Airway mucus obstruction, a major cause of morbidity and mortality in cystic fibrosis (CF) patients, is largely attributed to overproduction and hypersecretion of mucin glycoproteins (mucins). Sustained mucin hypersecretion requires increased expression of mucin (MUC) genes that encode the protein backbone of mucins. Recent reports have indicated that CF airways have an intrinsically high inflammatory milieu, prior to bacterial infection, that promotes mucin overproduction. We have recently shown that IL8 increases in vitro expression of two airway mucin genes, MUC5AC and MUC5B, by increasing mucin mRNA stability. Importantly, elevated levels of IL8 in the airways of CF patients prior to and after infection suggest that IL8 can initiate and maintain increased mucin gene expression in vivo. Glucocorticoids decrease airway inflammation and mucin production in vivo by multi-factorial mechanisms, including alterations in expression of anti-inflammatory proteins or cytokines. GC also decreases mucin gene expression in vitro. A better understanding of mucin gene regulation by pro-and anti-inflammatory mediators, as well as the ontogeny of inflammatory mediators in CF lungs, should ultimately result in therapies that circumvent increased mucin gene expression and mucus obstruction in CF airways.
Three aims are proposed to address this objective.
In Aim 1, the hypothesis that IL8 increases mucin mRNA stability in airway epithelial cells by altering expression of RNA-binding proteins (RBP) that recognize specific cis-sequences in the 3' untranslated region of mucin genes or by mediating expressions of genes that regulate or encode RBP will be tested by EMSA, mutation analysis, transfection assays and gene expression analyses.
In Aim 2, the hypothesis that GC decrease MUC5AC gene expression in airway epithelial cells by down-regulating transcriptions factors that interact at or near GC response elements in the 5'-upstream flanking sequences of MUC5AC and/or by altering expression of anti-or pro-inflammatory genes that mediate mucin gene expression will be investigated.
In Aim 3, candidate genes involved in airway mucin overproduction will be identified and assessed by gene expression array profiling of respiratory tract tissues from CF and non-CF patients to test the hypothesis that airway mucus obstruction in CF patients is a consequence of the defective CF gene, which results in altered expression of genes that mediate airway homeostasis, including mucin production.
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