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Is Mediated by a Protein Kinase C-Dependent Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Pathway in Bovine Luteal Cells1
The Women’s Research Institute (D.C., H.W.F., J.S.D.), Departments of Obstetrics and Gynecology, and Internal Medicine, University of Kansas School of Medicine-Wichita, and Research Service of the Department of Veterans Affairs (J.S.D.), 1010 North Kansas, Wichita, Kansas 67214
Address all correspondence and requests for reprints to: John S. Davis, Ph.D., The Women’s Research Institute, 1010 North Kansas, Wichita, Kansas 67214-3199. E-mail: jdavis3{at}kumc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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triggers the demise of the corpus luteum whereby progesterone
synthesis is inhibited, the luteal structure regresses, and the estrus
cycle resumes. Upon binding to its heterotrimeric G-protein-coupled
receptors, PGF2 initiates the phospholipase C/diacylglycerol and
inositol-1,4,5-trisphosphate/Ca2+-protein kinase C (PKC)
signaling pathway. More recently, we have demonstrated that PGF2
activates extracellular signal-regulated kinase (ERK) mitogen-activated
protein (MAP) kinase signaling through a Raf-dependent mechanism in
bovine luteal cells. However, the relationship between PKC and ERK
activation in PGF2 signaling has not been clearly defined. Moreover,
the signaling pathway that PGF2 uses to regulate gene expression is
unknown. In this report, primary cultures of bovine luteal cells were
used to address the role of PKC in ERK activation and the signaling
pathway for induction of c-fos and
c-jun messenger RNA (mRNA) expression in response to
PGF2. By using a PKC inhibitor and a PKC-deficient luteal cell
model, we observed that phorbol ester-responsive isoforms of PKC were
required for ERK phosphorylation and activation by PGF2 (1
µM) or phorbol 12-myristate 13-acetate (PMA) (20
nM). In PGF2- and PMA-treated cells, active ERK MAP
kinase was localized in the nucleus. PGF2-induced ERK
phosphorylation was dose-dependently inhibited by the MEK1 inhibitor
PD098059 (1–50 µM). The expression of
c-fos and c-jun mRNA in luteal cells was
markedly increased by treatment with PGF2 (1 µM) or
PMA (20 nM) for 30 min. We also observed that activation of
ERK MAP kinase was required for the expression of c-fos
and c-jun mRNA in response to PGF2 and PMA because it
was abrogated by blocking the ERK pathway with PD098059. In addition,
PGF2 and PMA-induced c-fos and c-jun
mRNA expression was abolished in the PKC-deficient cells. Taken
together, our data demonstrate that a PKC-dependent ERK MAP kinase
pathway mediates the expression of c-fos and
c-jun mRNA in PGF2-treated bovine luteal cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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triggers the demise of the corpus
luteum (luteolysis) in domestic farm animals whereby progesterone
synthesis is inhibited, the luteal structure regresses, and the estrous
cycle resumes (reviewed in 1, 2). Despite extensive
studies demonstrating the physiological role of PGF2 in the corpus
luteum, the cellular and molecular mechanisms of PGF2-induced
luteolysis remain poorly understood. It is well-known that, in bovine
luteal cells, PGF2 binds to specific G-protein-coupled receptors
(3) and activates phospholipase C (PLC), which leads to
the generation of two second messengers, i.e. diacylglycerol
(DAG) and inositol-1,4,5-trisphosphate (InsP3)
(4). Accumulation of InsP3 elevates
intracellular Ca2+ levels (4, 5).
PGF2 stimulates this signaling pathway presumptively to activate the
Ca2+- and phospholipid-dependent protein kinase C
(PKC). Indeed, PGF2 has been demonstrated to increase PKC activity
and PKC translocation in luteal cells (6, 7, 8, 9). Activation
of PKC is thought to, at least in part, mediate PGF2-induced
luteolysis in vivo (10).
Ligands that signal via G-protein-coupled receptors have been shown to
activate the mitogen-activated protein (MAP) kinase signaling cascade.
This cytoplasmic protein kinase cascade transduces signals initiated at
the plasma membrane to the nucleus, where it regulates the expression
of specific target genes (11, 12). In vertebrates,
multiple isoforms of MAP kinase have been identified and categorized
into three subfamilies, i.e. the extracellular
signal-regulated kinases (ERKs), p38mapk, and the
Jun N-terminal kinases (JNKs) or stress-activated protein
kinases. The classical ERKs, ERK2 or
p42mapk and ERK1 or
p44mapk, are positioned downstream of Raf-1 and
MEK1, and together comprise an orderly signaling cascade
(13). The MAP kinase signaling pathways have been
implicated in control of different, and even opposite, cellular
responses (including proliferation, differentiation, and cell death).
Such actions are elicited, at least in part, through translocation of
activated MAP kinase into the nucleus (14), where it
phosphorylates and thereby activates nuclear transcription factors,
including Elk-1 (15), Sap1 (16),
c-Jun (17), ATF2 (15, 17), and
others. These transcription factors stimulate the expression of the
immediate-early response oncogenes, i.e. c-fos
and c-jun (15, 17). Fos and Jun proteins are
constituents of the activator protein-1 (AP-1) transcription factors
that, in turn, regulate the transcription of numerous genes possessing
promoter AP-1 binding sites (18). In bovine luteal cells,
we have recently reported that PGF2 activates each component of the
Raf/MEK1/ERK signaling cascade (19), increases levels of
c-fos and c-jun messenger RNA (mRNA), and
activates the AP-1 transcription factors (20). It seems
likely that the Raf/MEK1/ERK signaling cascade constitutes a pivotal
intracellular network directly regulating the expression and activation
of the transcription factor AP-1 that may play an important role in
programming gene expression during PGF2-induced luteolysis. However,
the mechanisms regulating the expression of AP-1 transcription factors
in the corpus luteum are currently unknown.
The mechanism by which PGF2 regulates the activation of the
Raf/MEK1/ERK signaling cascade is also unclear. Watanabe et
al. (21) suggested that the mitogenic actions of
PGF2 in NIH3T3 cells are mediated via activation of
p21Ras, Raf, and subsequent activation of MEK1
and ERK2. Also of interest in this study were findings demonstrating
that ERK activation by PGF2 was independent of phorbol
ester-responsive isoforms of PKC (21).
G-protein-ß
-subunit-mediated tyrosine phosphorylation of Shc
and Sos has been recently implied in PGF2-induced Raf-1 activation
in myometrial cells (22). Additionally, Hakeda et
al. (23) reported that PGF2 activates the ERK MAP
kinase pathway in osteoblastic MC3T3-E1 cells via PKC-dependent
activation of Raf-1. Our recent data suggests that PKC is involved in
PGF2-induced activation of the Raf/MEK/ERK signaling cascade in
luteal cells, because the response to PGF2 was mimicked by the PKC
activator phorbol 12-myristate 13-acetate (PMA) (19). In
the present study, experiments were performed to determine: 1) the role
of PKC in regulating the activation of the ERK MAP kinase; and 2) the
role of PKC and ERK MAP kinase in the expression of c-fos
and c-jun by PGF2 in bovine luteal cells. Our results
indicate that activation of the c-fos and c-jun
protooncogenes by PGF2 in luteal cells is mediated by a
PKC-dependent ERK MAP kinase signaling pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The PKC-depleted luteal cells were obtained by chronic treatment with
high levels of PMA. After serum starvation for 24 h, luteal cells
were treated for an additional 24 h with 2.5 µM PMA.
Total cell extracts were prepared as described below. Western blot
analysis with specific anti-PKC-, -ßII, and -
antibodies was
used to verify depletion of phorbol ester-responsive isoforms of
PKC.
Chemicals, cell treatments, and total cell extracts
All chemicals, unless noted, were purchased from
Sigma (St. Louis, MO). The MEK-1 inhibitor PD098059 and
the PKC inhibitor bisindoylmaleimide (GF109203x) were purchased from
Calbiochem (La Jolla, CA). PGF2 was dissolved in
absolute ethanol at 2 mM. PMA, PD098059, and GF109203x
were prepared in dimethylsulfoxide at concentrations of 5
mM, 20 mM, and 10 mM, respectively.
Cell treatments were performed with serum-free M-199 plus 0.1% BSA
after 1-h equilibration at 37 C. Experiments were stopped by rapidly
rinsing twice with cold PBS. The cells were lysed with nondenaturing
lysis buffer A [10 mM Tris-HCl (pH 7.4), 100
mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton-100, 0.5% NP-40, 50 mM NaF, 1 mM
Na3VO4, 1 mM
phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 10 µg/ml pepstatin-A] on ice, with continuous shaking
for 30 min. The total cell extracts were collected using a disposable
cell scraper, vortexed vigorously, and clarified by centrifugation
(15,000 x g, 5 min). The protein content of the
samples was measured by a Bio-Rad Laboratories, Inc.
procedure using BSA as the standard. Aliquots of the extracts were
subjected to immunoprecipitation or frozen at -80 C until Western blot
analysis could be performed.
SDS-PAGE and Western blotting
The cell extracts were heat-denatured in Laemmili buffer and
subjected to discontinuous SDS-PAGE with 5% and 10% polyacrylamide in
the stacking and resolving gels, respectively. Proteins in the gel were
electrophoretically (100 V, 1.5 h) transferred to a polyvinylidene
difluoride (PVDF) membrane (Amersham Pharmacia Biotech,
Arlington Heights, IL). The membrane was preincubated in TBST [50
mM Tris-HCl (pH 7.5), 0.15 M NaCl, 0.05%
Tween-20] containing 5% fat-free milk, overnight at 4 C, and then
incubated with appropriate amounts of primary antibodies in TBST-3%
BSA (for monoclonal antibodies) or in TBST-5% fat-free milk (for
polyclonal antibodies) at room temperature for 1 h. After four
washes with 15 ml TBST, the membrane was incubated with corresponding
antimouse (1:2000) or antirabbit (1:4000) peroxidase-conjugated IgG for
1 h, respectively. The membrane was washed again with TBST, and
bound antibodies were detected by the Amersham Pharmacia Biotech enhanced chemiluminescence reagents.
Immunoprecipitation
Clarified cell extracts were precleared by coincubation with 1
µl normal goat serum and 20 µl protein-A conjugated agarose beads
for 1 h at 4 C. After centrifugation (15,000 x g,
4 C, 5 min), the precleared cell extracts were transferred to new
tubes. Equal amounts of protein were immunoprecipitated at 4 C, either
with the rabbit polyclonal anti-ERK2 or anti-ERK1 conjugated agarose
beads (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA) in 1 ml of lysis buffer A, with continuous rotation for 2 h.
The beads (immunocomplex) were collected by centrifugation. The
immunoprecipitates were washed three times with buffer A, three times
with LiCl solution (0.5 M LiCl, 100
mM Tris-HCl, pH 8.0), and two times with kinase
assay buffer B [50 mM ß-glycerophosphate (pH
7.3), 1.5 mM EGTA, 1 mM
dithiothreitol, and 0.09% Brig35]. The beads were resuspended in 40
µl kinase buffer B. Aliquots of the immunoprecipitates were prepared
in Laemmili buffer for subsequent Western blot analysis to verify that
the same amounts of immunoprecipitated MAP kinase were subjected to the
kinase assay (data not shown).
Immunocomplex kinase assay
The transcription activation domain of human Elk-1 (amino acids
307–428) was obtained by nucleotide sequence analysis
(24). The Elk-1 trans-activation domain was
cloned into pGEX3 to produce a GST-Elk-1 fusion gene. GST-Elk-1 fusion
protein was expressed in bacteria and partially purified as previously
described (25) and used as the substrate for ERK kinase
assay as reported (19). The kinase activity of ERK2 and
ERK1 was measured by in vitro phosphorylation of GST-Elk-1
as described (19, 24). To perform these kinase assays:
briefly, 20 µl of immunoprecipitates were coincubated with 0.5 µg
GST-Elk-1 in the kinase cocktail [30 µM ATP,
20 mM Tris-HCl (pH 7.4), 0.5
mM MnCl2, 5
mM MgCl2]. The reaction
was initiated by the addition of 10 µCi
[-32P]-ATP, incubated at 30 C for 30 min,
and stopped by the addition of Laemmili buffer. The reaction mixtures
were resolved on 10% SDS-PAGE, after which the gels were stained.
Autoradiography was performed on dried gels to visualize phosphorylated
GST-Elk-1. The corresponding bands of GST-Elk-1 were cut out and
quantified by liquid scintillation counting.
Fluorescence immunocytochemistry
Luteal cells were cultured in 8-well Lab-Tek glass chamber
slides as described above. After treatment with PGF2 (1
µM) and PMA (20 nM) for 5 min, the cells were
washed with PBS, and fixed with cold methanol for 10 min at -20 C. The
cells were air-dried and washed with 0.1% BSA-TBS [10 mM
Tris-HCl (pH7.4), 150 mM NaCl], and permeabilized with
0.02% Triton-100 in 0.1% BSA-TBS for 10 min. Nonspecific binding was
blocked by incubation with 3% BSA-TBS for 30 min at room temperature,
after which the phospho-MAP kinase antibody (5 µg/ml; Promega Corp., Madison, WI) or polyclonal rabbit
anti-3ß-hydroxysteroid dehydrogenase (anti-3ß-HSD) antibody
(1:100) diluted in 0.1% BSA-TBS was applied and incubated overnight at
4 C. After four washes (10 min each) with 0.1% BSA-TBS, the samples
were incubated with Cy3-conjugated antirabbit IgG (1:100) for
1 h at room temperature. After four washes (5 min each) with 0.1%
BSA-TBS, the samples were examined by confocal microscopy
(Bio-Rad Laboratories, Inc. MRC-1024, Laser Scanning
Confocal Imaging System using an argon/krypton lamp). Digital imaging
was captured by the Bio-Rad Laboratories, Inc. laser-shop
software. For negative controls, the same procedure described above was
performed either without primary antibody or without secondary antibody
(data not shown).
RNA extraction and Northern blot analysis
After cell stimulation for 30 min, the medium was discarded.
Total RNA was isolated by the single-step
guanidine-thiocyanate-phenol-chloroform procedure (26) and
quantified based upon the absorbance value at 260 nM by UV
spectrophotometer. RNA samples (15 µg/lane) were electrophoresed on a
denaturing gel of 1% agarose and 1.5% formaldehyde, transferred and
UV cross-linked to a zeta probe blotting membrane (Bio-Rad Laboratories, Inc., Richmond, CA). The membrane was
prehybridized in 500 mM NaHPO4 (pH
7.2), 7% SDS, and 1 mM EDTA, at 60 C for 4 h.
Hybridization was performed in the same buffer containing denatured
[32P]-labeled bovine c-fos (GenBank
accession number AF069575) and c-jun (GenBank
accession number 06951) complementary DNA (cDNA) probes (approximately
2.5 x 106 cpm/ml), overnight at 60 C. The
1.8-kb human ß-actin cDNA probe was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA) and served as a control for
sample loading. The cDNAs were radiolabeled with
[-32P] deoxycycidine triphosphate by using a
random-primed labeling kit (Roche Molecular Biochemicals,
Indianapolis, IN). After four washes (20 min each) in prewarmed (60 C)
buffer [40 mM NaHPO4 (pH
7.2), 1% SDS, 1 mM EDTA (pH 8.0)] at room
temperature, the membranes were subjected to autoradiography.
Statistical analysis
Data are presented as mean ± SEM. Each
experiment was performed at least three times. Data were analyzed using
the InStat program. One-way ANOVA was followed by Dunn’s t
test. P < 0.05 values were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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exposure is limited
and largely based on comparison with the response to phorbol esters. In
the corpus luteum, this comparison has been applied to evaluate the
possible role of phorbol ester-responsive isoforms of PKC in mediating
the luteolytic effects of PGF2 (8, 9, 10). Additionally,
we have recently demonstrated that PMA mimicked the stimulatory effects
of PGF2 on each component of the Raf-1/MEK1/ERK signaling cascade in
bovine luteal cells (19). Therefore, the PKC isoforms
involved in PGF2 action in the corpus luteum are presumptively the
PMA-sensitive conventional and novel isoforms of PKC. To test this
possibility, a PKC-deficient bovine luteal cell model was established
based on the fact that chronic treatment with PMA degrades phorbol
ester-responsive isoforms of PKC (27, 28). Luteal cells
were rendered PKC-deficient by pretreatment with 2.5 µM
PMA for 24 h (Fig. 1
). Western
blotting experiments, with subtype-specific antipeptide antibodies
raised against different isoforms of PKC, were performed to confirm the
depletion of PKC isoforms. A monoclonal antibody (Transduction Laboratories, Inc., Lexington, KY) against a C-terminal
domain epitope (270–427) of human PKC- recognized a single band
with an apparent molecular mass of 84 kDa in bovine luteal cells. The
specific PKC-ßII antibody (SC-210, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), raised against the C-terminal amino acids
657–673 of human PKC-ßII, recognized the predicted 84-kDa protein
but also cross-reacted with an unknown protein of approximately 95 kDa.
The specific PKC- antibody (Transduction Laboratories, Inc.), raised against the NH2-terminal amino acids 1–175 of
human PKC-, recognized a single protein with the predicted molecular
mass of 96 kDa. It is noteworthy that, compared with controls (Fig. 1, lanes 1–3), the levels of these PKC isoforms were greatly reduced
after exposure to 2.5 µM PMA for 24 h (Fig. 1, lanes
4–6). With the same samples, Western blotting experiments were also
performed with a specific antibody raised against human 3ß-HSD, the
enzyme that converts progenolene to progesterone (kindly provided by
Dr. Ian Mason, University of Texas Southwestern Medical Center). High
levels of the 40-kDa 3ß-HSD protein were observed in all samples and
were not altered after exposure to 2.5 µM PMA for 24
h (Fig. 1). Additionally, acute treatment with PGF2 and PMA for 5
min did not alter the cellular levels of PKC isoforms or 3ß-HSD (Fig. 1).
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and
PMA in bovine luteal cells exposure takes place within seconds
in ovarian cells (4, 5). Acute treatment with PGF2 and
PMA also results in a rapid activation of ERK MAP kinase in a number of
cell types (21, 22, 23), including ERK2 and ERK1 in primary
cultures of bovine luteal cells (19). To investigate
whether PKC is required for activation of ERK2 and ERK1 by PGF2 in
bovine luteal cells, we first assessed whether phosphorylation and
activation of ERK2 and ERK1 by PGF2 and PMA was mediated by phorbol
ester-responsive isoforms of PKC. For this purpose, a comparison of
phosphorylation and activation of ERK2 and ERK1 by PGF2 and PMA was
made by using cells with or without chronic PMA pretreatment (Fig. 2). Immunoblotting experiments with
monoclonal PanERK antibody (Transduction Laboratories, Inc.) showed that the cellular levels of ERK2 and ERK1 were not
altered by chronic pretreatment with 2.5 µM PMA (lanes
4–6) or by acute treatment with 1 µM PGF2 (lanes 2
and 5) and 20 nM PMA (lanes 3 and 6). In keeping with our
previous report (19), acute treatment with PGF2 and PMA
caused the gel-shift phosphorylation of ERK2 and ERK1 (lanes 2 and 3).
Notably, the PGF2 and PMA-induced gel shifts (lanes 2 and 3) were
not apparent in PMA-pretreated cells (lanes 5 and 6). Western blot
analysis of these samples was also performed using a phospho-MAP kinase
antibody. Figure 3 demonstrates that
treatment with PGF2 (1 µM) and PMA (20 nM)
for 5 min markedly stimulated the phosphorylation (Fig. 3A) of ERK2 and
ERK1 in bovine luteal cells. Compared with untreated cells (lanes
1–3), chronic PMA pretreatment (lanes 4–6) did not alter the basal
phosphorylation status of ERK2 and ERK1 (Fig. 3a). When cells were
pretreated with 2.5 µM PMA for 24 h, the
phosphorylation (Fig. 3A) of ERK2 and ERK1 by PGF2 and PMA was
completely attenuated. These data indicate an obligatory role of
phorbol ester-responsive isoforms of PKC in mediating ERK
phosphorylation in response to both PGF2 and PMA in bovine luteal
cells. When the cells were pretreated with a PKC inhibitor GF102903x
(100 nM, 1 h), which possesses high selectivity for
PKC-, -ß, -, -
, and - isozymes, the phosphorylation of
ERK2 and ERK1 by PGF2 was also completely blocked (Fig. 3B).
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) were performed to demonstrate that the
phosphorylation of ERK1 and ERK2 was associated with an increase in the
activity of the enzymes. Treatment with PGF2 (1 µM) or
PMA (20 nM) for 5 min increased the activities of both ERK2
(Fig. 4A) and ERK1 (Fig. 4B). Treatment with PMA (2.5 µM)
for 24 h had little effect on basal activities of ERK1 and ERK2
but inhibited the acute stimulatory actions of both PGF2 and PMA.
This result further supports a pivotal role of PKC in mediating ERK MAP
kinase activation by PGF2 and PMA in bovine luteal cells.
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and
PMA stimulation in bovine luteal cells and PMA stimulation in bovine luteal
cells, we localized the activated ERK by indirect fluorescence
immunocytochemistry using a specific phospho-MAP kinase antibody (Fig. 5). The active MAP kinase signal observed
either in the cytoplasm or the nucleus of untreated control cells was
weak (A). After treatment with PGF2 (C) and PMA (D) for 5 min,
fluorescence immunocytochemistry revealed a strong nuclear labeling of
activated MAP kinase, whereas the cytoplasmic signals of activated MAP
kinase labeling remained weak. In parallel, 3ß-HSD immunolabeling (
B) was primarily present in the cytoplasm of untreated control cells.
The majority of the cells were 3ß-HSD positive cells, demonstrating
their steroidogenic nature. In keeping with the evidence of various
gene expression upon PGF2 stimulation in bovine corpus luteum
(29), nuclear location of activated MAP kinase in response
to PGF2 may be a key step that transmits the initial PGF2-induced
PLC/InsP3-DAG/Ca2+-PKC
signaling pathway into the nucleus and further regulates the
transcription of PGF2-inducible genes in luteal cells.
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and
PMA-induced c-fos/c-jun mRNA expression and PMA. To test this, we
first examined the effects of the specific MEK1 inhibitor PD098059 on
phosphorylation of ERK2 and ERK1 by PGF2 in bovine luteal cells
(Fig. 6A). Western blot analysis with the
active MAP kinase antibody showed a marked increase of ERK2 and ERK1
phosphorylation with treatment of 1 µM PGF2
in bovine luteal cells. Pretreatment with increasing concentrations of
PD098059 for 1 h resulted in a concentration-dependent inhibition
of PGF2-induced ERK2 and ERK1 phosphorylation (Fig. 6A). Nearly
complete inhibition of ERK phosphorylation was observed with 50
µM PD098059, a concentration without cytotoxic
effects in a number of cell types (32). Treatment with
PD098059 had no effect on levels of ERK1 or ERK2 protein (data not
shown). Therefore, 50 µM PD098059 was chosen to
examine the role of ERK activation in PGF2 and PMA-induced
c-fos and c-jun mRNA expression.
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(1
µM) and PMA (20 nM) for 30 min induced the
maximal expression of c-fos and c-jun mRNA in
bovine luteal cells (20), experiments were performed to
determine whether blocking ERK activation attenuates the induction of
c-fos and c-jun mRNA expression by PGF2 and
PMA (Fig. 6B
). As expected, treatment with PGF2 and PMA for 30 min
significantly stimulated both c-fos and c-jun
mRNA expression (lanes 3 and 5, respectively). In comparison with cells
without PD098059 pretreatment (lane 3), the expression of
c-fos and c-jun mRNA induced by PGF2 was
completely blocked in cells pretreated with PD098059 (lane 4). This
result indicates that ERK activation is required for PGF2-induced
transcription of c-fos and c-jun protooncogenes.
Additionally, blocking ERK activation with PD098059 also inhibited
PMA-stimulated c-fos and c-jun mRNA expression
(lane 6). This result suggests that ERK MAP kinase is also involved in
c-fos and c-jun mRNA expression upon activation
of phorbol ester-responsive isoforms of PKC by PMA.
Stimulation of c-fos and c-jun mRNA by PGF2 and PMA was blocked
by chronic pretreatment with PMA
Having demonstrated that (1) PGF2 and PMA activate
a PKC-dependent ERK signaling pathway, and (2) ERK2 and
ERK1 mediate PGF2 and PMA stimulation of c-fos and
c-jun mRNA expression, we further tested whether PKC was
required for c-fos and c-jun expression in
response to PGF2 and PMA. For this purpose, the PKC-deficient cell
model was applied. As shown in Fig. 7, pretreatment with PMA (2.5 µM) for 24 h
did not alter the basal levels of c-fos and c-jun
mRNA expression. However, PGF2 and PMA failed to stimulate
c-fos and c-jun mRNA expression in PKC-deficient
cells (lanes 4–6), in comparison with cells without chronic PMA
pretreatment (lanes 1–3). These data suggest that phorbol
ester-responsive isoforms of PKC play an obligatory role in
PGF2-induced c-fos and c-jun mRNA
expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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exerts its
luteolytic effects (4, 5, 33). However, a gap exists in
our knowledge, between this initial signaling event and the
physiological sequelae observed in response to PGF2. In a recent
report, we demonstrated that PGF2 activates the Raf-1/MEK1/ERK
signaling cascade in bovine luteal cells (19). We now
extend these observations by showing that in bovine luteal cells: 1)
inhibition of PKC by a PKC inhibitor or by depletion of phorbol
ester-responsive PKC isoforms abrogated phosphorylation and activation
of ERK2 and ERK1 in response to both PGF2 and PMA; 2) activated ERKs
were present in the nucleus; 3) blocking the ERK MAP kinase pathway by
the specific MEK1 inhibitor PD098059 attenuated ERK activation and
expression of c-fos and c-jun mRNA in response to
PGF2 and PMA; and 4) depletion of phorbol ester-responsive isoforms
of PKC blocked PGF2 and PMA-induced expression of c-fos
and c-jun mRNA. These data indicate that a PKC-dependent ERK
MAP kinase pathway mediates the expression of c-fos and
c-jun transcription factors in response to PGF2. This
novel evidence supports the idea that a PKC-dependent ERK MAP kinase
pathway is, perhaps, the predominant cytoplasmic protein kinase cascade
that links the plasma membrane receptor-initiated signaling events to
transcription factor regulation of the expression of
PGF2-responsive genes.
Activation of the ERK MAP kinases involves a signaling cascade
comprising Raf, MEK1, and ERK2/ERK1 (11, 12, 13). Multiple
mechanisms exist for regulating Raf-1 activation, e.g.
membrane association upon Ras-binding (34, 35), tyrosine
phosphorylation by Src family of tyrosine kinases (36),
interaction with 14–3-3 proteins (37), and direct serine
phosphorylation by PKC (38, 39). Immediate upstream
activators other than Raf are also identified for MEK1, which represent
a Raf-independent mechanism for ERK activation, possibly involving
PKC-
(40). It is clear, therefore, that the ERK MAP
kinases can be activated through different mechanisms by different cell
surface receptors. Likewise, PGF2 seems to be able to activate ERK
differentially, based on the cell types and coupling to specific G
protein subunits. PGF2 is generally thought to activate PLC by
coupling to Gq. PGF2, possibly via Gq subunits, stimulated the
formation of p21Ras-GTP complexes and the
subsequent activation of Raf in NIH3T3 cells, suggesting a
Ras-dependent pathway for ERK activation by PGF2 (21).
In NIH3T3 cells, PGF2-induced ERK2 activation was also shown to be
independent of PMA-sensitive PKC isoforms (21).
Additionally, in rat myometrial cells,
G-protein-ß-subunit-mediated tyrosine phosphorylation of Shc and
Sos is thought to mediate PGF2-induced Raf-1 activation of ERKs
(22). In contrast, activation of the ERK MAP kinase
signaling cascade by PGF2 in osteoblastic MC3T3-E1 cells seems to be
mediated by PKC-dependent activation of Raf-1 (23).
Likewise, PKC has been implicated in the activation of the Raf/MEK1/ERK
signaling cascade by PGF2 in luteal cells, because a similar
activation pattern was observed in response to the PKC activator PMA
(19). Our present data further implicate PKC in the
activation of ERK MAP kinase signaling. Moreover, preliminary results
suggest that p21ras activation is not required
for PGF2-induced ERK activation in bovine luteal cells (Davis and
Obholz, unpublished observation). It seems, therefore, that PGF2 may
use cell-type-specific signaling pathways to activate ERKs in various
tissues and cell lines.
Multiple isoforms of PKC (41) are present in the corpus
luteum (33), including members of the conventional class
(cPKC-, -ßI, and -ßII), a novel class (nPKC- and -
), and
atypical class (aPKC- and -
/
). Although PGF2 activates PKC
in luteal cells (6, 7, 8, 9, 10), little is known about the
specific PKC isoform(s) and the physiological substrates lying
immediately downstream of PKC activation. The present study
demonstrates that PKC is required for phosphorylation and activation of
ERK2 and ERK1 by PGF2 and PMA in luteal cells. This is supported by
studies showing that a specific PKC inhibitor and depletion of phorbol
ester-responsive PKC isoforms completely blocked ERK phosphorylation
and activation in response to both PGF2 and PMA. The isoforms of PKC
involved in ERK activation and in response to PGF2 are most likely
the phorbol ester-sensitive isoforms of cPKCs and nPKCs. Whereas the
phorbol ester-unresponsive atypical PKC- and / isoforms are
present in bovine luteal cells (33), it seems unlikely
that these PKC isoforms mediate PGF2-induced ERK activation. Further
investigation is required to characterize the mechanisms by which PKC
mediates PGF2-induced ERK activation. It is tempting to speculate
that specific cPKCs or nPKCs serve as direct upstream kinases for Raf-1
activation (38, 39).
Transcription of the prototypic immediate-early response genes,
i.e. c-fos and c-jun, is rapidly
induced in cells on exposure to a wide variety of extracellular
stimuli. The data presented herein demonstrate that treatment with
PGF2 and PMA stimulated c-fos and c-jun mRNA
expression in primary cultures of bovine luteal cells. Our results
in vitro are in keeping with observations that treatment
with PGF2 in vivo stimulated c-jun mRNA
expression in bovine (42) and rat (43)
corpora lutea. Therefore, our present data underline the relevance of
expression of c-fos and c-jun protooncogenes as a
physiological criterion for PGF2 action in the corpus luteum.
The transcription of c-fos and c-jun is
controlled by the cis-acting elements in their promoter
regions, including the SRE and the
12-O-tetradecanoylphorbol 13-acetate response
element. The best-characterized SRE is that of the c-fos
gene (15, 44). Full-activation of the
c-fos SRE requires association with the ubiquitous
transcription factor SRF and formation of a ternary complex with
ternary complex factors, including the Ets family of transcription
factors Elk-1 and Sap1 (44, 45). The DNA binding domain at
the N terminus of Elk-1 and Sap1 facilitates ternary complex formation,
whereas the trans-activation domain at the C-terminus of
Elk-1 and Sap1 contains several conserved MAP kinase phosphorylation
sites. Upon cell stimulation, MAP kinase translocates into the nucleus
(14), where it phosphorylates the Elk-1 C-terminus, which
then cooperates with the C-terminus of the SRF activation domain to
initiate c-fos transcription (46). Our data
suggest that phosphorylation of Elk-1 by the ERK pathway is involved in
the activation of the c-fos gene by PGF2 in bovine luteal
cells. This notion is supported by the following evidence. First, ERK2
and ERK1 were phosphorylated and activated upon PGF2 and PMA
stimulation. Second, PGF2-activated ERK2 and ERK1 were able to
phosphorylate Elk-1 in vitro. Third, preincubation of cells
with the specific MEK1 inhibitor PD098059 attenuated the
phosphorylation of ERK2 and ERK1 by PGF2 and PMA. Fourth, active ERK
was observed in the nucleus of cells treated with PGF2 and PMA.
Last, preincubation of cells with PD098059 blocked the stimulatory
action of PGF2 on the expression of c-fos mRNA.
The regulatory mechanism for induction of the c-jun gene
seems more complicated and much less understood. Early studies
suggested that a putative AP-1 element in the c-jun promoter
mediates the positive autoinduction of c-jun by mitogens
(47). However, several consensus binding sites for
different transcription factors, including AP-1, MEF2, Sp-1, and CTF
are present in the c-jun promoter (17). Recent
studies demonstrated that the MEF2 site and the AP-1 site, but not the
Sp-1 and CTF sites, are required for induction of the c-jun
promoter in response to activators of epidermal growth factor (EGF)
receptors (48) and G-protein-coupled m1
acetylcholine receptors (49). Therefore, the AP-1
transcription factors seem to be important regulators of
c-jun expression and may be involved in PGF2 regulation
of c-jun mRNA expression in luteal cells.
Current evidence suggests that c-Jun activity can be regulated by
posttranslational modifications of preexisting c-Jun protein.
Phosphorylation of serine and threonine residues within the N-terminal
trans-activation domain (50, 51) results in
enhanced trans-activation, DNA-binding, and stability of the
c-Jun protein (51, 52). The direct upstream kinases that
phosphorylate the residues within c-Jun N terminus include members
of the two MAP kinase subfamilies, i.e. the ERKs, including
ERK2 and ERK1 (48, 53), and JNK (48, 54, 55).
Similar to the ERK pathway, JNK activation involves a parallel protein
kinase cascade comprising MEKK1, SEK, and JNKs (54, 55).
The small G proteins Rac1, cdc42, and Ras (56) have been
identified as upstream activators of MEKK1. In Hela cells, transfection
of active forms of Ras, RacI, cdc42Hs, and MEKK1 increased, whereas
transfection of dominant-negative mutants of Ras, RacI, and MEKK1
inhibited the expression of a c-jun promoter-driven
luciferase reporter gene in response to EGF (48). This
result suggested that the Rac-MEKK and JNK pathway was involved in the
activation of the c-jun promoter by EGF. However, in NIH3T3
cells expressing G-protein-coupled m1 acetylcholine receptors, the JNK
pathway did not seem to be involved in the activation of the
c-jun promoter in response to carbacol, because coexpression
of MEKK1 effectively increased JNK activity but only with limited
c-jun promoter activity (49). This suggests
that G-protein-coupled receptors can use mechanisms other than JNK to
regulate c-jun expression. In this regard, recent evidence
points to a role of ERK MAP kinase in regulating the nucleosomal
response associated with the induction of immediate-early response
genes (57, 58). In the present report, we demonstrate that
PGF2 and PMA rapidly stimulated the expression of c-jun
mRNA in bovine luteal cells. Moreover, enhanced c-jun mRNA
expression by PGF2 was attenuated by blocking the ERK pathway with
the specific MEK1 inhibitor PD098059. Although the exact mechanism(s)
remain to be established, our data suggest that ERK2 and ERK1 are
involved in the activation of the c-jun gene by PGF2 in
bovine luteal cells.
Lastly, we showed that depletion of PMA-responsive PKC isoforms by
chronic pretreatment with PMA blocked the expression of
c-fos and c-jun mRNA in response to PGF2 and
PMA. These results indicate that the phorbol ester-responsive
isoforms of PKC play a pivotal role in mediating the expression of the
AP-1 transcription factors by PGF2 in bovine luteal cells. Because
depletion of phorbol ester-responsive isoforms of PKC completely
inhibited ERK activation by PGF2 and PMA, and ERK activation was
required for the enhanced expression of c-fos and
c-jun mRNA by PGF2 and PMA, these data allow us to
conclude that induction of c-fos and c-jun mRNA
expression by PGF2 in bovine luteal cells is mediated by a
PKC-dependent ERK MAP kinase pathway.
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Acknowledgments |
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Footnotes |
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2 Current address: Perinatal Research Laboratories, Department of
Obstetrics and Gynecology, University of Wisconsin Medical School, 7E
Meriter Hospital/Park, 202 South Park Street, Madison, Wisconsin
53715.
Received September 8, 2000.
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References |
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