This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Despite significant improvement in control of point source emissions during the past 30 years, air pollution continues to contribute to thousands of premature deaths each year in the United States. Recent epidemiological studies also link O3 to acute coronary events in otherwise healthy, middle-aged adults. Asthmatics and other at-risk populations are known to be particularly sensitive to O3, which has recently (June 2007) prompted the EPA to recommend amending the federal standards from 80 ppb to 70 ppb (8 hour standard). Ozone (O3), produced at ground levels from an interaction between sunlight and vehicle exhaust emissions, is highly reactive with human respiratory tissues. Exposure leads to reduced respiratory and altered immune function. O3 is known to cause or exacerbate many pulmonary conditions, including asthma and chronic obstructive pulmonary disease. Ozone may induce its adverse affects directly by oxidizing cell surface proteins and lipids which act to promote cellular repair and inflammatory responses. Alternatively, or in combination, O3 may act indirectly via reactive oxygen species (ROS) pathways which leads to oxidative damage. In rodent models, the effects of O3 are strain-specific, suggesting genetic background is significant. Most studies also implicate modulation by cytokines/chemokines signaling among different cell types (resident macrophages, alveolar epithelial cells, etc.). Thus, we seek to elucidate how oxidant air pollutants may exacerbate or trigger proinflammatory molecular mechanisms that contribute to pulmonary and cardiovascular disease. We combine in vivo and in vitro approaches to analyze complex gene expression and regulation patterns between experimental and control subjects. All cells that respond to external stimuli share the same challenge; how to translate recognition of a stimulus into changes within the cell. Recognition of stimuli is typically via receptors on the outside of the cells, and extra cellular signals then must be transmitted across the cell membrane. These receptors, typically integral or membrane-bound proteins, are required for signal transduction, the conversion from one form of a signal into another. SAPK and p38 are responsive to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and regulate many cellular activities. SAPK and p38 affect transcription of genes via activation of transcription factors, with the transcription factor NF-kB perhaps the most essential (e.g., for IL-8 gene expression). The NF-kB family of transcription factors consists of homo- or hetero-dimeric subunits of the Rel family including p50, p52, p65/RelA, c-Rel, and Rel-B. Induction of NF-kB activity does not require protein synthesis. Instead, in unstimulated cells, most of NF-kB is inactive and localized in the cytoplasm bound by an inhibitory protein, IkB. When the cell is stimulated, both proinflammatory cytokines (e.g., TNF-a) and free radical intermediates (ROS and RNS), IkB is phosphorylate, then degraded, allowing NF-kB to move to the nucleus and bind to DNA targets, thus activating transcription of specific target genes. The result of ozone exposure on lung epithelial cells is rapid expression of 100's of genes; these genes can be grouped into clusters of known function within the lung cells, including those that affect innate immune function, cell adhesion, surfactant protein and lipid metabolism, and the antioxidant status of the cell. Ozone is among the numerous environmental agents that activates the transcription factor NF-kB nuclear binding activity in lung macrophage and epithelial cells. Many of the genes that are expressed within hours of ozone exposure are controlled by NF-kB (i.e., have binding regions in promoter or enhancer elements). However, it is unclear whether ozone activates NF-kB via SAPK or p38, or whether both are activated and essential; therefore, one of the primary goals of this proposed work is to test whether SAPK, p38, or both are essential for ozone-induced activation of NF-kB pathway in lung epithelial cells.
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