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Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia
Address all correspondence and requests for reprints to: Dr. Evdokia Dimitriadis, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: evdokia.dimitriadis{at}princehenrys.org.
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Introduction |
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IL-11 belongs to the IL-6 family of cytokines and signals via a heterodimeric complex of IL-11 receptor 
(IL-11R) and glycoprotein 130 (6). The cellular responses of IL-11 are induced by the activation of downstream Janus kinases that phosphorylate the latent cytoplasmic transcription factors, signal transducer and activator of transcription (STAT) (6). Phosphorylated (p) or activated STAT proteins translocate to the nucleus to modulate gene transcription (7). Cytokine signaling is tightly regulated by a variety of mechanisms (8). The inducible suppressor of cytokine signaling (SOCS) proteins, a family with eight members (SOCS1–SOCS7 and cytokine-inducible SH2 protein), are expressed in response to cytokine stimulation of STAT phosphorylation acting in a negative-feedback mechanism to hinder the activities of cytokine receptors. IL-11 signals via pSTAT3 in HESC and other cell types (9, 10). It is generally recognized that IL-11 stimulates SOCS3 in various cell types (11).
IL-11 is one of the few molecules identified to be obligatory for the progression of decidualization beyond the primary decidual zone in mice and is thus essential for successful embryo implantation (12, 13). In humans, IL-11 immunostaining is maximal in decidualized HESC, although weak staining is also apparent in nondecidualized HESC in secretory-phase endometrium, suggesting IL-11 may be involved in initiating and progressing differentiation (14). Sequential gene expression occurs as decidualization progresses, and importantly, IL-11 mRNA in HESC is up-regulated within 2 h after treatment with cAMP, suggesting IL-11 may have roles in initiating the differentiation process (15). IL-11 enhances progesterone-induced decidualization of HESC and its mRNA expression, and secretion from HESC is increased during cAMP- and progesterone-induced decidualization (2, 16). In addition, blocking endogenous IL-11 action reduces decidualization of HESC (15). Furthermore, relaxin and prostaglandin E2 enhance progesterone-induced decidualization of HESC by stimulating IL-11 via a cAMP/protein kinase A (PKA) pathway, indicating that IL-11 is a central player in the decidualization process (16). Importantly, a number of in vivo studies demonstrate that decreased local IL-11 production and secretion is associated with pregnancy disorders including infertility, miscarriage, and early abortion (17, 18, 19, 20).
Progesterone is undeniably a key regulator of embryo implantation and the main physiological inducer of decidualization in women (21). The effects of progesterone on inflammatory mediators are complex and involve modulation of cytokine production and signaling pathways in target cells. There is evidence of cross-talk between steroid hormone and cytokine receptor signaling pathways. Activated STATs associate with steroid hormone receptors to modulate gene transcription; STAT3 modulates transcription by direct association with progesterone receptor (PR) (22, 23). Interestingly, STAT3 interacts with PR by protein-protein association in rat decidua (24). Although progesterone is the main physiological inducer of decidualization in humans, evidence strongly suggests the cAMP/PKA pathway is also required for the process (25). It is likely that progesterone and cAMP/PKA pathways use both unique and common mechanisms to drive decidualization.
IL-11 is clearly important in progesterone- and cAMP-induced decidualization; however, little is known of IL-11’s downstream mechanisms of action. We hypothesized that IL-11, progesterone, and cAMP pathways interact to facilitate HESC decidualization. This study aimed to examine the role of IL-11 signaling mechanisms during HESC decidualization.
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Materials and Methods |
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Stromal cell isolation and culture
HESC were isolated from tissue by enzymatic digestion and filtration as described previously (2). This produces more than 95% pure stromal cell cultures, determined by analysis of vimentin and cytokeratin expression. Cells were plated at a density of 1 x 106 cells per well in six-well plates, grown to confluency in DMEM and Ham’s F12 medium (1:1) (Trace Biosciences, Sydney, Australia), supplemented with 1% penicillin, streptomycin, and fungizone (Commonwealth Serum Laboratories, Melbourne, Australia) and 10% charcoal-stripped fetal calf serum (Trace Biosciences). Experiments were conducted in medium containing either no serum or 2% charcoal-stripped fetal calf serum as indicated. At the end of each experiment, cell number and viability were assessed by trypan blue exclusion unless otherwise specified. All experiments were replicated at least three times using different preparations of HESC.
In vitro decidualization
HESC were decidualized as previously described (2). HESC (cultures from individual endometrial biopsies; n = 18) were incubated with combinations of either 0.5 mM 8-bromo-cAMP (Sigma Chemical Co., St. Louis, MO) or 10–8 M estradiol-17ß (E) (Sigma) plus 10–7 M medroxyprogesterone acetate (P) (Sigma) in duplicate wells for 8 d with media collection and replenishment every 48 h. Some cells were also cultured with an IL-11-neutralizing antibody (10 ng/ml) (2) (R&D Systems Inc., Minneapolis, MN) or the antiprogestin onapristone (a gift from Schering AG, Berlin, Germany).
HESC (cultures from individual endometrial biopsies; n = 6) in duplicate wells were treated with combinations of recombinant human IL-11 (100 ng/ml) (Genetics Institute, Cambridge, MA, and a gift from Dr. Lorraine Robb, Melbourne, Australia), IL-11-neutralizing antibody (10 ng/ml) (R&D Systems), and rabbit IgG (10 ng/ml) (Vector Laboratories, Burlingame, CA) for 24 h. Control wells received vehicle alone. Optimal concentrations of rhuIL-11 and IL-11 neutralizing antibody were previously determined by concentration-response studies (2).
Prolactin (PRL) assay
PRL secretion by HESC was assayed in duplicate by ELISA (Bioclone Australia Pty. Ltd., Sydney, Australia) to determine the extent of decidualization (2). Media collected at d 8 of decidualization were concentrated 5-fold for PRL measurement. A quality control sample (culture medium from a single endometrial cell culture) was included in every assay. The lower detection limit of the assay was 50 mIU/liter. The inter- and intraassay variabilities were 5.3 and 3.0%, respectively. Quality control samples were included in every assay to monitor precision. Results are expressed as mean ± SEM.
RNA extraction and purification
Total RNA was extracted from endometrial samples using the RNeasy Minikit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer’s instructions. All samples were treated with RNase-free DNase (Ambion, Austin, TX) to remove the possibility of genomic DNA contamination. RNA samples were then analyzed by spectrophotometry to determine RNA concentration, yield, and purity. RNA concentrations were also analyzed by Ribogreen fluorescence RNA assay (Molecular Probes, Eugene, OR). RNA samples were diluted to approximately 20 ng/µl based on spectrophotometric readings and analyzed in a 96-well plate, in conjunction with a standard curve of serially diluted rRNA (Molecular Probes) from 0–80 ng/well, by addition of Ribogreen fluorescent dye at a dilution of 1:500. This assay gave an intraassay coefficient of variation, as defined by the repeated analysis of a single RNA sample, of 3.3% (n = 6) and an interassay variation of 10.3% (n = 6).
Real-time RT-PCR
Standards for real-time PCR for SOCS3 were generated by conventional PCR. A representative endometrial RNA sample (1 µg) was reverse transcribed using AMV-RTase (Promega, Madison, WI) and 100 ng random hexanucleotide primers (Amersham Biosciences, Piscataway, NJ), and the cDNA generated was subsequently amplified by PCR (Hybaid Express Block Cycler): 95 C for 1 sec, 65 C for 1 sec, and 72 C for 1 sec for 40 cycles. The product was electrophoresed on a 2% agarose gel, the bands excised, and cDNA purified using UltraClean GelSpin columns (MoBio Laboratories Inc., Solana Beach, CA). The resultant PCR-generated cDNA was quantitated by spectrophotometry, sequenced to confirm identify, and thereafter used as a standard for real-time RT-PCR.
Real-time RT-PCR for SOCS3 was performed using a LightCycler (Roche, Mannheim, Germany) on RNA isolated from cells (duplicate wells) treated for either 24 h or 8 d with experimental treatments as indicated and medium-only control (n = 3 separate cultures per experiment). RNA samples were reverse transcribed as described above, in duplicate to minimize the inherent variability of this technique. PCR amplification of 4 µl cDNA (diluted 1:5) was performed with addition of a MasterMix (Roche Diagnostics Corp., Indianapolis, IN), which included SYBR Green I, dNTPs, Taq enzyme, and optimized concentrations of MgCl2 and SOCS3-specific primers. The specific primers for SOCS3 were 5'-TCCCCTCGCCACCTACTGA-3' and 5'-GGTCCAGGAACTCCCGAATG-3' (Sigma Genosys Australia Pty. Ltd., Castle Hill, New South Wales, Australia), used at a concentration of 0.5 pmol/µl. An initial denaturing step was performed for 10 min at 95 C, before 40 cycles of 95 C for 15 sec, 62 C for 5 sec, and 72 C for 10 sec. An additional stage at 70 C for 5 sec was included before acquisition of fluorescence to exclude quantitation of primer dimers.
mRNA expression was quantitated by performing the log-linear amplification phase with a four-point standard curve (standards serially diluted 1:10). At the end of each program, melting-curve analysis was carried out to ensure specificity of the reaction products. The sizes of the products were also confirmed by gel electrophoresis for selected samples. Data were normalized using the expression of a housekeeping gene (18S; 0.5 pmol/µl) with primers 5'-CGGCTACCACATCCAAGGAA-3' and 5'-GCTGGAATTACCGCGGCT-3' (Sigma). PCR amplification of 4 µl cDNA (diluted 1:200) was combined with MasterMix (Roche) and subjected to 35 cycles of 95 C for 15 sec, 60 C for 5 sec, and 72 C for 10 sec, at which point fluorescence was measured. Amplified SOCS3 mRNA in stimulated cells was calculated by normalizing to the amplification of 18S and then expressing the normalized values as percent change from unstimulated controls (controls defined as 100%).
Western blot analysis for STAT3, pSTAT3 and SOCS3 in HESC
Cells were grown to confluence, the medium aspirated, and cells washed with ice-cold sterile PBS, twice on ice. Cells were lysed and scraped in 150 µl/well ice-cold lysis buffer containing 50 mM Tris base, 150 mm NaCl, 2 mM EDTA, 2 mm EGTA, 25 mM NaF, 25 mm ß-glycerolphosphate (pH 7.5), and 2 µl/well protease inhibitors cocktail set III [4-(2-aminoethyl)benzenesulfonyl fluoride, 100 mM HCl, 80 µM aprotinin, 5 µM bestatin, 1.5 mm E-64, 2 mM leupeptin hemisulfate, and 1 mm pepstatin A (Calbiochem, San Diego, CA)]. Cell extracts were then centrifuged at 12,000 rpm for 30 min at 4 C, and supernatant protein wasquantified using the BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of total protein were then resolved on SDS-PAGE gels and transferred to nitrocellulose membranes. All membranes were incubated with Ponceau S (Sigma) to ensure equal protein loading in all lanes (results not shown). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) with 0.1% Tween and probed separately with antibodies specific for pSTAT3 (Tyr705; Cell Signaling Technology Inc., Beverly, MA) (1:1000), total STAT3 (Cell Signaling Technology) (1:1000), or SOCS3 (IBL Co. Ltd., Gunma, Japan). The membranes were washed in TBS with 0.1% Tween and then incubated for 1 h with horseradish peroxidase (HRP)-conjugated rabbit secondary antibody (DakoCytomation, Glostrup, Denmark) (1:1500). Finally, the HRP activity was detected using enhanced chemiluminescence reagent (Pierce). The membranes were exposed to autoradiographic film (Amersham Biosciences, Little Chalfont, UK) with the exposure time adjusted to keep the integrated OD within a linear and nonsaturated range.
Transient transfection of SOCS3 in HESC
Mammalian transfection vectors for SOCS3 and empty vector were obtained from Dr. Tracey Willson (Walter and Eliza Hall Institute of Medical Research, Victoria, Australia), and transient transfections were performed with FuGENE Transfection reagent (Roche Diagnostics) according to the manufacturer’s directions. Subconfluent HESC, grown in six-well plates as described above, were cotransfected with green fluorescent protein (GFP) vector (Clontech, Mountain View, CA) with a total vector concentration of 0.5 µg DNA per transfection in serum-free media for 24 h. All assays were performed using equimolar ratios of DNA, with the total amount of DNA per transfection adjusted according to the parent vector of SOCS3 to ensure that each well received 0.5 µg cDNA in serum-free media. After completion of the transfection, the culture media was changed and treatments were added as indicated. Each experimental condition was performed in duplicate in three independent experiments.
Immunohistochemistry for pSTAT3 and SOCS3 in human endometrium
Immunohistochemistry for pSTAT3 and SOCS3 were conducted using rabbit polyclonal antibodies raised against human peptides (R&D Systems) on endometrial sections from all stages of the menstrual cycle (n = 4–6 per group). Positive controls for pSTAT3 and SOCS3 were pregnant mouse uterus and human term placenta, respectively (26, 27). Briefly, 5-µm sections of formalin-fixed, paraffin-embedded tissues were dewaxed and rehydrated. Endogenous hydrogen peroxidase activity was quenched using 3% H2O2 in methanol for 10 min at room temperature. Nonspecific binding was prevented by preincubation of tissue sections with a nonimmune block containing 10% nonimmune horse serum (Sigma-Aldrich, Sydney, Australia), 2% normal human serum (in-house) in TBS and 0.1% Tween 20. Primary antibodies were applied overnight (17 h) at 4 C diluted to 2–4 µg/ml in nonimmune block. A nonimmune rabbit IgG (R&D Systems), diluted to a matching concentration as the primary antibody, was also included for each tissue. After stringent washing with TBS and 0.6% Tween 20, detection of positive binding was performed by the sequential application of biotinylated goat antirabbit IgG (Vector) 1:200 in nonimmune block and avidin-biotin-peroxidase conjugate (Dako), followed by the substrate diaminobenzidine (Dako) for between 3 and 5 min. Wherever possible, samples to be analyzed were included in the same run; otherwise, quality controls were included in each run. Sections were counterstained with Harris’ hematoxylin (Sigma), dehydrated, and mounted from Histosol with DPX mounting medium (BDH Laboratory Supplies, Poole, UK). Immunostaining was analyzed semiquantitatively by two independent observers blind to the stage of the menstrual cycle. Staining intensity and heterogeneity in each of the endometrial compartments (epithelium; stroma, including decidualized stromal cells; and vasculature) was assessed and allocated a score between 0 and 4 where numbers represent no stain (0), faint staining (1), moderate staining (2), strong staining (3), and very intense staining (4). For immunostaining in leukocytes infiltrating the endometrial stroma, a semiquantitative score between 0 and 3 was allocated based on their relative abundance where numbers represent absent (0), few positive leukocytes (1), abundant positive (2), and highly abundant leukocytes (3).
Statistical analysis
Data were expressed as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. A P value of <0.05 was considered significant.
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). HESC cultured in serum-free conditions do not secrete detectable levels of IL-11 (2). Addition of IL-11 to HESC stimulated pSTAT3 from 5 min. Coculture of cells with IL-11 and an IL-11-neutralizing antibody diminished the effect compared with cells treated with IL-11 alone for 15 min (Fig. 1, top). STAT3 protein abundance was not affected at any time point when IL-11 was added to HESC (Fig. 1, bottom).
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). To determine the effect of PR on HESC STAT3 abundance, P- or cAMP-treated cells were administered with a PR antagonist, onapristone, from d 6–8 of culture. This resulted in decreased STAT3 abundance at d 8 in both compared P- and cAMP-treated cells (Fig. 2A).
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). Similarly, at d 6 of culture, E+P- and cAMP-treated cells secreted PRL and were considered decidualized (data not shown). Importantly, PRL secretion levels, or the extent of decidualization, did not differ between cells treated with either cAMP or P (Fig. 2B). Cells treated with E+P, cAMP, or onapristone plus E+P or cAMP secreted more PRL than E-treated controls on d 8 (P < 0.05; Fig. 2B). However, onapristone treatment from d 6–8 attenuated PRL secretion during this time compared with cells cultured with either E+P or cAMP alone (P < 0.05; Fig. 2B). There was no difference in PRL release between cells cocultured with onapristone and either E+P or cAMP.
Effect of IL-11 on SOCS 3 expression in HESC
SOCS3 gene expression is mediated at least in part by the activation of STAT3 (8). The effect of IL-11 on SOCS3 mRNA expression and protein production in HESC was examined by quantitative real-time RT-PCR and Western blot, respectively. HESC cultured in serum-free conditions do not secrete detectable levels of IL-11 (2). SOCS3 mRNA was expressed at low levels in HESC before addition of IL-11 (Fig. 3A). SOCS3 mRNA increased at 30 min after addition of IL-11 of culture but returned to baseline levels by 2 h (Fig. 3A). To determine the effect of IL-11 on SOCS3 protein, HESC were treated with IL-11 for up to 240 min and SOCS3 abundance examined at 0 (before treatment), 30, 60, 120, and 240 min. SOCS3 protein in HESC was present at all time points examined, and its abundance increased after 30 min of treatment (Fig. 3B). Maximal abundance of SOCS3 protein was seen at 60 min of treatment with IL-11 (Fig. 3B).
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). SOCS3 mRNA expression was higher in cells decidualized with cAMP compared with E+P (P < 0.05; Fig. 4A). Interestingly, addition of onapristone from d 6–8 further increased SOCS3 mRNA in comparison with either E+P (P < 0.05; Fig. 4A). Similarly, SOCS3 protein increased in HESC after treatment with either P or cAMP compared with nondecidualized controls at 8 d of culture, although the effect was not as pronounced as that for mRNA expression levels (Fig. 4, B and C). Addition of onapristone to E+P- or cAMP-treated cells likewise further increased SOCS3 protein abundance (Fig. 4, B and C).
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). There was no difference in PRL secretion between cells treated with E+P or cAMP (Fig. 4D), but PRL secretion was reduced from cells treated with onapristone and E+P or cAMP compared with either E+P or cAMP alone (P < 0.05; Fig. 4D).
Effect of SOCS3 overexpression in HESC on decidualization
Given that enforced overexpression of SOCS3 in corticotrophs diminishes IL-11-induced gene expression (28), we determined the effect of SOCS3 overexpression in HESC on IL-11 signal transduction and decidualization. SOCS3 mRNA was 4-fold higher in cells transfected with SOCS3 expression vector compared with mock-transfected cells (P < 0.05; Fig. 5A). Similarly, HESC transfected with SOCS3 expression vector had a higher abundance of SOCS3 protein compared with mock-transfected cells (Fig. 5, B and C). Cells transfected in parallel with GFP expression vector demonstrated fluorescent GFP signal post transfection as visualized by fluorescent microscopy (Fig. 5D). To determine the effect of SOCS3 overexpression in HESC on IL-11 signaling, the effect on IL-11-induced pSTAT3 was investigated. Low levels of pSTAT3 were present in unstimulated HESC (Fig. 5E, top). Addition of IL-11 to mock-transfected cells increased pSTAT3 protein abundance compared with controls, whereas pSTAT3 abundance was not increased by IL-11 in cells transfected with SOCS3-expressing plasmid (Fig. 5E, top). Mock-transfected cells cocultured with IL-11 and anti-IL-11 had reduced pSTAT3 protein abundance compared with mock-transfected cells treated with IL-11. STAT3 abundance was not different between groups (Fig. 5E, bottom). To determine whether SOCS3 overexpression in HESC was able to regulate decidualization, SOCS3-overexpressing HESC were stimulated to decidualize with E+P or cAMP. In both cases, less PRL was secreted compared with mock-transfected cells (P < 0.05; Fig. 5F).
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and 7). The specificity of the antibodies was confirmed by optimization of the staining procedure on mouse endometrium for pSTAT3 and human term placenta for SOCS3; localization was similar to previous publications (27, 28). Nonspecific staining was not seen when the same concentration of rabbit IgG replaced primary antibodies (negative control) (Fig. 7, C and G, insets).
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). The epithelial compartment was a major source of pSTAT3 immunoreactivity with staining apparent in both luminal and glandular epithelium (Figs. 6A and 7, A–E). Interestingly, luminal epithelial pSTAT3 staining was minimal in the menstrual and proliferative phases and increased in the secretory phase, reaching maximal levels in the mid-secretory phase when the endometrium is receptive to blastocyst implantation (Figs. 6A and 7, A–E). Similarly, glandular epithelium was minimally stained for pSTAT3 in the menstrual phase, but this increased moderately in the proliferative phase, reaching maximal levels in the early- to mid-secretory phase (Figs. 6A and 7, A–E). There was very little endometrial stromal pSTAT3 staining throughout the menstrual cycle (Figs. 6A and 7, A–E). Similarly, vascular endothelial and smooth muscle cell pSTAT3 staining was of low intensity, but this increased moderately in endothelial cells during mid- to late-secretory phase of the cycle (Fig. 6A). Importantly, strong staining for pSTAT3 was seen in decidualized stromal cells in the late-secretory phase of the cycle (Figs. 6A and 7E). Subpopulations of leukocytes were strongly positive for pSTAT3 with a maximal number of stained leukocytes present during the menstrual phase (Figs. 6B and 7A). Positively stained leukocytes were also present in the secretory phase of the cycle (Figs. 6B and 7, D and E).
Overall, staining for SOCS3 was maximal in the secretory phase compared with the proliferative phase of the cycle (Figs. 6C and 7, F–J). SOCS3 staining in the luminal and glandular epithelial and decidualized stromal cells was intense in secretory-phase tissue; however, glandular epithelial cells also had moderate staining in the menstrual and proliferative phases (Figs. 6B and 7). Interestingly, SOCS3 staining in glandular epithelium was maximal in the mid-secretory phase and high in luminal epithelium in the late-secretory phase. Nondecidualized stromal cells did not stain for SOCS3 throughout the cycle (Figs. 6C and 7, F–J). Endothelial and associated perivascular cells stained minimally for SOCS3 in secretory-phase tissue, although no staining was found in menstrual and proliferative-phase tissue (Figs. 6C and 7, F and G). Similar to the pSTAT3 staining pattern in leukocytes, subpopulations of highly abundant infiltrating leukocytes were strongly positive for SOCS3 during the menstrual phase. SOCS3 staining in leukocyte subpopulations was of low to moderate intensity in the secretory phase (Figs. 6D and 7F).
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There is increasing evidence that IL-11 has an important function in implantation in humans. Both IL-11 and IL-11R mRNA and protein are present in decidual cells from late-secretory phase and early-pregnant endometrium (2, 20, 29), and nonhuman primates invasive trophoblast cells are a source of IL-11 and IL-11R during early pregnancy, suggesting an involvement in placentation (30). IL-11 secreted by HESC during decidualization advances the decidualization process (2, 16). It is likely to be an early initiator of decidualization because it is stimulated very early on in the process by cAMP and by generators of cAMP such as relaxin and prostaglandin E2, which are known to progress decidualization (15, 16). Importantly, endometrial disturbances in IL-11 secretion are associated with infertility. In vivo, immunoreactive IL-11 is undetectable in endometrium of some women with primary infertility and endometriosis during the implantation window, and IL-11 production is reduced in endometrium of women who have recurrent miscarriages compared with normal fertile women (17, 18). Furthermore, HESC isolated from women with primary infertility secrete lower amounts of IL-11 during cAMP-induced decidualization (19).
Decidualization is critical for successful implantation, although the mechanisms of the process are poorly understood. In vitro studies demonstrate that both cAMP- and PR-mediated pathways are necessary for decidualization, suggesting each pathway has a distinct function (31). The cAMP/PKA pathway is thought to sensitize HESC to the action of progesterone (5, 32). Conversely, treatment of HESC with antiprogestin inhibits cAMP-induced PRL expression (31). Although the importance of progesterone in decidualization in humans is unquestionable, it is also clear that decidualization involves numerous signaling pathways, and cross-talk is required to coordinate the differentiation process.
Activation of cytokine signaling pathways is limited both in magnitude and duration (33). The Janus kinase/STAT pathway is regulated at several levels, which is important physiologically because unregulated signal transduction can have serious consequences (11). In general, the transcription of genes encoding SOCS3 is rapidly induced by exposure to cytokines, and SOCS3 mRNA induction appears to be dependent on the activity of STAT3 (34). SOCS proteins act in a negative-feedback loop with their expression induced by the cytokines they subsequently inhibit (34). In the present study, both SOCS3 and STAT3 protein production increased during HESC decidualization, indicating both may have a role in the process. IL-11 stimulated the activation of STAT3 and SOCS3 mRNA and protein in HESC. Interestingly, SOCS3 mRNA and protein levels were stimulated predominantly by cAMP compared with P during decidualization, even though cAMP- and P-treated cells had decidualized to a similar extent. In contrast, cAMP stimulated STAT3 in HESC only minimally compared with P, even though the extent of decidualization of the cells did not differ between cAMP- or P-mediated decidualization. Interestingly, in cells that were decidualized with addition of either P or cAMP, STAT3 protein was mediated partly by PR. This further demonstrated a role for unliganded PR in cAMP-stimulated differentiation of HESC. That P stimulated STAT3 protein in HESC is in agreement with several studies demonstrating convergence between progesterone and growth factor signaling pathways (23). Progesterone treatment of rats during pregnancy enhances cytoplasmic STAT3 protein abundance in decidualized endometrium; this is inhibited by the progestin antagonist RU486 and is accompanied by an association between PR and STAT3 (24).
Although SOCS3 mRNA and protein increased during P- and cAMP-induced decidualization, SOCS3 levels increased further after the addition of PR antagonist to HESC and resulted in decreased PRL secretion or decidualization. This suggested that SOCS3 was partly regulated by PR and that the levels of SOCS3 mRNA and protein were important in regulating decidualization. In agreement, overexpression of SOCS3 in cells is known to inhibit cytokine signaling (35). In the present study, enforced overexpression of SOCS3 in HESC before the initiation of decidualization retarded the differentiation process, further strengthening the suggestion that SOCS3 is a critical regulator in initiating decidualization. In addition, SOCS3 overexpression in HESC reduced IL-11-induced pSTAT3, indicating the importance of activated STAT3 in initiating the decidualization process.
It is possible that other glycoprotein 130 cytokines such as leukemia inhibitory factor (LIF) and IL-6 regulate pSTAT3 in HESC; however, only IL-11 is known to enhance the differentiation or decidualization of HESC. Similarly, numerous cytokines and growth factors including LIF, IL-6, IL-10, interferon 
, GH, PRL, and leptin stimulate SOCS3 production in a cell-specific manner (11). It is possible that factors other than IL-11, known to be expressed by and involved in decidualization, such as PRL, also regulate SOCS3; this remains to be examined.
Importantly, in vivo evidence of a role for pSTAT3 and SOCS3 in HESC decidualization was also revealed in the present study. First and most relevant to the present study, immunoreactive SOCS3 and pSTAT3 were present in the stroma only in association with decidualization. pSTAT3 and SOCS3 were also detected in a number of cell types, particularly in glandular epithelium in the mid-secretory phase, indicating a function in uterine receptivity; given the importance of endometrial glandular LIF for implantation, it is likely that these molecules are involved in LIF signaling. Similarly, IL-11 immunolocalizes to luminal and glandular epithelium in mid-secretory-phase endometrium, suggesting pSTAT3 and SOCS3 may also be involved in IL-11 action in these cells. Both proteins also localized to blood vessels, both endothelial and vascular smooth muscle cells, but again predominantly in mid-secretory-phase endometrium, suggesting roles in preparing the endometrium for implantation.
Subpopulations of leukocytes in the endometrium produced SOCS3 and pSTAT3 with elevated numbers present in the menstrual phase and moderate amounts found in the secretory phase of the cycle. In particular, uterine natural killer cells and macrophages are increased in the mid-secretory phase, whereas inflammatory leukocytes including neutrophils, eosinophils, and mast cells are located in the endometrium premenstrually and during menstrual bleeding. It is likely that leukocyte subpopulations staining positive for pSTAT3 and SOCS3 during the mid-secretory phase have roles in decidualization, whereas those in the late-secretory or menstrual phases have a function in endometrial breakdown and/or repair. In agreement, inflammatory actions for SOCS3 and STAT3 have been proposed. STAT3 is activated in macrophages, neutrophils, natural killer cells, and mast cells (36, 37, 38, 39, 40, 41). Furthermore, SOCS3 is involved in macrophage and neutrophil cell function (42, 43).
This study confirmed that IL-11 has roles in initiation and progression of HESC decidualization and demonstrated for the first time that progesterone and cAMP pathways differentially regulated the IL-11 signaling components STAT3 and SOCS3 during decidualization. Furthermore, a regulatory role for SOCS3 in HESC decidualization was identified. The data strengthen the case that IL-11 is a critical driver of decidualization and support the notion that targeting IL-11, SOCS3, or pSTAT3 may be useful in either enhancing or blocking human embryo implantation.
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Acknowledgments |
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Footnotes |
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E.D., C.S., Y.-L.T., and L.A.S. have nothing to declare.
First Published Online May 18, 2006
Abbreviations: E, Estradiol-17ß; GFP, green fluorescent protein; HESC, human endometrial stromal cell; HRP, horseradish peroxidase; IL-11R, IL-11 receptor-; LIF, leukemia inhibitory factor; P, medroxyprogesterone acetate; PKA, protein kinase A; PR, progesterone receptor; PRL, prolactin; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TBS, Tris-buffered saline.
Received February 28, 2006.
Accepted for publication May 3, 2006.
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function is required for normal decidua and fetoplacental development in mice. Genes Dev 12:2234–2243 and leukemia inhibitory factor are dysregulated in endometrium of infertile women with endometriosis during the implantation window. J Reprod Immunol 69:53–64 and interleukin-11 protein in the endometrium of normal fertile women and women with recurrent miscarriage. J Reprod Immunol 64:145–155[CrossRef][Medline] immunolocalisation at primate implantation sites supports a role for IL-11 in placentation and fetal development. Reprod Biol Endocrinol 1:34[CrossRef][Medline] in human neutrophils. J Leukoc Biol 65:391–396[Abstract] regulates IL-21 and IL-21R expression in human NK and T cells. J Leukoc Biol 76:416–422This article has been cited by other articles:
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  B. Cloke, K. Huhtinen, L. Fusi, T. Kajihara, M. Yliheikkila, K.-K. Ho, G. Teklenburg, S. Lavery, M. C. Jones, G. Trew, et al. The Androgen and Progesterone Receptors Regulate Distinct Gene Networks and Cellular Functions in Decidualizing Endometrium Endocrinology, September 1, 2008; 149(9): 4462 - 4474. [Abstract] [Full Text] [PDF]
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 J. S. Fitzgerald, T. G. Poehlmann, E. Schleussner, and U. R. Markert Trophoblast invasion: the role of intracellular cytokine signalling via signal transducer and activator of transcription 3 (STAT3) Hum. Reprod. Update, April 17, 2008; (2008) dmn010v1. [Abstract] [Full Text] [PDF]
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 L. Bao, S. Devi, J. Bowen-Shauver, S. Ferguson-Gottschall, L. Robb, and G. Gibori The Role of Interleukin-11 in Pregnancy Involves Up-Regulation of {alpha}2-Macroglobulin Gene through Janus Kinase 2-Signal Transducer and Activator of Transcription 3 Pathway in the Decidua Mol. Endocrinol., December 1, 2006; 20(12): 3240 - 3250. [Abstract] [Full Text] [PDF]
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), proliferative phase (P, 
), early-secretory phase (ES,
), mid-secretory phase (MS,
), and late-secretory phase (LS,
). An immunohistochemical score for leukocytes ranging from 0–3 was allocated based on the abundance of positively stained leukocytes: 0 (absent), 1 (few positive), 2 (abundant positive), or 3 (highly abundant leukocytes). A, Staining for pSTAT3; B, pSTAT3 staining in leukocytes. C and D, An immunohistochemical score for leukocytes ranging from 0–4 was allocated based on the abundance of positively stained leukocytes. C, Staining for SOCS3; D, SOCS3 staining in leukocytes.