Osteoclast Lifespan Research Articles

Novel gene Merlot inhibits differentiation and promotes apoptosis of osteoclasts.

Extended osteoclast longevity is deeply involved in the pathogenesis of bone diseases such as osteoporosis and rheumatoid arthritis, though the mechanisms that determine osteoclast lifespan are not fully understood. Here we present findings indicating that the newly characterized gene Merlot, which encodes a highly conserved yet uncharacterized protein in vertebrates, is an important regulator for termination of osteoclastogenesis via induction of apoptosis. Mice lacking Merlot exhibited low bone mass due to increased osteoclast and bone resorption. Furthermore, osteoclast precursors overexpressing Merlot failed to differentiate into mature osteoclasts, while Merlot deficiency led to hyper-nucleation and prolonged survival of osteoclasts, accompanied by sustained nuclear localization of nuclear factor of activated T cell c1 (NFATc1) and derepression of glycogen synthase kinase-3β (GSK3β) activity, known to regulate NFATc1 activity and induce apoptosis. Merlot-deficient osteoclasts were found to represent suppression of caspase-3-mediated apoptosis and Merlot deficiency caused transcriptional downregulation of a proapoptotic cascade, including Bax, Bak, Noxa, and Bim, as well as the executor caspase members Casp-3, -6, and -7, and upregulation of anti-apoptotic Bcl2, resulting in a low apoptotic threshold. Thus, Merlot regulates osteoclast lifespan by inhibition of differentiation and simultaneous induction of apoptosis via regulation of the NFATc1-GSK3β axis.

Glucocorticoids Enhanced Osteoclast Autophagy Through the PI3K/Akt/mTOR Signaling Pathway.

Autophagy is an evolutionarily conserved dynamic process and present in variety of cells at basal levels to maintain homeostasis and to promote cell survival in response to stresses. The early bone loss with excessive glucocorticoids (GCs) was reported to be related with the extension of the life span of osteoclasts. However, the connection between GCs induced bone loss and osteoclast autophagy remains to be elucidated. Autophagy was detected in a Dexamethasone (Dex) induced osteoporotic mice model and primary osteoclast cultures by autophagosome detection kit, and autophagy-related proteins were assayed by Western blotting and Immunostaining. The bone morphology was examined by micro-CT and TRAP staining. The trabecular bone micro-architecture was deteriorated, and the osteoclast number and spread area were increased in the Dex-treated mice compared with the control group (P < 0.01). Meanwhile, autophagy in pre-osteoclasts was increased in mice under Dex administration evidenced by the increased number of autophagosome and up-regulation of autophagy-related protein levels. Further, the enhanced autophagy under Dex treatment was verified in primary cultured osteoclasts, as shown by the increased levels of Beclin 1 and LC3-II/LC3-I and the autophagy complex formation members including Atg1, Atg13, and Atg7. However, the expressions of PI3K, p-Akt and p-mTOR in primary cultured osteoclasts were inhibited under Dex induced autophagy. Using the selective PTEN inhibitor SF1670 to activate the PI3K/Akt/mTOR pathway reversed this osteoclast autophagy under Dex treatment. Our study suggests that osteoclast autophagy was enhanced in glucocorticoids induced bone loss, and the PI3K/Akt/mTOR signaling pathway mediated the increased autophagy in primary cultured osteoclasts under glucocorticoids treatment.

Developmental origin, functional maintenance and genetic rescue of osteoclasts

Osteoclasts are multinucleated giant cells that resorb bone, ensuring development and continuous remodelling of the skeleton and the bone marrow haematopoietic niche. Defective osteoclast activity leads to osteopetrosis and bone marrow failure1–9, whereas excess activity can contribute to bone loss and osteoporosis10. Osteopetrosis can be partially treated by bone marrow transplantation in humans and mice11–18, consistent with a haematopoietic origin of osteoclasts13,16,19 and studies that suggest that they develop by fusion of monocytic precursors derived from haematopoietic stem cells in the presence of CSF1 and RANK ligand1,20. However, the developmental origin and lifespan of osteoclasts, and the mechanisms that ensure maintenance of osteoclast function throughout life in vivo remain largely unexplored. Here we report that osteoclasts that colonize fetal ossification centres originate from embryonic erythro-myeloid progenitors21,22. These erythro-myeloid progenitor-derived osteoclasts are required for normal bone development and tooth eruption. Yet, timely transfusion of haematopoietic-stem-cell-derived monocytic cells in newborn mice is sufficient to rescue bone development in early-onset autosomal recessive osteopetrosis. We also found that the postnatal maintenance of osteoclasts, bone mass and the bone marrow cavity involve iterative fusion of circulating blood monocytic cells with long-lived osteoclast syncytia. As a consequence, parabiosis or transfusion of monocytic cells results in long-term gene transfer in osteoclasts in the absence of haematopoietic-stem-cell chimerism, and can rescue an adult-onset osteopetrotic phenotype caused by cathepsin K deficiency23,24. In sum, our results identify the developmental origin of osteoclasts and a mechanism that controls their maintenance in bones after birth. These data suggest strategies to rescue osteoclast deficiency in osteopetrosis and to modulate osteoclast activity in vivo. Multinucleated osteoclasts required for normal bone development and tooth eruption in the mouse originate from embryonic erythro-myeloid progenitors and are maintained after birth by fusion with circulating monocytes.

Glucocorticoid-Induced Bone Fragility Is Prevented in Female Mice by Blocking Pyk2/Anoikis Signaling.

Excess of glucocorticoids (GCs) is a leading cause of bone fragility, and therapeutic targets are sorely needed. We report that genetic deletion or pharmacological inhibition of proline-rich tyrosine kinase 2 (Pyk2) prevents GC-induced bone loss by overriding GC effects of detachment-induced bone cell apoptosis (anoikis). In wild-type or vehicle-treated mice, GCs either prevented osteoclast apoptosis or promoted osteoblast/osteocyte apoptosis. In contrast, mice lacking Pyk2 [knockout (KO)] or treated with Pyk2 kinase inhibitor PF-431396 (PF) were protected. KO or PF-treated mice were also protected from GC-induced bone resorption, microarchitecture deterioration, and weakening of biomechanical properties. In KO and PF-treated mice, GC increased osteoclasts in bone and circulating tartrate-resistant acid phosphatase form 5b, an index of osteoclast number. However, bone surfaces covered by osteoclasts and circulating C-terminal telopeptides of type I collagen, an index of osteoclast function, were not increased. The mismatch between osteoclast number vs function induced by Pyk2 deficiency/inhibition was due to osteoclast detachment and anoikis. Further, GC prolongation of osteoclast lifespan was absent in KO and PF-treated osteoclasts, demonstrating Pyk2 as an intrinsic osteoclast-survival regulator. Circumventing Pyk2 activation preserves skeletal integrity by preventing GC effects on bone cell survival (proapoptotic for osteoblasts/osteocytes, antiapoptotic for osteoclasts) and GC-induced bone resorption. Thus, Pyk2/anoikis signaling as a therapeutic target for GC-induced osteoporosis.

Developmental origin, functional maintenance and genetic rescue of osteoclasts.

Osteoclasts are multinucleated giant cells that resorb bone, ensuring development and continuous remodelling of the skeleton and the bone marrow haematopoietic niche. Defective osteoclast activity leads to osteopetrosis and bone marrow failure1-9, whereas excess activity can contribute to bone loss and osteoporosis10. Osteopetrosis can be partially treated by bone marrow transplantation in humans and mice11-18, consistent with a haematopoietic origin of osteoclasts13,16,19 and studies thatsuggest that they develop by fusion of monocytic precursors derived from haematopoietic stem cells in the presence of CSF1 and RANK ligand1,20. However, the developmental origin and lifespan of osteoclasts, and the mechanisms that ensure maintenance of osteoclast function throughout life in vivo remain largely unexplored. Here we report that osteoclasts that colonize fetal ossification centres originate from embryonic erythro-myeloid progenitors21,22. These erythro-myeloid progenitor-derived osteoclasts are required for normal bone development and tooth eruption. Yet, timely transfusion of haematopoietic-stem-cell-derived monocytic cells in newborn mice is sufficient to rescue bone development in early-onset autosomal recessive osteopetrosis. We also found that the postnatal maintenance of osteoclasts, bone mass and the bone marrow cavity involve iterative fusion of circulating blood monocytic cells with long-lived osteoclast syncytia. As a consequence, parabiosis or transfusion of monocytic cells results in long-term gene transfer in osteoclasts in the absence of haematopoietic-stem-cell chimerism, and can rescue an adult-onset osteopetrotic phenotype caused by cathepsin K deficiency23,24. In sum, our results identify the developmental origin of osteoclasts and a mechanism that controls their maintenance in bones after birth. These data suggest strategies to rescue osteoclast deficiency in osteopetrosis and to modulate osteoclast activity in vivo.

Glucocorticoid-induced abnormality in bone remodeling.

Glucocorticoid(GC)is widely used to treat a variety of autoimmune, inflammatory and allergic diseases. GC causes a number of adverse effects and among them osteoporosis and degenerative bone fracture are major complications of GC therapy. GC inhibits differentiation of mesenchymal stem cells to osteoblasts, induces apoptosis of osteoblasts and osteocytes and prolongs lifespan of osteoclast, resulting in low bone mass and micro-architectural deterioration of bone tissue, which leads to severe osteoporosis, increased bone fragility and susceptibility to bone fracture. Physicians who prescribe GC have to manage patients according to guidelines on the management and treatment of GC-induced osteoporosis.

Glucocorticoid Signaling and Bone Biology.

Since glucocorticoids remain an effective therapeutic option for the treatment of many inflammatory and autoimmune diseases, glucocorticoid-induced osteoporosis is the most common form of secondary osteoporosis. Fractures may occur in as many as 30-50% of patients receiving chronic glucocorticoid therapy. Under physiological conditions, glucocorticoids are required for normal bone development due to their regulation of osteoblast differentiation, possibly via the Wnt/β-catenin pathway and TSC22D3. However, serum levels of endogenous corticosterone are elevated in aged mice and glucocorticoids exert negative effects on the survival of osteoblasts and osteocytes as well as angiogenesis. Glucocorticoid treatments impair bone formation and enhance bone resorption. Excess glucocorticoids induce osteoblast and osteocyte apoptosis by increasing pro-apoptotic molecules, reactive oxygen species, and endoplasmic reticulum stress and suppressing the Wnt/β-catenin pathway. Autophagy protects osteocytes from glucocorticoid-induced apoptosis, but passed some threshold, the process of autophagy leads the cells to apoptosis. Excess glucocorticoids impair osteoblastogenesis by inducing Wnt antagonists, including Dkk1, Sost, and sFRP-1. However, the findings are controversial and the involvement of Wnt antagonists requires further study. Excess glucocorticoids reduce the phosphorylation of Akt and GSK3β, which enhances the degradation of β-catenin. Excess glucocorticoids have been shown to modulate the expression of miRNAs, including miR-29a, miR-34a-5p, and miR-199a-5p, which regulate the proliferation and differentiation of osteoblast lineage cells. Excess glucocorticoids also enhance bone resorption by reducing OPG expression, increasing Rankl expression and reactive oxygen species, and prolonging the life span of osteoclasts; however, they also suppress the bone-degrading capacity of osteoclasts by disturbing the organization of the cytoskeleton.

Conditional abrogation of Atm in osteoclasts extends osteoclast lifespan and results in reduced bone mass.

Ataxia-telangiectasia mutated (ATM) kinase is a central component involved in the signal transduction of the DNA damage response (DDR) and thus plays a critical role in the maintenance of genomic integrity. Although the primary functions of ATM are associated with the DDR, emerging data suggest that ATM has many additional roles that are not directly related to the DDR, including the regulation of oxidative stress signaling, insulin sensitivity, mitochondrial homeostasis, and lymphocyte development. Patients and mice lacking ATM exhibit growth retardation and lower bone mass; however, the mechanisms underlying the skeletal defects are not fully understood. In the present study, we generated mutant mice in which ATM is specifically inactivated in osteoclasts. The mutant mice did not exhibit apparent developmental defects but showed reduced bone mass due to increased osteoclastic bone resorption. Osteoclasts lacking ATM were more resistant to apoptosis and showed a prolonged lifespan compared to the controls. Notably, the inactivation of ATM in osteoclasts resulted in enhanced NF-κB signaling and an increase in the expression of NF-κB-targeted genes. The present study reveals a novel function for ATM in regulating bone metabolism by suppressing the lifespan of osteoclasts and osteoclast-mediated bone resorption.

Activation of dimeric glucocorticoid receptors in osteoclast progenitors potentiates RANKL induced mature osteoclast bone resorbing activity

Glucocorticoid (GC) therapy is the greatest risk factor for secondary osteoporosis. Pathogenic mechanisms involve an initial increase in bone resorption followed by decreased bone formation. To gain a better understanding of the resorptive activity of GCs, we have used mouse bone marrow macrophages (BMM) to determine if GCs can directly modulate RANKL stimulated osteoclast formation and/or activity. In agreement with previous studies, experiments performed in plastic wells showed that GCs (dexamethasone, hydrocortisone, and prednisolone) inhibited osteoclast number and size during the initial phases of RANKL stimulated osteoclastogenesis; however, in prolonged cultures, decreased apoptosis was observed and escape from GC induced inhibition occurred with an enhanced number of osteoclasts formed, many with an increased area. When BMM cells were seeded on bone slices, GCs robustly enhanced RANKL stimulated formation of resorption pits and release of CTX without affecting the number or size of osteoclasts formed and with no effect on apoptosis. Stimulation of pit formation was not associated with increased life span of osteoclasts or an effect on mRNA expression of several osteoclastic or osteoclastogenic genes. The potentiation of RANKL induced CTX release by dexamethasone was significantly less in BMM cells from mice with conditional knockout of the osteoclastic glucocorticoid receptor and completely absent in cells from GRdim mice, which carry a point mutation in one dimerizing interface of the GC receptor. These data suggest that: 1. Plastic is a poor medium to use for studying direct effects of GCs on osteoclasts 2. GCs can enhance bone resorption without decreasing apoptosis, and 3. A direct enhancement of RANKL mediated resorption is stimulated by the dimeric GC-receptor.

Osteoporosis treatment: a missed opportunity.

Osteoporosis affects 1.2 million Australians and, in 2012, fractures due to osteoporosis and osteopenia in Australians aged over 50 years cost $2.75 billion. Even minor minimal trauma fractures are associated with increased morbidity and mortality. Despite increasing therapeutic options for managing osteoporosis, fewer than 20% of patients with a minimal trauma fracture are treated or investigated for osteoporosis, so under-treatment is extremely common. Fracture risk assessment is important for selecting patients who require specific anti-osteoporosis therapy. Post-menopausal osteoporosis is frequently due to an imbalance in bone remodelling, with bone resorption exceeding bone formation. Antiresorptive drugs reduce the number, activity and lifespan of osteoclasts, and include bisphosphonates, oestrogen, selective oestrogen receptor-modulating drugs, strontium ranelate, and the human monoclonal antibody denosumab. Teriparatide is the only anabolic agent currently available that stimulates osteoblast recruitment and activity; its antifracture efficacy for non-vertebral fractures increases with the duration of therapy for up to 2 years when it is associated with persisting increases in bone formation rate at the tissue level. Newer anabolic agents are imminent and include an analogue of parathyroid hormone-related protein, abaloparatide, and a humanised monoclonal antibody to an inhibitor of bone formation, romosozumab. Selection of anti-osteoporosis therapy should be individualised to patients, and the duration of bisphosphonate therapy has been covered in recent guidelines. The benefits of treatment far outweigh any risks associated with long term treatment. General practitioners need to take up the challenge imposed by osteoporosis and become champions of change to close the evidence-treatment gap.

Glucocorticoid and Bone. Pathogenesis of glucocorticoid-induced osteoporosis

Glucocorticoid (GC) is widely used to treat a variety of inflammatory and allergic diseases and about one million people in Japan are known to take oral GC. GC causes a number of adverse effects and among them osteoporosis and degenerative bone fracture are major complications of GC therapy. GC induces apoptosis of osteoblasts and osteocytes and prolongs lifespan of osteoclast, resulting in low bone mass and microarchitectural deterioration of bone tissue, which leads to severe osteoporosis, increased bone fragility and susceptibility to bone fracture. Although GC-induced osteoporosis is the most common iatrogenic osteoporosis, few physicians fully manage it. Physicians who prescribe GC have to manage patients according to guidelines on the management and treatment of GC-induced osteoporosis, because recent multiple evidences strongly indicate that certain medication such as bisphosphonates is effective for treatment and prophylaxis of GC-induced osteoporosis.

OP0252 Effect of Glucocorticoids and Bisphosphonates on PPAR-Gamma and Hedgehog Pathway in Glucocorticoid-Induced Osteoporosis

BackgroundPrevious studies have investigated the molecular mechanisms involved in Glucocorticoid-induced osteoporosis (GIOP), which found that glucocorticoids (GCs) treatment increased bone resorption by prolonging of the lifespan of osteoclasts [1] and...

FRI0324 Dkk-1 and rankl/opg predict changes in bone mineral density and bone microstructure in sle on long-term glucocorticoid: a prospective study

BackgroundInflammation in SLE is associated with an increase in circulating levels of pro-inflammatory cytokines including receptor activator for nuclear factor-κB ligand (RANKL), inducing osteoclasts differentiation and bone resorption. On the...

Steroids and osteoporosis: the quest for mechanisms.

Advances made during the last 35 years have improved our understanding of the mechanisms of steroid hormone action on bone and how physiologic, pathologic, or iatrogenic changes in hormone levels can lead to increased fracture risk. Estrogens, androgens, and glucocorticoids alter the cellular composition of bone by regulating the supply and lifespan of osteoclasts and osteoblasts. Additionally, they influence the survival of osteocytes, long-lived cells that are entombed within the mineralized matrix and mediate the homeostatic adaptation of bone to mechanical forces. Altered redox balance is a proximal underlying mechanism of some of these effects, and sex steroid deficiency or glucocorticoid excess contributes to the aging of the skeleton.

Glucocorticoid and bone metabolism and disease

Glucocorticoid (GC) is widely used to treat a variety of inflammatory and allergic diseases and about one million people in Japan are known to take oral GC. GC causes a number of significant side effects and among them osteoporosis and degenerative bone fracture are major complications of GC therapy. GC induces apoptosis of osteoblasts and osteocytes and prolongs lifespan of osteoclast, resulting in severe osteoporosis. However, few physicians are aware of the increased risk of fracture caused by GC. Recent multiple evidence strongly indicates that bisphosphonate is effective for treatment and prophylaxis of GC-induced osteoporosis by improving both quantity and quality of bones, which results in reduction of the proportion of fractures. Recent findings regarding with anti-RANKL antibody and PTH (1-34) teriparatide for GC-induced osteoporosis will be also discussed.

Adverse Effects of Corticosteroids on Bone Metabolism: A Review

Glucocorticoid (GC) exposure is the most common etiology of drug-induced (secondary) osteoporosis. Twenty percent of all cases of osteoporosis have been attributed to GC exposure. Significant risk factors for the development of fractures after GC exposure include age older than 65 years, prolonged GC exposure (>3 months), positive family history of osteoporosis, and low calcium intake. GCs are known to inhibit bone remodeling and to increase fracture risk. GC exposure alters the fragile balance between osteoclast and osteoblast activity in bone metabolism. GC stimulates osteoclast-mediated bone resorption and reduces osteoblast-mediated bone formation, which results in increased overall net bone resorption. Specifically, the 2 main effects of GCs on bone metabolism are (1) inducing apoptosis in osteoblasts and osteocytes, thereby decreasing bone formation, and (2) prolonging the lifespan of osteoclasts and increasing bone resorption. The risk of fracture decreases 3 months after cessation of GC therapy; thus, a 3-month period may be ideal between GC exposures in patients at high risk for the development of osteoporosis. Patients managed with GCs who are at high risk for the development of secondary osteoporosis should have appropriate diagnostic testing; pre-GC exposure medication management (ie, use of bisphosphonates, human parathyroid hormone); and a limitation of GC therapy, with a wait period of 3 months between GC exposures if possible.

Intermittent Parathyroid Hormone Administration Counteracts the Adverse Effects of Glucocorticoids on Osteoblast and Osteocyte Viability, Bone Formation, and Strength in Mice

Glucocorticoids act directly on bone cells to decrease production of osteoblasts and osteoclasts, increase osteoblast and osteocyte apoptosis, and prolong osteoclast life span. Conversely, daily injections of PTH decrease osteoblast and osteocyte apoptosis and increase bone formation and strength. Using a mouse model, we investigated whether the recently demonstrated efficacy of PTH in glucocorticoid-induced bone disease results from the ability of this therapeutic modality to counteract at least some of the direct effects of glucocorticoids on bone cells. Glucocorticoid administration to 5- to 6-month-old Swiss-Webster mice for 28 d increased the prevalence of osteoblast and osteocyte apoptosis and decreased osteoblast number, activation frequency, and bone formation rate, resulting in reduced osteoid, wall and trabecular width, bone mineral density, and bone strength. In contrast, daily injections of PTH caused a decrease in osteoblast and osteocyte apoptosis and an increase in osteoblast number, activation frequency, bone formation rate, bone mineral density, and bone strength. The decreased osteocyte apoptosis was associated with increased bone strength. When the two agents were combined, all the adverse effects of glucocorticoid excess on bone were prevented. Likewise, in cultured osteoblastic cells, PTH attenuated the adverse effects of glucocorticoids on osteoblast survival and Wnt signaling via an Akt phosphorylation-dependent mechanism. We conclude that intermittent PTH administration directly counteracts the key pathogenetic mechanisms of glucocorticoid excess on bone, thus providing a mechanistic explanation of its efficacy against glucocorticoid-induced osteoporosis.

Effects and mechanism of aromatic aminoketone SY0916 on osteoclastic bone destruction.

To study the effects and mechanism of aromatic aminoketone (SY0916) on bone destruction in vitro. MC3T3-E1 cells and bone marrow cells were co-cultured to obtain purified osteoclasts. The proliferation of osteoclast-like cells (OCLs) was determined by MTT assay. The number of osteoclasts was measured by tartrate-resistant acid phosphatase (TRAP) staining. The functioning of osteoclasts was determined by measuring the area of bone resorption pits on bone slices. MMP-9 secretion by osteoclasts was measured by an ELISA kit. Osteoclast apoptosis was detected by flow cytometry using an AnnexinV-FITC kit. Gene expression of RANK and MMP-9 in osteoclasts as well as RANKL and OPG in MC3T3-E1 cells was determined by real-time PCR. SY0916 significantly inhibited the proliferation of OCLs, decreased both the total and average area of bone resorption pits, and dramatically inhibited the number of osteoclasts between concentrations of 0.01 and 10 micromol/L. Furthermore, SY0916 reversed IL-1 beta-mediated inhibition of osteoclast apoptosis and shortened osteoclast lifespan. In addition, SY0916 significantly inhibited the mRNA expression of RANK, RANKL, OPG, and MMP-9. However, the inhibition of OPG was weaker than that of RANKL. Accordingly, the ratio of RANKL to OPG mRNA expression in MC3T3-E1 cells was significantly decreased by SY0916. Meanwhile, the expression of MMP-9 protein in osteoclasts was inhibited by SY0916 between 0.01 and 10 micromol/L. SY0916 prevents osteoclastic bone destruction by inhibiting the proliferation and function of osteoclasts. The underlying mechanism for this effect involves the regulation of the RANKL-OPG-RANK axis, which determines the direction of bone metabolism.

Effects of SERMs on bone health. Mechanisms of bone mass control by selective estrogen receptor modulator

The bone mass, which is controlled by the balances between bone formation and bone resorption can be reduced by estrogen deficiency in post-menopausal osteoporosis. Reduced bone mass can be recovered by hormone replacement therapy (HRT) , however, HRT has various side effects. Although SERMs can rescue the bone mass with less side effect compared to HRT, the precise mechanisms of this effect is still elusive. From the results of the analyses for osteoclast specific estrogen receptor (ER) alphaknockout mice and the genome wide approach of ERalphabinding site, estrogen and SERMs can, at least in part, protect the bone mass by inducing the expression of Fas ligand and controling the life span of osteoclasts. More precise molecular mechanisms of the effect of SERM, especially in tissue/cell type specificity, may help to investigate new SERM, which is more specific and effective to treat post-menopausal osteoporosis.

Ascorbic acid accelerates osteoclast formation and death

Ascorbic acid (AA) plays a key role in bone formation. However controversy remains about the effect of AA on cells responsible for bone destruction, osteoclasts. We investigated the effect of AA on osteoclastogenesis using primary mouse bone marrow cultures and monocytic RAW 264.7 cells treated with osteoclastogenic factors RANKL and MCSF. Treatment with AA resulted in significant increase in osteoclast number, size and nucleation. To assess osteoclast oxidative stress level, a ratio of reduced (GSH) to oxidized (GSSG) glutathione and the total glutathione content (GSHt) were evaluated. Osteoclast differentiation was associated with a decrease in GSH/GSSG and GSHt. AA induced further decrease in both parameters, and resulted in significant production of H2O2, indicating its pro-oxidant action. At low concentration, H2O2 induced similar effects to AA, although less potently, and catalase partially inhibited AA-induced osteoclastogenesis. To assess the modification in osteoclast metabolism, the mitochondrial activity was evaluated using JC-1 and the ATP levels were assessed. Osteoclast formation was associated with the increase in mitochondrial activity and ATP concentration, which were further increased in the presence of AA. Importantly, the stimulatory effect of AA was only evident at early phase of osteoclastogenesis, whereas at the late stage AA significantly accelerated osteoclast death. Thus, during osteoclastogenesis AA acts as an oxidant, first stimulating osteoclast formation, but later limiting osteoclast lifespan. This duality of AA action allows reconciling the stimulatory action of AA on osteoclastogenesis observed in vitro with an overall attenuation of bone resorption in the presence of AA observed in vivo.