Abstract
Signaling through the receptor activator of nuclear factor kappa 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 kappa B kinase alpha (IKK alpha) have mammary gland defects similar to those of RANK-null mice yet have apparently normal osteoclast function. Because mice that completely lack IKK alpha have severe skin and skeletal defects that are not associated with IKK alpha-kinase activity, we wished to directly examine osteoclastogenesis in IKK alpha(-/-) mice. We found that unlike RANK-null mice, which completely lack osteoclasts, IKK alpha(-/-) 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 alpha(-/-) 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 alpha(-/-) cells, including osteoclast-specific genes such as TRAP, MMP-9, and c-Src. IKK alpha was not required for RANKL-mediated I kappa B alpha degradation or phosphorylation of mitogen-activated protein kinases but was required for RANKL-induced p100 processing. Treatment of IKK alpha(-/-) cells with tumor necrosis factor alpha (TNF alpha) in combination with RANKL led to partial rescue of osteoclastogenesis despite a lack of p100 processing. However, the ability of TNF alpha alone or in combination with transforming growth factor beta to induce osteoclast differentiation was dependent on IKK alpha, suggesting that synergy between RANKL and TNFalpha can overcome p100 processing defects in IKK alpha(-/-) cells.
Highlights
Skeletal mass homeostasis is maintained by the coupled activities of bone-building osteoblasts and bone-resorbing osteoclasts
Significantly fewer multinucleated TRAPϩ cells were identified in inhibitor of B kinase ␣ (IKK␣)Ϫ/Ϫ embryos (32%, p Ͻ 0.001 compared with ϩ/ϩ, Fig. 1B), and these cells had an altered morphology compared with wild type, often containing a reduced dense granulated cytoplasm
Unlike RANK or RANK ligand (RANKL) knock-out mice, which completely lack TRAPϩ osteoclasts in vivo, IKK␣Ϫ/Ϫ E18.5 dpc embryos did contain multinucleated TRAPϩ osteoclasts, the frequency of these cells was less than that observed in wild-type embryos
Summary
Skeletal mass homeostasis is maintained by the coupled activities of bone-building osteoblasts and bone-resorbing osteoclasts. Mice expressing a kinase-inactive allele of the inhibitor of B kinase ␣ (IKK␣AA) had defects in mammary gland differentiation similar to those in RANK or RANKL-null mice [18] These defects were linked to a lack of NF-B activation and subsequent up-regulation of cyclin D1 in mammary epithelial cells. Because these mice die at birth, we examined osteoclast differentiation in vivo in embryonic day 18.5 postcoitus (E18.5 dpc) IKK␣Ϫ/Ϫ embryos and in vitro by culturing IKK␣Ϫ/Ϫ hematopoietic cells with CSF-1 and RANKL Using this system, we determined that the ability of RANKL to induce the formation of large multinucleated osteoclasts in vitro is impaired in the absence of IKK␣. We observed a partial absence of multinucleated osteoclasts in vivo in IKK␣Ϫ/Ϫ embryos, indicating that a lack of IKK␣ in osteoclast precursor cells in vivo may be overcome by cooperation between RANKL and other cytokines, such as TNF␣
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