1,25-Dihydroxyvitamin D3 and the aging-related forkhead box O and sestrin proteins in osteoblasts.

Forkhead Box O (FoxO) transcription factors and Sestrins (SESN) are highly conserved and related stress-responsive proteins that protect the organism against age-related pathologies. For FoxOs, growing evidence shows a crucial role in osteoblast function. Here we investigated the role of different FoxO and SESN isoforms in 1,25(OH)2D3-treated MC3T3-E1 osteoblasts. 1,25(OH)2D3 rapidly and strongly induced the expression of SESN1 and FoxO3a but down-regulated the expression of SESN3 and FoxO1. SESN2 and FoxO4 levels were hardly affected by 1,25(OH)2D3. Chromatin Immunoprecipitation (ChIP)-sequencing revealed significant VDR/RXR binding to a DR3-type VDRE in SESN1 but not in the genomic region where FoxO3a is located. Mutation of the SESN1 VDRE abolished responsiveness to 1,25(OH)2D3 in luciferase-based transfection assays. siRNA-mediated knock-down of SESN1, SESN3, FoxO1 or FoxO3a did not prevent 1,25(OH)2D3 from reducing the expression of cell cycle markers like Cyclin D1 and Cdc6 and from exerting its characteristic antiproliferative effect on MC3T3-E1 osteoblasts. Accordingly, the 1,25(OH)2D3-induced reduction in the number of S-phase cells was also maintained. The antiproliferative effect was still present in primary osteoblast in which all three FoxO isoforms were deleted (TKOpOB). Interestingly, both MC3T3-E1 osteoblasts in which FoxO1 was knocked-down and TKOpOBs accumulated significantly more reactive oxygen species (ROS) after treatment with 1,25(OH)2D3 than control cells. siRNA-mediated knock-down of individual SESN isoforms did not result in significant differences in ROS levels. In conclusion, 1,25(OH)2D3 directly and indirectly alters the expression levels of different FoxO and SESN isoforms in osteoblasts, presumably not to exert its antiproliferative action but to control ROS levels. This article is part of a Special Issue entitled 'Vitamin D Workshop'.