Figure 1.
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.
Figure 2.
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.
Figure 3.
TRAIL induces activation of MAP Kinases is dependent on TRAF6.
RAW264.7 cells were treated with TRAIL (500 ng/ml), M-CSF (20 ng/ml) and RANKL (50 ng/ml) in the presence or absence of TRAF6 siRNA. After stimulation, cells were solubilized, and cell lysates were subjected to Western blot analysis of TRAF6, p38 MAPK, JNK, and ERK1/2. The trace shown in the top panel for each group indicates the immunoreactivity of the phosphorylated kinase. The same membrane was then stripped and reprobed with the kinase antibody recognizing the total protein level of kinase (bottom panel). The results are representative of five separate experiments.
Figure 4.
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.
Figure 5.
TRAIL-induced osteoclast differentiation was abolished in TRAF6 knock out bone marrow-derived macrophages.
(A) Bone marrow-derived macrophages isolated from TRAF6 knock out (TRAF6 KO) mice were treated with TRAIL (500 ng/ml) or RANKL (100 ng/ml) with M-CSF (200 ng/ml) for 7 days. 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. (B). TRAIL-induced formation of osteoclast-like multinucleated cells from bone marrow-derived macrophages isolated from wild type and TRAF6 KO mice. Data in (B) represent the mean±SD from three independent experiments. *p < 0.005.
Figure 6.
A model for TRAIL-induced activation of the TRAF6 associated signaling pathway in osteolcast differentiation.
Upon receptor engagement, in addition to transduce apoptosis signaling pathway, TRAIL induces activation of TRAF6, at least in part through the E3 ubiquitin ligase activity of the TRAF6 RING finger domain via a distinct signaling pathway. In osteoclast precursors, TRAIL is not able to induce sufficient amount of signals to activate the downstream apoptosis signaling to induce apoptosis; instead, TRAIL induces signaling to activate TRAF6 after receptor engagement. TRAF6 is the key molecule linking cytoplasmic signaling to the nuclear transcriptional program in osteoclast differentiation and bone resorption. These signals include activation of NF-κB by a process of ubiquitination in which TRAF6 functions as an E3 ligase [36] and recruitment of the TAB1-TAB2-TAK1 complex, which lead to the osteoclast-specific event, that is, autoamplification of NFATc1, the master transcription factor for osteoclast differentiation [29], [30], [31].