Ceramides in mammalian stratum corneum comprise a heterogeneous mixture of molecular species that subserve the epidermal permeability barrier, an essential function for survival in a terrestrial environment. In addition to a variation of sphingol species, hydroxylation at the alpha(2)- and omega-positions of the amide-linked fatty acids contribute to the diversity of epidermal ceramides. Fatty acid 2-hydroxylase, encoded by the gene FA2H, the mammalian homologue of FAH1 in yeast, catalyzes the synthesis of 2-hydroxy fatty acid-containing sphingolipids. We assessed here whether FA2H accounts for 2-hydroxyceramide/2-hydroxyglucosyl-ceramide synthesis in epidermis. RT-PCR and Western immunoblots demonstrated FA2H is expressed in cultured human keratinocytes and human epidermis. FA2H expression, and fatty acid 2-hydroxylase activity, increased with differentiation. FA2H-siRNA significantly suppressed 2-hydroxylase activity and decreased 2-hydroxyceramide/2-hydroxyglucosyl-ceramide levels, demonstrating that FA2H accounts for synthesis of these sphingolipids in keratinocytes. Whereas FA2H expression and 2-hydroxy free fatty acid production began to increase early in keratinocyte differentiation, production of 2-hydroxyceramides/2-hydroxyglucosylceramides with longer chain amide-linked fatty acids (>C24) increased later in differentiation. Keratinocytes transduced with FA2H-siRNA contained the abnormal epidermal lamellar bodies and did not form the normal extracellular lamellar membranes required for epidermal permeability barrier formation. These results reveal that 1) differentiation-dependent upregulation of ceramide synthesis and fatty acid elongation is accompanied by upregulation of FA2H, leading to the formation of unique 2-hydroxyceramides/2-hydroxyglucosyl-ceramides; 2) 2-hydroxylation of fatty acid by FA2H occurs prior to generation of ceramides/glucosylceramides; and 3) 2-hydroxyceramides/2-hydrooxyglucosylceramides are required for epidermal lamellar membrane formation. Thus, late-differentiation-linked increases in FA2H expression are essential for epidermal permeability barrier homeostasis.
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|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
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