RANK-mediated amplification of TRAF6 signaling leads to NFATc1 induction during osteoclastogenesis

RANK and CD40 activate NF-kappaB 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 extracellular 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.