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Endocrinology Vol. 142, No. 8 3483-3492
Copyright © 2001 by The Endocrine Society

ARTICLES

Transcriptional Regulation of the Human GnRH II Gene Is Mediated by a Putative cAMP Response Element

Alon Chen, Orly Laskar-Levy, Nurit Ben-Aroya and Yitzhak Koch

Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel

Address all correspondence and requests for reprints to: Dr. Y. Koch, Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: y.koch{at}weizmann.ac.il


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human neuronal medulloblastoma cells (TE-671) were recently demonstrated to express the two forms of GnRH (GnRH-I and GnRH-II). We have used this cell line as a model system to demonstrate regulation of the human GnRH-II gene by cAMP. RT-PCR and Southern hybridization demonstrated that GnRH-II mRNA is strongly up-regulated (~6-fold) by (Bu)2cAMP. The concentration of GnRH-II that was released into the medium of TE-671 cells treated with the cAMP analog was significantly higher than that of the untreated cells. TE-671 cells that were stimulated by (Bu)2cAMP demonstrated morphological changes and strong immunoreactive GnRH-II staining in part of the cell population. After screening of the GnRH-II promoter sequence, we identified a putative cAMP response element consensus site. The GnRH-I and GnRH-II promoters were isolated by PCR using human genomic DNA and cloned into the luciferase reporter plasmid. By measuring the basal activity of the promoters that were transfected to TE-671 cells, we found a much stronger basal activity of the GnRH-II promoter compared with that of GnRH-I. Treatment of transfected TE-671 cells with (Bu)2cAMP resulted in a strong activation of the GnRH-II promoter compared with a modest activation of the GnRH-I promoter. To determine the functionality of this putative cAMP response element site, we mutated this site. TE-671 cells that were transfected with cAMP response element mutant constructs demonstrated a diminished basal activity of the GnRH-II promoter. Treatment of the transfected cells with the cAMP analog demonstrated a decrease to 0.03% of the activity of the mutated promoter compared with that of the wild type. These results clearly demonstrate the importance of the putative cAMP response element site for the basal activity as well as for induction of the GnRH-II promoter by cAMP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH-I (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), originally isolated from the mammalian hypothalamus, plays a pivotal role as the physiological regulator of reproduction (1, 2). This peptide is synthesized and released by hypothalamic neurosecretory cells and reaches the pituitary gland by way of a specialized portal system to induce the synthesis and secretion of the gonadotropic hormones that regulate gonadal function (3). Recently, several groups have identified a second form of GnRH (GnRH-II; His5,Trp7,Tyr8-GnRH-I) in the brain of mammalian species (4, 5, 6, 7, 8). The GnRH-II gene was cloned from human (9) and monkey (10) brains. Originally, GnRH-II was isolated as a second form of GnRH from the chicken brain (11) and was initially termed chicken GnRH-II. Since then, it was found to be present in cartilaginous and bony fish (12, 13), amphibians (14), reptiles (15), and metatherian mammals (16). The wide distribution of this neuropeptide over all vertebrate classes demonstrates its conservation over the years of evolution and may imply that its physiological functions are most important. In the mammalian brain, the localization of GnRH-II neurons is restricted mainly to the brainstem and hypothalamic structures. The cells are scattered mainly in the periaqueductal and central regions of the midbrain and in the paraventricular, supraoptic, and medial-basal nuclei of the hypothalamus (4, 5, 6, 7, 8, 10). Recently, a third isoform of GnRH in the human, calf, and rat brain has been demonstrated (17).

After screening of several human neuronal cell lines we have recently demonstrated two cell lines, TE-671 medulloblastoma and LAN-1 neuroblastoma cells, that coexpress mRNA encoding the two forms, GnRH-I and GnRH-II (18). Nucleotide sequencing indicated that the cDNA fragments are identical to those of the known human (h) GnRH-I and GnRH-II sequences. These cell lines also contain immunoreactive GnRH-I and GnRH-II that have exhibited an elution profile on HPLC identical to the synthetic forms of GnRH. These neuronal cell lines therefore provide ideal tools for studying the differential regulation of gene expression and secretion as well as the interactions between the two forms.

In this study we demonstrate for the first time that the hGnRH-II, but not the hGnRH-I, gene is strongly up-regulated by a cAMP analog. By screening the hGnRH-II gene promoter, we have also identified a putative cAMP response element (CRE) site. The GnRH-II promoter construct was cloned upstream of a luciferase reporter plasmid and was transfected into the TE-671 neuronal cells. The luciferase activity of mutated CRE has demonstrated the importance of the CRE site not only for its activation by (Bu)2cAMP, but also for full expression of the basal activity of the promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of luciferase reporter gene plasmids
Genomic DNA was extracted from human tissue using the Wizard Genomic DNA Purification Kit (catalogue no. A1120, Promega Corp., Madison, WI). The human GnRH-II promoter (9) construct was cloned by PCR using human genomic DNA. The primers (Table 1Go) that were used for the construct were designed to include an artificial restriction endonuclease site. The PCR products were analyzed by agarose gel electrophoresis in 1 x TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA) and eluted from the gel. After digestion by the appropriate restriction enzyme (Table 1Go), the DNA fragment was cloned into the luciferase reporter plasmid pGL3 (catalogue no. E1751, Promega Corp.), and its sequence was verified using automated direct DNA sequencing, according to the manufacturer’s recommendations (model 377, PE Applied Biosystems, Perkin-Elmer Corp., Foster City, CA). The human GnRH-I promoter construct was prepared at our laboratory using the same methodology.


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  		Table 1. Primers used for human GnRH-II promoter construct and promoter mutagenesis

 
Cell culture, transfections, and luciferase assay
TE-671 cells, a human neuronal medulloblastoma cell line, were grown in phenol red-free DMEM containing 10% charcoal-treated FCS supplemented with 100 µg/ml penicillin/streptomycin (Life Technologies, Inc., Paisley, Scotland). For transfection, TE-671 cells (3 x 105) were plated on 60-mm plates. On the next day, the cells were transfected with 2.5 µg of the reporter plasmid DNA using the calcium phosphate method (19). Each experiment contained a transfection with a positive control reporter plasmid containing the cytomegalovirus (CMV) promoter to permit estimation of transfection efficiencies and comparison of results obtained from different experiments. Cells were treated with 1 mM (Bu)2cAMP (catalogue no. D-0627, Sigma, St. Louis, MO) for different periods of time and harvested 48 or 72 h after transfection, and luciferase reporter activity was assayed (20). In brief, cells were washed three times with PBS and lysed by freeze-thawing in 100 µl 100 mM KPO4 buffer (pH 7.8) containing 1 mM dithiothreitol and 0.5% (vol/vol) Triton X-100. The luciferase assay buffer contains 100 mM Tris acetate (pH 7.8), 10 mM MgOAc, 100 mM EDTA, 2 mM ATP (pH 7.0), and 74 µM luciferin. Activity tests were performed, and luminescence was measured in a Biocounter M2500 luminometer (Lumac, Landgraaf, The Netherlands) for 20 sec immediately after addition of luciferin assay buffer. Transfections were performed at least six times (in triplicate) for each construct or treatment that was tested.

RT-PCR and Southern analysis
Total RNA was extracted from TE-671 cells treated with 1 mM (Bu)2cAMP for different periods of time (6, 12, 24, and 48 h) as well as from untreated cells using TRIzol RNA isolation reagent (Molecular Research Center, Inc., Cincinnati, OH) based on the acid guanidinium thiocyanate-phenol-chloroform extraction method, according to the manufacturer’s recommendations. We used RT-PCR (21) to amplify the levels of endogenous GnRH-I and GnRH-II mRNA that may be present in the human TE-671 cell samples. The expression of ribosomal protein S-14 (22) served as an internal control. Each reaction contained four oligonucleotide primers, two for the GnRH form (GnRH-I or GnRH-II) and two for the S-14 internal control (18). Equivalents of 500 ng RNA were reversed transcribed and amplified by PCR for 35 cycles; the annealing temperature was 62 C, and the final MgCl2 concentration was 2.5 mM. The PCR products were transferred onto a nylon membrane (Nytran 0.45, Schleicher & Schuell, Inc., Dassel, Germany) by overnight capillary blotting in 20 x SSC solution, and the nylon was baked in a vacuum oven at 80 C for 2 h. Prehybridization was performed in the presence of 6 x SSC (standard saline citrate), 5 x Denhardt’s solution, 5 mM EDTA, and 0.2 mg/ml salmon sperm DNA for 3 h at 64 C. Overnight hybridizations were performed sequentially on the same membrane in the presence of a 32P-labeled probe that was specific to GnRH-I, GnRH-II, or S-14 cDNA at 64 C. The corresponding bands could be seen after exposure of the membranes to PhosphorImager plates (445 SI, Molecular Dynamics, Inc., Jersey City, NJ). Gels were also exposed to x-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan) for 2–16 h at -80 C and were developed in CURIX 60 processor (AGFA, Koln, Germany).

Oligonucleotide primers
For the PCR reactions the following specific GnRH-I, GnRH-II, and S-14 oligonucleotide primers were used: GnRH-I, 5'-AGTACTCAACCTACTTCAAG-3' and 5'-CATTCAAAGCGTTGGGTTTCT-3' [corresponding to nucleotides 1134–1153 (sense) and 3746–3766 (antisense), respectively (23); the predicted size of the band is 248 bp]; GnRH-II, 5'-CTGCAGCTGCCTGAAGGAG-3' and 5'-CTAAGGGCATTCTGGGGAT-3' [corresponding to nucleotides 1312–1330 (sense) and 2232–2250 (antisense), respectively (9); the predicted size of the band is 197 bp]; and S-14, 5'-GGCAGACCGAGATGAATCCTCA-3' and 5'-CAGGTCCAGGGGTCTTGGTCC-3' [corresponding to nucleotides 2941–2962 (sense) and 4166–4186 (antisense), respectively (24); the predicted size of the band is 143 bp]. The oligonucleotide probes for hybridization were: GnRH-I, 5'-CCAAGTCAGTAGAATAAGGCC-3' (corresponding to nucleotides 2091–2111); GnRH-II, 5'-GCAGGAGGCCTCGCCTGGAGCTGGCCATGGCTGCT-3' (corresponding to nucleotides 2098–2132); and S-14, 5'-ATATGCTGCTATGTTGGCTGC-3' (corresponding to nucleotides 2965–2985).

Mutagenesis
Mutants in the putative CRE site were obtained by PCR mutagenesis using the overlap extension procedure described by Ho et al. (25). For each mutation a set of two overlapping oligonucleotide primers containing the desired mutation was constructed (Table 1Go). The outside primers were homologous to those used in isolation of the human GnRH-II promoter (Table 1Go). The resulting mutagenized PCR products were digested with XhoI and HindIII and were cloned directly into the luciferase reporter vector pGL3. All constructs were cleaved with the restriction enzymes HindIII and AatII to verify generation of the mutation. The AatII enzyme recognizes only the wild-type sequence. In addition, the mutagenized fragments were sequenced to verify the mutation and to ensure that no other mutation occurred during the PCR amplification process.

GnRH determination
TE-671 cells were incubated with or without 1 mM (Bu)2cAMP for 24 h. The media were collected, boiled, and pumped onto columns of Sep-Pak C18 cartridges (Waters Corp., Milford, MA), washed by 0.1% trifluoroacetic acid (TFA), eluted by methanol, and evaporated by nitrogen. After reconstitution in 0.1 M phosphate buffer (PB; pH 7.4) containing 0.1% of bovine {gamma}-globulin, the concentration of GnRH-II was determined by RIA (26) using a GnRH-II-specific antiserum that was developed in our laboratory (18). Iodination of synthetic GnRH-II (Phoenix Pharmaceuticals, Inc., Mountain View, CA) was carried out using the chloramine-T method (27). Free iodine was removed on a Sep-Pak C18 cartridge, and the 125I-labeled peptide was separated from the unlabeled peptide by HPLC (Waters Corp.) using the following elution program: eluent A, 0.1% TFA in water; and eluent B, 75% CH3CN in 0.1% TFA. The gradient program consisted of a linear gradient of 0–50% eluent B for 50 min at a flow rate of 1 ml/min.

Antibodies
The following antisera were used throughout this study. Two polyclonal antibodies against GnRH-II were used. One antibody, aCII6, was provided by Dr. K. Okuzawa, and its specificity was previously defined (7, 28). Additional specificity tests in our laboratory demonstrated that GnRH-I did not displace any of the bound [125I]GnRH-II, even at a concentration that exceeded 2500 times the GnRH-II concentration needed to displace 50% of the tracer (20 ng vs. 8 pg). We used dilutions ranging from 1:4,000 to 1:10,000 of this antibody for the immunohistochemical studies. The other antiserum, KLII-2, was prepared and characterized in our laboratory. Specificity tests of this antibody demonstrated that GnRH-I did not displace any of the bound [125I]GnRH-II, even at a concentration that exceeded 1,000 times the GnRH-II concentration needed to displace 50% of the tracer (30 ng vs. 30 pg). We used dilutions ranging from 1:4,000 to 1:10,000 of the GnRH-II antibodies for the immunohistochemical studies.

Fluorescence immunocytochemical analysis
TE-671 cells were plated on round glass coverslips (13 mm) coated with poly-L-lysine (15 µg/ml) in 24-well culture plats. One day later, half of the cells were treated with 1 mM (Bu)2cAMP for 24 h. The cells were then fixed by the addition of 4% paraformaldehyde in 0.1 M PB, pH 7.4 (30 min), washed with PB (three times, 5 min each time), and permeabilized for 3 min with 0.5% Triton X-100. After washing (three times), the cells were incubated for 2 h at room temperature in a blocking medium (PBS containing 10% normal goat serum, 2% BSA, 1% glycine, and 0.5% Triton X-100) to saturate nonspecific binding sites for IgG. The primary antibody was added for 12–15 h at 4 C, and the cells were washed (three times, 5 min each time) with 0.1 M PB. The cells were then incubated for 2 h at room temperature with a secondary antibody (goat antirabbit) conjugated to Oregon Green 488 (Molecular Probes, Inc., Eugene, OR). Fluorescence was visualized by fluorescence microscopy using the appropriate filter. To determine the specificity of the signals, we included several control groups in which the antibody was preabsorbed with an excess (10–100 µg) of GnRH-II or GnRH-I for 24 h. Additional controls were incubated with normal rabbit serum instead of the first antibody.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Up-regulation of GnRH-II mRNA levels by (Bu)2cAMP treatment
Total RNA preparations derived from untreated or (Bu)2cAMP-treated TE-671 cells for different periods of time were reverse transcribed to generate cDNA. The cDNA products were used as templates for the quantitative PCR using specific primers for GnRH-I, GnRH-II, and ribosomal protein S14, which served as an internal control. Sense and antisense primers were selected to be located on different exons of GnRH-I, GnRH-II, and S14 to avoid false positive results caused by DNA contamination (Fig. 1AGo). To calibrate the experimental conditions for the quantitative RT-PCR assay and to confirm that the assay is carried out in the linear phase of amplification, we performed PCR for increasing numbers of amplification cycles using cDNA that is equivalent to 500 ng total RNA (Fig. 1BGo). The average relative signal of GnRH-I, GnRH-II, and S14, correlated to the number of PCR cycles, is demonstrated in Fig. 1CGo. cDNA equivalent to 500 ng RNA was amplified by PCR for 35 cycles using specific oligonucleotide primers for GnRH-I and GnRH-II. Each PCR tube contained 4 oligonucleotide primers, 2 for the GnRH-I or GnRH-II and 2 for the internal control, S14. Southern hybridizations were performed sequentially on the same membrane, using 32P-labeled oligonucleotide probes specific to GnRH-I, GnRH-II, or S14. The RT-PCR and Southern hybridization demonstrate that GnRH-II mRNA (Fig. 1DGo, middle panel) is strongly up-regulated by (Bu)2cAMP, whereas GnRH-I mRNA levels do not increase significantly (Fig. 1DGo, upper panel). The ribosomal protein S14 that served as an internal control was expressed practically unchanged, as expected, in all cDNA preparations (Fig. 1DGo, lower panel). Normalized results (adjusted according to S14 intensity) of three independent experiments are shown as the fold increase (Fig. 1EGo). This figure illustrates the strong activation of GnRH-II mRNA after treatment with a cAMP analog for 12–48 h. A similar time scale for the increased gene expression of the human ß-microseminoprotein by cAMP has been recently reported (29).



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  		Figure 1. Southern blot hybridization of amplified GnRH-I, GnRH-II, and the ribosomal protein S14 cDNA fragments after cAMP treatment. A, Scheme of the GnRH-I, GnRH-II, and the S14 transcripts. GnRH-I, GnRH-II, and S14 genes are shown with the introns (lines), exons (squares), poly(A) tail (wavy lines), and location of the PCR fragments (shaded squares). The lengths in base pairs of the introns, exons, and each of the PCR fragments are indicated. Sense and antisense primers for PCR were selected to be located on different exons to avoid false positive results caused by DNA contamination. B and C, Quantitative RT-PCR of GnRH-I, GnRH-II, and S14 transcripts obtained from human TE-671 cells. PCR was performed for increasing number of amplification cycles using cDNA that is equivalent to 500 ng total RNA. B, The Southern hybridization bands were quantified by a PhosphorImager. The average relative signals of GnRH-I, GnRH-II, and S14, correlated to the number of PCR cycles, are demonstrated in C. D, Amplified GnRH-I, GnRH-II, and S14 cDNA fragments from the human neuronal cell line TE-671, treated with or without 1 mM (Bu)2cAMP, were hybridized to 32P-labeled oligonucleotide probes of human GnRH-I (upper panel), GnRH-II (middle panel), and S14 (lower panel). The hybridizations were performed sequentially on the same membrane. The predicted sizes of GnRH-I, GnRH-II, and S14 fragments are 248, 197, and 143 bp, respectively. The results represent one of three experiments (D). The normalized results, corrected against S14 intensity, obtained from three independent experiments are presented in E as the fold increase compared with untreated cells (0 treatment).

 
GnRH-II levels are up-regulated by (Bu)2cAMP treatment
The concentrations of GnRH-II in the medium of untreated and (Bu)2cAMP-treated TE-671 cells were determined by a specific RIA for GnRH-II. Fig. 2AGo demonstrates that the concentrations of the peptide found in the medium of TE-671 cells that were incubated for 24 h with 1 mM (Bu)2cAMP were 3.6-fold higher than those in the medium of untreated cells (127 vs. 35 pg, respectively). Further demonstration of induction of the GnRH-II gene by (Bu)2cAMP is evident by immunofluorescence staining for GnRH-II in untreated (Fig. 2Go, B and D) and (Bu)2cAMP-treated (Fig. 2Go, C and E) TE-671 cells. Cells of the human medulloblastoma clonal line TE-671 exhibit polymorphism when grown in serum-supplemented medium (30). When (Bu)2cAMP is added (Fig. 2GGo), the cells rapidly (starting after 3 h of incubation) undergo a distinctive morphological transformation characterized by neurite extension and formation of cell-cell contacts (Fig. 2Go, compare F to G), and the appearance of a strong immunoreactive GnRH-II in a portion of the cell population (Fig. 2Go, C and E) compared with the basal expression of the peptide (Fig. 2Go, B and D). TE-671 cells reacted with normal rabbit serum followed by secondary antibodies did not result in any increased staining. Preabsorption of GnRH-II antibody with excess synthetic GnRH-II abolished the immunoreactive staining, whereas preabsorption with excess synthetic GnRH-I did not affect the signal (data not shown).



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  		Figure 2. Analysis of GnRH-II peptide expression. A, GnRH-II concentration in medium of TE-671 cells treated with 1 mM (Bu)2cAMP for 24 h. The GnRH-II concentrations in the media of the control and (Bu)2cAMP-treated groups were determined by RIA; there was a 3.6-fold increase in GnRH-II concentration in the medium of TE-671 cells that were treated with the cAMP analog. B–E, Immunofluorescence staining for GnRH-II in untreated (B and D) and (Bu)2cAMP-treated (C and E) TE-671 cells. Incubation of TE-671 cells with 1 mM of the cAMP analog for 24 h resulted in strong immunoreactive staining (arrows) for GnRH-II in part of the cell population (C and E) compared with the basal expression (B and D). F and G, Phase pictures of the control (F) and (Bu)2cAMP-treated cells (G), demonstrating the morphological transformation characterized by neurite extension and formation of cell-cell contacts after incubation with the cAMP analog (arrows in G). Scale bar, 10 µM.

 
Construction of GnRH-II promoter in luciferase reporter gene plasmid and its basal activity in neuronal cells
The hGnRH-II promoter was isolated by PCR using human genomic DNA and cloned into the luciferase reporter plasmid pGL3 using artificial restriction endonuclease sites for HindIII and XhoI that were designed in the primer sequences (Fig. 3AGo and Table 1Go). The human GnRH-II construct consisted of 1305 bp of the GnRH-II promoter, 44 bp of exon 1, and 25 bp from intron 1 (total size, 1374 bp). The construct was transfected into TE-671, human neuronal medulloblastoma cells, which express GnRH-II and GnRH-I endogenously (18). Ideally, the host cell line should express the endogenous gene of interest, because regulatory substances that affect the endogenous promoter will also control the transfected constructs. The activity of the promoter was evaluated by measuring luciferase activity. By measuring the basal activity of the GnRH-I and GnRH-II promoters, we could demonstrate that the GnRH-II promoter has a strong basal activity that is more than 200-fold higher than that of the GnRH-I promoter (Fig. 3BGo). Transfection of CMV promoter, cloned into the p20 luciferase plasmid, was used to permit estimation of transfection efficiencies and to compare the results obtained from different experiments.



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  		Figure 3. Basal activity of GnRH-I and GnRH-II promoters. A, Scheme of the hGnRH-II promoter construct. The 1374-bp construct is composed of 1305 bp of the GnRH-II promoter, 44 bp of exon 1, and of 25 bp from intron 1. The construct was cloned by PCR using human genomic DNA. The DNA fragment was cloned into the luciferase reporter vector (pGL3 Basic Vector) using artificial restriction endonuclease sites for XhoI and HindIII that were designed in the sequence of the primer. The location and sequence of the putative CRE site in the human GnRH-II promoter are indicated. The CRE site contains an endogenous endonuclease restriction site for the restriction enzyme AatII that was used, as an initial step, to screen and distinguish between the wild-type and the CRE mutants. B, Luciferase reporter gene assay of human GnRH-I, GnRH-II, and CMV promoter activity in TE-671 cells. The cells were cultured and transfected with 2.5 µg reporter plasmid DNA using the calcium phosphate method, as described in Materials and Methods. After 24 h, the luciferase basal activity of the promoters was determined and demonstrated a strong basal activity for the GnRH-II gene compared with that of the GnRH-I gene.

 
Up-regulation of the GnRH-II promoter by (Bu)2cAMP
TE-671 cells were transfected with an equal amount (2.5 µg) of either GnRH-I or GnRH-II promoter constructs. After transfection, the cells were treated with 1 mM (Bu)2cAMP for different periods of time, and luciferase activity was determined simultaneously for all samples. The results, summarized in Fig. 4Go, are presented as the fold increase over the basal activity of the respective promoter and demonstrate a strong activation of the GnRH-II promoter in response to 1 mM (Bu)2cAMP treatment, compared with a modest activation of the GnRH-I promoter. Activations of 4.5- and 6.8-fold were observed for the GnRH-II promoter after 24 and 48 h of 1 mM (Bu) 2cAMP treatment, respectively. The inset in Fig. 4Go demonstrates the background level of the pGL3 luciferase vector without or after treatment with 1 mM (Bu)2cAMP for 24 h.



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  		Figure 4. cAMP responsiveness and basal activity of human GnRH-I and GnRH-II promoter constructs. TE-671 cells were transfected with 2.5 µg GnRH-I or GnRH-II reporter plasmids, and parallel plates were treated with 1 mM (Bu)2cAMP for different periods of time. The cells of all groups were harvested at the same time, and luciferase activity was measured. The results (mean ± SD of six independent experiments) are presented as the fold increase compared with the basal activity of the specific promoter construct. Strong activation of the hGnRH-II promoter was observed after 24 and 48 h of cAMP treatment. The inset demonstrates the background level of the pGL3 luciferase vector without or after treatment with 1 mM (Bu)2cAMP for 24 h. *, P < 0.05 vs. basal promoter activity.

 
Putative CRE site in the GnRH-II promoter, mutagenesis, and (Bu)2cAMP activation
To determine whether the human GnRH-II and GnRH-I promoters contain a CRE consensus site, we screened the promoter sequences for a putative CRE element in the DNA sequences using Transcription Element Search Software (TESS) on the world-wide web (URL: http://www.cbil.upenn.edu/tess/index.html). We found a putative CRE consensus site, only in the GnRH-II promoter, at nucleotide sequence -67 to -60 (see Fig. 3AGo). To determine the functionality of this putative site to (Bu)2cAMP activation and to evaluate its contribution to the basal activity of the promoter, we performed a 3- or 4-bp mutation at this site by PCR mutagenesis using the overlap extension procedure (Table 1Go). The putative CRE site contains a restriction endonuclease site for the AatII enzyme (Fig. 3AGo). We used this enzyme to distinguish, as an initial step, between wild-type and mutant promoters. Thus, the product of 1380 bp derived from the HindIII+XhoI activity indicates the presence of the GnRH-II promoter in the plasmid construct. A product of 1249 bp derived from the activity of the restriction enzymes HindIII+AatII indicates a normal CRE site, whereas the mutations at the CRE site prevented its recognition by the AatII enzyme (Fig. 5AGo). The mutated fragments were sequenced to verify the mutation and to ensure that no other mutations occurred during the amplification process. The chromatographic sequences of the relevant fragments are presented in Fig. 5Go, B–D: the wild-type mutation (Fig. 5BGo), 3-bp mutation (Fig. 5CGo), and 4-bp mutation (Fig. 5DGo). Transfection of the CRE mutant constructs to TE-671 cells resulted in a strong decrease in the basal activity of the GnRH-II promoter (Fig. 5EGo). A decrease to 0.083% and 0.058% was observed for the 3- and 4-bp CRE mutants, respectively, compared with the basal activity of the wild-type promoter. Treatment of the transfected cells with 1 mM (Bu)2cAMP for 40 h demonstrated a decrease to 0.03% for both 3- and 4-bp mutations compared with the (Bu)2cAMP-treated wild-type mutation. These results demonstrate the importance of the putative CRE site for both basal activity and the (Bu)2cAMP response of the GnRH-II promoter.



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  		Figure 5. Analysis of mutations in the putative CRE site of the hGnRH-II promoter. A, Agarose gel electrophoresis and ethidium bromide staining of the wild-type (W. T) and mutated human GnRH-II promoter constructs, cleaved with two sets of restriction enzymes, HindIII+XhoI and HindIII+AatII. The products of 1380 bp derived from the HindIII+XhoI activity indicate the presence of the GnRH-II promoter in the plasmid constructs. A product of 1249 bp derived from the activity of the restriction enzymes HindIII+AatII indicates a normal CRE site, as the mutations at the CRE site prevent recognition by AatII. B–D, Sequence chromatography of relevant fragments of the wild-type (B), 3-bp (C), and 4-bp (D) mutations in the putative CRE of the hGnRH-II promoter. The wild-type and mutant CRE sites are indicated by squares. The location of the presented fragments on the gene is indicated in D. E, Basal activity and responsiveness to (Bu)2cAMP of the wild-type and mutated hGnRH-II promoter. Decreases to 0.083% and 0.058% compared with the basal activity of the wild-type promoter were observed for the 3- and 4-bp CRE mutants, respectively. Treatment of the transfected cells with 1 mM (Bu)2cAMP for 40 h demonstrated a decrease to 0.03% for both 3- and 4-bp mutations compared with the (Bu)2cAMP-treated wild-type. Values presented are the mean ± SD of six independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The availability of neuronal cell lines that express the two isoforms of GnRH provides an excellent model system to evaluate the mechanisms that mediate the specific differential expression of human GnRH-II or GnRH-I as well as the roles of various neurotransmitters, growth factors, hormones, and other biological substances that may regulate directly or indirectly the synthesis or secretion of the two GnRH forms. Moreover, the cloning of the human GnRH-II promoter into a luciferase reporter plasmid and the transfection of this construct into host cells that express GnRH-II endogenously enables study of regulation of the human GnRH-II gene at the promoter, mRNA, and peptide levels.

A number of hormones and growth factors have been shown to stimulate target cells via second messenger pathways that, in turn, regulate the phosphorylation of specific nuclear factors. Thus, cAMP regulates a striking number of physiological processes, including intermediary metabolism, cellular proliferation, and neuronal signaling, by influencing diverse gene expression (31). cAMP stimulates gene expression via a conserved CRE, consisting of an 8-bp palindrome (TGACGTCA) that may be slightly different in different genes (29, 32, 33, 34, 35, 36, 37, 38) (Table 2Go). The CRE site is typically found and is active within 100 nucleotides from the TATA box; it is less active at a more distal position (>500 nucleotides). The palindromic CRE can be separated into two CGTCA motifs, which may be configured on the same or opposite strands to function cooperatively in response to cAMP stimulation (39). Indeed, multimerization of the CRE strongly enhances cAMP inducibility, as revealed by the cooperative action of two tandems CREs located on the CG{alpha} gene promoter (37, 40).


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  		Table 2. Genes containing a cAMP-responsive element (CRE)

 
Several lines of evidence demonstrate that PKA is required for cAMP to stimulate transcription. For example, mutant PC12 phenochromocytoma cells that are deficient in PKA activity are unable to stimulate a CRE-chloramphenicol acetyltranferase reporter gene in transient transfection assays (33). Likewise, overexpression of protein kinase inhibitor, which inhibits PKA, specifically abolished cAMP- dependent transcription in transfected cells (41). Finally, microinjection of a purified PKA catalytic subunit into tissue culture cells was sufficient to induce the expression of cAMP-responsive genes without increased levels of cAMP (42). Taken together, these results suggest that cAMP induces gene expression via PKA. Three CRE- binding proteins were identified: CRE binding protein (43), activating transcription factor-1 (44), and CRE modulator (45). All three proteins of this family contain a consensus sequence for PKA phosphorylation sites and are regulated by cAMP in vivo. CRE-binding protein, activating transcription factor-1, and CRE modulator bind to DNA as dimers via a carboxyl-terminal basic region/leucine zipper motif (46) that is conserved in several nuclear factors, such as Jun, Fos, and Myc (47).

In this study we have showed, by using a variety of techniques, that the expression of the hGnRH-II gene is up-regulated by (Bu)2cAMP. The hGnRH-II promoter construct transfected into the TE-671 host cells was strongly up- regulated by (Bu)2cAMP via a CRE domain. The activation of GnRH-II by (Bu)2cAMP resulted in increased endogenous GnRH-II mRNA levels demonstrated by Southern blot hybridization (Fig. 1Go) and increased peptide concentration and release as determined by specific RIA and immunocytochemistry (Fig. 2Go). Mutations made in this region (Fig. 5Go) have clearly identified it as a functional CRE, as the effects of cAMP on gene expression were eliminated. The CRE domain in the GnRH-II promoter, -60AGACGTCA-67, has one discordance (A-60) with the CRE consensus sequence. A large number of studies have indeed demonstrated that certain modifications of the consensus CRE resulted in elements that are functional as CRE (Table 2Go). The GnRH-II element is involved not only in the induction of the gene by cAMP, but also in the basal activity of the promoter. The physiological signals (i.e. hormones and neurotransmitters) that activate adenylate cyclase and regulate the GnRH-II gene are still unknown. It is of interest to note that despite the dramatic decrease in the basal activity of the mutated promoter and in its response to cAMP, there is still a 2- to 3-fold increase in the level of the mutated promoters after treatment with (Bu)2cAMP (Fig. 5EGo). Similarly, although we could not identify a CRE site in the GnRH-I promoter, a similar magnitude of activation (2.3-fold) could be observed by the GnRH-I promoter after treatment with (Bu)2cAMP (Fig. 4Go). These activations may be attributed to the involvement of other transcription factors that can be activated by signals promoted by cAMP. Analysis of the putative transcription factor-binding sites of the promoter sequences of GnRH-I and GnRH-II show that the two GnRH isoforms possess essentially different putative transcription factor-binding sites, thus suggesting that the two genes are probably differentially regulated. Understanding the mode of regulation of the two genes by different substances may be helpful for elucidation of the possible biological functions of GnRH-II.

The basal activity of the GnRH-II promoter construct transfected into the TE-671 cells was demonstrated to be 200-fold more active than that of the GnRH-I gene (Fig. 3Go). However, the concentration of GnRH-II, as determined by RIA, in the brain of rodents and human is much lower than that of GnRH-I (7). This discrepancy could be explained by differences in the rate of the translation machinery or in the rate of enzymatic degradation (of the mRNA or the peptide) or by the fact that the constructs do not contain the whole size of the promoters.

The GnRH-I promoter was demonstrated to modulate GnRH-I gene transcription by steroid hormones and signaling pathways. The adenylyl cyclase is one of the signal transduction systems that is considered to take part in the regulation of the GnRH-I gene (for reviews, see Refs. 48, 49, 50, 51). However, the reports are controversial. Whereas forskolin has been reported to have no effect (52) or to repress the steady state GnRH-I mRNA levels in GT1 cells (53), another study (54) demonstrated that perfusion of hypothalamic fragments with forskolin stimulated GnRH-I release as well as its mRNA levels. A cAMP analog was reported (55) to mimic the down-regulation action of hCG on GnRH-I mRNA levels in GT1–7 cells. This report has also demonstrated that a PKA inhibitor, but not a PKC inhibitor, blocked the down-regulatory action of hCG as well as that of the cAMP analog. Treatment with the PKA inhibitor modestly decreased mRNA levels of GnRH-I, suggesting that PKA signaling also controls basal expression of the GnRH-I gene. In the present study TE-671 cells that were transfected with the GnRH-I promoter and treated with (Bu)2cAMP have demonstrated only a modest activation of the promoter compared with activation of the GnRH-II promoter (Fig. 4Go).

Conservation of the structure of the neuropeptide GnRH-II from the primitive fish to the human suggests that this neuropeptide possess vital bioactivities. However, the biological functions of GnRH-II are practically still unknown. In the bullfrog, application of exogenous GnRH-I has been reported to inhibit a specific voltage-dependent potassium current, leading to depolarization of sympathetic neurons and induction of a late, slow excitatory, postsynaptic potential (56). Later, it was found that GnRH-II is at least 1000 times more potent than GnRH-I in inducing this effect (57), and that indeed GnRH-II, but not GnRH-I, is present in the amphibian sympathetic ganglia (58). These findings imply that GnRH-II is the endogenous transmitter that mediates the excitatory postsynaptic potential in amphibian. Studies in mammals have indicated a role for GnRH-I in the process of sexual differentiation of the brain (59). Other studies demonstrated the involvement of GnRH-I in the induction of sexual behavior. Thus, GnRH-I that was administered sc or into the midbrain central gray has been demonstrated to facilitate sexual behavior (60, 61). However, the high doses of GnRH-I (microgram range) that were needed to induce this phenomenon and the discovery of GnRH-II in the midbrain of mammals raise the possibility that GnRH-II is the physiological regulator of mating behavior. Indeed, several studies have indicated that cAMP can facilitate lordosis behavior in estrogen-primed rats (62, 63). Therefore, it is possible that the facilitation of sexual behavior by cAMP is mediated by the activation of GnRH-II, that acts as a neurotransmitter in the midbrain central gray.


   Acknowledgments
 
The authors thank Drs. K. Okuzawa (Mie, Japan) for supplying the aCII6 antibody for GnRH-II.


   Footnotes
 
This work was supported by the Dr. Ernst Nathan Fund for Biomedical Research and the Israel Science Foundation, administered by the Israel Academy of Sciences and Humanities (412/99, to Y.K.).

Abbreviations: CMV, Cytomegalovirus; CRE, cAMP response element; hGnRH, human GnRH; PB, phosphate buffer; TFA, trifluoroacetic acid.

Received February 5, 2001.

Accepted for publication April 4, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Amoss M, Burgus R, Blackwell P, Vale W, Fellows R, Guillemin R 1971 Purification, amino acid composition and N-terminus of the hypothalamic luteinizing hormone-releasing factor (LRF) of ovine origin. Biochem Biophys Res Commun 44:205–210[CrossRef][Medline]
  2. Matsuo H, Baba Y, Nair RMG, Arimura A, Schally AV 1971 Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. Biochem Biophys Res Commun 43:1334–1339[CrossRef][Medline]
  3. Okon E, Koch Y 1976 Localisation of gonadotropin-releasing and thyrotropin-releasing hormones in human brain by radioimmunoassay. Nature 263:345–347[CrossRef][Medline]
  4. Rissman EF, Alones VE, Craig-Veit CB, Millam JR 1995 Distribution of chicken-II gonadotropin-releasing hormone in mammalian brain. J Comp Neurol. 357:524–531
  5. Kasten TL, White SA, Norton TT, Bond CT, Adelman JP, Fernald RD 1996 Characterization of two new preproGnRH mRNAs in the tree shrew: first direct evidence for mesencephalic GnRH gene expression in a placental mammal. Gen Comp Endocrinol 104:7–19[CrossRef][Medline]
  6. Lescheid DW, Terasawa E, Abler LA, et al. 1997 A second form of gonadotropin-releasing hormone (GnRH) with characteristics of chicken GnRH-II is present in the primate brain. Endocrinology 138:5618–5629[Abstract/Free Full Text]
  7. Chen A, Yahalom D, Ben-Aroya N, Kaganovsky E, Okon E, Koch Y 1998 A second isoform of gonadotropin-releasing hormone is present in the brain of human and rodents. FEBS Lett 435:199–203[CrossRef][Medline]
  8. Gestrin ED, White RB, Ferland RD 1999 Second form of gonadotropin- releasing hormone in mouse: immunocytochemistry reveals hippocampal and perivntricular distribution. FEBS Lett 448:289–91[CrossRef][Medline]
  9. White RB, Eisen JA, Kasten TL, Ferland RD 1998 Second gene for gonadotropin-releasing hormone in humans, Proc Natl Acad Sci. USA. 95:305–309
  10. Urbanski HF, White RB, Ferland RD, Kohama SG, Garyfallou VT, Densmore VS 1999 Regional expression of mRNA encoding a second form of gonadotropin- releasing hormone in the macaque brain. Endocrinology 140:1945–1948[Abstract/Free Full Text]
  11. Miyamoto K, Hasegawa Y, Nomura M, Igarashi M, Kangawa K, Matsuo H 1984 Identification of the second gonadotropin-releasing hormone in chicken hypothalamus: evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormone in avian species. Proc Natl Acad Sci USA 81:3874–3878[Abstract/Free Full Text]
  12. Powell RC, Millar RP, King JA 1986 Diverse molecular forms of gonadotropin-releasing hormone in an elasmobranch and teleost fish. Gen Comp Endocrinol 63:77–85[CrossRef][Medline]
  13. Lovejoy DA, Fischer WH, Ngamvongchon S, et al. 1992 Distinct sequence of gonadotropin-releasing hormone (GnRH) in goldfish brain provides insight into GnRH evolution. Proc Natl Acad Sci USA 89:6373–6377[Abstract/Free Full Text]
  14. King JA, Millar RP 1986 Identification of His5,Trp7,Tyr8-GnRH (chicken GnRH II) in amphibian brain. Peptides 7:827–834[CrossRef][Medline]
  15. Sherwood NM, Whittier JM 1988 Gonadotropin-releasing hormone from brains of reptiles: turtles (Pseudemys scripta) and snakes (Thamnophis sirtalis parietalis) Gen Comp Endocrinol. 69:319–327
  16. King JA, Steneveld AA, Curlewis JD, Rissman EF, Millar RP 1994 Identification of chicken GnRH-II in brains of metatherian and early evolved eutherian species of mammals. Regul Pept 54:467–477[CrossRef][Medline]
  17. Yahalom D, Chen A, Ben-Aroya N, et al. 1999 The gonadotropin-releasing hormone family of neuropeptides in the brain of human, bovine and rat: identification of a third isoform. FEBS Lett 463:289–294[CrossRef][Medline]
  18. Chen A, Yahalom D, Laskar-Levy O, Rahimipour S, Ben-Aroya N, and Koch Y 2001 Two isoforms of gonadotropin-releasing hormone are co-expressed in neuronal cell lines. Endocrinology 142:830–837[Abstract/Free Full Text]
  19. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory: Cold Spring Harbor Press
  20. Sadot E, Heicklen-Klein A, Barg J, Lazarovici P, Ginzburg I 1996 Identification of a Tau promoter region mediating tissue-specific-regulated expression in PC12 cells. J Mol Biol 256:805–812[CrossRef][Medline]
  21. Palmon A, Ben-Aroya N, Tel-Or S, Burstein Y, Fridkin M, Koch Y 1994 The gene for the neuropeptide gonadotropin-releasing hormone is expressed in the mammary gland of lactating rats. Proc Natl Acad Sci USA 91:4994–4996[Abstract/Free Full Text]
  22. Leonard M, Brice M, Engel JD, Papayannopulou T 1993 Dynamics of GATA transcription factor expression during erythroid differentiation. Blood 82:1071–1079[Abstract/Free Full Text]
  23. Hayflick JS, Adelman JP, Seeburg PH 1989 The complete nucleotide sequence of the human gonadotropin-releasing hormone gene. Nucleic Acids Res 17:6403–6404[Free Full Text]
  24. Rhoads DD, Dixit A, Roufa DJ 1986 Primary structure of human ribosomal protein S14 and the gene that encodes it. Mol Cell Biol 6:2774–2783[Abstract/Free Full Text]
  25. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR 1989 Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59[CrossRef][Medline]
  26. Koch Y, Wilchek M, Fridkin M, Chobsieng P, Zor U, Lindner HR 1973 Production and characterization of an antiserum to synthetic gonadotropin- releasing hormone. Biochem Biophys Res Commun 55:616–622[CrossRef][Medline]
  27. Hunter WM, Greenwood FC 1962 Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature 194:495–496[CrossRef][Medline]
  28. Okuzawa K, Amano M, Kobayashi M, Aida K, Hanyu I, Hasegawa Y, Miyamoto K 1990 Differences in salmon GnRH and chicken GnRH-II contents in discrete brain areas of male and female rainbow trout according to age and stage of maturity. Gen Comp Endocrinol 80:116–126[CrossRef][Medline]
  29. Ueyama H, Ohkubo I 1998 The expression of ß-microseminoprotein gene is regulated by cAMP. Biochem Biophys Res Commun 248:852–857[CrossRef][Medline]
  30. Siegel HN, Lukas RJ 1988 Morphological and biochemical differentiation of the human medulloblastoma cell line TE671. Dev Brain Res 44:269–280[CrossRef][Medline]
  31. Montiminy M 1997 Transcriptional regulation by cyclic AMP. Annu Rev Biochem 66:807–822[CrossRef][Medline]
  32. Fan NC, Peng C, Krisinger J, Leung PCK 1995 The human gonadotropin-releasing hormone receptor gene: complete structure including multiple promoters, transcription initiation sites, and polyadenylation signals. Mol Cell Enodocrinol 107:R1–R8
  33. Maya-Nunez G, Conn M 1999 Transcriptional regulation of gonadotropin-releasing hormone receptor gene is mediated in part by a putative repressor element and by the cyclic adenosine 3',5'-monophosphate response element. Endocrinology 140:3452–3458[Abstract/Free Full Text]
  34. Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH 1986 Identification of a cyclic-AMP responsive element within the rat somatostatin gene. Proc Natl Acad Sci USA 83:6682–6686[Abstract/Free Full Text]
  35. Quinn PG, Wong TW, Magnuson MA Shabb JB, Granner DK 1988 Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene. Mol Cell Biol 8:3467–3475[Abstract/Free Full Text]
  36. Tsukada T, Fink JS, Mendel G, Goodman RH 1987 Identification of a region in the human vasoactive intestinal polypeptide gene responsible for regulation by cyclic AMP. J Biol Chem 262:8743–8747[Abstract/Free Full Text]
  37. Delegeane AM, Ferland LH, Mellon PL 1987 Tissue-specific enhancer of the human glycoprotein hormone {alpha}-subunit gene: dependence on cyclic AMP-inducible elements. Mol Cell Biol 7:3994–4002[Abstract/Free Full Text]
  38. Comb M, Birnberg NC, Seascholtz A, Herbert E, Goodman HM 1986 A cyclic AMP and phorbol ester-inducible DNA element. Nature 323:353–356[CrossRef][Medline]
  39. Fink JS, Verhave M, Kasper S, Tsukada T, Mandel G, Goodman RH 1988 The CGTCA sequence motif is essential for biological activity of the vasoactive intestinal peptide gene cAMP-regulated enhacer. Proc Natl Acad Sci USA 85:6662–6666[Abstract/Free Full Text]
  40. Jameson JL, Jaffe RC, Gleason SL, Habener JF 1986 Transcriptional regulation of chorionic gonadotropin {alpha}- and ß-subunit gene expression by 8-bromo-adenosine 3',5'-monophosphate. Endocrinology 119:2560–2567[Abstract]
  41. Grove JR, Price DJ, Goodman HM, Avruch J 1987 Recombinant fragment of protein kinase inhibitor blocks cyclic AMP- dependent gene transcription. Science 283:530–533
  42. Riabowol KT, Fink JS, Gilman MZ, Walsh DA, Goodman RH, Feramisco JR 1988 The catalytic subunit of cAMP-dependent protein kinase induces expression of genes containing cAMP-responsive enhancer elements. Nature 336:83–86[CrossRef][Medline]
  43. Montiminy MR, Bilezikjian LM 1987 Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature 328:175–179[CrossRef][Medline]
  44. Hai TW, Liu F, Allegretto EA, Karin M, Green MR 1988 A family of immunologically related transcription factors that includes multiple forms of ATF and AP-1. Genes Dev 2:1216–1226[Abstract/Free Full Text]
  45. Foulkes NS, Borrelli E, Sassone-Corsi P 1991 CREM gene: use of alternative DNA-binding domains generates multiple antagonists of cAMP-induced transcription. Cell 64:739–749[CrossRef][Medline]
  46. Dwarki VJ, Montminy M, Verma IM 1990 Both the basic region and the ‘leucine zipper’ domain of the cyclic AMP response element binding (CREB) protein are essential for transcriptional activation. EMBO J 9:225–232[Medline]
  47. Landschulz WH, Jhonson PF, McKnight SL 1988 The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240:1759–1764[Abstract/Free Full Text]
  48. Gore AC, Roberts JL 1997 Regulation of gonadotropin-releasing hormone gene expression in vivo and in vitro. Front Neuroendocrinol 18:209–245[CrossRef][Medline]
  49. Wetsel WC 1995 Immortalized hypothalamic luteinizing hormone releasing (LHRH) neurons: a new tool for dissecting the molecular and cellular basis of LHRH physiology. Cell Mol Neurobiol 15:43–78[CrossRef][Medline]
  50. Wierman ME, Bruder JM, Kepa JK 1995 Regulation of gonadotropin releasing hormone (GnRH) gene expression in hypothalamic neuronal cells. Cell Mol Neurobiol 15:79–88[CrossRef][Medline]
  51. Nelson SB, Eraly SA, Mellon PL 1998 The GnRH promoter: target of transcription factors, hormones, and signaling pathways. Mol Cell Endocrinol 140:151–155[CrossRef][Medline]
  52. Wetsel WC Eraly SA, Whyte DB, Mellom PL 1993 Regulation of gonadotropin releasing hormone by protein kinase-A and -C in immortalized hypothalamic neurons. Endocrinology 132:2360–2370[Abstract]
  53. Yu KL, Yeo TTS, Dong KW, et al. 1994 Second messenger regulation of mouse gonadotropin- releasing hormone gene expression in immortalized mouse hypothalamic GT1–3 cells. Mol Cell Endocrinol 102:85–92[CrossRef][Medline]
  54. Lee BJ, Kim K, Cho WK 1990 activation of intracellular pathways with forskolin and phorbol ester in creases LHRH mRNA level in the rat hypothalamus superfused in vitro. Mol Brain Res 8:185–191[Medline]
  55. Lei ZM, Rao CV 1995 Signaling and transacting factors in the transcriptional inhibition of gonadotropin releasing hormone gene by human chorionic gonadotropin in immortaliazed hypothalamic GT1–7 neurons. Mol Cell Endocrinol 109:151–157[CrossRef][Medline]
  56. Jan YN, Jan LY, Kuffler SW 1979 A peptide as a possible transmitter in sympathetic ganglia of the frog. Proc Natl Acad Sci USA 76:1501–1505[Abstract/Free Full Text]
  57. Jones SW 1987 Chicken II luteinizing hormone-releasing hormone inhibits the M-current of bullfrog sympathetic neurons. Neurosci Lett 80:180–184[CrossRef][Medline]
  58. Troskie B, King JA, Millar RP, et al. 1997 Chicken GnRH II-like peptides and a GnRH receptor selective for chicken GnRH II in amphibian sympathetic ganglia. Neuroendocrinology 65:396–402[Medline]
  59. Kalcheim C, Szechtman H, Koch Y 1981 Bisexual behavior in male rats treated neonatally with antibodies to luteinizing hormone-releasing hormone. J Comp Physiol Psychol 95:36–44[CrossRef][Medline]
  60. Riskind P, Moss RL 1979 Midbrain central gray: LHRH infusion enhances lordotic behavior in estrogen-primed ovariectomized rats. Brain Res Bull 4:203–205[CrossRef][Medline]
  61. Sakuma Y, Pfaff DW 1980 LH-RH in the mesencephalic central gray can potentiate lordosis reflex of female rats. Nature 283:566–567[CrossRef][Medline]
  62. Mobbs CV, Rothfeld JM, Saluja R, Pfaff DW 1989 Phorbol esters and forskolin infused into midbrain central gray facilitate lordosis. Pharmacol Biochem Behav 34:665–667[CrossRef][Medline]
  63. Beyer C, Gonzalez-Flores O, Gonzalez-Mariscal G 1997 Progesterone receptor participates in the stimulatory effect of LHRH, prostaglandin E2, and cyclic AMP on lordosis and proceptive behaviours in rats. J Neuroendocrinol 9:609–614[CrossRef][Medline]



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