Of the known biologically active mediators generated by cells, both normally and during the inflammatory response, few have received more attention than those generated in the arachidonic acid cascade. Cytokines are established regulators of the arachidonic acid cascade in lung cells. The levels of various arachidonic metabolites distinguish between the normal and pathogenic states of the human lung. The arachidonyl-selective, cytosolic phospholipase A2 (cPLA2) is ubiquitously present in human lung and is most likely the rate-limiting step in eicosanoid generation. We therefore propose to study the molecular regulation of this pivotal gene in human lung fibroblasts and epithelial cells in response to glucocorticoids and pro-inflammatory cytokines. We will detect basal and stimulus-specific DNase I hypersensitive (HS) sites located in distant regions of the cPLA2 locus by utilizing a novel strategy involving field inversion gel electrophoresis (FIGE) to analyze altered regions of chromatin structure. A regulatory element trapping protocol will be employed to functionally identify regulatory sequences and complement our chromatin analysis. Deletion analysis will be used to precisely delineate cis-acting regulatory elements functionally by transient transfection in control, stimulated and repressed pulmonary cells. To identify, at single nucleotide resolution, the location of transcriptionally relevant protein-DNA contacts in living cells, we will employ in vivo footprinting using ligation mediated PCR (LMPCR) in conjunction with site-directed mutagenesis, EMSA and Transcription Factor Decoy (TFD) analysis. The knowledge gained by studying the molecular mechanisms controlling arachidonate levels will provide the basis for an understanding of the pathophysiology of acute lung injury that occurs in pulmonary diseases, such as asthma, acute respiratory distress syndrome, pulmonary fibrosis, and cystic fibrosis.
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