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TRAF-6 dependent signaling pathway is essential for TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation.

Yen ML, Hsu PN, Liao HJ, Lee BH, Tsai HF - PLoS ONE (2012)

Bottom Line: In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation.Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway.This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of General Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling.

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TRAIL-induced osteoclast bone resorption activity is TRAF6-dependent.(A). Human monocytes were plated on an artificial bone matrix slide (calcium phosphate apatite-coated plate; OAAS) and were cultured with TRAIL (500 ng/ml) as well as M-CSF (50 ng/ml) and RANKL (100 ng/ml) in the presence or absence of TRAF6 siRNA, control siRNA (Crtl siRNA), TRAF6 decoy peptide (T6DP), control peptide (Crtl peptide), or osteoprotegerin (OPG) as indicated. Cells were detached after 30 days of culture. The number of resorption pits and resorptive area in each well was observed and counted using a microscope. (B). The resorption pits were photographed and the resorptive area in each well was counted. Data in (B) represent the mean±SD from five independent experiments. *p < 0.05, **p < 0.005.
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pone-0038048-g002: TRAIL-induced osteoclast bone resorption activity is TRAF6-dependent.(A). Human monocytes were plated on an artificial bone matrix slide (calcium phosphate apatite-coated plate; OAAS) and were cultured with TRAIL (500 ng/ml) as well as M-CSF (50 ng/ml) and RANKL (100 ng/ml) in the presence or absence of TRAF6 siRNA, control siRNA (Crtl siRNA), TRAF6 decoy peptide (T6DP), control peptide (Crtl peptide), or osteoprotegerin (OPG) as indicated. Cells were detached after 30 days of culture. The number of resorption pits and resorptive area in each well was observed and counted using a microscope. (B). The resorption pits were photographed and the resorptive area in each well was counted. Data in (B) represent the mean±SD from five independent experiments. *p < 0.05, **p < 0.005.

Mentions: To further determine whether TRAF6 signaling pathway is essential for TRAIL-induced osteoclast differentiation and bone resorption activity, we performed functional assays for identification of osteoclast differentiation in an in vitro culture system. Using commercial calcium phosphate apatite as a resorption substrate, pit formation caused by TRAIL-treated human macrophages was compared to cultures of cells in the presence or absence of TRAF6 siRNA and the TRAF6 decoy peptide, T6DP (Figure 2). Lacunar resorption was observed in human macrophages treated with TRAIL on calcium phosphate apatite plates; however, the bone resoprtion activity was significantly reduced after treatment with TRAF6 siRNA or the TRAF6 decoy peptide, T6DP (Figure 2). Taken together, our results clearly demonstrated that TRAF6 is essential for TRAIL-induced osteoclast differentiation and bone resorption activity.


TRAF-6 dependent signaling pathway is essential for TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation.

Yen ML, Hsu PN, Liao HJ, Lee BH, Tsai HF - PLoS ONE (2012)

TRAIL-induced osteoclast bone resorption activity is TRAF6-dependent.(A). Human monocytes were plated on an artificial bone matrix slide (calcium phosphate apatite-coated plate; OAAS) and were cultured with TRAIL (500 ng/ml) as well as M-CSF (50 ng/ml) and RANKL (100 ng/ml) in the presence or absence of TRAF6 siRNA, control siRNA (Crtl siRNA), TRAF6 decoy peptide (T6DP), control peptide (Crtl peptide), or osteoprotegerin (OPG) as indicated. Cells were detached after 30 days of culture. The number of resorption pits and resorptive area in each well was observed and counted using a microscope. (B). The resorption pits were photographed and the resorptive area in each well was counted. Data in (B) represent the mean±SD from five independent experiments. *p < 0.05, **p < 0.005.
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Related In: Results  -  Collection

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pone-0038048-g002: TRAIL-induced osteoclast bone resorption activity is TRAF6-dependent.(A). Human monocytes were plated on an artificial bone matrix slide (calcium phosphate apatite-coated plate; OAAS) and were cultured with TRAIL (500 ng/ml) as well as M-CSF (50 ng/ml) and RANKL (100 ng/ml) in the presence or absence of TRAF6 siRNA, control siRNA (Crtl siRNA), TRAF6 decoy peptide (T6DP), control peptide (Crtl peptide), or osteoprotegerin (OPG) as indicated. Cells were detached after 30 days of culture. The number of resorption pits and resorptive area in each well was observed and counted using a microscope. (B). The resorption pits were photographed and the resorptive area in each well was counted. Data in (B) represent the mean±SD from five independent experiments. *p < 0.05, **p < 0.005.
Mentions: To further determine whether TRAF6 signaling pathway is essential for TRAIL-induced osteoclast differentiation and bone resorption activity, we performed functional assays for identification of osteoclast differentiation in an in vitro culture system. Using commercial calcium phosphate apatite as a resorption substrate, pit formation caused by TRAIL-treated human macrophages was compared to cultures of cells in the presence or absence of TRAF6 siRNA and the TRAF6 decoy peptide, T6DP (Figure 2). Lacunar resorption was observed in human macrophages treated with TRAIL on calcium phosphate apatite plates; however, the bone resoprtion activity was significantly reduced after treatment with TRAF6 siRNA or the TRAF6 decoy peptide, T6DP (Figure 2). Taken together, our results clearly demonstrated that TRAF6 is essential for TRAIL-induced osteoclast differentiation and bone resorption activity.

Bottom Line: In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation.Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway.This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of General Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling.

Show MeSH
Related in: MedlinePlus