Analysis of ceramide metabolites in differentiating epidermal keratinocytes treated with calcium or vitamin C

Ceramides (Cer) comprise the major constituent of sphingolipids in the epidermis and are known to play diverse roles in the outermost layers of the skin including water retention and provision of a physical barrier. In addition, they can be hydrolyzed into free sphingoid bases such as C18 sphingosine (SO) and C18 sphinganine (SA) or can be further metabolized to C18 So-1-phosphate (S1P) and C18 Sa-1-phosphate (Sa1P) in keratinocytes. The significance of ceramide metabolites emerged from studies reporting altered levels of SO and SA in skin disorders and the role of S1P and Sa1P as signaling lipids. However, the overall metabolism of sphingoid bases and their phosphates during keratinocyte differentiation remains not fully understood. Therefore, in this study, we analyzed these Cer metabolites in the process of keratinocyte differentiation. Three distinct keratinocyte differentiation stages were prepared using 0.07 mM calcium (Ca2+) (proliferation stage), 1.2 mM Ca2+ (early differentiation stage) in serum-free medium, or serum-containing medium with vitamin C (50 microL/mL) (late differentiation stage). Serum-containing medium was also used to determine whether vitamin C increases the concentrations of sphingoid bases and their phosphates. The production of sphingoid bases and their phosphates after hydrolysis by alkaline phosphatase was determined using high-performance liquid chromatography. Compared to cells treated with 0.07 mM Ca2+, levels of SO, SA, S1P, and SA1P were not altered after treatment with 1.2 mM Ca2+. However, in keratinocytes cultured in serum-containing medium with vitamin C, levels of SO, SA, S1P, and SA1P were dramatically higher than those in 0.07- and 1.2-mM Ca2+-treated cells; however, compared to serum-containing medium alone, vitamin C did not significantly enhance their production. Taken together, we demonstrate that late differentiation induced by vitamin C and serum was accompanied by dramatic increases in the concentration of sphingoid bases and their phosphates, although vitamin C alone had no effect on their production.

Royal jelly enhances migration of human dermal fibroblasts and alters the levels of cholesterol and sphinganine in an in vitro wound healing model

Oral administration of royal jelly (RJ) promotes wound healing in diabetic mice. Concerns have arisen regarding the efficacy of RJ on the wound healing process of normal skin cells. In this study, a wound was created by scratching normal human dermal fibroblasts, one of the major cells involved in the wound healing process. The area was promptly treated with RJ at varying concentrations of 0.1, 1.0, or 5 mg/ml for up to 48 hrs and migration was analyzed by evaluating closure of the wound margins. Furthermore, altered levels of lipids, which were recently reported to participate in the wound healing process, were analyzed by HPTLC and HPLC. Migration of fibroblasts peaked at 24 hrs after wounding. RJ treatment significantly accelerated the migration of fibroblasts in a dose-dependent manner at 8 hrs. Although RJ also accelerated the migration of fibroblasts at both 20 hrs and 24 hrs after wounding, the efficacy was less potent than at 8 hrs. Among various lipid classes within fibroblasts, the level of cholesterol was significantly decreased at 8 hrs following administration of both 0.1 ug/ml and 5 mg/ml RJ. Despite a dose-dependent increase in sphinganines, the levels of sphingosines, ceramides, and glucosylceramides were not altered with any concentration of RJ. We demonstrated that RJ enhances the migration of fibroblasts and alters the levels of various lipids involved in the wound healing process.