There is compelling evidence for a strong proresorptive function of TNF, which has long been implicated in the pathogenesis of bone loss and inflammation in a variety of common bone diseases (1
) and, more recently, cherubism (46
). TNF induces bone loss by indirect and direct mechanisms, including promotion of RANKL expression by accessory cells (4
) and induction of OCP proliferation and differentiation (47
). These cells can then secrete proinflammatory osteoclastogenic cytokines and thus lead to the initiation of autocrine and paracrine self-amplifying cycles that increase bone loss (48
). A pivotal role of TNF in pathologic bone loss is evidenced by the efficacy of anti-TNF therapy to limit disease progression in approximately 70% of patients with RA (49
) and to reduce bone erosion soon after menopause (2
Despite this evidence, TNF does not induce osteoclast formation when administered in vivo to Rankl–/–
), although it can induce osteoclastogenesis directly from Rank–/–
OCPs in vitro when costimulated with TGF-β (18
), leading some to suggest that prior priming of OCPs by RANKL (35
) or IL-1 expression by stromal cells (50
) is necessary for TNF-induced bone resorption. Here we explain these discrepant findings by showing that locally injected TNF fails to induce osteoclastogenesis in Rankl–/–
mice because of the inhibitory effects NF-κB p100 and provide conclusive evidence that TNF can induce osteoclastogenesis in these mice when they are deficient also in NF-κB p100.
The osteoclastogenesis induced locally by TNF in calvariae of Rank–/–/Nfkb2–/–
mice was associated with systemic induction of small numbers of mainly mononuclear TRAP+
cells along epiphyseal growth plates. We have reported that RANKL induced by BMP2 in hypertrophic chondrocytes at the growth plate attracts OCPs to this site to remove bone (51
), which is formed rapidly and must be removed to prevent development of osteopetrosis during development. Our findings suggest that the administered TNF directed OCPs to this site independently of RANKL. However, further studies are required to determine whether this was by a direct or indirect mechanism and whether TNF induces expression of chemokines here as it also does to attract circulating OCPs to inflamed joints (52
Our observation that TRAP+ cells do not form in untreated Rank–/–/Nfkb2–/– or Rankl–/–/Nfkb2–/– mice is important because it suggests that TNF levels at the growth plate during endochondral ossification are too low to induce OCP differentiation to osteoclasts and that TNF does not have an important positive or negative regulatory role in this physiologic process. However, TRAP5b released from osteoclasts induced by locally injected TNF resulted in slightly increased serum TRAP5b levels in Rank–/–/Nfkb2+/– mice and significantly increased levels in Rank–/–/Nfkb2–/– mice, supporting our conclusion that TNF-induced resorption in pathologic bone remodeling is attenuated by NF-κB2. The inhibitory role for NF-κB p100 in osteoclastogenesis is further supported by the development of erosive arthritis and systemic bone loss in TNF-Tg/Nfkb2–/– mice much earlier than TNF-Tg/Nfkb2+/– littermates.
The reports that TNF induces RANKL expression in the joints of TNF-Tg mice (53
) led us to consider whether NF-κB p100 also limits RANKL-induced resorption in TNF-Tg mice. Indeed, we found that TNF limits RANKL-induced differentiation of WT OCPs in vitro through induced NF-κB p100 and that retroviral expression of NF-κB2 in OCPs reduced RANKL-induced osteoclastogenesis. Interestingly, inhibition of osteoclastogenesis alone does not prevent TNF-induced synovial inflammation, since TNF-Tg/c-Fos–/–
hybrid mice lack osteoclasts and joint destruction but still have synovial inflammation (55
). Therefore, inhibition of osteoclasts alone is likely to have a limited role in the treatment of RA. Unexpectedly and importantly, we found that the TNF-Tg/Nfkb2–/–
mice also had significantly increased inflammation in their joints, indicating that NF-κB p100 limits not only OCP differentiation but also the number of inflammatory cells attracted to the joints of the mice in response to TNF.
Deficiency of Nfkb2 dramatically accelerated TNF-Tg–induced arthritic bone erosion and inflammation, but this was not associated with an increase in their serum concentrations of either human or murine TNF, suggesting that deficiency of NF-κB p100 could be associated with more severe joint inflammation in RA patients. This might be of great importance in the clinical setting of arthritis, but it will require further study to determine whether there are variations in the transcription, function, or degradation of p100 that could increase the susceptibility of RA patients to TNF. Similarly, further study is also required to determine the precise mechanism whereby p100 limits the inflammatory infiltration, and specifically whether NF-κB p100 can be further increased locally, for example by using local adenoviral gene delivery, to inhibit inflammation and bone resorption in these mice.
TNF stimulates Nfkb2
mRNA expression through the canonical NF-κB p65/p50 pathway to increase the total amount of NF-κB p100 transcripts and protein (24
). At the same time, TNF activates the noncanonical pathway leading to some processing of NF-κB p100 to NF-κB p52 (24
), but the molecular mechanisms have not been identified. We found that TNF stabilized TRAF3, resulting in its accumulation in OCPs in parallel with p100 levels, a finding that, to our knowledge, has not been reported previously in any cell type. This could account for limited NF-κB p100 degradation by NIK because TRAF3 induces degradation of NIK in B cells (26
). These effects of TNF differ from those of RANKL, which does not increase TRAF3 protein levels and activates NIK to induce efficient processing of p100 to p52 (23
). TRAF3 negatively regulates p100 processing to p52 by promoting proteasomal degradation of NIK through its physical association with the TRAF3 binding motif in NIK (25
). Loss of TRAF3 results in accumulation of NIK and constitutive p100 processing in TRAF3–/–
B cells (25
). TRAF6, which mediates RANKL/RANK-activated canonical NF-κB signaling, cannot associate with NIK directly and therefore does not inhibit it (56
TRAF3 siRNA reduced TRAF3 protein levels in WT OCPs associated with increased NIK levels and p100 processing, which released more RelB to go to nuclei in the cells and increased osteoclastogenesis. TNF induced only a slight increase NIK levels in the OCPs, but this in part may reflect the difficulty in detecting low levels of NIK in cells with currently available reagents (57
) and the fact that most published studies were able to detect changes in NIK levels only when NIK was overexpressed (26
). Although some investigators have suggested that TRAF2 is required for TNF-induced osteoclastogenesis (14
) and TRAF5 is involved in both RANKL- and TNF-induced osteoclastogenesis (15
), others have found that TRAF2- or TRAF5-deficient OCPs can differentiate into mature osteoclasts in response to TNF (18
). TRAF2 functions along with TRAF3 in B cells to degrade NIK (26
). However, TRAF2, -5, and -6 do not appear to play a major role in p100 processing in OCPs, based on our observation that their protein levels are similar in TNF- and RANKL-treated cells. Currently, we do not know the precise molecular mechanism by which TNF increases TRAF3 protein levels in OCPs. TNF does not affect TRAF3 mRNA, but it clearly prevents its degradation. It will be important to work this out in further studies, since stabilization of TRAF3 could potentially be one mechanism to limit bone resorption and possibly inflammation in inflammatory arthritis.
Our findings, coupled with the fact that TNF, RANKL, and IL-1 can all induce TNF expression by OCPs (40
), support an important direct role for TNF in osteoclastogenesis in conditions such as RA and postmenopausal osteoporosis, in which production of these cytokines is increased beyond physiologic levels (58
), while physiologic levels of the cytokines are unable to induce osteoclastogenesis in Rankl–/–
mice. However, they also identify an important negative regulatory role for TNF to limit its effects and those of RANKL. Given the important role that TNF has in inducing inflammation and bone destruction in many common bone diseases, we propose that this negative regulatory role for TNF in limiting bone resorption and inflammation might be harnessed to help reduce the high morbidity associated with many common diseases in which its expression is increased. These might include increasing the stability or expression of p100 or TRAF3 and inhibiting NIK in OCPs.