PI3-kinase and mTOR in NO production by macrophages

PI3-激酶和 mTOR 在巨噬细胞产生 NO 中的作用

• 批准号: 6415094
• 负责人: STEVEN LEE WEINSTEIN
• 金额: $ 14.36万
• 依托单位: SAN FRANCISCO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-15 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6415094
• 关键词: bacterial polysaccharides; biological signal transduction; enzyme activity; enzyme linked immunosorbent assay; immunofluorescence technique; immunoprecipitation; interferon beta; laboratory mouse; lipopolysaccharides; macrophage; macrophage activating factor; messenger RNA; nitric oxide synthase; nitrogen oxides; northern blottings; phosphatidylinositol 3 kinase; phosphorylation; posttranslational modifications; sirolimus; western blottings

DESCRIPTION (provided by applicant): Macrophages have evolved the capacity to recognize conserved structural components of pathogens such as the LPS moiety of Gram negative bacteria to facilitate making effective anti-microbial responses. The long-term objective of this research is to understand how LPS-elicited responses of macrophages are mediated by intracellular events. The production of nitric oxide (NO) by LPS-activated macrophages is known to play a central role in the host defense against infection, but in pathological immune responses, this molecule mediates severe tissue damage. For this reason, there is considerable interest in understanding the mechanisms underlying LPS regulation of NO production. To trigger this response, LPS requires a cytokine co-factor, which is believed to be macrophage-derived interferon-beta during the early stages of an Gram-negative infection. Recently, it has been observed that phosphatidylinositol 3-kinase (PI3-K) and mTOR are constituents of a signaling pathway that regulates interferon-beta synthesis and NO production. Thus, the proposed research tests the hypothesis that PI3-K and mTOR-mediate NO production in LPS-treated macrophages by activating a downstream signaling component, p70 S6 kinase, and this kinase regulates the synthesis of interferon-beta mRNA and enhances the expression and activity of NOS2, the NO-producing enzyme. The specific aims are designed to test this hypothesis by: (1) determining how PI3-K and mTOR regulate NOS2; (2) determining how PI3-K and mTOR regulate interferon-beta; and (3) determining whether p70 S6 kinase is a downstream effector of PI3-K and mTOR leading to interferon-beta and NO production. The effect of pharmacologically-inhibiting PI3-K or mTOR on the LPS-stimulated levels of NOS2 mRNA, protein and enzyme activity will be evaluated by Northern blotting, Western blotting and by an in vitro activity assay measuring the rate of conversion of radiolabeled L-arginine to L-citrulline (and NO), respectively. The effect of pharmacologically-inhibiting PI3-K or mTOR on LPS-modulation of interferon-beta mRNA stability will be determined by measuring the rate of mRNA degradation by ribonuclease protein assay. The activity of p70 S6 kinase will be perturbed with a transfected mutant p70 S6 kinase allele and the consequence of this manipulation on the synthesis of interferon-beta and NO will be determined.

描述（由申请人提供）：巨噬细胞已经进化出以下能力： 识别病原体的保守结构成分，例如 LPS 部分 革兰氏阴性菌有助于制造有效的抗菌剂 回应。这项研究的长期目标是了解如何 LPS 引发的巨噬细胞反应是由细胞内事件介导的。这 LPS 激活的巨噬细胞产生一氧化氮 (NO) 的作用已知 在宿主抵御感染的防御中发挥核心作用，但在病理免疫中也发挥着核心作用 响应，该分子介导严重的组织损伤。为此，有 对了解 LPS 的机制非常感兴趣 NO 产生的调节。为了触发这种反应，LPS 需要细胞因子 辅助因子，被认为是巨噬细胞衍生的干扰素-β 革兰氏阴性菌感染的早期阶段。近日，观察到 磷脂酰肌醇 3-激酶 (PI3-K) 和 mTOR 是 调节干扰素β合成和NO产生的信号通路。 因此，本研究测试了 PI3-K 和 mTOR 介导 NO 的假设 通过激活下游信号在 LPS 处理的巨噬细胞中产生 成分，p70 S6 激酶，该激酶调节 干扰素-β mRNA 并增强 NOS2（NOS2）的表达和活性 产生NO的酶。具体目标旨在通过以下方式检验这一假设： (1)确定PI3-K和mTOR如何调节NOS2； (2) 确定 PI3-K 和 mTOR 调节干扰素-β； (3) 确定 p70 S6 激酶是否是 PI3-K 和 mTOR 的下游效应子，导致干扰素-β 和 NO 生产。 药理学抑制 PI3-K 或 mTOR 对 LPS 刺激的影响 NOS2 mRNA、蛋白质和酶活性的水平将由 Northern 评估 印迹法、蛋白质印迹法以及通过体外活性测定测量速率 放射性标记的 L-精氨酸转化为 L-瓜氨酸（和 NO）， 分别。药理学抑制 PI3-K 或 mTOR 对 LPS 对干扰素-β mRNA 稳定性的调节将通过以下方式确定 通过核糖核酸酶蛋白测定法测量 mRNA 降解率。这 p70 S6 激酶的活性将受到转染突变体 p70 S6 的干扰 激酶等位基因以及这种操作对合成的影响 将测定干扰素-β和NO。