The """"""""free radical theory of oxygen toxicity"""""""" links the deleterious pulmonary effects of hyperoxia, cellular oxygen metabolism and the respiratory burst of activated inflammatory cells to highly reactive metabolic products of oxygen. These reactive oxygen species can inactivate cellular enzymes, damage DNA and destroy lipid bilayers. To protect cells from these cytotoxic oxygen metabolites, a system of cooperative antioxidants have evolved with the primary defense being the superoxide dismutases (SODs). It has been shown repeatedly that elevated levels of the manganese SOD (MnSOD) provide an effective antioxidant defense which is strongly associated with the cell's tolerance to superoxide induced injury and survival. Therefore, understanding the lung's normal mechanisms for stimulating endogenous antioxidant defenses may lead to logical steps in the development of therapeutiC regimens that are.effective in preventing or ameliorating free radical mediated pulmonary toxicity. To this end, the goals of this proposal are to understand the molecular mechanisms which control stimulus-dependent gene expression and mRNA stability of the MnSOD gene in pulmonary epithelial cells. We plan to evaluate the interaction of promoter and enhancer elements, relative to their role in mediating the MnSOD gene's response to the inflammatory mediators: LPS, IL-I, and TNF. Using in vivo footprinting, we plan to delineate, at single nucleotide resolution, the position of cis-acting regulatory sequences in the enhancer. The nucleotide contacts defined by in vivo footprinting will be utilized to identify and clone the appropriate stimulus-linked transacting factors and their coding sequences. Antisense and dominant negative strategies will be used to demonstrate functional relevance of each trans-acting factor to MnSOD gene expression. Finally, we will identify mRNA binding proteins involved in controlling MnSOD mRNA half-life using RLPCR and UV cross-linking.
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