The interaction of reactive nitrogen species (ROS/RNS) with lung epithelium, the first cell in contact with inhaled toxicants, may be critical in the initiation of pulmonary disease. Activation of signalling cascades and transcription factors may be important in determining the phenotypic outcome of exposure to these reactive metabolites and the activation of an inflammatory response. In this proposal we focus on the transcription factor, nuclear factor kappa B (Nf-kappaB) in a rat alveolar type II epithelial cell line (RLE) exposed to ROS or RNS. We hypothesize that activation of NF-kappaB by critical ROS/RNS occurs through unique signalling pathways that include mitogen- activated protein kinases (MAPK) and tyrosine kinases, and that the balance of these events is pivotal in the initiation of lung injury. NF-kappaB is critical in the regulation of genes with multiple functions that are involved in inflammation, immune immodulation or survival. Prevention of apoptosis by NF-kappaB may lead to activation of inflammatory genes, a hallmark of oxidant lung injury. Due to its' complex functions, the phenotypic implications of NF-kappaB activation in lung epithelium are obscure to date. In this proposal, we seek to investigate the mechanisms by which ROS or RNS activate NF-kappaB, the critical signalling pathways and the functional implications of NF- kappaB activation in RLE cells. We will investigate two critical RNS, hydrogen peroxide, or peroxynitrite to determine the implications of NF- kappaB activation and phenotypic endpoints (apoptosis and expression of inflammatory genes).Modulation of NF-kappaB by overexpression or prevention of its activation will allow us to determine the implications of NF-kappaB activation in lung epithelium by ROS and RNS. Furthermore, assessment of upstream signalling cascades and MAPKs as well as the modulation of various MAPKs will elucidate the critical signalling pathways by which ROS/RNS induce NF-kappaB. Combined approaches in specific aims 1-4 will lead to a better understanding of the role of NF- kappaB in pulmonary disease associated with exposure to ROS/RNS and may provide strategies for therapeutic intervention.
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