Divergent and Composite Gonadotropin-Releasing Hormone-Responsive Elements in the Rat Luteinizing Hormone Subunit Genes

Jennifer Weck1, Alice C. Anderson, Shannon Jenkins, Patricia C. Fallest and Margaret A. Shupnik

Division of Endocrinology Department of Internal Medicine (A.C.A., S.J., P.C.F., M.A.S.) Department of Molecular Physiology and Biological Physics (J.W., M.A.S.), and The National Science Foundation Center for Biological Timing University of Virginia Charlottesville, Virginia 22903


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH pulses regulate gonadotropin subunit gene transcription in a frequency-dependent, subunit-specific manner. The {alpha}-subunit gene is stimulated by constant GnRH and by rapid to intermediate pulse frequencies, while stimulation of LHß subunit gene transcription requires intermediate frequency pulses. We have defined the GnRH-responsive elements of the rat LH subunit gene promoters by deletion/mutation analysis and transfection studies in rat pituitary cells and two clonal gonadotrope cell lines. The {alpha}-subunit gene GnRH-responsive region lies between -411 and -375 bp. The region contains two Ets-domain protein binding sites, and mutating either site obliterates the response. DNA protein binding studies demonstrate the two sites are not equivalent, and that Ets-1 does not mediate this response. Studies of the LHß promoter reveal a major GnRH-responsive region between -456 and -342 bp. Within this region, two Sp1 binding sites contribute to the GnRH response, and the 3'Sp1 site is also critical for basal expression. The 5'Sp1 site partially overlaps a CArG box, and mutating the CArG element specifically eliminates the response to pulsatile GnRH. DNA containing this mutation cannot form intermediate mobility complexes with nuclear proteins, but retains Sp1 binding. Mutation of the 3'Sp1 site and either the 5'Sp1 or CArG element partially restores GnRH stimulation, suggesting a downstream element contributes to the full GnRH response. These studies demonstrate that unique composite elements and transcription factors are responsible for GnRH stimulation of the LH subunit genes and may contribute to their differential responses to GnRH pulses.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The gonadotropins LH and FSH act on the ovaries and testes to stimulate steroidogenesis and gametogenesis. Synthesis and secretion of the gonadotropins are dependent on appropriate secretion of the hypothalamic peptide GnRH. GnRH is secreted in pulses that vary in frequency and amplitude as a function of hormonal status and reproductive cycle stage (1, 2, 3, 4, 5). The intermittent pattern of release is critical for normal sexual development and gametogenesis, as interruption of GnRH pulses or administration of long-acting GnRH analogs and antagonists results in suppression of gonadotropins and sex steroid production, thus resulting in infertility (1, 2, 3, 6). GnRH pulse frequency differentially affects gonadotropin secretion, with higher frequency pulses favoring LH and lower frequency pulses favoring FSH in both humans and animal models (1, 2, 3, 4, 5). Frequency-dependent effects are also noted at the level of steady-state mRNAs encoding the gonadotropin subunits and the GnRH receptor (7, 8, 9, 10, 11). Administration of pulsatile GnRH in vivo to male rats in which endogenous GnRH pulses have been eliminated, and in vitro to cultured pituitary cells, differentially stimulates subunit mRNA levels. In general, the {alpha}-subunit is stimulated by rapid to intermediate pulse intervals (8–30 min) in vivo and by rapid pulses or constant GnRH in vitro, the LHß subunit is favored by intermediate pulses (30–60 min), and the FSHß subunit and GnRH receptor are favored by longer interval pulses (7, 8, 9). These results are mirrored at the transcriptional level in vivo, and treatment of perifused pituitary cells with GnRH emphasizes the requirement for intermittent GnRH for stimulated transcription of the LHß and FSHß genes (11, 12). Changes in the amplitude of the GnRH signal can modulate the GnRH response but, in general, appear to have much less impact than changes in frequency (9, 13).

The mechanisms by which GnRH differentially modulates gene expression have been postulated to occur through at least two pathways, including differential sensitivity of specific genes to the calcium influx and protein kinase C (PKC) arms of the intracellular GnRH signaling pathway (14, 15, 16, 17), and different gene targets for the GnRH response. Several investigators have examined individual promoters of the gonadotropin subunits or the GnRH receptor for sensitivity to GnRH signaling cascades, and a few studies have compared the calcium and PKC sensitivity of the gonadotropin subunit promoters to each other (15, 16, 17, 18, 19, 20). These data suggest preferential sensitivity of different genes to either calcium influx or stimulation through the PKC/mitogen-activated protein kinase (MAPK) cascade (18, 19, 20, 21, 22). However, at this time no clear consensus has emerged on either the subunit specificity of the signaling responses or the mechanisms by which GnRH pulses might preferentially activate individual pathways. Information on the GnRH-responsive elements in the gonadotropin subunit and GnRH receptor genes from several species indicates there is considerable diversity in both the DNA sequence elements and the potential transcription factors identified. Some GnRH-responsive regions for the human (18, 23) and mouse (22) {alpha}-subunits have been identified and include consensus binding sites for Ets-domain proteins. Several other genes tested by transfection analysis, including those for the rat LHß (24), ovine FSHß (25), and mouse GnRH receptor (26), have composite GnRH-responsive regions containing several different individual binding sites for multiple transcription factors. At present, little is known about how GnRH pulses, as opposed to static GnRH treatments of cultured cells, regulate gene transcription. The majority of investigations have also concentrated on responsive areas in individual genes, and several GnRH-responsive genes have not been compared in the same experiments. Previously, such studies have been hampered due to the lack of an appropriate clonal gonadotropin cell line capable of expressing the promoters of multiple GnRH-responsive genes.

We have used the LßT2 clonal gonadotrope cell line (27) to analyze the GnRH responses of the rat LH subunit gene promoters to static and pulsatile GnRH. We have found that the GnRH response of the rat {alpha}-subunit gene with static GnRH treatment is modulated through paired Ets-factor binding sites. In contrast, the rat LHß gene is activated by pulsatile GnRH, acting in part through a large upstream composite element containing binding sites for Sp1 and CArG proteins. This element acts to modulate and integrate the response of additional downstream regions containing binding sites for steroidogenic factor-1 (SF-1) and early growth response protein-1 (Egr-1). Both upstream and downstream elements are required for the maximal response to pulsatile GnRH.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH Responsiveness of the Rat {alpha}-Subunit Gene Requires Two Ets-Domain Protein Consensus Binding Sites
We previously cloned the rat {alpha}-subunit promoter from -479 to +77 bp and showed it to be responsive to GnRH in transfected {alpha}T3 cells (20). Deletion/mutation analysis of transfected promoter-luciferase constructs in LßT2 cells treated with static 10 nM GnRH (Fig. 1Go) demonstrated that the major GnRH-responsive region was located between -411 and -287 bp. Examination of the rat {alpha}-subunit gene promoter sequence between -411 and -375 bp (Fig. 2Go), corresponding to similar GnRH-responsive regions in the human and mouse genes, revealed the existence of two putative Ets-domain protein consensus binding sites, characterized by the core sequences GGAA, underlined in Fig. 2Go. Comparison with the human and mouse gene sequences shows that these promoters have only one consensus Ets site, and other groups have demonstrated that these sites are part of the responsive regions for GnRH or second messengers such as PKC (18, 19). Transfer of a single copy of the -411/-375 bp region to the heterologous thymidine kinase (tk) promoter (Fig. 3Go) or the -106 {alpha}-subunit minimal promoter, conferred GnRH responsiveness in both transfected LßT2 cells (Fig. 1Go) and {alpha}T3 cells (Table 1Go). Mutation of either the distal (tkmut5') or proximal (tkmut3') Ets sites, or both sites (tkmut5',3') eliminated GnRH stimulation of the heterologous promoter, suggesting that both sites are required for GnRH stimulation of the {alpha}-subunit gene. In agreement, mutation of either the distal site (411 mut5') or both sites (411 mut5',3') within the context of the entire -411/+77 promoter completely abolished the GnRH response. These mutations in the -411/+77 {alpha}-promoter also suppressed basal expression of the promoter 50% and 65%, respectively (Fig. 1Go). The data suggest that, in addition to playing a critical role in the stimulated response, the sites contribute to basal expression. Mutations in these regions would be expected to disrupt binding of proteins important for the transcriptional responses.



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Figure 1. Mutational Analysis of the Rat {alpha}-Subunit Gene in LßT2 Cells

Luciferase constructs (3 µg) were introduced into cells during a 16-h transfection period, followed by treatment with 100 nM GnRH for 6 h. The homologous -411 bp to +77 bp promoter had mutations in either the 5' (M5') or both (M5', 3') Ets sites, as designated by X in the bars along the Y axis. Normalized luciferase activity is expressed relative to basal expression of the wild-type promoter sequence. The heterologous thymidine kinase (tk) promoter was fused to the GnRH-responsive region of the {alpha}-subunit promoter, and mutations were made at either the 5'(M5') or 3'(M3') site, or both (M5', 3') sites. Normalized luciferase values are expressed relative to the untreated controls. Values represent the mean ± SEM of three experiments with three to four samples per group. *, P < 0.05 vs. untreated controls. Sequences of the mutated oligonucleotides are shown in Materials and Methods and in Fig. 3Go.

 


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Figure 2. DNA Sequences Representing the GnRH-Responsive Regions of the Rat LH Subunit Genes

The rat {alpha}-subunit responsive region is compared with similar gene regions from the human and mouse genes. The putative Ets-domain protein binding sites are shown in italics and underlined in each gene. The LHß gene GnRH-responsive region from -456 bp to -342 bp is shown. The 5'Sp1 and 3'Sp1 binding regions are italicized and underlined. The CArG box element is indicated by a double underline, and a third putative Sp1 site is shown by a dotted line.

 


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Figure 3. Binding of Nuclear Proteins to the {alpha}-Subunit GnRH-Responsive Region

Upper panel, The wild-type -411 to -375 bp {alpha}-subunit promoter region was labeled, and probe (50,000 cpm) was incubated with 5 µg of untreated LßT2 proteins and the indicated unlabeled competitor oligonucleotides at 200-fold excess molar concentrations. Competitor DNA included the homologous wild-type (WT) {alpha}-subunit promoter sequence, {alpha}-subunit DNA mutated at the 5'Ets (M5'), 3'Ets (M3') sites, or both Ets (M5', 3') sites, or consensus binding sites for CREB, Sp1, or Ets-domain proteins. In the far right portion of the panel, the consensus DNA sequence for Ets-domain proteins was labeled, and the probe (50,000 cpm) was incubated with 5 µg of LßT2 proteins in the absence or presence of the Ets-1 antibody. Lower panel, Individual double-stranded oligonucleotides corresponding to wild-type or mutant {alpha}-subunit probes as shown were labeled and incubated with 5 µg LßT2 proteins in the absence or present of Ets-1 antibody before electrophoresis on a nondenaturing gel. The positions of specific complexes lost upon mutation of the Ets sites are shown by the arrowheads.

 

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Table 1. Basal and GnRH-Stimulated Activity of Heterologous Promoter Luciferase Constructs Containing Portions of the Rat LH Subunit Genes Transfected into {alpha}T3 Cells

 
Nuclear Protein Binding to the {alpha}-Subunit GnRH-Responsive Region
Wild-type and mutated oligonucleotides corresponding to the GnRH-responsive region of the {alpha}-subunit gene, from -411 to -375 bp, were used in electrophoretic mobility shift assay (EMSA) studies to characterize nuclear proteins bound to this region. Experiments using nuclear proteins from LßT2 cells demonstrated four groups of DNA-protein complexes with wild-type DNA (Fig. 3Go, upper panel). Addition of unlabeled competitor DNA representing wild-type or mutated {alpha}-subunit promoter DNA or an Ets protein consensus binding site effectively competed for the formation of these complexes, but addition of unlabeled DNA representing binding sites for Sp1 and CREB had no effect. Labeled DNA containing mutations corresponding to mut 5', mut 3' and mut 5',3' in Fig. 3Go showed subtle differences in protein binding (Fig. 3Go, lower panel). Mutation of the distal Ets site (mut 5') inhibited formation of the most slowly migrating (upper) complex, whereas mutation of the proximal Ets site (mut 3') inhibited formation of the most rapidly migrating (lowest) complex. DNA with both mutated sites (mut 5',3') could not form either the upper or lower complex, but did form the two DNA-protein complexes of intermediate mobility. Addition of antibody specific for Ets-1 did not interfere with the formation or alter the mobility of any complex with any probe measured. Addition of a pan-Ets antibody that binds both Ets-1 and Ets-2 likewise had no effect (not shown). However, treatment with the Ets-1 antibody effectively eliminated the formation of protein complexes with the consensus Ets-domain DNA binding element (Fig. 3Go, upper panel), indicating that the antibody was effective. Thus, it appears that the two putative Ets sites are not equivalent, although both are critical for stimulated promoter activity, and form slightly different protein complexes. Because unlabeled mutant DNA can effectively compete with wild-type DNA for the formation of all four complexes, they may permit binding of "core" proteins, while prohibiting the formation of or destabilizing other heteromeric complexes necessary for transcription. None of the binding proteins appear to be Ets-1 or Ets-2. No significant differences were noted with nuclear proteins from control or GnRH-treated cells (not shown). Similar results were observed in studies with nuclear proteins from {alpha}T3 cells (not shown).

GnRH-Responsive Elements in the Rat LHß Subunit Gene
We performed initial studies in primary cultures of dispersed female rat pituitary cells treated with either static or intermittent 10 nM GnRH (1 pulse/h). Under these conditions (Fig. 4Go), the transfected LHß promoter was stimulated 2- to 3-fold by pulsatile but not static GnRH. The data suggest that in normal gonadotrope cells the nature of the GnRH signal is critical in LHß gene regulation and agrees with previous transcriptional run-off data of pituitary cells treated with pulsatile and static GnRH (11). Deletions of the gene between -2.0 kb and -617 bp relative to the transcriptional start site slightly increased basal activity, but subsequent deletions decreased basal activity. GnRH responsiveness was eliminated when the region from -495 and -245 bp was removed, indicating this area contained at least one physiologically relevant GnRH response element. Transfer of either the -495 to -342 bp region, or -450 to -342 bp region of the LHß promoter to either the viral thymidine kinase promoter or the -106{alpha}-subunit minimal promoter was sufficient to confer a GnRH response to the heterologous promoters in {alpha}T3 cells (Table 1Go). These data are in agreement with results from other investigators who used a heterologous sommatolactotrope pituitary cell line and identified a major GnRH responsive element that resided in this region of the LHß gene (24).



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Figure 4. Deletion Analysis of the Rat LHß Subunit Gene Promoter Identifying the Region Responsive to Pulsatile GnRH

Luciferase constructs (15 µg) containing portions of the rat LHß promoter and CMV-ß-galactosidase (1 µg) were transfected into primary cultures of female rat pituitary cells for 4 h, followed by a 15% glycerol shock. Constructs included promoter regions from the 5'-base indicated, to the common 3'-end at +41 bp. After incubation in culture media for 16 h, transfected cells were treated with 10 nM GnRH for 8 h either continuously (static), or as a series of 10-min pulses once per h (pulse) and compared with untreated (control) cells. After treatment, luciferase activity was measured and normalized for both protein content and ß-galactosidase activity. Normalized luciferase activity (ALU/mg protein) is expressed as the mean ± SEM for two experiments, with four culture dishes per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. untreated controls.

 
We next tested the response of several wild-type and mutated constructs in LßT2 cells, which express the endogenous LHß gene. Because the endogenous rat LHß gene (11) and LHß promoter constructs transfected into primary cultures of rat pituitary cells require pulsatile GnRH to stimulate promoter expression, we tested each construct for 6 h with hourly GnRH pulses, each with matched controls. Within this responsive region (Fig. 2Go.) are several putative binding sites for Sp1 at both the 5'- and 3'-ends. The 3'-region contains up to three such Sp1 sites, with two sites almost completely overlapping (shown by the underlined and italicized 3'-site in Fig. 2Go), while the 5' Sp1 site at -450 bp overlaps a CArG box at -443 to -434 bp. CArG elements [CC(A/T)6GG] are contained in many promoter enhancer regions, including the serum response element (SRE) of the c-fos gene, and are often part of composite elements involved in rapid and transient transcriptional responses (29, 30, 31). In LßT2 cells, pulsatile GnRH treatments, previously shown to be required for LHß mRNA stimulation (27), also stimulated the intact LHß promoter constructs 5- to 6-fold (Fig. 5Go). The degree of stimulation was greater than that observed with GnRH treatment of {alpha}T3 cells transfected with heterologous promoter constructs, and larger than responses observed with the native promoter and pulsatile GnRH treatment of transfected primary pituitary cells (Fig. 4Go). Hourly GnRH pulses, while stimulating the LHß gene promoter, were actually ineffective in stimulating {alpha}-subunit promoter activity (Control = 60,149 ± 5,948 vs. pulsed GnRH = 56, 908 ± 17, 444 arbitrary luciferase units (ALU)/100 µg protein) compared with a 4-fold stimulation with static GnRH in the same experiment (compare with Fig. 1Go).



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Figure 5. Deletion/Mutational Analysis of the LHß Promoter in LßT2 Cells with Pulsatile GnRH Treatment

Cells were transfected with the indicated promoter constructs (3 µg) for 16 h, followed by 6 h of treatment of 100 nM GnRH for 6 h with one 10-min pulse of GnRH per h. Each control group had media changes identical to treatment groups. The sites of the 5'Sp1, CArG box, 3'Sp1, and Egr-1 sites are shown, as well as a mutation in an A-T rich region at -340 bp. Mutated sites in the upstream responsive region are indicated by an X in the bar symbols along the Y axis. Normalized luciferase activity is represented as the mean ± SEM for six experiments, with three to four samples per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. untreated controls.

 
Mutational analysis of the LHß gene suggested a complex interrelationship between multiple elements in the -450 to -342 bp responsive region. Although the GnRH response was significantly reduced when the promoter region between -456 and -345 bp was removed, and the upstream region from -450 to -342 bp conferred a GnRH response to heterologous promoters (Table 1Go), the -345 bp construct was still stimulated by GnRH in LßT2 cells. This suggests that an additional GnRH-responsive site or sites are present in the -345 bp promoter. Mutation of an AT-rich site at -340 bp or deletion of the region between -375 and -245 bp, which contains several additional Sp1 consensus binding sites, had little impact on either basal or stimulated promoter activity. Individual mutations of either the CArG box or the -365 bp 3'Sp1 site within the context of the entire homologous promoter decreased basal stimulation, while mutation of the 5' Sp1 site actually increased basal expression. However, under pulsatile GnRH treatment conditions, single mutations in either the CArG box, the 5'Sp1, or 3'Sp1 sites effectively eliminated the GnRH response (Fig. 5Go), indicating that these mutations restrict the activity of GnRH-responsive sites in the -345 bp promoter.

Additional mutational analysis suggested that the two Sp1 and CArG sites interact in the intact promoter. Mutation of both the 3' and 5'Sp1 sites, or mutation of the 3'Sp1 site and the CArG box in the CG/SP mutant, partially restored GnRH stimulation. Similarly, mutation of the 5'Sp1 site in the presence of both the 3'Sp1 and CArG mutations did not significantly change the response to GnRH compared with the double mutant. These data demonstrate that stimulation of full-length promoter activity by pulsatile GnRH treatment requires at least three distinct upstream elements, including the two Sp1 sites that are involved in the response to static GnRH (24), and an additional site in the CArG box. Removal of this entire region, or mutation of sites at both the 5' and 3' ends of this region, results in 2- to 3-fold GnRH stimulation, equal to that observed with the -345 bp construct.

Recently, two binding sites for Egr-1, at approximately -112 and -50 bp, have been described in the rat LHß promoter and documented to be responsive to pulsatile GnRH (41, 42, 43, 44, 45, 46). To test the possibility that the Egr-1 sites contribute to the GnRH response, the -112 and -50 bp sites were mutated individually within the context of the -456 to +44 promoter, with and without mutation of the upstream GnRH-responsive element (Fig. 6Go). Mutation of the -112 Egr-1 site had only a slight effect on either basal or GnRH-stimulated expression, and inclusion of the -112 Egr-1 mutation in the CG/SP double mutant had little effect on GnRH stimulation. Mutation of the -50 Egr-1 site, however, reduced basal expression by 50% and GnRH-stimulated promoter activity to 2.3-fold, compared with 5-fold for the intact wild-type promoter. Furthermore, mutation of the -50 Egr-1 site within the context of the CG/SP mutant in which the CArG box and the 3'Sp1 site are mutated effectively obliterates GnRH stimulation. Thus, the upstream composite element cooperates with the Egr-1 binding site at -50 bp to confer the full GnRH response to the LHß promoter.



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Figure 6. Mutational Analysis of Egr-1 Sites with or without Mutation of the Upstream GnRH-Responsive Region of the LHß Promoter in LßT2 Cells Treated with Pulsatile GnRH

Cells were transfected with the indicated promoter constructs (3 µg) for 16 h, followed by 6 h of treatment of 100 nM GnRH for 6 h with one 10-min pulse of GnRH per h. Each control group had media changes identical to treatment groups. Mutated sites in each construct are designated by an X in the symbols along the Y axis. Normalized luciferase activity is represented as the mean ± SEM for four experiments with three samples per group. *, P < 0.05; **, P < 0.01 vs. untreated controls.

 
Although pulsatile GnRH treatment is required for LHß gene stimulation in vivo, treatment of LßT2 cells with acute 100 nM GnRH for 4 h can also stimulate LHß promoter activity to some extent. Under these conditions, single mutations of the 3'Sp1 and 5'Sp1 sites suppress the GnRH response (5'SP1mut = 2.2-fold stimulation, and 3'SP1mut = 1.3-fold stimulation vs. 4.8-fold stimulation for the wild-type promoter), but mutation of the CArG box has no effect (CArGmut = 5.0-fold, and was no different from the wild-type construct in five of five experiments). The loss of this sensitive interplay of gene elements with static GnRH treatment suggests that proteins binding to the GArG box are important for pulsatile GnRH responsiveness, which is the relevant stimulating paradigm in vivo.

Nuclear Protein Binding to GnRH-Responsive Regions of the LHß Gene
Oligonucleotides representing the portions of the upstream composite GnRH- responsive region of the rat LHß gene formed several DNA protein complexes with nuclear proteins from LßT2 gonadotrope cells. Similar results were obtained with proteins from control or GnRH-treated cells (not shown). An oligonucleotide representing the 3'Sp1 site at -365 bp binds nuclear proteins that include Sp1, based on the mobility of the complex, the addition of pure Sp1 protein (Fig. 7Go, top panel, Sp1 complex shown by an arrowhead), and the inhibition of complex formation by the addition of Sp1 antibody. The mutated 3'Sp1 oligonucleotide, corresponding to the transcriptionally inactive construct (Fig. 5Go), did not bind Sp1. Similar studies with the wild-type 5'Sp1/CArG box oligonucleotide (Fig. 7Go, bottom panel) demonstrated that at least three to four complexes were formed with nuclear proteins from gonadotrope cells. Individual mutations of the 5'Sp1 site (5'SP1mut) or the CArG box (CarGmut) site revealed that the two areas were functionally different when comparing protein binding to these labeled probes. The 5'Sp1mut probe does not form the upper complex corresponding to Sp1 from LßT2 nuclear proteins and could not bind pure Sp1 (not shown). In contrast, the oligonucleotide corresponding to the mutated CArG box, which effectively eliminated stimulation specifically by pulsatile GnRH, still binds Sp1 effectively. These data suggest that Sp1 binding alone is insufficient for pulsatile GnRH stimulation of transcription. Two additional mutated oligonucleotides, representing our double mutation of the 5'Sp1 and CArG box (5'SP1mCGm) and a previously described Sp1 mutation (24) with several widely spaced mutated bases (5'SP1m2CG), were also tested. The double mutant 5'Sp1mCGm oligonucleotide does not bind proteins effectively, while the previously described mutant (5'Sp1m2CG) fails to bind Sp1 but retains some binding to the intermediate mobility complexes, designated by a bracket.



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Figure 7. Nuclear Protein Binding to Labeled Wild-Type and Mutant DNA Oligonucleotides Representing Portions of the Upstream GnRH-Responsive Region of the Rat LHß Gene

The position of the Sp1 complex is shown by an arrowhead, and the positions of intermediate migrating complexes are designated by brackets. Top panel, Labeled DNA representing the wild-type and mutated 3'Sp1 LHß sequence was incubated with either 6 µg of untreated LßT2 cell nuclear protein or 1 footprinting unit of recombinant human Sp1. In one set of reactions, nuclear proteins were incubated with wild-type 3'Sp1 probe in the absence or presence of antibody to Sp1. Bottom panel, Labeled probes representing the wild-type and mutated 5'Sp1CArG region of the LHß promoter, with individual or double mutations in the Sp1 or CArG region as shown, were incubated in the absence or presence of 6 µg of LßT2 nuclear proteins and run on a nondenaturing gel. In the right three lanes of the panel, the 5'Sp1CGm (CArG mutation) probe is shown next to a wild-type (WT) probe run on the same gel for direct comparison.

 
Competition studies performed with the wild-type labeled probe (Fig. 8Go) were in essential agreement with the above data. Addition of unlabeled wild-type oligonucleotide (WT 5'Sp1CG) inhibited binding to all protein complexes. In comparison, addition of the previously described Sp1 mutation (5'Sp1m2CG) had no effect on the formation of the Sp1 complex and moderately inhibited formation of the intermediate complexes. Addition of the double mutant 5'Sp1mCGm oligonucleotide did not inhibit formation of any complexes. Addition of unlabeled WT 3'Sp1 binding site inhibits formation of only the Sp1-DNA complex, and similar results were obtained with an unlabeled consensus Sp1 site. In contrast, addition of an unlabeled oligonucleotide representing the CArG region, but not the entire 5'Sp1 site (WT CG), prevented formation of only the intermediate mobility complexes. This result is similar to the binding profile observed with competition by a full-length oligonucleotide mutated in only the CArG region (5'Sp1CGm), which specifically inhibits transcriptional stimulation by pulsatile GnRH. Addition of unlabeled consensus binding sites for CREB or Ets-domain proteins had no effect on nuclear protein binding (not shown). Thus, the functional interplay between the 5'Sp1 and CArG-box binding sites required for GnRH-stimulated transcription via the upstream response region occurs by interaction between at least two distinct transcription factors, one of which is Sp1. The second factor or factors bind to the CArG box and are critical for maintaining the transcriptional response to pulsatile GnRH signals. This complex is required for optimal GnRH responsiveness and appears to cooperate with downstream elements binding Egr-1.



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Figure 8. Competition Studies of Untreated LßT2 Nuclear Protein Binding to the Labeled Wild-Type 5'Sp1CArG Region Probe in the Presence of Unlabeled Competitor DNA

Nuclear proteins (4 µg) were preincubated with a 200-fold molar excess of unlabeled DNA before incubation with labeled probe. Competitor oligonucleotides included the 3'Sp1 site and wild-type and three different mutated 5'Sp1CArG regions as shown in Fig. 6Go. An additional oligonucleotide that included the 5'CArG region but not the entire 5'Sp1 site (WT CCG, 5'-cacccatttttggacccaatcc-3') was also added. The middle lane shows binding of the wild-type probe to recombinant human Sp1 for comparison.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pulsatile hypothalamic release of GnRH plays an integral role in regulating gonadotropin secretion and gene expression, and thus normal sexual development and reproductive function. Specific pulse frequencies of GnRH have differential effects on LH and FSH secretion, as well as on the transcription of the three gonadotropin subunit genes in vivo and in vitro. The common {alpha}-subunit gene can be stimulated by either rapid to intermediate pulses or by static GnRH, whereas the LHß gene requires intermittent GnRH stimulation (9, 10, 11, 12, 13). Given that all three genes are expressed in the same cell type and are stimulated by the same ligand and receptor, such subunit-specific effects are potentially due to differential sensitivity to GnRH-regulated signaling pathways and different nuclear targets responsive to GnRH. Emerging evidence from several groups suggest contributions from both possible mechanisms.

GnRH stimulates both calcium influx and phospholipase C/protein kinase C signaling with PKC stimulation linked to subsequent activation of MAPK activity (14, 15). No clear consensus on sensitivity to signaling pathways has emerged, although genes from several species have been tested in a variety of transfection systems. However, the relative sensitivity of the LH subunit genes to the two arms of the signaling pathways has differed in studies where the two mRNAs and promoters have been tested simultaneously (16, 17, 20, 21). Transfection studies performed in the heterologous GGH3 cell line suggest that the rat LHß promoter is more sensitive to the PKC pathway, while the human {alpha}-subunit promoter is stimulated by both calcium and PKC (24). Studies of GnRH stimulation of gonadotropin mRNA levels in primary rat pituitary cultures have shown that GnRH-stimulated {alpha}-subunit, FSHß, and GnRH-R mRNA levels are suppressed by inhibitors of MAPK activity, but LHß mRNA stimulation is not (17). We have used nuclear run-off assays in normal rat pituitary cells, transfection into {alpha}T3 gonadotrope cells, and transgenic animal studies and have found that the rat {alpha}-subunit promoter is relatively more sensitive to MAPK stimulation, while stimulation of the rat LHß gene is relatively more sensitive to calcium influx (20). Such apparent discrepancies may be a result of species differences in the gene promoters used, different transfection systems and cell lines, or slightly different promoter constructs used, including heterologous promoter/response element constructs. It is also probable that cross-talk between signaling pathways exists, and that the relative amount of signaling through each pathway could vary under physiological conditions. Thus, although the exact signaling pathways that are most critical for stimulation of each responsive gene are not consistent in all systems, there is clear divergence in the pathways’ importance among the GnRH-responsive genes. Recent studies suggest that stimulation of the ovine FSHß promoter (25) and the murine GnRH-R promoter by GnRH (26) occur via the PKC/MAPK pathway acting through multiple promoter regions including AP-1 sites. However, AP-1 sites do not appear to play a role in stimulation of the rat {alpha}-subunit or LHß promoters by GnRH.

Studies with the mouse {alpha}-subunit gene promoter transfected into clonal {alpha}T3 cells (19) or the human gene transfected into either rat pituitary cells (23) or {alpha}T3 cells (18) have demonstrated that the GnRH stimulation is tightly linked to PKC and MAPK kinase stimulation, similar to our observations with the rat {alpha}-subunit gene in normal pituitary or transfected {alpha}T3 cells (20). Stimulation of the human promoter in the heterologous GGH3 line appears to occur primarily through a calcium-mediated cascade (24). The mouse {alpha}-subunit promoter requires two cooperating regions for complete GnRH stimulation (22). One area from -337 to -330 bp is necessary for pituitary-specific promoter expression and another between -416 to -385 bp (the GnRH-RE) confers a GnRH response to heterologous promoters. This GnRH-RE region is depicted in Fig. 2Go and shares considerable sequence homology with the GnRH-responsive region in the rat {alpha}-promoter, which lies between -411 and -375 bp. The GnRH-responsive region of the human gene is much less well defined and appears to require multiple interacting regions, while second messenger specificity studies have been performed with the -846 bp construct (19). Alignment of the human {alpha}-subunit gene region from -428 to -397 bp with the rat GnRH-responsive region from -411 to -375 bp, and the mouse gene from -416 to -385 bp (Fig. 2Go) reveals significant DNA sequence homology and some important differences. All three genes contain GGAA sequence motifs, which are core consensus binding sites for Ets-domain proteins; however, the rat gene contains two closely aligned sequences on opposite DNA strands, and the mouse and human genes have only one (upstream) site. Ets factors have been shown to be phosphorylated directly by or in response to activated MAPK and thus could be direct mediators of GnRH stimulation (32, 33). Studies with the transfected rat {alpha}-subunit promoter show that inhibition of the MAPK pathway suppresses basal as well as stimulated promoter activity (20), as is observed with mutation of the putative Ets-factor binding sites, suggesting that these sites modulate promoter activity through the MAPK pathway. Disruption of either site in the rat gene suppresses basal and eliminates GnRH stimulation of promoter activity.

A GAL4-Elk-1 fusion protein directly stimulated the mouse {alpha}-subunit gene promoter, and both functional and DNA binding studies show that Ets-2 can bind the mouse gene region; however, the gonadotrope factor(s) binding directly to this region has not been identified (19). Because the addition of an antibody to Ets1/Ets-2 that recognizes a spectrum of Ets factor family members did not interfere with the binding of LßT2 cell proteins to the rat {alpha}-subunit promoter, but did recognize proteins binding to a consensus Ets site, a novel protein or proteins may participate in the GnRH response. A consensus Ets binding site oligonucleotide did compete for binding to this region, suggesting that a transcription factor with similar DNA sequence binding properties is involved. The unique positioning of two putative Ets binding sites on the GnRH-responsive region of the rat {alpha}-subunit gene, and the requirement for both intact sites for GnRH stimulation, suggests functional cooperation between the sites. GABP, which confers insulin sensitivity to the PRL gene promoter (34), is a heterodimeric protein in which one subunit is an Ets-like protein that binds DNA, and the other subunit contains a series of ankyrin repeats (35). Although the {alpha}-subunit promoter does not contain a consensus site for GABP binding, a similar type of protein or proteins, capable of forming functional protein-protein interactions, may be involved in conferring the GnRH response.

The GnRH-responsive region for the rat LHß gene is highly divergent from that of the {alpha}-subunit promoter, in keeping with the differential sensitivity of the two genes in vivo to different GnRH pulse regimens, and different responses of the transfected subunit promoters to various signaling pathways. Instead of relying on Ets-like binding sites, the LHß gene upstream GnRH-responsive region contains a number of interacting transcription factor binding sites, including two Sp1 binding regions and a CArG box element. In these studies, we have demonstrated the importance of the Sp1 binding sites in both GnRH-stimulated and basal expression in gonadotrope cells, as others have previously demonstrated in heterologous cell lines (24, 36). However, Sp1 binding alone is insufficient for the GnRH response, as mutations in the newly defined CArG element permit Sp1 binding, yet abolish the response to pulsatile GnRH (Figs. 5Go and 7Go). Proteins that bind to CArG boxes, such as serum response factor (SRF) and related proteins, often form multiprotein complexes and are involved in rapid and transient transcriptional responses (29, 30, 31). The smooth muscle myosin heavy chain gene promoter contains a negative-acting GC-rich region located between two stimulatory CArG elements that bind SRF (30, 31). The GC-rich region in the myosin promoter binds both Sp1 and Sp3 and modifies the gene response to SRF without interfering with SRF binding. In the LHß gene, the 5'-Sp1 binding site and CArG element overlap, but Sp1 and CArG box protein binding can be differentially inhibited by mutation analysis. Protein binding to both sites appears to be required for complete GnRH stimulation. The role of SRF and related proteins in rapid and transient transcriptional responses is intriguing in light of the preferential response of LHß to GnRH pulses, and the unique role of the CArG element in that response. It is unlikely that SRF, a muscle protein, is contained in gonadotropes, but a similar type of protein may be involved.

There is an interesting and complex relationship between the composite 5'Sp1/CArG element and the 3'Sp1 binding site. Although mutations in any of these three sites completely suppressed the GnRH response, mutation of the 3'Sp1 site in combination with the 5'Sp1 and/or the CArG box restores responsiveness to the level observed with the -345 bp construct. These data suggest that interactions between the two Sp1 sites occur and restrict the responsiveness of the gene promoter. The -345 bp construct contains two composite binding sites for two transcription factors found to have a critical role in gonadotropin expression, SF-1 and Egr-1 (37, 38, 39, 40, 41), and several groups have now described cooperative functional interactions between these two proteins on the rat and equine LHß promoters (42, 43, 44, 45). Our studies used a larger promoter region than the constructs used in other studies, and the data demonstrate that Egr-1 binding sites can cooperate with other upstream GnRH-responsive regions as well. In the context of the larger LHß promoter, both the upstream Sp1/CArG regions and the Egr-1 sites are required for the full response to pulsatile GnRH.

Disruption of either the SF-1 (40) or Egr-1 (41) genes in mice severely impairs LHß gene expression, but the relationships are not completely straightforward. For example, SF-1 gene disruption eliminates expression of all gonadotropin subunit genes, but injection of the animals with GnRH partially restores expression (40). Disruption of the Egr-1 gene completely eliminates Egr-1 protein expression in both males and females, but only in females is LHß gene expression completely eliminated with resulting infertility (41). In a separate study, target disruption of the Egr-1 gene at a different position completely eliminated LHß protein expression and resulted in infertility in both sexes (46). Both SF-1 (39) and Egr-1 mRNA (43, 44, 45) can be stimulated by GnRH treatment, and Egr-1 may also be stimulated by PKC. Particularly dramatic increases in Egr-1 mRNA have been observed in LßT2 cells with pulsatile GnRH (43), although increased Egr-1 expression has also been noted in {alpha}T3 cells (43, 44). The resulting increase in Egr-1 protein levels thus stimulates expression of the LHß promoter, and mutations in Egr-1 binding sites have a larger effect on basal and GnRH-stimulated promoter activity than do mutations in SF-1 sites. Our studies, and previous studies in a heterologous cell line, suggest that GnRH stimulation of the rat LHß promoter requires an integrated response of multiple transcription factors binding to several discrete promoter elements, distinct from and in addition to the promoter regions binding SF-1 and Egr-1 (19, 20). Studies with the isolated LHß response elements on two heterologous promoters (Table 1Go and Ref. 20) demonstrate that the region between -456 to -342 bp is capable of conferring a GnRH response separate from that conferred by the SF-1/Egr-1 regions and may help explain some differences in cell signaling transfection studies. The GnRH response of the promoter may thus be governed partly by Egr-1 levels that are modulated by GnRH, and partly by other GnRH-mediated pathways that stimulate the Sp1/CArG containing upstream region. Relatively high or static Egr-1 levels may permit the upstream region of the gene to play a larger role in GnRH regulation and might explain the very low response of the smallest LHß promoter construct in primary cultures. Overall, these studies indicate that the transcription factors involved are distinct from those conferring GnRH stimulation to the rat {alpha}-subunit gene and represent the first direct comparison of these two subunits from the same species. There may be physical as well as functional interactions between these factors, in addition to modification of transcription factors by calcium and PKC-activated kinases as well as MAPK. Direct interaction between Sp1 and Egr-1 has been demonstrated in osteoclast cells, with Egr-1 limiting the amount of free Sp1 available to activate gene transcription. (46). Functional interactions between Egr-1 and Sp1 could occur in the gonadotrope cell, either in solution or bound to DNA and help explain the restrictive influence of Sp1 mutations on the GnRH stimulation of the LHß promoter.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Vector Preparation
The rat {alpha}-subunit gene promoter from -479 to +77 relative to the transcriptional start site was cloned from rat genomic DNA and inserted into the luciferase reporter vector as previously described (18). Deletion constructs corresponding to 5'-ends of -411, -354, -245, and -106 bp were obtained from this construct by PCR amplification and cloning into the PCRII vector (Invitrogen, San Diego, CA). After DNA sequence verification of both strands, each promoter was inserted into the LUCII expression vector (18). Primers used for constructing promoter regions included intronic primer from +77 to +59 bp (5'-ttccccaaatgatttcatc-3'), and the 5'flanking sequence primers at -411 bp (5'-atggatccacttttctgtttcctgttc-3'), -354 bp (5'-ccgaaaacatcaggcac-3'), -245 bp (5'-cccttttctggccaag-3'), -179 bp (5'-ccccaaaatgtctaaaagc-3'), and -106 bp (5'-ccataccaagtaccatc-3').

The rat LHß gene promoter region from the BamHI site at -2 kb to the KpnI site at +41 bp relative to the transcriptional start site was obtained from a rat genomic DNA clone and inserted into the Luciferase expression vector as described (20, 48). Deletion constructs of this region were made by using the 3'-end KpnI site at +44 bp and restriction enzyme sites corresponding to 5'-ends at -617 (EcoRI) and -245 bp (PstI). Additional deletion constructs were generated by PCR amplification of the genomic clone using primers corresponding to the 3'-KpnI site and 5'ends at -456 bp (5'-gctgaaaccacacccatttttggaccc-3'), and -345 bp (5'-gaggatccgaacaccgaagctgtgc-3'). All amplified promoter regions were first cloned into the PCRII vector, and then inserted into the luciferase expression vector at the BamHI polylinker site. All constructs were sequenced to ensure fidelity. A construct containing the promoter region from -617 to +44 bp, but with a -370 to -246 bp deletion ({Delta}370/246), was made from a -617 to +44 bp LHß promoter clone in a pGEM3Z plasmid in which the PstI site was eliminated. This construct was cut with PstI and KpnI to remove the -617 to -245 bp region. This gene fragment was then cut with DraI, and the resultant 250 bp fragment from -617 to -371 bp was isolated and inserted by blunt end ligation into the pGEM3Z vector containing the remaining gene region, before cloning into the luciferase reporter.

Isolated putative GnRH-responsive regions were fused to two heterologous promoters that were not responsive to GnRH, including the -106 {alpha}-subunit luciferase construct, and the Herpes simplex virus thymidine kinase (tk) promoter construct, as previously described. For the {alpha}-subunit gene, the region between -411 and -375 was generated by PCR amplification. The amplified fragment was excised from the PCRII vector with BamHI and inserted into the BamHI site at the 5'-end of the -106 {alpha}-subunit promoter region, or in the polylinker region of the tkLUC vector. A similar strategy was followed for the LHß gene, in which the gene regions between -495 and -342, and between -450 and -342 bp, were amplified and fused to the heterologous promoters.

Mutagenesis
Two separate mutagenesis strategies were used, depending on the location of the altered region. For internal mutations within the context of the homologous promoter, the Altered Sites II in vitro mutagenesis kit (Promega Corp., Madison, WI) was used. Briefly, a template was prepared in which the -617 to +44 bp region of the rat LHß gene was inserted at the KpnI and BamHI sites into the pAlter vector, which contains potential ampicillin and tetracycline resistance regions. This construct was denatured to anneal with mutagenic oligonucleotides, including mutations for the identified 3'Sp1 site at -365 bp (5'-ggggctgggcgagggTTTgcgcccacctctgg-3'), the CArG-box region at -436 bp (5'-aaccacacccattttCCgacccaatccaggcatc-3'), an AT-rich region at -340 bp (5'-cacctctggttgtatCCCaagcaaatttggaggc-3'), the Egr-1 site at -112 bp (5'-ctgaccttgtctgtctCgCCCcaaagattagtgtc-3''), and the Egr-1 site at -50 bp (5'-gtggccttgccacccCCaCaaccCgcaggtataaagcc-3''). Simultaneously, ampicillin repair and tetracycline knock-out oligonucleotides were added to change the antibiotic resistance as a marker for successful mutagenesis. Each of these single mutations was introduced, and the resultant constructs were sequenced and inserted into the luciferase expression vector. The mutated 3'Sp1 site plasmid (3'SP1 mut) was used as a template to introduce the CarG box mutation (CArGm) and to form a double mutant (CGm3'SP1m). Alternatively, for mutations in the LHß promoter -456 to +44 bp constructs, PCR amplification was performed with wild-type or mutated -617 to +44 bp pAlter templates to introduce mutations at the 5'Sp1 site (5'SP1mut with oligonucleotide 5'-taggtaccacacAAatttttggacccaatcc-3'), alone or in combination with other mutations. The oligonucleotide 5'SP1mCGm (5'-taggtaccacacAAattttCCgacccaatcc-3') was used with the mutated 3'Sp1 site template to produce a triple mutant construct (5'SP1mCGm3'SP1m). Triple mutants containing single Egr-1 mutations in combination with the 5'CArG site and 3'SP1 site were similarly created. All mutated bases are shown in capital letters.

A similar strategy was used to introduce mutations into the potential Ets-domain binding sites of the rat {alpha}-subunit promoter. A mutation in the 5'-Ets site ({alpha}Mut5', with the primer 5'- atccatcttttctgttGActgttggaata-3') was made by PCR amplification from the -479 to +71 bp construct, and the 3'-genomic primer. The {alpha}Mut5' construct was then used with the {alpha}Mut5',3' primer (5'- atccatcttttctgttGActgttgTGataacgtaga-3') to prepare a construct with mutations in both Ets sites. Mutant {alpha}-subunit GnRH-responsive elements obtained by chemical synthesis of appropriate complementary oligonucleotides were annealed and inserted into tkLUC or -{alpha}106LUC vectors as described. Single mutations were incorporated into each Ets site with the following oligonucleotide sequences that contained 5'KpnI and 3'BamHI sites: MUT5' (5'-cttttctgttGActgttggaataacgtagag-3') and MUT3' (5'-tttctgtttcctgttgTGataacgtagag-3') and both mutations.

Cell Cultures and Transfections
For {alpha}-subunit gene constructs, and studies of both LH subunit gene isolated elements in heterologous promoter constructs, transfection studies were first performed in the {alpha}T3 cell line, and subsequently in the LßT2 cell line. The {alpha}T3 precursor gonadotrope cell line (28) contains functional, well characterized GnRH receptors and expresses the {alpha}-subunit gene, but neither the endogenous nor exogenous transfected LHß promoter. Cells were grown in DMEM containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were plated at a density of 0.5–0.7 x 106/60 mm well 36 h before a 16 h CaPO4 transfection with 5 µg DNA per well. After transfection, cells were washed and treated for 6 h with 10-7 M GnRH or the des-gly agonist (GnRHa). For all transfection studies, cells were then washed and collected in 250 µl lysis buffer (Promega Corp.), vortexed, spun for 1 min, and assayed in a Turner 20e luminometer (Turner Designs, Mountain View CA). The same lysates were used for protein determination, using the colorimetric protein assay system (Bio-Rad Laboratories, Inc., Hercules, CA). Luciferase activity was expressed as ALU per 100 µg protein. In each experiment, treatment groups contained three to four wells, and values represent the results of three to six experiments. Control and GnRH treatment groups were compared for each construct, and statistical significance was assigned by Student’s t test. In some mutational analysis studies, values are expressed relative to untreated controls for each construct, set at 100% or 1.0.

LHß promoter constructs were first tested in primary cultures of rat pituitary cells. Primary cultures of dispersed randomly cycling female rat pituitary cells were prepared from mascerated glands as previously described (49) by digestion with 0.125% trypsin at 37 C for 1.5 h, and collagenase (2 mg/ml, Worthington Biochemical Corp., Freehold NJ) for 30–45 min at 37 C. Cells were plated in 60-mm wells (1.5–2.0 x 106 cells per well) in DMEM/10% FBS media. After 20–24 h, cells were transfected by the CaPO4 method for 4 h, followed by a 5-min incubation with 15% glycerol in PBS. Each well received 15 µg LHß construct and 1.0 µg cytomegalovirus (CMV)-ß-galactosidase. After transfection, wells were washed and incubated for 16 h in culture media before treatment with 10- 8 M GnRH either continuously for 8 h, or in a regimen of 1 pulse of 10-8 M GnRH for 10 min per h. Luciferase activity was measured and normalized for protein and ß-galactosidase activity. Data are represented as the mean ± SEM for six wells per group, compared with untreated controls.

All wild-type and mutated LHß constructs were subsequently analyzed in the murine LßT2 gonadotrope cell line (27). These cells express both endogenous mouse and transfected rat LH subunit genes, secrete LH, and contain functional GnRH receptors that respond to pulsatile GnRH stimulation. Cells were grown in DMEM with 10% FBS and antibiotics and were plated in 35-mm wells at a density of 1.5 x 106 cells per well 16–24 h before CaPO4 transfection. Each well was transfected with 3 µg of vector for 16 h, washed, and treated with 10-7 M GnRH for either 6 h continuously (acute static treatment), or with 10-7 M GnRH for 10 min each hour for 6 h (pulsatile treatment). When acute and pulsatile treatment regimens were compared, separate controls (no GnRH) were used for the acute and pulsed treatment groups. The controls for the pulsatile GnRH treatment were handled identically to the treatment groups with respect to time out of the incubator and media changes with control (no treatment) media, to ensure that the observed responses were not due to experimental artifacts. In several experiments, a CMV-ßgalactosidase reporter was included (0.3 µg/well) to normalize luciferase activity. After treatment, cells were collected and assayed as above. Each experiment includes three to four wells per treatment group. Data are represented as the mean ± SEM and represents data from three to six experiments as indicated.

Nuclear Proteins and Electrophoretic Mobility Shift Assays
Nuclear proteins were isolated from LßT2 cell nuclei by the method of Dignam et al. (50). The extraction buffer [20 mM HEPES pH 7.3, 0.6 M KCl, 20 µg/ml ZnCl2, 0.2 mM EGTA, 0.5 mM dithiothreitol (DTT)] contained protease inhibitors (10 µg/ml each of aprotonin, antipain, chymostatin, leupeptin, and 1 µg/ml pepstatin). After ultracentrifugation at 100,000 x g, supernatant proteins were subjected to chromatography on a Sephadex G column with buffer (20 mM HEPES, pH 7.3, 1.5 mM MgCl2, 0.15 M KCl, 0.5 mM DTT, and protease inhibitors). For electrophoretic mobility shift (gel shift) assays, nuclear protein (4–6 µg) was incubated with labeled DNA 50–100(50–100,000 cpm) and buffer containing final concentrations of 10 mM Tris-HCl, pH 7.5, 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, and 1 µg poly(dI-dC). Final volumes were 15–20 µl, and final salt concentrations were adjusted to 100–125 mM KCl. Samples were incubated on ice for 45 min. For competition experiments, protein and buffer were incubated with unlabeled oligonucleotides, at 200-fold excess molar concentrations, on ice for 30 min before addition of labeled DNA. A similar preincubation step was included in studies with antibody addition. After incubation, reactions were subjected to electrophoresis on 5% acrylamide (19:1 acrylamide-bisacrylamide) 1 x Tris-borate-EDTA gel. Gels were run for 1 h at 100 V at 4 C, before sample loading, and samples were subjected to electrophoresis at 100–120 V for an additional 3–4 h.

Complimentary oligonucleotides representing {alpha}-subunit gene sequences in EMSA included the wild-type sequence {alpha}WT (5'-cttttctgtttcctgttggaataacgtacc-3') and mutated sequences {alpha}M5' (5'-cttttctgttGActgttggaataacgtacc-3'), {alpha}M3' (5'-cttttctgtttcctgttgTGataacgtacc-3') and {alpha}M5',3' with both mutated sites. Mutated bases are designated by capital letters. In some experiments, competitor DNA representing consensus binding sites for Ets factors (5'-cggctgcttgaggaagtataagag-3'), CREB, and SP1 were also included. Antibodies specific for Ets-1, or for Ets-1/Ets-2 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Complimentary oligonucleotides representing rat LHß gene sequences for EMSA included wild-type and several mutant sequences representing the 5'Sp1/CArG box region. The wild-type sense strand sequences include the entire region as WT 5'Sp1CG (5'-gctgaaaccacacccatttttggacccaatccaggcatcc-3') and the CArG region without the entire 5'Sp1 site as WT CG (5'-cacccatttttggacccaatcc-3'). Mutated sequences include a previously described 5'Sp1 binding mutation with two separate mutated sites 5'Sp1m2CG (5'-gctgaaaccacacAAatttttggaTccaatccaggcatcc-3'); a mutation in the CArG box only as 5'Sp1CGm (5'-gctgaaaccacacccattttCCgacccaatccaggcatcc-3'); and a mutation in both Sp1 and CArG sites as 5'Sp1mCGm (5'-gctgaaaccacacAAattttCCgacccaatccaggcatcc-3'). The oligonucleotide representing the wild-type 3'Sp1 site was 3'Sp1 (5'-gctgggcgaggggcggcgcccacctc-3') and the oligonucleotide representing the mutated site was 3'Sp1m (5'-gctgggcgagggTTTgcgcccacctc-3'). Pure human recombinant Sp1 was obtained from Promega Corp..


    ACKNOWLEDGMENTS
 
The authors would also like to thank Drs. Gary Owens and Chris Mack for many helpful discussions during presentations of this work and Dr. Pamela Mellon for the cell lines.


    FOOTNOTES
 
Address requests for reprints to: Dr. Margaret A. Shupnik, Box 578 HSC, 7141 Multistory Building, University of Virginia Medical Center, Charlottesville, Virginia 22903.

This work was supported by NIH Grant RO1-HD25719 (to M.A.S.), the National Science Foundation Center for Biological Timing (Grant DIR 890162), and as part of the Specialized Cooperative Centers Program in Reproductive Research (Grant NIH/NICHHD U54-HD-28934).

1 Current Address: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois. Back

Received for publication June 14, 1999. Revision received December 15, 1999. Accepted for publication January 3, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Beltchetz PE, Plant TM, Nakai Y, Keough EJ, Knobil E 1978 Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202:631–633[Medline]
  2. Crowley Jr WF, Filicori M, Spratt D, Santoro N 1985 The physiology of gonadotropin-releasing hormone (GnRH) in men and women. Recent Prog Horm Res 41:473–526[Medline]
  3. Marshall JC, Dalkin AC, Haisenleder DJ, Paul SJ, Ortolano GA, Kelch RP 1991 Gonadotropin-releasing hormone pulses: regulator of gonadotropin synthesis and ovulatory cycles. Recent Prog Horm Res 47:155–187[Medline]
  4. Levine JE, Ramirez VD 1982 Luteinizing hormone-releasing hormone release during the rat estrous cycle and after ovariectomy as estimated with push-pull cannulae. Endocrinology 111:1439–1448[Medline]
  5. Christman GM, Randolph JF, Kelch RP, Marshall JC 1991 Reduction of gonadotropin-releasing hormone pulse frequency is associated with subsequent selective follicle-stimulating hormone secretion in women with polycystic ovarian disease. J Clin Endocrinol Metab 71:1278–1285
  6. Fallest PC, Trader GL, Darrow JM, Shupnik MA 1995 Regulation of rat luteinizing hormone ß gene expression in transgenic mice by steroids and a GnRH antagonist. Biol Reprod 53:103–109[Abstract]
  7. Kaiser UB, Jakubowiak A, Steinberger A Chin WW 1997 Differential effects of gonadotropin-releasing hormone (GnRH) pulse frequency on gonadotropin subunit and GnRH receptor messenger ribonucleic acid levels in vitro. Endocrinology 138:1224–1231[Abstract/Free Full Text]
  8. Wierman ME, Rivier JE, Wang C 1989 GnRH-dependent regulation of gonadotropin subunit mRNA levels in the rat. Endocrinology 124:272–278[Abstract]
  9. Dalkin AC, Haisenleder DJ, Ortolano FGA, Ellis TR, Marshall JC 1989 The frequency of gonadotropin-releasing hormone stimulation and gonadotropin subunit messenger ribonucleic acid expression. Endocrinology 125:917–924[Abstract]
  10. Weiss J, Jameson LJ, Burrin JM, Crowley Jr WF 1990 Divergent responses of gonadotropin subunit messenger RNAs to continuous vs. pulsatile gonadotropin-releasing hormone in vitro. Mol Endocrinol 4:557–564[Abstract]
  11. Shupnik MA 1990 Effects of gonadotropin-releasing hormone on rat gonadotropin gene transcription in vitro:requirement for pulsatile administration for luteinizing hormone-ß gene stimulation. Mol Endocrinol 4:1444–1450[Abstract]
  12. Haisenleder DJ, Dalkin AC, Ortolano GA, Marshall JC, Shupnik MA 1991 A pulsatile gonadotropin-releasing hormone stimulus is required to increase transcription of the gonadotropin subunit genes: evidence for differential regulation of transcription by pulse frequency in vivo. Endocrinology 128:509–517[Abstract]
  13. Gharib SD, Wierman ME, Shupnik MA, Chin WW 1990 Molecular biology of pituitary gonadotropins. Endocr Rev 11:177–199[Medline]
  14. Stojilkovic SS, Reinhart J, Catt KJ 1994 Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr Rev 15:462–499[Medline]
  15. Conn PM, Janovick JA, Stanislaus D, Kuphal D, Jennes L 1995 Molecular and cellular basis of gonadotropin-releasing hormone action in the pituitary and central nervous system. Vitam Horm 50:151–214[Medline]
  16. Haisenleder DJ, Yasin M, Marshall JC 1997 Gonadotropin subunit and gonadotropin-releasing hormone receptor gene expression are regulated by alterations in the frequency of calcium pulsatile signals. Endocrinology 138:5227–5230[Abstract/Free Full Text]
  17. Haisenleder DJ, Cox ME, Parsons SJ, Marshall JC 1998 Gonadotropin-releasing hormone pulses are required to maintain activation of mitogen-activated protein kinase: role in stimulation of gonadotropin gene expression. Endocrinology 139:3104–3111[Abstract/Free Full Text]
  18. Sundaresan S, Colin IM, Pestell RG, Jameson JL 1996 Stimulation of mitogen-activated protein kinase by gonadotropin-releasing hormone: evidence for the involvement of protein kinase C. Endocrinology 137:304–311[Abstract]
  19. Roberson MS, Misra-Press A, Laurence MS, Maurer RA 1993 A role for mitogen-activated protein kinase in mediating activation of the glycoprotein {alpha}-subunit promoter by gonadotropin-releasing hormone. Mol Cell Biol 15:3531–3539[Abstract]
  20. Weck J, Fallest PC, Pitt LK, Shupnik MA 1998 Differential gonadotropin-releasing hormone stimulation of rat luteinizing hormone subunit gene transcription by calcium influx and mitogen-activated protein kinase-signaling pathways. Mol Endocrinol 12:451–457[Abstract/Free Full Text]
  21. Saunders BD, Sabbagh E, Chin WW, Kaiser UB 1998 Differential use of signal transduction pathways in the gonadotropin-releasing hormone-mediated regulation of gonadotropin subunit gene expression. Endocrinology 139:1835–1843[Abstract/Free Full Text]
  22. Schoderbek WE, Roberson MS, Maurer RA 1993 Two different DNA elements mediate gonadotropin releasing hormone effects on expression of the glycoprotein hormone {alpha}-subunit gene. J Biol Chem 268:3903–3910[Abstract/Free Full Text]
  23. Kay TWH, Jameson LJ 1992 Identification of a gonadotropin-releasing hormone-responsive region in the glycoprotein hormone {alpha}-subunit promoter. Mol Endocrinol 6:1767–1773[Abstract]
  24. Kaiser UB, Sabbagh E, Chen MT, Chin WW, Saunders BD 1998 Sp1 binds to the rat luteinizing hormone ß (LHß) gene promoter and mediates gonadotropin-releasing hormone-stimulated expression of the LHß subunit gene. J Biol Chem 273:12943–12951[Abstract/Free Full Text]
  25. Strahl BD, Huang HJ, Sebastian J, Ghosh BR, Miller WL 1998 Transcriptional activation of the ovine follicle-stimulating hormone ß-subunit gene by gonadotropin-releasing hormone: involvement of two activating protein-1 binding sites and protein kinase C. Endocrinology 139:4455–4465[Abstract/Free Full Text]
  26. White BR, Duval DL, Mulvaney JM, Roberson MS, Clay CM 1999 Homologous regulation of the gonadotropin-releasing hormone gene is partially mediated by protein kinase C activation of an activator protein-1 element. Mol Endocrinol 13:566–577[Abstract/Free Full Text]
  27. Turgeon JL, Kimura Y, Waring DW, Mellon PL 1996 Steroid and pulsatile gonadotropin-releasing hormone (GnRH) regulation of luteinizing hormone and GnRH receptor in a novel gonadotrope cell line. Mol Endocrinol 10:439–450[Abstract]
  28. Horn F, Bilezikjian LM, Perrin MH, Bosma MM, Windle JJ, Huber KS, Blout AL, Hille B, Vale W, Mellon PL 1991 Intracellular responses to gonadotropin-releasing hormone in a clonal cell line of gonadotropin lineage. Mol Endocrinol 5:347–355[Abstract]
  29. Liu S, Liu P, Borras A, Chatila T, Speck M 1997 Cyclosporin A-sensitive induction of the Epstein-Barr virus lytic switch is mediated via a novel pathway involving a MAF2 family member. EMBO J 16:143–153[Abstract/Free Full Text]
  30. Madsen CS, Regan CP, Owens GK 1997 Interaction of CArG elements and GC-rich repressor element in transcriptional regulation of the smooth muscle myosin heavy chain gene in vascular smooth muscle cells. J Biol Chem 272:29842–29851[Abstract/Free Full Text]
  31. Madsen CS, Hershey JC, Hautmann MB, White SL, Owens GK 1997 Expression of the smooth muscle myosin heavy chain gene is regulated by a negative-acting GC-rich element located between two positive-acting serum response factor-binding elements. J Biol Chem 272:6332–6340[Abstract/Free Full Text]
  32. Coffer P, de Jonge M, Mettouchi A, Binetroy B, Ghysdeal J, Kruiher N 1993 jun B promoter regulation: Ras mediated transactivation by c-Ets-1 and c-Ets-2. Oncogene 9:911–921
  33. O’Hagan RC, Tozer RG, Symons M, McCormick F, Hassel HA 1996 The activity of the Ets transcription factor PEA3 is regulated by two distinct MAPK cascades. Oncogene 13:1323–1333[Medline]
  34. Ouyang L, Jacob KK, Stanley FM 1996 GABP mediates insulin-increased prolactin gene transcription. J Biol Chem 271:10425–10428[Abstract/Free Full Text]
  35. Batchelor AH, Piper DE, de la Brousse FC, McKnight SL, Wolberger C 1998 The structure of GABP{alpha}/ß; an ETS domain-ankyrin repeat heterodimer bound to DNA. Science 279:1037–1041[Abstract/Free Full Text]
  36. Kaiser UB, Sabbagh E, Saunders BD, Chin WW 1998 Identification of cis-acting deoxyribonucleic acid elements that mediate gonadotropin-releasing hormone-stimulation of the rat luteinizing hormone ß-subunit gene. Endocrinology 139:2443–2451[Abstract/Free Full Text]
  37. Barnhardt KM, Mellon PL 1994 The orphan nuclear receptor, steroidogenic factor-1, regulates the glycoprotein hormone {alpha}-subunit gene in pituitary gonadotropes. Mol Endocrinol 8:878–885[Abstract]
  38. Keri RA, Nilson JH 1996 A steroidogenic factor-1 binding site is required for activity of the luteinizing hormone ß subunit promoter in gonadotropes of transgenic mice. J Biol Chem 271:10782–10785[Abstract/Free Full Text]
  39. Haisendleder DJ, Yasin M, Dalkin AC, Gilrain J, Marshall JC 1996 GnRH regulates steroidogenic factor-1 (SF-1) expression in the pituitary. Endocrinology 137:5719–5722[Abstract]
  40. Ikeda Y, Luo X, Abbud R, Nilson JH, Parker KL 1995 The nuclear receptor steroidogenic factor-1 is essential for the formation of the ventromedial hypothalamic nucleus. Mol Endocrinol 9:478–486[Abstract]
  41. Lee SL, Sadovsky Y, Swirnoff AH, Polish JA, Goda P, Gavrilina G, Millbrandt J 1996 Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (EGR-1). Science 273:1219–1221[Abstract]
  42. Halvorson LM, Kaiser UB, Chin WW 1999 The protein kinase C system acts through the early growth response protein-1 to increase luteinizing hormone ß gene expression in synergy with SF-1. Mol Endocrinol 13:106–111[Abstract/Free Full Text]
  43. Tremblay JJ, Drouin J 1999 Egr-1 is a downstream effector of GnRH and synergizes by direct interaction with Ptx-1 and SF-1 to enhance luteinizing hormone ß gene transcription. Mol Cell Biol 19:2567–2576[Abstract/Free Full Text]
  44. Wolfe M, Call GB 1999 Early growth response protein 1 binds to the luteinizing hormone-ß promoter and mediates gonadotropin-releasing hormone-stimulated gene expression. Mol Endocrinol 13:752–763[Abstract/Free Full Text]
  45. Dorn C, Ou Q, Svaren J, Crawford PA, Sadovsky Y 1999 Activation of luteinizing hormone ß gene by gonadotropin-releasing hormone requires the synergy of early growth response-1 and steroidogenic factor-1. J Biol Chem 274:13870–13876[Abstract/Free Full Text]
  46. Topilko P, Schneider-Maurnory S, Levi G, Trebleau A, Gourdji D, Driancourt M-A, rao CV, Charnay P Multiple pituitary, ovarian Defects in Krox- 24 (NGFI-A, Egr-1)-targeted mice. Mol Endocrinol 12:107–122
  47. Srivvastava S, Weitzmann MN, Kimble RB, Rizzo M, Zahner M, Millbrandt J, Ross FP, Pacifici R 1998 Estrogen blocks M-CSF gene expression and osteoclast formation by regulating phosphorylation of Egr-1 and its interaction with Sp-1. J Clin Invest 102:1850–1859[Abstract/Free Full Text]
  48. Shupnik MA, Rosenzweig BA 1991 Identification of an estrogen-response element in the rat LHß gene: DNA-receptor interactions and functional analysis. J Biol Chem 266:17084–17091[Abstract/Free Full Text]
  49. Shupnik MA, Rosenzwieg BA, Shower MO 1990 Interactions of thyrotropin-releasing hormone, phorbol ester, and forskolin-sensitive regions of the rat thyrotropin ß gene. Mol Endocrinol 4:829–836[Abstract]
  50. Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489[Abstract]