Functions common to Clara cells from different mammalian species are their ability to serve as a progenitor for renewal of the airway epithelium and their role in production of airway secretions. Both have potential to impact airway integrity yet surprisingly little is known of physiological roles for Clara cell secretion in airway homeostasis and disease. Goals of this competing renewal application are to investigate roles for Clara cells and their major secretory product, CCSP, in airway defense against oxidant injury. Key findings from our previous studies are that CCSP deficiency severely compromises Clara cell secretory function and elevates susceptibility of the epithelium to oxidative stress. Related findings are that ozone rapidly triggers the release of intracellular stores of CCSP, suggesting that secretion is an immediate cytoprotective response, and that CCSP deficiency is associated with altered post-translational processing of annexin A1 (ANXA1), a potential regulator of ciliary function. Based upon these data, continuing studies will address the hypothesis that CCSP functions directly in the cytoprotection of epithelial cells from the harmful effects of oxidative stress. We propose three specific aims to address this hypothesis. Air-liquid interface culture models will be used in Aim 1 to determine whether functional properties intrinsic to Clara cells and Clara cell secretory protein confer cytoprotection against oxidative stress. Experiments in Aim 2 will use a combination of in vitro and regulated in vivo models to determine contributions made by the highly conserved calcium and lipophilic ligand binding sites of CCSP in regulated secretion and cytoprotection of epithelial cells from oxidant injury. Goals of experiments in Aim 3 are to build upon exciting new data demonstrating that CCSP deficiency results in altered post-translational processing of ANXA1, a calcium and phospholipid binding protein that localizes to the apical membrane and cilia of ciliated cells. These data suggest a previously unappreciated link between secretory and ciliated cell function. We will determine post-translational modifications of ANXA1 in CCSP-/- mice to reveal CCSP-dependent signaling pathways that are compromised with CCSP deficiency. We will also use ANXA1-/- mice to determine contributions made by ANXA1 modifications to oxidant sensitivity of CCSP-/- mice and cultures epithelia. Completion of these aims will provide new insights into pathobiological mechanisms of chronic airway diseases that have the consistent feature of compromised Clara cell secretory function. ? ?
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