15-Lipoxygenase is a lipid-peroxidating enzyme implicated in the pathogenesis of asthma and atherosclerosis. It's expression in atherosclerotic lesions may contribute to the oxidation modification of low density lipoprotein. 15-lipoxygenase activity is upregulated in asthmatic airways and its metabolites may contribute to asthma pathophysiology by stimulating airway secretion, altering ion transport, contracting bronchial smooth muscle and promoting neutrophil/eosinophil chemotaxis. Interleukin- 4, a TH2 cytokine mediating allergic inflammation, induces 15-lipoxygenase in cultured monocytes and airway epithelial cells while gamma interferon, dexamethasone and phorbol esters inhibit this induction. Preliminary data suggests the induction of 15-lipoxygenase is controlled by transcriptional and post transcriptional processes. The proposed work will determine the contribution of transcriptional and post transcriptional processes to the regulation of 15-lipoxygenase expression, identify the genetic elements mediating these events and finally characterize trans acting factors interacting with these genetic elements. The effects of these regulatory stimuli on 15-lipoxygenase gene transcription will be characterized by nuclear run on and DNAase I hypersensitivity assays. The genetic elements regulating gene transcription will be further delineated by expression of reporter genes driven by serial deletions of the 5' flanking sequence of the 15-lipoxygenase gene. Sequences mediating post transcriptional regulation will be identified by stably transfecting cells with 15- lipoxygenase exon, intron, 3' untranslated region and 3' flanking sequences in fusion genes and then assessing 15-lipoxygenase mRNA after stimulation with the relevant regulatory factors. Finally, the trans acting factors interacting with these sequences will be characterized by gel mobility shift studies and purified by affinity chromatography.
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