Extracellular processing of phospholipids is required for permeability barrier homeostasis

Three key lipid types, cholesterol, ceramides (CER), and free fatty acids (FA), form the stratum corneum (SC) lamellar bilayers that mediate barrier function. Prior studies have shown that barrier requirements regulate CER generation from glucosylCER, and that this catabolic step takes place within the SC interstices. Here, we addressed whether extracellular processing of phospholipids (PL), the FA precursor delivered to the SC interstices with glucosylCER and cholesterol during exocytosis of lamellar body contents, is also required for barrier homeostasis. We applied two chemically unrelated inhibitors of phospholipase A2 (PLA2-I), BPB (irreversible) and MJ33 (reversible), topically to hairless mice after barrier perturbation with acetone. Both inhibitors delayed barrier recovery at non-cytotoxic doses, while MJ45, an analogue known to inhibit PLA2-II but not PLA2-I, had no effect. Moreover, the delays in barrier recovery induced by BPB and MJ33 could be overridden by co-applications of palmitic acid, but not linoleic acid or lysolecithin. Furthermore, inhibitor-treatment resulted in accumulation of PL and depletion of FA in the SC by 4 h, as well as the persistence of "immature" lamellar body-derived membrane structures in the SC interstices. Finally, these changes in membrane structure were reversed when inhibitor-treated SC was incubated in vitro with palmitic acid. These studies show that: 1) inhibition of PLA2 results in abnormalities in lipid composition and SC structure that alter barrier homeostasis; and 2) the functional defect can be attributed to a deficiency of nonessential FA within the SC. Thus, extracellular processing of PL into FA is required for normal barrier function.