Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption.

Bone resorption is highly dependent on the dynamic rearrangement of the osteoclast actin cytoskeleton to allow formation of actin rings and a functional ruffled border. Hem1 is a hematopoietic-specific subunit of the WAVE-complex which regulates actin polymerization and is crucial for lamellipodia formation in hematopoietic cell types. However, its role in osteoclast differentiation and function is still unknown. Here, we show that although the absence of Hem1 promotes osteoclastogenesis, the ability of Hem1-/- osteoclasts to degrade bone was severely impaired. Global as well as osteoclast-specific deletion of Hem1 in vivo revealed increased femoral trabecular bone mass despite elevated numbers of osteoclasts in vivo. We found that the resorption defect derived from the morphological distortion of the actin-rich sealing zone and ruffled border deformation in Hem1-deficient osteoclasts leading to impaired vesicle transport and increased intracellular acidification. Collectively, our data identify Hem1 as a yet unknown key player in bone remodeling by regulating ruffled border formation and consequently the resorptive capacity of osteoclasts.

Exercise and osteoimmunology in bone remodeling.

Bones can form the scaffolding of the body, support the organism, coordinate somatic movements, and control mineral homeostasis and hematopoiesis. The immune system plays immune supervisory, defensive, and regulatory roles in the organism, which mainly consists of immune organs (spleen, bone marrow, tonsils, lymph nodes, etc.), immune cells (granulocytes, platelets, lymphocytes, etc.), and immune molecules (immune factors, interferons, interleukins, tumor necrosis factors, etc.). Bone and the immune system have long been considered two distinct fields of study, and the bone marrow, as a shared microenvironment between the bone and the immune system, closely links the two. Osteoimmunology organically combines bone and the immune system, elucidates the role of the immune system in bone, and creatively emphasizes its interdisciplinary characteristics and the function of immune cells and factors in maintaining bone homeostasis, providing new perspectives for skeletal-related field research. In recent years, bone immunology has gradually become a hot spot in the study of bone-related diseases. As a new branch of immunology, bone immunology emphasizes that the immune system can directly or indirectly affect bones through the RANKL/RANK/OPG signaling pathway, IL family, TNF-α, TGF-ß, and IFN-γ. These effects are of great significance for understanding inflammatory bone loss caused by various autoimmune or infectious diseases. In addition, as an external environment that plays an important role in immunity and bone, this study pays attention to the role of exercise-mediated bone immunity in bone reconstruction.

The effect of Schizophyllan on the differentiation of osteoclasts and osteoblasts.

Schizophyllan (SPG), a ß-glucan from Schizophyllum commune, is recognized for its antioxidant, immunoregulatory, and anticancer activities. In this study, its effects on bone cells, particularly osteoclasts and osteoblasts, were examined. We demonstrated that SPG dose-dependently inhibited osteoclastogenesis and reduced gene expression associated with osteoclast differentiation. SPG also decreased bone resorption and F-actin ring formation. This inhibition could have been due to the downregulation of transcription factors c-Fos and nuclear factor of activated T cells 1 (NFATc1) via the MAPKs (JNK and p38), IκBα, and PGC1ß/PPARγ pathways. In coculture, SPG lowered osteoclastogenic activity in calvaria-derived osteoblasts by reducing macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) expression. In addition, SPG slightly enhanced osteoblast differentiation, as evidenced by increased differentiation marker gene expression and alizarin red staining. It also exhibited antiresorptive effects in a lipopolysaccharide-induced calvarial bone loss model. These results indicated a dual role of SPG in bone cell regulation by suppressing osteoclastogenesis and promoting osteoblast differentiation. Thus, SPG could be a therapeutic agent for bone resorption-related diseases such as osteoporosis, rheumatoid arthritis, and periodontitis.

Systematic review of the osteogenic effect of rare earth nanomaterials and the underlying mechanisms.

Rare earth nanomaterials (RE NMs), which are based on rare earth elements, have emerged as remarkable biomaterials for use in bone regeneration. The effects of RE NMs on osteogenesis, such as promoting the osteogenic differentiation of mesenchymal stem cells, have been investigated. However, the contributions of the properties of RE NMs to bone regeneration and their interactions with various cell types during osteogenesis have not been reviewed. Here, we review the crucial roles of the physicochemical and biological properties of RE NMs and focus on their osteogenic mechanisms. RE NMs directly promote the proliferation, adhesion, migration, and osteogenic differentiation of mesenchymal stem cells. They also increase collagen secretion and mineralization to accelerate osteogenesis. Furthermore, RE NMs inhibit osteoclast formation and regulate the immune environment by modulating macrophages and promote angiogenesis by inducing hypoxia in endothelial cells. These effects create a microenvironment that is conducive to bone formation. This review will help researchers overcome current limitations to take full advantage of the osteogenic benefits of RE NMs and will suggest a potential approach for further osteogenesis research.

Current perspectives on the multiple roles of osteoclasts: Mechanisms of osteoclast-osteoblast communication and potential clinical implications.

Bone remodeling is a complex process involving the coordinated actions of osteoblasts and osteoclasts to maintain bone homeostasis. While the influence of osteoblasts on osteoclast differentiation is well established, the reciprocal regulation of osteoblasts by osteoclasts has long remained enigmatic. In the past few years, a fascinating new role for osteoclasts has been unveiled in promoting bone formation and facilitating osteoblast migration to the remodeling sites through a number of different mechanisms, including the release of factors from the bone matrix following bone resorption and direct cell-cell interactions. Additionally, considerable evidence has shown that osteoclasts can secrete coupling factors known as clastokines, emphasizing the crucial role of these cells in maintaining bone homeostasis. Due to their osteoprotective function, clastokines hold great promise as potential therapeutic targets for bone diseases. However, despite long-standing work to uncover new clastokines and their effect in vivo, more substantial efforts are still required to decipher the mechanisms and pathways behind their activity in order to translate them into therapies. This comprehensive review provides insights into our evolving understanding of the osteoclast function, highlights the significance of clastokines in bone remodeling, and explores their potential as treatments for bone diseases suggesting future directions for the field.

p-Smad3 differentially regulates the cytological behavior of osteoclasts before and after osteoblasts maturation.

BACKGROUND: A series of previous investigations have revealed that p-Smad3 plays a facilitative role in the differentiation and maturation of osteoblasts, while also regulating the expression of certain intercellular communication factors. However, the effects of p-Smad3 in osteoblasts before and after maturation on the proliferation, migration, differentiation, apoptosis and other cellular behaviors of osteoclasts have not been reported. METHODS: MC3T3-E1 cells were cultured in osteogenic induction medium for varying durations, After that, the corresponding conditioned medium was collected and the osteoclast lineage cells were treated. To elucidate the regulatory role of p-Smad3 within osteoblasts, we applied the activator TGF-ß1 and inhibitor SIS3 to immature and mature osteoblasts and collected corresponding conditioned media for osteoclast intervention. RESULTS: We observed an elevation of p-Smad3 and Smad3 during the early stage of osteoblast differentiation, followed by a decline in the later stage. we discovered that as osteoblasts mature, their conditioned media inhibit osteoclasts differentiation and the osteoclast-coupled osteogenic effect. However, it promotes apoptosis in osteoclasts and the angiogenesis coupled with osteoclasts. p-Smad3 in immature osteoblasts, through paracrine effects, promotes the migration, differentiation, and osteoclast-coupled osteogenic effects of osteoclast lineage cells. For mature osteoblasts, p-Smad3 facilitates osteoclast apoptosis and the angiogenesis coupled with osteoclasts. CONCLUSIONS: As pre-osteoblasts undergo maturation, p-Smad3 mediated a paracrine effect that transitions osteoclast cellular behaviors from inducing differentiation and stimulating bone formation to promoting apoptosis and coupling angiogenesis.

[Latest Findings on the Role of RUNX1 in Bone Development and Disorders]. / RUNX1åœ¨éª¨å‘è‚²åŠéª¨ç›¸å ³ç–¾ç— ä¸­çš„ä½œç”¨ç ”ç©¶è¿›å±•.

Runt-related transcription factor (RUNX1) is a transcription factor closely involved in hematopoiesis. RUNX1 gene mutation plays an essential pathogenic role in the initiation and development of hematological tumors, especially in acute myeloid leukemia. Recent studies have shown that RUNX1 is also involved in the regulation of bone development and the pathological progression of bone-related diseases. RUNX1 promotes the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts and modulates the maturation and extracellular matrix formation of chondrocytes. The expression of RUNX1 in mesenchymal stem cells, chondrocytes, and osteoblasts is of great significance for maintaining normal bone development and the mass and quality of bones. RUNX1 also inhibits the differentiation and bone resorptive activities of osteoclasts, which may be influenced by sexual dimorphism. In addition, RUNX1 deficiency contributes to the pathogenesis of osteoarthritis, delayed fracture healing, and osteoporosis, which was revealed by the RUNX1 conditional knockout modeling in mice. However, the roles of RUNX1 in regulating the hypertrophic differentiation of chondrocytes, the sexual dimorphism of activities of osteoclasts, as well as bone loss in diabetes mellitus, senescence, infection, chronic inflammation, etc, are still not fully understood. This review provides a systematic summary of the research progress concerning RUNX1 in the field of bone biology, offering new ideas for using RUNX1 as a potential target for bone related diseases, especially osteoarthritis, delayed fracture healing, and osteoporosis.

[Research Progress in the Regulatory Role of circRNA-miRNA Network in Bone Remodeling]. / circRNA-miRNAç½‘ç»œè°ƒæŽ§éª¨é‡å¡‘çš„ç ”ç©¶è¿›å±•.

The dynamic balance between bone formation and bone resorption is a critical process of bone remodeling. The imbalance of bone formation and bone resorption is closely associated with the occurrence and development of various bone-related diseases. Under both physiological and pathological conditions, non-coding RNAs (ncRNAs) play a crucial regulatory role in protein expression through either inhibiting mRNAs translation or promoting mRNAs degradation. Circular RNAs (circRNAs) are a type of non-linear ncRNAs that can resist the degradation of RNA exonucleases. There is accumulating evidence suggesting that circRNAs and microRNAs (miRNAs) serve as critical regulators of bone remodeling through their direct or indirect regulation of the expression of osteogenesis-related genes. Additionally, recent studies have revealed the involvement of the circRNAs-miRNAs regulatory network in the process by which mesenchymal stem cells (MSCs) differentiate towards the osteoblasts (OB) lineage and the process by which bone marrow-derived macrophages (BMDM) differentiate towards osteoclasts (OC). The circRNA-miRNA network plays an important regulatory role in the osteoblastic-osteoclastic balance of bone remodeling. Therefore, a thorough understanding of the circRNA-miRNA regulatory mechanisms will contribute to a better understanding of the regulatory mechanisms of the balance between osteoblastic and osteoclastic activities in the process of bone remodeling and the diagnosis and treatment of related diseases. Herein, we reviewed the functions of circRNA and microRNA. We also reviewed their roles in and the mechanisms of the circRNA-miRNA regulatory network in the process of bone remodeling. This review provides references and ideas for further research on the regulation of bone remodeling and the prevention and treatment of bone-related diseases.

Toll-like receptor-2 induced inflammation causes local bone formation and activates canonical Wnt signaling.

It is well established that inflammatory processes in the vicinity of bone often induce osteoclast formation and bone resorption. Effects of inflammatory processes on bone formation are less studied. Therefore, we investigated the effect of locally induced inflammation on bone formation. Toll-like receptor (TLR) 2 agonists LPS from Porphyromonas gingivalis and PAM2 were injected once subcutaneously above mouse calvarial bones. After five days, both agonists induced bone formation mainly at endocranial surfaces. The injection resulted in progressively increased calvarial thickness during 21 days. Excessive new bone formation was mainly observed separated from bone resorption cavities. Anti-RANKL did not affect the increase of bone formation. Inflammation caused increased bone formation rate due to increased mineralizing surfaces as assessed by dynamic histomorphometry. In areas close to new bone formation, an abundance of proliferating cells was observed as well as cells robustly stained for Runx2 and alkaline phosphatase. PAM2 increased the mRNA expression of Lrp5, Lrp6 and Wnt7b, and decreased the expression of Sost and Dkk1. In situ hybridization demonstrated decreased Sost mRNA expression in osteocytes present in old bone. An abundance of cells expressed Wnt7b in Runx2-positive osteoblasts and ß-catenin in areas with new bone formation. These data demonstrate that inflammation, not only induces osteoclastogenesis, but also locally activates canonical WNT signaling and stimulates new bone formation independent on bone resorption.

Lactobacillus plantarum 45 activates SHP2 through inhibition of oxidative stress to regulate osteoblast and osteoclast differentiation.

BACKGROUND: The purpose of this study is to observe LP45 (Lactobacillus plantarum 45) to investigate the mechanism by which LP45 attenuates oxidative stress-induced damage and regulates the osteoblast-osteoclast balance. MATERIALS AND METHODS: The oxidative stress level and osteoblast- and osteoclast-related proteins were detected by immunofluorescence staining, Western blotting, ROS fluorescent probe and ELISA. Osteoblast cell proliferation capacity was determined by the CCK-8 assay. X-ray observation and HE staining were used to detect the effect of LP45 on osteoporosis. RESULTS: The expression level of SHP2 and Src was significantly increased, and the expression levels of NOX4, P22, P47, IL-1ß, NLRP3, IRF3, RANK, ß-catenin and INF-ß were inhibited in LP45 group and LPS + LP45 group as compared to those in LPS group. Compared with that in LPS group, the concentration of SOD was increased and the concentration of MDA was decreased in LPS + LP45 group. The protein expressions of OPG, RANKL, RUNX3, RANK and ß-catenin in LP45 group and LPS + LP45 group increased. The protein expressions of NF-κB, CREB and AP-1 in LP45 group and LPS + LP45 group decreased significantly. The results were also confirmed by immunofluorescence staining and ROS fluorescent probe. X-ray observation and HE staining showed that LP45 could inhibit the progression of osteoporosis. CONCLUSION: LP45 can exert its antioxidant effect by inhibiting the production of oxidative stress to activate the SHP2 signaling pathway, thus promoting osteoblast differentiation and repressing osteoclast formation to maintain bone homeostasis and improve bone metabolism.

The abortive SARS-CoV-2 infection of osteoclast precursors promotes their differentiation into osteoclasts.

The Coronavirus Disease 2019 (COVID-19) pandemic has resulted in the loss of millions of lives, although a majority of those infected have managed to survive. Consequently, a set of outcomes, identified as long COVID, is now emerging. While the primary target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the respiratory system, the impact of COVID-19 extends to various body parts, including the bone. This study aims to investigate the effects of acute SARS-CoV-2 infection on osteoclastogenesis, utilizing both ancestral and Omicron viral strains. Monocyte-derived macrophages, which serve as precursors to osteoclasts, were exposed to both viral variants. However, the infection proved abortive, even though ACE2 receptor expression increased postinfection, with no significant impact on cellular viability and redox balance. Both SARS-CoV-2 strains heightened osteoclast formation in a dose-dependent manner, as well as CD51/61 expression and bone resorptive ability. Notably, SARS-CoV-2 induced early pro-inflammatory M1 macrophage polarization, shifting toward an M2-like profile. Osteoclastogenesis-related genes (RANK, NFATc1, DC-STAMP, MMP9) were upregulated, and surprisingly, SARS-CoV-2 variants promoted RANKL-independent osteoclast formation. This thorough investigation illuminates the intricate interplay between SARS-CoV-2 and osteoclast precursors, suggesting potential implications for bone homeostasis and opening new avenues for therapeutic exploration in COVID-19.

Mechanistic study on the alleviation of postmenopausal osteoporosis by Lactobacillus acidophilus through butyrate-mediated inhibition of osteoclast activity.

In China, traditional medications for osteoporosis have significant side effects, low compliance, and high costs, making it urgent to explore new treatment options. Probiotics have demonstrated superiority in the treatment of various chronic diseases, and the reduction of bone mass in postmenopausal osteoporosis (PMOP) is closely related to the degradation and metabolism of intestinal probiotics. It is crucial to explore the role and molecular mechanisms of probiotics in alleviating PMOP through their metabolites, as well as their therapeutic effects. We aim to identify key probiotics and their metabolites that affect bone loss in PMOP through 16srDNA sequencing combined with non-targeted metabolomics sequencing, and explore the impact and possible mechanisms of key probiotics and their metabolites on the progression of PMOP in the context of osteoporosis caused by estrogen deficiency. The sequencing results showed a significant decrease in Lactobacillus acidophilus and butyrate in PMOP patients. In vivo experiments confirmed that the intervention of L. acidophilus and butyrate significantly inhibited osteoclast formation and bone resorption activity, improved intestinal barrier permeability, suppressed B cells, and the production of RANKL on B cells, effectively reduced systemic bone loss induced by oophorectomy, with butyric acid levels regulated by L. acidophilus. Consistently, in vitro experiments have confirmed that butyrate can directly inhibit the formation of osteoclasts and bone resorption activity. The above research results indicate that there are various pathways through which L. acidophilus inhibits osteoclast formation and bone resorption activity through butyrate. Intervention with L. acidophilus may be a safe and promising treatment strategy for osteoclast related bone diseases, such as PMOP.

A novel proteomic signature of osteoclast differentiation unveils the deubiquitinase UCHL1 as a necessary osteoclastogenic driver.

Bone destruction, a major source of morbidity, is mediated by heightened differentiation and activity of osteoclasts (OC), highly specialized multinucleated myeloid cells endowed with unique bone-resorptive capacity. The molecular mechanisms regulating OC differentiation in the bone marrow are still partly elusive. Here, we aimed to identify new regulatory circuits and actionable targets by comprehensive proteomic characterization of OCgenesis from mouse bone marrow monocytes, adopting two parallel unbiased comparative proteomic approaches. This work disclosed an unanticipated protein signature of OCgenesis, with most gene products currently unannotated in bone-related functions, revealing broad structural and functional cellular reorganization and divergence from macrophagic immune activity. Moreover, we identified the deubiquitinase UCHL1 as the most upregulated cytosolic protein in differentiating OCs. Functional studies proved it essential, as UCHL1 genetic and pharmacologic inhibition potently suppressed OCgenesis. Furthermore, proteomics and mechanistic dissection showed that UCHL1 supports OC differentiation by restricting the anti-OCgenic activity of NRF2, the transcriptional activator of the canonical antioxidant response, through redox-independent stabilization of the NRF2 inhibitor, KEAP1. Besides offering a valuable experimental framework to dissect OC differentiation, our study discloses the essential role of UCHL1, exerted through KEAP1-dependent containment of NRF2 anti-OCgenic activity, yielding a novel potential actionable pathway against bone loss.

Ethanol extract of Cyathulae Radix inhibits osteoclast differentiation and bone loss.

Cyathulae Radix, a traditional Chinese medicine and a common vegetable, boasts a history spanning millennia. It enhances bone density, boosts metabolism, and effectively alleviates osteoporosis-induced pain. Despite its historical use, the molecular mechanisms behind Cyathulae Radix's impact on osteoporosis remain unexplored. In this study, we investigated the effects and mechanisms of Cyathulae Radix ethanol extract (CEE) in inhibiting osteoporosis and osteoclastogenesis. Eight-week-old female mice underwent ovariectomy and were treated with CEE for eight weeks. Micro-computed tomography (micro-CT) assessed histomorphometric parameters, bone tissue staining observed distal femur histomorphology, and three-point bending tests evaluated tibia mechanical properties. Enzyme-linked immunosorbent assay (ELISA) measured serum estradiol (E2), receptor activator for nuclear factor B ligand (RANKL), and osteoprotegerin (OPG) levels. Osteoclastogenesis-related markers were analyzed via Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, CEE effects on RANKL-induced osteoclast formation and bone resorption were investigated in vitro using tartrate-resistant acid phosphatase (TRAP) staining, qRT-PCR, and WB assay. Compared with the ovariectomy (OVX) group, CEE treatment enhanced trabecular bone density, maximal load-bearing capacity, and various histomorphometric parameters. Serum E2 and OPG levels significantly increased, while Receptor activator of nuclear factor-κB (RANK) decreased in the CEE group. CEE downregulated matrix metallopeptidase 9 (MMP-9), Cathepsin K (CTSK), and TRAP gene and protein expression. In bone marrow macrophages (BMMs), CEE reduced mature osteoclasts, bone resorption pit areas, and MMP-9, CTSK, and TRAP expression during osteoclast differentiation. Compared with DMSO treatment, CEE markedly inhibited RANK, TNF receptor associated factor 6 (TRAF6), Proto-oncogene c-Fos (c-Fos), Nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expressions, and Extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), NF-kappa B-p65 (p65) phosphorylation in osteoclasts. In conclusion, CEE significantly inhibits OVX-induced osteoporosis and RANKL-induced osteoclastogenesis, potentially through modulating the Estrogen Receptor (ER)/RANK/NFATc1 signaling pathway.

The critical role of toll-like receptor 4 in bone remodeling of osteoporosis: from inflammation recognition to immunity.

Osteoporosis is a common chronic metabolic bone disorder. Recently, increasing numbers of studies have demonstrated that Toll-like receptor 4 (TLR4, a receptor located on the surface of osteoclasts and osteoblasts) plays a pivotal role in the development of osteoporosis. Herein, we performed a comprehensive review to summarize the findings from the relevant studies within this topic. Clinical data showed that TLR4 polymorphisms and aberrant TLR4 expression have been associated with the clinical significance of osteoporosis. Mechanistically, dysregulation of osteoblasts and osteoclasts induced by abnormal expression of TLR4 is the main molecular mechanism underlying the pathological processes of osteoporosis, which may be associated with the interactions between TLR4 and NF-κB pathway, proinflammatory effects, ncRNAs, and RUNX2. In vivo and in vitro studies demonstrate that many promising substances or agents (i.e., methionine, dioscin, miR-1906 mimic, artesunate, AEG-1 deletion, patchouli alcohol, and Bacteroides vulgatus) have been able to improve bone metabolism (i.e., inhibits bone resorption and promotes bone formation), which may partially attribute to the inhibition of TLR4 expression. The present review highlights the important role of TLR4 in the clinical significance and the pathogenesis of osteoporosis from the aspects of inflammation and immunity. Future therapeutic strategies targeting TLR4 may provide a new insight for osteoporosis treatment.

Subchondral osteoclasts and osteoarthritis: new insights and potential therapeutic avenues.

Osteoarthritis (OA) is the most common joint disease, and good therapeutic results are often difficult to obtain due to its complex pathogenesis and diverse causative factors. After decades of research and exploration of OA, it has been progressively found that subchondral bone is essential for its pathogenesis, and pathological changes in subchondral bone can be observed even before cartilage lesions develop. Osteoclasts, the main cells regulating bone resorption, play a crucial role in the pathogenesis of subchondral bone. Subchondral osteoclasts regulate the homeostasis of subchondral bone through the secretion of degradative enzymes, immunomodulation, and cell signaling pathways. In OA, osteoclasts are overactivated by autophagy, ncRNAs, and Rankl/Rank/OPG signaling pathways. Excessive bone resorption disrupts the balance of bone remodeling, leading to increased subchondral bone loss, decreased bone mineral density and consequent structural damage to articular cartilage and joint pain. With increased understanding of bone biology and targeted therapies, researchers have found that the activity and function of subchondral osteoclasts are affected by multiple pathways. In this review, we summarize the roles and mechanisms of subchondral osteoclasts in OA, enumerate the latest advances in subchondral osteoclast-targeted therapy for OA, and look forward to the future trends of subchondral osteoclast-targeted therapies in clinical applications to fill the gaps in the current knowledge of OA treatment and to develop new therapeutic strategies.

Bub1 suppresses inflammatory arthritis-associated bone loss in mice through inhibition of TNFα-mediated osteoclastogenesis.

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-1ß 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.

Rheumatoid arthritis (RA) is a disease caused by an abnormal immune system, resulting in inflammation, swelling, and bone destruction in the joints, along with systemic bone loss. While new medications have dramatically improved treatment efficacy, these therapies are not universally effective for all patients. Therefore, we need to understand the regulatory mechanisms behind RA, including associated bone loss, to develop better therapies. In this study, we found that Budding uninhibited by benzimidazoles 1 (Bub1) was highly expressed in inflamed joints, especially in myeloid cells, which are a type of immune cells. To explore its role, we created myeloid cell­specific Bub1 conditional knockout (cKO) mice and induced arthritis to analyze its role during arthritis. The cKO mice exhibited lower bone mineral density when compared with control mice under inflammatory arthritis because of increased osteoclastic bone resorption, without significant differences in joint inflammation or bone erosion. Further investigation showed that Bub1 prevents excessive osteoclast differentiation induced by inflammation in bone marrow macrophages. These data suggest that Bub1 in macrophages protects against bone loss caused by inflammatory arthritis, offering potential insights for developing treatments that focus on bone health.

Imbalanced EphB4/EphrinB2 Signaling Modulates Bone Resorption in Periodontitis Induced by Porphyromonas gingivalis.

Periodontitis, a chronic infectious disease in periodontal tissues, is characterized by an imbalance of alveolar bone resorption and remodeling, which eventually results in tooth loosening and even tooth loss. The etiology of periodontitis is polymicrobial synergy and dysbiosis, in which Porphyromonas gingivalis (P. gingivalis) is one of the primary pathogens responsible for periodontitis progression. The interplay of EphrinB2/EphB4 is crucial for osteoblast-osteoclast communication during bone remodeling and healing. This study investigates the mechanism of EphB4/EphrinB2 transduction modulating osteogenesis inhibition and bone resorption in periodontitis induced by P. gingivalis. An in vivo model of chronic periodontitis provoked by P. gingivalis was constructed, the inflammation and bone resorption were evaluated. The expression of EphB4 and EphrinB2 proteins in periodontal tissues was detected, which was also evaluated, respectively, in osteoblasts and osteoclasts infected with P. gingivalis in vitro. Then, a simulated coculture model of osteoblasts and osteoclasts was established to activate the forward and reverse pathways of EphB4/EphrinB2 with P. gingivalis infection. This study showed that P. gingivalis infection promoted alveolar bone resorption in rats and enhanced EphB4 and EphrinB2 expression in periodontal tissues. EphB4 and molecules associated with osteogenesis in osteoblasts infected with P. gingivalis were inhibited, while EphrinB2 and osteoclast differentiation-related markers in osteoclasts were activated. In conclusion, this study suggested that EphB4/EphrinB2 proteins were involved in alveolar bone remodeling in the process of periodontitis induced by P. gingivalis infection. Moreover, attenuated EphB4/EphrinB2 with P. gingivalis infection weakened osteoblast activity and enhanced osteoclast activity.

De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis.

Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitro but does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB.

Long noncoding RNA Malat1 protects against osteoporosis and bone metastasis.

MALAT1, one of the few highly conserved nuclear long noncoding RNAs (lncRNAs), is abundantly expressed in normal tissues. Previously, targeted inactivation and genetic rescue experiments identified MALAT1 as a suppressor of breast cancer lung metastasis. On the other hand, Malat1-knockout mice are viable and develop normally. On a quest to discover the fundamental roles of MALAT1 in physiological and pathological processes, we find that this lncRNA is downregulated during osteoclastogenesis in humans and mice. Remarkably, Malat1 deficiency in mice promotes osteoporosis and bone metastasis of melanoma and mammary tumor cells, which can be rescued by genetic add-back of Malat1. Mechanistically, Malat1 binds to Tead3 protein, a macrophage-osteoclast-specific Tead family member, blocking Tead3 from binding and activating Nfatc1, a master regulator of osteoclastogenesis, which results in the inhibition of Nfatc1-mediated gene transcription and osteoclast differentiation. Notably, single-cell transcriptome analysis of clinical bone samples reveals that reduced MALAT1 expression in pre-osteoclasts and osteoclasts is associated with osteoporosis and metastatic bone lesions. Altogether, these findings identify Malat1 as a lncRNA that protects against osteoporosis and bone metastasis.