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
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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 2

NF-κB2 deficiency enhances TNF-induced osteoclastogenesis in Rank–/– or Rankl–/– mice in vitro and in vivo.

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NF-κB2 deficiency enhances TNF-induced osteoclastogenesis in Rank–/– or ...
(A) NF-κB p100 and p52 were analyzed by Western blot in whole-cell lysates of PBS-, RANKL-, or TNF-treated (8 hours) OCPs from Rank–/– or Rankl–/– mice. (B) Left: OCPs from Rank–/–/Nfkb2–/– or Rankl–/–/Nfkb2–/– mice and their Nfkb2+/– littermates were treated with TNF for 2 days to evaluate osteoclast formation using TRAP staining (*P < 0.05 vs. Nfkb2+/–). Right: OCPs from Rank–/–/Nfkb2+/+ and Rankl–/–/Nfkb2+/+ mice were treated with RANKL or TNF for comparison with Rank–/–/Nfkb2+/– and Rankl–/–/Nfkb2+/– mice to determine the effects of haploinsufficiency of Nfkb2. (C) Murine TNF (0.5 μg in 10 μl PBS) or 10 μl PBS was injected twice daily over the calvariae of Rank–/–/Nfkb2–/– or Rankl–/–/Nfkb2–/– mice and Rank–/– or Rankl–/– littermates. Top: TRAP-stained sections show numerous actively resorbing TRAP+ osteoclasts locally in calvarial sections (original magnification, ×20) from TNF-injected Rank–/–/Nfkb2–/– or Rankl–/–/Nfkb2–/– mice. Bottom: Numbers and surface extent of osteoclasts (n = 3/genotype). Occasional osteoclasts induced by TNF from a Rank–/–/Nfkb2+/+ mouse are illustrated in the left panels. *P < 0.05 vs. single KO mice. (D) Left: Occasional binucleate (arrowhead), but mainly mononuclear (arrows), TRAP+ cells (left panel) formed beneath hypertrophic chondrocytes in the growth plate of the tibia of a Rank–/–/Nfkb2–/– mouse (original magnification, ×40), but not of Rank–/–/Nfkb2+/– littermates injected with TNF as described in C. Right: Osteoclast numbers (expressed per mm of length of growth plate) counted in representative sections. (E) Serum TRAP5b levels were tested with ELISA from TNF- or PBS-injected Rank–/–/Nfkb2–/– and Rank–/–/Nfkb2+/– mice (n = 3/group; *P < 0.05).