Significant progress has been made towards defining the mechanisms whereby mutations in the CF gene lead to airway infection and bronchiectasis, but the potential role of aberrant mucin processing in the pathogenesis of CF lung disease remains unclear. The mucin MUC1 is the only known transmembrane mucin the human lung, and is expressed in differentiated surface epithelial cells and in the serous cells of submucosal glands, which previously have bene shown to express high levels of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The cellular co-localization of MUC1 and CFTR, and the reported role of CFTR in the post-translational processing of glycoconjugates, suggest that CFTR mutations may alter the processing of MUC1. Moreover, the structure and spatial distribution of MUC1 implicate it in the interaction between the apical membrane of airway epithelia and luminal bacteria. The central hypotheses of this proposal re therefore that CF mutations impair the post- translational processing and release of MUC1, and that this impairs the contribution of MUC1 to normal airway defense against bacterial infection. Accordingly, specific aims are: 1. To define the cellular localization of the transmembrane mucin MUC1 in normal and diseased human airway epithelium and secretions. The expression and cellular distribution of MUC1 in human airway and in a clinically relevant in vitro system of primary CF and non-CF human airway epithelium will be determined using immunocytochemistry, flow cytometry, and immunoelectron microscopy. 2. To determine whether CFTR influences the post-translational processing of MUC1. The sulfation and sialylation of MUC1 will be determined in CALU-3 cells and in primary cultures of CF and non-CF epithelia. CFTR expression will be down regulated using an anti-sense approach in CALU-3 cells. 3. To determine whether MUC1 is aberrantly processed and released in CF airway epithelia. The kinetics of MUC1 trafficking and release in the presence and absence of cAMP stimulation will be determined in non-CF and CF airway epithelia. The kinetics of MUC1 trafficking and release in the presence and absence of cAMP stimulation will be determined in non- CF and CF airway epithelia, and in CALU-3 cells expressing various levels of CFTR. 4. To determine the impact of CF mutations in the interaction between MUC1 and Pseudomonas aeruginosa.. Bacterial adhesion will be compare din primary airway cells, and in CALU-3 cells in which CFTR and/or MUC1 expression have been down regulated. Completion of these aims will define the influence of CFTR on the processing and function of a specific mucin in human airway and thereby improve the understanding of the pathogenesis of airway infection in CF.
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