Regulation of Proinflammatory Signaling Pathways in CFTR

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病，由CFTR基因突变引起。CF的特点是气道上皮细胞成形性分泌促炎IL-8，慢性气道炎症和肺功能丧失通常导致死亡。因此，我们的长期目标是确定CFTR突变导致CF肺上皮细胞分泌组成性IL-8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，并确定哪一个(s)可能负责高增殖IL-8表型。初步数据显示NFkappaB信号对CF细胞中IL-8启动子活性至关重要。此外，我们发现hnRNP蛋白[A2B1]与IL-8启动子结合，影响NFkappaB的作用，进而激活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的组成性、nfkappab依赖性高表达。为了在体外和体内验证这一假设，我们提出以下具体目的：特异性目的1：鉴定CF肺上皮细胞中与IL-8启动子结合的蛋白，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。特异性目的2：定位CF肺上皮细胞中与IL-8启动子结合的蛋白的位置并确定其相互作用的分子机制。我们将测试自由野生型和突变型IL-8启动子DNA序列竞争性取代固定IL-8启动子DNA中的蛋白质的能力。特异性目的3：研究CF患者支气管肺上皮细胞中与IL-8启动子相关的蛋白集合。我们将从CF患者的CF扩增培养和非CF疾病控制支气管刷活检中制备核提取物，并确定与IL-8 DNA启动子序列结合的蛋白。意义、创新和独特性：该建议具有重要意义，因为成功完成将确定那些直接或间接负责CF促炎肺表型的功能失调的信号通路蛋白。该建议既独特又创新，将研究导向CF肺上皮细胞中IL-8启动子的生物学和化学，无论是体外还是体内。

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.