Intracellular signalling by arachidonic acid metabolites

Abstract

In intrauterine tissues, pro-inflammatory cytokines and prostaglandins (PGs) have been identified as key mediators in the maintenance of pregnancy and parturition. The rise in PGD2 detected in the amniotic fluid before labour prompted the research presented in this thesis which describes the effects of 15-deoxy Δ12,14 -prostaglandin J2 (15d-PGJ2), a non-enzymatic metabolite of PGD2, on amnion-derived WISH and JEG3 choriocarcinoma cells as models of the amnion and chorion trophoblasts, respectively.15d-PGJ2 induced apoptosis in both cell lines in a concentration-dependent fashion (2.5-10 µM). Apoptosis was characterised by condensation of chromatin (visualised after Hoechst 33342 staining), appearance of nucleosomal DNA fragmentation upon electrophoresis and flow cytometry analysis, and activation of caspase-3. Apoptotic cell death was inhibited in the presence of serum (0.5% w/v) and albumin, not serumderived growth factors (insulin growth factor (IGF)-1, IGF-2 or epidermal growth factor (EGF), was determined as the key survival factor. Since 15d-PGJ2 is reported to activate peroxisome proliferator activated receptor (PPAR)-γ, the activities of PPARs were assessed using JEG3 cells transfected with a PPAR-response element reporter construct (pTK-PPREx3-luc). The PPAR-γ-specific ligand, rosiglitazone, induced PPRE mediated activity in a concentration-dependent manner, while the PPAR-γ-specific irreversible inhibitor, GW9662, fully inhibited this induction. However, GW9662 only partially inhibited 15d-PGJ2-induced luciferase activity, suggesting that 15d-PGJ2 may also activate either of the other isoforms. The expressions of PPAR-α and -δ were identified in amnion, choriodecidua and placental membranes and PPAR-δ was significantly increased all tissues with labour. PPAR-α expression was reduced in chorio-decidua, but was significantly higher in placenta with labour. The changes observed with labour suggest that regulation of PPAR expression and function may have a role in the mechanisms that maintain pregnancy or initiate labour. The anti-inflammatory effects of 15d-PGJ2 were also investigated by measuring interleukin (IL)-1β-stimulated prostaglandin and cytokine productions by WISH cells after treatment with 15d-PGJ2 for 3 hours. 15d-PGJ2 exerted differential effects that were dependent upon its concentration. At low nanomolar physiologic concentrations (1-10 nM), 15d-PGJ2 inhibited IL-1β-stimulated PGE2, but not cytokine (IL-6/IL-8) production or cyclooxygenase (COX)-2 expression. This effect was attenuated by GW9662, by transfection with dominant negative PPAR constructs, and was reproduced by rosiglitazone. At micromolar (1-10 µM) concentrations, 15d-PGJ2 inhibited IL-1β-stimulated COX-2, PGE2 and cytokine productions and these effects were not blocked by GW9662 or mimicked by rosiglitazone. GW501516 (PPAR-δ agonist) also inhibited IL-1β-stimulated PGE2 production, but only at high concentrations (1 µM). IL-1β-induced NF-kB DNA binding activity was significantly inhibited by 15d-PGJ2 (10 µM) and GW501516 (1 µM), but increased by rosiglitazone (10 µM). In conclusion, this is the first report of an effect of 15d-PGJ2 at low nanomolar physiologic concentrations and 15d-PGJ2 mediates its actions through PPAR-γ (<0.1 µM) and PPAR-γ-independent(1-10 µM) pathways, the latter through inhibition of NF-kB and/or activation of PPAR-δ. Further studies on the effect of physiologic concentrations of 15d-PGJ2 on primary gestational tissues will provide understanding on the role(s) 15d-PGJ2 plays in fetal membrane remodelling and its involvement in the inflammatory processes associated with labour and parturition.