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Biology of Reproduction 61, 715-723 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Gonadotropin-Releasing Hormone Activates the Equine Luteinizing Hormone ß Promoter Through a Protein Kinase C/Mitogen-Activated Protein Kinase Pathway1

Gerald B. Calla, and Michael W. Wolfe2,a

a Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7401


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GnRH regulation of LH secretion is well understood and involves Ca2+ mobilization. However, the mechanism by which GnRH activates transcription of the LHß gene is controversial. GnRH is known to elevate intracellular calcium and activate the protein kinase C (PKC) pathway. The present study evaluated the pathway(s) involved in GnRH induction of LHß transcription. We have previously reported that the equine LHß (eLHß -448/+60) promoter is active in {alpha}T3-1 cells. Therefore, we created a clonal, stably transfected {alpha}T3-1 gonadotroph cell line harboring the eLHß promoter (-448/+60) fused to the luciferase reporter gene. Administration of a GnRH agonist resulted in induction of promoter activity that was completely inhibited by the antagonist antide. Various calcium-affecting drugs had no effect on the promoter. Administration of phorbol 12-myristate 13-acetate (PMA) elicited an activation similar to, albeit lower than, that with GnRH. Down-regulation or pharmacological inhibition of PKC completely blocked PMA's induction of the promoter, while GnRH induction was only partly attenuated. Treatment with the mitogen-activated protein kinase (MAPK) kinase inhibitor, PD98059, completely inhibited the activation of eLHß by PMA but only partly diminished GnRH's induction. Expression of the transcription factor, early growth response protein 1 (Egr1), correlated completely with activation of MAPK, suggesting that Egr1 is the factor through which PKC/MAPK acts. Our data suggest that GnRH induces activity of the eLHß promoter by activating a signal transduction cascade involving PKC-MAPK-Egr1 but that has no significant requirement for calcium.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GnRH is a decapeptide that is released from the hypothalamus in a pulsatile manner and is the key hormone responsible for the regulation of fertility [1]. Upon binding to its receptor on gonadotrophs, GnRH elicits a cascade of intracellular events responsible for the synthesis and release of the gonadotropins, LH and FSH. The GnRH receptor is a member of the seven-transmembrane G protein-coupled family of receptors [2]. The GnRH receptor couples to the Gq/11 class of G proteins [3, 4], which leads to activation of phospholipase C ß (PLCß) and subsequent hydrolysis of phosphatidylinositol bisphosphate to generate diacylglycerol and inositol trisphosphate [5, 6]. Inositol trisphosphate binds to receptors found on organelles within the cell and elicits a rapid rise in intracellular calcium. Diacylglycerol binds to specific isotypes of protein kinase C (PKC) and leads to their activation. Thus, GnRH can activate at least two signaling pathways within the gonadotrophs: calcium and PKC [7–9]. It is well documented that the rise in intracellular calcium is responsible for the release of the gonadotropins [10, 11], yet it is less clear which pathway(s) is responsible for the expression of the specific genes that encode the gonadotropins [11–14]. Even though it has been demonstrated that GnRH increases the expression of early immediate transcription factors, such as c-fos, Jun, and early growth response protein 1 (Egr1) [15, 16], to date there has been no signal transduction study correlating GnRH induction of expression of these genes with transcriptional activation of the gonadotropin subunits.

LH and FSH are part of a glycoprotein hormone family that also includes thyroid-stimulating hormone and chorionic gonadotropins. All these hormones are heterodimeric glycoproteins consisting of a common {alpha} subunit noncovalently linked to a unique ß subunit. The ß subunit is wholly responsible for conferring each hormone's specificity in binding to its receptors and hence its unique biological activity [17]. The signal transduction pathway responsible for the expression of the {alpha} subunit has been well characterized and involves the activation of PKC and mitogen-activated protein kinase (MAPK) [18–20]. These studies have been possible due to the generation of the murine {alpha} subunit-expressing cell line, {alpha}T3-1 [21]. Unfortunately, the {alpha}T3-1 cell line does not express its endogenous gonadotropin ß subunits and does not express reporter genes driven by the ß-subunit promoters in transient transfections. Therefore, knowledge concerning the expression of the ß subunits has not progressed as rapidly as it has for the {alpha} subunit. In this study we have examined GnRH regulation of the equine LHß (eLHß) promoter in {alpha}T3-1 cells. Previous studies from our laboratory have shown that the eLHß promoter is basally active and responds to GnRH in this cell line [16]. Thus, clonal, stably transfected {alpha}T3-1 cells that harbor the eLHß promoter linked to luciferase were used to characterize GnRH activation of LHß expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Plasmids

The GnRH agonist des-Gly10,[D-Ala6]-GnRH ethylamide, antide, phorbol 12-myristate 13-acetate (PMA), nifedipine, thapsigargin, and Bapta AM were purchased from Sigma (St. Louis, MO). PD98059 (PD) was purchased from New England Biolabs (Beverly, MA). Bisindolylmaleimide I (Bis I) and BayK 8644 were purchased from Calbiochem (La Jolla, CA). All radionuclides were purchased from New England Nuclear Life Science Products (Boston, MA). All other chemicals and reagents were obtained from Sigma and Fisher (Pittsburgh, PA). The human {alpha}-subunit promoter (-1500) linked to luciferase in pGL2 was a gift from Dr. John Nilson, Dept. of Pharmacology, School of Medicine, Case Western Reserve University Cleveland, Ohio. The eLHß (-448/+60) promoter fused to luciferase in pGL2 was derived in our laboratory.

Stable Cell Generation and Culture

{alpha}T3-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Grand Island, NY) with 5% fetal bovine serum, 5% horse serum (Atlanta Biologicals, Norcross, GA), and antibiotics. The cells were transfected with 1 µg of eLHß promoter (-448/+60) fused to luciferase and 0.5 µg of eLHß promoter (-448/+60) linked to the neomycin resistance gene as detailed in the transient transfection section. Selection was carried out in normal media with G418 (200 µg/ml; Life Technologies). After selection, all cells were maintained in the G418-containing media. Cells were plated in 96-well plates at a limiting dilution of 0.5 cells per well, and clonal colonies were selected and grown after determination of basal luciferase activity. All clonal lines with basal luciferase activity were screened for their responsiveness to GnRH stimulation, and all lines showed similar responsiveness to 10 nM GnRH at 4 h. Two clonal lines (4.1 and 4.7) were selected for the experiments described in this study, with all experiments being repeated at least three times with each line.

Experimental Procedure for Stable Cells

Prior to each experiment, cells were plated at 2 x 105 cells per well in 12-well plates in non-G418-containing medium. Approximately 24 h later, the medium was replaced with serum-free DMEM, and the cells remained serum starved until (16–18 h) the experiment was initiated. Cells that were given PMA overnight were administered either PMA or dimethyl sulfoxide (DMSO) at the time of serum starvation, which was replaced by treatments at the initiation of the experiment. Cells with pretreatments (PD, 1 h; nifedipine, 15 min; Bis I, 15 min) received coadministration of the inhibitor and the experimental treatment. Bapta AM was the only inhibitor given as a pretreatment only (for 90 sec), after which the medium was replaced by the experimental treatment. All control cells received DMSO at the same concentration for an equal pretreatment time for all experiments. All experiments were performed for 4 h unless otherwise noted.

At the end of the experiment, the cells were washed with ice-cold PBS and lysed with 100 µl of reporter lysis buffer (Promega, Madison, WI). The cells were then harvested, and 20 µl of lysate was assayed separately for luciferase and ß-galactosidase activity. Luciferase assays were performed according to the protocol for the Promega luciferase assay system. Briefly, 100 µl of luciferase assay buffer (Promega) was added to the 20-µl lysate, and light output was measured for 10 sec in a Berthold Lumat LB 9501 luminometer (Wallac, Gaithersburg, MD). The Galacto-Light ß-galactosidase reporter gene assay system (Tropix, Bedford, MA) was used to analyze ß-galactosidase activity. The reaction buffer (100 µl) was added to the 20-µl lysate, and incubation was carried out at room temperature for 1 h. Light emission accelerator (150 µl) was added, and light emission was measured for 5 sec in the luminometer identified above. The {alpha}T3-1 cells endogenously produce a small yet measurable level of ß-galactosidase. This was measured and used as an indicator of cell number. During all the experiments performed, the ß-galactosidase activity did not change significantly with any of the treatments from the mean ß-galactosidase of that experiment. It was determined that a high level of correlation (p < 0.001) existed between the level of ß-galactosidase and total protein in the lysate. Thus to correct for cell density, the luciferase value for each sample was divided by its corresponding ß-galactosidase level to give an adjusted luciferase activity as seen in the graphs.

Transient Transfections

On the day before transfection, {alpha}T3-1 cells were plated at a density of 1.8 x 105 cells per well in 6-well plates. Cells were transfected with 400 ng of h{alpha} (-1500) Luc plasmid DNA and 7 µl of LipofectAmine (Life Technologies) according to the manufacturer's recommendations. Briefly, DNA and LipofectAmine were diluted separately in OptiMEM (Life Technologies), combined, and incubated at room temperature for approximately 45 min. Medium was aspirated from the cells and replaced with the DNA/LipofectAmine mix. The plates were then returned to the CO2 incubator. After an overnight incubation, the DNA/LipofectAmine mix was removed, fresh medium was added, and the plates were returned to the incubator. That evening, the medium was replaced with serum-free DMEM prior to initiation of the experiments the next morning. Except for a few differences, the experimental procedure was the same as that used with the stable cells. The main differences were that incubation with treatment was for 8 h instead of 4 h and that instead of ß-galactosidase assays for cell number, total protein was used to adjust luciferase activity. Protein was quantitated using the Bradford method [22] and Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA).

RNA Isolation and Northern Blots

Cells were grown to 70–90% confluency in 10-cm culture plates and received treatments as described for stable cells. At various time points as indicated in the figures, the cells were quickly washed with cold PBS and harvested in 5 ml of Trizol reagent (Life Technologies). Total RNA was isolated using the Trizol protocol and fractionated (10 µg) in a 1% agarose/formaldehyde gel. The RNA was transferred to a nylon membrane and stained with methylene blue to assure equivalent loading of the wells. The blots were hybridized at 42°C in a 40% formamide/PIPES buffer (20 mM PIPES, 8 mM NaCl, 2 mM EDTA, 0.5% SDS, 100 µg/ml sheared salmon sperm DNA) to random primer-generated 32P-labeled cDNA probe for either Egr1 or luciferase. After washing at high stringency, the membranes were exposed to x-ray film. The luciferase blot was then stripped and rehybridized with a 32P-labeled cDNA probe for mouse ribosomal protein L7 for quantitative determination of equal loading of the wells [23]. Where shown, the blots were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and the data obtained were adjusted by dividing the arbitrary units of the probe by its corresponding L7 arbitrary units. The blots shown are representative of the results from three different experiments.

Protein Isolation and Western Blotting

Cells were grown to 70–90% confluency in 10-cm culture plates and received treatments as described for stable cells. After 15 min the cells were quickly washed with cold PBS and harvested in 1 ml of RIPA buffer (20 mM Tris pH 8.0, 137 mM NaCl, 10% glycerol, 1% igepal, 0.1% SDS, 0.5% deoxycholate, 2 mM EDTA, 5 mM sodium vanadate, 2.5 µg/ml pepstatin, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 mM benzamidine, 2.5 µg/ml leupeptin). The proteins were quantitated (as described in the transient transfection section), and 75 µg of protein was run in a 10% SDS-PAGE gel and transferred to an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was then immunoblotted using an anti-active MAPK antibody (Promega; 1:10 000) and subsequently incubated with a secondary antibody conjugated to horseradish peroxidase. Immunoglobulin complexes were detected using the ECL chemiluminescence protocol (Amersham, Arlington Heights, IL). The blots were then stripped and detected with an ERK2 antibody (Promega; 0.1 µg/ml) to determine total amount of ERK present. The blots shown are representative of the results from three different experiments.

Statistics

All experiments were performed in triplicate and repeated at least three times for both clonal lines tested. Statistics were performed where noted, using one-way ANOVA and Student's t-tests. In the event of unequal variances between groups, nonparametric tests were performed, including the Kruskal-Wallis test and Mann-Whitney test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of GnRH Responsiveness

{alpha}T3-1 cell lines stably harboring the eLHß promoter were used to characterize the eLHß promoter's response to GnRH (Fig. 1). A time-dependent increase in eLHß promoter activity was observed with maximal (2.6-fold, p < 0.001) activation at 4 h after administration of GnRH. This was followed by a rapid decrease in promoter activity that returned to basal levels by 10 h. Analysis of the mRNA (Fig. 1, B and C) for luciferase indicated a plateau (2-fold) in expression of eLHß at 1–3 h after GnRH treatment. The involvement of the PKC pathway was assessed after administration of PMA, a pharmacological activator of PKC. Treatment with 100 nM PMA activated the eLHß promoter with a maximum occurring at 3 h (1.7-fold, p < 0.001). Interestingly, at all time points after 1 h, PMA failed to reach the full level of activation (p < 0.01) that was elicited by 10 nM GnRH. In addition, cotreatment with PMA and GnRH had no additional effect over treatment with GnRH alone (data not shown). Thus activation of the PKC pathway partially mimicked GnRH induction of the eLHß promoter.



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  		FIG. 1. Time course of GnRH and PMA induction of eLHß promoter. A) GnRH induction of eLHß was significantly greater than control (p < 0.001) and PMA (p < 0.01) levels at every time point past 1 h. PMA induction of eLHß was significantly (p < 0.001) elevated above the DMSO control from 2 to 6 h. Values represent the mean ± SEM for four separate experiments. B) A single Northern blot for luciferase was performed with RNA from 4.7 cells exposed to GnRH at various time points. C) The graph represents luciferase adjusted for the amount of L7 activity in each lane from the Northern blot in B.

This disparity between PMA and GnRH in their ability to activate the eLHß promoter was further evaluated by performing dose-response experiments. A concentration-dependent increase in activation of the eLHß promoter was observed for both PMA and GnRH (Fig. 2). Full activation occurred at 1 nM GnRH and 10 nM PMA. To ensure maximal stimulation of the eLHß promoter with GnRH and PMA, we chose to use a concentration one order of magnitude greater (10 nM GnRH and 100 nM PMA) than those found to elicit maximal stimulation. To determine whether the response of these stable cell lines to GnRH was elicited through the GnRH receptor, a competitive antagonist of GnRH, antide, was used to block the response of GnRH. Coadministration of antide (1 µM) completely blocked the ability of GnRH to activate the eLHß promoter (Fig. 3). Conversely, induction of the promoter by PMA was unaffected, as PMA works at a level downstream of the GnRH receptor.



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  		FIG. 2. Dose-dependent activation of eLHß by GnRH and PMA. Treatment with GnRH (0.1–1000 nM) significantly induced activity of the eLHß promoter at concentrations as low as 1 nM (p < 0.001), with full activation occurring between 10 and 1000 nM. Activation of eLHß by PMA (0.1–1000 nM) occurred at 1 nM (p < 0.01) and significantly increased at higher concentrations (p < 0.001). Values represent the mean ± SEM for three separate experiments.



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  		FIG. 3. Competitive inhibition of GnRH induction of the eLHß promoter. GnRH induced eLHß 2.5-fold (p < 0.001) over control values. PMA induced the eLHß promoter 1.8-fold (p < 0.001) above control levels. The GnRH antagonist, antide, completely blocked the induction of GnRH. Values represent the mean ± SEM for three separate experiments.

Signal Transduction Pathways: Role of Calcium

Knowing that the PKC and calcium pathways are both activated by the GnRH receptor and that specific PKC isotypes are calcium responsive, we set out to further examine their involvement in GnRH activation of eLHß. Experiments were undertaken to determine whether calcium played a role in the activation of the eLHß promoter by GnRH. BayK 8644, an activator of L-type calcium channels, was used to stimulate calcium oscillations similar to those elicited by GnRH. The effects of BayK 8644 have been characterized extensively in {alpha}T3-1 cells [10–14, 24]. BayK 8644 (1 µM) had no effect on eLHß activity in the stable cells. In contrast, BayK 8644 increased (to 2.1-fold; p < 0.01) the activity of a human {alpha}-subunit (h{alpha}) promoter linked to luciferase in transient transfection assays (Fig. 4). Similar results were observed in experiments utilizing additional drugs that affect intracellular levels of calcium. Nifedipine (1 µM) was used to block external calcium influx through GnRH-activated calcium channels on the cell membrane (Fig. 5A). Blocking extracellular calcium entry had no detrimental effect on GnRH's ability to stimulate the eLHß promoter (2.1-fold vs. 2.0-fold), while the induction of the h{alpha} promoter by GnRH was attenuated (12.4-fold vs. 2.5-fold; p < 0.001) by nifedipine treatment. Similar results were seen when 2 mM EGTA was added to the medium to chelate external calcium (Fig. 5B). The induction of the eLHß promoter by GnRH was unaffected (2.1-fold vs. 2.2-fold), yet chelation of extracellular calcium inhibited (p < 0.001) the activation of h{alpha} promoter by GnRH from a 12.4- to a 1.7-fold increase. Finally, Bapta AM, an intracellular chelator of calcium, was used to determine the role of intracellular calcium on GnRH induction of the eLHß promoter. Pretreatment with Bapta AM (Fig. 5C) decreased (p < 0.001) basal eLHß promoter activity in the cells by 20% and decreased the activation of the eLHß promoter by GnRH from 2.2- to 1.9-fold (p < 0.001). This inhibition was also seen with the h{alpha} promoter but, to a much greater extent, reducing GnRH's stimulation from a 12.4- to a 5.8-fold increase. Similar results were observed using higher concentrations of Bapta AM (data not shown). It is important to realize that some of the early enzymatic events common to both the PKC and calcium pathways are dependent on calcium for their full activity (e.g., PLCß). This could explain the slight attenuation of GnRH's induction of the eLHß promoter after chelation of intracellular calcium by the Bapta AM treatment. Treatment with thapsigargin (1 µM) to deplete intracellular calcium stores produced results similar to those seen with Bapta AM, and combinatorial experiments utilizing thapsigargin or Bapta AM pretreatment and EGTA cotreatment with GnRH had little effect on GnRH induction of eLHß (data not shown). Collectively these data suggest that intracellular calcium may play a role in maintaining basal activity of the eLHß promoter, but that mobilization of extracellular and intracellular calcium is not required for GnRH induction of the eLHß promoter.



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  		FIG. 4. Calcium induction of the eLHß and h{alpha} promoters. GnRH induced the eLHß promoter 2-fold (p < 0.001) and the h{alpha} promoter 12.4-fold (p < 0.001). The L-type calcium channel activator BayK 8644 induced the h{alpha} promoter 2.1-fold over basal level (p < 0.01), yet failed to induce the eLHß promoter. Values represent the mean ± SEM for three separate experiments.



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  		FIG. 5. Role of calcium in GnRH induction of the eLHß and h{alpha} promoters. A) Basal activity or GnRH induction of eLHß (p < 0.001) was unaffected by treatment with the calcium channel blocker, nifedipine. In contrast, h{alpha} basal activity was diminished 35% (p < 0.01), and GnRH activation of h{alpha} was attenuated from a 12.4- to a 2.5-fold increase (p < 0.001). B) Chelation of calcium with EGTA had no effect on basal or induced eLHß promoter activity. GnRH induction of the h{alpha} promoter was attenuated (p < 0.001) from a 12.4- to a 1.7-fold increase above control values by EGTA treatment. C) Intracellular calcium chelation by Bapta AM decreased basal activity of the eLHß promoter by 20% (p < 0.001). GnRH induction of the promoter was blunted from a 2.2- to a 1.9-fold increase above the respective control levels (p < 0.001). This same inhibition of GnRH activation was seen with the h{alpha} promoter but to a much greater extent (12.4 vs. 5.8; p < 0.001), while there was no inhibition of basal h{alpha} promoter activity. Values represent the mean ± SEM for three separate experiments.

Signal Transduction Pathways: Role of PKC

The importance of the PKC pathway in GnRH activation of the eLHß promoter was further explored using two different paradigms: PKC down-regulation by overnight treatment with 1 µM PMA or treatment with the PKC inhibitor, Bis I. Administration of GnRH or PMA resulted in a 2.1- and 1.5-fold induction, respectively, of promoter activity over control levels (Fig. 6). Cotreatment with antide completely inhibited the GnRH induction. Treatment with PMA overnight or 1 µM Bis I had no significant effect on basal levels of eLHß expression, yet these two treatments completely blocked activation of the promoter by PMA. A similar, but incomplete, inhibition of GnRH induction occurred with these two treatments. Following PMA down-regulation, treatment with GnRH resulted in a 1.3-fold induction of eLHß promoter activity over control levels (p < 0.01). Similarly, Bis I treatment reduced GnRH activation of eLHß (1.7-fold vs. 2.1-fold; p < 0.005) but did not completely block the induction (p < 0.001). Interestingly, coadministration of antide with GnRH in the presence of PKC inhibition (Bis I) or down-regulation (PMA overnight [O/N]) completely inhibited GnRH induction of the eLHß promoter. These data suggest that GnRH induction of the eLHß promoter may involve an additional PKC-independent pathway.



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  		FIG. 6. Effect of PKC inhibition or down-regulation on GnRH or PMA activation of the eLHß promoter. Down-regulation of PKC by exposure to PMA O/N inhibited GnRH induction of the eLHß promoter by 69% (p < 0.001), yet this was still above basal levels (p < 0.01). This attenuation was mimicked by treatment with the PKC inhibitor, Bis I. GnRH induction was reduced by 36% (p < 0.005), yet this attenuated stimulation was significantly greater than basal levels (p < 0.001). Values represent the mean ± SEM for three separate experiments.

Signal Transduction Pathways: Role of MAPK

It has been previously reported that treatment of {alpha}T3-1 cells with GnRH leads to activation of PKCs [25–27] and MAPK [19, 28]. Furthermore, PKC is responsible for activating MAPK following GnRH treatment in {alpha}T3-1 cells [19, 29]. It has also been demonstrated that MAPK is involved in activation of the {alpha}-subunit promoter [18–20]. To date, there are no data suggesting that MAPK is involved in the expression of the LHß subunit. We explored this possibility by using PD, a specific MEK1/2 (MAPK kinase) inhibitor that has been shown to inhibit MAPK activation in vitro [30]. Pretreatment with PD (50 µM) had no effect on basal activity of the promoter (Fig. 7) but reduced GnRH induction of the eLHß promoter by 68% (2.1-fold vs. 1.4-fold; p < 0.001). Interestingly, PMA's activation of the promoter (1.5-fold, p < 0.001) was completely attenuated by PD pretreatment. Treatment with antide completely blocked GnRH induction of the eLHß promoter with or without PD treatment. The fact that PD completely inhibited PMA induction of the promoter would suggest that PKC-dependent activation of eLHß must occur through its activation of the MAPK pathway. Furthermore, the partial activation (1.4-fold; p < 0.001) of the promoter that remained after pretreatment with PD and stimulation by GnRH must be independent of MEK/MAPK.



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  		FIG. 7. Role of the MAPK pathway in GnRH and PMA stimulation of eLHß. Treatment with PD, a MAPK kinase inhibitor, suppressed GnRH stimulation of the eLHß promoter from a 2.1- to a 1.4-fold increase over control levels (p < 0.001). This decreased activation of the promoter was still significantly higher than control levels (p < 0.001). The residual activation of the promoter by GnRH after PD treatment was completely suppressed by cotreatment with antide. Induction of the promoter by PMA (1.5x, p < 0.001) was completely inhibited with PD treatment. Values represent the mean ± SEM for three separate experiments.

An active MAPK Western blot was performed to determine the effectiveness of the previous treatments of PD, PMA O/N, and Bis I on inhibiting the activity of the ERK1/ERK2 MAPKs. ERK1 and ERK2 were not active under the serum-free control conditions, while activity was induced after administration of GnRH or PMA (Fig. 8). Treatment of {alpha}T3-1 cells with PMA O/N effectively inhibited GnRH- and PMA-stimulated activation of MAPK. PD treatment completely blocked the PMA-induced activation of MAPK and blocked practically all GnRH-induced MAPK activity. This reinforced our hypothesis that activation of MAPK by GnRH in {alpha}T3-1 cells was primarily, if not completely, through the PKC pathway. Bis I, at 1 µM, attenuated the activation of MAPK after stimulation by GnRH and PMA, but not completely. This partial MAPK activity suggests that the concentration of Bis I utilized in these experiments was not completely effective at inhibiting PKC activation. This could explain the higher residual activity observed in the Bis I group when stimulated with GnRH as compared to the PMA O/N group after GnRH administration (Fig. 6). It is likely that this would be overcome by a higher concentration of Bis I. The transcription factor Egr1 has been shown to be critical for LHß expression in vivo in two separate gene disruption experiments [31, 32]. Furthermore, activation of the MAPK pathway rapidly induces expression of Egr1 [33]. On the basis of this information, the ability of GnRH and PMA to induce transcription of Egr1 in {alpha}T3-1 cells was examined. Not only did GnRH and PMA induce Egr1 expression (Fig. 8C): the pattern of expression mimicked the data for activation of MAPK (Fig. 8A). These data suggest that Egr1 could be one of the transcription factors that GnRH works through in activating the eLHß promoter.



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  		FIG. 8. Activity of MAPK (ERK1 and ERK2) and expression of Egr1 in response to GnRH and PMA stimulation with various inhibitor treatments. Total RNA or total protein was isolated from the 4.7 cells following the various treatments. A and B) Proteins were detected using an antibody directed against active MAPK (A) and ERK2 (B); C and D) mRNA was probed for Egr1 (C) using a 32P-labeled cDNA probe. Equivalent loading was determined by staining the membrane with methylene blue (D) prior to hybridization. The results shown are representative of three separate experiments performed for both the Western and Northern blots.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The overall goal of these experiments was to determine the signal transduction pathway(s) utilized by GnRH in activating the eLHß promoter. Alterations in the frequency of GnRH pulses have been demonstrated to be responsible for differential transcriptional activation of the various gonadotropin subunit genes [34]. Furthermore, the density of GnRH receptors on the gonadotroph appears to differentially regulate the expression of the gonadotropin subunits [35]. A high density of GnRH receptors preferentially leads to stimulation of LHß transcription. In the present study we used a set paradigm for GnRH administration (4-h static culture) with {alpha}T3-1 cells; thus changes in pulse frequency and receptor density were held to a minimum and are unlikely to be responsible for the differential activation of the promoters examined. From our study as well as those of others [12, 13], it appears that GnRH can differentially regulate the {alpha}- and LHß-subunit genes by activation of different intracellular signaling pathways. This occurs in the face of a single pattern of GnRH administration and presumably no change in receptor density.

Our results suggest that calcium is not involved in GnRH induction of the eLHß promoter, while PKC plays a substantial role. To ascertain that our calcium treatments were effective, we performed parallel experiments with the human {alpha}-subunit promoter, which is known to be calcium responsive [13, 24]. Our data confirm that GnRH induction of the h{alpha} promoter is dependent upon calcium to achieve a maximal response. Yet dependence of the h{alpha} promoter on calcium must be accompanied by activation of PKC to achieve maximal stimulation of the promoter. An increase in calcium alone (BayK 8644) only activated the h{alpha} promoter approximately 2-fold. PMA was also a very weak stimulator of the h{alpha} promoter when administered alone (~2-fold above basal, data not shown). However, when BayK 8644 was given in conjunction with PMA, activation of the h{alpha} promoter approached the activation achieved following GnRH administration—similar to the results seen in a previous study (data not shown) [24]. In support of this hypothesis, when GnRH was added to the medium and calcium oscillations were blocked (Fig. 5, A and B), promoter activity was attenuated to very low levels, similar to those seen with induction by PMA alone. This synergy between calcium and PKC activation was not seen with the eLHß promoter; in fact, when BayK 8644 and PMA were given simultaneously, the promoter was never activated above the levels induced by PMA alone (data not shown).

Pharmacological stimulation of PKC by PMA induced activity of the eLHß promoter, thus leading to the hypothesis that the PKC pathway may be partially responsible for GnRH signal transduction. In support of this, our results demonstrate that GnRH stimulation of the eLHß promoter could be attenuated by specific inhibition (or down-regulation) of PKC. A similar attenuation of GnRH activation of eLHß was observed after treatment with the MAPK kinase inhibitor, PD. Importantly, treatment with PD completely blocked PMA-induced activation of the eLHß promoter. Thus our data indicate that the MAPK pathway is downstream of PKC and partly responsible for GnRH induction of the eLHß promoter. Furthermore, our data indicate that GnRH-induced expression of Egr1 occurs through the PKC/MAPK pathway (Fig. 8). In support of this, the Egr1 promoter contains multiple serum response elements that are required for full induction of expression and are involved in regulation by MAPK [36]. Recent data have also shown that Egr1 can be activated by the transcription factor ETS [33], which is known to be activated by MAPK. Expression of the c-fos gene, another MAPK-inducible immediate-early gene whose expression is regulated by GnRH in {alpha}T3-1 cells [15], was induced by PMA and GnRH and correlated completely with the expression pattern of Egr1 (data not shown). However, AP-1 sites are not present within the eLHß -448/+60 promoter, negating a direct involvement of c-fos. Data from our laboratory demonstrate that the eLHß promoter harbors two Egr1 sites and that mutation of these sites abrogates GnRH activation of the promoter [16]. Similar findings have been reported for GnRH activation of the rat LHß promoter [37]. Thus, the current data reinforce our contention that GnRH activates the PKC and MAPK pathways leading to induction of the immediate-early gene, Egr1, and subsequent activation of the LHß promoter.

Recently, two studies have identified signal transduction pathways involved in GnRH activation of the gonadotropin promoters [12, 13]. Yet these studies are contradictory in their findings. One study suggested that the rat {alpha}-subunit promoter was regulated primarily by PKC and MAPK and that calcium played little role in promoter transactivation, while rat LHß was regulated predominantly through calcium and not PKC [12]. Another group found the opposite to be true [13]. The h{alpha} promoter was more responsive to calcium than PKC, while rat LHß was insensitive to changes in intracellular calcium concentration but was dependent upon PKC for GnRH activation of the promoter. The conflict with regard to the {alpha} promoters could be due to species differences, especially since the h{alpha} promoter contains two cAMP response element (CRE) sites that could render it more responsive to calcium [13, 24, 38] than the rat {alpha}, which has a single CRE-like element. The differences in the data for LHß can also be attributed to promoter differences. Both groups studied the rat LHß promoter, but they utilized different regions of the promoter. The rat LHß promoter contains two Egr1 sites (-112 and -50) that have been shown to be necessary for GnRH activation [37]. These sites were lost upon truncation of the promoter in the study by Weck et al. [12]. These investigators also used primary pituitary cultures and transcription run-off assays, which should have overcome the shortcomings of the truncated promoter construct. Unfortunately, the run-off assays were performed too acutely to have any Egr1 protein synthesized [16] and in turn stimulate LHß transcription. As shown by separate targeted mutagenesis experiments [31, 32], as well as in in vitro transfection experiments [16, 37], Egr1 plays a critical role in regulating expression of LHß. Our results and those of Saunders et al. [13] indicate that PKC is involved in GnRH induction of LHß, while calcium is not. Furthermore, our data provide evidence that GnRH activation of PKC leads to an induction of Egr1.

An interesting finding from the current study was that stimulation of the eLHß promoter by 100 nM PMA was less effective (47%) than that achieved by 10 nM GnRH (p < 0.001). This suggests that PKC is only partially responsible for activation of eLHß. A similar residual activation of the rat LHß promoter by GnRH was seen following inhibition of PKC [13]. Since it is apparent that calcium plays no role in GnRH's induction of the eLHß promoter, there must be another secondary pathway activated by GnRH. Data exist suggesting that cAMP and the PKA pathway are activated by GnRH and play a role in gonadotropin gene expression [39–42]; however, the current thought is that PKA is not involved in gonadotropin expression and release in gonadotrophs [3, 4, 10]. In our model we see no increase in expression of eLHß above basal levels after treatment with 8-bromo-cAMP or forskolin, or any diminution of GnRH induction by the PKA inhibitor H-89 (data not shown). Evidence exists that a pertussis toxin-sensitive G protein of the Go/i family [28, 40] can be activated by GnRH. Activation of Gi leads to inactivation of adenylate cyclase and reduces cAMP accumulation in cells; however, we have already determined that cAMP is unimportant for GnRH induction of eLHß. Interestingly, activation of Go leads to PKC-independent stimulation of MAPK via activation of Ras [26]. An additional signaling pathway that is a likely candidate for activation by the classical GnRH signal transduction cascade is the heterotrimeric G protein ß{gamma} subunits. There are many instances of ß{gamma} subunits activating various second messenger pathways [43–46]. In pituitary thyrotropes, thyrotropin-releasing hormone signals through the ß{gamma} subunits to activate MAPK via a Ras-dependent and PKC-independent mechanism [46]. However, if the ß{gamma} subunits in gonadotrophs lead to a PKC-independent activation of MAPK, similar to the aforementioned Go pathway, the PD treatment in our experiments would have blocked subsequent activation of the eLHß promoter. Thus alternate pathways that ultimately lead to activation of MAPK are probably not candidates, since residual induction of the eLHß promoter was still observed after PD inhibition of MAPK activity (Fig. 7). It has also been demonstrated that GnRH activates the non-receptor tyrosine kinase, c-Src, and the Jun N-terminal kinase (JNK) pathway [47]. However, activation of the JNK pathway required PKC, thus ruling out this as a candidate for our second pathway. It is important to elucidate alternate pathways that are activated by GnRH, since such a pathway(s) is responsible for 50% of GnRH's induction of eLHß.

In conclusion, GnRH initiates a series of intracellular events that culminates in increased activity of the eLHß promoter, which include 1) activation of PLCß and liberation of diacylglycerol, 2) diacylglycerol activation of PKC, 3) PKC activation of the MAPK pathway, 4) MAPK activation of Egr1 expression, and 5) Egr1 activation of the LHß promoter. Finally, our data suggest the involvement of an additional pathway(s) that is PKC- and MAPK-independent.


   ACKNOWLEDGMENTS
 
We would like to thank Dr. Pamela Mellon, Department of Reproductive Medicine, School of Medicine, University of California, San Diego, for the {alpha}T3-1 cell line; Dr. Leslie Heckert, Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, for the mouse L7 cDNA; and Dr. Mark Roberson, Department of Physiology, College of Veterinary Medicine, Cornell University, Ithaca, New York, for helpful insights and comments while this study was performed. This work was completed with assistance from the Cell Culture Core and Imaging/Photography Core of the NIH-supported Center of Reproductive Sciences (P30 HD 33994) at the University of Kansas Medical Center.


   FOOTNOTES
 
1 Supported by NIH Grant R29-DK-50668 to M.W.W. Back

2 Correspondence: Michael W. Wolfe, Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160–7401. FAX: 913 588 7430; mwolfe2{at}kumc.edu Back

Accepted: April 26, 1999.

Received: March 15, 1999.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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