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Vitamin C stimulates sphingolipid production and markers of barrier formation in submerged human keratinocyte cultures

Author(s): Uchida Y, Behne M, Quiec D, Elias PM, Holleran WM

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 µg 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 α- and ω-hydroxylated sphingolipids, increased preferentially [ceramide 4 (6-hydroxy-acylceramide), ceramide 5 (α-hydroxyceramide), ceramide 6 (4-hydroxy-α-hydroxyceramide), and ceramide 7 (6-hydroxy-α-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 ω-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.

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