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J. Biol. Chem., Vol. 277, Issue 1, 251-258, January 4, 2002

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Melatonin Receptor Activation Regulates GnRH Gene Expression and Secretion in GT1-7 GnRH Neurons

SIGNAL TRANSDUCTION MECHANISMS^*

Deboleena Roy and Denise D. Belsham^§¶

From the  Institute for Medical Sciences and the ^§ Department of Physiology, University of Toronto, and the ^¶ Division of Cellular and Molecular Biology, University Health Network, Toronto, Ontario M5S 1A8, Canada

Received for publication, September 14, 2001, and in revised form, October 25, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Melatonin plays a significant role in the control of the hypothalamic-pituitary-gonadal axis. Using the GT1-7 cell line, an in vitro model of GnRH-secreting neurons of the hypothalamus, we examined the potential signal transduction pathways activated by melatonin directly at the level of the GT1-7 neuron. We found that melatonin inhibits forskolin-stimulated adenosine 3'-, 5'-cyclic monophosphate accumulation in GT1-7 cells through an inhibitory G protein. Melatonin induced protein kinase C activity by 1.65-fold over basal levels, increased the phosphorylation of extracellular signal-regulated kinase 1 and 2 proteins, and activated c-fos and junB mRNA expression in GT1-7 cells. Using the protein kinase A inhibitor H-89, the protein kinase C inhibitor bisindolylmaleimide, and the mitogen-activated protein kinase kinase inhibitor PD98059, we found that the melatonin-mediated cyclical regulation of GnRH mRNA expression may involve the protein kinase C and the extracellular signal-regulated kinase 1 and 2 pathways, but not the protein kinase A pathway. We found that melatonin suppresses GnRH secretion by ~45% in the GT1-7 neurons. However, in the presence of the inhibitors H-89, bisindolylmaleimide, and PD98059 melatonin was unable to suppress GnRH secretion. These results provide insights into the potential signal transduction mechanisms involved in the control of GnRH gene expression and secretion by melatonin.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The pineal hormone melatonin plays an important role in the regulation of reproduction as well as circadian rhythm control in mammals. Melatonin is thought to mediate its circadian and reproductive effects through specific high affinity receptors (1). The mammalian melatonin receptors mt1 and MT2 have been cloned and characterized to be members of the G protein-coupled receptor superfamily (2, 3). Melatonin has been shown to regulate cell function via intracellular second messengers such as cAMP, Ca²⁺, cGMP, diacylglycerol, protein kinase C (PKC),¹ and arachidonic acid; however, the signaling pathways activated by melatonin appear to be differentially regulated and tissue-specific (reviewed in Ref. 4).

A number of studies have investigated the signal transduction mechanisms in tissues associated with the hypothalamic-pituitary-gonadal axis. At the level of the pituitary, melatonin inhibits adenylate cyclase through both PTX (pertussis toxin)-sensitive and PTX-insensitive G-proteins in ovine pars tuberalis cells (5, 6). Melatonin also inhibits gonadotropin-releasing hormone (GnRH)-induced increases in cAMP, diacylglycerol, [Ca²⁺]_i, and c-Fos in neonatal rat gonadotrophs (7, 8). At the level of the gonads, melatonin modulates androgen production in rat Leydig cells though a PTX-sensitive mechanism and also through a decrease in GnRH-induced [Ca²⁺]_i release (9-11). In prostate epithelial cells, melatonin suppresses cGMP levels through a process that involves the activation of PKC (12). Melatonin also reduces cAMP production by preovulatory follicles in the hamster ovary, but stimulates cGMP in porcine granulosa cells (13, 14).

Some evidence suggests that melatonin regulates reproduction by exerting inhibitory effects on the reproductive axis at the level of the hypothalamus (15-17). As such, melatonin uptake and binding have been demonstrated in rat and hamster hypothalamus (1, 18, 19). Furthermore, mt1 and MT2 mRNA expression have been detected in the hypothalamic suprachiasmatic nucleus (SCN) in several mammals including humans and rats (2, 3, 20). At the level of the hypothalamus, melatonin has been shown to alter SCN cellular function via a PTX-sensitive G protein pathway that activates PKC and reduces norepinephrine-induced activation of prostaglandin E₂ and cAMP production in the medial basal hypothalamus (21, 22). The signaling mechanisms directly involved in melatonin-mediated regulation of GnRH, a central component of the hypothalamic-pituitary-gonadal axis, have not yet been determined.

In an attempt to produce a suitable model to study the GnRH gene, a targeted tumorigenesis approach whereby expression of the SV-40 T-Antigen oncogene, driven by the rat GnRH 5'-regulatory region, was used to develop a murine immortal cell line of GnRH-secreting neurons (GT1-7 cells) (23). Using the GT1-7 GnRH-secreting cell line, we recently provided the first evidence that melatonin may act directly at the level of the GnRH neuron to regulate reproductive function. We have shown that these cells express both mt1 and MT2 G protein-coupled receptors and that melatonin down-regulates GnRH gene expression in a 24-h cyclical pattern (24). In the present study we have demonstrated that melatonin inhibits forskolin-induced cAMP accumulation though a PTX-sensitive mechanism in GT1-7 cells. In addition, we have provided evidence that in the short term, melatonin increases PKC activity in GT1-7 cells. We demonstrated that melatonin activates the mitogen-activated protein kinase (MAPK) pathway by increasing the phosphorylation of extracellular signal-regulated kinase (ERK 1/2) proteins in GT1-7 cells. Melatonin also increased the mRNA expression of the immediate early genes c-fos and junB in GT1-7 cells. In accordance with studies in the hypothalamus, we observed a melatonin-mediated suppression of GnRH secretion in GT1-7 cells within 1 h of treatment. Furthermore, using inhibitors of the protein kinase A (PKA), PKC, and ERK 1/2 pathways we have examined some of the signal transduction pathways that may be involved in melatonin-mediated regulation of GnRH gene expression and secretion in GT1-7 cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Reagents-- GT1-7 cells were grown in monolayer in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (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 as previously described (23). Melatonin, dimethyl sulfoxide (Me₂SO), aprotinin, leupeptin, pepstatin, EDTA, PTX, isobutylmethylxanthine, PKA inhibitor N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89) and mitogen-activated protein kinase kinase (MEK) inhibitor PD 098,059 were obtained from Sigma. 12-0-tetradecanoylphorbol-13-acetate (TPA) and PKC inhibitor bisindolylmaleimide (BIS) were obtained from Calbiochem (San Diego, CA). GnRH RIA kit was obtained from Peninsula Laboratories (San Carlos, CA). cAMP kit was obtained from Biomedical Technologies Inc., (Stoughton, MA). Biotrak PKC Enzyme assay kit was obtained from Amersham Biosciences, Inc. Pierce BCA Protein Assay Reagent kit was obtained from Pierce Ltd. ERK 1/2, phospho-ERK, JNK, phospho-JNK, p38 and phosph-p38 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Data were analyzed using the statistical program package SPSS 6.1.4 for Power Macintosh (University of Toronto site license).

cAMP Studies-- GT1-7 cells were split into 24-well plates. In the case of the dose-response curve, medium was replaced with 0.5 ml of fresh DMEM (without FBS) with melatonin (1 nM, 10 nM, 100 nM, or 10 µM) or vehicle alone in the absence or presence of forskolin (100 nM). In the experiment with the melatonin receptor antagonist luzindole, cells were treated with melatonin (10 µM), luzindole (10 µM), or vehicle alone in the absence or presence of forskolin (100 nM). In the experiment with PTX, medium was replaced with 0.5 ml of fresh DMEM (without FBS) with either 200 ng/ml PTX or vehicle alone (14 nM Me₂SO). Following a 6-h PTX treatment, cells were washed twice with serum-free DMEM. All drugs were diluted in DMEM containing IBMX (100 µM). Cells were treated with 10 µM melatonin, and ± 100 nM forskolin, or with vehicle alone (Me₂SO), 20 nM). After a 15-min incubation at 37 °C, 1 ml of ice-cold ethanol was added to each well. Cells were scraped from the plate and kept at 20 °C until the amounts of intracellular cAMP were determined in triplicate by RIA (Biotechnologies Inc., Stoughton, MA) according to the manufacturer's instructions.

PKC Enzyme Assay-- PKC activity was analyzed as previously described (25). Briefly, GT1-7 cells were seeded into 35-mm² Petri dishes as described above and then serum starved (0.1% FBS) for 12 h before stimulation. Cells were then stimulated by addition of serum (10% FBS), TPA (100 nM), and/or melatonin (10 nM). After 30 min stimulation, cells were rinsed twice with 2 ml of ice-cold PBS, then lysed with 200 µl of ice-cold lysis buffer (50 mM Tris/HCl, pH 7.5, 10 mM EGTA, 5 mM EDTA, 20 mM NaF, 0.2 mM Na₃VO₄, 20 mM -glycerophosphate, 10 mM benzamidine, 0.3% (v/v) -mercaptoethanol, 50 µg/ml phenylmethylsulfonyl fluoride, and 0.5 µg/ml leupeptin) and harvested using a rubber scraper. After transfer to microcentrifuge tubes, the lysates were passed seven times through a 25-gauge needle and maintained on ice. PKC activity was determined using a Biotrak PKC assay kit following the manufacturer's protocol (Amersham Biosciences, Inc.). Assay tubes contained 25 µl of component mixture, 25 µl of lysate, and 0.2 µCi [³²P]ATP (450,000 cpm ± 20,000 cpm), and reactions were performed for 15 min.

Analysis of MAPK Activity-- GT1-7 cells were grown in 100-mm² tissue culture plates as described above. Cells were serum-starved (0.1% FBS) for 2 h prior to treatment with melatonin (10 nM) or vehicle alone. Cells were stimulated with melatonin (10 nM) for 10 or 30 min and then washed in cold 1× PBS supplemented with proteinase inhibitors (1 µg/ml aprotinin, pepstatin, and leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA). Total protein from GT1-7 cells was then isolated. Protein concentration was measured using the Pierce BCA Protein Assay Reagent (Pierce Ltd.). Samples were stored at 20 °C until the time of assay. Total protein (50 µg) was resolved on a 12.5% SDS-polyacrylamide gel (26) and transferred to Immobilon-P membranes (Millipore Corporation, Bedford, MA). Membranes were washed briefly in PBS and then incubated in PBST (PBS, pH 7.4, 0.2% Tween 20, 5% powdered skim milk) for 16 h with 1 µg/ml goat polyclonal ERK 1/2 (K-23), mouse monoclonal phospho-ERK (E-4), rabbit monoclonal JNK, mouse monoclonal phospho-JNK, rabbit polyclonal p38, and mouse monoclonal phospho-p38 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoreactive bands were visualized with horseradish peroxidase-labeled secondary antibody antisera at 1:10,000 dilution and enhanced chemiluminescence (Amersham Biosciences, Inc.), as described by the manufacturer.

Analysis of Immediate Early Gene Expression-- GTI-7 cells were grown in 100-mm² tissue culture plates as described above. Cells were serum-starved for 12 h and then treated with melatonin (10 nM) or vehicle alone for 30 min, 60 min, or 120 min. Total cellular RNA was isolated by the guanidinium thiocyanate phenol chloroform extraction method (27). Ten micrograms of total RNA were electrophoresed in 1% formaldehyde agarose gels and transferred to Genescreen membranes (Dupont-New England Nuclear) by capillary blotting (28). The filters were probed with rat c-fos cDNA (29) or junB cDNA (30). Membranes were prehybridized for 2-6 h and hybridized for 16 h in hybridization buffer (1% w/v bovine serum albumin, 1 mM EDTA, 0.5 M Na₂HPO₄, 5% w/v SDS, 25% formamide) at 50 °C. The cDNA probes were labeled using random hexamers and [³²P]dATP (6000 Ci/mmol, PerkinElmer Life Sciences) incorporated with the Klenow fragment of DNA polymerase I (31). Blots were washed at high stringency (0.5 × SSC, 0.1% SDS; 50 °C) and exposed to Fuji film at 70 °C with intensifying screens for 4-24 h. Autoradiographs were scanned with a Hewlett Packard ScanJet 3p Scanner, and c-fos and junB mRNA signals were quantified by densitometry using the NIH Image program. Blots were stripped and then probed with -actin cDNA (32) as a loading control.

GnRH Transcription Studies-- GT1-7 cells were grown in 100-mm² tissue culture plates as described above. Cells were pretreated with PKA, PKC, or MAPK inhibitor (10 µM H-89, 20 nM BIS, and 20 µM PD98050, respectively) for 2 h prior to treatment with melatonin (10 nM), inhibitor, or vehicle alone (0, 12, 24 h). Total cellular RNA was isolated by the guanidinium thiocyanate phenol chloroform extraction method (27). Northern blot analysis was performed as described above. The filters were probed with rat GnRH cDNA (33) and human -actin cDNA (32) as a loading control. Autoradiographs were scanned with a Hewlett Packard ScanJet 3p Scanner and GnRH, and actin mRNA signals were quantified by densitometry using the NIH Image program.

GnRH Secretion Studies-- GT1-7 cells were split into 24-well plates. Medium was replaced 1 h prior to treatment with 0.5 ml of fresh DMEM (0.1% FBS), with either 1 nM melatonin, 1 nM melatonin + 10 µM luzindole, 10 µM luzindole, 10 µM forskolin (positive control) or vehicle alone (Me₂SO, 14 nM) after washing with PBS. In the case of inhibitor studies, cells were pretreated with PKA inhibitor, PKC inhibitor, or MAPK inhibitor (10 µM H-89, 20 nM BIS, and 20 µM PD98050, respectively) for 1 h prior to treatment with melatonin (10 nM). Medium was collected at 1 h for all treatments except for the forskolin-positive control, which was collected after 2 h. Protein content per well was assayed using the Pierce BCA Protein Assay Reagent (Pierce). Protein content was consistently equivalent between wells (10% variation). GnRH content in 200-µl aliquots of the cell culture medium samples was assayed in triplicate with a GnRH RIA kit (Peninsula Laboratories, Inc., Belmont, CA) according to the manufacturer's instructions.

Statistical Analysis-- For assessment of statistical significance, data were analyzed using analysis of variance. Comparisons between individual pairs were determined using Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Melatonin Receptors in GT1-7 Cells Are Coupled to G_i/o Proteins-- In the vast majority of cell types examined, previous studies have shown that 10 µM melatonin inhibits forskolin-induced cAMP accumulation and furthermore, the effects of melatonin are inhibited by PTX pretreatment (4). As such, most studies have demonstrated melatonin receptor coupling to G proteins belonging to the G_i/o family (reviewed in Ref. 4). In the GT1-7 cells, we used radioimmunoassays to determine the effect of melatonin on cAMP accumulation. Our results indicated that melatonin did not alter basal 3'-, 5'-cyclic monophosphate (cAMP) levels (vehicle, 1.0 ± 0; 1 nM melatonin, 1.08 ± 0.17; 10 nM melatonin, 0.99 ± 0.14; 100 nM melatonin, 0.8 ± 0.04; 10 µM melatonin, 0.8 ± 0.14), but caused an inhibition of forskolin-stimulated (100 nM) accumulation of cAMP in a dose-dependent manner (vehicle, 6.66 ± 1.7; 1 nM melatonin, 6.4 ± 0.82; 10 nM melatonin, 6.2 ± 0.8; 100 nM melatonin, 4.0 ± 1.1; 10 µM melatonin, 3.6 ± 0.6 (p < 0.005, Student's t test) (Fig. 1A). In the presence of luzindole (10 µM), a specific melatonin receptor antagonist (34), the effect of melatonin on forskolin-induced cAMP accumulation was not observed (100 nM forskolin, 7.03 ± 0.26; 100 nM forskolin + 10 µM melatonin, 3.8 ± 1.04; 100 nM forskolin + 10 µM melatonin + 10 µM luzindole, 6.68 ± 0.28). This result suggested that inhibition of cAMP by melatonin was specifically melatonin receptor-mediated (Fig. 1B). Furthermore, PTX pretreatment (100 ng/ml) for 6 h completely abolished the ability of melatonin to inhibit the forskolin-induced cAMP accumulation (10 µM forskolin, 25.01 ± 2.66; 10 µM forskolin + 10 µM melatonin, 19.43 ± 0.6; 10 µM forskolin + 10 µM melatonin + 100 ng/ml PTX, 24.55 ± 4.95) (Fig. 1C). This indicates that the membrane-bound melatonin receptors are coupled to G_i proteins and that melatonin inhibits adenylyl cyclase through a PTX-sensitive mechanism in the GT1-7 cells.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Melatonin inhibits forskolin-induced cAMP accumulation in GT1-7 cells. A, GT1-7 cells were treated for 15 min with 1 nM, 10 nM, 100 nM, 10 µM melatonin (Mel) or vehicle alone, in the presence or absence of forskolin (100 nM). Cell culture medium was then collected and assayed for cAMP immunoreactivity by radioimmunoassay. Melatonin alone did not alter basal cAMP accumulation however 100 nM and 10 µM melatonin inhibited forskolin (100 nM)-induced cAMP levels. B, GT1-7 cells were treated for 15 min with 10 µM melatonin (Mel), 10 µM luzindole (Luz), 100 nM forskolin (Forsk), and/or vehicle alone. In the GT1-7 cells, melatonin did not alter basal cAMP levels, but did cause an inhibition of forskolin-induced cAMP accumulation. Luzindole blocked the melatonin-mediated inhibition of forskolin-induced cAMP accumulation. C, GT1-7 cells were treated for 15 min with 10 µM melatonin (Mel), 10 µM forskolin (Forsk), (100 ng/ml) pertussis toxin (PTX), or vehicle alone. PTX pretreatment (100 ng/ml) for 6 h completely abolished the ability of melatonin to inhibit the forskolin-induced cAMP accumulation. Results shown are mean ± S.E. (n = three independent experiments each in triplicate; **, p < 0.005).

Melatonin Increases PKC Activity in GT1-7 Cells-- Melatonin has previously been shown to alter diacylglycerol levels and PKC activity, both of which are key elements in the phospholipase C (PLC) pathway (7). Melatonin-induced phase shifts of the circadian pacemaker in the SCN have previously been shown to be mediated by the activation of PKC (21). This study also demonstrated that in SCN tissue, melatonin increased PKC activity by nearly 2-fold in a rapid and transient manner (21). In our study, we examined the effect of melatonin on PKC activity in GT1-7 cells using an assay based upon the PKC catalyzed transfer of the -phosphate group of adenosine-5'-triphosphate to a peptide that is specific for PKC (Amersham Biosciences, Inc.). Cells were serum-starved for 12 h to remove any stimulants from the media and then treated with vehicle alone, serum (10% FBS) (positive control), an activator of PKC (TPA, 100 nM) (positive control), serum + melatonin (10 nM), TPA + melatonin (10 nM), or melatonin alone. Measurement of PKC activity in cell lysates prepared from GT1-7 cells stimulated with serum (10% FBS) and TPA (100 nM) showed enhanced PKC activity within 30 min (vehicle, 1.0 ± 0.08; serum 1.38 ± 0.12; TPA, 1.71 ± 0.12) (Fig. 2). Activation of PKC activity by melatonin (10 nM, 30 min) was modest but significant (vehicle, 1.0 ± 0; 10 nM melatonin, 1.64 ± 0.10) (p < 0.005, Student's t test) (Fig. 2). Serum or TPA-induced PKC activity did not increase further in the presence of melatonin (serum + melatonin, 1.65 ± 0.24; TPA + melatonin, 1.64 ± 0.20).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Melatonin increases PKC activity in GT1-7 cells. GT1-7 neurons were serum-starved (0.1% FBS) for 12 h before stimulation with melatonin (10 nM), serum (10% FBS) (positive control), TPA (100 nM, positive control), melatonin + serum, melatonin + TPA, or vehicle alone (Control, C). Following a 30-min stimulation with serum, TPA, and melatonin PKC activity was measured using a PKC enzyme assay kit. Serum replacement induced a 1.38-fold increase in PKC activity. The PKC activator TPA (positive control) induced a 1.71-fold increase in PKC activity, whereas melatonin induced a 1.64-fold increase in PKC activity in GT1-7 cells. Results shown are mean ± S.E. (n = three independent experiments each in triplicate; **, p < 0.005).

Melatonin Activates the MAPK Signaling Pathway in GT1-7 Cells-- There have been few studies investigating the effect of melatonin on the MAPK signaling pathway. In the ovine pars tuberalis, the MAPK signaling pathway does not contribute to the melatonin-mediated inhibition of c-fos mRNA expression (25), whereas melatonin was consistently found to be a highly potent inhibitor of the activation of MAPK induced by forskolin but not by IGF-1 (35). In estrogen receptor-positive MCF-7 breast cancer cells, the modulation of estrogen receptor transactivation by melatonin has been associated with changes in MAPK activity (36). We used Western blot analysis to determine whether melatonin induced the activation of the MAPK pathways in GT1-7 cells by observing the phosphorylation of the ERK 1/2, JNK/SAPK, and p38 proteins. Cells were first serum-starved for 2 h to remove any stimulants from the media and then treated with either vehicle alone or melatonin (10 nM) (10, 30 min). Western blot analysis was performed on total protein using an ERK 1 (p44)- and ERK 2 (p42)-specific polyclonal antibody (K-23) or a specific monoclonal antibody (E-4) reactive with tyrosine 204-phosphorylated (phospho)-ERK 1 and phospho-ERK 2. Similar immunoreactive bands of 44 kDa and 42 kDa were observed in vehicle and melatonin-treated GT1-7 total cell extracts over the entire time course (Fig. 3A). However melatonin treatment alone rapidly increased the amount of phospho-ERK 1 and phospho-ERK 2 immunoreactivity (~2-fold following 10 min of stimulation and 4-fold following 30 min of stimulation), suggesting that melatonin induces the activation of the ERK 1/2 signaling pathway in the GT1-7 cells (Fig. 3B). Vehicle alone (Me₂SO) did not change the amount of phospho-ERK 1/2 immunoreactivity over time. Western blot analysis was also performed on GT1-7 total protein using JNK, phospho-JNK, p38 and phospho-p38-specific antibodies. Similar immunoreactive bands of 54 kDa, 46 kDa, and 38 kDa (JNK2, JNK1, and p38, respectively) were observed in vehicle and melatonin (10 nM) (10, 30, 45 min)-treated GT1-7 total cell extracts. However, we were unable to detect any effect of melatonin on the phosphorylation of JNK and p38 proteins in the GT1-7 cells (data not shown).

View larger version (54K): [in this window] [in a new window]   Fig. 3.   Melatonin induces phosphorylation of ERK 1/2 proteins in GT1-7 cells. GT1-7 neurons were serum-starved (0.1% FBS) for 2 h prior to treatment with melatonin (10 nM) (+) or vehicle alone (). Cell lysates were resolved on 12.5% SDS-PAGE before transfer to nitrocellulose and immunoblotting with antisera that specifically recognized ERK 1/2 (A) or phosphorylated phospho-ERK 1/2 (B). The antisera used in each case recognized both the p44 (ERK 1) and p42 (ERK 2) proteins. A representative Western blot is shown (n = 3).

Melatonin Induces Immediate Early Gene Expression in GT1-7 Cells-- The effects of melatonin on immediate early gene expression such as c-fos and junB have also been shown to be tissue-specific (4). In the neonatal rat pituitary and ovine pars tuberalis, melatonin down-regulates c-fos mRNA and protein expression (8, 37). In the rat SCN, melatonin either induces or has no effect on c-fos mRNA expression (38, 39). To determine whether melatonin induced immediate early gene expression in GT1-7 cells, we used Northern blot analysis to examine c-fos and junB mRNA expression. Cells were serum-starved for 12 h to remove any stimulants in the medium and then treated with vehicle or with melatonin (10 nM) (30, 60, and 120 min). Using 10 µg of total RNA, c-fos and junB mRNA were detected in GT1-7 cells. In the presence of melatonin, c-fos mRNA expression increased significantly (p < 0.005, Student's t test) within 30 min but returned to basal levels by 60 min (10 nM melatonin; 30 min, 3.5 ± 0.8; 60 min, 1.2 ± 0.2; 120 min, 1.2 ± 0.1) (Fig. 4A). In the presence of melatonin, junB mRNA expression increased significantly (p < 0.05, Student's t test) within 30 min but also returned to basal levels by 60 min (10 nM melatonin; 30 min, 1.65 ± 0.3; 60 min, 0.9 ± 0.2) (Fig. 4B). -actin mRNA levels, a loading control, were not affected by treatment with melatonin (data not shown). Vehicle alone (Me₂SO) did not change the amount of c-fos or junB mRNA expression over time.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Melatonin induces immediate early gene expression in GT1-7 cells. GT1-7 cells were serum-starved for 12 h prior to treatment with 10 nM melatonin (+) or vehicle alone () for 30 min, 60 min (c-fos and junB), and 120 min (c-fos). At the indicated time points, total RNA was extracted and 10 µg of each sample were subjected to Northern blot analysis. Blots were probed first with rat c-fos cDNA (A) or rat junB cDNA (B), stripped, and reprobed with -actin (loading control). c-fos and junB mRNA levels were quantified by scanning densitometry of autoradiographs and normalized to loading control. Data shown are relative to c-fos or junB mRNA levels at 30 min (vehicle treated control) and are expressed as mean ± S.E. (n = three independent experiments; **, p < 0.005; *, p < 0.05).

Melatonin-mediated Regulation of GnRH Gene Expression in GT1-7 Cells Involves the PKC and MAPK Pathways but Not the PKA Pathway-- Although it has been shown previously that melatonin can alter the transcription of a number of genes including its own receptor (40-42), little is known of the signaling mechanisms involved in the regulation of gene expression originating through activation of the membrane melatonin receptors. In ovine pars tuberalis cells, it has been shown that melatonin does not act through PKC activation to reduce mt1 mRNA expression and furthermore it has been suggested that other as yet undefined pathways must play an important role in the regulation of mt1 receptor by melatonin (6). Using GT1-7 cells, we have previously demonstrated that melatonin (1 nM) down-regulates GnRH gene expression in a 24-h cyclical pattern (24). We have shown that following a 12-h melatonin treatment, GnRH mRNA levels in GT1-7 cells are down-regulated by ~40% compared with basal levels. Furthermore, GnRH mRNA expression returned to basal levels at 24 h (24).

To begin to understand the signaling pathways involved in melatonin-mediated regulation of GnRH gene expression in GT1-7 cells, we pretreated the cells with PKA, PKC, or MEK inhibitors for 2 h prior to treatment with melatonin and then measured GnRH gene expression by Northern blot analysis (Fig. 5). Inhibition of PKA activity by H-89 had no effect on basal GnRH gene expression (10 µM H-89, 12 h, 1.38 ± 0.45; 24 h, 0.98 ± 0.11), or melatonin-mediated regulation of GnRH gene expression (10 µM H-89, 10 nM melatonin, 12 h, 0.59 ± 0.08; 24 h, 1.23 ± 0.35). Inhibition of PKC activity by BIS decreased basal GnRH gene expression following 12 h treatment but returned to basal levels by 24 h (20 nM BIS, 12 h, 0.45 ± 0.03; 24 h, 1.11 ± 0.17). The PKC inhibitor had no effect on melatonin-mediated down-regulation of GnRH gene expression (20 nM BIS, 10 nM melatonin, 12 h, 0.25 ± 0.09; 24 h, 1.15 ± 0.16). Inhibition of MEK activity by PD98059 had no effect on basal GnRH gene expression (20 µM PD98059, 12 h, 0.57 ± 0.18; 24 h, 1.38 ± 0.11). In the presence of the MEK inhibitor, however, melatonin-mediated down-regulation of GnRH gene expression continued into the 24-h time point (20 µM PD98059, 10 nM melatonin, 12 h, 0.34 ± 0.08; 24 h, 0.39 ± 0.16) (p < 0.005, Student's t test), indicating that a component of the ERK 1/2 pathway is required for release of repression by melatonin.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Melatonin-mediated regulation of GnRH gene expression in GT1-7 cells involves the PKC and MAPK pathways but not the PKA pathway. GT1-7 cells were pretreated (2 h) with the PKA inhibitor (10 µM H-89), PKC inhibitor (20 nM BIS), MEK inhibitor (20 µM PD98050), or with vehicle alone (Control). Cells were then treated (0 h) with 10 nM melatonin (+) or vehicle alone () in the presence of inhibitor for 12 or 24 h. At the indicated time points, total RNA was extracted and 10 µg of each sample were subjected to Northern blot analysis. Blots were simultaneously probed with rat GnRH cDNA and -actin (loading control). GnRH and -actin mRNA levels were quantified by scanning densitometry of autoradiographs and normalized to loading control. Data shown are relative to GnRH mRNA levels at 0 h (vehicle alone treatment) and are expressed as mean ± S.E. (n = three independent experiments; **, p < 0.005).

Melatonin Suppresses GnRH Secretion in GT1-7 Cells-- The effect of melatonin on GnRH secretion in GT1-7 neurons was tested following a short-term exposure to melatonin. GnRH secretion has previously been shown to be affected by a number of neuromodulators within a 2-h time frame (43). Most hormones and neurotransmitters studied have been shown to increase GnRH secretion in GT1-7 cells (reviewed in Ref. 43). Only prolactin and glucocorticoids have been shown to decrease GnRH secretion in GT1-7 cells (44, 45). By radioimmunoassays, we determined that GnRH-peptide levels in the cell culture media were reduced to ~45% of basal secretion levels following 1 nM melatonin treatment over a 1-h period (1 nM melatonin, 1 h, 0.55 ± 0.14) (p < 0.005, Student's t test) (Fig. 6A). In contrast, treatment with forskolin increased GnRH secretion 2-fold (100 µM forskolin, 2 h, 2.3 ± 0.17) (p < 0.005), as has been previously reported (43) (Fig. 6A, inset). Suppression of GnRH secretion was blocked by luzindole, a specific antagonist of membrane-bound melatonin receptors (34), whereas luzindole alone did not affect basal GnRH secretion (1 nM melatonin + 10 µM luzindole, 1 h, 0.88 ± 0.06; 10 µM luzindole, 1 h, 0.80 ± 0.07) (Fig. 6A). These results indicate that in the short-term melatonin can suppress GnRH release in GT1-7 neurons.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Melatonin suppresses GnRH secretion from GT1-7 cells. A, GT1-7 neurons were treated for 1 h with 1 nM melatonin, 1 nM melatonin + 10 µM luzindole, 10 µM luzindole, or vehicle alone (Control). Cells were treated with forskolin (10 µM, 2 h) (positive control, inset). B, GT1-7 cells were pretreated (1 h) with the PKA inhibitor (10 µM H-89), PKC inhibitor (20 nM BIS), MEK inhibitor (20 µM PD98050), or with vehicle alone prior to treatment with melatonin (10 nM) (+) or vehicle alone () for 1 h. Cells were treated with forskolin (10 µM, 2 h) (positive control). Cell culture medium was then collected and assayed for GnRH-like immunoreactivity (GnRH-like IR) by radioimmunoassay. Results shown are mean ± S.E. (n = three independent experiments each in triplicate; **, p < 0.005).

To begin to understand the signaling pathways involved in the melatonin-mediated suppression of GnRH secretion in GT1-7 cells, we pretreated the cells with PKA, PKC, and MEK inhibitors (10 µM H-89, 20 nM BIS, and 20 µM PD98050, respectively) before treatment with melatonin and then measured GnRH-peptide levels by radioimmunoassay. Cells were also treated with forskolin (10 µM) as a positive control. Treatment with H-89 alone, a specific PKA inhibitor, has previously been demonstrated to decrease GnRH mRNA levels and increase GnRH secretion in GT1-7 cells (46, 47). To date, there have been no reports of the effects of PKC and MEK inhibitors on GnRH secretion in GT1-7 cells. In our study, treatment with H-89, BIS, or PD98059 alone resulted in a significant increase in basal GnRH secretion in GT1-7 cells (10 µM H-89, 2 h, 1.55 ± 00.02; 20 nM BIS, 2 h, 1.9 ± 0.26; 20 µM PD98059, 1.34 ± 0.19) (p < 0.005, Student's t test) (Fig. 6B). Melatonin was unable to suppress GnRH secretion in the presence of any of the inhibitors (Fig. 6B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Melatonin exerts its biological effects primarily through pharmacologically specific, high affinity receptors that belong to the G protein-coupled receptor superfamily. G proteins are heterotrimeric, consisting of , , and  subunits (48). Upon activation, the heterotrimer dissociates into  and dimers. Both G and G dimers interact with effector molecules to transmit a signal. G proteins can be divided into four subtypes including the G_i/o proteins whose members are typically PTX-sensitive and inhibit adenylyl cyclase, G_s proteins that stimulate adenylyl cyclase and phosphodiesterases, G_q/11 that stimulate phospholipase C, and G_12/13 proteins that activate small G proteins (reviewed in Ref. 49). Melatonin receptor studies have mainly focused on mt1 signaling through the adenylyl cyclase pathway. In several tissues, activated mt1 receptors have been shown to mediate the inhibition of cAMP accumulation via PTX-sensitive receptor coupling to inhibitory G_i/o proteins (reviewed in Ref. 4). However, mt1 receptors have also been shown to increase prostaglandin F (2)-promoted stimulation of PLC and to modulate PKC and phospholipase A₂ via G protein subunits released during G_i/o protein activation (21, 50). Interestingly, two subtypes of the G_i/o proteins, G_i2 and G_i3, as well as G_q/11 proteins have all been shown to couple to the mt1 receptor in an agonist-dependent and guanine nucleotide-sensitive manner and furthermore, coupling to PTX-insensitive G_q/11 proteins has been shown to increase [Ca²⁺]_i via activation of PLC (51). To date, studies of MT2 signal transduction mechanisms have mostly demonstrated coupling to PTX-sensitive G_i proteins (3, 52). There is very little known about subtype-specific differences in melatonin receptor signal transduction. However, in a recent study of human embryonic kidney cells expressing both melatonin receptors, MT2 receptors, but not mt1 receptors were shown to modulate cGMP levels (53). In the GT1-7 cells, melatonin did not affect the basal levels of cAMP but did however decrease forskolin-induced cAMP accumulation through a PTX-sensitive mechanism, suggesting receptor coupling to G_i proteins. Similar to results previously shown in the rat SCN, our study also demonstrates the activation of PKC by melatonin, suggesting the coupling of melatonin receptors to G_q/11 proteins, as well as G_i/o proteins in GT1-7 cells.

Since both G_i and G_q/11 proteins have been shown to activate the MAPK pathway (54-56), we were interested in investigating the possible involvement of this signaling pathway in melatonin-mediated regulation of GT1-7 cell function. The MAPK signaling pathway is comprised of a series of serine/threonine kinases that phosphorylate nuclear and cytoplasmic proteins that are ultimately involved in the transduction of mitogenic signals such as cell growth, division, and differentiation. There are currently three MAPK pathways known to be involved in cellular signaling including the ERK 1/2 pathway, the JNK/SAPK pathway, and the p38 pathway. Melatonin has been shown to alter cytoskeletal organization in Chinese hamster ovary cells stably expressing the human mt1 through a signaling process that involves the increased phosphorylation of ERK 1/2 protein and of its precursor MEK 1/2 protein (57). In MCF-7 breast cancer cells, melatonin in combination with epidermal growth factor has been shown to modulate estrogen receptor expression via changes in MAPK activity (36). In ovine pars tuberalis cells, melatonin was found to have both inhibitory effects as well as no effect on the activation of the MAPK pathway (35, 50). In the GT1-7 cells, we have demonstrated that melatonin induces rapid phosphorylation of ERK 1/2 proteins, suggesting the activation of this MAPK signaling pathway. Further studies are required to distinguish mt1- and MT2-mediated signaling events, as well as whether G_i, G and/or G_q/11 are involved in the activation of the ERK 1/2 signaling pathway in the GT1-7 cells.

Extracellular signals are transduced to the nucleus through a variety of signaling mechanisms, such as those regulated by cAMP, PKC, and MAPK to alter gene expression. Members of the Fos family and functionally related Jun family of immediate early genes function as nuclear messengers that appear to form the link between transient intracellular signals and long term cellular responses (58). Protein products of these immediate early genes assemble to form the transcription factor complex known as activator protein-1 (AP-1), which is involved in the transcriptional regulation of a number of genes (59). Melatonin has been shown to inhibit forskolin-stimulated induction of c-fos and junB mRNA as well as c-Fos protein in ovine pars tuberalis cells (37) and to inhibit GnRH-induced c-Fos immunoreactivity in neonatal rat pituitary cells (8). The only tissue in which melatonin has been shown to increase c-fos immediate early gene expression is the rat SCN (38). We have shown that melatonin induces a rapid but transient increase in both c-fos and junB mRNA expression in the GT1-7 cells. Previous studies in GT1-7 cells have shown that increases in c-fos gene expression plays a major role in the repression of GnRH gene expression by phorbol esters (60). Further studies are required to determine the potential role of AP-1 activity in the melatonin-mediated control of GnRH gene expression in GT1-7 cells.

The PKA, PKC, and cGMP second messengers have been implicated in the regulation of GnRH gene expression in GT1-7 cells. Activation of PKA activity has been reported to have no effect (61) as well as inhibit (46) GnRH gene expression in GT1-7 cells. In this study, treatment with the PKA inhibitor alone did not alter basal GnRH gene expression, suggesting that this signaling pathway may not be involved in the regulation of basal GnRH gene expression in GT1-7 cells. These results support previous findings which showed that activation of adenylyl cyclase by forskolin to raise cAMP levels also had no effect on steady state GnRH mRNA levels over a 48-h period (61). In the presence of PKA inhibitor, there was no effect on melatonin-mediated regulation of GnRH gene expression, suggesting that this pathway is not required for the cyclical regulation of GnRH gene expression by melatonin. PKC signaling has been shown to down-regulate GnRH gene expression (60-62), although there have been conflicting opinions as to whether or not this is due to the decrease or increase in PKC activity (60, 61). We have previously shown that melatonin down-regulates GnRH gene expression in GT1-7 cells in a 24-h cyclical pattern (24). Inhibition of the PKC pathway did not alter the melatonin-mediated cyclical regulation of GnRH gene expression over a 24-h period. Interestingly, treatment with the PKC inhibitor alone resulted in a decrease of GnRH mRNA expression following 12 h of treatment. These results are similar to those previously found in GT1-7 cells treated with the PKC inhibitor NPC 15437 (61) and support the suggestion that activity of the PKC pathway is necessary for basal GnRH mRNA expression. Inhibition of the PKC pathway may also therefore be involved in the melatonin-mediated down-regulation of GnRH gene expression at 12 h. GnRH expression returned to basal level following 24 h of treatment with the PKC inhibitor, which may reflect either degradation of the inhibitor or rescue of GnRH gene expression by alternative signaling mechanisms. Further studies are underway to address this observation. Interestingly, in the presence of melatonin and the MEK inhibitor, GnRH mRNA levels did not return to basal at 24 h, as expected. This result suggests that the activity of the ERK 1/2 pathway may be required for the maintenance of melatonin-mediated repression of GnRH mRNA levels in GT1-7 cells. We speculate that a transcription factor involved in the repression of GnRH gene expression may be modified by one of the elements of the ERK 1/2 pathway downstream of the MEK proteins in order for GnRH gene expression to return to basal levels. Further analysis of the downstream elements of the ERK 1/2 pathway are necessary to understand the mechanisms involved in the attenuation of melatonin-mediated repression of GnRH.

The signaling mechanisms involved in the complex regulation of GnRH pulsatile secretion in GT1-7 cells are yet to be completely understood. In the regulation of GnRH secretion from GT1-7 cells, the role of the cAMP/PKA signaling pathway has been investigated. Treatment of GT1-7 cells with dopamine or norepinephrine has been shown to increase intracellular cAMP levels and stimulate GnRH secretion (63, 64). Furthermore, oscillations in cAMP levels, regulated by cyclic nucleotide phosphodiesterases as well as activation of PKA have been implicated in a negative feedback mechanism involved in timing the pulsatile release of GnRH from GT1-7 cells (47, 65). It has been suggested that acetylcholine modulates GnRH release from GT1-7 cells via increases in PLC and inhibition of cAMP through both muscarinic (M)₁ and M₂ receptors that are coupled to G_q/11 and G_i proteins respectively (66). Stimulation of PKC activity (61, 62, 67) and elevation of intracellular cGMP levels (68) have also been shown to enhance GnRH secretion from GT1-7 cells.

The antigonadal action of melatonin has been thought to involve the suppression of the release of GnRH from its neurons of the mouse hypothalamus (69, 70). Short photoperiods, as well as melatonin treatment, have been shown to have a modulatory effect on GnRH secretion in hypothalamic fragments from adult rodents (71). In vitro incubations of male rat hypothalamic tissue with melatonin suggest diurnal mechanisms by which melatonin can either facilitate or suppress GnRH release (72). Our results indicate that in the short term (1 h), melatonin causes a 45% suppression of GnRH secretion in GT1-7 neurons. Although previous studies have not observed the direct effects of melatonin on GnRH neurons, our data concurs with the inhibitory nature of melatonin on GnRH secretion. The effect of melatonin on GnRH secretion in GT1-7 cells is specifically mediated through melatonin receptors as treatment with luzindole, a melatonin receptor antagonist, blocked the effect. We were also interested in investigating the signaling pathways involved in the melatonin-mediated repression of GnRH release in GT1-7 cells. It is of interest that pharmacological inhibition of the PKA, PKC, and ERK 1/2 pathways resulted in an increase in the basal secretion of GnRH in the GT1-7 neurons. This observation is consistent with another recent study that demonstrated that pharmacological inhibition of PKA activity caused an increase in the basal secretion of GnRH from GT1-7 cells (47). These results suggest that each of these pathways may be involved in maintaining basal levels of GnRH release in the GT1-7 cells. Furthermore, our findings also suggest that the activation of each of the PKA, PKC as well as the MAPK signaling pathways, is required for melatonin-mediated suppression of GnRH secretion.

The down-regulation of GnRH gene expression and secretion in GT1-7 cells by melatonin supports the hypothesis that the hypothalamus is a major target tissue for the antigonadal action of melatonin. The effects of melatonin on GT1-7 cell function appear to occur partly through G protein-coupled receptor-mediated signaling mechanisms that, in the short term, result in the inhibition of cAMP, the activation of PKC and MAPK, and the induction of the immediate early genes c-fos and junB. This study demonstrates that the regulation of GnRH gene expression and secretion by melatonin in GT1-7 cells appears to be under coordinated control by the PKA, PKC, and MAPK pathways.

	ACKNOWLEDGEMENTS

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

	FOOTNOTES

^* This work was supported by a Natural Science and Engineering Research Council Operating Grant (to D. D. B.) and an Ontario Graduate Scholarship in Science and Technology (to D. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Canadian Institutes of Health Research Scholar and a Canada Foundation for Innovation Researcher. To whom correspondence should be addressed: Dept. of Physiology, Univ. of Toronto, Medical Sciences Bldg., Rm. 3247A, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-946-7646; Fax: 416-978-4940; E-mail: d.belsham@utoronto.ca.

Published, JBC Papers in Press, October 29, 2001, DOI 10.1074/jbc.M108890200

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; PTX, pertussis toxin; SCN, suprachiasmatic nucleus; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PKA, protein kinase A; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MEK, mitogen-activated protein kinase kinase; TPA, 12-0-tetradecanoylphorbol-13-acetate; BIS, bisindolylmaleimide; RIA, radioimmunoassay; PBS, phosphate-buffered saline; PLC, phospholipase C.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES