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Biology of Reproduction 65, 561-567 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Cyclic Adenosine 3',5'-Monophosphate (cAMP) and cAMP Responsive Element-Binding Protein Are Involved in the Transcriptional Regulation of Gonadotropin-Releasing Hormone (GnRH) Receptor by GnRH and Mitogen-Activated Protein Kinase Signal Transduction Pathway in GGH3 Cells1

Guadalupe Maya-Núñeza, and P. Michael Conna

a Oregon Regional Primate Research Center and Department of Physiology and Pharmacology, Oregon Health and Sciences University, Portland, Oregon 97201

ABSTRACT

Stimulation of mouse GnRH receptor promoter by a GnRH agonist (Buserelin), or by a cAMP analogue, significantly increased reporter (luciferase) activity. Overexpression of Raf-1, ERK1, or ERK2 partially blocked Buserelin-stimulated luciferase activity. In contrast, treatment with a mitogen-activated protein kinase (MAPK) kinase inhibitor (PD 98059) activated basal and Buserelin-stimulated luciferase activity in a dose-dependent manner. Transient transfection of the deleted cAMP response element expression vector followed by pretreatment with PD98059 prior to Buserelin stimulation showed that the transcriptional response was decreased compared to wild-type promoter. A gel-mobility shift assay using a probe containing the cAMP response element showed the presence of two specific protein-DNA complexes that contain one or more members of the cAMP responsive element-binding (CREB) protein family. These results suggest that cAMP and CREB participate in the GnRH activation of GnRH receptor promoter activity and that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRH receptor transcriptional activity.

cyclic adenosine monophosphate, gonadotropin-releasing hormone receptor, neuroendocrinology, neuropeptides, signal transduction

INTRODUCTION

GnRH is synthesized in the hypothalamus, binds to a specific pituitary receptor, and then stimulates the synthesis and release of the gonadotropins LH and FSH [1]. The GnRH receptor (GnRHR) is a member of the G protein-coupled receptor superfamily [2]. Agonist occupancy of GnRHR rapidly stimulates an increase in phosphoinositide turnover and a rise in intracellular diacylglycerol levels, which results to activation of protein kinase C (PKC) [3–6]. GnRHR also stimulates increases in intracellular Ca2+ [6] and cAMP levels [7, 8], suggesting that multiple signal transduction pathways may mediate GnRH actions. In rat pituitary GGH3 cells (a GH3-derived cell line stably expressing rat GnRHR) [9], GnRHR is coupled to Gq/11, resulting in activation of phospholipase C and inositol phospholipid (IP) turnover [6, 10]. In addition, GnRHR appears to be coupled to Gs, which activates adenylate cyclase, leading to production of cAMP and activation of protein kinase A (PKA) [8, 10–12]. Recent studies relying on palmitoylation of G-proteins and overexpression of G-protein {alpha} subunit cDNAs showed that GnRHR was able to couple Gq/11 to Gs and Gi in pituitary gonadotropes and GGH3 cells [5, 13], suggesting that similar signal transduction pathways are employed to mediate GnRH action in GGH3 cells and in pituitary cells, and emphasizing the promiscuity of GnRHR as a function of the G-protein microenvironment in the target cells [6].

It has been shown that GnRH is capable of activating mitogen-activated protein kinase (MAPK) in primary pituitary cells as well as in pituitary-derived cell lines, including GH3 and {alpha}T3-1 cells [14–16]. In {alpha}T3-1 cells, ERK activation in response to GnRH is mediated by PKC and Ca2+, which seem to act in a Ras-independent manner [17]. Recently, it has been shown that GnRH also activates the Jun N-terminal kinase (JNK, an ERK-like cascade) signaling cascade through PKC, cSrc, and CDC42, and acts independently of the ERK cascade in {alpha}T3-1 cells [18]. In GGH3 cells MAPK activation occurs through both PKA and PKC signal transduction pathways [19]; GnRH also activates the MAPK cascade, which is involved in regulation of gene expression of the gonadotropin {alpha}-subunit [20–22].

The number of pituitary GnRH receptors (GnRHRs) [6] and the levels of GnRHR mRNA [23, 24] change during the estrous cycle and are associated with changes in the sensitivity of gonadotropes to GnRH and levels of serum gonadotropins. This suggests that GnRHR is an important site for the regulation of gonadotropin release, and that GnRH is involved in the regulation of its own receptor [6]; however, the cellular mechanism or mechanisms by which GnRH regulates transcription of this and other genes remains to be elucidated. In addition, previous studies in GGH3 cells showed that GnRH activates the transcriptional activity in part through the cAMP signal transduction pathway [25, 26], and that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRHR promoter activity in GGH3 cells [27]. In the present study we explored the possible involvement of the cAMP signal transduction pathway in the negative regulation of the GnRHR gene by MAPK cascade in the GGH31' cell line.

MATERIALS AND METHODS

Materials

Natural sequence GnRH was provided by the National Pituitary Agency (NIDDK, NIH, Bethesda, MD). A GnRH agonist, Buserelin (D-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH) was a kind gift from Hoeschst-Roussel Pharmaceuticals (Somerville, NJ). PD 98059 (2'-amino-3'-methoxyflavonate) and an anti-CREB polyclonal antibody were from Calbiochem (La Jolla, CA); dBcAMP and forskolin were from Sigma (St. Louis, MO). A rabbit anti-c-Fos polyclonal antibody was produced in our laboratory as described previously [5]. Dulbecco modified Eagle medium (DMEM), OPTI-MEM, lipofectamine, and polymerase chain reaction (PCR) reagents were purchased from Life Technologies (Grand Island, NY). Restriction enzymes, modified enzymes, and competent cells for subcloning were purchased from Promega (Madison, WI). Other reagents were of the highest degree of purity available from commercial sources.

Vector Construction

Luciferase reporter gene vector (GnRHR-pXP2) with a 1226-base pair (bp) promoter fragment (-1164 to +62 relative to the major transcriptional start site) of mouse GnRHR gene and an expression vector (pCIS-LacZ) expressing ß-galactosidase driven by cytomegalovirus (CMV) promoter were provided by Dr. W.W. Chin (Harvard Medical School, Boston, MA) and Dr. T.H. Ji (University of Wyoming, Laramie, WY), respectively. The construction of the promotorless pXP2 vector and the GnRHR-pXP2 without the cAMP response element (GnRHR-{Delta}CRE) have been described [25, 26]. Expression vectors for human ERK1 and ERK2 were generously provided by Dr. M. Cobb (University of Texas, Dallas, TX). Wild-type human Raf-1 cDNA in pUSE was subcloned into pcDNA3.1 at XhoI and KpnI restriction enzymes sites. An empty expression vector, pcDNA3.1, was obtained from Invitrogen (Carlsbad, CA) and used as a control plasmid.

Transient Transfection of GGH31' Cells

GnRHR-pXP2 reporter gene vector or control vector pXP2 were transiently expressed in GGH31' cells [9]. GGH31' cells were maintained in growth medium (DMEM containing 10% fetal calf serum [FCS; Hyclone Laboratories, Logan, UT] and 20 µg/ml gentamicin [Gemini Bioproducts, Calabasas, CA]) in a humidified atmosphere (37°C) containing 5% CO2. Cells (5 x 105 cells/well) were seeded in 6-well plates (Costar, Cambridge, MA). Twenty-four h after plating, the cells were transfected with 1.5 µg GnRHR-pXP2 or pXP2 plus 0.5 µg of pCIS-LacZ per well using 5 µl of lipofectamine in 1 ml of OPTI-MEM. Five h later, 1 ml of DMEM containing 20% FCS was added to each well. Twenty-four h after the start of transfection, the medium was replaced with fresh growth medium, and the cells were allowed to grow for another 24 h before treatment and functional assays (luciferase assay and ß-galactosidase assay) were performed.

Luciferase and ß-Galactosidase Assays

After treatment of transiently transfected GGH31' cells with GnRH agonist or other compounds for the indicated times, the cells were washed twice with PBS and lysed in 150 µl of Reporter Lysis Buffer (Promega). Luciferase activity in 20 µl of the cell lysate was determined using the Luciferase Assay System (Promega) in a LuciCount microplate luminometer (Packard, Meriden, CT). ß-Galactosidase activity in 30 µl of the cell lysate was also measured using the ß-Galactosidase Enzyme Assay System (Promega) in a SpectraCount photometric microplate counter (Packard) and was used as an internal control. The luciferase activity was normalized for transfection efficiency of each well by dividing the luciferase activity by ß-galactosidase activity.

Gel-Mobility Shift Assay

The oligonucleotides containing the region -55/-30 bp in the GnRHR-pXP2 (forward, 5'-CTAACCTGTGACGTTTCCATCTAAAG-3'; reverse, 5'-CTTTAGATGGAAACGTCACAGGTTAG-3') were chemically synthesized. These oligonucleotides were 3'-end labeled with digoxigenin-11-ddUTP using terminal transferase in the presence of Co2+ according to the manufacturer's instructions (Boehringer-Mannheim, Indianapolis, IN). Labeled oligonucleotides were purified by precipitation with ethanol and lithium chloride.

Nuclear extracts were prepared from GGH31' cells as reported previously [28]. The standard binding assay (20 µl) contained 5 µg of nuclear protein extract, 1 µg of poly[d(I-C)], 2 µg of poly[d(A-T)] and 0.5 pmol digoxigenin-labeled oligonucleotide in 10 mM Hepes (pH 7.6), 1 mM EDTA, 10 mM (NH4)2SO4, 1 mM dithiothreitol, 0.2% Tween 20 (v/v), and 30 mM KCl. The binding assay mixture was incubated at room temperature for 30 min, the samples were separated in 5% polyacrylamide gel (acrylamide:bisacrylamide 29:1) containing 0.25x TBE (Tris-borate-EDTA, 10x concentrate: 890 mM Tris, 890 mM boric acid, and 20 mM EDTA, pH 8.0). Prior to loading the samples, the gel was prerun for 1.5 h at 100 V and 4°C. Electrophoresis was then carried out at 100 V until the bromophenol blue reached the bottom of the gel. The probe was electroblotted onto a positively charged nylon membrane (Boehringer-Mannheim) in 0.25x TBE using a Hoefer Transphor (Hoefer Scientific Instruments, San Francisco, CA) at 4 mA/cm2 at 4°C for 30 min, then fixed to the membrane by heating at 80°C for 2 h. Detection of digoxigenin-labeled probe was carried out by the chemiluminescent detection system using the Boehringer-Mannheim kit according to the instruction manual. Imaging of the chemiluminescence was performed on Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY) by exposure for 1–4 h. Competition reactions were performed by preincubating the reactions with the specified amount of excess, unlabeled oligonucleotides before the addition of probe. Antibody experiments were performed by adding anti-CREB or anti-c-Fos antibodies to the nuclear extract, and incubating at room temperature for 15 min before adding labeled oligonucleotide probe and incubated at room temperature for an additional 30 min prior to the gel electrophoresis.

Data Analysis

Data shown are the means of triplicate assay wells and are presented as the means ± SEM of replicates in each experiment. The SEM was typically less than 10% of the mean. The data were analyzed by one-way ANOVA followed by Duncan multiple range test, P < 0.05 being considered significant. Each experiment was repeated three or more times to ensure the reproducibility of the findings.

RESULTS

Activation of GnRHR Gene Transcriptional Activity by GnRH and cAMP

The transcriptional activity of the GnRHR promoter in the transiently transfected GGH31' cells was assessed by expression of GnRHR-pXP2, a luciferase reporter gene vector containing a 1.2-kilobase (kb) promoter fragment (-1164/+62) of the mouse GnRHR gene.

To examine GnRH regulation of the GnRHR gene transcriptional activity, GGH31' cells transfected with GnRHR-pXP2 + pCIS-LacZ or pXP2 + pCIS-LacZ were treated with medium alone, a GnRH agonist (10-7 M Buserelin) for 3 or 6 h, respectively, before harvesting. The cells were harvested and luciferase and ß-galactosidase reporter activities were measured. Buserelin significantly increased luciferase activity at both 3 and 6 h, with higher stimulation at 6 h (Table 1). In addition, GGH31' cells transfected with GnRHR-pXP2 plus pCIS-LacZ or with pXP2 plus pCIS-LacZ were treated with medium alone, 0.1 µM Buserelin, 10 µM forskolin, or 5 mM dBcAMP for 6 h before harvesting. Buserelin stimulation resulted in a significant increase in luciferase activity (Fig. 1). Luciferase activity was also stimulated by dBcAMP and forskolin; however, the responses with these effectors were lower than that observed with Buserelin stimulation (Fig. 1).


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  		TABLE 1. Activation of GnRHR gene transcriptional activity by GnRH.*



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  		FIG. 1. Effect of cAMP on the GnRHR gene transcriptional activity by GnRH. Forty-eight h after transfection of GGH31' cells with GnRHR-pXP2 + pCIS-LacZ or with pXP2 + pCIS-LacZ, the cells were treated with medium alone, 0.1 µM Buserelin, 5 mM dBcAMP, or 10 µM forskolin for 6 h before harvesting. The cells were then lysed, and luciferase and ß-galactosidase reporter activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as fold induction as a percentage over that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences at P < 0.05 from values in the immediately adjacent groups are designated by different lowercase letters above the bars

Negative Regulation of the MAPK Pathway in GnRH Activation of GnRHR Gene Transcriptional Activity

GGH31' cells were cotransfected with GnRHR-pXP2 or pXP2 and Raf-1, ERK1, or ERK2, and were then treated with medium alone or 10-7 M Buserelin for 6 h before luciferase and ß-galactosidase reporter activities were measured. Overexpression of Raf-1 partially blocked Buserelin-stimulated luciferase activity, whereas overexpression of ERK1 or ERK2 partially blocked basal and Buserelin-stimulated luciferase activity (Fig. 2).



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  		FIG. 2. Effect of overexpression of Raf-1, ERK1, or ERK2 on activation of GnRHR transcriptional activity by GnRH. GGH31' cells were cotransfected with GnRHR-pXP2 or pXP2 and control vector (pcDNA3.1, 1 µg/well), Raf-1 (1 µg/well), ERK1 (1 µg/well), or ERK2 (1 µg/well), and treated with medium alone or 10-7 M Buserelin for 6 h before harvesting. The cells were then lysed, and luciferase and ß-galactosidase reporter activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as fold induction as a percentage over that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences at P < 0.05 from values in the immediately adjacent groups are designated by different lowercase letters above the bars

PD 98059 selectively blocks the activity of MEK by inhibiting the activation of MAPK and subsequent phosphorylation of MAPK substrates both in vitro and in intact cells [29, 30]. GGH31' cells transfected with GnRHR-pXP2 + pCIS-LacZ or with pXP2 + pCIS-LacZ were pretreated with different concentrations of PD 98059 (0, 0.1, 1, 10, or 100 µM) for 1 h prior to stimulation with medium alone or 10-7 M Buserelin for 6 h before harvesting. Pretreatment with PD 98059 significantly stimulated basal luciferase activity in a dose-dependent manner (Fig. 3).



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  		FIG. 3. Effect of MAPK kinase inhibitor on activation of GnRHR transcriptional activity by GnRH. GGH31' cells transfected with GnRHR-pXP2 plus pCIS-LacZ or with pXP2 plus pCIS-LacZ were pretreated with different concentrations of PD 98059 (0, 0.1, 1, 10, or 100 µM) for 1 h prior to stimulation with medium alone or 10-7 M Buserelin for 6 h before harvesting. The cells were then lysed, and luciferase and ß-galactosidase reporter activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as fold induction as a percentage over that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences at P < 0.05 from values in the immediately adjacent groups are designated by different lowercase letters above the bars

cAMP Response Element Is Required for the Regulation of the MAPK Pathway in GnRHR Gene Transcriptional Activity

The involvement of the cAMP response element in transcriptional regulation of the GnRHR promoter in GGH31' cells was assessed by expression of the GnRHR-pXP2 without the cAMP response element (GnRHR-{Delta}CRE) located at -47/-40 bp in the mGnRHR promoter (Fig. 4A).



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  		FIG. 4. Effect of the cAMP response element on activation of GnRHR transcriptional activity by GnRH. A) Schematic model of the 5'-flanking region of the mouse GnRHR gene showing the presence of the cAMP response element (CRE) located at -47/-40 bp. B) GGH31' cells transfected with GnRHR-pXP2 + pCIS-LacZ, GnRHR-{Delta}CRE (GnRHR-pXP2 without cAMP response element) + pCIS-LacZ, or pXP2 + pCIS-LacZ were pretreated with different concentrations of PD 98059 (0, 0.1, or 100 µM) for 1 h prior to stimulation with 10-7 M Buserelin for 6 h before harvesting. The cells were then lysed, and luciferase and ß-galactosidase reporter activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as a percentage of fold induction over that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. {Delta} = Deletion. Significant differences at P < 0.05 from values in the immediately adjacent groups are designated by different lowercase letters above the bars

GGH31' cells transfected with GnRHR-pXP2 + pCIS-LacZ, GnRHR-{Delta}CRE + pCIS-LacZ, or pXP2 + pCIS-LacZ were pretreated with different concentrations of PD 98059 for 1 h prior to stimulation with 10-7 M Buserelin for 6 h. Pretreatment with PD 98059 significantly increased basal and Buserelin-stimulated luciferase activity in both GnRHR-pXP2 and GnRHR-{Delta}CRE (Fig. 4B); however, the GnRHR-{Delta}CRE transcriptional responses were significantly decreased compared with that of the wild-type promoter.

CREB Binds to the cAMP Response Element in the GnRHR Promoter

Using nuclear extracts from GGH31' cells and digoxigenin-end-labeled probe (comprising -55/-30 bp of the mGnRHR gene promoter), two protein-DNA bands were identified on gel mobility-shift assays that were not present with probe alone (Fig. 5). The formation of these complexes could be competed by a 20-, 30-, or 50-fold excess of unlabeled -55/-30 probe, but not by other unrelated DNA sequences.



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  		FIG. 5. Gel-mobility shift assay of cAMP response element region of the mGnRHR promoter. A DNA fragment corresponding to -55/-30 of the mGnRHR promoter was digoxigenin-end-labeled and incubated with 5 µg of nuclear extract from GGH31' cells. Competition with 20-, 30-, or 50-fold excess of unlabeled -55/-30 probe (lanes 3–5), but not by the same fold excess of other, nonspecific DNA sequences (lanes 6–8). Two specific protein-DNA bands were detected, as indicated by the arrows

Because the -55/-30 region contains the CREB binding site, a rabbit anti-CREB polyclonal antibody was used in the gel mobility-shift assay. This "gel mobility-shift abrogation," relies on the ability of the anti-CREB antibody to effectively block accessibility of the DNA to the CREB DNA-binding domain. Addition of the anti-CREB antibody blocked binding of the protein complexes (Fig. 6); an equal amount of anti-cFos or immunoglobulin G failed to attenuate binding of the protein complexes.



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  		FIG. 6. CREB protein binds the cAMP response element in mGnRHR promoter. GGH31' nuclear extracts were incubated with 1 or 2 µl of anti-CREB antibody (lanes 2 and 3), or 1 or 2 µl of anti-cFos (lanes 4 and 5) prior to the addition of the labeled probe. The results showed that the addition of anti-CREB antibody blocked binding of the two protein complexes (lane 3, see arrows). An equal amount of anti-cFos failed to attenuate binding of the protein complexes (lanes 3 and 4)

DISCUSSION

In this study, a luciferase reporter gene vector (GnRHR-Luc) containing a 1.2-kb promoter fragment (-1164/+62) of the mGnRHR [31] was used to examine the regulation of GnRHR gene transcription by GnRH and cAMP. The results obtained show that stimulation by Buserelin, dBcAMP, or forskolin significantly increased luciferase reporter activity. In addition, overexpression of Raf-1, ERK1, or ERK2 partially blocked basal and Buserelin-stimulated luciferase activity. In contrast, inhibition of MAPK activity by PD 98059 activated basal and Buserelin-stimulated luciferase activity in a dose-dependent manner. Transient transfection of the deleted cAMP response element (GnRHR-{Delta}CRE) expression vector followed by pretreatment with PD 98059 prior to Buserelin stimulation showed that the transcriptional response was decreased compared with that of the wild-type promoter. A gel-mobility shift assay using a probe containing the cAMP response element showed the presence of two specific protein-DNA complexes. Using an antibody blocking technique we showed that these bands are protein complexes containing one or more members of the CREB protein family. These results suggest that cAMP positively regulates GnRHR transcriptional activity. It also indicates that the CRE response element and CREB participate in the regulation of GnRHR promoter activity. Coexpression of Raf-1, ERK1, or ERK2 also resulted in markedly suppressed GnRHR transcriptional activity. This inhibitory effect of the wt promoter was not observed when the cells were stimulated with an MEK inhibitor, PD 98059. The negative action of these MAP kinases on the wt promoter transcriptional activity can be explained by the assumption of a direct and specific action of the MAPK kinase cascade in the transcriptional regulation of the GnRH receptor, probably mediated through CREB proteins (Fig. 7).



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  		FIG. 7. Proposed model for the transcriptional regulation of GnRHR by PKA, PKC, and CREB. Binding of GnRH to the receptor activates both Gq-dependent and Gs-dependent signaling pathways. The activated Gq protein contributes to the activation of PKC, switching on the MAPK (ERK1/2) cascade. Activation of Gs helps activate the cAMP/PKA pathway, which positively influences transcriptional activity of the GnRHR, likely via CREB. On the contrary, activation of the MAPK cascade may potentially exert a negative influence on the regulation of the transcriptional activity of the receptor, also through CREB

These studies used GGH31' cells, a well-characterized rat pituitary cell line that stably express the rat GnRHR [9], and have been shown [32] to be a good model for studies of pituitary GnRH action. For example, GGH3 cells and gonadotropes both bind GnRH and GnRH analogues specifically and with similar affinities [32]. In response to GnRH and its agonists, GGH3 cells produce IP and cAMP via Gq/11-mediated and Gs-mediated signal transduction pathways, respectively [10, 11]. Studies relying on palmitoylation of G proteins and overexpression of G protein {alpha} subunit cDNAs have shown that the GnRHR couples toGq/11 as well as Gs and Gi in both pituitary gonadotropes and GGH3 cells [5, 13]. These observations suggest that similar signal transduction pathways are regulated in both cell types. Other similarities of GGH3 cells and primary pituitary cells include the degree of stimulation of {alpha}, LHß, and FSHß gene promoter activities and differential regulation of the gonadotropin subunit gene promoter activities by GnRH [33–36]. GGH3 cells have been also used successfully for the study of the GnRHR gene expression in response to GnRH [25–27, 31].

Buserelin significantly stimulated luciferase activity at both 3 and 6 h, with higher stimulation at 6 h. Forskolin and dBcAMP increased GnRHR promoter activity, suggesting that GnRH activates GnRHR transcriptional activity, in part, through the cAMP signal transduction pathway. Although it is well established that many of the biological actions of GnRH are mediated by Gq-coupled pathways, increased cAMP signaling may also mediate physiological responses in gonadotropes. The activation of the cAMP signal transduction pathway in the pituitary mediates the movement of LH from a nonreleasable pool to a releasable pool [37]. In hypothalamic neuronal GT1-7 cells, the elevation of intracellular cAMP levels by forskolin or 8-bromo-cAMP mimicked GnRH receptor activation by stimulating neurosecretion [38]. GnRHR is coupled to Gq/11{alpha} as well as to Gs{alpha}, which activates adenylate cyclase, leading to the production of cAMP in both GGH3 cells and pituitary gonadotropes. This indicates that the adenylate cyclase-cAMP signal transduction pathway is involved in GnRH action [13]. Also, it was demonstrated that the specific residues in the first intracellular loop are important for agonist-induced cAMP responses [39]. Recent studies [25, 26] reported the transcriptional activation of the mGnRHR gene by both GnRH and cAMP in GGH3 cells. Furthermore, dBcAMP or forskolin stimulated the luciferase activity, supporting the view that GnRH activates GnRHR promoter activity, in part through the cAMP signal transduction pathway. However, other studies in {alpha}T3-1 cells suggest that the response of the messenger GnRHR gene to GnRH agonist-stimulation is mediated by PKC and not by PKA [40].

The MAPK gene family signal activation is coupled to both G-protein-coupled receptors (GPCRs) and to growth factor receptor tyrosine kinases (RTKs), leading to cellular responses such as differentiation and proliferation [17]. GnRH activates MAPK in both primary pituitary cells and in pituitary-derived cell lines, including GH3 and {alpha}T3-1 cells [14–16]. In {alpha}T3-1 cells, ERK activation in response to GnRH is mediated by PKC and Ca2+, which act in a Ras-independent manner [17]. GnRH also activates the Jun N-terminal kinase (JNK, an ERK-like cascade) signaling cascade through PKC, c-Src, and CDC42, and acts independently of the ERK cascade in {alpha}T3-1 cells [18]. In GGH3 cells it has been demonstrated that MAPK activation occurs through both PKA and PKC signal transduction pathways [19]. GnRH also activates the MAPK cascade, which in turn, is involved in regulation of gene expression of the gonadotropin {alpha}-subunit [20–22]. In the present study, activation of MAPK activity by overexpression of Raf-1, ERK1, or ERK2 partially blocked both basal and Buserelin-stimulated luciferase activity compared with a control vector, and inhibition of MAPK activity by PD 98059 increased basal and Buserelin-stimulated luciferase activity in a dose-dependent manner. These results confirm a previous report suggesting that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRHR promoter activity [27]. However, our results are different from those obtained in {alpha}T3-1 cells [40, 41]; this may be due to use of a different cell line ({alpha}T3-1 cells), or may reflect differences in cell culture conditions. In addition, a recent report demonstrated MAPK-dependent induction of GnRHR mRNA levels by GnRH in primary cultures of rat pituitary cells [42]. The mechanism of negative regulation of GnRHR gene transcriptional activity by MAPK remains unknown; however, it has been shown that MAPK represses transcriptional activation in c-Fos, c-Myb, and heat shock factor-1 [43–45].

Analysis of {alpha}T3-1 cells transiently transfected with the 1.2-kb 5'-flanking region of the mGnRH gene (-1164/+62) has identified a tripartite enhancer that appears to be responsible for regulating cell-specific basal expression of the GnRHR gene [46]. Individual elements of this putative enhancer include binding sites for steroidogenic factor-1 (SF-1), AP-1, and a novel element designated "GnRH receptor activating sequence." GnRH-stimulated activity of the mGnRHR gene promoter is regulated by two distinct elements, and the key component of this mechanism involves the AP-1 protein complex, which activates transcription in a cell-specific fashion [40]. Our laboratory suggested the presence and functionality of the cAMP response element located at -47/-40 bp in the mGnRHR promoter in GGH3 cells [26]. In the present study, GGH3 cells transfected with GnRHR-{Delta}CRE (GnRHR-pXP2 without cAMP response element) pretreated with different concentrations of PD 98059, showed a diminished Buserelin-stimulated luciferase activity compared with the wild-type promoter. The results suggest that the cAMP response element participates in the regulation of the mGnRHR gene transcriptional activity by MAPK, which is consistent with previous reports in which the GnRHR couples to Gs{alpha} to activate adenylate cyclase and subsequent production of cAMP [13]. Similarly, a recent report has demonstrated that MAPK activation is, in part, mediated through the PKA signal transduction pathway in GGH3 cells [19].

Analysis of protein-DNA interactions using DNA probe corresponding to -55/-30 bp of the mGnRHR promoter, which contains the cAMP response element (-47/-40) sequence, identified two specific protein-DNA complexes present in GGH31' nuclear extracts. This suggested that several distinct proteins or protein complexes interact with sequences in the -55/-30 region, which may play a role in GnRHR transcriptional activity following Buserelin or dBcAMP stimulation. Using antibody-blocking experiments, we have demonstrated that those bands represent a complex containing one or more members of the CREB protein family. CREB activity is directly regulated by PKA [47]. These results suggest that CREB could be involved in the negative regulation of the GnRHR transcriptional activity by MAPK. Although our results do not rule out the potential involvement of the PKC pathway in the activation of MAPK, these results suggest participation of the PKA pathway in both positive and negative regulation of the GnRHR transcriptional activity by MAPK; however, this apparently needs to be confirmed using pituitary cells.

FOOTNOTES

First decision: 12 December 2000.

1 Supported by National Institutes of Health grants HD-19899, RR-00163, and HD-18185. G.M.N. received support from Fogarty Grant TW/HD00668 and from Unidad de Investigación Médica en Biología del Desarrollo, IMSS, México. Back

2 Correspondence: P. Michael Conn, 505 NW 185th Avenue, Beaverton, OR 97006. FAX: 503 690 5569; connm{at}ohsu.edu Back

Accepted: March 27, 2001.

Received: October 27, 2000.

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