Alexidine Dihydrochloride Attenuates Osteoclast Formation and Bone Resorption and Protects Against LPS‐Induced Osteolysis

Aseptic loosening and periprosthetic infection leading to inflammatory osteolysis is a major complication associated with total joint arthroplasty (TJA). The liberation of bacterial products and/or implant‐derived wear particles activates immune cells that produce pro‐osteoclastogenic cytokines that enhance osteoclast recruitment and activity, leading to bone destruction and osteolysis. Therefore, agents that prevent the inflammatory response and/or attenuate excessive osteoclast (OC) formation and bone resorption offer therapeutic potential by prolonging the life of TJA implants. Alexidine dihydrochloride (AD) is a bisbiguanide compound commonly used as an oral disinfectant and in contact lens solutions. It possesses antimicrobial, anti‐inflammatory and anticancer properties; however, its effects on OC biology are poorly described. Here, we demonstrate that AD inhibits OC formation and bone resorption in vitro and exert prophylatic protection against LPS‐induced osteolysis in vivo. Biochemical analysis demonstrated that AD suppressed receptor activator of NF‐κB ligand (RANKL)‐induced activation of mitogen‐activated protein kinases (ERK, p38, and JNK), leading to the downregulation of NFATc1. Furthermore, AD disrupted F‐actin ring formation and attenuated the ability of mature OC to resorb bone. Collectively, our findings suggest that AD may be a promising prophylactic anti‐osteoclastic/resorptive agent for the treatment of osteolytic diseases caused by excessive OC formation and function. © 2015 American Society for Bone and Mineral Research.     

Siglec‐15 Regulates Osteoclast Differentiation by Modulating RANKL‐Induced Phosphatidylinositol 3‐Kinase/Akt and Erk Pathways in Association With Signaling Adaptor DAP12

Siglecs are a family of sialic acid–binding immunoglobulin‐like lectins that regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. Here we show that Siglec‐15 regulates osteoclast development and bone resorption by modulating receptor activator of nuclear factor κB ligand (RANKL) signaling in association with DNAX‐activating protein 12 kDa (DAP12), an adaptor protein bearing an immunoreceptor tyrosine‐based activation motif (ITAM). Among the known Siglecs expressed in myeloid lineage cells, only Siglec‐15 was upregulated by RANKL in mouse primary bone marrow macrophages. Siglec‐15–deficient mice exhibit mild osteopetrosis resulting from impaired osteoclast development. Consistently, cells lacking Siglec‐15 exhibit defective osteoclast development and resorptive activity in vitro. RANKL‐induced activation of phosphatidylinositol 3‐kinase (PI3K)/Akt and Erk pathways were impaired in Siglec‐15–deficient cells. Retroviral transduction of Siglec‐15–null osteoclast precursors with wild‐type Siglec‐15 or mutant Siglec‐15 revealed that the association of Siglec‐15 with DAP12 is involved in the downstream signal transduction of RANK. Furthermore, we found that the ability of osteoclast formation is preserved in the region adjacent to the growth plate in Siglec‐15–deficient mice, indicating that there is a compensatory mechanism for Siglec‐15–mediated osteoclastogenesis in the primary spongiosa. To clarify the mechanism of this compensation, we examined whether osteoclast‐associated receptor (OSCAR)/Fc receptor common γ (FcRγ) signaling, an alternative ITAM‐mediated signaling pathway to DAP12, rescues impaired osteoclastogenesis in Siglec‐15–deficient cells. The ligands in type II collagen activate OSCAR and rescue impaired osteoclastogenesis in Siglec‐15–deficient cells when cultured on bone slices, indicating that Siglec‐15–mediated signaling can be compensated for by signaling activated by type II collagen and other bone matrix components in the primary spongiosa. Our findings indicate that Siglec‐15 plays an important role in physiologic bone remodeling by modulating RANKL signaling, especially in the secondary spongiosa. © 2013 American Society for Bone and Mineral Research.     

Disruption of the dynein‐dynactin complex unveils motor‐specific functions in osteoclast formation and bone resorption

Osteoclastic bone resorption requires strict interplay between acidified carrier vesicles, motor proteins, and the underlying cytoskeleton in order to sustain the specialized structural and functional polarization of the ruffled border. Cytoplasmic dynein, a large processive mechanochemical motor comprising heavy, intermediate, and light chains coupled to the dynactin cofactor complex, powers unilateral motility of diverse cargos to microtubule minus‐ends. We have recently shown that regulators of the dynein motor complex constitute critical components of the osteoclastic bone resorptive machinery. Here, by selectively modulating endogenous dynein activity, we show that the integrity of the dynein‐dynactin motor complex is an essential requirement for both osteoclast formation and function. Systematic dissection of the osteoclast dynein‐dynactin complex revealed that it is differentially localized throughout RANKL‐induced osteoclast formation and activation, undergoing microtubule‐coupled reorganization upon the establishment of cellular polarization. In osteoclasts actively resorbing bone, dynein‐dynactin intimately co‐localizes with the CAP‐Gly domain‐containing microtubule plus‐end protein CLIP‐170 at the resorptive front, thus orientating the ruffled border as a microtubule plus‐end domain. Unexpectedly, disruption of the dynein‐dynactin complex by exogenous p50/dynamitin expression retards osteoclast formation in vitro, owing largely to prolonged mitotic stasis of osteoclast progenitor cells. More importantly, loss of osteoclastic dynein activity results in a drastic redistribution of key intracellular organelles, including the Golgi and lysosomes, an effect that coincides with impaired cathepsin K secretion and diminished bone resorptive function. Collectively, these data unveil a previously unrecognized role for the dynein‐dynactin motor complex in osteoclast formation and function, serving not only to regulate their timely maturation but also the delivery of osteolytic cargo that is essential to the bone resorptive process. © 2013 American Society for Bone and Mineral Research     

Osteoclast TGF‐β Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation

Osteoblast‐mediated bone formation is coupled to osteoclast‐mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age‐related bone loss. Osteoclasts release and activate TGF‐β from the bone matrix. Here we show that osteoclast‐specific inhibition of TGF‐β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF‐β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF‐β receptor signaling. Osteoclasts in aged murine bones had lower TGF‐β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF‐β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF‐β availability with age. Therefore, osteoclast responses to TGF‐β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age‐related bone loss. © 2015 American Society for Bone and Mineral Research.     

Reversing LRP5‐Dependent Osteoporosis and SOST Deficiency–Induced Sclerosing Bone Disorders by Altering WNT Signaling Activity

The bone formation inhibitor sclerostin encoded by SOST binds in vitro to low‐density lipoprotein receptor‐related protein (LRP) 5/6 Wnt co‐receptors, thereby inhibiting Wnt/β‐catenin signaling, a central pathway of skeletal homeostasis. Lrp5/LRP5 deficiency results in osteoporosis‐pseudoglioma (OPPG), whereas Sost/SOST deficiency induces lifelong bone gain in mice and humans. Here, we analyzed the bone phenotype of mice lacking Sost (Sost^?/?), Lrp5 (Lrp5^?/?), or both (Sost^?/?;Lrp5^?/?) to elucidate the mechanism of action of Sost in vivo. Sost deficiency–induced bone gain was significantly blunted in Sost^?/?;Lrp5^?/? mice. Yet the Lrp5 OPPG phenotype was fully rescued in Sost^?/?;Lrp5^?/? mice and most bone parameters were elevated relative to wild‐type. To test whether the remaining bone increases in Sost^?/?;Lrp5^?/? animals depend on Lrp6, we treated wild‐type, Sost^?/?, and Sost^?/?;Lrp5^?/? mice with distinct Lrp6 function blocking antibodies. Selective blockage of Wnt1 class–mediated Lrp6 signaling reduced cancellous bone mass and density in wild‐type mice. Surprisingly, it reversed the abnormal bone gain in Sost^?/? and Sost^?/?;Lrp5^?/? mice to wild‐type levels irrespective of enhancement or blockage of Wnt3a class‐mediated Lrp6 activity. Thus, whereas Sost deficiency–induced bone anabolism partially requires Lrp5, it fully depends on Wnt1 class–induced Lrp6 activity. These findings indicate: first, that OPPG syndrome patients suffering from LRP5 loss‐of‐function should benefit from principles antagonizing SOST/sclerostin action; and second, that therapeutic WNT signaling inhibitors may stop the debilitating bone overgrowth in sclerosing disorders related to SOST deficiency, such as sclerosteosis, van Buchem disease, and autosomal dominant craniodiaphyseal dysplasia, which are rare disorders without viable treatment options. © 2014 American Society for Bone and Mineral Research.     

Interleukin‐33 is expressed in differentiated osteoblasts and blocks osteoclast formation from bone marrow precursor cells

Since the hematopoetic system is located within the bone marrow, it is not surprising that recent evidence has demonstrated the existence of molecular interactions between bone and immune cells. While interleukin 1 (IL‐1) and IL‐18, two cytokines of the IL‐1 family, have been shown to regulate differentiation and activity of bone cells, the role of IL‐33, another IL‐1 family member, has not been addressed yet. Since we observed that the expression of IL‐33 increases during osteoblast differentiation, we analyzed its possible influence on bone formation and observed that IL‐33 did not affect matrix mineralization but enhanced the expression of Tnfsf11, the gene encoding RANKL. This finding led us to analyze the skeletal phenotype of Il1rl1‐deficient mice, which lack the IL‐33 receptor ST2. Unexpectedly, these mice displayed normal bone formation but increased bone resorption, thereby resulting in low trabecular bone mass. Since this finding suggested a negative influence of IL‐33 on osteoclastogenesis, we next analyzed osteoclast differentiation from bone marrow precursor cells and observed that IL‐33 completely abolished the generation of TRACP⁺ multinucleated osteoclasts, even in the presence of RANKL and macrophage colony‐stimulating factor (M‐CSF). Although our molecular studies revealed that IL‐33 treatment of bone marrow cells caused a shift toward other hematopoetic lineages, we further observed a direct negative influence of IL‐33 on the osteoclastogenic differentiation of RAW264.7 macrophages, where IL‐33 repressed the expression of Nfatc1, which encodes one of the key transciption factors of osteoclast differentiation. Taken together, these findings have uncovered a previously unknown function of IL‐33 as an inhibitor of bone resorption. © 2011 American Society for Bone and Mineral Research.     

Novel RANK Antagonists for the Treatment of Bone‐Resorptive Disease: Theoretical Predictions and Experimental Validation

Receptor activator of nuclear factor‐κB (RANK) and RANK ligand (RANKL) play a pivotal role in bone metabolism, and selective targeting of RANK signaling has become a promising therapeutic strategy in the management of resorptive bone diseases. Existing antibody‐based therapies and novel inhibitors currently in development were designed to target the ligand, rather than the membrane receptor expressed on osteoclast precursors. We describe here an alternative approach to designing small peptides able to specifically bind to the hinge region of membrane RANK responsible for the conformational change upon RANKL association. A nonapeptide generated by this method was validated for its biological activity in vitro and in vivo and served as a lead compound for the generation of a series of peptide RANK antagonists derived from the original sequence. Our study presents a structure‐ and knowledge‐based strategy for the design of novel effective and affordable small peptide inhibitors specifically targeting the receptor RANK and opens a new therapeutic opportunity for the treatment of resorptive bone disease. © 2014 American Society for Bone and Mineral Research.     

Direct Crosstalk Between Cancer and Osteoblast Lineage Cells Fuels Metastatic Growth in Bone via Auto‐Amplification of IL‐6 and RANKL Signaling Pathways

The bone microenvironment and its modification by cancer and host cell interactions is a key driver of skeletal metastatic growth. Interleukin‐6 (IL‐6) stimulates receptor activator of NF‐κB ligand (RANKL) expression in bone cells, and serum IL‐6 levels are associated with poor clinical outcomes in cancer patients. We investigated the effects of RANKL on cancer cells and the role of tumor‐derived IL‐6 within the bone microenvironment. Using human breast cancer cell lines to induce tumors in the bone of immune‐deficient mice, we first determined whether RANKL released by cells of the osteoblast lineage directly promotes IL‐6 expression by cancer cells in vitro and in vivo. We then disrupted of IL‐6 signaling in vivo either via knockdown of IL‐6 in tumor cells or through treatment with specific anti‐human or anti‐mouse IL‐6 receptor antibodies to investigate the tumor effect. Finally, we tested the effect of RANK knockdown in cancer cells on cancer growth. We demonstrate that osteoblast lineage‐derived RANKL upregulates secretion of IL‐6 by breast cancers in vivo and in vitro. IL‐6, in turn, induces expression of RANK by cancer cells, which sensitizes the tumor to RANKL and significantly enhances cancer IL‐6 release. Disruption in vivo of this auto‐amplifying crosstalk by knockdown of IL‐6 or RANK in cancer cells, or via treatment with anti‐IL‐6 receptor antibodies, significantly reduces tumor growth in bone but not in soft tissues. RANKL and IL‐6 mediate direct paracrine‐autocrine signaling between cells of the osteoblast lineage and cancer cells, significantly enhancing the growth of metastatic breast cancers within bone. © 2014 American Society for Bone and Mineral Research.     

Genomewide Comprehensive Analysis Reveals Critical Cooperation Between Smad and c‐Fos in RANKL‐Induced Osteoclastogenesis

We have previously reported that transforming growth factor β (TGF‐β) plays an essential role in receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis. However, the detailed underlying molecular mechanisms still remain unclear. Formaldehyde‐assisted isolation of regulatory elements (FAIRE) and chromatin immunoprecipitation (ChIP) followed by sequencing (FAIRE‐seq and ChIP‐seq) analyses indicated the cooperation of Smad2/3 with c‐Fos during osteoclastogenesis. Biochemical analysis and immunocytochemical analysis revealed that physical interaction between Smad2/3 and c‐Fos is required for their nuclear translocation. The gene expression of nuclear factor of activated T‐cells, cytoplasmic 1 (Nfatc1), a key regulator of osteoclastogenesis, was regulated by RANKL and TGF‐β, and c‐Fos binding to open chromatin sites was suppressed by inhibition of TGF‐β signaling by SB431542. Conversely, Smad2/3 binding to Nfatc1 was impaired by c‐Fos deficiency. These results suggest that TGF‐β regulates RANKL‐induced osteoclastogenesis through reciprocal cooperation between Smad2/3 and c‐Fos. © 2014 American Society for Bone and Mineral Research.     

Understanding Age‐Induced Cortical Porosity in Women: The Accumulation and Coalescence of Eroded Cavities Upon Existing Intracortical Canals Is the Main Contributor

Intracortical bone remodeling normally ensures maintenance of the cortical bone matrix and strength, but during aging, this remodeling generates excessive porosity. The mechanism behind the age‐induced cortical porosity is poorly understood and addressed in the present study. This study consists of a histomorphometric analysis of sections of iliac bone specimens from 35 women (age 16–78 years). First, the study shows that the age‐induced cortical porosity reflects an increased pore size rather than an increased pore density. Second, it establishes a novel histomorphometric classification of the pores, which is based on the characteristics of the remodeling sites to which each pore is associated. It takes into consideration (i) the stage of the remodeling event at the level where the pore is sectioned, (ii) whether the event corresponds with the generation of a new pore through penetrative tunneling (type 1 pores) or with remodeling of an existing pore (type 2 pores), and (iii) in the latter case, whether or not the new remodeling event leads to the coalescence of pores. Of note, the advantage of this classification is to relate porosity with its generation mechanism. Third, it demonstrates that aging and porosity are correlated with: a shift from type 1 to type 2 pores, reflecting that the remodeling of existing pores is higher; an accumulation of eroded type 2 pores, reflecting an extended resorption‐reversal phase; and a coalescence of these eroded type 2 pores into enlarged coalescing type 2 cavities. Collectively, this study supports the notion, that age‐related increase in cortical porosity is the result of intracortical remodeling sites upon existing pores, with an extended reversal‐resorption phase (eroded type 2 pores) that may likely result in a delayed or absent initiation of the subsequent bone formation. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.     

JNK/c‐Jun Signaling Mediates an Anti‐Apoptotic Effect of RANKL in Osteoclasts

Introduction: RANKL is known to be important not only for differentiation and activation of osteoclasts but also for their survival. Experimentally, apoptosis of osteoclasts is rapidly induced by the deprivation of RANKL. RANKL activates Elk‐related tyrosine kinase (ERK), p38, c‐Jun N‐terminal kinase (JNK), and NF‐κB pathways through TRAF6 in osteoclasts and the precursor cells. It has been shown that ERK is critical for regulation of osteoclast survival. However, an involvement of other RANKL signaling pathways such as JNK signaling in survival of osteoclasts has not been fully understood yet. Materials and Methods: Osteoclasts derived from primary mouse bone marrow cells by soluble RANKL (sRANKL) were treated with a JNK inhibitor, SP600125, or infected with adenovirus carrying dominant‐negative (DN)‐c‐jun, DN‐c‐fos, mitogen‐activated protein kinase kinase 1 (MEKK1), I‐κBα mutant, or NF‐κB components, p50 and p65. Osteoclasts were cultured with or without sRANKL, and apoptotic phenotype was determined by TUNEL assay, DAPI staining, and expression of cleaved caspase 3 followed by TRACP staining. Results: Overexpression of TRAF6 activated JNK and NF‐κB signaling pathways and clearly prevented osteoclasts from apoptosis caused by abrogation of sRANKL. An anti‐apoptotic effect of RANKL/RANK/TRAF6 signaling on osteoclast was inhibited by JNK‐specific inhibitor SP600125 and by overexpression of dominant‐negative JNK1, c‐jun, and c‐fos. Also, overexpression of MEKK1 inhibited apoptosis of osteoclasts even in the absence of sRANKL along with activation of JNK/c‐jun signaling. On the other hand, blockade of NF‐κB signaling by I‐κBα mutant or overexpression of NF‐κB components showed a marginal effect on apoptosis of osteoclasts. Conclusions: An important role of RANKL‐induced activation of MEKK1/JNK/c‐jun signaling in the regulation of apoptosis in osteoclasts was shown. Our study suggests that c‐fos plays a role as a partner of activator protein‐1 factor, c‐jun, during the regulation of apoptosis in osteoclasts.     

Protection From Glucocorticoid‐Induced Osteoporosis by Anti‐Catabolic Signaling in the Absence of Sost/Sclerostin

Excess of glucocorticoids, either due to disease or iatrogenic, increases bone resorption and decreases bone formation and is a leading cause of osteoporosis and bone fractures worldwide. Improved therapeutic strategies are sorely needed. We investigated whether activating Wnt/β‐catenin signaling protects against the skeletal actions of glucocorticoids, using female mice lacking the Wnt/β‐catenin antagonist and bone formation inhibitor Sost. Glucocorticoids decreased the mass, deteriorated the microarchitecture, and reduced the structural and material strength of bone in wild‐type (WT), but not in Sost^–/– mice. The high bone mass exhibited by Sost^–/– mice is due to increased bone formation with unchanged resorption. However, unexpectedly, preservation of bone mass and strength in Sost^–/– mice was due to prevention of glucocorticoid‐induced bone resorption and not to restoration of bone formation. In WT mice, glucocorticoids increased the expression of Sost and the number of sclerostin‐positive osteocytes, and altered the molecular signature of the Wnt/β‐catenin pathway by decreasing the expression of genes associated with both anti‐catabolism, including osteoprotegerin (OPG), and anabolism/survival, such as cyclin D1. In contrast in Sost^–/– mice, glucocorticoids did not decrease OPG but still reduced cyclin D1. Thus, in the context of glucocorticoid excess, activation of Wnt/β‐catenin signaling by Sost/sclerostin deficiency sustains bone integrity by opposing bone catabolism despite markedly reduced bone formation and increased apoptosis. This crosstalk between glucocorticoids and Wnt/β‐catenin signaling could be exploited therapeutically to halt resorption and bone loss induced by glucocorticoids and to inhibit the exaggerated bone formation in diseases of unwanted hyperactivation of Wnt/β‐catenin signaling. © 2016 American Society for Bone and Mineral Research.     

Mast Cells Are Critical Regulators of Bone Fracture–Induced Inflammation and Osteoclast Formation and Activity

Mast cells, important sensor and effector cells of the immune system, may influence bone metabolism as their number is increased in osteoporotic patients. They are also present during bone fracture healing with currently unknown functions. Using a novel c‐Kit‐independent mouse model of mast cell deficiency, we demonstrated that mast cells did not affect physiological bone turnover. However, they triggered local and systemic inflammation after fracture by inducing release of inflammatory mediators and the recruitment of innate immune cells. In later healing stages, mast cells accumulated and regulated osteoclast activity to remodel the bony fracture callus. Furthermore, they were essential to induce osteoclast formation after ovariectomy. Additional in vitro studies revealed that they promote osteoclastogenesis via granular mediators, mainly histamine. In conclusion, mast cells are redundant in physiologic bone turnover but exert crucial functions after challenging the system, implicating mast cells as a potential target for treating inflammatory bone disorders. © 2017 American Society for Bone and Mineral Research.     

TGFβ Inducible Early Gene‐1 Plays an Important Role in Mediating Estrogen Signaling in the Skeleton

TGFβ Inducible Early Gene‐1 (TIEG1) knockout (KO) mice display a sex‐specific osteopenic phenotype characterized by low bone mineral density, bone mineral content, and overall loss of bone strength in female mice. We, therefore, speculated that loss of TIEG1 expression would impair the actions of estrogen on bone in female mice. To test this hypothesis, we employed an ovariectomy (OVX) and estrogen replacement model system to comprehensively analyze the role of TIEG1 in mediating estrogen signaling in bone at the tissue, cell, and biochemical level. Dual‐energy X‐ray absorptiometry (DXA), peripheral quantitative computed tomography (pQCT), and micro‐CT analyses revealed that loss of TIEG1 expression diminished the effects of estrogen throughout the skeleton and within multiple bone compartments. Estrogen exposure also led to reductions in bone formation rates and mineralizing perimeter in wild‐type mice with little to no effects on these parameters in TIEG1 KO mice. Osteoclast perimeter per bone perimeter and resorptive activity as determined by serum levels of CTX‐1 were differentially regulated after estrogen treatment in TIEG1 KO mice compared with wild‐type littermates. No significant differences were detected in serum levels of P1NP between wild‐type and TIEG1 KO mice. Taken together, these data implicate an important role for TIEG1 in mediating estrogen signaling throughout the mouse skeleton and suggest that defects in this pathway are likely to contribute to the sex‐specific osteopenic phenotype observed in female TIEG1 KO mice. © 2014 American Society for Bone and Mineral Research.     

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.     

Inhibition of Cathepsin K Increases Modeling‐Based Bone Formation,and Improves Cortical Dimension and Strength in Adult Ovariectomized Monkeys

Treatment with the cathepsin K (CatK) inhibitor odanacatib (ODN) protects against bone loss and maintains normal biomechanical properties in the spine and hip of ovariectomized (OVX) preclinical models. Here, we characterized the effects of ODN on the dynamics of cortical modeling and remodeling, and dimension and strength of the central femur in adult OVX‐rhesus monkeys. Animals were treated with vehicle or ODN (6 or 30 mg/kg, once per day [q.d., p.o.]) in prevention mode for 21 months. Calcein and tetracycline double‐labeling were given at 12 and 21 months, and the femoral cross‐sections were subjected to dynamic histomorphometric and cement line analyses. ODN treatment significantly increased periosteal and endocortical bone formation (BFR/BS), accompanied with an increase in endocortical mineralizing surface (102%, p < 0.01) with the 6 mg/kg dose. ODN at both doses reduced remodeling hemiosteon numbers by 51% and 66% (p < 0.05), respectively, and ODN 30 mg/kg numerically reduced activation frequency without affecting wall thickness. On the same endocortical surface, ODN increased all modeling‐based parameters, while reducing intracortical remodeling, consistent with the observed no treatment effects on cortical porosity. ODN 30 mg/kg markedly increased cortical thickness (CtTh, p < 0.001) and reduced marrow area (p < 0.01). Lastly, ODN treatment increased femoral structural strength (p < 0.001). Peak load was positively correlated with the increases in bone mineral content (BMC) (r² = 0.9057, p < 0.0001) and CtTh (r² = 0.6866, p < 0.0001). Taken together, by reducing cortical remodeling‐based and stimulating modeling‐based bone formation, ODN significantly improved cortical dimension and strength in OVX monkeys. This novel mechanism of CatK inhibition in stimulating cortical formation suggests that ODN represents a novel therapeutic approach for the treatment of osteoporosis. © 2014 American Society for Bone and Mineral Research.     

The Primary Function of gp130 Signaling in Osteoblasts Is To Maintain Bone Formation and Strength,Rather Than Promote Osteoclast Formation

Interleukin‐6 (IL‐6) family cytokines act via gp130 in the osteoblast lineage to stimulate the formation of osteoclasts (bone resorbing cells) and the activity of osteoblasts (bone forming cells), and to inhibit expression of the osteocyte protein, sclerostin. We report here that a profound reduction in trabecular bone mass occurs both when gp130 is deleted in the entire osteoblast lineage (Osx1Cre gp130 f/f) and when this deletion is restricted to osteocytes (DMP1Cre gp130 f/f). This was caused not by an alteration in osteoclastogenesis, but by a low level of bone formation specific to the trabecular compartment. In contrast, cortical diameter increased to maintain ultimate bone strength, despite a reduction in collagen type 1 production. We conclude that osteocytic gp130 signaling is required for normal trabecular bone mass and proper cortical bone composition. © 2014 American Society for Bone and Mineral Research.