Sp1, Steroidogenic Factor 1 (SF-1), and Early Growth Response Protein 1 (Egr-1) Binding Sites Form a Tripartite Gonadotropin-Releasing Hormone Response Element in the Rat Luteinizing Hormone-ß Gene Promoter: an Integral Role for SF-1

Ursula B. Kaiser, Lisa M. Halvorson and Marian T. Chen

Endocrine-Hypertension Division (U.B.K.) and Genetics Division (M.T.C.) Department of Medicine Brigham and Women’s Hospital and Harvard Medical School Boston, Massachusetts 02115
Department of Obstetrics and Gynecology (L.M.H.) New England Medical Center Tufts University School of Medicine Boston, Massachusetts 02111


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recently, several cis-regulatory elements that play roles in LHß gene expression, and their cognate DNA-binding transcription factors, have been identified. These factors include Sp1, steroidogenic factor-1 (SF-1), and early growth response protein 1 (Egr-1). Using the GH3 pituitary cell line (which lacks SF-1) as a model, we demonstrate that expression of SF-1 or Egr-1 increases rat LHß gene promoter activity but has little effect on the fold response to GnRH. However, expression of both SF-1 and Egr-1 synergistically enhances LHß gene promoter activity and prevents further stimulation of activity by GnRH. Mutations in the Sp1 binding sites of the rat LHß gene promoter decrease GnRH responsiveness, whereas mutations in the SF-1 and/or Egr-1 binding sites alone have little effect on the GnRH response. Combinatorial mutations in both the Sp1 and Egr-1 binding elements result in almost complete loss of the GnRH response. In contrast, in GH3 cells cotransfected with SF-1, mutations in the Sp1, SF-1, or Egr-1 binding elements independently decrease GnRH responsiveness. In LßT2 cells, a gonadotrope-derived cell line that expresses SF-1 endogenously, mutations in either the Sp1 or Egr-1 binding elements decrease GnRH responsiveness. These data suggest that the Sp1, SF-1, and Egr-1 binding sites form a tripartite GnRH response element in the rat LHß gene promoter. Changes in the spacing between the upstream Sp1 binding sites and the downstream SF-1/Egr-1 binding elements reduce the response to GnRH. SF-1, while having little direct effect on GnRH responsiveness, has a critical role in integrating the effects of Sp1 and Egr-1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The production and secretion of the pituitary gonadotropins, LH and FSH, are controlled by the complex interaction of multiple factors originating from the hypothalamus, pituitary, and gonads. The hypothalamic peptide, GnRH, is one of the most critical factors, effecting regulation of secretion of LH and FSH as well as of the transcription and biosynthesis of the gonadotropin subunits, {alpha}, LHß, and FSHß (1). Recent studies have identified several transcription factors that have profound effects on basal and/or GnRH-stimulated LHß gene promoter activity.

Steroidogenic factor-1 (SF-1), a member of the nuclear hormone receptor superfamily, is selectively expressed in the gonadotrope subpopulation of the anterior pituitary gland, as well as in the adrenal gland and the gonads (2). SF-1 binding elements have been identified in a range of genes important for steroidogenesis, sexual differentiation, and reproductive function (3). An SF-1 binding site was identified in the glycoprotein {alpha}-subunit gene (referred to as the gonadotrope-specific element, or GSE) and SF-1 binding was shown to result in transcriptional activation of this gene (4). Studies of the rat LHß gene have identified and characterized two SF-1 binding elements, located at positions -127 and -59 relative to the transcriptional start site (Fig. 1Go) (5, 6). The functional role of SF-1 in trans-activation of the LHß gene has been demonstrated in vivo and in vitro (5, 6, 7, 8). However, the role of SF-1 in the regulation of GnRH responsiveness of the LHß gene has not been fully characterized. Pituitary SF-1 mRNA and protein levels have been reported by some to increase in response to GnRH (9), whereas others have not observed this regulation (10, 11, 12). SF-1 has been found to have no effect on the fold activation of LHß gene promoter activity by GnRH (11, 13). Transgenic mice null for the SF-1 gene have reduced levels of gonadotropins, yet remain capable of responding to GnRH with an increase in gonadotropin expression (2, 14). Thus, the role of SF-1 in the regulation of LHß gene expression by GnRH remains controversial.



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Figure 1. Schematic Diagram of the Organization of the Rat LHß Gene Promoter

Two Sp1 binding sites are located upstream between positions -451/-386; two composite SF-1 and Egr-1 binding sites are located between -127/-106 and -59/-42. All numbering is relative to the transcriptional start site of the rat LHß gene. GTF, General transcription factors.

 
Early growth response protein 1 (Egr-1) has also been implicated in the tissue-specific expression of the LHß gene. Egr-1 is a member of the immediate early gene family characterized by zinc finger domains with Cys2-His2 motifs that recognize GC-rich nucleotide sequences (15). In vitro studies have demonstrated two functional Egr-1 binding sites in the rat LHß gene promoter, located at positions -112 and -50 relative to the transcriptional start site (Fig. 1Go) (6, 16). Synergy between SF-1 and Egr-1 has been demonstrated in the activation of the LHß gene (6, 11, 12, 17). Transgenic mice null for Egr-1 demonstrated specific loss of LHß gene expression and failed to respond to GnRH (16, 18). GnRH is known to activate protein kinase C (PKC) in gonadotropes, and we have demonstrated previously that activation of PKC increases Egr-1 expression levels and also activates rat LHß gene promoter activity, suggesting a role for Egr-1 in mediating the LHß gene response to PKC (17, 19). Furthermore, GnRH increases Egr-1 expression in pituitary gonadotrope-derived cell lines, and mutations of the Egr-1 binding sites almost completely prevent stimulation of the proximal LHß gene promoter by GnRH, implicating Egr-1 as a critical transcription factor involved in effecting this GnRH response (11, 12, 17, 20).

Our previous studies have identified additional DNA sequences further upstream, between -490/-352, in the rat LHß gene promoter that are also important for LHß gene expression and GnRH responsiveness (13). More detailed analyses demonstrated several Sp1 binding sites within this region (Fig. 1Go). Mutations of these elements, which block binding of Sp1, reduce basal LHß gene promoter activity as well as lessen the stimulation by GnRH (21, 22). Like Egr-1, Sp1 is a member of the Cys2-His2 zinc finger family of transcription factors and recognizes a similar but distinct GC-rich nucleotide sequence (23). These data suggest that Sp1 also plays an important role in conferring GnRH responsiveness and bring into question the requisite role of Egr-1 in this response.

The relative importance of these three transcription factors, Sp1, Egr-1, and SF-1, and possible interactions among them in mediating GnRH responsiveness have not been characterized. In this report, we demonstrate that all three transcription factors contribute to GnRH responsiveness and that the cognate binding sites form a tripartite GnRH response element. SF-1, while having little direct effect on GnRH responsiveness, has a critical role in integrating the effects of Sp1 and Egr-1 on the GnRH response.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SF-1 and Egr-1 Independently and Synergistically Increase LHß Gene Expression and Block GnRH Responsiveness When Present in Combination
A systematic approach to identifying mechanisms of hormonal regulation of gonadotropin subunit gene expression has been hampered by the lack of available cell lines that express either the endogenous or transfected LHß and FSHß genes in a regulated manner. In our previous studies, we have used GH3 cells, a well characterized rat somatolactotropic cell line, as a model for the analysis of cis-regulatory elements in the rat LHß gene (5, 13, 21). We, therefore, initially sought to determine the effects of SF-1 and Egr-1 on GnRH responsiveness of the LHß gene using this cell model.

GGH3-1' cells (GH3 cells stably transfected with rat GnRHR cDNA) were transfected with a reporter construct containing region -797 to +5 of the rat LHß gene promoter fused to a luciferase reporter (-797/+5LHßLUC) (Fig. 2Go). Treatment with a GnRH agonist (GnRHAg) for 6 h increased luciferase activity 6.6 ± 0.4-fold, consistent with our previous reports. Cotransfection with the cytomegalovirus (CMV)-driven SF-1 expression vector resulted in a 2.4 ± 0.1-fold increase in luciferase activity. Further stimulation with GnRHAg in the presence of SF-1 resulted in a 5.7 ± 0.2-fold stimulation in -797/+5LHßLUC activity (relative to vehicle-treated cells expressing SF-1), statistically slightly decreased from the fold GnRH response in the absence of SF-1 (P < 0.05). Cotransfection with the CMV-driven Egr-1 expression vector also increased luciferase activity by 6.5 ± 0.8-fold. Stimulation with GnRHAg in the presence of Egr-1 resulted in a 4.4 ± 0.2-fold stimulation of luciferase activity, again slightly decreased from the fold GnRH response in the absence of SF-1 and Egr-1 (P < 0.001). As previously reported, marked synergy was observed in the presence of both SF-1 and Egr-1, with a 180 ± 17-fold increase. Interestingly, the fold response to GnRH was almost completely abrogated (1.5 ± 0.1-fold) in the presence of both Egr-1 and SF-1. The loss of GnRH responsiveness after overexpression of both SF-1 and Egr-1 supports the possibility that these factors may have critical roles in mediating the GnRH response. If GnRH stimulates LHß gene expression by increasing levels of functionally active SF-1 and Egr-1, then we would expect that no further GnRH response could be elicited in the presence of high levels of both SF-1 and Egr-1.



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Figure 2. Effects of SF-1 and Egr-1 on Rat LHß Gene Promoter Activity

GGH3-1' cells were transiently transfected with -797/+5LHßLUC. Cells were cotransfected with expression plasmids encoding either SF-1, Egr-1, or both, or the appropriate control expression vector, and with an RSV-ß-Gal expression vector. Cells were treated with ± 100 nM GnRHAg for 6 h before harvesting. Luciferase activity was normalized to ß-galactosidase activity. Results are shown as the mean ± SEM of at least nine samples in three independent experiments. Fold response to GnRHAg relative to vehicle-treated controls was calculated for each transfection group.

 
Mutations in the SF-1 or Egr-1 Binding Sites Do Not Decrease GnRH Responsiveness of -797/+5LHßLUC in GGH3-1' Cells
If GnRH does stimulate LHß gene expression by increasing levels of functionally active SF-1 and Egr-1, as hypothesized above, then mutations in the DNA binding sites for SF-1 and Egr-1 in the rat LHß gene promoter, which block binding of these factors, would be predicted to prevent stimulation by GnRH. We generated constructs containing point mutations in either the two SF-1 DNA binding sites, the two Egr-1 DNA binding sites, or both, in the context of -797/+5LHßLUC. These mutations have been shown previously to eliminate binding by SF-1 and/or Egr-1 by electrophoretic mobility shift assay (EMSA) (6). These mutant constructs were first transfected into GGH3-1' cells. GGH3-1' cells lack endogenous SF-1, based on the failure of GH3 nuclear extracts to produce specific protein-DNA complexes with a GSE-containing oligonucleotide probe by EMSA (data not shown). However, these cells do have endogenous Sp1 and Egr-1 (17, 21). The mutations had little effect on basal luciferase activity. Interestingly, mutations in the SF-1 and Egr-1 binding sites of the LHß gene promoter also had little effect on the fold response to stimulation with GnRHAg [-797/+5LHßLUC: 7.1 ± 0.4-fold; LHSF1MLUC: 6.5 ± 0.3-fold; LHEgr1MLUC: 6.3 ± 0.3-fold; LHSF1MEgr1MLUC: 6.6 ± 0.6-fold] (Fig. 3Go). Therefore, these results do not suggest a major contribution of the SF-1 and Egr-1 binding sites in the rat LHß gene promoter in mediating GnRH responsiveness in GGH3-1' cells.



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Figure 3. Effects of Mutations in the SF-1 and Egr-1 Binding Sites on the Stimulation of Rat LHß Gene Promoter Activity by GnRH

Transient transfections were performed in GGH3-1' cells with either wild-type -797/+5LHßLUC or mutant constructs harboring mutations in the SF-1 and/or Egr-1 binding sites, as indicated. Cells were cotransfected with an RSV-ß-Gal expression vector. Cells were treated with ±100 nM GnRHAg for 6 h before harvesting. Luciferase activity was normalized to ß-galactosidase activity. The results are expressed graphically as the fold stimulation by GnRH over control. Results are shown as the mean ± SEM of at least nine samples in three independent experiments.

 
Combinatorial Mutations in Sp1, SF-1, and Egr-1 Binding Sites in the LHß Gene Promoter Are Necessary to Block GnRH Responsiveness
Our previous studies suggested that Sp1 is also involved in mediating the stimulation of the rat LHß gene in response to GnRH (13, 21). Mutation of the Sp1 binding sites in the rat LHß gene promoter reduced, but did not entirely eliminate, the GnRH response. We generated a new panel of constructs containing point mutations in the SF-1 DNA binding sites, the Egr-1 DNA binding sites, or both, in the context of LHßSp1MLUC, a mutant variant of -797/+5LHßLUC with mutations in the Sp1 binding sites (21). Transfections were performed with these constructs in GGH3-1' cells (Fig. 4Go). Similar to our previous findings, mutations in the Sp1 binding sites decreased the GnRH response [7.1 ± 0.4-fold to 3.4 ± 0.2-fold (P < 0.001)]. Additional mutations in the SF-1 binding sites had no further effect on the fold response to GnRH, but mutations in the Egr-1 binding sites almost completely blocked GnRH responsiveness [2.0 ± 0.2-fold]. In sum, whereas blockade of Egr-1 binding had no effect on GnRH response in the context of the wild-type LHß gene promoter in GGH3-1' cells, mutations that prevent the upstream binding of Sp1 "unmask" a role for Egr-1 in mediating GnRH responsiveness.



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Figure 4. Effects of Combinatorial Mutations in the Sp1 and SF-1 and/or Egr-1 Binding Sites on the Stimulation of Rat LHß Gene Promoter Activity by GnRH

Transient transfections were performed in GGH3-1' cells with luciferase reporter plasmids encoding -797/+5 of the rat LHß gene promoter harboring mutations in the Sp1, SF-1, and/or Egr-1 binding sites, as indicated. Cells were cotransfected with an RSV-ß-Gal expression vector. Cells were treated with ± 100 nM GnRHAg for 6 h before harvesting. Luciferase activity was normalized to ß-galactosidase activity. The results are expressed graphically as the fold stimulation by GnRH over control. Results are shown as the mean ± SEM of at least nine samples in three independent experiments. *, P < 0.005 compared with the fold response of the reporter construct harboring mutations in the Sp1 sites only.

 
The Sp1, SF-1, and Egr-1 Binding Sites All Contribute to GnRH Responsiveness of the LHß Gene Promoter in the Presence of SF-1
As mentioned above, GH3 cells lack SF-1. Therefore, it is not surprising that mutations in the SF-1 binding sites of the rat LHß gene had no effect on basal or GnRH-stimulated luciferase activity. The use of an SF-1-deficient cell line allows investigation of GnRH effects on LHß gene expression both in the absence and presence of SF-1. We repeated transfections of the wild-type and mutant -797/+5LHßLUC constructs in GGH3-1' cells, this time in the presence of a cotransfected CMV promoter-driven SF-1 expression vector, and studied the effects of the mutations on basal and GnRHAg-stimulated luciferase activity (Fig. 5Go). In the presence of SF-1, mutations in the Sp1, SF-1, or Egr-1 DNA binding elements independently reduced GnRH responsiveness [-797/+5LHßLUC: 8.8 ± 1.5-fold; LHSp1MLUC: 5.6 ± 0.8-fold; LHSF1MLUC: 5.2 ± 1.1-fold; LHEgr1MLUC: 4.1 ± 0.5-fold; LHSF1MEgr1MLUC: 5.4 ± 0.9-fold; P < 0.01 compared with -797/+5LHßLUC]. In addition, combined mutations in the Sp1 and either the SF-1 or Egr-1 binding elements almost completely abrogated GnRH responsiveness [LHSp1MSF1MLUC: 2.6 ± 0.3-fold; LHSp1MEgr1MLUC: 1.9 ± 0.2-fold; LHSp1MSF1-MEgr1MLUC: 2.2 ± 0.1-fold]. In the presence of SF-1, all three elements appear to contribute to mediate the full GnRH response.



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Figure 5. Effects of Mutations in the Sp1, SF-1, and/or Egr-1 Binding Sites on the Stimulation of Rat LHß Gene Promoter Activity by GnRH in the Presence of SF-1

Transient transfections were performed in GGH3-1' cells with either wild-type -797/+5LHßLUC or mutant constructs harboring mutations in the Sp1, SF-1, and/or Egr-1 binding sites, as indicated. Cells were cotransfected with an SF-1 expression vector and with an RSV-ß-Gal expression vector. Cells were treated with ±100 nM GnRHAg for 6 h before harvesting. Luciferase activity was normalized to ß-galactosidase activity. The results are expressed graphically as the fold stimulation by GnRH over control. Results are shown as the mean ± SEM of at least nine samples in three independent experiments. *, P < 0.01 compared with the fold response of -797/+5LHßLUC; **, P < 0.05 compared with the reporter construct harboring mutations in the Sp1 sites only.

 
The Roles of the Sp1, SF-1, and Egr-1 Binding Sites in the LHß Gene Promoter in Mediating GnRH Responsiveness in the LßT2 Cell Line
Our studies in GGH3-1' cells suggested that the Egr-1 binding sites played a role in mediating GnRH responsiveness of the rat LHß gene promoter in the presence of SF-1 or, alternatively, in the absence of Sp1 binding. However, GH3 cells lack endogenous SF-1, and cotransfection of the SF-1 expression vector may not accurately mimic SF-1 levels or activity present in gonadotropes. Therefore, we turned to the LßT2 cell line to confirm our findings. LßT2 cells are an immortalized, gonadotrope-derived cell line generated from a pituitary tumor occurring in a transgenic mouse expressing an LHß gene promoter-SV40 virus T antigen fusion gene (24). These cells express both {alpha}- and ß- subunits of LH as well as GnRHR and SF-1 (24, 25). LßT2 cells respond to pulsatile GnRH administration with an increase in GnRHR and LHß mRNA levels (25). We have transfected LßT2 cells with -797/+5LHßLUC by electroporation and demonstrated a 3.2 ± 0.2-fold increase in luciferase activity in response to GnRHAg (Fig. 6Go). In LßT2 cells, mutation of the Sp1 binding elements in the rat LHß gene decreased the fold response to GnRH [2.3 ± 0.1-fold, P < 0.001]; mutation of the SF-1 binding elements had no effect on the fold response to GnRH [2.9 ± 0.3-fold, P = 0.4, NS]; and mutation of the Egr-1 binding sites almost completely abolished stimulation of the LHß gene promoter by GnRH [1.4 ± 0.1-fold]. Thus, as anticipated from our results in GGH3-1' cells, both the Sp1 and the Egr-1 binding elements appear to contribute to mediating the full GnRH response in LßT2 cells (i.e. in the presence of SF-1).



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Figure 6. Effects of Mutations in the Sp1, SF-1, and/or Egr-1 Binding Sites on the Stimulation of Rat LHß Gene Promoter Activity by GnRH in the LßT2 Cell Line

LßT2 cells were transfected with either wild-type -797/+5LHßLUC or mutant constructs harboring mutations in the Sp1, SF-1, and/or Egr-1 binding sites, as indicated. Cells were cotransfected with an RSV-ß-Gal expression vector. Cells were treated with ± 100 nM GnRHAg for 6 h before harvesting. Luciferase activity was normalized to ß-galactosidase activity. The results are expressed graphically as the fold stimulation by GnRH over control. Results are shown as the mean ± SEM of at least nine samples in three independent experiments. *, P < 0.001 compared with the fold response of -797/+5LHßLUC.

 
DNA Spacing between the Sp1 Binding Sites and the SF-1 and Egr-1 Binding Sites Is Important for Full GnRH Responsiveness of the LHß Gene
The SF-1 and Egr-1 DNA binding sites form two composite elements in the proximal LHß gene promoter within 130 bp of the transcriptional start site. In contrast, the Sp1 binding sites are further upstream, between positions -451/-386. For Sp1 bound to the LHß gene promoter to interact with downstream SF-1 and Egr-1, DNA bending or looping and/or interacting proteins need to be hypothesized. We, therefore, wondered whether the spacing between the upstream Sp1 sites and the downstream SF-1/Egr-1 binding elements had an effect on the ability of GnRH to stimulate LHß gene expression. We generated a series of internal deletion constructs of the LHß gene promoter fused to a luciferase reporter. These internal deletions removed either the Sp1 binding sites (LH{Delta}A, -490/-353 deleted), one of the SF-1/Egr-1 composite elements (LH{Delta}B, -207/-83 deleted), or the intervening 135 bp (LH{Delta}C, -352/-208 deleted). These internal deletion constructs were transfected into GGH3-1' cells, along with either the SF-1 expression vector or the empty control vector (Fig. 7Go).



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Figure 7. Effects of Spacing between the Sp1, SF-1, and Egr-1 Binding Sites on the Response of the Rat LHß Gene to GnRH

Transient transfections were performed in GGH3-1' cells with either wild-type -797/+5LHßLUC or mutant constructs harboring internal deletions that remove Sp1 binding sites [LH{Delta}A (-490/-353 deleted)], SF-1 and Egr-1 binding sites [LH{Delta}B (-207/-83 deleted)], or intervening sequences [LH{Delta}C, -358/-208 deleted)], as indicated, either in the absence of SF-1 (upper panel) or in the presence of cotransfected SF-1 expression vector (lower panel). Cells were cotransfected with an RSV-ß-Gal expression vector. Cells were treated with ± 100 nM GnRHAg for 6 h before harvesting. Luciferase activity was normalized to ß-galactosidase activity. The results are expressed graphically as the fold stimulation by GnRH over control. Results are shown as the mean ± SEM of at least nine samples in three independent experiments. *, P < 0.005 compared with the fold response of -797/+5LHßLUC in the absence of SF-1; **, P < 0.005 compared with the fold response of -797/+5LHßLUC in the presence of SF-1.

 
As we would predict from our studies with point mutations, internal deletion of the Sp1 binding sites decreased the fold response to GnRH both in the absence and presence of SF-1 [-SF-1: -797/+5LHßLUC: 11.6 ± 1.1-fold, LH{Delta}A: 4.2 ± 0.2-fold, P < 0.001; +SF-1: -797/+5LHßLUC: 7.8 ± 0.9-fold, LH{Delta}A: 5.3 ± 0.2-fold, P < 0.005]. Internal deletion of the SF-1 and Egr-1 binding sites resulted in a small but significant decrease in the fold response to GnRH in the absence of SF-1, but a more marked decrease in response to GnRH in the presence of SF-1 [-SF-1: LH{Delta}B: 8.1 ± 0.6-fold, P < 0.001 compared with -797/+5LHßLUC; +SF-1: LH{Delta}B: 1.6 ± 0.2-fold, P < 0.001 compared with -797/+5LHßLUC], again consistent with our observations using point mutations in these elements (Figs. 3Go and 5Go). Interestingly, internal deletion of the intervening sequence (-359/-208) resulted in a decrease in the fold response to GnRH, both in the absence and presence of SF-1, even though all of the known binding sequences remain intact [-SF-1: LH{Delta}C: 4.9 ± 0.3-fold, P < 0.001 compared with -797/+5LHßLUC; +SF-1: LH{Delta}C: 4.1 ± 0.4-fold, P < 0.001 compared with -797/+5LHßLUC]. In contrast, our previous studies have demonstrated that 5'-deletion of this region has no effect on GnRH responsiveness (13). The possibility that this deleted segment may contain transcriptionally functional domains despite the lack of effect of 5'-deletion needs to be considered. Nonetheless, these findings are consistent with the hypothesis that the spacing between the Sp1 sites and the SF-1/Egr-1 sites is important for their interaction.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The importance of GnRH in the regulation of LH and FSH secretion as well as of gonadotropin subunit gene expression is firmly established. The recent development of GnRH-responsive cell lines and of transgenic animal models has begun to lead to new insights into the molecular mechanisms of this regulation, particularly for the LHß gene. Using these approaches, several transcription factors important for LHß gene expression have been identified. While each of these factors has been implicated individually in mediating GnRH responsiveness, their relative importance and possible interactions among them have not been fully characterized.

SF-1 and Egr-1 were each able to transactivate the rat LHß gene promoter but individually had little effect on the fold stimulation by GnRH. In contrast, when both factors were present, basal LHß activity was markedly and synergistically increased, and the response to GnRH was almost completely abrogated (Fig. 2Go). Although squelching cannot be ruled out, these results suggest that the GnRH stimulation of LHß gene expression may be effected by increased levels of functionally active SF-1 and Egr-1. To explore this observation further, we used the approach of mutagenesis of the cognate binding sites.

Mutagenesis of the Egr-1 binding elements had no effect on the GnRH response in GGH3-1' cells in the absence of SF-1 (Fig. 3Go). This was initially quite surprising in view of previous reports. The Egr-1-/- mouse exhibits LHß deficiency and fails to respond to gonadectomy with an increase in LHß mRNA, suggesting a role for Egr-1 in mediating the transcriptional regulation of the LHß gene by GnRH (16, 18). This role was confirmed by in vitro studies (11, 12, 17, 20). In these studies, mutations of the Egr-1 binding sites reduced or almost completely abrogated GnRH responsiveness of the LHß gene promoter. Closer evaluation reveals that the LHß promoter constructs used in these studies did not include the upstream Sp1 binding sites. Disruption of Sp1 binding to the LHß gene promoter, either by mutation (Fig. 4Go), by internal deletion (Fig. 7Go), or by 5'-deletion (11, 12, 17, 20) restores the importance of Egr-1 binding for the mediation of GnRH responsiveness. These findings reconcile previous reports of the roles of Sp1 and Egr-1 and indicate a novel functional relationship between Sp1 and Egr-1 in mediating the GnRH response.

The role of SF-1 in the regulation of GnRH responsiveness of the LHß gene has been controversial. While SF-1-/- mice are deficient in LHß production (3), they remain responsive to GnRH with an increase in LHß levels (2, 14). In contrast, mutation of the SF-1 binding sites in the bovine LHß gene promoter prevented GnRH stimulation in transgenic mice (7). Using GGH3-1' cells that lack SF-1, we demonstrated previously that exogenous SF-1 increased both basal and GnRH-stimulated LHß gene promoter activity; however, the fold response to GnRH was unaffected (13). Interestingly, as shown here, in the presence of SF-1, both Egr-1 and Sp1 binding elements contribute to GnRH responsiveness, whereas only the Sp1 site appears important in the absence of SF-1. Thus, it appears that SF-1 plays an important indirect role by integrating the effects of Sp1 and Egr-1. One could speculate that levels of SF-1 in gonadotropes may modulate the relative roles in vivo of Sp1 and Egr-1 in mediating the GnRH response.

In LßT2 cells, mutation of the SF-1 sites somewhat surprisingly did not affect the GnRH response (Fig. 6Go). The reason for this result is not entirely clear. It may be due to differences in SF-1 levels between GGH3-1' and LßT2 cells, other differences between the two cell lines, or possibly due to the technical limitations imposed by the comparatively small GnRH response observed in LßT2 cells, making it more difficult to detect reductions in responsiveness.

In the studies presented, we have not distinguished between the 5' (-127/-106) and 3' (-59/-42) SF-1/Egr-1 composite elements. The internal deletion studies in Fig. 7Go indicate that the more 5'-site does contribute to the GnRH response, but these studies do not examine the 3' site in isolation. We have individually mutated each of the SF-1 and Egr-1 binding sites and shown that each element contributes to the overall GnRH response (data not shown). The 3' Egr-1 binding site appeared to have a slightly greater effect than the 5' Egr-1 site, consistent with observations that the 3'-site has a higher binding affinity for Egr-1 (L. Halvorson, unpublished data).

Our studies indicate that Sp1, SF-1, and Egr-1 act and interact to contribute to GnRH responsiveness. Our results have led us to propose a model in which these factors may interact with a common, as yet unidentified, transcriptional cofactor (Fig. 8Go). According to our proposed model, Sp1, when bound to its cognate elements in the LHß gene promoter, is able to interact with this putative cofactor to mediate GnRH responsiveness. The loss of Sp1 binding to the LHß gene promoter therefore results in a decrease in the GnRH responsiveness of this gene. The decrease in GnRH responsiveness observed upon deletion of intervening sequences could be explained by disruption of interaction of Sp1 with the putative cofactor caused by alterations in spacing.



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Figure 8. Model for the Interaction of Sp1, SF-1, and Egr-1 with a Putative Transcriptional Cofactor in the Regulation of Rat LHß Gene Expression in Response to GnRH

A, In the absence of SF-1, GnRH induces Egr-1 binding to the LHß gene promoter, but Egr-1 is unable to interact with the transcriptional cofactor, so mutations in the Egr-1 binding sites have no effect on GnRH responsiveness. It is the interaction of Sp1 with this cofactor that mediates the GnRH response. B, If mutations are introduced into the rat LHß gene promoter that prevent Sp1 binding, then the cofactor is free to interact with Egr-1, and this interaction mediates the residual GnRH response. C, In the presence of SF-1, all three cofactors interact with the transcriptional cofactor to mediate the full response to GnRH.

 
We hypothesize that SF-1 is necessary for Egr-1 interaction with the putative transcriptional cofactor in the context of the wild-type rat LHß gene promoter. In the absence of SF-1, GnRH induces Egr-1 levels and Egr-1 binding to the cognate sites in the LHß gene promoter, but Egr-1 is unable to interact with the putative transcriptional cofactor (Fig. 8Go). As a result, mutation of the Egr-1 binding elements has no effect on the GnRH response in the absence of SF-1 (Fig. 3Go). However, if Sp1 binding is prevented, then the transcriptional cofactor is able to interact with Egr-1 even in the absence of SF-1, enabling Egr-1 to mediate GnRH responsiveness under these conditions (Fig. 4Go). We postulate that the stoichiometry of the interaction is such that in the absence of SF-1, the cofactor can interact with only Sp1 or Egr-1, but not both, with a preferential interaction with Sp1 (Fig. 8Go). Such a preferential interaction is not unprecedented. For example, NCoR (nuclear receptor corepressor) preferentially binds to thyroid hormone receptor when it is a homodimer rather than when it forms a heterodimer with retinoid X receptor (26).

In our model, Sp1, Egr-1, and SF-1 are shown interacting with a non-DNA-binding (classical) cofactor. These three transcription factors are known to interact variously with the coactivators SRC-1 and CBP/p300 (27, 28, 29, 30, 31), which were therefore tested for their role in mediating the GnRH response. In preliminary experiments in GGH3-1' and LßT2 cells, no effect on basal or GnRH-stimulated LHß gene promoter activity was observed with the addition of either cofactor (data not shown). While the interpretation of these experiments must be cautious, these results raise the intriguing possibility that a novel, possibly gonadotrope-specific or GnRH-regulated transcriptional coactivator may be involved. Efforts to identify such a factor are underway.

The cofactor depicted in the model could also be a transcription factor, binding directly to an as-yet- unidentified element in the LHß gene, or there may be more than one cofactor involved. Alternatively, it is possible that no cofactor is necessary, but rather Sp1, SF-1, and Egr-1 interact directly. Sp1 and SF-1 have been shown to interact directly to modulate CYP11A gene transcription (32), and direct protein-protein interaction between SF-1 and Egr-1 has also been reported (6, 11).

We have not yet addressed the role of Ptx1, a homeobox-containing transcription factor affecting the expression of multiple pituitary hormone genes, in the regulation of LHß gene expression (33). Ptx-1 has been shown to enhance LHß gene expression and to act in synergism with SF-1 and Egr-1 through direct protein-protein interaction in the stimulation of LHß gene expression (11). The role of Ptx-1 in GnRH responsiveness remains to be studied.

Physiologically, all three of these transcription factors are present in gonadotropes and are likely to interact to mediate the response to GnRH. Therefore, the cognate binding sites for these factors can be considered to form a tripartite GnRH response element. It is possible that such a composite GnRH response element provides a level of redundancy to protect such a critical point of regulation of reproductive function.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
The GnRH agonist des-Gly10, [D-Ala6]-GnRH ethylamide (GnRHAg) was purchased from Sigma (St. Louis, MO).

Reporter Plasmids and Expression Vectors
The wild-type reporter construct used for these studies contained 797 bp of the 5'-flanking sequence of the rat LHß gene and the first 5 bp of the 5'-untranslated region, cloned into the pXP2 luciferase reporter vector (-797/+5LHßLUC) (34, 35). The Transformer site-directed mutagenesis kit (CLONTECH Laboratories, Inc., Palo Alto, CA) was used to introduce point mutations into the Sp1, SF-1, and Egr-1 binding sites in -797/+5LHßLUC. Oligonucleotides used have been described previously (17, 21) and are summarized in Table 1Go. Generation of multiple mutations was performed either simultaneously or sequentially and required the use of two selection primers (mpXP2 and mpXP2Rev), as described previously (17). All mutagenic constructs were confirmed by dideoxysequencing.


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Table 1. Oligonucleotides Used for Site-Directed Mutagenesis

 
Constructs with internal deletions within the rat LHß gene promoter were generated by PCR using primers with ends modified to cause deletion of the indicated sequences during amplification. LH{Delta}A (-490/-353 deleted) and LH{Delta}B (-207/-83 deleted) were generated as previously reported (13). Additional primers, LHß56S (5'-GTGTTTAAAGCAAATTTGAGCCAATGTCAGTTAAGCTCAG-3') and LHß65AS (5'-CTGAGCTTAACTGACATTGGCTCAAATTTGCTTTAAACAC-3'), were used to generate LH{Delta}C (-359/-208 deleted). All constructs were confirmed by dideoxysequencing.

The SF-1 expression vector contained 2.1 kb of the murine SF-1 cDNA driven by CMV promoter sequences in the vector, pCMV5 (kindly provided by K. L. Parker, Southwestern School of Medicine, Dallas, TX) (36). The Egr-1 expression vector was generated by cloning the murine Egr-1 cDNA (provided by D. Nathans, John Hopkins University, Baltimore, MD) (37) into pCMV5 at BamHI and HindIII restriction enzyme sites. The F-SRC-1 expression vector, encoding the full-length human SRC-1 cDNA in pcDNA1/Amp, was generously provided by Dr. Akira Takeshita (Brigham and Women’s Hospital, Harvard Medical School, Boston, MA) (38). An expression vector containing p300 cDNA under the control of CMV promoter sequences was the generous gift of Dr. D. M. Livingston (Dana Farber Cancer Institute, Harvard Medical School, Boston, MA) (39). An expression vector expressing ß-galactosidase driven by the Rous sarcoma virus promoter (RSV-ß-Gal) was used as an internal standard and control (40).

Cell Culture and Transfection
GGH3-1' cells were prepared by stably transfecting GH3 cells with the rat GnRHR cDNA, as described previously (41). LßT2 cells were generously provided by Dr. P. L. Mellon (University of California, San Diego, CA) (24, 25). GGH3-1' and LßT2 cells were maintained in monolayer culture in DMEM (GGH3-1' cells in low glucose media and LßT2 cells in high glucose media) supplemented with 10% (vol/vol) FBS at 37 C in humidified 5% CO2/95% air. For transient transfection studies, cells were cultured to 50–70% confluence and transfected by electroporation. In each experiment, approximately 5 x 106 cells were suspended in 0.4 ml of Dulbecco’s PBS plus 5 mM glucose containing the DNA to be transfected. The cells received a single electrical pulse of 240 V from a total capacitance of 1000 microfarads, using an Electroporator II apparatus (Invitrogen, San Diego, CA). Cells received 2 µg/well of the reporter constructs. Where appropriate, cells also received 1 µg/well of SF-1, Egr-1, F-SRC-1, and/or p300 expression vectors or an equivalent amount of the empty pCMV5 expression vector. Cells were cotransfected with RSV-ß-Gal (1 µg/well). After electroporation, cells were plated in serum-containing medium. Medium was replaced after 24 h, and cells were harvested 48 h after transfection. Cells were treated with 100 nM GnRHAg or vehicle for 6 h immediately before harvesting. Cell extracts were prepared and analyzed for luciferase and ß-galactosidase activities as described previously (40, 42). Luciferase activity was normalized to the level of ß-galactosidase activity. Data are shown as mean ± SEM.

Statistical Analysis
Transfections were performed in triplicate and repeated at least three times. Data were combined across transfection experiments to determine the mean ± SEM of the corrected luciferase activity for basal and GnRHAg-treated cells, and fold stimulation in response to GnRH was calculated. Two-way ANOVA followed by post hoc comparisons with Fisher’s protected least significant difference test was used to assess whether changes in GnRH responsiveness were statistically significant between the indicated groups. Significant differences were established as P < 0.05.


    ACKNOWLEDGMENTS
 
We would like to thank Dr. William W. Chin for thoughtful and helpful discussions throughout the course of these investigations.


    FOOTNOTES
 
Address requests for reprints to: Dr. Ursula B. Kaiser, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: ukaiser{at}rics.bwh.harvard.edu

This work was supported in part by NIH Grants R29-HD-33001 and R01-HD-19938 (U.B.K.) and R01-HD-38089 and R03-HD-34692 (L.M.H.) and by the George W. Thorn Center (U.B.K.).

Received for publication December 13, 1999. Revision received April 25, 2000. Accepted for publication May 1, 2000.


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 MATERIALS AND METHODS
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