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. 2005 May 16;201(10):1677-87.
doi: 10.1084/jem.20042081.

I{kappa}B kinase (IKK){beta}, but not IKK{alpha}, is a critical mediator of osteoclast survival and is required for inflammation-induced bone loss

Maria Grazia Ruocco  1 , Shin Maeda, Jin Mo Park, Toby Lawrence, Li-Chung Hsu, Yixue Cao, Georg Schett, Erwin F Wagner, Michael Karin
Affiliations

I{kappa}B kinase (IKK){beta}, but not IKK{alpha}, is a critical mediator of osteoclast survival and is required for inflammation-induced bone loss

Maria Grazia Ruocco et al. J Exp Med. .

Abstract

Transcription factor, nuclear factor kappaB (NF-kappaB), is required for osteoclast formation in vivo and mice lacking both of the NF-kappaB p50 and p52 proteins are osteopetrotic. Here we address the relative roles of the two catalytic subunits of the IkappaB kinase (IKK) complex that mediate NF-kappaB activation, IKKalpha and IKKbeta, in osteoclast formation and inflammation-induced bone loss. Our findings point out the importance of the IKKbeta subunit as a transducer of signals from receptor activator of NF-kappaB (RANK) to NF-kappaB. Although IKKalpha is required for RANK ligand-induced osteoclast formation in vitro, it is not needed in vivo. However, IKKbeta is required for osteoclastogenesis in vitro and in vivo. IKKbeta also protects osteoclasts and their progenitors from tumor necrosis factor alpha-induced apoptosis, and its loss in hematopoietic cells prevents inflammation-induced bone loss.

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Figures

Figure 1.
Figure 1.
IkkαAA and IkkβD BM cells exhibit defective RANKL- mediated osteoclastogenesis in vitro. Equal numbers of BM cells were isolated from WT, IkkαAA, and IkkβΔ mice and cultured in the presence of M-CSF (10 ng/ml) alone or M-CSF plus RANKL (50 ng/ml). Osteoclastogenesis was monitored after 7 d by TRAP staining (red color). Note the giant cells that appear after incubation of WT BM with M-CSF and RANKL.
Figure 2.
Figure 2.
Biochemical analysis of RANKL signaling to NF-κB in IkkαAA and IkkβD osteoclast progenitors. (A) Western blot analysis of protein extracts from WT, IkkαAA, and IkkβΔ osteoclast precursors stimulated with RANKL for the indicated times. Immunoblot analysis was performed with anti-IKKβ, anti-IKKα, anti-IκBα, and anti-p38 (as loading control) antibodies. (B) NF-κB DNA binding activity was assayed at the indicated times in RANKL-stimulated WT, IkkαAA, and IkkβΔ osteoclast progenitors by electrophoretic mobility shift assay using a κB site oligonucleotide probe or a nuclear factor-y probe to control for loading and extract quality. (C) IKK activation by RANKL. Total protein lysates were prepared and IKK activity was measured by an immunocomplex kinase assay before and after RANKL stimulation of BM cells. IκBα(1–54) was used as a substrate. (D) Nuclear translocation of NF-κB subunits. Osteoclast progenitors from WT, IkkαAA, and IkkβΔ mice were incubated with RANKL for the indicated durations in the presence of M-CSF. Nuclear (nuc) and cytoplasmic (cyt) extracts were analyzed by immunoblotting using antibodies directed against NF-κB family members and an anti–poly(ADP-ribose)-polymerase antibody as a loading control. (E) p100 processing. Osteoclast progenitors from WT, IkkαAA, and IkkβΔ mice were incubated with RANKL for the indicated durations in the presence of M-CSF. Total protein extracts were analyzed for the presence of full-length p100 and its processed form, p52.
Figure 3.
Figure 3.
IkkβD, but not IkkαAA, mice are osteopetrotic. (A) Sections of the metaphyseal regions of the proximal tibia of 4-mo-old WT, IkkαAA, and IkkβΔ mice were subjected to H&E staining. (B) Decreased numbers of TRAP-positive (red-stained) multinucleated cells below the growth plate of IkkβΔ mice. TRAP-positive cells are marked by arrows. (C) Histomorphometric analysis of structural bone parameters in IkkβΔ (n = 6), IkkαAA(n = 6), and F/F (IkkβF/F, n = 6 ) mice at 4 and 7 mo of age. OcS/BS, osteoclast surface/bone surface; NOc/B.Pm, number of osteoclasts/bone perimeter/mm; Tr.Th, trabecular thickness (mm); Tr.N trabecular number/mm; Tr.S, trabecular separation/mm3; NOb/B.Pm, number of osteoblasts/bone perimeter/mm; ObS/BS, osteoblast surface/bone surface; MAR, mineral apposition rate. *P < 0.05.
Figure 4.
Figure 4.
Osteoclastogenesis is impaired in IkkβD, but not in IkkαAA, BM cells that are cocultured with osteoblasts. (A) Equal numbers of BM cells from WT, IkkαAA, and IkkβΔ mice were plated in the presence of 5 × 105 cells/ml of primary calvarial osteoblasts (Ob) from WT or IkkαAA mice and cultured in the presence of 1,25(OH)2 vitamin D3 and dexamethasone for 6 d. Osteoclastogenesis was assayed by TRAP staining. (B) The extent of osteoclastogenesis (expressed as staining intensity ×10−5) was determined by TRAP staining, quantified, and expressed as staining intensity ×10−5.
Figure 5.
Figure 5.
IL-1 or TNFα rescue RANKL-induced osteoclast differentiation of IkkαAA, but not IkkβD, osteoclast progenitors. (A) Equal numbers of BM cells from WT, IkkαAA, IkkβΔ, and IkkβΔ :Tnfr1 − / − mice were cultured in the presence of M-CSF (20 ng/ml) and RANKL (50 ng/ml) for 7 d. TNFα (20 ng/ml) or IL-1 (10 ng/ml) alone or in combination with RANKL were added. Osteoclastogenesis was assayed by TRAP staining. Incubation of IkkβΔ BM cells with TNFα resulted in death of all cells within 48 h. (B) Numbers of osteoclasts per field (%). Results shown are averages ± SD (n = 4). (C) The bone-resorbing activity of WT, IkkαAA, IkkβΔ, Tnfr1 − / −, and IkkβΔ :Tnfr1 − / − osteoclasts was analyzed in vitro by quantification of resorption pit areas on calcium phosphate films. (D) TUNEL assay of BM cells from WT and IkkβΔ mice that were incubated for the indicated times with TNFα (1 ng/ml). (E) Quantification of the TUNEL assay results. The percentage of TUNEL positive cells in four representative fields was determined by cell counting. n.d., not depicted.
Figure 6.
Figure 6.
Osteoclast precursors that lack IKKβ are sensitive to TNFα-mediated apoptosis. (A) Sections of the metaphyseal regions of femurs of 4-mo-old Tnfr1 − / − and IkkβΔ :Tnfr1 − / − mice were subjected to H&E staining. (B) TRAP staining of the metaphyseal regions of 4-mo-old WT, IkkβΔ, Tnfr1 − / −, and IkkβΔ :Tnfr1 − / − mice. The numbers refer to osteoclasts per field. (C) Deoxypiridinoline (DPD) cross-links in the urine of WT, IkkβΔ, Tnfr1 − / −, and IkkβΔ :Tnfr1 − / −mice were measured as an indicator of in vivo osteoclast activity. The values were normalized for muscle creatinine that also is excreted in the urine to account for urine concentration (n = 6). (D) H&E, F4/80 (red), and DAPI (blue) staining of sections from long bones of 4-mo-old WT, IkkβΔ, Tnfr1 − / −, and IkkβΔ :Tnfr1 − / − mice. (E) F4/80 (red) and TUNEL (green) double staining and DAPI (blue) staining of sections from long bones of WT and IkkβΔ mice. (F) F4/80 staining of liver sections of WT and IkkβΔ mice.
Figure 7.
Figure 7.
Lack of IKKβ prevents inflammation-induced bone loss. 2-mo-old WT (n = 11), IkkβΔ (n = 7), Tnfr1 − / − (n = 4), and IkkβΔ :Tnfr1 − / − (n = 4) mice were injected once with LPS (500 μg/ml) or saline control into the synovial space of their hind leg joints. (A) After 5 d the mice were killed and the limbs were fixed, decalcified, sectioned, and analyzed by H&E staining. The arrows show regions of bone loss. (B) Higher magnification of the H&E staining of the joints of WT and IkkβΔ mice. B, bone; I, inflammation; S, synovial space. (C) The presence of osteoclasts (arrows) in WT and IkkβΔ mice that were treated with LPS was analyzed by TRAP staining. (D) F4/80 (red) and DAPI (blue) staining of sections from joints of WT and IkkβΔ mice that were treated with LPS. (E) Schematic model of RANKL and TNFα signaling during osteoclastogenesis and inflammation-induced bone loss. X represents a pathway other than IKK/NF-κB that is activated by RANKL binding to RANK and is essential for production of functional osteoclasts. This pathway is not activated by TNFα binding to TNFR1.
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