Involvement of endogenously produced 1,25-dihydroxyvitamin D-3 in the growth and differentiation of human keratinocytes

Abstract

In this study, we investigated the possibility that cultured keratinocytes from normal human adult skin produce 1,25-dihydroxyvitamin D-3 (1,25(OH) 2D 3, a biologically active form of vitamin D-3) from 25-hydroxyvitamin D-3 [25(OH)D 3], and that 1,25(OH) 2D 3 endogenously produced by keratinocytes is involved in the self regulation of their growth and differentiation. To determine whether 1,25(OH) 2D 3 is produced from 25(OH)D 3 by skin keratinocytes, 25(OH)[ 3H]D 3 was added to keratinocyte cultures and incubated for 1 h and 5 h. The intracellular and extracellular metabolites were analyzed by three chromatographic systems. The three chromatograms revealed that the major metabolite produced from 25(OH) 2D 3 was 1,25(OH) 2D 3. Most of the 1,25(OH) 2D 3 endogenously produced from 25(OH)D 3 remained within the cells. To examine the time course of 1,25(OH) 2D 3 production, the amount of 1,25(OH)[ 3H]D 3 was measured at 15 min, 1 h, 5 h and 10 h, being at a maximum 1 h after the addition of 25(OH)D 3. These data indicate that keratinocytes rapidly convert 25(OH)D 3 to 1,25(OH) 2D 3 and that 1,25(OH) 2D 3 is not released into the medium. To determine whether endogenously produced 1,25(OH) 2D 3 is involved in the regulation of growth and differentiation of normal human keratinocytes, we examined the effects of 1,25(OH) 2D 3 and 25(OH)D 3 on their growth and differentiation. Keratinocyte growth was inhibited to 52.6% and 23.4% by 10 −8 M and 10 −7 M 1,25(OH) 2D 3 and to 80.5% and 23.9% by 10 −8 M and 10 −7 M 25(OH)D 3, respectively. Differentiation of these cells was evaluated by quantifying the number which express involucrin, a precursor protein of cornified envelope. The population of involucrin expressing cells (differentiated cells) increased from 6.2% to 14.5% by 2.5·10 −7 M 1,25(OH) 2D 3, and to 11.8% by 2.5·10 −7 M 25(OH)D 3. These results clearly indicate that 25(OH)D 3 is as effective on human keratinocytes as 1,25(OH) 2D 3 in inhibiting growth and inducing differentiation, although to a slightly lesser extent than 1,25(OH) 2D 3. The possibility that the effect of 25(OH)D 3 is mediated through binding to the 1,25(OH) 2D 3 receptor can be excluded, since a competitive binding assay revealed that the affinity of 25(OH)D 3 for the 1,25(OH) 2D 3 receptor in a cytosolic extract of keratinocytes was 100-times lower than that of 1,25(OH) 2D 3. Thus, these results suggest that 1,25(OH) 2D 3 endogenously produced in keratinocytes from 25(OH)D 3 is involved in the regulation of their growth and differentiation in vitro.