NF-κB p100 limits TNF-induced bone resorption in mice by a TRAF3-dependent mechanism (original) (raw)
TNF induces expression of noncanonical NF-κB proteins differently from RANKL in OCPs. To investigate the possibility that TNF induces fewer osteoclasts from OCPs than RANKL by promoting accumulation of the inhibitory NF-κB p100 protein, we examined the expression pattern of p100 in WT OCPs. Both RANKL and TNF increased p100/p52 mRNA levels within 4 hours of treatment by 11- and 7-fold, respectively, and these remained elevated by 7- and 5-fold, respectively, at 8 hours (data not shown). We examined p100 and p52 protein expression levels during our typical culture period (1–96 hours). TNF induced sustained accumulation of p100 between 4 and 72 hours (Figure 1A). In contrast, RANKL more efficiently processed p100 to p52 during this period (Figure 1A), and this was associated with the formation of larger numbers of osteoclasts (Figure 1B). p100 levels in TNF-treated cells were not different from those of PBS-treated controls at 96 hours, by which time osteoclast formation has peaked in these cultures (11). TNF also induced increased p52 levels, but to a lesser extent than RANKL between 8 and 48 hours, confirming that p100 is processed in response to TNF (24), especially at 72 and 96 hours, when TNF induces peak osteoclast formation (11). Both TNF and RANKL markedly increased RelB levels, but the effect of TNF was greater than that of RANKL from 48 to 96 hours (Figure 1A). Deficiency of p100 did not affect either TNF- or RANKL-mediated nuclear translocation of RelB (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI38716DS1), presumably because RelB also can associate with p50 and translocate into nuclei (36). In contrast, TNF and RANKL overall had similar stimulatory effects on the expression levels of the canonical NF-κB p50 and p65 proteins during osteoclastogenesis (24–96 hours), with some variation at each time point (Figure 1A). Of note, although both TNF and RANKL mediated rapid NF-κB p65 nuclear translocation, the effect of TNF was more sustained than that of RANKL (Supplemental Figure 1, B and C). We had observed this previously in OCPs from NF-κB1/2 double-knockout mice that do not form osteoclasts (11).
TNF-induced expression of NF-κB p100 inhibits osteoclastogenesis. (A) WT mouse OCPs, cultured from splenocytes with M-CSF for 3 days, were treated with RANKL or TNF for the indicated times. NF-κB proteins in whole-cell lysates were determined by Western blot. Experiments were repeated at least twice with similar results. P, PBS; R, RANKL 10 ng/ml; T, TNF 20 ng/ml. (B) WT or Nfkb2–/– OCPs were treated with RANKL or TNF directly on plastic or bone slices in 96-well plates in the presence of M-CSF for 2 and 5 days, respectively, to induce osteoclasts (OCs) and resorption pits. Top: Representative TRAP-stained osteoclasts (original magnification, ×4) and toluidine blue–stained pits (original magnification, ×20). Bottom: Osteoclast number and resorption pit area (n = 4/group; *P < 0.05 vs RANKL). (C) Nfkb2–/– or WT OCPs were infected with GFP, p100, or p52 retroviruses for 2 days and treated with TNF for 2 more days. Osteoclast numbers were counted (left panel; *P < 0.05 versus GFP), and the infection efficiency was confirmed by Western blot from the infected WT OCPs (right panels). (D) Murine TNF (0.5 μg in 10 μl PBS) or 10 μl PBS were injected twice daily over the calvariae of 4-week-old Nfkb2–/– or Nfkb2+/– control mice for 5 days (n = 4/group). The number of osteoclasts/mm bone surface, percentage of osteoclast surface/bone surface, and percentage of eroded surface/bone surface were measured in TRAP-stained calvarial bone sections, and serum TRAP5b was tested with ELISA.
TNF-induced NF-κB p100 limits osteoclastogenesis. To determine whether TNF-induced NF-κB p100 limits the number of osteoclasts formed in response to TNF, we treated Nfkb2–/– and WT OCPs with TNF and found significantly more osteoclasts from Nfkb2–/– than from WT cells, the numbers being similar to those in RANKL-treated WT or Nfkb2–/– cells (Figure 1B). This effect was not associated with induction in vitro or in vivo in OCPs of IFN-β (data not shown), which limits RANKL-induced osteoclastogenesis by downregulating c-Fos expression in OCPs (37). Importantly, osteoclasts induced by TNF from Nfkb2–/– OCPs formed resorption pits as effectively as those induced by RANKL (Figure 1B). TNF activates c-Fos and NFATc1, the same critical transcriptional factors activated by RANKL to induce osteoclast differentiation, but TNF induces these to a lesser extent than RANKL when mature osteoclasts are formed (11). However, we found that TNF induced a fold induction of c-Fos and NFATc1 similar to that of RANKL when osteoclasts were forming in Nfkb2–/– cells (data not shown). Erk and p38 signaling are also involved in osteoclast differentiation and are activated in response to RANKL and TNF (38). We found that TNF induced stronger activation of both Erk and p38 than RANKL, and this was observed in both WT and Nfkb2–/– cells (data not shown).
Nfkb2–/– OCPs lack both p100 and p52. To determine whether p100 or p52 is responsible for the inhibitory effect of TNF, we infected Nfkb2–/– and WT OCPs with p100, p52, or GFP control retrovirus and treated them with TNF. p100 inhibited TNF- and RANKL-induced osteoclastogenesis significantly in both Nfkb2–/– and WT OCPs (Figure 1C). p52 also caused a small but significant reduction in osteoclast numbers (Figure 1C), which may be due to a minor inhibitory effect of p52 homodimers, but this effect was much less than that of p100. High expression of these retrovirally induced proteins was confirmed in infected WT OCPs (Figure 1C).
To determine whether p100 limits TNF-induced osteoclastogenesis in vivo, we injected TNF or PBS vehicle into the supra-calvarial subcutaneous tissues of _Nfkb2_–/– and Nfkb2+/– control mice twice daily for 5 days and examined its effects on osteoclastogenesis and resorption. Basal osteoclast numbers as well as osteoclast surface and eroded surface were similar in _Nfkb2_–/– and Nfkb2+/– mice (Figure 1D). Mean values for these parameters of bone resorption increased significantly in the Nfkb2+/– and _Nfkb2_–/– mice in response to TNF, but the increase was significantly greater in the _Nfkb2_–/– mice (Figure 1D). Serum levels of tartrate-resistant acid phosphatase 5b (TRAP5b), a specific marker of bone resorption released by osteoclasts, were significantly higher in _Nfkb2_–/– mice than in control mice treated with TNF (7.3 ± 0.5 vs. 5.4 ± 0.8 U/l; P < 0.01), confirming that Nfkb2 deficiency enhances TNF-induced bone resorption. We also found that basal TRAP5b levels were higher in _Nfkb2_–/– mice than in their littermate controls (6.2 ± 0.1 vs 3.6 ± 0.4 U/l; P < 0.01), which suggests that osteoclasts in _Nfkb2_–/– mice are more active than WT osteoclasts, as we did not observe any difference in the number of osteoclasts at this time point. This effect differs from those we reported previously with IL-1, which induced similarly increased numbers of osteoclasts in Nfkb2+/– and _Nfkb2_–/– mice (39). IL-1 does not induce osteoclast formation from WT OCPs in the absence of RANKL but can do so when the cells overexpress c-Fos, which is activated by, and downstream of, NF-κB (40).
TNF induces osteoclastogenesis in Rank–/– or Rankl–/– mice in the absence of NF-κB2. TNF induces p100 expression through canonical NF-κB signaling (41). To test the hypothesis that p100 induced by TNF prevents osteoclast formation in vivo in Rank–/– mice, we first examined whether TNF increases the expression of NF-κB p100 protein in Rank–/– or Rankl–/– OCPs. Similar to its effects in WT cells, TNF induced p100 accumulation but not p52 protein in Rank–/– and Rankl–/– cells (Figure 2A), indicating that TNF-induced p100 protein expression is independent of RANK signaling. There was some variability in basal levels of p100 expression from one experiment to another, which is a recognized feature of currently available antibodies to this protein, but basal levels of expression overall did not vary significantly among WT, Rank–/–, and Rankl–/– OCPs. As expected, RANKL did not have any effect on p100/p52 protein in Rank–/– cells, while it clearly increased p52 in Rankl–/– cells (Figure 2A). Of note, different from WT cells, RANKL did not increase p100 levels in Rankl–/– OCPs, possibly because they had not encountered RANKL previously and therefore might be more sensitive to RANKL and completely process p100 to p52.
NF-κB2 deficiency enhances TNF-induced osteoclastogenesis in Rank–/– or Rankl–/– mice in vitro and in vivo. (A) NF-κB p100 and p52 were analyzed by Western blot in whole-cell lysates of PBS-, RANKL-, or TNF-treated (8 hours) OCPs from Rank–/– or Rankl–/– mice. (B) Left: OCPs from Rank–/–/Nfkb2–/– or Rankl–/–/Nfkb2–/– mice and their Nfkb2+/– littermates were treated with TNF for 2 days to evaluate osteoclast formation using TRAP staining (*P < 0.05 vs. Nfkb2+/–). Right: OCPs from Rank–/–/Nfkb2+/+ and Rankl–/–/Nfkb2+/+ mice were treated with RANKL or TNF for comparison with Rank–/–/Nfkb2+/– and Rankl–/–/Nfkb2+/– mice to determine the effects of haploinsufficiency of Nfkb2. (C) Murine TNF (0.5 μg in 10 μl PBS) or 10 μl PBS was injected twice daily over the calvariae of Rank–/–/Nfkb2–/– or Rankl–/–/Nfkb2–/– mice and Rank–/– or Rankl–/– littermates. Top: TRAP-stained sections show numerous actively resorbing TRAP+ osteoclasts locally in calvarial sections (original magnification, ×20) from TNF-injected Rank–/–/Nfkb2–/– or Rankl–/–/Nfkb2–/– mice. Bottom: Numbers and surface extent of osteoclasts (n = 3/genotype). Occasional osteoclasts induced by TNF from a Rank–/–/Nfkb2+/+ mouse are illustrated in the left panels. *P < 0.05 vs. single KO mice. (D) Left: Occasional binucleate (arrowhead), but mainly mononuclear (arrows), TRAP+ cells (left panel) formed beneath hypertrophic chondrocytes in the growth plate of the tibia of a Rank–/–/Nfkb2–/– mouse (original magnification, ×40), but not of Rank–/–/Nfkb2+/– littermates injected with TNF as described in C. Right: Osteoclast numbers (expressed per mm of length of growth plate) counted in representative sections. (E) Serum TRAP5b levels were tested with ELISA from TNF- or PBS-injected Rank–/–/Nfkb2–/– and Rank–/–/Nfkb2+/– mice (n = 3/group; *P < 0.05).
We next generated Rank–/–/Nfkb2–/– and Rankl–/–/Nfkb2–/– mice to determine whether TNF could induce osteoclastogenesis in the absence of NF-κB2 and either RANK or RANKL. First, we treated OCPs from Rank_–/–/Nfkb2_+/– and Rankl_–/–/Nfkb2_+/– mice with TNF and found that they formed slightly but significantly more osteoclasts than did OCPs from Nfkb2+/– littermates (Figure 2B). This likely reflects the fact that Rank–/– (42) and Rankl–/– (data not shown) mouse spleens contain more OCPs than do spleens of WT mice because of extramedullary hematopoiesis that results from their lack of an adequate marrow cavity. OCPs from Rank–/–/Nfkb2–/– and Rankl–/–/Nfkb2–/– mice treated with TNF formed significantly more osteoclasts than did OCPs from Rank_–/–/Nfkb2_+/– and Rankl_–/–/Nfkb2_+/– mice, the numbers being slightly higher than those from Nfkb2_–/– OCPs (Figure 2B). We also found that TNF induced the formation of bone-resorbing osteoclasts from Rank_–/–/Nfkb2+/– and Rankl_–/–/Nfkb2_+/– OCPs without the addition of TGF-β, which was suggested to be necessary as a pretreatment (18). A neutralizing TGF-β antibody did not prevent these effects, nor did the addition of TGF-β increase TNF-induced osteoclast numbers (data not shown). To exclude the possibility that haploinsufficiency of Nfkb2 affects the status of Rank–/– and Rankl–/– osteoclast differentiation, we treated spleen cells from Rank–/– and Rankl–/– mice with TNF in the presence of M-CSF and found that similar osteoclast numbers were formed as with OCPs from _Rank_–/– and _Rankl_–/– mice with Nfkb2 haploinsufficiency (Figure 2B, right panel). We believe that we were able to induce osteoclastogenesis in our cultures because we used lower numbers of M-CSF–dependent OCPs from these KO mice than we used from WT controls. The KO mice had higher numbers of OCPs in their spleens, as described above, and when we used similar numbers of OCPs from _Rankl_–/– or _Rank_–/– mice as from WT mice, TNF did not induce osteoclastogenesis (data not shown), presumably because their increased density inhibits differentiation.
Although absence of Nfkb2 itself did not induce any osteoclasts in Rank–/– or Rankl–/– mice (PBS injection; data not shown), TNF induced many osteoclasts and resorption lacunae in the calvarial bones of the Rank–/–/Nfkb2–/– and Rankl–/–/Nfkb2–/– mice following local injection, associated with increased osteoclast and eroded surfaces (Figure 2, C and D). Only occasional osteoclasts were observed in the sections of TNF-injected Rankl_–/– or Rank_–/– mice, as reported previously (34). We also observed small numbers of TRAP+ osteoclasts in the long bones of the TNF-injected Rank–/–/Nfkb2–/– and Rankl–/–/Nfkb2–/– mice, although in contrast to those formed in the calvariae, these were mainly mononuclear cells located predominantly along the edge of the growth plates (Figure 2D) or in the centers of the physes. These cells had no effect on the increased bone volume in these osteopetrotic mice, presumably reflecting the short period of 5-day administration and their small size. To assess the possible function of these cells further, we measured serum TRAP5b levels. TRAP5b was undetectable in Rank_–/–/Nfkb2+/–and Rank_–/–/Nfkb2_–/– mice and was slightly but significantly increased by TNF injection in Rank_–/–/Nfkb2+/–mice (0.61 ± 0.25 U/l). These values were increased further in Rank_–/–/Nfkb2_–/– mice after TNF injection (1.77 ± 0.34 U/l; Figure 2E), confirming that osteoclasts induced by TNF in Rank–/– mice are functional and that NF-κB2 deficiency enhances TNF-induced osteoclastogenesis and bone resorption in the mice lacking RANK signaling. TRAP5b was not observed in serum or osteoclasts in bone sections from vehicle-treated Rank–/–/Nfkb2–/– or Rankl–/–/Nfkb2–/– mice, presumably because the concentration of endogenous TNF in the marrow cavities of these mice is low and not sufficiently high to induce OCP differentiation even in the absence of p100.
TNF-Tg mice lacking NF-κB p100 have more severe joint erosion and inflammation and systemic bone loss than TNF-Tg mice. To determine whether the absence of NF-κB2 would enhance joint erosion in TNF-Tg mice, we generated TNF-Tg/Nfkb2–/– mice and found that they developed joint deformity earlier than their TNF-Tg littermates, that is, at 8 weeks versus 12 weeks of age (Figure 3A). At 12 weeks of age, TNF-Tg/Nfkb2–/– mice had significantly increased areas of inflammation and osteoclast numbers in their forepaw joints assessed histomorphometrically (Figure 3B). However, we observed no significant difference in the types of inflammatory cells or in the appearance of the hyperplastic synoviocytes in the TNF-Tg/Nfkb2–/– mice compared with control mice upon histologic analysis. FACS analysis showed that _Nfkb2_–/– mice had significantly reduced numbers of B220+ B cells in their spleens and peripheral blood compared with Nfkb2+/– control mice. This feature was also present in TNF-Tg/_Nfkb2_–/– mice. In contrast, there was no difference in numbers of CD3+, CD4+, or CD8+ cells among the Nfkb2+/–, _Nfkb2_–/–, TNF-Tg/Nfkb2+/–, and TNF-Tg/_Nfkb2_–/– mice (data not shown).
Increased joint inflammation and osteoclastogenesis in TNF-Tg_/Nfkb2–/–_ mice. (A) Age-related changes in clinically assessed joint deformation scores showed that joint deformation occurred earlier in the TNF-Tg_/Nfkb2–/–_ mice (n = 7) than in their TNF-Tg_/Nfkb2+/–_ littermates (n = 8). (B) Representative TRAP-stained sections (original magnification, ×20) from 12-week-old animals showed more severe wrist joint inflammation (green arrows) and more osteoclasts (yellow arrowheads) in a TNF-Tg_/Nfkb2–/–_ mouse than in a TNF-Tg_/Nfkb2+/–_ mouse. Histomorphometric analysis showed that the area of inflammatory tissue (upper panel) and osteoclast numbers (lower panel) were increased in the wrists of TNF-Tg/Nfkb2–/– mice. *P < 0.05. (C) The percentage of cartilage eroded surface/total joint surface was measured in carpal bones of 6-week-old mice (n = 5/group). (D) Serum levels of murine TNF (black bars) and human TNF (red bars) were tested with ELISA at 6 and 12 weeks of age (*P < 0.05 vs. TNF-Tg/Nfkb+/– littermates).
TNF-Tg/Nfkb2_–/– mice also had reduced long bone trabecular bone volume and cortical thickness compared with TNF-Tg/Nfkb2+/–_ mice (Figure 4A), which we confirmed morphometrically by 3-dimensional μCT imaging (Figure 4B). These TNF-Tg_/Nfkb2–/–_ mice had lost almost all of their metaphyseal trabecular bone (Figure 4A), making it difficult to quantify and normalize osteoclast parameters histomorphometrically. However, in 6-week-old TNF-Tg_/Nfkb2–/–_ mice, which had slightly but not significantly lower trabecular bone volume/tissue volume ratios than TNF-Tg mice, metaphyseal osteoclast numbers and surfaces were increased significantly (Figure 4C). These mice also had enhanced erosion of cartilage on their carpal bone joint surfaces compared with TNF-Tg/Nfkb2+/– littermates (Figure 3C). Bone formation rates assessed in undecalcified bone sections following double calcein labeling were similar in the TNF-Tg/Nfkb2–/– and control mice (data not shown).
More severe systemic bone loss in TNF-Tg/Nfkb2–/– mice. (A) Tibiae from 12-week-old Nfkb+/– (n = 4), Nfkb2–/– (n = 4), TNF-Tg/Nfkb2+/– (n = 7), and TNF-Tg/Nfkb2–/– mice (n = 8) were subjected to μCT scanning. Representative images (left) and data analysis (right) showed reduced trabecular bone volume and cortical bone thickness in TNF-Tg/Nfkb2–/– mice. (B) H&E-stained sections of tibiae (original magnification, ×2) showed decreased trabecular bone in the metaphyseal regions and thinner cortices (green arrow) in TNF-Tg/Nfkb2–/– mice. (C) TRAP-stained sections of 6-week-old mice showed increased numbers of osteoclasts in the secondary spongiosa of the proximal tibia of a TNF-Tg/Nfkb2–/– mouse (left panels), which was confirmed by histomorphometric analysis (right panels). BV/TV, bone volume/tissue volume. *P < 0.05 vs. TNF-Tg/Nfkb2+/– mice. Original magnification, ×2 (top), ×40 (bottom).
We next determined whether NF-κB2 deficiency influences human (Tg) or mouse serum TNF concentrations in these mice. Murine TNF values were similar among the groups in both 6- and 12-week-old mice. NF-κB2 deficienc0y did not affect human TNF concentrations in 6-week-old TNF-Tg mice, but mean values were significantly lower in TNF-Tg/Nfkb2–/– mice than in TNF-Tg/Nfkb2+/– littermates aged 12 weeks. We do not have an explanation for the reduction of TNF in these mice, but by this age TNF-Tg/_Nfkb2–/–_mice were much smaller than control mice, and some had already died. The cause of this early mortality requires further study.
TNF attenuates RANKL-induced osteoclastogenesis in vitro through NF-κB p100. Many cytokines, including RANKL, TNF, IL-1, and M-CSF, are involved in bone destruction in pathological conditions. Of these, RANKL and TNF can support the final stages of OCP differentiation to osteoclasts. Therefore, it is important to study how TNF and RANKL work together to control osteoclastogenesis. A previous study reported that TNF synergizes with RANKL to stimulate osteoclastogenesis in vitro (35) when both cytokines are added at the beginning of the culture period. However, the synergistic effect occurs only in RANKL pretreated cells (43), which we confirmed (data not shown). We then used a different osteoclastogenesis protocol in which spleen cells are first cultured with M-CSF for 3 days, to enrich for OCPs, and then TNF with or without RANKL was added to these cells for a further 2–3 days. TNF inhibited RANKL-induced osteoclastogenesis in a dose-dependent (0.8–20 ng/ml) manner (Figure 5A) at both optimal (10 ng/ml) and low (1 ng/ml) RANKL concentrations. Of note, although the number of osteoclasts induced by 1 ng/ml RANKL was just slightly less than the optimal dose, osteoclasts formed later and were smaller in these cultures, and this was evident as a reduced osteoclast area (Figure 5A). TNF alone (4 and 20 ng/ml) induced small numbers of osteoclasts in the absence of RANKL, as expected. In these experiments, NF-κB p100 levels in WT OCPs treated with TNF (20 ng/ml) plus RANKL (10 ng/ml) were significantly higher than in cells treated with RANKL alone, but the levels were less than those in cells treated with TNF alone, presumably because RANKL induced some proteasomal degradation of p100 through NIK, despite the inhibitory effect of TNF (Figure 5A). Accordingly, p52 expression was higher in the cultures of RANKL plus TNF than in those treated with TNF alone.
TNF-induced NF-κB p100 inhibits RANKL-induced osteoclastogenesis. (A) WT mouse spleen cells were cultured with M-CSF for 3 days, and RANKL and/or TNF were added at the indicated doses for 2 more days to generate osteoclasts. Top: TNF dose-dependently inhibited RANKL-induced osteoclastogenesis, assessed by osteoclast number (black bars) and area (red bars) per well. Bottom: The protein levels of NF-κB p100 and p52 were analyzed with Western blot and assessed in whole-cell lysates extracted from RANKL-treated (10 ng/ml) and/or TNF-treated (20 ng/ml) WT mouse OCPs at 8 hours. (B) The inhibitory effect of TNF on RANKL-induced osteoclastogenesis was abolished in Nfkb2–/– OCPs (*P < 0.05 vs. RANKL treatment alone; 4 wells/group). The same experiments were repeated at least twice with similar results.
We next treated WT and _Nfkb2_–/– OCPs with low but effective doses of TNF with or without RANKL and found that TNF (4 ng/ml) significantly reduced osteoclastogenesis induced by RANKL (1 ng/ml) in WT cultures but had no inhibitory effect in _Nfkb2_–/– cultures in which both cytokines had nearly maximal osteoclastogenic effects either alone or in combination (Figure 5B), confirming that NF-κB p100 mediates this TNF-induced inhibition of osteoclastogenesis.
TNF induction of NF-κB p100 and inhibition of osteoclastogenesis is mediated by TRAF3. To determine the mechanism whereby TNF-induced NF-κB p100 accumulation limits osteoclastogenesis, we examined the effects of TNF and RANKL on the expression of TRAFs in OCPs. TRAFs directly interact with TNF superfamily receptors and trigger intracellular signaling events, including NIK-mediated processing of p100 to p52 (7). In addition, TRAF2 and -3 can also mediate proteasomal degradation of signaling molecules (26, 27). We found that neither RANKL nor TNF affected the mRNA expression levels of TRAF3 or -6 (data not shown). However, both RANKL and TNF increased the protein levels of TRAF6, which activates NIK, and this is consistent with our observation that both cytokines slightly elevated NIK protein levels (Figure 6A). Importantly, TNF induced significantly higher levels of TRAF3, paralleling the elevated NF-κB p100 levels (Figure 6A). RANKL alone slightly reduced TRAF3, but it significantly decreased TNF-induced TRAF3 protein levels, which was matched by relatively lower levels of p100 (Figure 6A). Neither TNF nor RANKL affected protein levels of TRAF2 or -5 significantly in these cultures (data not shown).
TNF-induced TRAF3 negatively regulates osteoclastogenesis through NIK. (A) WT mouse OCPs were treated with RANKL and/or TNF for 8 hours, and whole-cell lysate protein was extracted and subjected to Western blotting for TRAF3, TRAF6, NIK, and NF-κB p100 and p52. (B) Cycloheximide (10 μM) was added to WT mouse OCPs treated with RANKL (10 ng/ml), TNF (20 ng/ml), or PBS control for the indicated times. Whole-cell lysates were subjected to Western blotting for TRAF3. (C) WT mouse OCPs were transfected with TRAF3 siRNA or a nonspecific control siRNA for 8 hours. The cells were treated with TNF (20 ng/ml) or PBS for an additional 8 hours. Whole-cell lysates were subjected to Western blotting to determine the levels of cytoplasmic TRAF3, NIK, and NF-κB p100 (left panel) and either nuclear p52 or cytoplasmic p100 and RelB (right panels). (D) TRAF3 siRNA–transfected cells were treated with TNF and/or RANKL (10 ng/ml) for 3 days in the presence of M-CSF to generate osteoclasts. *P < 0.05 vs. control).
To determine how TRAF3 is regulated by TNF, we added cycloheximide to the cultures of WT OCPs treated with PBS, RANKL, or TNF to prevent the synthesis of new protein and thus observe degradation of TRAF3 by Western blot. RANKL accelerated the degradation of TRAF3, starting at 1 hour and continuing through 4 hours (Figure 6B). In contrast, TNF prevented TRAF3 degradation, suggesting that TNF induction of TRAF3 results in the accumulation of NF-κB p100 and inhibition of osteoclastogenesis. To test this possibility, WT OCPs were transfected with TRAF3 siRNA to examine its effects on expression of NIK and p100 and on osteoclastogenesis. Transfection of TRAF3 siRNA reduced TRAF3 protein levels, and this was associated with higher levels of NIK and lower p100 levels in the TNF-treated cells compared with control siRNA-treated cells (Figure 6C). Surprisingly, we did not detect a significant change in p52 levels in either cytoplasmic or nuclear extracts using TRAF3 siRNA. This was a consistent finding that will require further study to determine whether TRAF3 also regulates p52 expression. TRAF3 siRNA significantly increased TNF-induced nuclear and cytoplasmic RelB, the typical partner of p52, which was reported recently to be involved in RANKL-induced osteoclastogenesis in vitro (44). Consistent with these results, TRAF3 siRNA significantly increased TNF-induced osteoclastogenesis (Figure 6D), although it had no effect on osteoclast numbers in RANKL-treated cultures, presumably because RANKL can degrade TRAF3 and TRAF3 siRNA likely would not have an additional effect on TRAF3 degradation. Of note, inhibition of TRAF3 attenuated the TNF-induced reduction of RANKL-mediated osteoclastogenesis.