Figure 3From: Phytosphingosine Stimulates the Differentiation of Human Keratinocytes and Inhibits TPA-Induced Inflammatory Epidermal Hyperplasia in Hairless Mouse SkinThe effect of phytosphingosine on the proliferation and differentiation of keratinocytes. (A) For the measurement of cornified envelope (CE) content, normal human epidermal keratinocytes were treated for 7 days with Ca2+ (1.5 mM), phytosphingosine (PS), or troglitazone (TGZ) at the concentrations indicated. CEs were extracted by exhaustive boiling followed by sonication and quantification by spectrophotometry at 310 nm. Data are means ± SD of 3 independent experiments. *Significant at P < 0.05. (B and C) To measure epidermal differentiation marker proteins, normal human epidermal keratinocytes were treated for 4 days with high Ca2+ (1.5 mM), 5 pM phytosphingosine (PS), or 5 µM troglitazone (TGZ). The level of involucrin was analyzed by ELISA. Data are means ± SD of 3 independent experiments. *Significant at P < 0.05. The expression of loricrin and keratin1 was assessed by immunoblotting with antiloricrin or antikeratin1 antibody. The relative densitometric ratios are indicated. The data are representative of 3 independent blots. (D) Normal human epidermal keratinocytes in 24-well plates were treated with the indicated concentrations of phytosphingosine (PS). [3H]thymidine (1 µCi) was added to the wells 20 h after the addition of PS, the plates were incubated for 4 h, and the amount of cell-associated radioactivity was quantified. The indicated DNA synthesis levels are the means of the levels of triplicate wells ± SD expressed as a percent of the control. The DNA synthesis rate for the control was 1,570,072 ± 141,322 cpm/well. *Significant at P < 0.01.Back to article page