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Prince Henry’s Institute of Medical Research (P.P., L.A.S., A.T., E.D.), and Department of Obstetrics and Gynecology (P.P., U.M., C.W., E.M.W.), Monash Medical Centre, Clayton, Victoria 3168, Australia
Address all correspondence and requests for reprints to: Premila Paiva, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: premila.paiva{at}princehenrys.org.
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Abstract |
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(IL-11R) are maximally expressed in the decidua and chorionic villi during early pregnancy; however, the role of IL-11 in trophoblast function is unknown. Therefore, we examined whether IL-11R is expressed in human first trimester implantation sites, and whether IL-11 influences proliferation and migration of a human extravillous trophoblast (EVT)-hybridoma cell line and primary EVT cells, used as models for EVT. Immunoreactive IL-11R localized to subpopulations of interstitial and endovascular EVT cells in vivo. In EVT cells in vitro, IL-11: 1) stimulated phosphorylation of signal transducer and activator of transcription-3; 2) was without effect on EVT cell proliferation; and 3) stimulated significant migration of EVT-hybridoma cells (no endogenous IL-11), whereas in primary EVT, blocking endogenous IL-11 inhibited EVT migration by 30–40%. These data demonstrate that IL-11 stimulates human EVT migration, but not proliferation, likely via signal transducer and activator of transcription-3, indicating an important role for IL-11 in placentation. |
Introduction |
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IL-11 belongs to the IL-6-type family of cytokines that includes leukemia inhibitory factor, IL-6, oncostatin M, ciliary neurotrophic factor, and cardiotropin-1. IL-11 signals via a heterodimeric receptor complex comprising the specific IL-11 receptor (IL-11R) chain and the common signaling component gp130. IL-11 acts primarily through the janus kinase/signal transducers and activators of transcription (STATs) signal transduction pathway. IL-11 has effects on both cellular proliferation and differentiation via its ability to modulate the expression of genes involved in the G1-S phase cell cycle transition, apoptosis, and terminal differentiation (4, 5, 6, 7).
IL-11 signaling is unequivocally required for blastocyst implantation in mice (8, 9). Female mice with a null mutation in the IL-11R gene are infertile due to a defective decidualization response, resulting in dysregulated trophoblast invasion and proliferation, and necrotic loss of the fetus. In the cynomolgus monkey, IL-11 and IL-11R are expressed in subsets of invasive EVTs in very early implantation sites, suggesting an important role for IL-11 in trophoblast invasion in primates (10). In humans, during early pregnancy, IL-11 and its receptor IL-11R are maximally expressed in both the decidua and in the chorionic villi of the developing placenta. Ligand and receptor colocalize in villous CT and ST cells (10). Importantly, there are abnormalities in decidual and villous trophoblast IL-11 expression in women with anembryonic pregnancies, leading to early pregnancy loss (11). Furthermore, IL-11 is reduced in the serum of women with early spontaneous abortions (12). This suggests an important autocrine/paracrine action of IL-11 on both human decidual and trophoblast cells during placental development. Although a role for IL-11 in decidualization of human stromal cells during pregnancy is well established (13, 14), its effect on trophoblast function is unknown.
We hypothesized that decidual and/or trophoblast IL-11 regulates human EVT function. We confirmed that IL-11R is expressed by EVTs in first trimester human implantation sites. To elucidate the functional significance of IL-11R on human EVTs, we examined the effects of IL-11 on the proliferative and migratory properties of these cells. Due to the restricted availability of primary human EVT cells, extended studies were performed using the human EVT-hybridoma cell line, ACI.88, a JEG-3-primary trophoblast hybrid (15, 16), followed by more limited studies using primary EVTs isolated from human chorionic villous explant cultures, to verify the findings.
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Materials and Methods |
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Dual immunohistochemistry
Immunohistochemistry for IL-11R and cytokeratin was performed in human first trimester implantation sites. Term placenta was used as a positive control (10). Paraffin sections (5 µm) were dewaxed in Histosol (Sigma-Aldrich, St. Louis, MO) and rehydrated before digestion with 0.2% trypsin (Sigma-Aldrich) in 0.2% calcium chloride/Tris-buffered saline (TBS) (pH 7.6) for 10 min at 37 C. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide. Tissues were incubated with nonimmune block (10% normal horse serum; 2% normal human serum) for 30 min, followed by mouse antihuman IL-11R (clone 4D12) (7.5 µg/ml) or nonimmune mouse IgG (negative control) at 4 C for 16 h, then biotinylated horse antimouse IgG (1:200; Vector Laboratories Inc., Burlingame, CA) for 30 min, and streptavidin-biotin-peroxidase complex ABC (DakoCytomation, Glostrup, Denmark). IL-11R protein was visualized as a brown precipitate using diaminobenzidine tetrahydrochloride substrate (DakoCytomation). Unbound avidin-biotin sites were blocked using avidin/biotin blocking solution (Zymed Laboratories, San Francisco, CA). Mouse antihuman cytokeratin (CAM 5.2; Becton Dickinson Immunocytochemistry Systems, San Jose, CA), which detects cytokeratins 7 and 8, was biotinylated in vitro using the Animal Research Kit (DakoCytomation) and applied to sections at 6 µg/ml for 16 h at 4 C. Sections were washed and incubated with streptavidin-biotin-alkaline phosphatase (DakoCytomation). Alkaline phosphatase activity was visualized in blue using Tris-HCl buffer [100 mM (pH 8.5)], Naphthol-AS-MX-phosphate as substrate (0.2 mg/ml; Sigma-Aldrich), and 1 mM levamisole (0.12 mg/ml). Fast Blue BB (Sigma-Aldrich) was added as chromogen (0.2 mg/ml). Sections were rinsed and mounted using Faramount aqueous mounting medium (DakoCytomation).
Placental villous explant culture and EVT cell isolation
Primary EVT cells were prepared from tips of placental terminal villi (five to eight samples) as previously described (17). Small pieces of terminal villi were coated with Matrigel (BD Biosciences, Franklin Lakes, NJ) diluted 1:3, allowed to adhere to tissue culture flasks, and maintained in DMEM/F12 (Invitrogen) culture media containing 10% fetal calf serum (FCS) (Invitrogen) and 1% antibiotics to allow extensive outgrowth. Outgrowing cells were detached by digestion with 0.03% trypsin-EDTA (Invitrogen) in PBS (pH 7.6) for 5 min at 37 C, followed by neutralization of trypsin activity with 10% FCS in M199 media. The dispersed cells were separated from tissue remnants using a 40 µm filter (BD Biosciences), placed in culture, and expanded. Cells were incubated with mouse antihuman cluster of differentiation antigen-9 (CD9) antibody (DakoCytomation) diluted 1:50, for 15 min at 4 C. Cells were then washed and incubated with mouse IgG-coated magnetic microbeads (MACS kit; Miltenyl Biotech, Bergisch-Gladbach, Germany) for a further 15 min at 4 C, after which trophoblast cells were separated from the mixed population of cells by negative selection via immunomagnetic column purification.
EVT cell culture
EVT cells were grown to 60–70% confluency in RPMI (ACI.88; Sigma-Aldrich) (18, 19) or DMEM/F12 (primary EVT; Invitrogen) containing 10% FCS and 1% antibiotics (penicillin, streptomycin, and Fungizone; Commonwealth Serum Laboratories, Melbourne, Australia). Cells were washed with PBS free of Ca2+ and Mg2+ (PBS–) and transferred to media containing 0.2% FCS (experimental media) for 48 h. At the end of the experiment, cell numbers were determined using a hemocytometer, conditioned media collected, and RNA extracted from the cells. Each experiment was performed two to three times.
Immunocytochemistry
EVT cells were grown on chamber slides (BD Biosciences), then washed with cold PBS–, and fixed in ice-cold ethanol for 10 min. To confirm trophoblast cell identity and purity of isolated EVTs, cells were immunostained using mouse antihuman cytokeratin 7 (clone OV-TL 12/30, diluted 1:250; DakoCytomation), a marker of trophoblast cells (20), and mouse-antihuman CD9 (diluted 1:50; DakoCytomation) to evaluate the proportion of contaminating villous stromal cells within the isolated EVT population. Nonimmune mouse IgG (DakoCytomation) substituted for primary antibody at the same protein concentration was used as a negative control. To examine the expression of IL-11R in EVT cells, cells were stained with mouse antihuman IL-11R (10 µg/ml). Primary human endometrial stromal cells were used as a positive control (14).
For all immunocytochemistry, rehydrated cells were treated with 3% hydrogen peroxide for 10 min washed with TBS and incubated with nonimmune block for 30 min. Primary antibody was applied to sections at 4 C for 16 h, followed by biotinylated horse antimouse IgG (1:200) for 30 min and streptavidin-biotin-peroxidase complex ABC. Peroxidase activity was visualized using diaminobenzidine tetrahydrochloride substrate. Cells were counterstained with Harris hematoxylin (Sigma-Aldrich), air dried, and mounted.
RNA extraction and purification
Total cellular RNA was extracted with the RNeasy Minikit (QIAGEN Sciences, Germantown, MD), according to the manufacturer’s instructions. All samples were treated with RNase-free DNase (Ambion, Austin, TX) to remove any genomic DNA. RNA samples (1 µg) were reverse transcribed at 46 C for 1.5 h in a 20-µl reaction mixture using avian myeloblastosis mosaic virus reverse transcriptase (Promega, Madison, WI) and 100 ng random hexanucleotide primers (Amersham Biosciences, Piscataway, NJ). Negative controls were included in which either RNA or the reverse transcriptase was omitted. cDNA generated was subsequently amplified by PCR (Hybaid Express Block Cycler; ThermoHybaid, Ashford, UK) using specific primers (Sigma-Aldrich) at 0.25 pmol/µl for IL-11, IL-11R, gp130, and 18S, as previously described, with term placenta and primary human endometrial stromal cells used as positive controls (13, 14). RT-PCR was performed in a total volume of 40 µl containing 1 µl cDNA, single-strength PCR buffer, 20 µM deoxynucleotide triphosphates, 0.25 pmol/µl forward and reverse primers, and 2.5 U Taq DNA polymerase (Roche Diagnostics, Laval, Québec, Canada). PCR products were electrophoresed on a 2% agarose gel to establish the specificity of the PCR reaction and product size.
IL-11 ELISA
IL-11 secreted from ACI.88 and primary EVT cells was assayed by ELISA (R&D Systems Inc., Minneapolis, MN). Media from primary human endometrial stromal cells were included as quality control (to monitor precision) and positive control samples (14). The lower detection limit of the assay was 15 pg/ml, and interassay and intraassay variabilities were 7.1 and 2.8%, respectively (21).
SDS-PAGE and Western blotting
Phosphorylated STAT-3 (pSTAT-3) was investigated by Western blot. EVT cells were grown to confluence, transferred to experimental medium for 24 h, and then treated with recombinant human (rh) IL-11 (a gift from Lorraine Robb, Walter and Eliza Hall Institute, Melbourne, Victoria, Australia) at 1, 10, and 100 ng/ml, or vehicle for 10 min. Cells were washed with ice-cold PBS–, lysed in 250 µl/flask ice-cold lysis buffer [50 mM Tris Base, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, and 25 mM B glycerolphosphate (pH 7.5)], and 2 µl/well protease inhibitors cocktail set (Calbiochem, San Diego, CA) was added. Cell extracts were centrifuged, and supernatant protein was quantified using the BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of protein were resolved on 10% SDS-PAGE gels and transferred to nitrocellulose membranes then blocked with 5% nonfat dry milk in TBS with 0.1% Tween 20 (Bio-Rad Laboratories, Hercules, CA), and probed separately with antibodies specific for phospho-STAT-3 and total STAT-3 (Tyr705, 5.6 ng/ml; Cell Signaling Technology Inc., Beverly, MA) overnight at 4 C. The membranes were washed in TBS with 0.1% Tween 20 and incubated for 1 h with horseradish peroxidase (HRP)-conjugated rabbit secondary antibody (1:1500; DakoCytomation). HRP activity was detected using enhanced chemiluminescence reagent (Pierce). 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. Membranes were stripped and incubated with anti-β-tubulin (1:5000; Sigma-Aldrich) as loading control.
Modified (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay-WST-1 assay
WST-1 (Roche) was assayed according to the manufacturer’s instructions. Cells were seeded at 5 x 104 cells per well in 12-well plates (Nunc GmbH and Co. KG, Weisbaden, Germany) and grown to preconfluence (30–40% confluence). Cells were rinsed twice with PBS–, and medium was replaced with experimental media for 24 h to induce growth arrest. Thereafter, five or more wells were incubated with rh IL-11 (1, 10, and 100 ng/ml), vehicle, 0.75 µM insulin, or 15% FCS (positive controls) for 48 h at 37 C. WST-1 dye (diluted 1:10) was then added to each well and incubated for 4 h at 37 C. The number of viable cells before and at the end of treatment was evaluated by measuring the absorbance at 450 nm. The experiment was repeated three times.
5-Bromo-2'-deoxyuridine (BrdU) incorporation assay
5 x 103 ACI.88 or primary EVT cells were seeded in 96-well plates (Nunc), grown to preconfluence (30–40%), washed with PBS–, and then transferred into experimental media for 24 h. Thereafter, five or more wells were incubated with vehicle, 100 ng/ml rh IL-11, or 15% FCS (positive control) for 48 h at 37 C. BrdU (10 mM) was added to each well and incubated for 2 h. BrdU incorporation was measured at 450 nm. The experiment was repeated three times (ACI.88) and twice (primary EVT).
Migration assay
Directed cell migration (chemotaxis) of EVT cells was assessed using an in vitro migration assay (Chemicon, Melbourne, Australia), as previously described (19). Briefly, AC.I88 cells or primary EVT cells were serum starved overnight, trypsinized, and cultured at a density of 5 x 104 cells in migration chamber inserts (upper wells). The lower wells contained vehicle, rh IL-11 (1, 10, and 100 ng/ml), 100 nM CX3CL1 (R&D Systems), or 10% FCS (positive controls) (n 
5 wells per treatment). Primary EVTs were also incubated with antihuman IL-11 neutralizing antibody (100 ng/ml; R&D Systems) or goat IgG (100 ng/ml; Sigma-Aldrich) before addition to upper wells. After 19–22 h, media containing nonmigrated cells were removed from the upper wells. Cells at the lower surface of the inserts were detached, lysed, and quantitated using CyQuant GR dye. Each migration assay was repeated two to three times.
Statistical analyses
Data are expressed as mean ± SEM fold change for each treatment compared with control. Statistical analysis was performed on raw data using one-way ANOVA, followed by Tukey’s post hoc test (P < 0.05 taken as significant) after testing for normal distribution using PRISM version 3.00 (GraphPad Software, San Diego, CA) for Windows.
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Results |
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in human EVT was localized in both ST and CT cells of anchoring villi (Fig. 1
, A and B). IL-11R was also localized in EVT in the cell column in both proximal and distal regions (Fig. 1, A and B), in subsets of iEVT invading the decidua (Fig. 1C), and in perivascular EVT (pEVT) and eEVT (Fig. 1D).
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) but negative for CD9 (Fig. 2B). ACI.88 cells stained positive for cytokeratin 7 (Fig. 2C). In both cases, nonimmune mouse IgG produced no staining (Fig 2, A and C, inset).
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, gp130, and IL-11 in EVT cells (Fig. 3A) and gp130 (Fig. 3B) mRNA were similarly expressed by both primary EVT and ACI.88. Immunoreactive IL-11R was likewise detected in both primary EVT (n = 3) (Fig. 3C) and ACI.88 cells (Fig. 3D). No immunostaining was observed with nonimmune mouse IgG (Fig. 3C, inset).
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). Although IL-11 was secreted by primary EVT (Fig. 3F), there was no detectable IL-11 secreted by ACI.88 cells.
Western blot analysis of tyrosine pSTAT-3 in EVT cells
IL-11 stimulated pSTAT-3 in both primary EVT (Fig. 4A) and ACI.88 cells (Fig. 4B) In primary EVT, pSTAT-3 was detected after vehicle treatment but was up-regulated upon stimulation with IL-11 (Fig. 4A). In ACI.88 cells, pSTAT-3 was absent after vehicle-treatment but was similarly up-regulated after IL-11 treatment at doses from 1–100 ng/ml. IL-11 had no effect on STAT-3 protein abundance (Fig. 4, A and B, bottom panels).
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) and DNA synthesis (BrdU assay) (Fig. 5, B and C). The total number of viable ACI.88 cells increased by 25% over the 48-h treatment (compared with 0 h; P < 0.05) (Fig. 5A). However, addition of IL-11 at 1–100 ng/ml to culture media had no effect on viable ACI.88 cell number compared with controls, whereas 15% FCS or insulin (0.75 µm) (positive controls) stimulated a 75 and 25% increase, respectively, in viable cell numbers compared with controls [P < 0.01]).
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), although the positive control (15% FCS) stimulated a 30% increase in the rate of DNA synthesis compared with controls (P < 0.01). Similarly, IL-11 did not affect primary EVT cell proliferation (Fig. 5C).
Effect of IL-11 on EVT cell migration
Treatment of ACI.88 cells with IL-11 (1–100 ng/ml) resulted in a dose-dependent increase in cell migration (Fig. 6A) compared with controls, with maximal migration observed at the highest dose (P < 0.001). The chemokine CX3CL1 and 10% FCS acted as positive controls. Exogenous IL-11 had no effect on primary EVT migration compared with controls (Fig. 6B). However, IL-11 neutralizing antibody significantly reduced EVT cell migration by 30–40% in comparison to IgG-treated controls (P < 0.01).
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Discussion |
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IL-11R was detected in vivo for the first time in first trimester EVT cells of the cell column and subpopulations of iEVTs and pEVTs/eEVTs. This pattern of EVT subset-specific localization of IL-11R is consistent with that in early implantation sites of the nonhuman primate (cynomolgus monkey) (10). IL-11 mRNA and protein are expressed in human endometrium throughout the menstrual cycle (11, 14, 22). IL-11 increases in luminal epithelium and glands in the mid-late secretory phase (the receptive phase) (13) but is maximal in decidua and chorionic villi during early pregnancy (first trimester) (11), with its expression declining by the beginning of the second trimester (23). Interestingly, abnormalities in first trimester decidual and villous trophoblast IL-11 are identified in anembryonic pregnancies that lead to pregnancy loss (11), whereas IL-11 is reduced in the serum of women with early spontaneous abortions (12). These reports suggest a critical role for IL-11 signaling during early pregnancy. IL-11 has been shown to progress human endometrial stromal cell decidualization in vitro (14, 22). Although it has been suggested that IL-11 may regulate trophoblast function, this is the first study to extend the known functions of IL-11 (of either decidual or trophoblast origin) to include an influence on EVT migration during early human placental development.
Our results indicate that IL-11 does not regulate EVT proliferation within the cell column of anchoring villi, therefore, the functional significance of IL-11R expression by EVT cells of the cell column is still unclear. Although IL-11 was initially described as a growth factor regulating hematopoiesis, particularly the proliferation of hematopoietic stem cells (4), increasing evidence suggests that IL-11 affects cellular differentiation, although this is cell-type dependent. IL-11 inhibits adipocyte differentiation (7) but stimulates osteoblast (24) and neuronal differentiation (6), and endometrial stromal cell decidualization (14).
While migration of human EVT-hybridoma cells significantly increased in response to exogenous IL-11, it was necessary to block endogenous IL-11 production by primary EVT to demonstrate an effect of IL-11 on cell migration, suggesting likely autocrine and paracrine actions of IL-11 in EVT migration. In agreement, IL-11 stimulates migration of human breast cancer (25) and T cells (26).
EVT function during placental development is clearly regulated by a large number of factors produced at the fetal-maternal interface. Our findings indicate that IL-11 is one such factor regulating migration, but not proliferation, of EVT. These cellular events are often independently regulated during placental development. Whereas TGF-β down-regulates both trophoblast proliferation (27) and migration (28), TGF- up-regulates trophoblast proliferation with no effect on migration (29). Similarly, IGF-2 stimulates trophoblast migration, but not proliferation (29), as demonstrated here for IL-11.
In most cell types, IL-11 mediates its effects through janus kinase/STAT pathway (30), with STAT-3 being the predominant signaling mediator. However, IL-11 signaling can also involve the Ras-MAPK pathway (31), and cross talk between STAT/MAPK signaling pathways has been demonstrated (31). This study indicates that IL-11 exerts its biological effect in EVT cells via pSTAT-3. pSTAT-3 is important for mouse reproduction: STAT-3 deficiency leads to pregnancy loss due to a failure of decidualization (32). Trophoblast migration/invasion is often likened to that exhibited by malignant tumor cells during tumorigenesis, although in contrast to tumor migration/invasion, that of trophoblast is precisely regulated both spatially and temporally (2). STAT-3 is of special interest in the context of malignant cellular properties because aberrant STAT-3 activity is directly linked with oncogenesis (33). Furthermore, analysis of primary tumors and cell lines derived from tumors indicates inappropriate STAT-3 activation in a variety of human cancers, likely contributing to their migratory/invasive properties. Constitutive STAT-3 activity is detected in invasive ex vivo human first trimester trophoblast cells and choriocarcinoma cell lines, whereas being absent in term placenta (34). This supports our data suggesting an important correlation between STAT-3 activity and trophoblast migratory ability/invasiveness. However, other factors, including leukemia inhibitory factor (35), hepatocyte growth factor (36, 37, 38), and leptin, may also regulate trophoblast migration via STAT-3.
In summary, this study demonstrates a role for IL-11 acting via STAT-3 in human EVT migration. IL-11 may regulate EVT migration through the decidua and into the spiral arterioles, thereby facilitating anchorage into the uterus and remodeling of the spiral arterioles. Failure of trophoblast migration/invasion and arterial remodeling leads to spontaneous abortion during early pregnancy, whereas defects in these processes are hallmarks of preeclampsia and intrauterine fetal growth restriction. Dysregulated trophoblast migration/invasion due to defective IL-11 production may contribute to the pathophysiology of such conditions. Thus, IL-11, via its effects on EVT migration, may play an important regulatory role in the initiation of a successful pregnancy. The downstream target genes involved in IL-11-mediated EVT migration remain to be identified.
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Acknowledgments |
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-antibody, Ms. Natalie Hannan for excellent technical assistance, and the patients who generously donated the tissues. |
Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online August 16, 2007
Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; CT, cytotrophoblast; eEVT, endovascular extravillous trophoblast; EVT, extravillous trophoblast; FCS, fetal calf serum; HRP, horseradish peroxidase; iEVT, interstitial extravillous trophoblast; IL-11R, IL-11 receptor ; pEVT, perivascular extravillous trophoblast; pSTAT-3, phosphorylated signal transducer and activator of transcription-3; rh, recombinant human; ST, syncytiotrophoblast; STAT, signal transducer and activator of transcription; TBS, Tris-buffered saline.
Received April 23, 2007.
Accepted for publication August 8, 2007.
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References |
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function is required for normal decidua and fetoplacental development in mice. Genes Dev 12:2234–2243 immunolocalisation at primate implantation sites supports a role for IL-11 in placentation and fetal development. Reprod Biol Endocrinol 1:34[CrossRef][Medline]This article has been cited by other articles:
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  P. Paiva, L. A. Salamonsen, U. Manuelpillai, and E. Dimitriadis Interleukin 11 Inhibits Human Trophoblast Invasion Indicating a Likely Role in the Decidual Restraint of Trophoblast Invasion During Placentation Biol Reprod, February 1, 2009; 80(2): 302 - 310. [Abstract] [Full Text] [PDF]
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 A. Tapia, L. A. Salamonsen, U. Manuelpillai, and E. Dimitriadis Leukemia inhibitory factor promotes human first trimester extravillous trophoblast adhesion to extracellular matrix and secretion of tissue inhibitor of metalloproteinases-1 and -2 Hum. Reprod., August 1, 2008; 23(8): 1724 - 1732. [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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