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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
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 IB kinase (IKK) complex that mediate NF-B activation, IKK and IKK, in osteoclast formation and inflammation-induced bone loss. Our findings point out the importance of the IKK 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, IKK is required for osteoclastogenesis in vitro and in vivo. IKK 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; IB, inhibitor of NF-B; IKK, IB kinase; M-CSF, macrophage-colony stimulating factor; NIK, NF-B–inducing kinase; poly(IC), polyinosinic-polycytidylic acid; RANKL, receptor activator of NF-B ligand; TNFR, TNF receptor; TRAP, tartrate-resistant acid phosphatase; TUNEL, terminal deoxynucleotidyl transferase–mediated 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 factor–related 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 (12–15). The relevance of these pathways to osteoclastogenesis is underscored by the osteopetrotic phenotypes of mice lacking TNF receptor–associated 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 IB kinase (IKK) complex that phosphorylates the inhibitors of NF-B (IBs) and targets them to ubiquitin-dependent degradation (21). The IBs 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 IKK, and a regulatory subunit, IKK/NF-B essential modulator (22). Gene disruption experiments demonstrated that IKK and IKK are important for IB 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-B–inducing 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 IKK 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 Ikk genes. We found that IKK, but not IKK, is essential for inflammation-induced bone loss and is required for osteoclastogenesis in vivo. However, the main function of IKK in osteoclastogenesis is to prevent TNF-induced apoptosis of osteoclast precursors. Once TNF-induced apoptosis is prevented through deletion of the Tnfr1 gene, IKK is no longer required for induction of inflammation-induced bone loss, but it is still needed for basal osteoclast function.
RESULTS
RANKL-induced in vitro osteoclastogenesis requires IKK and IKK
To determine the roles of the IKK catalytic subunits in osteoclastogenesis, we used mice that carry mutant forms of either subunit—IkkAA and Ikk mice. IkkAA 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). IkkAA 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. Ikk mice were generated by crossing IkkF/F mice, homozygotes for a "floxed" Ikk 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 IkkF/F:Mx1-Cre mice results in efficient deletion of the floxed third exon of the Ikk gene and generation of IKK-deficiency in IFN-responsive cells, including myeloid cells (34). To determine whether defective IKK activation or complete loss of IKK affect osteoclastogenesis, we first used an in vitro differentiation system. BM hematopoietic progenitors that were isolated from WT, IkkAA, and Ikk 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 IkkAA and Ikk cultures (Fig. 1). Expression of several other osteoclast markers, including CatK and matrix metalloproteinase-9, also was defective in RANKL-treated IkkAA and Ikk BM cultures (unpublished data). These results suggest that IKK and IKK are required for, and play nonredundant roles in, M-CSF and RANKL-induced osteoclastogenesis in vitro.
RANKL-mediated NF-B activation is impaired in Ikk, but not IkkAA, osteoclast progenitors
To gain insights into the possible mechanisms by which IKK and IKK promote osteoclastogenesis in vitro, we examined RANKL-induced NF-B activation in IkkAA and Ikk BM cultures. BM-derived precursors from WT, IkkAA, and Ikk mice were incubated with M-CSF and RANKL and analyzed for expression of IKK and IKK by immunoblot analysis (Fig. 2 A). As expected, no IKK protein was detectable in Ikk BM cells. Interestingly, the deletion of IKK 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:IKK:IKK complexes (22). Electrophoretic mobility shift assays revealed marked induction of NF-B DNA binding activity in response to RANKL treatment in WT and IkkAA cultures, but only a weak response in Ikk cultures (Fig. 2 B). Similarly, RANKL activated IKK in WT and IkkAA cultures, but no IKK activity could be detected before or after RANKL treatment of Ikk 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 IkkAA 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 Ikk cultures. Although IKK plays a minor role in NF-B activation, other experiments that were conducted with IkkAA 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).
Defective osteoclastogenesis in Ikk but not IkkAA mice
To determine if IKK and IKK play a role in osteoclastogenesis in vivo, bones from 4 mo-old IkkAA and Ikk mice were analyzed. To study the role of IKK in bone development we induced deletion of the Ikk gene in IkkF/F:Mx1-Cre mice 9 d after birth. Histologic and histomorphometric analyses of long bone (tibias) sections from IkkAA 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 A–C). These results suggest that basal osteoclastogenesis is not affected by the loss of IKK activation in vivo. By contrast, Ikk mice showed an osteopetrotic phenotype. Ikk mice are smaller and have a hunched back compared with their IkkF/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 IkkF/F littermates or WT mice of the same age (Fig. 3 A). TRAP staining showed a greatly reduced number of osteoclasts in Ikk bones (Fig. 3, B and C). Furthermore, quantitative histomorphometric analyses of Ikk 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 Ikk 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 Ikk mice becomes more dramatic with age (Fig. 3 C). These results suggest that Ikk mice develop osteopetrosis that is due to defective osteoclast formation, and furthermore, indicate a critical role for Ikk in bone development.
Osteoblasts can rescue defective in vitro osteoclastogenesis of IkkAA but not Ikk 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 IkkAA and Ikk BM cultures are caused by cell autonomous defects, we performed osteoclasts/osteoblast cocultures. Wt osteoblasts fully supported osteoclastogenesis of IkkAA osteoclast precursors, but not Ikk osteoclast precursors; this indicates a cell–autonomous osteoclast differentiation defect in Ikk BM cells (Fig. 4 A, upper panels). Reciprocal cocultures of IkkAA or WT BM cells with IkkAA osteoblasts showed that IkkAA osteoblasts had the same capacity as WT osteoblasts for supporting osteoclast differentiation (Fig. 4, A [lower panels] and B). Thus, IkkAA 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 IkkAA, but not Ikk osteoclast progenitors
To identify potential signals that may compensate for the defect in RANKL signaling of IkkAA 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, IkkAA, and Ikk 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 IkkAA BM, although they did not induce differentiation on their own (Fig. 5 A and not depicted). However, the defect in osteoclast differentiation of Ikk 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. IkkAA, but not Ikk, 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 Ikk osteoclast precursors were dead within 48 h (Fig. 5 A). This prompted us to examine whether Ikk BM cells undergo apoptosis in response to TNF. Terminal deoxynucleotidyl transferase–mediated 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 Ikk 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.
Deletion of Tnfr1 rescues Ikk 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 Ikk-deficient preosteoclasts, we crossed Ikk mice with Tnfr1–/– mice to generate IkkF/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 Ikk osteoclast progenitors (Fig. 5 A, bottom). Furthermore, the absence of TNFR1 rescued the inability of Ikk 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, IKK 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, Ikk, Tnfr1–/–, and Ikk:Tnfr1–/–mice. Ikk deletion in both strains was induced as early as 9 d after birth. Histochemical staining revealed osteopetrosis in Ikk:Tnfr1–/– bones (Fig. 6 A). However, TRAP-positive cells could be detected easily in Ikk:Tnfr1–/– mice, whereas they were scarce in Ikk mice (Fig. 6 B). Analysis of deoxypiridinoline cross-links in urine samples—which reflects osteoclast activity in vivo—revealed that in Ikk:Tnfr1–/– mice, osteoclast resorption activity remained severely attenuated, as in Ikk mice. Normal levels of deoxypiridinoline cross-links were found in urine samples from IkkAA or Tnfr1–/– mice.
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 Ikk mice, whereas many more such cells were seen in WT mice (Fig. 6 D). Remarkably, the loss of TNFR1 in Ikk: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 Ikk 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 Ikk 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 Ikk (Fig. 6 F). Despite the decreased apoptosis of osteoclast precursors and restoration of TRAP-positive cells, the Ikk:Tnfr1–/– double mutant mice remain osteopetrotic. This suggests that osteoclasts that lack IKK 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 IKK protects mice from inflammation-induced bone loss, in a manner dependent on TNFR1
IKK plays a crucial role in osteoclast differentiation under physiologic conditions. We next analyzed whether IKK or IKK is involved in inflammation-induced bone loss. We used an established model of endotoxin-induced bone resorption (39, 40). IkkAA, Ikk, Tnfr1–/–, Ikk: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. Ikk 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 Ikk mutant mice compared with WT controls or IkkAA mice (Fig. 7, A–D; not depicted for IkkAA). Although LPS-injected WT, IkkAA, and Tnfr1–/– mice completely lost the ability to flex the hind limb, no such aberrations were evident in Ikk 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 Ikk (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 Ikk:Tnfr1–/– and Tnfr1–/– mice. These results suggest that under strong inflammatory conditions, which are likely to result in massive cytokine production, IKK is no longer needed for osteoclastogenesis once its survival function has been rendered unnecessary by ablation of TNFR1.
DISCUSSION
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 IKK 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 IKK, but not IKK, in basic osteoclastogenesis in vivo and in inflammation-induced bone loss.
Although IKK and IKK 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, IkkAA mice were fully sensitive to inflammation-induced bone loss (unpublished data). These results are consistent with the small effect of the IkkAA 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, IkkAA BM progenitors do not differentiate in response to RANKL when cultured in the absence of osteoblasts. However, either osteoblasts or the proinflammatory cytokines, IL-1 and TNF, together with RANKL induce osteoclastogenesis of IkkAA 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 IKK-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-1.
In contrast with IKK, our findings illustrate a critical function for IKK. IKK-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 Ikk mice are osteopetrotic. IKK-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 IKK-deficient preosteoclasts is prevented by the loss of TNFR1, we propose that one of the mechanisms by which IKK-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 IKK, such cells become very sensitive to TNF and are eliminated when TNF is produced in sufficiently large amounts. Nonetheless, although the death of Ikk-deficient osteoclast progenitors is prevented by loss of TNFR1 and Ikk: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 Ikk deletion is induced early during bone development. The explanation for these results is that IKK-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 Ikk: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, IKK is required for terminal osteoclast differentiation. Although IKK-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 on—along with IKK and NF-B—for terminal osteoclast differentiation to take place. Nonetheless, our results illustrate the potential ability of IKK inhibition to prevent inflammation-driven bone destruction. Again, the mechanism through which IKK inhibition prevents inflammation-induced bone loss involves sensitization of osteoclast progenitors to TNF-induced apoptosis, because Ikk:Tnfr1–/– mice are fully susceptible to inflammation-induced bone loss. The bone-resorbing ability of osteoclasts in Ikk: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-1, which is known to be induced by LPS administration (44). However, in vitro IL-1 is unable to induce the formation of multinucleated bone-resorbing cells when given together with RANKL once IKK is absent (Fig. 5 A). Alternatively, Ikk: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 IKK 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 IKK 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 IKK and at least one more factor—that together with IKK—is required for induction of terminal osteoclast differentiation. During inflammation, proinflammatory cytokines, such as TNF and IL-1, 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 IKK-dependent NF-B activation (46). Once IKK is inhibited, TNF-induced apoptosis results in elimination of Ikk-deficient osteoclast progenitors, and thereby, prevents inflammation-induced bone destruction. Thus, IKK inhibition presents a logical strategy for prevention of numerous bone-resorbing disorders that are triggered by inflammation, such as rheumatoid arthritis.
MATERIALS AND METHODS
Mice.
IkkAA and IkkF/F mice were generated as described (29, 32). To delete IKK in hematopoietic cells, IkkF/F mice were crossed with Mx1-Cre transgenic mice (33), and IkkF/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 Ikk was confirmed by PCR, whereas the absence of IKK protein was examined by immunoblotting. These mice are referred to as Ikk 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 3–4-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).
Osteoblast–osteoclast cocultures.
Primary osteoblasts were isolated from calvarias of neonatal (2–4-d-old) WT and IkkAA 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, 10–8 M 1,25(OH)2-vitamin D3, and 10–7 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 -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-IKK (UBI), anti-RelA, anti-RelB, and anti-c-Rel (Santa Cruz Biotechnology, Inc.) antibodies. p100 processing in total extracts was assayed using anti–NF-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
IkkF/F (Video 1) and Ikk (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 Research–Genentech 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.
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