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Endocrinology Vol. 139, No. 5 2443-2451
Copyright © 1998 by The Endocrine Society

ARTICLES

Identification of cis-Acting Deoxyribonucleic Acid Elements That Mediate Gonadotropin-Releasing Hormone Stimulation of the Rat Luteinizing Hormone ß-Subunit Gene1

Ursula B. Kaiser, Elena Sabbagh, Brian D. Saunders and William W. Chin

Division of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Dr. Ursula B. Kaiser, G. W. Thorn Research Building, Room 1009, Brigham and Women’s Hospital, 20 Shattuck Street, Boston, Massachusetts 02115. E-mail: kaiser{at}rascal.med.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH plays a critical role in reproductive development and function by regulating the biosynthesis and secretion of the pituitary gonadotropins, LH and FSH. Although it is known that GnRH induces gonadotropin subunit gene transcription, the mechanism by which this occurs has not been elucidated. Studies have been hindered by the lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have transfected the rat pituitary GH3 cell line with the rat GnRH receptor complementary DNA. These cells, when cotransfected with regulatory regions of the LH or FSH subunit genes fused to a luciferase reporter gene, respond to GnRH with an increase in promoter activity comparable to that seen in primary rat pituitary cells. In this study, we have used this cell model to identify cis-acting elements of the LHß gene that mediate stimulation by GnRH. Analysis of a series of 5'-deletion and internal deletion constructs has revealed two regions of the rat LHß gene promoter involved in mediating the response to GnRH, region A (-490/-352) and region B (-207/-82). Fusion of region A upstream of a heterologous minimal promoter linked to the luciferase gene conferred GnRH responsiveness to the promoter, whereas region B did not. However, the presence of both regions A and B conferred a greater GnRH response than region A alone. Electrophoretic mobility shift assay revealed the presence of a protein(s) binding to region A using GH3 as well as {alpha}T3–1 nuclear extracts. Thus, region A (-490/-352) confers GnRH responsiveness to the LHß subunit gene and binds to a protein(s) present in pituitary cell lines. DNA sequences in region B (-207/-82) also contribute to GnRH responsiveness. The identification of putative GnRH response elements in the rat LHß gene promoter will aid in elucidation of the mechanisms of regulation of gene expression by GnRH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PITUITARY gonadotropins, LH and FSH, play an integral role in the regulation of normal reproductive development and function. The synthesis and release of these two pituitary glycoproteins are controlled by the complex interaction of multiple factors, with GnRH, a hypothalamic decapeptide, being critical (1). Pulsatile GnRH stimulates not only the secretion of LH and FSH, but also the biosynthesis of their subunits, {alpha}, LHß, and FSHß (2, 3). These effects of GnRH involve changes at the transcriptional level (4). The regulation of LHß gene expression and LH secretion is dependent on GnRH pulse amplitude and frequency and is regulated differentially compared with FSHß gene expression and FSH secretion (4, 5, 6, 7, 8). An understanding of the mechanisms of regulation of LHß gene expression by GnRH is an important first step in understanding the role and mechanisms of physiological differential regulation of LH and FSH by GnRH.

Studies of the gonadotropin {alpha}-subunit gene have identified a number of DNA elements in the 5'-flanking region that mediate tissue-specific and regulated expression (9, 10, 11, 12). An element located at positions -225/-208 of the mouse gene is important for expression in {alpha}T3–1 cells (an immortalized murine gonadotrope precursor cell line) and has been designated the gonadotrope-specific element (GSE) (13, 14). This element binds steroidogenic factor-1 (SF-1), a member of the nuclear receptor family (15, 16, 17). In addition to elements directing tissue-specific expression, several elements that appear to mediate GnRH stimulation of {alpha}-subunit gene expression have been identified (11, 18, 19, 20).

In contrast to the {alpha}-subunit gene, little is known about DNA elements that direct pituitary-specific or hormonally regulated expression of the LHß and FSHß subunit genes. Several transcription factors that appear to be involved in gonadotrope-specific expression of the gonadotropin subunit genes have been identified serendipitously, based on observations in vivo in transgenic mice generated with targeted disruptions of genes encoding these factors. Targeted disruption of the SF-1 gene in mice resulted in low levels of expression of LHß, FSHß, and GnRH receptor (GnRHR) as well as reduced levels of {alpha}-subunit gene expression in the pituitary (15, 16). It was subsequently demonstrated that SF-1 can bind directly to 5'-flanking sequences and activate the LHß gene, and that mutations in these binding sites reduce LHß gene promoter activity in vivo and in vitro (21, 22). In addition, targeted disruption of a zinc finger transcription factor, Egr-1, led to the specific decrease in LHß gene expression, but preservation of FSHß gene expression. Induction of GnRH by gonadectomy increased FSHß messenger RNA (mRNA) levels appropriately, but failed to increase LHß mRNA levels (23).

Despite this progress, a systematic approach to identifying mechanisms of hormonal regulation of LHß and FSHß subunit gene expression has been hampered by the lack of cell lines that express endogenous or transfected LHß and FSHß genes in a regulated manner. In this study, we have used the rat pituitary somatolactotropic cell line, GH3 cells, as a model for the analysis of cis-regulatory elements of the rat LHß subunit gene. GH3 cells are a well characterized cell line, expressing PRL, GH, and the TRH receptor (TRHR). Like the GnRHR, the TRHR is coupled to pertussis toxin-insensitive G proteins of the Gq/11 family (24). Thus, the effects of TRH and GnRH are likely to be mediated through similar intracellular signal transduction pathways (25). We have demonstrated previously that GH3 cells, when transfected with rat GnRHR complementary DNA (cDNA), bind and respond to GnRH (25, 26, 27, 28). Cotransfection with the regulatory region of the {alpha}, LHß, or FSHß subunit gene fused to a luciferase reporter results in the expression of the luciferase enzyme and a stimulation of luciferase activity in response to GnRH (29). Characterization of this cell model has demonstrated many similarities in the GnRH response compared with that in primary pituitary cells, including similarities in the intracellular signal transduction pathways activated, the degree of stimulation of the gonadotropin subunit promoter activities, and the presence of differential regulation of LHß and FSHß promoter activities by GnRH. GH3 cells thus appear to be a useful model for study of the regulation of expression of the gonadotropin subunit genes by GnRH (25, 26, 27, 28, 29). The aim of the current study is to localize cis-regulatory elements in the promoter of the rat LHß gene that mediate GnRH stimulation. We have identified two regions in the rat LHß 5'-flanking sequence that contain elements involved in mediating GnRH responsiveness.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The GnRH agonist, des-Gly10-[D-Ala6]GnRH ethylamide (GnRHAg), was purchased from Sigma Chemical Co. (St. Louis, MO).

Reporter plasmids and expression vectors
An expression vector encoding the rat GnRHR was prepared by subcloning the rat GnRHR cDNA sequence into pcDNA1 (Invitrogen, San Diego, CA), as described previously (25). The reporter constructs used had sequences of the rat LHß gene promoter cloned into the pXP2 luciferase reporter vector (29, 30). The rat LHß gene promoter was sequenced from rat genomic DNA by dideoxy sequencing. This sequence has been submitted to GenBank (accession no. AF020505). The nucleotide sequence of the rat LHß gene promoter used in these studies is based on our recent findings, with position -1 assigned to the nucleotide immediately 5' to the transcriptional start site. The LHß 5'-deletion constructs were prepared by PCR amplification of the designated LHß gene promoter sequences (with BamHI/HindIII sites introduced by the primers), all terminating at +5 relative to the transcriptional start site at the 3'-end (Table 1Go). The PCR products were then subcloned into BamHI/HindIII polylinker restriction sites in pXP2. The GH50-pXP1 construct was prepared by subcloning the rat GH gene minimal promoter (GH50) into BglII/SacI polylinker restriction sites in pXP1 (21, 30, 31). The indicated rat LHß gene 5'-flanking sequences were then amplified by PCR using primers that incorporated BamHI/BglII restriction enzyme sites (Table 1Go) and subcloned into BamHI/BglII polylinker restriction sites upstream of GH50 in GH50-pXP1. 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. To generate LH{Delta}A, PCR primers LHß45S and LHß54AS were used. Primer LHß54AS was used in a PCR reaction with primer LHß-797S, and primer LHß45S was used in combination with primer LHß+5AS. Aliquots from each of these PCR reactions were combined, denatured, annealed, and used as a template for a second PCR reaction using LHß-797S and LHß+5AS primers. The resultant PCR product was subcloned into BamHI/HindIII polylinker restriction sites in pXP2, resulting in the generation of LH{Delta}A. Additional primers, LHß67S and LHß76AS, were used to generate LH{Delta}B and LH{Delta}A{Delta}B in a similar fashion. All reporter constructs were confirmed by dideoxysequencing. An expression vector expressing ß-galactosidase driven by the Rous sarcoma virus promoter (RSV-ßGal) was used as an internal standard and control (32). A pCMV5-derived expression vector containing the SF-1 cDNA was provided by Dr. K. L. Parker (Duke University, Durham, NC).


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  		Table 1. Primers used for generation of rat LHß gene promoter sequences

 
Cell culture and transfection
GH3 cells, GGH3-1' cells (GH3 cells stably transfected with an expression vector containing the rat GnRHR cDNA sequence) (27), and {alpha}T3–1 cells were maintained in monolayer culture in DMEM 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 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 µF, using the Invitrogen Electroporator II apparatus (Invitrogen). After electroporation, cells were plated in serum-containing medium. Medium was replaced 24 h after transfection. Cells were treated with 100 nM GnRHAg or vehicle for 6 h immediately before harvesting and were analyzed 48 h after transfection. These conditions have been tested and optimized previously to give maximal levels of basal expression and GnRH stimulation (25, 29). Cells were harvested in lysis buffer [125 mM Tris (pH 7.6), and 0.5% (vol/vol) Triton X-100]. Supernatants were collected by centrifugation at 14,000 x g for 15 min at 4 C. Luciferase activity was measured using an LB 953 Autolumat (EG&G Berthold, Nashua, NH) by standard protocols and was normalized for expression of RSV-ßGal. ß-Galactosidase activity was assayed colorimetrically by standard protocols, as reported previously (29).

Preparation of nuclear extracts
GH3, GGH3-1', and {alpha}T3–1 cells were grown to approximately 70% confluence and treated with GnRHAg (100 nM) or vehicle for varying time intervals, then cells were harvested, and nuclear extracts were prepared by the method of Andrews and Faller (33).

Electrophoretic mobility shift assay (EMSA)
DNA sequences corresponding to region A or B (-490/-334 or -207/-64, respectively) were amplified by PCR, using primers similar to those used for generation of the luciferase fusion constructs, but modified to include BamHI/HindIII restriction sites, and subcloned into BamHI/HindIII polylinker restriction sites in pBluescript KS+ (Stratagene, La Jolla, CA; LHA-pBS and LHB-pBS, respectively). Sequences were confirmed by dideoxysequencing. 32P-Labeled DNA fragments for use in this assay were prepared by digesting LHA-pBS and LHB-pBS with XbaI or XhoI, and 3'-end labeling with [32P]deoxy-CTP and the Klenow fragment of DNA polymerase I, followed by excision with a second restriction enzyme, XhoI or XbaI. The 32P-labeled DNA fragment was purified on a 9% polyacrylamide gel in 1 x Tris-borate-EDTA buffer. Oligonucleotides used in competition studies include Pit-1, corresponding to -137/-65 of the rat GH gene promoter and containing two Pit-1-binding sites (sense strand sequence 5'-GGGAGGAGCTTCTAAATTATCCATCAGCACAAGCTGTCAGTGGCTCCAGCCATGAATAAATGTATAGGGAAA-3'), and malic enzyme-thyroid hormone response (ME-TRE), corresponding to -288/-254 of the rat malic enzyme gene and containing two thyroid hormone response elements (sense strand sequence 5'-AGCTAGGACGTTGGGGTAAGGGGAGGACAGTGGACGAG-3') (31, 34).

The binding reaction for EMSA was performed by incubating 50,000 cpm DNA probe with 5 µg nuclear extract and 2 µg salmon sperm DNA in reaction buffer [20 mM HEPES (pH 7.9), 60 mM KCl, 5 mM MgCl2, 10 mM phenylmethylsulfonylfluoride, 10 mM dithiothreitol, 1 mg/ml BSA, and 5% (vol/vol) glycerol] for 30 min at 4 C. For competition studies, excess unlabeled DNA was added 5 min before the addition of probe. Protein-DNA complexes were resolved by 4% low ionic strength nondenaturing PAGE in 0.5 x Tris-borate-EDTA buffer. Gels were then dried and subjected to autoradiography.

Statistical analysis
Transfections were performed in triplicate and repeated multiple times. Data in each experiment were normalized to the basal levels of activity of -797/+5LHßLUC or GH50-pXP1. They were then combined across experiments to provide the mean ± SEM for basal and GnRH-stimulated activities for each construct, and fold stimulation in response to GnRH was calculated. One-way ANOVA followed by post-hoc comparisons with Fisher’s protected least significant difference test were used to assess whether changes in GnRH responsiveness among different LHß promoter-luciferase reporter constructs were significant. Student’s t test was used for statistical analysis of the effects of SF-1 on basal and GnRH-stimulated luciferase activities. A significant difference was established as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of GnRH-responsive regions in the LHß gene promoter using 5'-deletion mutants
To delineate the region(s) of the rat LHß gene 5'-flanking sequence that mediates stimulation of the LHß gene by GnRH, we first prepared a series of 5'-deletion mutants of the rat LHß gene promoter fused to a luciferase reporter gene. These constructs were cotransfected with rat GnRHR cDNA into GH3 cells by electroporation. The cells were harvested, and luciferase activity was measured 48 h after transfection, after stimulation with 100 nM GnRHAg or vehicle for 6 h immediately before harvesting. Basal levels (i.e. in the absence of GnRHAg) were similar for all of the constructs tested, varying by no more than 50% and with no systematic change with progressive 5'-deletions (Fig. 1Go). Levels were approximately 10- to 15-fold above the expression levels of the promoterless control.



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  		Figure 1. Stimulation by GnRH of activity of 5'-deletion constructs of the rat LHß gene promoter fused to the luciferase reporter gene. Each construct or pXP2 (2 µg/well) was cotransfected into GH3 cells along with rat GnRHR cDNA (2 µg/well) and RSV-ßGal (1 µg/well). Cells were harvested 48 h after transfection and treated with or without 100 nM GnRHAg for 6 h immediately before harvesting. Levels of luciferase activity are internally standardized according to levels of activity of RSV-ß-galactosidase. Each bar represents the mean ± SEM of at least 11 samples. *, P < 0.001 compared with -490/+5LHßLUC; **, P < 0.001 compared with -207/+5LHßLUC.

 
The degree of stimulation of luciferase activity in response to GnRHAg was evaluated for the sequential 5'-deletion constructs. The largest construct tested, -797/+5LHßLUC, responded to stimulation by GnRHAg with a 10-fold increase in luciferase activity. There was a significant decrease in the fold stimulation by GnRHAg when the 5'-flanking sequence was deleted from position -490 to -352 (10.9 ± 0.7-fold to 6.4 ± 0.5-fold; P < 0.001; n = 20), and there was a further decrease with deletion from -207 to -82 (6.2 ± 0.3-fold to 3.5 ± 0.2-fold; P < 0.001; n = 16; Fig. 1Go). We refer to the region of the rat LHß gene 5'-flanking sequence from -490 to -352 as region A, and the region from -207 to -82 as region B. In addition, a small, but significant, residual GnRH responsiveness was observed with our shortest LHß-luciferase construct, -82/+5LHßLUC, compared with the promoterless control vector, pXP2 (3.5 ± 0.2-fold vs. 1.8 ± 0.1-fold; P < 0.05; n = 20). However, this was not observed in all transfection experiments.

Characterization of the GnRH-responsive regions in the LHß gene promoter using internal deletion mutants
Based on the data generated using the 5'-deletion LHß promoter-luciferase fusion constructs, we hypothesized that the rat LHß gene has at least two elements responsible for mediating GnRH stimulation: one located in region A, and a second in region B. These two elements may form a composite element, with the presence of both elements necessary for a full GnRH response. To confirm and test further the roles of these two regions in mediating GnRH stimulation, a series of internal deletion constructs was generated, deleting -490/-353 (region A), -207/-83 (region B), or both from -797/+5LHßLUC (Fig. 2Go). These constructs were cotransfected with rat GnRHR cDNA into GH3 cells, and the cells were exposed to GnRHAg, as described above. Basal levels of luciferase activity were essentially unchanged by deletion of region A, but were enhanced 2-fold by deletion of region B and 5-fold when both regions A and B were deleted (Fig. 2Go). This was observed consistently with different plasmid preparations. This suggests that elements that repress the activity of the rat LHß gene promoter in GH3 cells are present in the deleted regions, or that elements that enhance this promoter activity are present in the remaining sequences and act in a position-dependent manner, having been brought into closer proximity to the minimal LHß gene promoter.



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  		Figure 2. Stimulation by GnRH of activity of internal deletion mutants of the rat LHß gene promoter fused to the luciferase reporter gene. Each construct or pXP2 (2 µg/well) was cotransfected into GH3 cells along with rat GnRHR cDNA (2 µg/well) and RSV-ßGal (1 µg/well). Cells were harvested 48 h after transfection and treated with or without 100 nM GnRHAg for 6 h immediately before harvesting. Levels of luciferase activity are internally standardized according to levels of activity of RSV-ß-galactosidase. Each value represents the mean ± SEM for nine samples, from three independent experiments. *, P < 0.005 compared with -797/+5LHßLUC.

 
Analysis of the response of the internal deletion constructs to GnRHAg revealed that deletion of region A resulted in a significant decrease (8.7 ± 1.1-fold to 5.7 ± 0.4-fold; P < 0.005; n = 9) in the degree of stimulation by GnRH. In contrast, deletion of region B does not cause a change in GnRH stimulation (9.1 ± 0.8-fold; P = NS; n = 9). Deletion of both regions A and B caused a slight, but not statistically significant, further decrease in GnRH responsiveness compared with the deletion of region A alone (5.7 ± 0.4-fold to 4.7 ± 0.2-fold; P = NS; n = 9) and was essentially the same as the responsiveness to GnRHAg of -82/+5LHßLUC alone (4.0 ± 0.4; P = NS; n = 9; Fig. 2Go). This suggests that all cis-acting DNA elements present between positions -797/-82 that mediate GnRH stimulation of the rat LHß gene lie within regions A and B.

Characterization of the GnRH-responsive regions in the LHß gene promoter using heterologous promoter fusion constructs
Data generated using the 5'-deletion LHß gene promoter-luciferase constructs support the presence of at least two GnRH-responsive elements (GnRH-REs) in the rat LHß 5'-flanking sequence: one located in region A, and a second in region B. In contrast, although data generated using the internal deletion LHß gene promoter-luciferase constructs confirm the importance of DNA sequences in region A for conferring GnRH responsiveness, deletion of region B in this paradigm had no effect on the GnRH response. To assess whether regions A and/or B are capable of conferring GnRH responsiveness to a heterologous promoter and to determine whether they are individually or in combination sufficient to function as a GnRH-RE, sequences corresponding to regions A and B were fused upstream of the rat GH minimal promoter, GH50. The GH minimal promoter was chosen because the GH gene is expressed in GH3 cells. Our preliminary studies showed that GH50-pXP1 was not responsive to GnRH in GH3 cells, even in the presence of cotransfected GnRHR (Fig. 3Go). In addition, GH mRNA levels were not stimulated by GnRH in this cell model (data not shown). Analysis of constructs generated by fusing sequences corresponding to rat LHß region A (-490/-334), region B (-207/-64), or encompassing both (-490/-64) upstream of GH50 was performed. These constructs were cotransfected with rat GnRHR cDNA into GH3 cells, and the cells were exposed to GnRHAg, as described previously. The basal levels of expression varied considerably for the different constructs (Fig. 3Go). Region B conferred a 3-fold increase in expression of the GH minimal promoter in the absence of GnRHAg (compared with GH50-pXP1), whereas region A conferred a 116-fold increase. The combination of regions A and B increased basal luciferase activity by 21-fold compared with GH50-pXP1. This suggests that sequences in region A may be able to confer activation of basal expression of the LHß gene, at least in GH3 cells.



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  		Figure 3. Regions A and B of the rat LHß gene promoter confer GnRH stimulation to the rat GH gene minimal promoter. DNA sequences corresponding to regions A and B of the LHß 5'-flanking region were fused upstream of the rat GH gene minimal promoter (GH50), which, in turn, was fused upstream of the luciferase reporter gene (pXP1), to assess whether these regions could confer GnRH responsiveness to this heterologous promoter. Each construct (2 µg/well) was cotransfected into GH3 cells along with rat GnRHR cDNA (2 µg/well) and RSV-ßGal (1 µg/well). Cells were harvested 48 h after transfection and treated with or without 100 nM GnRHAg for 6 h immediately before harvesting. Levels of luciferase activity are internally standardized according to levels of activity of RSV-ß-galactosidase. Each value represents the mean ± SEM for 9–12 samples from 4 independent experiments. *, P < 0.005 compared with GH50-pXP1; **, P < 0.001 compared with -490/-334LHß-GH50-pXP1.

 
Analysis of the response of these heterologous fusion constructs to GnRHAg indicates that DNA sequences in region A are able to confer GnRH responsiveness to the GH promoter (4.1 ± 0.2-fold stimulation by GnRHAg vs. 1.7 ± 0.2-fold for GH50-pXP1; P < 0.005; n = 9), whereas sequences from region B alone cannot (1.0 ± 0.1-fold stimulation by GnRHAg; P = NS; n = 9). The presence of both regions A and B confers a greater GnRH response to the GH promoter than does region A alone (7.8 ± 1.8-fold compared with 4.1 ± 0.2-fold; P < 0.001; n = 9; Fig. 3Go). These studies indicate that region A contains DNA sequences that are sufficient to confer GnRH responsiveness to an unrelated promoter, supporting the hypothesis that region A has an important role in mediating GnRH stimulation of the LHß gene, and that a sequence(s) within region A acts as a GnRH-RE. DNA sequences in region B are not sufficient to confer GnRH responsiveness to a heterologous promoter, but, rather, appear to act in conjunction with region A to augment GnRH responsiveness in this context.

Characterization of protein interactions with GnRH-responsive regions of the LHß gene promoter by EMSA
The rat LHß gene promoter sequence -490/-352 (region A) appears to be functionally important in mediating GnRH responsiveness of the rat LHß gene based on our transfection studies. We were, therefore, interested in determining whether nuclear proteins present in GH3 cells could bind to DNA sequences within region A and, furthermore, whether a similar protein(s) was present in gonadotrope-derived cell lines. A DNA fragment corresponding to the rat LHß gene 5'-flanking sequence -490/-334 (LHA) was 32P labeled and used in EMSA with GH3 nuclear extracts to address this question. This study revealed the presence of three specific protein-DNA complexes (Fig. 4AGo). The formation of these complexes could be competed by an excess of unlabeled LHA, but not by other nonspecific DNA sequences such as Pit-1, which contains binding sites for the pituitary transcription factor, Pit-1, or ME-TRE, which contains binding sites for thyroid hormone receptor. A similar pattern of DNA-protein complexes was seen using nuclear extracts derived from {alpha}T3–1 cells, a gonadotrope-derived cell line, suggesting that the proteins that bind to rat LHß region A are common at least to these two pituitary cell lines. The DNA-protein binding patterns were unaffected by using nuclear extracts derived from GH3 cells (transfected with the rat GnRHR) or {alpha}T3–1 cells that had been treated with GnRHAg for 30 min, 2 h, or 6 h compared with the binding pattern in untreated cells (data not shown).



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  		Figure 4. EMSA of regions A and B of the rat LHß gene 5'-flanking sequence. A, A DNA fragment corresponding to region A (-490/-334) of the rat LHß gene promoter was 32P end-labeled and incubated with 5 µg nuclear extract from GH3 (lanes 1–7) or {alpha}T3–1 cells (lane 8) in an EMSA. Competition with a 20- to 200-fold excess of oligonucleotides corresponding to unlabeled LHß region A (lanes 2 and 3), Pit-1 (lanes 4 and 5), and ME-TRE (lanes 6 and 7) elements is shown. Three specific DNA-protein complexes were detected, as indicated by the arrows. The migration pattern of probe in the absence of nuclear extract is shown in lane 9. N.S., Nonspecific complexes. B, A DNA fragment corresponding to region B (-207/-64)) of the rat LHß gene promoter was 32P end-labeled and incubated with 5 µg nuclear extract from GH3 cells in an EMSA. No specific DNA-protein complexes were detected.

 
The rat LHß gene promoter sequence -207/-82 (region B) also appeared to contribute to conferring GnRH responsiveness to the rat LHß gene based on our transfection studies. We were, therefore, also interested in determining whether nuclear proteins present in GH3 cells could bind to DNA sequences within region B. However, in contrast to the results obtained using region A, EMSA analysis of the 32P-labeled rat LHß promoter sequence -207/-64 (LHB) did not reveal evidence of any specific protein-DNA interactions (Fig. 4BGo). Furthermore, LHB was not able to compete for nuclear protein binding to LHA (data not shown).

SF-1 augments expression of the rat LHß gene promoter but does not affect GnRH responsiveness
We have reported previously that the rat LHß gene promoter contains a consensus GSE, and that SF-1 is able to bind to this DNA sequence and trans-activate the rat LHß gene promoter (21). This GSE lies within region B at positions -125/-117. Although GH3 cells do not express SF-1, we wondered whether SF-1 might contribute to GnRH responsiveness to the LHß gene. To assess whether SF-1 is capable of conferring GnRH responsiveness to the LHß gene, -797/+5LHßLUC was cotransfected with either an expression vector containing the cDNA for SF-1 or a control expression vector (pCMV5) into GGH3-1' cells, and the effects on basal and GnRH-stimulated luciferase activities were observed (Fig. 5Go). SF-1 increased expression of -797/+5LHßLUC in the absence of GnRH by approximately 2-fold (2.2 ± 0.1-fold; P < 0.0001; n = 17). However, although the absolute luciferase activity of -797/+5LHßLUC was greater in the presence than in the absence of SF-1, the fold stimulation in response to GnRHAg was the same in the presence or absence of SF-1 (without SF-1, 6.4 ± 0.3-fold; with SF-1, 5.9 ± 0.2-fold; P = NS; n = 17). Thus, SF-1 appears to augment rat LHß gene promoter activity independently of stimulation by GnRH.



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  		Figure 5. SF-1 augments expression of the rat LHß gene promoter, but does not affect GnRH responsiveness. -797/+5LHßLUC (2 µg/well) was cotransfected into GGH3-1' cells along with either pCMV5 or the SF-1 cDNA (1 µg/well) and RSV-ßGal (1 µg/well). Cells were harvested 48 h after transfection and treated with or without 100 nM GnRHAg for 6 h immediately before harvesting. Levels of luciferase activity are internally standardized according to levels of activity of RSV-ß-galactosidase. Each value represents the mean ± SEM for 17 or 18 samples from 6 independent experiments. *, P < 0.0001 compared with cells treated with vehicle only; **, P < 0.0001 compared with cells transfected with pCMV5 and treated with vehicle.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified two regions of the rat LHß 5'-flanking sequence that are important for mediating the stimulation of LHß gene expression by GnRH. To our knowledge, this is the first description of putative cis-acting GnRH response elements in the LHß subunit gene. Expression of a rat LHß-luciferase construct similar to ours in primary pituitary cells has been reported previously; however, treatment of transfected cells with a GnRH agonist resulted in only a modest (<2-fold) induction of luciferase activity (35). The difficulty in obtaining adequate levels of expression of LHß-luciferase fusion reporters in cultured primary pituitary cells and the heterogeneity of cell types present in the pituitary have frustrated analyses of the LHß gene promoter. The {alpha}T3–1 cell line does not express the LHß gene or support LHß gene promoter activity, limiting its utility for such studies (36). RC-4B/C cells are a heterogeneous cell line generated from a rat pituitary tumor, containing cells from all pituitary cell types, but enriched for gonadotropes, containing 20–25% LHß- or FSHß-staining cells (37). These cells also do not support the expression or regulation of transfected LHß-luciferase genes (36) (data not shown). Fallest et al. generated transgenic mice bearing 2 kilobases of the rat LHß gene 5'-flanking sequence fused to a luciferase reporter gene. This trans-gene was expressed in the pituitary, luciferase activity in the pituitary was increased by gonadectomy, and the increase was blocked by a GnRH antagonist, antide (38). This suggests that the region of the rat LHß gene within 2 kilobases upstream of the transcriptional start site contains sequences that confer GnRH responsiveness. Similarly, Keri et al. have shown in a transgenic mouse model that 776 bp of the bovine LHß promoter were sufficient to direct expression to gonadotropes and to mediate responsiveness to GnRH (36).

The use of GH3 cells for the study of the LHß gene is not unprecedented. The expression of the LHß promoter fused to a chloramphenicol acetyltransferase reporter in GH3 cells, with stimulation by increases in intracellular cAMP, has been reported previously, and transcription initiation from the authentic start site in this cell line was confirmed (39). An estrogen-responsive element was identified at positions -1173/-1159 of the rat LHß promoter using GH3 cells (40, 41); this region was not included in our studies. Indeed, we have not observed estrogen regulation of our rat LHß gene promoter-luciferase constructs in our GH3 cell model (data not shown). In a paradigm similar to ours, GH3 cells were used to characterize TRH-responsive elements in the TSHß subunit gene (42).

Our data suggest that there are two cis-acting DNA elements in the LHß gene that mediate a response to GnRH: region A (-490/-352) and region B (-207/-82). Region A appears to be of primary importance in mediating a GnRH response. If this sequence is deleted from LHß-luciferase constructs, the degree of stimulation by GnRH is significantly decreased, and this sequence can confer a GnRH response to a heterologous promoter. The role of region B in mediating a GnRH response is less clear. Although 5'-deletion mutants suggest that deletion of region B results in a decrease in GnRH responsiveness, internal deletion of this element did not affect GnRH response, and fusion of region B upstream of the GH promoter did not confer GnRH responsiveness. The possibility that regions A and B act together as a composite GnRH-RE is supported by the observation that fusion of regions A and B upstream of the GH promoter resulted in greater stimulation by GnRH than did region A alone. Such composite elements are being increasingly recognized in other genes for other regulatory stimuli (11). In view of the complexity of the differential GnRH responses of the gonadotropin subunit genes, it would not be surprising for GnRH to act through such composite elements.

The precise DNA sequences within region A that act as a GnRH-RE have not yet been defined. Region A was analyzed for the presence of DNA consensus sequences (Fig. 6AGo). The sequence from -419/-412 had 100% homology with a conserved motif in the PRL promoter (43). In addition, an overlapping region (-421/-414) shares 75% homology with a region characterized as a GnRH-RE in the murine {alpha}-subunit gene (11). This sequence also overlaps with a palindromic sequence (ATCCTGAT), a feature of many transcription factor-binding sites. Further analysis of region A also reveals a sequence (-375/-361) with 75% homology to DNA sequences within the putative GnRH-RE in the human {alpha}-subunit gene (20). Analysis of the roles of these sequences within region A in protein binding and in mediating a functional GnRH response is currently underway.



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  		Figure 6. A, Comparison of the rat LHß gene region A (-490/-353) with known DNA consensus sequences. mGnRHRE{alpha}, GnRH response element in the murine {alpha}-subunit gene; hGnRHRE{alpha}, GnRH response element in the human {alpha}-subunit gene. B, Comparison of the rat LHß gene region B (-207/-82) with known DNA consensus sequences. CRE, cAMP response element; AP1, activating protein-1 consensus sequence; GSE, gonadotrope-specific element.

 
Analysis of region B also reveals some homology with DNA consensus sequences for several known transcription factors (Fig. 6BGo). A sequence with 85% homology to the activating protein-1 consensus sequence, at -157/-151, was identified (44). There is also a region with 75% homology with the consensus cAMP-responsive element at -171/-164 (45). Unlike region A, no significant homology with the putative GnRH-REs identified in the murine and human {alpha}-subunit genes was found. Interestingly, 100% homology was found at positions -125/-117 with a putative GSE conserved in the human, bovine, murine, rat, and equine {alpha}-subunit promoters (14). We have shown previously that SF-1 can bind directly to this GSE and activate expression of the LHß gene, analogous to the role of SF-1 in the gonadotrope-specific expression of the {alpha}-subunit gene (21). However, GH3 cells do not express SF-1 (14) (data not shown). The ability of {alpha} and LHß subunit gene promoters to respond to GnRH in GH3 cells transfected with the GnRHR suggests that SF-1 is not essential for GnRH responsiveness. Indeed, coexpression of SF-1 with -797/+5LHßLUC led to an increase in basal luciferase activity, but no change in the fold response to GnRH. In support of this, GnRH replacement was able to restore gonadotropin expression in SF-1-disrupted mice. In contrast, mutation of the SF-1-binding site in the bovine LHß gene nearly eliminated promoter activity in a transgenic mouse model, and the mutant trans-gene remained inactive after induction of GnRH by gonadectomy (22). Thus, the role of SF-1 as a mediator of GnRH responsiveness remains uncertain. One possible explanation is that another transcription factor(s) present in gonadotropes is able to bind to this site in the LHß gene and mediate the response to GnRH; this factor may also be present in GH3 cells to account for the role of region B in the GnRH response.

Analysis of protein-DNA interactions using DNA sequences corresponding to region A of the LHß 5'-flanking sequence identified three specific protein-DNA complexes, present in both GH3 and {alpha}T3–1 nuclear extracts. This suggests that several distinct proteins or protein complexes interact with sequences within region A, one or more of which may play a role in mediating the stimulation of LHß gene transcription in response to GnRH. We have not detected any change in the DNA-binding pattern of GH3 or {alpha}T3 nuclear extract proteins prepared from cells treated with GnRH compared with those of untreated extracts. This suggests that the mechanism by which region A acts as a GnRH-RE may not involve GnRH-regulated changes in the extent of protein binding to these DNA sequences. GnRH treatment may affect other functions of these binding proteins, such as transcriptional activation or interaction with other proteins.

Despite the presence of sequences in region B with homology to known DNA consensus protein-binding sites, no protein-DNA complexes were observed by EMSA using LHß region B as a probe, suggesting that these may not be functional sites within the context of the surrounding sequences of the LHß promoter. GH3 cells do not express SF-1, accounting for the absence of binding to this element. However, even when {alpha}T3–1 cell nuclear extracts, which do contain SF-1 (14), were used in EMSA, no complex was observed (data not shown), suggesting that the levels of SF-1 binding to this probe were below the limits of detection by our EMSA assay or, alternatively, that the conformation of this 130-bp probe did not allow SF-1 binding.

Some residual response to GnRH was observed with our shortest construct, -82/+5LHßLUC, suggesting that there may be an additional GnRH-RE within close proximity to the TATAA box and the minimal promoter. The Egr-1 sequence lies within this region and is a candidate cis-acting element within this sequence.

Our ultimate goal is to identify the cis elements and trans factors important in mediating GnRH regulation of LHß gene in gonadotropes. We have used GH3 cells for these studies. These cells, when cotransfected with the rat GnRHR, respond to GnRH in a manner similar to that of primary pituitary cells. Using this model, we have identified two putative cis-acting DNA sequences involved in mediating the GnRH responsiveness of the LHß gene. Recently, targeted expression of the simian virus 40 T antigen with the rat LHß subunit gene regulatory region was used to generate transgenic mice (46). An immortalized gonadotrope-derived cell line, LßT2 cells, was generated from a pituitary tumor arising in one of these mice. These cells express the {alpha} and LHß subunit genes and the GnRHR, and respond to GnRH with a 4- to 5-fold increase in LHß mRNA levels (47). These cells need to be characterized further to show that the stimulation of LHß mRNA levels by GnRH occurs at the level of transcriptional regulation, and that these cells can support the expression of transfected LHß-luciferase fusion genes and respond to GnRH stimulation with an increase in luciferase activity. We are currently in the process of performing these studies, and, if successful, will attempt to confirm our results in this cell model. In addition, studies are underway to define the GnRH-RE within regions A and B more precisely. When these are defined, our results can be confirmed in gonadotrope-derived cell lines, in primary pituitary cell cultures, or, perhaps most importantly, in vivo in transgenic mouse models to confirm their physiological importance.


   Footnotes
 
1 This work was supported in part by NIH Grants HD-19938 (to W.W.C.) and HD-33001 (to U.B.K.) and an American Society for Reproductive Medicine-Serono Research Grant (to U.B.K.). Back

Received October 13, 1997.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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