RANKL Cytokine Enhances Tnf-induced Osteoclastogenesis Independently Of TNF Receptor Associated Factor(TRAF)6 By Degrading TRAF3 In Osteoclast Precursors

BackgroundBone formation of normal bones is coupled with bone resorption,maintaining a dynamic balance.The imbalance of reconstruction of bone,increased bone resorption and/or reduced bone formation,can cause loss of bone mass,which is common in diseases such as osteoporosis,rheumatoid arthritis,bone metastases,aseptic loosening of joints and periodontitis.The enhancement of osteoclastic differentiation and function are responsible for bone destruction of these diseases.Currently,there is not a kind of drug that can inhibits bone resorption while promoting bone formation.Drugs that are resistant to bone resorption and enhanced to bone formation,especially those with dual effects of absorption and promotion,have great promise for the prevention and treatment of osteoporosis.Numerous studies have confirmed that cytokines,including receptor activator of nuclear factor-κB ligand（RANKL）and tumor necrosis factor（TNF）,induce increased osteoclast formation and bone loss in postmenopausal osteoporosis and inflammatory arthritid.RANKL and TNF can independently induce osteoclast formation in vitro from WT osteoclast precursors using 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.PurposeWe further investigated the role of TRAF6 and TRAF3 in TNF-and RANKL-induced OC formation and activation and examined if TRAF6 is involved in the degradation of TRAF3.Methods（1）The experimental animals we used were TRAF6-/-mice,which produced by TRAF6+/-mice hybridized with 129 mice.The number and area of the osteoclasts in secondary ossification centers and metaphysis osteoclasts in both TRAF6-/-mice and WT littermates mice were observed.（2）The OCPs,which were collected form 9-16 days-old of TRAF6-/-and WT littermates,were used for in vitro OC cultures.RANKL or TNF or both+/-different inhibitors were added,and the cells were cultured for 2-4 d when mature OCs typically are observed under inverted microscopy.We observed the number and area of the osteoclasts and bone resorption pits.（3）The OCPs,which were collected form 9-16 days old of TRAF6-/-and WT littermates,were used for in vitro OCs cultures.RANKL,TNF,RANKL+TNF,TNF+TGFβ1 or RANKL+TGFβ1 were added,followed by treatment with Chloroquine（CQ）or PBS,then observed the number and area of the osteoclasts;The TRAP3 levels of osteoclast precursors stimulated by different factors by Western Blot;The levels of TNF and TGFβ1 in the cell culture medium were detected by ELISA;T he levels of TNF and TGFβ1 in bone lysates of TRAF6-/-and WT littermates mice were detected by ELISA.To investigate if TRAF3 is a key inhibitor of TNF-induced OC formation in TRAF6-/-mice.The OCPs,which were collected form LyM^cre-TRAF3^f/and WT littermates mice,were used for in vitro OCs cultures,different stimulating factors were added,then observed the number and area of the osteoclasts.In addition,we infected TRAF6-/-and WT OCPs with either GFP or TRAF3 retroviral constructs,then added different stimulating factors and observed the number and area of the osteoclasts.（4）Each experiment was repeated three times.All results are given as the mean±S.D.Comparisons between two groups were analyzed using Student’s two-tailed unpaired t test and those among 3 or more groups using one-way analysis of variance and Dunnett’s post-hoc multiple comparisons.p values<0.05 were considered statistically significant.Results（1）We observed TRAP+multinucleated OCs on trabecular surfaces of this line of TRAF6-/-mice,although the numbers were⁴-fold lower than in WT mice;RANKL failed to induce any OCs from the TRAF6-/-cells on plastic plates.TNF induced OC formation from the TRAF6-/-spleen cells,and the number was similar to that derived from WT littermate cells.RANKL enhanced TNF-induced OC formation.（2）RANKL induced OC formation from the TRAF6-/-cells cultured on bone slices,although the numbers of OCs were 3-4-fold lower than from WT OCPs.OCs induced by RANKL from TRAF6-/-spleen cells were able to resorb bone,although the resorption pit area was much less than those from WT cells.（3）The TNF inhibitor and the TGFβAb,significantly reduced RANKL-induced OC formation from TRAF6-/-OCP on bone slices;TNF levels in the media from both knockout and WT OCPs cultured on bone slices with PBS were significantly higher than those from cells cultured on plastic,but the levels were not affected significantly by the addition of RANKL.Levels of the active form of TGFβin the conditioned medium in the RANKL-treated WT cells were higher than in PBS controls;TNF protein levels in the lysates of bone tissue from10-day-old TRAF6-/-and WT mice were similar and were around the lower limit of detection.The levels of active TGFβin bone samples from TRAF6-/-mice were significantly higher than those in bone from WT mice.（4）Basal levels of TRAF3 were similar in WT and-/-OCPs,and TNF increased TRAF3levels similarly in both cell types;RANKL reduced TRAF3 protein levels in WT OCPs;Addition of RANKL reduced the increase in TNF-induced TRAF3 levels in TRAF6-/-as well as in WT cells.CQ elevated basal TRAF3 levels and prevented the reduction in TRAF3 levels in response to RANKL to a similar extent in WT and TRAF6-/-OCPs pretreated with TNF,CQ can block Chloroquine could completely blocked osteoclastogenesis induced by TNF,TNF+TGFβ1 or RANKL+TNF from TRAF6-/-OCPs..（5）RANKL induced more OCs from TRAF3 cKO OCPs than from WT OCPs;TNF alone induced more OCs with a significantly higher area from LyMcre-TRAF3f/f OCPs than from WT littermate cells;Over-expression of TRAF3 significantly reduced OC formation induced by RANKL,TNF and RANKL+TNF from WT OCPs and it completely blocked osteoclastogenesis induced by TNF or RANKL+TNF from TRAF6-/-OCPs.Conclusion1.RANKL enhances OC formation and resorption induced by TNF from TRAF6-/-OCPs.2.TRAF6-/-OCPs form osteoclasts and bone resorption pits in response to RANKL through the effects of TNF and TGF.3.TRAF3 limits OC formation induced by TNF from TRAF6-/-OCPs.