Vitamin D inhibits Fas ligand-induced apoptosis in human osteoblasts by regulating components of both the mitochondrial and Fas-related pathways

Apoptosis plays an important role in the regulation of bone turnover. Previously, we showed that 1,25(OH)(2)D-3, the active form of vitamin D, may increase osteoblast survival by inhibiting apoptosis induced by serum deprivation. Human osteoblasts express the Fas receptor on their surface and its interaction with Fas ligand has been closely associated with human osteoblast apoptosis. To investigate the mechanism of 1,25(OH)(2)D-3 inhibition of apoptosis in ostcoblasts isolated from human calvaria, cells were exposed to Fas antibody. Visualization of apoptotic cells using annexin V revealed a significant decrease in apoptosis at 48 h in the presence of 1,25(OH)(2)D-3 (14 +/- 4%, P < 0.04) compared with non-treated cells (52 4%). Furthermore, flow cytometric analysis of TUNEL-labeled ostcoblasts showed a significant decrease in apoptotic cells in 1,25(OH)(2)D-3-treated cultures (12 +/- 2%) at 48 h compared with non-treated cultures (44 +/- 3%, P < 0.04). Additionally, cells treated with 1,25(OH)(2)D-3 survived longer as found by NITS analysis. To further explore the mechanism of 1,25(OH)(2)D(3-)mediated inhibition of apoptosis, we examined the changes in activation of death domain proteins, cleavage of caspases and mitochondrial regulators of apoptosis by Western blot analysis. A significant inhibition of caspase-8 cleavage and activity in 1,25(OH)(2)D-3-treated cells was observed in conjunction with a decrease in the expression of the proapoptotic protein Bax with a significant increase in the expression of antiapoptotic protein Bcl-2. Furthermore, the levels of p21(Cipi/WAF1), which inhibits the cleavage of caspase-8, was found to be highly induced in 1,25(OH)(2)D-3-treated cells. In summary, these results demonstrate that the anti-apoptotic effect of 1,25(OH)(2)D-3 in human osteoblasts after the activation of Fas-ligand is mediated by the regulation of components of both the mitochondrial and Fas-related pathways. (C) 2004 Elsevier Inc. All rights reserved.