1,25-Dihydroxyvitamin D insufficiency accelerates age-related bone loss by increasing oxidative stress and cell senescence.

We investigated the role of insufficiency of the active form of vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)2D] in age-related bone loss. We employed mice with heterozygous deletion of Cyp27b1, the gene encoding the enzyme that synthesizes 1,25(OH)2D, as a model for 1,25(OH)2D insufficiency and compared the phenotype of lumber vertebrae from 3-, 9- and 18-month-old Cyp27b1+/- mice and their wild-type littermates. We found that in wild-type mice, bone mineral density, bone volume, and Cyp27b1 protein expression levels decreased progressively with age, accompanied by declining osteoblastic bone formation and increasing osteoclastic bone resorption, however these age-related skeletal alterations were more severe in Cyp27b1+/- mice which had significantly lower serum 1,25(OH)2D levels. We then assessed the effect of 1,25(OH)2D haploinsufficiency on oxidative stress and DNA damage, cell senescence and senescence-associated secretory phenotype (SASP) in 9-month-old wild-type and Cyp27b1+/- mice. Our results demonstrated that, in Cyp27b1+/- mice compared with their wild-type littermates, the parameters of oxidative stress and DNA damage were significantly increased, whereas the expression levels of antioxidant enzymes were significantly down-regulated; the percentage of senescent osteocytes and bone marrow mesenchymal stem cells, and the expression levels of SASP molecules and p16, p19 and p53 proteins were all significantly increased in bone tissues. Taken together, the results of this study indicate that 1,25(OH)2D insufficiency accelerates age-related bone loss by increasing oxidative stress and DNA damage, inducing bone cell senescence and SASP, and subsequently inhibiting osteoblastic bone formation while stimulating osteoclastic bone resorption.

The Polycomb Protein Bmi1 Plays a Crucial Role in the Prevention of 1,25(OH)2 D Deficiency-Induced Bone Loss.

We analyzed the skeletal phenotypes of heterozygous null Cyp27b1 (Cyp27b1+/- ) mice and their wild-type (WT) littermates to determine whether haploinsufficiency of Cyp27b1 accelerated bone loss, and to examine potential mechanisms of such loss. We found that serum 1,25-dihydroxyvitamin D [1,25(OH)2 D] levels were significantly decreased in aging Cyp27b1+/- mice, which displayed an osteoporotic phenotype. This was accompanied by a reduction of expression of the B lymphoma Moloney murine leukemia virus (Mo-MLV) insertion region 1 (Bmi1) at both gene and protein levels. Using chromatin immunoprecipitation (ChIP)-PCR, electrophoretic mobility shift assay (EMSA) and a luciferase reporter assay, we then showed that 1,25(OH)2 D3 upregulated Bmi1 expression at a transcriptional level via the vitamin D receptor (VDR). To determine whether Bmi1 overexpression in mesenchymal stem cells (MSCs) could correct bone loss induced by 1,25(OH)2 D deficiency, we overexpressed Bmi1 in MSCs using Prx1-driven Bmi1 transgenic mice (Bmi1Tg ) mice. We then compared the bone phenotypes of Bmi1Tg mice on a Cyp27b1+/- background, with those of Cyp27b1+/- mice and with those of WT mice, all at 8 months of age. We found that overexpression of Bmi1 in MSCs corrected the bone phenotype of Cyp27b1+/- mice by increasing osteoblastic bone formation, reducing osteoclastic bone resorption, increasing bone volume, and increasing bone mineral density. Bmi1 overexpression in MSCs also corrected 1,25(OH)2 D deficiency-induced oxidative stress and DNA damage, and cellular senescence of Cyp27b1+/- mice by reducing levels of reactive oxygen species (ROS), elevating serum total superoxide dismutase levels, reducing the percentage of γH2 A.X, p16, IL-1ß, and TNF-α-positive cells and decreasing γH2A.X, p16, p19, p53, p21, IL-1ß, and IL-6 expression levels. Furthermore, 1,25(OH)2 D stimulated the osteogenic differentiation of MSCs, both ex vivo and in vitro, from WT mice but not from Bmi1-/- mice and 1,25(OH)2 D administration in vivo increased osteoblastic bone formation in WT, but not in Bmi1 -/- mice. Our results indicate that Bmi1, a key downstream target of 1,25(OH)2 D, plays a crucial role in preventing bone loss induced by 1,25(OH)2 D deficiency. © 2019 American Society for Bone and Mineral Research.

1,25-Dihydroxyvitamin D exerts an antiaging role by activation of Nrf2-antioxidant signaling and inactivation of p16/p53-senescence signaling.

We tested the hypothesis that 1,25-dihydroxyvitamin D3 [1α,25(OH)2 D3 ] has antiaging effects via upregulating nuclear factor (erythroid-derived 2)-like 2 (Nrf2), reducing reactive oxygen species (ROS), decreasing DNA damage, reducing p16/Rb and p53/p21 signaling, increasing cell proliferation, and reducing cellular senescence and the senescence-associated secretory phenotype (SASP). We demonstrated that 1,25(OH)2 D3 -deficient [1α(OH)ase-/- ] mice survived on average for only 3 months. Increased tissue oxidative stress and DNA damage, downregulated Bmi1 and upregulated p16, p53 and p21 expression levels, reduced cell proliferation, and induced cell senescence and the senescence-associated secretory phenotype (SASP) were observed. Supplementation of 1α(OH)ase-/- mice with dietary calcium and phosphate, which normalized serum calcium and phosphorus, prolonged their average lifespan to more than 8 months with reduced oxidative stress and cellular senescence and SASP. However, supplementation with exogenous 1,25(OH)2 D3 or with combined calcium/phosphate and the antioxidant N-acetyl-l-cysteine prolonged their average lifespan to more than 16 months and nearly 14 months, respectively, largely rescuing the aging phenotypes. We demonstrated that 1,25(OH)2 D3 exerted an antioxidant role by transcriptional regulation of Nrf2 via the vitamin D receptor (VDR). Homozygous ablation of p16 or heterozygous ablation of p53 prolonged the average lifespan of 1α(OH)ase-/- mice on the normal diet from 3 to 6 months by enhancing cell proliferative ability and reducing cell senescence or apoptosis. This study suggests that 1,25(OH)2 D3 plays a role in delaying aging by upregulating Nrf2, inhibiting oxidative stress and DNA damage，inactivating p53-p21 and p16-Rb signaling pathways, and inhibiting cell senescence and SASP.

Overexpression of Sirt1 in mesenchymal stem cells protects against bone loss in mice by FOXO3a deacetylation and oxidative stress inhibition.

OBJECTIVE: B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) deficiency (Bmi-1-/-) leads to an osteoporotic phenotype with a significant downregulation of Sirt1 protein expression. Sirtuin 1 (Sirt1) haploinsufficiency results in a bone loss by decreased bone formation; however, it is unclear whether Sirt1 overexpression in mesenchymal stem cells (MSCs) plays an anti-osteoporotic role. The aim of the study is to identify whether the overexpression of Sirt1 in MSCs could restore skeletal growth retardation and osteoporosis in Bmi-1 deficient mice. METHODS: We used our new generated transgenic mouse model that overexpresses Sirt1 in its MSCs (Sirt1TG) to cross with Bmi-1-/- mice to generate Bmi-1-/- mice with Sirt1 overexpression in MSCs, and compared their skeletal metabolism with those of their Bmi-1-/- and wild-type (WT) littermates (6 mice for each genotype) at 4 weeks of age using imaging, histopathological, immunohistochemical, histomorphometric, cellular, and molecular methods. RESULTS: The levels of expression for Sirt1 were noticeably higher in the skeletal tissue of Sirt1TG mice than in those of WT mice. In Comparison to WT mice, the body weight and size, skeletal size, bone volume, osteoblast number, alkaline phosphatase and type I collagen positive areas, osteogenic related gene expression levels were all significantly increased in the Sirt1TG mice. Overexpression of Sirt1 in Bmi-1-/- mouse MSCs resulted in a longer lifespan, improved skeletal growth and significantly increased bone mass by stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption in the Bmi-1-/- mice, although the defects were not completely restored. Furthermore, Sirt1 overexpression in MSCs reduced the acetylation level of FOXO3a (Forkhead box O3a), increasing levels of expression for FOXO3a and SOD2 (Superoxide dismutase 2) in bony tissue, enhanced osteogenesis and reduced osteogenic cell senescence. We also demonstrated that nicotinamide, a Sirt1 inhibitor, blocks the effect of overexpression of Sirt1 in MSCs on osteogenesis and osteogenic cell senescence. CONCLUSIONS: Taken together, these results demonstrate that Sirt1 overexpression in MSCs increased the osteoblastic bone formation and partially restores the defects in skeletal growth and osteogenesis in Bmi-1-/- mice by FOXO3a deacetylation and oxidative stress inhibition. Our data support the proposal that Sirt1 is a target for promoting bone formation as an anabolic approach for the treatment of osteoporosis.

1,25-Dihydroxy vitamin D prevents tumorigenesis by inhibiting oxidative stress and inducing tumor cellular senescence in mice.

Human epidemiological studies suggest that 1,25(OH)2 D3 deficiency might increase cancer incidence, but no spontaneous tumors have been reported in mice lacking 1,25(OH)2 D3 or deficient in its receptor. In our study, we detected, for the first time, diverse types of spontaneous tumors in l,25(OH)2 D3 deficient mice more than 1 year of age. This was associated with increased oxidative stress, cellular senescence and senescence-associated secretory phenotype molecules, such as hepatocyte growth factor, mediated via its receptor c-Met. Furthermore, 1,25(OH)2 D3 prevented spontaneous tumor development. We also demonstrated that l,25(OH)2 D3 deficiency accelerates allograft tumor initiation and growth by increasing oxidative stress and DNA damage, activating oncogenes, inactivating tumor suppressor genes, stimulating malignant cell proliferation and inhibiting their senescence; in contrast, supplementation with exogenous l,25(OH)2 D3 or antioxidant, or knock-down of the Bmi1 or c-Met oncogene, largely rescued the phenotypes of allograft tumors. Results from our study suggest that 1,25(OH)2 D3 deficiency enhances tumorigenesis by increasing malignant cell oxidative stress and DNA damage, stimulating microenvironmental cell senescence and a senescence-associated secretory phenotype, and activating oncogenes and inactivating tumor suppressor genes, thus increasing malignant cell proliferation. Our study provides direct evidence supporting the role of vitamin D deficiency in increasing cancer incidence. Conversely, 1,25(OH)2 D3 prevented spontaneous tumor development, suggesting that this inhibitory effect prevents the initiation and progression of tumorigenesis, thus provides a mechanistic basis for 1,25(OH)2 D3 to prevent tumorigenesis in an aging organism.

1, 25-dihydroxy-vitamin D3 with tumor necrosis factor-alpha protects against rheumatoid arthritis by promoting p53 acetylation-mediated apoptosis via Sirt1 in synoviocytes.

Impaired apoptosis of fibroblast-like synoviocytes (FLSs) causes synovial hyperplasia, facilitating destruction of cartilage and bone in rheumatoid arthritis (RA). Tumor necrosis factor (TNF)-α, a dominant inflammatory mediator in RA pathogenesis, promotes progression of RA symptoms. Prevalence of 1, 25-dihydroxy-vitamin D3 (hereafter termed VD) deficiency is 30-63% in patients with RA. Whether VD leads to apoptosis or enhances TNF-α-mediated apoptosis in FLSs to ameliorate RA is unclear. To determine this, 10-week-old CYP27B1-deficient (CYP27B1-/-) mice with collagen-induced arthritis (CIA) were intraperitoneally treated with 1 µg/kg VD every other day for 9 weeks. RA phenotypes were compared between vehicle-treated CYP27B1-/- and wild-type CIA mice. Human rheumatoid FLS-MH7A cells were treated with Dulbecco's modified Eagle's medium (DMEM) without fetal bovine serum (FBS) for 24 h, then with different concentrations of VD and TNF-α, human vitamin D receptor (VDR) siRNA or the p53 pro-apoptotic inhibitor pifithrin-α. Apoptosis and p53 pro-apoptotic signaling were analyzed. The 19-week-old vehicle-treated CYP27B1-/- CIA mice had increased cumulative arthritis scores and levels of serous rheumatoid factors and C-reactive protein. They had exacerbated articular cartilage and bone destruction, joint space narrowing, joint stiffness, deformity and dysfunction, synovitis and TNF-α secretion, FLS hyperplasia with increased proliferation and decreased apoptosis compared to CIA mice. These RA phenotypes that were aggravated in CIA mice by CYP27B1 deficiency were largely rescued by VD treatment. In vitro, VD with TNF-α treatment upregulated p53 acetylation-mediated apoptosis in MH7A cells by promoting Sirt1 translocation from the nucleus to the cytoplasm. These findings indicated that VD with TNF-α protected against RA by promoting apoptosis of FLSs. The results indicated that clinical administration of VD could be a specific therapy to promote FLS apoptosis and prevent RA progression.