Effects of vitamin D metabolites on proliferation and differentiation of cultured human epidermal keratinocytes grown in serum-free or defined culture medium

We examined the effects of 1, 25-dihydroxyvitamin D₃ [1, 25(OH)₂D₃], 25-hydroxyvitamin D₃ (25OHD₃), and vitamin D3 on human keratinocyte proliferation and differentiation in a serum-free or defined culture system. Concentrations greater than 10⁻⁸ M 1, 25-(OH)₂D₃ or 10⁻⁷ M 25(OH)₂D₃ caused marked inhibition of cell growth. Growth inhibition with high doses of 1, 25-(OH)₂D₃ was not stringent, but was mainly exerted in the G₁ phase of the cell cycle. Early release from the cell cycle block restored the proliferation of human keratinocytes. The calcium concentration in the medium had no significant effect on the antiproliferative action of 1, 25-(OH)₂D₃, 25OHD3, and vitamin D3. We also show that human keratinocyte proliferation is enhanced at doses of 1, 25-(OH)₂D₃ and 25OH2D3 of 10(-9) M or less. Enhanced proliferation of human keratinocytes with physiological concentrations of 1, 25-(OH)₂D₃ could only be shown in fully defined medium that contained no vitamin D3, related sterols, or bovine pituitary extract. Human keratinocyte differentiation was enhanced with higher doses of 1, 25-(OH)₂D₃ when cells were grown in the presence of high calcium concentrations. These studies demonstrate that the lower, physiological concentrations of vitamin D3 metabolites are capable of stimulating the proliferation of epidermal keratinocytes grown under selected conditions that eliminate confounding or unidentified medium culture factors. Vitamin D3 metabolites are shown to exert mitogenic trophic effects in cultured human epithelial cells similar to their established activities in vivo.