1a-Hydroxylation of 25-hydroxyvitamin D3 is believed to be essential for its biological effects. In this
study, we evaluated the biological activity of 25(OH)D3 itself comparing with the effect of cell-derived
1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3). First, we measured the cell-derived 1a,25(OH)2D3 level in
immortalized human prostate cell (PZ-HPV-7) using [3H]-25(OH)D3. The effects of the cell-derived
1a,25(OH)2D3 on vitamin D3 24-hydroxylase (CYP24A1) mRNA level and the cell growth inhibition were
significantly lower than the effects of 25(OH)D3 itself added to cell culture. 25-Hydroxyvitamin D3 1ahydroxylase
(CYP27B1) gene knockdown had no significant effects on the 25(OH)D3-dependent effects,
whereas vitamin D receptor (VDR) gene knockdown resulted in a significant decrease in the 25(OH)D3-
dependent effects. These results strongly suggest that 25(OH)D3 can directly bind to VDR and exerts
its biological functions. DNA microarray and real-time RT–PCR analyses suggest that semaphorin 3B,
cystatin E/M, and cystatin D may be involved in the antiproliferative effect of 25(OH)D3