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

Human keratinocytes differentiate in vitro in response to a variety of stim uli, but neither the levels nor the spectrum of ceramides approach those se en in vivo. Ceramide production increases when human keratinocytes are grow n at an air-liquid interface, and alterations in ceramide content occur whe n 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 s timulates sphingolipid production in human keratinocytes independent of dif ferentiation and air exposure. When submerged, human keratinocytes were gro wn in 1.2 mm calcium and serum-containing medium with vitamin C (50 mug per ml) for 9 d, total lipid content remained unchanged, but both glucosylcera mide and ceramide content increased. Moreover, selected ceramide and glucos ylceramide 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 acylglucosyl-ceramide, glucosylceramide-B, and glucosylceramide-D], wh ereas ceramide 1, ceramide 3, glucosylceramide-C, and sphingomyelin remaine d unchanged. Synthesis of the corresponding ceramide and glucosylceramide f ractions was enhanced by vitamin C, attributable, in part, to increased cer amide synthase activity (over 2-fold, p=0.01); both serine palmitoyltransfe rase 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 c ovalently bound omega -hydroxyceramide, and the appearance of prominent, co rneocyte-bound lipid envelopes, whereas cornified envelope formation was un changed. Thus, in submerged human keratinocytes, vitamin C induces both inc reased sphingolipid production and enhancement of permeability barrier stru ctural markers.