title: Vitamin C stimulates sphingolipid production and markers of barrier formation in submerged human keratinocyte cultures.
abstract: Human keratinocytes differentiate in vitro in response to a variety of stimuli, but neither the levels nor the spectrum of ceramides approach those seen in vivo. Ceramide production increases when human keratinocytes are grown at an air-liquid interface, and alterations in ceramide content occur when vitamin C is added to air-exposed, organotypic culture systems (Ponec et al. J Invest Dermatol 109:348, 1997). Here, we assessed whether vitamin C stimulates sphingolipid production in human keratinocytes independent of differentiation and air exposure. When submerged, human keratinocytes were grown in 1.2 mM calcium and serum-containing medium with vitamin C (50 microg per ml) for 9 d, total lipid content remained unchanged, but both glucosylceramide and ceramide content increased. Moreover, selected ceramide and glucosylceramide species: i.e., nonhydroxy ceramide 2 and both alpha- and omega-hydroxylated sphingolipids, increased preferentially [ceramide 4 (6-hydroxy-acylceramide), ceramide 5 (alpha-hydroxyceramide), ceramide 6 (4-hydroxy-alpha-hydroxyceramide), and ceramide 7 (6-hydroxy-alpha-hydroxyceramide); and acylglucosylceramide, glucosylceramide-B, and glucosylceramide-D], whereas ceramide 1, ceramide 3, glucosylceramide-C, and sphingomyelin remained unchanged. Synthesis of the corresponding ceramide and glucosylceramide fractions was enhanced by vitamin C, attributable, in part, to increased ceramide synthase activity (over 2-fold, p = 0.01); both serine palmitoyltransferase and glucosylceramide synthase activities remained unaltered. Finally, increased vitamin C-stimulated sphingolipid production correlated with the presence of lamellar bodies with mature internal contents, an increase in covalently bound omega-hydroxyceramide, and the appearance of prominent, corneocyte-bound lipid envelopes, whereas cornified envelope formation was unchanged. Thus, in submerged human keratinocytes, vitamin C induces both increased sphingolipid production and enhancement of permeability barrier structural markers.
authors.affiliation: Dermatology Service and Research Unit, Department of Veterans Affairs Medical Center, University of California, San Francisco 94121, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Uchida
authors.forename: M
authors.initials: M
authors.lastname: Behne
authors.forename: D
authors.initials: D
authors.lastname: Quiec
authors.forename: P M
authors.initials: PM
authors.lastname: Elias
authors.forename: W M
authors.initials: WM
authors.lastname: Holleran
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology*; D015415:Biomarkers; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism; D005963:Glucosylceramides / Q000378:metabolism; D006801:Humans; D007101:Immersion; D015603:Keratinocytes / Q000378:metabolism* / Q000502:physiology; D010088:Oxidoreductases / Q000378:metabolism; D013107:Sphingolipids / Q000096:biosynthesis*
chemical_list: D015415:Biomarkers; D002518:Ceramides; D005963:Glucosylceramides; D013107:Sphingolipids; D010088:Oxidoreductases; C108138:dihydroceramide desaturase; D001205:Ascorbic Acid