REGULATION OF PHOSPHOLIPASE A2 IN HUMAN AMNION

人羊膜中磷脂酶 A2 的调节

• 批准号: 6629086
• 负责人: LESLIE MYATT
• 金额: $ 26.43万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-02-01 至 2005-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6629086
• 关键词: amnion; arachidonate; biological signal transduction; birth; clinical research; cytokine; eicosanoid metabolism; embryo /fetus membrane; enzyme activity; epithelium; gene expression; human pregnant subject; human tissue; interleukin 1; lipopolysaccharides; mesenchyme; mitogen activated protein kinase; nuclear runoff assay; phospholipase A2; phosphorylation; premature labor; prostaglandin E; prostaglandin endoperoxide synthase; protein kinase C; tissue /cell culture

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.

促炎和抗炎细胞因子对自分泌/旁分泌的影响 胎膜中的生长因子似乎是合成的中心。 多种效应物在足月分娩和感染诱导中的作用 早产。我们已经证明II-1β可以协同诱导 胞浆磷脂酶A2和前列腺素H的表达 羊膜源性Wish细胞及其两种酶的合酶-2(PGHS-2) 定位于核膜上。这两种酶可以偶联到 进行局部花生四烯酸动员和PG合成。 抑制cPLA2活性可阻断细胞因子刺激的PG合成和 CPLA2基因敲除小鼠未能足月分娩，显示出 CPLA2介导分娩过程中PG的释放。催化剂 CPLA2的活性被激动剂如 EGF。我们有初步证据表明p38MAPK参与了 这条路。羊膜上皮细胞和间充质细胞的表达 足月分娩时的PGHS-2，但对糖皮质激素的反应不同。两者都有 表达cPLA2和间充质细胞表达诱导型一氧化氮 合成酶强调了它们在发出发病信号中的潜在作用 劳动力的问题。我们的目标是：1.确定cPLA2的磷酸化是否通过 Wish细胞中的11-1β或EGF与特定的核相关 组分(核膜或内质网)引起 花生四烯酸的动员和与PGHS-2的共定位。2. 利用特异性抑制物和免疫印迹法检测II-1β 或EGF刺激cPLA2磷酸化和cPLA2/PGHS-2mRNA表达 在Wish细胞中通过p38蛋白激酶、ERK-1、2或蛋白激酶发生 C路径。3.确定刺激细胞的脂多糖(LPS)是否 Wish细胞合成PGE2，通过p38使cPLA2磷酸化 MAPK和IF内毒素处理细胞为后续细胞启动 细胞因子的作用。4.确定促炎和抗炎的作用 细胞因子(II-1β、II-4和II-10)和生长因子(TGFbeta)在 Wish细胞中cPLA2的磷酸化和激活及其相互作用 这些细胞因子与内毒素的结合。5.确定表达式中的变化， CPLA2的磷酸化(激活)及其与核的结合 足月或足月患者羊膜组织中PGHS-2的表达 早产，无论是否处于分娩中。6.确定p38 MAP激酶是否 羊膜上皮和羊膜上皮细胞中cPLA2的激活磷酸化 间充质细胞和糖皮质激素对cPLA2的不同影响 在这些细胞中表达，就像它们对PGHS-2所做的那样。 这项研究将提供有关磷酸化和激活的新数据。 CPLA2在人羊膜组织中的表达，并进一步确定这一至关重要的作用 胎膜中二十烷类化合物合成的调节步骤 分娩。