GnRH might regulate selective gonadotropin subunit genes, as in signal specificity in general (18).

Interestingly, although GnRH does not play a major role in proliferation or differentiation and is not a stress signal in pituitary gonadotrophs (35), nevertheless, GnRH stimulates MAPK cascades, which specialize in the above functions. We therefore suggest that GnRH operates via MAPK in differentiated functions, such as the selective regulation of gonadotropin subunit synthesis.

	Acknowledgments

 
We thank Dr. O. Benard for his interest and help during the study. We also thank Dr. P. Mellon for the LßT2 cells, and Dr. W. Chin for the LHß-CAT.

	Footnotes

 
This work was supported by the Israel Science Foundation of the Israel Academy of Sciences and Humanities and the Adams Super Center for Brain Studies at Tel Aviv University.

Abbreviations: CAT, Chloramphenicol acetyl transferase; CSK, C-terminal Src kinase; Egr, early growth response; GnRH-A, GnRH agonist; GnRHR, GnRH receptor; JNK, Jun N-terminal kinase; MEK, MAPK/ERK kinase; MAP3K/MEKK, MEK kinase; SF-1, steroidogenic factor-1; TPA, 12-O-tetradecanoylphorbol-13-acetate.

Received July 10, 2001.

Accepted for publication November 7, 2001.

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