1 Department of Obstetrics and Gynecology and 3 Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1062; and 2 Department of Medicine, Dorothy Crowfoot Hodgkin Laboratories, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom
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ABSTRACT |
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Oxytocin (OT) induces PG synthesis by both uterine endometrial and amnion cells. We showed previously that CHO cells stably transfected with the rat oxytocin receptor (CHO-OTR cells) also synthesize PGE2 in response to OT. In the present work we have demonstrated that OTRs are coupled to both Gi and Gq/11, using immunoprecipitation of solubilized OTR complexes and ADP ribosylation. OT treatment caused the rapid phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2 or p42MAPK), which was partially inhibited by pertussis toxin (PTX), consistent with OTR-Gi coupling. The PTX-insensitive portion of ERK2 phosphorylation was linked to Gq, as inhibitors of both phospholipase C (U-73122) and protein kinase C (GF-109203X) blocked OT-induced ERK2 phosphorylation. OT-stimulated c-fos expression was also mediated by ERK2 phosphorylation. The ERK-c-fos pathway has been shown to be associated with cell proliferation, but OT had no effect on [3H]thymidine uptake by CHO-OTR cells. However, inhibition of OT-induced ERK2 phosphorylation with an ERK kinase inhibitor (PD-98059) markedly reduced OT-stimulated PGE2 synthesis, pointing to the importance of ERK2 activation in OT action.
mitogen-activated protein kinase, prostaglandin E2, oxytocin receptor, G proteins, protein kinase C
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INTRODUCTION |
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IN RODENTS AND OTHER ANIMALS pregnancy terminates after
luteolysis, which occurs naturally at term (29) or can be induced experimentally with PGF2
(4). Recent work with
PGF2
receptor knockout mice
illustrates the critical relationship between PGF2
, progesterone, and the
initiation of parturition (29). These mice appear normal except for
their inability to deliver pups at term. Blood progesterone levels,
which normally decline before the end of term, remain elevated, and the
upregulation of oxytocin (OT) receptor (OTR) mRNA in the myometrium
that normally occurs immediately before term is lacking. The knock-out
phenotype could be reversed by ovariectomy on day
19 of pregnancy, which causes a sharp fall in serum
progesterone, induction of OTR mRNA, and parturition. It is thought
that OT is the signal for PGF2
release from the decidua in the rat (8) and other species. OT-induced
luteolysis leads to the upregulation of myometrial OTR, which
sensitizes the uterus to basal levels of OT in the blood, resulting in
the initiation of labor contractions (3).
OT action, which is mediated by Gq/11, stimulates phosphoinositol-specific phospholipase C (PI-PLC), leading to increased hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) (17, 22). Both IP3 and DAG play important roles as intracellular second messengers that increase intracellular Ca2+ concentrations ([Ca2+]i) and activate protein kinase C (PKC), respectively. To better understand the signaling pathways involved in OT-stimulated synthesis of PGs, we stably transfected CHO cells with the rat OTR (CHO-OTR cells) and showed that OT elicited PGE2 synthesis (20). OT treatment also caused a rapid increase in [Ca2+]i and increased production of inositol phosphates and arachidonic acid release (20). Because these responses to OT are the same as those in primary cultures of endometrial tissue (17) and amnion (22) cells, CHO-OTR cells are useful for studying signal pathways in OT-induced PGE2 synthesis. A distinct advantage of CHO-OTR cells over primary cultures is their uniformity and ability to give reproducible results.
Mitogen-activated protein kinases (MAPKs) have been shown to be vital signal pathway components in an increasing number of hormonally responsive cells. These enzymes function as integrators of mitogenic and other signals originating from distinct classes of cell surface receptors, such as tyrosine kinase and G protein-coupled receptors (31). Three subgroups of the MAP kinase family have been identified and cloned: extracellular signal-regulated protein kinase (ERK), stress-activated protein kinase, or c-Jun NH2-terminal kinase, and p38 MAP kinase (p38MAPK). These kinases are structurally related, dually phosphorylated on tyrosine and threonine residues, and activated by upstream kinases (11). The various members of the MAP kinase families differ in their substrate specificity, and they are also activated by distinct upstream regulators and extracellular stimuli. In their activated forms, ERK1 and ERK2 transmit extracellular stimuli by phosphorylating a variety of substrates, including transcriptional factors and other kinases (6). ERKs also mediate transcriptional activation of immediate early genes such as c-fos and c-jun.
The pathways originating from many G protein-coupled receptors in the activation of MAPKs are still being defined, and very little is known about the role of MAPK in OT action. Ohmichi and co-workers (24) showed that OT activated p42MAPK (ERK2) phosphorylation and activity in human myometrial cells. The major effect of OT on myometrial cells is activation of contraction, but the effects of OT stimulation of p42MAPK were not reported. Several studies have indicated that ERKs phosphorylate and thereby activate cytoplasmic phospholipase A2 (cPLA2) to produce arachidonic acid, the substrate for PG synthesis (21, 26). These findings are not general, however, because PG synthesis in other cell types is ERK independent (7). One of the aims of the present studies was therefore to clarify the role of ERK in OT-stimulated PGE2 synthesis in CHO-OTR cells.
Recently, we showed that OTRs in the rat myometrium are coupled to
Gi, as well as
Gq/11 (28). Several G
protein-coupled receptors phosphorylate Raf-1 by a tyrosine
kinase/Ras-dependent activation through the -subunits of
Gi, independent of PKC (31). This
pathway has been shown to function with acetylcholine muscarinic receptors (13), lysophosphatidic acid receptors (18, 32),
2-adrenergic receptors (1), and thrombin receptors (32). There is also strong evidence from work with several G protein receptors that Gq/11-mediated PKC
activity stimulates MAPK kinase kinase (Raf-1) by a Ras-independent
pathway (31). A third possible mode of MAPK activation is through
increases in intracellular Ca2+,
which can result in phosphorylation of Pyk2. Phosphorylated Pyk2 then
forms a complex with activated Src and Grb2-Sos in the Ras/MAPK
signaling pathway (14). Eguchi and co-workers (15) have also shown that
the activation of MAPK by angiotensin II in cultured rat vascular
smooth muscle cells is PKC-independent, and possibly involves
Gq-mediated
p21ras activation via a
Ca2+-calmodulin-sensitive tyrosine
kinase. The present studies clarify the contributions of
Gi and
Gq to ERK phosphorylation in
OT-stimulated CHO-OTR cells. In addition, we have examined the role of
intracellular Ca2+ in OT
activation of ERK phosphorylation. We found that the principal pathway
involved in OT stimulation of PGE2
synthesis was Gq-PKC-mediated ERK2
phosphorylation.
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MATERIALS AND METHODS |
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Reagents.
Reagents were obtained from the following sources: OT and OT antagonist
(OTA)
[d(CH2)5,
Tyr(Me)2,
Thr4,
Tyr-NH29]ornithine
vasotocin, Peninsula Laboratories (Belmont, CA); pertussis toxin (PTX),
U-73122,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) · HCl, antibiotic
A-23187 (Calcimycin), GF-109203X, Biomol Research Laboratories
(Plymouth Meeting, PA); PD-98059, New England Biolabs (Beverly, MA);
[methyl-3H]thymidine
(25 Ci/mmol) and
Na-125I, Amersham Life
Science (Arlington Heights, IL);
[-32P]dCTP, 3,000 Ci/mmol, and
[
-32P]ATP, 3,000 Ci/mmol, NEN (Boston, MA); antibodies specific for ERK2/1 (C-14), Santa
Cruz Biotechnology (Santa Cruz, CA).
Cell lines.
Chinese hamster ovary cells (CHO-K1, ATCC CCL61) were maintained in
-MEM (GIBCO BRL, Grand Island, NY) containing 5% fetal bovine serum
(FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. CHO-K1 cells
stably transfected with cDNA for rat OTR (CHO-OTR) (20) were cultured
as above, with the addition of Geneticin (GIBCO), 400 µg/ml. All
cells were maintained at 37°C, under an atmosphere of 5%
CO2.
Iodination of OTA, preparation of membranes, solubilization of
occupied receptor, size exclusion chromatography, immunoadsorption with
G protein -subunit antibodies, inhibition of PTX
catalyzed ADP-ribosylation by OT.
These procedures were carried out as described previously (28).
Membranes isolated from CHO-OTR cells were solubilized after incubation
with 125I-OTA. Solubilized
proteins were separated by gel filtration on a fine-performance liquid
chromatography Superose 12 column into two major
125I-OTA-binding fractions:
1) a macromolecular complex (~400
kDa) and 2) a peak, ~50-60
kDa, corresponding in size to the OTR. The 400-kDa
fraction, containing OTR complexed to signal transduction components,
was incubated with specific anti-G-protein antibodies (10 µg) or
preimmune IgG (PI) as a control. Samples were then adsorbed to protein
A-Sepharose columns, and the amount of radioactivity remaining on the
columns after rinsing was determined. The percent of counts per minute
bound is expressed relative to the total radioactivity applied to each
column. To demonstrate specificity, 10 µg of peptide hapten were
coincubated with membrane extracts and antibodies.
Preparation of cell lysates.
Cells were grown to confluence on 35-mm (diameter) dishes and
maintained in serum-free medium for 18 h. After different drug treatments, the cells were rinsed twice with ice-cold PBS, pH 7.4, and
lysed on ice with 150 µl of lysis buffer (25 mM
Tris · HCl, pH 7.5, 25 mM NaCl, 1 mM sodium
orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 20 nM
okadaic acid, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1% Triton X-100, and 0.1% SDS). The
extracts were centrifuged at 12,000 g
for 20 min at 4°C, and the supernatant fractions were used either
to measure kinase activity or frozen at 20°C for subsequent
immunoblotting.
Immunoblotting. Cell lysates were subjected to 10% SDS-PAGE and were transferred into polyvinylidene difluoride (PVDF) membranes. The membranes were incubated in PBS containing 3% BSA and 0.1% Tween 20 for 1 h, followed by incubation with primary antibody (see legends in Figs. 1-9 for specific antibodies) for 45 min and secondary IgG antibody for 1 h. Immunocomplexes were visualized by enhanced chemiluminescence (Amersham). All operations were carried out at room temperature. Blots were densitometrically scanned and analyzed using a Dekmate III scanner and PDI ID software package (PDI, Hunting Station, NY). Quantification of pp42 (phosphorylated ERK2) was carried out by expressing its absorbance relative to the total absorbance (integrated area) of both pp42 and p42 bands. Maximal OT stimulation usually resulted in phosphorylation of 50-60% of total ERK2.
MAP kinase in vitro activity assay.
Cell lysates (5 µg of protein in 5 µl) were incubated with 40 µl
of kinase buffer {25 mM Tris · HCl, pH 7.4, 10 mM MgCl2, 2 mM
MnCl2, 1 mM dithiothreitol, 40 µM ATP, 1 µM staurosporine, 15 µg myelin basic protein (MBP), 0.5 mM EGTA, 0.2 µCi
[-32P]ATP}
at 30°C for 12 min (18). The reaction was stopped by adding 70 µl
of Laemmli sample preparation solution. Samples were separated on
15% minigels and transferred onto PVDF membranes, and radioactivity
was quantified using a PhosphorImager Scanner (Molecular Dynamics) and
the ImageQuant program. Autoradiography was carried out by exposure of
the gels to BIOMAX MS film (Kodak).
[3H]thymidine incorporation.
CHO-OTR cells (10,000/well) were seeded into wells of 12-well plates
and grown for 4 days until confluent. The cells were then incubated for
16 h with -MEM and antibiotics alone (basal) or with either 50 nM
OT, 5% FBS, or basic fibroblast growth factor (bFGF), 100 ng/ml.
[Methyl-3H]thymidine
(1 µCi) and thymidine (3 µM) were then added to each well, and the
incubation was continued for another 4 h. Cells were rinsed with
ice-cold PBS twice and were treated with ice-cold 10% TCA. The cells
were then rinsed twice with PBS and solubilized in 400 µl of a
solution composed of 0.03% SDS in 0.3 N NaOH. The extracts were
neutralized with HCl, and radioactivity incorporated into the cells was
quantified by liquid scintillation spectrometry.
Northern blot analysis.
RNA was isolated from cells using the method of Chomczynski and Sacchi
(9). The poly(A)+ RNA fraction was
prepared using the PolyATract mRNA Isolation System (Promega, Madison,
WI) and fractionated. Poly(A)+ RNA
(400 ng) was fractionated, using denaturing MOPS-formaldehyde-1% agarose gels, transferred to Biotrans nylon membranes (ICN, Irvine, CA)
by blotting, and fixed by baking the filters at 80°C for 2 h under
vacuum. Human c-fos (33) and chicken
-actin probes (10), both containing the entire coding region, were
labeled by random priming with
[
-32P]dCTP, using
the Megaprime DNA labeling system (Amersham). After hybridization, the
nylon membranes were exposed to Kodak XAR-5 film (Eastman Kodak,
Rochester, NY) at
70°C. The membranes were then stripped of
the c-fos probe by incubation in
buffer containing 10 mM Tris · HCl, pH 7.5, 1 mM
EDTA, and 0.5% (wt/vol) SDS for 15 min at 80°C. The membranes were
then rehybridized with the
-actin probe and exposed again to X-ray
film. The intensity of the c-fos and
-actin bands was determined by densitometry (see above), and the
signal intensity of c-fos mRNA was
expressed relative to that of
-actin mRNA.
PGE2 assay. The PGE2 concentration in cell culture medium was estimated using a PGE2 enzyme immunoassay kit from Amersham Life Sciences (Buckinghamshire, UK). The sensitivity of assay was 2.5 pg/ml of medium.
Statistical analysis. One-factor ANOVA was used to test the overall hypothesis of no group differences, followed by two-sample t-tests for pairwise comparisons where the overall hypothesis was rejected. All tests were made at the 0.05 level of significance.
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RESULTS |
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Validation of Gi-OTR coupling in CHO-OTR
cells.
Because the -subunits of Gi
are major activators of MAPK in some cell types, it was important to
determine whether OTRs are coupled to
Gi in CHO-OTR cells.
Gi coupling was established by two
independent approaches. Physical association of
Gi with the OTR was shown by
coprecipitation experiments, using antibody to
Gi3
-subunit and
125I-OTA-labeled OTR (Fig.
1A).
As in the case of rat uterine myometrial membranes, both
Gq/11 and
Gi were associated with the OTR in
approximately equal amounts (28). The addition of PTX to CHO-OTR
membranes without OT caused ADP ribosylation of 41 kDa
G
i, as demonstrated by SDS-PAGE
and autoradiography (Fig. 1B).
Ribosylation was inhibited by increasing concentrations of OT (Fig.
1B), due to OT-induced dissociation
of G
i from the heterotrimeric G
protein complex. Free G
i is not
a substrate for PTX-stimulated ADP ribosylation. The addition of 10 nM
OT resulted in about a 50% decrease in the ADP ribosylated
-subunit
of Gi. These results show that the
CHO-OTR cell line is a valid system in which to study both
PTX-dependent and -independent regulation of MAPK activity.
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OT-stimulated ERK2 phosphorylation. CHO-OTR cells were treated with OT for increasing times, and cell lysates were analyzed for phosphorylation of ERK1 and ERK2. OT caused the rapid phosphorylation of ERK2, as evidenced by the electrophoretic mobility shift of a fraction of total ERK2 on immunoblots (Fig. 2A). Phosphorylation gel shifts were detected with antibody cross-reacting to both ERKs, but ERK1 was not phosphorylated after OT addition. The level of phosphorylation of ERK2 peaked around 2 min and returned to basal values by 15 min (Fig. 2A). Comparable results were obtained by assaying ERK activity, which results in the phosphorylation of MBP in vitro (Fig. 2B). MBP phosphorylation was increased about ninefold by 3 min after OT treatment. OT had no effect on ERK2 phosphorylation in CHO cells lacking the OTR (Fig. 3A). Treatment of CHO-OTR cells with OTA (1 µM) had no effect on ERK2 phosphorylation in CHO-OTR cells, but pretreatment with OTA for 15 min completely blocked the stimulation by 50 nM OT (Fig. 3B).
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Roles of Gi and PLC in OT-induced ERK2 phosphorylation. Pretreatment of CHO-OTR cells for 18 h with PTX (200 ng/ml) completely inhibited PTX-stimulated ADP ribosylation of CHO-OTR cell membranes, without any apparent effect on cell viability (data not shown). This same dose of PTX inhibited OT-induced phosphorylation of ERK2 by 25.6 ± 8% (n = 3) by 3 min after addition of OT (Fig. 4A). These results indicate that the OTR-Gi coupled pathway accounts for part of the amount of ERK2 phosphorylated. To determine whether OT-induced ERK2 phosphorylation is primarily mediated by Gq/11, we utilized U-73122, which blocks the activity of PI-PLC (27), an effector enzyme that is coupled to Gq/11. U-73122 (2.5 µM) inhibited ERK2 phosphorylation by 75.1 ± 2.2% (n = 3) by 3 min after OT stimulation (Fig. 4B). Because the level of inhibition by U-73122 was greater than that seen with PTX, it is likely that Gq primarily mediates OT stimulation of ERK2 phosphorylation.
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Effects of Ca2+ and PKC on OT-elicited ERK2 phosphorylation. Activation of PI-PLC results in an increase in inositol trisphosphate concentration, resulting in increased release of Ca2+ from intracellular stores, and in increased DAG concentrations. Rises in both intracellular Ca2+ and DAG lead to activation of PKC. Elevation of [Ca2+]i by treatment of CHO-OTR cells with the Ca2+ ionophore A-23187 (Calcimycin) for 5 min resulted in the same level of ERK2 phosphorylation as seen after OT treatment (Fig. 5A). These findings suggest that elevation of [Ca2+]i after OT stimulation might result in ERK2 phosphorylation. Indeed, incubation of cells with intracellular Ca2+ sponges, such as BAPTA-AM (10 µM) and TMB-8 · HCl (10 µM) inhibited OT-induced ERK2 phosphorylation by 51 and 57%, respectively (Fig. 5B).
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Effect of OT on [3H]thymidine incorporation into CHO-OTR cells. Although OT has not been considered to be a growth-promoting agent, activation of the ERK is generally associated with cell proliferation. To determine if OT stimulates mitogenesis, [3H]thymidine incorporation into CHO-OTR cells was measured after addition of OT. This work was carried out under conditions where OT stimulates ERK2 phosphorylation (in virtually confluent cells). Known mitogens such as 5% FBS and bFGF caused 5.7 ± 0.3- and 1.8 ± 0.2-fold (n = 3) increases in the incorporation of [3H]thymidine by 4 h, respectively (Fig. 6). In contrast, the addition of OT under the same conditions had no effect on [3H]thymidine incorporation (Fig. 6).
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MAPK kinase (MEK1/2) inhibition completely blocks OT-induced ERK2
phosphorylation, transcription of c-fos mRNA, and
PGE2 synthesis.
In view of the lack of effect on OT on CHO-OTR cell proliferation, we
examined other possible sites of action. Ternary complex factors, one
of which is Elk-1, have been implicated in mediation of
c-fos induction (19). MAP kinase
catalyzed phosphorylation of Elk-1 allows the Elk-1-serum response
factor complex to bind to serum response elements and thereby to
activate c-fos expression. OT
treatment of CHO-OTR cells induced synthesis of
c-fos mRNA within 30 min of treatment
(Fig.
7B). To
determine the importance of OT-activated ERK2 phosphorylation on
c-fos mRNA levels, we used PD-98059, a
highly selective inhibitor of MAP kinase kinase (MEK1/2) activation and
the MAPK cascade. This agent binds to the inactive forms of MEK and
prevents its phosphorylation by either c-Raf or MEKK1 with
IC50 values of 5-10 µM
(25). Complete inhibition of OT-induced ERK2 phosphorylation was
obtained with 10 µM PD-98059 (Fig.
7A). Treatment of CHO-OTR cells with
20 µM PD-98059 resulted in 90 and 97.2% reductions in
c-fos mRNA levels (normalized to
-actin mRNA levels) at 30 and 60 min after addition of OT,
respectively (Fig. 7B).
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DISCUSSION |
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Our previous studies and those of others have shown that the OTR is
functionally coupled to both Gi
and Gq in rat myometrium (28). Our
present findings show that in CHO cells transfected with the rat OTR,
both Gi and
Gq interact with the receptor. The two are coupled in about the same proportion as seen in rat myometrium. Other studies have shown that OTR in PG responsive cells such as
endometrium and amnion are coupled to G proteins involved in PLC and
PKC activation (17, 22). The CHO-OTR cell system thus represents a good
model for studying OT action, both from the standpoint of G protein
isoforms coupled to the OTR and the cellular response elicited by OT.
Stimulation of these cells with OT resulted in the specific
phosphorylation and activation of ERK2. In a given cell, several signal
transduction pathways involving different heterotrimeric G proteins
operate in parallel, and there is increasing evidence that the G
protein-mediated signal transduction system undergoes adaptation and
cross-regulation at many different levels. Our findings show that both
Gi and
Gq mediate the effects of OT on
ERK2 phosphorylation. Whereas OT-stimulated
p42MAPK activity in human
myometrial cells was completely abolished by pretreatment of the cells
with PTX (100 ng/ml) for 4 h (24), we found that in CHO-OTR cells the
Gi-specific pathway (PTX
sensitive) accounted for only about one-quarter of phosphorylated ERK2.
Gq activation likely is
responsible for the rest, as the PLC inhibitor U-73122 blocked ERK2
phosphorylation by about 75% at a concentration of 2.5 µM. This dose
is near that reported for the
IC50, at least in some systems
(35), suggesting that complete inhibition might have been obtained with
greater doses. Unfortunately, because of the limited solubility of the
drug, we could not obtain greater concentrations without getting a
nonspecific vehicle-related inhibition of ERK2 phosphorylation.
Although we have not carried out detailed studies, it appears that the
Gi- and
Gq-mediated pathways converge at
PLC, because inhibition of PKC activity (resulting from PLC activation)
with GF-109203X completely blocked OT-stimulated ERK2 phosphorylation.
Thus the potential for -subunits of
Gi to activate a
p21ras-dependent pathway,
independent of PKC, appears to be less important in CHO-OTR cells after
OT treatment. The findings are in accord with the demonstration by
several laboratories that Gi
-subunits activate PLC directly (16). GF-109203X is highly
specific for the
,
, and
isoforms of PKC, but it has recently
been shown that it also inhibits MAPKAP kinase-1
(Rsk-2) and p70 S6
kinase, both members of the MAPK cascade (2). Because both these
activities are downstream from ERK2, it is more likely that the
inhibition of OT-stimulated ERK2 phosphorylation by GF-109203X was due
to the inhibition of PKC activity. Inhibition of increases in
[Ca2+]i
with the Ca2+ sponges partially
blocked OT-stimulated ERK2 phosphorylation. Conversely, elevation of
[Ca2+]i
with Calcimycin caused ERK2 phosphorylation, indicating that intracellular Ca2+ is important in
activating ERK2 in CHO-OTR cells. The effects of Calcimycin on ERK2
were blocked by PKC inhibition, suggesting that rather than
[Ca2+]i
causing ERK2 phosphorylation via a PKC-independent,
p21ras-mediated process (14, 15),
Ca2+ activates PKC.
The transcription factor Elk-1 (ternary complex factor-1), which participates in formation of a ternary complex with the serum response element allowing transcription of c-fos, is phosphorylated by ERKs (19). Because OT activated ERK2, it was not surprising that OT also induced increases in c-fos mRNA levels. Indeed, OT induction of c-fos mRNA was virtually eliminated by pretreating CHO-OTR cells with the MEK1/2 inhibitor PD-98059. AP1 activity often is induced by mitogens, and the ERK pathway is generally considered to be involved in cell proliferation or differentiation, depending on cellular context (12). However, OT had no effect on [3H]thymidine incorporation by CHO-OTR cells, unlike FBS or bFGF. Thus the role of OT-induced synthesis of c-fos mRNA in CHO-OTR cells remains to be determined. The phosphorylation of ERK2 is critical for OT-induced PGE2 synthesis, as the effects of OT on PGE2 were completely blocked by PD-98059. Inhibition of ERK2 phosphorylation by PTX and GF-109203X gave proportional effects on OT-stimulated PGE2 release, indicating a clear causal relationship between ERK2 activation and PGE2 synthesis. Although it has been demonstrated that MAPK is involved in cPLA2 phosphorylation (23), there is evidence to suggest that the rise in intracellular Ca2+, and not phosphorylation of cPLA2, is essential for activation of the arachidonic acid cascade in rat liver macrophages (5). In other studies, PD-98059 inhibited p42/p44MAPK activation in thrombin-, collagen- and phorbol ester-stimulated platelets, but did not interfere with the release of arachidonic acid or with cPLA2 phosphorylation (7). Our findings, which indicate a causal relationship between inhibition of ERK2 phosphorylation and inhibition of PGE2 production, are more consistent with the conclusions of other studies (34). In summary, we have shown that PKC activation through G proteins and ERK2 phosphorylation are vital steps in the process of OT stimulation of PGE2 and c-fos synthesis (Fig. 9). PKC-independent steps involving p21ras appear to be less important in OT signaling in CHO-OTR cells (Fig. 9). Our studies also establish these cells as a valid model to study additional signaling pathways involved in OT action.
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ACKNOWLEDGEMENTS |
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We thank Solweig Soloff for expert technical assistance.
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FOOTNOTES |
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This research was supported in part by National Institute of Child Health and Human Development Grant HD-26168 (M. S. Soloff).
Address for reprint requests: M. Soloff, Dept. of Obstetrics and Gynecology, Univ. of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1062.
Received 1 October 1997; accepted in final form 12 December 1997.
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