Nutr Res Pract.  2011 Oct;5(5):396-403.

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

• Kim J
• Yun H
• Cho Y

Abstract

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.

Keyword

Keratinocyte differentiation; calcium; vitamin C; sphingoid bases; sphingoid base 1 phosphates

MeSH Terms

Alkaline Phosphatase
Ascorbic Acid
Calcium
Ceramides
Chromatography, Liquid
Epidermis
Hydrolysis
Keratinocytes
Phosphates
Retention (Psychology)
Skin
Sphingolipids
Sphingosine
Vitamins
Water
Alkaline Phosphatase
Ascorbic Acid
Calcium
Ceramides
Phosphates
Sphingolipids
Sphingosine
Vitamins
Water

Figure

• 

  Fig. 1 Morphological characteristics of keratinocytes in the proliferative and early and late different differentiation stages. Cultured human keratinocytes grown in three different types of medium were observed under the microscope at a magnification 100×. Cells in the proliferation stage were grown in serum-free medium with 0.07 mM Ca2+ for six days and either harvested or induced to the early differentiation stage with 1.2 mM Ca2+. Late differentiation was induced by culturing cells in serum-containing medium with 1.2 mM Ca2+ and vitamin C (50 µL/mL). Late differentiated cells were maintained for 11 days, underwent media changes three times per week, and were then harvested.

• 

  Fig. 2 Altered levels of sphingoid bases associated with keratinocyte differentiation. Panel A: Sphingosine; Panel B: Sphinganine. Sphingoid bases were extracted from keratinocytes grown in culture medium with 0.07 mM Ca2+ (proliferation), 1.2 mM Ca2+ (early diff.), and control medium containing vitamin C (late diff.) and analyzed using high-performance liquid chromatography as described in Materials and Methods. Values are presented as means ± SEM (n = 3) Means with different letters are significantly different (P < 0.001) according to Duncan's multiple range test.

• 

  Fig. 3 Altered levels of sphingoid base phosphates associated with keratinocyte differentiation. Panel A: Sphingosine-1-phosphate; Panel B: Sphinganine-1-phosphate. Sphingoid base phosphates were extracted and dephosphorylated using an alkaline phosphatase assay from keratinocytes grown in culture medium with 0.07 mM Ca2+ (proliferation), 1.2 mM Ca2+ (early diff.), and control medium containing vitamin C (late diff.) and then analyzed using high-performance liquid chromatography as described in Materials and Methods. Values are presented as means ± SEM (n = 3) Means with different letters are significantly different (P < 0.001) according to Duncan's multiple range test.

• 

  Fig. 4 Effect of vitamin C on production of ceramides metabolites. Sphingoid bases and their phosphates were extracted from keratinocytes grown in serum-containing culture medium with 50 µg/mL of vitamin C (Vit C) or without (control) and were then analyzed by high-performance liquid chromatography. Values are presented as means ± SEM (n = 3).

• 

  Fig. 5 The ratio of sphingosine (SO) to sphinganine (SA) during keratinocyte differentiation. C18 sphingoid bases were extracted from keratinocytes grown in culture medium with 0.07 mM Ca2+ (proliferation), 1.2 mM Ca2+(early diff.), and control medium containing vitamin C (late diff.) and were then analyzed using high-performance liquid chromatography. The ratios of SO to SA were compared during differentiation. Values are presented as means ± SEM (n = 3). Means with different letters are significantly different (P < 0.05) according to Duncan's multiple range test.

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