Nitric Oxide Regulation of Inflammatory Responses and Ge

一氧化氮对炎症反应和 Ge 的调节

• 批准号: 6825010
• 负责人: ROBERT L DANNER
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6825010
• 关键词: biological signal transduction; cell population study; chemical structure function; cytokine; gene expression; genetic promoter element; genetic regulation; human tissue; immunogenetics; immunoregulation; inflammation; interleukin 8; leukocytes; microarray technology; nitric oxide; tissue /cell culture

Introduction and Objective: Nitric oxide is an important inter- and intracellular messenger implicated in the pathogenesis of septic shock. Inhibition of nitric oxide synthase is under investigation as a treatment for hypotension in septic shock. Furthermore, inhaled nitric oxide is under investigation as a therapy for respiratory distress syndrome in neonates and adults. In addition to its vasodilating effects on the systemic and pulmonary vasculatures, nitric oxide also modulates immune responses and regulates gene expression. These latter attributes may have implications for the use of nitric oxide synthase inhibitors or nitric oxide itself for syndromes in which the host?s inflammatory response plays a pathogenic role. In this project, we are examining the role of nitric oxide as a modulator of inflammation and gene expression. Progress: Human phagocytes, in particular neutrophils, were demonstrated to lack the capacity to endogenously produce nitric oxide under a number of ex vivo and in vivo conditions. Therefore, the ability of these cells to be regulated in a paracrine manner by exogenous sources of NO such as the endothelium has been explored. In addition to upregulating TNFa production, nitric oxide was found to modulate IL-8 mRNA levels and IL-8 production in human neutrophil preparations. We have confirmed that endogenously produced nitric oxide also up-regulates TNFa production using human U937 cells, a monoblastoid cell line, transfected to express murine inducible nitric oxide synthase. Investigation of TNFa regulation by nitric oxide resulted in the description of a cGMP-independent signaling pathway that utilizes cAMP downregulation as a signal transduction event. A nitric oxide-responsive Sp1 binding site was identified in the proximal TNFa promoter. NO-mediated decreases in cAMP leads to reduced Sp1 binding to the TNFa promoter with subsequent increases in TNFa transcription. Recent work has shown that NO downregulates the eNOS promoter through effects on Sp1 identical to those that cause TNFa upregulation. For TNFa, an AP1 site upstream to Sp1 serves to reverse the direction of the nitric oxide response. Mutation of the AP1 site converts the effect of nitric oxide in TNFa from up to down regulation (eNOS-like). The IL-8 promoter lacks a canonical Sp1 site. Unlike TNFa, IL-8 regulation by nitric oxide is both cGMP and cAMP-independent. Further, nitric oxide p38 MAPK-dependent effect on protein binding to regulation of IL-8 has been found to be post-transcriptional and mediated via AU-rich elements in its mRNA 3? UTR. An oligonucleotide microarray analysis in differentiated U937 cells has identified more than 100 additional nitric oxide regulated genes. Proposed Course of Work: Current work is focused on identifying all major pathways of nitric oxide gene regulation using microarray and molecular biology approaches. Part of this effort is directed toward characterizing nitric oxide effects on the cell-cycle and the respective roles of p38 MAPK, proteasome inhibition, and mRNA stability on gene regulation by nitric oxide. Separate experiments are directed at profiling the effects of nitric oxide and inflammatory mediators such as endotoxin, TNFa, and HMG-B1 on a variety of primary cell types including neutrophils, elutriated monocytes, and endothelial cells.

简介和目的：一氧化氮是重要的细胞间和细胞内信使，与感染性休克的发病机制有关。目前正在研究抑制一氧化氮合酶作为治疗感染性休克低血压的方法。此外，正在研究吸入一氧化氮作为新生儿和成人呼吸窘迫综合征的治疗方法。除了对全身和肺血管系统具有血管舒张作用外，一氧化氮还可以调节免疫反应并调节基因表达。后面这些属性可能对使用一氧化氮合酶抑制剂或一氧化氮本身治疗宿主炎症反应起致病作用的综合征有影响。 在这个项目中，我们正在研究一氧化氮作为炎症和基因表达调节剂的作用。 进步： 人类吞噬细胞，特别是中性粒细胞，被证明在许多离体和体内条件下缺乏内源性产生一氧化氮的能力。因此，已经探索了这些细胞通过外源NO（例如内皮细胞）以旁分泌方式调节的能力。 除了上调 TNFa 的产生外，一氧化氮还可以调节人中性粒细胞制剂中的 IL-8 mRNA 水平和 IL-8 的产生。 我们使用人 U937 细胞（一种单胚细胞系，转染表达鼠诱导型一氧化氮合酶）证实内源性一氧化氮也会上调 TNFa 的产生。 对一氧化氮调节 TNFa 的研究导致了对 cGMP 独立信号通路的描述，该通路利用 cAMP 下调作为信号转导事件。 在近端 TNFa 启动子中鉴定出一氧化氮响应性 Sp1 结合位点。 NO 介导的 cAMP 减少导致 Sp1 与 TNFa 启动子的结合减少，随后 TNFa 转录增加。 最近的研究表明，NO 通过对 Sp1 的影响来下调 eNOS 启动子，这与引起 TNFa 上调的作用相同。对于 TNFa，Sp1 上游的 AP1 位点用于​​逆转一氧化氮反应的方向。 AP1 位点的突变将 TNFa 中一氧化氮的作用从上调转为下调（类似 eNOS）。 IL-8 启动子缺少典型的 Sp1 位点。与 TNFa 不同，一氧化氮对 IL-8 的调节不依赖于 cGMP 和 cAMP。此外，一氧化氮 p38 MAPK 对蛋白质结合与 IL-8 调节的依赖效应已被发现是转录后的，并通过其 mRNA 3? 中富含 AU 的元件介导。 UTR。 对分化的 U937 细胞进行的寡核苷酸微阵列分析已鉴定出 100 多个额外的一氧化氮调节基因。 拟议的工作过程： 目前的工作重点是使用微阵列和分子生物学方法确定一氧化氮基因调控的所有主要途径。这项工作的一部分旨在表征一氧化氮对细胞周期的影响以及 p38 MAPK、蛋白酶体抑制和一氧化氮基因调节的 mRNA 稳定性各自的作用。 单独的实验旨在分析一氧化氮和炎症介质（例如内毒素、TNFa 和 HMG-B1）对各种原代细胞类型（包括中性粒细胞、淘析单核细胞和内皮细胞）的影响。