Molecular Human Reproduction, Vol. 7, No. 9, 867-874,
September 2001
© 2001 European Society of Human Reproduction and Embryology
Uterine physiology |
The Fas/FasL apoptotic pathway is involved in 
-opioid-induced apoptosis of human endometrial stromal cells
1 Departments of Pharmacology and 2 Clinical Chemistry, School of Medicine, University of Crete, Heraklion GR-711 10, Crete, Greece
Abstract
Human endometrium expresses specific 
-opioid binding sites and their endogenous ligands, the dynorphins. In neural crest-derived tissues, -opioids affect apoptosis, a phenomenon of major significance in endometrial stroma physiology. Our hypothesis was that endometrial -opioids may play a role in endometrial stromal cell apoptosis. Thus, we examined the effect of the synthetic -opioid agonist, U69593, on the apoptotic rate of human endometrial stromal cells in primary culture. Apoptosis of endometrial stromal cells was elevated after 3 h exposure to 100 nmol/l U69593, and remained elevated for up to 3 days. This effect was dose-dependent and was reversed by the general opioid antagonist, naloxone, suggesting that it is mediated via opioid receptors. In parallel, semi-quantitative Western blot and flow cytometry analysis showed that U69593 caused a rapid but transient up-regulation of Fas protein, suggesting that its effect on apoptosis is mediated by activation of the Fas/FasL apoptotic pathway. Additionally, U69593 increased the content of the anti-apoptotic members of the Bcl-2 family of proteins, the Bcl-2 and Bcl-xL, whereas it had no significant effect on the apoptosis-promoting homologues Bax, Bcl-xS and Bak. This implies that a transient survival mechanism is activated in stromal cells as a parallel rescue response to the apoptosis-inducing factor. In conclusion, our data suggest that endometrial opioid dynorphins may participate in the apoptotic processes related to endometrial tissue remodelling during early pregnancy or menstruation.
apoptosis/Bcl-2/endometrium/Fas/FasL/-opioids
Introduction
Human endometrium expresses specific -opioid binding sites (Hatzoglou et al., 1995
) and produces their main endogenous ligand, the opioid dynorphin (Chatzaki et al., 2000). Therefore, endogenous -opioids may play a paracrine role within the endometrial tissue. However, the biological place of -opioids in the endometrial physiology has not yet been determined.
A growing number of studies report a role of opioid peptides in regulating the apoptotic machinery. Thus, µ- and 
-opioids induce apoptosis in several tissues, within hours of their administration (Fuchs and Pruett, 1993; Maneckjee and Minna, 1994; Ientile et al., 1997; Kugawa et al., 1998; Singhal et al., 1998a,b; Sueoka et al., 1998). -Opioids have also been implicated in the apoptotic sensitivity of neuronal sites. Indeed, pre-treatment with the selective -agonist, U69593, has been shown to enhance staurosporine- and wortmannin-induced apoptosis in primary neuronal cultures of chick embryonic cerebral hemispheres and of the F-117 cell line, an immortalized mouse neuroblastomaxdorsal root ganglion hybrid stably transfected to overexpress -opioid receptors (Dawson et al., 1997; Goswami et al., 1998). In contrast, in a peripheral system, the rat pheochromocytoma cell line, PC12, -opioid agonists can transiently prevent activation of apoptotic mechanisms following short periods of serum withdrawal (Dermitzaki et al., 2000).
Apoptosis is a phenomenon of major significance in endometrial physiology, regulating the remodelling of this tissue during implantation or menstruation. Expression of apoptosis regulatory molecules is reported in human endometrial stroma and they are believed to fine-tune this process. The Fas antigen, a member of tumour necrosis factor receptor (TNFR) family and a type-I membrane protein (Itoh et al., 1991), induces apoptosis via cross-linking to Fas ligand (FasL) (Otsuki et al., 1994; Yamashita et al., 1999). Members of the Bcl-2 family, such as the apoptosis-inhibiting proteins, Bcl-2 and Bcl-xL, and their apoptosis-promoting homologues, Bax and Bak, have also been found in human endometrium (Gompel et al., 1994; Otsuki et al., 1994; Tabibzadeh et al., 1995; Tao et al., 1997, 1998).
The aim of the present study was to examine the role of -opioids in the apoptosis machinery of endometrial stroma. To achieve this aim, we have used purified primary cultures of human endometrial stromal cells that were exposed to the synthetic selective -opioid receptor agonist, U69593. Apoptosis was estimated by measuring nucleosomal DNA as an index of DNA fragmentation. In parallel, the effect of U69593 on the cellular content of the proteins of the Fas and Bcl-2 apoptotic systems was examined using semi-quantitative Western blot and flow cytometry analysis.
Materials and methods
Primary culture of isolated stromal cells from human endometrium
Endometrial specimens were obtained from women of reproductive age undergoing diagnostic curettage for fertility evaluation or hysterectomy for benign disease. Informed consent was obtained and the study was approved by the Ethics Committee of the University of Crete, School of Medicine. The tissues used for the experiments were derived from women with no histological signs of endometriosis or malignancy and from the early secretory stage of the cycle, as judged by conventional pathological examination. Purified stromal primary endometrial cell cultures were obtained as described previously (Chatzaki et al., 1994). Briefly, tissues were collected in Dulbecco's modified Eagle's medium (Gibco BRL, Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum and 1% antibiotic/antimycotic solution, trimmed and minced mechanically and digested for 90 min at 37°C using 0.25% of Type I collagenase (Sigma Chemicals Co., St Louis, MO, USA). Separation of the stromal fraction from the epithelial fraction was carried out by filtration through a 45 µm stainless steel sieve permitting passage of stromal but not epithelial cell suspension. Stromal cells were then plated in 24-well plates or 25 cm2 flasks (Corning Inc., NY, USA) with 1 ml or 5 ml of culture medium respectively and incubated in a humidified atmosphere of 5% CO2 at 37°C in the above-mentioned medium for at least 1 week before experimentation. The conditions of endometrial cell separation have been previously validated (Satyaswaroop et al., 1979; Chatzaki et al., 1994), allowing highly purified stromal cell cultures containing <5% of epithelial cells or macrophages, as judged by immunocytochemistry and fluorescence-activated cell sorting (FACS) analysis using cytokeratin as a marker for epithelial cells and 3C10 as a marker for macrophages.
Experiments were performed in primary stromal cell cultures at 100% confluency. The specific 1-opioid agonist, U69593, or the general opioid antagonist, naloxone (Upjohn Co., Kalamazoo, MI, USA), were diluted in Phenol red and serum-free Roswell Park Memorial Institute 1640 medium (Biochrom Co., Berlin, Germany) supplemented with 0.25% bovine serum albumin fraction V (Sigma), insulin from bovine pancreas (5 mg/l), transferin (5 mg/l) and sodium selenite (5 ng/l) (ITS cell culture supplement; Sigma), 2 mmol/l L-glutamine and 1% antibiotic/antimycotic solution. The dose of the compounds was replaced daily.
Quantitative measurement of apoptotic index
Apoptosis was quantified by direct determination of nucleosomal DNA fragmentation with the `cell death detection' enzyme-linked immunosorbent assay plus kit (Boehringer Mannheim GmbH, Mannheim, Germany). The assay used specific monoclonal antibodies directed against histones from fragmented DNA, allowing the determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. Briefly, after treatment, stromal cells in 24-well plates were centrifuged (200 g) and lysed according to the manufacturer's manual. Cell lysates were centrifuged again (200 g). The monoand oligonucleosomes contained in the supernatants were determined using an anti-histone–biotin antibody. The concentration of nucleosomes-antibody was determined photometrically using 2,2'-azino-di(3-ethylbenzthiazolin-sulphonate) as substrate. The optical density was read on a Dynatech MicroElisa reader (Chantilly, VA, USA) at a wavelength of 405 nm. The data are expressed in photometric units. Each unit corresponds to ~12 500 apoptotic cells, and the detection limit is 625 apoptotic cells. At the beginning of the experiments, cells had reached 100% confluency, therefore proliferation was considered minimal.
Semi-quantitative Western blot analysis
Following treatment, cells in flasks were washed twice with phosphate-buffered saline (PBS), removed by scraping and centrifuged at 800 g. Cell lysis was completed at 4°C by vigorous shaking of the pellet for 30 min reconstituted in a lysis buffer composed of 50 mmol/l Tris–HCl pH 8, 150 mmol/l NaCl, 0.1% sodium dodecyl sulphate (SDS), 0.5% sodium deoxycholate, 1% NP-40 and freshly added protein inhibitors 10 µg/ml PMSF and 1 µg/ml aprotinin. Solid cellular debris was removed by centrifugation at 12 000 g for 15 min. The cytoplasmic fractions were collected and stored at –80°C. Protein concentration was measured by the Bio-Rad Protein Assay Kit II (Bio-Rad Laboratories, Hertfordshire, UK) following the instructions of the manufacturer. Equal protein samples (20 µg) were solubilized with SDS polyacrylamide gel electrophoresis sample buffer and electrophoresed through a 12% SDS gel. The resulting protein bands were transferred to nitrocellulose membranes, using an LKB electroblot apparatus (LKB, Bromma, Sweden). Standard Western blotting procedures were employed. Nitrocellulose membranes were subjected to incubation with the first antibody diluted in 1% non-fat milk, followed by incubation with the appropriate secondary antibody, and use of the ECL Western blotting kit (ECL Amersham Corp., Amersham, UK). The membranes were then exposed to Kodak X-omat AR films. Band intensities were quantified by a PC-based Image Analysis System (Image Analysis Inc., Ontario, Canada). The following antibodies were used: as primary antibodies, anti-human Bcl-2 monoclonal antibody (1:200, clone 124; Dako A/S, Denmark), the rabbit polyclonal anti-sera against Bax, Bak, Bcl-xS/L and Bad (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), the anti-Fas (1:2500) and anti-FasL (1:1000; Transduction Laboratories, Lexington, KY, USA) antibodies and, as secondary antibodies, goat peroxidase conjugated anti-mouse IgG (1:10 000; Chemicon International Inc., CA, USA) or anti-rabbit IgG (1:4000; Immunotech, Marseille, France). For purposes of normalization, the membranes were also stained with a monoclonal anti-actin antibody in a dilution of 1:400 (Amersham). Protein content data were also analysed after normalization per total cellular protein, but there was no significant difference compared to the presented data normalized per total actin.
Flow cytometric analysis of human endometrial stromal cells
Flow cytometry was performed as previously described (Makrigiannakis et al., 1999). The isolated human stromal cells were washed in PBS with 0.1% bovine serum albumin (BSA; Sigma), precipitated by centrifugation and incubated with the anti-FasL or anti-Fas antibodies described above, at 4°C for 1 h. After washing with PBS, cells were incubated with anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary IgG (Sigma), at 4°C in the dark for 30 min. After washing in PBS, the cells were analysed using an Epics XL flow cytometer (Coulter Corporation, Hialeah, FL, USA). Negative controls included pre-absorption of the primary antibody with FasL or Fas (Santa Cruz Biotechnology, Inc.) or substitution of the anti-FasL or anti-Fas primary antibody with non-specific isotype-matched antibodies at the same concentrations.
Indirect immunofluorescence
Immunofluorescent staining was performed as described previously (Chatzaki et al., 2000). In brief, cells were grown on 22x22 mm coverslips were fixed and permeabilized by exposure to 4% formaldehyde for 10 min followed by exposure to 0.2% Triton for 10 min. The cells were then incubated overnight at 4°C with the primary antibody at a dilution of 1:100. Parallel negative controls were incubated with non-specific IgG. After washing, the FITC-conjugated anti-rabbit IgG was added (1:150; Sigma) for 1 h at room temperature. Specimens were visualized in a Zeiss Axioscope microscope and photographed using Kodak film. The primary antibodies used were those described above for the Western blot.
Statistical analysis
All apoptosis quantification experiments were performed in triplicates and in at least three stromal cultures from different biopsies (n denotes the number of different cultures). Results are presented as the means of these independent experiments expressed as percentage changes of parallel non-treated controls. In semi-quantitative Western blotting experiments, the concentration of the antigen was normalized per actin (Dermitzaki et al., 2000). Each treatment was done in duplicate and the mean value was used for further statistical analysis. The FACS and Western experiments were repeated at least three times using cultures from different biopsies. For statistical evaluation of data we used ANOVA, post-hoc comparison of means followed by non-parametric Kruskal–Wallis test for the evaluation of percentage changes for the n independent experiments.
Results
Effect of -opioid agonist U69593 on apoptosis of endometrial stromal cells in vitro
The primary cultures of purified human endometrial stromal cells were exposed to the specific -opioid agonist U69593 in concentrations ranging from 0.001 to 1000 nmol/l for a period of 3 days and the rate of apoptosis was quantified as described in the Materials and methods. At 100 nmol/l, U69593 caused an acute increase in the rate of endometrial stromal cell apoptosis that was 178 ± 32% of that for non-treated controls (n = 4, P < 0.05) after 3 h of exposure. The rate of stromal cell apoptosis was still almost doubled at the end of the first day of exposure (180 ± 39% of non-treated controls n = 4, P < 0.05), and remained high for the following 2 days (Figure 1A). The above effect was dose-dependent (Figure 1B) and was observed with U69593 concentrations down to 1 nmol/l, i.e. proximal to the Kd of the agonist for -opioid receptors. In order to examine if this effect was mediated through specific opioid receptors, stromal cell apoptosis was measured after 1 day of exposure to 100 nmol/l U69593 in the presence or absence of a 10-fold excess of the general opioid antagonist naloxone (1 µmol/l). As shown in Figure 1C, the increase in apoptosis caused by U69593 alone was reversed in the presence of naloxone, suggesting action through opioid receptors. Compared with the agonist, naloxone had the opposite effect on stromal cells, causing a statistically significant inhibition of apoptosis (79.8 ± 8.3% of non-treated control, n = 3, P < 0.05). This action is probably attributed to the blockage of the endogenously secreted -opioid peptides, dynorphins (Chatzaki et al., 2000).
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Effect of
-opioid agonists on the Fas/FasL system of endometrial stromal cellsThe effect of
-opioids on the Fas/FasL system of endometrial cells was examined using semi-quantitative Western analysis. Cells were exposed to 100 nmol/l of U69593 for 3 and 24 h and the content of Fas and FasL was estimated in cell homogenates. The duration of exposure was selected based on previous observations (Yin et al., 1999). The results are presented in Figure 2. Blotting with anti-Fas and anti-FasL antibodies revealed bands at 45 and at 37 kDa corresponding to the Fas and FasL proteins respectively. Band density was normalized per actin content. U69593 caused a statistically significant elevation in the Fas content at 3 h (154 ± 17% of non-treated controls, n = 3, P < 0.05). The stimulatory effect of U69593 could not be observed after 24 h (106 ± 11% of non-treated controls, n = 3), suggesting that it was fast but transient. No statistically significant effect was observed on FasL protein content (119 ± 23% and 108 ± 19% of non-treated controls for 3 and 24 h respectively, n = 4).
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The Western blot data on the effect of U69593 on Fas/FasL were confirmed by flow cytometry experiments. Fas and bound FasL can be detected on the cell membrane by specific anti-Fas or anti-FasL antibodies and then quantified by FACS. Purified stromal cell cultures were exposed to 100 nmol/l of U69593 for 3 and 24 h and the content of Fas and FasL was estimated by flow cytometry as described in Materials and methods. The results are presented in Figure 3
. U69593 caused a statistically significant elevation in the Fas protein content at 3 h (223 ± 7% of non-treated controls, n = 4, P < 0.005). This effect was lost at 24 h (125 ± 9% of non-treated controls, n = 4). No statistically significant effect was observed on FasL. These data corroborate the Western analysis results, showing a fast and transient positive effect of U69593 on the Fas content in the membrane of the stromal cells.
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Effect of
-opioid agonists on the Bcl-2 family of proteins of endometrial stromal cellsThe expression of the members of the Bcl-2 family of proteins was examined in vitro in purified cultures of endometrial stromal cells by immunofluorescence. Cells on coverslips were stained with primary antisera raised against Bcl-2, Bcl-xS/L, Bak, Bax and Bad and the immunofluorescence results are presented in Figure 4
. Positive staining was observed for all but the Bad proteins, in contrast with the negative control (cells treated without primary antibody). Bcl-2, Bcl-xS/L, Bak and Bax protein expression was localized in the cytoplasm. With the exception of Bcl-2, the proteins were also present in the nuclei of the cells. Our results show that endometrial stromal cells express all the members of the Bcl-2 family of proteins, except Bad, in vitro.
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The effect of the
-opioid agonist U69593 on the cellular content of those proteins was tested in primary cultures of isolated stromal cells exposed to 100 nmol/l of U69593 in parallel with non-treated controls. Cells were lysed after 3 and 24 h treatment. The duration of treatment was chosen based on previously published observations (Dermitzaki et al., 2000). Cell homogenates were subjected to semi-quantitative Western blot analysis (Figure 5), using antibodies against Bcl-2, Bcl-xS/L, Bax, Bak and Bad. In agreement with the immunofluorescence findings, all antibodies revealed a band of the predicted size for the respective protein (Figure 5A and C) except the anti-Bad antiserum that showed negative staining (not shown). Data were corrected for protein content by staining with an anti-actin antibody. After quantification, it was shown that U69593 stimulated the production of the anti-apoptotic proteins Bcl-2 (247 ± 47% and 148 ± 16% of non-treated controls, n = 4, P < 0.05) and Bcl-xL (156 ± 20% and 186 ± 21% of non-treated controls, n = 4, P < 0.05) at 3 and 24 h respectively (Figure 5B), whereas there was no statistically significant effect on the apoptosis-inducing homologues of the Bcl-2 family, Bax, Bcl-xS and Bak (Figure 5D).
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Discussion
Apoptosis is an important function of human endometrium, being involved in the process of decidualization and blastocyst implantation. Decidualization in the immediate vicinity of the implanting blastocyst involves the entire endometrial stroma. After a period of intense growth and differentiation, the antimesometrial decidua in the area adjacent to the trophoblast begins to degenerate by a process that has been characterized morphologically as apoptosis (Welsh and Enders, 1985). Progression of apoptosis through the antimesometrial decidua enables trophoblast giant cells to gain access to maternal vessels. The controlled cell death of large numbers of decidual cells in the antimesometrial decidua allows remodelling of the implantation chamber without disrupting the growth and development of the embryo or the integrity of the tissue (Pampfer and Donnay, 1999). Apoptotic cell death in specific regions of the decidualized endometrium with or without the presence of trophoblast cells suggests that uterine cells control apoptosis by paracrine and/or autocrine mechanisms. However, the exact molecular events of these processes remain largely unknown. Our findings suggest that locally secreted -opioid peptides may take part as paracrine modulators in the regulation of endometrial stromal apoptosis, being possibly involved in major phenomena of endometrial physiology, such as decidualization and implantation or menstruation.
Human endometrium produces opioid peptides and expresses their receptors, suggesting that these compounds serve as local autocrine/paracrine factors. Transcripts of endogenous opioid precursors, proopiomelanocortin and proenkephalin and their peptide end products have been reported in human uterus (Wahlstrom, 1985; Petraglia et al., 1986; Douglass et al., 1987; Makrigiannakis et al., 1992) in parallel with multiple types of opioid binding sites (Hatzoglou et al., 1995). The abundance and low affinity (Kd, 10.8 nmol/l) of -type opioid binding sites suggests the local expression and physiological function of the main endogenous ligands, the dynorphins. Indeed, we have recently shown expression of the prodynorphin gene as well as immunoreactive dynorphins by human endometrium (Chatzaki et al., 2000). The main source of endometrial -opioids was shown to be the epithelial cell fraction of the tissue, i.e. the glandular and luminal cells. In the present study, we report a distinct paracrine biological action of these agents in the endometrium, the induction of the apoptotic machinery in endometrial stromal cells. Using purified primary cultures of human endometrial stromal cells, we showed that a synthetic -opioid agonist (U69593) caused an increase in the apoptotic rate of these cells. This effect was immediate, since it could be observed after only 3 h of exposure, but was also still prominent for a period of 3 days. Induction of apoptosis by U69593 was dose dependent and could be detected using concentrations as low as 1 nmol/l, proximal to the Kd of -opioid receptors. Furthermore, the effect was reversed by the general opioid receptor antagonist, naloxone, confirming that it was an opioid receptor-mediated action. Interestingly, naloxone alone caused a reduction of the apoptotic rate in endometrial stromal primary cultures, probably by blocking the inducing effect of the endogenously secreted dynorphins.
-Opioid agonists have been previously shown to stimulate apoptosis in neuronal cells. Specifically, pre-treatment with the -agonist, U69593, enhances staurosporine- and wortmannin-induced apoptosis in primary neuronal cultures from embryonic cerebral hemispheres of chick brain (E7CH) and the F-117 cell line, an immortalized mouse neuroblastomaxdorsal root ganglion hybrid stably transfected to overexpress -opioid receptors (Dawson et al., 1997; Goswami et al., 1998). However, in both studies U69593 used as a single agent had no effect on non-induced DNA fragmentation. We have previously shown that, on the PC12 rat pheochromocytoma cell line, U69593 exerts an anti-apoptotic action which is rapid in onset and transient, beginning at 3 h and lasting no more than 5 h (Dermitzaki et al., 2000). We now report that U69593 directly induces apoptosis in a non-neuronal tissue via opioid receptor activation. Our results lead to the conclusion that the effects of -opioids on cellular apoptosis are tissue-specific and are likely to participate in different physiological phenomena through activation of alternative apoptotic pathways.
µ- and -opioid peptides induce apoptosis in several non-neuronal crest-deriving tissues, similar to our findings, in an acute manner. Thus, morphine, a µ-opioid receptor agonist, increases apoptosis in a human lung cancer cell line within 2 h of treatment in a dose-dependent and naloxone-reversible manner (Maneckjee and Minna, 1994), in murine thymocytes (Fuchs and Pruett, 1993) and macrophage and kidney fibroblasts (Singhal et al., 1998a,b). Furthermore, morphine alkaloid buprenorphine induced apoptosis in NG108-15 cells within 4 h (Kugawa et al., 1998). Similarly, ß-endorphin, an endogenous opioid with affinity towards µ- and -opioid receptors, induced apoptosis in T-lymphocytes (Ientile et al., 1997). It is possible that the changes in the fragmentation rate of DNA within a few hours of exposure may involve activation of stored apoptotic intracellular agents followed by transcriptional regulation of specific genes responsible for the more long-lasting effects.
The Fas/FasL system represents a major pathway for the induction of apoptosis. Upon binding to its ligand, Fas delivers an apoptotic signal that induces cell death (Nagata and Goldstein, 1995). Fas expression has been previously reported in human endometrium (Watanabe et al., 1997). Fas and its ligand are present in endometrial biopsies from pre-menopausal non-pregnant women throughout the menstrual cycle (Yamashita et al., 1999). Our data confirm these observations documenting the expression of Fas/FasL system in human endometrial stromal cells in vitro. The induction of apoptosis by -opioids is followed by an acute and transient increase in the content of Fas, implying activation of the Fas system.
An opioid-induced increase of Fas has been previously documented in a T-cell hybridoma, in mouse splenocytes and in human peripheral lymphocytes (Yin et al., 1999). The response of these immune cells to morphine was also fast, beginning at 2 h. In our experiments, Fas response to -opioids was equally fast, starting at 3 h, but it became undetectable at 24 h. A similar immediate and transient effect has been described for another inducer of endometrial apoptosis in the rat, TGF-ßL increasing FasL expression in stromal cells after 3 h administration, an effect not detectable at 6 h (Molulton, 1994; Garcia-Velasco et al., 1999). Such a fast response of Fas protein to opioids suggests an effect at a post-transcriptional level rather than one at the level of gene expression requiring more lengthy molecular events. The exact molecular mechanism of Fas regulation by opioids is currently under investigation.
Increased numbers of Fas-expressing cells in the stroma would lead to a higher rate of programmed cell death due to activation of the Fas/FasL apoptotic pathway by the stromal-derived ligand but also by ligand expressed by other endometrial cells that are sources of FasL in uterus, such as glandular epithelial cells (Yamashita et al., 1999). Thus, in vivo the Fas/FasL system may contribute not only to self-regulation of apoptosis in the stroma but also to a paracrine stimulus. Following exposure to dynorphins, mainly secreted by endometrial epithelium, the Fas/FasL system may undertake mediation of apoptosis induction as an early response mechanism of the stromal cells. After that, activation of the Fas/FasL system may no longer be necessary for the further apoptotic actions that could require activation of other apoptosis-related genes, representing a long-term mechanism of action. The identification of the molecules involved in this mechanism needs further investigation.
The expression of the Bcl-2 related proteins has a cyclic variability in human endometrium (Gompel et al., 1994; Otsuki et al., 1994; Pecci et al., 1997; Tao et al., 1997, 1998). It has been suggested that coordinated shifts in the balance between the anti-apoptotic (Bcl-2 and Bcl-xL) and the pro-apoptotic (Bax, Bak and Bcl-xS) homologues are responsible for the cyclic changes of endometrial growth and regression. Most of these studies are based on immunohistochemical analysis and show localization of the Bcl-2-related antigens in endometrial epithelium. Our experiments using immunofluorescence and Western blotting analysis indicate that Bcl-2, Bcl-xL, Bcl-xS, Bax and Bak are also expressed in the stromal compartment of human endometrium. Our findings suggest that the Bcl-2 family of proteins also plays a role in stromal apoptosis that is closely related to the process of decidualization and thus preparation of a receptive endometrium to fetus implantation (Akcali et al., 1996). Furthermore, we showed that -opioids, in parallel with their apoptosis-inducing action in the stroma, participate in the regulation of these proteins, causing an elevation in the protein content of the apoptosis-preventing homologue, Bcl-2. It is interesting that other apoptosis-inducing substances, such as rapamycin, up-regulate anti-apoptotic Bcl-2 proteins in human medullary thyroid carcinoma cell lines (Pfrangner et al., 2000). It is possible that a temporal Bcl-2-related mechanism is activated to counteract the stress signals generated by the apoptosis-inducing factors in order to rescue the cells from programmed death. A similar phenomenon has also been described in PC12 cells, where prevention of apoptosis caused by -opioids is accompanied by decreased Bcl-2 protein content (Dermitzaki et al., 2000).
In conclusion, our data indicate that -opioids induce apoptosis of human endometrial stromal cells via a mechanism that at least in part involves regulation of the Fas/FasL system. These findings suggest that endogenous endometrial dynorphins secreted mainly by the glandular and luminal epithelial cells participate in the control of programmed cell death of the proximal stroma, a process that is closely related to the differentiation of the latter cell type to decidua. Thus, dynorphins may be considered as molecular modulators of the endometrial microenvironment, contributing to the complex paracrine network of cross-talk between endometrial epithelium and stroma, controlling tissue remodelling during early pregnancy or menstruation.
Acknowledgements
We would like to thank Dr Chris Tsatsanis for his very helpful and constructive remarks. This work was supported by the EKBAN 99-66 grant from the European Union.
Notes
3 To whom correspondence should be addressed. E-mail: gravanis{at}med.uoc.gr 
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Submitted on March 19, 2001; accepted on July 19, 2001.
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