MOUSE PRIMARY OSTEOBLASTS EXPRESS VITAMIN-D-3 25-HYDROXYLASE MESSENGER-RNA AND CONVERT 1-ALPHA-HYDROXYVITAMIN-D-3 INTO 1-ALPHA,25-DIHYDROXYVITAMIN-D-3

We examined whether 1 alpha-hydroxyvitamin D-3 (1 alpha(OH)D-3) is met abolized into 1 alpha,25-dihydroxyvitamin D-3 (1 alpha,25(OH)(2)D-3) i n bone. Northern blot analysis indicated that the expression of vitami n D-3 25-hydroxylase mRNA was highest in the liver, followed by the du odenum, 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 mo stly osteoblastic cells, showed the highest expression of vitamin D-3 25-hydroxylase mRNA. When either cultured bone cells of fraction 3 or mouse calvaria were incubated with [H-3]-1 alpha(OH)D-3, a radioactive peak which comigrated at the same position as authentic 1 alpha,25(OH )(2)D-3 was found on an HPLC chromatogram. The radioactive fraction ob tained from the conditioned media of fetal mouse calvaria was tentativ ely identified as 1 alpha,25(OH)(2)D-3 by cochromatography with authen tic 1 alpha,25(OH)(2)D-3 on three different HPLC systems and a thermal isomerization analysis. These results indicate that 1 alpha(OH)D-3 is hydroxylated at the 25-position in bones, resulting in the local synt hesis of 1 alpha,25(OH)(2)D-3 from 1 alpha(OH)D-3 in the skeletal tiss ues.