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Laboratory of Developmental Chronobiology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Dr. Catherine Godson, Laboratory of Developmental Chronobiology, Jackson 1226, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: godson{at}helix.mgh.harvard.edu
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Abstract |
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stimulation on phospholipase C activation. Melatonin potentiates
arachidonate release stimulated by PGF2 and by
ionomycin. The effects of melatonin on arachidonate release are
sensitive to inhibition of protein kinase C. They are independent of
the effects of melatonin on cAMP and do not appear to involve
activation of mitogen-activated protein kinase. The effects of
melatonin on both phosphoinositide hydrolysis and arachidonate release
are sensitive to pertussis toxin treatment. Thus, we show that the
melatonin signal is transduced by parallel pathways involving
inhibition of adenylyl cyclase and potentiation of phospholipase
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies of the endogenous receptor in vertebrates indicate high affinity melatonin receptors negatively coupled to adenylyl cyclase by a pertussis toxin (PTX)-sensitive G protein (1). Using NIH 3T3 cells expressing the human Mel1a receptor, we investigated whether the receptor is coupled to inhibition of adenylyl cyclase activity and/or phosphoinositide hydrolysis and arachidonate (AA) release. Our data indicate that the human Mel1a receptor is coupled to inhibition of forskolin-stimulated cAMP accumulation via a PTX-sensitive G protein. Although melatonin alone does not stimulate phosphoinositide phospholipid hydrolysis or AA release in the Mel1a-expressing cells, it markedly potentiates the effects of calcium-mobilizing agents on these phenomena. These effects of melatonin are sensitive to PTX. The potentiation of AA release by melatonin is 1) sensitive to the protein kinase C (PKC) inhibitor GF 102903X and to PKC down-regulation, 2) independent of the effects of melatonin on cAMP, and 3) does not appear to involve activation of mitogen-activated protein (MAP) kinase. We conclude that the melatonin signal may be transduced by activation of parallel pathways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stable transfection and culture of NIH 3T3 cells
Cloning of the human melatonin receptor by RT-PCR has previously
been described (3). The complementary DNA encoding the
Mel1a receptor was subcloned into pcDNA-Neo (Invitrogen,
San Diego, CA) and transfected into NIH 3T3 cells using Lipofectin
(Life Technologies, Gaithersburg, MD). Colonies of transfected cells
were selected by culture in 1 mg/ml G418 (Geneticin (Life
Technologies). Cell lines expressing high levels of receptor were
selected on the basis of 2-[125I]melatonin binding. The
cell line used in the studies described here specifically bound more
than 500 fmol [125I]melatonin/mg protein, with a
Kd of 30 pM.
NIH 3T3 cells were routinely cultured in DMEM supplemented with 10% FCS, penicillin (50 U/ml), and streptomycin (50 µg/ml) at 37 C in a humidified atmosphere of 5% CO2. Culture medium for transfected stocks was supplemented with 1 mg/ml G418.
cAMP assay
Cells were plated at 1 x 105/35-mm plate and
harvested 48 h later. After washing twice with DMEM, cells were
preincubated for 10 min with 250 µM isobutylmethyxanthine
and then stimulated with vehicle, forskolin (10 µM),
melatonin (1 µM unless otherwise indicated), or both
forskolin and melatonin in isobutylmethyxanthine as described above.
After 10 min at 37 C, the medium was aspirated, and the cells were
lysed by boiling in 50 mM CH3COOH. The lysates
were centrifuged at 13,500 x g for 15 min, and cAMP in
the supernatant was determined by RIA (DuPont-New England Nuclear,
Boston, MA). All determinations were made in triplicate. The effects of
PTX on cAMP accumulation were measured after overnight treatment of the
cells with 100 ng/ml PTX (List Biologicals, Campbell, CA).
Inositol phosphate (InsP) accumulation
Cells were plated at 1 x 105/35-mm plate.
Twenty-four hours later the cells were washed twice with DMEM and
labeled overnight with 2 µCi/ml [3H]myo-inositol
(DuPont-New England Nuclear) in DMEM containing 0.1% BSA. The cultures
were washed twice with DMEM and preincubated for 10 min in 10
mM LiCl DMEM before the addition of vehicle,
PGF2 (final concentration, 1 µM), or
melatonin (1 µM) in 10 mM LiCl. After 30 min
at 37 C, medium were aspirated, and the cells were scraped into 500
µl MeOH-HCl (100:1) and kept on dry ice for 15 min before extraction
with 2 ml CHCl3, 800 µl H2O, and 1 ml
MeOH-HCl (100:1). The resulting aqueous phase was diluted before
applying it to anion exchange columns (AG-1X8, formate form, Bio-Rad
Laboratories, Richmond, CA). After washing with water and 0.1
M formic acid, InsP were eluted with 2 M
ammonium formate and 0.1 M formic acid.. Aliquots of the
eluate were taken for liquid scintillation counting. All measurements
were made in triplicate (elution profiles from the columns were
verified using purified [3H]InsP; DuPont-New England
Nuclear). The effects of PTX were determined as described above.
[3H]AA release
Cells were plated at 0.5 x 105/35-mm
plate. After 48 h in culture, cells were labeled with 0.2 µCi
[3H]AA/ml 0.5% FCS-DMEM. Cells were washed twice with
DMEM before stimulation with various reagents in DMEM-1 mg/ml fatty
acid-free BSA,(6) as indicated. After incubation at 37 C for the
indicated times, free AA release into the extracellular medium was
quantitated by liquid scintillation counting. The effect of PTX was
determined as described above. The effects of cAMP-modulating agents
(8-Br-cAMP and Rp-cAMP; Calbiochem, La Jolla, CA) were determined in
cells treated with these agents (1 µM) in the presence of
PGF2 and/or melatonin as indicated.
The effect of PKC down-regulation was investigated by pretreating the cells for 18 h with 500 nM PMA before measuring AA release. This treatment did not compromise [3H]AA incorporation into the cells. In experiments using the specific PKC inhibitor GF 109203X, bisindolylmaleimide (7) (Calbiochem) cells were pretreated with this compound (10 µM) or vehicle (0.1% dimethylsulfoxide) for 15 min before stimulation with various agents as indicated.
MAP kinase immunoblotting
Subconfluent cell cultures were rendered quiescent by serum
starvation for 24–36 h. After this period, cells were stimulated with
various agents in DMEM for 10 min as indicated. After washing with PBS
(4 C), lysates were harvested in RIPA buffer containing protease
inhibitors (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS
containing 2 mM PMSF, 20 µM
NaVO3, 50 mM NaF, 20 µM
leupeptin, and 10 µg/ml aprotinin). The lysates were clarified by
centrifugation at 15,500 x g for 10 min. Aliquots of
the supernatant were removed for protein assay (Bio-Rad), and the
remainder was denatured by incubation for 5 min at 95 C in SDS-PAGE
sample buffer. The cell lysates (25 µg protein/well) were resolved on
a 12% SDS-PAGE gel before blotting onto nitrocellulose membranes.
Nonspecific sites on the membranes were blocked by incubation in 3%
nonfat milk TBS. The blots were incubated with primary antibody
(anti-ERK-2, Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:500 in
blocking buffer containing 0.05% Tween-20 overnight at 4 C. After
several washes, incubation with an alkaline phosphate-conjugated
secondary antibody, and subsequent washing, specific antibody binding
was detected with a colorimetric substrate (4-nitro blue tetrazolium
chloride and 5-bromo-4-chloro-3-indolyl-phospate, Kirkegaard and Perry,
Gaithersburg, MD).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Melatonin
was without effect on forskolin-stimulated cAMP accumulation in
wild-type NIH 3T3 cells or in cells transfected with the pcDNA vector
(data not shown). Melatonin has also been shown to inhibit
forskolin-stimulated cAMP accumulation in cells expressing the
recombinant Mel1a, Mel1b, and Mel1c
receptors from different species with comparable IC50
values (3, 4, 5).
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). This suggests that
the human Mel1a receptor is coupled through
Gi, the only PTX substrate expressed in these
cells (8). PTX sensitivity of the effects of melatonin on cAMP has also
been demonstrated on expression of the other recombinant receptors and
on the endogenous receptors of vertebrates (1, 9).
Melatonin potentiates phosphoinositide hydrolysis
Melatonin stimulation of NIH 3T3 cells expressing the
Mel1a receptor did not alter phosphoinositide phospholipid
hydrolysis. However, PGF2-stimulated phosphoinositide
phospholipid hydrolysis was potentiated by costimulation with melatonin
(PGF2 alone, 2.5 ± 0.3-fold basal;
PGF2 and melatonin, 7.4 ± 0.9-fold basal;
P < 0.001; Fig. 2). Melatonin was
without effect on PGF2-stimulated phosphoinositide
metabolism in wild-type NIH 3T3 cells (data not shown).
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). It was insensitive to inhibition of PKC with GF 109203X (data
not shown). In PTX-treated cells, we consistently observed an increase
in PG-stimulated inositide production. The reason for this is not
clear, but it suggests possible tonic inhibition of
Gq-activated phospholipase Cß (PLCß)
(10).
Melatonin potentiates AA release
Although melatonin alone was without effect on AA release from the
Mel1a-transfected cells, melatonin potentiated the effects
of PGF2 on AA release (PGF2, 1.85 ±
0.07-fold basal; PGF2 and melatonin, 5.8 ±
0.2-fold basal; Fig. 3). The EC50 for
melatonin potentiation of AA release was approximately 0.5 x
10-9 M (data not shown). Melatonin also
potentiated ATP- and thrombin-stimulated AA release (data not shown).
In cells pretreated with PTX, melatonin potentiation of AA release was
not observed (Fig. 4A). PTX treatment enhanced the
sensitivity of the cells to PGF2 stimulation of AA
release, similar to its enhancement of PGF2-stimulated
InsP hydrolysis. (That we did not observe potentiation of AA release on
costimulation with melatonin was not because PGF2 alone
generated the maximal AA response, because in parallel experiments
combined ionomycin and PMA stimulation generated greater fold basal AA
release.)
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). These data indicate that melatonin can
potentiate AA release in combination with agents that elevate
intracellular calcium.
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-, melatonin-, or
combined melatonin- and PGF2-stimulated AA release (Fig. 4B).
Melatonin potentiation of AA release is PKC dependent
To investigate whether functional PKC was required for the
observed effects of melatonin, we used two approaches: pretreatment
with the PKC inhibitor GF 109203X and down-regulation of the kinase by
overnight incubation with phorbol ester. Both of these manipulations
resulted in an inhibition of the effect of melatonin on potentiation of
AA release (Fig. 4, C and D). PGF2 and melatonin
treatment of PKC-down-regulated cells resulted in 1.7 ± 0.1-fold
basal AA release, whereas under control conditions, the fold basal AA
release was 5.6 ± 0.2 (P < 0.001). GF 102903X
pretreatment of cells reduced the fold basal AA release stimulated by
PGF2 and melatonin from 4.9 ± 0.5 to 3 ±
0.27 (P < 0.05). The specificity of GF 109203X as a
PKC inhibitor has previously been demonstrated in intact 3T3 cells (7).
These data suggest that functional PKC is required for the observed
effects of melatonin on potentiation of AA release.
To control for the possibility that the loss of responsiveness in the PKC-down-regulated cells was not attributable to a loss in receptor number, as has been shown for some other GPCR (12), we determined [125I]melatonin binding in phorbol ester-treated and control cells. [125I]Melatonin binding was unchanged by PKC down-regulation (data not shown).
Melatonin potentiation of AA release is independent of MAP kinase
activation
Recent evidence suggests a role for MAP kinase in the activation
of hormone-stimulated AA release. Several
Gi-coupled receptors have been shown to activate
MAP kinase (13, 14). To determine whether the potentiation of AA
release by melatonin is related to a change in MAP kinase activation,
we used immunoblotting with an anti-MAP kinase-specific antibody in an
electrophoretic mobility gel shift assay (Fig. 6). Our
data indicate that PGF2 stimulation is associated with a
shift in the mobility of both p42 and p44 forms of MAP kinase,
consistent with the modification of electrophoretic mobility of the
activated phosphorylated form compared with the nonphosphorylated
inactive form (15). In contrast, melatonin treatment did not alter the
electrophoretic mobility of either the p42 or p44 form of the kinase.
Furthermore, when cells were stimulated with both melatonin and
PGF2, we did not detect any potentiation of the effects
observed with PGF2 alone. The observed effects on MAP
kinase hyperphosphorylation were PTX insensitive.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The physiological relevance of our findings is supported by several
observations. First, the reported effects on cAMP and phospholipases
can be achieved at physiological concentrations of melatonin. Data on
the endogenous receptor obtained from vertebrate tissue consistently
shows that the receptor is coupled to inhibition of adenylyl cyclase
(1, 9). Second, there is evidence of melatonin potentiating a
PLC-mediated phenomenon; studies from rat caudal artery indicate that
1-adrenergic receptor-stimulated vasoconstriction is
potentiated by physiological concentrations of melatonin (16). Third,
the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN)
has been identified as a major target of melatonin’s action (1).
Interestingly, in the context of the data presented here, recent
evidence suggests a role for PKC in modulating the effects of melatonin
on phase shifting of electrical activity in the SCN (17). Furthermore,
melatonin has been proposed to activate a K+ current in SCN
neurons (18). AA modulation of several K+ currents has also
been demonstrated previously (14, 19, 20).
GPCR activation of phospholipase C can be mediated through PTX-insensitive and sensitive G proteins (e.g. Gq and Gi/Go, respectively) (reviewed in Ref.21). Activation of phosphoinositide-specific PLC generates a bifurcating second messenger system: inositol 1,4,5-trisphosphate (InsP3), which releases Ca2+ from intracellular stores, and 1,2-diacylglcerol (DAG), which activates PKC. Hormone-sensitive AA release is modulated by the recently described cytosolic PLA2 (cPLA2) (22). This protein is a member of the Ca2+-dependent phospholipid binding domain family of pro-teins (23, 24). It requires elevated calcium to promote its translocation to the membrane, and phosphorylation is required for maximal catalytic activity (23, 25). cPLA2 activation may occur subsequent to PLC and protein kinase activation. Activation of the enzyme on phosphorylation by both MAP kinase and PKC has been shown (26, 27, 28). Direct coupling of the enzyme to a PTX-sensitive G protein has been suggested in some systems (29). AA release is the rate-determining step in the synthesis of PGs and leukotrienes. Recent evidence suggests an important role for the eicosanoids in ion channel regulation and synaptic transmission (reviewed in Refs. 19 and 20).
To date, evidence for a role of melatonin in modulation of phospholipid
metabolism has been inconclusive. In neonatal rat gonadotrophs,
melatonin is without effect on DAG and AA release, but can inhibit
LHRH-dependent activation of these pathways (30). In ovine pars
tuberalis melatonin is without effect on phospholipid metabolism (31, 32). In chick retina, dopaminergic increases in cAMP can be inhibited
by melatonin, which is without effect on the D1 receptor-induced
increase InsP production (33). The data presented here demonstrate that
melatonin can potentiate PGF2 activation of PLC. This
effect is PTX sensitive. Melatonin also potentiates
PGF2- and ionomycin-stimulated AA release. Melatonin
potentiation of AA release is PTX sensitive and is dependent on
functional PKC.
The potentiation of hormone-stimulated AA release in response to
stimulation of several Gi-coupled receptors has
previously been shown. D2 dopaminergic, 2-adrenergic,
and M2 and M4 muscarinic receptors act synergistically with
Ca-mobilizing agents to promote AA release. These phenomena are
sensitive to PTX and can be mimicked by guanosine
5'-O-(3-thiotriphosphate) (11, 34), but are independent of
effects on PLC activation (11, 35). In contrast, adenosine potentiation
of muscarinic receptor-stimulated AA release appears to involve
activation of PLC in some systems (36). Receptor-dependent potentiated
AA release has also been shown after stimulation of endogenous
receptors in neurons and astrocytes (37, 38).
The sensitivity of melatonin-facilitated AA release to PKC inhibition
and down-regulation that we report here suggests that melatonin may
activate PKC. This activation may be isoform selective (6, 39, 40), as
melatonin alone does not stimulate AA release, whereas PMA stimulation
does. Differential regulation of PLC and AA metabolism by PKC and
-
has been demonstrated in NIH 3T3 cells (40, 41). The activation of
conventional PKC isoforms is dependent on DAG. That melatonin alone
does not stimulate phosphoinositide hydrolysis argues against the
hormone acting as a classical PKC activator. However, DAG produced by
hydrolysis of other phospholipids, e.g. phosphatidylcholine,
has frequently been shown to activate PKC (42). Further evidence
supporting possible PKC activation by melatonin is suggested by our
observation that melatonin cannot potentiate AA release stimulated by
the PKC activator PMA. In contrast, melatonin potentiates AA release
stimulated by the calcium ionophore ionomycin. Thus, one might envisage
that maximal AA release could be facilitated by ionomycin-induced
increases in intracellular calcium, resulting in cPLA2
translocation in combination with melatonin-activated PKC and
subsequent cPLA2 phosphorylation.
cPLA2 can be activated by MAP kinase. Activation of p42 and
p44 MAP kinase by Gi-coupled receptors has
previously been shown (13, 14). PGF2 activation of MAP
kinase by a PTX-insensitive pathway has recently been demonstrated in
NIH 3T3 cells. (43). This suggests that MAP kinase activation might be
an upstream locus at which the PGF2 and melatonin
signals converge under permissive conditions to activate
cPLA2. However, the data presented here do not support this
hypothesis: We have demonstrated PGF2 activation of both
p42 and p44 MAP kinases, but we did not discern any effect of melatonin
on either form of MAP kinase when it was added alone or in combination
with PGF2. Recent evidence from ovine pars tuberalis
also indicates that melatonin does not activate either p42 or p44 MAP
kinase (44). A further possibility is that melatonin might activate a
p38 MAP kinase that has recently been described in thrombin-activated
platelets. cPLA2 is a downstream target of this kinase
(45).
That melatonin alone is without effect on AA release or PLC activation
raises the question of what do PGF2 and ionomycin do to
sensitize the system to the effects of the hormone? A hypothetical
scenario is summarized in Fig. 7. The lack of effect of
melatonin on AA release argues against direct coupling of the receptor
to cPLA2. In NIH 3T3 cells, the PGF2
receptor is coupled through Gq to PLC activation
(46). The effects of melatonin that we report here are coupled through
the PTX-sensitive G protein, G. Gß
activation of PLCß has
previously been shown (47, 48, 49). Activation of some forms of PLCß by
ß are calcium dependent (50). We hypothesize that the G protein
ß-subunits released after melatonin receptor stimulation can
conditionally activate PLC subsequent to PGF2
stimulation. This may explain how melatonin potentiates both PLC and
cPLA2 activation. Thus, the increase in intracellular
Ca2+ produced subsequent to PGF2-mediated
Gq activation of PLC and InsP3
generation facilitates ß activation of PLCß. Activation of PLC,
in turn, generates InsP3 and DAG. This may involve
superactivation of the PLCß species initially activated by
Gq or activation of an alternative form of PLC
(10). As PTX inhibits receptor-mediated dissociation of
Gi/Go into its - and
ß-subunits the sensitivity of PLC activation and AA release to PTX
can be explained by inhibition of ß production. The finding that
the ß-subunits produced from the Gi
heterotrimer may activate PLC in preference to the ß-subunits of
the Gq complex might be explained by the relative
abundance of the Gi complex, which is typically
found in a 10-fold greater abundance than the Gq
complex (10). Some preliminary data support this hypothesis. Melatonin
potentiates intracellular calcium mobilization of
PGF2-stimulated cells loaded with fura-2. The
melatonin-induced increase in Ca2+ is PTX sensitive and
independent of extracellular calcium.2
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Acknowledgments |
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Footnotes |
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2 Vanecek, J., C. Godson, and S. M. Reppert,
unpublished.
3 Godson, C., and S. M. Reppert,
unpublished.
Received July 15, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 J. VANECEK Cellular Mechanisms of Melatonin Action Physiol Rev, July 1, 1998; 78(3): 687 - 721. [Abstract] [Full Text] [PDF]
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A. W. Ross, C. A. Webster, M. Thompson, P. Barrett, and P. J. Morgan A Novel Interaction Between Inhibitory Melatonin Receptors and Protein Kinase C-Dependent Signal Transduction in Ovine Pars Tuberalis Cells Endocrinology, April 1, 1998; 139(4): 1723 - 1730. [Abstract] [Full Text] [PDF]
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E. Gilad, H. Matzkin, and N. Zisapel Inactivation of Melatonin Receptors by Protein Kinase C in Human Prostate Epithelial Cells Endocrinology, October 1, 1997; 138(10): 4255 - 4261. [Abstract] [Full Text] [PDF]
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B. McMahon, C. Stenson, F. McPhillips, A. Fanning, H. R. Brady, and C. Godson Lipoxin A4 Antagonizes the Mitogenic Effects of Leukotriene D4 in Human Renal Mesangial Cells. DIFFERENTIAL ACTIVATION OF MAP KINASES THROUGH DISTINCT RECEPTORS J. Biol. Chem., September 1, 2000; 275(36): 27566 - 27575. [Abstract] [Full Text] [PDF]
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