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Endocrinology Vol. 140, No. 8 3452-3458
Copyright © 1999 by The Endocrine Society

ARTICLES

Transcriptional Regulation of the Gonadotropin-Releasing Hormone Receptor Gene Is Mediated in Part by a Putative Repressor Element and by the Cyclic Adenosine 3',5'-Monophosphate Response Element1

Guadalupe Maya-Núñez and P. Michael Conn

Oregon Regional Primate Research Center (G.M.N., P.M.C), Beaverton, Oregon 97006; and the Department of Physiology and Pharmacology, (P.M.C.), Oregon Health Sciences University, Portland, Oregon 97201

Address all correspondence and requests for reprints to: Dr. P. Michael Conn, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: connm{at}ohsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The levels of the GnRH receptor (GnRHR) and its messenger RNA depend on the pattern of administration of GnRH. In this study, internal deletion mutants in a luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment of mouse GnRHR gene were used to examine the regulation of GnRHR gene transcription in GGH3 cells. Our results indicate that the mouse GnRHR promoter contains one putative repressor element located at position -343/-335. When this sequence was deleted, the GnRHR promoter activity was significantly increased in both basal and GnRH agonist (Buserelin)-, phorbol ester-, and forskolin-stimulated cells. Gel mobility shift assay showed that the sequence -343/-335 is capable of binding GGH3 nuclear proteins. With deletion of the cAMP response element (-107/-100), basal and Buserelin-stimulated transcription was decreased. The same response was observed after stimulation with forskolin. Stimulation with (Bu)2cAMP did not alter transcription above basal levels. The stimulation with phorbol ester resulted in an attenuated increase in transcriptional activity, suggesting that this sequence of the GnRHR promoter is a cAMP response element. These results suggest that the transcriptional activity of the GnRHR gene is mediated in part by a putative repressor element and by the cAMP response element.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH IS SYNTHESIZED in the hypothalamus and stimulates the synthesis and release of the gonadotropins, LH and FSH, acting through a specific receptor (GnRHR) on the plasma membrane (1). The responsiveness of gonadotropes to GnRH correlates with changes in GnRHR number (2), which are mediated in part at the level of gene transcription (3, 4).

GnRH regulates the expression of its own receptor in pituitary gonadotropes (5). Pulsatile stimulation with low concentrations of GnRH causes up-regulation of its receptors, whereas continuous exposure with high concentrations of agonist results in desensitization and down-regulation of receptors (2, 5, 6, 7, 8). In addition, administration of exogenous GnRH in a continuous fashion results in the down-regulation of its own messenger RNA (mRNA) receptor, whereas pulsatile treatment stimulates GnRHR mRNA levels (9, 10, 11, 12, 13). Regulation of the receptor gene is clearly an important determinant of the density of cell surface receptors in gonadotropes.

Gene expression is a multistep process regulated at different levels, involving multiple regulatory elements. Recently, isolation and characterization of the 5'-flanking region of GnRHR gene from human, mouse, rat, and sheep were reported (14, 15, 16, 17, 18, 19) and revealed the presence of consensus sequences that may be involved in controlling gene expression (14, 15, 16, 17, 18, 19). The basal activity of the mouse GnRHR (mGnRHR) promoter is dependent on the steroidogenic factor-1-binding site, the activator protein-1-binding site, and the GnRHR-activating sequence (20, 21). In addition, two consensus sequences similar to the GnRHR response elements found in the gonadotropin {alpha}-subunit promoter were identified in the mGnRHR gene (14).

A cAMP response element (CRE)-like sequence has been identified in rat and human GnRHR gene (16, 18). A CRE-like sequence has not been identified in mGnRHR; however, a recent study (22) demonstrated that cAMP activates transcriptional activity of the GnRHR gene. The consensus CRE site has an 8-bp palindromic sequence (TGACGTCA) (23). A comparison of the CRE sequences identified to date shows that the 5'-half of the palindrome is conserved, whereas the 3'-motif is less constant (23). In the present study we report that basal activity of the proximal promoter of the mGnRHR is dependent on a putative repressor element and a CRE-like sequence (5'-TGACGTTT-3') in the GGH3 cell line (GH3 cells stably transfected with GnRHR).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Luciferase reporter gene vector (GnRHR-pXP2) with a 1226-bp promoter fragment (-1227 to -1 relative to the start codon) of mouse GnRHR gene (14) and an expression vector (pCIS-LacZ) expressing ß-galactosidase driven by cytomegalovirus (CMV) promoter (24) were provided by Drs. W. W. Chin and Tae H. Ji, respectively. Natural sequence GnRH was provided by the National Pituitary Agency. A GnRH agonist, buserelin (D-ter-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH), was a gift from Hoeschst-Roussel Pharmaceuticals (Somerville, NJ). (Bu)2cAMP and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma Chemical Co. (St. Louis, MO). DMEM, OPTI-MEM, lipofectamine, and PCR reagents were purchased from Life Technologies (Grand Island, NY). Restriction enzymes, modified enzymes, and competent cells for subcloning were purchased from Promega Corp. (Madison, WI). Other reagents were of the highest degree of purity available from commercial sources.

Vector construction
A 1226-bp fragment (-1226 to -1 relative to the start codon) of 5'-flanking region of mouse GnRHR gene (14) was fused into a luciferase reporter gene vector (pXP2) (24) and designated the GnRHR-pXP2 reporter gene construct. The construction of promotorless pXP2 vector has been described previously (22). An expression vector (pCIS-LacZ) expressing ß-galactosidase driven by CMV promoter was used as an internal control (24).

Constructs with internal deletions within the promoter region were generated by PCR using primers with ends modified to cause deletion of the indicated sequences during amplification. To generate deletion -343 to -335 PCR primers S1 (5'-CAATTTGGTATGATAATAAC-3') and AS1 (5'-TTGCTCTCCAGCGGTTCCAT-3') were used. Primer AS13 (5'-GTGACTCATAATACTTAATGAATAGGTTTTC-3') was used in a PCR reaction with primer S1, and primer S13 (5'-GAAAACCTATTCATTAAGTATTATGAGTCACTTTC-3') was used in combination with primer AS1. PCR products of these reactions were combined, denatured, annealed, and used as a template for a second PCR reaction using S1 and AS1 primers. The resultant PCR product was digested with XbaI and XhoI and their 5'- and 3'-ends, respectively, and subcloned into the same sites in the pXP2 vector. Additional primers S7 (5'-GCTTGGCATGTTCTGTCTTTTAGATTATAAAC-3') and AS7 (5'-GTTTATAATCTAAAAGACAGAACATGCCAAGC-3') were used to generate deletion -212 to -206, primers S9 (5'-GATAAAAAGACGGGCCAGGGCTACGGTTAC-3') and AS9 (5'-GTAACCGTAGCCCTGGCCCGTCTTTTTATC-3') were used to generate deletion -268 to -259, and primers S8 (5'-CAGAAATGCTAACCTGCCATCTAAAGGAGGCAG-3') and AS8 (5'-CTCCTTTAGATGGCAGGTTAGCATTTCTGC-3') were used to generate deletion of the CRE element in a similar fashion.

The identities of all reporter gene constructs and the correctness of all PCR-derived sequences were verified by Dye Terminator Cycle Sequencing according to the manufacturer’s instructions (Perkin-Elmer, Foster City, CA). For transfection, large scale plasmid DNAs were prepared by double banded CsCl gradient centrifugation. The purity and identity of plasmid DNAs were further verified by restriction enzyme analysis.

Transient transfection of GGH31' cells
GnRHR-pXP2 reporter gene vector or control vector pXP2 were transiently expressed in GGH31' cells (25). GGH31' cells were maintained in growth medium [DMEM containing 10% FCS (HyClone Laboratories, Inc., Logan, UT) and 20 µg/ml gentamicin (Gemini Bioproducts, Calabasas, CA)] in a humidified atmosphere (37 C) containing 5% CO2. Cells (5 x 105 cells/well) were seeded in six-well plates (Costar, Cambridge, MA). Twenty-four hours after plating, the cells were transfected with 1.5 µg GnRHR-pXP2 or promoterless pXP2 plus 0.5 µg pCIS-LacZ/well using 5 µl lipofectamine in 1 ml OPTI-MEM. Five hours later, 1 ml DMEM containing 20% FCS was added to each well. Twenty-four hours after the start of transfection, the medium was replaced with fresh growth medium, and the cells were allowed to grow for another 24 h before treatment and functional assays (luciferase assay and ß-galactosidase assay) were performed.

Luciferase and ß-galactosidase assays
After treatment of transiently transfected GGH31' cells with GnRH or other compounds for the indicated times, the cells were washed twice with PBS and lysed in 150 µl Reporter Lysis Buffer (Promega Corp.). Luciferase activity in 20 µl cell lysate was determined using the Luciferase Assay System (Promega Corp.) in a LuciCount microplate luminometer (Packard, Meriden, CT). ß-Galactosidase activity in 30 µl of the cell lysate was also measured using ß-Galactosidase Enzyme Assay System (Promega Corp.) in a SpectraCount photometric microplate counter (Packard) and was used as an internal control. Luciferase activity was normalized for transfection efficiency of each well by dividing luciferase activity by ß-galactosidase activity.

Gel mobility shift assay
The oligonucleotides containing the regions -343/-335 (GSF13: forward, 5'-TTAAGGCTAATTGGATGATATT-3'; reverse, 5'- AATATCATCCAATTAGCCTTAA-3') and -212/-206 (GSA7: forward, 5'-ATGTTCTGTTAGCACTCTTTTA-3'; reverse, 5'-TAAAAGAGTGCTAACAGAACAT-3') were chemically synthesized. These oligonucleotides were 3'-end labeled with digoxigenin-11-dideoxy-UTP using terminal transferase in the presence of Co2+ according to the manufacturer’s instructions (Boehringer Mannheim, Indianapolis, IN). Labeled oligonucleotides were purified by precipitation with ethanol and lithium chloride. Labeling efficiency was determined by means of direct detection of dot blots on nylon membranes (26).

Nuclear extracts were prepared from GGH31' cells according to the method of Andrews and Faller (27). The standard binding assay (20 µl) contained 5 µg nuclear protein extract, 2 µg poly[d(I-C)], and 0.5 pmol digoxigenin-labeled oligonucleotide in 10 mM HEPES (pH 7.6), 1 mM EDTA, 10 mM (NH4)2SO4, 1 mM dithiothreitol, 0.2% Tween-20 (vol/vol), 30 mM KCl, and 2 mg/ml BSA. The binding assay mixture was incubated at room temperature for 30 min, the samples were separated in 5% polyacrylamide gel (acrylamide-bisacrylamide, 39:1) containing 0.25 x TBE (Tris-borate-EDTA, 10-fold concentrated; 890 mM Tris, 890 mM boric acid, and 20 mM EDTA, pH 8.0). Before loading the samples, the gel was run for 2 h at 100 V and 4 C. Electrophoresis was carried out at 100 V until the bromophenol blue reached the bottom of the gel. The probe DNA was then electroblotted to nylon membrane positively charged (Boehringer Mannheim, Indianapolis, IN) in 0.25 x TBE using a Hoefer Transphor (Hoefer Scientific, St. Francisco, CA) at 4 mA/cm2 at 4 C for 30 min, then fixed to the membrane by heating at 80 C for 2 h. Detection of digoxigenin-labeled probe was carried out by the chemiluminescent detection system using the Boehringer Mannheim kit according to the instruction manual. Imaging of the chemiluminescence was performed on Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY) by exposure for 1–16 h (26, 28).

Data analysis
The data shown are the means of triplicate assay wells and are presented as the mean ± SEM of replicates in each experiment. The SEM was typically less than 10% of the mean. The data were analyzed by one-way ANOVA followed by Duncan’s multiple range test, P < 0.05 was considered significant. Each experiment was repeated three or more times to ensure the reproducibility of the findings.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of sequences required for transcriptional activity of mGnRHR gene using internal deletion mutants
To delineate the responsive sequences of the mGnRHR, three series of internal deletion constructs were generated, deleting -343/-335, -268/-259, and -212/-206 (relative to the start codon), respectively (Fig. 1Go). These sequences are highly conserved among rat, human, mouse, and ovine. Transient expression of the constructs containing either one of the internal deletion sequences or the original promoter (1226 bp) in GGH31' cells revealed that basal GnRHR-Luc activity was significantly increased in the -268/-259, -343/-335, and -212/-206 deletions (Fig. 1Go). Similarly, stimulation of GnRHR activity by Buserelin (10-7 M; 6 h) was retained in the -268/-259 deletion and was significantly increased in the -343/-335 and -212/-206 deletions (Fig. 1Go). When these deletion mutants were stimulated with PMA (100 nM) or forskolin (10 µM), transcriptional activity increased for all three deletions (Fig. 2Go). These results indicate that -343/-335 and -212/-206 elements may repress the GnRHR-Luc activity in GGH31' cells.



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  		Figure 1. Characterization of sequences required for transcriptional activity of mGnRHR gene promoter using internal deletion mutants. Three series of internal deletion constructs were generated in the 1226-bp promoter fragment (-1127 to -1, relative to the start codon) of mGnRHR, deleting -335/-343, -268/-259, and -212/-206 (relative to the start codon), respectively. The start codon site is indicated by a bent arrow. Forty-eight hours after transfection of GGH31' cells with GnRHR-pXP2 containing one of the three internal deletion promoters or original promoter (1226 bp) plus pCIS-LacZ or pXP2 plus pCIS-LacZ, the cells were treated with medium alone or buserelin (10-7 M) for 6 h. The cells were then lysed, and luciferase and ß-galactosidase activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as fold induction over that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences (P < 0.05) from values in the immediately adjacent groups are designated by different lowercase letters above the bars.

 


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  		Figure 2. Stimulation by PMA (upper panel) and forskolin (lower panel) of activity of internal deletion mutants of the mGnRHR gene promoter. Forty-eight hours after transfection of GGH31' cells with GnRHR-pXP2 containing one of the three internal deletion promoters or original promoter (1226 bp) plus pCIS-LacZ or pXP2 plus pCIS-LacZ, the cells were treated with medium, PMA (100 nM), or forskolin (10 µM) for 6 h. The cells were then lysed, and luciferase and ß-galactosidase activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as fold induction over that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences (P < 0.05) from values in the immediately adjacent groups are designated by different lower case letters above the bars. {Delta}, Deletion.

 
Analysis of DNA-binding protein activity in GGH31' cell nuclear extracts
Based on the functional data described above, suggesting the presence of potential repressor elements in -343/-335 and -212/-206, we sought to determine whether these same regions of DNA could bind one or more proteins specific to GGH31' cells. Overlapping 22-bp oligodeoxyribonucleotides spanning the sequences -343/-335 and -212/-206 (GSA13 and GSA7, respectively) served as either digoxigenin-labeled probes or nonlabeled competitors in gel mobility shift assays using GGH31' nuclear extracts. Probe GSA7 failed to show a shift (data not shown). In contrast, probe GSA13 exhibited binding to protein in GGH31' cells (Fig. 3Go). The formation of this complex could be competed by an excess of unlabeled GSA13 probe, but not by other nonspecific DNA sequences. These results indicate that the -343/-335 sequence appeared to contribute to conferring transcriptional activity to mGnRHR.



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  		Figure 3. Gel shift mobility assay of the region -335/-343 using the GGH31' nuclear extract. Digoxigenin-labeled probe GSA13 was incubated with 5 µg nuclear extract in a gel mobility shift assay. Homologous competition with a 20-fold molar excess of unlabeled probe GSA13 (lane 3) is shown. The specificity of the DNA-protein interaction was assessed by competition with a 20-fold molar excess of an unrelated DNA sequence (lane 4). The specific DNA-protein complex was detected, as indicated by the arrow. NS, Nonspecific complex.

 
Deletion analysis of CRE required for transcriptional activity of mGnRHR gene
To locate the CRE, one internal deletion in the 1226-bp 5'-flanking region of mGnRHR gene was generated deleting -107/-100 (relative to the start codon; Fig. 4Go). The promoter sequence of the mGnRHR did not contain the classical CRE 5'-TGACGTCA-3', although a 5'-TGACGTTT-3' sequence is present at the position mentioned above. Transient expression of the construct containing the internal deletion sequence or original promoter (1226 bp) in GGH31' cells revealed that basal GnRHR-Luc activity was significantly decreased in the CRE deletion (Fig. 4Go). Similarly, stimulation of GnRHR activity by Buserelin (10-7 M; 6 h) was significantly decreased in the CRE deletion (Fig. 4Go). When this deletion mutant was stimulated with (Bu)2cAMP (5 mM; 6 h), the fold activity over the untreated cells remained the same. In both basal and stimulated cells, GnRHR-Luc activity was lower than that of the wild-type promoter. After stimulation with PMA (100 nM) and forskolin (10 µM), GnRHR activity decreased (Fig. 5Go), and basal levels were lower in both cases. These results indicate that the element located in -107/-100 is important for transcriptional activity of GnRHR gene in GGH31' cells and that it responds to cAMP.



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  		Figure 4. Deletion analysis of CRE required for transcriptional activity of mGnRHR gene. One internal deletion in the 1226-bp promoter fragment (-1227 to -1, relative to the start codon) of the mGnRHR gene was generated, deleting -107/-100 (relative to the start codon). The start codon site is indicated by a bent arrow. Forty-eight hours after transfection of GGH31' cells with GnRHR-pXP2 containing the internal deletion promoter or original promoter (1226 bp) plus pCIS-LacZ or pXP2 plus pCIS-LacZ, the cells were treated with medium alone, Buserelin (10-7 M), or (Bu)2cAMP (5 mM) for 6 h. The cells were then lysed, and luciferase and ß-galactosidase activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as fold induction over that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences (P < 0.05) from values in the immediately adjacent groups are designated by different lower case letters above the bars. {Delta}, Deletion.

 


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  		Figure 5. Stimulation by forskolin and PMA of activity of CRE deletion of the mGnRHR gene promoter. Forty-eight hours after transfection of GGH31'cells with GnRHR-pXP2 containing the internal deletion or original promoter (1226 bp) plus pCIS-LacZ or pXP2 plus pCIS-LacZ, the cells were treated with medium, PMA (100 nM), or forskolin (10 µM) for 6 h. The cells were then lysed, and luciferase and ß-galactosidase activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as fold induction over that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences (P < 0.05) from values in the immediately adjacent groups are designated by different lower case letters above the bars. {Delta}, Deletion.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Gene expression is a multistep process regulated at different levels, involving multiple regulatory elements. There is evidence that the expression of the ß2-adrenergic receptor can be regulated at the level of its transcription (29), posttranscriptionally at the level of mRNA stability (30), or at the level of translation (31). Recent studies show the presence of both positive and negative cis-acting regulatory sequences in the 5'-flanking region of the rat ß2-adrenergic receptor gene (32, 33). Results from studies using GnRHR complementary DNA from several species indicate that pulsatile GnRH up-regulates the expression of its own receptor mRNA (5, 6, 7, 8, 9), whereas high amplitude pulsatile or continuous treatment with GnRH generally down-regulates the levels of GnRHR mRNA (10, 11, 12, 13, 14, 34, 35, 36, 37). However, it is unclear whether homologous regulation of GnRHR gene expression occurs at transcriptional and/or post-transcriptional levels. Sequence analysis of 5'-flanking regions of GnRHR gene from several species revealed the presence of consensus sequences that may have significance in controlling the expression of the gene. In addition, two consensus sequences similar to the GnRH response elements (70% and 75% identities, respectively) found in the gonadotropin {alpha}-subunit promoter were identified in the mGnRHR gene at positions -416/-397 and -677/-662 (relative to the start codon) (14). Also, the basal activity of the mGnRHR promoter is dependent on the steroidogenic factor-1-binding site (-245 to -237, relative to the start codon), the activator protein-1 binding site (-360 to -330, relative to the start codon), and a GnRHR-activating sequence (-391 to -380, relative to the start codon) (20, 21). However, repressor elements have not been identified in the GnRHR promoter; the present results show that the deletion of -343 to -335 (relative to the start codon) resulted in a small, but significant, increase in GnRHR transcriptional activity after the stimulation with Buserelin. The interaction of both positive acting and potentially negative acting nuclear factor sequences suggests a complex mechanism of transcriptional regulation that may depend on the activities of competing suppressor or activator elements. Deletion of the C/EBP{alpha} sequence of the ß2-adrenergic receptor, for example, resulted in a small increase in the transcriptional activity of the receptor; however, the negative regulatory role of this sequence has also been confirmed (38). Based on these previous results, we do not rule out the biological significance of the -343/-335 sequence. When this sequence was deleted, stimulation with PMA and forskolin resulted in an increase in GnRHR transcriptional activity, suggesting that the deletion of this sequence in part contributed to increased responses through PKC and cAMP signal transduction. These results suggest that this sequence binds a protein(s) that partially inhibits the GnRHR transcriptional activity. A gel mobility shift assay shows specific binding to element -343 to -335 (relative to the start codon) in GGH31' nuclear extracts; this result demonstrates that there exists specific interaction between this putative repressor element and protein. We have been unable to demonstrate specific binding for another element (-212 to -206, relative to the start codon), suggesting that this sequence may not be a functional site within the context of the surrounding sequences of the mGnRHR gene promoter. Another possibility is that the absence of sequence-specific binding using a standard gel mobility shift assay may be due to an unstable DNA-protein complex; this problem could be resolved using specific DNA-binding proteins from crude nuclear extract by UV cross-linking of aryl azide-derived oligonucleotides (39).

The GnRHR appears to couple to multiple G proteins (40). In GGH3 cells, the GnRHR is coupled to Gq/11{alpha}, resulting in activation of phospholipase C and inositol phospholipid turnover (25, 40, 41, 42). Also, the receptor is coupled to Gs{alpha}, which activates adenylate cyclase, leading to cAMP production (43, 44). Recently, a study relying on palmitoylation of G proteins and overexpression of different G protein {alpha}-subunit complementary DNAs showed that the GnRHR couples to Gq/11{alpha} as well as Gs{alpha} and Gi{alpha} in both GGH3 cells and pituitary gonadotropes, suggesting that similar signal transduction pathways are employed to mediate GnRH action in GGH3 cells and pituitary cells, and that the adenylate cyclase-cAMP signal transduction pathway is involved in GnRH action (45). A CRE-like sequence has been identified in rat and human GnRHR genes (16, 18). A CRE-like sequence has not been identified in mGnRHR. The consensus CRE site has an 8-bp palindromic sequence (TGACGTCA) (23). A comparison of the CRE sequences identified to date shows that the 5'- half of the palindrome is conserved, whereas the 3'-motif is less constant (23). The promoter sequence of the rat GnRHR does not contain the classical CRE; a 5'-ACGCCA-3' sequence is present at position -192/-187 (16). Also, the protooncogene c-fos contains a powerful CRE at positionA -66/-59 (46, 47), the 3'-sequence contains TTT instead of TCA. The present results show that the mGnRHR promoter contains the same CRE element as c-fos in position -107/-100 (relative to the start codon). This is consistent with other work in our laboratory (Maya Núñez, G., and P. M. Conn, manuscript submitted), which shows the presence of a member(s) of the CRE-binding protein associated with this region.

When this region was deleted, both basal and Buserelin-stimulated transcription decreased. Therefore, cAMP may have a critical role in maintaining the basal expression of the GnRHR gene. The regulation of basal gene expression by cAMP has been described for other genes, including the cystic fibrosis transmembrane conductance regulator gene (48) and the 3'-aldehyde dehydrogenase gene (49). The deletion of -343/-335 sequence was deleted both basal and forskolin-stimulated transcription was decreased. Stimulation with (Bu)2cAMP did not alter transcription above basal levels. Stimulation with PMA resulted in an attenuated increase in transcriptional activity, suggesting that the sequence element of the GnRHR promoter is important for transcriptional activity of mGnRHR gene specifically by cAMP in GGH31' cells. However, these results suggest that cAMP could be involved in the transcriptional regulation of GnRHR, although we did not rule out the potential involvement of the protein kinase C pathway. The present results are consistent with a recent study (22) in which the response elements on the mouse GnRHR gene for cAMP reside in -318 to -1. The present data suggest the presence of a CRE-like sequence in the mGnRHR gene that is responsible for the transcriptional activation of the GnRHR gene by cAMP and GnRH, and identify one putative repressor element in positions -343/-335 (relative to the start codon) and a cAMP response element (-107/-100, relative to the start codon) that regulate basal activity of the GnRHR gene promoter in GGH31' cells.


   Acknowledgments
 
We thank Dr. Xinwei Lin and Jo Ann Janovick for their help.


   Footnotes
 
1 This work was supported by NIH Grants HD-19899, RR-00163, and HD-18185; Fogarty Grant TW/HD00668; and Unidad de Investigación Médica en Biología del Desarrollo, Mexico. Back

Received January 12, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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