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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 activation of NFATc1 in osteoclast differentiation is dependent on TRAF6.(A). RAW264.7 cells were treated with TRAIL, M-CSF (20 ng/ml) and RANKL (50 ng/ml) in the presence or absence of TRAF6 siRNA, TRAIL receptor (TRAIL-R) siRNA, anti-TRAIL-R antagonist Ab, or OPG as indicated in the figure. After stimulation, cells lysates of the nuclear fraction were prepared, and immunoblotted with anti-NFATc1 and anti-HDAC antibodies. The results are representative of three separate experiments. (B).TRAF6 is ubiquitinated after TRAIL stimulation. Ubiquitin transfected RAW 264.7 cells were treated with TRAIL in the presence or absence of TRAF6 siRNA, TRAIL-R siRNA, anti-TRAIL-R antagonist Ab, or OPG as indicated in the figure. The cell lyses were immunoprecipitated with anti-TRAF6 mAb. Bound proteins were subjected to SDS–PAGE and immunoblotted with anti-Ubiquitin mAb, and then the membrane was stripped and reprobed with anti-TRAF6 mAb. The results are representative of three separate experiments.
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pone-0038048-g004: TRAIL-induced activation of NFATc1 in osteoclast differentiation is dependent on TRAF6.(A). RAW264.7 cells were treated with TRAIL, M-CSF (20 ng/ml) and RANKL (50 ng/ml) in the presence or absence of TRAF6 siRNA, TRAIL receptor (TRAIL-R) siRNA, anti-TRAIL-R antagonist Ab, or OPG as indicated in the figure. After stimulation, cells lysates of the nuclear fraction were prepared, and immunoblotted with anti-NFATc1 and anti-HDAC antibodies. The results are representative of three separate experiments. (B).TRAF6 is ubiquitinated after TRAIL stimulation. Ubiquitin transfected RAW 264.7 cells were treated with TRAIL in the presence or absence of TRAF6 siRNA, TRAIL-R siRNA, anti-TRAIL-R antagonist Ab, or OPG as indicated in the figure. The cell lyses were immunoprecipitated with anti-TRAF6 mAb. Bound proteins were subjected to SDS–PAGE and immunoblotted with anti-Ubiquitin mAb, and then the membrane was stripped and reprobed with anti-TRAF6 mAb. The results are representative of three separate experiments.

Mentions: RANKL induces activation of the TRAF6 and c-Fos pathways, which lead to the osteoclast-specific event of autoamplification of nuclear factor of activated T cells (NFAT)c1, the master transcription factor for osteoclast differentiation [29], [30], [31]. However, it is not clear whether TRAIL induces activation of NFATc1 in osteoclastogenesis; or if TRAF6 is essential for activation of NFATc1 in TRAIL-induced osteoclast differentiation. For further support TRAIL induces osteoclast differentiation through direct engagement with TRAIL receptor and to transduce signaling via TRAF6, we used the TRAIL receptor (TRAIL-R) siRNA to knock down the expression of TRAIL-R and also used the anti-TRAIL-R antagonist Ab (R&D System, Inc., Minneapolis, MN) to neutralize the interaction between TRAIL and its receptor. The results in Figure 4 demonstrated that TRAIL-induced activation of NFATc1 was significantly inhibited by transfection of TRAF6 siRNA, TRAIL-R siRNA, or treatment with anti-TRAIL-R antagonist Ab, indicating that TRAIL-induced NFATc1 activation and osteoclast differentiation activity is dependent on TRAF6 associated signaling (Figure 4).


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 activation of NFATc1 in osteoclast differentiation is dependent on TRAF6.(A). RAW264.7 cells were treated with TRAIL, M-CSF (20 ng/ml) and RANKL (50 ng/ml) in the presence or absence of TRAF6 siRNA, TRAIL receptor (TRAIL-R) siRNA, anti-TRAIL-R antagonist Ab, or OPG as indicated in the figure. After stimulation, cells lysates of the nuclear fraction were prepared, and immunoblotted with anti-NFATc1 and anti-HDAC antibodies. The results are representative of three separate experiments. (B).TRAF6 is ubiquitinated after TRAIL stimulation. Ubiquitin transfected RAW 264.7 cells were treated with TRAIL in the presence or absence of TRAF6 siRNA, TRAIL-R siRNA, anti-TRAIL-R antagonist Ab, or OPG as indicated in the figure. The cell lyses were immunoprecipitated with anti-TRAF6 mAb. Bound proteins were subjected to SDS–PAGE and immunoblotted with anti-Ubiquitin mAb, and then the membrane was stripped and reprobed with anti-TRAF6 mAb. The results are representative of three separate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375273&req=5

pone-0038048-g004: TRAIL-induced activation of NFATc1 in osteoclast differentiation is dependent on TRAF6.(A). RAW264.7 cells were treated with TRAIL, M-CSF (20 ng/ml) and RANKL (50 ng/ml) in the presence or absence of TRAF6 siRNA, TRAIL receptor (TRAIL-R) siRNA, anti-TRAIL-R antagonist Ab, or OPG as indicated in the figure. After stimulation, cells lysates of the nuclear fraction were prepared, and immunoblotted with anti-NFATc1 and anti-HDAC antibodies. The results are representative of three separate experiments. (B).TRAF6 is ubiquitinated after TRAIL stimulation. Ubiquitin transfected RAW 264.7 cells were treated with TRAIL in the presence or absence of TRAF6 siRNA, TRAIL-R siRNA, anti-TRAIL-R antagonist Ab, or OPG as indicated in the figure. The cell lyses were immunoprecipitated with anti-TRAF6 mAb. Bound proteins were subjected to SDS–PAGE and immunoblotted with anti-Ubiquitin mAb, and then the membrane was stripped and reprobed with anti-TRAF6 mAb. The results are representative of three separate experiments.
Mentions: RANKL induces activation of the TRAF6 and c-Fos pathways, which lead to the osteoclast-specific event of autoamplification of nuclear factor of activated T cells (NFAT)c1, the master transcription factor for osteoclast differentiation [29], [30], [31]. However, it is not clear whether TRAIL induces activation of NFATc1 in osteoclastogenesis; or if TRAF6 is essential for activation of NFATc1 in TRAIL-induced osteoclast differentiation. For further support TRAIL induces osteoclast differentiation through direct engagement with TRAIL receptor and to transduce signaling via TRAF6, we used the TRAIL receptor (TRAIL-R) siRNA to knock down the expression of TRAIL-R and also used the anti-TRAIL-R antagonist Ab (R&D System, Inc., Minneapolis, MN) to neutralize the interaction between TRAIL and its receptor. The results in Figure 4 demonstrated that TRAIL-induced activation of NFATc1 was significantly inhibited by transfection of TRAF6 siRNA, TRAIL-R siRNA, or treatment with anti-TRAIL-R antagonist Ab, indicating that TRAIL-induced NFATc1 activation and osteoclast differentiation activity is dependent on TRAF6 associated signaling (Figure 4).

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