DESCRIPTION (provided by applicant): Mammalian epidermis is poised at the interface with the environment, and it must both prevent excess transcutaneous water loss in a desiccating terrestrial environment, and protect underlying tissues from toxic mechanical and chemical insults. In addition to blocking the penetration of xenobiotics and chemical toxins, it mitigates the adverse effects of ultraviolet radiation and other form of oxidative stress (OS). Ceramide (Cer), as members of the nitrogen-containing lipid class (sphingolipids), represent one of the primary lipid constituents of the outermost layer of the epidermis, the stratum corneum, in which this critical permeability/xenobiotic/ antioxidant barrier resides. In addition to their critical role as bulk membrane constituents in barrier function, various Cer metabolites also have emerged as critical for numerous other cellular functions. Pertinently, OS from a variety of stressors increases cellular Cer levels, thereby triggering increased (potentially premature) apoptosis, and excessive apoptosis or premature differentiation can impact epidermal function negatively. It is our hypothesis that epidermal keratinocytes deploy two classes of protective mechanisms that restrict levels of intracellular Cer, both from the ever-present risk of exposure to oxidative stressors, and from the high levels of Cer production required to meet barrier requirements during epidermal differentiation: 1) distal metabolism/catabolism of excess Cer to non-apoptotic metabolites; and 2) rapid sequestration/externalization of Cer, as glucosylceramide and sphingomyelin precursors, within the epidermal lamellar body secretory system. In our first aim, we will delineate the mechanisms that protect keratinocytes from Cer generated in response to oxidative stress. In our second aim, we will determine the mechanisms that protect the epidermis from upregulated Cer production for maintenance of the permeability barrier. Because epidermis is inherently at higher potential risk than other tissues, due to its exposure to external oxidative stressors, and because it generates abundant amounts of Cer for the epidermal barrier, it is important to understand those mechanism(s) that regulate intracellular Cer levels, thereby protecting epidermis from premature Cer-induced apoptosis.