Proliferation and differentiation of cultured human keratinocytes is modulated by 1,25(OH)2D3 and synthetic vitamin D3 analogues in a cell density-, calcium- and serum-dependent manner.

The natural form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) decreases proliferation and promotes terminal differentiation of cultured human epidermal keratinocytes. The purpose of this study was to find out to what extent the culture conditions determine the sensitivity of keratinocytes to 1,25(OH)2D3 and synthetic vitamin D analogues. Human keratinocytes were grown in microplates. Cell proliferation (MTT-assay) and differentiation (quantity of transglutaminase type I) were measured consecutively in the same monolayer. When vitamin D3 analogues were added to 50-60% confluent keratinocytes grown in serum-free keratinocyte growth medium with 0.09 mM Ca2+, stimulation of the proliferation was either minimal or non-existent, while differentiation was unaffected or slightly inhibited. There was no difference in the sensitivity to 1,25(OH)2D3 and the vitamin D3 analogues. When 1,25(OH)2D3 was added to less confluent keratinocytes (30%) a marked antiproliferative effect was observed. Addition of 3% charcoal stripped foetal calf serum further enhanced the antiproliferative effect of 1,25(OH)2D3, and a difference in the sensitivity of the vitamin D3 analogues was noted. If, finally, the Ca2+ concentration was raised to 0.3 mM, 1,25(OH)2D3 and the vitamin D3 analogues stimulated differentiation. Also, a biphasic effect on proliferation occurred: stimulation at low vitamin D concentrations and inhibition at higher concentrations. Furthermore, keratinocytes became more sensitive to the synthetic vitamin D3 analogues than to 1,25(OH)2D3: KH1060 > EB1089 > GS1500 > or = EB1213 > calcipotriol > 1,25(OH)2D3. For all compounds tested differentiation occurred at concentrations 10 to 30 times lower than for proliferation. These results indicate that the sensitivity to vitamin D3 analogues as well as the direction of the response to vitamin D3 analogues is dependent on the keratinocyte density, the availability of serum and Ca2+ concentrations.