Joanne Yap

Leukemia Inhibitory Factor Enhances Endometrial Stromal Cell Decidualization in Humans and Mice

Adequate differentiation or decidualization of endometrial stromal cells (ESC) is critical for successful pregnancy in humans and rodents. Here, we investigated the role of leukemia inhibitory factor (LIF) in human and murine decidualization. Ex vivo human (H) ESC decidualization was induced by estrogen (E, 10−8 M) plus medroxyprogesterone acetate (MPA, 10−7 M). Exogenous LIF (≥50 ng/ml) induced STAT3 phosphorylation in non-decidualized and decidualized HESC and enhanced E+MPA-induced decidualization (measured by PRL secretion, P<0.05). LIF mRNA in HESC was down-regulated by decidualization treatment (E+MPA) whereas LIF receptor (R) mRNA was up-regulated, suggesting that the decidualization stimulus ‘primed’ HESC for LIF action, but that factors not present in our in vitro model were required to induce LIF expression. Ex vivo first trimester decidual biopsies secreted >100 pg/mg G-CSF, IL6, IL8, and MCP1. Decidualized HESC secreted IL6, IL8, IL15 and MCP1. LIF (50 ng/ml) up-regulated IL6 and IL15 (P<0.05) secretion in decidualized HESC compared to 0.5 ng/ml LIF. In murine endometrium, LIF and LIFR immunolocalized to decidualized stromal cells on day 5 of gestation (day 0 = day of plug detection). Western blotting confirmed that LIF and the LIFR were up-regulated in intra-implantation sites compared to inter-implantation sites on Day 5 of gestation. To determine the role of LIF during in vivo murine decidualization, intra-peritoneal injections of a long-acting LIF antagonist (PEGLA; 900 or 1200 µg) were given just post-attachment, during the initiation of decidualization on day 4. PEGLA treatment reduced implantation site decidual area (P<0.05) and desmin staining immuno-intensity (P<0.05) compared to control on day 6 of gestation. This study demonstrated that LIF was an important regulator of decidualization in humans and mice and data provides insight into the processes underlying decidualization, which are important for understanding implantation and placentation.

Decidual-Secreted Factors Alter Invasive Trophoblast Membrane and Secreted Proteins Implying a Role for Decidual Cell Regulation of Placentation

Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the ‘extravillous trophoblast’ (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10−8 M), medroxyprogesterone acetate (10−7 M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN) before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition. Overall, we have demonstrated the potential of a proteomics approach to identify novel proteins expressed by EVT and to uncover the mechanisms leading to disease states.

Interleukin 11 regulates endometrial cancer cell adhesion and migration via STAT3

Endometrial carcinoma is the most common gynaecological malignancy. There is however a lack of curative therapies, especially for patients diagnosed with late stage, recurrent or aggressive disease, who have a poor prognosis. Interleukin (IL) 11 is a pleiotropic cytokine that has a role in a number of cancers including colon and breast cancer. IL11 was recently found to be upregulated in endometrial cancers, however the function of IL11 in endometrial cancer is not known. This study aimed to determine the effects of IL11 on endometrial cancer cell proliferation, adhesion and migration. Three endometrial cancer cell lines, Ishikawa, HEC-1A and AN3CA (derived from endometrial cancers grade I, II and III, respectively), were used to determine the effect of IL11 on endometrial cancer cell function. Cell proliferation and viability were assessed by BrdU and Wst-1 assays. Cell adhesion to the extracellular matrix proteins fibronectin, collagen I and IV, vitronectin and laminin was assessed. Modified boyden chambers were utilized to access IL11 action on migration and invasion, respectively. The specific effect of IL11 action on these processes was determined using a unique IL11 inhibitor. IL11 phosphorylated (p)-STAT3 protein abundance in all 3 cell lines but had no effect on pERK and pAKT abundance. Similarly, IL11 had no effect on cell proliferation and viability but increased adhesion of ANC3A cells to fibronectin while having no effect on the other extracellular matrix proteins. IL11 did not alter the adhesive properties of the Ishikawa and HEC-1A cells. In the AN3CA cells, IL11 treatment resulted in a 50% increase in migration and co-treatment with the specific IL11 inhibitor or a STAT3 inhibitor abolished the effect. This study shows a role for IL11 in endometrial cancer and suggests IL11 may be involved in endometrial cancer development and thus may be useful as a therapeutic target.

Interleukin (IL)11 mediates protein secretion and modification in human extravillous trophoblasts

BACKGROUND Human trophoblast invasion and differentiation are essential for a successful pregnancy outcome. Dysregulation of these processes can lead to placental pathologies such as pre-eclampsia. The molecular mechanisms; however, are poorly understood. Interleukin (IL)11--a cytokine that regulates endometrial epithelial cell adhesion, trophoblast motility and invasion during implantation--may be involved in some of these processes. METHODS AND RESULTS The effect of IL11 on protein expression was investigated in trophoblastic HTR8/SVneo cells and primary extravillous trophoblasts (EVTs) purified from first- trimester placentas. Two-dimension (2D)-differential in-gel electrophoresis analyses revealed that 731 spots were significantly differentially regulated by IL11 in HTR8/SVneo cells: seven spots were analyzed by liquid chromatography-tandem mass spectrometry and 14 unique proteins identified. Protein disulfide isomerase family A, member 3 (PDIA3; endoplasmic reticulum p57) and glucose-regulated protein 78 (GRP78) were further validated to be regulated by IL11 in HTR8/SVneo and primary EVT. One dimension western blot analysis confirmed that PDIA3 was down-regulated in EVT. 2D western blot analysis revealed that GRP78 was post-translationally modified following IL11 treatment. Moreover, IL11 stimulated the secretion of GRP78 in EVT. CONCLUSIONS Data suggest that IL11, possibly via signal transducers and activators of transcription 3 signaling pathway, regulates PDIA3 protein expression and modification/secretion of GRP78. This is the first study to identify PDIA3 and GRP78 as IL11 targets in invasive trophoblasts and identifies a possible mechanism by which IL11 regulates trophoblast function.

Interleukin-11 alters placentation and causes preeclampsia features in mice

Significance Preeclampsia is an insidious disease, unique to humans, affecting ∼8% of pregnancies. There are no early detection tests or pharmacological treatments. Impaired placentation is widely accepted to contribute to the pathogenesis. However, the mechanisms remain elusive, given the complications of studying first-trimester placental development in women. A major limitation for the study of new treatments is the lack of available animal models that recapitulate the full spectrum of preeclampsia features. We have developed a mouse model characterized by elevated levels of the cytokine Interleukin-11 (IL11). This study provides evidence of a novel pathway causative of preeclampsia features in vivo. It also provides a novel in vivo mouse model that is useful for preclinical studies to test potential therapeutics. Preeclampsia (PE) is a pregnancy-specific disorder characterized by hypertension and proteinuria after 20 wk gestation. Abnormal extravillous trophoblast (EVT) invasion and remodeling of uterine spiral arterioles is thought to contribute to PE development. Interleukin-11 (IL11) impedes human EVT invasion in vitro and is elevated in PE decidua in women. We demonstrate that IL11 administered to mice causes development of PE features. Immunohistochemistry shows IL11 compromises trophoblast invasion, spiral artery remodeling, and placentation, leading to increased systolic blood pressure (SBP), proteinuria, and intrauterine growth restriction, although nonpregnant mice were unaffected. Real-time PCR array analysis identified pregnancy-associated plasma protein A2 (PAPPA2), associated with PE in women, as an IL11 regulated target. IL11 increased PAPPA2 serum and placental tissue levels in mice. In vitro, IL11 compromised primary human EVT invasion, whereas siRNA knockdown of PAPPA2 alleviated the effect. Genes regulating uterine natural killer (uNK) recruitment and differentiation were down-regulated and uNK cells were reduced after IL11 treatment in mice. IL11 withdrawal in mice at onset of PE features reduced SBP and proteinuria to control levels and alleviated placental labyrinth defects. In women, placental IL11 immunostaining levels increased in PE pregnancies and in serum collected from women before development of early-onset PE, shown by ELISA. These results indicate that elevated IL11 levels result in physiological changes at the maternal–fetal interface, contribute to abnormal placentation, and lead to the development of PE. Targeting placental IL11 may provide a new treatment option for PE.

Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

BackgroundDuring the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL) 11 regulates human endometrial epithelial cells (hEEC) adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach.MethodsUsing a 2D-differential in-gel electrophoresis (DIGE) electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2) and flotillin-1 (FLOT1), were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa) and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle).Results2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary hEEC and ECC-1 whilst, the lipid-raft protein FLOT1 demonstrated punctate staining in the apical surface of ECC-1 plasma membranes and was upregulated in the epithelium in the receptive phase of the menstrual cycle (p lower or equal 0.05).ConclusionsThis is the first study to use a proteomics approach to identify hEEC plasma membrane proteins that may be useful as infertility markers or pharmacological targets for fertility regulation.

Interleukin 11 is upregulated in uterine lavage and endometrial cancer cells in women with endometrial carcinoma.

BackgroundInterleukin (IL) 11 is produced by human endometrium and endometrial cancer tissue. It has roles in endometrial epithelial cell adhesion and trophoblast cell invasion, two important processes in cancer progression. This study aimed to determine the levels of IL11 in uterine lavage fluid in women with endometrial cancer and postmenopausal women. It further aimed to determine the levels of IL11 protein and its signaling molecules in human endometrial cancer of varying grades, and endometrium from postmenopausal women and IL11 signalling mechanisms in endometrial cancer cell lines.MethodsIL11 levels in uterine lavage were measured by ELISA. IL11, IL11 receptor(R) α, phosphorylated (p) STAT3 and SOCS3 were examined by immunohistochemistry in endometrial carcinomas and in control endometrium from postmenopausal women and normal cycling women. The effect of IL11 on pSTAT3/STAT3 and SOCS3 protein abundance in endometrial cancer cell lines and non-cancer endometrial epithelial cells was determined by Western blot.ResultsIL11 was present in uterine flushings and was significantly higher in women with Grade 1 carcinomas compared to postmenopausal women (p < 0.05). IL11 immunostaining was significantly elevated in the endometrial tumour epithelial cells from Grade 1 and 3 compared to endometrial epithelium from postmenopausal and cycling women. IL11Rα immunostaining intensity was increased in cancer epithelium in the Grades 1 and 2 tumours compared to epithelium from postmenopausal women. Both IL11 and IL11Rα localized to vascular endothelial and smooth muscle cells while IL11 also localized to subsets of leucocytes in the cancer tissues. pSTAT3 was found in both the tumour epithelial and stromal compartments but was maximal in the tumour epithelial cells, while SOCS3 was predominantly found in the tumour epithelial cells. pSTAT3 staining intensity was significantly higher in Grade 1 and 2 tumour epithelial cells compared to epithelial cells from cycling and postmenopausal women. SOCS3 staining intensity did not differ between between each tumour and postmenopausal endometrial epithelium but SOCS3 in cycling endometrium was significantly higher compared to postmonopausal and Tumour Grades 2 and 3. IL11 increased pSTAT3/STAT3 in all tumour cell lines, while SOCS3 abundance was increased only in one tumour cell line.ConclusionsThe present study suggests that IL11 in uterine washings may be useful as a diagnostic marker for early stage endometrial cancer. It indicates that IL11, along with its specific receptor, IL11Rα, and downstream signalling molecules, STAT3 and SOCS3, are likely to play a role in the progression of endometrial carcinoma. The precise role of IL11 in endometrial cancer remains to be elucidated.

Galectin-7 is important for normal uterine repair following menstruation

Menstruation involves the shedding of the functional layer of the endometrium in the absence of pregnancy. At sites where tissue shedding is complete, re-epithelialization of the tissue is essential for repair and termination of bleeding. The complement of growth factors that mediate post-menstrual endometrial repair are yet to be completely elucidated. Galectins regulate many cell functions important for post-menstrual repair, such as cell adhesion and migration. Galectin-7 has a well characterized role in re-epithelialization and wound healing. We hypothesized that galectin-7 would be important in re-epithelialization during post-menstrual repair. We aimed to identify endometrial expression of galectin-7 in women undergoing normal endometrial repair and in women with amenorrhoea who do not experience endometrial breakdown and repair, and to determine whether galectin-7 enhances endometrial re-epithelialization in vitro. Galectin-7 immunolocalized to the endometrial luminal and glandular epithelium during the late secretory and menstrual phases, and to decidualized stroma in regions exhibiting tissue breakdown. Immunostaining intensity was significantly reduced in the endometrium of women with amenorrhoea compared with normally cycling woman. ELISA identified galectin-7 in menstrual fluid at significantly elevated levels compared with matched peripheral plasma. Exogenous galectin-7 (2.5 µg/ml) significantly enhanced endometrial epithelial wound repair in vitro; this was abrogated by inhibition of integrin binding. Galectin-7 elevated epithelial expression of extracellular matrix-related molecules likely involved in repair including β-catenin, contactin and TGF-β1. In conclusion, galectin-7 is produced by the premenstrual and menstrual endometrium, where it accumulates in menstrual fluid and likely acts as a paracrine factor to facilitate post-menstrual endometrial re-epithelialization.