© Rockefeller University Press, 0022-1007 $8.00
JEM, Volume 201, Number 10, 1677-1687
I
B kinase (IKK)ß, but not IKK
, is a critical mediator of osteoclast survival and is required for inflammation-induced bone loss
Maria Grazia Ruocco1,
Shin Maeda1,
Jin Mo Park1,
Toby Lawrence1,
Li-Chung Hsu1,
Yixue Cao1,
Georg Schett2,
Erwin F. Wagner3, and
Michael Karin1
1 Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA 92093
2 Department of Internal Medicine III, Division of Rheumatology, University of Vienna, A-1090 Vienna, Austria
3 Research Institute of Molecular Pathology, A-1030 Vienna, Austria
CORRESPONDENCE Michael Karin: karinoffice{at}ucsd.edu
Transcription factor, nuclear factor
B (NF-
B), is required for osteoclast formation in vivo and mice lacking both of the NF-
B p50 and p52 proteins are osteopetrotic. Here we address the relative roles of the two catalytic subunits of the I
B kinase (IKK) complex that mediate NF-
B activation, IKK
and IKKß, in osteoclast formation and inflammation-induced bone loss. Our findings point out the importance of the IKKß subunit as a transducer of signals from receptor activator of NF-
B (RANK) to NF-
B. Although IKK
is required for RANK ligand-induced osteoclast formation in vitro, it is not needed in vivo. However, IKKß is required for osteoclastogenesis in vitro and in vivo. IKKß also protects osteoclasts and their progenitors from tumor necrosis factor
induced apoptosis, and its loss in hematopoietic cells prevents inflammation-induced bone loss.
Abbreviations used: H&E, hematoxylin-eosin; I
B, inhibitor of NF-
B; IKK, I
B kinase; M-CSF, macrophage-colony stimulating factor; NIK, NF-
Binducing kinase; poly(IC), polyinosinic-polycytidylic acid; RANKL, receptor activator of NF-
B ligand; TNFR, TNF receptor; TRAP, tartrate-resistant acid phosphatase; TUNEL, terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling.
Bone development and remodeling are highly regulated processes that involve synthesis of bone matrix by osteoblasts and coordinated bone resorption by osteoclasts (1). Osteoblasts originate from mesenchymal stem cells, whereas osteoclasts are derived from hematopoietic monocyte/macrophage precursors (1). Imbalanced osteoclast and osteoblast formation, activity, or survival can be caused by a variety of hormonal changes or perturbed production of inflammatory cytokines and growth factors, and result in skeletal abnormalities that are characterized by decreased (osteoporosis) or increased (osteopetrosis) bone mass (1). Increased osteoclast formation and activity is observed in many osteopenic disorders, including postmenopausal osteoporosis (2), lytic bone metastasis, or rheumatoid arthritis (3), and leads to accelerated bone resorption and crippling bone damage.
During osteoclast differentiation, osteoblastic/stromal cells provide a physical support for nascent osteoclasts and produce soluble and membrane-associated factors, such as macrophage-colony stimulating factor (M-CSF), and receptor activator of NF-
B ligand (RANKL) (4). RANKL (also called tumor necrosis factorrelated activation-induced cytokine, osteoclast differentiation factor, osteoprotegerin ligand) is a member of the TNF cytokine family and an essential inducer of osteoclastogenesis and bone remodeling through its receptor RANK, a TNF-receptor (TNFR) family member (5, 6). Mice with a disrupted Rankl gene exhibit severe osteopetrosis (6, 7). Disruption of the Rank gene also results in lack of osteoclasts and ensuing osteopetrosis (8). Similar to RANKL, TNF-
is a potent osteoclastogenic factor that enhances proliferation and differentiation of osteoclast precursors through its type I receptor (TNFR1; reference 9). However, it remains controversial whether TNF
promotes osteoclastogenesis independently of RANKL (10, 11). RANK, like most other TNFR family members, including TNFR1, transduces its biochemical signals through recruitment of intracellular signal transducers, called TNF receptor-associated factors, which lead to activation of NF-
B and mitogen-activated protein kinase effector pathways (1215). The relevance of these pathways to osteoclastogenesis is underscored by the osteopetrotic phenotypes of mice lacking TNF receptorassociated factor 6 (16); the NF-
B1/p50 and NF-
B2/p52 subunits of NF-
B (15, 17); or c-Fos (18), a component of the AP-1 transcription factor, whose expression is mitogen-activated protein kinase dependent (19).
NF-
B is a collection of dimeric transcription factors that recognize similar DNA sequences called
B sites. In mammals there are five NF-
B proteins: cRel, RelA and RelB, as well as NF-
B1/p50 and NF-
B2/p52. Although the Rel proteins contain transcriptional activation domains, such domains are absent in p50 and p52, whose activation function depends on heterodimerization with any of the three Rel proteins (20). As mentioned above, ablation of p50 and p52 results in a severe osteopetrotic phenotype, which most likely is due to the poor DNA binding activity of the remaining NF-
B subunits (15). NF-
B proteins reside in the cytoplasm of nonstimulated cells but rapidly enter the nucleus upon cell stimulation (21). This process, called NF-
B activation, depends on two pathways. The classic NF-
B signaling pathway involves activation of the I
B kinase (IKK) complex that phosphorylates the inhibitors of NF-
B (I
Bs) and targets them to ubiquitin-dependent degradation (21). The I
Bs retain most NF-
B dimers, with the exception of p52:RelB dimers, in the cytoplasm by masking their nuclear localization signals (21). The alternative NF-
B signaling pathway is responsible for activation of p52:RelB dimers, which are generated by processing of cytoplasmic p100:RelB dimers (21).
Currently, it is not entirely clear which of the two NF-
B activation pathways plays the dominant role in osteoclastogenesis. The IKK complex that is responsible for activation of the canonical NF-
B pathway consists of two catalytic subunits, IKK
and IKKß, and a regulatory subunit, IKK
/NF-
B essential modulator (22). Gene disruption experiments demonstrated that IKKß and IKK
are important for I
B phosphorylation and degradation, whereas IKK
has different and nonoverlapping functions (21). Importantly, IKK
forms homodimers, not associated with IKK
, that are required for phosphorylation-induced p100 processing and activation of the alternative pathway (23). Activation of the alternative pathway also depends on the IKK
-phosphorylating kinase, NF-
Binducing kinase (NIK; refereneces 23, 24). It was observed that NIK-deficient osteoclast precursors do not respond to RANKL in an in vitro differentiation system that is devoid of osteoblasts (25). However, aly mice, which carry a point mutation in the Nik gene that prevents NIK activation, are not osteopetrotic (26); osteopetrosis also was not reported for Nik/ mice (25). More recently, a peptide inhibitor of IKK, which prevents the association of IKKß with IKK
, and therefore, blocks activation of the classic pathway without affecting the alternative pathway, was shown to prevent inflammation-induced bone loss in vivo (27). These results suggest that the classic pathway is of greater importance for osteoclastogenesis. To determine the relative roles of the two pathways in osteoclastogenesis and inflammation-induced bone loss, we undertook a genetic approach based on the use of mouse strains that carry specific mutations in the Ikk
and Ikkß genes. We found that IKKß, but not IKK
, is essential for inflammation-induced bone loss and is required for osteoclastogenesis in vivo. However, the main function of IKKß in osteoclastogenesis is to prevent TNF
-induced apoptosis of osteoclast precursors. Once TNF
-induced apoptosis is prevented through deletion of the Tnfr1 gene, IKKß is no longer required for induction of inflammation-induced bone loss, but it is still needed for basal osteoclast function.
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RESULTS
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RANKL-induced in vitro osteoclastogenesis requires IKK
and IKKß
To determine the roles of the IKK catalytic subunits in osteoclastogenesis, we used mice that carry mutant forms of either subunitIkk
AA and Ikkß
mice. Ikk
AA mice are homozygous for a knock-in mutant allele, in which the activation loop serines of IKK
whose phosphorylation is required for its activation (28)were replaced with alanines; this prevents IKK
activation by upstream stimuli, including RANK (29) and NIK (23). Ikk
AA mice are viable, healthy, and fertile. These mice exhibit defective organization of secondary lymphoid organs and B cell maturation (30), a defect that is very similar to what was found in Nik/ (31) or Nikaly/aly (26) mice. Ikkß
mice were generated by crossing IkkßF/F mice, homozygotes for a "floxed" Ikkß allele (32), with Mx1-Cre transgenic mice that express Cre recombinase from the IFN-inducible Mx-1 promoter (33). Injection of polyinosinic-polycytidylic acid (poly[IC]), which induces IFN production, into IkkßF/F:Mx1-Cre mice results in efficient deletion of the floxed third exon of the Ikkß gene and generation of IKKß-deficiency in IFN-responsive cells, including myeloid cells (34). To determine whether defective IKK
activation or complete loss of IKKß affect osteoclastogenesis, we first used an in vitro differentiation system. BM hematopoietic progenitors that were isolated from WT, Ikk
AA, and Ikkß
mice were incubated with M-CSF and RANKL for 7 d, and stained for tartrate-resistant acid phosphatase (TRAP), a marker for osteoclasts. Wt cultures showed robust osteoclastogenesis and formed giant TRAP-positive cells, whereas osteoclast formation was absent in Ikk
AA and Ikkß
cultures (Fig. 1). Expression of several other osteoclast markers, including CatK and matrix metalloproteinase-9, also was defective in RANKL-treated Ikk
AA and Ikkß
BM cultures (unpublished data). These results suggest that IKK
and IKKß are required for, and play nonredundant roles in, M-CSF and RANKL-induced osteoclastogenesis in vitro.
RANKL-mediated NF-
B activation is impaired in Ikkß
, but not Ikk
AA, osteoclast progenitors
To gain insights into the possible mechanisms by which IKK
and IKKß promote osteoclastogenesis in vitro, we examined RANKL-induced NF-
B activation in Ikk
AA and Ikkß
BM cultures. BM-derived precursors from WT, Ikk
AA, and Ikkß
mice were incubated with M-CSF and RANKL and analyzed for expression of IKK
and IKKß by immunoblot analysis (Fig. 2 A). As expected, no IKKß protein was detectable in Ikkß
BM cells. Interestingly, the deletion of IKKß also resulted in slightly decreased levels of IKK
(Fig. 2 A); this suggests that IKK
:IKK
complexes or IKK
homodimers are not as stable as the heterotrimeric IKK
:IKKß:IKK
complexes (22). Electrophoretic mobility shift assays revealed marked induction of NF-
B DNA binding activity in response to RANKL treatment in WT and Ikk
AA cultures, but only a weak response in Ikkß
cultures (Fig. 2 B). Similarly, RANKL activated IKK in WT and Ikk
AA cultures, but no IKK activity could be detected before or after RANKL treatment of Ikkß
BM cells (Fig. 2 C). In addition, RANKL induced the nuclear translocation of all three Rel proteins in WT cells; this response was slightly reduced for RelB and cRel, but not for RelA, in Ikk
AA cells (Fig. 2 D). By contrast, the basal level of all three Rel proteins in the nucleus and their RANKL-induced nuclear translocation were diminished severely in Ikkß
cultures. Although IKK
plays a minor role in NF-
B activation, other experiments that were conducted with Ikk
AA BM cells revealed its requirement for induction of NF-
B2/p100 processing to p52 in response to RANKL (Fig. 2 E), as previously shown for several other TNF family members (30).

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Figure 2. Biochemical analysis of RANKL signaling to NF- B in Ikk AA and IkkßD osteoclast progenitors. (A) Western blot analysis of protein extracts from WT, Ikk AA, and Ikkß osteoclast precursors stimulated with RANKL for the indicated times. Immunoblot analysis was performed with anti-IKKß, anti-IKK , anti-I B , and anti-p38 (as loading control) antibodies. (B) NF- B DNA binding activity was assayed at the indicated times in RANKL-stimulated WT, Ikk AA, and Ikkß osteoclast progenitors by electrophoretic mobility shift assay using a B 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. I B (154) was used as a substrate. (D) Nuclear translocation of NF- B subunits. Osteoclast progenitors from WT, Ikk AA, and Ikkß 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- B family members and an antipoly(ADP-ribose)-polymerase antibody as a loading control. (E) p100 processing. Osteoclast progenitors from WT, Ikk AA, and Ikkß 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.
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Defective osteoclastogenesis in Ikkß
but not Ikk
AA mice
To determine if IKK
and IKKß play a role in osteoclastogenesis in vivo, bones from 4 mo-old Ikk
AA and Ikkß
mice were analyzed. To study the role of IKKß in bone development we induced deletion of the Ikkß gene in IkkßF/F:Mx1-Cre mice 9 d after birth. Histologic and histomorphometric analyses of long bone (tibias) sections from Ikk
AA mice revealed no differences compared with WT bones, and no alterations were found in trabecular size and distribution or in the number of osteoclasts as determined by TRAP staining (Fig. 3 AC). These results suggest that basal osteoclastogenesis is not affected by the loss of IKK
activation in vivo. By contrast, Ikkß
mice showed an osteopetrotic phenotype. Ikkß
mice are smaller and have a hunched back compared with their IkkßF/F littermates (unpublished data). Histologic analysis of tibias from these mice showed increased trabecular size and distribution which resulted in obliteration of the bone marrow cavity compared with IkkßF/F littermates or WT mice of the same age (Fig. 3 A). TRAP staining showed a greatly reduced number of osteoclasts in Ikkß
bones (Fig. 3, B and C). Furthermore, quantitative histomorphometric analyses of Ikkß
bones revealed a significant increase in bone volume due to increased trabecular number compared with bones of WT littermates (Fig. 3 C); this is diagnostic of reduced osteoclast-mediated bone resorption. The osteoclast parameters, number and surface, are reduced significantly in Ikkß
mice (Fig. 3 C). It also seems that osteoblast number is reduced at 7 mo in the mutant mice, a defect that was observed in other osteopetrotic mouse models (35). The osteopetrotic phenotype of Ikkß
mice becomes more dramatic with age (Fig. 3 C). These results suggest that Ikkß
mice develop osteopetrosis that is due to defective osteoclast formation, and furthermore, indicate a critical role for Ikkß in bone development.

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Figure 3. IkkßD, but not Ikk AA, mice are osteopetrotic. (A) Sections of the metaphyseal regions of the proximal tibia of 4-mo-old WT, Ikk AA, and Ikkß mice were subjected to H&E staining. (B) Decreased numbers of TRAP-positive (red-stained) multinucleated cells below the growth plate of Ikkß mice. TRAP-positive cells are marked by arrows. (C) Histomorphometric analysis of structural bone parameters in Ikkß (n = 6), Ikk AA (n = 6), and F/F (IkkßF/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.
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Osteoblasts can rescue defective in vitro osteoclastogenesis of Ikk
AA but not Ikkß
BM cells
Osteoclasts also can be generated in vitro from BM hematopoietic precursors cultured in the presence of 1,25(OH)2-vitamin D3 and dexamethasone together with osteoblastic/stromal cells from mouse calvarias (36). In such a system, activated osteoblasts play the major role in osteoclast differentiation and provide M-CSF and RANKL directly. To examine whether the osteoclastogenic defects that are observed in Ikk
AA and Ikkß
BM cultures are caused by cell autonomous defects, we performed osteoclasts/osteoblast cocultures. Wt osteoblasts fully supported osteoclastogenesis of Ikk
AA osteoclast precursors, but not Ikkß
osteoclast precursors; this indicates a cellautonomous osteoclast differentiation defect in Ikkß
BM cells (Fig. 4 A, upper panels). Reciprocal cocultures of Ikk
AA or WT BM cells with Ikk
AA osteoblasts showed that Ikk
AA osteoblasts had the same capacity as WT osteoblasts for supporting osteoclast differentiation (Fig. 4, A [lower panels] and B). Thus, Ikk
AA osteoclasts can form normally as long as they receive osteoblast-derived signals; this suggests that these signals compensate for the defect in RANKL signaling.
IL-1 and TNF
rescue the osteoclastogenic defect of Ikk
AA, but not Ikkß
osteoclast progenitors
To identify potential signals that may compensate for the defect in RANKL signaling of Ikk
AA osteoclast precursors, we examined the effect of the proinflammatory cytokines, IL-1 and TNF
. IL-1 and TNF
are potent osteoclastogenic factors and are likely to be involved with RANKL in inflammation-induced bone loss (10, 37). We cultured osteoclast progenitors from WT, Ikk
AA, and Ikkß
mice in the presence of IL-1 or TNF
, alone or together with RANKL. IL-1 and TNF
strongly augmented the osteoclastogenic response of WT BM to RANKL and led to the formation of numerous TRAP-positive giant cells (Fig. 5 A). Either IL-1 or TNF
, when combined with RANKL, completely rescued the osteoclastogenic defect of Ikk
AA BM, although they did not induce differentiation on their own (Fig. 5 A and not depicted). However, the defect in osteoclast differentiation of Ikkß
BM cultures could not be rescued by IL-1 or TNF
(Fig. 5, A and B). The bone-resorbing activity of these cells was assayed by an in vitro resorption pit assay and further quantified. Ikk
AA, but not Ikkß
, BM cells were able to resorb bone, once their differentiation defect was rescued by IL-1 (not depicted) or TNF
(Fig. 5 C). Furthermore, we found that when incubated with TNF
, either in the absence or presence of RANKL, most Ikkß
osteoclast precursors were dead within 48 h (Fig. 5 A). This prompted us to examine whether Ikkß
BM cells undergo apoptosis in response to TNF
. Terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) assay revealed the appearance of cells with distinct apoptotic morphology and fragmented DNA within 24 h of TNF
addition to Ikkß
osteoclast precursors (Fig. 5, D and E). Very few such cells were detected in cultures that were not exposed to TNF
or in WT osteoclast precursors that were incubated with TNF
.

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Figure 5. IL-1 or TNF rescue RANKL-induced osteoclast differentiation of Ikk AA, but not IkkßD, osteoclast progenitors. (A) Equal numbers of BM cells from WT, Ikk AA, Ikkß , and Ikkß :Tnfr1/ mice were cultured in the presence of M-CSF (20 ng/ml) and RANKL (50 ng/ml) for 7 d. TNF (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 Ikkß BM cells with TNF 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, Ikk AA, Ikkß , Tnfr1/, and Ikkß :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 Ikkß mice that were incubated for the indicated times with TNF (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.
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Deletion of Tnfr1 rescues Ikkß
osteoclast progenitors from TNF
-induced apoptosis, but does not prevent osteopetrosis
TNFR1 contains a death domain and is capable of engaging the apoptotic machinery (38). To further examine the mechanism that underlies TNF
-induced death of Ikkß-deficient preosteoclasts, we crossed Ikkß
mice with Tnfr1/ mice to generate IkkßF/F:Mx1-Cre:Tnfr1/ double mutants. After poly(IC) injection, BM cultures that were isolated from these mice were stimulated with RANKL alone or with RANKL plus TNF
; osteoclast differentiation was analyzed by TRAP staining. Loss of TNFR1 prevented TNF
-induced death of Ikkß
osteoclast progenitors (Fig. 5 A, bottom). Furthermore, the absence of TNFR1 rescued the inability of Ikkß
cells to become TRAP-positive, but did not allow them to differentiate fully into multinucleated giant osteoclasts (Fig. 5 A) with the ability to resorb bone (Fig. 5 C). Thus, IKKß is required for the prevention of TNF
-induced death and is needed for formation of fully functional bone-resorbing osteoclasts.
Next we analyzed femurs of 4-mo-old WT, Ikkß
, Tnfr1/, and Ikkß
:Tnfr1/mice. Ikkß deletion in both strains was induced as early as 9 d after birth. Histochemical staining revealed osteopetrosis in Ikkß
:Tnfr1/ bones (Fig. 6 A). However, TRAP-positive cells could be detected easily in Ikkß
:Tnfr1/ mice, whereas they were scarce in Ikkß
mice (Fig. 6 B). Analysis of deoxypiridinoline cross-links in urine sampleswhich reflects osteoclast activity in vivorevealed that in Ikkß
:Tnfr1/ mice, osteoclast resorption activity remained severely attenuated, as in Ikkß
mice. Normal levels of deoxypiridinoline cross-links were found in urine samples from Ikk
AA or Tnfr1/ mice.

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Figure 6. Osteoclast precursors that lack IKKß are sensitive to TNF -mediated apoptosis. (A) Sections of the metaphyseal regions of femurs of 4-mo-old Tnfr1/ and Ikkß :Tnfr1/ mice were subjected to H&E staining. (B) TRAP staining of the metaphyseal regions of 4-mo-old WT, Ikkß , Tnfr1/, and Ikkß :Tnfr1/ mice. The numbers refer to osteoclasts per field. (C) Deoxypiridinoline (DPD) cross-links in the urine of WT, Ikkß , Tnfr1/, and Ikkß :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, Ikkß , Tnfr1/, and Ikkß :Tnfr1/ mice. (E) F4/80 (red) and TUNEL (green) double staining and DAPI (blue) staining of sections from long bones of WT and Ikkß mice. (F) F4/80 staining of liver sections of WT and Ikkß mice.
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Immunohistochemical analyses by hematoxylin-eosin (H&E) staining revealed a small number of osteoclast precursors in the vicinity of the growth plate that were positive for F4/80 staining in Ikkß
mice, whereas many more such cells were seen in WT mice (Fig. 6 D). Remarkably, the loss of TNFR1 in Ikkß
:Tnfr1/ double mutant mice restored the presence of F4/80 positive cells next to the growth plate (Fig. 6 D). Double staining experiments revealed that in Ikkß
mice, most of the few F4/80-positive cells that were present were apoptotic, based on TUNEL staining (Fig. 6 E). This further supports the hypothesis that the decreased osteoclast number in Ikkß
mice is due to increased apoptosis of their precursors. Control experiments showed that in other tissues, such as the liver, the number of F4/80-positive cells did not change in the absence of Ikkß (Fig. 6 F). Despite the decreased apoptosis of osteoclast precursors and restoration of TRAP-positive cells, the Ikkß
:Tnfr1/ double mutant mice remain osteopetrotic. This suggests that osteoclasts that lack IKKß and TNFR1 are not fully functional under basal conditions; this interpretation is consistent with the severely reduced bone-resorbing activity in these mice (Fig. 6 C).
Absence of IKKß protects mice from inflammation-induced bone loss, in a manner dependent on TNFR1
IKKß plays a crucial role in osteoclast differentiation under physiologic conditions. We next analyzed whether IKKß or IKK
is involved in inflammation-induced bone loss. We used an established model of endotoxin-induced bone resorption (39, 40). Ikk
AA, Ikkß
, Tnfr1/, Ikkß
:Tnfr1/, and WT mice were injected with 500 µg LPS in saline into the synovial space of the hind limb knee joint, whereas the contralateral knee was injected with saline alone; mice were killed 5 d after injection. Ikkß deletion was induced 10 d before knee injection. Before sacrificing the mice, their ability to move and flex their hind limb was assessed by video cinematography (Videos 1 and 2, available at http://www.jem.org/cgi/content/full/jem.20042081/DC1). The staining of the joint bones (femurs and tibia) with H&E, TRAP, and F4/80 showed a considerably lower number of osteoclasts and osteoclast precursors in Ikkß
mutant mice compared with WT controls or Ikk
AA mice (Fig. 7, AD; not depicted for Ikk
AA). Although LPS-injected WT, Ikk
AA, and Tnfr1/ mice completely lost the ability to flex the hind limb, no such aberrations were evident in Ikkß
mice, which retained normal flexibility and movement of the LPS-treated hind limb (Videos 1 and 2). The loss of TNFR1 restored inflammation-induced bone loss in mice that lack Ikkß
(Fig. 7), although the bone damage is not as extensive as in WT mice (compare resorption sites indicated by arrows in Fig. 7 A). The degree of inflammation-induced bone loss was quite similar in Ikkß
:Tnfr1/ and Tnfr1/ mice. These results suggest that under strong inflammatory conditions, which are likely to result in massive cytokine production, IKKß is no longer needed for osteoclastogenesis once its survival function has been rendered unnecessary by ablation of TNFR1.
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DISCUSSION
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Previous studies that were based on ablation of the p50 and p52 NF-
B members outlined an important role for these transcription factors in osteoclast differentiation (15). NF-
B is activated by RANKL, a cytokine whose expression and binding to the receptor, RANK, are essential for osteoclastogenesis. NF-
B also is activated by the proinflammatory cytokines, TNF
and IL-1 (21), which enhance inflammation-induced bone loss, although they are not essential for developmental osteoclastogenesis (41, 42). The activation of NF-
B by all of these cytokines depends on integrity of the IKK complex (21); recent results show that a small peptide that can prevent binding of the IKK
and IKKß catalytic subunits to the IKK
regulatory subunit can inhibit inflammation-induced bone loss (27). Although these results provide further support for the role of NF-
B in osteoclastogenesis, it has not been established which of the two IKK catalytic subunits plays a more critical role in basal osteoclastogenesis and inflammation-induced bone loss. For instance, it recently was described that the NIK- and IKK
-dependent alternative pathway is required for RANKL-induced osteoclast differentiation in vitro (25). Our results demonstrate a critical role for IKKß, but not IKK
, in basic osteoclastogenesis in vivo and in inflammation-induced bone loss.
Although IKK
and IKKß are important transducers of osteoclastogenic signals which emanate from RANKL in vitro, the mutation that prevents IKK
activation by its upstream kinase, NIK (23), had no effect on osteoclast formation in vivo and did not increase bone density. Furthermore, Ikk
AA mice were fully sensitive to inflammation-induced bone loss (unpublished data). These results are consistent with the small effect of the Ikk
AA mutation on RANKL-induced translocation of NF-
B proteins to the nucleus, as well as the absence of an osteopetrotic phenotype in mice defective in NIK, the upstream activator of IKK
(25). Like BM progenitors from Nik/ mice, Ikk
AA BM progenitors do not differentiate in response to RANKL when cultured in the absence of osteoblasts. However, either osteoblasts or the proinflammatory cytokines, IL-1ß and TNF
, together with RANKL induce osteoclastogenesis of Ikk
AA BM progenitors. Thus, although IKK
contributes to RANKL-induced differentiation in vitro, its function in vivo is dispensable because of the action of other factors that activate the IKKß-driven classic NF-
B pathway. During normal bone development, these factors are likely to be derived from osteoblasts, whereas during inflammation these factors could be TNF
and IL-1ß.
In contrast with IKK
, our findings illustrate a critical function for IKKß. IKKß-deficient BM progenitors do not form osteoclasts in vitro in response to RANKL or when cocultured with osteoblasts. Furthermore, their inability to respond to RANKL cannot be complemented by IL-1 or TNF
, and Ikkß
mice are osteopetrotic. IKKß-deficient BM progenitors are extremely sensitive to TNF
and undergo extensive apoptosis, despite the presence of the myeloid survival factor, M-CSF. Because TNF
-induced apoptosis of IKKß-deficient preosteoclasts is prevented by the loss of TNFR1, we propose that one of the mechanisms by which IKKß-dependent NF-
B activation contributes to osteoclastogenesis in vivo, especially during inflammation, is through prevention of TNF
-induced apoptosis of osteoclast progenitors. In the absence of IKKß, such cells become very sensitive to TNF
and are eliminated when TNF
is produced in sufficiently large amounts. Nonetheless, although the death of Ikkß-deficient osteoclast progenitors is prevented by loss of TNFR1 and Ikkß
:Tnfr1/ double mutants display close to normal numbers of TRAP-positive cells in their bones (Fig. 6 B), these mice become osteopetrotic (Fig. 6 A) if Ikkß deletion is induced early during bone development. The explanation for these results is that IKKß-deficient osteoclasts remain defective in bone resorption, even when their TNF
-induced elimination does not take place. Our in vitro results (Fig. 5 A) suggest that Ikkß
:Tnfr1/ progenitors cannot give rise to fully differentiated osteoclasts, although they do become TRAP-positive in response to RANKL. Morphologically, such cells look like monocytes, rather than multinucleated giant cells; this suggests that their ability to undergo cell fusion is eliminated. Similar defects in basal osteoclast functions were observed in Traf6/ and Src/ mice (16, 43). These mice are osteopetrotic as a result of the presence of osteoclasts that are unable to form ruffled borders, and therefore, are defective in bone resorption.
Thus, in addition to the prevention of TNF
-induced apoptosis, IKKß is required for terminal osteoclast differentiation. Although IKKß-dependent NF-
B activation is essential for this process, it is not sufficient; potent NF-
B activating cytokines, such as TNF
, cannot substitute for RANKL (Fig. 7 E). Most likely, another pathway or factor, designated X in Fig. 7 E, needs to be switched onalong with IKKß and NF-
Bfor terminal osteoclast differentiation to take place. Nonetheless, our results illustrate the potential ability of IKKß inhibition to prevent inflammation-driven bone destruction. Again, the mechanism through which IKKß inhibition prevents inflammation-induced bone loss involves sensitization of osteoclast progenitors to TNF
-induced apoptosis, because Ikkß
:Tnfr1/ mice are fully susceptible to inflammation-induced bone loss. The bone-resorbing ability of osteoclasts in Ikkß
:Tnfr1/ mice seems to be restored after LPS injection. Because these cells do not respond to TNF
(as a result of the loss of TNFR1), the inflammation-induced factor that may stimulate their bone-resorbing activity could be IL-1ß, which is known to be induced by LPS administration (44). However, in vitro IL-1ß is unable to induce the formation of multinucleated bone-resorbing cells when given together with RANKL once IKKß is absent (Fig. 5 A). Alternatively, Ikkß
:Tnfr1/ osteoclast progenitors may be only partially defective in their ability to respond to RANKL; this defect may be eliminated when high levels of RANKL are present along with other proinflammatory cytokines. LPS-induced inflammation results in induction of RANKL along with other cytokines (45).
Our results support a role for IKKß as an important regulator of bone homeostasis and a mediator of inflammation-induced bone loss. Our results also suggest that the major mechanism through which deletion or inhibition of IKKß exerts its therapeutic effect in inflammation-induced bone loss is by predisposing osteoclast precursors to TNF
-induced apoptosis. A schematic model that summarizes our findings is presented in Fig. 7 E. Binding of RANKL to its receptor, RANK, induces a cascade of events that leads to activation of IKKß and at least one more factorthat together with IKKßis required for induction of terminal osteoclast differentiation. During inflammation, proinflammatory cytokines, such as TNF
and IL-1ß, are induced and strongly potentiate RANKL-induced osteoclastogenesis, although such factors cannot induce osteoclast differentiation on their own. TNF
signaling through TNFR1 has the potential to induce apoptosis through caspase 8, a process that is prevented by IKKß-dependent NF-
B activation (46). Once IKKß is inhibited, TNF
-induced apoptosis results in elimination of Ikkß-deficient osteoclast progenitors, and thereby, prevents inflammation-induced bone destruction. Thus, IKKß inhibition presents a logical strategy for prevention of numerous bone-resorbing disorders that are triggered by inflammation, such as rheumatoid arthritis.
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MATERIALS AND METHODS
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Mice.
Ikk
AA and IkkßF/F mice were generated as described (29, 32). To delete IKKß in hematopoietic cells, IkkßF/F mice were crossed with Mx1-Cre transgenic mice (33), and IkkßF/F:Mx1-Cre progeny were injected three times with poly(IC) every 2 d. Injections started at day 9 after birth for in vivo analysis, whereas injections were performed 10 d before killing mice or collecting BM cells, respectively. Deletion of Ikkß was confirmed by PCR, whereas the absence of IKKß protein was examined by immunoblotting. These mice are referred to as Ikkß
mice. All experimental procedures were approved by the Animal Subjects Committee at the University of California San Diego, according to U.S. National Institutes of Health guidelines.
Histologic and histomorphometric analyses.
Tissues were fixed in PBS-buffered 4% formaldehyde, embedded in paraffin, sectioned at 5 µm, and stained as indicated using standard techniques. Calcified tissues were decalcified in EDTA (0.5 M, pH 8) for 12 d before embedding. TRAP staining was performed using a leukocyte acid phosphatase kit (Sigma-Aldrich). For histomorphometry, tibiae were embedded in methacrylate (Echnovit; Heraeus Kulzer) without previous decalcification and 34-µm sections were stained with Goldner trichrome. Histomorphometry of metaphyses was performed using an Axioskop 2 microscope (Carl Zeiss MicroImaging, Inc.) and OsteoMeasure Analysis System (OsteoMetrics) according to international standards (47). TUNEL assay and immunohistochemistry were as described previously (48, 49).
Osteoclast culture and activity assay.
BM cells from 6-wk-old mice were plated in the presence of 5 ng/ml recombinant M-CSF for 24 h Nonadherent cells were replated in the presence of recombinant M-CSF (10 ng/ml) and recombinant-RANKL (50 ng/ml) for 7 d and then fixed and stained for TRAP activity. For biochemical analysis, BM cells were plated in the presence of M-CSF (10 ng/ml) for 6 d, collected, and counted. Osteoclast activity was assayed in vitro after differentiation on a calcium phosphate film (BioCoat Bone Cell Culture System, Osteologic) and the resorpted area was quantified using AxioVision 4.3 software. Osteoclast activity in vitro was measured in urine samples using RatLaps ELISA (Nordic Bioscience Diagnostics).
Osteoblastosteoclast cocultures.
Primary osteoblasts were isolated from calvarias of neonatal (24-d-old) WT and Ikk
AA mice and were digested for 10 min in modified Eagle's medium (
-MEM) which contained 0.1% collagenase and 0.2% dispase. Cells from two to five mice were combined as an osteoblastic cell population and plated at a density of 5 x 105 cells/ml in
-MEM with 10% FCS for 24 h. Cocultures were performed as described (36, 50). Briefly, BM cells (106 per well) were added to primary osteoblasts (5 x 105 cells per well) and cultured in
-MEM which contained 10% FCS, 108 M 1,25(OH)2-vitamin D3, and 107 M dexamethasone in 24-well plates. Intensity of TRAP staining was measured by Image Pro Plus 5.1.
Subcellular fractionation and immunoblot analysis.
Cells were resuspended in buffer L1 (50 mM Tris-Cl, pH 8.0, 2 mM EDTA, 0.1% NP-40, 10% glycerol) that contained protease inhibitors, incubated for 5 min at 4°C, and centrifuged for 5 min at 4,000 revolutions/min in a microcentrifuge. Cytoplasmic supernatants were stored and nuclear pellets were extracted further in buffer L2 (20 mM Hepes-KOH, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 25 mM ß-glycerophosphate) which contained protease inhibitors. After lysis, nuclei were centrifuged at 15,000 revolutions/min and the supernatant was collected for further analysis. Nuclear (5 µg) and cytoplasmic (20 µg) extracts were electrophoresed, transferred to nitrocellulose membranes, and immunoblotted with anti-IKK
(Imgenex), anti-IKKß (UBI), anti-RelA, anti-RelB, and anti-c-Rel (Santa Cruz Biotechnology, Inc.) antibodies. p100 processing in total extracts was assayed using antiNF-
B/p52 K-27 antibody (Santa Cruz Biotechnology, Inc.).
IKK and gel shift assays.
IKK immunocomplex kinase assay was as described (51), except that an IKK
antibody (BD Biosciences) was used for immunoprecipitation. Electrophoretic mobility shift assay for NF-
B was described previously (23).
Inflammation-induced bone loss.
2- to 3-mo-old mice were given an intrajoint injection of Escherichia coli LPS (Sigma-Aldrich), 500 µg in saline, and a vehicle control into the contralateral joint, 5 d after the last poly(IC) treatment. 5 d later, mice were killed and joint histology was examined in the Histologic and histomorphometric analyses section.
Statistical analysis.
Data are expressed as mean ± SEM. Differences were analyzed by Student's t test.
Online supplemental material
IkkßF/F (Video 1) and Ikkß
(Video 2) mice were given an intrajoint injection of E. coli LPS (Sigma-Aldrich), 500 µg in saline, (left hind limb) and a vehicle control (right hind limb). The two videos show the ability of the mice to move and stretch their hind limbs. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20042081/DC1.
 |
Acknowledgments
|
---|
The authors would like to thank Drs. A. Hoebertz and L. Kenner for helpful discussion and A. Chang for LPS knee joint injections.
S. Maeda, J.M. Park, L.-C. Hsu, and Y. Cao were supported by postdoctoral fellowships from the Japan Society for the Promotion of Science, Bristol-Myers Squibb Foundation Research Fellowship at the Irvington Institute, the Cancer Research Institute, and an American Association for Cancer ResearchGenentech BioOncology Career Development Award for Cancer Research. Support was provided by National Institutes of Health grants nos. ES06376 and AI43477 (to M. Karin). In addition, M. Karin is an American Cancer Society Research Professor.
The authors have no conflicting financial interests.
Submitted: 8 October 2004
Accepted: 7 April 2005
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References
|
---|
- Karsenty, G., and E.F. Wagner. 2002. Reaching a genetic and molecular understanding of skeletal development. Dev. Cell. 2:389406.[CrossRef][Medline]
- Manolagas, S.C., and R.L. Jilka. 1995. Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. N. Engl. J. Med. 332:305311.[Free Full Text]
- Gravallese, E.M. 2002. Bone destruction in arthritis. Ann. Rheum. Dis. 61(Suppl 2):ii8486.[Abstract/Free Full Text]
- Suda, T., N. Takahashi, N. Udagawa, E. Jimi, M.T. Gillespie, and T.J. Martin. 1999. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr. Rev. 20:345357.[Abstract/Free Full Text]
- Kong, Y.Y., H. Yoshida, I. Sarosi, H.L. Tan, E. Timms, C. Capparelli, S. Morony, A.J. Oliveira-dos-Santos, G. Van, A. Itie, et al. 1999. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature. 397:315323.[CrossRef][Medline]
- Lacey, D.L., E. Timms, H.L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 93:165176.[CrossRef][Medline]
- Anderson, D.M., E. Maraskovsky, W.L. Billingsley, W.C. Dougall, M.E. Tometsko, E.R. Roux, M.C. Teepe, R.F. DuBose, D. Cosman, and L. Galibert. 1997. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature. 390:175179.[CrossRef][Medline]
- Dougall, W.C., M. Glaccum, K. Charrier, K. Rohrbach, K. Brasel, T. De Smedt, E. Daro, J. Smith, M.E. Tometsko, C.R. Maliszewski, et al. 1999. RANK is essential for osteoclast and lymph node development. Genes Dev. 13:24122424.[Abstract/Free Full Text]
- Zhang, Y.H., A. Heulsmann, M.M. Tondravi, A. Mukherjee, and Y. Abu-Am. 2001. Tumor necrosis factor-alpha (TNF) stimulates RANKL-induced osteoclastogenesis via coupling of TNF type 1 receptor and RANK signaling pathways. J. Biol. Chem. 276:563568.[Abstract/Free Full Text]
- Kobayashi, K., N. Takahashi, E. Jimi, N. Udagawa, M. Takami, S. Kotake, N. Nakagawa, M. Kinosaki, K. Yamaguchi, N. Shima, et al. 2000. Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J. Exp. Med. 191:275286.[Abstract/Free Full Text]
- Lam, J., S. Takeshita, J.E. Barker, O. Kanagawa, F.P. Ross, and S.L. Teitelbaum. 2000. TNF-alpha induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand. J. Clin. Invest. 106:14811488.[Abstract/Free Full Text]
- Tsao, D.H., T. McDonagh, J.B. Telliez, S. Hsu, K. Malakian, G.Y. Xu, and L.L. Lin. 2000. Solution structure of N-TRADD and characterization of the interaction of N-TRADD and C-TRAF2, a key step in the TNFR1 signaling pathway. Mol. Cell. 5:10511057.[CrossRef][Medline]
- Inoue, J., T. Ishida, N. Tsukamoto, N. Kobayashi, A. Naito, S. Azuma, and T. Yamamoto. 2000. Tumor necrosis factor receptor-associated factor (TRAF) family: adapter proteins that mediate cytokine signaling. Exp. Cell Res. 254:1424.[CrossRef][Medline]
- Baud, V., Z.G. Liu, B. Bennett, N. Suzuki, Y. Xia, and M. Karin. 1999. Signaling by proinflammatory cytokines: oligomerization of TRAF2 and TRAF6 is sufficient for JNK and IKK activation and target gene induction via an amino-terminal effector domain. Genes Dev. 13:12971308.[Abstract/Free Full Text]
- Iotsova, V., J. Caamano, J. Loy, Y. Yang, A. Lewin, and R. Bravo. 1997. Osteopetrosis in mice lacking NF-kappaB1 and NF-kappaB2. Nat. Med. 3:12851289.[CrossRef][Medline]
- Lomaga, M.A., W.C. Yeh, I. Sarosi, G.S. Duncan, C. Furlonger, A. Ho, S. Morony, C. Capparelli, G. Van, S. Kaufman, et al. 1999. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev. 13:10151024.[Abstract/Free Full Text]
- Franzoso, G., L. Carlson, L. Xing, L. Poljak, E.W. Shores, K.D. Brown, A. Leonardi, T. Tran, B.F. Boyce, and U. Siebenlist. 1997. Requirement for NF-kappaB in osteoclast and B-cell development. Genes Dev. 11:34823496.[Abstract/Free Full Text]
- Grigoriadis, A.E., Z.Q. Wang, M.G. Cecchini, W. Hofstetter, R. Felix, H.A. Fleisch, and E.F. Wagner. 1994. c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science. 266:443448.[Medline]
- Karin, M. 1995. The regulation of AP-1 activity by mitogen-activated protein kinases. J. Biol. Chem. 270:1648316486.[Free Full Text]
- May, M.J., and S. Ghosh. 1997. Rel/NF-kappa B and I kappa B proteins: an overview. Semin. Cancer Biol. 8:6373.[CrossRef][Medline]
- Ghosh, S., and M. Karin. 2002. Missing pieces in the NF-kappaB puzzle. Cell. 109(Suppl):S8196.[CrossRef][Medline]
- Rothwarf, D.M., and M. Karin. 1999. The NF-kappa B activation pathway: a paradigm in information transfer from membrane to nucleus. Sci STKE:RE1.
- Senftleben, U., Y. Cao, G. Xiao, F.R. Greten, G. Krahn, G. Bonizzi, Y. Chen, Y. Hu, A. Fong, S.C. Sun, and M. Karin. 2001. Activation by IKKalpha of a second, evolutionary conserved, NF-kappa B signaling pathway. Science. 293:14951499.[Abstract/Free Full Text]
- Xiao, G., E.W. Harhaj, and S.C. Sun. 2001. NF-kappaB-inducing kinase regulates the processing of NF-kappaB2 p100. Mol. Cell. 7:401409.[CrossRef][Medline]
- Novack, D.V., L. Yin, A. Hagen-Stapleton, R.D. Schreiber, D.V. Goeddel, F.P. Ross, and S.L. Teitelbaum. 2003. The IkappaB function of NF-kappaB2 p100 controls stimulated osteoclastogenesis. J. Exp. Med. 198:771781.[Abstract/Free Full Text]
- Shinkura, R., K. Kitada, F. Matsuda, K. Tashiro, K. Ikuta, M. Suzuki, K. Kogishi, T. Serikawa, and T. Honjo. 1999. Alymphoplasia is caused by a point mutation in the mouse gene encoding Nf-kappa b-inducing kinase. Nat. Genet. 22:7477.[CrossRef][Medline]
- Jimi, E., K. Aoki, H. Saito, F. D'Acquisto, M.J. May, I. Nakamura, T. Sudo, T. Kojima, F. Okamoto, H. Fukushima, et al. 2004. Selective inhibition of NF-kappa B blocks osteoclastogenesis and prevents inflammatory bone destruction in vivo. Nat. Med. 10:617624.[CrossRef][Medline]
- Delhase, M., M. Hayakawa, Y. Chen, and M. Karin. 1999. Positive and negative regulation of IkappaB kinase activity through IKKbeta subunit phosphorylation. Science. 284:309313.[Abstract/Free Full Text]
- Cao, Y., G. Bonizzi, T.N. Seagroves, F.R. Greten, R. Johnson, E.V. Schmidt, and M. Karin. 2001. IKKalpha provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development. Cell. 107:763775.[CrossRef][Medline]
- Bonizzi, G., M. Bebien, D.C. Otero, K.E. Johnson-Vroom, Y. Cao, D. Vu, A.G. Jegga, B.J. Aronow, G. Ghosh, R.C. Rickert, and M. Karin. 2004. Activation of IKKalpha target genes depends on recognition of specific kappaB binding sites by RelB:p52 dimers. EMBO J. 23:42024210.[Abstract/Free Full Text]
- Yin, L., L. Wu, H. Wesche, C.D. Arthur, J.M. White, D.V. Goeddel, and R.D. Schreiber. 2001. Defective lymphotoxin-beta receptor-induced NF-kappaB transcriptional activity in NIK-deficient mice. Science. 291:21622165.[Abstract/Free Full Text]
- Li, Z.W., S.A. Omori, T. Labuda, M. Karin, and R.C. Rickert. 2003. IKK beta is required for peripheral B cell survival and proliferation. J. Immunol. 170:46304637.[Abstract/Free Full Text]
- Kuhn, R., F. Schwenk, M. Aguet, and K. Rajewsky. 1995. Inducible gene targeting in mice. Science. 269:14271429.[Medline]
- Hsu, L.C., J.M. Park, K. Zhang, J.L. Luo, S. Maeda, R.J. Kaufman, L. Eckmann, D.G. Guiney, and M. Karin. 2004. The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4. Nature. 428:341345.[CrossRef][Medline]
- Amling, M., L. Neff, M. Priemel, A.F. Schilling, J.M. Rueger, and R. Baron. 2000. Progressive increase in bone mass and development of odontomas in aging osteopetrotic c-src-deficient mice. Bone. 27:603610.[CrossRef][Medline]
- Jimi, E., I. Nakamura, H. Amano, Y. Taguchi, T. Tsurukai, M. Tamura, N. Takahashi, and T. Suda. 1996. Osteoclast function is activated by osteoblastic cells through a mechanism involving cell-to-cell contact. Endocrinology. 137:21872190.[Abstract]
- Abu-Amer, Y., J. Erdmann, L. Alexopoulou, G. Kollias, F.P. Ross, and S.L. Teitelbaum. 2000. Tumor necrosis factor receptors types 1 and 2 differentially regulate osteoclastogenesis. J. Biol. Chem. 275:2730727310.[Abstract/Free Full Text]
- Baud, V., and M. Karin. 2001. Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol. 11:372377.[CrossRef][Medline]
- Chiang, C.Y., G. Kyritsis, D.T. Graves, and S. Amar. 1999. Interleukin-1 and tumor necrosis factor activities partially account for calvarial bone resorption induced by local injection of lipopolysaccharide. Infect. Immun. 67:42314236.[Abstract/Free Full Text]
- Takayanagi, H., K. Ogasawara, S. Hida, T. Chiba, S. Murata, K. Sato, A. Takaoka, T. Yokochi, H. Oda, K. Tanaka, et al. 2000. T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma. Nature. 408:600605.[CrossRef][Medline]
- Li, P., H. Allen, S. Banerjee, S. Franklin, L. Herzog, C. Johnston, J. McDowell, M. Paskind, L. Rodman, J. Salfeld, et al. 1995. Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell. 80:401411.[CrossRef][Medline]
- Rothe, J., W. Lesslauer, H. Lotscher, Y. Lang, P. Koebel, F. Kontgen, A. Althage, R. Zinkernagel, M. Steinmetz, and H. Bluethmann. 1993. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature. 364:798802.[CrossRef][Medline]
- Soriano, P., C. Montgomery, R. Geske, and A. Bradley. 1991. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 64:693702.[CrossRef][Medline]
- Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol. 21:335376.[CrossRef][Medline]
- Wada, N., H. Maeda, Y. Yoshimine, and A. Akamine. 2004. Lipopolysaccharide stimulates expression of osteoprotegerin and receptor activator of NF-kappa B ligand in periodontal ligament fibroblasts through the induction of interleukin-1 beta and tumor necrosis factor-alpha. Bone. 35:629635.[CrossRef][Medline]
- Karin, M., and A. Lin. 2002. NF-kappaB at the crossroads of life and death. Nat. Immunol. 3:221227.[CrossRef][Medline]
- Parfitt, A.M., M.K. Drezner, F.H. Glorieux, J.A. Kanis, H. Malluche, P.J. Meunier, S.M. Ott, and R.R. Recker. 1987. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res. 2:595610.[Medline]
- Park, J.M., F.R. Greten, Z.W. Li, and M. Karin. 2002. Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science. 297:20482051.[Abstract/Free Full Text]
- Chen, L.W., L. Egan, Z.W. Li, F.R. Greten, M.F. Kagnoff, and M. Karin. 2003. The two faces of IKK and NF-kappaB inhibition: prevention of systemic inflammation but increased local injury following intestinal ischemia-reperfusion. Nat. Med. 9:575581.[CrossRef][Medline]
- Udagawa, N., N. Takahashi, T. Akatsu, H. Tanaka, T. Sasaki, T. Nishihara, T. Koga, T.J. Martin, and T. Suda. 1990. Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc. Natl. Acad. Sci. USA. 87:72607264.[Abstract/Free Full Text]
- DiDonato, J.A., M. Hayakawa, D.M. Rothwarf, E. Zandi, and M. Karin. 1997. A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB. Nature. 388:548554.[CrossRef][Medline]
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