Airway hyperresponsiveness and mucous cell metaplasia are essential features of inflammatory airway diseases (including asthma and COPD), but the cellular and molecular pathways leading to these disease traits still need to be better defined. Previous work by others suggested that a goblet cell-specific member of the calcium activated chloride channel family of proteins (mClca3 in mouse and hCLCAl in human) is necessary and sufficient for allergen-induced airway hyperresponsiveness and goblet cell metaplasia in mice and is overexpresssed in allergic asthma in humans. However, we found that a new /nC/ca3-null mouse still fully develops these asthma traits after viral infection or allergen challenge. These and other preliminary results suggest functional redundancy in the CLCA gene family. Indeed, we find that the CLCA locus consists of 6 distinct genes in the mouse and 4 in the human, and each exhibits a high degree of intra- and inter-species sequence homology but a distinct pattern of expression in tissues. We therefore propose that select members of the CLCA family of proteins play tissue-specific but convergent roles in the development and maintenance of airway hyperresponsiveness and goblet cell metaplasia. Thus, we propose to: I. Determine the patterns of gene expression for mClca family members in the setting of experimental airway disease driven by viral infection or allergen challenge, using real-time quantitative RT-PCR assays to precisely monitor the levels of mC/ca family gene expression in wild-type and mClca3-null mice. Studies will be performed in vivo using our mouse models of viral bronchiolitis and allergen challenge and in vitro using primary-culture mouse airway epithelial cells grown at air-liquid interface and stimulated with cytokines (IL-4, IL-9, and IL-13) to induce goblet cell metaplasia. II. Determine the effects of selectively expressing mClca family members on experimental airway disease phenotypes (i.e.goblet cell metaplasia;and airway hyperresponsiveness) using adeno-associated virus (AAV) gene transfer vectors. In vivo studies will again be compared to the actions of mClca expression done in vitro using primary-culture mouse and/or human airway epithelial cells. III. Determine the effects of selectively blocking expression of mClca family members on experimental airway disease phenotypes driven by viral infection or allergen challenge, using AAV vectors to deliver antisense oligonucleotides that selectively inhibit mClca gene expression. This will be compared to effects of targeted mutagenesis (i.e., our mC/ca3-null mouse) and of treatment with taniflumate (an inhibitor of calcium-dependent chloride flux). In vivo studies will again be compared to the actions of these inhibitors in vitro using primary-culture mouse and/or human airway epithelial cells. These proposed experiments should serve to define CLCA function in experimental asthma and thereby provide for new therapeutic targets in humans with airway disease.
