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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 differentiation is dependent on TRAF6.(A) Human peripheral blood mononuclear cells (PBMCs) were plated in 96-well plates at 1.5 × 105 cells/well, and the next day the adherent monocytes were treated 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 in the figure for 30 days. The transfection of siRNAs into human macrophages cells was performed by electroporation. The TRAF6 inhibitory peptide, T6PD and its control peptide were used to examine its effects on TRAIL-mediated osteoclast differentiation. After incubation, cells were subjected to the TRAP assay. Cell morphology was examined by light microscopy, and the number of TRAP-positive multinuclear cells was quantified. The bar in each figure represents 100 µM. (B). TRAIL-induced formation of osteoclast-like multinucleated cells from human monocytes. Data in (B) represent the mean±SD from three to six independent experiments. *p < 0.05, **p < 0.005.
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pone-0038048-g001: TRAIL induced osteoclast differentiation is dependent on TRAF6.(A) Human peripheral blood mononuclear cells (PBMCs) were plated in 96-well plates at 1.5 × 105 cells/well, and the next day the adherent monocytes were treated 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 in the figure for 30 days. The transfection of siRNAs into human macrophages cells was performed by electroporation. The TRAF6 inhibitory peptide, T6PD and its control peptide were used to examine its effects on TRAIL-mediated osteoclast differentiation. After incubation, cells were subjected to the TRAP assay. Cell morphology was examined by light microscopy, and the number of TRAP-positive multinuclear cells was quantified. The bar in each figure represents 100 µM. (B). TRAIL-induced formation of osteoclast-like multinucleated cells from human monocytes. Data in (B) represent the mean±SD from three to six independent experiments. *p < 0.05, **p < 0.005.

Mentions: To determine role of TRAF6 –dependent signaling in TRAIL-induced osteoclast differentiation, we first investigated the influence of TRAF6 in TRAIL-induced osteoclast differentiation in human preosteoclastic precursors. For this purpose, human PBMCs were allowed to adhere for 3∼5 days. The adherent PBMCs were positive for the monocytic/macrophagic markers CD14, CD36, and CD64 at this time point (data not shown). Monocyte precursors can differentiate into osteoclast-like cells in the presence of RANKL (100 ng/ml) and M-CSF (50 ng/ml) or TRAIL (500 ng/ml). This differentiation was verified not only by the appearance of multinuclear cells but also by the positive staining with TRAP. The formation of large, multinucleated TRAP+ cells was observed, characterized by the appearance of TRAP-positive giant multinucleated cells with more than three nuclei. In our previous study, TRAIL in this does range did not induce apoptosis; instead, TRAIL induced osteoclast differentiation in preosteoclastic precursors [14] (Figure S1). We then examined whether the TRAIL-induced osteoclast differentiation activity is dependent on TRAF6 signaling pathway after TRAIL engagement. The monocyte precursors were treated 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, TRAF6 decoy peptide (T6DP) or osteoprotegerin (OPG). In addition to being an inhibitor of RANKL, OPG is also a decoy receptor for the cytotoxic ligand TRAIL [25]. OPG can bind to TRAIL to neutralize the osteoclast differentiation effects induced by TRAIL [14]. In Figure 1, our results demonstrated that both RANKL- and TRAIL-induced osteoclast differentiation ability was significantly inhibited after silencing the expression of TRAF6 by using TRAF6 siRNA, indicating that TRAIL-induced osteoclast differentiation activity is dependent on TRAF6 (Figure 1). Both RANKL- and TRAIL-induced osteoclast differentiation was also significantly reduced when treated with OPG.


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 differentiation is dependent on TRAF6.(A) Human peripheral blood mononuclear cells (PBMCs) were plated in 96-well plates at 1.5 × 105 cells/well, and the next day the adherent monocytes were treated 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 in the figure for 30 days. The transfection of siRNAs into human macrophages cells was performed by electroporation. The TRAF6 inhibitory peptide, T6PD and its control peptide were used to examine its effects on TRAIL-mediated osteoclast differentiation. After incubation, cells were subjected to the TRAP assay. Cell morphology was examined by light microscopy, and the number of TRAP-positive multinuclear cells was quantified. The bar in each figure represents 100 µM. (B). TRAIL-induced formation of osteoclast-like multinucleated cells from human monocytes. Data in (B) represent the mean±SD from three to six independent experiments. *p < 0.05, **p < 0.005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375273&req=5

pone-0038048-g001: TRAIL induced osteoclast differentiation is dependent on TRAF6.(A) Human peripheral blood mononuclear cells (PBMCs) were plated in 96-well plates at 1.5 × 105 cells/well, and the next day the adherent monocytes were treated 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 in the figure for 30 days. The transfection of siRNAs into human macrophages cells was performed by electroporation. The TRAF6 inhibitory peptide, T6PD and its control peptide were used to examine its effects on TRAIL-mediated osteoclast differentiation. After incubation, cells were subjected to the TRAP assay. Cell morphology was examined by light microscopy, and the number of TRAP-positive multinuclear cells was quantified. The bar in each figure represents 100 µM. (B). TRAIL-induced formation of osteoclast-like multinucleated cells from human monocytes. Data in (B) represent the mean±SD from three to six independent experiments. *p < 0.05, **p < 0.005.
Mentions: To determine role of TRAF6 –dependent signaling in TRAIL-induced osteoclast differentiation, we first investigated the influence of TRAF6 in TRAIL-induced osteoclast differentiation in human preosteoclastic precursors. For this purpose, human PBMCs were allowed to adhere for 3∼5 days. The adherent PBMCs were positive for the monocytic/macrophagic markers CD14, CD36, and CD64 at this time point (data not shown). Monocyte precursors can differentiate into osteoclast-like cells in the presence of RANKL (100 ng/ml) and M-CSF (50 ng/ml) or TRAIL (500 ng/ml). This differentiation was verified not only by the appearance of multinuclear cells but also by the positive staining with TRAP. The formation of large, multinucleated TRAP+ cells was observed, characterized by the appearance of TRAP-positive giant multinucleated cells with more than three nuclei. In our previous study, TRAIL in this does range did not induce apoptosis; instead, TRAIL induced osteoclast differentiation in preosteoclastic precursors [14] (Figure S1). We then examined whether the TRAIL-induced osteoclast differentiation activity is dependent on TRAF6 signaling pathway after TRAIL engagement. The monocyte precursors were treated 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, TRAF6 decoy peptide (T6DP) or osteoprotegerin (OPG). In addition to being an inhibitor of RANKL, OPG is also a decoy receptor for the cytotoxic ligand TRAIL [25]. OPG can bind to TRAIL to neutralize the osteoclast differentiation effects induced by TRAIL [14]. In Figure 1, our results demonstrated that both RANKL- and TRAIL-induced osteoclast differentiation ability was significantly inhibited after silencing the expression of TRAF6 by using TRAF6 siRNA, indicating that TRAIL-induced osteoclast differentiation activity is dependent on TRAF6 (Figure 1). Both RANKL- and TRAIL-induced osteoclast differentiation was also significantly reduced when treated with OPG.

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