Effects of 1α,25-(OH)2D3 on the formation and activity of osteoclasts in RAW264.7 cells

The hormonally active form of vitamin D3, 1 alpha,,25-(OH)(2)D-3, has an important role in bone metabolism. This study examined the effects of 1 alpha,,25-(OH)(2)D-3 on the ability of two cytokines, receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), to induce RAW 264.7 cells to form osteoclasts. A TRAP histochemical staining assay and bone resorption analysis were used to identify the rate of formation and activity of osteoclasts. The numbers of osteoclasts formed, and their bone resorption activity, was enhanced by the addition of 1a,25-(OH)(2)D-3. The expression levels of osteoclast-specific proteins that are essential for bone resorption, integrin beta(3), V-ATPase, CAII, CTSK, TRAP and MMP-9, were detected by western blotting. During 48 h, the expression levels of all these proteins significantly increased. Quantitative real-time polymerase chain reaction was used to determine the expression levels of the transcription factors, c-Fos and NFATcl. The expression levels of c-Fos and NFATcl also increased 24h after treatment with 1 alpha,25-(OH)(2)D-3. These results suggest that 1 alpha,25-(OH)(2)D-3 can regulate bone metabolism by directly enhancing the formation and maturation of osteoclasts. (C) 2015 Elsevier Ltd. All rights reserved.