Regulation of Proinflammatory Signaling Pathways in CFTR

CFTR 中促炎信号通路的调节

• 批准号: 7143989
• 负责人: Harvey Bruce Pollard
• 金额: $ 31.02万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-15 至 2011-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7143989
• 关键词: Pseudomonas aeruginosa; biological signal transduction; cell line; chloride channels; clinical research; confocal scanning microscopy; cystic fibrosis; genetic promoter element; human tissue; immunogenetics; immunopharmacology; immunoregulation; inflammation; interleukin 8; intracellular transport; microarray technology; molecular pathology; nucleic acid sequence; pharmacogenetics; protein structure function; respiratory infections; transfection

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is the most common fatal autosomal recessive disease in the U.S., and is due to mutations in the CFTR gene. CF is characterized by constitutive hypersecretion of proinflammatory IL-8 from airway epithelial cells, and death usually ensues from chronic airway inflammation and loss of lung function. Our long term goal has therefore been to identify the mechanisms by which CFTR mutations cause constitutive IL-8 secretion from CF lung epithelial cells. Our approach is to identify the proteins which bind to the IL-8 promoter in CF lung epithelial cells, and to determine which one(s) might be responsible for the hyper-productive IL-8 phenotype. Preliminary data show that NFkappaB signaling is critical for IL-8 promoter activity in CF cells. In addition, we find that the protein hnRNP[A2B1] binds to the IL-8 promoter, affects NFkappaB action, and further activates IL-8 expression in CF cells. Based on these and other preliminary data we have hypothesized that hnRNP[A2B1], and possibly other proteins binding to the IL-8 promoter, are responsible for regulating constitutive, NFkappaB-dependent hyperexpression of IL-8 in CF lung epithelial cells. To test this hypothesis in vitro and in vivo, we propose the following specific aims: Specific Aim1: To identify proteins which bind to the IL-8 promoter in CF lung epithelial cells, and to determine their mechanisms of action on IL-8 expression. We will use mass spectrometry and crosslinking studies to identify and chemically validate proteins in nuclear extracts of CF lung epithelial cells which bind to the 167 bp IL-8 promoter DNA. Specific Aim 2: To map the locations and determine the molecular mechanism of interaction for proteins binding to the IL-8 promoter in CF lung epithelial cells. We will test the ability of free wildtype and mutant IL-8 promoter DNA sequences to competitively displace proteins from immobilized IL-8 promoter DNA. Specific Aim 3: To investigate the ensemble of proteins associated with the IL-8 promoter in bronchial lung epithelial cells from CF patients. We will prepare nuclear extracts from expanded cultures of CF and non-CF disease control bronchial brush biopsies of CF patients, and determine the proteins which bind to the IL-8 DNA promoter sequence. Significance, innovation, and uniqueness: This proposal is significant in that successful completion will determine those dysfunctional signaling pathway proteins which are responsible, directly or indirectly, for the proinflammatory lung phenotype of CF. The proposal is both unique and innovative by directing the investigation to the biology and chemistry of the IL-8 promoter in CF lung epithelial cells, both in vitro and in vivo.

描述（由申请人提供）：囊性纤维化 (CF) 是美国最常见的致命性常染色体隐性遗传疾病，由 CFTR 基因突变引起。 CF 的特征是气道上皮细胞促炎性 IL-8 的组成性过度分泌，慢性气道炎症和肺功能丧失通常会导致死亡。因此，我们的长期目标是确定 CFTR 突变导致 CF 肺上皮细胞组成型 IL-8 分泌的机制。我们的方法是鉴定 CF 肺上皮细胞中与 IL-8 启动子结合的蛋白质，并确定哪些蛋白质可能导致 IL-8 高产表型。初步数据表明，NFkappaB 信号传导对于 CF 细胞中 IL-8 启动子活性至关重要。此外，我们发现蛋白质hnRNP[A2B1]与IL-8启动子结合，影响NFkappaB的作用，并进一步激活CF细胞中IL-8的表达。基于这些和其他初步数据，我们假设 hnRNP[A2B1] 以及可能与 IL-8 启动子结合的其他蛋白质负责调节 CF 肺上皮细胞中 IL-8 的组成型、NFκB 依赖性超表达。为了在体外和体内检验这一假设，我们提出以下具体目标： 具体目标1：鉴定CF肺上皮细胞中与IL-8启动子结合的蛋白质，并确定其对IL-8表达的作用机制。我们将使用质谱和交联研究来鉴定和化学验证 CF 肺上皮细胞核提取物中与 167 bp IL-8 启动子 DNA 结合的蛋白质。具体目标 2：绘制 CF 肺上皮细胞中与 IL-8 启动子结合的蛋白质的位置图并确定相互作用的分子机制。我们将测试游离野生型和突变型 IL-8 启动子 DNA 序列竞争性取代固定化 IL-8 启动子 DNA 中的蛋白质的能力。具体目标 3：研究 CF 患者支气管肺上皮细胞中与 IL-8 启动子相关的蛋白质集合。我们将从 CF 患者的 CF 和非 CF 疾病控制支气管刷活检的扩大培养物中制备核提取物，并确定与 IL-8 DNA 启动子序列结合的蛋白质。意义、创新性和独特性：该提案具有重要意义，因为成功完成将确定那些直接或间接导致 CF 促炎性肺表型的功能失调的信号通路蛋白。该提案通过在体外和体内研究 CF 肺上皮细胞中 IL-8 启动子的生物学和化学特性，既独特又具有创新性。