Nitrogen dioxide (NO2) is a toxic free radical gas and a major air pollutant. Importantly, NO2 can also be formed in lung as a result of metabolism of nitric oxide. NO2 causes acute injury, but also is critical to the aggravation of asthma in children. Currently, the molecular targets and pathways, by which NO2 induces acute lung injury are unknown. The central hypothesis of this proposal is that NO2 causes apoptosis in lung epithelial cells following activation of c-Jun-N-terminal kinases (JNK), the JNK substrate, c-Jun and the downstream induction of Fas ligand. This hypothesis will be tested in four specific aims that utilize both cell culture models as well as mouse inhalation studies. In the first Specific Aim, we will determine the activation of JNK, c-Jun-apoptosis in rodent lung epithelial cells and normal human bronchiolar epithelial cells exposed to NO2, We will use dominant negative constructs to block the JNK pathway to determine whether this blocks apoptosis in lung epithelial cells exposed to NO2.
In Specific Aim #2 we will determine whether the activity of c-Jun as a transcription factor is required for NO2- induced apoptosis in vitro.
In Specific Aim #3, we will utilize mice containing a mutated c-jun gene that cannot be activated by JNK. In these mice and their wild type counterparts, we will determine patterns of acute lung damage, apoptosis, fibrosis and alterations in pulmonary function after exposure to NO2.
In Specific Aim #4, we will create a transgenic mouse that expresses a dominant negative Fas construct selectively in bronchiolar epithelium in order to confirm the causality of Fas-induced apoptosis within bronchiolar epithelium of mice exposed to NO2. The combined strategies of cell culture models and transgenic animals will allow elucidation of the mechanisms by which NO2 initiates pulmonary damage. Understanding of molecular targets and pathways of injury after exposure to NO2 will provide strategies for intervention that may benefit large populations, including patients with asthma.
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