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INSERM,3 U767, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, 75006 Paris, France
Maternité Port-Royal,4 Hôpital Cochin, Université René Descartes, 75014 Paris, France
ABSTRACT
The present study investigated the ability of human choriodecidua to induce myometrial cell apoptosis through the secretion of tumor necrosis factor alpha (TNF). The secretion of TNF was evaluated in the culture supernatants of amnion and choriodecidua explants that were exposed to the bacterial endotoxin lipopolysaccharide (LPS) to mimic inflammation. The choriodecidua explants produced more TNF than the amnion explants in response to LPS stimulation, despite the fact that the choriodecidua had lower levels of TLR4 expression. Moreover, conditioned medium obtained from LPS-treated choriodecidua explants, but not that from amnion explants, decreased the number of viable cultured myometrial cells and induced cell apoptosis by inducing the overexpression of the proapoptotic protein BAX and by decreasing the expression of the anti-apoptotic protein BCL2. Neutralization of TNF in the choriodecidua-conditioned medium reversed this effect. Exogenous TNF mimicked LPS-treated choriodecidua-conditioned medium in that it induced myometrial cell apoptosis, reduced BCL2 expression, and increased BAX expression. Using neutralizing antibodies against both subtypes of TNF receptors, we found that only TNFRSF1A participates in TNF-induced myometrial cell apoptosis. Our in vitro model of LPS-induced inflammation of human fetal membrane explants suggests a mechanism by which TNF secreted by choriodecidua governs human myometrial cell apoptosis at the end of pregnancy. These data support the hypothesis that TNF participates in the complex network of signaling processes associated with uterine involution.
apoptosis, cytokines, placenta, pregnancy, uterus
The physiology and pathology of human labor are far from being fully understood owing to the multiplicity of hormonal, mechanical, genetic, and environmental factors involved. The quiescence of the uterine smooth muscle, the myometrium, is essential for the support and accommodation of fetal growth, whereas the onset of regular strong contractions is the essence of parturition. Accumulated data indicate that human parturition is associated with an inflammatory process. Indeed, leukocytes (predominantly neutrophils and macrophages) infiltrate uterine tissues at the end of pregnancy. Furthermore, the levels of proinflammatory cytokines, such as interleukin-1beta (IL1B), IL6, IL8, and tumor necrosis factor alpha (TNF), increase within the tissues of the laboring human uterus in a feed-forward phenomenon that ultimately leads to the expulsion of the fetus [1]. The cytokine-mediated inflammatory process is directly involved in the release of uterotonic agents. A classic example is the upregulation of cyclooxygenase-2 (also known as prostaglandin-endoperoxide synthase 2, PGTGS2) [2] and prostaglandin production, which have been shown to induce cervical ripening [3], myometrial contractions [4, 5], and rupture of the fetal membranes [6], leading to delivery and birth [7].
Recently, a novel concept regarding the mechanisms that govern parturition has emerged from the functional genomic study of Charpigny et al. [8]. These authors have described differential gene expression in the preterm and term human myometrium, and they have reported that parturition is characterized by massive downregulation of a large panel of developmental, cell adhesion molecule, and proliferation-related genes, along with the upregulation of inflammatory, contraction-associated, and apoptosis-associated genes. Several lines of evidence support this new concept. For instance, an increase in apoptosis has been reported in the rat uterus during late pregnancy [9]. Mechanical stretching induced by the growing fetus has been proposed to explain uterine growth in terms of a combination of hyperplasia and hypertrophy in the pregnant rat [10]. More recently, Shynlova et al. [11] have described two distinct stages of rat uterine growth: an early stage characterized by hyperplasia associated with the increased expression of antiapoptotic proteins, which is followed by a hypertrophic stage later in gestation. The transition between these two stages is associated with transient activation of a caspase cascade, which allows the differentiation of myometrial cells. Although the proliferation of smooth muscle cells decreases as the uterus nears parturition, the extent of apoptosis of human myometrial cells during the last trimester of pregnancy remains unknown.
The implication of TNF in the normal process of human pregnancy has been extensively documented [12]. TNF is a pleiotropic cytokine that regulates multiple cellular responses, including inflammation and cell survival [13]. TNF action begins with binding to one of the two cell surface receptors, p55/p60 (Type 1, TNFRS1A, abbreviated as TNFR1) or p75/p80 (Type 2, TNFRS1B, abbreviated as TNFR2). An important difference between these receptors is that the conserved TNF receptor family death domain is limited to TNFR1 (reviewed in [14]). Briefly, the binding of TNF to TNFR1 results in receptor trimerization and recruitment of a series of intracellular proteins that bind caspase-8 (also known as CASP8), leading to its activation. Activated CASP8 initiates a proteolytic cascade that leads to activation of executioner caspases, typically CASP3. This activation cleaves multiple cellular proteins, resulting ultimately in DNA fragmentation, which is a characteristic feature of apoptotic cell death.
Studies of the mechanism of apoptotic cell death induced by cytokines in the uterus have focused primarily on endometrial epithelial and stromal cells, endometrial adenocarcinoma cell lines [15–17], and cells cultured from human leiomyoma and matched myometrium [18, 19]. Since the labor regulatory process may involve cross-talk between the maternal and fetal compartments, we explored the hypothesis that the local actions of inflammatory signaling factors, which originate in the fetal membranes, may control myometrial functions. Recent studies have documented fetal membranes as potential sources of paracrine regulators of myometrial contractions [20, 21]. Whether the cytokines that originate in the fetal membranes induce apoptosis in pregnant human myometrial cells remains unclear.
In the present study, we first examined whether TNF released from human fetal membranes challenged with the bacterial endotoxin lipopolysaccharide (LPS) contributes to the apoptosis of cultured pregnant human myometrial cells. We then investigated how exogenous TNF induces myometrial cell apoptosis, and we determined the TNF receptor subtype involved in this process.
Dulbecco modified Eagle medium (DMEM), trypsin-EDTA, penicillin-streptomycin mixture, PBS with or without calcium, and fetal calf serum (FCS) were obtained from Invitrogen Life Technologies (Cergy-Pontoise, France). Lipopolysaccharide from Escherichia coli 127:B8 was obtained from Sigma Aldrich (St Louis, MO). TNF was from Biosource-Europe (Clinisciences, Montrouge, France). Electrophoresis reagents were obtained from Bio-Rad (Richmond, CA). Hybond-P membranes, the enhanced chemiluminescence detection system (ECL), and x-ray film were obtained from Amersham International (Chalfont, Buckinghamshire, UK).
Antibodies against BAX, BCL2, ACTB (also known as beta-actin) and Toll-like receptor 4 (TLR4) were purchased from Santa Cruz Biotechnology (Tebu-Bio, Le Perray en Yvelines, France) and the antibody against cleaved CASP3 was from Ozyme (Saint Quentin en Yvelines, France). The anti-human TNFR1, TNFR2, and TNF antibodies were obtained from R&D Systems Europe (Lille, France). Secondary antibodies conjugated to horseradish peroxidase were from Amersham International. The other drugs and chemicals used were of the highest quality available from Sigma-Aldrich.
Placentas with attached fetal membranes from term, normal, uncomplicated, singleton pregnancies were collected within 30 min of elective cesarean section at the Port-Royal Cochin Hospital (Paris, France). Fetal membranes were dissected free of the placenta under sterile conditions and washed with PBS to eliminate blood clots. Biopsies of the myometrium were obtained from pregnant women who delivered by elective cesarean section performed before the onset of labor (range 38.5–40 wk of gestation) because of diagnosed cephalopelvic disproportion. Myometrial strips were taken from the upper edge of the hysterotomy in the transverse lower uterine segment during caesarean section. Myometrial tissue from the outer uterine wall at an extraplacental site was removed by sharp dissection, leaving behind the decidua. The tissue was then carefully minced with fine scissors before rapid placement in culture medium. Patient consent and ethical approval were obtained in accordance with the Comité Consultatif de Protection des Personnes pour la Recherche Biomédicale (Paris-Cochin, France).
The amnion and choriodecidua were manually separated and cut into 1-cm squares. Two pieces of tissue per well were placed in 24-well plates that contained 1 ml of culture medium (DMEM supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin) for determinations of TNF release. Ten explants per well were pooled and distributed into 6-well plates that contained 2 ml culture medium, for the collection of amnion- and choriodecidua-conditioned media. The explants were incubated in 5% CO2/95% air at 37°C for 48 h to allow them to stabilize, as described previously [22, 23]. The explants were then treated with the indicated concentrations of LPS for 24 h in serum-free medium. At the end of the incubation period, the culture media were either stored at –80°C or used freshly as amnion-conditioned and choriodecidua-conditioned media. Explant tissue wet weights (mg/well) were recorded at the end of the experiment for normalization purposes. The cell viability of the explants, which was checked at the end of the experiments by trypan blue exclusion after trypsin and collagenase digestion of the membranes, was 96–99% (data not shown).
After collection, the myometrial biopsies were placed in DMEM that was supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. Human myometrial cells were obtained by the explant method, as described previously [24]. Cells were cultured in DMEM supplemented with antibiotics and 10% FCS, and routinely passaged when the cells were 90% to 95% confluent. Confluent myometrial cells were identified by their typical "hill and valley" microscopic appearance and by their positive reaction to a monoclonal antibody raised against ACTA2 (also known as smooth muscle alpha-actin). Cells at subconfluent density were incubated for 72 h in serum-free medium before incubation with or without TNF for the indicated time periods. Alternatively, in the TNF receptor blocking experiments, cells were incubated with neutralizing antibodies against TNFR1 or TNFR2 for 2 h prior to the addition of TNF. The experiments described in the present study were performed with cells between the third and fifth passages, with no noticeable differences noted for results obtained with cells from individual passages and with cells obtained from different uteri. Each population of myometrial cells studied was obtained from a different patient. In another set of studies, subconfluent myometrial cells were cultured in serum-free medium, after which either LPS-activated or resting amnion-conditioned medium and choriodecidua-conditioned medium were added (50% v/v) for 72 h in the absence or presence of 0.8 µg/ml neutralizing anti-human TNF antibody. The concentration of the antibody used was based on the manufacturer's recommendations.
Myometrial cell viability was assessed by trypan blue exclusion. At the indicated times after treatment, cells were detached by trypsin treatment, pelleted, and resuspended in DMEM that was supplemented with 10% FCS. After staining with trypan blue, viable cells were counted with a hemocytometer. Six replicate wells were used for each test condition. All the experiments were repeated at least three times with cells from different uteri. The percentage of treated viable cells was calculated as follows: % cell viability = (number of viable cells) / (total number of cells) x 100.
The TNF concentrations in the culture supernatants of the amnion and choriodecidua explants were determined using commercial kits (Perbio Science, Brebières, France). The detection limit for the assay was <2 pg/ml for TNF. The TNF levels are expressed as pg/mg of wet weight of tissue per 24 h.
In situ detection of apoptotic cells was performed using terminal deoxynucleotidyl transferase (TdT) TUNEL with the ApopTag Plus Fluorescein In situ Apoptosis Detection kit (Chemicon, Temecula, CA). Briefly, myometrial cells were cultured in 24-well dishes and incubated with or without TNF for 72 h in serum-free medium. After treatment, the cells were washed in PBS and fixed in 1% PFA. DNA fragments were tailed with digoxigenin-deoxy-UTP and then bound to FITC-conjugated anti-digoxigenin antibody. The nuclei were counterstained with Hoechst 33342 (2 µg/ml), to determine the percentage of TUNEL-positive nuclei relative to the total number of Hoechst-stained nuclei (x 100). All TUNEL-stained nuclei were scored as positive for apoptosis. Microscopic examination was carried out by two investigators in a double-blinded fashion. An average of 1000 nuclei was assessed in each experimental group.
Myometrial cells treated as indicated were scraped from plates, collected by centrifugation, and the cell pellet was suspended in a lysis buffer [50 mM NaCl, 25 mM Hepes (pH 7.6), 2.5 mM EDTA, 10% glycerol, 1% Nonidet NP40, 50 mM NaF, 30 mM Na4P2O7, 5 mM Na3VO4, and protease inhibitor cocktail]. The lysates were subsequently clarified by centrifugation at 13 000 x g for 30 min at 4°C, and the supernatants were collected. The protein concentrations were determined by the method of Bradford, using BSA as standard. Equal amounts of proteins were separated by SDS-PAGE on 10% or 12% gels and transferred onto a nitrocellulose membrane. Nonspecific binding sites were blocked by incubating the membranes with 5% low-fat dried milk in TBST [10 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 0.1% Tween-20]. The membranes were then incubated overnight at 4°C in TBST/1% milk with the indicated antibodies at the appropriate concentrations: BAX (1:1000), BCL2 (1:200), and cleaved CASP3 (1:1000). The membranes were washed with TBST and incubated with either donkey anti-rabbit or anti-mouse secondary antibody conjugated with horseradish peroxidase. The blots were developed with ECL reagents and visualized on Kodak x-ray films. Molecular weight markers were run in parallel.
The immunodetection of TLR4 in fetal membranes (amnion and choriodecidua) was performed as described above. Tissues were homogenized (100 mg/ml) with an Ultra-Turrax apparatus (IKA-WERKE) in ice-cold homogenization buffer [100 mM Tris-HCl (pH 7.4), 2 mM MgSO4, 2 mM EDTA, 10% glycerol, and protease inhibitor cocktail]. A polyclonal antibody directed against TLR4 was used at a 1:200 dilution.
For standardization, the membranes were stripped with a buffer that contained 62.5 mM Tris (pH 6.2), 2% SDS, and 100 mM beta-mercaptoethanol at 50°C for 30 min and reprobed with a polyclonal antibody raised against ACTB. Densitometric analysis was performed using the National Institutes of Health Image J software (http://rsb.info.nih.gov/ij) developed by W.S. Rasband (NIH, Bethesda, MD).
The results are expressed as the mean ± SEM. Statistical significance was determined using one-way ANOVA, followed by post hoc tests with StatView software (SAS Institute, Cary, NC). Values of P < 0.05 were considered statistically significant.
Production of TNF by Human Fetal Membranes after LPS Treatment
In order to determine the optimum concentration of LPS required for maximal release of TNF from fetal membranes, we established a concentration-response curve for doses of up to 10 µg/ml LPS for the choriodecidua and amnion tissues. As shown in Figure 1, LPS induced the release of TNF in the supernatants of the membrane explants in a concentration-dependent manner, peaking at 1 µg/ml after 24 h of treatment. The level of TNF release induced by LPS was higher in the choriodecidua than in the amnion explants at all the concentrations tested. The concentrations of TNF in cultures stimulated with 10 µg/ml LPS were 27.8 ± 5.9 pg/mg tissue and 2.21 ± 0.32 pg/mg tissue for the choriodecidua and amnion explants, respectively.
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The Amnion and Choriodecidua Express TLR4
Since TLR4 acts as an innate immune recognition receptor of LPS, we evaluated the levels of TLR4 in fetal membranes by Western blotting. As shown in Figure 2A, a protein of the predicted size, approximately 96 kDa, was present in the amnion and choriodecidua tissues. The intensity of the TLR4 band was determined by densitometry, and the results are expressed as the TLR4/ACTB band intensity. TLR4 expression was significantly lower in the choriodecidua than in the amnion (Fig. 2B).
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Effects of Amnion- and Choriodecidua-Conditioned Media on Myometrial Cell Viability and Apoptosis
Amnion and choriodecidua explants were incubated with or without LPS (10 µg/ml) for 24 h, and the conditioned media were collected. Myometrial cells in culture were treated for 72 h with the conditioned media and cell viability was measured (Fig. 3). The addition of conditioned media from LPS-activated choriodecidua explants caused a significant decrease in cell viability. In contrast, a slight but nonsignificant decrease in cell viability was noticed after the addition of conditioned media from LPS-activated amnion explants. No difference in myometrial cell viability was observed when cells were treated with the conditioned media from either untreated choriodecidua or amnion.
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We also examined the effect of conditioned media from choriodecidua explants on the expression of the myometrial BCL2 family. BCL2 protein was barely detected after myometrial cell incubation with conditioned media from LPS-activated choriodecidua explants, while abundant expression of BAX protein was observed, as compared to cells cultured in the presence of conditioned media from untreated choriodecidua explants (Fig. 4).
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To evaluate the contribution of TNF released from the choriodecidua explants on myometrial cell viability, cells were incubated with conditioned media from LPS-treated choriodecidua explants in the presence or absence of anti-TNF neutralizing antibody. TNF-neutralizing antibody pretreatment partially inhibited the effect on myometrial cell viability of the conditioned medium from LPS-treated choriodecidua (Fig. 3) and affected the expression of the BCL2 family. Densitometric analyses of immunoblots revealed that exposure to the anti-TNF antibody reduced the ability of the conditioned media from LPS-treated choriodecidua to decrease the BCL2 protein level and to increase the BAX protein level (Fig. 4).
Exogenous TNF Decreases Myometrial Cell Viability
As shown in the time-course study (Fig. 5A), myometrial cells were maintained for up to 3 days in serum-free medium in the absence of TNF without a significant change in cell viability. Treatment with TNF (200 ng/ml) had no significant effect on the number of viable cells at 24 h and 48 h after treatment. However, a significant decrease was observed at 72 h poststimulation with TNF. The concentration-dependent effect of TNF (2–200 ng/ml) after 72 h of stimulation was also determined. A significant decrease in the number of viable cells was detected at 20 ng/ml TNF and the effect was more pronounced at 200 ng/ml TNF (Fig. 5B). To determine which TNF receptor promotes this effect, we compared the actions of anti-TNFR1 and anti-TNFR2 neutralizing antibodies on cell viability after TNF treatment (200 ng/ml) for 72 h (Table 1). Anti-TNFR1, but not anti-TNFR2, suppressed the decrease in myometrial cell viability induced by TNF.
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Exogenous TNF Induces Myometrial Cell Apoptosis
TNF induces nuclear morphological changes in myometrial cells and nuclear fragmentation. Hoechst 33342 staining was used to assess changes in the nuclear morphology of myometrial cells following TNF treatment. The nuclei of the untreated cells were larger and exhibited homogeneous staining of chromatin (Fig. 6A). In contrast, the nuclei of myometrial cells exposed to 200 ng/ml TNF for 72 h displayed morphological changes that were characteristic of apoptosis, such as shrinkage and condensation of chromatin.
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DNA fragments were visualized in situ using the TUNEL technique. Few of the untreated cells (2.6%) showed TUNEL staining (Fig. 6B). Stimulation of myometrial cells with TNF (2–200 ng/ml) for 72 h resulted in a dose-dependent increase in the number of TUNEL-positive nuclei, with a significant effect noted at 20 ng/ml TNF.
TNF affects the expression of cleaved CASP3 and BCL2 family proteins. To determine whether the treatment of myometrial cells with TNF (200 ng/ml) for 72 h leads to caspase activation, we performed Western blot analysis for the executioner CASP3 using a specific antibody that recognizes the activated cleaved form (17 kDa). As shown in Figure 7, treatment of myometrial cells with TNF resulted in the presence of the proteolytic 17-kDa fragment of CASP3. In addition, after TNF treatment, the expression of BCL2 protein (26 kDa) was clearly decreased, whereas the level of BAX protein (23 kDa) was increased.
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Finally, we determined whether TNFR1 or TNFR2 was involved in TNF-induced myometrial cell apoptosis by pretreating myometrial cells with neutralizing anti-TNF receptor antibodies before the addition of TNF. The anti-TNFR1 antibody significantly reduced the increase in expression of cleaved CASP3 induced by TNF (Fig. 7). Densitometric analyses of immunoblots revealed that exposure to the anti-TNFR1 neutralizing antibody slightly reduced the ability of TNF to decrease the level of BCL2 protein and to increase the level of BAX protein. In contrast, the anti-TNFR-2 antibody had no effect on the TNF-induced changes in the levels of the cleaved CASP3 and BCL2 proteins.
The present study examined the hypothesis that TNF produced by human choriodecidua in response to LPS is a modulator of human myometrial cell apoptosis. Explants from both amnion and choriodecidua were found to produce TNF upon LPS stimulation, albeit with distinct patterns of secretion. Choriodecidua explants produced 10-fold higher levels of TNF than amnion explants, which is consistent with a previous report by Zaga et al. [25]. Our findings regarding the expression of the main LPS receptor TLR4 in fetal membranes revealed certain differences. Previous immunohistochemical analyses have revealed strong expression of TLR4 in the amnion epithelium, with a discrete signal detected in the choriodecidua [26]. The results of the present study are consistent with these findings. Interestingly, we found TLR4 expression in both fetal membranes, although the TLR4 signal was clearly weaker in the choriodecidua than in the amnion. Given the strategic location of the choriodecidua in close proximity to the maternal uterine tissues, we believe that the myometrium is a major target for the action of TNF secreted from the choriodecidua.
TNF is a pleiotropic cytokine that regulates multiple cellular responses, such as inflammatory and immunoregulatory responses and modulation of apoptotic cell death. Depending on the cell type, TNF is thought to activate signaling pathways linked to both cell survival and programmed cell death (reviewed in [13]). Previous investigations using smooth muscle cell cultures have shown conflicting effects of TNF. Some studies have reported that TNF promotes either growth or apoptosis depending on the phenotype of the cell [27, 28], whereas others have demonstrated that TNF has little or no effect in these processes [29–32]. To demonstrate the functional activity of TNF, myometrial cells were cultured with conditioned media from LPS-treated choriodecidua explants in the presence of neutralizing antibody specific for TNF. We found that only conditioned media obtained from LPS-treated choriodecidua explants were able to induce myometrial cell apoptosis by upregulating proapoptotic BAX protein expression and downregulating antiapoptotic BCL2 protein expression. Neutralization of TNF in the choriodecidua-conditioned medium partially reversed the cell death process. This incomplete effect suggests that other humoral factor(s) secreted from choriodecidua may regulate myometrial cell apoptosis. However, the possibility that this incomplete effect results from imperfect penetration of the anti-TNF antibody cannot be excluded.
Since immunoneutralization of TNF bioactivity results in a significant reduction in LPS-stimulated production of IL1B, IL10, and PGE2 by human choriodecidual explants [33], it is possible that an imbalance in these factors may influence myometrial cell growth properties. However, our data strongly suggest that the myometrial cell apoptotic activity that results from the LPS-choriodecidua-conditioned medium is largely attributable to TNF. To confirm this notion, we examined whether or not exogenous TNF participates in the myometrial cell death process. Prolonged exposure to exogenous TNF effectively decreased the number of viable myometrial cells that displayed the basic characteristics of apoptosis, i.e., cell shrinkage and nuclear shape changes associated with molecular alteration of DNA. We detected only a moderate number of TUNEL-positive cells after TNF treatment. However, it should be pointed out that TUNEL nuclear labeling was performed on attached cells, which may have led to an underestimation of the incidence of apoptosis. Since the TUNEL assay is not a perfect measurement of apoptosis of adherent cells [34], additional studies were performed with classical markers of apoptosis, which included the balance between BCL2 family proteins and CASP3 protein. Apoptotic stimuli have been shown to activate the 32-kDa pro-CASP3 protein by cleavage to the mature CASP3 form, which is composed of active 17-kDa fragments (reviewed in [35]). In the present study, we observed that TNF induced the cleavage of CASP3 in myometrial cells and reduced the expression of BCL2, together with an increase in the level of BAX protein.
TNF traditionally signals via TNFR1 and TNFR2. TNFR1 is expressed constitutively on a broad spectrum of different cell types and has been shown to mediate most of the commonly known biological effects of TNF. Only TNFR1 contains the death domain motif in its intracellular domain [13]. TNF plays a role in triggering the apoptosis of mouse uterine epithelial WEG-1 cells [15], and TNFR1 has previously been described in the mouse uterus [36]. Using neutralizing antibodies against both selective TNF receptor subtypes, we demonstrated that only the TNFR1 subtype is functionally involved in TNF-induced myometrial cell apoptosis. TNFR1 neutralization, but not TNFR2 neutralization, attenuated the TNF-induced decrease in BCL2 protein, as well as the TNF-induced expression of BAX. In addition, pretreatment of myometrial cells with the anti-TNFR1 neutralizing antibody successfully blocked the activation of CASP3 induced by TNF, which indicates that TNFR1 responds to TNF stimulation and activates the signaling cascade that leads to apoptotic cell death. Despite the general consensus that caspases play central roles in apoptotic cell death [35], caspase signaling may also be necessary for the initiation of cellular differentiation (reviewed in [37]). Thus, the fusion of myoblasts with myotubes [38] and the fusion of cytotrophoblasts with placental syncytiotrophoblasts [39] require caspase activation. Shynlova et al. [11] have recently reported that activation of the caspase cascade (CASP3, CASP6, CASP7, and CASP9) occurs transiently in pregnant rat myometrium at midgestation. This may trigger the differentiation of myometrium from a state of hyperplasia to hypertrophy, thus contributing to myometrial growth during gestation. Such a broad hypothesis has not been examined directly in the case of the human myometrium.
In the present study, we observed that the effect of exogenous TNF on myometrial cell apoptosis mimics the effect of conditioned medium from LPS-activated choriodecidua, which suggests that TNF released from the choriodecidua is capable of acting in a paracrine fashion. Recent experimental evidence also indicates that TNF, acting as an autocrine factor, controls critical events in the apoptosis of fetal membranes [6, 40, 41]. Our present results indicate that besides its critical role in fetal membranes, TNF that originated from the choriodecidua is an important regulator of myometrial cell apoptosis. Since the secretion of TNF by choriodecidua is much more prominent in response to bacterial or viral infection [25, 40], it seems reasonable to suggest that such specific pathologic situations generate a prominent apoptosis process in the human myometrium.
These compelling results are consistent with previous functional genomic studies, which have indicated that groups of genes related to apoptosis are differentially regulated in the human myometrium at the end of pregnancy. Among the most relevant genes associated with the inhibition of cell proliferation are the TNF receptor super family member FAS and its cognate ligand (FASLG), as well as TNF and its receptors [8, 42]. The arrest of myometrial growth may be a prerequisite for the uterus to develop full contractile activity. However, it cannot be ruled out that the process of postpartum uterine involution is programmed to start before the onset of labor. Uterine involution, which has been studied most intensely in the rat, is characterized by remodeling of the extracellular matrix in association with cell proliferation and apoptosis, as the uterus returns to the nonpregnant state [43]. Given the difficulties associated with obtaining myometrial biopsies after parturition, little is known about the regulation of uterine involution processes in women.
In conclusion, although further studies are necessary to elucidate additional mechanisms involved in the potential cross-talk between the fetal membranes and the myometrium, the present results indicate that TNF participates in the complex network of signaling processes associated with uterine involution.
ACKNOWLEDGMENTS
We are very grateful to Céline Méhats and Marie-Noelle Raymond for constructive and helpful discussions and to Carol Sable for editing the English text.
FOOTNOTES
1Supported by grants from the Fondation de la Recherche Médicale. 
Correspondence: 2Michelle Breuiller-Fouché, INSERM, U767, Faculté de Pharmacie, 4 avenue de l'Observatoire, 75270 Paris cedex 06, France. FAX: 33 1 40 48 83 94; e-mail: breuiller-fouche{at}cochin.inserm.fr
Received: 12 October 2006.
First decision: 6 November 2006.
Accepted: 10 January 2007.
REFERENCES
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P < 0.05, significant difference compared to the CM-choriodecidua + LPS.
