The 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.