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BOR - Papers in Press, published online ahead of print September 8, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.033852
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BIOLOGY OF REPRODUCTION 72, 69–77 (2005)
DOI: 10.1095/biolreprod.104.033852
© 2005 by the Society for the Study of Reproduction, Inc.

Contribution of Phospholipase D in Endothelin-1-Mediated Extracellular Signal-Regulated Kinase Activation and Proliferation in Rat Uterine Leiomyoma Cells1

Philippe Robin, Sondes Chouayekh, Christine Bole-Feysot, Denis Leiber, and Zahra Tanfin2

Laboratoire de signalisation et régulations cellulaires, IBBMC, CNRS UMR 8619, Bat 430 Université Paris Sud, 91 405 Orsay Cedex, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endothelin (ET)-1 is a mitogenic factor in numerous cell types, including rat myometrial cells. In the present study, we investigated the potential role of ET-1 in the proliferation of tumoral uterine smooth muscle cells (ELT-3 cells). We found that ET-1 exerted a more potent mitogenic effect in ELT-3 cells than in normal myometrial cells, as indicated by the increase in [3H]thymidine incorporation, cell number, and bromodeoxyuridine incorporation. The ET-1 was more efficient than platelet-derived growth factor and epidermal growth factor to stimulate proliferation. The ET-1-mediated cell proliferation was inhibited in the presence of U0126, a specific inhibitor of (mitogen-activated protein kinase ERK kinase), indicating that extracellular signal-regulated kinase (ERK) activation is involved. Additionally, ET-1 induced the activation of phospholipase (PL) D, leading to the synthesis of phosphatidic acid (PA). The ET-1-induced activation of PLD was twofold higher in ELT-3 cells compared to that in normal cells. The two cell types expressed mRNA for PLD1a and PLD2, whereas PLD1b was expressed only in ELT-3 cells. The exposure of cells to butan-1-ol reduced ET-1-mediated production of PA by PLD and partially inhibited ERK activation and DNA synthesis. Addition of exogenous PLD or PA in the medium reproduced the effect of ET-1 on ERK activation and cell proliferation. Collectively, these data indicate that ET-1 is a potent mitogenic factor in ELT-3 cells via a signaling pathway involving a PLD-dependent activation of ERK. This highlights the potential role of ET-1 in the development of uterine leiomyoma, and it reinforces the role of PLD in tumor growth.

female reproductive tract, growth factors, kinases, signal transduction, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Uterine leiomyoma, or "fibroids," are the most common benign tumor of the uterine smooth muscle. Common symptoms associated with these tumors are dysmenorrhea, menorrhagia, infertility, and fetal morbidity [1, 2]. The Eker rat is a well-characterized animal model for spontaneous uterine leiomyoma [3] histologically similar to human leiomyoma. An Eker leiomyoma tumor-derived cell line (ELT-3) was characterized and has been successfully used to investigate the hormonal modulation in association with the disease pathogenesis [4, 5]. This cell model was also developed for preclinical studies to identify novel therapeutic agents for fibroids.

Leiomyoma growth is controlled by ovarian steroids and growth factors [6]. Nonetheless, little is known concerning the role of G protein-coupled receptor (GPCR) ligands in this disease. Recently, we reported that in normal rat myometrial smooth muscle cells, proliferation is regulated by growth factors acting via tyrosine kinase receptors (i.e., platelet-derived growth factor [PDGF] or epidermal growth factor [EGF]) but also by endothelin (ET)-1, which activates G protein-coupled receptors [7].

ET-1, a 21-amino acid peptidic hormone, belongs to a family of structurally homologous peptides that includes ET-1, ET-2, and ET-3. Endothelins mediate their actions via two receptor types that have been cloned and classified as the ETA and ETB receptors. The ET receptors modulate the generation of multiple second messengers, including inositol-1,4,5 trisphosphate (InsP3), diacylglycerol (DAG), arachidonic acid, and cAMP [8]. In rat myometrial strips [9] as well as in rat myometrial cells [10], ET-1, via ETA receptors, is able to stimulate phospholipase (PL) C to produce InsP3 and DAG, which are required for the release of Ca2+ from intracellular stores and the activation of protein kinase (PK) C, respectively. Stimulation of specific PKC isoforms contributes to uterine contractility [11] but also plays a critical role in rat [7, 10] and human [12] myometrial cell proliferation. In rat myometrial cells, DNA synthesis triggered by ET-1 involves a PKC-dependent activation of mitogen-activated protein (MAP) kinases of the extracellular signal-regulated kinase (ERK) type [7, 10]. Additionally, ET-1 is also involved in the development of several cancer tumors, such as prostate, colorectal, and ovarian cancers [13–16].

Data from our group have demonstrated that ET-1 also activates PLD in rat myometrium, and the regulatory mechanisms involved have been studied [17–19]. PLD catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline. PA has a number of biological activities by itself but can also be deacylated into lysoPA (LPA) by PLA2 [20, 21]. Two isoforms of mammalian PLDs (PLD1 and PLD2) have been cloned and characterized [22, 23]. The PLD1 presents two splice variants (PLD1a and PLD1b) [24], and three splice variants of PLD2 have been described [25]. In rat myometrium, PLD1 and PLD2 proteins are expressed [17], but their physiological role is unknown. PLD has been implicated in diverse physiological functions, including proliferation, migration, inflammation, and secretion [23, 26]. Moreover, the enhancement of PLD activity has been described in association with some cancer tumors [27].

The present study was designed to gain insight regarding the possible role of ET-1 in leiomyoma cell proliferation and to analyze the signaling pathways involved. Our findings reveal the potent, ERK-dependent mitogenic effect of ET-1, and they highlight the contribution of PA produced by PLD in this process.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

The [9,10-3H(N)]myristic acid (30–40 Ci/mmol) and [3H]thymidine (6.7 Ci/mmol) were purchased from N.E.N. (Les Ulis, France). The myo-[3H]inositol (20 Ci/mmol) was from Amersham Pharmacia Biotechnology (Les Ulis, France). The 4ß-phorbol 12,13-dibutyrate (PDBu), leupeptin, aprotinin, PMSF, Streptomyces chromofuscus PLD, and EGF were obtained from Sigma Chemicals (St. Louis, MO). All media and reagents for the cell culture were from Invitrogen (Cergy Pontoise, France). The Ro-31-8220 was from Calbiochem (Meudon, France). The ET-1 was from Neosystem (Strasbourg, France). The PDGF was from Peprotech (Tebu, Le Perray-en-Yvelynes, France). The bromodeoxyuridine (BrdU) and anti-BrdU antibody were from Roche Diagnostics (Meylan, France). Anti-active ERK1/2, deoxyribonucleotides, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, and Taq polymerase were from Promega (Madison, WI). The anti-ERK1/2 antibodies were from Zymed Laboratories (San Francisco, CA). The anti-rabbit immunoglobulin (Ig) G horseradish peroxidase (HRP)-conjugate was from DAKO (Trappes, France). The RNA purification reagent Insta-Pure was from Eurogentec (Angers, France). All other chemicals were of the highest grade available.

Cell Lines

The Eker rat tumor-derived ELT-3 uterine leiomyoma cell line was kindly provided by Dr C. Walker (University of Texas, Smithville, TX). The ELT-3 cells were maintained in DF8 medium supplemented with 10% (v/v) fetal calf serum (FCS) as described previously [4] at 37°C and 5% CO2/95% humidified air. Cells were placed in serum-free medium for 24 h before experiments.

Animals

Prepubertal Wistar female rats (Janvier, France; age, 21 days) 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 at 28 days of age by 1-min inhalation of carbon dioxide. All treatments were performed in accordance with the principles and procedures outlined in the European guidelines for the care and use of experimental animals.

Myometrial Cell Preparation and Culture

Primary cultures of myometrial cells were prepared by collagenase digestion as previously described [7]. The myometrial cells were cultured in minimum essential medium (MEM) supplemented with 10% (v/v) FCS at 37°C in an atmosphere of 5% CO2/95% (v/v) humidified air. The medium was changed every 2 days, and the cells were kept in serum-free medium for 24 h before experiments.

PLD Assay

Phospholipase D catalyzes formation of PA via transphosphatidylation reaction. Primary short-chain alcohols are able to competitively substitute for water in this reaction, resulting in the generation of a phosphatidylalcohol [28]. The PLD activity was determined in [3H]myristic acid-labeled ELT-3 cells in the presence of butan-1-ol and by measuring the accumulation of [3H]phosphatidylbutanol (Pbut), which is the product of the specific transphosphatidylation reaction catalyzed by PLD. Incubations were carried out as described previously [19] with minor modifications. Briefly, confluent ELT-3 cells in 24-well plates were incubated overnight in 1 ml of MEM without FCS but containing 4 µCi [3H]myristic acid and 1 mg/ ml of BSA. The cells were then washed once with Hanks balanced salt solution containing 20 mM Hepes (pH 7.5) and 1 mg/ml of BSA, followed by two successive washings using the same buffer but without BSA. The cells were incubated in 1 ml of fresh buffer containing 0.3% (v/v) butan-1-ol for 10 min before exposure to the agents tested for the time and at the concentration indicated for each experiment. Reactions were stopped by aspiration of the incubation medium followed by the addition of 1 ml of cold (–20°C) methanol. The cells were detached by scraping on ice, and 800 µl of cold (4°C) chloroform:methanol:HCl (149:48:3, v/v/v) were added. After a vigorous mixing, the lipids were extracted for 30 min at 4°C. Then, 0.5 ml of H2O was added, and the monophase was split by the addition of 0.6 ml of 2 M KCl and 0.6 ml of chloroform. The chloroform extract was dried under vacuum, suspended in 50 µl of methanol:chloroform (95:5, v/v) and spotted on thin-layer chromatographic silica gel plates. Plates were developed in the organic phase of a mixture of ethyl acetate:2,2,4-trimethylpentane:acetic acid:water (13:2:3:10, v/v/v/v) [29] and analyzed with a computerized Berthold radiochromascanner. The production of [3H]Pbut was expressed as a percentage of the total lipid-associated radioactivity.

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% [v/v] glycerol, 10 mM Na4P2O7, 200 µM Na3VO4, 10 mM EDTA, 1% [v/v [Triton-X100, 10 µg/ml of aprotinin and 10 µg/ml leupeptin, and 0.5 mM PMSF). Cells were detached by scraping on ice and centrifuged at 10 000 x g for 10 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% (w/v) SDS-PAGE. The separated proteins were transferred to nitrocellulose sheets and probed with polyclonal antiactive ERK1/2 antibodies (1:5000, v/v). The immunoreactive bands were visualized by enhanced chemiluminescence system after incubation with HRP-conjugated anti-rabbit IgG. Quantification of the developed blots was performed with a densitometer (Molecular Dynamics, Sunnyvale, CA).

Polymerase Chain Reaction of PLD Isoform Expression

Total cellular RNA from ELT-3 cells was isolated with Insta-Pure reagent (Eurogentec) according to the manufacturer's protocol, and 5 µg of total RNA were reverse transcribed into cDNA using 200 U of MMLV reverse transcriptase, 0.2 mM dNTPs, and 10 µM of random hexamer primers. Target cDNA was amplified using 1/10 of the reverse-transcribed cDNA prepared, 0.2 mM dNTPs, 2 mM MgCl2, 2.5 U of Taq polymerase, and 100 pmol of primers in polymerase chain reaction (PCR) buffer. The following primers were used: to amplify PLD1a (expected size, 493 base pairs [bp]) and PLD1b (expected size, 379 bp), 5'-AGCCTCTATCGCCAACTTCA-3' (sense) and 5'-CTTGAGACTTTGGGAGCAGG–3' (antisense); to amplify PLD2a (expected size, 454 bp) and PLD2b (expected size, 421 bp), 5'-ACCTACAGGACCCTGTGTCG-3' (sense) and 5'-AGATGGTGGCATTGTTCTCC–3' (antisense). The mixture was amplified in a thermal cycler (iCycler; Bio-Rad) under the following conditions: 94°C for 15 sec, 55°C for 30 sec, and 72°C for 30 sec for 30 cycles. The resulting PCR products were analyzed by electrophoresis on a 2% (w/v) agarose gel and stained with ethidium bromide.

[3H]Thymidine Incorporation

Serum-starved ELT-3 cells (50% confluent) in a 24-well plate were incubated for 48 h with the various agents to be tested, and 2 µCi/ml of [3H]thymidine were added 24 h before the end. Reactions were terminated by aspiration of the incubation medium and addition of 0.5 ml of cold trichloroacetic acid (TCA; 10%, w/v). Radioactivity incorporated into TCA-precipitable material was recovered with 0.5 ml of 0.1 N NaOH and quantified by liquid scintillation counting.

Cell Counting

Serum-starved cells (50% confluent) were treated for 48 h with the agents to be tested and then detached by trypsinization and counted in a Malassez hemocytometer.

BrdU Incorporation

Serum-starved ELT-3 cells (50% confluent) grown on coverslips in a 24-well plate were incubated with the agents to be tested for 48 h. The BrdU (final concentration, 1 µM) was added to the culture medium 16 h before the end. Cells were then fixed in methanol at –20°C for 5 min and incubated for 30 min in 1.5 N HCl. After two washes in 0.1 M sodium borate (pH 8.5) and two washes in PBS, BrdU-positive cells were stained by sequential incubation with anti-BrdU antibody (1:10, v/v) for 1 h and anti-mouse IgG fluorescein isothiocyanate (FITC)-conjugated antibody (1: 160, v/v) for 1 h. A counter-staining with 0.05 µg/ml of Hoechst 33342 was performed for 5 min after the secondary-antibody incubation. Coverslips were mounted on slides and observed on an Axiophot II fluorescence microscope equipped with an Axiocam digital camera (Zeiss). At least 500 cells were counted in each experimental condition.

Data Analysis

The results are expressed as the mean ± SEM. Means were compared using the Student t-test. Differences at P < 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ET-1-Induced Proliferation of ELT-3 Leiomyoma Cells

The mitogenic effect of ET-1 was analyzed by different approaches. As illustrated in Figure 1A, cells incubated in the presence of ET-1 for 48 h exhibited a strong (i.e., fourfold) thymidine incorporation compared to serum-deprived cells (control). Recent data demonstrated that PDGF receptors are expressed in ELT-3 cells, where they activate ERK and cell proliferation [30]. Our data confirmed that PDGF stimulated thymidine incorporation, though to a lesser extent compared with ET-1 (Fig. 1A). By contrast, although EGF receptors are expressed in these cells (data not shown), EGF failed to stimulate cell proliferation. In these comparative experiments, all agonists were used at maximal concentrations determined in preliminary dose-response experiments (data not shown). These results obtained in ELT-3 cells contrasted with our recent data demonstrating that in normal myometrial cells, ET-1, which was less efficient than PDGF, only induced a twofold increase in thymidine incorporation [7]. The ET-1-induced ELT-3 cell proliferation was confirmed by experiments based on BrdU incorporation. Indeed, after 48-h treatment of the cells in the presence of ET-1, the percentage of cells that incorporated BrdU increased by threefold compared to control (Fig. 1B), which is consistent with the proliferative effect of ET-1 as observed using the thymidine-incorporation method. Moreover, the mitogenic effect of ET-1 was also characterized by an increase in cell number. Indeed, incubation of ELT-3 cells in the presence of ET-1 for 48 h resulted in a twofold increase in cell number (Fig. 1C).



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  		FIG. 1. Mitogenic effect of ET-1 in ELT-3 cells. A) Serum-starved cells (50% confluent) were treated in the absence or the presence of 50 nM ET-1, 25 ng/ml of PDGF, or 25 ng/ml of EGF for 48 h. [3H]Thymidine (2 µCi/ml) was present for the last 24 h. [3H]Thymidine incorporation was measured as described in Materials and Methods. Results are expressed as cpm/well. B) Quiescent cells were treated for 48 h with or without 50 nM ET-1. The BrdU-positive cells were stained as described in Materials and Methods and then counted. Data are expressed as the percentage of positive cells over the total. C) ELT-3 cells in control and ET-1-stimulated groups were counted. Data are the mean ± SEM of three to four experiments, each carried out in duplicate. *Significant difference (P < 0.05) from control values

Involvement of ERK in the Mitogenic Effect of ET-1 in ELT-3 Cells

Activation of ERK is required for the mitogenic effect of ET-1 in normal myometrial cells [10]. In ELT-3 cells, the proliferative effect of ET-1 was markedly reduced in the presence of 5 µM U0126, a selective inhibitor of MAP kinase ERK kinase (MEK) (Fig. 2A), demonstrating involvement of the ERK cascade in the mitogenic response of ET-1. Activation of ERK by ET-1 was determined with an antiactive ERK1/2 antibody that recognizes the diphosphorylated active form of the enzymes. Incubation of ELT-3 cells in the presence of 50 nM ET-1 led to a transient activation of ERK1/2 (Fig. 2B). This effect was maximal at 3 min and then declined rapidly to a very low level.



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  		FIG. 2. Involvement of ERK activation in ET-1-mediated DNA synthesis. A) Quiescent cells were treated in the absence (control) or presence of 50 nM ET-1 with or without 5 µM U0126 for 48 h. [3H]Thymidine (2 µCi/ ml) was present for the last 24 h. Results are expressed as cpm/well and are the mean ± SEM of three experiments, each carried out in duplicate. *Significant difference (P < 0.05) from values obtained with ET-1 alone. B) ELT-3 cells were incubated in the absence or presence of 50 nM ET-1 for the time indicated. Detergent-extracted proteins were analyzed by 10% SDS-PAGE and immunoblotted with antiactive ERK1/2 (top).The blot was stripped and reprobed with anti-ERK1/2 antibodies (bottom). C) Phosphorylated ERK2 and total ERK2 shown in B were quantified with a densitometer. The level of ERK2 phosphorylation was normalized with respect to total ERK2 amount in each sample and expressed as arbitrary units (AU). Data are from one of three independent experiments

ET-1 Stimulated PLD Activity

Data from our laboratory demonstrated that ET-1 activates PLD in rat myometrial strips [18]. PLD hydrolyzes PC with the production of PA, which may serve as a potential lipid second messenger and constitutes a source for DAG, an activator for PKC. When ELT-3 cells labeled with [3H]myristic acid were incubated in the presence of butan-1-ol, ET-1 induced a rapid increase of [3H]Pbut, which reflected the specific PLD-catalyzed transphosphatidylation reaction (Fig. 3A). Production of Pbut reached a maximum at 10 min (Fig. 3A) and persisted for at least 30 min (data not shown). These results indicated that ELT-3 cells were endowed with the PLD activity that was stimulated by ET-1.



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  		FIG. 3. Stimulation of PLD by ET-1: up-regulation of PLD activation in ELT-3 cells compared to normal myometrial cells. A) [3H]Myristic acid-labeled ELT-3 cells were incubated with 0.3% butan-1-ol for 10 min. Incubations were further continued in the presence of 50 nM ET-1 for the time indicated. Production of [3H]Pbut production was expressed as a percentage of total labeled phospholipids. Data represent the mean ± SEM of three independent experiments, each performed in duplicate. B The cells labeled with [3H]myristic acid were exposed to 0.3% butan-1-ol for 10 min. Cells were then stimulated with or without 50 nM ET-1 for 10 min. The production of [3H]Pbut was expressed as the percentage of total labeled phospholipids. Data represent the mean ± SEM of six independent experiments, each performed in duplicate. The letters a, b, and c denote significantly different groups (P < 0.01)

The results in Figure 3B illustrate the effect of maximal concentration of ET-1 on PLD activity in ELT-3 and normal myometrial cells. The data clearly demonstrated that in ELT-3 cells, ET-1-mediated Pbut production was twofold higher than production in normal myometrial cells.

ELT-3 Cells Expressed Different PLD Isoforms

The expression pattern of PLD isoforms was examined in ELT-3 cells in comparison with normal myometrial cells. By reverse transcription-PCR analysis, we detected mRNA corresponding to PLD2a in ELT-3 and normal myometrial cells (Fig. 4). Interestingly, a differential expression of PLD1 isoforms was observed. Indeed, ELT-3 cells expressed PLD1a and PLD1b mRNA, whereas only PLD1b mRNA was detected in normal myometrial cells.



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  		FIG. 4. Differential expression of PLD isoforms in myometrial cells and ELT-3. The expression of PLD isoforms in normal myometrial and pathological cells was examined by reverse transcription-PCR as described in Materials and Methods. This image is representative of three independent experiments. The letters a and b correspond to PLD1a and PLD1b or PLD2a, respectively

ET-1-Mediated PLD and ERK Stimulation Triggered by ET-1 Involved PKC

Protein kinase C is a major regulator of PLD activity in numerous cell models and tissues, including rat myometrium [18]. The involvement of PKC in PLD activation was investigated in ELT-3 cells. The production of Pbut was strongly increased when the cells were incubated in the presence of PDBu, a direct activator of PKC (Fig. 5A), indicating that PKC regulated PLD activity in these cells. Furthermore, treatment of ELT-3 cells with the PKC inhibitor Ro-31-8220 strongly reduced (by >70%) the generation of Pbut induced by ET-1 and PDBu (Fig. 5A). These data demonstrated that stimulation of PLD by ET-1 in ELT-3 cells involved PKC.



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  		FIG. 5. Involvement of PKC in ET-1-mediated PLD activation and ERK activation in ELT-3 cells. A) [3H]Myristic acid-labeled ELT-3 cells were exposed to 0.3% butan-1-ol in the absence or presence of 5 µM Ro-31-8220. Cells were then stimulated with 1 µM PDBu or 50 nM ET-1 for 10 min. The production of [3H]Pbut was expressed as the percentage of total labeled phospholipids. B) ELT-3 cells were incubated with or without 5 µM Ro-31-8220 for 10 min. The incubations were continued in the absence or presence of 50 nM ET-1 for 10 min. Activation of ERK2 was analyzed as described in Figure 2. Values are the mean ± SEM of three separate experiments. *Significant difference (P < 0.05) from corresponding values obtained in the absence of Ro-31-8220

The involvement of PKC in ET-1-stimulated proliferation and ERK activation has been described in human and rat myometrial cells, respectively [7, 10, 12]. In ELT-3 cells, the activation of ERK by ET-1 was markedly reduced (by 85%) when the incubations were performed in the presence of Ro-31-8220, (Fig. 5B). This observation is consistent with the involvement of PKC in ET-1-mediated ERK stimulation. Our data suggested that PLD and ERK are involved in a common, PKC-dependent signaling pathway.

PLD Is Involved in ET-1-Induced ERK Activation

We examined whether PLD activation triggered by ET-1 contributed to ET-1-mediated ERK activation in ELT-3 cells. This question was addressed by reducing intracellular PA level by the use of butan-1-ol, which induces the production of Pbut by PLD at the expense of PA. Incubation (5 min) of cells with butan-1-ol before stimulation with ET-1 resulted in a 55% inhibition of PA production (Fig. 6A). Similarly, in the presence of butan-1-ol, the activation of ERK by ET-1 was reduced by approximately 55% (Fig. 6B). These inhibitory effects were not observed when cells were incubated with butan-3-ol, which is not a substrate of the transphosphatidylation reaction (Fig. 6). Additionally, the specificity of butan-1-ol on PA production via transphosphatidylation was reinforced by our results demonstrating that ET-1-induced PLC pathway activation remained unaltered in the presence of 0.3% butan-1-ol (data not shown). Thus, our results strongly suggest that ET-1-induced ERK activation involves PLD-dependent production of PA.



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  		FIG. 6. Involvement of PLD/PA in ET-1-mediated ERK activation in ELT-3 cells. A) [3H]Myristic acid-labeled ELT-3 cells were preincubated with or without 0.3% butan-1-ol or 0.3% butan-3-ol for 10 min. Incubations were further continued in the presence of 50 nM ET-1 for 10 min. Production of [3H]PA was expressed as the percentage of total labeled phospholipids over basal (mean ± SEM: 1% ± 0.05%, 1.07% ± 0.04%, and 1.05% ± 0.06% for control, butan-1-ol, and butan-3-ol, respectively). Data represent the mean ± SEM of four independent experiments, each performed in duplicate. *Significant difference (P < 0.05) from that obtained with ET-1 alone. B) Cells were incubated with or without 0.3% butan-1-ol or 0.3% butan-3-ol for 10 min before be treated with or without 50 nM ET-1 for 10 min. Activation of ERK2 was analyzed as described in Figure 2. Values are the mean ± SEM of three separate experiments. *Significant difference (P < 0.05) from that obtained with ET-1 alone

To confirm the involvement of PLD and PA in ERK activation and proliferation, we tested the direct effect of exogenous PLD extracted from S. chromofuscus, which hydrolyzes endogenous PC into PA. Incubation of ELT-3 cells in the presence of exogenous PLD led to transient ERK activation comparable to that observed with ET-1 (Fig. 7). However, the maximal effect of exogenous PLD was lower than that obtained with ET-1 (Fig. 7).



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  		FIG. 7. Exogenous PLD stimulated ERK activation. Cells were incubated in the presence of 10 U/ml of exogenous PLD for the indicated times. Activation of ERK2 activation was analyzed as described in Figure 2. Values are the mean ± SEM of three separate experiments

PLD Is Involved in ET-1-Induced Proliferation

To test whether PLD activation also contributed to ET-1-induced proliferation, ELT-3 cells were incubated in the presence of ET-1 with or without butan-1-ol. When the cells were treated with butan-1-ol, the effect of ET-1 on [3H]thymidine incorporation was reduced by 45% (Fig. 8). Butan-3-ol, which does not interfere with PLD activity, was without effect on the proliferative effect of ET-1. This result indicated that PLD activation contributed to ET-1-induced cell proliferation. Furthermore, exogenous PLD was also able to increase DNA synthesis in ELT-3 cells, though at a lesser extent than ET-1 (Fig. 8). Indeed, treatment of the cells in the presence of bacterial PLD increased [3H]thymidine incorporation by approximately 2.4-fold. This effect was not inhibited by butan-1-ol treatment, because this enzyme does not catalyze the transphosphatidylation reaction. Taken together, these data indicate that PA generated by PLD participates in the ERK activation and DNA synthesis induced by ET-1.



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  		FIG. 8. Involvement of PLD in ET-1-mediated cell proliferation. Cells were treated in the absence or presence of 50 nM ET-1 or 1 U/ml of exogenous PLD for 48 h with or without 0.3% butan-1-ol or butan-3-ol. [3H]Thymidine (2 µCi/ml) was present for the last 24 h. [3H]Thymidine incorporation was measured as described in Materials and Methods. Results are expressed as the fold-increase compared to control values without butanol. Values are the mean ± SEM of four separate experiments. +Significant difference (P < 0.05) from that obtained with ET-1 alone, *significant difference (P < 0.05) from that obtained in the control


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we show that ET-1 exerts a potent, ERK-dependent, mitogenic effect in ELT-3 leiomyoma cells in which PLD, via the production of PA, contributes to ERK activation. This effect is more pronounced than in normal rat myometrial cells [10]. The mitogenic effect of ET-1 was characterized by an increase in DNA synthesis and cell number. Interestingly, using the maximal dose of ET-1 and PDGF, we observed that in ELT-3 cells, ET-1 was more effective than PDGF at increasing cell proliferation, whereas in normal myometrial cells, proliferative action of ET-1 was twofold lower [7, 10]. These observations revealed an up-regulation of the ET-1 mitogenic effect in ELT-3 cells, which may indicate that this peptide plays an important role in the pathology of myometrial leiomyoma. A possible involvement of ET-1 has also been reported in the pathogenesis of human leiomyoma [31]. In these cells, ET-1 potentiated proliferation induced by different growth factors (EGF, insulin-like growth factor [IGF]-1, IGF-II, basic fibroblast growth factor). In parallel with up-regulation of the ET-1 mitogenic effect in ELT-3 cells, we observed that ET-1-induced PLD activation was twofold greater. We also found that in addition to PLD1a and PLD2 mRNA, which are expressed in both ELT-3 and normal myometrial cells, ELT-3 cells express mRNA coding for PLD1b. This may represent one potential explanation for the up-regulation of PLD-pathway responsiveness.

Phospholipase D has been implicated in diverse cellular functions, including proliferation (for review, see [23]), and increased PLD activity has been described in some cancer tumors [27]. We thus investigated a potential role of the PLD pathway in ET-1-mediated cell proliferation and ERK activation in ELT-3 cells. This question was addressed using the "alcohol trap" assay, which is widely used to block the production of PA catalyzed by PLD [28]. When ELT-3 cells were incubated in the presence of butan-1-ol, ET-1-mediated PA production was reduced by approximately 50%, indicating that PLD is certainly not the unique source of PA in ELT-3 cells. In these experimental conditions, ERK activation as well as cell proliferation caused by ET-1 was also decreased by approximately half, suggesting the existence of a close relationship between PA level, ERK activation, and proliferation. This hypothesis was reinforced by the results demonstrating that bacterial PLD, which produced PA, was able to activate ERK with kinetics similar to those of ET-1. Furthermore, the effect of exogenous PLD was accompanied by an increased [3H]thymidine incorporation. These data pointed out a physiological role of PLD in leiomyoma cells.

These results are in line with reports demonstrating the role of PLD and PA production in ERK activation [32, 33]. Ghosh et al. [32] reported that PA can interact directly with the serine-threonine kinase Raf-1, a major component of the ERK signaling cascade. Moreover, Rizzo et al. [34] showed that stimulation of the ERK pathway by insulin was dependent on PLD activation, and this effect was mediated through the induction of Raf-1 translocation to the plasma membrane by PA. An alternative mechanism has been described by Wang et al. [35], in which PLD activation induced by LPA leads to transactivation of the PDGF receptor and subsequent ERK phosphorylation. This hypothesis was attractive, because ELT-3 cells are able to synthesize and secrete PDGF-BB in particular conditions [30]. However, this possibility was eliminated, because we determined that inhibition of the PDGF-receptor tyrosine kinase by AG1296 failed to prevent ET-1-induced ERK activation (data not shown). Transactivation of the EGF receptor, which has been widely described [36], was not investigated, because EGF by itself did not stimulate DNA synthesis in ELT-3 cells. Although EGF- and PDGF-receptor transactivation is not involved in the ET-1 response, we cannot exclude the possibility that other receptor tyrosine kinases expressed in ELT-3 cells, such as transforming growth factor ß, fibroblast growth factor, vascular endothelial growth factor, and IGF, could participate [37].

Other mechanisms by which PA activates the ERK pathway have been proposed. The PA could directly activate PKC{zeta} [38]. We have recently demonstrated in rat myometrial cells that PKC{zeta} contributes to ERK activation in response to ET-1 [7]. The involvement of this isoform would merit testing in ELT-3 cells. Additionally, PA may interact with and activate PLC{gamma} [39], which is involved in ERK activation by various growth factors. We demonstrated in rat myometrial cells that PDGF-induced ERK activation via a PLC{gamma}- and PKC-dependent process [7, 40]. In ELT-3 cells, such a possibility was excluded, because PA failed to induce any significant InsP3 production (data not shown).

It is proposed that PA produced by PLD may influence membrane topology to promote formation of vesicles [41] and may contribute to the endocytosis mechanisms of GPCR, such as mu-opioid and angiotensin II receptors [42, 43]. Several GPCR as well as growth factor receptors [44] require endocytosis to trigger ERK activation. In this context, PA produced by PLD modulates EGF-receptor internalization and signaling [33]. Because ET receptors undergo endocytosis on activation [45], it is conceivable that PLD participates in this phenomenon, which may be required for mitogen-activated protein kinase activation.

Recently, the idea that PA may act via a transmembrane receptor has emerged. Indeed, data have demonstrated that exogenous PA is an agonist for orphan-receptor GPR63/ PSP24ß [46]. Furthermore, PA has been described to signal via a pertussis toxin-sensitive G protein to stimulate the migration of metastatic human breast cancer cells [46] and to activate ERK in HEK 293 cells [47].

The PA is also an activator of mammalian target of rapamycin (mTOR) [48]; mTOR is a serine-threonine kinase that plays a central role in the regulation of cell proliferation, growth, and differentiation via ribosomal S6 kinase activation. However, ELT-3 cells are characterized by a deficiency in the TSC2 gene product tuberin, a repressor of mTOR activation; thus, mTOR is constitutively activated in these cells [49, 50]. These observations indicate that PA does not produce its mitogenic effect through mTOR activation in leiomyoma cells; however, we cannot exclude the possibility that constitutively activated mTOR could participate to ELT-3 cell proliferation.

Additionally, PA can be deacylated into LPA by PLA2 or PLA1 [21, 51]. The LPA is a mitogenic lipidic mediator found in serum that acts via its cognate receptors coupled to G proteins [52]. It is interesting to note that the receptor expression and mitogenic effect of LPA have been reported in human myometrial cells [53]. It is thus conceivable that ET-1 may induce the production of LPA from PA, which could contribute to its proliferative response.

In conclusion, our findings demonstrate, to our knowledge for the first time, that PA produced by ET-1 through PLD stimulation is an important messenger in ET-1-stimulated ERK activation and cell proliferation in leiomyoma cells. The mechanisms by which PA exerts its mitogenic effect on ELT-3 cells deserve further investigation. Emerging evidence reveals that the ET-1 axis and PLD pathway play an important role in various cancer tumors [16, 27, 52]. Our data indicate that these two elements need to be considered in the context of leiomyoma growth and provide new perspectives for therapeutic strategies to treat this tumor.


   ACKNOWLEDGMENTS
 
We thank Dr C. Walker (University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX) for providing us the ELT-3 cell line. We are grateful to G. Delarbre for her helpful technical assistance.


   FOOTNOTES
 
1 Supported by grants from the Centre National de la Recherche Scientifique and Université Paris Sud. Back

2 Correspondence: Zahra Tanfin, CNRS UMR 8619, Bat 430 Université Paris Sud, 91 405 Orsay Cedex, France. FAX: 33 16 985 3715; zahra.tanfin{at}erc.u-psud.fr Back

Received: 30 June 2004.

First decision: 21 July 2004.

Accepted: 23 August 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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