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）是美国最常见的致死性常染色体隐性遗传病，这是由于CFTR基因的突变。CF的特征在于气道上皮细胞的促炎性IL-8的组成性高分泌，并且死亡通常由慢性气道炎症和肺功能丧失引起。因此，我们的长期目标是确定CFTR突变引起CF肺上皮细胞组成性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，并确定哪种蛋白质可能是导致IL-8高产表型的原因。初步数据显示NF κ B信号传导对于CF细胞中的IL-8启动子活性至关重要。此外，我们发现蛋白hnRNP[A2 B1]与IL-8启动子结合，影响NF κ B作用，并进一步激活CF细胞中的IL-8表达。基于这些和其他初步数据，我们假设hnRNP[A2 B1]和可能与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的组成性NF κ B依赖性过表达。为了在体外和体内验证这一假设，我们提出了以下具体目标：具体目标1：确定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肺上皮细胞的核提取物中与167 bp IL-8启动子DNA结合的蛋白质。具体目标二：目的定位CF肺上皮细胞中IL-8启动子结合蛋白的位置并确定其相互作用的分子机制。我们将测试游离的野生型和突变型IL-8启动子DNA序列从固定的IL-8启动子DNA竞争性置换蛋白质的能力。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的集合。我们将从CF患者的CF和非CF疾病控制支气管刷活检的扩增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。重要性、创新性和独特性：该提议的重要性在于，成功完成将确定那些直接或间接负责CF的促炎性肺表型的功能失调的信号通路蛋白。该提案是独特的和创新的，通过指导调查CF肺上皮细胞中IL-8启动子的生物学和化学，在体外和体内。