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

We examined whether 1α-hydroxyvitamin D3 (1α(OH)D3) is metabolized into 1α,25-dihydroxyvitamin D3 (1α,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]-1α(OH)D3, a radioactive peak which comigrated at the same position as authentic 1α,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 1α,25(OH)2D3 by cochromatography with authentic 1α,25(OH)2D3 on three different HPLC systems and a thermal isomerization analysis. These results indicate that 1α,(OH)D3 is hydroxylated at the 25-position in bones, resulting in the local synthesis of 1α,25(OH)2D3 from 1α(OH)D3 in the skeletal tissues.