Acylceramides and lanosterol-lipid markers of terminal differentiation in cultured human keratinocytes: Modulating effect of retinoic acid

Epidermal differentiation is accompanied by profound changes in the synthesis of a variety of intracellular proteins and intercellular lipids. In conventional, submerged culture keratinocytes have been shown to lose the ability to synthesize the protein markers of differentiation. They re-express them, however, when they are cultured in medium supplemented with delipidized retinoic acid (RA)-depleted] serum or in air-exposed cultures using de-epidermized dermis (DED) as a substrate. Recent studies have revealed that acylceramides (AC) and lanosterol (LAN), which are present only in trace amounts in cultures of keratinocytes grown under submerged conditions on DED in medium supplemented with normal serum, become expressed in significant amounts when the culture is lifted to the air-liquid interface. Inasmuch as culture conditions may markedly affect the extent of keratinocyte differentiation, the present study aimed to investigate the effect of normal (RA-containing) or delipidized (RA-depleted) serum and of RA administration on lipid composition (especially of the AC and LAN contents) in cells cultured under submerged and air-exposed conditions. To test a possible effect of dermal substrate (used in the air-exposed model), the lipid composition of keratinocytes grown under submerged conditions on a plastic and on a dermal substrate (de-epidermized dermis, DED) has also been compared. The results revealed that under all culture conditions, RA deprivation of fetal bovine serum resulted in a marked increase of total ceramide content. Even under submerged conditions, the presence of both AC and LAN could be detected. In air-exposed culture, the content of these lipids was markedly increased. Addition of RA at 1 μM concentration to cultures grown in RA-depleted medium induced marked changes in lipid composition under all culture conditions tested. In cells grown under submerged conditions (both on plastic and on DED) AC and LAN were no longer present in detectable amounts. Also in air-exposed culture, a marked decrease in the content of these lipids was observed. These results suggest that liposoluble serum components, like RA, control the synthesis of lipids that are present in later stages of epidermal differentiation.