The synthesis of vitamin D metabolites by human melanoma cells

Two melanin-producing human melanoma cell lines originally established from fresh surgical specimens were incubated with 25 hydroxyvitamin D3 (25 OHD3). Both cell lines produced material comigrating with 1,25 dihydroxy-vitamin D3 (1,25(OH)2D3) and 24,25 dihydroxyvitamin D3 (24,25(OH)2D3) in straight and reverse phase high performance liquid chromatography systems and displacing the relevant labeled ligands in competitive binding assays. The material designated 1,25(OH)2D3 was found almost entirely within the cells, whereas 24,25(OH)2D3 was evenly distributed between cells and medium. The synthesis of dihydroxylated materials was time dependent and was not observed if the cells were boiled before incubation with 25 OHD3. Preincubation with 1,25(OH)2D3 caused an increase in the synthesis of 24,25(OH)2D3 and a decrease in the synthesis of 1,25(OH)2D3. Michaelis-Menten constant (Km) values were 1.4 X 10(-9) mol/liter 25 OHD3 for the 1-alpha-hydroxylase enzyme and 72 X 10(-9) mol/liter for 24-hydroxylase. These studies constitute further evidence for the extrarenal synthesis of 1,25(OH)2D3. The suppressibility of 1 alpha-hydroxylase by preincubation with 1,25(OH)2D3 suggests a regulatory function for this system in the skin.