Cyclooxygenase-2 and prostaglandins in human endometrial function

A role for cyclooxygenase enzymes and prostaglandins (such as prostaglandin E2) has been observed in benign endometrial pathologies (endometriosis, excessive blood loss and dysmenorrhoea) and in adenocarcinoma. Cyclooxygenase and prostaglandin E synthase enzymes catalyse the conversion of arachidonic acid to prostaglandin E2. Once synthesised, prostaglandin E2 mediates its effects via four G protein coupled receptors namely EP1, EP2, EP3 and EP4. These receptors signal via alternate and sometimes opposing pathways. The initial aim of the research presented in this thesis was to investigate the temporal expression and signalling of the prostaglandin E2 pathway in the normal human endometrium across the menstrual cycle. Prostaglandin E synthase and prostaglandin E2 were localised to glandular epithelial and endothelial cells. Similarly, expression of the prostaglandin E2 receptors, namely EP2 and EP4, was temporally up regulated and co-localised to the glandular and vascular compartments. This was associated with enhanced cAMP turnover in response to exogenous prostaglandin E2. In order to investigate further the role of cyclooxygenase-2 and prostaglandin E2 in glandular epithelial cells we generated a stably transfected endometrial epithelial cell line (Ishikawa) overexpressing cyclooxygenase-2 in either the sense or antisense directions. Using these cell lines we observed enhanced secretion of prostaglandins E2 and F2tt into the culture media of the cyclooxygenase-2 sense cells compared with the cyclooxygenase-2 antisense and wild type cells in response to exogenous arachidonic acid. Co-incubation of the cells with NS398 (specific cyclooxygenase-2 inhibitor) abolished the increase in prostaglandin synthesis. Overexpression of cyclooxygenase-2 was accompanied with significantly elevated EP2/EP3 receptor expression. No differences were detected for EP1, EP4 and FP receptors. These results indicate a possible autocrine/paracrine action of cyclooxygenase-2 enzyme products on expression of prostanoid receptors such as EP2/EP3. During the course of my PhD, numerous reports were published implicating cyclooxygenase-2 and its products in angiogenesis through the expression of angiogenic factors such as vascular endothelial growth factor, basic fibroblast growth factor and angiopoietins. To investigate the potential role of cyclooxygenase-2 in regulation of endometrial angiogenesis, cDNA array technology was employed to identify differentially expressed genes that may be involved in vascular function. Using this technique, a total of 81 genes were differentially regulated including cathepsin D. Cathepsin D mRNA and protein expression were elevated in the cyclooxygenase-2 antisense cells compared with the sense and wild type cells. Cathepsin D is known to proteolytically cleave plasminogen to the antiangiogenic factor angiostatin. Hence, we investigated the generation of angiostatin from plasminogen in conditioned media collected from cyclooxygenase-2 sense, cyclooxygenase-2 antisense and wild type cells. The cleavage of angiostatin from plasminogen was markedly enhanced in conditioned media from cyclooxygenase-2 antisense cells compared with cyclooxygenase-2 sense and wild type cells. Co-incubation of plasminogen with pepstatin A, a selective cathepsin D inhibitor, markedly reduced the cleavage of angiostatin from plasminogen thus further implicating cathepsin D in the differential angiostatin production by the cyclooxygenase-2 sense and antisense cell lines.

Grantor dc:publisher

The University of Edinburgh

Year dc:date.issued

2006

Author dc:creator

• Perchick, Gabrielle Beth

Handle dc:identifier.uri

http://hdl.handle.net/1842/29319

OAI identifier oai:identifier

oai:era.ed.ac.uk:1842/29319