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*ESTRADIOL
Endocrinology Vol. 139, No. 9 3796-3802
Copyright © 1998 by The Endocrine Society

ARTICLES

Estradiol Sensitization of Rat Pituitary Cells to Gonadotropin-Releasing Hormone: Involvement of Protein Kinase C- and Calcium-Dependent Signaling Pathways1

Ides M. Colin2 and J. Larry Jameson

Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Tarry 15-709, 303 East Chicago Avenue, Chicago, Illinois 60611-3008. E-mail: ljameson{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During the female reproductive cycle, estrogen enhances the actions of GnRH on the gonadotrope cell. Recently, we reported that in vivo exposure to estradiol causes a marked enhancement GnRH-induced transcription of the {alpha} gene promoter in primary cultures of pituitary cells. In the present study, we analyzed the GnRH signaling pathways that mediate the sensitizing effects of estradiol on the {alpha} promoter. Primary cultures of male and female rat pituitary cells were transfected with the -420{alpha}LUC reporter gene and treated with agonists or antagonists for 24 h. As found previously, the degree of GnRH (1 nM) stimulation was 15-fold greater in females (157-fold) than in males (9-fold). When cells were treated with phorbol esters [phorbol 12-myristate 13-acetate (PMA); 10 nM], the level of stimulation was half that observed with GnRH, but the sexual dimorphism was preserved. When protein kinase C (PKC) activity was either depleted by long term treatment with phorbol esters (1 µM PMA for 24 h) or inhibited with staurosporine, the stimulatory effect of GnRH was minimally affected in males, but was markedly reduced in females. The reduced threshold of GnRH responsiveness after inhibition of PKC suggests that the actions of estrogen involve this pathway. Coexpression of c-jun and c-fos, which are increased by GnRH and PMA, suppressed basal {alpha}LUC activity, but did not alter the sensitivity to GnRH in a sexually dimorphic manner. Dominant negative mutants of the mitogen-activated protein kinase pathway, which is also activated by GnRH and PMA, failed to reveal sexually dimorphic alterations in GnRH responsiveness. These findings indicate that the mitogen-activated protein kinase pathway and activating protein-1 are probably not involved in estrogen sensitization of transcriptional responses to GnRH. The involvement of Ca2+-dependent pathways was analyzed either by chelating extracellular Ca2+ with EGTA (5 mM) or by using a Ca2+ channel blocker, methoxyverapamil (D600; 1 µM). Depletion of extracellular Ca2+ markedly reduced GnRH action in females, but not in males. Treatment with the Ca2+ channel blocker D600 did not alter GnRH-induced stimulation of -420{alpha}LUC in males, but in females, GnRH stimulation was significantly impaired (208- vs. 23-fold). Estrogen replacement in ovariectomized females reconstituted GnRH sensitivity and the inhibitory effect of methoxyverapamil (84- vs. 13-fold). We conclude that both PKC- and Ca2+-dependent signaling pathways are involved in estradiol-induced sensitization of female pituitary cells to GnRH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH IS A hypothalamic releasing factor that controls the synthesis and secretion of the two pituitary gonadotropins, LH and FSH. In addition to GnRH, gonadotropin levels are regulated by gonadal sex steroids and peptides (1). Removal of sex steroids (e.g. castration) increases GnRH pulse frequency and reduces feedback inhibition at the level of the pituitary gland, resulting in large increases in LH (2). In the case of FSH, synthesis is enhanced by relatively slow GnRH pulse frequencies, and in addition to sex steroids, ambient levels of activin, inhibin, and follistatin determine the levels of FSH (3, 4, 5).

The mechanisms of feedback regulation by sex steroids are complex and include effects at both the hypothalamic and pituitary levels. During the female reproductive cycle, sex steroids are thought to sensitize the pituitary to GnRH, providing part of the basis for the LH surge (6, 7, 8, 9). In some species (e.g. sheep), there is also a marked increase in GnRH at the time of the gonadotropin surge (10). In other species (e.g. monkeys and humans), there is evidence for reduced production of GnRH at the time of the LH surge, placing even greater importance on gonadotrope sensitivity to GnRH (11, 12).

Although the pathways for GnRH signaling have been characterized extensively (13, 14, 15), the cellular mechanisms by which estradiol enhances gonadotrope responses are not well understood. GnRH acts through a seven-transmembrane, G protein-coupled receptor (16). After stimulation by GnRH, multiple signaling pathways are activated. There is a biphasic spike (release of intracellular Ca2+) and plateau (influx of extracellular Ca2+) pattern of intracellular Ca2+ that parallels hormone secretion (17). In addition, GnRH stimulates phosphoinositol turnover, generates diacylglycerol, activates protein kinase C (PKC), increases ryanodine receptors, and activates mitogen-activated protein kinase (MAPK) (13, 14, 18, 19, 20, 21) among other pathways. Estrogen may act to enhance the actions of one or more of these pathways.

Recently, we developed an experimental system in which treatment with estrogen had a profound effect on GnRH stimulation of the gonadotropin {alpha}-subunit promoter that was transfected into primary cultures of rat pituitary cells (22). In this model, the degree of GnRH responsiveness was about 50-fold greater in female vs. male pituitary cells. Hormone replacement experiments performed in gonadectomized animals indicated that the enhanced effects of GnRH in females could be accounted for by several days of estrogen treatment. In the present study, we investigated the GnRH signaling pathways that mediate the ability of estrogen to enhance GnRH action in this model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and hormonal treatment
All surgical and experimental procedures were conducted in accordance with the policies of Northwestern University’s animal care and use committee. Male and female Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were housed in groups of five animals in a temperature-controlled room, with lights on from 0500–1900 h. They had free access to tap water and standard laboratory chow. Animals were kept in the animal facility for at least 1 day before being used for experiments. In experiments in which the effects of estrogens were investigated, ovariectomized (OVX) females (7–10 days before the experiment) received either blank or 17ß-estradiol-filled SILASTIC brand capsules (Dow Corning, Midland, MI; id, 0.062 in.; od, 0.125 in.; 5-mm length) for 2–3 days.

Cell cultures
Animals were killed by decapitation. Anterior pituitary glands were rapidly excised, and the posterior lobe was discarded. The pituitaries were cut into 15–20 small pieces, rinsed twice in incomplete PBS (pH 7.1; 2.7 mM KCl, 1.2 mM K2HPO4, 138 mM NaCl, and 8.1 mM Na2HPO4·7H2O), and digested for two 15-min periods in a solution containing 0.125% trypsin (TRLS, Worthington, Freehold, NJ) in PBS followed by a 2-min digestion in a solution containing 10 U/ml deoxyribonuclease I (Sigma Chemical Co., St. Louis, MO) in PBS. Cells were then incubated for 10 min in a solution of 0.125% collagenase (type IV, Sigma) and dispersed mechanically for 5 min by pipetting through a 25-ml pipette. They were rinsed twice and resuspended in DMEM (Life Technologies, Grand Island, NJ) containing 10% FBS (Life Technologies), penicillin (50 U/ml; Life Technologies), streptomycin (50 µg/ml; Life Technologies, and fungizone (2.5 µg/ml; Biologos, Naperville, IL). Cellular yields were approximately 1.5–2 x 106 cells/pituitary. Cells were plated in 24-well dishes (Corning, Oneonta, NY) at 3.5–4 x 105 cells/well in a humidified atmosphere of 95% air-5% CO2 at 37 C. After recovery overnight, cells were washed, incubated for 24 h in DMEM containing 1% FBS (referred to as culture medium), and then transfected.

Transfection and luciferase assay
Cells were transfected as described previously (22) using a reporter gene containing 420 bp of 5'-flanking sequence and 44 bp of exon 1 of the human glycoprotein {alpha} gene linked to the luciferase gene in the plasmid pA3 Luc (15 µg/well). Transfected cells were treated with a GnRH analog (des-Gly10,D-Ala6-GnRH ethylamide; Sigma) or other agonists or antagonists as indicated. Unless otherwise specified, all chemicals were obtained from Sigma. After 24 h, the cells were harvested for assays of luciferase activity as described previously (22). In experiments involving cotransfection of other expression plasmids, controls were transfected with an equal amount of an empty expression vector. The plasmids, cytomegalovirus (CMV)-c-jun and c-fos vectors have been described previously (23), and dominant mutants of MAP kinase (MAPK; ERK1MUT and ERK2MUT) were provided by Dr. M. Cobb (Southwestern Medical Center, Dallas, TX).

Data analysis
Luciferase data are presented as the mean ± SEM of triplicate transfections. Statistical evaluation of experimental data used ANOVA. Post-hoc pairwise comparisons used the Scheffe method. Calculations were performed with Data Desk software (version 4.2, Data Description, Ithaca, NY).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PKC-dependent pathways are involved in the sensitizing effects of estradiol on GnRH action
Male and female pituitary cells were transfected with 15 µg -420{alpha}LUC and then treated for 24 h with either GnRH or phorbol 13-myristate 12-acetate (PMA). As found previously (22), basal {alpha}-gene expression was very low in random cycling female rats and represented less than 16% of that in males (P < 0.05). After treatment with 1 nM GnRH, a 157-fold stimulation of {alpha} promoter activity was observed in females, in contrast to 9-fold stimulation in males. The phorbol ester, PMA, mimics some of the effects of GnRH (24, 25). When cells were treated with 10 nM PMA, the degree of stimulation in each group was half that observed with GnRH, but the sexually dimorphic response was maintained (Fig. 1Go).



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  		Figure 1. Effects of phorbol esters (PMA) and GnRH on {alpha} promoter activity. Primary pituitary cells from male and randomly cycling female rats were transfected with 15 µg -420 {alpha}LUC. Six hours after transfection, the cells were treated with GnRH (1 nM) or PMA (10 nM). Luciferase activity was measured 24 h after treatment. Values shown are the mean ± SEM of triplicate transfections.

 
The potential role of the PKC pathway in the potentiating effects of GnRH in females was examined by depleting PKC with PMA and by using an inhibitor of PKC. To deplete phorbol ester-sensitive forms of PKC, cells were pretreated with a high dose of PMA (1 µM) for 24 h before transfection (20). Cells were then transfected with 15 µg {alpha}LUC and treated for 24 h with GnRH (1 nM). Pretreatment with PMA reduced basal {alpha}LUC activity in both sexes similarly (70–80% decrease). In contrast, the stimulatory effect of GnRH was unchanged in males (9- vs. 8-fold), but was markedly reduced in females (157- vs. 32-fold; P < 0.05) after pretreatment with PMA (Fig. 2Go).



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  		Figure 2. Effects of phorbol ester (PMA) pretreatment on basal and GnRH-induced {alpha} promoter activity. Primary pituitary cells from male and female rats were pretreated (pt) with PMA (1 µM) 24 h before transfection to deplete phorbol ester-sensitive forms of PKC. Cells were then transfected with 15 µg -420 {alpha}LUC. Six hours after transfection, the cells were treated with GnRH (1 nM), and luciferase activity was measured 24 h after treatment. Values shown are the mean ± SEM of triplicate transfections.

 
Cells were also treated with a PKC inhibitor, staurosporine (10 nM). In the presence of the inhibitor, there was little effect on basal activity. However, the degree of GnRH responsiveness was significantly reduced in females (355- vs. 13-fold; P < 0.05). Basal activity is variable in males, but there was no apparent effect of staurosporine in males (Fig. 3Go).



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  		Figure 3. Effects of the PKC inhibitor, staurosporine, on basal and GnRH-mediated {alpha} promoter activity. Primary pituitary cells from male and female rats were transfected with 15 µg -420 {alpha}LUC. Six hours after transfection, the cells were treated with GnRH (1 nM) alone or with GnRH (1 nM) and staurosporine (10 nM), a PKC inhibitor. Luciferase activity was measured 20–24 h after treatment. Values shown are the mean ± SEM of triplicate transfections.

 
GnRH and PMA stimulate early response genes, including c-jun and c-fos that encode the activating protein-1 (AP-1) transcription factor complex (26). The effects of c-jun and c-fos were therefore analyzed with respect to basal and GnRH-stimulated {alpha} promoter activities (27). Cotransfection with c-jun alone or together with c-fos caused a significant reduction in the basal activity of the reporter gene in both sexes (P < 0.05). In contrast, the degree of GnRH responsiveness was unchanged or slightly enhanced, but this effect was not significant (Fig. 4Go). These results suggest that the AP-1 complex represses basal promoter activity, but does not influence the selective enhancement of GnRH action in females.



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  		Figure 4. Effects of c-jun and c-fos on basal and GnRH-induced {alpha} promoter activity. Primary pituitary cells from male and female rats were cotransfected with 15 µg -420 {alpha}LUC along with CMV-jun (100 ng) and/or CMV-jun (100 ng) plus CMV-Fos (1 µg). Six hours after transfection, the cells were treated with GnRH (1 nM). Controls were transfected with equal amounts of control plasmids. Luciferase activity was measured 20–24 h after treatment. Values shown are the mean ± SEM of triplicate transfections.

 
Ca2+-dependent signaling pathways are involved in the sensitizing effects of estradiol on GnRH action
Several different experimental approaches were used to investigate the potential role of Ca2+-dependent pathways on the sensitizing effects of GnRH in females. Initially, transfected pituitary cells were treated with GnRH after depletion of extracellular Ca2+ from the culture medium using the chelator EGTA (5 mM) for 20–24 h before luciferase assay. When cells were treated with EGTA, GnRH-induced {alpha} gene activity was minimally altered in males, but was significantly reduced in females (from 133- to 20-fold; P < 0.05; Fig. 5Go).



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  		Figure 5. Effects of Ca2+ deprivation on basal and GnRH-induced {alpha} promoter activity. Primary pituitary cells from male and female rats were transfected with 15 µg -420 {alpha}LUC. Six hours after transfection, the cells were treated with GnRH (1 nM). Luciferase activity was measured 20–24 h after treatment. To deplete culture medium of Ca2+, cells were incubated with EGTA (5 mM) 20–24 h before luciferase assays. Values shown are the mean ± SEM of triplicate transfections.

 
Methoxyverapamil (D600; 1 µM), a T- and L-type Ca2+ channel inhibitor (28), also markedly inhibited the degree of GnRH responsiveness in females (from 208- to 23-fold; P < 0.05; Fig. 6Go). After ovariectomy, the effect of D600 on GnRH stimulation in females was less pronounced (Fig. 7Go). As found previously (22), replacement with estradiol for 10 days in OVX females restored GnRH responsiveness and also reconstituted inhibition by the Ca2+ channel inhibitor (Fig. 7Go).



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  		Figure 6. Effects of methoxyverapamil (D600) on basal and GnRH-induced {alpha} promoter activity. Primary pituitary cells from male and female rats were transfected with 15 µg -420 {alpha}LUC. Six hours after transfection, the cells were treated with GnRH (1 nM) alone or with GnRH (1 nM) plus D600 (1 µM), a T- and L-type Ca2+ channel inhibitor. Luciferase activity was measured 20–24 h after treatment. Values shown are the mean ± SEM of triplicate transfections.

 


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  		Figure 7. Effects of methoxyverapamil (D600) on basal and GnRH-induced {alpha} promoter expression in primary pituitary cells from OVX and OVX estrogen-treated (OVX+Est) female rats. Primary pituitary cells from intact females, OVX females (OVX), and estrogen-primed females (OVX/E) were transfected with 15 µg -420 {alpha}LUC. Six hours after transfection, the cells were treated with GnRH (1 nM) alone or with GnRH (1 nM) plus D600 (1 µM), a T- and L-type Ca2+ channel inhibitor. Luciferase activity was measured 20–24 h after treatment. Values shown are the mean of triplicate transfections ± SEM.

 
Two additional Ca2+-dependent pathways (Ca2+ channel-calmodulin kinase-dependent pathway and mobilization of Ca2+ from intracellular stores) were investigated using pimozide and thapsigargin, respectively (29). After treatment with thapsigargin (10 nM), the degree of GnRH responsiveness was unchanged in both sexes. In contrast, pimozide (1 µM) decreased the effects of GnRH, especially in females (133- to 45-fold; P < 0.05; Fig. 8Go).



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  		Figure 8. Effects of thapsigargin and pimozide on basal and GnRH-induced {alpha} promoter activity. Primary pituitary cells from male and female rats were transfected with 15 µg -420 {alpha}LUC. Six hours after transfection, the cells were treated with GnRH (1 nM) alone; with GnRH (1 nM) plus thapsigargin (10 nM), an inhibitor of the endoplasmic reticulum Ca2+ pump; or with GnRH (1 nM) plus pimozide (1 µM), a Ca2+-channel-calmodulin kinase inhibitor. Luciferase activity was measured 20–24 h after treatment. Values shown are the mean ± SEM of triplicate transfections.

 
Effect of dominant negative inhibitors of MAPK isoforms (ERK1 and ERK2) on basal and GnRH-induced {alpha} gene expression in pituitary cells from male and female rats
Because MAPK is partially involved in transducing the transcriptional effects of GnRH (19, 20, 21), we analyzed whether mutant forms of this pathway (ERK1MUT and ERK2MUT) might influence the transcriptional effects of GnRH. After cotransfection with mutant forms of the MAPK pathway, the degree of GnRH responsiveness was reduced in both sexes, but the sexual dimorphism was conserved, suggesting that MAPK-dependent pathways are not directly involved in the sensitizing effects of estradiol (Fig. 9Go).



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  		Figure 9. Effects of dominant negative inhibitors of MAPK isoforms (ERK1 and ERK2) on basal and GnRH-induced {alpha} promoter activity. Primary pituitary cells from male and female rats were cotransfected with 15 µg -420 {alpha}LUC along with CMV-ERK1MUT (10 µg) and/or CMV-ERK1MUT (10 µg) as indicated. Six hours after transfection, the cells were treated with GnRH (1 nM). Controls were transfected with equal amounts of control plasmids. Luciferase activity was measured 20–24 h after treatment. Values shown are the mean ± SEM of triplicate transfections.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Feedback regulation by estradiol at the level of the hypothalamus and pituitary is of fundamental importance for controlling reproduction in females. One effect of estradiol is to sensitize the pituitary to hypothalamic signals such as GnRH (13, 14, 30). Estradiol increases GnRH receptor messenger RNA (mRNA) and receptors (31, 32), enhances voltage-sensitive calcium currents (33) and GnRH-stimulated calcium influx (34), and increases PKC (35). In this report, we provide evidence that two previously characterized pathways for GnRH-mediated transcription (PKC and calcium) are enhanced by estrogen.

Recently, we developed an experimental model that allowed the detection of marked estrogen-dependent effects on GnRH signaling pathways (22). This model used primary cultures of pituitary cells that have been transiently transfected with the GnRH-responsive reporter gene, {alpha}-LUC. Several key features of this model should be emphasized. 1) The GnRH-induced responses of {alpha}-LUC are strikingly sexually dimorphic, primarily reflecting the effects of estrogen. 2) The effects of estrogen appear to require in vivo exposure for more than 3 days, 3) Estrogen treatment reduces the basal activity of {alpha}-LUC as well as enhancing the GnRH-dependent responses of the promoter. Although this is admittedly an artificial experimental system, the model is useful for analyzing the transcriptional effects of estrogen in the gonadotrope. In practical terms, the large degree of estrogen enhancement of GnRH-stimulated {alpha}-LUC activity (from ~5-fold to greater than ~100-fold) allows the effects of inhibitors to be analyzed with greater confidence.

For several reasons, the effects of GnRH on the {alpha}-LUC reporter gene appear to reflect its actions on the endogenous gonadotropin genes. The gonadotropin {alpha} and LHß mRNAs are increased by treatment with GnRH, and this effect is mimicked by the addition of PMA or the Ca2+ ionophore, ionomycin. In addition, the stimulatory effect of GnRH on gonadotropin subunit mRNA levels is blocked by the PKC inhibitors, incubation in Ca2+-free medium, or Ca2+ channel blockers (24, 32). Analogous experiments have been performed using the human {alpha}-LUC reporter gene transfected into {alpha}T3 gonadotrope cells (36, 37). As with the endogenous genes, these studies reveal a critical role for PKC and extracellular Ca2+ for transcriptional stimulation of the {alpha} promoter.

We used several different pharmacological approaches to help define the pathways involved in estrogen-enhanced transcription by GnRH. Although the interpretation of this type of experiment is inherently limited by possible nonspecific effects, we attempted to use independent approaches whenever possible. In the case of the PKC pathway, one paradigm used depletion of the enzyme by treatment with phorbol esters, whereas another used inhibition with staurosporine. With either approach, inhibition of the PKC pathway markedly reduced GnRH-induced {alpha}-LUC activity in females, but had little effect in males. Other studies have demonstrated effects of estradiol on the PKC pathway in gonadotropes. PKC activity is higher in female pituitary cells than in those from males, and chronic treatment of OVX females with estradiol increases total PKC activity as well as GnRH- and PMA-induced LH synthesis and release (35). Increased PKC activity is also found in pituitaries induced to undergo hyperplasia by long term treatment with estradiol (38). Whether these estrogen-induced increases in PKC activity account for its ability to enhance GnRH-induced transcription of the {alpha} promoter will require further studies, perhaps using strategies that overexpress PKC isoforms.

GnRH stimulates the expression of c-jun and c-fos (AP-1) in gonadotrope cells (26). This effect of GnRH is mimicked by PMA, and depletion of PKC reduces GnRH- and PMA-induced expression of these early response genes. The PKC inhibitor, staurosporine, also attenuates GnRH stimulation of c-jun and c-fos (26). Because the transcription factor AP-1 (c-Jun/c-Fos) may mediate some of the transcriptional effects of the GnRH/PKC pathway (26, 39, 40), we examined whether expression of c-jun and c-fos alters basal or GnRH-activated {alpha} gene activity in male and female pituitary cells. AP-1 repressed basal activity in both sexes, but it had little effect on the degree of stimulation by GnRH. The basal suppression by AP-1 is consistent with previous studies showing that c-jun represses {alpha} promoter expression in JEG-3 cells (27). Because basal suppression by AP-1 was seen in both males and females, it seems unlikely to account for the ability of estradiol to reduce basal {alpha} promoter activity. Moreover, the inability of AP-1 to enhance transcriptional responses to GnRH suggests that this effect probably involves other transcription factor pathways.

The MAPK cascade is activated by GnRH (19, 20, 21) and represents a possible pathway for transcriptional stimulation of the {alpha} promoter. Previous studies showed that GnRH stimulation of MAPK activity was dependent upon PKC and that depletion of PKC impaired GnRH-stimulated {alpha}-LUC activity (20, 21). In this report, dominant negative MAPK mutants reduced GnRH stimulation of the {alpha} promoter. However, this effect was modest, and the degrees of inhibition were similar between sexes. These findings are consistent with a role for MAPK in regulation of the {alpha} promoter (19, 20), but suggest that it does not account for the estrogen-dependent sexual dimorphism.

GnRH induces striking changes in cytosolic Ca2+, reflecting the release of intracellular Ca2+ stores as well as the influx of extracellular Ca2+ (13, 41). In addition to well characterized effects on hormone secretion, changes in Ca2+ play an important role in transcription (42, 43, 44). With respect to the {alpha} promoter, several previous studies have demonstrated that increases in intracellular Ca2+ stimulate transcriptional activity (25, 37, 45, 46). The transcription factor, cAMP response element (CRE)-binding protein (CREB), is a major regulator of the {alpha} promoter (47) and is activated by Ca2+ as well as cAMP pathways (48, 49). Therefore, we assessed whether Ca2+ pathways might also mediate estrogen-dependent effects on GnRH signaling. Like inhibition of PKC, substances (D600 and EGTA) that directly or indirectly block the influx of extracellular Ca2+ markedly inhibited the transcriptional effects of GnRH. The inhibitory action of the calcium channel inhibitor, D600, was more pronounced in intact females and in estrogen-primed OVX females than in males or OVX females. Treatment with pimozide, an inhibitor of Ca2+ channels and calmodulin-dependent kinases, blunted the estrogen effect, suggesting that this enzyme pathway may mediate some of the effects of calcium.

The finding that extracellular Ca2+ influx may play a role in estrogen’s effects on transcription is reminiscent of the positive effect of estrogen on GnRH-induced LH release, which is also linked to greater influxes of Ca2+ in female gonadotrope cells (50, 51, 52, 53). It is possible that increased GnRH responsiveness in females might be mediated by estradiol-induced effects on the activity of Ca2+ channels. For example, estrogen increases the number of functional Ca2+ channels in the plasma membranes of GH3 cells (53).

In contrast to the results with D600 and pimozide, thapsigargin, an inhibitor of Ca2+ pumps in the endoplasmic reticulum (54, 55), had no apparent effect on GnRH-induced {alpha} gene expression. The finding that thapsigargin has minimal effects on the estrogen-dependent enhancement of GnRH action in females is consistent with previous studies showing that thapsigargin does not inhibit GnRH stimulation of {alpha}-LUC activity in {alpha}T3 cells (37). Although additional studies are required to unravel the relative contributions of extracellular and intracellular Ca2+ stores, these results suggest that the influx of extracellular Ca2+ may be more important for the transcriptional effects of estrogen and GnRH.

A challenge for future studies is to identify the genes and cellular targets that are activated by estrogen in the gonadotrope. Because the effects of estrogen require relatively long treatments (>3 days), it is possible that some its effects are indirect and may involve the activation of a cascade of genetic events. It is also of interest to consider how the PKC and Ca2+ signaling pathways stimulate the transcription of genes such as the {alpha} promoter. Although transcription factor CREB is a likely target for these pathways, there is also evidence that regulatory elements upstream of the cAMP response element are involved in stimulation by GnRH and Ca2+ signaling pathways (37, 45, 56, 57).


   Acknowledgments
 
We are grateful to Dr. A. C. Bauer-Dantoin and V. Sundaresan for helpful suggestions.


   Footnotes
 
1 This work was conducted as a part of the National Cooperative Program on Infertility Research and was supported by NIH Grant U54-HD-29164 and core facilities from NIH Grant P30-HD-28048. Back

2 Recipient of grants from NATO, the Fogarty International Center (NIH), and the Belgian National Fund for Scientific Research. Back

Received February 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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