Mouse primary osteoblasts express vitamin D3 25-hydroxylase mRNA and convert 1 alpha-hydroxyvitamin D3 into 1 alpha,25-dihydroxyvitamin D3.

We examined whether 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3) is metabolized into 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) in bone. Northern blot analysis indicated that the expression of vitamin D3 25-hydroxylase mRNA was highest in the liver, followed by the duodenum, calvaria, lung, kidney, skin and long bone, and lowest in the spleen. Of the bone cell fractions isolated from fetal mouse calvaria by a sequential enzymatic digestion, fraction 3, which consisted of mostly osteoblastic cells, showed the highest expression of vitamin D3 25-hydroxylase mRNA. When either cultured bone cells of fraction 3 or mouse calvaria were incubated with [3H]-1 alpha (OH)D3, a radioactive peak which comigrated at the same position as authentic 1 alpha,25(OH)2D3 was found on an HPLC chromatogram. The radioactive fraction obtained from the conditioned media of fetal mouse calvaria was tentatively identified as 1 alpha,25(OH)2D3 by cochromatography with authentic 1 alpha,25(OH)2D3 on three different HPLC systems and a thermal isomerization analysis. These results indicate that 1 alpha(OH)D3 is hydroxylated at the 25-position in bones, resulting in the local synthesis of 1 alpha,25(OH)2D3 from 1 alpha(OH)D3 in the skeletal tissues.