Sheila C. Boddy

Expression of COX-2 and PGE synthase and synthesis of PGE(2)in endometrial adenocarcinoma: a possible autocrine/paracrine regulation of neoplastic cell function via EP2/EP4 receptors.

This study was designed to investigate the possible role of cyclo-oxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in endometrial adenocarcinoma. COX-2 RNA expression was confirmed in various grades of adenocarcinoma by ribonuclease protection assay. COX-2 and microsomal glutathione-dependent prostaglandin E synthase (mPGES) expression and PGE2 synthesis were localised to the neoplastic epithelial cells and endothelial cells. In order to establish whether PGE2 has an autocrine/paracrine effect in adenocarcinomas, we investigated the expression of 2 subtypes of PGE2 receptors, namely EP2 and EP4, by real time quantitative PCR. Expression of EP2 and EP4 receptors was detected in adenocarcinomas from all grades of differentiation and was significantly higher than that detected in normal secretory phase endometrium (P < 0.01). The fold induction of expression in adenocarcinoma compared with normal secretory phase endometrium was 28.0 ± 7.4 and 52.5 ± 10.1 for EP2 and EP4 receptors respectively. Immunohistochemistry localised the site of expression of EP4 receptor in neoplastic epithelial cells and in the endothelium of carcinomas of all grades of differentiation. Finally, the functionality of the EP2/EP4 receptors was assessed by investigating cAMP generation following in vitro culture of adenocarcinoma tissue in the presence or absence of 300 nM PGE2. cAMP production in response to PGE2 was significantly higher in carcinoma tissue than that detected in normal secretory phase endometrium (3.42 ± 0.46 vs 1.15 ± 0.05 respectively; P < 0.001). In conclusion, these data suggest that PGE2 may regulate neoplastic cell function in an autocrine/paracrine manner via the EP2/EP4 receptors.

A Novel Angiogenic Role for Prostaglandin F2α-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.

Pattern and localisation of expression of vascular endothelial growth factor and its receptor flt-1 in the ovine pituitary gland: expression is independent of hypothalamic control

The pituitary gland, a highly vascularised endocrine organ, contains permeable fenestrated endothelium that allows direct access of endocrine cells to the hemal milieu. Vascular endothelial growth factor (VEGF) has a mitogenic effect on endothelial cells and renders the endothelium more permeable. The following study investigated the expression of VEGF and its receptor flt-1 mRNA and protein in the pituitary gland of sheep. VEGF expression was localised, by in situ hybridisation and immunocytochemistry, mainly to the pars tuberalis/zona tuberalis (PT/ZT) region of the gland. No hybridisation signal was observed in the pars intermedia or pars nervosa. Reverse transcriptase-polymerase chain reaction (RT-PCR) Southern blotting confirmed the predominant expression of VEGF in the PT/ZT compared with the pars distalis (PD). Western blot analysis with the VEGF antibody revealed major (48 kDa) and minor (24 kDa) bands representing the monomer and dimer forms of VEGF and also confirmed the differential expression of VEGF in the PT/ZT compared with the PD. Double immunocytochemistry with VEGF and prolactin or luteinising hormone-beta (LH-beta) antibodies demonstrated that the VEGF-secreting cells are not lactotrophs or gonadotrophs. However, co-localisation of VEGF with S-100 was observed in a proportion of cells suggesting that some VEGF secreting cells are follicular stellate. Immunocytochemistry with a flt-1 antibody confirmed the expression of this high affinity receptor for VEGF in endothelial cells across the pituitary gland. Immunocytochemistry with the VEGF antibody using pituitary glands from intact and hypothalamo-pituitary disconnected sheep demonstrated comparable expression patterns suggesting that the regulation of blood flow and vascular permeability in the pituitary gland is under local regulation and is independent of hypothalamic input.

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 PGF2α synthases identified AKR1B1 and CBR1 as the likely regulators of PGF2α 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 A4 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 A4 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 A4 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 A4 was regulated by human chorionic gonadotrophin (hCG) during early pregnancy, because treatment of human decidua tissue with hCG increased lipoxin A4 release (P<0.01). Finally, we have shown that lipoxin A4 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 A4 and its receptor FPR2/ALX can regulate inflammatory events in the human endometrium and decidua of early pregnancy.

F-prostanoid receptor alters adhesion, morphology and migration of endometrial adenocarcinoma cells

Cellular adhesion to extracellular matrix is a central phenomenon for the maintenance of tissue integrity and cellular movement. Collectively, these processes are regulated by a fine-tuned balance between the formation and loosening of adhesive contacts, a process involving integrins, and the elevation and diminution of cytoplasmic signalling molecules. We demonstrate that prostaglandin (PG) F2α stimulation rapidly increases the capacity of Ishikawa cells stably expressing the F-prostanoid receptor (FPS) to adhere to vitronectin. Coincident with this elevation in matrix adhesion, we demonstrate a profound PGF2α-induced alteration in cytoskeletal remodelling, characterized by polymerization of the actin cytoskeleton and recruitment of focal adhesion kinase at focal adhesions and enhanced cell migration. Moreover, we show that these PGF2α-induced alterations in adhesion and morphology on vitronectin and migration could be abolished by cultivating FPS cells in the presence of integrin αvβ3 antibody or αvβ3-directed tetrapeptide arg–gly–asp–ser or inhibition of FP receptor signalling with the FP receptor antagonist, chemical disruptors of the phospholipase C-β, protein kinase A, c-Src and epidermal growth factor receptor kinase pathways or inhibition of the monomeric G proteins Rho, Rac and CDC42. These results reveal a mechanism by which prostanoids regulate cell movement, which may be relevant to pathologies of the endometrium.

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) E2. PTGS2 expression and PGE2 biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE2 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 PGE2 and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE2 and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.