The generation of reactive oxygen species (ROS) has been linked to numerous diseases and processes, including cancer, aging, and cell death. ROS, which are generated in cells through normal processes and increased by oxidative stress, are removed from cells by superoxide dismutases (SOD). Only the manganous SOD (MnSOD) is inducible by agents that stimulate cellular activation and inflammation, such as tumor necrosis factor (TNF). Despite its clear importance, little is known about how MnSOD is regulated. The murine MnSOD gene is regulated by two response elements. A promoter proximal region (PPR), which is sufficient for basal but not induced expression. A complex TNF and IL-1 responsive enhancer (TNFRE), discovered in the second intron of the MnSOD gene encodes numerous cis-elements that could potentially bind C/EBPBeta NFKB, and NP-1 The role that these elements play in controlling TNF induced expression has not been fully discerned. Using a series of cell lines deficient for Sp1 and NF-KappaB p65 and p50, we propose to determine how this intronic enhancer functions and mediates the activity of its upstream promoter. We will also determine how the TNFRE influences the chromatin architecture of the surrounding DNA and how this may contribute to the regulation of the MnSOD gene. To further define the role of the TNFRE in regulating MnSOD in vivo, a series of mice containing conditional knock-outs of the regulatory elements will be created and analyzed. To provide an in vivo correlate to the study, we will study the role of these mutations on the development of T cell immune responses. The immune system is extraordinarily dynamic, with tremendous cellular proliferation and apoptosis of antigen specific T cells occurring during the course of infection. Preliminary data indicates that MnSOD mRNA expression increases with the expansion of T cells during infection but returns to basal levels after the virus is cleared and the T cell population crashes, suggesting that MnSOD and its regulation may play a critical role in the dynamics of T cell responses. Understanding the role of this enhancer and MnSOD in immune processes will provide the groundwork for the potential manipulation of MnSOD expression in 1) disease states where ROS are abundantly produced and 2) vaccine development , where the need to maximize immune responses is critical.
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