NF-κB p100 limits TNF-induced bone resorption in mice by a TRAF3-dependent mechanism
Zhenqiang Yao, … , Lianping Xing, Brendan F. Boyce
Zhenqiang Yao, … , Lianping Xing, Brendan F. Boyce
Published September 21, 2009
Citation Information: J Clin Invest. 2009;119(10):3024-3034. https://doi.org/10.1172/JCI38716.
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Research Article

NF-κB p100 limits TNF-induced bone resorption in mice by a TRAF3-dependent mechanism

Abstract

TNF and RANKL mediate bone destruction in common bone diseases, including osteoarthritis and RA. They activate NF-κB canonical signaling directly in osteoclast precursors (OCPs) to induce osteoclast formation in vitro. However, unlike RANKL, TNF does not activate the alternative NF-κB pathway efficiently to process the IκB protein NF-κB p100 to NF-κB p52, nor does it appear to induce osteoclast formation in vivo in the absence of RANKL. Here, we show that TNF limits RANKL- and TNF-induced osteoclast formation in vitro and in vivo by increasing NF-κB p100 protein accumulation in OCPs. In contrast, TNF induced robust osteoclast formation in vivo in mice lacking RANKL or RANK when the mice also lacked NF-κB p100, and TNF-Tg mice lacking NF-κB p100 had more severe joint erosion and inflammation than did TNF-Tg littermates. TNF, but not RANKL, increased OCP expression of TNF receptor–associated factor 3 (TRAF3), an adapter protein that regulates NF-κB p100 levels in B cells. TRAF3 siRNA prevented TNF-induced NF-κB p100 accumulation and inhibition of osteoclastogenesis. These findings suggest that upregulation of TRAF3 or NF-κB p100 expression or inhibition of NF-κB p100 degradation in OCPs could limit bone destruction and inflammation-induced bone loss in common bone diseases.

Authors

Zhenqiang Yao, Lianping Xing, Brendan F. Boyce

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Figure 1

TNF-induced expression of NF-κB p100 inhibits osteoclastogenesis.

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TNF-induced expression of NF-κB p100 inhibits osteoclastogenesis. (A) WT...
(A) WT mouse OCPs, cultured from splenocytes with M-CSF for 3 days, were treated with RANKL or TNF for the indicated times. NF-κB proteins in whole-cell lysates were determined by Western blot. Experiments were repeated at least twice with similar results. P, PBS; R, RANKL 10 ng/ml; T, TNF 20 ng/ml. (B) WT or Nfkb2–/– OCPs were treated with RANKL or TNF directly on plastic or bone slices in 96-well plates in the presence of M-CSF for 2 and 5 days, respectively, to induce osteoclasts (OCs) and resorption pits. Top: Representative TRAP-stained osteoclasts (original magnification, ×4) and toluidine blue–stained pits (original magnification, ×20). Bottom: Osteoclast number and resorption pit area (n = 4/group; *P < 0.05 vs RANKL). (C) Nfkb2–/– or WT OCPs were infected with GFP, p100, or p52 retroviruses for 2 days and treated with TNF for 2 more days. Osteoclast numbers were counted (left panel; *P < 0.05 versus GFP), and the infection efficiency was confirmed by Western blot from the infected WT OCPs (right panels). (D) Murine TNF (0.5 μg in 10 μl PBS) or 10 μl PBS were injected twice daily over the calvariae of 4-week-old Nfkb2–/– or Nfkb2+/– control mice for 5 days (n = 4/group). The number of osteoclasts/mm bone surface, percentage of osteoclast surface/bone surface, and percentage of eroded surface/bone surface were measured in TRAP-stained calvarial bone sections, and serum TRAP5b was tested with ELISA.