TNF receptor-associated factors (TRAFs) −6 and −3 regulate RANKL and TNF signaling in osteoclast precursors (OCPs), but they can have opposing effects, and it is not known if their functions are inter-dependent. For example, TRAF6 is required for RANKL/RANK-induced osteoclastogenesis, while TRAF3 limits both RANKL- and TNF-induced osteoclastogenesis through proteasomal degradation of NF-κB-inducing kinase; and inhibition of autophagic degradation of TRAF3 by chloroquine prevents ovariectomy-induced osteoporosis. TRAF6−/− and TRAF3−/− mice die before or within ~2 weeks of birth, limiting in vivo studies. To further examine the relationship between these cytokines and TRAFs, we treated TRAF6−/− OCPs with RANKL, TNF, or RANKL+TNF on plastic. As expected, RANKL did not induce osteoclastogenesis, but TNF induced small numbers of osteoclasts (51±6/well), while RANKL significantly increased TNF-induced osteoclastogenesis (276±18/well). Surprisingly, RANKL induced functional osteoclasts from TRAF6−/− OCPs when they were cultured on bone slices (186±25/slice), which was inhibited by TNFR:Fc, but not by IL-1R antagonist or TGFβ1 neutralizing antibody. WT or TRAF6−/− OCPs cultured on bone slices secreted similar concentrations of TNF (169±43 and 151±17 pg/ml), which were significantly higher than those from cells cultured on plastic (26±4 and 19±2 pg/ml). TNF increased TRAF3 protein levels in TRAF6−/− OCPs, and addition of RANKL degraded TRAF3, while over-expression of TRAF3 inhibited osteoclastogenesis induced by RANKL+TNF. Finally, over-expression of NFATc1 significantly increased osteoclastogenesis induced by TNF in WT OCPs, but not by RANKL or by TNF in TRAF6−/− OCPs; however, it induced similar numbers of osteoclasts from TRAF6−/− and WT OCPs treated with RANKL+TNF (174±14 and 196±21/well). We conclude that interaction with bone matrix increases OCP expression of TNF, and that RANKL enhances TNF-induced osteoclastogenesis by promoting degradation of TRAF3 through a TRAF6-independent mechanism. Thus, strategies to increase TRAF3 levels in OCPs should inhibit RANKL- and TNF-induced osteoclastogenesis.
17 - 20 May 2014
European Calcified Tissue Society