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Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
Address all correspondence and requests for reprints to: Dr. Mark S. Roberson, Department of Biomedical Sciences, T6–008a Veterinary Research Tower, Cornell University, Ithaca, New York 14853. E-mail: msr14{at}cornell.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cells resulted in
tyrosine phosphorylation of several intracellular proteins. Separation
of phosphorylated proteins by ion exchange chromatography suggested
that GnRH receptor stimulation can activate the p38 MAPK pathway.
Immunoprecipitation studies using a phospho-tyrosine antibody resulted
in increased amounts of immunoprecipitable p38 MAPK from T3–1 cells
treated with GnRH. Immunoblot analysis of whole cell lysates using a
phospho-specific antibody directed against dual phosphorylated p38
kinase revealed that GnRH-induced phosphorylation of p38 kinase was
dose and time dependent and was correlated with increased p38 kinase
activity in vitro. Activation of p38 kinase was blocked
by chronic phorbol ester treatment, which depletes protein kinase C
isozymes and 
. Overexpression of p38 MAPK and an activated form
of MAPK kinase 6 resulted in activation of c-jun and
c-fos reporter genes, but did not alter the expression
of the glycoprotein hormone -subunit reporter. Inhibition of p38
activity with SB203580 resulted in attenuation of GnRH-induced
c-fos reporter gene expression, but was not sufficient
to reduce GnRH-induced c-jun or glycoprotein hormone
-subunit promoter activity. These studies provide evidence that the
GnRH signaling pathway in T3–1 cells includes protein kinase
C-dependent activation of the p38 MAPK pathway. GnRH integration of
c-fos promoter activity may include regulation by p38
MAPK. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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q/11-mediated activation of phospholipase C,
leading to the production of inositol 1,4,5-trisphosphate and
diacylglycerol, increased intracellular calcium, and activation of
protein kinase C (PKC) (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). The consequences of GnRH receptor
occupancy include increased synthesis and secretion of the gonadotropic
hormones, LH and FSH. Ablation of GnRH stimulation of the anterior
pituitary results in decreased glycoprotein hormone subunit messenger
RNA concentrations and diminished secretion of the gonadotropic
hormones (13, 14). In addition to the glycoprotein hormone subunit
genes, GnRH stimulates increased c-fos (15, 16),
c-jun (15), mitogen-activated protein kinase phosphatase 2
messenger RNA and transcriptional activation of the ternary complex
factor Elk-1 (17). GnRH-dependent intracellular signaling events downstream of PKC have only recently been characterized. Several groups have demonstrated that GnRH receptor occupancy results in activation of extracellular signal regulated protein kinases (ERK) (17, 18, 19, 20) and c-jun N-terminal protein kinase (21) (Mulvaney, J. M., T. Zhang, and M. S. Roberson, in preparation), members of the mitogen-activated protein kinase (MAPK) superfamily. Acute phorbol ester administration can mimic GnRH action on ERKs (17, 18, 19). Chronic phorbol ester treatment to deplete PKC isozymes reduces GnRH-induced ERK activity, suggesting a requirement for PKC in GnRH activation of the ERK pathway (18, 19). GnRH activation of the JNK cascade is dependent on the low mol wt GTP-binding protein, Cdc42 (21), and appears to be activated independently of diacylglycerol-dependent PKC isozymes, which differentiates GnRH modulation of the ERK and JNK cascades (Mulvaney, J. M., T. Zhang, and M. S. Roberson, in preparation).
In addition to the ERK and JNK cascades, the MAPK superfamily includes
the p38 kinase pathway (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). Studies of other
Gq-coupled receptors have shown that ligand association
results in activation of all three MAPK pathways (36, 37). Activation
of the p38 signaling pathway has been linked to activation of Elk-1
ternary complex factor, which binds to the c-fos promoter
(38), and myocyte enhancer family (MEF-2)-dependent c-jun
gene expression (39), suggesting a potential role for p38 MAPK in the
modulation of immediate early gene expression in pituitary
gonadotropes. The present studies investigated the possibility that
GnRH induces activation of p38 MAPK in the T3–1 gonadotrope cell
line. We report here that GnRH stimulates activation of the p38 kinase
pathway. Activation of the p38 kinase by GnRH requires PKC. Our studies
suggest that GnRH-induced p38 MAPK activation may selectively
contribute to the regulation of c-fos protooncogene
expression, but not c-jun or the glycoprotein hormone
-subunit gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit promoter linked to luciferase has been described previously
(17). Expression vector for Gal4-Elk-1 was prepared as
previously described (17). Expression vector for MAPK kinase-6 (MKK-6)
Glu was a gift from Dr. R. Davis (University of Massachusetts, Boston,
MA). Bacterial expression vector for
glutathione-S-transferase (GST)-activating transcription
factor-2 (ATF-2) was a gift from Dr. M. Green (University of
Massachusetts). Bacterial expression and partial purification of GST
fusion proteins were accomplished as previously described (41).
Expression vector for c-jun-luciferase reporter was a gift
from Dr. Ron Prywes (Columbia University, New York, NY) and has been
described previously (42). All plasmid DNA was prepared by two cycles
of centrifugation through cesium chloride.
Cell culture and transfection
T3–1 cells (provided by Dr. P. Mellon, University of
California-San Diego) (17, 43, 44) were maintained in monolayer culture
in DMEM supplemented with 5% FBS and 5% horse serum (Life Technologies, Grand Island, NY). For immunoblotting and
transient transfection studies, cells were grown to approximately 60%
confluence before transfection. For immunoblot studies, cells were
serum deprived for 2 h before hormone treatments. Agonists
included the GnRH analog buserelin
([D-Ser(tBu)6,Pro9-ethylamide]GnRH;
this analog is referred to as GnRHa), phorbol myristate acetate (PMA),
or sorbitol (0.3 M) in DMEM. In some experiments, the
GnRH antagonist
([N-Ac-D-Nal(2)1-pCl-D-Phe2-D-Pal(3)3-Lys(Nic)5-D-Lys(Nic)6-Lys(iPr)8-D-Ala10]GnRH;
antide, Bachem, Torrance, CA) was added to the medium 30 min before and
during treatment with buserelin. For transient transfection studies,
cells were transfected by electroporation using a single electrical
pulse at 220 V and 950 µF, as described previously (17). In
transfection studies, T3–1 cells were treated with buserelin at a
concentration of 10 nM for 16 h before collection (18
h after electroporation) to allow for accumulation of luciferase
activity. Some transfected cells were given 20 µM
SB203580 (Calbiochem, La Jolla, CA) beginning 2 h
before buserelin administration. SB203580 remained on transfected cells
for the duration of hormone treatment. After cell collection, lysates
were prepared by three freeze-thaw cycles and clarified by
centrifugation, and luciferase activity was determined in equal amounts
for cellular protein (45).
Mono Q chromatography
For preparation of large scale T3–1 cell lysate for
fractionation by Mono Q chromatography, cells were cultured in 150-mm
dishes to approximately 50% confluence. Cells were serum starved for
2 h, then treated (where indicated) with control solution or the
GnRH analog buserelin (10 nM) for 15 min. The dishes were
then placed on ice, washed three times with ice-cold buffer containing
0.15 M NaCl and 10 mM HEPES (pH 7.5), and lysed
with gentle agitation for 15 min at 4 C. The lysis buffer contained 70
mM ß-glycerophosphate (pH 7.2), 2 mM sodium
vanadate, 2 mM MgCl2, 1 mM EGTA,
0.5% Triton X-100, 2.5 µg leupeptin/ml, 2.5 µg pepstatin/ml, 0.2
mM phenylmethylsulfonylfluoride, 5 mM
benzamidine, and 1 mM dithiothreitol (buffer A). Cell
debris was removed by centrifugation, and clarified supernatants (equal
amounts of total protein, 1.0–1.5 mg) were loaded onto a Mono Q ion
exchange chromatography column (Pharmacia Biotech,
Piscataway, NJ) maintained at 4 C. During all procedures, the column
was maintained at a flow rate of 0.3 ml/min, and fractions were
collected in 1.0-ml volumes. After loading of the cell lysate, the
column was washed with 5 ml buffer A and eluted in a NaCl gradient
(buffer A plus 1.0 M NaCl) from 0–0.4 M NaCl
delivered over a 25-ml column elution. For experiments with cells
treated without or with buserelin, control cell lysates were processed
first, followed immediately (column was washed and reequilibrated
between lysate preparations) by lysates from cells receiving buserelin
treatment. Fractions (50 µl) were then subjected to immunoblot
analysis as described above.
Antibodies, immunoprecipitation, immunoblotting, and kinase
assays
Monoclonal antibodies directed against phosphotyrosine (4G10;
3.9 mg/ml) were provided by Dr. B. Drucker (Oregon Health Sciences
University, Portand, OR). An antibody dilution of 1:12,500 was
used for all phosphotyrosine blots, and 5% BSA was used as a blocking
agent. AU1 monoclonal antibody was purchased from BAbCO (Berkeley, CA).
Specific polyclonal antibodies directed against ERKs and p38 kinase,
horseradish peroxidase-coupled secondary antibodies (antimouse and
antirabbit), and protein A/G-agarose were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and used according to
the manufacturer’s instruction. Phospho-specific antibodies for ERK
and p38 kinase were purchased from New England Biolabs, Inc. (Beverly, MA), and used according to the manufacturer’s
instructions. The antibodies for PKC isozymes, , , and 
were
obtained from Life Technologies. For immunoprecipitations
(IP) and immunoblotting, T3–1 cells were treated for the specified
time periods, then washed in ice-cold buffer containing 0.15
M NaCl and 10 mM HEPES (pH 7.5). The cells were
lysed in a buffer containing 25 mM HEPES (pH 7.5), 3
mM ß-glycerophosphate, 3 mM EDTA, 3
mM EGTA, 250 mM NaCl, 2 mM sodium
vanadate, 1% Triton X-100, 1 mM dithiothreitol, 1
mM phenylmethylsulfonylfluoride, and 5 mM
benzamidine (referred to as lysis buffer) at 4 C for 10 min with gentle
rocking. The cell lysates were scraped from the dishes and clarified by
centrifugation. Methods for IP and subsequent washes have been
described previously (17). For whole cell lysates (40–80 µg total
protein) or IPs, proteins were resolved on denaturing polyacrylamide
gels and transferred to polyvinylidene difluoride (PVDF) membrane by
electroblotting. After immunostaining, specific proteins or
phosphotyrosine accumulation was visualized with enhanced
chemiluminescence reagents using protocols described by the supplier
(New England Nuclear-DuPont, Boston, MA). Stripping of PVDF membranes
was accomplished by placing the membrane in a solution containing 62.5
mM Tris (pH 6.8), 2% SDS, and 100 mM
2-mercaptoethanol and heating to 55 C for 30 min. The membrane was then
washed three times (10 min each) in Tris-buffered saline (pH 7.5)
containing 0.1% Tween-20. For p38 kinase assays, IP complexes were
washed four times in 1 ml lysis buffer followed by one wash in a kinase
buffer containing 20 mM HEPES (pH 7.5), 20 mM
MgCl2, 25 mM ß-glycerol phosphate, 100
µM sodium vanadate, 50 mM ATP, and 2
mM dithiothreitol. The IP agarose beads were resuspended in
kinase buffer, and [
-32P]ATP (5 µCi) and a specific
p38 substrate (recombinant GST-ATF-2) were added last. Samples were
subjected to kinase reaction for 30 min at 30 C. The reaction was
stopped by the addition of SDS loading buffer. Samples were then boiled
for 2 min and resolved by SDS-PAGE. Kinase activity was visualized by
autoradiography and analyzed by scanning densitometry.
Statistical analysis
Transfection data were subjected to ANOVA, and treatment
differences were determined by either pairwise t test or
Tukey’s Studentized range test. Differences were considered
statistically significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cells results in induction of
tyrosine-phosphorylated proteins that include p38 kinaseT3–1 cells (Fig. 1
). T3–1
cells are a clonal cell line derived from targeted oncogenesis of the
gonadotrope cell lineage of the anterior pituitary (43, 44). T3–1
cells were treated with control solution or the GnRH agonist buserelin
(GnRHa; Fig. 1). Administration of GnRHa for 15–30 min resulted in
phosphotyrosine accumulation of at least three proteins in the
approximate molecular mass range of 43 and 29 kDa. We have previously
demonstrated that GnRH receptor occupancy activates the ERK signaling
pathway (17), and p44/p42 ERKs probably accounted for two of the
phosphotyrosine-containing proteins. The presence of multiple
phosphotyrosine-containing bands suggested the possibility that GnRH
also induced phosphorylation of other members of the MAPK family.
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T3–1
cells was used to identify other MAPK family members that may play a
role in GnRH action. Cell extracts from GnRHa-treated T3–1 cells
were separated by ion exchange chromatography on a Mono Q column,
fractions were further resolved by denaturing PAGE, and
phosphotyrosine-containing proteins were identified by immunoblotting
(Fig. 2A). The results of these studies
revealed that phosphotyrosine-containing proteins in the 43–29 kDa
range could be resolved into two groups of fractions (fractions 38–44
and fractions 48–50). Analysis with antibodies for specific members of
the MAPK family indicated that ERK-1 and ERK-2 elute with fractions
38–44 (Fig. 2B), whereas p38 elutes in fractions 48–50 (Fig. 2C). In
a second study, T3–1 cells were treated with control solution (Fig. 2D) or GnRHa (Fig. 2E) for 15 min, cells were lysed, and lysates were
subjected to Mono Q fractionation. Fractions expected to contain p38
immunoactivity were then assayed by phosphotyrosine immunoblotting. A
marked accumulation of a phosphotyrosine-containing band with the
appropriate size and elution profile for p38 kinase was observed after
GnRH treatment. These studies suggest that GnRH treatment may activate
the p38 MAPK.
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T3–1 cells
would result in IP of increased amounts of the p38 MAPK. T3–1 cells
were treated with GnRHa for 15 min, and whole cell lysates were
prepared. An antiphosphotyrosine antibody was used to IP
tyrosine-phosphorylated proteins induced by GnRHa. The proteins present
in the IP were resolved by SDS-PAGE and transferred to PVDF membrane.
Immunoblot analysis was performed using antibodies directed against p38
kinase (Fig. 3). Consistent with our
prediction, a greater amount of p38 kinase was present in the IP from
T3–1 cells treated with GnRHa compared with control cells. This
study is consistent with observations from Mono Q chromatography and
provides additional evidence that mechanisms of GnRH signaling include
phosphorylation of p38 MAPK.
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). GnRHa induced activation of p38 MAPK
at doses as low as 0.1 nM (4.3-fold) and appeared to have a
modest peak in activation at 10 nM GnRHa (5.5-fold).
Time-course studies revealed that increased phospho-specific staining
of the p38 MAPK was evident after 15 min (5.2-fold) of GnRHa treatment,
began to decline at 30 min (4.4-fold), and had essentially returned to
baseline 2 h after hormone administration (Fig. 4A). Similar
amounts of p38 kinase (independent of the phosphorylation state) were
present in all lanes. The second approach to assay p38 kinase
activation was an immune complex kinase assay using GST-ATF-2 as a
substrate (Fig. 4B). GnRHa administration for 15 or 30 min stimulated
an increase (2.5- and 3.5-fold for 15 and 30 min, respectively) in p38
kinase activity consistent with results obtained with phospho-specific
p38 antiserum. Sorbitol was administered in these studies to induce
osmotic shock, and p38 kinase activity was used as a positive control
(40, 46). Concurrent administration of the specific GnRH receptor
antagonist, antide, at a 10-fold molar excess blocked GnRHa-induced p38
MAPK activation (data not shown). These studies provide direct evidence
that GnRH receptor occupancy results in activation of p38 kinase in
T3–1 cells.
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T3–1 cells (18, 19). Further, phorbol ester treatment has been
shown to activate the p38 kinase pathway in some cell types (47). We
tested the hypothesis that GnRH-induced activation of p38 kinase was
PKC dependent. Chronic treatment of T3–1 cells with PMA (100
nM for 20 h) was used to deplete PKC isozymes. After
PKC depletion, T3–1 cells were treated with GnRHa for the specified
times (Fig. 5A). Phospho-specific p38 and
phospho-specific ERK immunoblots were conducted on T3–1 whole cell
lysates to determine the activation state of MAPKs. In the absence of
chronic PMA treatment, GnRHa induced activation of p38 kinase and ERKs
(Fig. 5A). PKC depletion by chronic PMA administration resulted in a
marked attenuation of GnRHa-induced phosphorylation of p38 kinase and
ERKs. PKC isozyme depletion in this paradigm did not appreciably alter
the total amounts of p38 or ERKs present in T3–1 cells (Fig. 5A).
To confirm the efficacy of our PKC depletion studies, amounts of PKC
isozymes present before and after chronic administration of PMA were
investigated by immunoblot analysis (Fig. 5B). T3–1 cells express
at least three different isozymes of the PKC family (, , and )
(48). Chronic phorbol ester administration at 100 nM PMA
for 20 h resulted in a near-complete loss of the and
isozymes (Fig. 5B). Additional immunoblot analysis for the PKC
isozyme revealed that this isozyme remained unchanged after chronic
phorbol ester treatment in T3–1 cells (data not shown). These
findings suggest a requirement for diacylglycerol-dependent PKC
isozymes in GnRH-mediated p38 kinase activation similar to that
demonstrated for GnRH-induced activation of ERKs (18, 19).
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T3–1 cells-subunit promoter (15, 16, 17, 38). The -subunit promoter is
regulated by the ERK pathway (17, 18, 19); however, regulation by other
MAPK pathways has not been examined. Studies examined whether p38
activation was sufficient and/or required for the regulation of
protooncogene expression or the -subunit promoter. Initial
investigation focused on isolating the activation of the p38 signaling
cascade in the absence of ERK or JNK activation in T3–1 cells to
determine whether activation of the p38 pathway alone was sufficient to
activate the various reporter genes. The MKK-6 has been reported as a
specific activator of p38 kinase (47, 49). Substitution mutations at
Ser207 and Thr211 with Glu results in partial
activation of MKK-6 (referred to as MKK-6 Glu). Cotransfection of p38
and MKK-6 Glu expression vectors resulted in a dramatic increase in p38
kinase activity in COS-1 cells (47). Based upon this approach, T3–1
cells were transfected with expression vector for epitope-tagged (AU1)
p38 kinase alone or in combination with MKK-6 (Glu). p38 kinase
activity was then isolated by IP using the AU1 monoclonal antibody and
subjected to kinase assay using GST-ATF-2 as substrate. AU1-p38 kinase
protein levels were similar in the absence or presence of MKK-6 Glu
expression vector in transfected T3–1 cells (Fig. 6A). Transfection of MKK-6 Glu expression
alone was not sufficient to activate endogenous p38 kinase (not shown).
Cotransfection of AU1-p38 with MKK-6 Glu expression vector resulted in
marked activation of p38 kinase. MKK-6 has been shown to be a specific
activator of p38 kinase, but not ERKs or JNK, and our results were
consistent with previous studies with activation of p38 kinase by
overexpression of MKK-6 Glu in COS-1 cells (47). This approach provided
an overexpression paradigm to specifically activate the p38 kinase
without activation of parallel MAPK (ERK and JNK) pathways. Based upon
these experimental conditions, we examine the effects of specific p38
kinase activation on expression of the -subunit, c-jun,
and c-fos reporter genes (Fig. 6B). Specific activation of
p38 kinase did not alter expression of the glycoprotein hormone
-subunit reporter gene activity. Overexpression and activation of
p38 kinase was sufficient to activate the c-jun and
c-fos reporter genes. Replacement of the p38 expression
vector with expression vectors for wild-type ERK-2 or JNK in additional
transfection studies did not result in activation of the
c-jun or c-fos reporter constructs (data not
shown), suggesting that the effects of overexpression of MKK-6 Glu and
p38 were specific.
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-subunit or protooncogene expression. In these studies,
we used the specific p38 kinase inhibitor, SB203580 (50). SB203580 is a
pyridinyl imidazole that specifically inhibits the p38 MAPK in a
dose-dependent manner, but does not inhibit ERK or JNK activity (50).
T3–1 cells were transfected with the various reporter genes and
cultured in the absence or presence of 20 µM SB203580 for
2 h. Some cells then received GnRHa for approximately 16 h.
All cells were collected approximately 18 h after electroporation
and assayed for luciferase activity. Administration of SB203580
resulted in a reduction of basal levels of -subunit reporter
activity. GnRHa administration induced the -subunit reporter
approximately 6-fold, and administration of SB203580 did not alter this
effect (Fig. 7). Thus, the reduction in
the absolute magnitude of GnRHa-induced -subunit reporter activity
with SB203580 pretreatment was attributable to changes in basal level
of -subunit reporter activity. SB203580 administration did not alter
basal or GnRH-induced c-jun reporter gene expression
levels (Fig. 7). In contrast, pretreating transfected cells with
SB203580 resulted in a partial loss (
50%) of GnRHa-induced
activation of the c-fos protooncogene (Fig. 7). Similar
partial inhibition was obtained in experiments using 10
µM SB203580 (not shown), suggesting that 20
µM SB203580 was saturating. Collectively, these studies
provide evidence that GnRH induction of the p38 MAPK cascade is
probably not required for the activation of the -subunit or
c-jun promoters, but can selectively contribute to
GnRH-induced transcriptional activation of the c-fos
protooncogene.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cells resulted
in an accumulation of tyrosine-phosphorylated p38 kinase within 15 min
of treatment. Tyrosine phosphorylation of p38 kinase was correlated
with increased enzyme activity in vitro. The time course of
p38 activation was consistent with activation of the ERK cascade, with
peak activity at 15 min and a return to baseline by 1–2 h after
hormone administration (Mulvaney, J. M., T. Zhang, and M. S.
Roberson, unpublished observations). In contrast, GnRH induction of the
JNK cascade is slightly delayed, with peak activity occurring at 30 min
after hormone treatment (21) (Mulvaney, J. M., T. Zhang, and
M. S. Roberson, unpublished observations). GnRH receptor occupancy
resulting in activation of all three MAPK pathways is consistent with
other serpentine receptors that couple to Gq/11.
Endothelin (ET) interaction with the ET-B receptor results in ERK, JNK,
and p38 activation (51). In studies of the ET-B receptor, the
carboxyl-terminal 146 amino acids of the cytoplasmic tail were required
to couple to multiple MAPK pathways (37). The GnRH receptor has only
two amino acids associated with the carboxyl-terminal cytoplasmic tail
(for review, see Ref. 52). Recent studies (53) have shown that
overexpression of the third intracellular loop from the GnRH receptor
appears to interfere with inositol phosphate production in
GH3 cells stably transfected with the GnRH receptor
complementary DNA; however, the role of intracellular loops within the
GnRH receptor in modulating multiple MAPK signaling has not yet been
determined. Thus, the actual mechanism(s) of coupling of serpentine
receptors to multiple MAPK pathways may vary with structural
differences among receptor subtypes.
The p38 MAPK is a mammalian homolog of the yeast protein HOG-1. HOG-1
was originally identified as an intracellular mediator of osmotic
stress (28, 40, 46). The p38 kinase has been shown to be activated by
osmotic manipulation, UV irradiation, endotoxins, and cytokines (28, 46, 47, 54, 55). The present studies demonstrate that the effects of
osmotic stress (induced by sorbitol treatment) on p38 kinase activity
were greater than activation induced by GnRHa in T3–1 cells.
Osmotic stress-induced p38 kinase activity also does not appear to
require PKC (Roberson, M. S., unpublished observation),
demonstrating a possible divergence in upstream regulators of the p38
kinase. The reasons for variable magnitude in p38 kinase activity
induced by stress or GnRH are not presently known. However, it is
possible that in response to adverse environmental conditions, high
level or prolonged activation of p38 kinase (combined perhaps with
other MAPK pathways) may be required to regulate critical mechanisms of
cell survival (or cell death). Activation of p38 kinase by GnRH appears
to support cellular mechanisms that contribute to immediate early gene
expression.
Previous studies have demonstrated a requirement for PKC isozymes on
GnRH-mediated ERK activation (18). The present studies extend these
observations, demonstrating that PKC isozyme depletion can block
GnRH-induced p38 activation. Our studies and those of others also using
T3–1 cells (56) have shown that the cellular content of both PKC
and PKC are depleted by chronic phorbol treatment. Interestingly,
recent studies (56) failed to detect redistribution of PKC to the
membrane after GnRH stimulation of T3–1 cells, raising the
possibility that this isozyme is not involved in GnRH signaling.
However, as with other cell systems (57), more extensive time-course
studies will be required to confirm these results and investigate the
possibility the PKC may have translocated to the membrane more
rapidly than the reported time course. Thus, it may be premature to
discount a role for PKC in mediating the effects of GnRH on
activation of ERKs or p38 MAPK. In contrast to the isozyme, PKC
was found to redistribute to the membrane after GnRH administration
(56) consistent with a role for the isozyme in mediating responses
to GnRH, including activation of the ERK and p38 cascades. More
definitive studies of the role of specific PKC isozymes in mediating
GnRH effects will require the development of reagents that can
selectively block the action of specific PKC isozymes.
To date, several downstream targets of p38 kinase have been identified. Inflammatory cytokines and heat stress result in p38 kinase-dependent activation of MAPK-activated protein kinase-2 (MAPKAP-2) and MAPKAP-3, leading to the phosphorylation of the small heat shock protein, Hsp27 (58, 59, 60, 61). Two additional targets of p38 kinase are transcription factors that are presumably activated by p38 kinase-dependent phosphorylation, including the ternary complex factor, Elk-1 (38, 47) and MEF-2 (39). In the present studies, both MKK-6 Glu/p38 overexpression and studies using SB203580 demonstrated that p38 activation was sufficient and at least partially required for GnRH signaling to the c-fos reporter. These observations are consistent with studies of p38 activation of the c-fos serum response element and activation of the ternary complex factor Elk-1 (38, 61). Our present studies suggest that while p38 may contribute to c-fos gene expression, it is probably not the primary mechanism for GnRH action on this immediately early gene. This is based on two observations. First, overexpression studies using MKK-6 Glu and p38 did increase c-fos promoter activity. We used this overexpression paradigm simply to isolate p38 kinase activity in the absence of other related MAPK family members. The caveat to these studies is that the level of p38 kinase activity induced by MKK-6 overexpression far exceeded that induced by GnRH. This may suggest that high levels of p38 activity are sufficient to activate the c-fos reporter. The possibility exists that lower levels of p38 activity (such as that induced by GnRH) may contribute less to c-fos expression probably relative to more robust contributions of GnRH-induced ERK and JNK activities. Second, only partial attenuation of GnRH-induced c-fos reporter gene activity by SB203580 was observed. In 3T3 cells, 10 µM SB203580 was sufficient to block 95% of p38 kinase activity without significant alteration of ERK or JNK activity (50). This suggests that the conditions used in the present study (20 µM SB203580) were sufficient to block more than 95% of GnRH-induced p38 enzyme activity. Probably other signaling mechanisms, such as GnRH-induced ERK or JNK activation, may play a primary role in up-regulation of the c-fos protooncogene, whereas GnRH-induced p38 activity may represent a contributory, but secondary, mechanism.
The p38 MAPK has been linked to transcriptional regulation of
c-jun via an MEF-2-binding site present on the
c-jun promoter (39). In those studies, MEF-2C was shown to
be a specific target of the p38 MAPK pathway. Specific activation of
p38 kinase by overexpression of MKK-6 Glu/p38 MAPK in the present
studies did result in a 2-fold activation of the c-jun
promoter in T3–1 cells. However, administration of SB203580 was not
sufficient to block GnRH-induced activation of the c-jun
reporter, suggesting that p38 kinase activation was not required for
GnRH action on c-jun. These data support the conclusion that
overexpression of activated p38 MAPK at high levels was sufficient to
activate the c-jun reporter probably in a manner not
consistent with GnRH action. Lower levels of p38 MAPK activity induced
by GnRH are not apparently required for GnRH-induced c-jun
promoter activity in T3–1 cells.
The studies presented have identified the p38 MAPK signaling pathway as
a target of GnRH hormone action. Activation of p38 kinase by GnRH
requires PKC isozymes. One possible consequence of GnRH action on p38
kinase appears to be contribution to the transcriptional regulation of
the c-fos protooncogene. These studies also suggest that
GnRH action on the tissue-specific promoter for the glycoprotein
hormone -subunit gene does not require activation of p38 kinase.
Activation of the ERK, JNK, and p38 MAPK pathways by GnRH receptor
activation is consistent with other serpentine receptors and provides a
mechanism to modulate the expression and activity of multiple
transcriptional activators.
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Acknowledgments |
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Footnotes |
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Received September 8, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit, growth hormone, and prolactin after
hypothalamic pituitary disconnection in ovariectomized ewes.
Endocrinology 119:2704–2710-subunit promoter
by gonadotropin-releasing hormone. Mol Cell Biol 15:3531–3539[Abstract]
T3–1 cell line is mediated by protein kinase C, c-src
and CDC42. Mol Endocrinol 12:815–824 activation of
p21ras. Proc Natl Acad Sci USA 91:12706–12710T3–1 gonadotroph-derived cell line. FEBS Lett 333:67–72[CrossRef][Medline]
T3–1 cell line. Mol Cell Endocrinol 118:103–111[CrossRef][Medline]
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O. Benard, Z. Naor, and R. Seger Role of Dynamin, Src, and Ras in the Protein Kinase C-mediated Activation of ERK by Gonadotropin-releasing Hormone J. Biol. Chem., February 9, 2001; 276(7): 4554 - 4563. [Abstract] [Full Text] [PDF]
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