NF-kB Regulation, ICAM-1 Expression, and Lung Injury

NF-kB 调节、ICAM-1 表达和肺损伤

• 批准号: 8005126
• 负责人: CHINNASWAMY TIRUPPATHI
• 金额: $ 28.73万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8005126
• 关键词: 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one; A20 protein; Acute Lung Injury; Address; Adhesions; Binding; Binding Proteins; Biological Assay; CabP2; Cells; Cloning; Complement; Complementary DNA; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA Binding; Data; Dominant-Negative Mutation; Dreams; EF Hand Motifs; Ectopic Expression; Elements; Endothelial Cells; Esthesia; Figs - dietary; Gene Expression; Gene Targeting; Genes; Genetic Models; Genetic Transcription; Host Defense; Human; Hydrogen Peroxide; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Injury; Instruction; Intercellular adhesion molecule 1; Intervention; Knock-out; Ligation; Link; Lung; Mediating; Modeling; Molecular; Mus; NADPH Oxidase; NF-kappa B; Natural Immunity; Nucleic Acid Regulatory Sequences; Oxidants; Pain; Phenotype; Phosphorylation; Positioning Attribute; Prevention; Promoter Regions; Protein Biosynthesis; Proteins; Puncture procedure; RIPK2 gene; Reactive Oxygen Species; Regulation; Reporter; Repression; Repressor Proteins; Research Proposals; Role; Seminal; Sepsis; Signal Pathway; Signal Transduction; Signaling Pathway Gene; Stimulus; Testing; Therapeutic; Thrombin; Time; Transcription Repressor/Corepressor; Vascular Endothelial Cell; Wild Type Mouse; base; chromatin immunoprecipitation; derepression; gene repression; genetic regulatory protein; glucosylthiazolidine-4-carboxylic acid; in vivo; inhibitor/antagonist; intraperitoneal; knock-down; lung injury; lung vascular injury; mortality; neutrophil; novel; prevent; promoter; research study; response; stem; sulfated glycoprotein 2; transcription factor

PROJECT SUMMARY (See instructions): Transcription factor NF-KB activation-dependent ICAM-1 expression in pulmonary endothelial cells results in neutrophil (PMN) adhesion-mediated lung vascular injury. NF-KB-dependent expression of the NF-KB negative regulatory protein A20 inhibits unrestrained activation of NF-KB and thereby controls the inflammatory response. The pre-transcriptional mechanisms involved in regulating A20 gene expression are poorly understood. This research proposal stems from our seminal discovery of multiple "GTCA" sequences (ORE elements) forthe Ca2+ binding transcriptional repressor protein Dream in the 5'-regulatory region of both human and mouse A20 genes. Thus, in Project 3, we will address the crucial role of Dream in regulating the expression of A20 in pulmonary microvessels and thereby controlling NF-KB activation, ICAM-1 expression, and PMN adhesion-mediated lung vascular injury. We cloned a 1.5 kb human A20 promoter sequence linked to reporter and showed robust promoter activity in response to thrombin and TNF-a which was blocked by co-expression of Dream. In addition, we showed that Ca2+ influx through TRPC4 channels or oxidant-sensitive TRPM2 channels induced the de-repression of A20 transcription in response to thrombin and H2O2 respectively. Importantly, we observed that in Dream knock-out (Dream -/-) mice, basal A20 expression was enhanced and this was associated with prevention of thrombin-TNF-a- or LPS-induced ICAM-1 expression in lungs of Dream''' mice. Further, LPS-induced acute lung injury was markedly reduced in Dream -/- mice. In addition, we observed that NF-KB activation in response to inflammatory stimuli was suppressed in Dream-/- mice. Based on these novel findings, in Aim 1, we will test the hypothesis that repressor Dream regulates the expression of the NF-KB inhibitor A20 by interacting with 5'-regulatory region of the A20 gene. In Aim 2, we will test the hypothesis that H2O2 activation of oxidant-sensitive TRPM2 channels and induces Ca2+ influx leading to de-repression of A20 transcription and its consequent suppression of NF-KB activity. In Aim 3, we will test the hypothesis that Dream in vivo regulates the basal expression of A20 and thereby suppresses unrestrained activation of NF-KB utilizing Dream-/- mice. With this understanding of Dream, we will be in the position to develop therapeutic strategies that can target Dream function and thereby prevent acute lung injury without compromising innate immunity.

项目摘要（参见说明）： 肺内皮细胞中转录因子 NF-KB 激活依赖性 ICAM-1 表达导致中性粒细胞 (PMN) 粘附介导的肺血管损伤。 NF-KB 负调节蛋白 A20 的 NF-KB 依赖性表达抑制 NF-KB 的无限制激活，从而控制炎症反应。人们对调节 A20 基因表达的转录前机制知之甚少。这项研究提案源于我们在人类和小鼠 A20 基因 5' 调控区中对 Ca2+ 结合转录抑制蛋白 Dream 的多个“GTCA”序列（ORE 元件）的开创性发现。因此，在项目 3 中，我们将探讨 Dream 在调节肺微血管中 A20 表达从而控制 NF-KB 激活、ICAM-1 表达和 PMN 粘附介导的肺血管损伤中的关键作用。我们克隆了与报告基因连接的 1.5 kb 人 A20 启动子序列，并显示出响应凝血酶和 TNF-α 的强大启动子活性，该活性被 Dream 的共表达所阻断。此外，我们还发现，Ca2+ 通过 TRPC4 通道或氧化剂敏感的 TRPM2 通道流入，分别响应凝血酶和 H2O2 诱导 A20 转录去抑制。重要的是，我们观察到，在 Dream 敲除 (Dream -/-) 小鼠中，基础 A20 表达增强，这与防止 Dream 肺部凝血酶 TNF-a 或 LPS 诱导的 ICAM-1 表达相关。 ' 老鼠。此外，Dream -/- 小鼠中 LPS 诱导的急性肺损伤显着减少。此外，我们观察到 Dream-/- 小鼠中响应炎症刺激的 NF-KB 激活受到抑制。基于这些新发现，在目标 1 中，我们将检验阻遏蛋白 Dream 通过与 A20 基因的 5' 调节区相互作用来调节 NF-KB 抑制剂 A20 表达的假设。在目标 2 中，我们将测试以下假设：H2O2 激活氧化剂敏感的 TRPM2 通道并诱导 Ca2+ 流入，从而导致 A20 转录去抑制，从而抑制 NF-KB 活性。在目标 3 中，我们将利用 Dream-/- 小鼠测试 Dream 在体内调节 A20 的基础表达，从而抑制 NF-KB 不受限制的激活的假设。有了对 Dream 的了解，我们将能够开发针对 Dream 功能的治疗策略，从而在不损害先天免疫的情况下预防急性肺损伤。