Transcriptional Elongation in NF-kB Mediated Inflammation

NF-kB 介导的炎症中的转录延伸

• 批准号: 8710493
• 负责人: Allan R. Brasier
• 金额: $ 28.18万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8710493
• 关键词: Acetylation; Acute; Affect; Ambrosia; American; Antioxidants; Antiviral Agents; Antiviral Response; Asthma; Binding; Biological Assay; Breathing; Bromodomain; Bronchiolitis; C-terminal; CDK9 Protein Kinase; Cells; Complex; Cyclin-Dependent Kinases; DNA Damage; DNA-Directed RNA Polymerase; Disease; Elongation Factor; Embryo; Epithelial Cells; Fibroblasts; Foundations; Gene Targeting; Genes; Goals; Histone H4; Host Defense; IRF3 gene; Immune response; Immunity; Inbred BALB C Mice; Infection; Inflammation; Inflammatory; Inflammatory Response; Interferons; Kinetics; Knowledge; Lead; Lung; Measures; Mediating; Mediator of activation protein; Monitor; Mucous Membrane; Mus; NADPH Oxidase; NF-kappa B; Nuclear; Pathology; Pathway interactions; Peptides; Phosphorylation; Phosphotransferases; Play; Pollen; Process; Production; Proteins; RNA; Reaction; Reactive Oxygen Species; Regulation; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Structure of parenchyma of lung; Tertiary Protein Structure; Testing; Time; Transcription Factor 3; Viral; Virus; Virus Diseases; Work; adapter protein; airway epithelium; airway hyperresponsiveness; airway inflammation; base; chromatin immunoprecipitation; cytokine; design; in vivo; inhibitor/antagonist; mouse model; mutant; novel; novel strategies; particle; programs; promoter; reconstitution; research study; response; small molecule

Inducible reactive oxygen species (ROS) play a critical regulatory role in the innate immune response (MR) by controlling the phosphorylation of both 1RF3 and NF-KB/ROIA. RelA phosphorylation is required for its inducible acetylation at Lys and binding to the positive elongation factor-b (PTEF-b) complex. The PTEF-b complex of cyclin-dependent kinase (CDK)-9 and bromodomain-4 (Brd4) proteins induces RNA Pol 11 Ser2 phosphorylation to induce transcriptional elongation in downstream genes. P1 will test the hypothesis that ROS modulate innate inflammatory and antiviral activities by modulating the activation states of NFKappaB/ RelA and iRF3 in the airway epithelium in three specific aims. Aim 1 will examine how ROS modify NF-KappaB/RelA to induce recruitment of the Brd4-CDK9 complex mediating inflammatory cytokine production. The requirement of RelA Lys310 for ROS-induced PTEF-b recruitment will be tested by chromatin immunoprecipitation (ChIP) assays in RelA''' mouse embryonic fibroblasts reconstituted with WT RelA or its non-acetylated Lys310 Arg mutant in response to TNFa, RSV and ragweed pollen particle NADPH oxidase. The role of ROS in inducing promoter-associated inactive CDK9 clearance and replacement with activated CDK9 will be determined in small airway epithelial cells (SAEs). The role of Brd4 as an adapter protein will be examined using siRNA knockdown or in vivo competition using Lys-acetylated histone H4 peptides. Aim 2 will examine how ROS mediate IRF3 pathway signaling. We will modulate inducible ROS to examine the kinetics of 1RF3 translocation in SAEs, using our novel selected reaction monitoring (SRM) assay. The requirement of ROS for 1RF3 enhanceosome formation will be tested by ChIP assay on IFNp and interferon-stimulated gene (ISG) promoters ¿ antioxidant modulation. We will determine whether IRF3 induces antiviral transcriptional initiation or elongation by siRNA depletion of CDK-7 and -9. Aim 3 will examine how transcriptional elongation mediates the effect of the ilR in RSV infection in vivo. How inhibiting NF-KB-PTEF-b recruitment and CDK9 affects cellular inflammation, anti-viral immunity, airway pathology and airway hyper-reactivity (AHR) will be tested in a BALB/c mouse model using an NF-KB inhibitory IKKy/NEMO binding domain (NBD) peptide and small molecule CDK9 inhibitor. These studies will establish the role of the ROS-initiated transcriptional elongation pathway in the MR and so lay the foundation for novel approaches to selectively modulate inflammation in airway mucosa and RSV bronchiolitis.

诱导型活性氧（ROS）在先天免疫应答（MR）中起着重要的调节作用 通过控制1 RF 3和NF-κ B/ROIA的磷酸化。RelA磷酸化是其 在Lys处的诱导型乙酰化和与正延伸因子-b（PTEF-b）复合物的结合。PTEF-B 细胞周期蛋白依赖性激酶（CDK）-9和溴结构域-4（Brd 4）蛋白的复合物诱导RNA Pol 11 Ser 2 磷酸化以诱导下游基因中的转录延伸。P1将检验假设， ROS通过调节NF κ B/κ B的活化状态来调节先天性炎症和抗病毒活性。 RelA和iRF 3在三个特定目的中在气道上皮中的表达。目标1将研究ROS如何修改 NF-κ B/RelA诱导介导炎性细胞因子的Brd 4-CDK 9复合物的募集 生产将通过以下方法检测RelA Lys 310对ROS诱导的PTEF-b募集的要求： 用WT重建的RelA小鼠胚胎成纤维细胞中的染色质免疫沉淀（ChIP）测定 RelA或其非乙酰化Lys 310 Arg突变体对TNF α、RSV和豚草花粉颗粒NADPH的响应 氧化酶。ROS在诱导启动子相关的失活CDK 9清除和替换中的作用 将在小气道上皮细胞（SAE）中测定活化的CDK 9。Brd 4作为适配器的作用 将使用siRNA敲低或使用Lys-乙酰化组蛋白H4的体内竞争来检查蛋白 缩氨酸目的2将研究ROS如何介导IRF 3信号通路。我们将调节可诱导的 使用我们的新型选择性反应监测，检查SAE中1 RF 3易位的动力学 (SRM)比色法ROS对1 RF 3增强体形成的需求将通过ChIP测定来测试。 IFN β和干扰素刺激基因（ISG）启动子抗氧化调节。我们将决定 IRF 3通过siRNA耗尽CDK-7和-9诱导抗病毒转录起始或延长。目标3将 研究转录延伸如何介导ilR在体内RSV感染中的作用。如何 抑制NF-KB-PTEF-b募集和CDK 9影响细胞炎症、抗病毒免疫、气道炎症和呼吸道炎症。 病理学和气道高反应性（AHR）将在BALB/c小鼠模型中使用NF-κ B 抑制性IKK γ/NEMO结合结构域（NBD）肽和小分子CDK 9抑制剂。这些研究将 确立ROS启动的转录延伸途径在MR中的作用，从而奠定基础 用于选择性调节气道粘膜炎症和RSV细支气管炎的新方法。