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Bone remodeling mediated by bone-forming osteoblasts (OBs) and bone-resorbing osteoclasts (OCs) maintains bone structure and function. Excessive OC activation leads to bone-destroying diseases such as osteoporosis and bone erosion of rheumatoid arthritis (RA). Differentiation of OCs from bone marrow cells (BMCs) is regulated by the bone microenvironment. The proinflammatory cytokine interleukin (IL)-1β reportedly enhances osteoclastogenesis and plays important roles in RA-associated bone loss. The present study investigated the effect of IL-1β on OC formation via microenvironmental cells. Treating mouse BMCs with IL-1β in the presence of receptor activator of NF-κB ligand and macrophage colony-stimulating factor increased the number of OCs. Real-time RT-PCR revealed increased expression of the IL-1β, IL-1RI, and IL-1RII genes in non-OCs compared with OCs. Removing CD45

Breast carcinomas (BC) with osteoclast-like giant cells (OGC) are rare. Despite their distinct stromal features, their molecular characteristics remain unknown. Here, we report comprehensive clinico-pathological and molecular findings for 27 patients diagnosed with BC-OGC at Institut Curie between 2000 and 2021. Seventeen (63%) cases were invasive carcinomas of no special type (IC NST) with OGC (OGC-IC NST), four (15%) were mixed or multifocal cases with and without OGC (OGC-Mixed), and six (22%) were metaplastic carcinomas with OGC (OGC-MC). All OGC-IC NST and OGC-Mixed cases were ER+ HER2- tumors (most being luminal A based on transcriptomic subtyping, when available), while all OGC-MC were triple-negative. The median age at diagnosis was 46, 45 and 62 years for OGC-IC NST, OGC-Mixed and OGC-MC, respectively. Three patients developed distant metastases (one OGC-IC NST, two OGC-Mixed), one of whom died of metastatic disease (OGC-Mixed), and one other patient died of locally advanced disease (OGC-MC). Histopathological evaluation comparing 13 OGC-IC NST and 19 control IC NST without OGC confirmed that OGC-IC NST showed significantly higher density of vessels (by CD34 immunohistochemistry (IHC)), iron deposits (Perls stain), and CD68 and CD163-positive cell infiltrates. Genomic findings for nine OGC-IC NST and four OGC-MC were consistent with the underlying histologic subtype, including activating alterations of the PI3K/AKT/mTOR pathway in 7/13 cases. Using RNA-seq data, differential gene expression analysis between OGC-IC NST (n = 7) and control IC NST without OGC (n = 7) revealed significant overexpression of TNFSF11 (RANK-L), TNFRSF11A (RANK), CSF1 (M-CSF), CSF1R, and genes encoding osteoclastic enzymes (MMP9, ACP5, CTSK, CTSB) in OGC-IC NST, while OPG (osteoprotegerin) was underexpressed. We also confirmed for the first time RANK-L expression in BC with OGC by IHC (seen in 15 out of 16 cases, and only in 2 of 16 controls without OGC). These findings could offer a rationale for further investigating RANK-L as a therapeutic target in BC with OGC.

Bone remodels via resorption and formation, two phenomena that continuously occur in bone turnover. The RANKL/RANK/OPG pathway is one of the several mechanisms that affect bone turnover. The RANKL/OPG ratio has a substantial role in bone resorption. An imbalance between formation and resorption is related to an increased RANKL/OPG balance. OPG, a member of this system, can bind to RANKL and suppress RANK-RANKL interaction, and subsequently, inhibit further osteoclastogenesis. The serum levels of RANKL and OPG in the bone microenvironment are vital for osteoclasts formation. The RANK/RANKL/OPG system plays a role in the pathogenesis of bone disorders. This system can be considered a new treatment target for bone disorders. Soy isoflavones affect the RANK/RANKL/OPG system through numerous mechanisms. Soy isoflavones decrease RANKL levels and increase OPG levels. Therefore, isoflavones improve bone metabolism and decrease bone resorption. Soy isoflavones decrease serum markers of bone resorption and improve bone metabolism. However, while the available data are promising, the results of several studies reported no change in RANKL and OPG levels with isoflavones supplementation. In this regard, current evidence is insufficient for conclusive approval of the efficacy of isoflavones on RANKL/RANK/OPG and further research, including animal and human studies, are needed to confirm the effect of soy isoflavones on the RANKL/RANK/OPG pathway. This study was a review of available evidence to determine the role of isoflavones in bone hemostasis and the RANK/RANKL/OPG pathway. The identification of the effects of isoflavones on the RANKL/RANK/OPG pathway directs future studies and leads to the development of effective treatment strategies for bone disorders.

Extracellular vesicles (EVs) are potent means of cell-to-cell communication. They are released in biological fluids, including blood, urine, and saliva, and can be exploited to identify new biomarkers of diseases. We hypothesized that EVs contain molecular cargos involved in bone metabolism, possibly mirroring biological differences between postmenopausal and disuse osteoporosis. We tested this hypothesis in primary murine osteoblasts subjected to steroid depletion or to unloading, and in the serum of animal models of osteoporosis induced by ovariectomy or hindlimb tail suspension. EVs were isolated by ultracentrifugation and analysed by transmission electron microscopy, cytofluorimetry, immunoblotting and RT-PCR. Large-scale analyses were performed by Real-Time arrays and Proteome Profiler™ Antibody arrays. Finally, precise titration of analytes was carried out by ELISA assay. In vitro, we confirmed an increased release of EVs enriched in surface RANKL by primary mouse osteoblasts subjected to steroid depletion or simulated microgravity compared to controls. In vivo, circulating EVs isolated from the sera of control female mice expressed RANKL along with other genes associated with bone metabolism. Serum EVs from ovariectomized or hindlimb tail-suspended mice showed distinct molecular profiles. They expressed RANKL with different kinetics, while transcriptomic and proteomic profiles uncovered unique molecular signatures that discriminated the two conditions, unveiling exclusive molecules expressed in time- and osteoporosis type-dependent manner. These results suggest that circulating EVs could represent a new tool for monitoring the onset and the progression of diverse types of the disease in mice, paving the way for their exploitation to diagnose human osteoporosis in liquid biopsies.

Cystatin A (CSTA) is a cysteine protease inhibitor that is expressed highly during osteoporosis. However, the exact role of CSTA in osteoporosis remains unknown. In this study, we examined the role of CSTA in the formation, differentiation, and bone resorption of osteoclasts. We extracted bone marrow cells from 8-week-old wildtype mice to obtain RANKL and M-CSF-induced osteoclasts. We performed CSTA overexpression and knockdown experiments in the cells. We analyzed the role of CSTA in the process of osteoclasts by trap staining. In addition, we studied the contribution of CSTA to osteogenesis through the DAP12/TREM2 (DNAX-activating protein of 12 kDa/Triggering receptor expressed on myeloid cells-2) complex. We analyzed the role of CSTA in postmenopausal osteoporosis using OVX mouse models. We found that the silencing of CSTA inhibited the differentiation and formation of osteoclasts. The loss of CSTA weakened the expression of osteoclast marker genes. In contrast, overexpression of CSTA significantly increased differentiation and formation of osteoclasts and enhanced bone resorption. Immunofluorescence staining indicated that CSTA and DAP12 are co-expressed in osteoclasts, and the loss of either DAP12 or TREM2 inhibited osteoclast differentiation and bone resorption. Suppression of CSTA decreased DAP12 and TREM2 expression, whereas overexpression of CSTA rescued the loss of TREM2 expression caused by DAP12 knockdown. Co-immunoprecipitation and co-localization experiments indicated that CSTA interacted with DAP12. In addition, we found that injection of si-CSTA into OVX mice significantly improved bone parameters. Our research indicates that CSTA interacts with the DAP12/TREM2 complex and could be a potential targeted therapy for osteoporosis management.

RANKL induces NFATc1, a key transcriptional factor to induce osteoclast-specific genes such as cathepsin K, whereas transcriptional control of osteoclast survival is not fully understood. Leukemia/lymphoma-related factor (LRF) in mouse and osteoclast zinc finger protein (OCZF) in rat are zinc finger and BTB domain-containing protein (zBTB) family of transcriptional regulators, and are critical regulators of hematopoiesis. We have previously shown that differentiation and survival were enhanced in osteoclasts from OCZF-Transgenic (Tg) mice. In the present study, we show a possible mechanism of osteoclast survival regulated by LRF/OCZF and the role of OCZF overexpression in pathological bone loss. In the in vitro cultures, LRF was highly colocalized with NFATc1 in cells of early stage in osteoclastogenesis, but only LRF expression persisted after differentiation into mature osteoclasts. LRF expression was further enhanced in resorbing osteoclasts formed on dentin slices. Osteoclast survival inhibitor such as alendronate, a bisphosphonate reduced LRF expression. Micro CT evaluation revealed that femurs of OCZF-Tg mice showed significantly lower bone volume compared to that of WT mice. Furthermore, OCZF overexpression markedly promoted bone loss in ovariectomy-induced osteolytic mouse model. The expression of anti-apoptotic Bcl-xl mRNA, which is formed by alternative splicing, was enhanced in the cultures in which osteoclasts are formed from OCZF-Tg mice. In contrast, the expression of pro-apoptotic Bcl-xs mRNA was lost in the culture derived from OCZF-Tg mice. We found that the expression levels of RNA binding splicing regulator, Src substrate associated in mitosis of 68 kDa (Sam68) protein were markedly decreased in OCZF-Tg mice-derived osteoclasts. In addition, shRNA-mediated knockdown of Sam68 expression increased the expression of Bcl-xl mRNA, suggesting that SAM68 regulates the expression of Bcl-xl. These results indicate that OCZF overexpression reduces protein levels of Sam68, thereby promotes osteoclast survival, and suggest that LRF/OCZF is a promising target for regulating pathological bone loss.

Osteoclast generation from monocyte/macrophage lineage precursor cells needs to be tightly regulated to maintain bone homeostasis and is frequently over-activated in inflammatory conditions. PARK2, a protein associated with Parkinson's disease, plays an important role in mitophagy via its ubiquitin ligase function. In this study, we investigated whether PARK2 is involved in osteoclastogenesis. PARK2 expression was found to be increased during the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. PARK2 gene silencing with siRNA significantly reduced osteoclastogenesis induced by RANKL, LPS (lipopolysaccharide), TNFα (tumor necrosis factor α), and IL-1β (interleukin-1β). On the other hand, overexpression of PARK2 promoted osteoclastogenesis. This regulation of osteoclastogenesis by PARK2 was mediated by IKK (inhibitory κB kinase) and NF-κB activation while MAPK (mitogen-activated protein kinases) activation was not involved. Additionally, administration of PARK2 siRNA significantly reduced osteoclastogenesis and bone loss in an

The potential role of the transcription factor Differentiated embryo-chondrocyte 2 (Dec2) in the progression of inflammatory diseases such as periodontitis has been unclear. Here, the effect of Dec2 on the expression of RANKL and on osteoclastogenesis was determined.Wild-type (WT) and Dec2 knockout (KO) mice as a model for periodontitis were used to assess alveolar bone resorption by microcomputed tomography (CT). Western blot, flow cytometry, quantitative real-time PCR, and immunohistochemical analyses were utilized to detect inflammation and osteoclasts. Luciferase reporter and Chromatin immunoprecipitation (ChIP) assays examined the interaction between Dec2 and RANKL.Micro-CT showed that the alveolar bone resorption of Dec2KO mice was more severe than WT mice after treatment with P. gingivalis. Immunohistochemistry and Tartrate-resistant acid phosphatase staining showed active osteoclast differentiation in Dec2KO mice. There was an increase in CD11bDec2 has an immune regulation ability that modulates P. gingivalis-induced periodontitis via RANKL.

Mouse strains can have divergent basal bone mass, yet this phenotype is seldom reflected in the design of studies seeking to identify new modulators of bone resorption by osteoclasts. Sulforaphane exerts inhibitory effects on in vitro osteoclastogenesis in cells from C57BL/6 mice. Here, we explore whether a divergent basal bone mass in different mouse strains is linked both to in vitro osteoclastogenic potential and to SFX-01 sensitivity. Accordingly, osteoclasts isolated from the bone marrow (BM) of C57BL/6, STR/Ort and CBA mice with low, high, and intermediate bone mass, respectively, were cultured under conditions to promote osteoclast differentiation and resorption; they were also treated with chemically stabilised sulforaphane (SFX-01) and respective sensitivity to inhibition evaluated by counting osteoclast number/resorption activity on dentine discs. We observed that osteoclastogenesis exhibited different macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-Β ligand sensitivity in these mouse strains, with cells from C57BL/6 and CBA generating higher osteoclast numbers than STR/Ort; the latter formed only half as many mature osteoclasts. We found that 100 nM SFX-01 exerted a potent and significant reduction in osteoclast number and resorptive activity in cells derived from C57BL/6 mice. In contrast, 10-fold higher SFX-01 concentrations were required for similar inhibition in CBA-derived cells and, strikingly, a further 2.5-fold greater concentration was required for significant restriction of osteoclast formation/function in STR/Ort. These data are consistent with the notion that the BM osteoclast precursor population contributes to the relative differences in mouse bone mass and that mice with higher bone mass exhibit lower in vitro osteoclastogenic potential as well as reduced sensitivity to inhibition by SFX-01.

We employed the co-culture of CD34Mature HMC and osteoclast precursors were cultured from monocytes isolated from human buffy coat. The osteoclast precursors were incubated with HMC or receptor activator of nuclear factor kappa-B ligand (RANKL) for a week prior to determination of osteoclast maturation through characterization by their morphology and tartrate resistant acid phosphatase (TRAP) expression. The bone absorption activity was determined by pit formation on osteo-assay plate.Mature osteoclasts were identified following co-culture of osteoclast precursors with HMC for one week in the absence of RANKL and they were capable of bone resorption. These actions of HMC on osteoclasts were not affected by mast cell activators such anti-IgE or substance P but could be reversed by osteoprotegerin (OPG) in the co-culture system suggesting the involvement of RANKL. The expression of RANKL on the cell surface of HMC was confirmed by flow cytometry and the density was not affected by activation of HMC.Our study provided direct evidence confirming the initiation of osteoclast proliferation and activation by mast cells through cell surface RANKL suggesting that mast cells may contribute to bone destruction in pathological conditions such as osteoporosis.

The basic helix-loop-helix transcriptional factor, Bhlhe40 has been shown as a crucial regulator of immune response, tumorigenesis, and circadian rhythms. We identified Bhlhe40 as a possible regulator of osteoclast differentiation and function by shRNA library screening and found that Bhlhe40 was required for osteoclast activation. Bhlhe40 expression was induced in bone marrow macrophages (BMMs) by RANKL, whereas the expression of its homolog Bhlhe41 was decreased in osteoclastogenesis. μCT analysis of tibias revealed that Bhlhe40 knockout (KO) mice exhibited increased bone volume phenotype. Bone morphometric analysis showed that osteoclast number and bone resorption were decreased in Bhlhe40 KO mice, whereas significant differences in the osteoblast parameters were not seen between wild-type (WT) and Bhlhe40 KO mice. In vitro culture of BMMs showed that Bhlhe40 deficiency did not cause difference in osteoclast formation. In contrast, bone resorption activity of Bhlhe40 KO osteoclasts was markedly reduced in comparison with that of WT osteoclasts. Analysis of potential target genes of Bhlhe40 using data-mining platform ChIP-Atlas (http://chip-atlas.org) revealed that predicted target genes of Bhlhe40 were related to proton transport and intracellular vesicle acidification. We then analyzed the expression of proton pump, the vacuolar (V)-ATPases which are responsible for bone resorption. The expression of V-ATPases V1c1 and V0a3 was suppressed in Bhlhe40 KO osteoclasts. In addition, Lysosensor yellow/blue DND 160 staining demonstrated that vesicular acidification was attenuated in vesicles of Bhlhe40 KO osteoclasts. Furthermore, analysis with pH-sensitive fluorescent probe showed that proton secretion was markedly suppressed in Bhlhe40 KO osteoclasts compared to that in WT osteoclasts. Our findings suggest that Bhlhe40 plays a novel important role in the regulation of acid production in osteoclastic bone resorption.

Recently, the potential of β-cyclodextrin-thread acid-degradable polyrotaxane (AdPRX) has been emphasized as a therapeutic agent for cholesterol-related metabolic disorders. In this study, we investigated whether carboxymethyl carbamate-modified AdPRX (CMC-AdPRX) can be used for adsorption to calcium phosphate to treat bone diseases. We first synthesized CMC-AdPRX and used it to coat the calcium phosphate plate. RAW264.7 cells were then differentiated into osteoclasts via a receptor activator of nuclear factor-κB ligand, and the number of osteoclasts and the area of absorption lacunae were determined. The number of tartrate-resistant acid phosphatase-positive multinucleated cells was reduced on the CMC-AdPRX-coated plate. The area of the absorption lacunae was smaller with CMC-AdPRX than with AdPRX, which was not carboxy-modified. Our results suggest that CMC-AdPRX can adsorb to calcium phosphate and act on differentiated osteoclasts to suppress their functional expression.

Diabetes osteoporosis is a chronic complication of diabetes mellitus (DM) and is associated with osteoclast formation and enhanced bone resorption. Specnuezhenide (SPN) is an active compound with anti-inflammatory and immunomodulatory properties. However, the roles of SPN in diabetic osteoporosis remain unknown. In this study, primary bone marrow macrophages (BMMs) were pretreated with SPN and were stimulated with receptor activator of nuclear factor kappa B ligand (RANKL; 50 ng/mL) to induce osteoclastogenesis. The number of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. The protein levels of cellular oncogene fos/nuclear factor of activated T cells c1 (c-Fos/NFATc1), nuclear factor kappa-B (NF-κB), and mitogen-activated protein kinases (MAPKs) were evaluated by western blot analysis. NF-κB luciferase assays were used to examine the role of SPN in NF-κB activation. The DM model group received a high-glucose, high-fat diet and was then intraperitoneally injected with streptozotocin (STZ). Micro-CT scanning, serum biochemical analysis, histological analysis were used to assess bone loss. We found that SPN suppressed RANKL-induced osteoclast formation and that SPN inhibited the expression of osteoclast-related genes and

Rat spine fusion model.The present study aimed to determine whether administration of osteoprotegerin (OPG) in a rat model of spinal fusion increases bone volume, bone density, and decreases osteoclasts in the fusion mass.OPG is a soluble RANK-ligand inhibitor that blocks osteoclast differentiation and activation. This makes it a potential agent to control the remodeling process and enhance bone mass during spinal fusion.Forty-eight male Sprague-Dawley rats received a one-level spinal fusion of L4-L5 with bone allograft. Rats were then divided into four groups according to initiation of treatment: (1) saline on day 0 (saline), (2) OPG on day 0 (OPG D0), (3) OPG on day 10 (OPG D10), and (4) OPG on day 21 (OPG D21) postsurgery. After their initial injection, rats received weekly subcutaneous injections of OPG (10 mg/kg) and were euthanized six weeks postsurgery. MicroCT analysis of the fusion site and histological analysis of bone surface for quantification of osteoclast lining was performed.Increased bone volume in the fusion site and around the spinous process was seen in OPG D0 and OPG D10 when compared with saline. Mean trabecular thickness was greater in all groups receiving OPG compared with saline, with OPG D0 and OPG D10 having significantly greater mean trabecular thickness than OPG D21. All OPG groups had less bone surface lined with osteoclasts when compared with Saline, with OPG D0 and OPG D10 having fewer than OPG D21.This study indicates that OPG inhibited osteoclast bone resorption, which led to greater bone at the fusion site. Future studies investigating OPG on its own or in combination with an osteogenic factor to improve spinal fusion outcomes are warranted to further elucidate its potential therapeutic effect.

Ganoderma mushrooms have been used to treat rheumatoid arthritis (RA) in East Asia. Whether Ganoderic acid A (GAA), the natural product extracted from Ganoderma, could be utilized to alleviate osteoarthritis (OA) is investigated in this study. Destabilization of the medial meniscus (DMM) model was constructed to reveal the in vivo effect of GAA. We found that GAA could significantly alleviate the pathology of DMM, as confirmed by the diminished maximum histologic scores. On the contrary, GAA could down-regulate the relative expression of osteoprotegerin (OPG) and up-regulate the relative expression of nuclear factor kappa-B ligand (RANKL) in DMM cartilage and human articular chondrocytes (HC-A) cells with diminished matrix metallopeptidase 13 (MMP-13) secretion in the synovial fluid. It was further demonstrated that the serum concentration of OPG was correlated with the severity of osteoarthritis. All these data reveal that GAA could improve OA by regulating the RANKL/OPG ratio to inhibit the secretion of MMP-13.

Branched-chain aminotransferase 1 (BCAT1) transfers the amine group on branched-chain amino acids (BCAAs) to alpha-ketoglutarate. This generates glutamate along with alpha-keto acids that are eventually oxidized to provide the cell with energy. BCAT1 thus plays a critical role in sustaining BCAA concentrations and availability as an energy source. Osteoclasts have high metabolic needs during differentiation. When we assessed the levels of amino acids in bone marrow macrophages (BMMs) that were undergoing receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation, we found that the BCAA levels steadily increase during this process. In vitro analyses then showed that all three BCAAs but especially valine were needed for osteoclast maturation. Moreover, selective inhibition of BCAT1 with gabapentin significantly reduced osteoclast maturation. Expression of enzymatically dead BCAT1 also abrogated osteoclast maturation. Importantly, gabapentin inhibited lipopolysaccharide (LPS)-induced bone loss of calvaria in mice. These findings suggest that BCAT1 could serve as a therapeutic target that dampens osteoclast formation.

Diabetic osteoporosis is secondary osteoporosis and a serious complication of diabetes with a high incidence rate and poor prognosis. The specific mechanism of diabetic osteoporosis is unclear, and prevention and treatment options are limited. Recently, melatonin has been found to prevent and treat diabetic osteoporosis. Herein, we investigated the mechanism whereby melatonin inhibits osteoclastogenesis and identified a new target for osteoporosis treatment. We established an in vitro osteoblast-osteoclast co-culture system as a diabetic osteoporosis model. Osteoclastogenesis was determined using tartrate-resistant acid phosphatase staining and cathepsin K expression. Real-time PCR was used to ascertain expression of microRNA mir-882, targeting Rev-Erbα. Western blotting was performed to detect the expression of Rev-Erbα, receptor activator of NF-kB ligand (RANKL), and osteoprotegerin (OPG), and ELISA was utilized to analyse the secreted form of RANKL. High glucose promoted osteoclastogenesis and elevated the RANKL/OPG ratio in osteoblasts, while melatonin reversed these effects. High glucose inhibited Rev-Erbα expression, while melatonin promoted its expression. Conversely, high glucose promoted mir-882 expression, while melatonin inhibited it. We infer that melatonin inhibits RANKL expression in osteoblasts via the mir-882/Rev-Erbα axis, thus inhibiting osteoclastogenesis. Our findings provide insights into diabetic osteoporosis and identify a new therapeutic target for osteoporosis.

This study investigated whether a vitronectin-derived peptide (VnP-16) prevents and/or reverses alveolar bone resorption induced by ligature-induced periodontitis in rodents and identified the underlying mechanism.We evaluated the effects of VnP-16 on osteogenic differentiation in human periodontal ligament cells (hPDLCs), lipopolysaccharide-induced inflammatory responses in gingival fibroblasts, and immune response in T lymphocytes. Ligature-induced periodontitis was induced by ligating the bilateral mandibular first molars for 14 days in rats and for 7 days in mice (n = 10/group). VnP-16 (100 μg/10 μl) was applied topically into the gingival sulcus of rats via intra-sulcular injection, whereas the peptide (50 μg/5 μl) was administered directly into the gingiva of mice via intra-gingival injection. To evaluate the preventive and therapeutic effects of VnP-16, micro-computed tomography analysis and histological staining were then performed.VnP-16 promoted osteogenic differentiation of periodontal ligament cells and inhibited the production of lipopolysaccharide-induced inflammatory mediators in gingival fibroblasts. Concomitantly, VnP-16 modulated the host immune response by reducing the number of receptor activator of NF-κB ligand (RANKL)-expressing lipopolysaccharide-stimulated CD4Our findings suggest that VnP-16 acts as a potent therapeutic agent for preventing and treating periodontitis by regulating bone re-modelling and immune and inflammatory responses.

Circular RNAs (circRNAs) have been verified as vital regulators in various diseases, including membranous nephropathy (MN). Therefore, the role of circ_CDYL in podocyte apoptosis and MN was investigated. The real-time quantitative polymerase chain reaction was performed to measure the expression of circ_CDYL, microRNA-149-5p (miR-149-5p), and tumor necrosis factor superfamily member 11 (TNFSF11) in podocytes. In addition, angiotensin II (Ang II) was used to induce apoptosis of podocytes. The apoptosis-related protein expression was quantified by western blot assay. The apoptosis of podocytes was evaluated by flow cytometry assay. The interaction relationship between miR-149-5p and circ_CDYL or TNFSF11 was confirmed by dual-luciferase reporter assay. Circ_CDYL was significantly overexpressed in MN patients and Ang II-induced podocytes compared with control groups. Importantly, loss-of-functional experiments indicated that knockdown of circ_CDYL protected podocytes from Ang II-induced apoptosis. MiR-149-5p was verified as target of circ_CDYL and negatively correlated with circ_CDYL expression in MN patients. Knockdown of circ_CDYL-mediated effects on Ang II-induced podocyte cells were abolished by silencing miR-149-5p. Besides, the upregulation of miR-149-5p could suppress apoptosis in Ang II-induced podocyte cells by targeting TNFSF11. Under Ang II stimulation, the upregulation of TNFSF11 could increase the expression of TNFSF11 and induce apoptosis in circ_CDYL-silencing podocytes. Our results confirmed that circ_CDYL specifically targeted miR-149-5p/TNFSF11 pathway to regulate Ang II-induced apoptosis in podocytes, which might be useful diagnostic biomarkers in MN.

The exact role of IL-6 in inflammatory osteoclast formation is still under debate. Our previous study demonstrated that IL-6 in the combination of sIL-6R significantly promoted low level of RANKL-induced osteoclast differentiation which was not affected by IL-6 alone. However, the precise molecular mechanisms underlying the regulation of sIL-6R-induced trans-signaling on osteoclast differentiation remains to be elucidated. Mouse bone marrow‑derived monocytes (BMMs) were isolated and cultured with RANKL and IL-6/sIL-6R in the presence or absence of sgp130. TRAP staining and pit formation assay were used to visualize multinucleated giant osteoclasts and evaluate their bone resorption ability. Western blot and real time-PCR were applied to determine the activations of IL-6 signaling pathway and osteoclastogenesis- associated signaling pathways. The results showed that sIL-6R activation of IL-6 trans-signaling enhanced IL-6 signaling cascades and promoted low concentration of RANKL-induced osteoclasts formation and bone resorption by mouse BMMs. Furthermore, blocking IL-6 trans-signaling with sgp130 abrogated this promotive effect by suppressing NF-κB and JNK signaling pathways. In conclusion, sIL-6R-mediated trans-signaling pathway plays a decisive role in promotion of low level of RANKL-induced osteoclastic differentiation by IL-6/sIL-6R and targeting the IL-6 trans-signaling pathway may represent a potential strategy for inflammatory diseases with pathological bone resorption.

Osteoporosis is a bone disease that is caused by disorder of the skeletal microenvironment, and it characterized by a high disability rate and the occurrence of low energy fractures. Studies on osteoporosis and related treatment options have always been hot spots in the field of bone biology. In the past, the understanding of osteoporosis has been rather limited; research has only shown that osteoporosis involves the imbalance of bone resorption and bone formation, and recent studies have not provided cutting-edge theories of the basic understanding of osteoporosis. Recent studies have shown crosstalk between bone and immune responses. RANKL, an essential factor for osteoclasts (OCs), is associated with the immune system. T helper (Th17)/regulatory T (Treg) cells are two different kinds of T cells that can self-interact and regulate the differentiation and formation of OCs. Therefore, understanding the correlation between the skeletal and immune systems and further revealing the roles and the cooperation between RANKL and the Th17/Treg balance will help to provide new insights for the treatment of osteoporosis.

Aseptic loosening of the prosthesis caused by wear-particle-induced osteolysis is a long-term complication and one of the most common reasons for the failure of joint implants. The primary cause of aseptic loosening of the prosthesis is overactive bone resorption caused by wear-particle-activated osteoclasts in both direct and indirect ways. Therefore, drugs that can inhibit differentiation and bone resorption of osteoclasts need investigation as a potential therapeutic strategy to prevent and treat peri-prosthetic osteolysis and thereby prolong the service life of the prosthesis. This study has verified the potential inhibitory effect of LY450139 on inflammatory osteolysis induced by titanium particles in a mice skull model. In addition, we found that LY450139 inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis, bone resorption, and podosomal actin belt formation in a dose-dependent manner without evidence of cytotoxicity in vitro. In addition, LY450139 significantly decreased the expression of osteoclast-specific markers, including TRAP, CTSK, V-ATPase d2, CTR, DC-STAMP, NFATc1, and the downstream target gene Hes1 in Notch signaling pathway. Further investigation of the molecular mechanism demonstrated that LY450139 inhibited the formation of osteoclasts via inhibition of the NF-κB and Notch signaling pathways. In summary, LY450139 inhibited the formation of RANKL-mediated osteoclasts via NF-κB and Notch signaling and inhibited Ti particle-induced inflammatory osteolysis in vivo. LY450139 is a potential targeted drug for the treatment of peri-prosthetic osteolysis and other osteolytic disease associated with overactive osteoclasts.

A major cause of osteoporosis is impaired coupled bone formation. Mechanistically, both osteoclast-derived and bone-derived growth factors have been previously implicated. Here, we hypothesize that the release of bone calcium during osteoclastic bone resorption is essential for coupled bone formation. Osteoclastic resorption increases interstitial fluid calcium locally from the normal 1.8 mM up to 5 mM. MC3T3-E1 osteoprogenitor cells, cultured in a 3.6 mM calcium medium, demonstrated that calcium signaling stimulated osteogenic cell proliferation, differentiation, and migration. Calcium channel knockdown studies implicated calcium channels, Cav1.2, store-operated calcium entry (SOCE), and calcium-sensing receptor (CaSR) in regulating bone cell anabolic activities. MC3T3–E1 cells cultured in a 3.6 mM calcium medium expressed increased gene expression of Wnt signaling and growth factors platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and bone morphogenic protein-2 (BMP 2). Our coupling model of bone formation, the receptor activator of nuclear factor-κΒ ligand (RANKL)-treated mouse calvaria, confirmed the role of calcium signaling in coupled bone formation by exhibiting increased gene expression for osterix and osteocalcin. Critically, dual immunocytochemistry showed that RANKL treatment increased osterix-positive cells and increased fluorescence intensity of Cav1.2 and CaSR protein expression per osterix-positive cell. The above data established that calcium released by osteoclasts contributed to the regulation of coupled bone formation. CRISPR/Cas-9 knockout of Cav1.2 in osteoprogenitor cells cultured in basal calcium medium caused a >80% decrease in the expression of downstream osteogenic genes, emphasizing the large magnitude of the effect of calcium signaling. Thus, calcium signaling is a major regulator of coupled bone formation.

As the main cause of osteoporosis, abnormal activity of osteoclasts could disrupt the balance between bone resorption and formation. Moreover, up-regulation of nuclear factor-kappa ligand (RANKL) expression by chronic inflammation-mediated inflammatory factors might contribute to the differentiation of osteoclast precursor cells. Therefore, an anti-inflammatory agent named yangonin was presented for inhibiting osteoclast and relieving inflammatory osteoporosis through down-regulating inflammatory factors.We established a model of macrophage inflammation and then verified the anti-inflammatory effect of yangonin. The inhibitory effect of yangonin on osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining, Western blotting and quantitative real-time PCR (qRT-PCR). Finally, micro-CT, TRAP and hematoxylin-eosin (HE) staining were used to show the effect of yangonin on inflammatory osteoporosis in vivo.Our results suggested that yangonin was able to reduce the secretion of inflammatory factors, down-regulate osteoclast-related genes such as TRAP, RANKL, cathepsin K (CTSK) and nuclear factor-activated T-cell 1 (NFATc1). Furthermore, it was demonstrated that yangonin could suppress the function of inflammatory cytokines in osteoclast differentiation and reporting, wherein NF-κB, AKT and downstream c-Fos/NFATc1 signaling pathways were involved. In an in vivo study, we implied that yangonin has a relieving effect on inflammatory osteoporosis.Our research shows that yangonin down-regulates inflammatory factors and inhibits the bone-breaking effect of inflammation through NF-κB, AKT and downstream c-Fos/NFATc1 signaling pathways to achieve the purpose of treating inflammatory osteoporosis.

High serum levels of osteoprotegerin (OPG) are found in patients with obesity, type 2 diabetes, sepsis, or septic shock and are associated with a high mortality rate in stroke. The primary known function of OPG is to bind to the receptor activator of NF-κB ligand (RANKL), and by doing so, it inhibits the binding between RANKL and its receptor (RANK). TLR4 signaling in macrophages involves TRAF6 recruitment and contributes to low-grade chronic inflammation in adipose tissue. LPS is a classical activator of the TLR4 pathway and induces the expression of inflammatory cytokines in macrophages. We have previously observed that in the presence of RANKL, there is no LPS-induced activation of TLR4 in macrophages. In this study, we investigated the crosstalk between RANK and TLR4 pathways in macrophages stimulated with both RANKL and LPS to unveil the role of OPG in inflammatory processes. We found that RANKL inhibits TLR4 activation by binding to RANK, promoting the binding between TRAF6 and RANK, lowering TLR4 activation and the expression of proinflammatory mediators. Furthermore, high OPG levels aggravate inflammation by inhibiting RANKL. Our findings elect RANKL as a candidate for drug development as a way to mitigate the impact of obesity-induced inflammation in patients.

Icariin (ICA), isolated from Herba Epimedii, is a natural flavonoid glycoside that possesses antioxidant properties and inhibits osteoclastogenesis. However, the mechanism underlying osteoclastogenesis inhibition by ICA remains unclear. Here, we investigated the effects of ICA on receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells. ICA inhibited the expression of osteoclastogenesis-related genes in RAW264.7 cells induced by RANKL. ICA could inhibit osteoclastogenesis without inhibiting the viability of RAW264.7 cells. In addition, ICA inhibited reactive oxygen species production in RANKL-induced RAW264.7 cells. ICA reduced the expression of nuclear factor in activated T cells, cytoplasmic 1, and tartrate-resistant acid phosphatase, which are osteoclast-related molecules. Moreover, ICA decreased the expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX), specifically NOX1 and NOX4, in RANKL-induced RAW264.7 cells. Our findings suggest that ICA can be used as a potential therapeutic agent for osteolytic diseases such as osteoporosis.

In this experimental study, we aimed to investigate the specific value of receptor activator of nuclear factor kappa-Β ligand (RANKL) plasma level in osteomyelitis to show the bone destruction and to determine its correlation with classical markers of infection in mice model of osteomyelitis.Sixty Balb/c female mice (30 to 40 g weight, 3.5 to 4 month-old) were divided into two groups: Controls (n=15) and study group (n=45). All mice underwent tibial decortication and received an injection of sclerosing agent into the intramedullary cavity. The next process was proceeded in two steps to observe the detectability of osteomyelitis-induced bone destruction (step 1) and treatment response (step 2) using the variables examined in our study. In step 1, the study group received 1 mL solution containing Staphylococcus aureus (S. aureus) bacteria (2X108 per mL) into the intramedullary cavity. Five mice from each group were sacrificed every seven days for three weeks and tibia and blood samples were obtained. In step 2, the remaining 30 infected mice were further divided into two groups to investigate the possible value of RANKL plasma level as a marker of treatment response. Fifteen of these mice received teicoplanin 20 mg/kg for four weeks, while the rest did not receive antibiotics. Eight mice from each group were sacrificed at the end of the second week and the remaining 14 mice were sacrificed at the end of four weeks. Complete blood count, procalcitonin level, C-reactive protein (CRP), and RANKL concentrations were measured from blood samples of each sacrificed mouse.Median RANKL concentration of the control subjects was significantly higher than recipients of intervention at the first and third weeks in step 1 where bone destruction of osteomyelitis was examined. No significant changes occurred in groups receiving and not receiving antimicrobial treatment in terms of RANKL, CRP, and procalcitonin levels throughout four weeks in step 2. The RANKL concentration was significantly correlated with colony growth in subjects allocated to the S. aureus inoculation group (r=-0.547, p=0.035).The RANKL levels in mice with S. aureus osteomyelitis are not correlated with colony growth or other markers of inflammation and not useful for monitoring the response to antimicrobial treatment during osteomyelitis.

The capability of mesenchymal stem cells (MSCs) to repair bone damage and defects has long been investigated. The receptor activator of nuclear factor-kappa B (RANK), its ligand (RANKL) and the decoy receptor osteoprotegerin (OPG) axis is crucial to keep the equilibrium between osteoblastic and osteoclastic activity. Exendin-4 utilization increased bone formation and enhanced bone integrity. This study aimed to investigate the mentioned axis and determine the effect of exendin-4 upon adipose mesenchymal stem cells (Ad-MSCs) osteogenic differentiation. Ad-MSCs were isolated from rat epididymal fat, followed by characterization and then differentiation into osteocytes both in the presence or absence of exendin-4. Osteogenic differentiation was evaluated by alizarin red staining and the expression of osteogenic markers; using reverse transcriptase-quantitative polymerase chain reaction, western blotting and enzyme-linked immunoassay. MSCs derived from rat epididymal fat were isolated and characterized, along with their differentiation into osteocytes. The differentiated cells were alizarin red-stained, showing increased staining intensity upon addition of exendin-4. Moreover, the addition of exendin-4 elevated the messenger RNA expression levels of osteogenic markers; runt-related transcription factor-2 (RUNX-2), osteocalcin, and forkhead box protein O-1 while reducing the expression of the adipogenic marker peroxisome-proliferator-activated receptor-gamma. Exendin-4 addition elevated OPG levels in the supernatant of osteogenic differentiated cells. Moreover, exendin-4 elevated the protein levels of glucagon-like peptide-1 receptor and RUNX-2, while decreasing both RANK and RANKL. In conclusion, osteogenic differentiation of Ad-MSCs is associated with increased osteoblastic rather than osteoclastic activity. The findings of this study suggest that exendin-4 can enhance Ad-MSCs osteogenic differentiation partially through the RANK/RANKL/OPG axis.

Summary Background and objectives Cementoblasts can communicate with osteoclasts by synthesis and secretion of cytokines, such as RANKL, OPG, and M-CSF. Previously, we reported that irisin promotes the differentiation of cementoblasts, while the effect of irisin on cementoblast-mediated osteoclastogenesis remains inconclusive. This study aimed to explore the effect of irisin on the expression of osteoclastogenesis-related cytokines in cementoblasts. Material and methods An immortalized murine cementoblast cell line OCCM-30 was used. Immunofluorescence and Western Blot were performed to identify the expression of irisin receptor integrin alphaV and the activation of its downstream signals in OCCM-30 cells. Cells were treated with irisin (100 ng/ml) for various time lengths ranging from 0 to 72 hours, and then qRT-PCR was used to detect the expression of osteoclastogenesis-related genes, including RANKL, IL-6, M-CSF, OPG, Wnt5A, Sema3A. Cells were also incubated with irisin in a series of concentrations (0–200 ng/ml) for 24 hours, and then qRT-PCR and ELISA were performed to examine the above osteoclastogenesis-related cytokines. Results Irisin receptor integrin alphaV was expressed in OCCM-30 cells and its downstream signaling pathways were markedly activated by irisin. Both qRT-PCR and ELISA results revealed that RANKL and IL-6 were up-regulated by irisin while M-CSF, OPG, Wnt5A, Sema3A remained unaffected. Conclusions OCCM-30 cells were responsive to the stimulation of irisin. The expression of RANKL and IL-6 was significantly enhanced by irisin, suggesting a possible promotive effect on cementoblast-mediated osteoclastogenesis.

Accumulating evidence suggests that Rab GTPases representing the largest branch of Ras superfamily have recently emerged as the core factors for the regulation of osteoclastogenesis through modulating vesicular transport amongst specific subcellular compartments. Among these, Rab34 GTPase has been identified to be important for the post-Golgi secretory pathway and for phagocytosis; nevertheless, its specific role in osteoclastogenesis has been completely obscure. Here, upon the in vitro model of osteoclast formation derived from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we reveal that Rab34 regulates osteoclastogenesis bidirectionally. More specifically, Rab34 serves as a negative regulator of osteoclast differentiation by promoting the lysosome-induced proteolysis of two osteoclastogenic surface receptors, c-fms and RANK, via the axis of early endosomes-late endosomes-lysosomes, leading to alleviate the transcriptional activity of two of the master regulator of osteoclast differentiation, c-fos and NFATc-1, eventually attenuating osteoclast differentiation and bone resorption. Besides, Rab34 plays a crucial role in modulating the secretory network of lysosome-related proteases including matrix metalloprotease 9 and Cathepsin K across the ruffled borders of osteoclasts, contributing to the regulation of bone resorption.

Inflammation is a major risk factor for osteoporosis, and reducing inflammatory levels is important for the prevention of osteoporosis. Although nuclear receptor 77 (Nur77) protects against inflammation in a variety of diseases, its role in osteoporosis is unknown. Therefore, the main purpose of this study was to investigate the osteoprotective and anti-inflammatory effects of Nur77. The microCT and haematoxylin and eosin staining results indicated that knockout of Nur77 accelerated femoral bone loss in mice. The enzyme-linked immunosorbent assay (ELISA) results showed that knockout of Nur77 increased the serum levels of hsCRP and IL-6. The expression levels of NF-κB, IL-6, TNF-α and osteoclastogenesis factors (TRAP, NFATC1, Car2, Ctsk) in the femurs of Nur77 knockout mice were increased significantly. Furthermore, in vitro, shNur77 promoted the differentiation of RAW264.7 cells into osteoclasts by activating NF-κB, which was confirmed by PDTC treatment. Mechanistically, Nur77 inhibited osteoclast differentiation by inducing IκB-α and suppressing IKK-β. In RAW264.7 cells, overexpression of Nur77 alleviated inflammation induced by siIκB-α, while siIKK-β alleviated inflammation induced by shNur77. Consistent with the in vivo studies, we found that compared with control group, older adults with high serum hsCRP levels were more likely to suffer from osteoporosis (OR = 1.76, p 0.001). Our data suggest that Nur77 suppresses osteoclast differentiation by inhibiting the NF-κB signalling pathway, strongly supporting the notion that Nur77 has the potential to prevent and treat osteoporosis.

Objective: Enamel matrix derivative (EMD) is widely used under the brand name Emdogain® to promote periodontal regeneration in surgical treatment of periodontitis and peri-implantitis. The molecular mechanisms are unclear, but it has been proposed that EMD has stimulatory effects on the root cementum and periodontal ligament cells. Since dental implants lack these structures, we hypothesized that EMD-induced bone gain involve interactions with osteoclast precursor cells, with consequent inhibitory effect on osteoclast formation and/or activity. The aim was to evaluate this hypothesis. Material and methods: Primary mouse bone marrow macrophages (BMMs) and human peripheral blood monocytes were cultured in the presence of receptor activator nuclear factor-κB ligand (RANKL) to stimulate osteoclast formation. A purified Emdogain® fraction was added to the cell cultures and the effect on number and size of newly formed osteoclasts were evaluated. In cultures on natural bone slices, bioanalytical methods were used to assay osteoclast number and bone resorption. Results: EMD had a negative effect on osteoclastogenesis in mouse cultures on plastic surface, whereas addition of EMD to osteoclast precursor cells on bone substrate did not affect osteoclast formation or bone resorption. Conclusions: The results on natural bone matrix contradict a direct effect of EMD on osteoclast precursor cells. Originally published in thesis with title: Enamel matrix derivative does not affect osteoclast formation or bone resorption in mouse bone marrow macrophage cultures.

The fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling pathway plays important roles in the development and growth of the skeleton. Apert syndrome caused by gain-of-function mutations of FGFR2 results in aberrant phenotypes of the skull, midface, and limbs. Although short limbs are representative features in patients with Apert syndrome, the causative mechanism for this limb defect has not been elucidated. Here we quantitatively confirmed decreases in the bone length, bone mineral density, and bone thickness in the Apert syndrome model of gene knock-in Fgfr2

The long-term inhibition of bone resorption suppresses new bone formation because these processes are coupled during physiological bone remodeling. The development of anti-bone-resorbing agents that do not suppress bone formation is urgently needed. We previously demonstrated that Wnt5a-Ror2 signaling in mature osteoclasts promoted bone-resorbing activity through protein kinase N3 (Pkn3). The p38 MAPK inhibitor SB202190 reportedly inhibited Pkn3 with a low Ki value (0.004 μM). We herein examined the effects of SB202190 on osteoclast differentiation and function in vitro and in vivo.Bone marrow cells were cultured in the presence of M-csf and GST-Rankl to differentiate into multinucleated osteoclasts. Osteoclasts were treated with increasing concentrations of SB202190. For in vivo study, 10-week-old female mice were subjected to ovariectomy (OVX). OVX mice were intraperitoneally administered with a Pkn3 inhibitor at 2 mg/kg or vehicle for 4 weeks, and bone mass was analyzed by micro-CT.SB202190 suppressed the auto-phosphorylation of Pkn3 in osteoclast cultures. SB202190 significantly inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation. SB202190 reduced c-Src activity in osteoclast cultures without affecting the interaction between Pkn3 and c-Src. A treatment with SB202190 attenuated OVX-induced bone loss without affecting the number of osteoclasts or bone formation by osteoblasts.Our results showed that Pkn3 has potential as a therapeutic target for bone loss due to increased bone resorption. SB202190 is promising as a lead compound for the development of novel anti-bone-resorbing agents.

A number of studies demonstrated that some tea extracts exert inhibitory effects on osteoclastogenesis induced by receptor activator of nuclear factor κB ligand (RANKL). However, the effect of purple tea, a famous tea in China, on osteoclastogenesis remains unclear. In this study, a water-based purple tea extract (PTE) was found to suppress osteoclast formation, osteoclastic resorption pit area formation, and F-actin ring formation within RANKL-stimulated bone marrow macrophages (BMMs). Furthermore, our results demonstrated that PTE could inhibit expression of master transcription factors NFATc1 and c-Fos and their target genes

Free cholesterol acts as an endogenous agonist for estrogen-related receptor α (ERRα), a nuclear receptor that regulates osteoclastogenesis. Because stimulation of macrophages with receptor activator of nuclear factor κB ligand (RANKL) induces an overload of free cholesterol and activates ERRα, we hypothesized that direct removal of cellular cholesterol would suppress osteoclastogenesis. In this study, the effect of 2-hydroxypropyl β-cyclodextrin (HP-β-CD), a highly water-soluble cyclic glucopyranose, and β-CD-threaded polyrotaxanes (PRXs), supramolecular polymers designed to release threaded β-CDs in acidic lysosomes, on RANKL-induced cholesterol overload and osteoclast differentiation of murine macrophage-like RAW264.7 cells were investigated. PRXs suppressed RANKL-induced cholesterol overload. Additionally, RANKL-induced osteoclast differentiation of RAW264.7 cells was inhibited by PRXs. In contrast, HP-β-CD did not reduce cholesterol levels or inhibit osteoclast differentiation in RAW264.7 cells. Gene expression analysis of osteoclast markers suggested that PRXs suppress only the early stage of osteoclast differentiation, as PRXs cannot be internalized into multinucleated osteoclasts. However, modification of PRXs with cell-penetrating peptides facilitated their cellular uptake into multinucleated osteoclasts and inhibited osteoclast maturation. Thus, PRXs are promising candidates for inhibiting osteoclast differentiation by suppressing cholesterol overload and may be useful for treating osteoporosis or other bone defects caused by the overactivity of osteoclasts.

By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa-Β ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of −6.9, −7.6, −7.1, −6.9, −6.7, and −7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 μg / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 μg / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss. Resumo Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa-Β [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de −6,9, −7,6, −7,1, −6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow-derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 μg / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 μg / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.

Background: Dickkopf-1 (DKK1) regulates bone formation by inhibiting canonical Wnt/β-catenin pathway signaling, and indirectly enhances osteoclastic activity by altering the expression ratio of receptor activator of nuclear factor-κB ligand (RANKL) relative to osteoprotegerin (OPG). However, it is difficult to explain continued bone loss after allogeneic stem cell transplantation (allo-SCT) in terms of changes in only RANKL and OPG. Few studies have evaluated changes in DKK1 after allo-SCT.Methods: We prospectively enrolled 36 patients with hematologic malignancies who were scheduled for allo-SCT treatment. Serum DKK1, OPG, and RANKL levels were measured before (baseline), and at 1, 4, 12, 24, and 48 weeks after allo-SCT treatment. Bone mineral density (BMD) was assessed using dual-energy X-ray absorptiometry before (baseline) and 24 and 48 weeks after allo-SCT treatment.Results: After allo-SCT treatment, the DKK1 level decreased rapidly, returned to baseline during the first 4 weeks, and remained elevated for 48 weeks (PPP

Osteoporosis is a systemic skeletal disorder characterized by reduced bone mineral density (BMD) and bone quality and increased bone porosity, which increase the risk of bone fracture. Inflammation, one of the important mechanisms related to aging, is associated with osteoporosis. Treatment with anti-inflammatory agents is effective for alleviating senile osteoporosis. Alginate oligosaccharide (AOS) can prevent and treat diseases related to inflammation, oxidative stress, and immunity. This study evaluates the effect of AOS on osteoporosis and investigates the underlying mechanism. Osteoporosis model was induced by D-galactose (D-gal) (200 mg kg-1 day-1 ) for eight weeks. Three groups were administered via AOS (50, 100, and 150 mg kg-1 day-1 ) for four weeks, while a control group received sterile water (5 ml kg-1 day-1 ) for 8 weeks. The results showed that AOS improved bone density and bone microstructure in D-gal-induced osteoporosis mice. AOS inhibited osteoclast proliferation, probably through the suppression of receptor activator of nuclear factor-kappa B ligand (RANKL)-associated nuclear factor kappa B (NF-κB) and c-Fos signaling pathway. AOS also increased osteoprotegerin (OPG) expression and competitively inhibited the binding between RANK and RANKL in senile osteoporosis. Further, AOS decreased the secretion of serum osteocalcin and reduced bone conversion. Together, these results demonstrate the anti-osteoporosis activity of AOS in mice with osteoporosis.

The possible mechanisms underlying the quantitative and qualitative effects of cinacalcet on bone were explored in a chronic kidney disease-mineral and bone disorder (CKD-MBD) mouse model in relation to the influence of the interactions among the osteoclast (OC) endoplasmic reticulum (ER) stress, autophagy and apoptosis pathways on OC differentiation. Body weight and biochemical parameters improved significantly in the CKD + cinacalcet groups compared to the CKD group. Micro-computed tomography (μCT) revealed both cortical and trabecular parameters deteriorated significantly in the CKD group and were reversed by cinacalcet in a dose-dependent manner. Nanoindentation analysis of bone quality proved that both cortical hardness and elastic modulus improved significantly with high dose cinacalcet treatment. In vitro studies revealed that cinacalcet inhibited receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB (RANK)-induced OC differentiation in a concentration-dependent manner through a close interaction between activation of caspase-related apoptosis, reversal of OC autophagy through the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK) pathways, and attenuation of the OC ER stress/CREBH/NFATc1 signaling pathway. Cinacalcet improves both bone quantity and bone quality in CKD mouse model and inhibits OC differentiation through regulation of the interactions among the apoptosis, ER stress, and autophagy pathways within OCs. © 2021 American Society for Bone and Mineral Research (ASBMR).

Cadmium (Cd) contamination in the environment is a major public health concern since it has been linked to osteoporosis and other bone deformities. Linarin is a flavonoid glycoside, and it can promote osteoblastogenesis. This research aimed to investigate the potential role of linarin against Cd-exposed bone deformations in mice model. In our research, male mice were randomly allocated into four groups: control, Cd-exposed, and Cd + linarin (20 and 40mg/kg/bw, respectively). Linarin prevented body weight loss, increased serum calcium (Ca) and phosphorus (P), and bone alkaline phosphatase (BAP) levels in Cd-exposed groups. Furthermore, linarin treatment at 20 and 40mg/kg/bw significantly decreased RANK and OPG, resulting in an increase in RANKL mRNA levels and protein distribution in the bone of Cd-exposed mice. In addition, the bone of Cd-exposed mice administered with linarin showed higher TRAP, NFATc1, MMP9, and RUNX2 mRNA levels and protein distribution. Linarin significantly decreased oxidative stress in Cd-exposed mice bone by decreasing MDA, a lipid peroxidation product. Moreover, linarin protects Cd-exposed mice antioxidant enzymes by increasing bone SOD, CAT, and GPx levels. Besides, linarin suppresses alterations in the inflammatory system, i.e., NF-κB p65/IKKβ, by reducing NF-κB p65, IKKβ, IL-6, and TNF-α in the bone of Cd-exposed animals. This study concluded that linarin has potential to cure osteoporosis in Cd-exposed mice by reducing oxidative stress and inflammation and modulating the RANK/RANKL/OPG pathway.

Osteoporosis is an age‐related disease characterized by low mineral density, compromised bone strength and increased risk of fragility fracture. Most agents for treating osteoporosis focus primarily on anti‐resorption by inhibiting osteoclast activity. Bisphosphonate (BP) is a potent anti‐resorptive agent that has been used clinically for decades and is proven to be effective. However, BP has a variety of side effects and is far from being an ideal anti‐osteoporosis agent. BP selectively binds to calcium crystals, which are subsequently taken up or released by osteoclasts. Based on the action of BP, we previously demonstrated the inhibitory effect of a novel bone‐targeting BP derivative, bisphosphonate‐enoxacin (BE). In the current study, we used bone marrow‐derived osteoclast cultures to further assess the inhibitory effect of BE on osteoclastogenesis and employed reverse transcription PCR and real‐time PCR to examine expression of osteoclast‐specific genes. Additionally, we used bone resorption and F‐actin immunofluorescence assays to evaluate the effect of BE on osteoclast function and investigated the potential mechanisms affecting osteoclast differentiation and function in vitro. Furthermore, an ovariectomized (OVX) rat model was established to evaluate the therapeutic effects of BE on preventing bone loss. Results showed that BE exerted potent inhibitory effects on osteoclast formation and bone resorption by specifically abrogating RANKL‐induced JNK signalling, and that it preserved OVX rat bone mass in vivo without any notable side effects. Collectively, these results indicated that the BP derivative BE may have significant potential as a treatment for osteoporosis and other osteolytic diseases.

Peri‑implantitis, which is characterized by peri‑implant mucositis and alveolar bone resorption, significantly shortens the service life of dental implants. Melatonin is well-known for its anti-inflammatory and osteoprotective activities. Nevertheless, the effects and mechanisms of melatonin to prevent peri‑implantitis remain unknown. In this study, the lipopolysaccharide-induced peri‑implantitis model was established after the titanium implants were osseointegrated, and the rats received daily administrations of melatonin. The gingival fibroblasts and osteoclasts/osteoblasts were also co-cultured to simulate the inflammatory environment in vitro. We found that prophylactic administration of melatonin decreased proinflammatory cytokine levels and osteoclast numbers, attenuated alveolar bone resorption, and reduced the incidence of peri‑implantitis in vivo. Furthermore, melatonin suppressed osteoclastic formation and function in the inflammatory co-culture environment, while melatonin promoted osteoblastic differentiation and function in the in vitro model. Mechanistically, melatonin reduced TLR4 protein levels, and inhibited activation of NF-κB to downregulate the levels of TNF, IL-1β, and IL-6. These data showed that melatonin was a potent agent to prevent peri‑implantitis through inhibiting TLR4/NF-κB signaling. Our findings provide a novel strategy to prevent peri‑implantitis, and expand the applications of melatonin. Statement of significance Dental implants have become the first choice for restoring partial and full edentulism, but its service life is seriously affected by peri‑implantitis. Exploration of novel and effective approaches to prevent peri‑implantitis is an important and urgent need. In the present study, we have reported for the first time that prophylactic administration of melatonin delayed the occurrence and reduced the incidence of peri‑implantitis by decreasing proinflammatory cytokine levels, inhibiting osteoclastogenesis, and promoting osteogenesis. The study is expected to have an important significance on the prevention of peri‑implantitis.

BACKGROUND The gut microbiota (GM)-bone axis has emerged as a crucial mediator of bone homeostasis. Estrogen deficiency-induced bone loss is closely associated with an altered GM. However, the underlying mechanisms remain unclear. OBJECTIVES We sought to explore the putative effects of GM on estrogen deficiency-induced bone loss and determine a potential mechanism. METHODS Fecal samples collected from postmenopausal women with osteoporosis (PMO) and with normal bone mass (PMN) were examined by 16S ribosomal RNA (rRNA) gene sequencing and analysis. Prevotella histicola, a typical species of Prevotella, was orally given to female C57BL6/J mice after ovariectomy [ovariectomized (OVX)]. The primary outcomes were changes in bone microstructures as measured by micro-computed tomography scanning and bone histomorphometry analysis. Secondary outcomes included changes in osteoclast activity, the expression of osteoclastogenic cytokines, and gut permeability, which were measured by ELISA, qRT-PCR, western blot, and immunofluorescence. RESULTS As demonstrated through 16S rRNA gene sequencing and analysis, the GM in the PMO group featured a significantly decreased proportion of the genus Prevotella in comparison with that in the PMN group (∼60%, P

Purpose Emerging evidence suggests that the progesterone-mediated receptor activator of nuclear factor κB (RANK)/soluble RANK ligand (sRANKL)/osteoprotegerin (OPG) pathway plays an important role in mammary carcinogenesis and is hyperactivated in germline (g)BRCA1/2 mutation carriers. We analyzed the effects of a 3-month intensive lifestyle intervention within the LIBRE-1 study on the serum levels of OPG and sRANKL and hypothesized that the intervention program provides a beneficial impact on the biomarkers by increasing OPG and reducing sRANKL serum concentrations. Methods Serum levels of OPG and sRANKL of 49 gBRCA1/2 mutation carriers were quantified using enzyme-linked immunosorbent assays. We used previously collected blood samples from participants of the prospective LIBRE-1 study, who were randomized into an intervention group (IG), increasing physical activity and adherence to the Mediterranean diet (MedD) through supervised sessions from study entry to the first study visit after 3 months and a usual-care control group (CG). Differences in biomarker levels before and after the 3-month intervention were tested within and between study groups. Results The lifestyle intervention resulted in a significant increase in OPG for participants in both the IG (q = 0.022) and CG (q = 0.002). sRANKL decreased significantly in the IG (q = 0.0464) and seemed to decrease in the CG (q = 0.5584). An increase in the intake of Omega-3 polyunsaturated fatty acids was significantly associated with an increase in OPG (r = 0.579, q = 0.045). Baseline serum levels of sRANKL were a strong predictor for the change of sRANKL in the course of the intervention (ß-estimate = − 0.70; q = 0.0018). Baseline physical fitness (assessed as VO2peak) might predict the change of OPG in the course of the intervention program (ß-estimate = 0.133 pg/ml/ml/min/kg; p = 0.0319; q = 0.2871). Conclusion Findings from this pilot study seem to confirm our hypothesis by showing an increase in OPG and decrease in sRANKL over a 3-month lifestyle intervention and suggest that increased physical activity and adherence to the MedD are potent modulators of the biomarkers OPG and potentially sRANKL.

Objectives To evaluate the effects of submucosally administered platelet-rich plasma (PRP) on the rate of maxillary canine retraction. Levels of soluble receptor activator of nuclear factor-κb ligand (sRANKL) and osteoprotegerin (OPG) in the gingival crevicular fluid (GCF) were also measured over 2 months. Materials and Methods This split-mouth trial involved 20 sites in 10 subjects randomly assigned to PRP (experimental) side and control side. After alignment, the freshly prepared PRP was injected submucosally distal to the experimental side maxillary canine, and retraction was performed using NiTi closed-coil springs (150 g) on 0.019 × 0.025-inch stainless steel wire. The rate of canine movement was assessed using digital model superimposition at 0, 30, and 60 days. The OPG and sRANKL were assayed using enzyme-linked immunosorbent assay from GCF collected at 0, 1, 7, 21, 30, and 60 days. Results Twenty sites were analyzed using paired t test. The rate of tooth movement increased significantly by 35% on the PRP side compared with the control side in the first month (P = .0001) and by 14% at the end of the second month (P = .015). Using the Mann–Whitney U test, OPG levels were found to be significantly decreased on the 7th (P = .003) and 30th day on the PRP side (P = .01), while sRANKL became detectable by the third week postinjection on the PRP side (P = .069). Conclusions Submucosal injection of platelet-rich plasma significantly increased tooth movement during the 60-day observation period. Local injection of PRP significantly altered the levels of OPG and sRANKL in GCF.

OBJECTIVE: The expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in human periodontal ligament cells (HPDLCs) was investigated by cell culture under continuous static pressure. METHODS: HPDLCs were primarily cultured by tissue explant method and divided into three groups: group A (13-18 years old), group B (19-29 years old), and group C (30-44 years old). CCK-8 was used to detect the proliferation of HPDLCs. The senescence of HPDLCs was detected by senescence-associated β-galactosidase staining. Cells in the three groups were respectively given 0, 1.5, 3, 6, 12, 24, 48, and 72 h of continuous static pressure in vitro. The expression of OPG and RANKL in the supernatant was detected by enzyme-linked immunosorbent assay. RESULTS: After continuous static pressure in vitro for stimulation, the expression of OPG and RANKL changed. The expression of OPG increased with time and age (P

Isoorientin has many biological activities, including antioxidant, anti-inflammatory, antitumor. However, the effect of isoorientin on postmenopausal osteoporosis remains unclear.To evaluate the effect of isoorientin on postmenopausal osteoporosis.Sprague-Dawley rats were divided into five groups (Isoorientin improved the bone mineral density of the lumbar vertebrae (2.01 ± 0.05 g/cmIsoorientin ameliorates osteoporosis

The expression of GPR84 in bone marrow-derived monocytes/macrophages (BMMs) can inhibit osteoclast formation; however, its role in bone metastasis of colorectal cancer (CRC) is still unknown. To investigate the effects of GPR84 on bone metastasis of CRC, the murine CRC cell line MC-38 was injected into tibial bone marrow. We found that the expression of GPR84 in BMMs was gradually downregulated during bone metastasis of CRC, and the activation of GPR84 significantly prevented osteoclastogenesis in the tumor microenvironment. Mechanistically, the MAPK pathway mediated the effects of GPR84 on osteoclast formation. Moreover, we found that IL-11 at least partly inhibited the expression of GPR84 in the tumor microenvironment through the inactivation of STAT1. Additionally, activation of GPR84 could prevent osteolysis during bone metastasis of CRC. Our results suggest that CRC cells downregulate the expression of GPR84 in BMMs to promote osteoclastogenesis in an IL-11-dependent manner. Thus, GPR84 could be a potential therapeutic target to attenuate bone destruction induced by CRC metastasis.

Background Postmenopausal bone loss, mainly caused by excessive bone resorption mediated by osteoclasts, has become a global public health burden. Metformin, a hypoglycemic drug, has been reported to have beneficial effects on maintaining bone health. However, the role and underlying mechanism of metformin in ovariectomized (OVX)-induced bone loss is still vague. Results In this study, we demonstrated for the first time that metformin administration alleviated bone loss in postmenopausal women and ovariectomized mice, based on reduced bone resorption markers, increased bone mineral density (BMD) and improvement of bone microstructure. Then, osteoclast precursors administered metformin in vitro and in vivo were collected to examine the differentiation potential and autophagical level. The mechanism was investigated by infection with lentivirus-mediated BNIP3 or E2F1 overexpression. We observed a dramatical inhibition of autophagosome synthesis and osteoclast formation and activity. Treatment with RAPA, an autophagy activator, abrogated the metformin-mediated autophagy downregulation and inhibition of osteoclastogenesis. Additionally, overexpression of E2F1 demonstrated that reduction of OVX-upregulated autophagy mediated by metformin was E2F1 dependent. Mechanistically, metformin-mediated downregulation of E2F1 in ovariectomized mice could downregulate BECN1 and BNIP3 levels, which subsequently perturbed the binding of BECN1 to BCL2. Furthermore, the disconnect between BECN1 and BCL2 was shown by BNIP3 overexpression. Conclusion In summary, we demonstrated the effect and underlying mechanism of metformin on OVX-induced bone loss, which could be, at least in part, ascribed to its role in downregulating autophagy during osteoclastogenesis via E2F1-dependent BECN1 and BCL2 downregulation, suggesting that metformin or E2F1 inhibitor is a potential agent against postmenopausal bone loss.