Molecular Human Reproduction, Vol. 7, No. 8, 755-763,
August 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Nitric oxide induces extensive apoptosis in endometrial epithelial cells in the presence of progesterone: involvement of mitogen-activated protein kinase pathways
1 Institute of Clinical Medicine, School of Medicine and 2 Institute of Anatomy and Cell Biology, School of Life Science, National Yang-Ming University, Taipei, Taiwan, ROC and 3 Department of Obstetrics and Gynecology, Veterans General Hospital, Taipei, Taiwan, ROC
Abstract
During trophoblast invasion, luminal and glandular endometrial epithelial cells (EEC) have been found to undergo apoptosis through undetermined mechanisms. We postulate that nitric oxide (NO) and progesterone may mediate apoptosis in EEC because they are produced by trophoblasts at concentrations that can cause apoptosis in non-uterine cells. Using a cultured EEC line, RL95-2, we found that sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP), two commonly used NO-releasing agents, caused the death of EEC in a dose-dependent manner and progesterone markedly enhanced NO-induced cytotoxicity. Cells treated with NO/progesterone showed a significant increase in the percentage of condensed nuclei, as detected by DAPI staining, and in caspase-3 activity, indicating that these cells underwent apoptosis. Immunoblot analysis revealed that SNP/NO could activate extracellular signal-regulated kinase (ERK) and, to a lesser extent, p38 mitogen-activated protein kinase (MAPK). While pretreatment with PD98059 (an ERK inhibitor) did not prevent cell death, the addition of SB203580 (a p38 MAPK inhibitor) effectively rescued the cells from NO/progesterone treatment. Moreover, SNP/NO-induced p38 MAPK activation was significantly up-regulated by progesterone. Our results demonstrate that NO and progesterone may synergistically activate p38 MAPK to induce apoptosis in EEC, a process that may facilitate implantation.
apoptosis/endometrium/MAP kinase/nitric oxide/progesterone
Introduction
Embryo implantation is a complex process that can be divided into three stages: apposition, attachment, and invasion (Klentzeris, 1997
). In the invasion stage, the trophoblasts intrude between the luminal epithelial cells of endometrium, invade the endometrial stroma, and erode the maternal vessels and glands (Moore and Persaud, 1998). Signs of apoptosis can be detected in glandular epithelial cells during the implantation window of menstrual cycle (von Rango et al., 1998). In rodent models, uterine epithelial cells surrounding the embryos were also found to undergo apoptosis, leading to their phagocytosis by trophoblasts (Parr et al., 1987). Therefore, apoptotic cell death of endometrial epithelial cells (EEC) may represent an essential cellular event for successful implantation. Mechanisms of apoptosis in EEC during trophoblast invasion, however, remain to be established (Pampfer and Donnay, 1999).
Nitric oxide (NO), a free radical molecule, is generated by the enzyme nitric oxide synthase (NOS) that converts L-arginine into L-citrulline and NO in equimolar amounts (Palmer and Moncada, 1989). At least three isoforms of NOS, namely, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), have been isolated (Knowles and Moncada, 1994). Previous studies have suggested that NO may play an important role in embryo implantation. For example, intrauterine injection of NOS inhibitor during the peri-implantation period decreases embryo implantation rate in the rat (Novaro et al., 1997; Biswas et al., 1998). Furthermore, it has been shown that early embryos can generate NO (Gouge et al., 1998) and that trophoblasts express eNOS and iNOS (Ariel et al., 1998; Gagioti et al., 2000). The endometrial epithelial glands from the secretory phase, which encompasses the implantation window, contain more eNOS mRNA than those from the proliferative phase (Tseng et al., 1996). Immunoreactivity for iNOS is not present in endometrial stroma throughout the menstrual cycle; however, significant iNOS expression is detected in decidualized stromal cells obtained from the first trimester of pregnancy (Telfer et al., 1997). These observations suggest that the local concentration of NO, being actively generated by various forms of NOS, may be considerably high at the trophoblast–decidual interface during the implantation phase. Since induction of apoptosis is a major biological effect of NO (Brune et al., 1999), it is likely that NO may induce or facilitate death of EEC and thereby promote implantation. Previous studies have also shown that NO can activate mitogen-activated protein kinases (MAPK) (Kim et al., 1997; Jun et al., 1999), including extracellular signal-regulated kinase (ERK or p44/42 MAPK), c-Jun NH2-terminal kinase (JNK) and p38 kinase, and these may serve as regulators for apoptosis (Herlaar and Brown, 1999; Cross et al., 2000). NO has been demonstrated to induce apoptosis in human leukaemia cells (HL-60), at least in part through p38 kinase activation (Jun et al., 1999). However, to date there has been no report that relates apoptosis in EEC during implantation with NO.
The successful use of antiprogestins in postcoital contraception and early pregnancy termination clearly shows that progesterone is essential for endometrial receptivity and maintenance of pregnancy (Heikinheimo and Archer, 1996; Spitz et al., 1998). Although progesterone has been shown to regulate the expression of certain implantation-related molecules, such as leukaemia inhibitory factor (Stewart et al., 1992; Liu et al., 1999), calcitonin (Kumar et al., 1998; Zhu et al., 1998), and integrins (Lessey et al., 1994,1996), the exact mechanism for progesterone in facilitating implantation remains to be elucidated. Because trophoblasts can secrete progesterone as early as 4 weeks after fertilization (Winkel et al., 1981; Scott et al., 1991), the local concentration of progesterone at the trophoblast–decidual interface may be exceedingly high. The accurate concentration of progesterone at the trophoblast–decidual interface is difficult to measure; however, the tissue concentration of progesterone in the first trimester placenta has been determined to be as high as 8–16 µmol/l (Runnebaum et al., 1975; Laatikainen et al., 1982). Noticeably, progesterone at this concentration range has been shown to induce apoptosis in sex hormone-sensitive cells (Bu et al., 1997; Formby and Wiley, 1998). Therefore, whether the high progesterone concentration at the trophoblast-decidual interface contributes to apoptosis in EEC warrants further investigation.
The purpose of this study is to examine the effects of NO and progesterone on EEC death and the possible involvement of MAPK in the transduction of NO/progesterone signals. Our results may have important clinical ramifications as they may shed light on the role of NO and progesterone in early pregnancy. We postulate that high concentrations of NO at the trophoblast–decidual interface may participate in the initiation of EEC apoptosis and thereby facilitate trophoblast invasion; once the trophoblasts acquire the ability to secrete progesterone, NO-mediated apoptosis may be greatly enhanced to accommodate the expanding placenta.
Materials and methods
Cell culture
Human endometrial carcinoma cell line RL95-2 (CRL 1671) was purchased from the American Type Culture Collection (Rockville, MD, USA) through the National Health Research Institute Cell Bank (Hsin-Chu, Taiwan) (Way et al., 1983). Cells were maintained at 5% CO2 and 37°C in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) and Ham's F12 (Gibco) supplemented with 10% fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), 10 mmol/l of HEPES (BDH, Poole, UK), and 5 µg/ml of bovine insulin (Sigma Chemical Co., St Louis, MO, USA). The growth medium was changed every 3 days, and cells were subcultured by trypsinization (trypsin–EDTA solution; Gibco) when they became confluent.
Reagents for the treatment of cultured cells were prepared as follows. Sodium nitroprusside (SNP; Sigma) potassium ferricyanide (KFC; Sigma) and progesterone as the water-soluble cyclodextrin complex (Sigma), 2-hydroxypropyl-ß-cyclodextrin were dissolved and diluted appropriately with culture medium to give the desired final concentration in cell culture. S-Nitroso-N-acetylpenicillamine (SNAP; Tocris, Bristol, UK) was dissolved in dimethylsulphoxide (DMSO; Sigma). The p38 MAPK inhibitor, SB203580 (Calbiochem-Novabiochem, San Diego, CA, USA), and the ERK inhibitor, PD98059 (Calbiochem-Novabiochem), were also dissolved in DMSO and added to cell culture 1 h before treatments with NO donors and progesterone. Final concentrations of DMSO in all cell cultures were <1%, and this had no significant effect on the viability of RL95-2 cells.
Cell survival analysis
Cell survival was assayed by measuring the conversion of the yellow, water-soluble tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma), to the blue, water-insoluble formazan. The rate of this reaction, catalysed by cellular mitochondrial enzymes, is proportional to the number of surviving cells; therefore, the MTT assay has been widely used to quantify viable cells (Mosmann, 1983). For the cell survival study, RL95-2 cells were plated in 96-well plates at 1.2x104/100 µl/well. After 4 h of incubation to allow cell adherence, various drug treatments were added and the cells were incubated at 37°C, 5% CO2 for another 24, 48 or 72 h. At the end of cell culture, the supernatant was discarded and 100 µl fresh medium was dispensed to each well. Then MTT was added to each well at a final concentration of 0.5 mg/ml. After an additional 4 h of incubation, 50 µl of 10% sodium dodecyl sulphate (SDS) in 0.04 N HCl was added to each well. The reduced MTT–formazan product was dissolved by incubating the SDS–MTT–medium mixture at 37°C overnight. Quantification of MTT reduction was accomplished by measuring absorbance at 570 nm against a 650 nm reference using the microplate reader (BioTek EL311sx). Data were presented as the percentage of survival relative to that in vehicle-treated control cultures. All MTT assays were performed in triplicate.
Nitrite assay
Nitrites are stable metabolites of NO that have served as a reliable indicator of NO production in cell cultures (Stuehr and Nathan, 1989). Nitrite assays were performed in 96-well plates using equal volumes of Griess reagent (0.5% sulphanilamide, 0.05% naphthalene diamine hydrochloride in 2.5% orthophosphoric acid) and sample. After 10 min of reaction at room temperature, the plates were read at 550 nm absorbance. Nitrite concentrations were calculated from a precalibrated standard curve of NaNO2.
Detection of apoptotic cells with DAPI staining
Cells were seeded at a concentration of 1.2x105/ml and 3 ml of cell suspension was dispensed to each well of 6-well plates. After 4 h of incubation to allow adherence, various treatments were added. At the end of the experiments, cells that had detached from the culture plate were collected by aspiration. The remaining attached cells were collected by trypsinization and pooled with the detached cells. All collected cells were then centrifuged at 120g for 5 min and fixed with 2% paraformaldehyde for 10 min. The cells were washed twice and resuspended with 200 µl PBS. The cell concentration was adjusted to 4x105/ml and 100 µl of cell suspension was placed into a Shandon cytospin chamber. After centrifugation for 5 min at 45 g cells were stained with 2 µg/ml DAPI (Sigma) for 30 min. The slides were washed three times with PBS and examined under a fluorescence microscope.
Caspase-3 activity measurement
Cells were seeded in 96-well plates at 1x105/100 µl/well and cultured for 48 h at 37°C. SNP and/or progesterone were then added and incubation was continued for an additional 24 h. At the end of cell culture, the supernatant was discarded and cells were gently washed twice with PBS. Then 25 µl of 1% Triton X-100 (Sigma) was added to each well and the cells were subjected to four cycles of freezing and thawing. Protein concentration of cell extract was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). An equal amount of cellular protein from extract of each group was added to a reaction mixture containing 50 µmol/l of the fluorogenic caspase-3 substrate acetyl-Asp-Glu-Val-Asp-
-(7-amino-4-methyl coumarin) (Ac-DEVD-AMC; Promega, Madison, WI, USA), followed by incubation at 30°C for 60 min. Free 7-amino-4-methyl coumarin (AMC) liberated upon enzymatic cleavage was detected fluorometrically by Cytofluor 2300 with excitation and emission wavelengths of 360 and 460 nm respectively. Caspase-3 activity correlated with the concentration of free AMC generated in the reaction. Data are presented as fold of activation relative to vehicle treated control cultures.
Immunoblotting assay for MAPK
Assessment of the phosphorylation of MAPK was accomplished by immunoblotting. Cell concentration was adjusted to 1x106/ml and 5 ml of cell suspension was seeded to 60 mm culture dishes. After 48 h of incubation, the culture medium was replaced by serum-free medium supplemented with 1 mg/ml bovine serum albumin (USBTM, Cleveland, OH, USA
) and serum starvation was continued for 16–18 h, followed by the addition of various drug treatments. The treatments were terminated after the indicated intervals by aspirating the supernatant and washing the dishes with PBS. Fifty µl of lysis buffer (20 mmol/l Tris, pH 7.5, 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 1% Triton, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l ß-glycerophosphate, 1 mmol/l Na3VO4, 1 µg/ml leupeptin and 1 mmol/l PMSF
) was added to the cells and the dish was kept on ice for 5 min. The lysed cells were scraped off the dish, transferred to microcentrifuge tubes, and sonicated. The cell lysates were centrifuged to remove insoluble material and ~20 or ~40 µg of supernatant protein was used in 10% SDS–polyacrylamide gel electrophoresis. After the cell lysates were fractionated by electrophoresis and transferred to PVDF membranes (NENTM Life Science Products, Inc., Boston, MA, USA
), blocking with 5% non-fat milk in TBST [25 mmol/l Tris, pH 7.5, 150 mmol/l NaCl, 2.7 mmol/l KCl, 0.1% (v/v) Tween 20] was performed, followed by incubation overnight with the primary antibody (rabbit anti-human phospho-ERK antibody, rabbit anti-human ERK antibody, rabbit anti-human phospho-JNK antibody, rabbit anti-human JNK antibody, rabbit anti-human phospho-p38 MAPK antibody, or rabbit anti-human p38 MAPK antibody; all from Cell Signaling Technology, Beverly, MA, USA). The membranes were then extensively washed with TBST and incubated for 60 min with the secondary antibody (goat anti-rabbit antibody conjugated with horseradish peroxidase; Transduction Laboratories, Lexington, KY, USA). After extensive washing with TBST, the immune complexes were detected by chemiluminescence using the Western blotting kit from NENTM Life Science Products, Inc.
Statistical analysis
Data of cell survival analyses, nitrite assays and caspase-3 activity measurements were presented as means ± SEM. Statistical significance between groups was determined by one-way analysis of variance, followed by Fisher's post-hoc least significant difference (LSD) test. Simple linear regression was used to analyse slope changes between different survival curves. All analyses were performed using the SAS software package on a Pentium III-based personal computer.
Results
Effect of NO on the survival of endometrial epithelial cells
To examine whether NO can modulate the survival of EEC, the RL95-2 cells were cultured in the presence or absence of SNP or SNAP, the two most commonly used NO donors, that had been used in previous studies (Messmer et al., 1995; Jea et al., 1998). Exposure of RL95-2 cells to SNP for 24 h caused a dose-dependent decrease in cell viability, as measured by the MTT metabolism assay (Figure 1). While 0.1 mmol/l SNP treatment showed no significant effect on cell survival, SNP concentrations >0.5 mmol/l significantly reduced MTT metabolism as compared with the controls in a dose-dependent manner (P < 0.05; n = 3
). Potassium ferricyanide (KFC), which is structurally similar to SNP except for the absence of a nitroso group (Takeda et al., 1999), had no adverse effect on cell survival at concentrations comparable with those of SNP, suggesting that the effect of SNP on cell survival did not occur through the cyanoid moiety (Figure 1). SNAP also caused the death of RL95-2 cells in a dose-dependent manner, with an apparent cytotoxic concentration >0.2 mmol/l (Figure 1; P < 0.05; n = 3
).
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Effect of progesterone–NO interaction on the survival of EEC
Both progesterone and NO can be produced by trophoblasts during the invasion process (Winkel et al., 1981
; Gagioti et al., 2000); however, the role of interactions between progesterone and NO in EEC death has not been completely elucidated. To study the possible regulatory effects of progesterone on NO-induced death of EEC, progesterone was added to RL95-2 cells in the presence or absence of SNP or SNAP. When added alone at a concentration of 1 or 10 µmol/l, progesterone did not induce RL95-2 cell death (Figure 2)
. In contrast, administration of progesterone at these concentrations in combination with 1 mmol/l SNP to RL95-2 cells for 24 h resulted in markedly enhanced cell death as compared with SNP treatment alone (Figure 2A; P < 0.05; n = 3
). Higher concentrations of progesterone treatment alone (for example, 100 µmol/l) could cause extensive cell death; however, an enhancing effect was still apparent when 100 µmol/l progesterone and 1 mmol/l SNP were added together. Simple linear regression analysis on cell survival data showed that progesterone increased the slopes of SNP/NO-treated survival curves (from –0.05 ± 0.01 to –0.14 ± 0.02), indicative of a synergistic effect. The synergistic effect became more prominent after 72 h culture when 1 or 10 µmol/l progesterone was added together with 1 mmol/l SNP (Figure 2B). Since an apparent synergistic effect was observed at both 24 and 72 h of treatment with 10 µmol/l progesterone and the tissue progesterone concentration of first trimester placenta was ~8–16 µmol/l (Runnebaum et al., 1975; Laatikainen et al., 1982), we used 10 µmol/l progesterone in all subsequent experiments. When various concentrations (0, 0.1, 0.5 and 1 mmol/l) of SNP were administered with 10 µmol/l progesterone for 72 h, the synergistic effect on cell death appeared to be most prominent with 1 mmol/l SNP and 10 µmol/l progesterone (Figure 2C). Similar synergistic effects on cell death were observed when SNAP was added as the NO donor in the presence of progesterone (Figure 2B,D). The synergistic effect was most pronounced with 0.2 mmol/l SNAP and 10 µmol/l progesterone. In subsequent experiments, therefore, 1 mmol/l SNP or 0.2 mmol/l SNAP in combination with 10 µmol/l progesterone were employed for studies on synergism.
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P < 0.05, as compared with SNP treatment alone. 
P < 0.05, as compared with SNAP treatment alone. ¶P < 0.05, as compared with progesterone treatment alone (n = 3
).Mode of NO/progesterone-induced cell death
To assess whether EEC death caused by progesterone and SNP/NO was due to apoptosis, we examined two hallmarks of apoptosis: nuclear condensation and caspase-3 activation. Nuclear condensation was detected by the fluorescent DNA staining method using DAPI (Barber et al., 1998
). After treatment with 10 µmol/l progesterone and/or 1 mmol/l SNP for 72 h, all adherent and non-adherent cells were collected, subjected to cytospin, and stained with DAPI for observation of nuclear morphology under a fluorescence microscope. Nuclei of cells from the vehicle-treated control group were mostly round and faintly stained (Figure 3A)
. A small percentage of cell nuclei treated with progesterone or SNP alone displayed signs of apoptosis: shrunken and intensely stained (Figure 3B,C). In contrast, the percentage of apoptotic nuclei increased substantially when progesterone and SNP/NO were added together (Figure 3D). Likewise, we also observed a marked synergistic effect of the SNAP–progesterone combination on the increase in apoptotic nuclei as detected by DAPI staining (data not shown).
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Caspase-3 has been demonstrated to function as an executioner of apoptosis (Villa et al., 1997
); therefore, its activity should reflect the percentage of apoptotic cells. Caspase-3 activity was determined by measuring the proteolytic cleavage of a fluorogenic substrate, Ac-DEVD-AMC (Ciriolo et al., 2000). As shown in Figure 4, a slight, but statistically insignificant, increase in caspase-3 activity was observed in cells receiving 10 µmol/l progesterone or 1 mmol/l SNP alone for 24 h (Figure 4). In contrast, a more than 5-fold increase in caspase-3 activity was observed in cells treated with progesterone and SNP/NO simultaneously (Figure 4; P < 0.05 as compared with the other three groups; n = 3
).
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Involvement of mitogen-activated protein kinases (MAPK) in NO/progesterone-induced apoptosis
Previous studies have shown that NO can activate MAPK in the signalling pathway leading to apoptosis (Jun et al., 1999
; Oh-hashi et al., 1999). The MAPK are activated by phosphorylation of specific tyrosine and threonine residues and the relative levels of phosphorylated MAPK in total MAPK represent the degree of MAPK activation. The level of phosphorylated ERK (phospho-ERK1 and ERK2) increased significantly within 10–30 min following SNP addition, declined at 1 h, elevated again after 4 h, and continued to rise thereafter (Figure 5A)
. On the other hand, two bands of phosphorylated p38 MAPK (phospho-p38) were detected by the anti-human phospho-p38 MAPK antibody used in this study. These bands corresponded to two of the four isoforms [, ß, 
and 
of p38 MAPK that have been identified in the literature (Wang et al., 1997; Herlaar and Brown, 1999)]. Exposure to SNP/NO seemed to increase the intensity of the upper band of phospho-p38 MAPK at 30 min and 24 h (Figure 5B). JNK did not appear to respond to SNP/NO treatment (Figure 5C).
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To further examine whether MAPK activation is involved in NO/progesterone-induced death of EEC, inhibitors of ERK (PD98059) and p38 MAPK (SB203580) were administered 1 h prior to the addition of progesterone and SNP. MTT metabolism was measured 24 h and 72 h after treatment. While PD98059 was not able to block cell death, pre-treatment with SB203580 significantly, though not completely, rescued RL95-2 cells from SNP/progesterone-induced death at both 24 and 72 h (Figure 6A and B;
P < 0.05; n = 3
). A comparable protection of RL95-2 cells by SB203580 from SNAP/ progesterone-induced death was also found (data not shown). Furthermore, significantly enhanced activation of p38 MAPK was observed in cells receiving simultaneous stimulation of progesterone and SNP/NO for 24 h (Figure 6C).
|
Discussion
Glandular and luminal epithelial cells of the peri-implantation phase endometrium have been shown to undergo apoptosis (Parr et al., 1987; von Rango et al., 1998). Although apoptosis of EEC is enhanced by transforming growth factor-ß1 (Tanaka et al., 1998a), interleukin-1ß (Tanaka et al., 1998b) and epidermal growth factor (Tanaka et al., 1999) in a Fas-dependent manner, microenvironmental mechanisms that regulate the apoptotic process of these cells during the peri-implantation period remain elusive. Previous studies have established that both NO (Brune et al., 1999) and progesterone (Formby and Wiley, 1998) can induce apoptosis in various cell types of non-uterine origin. Since trophoblasts express eNOS and iNOS (Thomson et al., 1997; Gagioti et al., 2000) and begin to secrete progesterone as early as 4 weeks after conception (Scott et al., 1991), local concentrations of NO and progesterone may be high at the trophoblast–decidual interface. Questions then arise as to whether NO and progesterone at such concentrations can cause apoptosis in EEC and how these two agents interact to induce cell death. Our results demonstrated that NO, as liberated by SNP or SNAP, dose-dependently causes death of cultured EEC. Synergistic enhancement of cell death was observed when progesterone was introduced in combination with SNP or SNAP to the cell culture, as evidenced by increased slopes of the cell survival curves. Treatment of EEC with NO plus progesterone also caused a marked increase in nuclear condensation, as detected by DAPI staining, as well as the activation of caspase-3, a major pro-apoptotic protease, indicating that these cells underwent apoptosis. Exposure to SNP alone activated both ERK and p38 MAPK, but not JNK, over a 24 h period. Pretreatment of cells with an ERK inhibitor, PD98059, did not prevent NO/progesterone-induced cell death, whereas inhibition of p38 MAPK by SB203580 significantly, though not completely, blocked cell death resulting from NO/progesterone treatment. In addition, the NO-induced activation of p38 MAPK was markedly increased in the presence of progesterone. Our data establish that NO and progesterone synergistically induce apoptosis in EEC through mechanisms involving activation of p38 MAPK pathway.
While NO has been shown to be essential for implantation in animal models (Novaro et al., 1997; Biswas et al., 1998), there have been few studies on the mechanistic interaction of NO in this process. To the best of our knowledge, our study demonstrates for the first time that NO can induce apoptosis of EEC, especially in the presence of a high concentration of progesterone. We propose that the high concentration of NO at the trophoblast–decidual interface may initiate apoptotic death of EEC to give way to the invading trophoblasts. As soon as the trophoblasts start secreting progesterone, NO-induced apoptosis is markedly augmented to accommodate the increasing number of invading trophoblasts. This may account for the synergistic effect of NO and progesterone on implantation observed in a previous study where treatment of rats during the peri-implantation period with NOS inhibitor and antiprogestin in combination prevented pregnancy in a much higher percentage of dams than did single agent treatment (Chwalisz et al., 1999).
It has been shown that MAPK play pivotal roles in the signal transduction of apoptosis induced by various agents (Cross et al., 2000). In different cell types, however, specific MAPK pathways may be preferentially utilized after stimulation by NO. For example, exposure of human leukaemia cells (HL-60) to a NO-releasing agent results in apoptosis with the activation of p38 MAPK but not JNK or ERK (Jun et al., 1999). In contrast, NO activates JNK in human embryonic kidney (HEK293) cells (Kim et al., 1997) and ERK in rat mesangial cells (Callsen et al., 1998). Thus far there has been no report on the activation of MAPK by NO in EEC. Our data showed that NO could activate ERK and slightly activate p38 MAPK in a biphasic manner, whereas no effect on JNK was observed. In addition, progesterone was shown to significantly increase the SNP-stimulated p38 MAPK activation, suggesting that both NO and progesterone signal through the p38 MAPK pathway. Our data may also explain the synergistic role of NO and progesterone in the induction of apoptosis in EEC. The anti-phospho-p38 antibody used in this study was generated by immunization of rabbits with synthetic peptides containing phosphorylated Thr(180) and Tyr(182) based on the human p38 MAPK sequence (Cell Signaling Technology). This antibody detected two bands of phospho-p38 on electrophoresed EEC lysates, probably representing two of the four p38 MAPK isoforms (, ß, and ) reported in the literature (Wang et al., 1997; Herlaar and Brown, 1999). The density of the upper band of phospho-p38 MAPK slightly increased after SNP treatment and was more significantly enhanced by the combination of SNP and progesterone. The role of p38 MAPK in the signalling for NO/progesterone-induced EEC death was further established by the fact that inhibition of p38 MAPK by SB203580 could significantly, though not completely, prevent cells from dying. The present study focuses mainly on the modulation of the NO signalling pathway by progesterone. Further studies are needed to investigate whether NO can regulate the biochemical effects of progesterone, such as changes in numbers of oestrogen and progesterone receptors. Numerous studies have indicated that activation of various classes of MAPK results in distinct cell fates. For instance, whereas activation of p38 MAPK leads to apoptosis, activation of ERK can promote cell survival (Herlaar and Brown, 1999; Chuang et al., 2000; Deng et al., 2000). Our data showed that both p38 and ERK were activated by SNP/progesterone; however, only inhibition of p38 by SB203580 rescued the cells. This suggests that p38 MAPK is preferentially utilized in the NO-induced death signalling pathways. Activation of ERK may be a secondary event to provide self-protection against NO. When this self-protection mechanism was blocked by PD98059, NO/progesterone-induced cell death appeared to increase to a greater extent, though the result was not statistically significant.
Concentrations of nitrite, a stable metabolite of NO that is released from SNP, were 15.8 ± 1.7 and 22.2 ± 2.1 µmol/l in our samples receiving 24 h treatment with 0.5 mmol/l and 1 mmol/l SNP respectively (n = 3
). The average nitrite concentration of blood samples taken from the uterine vein draining the placental site is ~22 µmol/l (Norris et al., 1999), which approximates the nitrite concentration accumulated 24 h after treatment with 1 mmol/l SNP. The concentration of progesterone during pregnancy is ~12 times higher in the placenta than in maternal plasma (Khan-Dawood and Dawood, 1984) and the tissue concentration of progesterone in first trimester placenta is as high as 8–16 µmol/l (Runnebaum et al., 1975; Laatikainen et al., 1982). In this study, the concentrations of NO and progesterone used correspond to the possible concentrations in the embryonic milieu, raising the likelihood that NO and progesterone may have roles in the manifestation of apoptosis as observed in prior studies (Parr et al., 1987; von Rango et al., 1998). The fact that the effect of progesterone to enhance NO-mediated apoptosis was more significant after 72 h than 24 h of treatment implies that a prolonged period of exposure to NO and progesterone may recruit more cellular elements for overt activation of apoptosis. In agreement with this argument, we found that p38 MAPK was not activated 10 min after combined NO and progesterone treatment (data not shown); rather, it was significantly activated 24 h later. The delayed effect of progesterone on p38 MAPK augmentation can be further reconciled by the finding that treatment of breast cancer cells with progestin for 48 h enhances the activation of p38 MAPK by epidermal growth factor (Lange et al., 1998).
In summary, this study established that NO can induce apoptosis in EEC, and that progesterone can synergistically enhance the apoptotic susceptibility to NO. Signalling for apoptosis induced by NO/progesterone appears to involve the p38 MAPK pathway. We propose that the high concentrations of NO and progesterone at the trophoblast–decidual interface may contribute to apoptosis in glandular epithelial cells of endometrium and facilitate trophoblast invasion. The relevance of NO/progesterone-induced apoptosis in EEC to implantation deserves further research.
Acknowledgements
The authors thank Dr Yen-Chang Chen at the Center for General Education, Biology Division, National Yang-Ming University for his technical assistance. This work has been supported, in part, by grants from the National Science Council, Taiwan (NSC89-2320-B-010-144), and the Veterans General Hospital, Taipei (VTY89-P5-43).
Notes
4 To whom correspondence should be addressed at: Institute of Anatomy and Cell Biology, National Yang-Ming University, 155 Section 2, Li-Nong Street, Taipei, Taiwan 112. E-mail: yjsung{at}ym.edu.tw 
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Submitted on November 27, 2000; accepted on June 12, 2001.
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