Elucidation of molecular mechanisms of osteoclastogenesis and its application for bone disease therapy

RANK和CD 40通过TRAF 6将NF-κ B和MAPK激活至相似水平。尽管TRAF 6过表达导致破骨细胞形成，但RANK而不是CD 40促进破骨细胞生成。为了了解破骨细胞生成中RANK特异性活性的分子基础，我们创建了一个由抗人CD 40抗体介导的嵌合受体h40/mRK刺激驱动的破骨细胞形成系统，该受体由人CD 40的细胞外结构域和小鼠RANK的跨膜和胞质结构域组成。通过在RANK的三个TRAF 6结合位点中引入突变，我们发现具有单个TRAF 6结合位点的h40/mRK有效诱导Ca 2+振荡和NFATc 1（破骨细胞生成的主开关）的表达，而携带单个TRAF 6结合位点的CD 40则不然。然而，CD 40的表达比h40/mRK高约100倍，导致破骨细胞形成，表明RANK-TRAF 6信号在NFATc 1激活和破骨细胞生成方面比CD 40-TRAF 6信号更有效。这些结果表明，RANK可能具有放大TRAF 6信号传导的特定结构域。为了鉴定特定的结构域，我们在RANK的胞质结构域中引入了各种缺失。我们发现了一个缺失突变体，可以激活NF-κ B和MAPK，但不能产生破骨细胞。该突变体中缺失的结构域在不同物种中高度保守，表明该结构域可能在破骨细胞生成所需的TRAF 6活化中至关重要。我们目前正专注于这个领域。

RANK and CD40 activate NF-kB and MAPKs to similar levels via TRAF6. Even though overexpression of TRAF6 results in osteoclast formation, RANK but not CD40 promotes osteoclastogenesis. To understand the molecular basis for RANK-specific activity in osteoclastogenesis, we created an osteoclast formation system driven by antihuman CD40 antibody-mediated stimulation of a chimeric receptor, h40/mRK, which consists of the extra cellular domain of human CD40 and the transmembrane and cytoplasmic domains of mouse RANK. By introducing mutations into three TRAF6-binding sites of RANK, we found that h40/mRK with a single TRAF6-binding site efficiently induced Ca2+ oscillation and expression of NFATc1, a master switch in osteoclastogenesis, whereas CD40 carrying a single TRAF6-binding site did not. However, expression of CD40 that was approximately 100 times greater than that of h40/mRK resulted in osteoclast formation, indicating that the RANK-TRAF6 signal is more potent than the CD40-TRAF6 signal in terms of NFATc1 activation and osteoclastogenesis. These results suggest that RANK may harbor a specific domain that amplifies TRAF6 signaling. To identify the specific domain, we have introduced various deletion into the cytoplasmic domain of RANK. We found a deletion mutant that can activate NF-kB and MAPKs but can not generate ostesoclasts. The domain missing in this mutant is highly conserved among various species, suggesting that the domain could be critical in TRAF6 activation required for osteoclastogenesis. We are currently focusing on this domain.