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Endocrinology Vol. 139, No. 1 163-171
Copyright © 1998 by The Endocrine Society

ARTICLES

Mel 1a Melatonin Receptor Expression Is Regulated by Protein Kinase C and an Additional Pathway Addressed by the Protein Kinase C Inhibitor Ro 31–8220 in Ovine Pars Tuberalis Cells1

Perry Barrett, Gary Davidson2, David G. Hazlerigg3, Marie A. Morris, Alexander W. Ross and Peter J. Morgan

Molecular Neuroendocrinology Group, Rowett Research Institute, Bucksburn, Aberdeen, Scotland AB21 9SB

Address all correspondence and requests for reprints to: Dr. Perry Barrett, Molecular Neuroendocrinology Group, Rowett Research Institute, Bucksburn, Aberdeen, Scotland AB21 9SB. E-mail: pb{at}rri.sari.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression of the melatonin receptor is positively regulated by cAMP and negatively regulated by melatonin in the ovine pars tuberalis (PT). Furthermore, when PT cells are dispersed in primary culture, both messenger RNA (mRNA) and protein levels spontaneously increase through a process that can be blocked by melatonin, but does not involve cAMP. This suggests that other second messengers may be regulated by melatonin, which, in turn, regulates melatonin receptor mRNA and protein levels. In this study using ribonuclease protection assays, ligand binding, protein kinase C (PKC), and cAMP analysis, we demonstrate that the levels of Mel 1a mRNA and protein expression in ovine PT are reduced by phorbol 12-myristate 13-acetate in a cAMP-independent process. This is indicative of an inhibitory role for PKC in receptor regulation. Melatonin, however, does not act through PKC activation to reduce Mel 1a mRNA or protein levels. Basal PKC activity in PT cells can be inhibited by the PKC inhibitor Ro 31–8220, and this suggests that basal PKC activity may suppress Mel 1a receptor expression. Paradoxically, however, Ro 31–8220 also inhibits melatonin receptor mRNA and protein levels in PT cells by a cAMP-independent mechanism. This suggests that other undefined pathways must play an important role in the physiological self-regulation of Mel 1a receptor expression by melatonin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DAILY release of melatonin from the pineal gland occurs during the hours of darkness to provide a sustained hormonal signal of between 4–16 h in a 24-h period. The action of melatonin is mediated by a G protein-coupled receptor (1, 2) at central sites within the brain or peripheral tissues and, in particular, the pars tuberalis (PT) of the anterior pituitary gland (3, 4). The effects of melatonin are to entrain the circadian clock on a daily basis to a 24-h light-dark cycle (5, 6) and, in particular through a change in the length of the melatonin signal during successively lengthening nights of the fall-winter seasons, initiate a number of circannual physiological, metabolic, and behavioral changes required for optimal survival (7, 8, 9, 10).

The melatonin receptor expressed in the plasma membrane of the cells of the ovine PT appears to mediate one of several key events in response to a lengthening melatonin signal, i.e. a reduction in PRL output from the pars distalis (11). Recent evidence suggests that the physiological function of melatonin receptors within the PT may be to modulate the output of a factor called tuberalin (12, 13). This factor can stimulate the release of PRL from pars distalis cells and probably contributes to the seasonal changes in plasma PRL levels, which can occur in the absence of neuroendocrine factors (11). In the ovine PT, the melatonin receptor is present as a part of a large protein complex (14) that mediates the inhibition of the cAMP second messenger pathway via pertussis toxin-sensitive and insensitive mechanisms (15). This inhibition of cAMP synthesis can have consequences for gene expression in the PT (16, 17).

As the duration of the melatonin signal determines the nature of the seasonal response (7, 8, 9, 10), the regulation of melatonin receptor sensitivity, function, and expression becomes a critical issue in understanding how melatonin-responsive cells can decode the daily and seasonal changes in the duration of the melatonin profile. Studies to date have demonstrated daily variation in melatonin receptor density in the rat suprachiasmatic nucleus (SCN) and PT and also in the ovine PT in vivo (18, 19, 20, 21). In a more detailed study we have begun to address melatonin receptor regulation at the cellular and molecular levels. We have demonstrated that transcriptional control and/or messenger RNA (mRNA) stability of melatonin receptor mRNA are at least one level of control through which the receptor is regulated (22). In primary culture of PT cells, the mRNA for the Mel 1a receptor is elevated by an increase in cAMP. This increase can be blocked by melatonin (22). We have also shown that there is a spontaneous increase in mRNA and protein levels for the receptor after the release of PT cells into primary culture, which is not mediated through cAMP. Significantly, this increase, which occurs in the absence of serum in the culture medium, can be inhibited by melatonin (22). This indicates that second messengers other than cAMP, which are under the control of melatonin, are important to the regulation of Mel 1a receptor expression in the PT cell.

With the exception of insulin-like growth factor I, no endocrine hormone has been shown to provide a stimulatory input to melatonin-responsive cells of the PT (3, 23). The insulin-like growth factor I receptor is probably present in melatonin-responsive cells of the PT, where it can mediate activation of mitogen-activated protein kinase, but this activation is not modified by melatonin (23). Currently, therefore, we can only address the action of melatonin through the use of pharmacological tools such as the adenylate cyclase activator, forskolin, and the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA). These tools have demonstrated the existence of functional signal transduction pathways in the PT that are available for activation by the appropriate endocrine hormones, but these pathways have yet to be elucidated.

In this study we have demonstrated that the activation of PMA-responsive isoforms of PKC can negatively regulate Mel 1a mRNA and protein levels in the PT in a cAMP-independent manner. However, melatonin cannot activate PKC and, therefore, does not use PKC activation to regulate Mel 1a gene expression in the PT. In addition to this, the PKC inhibitors Ro 31–8220 and GF109203X are shown to down-regulate melatonin receptor mRNA and protein levels in the PT, and similar to the action of PMA, these effects are independent of changes in cAMP levels. This suggests that Ro 31–8220 and GF 109203X address another pathway to down-regulate Mel 1a mRNA expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Reagents used in this study were obtained from the following suppliers: media, serum and other reagents for cell culture were purchased form Life Technologies (Paisley, Scotland); PMA and 4{alpha}PMA were obtained from Alexis Corp., LC Laboratories (Nottingham, UK); GF 109203X, Ro 31–6045, and Ro 31–8220 (also known as bisindoylmalemide I, bisindoylmalemide V, and bisindoylmalemide IX, respectively) were obtained from Calbiochem (Nottingham, UK); ribonuclease (RNase) protection assay reagents were purchased from AMS Biotechnology (Ambion, Oxon, UK); and RNeasy RNA isolation kits were purchased from Qiagen (Surrey, UK). Monoclonal anti-PKC antibodies were obtained from Affiniti Research Products (Transduction Laboratories, Exeter, UK). The ECL chemiluminescent detection kit and Biotrak PKC assay kit were purchased from Amersham (Aylesbury, UK).

Preparation of ovine PT cells and cell stimulations
PTs from sheep of mixed breed and sex were collected from a local abattoir. Primary cell cultures were prepared as described previously (22) and seeded into 140-mm cell culture petri dishes at a density of 80–120 x 106 cells/dish. The following day cells were harvested using a cell scraper and collected by centrifugation at 500 x g for 15 min. The cells were resuspended in supplemented DMEM (DMEM containing 12.5% lamb serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B) and counted. Ten million cells required for each treatment point were aliquoted into 60-mm cell culture petri dishes in a volume of 5 ml supplemented DMEM. The cells were allowed to equilibrate for 1 h before the commencement of the experiment. Cells that required pretreatment with PMA were seeded on the first day of cell preparation in an appropriate petri dish. PMA to a final concentration of 1 µM was added to the culture medium, and cells were incubated overnight (16 h) at 37 C. PMA-pretreated cells were harvested with a cell scraper and washed twice with supplemented DMEM before aliquoting 107 cells for subsequent treatments. Unless otherwise stated, the concentrations of drugs used in this study were 10 µM forskolin, 10 nM PMA, 10 nM 4{alpha}PMA, 10 nM melatonin, 1 µM Ro 31-6045 and the PKC inhibitor Ro 31–8220, and 1 µM GF 109203X, a concentration shown to inhibit PKC (24, 25).

Stimulation of the cells was terminated by scraping the cells from the petri dishes, then transferring them to a 14-ml polypropylene tube for centrifugation at 2000 x g for 5 min. The medium was aspirated, and the cells were resuspended in 1 ml 1 x PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.47 mM KH2PO4, pH 7.3) and pelleted again at 12,000 x g for 1 min. The PBS was removed, and the cell pellet was frozen on dry ice and held at -70 C until the RNA was extracted. All treatments were performed in duplicate, and all experiments were repeated at least three times.

Preparation of total RNA and RNase protection assay
Total RNA was prepared as described previously (22). In addition to the standard guanidinium method of RNA preparation, some total RNA samples were prepared using the RNeasy kit (Qiagen) and required no further processing to remove contaminating DNA.

Ten micrograms of total RNA were used in the RNase protection assay. This was carried out as described previously using the Ambion RNase protection assay kit (22), except for the inclusion of an antisense riboprobe for ovine glyceraldehyde-3-phosphate dehydrogenase (G3PDH; Barrett, P., unpublished data), which was used to standardize sample loading. The protected RNA fragments were separated on an 8 M urea-5% polyacrylamide gel in 1 x TBE (90 mM Tris-borate and 2 mM EDTA). Gels were exposed to Kodak X-Omat LS film (Eastman Kodak, Rochester, NY) for 1–3 days. Densitometric quantification of the bands on the autoradiographs was carried out using Bioimage software (Bioimage, Watford, UK).

Northern blot analysis of c-fos expression
PT cells were prepared as described above. On day 2, cells were harvested by scraping and collected by centrifugation. Cells were then washed twice by resuspension and centrifugation in an equal volume of DMEM containing 0.1% BSA (serum-free medium). After the final wash cells were resuspended in serum-free medium and plated out in 60-mm petri dishes at 107 cells/dish in 4.5 ml serum-free medium. Cells were left for 20–24 h before commencement of treatments.

The PKC inhibitor Ro 31–8220 [dissolved in dimethylsulfoxide (DMSO)] was added to 0.5 ml prewarmed serum-free medium to give a concentration of 1 µM. Control cells received an equivalent amount of DMSO in 0.5 ml serum-free medium. Cells were incubated at 37 C for 1 h. The treatments were terminated as described above.

RNA was extracted using the RNeasy kit (Qiagen). Ten micrograms of total RNA were run on a denaturing 1% agarose gel (26) and transferred to nylon (GeneScreen, New England Nuclear-DuPont, Boston, MA). The nylon filter was probed with a 32P-labeled v-fos probe and reprobed with a 32P-labeled G3PDH probe to check RNA loading (17). Densitometric quantification of the bands on the autoradiographs was carried out using the Millipore Bioimage software.

Western blotting
Aliquots of 2 x 106 PT cells were seeded in 60-mm petri dishes and treated with PMA (1 µM) or Ro 31–8220 (1 µM) or left unstimulated for 16 h. Particulate fractions of PT cells were prepared as previously described (27). Cells were lysed in the petri dish by the addition of 200 µl lysis buffer (20 mM Tris-HCl, pH 7.5; 0.25 M sucrose, 10 mM EGTA; 2 mM EDTA; 1 mM phenylmethylsulfonylfluroride; and 20 µg/ml leupeptin), scraping, and trituration through a 25 gauge syringe needle. The membrane fraction was pelleted by centrifugation at 100,000 x g for 1 h at 4 C. The supernatant was removed, and the membrane pellet was resuspended in lysis buffer containing 1% Triton X-100. After an additional 60-min incubation on ice, nonsolubilized material was pelleted at 100,000 x g for 30 min at 4 C. The supernatant from this centrifugation step was removed, and an aliquot was used for protein determination by the method of Bradford (28). Ten micrograms of solubilized membrane protein diluted with 2 x Laemmli SDS-PAGE loading buffer were loaded onto a 10% SDS-acrylamide gel (29). After electrophoresis, the proteins were transferred to an Immobilon-P polyvinylidene difluoride membrane using the Bio-Rad transblotter in Towbin transfer buffer (25 mm Tris, 190 mM glycine, and 20% methanol, pH 7.5) (30).

The polyvinylidene difluoride membrane was blocked for 1 h in Blotto (10 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1% Tween 20; and 5% fat-free dried skimmed milk) before incubation with the primary antibodies diluted in the same buffer. Anti-PKC monoclonal antibodies were used at the following dilutions; anti-PKC{alpha}, 1:2500; anti-PKC{gamma}, 1:5000; anti-PKC{epsilon}, -{lambda}, and -{iota}, 1:250; and anti-PKCµ, 1:1000. The specificity of these antibodies has been demonstrated previously (31, 32, 33). After a 1-h incubation with the primary antibody, membranes were washed five times, for 5 min each wash, in Blotto without fat-free dried skimmed milk. The second antibody was an antimouse horseradish peroxidase-conjugated antibody used at a 1:5000 dilution in Blotto and incubated with the membranes for 1 h. The membranes were then washed as described above. Detection of the immune complexes was made using the ECL chemiluminescent detection system.

PKC assay
PT cells were prepared as described above. On day 2 of culture, the cells were harvested, collected by centrifugation, and washed twice with serum-free medium. Cells were then replated at a density of 2.5 x 106 cells in 1.8 ml in poly-D-lysine-coated 35-mm petri dishes. Cells were incubated for 16–20 h before commencement of treatments. The PKC inhibitor Ro 31–8220 at a final concentration of 1 µM or DMSO vehicle was added, and incubation proceeded for 30 min before additional drugs were added. Additional drugs, diluted in serum-free medium and prewarmed to 37 C, were added in a volume of 200 µl. Cells were further stimulated for 30 min. At the end of the stimulation period, cells were placed on ice. The medium was aspirated, and the cells were washed three times with ice-cold 1 x PBS. After the last wash, cells were scraped off the plate in 200 µl homogenization buffer (50 mM Tris-HCl, pH 7.5; 10 mM EGTA; 5 mM EDTA, pH 8; 20 mM NaF; 0.2 mm sodium vanadate; 20 mM ß-glycerophosphate; 10 mM benzamidine; 0.3% ß-mercaptoethanol; 50 µg/ml phenylmethylsulfonylfluoride; and 0.5 µg/ml leupeptin). Cells were transferred to a chilled Eppendorf tube, then passed through a 25-gauge syringe seven times to lyse the cells. Lysates were kept on ice until assayed.

The PKC assay was performed with the Amersham Biotrak PKC assay kit according to the manufacturer’s instructions using 25 µl of the PT cell homogenate. Values for PKC activity were normalized for protein content, which was assessed using an aliquot of the cell homogenates in conjunction with the Bradford protein assay (28).

[125I]2-Iodomelatonin binding assays and measurement of cAMP
[125I]2-Iodomelatonin binding assays using 200 pM [125I]2-iodomelatonin and measurement of intracellular cAMP levels were performed as previously described (22).

Statistics
The effects of treatments in the PKC assay and c-fos expression by Northern analysis were analyzed by one-way ANOVA using the SigmaStat software package (Jandel, Erkrath, Germany). Differences were assessed using the Student-Newman-Keuls method for pairwise multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of PMA on Mel 1a mRNA and protein levels
Treatment of ovine PT cells in culture with 10 µM forskolin resulted in a 2- to 10-fold linear increase in mRNA levels for the Mel 1a receptor. This increase was observable by 2 h postforskolin stimulation, reached a maximal response after 4 h, and remained elevated in the presence of forskolin (Fig. 1Go) (22). When PT cells were treated with the PKC activator PMA after a 4-h stimulation by forskolin, the level of Mel 1a mRNA was reduced. This was dependent on the concentration of PMA in the culture medium, being completely ineffective at concentrations below 100 pM (Fig. 2AGo). In addition to its inhibitory effect on Mel 1a mRNA levels previously elevated by forskolin, PMA could prevent the increase caused by forskolin when it was added 15 min before forskolin treatment (Fig. 2BGo). Furthermore, PMA in the absence of forskolin was able to reduce basal mRNA levels to values approaching the limit of detection (data not shown). The specificity of the action of PMA was verified by the substitution of PMA by its inactive analog, 4{alpha}PMA. This compound failed to prevent Mel 1a mRNA levels from increasing when added 15 min before forskolin treatment or to reverse the mRNA levels after 4-h forskolin stimulation (Fig. 2BGo).



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  		Figure 1. Time course of Mel 1a induction by forskolin over the first 4 h of forskolin treatment. PT cells were incubated with or without 10 µM forskolin for the times indicated. The cells were harvested, and RNA was prepared, then assayed for Mel 1a mRNA by RNase protection analysis. The upper panel shows the autoradiograph from the RNase protection experiment, and the lower panel is a densitometric quantification of the Mel 1a mRNA bands normalized to the level of G3PDH mRNA. Shown is a representative experiment. The experiment was repeated three times with similar results.

 


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  		Figure 2. The effect and specificity of PMA treatment on Mel 1a mRNA levels in PT cells. A, PT cells were stimulated for 4 h with 10 µM forskolin before subsequent addition of various concentrations of PMA and incubation for an additional 4 h. There was a dose-dependent inhibition by PMA of Mel 1a mRNA levels. B, The specificity of the effect of PMA was verified by the inability of 10 nM 4{alpha}PMA to inhibit or reverse the effect of forskolin on Mel 1a mRNA levels. The effect of 10 nM PMA was demonstrated whether PMA was added 15 min before or 4 h after forskolin stimulation of the PT cells. The upper panel shows the autoradiograph from the RNase protection experiment, and the lower panel is a densitometric quantification of the Mel 1a mRNA bands normalized to the level of G3PDH mRNA. Shown is a representative experiment. The experiment was repeated three times with similar results.

 
The time course for the reversal of the forskolin-stimulated increase in Mel 1a mRNA levels by PMA was investigated. Although during the 4-h period following forskolin stimulation there was some decline in Mel 1a mRNA levels in non-PMA-treated cells, the addition of 10 nM PMA to the culture medium 4 h after the initial forskolin stimulation caused a more rapid decline and reduced Mel 1a mRNA levels to basal (time zero) values within this time (Fig. 3Go).



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  		Figure 3. Time-dependent reversal of forskolin-stimulated Mel 1a mRNA levels by PMA. Aliquots of 107 PT cells were stimulated for 4 h with 10 µM forskolin before the addition of 10 nM PMA to half of the culture dishes. Cells were harvested at the times indicated relative to the start of the experiment (time zero). Within 4 h, there was a return to basal values of Mel 1a mRNA levels in the PMA-treated cell cultures. The upper panel shows the autoradiograph from the RNase protection experiment, and the lower panel is a densitometric quantification of the Mel 1a mRNA bands normalized to the level of G3PDH mRNA. Shown is a representative experiment. The experiment was repeated three times with similar results.

 
The effect of PMA on Mel 1a mRNA levels was reflected in the levels of melatonin receptor protein in PT cells. Figure 4Go shows the spontaneous increase in receptor protein when PT cells were cultured for 24 h after their recovery from cell dispersion. Melatonin or PMA applied for this 24-h period attenuated this spontaneous increase. Treatment with forskolin, which increases Mel 1a mRNA levels, also produced an increase in receptor protein in PT cells similar to the level obtained in control cells. Paradoxically, the PKC inhibitor Ro 31–8220 inhibited the spontaneous increase in receptor protein in PT cells (see below).



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  		Figure 4. The effects of PMA and Ro 31–8220 on the spontaneous increase in receptor density in PT cells. [125I]2-Iodomelatonin binding experiments were used to determine the receptor density in response to treatments as indicated. Time zero was the beginning of the experiment, when forskolin (Fsk; 10 µM), melatonin (Mel; 10 nM), PMA (10 nM), or Ro 31–8220 (Ro; 1 µM) was added to the cultures and incubated for an additional 24 h. Shown are the mean ± SEM for a representative experiment.

 
One possible explanation for the effect of PMA is that it acts through a decrease in cAMP levels in PT cells. However, analysis of intracellular levels of cAMP 30 min and 4 h after the addition of PMA to cells previously stimulated with forskolin for 4 h did not show a greater reduction than those in control cells stimulated with forskolin alone (Fig. 5AGo). In addition, there was no effect on basal levels of cAMP (Fig. 5BGo). Thus, PMA through activation of isoforms of PKC, exerts a cAMP-independent action to inhibit transcription of the Mel 1a gene and/or decrease mRNA stability.



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  		Figure 5. The effects of PMA and Ro 31–8220 on basal and forskolin-stimulated levels of intracellular cAMP. A, PT cells were stimulated for 4 h with 10 µM forskolin before the addition of 10 nM PMA, 1 µM Ro 31–8220 (Ro), or 10 nM melatonin (Mel). Cells were harvested 30 min or 4 h after the additional treatment and washed three times with PBS. Cell pellets were frozen at -70 C until cAMP measurements were made. Cells were lysed, then heated to 100 C in cAMP assay buffer before an aliquot was used for cAMP determination. B, PMA or Ro 31–8220 was added to resting cells after 4 h in culture. Cells were harvested and assayed for intracellular cAMP levels 30 min or 4 h after addition of the PKC stimulant or inhibitor. Shown are the mean ± SEM of a representative experiment.

 
The similarity between the effects of PMA and melatonin on Mel 1a mRNA and protein levels (Figs. 2BGo and 4Go) suggested a possible mechanism through which melatonin may act, i.e. via activation of PMA-responsive PKC isoforms. To test this possibility, we examined the effect of melatonin on Mel 1a mRNA levels in PKC down-regulated cells and determined whether melatonin could activate PKC.

PT cells were pretreated with 1 µM PMA for 16 h to down-regulate the known isoforms of PKC affected by this treatment. The effectiveness of this PMA treatment was assessed by Western blotting with antibodies to a range of PKC isoforms, which include representatives of the three classes of PKC enzymes: 1) the classical PMA/Ca2+-responsive class, {alpha}; 2) the novel, mainly PMA-responsive, but Ca2+-independent class, {delta}, {epsilon}, and µ (µ is not PMA responsive); and 3) the atypical, PMA- and Ca2+-independent class, {iota} and {lambda}. Figure 6AGo shows that after a 16-h PMA exposure, PMA-responsive PKC isoforms {alpha} and {delta} that were present in untreated PT cells were undetectable. The PMA-responsive isoform PKC{epsilon} was partially down-regulated (~40% reduction) with this treatment. PMA-nonresponsive PKC isoforms µ, {iota}, and {lambda} were present in the PT, but the amounts of these enzymes were not reduced by prolonged PMA exposure. The PKC inhibitor Ro 31–8220 was also tested and did not have an effect on the levels of any of these PKC isoforms.



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  		Figure 6. PKC down-regulation does not impede the effect of melatonin on forskolin stimulation of Mel 1a mRNA levels. A, Western blot analysis of PKC isoforms present in PT membranes. PT cells were treated for 16 h with 1 µM PMA or 1 µM Ro 31–8220 (Ro) or were left untreated (Con). PT membranes were prepared as described in Materials and Methods. Ten micrograms of solubilized membrane proteins were separated on a 10% SDS-polyacrylamide gel and Western blotted with antibodies to isoforms of PKC. At least 6 of the 12 known isoforms were present in the ovine PT. PKC{alpha} and -{delta} were completely down-regulated by prolonged PMA exposure. B, PKC isoforms were depleted by a 16-h incubation with 1 µM PMA. Using the RNase protection assay, cells were then assayed for the ability to respond to 10 µM forskolin and subsequent inhibition of Mel 1a mRNA levels by melatonin and 10 nM PMA. The upper panel shows the autoradiograph from the RNase protection experiment, and the lower panel is a densitometric quantification of the Mel 1a mRNA bands normalized to the level of G3PDH mRNA. Shown is a representative experiment. The experiment was repeated 3 times with similar results.

 
PT cells treated for 16 h with PMA were assayed for Mel 1a mRNA (Fig. 6BGo). The level of Mel 1a mRNA in PMA-pretreated cells was reduced relative to that in untreated control cells. Stimulation with forskolin induced at least a 2- to 3-fold increase in Mel 1a mRNA levels, which after 4 h begins to exceed the basal level in the untreated control cells. Treatment of these PMA-pretreated cells with PMA (10 nM) added 15 min before forskolin treatment (data not shown) or after a 4-h stimulation by forskolin was unable to inhibit or reverse Mel 1a mRNA levels. In contrast, melatonin (10 nM) reversed the forskolin-stimulated levels to basal values within 4 h. This experiment supports the specificity of action of PMA. In addition, it can be concluded that activation of a PMA-responsive isoform of PKC is not the primary pathway through which melatonin is able to reverse the forskolin-induced increase in receptor mRNA and protein levels. Furthermore, although PKC{epsilon} is activated by PMA, PKC{epsilon} is unlikely to be the mediator of PMA action, as this PKC isoform was only partially down-regulated after prolonged PMA treatment, but no effect was seen on Mel 1a mRNA levels after subsequent exposure to PMA.

PKC activity in PT cells
To determine whether PKC activity may have a physiological significance to the regulation of the melatonin receptor mRNA and protein levels, we assayed PKC activity in PT cells.

PT cells were serum depleted, then treated as indicated in Fig. 7Go, before assaying for PKC activity. These data demonstrate a basal level of PKC activity in serum-depleted cells. This PKC activity can be reduced by the PKC inhibitor Ro 31–8220 by at least 50%. PMA (10 nM) increased PKC activity in PT cells by approximately 50%, and this could be inhibited by pretreatment with Ro 31–8220. In this study we also observed an inhibition of PKC activity in the presence of 10 nM melatonin (28 ± 5.1%; n = 4; P < 0.05). This latter result rules out a common pathway for Mel 1a receptor regulation by PMA and melatonin through activation of classical PKC isoforms.



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  		Figure 7. Percent change in PKC activity in PT cells in response to PMA, the PKC inhibitor Ro 31–8220, and melatonin. Serum-depleted PT cells were treated with the drugs indicated as described in Materials and Methods. PKC activity was determined from an aliquot of the cell lysate and expressed as a percentage of the value in control unstimulated cells (Con). The final concentration of Ro 31–8220 (Ro) was 1 µM; that of both PMA and melatonin (Mel) was 10 nM. *, Statistically significant difference relative to the control; **, statistically significant difference relative to PMA-treated PT cells (P < 0.05).

 
That this basal PKC activity in PT cells may be significant and have a functional consequence is reflected in the observation that c-fos expression is detectable in serum-depleted cells and can be reduced (28 ± 4%; n = 4; P < 0.05) by the PKC inhibitor Ro 31–8220.

An additional pathway involved in receptor regulation
The above data confirm that activation of a PMA-responsive PKC isoform is involved in Mel 1a receptor regulation in PT cells. However, in initial studies we set out to confirm the specificity of PMA action on PKC through use of the potent PKC inhibitor Ro 31–8220 (also referred to as bisindolymalemide IX), whose structure is based upon that of another PKC inhibitor, staurosporine.

Paradoxically, we found Ro 31–8220 to have an inhibitory effect on Mel 1a mRNA levels similar to that of PMA. Like PMA, Ro 31–8220 added to the culture medium 15 min before forskolin or added 4 h after forskolin treatment was able to inhibit or reverse Mel 1a mRNA levels with a time course similar to that of melatonin (Fig. 8AGo). As with PMA, in the absence of forskolin, Ro 31–8220 reduced basal levels of Mel 1a mRNA levels in unstimulated cells (Fig. 8AGo). Furthermore, consistent with the effect of this inhibitor on Mel 1a mRNA levels, the spontaneous increase in receptor density was also prevented (Fig. 4Go). The effect of staurosporine-like PKC inhibitors on Mel 1a mRNA levels is not limited to Ro 31–8220, as a similar effect was observed with another member of this family of PKC inhibitors, GF-109203X (bisindolymalemide I; Fig. 8BGo). However, the inhibitory effect of these compounds does not extend to all members of this group, as Ro 31–6045 (bisindolymalemide V) does not reduce Mel 1a mRNA levels (data not shown).



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  		Figure 8. Effects of the PKC inhibitors Ro 31–8220 and GF 109203X on Mel 1a mRNA levels in PT cells. Aliquots of 107 PT cells were treated as indicated, and Mel 1a mRNA levels were assessed using the RNase protection assay. A, The effect of Ro 31–8220 (1 µM) on Mel 1a mRNA levels when added to PT cells 15 min before or 4 h after forskolin (10 µM) stimulation. B, The effect of GF 109203X (1 µM) on Mel 1a mRNA levels when added to PT cells 4 h after stimulation with forskolin. The upper panel shows the autoradiograph from the RNase protection experiment, and the lower panel is a densitometric quantification of the Mel 1a mRNA bands normalized to the level of G3PDH mRNA. Shown is a representative experiment. The experiment was repeated three times with similar results.

 
The PKC inhibitor Ro 31–8220 was applied to PT cells in culture that had been depleted of specific PKC isoforms ({alpha} and {delta}, completely; {epsilon}, 40%; by Western analysis) by prolonged exposure to PMA. Like melatonin, but in contrast to PMA, Ro 31–8220 was able to reverse forskolin-stimulated levels of Mel 1a mRNA (Fig. 6BGo).

The effect of Ro 31–8220 was not mediated through an inhibitory effect on forskolin-stimulated or basal levels of cAMP (Fig. 5Go), ruling out an effect on this signal transduction event. Similar results were also obtained for GF-109203X (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study demonstrate that the activation of PKC has profound effects on the expression of both Mel 1a receptor mRNA and protein. The inducibility of this response by the phorbol ester PMA implicates the PMA-stimulated isoforms as mediators of this response. The question of whether these same isoforms might provide the mechanism for the cAMP-independent suppression of Mel 1a expression by melatonin, described previously (22), was a potentially attractive hypothesis, as melatonin has been reported to activate PKC in the SCN (34). However, this hypothesis can be discounted, as stimulation of PT cells with melatonin does not stimulate PKC activity. In addition, in cells depleted of some forms of PKC, melatonin was still effective at reducing forskolin-stimulated Mel 1a mRNA levels. This latter response is likely to be attributable to cAMP inhibition by melatonin with or without a contribution from the cAMP-independent mechanism of Mel 1a mRNA regulation (22). This further emphasizes the independent nature of the melatonin and PMA responses. The mechanism by which activation of PKC could lead to the suppression of forskolin-stimulated Mel 1a mRNA expression is presently unclear. Perhaps the most plausible mechanism is phosphorylation by PKC of proteins involved in transcriptional regulation or mRNA stability, of which the latter mechanism is known to be involved in regulating the expression of other receptor mRNAs through PKC activation (35, 36, 37, 38). In addition to a reduction in Mel 1a mRNA levels, PKC activation could have a direct effect on the receptor protein, as a number of PKC phosphorylation sites exist in the C-terminal domain of the sheep Mel 1a receptor and could contribute to receptor desensitization. However, this may be an unlikely mechanism for melatonin receptor regulation, as melatonin can still inhibit forskolin-induced cAMP levels in PT cells in the presence of PMA (39), and PKC activation does not change receptor density in transfected NIH-3T3 cells (40).

The effect of Ro 31–8220 and GF 109203X was unexpected and paradoxical, as both of these compounds, which are staurosporine analogs, are well characterized PKC inhibitors, and PKC assays demonstrate that Ro 31–8220 is an effective inhibitor of PKC activity in PT cells. The mechanism by which these compounds exert their cAMP-independent action could be through inhibition of a PKC enzyme involved in maintaining transcriptional activity or mRNA integrity. This is supported by the inability of another staurosporine analog (Ro 31–6045) that does not inhibit PKC enzymes to decrease Mel 1a mRNA levels. Several PKC enzymes could be ruled out in this mechanism, including PKC{alpha} and -{delta}, as Ro 31–8220 is still effective in PT cells in which these enzymes have been down-regulated. However, this mechanism of Mel 1a mRNA down-regulation may be an oversimplistic interpretation, as Ro 31–8220 has now been demonstrated to have inhibitory and stimulatory effects on gene expression. The new pharmacological effects of Ro 31–8220, which have been reported recently in Rat-1 fibroblasts, include inhibition of c-fos expression and stimulation of mitogen-activated protein kinase phosphatase-1 and c-Jun protein levels. These effects are likely (but not conclusively proven) to be independent of PKC (41). Although we cannot draw any parallel between the cAMP-independent actions of Ro 31–8220 and melatonin on Mel 1a mRNA levels, the effect of Ro 31–8220 to stimulate phosphatase activity in Rat-1 fibroblasts (41) raises the possibility that melatonin may be exerting its cAMP-independent action through a similar mechanism.

In this study we observed a small inhibitory effect of melatonin on basal PKC activity in four consecutive independent experiments. However, we cannot unambiguously conclude that melatonin inhibits PKC activity, as in other experiments PKC inhibition in response to melatonin was not observed (Ross, A. W., C. A. Webster, M. H. Thompson, P. Barrett, and P. J. Morgan, unpublished observations). This variability may reflect variations in cell preparation, the breed of sheep from which the PTs were collected, or even the time of year the PTs were collected and experiments were performed. Therefore, we do not make a correlation between potential inhibition by melatonin and Ro 31–8220 as the common cAMP-independent mechanism to inhibit Mel 1a mRNA levels.

Thus, together with the data in this study, we have identified four potential mechanisms to regulate Mel 1a receptor expression. The first two mechanisms, which involve melatonin, include either a reversal of elevated cAMP levels or a mechanism that is cAMP independent. A third mechanism invoked by Ro 31–8220 (and GF 109203X) is undefined, but the possibility does exist that certain isoforms of PKC are involved. The fourth mechanism of activation involves PMA-responsive isoforms of PKC, which leads to Mel 1a mRNA down-regulation. This latter mechanism may be physiologically important to regulation of the melatonin receptor in specific tissues. For example, melatonin has been reported to activate PKC in the SCN (34) and thus may be involved in an autoregulatory mechanism of expression. Regulation of melatonin receptors by PKC activation may also play an important role in the neonatal pars distalis. In this tissue there is the potential for PKC activation through LHRH-induced elevation in diacylglycerol and Ca2+ (42, 43), which could result in a down-regulation of melatonin receptor mRNA. In the neonatal pituitary, activation of PKC by LHRH may be the mechanism by which the receptor is developmentally down-regulated by day 30 after birth (44). In the PT, elevation of diacylglycerol through turnover of inositol phosphates and phosphatidylcholine and an increase in Ca2+ can occur with the aid of pharmacological tools, but no stimulatory input to these pathways is known at the present time (45, 46). Moreover, these second messengers are not regulated by melatonin in PT cells (45, 46). However, the ability of Mel 1a mRNA to be regulated by PKC in the PT may be physiologically relevant. We have observed in the absence of serum, a basal PKC activity that has the functional consequence of contributing to the constitutive expression of c-fos mRNA. This basal PKC activity may have a negative effect on Mel 1a mRNA levels, as a further 50% increase in PKC activity is sufficient to reverse forskolin-stimulated levels or reduce basal levels of Mel 1a mRNA.

In summary, activation of the classical group of PKC isoforms by PMA causes a reduction in mRNA levels for the Mel 1a receptor and inhibits a subsequent spontaneous increase in receptor levels at the cell membrane. Although melatonin does not act through the classical isoforms of PKC in PT cells, the endogenous level of PKC activity in PT cells may have a physiological significance.


   Acknowledgments
 
We thank Keith Pennie for his assistance with the collection of tissue samples, and Michael Thompson for the initial supply of the PKC inhibitor and his comments on the manuscript.


   Footnotes
 
1 This work was supported by the Scottish Office Agricultural Environment and Fisheries Department. Back

2 Present address: EMBL, Meyerhof Strasse, Postfach 10 22 09, 69012 Heidelberg, Germany. Back

3 Present address: Department of Agriculture, University of Aberdeen, King Street, Aberdeen, Scotland AB24 5UA. Back

Received June 27, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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