Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption.

Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.

Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1.

Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

RANK ligand converts the NCoR/HDAC3 co-repressor to a PGC1ß- and RNA-dependent co-activator of osteoclast gene expression.

The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1ß with the NCoR/HDAC3 complex, resulting in the activation of PGC1ß and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation in vitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts.

A Zeb1/MtCK1 metabolic axis controls osteoclast activation and skeletal remodeling.

Osteoclasts are bone-resorbing polykaryons responsible for skeletal remodeling during health and disease. Coincident with their differentiation from myeloid precursors, osteoclasts undergo extensive transcriptional and metabolic reprogramming in order to acquire the cellular machinery necessary to demineralize bone and digest its interwoven extracellular matrix. While attempting to identify new regulatory molecules critical to bone resorption, we discovered that murine and human osteoclast differentiation is accompanied by the expression of Zeb1, a zinc-finger transcriptional repressor whose role in normal development is most frequently linked to the control of epithelial-mesenchymal programs. However, following targeting, we find that Zeb1 serves as an unexpected regulator of osteoclast energy metabolism. In vivo, Zeb1-null osteoclasts assume a hyperactivated state, markedly decreasing bone density due to excessive resorptive activity. Mechanistically, Zeb1 acts in a rheostat-like fashion to modulate murine and human osteoclast activity by transcriptionally repressing an ATP-buffering enzyme, mitochondrial creatine kinase 1 (MtCK1), thereby controlling the phosphocreatine energy shuttle and mitochondrial respiration. Together, these studies identify a novel Zeb1/MtCK1 axis that exerts metabolic control over bone resorption in vitro and in vivo.

Dancr-BRG1 regulates Nfatc1 transcription and Pgc1ß-dependent metabolic shifts in osteoclastogenesis.

Long non-coding RNA (lncRNA) serves as a vital regulator of bone metabolism, but its role in pathologically overactive osteoclast differentiation remains elusive. Here, we identify lncRNA Dancr (Differentiation Antagonizing Non-protein Coding RNA) as a critical suppressor of osteoclastogenesis and bone resorption, which is down-regulated in response to estrogen deficiency. Global or osteoclast-specific Dancr Knockout mice display significant trabecular bone deterioration and enhanced osteoclast activity, but minimal alteration of bone formation. Moreover, the bone-targeted delivery of Dancr by Adeno-associated viral remarkably attenuates ovariectomy-induced osteopenia in mice. Mechanistically, Dancr establishes a direct interaction with Brahma-related gene 1 to prevent its binding and preserve H3K27me3 enrichment at the nuclear factor of activated T cells 1 and proliferator-activated receptor gamma coactivator 1-beta promoters, thereby maintaining appropriate expression of osteoclastic genes and metabolic programs during osteoclastogenesis. These results demonstrate that Dancr is a key molecule maintaining proper osteoclast differentiation and bone homeostasis under physiological conditions, and Dancr overexpression constitutes a potential strategy for treating osteoporosis.

Hypoxia-Sensitive COMMD1 Integrates Signaling and Cellular Metabolism in Human Macrophages and Suppresses Osteoclastogenesis.

Hypoxia augments inflammatory responses and osteoclastogenesis by incompletely understood mechanisms. We identified COMMD1 as a cell-intrinsic negative regulator of osteoclastogenesis that is suppressed by hypoxia. In human macrophages, COMMD1 restrained induction of NF-κB signaling and a transcription factor E2F1-dependent metabolic pathway by the cytokine RANKL. Downregulation of COMMD1 protein expression by hypoxia augmented RANKL-induced expression of inflammatory and E2F1 target genes and downstream osteoclastogenesis. E2F1 targets included glycolysis and metabolic genes including CKB that enabled cells to meet metabolic demands in challenging environments, as well as inflammatory cytokine-driven target genes. Expression quantitative trait locus analysis linked increased COMMD1 expression with decreased bone erosion in rheumatoid arthritis. Myeloid deletion of Commd1 resulted in increased osteoclastogenesis in arthritis and inflammatory osteolysis models. These results identify COMMD1 and an E2F-metabolic pathway as key regulators of osteoclastogenic responses under pathological inflammatory conditions and provide a mechanism by which hypoxia augments inflammation and bone destruction.

Schnurri-3 inhibition suppresses bone and joint damage in models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to systemic and articular bone loss by activating bone resorption and suppressing bone formation. Despite current therapeutic agents, inflammation-induced bone loss in RA continues to be a significant clinical problem due to joint deformity and lack of articular and systemic bone repair. Here, we identify the suppressor of bone formation, Schnurri-3 (SHN3), as a potential target to prevent bone loss in RA. SHN3 expression in osteoblast-lineage cells is induced by proinflammatory cytokines. Germline deletion or conditional deletion of Shn3 in osteoblasts limits articular bone erosion and systemic bone loss in mouse models of RA. Similarly, silencing of SHN3 expression in these RA models using systemic delivery of a bone-targeting recombinant adenoassociated virus protects against inflammation-induced bone loss. In osteoblasts, TNF activates SHN3 via ERK MAPK-mediated phosphorylation and, in turn, phosphorylated SHN3 inhibits WNT/ß-catenin signaling and up-regulates RANKL expression. Accordingly, knock-in of a mutation in Shn3 that fails to bind ERK MAPK promotes bone formation in mice overexpressing human TNF due to augmented WNT/ß-catenin signaling. Remarkably, Shn3-deficient osteoblasts are not only resistant to TNF-induced suppression of osteogenesis, but also down-regulate osteoclast development. Collectively, these findings demonstrate SHN3 inhibition as a promising approach to limit bone loss and promote bone repair in RA.

14-3-3ζ Suppresses RANKL Signaling by Destabilizing TRAF6.

Macrophages are essential regulators of inflammation and bone loss. RANKL, a pro-inflammatory cytokine, is responsible for macrophage differentiation to osteoclasts and bone loss. We recently showed that 14-3-3ζ-knockout (YwhazKO) rats exhibit increased bone loss in the inflammatory arthritis model. 14-3-3ζ is a cytosolic adaptor protein that actively participates in many signaling transductions. However, the role of 14-3-3ζ in RANKL signaling or bone remodeling is unknown. We investigated how 14-3-3ζ affects osteoclast activity by evaluating its role in RANKL signaling. We utilized 14-3-3ζ-deficient primary bone marrow-derived macrophages (BMDMs) obtained from wildtype (Wt) and YwhazKO animals, and RAW cells generated using CRISPR-Cas9. Our results showed that 14-3-3ζ-deficient macrophages, upon RANKL stimulation, have bigger and stronger TRAP-positive multinucleated cells and increased bone resorption activity. The presence of 14-3-3ζ suppressed RANKL-induced MAPK and AKT phosphorylation, transcription factors (NFATC1 and p65) nuclear translocation, and subsequently, gene induction (Rank, Acp5, and Ctsk). Mechanistically, 14-3-3ζ interacts with TRAF6, an essential component of the RANKL receptor complex. Upon RANKL stimulation, 14-3-3ζ-TRAF6 interaction was increased, while RANK-TRAF6 interaction was decreased. Importantly, 14-3-3ζ supported TRAF6 ubiquitination and degradation by the proteasomal pathway, thus dampening the downstream RANKL signaling. Together, we show that 14-3-3ζ regulates TRAF6 levels to suppress inflammatory RANKL signaling and osteoclast activity. To the best of our knowledge, this is the first report on 14-3-3ζ regulation of RANKL signaling and osteoclast activation.

Innate lymphoid cells: New players in osteoimmunology.

Innate lymphoid cells (ILCs) are the most recently identified immune cell types existing in lymphoid and nonlymphoid organs. Albeit they lack the expression of antigen receptors, ILCs play vital roles in innate immune responses by producing multiple effector cytokines. The ILC family includes conventional natural killer cells and cytokine-producing ILCs, which are divided into group 1, group 2, and group 3 ILCs based on their effector cytokines and developmental requirements. Emerging evidence has indicated that ILCs are essential immune regulators of bone homeostasis, playing a critical role in osteoimmunology. In this mini-review, we discuss recent advances in the understanding of ILC functions in bone homeostasis under physiological and pathological conditions, with an emphasis on the communication between ILCs and bone cells including osteoclasts and osteoblasts, as well as the underlying immunoregulatory networks involving ILC-derived cytokines and growth factors. This review also discusses future research directions and the potential of targeting ILCs for the treatment of inflammation-associated bone disorders.

The origins and roles of osteoclasts in bone development, homeostasis and repair.

The mechanisms underlying bone development, repair and regeneration are reliant on the interplay and communication between osteoclasts and other surrounding cells. Osteoclasts are multinucleated monocyte lineage cells with resorptive abilities, forming the bone marrow cavity during development. This marrow cavity, essential to hematopoiesis and osteoclast-osteoblast interactions, provides a setting to investigate the origin of osteoclasts and their multi-faceted roles. This Review examines recent developments in the embryonic understanding of osteoclast origin, as well as interactions within the immune environment to regulate normal and pathological bone development, homeostasis and repair.

Cortactin controls bone homeostasis through regulating the differentiation of osteoblasts and osteoclasts.

Cortactin (CTTN), a cytoskeletal protein and substrate of Src kinase, is implicated in tumor aggressiveness. However, its role in bone cell differentiation remains unknown. The current study revealed that CTTN was upregulated during osteoblast and adipocyte differentiation. Functional experiments demonstrated that CTTN promoted the in vitro differentiation of mesenchymal stem/progenitor cells into osteogenic and adipogenic lineages. Mechanistically, CTTN was able to stabilize the protein level of mechanistic target of rapamycin kinase (mTOR), leading to the activation of mTOR signaling. In-depth investigation revealed that CTTN could bind with casitas B lineage lymphoma-c (c-CBL) and counteract the function of c-CBL, a known E3 ubiquitin ligase responsible for the proteasomal degradation of mTOR. Silencing c-Cbl alleviated the impaired differentiation of osteoblasts and adipocytes caused by CTTN siRNA, while silencing mTOR mitigated the stimulation of osteoblast and adipocyte differentiation induced by CTTN overexpression. Notably, transplantation of CTTN-silenced bone marrow stromal cells (BMSCs) into the marrow of mice led to a reduction in trabecular bone mass, accompanied by a decrease in osteoblasts and an increase in osteoclasts. Furthermore, CTTN-silenced BMSCs expressed higher levels of receptor activator of nuclear factor κB ligand (RANKL) than control BMSCs did and promoted osteoclast differentiation when cocultured with bone marrow-derived osteoclast precursor cells. This study provides evidence that CTTN favors osteoblast differentiation by counteracting the c-CBL-induced degradation of mTOR and inhibits osteoclast differentiation by downregulating the expression of RANKL. It also suggests that maintaining an appropriate level of CTTN expression may be advantageous for maintaining bone homeostasis.

The G9a histone methyltransferase represses osteoclastogenesis and bone resorption by regulating NFATc1 function.

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.

M2 macrophages secrete glutamate-containing extracellular vesicles to alleviate osteoporosis by reshaping osteoclast precursor fate.

Osteoclast precursors (OCPs) are thought to commit to osteoclast differentiation, which is accelerated by aging-related chronic inflammation, thereby leading to osteoporosis. However, whether the fate of OCPs can be reshaped to transition into other cell lineages is unknown. Here, we showed that M2 macrophage-derived extracellular vesicles (M2-EVs) could reprogram OCPs to downregulate osteoclast-specific gene expression and convert OCPs to M2 macrophage-like lineage cells, which reshaped the fate of OCPs by delivering the molecular metabolite glutamate. Upon delivery of glutamate, glutamine metabolism in OCPs was markedly enhanced, resulting in the increased production of α-ketoglutarate (αKG), which participates in Jmjd3-dependent epigenetic reprogramming, causing M2-like macrophage differentiation. Thus, we revealed a novel transformation of OCPs into M2-like macrophages via M2-EVs-initiated metabolic reprogramming and epigenetic modification. Our findings suggest that M2-EVs can reestablish the balance between osteoclasts and M2 macrophages, alleviate the symptoms of bone loss, and constitute a new approach for bone-targeted therapy to treat osteoporosis.

NOTCH2 Hajdu-Cheney Mutations Escape SCFFBW7-Dependent Proteolysis to Promote Osteoporosis.

Hajdu-Cheney syndrome (HCS), a rare autosomal disorder caused by heterozygous mutations in NOTCH2, is clinically characterized by acro-osteolysis, severe osteoporosis, short stature, neurological symptoms, cardiovascular defects, and polycystic kidneys. Recent studies identified that aberrant NOTCH2 signaling and consequent osteoclast hyperactivity are closely associated with the bone-related disorder pathogenesis, but the exact molecular mechanisms remain unclear. Here, we demonstrate that sustained osteoclast activity is largely due to accumulation of NOTCH2 carrying a truncated C terminus that escapes FBW7-mediated ubiquitination and degradation. Mice with osteoclast-specific Fbw7 ablation revealed osteoporotic phenotypes reminiscent of HCS, due to elevated Notch2 signaling. Importantly, administration of Notch inhibitors in Fbw7 conditional knockout mice alleviated progressive bone resorption. These findings highlight the molecular basis of HCS pathogenesis and provide clinical insights into potential targeted therapeutic strategies for skeletal disorders associated with the aberrant FBW7/NOTCH2 pathway as observed in patients with HCS.

IFT80 negatively regulates osteoclast differentiation via association with Cbl-b to disrupt TRAF6 stabilization and activation.

Excess bone loss due to increased osteoclastogenesis is a significant clinical problem. Intraflagellar transport (IFT) proteins have been reported to regulate cell growth and differentiation. The role of IFT80, an IFT complex B protein, in osteoclasts (OCs) is completely unknown. Here, we demonstrate that deletion of IFT80 in the myeloid lineage led to increased OC formation and activity accompanied by severe bone loss in mice. IFT80 regulated OC formation by associating with Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) to promote protein stabilization and proteasomal degradation of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). IFT80 knockdown resulted in increased ubiquitination of Cbl-b and higher TRAF6 levels, thereby hyperactivating the receptor activator of nuclear factor-κß (NF-κß) ligand (RANKL) signaling axis and increased OC formation. Ectopic overexpression of IFT80 rescued osteolysis in a calvarial model of bone loss. We have thus identified a negative function of IFT80 in OCs.

Exploring the impact of osteoprotegerin on osteoclast and precursor fusion: Mechanisms and modulation by ATP.

Osteoclast (OC) differentiation, vital for bone resorption, depends on osteoclast and precursor fusion. Osteoprotegerin (OPG) inhibits osteoclast differentiation. OPG's influence on fusion and mechanisms is unclear. Osteoclasts and precursors were treated with OPG alone or with ATP. OPG significantly reduced OC number, area and motility and ATP mitigated OPG's inhibition. However, OPG hardly affected the motility of precusors. OPG downregulated fusion-related molecules (CD44, CD47, DC-STAMP, ATP6V0D2) in osteoclasts, reducing only CD47 in precursors. OPG reduced Connexin43 phosphorylated forms (P1 and P2) in osteoclasts, affecting only P2 in precursors. OPG disrupted subcellular localization of CD44, CD47, DC-STAMP, ATP6V0D2, and Connexin43 in both cell types. Findings underscore OPG's multifaceted impact, inhibiting multinucleated osteoclast and mononuclear precursor fusion through distinct molecular mechanisms. Notably, ATP mitigates OPG's inhibitory effect, suggesting a potential regulatory role for the ATP signaling pathway. This study enhances understanding of intricate processes in osteoclast differentiation and fusion, offering insights into potential therapeutic targets for abnormal bone metabolism.

Novel insights into the coupling of osteoclasts and resorption to bone formation.

Bone remodeling consists of resorption by osteoclasts (OCs) and formation by osteoblasts (OBs). Precise coordination of these activities is required for the resorbed bone to be replaced with an equal amount of new bone in order to maintain skeletal mass throughout the lifespan. This coordination of remodeling processes is referred to as the "coupling" of resorption to bone formation. In this review, we discuss the essential role for OCs in coupling resorption to bone formation, mechanisms for this coupling, and how coupling becomes less efficient or disrupted in conditions of bone loss. Lastly, we provide perspectives on targeting coupling to treat human bone disease.

Exercise to Mend Aged-tissue Crosstalk in Bone Targeting Osteoporosis & Osteoarthritis.

Aging induces alterations in bone structure and strength through a multitude of processes, exacerbating common aging- related diseases like osteoporosis and osteoarthritis. Cellular hallmarks of aging are examined, as related to bone and the marrow microenvironment, and ways in which these might contribute to a variety of age-related perturbations in osteoblasts, osteocytes, marrow adipocytes, chondrocytes, osteoclasts, and their respective progenitors. Cellular senescence, stem cell exhaustion, mitochondrial dysfunction, epigenetic and intracellular communication changes are central pathways and recognized as associated and potentially causal in aging. We focus on these in musculoskeletal system and highlight knowledge gaps in the literature regarding cellular and tissue crosstalk in bone, cartilage, and the bone marrow niche. While senolytics have been utilized to target aging pathways, here we propose non-pharmacologic, exercise-based interventions as prospective "senolytics" against aging effects on the skeleton. Increased bone mass and delayed onset or progression of osteoporosis and osteoarthritis are some of the recognized benefits of regular exercise across the lifespan. Further investigation is needed to delineate how cellular indicators of aging manifest in bone and the marrow niche and how altered cellular and tissue crosstalk impact disease progression, as well as consideration of exercise as a therapeutic modality, as a means to enhance discovery of bone-targeted therapies.

Sesamin inhibits RANKL-induced osteoclastogenesis and attenuates LPS-induced osteolysis via suppression of ERK and NF-κB signalling pathways.

Infection by bacterial products in the implant and endotoxin introduced by wear particles activate immune cells, enhance pro-inflammatory cytokines production, and ultimately promote osteoclast recruitment and activity. These factors are known to play an important role in osteolysis as well as potential targets for the treatment of osteolysis. Sesamin has been shown to have a variety of biological functions, such as inhibiting inflammation, anti-tumour and involvement in the regulation of fatty acid and cholesterol metabolism. However, the therapeutic effect of sesamin on osteolysis and its mechanism remain unclear. Present studies shown that in the condition of in vitro, sesamin could inhibit osteoclastogenesis and bone resorption, as well as suppressing the expression of osteoclast-specific genes. Further studies on the mechanism suggest that the effect of sesamin on human osteoclasts was mediated by blocking the ERK and NF-κB signalling pathways. Besides, sesamin was found to be effective in treating LPS-induced osteolysis by decreasing the production of pro-inflammatory cytokines and inhibiting osteoclastogenesis in vivo. Sesamin was non-toxic to heart, liver, kidney, lung and spleen. Therefore, sesamin is a promising phytochemical agent for the therapy of osteolysis-related diseases caused by inflammation and excessive osteoclast activation.

AMPKα1 negatively regulates osteoclastogenesis and mitigates pathological bone loss.

Osteoclasts are specialized cells responsible for bone resorption, a highly energy-demanding process. Focus on osteoclast metabolism could be a key for the treatment of osteolytic diseases including osteoporosis. In this context, AMP-activated protein kinase α1 (AMPKα1), an energy sensor highly expressed in osteoclasts, participates in the metabolic reconfiguration during osteoclast differentiation and activation. This study aimed to elucidate the role of AMPKα1 during osteoclastogenesis in vitro and its impact in bone loss in vivo. Using LysMcre/0AMPK⍺1f/f animals and LysMcre/0 as control, we evaluated how AMPKα1 interferes with osteoclastogenesis and bone resorption activity in vitro. We found that AMPKα1 is highly expressed in the early stages of osteoclastogenesis. Genetic deletion of AMPKα1 leads to increased gene expression of osteoclast differentiation and fusion markers. In addition, LysMcre/0AMPK⍺1f/f mice had an increased number and size of differentiated osteoclast. Accordingly, AMPKα1 negatively regulates bone resorption in vitro, as evidenced by the area of bone resorption in LysMcre/0AMPK⍺1f/f osteoclasts. Our data further demonstrated that AMPKα1 regulates mitochondrial fusion and fission markers upregulating Mfn2 and downregulating DRP1 (dynamics-related protein 1) and that Ctskcre/0AMPK⍺1f/f osteoclasts lead to an increase in the number of mitochondria in AMPK⍺1-deficient osteoclast. In our in vivo study, femurs from Ctskcre/0AMPK⍺1f/f animals exhibited bone loss associated with the increased number of osteoclasts, and there was no difference between Sham and ovariectomized group. Our data suggest that AMPKα1 acts as a negative regulator of osteoclastogenesis, and the depletion of AMPKα1 in osteoclast leads to a bone loss state similar to that observed after ovariectomy.