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.

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.

F-prostanoid receptor regulation of fibroblast growth factor 2 signaling in endometrial adenocarcinoma cells.

Prostaglandin (PG) F(2alpha) is a potent bioactive lipid in the female reproductive tract, and exerts its function after coupling with its heptahelical G-protein-coupled receptor [F-series-prostanoid (FP) receptor] to initiate cell signaling and target gene transcription. In the present study, we found elevated expression of fibroblast growth factor (FGF) 2, FGF receptor 1 (FGFR1), and FP receptor, colocalized within the neoplastic epithelial cells of endometrial adenocarcinomas. We investigated a role for PGF(2alpha)-FP receptor interaction in modulating FGF2 expression and signaling using an endometrial adenocarcinoma cell line stably expressing the FP receptor to the levels detected in endometrial adenocarcinomas (FPS cells) and endometrial adenocarcinoma tissue explants. PGF(2alpha)-FP receptor activation rapidly induced FGF2 mRNA expression, and elevated FGF2 protein expression and secretion into the culture medium in FPS cells and endometrial adenocarcinoma explants. The effect of PGF(2alpha) on the expression and secretion of FGF2 could be abolished by treatment of FPS cells and endometrial tissues with an FP receptor antagonist (AL8810) and inhibitor of ERK (PD98059). Furthermore, we have shown that FGF2 can promote the expression of FGF2 and cyclooxygenase-2, and enhance proliferation of endometrial adenocarcinoma cells via the FGFR1 and ERK pathways, thereby establishing a positive feedback loop to regulate neoplastic epithelial cell function in endometrial adenocarcinomas.

A novel angiogenic role for prostaglandin F2alpha-FP receptor interaction in human endometrial adenocarcinomas.

Prostaglandins have been implicated in several neovascular diseases. In the present study, we found elevated FP receptor and vascular endothelial growth factor (VEGF) expression colocalized in glandular epithelial and vascular cells lining the blood vessels in endometrial adenocarcinomas. We investigated the signaling pathways activated by the FP receptor and their role in modulating VEGF expression in endometrial adenocarcinoma (Ishikawa) cells. Ishikawa cells were stably transfected with FP receptor cDNA in the sense or antisense orientations. Treatment of Ishikawa cells with prostaglandin F2alpha (PGF2alpha) rapidly induced transphosphorylation of the epidermal growth factor receptor (EGFR) and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 via the FP receptor. Activation of EGFR-Ras-mitogen-activated protein kinase/ERK kinase (MEK) signaling via the FP receptor resulted in an increase in VEGF promoter activity, expression of VEGF mRNA, and secretion of VEGF protein. These effects of PGF2alpha on the FP receptor could be abolished by treatment of cells with a specific FP receptor antagonist, chemical inhibitors of c-Src, matrix metalloproteinase, and EGFR kinase or by inactivation of signaling with dominant-negative mutant isoforms of EGFR, Ras, or MEK or with small inhibitory RNA oligonucleotides targeted against the EGFR. Finally, we confirmed that PGF2alpha could potentiate angiogenesis in endometrial adenocarcinoma explants by transactivation of the EGFR and induction of VEGF mRNA expression.

Prostaglandin receptors are mediators of vascular function in endometrial pathologies.

Prostaglandins are bioactive lipids produced from arachidonic acid by cyclooxygenase enzymes and specific terminal prostanoid synthase enzymes. Following biosynthesis, prostaglandins exert an autocrine/paracrine function by coupling to specific prostanoid G protein-coupled receptors to activate intracellular signaling and gene transcription. For many years prostaglandins have been recognised as key molecules in reproductive biology by regulating ovulation, endometrial physiology and proliferation of endometrial glands and menstruation. More recently a role for COX enzymes and prostaglandins has been ascertained in reproductive tract pathology, including dysmenorrhea, endometriosis, menorrhagia and cancer. Emerging evidence supports a role for COX enzymes, prostaglandins and prostaglandin receptor signaling pathways in a multitude of phenotypic changes in reproductive tissues including the promotion of angiogenesis and vascular function. Here we provide an overview of some of the findings from these studies with specific emphasis on the role of cyclooxygenase enzymes, prostaglandins and their receptors in benign and neoplastic pathologies of the human endometrium.

A positive feedback loop that regulates cyclooxygenase-2 expression and prostaglandin F2alpha synthesis via the F-series-prostanoid receptor and extracellular signal-regulated kinase 1/2 signaling pathway.

Cyclooxygenase (COX) enzymes catalyze the biosynthesis of eicosanoids, including prostaglandin (PG) F2alpha. PGF2alpha exerts its autocrine/paracrine function by coupling to its G protein-coupled receptor [F-series-prostanoid (FP) receptor] to initiate cell signaling and target gene transcription. In the present study, we found elevated expression of COX-2 and FP receptor colocalized together within the neoplastic epithelial cells of endometrial adenocarcinomas. We investigated a role for PGF2alpha-FP receptor interaction in modulating COX-2 expression and PGF2alpha biosynthesis using an endometrial adenocarcinoma cell line stably transfected with the FP receptor cDNA (FPS cells). PGF2alpha-FP receptor activation rapidly induced COX-2 promoter, mRNA, and protein expression in FPS cells. These effects of PGF2alpha on the expression of COX-2 could be abolished by treatment of FPS cells with an FP receptor antagonist (AL8810) and chemical inhibitor of ERK1/2 kinase (PD98059), or by inactivation of ERK1/2 signaling with dominant-negative mutant isoforms of Ras or ERK1/2 kinase. We further confirmed that elevated COX-2 protein in FPS cells could biosynthesize PGF2alpha de novo to promote a positive feedback loop to facilitate endometrial tumorigenesis. Finally, we have shown that PGF2alpha could potentiate tumorigenesis in endometrial adenocarcinoma explants by inducing the expression of COX-2 mRNA.

Expression and localization of endothelial monocyte-activating polypeptide II in the human endometrium across the menstrual cycle: regulation of expression by prostaglandin E(2).

A role for prostaglandins (PGs) in the regulation of endometrial functions, such as menstruation, has been established, although the mechanisms by which this is achieved are not fully elucidated. In the present study, cDNA array analysis has identified endothelial monocyte-activating polypeptide II (EMAP II) as a PGE(2)-regulated gene in endometrial epithelial cells. Incubation of endometrial epithelial cells with 100 nM PGE(2) for 4 and 24 h resulted in a 2.3- and 16-fold decrease in EMAP II expression, respectively. In endometrial tissue collected across the menstrual cycle, a significant increase in EMAP II mRNA was observed during the late secretory phase, compared with the proliferative and early-midsecretory phases. The temporal pattern of EMAP II expression was confirmed further by Western blotting; EMAP II protein expression was detected as a 43-kDa band. In situ hybridization and immunohistochemistry localized EMAP II mRNA and protein expression in glandular epithelial, endothelial, and stromal cells in the functionalis and basalis layers of the endometrium. Finally, the role of PGE(2) in the regulation of EMAP II expression in the endometrium was assessed. Incubation of fresh endometrial tissue (n = 5) with 3 micro g/ml indomethacin resulted in an increase in EMAP II protein expression, compared with control untreated tissue. However, cotreatment of the cells with 100 nM PGE(2) resulted in a significant decrease in EMAP II protein expression, compared with tissue incubated with indomethacin alone (P < 0.05). These data confirm temporal variation in EMAP II expression in the human endometrium across the menstrual cycle and localize expression to glandular epithelial, endothelial, and stromal cells. Moreover, EMAP II expression is negatively regulated by PGE(2).

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.