Prokineticin Ligands and Receptors Are Expressed in the Human Fetal Ovary and Regulate Germ Cell Expression of COX2.

CONTEXT: Fetal ovarian development and primordial follicle formation underpin future female fertility. Prokineticin (PROK) ligands regulate cell survival, proliferation, and angiogenesis in adult reproductive tissues including the ovary. However, their expression and function during fetal ovarian development remains unclear. OBJECTIVE: This study aimed to investigate expression and localization of the PROK ligands, receptors, and their downstream transcriptional targets in the human fetal ovary. SETTING: This study was conducted at the University of Edinburgh. PARTICIPANTS: Ovaries were collected from 37 morphologically normal human fetuses. DESIGN AND MAIN OUTCOME MEASURES: mRNA and protein expression of PROK ligands and receptors was determined in human fetal ovaries using qRT-PCR, immunoblotting, and immunohistochemistry. Functional studies were performed using a human germ cell tumor line (TCam-2) stably transfected with Prokineticin receptor 1 (PROKR1). RESULTS: Expression of PROK1 and PROKR1 was significantly higher in mid-gestation ovaries (17-20 wk) than at earlier gestations (8-11 and 14-16 wk). PROK2 significantly increased across the gestations examined. PROKR2 expression remained unchanged. PROK ligand and receptor proteins were predominantly localized to germ cells (including oocytes within primordial follicles) and endothelial cells, indicating these cell types to be the targets of PROK signaling in the human fetal ovary. PROK1 treatment of a germ cell line stably expressing PROKR1 resulted in ERK phosphorylation and elevated COX2 expression. CONCLUSIONS: Developmental changes in expression and regulation of COX2 and phosphorylated ERK (pERK) by PROK1 suggest that PROK ligands may be novel regulators of germ cell development in the human fetal ovary, interacting within a network of growth and survival factors prior to primordial follicle formation.

Autocrine and paracrine mechanisms of prostaglandin E2 action on trophoblast/conceptus cells through the prostaglandin E2 receptor (PTGER2) during implantation.

The conceptus and endometrium secrete large amounts of prostaglandin E2 (PGE2) into the porcine uterine lumen during the periimplantation period. We hypothesized that PGE2 acts on conceptus/trophoblast cells through auto- and paracrine mechanisms. Real-time RT-PCR analysis revealed that PGE2 receptor (PTGER)2 mRNA was 14-fold greater in conceptuses/trophoblasts on days 14-25 (implantation and early placentation period) vs preimplantation day 10-13 conceptuses (P < .05). Similarly, expression of PTGER2 protein increased during implantation. Conceptus expression of PTGER4 mRNA and protein did not differ on days 10-19. PGE2 stimulated PTGER2 mRNA expression in day 15 trophoblast cells through PTGER2 receptor signaling. PGE2 elevated aromatase expression and estradiol-17ß secretion by trophoblast cells. Moreover, PGE2 and the PTGER2 agonist, butaprost, increased the adhesive capacity of both human HTR-8/SVneo trophoblast and primary porcine trophoblast cells to extracellular matrix. This PGE2-induced alteration in trophoblast cell adhesion to extracellular matrix was abolished by incubation of these cells with AH6809 (PTGER2 antagonist), ITGAVB3-directed tetrapeptide arg-gly-asp-ser or integrin ITGAVB3 antibody. PGE2 stimulated adhesion of porcine trophoblast cells via the estrogen receptor and MEK/MAPK signaling pathway. PGE2 induced phosphorylation of MAPK1/MAPK3 through PTGER2 and up-regulated expression of cell adhesion proteins such as focal adhesion kinase and intercellular adhesion molecule-1. Our study indicates that elevated PGE2 in the periimplantation uterine lumen stimulates conceptus PTGER2 expression, which in turn promotes trophoblast adhesion via integrins, and synthesis and secretion of the porcine embryonic signal estradiol-17ß. Moreover, the mechanism through which PGE2 increases trophoblast adhesion is not species specific because it is PTGER2- and integrin-dependent in both porcine and human trophoblast cells.

Seminal plasma induces angiogenic chemokine expression in cervical cancer cells and regulates vascular function.

Cervical cancer is one of the leading gynecological malignancies in women. We have recently shown that seminal plasma (SP) can regulate the inflammatory cyclooxygenase-prostaglandin pathway and enhance the growth of cervical epithelial tumours in vivo by promoting cellular proliferation and alteration of vascular function. This study investigated the molecular mechanism whereby SP regulates vascular function using an in vitro model system of HeLa cervical adenocarcinoma cells and human umbilical vein endothelial cells (HUVECs). We found that SP rapidly enhanced the expression of the angiogenic chemokines, interleukin (IL)-8 and growth regulated oncogene alpha (GRO) in HeLa cells in a time-dependent manner. We investigated the molecular mechanism of SP-mediated regulation of IL-8 and GRO using a panel of chemical inhibitors of cell signalling. We found that treatment of HeLa cells with SP elevated expression of IL-8 and GRO by transactivation of the epidermal growth factor receptor, activation of extracellular signal-regulated kinase and induction of cyclooxygenase enzymes and nuclear factor kappa B. We investigated the impact of IL-8 and GRO, released from HeLa cells after treatment with SP, on vascular function using a co-culture model system of conditioned medium (CM) from HeLa cells, treated with or without SP, and HUVECs. We found that CM from HeLa cells induced the arrangement of endothelial cells into a network of tube-like structures via the CXCR2 receptor on HUVECs. Taken together our data outline a molecular mechanism whereby SP can alter vascular function in cervical cancers via the pro-angiogenic chemokines, IL-8 and GRO.

Seminal plasma enhances cervical adenocarcinoma cell proliferation and tumour growth in vivo.

Cervical cancer is one of the leading causes of cancer-related death in women in sub-Saharan Africa. Extensive evidence has shown that cervical cancer and its precursor lesions are caused by Human papillomavirus (HPV) infection. Although the vast majority of HPV infections are naturally resolved, failure to eradicate infected cells has been shown to promote viral persistence and tumorigenesis. Furthermore, following neoplastic transformation, exposure of cervical epithelial cells to inflammatory mediators either directly or via the systemic circulation may enhance progression of the disease. It is well recognised that seminal plasma contains an abundance of inflammatory mediators, which are identified as regulators of tumour growth. Here we investigated the role of seminal plasma in regulating neoplastic cervical epithelial cell growth and tumorigenesis. Using HeLa cervical adenocarcinoma cells, we found that seminal plasma (SP) induced the expression of the inflammatory enzymes, prostaglandin endoperoxide synthase (PTGS1 and PTGS2), cytokines interleukin (IL) -6, and -11 and vascular endothelial growth factor-A (VEGF-A). To investigate the role of SP on tumour cell growth in vivo, we xenografted HeLa cells subcutaneously into the dorsal flank of nude mice. Intra-peritoneal administration of SP rapidly and significantly enhanced the tumour growth rate and size of HeLa cell xenografts in nude mice. As observed in vitro, we found that SP induced expression of inflammatory PTGS enzymes, cytokines and VEGF-A in vivo. Furthermore we found that SP enhances blood vessel size in HeLa cell xenografts. Finally we show that SP-induced cytokine production, VEGF-A expression and cell proliferation are mediated via the induction of the inflammatory PTGS pathway.

Developmentally regulated IL6-type cytokines signal to germ cells in the human fetal ovary.

Fetal ovarian development and primordial follicle formation are imperative for adult fertility in the female. Data suggest the interleukin (IL)6-type cytokines, leukaemia inhibitory factor (LIF), IL6, oncostatin M (OSM) and ciliary neurotrophic factor (CNTF), are able to regulate the survival, proliferation and differentiation of fetal murine germ cells (GCs) in vivo and in vitro. We postulated that these factors may play a similar role during early human GC development and primordial follicle formation. To test this hypothesis, we have investigated the expression and regulation of IL6-type cytokines, using quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Expression of transcripts encoding OSM increased significantly across the gestational range examined (8-20 weeks), while expression of IL6 increased specifically between the first (8-11 weeks) and early second (12-16 weeks) trimesters, co-incident with the initiation of meiosis. LIF and CNTF expression remained unchanged. Expression of the genes encoding the LIF and IL6 receptors, and their common signalling subunit gp130, was also found to be developmentally regulated, with expression increasing significantly with increasing gestation. LIF receptor and gp130 proteins localized exclusively to GCs, including oocytes in primordial follicles, indicating this cell type to be the sole target of IL6-type cytokine signalling in the human fetal ovary. These data establish that IL6-type cytokines and their receptors are expressed in the human fetal ovary and may directly influence GC development at multiple stages of maturation.

The regulation of vascular endothelial growth factor by hypoxia and prostaglandin F2α during human endometrial repair.

CONTEXT: The human endometrium has an exceptional capacity for repeated repair after menses, but its regulation remains undefined. Premenstrually, progesterone levels fall and prostaglandin (PG) F2α synthesis increases, causing spiral arteriole constriction. We hypothesized that progesterone withdrawal, PGF2α, and hypoxia increase vascular endothelial growth factor (VEGF), an endometrial repair factor. DESIGN AND RESULTS: Endometrial biopsies were collected (n = 47) with ethical approval and consent. VEGF mRNA, quantified by quantitative RT-PCR, was increased during menstruation (P < 0.01).VEGF protein was maximally secreted from proliferative endometrial explants. Treatment of an endometrial epithelial cell line and primary human endometrial stromal cells with 100 nm PGF2α or hypoxia (0.5% O2) resulted in significant increases in VEGF mRNA and protein. VEGF was maximal when cells were cotreated with PGF(2α) and hypoxia simultaneously (P < 0.05-0.001). Secretory-phase endometrial explants also showed an increase in VEGF with cotreatment (P < 0.05). However, proliferative-phase explants showed no increase in VEGF on treatment with PGF2α and/or hypoxia. Proliferative tissue was induced to increase VEGF mRNA expression when exposed to progesterone and its withdrawal in vitro but only in the presence of hypoxia and PG. Hypoxia-inducible factor-1α (HIF-1α) silencing with RNA interference suppressed hypoxia-induced VEGF expression in endometrial cells but did not alter PGF2α-induced VEGF expression. CONCLUSIONS: Endometrial VEGF is increased at the time of endometrial repair. Progesterone withdrawal, PGF2α, and hypoxia are necessary for this perimenstrual VEGF expression. Hypoxia acts via HIF-1α to increase VEGF, whereas PGF2α acts in a HIF-1α-independent manner. Hence, two pathways regulate the expression of VEGF during endometrial repair.

Hypoxia and prostaglandin E receptor 4 signalling pathways synergise to promote endometrial adenocarcinoma cell proliferation and tumour growth.

The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E(2). PTGS2 expression and PGE(2) biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE(2) regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1-4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE(2) and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE(2) and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.

Prokineticin 1 induces Dickkopf 1 expression and regulates cell proliferation and decidualization in the human endometrium.

Prokineticin 1 (PROK1) signalling via prokineticin receptor 1 (PROKR1) regulates the expression of several genes with important roles in endometrial receptivity and implantation. This study investigated PROK1 regulation of Dickkopf 1 (DKK1) expression, a negative regulator of canonical Wnt signalling, and its function in the non-pregnant endometrium and first trimester decidua. DKK1 mRNA expression is elevated during the mid-secretory phase of the menstrual cycle and expression increases further in first trimester decidua. DKK1 protein expression is localized to glandular epithelial and stromal cells during the proliferative, early- and mid-secretory phases, whereas expression is confined to the stroma in the late-secretory phase and first trimester decidua. PROK1 induces the expression of DKK1 in endometrial epithelial cells stably expressing PROKR1 and in first trimester decidua explants, via a Gq-calcium-calcineurin-nuclear factor of activated T-cells-mediated pathway. Endometrial epithelial cell proliferation is negatively regulated by PROK1-PROKR1 signalling. We demonstrate that this effect on cell proliferation occurs via DKK1 expression, as siRNA targeted against DKK1 reduces the PROK1-induced decrease in proliferation. Furthermore, decidualization of primary human endometrial stromal cells with progesterone and cyclic adenosine monophosphate is inhibited by miRNA knock down of PROK1 or DKK1. These data demonstrate important roles for PROK1 and DKK1 during endometrial receptivity and early pregnancy, which include regulation of endometrial cell proliferation and decidualization.

The expression and regulation of adrenomedullin in the human endometrium: a candidate for endometrial repair.

After menstruation, the endometrium has a remarkable capacity for repair, but the factors involved remain undefined. We hypothesize adrenomedullin (AM) plays a role in this process. Premenstrually progesterone levels decline, stimulating prostaglandin (PG) synthesis, vasoconstriction, and hypoxia. This study aimed to determine 1) AM expression throughout the menstrual (M) cycle and 2) its regulation by PG and hypoxia. Human endometrial biopsies (n = 51) were collected with ethical approval and consent. AM mRNA expression was examined by quantitative RT-PCR and was found to be selectively elevated in endometrium from the menstrual (M) phase (P < 0.001). AM immunohistochemical staining was maximal in M and proliferative (P) endometrium. Culture of secretory, but not P, explants with 100 nm PGF(2α) or hypoxia (0.5% O2) increased AM mRNA (P < 0.05). P explants were induced to increase AM expression using in vitro progesterone withdrawal but required the presence of hypoxia (P < 0.05). Short hairpin sequences against hypoxia-inducible factor-1α (HIF-1α) inhibited AM hypoxic up-regulation but did not alter PGF(2α)-induced expression. The AM receptor was immunolocalized to endothelial cells in both lymphatic and blood vessels. Conditioned medium from PGF(2α)-treated cells increased endothelial cell proliferation and branching (P < 0.05). This was abolished by AM receptor antagonists. In conclusion, AM is elevated at the time of endometrial repair and induces both angiogenesis and lymphangiogenesis by stimulating endothelial cell proliferation and tube formation. In the human endometrium, AM expression is up-regulated by two mechanisms: a HIF-1α-mediated hypoxic induction and a HIF-1α-independent PGF(2α) pathway. These physiological mechanisms may provide novel therapeutic targets for disorders such as heavy menstrual bleeding.

A role for lipoxin A4 as an anti-inflammatory mediator in the human endometrium.

Lipoxin A(4) is a lipid mediator that elicits anti-inflammatory and pro-resolution actions via its receptor, formyl peptide receptor 2 (FPR2/ALX). In this study, we aimed to investigate the expression and potential role of lipoxin A(4) and FPR2/ALX in the regulation of inflammation associated with cyclical remodeling of the human endometrium across the menstrual cycle and during early pregnancy. Using quantitative RT-PCR analysis, we found that FPR2/ALX expression is upregulated during the menstrual phase of the cycle and in decidua tissue from the first trimester of pregnancy. We localized the site of expression of FPR2/ALX in menstrual phase endometrium and first-trimester decidua tissue to glandular epithelial cells and cells within the stromal compartment, including cells lining the blood vessels and immune cells. Measurement of serum lipoxin A(4) by ELISA revealed no difference in its levels across the menstrual cycle but an elevation in early pregnancy (P<0.001). We found that lipoxin A(4) was regulated by human chorionic gonadotrophin (hCG) during early pregnancy, because treatment of human decidua tissue with hCG increased lipoxin A(4) release (P<0.01). Finally, we have shown that lipoxin A(4) can suppress phorbol myristate acetate-induced expression of the inflammatory cytokines interleukin 6 and 8 in human endometrium and decidua tissue. These results demonstrate for the first time that lipoxin A(4) and its receptor FPR2/ALX can regulate inflammatory events in the human endometrium and decidua of early pregnancy.

Novel roles for hypoxia and prostaglandin E2 in the regulation of IL-8 during endometrial repair.

The endometrium has a remarkable capacity for efficient repair; however, factors involved remain undefined. Premenstrual progesterone withdrawal leads to increased prostaglandin (PG) production and local hypoxia. Here we determined human endometrial expression of interleukin-8 (IL-8) and the roles of PGE(2) and hypoxia in its regulation. Endometrial biopsy specimens (n = 51) were collected. Endometrial cells and explants were exposed to 100 nmol/L of PGE(2) or 0.5% O(2). The endometrial IL-8 concentration peaked during menstruation (P < 0.001) and had a significant proangiogenic effect. IL-8 was increased by PGE(2) and hypoxia in secretory but not proliferative explants, which suggests that exposure to progesterone is essential. In vitro progesterone withdrawal induced significant IL-8 up-regulation in proliferative explants primed with progestins, but only in the presence of hypoxia. Epithelial cells treated simultaneously with PGE(2) and hypoxia demonstrated synergistic increases in IL-8. Inhibition of HIF-1 by short hairpin RNA abolished hypoxic IL-8 induction, and inhibition of NF-κB by an adenoviral dominant negative inhibitor decreased PGE(2)-induced IL-8 expression (P > 0.05). Increased menstrual IL-8 is consistent with a role in repair. Progesterone withdrawal, hypoxia, and PGE(2) regulate endometrial IL-8 by acting via HIF-1 and NF-κB. Hence, progesterone withdrawal may activate two distinct pathways to initiate endometrial repair.

Chlamydia trachomatis infection increases fallopian tube PROKR2 via TLR2 and NFκB activation resulting in a microenvironment predisposed to ectopic pregnancy.

Chlamydia trachomatis and smoking are major risk factors for tubal ectopic pregnancy (EP), but the underlying mechanisms of these associations are not completely understood. Fallopian tube (FT) from women with EP exhibit altered expression of prokineticin receptors 1 and 2 (PROKR1 and PROKR2); smoking increases FT PROKR1, resulting in a microenvironment predisposed to EP. We hypothesize that C. trachomatis also predisposes to EP by altering FT PROKR expression and have investigated this by examining NFκB activation via ligation of the Toll-like receptor (TLR) family of cell-surface pattern recognition receptors. PROKR2 mRNA was higher in FT from women with evidence of past C. trachomatis infection than in those without (P < 0.05), and was also increased in FT explants and in oviductal epithelial cell line OE-E6/E7 infected with C. trachomatis (P < 0.01) or exposed to UV-killed organisms (P < 0.05). The ability of both live and dead organisms to induce this effect suggests ligation of a cell-surface-expressed receptor. FT epithelium and OE-E6/E7 were both found to express TLR2 and TLR4 by immunohistochemistry. Transfection of OE-E6/E7 cells with dominant-negative TLR2 or IκBα abrogated the C. trachomatis-induced PROKR2 expression. We propose that ligation of tubal TLR2 and activation of NFκB by C. trachomatis leads to increased tubal PROKR2, thereby predisposing the tubal microenvironment to ectopic implantation.

Chemokine (C-C) motif ligand 20 is regulated by PGF(2α)-F-prostanoid receptor signalling in endometrial adenocarcinoma and promotes cell proliferation.

Prostaglandin F(2α) (PGF(2α)) is an inflammatory mediator which signals through a G-protein coupled receptor, the F-prostanoid (FP) receptor. We have previously shown elevated FP receptor expression in endometrial adenocarcinoma, a common gynaecological malignancy in Western countries. In this study, the expression of the chemokine CC motif Ligand 20 (CCL20) was determined to be regulated by PGF(2α)-FP receptor signalling in endometrial adenocarcinoma explants and cell line, and expression of CCL20 and its receptor CCR6 was elevated in endometrial adenocarcinoma compared to non-malignant endometrium. Both CCL20 and CCR6 were localised to neoplastic endometrial epithelial cells. The induction of CCL20 expression by PGF(2α)-FP signalling in an endometrial adenocarcinoma cell line stably expressing the FP receptor (FPS cells) was found to be dependent on the intracellular signalling of Gq, EGFR, ERK, calcineurin and nuclear factor of activated T-cells (NFAT) proteins. The treatment of FPS cells with recombinant CCL20 caused a significant increase in proliferation. Therefore these data demonstrate a role for the FP receptor in regulation of the chemokine CCL20, which can mediate proliferation of endometrial adenocarcinoma epithelial cells.

Placental growth factor: a promising diagnostic biomarker for tubal ectopic pregnancy.

CONTEXT: Tubal ectopic pregnancy is common, but accurate diagnosis is difficult and costly. There is currently no serum test to differentiate tubal from intrauterine implantation, and an effective biomarker of ectopic pregnancy would be a major clinical advance. OBJECTIVE: A key feature of successful intrauterine implantation is the establishment of a supportive vascular network, and this has been associated with the activity of placental growth factor (PIGF). We hypothesized that the local decidual environment facilitates PIGF-dependent angiogenesis and that this pathway is not active in tubal implantation. We aimed to determine whether tubal implantation is manifest by an attenuation of the normal trophoblast PIGF response and whether serum PIGF levels are different in ectopic compared with intrauterine pregnancy. DESIGN AND SETTING: Tissue and serum analysis was done at a large United Kingdom teaching hospital. PATIENTS: Tissue and sera were collected from gestation-matched pregnant women undergoing surgical termination of pregnancy (viable intrauterine) (n = 15), evacuation of uterus for embryonic missed miscarriage (nonviable intrauterine) (n = 10) and surgery for tubal ectopic pregnancy (n = 15). INTERVENTIONS: Trophoblast was examined by immunohistochemistry and quantitative RT-PCR, and serum was analyzed by ELISA. RESULTS: PIGF was localized to the cytotrophoblast cells. Expression of PIGF mRNA was reduced in trophoblast isolated from women with ectopic compared with intrauterine pregnancies (P < 0.05). Serum PIGF was undetectable in women with tubal ectopic pregnancies and reduced, or undetectable, in miscarriage compared with viable intrauterine pregnancies (P < 0.01). CONCLUSIONS: Serum PIGF is a promising novel diagnostic biomarker for early pregnancy location and outcome, and large-scale studies are now required to determine its clinical utility.

Comprehensive expression analysis of prostanoid enzymes and receptors in the human endometrium across the menstrual cycle.

Prostanoids are well-described primary mediators of inflammatory processes and are essential for the normal physiological function of the female reproductive system. The aim of this study was to determine the temporal expression of the prostanoid biosynthetic enzymes (PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, AKR1C3, CBR1, HPGDS, PTGDS, PTGIS, TBXAS1 and HPGD) and the prostanoid receptors (PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PTGDR, GPR44, PTGIR and TBXA2R) in the human endometrium throughout the menstrual cycle. The analysis identified PTGFR to have a distinct expression profile compared with other components of the prostanoid system, as expression is maximal during the proliferative phase. Immunohistochemical analysis for PTGER1 suggests a dual function for this receptor depending on its temporal (proliferative versus secretory) and spatial (nuclear versus cell membrane) expression. The expression profiles of the PGF(2α) synthases identified AKR1B1 and CBR1 as the likely regulators of PGF(2α) production during the menstrual phase. Immunohistochemical analysis for AKR1B1, CBR1 and AKR1C3 suggest expression to be in the glandular epithelium and vasculature. This study represents the first comprehensive analysis of the components of prostanoid biosynthetic and signalling pathway in the human endometrium. The expression profiles described have the potential to identify specific prostanoid components that may be dysregulated in inflammatory-associated disorders of the endometrium.

Chorionic gonadotrophin regulates CXCR4 expression in human endometrium via E-series prostanoid receptor 2 signalling to PI3K-ERK1/2: implications for fetal-maternal crosstalk for embryo implantation.

Murine knock-out models and blastocyst co-culture studies have identified prostaglandin-endoperoxide synthase (PTGS) 2, prostaglandin (PG) E receptor 2 (PTGER2) and the chemokine receptor CXCR4 as important regulators of early pregnancy events. In vitro studies and studies in non-human primates have shown that these proteins are regulated in the endometrium by the early embryonic signal, chorionic gonadotrophin (CG). Here we show that expressions of PTGER2 and CXCR4 are elevated during the mid-secretory phase of the menstrual cycle and decidua of early pregnancy in humans. Using first trimester decidua explants, we show that CG induces expression of PTGS2 and biosynthesis of PGE2, and expression of PTGER2. Subsequently, PGE2via PTGER2 induces expression of CXCR4. Using an in vitro model system of Ishikawa endometrial epithelial cells stably expressing PTGER2 and human first trimester decidua explants, we demonstrate that CXCR4 expression is regulated by PTGER2 via the epidermal growth factor receptor (EGFR)-phosphatidylinositol-3-kinase (PI3K)-extracellular signal-regulated kinase (ERK1/2) pathway.Taken together, our data suggest that early embryonic signals may regulate fetal-maternal crosstalk in the human endometrium by inducing CXCR4 expression via the PGE2-PTGER2-mediated induction of the EGFR, PI3K and ERK1/2 pathways.

Inflammatory pathways in endometrial disorders.

Complex interactions between the endocrine and immune systems govern the key endometrial events of implantation and menstruation. In contrast to other tissue sites, cyclical endometrial inflammation is physiological. However, dysregulation of this inflammatory response can lead to endometrial disorders. This review examines the inflammatory processes occurring in the normal endometrium during menstruation and implantation, highlighting recent advances in our understanding and gaps in current knowledge. Subsequently, the role of inflammatory pathways in the pathology of various common endometrial conditions is discussed, including heavy menstrual bleeding, dysmenorrhoea (painful periods), uterine fibroids, endometriosis and recurrent miscarriage.

PGF2α-F-prostanoid receptor signalling via ADAMTS1 modulates epithelial cell invasion and endothelial cell function in endometrial cancer.

BACKGROUND: An increase in cancer cell invasion and microvascular density is associated with a poorer prognosis for patients with endometrial cancer. In endometrial adenocarcinoma F-prostanoid (FP) receptor expression is elevated, along with its ligand prostaglandin (PG)F2α, where it regulates expression and secretion of a host of growth factors and chemokines involved in tumorigenesis. This study investigates the expression, regulation and role of a disintegrin and metalloproteinase with thrombospondin repeat 1 (ADAMTS1) in endometrial adenocarcinoma cells by PGF2α via the FP receptor. METHODS: Human endometrium and adenocarcinoma tissues were obtained in accordance with Lothian Research Ethics Committee guidance with informed patient consent. Expression of ADAMTS1 mRNA and protein in tissues was determined by quantitative RT-PCR analysis and immunohistochemistry. Signal transduction pathways regulating ADAMTS1 expression in Ishikawa cells stably expressing the FP receptor to levels seen in endometrial cancer (FPS cells) were determined by quantitative RT-PCR analysis. In vitro invasion and proliferation assays were performed with FPS cells and human umbilical vein endothelial cells (HUVECs) using conditioned medium (CM) from PGF2α-treated FPS cells from which ADAMTS1 was immunoneutralised and/or recombinant ADAMTS1. The role of endothelial ADAMTS1 in endothelial cell proliferation was confirmed with RNA interference. The data in this study were analysed by T-test or ANOVA. RESULTS: ADAMTS1 mRNA and protein expression is elevated in endometrial adenocarcinoma tissues compared with normal proliferative phase endometrium and is localised to the glandular and vascular cells. Using FPS cells, we show that PGF2α-FP signalling upregulates ADAMTS1 expression via a calmodulin-NFAT-dependent pathway and this promotes epithelial cell invasion through ECM and inhibits endothelial cell proliferation. Furthermore, we show that CM from FPS cells regulates endothelial cell ADAMTS1 expression in a rapid biphasic manner. Using RNA interference we show that endothelial cell ADAMTS1 also negatively regulates cellular proliferation. CONCLUSIONS: These data demonstrate elevated ADAMTS1 expression in endometrial adenocarcinoma. Furthermore we have highlighted a mechanism whereby FP receptor signalling regulates epithelial cell invasion and endothelial cell function via the PGF2α-FP receptor mediated induction of ADAMTS1.

Cotinine exposure increases Fallopian tube PROKR1 expression via nicotinic AChRalpha-7: a potential mechanism explaining the link between smoking and tubal ectopic pregnancy.

Tubal ectopic pregnancy (EP) is the most common cause of maternal mortality in the first trimester of pregnancy; however, its etiology is uncertain. In EP, embryo retention within the Fallopian tube (FT) is thought to be due to impaired smooth muscle contractility (SMC) and alterations in the tubal microenvironment. Smoking is a major risk factor for EP. FTs from women with EP exhibit altered prokineticin receptor-1 (PROKR1) expression, the receptor for prokineticins (PROK). PROK1 is angiogenic, regulates SMC, and is involved in intrauterine implantation. We hypothesized that smoking predisposes women to EP by altering tubal PROKR1 expression. Sera/FT were collected at hysterectomy (n=21). Serum levels of the smoking metabolite, cotinine, were measured by enzyme-linked immunosorbent assay. FTs were analyzed by q-RT-PCR, immunohistochemistry, and Western blotting for expression of PROKR1 and the predicted cotinine receptor, nicotinic acetylcholine receptor α-7 (AChRα-7). FT explants (n=4) and oviductal epithelial cells (cell line OE-E6/E7) were treated with cotinine and an nAChRα-7 antagonist. PROKR1 transcription was higher in FTs from smokers (P<0.01). nAChRα-7 expression was demonstrated in FT epithelium. Cotinine treatment of FT explants and OE-E6/E7 cells increased PROKR1 expression (P<0.05), which was negated by cotreatment with nAChRα-7 antagonist. Smoking targets human FTs via nAChRα-7 to increase tubal PROKR1, leading to alterations in the tubal microenvironment that could predispose to EP.

Inflammatory events in endometrial adenocarcinoma.

Endometrial adenocarcinoma is the most common gynaecological malignancy in western countries. Many of the established risk factors for developing endometrial cancer are associated with excess exposure to oestrogen unopposed by progesterone. These include nulliparity, late onset of the menopause, post-menopausal hormone replacement therapy and obesity. However, a number of risk factors also promote inflammation, another feature proposed to influence cancer development. The human cycling endometrium undergoes regular and cyclical episodes of inflammation. Moreover, hormonal and genetic changes that occur early in the development of endometrial adenocarcinoma can exacerbate the local inflammatory environment. Via alterations in the expression of local mediators and immune cell function, these may contribute to the development of endometrial cancer. This review discusses the contribution of inflammation to the initiation and progression of endometrial adenocarcinoma. Manipulation of inflammatory pathways may therefore represent a therapeutic target in endometrial adenocarcinoma.