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Endocrinology, doi:10.1210/en.2007-1756
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Endocrinology Vol. 149, No. 9 4669-4679
Copyright © 2008 by The Endocrine Society

Signaling Pathways Involved in Sphingosine Kinase Activation and Sphingosine-1-Phosphate Release in Rat Myometrium in Late Pregnancy: Role in the Induction of Cyclooxygenase 2

Martin Serrano-Sanchez, Zahra Tanfin and Denis Leiber

Signalisation et Régulations Cellulaires, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Unité Mixte de Recherche 8619, Centre National de la Recherche Scientifique, Université Paris-Sud, 91405 Orsay, France

Address all correspondence and requests for reprints to: Dr. Zahra Tanfin, Signalisation et Régulations Cellulaires, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Unité Mixte de Recherche 8619, Centre National de la Recherche Scientifique, Batiment 430, Université Paris-Sud, 91405 Orsay, France. E-mail: zahra.tanfin{at}u-psud.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the regulation of the sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) axis and its role during pregnancy in the rat myometrium. SphK1 and SphK2 were coexpressed in myometrium during gestation. The levels and activity of SphK1/2 were modest at midgestation (d 12), increased at d 19 and progressively declined to low at postpartum. Similar patterns were observed for the phosphorylation of ERK and protein kinase C (PKC). Inhibition of PKC and ERK reduced SphK1/2 activity. In late pregnancy, levels of cyclooxygenase 2 (COX2) increased in parallel to SphK levels. Using a pharmacological approach, we demonstrated that in primary cultures of myometrial cells from d-19 pregnant rats, induction of COX2 was mediated by 4β-phorbol 12,13-dibutyrate and IL-1β through sequential activation of PKC, ERK1/2, and SphK1. S1P produced by SphK1 was released in the medium. Addition of S1P, IL-1β or 4β-phorbol 12,13-dibutyrate enhanced COX2 levels via Gi protein. Interestingly, S1P was also released by myometrial tissues at late gestation. This event was dependent on PKC/ERK/SphK1. By contrast, in d-12 myometrial tissues, the release of S1P was markedly reduced in association with low levels of SphK1 and COX2. However, prolonged incubation of myometrium from midgestation led to the induction of COX2. This effect was blocked by SphK inhibitors, providing evidence of the close relationship between SphK activity and COX2 induction in rat myometrium. Overall, our findings provided insight into the physiological relevance of the SphK activation and S1P release in uterine smooth muscle during gestation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING GESTATION, myometrium (the smooth muscle tissue of the uterus) is the site of diverse physiological processes such as hypertrophy, hyperplasia, quiescence, contraction, and apoptosis. Myometrial contraction is the most widely studied. The myometrium contracts strongly at parturition for the expulsion of the fetus. Our laboratory and other groups have investigated the various signaling pathways involved in myometrial contractility induced by agents such as prostaglandins (PGs), carbachol, and peptide molecules oxytocin, bombesin and endothelin-1 (ET-1) (1, 2, 3, 4, 5). Smooth muscle contractility is also regulated by sphingosine-1-phosphate (S1P) (6). We recently reported that in nonpregnant rat myometrium, contraction is stimulated by exogenous S1P or by ET-1-induced endogenous S1P production, through sphingosine kinase 1 (SphK1) activation (7).

SphK promotes the phosphorylation of sphingosine to form S1P. Two isoforms of SphK (SphK1 and SphK2) have been characterized (8, 9). Many agonists activate and induce translocation of SphK1 to the plasma membrane, causing a rapid increase in S1P production. Much less is known about the function and the regulation of SphK2. S1P interacts with five specific S1P receptors belonging to the G protein- coupled receptors (GPCR) family (8, 9). S1P triggers several different signaling processes involved in smooth muscle contraction (6, 7) as well as numerous target cell processes including cell proliferation, cell growth, migration, adhesion, generation of certain molecules, morphogenesis, cell survival, and apoptosis (8, 9, 10). Additionally, SphK activation can exert intracellular effects, independently from the interaction between S1P and S1P membrane receptors (11). SphK1 is activated through phosphorylation by ERKs that are stimulated by protein kinase C (PKC). Recent studies demonstrated that SphK2 is also phosphorylated by ERK (10).

SphK activation can increase intracellular calcium levels (12), which play a prominent role in smooth muscle contractility. Furthermore, SphK activity is involved in the induction of cyclooxygenase 2 (COX2) or PGH2 synthase (13, 14). COX2 is a key enzyme in the biosynthesis of PGs. COX exists as two distinct isoforms: COX1 and COX2. COX1 is a housekeeping enzyme that is constitutively expressed in nearly all tissues. COX1 mediates physiological responses such as platelet aggregation (15). In contrast, COX2 is induced by several different agonists, including growth factors, GPCR agonists, and ILs (16, 17, 18). In the uterus, the levels of ILs, including IL-1β, increase at the end of gestation and are involved in normal and pathological parturition, by increasing COX2 production and PG biosynthesis (19, 20). Interaction of PGs with their cognate receptors also induces oxytocin secretion, further increasing the contractile response triggered by PGs (21). COX2, but not COX1, strongly increases the intracellular concentration of PGs in uterus (21, 22). COX2 levels are regulated by PKC, MAPKs, ERK, and p38 (22). Recently, Jeng et al. (23) demonstrated that SphK1 is up-regulated by progesterone, a steroid hormone playing an important role in uterus physiology during gestation.

We recently demonstrated the rapid effect of exogenous S1P or ET-1-induced SphK1 activation on rat myometrial contractility. This effect is associated with Rho kinase activation (7), which is involved in Ca2+ sensitization (6), representing one mechanism involved in normal parturition (24, 25). In this study, we investigated the potential link between SphK activity, subsequent SIP release, and COX2 levels in the rat myometrium during pregnancy. This is the first study to demonstrate that SphK activation and S1P release are required for the induction of COX2 in rat myometrium during late pregnancy. We analyzed the signaling pathway involved in the activation of SphK associated with up-regulation of COX2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
[{gamma}-32P]ATP (2000 Ci/mmol) and [3H]sphingosine (21.2 Ci/mmol) were purchased from PerkinElmer (Les Ulis, France). Goat anti-actin antibodies, (Santa Cruz Biotechnology, Santa Cruz, CA), S1P, dimethysphingosine (DMS) and DL-threo-dihydrosphingosine (t-DHS) were from Biomol (Tebu, Le Perray-en-Yvelines, France). Pertussis toxin (PTX), sphingosine, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), 4β-phorbol 12,13-dibutyrate (PDBu), and IL-1β were from Sigma Chemical Co. (St. Louis, MO). Ro-318220, SKI (SphK1 inhibitor), and antigoat IgG conjugated to horseradish peroxidase were from Calbiochem (Meudon, France). Type II collagenase, all the media, and reagents for cell culture were obtained from Invitrogen (Carlsbad, CA). Rabbit anti-SphK2 antibodies were a generous gift from Dr. Taro Okada (Dr. Shunichi Nakamura laboratory, Kobe University Graduate School of Medicine, Kobe, Japan). Rabbit anti-SphK1 antibodies were a generous gift from Dr. Yoshiko Banno (Department of Cell Signaling, Gifu University Graduate School of Medicine, Gifu, Japan). Rabbit anti-COX2 antibodies were from Cayman Chemical Co. (Ann Arbor, MI). Anti-phospho-ERK1/2 antibodies and U0126 were from Promega (Madison, WI). Secondary horseradish peroxidase-conjugated antirabbit antibodies were from Cell Signaling (Ozyme, Saint-Quentin en Yvelines, France). Silica G60 (10 x 20) thin-layer chromatography (TLC) plates were from Merck (VWR, Fontenay sous Bois, France). All other chemicals were of the highest grade available.

Animals
Pregnant (gestation d 12, 19, and 21), postpartum (PP, just after delivery), and late postpartum (LPP, 24 h after delivery). Wistar rats (Janvier, France) were housed in an environmentally controlled room before use. Chow and water were available ad libitum. Rats were killed by carbon dioxide inhalation. All treatments were performed in accordance with the European guidelines for the care and use of experimental animals. Uteri were removed immediately from the animals. The fetus, placenta, fetal membranes, and areas of contact between trophoblasts and the endometrium were excluded, and the myometrium was prepared free of endometrium as previously described (26).

Myometrial cell preparation and culture
Primary cultures of myometrial cells were prepared by collagenase digestion as previously described for nonpregnant rat myometrium (27). The cells were cultured in MEM supplemented with penicillin, fungizone, glutamine, and 10% fetal calf serum at 37 C in 5% CO2 and 95% humidified air. The medium was changed every 2 d until cells were confluent. Cells were then incubated for 16 h in serum-free medium before experiments.

Cell treatment and membrane preparation
Cells were treated with 1 µM PDBu or 10 ng/ml IL-1β for the indicated times. Cells were washed twice with ice-cold PBS and then scraped in 500 µl lysis buffer [20 mM Tris (pH 7.5), 10 nM EDTA, 2 mM EGTA, 250 mM sucrose, 1 mM PMSF, 10 nM okadaic acid, 1 mM dithiothreitol, 5 mM NaF, 1 mM Na3VO4, 0.5 mM 4-deoxypyridoxine, and 10 µg/ml leupeptin and aprotinin). Cell suspensions were freeze/thawed several times, and cell homogenates were clarified by centrifugation at 700 x g for 10 min. Homogenates were separated into membrane and cytosolic fractions by centrifugation at 100, 000 x g for 60 min at 4 C. The membrane fractions were resuspended in SphK buffer. Protein concentrations in homogenates and membranes were determined by the Bradford assay.

Preparation of myometrial homogenates
Myometrial strips from rats at d 12, 19, and 21 of pregnancy and at postpartum were pooled and divided into equal parts of 50 mg. Tissues were allowed to equilibrate 30 min in Krebs-Ringer bicarbonate buffer containing 0.8 mM CaCl2 and 5 mM glucose at 37 C under constant agitation (gas phase 95% O2 and 5% CO2). Myometrium samples were then incubated in the presence of the different molecules to be tested for the times indicated in figure legends. Tissues were exposed to liquid N2 and homogenized with an Ultra Turrax homogenizer. To determine SphK activity, tissues were homogenized in the SphK buffer containing 20 mM Tris (pH 7.4), 20% glycerol, 1 mM mercaptoethanol, 1 mM EDTA, phosphatase inhibitors (15 mM NaF, 1 mM sodium orthovanadate, and 40 mM glycerol phosphate), protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml trypsin inhibitor, and 1 mM PMSF), and 0.5 mM pyridoxal phosphate analog 4-deoxypyridoxine for inhibition of the pyridoxal-dependent S1P lyase. Homogenates were clarified by centrifugation at 700 x g for 5 min at 4 C. Protein concentrations were determined by the Bradford assay.

Measurement of SphK1 and SphK2 activity
SphK1 or SphK2 activity in rat myometrial homogenates (25 µg proteins) and cell membranes (5 µg proteins) was analyzed, as described (28). Briefly, 200 µl SphK buffer containing 50 µM sphingosine, [{gamma}-32P]ATP (5 µCi, 1 mM), and 100 mM MgCl2 was added to protein samples in the presence of either 0.5% Triton X-100, which abolishes SphK2 activity, or 0.5 or 1 M KCl, which abolishes SphK1 activity (28). After 30 min incubation at 37 C, the reactions were stopped by addition of 800 µl chloroform, methanol, concentrated HCl (100:200:1, vol/vol); 250 µl chloroform and 250 µl 2 M KCl were added for phase separation. The samples were vortexed and centrifuged 5 min at 700 x g. Aliquots (100 µl) of the organic phase containing labeled lipids were separated by TLC on silica gels with chloroform, acetone, methanol, acetic acid, and water (50:20:15:10:5, vol/vol). The radioactive S1P was visualized by exposing the plate overnight on a phosphor screen. SphK activity was expressed in picomoles S1P per minute per milligram protein. We determined the optimal conditions for analysis of SphK1 and SphK2 activity levels in rat myometrial fractions. In the myometrium from pregnant rats, sphingosine phosphorylation by SphK1/K2, measured at 37 C, was linear for at least 60 min for up to 50 µg protein (data not shown).

Western blot analysis
For the immunodetection of COX2, ERK, PKC, SphK1/2, and actin, myometrial cells and tissues were homogenized in lysis buffer containing 20 mM Tris (pH 7.5), 10 mM EDTA, 2 mM okadaic acid, 1 mM dithiothreitol, 5 mM NaF, 1 mM Na3VO4, 0.5 mM 4-deoxypyridoxine, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, and 1% Triton X-100. Detergent-extracted proteins (25 µg) were heated for 10 min at 95 C in Laemmli sample buffer and analyzed by 10% (wt/vol) SDS-PAGE. To detect SphK1 and SphK2 proteins, we used two specific antibodies that did not cross-react. The separated proteins were transferred to nitrocellulose sheets and probed with rabbit polyclonal anti-SphK1 (dilution 1:1000, vol/vol), anti-SphK2 (1:5000, vol/vol), anti-COX2 (1:5000, vol/vol), anti-phospho-ERK (1:5000, vol/vol), anti-total ERK (1:5000, vol/vol), and anti-phospho-PKC (1:1000 vol/vol) antibodies or with goat anti-actin (dilution 1:1000, vol/vol) antibodies. Immunoreactive bands were visualized using the enhanced chemiluminescence system after incubation with horseradish peroxidase-conjugated antirabbit IgG or horseradish peroxidase-antigoat IgG for actin. Band intensity was quantified with a densitometer (Molecular Dynamics, Sunnyvale, CA).

Production and release of S1P in the incubation medium
Myometrial serum-starved cells or tissues (50 mg) were washed and incubated in MEM in the presence or the absence of various inhibitors for indicated times. Tissues and cells were then incubated in the presence of 50 nM [3H]sphingosine for 20 min. For the cells, PDBu or IL-1β was added in the medium for the last 15 min. S1P present in the incubation medium was extracted as previously described (29). Briefly, chloroform, methanol, KCl, and aqueous disodium EDTA were used to separate the substrate [3H]sphingosine from the product [3H]S1P. The medium was transferred into methanol to stop the reaction, and chloroform was added. The mixture was separated into two phases by the addition of 2 M KCl and 25 mM trisodium EDTA (pH 9) followed by vortexing and a centrifugation for 5 min. The upper aqueous phase containing [3H]S1P was quantified by scintillation counting. Data are expressed in counts per minute per well or counts per minute per 50 mg tissue. We confirmed that the upper aqueous phase contained only S1P and that the lower organic phase contained only sphingosine using the following procedure: the volume of the organic phase was evaporated, dissolved in chloroform and methanol (2:1, vol/vol), and spotted on TLC silica gel plates. The aqueous phase was evaporated, dissolved in chloroform and methanol (2:1, vol/vol), sonicated, and spun at 700 x g for 10 min to remove salts. The solution was transferred into a new tube, concentrated by evaporation, and spun for maximal removal of salts. The solution was transferred into a new tube and evaporated. This evaporate was dissolved in chloroform and methanol (2:1, vol/vol) and analyzed by TLC. TLC plates were developed in a mixture of chloroform, methanol, acetone, acetic acid, and water (40:20:15:10:5, vol/vol) and analyzed with a computerized Berthold radioscanner (Trace Master, LB285; Berthold, Bad Wilbad, Germany). Radioactive spots were located by comparing their migration patterns to those of with authentic standards (S1P and sphingosine) visualized with ninhydrin.

Statistical analysis
Results are expressed as the means ± SEM of at least three independent experiments, each performed in duplicate or triplicate. Statistical analysis was performed using one- or two-way ANOVA followed by post hoc comparisons with 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
 
Pregnancy-related changes in SphK1 and SphK2 activity in rat myometrium during pregnancy and postpartum
We previously demonstrated that SphK1 is present in nonpregnant rat myometrium and its activation contributes to the contractile effect of ET-1 (7). Our data demonstrated that SphK1 (Fig. 1AGo) and SphK2 (Fig. 1BGo) activities were modulated during pregnancy. In the rat myometrium at midgestation (d 12), SphK1 and SphK2 activities were low and markedly increased at the end of gestation (d 19) and then declined at d 21 to low levels during postpartum and late postpartum. SphK1 and SphK2 profiles followed similar patterns during pregnancy and postpartum periods. SphK2 activity was measured in the presence of 1 M KCl, which abolishes SphK1 activity (28). To confirm that the kinase activity measured in the presence of KCl corresponded to that of SphK2, the phosphorylation of phytosphingosine, a specific substrate for SphK2 (28), was investigated in different conditions. In the presence of BSA, in which both SphK1 and SphK2 are active, phytosphingosine was phosphorylated (Fig. 1CGo). In the presence of 0.5% Triton X-100, which favors SphK1 activity (28), phytosphingosine was not phosphorylated. In contrast, in the presence of 0.5 or 1 M KCl, which abolishes SphK1 but not SphK2 activity (28), phytosphingosine was phosphorylated. The degree of phosphorylation of phytosphingosine in these conditions was higher than the phosphorylation observed in the presence of BSA. This effect is due to the presence of KCl in the buffer assay, which increases the activity of SphK2 (28). Our findings confirmed that SphK2 activity was present in pregnant rat myometrium and that the levels of SphK1 and SphK2 activity were similarly regulated during pregnancy and postpartum.


Figure 1
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  		FIG. 1. Changes in SphK1/SphK2 activity during pregnancy and postpartum. Myometrial tissues from rats at different stages of gestation and at postpartum were homogenized in SphK buffer. SphK1 (A) and SphK2 (B) activity was determined and expressed in picomoles per minute per milligram protein. Data are the means ± SEM of three separate experiments performed in duplicate. #, P < 0.05 vs. d 12; *, P < 0.05 vs. d 19; **, P < 0.05 vs. d 21; ***, P < 0.05 vs. postpartum (PP). D, Day of gestation; LPP, late postpartum. C, SphK-induced phosphorylation of phytosphingosine (phyto-S1P) or sphingosine (S1P) in rat myometrial homogenates at d 19 of gestation was analyzed in several different conditions described in the figure. Phosphorylation of the substrates was determined as described in Materials and Methods.

 
Pregnancy-related changes in SphK1 and SphK2 protein levels during pregnancy and postpartum
The pattern of SphK1 and SphK2 activity during pregnancy and postpartum was correlated with the level of their expression, as demonstrated by Western blot analysis using specific SphK1 (Fig. 2AGo) or SphK2 antibodies (Fig. 2BGo). Protein levels were low at midgestation (d 12), markedly increased at d 19, and declined to low levels in the postpartum period. The up-regulation of SphK1 in myometrium at d 19 of gestation is consistent with findings from the recent study by Jeng et al. (23) performed in rat uterus.


Figure 2
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  		FIG. 2. Changes in SphK1/SphK2 levels in myometrium during pregnancy and postpartum. A, Myometrial tissues were homogenized in lysis buffer. SphK1 was detected using specific anti-SphK1 antibodies. The blot was stripped and reprobed with anti-SphK2 and anti-actin antibodies. B, The intensity of the bands was quantified with a densitometer. The levels of SphK1 and SphK2 were normalized with respect to actin level in each sample. Data represented the mean ± SEM of three independent experiments performed in duplicate. #, P < 0.05 vs. d 12; *, P < 0.05 vs. d 19; **, P < 0.05 vs. postpartum (PP). LPP, Late postpartum.

 
The role of PKC and ERK in the up-regulation of SphK1 and SphK2 activity at the end of pregnancy
Several studies have previously shown that SphK activity can be regulated by active forms of PKC and ERK (30, 31). The presence of specific PKC isoforms, implicated in myometrial contractility (32), is related to hormonal status (33). We used anti-phospho-pan-PKC antibodies recognizing the phosphorylated forms of PKC, considered to be active. Data (Fig. 3AGo) showed that the level of phospho-PKC was low at d 12 and increased at the end of gestation (d 19 and 21). The signal was substantially reduced in the late postpartum period (LPP). Our findings demonstrated that the level of activated phospho-ERK in the myometrium was regulated as a function of the stage of pregnancy, as previously described (34). Indeed, the level of active ERK2 (Fig. 3BGo), which was undetectable at midgestation (d 12), increased at the end of gestation (d 19 and 21). At postpartum, the level of active ERK was strongly attenuated. The levels of phospho-PKC and phospho-ERK during pregnancy followed similar trends to those observed for SphK1 and SphK2 activities, suggesting that phospho-PKC and/or phospho-ERK may regulate SphK activity in the myometrium of pregnant rats. In the presence of Ro-318220 or U0126, inhibitors of PKC and ERK1/2 activation, respectively, both SphK1 and SphK2 activities were reduced (Fig. 3Go, C and D). PKC and ERK had equivalent effects on the regulation of the two SphK activities. Our findings suggested that the activation of PKC and ERK was involved in the increase of SphK1 and SphK2 activity in rat myometrium at the end of pregnancy. The inhibitory effects of Ro-318220 and U0126 were partial; residual activity (basal activity) may be due to PKC- and ERK-independent SphK activity.


Figure 3
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  		FIG. 3. The levels of phospho-PKC and phospho-ERK increased at the end of gestation and contributed to the regulation of SphK1/SphK2 activity. Phospho-PKC (P-PKC) (A), phospho-ERK2 (P-ERK2) (B), and actin (A and B) were detected with specific antibodies. The levels of P-PKC and P-ERK2 were normalized with respect to the level of actin in each sample. Data represented the mean ± SEM of three independent experiments performed in duplicate. #, P < 0.05 vs. d 12; *, P < 0.05 vs. d 19; **, P < 0.05 vs. postpartum (PP) (A). D, Day of gestation; LPP, late postpartum. (C and D) Day-19 Myometrial tissues were treated for 1 h in the presence or the absence of 5 µM Ro-318220 or 10 µM U0126. Tissues were homogenized in SphK buffer, and SphK1 (C) or SphK2 (D) activity was determined in the presence of 0.5% Triton X-100 or 1 M KCl, respectively. Data represented the mean ± SEM of three independent experiments performed in duplicate. *, P < 0.05 vs. respective control.

 
SphK activation and exogenous S1P up-regulated COX2 expression through a Gi-dependent pathway
COX2 is up-regulated in mouse and human myometrium and contributes to the onset of parturition (35, 36). We showed that COX2 was present only at the end of gestation and at postpartum, but declined at late postpartum in rat myometrium (Fig. 4AGo). COX2 and SphK1/2 levels exhibited similar profiles in rat myometrium during pregnancy. The SphK1/S1P pathway increases COX2 levels in L929 fibroblasts (37). We investigated the possible relationship between the SphK/S1P axis and COX2 expression and the signaling pathway involved in this process. These mechanistic studies cannot be performed in tissues because basal SphK activity is markedly (constitutively) enhanced in the myometrium at late gestation. Therefore, exogenous stimuli cannot be used to analyze the sequential events involved in S1P release in this tissue. We therefore used myometrial cells in primary culture. Incubation of myometrial cells for 4 h in the presence of increasing concentrations of exogenous S1P induced a dose-dependent increase of COX2 (EC50 = 1 µM, and maximal response = 10 µM). The maximal increase in COX2 was about 6-fold the basal value (Fig. 4BGo). S1P can interact with membrane receptors coupled to various G proteins including Gi (38). We analyzed the effect of PTX on S1P-triggered COX2 up-regulation. Treatment of cells overnight in the presence of PTX abolished the S1P-induced increase in COX2 expression (Fig. 4CGo). We used IL-1β, a cytokine associated with parturition (19) and COX2 up-regulation in different cell types (39, 40). IL-1β increased COX2 levels in rat myometrial cells (Fig. 4CGo). We showed in a previous study that PKC is involved in SphK1 activation in nonpregnant rat myometrium (7) and in rat myometrium during late pregnancy (Fig. 3DGo). Treatment of cells in the presence of PDBu, a direct PKC activator, also increased COX2 levels (Fig. 4CGo). Treatment of cells with PTX completely abolished the effect observed after 4 h incubation with IL-1β and markedly reduced the effect observed after 4 h incubation with PDBu (Fig. 4CGo). These findings suggested that the up-regulation of COX2 by IL-1β, PDBu, and exogenous S1P, was mediated through the activation of Gi.


Figure 4
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  		FIG. 4. COX2 was up-regulated in myometrium at the end of gestation, and S1P induced COX2 in myometrial cells from late pregnant rats through interaction with Gi proteins. A, The presence of COX2 in myometrial homogenates from rats at different stages of gestation and at postpartum was determined by Western blot with anti-COX2 and -actin antibodies. B, Myometrial cells from pregnant rats (d 19) in primary culture were incubated for 4 h with the indicated concentrations of S1P. The presence of COX2 in the lysate was analyzed by blotting with anti-COX2 antibodies. The nitrocellulose membrane was stripped and reprobed with anti-actin antibodies. Immunoreactive bands were quantified densitometrically, and the levels of COX2 were normalized with respect to actin level in each sample. Data are expressed in fold over basal values and represent the mean ± SEM of three independent experiments performed in duplicate. *, P < 0.05 vs. COX2 production observed in the presence of 10–11 M S1P. C, Myometrial cells from d-19 rats were treated overnight with or without 200 ng/ml PTX. The cells were then washed and stimulated during 4 h with or without 10 ng/ml IL-1β, 10 µM S1P, or 1 µM PDBu. COX2 was detected with anti-COX2 antibodies. The levels of COX2 were normalized with respect to actin. Results are expressed in fold over basal values. Data represent the mean ± SEM of three independent experiments performed in duplicate. #, P < 0.05 vs. control in the absence of PTX; *, P < 0.05 vs. the values obtained in the presence of the corresponding stimulators in the absence of PTX.

 
SphK1 activation is involved in PDBu- and IL-1β-induced up-regulation of COX2 in myometrial cells
Treatment of myometrial cells in the presence of DMS, an inhibitor of SphK1 and SphK2, reduced the PDBu-induced increase in COX2 by 75% (Fig. 5AGo). A similar level of inhibition was observed upon incubation with t-DHS or SKI, two SphK1 inhibitors (41, 42). The inhibitory effect of SKI seemed to be specific because it did not block S1P-dependent COX2 induction (Fig. 5AGo). IL-1β-triggered COX2 up-regulation was also markedly reduced in the presence of DMS and SKI (Fig. 5BGo). These data indicated that SphK activation (most probably SphK1) was involved in PDBu- and IL-1β-mediated COX2 up-regulation.


Figure 5
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  		FIG. 5. SphK activation by PDBu and IL-1β increased COX2 levels in myometrial cells from d-19 pregnant rats. Myometrial cells were incubated in the presence or absence of 20 µM t-DHS (A) and 5 µM DMS or 5 µM SKI (A and B). After 20 min, the cells were incubated for 4 h in the presence or absence of 1 µM PDBu or 10 µM S1P (A) or 10 ng/ml IL-1β (B). The levels of COX2 were normalized with respect to actin level. Results are expressed in fold over basal values. Data represent the mean ± SEM of three independent experiments performed in duplicate. #, P < 0.05 vs. control without inhibitors; *, P < 0.05 vs. PDBu alone (A) or IL-1β alone (B); {dagger}, no significant difference vs. S1P alone.

 
PKC and ERK acted upstream from SphK to increase COX2 levels in myometrial cells
Our data (Fig. 3Go, C and D) indicated that SphK activity was regulated by PKC and ERK1/2 in rat myometrium at late gestation. In addition, we demonstrated that SphK was involved in the increase in COX2 levels. We therefore investigated the sequential involvement of PKC, ERK1/2, and SphK in the regulation of COX2 levels. Incubation of the cells in the presence of Ro-318220 or U0126 abolished the PDBu- and IL-1β-mediated increase in COX2 levels (Fig. 6AGo), indicating that PKC and ERK1/2 acted upstream from COX2 induction. Our findings also demonstrated that PKC is upstream of ERK1/2 (Fig. 6BGo). Indeed, Ro-318220 substantially reduced the activation of ERK by PDBu and IL-1β. Inhibition of SphK1 by DMS or SKI, which reduced the PDBu- and IL-1β-mediated increase in COX2 levels (Fig. 5Go, A and B), had no effect on PDBu- and IL-1β-mediated ERK activation (Fig. 6BGo). These findings suggested that the sequential activation of PKC->ERK->SphK is required for up-regulation of COX2 in rat myometrial cells. The activation of ERK by IL-1β is PTX sensitive (43); thus, we did not use this agonist to investigate whether Gi acts between ERK activation and induction of COX2. PDBu-mediated activation of ERK1/2 was insensitive to PTX pretreatment (Fig. 6CGo), whereas the increase in COX2 levels was markedly reduced by PTX (Fig. 4CGo). These data suggested that the PDBu-induced increase in COX2 requires the production of a mediator acting through a Gi protein.


Figure 6
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  		FIG. 6. The up-regulation of COX2 in myometrial cells involved sequential activation of PKC->ERK->SphK->Gi. A, Cells were treated in the presence or absence of 5 µM Ro-318220 (Ro) or 10 µM U0126 (U). After 20 min, the cells were incubated for 4 h in the presence or absence of 1 µM PDBu or 10 ng/ml IL-1β. The levels of COX2 and actin were determined as described in Fig. 4Go. Results are expressed in fold over basal values. Data represent the mean ± SEM of three independent experiments performed in duplicate. #, P < 0.05 vs. control in the absence of inhibitor; *, P < 0.05 vs. corresponding values in the absence of inhibitors. B, Cells were treated in the presence or absence of 5 µM SKI, 5 µM DMS, or 5 µM Ro-318220 (Ro). After 20 min, the cells were incubated for 5 min in the presence or absence of 10 ng/ml IL-1β or 1 µM PDBu. Data represent the mean ± SEM of three independent experiments each performed in duplicate. #, P < 0.05 vs. control in the absence of inhibitors; *, P < 0.05 vs. corresponding values in the presence of PDBu or IL-1β added alone; {dagger}, not significantly different from corresponding values in the presence of PDBu or IL-1β added alone. C, Cells were treated overnight with or without 200 ng/ml PTX. Cells were then incubated for 5 min with or without 1 µM PDBu. Active ERK was detected with anti-phospho-ERK2 antibodies. The intensity of the bands was quantified and the levels of phospho-ERK (P-ERK) were normalized with respect to total ERK2 level. Data are expressed in fold over basal values and represent the mean ± SEM of three independent experiments performed in duplicate. *, P < 0.05 vs. respective control; {dagger}, not significantly different from the value obtained with PDBu added alone.

 
PDBu and IL-1β increased membrane SphK1 activity and S1P release from rat myometrial cells
We hypothesized that S1P, produced by SphK activation in response to PDBu or IL-1β, may be released into the medium, where it interacts with membrane Gi-coupled receptor to increase COX2 expression. To determine whether myometrial cells do release S1P in response to PDBu and IL-1β, cells were labeled with [3H]sphingosine. [3H]S1P released was extracted from the medium under alkaline condition as described in Materials and Methods. TLC analysis of lipids present in aqueous phase confirmed that the radioactivity detected corresponded mainly to S1P (Fig. 7AGo), whereas sphingosine was present exclusively in the organic phase (Fig. 7BGo), as previously described (29). Treatment of the cells in the presence of PDBu or IL-1β increased the amount of S1P released into the medium (1.8- to 1.9-fold increase) (Fig. 7CGo). This effect was markedly reduced by pretreatment of cells with DMS or SKI, suggesting that SphK activity, most probably SphK1, was responsible for the release of S1P. Treatment of cells with Ro-318220 or U0126 abolished PDBu- and IL-1β-induced [3H]S1P release (Fig. 7CGo). This suggested that stimulation of PKC, ERK, and SphK1 by PDBu and IL-1β increased the release of S1P into the incubation medium. We previously reported that in nonpregnant rat myometrium, the increase in SphK1 activity is observed in the membrane fractions (7). In d-19 myometrial cells, the total SphK1 activity stimulated by PDBu or IL-1β increased about 80 and 65%, respectively, in the membrane fraction (Fig. 7CGo, inset). The increase of SphK activity in membrane may be explained by SphK1 translocation. However, due to the high amount of membrane SphK1 in basal conditions, the translocation of SphK1 by PDBu or IL-1β was hardly detectable (data not shown).


Figure 7
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  		FIG. 7. PDBu and IL-1β increased levels of S1P released from myometrial cells through activation of PKC, ERK, and SphK1. A and B, Myometrial cells were incubated for 5 min in the presence of 50 nM [3H]sphingosine and then for 15 min in the presence of 1 µM PDBu. The radioactive material present in the incubation medium was separated as described in Materials and Methods. Radioactivity (cpm) in the alkaline upper aqueous phase (A) or in the organic phase (B) was analyzed by TLC and quantified with a computerized Berthold radioscanner. C, Myometrial cells were treated in the presence or absence of 5 µM DMS, 5 µM SKI, 5 µM Ro-318220 (Ro), or 10 µM U0126 (U). After 15 min, the cells were labeled for 5 min with 50 nM [3H]sphingosine and then treated in the presence or absence (control) of 1 µM PDBu or 10 ng/ml IL-1β for 15 min. The reactions were stopped and radioactive S1P (cpm/well) was determined by scintillation counting. Results are the means ± SEM of four independent experiments performed in duplicate. #, P < 0.05 vs. control in the absence of inhibitors; *, P < 0.05 vs. values obtained in the presence of PDBu or IL-1β added alone. Inset, Myometrial cells were treated with or without 1 µM PDBu or 10 ng/ml IL-1β for 15 min, and total SphK1 activity in membrane fractions was determined. The data represent the mean ± SEM of three independent experiments. *, P < 0.05 vs. control.

 
The amount of S1P released from myometrial cells was modest. This can be explained by the low increase of SphK1 membrane activity (Fig. 7CGo, inset). Moreover, the amount of labeled S1P detected in the medium may be underestimated due to the low amount of radioactive sphingosine used in our experimental conditions. Alternatively, this low effect may reflect the rapid degradation of S1P by specific phosphatases (44, 45).

The d-19, but not d-12, myometrial tissues released S1P by the activation of PKC, ERK, and SphK1
Data in Fig. 1Go demonstrated that SphK activity was markedly increased at d 19 compared with d 12. Furthermore, myometrial cells are able to release S1P. Therefore, we investigated the potential ability of myometrial tissues from d 19 to release SIP. The d-19, but not d-12, myometrium a released substantial amount of S1P (Fig. 8AGo). SphK activity was regulated by PKC and ERK (Fig. 3Go, C and D). Treatment of d-19 myometrium with Ro-318220 or U0126 led to a marked reduction in the release of S1P (Fig. 8BGo). Moreover, treatment of d-19 myometrium with DMS or SKI substantially reduced the release of S1P, suggesting that SphK1 is involved in this response. Thus, the release of S1P detected in myometrial cells was also observed for myometrial tissue from d-19 pregnant rats. This release of S1P also required the activation of PKC, ERK, and SphK1 and was related to SphK protein/activity levels.


Figure 8
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  		FIG. 8. Myometrium from d 19, but not d 12, released S1P through activation of PKC, ERK, and SphK1. A, Myometrium from d-12 and -19 pregnant rats were labeled with 50 nM [3H]sphingosine for 20 min. B, Myometrium from d-19 pregnant rats was incubated (1 h) in the presence or absence (control, C) of 10 µM DMS, 10 µM SKI, 5 µM Ro-318220 (Ro), or 10 µM U0126 (U) before addition of 50 nM [3H]sphingosine for 20 min. [3H]S1P accumulated in the incubation medium was determined as described in Fig. 7CGo. Data are expressed in cpm [3H]S1P/50 mg tissue and represent the mean ± SEM of three independent experiments performed in duplicate. *, P < 0.05 vs. d 12 (A) or control d 19 (B). D, Day of gestation.

 
The induction of COX2 involved SphK and S1P in myometrial tissues
In rat myometrium at midgestation, the level of expression of COX2 was very modest (Fig. 4AGo). When the myometria were removed and their levels of COX2 expression analyzed (time zero; Fig. 9Go), the tissues did not express COX2 as in Fig. 4AGo. In contrast, when the myometrial tissues were incubated for 8 h, a marked induction of COX2 was observed. The effect was abolished when SphK1 was inhibited by DMS or SKI. The inhibitory effect was overcome by S1P. It is interesting to note that the response of S1P was not altered by the DMS and SKI because it remained similar to that obtained in the absence of inhibitors. These data provided evidence for a link between SphK1, S1P, and COX2 induction in uterine smooth muscle.


Figure 9
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  		FIG. 9. The induction of COX2 by SphK1 activation and S1P in myometrial tissues. Day-12 myometrial tissues were homogenized immediately (0 h) or were incubated for 8 h with or without 10 µM DMS or 10 µM SKI in the presence or absence of 10 µM S1P. COX2 and actin were detected and expressed as described in Fig. 4Go. Data represent the mean ± SEM of three independent experiments performed in duplicate. #, P < 0.05 vs. time 0 h; *, P < 0.05 vs. control (8 h); {dagger}, not significantly different from S1P plus inhibitors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study is the first to demonstrate that SphK activation and S1P release are up-regulated in rat myometrium at the end of gestation and involved in the induction of COX2. Both SphK1 and SphK2 are present in rat myometrium and undergo similar pregnancy-related changes. Indeed, at midgestation, SphK1 and SphK2 were barely detectable by immunostaining. By contrast, their protein and activity levels were markedly enhanced at d 19 of pregnancy. These findings suggested that SphK1/2 protein synthesis may be similarly controlled in the myometrium at the end of gestation. A recent study showed that in uterus, SphK1 mRNA and protein increased at late pregnancy (23). However, no significant pregnancy-related changes in SphK2 mRNA levels were observed in this study (23). By contrast, we demonstrated that in myometrial tissues, not only the levels of SphK2 protein, but also its activity, increased at the end of gestation. This apparent discrepancy may be explained by posttranscriptional regulation of SphK2. Alternatively, this difference can be due to the use of uterine (endometrium plus myometrium) tissues by Jeng et al. (23), whereas we used only myometrium. We observed that the up-regulation of SphK1/SphK2 coincided with an elevated progesterone/estradiol ratio. This is consistent with previous findings that the pretreatment of rats with progesterone up-regulates SphK1 mRNA levels in the myometrium (23). Notably, we observed the maximal increase of SphK at around d 19. SphK1/SphK2 protein and activity levels then declined at parturition and continued to decrease at postpartum. Our previous findings, based on global analysis of the human myometrial transcriptome at term, demonstrated that late human pregnancy is associated with a marked down-regulation of genes related to cell growth, proliferation, and development (46). Unfortunately, sphingosine kinase genes were not represented in the human membrane microarrays used. Thus, these down-regulated genes at term may have included SphK1/2. Furthermore, the up-regulation of SphK (d 19) may be linked to the processes involved in the switch from the synthetic to the contractile phenotype of the myometrium (47). This interpretation is in line with findings showing that S1P regulates the differentiation of skeletal muscle cells (48) and smooth muscle cells (49), including those of the myometrium (23). The marked down-regulation of SphK1/2 observed in the myometrium from postpartum rats may be linked to the initiation of apoptosis associated with involution during postpartum. This observation is consistent with our previous study demonstrating that SphK/S1P axis contributes to survival of leiomyoma smooth muscle cells (50). SphK1 and SphK2 may exert opposite effects on cellular functions. In particular, SphK1 mediates proliferation and growth, whereas SphK2 is involved in apoptotic events (51). However, SphK2 can fulfill an antiapoptotic role in human macrophages (52). Mice deficient in either SphK1 or SphK2 do not display any significant abnormalities demonstrating the ability of SphK1 and SphK2 to substitute for each other (53, 54). In myometrium, we showed that SphK1 and SphK2 are similarly regulated during pregnancy. However, only SphK1 appeared to be involved in COX2 induction. The role of SphK2 remains to be determined.

Recent studies indicate that SphK1 and SphK2 activities can be regulated by phosphorylation (31, 55). We have previously shown that SphK is activated by PKC (7) in nonpregnant rat myometrium and that PKC is involved in ERK activation in myometrial cells (56). Our findings demonstrated that PKC and ERK were present in active forms in the myometrium in late pregnancy. The levels of diverse endogenous stimuli, including agonists acting through GPCR, growth factors, cytokines (46), and estradiol (57), increased at late gestation and may contribute to the up-regulation of PKC and ERK activities. The activated kinases subsequently stimulate SphK1/2 activity.

The pregnancy-related changes observed for SphK1 and SphK2 were also observed for COX2 which plays an important role in parturition (19, 22). Using myometrial cells in primary culture we demonstrated COX2 induction was consecutive to the sequential activation of PKC, ERK, SphK and subsequent release of S1P which as exogenous S1P acted via Gi (Fig. 10Go). We demonstrated that SKI blocked the release of S1P, suggesting that SphK1, but not SphK2, is involved in this signaling pathway. S1P release may be facilitated by the presence of SphK1 in the membrane where both sphingosine and S1P receptors coupled to Gi are located (30).


Figure 10
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  		FIG. 10. Schematic representation of the signaling pathway involved in PDBu-, IL-1β-, and S1P-mediated induction of COX2 in myometrial cells from d-19 pregnant rats. In late pregnant myometrial cells, PDBu- or IL-1β-mediated PKC activation induced the sequential activation of ERK and SphK1, increasing production and release of S1P. The COX2 induction by PDBu, S1P, and IL-1β occurred through activation of Gi proteins. Sph, Sphingosine.

 
Interestingly, S1P release also occurs in myometrial tissue and is related to SphK levels and activity. Indeed, almost no S1P was detected in the medium of d-12 myometrium, in which the activity levels of SphK1 were low. In contrast, S1P release from d-19 myometrium, which has a high level of SphK1 activity, was enhanced. Moreover, S1P production and release are regulated by PKC, ERK, and SphK1 in the myometrium, as observed in rat myometrial cells.

The mechanisms underlying the secretion of SphK-induced intracellular S1P and its interaction with membrane receptors remain unclear. A previous study suggested that the presence of S1P in the medium could be explained by the export of SphK1 (58). However, findings from recent studies implicate the ATP binding cassette (ABC) transporter in S1P release (59). This transporter catalyzes the movement of lipids from the inner to the outer leaflet of the plasma membrane (60).

Exported S1P may regulate cellular responses by paracrine and autocrine mechanisms through S1P membrane receptors. In this context, Jeng et al. (23) identified mRNA for three S1P receptors (S1P1, S1P2, and S1P3) in late pregnant rat uterus. The mechanisms by which S1P induces COX2 are not fully elucidated. All S1P receptors can couple to Gi proteins. COX2 induction by S1P seems to be mediated by the S1P1 and S1P3 receptors in amnion Wish cells (61) and by S1P2 receptors in angiogenesis in the mouse retina (62).

The involvement of SphK1/S1P axis in the induction of COX2 was strengthened by our data obtained when d-12 myometrial strips were incubated for a long time (Fig. 9Go). Our findings demonstrated that SphK/S1P release contributed to COX2 induction in myometrial cells and myometrial tissues. Therefore, myometrial cells in primary culture, which we established for this analysis, appear to be an appropriated model to study the mechanisms involved in the other events associated with the end of gestation.

We previously demonstrated that S1P and SphK exert a rapid effect on myometrial contraction (7). This study shows that the SphK/S1P axis was also able to control, through a long-lasting mechanism, the induction of COX2, which plays a key role during labor. This is consistent with a previous study demonstrating that S1P may stimulate myometrial contractility by stabilizing oxytocin receptor mRNA in human myometrial cells (63).

In conclusion, our findings reveal that S1P produced by uterine smooth muscle has an autocrine role in myometrial tissues but may exert a paracrine effect in neighboring tissues such as endometrium. Disruption of this complex signaling network may be associated with different pathologies including preterm birth. Disruption of the genes encoding SphK leads to defective decidualization impairing implantation and causing pregnancy loss (64). Thus, the SphK/S1P axis may be considered as an important regulator of uterine function during pregnancy.


   Acknowledgments
 
We thank Dr. Taro Okada for the generous gift of rabbit anti-SphK2 antibody (Dr. Shunichi Nakamura laboratory, Kobe University Graduate School of Medicine, Kobe, Japan) and Dr. Yoshiko Banno for the generous gift of rabbit anti-SphK1 antibody (Department of Cell Signaling, Gifu University Graduate School of Medicine, Gifu, Japan). We are grateful to Ginette Vilain for expert technical assistance.


   Footnotes
 
This work was supported by grants from the Centre National de la Recherche Scientifique and Université Paris-Sud. M.S.-S. was the recipient of a fellowship from the Mexican National Council for Science and Technology.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 29, 2008

Abbreviations: COX2, Cyclooxygenase 2; DMS, dimethysphingosine; ET-1, endothelin-1; GPCR, G protein-coupled receptor; PDBu, 4β-phorbol 12,13-dibutyrate; PG, prostaglandin; PKC, protein kinase C; PMSF, phenylmethylsulfonyl fluoride; PTX, pertussis toxin; S1P, sphingosine-1-phosphate; SKI, sphingosine kinase inhibitor; SphK, sphingosine kinase; t-DHS, DL-threo-dihydrosphingosine; TLC, thin-layer chromatography.

Received December 19, 2007.

Accepted for publication May 22, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
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