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Title: Prostaglandin signalling and control of MMP production in human fetal membranes
Author: Carr, Nancy J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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The aim of this thesis is to investigate the cellular pathways by which PGE2 and PGF act via their receptors in human fetal membranes and the effect that these responses have on the production and activation of MMP-2 and MMP-9. The JEG3 choriocarcinoma cell-line was used alongside human tissue as a model of chorion trophoblast cells. PCR and immunohistochemistry were used to identify and localise the prostaglandin receptors, EP2, EP4 and FP. These receptors were localised to the amnion epithelium and fibroblast layer, chorion trophoblast and reticular layer, glandular epithelial cells, stromal cells and vascular endothelial cells of the deciduas, placental syncytiotrophoblasts, smooth muscle cells of the myometrium and also JEG3 cells. To investigate intracellular signalling, amnion and chorio-decidua were collected immediately after term elective caesarean sections and treated with PGE2 and PGF, and selective inhibitors and antagonists, as were the JEG3 cells. Samples were homogenised and assayed for cyclic AMP (cAMP) and analysed for extracellular signal-regulated kinase (ERK) phosphorylation by immunoblotting. PGE2 elevated cAMP levels over 2-fold after 10 min in amnion and JEG3 cells, with no effect on chorio-decidua. These effects were inhibited by the addition of an EP2 antagonist in amnion and an EP4 antagonist in JEG3 cells. PGE2 and PGF had no effect on phosphorylation of ERK in amnion or chorio-decidua, though in JEG3 cells, PGE2 and PGF increased it by 9-fold and 2-fold respectively after 10 min. Pre-treatment with an inhibitor of MEK totally inhibited this stimulation, and an inhibitor of PLC and EGFR kinase had no effect. The ERK phosphorylation stimulations by PGE2 and PGF were inhibited by an EP4 and an FP antagonist, respectively. To investigate the effects of prostaglandins on MMP production and activation, amnion, chorio-decidua and placenta were collected after term elective caesarean sections and stimulated with PGE2 and PGF, as were JEG3 cells. Quantitative PCR was carried out to determine mRNA levels of MMP-2 and MMP-9 after this stimulation. Zymography was performed to reveal latent and active forms of MMP-3 and MMP-9, and reverse zymography and Western blotting carried out to quantify. TIMP levels. Results show that although there were no major PG-mediated changes in latent or active forms of MMP protein, MMP-2 mRNA production was up-regulated by both PGs. These results suggest that prostaglandins are acting via the EP2 and FP receptors to activate intracellular signalling pathways which up-regulate the production of MMPs. This mechanism could be involved in rupture of the fetal membranes, and the PG receptor antagonists could potentially be used to block this process to prevent preterm rupture of the membranes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available