25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand

25-Hydroxyvitamin D-3 1 alpha-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D-3 into 1 alpha,25-dihydroxyvitamin D-3, a vitamin D receptor ligand. 25-Hydroxyvitamin D-3 has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1 alpha-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D-3 has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D-3 internalisation, in Cyp27b1(-/-) cells explains their higher sensitivity to 25-hydroxyvitamin D-3. 25-Hydroxyvitamin D-3 action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D-3 and 1 alpha,25-dihydroxyvitamin D-3 with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D-3. Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D-3 in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D-3 with 1 alpha,25-dihydroxyvitamin D-3 in Cyp27b1(-/-) cells further demonstrates the agonistic action of 25-hydroxyvitamin D-3 and suggests that a synergism between 25-hydroxyvitamin D-3 and 1 alpha,25-dihydroxyvitamin D-3 might be physiologically important. In conclusion, 25-hydroxyvitamin D-3 is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties. (C) 2009 Elsevier Ltd. All rights reserved.