Intracellular free calcium and growth changes in single human keratinocytes in response to vitamin D and five 20-epi-analogues

Vitamin D, 1,25(OH)₂D₃, decreases proliferation and promotes differentiation of keratinocytes, and other keratinocyte differentiation stimuli have been associated with an early rise in intracellular free calcium, [Ca²⁺]_i. We therefore investigated the effect of 1,25(OH)₂D₃, its precursor D₃ and five 20-epi-analogues (EB1089, KH1060, KH1139, MC1288, MC1301) on growth and [Ca²⁺]_i levels of normal human keratinocytes. Cells were cultured in medium MCDB153 with an extracellular calcium concentration of 70 M or 1 mM. All the analogues were more potent than 1,25(OH)₂D₃ at inducing the morphological changes of differentiation, but D₃ was inactive. At concentrations down to 10^–8M 1,25(OH)₂D₃, caused significant inhibition of growth, as assessed by counting cells and measurement of thymidine labelling. At 5 days 50% inhibition of growth occurred with 64 nM 1,25(OH)₂D₃ and 3330 nM D₃. All the analogues were more potent than 1,25(OH)₂D₃, and KH1060 inhibited growth at 10^–10M. In single keratinocytes [Ca²⁺]_i was measured by micro-spectrofluorimetric techniques using the dye fura-2. No immediate rise in [Ca²⁺]_i was observed following addition of 1,25(OH)₂D₃ or the analogues up to 10^–6M. However 10^–7M 1,25(OH)₂D₃ or the analogues induced a gradual increase in [Ca²⁺]_i, significant at 4 h (P<0.001), which increased further over 2–3 days. D₃ had no effect on [Ca²⁺]_i. Increases in [Ca²⁺]_i following the differentiation stimuli of either 2 mM extracellular calcium or 1,25(OH)₂D₃ were similar at 48 h, increasing from 100 ± 3 nM (mean ± SEM) in control cells to 150 ± 3 nM with 2 mM calcium and 144 ± 6 nM with 10^–7M 1,25(OH)₂D₃. The effect of extracellular calcium in raising [Ca²⁺]_i within minutes was more rapid than 1,25(OH)₂D₃, but in combination the two were not additive.