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Biology of Reproduction 65, 496-506 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Platelet-Derived Growth Factor Stimulates Phospholipase C-{gamma}1, Extracellular Signal-Regulated Kinase, and Arachidonic Acid Release in Rat Myometrial Cells: Contribution to Cyclic 3',5'-Adenosine Monophosphate Production and Effect on Cell Proliferation1

Isaline Boulvena, Bruno Palmiera, Philippe Robina, Monique Vachera, Simone Harbona, and Denis Leibera

a Signalisation et Régulations Cellulaires, Centre National de la Recherche Scientifique, UMR 8619, Université Paris-Sud, 91405 Orsay Cedex, France

ABSTRACT

In the present study, we examined downstream signaling events that followed exposure of cultured rat myometrial cells to platelet-derived growth factor (PDGF) and their effect on cell proliferation. PDGF-BB induced tyrosine phosphorylation of PDGF-ß receptors and increased inositol trisphosphate production via the tyrosine phosphorylation of phospholipase (PL)C-{gamma}1. PDGF-BB also increased cAMP synthesis. This increase was potentiated by forskolin and reduced by indomethacin, a cyclooxygenase inhibitor, reflecting a Gs protein-mediated process via prostaglandin biosynthesis. The prostaglandin produced by PDGF was characterized as prostacyclin (PGI2). PDGF-BB increased arachidonic acid (AA) release, which, similarly to cAMP accumulation, was abolished in the presence of AACOCF3, a cytosolic PLA2 inhibitor, and in the absence of Ca2+. U-73122, a potent inhibitor of PLC activity, blocked both the production of inositol phosphates and the AA release triggered by PDGF-BB. Extracellular signal-regulated kinases (ERKs) 1 and 2 are expressed in myometrial cells, and PDGF-BB selectively activated ERK2. PD98059, an inhibitor of the ERK-activating kinase, blocked PDGF-BB-mediated ERK2 activation, AA release, and cAMP production. The results demonstrate that PDGF-BB stimulated cAMP formation through both PLC activation and ERK-dependent AA release and PGI2 biosynthesis. PDGF-BB also increased cell proliferation and [3H]thymidine incorporation. This was abolished by PD98059, demonstrating that the ERK cascade is required for the mitogenic effect of PDGF-BB. Forskolin, which potentiated the cAMP response to PDGF-BB, attenuated both DNA synthesis and ERK activation triggered by PDGF-BB, suggesting the presence of a negative feedback regulation.

cyclic adenosine monophosphate, growth factors, kinases, signal transduction, uterus

INTRODUCTION

In rat myometrium, various signaling processes are regulated by G protein-coupled receptors. The stimulation of ß-adrenergic and prostaglandin (PGE2 and prostacyclin) receptors by their specific ligands increases adenylyl-cyclase activity and cAMP production [1–3] via Gs activation. Cyclic AMP has been shown to contribute to uterine relaxation [4]. On the other hand, the necessity of Ca2+ for uterine contraction has long been recognized [5]. Various contractile agonists are associated with the stimulation of phospholipase (PL)C-ß3 via specific Gq-protein-coupled receptors [3, 6–9] that stimulate phosphatidyl inositol 4,5-bisphosphate (PIP2) hydrolysis. This stimulatory process induces an increase in inositol 1,4,5-trisphosphate (InsP3) production, which triggers Ca2+ release from intracellular stores, followed by an increase in Ca2+ entry [10]. Protein tyrosine kinase activities also regulate myometrial signaling processes [11, 12]. Palmier et al. [11] reported that in rat myometrium, pervanadate, a protein tyrosine phosphatase inhibitor, increased InsP3 production by tyrosine phosphorylation and stimulation of PLC-{gamma}1.

Growth factors, including platelet-derived growth factor (PDGF), mediate their effects via receptor tyrosine kinases and have been reported to play a central role in regulating female reproductive tract function [13]. PDGF is a dimer of homologous A- and B-polypeptide chains, which are assembled as homodimers (PDGF-AA and PDGF-BB) or heterodimer (PDGF-AB). PDGF exerts its effect on target cells by binding to two structurally related receptor tyrosine kinases, denoted {alpha} and ß. Upon ligand binding, the PDGF receptor dimerizes and autophosphorylates on a number of tyrosine residues. Tyrosine phosphorylated sites are used by PDGF receptors as anchor sites for various SH2 domain-containing proteins. These proteins fall into two categories: 1) signaling enzymes such as PLC-{gamma}1, phosphatidylinositol 3-kinase, Src family members, the GTPase-activating protein of Ras (RasGAP), and the tyrosine phosphatase (SHP-2); and 2) adaptor molecules, including Shc, and Grb2 that is associated with Sos, the nucleotide exchange factor for Ras (reviewed in [14, 15]). The recruitment of the Grb2-Sos complex by the activated PDGF receptor allows the conversion of the inactive Ras-GDP into active Ras-GTP. Ras activation leads to the stimulation of the mitogen-activated protein kinases (MAPKs) of the ERK (i.e., extracellular signal-regulated kinase) type, which occurs by sequential activation of Raf and MEK (MAPK/ERK kinase) [16–18]. Activated ERKs 1 and 2 provide a link between plasma membrane receptors and the nucleus, where they are translocated and serve as important regulators of nuclear transcriptional activity [16, 19].

Both the PDGF {alpha} and ß receptors are expressed in mouse uterus [20, 21], whereas human myometrial tissue and myometrial smooth muscle cells in primary culture contain the PDGF-ß receptor, but not the {alpha} receptor [22]. The presence of the two isoforms of PDGF has been reported in mouse uterus [20, 21] as well as in human myometrium [22]. In mouse uterus, changes have been described in the expression of both PDGF and PDGF receptors by estradiol [21] and at mid-gestation [20]. Expression of PDGF-A chain also increases in human uterine smooth muscle cells during the physiological hypertrophy of pregnancy [23]. In rat uterine cells, PDGF stimulates DNA synthesis [24]. However, no information is yet available about the expression of PDGF and its specific receptor in rat myometrium. Furthermore, nothing is yet known about the role of the growth factor in the regulation of signaling pathways controlling proliferation in the myometrial cells of the different species so far examined.

Considering the importance of cell hypertrophy and proliferation in myometrium under specific physiological or pathological conditions, the current study was designed to evaluate in cultured rat myometrial cells, the expression of PDGF-ß receptors and the downstream signaling events, including PLC/InsP3 cascade, ERK activation, and cAMP metabolism that followed exposure to PDGF. We further analyzed the effect of these signaling pathways on cell proliferation.

MATERIALS AND METHODS

Materials

PDGF-BB and arachidonic acid (AA) were from Peprotech (Tebu, France). Iloprost was generously donated by Dr. N. Sprzagala (Schering, Berlin, Germany). 3-Isobutyl-1-methylxanthine (IBMX) was from Aldrich Chemical (Milwaukee, WI). Cyclic AMP was from P-L Biochemicals Inc. (Milwaukee, WI). ß-Estradiol-3-benzoate, leupeptin, aprotinin, LiCl, forskolin, and indomethacin were from Sigma (St. Louis, MO). [{gamma}-32P]ATP (3000 Ci/mmol), [5,6,8,9,11,12,14,15-3H–]AA (60–100 Ci/mmol), [3H]cAMP (37 Ci/mmol), methyl-[3H]thymidine, and Western blotting detection reagents were obtained from Du Pont New England Nuclear Products Division (Paris, France). Myo-[23H]inositol (10–20 Ci/mmol) was obtained from Amersham International (Les Ulis, France). AACOCF3, PD98059, U-73122, and U-73343 were from Biomol (Plymouth Meeting, PA). Forskolin and tyrphostin AG1296 were from Calbiochem (Los Angeles, CA). Collagenase was from Boehringer-Mannheim S.A. (Meylan, France). The monoclonal 4G10 antiphosphotyrosine antibodies and the polyclonal antibodies to PLC-{gamma}1 were from Upstate Biotechnology (Lake Placid, NY). Polyclonal antibodies to ERK1/2 were from Zymed Laboratories Inc. (San Francisco, CA), and polyclonal antiactive ERK1/2 antibodies were from Promega Corporation (Madison, WI). Polyclonal antibodies to PDGF-ß receptor ({alpha}-PR3), raised against a synthetic peptide corresponding to amino acids 981–994 in the deduced amino acid sequence of the murine PDGF-ß receptor, were kindly provided by Dr. Serge Roche (CNRS, UPR 1086, Montpellier, France). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were from Dako (Trappes, France) and Bio-Rad (Ivry sur Seine, France), respectively. All other reagents were of the highest grade commercially available.

Animals

Animals were treated in accordance with the principles and procedures outlined in the European Guidelines for the Care and Use of Experimental Animals. Prepubertal Wistar female rats (Janvier, France), 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 the next day by 1 min of carbon dioxide inhalation.

Myometrial Cell Preparation and Culture

Ten rats were used for each cell preparation. Immediately after the death of the animals, uteri were removed and immersed in Eagle modified minimum essential medium (MEM), which contained Earle salts, 100 µg/ml penicillin-streptomycin, 2.5 µg/ml fungizone amphotericin B, 2 mM glutamine, and 25 mM Hepes pH 7.4 at room temperature. Myometria were prepared free of endometrium [1], pooled, and minced in MEM-supplemented medium. Minced tissues were incubated for 20 min at 37°C with agitation in the dissociation medium (MEM-supplemented medium that contained 0.05% collagenase, 0.02% DNase I, and 0.1% trypsin inhibitor) under constant gas flow (5% CO2/95% humidified air). The cell suspension obtained from this first digestion, which could be contaminated by endometrial cells, was discarded. Myometrial fragments were then subjected to three successive 40-min digestions in the dissociation medium. The cell suspensions were centrifuged at 300 x g for 10 min, and the pellets were resuspended in MEM-supplemented medium containing 10% fetal calf serum (FCS). The myometrial cells were cultured in MEM-supplemented medium plus 10% FCS in Petri dishes at 37°C in an atmosphere of 5% CO2/95% humidified air for 20 min to eliminate fibroblasts. Cell suspension (typically 6 x 106 myometrial cells) was then cultured in the same conditions at a plating density of 15 x 103 cells/cm2. The medium was changed every 2 days and, 48 h prior to experiments, the cells were incubated in serum-free medium. Experiments comparing the effects of different agents were systematically performed with the same cell preparation and repeated at least on three different cell preparations.

It was verified that the cells had structural and morphological characteristics of smooth muscle cells. The presence of desmin and smooth muscle {alpha}-actin, and the absence of cytokeratin were analyzed by immunofluorescence with specific antibodies (monoclonal anti-smooth muscle {alpha}-actin clone 1A4, monoclonal antidesmin clone DE-U-10 from Sigma; and monoclonal anticytokeratin 18 clone DC10 from Novo Castra, Tébu, Le Perray-en-Yvelines, France), confirming the muscle origin of the cells (data not shown).

Measurement of [3H]Inositol Phosphates

Serum-starved confluent myometrial cells plated in 24-well plates were labeled by incubation for 24 h with 5 µCi/ml myo-[23H]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 concentration, 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% w/v). The cells were detached by scraping on ice and centrifuged at 10 000 x g for 15 min at 4°C. The TCA-soluble supernatants were extracted with diethyl ether, neutralized with Tris-[hydroxymethyl] aminomethane (Tris) base, and applied to a column of anion-exchanged resin (AG 1-X8; formate form; 200–400 mesh) for the separation of the individual inositol phosphates as described previously [6]. Alternatively, total inositol phosphates (InsPs) (i.e., inositol trisphosphate [InsP3] + inositol bisphosphate [InsP2] + inositol monophosphate [InsP1]) were eluted together in a single step with 12 ml of 1 M ammonium formate + 0.1 M formic acid. The 3H content of the fractions was determined by scintillation counting. Results were expressed as cpm/well or, alternatively, as a percentage of stimulation over the basal values obtained before the addition of the stimulatory agonist.

[3H]AA Release

Serum-starved confluent myometrial cells seeded in 24-well plates were labeled by incubation for 24 h with 0.4 µCi [3H]AA, final concentration 4 nM. Cells were washed four times with Hanks balanced salt solution supplemented with 5 mg/ml fatty acid-free BSA and 20 mM Hepes (pH 7.5). Cells were then allowed to equilibrate at 37°C in the same medium containing 1 mg/ml fatty acid-free BSA. After 15 min, the agents to be tested were added at the indicated concentrations, and incubation was continued for the time indicated. Reactions were stopped by aspiration of the incubation medium and the cells were washed with 1 ml of medium. The incubation and washing media were combined, transferred to ice-cold tubes containing 100 µl of EGTA and EDTA (final concentration, 5 mM each) as described [25], and centrifuged to eliminate cell debris. The radioactivity in the supernatants was determined by scintillation counting. Cells left attached to the plate were scraped with 0.2% Triton X-100 and counted for radioactivity. The release of [3H]AA was normalized as a percentage of total incorporated radioactivity obtained from the same sample, which corresponds to the total released radioactivity plus the total cell-associated radioactivity. An average of 175 000 to 220 000 cpm/well of [3H]AA were incorporated in phospholipids after 24 h of labeling.

Analysis of Prostaglandin Synthesis

Serum-starved confluent myometrial cells plated in 5-cm-diameter Petri dishes were washed four times with Hanks balanced salt solution supplemented with 20 mM Hepes (pH 7.5). Cells were then allowed to equilibrate at 37°C in 1 ml of the same medium. After 15 min, PDGF-BB (25 ng/ml) was added for 20 min. The release of 6-keto-PGF1{alpha} and PGE2 in incubation medium was assayed by a specific enzyme immunoassay as described by Pradelles et al. [26].

Incubation Experiments for Assay of cAMP Levels

Serum-starved confluent myometrial cells plated in 5-cm Petri dishes were rinsed twice with Hanks balanced salt solution containing 20 mM Hepes (pH 7.5). The cells were then incubated in 2 ml of fresh medium and allowed to equilibrate for 10 min in a 37°C water bath. Cells were incubated with IBMX at 200 µM for 5 min before exposure to the agents tested. Reactions were stopped by aspiration of the incubation medium followed by the addition of 1 ml of cold, 7% (w/v) TCA. The cells were detached by scraping on ice and centrifuged at 10 000 x g for 20 min at 4°C. The TCA-supernatants were extracted by diethyl ether, and cAMP was measured as detailed previously [1]. Cyclic AMP levels were expressed as picomoles per dish.

Immunoprecipitation and Western Blot Analysis of PDGF-ß Receptor and PLC-{gamma}1

Immunoprecipitation experiments were carried out essentially as in [11, 12]. Serum-starved confluent myometrial cells plated in 5-cm Petri dishes were rinsed twice with Hanks balanced salt solution containing 20 mM Hepes (pH 7.5). The cells were incubated in 2 ml of fresh buffer and allowed to equilibrate for 10 min in a 37°C water bath. The cells were then exposed to the various agents. Reactions were stopped by aspiration of the incubation medium followed by the addition of 200 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 phenylmethanesulfonyl fluoride). Cells were detached by scraping on ice and centrifuged at 10 000 x g for 20 min at 4°C. The amount of detergent-extracted proteins in the supernatant was determined using the bicinchonimic acid protein assay reagent kit. The equivalent of 250 µg of proteins was incubated with anti-PLC-{gamma}1 antibody (1:250) or anti-PDGF-ß receptor antibody ({alpha}-PR3; 1:250) overnight at 4°C and then with protein A-sepharose (10 mg) for 2 h at 4°C. Immune complexes were collected by centrifugation and subjected to 7.5% SDS-PAGE. The separated proteins were then transferred to nitrocellulose and blotted with the antiphosphotyrosine antibody (4G10; 1:5000), overnight at 4°C. In certain experiments, the blot was stripped in 62.5 mM Tris-HCl pH 6.7, 100 mM ß-mercaptoethanol, and 2% SDS for 60 min at 60°C, and reprobed with anti-PLC-{gamma}1 antibody (1:2000) or anti-PDGF-ß receptor antibody ({alpha}-PR3; 1:2000). The immunoreactive bands were visualized with an enhanced chemiluminescence system. In preliminary experiments, we verified that for all treatments, the immunoprecipitations resulted in the recovery of identical amount of proteins. Also see Figure 1.



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  		FIG. 1. PDGF-BB-induced tyrosine phosphorylation of the PDGF-ß receptor. Inhibitory effect of AG1296. A) Myometrial cells were treated for 5 min in the absence or presence of 25 ng/ml PDGF-BB or 50 ng/ml PDGF-AA. Detergent-extracted proteins were analyzed by Western blot using antiphosphotyrosine antibodies. B) Myometrial cells were treated for 10 min in the absence or presence of 20 µM AG1296 and were then incubated for 5 min without or with 25 ng/ml PDGF-BB. Detergent-extracted proteins were immunoprecipitated with anti-PDGF-ß receptor antibodies. Immunoblotting was performed using antiphosphotyrosine antibodies (upper panel). The blot was stripped and reprobed with anti-PDGF-ß receptor antibodies (lower panel). The positions of the molecular mass markers (Mr x 10-3) are shown. Data are from one of three experiments

Western Blot Analysis of Phosphorylated ERK1/2

Serum-starved confluent myometrial cells seeded in 6-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. Cells were detached by scrapping on ice and centrifuged at 10 000 x 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 were probed with either antiactive ERK1/2 antibodies (1:5000) or with anti-ERK1/2 antibodies (1:5000) overnight at 4°C. The immunoreactive bands were visualized with an enhanced chemiluminescence system. Quantification of the developed films was performed using a densitometer (Molecular Dynamics, Sunnyvale, CA).

[3H]Thymidine Incorporation and Cell Proliferation

Serum-starved myometrial cells (50% confluent) in 24-well dishes were incubated with PDGF-BB (25 ng/ml) in the absence or the presence of the various agents to be tested for 24 h, 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 the addition of 0.5 ml cold TCA (10% w/v). Radioactivity incorporated into TCA-precipitable material was recovered with 0.5 ml NaOH (1 N) and quantified by liquid scintillation counting. These experimental conditions have been optimized in preliminary experiments performed with 10% FCS, which induces a 10-fold stimulation of [3H]thymidine incorporation. In a separate cell proliferation assay, serum-starved myometrial cells were treated with PDGF-BB for 72 h, and then detached by trypsin treatment and counted with a hemocytometer.

Data Analysis

The results are expressed as means ± SEM. The experiments were analyzed with the general linear model procedure of SAS followed by the Scheffé test for multiple comparisons. A P value of less than 0.05 was considered statistically significant.

RESULTS

PDGF-BB Increased Tyrosine Phosphorylation of PDGF-ß Receptors in Rat Myometrial Cells in Primary Culture

Figure 1A shows an immunoblot of detergent-extracted proteins with an antiphosphotyrosine antibody. The treatment of rat myometrial cells in primary culture with PDGF-BB increased the tyrosine phosphorylation of a single protein of 180 kDa, a protein with similar electrophoretic mobility to the PDGF-ß receptor [23, 27]. PDGF-AA, which interacts only with the PDGF-{alpha} receptor, did not stimulate phosphorylation of any protein. Proteins from detergent-extract of PDGF-BB-treated and untreated myometrial cells were immunoprecipitated with anti-PDGF-ß receptor antibodies. Western blot analyses were then performed using anti-PDGF-ß receptor or antiphosphotyrosine antibodies. In immunoprecipitates from both control and PDGF-BB-stimulated cells, the anti-PDGF-ß receptor antibodies recognized equal amounts of a single protein band at 180 kDa (Fig. 1B, lower panel). Immunoblot analysis of the precipitated proteins with antiphosphotyrosine antibodies showed that PDGF-BB induced an increase in tyrosine phosphorylation of PDGF-ß receptor (Fig. 1B, upper panel) that was strongly reduced in the presence of 20 µM tyrphostin AG1296, a specific inhibitor of the intrinsic tyrosine kinase of the PDGF receptor [28].

PDGF-BB-Stimulated cAMP Generation via AA Release and Prostaglandin Synthesis

PDGF-BB increased cAMP production in rat myometrial cells (Fig. 2). The PDGF-BB-mediated cAMP accumulation was compared with that obtained with iloprost, a stable analogue of prostacyclin (PGI2), which is a well-known activator of adenylyl-cyclase in rat myometrium [2, 3]. Iloprost increased cAMP in myometrial cells (Fig. 2). A suboptimal concentration (10 µM) of forskolin-stimulated cAMP accumulation and markedly potentiated the production of cAMP, which was triggered by iloprost and PDGF-BB. This potentiation reflects the interaction of forskolin with the catalytic unit of adenylyl cyclase when it is associated with the activated G{alpha}s subunit [29]. Thus, similar to iloprost, the PDGF-BB-induced cAMP response appeared to be mediated via activation of the Gs protein. PDGF-AA had no effect, even in the presence of forskolin.



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  		FIG. 2. Effect of PDGF on cAMP production. Potentiation by forskolin. Inhibitory effect of indomethacin. Myometrial cells were exposed for 5 min to 200 µM IBMX and were then incubated for 10 min in the absence (basal) or presence of 25 ng/ml PDGF-BB, 50 ng/ml PDGF-AA, or 4 µM iloprost without (hatched bars) or with (open bars) 10 µM forskolin (FK). When used, 10 µM indomethacin (indo, black bars) was added 5 min before IBMX. Cyclic AMP concentration was determined as described in Materials and Methods. Data are expressed as pmol cAMP/well. Values are means ± SEM of five separate experiments. *Significantly different from basal - FK (P < 0.05). **Significantly different from basal + FK (P < 0.05). {dagger}Not significantly different from iloprost + FK

The PDGF-BB-mediated increase in cAMP generation was strongly reduced if the cells were incubated in the presence of indomethacin, a specific inhibitor of cyclooxygenase that is involved in the prostaglandin biosynthesis pathway (Fig. 2). Cyclic AMP production induced by iloprost was not significantly affected by indomethacin. This strongly suggested that PDGF-BB stimulated cAMP production via a prostaglandin-dependent pathway. It has been previously shown that PGI2 and PGE2 are able to stimulate cAMP production in rat myometrium [2]. We thus analyzed the production of these two prostaglandins in response to PDGF-BB. As shown in Table 1, PDGF-BB induced a twofold increase in 6-keto-PGF1{alpha}, the stable metabolite of PGI2, whereas no effect on PGE2 synthesis was detected (not shown). As expected, this increase in 6-keto-PGF1{alpha} was completely inhibited by pretreatment with indomethacin (Table 1).


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  		TABLE 1. Effect of PDGF-BB on 6-keto-PGF1{{alpha}} synthesis.*

Because AA release is believed to be the rate-limiting step in prostaglandin synthesis, the ability of PDGF-BB to stimulate the release of AA was examined. Treatment of [3H]AA-labeled myometrial cells with PDGF-BB induced a rapid release of AA that reached a plateau at 15 min (data not shown). The effect of PDGF-BB at 10 min was dose-dependent, with an EC50 equal to 2.5 ng/ml, and a maximal response at 25 ng/ml (Fig. 3A). Thus, PDGF-BB stimulated AA release as efficiently as it stimulated cAMP production (EC50 = 3 ng/ml and maximal effect at 30 ng/ml; Fig. 3B). The release of AA and the production of cAMP mediated by PDGF-BB were completely inhibited by AG1296 (Fig. 3, A and B).



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  		FIG. 3. Dose-dependent effects of PDGF-BB on AA release and cAMP accumulation. A) [3H]AA-labeled myometrial cells were exposed to the indicated concentrations of PDGF-BB for 10 min. When used, 20 µM AG1296 (black bar) was added 10 min prior to stimulation with PDGF-BB. [3H]AA release was determined as described in Materials and Methods and is expressed as a percentage of total incorporated radioactivity. Values are means ± SEM of three separate experiments each carried out in duplicate. B) IBMX-treated myometrial cells were exposed for 10 min to various concentrations of PDGF-BB combined with 10 µM forskolin. When used, 20 µM AG1296 (black bar) was added 10 min before stimulation with PDGF-BB. Data are expressed as pmol cAMP/well. Values are means ± SEM of four separate experiments

PDGF-BB Increased AA Release and cAMP Production by Stimulating cPLA2

The cytosolic phospholipase A2 (cPLA2), which preferentially catalyses the hydrolysis of AA from phospholipids, can be activated by growth factor [30]. Treatment of the cells with arachidonyl trifluoromethyl ketone (AACOCF3), a specific inhibitor of the cPLA2 [31], strongly decreased (>80%) both the AA release (Fig. 4A) and cAMP production (Fig. 4B) mediated by PDGF-BB. The inhibitor failed to reduce cAMP accumulation mediated by iloprost (Fig. 4B). As activation of cPLA2 requires Ca2+ for membrane association and full activity [32], we tested the effect of Ca2+ depletion on AA release and cAMP production. The accumulation of cAMP triggered by PDGF-BB was strongly reduced when the cells were depleted of Ca2+ by a 15-min incubation in a Ca2+-free medium supplemented with 1 mM EGTA (Fig. 4B). The production of cAMP stimulated by iloprost was unaffected by the absence of Ca2+, indicating that Ca2+ is not required for activation of the prostaglandin receptor-Gs protein-adenylyl cyclase system. The release of AA induced by PDGF-BB was blocked by Ca2+ depletion (Fig. 4A), suggesting that the PDGF-BB-mediated AA release, involving cPLA2, was the Ca2+-dependent step in the cAMP production.



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  		FIG. 4. Inhibitory effects of AACOCF3 and Ca2+ depletion on AA release and cAMP production due to PDGF-BB. A) [3H]AA-labeled myometrial cells were incubated in a normal Ca2+ medium in the absence (open bars) or presence of 10 µM AACOCF3 (hatched bars) for 10 min and then stimulated by incubation for 10 min with 25 ng/ml PDGF-BB. In some experiments, [3H]AA-labeled myometrial cells were incubated in Ca2+-depleted Hanks solution containing 1 mM EGTA (black bars), and were allowed to equilibrate for 15 min. Cells were then treated for 10 min with PDGF-BB. [3H]AA release is expressed as a percentage of total incorporated radioactivity. Values are means ± SEM of five separate experiments, each carried out in duplicate. *Significantly different from basal (P < 0.05). B) Myometrial cells were treated as described for AA release in the presence of Ca2+ without or with 10 µM AACOCF3, or in the absence of Ca2+ plus 1 mM EGTA. The cells were then incubated with 10 µM forskolin added alone (basal) or in combination with 25 ng/ml PDGF-BB or 4 µM iloprost for 10 min. Results, expressed as pmol of cAMP/well, are means ± SEM of five separate experiments. *Significantly different from basal (P < 0.05). {dagger}Not significantly different from iloprost (P < 0.05)

PDGF-BB Increased the Production of InsPs in Rat Myometrial Cells. Association with Tyrosine Phosphorylation of PLC-{gamma}1

The production of InsP3 plays a critical role in the increase of intracellular Ca2+ concentration in myometrium. The treatment of [3H]inositol-labeled myometrial cells with PDGF-BB produced a time-dependent increase in the InsPs (InsP1, InsP2, InsP3; Fig. 5A), which was markedly reduced when the tyrosine kinase of the PDGF receptor was inhibited by AG1296 (data not shown). The accumulation of InsP2, and particularly of InsP3, was a rapid process that reached a plateau at 20 min and 5 min, respectively. The increased production of InsP1 was delayed and was not stabilized up to 20 min. The sequential generation of InsPs in the order InsP3, InsP2, and InsP1 provided evidence that PDGF-BB stimulates the activity of a PLC that degrades PIP2. We investigated whether tyrosine phosphorylation of PLC-{gamma}1 could account for the production of InsPs mediated by PDGF-BB. Immunoblots of the proteins immunoprecipitated with anti-PLC-{gamma}1 antibodies revealed a single, 150-kDa protein identified as PLC-{gamma}1 (Fig. 5B, lower panel). Similar amounts of PLC-{gamma}1 were immunodetected in PDGF-BB-treated and -untreated cells. The antiphosphotyrosine antibodies (Fig. 5B, upper panel) detected tyrosine phosphorylated PLC-{gamma}1 only in PLC-{gamma}1 immunoprecipitates from PDGF-BB-treated cells. Thus, PDGF-BB stimulated the phosphorylation of tyrosine residues on the PDGF-ß receptor (Fig. 1B), which was associated with the phosphorylation of PLC-{gamma}1 and the increased production of InsPs.



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  		FIG. 5. Stimulatory effects of PDGF-BB on the accumulation of inositol phosphates and the tyrosine phosphorylation of PLC-{gamma}1. A) [3H]Inositol-prelabeled myometrial cells were incubated for 10 min in the presence of 10 mM LiCl. Cells were then treated with 25 ng/ml PDGF-BB for the time indicated and the individual [3H]inositol phosphates (i.e., InsP1 [{diamondsuit}], InsP2 [{block}], and InsP3 [{blacktriangleup}]) were separated on AG1-X8 columns as described in Materials and Methods. Results are expressed as the percentage of stimulation over basal values (1048 ± 108, 232 ± 22, 112 ± 10 cpm/well for InsP1, InsP2, and InsP3, respectively). Values are means ± SEM of five separate experiments each performed in duplicate. B) Serum-starved myometrial cells were incubated for 5 min with 25 ng/ml PDGF-BB. Detergent-extracted proteins were immunoprecipitated with anti-PLC-{gamma}1 antibodies. Immunoblotting was performed using antiphosphotyrosine (anti-P-Tyr) antibodies (upper panel). The blot was stripped and reprobed with an anti-PLC-{gamma}1 antibody (lower panel). The positions of the molecular mass markers (Mr x 10-3) are shown. Data are from one of three separate experiments

PDGF-BB-Induced AA Release Is Mediated by PLC-{gamma}1 Activation

Compound U-73122 has been described as a potent inhibitor of the activation of different PLC isoforms [33]. Treatment of the cells with U-73122 abolished InsPs production triggered by PDGF-BB. U-73343, the inactive analogue, had no effect (Fig. 6A). Inhibition of AA release due to PDGF-BB was similarly observed with U-73122, but not with the inactive U-73343 (Fig. 6B). The data demonstrated that in rat myometrial cells, the stimulation of the PLC was a prerequisite for the Ca2+-dependent release of AA induced by PDGF-BB.



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  		FIG. 6. Inhibitory effect of U-73122 on inositol phosphates production and AA release triggered by PDGF-BB. A) [3H]Inositol-prelabeled myometrial cells were incubated for 10 min in the presence of 10 mM LiCl, without (open bars) or with 2.5 µM U-73122 (black bars) or 2.5 µM U-73343 (hatched bars). Cells were then treated with 25 ng/ml PDGF-BB for 10 min. Total inositol phosphates were determined as described in Materials and Methods. Values are means ± SEM of three separate experiments each performed in duplicate. B) [3H]AA-labeled myometrial cells were incubated in the absence or presence of 2.5 µM U-73122 or 2.5 µM U-73343 for 10 min. Cells were then stimulated by incubation for 10 min with 25 ng/ml PDGF-BB. [3H]AA release is expressed as a percentage of total incorporated radioactivity. Values are means ± SEM of three separate experiments, each carried out in duplicate. *Significantly different from basal (P < 0.05). {dagger}Not significantly different from PDGF-BB alone

PDGF-BB-Induced AA Release and cAMP Production Are Mediated by ERK Activation

A full activation of cPLA2 requires not only an increase of intracellular Ca2+ concentration, but also phosphorylation by ERKs [30, 34]. Treatment of the cells with PD98059, an inhibitor of MEK activation [35], markedly affected both the AA release (89% inhibition; Fig. 7A) and cAMP production (75% inhibition; Fig. 7B) stimulated by PDGF-BB. In contrast, incubation of the cells with PD98059 did not significantly affect the cAMP production induced by iloprost (Fig. 7B). The data suggested that ERK activation was required for both the production of cAMP and the release of AA triggered by PDGF-BB.



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  		FIG. 7. Inhibitory effect of PD98059 on AA release and cAMP production due to PDGF-BB. A) [3H]AA-labeled myometrial cells were incubated in the absence (open bars) or presence of 20 µM PD98059 (hatched bars) for 20 min and then stimulated by incubation for 10 min with 25 ng/ml PDGF-BB. [3H]AA release is expressed as a percentage of total incorporated radioactivity. Values are means ± SEM of five separate experiments, each carried out in duplicate. *Significantly different from basal (P < 0.05). B) Myometrial cells were treated as described for AA release and then incubated for 10 min with 10 µM forskolin added alone (basal) or in combination with 25 ng/ml PDGF-BB or 4 µM iloprost. Results, expressed in pmol of cAMP/well, are means ± SEM of five separate experiments. *Significantly different from basal (P < 0.05). {dagger}Not significantly different from iloprost

ERK1 and ERK2 were both present in detergent extracts from rat myometrial cells (Fig. 8A, lower panels). The expression of ERK1 and ERK2 was not affected by stimulation of the cells with PDGF-BB. Immunoblot analysis of the detergent-extracted proteins with the antiactive ERK1/2 antibody demonstrated (Fig. 8A, upper panels) that PDGF-BB mediated the selective activation of ERK2. PDGF-BB caused a rapid rise in ERK2 phosphorylation, which peaked at 5 min. Activation gradually declined, with a sustained phase that persisted for at least 90 min (Fig. 8, A and B). PD98059 strongly reduced the phosphorylation of ERK2 (Fig. 8, A and B). The activation of ERK2 by PDGF-BB was confirmed by measuring ERK activity in an ERK2 immunocomplex. A 5-min stimulation of myometrial cells with PDGF-BB produced a threefold increase in ERK2 activity, as determined by its ability to phosphorylate an exogenous substrate, the myelin basic protein (MBP). The stimulatory effect was abolished in the presence of PD98059 (data not shown).



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  		FIG. 8. Effect of PDGF-BB on ERK activation. A) Myometrial cells were incubated in the presence of 25 ng/ml PDGF-BB for the time indicated, in the absence (lanes 1–9) or presence (lane 10) of 20 µM PD98059 added 20 min before PDGF-BB. Detergent-extracted proteins were analyzed by SDS-PAGE. Immunoblotting was performed using antiactive ERK1/2 antibody (upper panels) or anti-ERK1/2 antibody (lower panels). Data are from one of three separate experiments. B) Cells were treated with PDGF-BB (25 ng/ml) for the times indicated, without ({block}) or with (•) PD98059, as described in A. Detergent-extracted proteins were analyzed by SDS-PAGE and immunoblotting using antiactive ERK1/2 antibodies and anti-ERK1/2 antibodies. Phosphorylated ERK2 and total ERK2 were quantified using a densitometer (Molecular Dynamics). The level of ERK2 phosphorylation is expressed as the fold stimulation over unstimulated control after normalization with respect to total ERK2 level. Values are means ± SEM of three separate experiments

Effect of PDGF-BB on [3H]Thymidine Incorporation and Cell Proliferation

We next examined whether PDGF-BB could exert a mitogenic effect in myometrial cells. The addition of PDGF-BB (25 ng/ml) for 72 h to quiescent myometrial cells cultured in serum-free medium caused a 2.8-fold increase in cell number (Table 2). Incubation of the cells with PDGF-BB for 48 h also induced a large increase (10-fold) in the incorporation of [3H]thymidine into DNA. The increase in [3H]thymidine incorporation induced by PDGF-BB was completely inhibited in cells preincubated with PD98059, suggesting that the ERK cascade is necessary for PDGF-induced DNA synthesis (Table 2).


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  		TABLE 2. Effect of PDGF-BB on [3H]thymidine incorporation and cell proliferation; inhibitory effect of PD98059.*

Inhibitory Effect of cAMP on [3H]Thymidine Incorporation and ERK Activation Triggered by PDGF-BB

Cyclic AMP has been described as a negative modulator of ERK activation in smooth muscle cells [36, 37]. Because PDGF-ß receptors are associated with elevated cAMP levels in myometrial cells, we investigated whether increases in intracellular cAMP concentration affected PDGF-induced ERK activation and DNA synthesis. Results in Figure 9A show that treatment of the cells with the cAMP elevating agent, iloprost, partially reduced (64%) PDGF-BB-mediated [3H]thymidine incorporation. The inhibitory effect of iloprost was greatly increased (100% inhibition) if the cells were incubated in the copresence of iloprost and IBMX, a phosphodiesterase inhibitor. When myometrial cells were stimulated with PDGF-BB in the copresence of forskolin and IBMX, which resulted in a marked elevation of intracellular cAMP, [3H]thymidine incorporation due to PDGF-BB was completely abolished. This inhibition of the PDGF-BB response was counteracted by prior indomethacin treatment, which strongly reduced the cAMP response triggered by the growth factor. As expected, the reduction of PDGF-BB-mediated DNA synthesis observed in the presence of iloprost plus IBMX was insensitive to indomethacin. Data in Figure 9B show that the activation of ERK2 by PDGF-BB was attenuated when the cells were stimulated in the copresence of forskolin and IBMX, with both the peak and the sustained phase affected.



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  		FIG. 9. Inhibitory effect of cAMP on [3H]thymidine incorporation and ERK activation triggered by PDGF-BB. A) Quiescent cells were incubated without (open bars) or with (hatched bars) 25 ng/ml PDGF-BB, in the absence or presence of 10 µM forskolin (FK), 4 µM iloprost, or 200 µM IBMX for 48 h. Where indicated, cells were first treated for 10 min with 10 µM indomethacin (indo). [3H]Thymidine (2 µCi/ml) was present for the last 24 h. [3H]Thymidine incorporation was estimated as described in Materials and Methods. Results are expressed as cpm/well and are means ± SEM of five separate experiments, each carried out in duplicate. **Significantly different from basal (P < 0.05). *Significantly different from PDGF-BB alone (P < 0.05). {dagger}Not significantly different from PDGF-BB + iloprost (P < 0.05). B) Myometrial cells were incubated in the presence of 25 ng/ml PDGF-BB for the time indicated, without ({circ}) or with ({block}) 10 µM forskolin plus 200 µM IBMX. Detergent-extracted proteins were analyzed by SDS-PAGE and immunoblotting using antiactive ERK1/2 antibodies and anti-ERK1/2 antibodies. Phosphorylated ERK2 and total ERK2 were quantified using a densitometer (Molecular Dynamics). The level of ERK2 phosphorylation is expressed as the fold stimulation over unstimulated control after normalization with respect to total ERK2 level. Values are means ± SEM of three separate experiments

DISCUSSION

These results demonstrate that the PDGF-ß receptor is present on rat myometrial cells and is phosphorylated in response to PDGF-BB. The expression of PDGF-ß receptors has also been reported in mouse [20, 21] uterus and human myometrium [22]. However, nothing was known about the role of PDGF in the regulation of signaling pathways in myometrial cells. The data described herein are consistent with two cascades of events triggered by the activation of PDGF-ß receptors in a primary culture of rat myometrial cells. First, tyrosine phosphorylation of PLC-{gamma}1, which is then able to hydrolyse PIP2, resulting in an increased production of InsP3 and the attendant rise in cytosolic Ca2+ concentration; and second, sustained activation of ERK, particularly ERK2, which plays a key role in both the PDGF-BB-mediated increase in DNA synthesis and the PDGF-BB-mediated activation of cPLA2 with the subsequent generation of prostacyclin, contributing to cAMP production (Fig. 10).



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  		FIG. 10. Schematic representation of PDGF-mediated signaling pathways in rat myometrial cells. Activated PDGF-ß receptor (PDGFß-R) is associated with an increased activity of a) PLC-{gamma}/InsP3/Ca2+ pathway and b) the ERK cascade. The b) process is required for the growth factor-induced cell proliferation. Both a) and b) processes contribute to cPLA2 activation with an increased release of AA and biosynthesis of prostacyclin (PGI2), which stimulates the adenylyl cyclase (AC)/cAMP system. Subsequently, cAMP retroinhibits the ERK and proliferation cascades. COX, Cyclooxygenase; PL, phospholipids

PDGF-BB has been described as an activator of cAMP synthesis in arterial and airway smooth muscles [36, 37]. In myometrial cells, the stimulation of PDGF-ß receptors increased the generation of cAMP. Forskolin, which potentiated the cAMP responses triggered by iloprost, acting via a G-protein coupled receptor, similarly potentiated PDGF-BB-mediated cAMP accumulation. It is generally accepted that the synergistic cAMP response observed with forskolin reflects the interaction of the diterpene with adenylyl-cyclase when it is activated by the G{alpha}s subunit [29]. We found that the generation of cAMP induced by PDGF-BB was indeed regulated via the biosynthesis of prostaglandins that bind to cell surface receptors and activate the Gs-adenylyl cyclase system. The involvement of prostaglandins in PDGF-mediated cAMP production has also been reported in both arterial [38] and airway [37] smooth muscles and in myofibroblastic hepatic stellate cells [39]. Graves et al. [38] and Mallat et al. [39] noted a correlation between the increased synthesis of PGE2 and the production of cAMP triggered by PDGF. In myometrial cells, no PGE2 production could be detected under PDGF stimulation, but we observed an increased synthesis of 6-keto-PGF1{alpha}, the stable metabolite of PGI2. In rat myometrium, PGI2, which is the major prostaglandin generated from AA, strongly stimulates cAMP synthesis [2]. In addition, specific receptors for PGI2 are present in myometrium and are involved in PGI2-mediated and iloprost-mediated adenylyl cyclase activation [3]. These results together indicate that PGI2 is the prostaglandin involved in PDGF-induced cAMP production.

It is well known that the release of AA from membrane phospholipids is a limiting step in the biosynthesis of prostaglandins. PDGF-BB stimulated AA release in a dose-dependent manner with a pattern similar to that for the PDGF-BB-mediated increase in cAMP synthesis. In addition, supportive evidence is provided that cPLA2 is responsible for PDGF-BB-mediated AA release and cAMP production in myometrial cells. First, both the AA release and cAMP production triggered by PDGF-BB were sensitive to the cPLA2 inhibitor, AACOCF3. Second, Ca2+ depletion and inhibition of the PLC/InsP3 pathway blocked PDGF-BB-stimulated AA release and cAMP production. This is consistent with the release of AA by cPLA2, which requires micromolar concentrations of Ca2+ for binding to membranes and full activation [32]. Third, both the AA release and cAMP production triggered by PDGF-BB were abolished in the presence of PD98059, a specific inhibitor of ERK activation, which is known to contribute to cPLA2 stimulation via phosphorylation of serine 505 in the catalytic site of the enzyme [30, 32, 34].

Thus, our observations demonstrate that the activation of cPLA2 induced by PDGF-BB results from the coordinated effects of PDGF-mediated InsP3/Ca2+ and ERK cascades, resulting in the stimulation of the Gs-adenylyl cyclase system via AA release and PGI2 biosynthesis. Such cross-talk between signaling pathways provides a mean by which a receptor tyrosine kinase could stimulate a biochemical pathway (i.e., adenylyl cyclase/cAMP) that is activated by a G protein-coupled receptor.

PDGF is a potent mitogen for various cell types. Our data have shown that PDGF-BB induced cell proliferation and [3H]thymidine incorporation in myometrial cells. We demonstrated that iloprost increased cAMP production and attenuated PDGF-BB-mediated DNA synthesis. Incubation of the cells with a suboptimal concentration of forskolin inhibited the DNA synthesis triggered by the activated PDGF-ß receptors. This inhibition was reduced by indomethacin, indicating that the cAMP generated in response to PDGF-BB plays a key role in the inhibition of PDGF-BB-mediated DNA synthesis. PDGF-BB plus forskolin also inhibited the ERK activation triggered by PDGF-BB, suggesting that the inhibition of DNA synthesis by cAMP is due to an attenuation of ERK activation. Depending on the cell type examined, cAMP has been described as an activator or an inhibitor of the ERK pathway [40]. In keeping with our results, in myofibroblasts and smooth muscle cells [36, 37, 39], an increase in the level of cAMP production resulted in an inhibition of both ERK activation and DNA synthesis triggered by PDGF. Heldin et al. [41] and Graves et al. [38] reported that the PDGF-ß receptor is not the target for the inhibitory effect of cAMP on PDGF-mediated DNA synthesis in fibroblasts and arterial smooth muscle. We also found that elevation of cAMP in myometrial cells did not inhibit PDGF-induced receptor ß-subunit autophosphorylation or InsP accumulation (data not shown). It has been suggested that the phosphorylation of Raf-1, a major component of the ERK cascade, by the cAMP-dependent protein kinase, reduces the ability of this protein to interact with Ras, thereby preventing its Ras-dependent activation [42, 43]. The target for the inhibitory effect of cAMP on PDGF-BB-mediated ERK activation/DNA synthesis in myometrial cells remains to be identified.

In conclusion, this study demonstrates that in myometrial cells, PDGF-ß receptors are associated with the activation of specific signaling pathways that converge to regulate diverse intracellular cascades, including PLC-{gamma}1/InsP3 stimulation and ERK activation that contributes to both DNA synthesis and cAMP production by increasing the release of AA and PGI2 biosynthesis. The inhibition of ERK activation and DNA synthesis by cAMP constitutes a potentially important feedback loop for PDGF-BB-mediated responses, and may contribute to the development of homologous desensitization of the PDGF-ß receptor signaling (Fig. 10). In addition, the cross-talk that occurs between the adenylyl cyclase stimulating pathway, associated with G protein-coupled receptors, and the PDGF-ß receptor pathway, may serve to produce heterologous desensitization of PDGF-BB-mediated responses. It is reasonable to consider that a balance between the stimulatory and inhibitory pathways described in this study would play a major role in the control of myometrial activities, such as growth and proliferation, by PDGF operating through an autocrine/paracrine circuit. These concerns are the subjects of our current work.

ACKNOWLEDGMENTS

We thank G. Thomas and C. Desmyter for expert technical assistance. We also thank Dr. J. Coursol (Laboratoire de Mathématiques, Université de Paris-Sud, Orsay, France) for his kind assistance with statistical analysis. We are grateful to Dr. C. Creminon (Service de pharmacologie et d'immunologie, CEA Saclay, Gif sur Yvette, France) for performing the immuno enzymatic measurement of 6-keto-PGF1{alpha} and PGE2.

FOOTNOTES

First decision: 3 January 2001.

1 This work was supported by grants from the Centre National de la Recherche Scientifique (UMR 8619) and by a contribution from the Association de la Recherche contre le Cancer (contract 1355). Back

2 Correspondence: Denis Leiber, Laboratoire de Signalisation et Régulations Cellulaires, CNRS UMR 8619, Bâtiment 430, Université de Paris- Sud 91405 Orsay Cedex, France. FAX: 033 1 69 85 37 15;denis.leiber{at}erc.u-psud.fr Back

Accepted: March 29, 2001.

Received: October 23, 2000.

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Am J Physiol Cell Physiol, July 1, 2002; 283(1): C251 - C260.
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