Epidermal keratinocytes express unique forms of cytochrome P450 (CYP gene superfamily) that metabolize arachidonic acid, including murine CYP2B19 and rat CYP2B12 and CYP2B15. The regulation of the expression of these CYP genes, and thus production of CYP-derived eicosanoids, is highly cell type- and differentiation-specific. CYP2B19 utilizes endogenous arachidonic acid and generates lipid mediators having biological activity in human and murine keratinocytes: epoxyeicosatrienoic (EET) and hydroxyeicosatetraenoic acids. 11, 12-EET, a major CYP2B19 metabolite, mobilizes intracellular calcium and promotes cornification of keratinocyte cultures. Abnormalities in fatty acid and eicosanoid metabolism are associated with hyperproliferative skin phenotypes in humans and mice. Despite the enormous potential of eicosanoids to regulate keratinocyte differentiation, there is no unifying hypothesis how they function in normal epithelial differentiation or in aberrant differentation leading to hyperproliferation and cancers. Recently we identified CYP2B19-related (immunologically) proteins(s) in human keratinocytes. We hypothesize these are CYP2B19 homologue(s), that is, P450 epoxygenase(s) that participate in endogenous arachidonic acid metabolism. We will test this hypothesis by first proving that human epidermal keratinocytes express P450 epoxygenase activities during differentiation in vitro, by characterizing the regio- and stereochemistry of lipid mediators that they generate from [1-C14]-arachidonic acid. Second, we will prove that P450 epoxygenases in human epidermal keratinocytes participate in endogenous metabolism, by using a mass spectral assay to measure intracellular EET oncentrations arising from endogenous arachidonate. Third, we will prove that the human keratinocyte protein(s) binding to CYP2B19 antibodies is a CYP2B19 homologue, by peptide sequencing immunoisolated protein(s). Finally, we will prove that the CYP2B19-related protein(s) in human keratinocytes has arachidonic acid epoxygenase activities. We will generate cDNA, produce recombinant protein, characterize activities with arachidonate and demonstrate expression is skin by in situ hybridization.
