This application addresses the role of fatty acid metabolism in hyperproliferative skin diseases where the disease phenotype leads to abnormally high levels of eicosanoids and free fatty acids in skin lesions. While there is no unifying hypothesis explaining the roles of eicosanoids in epithelial differentiation and disease, these lipids are thought to mediate signals regulating cell proliferation and differentiation. Dr. Keeney's proposal deals with the roles of lipid mediators generated by cytochrome P450 epoxygenases in the control of epithelial cell differentiation. The applicant and colleagues have characterized keratinocyte-specific epoxygenases (CYP2B19, CYP2B12) that generate epoxyeicosatrienoic acid (EET) and hydroxyeicosatetraenoic acids from arachidonic acid. Their expression in rodent skin in epidermal keratinocyte cultures is highly differentiation-specific. Their lipid mediator (EETs) are present in normal skin, and the EETs are produced from endogenous arachidonic acid in differentiated keratinocytes. The applicant hypothesizes that P450-derived eicosanoids mediate signals regulating differentiated functions in epidermal granular cells and that ablation or overexpression of P450 epoxygenases will alter this differentiated phenotype of keratinocytes. This proposal will address 1) The endogenous CYP2B19 products that are biologically active by confirming that the EETs generated by CYP2B19 are present in murine epidermal keratinocyte cultures, coincident with CYP2B19 mRNA and protein. The applicant will prove that EETs are biologically active in murine epidermal keratinocyte cultures, coincident with mRNA and protein. 2) The applicant will demonstrate a functional relationship between endogenous EET production and the differentiated keratinocyte phenotype, and that ablation of EET production alters this phenotype. In the third specific aim, the applicant will prove that EET's are biologically active in murine epidermal keratinocyte cultures by demonstrating how the differentiation and the sensitivity of these cells to extracellular Ca2+ is altered by inappropriate overproduction of EETs. Endogenous EETs will be measured using a mass spectral assay. Chemical inhibitors and antisense cDNA will be used to ablate CYP-derived EET production. Recombinant adenoviruses expressing P450 epoxygenases (including CYP2B19) will be used to overproduce EETs intracellularly. Results of these studies will identify specific cellular pathways and granular cell functions regulated by CYP-derived eicosanoids and suggest potential mechanisms for their actions.
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