Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene Is Partially Mediated by Protein Kinase C Activation of an Activator Protein-1 Element

Brett R. White, Dawn L. Duval, Jennifer M. Mulvaney, Mark S. Roberson and Colin M. Clay

Animal Reproduction and Biotechnology Laboratory (B.R.W., D.L.D., C.M.C.) Department of Physiology College of Veterinary Medicine and Biomedical Sciences Foothills Campus, Colorado State University Fort Collins, Colorado 80523
Department of Biomedical Sciences (J.M.M., M.S.R.) Cornell University Ithaca, New York 14853


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Homologous regulation of GnRH receptor (GnRHR) gene expression is an established mechanism for controlling the sensitivity of gonadotropes to GnRH. We have found that expression of the GnRHR gene in the gonadotrope-derived {alpha}T3–1 cell line is mediated by a tripartite enhancer that includes a consensus activator protein-1 (AP-1) element, a binding site for SF-1 (steroidogenic factor-1), and an element we have termed GRAS (GnRHR-activating sequence). Further, in transgenic mice, approximately 1900 bp of the murine GnRHR gene promoter are sufficient for tissue-specific expression and GnRH responsiveness. The present studies were designed to further delineate the molecular mechanisms underlying GnRH regulation of GnRHR gene expression. Vectors containing 600 bp of the murine GnRHR gene promoter linked to luciferase (LUC) were transiently transfected into {alpha}T3–1 cells and exposed to treatments for 4 or 6 h. A GnRH-induced, dose-dependent increase in LUC expression of the -600 promoter was observed with maximal induction of LUC noted at 100 nM GnRH. We next tested the ability of GnRH to stimulate expression of vectors containing mutations in each of the components of the tripartite enhancer. GnRH responsiveness was lost in vectors containing mutations in AP-1. Gel mobility shift data revealed binding of fos/jun family members to the AP-1 element of the murine GnRHR promoter. Treatment with GnRH or phorbol-12-myristate-13-acetate (PMA) (100 nM), but not forskolin (10 µM), increased LUC expression, which was blocked by the protein kinase C (PKC) inhibitor, GF109203X (100 nM), and PKC down-regulation (10 nM PMA for 20 h). In addition, a specific MEK1/MEK2 inhibitor, PD98059 (60 µM), reduced the GnRH and PMA responses whereas the L-type voltage-gated calcium channel agonist, ±BayK 8644 (5 µM), and antagonist, nimodipine (250 nM), had no effect on GnRH responsiveness. Furthermore, treatment of {alpha}T3–1 cells with 100 nM GnRH stimulated phosphorylation of both p42 and p44 forms of extracellular signal-regulated kinase (ERK), which was completely blocked with 60 µM PD98059. We suggest that GnRH regulation of the GnRHR gene is partially mediated by an ERK-dependent activation of a canonical AP-1 site located in the proximal promoter of the GnRHR gene.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Upon binding to specific, high-affinity receptors on gonadotrope cells of the anterior pituitary gland, GnRH stimulates expression of genes encoding the common {alpha}-subunit and specific LHß- and FSHß-subunits that combine to produce LH or FSH (1, 2, 3, 4). GnRH also stimulates the secretion of these pituitary gonadotropins that are essential for normal gonadal function in both males and females (4, 5). Therefore, the interaction of GnRH with its cognate pituitary receptor serves as a central point for regulation of reproductive function.

Given the role of GnRH in stimulating gonadotropin synthesis and secretion, it is not surprising that changes in the secretory rate of LH are highly dependent on the level of hypothalamic GnRH secretion (6, 7). Additionally, changes in numbers of GnRH receptors (GnRHRs) are correlated with changes in LH secretion (8). Thus, regulation of LH secretion appears to be dependent not only on GnRH availability, but also on the number of GnRHRs and, consequently, the sensitivity of the pituitary gland to a given dose of GnRH (9). In this regard, a number of endocrine factors, including 17ß-estradiol, progesterone, testosterone, inhibin, activin, and GnRH itself, have been implicated as mediating dynamic changes in the numbers of GnRHRs in the pituitary gland (10, 11, 12, 13, 14, 15, 16, 17). Of these, perhaps the most dramatic effects are those mediated by GnRH and 17ß-estradiol. Stimulatory effects of these two hormones on GnRHR numbers have been demonstrated in several different species (14, 18, 19, 20). This regulation presumably represents a physiologically relevant avenue for increasing the sensitivity of the pituitary gland to GnRH during the periovulatory period (21). More recently, with the availability of cDNAs for the GnRHR, researchers have found that changes in GnRHR numbers associated with GnRH and/or 17ß-estradiol treatment largely correlate with concomitant fluxes in steady state levels of mRNA (22, 23, 24, 25, 26, 27, 28). Thus, regulation of GnRHRs by these two hormones may involve a transcriptional component.

To examine the molecular mechanisms underlying transcriptional regulation of the GnRHR gene, we have cloned the gene encoding the murine GnRHR (29) and have begun analyzing the regulatory regions within the promoter of this gene. We have found that expression of the murine GnRHR gene in the gonadotrope-derived {alpha}T3–1 cell line (30) is mediated by a tripartite enhancer located within 600 bp of proximal 5'-flanking region. The components of this enhancer include a binding site for the nuclear orphan receptor SF-1 (steroidogenic factor-1) (31), a consensus activator protein-1 (AP-1) element, and a noncanonical element we have termed the GnRHR-activating sequence or GRAS (32, 33). In addition, we have constructed transgenic mice harboring a transgene consisting of approximately 1900 bp of 5'-flanking sequence from the murine GnRHR gene linked to the cDNA encoding luciferase (LUC) (34). LUC expression in these animals was confined to the pituitary gland, brain, and testes, all established sites of expression of the endogenous GnRHR gene. We also found that pituitary expression of LUC in these transgenic mice was diminished by immunoneutralization with GnRH antisera and subsequently restored by administration of a non-cross-reactive GnRH agonist (34). Thus, we concluded that 1900 bp of proximal promoter from the murine GnRHR gene contains not only the elements that confer tissue-specific expression but also one or more regulatory elements that act to confer GnRH responsiveness in vivo.

Several GnRH responsive elements have been identified in other genes that are targets for GnRH activation, including the upstream GnRH response element (GnRH-RE) and pituitary glycoprotein hormone basal element (PGBE) in the murine {alpha}-subunit gene (35), the GnRH-RE of the human {alpha}-subunit gene (36), and two regions referred to as regions A and B in the rat LHß-subunit gene promoter (37). However, we were not able to identify homologies to any of these candidate elements in the GnRHR gene, suggesting that the GnRH-responsive element(s) in the GnRHR gene may be distinct from those previously defined in either the glycoprotein hormone {alpha}- or LHß-subunit genes. We were intrigued with the possibility that one or more of the elements comprising the basal, tripartite enhancer of the GnRHR gene (33) may also be involved with mediating GnRH responsiveness. For example, AP-1 is a well established mediator of several signal transduction pathways, including protein kinase C (PKC) (38, 39, 40). Furthermore, GnRH activates transcription of fos and jun in pituitary and {alpha}T3–1 cells (41, 42), and temporal patterns of expression of GnRHR and jun in the pituitary gland are similar during the ovine estrous cycle (41). Similarly, several lines of evidence implicate a role for SF-1 in at least partially mediating GnRH responsiveness. GnRH regulation of SF-1 mRNA has been demonstrated in the rat pituitary gland (43), and surgical disconnection of the hypothalamus and pituitary leads to a loss of SF-1 mRNA in the ovine pituitary gland (44). Also, disruption of the SF-1-binding site in the bovine LHß-subunit gene promoter leads to a loss of GnRH responsiveness of this promoter in transgenic mice (45). Finally, since the identity of the protein(s) binding to GRAS is not yet known, we cannot exclude a potential role for this element in mediating GnRH responsiveness.

In a similar vein, little is known as to the pathways involved in GnRH regulation of GnRHR gene expression. In {alpha}T3–1 cells, it appears that GnRH treatment activates PKC with little effect on intracellular concentrations of cAMP (46). Also, treatment of rat primary pituitary cultures with GnRH has revealed a crucial role for mitogen-activated protein kinase (MAPK) in regulation of GnRHR mRNA levels (47), an effect likely mediated by PKC (48). Recently, however, Lin and Conn (49) have suggested that GnRH activation of the GnRHR promoter occurs via cAMP and protein kinase A (PKA) in a GH3 cell line engineered to express GnRHRs (GGH3). Thus, the goals of the present studies were to investigate the relative roles of PKC, PKA, or calcium in mediating GnRH activation of the GnRHR gene promoter. Additionally, we sought to identify the GnRH-responsive element(s) located in the proximal promoter of the murine GnRHR gene. Herein, we report that GnRH responsiveness of the GnRHR gene promoter in {alpha}T3–1 cells is dependent on PKC activation of MAPK and is ultimately mediated at a canonical AP-1 site that binds members of the jun and fos family of transcription factors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GnRHR Promoter Is Responsive to Increasing Doses of GnRH in {alpha}T3–1 Cells
To establish the utility of the gonadotrope-derived {alpha}T3–1 cell line as a model for GnRH regulation, we examined the response of 600 bp of proximal promoter from the murine GnRHR gene to increasing doses of GnRH. A GnRH dose-dependent increase in expression of -600 LUC was observed (Fig. 1Go). The fold-induction of cells treated with 100, 1,000, and 10,000 nM GnRH was higher (P < 0.05) than in untreated cells, whereas treatment with 0.1, 1, and 10 nM GnRH was not different (P > 0.05) from controls. Activation at these concentrations is in range with established affinities of GnRH for its receptor (46). The somewhat higher concentration of GnRH necessary to activate the GnRHR promoter may reflect the lower number of receptors on {alpha}T3–1 cells as compared with bona fide gonadotropes (46). The specificity of the GnRH response was tested by addition of increasing doses of the competitive GnRH antagonist Antide (0.001, 0.1, 10, and 1,000 nM) in the presence of 100 nM GnRH. The ability of 100 nM GnRH to stimulate the -600 promoter was blocked by inclusion of 0.1 nM Antide (Fig. 1Go).



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Figure 1. Expression of Murine GnRHR -600 LUC with Increasing Doses of GnRH or Antide in {alpha}T3–1 Cells

A vector containing approximately 600 bp of 5'-flanking region from the murine GnRHR gene fused to LUC was cotransfected with RSV-ßgal using a calcium phosphate-DNA coprecipitation protocol as described in Materials and Methods. Cells were transfected and treated with either 0, 0.1, 1, 10, 100, 1,000, or 10,000 nM GnRH. For antagonist treatments, cells were transfected as above, treated for 30 min with 0, 0.001, 0.1, 10, or 1,000 nM Antide, and treated with 100 nM GnRH. At 4 h post transfection, cells were harvested and cellular lysates were assayed for LUC and ß-galactosidase activity. LUC activity was adjusted for ß-galactosidase activity, and values are expressed as fold increase over the untreated -600 LUC controls. Values represent the mean ± SEM. An asterisk indicates values that are greater (P < 0.05) than that of the untreated -600 LUC control.

 
AP-1 Is Critical for GnRH Responsiveness of the GnRHR Promoter
The retention of GnRH responsiveness to within 600 bp of 5'-flanking sequence is consistent with the possibility that one or more of the three elements comprising the cell-specific basal enhancer (33) may also serve to confer GnRH responsiveness. Thus, we next tested the ability of GnRH to stimulate expression of GnRHR promoters containing mutations in each of the three components of the tripartite, basal enhancer alone or in combination in the context of -600 LUC to determine whether any of these previously identified elements may mediate GnRH responsiveness. The fold-induction of the wild-type vector, µSF-1, µGRAS, and the µGRAS/µSF-1 double mutant by GnRH was greater (P < 0.05) than that of promoterless control (Fig. 2Go) and not different (P < 0.05) from the -600 wild-type construct. However, GnRH responsiveness of vectors containing the mutation in AP-1, either alone or in combination with GRAS or SF-1, was not different from promoterless control (P > 0.05; Fig. 2Go) and below (P < 0.05) that for the wild-type -600 vector. Finally, basal expression of the GnRHR promoter constructs was not different (P > 0.05) from promoterless control. These results indicated that GnRH responsiveness of the GnRHR gene promoter was acting through the consensus AP-1 element located between -336 and -330 relative to the start site of translation in the murine GnRHR gene promoter.



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Figure 2. Effect of Mutations in the Tripartite Enhancer on GnRH Responsiveness of the Murine GnRHR Gene

Vectors containing mutations in a single element, two elements, or all three elements of the tripartite, basal enhancer of the murine GnRHR gene were cotransfected with RSV-ßgal using a calcium phosphate-DNA coprecipitation protocol as described in Materials and Methods. Cells were transfected and treated with 100 nM GnRH. At 4 h post transfection, cells were harvested, and cellular lysates were assayed for LUC and ß-galactosidase activity. LUC activity was adjusted for ß-galactosidase activity, and values are expressed as fold increase over untreated for each mutation. Values represent the mean ± SEM. An asterisk indicates values that are greater (P < 0.05) than that of the promoterless LUC vector.

 
Members of the Fos/Jun Family of Transcription Factors Bind to the Murine GnRHR AP-1 Element
We determined whether a radiolabeled probe containing the consensus AP-1 element from the murine GnRHR gene promoter could bind to protein(s) in whole-cell extracts from {alpha}T3–1 cells. Competition with 10-, 100-, and 500-fold molar excess of homologous unlabeled DNA identified a sequence-specific complex, whereas the addition of heterologous competitor (10-, 100-, and 500-fold molar excess) did not compete for binding (Fig. 3Go). To determine whether the protein complex binding to the consensus AP-1 element was comprised of members of the fos/jun family of transcription factors, we tested broad-spectrum antibodies that recognize all members of either the jun or fos families of transcription factors for their ability to alter binding to this complex. The jun antibody is directed against the conserved DNA-binding domain of jun family members and thus blocks accessibility of the DNA to the jun DNA-binding domain resulting in attenuated binding. In contrast, binding of the fos antibody, which is directed against a conserved, non-DNA-binding region in each of the fos family members, results in a higher mol wt complex (i.e. supershift). The addition of antibodies for either jun (1, 2, or 4 µg) or fos (1 or 2 µg) family members resulted in attenuated binding or a supershifted complex, respectively (Fig. 3Go). Neither the addition of 1, 2, or 4 µg of an antibody directed against cAMP-regulatory element binding protein-1 (CREB-1) nor similar concentrations of rabbit IgG affected binding of cellular proteins to the radioactive murine AP-1 probe.



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Figure 3. Members of the fos and jun Families of Transcription Factors bind to the Consensus AP-1 Element in the Murine GnRHR Gene Promoter

Whole-cell extracts from {alpha}T3–1 cells were incubated with a radiolabeled probe consisting of the consensus AP-1 element from the murine GnRHR gene promoter. Specificity of DNA-protein interactions was assessed by competition with 10-, 100-, and 500-fold molar excess of homologous and heterologous unlabeled DNA. In addition, whole-cell extracts were incubated with a goat polyclonal antibody directed against the DNA-binding domain of mouse c-jun p-39 (1, 2, or 4 µg), a rabbit polyclonal antibody directed against a conserved domain of human c-fos p62 (1 or 2 µg), a mouse monoclonal antibody directed against the DNA-binding and dimerization domain of human CREB-1 (1, 2, or 4 µg), or an equal mass of rabbit IgG before the addition of radiolabeled probe. Binding reactions were subjected to electrophoresis through polyacrylamide gels as described in Materials and Methods.

 
Activation of the GnRHR Promoter by GnRH Is Mediated by Protein Kinase C
We next determined whether pharmacological activation of either PKC [100 nM phorbol-12-myristate-13-acetate (PMA)] or PKA (10 µM forskolin) could mimic the GnRH response of the -600 GnRHR promoter. Treatment with GnRH (100 nM), PMA, or GnRH and PMA in combination increased (P < 0.05) expression of -600 LUC (4.1-, 8.8-, and 18.4-fold, respectively) as compared with untreated controls (Fig. 4Go). Interestingly, while forskolin alone had no effect on expression of -600 LUC, the inclusion of forskolin essentially abrogated induction of LUC expression by GnRH, PMA, or the combined GnRH/PMA treatments (Fig. 4Go). None of the treatments resulted in any change (P > 0.05) in LUC expression from the promoterless control vector (data not shown).



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Figure 4. Effects of GnRH and Activators of the Protein Kinase A or C Second Messenger Pathways on Expression of Murine GnRHR -600 LUC in {alpha}T3–1 Cells

A vector containing approximately 600 bp of proximal 5'-flanking region from the murine GnRHR gene fused to LUC was cotransfected with RSV-ßgal using a calcium phosphate-DNA coprecipitation protocol as described in Materials and Methods. Cells were transfected and treated with 100 nM GnRH, 100 nM PMA, 10 µM forskolin (FSK), or each combination of these treatments. At 4 h post transfection, cells were harvested and cellular lysates were assayed for LUC and ß-galactosidase activity. LUC activity was adjusted for ß-galactosidase activity, and values are expressed as fold increase over untreated -600 LUC. Values represent the mean ± SEM. An asterisk represents values that are greater (P < 0.05) than that of the untreated -600 LUC control.

 
Activation of the -600 murine GnRHR promoter by PMA and not forskolin suggests a PKC-mediated pathway for induction of GnRHR gene expression. To further examine this possibility, we added 100 nM of GF109203X (Bisindolylmaleimide I), a PKC inhibitor, in the presence of 100 nM GnRH or PMA. Consistent with PKC-mediated activation, the inclusion of 100 nM GF109203X completely blocked both GnRH- and PMA-mediated induction of -600 LUC (Fig. 5Go). The specificity of this inhibition was tested by examining the response of 1500 bp of proximal promoter from the human glycoprotein hormone {alpha}-subunit gene, a well established PKA-responsive promoter (50), to forskolin in the presence of GF109203X. The fold-induction of human {alpha} -1500 LUC by forskolin was not affected by GF109203X, indicating that the inhibition of GnRH and PMA induction of the murine GnRHR promoter by GF109203X was specific to PKC (Fig. 5Go).



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Figure 5. Effect of GF109203X on GnRH and PMA Responsiveness of the Murine GnRHR Promoter

Vectors containing approximately 600 bp of 5'-flanking region from the murine GnRHR gene or 1500 bp of proximal 5'-flanking region from the human glycoprotein hormone {alpha}-subunit gene fused to LUC were cotransfected with RSV-ßgal using a calcium phosphate-DNA coprecipitation protocol as described in Materials and Methods. Cells were transfected, treated with or without 100 nM GF109203X (GFX) for 15 min, and treated with either 100 nM GnRH, 100 nM PMA, both GnRH and PMA, or 10 µM forskolin (FSK). At 4 h post transfection, cells were harvested and cellular lysates were assayed for LUC and ß-galactosidase activity. LUC activity was adjusted for ß-galactosidase activity, and values are expressed as fold increase over untreated -600 LUC. Values represent the mean ± SEM. An asterisk represents values that are greater (P < 0.05) than that of the untreated -600 LUC control.

 
As further confirmation of PKC dependence of the GnRHR gene response to GnRH, {alpha}T3–1 cells were treated with 10 nM PMA for 20 h to down-regulate PKC (51). After 20 h of 10 nM PMA, the GnRH, PMA, and combined responses were completely blocked (Fig. 6Go).



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Figure 6. Effect of PKC Down-Regulation on GnRH and PMA Responsiveness of the Murine GnRHR Promoter

Vectors containing approximately 600 bp of 5'-flanking region from the murine GnRHR gene fused to LUC were cotransfected with RSV-ßgal using a calcium phosphate-DNA coprecipitation protocol as described in Materials and Methods. Approximately 20 h before experimental treatments, one-half of the cells were treated with 10 nM PMA to induce PKC down-regulation, and the other half remained untreated for controls. Cells were transfected and treated with either 100 nM GnRH, 100 nM PMA, or both GnRH and PMA. At 6 h post transfection, cells were harvested and cellular lysates were assayed for LUC and ß-galactosidase activity. LUC activity was adjusted for ß-galactosidase activity, and values are expressed as fold increase over untreated -600 LUC. Values represent the mean ± SEM]. An asterisk represents fold increases that are greater (P < 0.05) than that of the untreated -600 LUC control.

 
GnRH Activation of the GnRHR Promoter Is Mediated by a Mitogen-Activated Protein Kinase Pathway
At least two possible pathways downstream of PKC have been identified as being involved in GnRH responsiveness. One pathway results in the activation of jun-N-terminal kinase (JNK), and the other results in the activation of extracellular signal-regulated kinase (ERK), also known as MAPK (51, 52). Since no specific inhibitors of JNK were available, we examined a specific MEK1/MEK2 (MAPK kinase family members) inhibitor (PD98059) to determine the role of the MAPK pathway on GnRH induction of GnRHR gene expression. As shown previously, GnRH and PMA, alone or in combination, stimulated (P < 0.05) LUC expression (Fig. 7Go). The addition of 60 µM PD98059 resulted in a dramatic reduction (P < 0.05) in the GnRH, PMA, and combined responses (Fig. 7Go). Thus, these results indicate that GnRH responsiveness may be conferred through PKC activation of a MAPK pathway. If correct, then GnRH should lead to ERK activation that in turn can be inhibited by PD98059.



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Figure 7. Effect of PD98059 on GnRH and PMA Responsiveness of the Murine GnRHR Promoter

Vectors containing approximately 600 bp of 5'-flanking region from the murine GnRHR gene fused to LUC were cotransfected with RSV-ßgal using a calcium phosphate-DNA coprecipitation protocol as described in Materials and Methods. Cells were transfected, treated with or without 60 µM PD98059 for 15 min, and treated with either 100 nM GnRH, 100 nM PMA, or both GnRH and PMA. In addition, cells were also treated with 60 µM PD98059 at 3 h after GnRH treatment. At 6 h post transfection, cells were harvested and cellular lysates were assayed for LUC and ß-galactosidase activity. LUC activity was adjusted for ß-galactosidase activity, and values are expressed as fold increase over untreated -600 LUC. Values represent the mean ± SEM. An asterisk represents values that are greater (P < 0.05) than that of the untreated -600 LUC control. A dagger represents values that are less (P < 0.05) than those for cells treated with GnRH, PMA, or both GnRH and PMA in the absence of PD98059.

 
To test this possibility, {alpha}T3–1 cells were treated with the GnRH agonist, buserelin (10 nM), for 0, 15, or 30 min in the presence or absence of 60 µM PD98059 and examined for ERK activation by Western blot analysis using an antibody recognizing phosphorylated p42 and p44 ERK. Phosphorylation of both the p42 and p44 forms of ERK was evident at 15 and 30 min after 10 nM buserelin treatment (Fig. 8Go). At each time point, phosphorylation of both forms of ERK was blocked by PD98059. To control for potential loading differences, the antiphospho-ERK immunoblot was stripped and reprobed using an antisera recognizing ERK protein independent of phosphorylation state (Fig. 8Go).



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Figure 8. The Specific MEK1/MEK2 Inhibitor, PD98059, Blocks GnRH Activation of ERK1 and ERK2 in {alpha}T3–1 Cells

{alpha}T3–1 cells were serum starved for approximately 2 h. Cells received dimethylsulfoxide (Control) or PD98059 (60 µM) 15 min before and for the duration of buserelin administration (0, 15, or 30 min). Cells were then lysed and debris cleared by centrifugation. Lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Initially, the blot was probed with a phospho-specific ERK antibody (p-ERK), which recognizes the dual phosphorylated (thus activated) forms of ERK1 and ERK2. The blot was then stripped and reprobed with an ERK antibody (ERK) that detects relative amounts of ERK protein independent of phosphorylation state. The arrows identify the p44 (upper arrow) and p42 (lower arrow) forms of ERK.

 
GnRH Activation of the GnRHR Promoter Is Not Affected by an Activator or an Inhibitor of L-Type Voltage-Gated Calcium Channels
In addition to MAPK, L-type voltage-gated calcium channels have also been implicated in mediating GnRH regulation of the common {alpha}- and specific LHß-subunit genes. To assess the potential role of L-type voltage-gated calcium channels, we tested the ability of either an agonist (±BayK 8644) or antagonist (nimodipine) of L-type voltage-gated calcium channels to alter the responsiveness of the -600 GnRHR promoter to GnRH in {alpha}T3–1 cells. Neither the addition of 5 µM ±BayK 8644 nor 250 nM nimodipine, alone or in combination with GnRH, affected (P < 0.05) the GnRH response of -600 LUC expression compared with that of controls (Fig. 9Go, A and B). In contrast, consistent with others (53, 54), treatment of the human {alpha} -1500 promoter with ±BayK 8644 increased LUC activity (Fig. 9AGo), whereas nimodipine decreased GnRH-induced LUC activity (Fig. 9BGo).



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Figure 9. Effect of ±BayK 8644 and Nimodipine on GnRH Responsiveness of the Murine GnRHR Promoter

Vectors containing approximately 600 bp of 5'-flanking region from the murine GnRHR gene or 1500 bp of proximal 5'-flanking region from the human glycoprotein hormone {alpha}-subunit gene fused to LUC were cotransfected with RSV-ßgal using a calcium phosphate-DNA coprecipitation protocol as described in Materials and Methods. Cells were transfected, and then treated with or without 5 µM ±BayK 8644 simultaneously with GnRH (100 nM) treatment (panel A) or 250 nM nimodipine for 30 min before GnRH treatment (panel B). At 6 h post transfection, cells were harvested, and cellular lysates were assayed for LUC and ß-galactosidase activity. LUC activity was adjusted for ß-galactosidase activity, and values are expressed as fold increase over untreated -600 LUC or human {alpha}-1500 LUC. Values represent the mean ± SEM. An asterisk represents values that are greater (P < 0.05) than that of the untreated -600 LUC or human {alpha}-1500 LUC controls. A dagger represents values, within each vector, that are different (P < 0.05) from cells treated with GnRH in the absence of either ±BayK 8644 (panel A) or nimodipine (panel B).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The binding of GnRH to its pituitary receptor not only stimulates but is obligatory for the synthesis and secretion of LH. Thus, the GnRHR is the site of the primary stimulatory input to gonadotrope cells and represents a potential control point in regulation of gonadotropin secretion and, consequently, gonadal function in mammals. In fact, changes in the number of pituitary GnRHRs have been implicated as an important mechanism underlying the regulation of LH secretion (8, 9, 10, 26, 27). Consistent with this, several hormones, most notably 17ß-estradiol and GnRH itself, have been shown to regulate both GnRHR numbers and mRNA in the pituitary gland (22, 23, 25, 26, 27, 28).

For the past several years, our laboratory has focused on the molecular mechanisms underlying regulation of GnRHR gene expression. Based on transient expression assays in the gonadotrope-derived {alpha}T3–1 cell line, we have suggested that cell-specific expression of the murine GnRHR gene is mediated by a tripartite enhancer located within 600 bp of proximal 5'-flanking region. The components of this enhancer include a consensus AP-1 element, a binding site for SF-1, and a noncanonical element we have termed GRAS (33). Furthermore, approximately 1900 bp of proximal promoter from the GnRHR gene are capable of conferring tissue-specific expression and GnRH responsiveness on a heterologous reporter gene in transgenic mice (34). In the present study, we have been able to recapitulate GnRH responsiveness in vitro in the {alpha}T3–1 cell line, thus allowing a more refined analysis of the region(s) of the GnRHR gene that confer GnRH regulation.

Based on several lines of evidence, we suggest that PKC-mediated activation of an AP-1 element in the proximal promoter of the GnRHR gene is an important component underlying GnRH regulation of GnRHR gene expression. First, retention of GnRH responsiveness to within 600 bp of proximal 5'-flanking region is consistent with the location of AP-1 between -336 and -330 relative to the start site of translation in the GnRHR gene promoter. Second, mutation of AP-1 alone or in combination with the other two components of the tripartite enhancer of the GnRHR gene leads to loss of GnRH responsiveness. Third, pharmacological activation of PKC, but not PKA, mimics GnRH induction of the -600 GnRHR gene promoter. Fourth, a specific PKC inhibitor (GF109203X) blocks both GnRH and PMA activation of the GnRHR promoter. Finally, down-regulation of the PKC second messenger system dramatically reduces the GnRH and PMA responses of the GnRHR promoter.

Our result indicating that GnRH-induced GnRHR gene expression is mediated by PKC is consistent with the proposed mechanism(s) of action of GnRH. In both gonadotropes and {alpha}T3–1 cells, GnRH-induced signal transduction partially occurs via coupling of the bound GnRHR with Gq{alpha}/G11{alpha}, leading to stimulation of multiple phospholipase activities, formation of inositol 1,4,5-trisphosphate and diacylglycerol, elevation of intracellular free calcium concentrations, and activation of PKC (46, 55). Recently, however, others have reported that GnRH regulation of the GnRHR gene in a heterologous cell line (GH3) is mediated by cAMP and PKA (49). In the present study, neither forskolin nor (Bu)2-cAMP (data not shown) had any detectable stimulatory effect on activity of the GnRHR promoter. In fact, the most striking effect of these compounds was a complete inhibition of both GnRH and PMA induction of the GnRHR gene promoter. Recently, investigators have reported that coupling of a G protein-coupled receptor to G{alpha}q/phospolipase C can be inhibited by cAMP (56). Thus, a relative lack of phospholipase C activity may represent a potential mechanism for attenuation of GnRH responsiveness by forskolin. Alternatively, inhibition of PKC signaling by forskolin could occur by inhibition of the MAPK pathway (57, 58) or phosphorylation of CREB and subsequent inhibition of c-jun (59). Certainly other possibilities exist for cross-talk among these signal transduction pathways (60); however, regardless of the precise mechanisms, it is clear that our results regarding the role of PKA in affecting GnRHR gene expression in {alpha}T3–1 cells is fundamentally different from those obtained in GH3 cells (49). As the mouse GnRHR promoter was common to both of these studies, it would seem that the discrepancy is most likely due to the different cell lines used to detect GnRH regulation.

It is abundantly clear that GnRH can activate a myriad of intracellular signaling pathways, including MAPK and changes in intracellular concentrations of calcium (46, 60). In fact, both of these pathways have been implicated in GnRH regulation of gene expression. Transcriptional induction of the murine glycoprotein hormone {alpha}-subunit gene by GnRH requires activated MAPK (61). In contrast, the GnRH response of the human {alpha}-subunit gene may be more dependent on calcium (32, 62, 63, 64). The picture in regard to the LHß-subunit gene is not entirely clear with conflicting reports as to the relative dependency of MAPK or voltage-gated calcium channels in mediating GnRH responsiveness of the rat LHß-subunit gene promoter (53, 54). Herein, we provide evidence that PKC activation of a MAPK pathway, and not L-type voltage-gated calcium channels, is largely involved in GnRH responsiveness of the GnRHR gene promoter. In support of this, we find that a specific MEK1/MEK2 inhibitor (PD98059) not only reduces the GnRH and PMA responses of the GnRHR promoter but also blocks GnRH-induced ERK phosphorylation in {alpha}T3–1 cells. In contrast, treatment with a calcium channel agonist (±BayK 8644) or antagonist (nimodipine) had little effect on GnRH responsiveness of the GnRHR gene promoter. Thus, these data are consistent with a recent report demonstrating MAPK-dependent induction of GnRHR mRNA levels by GnRH in primary cultures of rat pituitary cells (47).

In addition to activation of MAPK and calcium channels, GnRH has recently been shown to activate the JNK pathway in {alpha}T3–1 cells (51). Due to the absence of specific JNK inhibitors, we were not able to directly address the potential contribution of this pathway to GnRH activation of the GnRHR promoter. However, since GnRH induction of the GnRHR gene is mediated at a canonical AP-1 site that clearly binds one or more jun family members, it is not at all unlikely that the JNK pathway may also play a major role in conferring GnRH responsiveness of the GnRHR gene. If correct, then GnRH induction of GnRHR gene expression may require functional activation of both ERK- and JNK-mediated signaling cascades that ultimately converge at AP-1.

In contrast to GnRH regulation of the {alpha}- and LHß-subunit gene promoters in which multiple elements appear to serve as targets for GnRH signaling, our results indicate that GnRH responsiveness of the GnRHR gene may be largely conferred through a single element. GnRH responsiveness of the murine {alpha}-subunit gene is conferred by an element termed the pituitary glycoprotein basal element or PGBE and an upstream GnRH-RE (35). More specifically, binding sites for a LIM-homeodomain transcription factor (LH-2) within the PGBE (65) and an Ets factor-binding motif within the GnRH-RE (61) appear to be the operative sites for GnRH induction of murine {alpha}-subunit gene expression. The presence of the Ets-binding site is consistent with the involvement of MAPK in mediating GnRH regulation of the murine {alpha}-subunit gene. In regard to GnRH regulation of the LHß-subunit gene, two separate regions contained within 490 bp of proximal promoter appear to interact to confer GnRH responsiveness of the rat LHß-subunit gene in GH3 cells engineered to express GnRHRs; however, the identity of the functional GnRH response element(s) contained within these regions is not known (37). Using a transgenic mouse model, Keri et al. (66) found that 776 bp of 5'-flanking sequence from the bovine LHß-subunit gene were sufficient for GnRH responsiveness. Although the operative GnRH response elements in the bovine LHß-subunit gene have not been conclusively identified, mutation of the SF-1 binding site in the bovine gene led to a significant reduction in basal activity as well as GnRH responsiveness in transgenic mice (45). Thus, regulation of SF-1 binding activity may represent one avenue for GnRH regulation of LHß-subunit gene expression. We were not able, however, to detect any significant role for the SF-1-binding site in the GnRHR gene in mediating GnRH responsiveness. Rather, our data indicate that virtually all of the GnRH responsiveness of the GnRHR promoter is mediated at a single AP-1 site. Thus, while common pathways may underlie GnRH regulation of its primary target genes in gonadotropes, different elements in the promoters of the {alpha}-subunit, LHß-subunit, and GnRHR genes ultimately mediate the GnRH response.

In summary, the past several years have witnessed enormous progress in our understanding of the molecular mechanisms underlying GnRH regulation of {alpha}- and LHß-subunit gene expression (46). In contrast, GnRH regulation of the GnRHR gene has remained relatively unexplored. Results from the present studies suggest that GnRH regulation of GnRHR gene expression is partially mediated by PKC/MAPK activation of a canonical AP-1 site located in the proximal promoter of the GnRHR gene. Also, it is clear that both jun and fos family members are present in {alpha}T3–1cells and are capable of binding to the AP-1 site in the GnRHR promoter. In this regard, it is important to note that while both jun and fos are established targets for GnRH regulation both in vivo and in {alpha}T3–1 cells (41, 42), a clear role for these proteins in mediating GnRH responsiveness has been lacking. As such, these data provide a functional, candidate target element for jun and fos in a well established GnRH-responsive gene and contribute to our expanding knowledge of the repertoire of elements and factors used by GnRH to communicate with its primary target genes in gonadotropes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Forskolin, PMA, and GnRH antagonist (Antide) were purchased from Sigma Chemical Co. (St. Louis, MO). The GnRH was obtained from Bachem (Philadelphia, PA). The GF109203X (Bisindolylmaleimide I), nimodipine, ±BayK 8644, and PD98059 were purchased from Calbiochem (La Jolla, CA). Antibodies for jun (c-Jun/AP-1 [D]-G, catalog no. sc-44-G), fos (c-Fos [K-25], catalog no. sc-253), and CREB (CREB-1 [X-12], catalog no. sc-240) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Plasmids
The plasmid -600 LUC consisted of 600 bp of 5'-flanking region from the murine GnRHR gene fused to the cDNA encoding LUC in the pGL3 basic vector (Promega, Madison, WI) (29). The construction of vectors containing mutations in the individual elements of the tripartite basal enhancer (µGRAS, µAP-1, and µSF-1) were described previously (33). The mutant vectors contained either a NotI site (µGRAS and µSF-1) or an EcoRI site (µAP-1) in place of the wild-type sequence. Double and triple mutants that included all combinations of these mutated elements were also constructed (33). The human {alpha}-1500 LUC vector consisted of approximately 1500 bp of 5'-flanking region from the human {alpha}-subunit gene promoter linked to LUC (50). The control vector used to test for transfection efficiency in all experiments contained the Rous sarcoma virus promoter linked to the cDNA encoding ß-galactosidase (RSV-ßgal).

Cell Culture and Transient Transfections
Cultures of {alpha}T3–1 cells were maintained at 37 C in a humidified 5% CO2 in air atmosphere. Cells were cultured before transfection in high-glucose DMEM containing 2 mM glutamine, 5% FBS, 5% horse serum, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate (Mediatech, Herndon, VA). After transfection, the cells were cultured in the same medium without FBS. Transient transfections were carried out using a calcium phosphate/DNA coprecipitation method as previously described (50). Briefly, the day before transfection, 2 x 106 cells were plated in 100-mm tissue culture dishes. Complete media were removed, and calcium phosphate/DNA precipitates in a total volume of 1 ml were added to the plates. At 30 min, posttransfection media were added, and cells were treated for either 4 or 6 h with either GnRH or the treatment as indicated. Results of a 4-h treatment are depicted in Figs. 1Go, 2Go, 4Go, and 5Go; however, further analysis of the time course of the GnRH response revealed a greater response at 6 h; therefore, Figs. 6Go, 7Go, and 9Go show results of a 6-h treatment. In Fig. 1Go, the GnRH antagonist Antide (67) was added 30 min before GnRH. In Fig. 5Go, the PKC inhibitor GF109203X (68) was added 15 min before GnRH, PMA, or forskolin. In Fig. 7Go, the MEK1/MEK2 inhibitor PD98059 was added 15 min before GnRH or PMA and again at 3 h after treatment. In Fig. 9Go, the L-type calcium channel antagonist nimodipine was added 30 min before GnRH. Within each assay, treatments were performed in triplicate, and different plasmid preparations were used for each assay. After either 4 or 6 h of treatment, cells were washed twice with ice-cold PBS, harvested in 1 ml of ice-cold PBS containing 1 mM EDTA, concentrated by centrifugation at 300 x g for 5 min, and lysed in 200 µl of 25 mM glycyl-glycine (pH 7.8), 15 mM MgSO4, 1% Triton-X100, and 1 mM dithiothreitol. Lysates were cleared by centrifugation at 16,000 x g for 2 min. Lysates (20 and 50 µl for LUC and ß-galactosidase, respectively) were assayed according to manufacturer’s instructions for LUC (Promega, Madison, WI) and ß-galactosidase (Topix, Bedford, MA) activity using a Turner 20D luminometer (Turner Designs, Sunnyvale, CA). LUC values were divided by ß-galactosidase activity to normalize for transfection efficiency (33).

Gel-Shift Assays
Whole-cell extracts from {alpha}T3–1 cells were prepared by the method of Manley et al. (69). Gel-shift assays were conducted as previously described (29). Briefly, whole-cell extracts (5.1 µg of protein) were incubated for 10 min at 4 C in 20 µl of Dignam buffer D [20 mM HEPES (pH 7.9), 20% glycerol (vol/vol), 0.1 M KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol] containing 2 µg of poly(dI-dC) (Pharmacia Biotech, Piscataway, NJ) and, where indicated, either a goat polyclonal antibody directed against the DNA-binding domain of mouse c-jun p-39 (1, 2, or 4 µg), a rabbit polyclonal antibody directed against a conserved domain of human c-fos p62 (1 or 2 µg), a mouse monoclonal antibody directed against the DNA-binding and dimerization domain of human CREB-1 (1, 2 or 4 µg), or an equal mass of rabbit IgG. After incubation, the radiolabeled probe (100,000 cpm) was added, and, where indicated, unlabeled competitor. Reactions were incubated at room temperature for 30 min, and free probe was separated from bound probe by electrophoresis for 1–2 h at 35 mA in 6% polyacrylamide gels that were prerun at 100 V for 30 min in 25 mM Tris, 190 mM glycine, and 1 mM EDTA, pH 8. Gels were transferred to blotting paper, dried, and exposed to Hyperfilm MP (Amersham, Arlington Heights, IL) for approximately 16 h at -70 C with Dupont Cronex intensifying screens (Dupont, Boston, MA). Radiolabeled probes were prepared by labeling the antisense strand with [{gamma}-32P]ATP (4500 Ci/mmol; ICN, Irvine, CA) and T4 poly-nucleotide kinase followed by annealing to the complementary strand. Double-stranded DNA probes were purified by centrifugation on a G-25 Microspin column (Pharmacia Biotech, Piscataway, NJ).

ERK Activation Assays
{alpha}T3–1 cells were grown to approximately 70% confluence and serum starved for 2 h before drug treatment and lysis. The specific MEK1/MEK2 inhibitor PD98059 (60 µM) or control vehicle (dimethyl sulfoxide) was applied to the cells in DMEM 15 min before and during treatment with the GnRH agonist buserelin ([D-SER(tBU)6,Pro9-ethylamide]GnRH (10 nM). After treatment, cells were washed with ice-cold buffer containing 0.15 M NaCl and 10 mM HEPES (pH 7.5) and lysed in RIPA buffer containing 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, 2 mM EDTA, 5 mM sodium vanadate, 5 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride on ice. The cell lysates were collected and debris cleared by centrifugation. Proteins were resolved using denaturing PAGE followed by transfer to polyvinylidene difluoride membrane by electroblotting. Samples were analyzed for ERK phosphorylation by Western blotting using an antibody to the dual phosphorylated forms of ERK1 and ERK2 (Promega, Madison, WI). The blot was then stripped and reprobed with an antibody that detects relative amounts of ERK protein independent of phosphorylation state (Santa Cruz Biotechnology).

Statistical Analysis
Data were analyzed using SAS (70). Means for GnRH-treated cells were expressed as fold increases over nontreated cells. Means for LUC activity in Figs. 1Go, 7Go, and 9Go were logarithmically transformed due to nonnormality and then analyzed. In Figs. 1Go, 4Go, 5Go, and 6Go, means for LUC activity were analyzed by ANOVA and compared with control values with Dunnett’s two-tailed t-test. Least-squares means for LUC activity in Fig. 2Go were analyzed with the General Linear Models procedure and compared using least significant differences. Since the response of the pGL3 basic vector varied across assays, the mean GnRH response for the pGL3 basic vector within each assay was included as a covariable in the model used for calculation of least-squares means for all vectors in Fig. 2Go. In Figs. 7Go and 9Go, means for LUC activity were compared using Tukey’s studentized range test.


    ACKNOWLEDGMENTS
 
The {alpha}T3–1 cells were a generous gift from Dr. Pam Mellon (Salk Institute, La Jolla, CA). The authors would like to thank Ann Burns, Buffy Ellsworth, Anthony Guillen, Meredith Holtzen, Dr. Scott Nelson, and Mark Riccardi for their time and efforts toward completion of this study.


    FOOTNOTES
 
Address requests for reprints to: Dr. Colin M. Clay, Animal Reproduction and Biotechnology Laboratory, Department of Physiology, College of Veterinary Medicine and Biomedical Sciences, Foothills Campus, Colorado State University, Fort Collins, Colorado 80523.

This work was supported by NIH Grants R29HD-32416 to C.M.C. and R29HD-34722 to M.S.R. B.R.W. was supported by NIH Training Grant HD-07031, and D.L.D. was supported by NIH Postdoctoral Fellowship National Research Service Award 1F32HD-08169.

Received for publication June 11, 1998. Revision received December 21, 1998. Accepted for publication December 28, 1998.


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