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 reactivenitrogen species in the asthmatic airway. High output NO synthesis has been linked to airway inflammationin 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 thatinterferon gamma (IFNgamma) induces, and interleukin-4 augments, NOS2 expression in HAEC in vitro throughmechanisms that require physical interaction between activator prrotein (AP)-1 and IFNgamma-activated signaltransducer and activator of transcription (STAT)-1 prior to DNA binding. In the context that asthma resultsfrom inflammatory processes that injure or modify airway function, excessive NO may participate in thepathogenesis 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 lungepithelial cells after NOS2 induction, and in the human airway, e.g. Mn superoxide dismutase (MnSOD) andcatalase. Our preliminary data show that reactive nitrogen and oxygen species lead to loss of catalase andMnSOD activity, and consequent apoptosis. Taken together, we hypothesize that NO synthesis is increasedin asthma due to NOS2 gene induction by IFNgamma utilizing AP-1 and STAT-1 signaling mechanisms in theairway epithelial cell, and that the generation of excess NO leads to protein oxidation modifying proteinfunctions and contributing to airway injury/inflammation. To test this hypothesis, we will (1) define theregulation of NOS2 gene expression using HAEC in culture, and airway cells freshly obtained from asthmaticand healthy control lungs, and (2) determine the role of NO in asthma by a proteomic approach to identifynitrated proteins before and after NOS2 induction in HAEC in culture, in clinical samples from asthmatics incomparison to controls before and after an allergen challenge, and in an allergen-induced airwayinflammation mouse model. Quantitation of biological tyrosine nitration/oxidation will be performed incollaboration with Project 3. Experiments to assess biologic effects of NO/reactive nitrogen species onextracellular matrix formation will be done in concert with Project 2. All Cores will be used extensively byProject 1. Together, these studies will provide a valuable comprehensive picture of the mechanisms thatcontrol 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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