Trolox prevents osteoclastogenesis by suppressing RANKL expression and signaling

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.

FIGURE 1.

Effects of Trolox on IL-1-induced osteoclast formation in cocultures. A, mouse bone marrow cells and primary osteoblasts were cocultured in the presence of IL-1 (10 ng/ml) with or without Trolox (500 μm) and RANKL (100 ng/ml) for 6 days. After culturing, the generated osteoclasts were detected by TRAP staining, and TRAP-positive multinucleated cells containing three or more nuclei were counted as osteoclasts (^*, p < 0.01). B, primary osteoblasts were pretreated with Trolox or vehicle (dimethyl sulfoxide) for 24 h and were then stimulated with IL-1 (10 ng/ml) for 24 h. After culturing, total RNA was isolated, and the expression of mRNA for RANKL and OPG was analyzed by real time quantitative PCR with HPRT mRNA as an endogenous control. C, osteoblasts were treated as in B. The amounts of RANKL (^*, p < 0.01 versus untreated control; #, p < 0.01 versus group treated with IL-1 only) and OPG (^*, p < 0.01 versus untreated control) were determined by using enzyme-linked immunosorbent assay kits in cell lysates and in cell culture media, respectively.

FIGURE 2.

Effects of Trolox on IL-1-induced PGE₂ synthesis in osteoblasts. A, primary osteoblasts were pretreated with Trolox (500 μm) or vehicle (dimethyl sulfoxide) for 24 h and were then stimulated with IL-1 (10 ng/ml) for the indicated times. B, osteoblasts pretreated with Trolox (100–500 μm) were stimulated with IL-1 (10 ng/ml) for 24 h. The PGE₂ concentration (^*, p < 0.01 versus untreated control; #, p < 0.01 versus group treated with IL-1 only) in the culture medium was determined by enzyme immunoassay. C, primary osteoblasts were treated with or without IL-1 (10 ng/ml), Trolox (500 μm), and PGE₂ (1 nm) for 24 h. Expression of mRNA for RANKL and OPG was analyzed by real time PCR with HPRT mRNA as an endogenous control. D, osteoblasts were treated as in A. Western blotting was performed with the indicated antibodies. E, human recombinant COX-2 was incubated with the indicated Trolox doses, and the COX-2 activity assay was performed as described under “Experimental Procedures” (^*, p < 0.01 versus untreated control).

FIGURE 3.

Effects of Trolox on RANKL-induced osteoclast formation in BMMs. A, BMMs were cultured in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) with or without Trolox for 4 days. After culturing, cells were fixed, and the number of TRAP-positive multinucleated cells was counted (^*, p < 0.01 versus untreated control). B, BMMs were cultured for 48 h with M-CSF (30 ng/ml) and RANKL (100 ng/ml) at the indicated doses of Trolox. Then cell viability was determined by use of the XTT assay. C, as in A, except the cells were treated with 500 μm Trolox on the indicated days (^*, p < 0.01 versus untreated control). D, BMMs were treated with (panels ii and iii) or with out (panel i) Trolox at the beginning of the culture period, and Trolox was continued (panel ii) or withdrawn (panel iii) on day 2. E, cocultured osteoclasts were replated on OAAS plates as described under “Experimental Procedures,” incubated with or without Trolox (100 and 500 μm) for 1 h, and then further cultured in the presence of RANKL (100 ng/ml) for 24 h. Resorbed pits were photographed (left), and the pit areas were analyzed (right) after osteoclasts were removed. F, purified mature osteoclasts from the coculture were incubated with Trolox (100 and 500 μm) for 1 h were then and further cultured in the presence of RANKL (100 ng/ml) for 24 h. After that, surviving osteoclasts were detected by TRAP staining (left), and the number of osteoclasts was counted (right).

FIGURE 4.

Effects of Trolox on RANKL-induced early signaling in BMMs. A and B, BMMs were pretreated with Trolox for 3 or 24 h in the presence of M-CSF (30 ng/ml) and were then stimulated with RANKL (100 ng/ml) for the indicated time points. Whole-cell lysates were subjected to Western blotting with the indicated antibodies. C, BMMs were pretreated with Trolox or vehicle (dimethyl sulfoxide) for 24 h in the presence of M-CSF (30 ng/ml) and were then stimulated with RANKL (100 ng/ml) for 15 min. Cells were harvested, and nuclear extracts were prepared. The DNA binding activity of NF-κB was assessed by electrophoretic mobility shift assay. N.S., nonspecific band.

FIGURE 5.

Effects of Trolox on the RANKL-induced expression of c-Fos and NFAT2 in BMMs. A and B, BMMs were pretreated with Trolox or vehicle (dimethyl sulfoxide) in the presence of M-CSF for 24 h and were then stimulated with RANKL (100 ng/ml) for the indicated times. C and D, BMMs were pretreated with Trolox (100–500 μm) or vehicle for 24 h and were then stimulated with RANKL for 24 h. A and C, expression of mRNA for c-Fos and NFAT2 was analyzed by real time PCR using HPRT mRNA as an endogenous control. B and D, Western blotting was performed with the indicated antibodies. Actin served as an internal control.

FIGURE 6.

Trolox-induced inhibition of c-Fos translation in BMMs. A, BMMs were pretreated with or without Trolox in the presence of M-CSF for 24 h and were then stimulated with RANKL (100 ng/ml). After 20 h, 1 g/ml cycloheximide (CHX) and 7 μm MG132 (MG) were added to the cultures for 4 h before harvest. c-Fos protein levels were detected by Western blotting. B, BMMs were pretreated with or without Trolox for 24 h and were then stimulated with RANKL (100 ng/ml) for 23 h. The cells then were metabolically radiolabeled with l-[³⁵S]methionine/cysteine for 1 h and collected after a 30-min chase time. The cell lysates were subjected to immunoprecipitation with anti-c-Fos antibody, and the immunoprecipitates were resolved by SDS-PAGE and detected by autoradiography. C, BMMs were infected with retroviruses expressing pMX-IRES-EGFP (GFP-vector), pMX-c-Fos-EGFP (c-Fos), and pMX-CA-NFAT2-EGFP (CA-NFAT2). Infected cells were cultured with or without Trolox in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 4 days. After culturing, the cells were fixed; the ectopic expression of each construct was detected by a fluorescence microscope (upper), and the cells were stained for TRAP. TRAP-positive multinucleated osteoclasts were counted (lower;^*, p < 0.01 versus untreated control).

FIGURE 7.

Effects of Trolox on IL-1-induced bone destruction in vivo. A–C, a collagen sponge treated with vehicle (PBS) or IL-1 (1.5 μg) was implanted over mouse calvaria. Trolox (60 mg/kg of body weight, intraperitoneal) or vehicle (dimethyl sulfoxide) was administered daily. The mice were killed 7 days after implantation. A, TRAP staining of whole calvaria. B, three-dimensional images of calvarial bone by micro-CT analysis. C, TRAP staining of histological sections of calvarial bone, with hematoxylin counterstaining.