Osteoclast Differentiation Is Impaired in the Absence of Inhibitor of κB Kinase α

Abstract

Signaling through the receptor activator of nuclear factor B (RANK) is required for both osteoclast differentiation and mammary gland development, yet the extent to which RANK utilizes similar signaling pathways in these tissues remains unclear. Mice expressing a kinase-inactive form of the inhibitor of B kinase ␣ (IKK␣) have mammary gland defects similar to those of RANKnull mice yet have apparently normal osteoclast function. Because mice that completely lack IKK␣ have severe skin and skeletal defects that are not associated with IKK␣-kinase activity, we wished to directly examine osteoclastogenesis in IKK␣ ؊/؊ mice. We found that unlike RANK-null mice, which completely lack osteoclasts, IKK␣ ؊/؊ mice did possess normal numbers of TRAP ؉ osteoclasts. However, only 32% of these cells were multinucleated compared with 57% in wild-type littermates. A more profound defect in osteoclastogenesis was observed in vitro using IKK␣ ؊/؊ hematopoietic cells treated with colony-stimulating factor 1 and RANK ligand (RANKL), as the cells failed to form large, multinucleated osteoclasts. Additionally, overall RANKL-induced global gene expression was significantly blunted in IKK␣ ؊/؊ cells, including osteoclastspecific genes such as TRAP, MMP-9, and c-Src. IKK␣ was not required for RANKL-mediated I B␣ degradation or phosphorylation of mitogen-activated protein kinases but was required for RANKL-induced p100 processing. Treatment of IKK␣ ؊/؊ cells with tumor necrosis factor ␣ (TNF␣) in combination with RANKL led to partial rescue of osteoclastogenesis despite a lack of p100 processing. However, the ability of TNF␣ alone or in combination with transforming growth factor ␤ to induce osteoclast differentiation was dependent on IKK␣, suggesting that synergy between RANKL and TNF␣ can overcome p100 processing defects in IKK␣ ؊/؊ cells.Skeletal mass homeostasis is maintained by the coupled activities of bone-building osteoblasts and bone-resorbing osteoclasts. Dysregulation of osteoclast differentiation or function underlies many diseases affecting the skeletal system, including osteoporosis, joint inflammation, tumor metastasis to bone, and Paget disease (1). Osteoclasts are highly specialized multinucleated cells that differentiate from monocyte/macrophage lineage hematopoietic precursors. The key factors regulating osteoclastogenesis are the TNF 1 receptor family member RANK, its ligand (RANKL), and the RANKL inhibitor, osteoprotegerin. Ablation of RANK or RANKL in mice results in a complete absence of osteoclasts and severe osteopetrosis, whereas lack of osteoprotegerin leads to excessive osteoclast activity and osteoporosis (2-5).Genetic studies in mice, as well as in vitro culture of osteoclasts using CSF-1 and RANKL, have elucidated many key components of RANK signaling during osteoclastogenesis. Binding of RANKL to RANK stimulates recruitment of TRAF2, -5, and -6 (6) followed by activation of mitogenactivated protein kinases and I B kinases, which ultimately lead to activation of the transcription factors AP-1 and NF-B. Osteo...

RANKL acts directly on RANK‐expressing prostate tumor cells and mediates migration and expression of tumor metastasis genes

“…We looked at genes upregulated in response to RANKL during osteoclastogenesis (identified in Chaisson et al [6]), and studied the regulation of a subset of these genes in PC3 cells. Using RT-PCR we confirmed that MMP-9 was upregulated approximately 25-fold in PC3 cells at 12 hr post-RANKL stimulation (Fig.…”

NF-κB and the regulation of hematopoiesis

“…These results, together with the result of the NIK studies, indicate that the NIK/IKK1 pathway is not involved in basal osteoclastogenesis, but is important for stimulated differentiation such as during inflammation or cancer. 18 The importance of IKK1 in in vitro differentiation of osteoclasts and in p100 processing to p52 upon RANKL stimulation has been confirmed using bone marrow cells expressing a kinase-dead IKK1. 19 However, this study did not confirm the in vivo role of IKK1.…”

“…In vitro experiments using IKK1-deficient fetal liver cells demonstrated the involvement of IKK1 in RANKL-mediated osteoclastogenesis. 18 Chaisson et al described a normal number of TRAP þ osteoclasts, but also showed a decrease in the number of multinucleated cells. Processing of p100 into p52 was also impaired after RANKL treatment.…”

Role of NF-κB in the skeleton

“…TNF can also induce the expression of c-fos and NFATc1, in a p50/p52-dependent manner, albeit at lower levels than RANKL [34], indicating a pro-differentiation role for TNFα as well. Furthermore, addition of TNFα with RANKL significantly rescues the ability of NIK-or IKKα-deficient precursors to form OCs in vitro, despite the fact that p100 levels are elevated [12,23,35], suggesting that the differentiation signal is independent of alternative NF-κB activation. However, in vivo, the osteoclastogenic response of relB −/− mice was limited in response to TNFα [22].…”

“…NIK −/− mice have a mild but significant increase in trabecular bone volume at baseline, but have a severely blunted OC response to RANKL administration in vivo [21,22]. In the absence of NIK or IKKα, p100 cannot be processed to p52, and accumulated p100 acts as a global IκB over time [23]. Thus, in such cases although RANKL-naïve BMMs have normal IκBα degradation and p65 activation acutely, preOCs differentiated in RANKL for 48 hours do not have significant levels of p65, p50, RelB or p52 in the nucleus.…”