LPS SIGNAL TRANSDUCTION IN NEUTROPHILS

中性粒细胞中的 LPS 信号转导

• 批准号: 6182394
• 负责人: JERRY A NICK
• 金额: $ 11.16万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6182394
• 关键词: CD14 molecule; biological signal transduction; enzyme activity; human subject; inflammation; ion exchange chromatography; laboratory mouse; lipopolysaccharides; mitogen activated protein kinase; neutrophil; phlebotomy; protein tyrosine kinase

DESCRIPTION (Adapted from applicant's abstract) Stimulation of neutrophils by lipopoly-saccharide (LPS) is central to the pathogenesis of sepsis and the Adult Respiratory Distress Syndrome. LPS binds to CD14, a cell surface protein, resulting in actin assembly and adhesion. The intracellular signaling pathway that links the LPS binding of CD14 with functional cellular responses is largely unknown. Based on studies of chemoattractant stimulation of neutrophils and signaling events in mammalian cell lines, a framework now exists to study intracellular signal transduction in human neutrophils by LPS. Several families of intracellular proteins are present in mammalian cells that function to phosphorylate other proteins. These kinases are inactive until phosphorylated, and then are capable of phosphorylating and activating a specific "downstream" kinase in turn. The applicants hypothesize that following LPS binding to CD14, one or more nonreceptor protein tyrosine kinases (NRPTKs) are activated. Through a series of sequential phosphorylation reactions the signal is passed through a branch of the mitogen-activated protein (MAP) kinase cascade. A member of the MAP/ERK kinase kinase (MEKK) family is activated, which in turn phosphorylates and activates a member of the MAP/ERK kinase (MEK) family which then phosphorylates and activates a member of the MAP kinase family. They have recently reported that the MAP kinase activated in human neutrophils in response to LPS is p38 MAP kinase. In this proposal members of the MEK, MEKK, and NRPTK-families utilized by the neutrophil in response to LPS stimulation will be identified. These kinases will be isolated by anion-exchange chromatography, assayed for activation in response to LPS, and identified by specific antibodies and protein sequencing. Simultaneously, these signaling events will be explored in murine neutrophils. Signaling proteins share a high degree of homology in all mammalian cells, and it is expected that signaling mechanisms utilized by murine and human neutrophils will be nearly identical. The link between LPS binding of CD14, intracellular signaling, and subsequent functional responses will then be established through the use of a specific inhibitor to p38 MAP kinase, genetically modified mice strains, and murine models of pulmonary neutrophil accumulation. The comprehensive study of this signal transduction pathway may lead to new approaches for modifying LPS induced inflammatory responses. (End of abstract)

描述 (改编自申请人的摘要)通过以下方式刺激中性粒细胞 脂多糖(LPS)在脓毒症的发病机制中起核心作用。 成人呼吸窘迫综合征。脂多糖与细胞表面CD14结合 蛋白质，导致肌动蛋白组装和粘连。细胞内 CD14的内毒素结合与功能相关的信号通路 细胞反应在很大程度上是未知的。基于对化学引诱剂的研究 哺乳动物细胞系中性粒细胞的刺激和信号事件 目前已有研究人类细胞内信号转导的框架 中性粒细胞由脂多糖诱导。存在几个胞内蛋白家族 在哺乳动物细胞中，它的功能是使其他蛋白质磷酸化。这些 在磷酸化之前，激酶是不活跃的，然后能够 依次磷酸化和激活特定的“下游”激酶。这个 申请者假设在脂多糖与CD14结合后，一个或多个 非受体蛋白酪氨酸激酶(NRPTKs)被激活。通过一个 一系列顺序的磷酸化反应信号通过 丝裂原活化蛋白(MAP)激酶级联的一个分支。成员之一 MAP/ERK激酶(MEKK)家族被激活，进而 磷酸化并激活MAP/ERK激酶(MEK)家族的一个成员 然后，它磷酸化并激活MAP激酶家族的一个成员。 他们最近报告说，在人类体内激活的MAP激酶 中性粒细胞对内毒素的反应是p38 MAP激酶。在本提案中，成员 中性粒细胞对MEK、MEKK和NRPTK家族的利用 对内毒素的刺激将被鉴定。这些激酶将通过以下方式分离 阴离子交换层析，检测对内毒素的反应激活， 并通过特定抗体和蛋白质测序进行鉴定。 同时，这些信号事件将在小鼠身上进行探索 中性粒细胞。所有信号蛋白都有高度的同源性 哺乳动物细胞，预计信号机制由 小鼠和人类的中性粒细胞将几乎相同。内毒素之间的联系 CD14的结合、细胞内信号转导及随后的功能 然后，通过使用特定的抑制剂来建立反应 P38 MAP激酶、转基因小鼠品系和小鼠模型 肺中性粒细胞聚集。对这一信号的综合研究 信号转导通路可能为内毒素诱导的修饰开辟新途径 炎症反应。(摘要结束)