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Endocrinology Vol. 147, No. 12 5873-5882
Copyright © 2006 by The Endocrine Society

Endothelin-1 Inhibits Apoptosis through a Sphingosine Kinase 1-Dependent Mechanism in Uterine Leiomyoma ELT3 Cells

Marie-Noëlle Raymond, Christine Bole-Feysot, Yoshiko Banno, Zahra Tanfin and Philippe Robin

Signalisation et Régulations Cellulaires (M.N.-R., C.B.-F., Z.T., P.R.), Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8619, Université Paris Sud, 91 405 Orsay Cedex, France; and Department of Cell Signaling (Y.B.), Gifu University, Graduate School of Medicine, Gifu 504-0838, Japan

Address all correspondence and requests for reprints to: Zahra Tanfin, Signalisation et Régulations Cellulaires, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8619, Bâtiment 430, Université Paris Sud, 91 S/R/C 405 Orsay Cedex, France. E-mail: zahra.tanfin{at}erc.u-psud.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Uterine leiomyomas, or fibroids, are the most common tumors of the myometrium. The ELT3 cell line, derived from Eker rat leiomyoma, has been successfully used as a model for the study of leiomyomas. We have demonstrated previously the potent mitogenic properties of the peptidic hormone endothelin (ET)-1 in this cell line. Here we investigated the antiapoptotic effect of ET-1 in ELT3 cells. We found that 1) serum starvation of ELT3 cells induced an apoptotic process characterized by cytochrome c release from mitochondria, caspase-3/7 activation, nuclei condensation and DNA fragmentation; 2) ET-1 prevented the apoptotic process; and 3) this effect of ET-1 was fully reproduced by ETB agonists. In contrast, no antiapoptotic effect of ET-1 was observed in normal myometrial cells. A pharmacological approach showed that the effect of ET-1 on caspase-3/7 activation in ELT3 cells was not dependent on phosphatidylinositol 3-kinase, ERK1/2, or phospholipase D activities. However, inhibitors of sphingosine kinase-1 (SphK1), dimethylsphingosine and threo-dihydrosphingosine, reduced the effect of ET-1 by about 50%. Identical results were obtained when SphK1 expression was down-regulated in ELT3 cells transfected with SphK1 small interfering RNA. Furthermore, serum starvation induced a decrease in SphK1 activity that was prevented by ET-1 without affecting the level of SphK1 protein expression. Finally, sphingosine 1-phosphate, the product of SphK activity, was as efficient as ET-1 in inhibiting serum starvation-induced caspase-3/7 activation. Together, these results demonstrate that ET-1 possesses a potent antiapoptotic effect in ELT3 cells that involves sphingolipid metabolism through the activation of SphK1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UTERINE LEIOMYOMAS, or fibroids, are the most common benign tumors of the uterine smooth muscle. Common symptoms associated with these tumors are dysmenorrhea, menorrhagia, infertility, and fetal morbidity (1), and they are the main indication for hysterectomy. It is now well established that leiomyoma growth is controlled by ovarian steroids and growth factors (2). Increased growth of leiomyomas results from an imbalance between the proliferation/apoptosis rates. Indeed, a main feature of leiomyoma cells is that they fail to undergo apoptosis in vivo (3). Apoptosis is a major programmed cell-death process characterized by a series of morphological and biochemical alterations including the fragmentation of nuclear chromatin, a decrease in cell volume, and alterations of the plasma membrane. These morphological and biochemical alterations lead to the recognition and phagocytosis of the apoptotic cells. Among the different events controlling apoptosis, activation of caspases represents a major control point (for review see Ref. 4).

To develop more specific treatments against leiomyomas, it is necessary to determine how hormones control fibroid development at a molecular level. The Eker rat strain is a well-characterized animal model for spontaneous uterine leiomyomas that are histologically similar to human leiomyomas. Eker rats have been used in many studies of hormonal control of fibroid development and apoptosis. An Eker leiomyoma tumor-derived cell line (ELT-3) has been characterized and used successfully to investigate the effects of hormonal modulations in association with the pathogenesis (5). For instance, Burroughs et al. (6) have shown that ELT3 cells could enter an apoptotic process after serum starvation but that estrogens were not able to rescue them from death, indicating that estrogens are not responsible for the resistance of leiomyomas to apoptosis observed in vivo. Therefore, it is important to determine the involvement of other hormones secreted at the level of uterine tissues that may participate in this phenomenon.

Among the hormones that regulate uterine functions and may have a role in the pathogenesis of leiomyomas, a good candidate is the peptidic hormone endothelin (ET)-1. This hormone exerts a mitogenic effect in human and rat myometrial cells (7, 8), but also exerts an antiapoptotic effect in different cell types (9, 10). Moreover, although ET-1 is produced primarily by endothelial cells, it is also synthesized in the uterus by endometrial and myometrial cells (9, 11). ETs comprise a family of structurally homologous 21-amino acid peptidic hormones that include ET-1, -2, and -3. ETs exert their effects by binding to the cell-surface ET receptors, ETA and ETB, which belong to the G protein-coupled receptor (GPCR) family. These two receptor subtypes are expressed in many tissues, including human and porcine myometrium (12, 13). Cumulative data indicate that the up-regulation of the ET axis is associated with tumor development and is observed in diverse cancer cell lines. For instance, it has been reported that ET-1 is a progression factor in many cancers and tumors, including prostate, ovarian, breast, renal, lung, colon and cervical cancer, melanoma, Kaposi’s sarcoma, central nervous system tumors, and bone malignancies (for reviews see Refs. 9 and 10). In many tumors, ET-1 acts as an autocrine/paracrine hormone that locally promotes the growth of cells. ET-1 has also been shown to inhibit apoptosis in various cell types through different pathways, including the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt/bcl-2 pathway (14, 15, 16), the ERK1/2 pathway (17), and the nuclear factor-{kappa}B (18) or calcineurin/nuclear factor of activated T cells (15) pathway.

In addition to these well-characterized molecular factors that affect apoptosis, the recently cloned sphingosine kinases (SphKs) have also been shown to modulate the apoptotic process (19, 20). SphK1 has a prosurvival effect, whereas SphK2 inhibits cell growth and enhances apoptosis (21). SphK1, is activated by diverse external stimuli including ET-1 (22) and catalyzes the synthesis of sphingosine 1-phosphate (S1P) by phosphorylating sphingosine (23). S1P is a polar sphingolipid that can act both as an extracellular mediator via a recently identified family of GPCRs and as an intracellular second messenger (for reviews see Refs. 24 and 25). S1P modulates several cellular functions and exerts an antiapoptotic effect in many cell types (23).

We have shown previously that ET-1-induced cell proliferation through a phospholipase D and ERK1/2 MAPK-dependent pathway in ELT3 cells (26). The mitogenic effect of ET-1 was more pronounced in the pathologic ELT3 cells than in the normal myometrial cells (26, 27). One possible explanation for this stronger effect of ET-1 in ELT3 cells is that, in addition to its mitogenic effect, ET-1 may exert an antiapoptotic effect only in ELT3 cells. In the present work, we tested this hypothesis and investigated the signaling pathway involved in the potential antiapoptotic effect of ET-1. A comparative analysis was conducted in parallel on normal myometrial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
[{gamma}-32P]ATP (2000 Ci/mmol) and ECL reagent were purchased from PerkinElmer (Boston, MA). Wortmannin, bongkrekic acid, sphingosine, reduced nicotinamide adenine dinucleotide, Hoechst 33342, pyruvate, and ribonuclease A were obtained from Sigma (St. Louis, MO). Type II collagenase, Lipofectamine 2000 and all the media and reagents for cell culture were obtained from Invitrogen (Carlsbad, CA). Dimethyl sphingosine (DMS), threo-dihydrosphingosine (t-DHS), Z-VAD and S1P were obtained from Biomol (Plymouth Meeting, PA). Sarafotoxin S6c and LY294002 were obtained from Merck Calbiochem (La Jolla, CA). ET-1 and IRL1620 were from NeoMPS (Strasbourg, France). Small interfering RNA (siRNAs) were synthesized by MWG-Biotech (Ebersberg, Germany). U0126 and Apo-One homogeneous caspase-3/7 assay were from Promega (Madison, WI). Bicinchoninic acid protein assay kit was from Pierce (Rockford, IL). Anti-cytochrome c (cyt c) antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-cleaved caspase 3 antibody was from Cell Signaling (Beverly, MA). Anti-{alpha}-tubulin antibody was from Amersham (Arlington Heights, IL). Anti-SphK1 antibody has been previously described (28). Anti-SphK2 antibody (29) was a gift from Dr. T. Okada (Kobe University Graduate School of Medicine, Kobe, Japan).

Cell line
The ELT-3 Eker rat uterine leiomyoma-derived cell line was kindly provided by Dr. C. Walker (Anderson Cancer Center, University of Texas, Smithville, TX). ELT-3 cells were maintained in DF8 medium supplemented with 10% fetal calf serum, as described previously (26), at 37 C and 5% CO2-95% humidified air. MEM was used for serum starvation.

Myometrial cell preparation and culture
Prepubertal Long Evans female rats (Elevage Janvier, France), 25 d old, were housed for 7 d in an environmentally controlled room before use. Food and water were available ad libitum. Rats were treated with 30 µg of estradiol for the last 2 d and were killed at 32 d of age by 1 min of carbon dioxide inhalation. All treatments were performed in accordance with the principles and procedures outlined in the European guidelines for the care and use of experimental animals. Primary cultures of myometrial cells were prepared by collagenase digestion as described previously (7). The myometrial cells were cultured in MEM supplemented with 10% fetal calf serum at 37 C in an atmosphere of 5% CO2-95% humidified air. Cells were used 5–7 d after seeding, when they reached confluence. Cells were serum starved in MEM and the experiments were carried out as described for ELT3 cells.

Cell counting
ELT3 and primary myometrial cells were seeded in 12-well plates (4 x 104 cells per well). After reaching confluence (about 48 h after seeding), cells were serum starved in the presence or absence of 200 nM ET-1 for the times indicated in the figures. This supra-maximal concentration of ET-1 was used to obtain a maximal effect even if a partial degradation of the hormone occurs in the culture medium of cells at 37 C. This concentration was used for all other experiments. Adherent cells were then detached by trypsinization and counted in a Malassez hemocytometer. For counting cells with apoptotic nuclei, ELT3 cells were treated as described above. After 48 h of serum starvation, cells pelleted from the culture medium were added to trypsinized cells. Cells were resuspended for 30 min in PBS in the presence of 0.1% Triton X-100 and 0.05 µg/ml Hoechst 33342 and counted in a Malassez hemocytometer.

DNA ladders
ELT3 cells were seeded in 10-cm diameter Petri dishes (2 x 105 cells per dish). After reaching confluence (about 48 h after seeding), cells were serum starved for 48 h in the presence or absence of ET-1 or serum. Adherent cells and cells pelleted from the corresponding culture medium were lysed by 500 µl of 10 mM Tris (pH 8) in the presence of 10 mM EDTA and 0.5% Triton X-100 for 30 min at 4 C and centrifuged at 20,000 x g. Nucleic acids were isolated from the supernatant by phenol-chloroform (500 µl) extraction and ethanol precipitation. RNA from the resulting pellet was removed by digestion in the presence of ribonuclease A for 1 h at 37 C. The resulting DNA was analyzed by electrophoresis on a 1.2% agarose gel.

Microscopic observations
ELT3 cells were grown in 4-cm diameter Petri dishes (4 x 105 cells per dish) for phase contrast microscopic observations. When confluence was reached (about 48 h after seeding), cells were serum starved (or not) in the presence or absence of 200 nM ET-1 for 48 h. Cells were observed directly in the Petri dishes.

ELT3 cells were seeded on glass coverslips (1.5 x 104 cells per well in 24-well plates) for immunofluorescence microscopic observations. After reaching 50% confluence, cells were serum starved (or not) for 24 h. The cells were fixed in 4% paraformaldehyde in PBS and permeabilized in 0.2% Triton X-100 in the presence of 1% BSA. Primary anti-cyt c antibody and fluorescein isothiocyanate-conjugated secondary antibody directed against mouse IgG were diluted 1:50 and 1:160 respectively. Hoechst 33342 (0.05 µg/ml) was used for nuclei staining. Observations were performed using an epifluorescence Zeiss Axioplan 2 microscope equipped with a Zeiss Axiocam digital camera (Carl Zeiss, Jena, Germany).

Caspase-3/7 activity assay
Caspase-3/7 activity assays were performed in 96-well plates with the Promega Apo-ONE Homogeneous Caspase-3/7 Assay kit. Cells were seeded at a density of 7000 cells per well and grown until confluence (about 48 h after seeding). Serum starvation was then performed in MEM (200 µl per well) in the presence or absence of different drugs or agonists. For caspase-3/7 activity assays, 150 µl of the medium was carefully removed from the upper part of the well (to avoid aspiration of floating cells). Fifty microliters of lysis buffer containing the fluorogenic Z-DEVD-R110 substrate were added to each well. Fluorescent product formation was measured every 6 min over a 120 min period using a Wallac 1420 PerkinElmer plate reader set at 37 C (excitation at 499 nm and emission at 521 nm). The lactate dehydrogenase content in each lysate was determined at the end of the caspase assay to evaluate the quantity of cells remaining in the wells at the end of the incubation period. Caspase-3/7-specific activity was expressed as the ratio of caspase activity (slope of the kinetic in arbitrary units) to lactate dehydrogenase activity (slope of the kinetic in arbitrary units). Each experimental condition tested was performed in triplicate.

SphK1 assay
ELT3 cells were seeded in a 12-well plate (1.8 x 105 cells per well). After reaching confluence (about 24 h after seeding), cells were serum starved for 28 h in the presence or absence of ET-1 and then lysed in the sphingosine kinase assay buffer [200 mM Tris (pH 7.4), 20% glycerol, 1 mM mercaptoethanol, 1 mM EDTA, 1 mM sodium orthovanadate, 40 mM ß-glycerophosphate, 15 mM NaF, 0.5 mM 4-deoxypyridoxine containing 0.5% Triton X-100 and protease inhibitors]. Aliquots of each fraction (10 µg protein) were incubated for 30 min at 37 C with 50 µM sphingosine in the presence of 0.95 mM [{gamma}-32P]ATP. Triton X-100 concentration was adjusted to 0.25%, a condition in which only SphK1 activity is determined (20). The resulting [32P]S1P was extracted with 0.8 ml chloroform/methanol/HCl (100/200/1, vol/vol/vol). After a vigorous mixing, the monophase was split by the addition of 0.25 ml chloroform and 0.25 ml 2 M KCl. [32P]S1P was then resolved on Silica gel thin-layer chromatography plates using chloroform/acetone/methanol/acetic acid/water (50/20/15/10/5, vol/vol/vol/vol/vol) and quantified using a Molecular Dynamics Storm Phosphor Imager. SphK1-specific activity was expressed as pmol S1P/min·mg protein (bicinchoninic acid microbiuret assay was used to determine protein concentration). Linearity of the enzymatic reaction with time of incubation and protein concentration was verified.

Western blot analysis of SphKs and cleaved caspase-3
For SphKs blotting, cells cultured in 12-well plates were lysed by addition of 200 µl of sphingosine kinase assay buffer described above. Lysates were centrifuged at 10,000 x g for 10 min at 4 C and the supernatants (10 µg of proteins) were analyzed by 10% SDS-PAGE. The separated proteins were transferred to nitrocellulose sheets and probed with polyclonal anti-SphK1 antibody (1:2000) or anti-SphK2 antibody (1:5000) and monoclonal antitubulin antibody (1:5000) for standardization.

For caspase-3 blotting, ELT3 cells (about 5 x 105 cells) were seeded in 10-cm diameter Petri dishes. After reaching confluence, cells were serum starved for 48 h in the presence or absence of ET-1. Cells pelleted from the culture medium were added to adherent cells and lysed by 500 µl of 50 mM HEPES buffer (pH 7.4) containing 150 mM NaCl, 10 mM EDTA, protease inhibitors and 1% Triton X-100. After a 10 min centrifugation at 10,000 x g, the supernatants (25 µg of proteins) were analyzed by 15% SDS-PAGE, transferred to nitrocellulose sheets and probed with anti-cleaved caspase-3 antibody (1:1000).

The immunoreactive bands were visualized by an enhanced chemiluminescence system after incubation with horseradish peroxidase-conjugated antirabbit or antimouse IgG. Quantification of the developed blots was performed with a Molecular Dynamics Personal Densitometer IIsi.

RNA interference
For the down-regulation of SphK1, sequence-specific siRNA targeting rat SphK1 (sense 5'-CUGGCCUACCUUCCUGUAGdTdT-3'; antisense 5'-CUACAGGAAGGUAGGCCAGdTdT-3') was used. The following siRNA (sense 5'-UUCUCCGAACGUGUCACGUdTdT-3'; antisense 5'-ACGUGACACGUUCGGAGAAdTdT-3') was used as a control (30). ELT3 cells were seeded into 12-well plates (about 5 x 104 cells per well) for the determination of SphK1 expression and in 96-well plates (3500 cells per well) for caspase-3/7 activity determination. Twenty-four hours after seeding, cells were transfected for 6 h with the 21-nucleotide duplexes using Lipofectamine 2000 as recommended by the manufacturer (30 pmol per well in 96-well plates and 300 pmol per well in 12-well plates). SphK1 expression was analyzed 48 h after transfection. For caspase-3/7 activity assays, cells were serum starved 48 h after transfection in the presence or absence of 200 nM ET-1 for 28 h. Caspase-3/7 activity was then determined as described above.

Statistical analysis
Results are expressed as the mean ± SEM of at least three independent experiments. Statistical analysis was performed using one- or two-way ANOVAs followed by post hoc comparisons with the Fisher’s least significant difference test. Values with P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ET-1 rescues ELT3 cells from death induced by serum starvation
We have recently shown that the peptidic hormone ET-1 induced the proliferation of ELT3 leiomyoma cells (26). Here we investigated whether ET-1 may also protect ELT3 cells from death induced by serum starvation. Figure 1AGo shows that after 48 h of serum starvation the number of viable ELT3 cells began to decrease and at 72 h, more than 60% of the cells were dead. Interestingly, when the cells were cultured in the presence of 200 nM ET-1 in serum-free medium, the number of viable cells did not decrease. Figure 1BGo shows the effect of serum starvation and ET-1 treatment on ELT3 cell morphology. In normal culture conditions (DF8 medium containing 10% serum), the cells were elongated and adherent, whereas after serum starvation they became round with very thin extensions and tended to lose their adherence to the plate. When cultured in the presence of ET-1, ELT3 cells remained in the same morphological state as when they were grown in the presence of serum. These results suggest that ET-1 is able to prevent ELT3 cell death induced by serum starvation.


Figure 1
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  		FIG. 1. ET-1 prevents ELT3 cells from death induced by serum starvation. A, Cells were serum starved for 48 or 72 h in the absence or presence of 200 nM ET-1. Adherent cells were then detached by trypsin digestion and counted in a Malassez hemocytometer. Values, expressed as number of adherent cells per well, are the mean ± SEM of three separate experiments performed in duplicate. *, P < 0.05 vs. both time 0 h and serum starved + ET-1 72 h. B, Phase contrast images of cells serum starved or not for 48 h in the absence or presence of 200 nM ET-1 (bars, 100 µm).

 
ET-1 prevents ELT3 cells from apoptosis induced by serum starvation
We characterized the pathway leading to cell death induced by serum starvation by testing different cellular events that are typical of apoptotic death and occur sequentially before detaching of cells from the plate. Thus, these experiments were performed at times shorter than 72 h of starvation: 24 h for cytochrome c release studies and 48 h for nuclei observations or DNA ladder experiments. These different optimal times were chosen based on the results of preliminary kinetic experiments (data not shown). Our results demonstrate that serum starvation induced DNA condensation in about 20% of the cells as determined by Hoechst 33342 staining (Fig. 2AGo). Moreover, serum starvation also induced DNA fragmentation leading to the formation of a typical DNA ladder pattern after electrophoresis on agarose gel (Fig. 2BGo). These molecular alterations of DNA were associated with nuclei shape changes (Fig. 3Go, A and B).


Figure 2
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  		FIG. 2. ET-1 prevents ELT3 cells from apoptosis induced by serum starvation. A, Cells were serum starved or not for 48 h in the absence or presence of 200 nM ET-1. Adherent cells were detached by trypsinization, added to floating cells, stained with Hoechst dye and counted in a Malassez hemocytometer. Results are expressed as the percentage of cells presenting a typical condensed apoptotic nucleus. Values are the mean ± SEM of three separate experiments performed in duplicate. *, P < 0.05 vs. both serum and serum starved + ET-1. B, Cells were serum starved or not for 48 h in the absence or presence of 200 nM ET-1. Adherent cells and cells pelleted from the medium were lysed. The DNA was extracted, analyzed by electrophoresis on agarose gel and stained with ethidium bromide. C, Cells were serum starved for the indicated times and caspase-3/7 activity was determined as described in Materials and Methods. Values are the mean ± SEM of three separate experiments performed in triplicate. *, P < 0.05 vs. caspase activity at 8 h. D, Cells were serum starved or not for 48 h in the absence or presence of 200 nM ET-1. Adherent cells and cells pelleted from the medium were lysed and proteins were analyzed by Western blot using an anticleaved caspase 3 antibody. E, Cells were serum starved or not for 28 h in the absence or presence of 50 µM bongkrekic acid (bong), 200 nM ET-1 or 50 µM Z-Val-Ala-Asp(OCH3)-fluoromethylketone (Z-VAD). Values are the mean ± SEM of three to six separate experiments performed in triplicate. *, P < 0.05 vs. untreated serum starved cells.

 

Figure 3
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  		FIG. 3. Effect of serum starvation on nuclei morphology and cyt c release. Cells cultured on glass coverslips were serum starved (B and D) or not (A and C) for 24 h, fixed and treated as described in Materials and Methods. Hoescht staining (A and B), cyt c staining (C and D) (bar, 15 µm).

 
The cysteine proteases caspases 3 and 7 are typical effectors of the apoptotic process. When ELT3 cells were serum starved, an increase in caspase-3/7 activity was observed as soon as 20 h after serum starvation (Fig. 2CGo). Maximal caspase-3/7 activity was observed 28–32 h after starvation. For further caspase-3/7 activity measurements, a serum starvation of 28 h was chosen. Caspase-3 activation after serum starvation was confirmed by the presence of the proteolytic 17-kDa fragment of caspase-3 detected by Western blot using a specific anticleaved caspase-3 antibody (Fig. 2DGo).

Caspase-3/7 activation is often controlled by cyt c released from mitochondria. Thus, we tested the effect of bongkrekic acid on cyt c release because this drug blocks this release by inhibiting the permeability transition pore. In ELT3 cells, bongkrekic acid significantly reduced caspase-3/7 activation induced by serum starvation (Fig. 2EGo), indicating that cyt c release is involved in caspase-3/7 activation. The release of cyt c from mitochondria was confirmed by immunofluorescence observations. Figure 3CGo shows that in control cells cyt c is localized in a juxta-nuclear region. This region was also stained with the mitochondrial dye Mito Traker Red CMX Ros (Invitrogen Molecular Probes, Carlsbad, CA) confirming that this area corresponded to a mitochondria enriched region of the cells (data not shown). After serum starvation, staining of cyt c was no longer concentrated in the juxta nuclear region (Fig. 3DGo), indicating that cyt c had been released from mitochondria. However, although these data implicate a mitochondrial step in the apoptotic process of ELT3 cells, the possibility of the involvement of a minor mitochondria-independent mechanism cannot be ruled out. Taken together, these observations indicate that serum starvation induced the death of ELT3 cells by an apoptotic process involving a mitochondrial pathway and caspase-3/7 activation.

We next investigated the mechanism by which ET-1 affected the apoptotic events triggered by serum starvation. Incubation of cells with ET-1 reduced the proportion of cells with condensed nuclei to 3.5% compared with 21% obtained in serum-free medium (Fig. 2AGo). Treatment with ET-1 also prevented DNA fragmentation; this was evident because the DNA ladder pattern observed with serum-starved cells was strongly reduced (Fig. 2BGo). Finally, ET-1 treatment prevented the proteolytic processing of caspase-3 (Fig. 2DGo) and reduced caspase-3/7 activity (Fig. 2EGo). This inhibitory effect of ET-1 was comparable to that observed in the presence of serum (Fig. 2EGo). Nevertheless, in non-serum-starved cells as well as in ET-1-treated cells, a residual hydrolysis of the fluorogenic substrate was detected. Figure 2EGo shows that this basal value, about 450 aU, was not due to caspase-3/7 activity because Z-VAD, a powerful general caspase inhibitor, did not significantly reduce this value.

Dose response experiments showed that a concentration of 10 nM of ET-1 was sufficient to induce the maximal antiapoptotic response (Fig. 4AGo). Interestingly, sarafotoxin S6c and IRL1620, two selective ETB agonists, inhibited caspase-3/7 activation as efficiently as ET-1, which acts on both ETA and ETB receptors (Fig. 4BGo). This suggests that ETB-agonist-sensitive receptors are involved in the ET-1 effect and are sufficient to give a full antiapoptotic response. Taken together, these results indicate that ET-1, acting at least through ETB receptors, is able to inhibit serum starvation-induced apoptosis at a step located upstream from caspase-3/7 activation and DNA fragmentation.


Figure 4
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  		FIG. 4. Dose-dependent effect of ET-1 on caspase-3/7 activity. A, ELT3 cells were serum starved for 28 h in the absence or presence of indicated ET-1 concentrations. Values are the mean ± SEM of three separate experiments performed in triplicate. *, P < 0.05 vs. no ET-1. B, ELT3 cells were serum starved for 28 h in the absence (control) or presence of the indicated concentrations of ET-1, sarafotoxin S6c (S6c) or IRL1620. Values are the mean ± SEM of three separate experiments performed in triplicate. *, P < 0.05 vs. control.

 
SphK1 is involved in the antiapoptotic effect of ET-1 in ELT3 cells
Antiapoptotic effects of ET-1 are mediated by different pathways in various cell types (14, 17, 31). Thus, we investigated the effects of some inhibitors of signaling enzymes that can be activated in response to ET-1. As shown in Fig. 5AGo, U0126, an inhibitor of ERK1/2 MAPK pathway, did not decrease the effect of ET-1 on caspase-3/7 activation. Wortmannin, a PI 3-kinase inhibitor also failed to alter the ET-1 antiapoptotic effect (Fig. 5AGo). LY294002, another PI 3-kinase inhibitor, also had no effect. ET-1 inhibited caspase-3/7 activation by 40.3 ± 2.8% in the absence of LY294002 compared with 36.5 ± 2.5% in the presence of LY294002 (P > 0.05). We have shown previously that ET-1 activated phospholipase D (PLD) in ELT3 cells (26). Because PLD has been suggested to play an antiapoptotic role in some cell types (32), we tested its contribution to the effect of ET-1. The results show that butanol-1, which prevents phosphatidic acid synthesis catalyzed by PLD, was not able to reduce the effect of ET-1 (Fig. 5BGo). As expected, butanol-3, used as a negative control for butanol-1, also had no effect. These results suggest strongly that ERK1/2, PI 3-kinase and PLD are not involved in the pathways mediating the antiapoptotic effect of ET-1.


Figure 5
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  		FIG. 5. SphK1 is involved in the ET-1 inhibition of caspase-3/7 activity. A, ELT3 cells were serum starved for 28 h with or without 200 nM ET-1 in the absence (control) or presence of 5 µM U0126 or 100 nM Wortmannin (W). Values are the mean ± SEM of three to four separate experiments performed in triplicate. *, P < 0.05 vs. respective controls in the absence of ET-1. B, ELT3 cells were serum starved for 28 h with or without 200 nM ET-1 in the absence (control) or presence of 0.3% butanol-1 (but-1) or butanol-3 (but-3). Values are the mean ± SEM of three separate experiments performed in triplicate. *, P < 0.05 vs. respective controls in the absence of ET-1. C, ELT3 cells were serum starved for 28 h with or without 200 nM ET-1 in the absence (control) or presence of 5 µM DMS or 20 µM t-DHS. Values are the mean ± SEM of four separate experiments performed in triplicate. *, P < 0.05 vs. all other treatments. D, Cells were serum starved for 28 h in the absence (control) or presence of 200 nM ET-1, 0.2 mg/ml BSA or 10 µM S1P in the presence of 0.2 mg/ml BSA, used as a vehicle for S1P. Caspase-3/7 activity was determined as described in Materials and Methods. Values are the mean ± SEM of three separate experiments performed in triplicate. *, P < 0.05 vs. control.

 
We next tested the involvement of SphK1, the isoform of SphKs that has been shown to be an antiapoptotic enzyme (21, 33). Treatment of cells with DMS and t-DHS, two inhibitors that reduce SphK1 activity, decreased the antiapoptotic effect of ET-1 by more than 50% (Fig. 5CGo). Furthermore, addition of S1P, the product of SphK1 activity, in the culture medium of serum starved cells, reduced caspase-3/7 activity as efficiently as ET-1 (Fig. 5DGo). We analyzed the expression and activity of SphK1 in response to serum starvation and ET-1 treatment. As shown in Fig. 6Go, A and B, serum starvation as well as ET-1 treatment of serum-starved cells had no significant effect on SphK1 expression. However, serum starvation reduced SphK1 activity, whereas ET-1 prevented this decrease (Fig. 6CGo). To further substantiate the role of SphK1, we tested the effect of its down-regulation using siRNA. Figure 7Go, A and B, shows that transfection of ELT3 cells with SphK1-specific siRNA resulted in a 35% inhibition of SphK1 expression compared with cells treated with control siRNA. SphK1 siRNA had no effect on SphK2 expression (Fig. 7Go, A and C). Finally, transfection with SphK1 siRNA reduced the ability of ET-1 to inhibit caspase-3/7 activation by about 60% (Fig. 7DGo), consistent with the results obtained with SphK inhibitors (Fig. 5CGo). These data confirm that SphK1 contributes to the antiapoptotic effect of ET-1. Nevertheless, the participation of a SphK1-independent pathway cannot be excluded because SphK inhibitors and SphK1-siRNA failed to fully reduce the antiapoptotic effect of ET-1.


Figure 6
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  		FIG. 6. ET-1 regulates the activity but not the expression of SphK1 in ELT3 cells. Cells were serum starved or not for 28 h in the absence or presence of 200 nM ET-1 and lysed for Western blot analysis and SphK1 activity assay. A and B, SphK1 expression was determined by Western blot with the anti SphK1 antibody. 10 µg of proteins were loaded in each lane. Tubulin (tub) was detected on the same nitrocellulose sheet and used as an internal standard. A, Result of a typical experiment performed in duplicate. B, Intensities of the SphK1 bands expressed in arbitrary units. Values are the mean ± SEM of four separate experiments performed in duplicate. P > 0.05. C, SphK1 activity was measured as described in Materials and Methods with 10 µg of proteins from the same lysates as those used for Western blots. Values, expressed as percentages of the serum value, are the mean ± SEM of four separate experiments performed in quadruplicate. 100% corresponds to 202 ± 32 pmol/min·mg. *, P < 0.05 vs. both serum and serum starved + ET-1.

 

Figure 7
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  		FIG. 7. SphK1 siRNA reduces the ET-1 inhibitory effect on caspase-3/7 activation. A–C, ELT3 cells were transfected with either control siRNA or SphK1 siRNA as described in Materials and Methods. Expression of SphK1 and SphK2 was analyzed by Western blot 48 h after transfection. Tubulin (tub) was used as an internal standard and quantified on the same nitrocellulose sheet. A, Result of a typical experiment. B and C, Intensities of the bands of SphK1 (B) and SphK2 (C) are expressed in arbitrary units. Values are the mean ± SEM of three separate experiments performed in duplicate. *, P < 0.05 vs. control siRNA. D, ELT3 cells were transfected with control siRNA or SphK1 siRNA. 48 h after transfection cells were serum starved for 28 h in the absence or presence of 200 nM ET-1. Caspase-3/7 activity was then determined as described in Materials and Methods. Values are the mean ± SEM of three separate experiments performed in triplicate. *, P < 0.05 vs. all other treatments.

 
ET-1 does not prevent apoptosis induced by serum starvation in normal myometrial cells
Because ELT3 cells derived from pathologic tissues, we further investigated whether ET-1 also prevented cell-death in normal myometrial cells. Figure 8Go, A–C, shows that, as in ELT3 cells, serum starvation of normal myometrial cells induced a decrease in cell number preceded by a cyt c release in the cytoplasm of the cells and an increase in caspase-3/7 activity. However, ET-1 did not counteract cell number decrease and caspase-3/7 activation (Fig. 8Go, A and C). This absence of ET-1 effect on normal myometrial cell apoptosis could have been the result of a defect in the activation pathway of SphK1 in these cells. As shown in Fig. 8DGo, SphK1 activity was reduced by serum starvation in normal myometrial cells, as observed in ELT3 cells. However, this decrease was not prevented by ET-1 treatment (Fig. 8DGo).


Figure 8
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  		FIG. 8. ET-1 does not prevent normal myometrial cells from apoptosis induced by serum starvation. A, Normal rat myometrial cells in primary culture were serum starved for 48 or 72 h in the absence or presence of 200 nM ET-1. Adherent cells were then detached by trypsin digestion and counted in a Malassez hemocytometer. Values, expressed as number of adherent cells per well, are the mean ± SEM of three separate experiments performed in duplicate. *, P < 0.05 vs. time 0 h. B, Cells cultured on glass coverslips were serum starved or not for 28 h, fixed and treated as described in Materials and Methods for cyt c staining (bar, 10 µm). C, Cells were serum starved or not for 28 h in the absence or presence of 200 nM ET-1 and caspase-3/7 activity was determined as described in Materials and Methods. Values are the mean ± SEM of four separate experiments performed in triplicate. *, P < 0.05 vs. both serum starved and serum starved + ET-1. D, Cells were serum starved or not as described in (C) and SphK1 activity was measured as described in Materials and Methods. Values, expressed as percentages of the serum value, are the mean ± SEM of four separate experiments performed in quadruplicate. 100% corresponds to 752 ± 200 pmol/min·mg. *, P < 0.05 vs. serum.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Eker leiomyoma tumor-derived cell line ELT-3 has been successfully used to investigate the hormonal modulation of tumor growth (5). Recently, we reported that ET-1 controlled cell proliferation in ELT3 cells (26). The results of the present study provide evidence that ET-1 inhibits ELT3 cell death induced by serum starvation, in addition to its mitogenic effect.

We found that serum starvation of ELT3 cells induced their death by a classical apoptotic process characterized by DNA fragmentation and condensation of nuclei, a result consistent with results described previously by Burroughs et al. (6). In addition, our results showed that serum starvation induced cyt c release from mitochondria and activation of caspase-3/7, two key factors controlling the apoptotic process. The sensitivity of caspase-3/7 activation to bongkrekic acid, an inhibitor of cyt c release, indicates that caspase-3/7 activation is mainly under mitochondrial control. These results clearly demonstrated that ELT3 cells are endowed with functional apoptotic machinery.

If the apoptotic process of normal myometrial cells is regulated by estrogens in vivo, these hormones cannot prevent apoptosis of ELT3 cells (6). In this study, we demonstrated that ET-1 is the first effective antiapoptotic factor for ELT3 cells. The survival effect induced by ET-1 has also been shown to occur in other cell types including cardiomyocytes (31), endothelial (34), human pericardial smooth muscle (35), human colon cancer (36), and ovarian carcinoma (14) cells. Moreover, this antiapoptotic effect of ET-1 seems to be independent of the type of apoptotic stimuli used because apoptosis triggered by serum starvation (31, 34), paclitaxel (14, 35), FasL (36), NO (17), or H2O2 (16, 37) were all reversed by ET-1.

In many cancer cells, as well as in fibroblasts, cardiomyocytes and vascular smooth-muscle cells, ET-1-induced survival is mediated by ETA receptors (10, 14, 17, 31). Surprisingly, in ELT3 cells, the antiapoptotic effect of ET-1 appears to mainly involve ETB receptors because sarafotoxin S6c and IRL1620, two specific ETB receptor agonists, were as effective in inducing the antiapoptotic effect as ET-1, which acts on both ETA and ETB receptors. However, the ETB antagonists BQ 788 and IRL 1038 unexpectedly failed to reduce the antiapoptotic effect of ET-1, although they were not degraded during the incubation (data not shown). Some instances of resistance of ET-1 responses to antagonists have been described previously (38, 39, 40, 41, 42). This phenomenon has been proposed to result from the existence of subpopulations of ETA and ETB receptors with different pharmacological properties (38, 41). More likely, the resistance of the ET-1 effect to antagonists may result from the low reversibility of ET-1 binding compared with the higher reversibility of antagonist binding (40, 42). This difference in binding properties may explain the decreasing potency of the antagonists observed after long term incubations with ET-1 (39, 40).

An antiapoptotic effect of ETB receptors has only been reported in endothelial cells (34). Furthermore, ETB receptors have been shown to mainly mediate a proapoptotic effect of ET-1 in cell types such as vascular smooth muscle cells and A375 human melanoma cells (43, 44). The involvement of ETB receptors in the inhibitory effect of ET-1 against ELT3 cell apoptosis was particularly unexpected. Indeed, results from our group have thus far demonstrated that the regulation of signaling enzymes (including phospholipase C, PLD, ERK1/2 MAPKs) and physiological responses (contraction and proliferation) triggered by ET-1 in rat myometrium or myometrial cells were mainly dependent on ETA receptors (7, 45, 46). Interestingly, ET-1 exerted an antiapoptotic action only in leiomyoma cells. Indeed, under serum starvation conditions, normal myometrial cells died through an apoptotic pathway as ELT3 cells, but ET-1 did not rescue them from death. These results led us to speculate that the absence of a prosurvival effect of ET-1 in normal myometrial cells may be the result of low expression of ETB receptors and/or their uncoupling from an antiapoptotic signaling pathway. Further experiments will be needed to test these hypotheses.

The antiapoptotic pathways triggered by survival factors such as growth factors or cytokines have often been shown to be dependent on Bcl-2-antagonist of cell death phosphorylation, which controls antiapoptotic Bcl-2 family members. Bcl-2-antagonist of cell death phosphorylation is regulated by either the PI 3-kinase-Akt pathway or by a Ras-MAPK pathway (47). It has been reported that ET-1 inhibits the apoptotic process via several pathways involving MAPK ERK kinase 1-ERK, PI 3-kinase-Akt-S6K, mammalian target of rapamycin (mTOR)-S6K, or calcineurin-NFAT-Bcl-2 (14, 15, 17). In ELT3 cells, the PI 3-kinase inhibitors (wortmannin and LY294002) had no effect on the inhibitory effect of ET-1 on caspase-3/7 activation. Although ET-1 is able to stimulate the ERK1/2 pathway in ELT3 cells (26), the MAPK kinase inhibitor (U0126) was also unable to inhibit the ET-1 antiapoptotic effect. These results indicate that the PI 3-kinase/Akt pathway as well as the ERK1/2 pathway are not involved in the antiapoptotic effect of ET-1 in ELT3 cells. The active participation of the mTOR-S6K pathway on the effect of ET-1 seems unlikely because this pathway is constitutively activated in ELT-3 cells. Indeed, ELT3 cells are characterized by a loss of tuberin expression (5), a protein whose function is to repress mTOR activation (48). This results in a constitutive activation of p70(S6K) and phosphorylation of the S6 ribosomal protein (49). Finally, the possible involvement of PLD, which has recently emerged as a critical regulator of survival signaling (32), was tested. However, although we previously demonstrated that ET-1 induced a strong activation of PLD in ELT-3 cells (26), results of the present work indicate that this enzyme is not involved in the antiapoptotic effect of ET-1. These findings indicate that, in ELT3 cells, different signaling pathways are involved in ET-1 mediated proliferation and survival that occur dependently and independently from ERK1/2, respectively.

Recently, SphK1 has emerged as a new prosurvival enzyme (21). Indeed, the overexpression of SphK1 in NIH 3T3, HEK293, PC12, and Jurkat T cells protected those cells from apoptosis induced by serum starvation (23, 50, 51). SphK1 plays a critical role in regulating the balance between the proapoptotic sphingolipid metabolite ceramide and the prosurvival S1P (25). The two isoforms of SphKs are inhibited by DMS, but only SphK1 is inhibited by t-DHS (20). We showed that, in ELT3 cells, these two inhibitors reduced the antiapoptotic effect of ET-1 by about 50%, suggesting participation of SphK1. The involvement of SphK1 was confirmed by the use of SphK1 siRNA, which reduced the antiapoptotic effect of ET-1 by about 60%. The regulation of caspase-3 (and/or caspase-7) activity by SphK1 has also been reported in PC12 cells (50). Furthermore, S1P, the product of SphK activity, reproduced the antiapoptotic effect of ET-1 on ELT3 cells.

In ELT3 cells, we observed that serum starvation decreased SphK1 activity and that this decrease was prevented by ET-1 treatment. Regulation of SphK1 activity may occur through both translational and posttranslational mechanisms. A translational regulation of SphK1 was certainly not involved in ELT3 cells because we found that serum starvation in the presence or absence of ET-1 did not significantly modified the level of SphK1 protein expression. These results are consistent with earlier results showing that many activators of SphK1 regulate the enzyme posttranslationally by affecting its phosphorylation state, its cellular localization, or its interaction with other proteins (52). However, so far we have no data concerning an eventual effect of ET-1 on the phosphorylation state or the localization of SphK1 in ELT3 cells. It has been shown previously that ERK2 can phosphorylate and activate SphK1 (53), and we have observed that ET-1 activated ERK1/2 in ELT3 cells (26). Thus, it could have been possible that, in ELT3 cells, ET-1 activated SphK1 through its phosphorylation by ERK2. However, this possibility is not supported by our observation that the ERK1/2 MAPK pathway inhibitor, U0126, did not reduce the inhibitory effect of ET-1 on caspase-3/7 activation.

The mechanisms by which SphK1 and S1P regulate apoptosis are not yet fully understood. Cuvillier and Levade (54) demonstrated that S1P prevents caspase-3 activation by inhibiting the translocation of cyt c and Smac/DIABLO from mitochondria to the cytosol and Goetzl et al. (55) showed that S1P suppressed cellular levels of the apoptosis-promoting protein Bax. Interestingly, SphK1 was reported to mediate cyclooxygenase (COX)-2 induction and prostaglandin E2 production (56, 57). In ELT3 cells, the addition of exogenous prostaglandin E2 rescued cells from serum starvation-induced apoptosis as efficiently as ET-1 (data not shown) but no data are available concerning COX-2 expression or activity in these cells. Further work will be needed to investigate a possible ET-1-SphK1-COX-2-prostaglandin axis in the control of apoptosis in ELT3 cells.

Our findings, obtained in ELT3 and normal myometrial cells, support the notion that SphK1 is an antiapoptotic enzyme. Indeed, we observed that apoptosis resulting from serum starvation was correlated with low SphK1 activity, whereas survival was correlated with a high SphK1 activity. The main difference between the two cell types is the ability of ET-1 to mimic the serum effect on SphK1 activity in ELT3 cells but not in normal cells. These observations reveal a difference in the ET-1-SphK1 axis between the normal and the pathological cells. This difference may be either at the level of the receptor, as discussed above, or in the signaling pathway triggering the activation of SphK1. Nevertheless, the mechanisms of SphK1 activation in response to GPCR agonists are still under investigation (23, 52).

In conclusion, the results described here demonstrate that, in ELT3 leiomyoma cells, ET-1 is a potent antiapoptotic factor that acts, at least partly, through a SphK1-dependent mechanism. This antiapoptotic effect of ET-1 on ELT3 cells may result from the presence of functional ETB-agonist sensitive receptors in these cells. Because ET-1 is known to act as an autocrine/paracrine factor in the uterus, it can be hypothesized that ET-1 may participate in the resistance to apoptosis of leiomyomas observed in vivo. Moreover, the data highlight the role of SphK1 in the control of leiomyoma cell apoptosis and suggest that virtually all the factors capable of inhibiting SphK1 could be potential proapoptotic factors for leiomyomas. Insofar as the data obtained with ELT3 cells reflect common features of leiomyoma cells, then SphK1 would appear as a suitable target for the development of new therapeutic strategies for the treatment of leiomyomas.


   Acknowledgments
 
We thank Dr. C. Walter (University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX) for providing us with the ELT3 cell line and Dr. T. Okada (Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA) for the gift of the antibody against SphK2. We are grateful to G. Delarbre from our laboratory for helpful technical assistance. We thank Dr. D. Leiber from our laboratory and Dr. H. Le Stunff (Laboratoire de Physiopathologie de la Nutrition, Université Paris 7, Paris, France) for helpful discussions.


   Footnotes
 
This work was supported by grants from the Centre National de la Recherche Scientifique and the Université Paris Sud.

Disclosure statement: The authors have nothing to disclose.

First Published Online September 7, 2006

Abbreviations: COX, Cyclooxygenase; cyt c, cytochrome c; DMS, dimethyl sphingosine; ELT-3, Eker leiomyoma tumor-derived cell line; ET, endothelin; GPCR, G protein-coupled receptor; mTOR, mammalian target of rapamycin; PI 3-kinase, phosphatidylinositol 3-kinase; PLD, phospholipase D; S1P, sphingosine 1-phosphate; siRNA, small interfering RNA; SphK1, sphingosine kinase-1; t-DHS, threo-dihydrosphingosine.

Received March 8, 2006.

Accepted for publication August 30, 2006.


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 Results
 Discussion
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