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Endometrial Stromal Cells Undergoing Decidualization Down-Regulate Their Properties to Produce Proinflammatory Cytokines in Response to Interleukin-1ß via Reduced p38 Mitogen-Activated Protein Kinase Phosphorylation

Department of Obstetrics and Gynecology (O.Y., Y.O., Y.H., K.K., T.H., T.Y., T.A., O.T., Y.T.), Faculty of Medicine, University of Tokyo, Tokyo 113-8655; Department of Obstetrics and Gynecology (T.A.), Teikyo University, Tokyo 173-8605; and CREST (O.T.), Japan Science and Technology, Kawaguchi 332-0012, Japan

Address all correspondence and requests for reprints to: Yutaka Osuga, M.D., Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: yutakaos-tky{at}umin.ac.jp.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
The decidualized endometrium plays a role in regulating trophoblast invasion for successful implantation and maintenance of pregnancy. IL-1ß, a proinflammatory cytokine, has been suggested to play a role in this process. Recently, several lines of evidence indicate the importance of p38 MAPK in various inflammatory responses. We investigated whether endometrial stromal cells (ESC) change their inflammatory responses to IL-1ß as related to p38 MAPK phosphorylation during the process of decidualization. ESC were decidualized by the treatment with progesterone for 9 d, as determined as such by an increase in the production of prolactin and cAMP by the cells. Whereas IL-1ß increased the production of IL-6, IL-8, and monocyte chemotactic protein-1, and expression of cyclooxygenase-2 mRNA in ESC cultured without treatment, the stimulatory effects of IL-1ß were reduced in the decidualized cells. Treatment with SB202190, a p38 MAPK inhibitor, also reduced the stimulatory effects of IL-1ß in nondecidualized ESC. P38 MAPK phosphorylation was increased by IL-1ß in nondecidualized ESC, whereas the IL-1ß-induced increase was suppressed in the decidualized cells. Treatment with 8-bromo-cAMP reduced IL-1ß-induced phosphorylation of p38 MAPK in nondecidualized ESC. In contrast, treatment with H89, a protein kinase A inhibitor, reversed a reduction in IL-1ß-induced p38 MAPK phosphorylation in the decidualized cells. In summary, decidualization seems to be a process during which endometrial cells diminish their response to IL-1ß, a known key factor for implantation, leading to the down-regulation of inflammation-like events, which may be relevant to controlled trophoblast invasion. The altered property of decidualized cells is thought to be caused by attenuation of IL-1ß-induced p38 MAPK phosphorylation in a way that involves the activation of the cAMP/protein kinase A pathway.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
THE ENDOMETRIUM BEGINS to alter its morphology and function (that is, decidual changes) around the time of implantation, the phenomenon being crucial for implantation and embryo development. Decidual tissues offer the sites for trophoblast invasion. The communication of decidual cells with trophoblasts is tightly regulated such that trophoblast invasion is spatiotemporally controlled, based on the balance between proinvasive and antiinvasive factors.

It is increasingly clear that the process of implantation entails inflammation-like events (1, 2, 3, 4). In this regard, several lines of evidence have implicated roles of a variety of cytokines in regulating decidual functions. Among others, IL-1ß is shown to stimulate endometrial stromal cells (ESC) to secrete various cytokines, thus playing a role in implantation.

Recent studies demonstrated the importance of p38 MAPK in the regulation of a variety of inflammatory responses, such as expression of proinflammatory mediators, leukocyte adhesion, and chemotaxis (5, 6). P38 MAPK, an intracellular signal-transducing molecule, transmits signals in response to proinflammatory molecules, including IL-1ß. On phosphorylation, it activates downstream signal transducers that induce the expression of a range of molecules.

Given the role of p38 MAPK in cellular inflammatory responses, we hypothesized that it might serve as a modulator of inflammatory responses occurring in the endometrium during implantation. In particular, in search for the mechanisms underlying temporally controlled regulation of endometrial cells by cytokines during the process of decidualization, we asked whether endometrial cells change their responses to IL-1ß during the process of decidualization, with special reference to the activity of p38 MAPK. We also examined whether cAMP, a potent mediator of decidualization, exerted any effect on p38 MAPK phosphorylation.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients and samples

Endometrial tissues were obtained from 46 patients undergoing hysterectomy for benign gynecological conditions. All patients had regular menstrual cycles, and none had received hormonal treatment for at least 6 months before surgery. All the samples were collected at the mid- to late-proliferative phase. The tissues collected under sterile conditions were processed for primary cell cultures. The experimental procedures were approved by the institutional review board of the University of Tokyo, and signed informed consent for use of the endometrium was obtained from each woman.

Isolation and culture of human ESC

Isolation and culture of human ESC were as described previously (7). Fresh endometrial biopsy specimens, collected in sterile medium, were rinsed to remove blood cells. The tissues were minced into small pieces and incubated in DMEM/Ham’s F-12 containing type I collagenase (0.25%; Sigma, St. Louis, MO) and deoxyribonuclease I (15 U/ml; Takara Shuzo, Tokyo, Japan) for 60 min at 37 C. The resultant dispersed endometrial cells were separated by filtration through a 40-µm nylon cell strainer (Becton Dickinson and Co., Franklin Lakes, NJ). Endometrial epithelial glands that remained intact were retained by the strainer, whereas dispersed ESC passed through the strainer into the filtrate. ESC in the filtrate were collected by centrifugation and resuspended in phenol-red free DMEM/Ham’s F-12 containing 10% charcoal-stripped fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin. ESC were plated in a 100-mm culture plate and kept at 37 C in a humidified 5% CO₂-95% air atmosphere. At the first passage, the cells were plated at a density of 2 x 10⁵ cells/ml into 60-mm or 24-well culture plates. The purity of the stromal preparations was confirmed by positive cellular staining for vimentin.

In vitro decidualization and cell treatment

In vitro decidualization was achieved as described previously (7, 8). Briefly, after 80% confluence in a 60-mm plate, the cells were rinsed and treated with 2.5% charcoal-stripped fetal bovine serum in the presence of 10 ng/ml (36.7 nM) estradiol (E), 100 ng/ml (318 nM) progesterone (P), E plus P (EP), or 0.1% ethanol vehicle (control). Culture media were collected and replenished every 3 d. Decidualization was assessed by measurement of prolactin (PRL) in the culture medium. To investigate the cellular responses to IL-1ß, the cultured cells were incubated with or without IL-1ß (5 ng/ml; Genzyme/Techne, Minneapolis, MN). After 24 h, the media were collected, centrifuged, and stored at -80 C for analysis of IL-6, IL-8, and monocyte chemotactic protein (MCP)-1 concentrations by specific ELISAs. For experiments to examine cyclooxygenase (COX)-2 mRNA expression, the cells were harvested after 4 h of the treatment. In some experiments, the cells were treated with SB202190 (Calbiochem, San Diego, CA) or H89 (5 µM; Calbiochem) for 1 h before IL-1ß treatment. In the experiments to see the effect of 8-bromo-cAMP (1 mM; Sigma), the cells were treated with 8-bromo-cAMP for 24 h before IL-1ß treatment.

Measurement of cAMP, PRL, and cytokines

Concentrations of PRL in conditioned media were measured using an SR1 analyzer (Stat Profile Ultra; Nova Biomedical, Boston, MA). The sensitivity was 500 pg/ml, and the intra-assay and interassay coefficients of variation were less than 10%. Concentrations of cAMP in conditioned media were measured using a cAMP immunoassay kit (R&D, Minneapolis, MN); and those of IL-6, IL-8, and MCP-1 were measured using a specific ELISA kit (Quantikine; Genzyme/Techne). The sensitivities of the assays were 0.3 pmol/ml, 3.12 pg/ml, 15.6 pg/ml, and 31.2 pg/ml for cAMP, IL-6, IL-8, and MCP-1, respectively. The intra-assay and interassay coefficients of variation were less than 5% in these assays.

Western blotting

Cultured cells were homogenized in the lysis buffer containing 50 mM TrisHCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue and diluted to 1 mg total protein/ml. Samples were resolved by 10% SDS-PAGE. Proteins were blotted onto a nitrocellulose membrane and incubated with rabbit antibody to total human p38 MAPK or rabbit antibody to phospho-specific (Thr180/Tyr182) p38 MAPK (1:1000; New England Biolabs, Inc. Beverly, MA) as a primary antibody, and antirabbit antibody (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as a secondary antibody. Phosphorylation of Thr180/Tyr182 is known to activate p38 MAPK (9, 10). Immune complexes were visualized by use of an ECL Western blotting system (Amersham Pharmacia Biotech, Little Chalfont, UK).

RT-PCR

Total RNA was extracted, using an RNeasy Mini Kit (QIAGEN, Hilden, Germany). One microgram of total RNA was reverse-transcribed in a 20-µl volume using an RT-PCR kit (TOYOBO, Osaka, Japan). Standard PCR was performed using ReverTra Dash (TOYOBO) according to the manufacturer’s instructions. The following PCR primers were used: COX-2 sense primer, 5'-TTCAAATGAGATTGTGGGAAAATTGCT-3'; COX-2 antisense primer, 5'-AGATCATCTCTGCCTGAGTATCTT-3' (11, 12); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense primer, 5'-ACCACAGTCCATGCCATCAC-3'; GAPDH antisense primer, 5'-TCCACCACCCTGTTGCTGTA-3'. Amplification was performed for COX-2 and GAPDH with 30 cycles of denaturing (98 C, 10 sec), annealing (60 C, 6 sec), and extension (74 C, 25 sec).

Statistical analysis

Data were evaluated using ANOVA with Scheffé’s post hoc analysis for multiple comparisons. P less than 0.05 was accepted as statistically significant.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
IL-1ß-induced p38 MAPK phosphorylation in cultured ESC

We determined p38 MAPK phosphorylation by IL-1ß in isolated ESC cultured without any hormone (Fig. 1). An increase in p38 MAPK phosphorylation was apparent at as early as 1 min. The phosphorylation level was maximal at 15 min, followed by a decrease to the level before treatment at 3 h. However, the amount of total p38 MAPK was unchanged during 24 h of culture. In parallel culture, the status of p38 MAPK phosphorylation was not altered without the addition of IL-1ß (data not shown). Based on this finding, we evaluated the IL-1ß-induced p38 MAPK phosphorylation by treating the cells with IL-1ß for 15 min in the following experiments.

View larger version (36K): [in this window] [in a new window]   Figure 1. Phosphorylation of p38 MAPK by IL-1ß in cultured endometrial stromal cells. Endometrial stromal cells were incubated with IL-1ß for the indicated times (0–24 h). Cell extracts were prepared and assayed for phosphorylated p38 MAPK (phospho-p38) or total p38 MAPK (total-p38) by Western blotting. The result is representative of three separate experiments.

Decidualization of ESC treated with EP for 9 d was confirmed as judged by an increase in PRL production (that is, more than a 70-fold higher PRL level in culture media over the detection level). In contrast, the PRL levels for ESC, cultured for 9 d without the hormones or cultured for 1 d with or without the hormones, were below the detection level (Fig. 2A).

View larger version (13K): [in this window] [in a new window]   Figure 2. Production of PRL and cAMP by cultured endometrial stromal cells treated with E and P. Endometrial stromal cells were cultured without hormone (control, C) or with 10 ng/ml E and 100 ng/ml P (EP) for 1 d and 9 d. After replacement with fresh culture media, the cells were incubated for 24 h to determine PRL and cAMP concentrations in conditioned media. A, PRL concentrations in conditioned media, measured by a specific assay kit, are shown with mean ± SEM of duplicate cultures. Values less than detection limits (<0.6) are shown as ND (not detected); P < 0.0001 C vs. EP on d 9. B, cAMP concentrations in conditioned media, measured by a specific assay kit, are shown with mean ± SEM of duplicate cultures; P < 0.0001 C vs. EP on d 9. Each result is representative of six separate experiments.

Responses to IL-1ß in the induction of p38 MAPK phosphorylation; production of IL-6, IL-8, and MCP-1; and expression of COX-2 mRNA in decidualized ESC

ESC were exposed to IL-1ß after incubation either with or without EP. IL-1ß-induced p38 MAPK phosphorylation in ESC cultured with EP for 9 d was suppressed, compared with those in ESC cultured without the hormones. In contrast, suppression of p38 MAPK phosphorylation by the hormone treatment was not observed when cultured for 1 d (Fig. 3A). IL-1ß-induced phosphorylation of p38 MAPK was significantly suppressed in ESC cultured for 9 d with P alone, whereas treatment with E did not result in the suppression of the phosphorylation, as was the case with untreated ESC (Fig. 3B). IL-1ß produced a significant increase in the levels of IL-6, IL-8, and MCP-1 in culture media of ESC cultured for 9 d in the absence of any hormone. Treatment with P or EP significantly suppressed IL-1ß-induced production of IL-6, IL-8, and MCP-1 by ESC, as compared with no treatment or treatment with E alone (Fig. 3C). IL-1ß stimulated the expression of mRNA of COX-2 in ESC cultured for 9 d in the absence of any hormone, which was suppressed by the treatment with P or EP. Treatment with E alone had no effect on the IL-1ß-stimulated expression of COX-2 mRNA (Fig. 3D).

View larger version (29K): [in this window] [in a new window]   Figure 3. Basal and IL-1ß-induced phosphorylation of p38 MAPK; production of IL-6, IL-8, and MCP-1; and expression of COX-2 mRNA in endometrial stromal cells cultured with different hormone treatments. Endometrial stromal cells were cultured without hormone (control, C) or with 10 ng/ml E, 100 ng/ml P, or EP for 1 and 9 d. Then, the cells were incubated, with or without IL-1ß (5 ng/ml), for 15 min to determine p38 MAPK phosphorylation; for 24 h to determine IL-6, IL8, and MCP-1 concentrations in conditioned media; and for 4 h to determine COX-2 mRNA expression in the cells. A, Extracts of cultured endometrial stromal cells treated with EP for 1 and 9 d were prepared and assayed for phosphorylated p38 MAPK (phospho-p38) or total p38 MAPK (total-p38), by Western blotting. B, Extracts of cultured endometrial stromal cells, treated with different hormone treatment for 9 d, were prepared and assayed for phosphorylated p38 MAPK (phospho-p38) or total p38 MAPK (total-p38), by Western blotting. Panel C, Concentrations of IL-6, IL-8, and MCP-1 in conditioned media measured by specific enzyme immunosorbent assays. Values are shown as percent of IL-1ß-induced levels in the control with mean ± SEM of duplicate cultures. *, P < 0.005; **, P < 0.0001 vs. control. D, Total RNA was extracted from the cells, and RT-PCR analysis was performed for COX-2 mRNA. M, DNA molecular weight standards. Each result is representative of at least three separate experiments.

To investigate whether p38 MAPK is involved in IL-1ß-induced production of IL-6, IL-8, and MCP-1 and the expression of COX-2 mRNA in ESC, the effect SB202190, a p38 MAPK specific inhibitor, was tested. Treatment with SB202190 suppressed IL-1ß-induced production of IL-6, IL-8, and MCP-1 by ESC in a dose-dependent manner (Fig. 4A). Similarly, treatment with SB202190 suppressed IL-1ß-induced expression of COX-2 mRNA by ESC (Fig. 4B).

View larger version (31K): [in this window] [in a new window]   Figure 4. Effects of SB202190 on IL-6, IL-8, and MCP-1 production and COX-2 mRNA expression induced by IL-1ß in endometrial stromal cells. Endometrial stromal cells were treated, with or without SB202190, for 1 h, and then stimulated with IL-1ß (5 ng/ml) for 24 h to determine IL-6, IL8, and MCP-1 concentrations in conditioned media and for 4 h to determine COX-2 mRNA expression in the cells. A, Concentrations of IL-6, IL-8, and MCP-1 in conditioned media were measured by respective specific enzyme immunosorbent assays. Values are shown as percent of IL-1ß-induced levels in the control with mean ± SEM of triplicate cultures. *, P < 0.001; **, P < 0.0001 vs. control. B, Total RNA was extracted from the cells, and RT-PCR analysis was performed for COX-2 mRNA. M, DNA molecular weight standards. Each result is representative of five separate experiments.

We further investigated whether cAMP, a known mediator of decidualization, could suppress IL-1ß-induced p38 MAPK phosphorylation in ESC. Treatment with 8-bromo-cAMP for 24 h suppressed IL-1ß-induced p38 MAPK phosphorylation in ESC (Fig. 5A). Then, to see whether the cAMP/PKA pathway is involved in decidualization-dependent suppression of p38 MAPK phosphorylation induced by IL-1ß, decidualized ESC were treated with H89, a PKA inhibitor. H89 treatment attenuated the suppression of IL-1ß-induced p38 MAPK phosphorylation in decidualized ESC (Fig. 5B), suggesting the involvement of the cAMP/PKA pathway in the altered phosphorylation status coupled with decidualization.

View larger version (64K): [in this window] [in a new window]   Figure 5. Effects of 8-bromo-cAMP and H89 on IL-1ß-induced p38 MAPK phosphorylation in endometrial stromal cells. A, Endometrial stromal cells were treated, with or without 1 mM 8-bromo-cAMP, for 24 h, and then stimulated with IL-1ß (5 ng/ml) for 15 min to determine p38 MAPK phosphorylation. B, Decidualized endometrial stromal cells, treated with 10 ng/ml E and 100 ng/ml P (EP) for 9 d, were treated, with or without 5 µM H89, for 1 h, and then stimulated with IL-1ß (5 ng/ml) for 15 min to determine p38 MAPK phosphorylation. Cell extracts were prepared and assayed for phosphorylated p38 MAPK (phospho-p38) or total p38 MAPK (total-p38) by Western blotting. Each result is representative of three separate experiments.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

At the implantation sites, IL-1ß is present in the villous trophoblast, syncytiotrophoblast, intermediate trophoblast, and maternal stromal decidual cell (13). IL-1ß has been shown to stimulate the expression of various molecules, such as cytokines, chemokines, growth factors, matrix proteases, prostaglandins, and adhesion molecules in the endometrium (1), suggesting roles for IL-1ß in cell growth, cell migration, and tissue remodeling, each being crucial for implantation. The present finding that IL-1ß stimulated the expression of COX-2 and secretion of IL-6, IL-8, and MCP-1 in ESC is consistent with earlier studies (14, 15, 16, 17).

IL-6 is a member of the proinflammatory cytokine family. IL-8 and MCP-1 are prototype chemokines, exerting a chemoattractive effect for neutrophils and macrophages, respectively. COX-2 is an inducible enzyme that synthesizes prostaglandins, and is expressed at the implantation site (18). Ample evidence suggests that prostaglandins produced by COX-2 increase capillary permeability for leukocytes in the decidualized endometrium, thus augmenting a chemoattractive effect of chemokines(19, 20). In this way, IL-1ß, prostaglandins, and chemokines seem to constitute the self-perpetuating system to generate proinflammatory cytokines. However, exaggerated inflammatory responses may perturb the integrity of endometrial function and lead to pathological conditions, including abortion and complicated pregnancies, such as preeclampsia and underdevelopment of the fetus. To render the endometrium favorable for implantation and ensuing fetal development, inflammatory responses of decidual cells seem to be spatiotemporally fine-tuned. The present observation that the production of proinflammatory molecules by IL-1ß was suppressed in decidualized cells may be one way to control inflammatory responses operating in the process of implantation.

To investigate the cellular mechanism for the attenuated IL-1ß-induced response in decidualized cells, we examined phosphorylation of p38 MAPK, a major signal transducer of proinflammatory stimuli, in the endometrium. P38 MAPK has been shown to be a mediator for IL-1ß to increase IL-6, IL-8, MCP-1, and COX-2 expression in certain cells (21, 22, 23, 24, 25, 26). The present study demonstrated that IL-1ß-induced production of IL-6, IL-8, MCP-1, and COX-2 was suppressed by p38 MAPK inhibitor SB 202190 in ESC, implying that p38 MAPK mediates the effects of IL-1ß to stimulate the production of these molecules. Interestingly, p38 MAPK phosphorylation was stimulated by IL-1ß in ESC, and the stimulatory effect of IL-1ß was significantly suppressed in decidualized cells. Collectively, p38 MAPK is suggested to be involved in the suppression of IL-1ß-induced IL-6, IL-8, MCP-1, and COX-2 expression in decidualized ESC.

Long-term treatment was required for P to exert its inhibitory effect on IL-1ß-induced p38 MAPK phosphorylation. The days needed were compatible with those required for decidualization, as determined by PRL secretion. Thus, alteration of p38 MAPK phosphorylation is unlikely to be ascribed to the direct effect of P but may be an attribute to decidualized cells. cAMP is suggested to be an important inducer for decidualization because molecules known to stimulate cAMP production, such as PGE2 (27) and relaxin (28) and cAMP derivatives (29), are capable of inducing decidualization. In addition, cAMP production increased with decidualization induced by P. Therefore, we hypothesized that cAMP could participate in the suppression of IL-1ß-induced p38 MAPK phosphorylation in decidualized cells. The findings that cAMP mimicked P in attenuating IL-1ß-induced p38 MAPK phosphorylation and that PKA inhibitor abrogated the effect of cAMP are in agreement with the hypothesis.

cAMP activates p38 MAPK in thyroid cells (30) and neural cells (31) in a PKA-dependent manner, whereas it does so in Th2 cells (32) in a PKA-independent manner. The present finding that cAMP suppressed the p38 MAPK activation is an as-yet-undescribed mode of regulation of the p38 MAPK pathway by cAMP. Thus, it may be that the manner in which cAMP modulates p38 MAPK signal cascade is distinct depending on the cell type. The molecular mechanism with which cAMP suppresses IL-1ß-induced p38 MAPK activation still remains to be elucidated. The low-molecular-weight G proteins Ras and Rap were reported to modulate the activation of p38 MAPK by IL-1, Ras having a stimulatory effect and Rap having an inhibitory effect (33). Because Ras and Rap can be activated by cAMP (34), these proteins may be involved in the modulation of IL-1-induced p38 MAPK response by cAMP in a cell-type-specific manner.

Endometriosis is thought to be initiated by implantation of eutopic endometrial tissues onto the pelvic peritoneum. Inflammatory environment associated with endometriosis is proposed to promote the progression of the disease. Recent studies demonstrated that IL-1ß induces the production of IL-8 and MCP-1 in endometriotic cells (35, 36). IL-8 and MCP-1 are able to recruit and activate immune cells that secrete p38 MAPK-activating cytokines. Overall, it is intriguing to speculate that proinflammatory cytokines and p38 MAPK may cooperate in a synergistic way in the progression of the disease.

Progestogens exert therapeutic effect on endometriosis. Assuming that progestogens suppress IL-1ß-induced p38 MAPK activation in ectopic endometriotic cells as well, the therapeutic effect of progestogens could be partly explained by down-regulation of inflammatory responses in endometriotic lesions. This concept could be extrapolated to the possibility that cytokine-specific antiinflammatory drugs, such as SB202190, which inhibit intracellular signaling cascades stimulated by proinflammatory cytokines, have the potential for treating endometriosis.

In summary, our study demonstrated that decidualized cells exhibited attenuated responses to IL-1ß in producing IL-6, IL-8, MCP-1, and COX-2. A reduction in IL-1ß-induced p38 MAPK phosphorylation via the activation of the cAMP/PKA pathway is a possible mechanism for this (Fig. 6). This may provide new ways to understand how inflammatory responses occurring in the implantation site are exquisitely regulated such that trophoblasts invade in partnership with maternal stromal cells.

View larger version (17K): [in this window] [in a new window]   Figure 6. A proposed mechanism underlying suppression of IL-1ß-induced production of proinflammatory mediators in decidualized endometrial stromal cells.

	Acknowledgments

 
We thank Emi Nose for technical assistance.

	Footnotes

 
Abbreviations: COX, Cyclooxygenase; E, estradiol; EP, estradiol plus progesterone; ESC, endometrial stromal cells; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MCP, monocyte chemotactic protein; P, progesterone; PKA, protein kinase A; PRL, prolactin.

Received November 14, 2002.

Accepted February 2, 2003.

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