Regulation of Proinflammatory Signaling Pathways in CFTR

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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.