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

RANK和CD40通过TRAF6将NF-kB和MAPK激活到相似的水平。尽管过表达TRAF6会导致破骨细胞的形成，但RANK而不是CD40会促进破骨细胞的形成。为了了解RANK在破骨细胞发生中特异性活性的分子基础，我们建立了一个破骨细胞形成系统，该系统由抗人CD40抗体介导的h40/mRK嵌合受体的刺激驱动，该嵌合受体由人CD40的胞外结构域和小鼠RANK的跨膜和胞浆结构域组成。通过将突变引入三个TRAF6结合位点，我们发现具有单一TRAF6结合位点的h40/mRK有效地诱导了钙振荡和破骨细胞形成中的主开关NFATc1的表达，而携带单一TRAF6结合位点的CD40则不能。然而，CD40的表达大约是h40/mRK的100倍，导致破骨细胞的形成，这表明RANK-TRAF6信号在NFATc1激活和破骨细胞形成方面比CD40-TRAF6信号更有效。这些结果表明，RANK可能含有一个特定的结构域来放大TRAF6信号。为了确定特定的结构域，我们在RANK的细胞质结构域中引入了不同的缺失。我们发现了一个缺失突变体，它可以激活NF-kB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，这表明该结构域可能在破骨细胞形成所需的TRAF6激活过程中起关键作用。我们目前正专注于这一领域。

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.