The overall goal of Project 1 of the Research Program is to identify how airway epithelial cells initiate, intensify and modulate the inflammatory response through the generation of nitric oxide (NO) and reactive nitrogen species in the asthmatic airway. High output NO synthesis has been linked to airway inflammation in asthma. NO is produced by human airway epithelial cells (HAEC) that have increased NO synthase 2 (NOS2) expression in asthma due to transcriptional activation of the gene. Our preliminary results show that interferon gamma (IFNgamma) induces, and interleukin-4 augments, NOS2 expression in HAEC in vitro through mechanisms that require physical interaction between activator prrotein (AP)-1 and IFNgamma-activated signal transducer and activator of transcription (STAT)-1 prior to DNA binding. In the context that asthma results from inflammatory processes that injure or modify airway function, excessive NO may participate in the pathogenesis of asthma through reactive nitrogen species formation and subsequent oxidation of proteins, modulating their biologic functions. In support of this, nitrated proteins are increased in the asthmatic airway. Using an innovative proteomic approach, we have identified tyrosine nitration of specific proteins in lung epithelial cells after NOS2 induction, and in the human airway, e.g. Mn superoxide dismutase (MnSOD) and catalase. Our preliminary data show that reactive nitrogen and oxygen species lead to loss of catalase and MnSOD activity, and consequent apoptosis. Taken together, we hypothesize that NO synthesis is increased in asthma due to NOS2 gene induction by IFNgamma utilizing AP-1 and STAT-1 signaling mechanisms in the airway epithelial cell, and that the generation of excess NO leads to protein oxidation modifying protein functions and contributing to airway injury/inflammation. To test this hypothesis, we will (1) define the regulation of NOS2 gene expression using HAEC in culture, and airway cells freshly obtained from asthmatic and healthy control lungs, and (2) determine the role of NO in asthma by a proteomic approach to identify nitrated proteins before and after NOS2 induction in HAEC in culture, in clinical samples from asthmatics in comparison to controls before and after an allergen challenge, and in an allergen-induced airway inflammation mouse model. Quantitation of biological tyrosine nitration/oxidation will be performed in collaboration with Project 3. Experiments to assess biologic effects of NO/reactive nitrogen species on extracellular matrix formation will be done in concert with Project 2. All Cores will be used extensively by Project 1. Together, these studies will provide a valuable comprehensive picture of the mechanisms that control NO synthesis in the human airway epithelium, alterations in those mechanisms that lead to asthma, and functional consequences of increased NO in the airway.
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