Alternative NF‐κB Regulates RANKL‐Induced Osteoclast Differentiation and Mitochondrial Biogenesis via Independent Mechanisms

Mitochondrial biogenesis, the generation of new mitochondrial DNA and proteins, has been linked to osteoclast (OC) differentiation and function. In this study we used mice with mutations in key alternative NF‐κB pathway proteins, RelB and NF‐κB–inducing kinase (NIK), to dissect the complex relationship between mitochondrial biogenesis and osteoclastogenesis. In OC precursors lacking either NIK or RelB, receptor activator of NF‐κB ligand (RANKL) was unable to increase mitochondrial DNA or oxidative phosphorylation (OxPhos) protein expression, which was associated with lower oxygen consumption rates. Transgenic OC precursors expressing constitutively active NIK showed normal RANKL‐induced mitochondrial biogenesis (OxPhos expression and mitochondria copy number) compared to controls, but larger mitochondrial dimensions and increased oxygen consumption rates, suggesting increased mitochondrial function. To deduce the mechanism for mitochondrial biogenesis defects in NIK‐deficient and RelB‐deficient precursors, we examined expression of genes known to control this process. PGC‐1β (Ppargc1b) expression, but not PGC‐1α, PPRC1, or ERRα, was significantly reduced in RelB^–/– and NIK^–/– OCs. Because PGC‐1β has been reported to positively regulate both mitochondrial biogenesis and differentiation in OCs, we retrovirally overexpressed PGC‐1β in RelB^–/– cells, but surprisingly found that it did not affect differentiation, nor did it restore RANKL‐induced mitochondrial biogenesis. To determine whether the blockade in osteoclastogenesis in RelB‐deficient cells precludes mitochondrial biogenesis, we rescued RelB^–/– differentiation via overexpression of NFATc1. Mitochondrial parameters in neither WT nor RelB‐deficient cultures were affected by NFATc1 overexpression, and bone resorption in RelB^–/– was not restored. Furthermore, NFATc1 co‐overexpression with PGC‐1β, although allowing OC differentiation, did not rescue mitochondrial biogenesis or bone resorption in RelB^–/– OCs, by CTX‐I levels. Thus, our results indicate that the alternative NF‐κB pathway plays dual, but distinct, roles in controlling the independent processes of OC differentiation and OC mitochondrial biogenesis. Furthermore, the inability of PGC‐1β to drive mitochondrial biogenesis in OCs without RelB indicates a cell‐type specificity in mitochondria regulation. © 2015 American Society for Bone and Mineral Research.     

The pivotal role of the alternative NF‐κB pathway in maintenance of basal bone homeostasis and osteoclastogenesis

The alternative NF‐κB pathway consists predominantly of NF‐κB‐inducing kinase (NIK), IκB kinase α (IKKα), p100/p52, and RelB. The hallmark of the alternative NF‐κB signaling is the processing of p100 into p52 through NIK, thus allowing the binding of p52 and RelB. The physiologic relevance of alternative NF‐κB activation in bone biology, however, is not well understood. To elucidate the role of the alternative pathway in bone homeostasis, we first analyzed alymphoplasic (aly/aly) mice, which have a defective NIK and are unable to process p100, resulting in the absence of p52. We observed increased bone mineral density (BMD) and bone volume, indicating an osteopetrotic phenotype. These mice also have a significant defect in RANKL‐induced osteoclastogenesis in vitro and in vivo. NF‐κB DNA‐binding assays revealed reduced activity of RelA, RelB, and p50 and no binding activity of p52 in aly/aly osteoclast nuclear extracts after RANKL stimulation. To determine the role of p100 itself without the influence of a concomitant lack of p52, we used p100^?/? mice, which specifically lack the p100 inhibitor but still express p52. p100^?/? mice have an osteopenic phenotype owing to the increased osteoclast and decreased osteoblast numbers that was rescued by the deletion of one allele of the relB gene. Deletion of both allele of relB resulted in a significantly increased bone mass owing to decreased osteoclast activity and increased osteoblast numbers compared with wild‐type (WT) controls, revealing a hitherto unknown role for RelB in bone formation. Our data suggest a pivotal role of the alternative NF‐κB pathway, especially of the inhibitory role of p100, in both basal and stimulated osteoclastogenesis and the importance of RelB in both bone formation and resorption. © 2010 American Society for Bone and Mineral Research     

Silent information regulator (Sir)T1 inhibits NF‐κB signaling to maintain normal skeletal remodeling

Silent information regulator T1 (SirT1) is linked to longevity and negatively controls NF‐κB signaling, a crucial mediator of survival and regulator of both osteoclasts and osteoblasts. Here we show that NF‐κB repression by SirT1 in both osteoclasts and osteoblasts is necessary for proper bone remodeling and may contribute to the mechanisms linking aging and bone loss. Osteoclast‐ or osteoblast‐specific SirT1 deletion using the Sirt^flox/flox mice crossed to lysozyme M‐cre and the 2.3 kb col1a1‐cre transgenic mice, respectively, resulted in decreased bone mass caused by increased resorption and reduced bone formation. In osteoclasts, lack of SirT1 promoted osteoclastogenesis in vitro and activated NF‐κB by increasing acetylation of Lysine 310. Importantly, this increase in osteoclastogenesis was blocked by pharmacological inhibition of NF‐κB. In osteoblasts, decreased SirT1 reduced osteoblast differentiation, which could also be rescued by inhibition of NF‐κB. In further support of the critical role of NF‐κB signaling in bone remodeling, elevated NF‐κB activity in IκBα^+/? mice uncoupled bone resorption and formation, leading to reduced bone mass. These findings support the notion that SirT1 is a genetic determinant of bone mass, acting in a cell‐autonomous manner in both osteoblasts and osteoclasts, through control of NF‐κB and bone cell differentiation. © 2013 American Society for Bone and Mineral Research.     

Cyclin‐Dependent Kinase Inhibitor‐1‐Deficient Mice are Susceptible to Osteoarthritis Associated with Enhanced Inflammation

Osteoarthritis (OA) is a multifactorial disease, and recent data suggested that cell cycle–related proteins play a role in OA pathology. Cyclin‐dependent kinase (CDK) inhibitor 1 (p21) regulates activation of other CDKs, and recently, we reported that p21 deficiency induced susceptibility to OA induced by destabilization of the medial meniscus (DMM) surgery through STAT3‐signaling activation. However, the mechanisms associated with why p21 deficiency led to susceptibility to OA by the STAT3 pathway remain unknown. Therefore, we focused on joint inflammation to determine the mechanisms associated with p21 function during in vitro and in vivo OA progression. p21‐knockout (p21^?/?) mice were used to develop an in vivo OA model, and C57BL/6 (p21^+/+) mice with the same background as the p21^?/? mice were used as controls. Morphogenic changes were measured using micro‐CT, IL‐1β serum levels were detected by ELISA, and histological or immunohistological analyses were performed. Our results indicated that p21‐deficient DMM‐model mice exhibited significant subchondral bone destruction and cartilage degradation compared with wild‐type mice. Immunohistochemistry results revealed p21^?/? mice susceptibility to OA changes accompanied by macrophage infiltration and enhanced MMP‐3 and MMP‐13 expression through IL‐1β‐induced NF‐κB signaling. p21^?/? mice also showed subchondral bone destruction according to micro‐CT analysis, and cathepsin K staining revealed increased numbers of osteoclasts. Furthermore, p21^?/? mice displayed increased serum IL‐1β levels, and isolated chondrocytes from p21^?/? mice indicated elevated MMP‐3 and MMP‐13 expression with phosphorylation of IκB kinase complex in response to IL‐1β stimulation, whereas treatment with a specific p‐IκB kinase inhibitor attenuated MMP‐3 and MMP‐13 expression. Our results indicated that p21‐deficient DMM mice were susceptible to alterations in OA phenotype, including enhanced osteoclast expression, macrophage infiltration, and MMP expression through IL‐1β‐induced NF‐κB signaling, suggesting that p21 regulation may constitute a possible therapeutic strategy for OA treatment. © 2017 American Society for Bone and Mineral Research.     

Loss of Cbl‐b Increases Osteoclast Bone‐Resorbing Activity and Induces Osteopenia

Cbl proteins are multifunctional adaptor molecules that modulate cellular activity by targeting the ubiquitylating system, endocytic complexes, and other effectors to a wide variety of regulatory proteins, especially activated receptor and nonreceptor tyrosine kinases. Cbl and Cbl‐b perform unique functions in various cells, in addition to redundant functions that are required for embryonic development. We previously showed that eliminating Cbl impaired osteoclast motility, which modestly delayed embryonic bone development. We now report that Cbl‐b^?/? mice are osteopenic, because of increased bone resorption with little compensating increase in bone formation. In vitro bone‐resorbing activity and differentiation of osteoclast‐like cells (OCLs) were increased, as were some RANKL‐induced signaling events (activation of NF‐κB and the mitogen‐activated protein kinases extracellular signal‐regulated kinase [ERK] and p38), suggesting that specific RANKL‐activated mechanisms contribute to the increased rate of differentiation and bone‐resorbing activity. Re‐expressing Cbl‐b in Cbl‐b^?/? OCLs normalized the increased bone‐resorbing activity and overexpressing Cbl‐b in wildtype OCLs inhibited bone resorption. Cbl was without effect in either wildtype or Cbl‐b^?/? OCLs. Functional tyrosine kinase binding (TKB) and RING finger domains were required for the rescue by Cbl‐b. Thus, both Cbl and Cbl‐b perform regulatory functions in osteoclasts that are unique to one or the other protein (i.e., functions that cannot be compensated by the other homolog). One of Cbl‐b's unique functions in osteoclasts is to downregulate bone resorption.     

Endogenous parathyroid hormone–related protein compensates for the absence of parathyroid hormone in promoting bone accrual in vivo in a model of bone marrow ablation

To assess the effect of hypoparathyroidism on osteogenesis and bone turnover in vivo, bone marrow ablation (BMXs) were performed in tibias of 8‐week‐old wild‐type and parathyroid hormone–null (PTH^?/?) mice and newly formed bone tissue was analyzed from 5 days to 3 weeks after BMX. At 1 week after BMX, trabecular bone volume, osteoblast numbers, alkaline phosphatase‐positive areas, type I collagen‐positive areas, PTH receptor–positive areas, calcium sensing receptor–positive areas, and expression of bone formation–related genes were all decreased significantly in the diaphyseal regions of bones of PTH^?/? mice compared to wild‐type mice. In contrast, by 2 weeks after BMX, all parameters related to osteoblastic bone accrual were increased significantly in PTH^?/? mice. At 5 days after BMX, active tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts had appeared in wild‐type mice but were undetectable in PTH^?/? mice, Both the ratio of mRNA levels of receptor activator of NF‐κB ligand (RANKL)/osteoprotegerin (OPG) and TRAP‐positive osteoclast surface were still reduced in PTH^?/? mice at 1 week but were increased by 2 weeks after BMX. The expression levels of parathyroid hormone–related protein (PTHrP) at both mRNA and protein levels were upregulated significantly at 1 week and more dramatically at 2 weeks after BMX in PTH^?/? mice. To determine whether the increased newly formed bones in PTH^?/? mice at 2 weeks after BMX resulted from the compensatory action of PTHrP, PTH^?/?PTHrP^+/? mice were generated and newly formed bone tissue was compared in these mice with PTH^?/? and wild‐type mice at 2 weeks after BMX. All parameters related to osteoblastic bone formation and osteoclastic bone resorption were reduced significantly in PTH^?/?PTHrP^+/? mice compared to PTH^?/? mice. These results demonstrate that PTH deficiency itself impairs osteogenesis, osteoclastogenesis, and osteoclastic bone resorption, whereas subsequent upregulation of PTHrP in osteogenic cells compensates by increasing bone accrual. © 2013 American Society for Bone and Mineral Research     

IKKβ activation is sufficient for RANK‐independent osteoclast differentiation and osteolysis

Monocytes differentiate into osteoclasts through stimulation of receptor activator of NF‐κB (RANK). Many downstream effectors of RANK play a positive role in osteoclastogenesis, but their relative importance in osteoclast differentiation is unclear. We report the discovery that activation of a single pathway downstream of RANK is sufficient for osteoclast differentiation. In this regard, introduction of constitutively activated IKKβ (IKKβ^SSEE) but not wild‐type IKKβ into monocytes stimulates differentiation of bona fide osteoclasts in the absence of RANK ligand (RANKL). This phenomenon is independent of upstream signals because IKKβ^SSEE induced the development of bone‐resorbing osteoclasts from RANK and IKKα knockout monocytes and in conditions in which NEMO‐IKKβ association was inhibited. NF‐κB p100 and p105, but not RelB, were critical mediators of this effect. Inflammatory autocrine signaling by tumor necrosis factor α (TNF‐α) and interleukin 1 (IL‐1) were dispensable for the spontaneous osteoclastogenesis driven by IKKβ^SSEE. More important, adenoviral gene transfer of IKKβ^SSEE induced osteoclasts and osteolysis in calvariae and knees of mice. Our data establish the sufficiency of IKKβ activation for osteolysis and suggest that IKKβ hyperactivation may play a role in conditions of pathologic bone destruction refractory to RANK/RANKL proximal therapeutic interventions. © 2010 American Society for Bone and Mineral Research     

Regulation of bone mass and osteoclast function depend on the F-actin modulator SWAP-70

Bone remodeling involves tightly regulated bone‐resorbing osteoclasts and bone‐forming osteoblasts. Determining osteoclast function is central to understanding bone diseases such as osteoporosis and osteopetrosis. Here, we report a novel function of the F‐actin binding and regulatory protein SWAP‐70 in osteoclast biology. F‐actin ring formation, cell morphology, and bone resorption are impaired in Swap‐70^?/? osteoclasts, whereas the expression of osteoclast differentiation markers induced in vitro by macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL) remains unaffected. Swap‐70^?/? mice develop osteopetrosis with increased bone mass, abnormally dense bone, and impaired osteoclast function. Ectopic expression of SWAP‐70 in Swap‐70^?/? osteoclasts in vitro rescues their deficiencies in bone resorption and F‐actin ring formation. Rescue requires a functional pleckstrin homology (PH) domain, known to support membrane localization of SWAP‐70, and the F‐actin binding domain. Transplantation of SWAP‐70–proficient bone marrow into Swap‐70^?/? mice restores osteoclast resorption capacity in vivo. The identification of the role of SWAP‐70 in promoting osteoclast function through modulating membrane‐proximal F‐actin rearrangements reveals a new pathway to control osteoclasts and bone homeostasis. © 2012 American Society for Bone and Mineral Research.     

Telomerase‐deficient mice exhibit bone loss owing to defects in osteoblasts and increased osteoclastogenesis by inflammatory microenvironment

Telomere shortening owing to telomerase deficiency leads to accelerated senescence of human skeletal (mesenchymal) stem cells (MSCs) in vitro, whereas overexpression leads to telomere elongation, extended life span, and enhanced bone formation. To study the role of telomere shortening in vivo, we studied the phenotype of telomerase‐deficient mice (Terc^?/?). Terc^?/? mice exhibited accelerated age‐related bone loss starting at 3 months of age and during 12 months of follow‐up revealed by dual‐energy X‐ray absorptiometric (DXA) scanning and by micro–computed tomography (µCT). Bone histomorphometry revealed decreased mineralized surface and bone‐formation rate as well as increased osteoclast number and size in Terc^?/? mice. Also, serum total deoxypyridinoline (tDPD) was increased in Terc^?/? mice. MSCs and osteoprogenitors isolated from Terc^?/? mice exhibited intrinsic defects with reduced proliferating cell number and impaired osteogenic differentiation capacity. In addition, the Terc^?/?‐MSC cultures accumulated a larger proportion of senescent β‐galactosidase⁺ cells and cells exhibiting DNA damage. Microarray analysis of Terc^?/? bone revealed significant overexpression of a large number of proinflammatory genes involved in osteoclast (OC) differentiation. Consistently, serum obtained from Terc^?/? mice enhanced OC formation of wild‐type bone marrow cultures. Our data demonstrate two mechanisms for age‐related bone loss caused by telomerase deficiency: intrinsic osteoblastic defects and creation of a proinflammatory osteoclast‐activating microenvironment. Thus telomerization of MSCs may provide a novel approach for abolishing age‐related bone loss. © 2011 American Society for Bone and Mineral Research.