Abstract
Excessive receptor activator of NF-kappaB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. Thus, down-regulation of RANKL expression or its downstream signals may be a therapeutic approach to the treatment of pathological bone loss. In this study, we investigated the effects of Trolox, a water-soluble vitamin E analogue, on osteoclastogenesis and RANKL signaling. Trolox potently inhibited interleukin-1-induced osteoclast formation in bone marrow cell-osteoblast coculture by abrogating RANKL induction in osteoblasts. This RANKL reduction was attributed to the reduced production of prostaglandin E(2) via a down-regulation of cyclooxygenase-2 activity. We also found that Trolox inhibited osteoclast formation from bone marrow macrophages induced by macrophage colony-stimulating factor plus RANKL in a reversible manner. Trolox was effective only when present during the early stage of culture, which implies that it targets early osteoclast precursors. Pretreatment with Trolox did not affect RANKL-induced early signaling pathways, including MAPKs, NF-kappaB, and Akt. We found that Trolox down-regulated the induction by RANKL of c-Fos protein by suppressing its translation. Ectopic overexpression of c-Fos rescued the inhibition of osteoclastogenesis by Trolox in bone marrow macrophages. Trolox also suppressed interleukin-1-induced osteoclast formation and bone loss in mouse calvarial bone. Taken together, our findings indicate that Trolox prevents osteoclast formation and bone loss by inhibiting both RANKL induction in osteoblasts and c-Fos expression in osteoclast precursors.
Highlights
Stimulation of RANKL in osteoclast precursors induces a dramatic up-regulation of c-Fos, which is a member of the AP-1 transcription factor family, and c-Fos-deficient mice fail to form osteoclasts, with an elevated number of bone marrow macrophages (BMMs), and develop osteopetrosis [13, 14]
Mice were intraperitoneally ways involved in IL-1-induced RANKL induction and the administered with the vehicle or Trolox effects of Trolox on these pathways in osteoblasts
Increased the secretion of PGE2 into the culture medium of osteo- Trolox Does Not Alter the Activation of MAPKs, AKT, and blasts, which was significantly inhibited by Trolox in a time- and NF-B Induced by RANKL—macrophage colony-stimulating factor (M-CSF) is required for the survival, dose-dependent manner (Fig. 2, A and B)
Summary
Reagents and Materials—Penicillin, streptomycin, ␣-MEM, and fetal bovine serum were purchased from Invitrogen. Mouse bone marrow cells were obtained from femurs and tibias of 5-week-old ICR mice and incubated in ␣-MEM complete media containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin on 10-cm culture dishes in the presence of M-CSF (10 ng/ml) overnight. Bone marrow cells (3 ϫ 105 cells/well) and primary osteoblasts were cocultured in 48-well (1 ml/well) tissue culture plates for 6 days in ␣-MEM complete medium. BMMs (1 ϫ 104 cells/well) were cultured with Trolox at various concentrations for 48 h in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) on 96-well plates (200 l/well). The survival assay was performed as follows; mature osteoclasts were incubated with various concentrations of Trolox for 1 h and further cultured in the presence of RANKL (100 ng/ml). All animal experiments were reviewed and approved by the Seoul National
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