LPS SIGNAL TRANSDUCTION IN NEUTROPHILS

中性粒细胞中的 LPS 信号转导

• 批准号: 6388395
• 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/6388395
• 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）中性粒细胞刺激 脂多糖（LPS）是脓毒症发病机制的核心， 成人呼吸窘迫综合征。 LPS与细胞表面CD 14结合 蛋白质，导致肌动蛋白组装和粘附。 细胞内 信号通路，其将CD 14的LPS结合与功能性 细胞反应在很大程度上是未知的。 基于化学引诱物的研究 刺激哺乳动物细胞系中的中性粒细胞和信号传导事件， 现在有一个研究人类细胞内信号转导的框架， 中性粒细胞LPS。 存在几个细胞内蛋白质家族 在哺乳动物细胞中磷酸化其他蛋白质。 这些 激酶在磷酸化之前是无活性的，然后能够 磷酸化并依次激活特定的“下游”激酶。 的 申请人假设在LPS与CD 14结合后，一种或多种 非受体蛋白酪氨酸激酶（NRPTKs）被激活。 通过 信号通过的一系列连续的磷酸化反应 丝裂原活化蛋白（MAP）激酶级联的分支。 的成员 MAP/ERK激酶激酶（MEKK）家族被激活， 磷酸化并激活MAP/ERK激酶（MEK）家族的成员 然后磷酸化并激活MAP激酶家族的成员。 他们最近报道，MAP激酶在人类中被激活， 中性粒细胞对LPS的反应是p38 MAP激酶。 在这项提案中， 中性粒细胞在应答中利用的MEK、MEKK和NRPTK家族 LPS刺激将被识别。 这些激酶将被分离， 阴离子交换层析，测定响应于LPS的活化， 并通过特异性抗体和蛋白质测序进行鉴定。 同时，这些信号事件将在小鼠中进行探索。 中性粒细胞 信号蛋白在所有细胞中具有高度的同源性。 哺乳动物细胞，并预计利用的信号传导机制， 鼠和人的嗜中性粒细胞几乎相同。 LPS之间的联系 CD 14的结合，细胞内信号传导，以及随后的功能 然后通过使用特定的抑制剂来建立反应 对p38 MAP激酶，遗传修饰的小鼠品系，和 肺中性粒细胞蓄积。 对这个信号的综合研究 转导途径可能导致新的方法来修饰LPS诱导的 炎症反应。 (End摘要）