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Extracellular Signal-Regulated Kinase, Jun N-Terminal Kinase, p38, and c-Src Are Involved in Gonadotropin-Releasing Hormone-Stimulated Activity of the Glycoprotein Hormone Follicle-Stimulating Hormone ß-Subunit Promoter

Departments of Biochemistry (D.B., D.C., Z.N.) and Cell Research and Immunology (D.S., I.F.), The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel; Department of Biological Regulation (S.K., R.S.), The Weizmann Institute of Science, Rehovot 76100, Israel; and Human Reproduction Sciences Unit, Medical Research Council (Z.N.), The University of Edinburgh, Edinburgh EH164SB, United Kingdom

Address all correspondence and requests for reprints to: Dr. Zvi Naor, Human Reproduction Sciences Unit, Medical Research Council, The University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH164SB, United Kingdom. E-mail: z.naor{at}hrsu.mrc.ac.uk.

	Abstract

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The role of ERK, Jun N-terminal kinase (JNK), p38, and c-Src in GnRH-stimulated FSHß-subunit promoter activity was examined in the LßT-2 gonadotroph cell line. Incubation of the cells with a GnRH agonist resulted in activation of ERK, JNK, p38, and c-Src. The peak of ERK activation was observed at 5 min, whereas that of JNK, p38, and c-Src at 30 min, declining thereafter. ERK activation by GnRH is dependent on protein kinase C (PKC), as evident by activation, inhibition, and depletion of 12-O-tetradecanoylphorbol-13-acetate-sensitive PKC subspecies. Ca²⁺ influx, but not Ca²⁺ mobilization, is required for ERK activation. GnRH signaling to ERK is partially mediated by dynamin and a protein tyrosine kinase, apparently c-Src. ERK activation by GnRH in LßT-2 cells does not involve transactivation of epidermal growth factor receptor or mediation via Gß or ß-arrestin. Once activated by GnRH, ERK translocates to the nucleus. We examined the role of ERK, JNK, p38, and c-Src in GnRH-stimulated ovine FSHß promoter, linked to a luciferase reporter gene (–4741oFSHß-LUC). The PKC activator 12-O-tetradecanoylphorbol-13-acetate, but not the Ca²⁺ ionophore ionomycin, stimulated FSHß-luciferase (LUC) activity. Furthermore, down-regulation of PKC, but not removal of Ca²⁺, inhibited the GnRH response. Cotransfection of FSHß-LUC and the constitutively active forms of Raf-1 and MEK stimulated FSHß-LUC activity, whereas the dominant negatives of Ras, Raf-1, and MEK and the selective MEK inhibitor PD98059, abolished GnRH-induced FSHß-LUC activity. The dominant negatives of CDC42 and JNK reduced the GnRH response by 36 and 49%, respectively. Incubation of the cells with the p38 or the c-Src inhibitors SB203580 and PP1 also reduced the GnRH response. Surprisingly, two proximal activator protein-1 sites contribute very little to the GnRH response. Thus, PKC, ERK, JNK, p38, and c-Src, but not Ca²⁺, are involved in GnRH induction of the ovine FSHß gene.

	Introduction

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GnRH IS SECRETED in a pulsatile manner from the hypothalamus to stimulate the synthesis and release of the heterodimeric glycoprotein hormones LH and FSH from pituitary gonadotrophs. The heterodimeric glycoprotein hormones of the pituitary, LH, FSH, and TSH have a common -subunit, which binds noncovalently to a specific ß-subunit (1, 2, 3, 4). GnRH stimulates phosphoinositide turnover via phospholipase Cß followed by sequential activation of phospholipase D and phospholipase A₂, Ca²⁺ mobilization and influx, activation of selective protein kinase C (PKC) isoforms such as PKC and PKC, and stimulation of the MAPK cascades ERK, Jun N-terminal kinase (JNK), and p38 (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). Although much is known about mechanisms involved in gonadotropin release, relatively little is known about gonadotropin subunit gene expression (2, 3, 4).

MAPK cascades consist of up to six tiers of protein kinases, which sequentially activate each other by phosphorylation (18, 21). At least four distinct MAPK cascades are known in mammals: ERK1/2 (p42 and p44), JNK (1/3), p38(, ß, , ), and ERK5 (big MAPK; BMK) (18, 21). A Ras family member of the small G-proteins initiates the activation of the cascade by activation of MAP3K (MEKK), MAPKK (MEK), and MAPK. ERK1/2 is activated by Raf (MEKK) and MEK1/2, and JNK1–3 is activated by MEKK1–4 and MKK4/7. The hallmark of the MAPK family is its ability to translocate to the nucleus and activate a variety of transcription factors, suggesting a role in the transcriptional machinery.

GnRH stimulates ERK, JNK, and p38 in T3–1, LßT-2, and GnRH receptor (GnRHR) transfected cells (7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27). The role of MAPKs in the regulation of gonadotropin gene expression is controversial. It was reported that ERK is involved in GnRH-stimulated transcription of the -subunit (8, 25, 28), whereas others reported that ERK is involved mainly in the basal regulation (9). Controversy also exists concerning the role of ERK in GnRH regulation of the LHß gene (22, 25, 26, 27).

Concerning the role of JNK in GnRH action, we anticipated that JNK will be important in common -subunit gene expression because Jun and activating transcription factor 2, which are known substrates of JNK, have been shown to contribute to -subunit gene transcription via binding to the cAMP response element domain of the promoter (29). Nevertheless, we recently found that ERK, but not JNK, is involved in GnRH-induced -subunit transcription (28). Yokoi et al. (22) recently reported that JNK, but not ERK, is involved in GnRH-induced LHß gene expression, whereas we reported that both ERK and JNK mediate GnRH-stimulation of LHß transcription (27).

Relatively little is known about signaling involved in GnRH stimulation of FSHß gene expression and the role of MAPK (3, 4). The important role of PKC in GnRH regulation of FSHß gene expression has been demonstrated (30, 31, 32). Ca²⁺ influx does not mimic the stimulatory effect of GnRH on FSHß gene expression (30, 31, 32), but its removal diminished the GnRH response (32). As to the role of MAPK, the MEK inhibitor PD98059 was shown to inhibit GnRH stimulation of -subunit and FSHß, but not LHß mRNA accumulation (33). Another MEK inhibitor, UO126, was recently reported to inhibit GnRH, but not 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced FSHß promoter activity in LßT-2 cells (32). We report here that PKC, ERK, JNK, p38, and c-Src but not Ca²⁺ are involved in GnRH-stimulated ovine (o)FSHß promoter activity in LßT-2 cells.

	Materials and Methods

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Materials
The stable GnRH agonist (GnRH-A), buserelin, was a kind gift from Dr. J. Sandow (Aventis Pharma, Hoechst, Frankfurt, Germany). Ionomycin, epidermal growth factor (EGF), pertussis toxin, and TPA were obtained from Sigma (St. Louis, MO). PD098059, SB203580, and PP1 were from Calbiochem (La Jolla, CA), and recombinant human activin A from R&D Systems (Minneapolis, MN). Mouse monoclonal antiactive (doubly phosphorylated) MAPKs (ERK, JNK, and p38) and polyclonal antibodies to general MAPKs were from Sigma (Rehovot, Israel). Anti-EGF receptor (EGFR) (1005), and anti-PY were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and antiphospho-c-Src (Tyr 418) was from Bio-Source International (Nivelles, Belgium). The selective EGFR inhibitor, AG 1478, was kindly provided by Drs. A. Gazit and A. Levitzky (Hebrew University, Jerusalem, Israel). The plasmids for the various ERK and JNK cascade members used here were recently described (13, 23, 24, 27). The Western blotting detection reagent, enhanced chemiluminescence was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Secondary horseradish peroxidase-conjugated goat antimouse antibodies or goat antirabbit antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). All media and sera were from Biological Industries (Beit-Hemeek, Israel).

Plasmids
Mammalian expression vectors containing dominant-negative (DN) dynamin (K44A-dynamin), DN ß-arrestin (V54D-ß-arrestin-2) in pCDNA3 were a gift from Dr. M. Caron (Duke University, Durham, NC). The DN plasmid for the EGFR (K721A) in pCDNA3 was provided by Dr. Y. Yarden (Weizmann Institute of Science, Rehovot, Israel). The DN plasmid for JNK in pCMV5 was provided by Dr. M. Cobb (University of Texas, Dallas, TX). CD8-tagged ß-adrenergic receptor kinase (ARK) was from Dr. Zvi Vogel (Weizmann Institute of Science). hemagglutinin (HA) epitope-tagged ERK (HA-ERK2) and N-17 Ras were previously described (23). The oFSHß promoter (–4741/+759 bp) and its mutant AP-1oFSHß, linked to luciferase (LUC) were provided by Dr. W. Miller (North Carolina State University, Raleigh, NC). Other plasmids used were recently described (23, 27).

Cell culture
LßT-2 cells were grown in monolayer cultured in DMEM supplemented with 10% fetal calf serum (FCS) and antibiotics in humidified 5% CO₂ at 37 C. Cells were starved overnight in 0.5% serum DMEM and then pretreated with the various inhibitors for 20 min. GnRH-A (buserelin) or TPA were then added for the appropriate time as indicated.

Activation of MAPK cascades
LßT-2 cells were grown in 6-well plates, serum starved (0.5% FCS) for 16 h, later stimulated with the various ligands, and 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, and 1 mM phenylmethylsulfonyl fluoride, followed by sonication (2 x 7 sec, 40 W) and centrifugation (15,000 x g, 15 min, 4 C). The supernatants were collected and aliquots (30 µg/sample) were separated on 10% SDS-PAGE, followed by Western blotting with mouse monoclonal anti-phospho MAPKs (ERK, JNK, and p38) (23, 27). Total MAPKs were detected with polyclonal antibodies for the various MAPKs as a control for sample loading. The blots were developed with alkaline phosphatase or horseradish peroxidase conjugated antimouse or antirabbit Fab antibodies (Jackson ImmunoResearch Laboratories). The blots were autoradiographed on X-100 films (Kodak, Rochester, NY), and the phosphorylation was quantitated by densitometry (690 densitometer, Bio-Rad Laboratories) (23, 27).

Cell culture and transfection
Subconfluent LßT-2 cells were cotransfected for 8 h with 5 µg each of FSHß-LUC and 10 µg of the plasmids as described in the legends, using the calcium phosphate method. The total amount of plasmid was adjusted to 20 µg with vector DNA for control. Cells were also cotransfected for 8 h with 5 µg HA-ERK2 and the examined plasmids (5 µg) using the calcium phosphate method. The total amount of plasmid was adjusted to 10 µg with vector DNA for control. Transfection efficiency (10–30%) was determined using Rous sarcoma virus-LUC for control. After the transfection (24–36 h), cells were serum starved (0.5% FCS, 16 h) and incubated with stimulants or inhibitors as described in the legends.

LUC and chloramphenicol acetyl transferase (CAT) assays
Cells were washed twice in buffer A and then 0.3 ml buffer H was added to each plate. The cells were harvested and transferred to microcentrifuge tubes followed by sonication (2 x 7 sec, 40 W) and centrifugation (15,000 x g, 15 min, 4 C). LUC activity was measured using an LKB luminometer (Wallac, Turku, Finland) by injecting reaction buffer containing 200 µM K-HEPS, 40 µM MgAc, 10 µM EDTA, 10 µg/ml BSA, 5 µM ATP, 2.7 µM Coenzyme A, and 4 µM luciferin. LUC activities were normalized to the level of TK-CAT activity (27). CAT activity was preformed as described (27). All transfection assays were performed at least three times.

Immunoprecipitation with anti-HA antibodies
For immunoprecipitation, protein A-Sepharose was mixed with HA antibodies at room temperature for 1 h, after which the beads were washed twice with PBS and twice with buffer H. Cell lysates (300 µg) were added to the beads and swirled end to end at 4 C for 2 h. The immunocomplexes were washed once with radioimmune precipitation buffer, washed twice with 0.5 M LiCl in 100 mM Tris (pH 8.0), and finally washed with buffer A.

Determination of EGFR phosphorylation
Cells were grown as described above to 75–85% confluency, the growth medium was replaced with the serum-free medium, and the cells were incubated for an additional 16 h. Then the cells were stimulated with GnRH-A or EGF in the presence or absence of AG1478, and EGFR phosphorylation was determined as recently described (24).

Fluorescent cell staining
Subconfluent cells were serum starved (16 h, 0.1% FCS) and treated with GnRH-A for various time points. Subsequently the cells were washed with PBS, fixed in 3% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 in PBS for 5 min. Staining was performed by 45 min incubation with polyclonal antigeneral ERK antibody (diluted 1:100), followed by 45 min incubation with rhodamine-conjugated goat-antirabbit IgG (diluted 1:100). Fluorescent staining was viewed with a fluorescence microscope (EFD-3, Nikon, Tokyo, Japan).

Statistical analysis
Statistical analysis was done between control and treatment groups using one-way ANOVA followed by a phosteriori Bonferroni t test, which simultaneously compares means of all examined groups. Three independent experiments were carried out and the results are presented as mean of the three experiments.

	Results

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ERK, JNK, and p38 activation by GnRH
Incubation of LßT-2 cells with GnRH-A resulted in a transient activation of ERK1 and 2 and a slower activation of JNK and p38 (Fig. 1A). MAPK activity was detected by Western blot using antibodies, which recognize the doubly phosphorylated, active forms of ERK, JNK, and p38 (23, 27). The peak of ERK activation was observed at 5 min, whereas that of JNK and p38 was detected at 30 min. Because ERK activation by GnRH was extensively studied in other cell types (7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 23, 24), we decided to further examine ERK activation by GnRH in LßT-2 cells.

View larger version (39K): [in this window] [in a new window]   FIG. 1. Effect of GnRH on ERK, JNK, and p38 activation in LßT-2 cells. A, Subconfluent LßT-2 cells were treated with GnRH-A (100 nM) for the indicated times and ERK, JNK, and p38 activities were determined by Western blotting with type-specific antibodies to the phosphorylated enzymes. Total MAPKs were detected with polyclonal antibodies as a control for sample loading. A representative blot is shown and similar results were observed in three other experiments. B, Subconfluent LßT-2 cells were pretreated (16 h) with pertussis toxin before stimulation with GnRH-A (100 nM) for 7 min and ERK activity was determined. C, Subconfluent LßT-2 cells were pretreated (15 min) with the PKC inhibitor GF109203 (GF) before stimulation with GnRH-A (100 nM) for 7 min, and ERK activity was determined. In this and subsequent figures, results from three experiments are shown as mean ± SEM. Means designated by different letters are significantly different for ERK1 and separately for ERK2 (P < 0.05).

Role of PKC in ERK activation by GnRH
As seen in Fig. 1C, GF109203X, a selective PKC inhibitor, produced a concentration-related inhibition of ERK activation by GnRH-A, with full inhibition at 2.5 µM. Incubation of the cells with the PKC activator TPA (100 ng/ml for 7 min) resulted in ERK activation, which was similar in its magnitude to the GnRH-A effect (Fig. 2A). Down-regulation of TPA-responsive PKC isoforms by prolonged treatment with TPA (100 ng/ml for 16 h) abolished ERK activation by GnRH-A (Fig. 2A). Combined treatment for 7 min with GnRH-A and TPA resulted in a small (20%) but significant enhancement of the separate response of both ligands. However, because the combined effect is less than additive, the data suggest that GnRH and TPA activate similar PKCs, whereas TPA is activating additional PKCs to those employed by GnRH.

View larger version (31K): [in this window] [in a new window]   FIG. 2. Role of PKC and Ca2+ in GnRH stimulation of ERK in LßT-2 cells A, Subconfluent LßT-2 cells were incubated with TPA (100 ng/ml), GnRH-A (100 nM), or both for 7 min. To remove endogenous PKC activity, cells were also down-regulated by incubation with TPA (100 ng/ml) for 16 h. Cells were then washed and treated with or without GnRH-A (100 nM) for 7 min, and ERK activity was determined. B, Subconfluent LßT-2 cells were preincubated with BAPTA-AM (50 µM) or EGTA (3 mM) for 15 min and later stimulated with or without GnRH-A (100 nM) or ionomycin (100 nM) for 7 min, and ERK activity was determined.

Role of protein tyrosine kinases (PTKs) in ERK activation by GnRH
To gain further insight into the mechanism of ERK activation by GnRH in LßT-2 cells, we used general and selective receptor tyrosine kinase and tyrosine kinase inhibitors. Because we previously implicated c-Src in MAPK activation by GnRH in T3–1 cells (13, 23), we examined here the effect of the selective c-Src inhibitor PP1. Indeed, PP1 partially inhibited ERK activation by GnRH-A (24 and 45% for ERK1 and ERK2, respectively; Fig. 3A). The general PTK inhibitor genistein also produced a partial (46%) inhibition, which was similar in its magnitude to that obtained with PP1, suggesting that c-Src might be the main tyrosine kinase involved in GnRH action. On the other hand, the selective MEK and PKC inhibitors PD98059 and GF109203X abolished GnRH-A-stimulated ERK activation, with PD98059 also affecting basal levels.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Effect of tyrosine kinase inhibitors on GnRH stimulation of ERK. A, Subconfluent LßT-2 cells were pretreated (15 min) with 2 µM PPI, 200 µM genistein (Gen), 2 µM AG1478 (AG), 25 µM PD98059 (PD), or 2 µM GF109203 (GF) before stimulation with GnRH-A (100 ng/ml) for 7 min, and ERK activity was determined. B, Subconfluent LßT-2 cells were pretreated (15 min) with 2 µM AG1478 before stimulation with or without GnRH-A (100 ng/ml) or EGF (100 ng/ml) for 7 min. EGFR phosphorylation and ERK activity were then determined as in Materials and Methods.

To further validate our findings concerning the transact-ivation of the EGFR and other signaling molecules, we used the approach of coexpression. We coexpressed HA-ERK and a DN plasmid of the EGFR (K721A) or other plasmids as below. We then stimulated the cells with or without GnRH-A, lysed the cells, immunoprecipitated HA-ERK with anti-HA antibodies, and blotted with anti-diphospho-ERK antibodies. As seen in Fig. 4A, the DN-EGFR produced only a small inhibition (18%) of HA-ERK activation by GnRH-A, which was similar in its magnitude to the inhibition obtained with AG1478 (Fig 3).

View larger version (35K): [in this window] [in a new window]   FIG. 4. Role of signaling molecules in GnRH stimulation of ERK. Subconfluent LßT-2 cells were cotransfected with HA-ERK2 and a DN plasmid of the EGFR (K721A); CD8-ARK-C, a plasmid of a chimera that acts as a scavenger of the Gß dimer (A); or a DN form of ß-arrestin (V54D-ß-arrestin2), dynamin (K44A), or with the DN form of c-Src (CSK) (B). Two days later, the cells were serum starved for 16 h and stimulated with or without GnRH-A (100 nM) for 7 min. We then lysed the cells, immunoprecipitated HA-ERK2 with anti-HA antibodies, and blotted with anti-diphospho-ERK antibodies as above.

Involvement of dynamin and c-Src but not ß-arrestin or Gß in ERK activation by GnRH

ß-Arrestin and dynamin, which are known to be involved in GPCR internalization and desensitization, have been implicated recently in the G-independent GPCR to ERK signaling (see Refs. 18 , 38 for reviews). ß-Arrestin is thought to act as a scaffold protein for some of the ERK cascade members (38). Because the GnRHR lacks the C-terminal carboxy tail and does not undergo rapid desensitization (11), it is interesting to evaluate the role of ß-arrestin and dynamin in GnRH to ERK signaling. We used a DN form of ß-arrestin (V54D-ß-arrestin2), which was shown to inhibit the activity of all endogenous ß-arrestins (41) and a DN form of dynamin (K44A, 42). We used coexpression of V54D-ß-arrestin2 or K44A-dynamin with HA-ERK and measured GnRH-A-stimulated ERK. As shown in Fig. 4B, the DN of dynamin exerted a partial inhibition (38%), whereas the DN of ß-arrestin had no effect. Similarly, the DN form of c-Src (CSK) exerted a partial inhibition of the GnRH effect, similar to the inhibition obtained with PP1 in Fig. 3A.

Taken together the results suggest that GnRHR signaling to ERK in LßT-2 cells is mainly mediated by PKC and to a lesser extent also by a PTK, which seems to be c-Src. Ca²⁺ influx is partially required for the GnRH response, with ERK1 being more sensitive to Ca²⁺ removal than ERK2. The results also suggest that ERK activation by GnRH does not involve transactivation of EGFR or mediation via Gß or ß-arrestin, unlike several other GPCRs (35, 36, 37, 38, 41, 42, 43).

Effect of GnRH-A on ERK translocation
To study the effect of GnRH-A on the subcellular localization of ERK, cells were treated with GnRH-A and later stained with the general polyclonal anti-ERK antibody (Fig. 5). The staining of nonstimulated cells with this antibody was detected primarily in the cytosol. GnRH-A treatment induced translocation of ERK to the nucleus, which was detected after 30 min and continued progressively to reach a peak at 120 min, in which all cells examined were stained. Thus, although ERK activation reached a peak at 5 min, declining thereafter (Fig. 1A), ERK translocation to the nucleus proceeded for at least 120 min.

View larger version (50K): [in this window] [in a new window]   FIG. 5. Effect of GnRH on ERK translocation in LßT-2 cells. Subconfluent LßT-2 cells were serum starved (16 h, 0.1% FCS) and treated in the absence or presence of GnRH-A (100 nM). After stimulation, the cells were washed, fixed, permeabilized, and then stained with polyclonal antigeneral ERK antibody (1:100) followed by rhodamine-conjugated secondary antibody (1:100) as in Materials and Methods. Control staining was detected primarily in the cytosol, whereas nuclear staining was detected progressively within 30–120 min of GnRH-stimulation. A representative experiment is shown.

View larger version (13K): [in this window] [in a new window]   FIG. 6. Effect of GnRH on FSHß-LUC activity. Subconfluent LßT-2 cells were transfected with 5 µg oFSHß-LUC for 8 h and 3 µg TK-CAT plasmid as an internal control. The transfected cells were washed several times and incubated for 24 h. GnRH-A was then added in different doses for 8 h (A) or for the indicated times (B, GnRH-A, 10 nM). In this and subsequent figures, we also included activin A (25 ng/ml) in all treatment groups for 8 h. FSHß-LUC activity was determined as described in Materials and Methods. Results for promoter activity in this and subsequent figures are expressed as fold increase relative to untreated controls and represent the average of three independent experiments, each performed in duplicate. Results are shown as mean ± SEM. *, P < 0.05; **, P < 0.02; ***, P < 0.001.

View larger version (14K): [in this window] [in a new window]   FIG. 7. Role of PKC in GnRH stimulation of oFSHß-LUC promoter activity. Subconfluent LßT-2 cells were transfected with 5 µg oFSHß-LUC and 3 µg TK-CAT for 8 h. The transfected cells were washed and incubated for 24 h. A, The cells were incubated with different doses of TPA for 8 h. B, To remove PKC activity, cells were down-regulated by including TPA (100 ng/ml) during the 24 h of incubation (D-reg) or left untreated. GnRH-A (100 nM) or TPA (100 ng/ml) was then added to untreated or down-regulated cells for 8 h, and promoter activity was determined.

View larger version (14K): [in this window] [in a new window]   FIG. 8. Role of Ca2+ in GnRH stimulation of oFSHß-LUC promoter activity. Subconfluent LßT-2 cells were transfected with 5 µg oFSHß-LUC and 3 µg TK-CAT for 8 h. The transfected cells were washed several times and incubated for 24 h. A, The cells were incubated with ionomycin in different doses for 8 h. B, The transfected cells were transferred to normal DMEM (+), Ca2+-free DMEM (–), or Ca2+-free DMEM also containing EGTA (0.25 M) and incubated with or without GnRH-A (100 nM) for 8 h. oFSHß-LUC activity was determined as described in Materials and Methods.

View larger version (14K): [in this window] [in a new window]   FIG. 9. Role of ERK in GnRH stimulation oFSHß-LUC promoter activity. Subconfluent LßT-2 cells were transfected with 5 µg oFSHß-LUC, 3 µg TK-CAT, and with or without: constitutively active (C.A) forms of Raf-1 or MEK (A) or the DN plasmids (D.N) of Ras, Raf-1, or MEK (B) for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (100 nM) was then added in the presence (C) or absence (A, B) of the MEK inhibitor PD98059 (25 µM) for 8 h, and oFSHß-LUC activity was determined.

View larger version (27K): [in this window] [in a new window]   FIG. 10. Activation and role of JNK in GnRH stimulation of oFSHß promoter activity. Subconfluent LßT-2 cells were incubated with GnRH-A (100 nM), TPA (100 ng/ml), or ionomycin (100 nM) for the time indicated, and JNK activity was determined as above (A). Subconfluent LßT-2 cells were transfected with 5 µg oFSHß-LUC, 3 µg TK-CAT, and with or without the DN plasmids of cDC42 and JNK for 8 h. Cells were washed and incubated for 36 h. GnRH-A (100 nM) or TPA (100 ng/ml) was then added for 8 h, and oFSHß-LUC activity was determined (B).

View larger version (31K): [in this window] [in a new window]   FIG. 11. Activation and role of p38 in GnRH stimulation of oFSHß promoter activity. A, Subconfluent LßT-2 cells were incubated with GnRH-A (100 nM), TPA (100 ng/ml), or ionomycin (100 nM), with or without the p38 inhibitor SB203580 (25 µM) for 30 min. p38 activity was determined by Western blotting as above. B, Subconfluent LßT-2 cells were transfected with 5 µg oFSHß-LUC and 3 µg TK-CAT plasmid for 8 h. The transfected cells were washed several times and incubated for 24 h. GnRH-A (100 nM) was then added for 8 h in the presence and absence of SB203580 (5–25 µM). oFSHß-LUC activity was determined as above.

View larger version (27K): [in this window] [in a new window]   FIG. 12. Activation and role of c-Src in GnRH stimulation of oFSHß promoter activity. A, Subconfluent LßT-2 cells were incubated with GnRH-A (100 nM), TPA (100 ng/ml), or ionomycin (100 nM) for the time indicated, and c-Src activity was determined by Western blotting with antiphospho-c-Src (Tyr-418) and anti-ERK as control for loading. B, Subconfluent LßT-2 cells were transfected with 5 µg oFSHß-LUC and 3 µg TK-CAT for 8 h. The transfected cells were washed several times and incubated for 24 h. GnRH-A (100 nM) was then added for 8 h in the presence and absence of the c-Src inhibitor PP1 (2 µM), and oFSHß-LUC activity was determined.

View larger version (13K): [in this window] [in a new window]   FIG. 13. Minimal role for AP-1 in GnRH stimulation oFSHß promoter activity. Subconfluent LßT-2 cells were transfected with 5 µg oFSHß-LUC or a mutated promoter, AP-1oFSHß-LUC, and 3 µg TK-CAT for 8 h. The transfected cells were washed and incubated for 24 h. GnRH-A (100 nM) was then added for 8 h in the presence and absence of activin A (25 ng/ml). Promoter activity was determined as described in Materials and Methods.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Note Added in Proof References

Binding of GnRH to its receptor (GnRHR) is followed by enhanced phosphoinositide turnover resulting in Ca²⁺ mobilization and influx, activation of PKC, in particular PKC and PKC (51, 52), activation of MAPK (ERK, JNK, and p38), and activation of phospholipase A₂ and phospholipase D (see Refs. 5 , 6 , 11 , 12 , 18, 19, 20 for reviews). Still, although relatively much is known about GnRH regulation of gonadotropin release (5, 6, 11), little is known about the signaling of GnRHR during gonadotropin biosynthesis (2, 3, 4). Signaling downstream of PKC was poorly understood until it was recognized that GPCRs like receptor tyrosine kinases are also using the MAPK cascades (18, 21). Because MAPKs translocate to the nucleus and activate transcription, it is an ideal messenger for activation of gene expression by GPCRs. The recent availability of gonadotroph cell lines expressing GnRHR, the common -subunit (T3–1) and LHß/FSHß (LßT-2) (53, 54), facilitated the studies directed to elucidate the molecular steps involved in GnRH regulation of gonadotropin synthesis. Indeed, GnRH activates MAPK (ERK, JNK, and p38) in T3–1 (7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 28) and LßT-2 cells (22, 27, 32, 34, 55 and present results).

Although MAPK is implicated in transcriptional control (18, 21), its role in GnRH-induced gonadotropin subunit gene expression is controversial. It was 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⁺ (25). Others reported opposite results, namely that Ca²⁺ mediates the GnRH effect on -subunit expression and PKC (31), or PKC and ERK mediate the effect on LHß (26). On the other hand, Yokoi et al. (22) found a role for JNK but not for PKC and ERK in regulation of LHß gene expression by GnRH. Still we have recently reported a role for PKC, ERK, and JNK in GnRH regulation of LHß gene expression (27). Some of the opposing results can be explained by different cell types, promoters, species, and growing conditions used. Very little is known about FSHß gene expression due to the absence, until recently, of a suitable cell line (32, 53, 54, 55). PKC has been implicated in GnRH regulation of FSHß gene expression (30, 31, 32), whereas the MEK inhibitors PD98059 and UO126 were reported to inhibit GnRH-stimulated FSHß mRNA and promoter activity (32, 33).

We report here a partial role for Gi but not for Gß in ERK activation by GnRH. The results seem to contradict a recent report in which Gq and Gs, but not Gi, were implicated in GnRH action (34). The discrepancy can be explained by the fact that the inhibition by pertussis toxin was mainly exerted on ERK1 but not ERK2, which is the major phospho-ERK in the cells, whereas Liu et al. (34) did not separate the ERKs. Our results suggest the interesting possibility that the two ERKs are differentially controlled. GnRHR signaling to ERK in LßT-2 cells is also partially mediated by a PTK, which seems to be c-Src acting downstream to Ca²⁺ and PKC because c-Src was also activated by ionomycin and TPA. However, ERK activation by GnRH does not involve transactivation of EGFR or mediation via ß-arrestin as in several other GPCRs (35, 36, 37, 38, 41, 42, 43). It was recently reported (24, 56) that activation of ERK by GnRH in GnRHR-transfected COS-7, and the hypothalamic GT1–7 cells is mediated by transactivation of the EGFR. Our findings in LßT-2 (present results) and T3–1 cells (23), that ERK activation by GnRH does not involve transactivation of the EGFR, suggest that activation of ERK by GnRH varies in the different cellular systems examined.

We also found that activation of ERK by GnRH in LßT-2 cells is dependent on PKC as evident by similar activation by TPA, inhibition of GnRH to ERK signaling by a selective inhibitor, GF109203X and down-regulation of PKC. Also, activation of ERK by GnRH is dependent on Ca²⁺ influx, but not Ca²⁺mobilization, as evident by inhibition of the GnRH-A effect by EGTA but not by BAPTA-AM. Interestingly, we previously reported that GnRH activates PKC and PKC (52), and PKC is now implicated in ERK activation by forming a signaling module with c-N-Ras and c-Raf (57). The observation that GnRH and TPA induced similar activation of the FSHß promoter activity in LßT-2 cells (about 3-fold) argues for a role for PKC in the transcriptional control of the FSHß gene. Nevertheless, down-regulation of PKCs abolished GnRH stimulation of ERK but only partially (40%) reduced GnRH-induced FSHß promoter activity. It is therefore possible that GnRH activates the FSHß promoter by additional PKC-independent mechanisms. Interestingly, down-regulation of PKCs abolished GnRH stimulation of LHß promoter activity but had only a minor inhibitory effect on GnRH-induced common -promoter activity (27, 28). We therefore suggest that PKC differentially regulates gonadotropin subunit genes: LHß>FSHß>common during the selective activation of LH and FSH by GnRH. Addition of ionomycin activated ERK but had no effect on FSHß promoter activity. It is therefore possible that elevation of Ca²⁺ by the ionophore also may have a negative effect on promoter activation, which will neutralize the effect of ERK. Also, addition of ionomycin or removal of Ca²⁺ had no effect on GnRH-induced FSHß (present results) and LHß promoter activities (27), but removal of Ca²⁺ abolished GnRH-induced -subunit gene expression (28). Similar differential Ca²⁺ requirements were also noticed at the mRNA levels (30). Hence, Ca²⁺, like PKC, may participate in mediating differential activation of gonadotropin subunit genes by GnRH. Because elevation of Ca²⁺ is required for the exocytotic response to GnRH (5, 6), activation of the FSHß and LHß promoter activities by GnRH in a Ca²⁺-independent manner suggests dissociation between gonadotropin release and synthesis and that exocytosis is not a prerequisite for LHß and FSHß gene expression as observed also for common -subunit (52).

ERK activation by GnRH can be blocked by the MEK inhibitor PD98059, and similar inhibition of GnRH-A-stimulated FSHßLUC activity was exerted by the drug. Interestingly, removal of extracellular Ca²⁺ reduced the ERK response to GnRH-A (50%) but had no effect on GnRH-A-stimulated FSHß-LUC activity. The data suggest that ERK contributes about 50% to FSHß promoter activation by GnRH. JNK and p38 contribute also to the GnRH response but to a lesser degree. Although the selective c-Src inhibitor PPI and CSK produced only a partial inhibition of GnRH to ERK signaling (30%), still PP1 abolished the stimulatory effect of GnRH on promoter activity. GnRH activates c-Src and c-Src is required for ERK activation by GnRH (13, 23). Thus, c-Src contributes to FSHß transcription via an ERK-dependent and -independent mechanism. Hence, common and separate GnRH enhancer regions on the FSHß promoter are activated by ERK and c-Src.

In all species FSHß gene expression is regulated by GnRH and members of the TGFß superfamily, which are also regulated by GnRH (58). Of those, activins and bone morphogenic proteins-6 and -7 are the most active, even more than GnRH, and are neutralized by follistatin (58). Still, GnRH stimulates a rapid (5 min, 4-fold) induction of FSHß nuclear primary transcripts in castrated testosterone-replaced male rats (59). Similarly, GnRH stimulates a rapid (30 min, 2.2-fold) elevation of FSHß mRNA levels in primary cultures of male rat pituitary cells (50). Nevertheless, perifusion of rat pituitary cell cultures with activin A increased FSHß mRNA levels by 25-fold and GnRH augmented the response by 2-fold (60). This and other studies led Miller et al. (58) to suggest that GnRH is not the prime regulator of FSHß gene expression and that GnRH controls only approximately 50–67% of overall FSHß mRNA levels.

Two AP-1-like enhancers located at –120 and –83 bp of the oFSHß promoter were found to confer a 2- to 3-fold transcriptional response to GnRH in transfected HeLa cells (45). Surprisingly, a mutant oFSHß promoter, which lacked functional AP-1 sites, responded to GnRH in transgenic mouse lines as in those harboring the wild-type oFSHßLuc promoter (45, 46). Unlike the in vivo data, the two AP-1 sites were crucial for activation by GnRH in pituitary cultures from the above transgenic mouse models (46). In contrast, a recent observation and the present results show that the mutant oFSHß promoter (lacking two functional AP-1 sites) responded to GnRH in LßT-2 cells (Ref. 32 and present results). Nevertheless, mutation of an AP-1 site, overlapping the basal transcription factor nuclear factor Y (NFY) binding site in the mouse proximal FSHß promoter, resulted in a 30% decrease in GnRH induction (61). Also, it was recently demonstrated that activation of the oFSHß promoter in LßT-2 cells involves two elements residing at –4152/–2878 and –2550/–1089 in association with elements within the proximal region of the promoter, apparently the AP-1 sites (32). It is likely that the various elements identified cooperate during promoter activation, and the relative contribution of the AP-1 sites might change in the various models described above and hence the conflicting data.

The mouse proximal FSHß promoter contains two potential binding sites for steroidogenic factor-1 (SF-1) and an overlapping binding site for the ubiquitously expressed basal transcription factor NFY and the induced factor AP-1 (62). SF-1 and NFY seem to act in concert to regulate basal FSHß gene expression specifically in LßT-2 cells (62). SF-1 and the early growth response protein Egr-1 were shown to participate in LHß gene expression, both acting in a synergistic manner (63). Indeed, Egr-1 expression is increased by GnRH in a PKC-dependent manner and is known to be modulated by MAPK (63). In addition, the pituitary homeobox 1 transcription factor, which is known to participate in pituitary organogenesis, was shown to regulate the basal expression of the rat FSHß promoter (64). We have anticipated that JNK, p38, and ERK will operate via the activation of c-Jun and c-Fos acting on AP-1 sites. However, the AP-1 sites in the ovine FSHß promoter were required only in two in vitro systems but not in vivo or in LßT-2 cells (32, 45, 46, 58), whereas in the mouse FSHß promoter, the AP-1 sites contributed about 30% to the GnRH response (61). We therefore propose that MAPK and c-Src are acting on additional response elements within the FSHß promoter.

	Note Added in Proof

Top Abstract Introduction Materials and Methods Results Discussion Note Added in Proof References

 
Although we found a minimal role (22%) for the two proximal AP-1 sites (–120 and –83 bp) in the GnRH response on the oFSHß promoter, a recent publication (65) identified a novel AP-1 half-site (–72/–69) juxtaposed to a CCAAT box, which binds nuclear factor-Y in the mouse FSHß promoter. ERK was shown to be involved in GnRH induction of the mouse FSHß gene, in part through the induction of JunB and c-Fos. Still, this novel AP-1 site contributes only 35% to the GnRH response, supporting our conclusion that MAPK is acting on additional response elelments within the FSHß promoter.

	Acknowledgments

 
We thank Drs. O. Benard and R. Millar for their interest and help during the study. We also thank Drs. P. Mellon for the LßT-2 cells and W. Miller for the FSHß-LUC and the AP-1oFSHßLUC promoters.

	Footnotes

 
This work was supported by the Adams Super Center for Brain Studies at Tel Aviv University.

Abbreviations: AP, Activator protein; ARK-C, C terminus of the ß-adrenergic receptor kinase; BAPTA-AM, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester; CAT, chloramphenicol acetyl transferase; CSK, DN form of c-Src; DN, dominant negative; EGF, epidermal growth factor; EGFR, EGF receptor; FCS, fetal calf serum; GnRH-A, GnRH agonist; GnRHR, GnRH receptor; GPCR, G protein-coupled receptor; HA, hemagglutinin; HA-ERK2, HA epitope-tagged ERK; JNK, Jun N-terminal kinase; LUC, luciferase; MEK, MAPK kinase; MEKK, MEK kinase; NFY, nuclear factor Y; o, ovine; PKC, protein kinase C; PTK, protein tyrosine kinase; SF-1, steroidogenic factor-1; TPA, 12-O-tetradecanoylphorbol-13-acetate.

Received October 22, 2003.

Accepted for publication January 14, 2004.

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