Epithelial Cell Cycle Regulation by Elastase

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DESCRIPTION (provided by applicant): Respiratory morbidity and mortality in patients with cystic fibrosis (CF), chronic bronchitis (CB), and pollution- or viral-triggered asthma results from chronic neutrophil-dominant inflammation. These patients are plagued by progressive airway obstruction, parenchyma! damage and scarring. Neutrophil elastase (NE), a serine protease released by neutrophils is present in the airways of these patients and injures the airway epithelium. We have reported that as part of the mechanism of NE-regulation of MUC5AC mucin gene expression, NE triggers the generation of intracellular reactive oxygen species (ROS). We examined the epithelial response to NE - mediated oxidative injury. Although, NE did not cause cell death, it did cause a G1 cell cycle arrest corresponding to a marked decrease in epithelial DNA synthesis and proliferation. Further, we demonstrate that NE- generated ROS mediate the decrease in DNA synthesis. In this proposal, we establish a previously unrecognized mechanism employed by the airway epithelium to reestablish homeostasis following NE-induced injury. The Hypothetical Schema to be addressed is: NE triggers the generation of intracellular reactive oxygen species (ROS) by NAD(P)H:quinone oxidoreductase 1 (NQ01), specifically superoxide and hydrogen peroxide. As a result of oxidant stress and consequential DNA damage, there is increased expression of the Cip/Kip family of mitotic inhibitors. Increased expression of these Cip/Kip proteins results in G1 arrest and inhibition of cell cycle progression and epithelial proliferation. This pause in the cell cycle allows for subsequent DNA repair followed by restoration of epithelial proliferation. The Specific Aims are: 1) To determine whether or not NE - induced DNA damage and G1 cell cycle arrest are mediated by reactive oxygen species; 2) To determine whether or not NE - induced molecular regulation and expression of the Cip/Kip family of mitotic inhibitors, p21, p27 and p57, is mediated by reactive oxygen species; 3) To determine whether or not NE - induced inhibition of DNA synthesis and cell cycle arrest are mediated by the Cip/Kip family of mitotic inhibitors - p21, p27, and p57. The information derived from these experiments will provide fundamental insights into epithelial response to injury, and provide a foundation to investigate normal epithelial regeneration versus pathologic remodeling.