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

NF-kB 调节、ICAM-1 表达和肺损伤

• 批准号: 8380084
• 负责人: CHINNASWAMY TIRUPPATHI
• 金额: $ 28.44万
• 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
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8380084
• 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; 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; Knockout Mice; Ligation; Link; Lung; Lung Inflammation; 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; TNF gene; 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.

项目总结(见说明)： 肺内皮细胞转录因子NF-KB激活依赖ICAM-1的表达导致中性粒细胞(PMN)黏附介导的肺血管损伤。依赖于核因子-kB的负性调节蛋白A20的表达抑制了核因子-kB的不受限制的激活，从而控制了炎症反应。参与调控A20基因表达的转录前机制还知之甚少。这项研究建议源于我们在人和小鼠A20基因的5‘-调控区为钙结合转录抑制蛋白Dream的多个“GTCA”序列(ORE元件)的开创性发现。因此，在项目3中，我们将阐述DREAM在调节肺微血管中A20的表达，从而控制核因子-KB的激活、ICAM-1的表达以及PMN黏附介导的肺血管损伤中的关键作用。我们克隆了一个与报告基因连锁的1.5kb的人A20启动子序列，并显示出对凝血酶和肿瘤坏死因子-α的强烈启动子活性，而凝血酶和肿瘤坏死因子-α被DREAM的共表达所阻断。此外，我们还发现，钙离子通过TRPC4通道或氧化剂敏感的TRPM2通道内流分别诱导凝血酶和H_2O_2抑制A20转录。重要的是，我们观察到，在Dream基因敲除(Dream-/-)小鼠中，基础A20的表达增强，这与防止Dream‘’小鼠肺内凝血酶-肿瘤坏死因子-α或脂多糖诱导的ICAM-1表达有关。此外，在Dream-/-小鼠中，脂多糖诱导的急性肺损伤明显减轻。此外，我们观察到，在Dream-/-小鼠中，对炎症刺激的反应中，核因子-KB的激活受到抑制。基于这些新的发现，在目标1中，我们将检验抑制子Dream通过与A20基因的5‘-调控区相互作用来调节核因子-KB抑制物A20的表达的假设。在目标2中，我们将验证这样的假设，即过氧化氢激活氧化剂敏感的TRPM2通道并诱导钙内流导致A20转录的下调，从而抑制核因子-KB的活性。在目标3中，我们将利用Dream-/-小鼠验证这一假设，即Dream在体内调节A20的基础表达，从而抑制NF-KB的自由激活。有了对Dream的了解，我们将能够开发针对Dream功能的治疗策略，从而在不损害天然免疫的情况下预防急性肺损伤。