Whereas prior studies have shown that the initial stages of permeability barrier homeostasis, which require lipid synthesis, are tightly regulated, this proposal will assess whether several subsequent stages of barrier maintenance also are regulated processes. First, the assembly and secretion of the epidermal lamellar body (LB), focusing on the subcellular site of origin of the LB and the control LB secretion by cytoskeleton and other regulatory factors (e.g., beta-adrenergic stimuli, growth factors, and cytokines). Second, we will examine the regulation of extracellular hydrolase activity, thought to transform secreted LB contents into the intercellular bilayers that subserve the barrier. Third, we will examine whether specific lipids or lipid mixtures are required for barrier homeostasis, and how this compositional profile is regulated. In these studies, we will employ well-characterized, hairless mouse models, subjected to various types of barrier disruption, as well as organotypic human keratinocyte cultures, which approach the epidermis in LB content, lipid composition, and barrier function. Ultrastructural, lipid biochemical, metabolic, fluorescence and isotopic tracers, immunocytochemical, and molecular biological approaches will be utilized. In addition, a wide variety of pharmacologic inhibitors will be employed to ascertain: a) the subcellular site of LB synthesis; b) the cellular basis for LB secretion; and c) the enzymes required for extracellular processing of LB contents. Interpretations will be based upon this multidisciplinary approach applied to each of these areas to be studied. Although our goal is to learn as much as possible about the basic mechanisms underlying permeability barrier homeostasis, this work has wide practical implications. Barrier function is lost in premature infants, extensive burn wounds, blistering disorders, exfoliative erythrodermas, and important dermatoses, such as psoriasis and atopic dermatitis. These studies could lead to new strategies to reinforce or stabilize barrier function in these conditions. Conversely, the range of drugs that can be delivered by the percutaneous route is currently limited by the seeming impregnability of the barrier. These studies could lead to novel approaches to enhance transdermal drug delivery, potentially allowing whole new classes of formerly excluded compounds to access the internal environment.
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