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 3

Rela–/– BMMs are sensitive to RANKL-induced cell death.

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Rela–/– BMMs are sensitive to RANKL-induced cell death. (A) Rela+/+...
(A) Rela+/+ or Rela–/– BMMs were cultured in M-CSF and RANKL or (B) cultured with wild-type calvarial osteoblasts in the presence of 1,25 vitamin D3, then fixed and TRAP stained. Scale bar: 500 μm. The number of OCs per field (mean ± SEM) is shown, demonstrating fewer OCs in the absence of RelA. (C) Cell number was assessed by MTT assay in BMMs cultured with M-CSF and RANKL for 3–48 hours, showing fewer cells after 48 hours in Rela–/– cultures relative to Rela+/+ (*P < 0.00001; n = 4). (D) Apoptosis was measured by DNA fragmentation assay after 18 or 36 hours in the presence of M-CSF, with (+) or without (–) RANKL. After 36 hours, Rela–/– BMMs cultured with M-CSF and RANKL, but not with M-CSF alone, had increased cell death. ^P < 0.0001 compared with Rela+/+ without RANKL at 36 hours; †P < 0.00001 relative to Rela+/+ with RANKL at 36 hours. (E) Caspase-3 (casp 3) activation was measured in Rela+/+ and Rela–/– BMMs cultured in M-CSF/RANKL for 36 and 48 hours, with or without ZVAD, and fold induction relative to the same cells in M-CSF alone was plotted. #P < 0.05 relative to Rela+/+ 36 hours; ‡P < 0.01, ΧP < 0.00001 relative to Rela+/+ at 36 or 48 hours. (F) OCs were generated with or without ZVAD during the first 48 hours. Scale bar: 500 μm. (G) OCs were generated as in F on cortical bone slices. Resorption pits (brown) were stained with horseradish peroxidase-conjugated wheat germ agglutinin. Scale bar: 100 μm.