title: Fatty acid 2-hydroxylase, encoded by FA2H, accounts for differentiation-associated increase in 2-OH ceramides during keratinocyte differentiation.
abstract: 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 of the amide-linked fatty acids contributes 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-hydroxyglucosylceramide synthesis in epidermis. Reverse transcription-PCR and Western immunoblots demonstrated that FA2H is expressed in cultured human keratinocytes and human epidermis, with FA2H expression and fatty acid 2-hydroxylase activity increased with differentiation. FA2H-siRNA suppressed 2-hydroxylase activity and decreased 2-hydroxyceramide/2-hydroxyglucosylceramide levels, demonstrating that FA2H accounts for synthesis of these sphingolipids in keratinocytes. Whereas FA2H expression and 2-hydroxy free fatty acid production increased early in keratinocyte differentiation, production of 2-hydroxyceramides/2-hydroxyglucosylceramides with longer chain amide-linked fatty acids (> or =C24) increased later. Keratinocytes transduced with FA2H-siRNA contained abnormal epidermal lamellar bodies and did not form the normal extracellular lamellar membranes required for the epidermal permeability barrier. These results reveal that 1) differentiation-dependent up-regulation of ceramide synthesis and fatty acid elongation is accompanied by up-regulation of FA2H; 2) 2-hydroxylation of fatty acid by FA2H occurs prior to generation of ceramides/glucosylceramides; and 3) 2-hydroxyceramides/2-hydroxyglucosylceramides are required for epidermal lamellar membrane formation. Thus, late differentiation-linked increases in FA2H expression are essential for epidermal permeability barrier homeostasis.
authors.affiliation: Department of Dermatology, School of Medicine, University of California, San Francisco, CA, USA. uchiday@derm.ucsf.edu
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Uchida
authors.forename: Hiroko
authors.initials: H
authors.lastname: Hama
authors.forename: Nathan L
authors.initials: NL
authors.lastname: Alderson
authors.forename: Sounthala
authors.initials: S
authors.lastname: Douangpanya
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Debra A
authors.initials: DA
authors.lastname: Crumrine
authors.forename: Peter M
authors.initials: PM
authors.lastname: Elias
authors.forename: Walter M
authors.initials: WM
authors.lastname: Holleran
mesh_terms: D002454:Cell Differentiation / Q000235:genetics; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D005227:Fatty Acids / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D005963:Glucosylceramides / Q000096:biosynthesis*; D006706:Homeostasis / Q000235:genetics; D006801:Humans; D015603:Keratinocytes / Q000201:enzymology* / Q000648:ultrastructure; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D010539:Permeability; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D005227:Fatty Acids; D005963:Glucosylceramides; D034741:RNA, Small Interfering; D006899:Mixed Function Oxygenases; C019081:fatty acid alpha-hydroxylase