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Ceramide is the most abundant lipid in the epidermis and plays a critical role in maintaining epidermal barrier function. Overall ceramide content in keratinocyte increases in parallel with differentiation, which is initiated by supplementation of calcium and/or vitamin C. However, the role of metabolic enzymes responsible for ceramide generation in response to vitamin C is still unclear. Here, we investigated whether vitamin C alters epidermal ceramide content by regulating the expression and/or activity of its metabolic enzymes. When human keratinocytes were grown in 1.2 mM calcium with vitamin C (50 μg/ml) for 11 days, bulk ceramide content significantly increased in conjunction with terminal differentiation of keratinocytes as compared to vehicle controls (1.2 mM calcium alone). Synthesis of the ceramide fractions was enhanced by increased
Mammalian epidermis consists of four different layers, stratum basale (SB), stratum spinosum (SP), stratum granulosum (SG), and stratum corneum (SC) (Elias and Menon, 1991; Uchida, 2014). Since the SC is the outermost layer and directly faces the external environment, it functions to serve as a barrier (Elias, 2005). Multiple factors are involved in maintaining epidermal barrier function, of which ceramide is of particular importance (Uchida, 2014). Precursors of ceramide are synthesized during keratinocyte (KC) differentiation and are packaged into lamellar bodies (LB) and eventually secrete their contents at the SG-SC interface (Uchida
While the overall level of ceramide in the epidermis is regulated by several metabolic enzymes,
Although previous studies have provided evidence for the importance of epidermal ceramide in maintaining normal barrier function, quantitative analysis looking into specific levels of metabolic enzymes responsible for ceramide synthesis in KC following Vit C is still unclear. In this study, we show that high Ca2+ (1.2 mM) and Vit C treatment stimulates production of ceramide. This stimulation is mediated by altered activity/ expression of metabolic enzymes involved in ceramide synthesis.
Human primary KC from Life Technologies (Carlsbad, CA, USA) were maintained in serum-free KC growth medium containing 0.07 mM Ca2+, as described previously (Park
Western blot analysis was performed as previously described (Park
To assess the levels of cellular ceramide, human KC were incubated with Vit C and washed with phosphate-buffered saline followed by extracting total lipids, as we reported previously (Shin
SPT activity was determined as previously described with a minor modification (Rutti
CerS activity assay was performed as described previously (Kim
Activities of SMases were assessed as described previously (Loidl
SPPase activity was determined as previously described with a minor modification (Johnson
Statistical analyses used the unpaired Student
Previous studies have shown high Ca2+ and/or Vit C to alter gene expression associated with KC differentiation (Eichner
We also assessed ceramide content in KC incubated with Vit C. Our lipid analysis data shows that Vit C treatment significantly increases ceramide content (Table 1). To investigate the metabolic step(s) at which Vit C exhibits its effects on ceramide synthesis, we next measured the expression and activity of key enzymes that are involved in ceramide metabolism, e.g., SPT, CerS, SMase, SPPase, following Vit C treatment. Western blot and enzyme assays showed that both expression and activity of SPT and CerS was significantly increased in cells treated with Vit C (Fig. 2A?2D). Whereas, acidic and neutral SMase activities were not affected by Vit C treatment (Fig. 2E). Furthermore, prior studies have suggested that SPPase expression is a critical factor in regulating ceramide levels. As such, we measured SPPase mRNA and protein expression in KC cultured in Vit C (Fig. 3). Our
Of the lipids enriched in the extracellular lamellar membranes, ceramide is the most dominant lipid, which ultimately comprise 50% of total lipid mass in the SC (Elias and Menon, 1991). Prior studies have highlighted the importance of the respective lipid in maintaining barrier function: i) epidermal ceramide synthesis is required for normal barrier recovery after barrier disruption (Holleran
It is well established that ceramide content rises in parallel with KC differentiation (Uchida
Prior studies have demonstrated that overall epidermal ceramide production is stimulated through
Moreover, we show here that Vit C-mediated increase in both expression and activity of SPPase, an enzyme which dephosphorylates S1P to SO, contributes, at least in part, to increased epidermal ceramide synthesis. Previous studies using HEK-293 (human embryonic kidney cells) and NIH 3T3 fibroblasts have provided evidence for the important role of SPPase in regulating ceramide levels; i) SPPase regulates ceramide levels in the endoplasmic reticulum (Le Stunff
In conclusion, the present studies show that Vit C supplementation increases epidermal ceramide production by three pathways (Fig. 4): 1) increased
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2012R1A1A3005669).
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