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 [
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Figure 3Rela–/– BMMs are sensitive to RANKL-induced cell death.
(
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 D
3, 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.
