Vitamin E decreases bone mass by stimulating osteoclast fusion

Journal name:
Nature Medicine
Volume:
18,
Pages:
589–594
Year published:
DOI:
doi:10.1038/nm.2659
Received
Accepted
Published online
Corrected online

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption1, 2, 3. Osteoclasts are multinucleated cells that are formed by mononuclear preosteoclast fusion1, 2, 4, 5. Fat-soluble vitamins such as vitamin D are pivotal in maintaining skeletal integrity. However, the role of vitamin E in bone remodeling is unknown. Here, we show that mice deficient in α-tocopherol transfer protein (Ttpa−/− mice), a mouse model of genetic vitamin E deficiency6, have high bone mass as a result of a decrease in bone resorption. Cell-based assays indicated that α-tocopherol stimulated osteoclast fusion, independent of its antioxidant capacity, by inducing the expression of dendritic-cell–specific transmembrane protein, an essential molecule for osteoclast fusion, through activation of mitogen-activated protein kinase 14 (p38) and microphthalmia-associated transcription factor, as well as its direct recruitment to the Tm7sf4 (a gene encoding DC-STAMP) promoter7, 8, 9. Indeed, the bone abnormality seen in Ttpa−/− mice was rescued by a Tm7sf4 transgene. Moreover, wild-type mice or rats fed an α-tocopherol–supplemented diet, which contains a comparable amount of α-tocopherol to supplements consumed by many people, lost bone mass. These results show that serum vitamin E is a determinant of bone mass through its regulation of osteoclast fusion.

At a glance

Figures

  1. Serum vitamin E regulates bone resorption.
    Figure 1: Serum vitamin E regulates bone resorption.

    (ac) Serum α-tocopherol concentrations (a), histological analysis of the vertebrae (b) and micro computed tomography analysis (c) of the femurs of 3-month-old WT and Ttpa−/− mice. Von Kossa staining is shown in b. BV/TV, bone volume per tissue volume. Scale bars, 500 μm. An increase in bone volume in the Ttpa−/− mice can be seen. (d,e) Histomorphometric analysis (d) and serum deoxypyridinoline (dpd) measurement (e). Oc.S/BS, osteoclast surface area over bone surface area; N.Oc/B.Pm, osteoclast number over bone perimeter; MAR, mineral apposition rate; BFR/BS, bone formation rate over bone surface area; Ob.S/BS, osteoblast surface area over bone surface area. A decrease in bone resorption in the Ttpa−/− mice can be seen. (f,g) Serum α-tocopherol concentrations (f) and histological analysis (g) in Ttpa−/− mice fed a diet supplemented with α-tocopherol (α-toc diet). Von Kossa staining is shown in g. Scale bars, 500 μm. A decrease in bone volume as a result of the α-toc diet can be seen. (h) Serum α-tocopherol affects osteoclast differentiation. BMCs from the femurs of WT or Ttpa−/− mice were differentiated into osteoclasts in the presence of serum from WT or Ttpa−/− mice without addition of FBS. TRAP staining (left) and the number of osteoclasts (right) are shown. Scale bars, 50 μm. A decrease in the number of osteoclasts from WT BMCs with Ttpa−/− serum can be seen, whereas Ttpa−/− BMCs differentiated into osteoclasts normally with WT serum. *P < 0.05, **P < 0.01 by Tukey-Kramer testing (b) or Student's t test (a, cg). All data are means ± s.e.m.

  2. Vitamin E stimulates osteoclast fusion independent of its antioxidant activity.
    Figure 2: Vitamin E stimulates osteoclast fusion independent of its antioxidant activity.

    (ac) The effect of α-tocopherol on osteoclast differentiation, proliferation and apoptosis. (a) BMCs were cultured with M-CSF, RANKL and 10% FBS. TRAP-stained cells (left) and the number of cells with more than three nuclei (right) are shown. An increase in osteoclasts after α-tocopherol treatment can be seen. (b) BrdU assay. BMCs were cultured with M-CSF, 10% FBS and α-tocopherol. (c) TUNEL assay. BMCs were cultured with M-CSF, RANKL and 10% FBS. (d) The effect of α-tocopherol on osteoblasts. An alkaline phosphatase (Alp) assay is shown. p-NP, p-nitrophenol. (e) The effect of α-tocopherol on osteoclast fusion. BMCs were cultured with M-CSF and RANKL, and α-tocopherol was added in the proliferation (1), differentiation (2 and 3) or maturation (3 and 4) phase. An increase in the proportion of multinucleated osteoclasts (3 and 4) can be seen. (f) The effect of α-tocopherol on bone resorption. A pit formation assay is shown. The eroded area (arrows, left) and the number of pits (right) are shown. BMCs were cultured on dentin with M-CSF and RANKL. α-tocopherol was added later. An increase in bone resorption can be seen. (g) Giant-cell progenitors from bone marrow were treated with α-tocopherol. (h,i) BMCs were cultured with M-CSF and RANKL. Vitamin E isoforms and antioxidants were added later. *P < 0.05, **P < 0.01 by Tukey-Kramer testing (a) or Student's t test (f,h,i). Scale bars, 50 μm. All data are means ± s.e.m.

  3. [alpha]-tocopherol regulates osteoclast fusion through DC-STAMP.
    Figure 3: α-tocopherol regulates osteoclast fusion through DC-STAMP.

    (ac) Quantitative RT-PCR analyses. (a) The expression of osteoclast marker genes (left) and osteoclast-fusion–related genes (right) in α-tocopherol–treated osteoclasts. BMCs were cultured with M-CSF and RANKL. α-tocopherol was added to the culture media after the BMCs were seeded. (b) The expression of Tm7sf4 after treatment with various isoforms of vitamin E. (c) Expression of osteoclast-marker genes in WT and Ttpa−/− femurs. A decrease of the expression of Tm7sf4 among the osteoclast-fusion–related genes can be seen. (df) DC-STAMP is essential for α-tocopherol–induced osteoclast fusion. (d) Retroviral overexpression of DC-STAMP. An increase in osteoclast fusion by DC-STAMP in the absence of α-tocopherol can be seen. Retro-Tm7sf4, retroviral overexpression of Tm7sf4; retro-control, retroviral overexpression of control vector. (e,f) The effect of α-tocopherol on siRNA-treated BMCs (e) and Tm7sf4−/− BMCs (f). si-control, non-targeting siRNA; si-Tm7sf4, siRNA to Tm7sf4; si-Nfatc1, siRNA to Nfatc1. A decrease in osteoclast fusion even in the presence of α-tocopherol can be seen. BMCs from WT (d,e) and Tm7sf4−/− (f) mice were cultured with M-CSF and RANKL. α-tocopherol was added to culture media after the BMCs were seeded. Scale bars, 50 μm. (g) Histological analysis of the vertebrae from WT, Tm7sf4 transgenic (Tm7sf4 tg), Ttpa−/− and Ttpa−/−; Tm7sf4 tg mice. Scale bars, 500 μm. *P < 0.05, **P < 0.01 by Tukey-Kramer testing (a,b,g) or Student's t test (c). All data are means ± s.e.m.

  4. [alpha]-tocopherol decreases bone mass through p38[alpha] and Mitf.
    Figure 4: α-tocopherol decreases bone mass through p38α and Mitf.

    (a) Protein analysis of the α-tocopherol–treated osteoclasts. BMCs cultured with M-CSF only (left, middle) or mature osteoclasts that were induced by M-CSF and RANKL (right) were stimulated with α-tocopherol (20 μM) (– or +) and RANKL (middle right). Phospho-, phosphorylated; SAPK/JNK, mitogen-activated protein kinase 9 or mitogen-activated protein kinase 8; p44/42, mitogen-activated protein kinase 3 or cyclin-dependent kinase 20; Pkcd, protein kinase C, δ; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Akt, thymoma viral proto-oncogene 1; Pkd, protein kinase D; Plcg2, phospholipase C, γ 2; Nfatc1, nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1; c-Src, Rous sarcoma oncogene. (b) Immunoprecipitation analysis. An increase in the phosphorylation of p38α after treatment with α-tocopherol in p38α-expressing HEK293 cells can be seen. (c) Chromatin immunoprecipitation assay. Three potential binding sites (boxes 1–3) in the Tm7sf4 promoter are shown (above). An antibody against Mitf (anti-Mitf) specifically immunoprecipitated the region containing the box 1 site of the Tm7sf4 promoter. IgG, immunoglobulin G; DW, distilled water. (d,e) Gene knockdown (d) and retroviral overexpression (e) in osteoclasts. BMCs derived from WT mice were cultured with M-CSF and RANKL. α-tocopherol was added later. Scale bars, 50 μm. A decrease in osteoclast fusion even in the presence of α-tocopherol in BMCs treated with siRNA to Mapk14 (si-Mapk14) (encoding p38α) or siRNA to Mitf (si-Mitf) (d) and an increase in osteoclast fusion in the absence of α-tocopherol in constitutively active p38α (Mapk14CA)-expressing BMCs (e) can be seen. (fh) Analyses of WT mice and rats fed an α-tocopherol–supplemented diet. Serum α-tocopherol concentrations in these animals (f) and histological (g) and histomorphometric analyses (h). Scale bars, 500 μm. AA decrease in bone mass and an increase in bone resorption after α-tocopherol treatment can be seen. (i) Dual-energy X-ray absorptiometry analysis. A decrease in bone mineral density resulting from a α-tocopherol–supplemented diet. (j) The proposed mechanism of vitamin E (VitE)-induced osteoclastic fusion. P, phosphorylated. *P < 0.05, **P < 0.01 by Student's t test. All data are means ± s.e.m.

Change history

Corrected online 04 May 2012

In the version of this article initially published, it was incorrectly stated that the mice were fed a diet supplemented with α-tocopherol at 600 mg per kg of body weight. Instead, the food itself contained 600 mg of α-tocopherol per kg. The error has been corrected in the HTML and PDF versions of the article.

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Author information

Affiliations

  1. Department of Orthopedic Surgery, Tokyo Medical and Dental University, Tokyo, Japan.

    • Koji Fujita,
    • Makiko Iwasaki,
    • Chengshan Ma,
    • Kenichi Shinomiya &
    • Atsushi Okawa
  2. Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan.

    • Koji Fujita &
    • Chengshan Ma
  3. Section of Nephrology, Endocrinology and Metabolism, Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.

    • Hiroki Ochi,
    • Toru Fukuda,
    • Satoko Sunamura,
    • Hiroshi Itoh &
    • Shu Takeda
  4. Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan.

    • Takeshi Miyamoto
  5. Department of Pediatrics, Osaka Medical College, Osaka, Japan.

    • Kimitaka Takitani &
    • Hiroshi Tamai
  6. Department of Cell Signaling, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

    • Takako Negishi-Koga &
    • Hiroshi Takayanagi
  7. Laboratory for Systems Biology and Medicine, University of Tokyo, Tokyo, Japan.

    • Tatsuhiko Kodama
  8. Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan.

    • Shigeaki Kato
  9. Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan.

    • Hiroyuki Arai

Contributions

K.F. conducted most of the experiments. M.I., H.O. and C.M. conducted mice analyses. T.F. and S.S. conducted in vitro experiments. T.M. provided DC-STAMP–related mice. K.T. and H. Tamai conducted the analyses of vitamin E serum concentrations. T.N.-K. performed western blots. H.A. provided Ttpa−/− mice. T.K. and H. Takayanagi conducted gene expression analyses. S.T., K.S., A.O. and H.I. designed the project. S.T. supervised the project and wrote most of the manuscript. S.K. designed the project.

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The authors declare no competing financial interests.

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