Autocrine/paracrine influences of pro and antiinflammatory cytokines and growth factors in the fetal membranes appear central to the synthesis of many effectors in the onset of labor at term and in infection-induced preterm labor. We have shown that II-1beta can coordinately induce expression of cytosolic phospholipase A2 (cPLA2) and prostaglandin H synthase-2 (PGHS-2) in amnion-derived WISH cells and both enzymes localize on the nuclear membrane. The two enzymes may be coupled to give localized arachidonic acid mobilization and PG synthesis. Inhibition of cPLA2 activity blocks cytokine-stimulated PG synthesis and cPLA2 knockout mice fail to deliver at term showing a central role for cPLA2 mediated PG release in the process of parturition. The catalytic activity of cPLA2 is stimulated by phosphorylation by agonists such as EGF. We have preliminary evidence for involvement of p38 MAP kinase in this pathway. Both epithelial and mesenchymal cells in amnion express PGHS-2 at term labor but respond differently to glucocorticoids. Both express cPLA2 and the mesenchymal cells express inducible nitric oxide synthase highlighting a potential role for them in signalling the onset of labor.
We aim to: 1. Determine if phosphorylation of cPLA2 by 11-1beta or EGF in WISH cells gives association with a specific nuclear fraction (nuclear membrane or endoplasmic reticulum) to cause arachidonic acid mobilization and co-localization with PGHS-2. 2. Utilize specific inhibitors and western blots to determine if II-1beta or EGF stimulated cPLA2 phosphorylation and cPLA2/PGHS-2 mRNA expression in WISH cells occurs via the p38 MAP kinase, ERK-1,2 or protein kinase C pathways. 3. Determine if lipopolysaccharide (LPS), which stimulates PGE2 Synthesis by WISH cells, causes phosphorylation of cPLA2 via p38 MAP kinase and if LPS treatment of cells primes cells for the subsequent action of cytokines. 4. Determine the role of pro and antiinflammatory cytokines, (II-1beta, II-4 and II-10) and growth factors (TGFbeta) on cPLA2 phosphorylation and activation in WISH cells and the interaction of these cytokines with LPS. 5. Determine changes in the expression, phosphorylation (activation) of cPLA2 and association with the nuclear membrane (with PGHS-2) in amnion tissue from patients at term or preterm, either in or not in labor. 6. Determine if p38 MAP kinase stimulated phosphorylation of cPLA2 occurs in amnion epithelial and mesenchymal cells and if glucocorticoids differentially affect cPLA2 expression in these cells as they have been shown to do for PGHS-2. This study will provide novel data on phosphorylation and activation of cPLA2 in human amnion tissue and further define the role of this crucial regulatory step in eicosanoid synthesis in the fetal membranes at parturition.
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