REGULATION OF PHOSPHOLIPASE A2 IN HUMAN AMNION

人羊膜中磷脂酶 A2 的调节

• 批准号: 6151149
• 负责人: LESLIE MYATT
• 金额: $ 24.53万
• 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 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6151149
• 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.

促炎细胞因子和抗炎细胞因子的自分泌/旁分泌影响 胎膜中的生长因子似乎是合成的核心 足月临产和感染引起的许多效应物的影响 早产。 我们已经证明 II-1beta 可以协调诱导 胞质磷脂酶 A2 (cPLA2) 和前列腺素 H 的表达 羊膜来源的 WISH 细胞中的合酶 2 (PGHS-2) 和两种酶 定位于核膜上。 这两种酶可以偶联至 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 敲除小鼠足月分娩失败，显示出其核心作用 cPLA2 介导分娩过程中 PG 的释放。 催化 cPLA2 的活性通过激动剂的磷酸化来刺激，例如 表皮生长因子。 我们有初步证据表明 p38 MAP 激酶参与 这条途径。 羊膜上皮细胞和间充质细胞均表达 足月分娩时的 PGHS-2 但对糖皮质激素的反应不同。 两个都 表达 cPLA2，间充质细胞表达诱导型一氧化氮 合酶强调了它们在发病信号中的潜在作用 的劳动。 我们的目标是： 1. 确定 cPLA2 的磷酸化是否通过 WISH 细胞中的 11-1beta 或 EGF 与特定的细胞核相关 部分（核膜或内质网）引起 花生四烯酸动员并与 PGHS-2 共定位。 2. 利用特定抑制剂和蛋白质印迹来确定 II-1beta 是否 或 EGF 刺激 cPLA2 磷酸化和 cPLA2/PGHS-2 mRNA 表达 在 WISH 细胞中，通过 p38 MAP 激酶、ERK-1,2 或蛋白激酶发生 C 途径。 3. 确定脂多糖 (LPS) 是否会刺激 WISH 细胞合成 PGE2，通过 p38 引起 cPLA2 磷酸化 MAP 激酶和 LPS 处理细胞可以为随后的细胞做好准备 细胞因子的作用。 4.确定促炎和抗炎作用 细胞因子（II-1β、II-4 和 II-10）和生长因子 (TGFbeta) WISH 细胞中的 cPLA2 磷酸化和激活及其相互作用 这些细胞因子与 LPS 的结合。 5. 确定表达式的变化， cPLA2 的磷酸化（激活）及其与核的关联 足月或足月患者的羊膜组织中的膜（带有 PGHS-2） 早产，无论是在临产还是未临产。 6. 确定 p38 MAP 激酶是否 cPLA2 的刺激磷酸化发生在羊膜上皮和 间充质细胞以及糖皮质激素是否对 cPLA2 有不同影响 在这些细胞中表达，正如它们对 PGHS-2 的作用一样。 这项研究将提供有关磷酸化和激活的新数据 cPLA2 在人类羊膜组织中的作用并进一步明确了这一关键的作用 胎膜中类二十烷酸合成的调节步骤 分娩。