Human Uterine Epithelial Cells Research Articles

Sex Hormones and Aging Modulate Interferon Lambda 1 Production and Signaling by Human Uterine Epithelial Cells and Fibroblasts.

Estradiol (E2) and progesterone (P) have potent effects on immune function in the human uterine endometrium which is essential for creating an environment conducive for successful reproduction. Type III/lambda (λ) interferons (IFN) are implicated in immune defense of the placenta against viral pathogens, which occurs against the backdrop of high E2 and P levels. However, the effect of E2 and P in modulating the expression and function of IFNλ1 in the non-pregnant human uterine endometrium is unknown. We generated purified in vitro cultures of human uterine epithelial cells and stromal fibroblast cells recovered from hysterectomy specimens. Poly (I:C), a viral dsRNA mimic, potently increased secretion of IFNλ1 by both epithelial cells and fibroblasts. The secretion of IFNλ1 by epithelial cells significantly increased with increasing age following poly (I:C) stimulation. Stimulation of either cell type with E2 (5x10-8M) or P (1x10-7M) had no effect on expression or secretion of IFNλ1 either alone or in the presence of poly (I:C). E2 suppressed the IFNλ1-induced upregulation of the antiviral IFN-stimulated genes (ISGs) MxA, OAS2 and ISG15 in epithelial cells, but not fibroblasts. Estrogen receptor alpha (ERα) blockade using Raloxifene indicated that E2 mediated its inhibitory effects on ISG expression via ERα. In contrast to E2, P potentiated the upregulation of ISG15 in response to IFNλ1 but had no effect on MxA and OAS2 in epithelial cells. Our results demonstrate that the effects of E2 and P on IFNλ1-induced ISGs are cell-type specific. E2-mediated suppression, and selective P-mediated stimulation, of IFNλ1-induced ISG expression in uterine epithelial cells suggest that the effects of IFNλ1 varies with menstrual cycle stage, pregnancy, and menopausal status. The suppressive effect of E2 could be a potential mechanism by which ascending pathogens from the lower reproductive tract can infect the pregnant and non-pregnant endometrium.

Proteinase Activated Receptors Mediate the Trypsin-Induced Ca2 + Signaling in Human Uterine Epithelial Cells.

Embryo implantation is a complex and tightly regulated process. In humans, uterine luminal epithelium functions as a biosensor gauging the embryo quality and transmitting this information to the underlying endometrial stromal cells. This quality control ensures that only high quality embryos are implanted, while aberrant ones are rejected. The mechanisms of the embryo-uterine mucosa crosstalk remain incompletely understood. Trypsin, a serine protease secreted by the blastocyst, has been implicated in the cross-signaling. Here we address the mechanisms by which trypsin triggers the intracellular calcium signaling in uterine epithelium. We found that protease-activated G-protein coupled receptors are the main mechanism mediating the effects of trypsin in human uterine epithelium. In addition, trypsin activates the epithelial sodium channels thus increasing the intracellular Na+ concentration and promoting Ca2+ entry on the reverse mode of the sodium/calcium exchanger.

Annexin A1/Formyl Peptide Receptor Pathway Controls Uterine Receptivity to the Blastocyst.

Embryo implantation into the uterine wall is a highly modulated, complex process. We previously demonstrated that Annexin A1 (AnxA1), which is a protein secreted by epithelial and inflammatory cells in the uterine microenvironment, controls embryo implantation in vivo. Here, we decipher the effects of recombinant AnxA1 in this phenomenon by using human trophoblast cell (BeWo) spheroids and uterine epithelial cells (Ishikawa; IK). AnxA1-treated IK cells demonstrated greater levels of spheroid adherence and upregulation of the tight junction molecules claudin-1 and zona occludens-1, as well as the glycoprotein mucin-1 (Muc-1). The latter effect of AnxA1 was not mediated through IL-6 secreted from IK cells, a known inducer of Muc-1 expression. Rather, these effects of AnxA1 involved activation of the formyl peptide receptors FPR1 and FPR2, as pharmacological blockade of FPR1 or FPR1/FPR2 abrogated such responses. The downstream actions of AnxA1 were mediated through the ERK1/2 phosphorylation pathway and F-actin polymerization in IK cells, as blockade of ERK1/2 phosphorylation reversed AnxA1-induced Muc-1 and claudin-1 expression. Moreover, FPR2 activation by AnxA1 induced vascular endothelial growth factor (VEGF) secretion by IK cells, and the supernatant of AnxA1-treated IK cells evoked angiogenesis in vitro. In conclusion, these data highlight the role of the AnxA1/FPR1/FPR2 pathway in uterine epithelial control of blastocyst implantation.

IL-27 Expression and Responsiveness in Human Uterine Epithelial Cells and Fibroblasts In Vitro and the Role of Estradiol.

Interleukin (IL)-27 is a pleiotropic cytokine that regulates multiple aspects of innate and adaptive immunity, but whose role in immune protection of the female reproductive tract is unknown. Although not constitutively expressed by human uterine epithelial cells and fibroblasts in culture, IL-27 secretion was upregulated after treatment with the viral ligand poly (I:C) in a type I interferon (IFN)-dependent manner, with higher levels measured in fibroblasts than epithelial cells. Estradiol increased poly (I:C)-induced IL-27 production by fibroblasts, but not epithelial cells. While both cell types expressed the IL-27 receptor, only fibroblasts responded to recombinant IL-27 with increased expression of the antiviral genes, APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) and MxA, and the tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase (IDO). Estradiol inhibited IL-27-mediated induction of IDO in fibroblasts through estrogen receptor alpha, but had no effect on APOBEC3G. IL-27 pretreatment also potentiated poly (I:C) upregulation of the antiviral genes, OAS2 and APOBEC3G, in fibroblasts. Thus, IL-27 is part of the antiviral response by uterine cells against potential pathogens. The effect of estradiol on IL-27 production and sensitivity by fibroblasts demonstrates a selective hormone action on individual cell types in the uterus and suggests that IL-27 may have differential effects during the menstrual cycle.

Chlamydia trachomatis regulates innate immune barrier integrity and mediates cytokine and antimicrobial responses in human uterine ECC-1 epithelial cells.

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide and known to increase the risk for HIV acquisition. Few studies have investigated how infection of epithelial cells compromises barrier integrity and antimicrobial response. ECC-1 cells, a human uterine epithelial cell line, were treated with live and heat-killed C.trachomatis. Epithelial barrier integrity measured as transepithelial resistance (TER), chemokines antimicrobial levels, and antimicrobial mRNA expression was measured by ELISA and Real-time RT-PCR. Epithelial barrier integrity was compromised when cells were infected with live, but not with heat-killed, C.trachomatis. IL-8 secretion by ECC-1 cells increased in response to live and heat-killed C.trachomatis, while MCP-1, HBD2 and trappin2/elafin secretion decreased with live C.trachomatis. Live C.trachomatis suppresses ECC-1 innate immune responses by compromising the barrier integrity, inhibiting secretion of MCP-1, HBD2, and trappin-2/elafin. Differential responses between live and heat-killed Chlamydia indicate which immune responses are dependent on ECC-1 infection rather than the extracellular presence of Chlamydia.

Regulatory function of β1,4-galactosyltransferase I expression on Lewis-Y glycan and embryo implantation

β1,4-Galactosyltransferase I (β1,4-GalT-I), a key enzyme in glycobiology, mediates several biological mechanisms. However, the correlation between β-1,4-GalT-I expression in the uterine endometrium and embryo implantation remains unclear. This study aims to elucidate the relationship between β1,4-GalT-I and LewisY (LeY) glycan during embryo implantation. So far, using green fluorescent protein as an indicator, β1,4-GalT-I interference plasmid (pcDNA3.0-siGalT I), overexpression plasmid (pcDNA3.0-HA-GalT I), interference control plasmid (control pcDNA3.0-siGalT I), and empty vector (pcDNA3.0) were transfected into human uterine epithelial RL95-2 cells that imitate the receptive endometrium. Invasive embryos at pre-implantation and treated RL95-2 cells were co-cultured to determine embryo attachment in each of the transfection groups. The results showed that plasmid transfection was successful in all the groups. β1,4-GalT-I and Fucosyltransferase 1 (FUT1) gene expression declined in the interference group, and the synthesis of LeY decreased accordingly, but the expression of this antigen increased in the overexpression group. After co-culturing of the embryos and 36h transfection of RL95-2, the results of these in vitro implantation models showed that the attachment rate was lower in the interference group (30.0±0.2%) than in the untreated group (50.0±0.6%), empty vector group (50.0±0.2%), and interference control group (46.7±0.6%), however, it was highest in the overexpression group (70.0±0.2%). These results indicated that β1,4-galactosyltransferase I possibly regulate mutual uterus–embryo adhesion and embryo implantation by regulating cell surface LeY glycan expression.

Erratum to: EMMPRIN is secreted by human uterine epithelial cells in microvesicles and stimulates metalloproteinase production by human uterine fibroblast cells

Braundmeier AG, Dayger CA, Mehrotra P, Belton RJ Jr, Nowak RA. EMMPRIN is secreted by human uterine epithelial cells in microvesicles and stimulates metalloproteinase production by human uterine fibroblast cells. Reprod Sci. 2012;19(12):1292–1301 (Original doi:10.1177/1933719112450332).

OPN induces FoxM1 expression and localization through ERK 1/2, AKT, and p38 signaling pathway in HEC-1A cells.

Mammalian embryo implantation is an extremely complex process and requires endometrial receptivity. In order to establish this receptivity, sequential proliferation and differentiation during the menstrual cycle is necessary. Forkhead box M1 (FoxM1) is described as a major oncogenic transcription factor in tumor initiation, promotion and progression. According to these functions, we believe that FoxM1 should also play an essential role in embryo implantation. Osteopontin (OPN), an adhesion molecule, has been studied extensively in reproduction. In this study, we observed the expression and distribution of FoxM1 during the proliferative-phase and secretory-phase human endometrium and the pre-implantation mouse uterus firstly. Then we observed the relationship between OPN and FoxM1. Our results showed that FoxM1 was mainly distributed in glandular epithelium. OPN increased the expression of FoxM1 in the human uterine epithelial cell line HEC-1A cells in a time- and concentration-dependent manner. OPN regulates FoxM1 to influence HEC-1A cell proliferation through extracellular regulated protein kinases (ERK 1/2), protein kinase B (PKB, AKT), and the p38 mitogen activated protein kinases (p38MAPK, p38) signaling pathway. Inhibition of ERK 1/2, AKT and p38 suppressed OPN-induced FoxM1 expression and location. Our data indicate that FoxM1 might be regulated by OPN to influence endometrial proliferation to establish endometrial receptivity.

High expression of CD147 in endometriosis and its role in regulating apoptosis and migration in human endometrial cells (1093.3)

Endometriosis is a common gynecological disease, which contributes to 40% of infertile women in reproductive age. CD147 has been found highly expressed in ectopic endometrium and stimulated the MMPs in human uterine fibroblasts. However, it is poorly understood if and how CD147 affects endometrium epithelial cell migration during the progression of endometriosis. We demonstrate here that the expression of human CD147 was significantly higher in ovarian ectopic endometrium than that of normal endometrium by real‐time PCR. Interfering CD147 function decreased cell migration and cell viability in the normal human uterine epithelial cells (HES). Surprisingly, MMP‐2 expression and activity were not changed when HES treated with anti‐CD147 antibody. Further studies revealed that immunodepletion of CD147 induced apoptosis in HES cells, which led to Caspase‐3 and poly ADP‐ribose polymerase (PARP) activation. The present study demonstrated that CD147 regulates apoptosis and the cell migration in a MMP‐2 independent manner in human endometrial epithelial cells and that abnormally high expression of CD147 in the ectopic endometriosis lesions may contribute to the pathogenesis of endometriosis.Grant Funding Source: Supported by NSFC (31100841) and Shenzhen Projects of Science and Technology (JCYJ20120614085604107)

Elevated expression of CD147 in patients with endometriosis and its role in regulating apoptosis and migration of human endometrial cells

To examine the expression of CD147 in 60 human endometriosis lesions and how CD147 regulates migration and apoptosis in human uterine epithelial (HESs) cells. Experimental clinical study and laboratory-based investigation. Hospital and academic research center. Sixty women with chocolate cysts and 16 control women without endometriosis. Human uterine epithelial cells were treated with anti-CD147 antibody. Real-time polymerase chain reaction for detecting CD147 expression in 60 human endometriosis lesions; migration assay and CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) assay for cell functional investigation; Western blot for detecting protein levels; gelatin zymography for evaluating the activity of matrix metalloproteinase-2 (MMP-2) in cultured cells. Expression of CD147 was significantly higher in ectopic endometrial tissues from patients with endometriosis than in normal endometrial tissues. Interference with CD147 function led to decreased migration and cell viability in HESs cells. Surprisingly, MMP-2 expression and activity were not changed after treating HESs cells with anti-CD147 antibody. Further examination revealed that immunodepletion of CD147 induced apoptosis in HESs cells, leading to the activation of caspase 3 and poly(ADP-ribose) polymerase. The results of the present study suggest that abnormally high expression of CD147 in ovarian endometriosis lesions with enhanced cell survival (reduced apoptosis) and migration, in an MMP-2-independent manner, may underlie the progression of endometriosis in humans.

Global gene expression analysis in human uterine epithelial cells defines new targets of glucocorticoid and estradiol antagonism.

In preparation for embryo implantation and pregnancy, the uterine epithelium undergoes a genomic and biological transition that mediates adhesion and invasion of the blastocyst. These events resemble an inflammatory response, and the immune system likely takes an active role in the establishment and maintenance of pregnancy. Although glucocorticoids are primary mediators of the immune system, the functional role of glucocorticoid signaling in the uterine epithelium is not well defined. To investigate the dynamic relationship between glucocorticoids and reproductive hormones, we performed whole-genome microarray analysis in a human uterine endometrial cancer cell line (ECC1 cells) treated with the synthetic glucocorticoid dexamethasone (Dex) alone or in combination with estradiol (E₂). Over 10,000 genes were significantly regulated in the presence of Dex and/or E₂. Surprisingly, unique targets of Dex and E₂ together represented the largest group of regulated genes. Ingenuity pathway analysis found both overlapping and independent regulated networks for each hormone. Several hundred genes were found to be coregulated by Dex and E₂, including several that were antagonistically regulated. The effects of glucocorticoids and E₂ are mediated primarily through the glucocorticoid receptor (NR3C1) and estrogen receptor (ESR1), respectively. In silico promoter analysis revealed that NR3C1 and ESR1 response elements are enriched in antagonistically regulated genes, and signaling through these receptors was required for antagonism. Glucocorticoid and E₂ antagonism of target genes may represent a critical junction between the immune system and female reproductive system. Moreover, identification and ontology analysis of glucocorticoid-regulated genes in a uterine epithelial-like cell line suggests that glucocorticoid signaling regulates important biological functions, including immune cell trafficking and embryonic development.

EMMPRIN Is Secreted by Human Uterine Epithelial Cells in Microvesicles and Stimulates Metalloproteinase Production by Human Uterine Fibroblast Cells

Endometrial remodeling is a physiological process involved in the gynecological disease, endometriosis. Tissue remodeling is directed by uterine fibroblast production of matrix metalloproteinases (MMPs). Several MMPs are regulated directly by the protein extracellular matrix metalloproteinase inducer (EMMPRIN) and also by proinflammatory cytokines such as interleukin (IL)1-α/β. We hypothesized that human uterine epithelial cells (HESs) secrete intact EMMPRIN to stimulate MMPs. Microvesicles from HES cell-conditioned medium (CM) expressed intact EMMPRIN protein. Treatment of HES cells with estradiol or phorbyl 12-myristate-13-acetate increased the release of EMMPRIN-containing microvesicles. The HES CM stimulated MMP-1, -2, and -3 messenger RNA levels in human uterine fibroblasts (HUFs) and EMMPRIN immunodepletion from HES-cell concentrated CM reduced MMP stimulation (P < .05). Treatment of HUF cells with low concentrations of IL-1β/α stimulated MMP production (P < .05). These results indicate that HES cells regulate MMP production by HUF cells by secretion of EMMPRIN, in response to ovarian hormones, proinflammatory cytokines as well as activation of protein kinase C.

Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus

Sex hormone signaling regulates a variety of functions in the uterine endometrium essential for embryo implantation and immunity. Epithelial cells of the uterine endometrium are the target of the coordinated actions of estradiol (E(2)) and progesterone. However, little information exists regarding the interplay of estrogens with glucocorticoids in this tissue. Using the human uterine epithelial cell line ECC1, E(2) was found to antagonize induction of the glucocorticoid-induced leucine zipper (GILZ) gene expression, which is associated with several of the immune-related functions of glucocorticoids. Interestingly, E(2) antagonizes glucocorticoid regulated nascent RNA GILZ expression within 1 h of hormone treatment. Repression of glucocorticoid-induced GILZ expression requires the estrogen receptor (ER), because both treatment with the ER-antagonist ICI 182,780 and small interfering RNA knockdown of ERα block E(2)'s ability to repress GILZ gene expression. Antagonism of glucocorticoid-induced GILZ expression may not be unique to ERα, as the ERβ agonist Liquiritigenin is also able to antagonize glucocorticoid signaling. Transcriptional regulation appears to be at the level of promoter binding. Both the glucocorticoid receptor and ERα are recruited to regions of the GILZ promoter containing glucocorticoid response elements and the transcriptional start site. Glucocorticoid receptor binding to these regions in the presence of dexamethasone decreases with E(2) treatment. GILZ gene expression was also found to be repressed in the whole mouse uterus treated with a combination of dexamethasone and E(2). Regulation of the antiinflammatory gene GILZ by glucocorticoids and E(2) suggests cross talk between the immune modulating functions of glucocorticoids and the reproductive actions of estradiol signaling.

Stimulation of GPR30 Increases Release of EMMPRIN-Containing Microvesicles in Human Uterine Epithelial Cells

Uterine remodeling is highly dependent on the glycosylated transmembrane protein extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN). Previous studies indicate estradiol can increase EMMPRIN expression in uterine cells and promote subsequent induction of MMP production. The aim of this study was to investigate the role of G protein-coupled receptor 30 (GPR30) stimulation on EMMPRIN microvesicle release in the human uterine epithelial cell line hTERT-EEC (EECs). We examined EMMPRIN release by human EECs in response to GPR30 stimulation by microvesicle isolation, Western blot, and immunocytochemistry. We employed a pharmacological approach using the GPR30-selective agonist G1 and the antagonist G15 to determine the receptor specificity of this response. We demonstrated GPR30 expression in EECs and release of EMMPRIN in microvesicles in response to stimulation of GPR30. G1, estradiol, and cholera toxin stimulated EMMPRIN release in microvesicles as detected by Western blot and immunocytochemistry, indicating that stimulation of GPR30 can induce EMMPRIN microvesicle release. These data indicate that EMMPRIN release in microvesicles can be mediated by stimulation of GPR30 in human EECs, suggesting that inappropriate stimulation or expression of this receptor may be significant in uterine pathology.

Differential expression of LeY and fucosyltransferase IV correlates with the receptivity of RL95‐2 and HEC‐1A human uterine epithelial cells

Adhesion molecules expressed on the uterine endometrium are potential receptive markers in embryo implantation. RL95-2 and HEC-1A cell lines represent the high- and low-receptive endometrial epithelium respectively. LeY (Lewis Y) is a difucosylated oligosaccharide highly expressed in the endometrium of some species during implantation. α1, 3 fucosylation of LeY is catalysed by FUT4 (fucosyltransferase IV), the key synthesis enzyme for LeY. We investigated whether the difference in receptivity between the 2 cell lines was related to different expressions of LeY and FUT4. RL95-2 cells expressed a higher level of LeY and FUT4 than HEC-1A cells, as shown by immunofluorescent staining, RT-PCR (reverse transcription-PCR) or Western blotting. FUT4-siRNA (small interfering RNA) transfection down-regulated FUT4 and LeY in RL95-2 cells, and inhibited the adhesion of the embryonic cells (JAR) to RL95-2 cell monolayer. FUT4-cDNA, however, increased the expression of FUT4 and LeY in HEC-1A cells, and increased the adhesion of embryonic cells to HEC-1A cell monolayer. Alterations of LeY level by up- or down-regulation of FUT4 also mediated EGFR (epidermal growth factor receptor)/MAPK (mitogen-activated protein kinase) signalling pathway. To conclude, the expression of LeY and FUT4 correlates with endometrial receptivity, making them potential new markers for the evaluation of endometrial receptivity.

Uterine Epithelial Cells Specifically Induce Interferon-Stimulated Genes in Response to Polyinosinic-Polycytidylic Acid Independently of Estradiol

Interferon β (IFNβ) is an antiviral cytokine secreted in response to pathogenic exposure that creates a restrictive intracellular environment through the action of downstream interferon-stimulated genes (ISG). The objective of this study was to examine the expression of IFNβ and ISG in both human uterine epithelial cells (UEC) and the ECC-1 uterine epithelial cell line and determine if expression changes with TLR stimulation and hormone exposure. Stimulation of primary uterine epithelial cells and ECC-1 cells with the TLR3 agonist poly (I∶C) induced the mRNA expression of IFNβ, MxA, OAS2 and PKR. Other TLR agonists including imiquimod and CpG had no effect on either IFNβ or ISG expression. In contrast to ECC-1 cell responses which were slower, maximal IFNβ upregulation in UEC occurred 3 hours post-stimulation and preceded the ISG response which peaked approximately 12 hours after poly (I∶C) exposure. Unexpectedly, estradiol, either alone or prior to treatment with poly (I∶C), had no effect on IFNβ or ISG expression. Blockade of the IFN receptor abrogated the upregulation of MxA, OAS2 and PKR. Furthermore, neutralizing antibodies against IFNβ partially inhibited the upregulation of all three ISG. Estradiol, directly and in the presence of poly (I∶C) had no effect on IFNβ and ISG expression. These results indicate that uterine epithelial cells are important sentinels of the innate immune system and demonstrate that uterine epithelial cells are capable of mounting a rapid IFN-mediated antiviral response that is independent of estradiol and is therefore potentially sustained throughout the menstrual cycle to aid in the defense of the uterus against potential pathogens.

A high-throughput in vitro model of human embryo attachment

To develop a simple, high-throughput and widely applicable in vitro human implantation model that assesses trophoblast spheroid attachment to human uterine epithelial cells. Experimental study to establish and validate the model. Cell culture. CELL(S): Lines BeWo, RL95-2, and AN3-CA. Labeling trophoblast spheroids with green fluorescence, selecting spheroids of size similar to implanting blastocysts by use of cell strainers, and assessing spheroid attachment by use of an automated microplate reader. Establishment of a simple, reliable, and high-throughput in vitro model to study human embryo implantation. The assay enabled rapid fluorometric assessment of spheroid attachment to human endometrial epithelial cells (RL95-2 and AN3-CA) under different experimental conditions. The high-throughput assay was confirmed by conventional counting method to be highly reproducible and accurate. The described methodology provides for the first time a high-throughput assay for the study of human embryo implantation and a promising tool for screening potential inhibitors or enhancers of embryo attachment.

Abstract 2032: Regulation of MUC1 expression by activated EGFR and PPARγ in human uterine and pancreatic cancer cell lines

Abstract Many epithelial tumors including endometrial and pancreatic cancers, overexpress the high molecular weight, transmembrane mucin, MUC1, on their cell surface. Overexpression and aberrant glycosylation of MUC1 makes tumor cells poorly adherent, increases drug resistance and promotes cancer cell metastasis. MUC1 has three distinct domains: an extracellular domain (ECD) composed of a tandem repeat region rich in serine, threonine and proline; a transmembrane domain (TM) and; a short cytoplasmic tail domain (CT) involved in a variety of signal transduction events. Previous studies (Wang et al. 2010. Mol. Endocrinol. 24: 1368-1379) demonstrated that the Peroxisome Proliferator Activated Receptor γ (PPARγ) agonist, rosiglitazone severely inhibits progesterone stimulated MUC1 expression. Two independent approaches were used to determine if rosiglitazone as well as inhibitors of Epidermal Growth Factor Receptor (EGFR) tyrosine kinase activity reduced MUC1 expression in the human uterine epithelial cell line, HES, the uterine adenocarcinoma cell line, HEC-1A, and in the human pancreatic adenocarcinoma cell lines, HPAFII and CAPAN2. EGFR activation by Epidermal Growth Factor (EGF) stimulated (1.5-2 fold) MUC1 protein expression 1.5-2.0 fold in both uterine and pancreatic cancer cells, while treatment with EGFR tyrosine kinase inhibitor, AG1478, reduced both basal and stimulated MUC1 protein expression. Furthermore, the PPARγ agonist, rosiglitatzone, markedly (&amp;gt; 90%) reduced EGFR-driven MUC1 expression detected by antibodies directed against the MUC1 ECD and CT. A combination of biochemical and molecular biological assays have been used to identify the transcriptional and signal transduction pathways activated by EGFR leading to increased MUC1 expression and the impact of AG1478 and rosiglitazone on these events. Understanding the molecular basis of how these agents reduce MUC1 expression may offer novel therapeutic avenues to improve cancer chemotherapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2032. doi:10.1158/1538-7445.AM2011-2032

SLeX/L-selectin mediates adhesion in vitro implantation model

The complex implantation process is initiated by the recognition and adhesion between the embryo and uterine endometrial epithelium. The expression and interactions between the adhesive molecules from both fetal and maternal sides are crucial for the successful implantation. In this study, we aimed to investigate the expression and adhesive function of sLeX on the trophoblasts and L-selectin on uterine epithelial cells mediated the adhesion at the fetal-maternal interface, and to further explore whether this adhesion system could induce endometrial apoptosis, using in vitro implantation model consisting of the human trophoblast cell line (JAR) and human uterine epithelial cell line (RL95-2). The results showed that sLeX was expressed on JAR cells by indirect immunofluorescence staining. After transfection of JAR cells with fucosyltransferase VII (FUT7) which is the key enzyme for sLeX synthesis, the expression of FUT7 and sLeX synthesis were increased, and the percent adhesion of trophoblast cells to RL95-2 cell monolayer was significantly increased (P<0.01). L-selectin was strongly expressed but not E- and P-selectin on epithelial RL95-2 cells by RT-PCR, Western blot. Blocking L-selectin with specific antibody or heparin pretreatment in RL95-2 cells inhibited the adhesion of JAR cells to RL95-2 cell monolayer. Furthermore, regulating the expression of sLeX on JAR cells or blocking L-selectin on RL95-2 cells could activate the apoptosis of uterine epithelial cells. These results suggest the sLeX/L-selectin adhesion system at fetal-maternal interface not only mediates the adhesion of embryo to uterine epithelium, but also effectively induces the apoptosis in uterine epithelium. The study supplies a molecular basis for the elucidation of the initial recognition and adhesion during embryo implantation.

Endogenous EMMPRIN Expression by Human Uterine Fibroblast Cells Regulates Metalloproteinase Production, Proliferation, and Decidualization.

The uterine endometrium undergoes extensive proliferation and remodeling in preparation for embryo implantation. This tissue remodeling occurs, in part, through upregulation of matrix metalloproteinases (MMPs) in uterine stromal fibroblasts. Extracellular matrix metalloproteinase inducer (EMMPRIN) stimulates MMP production in a number of carcinoma cell lines and also in human uterine fibroblast (HUF) cells. We have shown in previous studies that human uterine epithelial cells (HES) express the cell surface form of EMMPRIN and secrete full length EMMPRIN via microvesicle shedding. This soluble form of EMMPRIN acts in a paracrine manner on underlying stromal fibroblast cells (HUF) to stimulate MMPs. The goals of the present study were to determine 1) whether soluble EMMPRIN is the major secretory product of HES cells that stimulates MMP production by HUF cells; 2) whether expression of EMMPRIN by HUF cells is necessary for these cells to be able to respond to HES secreted EMMPRIN; and 3) whether EMMPRIN expression in HUF cells is important for cell proliferation and decidualization. We harvested conditioned medium from HES cells and confirmed that this contains the full length form of EMMPRIN via immunoblotting. Treatment of HUF cells with either unconcentrated or 50-fold concentrated HES-cell conditioned medium (p<0.05) increased MMP-1, -2, and -3 mRNA levels without altering endogenous EMMPRIN mRNA levels. Removal of the soluble form of EMMPRIN from HES-cell conditioned medium by immunodepletion using a specific anti-human EMMPRIN antibody caused a reduction in MMP stimulation (7.7 fold for MMP-1, 4.2 fold for MMP-2 and 12.4 fold for MMP-3 (p<0.05)). Our results also showed that endogenous expression of EMMPRIN by HUF cells was important for MMP stimulation by HES-cell conditioned medium. Transfection of HUF cells with EMMPRIN siRNAs resulted in a dramatic reduction in the stimulatory effect of HES-cell conditioned medium on MMP mRNA levels (57 fold MMP-1, 69 fold MMP-2) but a marked increase in MMP-3 mRNA levels (36.7 fold, p<0.05). Treatment of uterine fibroblasts with EMMPRIN siRNAs also decreased the proliferative response of these cells to serum by 40% as measured by DNA synthesis assays. Effects of EMMPRIN siRNA on decidualization of stromal cells was determined by treating cells for 10 days with estradiol, progesterone and cAMP in the presence or absence of EMMPRIN siRNAs. Immunoblotting showed that EMMPRIN protein levels were significantly reduced by day 4 of treatment and nondetectable on days 6-10. Levels of prolactin mRNA, a marker of decidualization, increased 150-200 fold in control cells by days 6-8 but were increased less than 50 fold in EMMPRIN siRNA transfected cells. MMP-7 mRNA levels were increased 20-30 fold in control cells but only 5-7 fold in EMMPRIN siRNA transfected cells. These data indicate that EMMPRIN expression by uterine fibroblast cells is important not only for regulation of MMP production but also appears to play a role in regulating proliferation and decidualization of these cells. Future studies will focus on the role of EMMPRIN in cell cycle regulation in uterine stromal cells. (Supported by SCCPRR U54HD20093). (platform)