24R, 25-Dihydroxyvitamin D3 Promotes the Osteoblastic Differentiation of Human Mesenchymal Stem Cells

Although 1 alpha,25-dihydroxyvitamin D-3 [1 alpha,25(OH)(2)D-3] is considered the most biologically active vitamin D3 metabolite, the vitamin D-3 prohormone, 25-hydroxyvitamin D3 [25(OH)D-3], is metabolized into other forms, including 24R, 25-dihydroxyvitamin D3 [24R, 25(OH)(2)D-3]. Herein we show that 24R, 25(OH)(2)D-3 is fundamental for osteoblastic differentiation of human mesenchymal stem cells (hMSCs). Our approach involved analyses of cell proliferation, alkaline phosphatase activity, and pro-osteogenic genes (collagen 1A1, osteocalcin, vitamin D receptor [VDR], vitamin D(3)hydroxylating enzymes [cytochrome P450 hydroxylases: CYP2R1, CYP27A1, CYP27B1 and CYP24A1]) and assessment of Ca2+ mineralization of extracellular matrix. 24R, 25(OH)(2)D-3 inhibited hMSC proliferation, decreased 1 alpha-hydroxylase (CYP27B) expression, thereby reducing the ability of hMSCs to convert 25(OH)D-3 to 1 alpha,25(OH)(2)D-3, and promoted osteoblastic differentiation through increased alkaline phosphatase activity and Ca2+ mineralization. 24R, 25(OH)(2)D-3 decreased expression of the 1 alpha,25(OH)(2)D-3 receptor, VDR. 24R, 25(OH)(2)D-3 but not 1 alpha,25(OH)(2)D-3 induced Ca2+ mineralization dependent on the absence of the glucocorticoid analog, dexamethasone. To elucidate the mechanism(s) for dexamethasone-independent 1 alpha,25(OH)(2)D-3 inhibition/24R, 25(OH)(2)D-3 induction of Ca2+ mineralization, we demonstrated that 1 alpha,25(OH)(2)D-3 increased whereas 24R, 25(OH)(2)D-3 decreased reactive oxygen species (ROS) production. 25(OH)D-3 also decreased ROS production, potentially by conversion to 24R, 25(OH)(2)D-3. Upon inhibition of the vitamin D-3-metabolizing enzymes (cytochrome P450s), 25(OH)D-3 increased ROS production, potentially due to its known (low) affinity for VDR. We hypothesize that vitamin D-3 actions on osteoblastic differentiation involve a regulatory relationship between 24R, 25(OH)(2)D-3 and 1 alpha,25(OH)(2)D-3. These results implicate 24R, 25(OH)(2)D-3 as a key player during hMSC maturation and bone development and support the concept that 24R, 25(OH)(2)D-3 has a bioactive role in the vitamin D-3 endocrine system.