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

Abstract

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.

Keywords: Nrf2; aging; cell senescence; p16 and p53; vitamin D.

Figure 1

The effects of a high calcium/phosphate diet, of 1,25(OH)₂D_3, and of antioxidant supplementation on lifespan, body weight, and skin morphology in 1α(OH)ase^−/− mice. After weaning, sex‐matched wild‐type (WT) and 1α(OH)ase^−/− (KO) mice were fed a normal diet (ND) or a “rescue diet” diet (RD) or received thrice weekly subcutaneous injections of vehicle (ND) or 1,25(OH)₂D₃ (1 μg/kg) (VD), or were fed a rescue diet with 1 mg/ml NAC in drinking water (RD + NAC). (a) Serum calcium, (b) phosphorus, (c) 1,25(OH)₂D₃, (d) 25(OH)D, and (e) PTH. (f) Survival rate of the mice; (g) body weight. Representative micrographs of skin sections stained (h) with H&E or (j) histochemically for total collagen (T‐Col). Scale bars represent 200 μm in c and e. (i) Skin thickness and (k) total collagen‐positive area (%). Each value is the mean ± SEM of determinations in five mice of each group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with WT mice. ^# p < 0.05; ^## p < 0.01 compared with ND KO mice. ^& p < 0.05; ^&& p < 0.01 compared with RD KO mice

Figure 2

The effects of a high calcium/phosphate diet, of 1,25(OH)₂D_3, and of antioxidant supplementation on oxidative stress, DNA damage, and protein expression of oncogenes and tumor suppressive genes in 1α(OH)ase^−/− mice. Mice from each group were treated as described in Figure 1. ROS levels in freshly isolated cells of (a) skin, (b) liver, and (c) kidney from 10‐week‐old wild‐type (WT) and 1α(OH)ase^−/− (KO) mice. Representative micrographs of skin sections stained immunohistochemically for (d) SOD2 and (f) γ‐H2AX. Scale bars represent 50 μm in d and f. (e) The percentage of SOD2‐positive cells of skin and (g) the percentage of γ‐H2AX‐positive cells of skin; (h) representative Western blots of skin extracts to determine Prdx I protein levels. β‐actin was used as loading control. (i) Skin Prdx I and Bmi1, p16, p53 and p21 protein levels in (j) skin and (k) liver relative to β‐actin protein levels were assessed by densitometric analysis and expressed as a percentage of the levels of vehicle‐treated wild‐type mice fed the normal diet (ND).Each value is the mean ± SEM of determinations in five mice of each group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with WT mice. ^# p < 0.05; ^## p < 0.01 compared with ND KO mice. ^& p < 0.05; ^&& p < 0.01 compared with RD KO mice

Figure 3

The effects of a high calcium/phosphate diet, of 1,25(OH)₂D_3, and of antioxidant supplementation on cell proliferation and senescence in 1α(OH)ase^−/− mice. Mice from each group were treated as described in Figure 1. (a) Representative micrographs of paraffin sections from 10‐week‐old wild‐type (WT) and 1α(OH)ase^−/− (KO) mice stained immunohistochemically for ki67 in skin (b) The percentage of ki67‐positive cell number relative to total cell number in skin. Representative micrographs of sections from 10‐week‐old WT and KO mice stained histochemically for senescence‐associated β‐galactosidase (SA‐β‐gal) in (c) skin and (e) kidney. Scale bars represent 50 μm in a, c, and e. The percentage of SA‐β‐gal‐positive area in (d) skin and (f) kidney. RT–PCR of (g) skin, and (h) kidney tissue extracts for expression of TNFα, IL‐1α and β, IL‐6, Mmp 3 and 13. Messenger RNA expression assessed by real‐time RT–PCR is calculated as a ratio relative to GAPDH, and expressed relative to ND WT mice. Each value is the mean ± SEM of determinations in five mice of each group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with WT mice. ^# p < 0.05; ^## p < 0.01; ^### p < 0.001 compared with ND KO mice. ^& p < 0.05; ^&& p < 0.01; ^&&& p < 0.001 compared with RD KO mice

Figure 4

1,25(OH)₂D₃ exerts an antioxidant role by transcriptional regulation of Nrf2 mediated through the vitamin D receptor. MEFs from wild‐type and VDR knockout (VDR KO) mice were treated with 10⁻⁹–10⁻⁷ M 1,25(OH)₂D₃ for 24 hr, and the mRNA relative expression levels of Nrf2 in MEFs were examined by real‐time RT–PCR. *p < 0.05; **p < 0.01; ***p < 0.001 compared with control cultures. (b) VDR‐like elements in mouse Nrf2 promoter region and the mutated VDRE sequence highlighted in red color (upper panels); structure schematic diagram of pGL3‐Nrf2 promoter reporter plasmid and mutant pGL3‐Nrf2 promoter reporter plasmid. (c) Luciferase activity driven by Nrf2 promoter, more dramatically by 1,25(OH)₂D₃treatment_,but not driven by Nrf2 luciferase reporter with mutated VDRE treated without/with 1,25(OH)₂D₃. **p < 0.01; ***p < 0.001 compared with negative control. ^### p < 0.001 compared with genotype‐matched cells. (d) Nrf2 promoter sequences could be recovered by PCR from VDR immunoprecipitates but not from pre‐immune IgG immunoprecipitates. (e) The mRNA level of Nrf2 in Vector (SiControl) or SiNrf2 transfected MEFs. ***p < 0.001 compared with SiControl. (f–i) Messenger RNA expression assessed by real‐time RT–PCR is calculated in Vector (real‐time RT–PCR is calculated in Vector (Si‐Control) or siNrf2 transfected MEFs as a ratio relative to GAPDH, and expressed relative to SiControl MEFs. Each value is the mean ± SEM of determinations in triplicated cultures. *p < 0.05; ***p < 0.001 compared with wild‐type MEFs. ^# p < 0.05; ^### p < 0.001 compared with SiControl. ^& p < 0.05; ^&&& p < 0.001 compared with SiNrf2

Figure 5

p16 deletion delays aging induced by 1,25(OH)₂D₃deficiency. (a) The survival rate of WT, p16^−/−, 1α(OH)ase^−/−, and 1α(OH)ase^−/−p16^−/− mice; (b) body weight; (c) ROS levels in freshly isolated cells of skin; representative micrographs of skin sections from 10‐week‐old mice stained with (d) HE, (f) histochemically for total collagen and (h) immunohistochemically for Ki67 and (j) histochemically for SA‐β‐gal. Scale bars represent 200 μm in (d) and (f) and 50 μm in (h) and (j). (e) Skin thickness; (g) total collagen‐positive area; the percentage of (i) BrdU‐positive cells of skin; (k) the percentage of SA‐β‐gal‐positive area in skin; (l) representative Western blots of skin extracts to determine p53, p21, cyclin E, cyclin D1, and SOD protein levels in skin. β‐actin was used as loading control. (m) Skin protein levels relative to β‐actin protein levels were assessed by densitometric analysis and expressed as percentage of the levels of wild‐type mice. Each value is the means ± SEM of determinations in five mice of each group. *p < 0.05; ^** p < 0.01; ***p < 0.001 compared with WT mice. ^#p < 0.05; ^## p < 0.01; ^### p < 0.001 compared with 1α(OH)ase^−/− mice

Figure 6

p53 haploinsufficiency delays aging induced by 1,25(OH)₂D₃deficiency. (a) The survival rates of WT, p53^+/−, 1α(OH)ase^−/−, and 1α(OH)ase^−/−p53^+/− mice; (b) body weight; (c) ROS levels in freshly isolated cells of skin; representative micrographs of skin sections from 10‐week‐old mice stained with (d) HE, histochemically for (f) total collagen, immunohistochemically for (h) γ‐H2AX and (j) Ki‐67, histochemically for (l) SA‐β‐gal and (o) with TUNEL. Scale bars represent 200 μm in (d) and (f) and 50 μm in h, j, l, and o. (e) Skin thickness; (g) total collagen‐positive area; the percentage of (i) γ‐H2AX‐ and (k) Ki‐67‐positive cells of skin; (m) the percentage of SA‐β‐gal‐positive area in skin; (p) the percentage of TUNEL‐positive cells in skin. (q) Representative Western blots of skin extracts to determine Wnt16, p53, p21, caspase3, cyclin D1, and cyclin E protein levels in skin. β‐actin was used as loading control. (r) Skin protein levels relative to β‐actin protein levels were assessed by densitometric analysis and expressed as percentage of the levels of vehicle‐treated wild‐type mice fed the normal diet (ND). Each value is the mean ± SEM of determinations in five mice of each group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with WT mice. ^#p < 0.05; ^##p < 0.01; ^### p < 0.001 compared with 1α(OH)ase^−/− mice

Figure 7

Model of mechanisms leading from 1,25(OH)₂D₃ deficiency to accelerated aging