help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ross, A. W.
Right arrow Articles by Morgan, P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ross, A. W.
Right arrow Articles by Morgan, P. J.
Endocrinology Vol. 139, No. 4 1723-1730
Copyright © 1998 by The Endocrine Society

ARTICLES

A Novel Interaction Between Inhibitory Melatonin Receptors and Protein Kinase C-Dependent Signal Transduction in Ovine Pars Tuberalis Cells1

Alexander W. Ross, Catriona A. Webster, Michael Thompson, Perry Barrett and Peter J. Morgan

Molecular Neuroendocrinology Unit, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, Scotland, AB21 9SB, United Kingdom

Address all correspondence and requests for reprints to: Peter J. Morgan, Molecular Neuroendocrinology Unit, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, Scotland, AB21 9SB, United Kingdom. E-mail: p.morgan{at}rri.sari.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study revealed an important and unexpected finding: namely, that inhibitory melatonin receptors can inhibit a phorbol 12,13 myristate acetate (PMA)-induced, protein kinase C (PKC)-dependent increase in c-fos messenger RNA expression in ovine pars tuberalis (PT) cells. PMA induces dose-dependent stimulation of c-fos expression that is attenuated by melatonin in a dose-dependent and pertussis toxin-sensitive manner. The effect of 100 nM PMA is blocked by Ro31–8220 (1 µM), yet is not mimicked by 4{alpha}-PMA (100 nM). PMA (100 nM) induces PKC activity in PT cells (P < 0.05) within 5 min, but melatonin has no effect on this response. PMA (100 nM) stimulates both phospholipase D and mitogen-activated protein kinase (MAPK) (p42/44) activities in PT cells, but melatonin has no effect on these responses. The results indicate that neither of these second-messenger activities contribute to the melatonin-sensitive pathway of c-fos activation. The MEK (MAPK kinase) inhibitor, PD98059 (50 µM), does not block the induction of c-fos by PMA, although at the same dose it inhibits PMA-mediated activation of p42/44 MAPK by 50–70%, and activation by forskolin or insulin-like growth factor-I by 100%. These data suggest that p42/44 MAPK may not be the primary mediator of PKC-dependent c-fos induction. In contrast to the effect of melatonin on PMA-mediated c-fos induction in PT cells, in L cells stably transfected with the sheep Mel1aß receptor, melatonin potentiates the c-fos response in a pertussis toxin-sensitive manner. These data indicate the tissue-specific nature of melatonin receptor signaling, and reveal that a pertussis toxin-sensitive pathway can block PKC-mediated c-fos induction in PT cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MELATONIN is an important hormone in photoperiodically sensitive species, as it programs major physiological changes through its action on the neuroendocrine system (1). In seasonal mammals, these changes include altered plasma PRL, ß-endorphin, FSH, and LH levels, which lead to changes in reproductive activity, body weight, appetite, and coat condition (2, 3, 4). A major neuroendocrine target site for melatonin is the pars tuberalis (PT), which expresses one or two allelic variants of the Mel1a receptor subtype (5). The biological importance of the PT appears to be in the photoperiodic regulation of PRL secretion and endocrine regulation of the pars distalis (6, 7, 8).

The PT has therefore become an important model tissue for the study of the cellular mode of action of melatonin in mammals. Studies of signal transduction in the PT have shown that melatonin acts to inhibit the production of forskolin-stimulated cAMP through a pertussis toxin-sensitive mechanism (9). It has also been confirmed that downstream processing of the cAMP signal both in terms of protein kinase A activation and the phosphorylation of the transcription factor cAMP-response element binding protein occur in response to forskolin, and that these responses are blocked by melatonin (10, 11). Cross-talk between the cAMPK cascade and the p42/44 mitogen-activated protein kinase (MAPK) pathway has also been demonstrated in ovine PT cells, indicating that melatonin can influence other signal transduction events through a primary effect on cAMP (12). These results suggest an important role for cAMP signal transduction in melatonin receptor function.

The functional relationship between melatonin receptors and the inhibition of cAMP has been confirmed through the stable expression of the recombinant Mel1a receptor protein in both and NIH3T3 and L cells (5, 13). However, several lines of evidence have indicated that melatonin may act through alternative pathways to influence cellular activity in other native tissues. In neonatal rat pituitary, melatonin has been shown to inhibit a number of signal transduction cascades including mobilization of calcium, the release of arachidonic acid, and the synthesis of diacylglycerol (14, 15). In rat caudal artery, melatonin potentiates noradrenaline-stimulated vasoconstriction through a cAMP-independent action (16), and in the suprachiasmatic nucleus (SCN), melatonin has been reported to stimulate protein kinase C (PKC) (17) and to activate potassium currents (18). Evidence from studies in which the recombinant human Mel1a receptor has been expressed in NIH3T3 cells also indicates that melatonin can act to potentiate PGF2{alpha}-stimulated arachidonic acid release (19). Thus the effects of melatonin are clearly not limited to the inhibition of cAMP, and suggest that the mechanisms of action at particular cellular targets may be tissue specific.

The effects of melatonin on signal transduction pathways other than cAMP have been studied in the sheep PT. These studies have shown that melatonin alone has no direct stimulatory or inhibitory effect on a number of second-messenger events, including calcium mobilization and the activities of phospholipases A2, D, and C and MAPK (12, 20, 21). Likewise, melatonin alone neither stimulates nor inhibits cAMP accumulation, rather it prevents or reverses forskolin-activated cAMP synthesis (22). Thus it is possible that melatonin acts in the ovine PT to inhibit or potentiate other signal transduction pathways, but it has not been possible to reveal these in the absence of a second stimulus. Nevertheless, we have recently shown that melatonin can prevent the increase in Mel1a mRNA, which occurs on release of primary PT cells into culture, through a cAMP-independent mechanism (23).

The present study reveals a novel interaction between an inhibitory G-protein-coupled receptor and PKC-mediated signal transduction. We demonstrate that melatonin inhibits phorbol ester-induced, PKC-dependent, c-fos expression in ovine PT cells through a pertussis toxin-sensitive mechanism. In contrast to the native tissue, melatonin potentiates phorbol ester-induced c-fos expression in L cells stably transfected with the sheep Mel1a receptor, indicating for the first time that the effects of melatonin are dependent on cellular phenotype. These data have importance to both signal transduction generally, and to our understanding of the cellular action of melatonin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
All liquid media were purchased from Life Technologies (Paisley, Scotland). Primary cell cultures were prepared as described previously (24). Cells were seeded at a density of approximately 2 x 106 cells/ml in tissue culture dishes and incubated at 37 C in a CO2 incubator (5% CO2) for 16–24 h. Cells were harvested using a rubber scraper, centrifuged at 500 x g for 10 min at 4 C, washed twice with DMEM/BSA (DMEM supplemented with 0.1% BSA and antibiotic-antimycotic (100 U/ml penicillin, 100 U/ml streptomycin, and 0.25 µg/ml fungizone, GIBCO, Paisley, UK), and resuspended in the same medium. Cells were counted then dispensed as appropriate (see below) into culture dishes.

Cell lines
L cells were cultured as described previously (5). L cells, stably expressing the sheep Mel1aß receptor, were transfected as described previously (5) and grown in DMEM supplemented with 10% FCS and G418 (0.5 mg/ml).

Measurement of c-fos mRNA expression
L cells transfected with the sheep melatonin receptor (Mel1aß) and nontransfected L cells were grown to subconfluency in 90-mm culture dishes, then washed with 2 x 5 ml DMEM/BSA and serum starved at 37 C in a CO2 incubator (5% CO2) for 18–20 h before stimulation. Serum-free PT cells were seeded into 35-mm culture dishes at a cell density of 107 cells in 4.5 ml DMEM/BSA, then similarly serum starved. In experiments that utilized PD98059, this MEK inhibitor [25 mM stock in dimethylsulfoxide (DMSO)] was added to give 50 µM final concentration in appropriate dishes and preincubated for 1 h before stimulations. When Ro31–8220 was used, the inhibitor was dissolved in DMSO (at 0.5 mM) and added to give 1 µM final concentration in appropriate dishes and preincubated for 20 min. Vehicle alone was added to control dishes. Cells were stimulated for 45 min with 10 x concentrates of drugs and hormones in 1 ml or 0.5 ml DMEM/BSA for L cells and PT cells, respectively, to give final concentrations as described in the figure legends.

Total RNA was isolated using Qiagen RNeasy Kits (Qiagen Ltd., Dorking, Surrey, UK). Typically, 107 cells yielded 25–30 µg total RNA. Twenty micrograms total RNA were electrophoresed, transferred to Genescreen Nylon membrane (New England Nuclear-DuPont, Boston, MA), and then fixed by UV illumination. A 684-bp mouse v-fos complementary DNA (cDNA) fragment (70 ng) and a human glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNA (Clontech, Palo Alto, CA) probe (25 ng) were radiolabeled using a random priming kit following the manufacturer’s protocol (Boehringer Mannheim, Indianapolis, IN). Hybridizations were performed using QuikHyb solution (Stratagene, Cambridge, UK) according to the manufacturer’s protocol and involved a final wash at 55 C with 0.5 x SSC-0.1% SDS for 30 min. RNA size markers (Promega, Southampton, UK) were used. Membranes were placed against Kodak X-OMAT-LS film (Eastman Kodak, Rochester, NY) with intensifying screens for 1–3 days. Integrated optical densities were measured using the Bioimage software (Bioimage, Watford, UK). The v-fos probe was kindly provided by Dr. David Gillespie, Beatson Institute, Glasgow, Scotland.

Measurement of PKC activity
PT cells were seeded into 35-mm Petri dishes at a density of 2.5 x 106 cells in 1.8 ml DMEM/BSA, then serum starved at 37 C in a CO2 incubator (5% CO2) for 18–20 h before stimulation. Stimulants were added as 10 x concentration in 0.2 ml DMEM/BSA prewarmed to 37 C giving final concentrations of 100 nM phorbol 12,13 myristate acetate (PMA) and 10 nM melatonin. After 30 min stimulation, cells were rinsed twice with 2 ml ice-cold PBS, then lysed with 200 µl 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 ß-glycerophosphate, 10 mM benzamidine, 0.3% (vol/vol) ß-mercaptoethanol, 50 µg/ml phenylmethylsulfonylfluoride, 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 International plc, Buckinghamshire, UK), except that the component mixture used with the cell lysates was prepared with the stimulants substituted by lysis buffer. Assay tubes contained 25 µl component mixture, 25 µl lysate, and 0.2 µCi [32P]ATP (450,000 cpm ± 20,000 cpm), and reactions were performed for 15 min. Ultima-Gold scintillant was used and 32P incorporation counted using a Minaxi ß-counter (Packard Instrument Co., Meriden, CT).

Assay of MAPK activity by in-gel renaturation assay
In-gel renaturation assays were conducted as described previously (12). PT cells were serum starved and stimulated as for the PKC assay described above.

Western blotting of cytosolic extracts and immunostaining for MAPK
PT cells were prepared and serum starved as described for the PKC assay above. In experiments that utilized PD98059, this MEK (MAPK kinase) inhibitor at 50 µM final concentration (25 mM stock in DMSO) was added to appropriate dishes, whereas DMSO alone was added to the remaining test dishes and preincubated for 1 h before stimulations. Cells were stimulated for 30 min with drugs and hormones as described in the figure legends. The cell stimulants were removed, cells briefly rinsed with 10 mM HEPES, pH 8.0, and 150 mM NaCl, and then harvested in 150 µl of this buffer and transferred to microcentrifuge tubes. To 90 µl of each cell suspension, 30 µl of 4 x SDS gel loading buffer were added, lysates boiled for 5 min, and then stored at -80 C. Total protein content of the cell suspensions was determined following the method of Lowry et al. (25). In preparation for electrophoresis, samples were thawed, sonicated for 5 sec, and then centrifuged at 17,000 x g for 5 min, and the supernatants transferred to new tubes. Aliquots containing 15 µg total protein were applied per lane. SDS-PAGE and Western blotting were performed as described previously (8, 12). Immunostaining was performed using PhosphoPlus MAPK Antibody kit [New England Biolabs (UK) Ltd., Hitchin, Hertfordshire, UK] according to the manufacturer’s protocol. This kit employs an antiserum that detects phosphorylated tyrosine 204 of p44 and p42 MAPKs but does not cross-react appreciably with the corresponding phosphorylated tyrosine of either Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) or p38 MAPK homologs [New England Biolabs (UK) Ltd.]. Integrated optical densities were measured by computing densitometry.

Assay of phospholipase D activity in PT cells
PT cells (2 x 106/well) were seeded in 6-well culture plates (GIBCO) in 2 ml of serum-free DMEM and were metabolically labeled with 5 µCi of [3H]oleic acid (10 Ci/mmol; Amersham) for 24 h. Radioactive medium was discarded, and the adherent cells were washed twice in DMEM. Medium in each well was replaced by DMEM containing 0.5% butanol and incubated for 10 min (37 C). Stimulants were then added at 10x concentration, and the incubation continued for 30 min at 37 C. After incubation, the medium was removed, and the cells were washed three times in ice-cold PBS. Cellular lipids from cells in each well were solubilized using methanol (2 ml), chloroform (1 ml), and 20 µl 12 M HCl followed by incubation on ice for 30 min. Separation of the aqueous and organic phases was achieved by the addition of 1.25 ml chloroform and 1.25 ml H2O followed by centrifugation at 3000 x g for 5 min. The lower layer was recovered and dried under nitrogen, and then resuspended in 40 µl chloroform/methanol (2:1, vol/vol) for separation by thin-layer chromatography using ethyl acetate; 2,2,4 trimethylpentane; acetic acid; H2O (55;25;10;50, vol/vol) as the solvent system. The level of [3H]phosphatidylbutanol production was measured by autoradiography, and recovery of the radioactive product from the chromatography plate followed by scintillation counting.

Statistical analyses. Statistical comparisons were made using using Sigmastat software (Jandel Scientific, Erkath, Germany). Data were analyzed by one-way ANOVA followed by Student Newman Keuls comparisons. Statistical differences were accepted where P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Incubation of the ovine PT cells with PMA increased c-fos mRNA expression in a dose-dependent manner (Fig. 1Go). In the presence of 10 nM melatonin, the stimulatory effect of 100 nM PMA was significantly attenuated (P < 0.05), although the inhibitory effect of melatonin was only partial (Fig. 1aGo). Melatonin inhibited PMA-induced c-fos induction in both a dose-dependent (Fig. 1bGo) and pertussis toxin-sensitive manner (Fig. 2Go). The increase of c-fos mRNA in response to PMA (100 nM) was completely blocked by the PKC inhibitor Ro31–8220 used at a concentration of 1 µM (P < 0.05) (Fig. 3Go). The PKC inhibitor alone had no effect on c-fos mRNA expression, nor did the phorbol ester analog 4{alpha}-PMA (Fig. 3Go). Because the latter is inactive on PKC, and Ro31–8220 totally blocks the effect of PMA, these data are consistent with a major role of PKC in the induction of c-fos by PMA. Measurement of PKC activity in cell lysates, prepared from ovine PT cells stimulated with PMA (100 nM), showed enhanced PKC activity within 5 min that was maintained over 30 min (Fig. 4aGo). However, melatonin (10 nM) had no effect on PKC activity, either alone, at the 30 min time point (Fig. 4bGo), or in the presence of 100 nM PMA between 5 and 30 min (Fig. 4Go, a and b). These data indicate that the effect of melatonin on PMA-induced c-fos is not due to direct action of melatonin at the level of PKC but indicate an interaction downstream of PKC activation.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 1. a, Dose-dependent stimulation of c-fos mRNA expression in ovine PT cells by PMA in presence and absence of 10 nM melatonin (Mel) over 45 min. b, Representative Northern blot showing dose-dependent effect of PMA on c-fos (upper band) band expression in presence and absence of melatonin and G3PDH (lower band) used to normalize for loading. c, Dose-dependent inhibition of PMA-induced c-fos expression in PT cells by melatonin. Control in a and c indicates level of c-fos mRNA in cells treated with vehicle. Data in a and c are relative integrated optical density measurements of c-fos levels as a ratio to G3PDH, determined by Northern blotting from three independent experiments. Data are mean ± SEM. *, Statistically significant effect of melatonin compared with PMA alone (P < 0.05).

 


View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. Effect of pertussis toxin (PTX) on ability of melatonin (10 nM) to inhibit PMA-induced (100 nM) c-fos mRNA expression in ovine PT cells. a, Cells treated with vehicle; b, cells pretreated with PTX (500 ng/ml) for 16 h. Data are relative integrated optical density measurements of c-fos levels expressed as a ratio to G3PDH, determined by Northern blotting from three independent experiments. Data are mean ± SEM. *, Statistically significant effect of melatonin compared with PMA alone (P < 0.05).

 


View larger version (20K):
[in this window]
[in a new window]
  		Figure 3. Effect of Ro31–8220 (Ro) inhibitor and PMA analog, 4{alpha}-PMA, on c-fos mRNA expression in ovine PT cells over 45 min. Control, Level of c-fos mRNA in cells treated with vehicle. Data are relative integrated optical density measurements of c-fos levels as a ratio to G3PDH, determined by Northern blotting from three independent experiments. Data are mean ± SEM. *, Statistically significant effect of melatonin (P < 0.05).

 


View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. a, Time-dependent effect of PMA and melatonin on PKC activity in ovine PT cells. b, Effect of melatonin alone at 30 min. Control, PKC activity in cells treated with vehicle. Data represent relative PKC activities obtained from three to four independent experiments. Data are mean ± SEM. *, Statistically significant effect of PMA compared with control (P < 0.05).

 
One of the potential mechanisms through which PMA and melatonin could induce c-fos is through activation of phospholipase D. PMA induced a dose-dependent increase in phospholipase D activity measured through the transphosphatidylation reaction (Fig. 5Go). However, melatonin (10 nM) failed to modulate this activity (Fig. 5Go). MAPK activity is also known to be altered in association with changes in PKC activity. Consistent with this, both the p42 and p44 isoforms of MAPK were activated within 5 min in response to PMA (P < 0.05) (Fig. 6Go). However, melatonin had no significant effects on these activities either alone (data not shown) or in combination with PMA (Fig. 6Go). These results suggest that neither the activation of phospholipase D nor the activation of MAPK contributes to the melatonin-sensitive pathway that leads to the activation of c-fos.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 5. Dose-dependent effect of PMA on phospholipase D activity, measured by production of phosphatidylbutanol (PtBuOH), in presence and absence of 10 nM melatonin. Cells were stimulated over 30 min. Data are from a single experiment repeated twice and show incorporation of [3H]oleic acid into PtBuOH as a ratio to incorporation into total phospholipids.

 


View larger version (19K):
[in this window]
[in a new window]
  		Figure 6. Time-dependent effect of PMA and melatonin on activation of p42 (a) and p44 (b) MAPK. Control, Level of MAPK activity in cells treated with vehicle. Data are relative integrated optical density measurements of MAPK activity determined by in-gel renaturation assay, from three independent experiments. Data are mean ± SEM.

 
Interference with MAPK activation using the selective MEK (MAPK kinase) inhibitor, PD98059, did not prevent the induction of c-fos by 100 nM PMA, and had no effect on the magnitude of the inhibitory response by melatonin (Fig. 7Go). The MEK inhibitor did block the activation of both the p42 and p44 isoforms of MAPK by PMA, although the inhibition was only 50% and 70% of the total response to 100 nM PMA for these two isoforms, respectively (Fig. 8Go). Nevertheless, PD98059 did fully block the activation of both p42 and p44 MAPK isoforms by forskolin (10 µM) and insulin-like growth factor-I (IGF-I) (10 nM) (Fig. 8Go).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 7. Effect of MEK inhibitor, PD98059 (PD), on induction of c-fos mRNA by PMA in presence and absence of melatonin. Control, Level of c-fos mRNA in cells treated with vehicle. Data are relative integrated optical density measurements of c-fos levels as a ratio to G3PDH, determined by Northern blotting from three independent experiments. Data are mean ± SEM. *, Statistically significant differences (P < 0.05).

 


View larger version (32K):
[in this window]
[in a new window]
  		Figure 8. Effect of MEK inhibitor, PD98059 (PD), on activation of p42 (a) and p44 (b) MAPK by PMA, forskolin, and IGF-1 in ovine PT cells over 30 min. Control, Level of MAPK activity in cells treated with vehicle. Data are relative integrated optical density measurements of MAPK activity determined by immunodetection of phosphorylated p42 and p44 proteins from three independent experiments. Data are mean ± SEM. *, Statistically significant differences (P < 0.05).

 
To test whether the effect of melatonin on PMA-induced c-fos induction is observed in cells expressing the recombinant sheep Mel1a receptor, the effect of melatonin on PMA-induced c-fos expression in L cells transfected with sheep Mel1a cDNA was examined. This showed that, in marked contrast to its effect on PT cells, melatonin potentiated the stimulatory effect of PMA on c-fos expression, but alone it had no effect (Fig. 9Go). This effect was absent both in nontransfected L cells and in L cells transfected with the Mel1a receptor and pretreated with pertussis toxin (100 ng/ml), indicating that this response was mediated via the Mel1a melatonin receptor (Fig. 9Go).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 9. Effect of PMA in presence and absence of melatonin on expression of c-fos mRNA in L cells stably expressing sheep Mel1aß receptor pretreated with and without pertussis toxin (PTX). a, Cells without PTX treatment; b, cells pretreated with PTX (100 ng/ml) for 16 h; and c, nontransfected L cells. Cells were treated with stimulants for 45 min. Control, Level of c-fos mRNA in cells treated with vehicle. Data are relative integrated optical density measurements of c-fos levels as a ratio to G3PDH, determined by Northern blotting from three independent experiments. Data are mean ± SEM. *, Statistically significant effect of melatonin compared with PMA (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study confirm our previous observations that the phorbol ester, PMA, can increase the levels of c-fos mRNA in PT cells (24). However, what was not anticipated was the ability of melatonin to attenuate this response. Phorbol esters are lipophilic and known to act by passing through the plasma cell membrane to act on PKC, where they mimic the action of endogenous diacylglycerol (26, 27). Although melatonin is also a lipophilic molecule, its primary activity on cell physiology is mediated via the Mel1 family of membrane-bound, G-protein-coupled receptors (28). Support for the involvement of high-affinity melatonin receptors in this study is provided first by the sensitivity to pertussis toxin, which indicates the role of a G-protein-coupled receptor, and second by the dose-response range over which melatonin inhibits the response. It is known that Mel1a receptor transcripts are expressed in the sheep PT cells (5, 13), and when the recombinant Mel1a receptor has been expressed in NIH3T3 cells, it has been shown to mediate pertussis toxin-sensitive inhibition of cAMP (13), as occurs in native PT cells (29). On this basis, it seems reasonable to suggest that the effects of melatonin observed in this study are mediated through Mel1a receptors.

The possibility of a direct inhibitory effect of melatonin on the activation of PKC by PMA can be discounted by the lack of effect of either melatonin alone or in combination with PMA on PKC activity. This contrasts with the stimulatory effect of melatonin on PKC, which has been reported for the suprachiasmatic nucleus (17). However, given that knock out of the Mel1a transcript abolishes the acute inhibitory effects of melatonin on SCN electrical activity but not the pertussis toxin-sensitive effects on entrainment, it appears that some of the effects of melatonin in the SCN are not mediated by the Mel1a receptor (30).

The inhibition of PMA-induced c-fos levels by melatonin is only partial, even at a dose high enough to saturate the Mel1a receptors (5, 13). This is not due to the use of an inadequate concentration of melatonin, because the inhibitory effect is still incomplete when higher concentrations of melatonin (1 µM) were used (data not shown). A more plausible explanation is that PMA activates PKC, which in turn induces c-fos expression through at least two parallel pathways. Only one of these can be inhibited by melatonin, leaving a residual effect that cannot be addressed via Mel1a receptors. PKC has been shown to cause the phosphorylation of many intracellular proteins (27), and therefore, it is potentially possible for the activation of PKC to affect cellular activity through a number of different routes. For example, PKC has been shown to regulate phospholipase D activity (31), to modulate voltage-gated calcium channel function (32), and to be involved in the activation of MAPK (33). Changes in activity by any of these signaling molecules could lead to c-fos expression. In addition, evidence is emerging that suggests that the translocation of PKC isoforms directly to the nucleus can occur, which potentially could be involved in transcriptional regulation (27).

Neither p42/44 MAPK nor phospholipase D activities appear to participate in the melatonin-sensitive pathway leading to PKC-dependent c-fos activation, because each of these activities can be increased in response to PMA without an effect of melatonin. Nevertheless, these second-messenger enzymes could be involved in the melatonin-insensitive activation of c-fos. The inhibition of PMA-induced MAPK activation by PD98059 suggests further that MEK participates, at least in part, in the activation of p42/44 MAPK by PKC. However, the incomplete inhibition of the PMA response by PD98059 indicates that other pathways are probably involved in the activation of p42/44 MAPK by PKC. Although these results could be explained by the use of a submaximal dose of the MEK inhibitor, the ability of PD98059 to fully block the effect of p42/44 MAPK activation by both forskolin and IGF-1 does not support this explanation.

Melatonin has been noted for its particular lack of effect on a wide range of second-messenger events when tested alone (9). For example, melatonin has a potent inhibitory effect on forskolin-stimulated cAMP accumulation presumed to involve regulation of adenylate cyclase by pertussis toxin-sensitive Gi{alpha}. Alone however, melatonin has no effect on basal cAMP levels (22). In the absence of obvious candidate second-messenger events regulated directly through melatonin receptors (12, 20, 21, 22), it seems most likely that the mechanism of interaction of melatonin with PKC is at the level of the signal transduction machinery within the plasma membrane. Such an interaction could involve the phosphorylation of a membrane-signaling protein (G-protein, effector, or receptor) leading to increased activity of an effector enzyme that in turn leads to c-fos activation. In a mechanism analogous to the regulation of adenylate cyclase, melatonin could regulate the activity of another unidentified effector enzyme through the release of Gi{alpha} or ß{gamma}. PKC has been shown to phosphorylate a number of membrane-signaling proteins, including adenylate cyclase (34). PMA, however, does not alter cAMP levels in ovine PT cells (22), indicating that this route of activation does not account for melatonin-sensitive, PMA-induced c-fos expression. Candidate proteins that could mediate PKC effects include the G-proteins, G12 and G13, both of which are phosphorylated by PKC in vitro and in vivo (35). It has been speculated that this event leads to increased signaling by these G-proteins (35). The nature of the signal transduction cascade triggered via G12 and G13 is not known, but some evidence suggests that the activation of Jun N-terminal kinase may be involved (36). As Jun N-terminal kinase can activate c-fos transcription via Elk-1 and the serum response element enhancer elements, this offers a potential melatonin-sensitive route of c-fos expression (33, 35). Therefore, these or similar events could provide the basis for the interaction between PKC and melatonin, although at this time the identity of such components or the nature of the mechanism has not been established.

In marked contrast to the inhibitory effect of melatonin on PMA-induced c-fos expression in ovine PT cells, melatonin potentiates PMA-induced c-fos expression in L cells stably transfected with the sheep melatonin receptor. This effect is pertussis toxin sensitive and absent in nontransfected cells, indicating that it is mediated via Mel1a receptors. These data emphasize the importance of cellular phenotype to the signal transduction response. Comparable effects of melatonin have been reported for NIH3T3 cells expressing Mel1a receptors, in which PGF2{alpha}-stimulated arachidonic acid release is potentiated by melatonin (19), although we have no evidence that there is any functional relationship between arachidonic acid release and c-fos expression between these two cell types. Despite the differences in the response between the native PT cells and the transfected cell lines, the ability of melatonin to inhibit forskolin-stimulated cAMP formation is common to each cell type.

Overall therefore, these results raise two important issues. First, they demonstrate the unexpected finding that an inhibitory G-protein-coupled receptor, such as the melatonin receptor, can inhibit a PKC-dependent signal transduction pathway leading to the inhibition of c-fos expression. To our knowledge, such an interaction has not been previously reported. Second, the data emphasize the importance of cell type and hence cell-specific signaling pathways to the mechanism of action of melatonin. This in turn has important ramifications to our understanding of the biological activities of melatonin at the physiological level. In the PT, a great deal of evidence supports an important role for the cAMP pathway to the biological function of melatonin. The data from this study highlight the importance of other second-messenger pathways to the function of melatonin within the PT.


   Footnotes
 
1 This work was funded by the Scottish Office for Agriculture, Environment and Fisheries Department. Back

Received June 20, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Morgan P, Mercer J 1994 Control of seasonality by melatonin. Proc Nutr Soc 53:483–493[CrossRef][Medline]
  2. Lincoln GA, Libre EA, Merriam GR 1989 Long-term reproductive cycles in rams after pinealectomy or superior cervical ganglionectomy. J Reprod Fertil 85:687–704[Abstract/Free Full Text]
  3. Lincoln GA, Short RV 1980 Seasonal breeding: nature’s contraceptive. Recent Prog Horm Res 36:1–52
  4. Ebling FJ, Lincoln GA 1987 ß-endorphin secretion in rams related to season and photoperiod. Endocrinology 120:809–818[Abstract/Free Full Text]
  5. Barrett P, Conway S, Jockers R, Strosberg A, Guardiola B, Delagrange P, Morgan PJ 1997 Cloning and functional analysis of a polymorphic variant of the ovine Mel1a melatonin receptor. Biochim Biophys Acta 1356:299–307[Medline]
  6. Lincoln GA, Clarke IJ 1994 Photoperiodically-induced cycles in secretion of prolactin in hypothalamo-pituitary disconnected rams. Evidence for translation of the melatonin signal in the pituitary gland. J Neuroendocrinol 6:251–260[Medline]
  7. Morgan P, Webster C, Mercer J, Ross A, Hazlerigg D, Maclean A, Barrett P 1996 The ovine pars tuberalis secretes a factor(s) that regulates gene expression in both lactotrophic and nonlactotrophic pituitary cells. Endocrinology 137:4018–4026[Abstract]
  8. Hazlerigg D, Hastings M, Morgan P 1996 Production of a prolactin releasing factor by the ovine pars tuberalis. J Neuroendocrinol 8:489–492[CrossRef][Medline]
  9. Morgan PJ, Barrett P, Howell HE, Helliwell R 1994 Melatonin receptors: localization, molecular pharmacology and physiological significance. Neurochem Int 24:101–146[CrossRef][Medline]
  10. Hazlerigg DG, Morgan PJ, Lawson W, Hastings MH 1991 Melatonin inhibits the activation of cyclic AMP-dependent kinase in cultured pars tuberalis cells from ovine pituitary. J Neuroendocrinol 3:597–603[CrossRef][Medline]
  11. McNulty S, Ross A, Barrett P, Hastings M, Morgan P 1994 Melatonin regulates the phosporylation of CREB in ovine pars tuberalis cells. J Neuroendocrinol 6:523–532[CrossRef][Medline]
  12. Hazlerigg D, Thompson M, Hastings M, Morgan P 1996 Regulation of mitogen-activated protein kinase in the pars tuberalis of the ovine pituitary:interactions between insulin like growth factor-1 and forskolin. Endocrinology 137:210–218[Abstract]
  13. Reppert SM, Weaver DR, Ebisawa T 1994 Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses. Neuron 13:1177–1185[CrossRef][Medline]
  14. Vanecek J 1991 Mechanism of melatonin action. Physiol Res 40:11–23[Medline]
  15. Vanecek J, Klein DC 1992 Melatonin inhibits gonadotropin-releasing hormone-induced elevation of intracellular Ca2+ in neonatal rat pituitary cells. Endocrinology 130:701–707[Abstract/Free Full Text]
  16. Viswanathan M, Laitinen JT, Saavedra JM 1990 Expression of melatonin receptors in arteries involved in thermoregulation. Proc Natl Acad Sci USA 87:6200–6203[Abstract/Free Full Text]
  17. McArthur A, Hunt A, Gillette M 1997 Melatonin action and signal transduction in the rat suprachiasmatic circadian clock: activation of protein kinase C at dusk and dawn. Endocrinology 138:627–634[Abstract/Free Full Text]
  18. Jiang ZG, Nelson CS, Allen CN 1995 Melatonin activates an outward current and inhibits I-h, in rat suprachiasmatic nucleus neurons. Brain Res 687:125–132[CrossRef][Medline]
  19. Godson C, Reppert SM 1997 The Mel(1a) melatonin receptor is coupled to parallel signal transduction pathways. Endocrinology 138:397–404[Abstract/Free Full Text]
  20. McNulty S, Morgan PJ, Thompson M, Davidson G, Lawson W, Hastings MH 1994 Phospholipases and melatonin signal transduction in the ovine pars tuberalis. Mol Cell Endocrinol 99:73–79[CrossRef][Medline]
  21. Morgan PJ, Hastings MH, Thompson M, Barrett P, Lawson W, Davidson G 1991 Intracellular signalling in the ovine pars tuberalis: an investigation using aluminium fluoride and melatonin. J Mol Endocrinol 7:137–144[Abstract/Free Full Text]
  22. Morgan PJ, Lawson W, Davidson G 1991 Interaction of forskolin and melatonin on cyclic AMP generation in pars tuberalis cells of ovine pituitary. J Neuroendocrinol 3:497–501
  23. Barrett P, Maclean A, Davidson G, Morgan P 1996 Regulation of the Mel1a melatonin receptor mRNA and protein levels in the ovine pars tuberalis: evidence for a cyclic adenosine 3',5'-monophosphate-independent Mel1a receptor coupling and an autoregulatory mechanism of expression. Mol Endocrinol 10:892–902[Abstract/Free Full Text]
  24. Ross AW, Barrett P, Mercer JG, Morgan PJ 1996 Melatonin suppresses the induction of AP-1 transcription factor components in the pars tuberalis of the pituitary. Mol Cell Endocrinol 123:71–80[CrossRef][Medline]
  25. Lowry O, Rosebrough N, Farr A, Randall R 1951 Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275[Free Full Text]
  26. Aschendel C 1985 The phorbol ester receptor: a phospholipid-regulated protein kinase. Biochim Biophys Acta 822:219–242[Medline]
  27. Buchner K 1995 Protein kinase C in the transduction of signals toward and within the cell nucleus. Eur J Biochem 228:211–221[Medline]
  28. Reppert SM, Weaver DR, Godson C 1996 Melatonin receptors step into the light: Cloning and classification of subtypes. Trends Pharmacol Sci 17:100–102[CrossRef][Medline]
  29. Morgan PJ, Davidson G, Lawson W, Barrett P 1990 Both pertussis toxin-sensitive and insensitive G-proteins link melatonin receptor to inhibition of adenylate cyclase in the ovine pars tuberalis. J Neuroendocrinol 2:773–776[CrossRef][Medline]
  30. Liu C, Weaver DR, Jin X, Shearman LP, Pieschi RL, Gribkoff VK, Reppert SM 1997 Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock. Neuron 19:1–20[CrossRef][Medline]
  31. Exton J 1997 Phospholipase D: enzymology, mechanisms of regulation and function. Physiol Rev 77:303–320[Abstract/Free Full Text]
  32. Zamponi G, Bourinet E, Nelson D, Nargeot J, Snutch T 1997 Crosstalk between G proteins and protein kinase C mediated by the calcium channel {alpha}1 subunit. Nature 385:442–445[CrossRef][Medline]
  33. Whitmarsh A, Davis R 1996 Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 74:589–607[CrossRef][Medline]
  34. Sunahara RK, Dessauer CW, and Gilman AG 1996 Complexity and diversity of mammalian adenylyl cyclases. Ann Rev Pharmacol Toxicol 36:461–480[CrossRef][Medline]
  35. Offermans S, Hu Y, Simon M 1996 G{alpha}12 and G{alpha}13 are phosphorylated during platelet activation. J Biol Chem 271:26044–26048[Abstract/Free Full Text]
  36. Vara Prasad M, Dermott J, Heasley L, Johnson G, Dhanasekran N 1995 Activation of Jun kinase/stress-activated protein kinase by GTPase-deficient mutants of G{alpha}12 and G{alpha}13. J Biol Chem 270:18655–18659[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
D. Roy and D. D. Belsham
Melatonin Receptor Activation Regulates GnRH Gene Expression and Secretion in GT1-7 GnRH Neurons. SIGNAL TRANSDUCTION MECHANISMS
J. Biol. Chem., January 4, 2002; 277(1): 251 - 258.
[Abstract] [Full Text]

Home page
 FASEB J.Home page
P. BARRETT, W.-S. CHOI, M. MORRIS, and P. MORGAN
A role for tyrosine phosphorylation in the regulation and sensitization of adenylate cyclase by melatonin
FASEB J, August 1, 2000; 14(11): 1619 - 1628.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ross, A. W.
Right arrow Articles by Morgan, P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ross, A. W.
Right arrow Articles by Morgan, P. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals