Blueberry metabolite-derived phenolic acids are thought to suppress bone resorption via interactions with the G protein-coupled receptor 109A (GPR109A). Previously, global GPR109A knockout (GPR109A ⁻/⁻ ) mice exhibited increased bone mass and a diminished bone-protective response to phenolic acids. While GPR109A is highly expressed in osteoclast precursor macrophages, its role in bone development remains unclear. To address this, we generated a myeloid cell-specific GPR109A knockout (GPR109A flox/flox /LysM-Cre⁺; CKO) mouse model and assessed bone phenotypes in male and female mice at 35 days, 3 months, 6 months, and 12 months using µCT. At 35 days, CKO males showed significantly improved tibia and vertebrae µCT parameters compared to controls (f/f, Cre⁺). However, at later time points (6 and 12 months), Cre recombinase effects were observed, with Cre⁺ males exhibiting similar bone parameters to CKO mice. In contrast, female CKO mice displayed significantly improved µCT parameters at 6 and 12 months. Notably, 12-month-old Cre⁺ males exhibited altered bone mechanical properties, while females did not. Gene expression analysis revealed increased Interferon regulatory factor 8 (Irf8), an osteoclastogenesis suppressor, in female CKO mice. These findings suggest that GPR109A regulates bone resorption through osteoclastogenic pathways in a sex-specific manner.

Disulfiram (DSF), known as an anti-alcoholism drug, has been reported to suppress osteoclast differentiation in vitro; however, it remains uncertain whether DSF is effective in preventing osteoclastogenesis in vivo. This study aimed to investigate the effect of DSF administration in osteoporotic mice and its contribution to osteoclastogenesis in vivo.

The bone phenotype of ovariectomized mice, both treated and untreated with DSF, was examined using microcomputed tomography analysis. Osteoclastic and osteoblastic parameters were assessed through bone morphometric analysis. The direct effect of DSF on osteoblastogenesis in vitro was evaluated via a primary osteoblast culture experiment. The expression of genes related to DSF targets (Nup85, Ccr2, and Ccr5) in osteoclast-lineage cells was examined using scRNA-seq analysis and flow cytometry analysis using the bone marrow cells from ovariectomized mice. The impact of DSF on osteoclast-lineage cells was assessed using primary cultures of osteoclasts.

DSF administration ameliorated ovariectomy-induced bone loss and mitigated the increase of osteoclasts without affecting osteoblastogenesis. The scRNA-seq data revealed that osteoclast precursor cells expressed Nup85, Ccr2, and Ccr5. CCR2 and CCR5-positive cells in osteoclast precursor cells within bone marrow increased following ovariectomy, and this increase was canceled by DSF administration. Finally, we found that DSF had a significant inhibitory effect on osteoclastogenesis in the early stage by suppressing Tnfrsf11a expression.

This study demonstrates that DSF could be a candidate for osteoporosis therapies because it suppresses osteoclastogenesis from an early stage in vivo.

Multiple Cervical Root Resorption (MCRR) is a rare condition characterized by the progressive destruction of the cervical region of multiple tooth roots, leading to significant tooth loss. The etiology and pathogenesis of MCRR remain poorly understood. Existing knowledge is largely derived from case reports/series. A comprehensive review of literature is crucial to identify potential systemic and dental factors that contribute to the development and progression of MCRR.

A scoping review was conducted following PRISMA guidelines. Five major health science databases were systematically searched to capture all reported cases of MCRR published to date. Potential etiological factors were identified and categorized based on their association with MCRRS.

A total of 65 reports documenting 101 patients and involving 921 teeth were included in the analysis. The review identified several potential etiological factors, including skeletal disorders, autoimmune diseases, viral infections, genetic diseases, specific genetic mutations, liver dysfunctions, the use of anti-resorptive medications, and endocrine disturbances. Each of these factors may influence osteoclast/odontoclast functioning, implicating them in the pathogenesis of MCRR.

Systemic diseases and medications that alter bone remodeling process or osteoclast/odontoclast function play a significant role in the development of a large proportion of MCRR cases. Given the complex and multifactorial nature of this condition, an interdisciplinary approach involving general dentists, specialists, and physicians is essential. Early detection, prevention, and personalized management of MCRR are critical in minimizing the risk of extensive tooth loss and improving patient outcomes.

Osteoclasts are specialized cells essential for bone resorption, a crucial process in bone remodeling, and dysregulation of osteoclastogenesis can lead to pathological bone loss such as osteoporosis and rheumatoid arthritis. Therefore, understanding the precise mechanisms governing osteoclast differentiation is crucial for developing effective therapies for skeletal diseases. In osteoclastogenesis, as well as other differentiated cells, it is well understood that cell cycle arrest is essential for terminal differentiation and is tightly regulated by CDK inhibitors such as Cip/Kip family and Ink4 family protein. In this manuscript, we identified p15Ink4b, a member of the Ink4 family, as a novel regulator of osteoclastogenesis by comprehensive single-cell RNA sequence data reanalyzing. Furthermore, histological analysis and in vitro osteoclast differentiation assay revealed that p15Ink4b functionally regulates osteoclastogenesis. Our findings may not only provide insights into the molecular mechanisms of osteoclast differentiation but also underscore the potential of harnessing cell cycle mechanisms to develop novel therapeutic strategies for bone diseases.

Osteoporosis is defined as a condition of increased risk of fracture due to decreased bone strength. In developed countries, the number of patients with osteoporosis and fragility fractures has been increasing in recent years due to the growing elderly population, posing a social challenge not only to fracture patients and their families but also to the social healthcare economy. Osteoporosis can be divided into two categories: primary osteoporosis caused by aging or menopause and secondary osteoporosis caused by metabolic or inflammatory diseases or drugs such as glucocorticoids. The majority of patients have primary osteoporosis, and the pathogenesis of postmenopausal osteoporosis and factors associated with fragility fractures in the elderly have been elucidated. On the other hand, rheumatoid arthritis (RA) is one of the causes of secondary osteoporosis. RA is a chronic inflammatory disease characterized by joint swelling and destruction. Most often, treatment focuses on suppressing these symptoms. However, physicians should be aware of the risk of osteoporosis in RA patients, because (1) RA is a chronic inflammatory disease, which itself can be a risk factor for osteoporosis; (2) glucocorticoids, which are sometimes administered to treat RA, can be a risk factor for osteoporosis; and (3) patients with RA are becoming older, and aging is an osteoporosis risk factor. A comprehensive understanding of the pathogenesis of osteoporosis and its fragility fractures requires elucidating the mechanisms underlying osteoclast activation, which drives their development. Furthermore, identifying the factors associated with fragility fractures is essential. This review summarizes the pathogenesis of osteoporosis, the factors associated with fragility fractures, and the associations between RA and osteoporosis development.

The IncRNA Malat1 was initially believed to be dispensable for physiology due to the lack of observable phenotypes in Malat1 knockout (KO) mice. However, our study challenges this conclusion. We found that both Malat1 KO and conditional KO mice in the osteoblast lineage exhibit significant osteoporosis. Mechanistically, Malat1 acts as an intrinsic regulator in osteoblasts to promote osteogenesis. Interestingly, Malat1 does not directly affect osteoclastogenesis but inhibits osteoclastogenesis in a non-autonomous manner in vivo via integrating crosstalk between multiple cell types, including osteoblasts, osteoclasts, and chondrocytes. Our findings substantiate the existence of a novel remodeling network in which Malat1 serves as a central regulator by binding to β-catenin and functioning through the β-catenin-OPG/Jagged1 pathway in osteoblasts and chondrocytes. In pathological conditions, Malat1 significantly promotes bone regeneration in fracture healing. Bone homeostasis and regeneration are crucial to well-being. Our discoveries establish a previous unrecognized paradigm model of Malat1 function in the skeletal system, providing novel mechanistic insights into how a lncRNA integrates cellular crosstalk and molecular networks to fine tune tissue homeostasis, remodeling and repair.

Osteoporosis is a degenerative bone disease highly prevalent in older women, causing high morbidity and mortality rates. Fourteen kinds of fucoidan were isolated from Sargassum fusiforme through acid (named as SFS), alkaline (SFJ) and water (SFW). SFW was passed through an anion exchange column to obtain SFW-0, SFW-0.5 and SFW-2. SFW-0.5 and SFW-2 were degraded to obtain different sulfate group contents SFW-x-M/S/O (x for 0.5 or 2). We further confirmed SFW-0.5-O was the most effective fraction of SFW. SFW-0.5-O may have alternating backbones of (Gal)n and (Fuc)n, and the main sulfation may be at C2/C3 of the Fuc/Gal residues. SFW-0.5-O inhibition of OC differentiation was associated with IRF-8 signaling; meanwhile, SFW-0.5-O promoted osteoblast differentiation and bone mineral nodule formation. SFW-0.5-O also effectively ameliorated osteoporosis symptom caused by estrogen deprivation in vivo. We uncovered that the fucoidan active fraction SFW-0.5-O demonstrated effective bone protection, may be exploited for osteoporosis therapy.

Pulsatilla koreana Nakai (P. koreana) is a perennial herb traditionally used to treat malaria and fever. Although the pharmacological properties of P. koreana have been explored in various contexts, its effects on bone diseases, such as osteoporosis, remain poorly studied. In this study, we investigated the effects of water extracts of P. koreana (WEPK) on osteoclasts, which play a crucial role in bone remodeling, and an ovariectomized (OVX) mouse model, which mimics osteoporosis. Phytochemical profiling of WEPK revealed several compounds that regulate bone or fat metabolism. WEPK suppressed osteoclast differentiation by downregulating the expression of receptor activator of nuclear factor-κB ligand (RANKL), a cytokine that induces osteoclastogenesis. Additionally, WEPK directly inhibited RANKL-induced differentiation of osteoclast precursors by downregulating nuclear factor of activated T cells 1 (NFATc1), the master transcription factor for osteoclastogenesis, by modulating its upstream regulators. In vivo, oral administration of WEPK suppressed bone loss, reduced weight gain, and mitigated fat accumulation in the liver and gonadal tissues of OVX mice. Given its positive impact on bone and fat accumulation under estrogen deficiency, WEPK may serve as a promising alternative therapy for postmenopausal osteoporosis, especially when accompanied by other metabolic disorders, such as obesity and fatty liver.

The IncRNA Malat1 was initially believed to be dispensable for physiology due to the lack of observable phenotypes in Malat1 knockout (KO) mice. However, our study challenges this conclusion. We found that both Malat1 KO and conditional KO mice in the osteoblast lineage exhibit significant osteoporosis. Mechanistically, Malat1 acts as an intrinsic regulator in osteoblasts to promote osteogenesis. Interestingly, Malat1 does not directly affect osteoclastogenesis but inhibits osteoclastogenesis in a non-autonomous manner in vivo via integrating crosstalk between multiple cell types, including osteoblasts, osteoclasts and chondrocytes. Our findings substantiate the existence of a novel remodeling network in which Malat1 serves as a central regulator by binding to β-catenin and functioning through the β-catenin-OPG/Jagged1 pathway in osteoblasts and chondrocytes. In pathological conditions, Malat1 significantly promotes bone regeneration in fracture healing. Bone homeostasis and regeneration are crucial to well-being. Our discoveries establish a previous unrecognized paradigm model of Malat1 function in the skeletal system, providing novel mechanistic insights into how a lncRNA integrates cellular crosstalk and molecular networks to fine tune tissue homeostasis, remodeling and repair.

The human skeleton is a multifunctional organ made up of multiple cell types working in concert to maintain bone and mineral homeostasis and to perform critical mechanical and endocrine functions. From the beginning steps of chondrogenesis that prefigures most of the skeleton, to the rapid bone accrual during skeletal growth, followed by bone remodeling of the mature skeleton, cell differentiation is integral to skeletal health. While growth factors and nuclear proteins that influence skeletal cell differentiation have been extensively studied, the role of cellular metabolism is just beginning to be uncovered. Besides energy production, metabolic pathways have been shown to exert epigenetic regulation via key metabolites to influence cell fate in both cancerous and normal tissues. In this review, we will assess the role of growth factors and transcription factors in reprogramming cellular metabolism to meet the energetic and biosynthetic needs of chondrocytes, osteoblasts, or osteoclasts. We will also summarize the emerging evidence linking metabolic changes to epigenetic modifications during skeletal cell differentiation.

Interferon Regulatory Factors (IRFs), a family of transcription factors, profoundly influence the immune system, impacting both physiological and pathological processes. This review explores the diverse functions of nine mammalian IRF members, each featuring conserved domains essential for interactions with other transcription factors and cofactors. These interactions allow IRFs to modulate a broad spectrum of physiological processes, encompassing host defense, immune response, and cell development. Conversely, their pivotal role in immune regulation implicates them in the pathophysiology of various diseases, such as infectious diseases, autoimmune disorders, metabolic diseases, and cancers. In this context, IRFs display a dichotomous nature, functioning as both tumor suppressors and promoters, contingent upon the specific disease milieu. Post-translational modifications of IRFs, including phosphorylation and ubiquitination, play a crucial role in modulating their function, stability, and activation. As prospective biomarkers and therapeutic targets, IRFs present promising opportunities for disease intervention. Further research is needed to elucidate the precise mechanisms governing IRF regulation, potentially pioneering innovative therapeutic strategies, particularly in cancer treatment, where the equilibrium of IRF activities is of paramount importance.

Tumour necrosis factor (TNF) superfamily member 11 (TNFSF11), also known as RANKL, plays a crucial role in regulating several physiological and pathological activities. Additionally, it is a vital factor in bone physiology, and the sex hormone progesterone regulates the expansion of stem cells and the proliferation of mammary epithelial cells. It is essential for animal growth and reproductive physiological processes. This study aimed to evaluate the tissue-specific expression characteristics and promoter activity of the TNFSF11 gene in pigs. As a result, the study examined the presence of TNFSF11 expression in the tissues of Xiangsu pigs at 0.6 and 12 months of age. Moreover, the core promoter region of TNFSF11 was also identified by utilizing a combination of bioinformatic prediction and dual-luciferase activity tests. Finally, the effect of transcription factors on the transcriptional activity of the core promoter region was determined using site-directed mutagenesis. TNFSF11 was uniformly expressed in all tissues; however, its expression in muscles was comparatively low. The core promoter region of TNFSF11 was located in the -555 to -1 region. The prediction of the transcription start site of TNFSF11 gene-2000 ∼ + 500bp showed that there was a CpG site in 17 ∼ + 487bp. Analysis of mutations in the transcription factor binding sites revealed that mutations in the Stat5b, Myog, Trl, and EN1 binding sites had significant effects on the transcriptional activity of the TNFSF11 gene, particularly following the EN1 binding site mutation (P < 0.001). This study provides insights into both the tissue-specific expression patterns of TNFSF11 in the tissues of Xiangsu pigs and the potential regulatory effects of transcription factors on its promoter activity. These results may be helpful for future research aimed at clarifying the expression and role of the porcine TNFSF11 gene.

Osteoclasts are multinucleated bone-resorbing cells, and their formation is tightly regulated to prevent excessive bone loss. However, the mechanisms by which osteoclast formation is restricted remain incompletely determined. Here, we found that sterol regulatory element binding protein 2 (SREBP2) functions as a negative regulator of osteoclast formation and inflammatory bone loss. Cholesterols and SREBP2, a key transcription factor for cholesterol biosynthesis, increased in the late phase of osteoclastogenesis. The ablation of SREBP2 in myeloid cells resulted in increased in vivo and in vitro osteoclastogenesis, leading to low bone mass. Moreover, deletion of SREBP2 accelerated inflammatory bone destruction in murine inflammatory osteolysis and arthritis models. SREBP2-mediated regulation of osteoclastogenesis is independent of its canonical function in cholesterol biosynthesis but is mediated, in part, by its downstream target, interferon regulatory factor 7 (IRF7). Taken together, our study highlights a previously undescribed role of the SREBP2-IRF7 regulatory circuit as a negative feedback loop in osteoclast differentiation and represents a novel mechanism to restrain pathological bone destruction.

Over-activated osteoclast (OC) is a major cause of diseases related to bone loss and bone metabolism. Both bone resorption inhibition and apoptosis induction of osteoclast are crucial in treating these diseases. X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) is an important interferon-stimulated and apoptotic gene. However, how XAF1 regulates bone formation and remodeling is unknown.

We generate global and chimeric Xaf1 knockout mouse models and utilize these models to explore the function and mechanism of XAF1 in regulating bone formation and remodeling in vivo and in vitro.

We show that XAF1 depletion enhances osteoclast generation in vitro. XAF1 knockout increases osteoclast number and bone resorption, thereby exacerbating bone loss in both OVX and osteolysis models. Activation of XAF1 with BV6 (a potent XIAP inhibitor) suppresses osteoclast formation. Mechanistically, XAF1 deletion decreases osteoclast apoptosis by facilitating the interaction between XIAP and caspase-3/7.

Our data illustrates an essential role of XAF1 in controlling osteoclastogenesis in both osteoporosis and osteolysis mouse models and highlights its underlying mechanism, indicating a potential role in clinical treatment.The translational potential of this article: The translation potential of this article is that we first indicated that osteoclast apoptosis induced by XAF1 contribute to the progression of osteoporosis and osteolysis, which provides a novel strategy in the prevention of osteoporosis and osteolysis.

Patients with multiple myeloma exhibit malignant osteolytic bone disease due to excessive osteoclast formation and function. We recently identified that osteoclastogenic stimulator selenoprotein W (SELENOW) is upregulated via ERK signaling and downregulated via p38 signaling during receptor activator of nuclear factor (NF)-κΒ ligand (RANKL)-induced osteoclast differentiation. In the intrinsic physiological process, RANKL-induced downregulation of SELENOW maintains proper osteoclast differentiation; in contrast, forced overexpression of SELENOW leads to overactive osteoclast formation and function.

We observed that SELENOW is highly expressed in multiple myeloma-derived peripheral blood mononuclear cells (PBMCs) and mature osteoclasts when compared to healthy controls. Also, the level of tumor necrosis factor alpha (TNFα), a pathological osteoclastogenic factor, is increased in the PBMCs and serum of patients with multiple myeloma. ERK activation by TNFα was more marked and sustained than that by RANKL, allowing SELENOW upregulation. Excessive expression of SELENOW in osteoclast progenitors and mature osteoclasts derived from multiple myeloma facilitated efficient nuclear translocation of osteoclastogenic transcription factors NF-κB and NFATc1, which are favorable for osteoclast formation.

Our findings suggest a possibility that feedforward signaling of osteoclastogenic SELENOW by TNFα derived from multiple myeloma induces overactive osteoclast differentiation, leading to bone loss during multiple myeloma.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by synovitis, bone and cartilage destruction, and increased fracture risk with bone loss. Although disease-modifying antirheumatic drugs have dramatically improved clinical outcomes, these therapies are not universally effective in all patients because of the heterogeneity of RA pathogenesis. Therefore, it is necessary to elucidate the molecular mechanisms underlying RA pathogenesis, including associated bone loss, in order to identify novel therapeutic targets. In this study, we found that Budding uninhibited by benzimidazoles 1 (BUB1) was highly expressed in RA patients' synovium and murine ankle tissue with arthritis. As CD45+CD11b+ myeloid cells are a Bub1 highly expressing population among synovial cells in mice, myeloid cell-specific Bub1 conditional knockout (Bub1ΔLysM) mice were generated. Bub1ΔLysM mice exhibited reduced femoral bone mineral density when compared with control (Ctrl) mice under K/BxN serum-transfer arthritis, with no significant differences in joint inflammation or bone erosion based on a semi-quantitative erosion score and histological analysis. Bone histomorphometry revealed that femoral bone mass of Bub1ΔLysM under arthritis was reduced by increased osteoclastic bone resorption. RNA-seq and subsequent Gene Set Enrichment Analysis demonstrated a significantly enriched nuclear factor-kappa B pathway among upregulated genes in receptor activator of nuclear factor kappa B ligand (RANKL)-stimulated bone marrow-derived macrophages (BMMs) obtained from Bub1ΔLysM mice. Indeed, osteoclastogenesis using BMMs derived from Bub1ΔLysM was enhanced by RANKL and tumor necrosis factor-α or RANKL and IL-1β treatment compared with Ctrl. Finally, osteoclastogenesis was increased by Bub1 inhibitor BAY1816032 treatment in BMMs derived from wildtype mice. These data suggest that Bub1 expressed in macrophages plays a protective role against inflammatory arthritis-associated bone loss through inhibition of inflammation-mediated osteoclastogenesis.

Interferon regulatory factor 8 (IRF8), a transcription factor expressed in immune cells, functions as a negative regulator of osteoclasts and helps maintain dental and skeletal homeostasis. Previously, we reported that a novel mutation in the IRF8 gene increases susceptibility to multiple idiopathic cervical root resorption (MICRR), a form of tooth root resorption mediated by increased osteoclast activity. The IRF8 G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. To investigate the molecular basis of MICRR and IRF8 function in osteoclastogenesis, we generated Irf8 knock-in (KI) mice using CRISPR/Cas9 technique modeling the human IRF8G388S mutation. The heterozygous (Het) and homozygous (Homo) Irf8 KI mice showed no gross morphological defects, and the development of hematopoietic cells was unaffected and similar to wild-type (WT) mice. The Irf8 KI Het and Homo mice showed no difference in macrophage gene signatures important for antimicrobial defenses and inflammatory cytokine production. Consistent with the phenotype observed in MICRR patients, Irf8 KI Het and Homo mice demonstrated significantly increased osteoclast formation and resorption activity in vivo and in vitro when compared to WT mice. The oral ligature-inserted Het and Homo mice displayed significantly increased root resorption and osteoclast-mediated alveolar bone loss compared to WT mice. The increased osteoclastogenesis noted in KI mice is due to the inability of IRF8G388S mutation to inhibit NFATc1-dependent transcriptional activation and downstream osteoclast specific transcripts, as well as its impact on autophagy-related pathways of osteoclast differentiation. This translational study delineates the IRF8 domain important for osteoclast function and provides novel insights into the IRF8 mutation associated with MICRR. IRF8G388S mutation mainly affects osteoclastogenesis while sparing immune cell development and function. These insights extend beyond oral health and significantly advance our understanding of skeletal disorders mediated by increased osteoclast activity and IRF8's role in osteoclastogenesis.

Epigenetic regulation plays a crucial role in the pathogenesis of autoimmune diseases such as inflammatory arthritis. DNA hypomethylating agents, such as decitabine (DAC), have been shown to dampen inflammation and restore immune homeostasis. In the present study, we demonstrate that DAC elicits potent anti-inflammatory effects and attenuates disease symptoms in several animal models of arthritis. Transcriptomic and epigenomic profiling show that DAC-mediated hypomethylation regulates a wide range of cell types in arthritis, altering the differentiation trajectories of anti-inflammatory macrophage populations, regulatory T cells, and tissue-protective synovial fibroblasts (SFs). Mechanistically, DAC-mediated demethylation of intragenic 5'-Cytosine phosphate Guanine-3' (CpG) islands of the transcription factor Irf8 (interferon regulatory factor 8) induced its re-expression and promoted its repressor activity. As a result, DAC restored joint homeostasis by resetting the transcriptomic signature of negative regulators of inflammation in synovial macrophages (MerTK, Trem2, and Cx3cr1), TREGs (Foxp3), and SFs (Pdpn and Fapα). In conclusion, we found that Irf8 is necessary for the inhibitory effect of DAC in murine arthritis and that direct expression of Irf8 is sufficient to significantly mitigate arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by inflammation of the synovial tissue and joint bone destruction, often leading to significant disability. The main pathological manifestation of joint deformity in RA patients is bone destruction, which occurs due to the differentiation and proliferation of osteoclasts. The transcription factor nuclear factor-activated T cell 1 (NFATc1) plays a crucial role in this process. The regulation of NFATc1 in osteoclast differentiation is influenced by three main factors. Firstly, NFATc1 is activated through the upstream nuclear factor kappa-B ligand (RANKL)/RANK signaling pathway. Secondly, the Ca2+-related co-stimulatory signaling pathway amplifies NFATc1 activity. Finally, negative regulation of NFATc1 occurs through the action of cytokines such as B-cell Lymphoma 6 (Bcl-6), interferon regulatory factor 8 (IRF8), MAF basic leucine zipper transcription factor B (MafB), and LIM homeobox 2 (Lhx2). These three phases collectively govern NFATc1 transcription and subsequently affect the expression of downstream target genes including TRAF6 and NF-κB. Ultimately, this intricate regulatory network mediates osteoclast differentiation, fusion, and the degradation of both organic and inorganic components of the bone matrix. This review provides a comprehensive summary of recent advances in understanding the mechanism of NFATc1 in the context of RA-related bone destruction and discusses potential therapeutic agents that target NFATc1, with the aim of offering valuable insights for future research in the field of RA. To assess their potential as therapeutic agents for RA, we conducted a drug-like analysis of potential drugs with precise structures.

Osteoclasts are the principal cells that efficiently resorb bone. Numerous studies have attempted to reveal the molecular pathways leading to the differentiation and activation of osteoclasts to improve the treatment and prevention of osteoporosis and other bone-destructive diseases. While the cumulative knowledge of osteoclast regulatory molecules, such as receptor activator of nuclear factor-kB ligand (RANKL) and nuclear factor of activated T cells 1 (NFATc1), contributes to the understanding of the developmental progression of osteoclasts, little is known about how the discrete steps of osteoclastogenesis modify osteoclast status but not the absolute number of osteoclasts. The regulatory mechanisms involved in osteoclast maturation but not those involved in differentiation deserve special attention due to their potential use in establishing a more effective treatment strategy: targeting late-phase differentiation while preserving coupled bone formation. Recent studies have shed light on the molecules that govern late-phase osteoclast differentiation and maturation, as well as the metabolic changes needed to adapt to shifting metabolic demands. This review outlines the current understanding of the regulation of osteoclast differentiation, as well as osteoclast metabolic adaptation as a differentiation control mechanism. Additionally, this review introduces molecules that regulate the late-phase osteoclast differentiation and thus minimally impact coupled bone formation.

Osteoporosis is a chronic disease that endangers the health of the elderly. Inhibiting osteoclast hyperactivity is a key aspect of osteoporosis prevention and treatment. Several studies have shown that interferon regulatory factor 9 (IRF9) not only regulates innate and adaptive immune responses but also plays an important role in inflammation, antiviral response, and cell development. However, the exact role of IRF9 in osteoclasts has not been reported. To elucidate the role of IRF9 in osteoclast differentiation, we established the ovariectomized mouse model of postmenopausal osteoporosis and found that IRF9 expression was reduced in ovariectomized mice with overactive osteoclasts. Furthermore, knockdown of IRF9 expression enhanced osteoclast differentiation in vitro. Using RNA sequencing, we identified that the differentially expressed genes enriched by IRF9 knockdown were related to ferroptosis. We observed that IRF9 knockdown promoted osteoclast differentiation via decreased ferroptosis in vitro and further verified that IRF9 knockdown reduced ferroptosis by activating signal transducer and activator of transcription 3 (STAT3) to promote osteoclastogenesis. In conclusion, we identified an essential role of IRF9 in the regulation of osteoclastogenesis in osteoporosis and its underlying mechanism.

Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1. In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow-derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis of Notch2tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2tm1.1Ecan BMMs treated with M-CSF or M-CSF and receptor activator of NF-κB ligand for 3 days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors, and mature cells. There were an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts, and affects cell cluster allocation in BMMs.

Poly (ADP-ribose) glycohydrolase (PARG) is an enzyme that mainly degrades poly (ADP-ribose) (PAR) synthesized by poly (ADP-ribose) polymerase (PARP) family proteins. Although PARG is involved in many biological phenomena, including DNA repair, cell differentiation, and cell death, little is known about the relationship between osteoclast differentiation and PARG. It has also not been clarified whether PARG is a valuable target for therapeutic agents in the excessive activity of osteoclast-related bone diseases such as osteoporosis. In the present study, we examined the effects of PARG inhibitor PDD00017273 on osteoclast differentiation in RANKL-induced RAW264 cells. PDD00017273 induced the accumulation of intracellular PAR and suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. PDD00017273 also downregulated osteoclast differentiation marker genes such as Trap, cathepsin K (Ctsk), and dendrocyte expressed seven transmembrane protein (Dcstamp) and protein expression of nuclear factor of activated T cells 1 (NFATc1), a master regulator of osteoclast differentiation. Taken together, our findings suggest that dysfunction of PARG suppresses osteoclast differentiation via the PAR accumulation and partial inactivation of the NFATc1.

Ubiquitination is a reversible post-translational modification implicated in cell differentiation, homeostasis, and organ development. Several deubiquitinases (DUBs) decrease protein ubiquitination through the hydrolysis of ubiquitin linkages. However, the role of DUBs in bone resorption and formation is still unclear. In this study, we identified DUB ubiquitin-specific protease 7 (USP7) as a negative regulator of osteoclast formation. USP7 combines with tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibits its ubiquitination by impairing the Lys63-linked polyubiquitin chain. Such impairment leads to the suppression of receptor activator of NF-κB ligand (RANKL)-mediated nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) activation without affecting TRAF6 stability. USP7 also protects the stimulator of interferon genes (STING) against degradation, inducing interferon-β (IFN-β) expression in osteoclast formation, thereby inhibiting osteoclastogenesis cooperatively with the classical TRAF6 pathway. Furthermore, USP7 inhibition accelerates osteoclast differentiation and bone resorption both in vitro and in vivo. Contrarily, USP7 overexpression impairs osteoclast differentiation and bone resorption in vitro and in vivo. Additionally, in ovariectomy (OVX) mice, USP7 levels are lower than those in sham-operated mice, suggesting that USP7 plays a role in osteoporosis. Altogether, our data reveal the dual effect of USP7-mediated TRAF6 signal transduction and USP7-mediated protein degradation of STING in osteoclast formation.

Radix Asteris, the root of Aster tataricus L. f., is historically significant in East Asian medicine for treating respiratory conditions. Yet, its implications on bone health remain uncharted. This research investigated the impact of an aqueous ethanol extract of Radix Asteris (EERA) on osteoclast differentiation and its prospective contribution to osteoporosis management. We discerned that EERA retards osteoclast differentiation by inhibiting receptor activator of nuclear factor kappa-B ligand (RANKL) expression and obstructing RANKL-induced osteoclastogenesis. EERA markedly suppressed RANKL-induced expression of NFATc1, a pivotal osteoclastogenic factor, via modulating early RANK signaling. EERA's therapeutic potential was underscored by its defense against trabecular bone degradation and its counteraction to increased body and perigonadal fat in ovariectomized mice, mirroring postmenopausal physiological changes. In the phytochemical analysis of EERA, we identified several constituents recognized for their roles in regulating bone and fat metabolism. Collectively, our findings emphasize the potential of EERA in osteoclast differentiation modulation and in the management of osteoporosis and associated metabolic changes following estrogen depletion, suggesting its suitability as an alternative therapeutic strategy for postmenopausal osteoporosis intertwined with metabolic imbalances.

Wear particle‑induced osteolysis is a serious complication that occurs in individuals with titanium (Ti)‑based implants following long‑term usage due to loosening of the implants. The control of excessive osteoclast differentiation and inflammation is essential for protecting against wear particle‑induced osteolysis. The present study evaluated the effect of britanin, a pseudoguaianolide sesquiterpene isolated from Inula japonica, on osteoclastogenesis in vitro and Ti particle‑induced osteolysis in vivo. The effect of britanin was examined in the osteoclastogenesis of mouse bone marrow‑derived macrophages (BMMs) using TRAP staining, RT‑PCR, western blotting and immunocytochemistry. The protective effect of britanin was examined in a mouse calvarial osteolysis model and evaluated using micro‑CT and histomorphometry. Britanin inhibited osteoclast differentiation and F‑actin ring formation in the presence of macrophage colony‑stimulating factor and receptor activator of nuclear factor kB ligand in BMMs. The expression of osteoclast‑specific marker genes, including tartrate‑resistant acid phosphatase, cathepsin K, dendritic cell‑specific transmembrane protein, matrix metallopeptidase 9 and nuclear factor of activated T‑cells cytoplasmic 1, in the BMMs was significantly reduced by britanin. In addition, britanin reduced the expression of B lymphocyte‑induced maturation protein‑1, which is a transcriptional repressor of negative osteoclastogenesis regulators, including interferon regulatory factor‑8 and B‑cell lymphoma 6. Conversely, britanin increased the expression levels of anti‑oxidative stress genes, namely nuclear factor erythroid‑2‑related factor 2, NAD(P)H quinone oxidoreductase 1 and heme oxygenase 1 in the BMMs. Furthermore, the administration of britanin significantly reduced osteolysis in a Ti particle‑induced calvarial osteolysis mouse model. Based on these findings, it is suggested that britanin may be a potential therapeutic agent for wear particle‑induced osteolysis and osteoclast‑associated disease.

Melia toosendan fructus, traditionally employed in traditional Chinese and Korean herbal medicine, exhibits diverse biological properties encompassing anti-tumor, anti-inflammatory, and anti-viral effects. However, its influence on bone metabolism remains largely unexplored. In this study, we investigated the impact of an ethanolic extract of Melia toosendan fructus (MTE) on osteoclast differentiation and characterized its principal active constituent in osteoclast differentiation and function, as well as its effects on bone protection. Our findings demonstrate that MTE effectively inhibits the differentiation of osteoclast precursors induced by receptor activator of nuclear factor κB ligand (RANKL). Utilizing a bioassay-guided fractionation approach coupled with UHPLC-MS/MS analysis, we isolated and identified the triterpenoid compound toosendanin (TSN) as the active constituent responsible for MTE's anti-osteoclastogenic activity. TSN treatment downregulated the expression of nuclear factor of activated T cells c1, a pivotal osteoclastogenic transcription factor, along with molecules implicated in osteoclast-mediated bone resorption, including tumor necrosis factor receptor-associated factor 6, carbonic anhydrase II, integrin beta-3, and cathepsin K. Furthermore, treatment of mature osteoclasts with TSN impaired actin ring formation, acidification, and resorptive function. Consistent with our in vitro findings, TSN administration mitigated trabecular bone loss and reduced serum levels of the bone resorption marker, C-terminal cross-linked telopeptides of type I collagen, in a mouse bone loss model induced by intraperitoneal injections of RANKL. These results suggest that TSN, as the principal active constituent of MTE with inhibitory effects on osteoclastogenesis, exhibits bone-protective properties by suppressing both osteoclast differentiation and function. These findings imply the potential utility of TSN in the treatment of diseases characterized by excessive bone resorption.

Anethum graveolens L., known as European dill, is a versatile herb widely used in both traditional medicine and culinary practices. Despite its long-standing history, the potential impact of the water extract of A. graveolens seeds (WEAG) on bone health remains unexplored. In this study, we investigated the influence of WEAG on osteoclast differentiation and assessed its potential as an anti-osteoporotic agent. WEAG hindered osteoclast differentiation through the suppression of receptor activator of nuclear factor-κB ligand (RANKL) expression in osteoclast-supporting cells and by directly targeting osteoclast precursor cells. WEAG significantly reduced the expression of key osteoclastogenic transcription factors, namely c-Fos and NFATc1, typically induced by RANKL in osteoclast precursors. This reduction was attributed to the suppression of both MAPKs and NF-κB pathways in response to RANKL. In vivo experiments further revealed that WEAG administration effectively reduces trabecular bone loss and weight gain triggered by ovariectomy, mimicking postmenopausal osteoporosis. Furthermore, our comprehensive phytochemical analysis of WEAG identified a range of phytochemical constituents, associated with bone health and weight regulation. Notably, we discovered a specific compound, isorhamnetin-3-O-glucuronide, within WEAG that exhibits anti-osteoclastogenic potential. Overall, this research elucidated the beneficial effects and mechanistic basis of WEAG on osteoclast differentiation and bone loss, indicating its potential as a viable alternative to address bone loss in conditions like postmenopause.

Spherical 50 nm silica-based nanoparticles (SiNPs) promote healthy bone homeostasis and maintenance by supporting bone forming osteoblast lineage cells while simultaneously inhibiting the differentiation of bone resorbing osteoclasts. Previous work demonstrated that an intraperitoneal injection of SiNPs in healthy mice - both young and old - increased bone density and quality, suggesting the possibility that SiNPs represent a dual action therapeutic. However, the underlying mechanisms governing the osteoclast response to SiNPs have yet to be fully explored and defined. Therefore, the goals of this study were to investigate the cellular and molecular mechanisms by which SiNPs inhibit osteoclastogenesis. SiNPs strongly inhibited RANKL-induced osteoclast differentiation within the first hours and concomitantly inhibited early transcriptional regulators such as Nfatc1. SiNPs simultaneously stimulated expression of autophagy related genes p62 and LC3β dependent on ERK1/2 signaling pathway. Intriguingly, SiNPs were found to stimulate autophagosome formation while inhibiting the autophagic flux necessary for RANKL-stimulated osteoclast differentiation, resulting in the inhibition of both the canonical and non-canonical NF-κB signaling pathways and stabilizing TRAF3. These results suggest a model in which SiNPs inhibit osteoclastogenesis by inhibiting the autophagic machinery and RANKL-dependent functionality. This mechanism of action defines a novel therapeutic strategy for inhibiting osteoclastogenesis.

Angelica dahurica radix has a long history of traditional use in China and Korea for treating headaches, cold-damp pain and skin diseases. Despite various pharmacological studies on A. dahurica, its impact on bones remains unclear. Hence, this study investigated the inhibitory effect of A. dahurica's radix water extract (WEAD) on osteoclast differentiation. In vitro experiments showed that WEAD effectively suppresses osteoclast differentiation. Treatment of an osteoclast precursor with WEAD significantly suppressed the expression of nuclear factor of activated T-cells 1 (NFATc1), essential transcription factor for osteoclastogenesis, while increasing the expression of negative regulators, interferon regulatory factor 8 (Irf8) and v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MafB). Consistent with the in vitro findings, the oral administration of WEAD (100 and 300 mg/kg/day) to mice subjected to surgical ovariectomy for a duration of six weeks alleviated bone loss, while also mitigating weight gain and liver fat accumulation. In addition, we also identified phytochemicals present in WEAD, known to regulate osteoclastogenesis and/or bone loss. These results suggest the potential use of WEAD for treating various bone disorders caused by excessive bone resorption.

Objectives: Bone immune disorders are major contributors to osteoporosis development. This study aims to identify potential diagnostic markers and molecular targets for osteoporosis treatment from an immunological perspective. Method: We downloaded dataset GSE56116 from the Gene Expression Omnibus database, and identified differentially expressed genes (DEGs) between normal and osteoporosis groups. Subsequently, differentially expressed immune-related genes (DEIRGs) were identified, and a functional enrichment analysis was performed. A protein-protein interaction network was also constructed based on data from STRING database to identify hub genes. Following external validation using an additional dataset (GSE35959), effective biomarkers were confirmed using RT-qPCR and immunohistochemical (IHC) staining. ROC curves were constructed to validate the diagnostic values of the identified biomarkers. Finally, a ceRNA and a transcription factor network was constructed, and a Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed to explore the biological functions of these diagnostic markers. Results: In total, 307 and 31 DEGs and DEIRGs were identified, respectively. The enrichment analysis revealed that the DEIRGs are mainly associated with Gene Ontology terms of positive regulation of MAPK cascade, granulocyte chemotaxis, and cytokine receptor. protein-protein interaction network analysis revealed 10 hub genes: FGF8, KL, CCL3, FGF4, IL9, FGF9, BMP7, IL17RA, IL12RB2, CD40LG. The expression level of IL17RA was also found to be significantly high. RT-qPCR and immunohistochemical results showed that the expression of IL17RA was significantly higher in osteoporosis patients compared to the normal group, as evidenced by the area under the curve Area Under Curve of 0.802. Then, we constructed NEAT1-hsa-miR-128-3p-IL17RA, and SNHG1-hsa-miR-128-3p-IL17RA ceRNA networks in addition to ERF-IL17RA, IRF8-IL17RA, POLR2A-IL17RA and ERG-IL17RA transcriptional networks. Finally, functional enrichment analysis revealed that IL17RA was involved in the development and progression of osteoporosis by regulating local immune and inflammatory processes in bone tissue. Conclusion: This study identifies the immune-related gene IL17RA as a diagnostic marker of osteoporosis from an immunological perspective, and provides insight into its biological function.

The pathophysiology of chronic implant-related bone infections is characterized by an increase in osteoclast numbers and enhanced bone resorption. Biofilms are a major reason for chronicity of such infections as the biofilm matrix protects bacteria against antibiotics and impairs the function of immune cells. Macrophages are osteoclast precursor cells and therefore linked to inflammation and bone destruction.

Investigations on the impact of biofilms on the ability of macrophages to form osteoclasts are yet missing and we, therefore, analyzed the effect of Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) planktonic and biofilm environments on osteoclastogenesis using RAW 264.7 cells and conditioned media (CM).

Priming with the osteoclastogenic cytokine RANKL before CM addition enabled the cells to differentiate into osteoclasts. This effect was highest in SE planktonic or SA biofilm CM. Simultaneous stimulation with CM and RANKL, however, suppressed osteoclast formation and resulted in formation of inflammation-associated multinucleated giant cells (MGCs) which was most pronounced in SE planktonic CM.

Our data indicate that the biofilm environment and its high lactate levels are not actively promoting osteoclastogenesis. Hence, the inflammatory immune response against planktonic bacterial factors through Toll-like receptors seems to be the central cause for the pathological osteoclast formation. Therefore, immune stimulation or approaches that aim at biofilm disruption need to consider that this might result in enhanced inflammation-mediated bone destruction.

This study was to investigate the functional role and mechanism of receptor activator of nuclear factor-kappa B ligand (RANKL) associated autophagy and chemoresistance in breast cancer.

Cell Counting Kit-8 (CCK-8) assay was used to detect the cell viability. Real-time polymerase chain reaction (PCR) was used for determining the relative mRNA levels of key genes and protein expression was assessed by Western blotting. Immunofluorescence was performed to evaluate the changes in the autophagy flux. Short hairpin (shRNA) was used to knockdown the expression of the target genes in breast cancer cells. Based on The Cancer Genome Atlas (TCGA) database, we explored the expression of receptor activator of nuclear factor-kappa B (RANK), autophagy and signal transducer and activator of transcription 3 (STAT3) signaling associated genes and analyzed their correlation with the prognosis of breast cancer patients.

The findings showed that receptor activator of nuclear factor-kappa B ligand (RANKL), the ligand of RANK, could effectively enhance the chemoresistance potential of breast cancer cells. Our results showed that RANKL induced autophagy and enhanced the expression of autophagy associated genes in breast cancer cells. The knockdown of RANK suppressed RANKL mediated autophagy induction in these cells. Furthermore, the inhibition of autophagy suppressed RANKL mediated chemoresistance in breast cancer cells. We found STAT3 signaling pathway was involved in RANKL-induced autophagy. Analysis of the expression of RANK, and autophagy and STAT3 signaling associated genes in breast cancer tissues showed that the expression of autophagy and STAT3 signaling associated genes was correlated with the prognosis of breast cancer patients.

The present study suggests that the RANKL/RANK axis may potentially mediate chemoresistance in breast cancer cells by inducing autophagy through the STAT3 signaling pathway.

Osteoporosis (OP) is a systemic bone degenerative disease characterized by low bone mass and deteriorated microarchitecture of bone tissue, causing high morbidity and mortality rates. Bone resorption by overactivated osteoclasts (OCs) is the main cause of osteoporosis. Glucuronomannan and its oligomers (Gs) and their sulfated derivatives (SGs) were previously prepared. The anti-osteoporosis activities of these glycans were evaluated. Firstly, we determined the viability of RAW264.7 by CCK-8 test. Nextly, we investigated the inhibitory effects of Gs and SGs on the differentiation of RAW264.7 cells into OCs using tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring staining, qualitative reverse-transcription polymerase chain reaction(qRT-PCR) and western blotting. TRAP staining revealed that Gs significantly blocked RANKL-induced OC generation while SGs did not exhibit this ability. F-actin staining assays demonstrated that Gs inhibits RANKL-induced actin ring formation. qRT-PCR analyses indicated that Gs dose-dependently inhibited the expression of OCs marker genes including Trap, NFATc1, c-Fos, DC-Stamp and ATP60 during the differentiation process, while SGs did not suppress. Regarding the mechanism of Gs, it was found that Gs suppressed osteoclastogenesis via inhibiting the degradation of IRF-8 and interfering with NF-κB pathway activation. Together, these results suggest that Gs have the ability to inhibit osteoclastogenesis by modulating IRF-8 signaling.

The past decade has seen significant advances in our understanding of skeletal homeostasis and the mechanisms that mediate the loss of bone integrity in disease. Recent breakthroughs have arisen mainly from identifying disease-causing mutations and modeling human bone disease in rodents, in essence, highlighting the integrative nature of skeletal physiology. It has become increasingly clear that bone cells, osteoblasts, osteoclasts, and osteocytes, communicate and regulate the fate of each other through RANK/RANKL/OPG, liver X receptors (LXRs), EphirinB2-EphB4 signaling, sphingolipids, and other membrane-associated proteins, such as semaphorins. Mounting evidence also showed that critical developmental pathways, namely, bone morphogenetic protein (BMP), NOTCH, and WNT, interact each other and play an important role in postnatal bone remodeling. The skeleton communicates not only with closely situated organs, such as bone marrow, muscle, and fat, but also with remote vital organs, such as the kidney, liver, and brain. The metabolic effect of bone-derived osteocalcin highlights a possible role of skeleton in energy homeostasis. Furthermore, studies using genetically modified rodent models disrupting the reciprocal relationship with tropic pituitary hormone and effector hormone have unraveled an independent role of pituitary hormone in skeletal remodeling beyond the role of regulating target endocrine glands. The cytokine-mediated skeletal actions and the evidence of local production of certain pituitary hormones by bone marrow-derived cells displays a unique endocrine-immune-skeletal connection. Here, we discuss recently elucidated mechanisms controlling the remodeling of bone, communication of bone cells with cells of other lineages, crosstalk between bone and vital organs, as well as opportunities for treating diseases of the skeleton.

Monocyte/macrophage lineage cells are highly plastic and can differentiate into various cells under different environmental stimuli. Bone-resorbing osteoclasts are derived from the monocyte/macrophage lineage in response to receptor activator of NF-κB ligand (RANKL). However, the epigenetic signature contributing to the fate commitment of monocyte/macrophage lineage differentiation into human osteoclasts is largely unknown. In this study, we identified RANKL-responsive human osteoclast-specific superenhancers (SEs) and SE-associated enhancer RNAs (SE-eRNAs) by integrating data obtained from ChIP-seq, ATAC-seq, nuclear RNA-seq and PRO-seq analyses. RANKL induced the formation of 200 SEs, which are large clusters of enhancers, while suppressing 148 SEs in macrophages. RANKL-responsive SEs were strongly correlated with genes in the osteoclastogenic program and were selectively increased in human osteoclasts but marginally presented in osteoblasts, CD4+ T cells, and CD34+ cells. In addition to the major transcription factors identified in osteoclasts, we found that BATF binding motifs were highly enriched in RANKL-responsive SEs. The depletion of BATF1/3 inhibited RANKL-induced osteoclast differentiation. Furthermore, we found increased chromatin accessibility in SE regions, where RNA polymerase II was significantly recruited to induce the extragenic transcription of SE-eRNAs, in human osteoclasts. Knocking down SE-eRNAs in the vicinity of the NFATc1 gene diminished the expression of NFATc1, a major regulator of osteoclasts, and osteoclast differentiation. Inhibiting BET proteins suppressed the formation of some RANKL-responsive SEs and NFATc1-associated SEs, and the expression of SE-eRNA:NFATc1. Moreover, SE-eRNA:NFATc1 was highly expressed in the synovial macrophages of rheumatoid arthritis patients exhibiting high-osteoclastogenic potential. Our genome-wide analysis revealed RANKL-inducible SEs and SE-eRNAs as osteoclast-specific signatures, which may contribute to the development of osteoclast-specific therapeutic interventions.