REGULATION OF PHOSPHOLIPASE A2 IN HUMAN AMNION

前和后细胞因子的自分泌/旁分泌影响， 胎膜中的生长因子似乎是合成的中心 在足月分娩和感染诱导的分娩中， 早产 我们已经证明，II-1 β可以协同诱导 胞浆磷脂酶A2（cPLA 2）和前列腺素H的表达 合成酶-2（PGHS-2）的表达，并且两种酶 定位在核膜上。 这两种酶可以偶联到 提供局部花生四烯酸移动和PG合成。 cPLA 2活性的抑制阻断了精氨酸刺激的PG合成， cPLA 2基因敲除小鼠未能在足月分娩，显示了cPLA 2基因的核心作用。 在分娩过程中，cPLA 2介导PG的释放。 催化 cPLA 2的活性通过激动剂如 EGF。 我们有初步证据表明p38 MAP激酶参与了 这条路。 羊膜上皮细胞和间充质细胞均表达 PGHS-2在足月分娩中，但对糖皮质激素的反应不同。 两 间充质细胞表达诱导型一氧化氮 合成酶，突出了它们在信号传导发病中的潜在作用。 劳动。 我们的目标是：1. 确定cPLA 2的磷酸化是否通过 11-1 WISH细胞中的β或EGF与特定的核 部分（核膜或内质网）引起 花生四烯酸动员和与PGHS-2共定位。 2. 利用特异性抑制剂和蛋白质印迹法确定II-1 β EGF刺激cPLA 2磷酸化和cPLA 2/PGHS-2 mRNA表达 在WISH细胞中，通过p38 MAP激酶、ERK-1、2或蛋白激酶 C路径。 3. 确定是否有脂多糖（LPS）， WISH细胞合成PGE 2，通过p38引起cPLA 2磷酸化 MAP激酶和如果LPS处理细胞，则引发细胞用于随后的免疫反应。 细胞因子的作用。 4. 确定赞成和反对的角色 细胞因子（II-1 β、II-4和II-10）和生长因子（TGF β）， WISH细胞中cPLA 2的磷酸化和活化及其相互作用 这些细胞因子与脂多糖。 5. 确定表情的变化， cPLA 2的磷酸化（活化）和与核 膜（含PGHS-2），来自足月或足月患者的羊膜组织， 早产，无论是否在分娩中。 6. 确定p38 MAP激酶 cPLA 2的刺激磷酸化发生在羊膜上皮细胞中， 间充质细胞，如果糖皮质激素差异影响cPLA 2 在这些细胞中的表达，因为它们已被证明为PGHS-2。 这项研究将提供新的数据磷酸化和激活的 cPLA 2在人羊膜组织中的作用，并进一步确定这一关键的 在胎膜中类花生酸合成的调节步骤. 分娩

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.