Osteoclasts are multinucleated giant cells that are formed by the fusion of precursor cells. Cell-cell fusion is mediated by membrane protrusion driven by actin reorganization, but the role of membrane mechanics in this process is unknown. Utilizing live-cell imaging, optical tweezers, manipulation of membrane-to-cortex attachment (MCA), and genetic interference, we show that a decrease in plasma membrane (PM) tension is a mechanical prerequisite for osteoclast fusion. Upon RANKL-induced differentiation, ezrin expression in fusion progenitor cells is reduced, resulting in a decrease in MCA-dependent PM tension. A forced elevation of PM tension by reinforcing the MCA conversely suppresses cell-cell fusion. Mechanistically, reduced PM tension leads to membrane protrusive invadosome formation driven by membrane curvature-inducing/sensing BAR proteins, thereby promoting cell-cell fusion. These findings provide insights into the mechanism of cell-cell fusion under the control of membrane mechanics.

Rheumatoid arthritis (RA) is an autoimmune chronic inflammatory disease that imposes a heavy economic burden on patients and society. Bone and cartilage destruction is considered an important factor leading to RA, and inflammation, oxidative stress, and mitochondrial dysfunction are closely related to bone erosion and cartilage destruction in RA. Currently, there are limitations in the clinical treatment methods for RA, which urgently necessitates finding new effective treatments for patients. Nuclear transcription factor-κB (NF-κB) is a signaling transcription factor that is widely present in various cells. It plays an important role as a stress source in the cellular environment and regulates gene expression in processes such as immunity, inflammation, cell proliferation, and apoptosis. NF-κB has long been recognized as a pathogenic factor of RA, and its activation can exacerbate RA by promoting inflammation, oxidative stress, mitochondrial dysfunction, and bone destruction. Conversely, inhibiting the activity of the NF-κB pathway effectively inhibits these pathological processes, thereby alleviating RA. Therefore, NF-κB may be a potential therapeutic target for RA. This article describes the physiological structure of NF-κB and its important role in RA through the regulation of oxidative stress, inflammatory response, mitochondrial function, and bone destruction. Meanwhile, we also summarized the impact of NF-κB crosstalk with other signaling pathways on RA and the effect of related drugs or inhibitors targeting NF-κB on RA. The purpose of this article is to provide evidence for the role of NF-κB in RA and to emphasize its significant role in RA by elucidating the mechanisms, so as to provide a theoretical basis for targeting the NF-κB pathway as a treatment for RA.

This study compared pharmacokinetic (PK) similarity, pharmacodynamic, safety, and immunogenicity of AVT03, a candidate biosimilar, with reference product (RP) denosumab (Prolia).

Healthy male participants (N = 209) were randomized 1:1 to receive one 60 mg dose of either AVT03 or RP. PK similarity was demonstrated if the 90% confidence intervals (CI) for the ratio of geometric means for the primary PK parameters (Cmax and AUC0-inf for EMA; Cmax and AUC0-t for FDA and PMDA) were within the prespecified margins of 80.00% and 125.00%. Secondary PK parameters assessed were AUC0-24, Tmax, Kel, t1/2, Vz/F, and CL/F. The serum biomarker of bone resorption, CTX-1 was evaluated to compare pharmacodynamic (PD) profiles. Safety and immunogenicity were also assessed.

The 90% CI for the ratio of geometric means for primary PK parameters was contained between the pre-specified margins of 80.00% and 125.00% (Cmax [102.23, 113.64]; AUC0-inf [107.17, 118.87]; AUC0-t [107.72, 120.42]), supporting demonstration of PK similarity between AVT03 and RP. Secondary PK parameters supported the analysis. PD, safety and immunogenicity profiles were comparable between the two arms.

Results supported a demonstration of PK similarity between AVT03 and RP denosumab. Comparable PD, safety and immunogenicity profiles were also shown.

The clinical trial is registered at https://www.clinicaltrials.gov under identifier NCT05126784.

This study aimed to investigate the impacts of prior anti-osteoporosis treatments on bone mineral density (BMD) changes in Chinese postmenopausal women with osteoporosis following 1-year Denosumab (Dmab) therapy.

This retrospective cohort study enrolled 381 postmenopausal women, all receiving a 1-year Dmab treatment. Participants were stratified into five groups based on prior anti-osteoporosis treatments: no treatment (NT), alendronate (ALN), zoledronic acid (ZOL), teriparatide (TPT), and raloxifene (RAL). Potential factors influencing BMD changes were screened using least absolute shrinkage and selection operator (LASSO). The selected variables were then incorporated into a multivariate regression model to identify independent risk factors. Finally, after adjusting for confounders, the impacts of prior anti-osteoporosis treatment on sequential Dmab responses were evaluated.

1) Further BMD increases were observed after sequential 1-year Dmab with prior use of other anti-osteoporosis drugs; 2) Compared to the NT group, ZOL significantly reduced BMD changes at the lumbar spine (LS), femoral neck (FN), and total hip (TH) (LS: β = -0.01, P = 0.016; FN: β = -0.01, P = 0.010; TH: β = -0.01, P = 0.011); Significant negative associations with FN BMD changes were observed for the ALN group (β = -0.01, P< 0.001), and the RAL group (β = -0.01, P = 0.010) compared to the NT group; TPT showed no significant differences with the NT group at all sites; 3) Multiple analysis revealed baseline BMD were independently associated with changes in BMD (LS: β = -0.04, P = 0.009; FN: β = -0.19, P <0.001; TH: β = -0.14, P <0.001).

These findings indicated that prior anti-osteoporosis treatments differentially influenced BMD responses to 1-year Dmab therapy. While patients who had previously been treated with ZOL had limited subsequent BMD improvement, patients who had previously used TPT and had lower baseline BMD benefited more.

Breast cancer is the leading cause of death in the female population. Hormone receptor-positive cancers are usually treated with surgery in combination with endocrine therapy. The latter is known to lower estrogen levels, contributing, therefore, to loss of bone density (BMD) and higher risk of fracture. Bone-modifying agents (BMAs) can regulate the bone-related adverse effects of cancer treatment. In premenopausal women, intravenous zoledronate effectively prevents bone loss. However, the evidence regarding its ability to reduce disease recurrence remains inconclusive. In postmenopausal women, denosumab demonstrates the most substantial evidence for fracture prevention, supported by one well-powered randomized controlled trial, but has not been shown to confer anticancer benefits. While bisphosphonates effectively prevent and reduce clinical vertebra fractures, their impact on overall fracture risk is unclear. In clinical practice, management of bone health in this group of patients starts with stratification for the risk of fracture. This can be done using the FRAX algorithm; measurements of bone mineral density can help to optimize stratification for individuals at higher fracture risk. Caution is advised when interpreting the results, as the FRAX algorithm has been considered to underestimate the true fracture risk in this population, given that the algorithm has not been adjusted for the effect of anti-cancer agents. Nowadays, clodronate, ibandronate, and zoledronic acid are recommended for bone protection in this group of patients, while denosumab is not. Further research is required to highlight the optimal BMA according to patient characteristics.

Osteocytes are terminally differentiated osteoblasts that secrete molecules that regulate bone-tissue homeostasis. Considering that the extracellular matrix protein agrin (AGRN) is secreted by osteoblasts and modulates their differentiation, we hypothesized that AGRN is also expressed by osteocytes and plays a role in their function and therefore in bone remodeling. To test this hypothesis, we deleted agrin specifically in osteocytes using dentin matrix acidic phosphoprotein 1 (DMP1)-Cre mice (C57/BL6 background) and silenced agrin in vitro using clustered regularly interspaced short palindromic repeats/associated nuclease Cas-9 in the Ocy454 osteocyte cell line. We found that osteocytes express agrin and its receptors, low-density lipoprotein receptor-related protein 4, and α-dystroglycan, and that mice with agrin-deficient osteocytes exhibited lower bone mass and impaired mechanical and chemical properties of bone tissue. Agrin knockdown in Ocy454 cells disrupted osteocyte differentiation and function, which reduced osteoblast and increased osteoclast differentiation in a cell co-culture model. Our results showed that agrin is expressed by osteocytes, which are key regulators of bone mass and its mechanical and chemical properties. These findings indicate that agrin may be a therapeutic target because it is important to maintain the balance of the osteocyte-osteoblast-osteoclast circuit, and consequently, bone tissue homeostasis.

Osteoporosis is the most common skeletal disease worldwide, characterized by low bone mineral density, resulting in weaker bones, and an increased risk of fragility fractures. The maintenance of bone mass relies on the precise balance between bone synthesis and resorption. The close relationship between the immune and skeletal systems, called "osteoimmunology", was coined to identify these overlapping "scientific worlds", and its function resides in the evaluation of the mutual effects of the skeletal and immune systems at the molecular and cellular levels, in both physiological and pathological states. Lipids play an essential role in skeletal metabolism and bone health. Indeed, bone marrow and its skeletal components demand a dramatic amount of daily energy to control hematopoietic turnover, acquire and maintain bone mass, and actively being involved in whole-body metabolism. Statins, the main therapeutic agents in lowering plasma cholesterol levels, are able to promote osteoblastogenesis and inhibit osteoclastogenesis. This review is meant to provide an updated overview of the pathophysiology of bone remodelling cycle, focusing on the interplay between bone, immune system and lipids. Novel therapeutic strategies for the management of osteoporosis are also discussed.

The diagnosis and treatment of malignant spinal tumors are complex and require an integrated approach known as Jaffe's triangle.　This review discusses recent topics in the diagnosis and treatment of primary and metastatic malignant spinal tumors.　Integrated diagnostic methods, including the development of a dumbbell scoring system for benign-malignant differentiation and the use of positron emission tomography and magnetic resonance imaging (PET-MRI), have improved diagnostic accuracy.　Curative resection techniques such as vertebrectomy, sagittal resection, and posterior resection are crucial for primary malignant tumors.　Heavy particle radiation therapy, such as carbon-ion radiotherapy, shows promise against radiation-resistant tumors, whereas novel drug therapies, such as denosumab, are effective for giant cell tumors of the bone arising in the spine.　For metastatic spinal tumors, the collaborative efforts of the Bone Metastasis Cancer Board and minimally invasive spine stabilization have expanded surgical indications and improved patient outcomes.　The treatment system has shifted towards preventive surgery and outpatient management, aiming to maintain quality of life and continue chemotherapy.　Interdisciplinary collaboration is essential for improving treatment outcomes in both primary and metastatic malignant spinal tumors.Primary malignant spinal cord tumors (PMST) and metastatic spinal tumors (MST) are among the most difficult areas of orthopedic surgery.　Their diagnosis and treatment require multidisciplinary diagnostic and therapeutic strategies that integrate knowledge and skills in orthopedics, pathology, and diagnostic radiology (the so-called Jaffe triangle), as well as in clinical oncology and tumor biology, which have made remarkable progress in recent years.　Here, we review recent topics related to the diagnosis and treatment of PMST and MST.

CD95, also known as Fas, belongs to the tumor necrosis factor (TNF) receptor superfamily. The main biological function of this receptor is to orchestrate and control the immune response since mutations in CD95 or deregulation of its downstream signaling pathways lead to auto-immunity and inflammation. Interestingly, more than twenty years ago, pioneer studies highlighted that like TNFR1, TRAILR1 or CD40, CD95 pre-associates at the plasma membrane in a ligand-independent fashion. This self-association occurs through a domain designated pre-ligand assembly domain or PLAD. Although the disruption of this pre-association prevents CD95 signaling, no drugs targeting this region have been generated because many questions remain on the stoichiometry and conformation of this receptor. Despite more than 40.000 publications, no crystal structure of CD95 alone or in combination with its ligand, CD95L, exists. Based on other TNFR members, we herein discuss the predicted conformation of CD95 at the plasma membrane and how these putative structures might account for the induction of the cell signaling pathways.

A range of biomarkers of bone metabolism are thought to mediate adipose tissue-bone crosstalk and fulfil a homeostatic role. While considered clinically relevant, their utility and application appears limited by lack of data characterising biological variability and reference intervals rather than by analytical issues. We have therefore studied the biological variation (BV) of these biomarkers. Concentrations of Dikkopf-related protein 1, leptin, osteoprotegerin, sclerostin, lipocalin2 (Lcn2) and resistin were measured by Luminex assays in serum samples from the EuBIVAS study. Samples were taken once per week, over 10 consecutive weeks, from 91 subjects in cohorts from 5 European countries. Estimates of analytical variation (CVA), within-subject (CVI) and between-subject (CVG) BV were calculated and analytical imprecision (CVAPS) and analytical bias (BAPS) specifications, index of individuality (II), reference change values (RCV) for increase and decrease and the number of samples required to estimate the homeostatic set points (NHSPs) were derived. Mean concentrations differed between males and females for leptin, osteoprotegerin, and sclerostin, and for osteoprotegerin and sclerostin between females in fertile and menopausal ages. No male-to-female differences were observed in CVI estimates. Index of individuality was below 0.6, for all measurands. Determination of reference intervals (RI) limits indicated that all, with the exception Lcn2, described data which were non-gaussian distributed and that only leptin differed between sexes. Availability of high-quality biological variation enables objective assessment of the bone metabolism biomarker results which may enhance their clinical utility. The data indicates that they exhibit significant individuality.

Originally identified for its involvement in bone remodeling, accumulating data emerged in the past years indicating that receptor activator of nuclear factor κB ligand (RANKL) actually acts as a multifunctional soluble molecule that influences various physiological and pathological processes. Regarding its role in carcinogenesis, while direct effects on tumor cell behavior have been precisely characterized, the impact of the RANKL/RANK system (and its inhibition) on the intratumoral immune landscape remains unclear.

After various in silico/in situ/in vitro analyses, the immunotherapeutic efficacy of RANKL blockade (alone and in combination with immune checkpoint inhibitors (anti-programmed cell death protein-1 (PD-1)) or doxorubicin/paclitaxel-based chemotherapy) was investigated using different syngeneic mouse models of triple-negative breast cancer (4T1, 67NR and E0771). Isolated from retrieved tumors, 14 immune cell (sub)populations, along with the activation status of antigen-presenting cells, were thoroughly analyzed in each condition. Finally, the impact of RANKL on the functionality of both dendritic cells (DC) and plasmacytoid dendritic cells (pDC) was determined.

A drastic tumor growth inhibition was reproductively observed following RANKL inhibition. Strikingly, this antitumor activity was not detected in immunocompromised mice, demonstrating its dependence on the adaptive immune responses and justifying the diverse enriched signatures linked to immune cell regulation/differentiation detected in RANKLhigh-expressing human neoplasms. Interestingly, neoadjuvant chemotherapy (but not PD-1 checkpoint inhibition) potentiated the anticancer effects of RANKL blockade by priming effector T cells and increasing their infiltration within the tumor microenvironment. Mechanistically, we highlighted that RANKL indirectly promotes regulatory T cell differentiation and suppressive function by inhibiting the mTOR signaling pathway on antigen-presenting cells.

Taken together, this study provides insight into the role of RANKL/RANK axis in immune tolerance, demonstrates the significant impact of RANKL-dependent impairment of T cell-DC/pDC crosstalk on tumor development and, ultimately, supports that this ligand could be an interesting actionable target for cancer immunotherapy.

Epithelial-mesenchymal transition (EMT) promotes several cancers by increasing tumor cell motility, disrupting epithelial cell phenotypes, apical-basal polarity, and intracellular connections, and enhancing tumor resistance to immunotherapy and chemotherapy. Mesenchymal-epithelial transition (MET), the opposite of EMT, causes tumor metastasis. EMT drives primary tumor cells, whereas MET inhibits them. Importantly, the complex network of EMT includes cell-cell interactions in the tumor microenvironment. Transcription factors, post-translational regulation, cytokine-mediated signaling, and microRNAs control EMT. In this review, we discussed how molecular mechanisms, signaling networks, and epithelial/mesenchymal states affect cancer treatment resistance and the tumor microenvironment. Research on immunotherapy and chemotherapy problems associated with EMT suggests that targeting EMT might be a potential cancer treatment resistance strategy.

In this study, a phytochemical investigation on the methanol extract of Potentilla chinensis led to the isolation of eleven triterpenoids including ursolic acid (1), pomolic acid (2), tormentic acid (3), 2-epi-corosolic acid (4), 3-epi-corosolic acid (ECA, 5), 3β-hydroxyurs-11-en-13β(28)-olide (6), euscaphic acid (7), 2-epi-tormentic acid (8), corosolic acid (9), uvaol (10), and 3-O-acetylpomolic acid (11). Among them, ECA (5) showed potential anti-osteoclastogenic activity. To the best of our knowledge, this represents the first isolation of ECA (5) from P. chinensis as well as the first investigation of its effects on osteoclast formation. Further study revealed that ECA inhibited RANKL-induced mature osteoclast formation in vitro without compromising cell viability. Mechanistically, ECA attenuated RANKL-induced mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) activation, leading to the inhibition of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) activation. Moreover, ECA protected against LPS-induced inflammatory bone loss and osteoclast formation in a mouse model. However, ECA did not inhibit LPS-induced inflammatory responses in macrophages. Our findings suggest that ECA mitigates LPS-induced inflammatory bone loss in mice by inhibiting RANKL-induced activation of key osteoclastogenic transcription factors, including c-Fos and NFATc1, and may be a potential natural triterpenoid for preventing or treating osteolytic diseases.

This study investigated the function effects of KMUP-3, a self-developed synthetic xanthine-based derivative, in suppressing Porphyromonas gingivalis (Pg-LPS)-aggravated osteoclastogenesis and pyroptosis as a potential treatment for periodontitis.

In vitro, the effects of Pg-LPS and KMUP-3 on osteoclast formation and macrophage pyroptosis were investigated using the receptor activator of nuclear factor-κB ligand (RANKL)-primed RAW264.7 macrophages. In vivo, the therapeutic effects of KMUP-3 were evaluated in a model of experimental periodontitis induced by gingival ligature placement.

We reveal that KMUP-3 suppressed osteoclastogenesis, inducible nitric oxide synthase activation, and reduced nitric oxide production enhanced by Pg-LPS in RANKL-primed RAW264.7 cells while also decreasing TLR4/NF-κB p65 pathway activation and decreased pro-inflammatory cytokine production; moreover, Pg-LPS promoted NLRP3 activation and exacerbated pyroptosis induction effects that were abolished by KMUP-3. Finally, KMUP-3 ameliorated alveolar bone loss and IL-1β levels in the gingival crevicular fluid in the rat ligature periodontitis model.

Our study demonstrated that KMUP-3 attenuates Pg-LPS-enhanced osteoclastogenesis and macrophage pyroptosis. Notably, KMUP-3 alleviates alveolar bone loss in experimental periodontitis rats and thus suggests its certain role in safeguarding against periodontal bone resorption.

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.

Radium-223 (223Ra) was the first radioactive isotope approved for treating castration-resistant prostate cancer (CRPC) with symptomatic bone metastases without visceral metastatic disease. To better understand the action of 223Ra, its role in the tumor microenvironment represents a crucial aspect. A literature search was conducted using the PubMed/MEDLINE database and studies regarding the relationship between 223Ra and the tumoral microenvironment were considered. The tumoral microenvironment is a complex setting in which complex interactions between cells and molecules occur. Radium-223, as an alpha-emitter, induces double-stranded DNA breaks; to potentiate this effect, it could be used in patients with genetic instability but also in combination with therapies which inhibit DNA repair, modulate the immune response, or control tumor growth. In conclusion, a few studies have taken into consideration the tumoral microenvironment in association with 223Ra. However, its understanding is a priority to better comprehend how to effectively exploit 223Ra and its action mechanism.

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.

Osteoporosis significantly increases the risk of vertebral fractures, particularly among postmenopausal women, decreasing their quality of life. These fractures, often undiagnosed, can lead to severe health consequences and are influenced by bone mineral density and abnormal loads. Management strategies range from non-surgical interventions to surgical treatments. Moreover, the interaction between immune cells and bone cells plays a crucial role in bone repair processes, highlighting the importance of osteoimmunology in understanding and treating bone pathologies.

This study aims to investigate the xCell signature-based immune cell profiles in osteoporotic patients with and without vertebral fractures, utilizing advanced predictive modeling through the XGBoost algorithm.

Our findings reveal an increased presence of CD4 + naïve T cells and central memory T cells in VF patients, indicating distinct adaptive immune responses. The XGBoost model identified Th1 cells, CD4 memory T cells, and hematopoietic stem cells as key predictors of VF. Notably, VF patients exhibited a reduction in Th1 cells and an enrichment of Th17 cells, which promote osteoclastogenesis and bone resorption. Gene expression analysis further highlighted an upregulation of osteoclast-related genes and a downregulation of osteoblast-related genes in VF patients, emphasizing the disrupted balance between bone formation and resorption. These findings underscore the critical role of immune cells in the pathogenesis of osteoporotic fractures and highlight the potential of XGBoost in identifying key biomarkers and therapeutic targets for mitigating fracture risk in osteoporotic patients.

Phospholipase C β (PLCβ) is involved in diverse biological processes, including inflammatory responses and neurogenesis; however, its role in bone cell function is largely unknown. Among the PLCβ isoforms (β1-β4), we found that PLCβ4 was the most highly upregulated during osteoclastogenesis. Here we used global knockout and osteoclast lineage-specific PLCβ4 conditional knockout (LysM-PLCβ4-/-) mice as subjects and demonstrated that PLCβ4 is a crucial regulator of receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation. The deletion of PLCβ4, both globally and in the osteoclast lineage, resulted in a significant reduction in osteoclast formation and the downregulation of osteoclast marker genes. Notably, male LysM-PLCβ4-/- mice presented greater bone mass and fewer osteoclasts in vivo than their wild-type littermates, without altered osteoblast function. Mechanistically, we found that PLCβ4 forms a complex with p38 mitogen-activated protein kinase (MAPK) and MAPK kinase 3 (MKK3) in response to RANKL-induced osteoclast differentiation, thereby modulating p38 activation. An immunofluorescence assay further confirmed the colocalization of PLCβ4 with p38 after RANKL exposure. Moreover, p38 activation rescued impaired osteoclast formation and restored the reduction in p38 phosphorylation caused by PLCβ4 deficiency. Thus, our findings reveal that PLCβ4 controls osteoclastogenesis via the RANKL-dependent MKK3-p38 MAPK pathway and that PLCβ4 may be a potential therapeutic candidate for bone diseases such as osteoporosis.

Fibroblast-like synoviocytes (FLSs) and osteoclasts are central cells in the maintenance of joint homeostasis. Rheumatoid arthritis (RA) is a chronic inflammatory disease of joints that induces cytokine-activated FLSs and progressive bone erosion. Interactions between FLSs and other cells, such as T cells and B cells, have been recognized in the development of RA. Here we hypothesized that calcium released from bone by mature osteoclasts might activate FLSs, which are also affected by inflammatory cytokines in the inflamed synovium. Osteoclastogenesis occurs in the presence of cytokine-stimulated FLS medium, and calcium released from the bone disc activates FLS migration. We first investigated the calcium and cytokine feedback loop between FLSs and osteoclast maturation. Moreover, by addressing the role of the sodium-bicarbonate cotransporter NBCn1 in osteoclastogenesis, we found that the inhibition of NBCn1 attenuated the infinite calcium and cytokine feedback loop between FLSs and osteoclasts. In a collagen-induced arthritis mouse model, the inhibition of NBC reduced the RA pathological phenotype and bone resorption area in the femur. These results suggest that modulation of the crosstalk between FLSs and osteoclasts by inhibiting the calcium and cytokine feedback loop could be considered to develop pioneering strategies to combat RA severity and dysregulated bone homeostasis.

Periodontitis is a multifactorial immune-mediated disease exacerbated by dysregulated alveolar bone homeostasis. Timely intervention is crucial for disease management to prevent tooth loss. To successfully manage periodontitis, it is imperative to understand the cellular and molecular mechanisms involved in its pathogenesis to develop novel treatment modalities. Non-surgical periodontal therapy (NSPT) such as subgingival instrumentation/debridement has been the underlying treatment strategy over the past decades. However, new NSPT approaches that target key signaling pathways regulating alveolar bone homeostasis have shown positive clinical outcomes. This narrative review aims to discuss endogenous bone homeostasis mechanisms impaired in periodontitis and highlight the clinical outcomes of preventive periodontal therapy to avoid invasive periodontal therapies. Although the anti-resorptive therapeutic adjuncts have demonstrated beneficial outcomes, adverse events have been reported. Diverse immunomodulatory therapies targeting the osteoblast/osteoclast (OB/OC) axis have shown promising outcomes in vivo. Future controlled randomized clinical trials (RCT) would help clinicians and patients in the selection of novel preventing therapies targeting key molecules to effectively treat or prevent periodontitis.

Bone is a unique organ crucial for locomotion, mineral metabolism, and hematopoiesis. It maintains homeostasis through a balance between bone formation by osteoblasts and bone resorption by osteoclasts, which is regulated by the basic multicellular unit (BMU). Abnormal bone metabolism arises from an imbalance in the BMU. Osteoclasts, derived from the monocyte-macrophage lineage, are regulated by the RANKL-RANK-OPG system, which is a key factor in osteoclast differentiation. RANKL activates osteoclasts through its receptor RANK, while OPG acts as a decoy receptor that inhibits RANKL. In trabecular bone, high turnover involves rapid bone formation and resorption, influenced by conditions such as malignancy and inflammatory cytokines that increase RANKL expression. Cortical bone remodeling, regulated by aged osteocytes expressing RANKL, is less understood, despite ongoing research into how Rett syndrome, characterized by MeCP2 abnormalities, affects RANKL expression. Balancing trabecular and cortical bone involves mechanisms that preserve cortical bone, despite overall bone mass reduction due to aging or oxidative stress. Research into genes like sFRP4, which modulates bone mass, highlights the complex regulation by BMUs. The roles of the RANKL-RANK-OPG system extend beyond bone, affecting processes such as aortic valve formation and temperature regulation, which highlight the interconnected nature of biological research.

Osteoporosis is a metabolic bone disease primarily caused by a decreased bone formation and increased bone resorption. Osteoclasts are a special class of terminally differentiated cells that play an important role in normal bone remodeling and bone loss in osteoporosis as well as in a variety of osteolytic diseases. Osteoclasts can be differentiated from monocyte-macrophage cells of the hematopoietic system; they are the key cells in bone resorption. Osteoclast formation and differentiation are regulated by various cytokines and transcription factors. In this review, we summarize recent advances in research on the regulation of osteoclast differentiation and function by factors such as M-CSF, RANKL, AP-1, NFATC1, MITF, and PU.1. Understanding these cytokines and transcription factors can not only help identify targets for osteoclast differentiation but also aid in intervening in the treatment of osteoclast-related diseases.

Osteoporosis is a metabolic skeletal disease characterized by low bone mass and strength, and increased risk for fragility fractures. It is a major health issue in aging populations, due to fracture-associated increased disability and mortality. Antiresorptive treatments are first line choices in most of the cases.

Bone homeostasis is complicated, and multiple factors can compromise skeletal health. Bone turnover is a continuous process regulated by the coupled activities of bone cells that preserves skeletal strength and integrity. Imbalance between bone resorption and formation leads to bone loss and increased susceptibility to fractures. Antiresorptives prevent bone loss and reduce fracture risk, by targeting osteoclastogenesis and osteoclast function and survival. Their major drawback is the coupling of osteoclast and osteoblast activity, due to which any reduction in bone resorption is followed by suppression of bone formation.

During the last couple of decades significant progress has been made in understanding of the genetic and molecular basis of osteoporosis. Critical pathways and key molecules that mediate regulation of bone resorption have been identified. These factors may underpin novel therapeutic avenues for osteoporosis, but their potential for translation into clinical applications is yet to be tested.

Orthodontic tooth movement (OTM) is a complex process involving bone remodeling, and is regulated by various molecular factors, including microRNAs (miRNAs). These small, non-coding RNAs are critical in post-transcriptional gene regulation and have been implicated in the modulation of osteoclast and osteoblast activity during OTM. This study aimed to explore the expression profiles of salivary exosome-derived miRNAs during OTM to identify potential biomarkers that could provide insights into the biological processes involved in orthodontic tooth movement. Saliva samples were collected from 15 patients at three time points: before treatment (Day 0), 7 days after the treatment's onset (Day 7), and 40 days after the treatment's onset (Day 40). The exosomes were isolated, and the miRNAs were extracted and sequenced. A differential expression analysis and gene ontology (GO) enrichment were performed to identify the miRNAs involved in osteoblast and osteoclast differentiation. Out of the 1405 detected miRNAs, 185 were analyzed. Several miRNAs were associated with bone-remodeling processes. The statistically significant finding was the downregulation of hsa-miR-4634 after 40 days of treatment. These findings contribute to the understanding of miRNA regulation in orthodontics and may have broader implications for skeletal disorders, such as osteoporosis.

Indole-3-propionic acid (IPA), a metabolite produced by gut microbiota through tryptophan metabolism, has recently been identified as playing a pivotal role in bone metabolism. IPA promotes osteoblast differentiation by upregulating mitochondrial transcription factor A (Tfam), contributing to increased bone density and supporting bone repair. Simultaneously, it inhibits the formation and activity of osteoclasts, reducing bone resorption, possibly through modulation of the nuclear factor-κB (NF-κB) pathway and downregulation of osteoclast-associated factors, thereby maintaining bone structural integrity. Additionally, IPA provides indirect protection to bone health by regulating host immune responses and inflammation via activation of receptors such as the Aryl hydrocarbon Receptor (AhR) and the Pregnane X Receptor (PXR). This review summarizes the roles and signaling pathways of IPA in bone metabolism and its impact on various bone metabolic disorders. Furthermore, we discuss the therapeutic potential and limitations of IPA in treating bone metabolic diseases, aiming to offer novel strategies for clinical management.

The study aimed to evaluate the potential protection against fractures of oral Q10 supplementation in the tibias of rats exposed to nicotine. Nicotine is known to negatively impact bone density and increase the risk of fractures, in addition to affecting other systems such as the gastrointestinal system, impairing its absorption capacity, negatively affecting bone health. To investigate this, eighty male rats were divided into four groups (n = 20) receiving either nicotine hemisulfate or saline solution (SS) for 28 days. Two daily subcutaneous applications were administered accordingly. Concurrently, vegetable glycerin and Q10 gavage began on day "0". SS: the animals in this group received two daily subcutaneous applications of sodium chloride solution during the entire trial period. 30 days after starting the SS applications subcutaneously, the animals received vegetable glycerin daily until the end of the experiment. SS-Q10: the animals received the SS protocol and daily supplementation with Q10 until the end of the experiment. NIC: The animals received the protocol for NIC and vegetable glycerin daily until the end of the experiment. NIC-Q10: The animals received the protocol for NIC and daily supplementation and Q10 until the end of the experiment. Euthanasia occurred at 7 and 28 days after the beginning the gavage. The tibiae collected were processed for morphometric, densitometric, mechanical, and microtomographic (micro-Ct) analysis. A complementary analysis of intestinal changes was performed. The groups that received Q10 showed slightly better results regarding the mechanical resistance and micro-Ct parameters and to intestinal histomorphometry, as compared with groups not supplemented with Q10. Thus, in rats, it can be concluded that coenzyme Q10 exhibited a protective property to the skeletal system and the gastrointestinal tract, even in the presence of nicotine.

Tanshinone, a lipophilic component of Salvia miltiorrhiza, is used to treat diseases like atherosclerosis, hypertension, Alzheimer's disease, and diabetes mellitus through its pharmacological activities like anti-inflammatory, anti-oxidant, and anti-tumor. Excessive inflammation is the primary cause of bone diseases such as osteoporosis and rheumatoid arthritis, affecting more than millions of people across the globe. Recently, tanshinone has shown potential benefits against bone diseases by modulating signaling pathways accountable for the proliferation and differentiation of bone cells. In vitro and in vivo studies reported that tanshinone promotes osteoblast formation and mineralization and suppresses excessive bone resorption during disease conditions. In this review, we have summarized the beneficial effects of tanshinone and other extracts of Salvia miltiorrhiza for bone health and their potential molecular targets in signaling.

The field of osteoimmunology has primarily focused on fracture healing in isolated musculoskeletal injuries. The innate immune system is the initial response to fracture, with inflammatory macrophages, cytokines, and neutrophils arriving first at the fracture hematoma, followed by an anti-inflammatory phase to begin the process of new bone formation. This review aims to first discuss the current literature and knowledge gaps on the immune responses governing single fracture healing by encompassing the individual role of macrophages, neutrophils, cytokines, mesenchymal stem cells, bone cells, and other immune cells. This paper discusses the interactive effects of these cellular responses underscoring the field of osteoimmunology. The critical role of the metabolic environment in guiding the immune system properties will be highlighted along with some effective therapeutics for fracture healing in the context of osteoimmunology. However, compared to isolated fractures, which frequently heal well, long bone fractures in over 30 % of polytrauma patients exhibit impaired healing. Clinical evidence suggests there may be distinct physiologic and inflammatory pathways altered in polytrauma resulting in nonunion. Nonunion is associated with worse patient outcomes and increased societal healthcare costs. The dysregulated immunomodulatory/inflammatory response seen in polytrauma may lead to this increased nonunion rate. This paper will investigate the differences in immune response between isolated and polytrauma fractures. Finally, future directions for fracture studies are explored with consideration of the emerging roles of newly discovered immune cell functions in fracture healing, the existing challenges and conflicting results in the field, the translational potential of these studies in clinic, and the more complex nature of polytrauma fractures that can alter cell functions in different tissues.

RANKL and its cognate receptor RANK are crucial regulators of bone metabolism in physiological as well as in pathological conditions. Here we go through the works that unveiled the paramount role of this signaling pathway. We focus on the RANKL cytokine, whose alterations are responsible for rare and common bone diseases. We describe recent insights on the regulation of RANKL expression, which provide new hints for the pharmacological regulation of this molecule. Based on the multiple functions exerted by RANKL (within and outside the bone tissue), we advise caution regarding the potential unintended consequences of its inhibition.

RANK pathway has attracted increasing interest as a promising target in breast cancer, given the availability of denosumab, an anti-RANKL drug. RANK signaling mediates progesterone-driven regulation of mammary gland development and favors breast cancer initiation by controlling mammary cell proliferation and stem cell fate. RANK activation promotes luminal mammary epithelial cell senescence, acting as an initial barrier to tumorigenesis but ultimately facilitating tumor progression and metastasis. Comprehensive analyses have demonstrated that RANK protein expression is an independent biomarker of poor prognosis in postmenopausal and estrogen receptor-negative breast cancer patients. RANK pathway also has multiple roles in immunity and inflammation, regulating innate and adaptive responses. In the tumor microenvironment, RANK and RANKL are expressed by different immune cell populations and contribute to the regulation of tumor immune surveillance, mainly driving immunosuppressive effects.Herein, we discuss the preventive and therapeutic potential of targeting RANK signaling in breast cancer given its tumor cell intrinsic and extrinsic effects. RANKL inhibition has been shown to induce mammary tumor cell differentiation and an antitumor immune response. Moreover, loss of RANK signaling increases sensitivity of breast cancer cells to chemotherapy, targeted therapies such as HER2 and CDK4/6 inhibitors, and immunotherapy. Finally, we describe clinical trials of denosumab for breast cancer prevention, such as those ongoing in women with high risk of developing breast cancer, large phase III clinical trials where the impact of adjuvant denosumab on disease-free survival has been assessed, and window trials to evaluate the immunomodulatory effects of denosumab in breast cancer and other solid tumors.

Bone is a frequent site for breast cancer metastasis. The vast majority of breast cancer-associated metastasis is osteolytic in nature, and RANKL (receptor activator for nuclear factor κB)-induced differentiation of bone marrow-derived macrophages to osteoclasts (OCLs) is a key requirement for osteolytic metastatic growth of cancer cells. In this study, we demonstrate that Myocardin-related transcription factor (MRTF) in breast cancer cells plays an important role in paracrine modulation of RANKL-induced OCL differentiation. This is partly attributed to MRTFs' critical role in maintaining the basal cellular expression of connective tissue growth factor (CTGF), findings that align with a strong positive correlation between CTGF expression and MRTF-A gene signature in the human disease context. Luminex analyses reveal that MRTF depletion in breast cancer cells has a broad impact on OCL-regulatory cell-secreted factors that extend beyond CTGF. Experimental metastasis studies demonstrate that MRTF depletion diminishes OCL abundance and bone colonization of breast cancer cells in vivo, suggesting that MRTF inhibition could be an effective strategy to diminish OCL formation and skeletal involvement in breast cancer. In summary, this study highlights a novel tumor-extrinsic function of MRTF relevant to breast cancer metastasis.

Bone non-union has always been a research hotspot in the field of orthopedics. Non-unions are often accompanied by symptoms such as pain, deformity, and dysfunction, which can significantly affect patients' quality of life and cause related socioeconomic problems. Clinically, there are various treatments available for non-unions, and the main treatment methods are divided into surgical and non-surgical treatments. At present, surgery is the most widely used treatment for bone non-unions and has a high healing rate. However, even after surgery, some patients still face the problem of bone non-union. Furthermore, a small number of patients have surgical contraindications and could not tolerate surgery. Therefore, alternative treatments are needed to improve outcomes for patients with bone fractures. Extracorporeal shock wave therapy (ESWT) is a non-invasive treatment method with similar efficacy and better safety compared with surgery. Nevertheless, the exact mechanism for ESWT to treat patients with bone non-union are still not well understood. This article reviews the mechanisms of ESWT in promoting bone fracture healing by regulating osteoblasts and osteoclasts, providing a theoretical foundation for the clinical application of ESWT. The Translational Potential of this Article: This review provides a comprehensive overview of the mechanisms underlying ESWT on promoting bone fracture healing by regulating osteoblasts and osteoclasts. The information provided in this article can offer a novel non-invasive method for clinicians to treat bone non-union.

Tissue regeneration involves dynamic dialogue between and among different cells and their surrounding matrices. Bone regeneration is specifically governed by reciprocity between osteoblasts and osteoclasts within the bone microenvironment. Osteoclast-directed resorption and osteoblast-directed formation of bone are essential to bone remodeling, and the crosstalk between these cells is vital to curating a sequence of events that culminate in the creation of bone tissue. Among bone biomaterial strategies, many have investigated the use of different material cues to direct the development and activity of osteoblasts. However, less attention has been given to exploring features that similarly target osteoclast formation and activity, with even fewer strategies demonstrating or integrating biomaterial-directed modulation of osteoblast-osteoclast coupling. This review aims to describe various biomaterial cues demonstrated to influence osteoclastogenesis and osteoclast function, emphasizing those that enhance a material construct's ability to achieve bone healing and regeneration. Additionally discussed are approaches that influence the communication between osteoclasts and osteoblasts, particularly in a manner that takes advantage of their coupling. Deepening our understanding of how biomaterial cues may dictate osteoclast differentiation, function, and influence on the microenvironment may enable the realization of bone-replacement interventions with enhanced integrative and regenerative capacities.

Septic arthritis, primarily caused by Staphylococcus aureus (S. aureus), is a severe joint infection that leads to joint and bone damage. S. aureus lipoproteins (LPPs) bind to Toll-like Receptor 2 (TLR2), inducing arthritis and localized bone loss. Aging affects TLR2 immune response to pathogens. While intra-articular injections of S. aureus LPPs induces local bone resorption in mice, the influence of aging and TLR2 expression on bone mineral density (BMD) after S. aureus bacteremia remains unclear.

We analyzed distal femoral BMD in young and old TLR2 knock-out and wild-type mice following intravenous S. aureus infection. BMD was measured in both total and trabecular bone in old and young mice to determine age and TLR2-dependent responses to infection.

In non-infected mice, BMD in both total and trabecular bone was mainly age-related and TLR2-independent. Following S. aureus bacteremia, young wild-type mice with TLR2 expression showed decreased combined cortical and trabecular BMD. This effect was absent in aged mice or TLR2 deficient mice. Focal septic arthritis, induced by S. aureus bacteremia, emerged as the primary cause to bone loss in the femur metaphysis. TLR2 appears to play a crucial role in focal septic arthritis-induced bone loss, as evidenced by in vitro findings demonstrating that staphylococcal LPPs, known TLR2 agonists, increase the Tnfsf11/Tnfrsf11b ratio in mouse pariosteal osteoblasts.

S. aureus bacteremia triggers local bone loss in murine arthritis, depending on both age and TLR2 expression.

Intracellular reactive oxygen species (ROS) accumulation is key to osteoclast differentiation. Plant-derived polyphenols that have reduced ROS production have been widely studied for the treatment of osteoporosis. However, these compounds are rarely absorbed in the small intestine and are instead converted to phenolic acids by the microbiota in the colon. These large quantities of low-molecular-weight phenolic acids can then be absorbed by the body. 4-Hydroxyphenylacetic acid (4-HPA) is an important metabolite of these polyphenols that is generated by the human intestinal microbiota. However, its potential mechanism is not fully understood. In this study, we aimed to elucidate the role of 4-HPA on osteoclastogenesis and treating osteoporosis. Our study showed that 4-HPA inhibited osteoclast differentiation and function and downregulated osteoclast-specific genes, including NFATc1, Atp6v0d2, MMP9, CTSK, Acp5, and c-Fos. As for further mechanism exploration, 4-HPA reduced ROS accumulation by regulating nuclear factor erythroid 2-related factor (Nrf2) and subsequently inhibited the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. To evaluate the effect of 4-HPA on postmenopausal osteoporosis, an ovariectomized (OVX) mouse model was used. The Micro-CT and histomorphometry analyses showed that 4-HPA effectively prevents bone loss. Encouragingly, 4-HPA demonstrated efficacy in treating osteoporosis induced by OVX. In conclusion, our study revealed that 4-HPA, a polyphenol metabolite produced by intestinal microorganisms, also inhibits osteoclast formation and treats osteoporosis, which provides a new experimental basis and candidate drug for the treatment of osteoporosis.

The OPG/RANKL/RANK framework, along with its specific receptors, plays a crucial role in bone remodeling and the functioning of the central nervous system (CNS) and associated disorders. Recent research and investigations provide evidence that the components of osteoprotegerin (OPG), receptor activator of NF-kB ligand (RANKL), and receptor activator of NF-kB (RANK) are expressed in the CNS. The CNS structure encompasses cells involved in neuroinflammation, including local macrophages, inflammatory cells, and microglia that cross the blood-brain barrier. The OPG/RANKL/RANK trio modulates the neuroinflammatory response based on the molecular context. The levels of OPG/RANKL/RANK components can serve as biomarkers in the blood and cerebrospinal fluid. They act as neuroprotectants following brain injuries and also participate in the regulation of body weight, internal body temperature, brain ischemia, autoimmune encephalopathy, and energy metabolism. Although the OPG/RANKL/RANK system is primarily known for its role in bone remodeling, further exploring deeper into its multifunctional nature can uncover new functions and novel drug targets for diseases not previously associated with OPG/RANKL/RANK signaling.

Cell-cell fusion is a vital biological process where the membranes of two or more cells merge to form a syncytium. This phenomenon is critical in various physiological and pathological contexts, including embryonic development, tissue repair, immune responses, and the progression of several diseases. Osteoclasts, which are cells from the monocyte/macrophage lineage responsible for bone resorption, have enhanced functionality due to cell fusion. Additionally, other multinucleated giant cells (MGCs) also arise from the fusion of monocytes and macrophages, typically during chronic inflammation and reactions to foreign materials such as prostheses or medical devices. Foreign body giant cells (FBGCs) and Langhans giant cells (LGCs) emerge only under pathological conditions and are involved in phagocytosis, antigen presentation, and the secretion of inflammatory mediators. This review provides a comprehensive overview of the mechanisms underlying the formation of multinucleated cells, with a particular emphasis on macrophages and osteoclasts. Elucidating the intracellular structures, signaling cascades, and fusion-mediating proteins involved in cell-cell fusion enhances our understanding of this fundamental biological process and helps identify potential therapeutic targets for disorders mediated by cell fusion.

The Gaussian graphical model (GGM) is a statistical network approach that represents conditional dependencies among components, enabling a comprehensive exploration of disease mechanisms using high-throughput multi-omics data. Analyzing differential and similar structures in biological networks across multiple clinical conditions can reveal significant biological pathways and interactions associated with disease onset and progression. However, most existing methods for estimating group differences in sparse GGMs only apply to comparisons between two groups, and the challenging problem of multiple testing across multiple GGMs persists. This limitation hinders the ability to uncover complex biological insights that arise from comparing multiple conditions simultaneously. To address these challenges, we propose the Omics Networks Differential and Similarity Analysis (ONDSA) framework, specifically designed for continuous omics data. ONDSA tests for structural differences and similarities across multiple groups, effectively controlling the false discovery rate (FDR) at a desired level. Our approach focuses on entry-wise comparisons of precision matrices across groups, introducing two test statistics to sequentially estimate structural differences and similarities while adjusting for correlated effects in FDR control procedures. We show via comprehensive simulations that ONDSA outperforms existing methods under a range of graph structures and is a valuable tool for joint comparisons of multiple GGMs. We also illustrate our method through the detection of neuroinflammatory pathways in a multi-omics dataset from the Framingham Heart Study Offspring cohort, involving three apolipoprotein E genotype groups. It highlights ONDSA's ability to provide a more holistic view of biological interactions and disease mechanisms through multi-omics data integration.

Bone is a frequent site for breast cancer metastasis. The vast majority of breast cancer-associated metastasis is osteolytic in nature, and RANKL (receptor activator for nuclear factor κB)-induced differentiation of bone marrow-derived macrophages (BMDMs) to osteoclasts (OCLs) is a key requirement for osteolytic metastatic growth of cancer cells. In this study, we demonstrate that Myocardin-related transcription factor (MRTF) in breast cancer cells plays an important role in paracrine modulation of RANKL-induced osteoclast differentiation. This is partly attributed to MRTF's critical role in maintaining the basal cellular expression of connective tissue growth factor (CTGF), findings that align with a strong positive correlation between CTGF expression and MRTF-A gene signature in the human disease context. Luminex analyses reveal that MRTF depletion in breast cancer cells has a broad impact on OCL-regulatory cell-secreted factors that extend beyond CTGF. Experimental metastasis studies demonstrate that MRTF depletion diminishes OCL abundance and bone colonization breast cancer cells in vivo , suggesting that MRTF inhibition could be an effective strategy to diminish OCL formation and skeletal involvement in breast cancer. In summary, this study highlights a novel tumor-extrinsic function of MRTF relevant to breast cancer metastasis.

MRTF, a transcriptional coactivator of SRF, is known to promote breast cancer progression through its tumor-cell-intrinsic function. Whether and how MRTF activity in tumor cells modulates other types of cells in the tumor microenvironment are not clearly understood.This study uncovers a novel tumor-cell-extrinsic function of MRTF in breast cancer cells in promoting osteoclast differentiation partly through CTGF regulation, and further demonstrates MRTF's requirement for bone colonization of breast cancer cells in vivo.Our studies suggest that MRTF inhibition could be an effective strategy to diminish osteoclast formation and skeletal involvement in metastatic breast cancer.

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.