Mucin Gene Regulation by Elastase and Oxidants

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A major pathologic feature contributing to pulmonary function decline in acute exacerbations of chronic bronchitis is airway obstruction by mucus secretions. In this application, we present a new paradigm in which a novel pathway links together COPD triggers, airway inflammation and mucus overproduction. It is known that triggers for acute exacerbations increase airway concentrations of neutrophil elastase (NE). We have previously shown in airway epithelial cells that NE treatment generates intracellular reactive oxygen species and these reactive oxygen species are required for NE-increased MUC5AC mucin expression. In this application, we present several key observations linking NE with activation of NAD(P)H:quinone oxidoreductase (NQO1) activation, increased generation of reactive oxygen species, and increased mucin MUC5AC mRNA stabilization due to binding of HuR, an RNA-binding protein that stabilizes mRNA. From these results, we propose the following hypothetical schema: During acute exacerbations of chronic bronchitis, neutrophil elastase (NE) activates NQO1; NQO1 generates reactive oxygen species (ROS); ROS alter the expression and/or the cytoplasmic localization of HuR, thus enhancing HuR binding to MUC5AC mRNA; HuR binding increases MUC5AC mRNA stability, leading to increased MUC5AC mucin glycoprotein production, and increased airway obstruction. To test these hypotheses, we will use both in vitro and in vivo model systems. The Specific Aims are: Specific Aim 1. To determine whether or not NQO1 is an integral component of the signaling pathway regulating NE-induced MUC5AC mRNA levels. a. To determine whether NE exposure affects NQO1 expression or activity. b. To determine whether NQO1 expression and activity are required for NE-regulated MUC5AC expression. Specific Aim 2. To determine whether or not NE increases MUC5AC mRNA stability via HuR binding. a. To determine whether NE and/or NQO1-generated ROS alter HuR expression or cytoplasmic localization in airway epithelial cells. b. To determine whether NE and/or NQO1-generated ROS affect HuR binding to MUC5AC. c. To determine whether HuR binding is sufficient for MUC5AC mRNA stabilization.