1,25-Dihydroxyvitamin D Deficiency Accelerates Aging-related Osteoarthritis via Downregulation of Sirt1 in Mice

Abstract

Emerging observational data suggest that vitamin D deficiency is associated with the onset and progression of knee osteoarthritis (OA). However, the relationship between vitamin D level and OA and the role of vitamin D supplementation in the prevention of knee OA are controversial. To address these issues, we analyzed the articular cartilage phenotype of 6- and 12-month-old wild-type and 1α(OH)ase^-/- mice and found that 1,25(OH)₂D deficiency accelerated the development of age-related spontaneous knee OA, including cartilage surface destruction, cartilage erosion, proteoglycan loss and cytopenia, increased OARSI score, collagen X and Mmp13 positive chondrocytes, and increased chondrocyte senescence with senescence-associated secretory phenotype (SASP). 1,25(OH)₂D₃ supplementation rescued all knee OA phenotypes of 1α(OH)ase^-/- mice in vivo, and 1,25(OH)₂D₃ rescued IL-1β-induced chondrocyte OA phenotypes in vitro, including decreased chondrocyte proliferation and cartilage matrix protein synthesis, and increased oxidative stress and cell senescence. We also demonstrated that VDR was expressed in mouse articular chondrocytes, and that VDR knockout mice exhibited knee OA phenotypes. Furthermore, we demonstrated that the down-regulation of Sirt1 in articular chondrocytes of 1α(OH)ase^-/- mice was corrected by supplementing 1,25(OH)₂D₃ or overexpression of Sirt1 in mesenchymal stem cells (MSCs) and 1,25(OH)₂D₃ up-regulated Sirt1 through VDR mediated transcription. Finally, we demonstrated that overexpression of Sirt1 in MSCs rescued knee OA phenotypes in 1α(OH)ase^-/- mice. Thus, we conclude that 1,25(OH)₂D_3, via VDR-mediated gene transcription, plays a key role in preventing the onset of aging-related knee OA in mouse models by up-regulating Sirt1, an aging-related gene that promotes articular chondrocyte proliferation and extracellular matrix protein synthesis, and inhibits senescence and SASP.

Keywords: Sirt1; Vitamin D deficiency; osteoarthritis; vitamin D receptor; vitamin D supplementation.

Figure 1

1,25(OH)₂D deficiency accelerates the development of age-related spontaneous knee OA. (A) Serum calcium, phosphorus, intact PTH, 25(OH)D and 1,25(OH)₂D₃ levels in 12-month-old wild-type (n=8) and 1α(OH)ase^-/- (n=8) mice on a normal diet (ND) or a rescue diet (RD). N.S.= not significant; N.D.= not detected. **: p< 0.01, ***: p<0.001. Representative sectioned images of articular cartilage stained for (B) safranin O and (C) the quantification of OARSI grade in 6- and 12-month-old WT and 1α(OH)ase^-/- mice on the rescue diet. n=8 mice per group. (D) Representative μCT images of knee joints and (E) the quantification of subchondral bone volume (BV/TV, %) in indicated groups of mice. (F) Western blot detection of 1α(OH)ase in articular cartilage isolated from 6- and 12-month-old WT mice (n=5 mice in each group). Kidney tissue from 6-month-old WT mice (n=2) were used as a positive control. (G) Quantification of 1α(OH)ase relative protein levels. *: p< 0.05, **: p< 0.01, ***: p<0.001.

Figure 2

Supplementation of 1,25(OH)₂D₃ rescues the knee OA phenotype caused by 1,25(OH)₂D deficiency. Representative images of articular cartilage stained for (A) safranin O and immunostained for (C) collagen X, (E) Mmp13 and (G) p16 in 12-month-old wild-type mice on the rescue diet (RD) (n=4), 1α(OH)ase^-/- mice on the RD (n=4), and 1α(OH)ase^-/- mice treated with 1,25(OH)₂D₃ (n=5). Quantification of (B) OARSI grade, (D) collagen X⁺ cells, (F) Mmp13⁺ cells and (H) p16⁺ cells. (I) Quantification of mRNA levels for SASP including p16, p21, IL-1β, IL-6 and Mmp13. **: p< 0.01, ***: p<0.001.

Figure 3

1,25(OH)₂D₃ rescues the reduction of human chondrocyte proliferation and the increases of oxidative stress and cellular senescence induced by IL-1β. Human articular chondrocytes were cultured in vitro in the presence or absence of IL-1β with or without 1,25(OH)₂D₃ and stained (A) with crystal violet, (D) immunocytochemically for EdU, (F) with DHE and (H) cytochemically for SA-β-gal. n = 3 wells per condition. (B) Clonal expansion analysis. (C) CCK8 assay showing cell viability. (E) EdU-positive cells. (G) ROS relative levels (relative intensity of DHE fluorescence analyzed by Image J). (I) SA-β-gal positive cells. (J) Western blot detection of the cellular senescence marker p16 and (K) the quantification of protein levels of p16. n = 3 wells per condition. *: p< 0.05, **: p< 0.01, ***: p<0.001.

Figure 4

VDR deficiency induces chondrocyte senescence and a knee OA phenotype. (A) Representative micrographs of articular cartilage sections of 6-month-old wild-type mice (n=5) immunostained for VDR using two sources of VDR antibody (Abcam 3508 and Santa Cluz D6). (B) Western blot detection of VDR in monolayer-cultured wild-type mouse chondrocytes. Proteins isolated from wild-type BM-BMSCs and VDR^-/- chondrocytes were loaded as positive and negative control, respectively. (C) Marked increase of Cyp24a1 and VDR mRNA levels in 1,25(OH)₂D₃-treated wild-type mouse chondrocytes, but not in VDR^-/- cells demonstrated by RT-PCR. GAPDH was used as a loading control. (D) Immunofluorescence staining for VDR in mouse chondrocytes after treatment with vehicle or 1,25(OH)₂D₃ for 24 h. 1,25(OH)₂D₃-treated cells showed an increased localization of VDR in the nucleus compared with vehicle-treated cells. (E-H) Representative images of sections from wild-type (n=3) and VDR^-/- (n=3) chondrocyte micromass cultures stained with (E) safranin O and immunostained for (F) collagen II, (G) Mmp13 and (H) p16 (scale bars, 200 µm). (I) Relative mRNA levels of col2a1, aggrecan, Mmp13 and p16 in macromass cultures above. n=3 wells per group. Representative micrographs of sections from 6-month-old wild-type (n=4) and VDR^-/- (n=4) mice on the rescue diet stained (J) with safranin O and immunostained for (K) Mmp13, (L) collagen X and (M) p16. (N) Statistical analysis of Mmp13⁺, collagen X⁺ and p16⁺ cells. (O) Relative mRNA levels of p16, IL-1β, IL-6, Mmp13, aggrecan and col2a1 in wild-type and VDR^-/- cartilage. *: p< 0.05, **: p< 0.01, ***: p<0.001.

Figure 5

1,25(OH)₂D₃ suppresses IL-1β-induced chondrocyte senescence by VDR-mediated transcriptional up-regulation of Sirt1. (A) Western blot detection of Sirt1 in articular chondrocytes isolated from WT (n=3) and VDR^-/- (n=3) mice. (B) Western blot detection of Sirt1 in human articular chondrocyte cultures treated with IL-1β alone or combined with 1,25(OH)₂D₃ for indicated times. (C) Relative Sirt1 mRNA levels in human articular chondrocytes cultured with 1,25(OH)₂D₃ for 3, 6 and 12 hours. (D) Upper: VDRE-like elements in human Sirt1 promoter region highlighted in red. Lower: Chromatin immunoprecipitation (ChIP) with IgG antibody, H3 antibody (positive control) or VDR antibody were performed in human articular chondrocytes and relative enrichment of human Sirt1 promoter was determined using RT-PCR assay. (E) Human Sirt1 promoter or Sirt1 promoter mutant Luc-plasmid were transfected into human articular chondrocytes followed by vehicle or 1,25(OH)₂D₃ treatment for 12 hours and (F) relative luciferase activity was analyzed after 48 hours. n=3 replicates per condition. Human articular chondrocytes were cultured with vehicle, 1,25(OH)₂D₃, or 1,25(OH)₂D₃ plus Ex527 (a Sirt1 inhibitor) in the presence of IL-1β and stained (G) with dihydroethidium (DHE) to determine relative ROS levels, (I) cytochemically for EdU and (K) cytochemically for SA-β-gal. n=3 replicates per condition. Statistical analysis of (H) DHE fluorescence intensity, (J) EdU-positive cells and (L) SA-β-gal-positive cells. (M) Protein expression levels of p16 in human articular chondrocytes treated with vehicle or the Sirt1 inhibitor (Ex527) in the presence or absence of IL-1β and 1,25(OH)₂D₃. n=3 wells per condition. (N) Quantification of (M). *: p< 0.05, ***: p<0.001.

Figure 6

Overexpression of Sirt1 in MSCs prevents 1,25(OH)₂D deficiency-induced development of knee OA. (A) Serum calcium, phosphorus, intact PTH and 1,25(OH)₂D₃ levels in 12-month-old wild-type (n=5), 1ɑ(OH)ase^-/- (n=5) and 1ɑ(OH)ase^-/-Sirt1^Tg mice (n=5) on the rescue diet (RD). (B) Representative micrographs of articular cartilage sections immunostained for Sirt1 in indicated groups of mice and (C) a quantitative analysis of the percentage of Sirt1-positive chondrocytes. Representative micrographs of knee sections stained with (D) safranin O, and immunostained for (F) collagen X, (H) Mmp13 and (J) p16. Evaluation for (E) OARSI score and quantification of (G) collagen X⁺, (I) Mmp13⁺ and (K) p16⁺ cells. (L) Relative mRNA levels of SASP factors including IL-1ɑ, IL-1β, IL-6, Mmp3, Mmp13 were analyzed using qPCR. n=5 mice per group. *: p< 0.05, **: p< 0.01, ***: p<0.001.

Figure 7

Model of mechanisms leading from 1,25(OH)₂D₃ to prevent age-related spontaneous knee osteoarthritis via VDR-mediated upregulation of Sirt1.