Laboratoire de Signalisation et Régulations Cellulaires, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8619, Université de Paris-Sud, 91405 Orsay cedex, France
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ABSTRACT |
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In this study, we analyzed in rat myometrial cells the signaling pathways involved in the endothelin (ET)-1-induced extracellular signal-regulated kinase (ERK) activation required for the induction of DNA synthesis. We found that inhibition of protein kinase C (PKC) by Ro-31-8220 abolished ERK activation. Inhibition of phospholipase C (PLC) by U-73122 or of phosphoinositide (PI) 3-kinase by wortmannin partially reduced ERK activation. A similar partial inhibition was observed after treatment with pertussis toxin or PKC downregulation by phorbol ester treatment. The effect of wortmannin was additive with that produced by PKC downregulation but not with that due to pertussis toxin. These results suggest that both diacylglycerol-sensitive PKC, activated by PLC products, and diacylglycerol-insensitive PKC, possibly activated by a Gi-PI 3-kinase-dependent process, are involved in ET-1-induced ERK activation. These two pathways were found to be activated mainly through the ETA receptor subtype. ET-1 and phorbol ester stimulated Src activity in a PKC-dependent manner, both responses being abolished in the presence of Ro-31-8220. Inhibition of Src kinases by PP1 abrogated phorbol ester- and ET-1-induced ERK activation. Finally, ET-1 activated Ras in a PP1- and Ro-31-8220-sensitive manner. Altogether, our results indicate that ET-1 induces ERK activation in rat myometrial cells through the sequential stimulation of PKC, Src, and Ras.
phosphoinositide 3-kinase; deoxyribonucleic acid synthesis; phospholipase C; Ras
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INTRODUCTION |
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MYOMETRIUM, the smooth muscle tissue of the uterus, is the site of diverse physiological processes such as hypertrophy, hyperplasia, contraction, and apoptosis, which are critical for uterine activities. These biological functions are under the control of multiple hormonal factors including growth factors, neurotransmitters, prostaglandins, steroid, and peptidic hormones.
Endothelin (ET)-1 is a peptidic hormone described first as a potent vasoconstrictor and later as an important physiological modulator of uterine activities. Indeed, ET-1 is synthesized in intrauterine tissues (40) and can affect myometrial cells in a paracrine fashion to induce contractility. In rat myometrium, our laboratory previously showed (23) that ET-1 activates the ETA receptors that are functionally coupled to Gq/11 protein to stimulate phospholipase C (PLC) activity and to Gi protein to inhibit adenylyl cyclase. The increase in Ca2+ concentration induced by inositol 1,4,5-trisphosphate (InsP3) and a decrease of the cAMP level are the major determinants of ET-1-induced uterine contraction. Additional data from our laboratory indicate that ET-1 also activates protein tyrosine kinases that contribute to stimulation of rat uterine contractility (38) and protein kinase C (PKC) (29), which has been involved in human uterine contractility at term (13).
ET-1 also has mitogenic properties in various cell types, including
human myometrial cells (2, 8). It is well established that
the initiation of proliferation relies on the activation of the
mitogen-activated protein (MAP) kinase cascade (18). The
activation of MAP kinases has been observed in human myometrial cells
stimulated by interleukin-1 and oxytocin (1, 37), in nonpregnant rat myometrial cells on platelet-derived growth factor (PDGF) stimulation (6), and in puerperal rat myometrial
cells in response to G protein-coupled receptors activation by ET-1, prostaglandin F2, and oxytocin (25, 35,
36). The mechanisms involved in the stimulation of this
signaling pathway by G protein-coupled receptors are complex and seem
to vary with the cell type and agonist used. These mechanisms may
require the transactivation of a receptor tyrosine kinase such as
epidermal growth factor (EGF) receptor or stimulation of nonreceptor
tyrosine kinases including Src family kinases (18, 39).
The activation of these tyrosine kinases results in the recruitment of
the adaptor protein Grb2 on tyrosine phosphorylated proteins. Grb2 is
associated with Sos, the specific exchange factor of Ras, which
catalyzes the conversion of inactive Ras-GDP into active Ras-GTP.
Activated Ras initiates the MAP kinase cascade constituted by the
kinases Raf-1, MAP kinase kinase (MEK), and extracellular
signal-regulated kinases (ERKs). Such a mechanism, mediated by
transactivation of the EGF receptor tyrosine kinase, has been observed
in vascular smooth muscles cells (21) and in human ovarian
carcinoma cells (52) stimulated by ET-1.
A role for PKC has also been described in the ERK activation process
induced by ET-1 (5, 20, 22, 54). PKC is a family of
serine/threonine kinases that is subdivided into three groups on the
basis of structural and biochemical properties, conventional (c; ,
1,
2,
), novel (n;
,
,
,
, µ,
), and atypical
(a;
,
/
) isoforms. c/nPKC isoforms are activated by
diacylglycerol (DAG), a product of phosphatidylinositol
4,5-bisphosphate (PIP2) hydrolysis, and by phorbol esters.
In addition to DAG, cPKC requires Ca2+ for full activation
(41). In contrast to c/nPKC, aPKCs are insensitive to DAG
and are activated by other pathways. Indeed, PKC-
is stimulated
through phosphorylation by phosphoinositide (PI)-dependent protein
kinase (PDK) 1 in a phosphatidylinositol 3,4,5-trisphosphate
(PIP3)-dependent manner (10, 28). This signaling pathway is activated by PI 3-kinase, which catalyzes the
phosphorylation of PIP2 into PIP3. The
mechanisms by which PKC could activate the MAP kinase cascade are not
fully elucidated and seem to be, in some extent, cell type specific. It
has been reported that PKC activates Raf-1 (26) or MEK
(34) through a direct phosphorylation. However, PKC may
also stimulate Ras through the activation of protein tyrosine kinases
(3, 14).
Given that proliferation is one of the main functions of myometrial cells in uterine physiology, we aimed to investigate whether ET-1 is able to induce proliferation of rat myometrial cells and to determine the mechanisms involved. Because ERKs are essential for mitogen-induced DNA synthesis, we focused our study on the characterization of the pathways controlling ERK activation in response to ET-1. The results presented here highlight the crucial role of the sequential activation of PKC and Src family tyrosine kinases in this process.
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MATERIALS AND METHODS |
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Materials.
ET-1 and FR-139317 were from NeoSystem (Strasbourg, France), and
PDGF-BB was from Peprotech (Tebu, Le Perray-en-Yvelynes, France). EGF,
wortmannin, pertussis toxin (PTX), -estradiol-3-benzoate, leupeptin,
aprotinin, LiCl, phorbol 12-myristate 13-acetate (PMA), and phorbol
12,13-dibutyrate (PDBu) were from Sigma (St. Louis, MO). Western
blotting detection reagents and [
-32P]ATP were
obtained from Du Pont New England Nuclear Products Division (Paris,
France). Myo-[2-3H]inositol (10-20
Ci/mmol), pGEX-2T vector, and glutathione-Sepharose CL-4B were obtained
from Amersham Pharmacia Biotechnology (Les Ulis, France). PP1, PP3,
PD-98059, U-73122, and U-73343 were from Biomol (Plymouth Meeting, PA).
Tyrphostins AG-1296 and AG-1478, Ro-31-8220, sarafotoxin S6c,
BQ-788, LY-294002, and U-0126 were from Calbiochem (Los Angeles, CA).
Collagenase was from Boehringer Mannheim (Meylan, France). Polyclonal
antibody to ERK1/2 was from Zymed Laboratories (San Francisco, CA), and
polyclonal anti-active ERK1/2 antibody was from Promega (Madison, WI).
Monoclonal anti-Src antibody (clone GD11), monoclonal anti-pan-Ras
antibody, and Src substrate peptide were from Upstate Biotechnology
(Lake Placid, NY). Horseradish peroxidase-conjugated anti-rabbit
antibodies were from Dako (Trappes, France). Restriction enzymes
BamHI and EcoRI were from Promega, and
oligonucleotides were synthesized by MWG Biotech (Les Ulis, France).
Protein G Plus-agarose was from Santa Cruz (Tebu, Le
Perray-en-Yvelynes, France). All other reagents were of the highest
grade commercially available.
Animals. Animals were treated in accordance with the principles and procedures outlined in European guidelines for the care and use of experimental animals. Prepubertal Wistar female rats (Janvier), 21 days old, were housed for 7 days in an environmentally controlled room before use. Chow and water were available ad libitum. Rats were treated with 30 µg of estradiol for the last 2 days and were killed when 28 days old by 1 min of carbon dioxide inhalation.
Myometrial cell preparation and culture. Primary cultures of myometrial cells were prepared by collagenase digestion as previously described (6). The myometrial cells were cultured in minimum essential medium (MEM) supplemented with 10% fetal calf serum at 37°C in an atmosphere of 5% CO2-95% humidified air at a plating density of 3.5 × 104 cells/ml. The medium was changed every 2 days, and the cells were kept in serum-free medium for 48 h before experiments.
Measurement of [3H]inositol phosphates. Confluent myometrial cells seeded in 24-well plates were labeled by incubation for 48 h in serum-free medium supplemented with 5 µCi/ml myo-[2-3H]inositol (final concentration 10 µM). The cells were washed twice with Hanks' balanced salt solution containing 20 mM HEPES (pH 7.5) and then incubated at 37°C in fresh buffer with 10 mM LiCl. After 10 min, the agents to be tested were added at the indicated concentrations and incubation was continued for the time indicated for the specific experiment. Reactions were stopped by aspiration of the incubation medium followed by the addition of 1 ml of cold trichloroacetic acid (TCA; 7% wt/vol). The cells were detached by scraping on ice and centrifuged at 10,000 g for 15 min at 4°C. Total inositol phosphates (InsPs) were quantified as previously described (6). Results were expressed as counts per minute per well.
Western blot analysis of phosphorylated ERK1/2. Serum-starved confluent myometrial cells seeded in six-well plates were rinsed twice with Hanks' balanced salt solution containing 20 mM HEPES (pH 7.5) and incubated in 2 ml of fresh medium for 10 min. Cells were then exposed to the agents tested. Reactions were stopped by aspiration of the incubation medium followed by the addition of 100 µl of cold solubilization buffer [50 mM HEPES, pH 7.4, 150 mM NaCl, 100 mM NaF, 10% glycerol, 10 mM Na4P2O7, 200 µM Na3VO4, 10 mM EDTA, 1% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)]. Cells were detached by scraping on ice and centrifuged at 10,000 g for 20 min at 4°C. Detergent-extracted proteins (40 µg) were heated for 10 min at 95°C with Laemmli sample buffer and analyzed by 10% SDS-PAGE. The separated proteins were transferred to nitrocellulose sheets and probed with polyclonal anti-active ERK1/2 antibodies (1:5,000). The blots were then stripped in glycine 0.1 M-HCl, pH 2.2, and reprobed with polyclonal anti-ERK1/2 antibodies (1:5,000). The immunoreactive bands were visualized by a enhanced chemiluminescence system after incubation with horseradish peroxidase-conjugated swine anti-rabbit IgG. Quantification of the developed blots was performed with a densitometer (Molecular Dynamics, Sunnyvale, CA).
[3H]thymidine incorporation. Serum-starved myometrial cells (50% confluent) in 24-well dishes were incubated with the various agents to be tested for 24 h, and then [3H]thymidine (2 µCi/ml) was added to each well. Cells were incubated for an additional 24 h, and then reactions were terminated by aspiration of the incubation medium and addition of 0.5 ml of cold TCA (10% w/vol). Radioactivity incorporated into TCA-precipitable material was recovered with 0.5 ml of 1 N NaOH and quantified by liquid scintillation counting.
Measurement of Src kinase activation.
Src activity was determined as previously described (7).
Serum-starved confluent myometrial cells seeded in 55-cm2
petri dishes were rinsed twice with Hanks' balanced salt solution containing 20 mM HEPES (pH 7.5) and incubated in 4 ml of fresh medium
for 10 min. Cells were then stimulated by incubation for 3 min with 50 nM ET-1. Reactions were stopped by aspiration of the incubation medium
followed by addition of 500 µl of cold solubilization buffer. Cells
were detached by scraping on ice and were centrifuged at 10,000 g for 10 min at 4°C. For immunoprecipitation, the
supernatants were each incubated with 2 µg of monoclonal anti-Src
antibodies for 1 h at 4°C. We then added 20 µl of protein G
Plus-Agarose, and the incubation was continued for another 1 h.
The immunoprecipitates were washed four times in solubilization buffer
and once in kinase buffer [50 mM Tris · HCl, pH 7.5, 50 mM
NaCl, 10 mM MgCl2, 10 mM MnCl2, 1 mM
dithiothreitol (DTT), and 100 µM Na3VO4].
Src activity was assayed at 30°C for 20 min in 50 µl of kinase
buffer supplemented with 100 µM Src substrate peptide
(KVEKIGEGTYGVVYK) (9) and 50 µM
[-32P]ATP (5 µCi). The reaction was stopped by
adding 25 µl of 50% acetic acid. The samples were centrifuged, and
the supernatants were spotted onto phosphocellulose paper strips. The
strips were washed in 0.4% H3PO4 three times
for 15 min each and once in acetone and then dried. The dried strips
were counted for radioactivity in the presence of scintillation fluid.
Ras activation assay. The DNA fragment coding for amino acids 51-131 of the Ras binding domain (RBD) of human c-Raf-1 was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from human cDNA with the following set of primers: forward 5'-CACAGATGGATCCAAGACAAGCAACAC-3'; reverse 5'-GGGAAGAATTCACAGGAAATCTAC-3'. The forward primer contains a BamHI restriction site that allows the in-frame insertion of the RBD coding sequence with the glutathione-S-transferase (GST) gene located in the pGEX-2T vector. The reverse primer was designed to introduce a stop codon followed by an EcoRI restriction site. The RT-PCR product was digested with BamHI and EcoRI restriction enzymes and inserted in the BamHI-EcoRI sites of pGEX-2T plasmid. The plasmid obtained (pGEX-RBD) was sequenced to verify good insertion of the RBD coding sequence and then introduced in Escherichia coli BL21 cells for production of the fusion protein GST-RBD. Bacteria from a 250-ml overnight culture were pelleted (2,500 g, 15 min, 4°C) and resuspended in 15 ml of ice-cold lysis buffer (50 mM Tris · HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.5 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). Cells were lysed by sonication, and NP-40 was added (0.5% final concentration). The lysate was cleared by centrifugation at 10,000 g for 15 min at 4°C, and the supernatant was incubated for 1 h in the presence of glutathione-Sepharose beads to recover the GST-RBD protein. The beads were then washed five times in the same buffer.
Ras activation was determined by Ras-GTP pull-down assay with GST-RBD. Serum-starved confluent myometrial cells seeded in 55-cm2 plates were rinsed twice with Hanks' balanced salt solution containing 20 mM HEPES (pH 7.5) and incubated in 4 ml of fresh medium for 10 min. Cells were then exposed to the agents to be tested. Reactions were stopped by aspiration of the medium and addition of 1 ml of cold Ras assay (RA) buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 100 mM NaF, 10% glycerol, 10 mM Na4P2O7, 200 µM Na3VO4, 10 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.5 mM PMSF) and centrifuged at 10,000 g for 5 min at 4°C. The supernatants were collected and incubated in the presence of 8 µg of GST-RBD bound to glutathione-Sepharose beads for 1 h at 4°C in a final volume of 2 ml. Beads were then washed four times with RA buffer and resuspended in 50 µl of Laemmli sample buffer. Samples were analyzed by 15% SDS-PAGE, and the separated proteins were transferred to nitrocellulose sheets and probed with monoclonal anti-pan-Ras antibodies (1:500). The immunoreactive bands were visualized by a enhanced chemiluminescence system after incubation with horseradish peroxidase-conjugated anti-mouse IgG.Data analysis.
Results are expressed as means ± SE and were analyzed
statistically with Student's t-test. A P value
of 0.05 was considered significant.
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RESULTS |
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Activation of ERK is involved in mitogenic effect of ET-1.
Treatment of rat myometrial cells in primary culture with ET-1 enhanced
incorporation of [3H]thymidine (Fig.
1). DNA synthesis triggered by ET-1 was
strongly reduced when the cells were pretreated with PD-98059, a
selective inhibitor of MEK. This result pointed out the critical role
played by the ERK cascade in the mitogenic effect of ET-1. Data in Fig. 2A show that ET-1 induced ERK2
activation. ET-1-mediated activation of ERK2 was time dependent (Fig.
2B), with a maximal response at 5 min that slowly declined
to a low but observable level that persisted for at least 60 min. The
stimulatory effect of ET-1 was dose dependent (EC50 = 1-2 nM), with a maximal effect at 10 nM (Fig. 2C).
Stimulation of ERK2 triggered by ET-1 was potently reduced after
incubation of the cells with PD-98059 (Fig. 2A). The
present observations demonstrate that ET-1 exerts a mitogenic effect on
rat myometrial cells that is consecutive to the activation of ERK.
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ET-1-induced ERK2 activation is not due to transactivation of EGF
and PDGF receptors.
Some reports have demonstrated that activation of ERK by ET-1 could
occur through transactivation of a growth factor receptor such as EGF
receptor (21, 52). We recently demonstrated
(6) that PDGF-BB also stimulated ERK2 activity in our cell
system, and this response was completely blocked by AG-1296, a
selective inhibitor of PDGF receptors (Fig.
3). EGF also stimulated ERK2 activation
in myometrial cells, and this response was abolished in the presence of
AG-1478, a specific inhibitor of the tyrosine kinase of EGF receptors
(Fig. 3). In contrast, ERK2 activation triggered by ET-1 was completely
insensitive to these two inhibitors (Fig. 3). Thus the activation of
ERK2 induced by ET-1 was mediated by biochemical mechanisms that do not
involve either PDGF or EGF receptor tyrosine kinase.
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ERK2 activation by ET-1 is mediated by PKC and PLC activation.
Phosphorylation of ERK2 in response to ET-1 was abolished by
pretreatment of cells with Ro-31-8220, a specific inhibitor of all
classes of PKC (Fig. 4A).
Similar inhibition was obtained with 5 µM calphostin
C and 5 µM bisindolylmaleimide I, two other PKC inhibitors (data not
shown). Downregulation of c/nPKC by incubation of cells with 1 µM PMA
for 6 h also attenuated the activation of ERK2 induced by ET-1,
but to a lesser extent (48%; Fig. 4A). When the incubation
in the presence of PMA was lengthened to 20 h, the level of
inhibition was not increased (data not shown). PDBu also stimulated the
phosphorylation of ERK2, which, as expected, was completely inhibited
by Ro-31-8220 and by c/nPKC downregulation (Fig. 4A).
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ET-1-stimulated ERK2 involved a PI 3-kinase- and PTX-sensitive G
protein-dependent pathway.
It has been reported that PKC-, an aPKC, is activated by a PI
3-kinase-dependent pathway (10, 34, 44, 50). We therefore analyzed the effect of wortmannin, an inhibitor of PI 3-kinase, on
ET-1-induced ERK2 activation. Treatment with wortmannin inhibited ET-1-dependent ERK2 activation by 54 ± 3% (Fig.
5A). A similar partial
inhibition (53 ± 11%) was obtained when the cells were incubated
in the presence of 20 µM LY-294002, another PI 3-kinase inhibitor.
Treatment of cells with PTX also partially reduced ERK activation
(52 ± 5% inhibition). This effect of PTX was not additive with
that produced by wortmannin (Fig. 5A), indicating that PI
3-kinase and Gi protein act in a common pathway to trigger ERK2 activation in response to ET-1.
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ET-1 stimulation of ERK2 involved activation of Src.
Treatment of the cells with PP1, a specific inhibitor of Src, abolished
ERK2 activation induced by ET-1 (Fig.
6A). PP3, the inactive analog
of PP1, failed to reduce the stimulatory effect of ET-1 (Fig.
6A). Interestingly, activation of ERK2 stimulated with PDBu
was also potently inhibited by PP1 (Fig. 6A), indicating that Src may act downstream of PKC. To confirm this
observation, we tested the ability of ET-1 and PDBu to stimulate Src
activity. The results in Fig. 6B show that ET-1 and PDBu are
both able to activate Src about twofold. Moreover, Ro-31-8220
inhibited the activation of Src stimulated by ET-1 and PDBu, confirming
that, in both cases, Src activation is consecutive to PKC activation.
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ET-1 stimulated Ras activation.
The potency of ET-1 to activate Ras was investigated with a GST fusion
protein including the RBD of Raf. ET-1 caused a rapid but transient
activation of Ras that peaked at 3 min (Fig.
7A). Activation of Ras by ET-1
was inhibited when the cells were treated with PP1 and Ro-31-8220,
indicating that the PKC-Src pathway was required for this process (Fig.
7B).
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DISCUSSION |
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In the present study, we demonstrated that ET-1 is able to stimulate DNA synthesis in rat myometrial cells in primary culture through the activation of ERK pathway. Considering the heterogeneity of the mechanisms involved in ERK activation by G protein-coupled receptors, we characterized, in rat myometrial cells, the different pathways operating in ET-1-dependent ERK activation.
It is well documented that G protein-coupled receptors may activate ERK through the stimulation of tyrosine kinases including growth factor receptors such as EGF or PDGF receptors or nonreceptor tyrosine kinases. In this context, it has been reported in vascular smooth muscle cells and ovarian carcinoma cells that ET-1 transactivates the EGF receptor to stimulate ERK signaling pathway (21, 52). In rat myometrial cells, we found that ERK activation was independent of either the EGF or the PDGF receptor but involved a nonreceptor tyrosine kinase of the Src family. Indeed, we found that ET-1 stimulated Src activity whose inhibition abolished ERK activation. These data are in line with various reports indicating that ET-1 is able to stimulate Src family tyrosine kinases, which may mediate ERK activation (4, 11, 49).
In other respects, we demonstrate here the major role played by PKC in
the signaling pathway triggered by ET-1 in rat myometrial cells.
Indeed, inhibition of PKC abrogated ERK activation, which is consistent
with previously published data (20, 32, 54). Interestingly, PKC inhibition also abolished Src activation induced by
ET-1, suggesting that Src is a downstream effector of PKC. This notion
was strengthened by the result that direct activation of PKC by a
phorbol ester similarly stimulated Src activity. Recent reports also
indicate that Src acts downstream of PKC in signaling pathways
triggered by gonadotropin-releasing hormone (GnRH) (3, 15,
30). However, the mechanisms by which PKC could activate Src are
not clearly established. As indicated by recent studies, PKC-, -
,
and -
are able to interact directly with Src family kinases,
increasing the catalytic activity of the tyrosine kinases (46-48, 53). Activation of Src by PKC may also occur
through focal adhesion kinase family kinases such as Pyk2.
Indeed, PKC, in conjunction with Ca2+, has been shown to
activate Pyk2 (17, 42), which in turn may recruit and
activate Src (12). Whatever the mechanism involved in the
stimulation of Src by PKC, our data demonstrated that the sequential
activation of PKC and Src is required for ET-1-induced ERK activation.
Src-dependent activation of ERK generally involves a Ras-dependent
pathway (12, 31, 43, 45), which occurs through the
tyrosine phosphorylation of Shc and recruitment of the Grb2-Sos complex. In rat myometrial cells, we found that ET-1 induced a transient activation of Ras. The stimulation of Ras was dependent on
Src activity and, as expected, on PKC activity. Together, our data
suggest that the sequential activation of PKC and Src by ET-1 leads to
ERK stimulation in a Ras-dependent manner. However, previously
published data show that PKC-dependent activation of ERK may occur via
a direct activation of Raf (26, 33, 54) or MEK (34,
50) by PKC. We therefore cannot exclude that such processes
could influence the Ras-dependent ERK activation. In this context,
Benard et al. (3) showed that, in T3-1 cells, GnRH
stimulated ERK activation through both PKC-Src-Ras-Raf and PKC-Raf
pathways. However, in contrast to what we found in myometrial cells,
the direct activation of Raf by PKC appears to be the main pathway in
this cell model.
In human myometrial cells, Eude et al. (13) demonstrated
that ET-l-induced proliferation was totally dependent on cPKC-. In
rat myometrial cells, we demonstrated that c/nPKCs, activated through a
Gq-PLC-
pathway, are involved in ERK activation but only
for ~50%. The remaining 50% have been attributed to an aPKC, possibly PKC-
. This isoform is expressed in rat myometrium
(24) and is a substrate for PDK1, a kinase regulated by
PIP3, a product of PI 3-kinase activity (10,
34). A role for PI 3-kinase in ERK activation by G
protein-coupled receptors, including lysophosphatidic acid, angiotensin
II, and ET receptors, has been described (4, 16, 50). In
rat myometrial cells, we showed that ET-1-induced ERK activation is
partially (50%) dependent on PI 3-kinase activity, strengthening the
hypothesis of the involvement of aPKC. We also demonstrated that the PI
3-kinase-dependent activation of ERK by ET-1 was sensitive to PTX. This
is consistent with previous studies that demonstrated the existence of
a Gi-PI 3-kinase pathway in the activation of ERK by
diverse G protein-coupled receptors such as lysophosphatidic acid and
2-adrenergic receptors (19, 50).
Together, our results allow us to propose a model for ET-1-induced ERK
activation and DNA synthesis (Fig. 8) in
which ET-l, mainly through ETA receptors, activates two
parallel pathways that contribute equally to ERK activation. The first
pathway involves Gq, PLC-, and c/nPKC and the second
pathway Gi, PI 3-kinase, and aPKC. Activated PKCs in turn
trigger the sequential stimulation of Src and Ras, leading to ERK
activation. This model is compatible with previous results from our
laboratory (23) that indicated that in intact myometrium,
ET-1, through ETA receptor subtype, activates both
PTX-sensitive and -insensitive G proteins, the latter being coupled to
PLC-
activation.
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Kimura et al. (25) described in rat puerperal myometrial cells that PTX blocked ET-1-mediated ERK activation and that PMA-sensitive PKC was not involved in this regulatory pathway. In myometrial cells from prepubertal rats, we also demonstrated that stimulation of ERK by ET-1 was regulated through the activation of a PTX-sensitive, PMA-insensitive process, suggesting the existence of a common pathway in the two cell models. Moreover, Kimura et al. (25) showed the involvement of Sos, the exchange factor of Ras, the latter being activated in our model. However, in myometrial cells from prepubertal rats, the PTX-sensitive pathway contributes only 50% to the stimulation of ERK triggered by ET-1, the remaining 50% being attributed to the Gq, PLC, and c/nPKC pathway. At the end of gestation in puerperal myometrial cells, this last pathway no longer contributes to ERK activation but is devoted to the activation of other components of the contractile machinery (25). This suggests that signaling pathways leading to ERK activation in response to ET-1 are altered during gestation. This is consistent with previous data from our laboratory (27, 51) showing that the expression and/or functions of G proteins and effectors such as PLC are the target for differential regulation, linked to the hormonal environment during gestation. Together, these data illustrate the complexity of the mechanisms of regulation of ERK, which may depend not only on the stimulus and the cell type but also on the physiological status of the tissue.
In summary, we present evidence that in rat myometrial cells, ET-1, which is an important physiological modulator of uterine activities, stimulates DNA synthesis. This occurs through the sequential activation of PKC, Src, and Ras, which leads to ERK activation. Our data strongly suggest that c/nPKC and aPKC, which are activated downstream of PLC and PI 3-kinase, respectively, contribute equally to ET-1-induced ERK activation. The differential regulation of these signaling pathways may be of physiological relevance for the precise coordination, from implantation to parturition, of the two major functions of uterine smooth muscle, namely, contraction and proliferation. The characterization of these diverse regulatory pathways is of particular interest in light of the importance of cell proliferation in the physiology of the myometrium during gestation. Moreover, deregulation of the pathways controlling cell proliferation is undoubtedly determinant for the presence of pathological states of myometrial cells such as leiomyoma.
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ACKNOWLEDGEMENTS |
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We thank G. Delarbre for expert technical assistance. We are very grateful to Dr. M. Breuiller Fouché for help in ET receptor characterization.
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FOOTNOTES |
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* P. Robin and I. Boulven contributed equally to this work.
This work was supported by grants from the Centre National de la Recherche Scientifique (UMR 8619).
Address for reprint requests and other correspondence: D. Leiber, Laboratoire de Signalisation et Régulations Cellulaires, CNRS UMR 8619, Bât. 430, Université de Paris-Sud, 91405 Orsay Cedex, France (E-mail: denis.leiber{at}erc.u-psud.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 6, 2002;10.1152/ajpcell.00601.2001
Received 19 December 2001; accepted in final form 5 March 2002.
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