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Endocrinology Vol. 142, No. 11 4711-4720
Copyright © 2001 by The Endocrine Society

ARTICLES

Cyclical Regulation of GnRH Gene Expression in GT1–7 GnRH-Secreting Neurons by Melatonin

Deboleena Roy, Nadia L. Angelini, Hiroki Fujieda, Gregory M. Brown and Denise D. Belsham

Institute for Medical Sciences (D.R., G.M.B., D.D.B.) and Department of Physiology (G.M.B., D.D.B), University of Toronto; Division of Cellular and Molecular Biology (D.R., N.L.A., D.D.B.), University Health Network; and Clarke Institute of Psychiatry (H.F., G.M.B.), Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: Denise D. Belsham, Ph.D., Department of Physiology/Division of Cellular and Molecular Biology, University of Toronto/University Health Network, Medical Sciences Building, Room 3247A, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: d.belsham{at}utoronto.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pineal hormone melatonin plays an important role in the neuroendocrine control of reproductive physiology, but its effects on hypothalamic GnRH neurons are not yet known. We have found that GT1–7 GnRH-secreting neurons express membrane-bound G protein-coupled melatonin receptors, mt1 (Mel-1a) and MT2 (Mel-1b) as well as the orphan nuclear receptors ROR{alpha} and RZRß. Melatonin (1 nM) significantly downregulates GnRH mRNA levels in a 24-h cyclical manner, an effect that is specifically inhibited by the melatonin receptor antagonist luzindole (10 µM). Repression of GnRH gene expression by melatonin appears to occur at the transcriptional level and can be mapped to the GnRH neuron-specific enhancer located within the 5' regulatory region of the GnRH gene. Using transient transfection of GT1–7 cells, downregulation of GnRH gene expression by melatonin was further localized to five specific regions within the GnRH enhancer including -1827/-1819, -1780/-1772, -1746/-1738, -1736/-1728, and -1697/-1689. Interestingly, the region located at -1736/-1728 includes sequences that correspond to two direct repeats of hexameric consensus binding sites for members of the ROR/RZR orphan nuclear receptor family. To begin to dissect the mechanisms involved in the 24-h cyclical regulation of GnRH transcription, we have found that melatonin (10 nM) induces rapid internalization of membrane-bound mt1 receptors through a ß-arrestin 1-mediated mechanism. These results provide the first evidence that melatonin may mediate its neuroendocrine control on reproductive physiology through direct actions on the GnRH neurons of the hypothalamus, both at the level of GnRH gene expression and through the regulation of G protein-coupled melatonin receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MELATONIN IS THE principal hormone produced by the vertebrate pineal gland and mainly regulates circadian rhythms and reproduction (1, 2). Evidence suggests that melatonin may exert its inhibitory effects at various levels of the hypothalamic-pituitary-gonadal axis (3). As the governing factor of this axis, GnRH is secreted in a pulsatile manner from a small number of neurons in the hypothalamus to control LH and FSH synthesis, which in turn regulate hormonal secretions from the gonads. The hypothalamus has been postulated as a potential site of melatonin-mediated regulation of gonadal activity (reviewed in Ref. 2). Melatonin uptake and binding have been detected in a number of brain regions, including the hypothalamus and median eminence (4, 5, 6).

Melatonin mediates both its circadian and reproductive effects through specific G protein-coupled receptors (GPCRs). The mt1 receptor mRNA is expressed mainly in the suprachiasmatic nucleus and hypophyseal pars tuberalis in several mammals (7), whereas MT2 receptor mRNA is expressed in retina and brain (8, 9). Previous studies have also reported specific melatonin binding and immunolocalization to the cell nucleus, mainly associated with the chromatin, indicating a genomic role for melatonin (10). Interestingly, retinoic acid receptor-related orphan (ROR){alpha} and retinoid Z receptor (RZR)ß receptors, both members of the ROR/RZR retinoid-related orphan nuclear receptor family, have been shown to bind melatonin with high affinity (11), although these findings are still being debated in the literature.

Studies on the mouse anterior hypothalamus and medial preoptic area suggest that the antigonadal action of melatonin on the brain involves the suppression of the release of GnRH (12), thought to occur through afferent neurons synapsing on GnRH neurons (13). To date, however, there have been no studies addressing the possible direct regulatory action of melatonin on the GnRH neuronal cell population. The scarcity of GnRH-secreting neurons in the hypothalamus led to the establishment of a more suitable model to study GnRH biosynthesis. Through targeted tumorigenesis by SV-40 T-antigen (T-Ag), a murine immortalized cell line of GnRH-secreting neurons (GT1–7 cells) was developed (14). The GT1–7 cell line has been shown to exhibit many of the known characteristics of hypothalamic GnRH neurons in situ (15, 16). We have used the GT1–7 cell line to investigate the direct effect of melatonin on GnRH gene expression. Our results provide the first evidence that melatonin may act directly at the level of the GnRH neuron to regulate GnRH transcription in what appears to be a cyclical pattern.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents
GT1–7 cells were grown in monolayer in DMEM (Life Technologies, Inc., Burlington, Ontario, Canada) supplemented with 10% FBS (HyClone Laboratories, Inc., Logan, UT), 4.5 mg/ml glucose and penicillin/streptomycin, and maintained at 37 C in an atmosphere of 5% CO2 (14). Human prostate LNCaP cells, DU145 cells, and NIH3T3 cells were grown in RPMI 1640 (Life Technologies, Inc.), supplemented with 5% FBS (Life Technologies, Inc.). Melatonin, aprotinin, leupeptin, and pepstatin were obtained from Sigma-Aldrich Corp. Canada, Ltd. (Oakville, Ontario, Canada). Luzindole was obtained from Tocris Cookson Inc. (Ballwin, MO). Disuccinimidyl suberate was obtained from Pierce Chemical Co. (Brockville, Ontario, Canada). Luciferin was obtained from ICN Biomedicals, Inc. (Montréal, Québec, Canada).

RT-PCR, subcloning, and sequencing
Total RNA was isolated from GT1–7 and DU145 cells by the guanidinium thiocyanate phenol chloroform extraction method (17). First-strand cDNA was synthesized from 1–10 µg of deoxyribonuclease I-treated RNA, using SuperScript II reverse transcriptase (RT), as described in the SuperScript II cDNA synthesis kit (Life Technologies, Inc.). The RT reaction was primed with oligo. The cDNA synthesis was followed by RNase H (Life Technologies, Inc., 180 U/ml) digestion of RNA in a total volume of 20 µl. The specificity of each amplification reaction was monitored in control reactions in which amplification was carried out on samples in which the RT was omitted (RT-). PCR amplifications were performed with 1.25 U Taq polymerase (Life Technologies, Inc.) in a 50-µl reaction for 30 cycles (1 min at 94 C, 1 min at 57 C, 1 min at 72 C). The primers selected are shown in Table 1Go. All primers were made by ACGT Corp. (Toronto, Ontario, Canada). PCR products were electrophoresed in 1.5% agarose gels, stained with 1 µg/ml ethidium bromide, and visualized under UV light. Corresponding DNA fragments were isolated and purified using Geneclean (BIO101 Inc., Vista, CA) and subcloned into pCR2.1-TOPO cloning vector using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). DNA was prepared for sequencing using the Wizard Plus miniprep kit (Promega Corp., Madison, WI) according to the manufacturer’s instructions. DNA sequencing was performed by the dideoxy-sequencing method of Sanger et al. (18) using the T7 sequencing kit (Pharmacia Biotech) and [35S] dATP (10 mCi/ml, Amersham Pharmacia Biotech Inc., Arlington Heights, IL). Sequencing reactions were run on 6% polyacrylamide gels for 3–6 h. Gels were then dried and exposed to X-600 autoradiography film (Kodak, Rochester, NY) for 16 h.


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  		Table 1. Primers used for PCR amplification

 
Western blot analysis
Membrane fractions of GT1–7, LNCaP, and NIH3T3 cells were obtained according to (19). Briefly, cells were homogenized in 0.5 M Tris-HCl (pH 7.4) supplemented with proteinase inhibitors (1 µg/ml aprotinin, pepstatin, and leupeptin, 1 mM PMSF, 1 mM EDTA) and centrifuged at 40,000 x g for 25 min. The pellets were extracted in PBS (pH 7.4) supplemented with 1% Triton X-100 (Fisher Scientific, Whitby, Ontario, Canada) and the above-mentioned proteinase inhibitors, and centrifuged at 20,000 x g for 20 min. The final supernatant, representing solubilized cell membranes, was collected and an aliquot was taken for protein assay. Protein was measured using BCA protein assay reagent (Pierce Chemical Co. Ltd., Rockford, IL). Samples were stored at -20 C until the time of assay. Membrane proteins (100 µg) were resolved on a 12.5% SDS-polyacrylamide gel (20) and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA). Membranes were washed briefly in PBS, then incubated in PBST (PBS, pH 7.4, 0.2% Tween-20, 5% powdered skim milk) for 16 h with 1 µg/ml polyclonal rabbit antihuman mt1 (19) or MT2 antisera in the presence or absence of the corresponding blocking peptides (0.5 µg/ml). Immunoreactive bands were visualized with horseradish peroxidase-labeled secondary goat antirabbit antisera at 1:10,000 dilution and enhanced chemiluminescence (Amersham Pharmacia Biotech Inc., Oakville, Ontario, Canada), as described by the manufacturer.

Northern blot analysis
Total cellular RNA was isolated as described above. Ten to fifteen micrograms of total RNA were electrophoresed in 1% formaldehyde agarose gels and transferred to Genescreen membranes (NEN Life Science Products, Boston, MA) by capillary blotting (21). The filters were probed with rat GnRH cDNA (22), SV-40 T-Ag cDNA (23), human {gamma}-actin cDNA (24), and a mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA generated by RT-PCR, as indicated above. Membranes were prehybridized 2–6 h and hybridized 16 h in hybridization buffer (1% w/vol BSA, 1 mM EDTA, 0.5 M Na2HPO4, 5% w/vol SDS, 25% formamide) at 55 C. The cDNA probes were labeled using random hexamers and [32P]dATP (6000 Ci/mmol, NEN Life Science Products) incorporated with the Klenow fragment of DNA polymerase I (25). Blots were washed at high stringency (0.5x SSC, 0.1% SDS; 55 C), and exposed to Fuji Photo Film Co., Ltd. (Stamford, CT) film at x70 C with intensifying screens for 4–24 h. Autoradiographs were scanned with a ScanJet 3p scanner (Hewlett-Packard Co., Boise, ID) and GnRH, T-Ag, actin, and GAPDH mRNA signals were quantified by densitometry using the NIH Image program. Statistical significance of the results was determined by using the t test.

Melatonin RIA
GT1–7 cells were split into 60-mm2 plates. Medium was replaced with 5 ml fresh DMEM with 10% FBS with either 10 nM melatonin or vehicle alone (dimethylsulfoxide, 14 nM). Medium was collected at different time points (0–48 h) following treatment. Protein content per well was assayed using the BCA protein assay reagent (Pierce Chemical Co. Ltd.). Protein content was consistently equivalent between plates (±10% variation). Melatonin content in 500-µl aliquots of the cell culture medium samples was assayed in triplicate with a melatonin RIA kit (produced by Dr. Greg Brown, University of Toronto).

Transient transfections
Transfections were performed using the calcium phosphate precipitate method as previously described (26) containing 15 µg of plasmid DNA and 5 µg of the internal control plasmid TK-luciferase. The cells were incubated 12–14 h with DNA, followed by three PBS rinses, and then treated with melatonin (10 nM) in DMEM 12 h before harvesting. Chloramphenicol acetyl transferase (CAT) assays were done using the CAT ELISA kit (Roche Molecular Biochemicals, Laval, PQ, Canada), according to the manufacturer’s instructions. Luciferase assays were done as previously described (27). Protein concentrations were determined using the BCA protein assay reagent kit (Pierce Chemical Co. Ltd.).

Subcellular cell fractionation
GT1–7 cells were stimulated with 10 nM melatonin for 15, 30, or 60 min at 37 C. Subcellular fractionation was performed as described (28). Briefly, the cells were washed twice with ice-cold PBS (pH 7.4), removed from plates by gentle scraping, and then pelleted at 500 rpm for 10 min. The cell pellet was resuspended in 1 ml of buffer A (10 mM Tris-HCl, pH 7.4, 2 mM EDTA), incubated on ice for 30 min, and homogenized using a Dounce homogenizer. Nuclei were removed by centrifugation at 500 rpm for 10 min. The supernatant was loaded on a stepwise sucrose cushion (35% and 5% sucrose in PBS) and centrifuged at 35,000 rpm for 90 min at 4 C. The supernatant was removed and the 35% sucrose interface fraction containing endosomes (the light vesicular fraction) was collected, diluted in 3 ml buffer A, and recentrifuged at 35,000 rpm for 45 min at 4 C. The pellets were resuspended in 30 µl of buffer A. One hundred micrograms of each protein sample were diluted in 3x SDS sample buffer and analyzed by SDS-PAGE.

Cross-linking and immunoprecipitation
GT1–7 cells were incubated at 37 C in serum-starved media for 2 h. Cells were then stimulated with melatonin (10 nM) or vehicle alone for 10 or 30 min. Proteins were cross-linked as described (29). Briefly, the medium was removed and the cells were incubated in 3 ml cross-linking buffer (10 mM HEPES, pH 7.4, 2.5 mM disuccinimidyl suberate, and 10% [vol/vol] DMSO in PBS) for 30 min at room temperature with occasional shaking to avoid drying the cells. The cells were placed on ice and the cross-linking buffer was replaced with 1 ml lysis buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.5, 10 mM NaCl, 1 mM EDTA, and 1 mM EGTA). Cells were collected by gentle scraping and sonicated briefly. Samples were then centrifuged for 10 min at 14,000 rpm at 4 C. Protein concentration of the supernatant was quantified using the BCA protein assay reagent (Pierce Chemical Co. Ltd.). Equal amounts of each protein were used for immunoprecipitation of ß-arrestin 1. Protein samples were incubated with Protein G Plus Agarose for 1 h at 4 C, with gentle shaking. The samples were then centrifuged for 10 min at 12,000 rpm, 4 C. The supernatant was transferred to a new microcentrifuge tube and incubated with ß-arrestin 1 goat polyclonal IgG antibodies (0.1 mg/ml) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and protein G plus agarose (Santa Cruz) for 16 h at 4 C, gently shaking. Samples were then centrifuged and the pellet was washed 5x in 1 ml lysis buffer and centrifuged for 10 min at 12,000 rpm at 4 C. The immunoprecipitate was then resuspended in 1x SDS loading buffer. Cross-linked and immunoprecipitated proteins were resolved on a 7.5% SDS-polyacrylamide gel (20) and transferred to Immobilon-P membranes (Millipore Corp.). Membranes were washed briefly in PBS and then blocked in PBST (PBS, pH 7.4, 0.2% Tween-20, 5% powdered skim milk) for 30 min. Membranes were then incubated for 16 h with 1 µg/ml polyclonal rabbit antihuman mt1 (19). Immunoreactive bands were visualized with horseradish peroxidase-labeled secondary goat antirabbit antisera at 1:5,000 dilution and enhanced chemiluminescence (Amersham Pharmacia Biotech Inc.), as described by the manufacturer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of mouse mt1, MT2, ROR{alpha}, and RZRß receptors in GT1–7 neurons
To investigate possible direct effects of melatonin on the GnRH neuron, the expression of melatonin receptors was analyzed in GT1–7 cells. RT-PCR performed on GT1–7 cDNA using mt1-, MT2-, ROR{alpha}-, and RZRß-specific primers produced PCR products of the expected sizes (Fig. 1AGo, B, and C). Because the PCR products obtained using the MT2 and RZRß external primers were not easily detectable by visual analysis of agarose gels, we also used a pair of internal or nested primers for each to reamplify the receptor fragments and to confirm specificity of the initial reaction. We then cloned and sequenced these PCR products. Because membrane-bound melatonin receptors have previously been reported in prostate tissue and cell lines (30), we used the prostate cell line DU145 as a positive control. DNA sequencing of the cloned PCR products confirmed that both the mouse membrane-bound G protein-coupled melatonin receptor subtypes mt1 and MT2, as well as the orphan nuclear receptors ROR{alpha} and RZRß, are expressed in GT1–7 cells.



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  		Figure 1. Identification of mt1 and MT2 receptors as well as the orphan nuclear receptors, ROR{alpha}, and RZRß in GT1–7 neurons. cDNA was synthesized with (+) or without (-) RT from total RNA isolated from GT1–7 and DU145 prostate cancer cells (positive control). The cDNA was used as a template for PCR with primers (Rec-PR) specifically designed to amplify (A) mt1, (B) MT2, or (C) ROR{alpha} and RZRß. PCR products were size fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining. Amplification of histone H3 (H3-PR) transcripts was used to control for the integrity of the RNA in the RT-PCR. The mt1 and MT2 receptor proteins were detected in GT1–7 membrane fractions. Membrane fractions from GT1–7 neurons, LNCaP cells (positive control), and NIH3T3 cells (negative control) were isolated and subjected to SDS-PAGE. Western blot analysis was performed with enhanced chemiluminescence using specific antibodies for the (D) mt1 and (E) MT2 receptors. Specificity of the immunoreactive bands was determined in parallel blots incubated with the antibody in conjunction with the peptide used to create the antibody (+ peptide panels).

 
To determine whether melatonin receptor mRNAs are translated into protein in GT1–7 neurons, Western blot analysis was performed on membrane fractions of GT1–7 neurons, LNCaP cells (prostate cell line, positive control), and NIH3T3 cells (negative control) using peptide-derived mt1 (19) and MT2 (Brown, G.M., unpublished data) receptor-specific polyclonal antibodies. An immunoreactive band migrating at 37 kDa was detected in GT1–7 and LNCaP membrane fractions using mt1 antibody (Fig. 1DGo). The immunoreactive bands were blocked by the specific mt1 peptide used to generate the mt1 antibody. Immunoreactive bands migrating at approximately 40 kDa and 80 kDa were observed in GT1–7 membrane fractions exposed to the MT2 antibody. Similar-sized bands were also detected in LNCaP membrane fractions (Fig. 1EGo). Although the anticipated molecular weight of the MT2 protein is approximately 40 kDa, the 80-kDa bands were blocked by the specific peptide used to generate the MT2 antibody in GT1–7 and LNCaP membrane fractions, but the 40-kDa band exhibited only minor competition. We have also found that melatonin inhibits forskolin-stimulated cAMP accumulation in the GT1–7 cells, indicative of functional melatonin receptors (data not shown, Roy, D. and D.D. Belsham, manuscript in preparation).

Effect of melatonin and luzindole on GnRH mRNA levels
GnRH steady-state mRNA levels were examined over an extended time course, to determine whether melatonin is involved in the transcriptional regulation of the GnRH gene. The GT1–7 cells were treated with a 1 nM dose of melatonin, alone or with luzindole (10 µM), over a 48-h period, with daily replacement of the compounds. A significant downregulation of GnRH mRNA levels was observed by Northern blot analysis at 12 and 36 h following melatonin treatment (Figs. 2Go and 3Go). Interestingly, although GnRH gene expression was reduced to approximately 40% of basal GnRH mRNA levels after 12- and 36-h treatments, the levels returned to normal at 24 and 48 h (1 nM melatonin, 6 h, 0.77 ± 0.09; 12 h, 0.38 ± 0.05; 18 h, 0.81 ± 0.07; 24 h, 0.92 ± 0.06; 36 h, 0.40 ± 0.05; 48 h, 0.90 ± 0.03) (Fig. 2AGo). It is important to note that the addition of melatonin at 12 h intervals did not change the cyclical pattern of GnRH mRNA repression, whereas a single does of melatonin at the 0 time point still revealed a downregulation of GnRH mRNA levels at 36 h, although not as robust as when melatonin was added every 24 h (data not shown). These controls were performed to ensure that the apparent cyclical effect was not simply owing to experimental technique. Furthermore, the half-life of melatonin in culture media, as assayed by a melatonin RIA, was found to exceed 48 h (Fig. 2BGo), therefore indicating that the effects of melatonin on GnRH mRNA levels are not owing to the degradation of melatonin.



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  		Figure 2. Effect of melatonin and luzindole on steady-state GnRH mRNA levels in GT1–7 neurons. A, GT1–7 cells were treated with 1 nM melatonin or vehicle alone over a 48-h time course. At the indicated time points, total RNA was extracted and 15 µg of each sample were subjected to Northern blot analysis. Blots were probed simultaneously with rat GnRH cDNA and human {gamma}-actin cDNA. GnRH mRNA levels were quantified by scanning densitometry of autoradiographs and normalized to loading control ({gamma}-actin). Data shown are relative to GnRH mRNA levels at time 0 and are expressed as mean ± SEM (n = five independent experiments; **, P < 0.005 vs. control time 0 and corresponding vehicle controls). B, GT1–7 cells were treated with melatonin over a 48-h period. Cell medium was collected and assayed for melatonin-like immunoreactivity by RIA. Results shown are mean ± SEM (n = three independent experiments). C, GT1–7 cells were treated with 1 nM melatonin, 1 nM melatonin + 10 µM luzindole, 10 µM luzindole, or vehicle alone for 12 or 24 h. RNA sample preparation and Northern blot analysis was conducted described above. Data shown are relative to GnRH mRNA levels at time 0 and are expressed as mean ± SEM (n = three independent experiments; **, P < 0.005 vs. control time 0 and corresponding vehicle controls). D, Representative Northern blot corresponding to the data shown.

 


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  		Figure 3. Effect of melatonin on SV-40 T-Ag steady-state mRNA levels. A, GT1–7 cells were treated with 1 nM melatonin or vehicle alone over a 48-h time course. At the indicated time points, total RNA was extracted and 15 µg of each sample were subjected to Northern blot analysis. Blots were probed sequentially with rat GnRH cDNA, SV-40 T-Ag cDNA and mouse GAPDH cDNA to monitor for variations in gel loading and transfer efficiency. Data shown are relative to T-Ag mRNA levels at time 0 and are expressed as mean ± SEM (n = three independent experiments; **, P < 0.005 vs. control time 0 and corresponding vehicle controls). B, Representative Northern blot corresponding to the data shown.

 
The competitive melatonin receptor antagonist luzindole shows a 25-fold higher affinity for the MT2 receptor isoform, although it also binds mt1. Nevertheless, its effect on the ROR/RZR nuclear receptors has not yet been studied, and structural analysis indicates that it is membrane permeable and could thereby act as a competitive antagonist to the ROR/RZR receptors as well. In the presence of the specific melatonin receptor antagonist luzindole, the melatonin-mediated downregulation of GnRH mRNA levels was inhibited (1 nM melatonin plus 10 µM luzindole, 12 h, 1.13 ± 0.17; 24 h, 1.24 ± 0.11) (Fig. 2CGo). Luzindole alone or vehicle alone had no effect on GnRH mRNA levels. {gamma}-actin mRNA levels, a loading control, were not affected by treatment with melatonin. These results again indicate that the GT1–7 cells, an in vitro model of the GnRH-secreting neuron, are directly influenced by melatonin, suggesting that GnRH hypothalamic neurons may also be a target of melatonin action in vivo.

Transcriptional regulation of GnRH mRNA levels by melatonin
Downregulation of GnRH gene expression at 12 and 36 h by melatonin in the GT1–7 neurons may occur at a number of levels. It is possible that melatonin affects gene transcription, either directly or indirectly, and/or may have an effect on GnRH mRNA stability. To determine at what level melatonin acts to regulate GnRH mRNA levels, we studied changes in the mRNA levels of the T-Ag oncogene used to create the GT1–7 cell line. In the GT1–7 neurons, T-Ag gene expression is under the control of 2.3 kb of the rat GnRH 5' regulatory region (14). Because T-Ag mRNA synthesis is driven by the 5' regulatory region of the rat GnRH gene, changes in T-Ag mRNA levels can be used as a reporter gene to monitor transcriptional regulation by the GnRH upstream regulatory region. On the same Northern blots used to establish the repression of GnRH mRNA levels by melatonin, T-Ag mRNA levels mimicked those of GnRH after hybridization with mouse T-Ag cDNA (Fig. 3Go, with 10 nM melatonin, 12 h, 0.37 ± 0.04; 24 h, 0.89 ± 0.07; 36 h, 0.4 ± 0.05; 48 h, 0.93 ± 0.07). We detected two T-Ag mRNA species (2200 and 2450 bp), as expected (31). These results suggest that melatonin mediates downregulation of GnRH mRNA synthesis at the transcriptional level.

Localization of regulatory elements within the neuron-specific enhancer of the GnRH gene
Previously, using transfection into GT1–7 cells, a neuron-specific enhancer, essential for GnRH gene expression, was found 1.6 kb upstream of the GnRH gene (32). This 300-bp enhancer (-1863 to -1571) is capable of restoring full activity to an otherwise silent, truncated (-173) GnRH promoter. Using the GT1–7 neurons, the basal promoter region (33) and a complex neuron-specific enhancer within the 5' regulatory region of the GnRH gene have been characterized (32). These regions have been found to bind a number of transcription factors from the GT1–7 cells (32), which include Oct-1 (34), GATA-4 (35), C/EBPß (36), Otx2 (37), and two homeodomain proteins (38). We have examined melatonin-mediated regulation of the GnRH enhancer and promoter regions using GT1–7 cells that were transiently transfected with an expression plasmid containing the enhancer (-1863 to -1571) on the -173 promoter directing expression of the reporter gene CAT (Fig. 4Go). Treatment of transiently transfected GT1–7 cells with 10 nM melatonin for 12 h resulted in an approximately 30% decrease in CAT activity. This result indicated that the sequences responsible for mediating melatonin regulation of GnRH gene expression are present within the enhancer and/or the promoter regions.



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  		Figure 4. Localization of the effect of melatonin to the GnRH gene neuron-specific enhancer. GT1–7 cells were transiently transfected with CAT expression vectors containing combinations of the GnRH enhancer (Enh) and promoter (Pro) regions as well as heterologous enhancer (CRE) and promoter (TK) regions and then treated with 10 nM melatonin in 10% DMEM. Treatments were for 12 h after previous transfection of the DNA for 16 h. Values are expressed as relative CAT expression of the nontreated vectors compared with the level of expression in the melatonin-treated vectors. TK-luciferase was included as an internal control in all transfections. Each value is an average of at least three experiments in duplicate or triplicate ± SEM corrected for the internal control (**, P < 0.005).

 
To further determine whether the enhancer or the promoter region was responsible for the melatonin-mediated repression of GnRH gene expression, hybrid CAT expression vectors were transfected into GT1–7 cells, including the GnRH enhancer coupled with the neutral thymidine kinase (TK) promoter, the -173 promoter coupled with three tandem repeats of the cAMP response element (CRE), and a vector containing the CRE enhancer linked to the TK promoter. The CAT activities of the -173 promoter coupled to the CRE enhancer and the control vector were not altered by treatment with melatonin. However, the CAT expression of the vector containing the GnRH enhancer coupled with the TK promoter was repressed by approximately 30% following melatonin treatment (Fig. 4Go, with 10 nM melatonin, Enh-Pro CAT 0.72 ± 0.02; Enh-TK CAT 0.71 ± 0.03; CRE-Pro CAT 1.02 ± 0.11; CRE-TK CAT 1.03 ± 0.03). The TK promoter was transfected alone to assess any regulation of the heterologous promoter sequence or to detect any reporter protein degradation by melatonin. There was no change in reporter gene activity using the TK CAT vector on melatonin treatment (Fig. 4Go, control, 0.41 ± 0.12; with 10 nM melatonin, 0.38 ± 0.05). These data suggest that melatonin mediates the downregulation of GnRH mRNA synthesis through DNA sequences located within the GnRH enhancer.

The 300-bp GnRH neuron-specific enhancer (-1863 to -1571 bp) has been previously shown to contain several protein-binding regions as determined by deoxyribonuclease I footprinting (32). To study basal activity of the 300-bp enhancer region of the GnRH 5' regulatory sequence, block replacement mutants were constructed within the major footprints located in the enhancer (32). Enhancer regions containing these block mutants were placed upstream of the truncated (-173) GnRH promoter and the CAT reporter gene (32). We used these block mutant constructs to determine which region(s) of the GnRH enhancer conferred melatonin-mediated repression of GnRH synthesis by transiently transfecting them into GT1–7 cells for 12–14 h and then treating the cells with melatonin (10 nM) or with vehicle alone for 12 h (Fig. 5Go). Treatment with melatonin repressed CAT reporter gene expression from many of the block mutant constructs, reproducing the repression observed with the intact GnRH enhancer (averaging approximately 70% relative reporter gene expression, compared with the wild-type enhancer). However, transfection with five of the block mutant constructs including -1697/-1689, -1736/-1728, -1746/-1738, -1780/-1772, and -1827/-1819 abolished or significantly decreased repression upon treatment with melatonin (-1697/-1689: 1.13 ± 0.36; -1736/-1728: 0.90 ± 0.06; -1746/-1738: 0.93 ± 0.14; -1780/-1772: 1.12 ± 0.26; -1827/-1819: 0.96 ± 0.06) (P < 0.05) (Fig. 5Go). All of these regions have previously been shown to bind proteins, some of which have already been studied, such as Oct1 and GATA (Fig. 6Go).



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  		Figure 5. Elements within the 300-bp GnRH enhancer confer repression by melatonin. GT1–7 cells were transiently transfected with 15 µg of one of the CAT expression vectors containing block replacement mutations within the enhancer region of the GnRH gene (GnRH Enh) and then treated with melatonin (10 nM). Treatments were for 12 h after previous transfection of the DNA for 12–14 h. Bars represent the relative CAT expression following melatonin treatment, compared with basal expression of each block mutant construct. TK-luciferase was included as an internal control for transfection efficiency. Each value is an average of at least three independent measurements in duplicate or triplicate ± SEM. Values were compared with the level of melatonin repression detected with the intact GnRH Enh construct (*P < 0.05).

 


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  		Figure 6. The neuron-specific GnRH enhancer contains two putative ROR/RZR response elements. A representative diagram of the 5' flanking region from -1863 to -1571 representing the GnRH enhancer is modified from (32 ). Footprinted regions previously found using GT1–7 cell nuclear extracts (32 ) are indicated by boxes. The two AT-rich regions, namely AT-a and AT-b, and the two GATA factor-binding sites, namely GATA-a and GATA-b, are located within footprinted regions of the neuron-specific enhancer. Oct-1 transcription factor binding has been demonstrated within the AT-a and AT-b regions. The GATA-a and GATA-b sites have been demonstrated to bind proteins including GATA-4. The five block mutants corresponding to specific areas within the enhancer that may confer repression of GnRH synthesis by melatonin are indicated (black bars). The block mutant region encompassing the sequences -1736 to -1728 overlaps two direct repeats of putative hexameric consensus transcription factor-binding sequences for the ROR/RZR nuclear receptor family (ROREs) (shown in bold print).

 
Internalization of membrane-bound melatonin receptors in GT1–7 cells
Many GPCRs have been shown to undergo agonist-promoted internalization by clathrin-coated vesicles, thereby attenuating receptor signaling (39). Because the mechanisms of melatonin-mediated downregulation of GnRH synthesis may initially involve melatonin receptor regulation, we therefore examined whether melatonin receptors undergo agonist-stimulated internalization. The mt1 receptor protein levels in vesicular fractions of melatonin-stimulated (10 nM) GT1–7 cells were measured by Western blot analysis (Fig. 7AGo). Over a 1-h period, mt1 receptor protein expression in endosomal vesicular fractions of GT1–7 cells increased in the first 10 min following melatonin treatment (10 nM) and then steadily decreased, suggesting rapid agonist-stimulated internalization of melatonin receptors, followed by receptor degradation and/or recycling.



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  		Figure 7. Effect of melatonin on the subcellular distribution of mt1 receptor in GT1–7 neurons. A, GT1–7 neurons were incubated in the presence of 10 nM melatonin (+) or with vehicle alone (-) for 15, 30, or 60 min. Light vesicular proteins isolated by subcellular fractionation were subjected to SDS-PAGE. Western blot analysis was performed with enhanced chemiluminescence using specific antibodies for the mt1 receptor. As a loading control, Western blot analysis was performed on the same blot using specific antibodies for actin. B, Coimmunoprecipitation of ß-arrestin 1 and mt1 receptor was performed by treating GT1–7 neurons in the presence of 10 nM melatonin (+) or with vehicle alone (-) for 10 min or 30 min, followed by an additional 30-min incubation in the presence of a chemical cross-linker. Cross-linked proteins were immunoprecipitated with monoclonal anti-ß-arrestin 1 antibodies and then subjected to SDS-PAGE. Western blot analysis was performed with enhanced chemiluminescence using specific antibodies for the mt1 receptor. All experiments were performed in triplicate and representative Western blots are shown.

 
Several studies show that GPCR kinase-catalyzed phosphorylation followed by ß-arrestin binding is crucial steps in the internalization of many GPCRs (40). To examine whether ß-arrestins were involved in melatonin receptor regulation, we first treated GT1–7 cells that had been exposed to melatonin (10 nM) for a brief time (10–30 min) with a protein cross-linking reagent. This was followed by Western blot analysis of mt1 receptor proteins bound to immunoprecipitated ß-arrestin 1 proteins (Fig. 7BGo). Results indicated the presence of an immunoreactive band at approximately 100 kDa, present only in melatonin-stimulated cells. Western blot analysis with immunoprecipitated ß-arrestin 2 proteins did not yield any mt1 receptor-specific immunoreactive bands (results not shown). More detailed studies are currently underway to determine the role of mt1 internalization in the cyclical regulation of GnRH gene expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although almost all of the specific melatonin binding in the brain has been attributed to the mt1 receptor, the recent mt1 receptor knockout mouse analysis has indicated that the major activities of melatonin, which include regulation of circadian rhythms and reproductive changes, may not result from the action of the mt1 receptor alone (41). We have found that both membrane receptors are expressed in the GT1–7 neurons. Using the MT2 antibody, we detected two immunoreactive bands by Western blot analysis. We speculate that the 80-kDa band may result from extensive glycosylation of the mature MT2 receptor protein or may be owing to protein dimerization resistant to the reducing agents present in our current method of Western blot analysis. Although still somewhat controversial, melatonin has been identified as a specific ligand for ROR{alpha} and RZRß, members of the ROR/RZR orphan nuclear receptor family (42, 43). The ROR{alpha} gene is ubiquitously expressed, and the RZRß gene has been shown to be abundantly expressed in the brain, retina, pineal gland, and spleen (44). Members of the ROR/RZR family are thought to regulate gene transcription bound either as monomers to the hexameric consensus motif A/GGGTCA or as homodimers to direct repeats or inverted palindromic consensus-binding sites (45). However, one study has demonstrated ROR{alpha} heterodimerization with the thyroid hormone receptor (46). Some researchers have questioned the validity of the melatonin-binding studies to ROR/RZR receptors and further suggest that these receptors may not be the correct or only nuclear receptors for melatonin (44). These findings add complexity to the known molecular signaling mechanisms of melatonin action and may provide a direct link to transcriptional control of melatonin-responsive genes. Our findings of both membrane and nuclear melatonin receptors in GT1–7 cells support the hypothesis that the GnRH neurons of the hypothalamus may be a direct target of melatonin.

We found that melatonin downregulates GnRH gene expression in a cyclical pattern over a 24-h period. When melatonin was replaced daily, which likely mimics the nocturnal secretion of melatonin from the pineal gland, GnRH mRNA levels were downregulated 12 h after melatonin exposure. GnRH biosynthesis then returned to basal levels at 24 h. Notably, when melatonin was replaced in the medium at 12 h, GnRH mRNA levels still returned to basal levels at 24 h, indicating that the cyclical pattern of downregulation was not simply owing to the metabolism or degradation of melatonin itself. In fact, RIA of melatonin in culture medium shows that the half-life of melatonin exceeds 48 h. Similarly, when melatonin was added only at time 0 h, repression of GnRH mRNA levels still occurred at 36 h, although not as robustly as when melatonin was replaced at 24 h.

The molecular mechanisms controlling the cyclical pattern of regulation of GnRH mRNA expression in GT1–7 neurons in the presence of melatonin may be owing to the downstream effects of signaling from the cell membrane to the transcriptional level and/or directly at the level of the GnRH gene. T-Ag biosynthesis in the GT1–7 cells is under the control of the GnRH 5' regulatory region (14). We have found that T-Ag mRNA levels mimic those of GnRH after melatonin exposure, which suggests that melatonin mediates its effect on GnRH mRNA expression at the transcriptional level. Downregulation by melatonin can be further localized to within the well-characterized 300-bp neuron-specific enhancer of the GnRH gene (32). In this study we have defined five specific regions within the GnRH neuron-specific enhancer that are critical for repression of GnRH gene expression by melatonin. Two of these elements, -1780/-1772 and -1697/-1689, lie in regions that have previously been shown to bind the Oct-1 transcription factor (34) and a third element, -1746/-1738, lies in a region that has previously been shown to bind the GATA-4 transcription factor (35). The Oct-1 and GATA-factor binding sites are all essential for basal activity of the GnRH enhancer (34, 35). Through careful sequence analysis, we found that one of the two remaining elements, namely -1736/-1728, lies within a region that overlaps two direct repeats of consensus binding sites for members of the ROR/RZR orphan nuclear receptor family.

It has previously been shown that melatonin can alter the transcription of a number of genes. Melatonin represses c-fos gene expression in both breast cancer cells and neonatal rat pituitary (47, 48). Furthermore, E receptor and progesterone receptor mRNA levels were downregulated, while steady-state mRNA levels of TGF {alpha}, c-myc, and pS2 were upregulated after melatonin treatment in breast cancer cells (47, 49). Melatonin also downregulates expression of the 5-lipoxygenase gene (11) and bone sialoprotein (50) through an ROR/RZR response element. It is therefore of particular interest that the GnRH enhancer contains direct repeats of a consensus transcription factor-binding sequence for the ROR/RZR nuclear receptor family and that we can localize the downregulation of GnRH gene expression to regions that contain these consensus sequences. Further studies are being done to determine whether the ROR/RZR family of nuclear receptors is involved in the regulation of GnRH mRNA levels by melatonin.

Many GPCRs including the ß2-adrenergic receptor undergo agonist-mediated internalization and desensitization (39). In dynamin-dependent endocytosis, ß-arrestin proteins have a central role in directing GPCRs by acting as adapter-like molecules for receptor trafficking (51). In addition to the melatonin-induced decrease in expression of its own receptor mRNA, our results demonstrate for the first time that mt1 receptors also undergo agonist-stimulated internalization through a ß-arrestin 1-mediated mechanism, which is likely followed by degradation or recycling. The melatonin-mediated downregulation of GnRH mRNA synthesis at 12 and 36 h, and the return of GnRH mRNA expression to basal levels at 24 h, may rely on new receptor synthesis and/or internalization/recycling to the cell surface to mediate melatonin responsiveness. These findings do not exclude the added complexity of direct transcriptional regulation of GnRH synthesis by nuclear transcription factors and changes in the stability of GnRH transcripts.

The apparent cyclical changes in GnRH mRNA levels may reflect the oscillatory nature of the GnRH neuron itself or the effect of a daily exposure to melatonin. Alternatively, melatonin receptor regulation may also be involved in the responsiveness of GT1–7 cells to melatonin. Studies on melatonin-mediated regulation of mt1 receptor mRNA expression in ovine pars tuberalis have demonstrated that melatonin can act via a cAMP-independent signal transduction pathway to repress transcription of its own receptor (52). Furthermore, mt1 receptor mRNA synthesis has been shown to follow a diurnal rhythm in the rat suprachiasmatic nucleus (53). Whether the change in mt1 and MT2 transcription affects GnRH gene expression is yet to be determined. It is also of interest to note that in female rats, GnRH release follows a 24-h periodicity (54) and that diurnal rhythms of GnRH gene expression have also been shown to occur in both cycling female rats and adult male rats (55). We have now demonstrated cyclicity in GnRH gene expression in GT1–7 cells following melatonin treatment and the mechanisms by which melatonin exerts these effects on the GnRH neuron are under investigation.

The direct action of melatonin on GT1–7 neurons presents evidence that the hypothalamus may represent one of the main sites of melatonin-mediated antigonadal activity in vivo. Nonetheless, the GT1–7 neurons are a transformed, clonal population of cells outside of their natural environment; thus, these results should be validated in situ. The effects of melatonin on the GT1–7 cells appear to occur by a specific receptor-mediated event, either through the membrane-bound G protein-coupled melatonin receptors or ROR/RZR nuclear receptors. These results do not challenge previous findings that demonstrate that melatonin acts on specific cell populations within the pituitary (56) or through interneurons that synapse on the GnRH neurons (13) but mainly add to the growing number of sites that may be controlled by melatonin. Although these effects are yet to be studied in situ, our data corroborate the hypothesis that the hypothalamus is a likely target of melatonin action and that melatonin may mediate its repression of reproductive function directly at the level of the GnRH neuron.


   Acknowledgments
 
We thank Dr. Pamela L. Mellon, University of California, San Diego, for generously providing the GT1-7 cells and plasmid constructs. Thanks to Dr. Bernardo Yusta for advice and critical reading of the manuscript and to Nina Umar for technical assistance.


   Footnotes
 
This work was supported by a Natural Science and Engineering Research Council Operating Grant (to D.D.B.), a Clarke Foundation Award (to G.M.B.), and an Ontario Graduate Scholarship in Science and Technology (to D.R.). D.D.B. is a Canadian Institutes of Health Research Scholar and a Canada Foundation for Innovation Researcher.

Abbreviations: CAT, Chloramphenicol acetyltransferase; CRE, cAMP response element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPCR, G protein-coupled receptors; ROR, retinoic acid receptor-related orphan receptor; RT, reverse transcriptase; RZR, retinoid Z receptor; T-Ag, T-antigen; TK, thymidine kinase.

Received April 18, 2001.

Accepted for publication July 9, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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