Fig. 1. 1,25(OH)₂D₃ and it analogs inhibit cell proliferation and stimulate differentiation of primary human epidermal keratinocytes (HPEKp cell line).

Inhibition of the growth of primary human epidermal keratinocyte by 1,25(OH)₂D₃ (A), 20(OH)D₃ (B) and 21(OH)pD (C) was measured at 48 h using the SRB protein assay. Concentration-response curves were plotted and the IC₅₀ value determined as the concentration of the secosteroid which caused a 50% decrease in cell proliferation, calculated using Graph Pad Prism 5. Real-time quantitative PCR analyses were carried out on the expression of key differentiation markers, cytokeratin 1 (KRT1, D), cytokeratin 14 (KRT14, E) and involucrin (IVN, F), in primary human epidermal keratinocytes stimulated with 0.1 µM 1,25(OH)₂D₃, 20(OH)D₃ or 21(OH)pD for 24 h. Immunofluorescence labeling for Ki67 in control and stimulated cells with 1,25(OH)₂D₃ (0.1 µM) indicated a decrease in number of cells which entered cell cycle (G). Data are presented as means ± SD. Involucrin immunofluorescent-stained primary human epidermal keratinocytes (H) displayed increases in cell differentiation between control and stimulated cells with 1,25(OH)₂D₃ (0.1 µM), Ca²⁺ (2.5 mM) or both. Magnification 400×. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control.