(A) WT mouse OCPs were treated with RANKL and/or TNF for 8 hours, and whole-cell lysate protein was extracted and subjected to Western blotting for TRAF3, TRAF6, NIK, and NF-κB p100 and p52. (B) Cycloheximide (10 μM) was added to WT mouse OCPs treated with RANKL (10 ng/ml), TNF (20 ng/ml), or PBS control for the indicated times. Whole-cell lysates were subjected to Western blotting for TRAF3. (C) WT mouse OCPs were transfected with TRAF3 siRNA or a nonspecific control siRNA for 8 hours. The cells were treated with TNF (20 ng/ml) or PBS for an additional 8 hours. Whole-cell lysates were subjected to Western blotting to determine the levels of cytoplasmic TRAF3, NIK, and NF-κB p100 (left panel) and either nuclear p52 or cytoplasmic p100 and RelB (right panels). (D) TRAF3 siRNA–transfected cells were treated with TNF and/or RANKL (10 ng/ml) for 3 days in the presence of M-CSF to generate osteoclasts. *P < 0.05 vs. control).

(A) Tibiae from 12-week-old Nfkb^+/– (n = 4), Nfkb2^–/– (n = 4), TNF-Tg/Nfkb2^+/– (n = 7), and TNF-Tg/Nfkb2^–/– mice (n = 8) were subjected to μCT scanning. Representative images (left) and data analysis (right) showed reduced trabecular bone volume and cortical bone thickness in TNF-Tg/Nfkb2^–/– mice. (B) H&E-stained sections of tibiae (original magnification, ×2) showed decreased trabecular bone in the metaphyseal regions and thinner cortices (green arrow) in TNF-Tg/Nfkb2^–/– mice. (C) TRAP-stained sections of 6-week-old mice showed increased numbers of osteoclasts in the secondary spongiosa of the proximal tibia of a TNF-Tg/Nfkb2^–/– mouse (left panels), which was confirmed by histomorphometric analysis (right panels). BV/TV, bone volume/tissue volume. *P < 0.05 vs. TNF-Tg/Nfkb2^+/– mice. Original magnification, ×2 (top), ×40 (bottom).

(A) WT mouse spleen cells were cultured with M-CSF for 3 days, and RANKL and/or TNF were added at the indicated doses for 2 more days to generate osteoclasts. Top: TNF dose-dependently inhibited RANKL-induced osteoclastogenesis, assessed by osteoclast number (black bars) and area (red bars) per well. Bottom: The protein levels of NF-κB p100 and p52 were analyzed with Western blot and assessed in whole-cell lysates extracted from RANKL-treated (10 ng/ml) and/or TNF-treated (20 ng/ml) WT mouse OCPs at 8 hours. (B) The inhibitory effect of TNF on RANKL-induced osteoclastogenesis was abolished in Nfkb2^–/– OCPs (*P < 0.05 vs. RANKL treatment alone; 4 wells/group). The same experiments were repeated at least twice with similar results.

(A) Age-related changes in clinically assessed joint deformation scores showed that joint deformation occurred earlier in the TNF-Tg/Nfkb2^–/– mice (n = 7) than in their TNF-Tg/Nfkb2^+/– littermates (n = 8). (B) Representative TRAP-stained sections (original magnification, ×20) from 12-week-old animals showed more severe wrist joint inflammation (green arrows) and more osteoclasts (yellow arrowheads) in a TNF-Tg/Nfkb2^–/– mouse than in a TNF-Tg/Nfkb2^+/– mouse. Histomorphometric analysis showed that the area of inflammatory tissue (upper panel) and osteoclast numbers (lower panel) were increased in the wrists of TNF-Tg/Nfkb2^–/– mice. *P < 0.05. (C) The percentage of cartilage eroded surface/total joint surface was measured in carpal bones of 6-week-old mice (n = 5/group). (D) Serum levels of murine TNF (black bars) and human TNF (red bars) were tested with ELISA at 6 and 12 weeks of age (*P < 0.05 vs. TNF-Tg/Nfkb^+/– littermates).

(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.

(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).