The overall objectives of the study are to determine whether essential fatty acids play a role in the process of keratinocyte differentiation. Preliminary evidence suggests that one essential fatty acid, linoleic acid, may act as a regulator of differentiated function. The current proposal seeks to understand the role of this polyunsaturated fatty acid in epidermal biology. The metabolic fate of the fatty acid, which cannot be biosynthesized and therefore must be exogenously supplied, will be traced. Incorporation of radiolabeled linoleic acid into all lipid classes will be examined by chromatographic analysis of cellular extracts, using TLC, GLC and HPLC methodologies. In this way we will be able to determine which lipid class may be the critical one to focus further studies on. The levels and distribution of linoleic acid in differentiating and non-differentiating keratinocyte cultures will be determined and correlated with known markers of differentiation. These include quantification of squame formation and nuclear dissolution, PAGE analysis of keratins, and assays of cell-associated plasminogen activator and transglutaminase activities. In a parallel approach, keratinocytes will be enriched in linoleic acid and examined for evidence of enhance differentiation. Lipids will be extracted from these cells to determine if there is preferential sequestration of linoleic acid in any particular lipid class. Finally, we will selectively block the metabolic pathways of eicosanoid synthesis in linoleate-enriched keratinocytes to determine whether the regulatory effect of linoleic acid is mediated by its prostaglandin or leukotriene metabolites. These studies will help elucidate the role of linoleic acid in the keratinocyte differentiative process and clarify the mechanism by which this regulation is achieved.
