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. 2017 Jun 16;292(24):10169-10179.
doi: 10.1074/jbc.M116.771816. Epub 2017 Apr 24.

RANKL cytokine enhances TNF-induced osteoclastogenesis independently of TNF receptor associated factor (TRAF) 6 by degrading TRAF3 in osteoclast precursors

Zhenqiang Yao  1   2 , Wei Lei  3   4 , Rong Duan  3 , Yanyun Li  3 , Lu Luo  3 , Brendan F Boyce  5   2
Affiliations

RANKL cytokine enhances TNF-induced osteoclastogenesis independently of TNF receptor associated factor (TRAF) 6 by degrading TRAF3 in osteoclast precursors

Zhenqiang Yao et al. J Biol Chem. .

Abstract

Cytokines, including receptor activator of nuclear factor κB ligand (RANKL) and TNF, induce increased osteoclast (OC) formation and bone loss in postmenopausal osteoporosis and inflammatory arthritides. RANKL and TNF can independently induce OC formation in vitro from WT OC precursors via TNF receptor-associated factor (TRAF) adaptor proteins, which bind to their receptors. Of these, only TRAF6 is required for RANKL-induced osteoclastogenesis in vitro However, the molecular mechanisms involved remain incompletely understood. Here we report that RANKL induced the formation of bone-resorbing OCs from TRAF6-/- OC precursors when cultured on bone slices but not on plastic. The mechanisms involved increased TNF production by TRAF6-/- OC precursors resulting from their interaction with bone matrix and release of active TGFβ from the resorbed bone, coupled with RANKL-induced autophagolysosomal degradation of TRAF3, a known inhibitor of OC formation. Consistent with these findings, RANKL enhanced TNF-induced OC formation from TRAF6-/- OC precursors. Moreover, TNF induced significantly more OCs from mice with TRAF3 conditionally deleted in myeloid lineage cells, and it did not inhibit RANKL-induced OC formation from these cells. TRAF6-/- OC precursors that overexpressed TRAF3 or were treated with the autophagolysosome inhibitor chloroquine formed significantly fewer OCs in response to TNF alone or in combination with RANKL. We conclude that RANKL can enhance TNF-induced OC formation independently of TRAF6 by degrading TRAF3. These findings suggest that preventing TRAF3 degradation with drugs like chloroquine could reduce excessive OC formation in diseases in which bone resorption is increased in response to elevated production of these cytokines.

Keywords: RANKL; TGF-β; TNF; TNF receptor-associated factor (TRAF); bone; osteoclast.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
TNF and TNF+RANKL induce OC formation from TRAF6−/− OCPs cultured on plastic. A, representative sections of tibiae from 12-day-old WT (+/+) and TRAF6 KO (−/−) mice stained for TRAP activity (red staining). OC numbers (Oc.N) in the tibial secondary ossification centers and metaphyses were counted. **, p < 0.01 for −/− mice (n = 7) versus +/+ mice (n = 5). B, 2 × 105 spleen cells from 12-day-old +/+ and −/− mice were treated with M-CSF for 2 days to recruit OCPs, followed by treatment with TNF (T, 20 ng/ml) or RANKL (R, 10 ng/ml) for 3 days in 96-well plates. TRAP+ OC numbers and areas (Oc.Ar) were counted. **, p < 0.01 versus the respective RANKL-treated cells; ##, p < 0.01 versus the respective TNF-treated cells; n = 4/group. C, OCPs were cultured with TNF and M-CSF for 10 days on bone slices in 96-well plates. The bone slices were fixed with 10% formalin, and the cells on them were brushed off followed by 0.1% toluidine blue staining. Resorption pit areas were counted. All in vitro experiments were repeated at least twice with similar results.
Figure 2.
Figure 2.
RANKL induces OC formation from TRAF6−/− OCPs cultured on bone slices. 2 × 105 spleen cells from 7- to 11-day-old WT (+/+) and TRAF6−/− mice were seeded on bone slices in 96-well plates and cultured with RANKL+M-CSF for 9 days. A, TRAP+ OCs are shown on bone slices (left panel) and on the plastic around the bone slices (right panel) in the same well. TRAP-positive osteoclast numbers (Oc.N) and areas (Oc.Ar) on the bone slices and on the plastic around the slices were counted and expressed per slice or per well after removal of the slices. B, the bone slices were brushed to remove cells and stained with 0.1% toluidine blue to highlight resorption pits (left panel). Pit areas were counted and normalized to slice area. **, p < 0.01 versus values for WT cells. All experiments were repeated at least twice with similar results.
Figure 3.
Figure 3.
Either TNF or TGFβ1 mediates RANKL-induced OC formation from TRAF6−/− OCPs on bone. A, TRAF6−/− spleen cells were cultured with TNF, IL-1 (10 ng/ml), or TGFβ1 (2 ng/ml) or with each of them in combination with RANKL plus M-CSF in 96-well plastic plates for 5 days to generate OCs. TRAP+ OCs were counted after TRAP staining. **, p < 0.01 versus cells treated with TNF alone. Oc.N, OC number. B, spleen cells from 11-day-old WT and TRAF6−/− mice were seeded on bone slices in 96-well plates and cultured with RANKL and M-CSF plus the TNF inhibitor TNFR:Fc, an IL-I receptor antagonist (IL-1Ra), or a TGFβ-neutralizing Ab for 5 days. TRAP+ OCs were counted on bone slices after TRAP staining. **, p < 0.01 versus PBS-treated culture. C, 2 × 105 spleen cells from 11-day-old WT and TRAF6−/− mice were seeded in 96-well plates on plastic or on bone slices and treated with PBS (P) or RANKL (R) plus M-CSF for 5 days. Levels of TNF (left panel) and the active form of TGFβ (right panel) in the culture media were tested by ELISA. **, p < 0.01 versus the respective level in medium from cells cultured on plastic (n = 3/group); #, p < 0.05; N.S, no significant difference between the two groups. D, the lower extremities of 10-day-old TRAF6−/− and WT mice were homogenized in 1 ml of tissue protein extraction reagent containing a proteasome inhibitor mixture. 50 μg of tissue lysate protein from each sample was used to test the levels of TNF (left panel) and the active form of TGFβ1 (right panel) by ELISA (n = 4). All experiments were repeated at least twice with similar results.
Figure 4.
Figure 4.
TRAF3 induced by TNF in OCPs is degraded by RANKL to enhance OC differentiation independently of TRAF6. A, spleen cells from 11-day-old WT and TRAF6−/− mice were cultured with M-CSF in 60-mm dishes for 3 days to recruit OCPs, which were then treated with the indicated cytokines for 8 h. Cell lysates were subjected to Western blot analysis of TRAF3, TRAF6, and β-actin. B, the autophagolysosomal inhibitor CQ or PBS was added to cultures similar to those in A along with cytokines for 8 h. Protein levels of TRAF3 and β-actin in the cell lysates were analyzed by Western blotting. C, 2 × 105 WT or TRAF6−/− spleen cells were cultured with M-CSF for 2 days in 96-well plates to recruit OCPs, followed by treatment with the indicated cytokines and CQ or PBS for an additional 3 days to generate OCs. OC numbers (Oc.N) were counted after TRAP staining (bottom panel). **, p < 0.01 between vehicle- and CQ-treated cells from WT and TRAF6−/− mice. All experiments were repeated at least twice with similar results. T, TNF, 20 ng/ml; R, RANKL, 10 ng/ml; Tβ1, TGFβ1, 1 ng/ml.
Figure 5.
Figure 5.
TRAF6−/− OCs forms actin ring similar to WT OCs. 7-day-old WT and TRAF6−/− spleen cells were cultured with M-CSF plus the indicated cytokines for 5 days when mature OCs were observed under inverted microscopy. The cells were fixed with 10% neutral formalin for 20 min and stained with Alexa Fluor 488 phalloidin for 30 min with DAPI counterstaining. Representative images of phalloidin- and DAPI-stained and merged images are shown. Arrows indicate actin rings in the cell membrane of OCs.
Figure 6.
Figure 6.
TRAF3 limits OC differentiation in the absence of TRAF6. A, BM cells from 2-month-old TRAF3f/f-LysMcre (TRAF3 conditional knock out in myeloid cells, −/−) mice and their WT (+/+) littermates were cultured with RANKL (R), TNF (T), TGFβ1 (Tβ1), or combinations of them in the presence of M-CSF for 5 days. TRAP staining was performed to evaluate OC numbers (Oc.N) and area (Oc.Ar). *, p < 0.05; **, p < 0.01 between cKO and WT cells. Western blotting was performed on cell lysates from PBS- and TNF-treated WT and TRAF3f/f-LysMcre OCPs and shows the absence of TRAF3 in the cells from TRAF3f/f-LysMcre (bottom right panel). B, 2 × 105 WT or TRAF6−/− spleen cells were cultured in 96-well plates with M-CSF for 2 days and then infected with GFP or TRAF3 retroviral supernatant. 1 day after infection, cytokines were added to the cultures for 3 days to generate OCs. OC numbers were counted after TRAP staining (top right panel). *, p < 0.05 versus its respective level in GFP-infected cells. TRAF3 protein levels were assessed in OCPs infected with GFP or TRAF3 retroviruses by Western blotting (bottom right panel) to confirm overexpression of TRAF3. All experiments were repeated at least twice with similar results. T, TNF, 20 ng/ml; R, RANKL, 10 ng/ml; Tβ1, TGFβ1, 1 ng/ml.
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