RelA/p65 promotes osteoclast differentiation by blocking a RANKL-induced apoptotic JNK pathway in mice
Sergio Vaira, … , Roberta Faccio, Deborah Veis Novack
Sergio Vaira, … , Roberta Faccio, Deborah Veis Novack
Published May 8, 2008
Citation Information: J Clin Invest. 2008;118(6):2088-2097. https://doi.org/10.1172/JCI33392.
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Research Article Bone biology

RelA/p65 promotes osteoclast differentiation by blocking a RANKL-induced apoptotic JNK pathway in mice

Abstract

Osteoclasts (OCs) function to reabsorb bone and are responsible for the bone loss associated with inflammatory arthritis and osteoporosis. OC numbers are elevated in most disorders of accelerated bone destruction, reflecting altered rates of precursor differentiation and apoptosis. Both of these processes are regulated by the JNK family of MAP kinases. In this study, we have demonstrated that the NF-κB subunit RelA/p65 inhibits JNK-mediated apoptosis during a critical period of commitment to the OC phenotype in response to the cytokine RANKL. This RelA/p65-mediated arrest of cell death led to enhanced OC differentiation. Hence, Rela–/– OC precursors displayed prolonged JNK activation in response to RANKL, and this was accompanied by an increase in cell death that prevented efficient differentiation. Although complete blockade of JNK activity inhibits osteoclastogenesis, both short-term blockade in RelA-deficient cultures and suppression of the downstream mediator, Bid rescued apoptosis and differentiation. These antiapoptotic effects were RelA specific, as overexpression of RelA, but not RelB, blocked apoptosis and rescued differentiation in Rela–/– precursors. Thus, RelA blocks a RANKL-induced, apoptotic JNK-Bid pathway, thereby promoting OC differentiation. Consistent with this, mice lacking RelA/p65 in the hematopoietic compartment were shown to have a deficient osteoclastogenic response to RANKL and were protected from arthritis-induced osteolysis.

Authors

Sergio Vaira, Muhammad Alhawagri, Imani Anwisye, Hideki Kitaura, Roberta Faccio, Deborah Veis Novack

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Figure 6

Enhanced RANKL-induced apoptosis is specific for RelA.

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Enhanced RANKL-induced apoptosis is specific for RelA. (A) BMMs from...
(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.