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 microM 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(-8) M 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(-10) M. In single keratinocytes [Ca2+]i was measured by microspectrofluorimetric 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(-6) M. However 10(-7) M 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(-7) M 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.