RelA/p65 promotes osteoclast differentiation by blocking a RANKL-induced apoptotic JNK pathway in mice
J. Clin. Invest. Sergio Vaira, et al. 118:2088 doi:10.1172/JCI33392 [Go to this article.]

Figure 6
Enhanced RANKL-induced apoptosis is specific for RelA. (A) BMMs from Rela^+/+ and Rela^–/– mice were cultured in the presence of M-CSF and RANKL for 0 or 24 hours. Equal amounts of nuclear extracts were incubated with biotinylated κB oligos bound to streptavidin-coated beads. Beads were washed to isolate κB-bound proteins, and these were analyzed by immunoblot for RelB or c-Rel. Unbound nuclear proteins were analyzed by immunoblot for Sp1 as loading control. (B) BMMs from Relb^+/+ and Relb^–/– mice were cultured with M-CSF and RANKL for 48 hours, and apoptosis was assessed by DNA fragmentation assay, showing no effect of RelB on apoptosis. (C) Rela^–/– BMMs retrovirally transduced with empty vector (EV), RelA, or RelB, and Rela^+/+ BMMs transduced with EV were selected in blastocydin, then analyzed by immunoblot. Viral-encoded proteins (both RelA and RelB), migrate more slowly than their endogenous (endo) counterparts, and are expressed at 2- to 3-fold over endogenous levels. (D) BMMs transduced in C were cultured with RANKL for 48 hours, and apoptosis was assessed by DNA fragmentation assay. While the EV control and RelB vectors do not abrogate the RANKL-induced cell death in Rela^–/– cultures, RelA reduces apoptosis to the same level as Rela^+/+ transduced with EV. *P < 0.001 Rela^+/+ EV. (E) Transduced BMMs were grown in osteoclastogenic conditions for 6 days and then were fixed and stained for TRAP. Reexpression of RelA restores Rela^–/– OC differentiation, while overexpression of RelB has no effect. Scale bar: 200 μm.