RANKL-induced Differentiation Research Articles

Breast cancer cells promote osteoclast differentiation in an MRTF-dependent paracrine manner.

Bone is a frequent site for breast cancer metastasis. Conditioning of the local tumor microenvironment (TME) through crosstalk between tumor cells and bone resident cells in the metastatic niche is a major driving force for bone colonization of breast cancer cells. The vast majority of breast cancer-associated metastasis is osteolytic in nature, and RANKL-induced differentiation of bone marrow-derived macrophages to osteoclasts (OCLs) is a key requirement for osteolytic metastatic growth of cancer cells. In this study, we demonstrate that breast cancer cell-secreted factors stimulate RANKL-induced OCL differentiation of BMDMs requiring the function of Myocardin-related transcription factor (MRTF) in tumor cells. This is partly attributed to the critical role of MRTF in maintaining the basal cellular expression of connective tissue growth factor (CTGF), a pro-osteoclastogenic matricellular factor known to promote bone metastasis in human breast cancer. Supporting these in vitro findings, bioinformatics analyses of multiple human breast cancer transcriptome datasets reveal a strong positive correlation between CTGF expression and MRTF gene signature further establishing the relevance of our findings in a human disease context. By Luminex analyses, we show that MRTF depletion in breast cancer cells has a broad impact on OCL-regulatory cell-secreted factors that extends beyond CTGF. These findings, taken together with demonstration of MRTF-dependence for bone colonization breast cancer cells in vivo, suggest that MRTF inhibition could be an effective strategy to diminish OCL formation and skeletal involvement in breast cancer. In summary, this study highlights a novel tumor-extrinsic function of MRTF relevant to breast cancer metastasis.

Epiberberine inhibits bone metastatic breast cancer-induced osteolysis

Ethnopharmacological relevanceThe anti-tumor related diseases of Coptidis Rhizoma (Huanglian) were correlated with its traditional use of removing damp-heat, clearing internal fire, and counteracting toxicity. In the recent years, Coptidis Rhizoma and its components have drawn extensive attention toward their anti-tumor related diseases. Besides, Coptidis Rhizoma is traditionally used as an anti-inflammatory herb. Epiberberine (EPI) is a significant alkaloid isolated from Coptidis Rhizoma, and exhibits multiple pharmacological activities including anti-inflammatory. However, the effect of epiberberine on breast cancer and the inflammatory factors of metastatic breast cancer-induced osteolysis has not been demonstrated clearly. Aim of the studyBone metastatic breast cancer can lead to osteolysis via inflammatory factors-induced osteoclast differentiation and function. In this study, we try to analyze the effect of epiberberine on breast cancer and the inflammatory factors of metastatic breast cancer-induced osteolysis. MethodsTo evaluate whether epiberberine could suppress bone metastatic breast cancer-induced osteolytic damage, healthy female Balb/c mice were intratibially injected with murine triple-negative breast cancer 4T1 cells. Then, we examined the inhibitory effect and underlying mechanism of epiberberine on breast cancer-induced osteoclastogenesis in vitro. Xenograft assay was used to study the effect of epiberberine on breast cancer cells in vivo. Moreover, we also studied the inhibitory effects and underlying mechanisms of epiberberine on RANKL-induced osteoclast differentiation and function in vitro. ResultsThe results show that epiberberine displayed potential therapeutic effects on breast cancer-induced osteolytic damage. Besides, our results show that epiberberine inhibited breast cancer cells-induced osteoclast differentiation and function by inhibiting secreted inflammatory cytokines such as IL-8. Importantly, we found that epiberberine directly inhibited RANKL-induced differentiation and function of osteoclast without cytotoxicity. Mechanistically, epiberberine inhibited RANKL-induced osteoclastogensis via Akt/c-Fos signaling pathway. Furthermore, epiberberine combined with docetaxel effectively protected against bone loss induced by metastatic breast cancer cells. ConclusionsOur findings suggested that epiberberine may be a promising natural compound for treating bone metastatic breast cancer-induced osteolytic damage by inhibiting IL-8 and is worthy of further exploration in preclinical and clinical trials.

Icariin regulates RANKL-induced osteoclast differentiation via the ERα/c-Src/RANK signaling

Osteoporosis (OP) is a common metabolic bone disease. Excessive osteoclastic activity significantly contributes to the development of OP. Icariin (ICA) is a flavonol glycoside derived from herbal plants and possesses curative effects on postmenopausal OP and bone fracture. This study aimed to investigate the effects of ICA on osteoclast differentiation induced by receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) and the involvement of estrogen receptor α (ERα) and RANK signaling cascade in this process. RANKL was used to induce the differentiation of RAW264.7 cells to into osteoclasts. Small interfering RNA technique was used to knockdown ERα in cells. Cell counting kit-8 assay was performed to determine the cytotoxicity of ICA. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells was quantified by TRAP staining. RANKL induced the differentiation of RAW264.7 cells into osteoclasts, while ICA abolished the pro-osteoporotic effect of RANKL. Moreover, ERα knockdown abolished the effects of ICA on RANKL-induced osteoclastogenesis. Further exploration revealed that ICA inhibited the phosphorylation of c-Src in osteoclasts via regulating ERα, while inactivation of c-Src reversed ERα knockdown-promoted osteoclastogenesis. Lastly, ICA inhibited the activation of the mitogen-activated protein kinase signaling pathway and downregulated the expressions of target osteoclastogenic proteins in RANKL-treated RAW 264.7 cells, while ERα knockdown almost completely diminished the effects of ICA. ICA inhibited RANKL-induced osteoclast differentiation via regulating the ERα/c-Src/RANK signaling. These findings elucidated a novel mechanism by which ICA exerts an anti-osteoporotic effect.

Myeloid zinc finger 1 knockdown promotes osteoclastogenesis and bone loss in part by regulating RANKL-induced ferroptosis of osteoclasts through Nrf2/GPX4 signaling pathway.

The overactivation of the osteoclasts is a crucial pathological factor in the development of osteoporosis. MZF1, belonging to the scan-zinc finger family, plays a significant role in various processes associated with tumor malignant progression and acts as an essential transcription factor regulating osteoblast expression. However, the exact role of MZF1 in osteoclasts has not been determined. In this study, the purpose of our study was to elucidate the role of MZF1 in osteoclastogenesis. First, we established MZF1-deficient female mice and evaluated the femur bone phenotype by micro-computed tomography and histological staining. Our findings indicate that MZF1-/- mice exhibited a low bone mass osteoporosis phenotype. RANKL could independently induce the differentiation of RAW264.7 cells into osteoclasts, and we found that the expression level of MZF1 protein decreased gradually. Then, the CRISPR/Cas 9 gene-editing technique was used to build a RAW264.7 cell model with MZF1 knockout, and RANKL was used to independently induce MZF1-/- and wild-type cells to differentiate into mature osteoclasts. Tartrate-resistant acid phosphatase staining and F-actin fluorescence results showed that the MZF1-/- group produced more tartrate-resistant acid phosphatase-positive mature osteoclasts and larger actin rings. The expression of osteoclast-associated genes (including tartrate-resistant acid phosphatase, CTSK, c-Fos, and NFATc1) was evaluated by reverse transcription quantitative polymerase chain reaction and Western blot. The expression of key genes of osteoclast differentiation in the MZF1-/- group was significantly increased. Furthermore, we found that cell viability was increased in the early stages of RANKL-induced cell differentiation in the MZF1-/- group cells. We examined some prevalent ferroptosis markers, including malondialdehyde, glutathione, and intracellular Fe, the active form of iron in the cytoplasm during the early stages of osteoclastogenesis. The results suggest that MZF1 may be involved in osteoclast differentiation by regulating RANKL-induced ferroptosis of osteoclasts. Collectively, our findings shed light on the essential involvement of MZF1 in the regulation of osteoclastogenesis in osteoporosis and provide insights into its potential underlying mechanism.

Epigenetic landscape analysis reveals the significance of early reduced chromatin accessibility in osteoclastogenesis.

Recently improved techniques could provide snapshots of chromatin structure generated based on chromatin accessibility. Since chromatin accessibility determines transcriptional potential, it has been attempted in a variety of cell systems. However, there has been no genome-wide analysis of chromatin accessibility for the entire murine osteoclast (OC) differentiation process. We performed an Assay for Transposase-Accessible Chromatin (ATAC)-sequencing (seq) during RANKL-induced OC differentiation and found that global chromatin accessibility decreased, especially early in OC differentiation. The global histone H3K27Ac level, an active histone modification mark, was diminished during OC differentiation by western blot and histone extract experiments. Its genomic enrichment was also reduced based on publicly available H3K27Ac chromatin immunoprecipitation (ChIP)-seq data. ATAC-seq and H3K27Ac ChIP-seq data demonstrated that RANKL induced a less accessible chromatin state during OC differentiation. Restoration of reduced H3K27Ac, presumably representing accessible states upon acetate treatment, suppresses OC differentiation by provoking immune-related gene expression. Subsequential integrative analysis of ATAC-seq, RNA-seq after acetate treatment, and H3K27Ac ChIP-seq reveals that Irf8 and its downstream targets are the most vulnerable to chromatin accessibility changes and acetate supplementation. Taken together, our study generated chromatin accessibility maps during the whole OC differentiation and suggested perturbation of chromatin accessibility might be a potential therapeutic strategy for excessive OC diseases.

Extracellular Vesicles from Human Adipose Tissue-Derived Mesenchymal Stem Cells Suppress RANKL-Induced Osteoclast Differentiation via miR122-5p.

Researchers are increasingly interested in cell therapy using mesenchymal stem cells (MSCs) as an alternative remedy for osteoporosis, with fewer side effects. Thus, we isolated and characterized extracellular vesicles (EVs) from human adipose tissue-derived MSCs (hMSCs) and investigated their inhibitory effects on RANKL-induced osteoclast differentiation. Purified EVs were collected from the supernatant of hMSCs by tangential flow filtration. Characterization of EVs included typical evaluation of the size and concentration of EVs by nanoparticle tracking analysis and morphology analysis using transmission electron microscopy. hMSC-EVs inhibited RANKL-induced differentiation of bone marrow-derived macrophages (BMDMs) into osteoclasts in a dose-dependent manner. F-actin ring formation and bone resorption were also reduced by EV treatment of osteoclasts. In addition, EVs decreased RANKL-induced phosphorylation of p38 and JNK and expression of osteoclastogenesis-related genes in BMDMs treated with RANKL. To elucidate which part of the hMSC-EVs plays a role in the inhibition of osteoclast differentiation, we analyzed miRNA profiles in hMSC-EVs. The results showed that has-miR122-5p was present at significantly high read counts. Overexpression of miR122-5p in BMDMs significantly inhibited RANKL-induced osteoclast differentiation and induced defects in F-actin ring formation and bone resorption. Our results also revealed that RANKL-induced phosphorylation of p38 and JNK and osteoclast-specific gene expression was decreased by miR122-5p transfection, which was consistent with the results of hMSC-EVs. These findings suggest that hMSC-EVs containing miR122-5p inhibit RANKL-induced osteoclast differentiation via the downregulation of molecular mechanisms and could be a preventive candidate for destructive bone diseases.

Wheat seedling extract and its constituents attenuate RANKL-induced differentiation and fusion of osteoclasts and bone resorption

The occurrence of osteoporosis gradually increases within the aging population. As the side effects of therapeutic agents currently used for osteoporosis are increasing, the development of preventive and therapeutic agents derived from natural products without any long-term side effects is important. Here, we investigated the effect of wheat seedling extract (WSE) on the RANKL-mediated differentiation, fusion, and function of osteoclasts. WSE inhibited the differentiation of RANKL-induced bone marrow macrophages and phosphorylation of AKT and ERK. Moreover, the protein and mRNA expression levels of c-Fos and NFATc1 as well as RANKL-induced transcription of TRAP and OSCAR were suppressed by WSE treatment. DC-STAMP and cathepsin K, which are essential for cell fusion and bone degradation, were also inhibited by WSE. Furthermore, eight components constituting WSE were confirmed to decrease the osteoclast TRAP activity. Taken together, WSE may have potential implications as a useful therapeutic or preventive agent for inhibition of bone loss.

Concomitant induction of SLIT3 and microRNA-218–2 in macrophages by toll-like receptor 4 activation limits osteoclast commitment

BackgroundToll-like receptor 4 (TLR4) conducts a highly regulated inflammatory process by limiting the extent of inflammation to avoid toxicity and tissue damage, even in bone tissues. Thus, it is plausible that strategies for the maintenance of normal bone-immunity to prevent undesirable bone damage by TLR4 activation can exist, but direct evidence is still lacking.MethodsOsteoclast precursors (OCPs) obtained from WT or Slit3-deficient mice were differentiated into osteoclast (OC) with macrophage colony-stimulating factor (M-CSF), RANK ligand (RANKL) and lipopolysaccharide (LPS) by determining the number of TRAP-positive multinuclear cells (TRAP+ MNCs). To determine the alteration of OCPs population, fluorescence-activated cell sorting (FACS) was conducted in bone marrow cells in mice after LPS injection. The severity of bone loss in LPS injected WT or Slit3-deficient mice was evaluated by micro-CT analysis.ResultWe demonstrate that TLR4 activation by LPS inhibits OC commitment by inducing the concomitant expression of miR-218–2-3p and its host gene, Slit3, in mouse OCPs. TLR4 activation by LPS induced SLIT3 and its receptor ROBO1 in BMMs, and this SLIT3-ROBO1 axis hinders RANKL-induced OC differentiation by switching the protein levels of C/EBP-β isoforms. A deficiency of SLIT3 resulted in increased RANKL-induced OC differentiation, and the elevated expression of OC marker genes including Pu.1, Nfatc1, and Ctsk. Notably, Slit3-deficient mice showed expanded OCP populations in the bone marrow. We also found that miR-218–2 was concomitantly induced with SLIT3 expression after LPS treatment, and that this miRNA directly suppressed Tnfrsf11a (RANK) expression at both gene and protein levels, linking it to a decrease in OC differentiation. An endogenous miR-218–2 block rescued the expression of RANK and subsequent OC formation in LPS-stimulated OCPs. Aligned with these results, SLIT3-deficient mice displayed increased OC formation and reduced bone density after LPS challenge.ConclusionOur findings suggest that the TLR4-dependent concomitant induction of Slit3 and miR-218–2 targets RANK in OCPs to restrain OC commitment, thereby avoiding an uncoordinated loss of bone through inflammatory processes. These observations provide a mechanistic explanation for the role of TLR4 in controlling the commitment phase of OC differentiation.CNR6qWg43XLt4z6Q5VLfxQVideo

Reduction of Osteoclastic Differentiation of Raw 264.7 Cells by EMF Exposure through TRPV4 and p-CREB Pathway.

In this study, we investigated the effect of EMF exposure on the regulation of RANKL-induced osteoclast differentiation in Raw 264.7 cells. In the EMF-exposed group, the cell volume did not increase despite RANKL treatment, and the expression levels of Caspase-3 remained much lower than those in the RANKL-treated group. TRAP and F-actin staining revealed smaller actin rings in cells exposed to EMF during RANKL-induced differentiation, indicating that EMF inhibited osteoclast differentiation. EMF-irradiated cells exhibited reduced mRNA levels of osteoclastic differentiation markers cathepsin K (CTSK), tartrate-resistant acid phosphatase (TRAP), and matrix metalloproteinase 9 (MMP-9). Furthermore, as measured by RT-qPCR and Western blot, EMF induced no changes in the levels of p-ERK and p-38; however, it reduced the levels of TRPV4 and p-CREB. Overall, our findings indicate that EMF irradiation inhibits osteoclast differentiation through the TRPV4 and p-CREB pathway.

Experimental Study on the Inhibition of RANKL-Induced Osteoclast Differentiation In Vitro by Metformin Hydrochloride.

Objective Establishment of an in vitro osteoclast induction model under nuclear factor-κB receptor activator ligand (RANKL) induction for investigating the effect of metformin hydrochloride (Met) on osteoclast differentiation. Methods RANKL induced the differentiation of mouse bone marrow macrophages (BMMs) into osteoclasts in vitro, and Met was added at different concentrations for intervention during the induction process. After 5 d of culture and fixation, the number of osteoclasts was counted by tartrate-resistant acid phosphatase (TRAP) staining and F-actin staining, and the function of osteoclasts was examined with hydroxyapatite-coated plates. Real-time fluorescence quantitative PCR was performed to detect the expression of Cathepsin K, osteoclast associated receptor (OSCAR), and TRAP, and the effect of Met on Mitogen-activated protein kinases (MAPK) signaling pathway was detected by Western blot. Results Met significantly reduced osteoclast formation, F-actin ring formation, bone resorption, and the expression of relevant genes Cathepsin K, OSCAR, and TRAP. The Western blotting study demonstrated that Met inhibited the MAPK signaling pathway by decreasing the phosphorylation of extracellular regulated protein kinase (ERK), which plays important roles in osteoclast formation. Conclusion Metformin hydrochloride inhibited the differentiation of osteoclasts, decreased the bone resorption area, and suppressed phosphorylation of ERK in vitro.

Mussaendoside O, a N-triterpene cycloartane saponin, attenuates RANKL-induced osteoclastogenesis and inhibits lipopolysaccharide-induced bone loss

BackgroundElevated activity of osteoclasts (OCs) is linked to osteolytic bone diseases, such as osteoporosis and rheumatoid arthritis. Developing natural anti-osteoclastogenic compounds with greater efficacy and fewer adverse effects is crucial for preventing or treating osteolytic bone diseases. N-triterpene cycloartane saponins (NTCSs) are rarely found in nature, and their inhibitory effects on OC differentiation in vitro and in vivo have not yet been explored. PurposeThis study was aimed to investigate the effect of mussaendoside O, an NTCS isolated from Mussaenda pubescens, on RANKL-induced OC differentiation and its underlying mechanism in vitro, and lipopolysaccharide (LPS)-induced bone resorption in a mouse model. MethodsThe content of mussaendoside O in methanol extract of M. pubescens was determined by HPLC. The inhibitory effects of mussaendoside O on RANKL-induced OC formation were assessed using TRAP staining, western blotting, immunofluorescence staining, and real-time qPCR. Meanwhile, the effects of mussaendoside O on LPS-induced inflammatory responses were assessed using a Griess reagent and qPCR. The effects of mussaendoside O on LPS-induced bone resorption in a mouse model were evaluated using micro-CT and immunohistochemical staining. ResultsMussaendoside O inhibited RANKL-induced TRAP-positive multinucleated OC formation in a concentration-dependent manner without affecting cell viability. However, mussaendoside O did not inhibit LPS-induced mRNA expression of COX-2, iNOS, and TNF-α. Mice orally administrated with mussaendoside O exhibited significant protection from LPS-induced bone resorption and OC formation. At the molecular level, mussaendoside O suppressed RANKL-activated phosphorylation of p38 MAPK and JNK, as well as c-Fos expression. In addition, mussaendoside O suppressed RANKL-induced NFATc1 activation and the expression of its target genes, including OSCAR, DC-STAMP, CtsK, and TRAP. ConclusionMussaendoside O attenuates OC differentiation in vitro and LPS-induced bone resorption in a mouse model by inhibiting the RANKL-activated c-Fos/NFATc1 signaling pathways. Therefore, mussaendoside O may be a valuable lead compound for preventing or treating of osteolytic bone diseases.

Protection against Osteoporosis by Fermented Mulberry Vinegar Supplementation via Inhibiting Osteoclastic Activity in Ovariectomized Rats and Osteoclastic Cells

Menopause increases the osteoporosis risk, to which phytoestrogen intake can be beneficial. This study hypothesized that mulberry vinegar had a preventive effect on osteoporosis by decreasing osteoclastic activity. The hypothesis was tested in ovariectomized (OVX) rats and RANKL-differentiated osteoclast cells. OVX rats were given 0(OVX-CON), 0.5(OVX-MVL), 1(OVX-MVM), and 2(OVX-MVH) fermented mulberry vinegar (MV) mL/kg body weight (BW) daily for 12 weeks. Sham-operated rats had no MV supplementation (Normal-CON). The osteoporosis-related biomarkers were measured, and Micro-CT determined the bone mass of the femur. RANKL-differentiated Raw 264.7 cells were treated with MV (0–100 μg/mL). The cell viability, osteoporosis-related mRNA expression, and protein contents were measured. MV contained Acetobacter pasteurianus (7.31 log CFU/mL), citric acid (106 mg/mL), lactic acid (19.2 mg/mL), acetic acid (15.0 mg/mL), and rutin (0.36 mg/mL). OVX-MVM elevated the serum 17β-estradiol concentration similar to the Normal-CON group, but it did not prevent the decrease in uterine weight. OVX-MVM prevented the increase in osteoclastic-related parameters, including cathepsin K(CtsK), receptor activator of NF-κB ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) in the circulation. OVX-MVH also lowered C-telopeptide of type Ⅰ collagen as much as the Normal-CON group (p < 0.05). By contrast, OVX-MVH increased the serum osteoprotegerin concentration, an inhibitor of osteoclasts, better than the Normal-CON group (p < 0.05). These changes were integrated to alter the bone mineral density (BMD) in Micro-CT analysis: OVX-MVM and OVX-MVH prevented BMD decrease after OVX as much as the Normal-CON. In RANKL-differentiated osteoclast cells, the MV treatment for 24 and 48 h decreased RANKL-induced differentiation in osteoclast cells dose-dependently up to 100 µg/mL. Its decrease was related to inhibiting the TRAP activity and reducing TRAP-positive multinucleated cells during the five-day administration of RANKL. MV treatments also decreased mRNA expression of osteoclast-related genes (TRAP, Ctsk, OSCAR, and NFATc1). MV suppressed the protein contents of NFATc1 and c-FOS-related osteoclast. In conclusion, MV intake (1 mg/kg bw) protected against BMD loss mainly by inhibiting the osteoclastic activity (RANKL/RANK/TRAP) in OVX rats. MV may develop as a functional food for anti-osteoporosis in menopausal women.

MyD88 Mediates Colitis- and RANKL-Induced Microfold Cell Differentiation.

Intestinal microfold (M) cells are critical for sampling antigens in the gut and initiating the intestinal mucosal immune response. In this study, we found that the oral administration of dextran sulfate sodium (DSS) and Salmonella infection induced colitis. In the process, the expression levels of M cell differentiation-related genes were synchronized with the kinetics of pro-inflammatory cytokines. Compared to wild-type (WT) mice, MyD88−/− mice exhibited significantly lower expression levels of M cell differentiation-related genes. However, DSS induced colitis in MyD88−/− mice but failed to promote the transcription of M cell differentiation related genes. Furthermore, the receptor activator of the Nuclear Factor-κB ligand (RANKL) upregulated the transcription of M cell differentiation related genes in murine intestinal organoids prepared from both WT and MyD88−/− mice. Meanwhile, fewer changes in M cell differentiation related genes were found in MyD88−/− mice as compared to WT mice. Hence, we concluded that myeloid differentiation factor 88 (MyD88) is an essential molecule for colitis- and RANKL-related differentiation of M cells.

Cell-free culture supernatant of Lactobacillus curvatus Wikim38 inhibits RANKL-induced osteoclast differentiation and ameliorates bone loss in ovariectomized mice.

This study was conducted to investigate the inhibitory effects of the cell-free culture supernatant of Lactobacillus curvatus Wikim 38 (LC38-CS) on RANKL-induced osteoclast differentiation and bone loss in a mice model of ovariectomy-induced post-menopausal osteoporosis. LC38-CS inhibited the RANKL-induced differentiation of bone marrow-derived macrophages (BMDMs) into osteoclasts in a dose-dependent manner. F-actin ring formation and bone resorption were also reduced by LC38-CS treatment of RANKL-treated BMDMs. In addition, LC38-CS decreased the RANKL-induced activation of the TRAF6/NF-κB/MAPKs axis at the early stage and the expression of osteoclastogenesis-related genes in BMDMs treated with RANKL. PRMT1 and ADMA levels, new biomarkers for osteoclastogenesis, were decreased by LC38-CS treatment. The administration of LC38-CS increased bone volume and bone mineral density in ovariectomized mice in μ-CT analysis. These findings suggest that LC38-CS inhibited RANKL-induced osteoclast differentiation by the downregulation of molecular mechanisms and exerted anti-osteoporotic effects.

D(-)-salicin inhibits RANKL-induced osteoclast differentiation and function in vitro

Osteoporosis is a disease, which causes huge economic and social burden. Using natural compound to treat such disease is beneficial for the fewer side effects and effectiveness. D-(-)-salicin (DSA) is a component extracted from the bark of Populus and Salix species. In our research, we discovered that DSA suppressed RANKL-induced differentiation of osteoclast in vitro in a dose-dependent manner. It was also found that the mineral resorbing activity by osteoclasts was depressed via DSA. For the mechanism, we confirmed the inhibitory effect, by which DSA suppressed osteoclast formation and function, was through the inhibition of ROS signaling, MAPK and NF-κB cascades. DSA also suppressed the expression and activity of NFATc1. Therefore, by inhibiting the ROS production, MAPK and NF-κB signal cascade, DSA inhibited the osteoclast differentiation and function in vitro.

Aplidin (Plitidepsin) Regulates Viability and Function of Myeloma Cells and Bone Cells in Combination with Other Anti-MM Drugs

In multiple myeloma (MM) the clonal expansion of malignant plasma cells within the bone marrow results in the development of lytic bone lesions due to exacerbated bone resorption. Despite recent progress in its treatment, MM remains incurable and the associated bone disease persists even after complete remission. Thus, identification of new therapeutic approaches that suppress MM cell growth and protect the bone is an unmet need. Aplidin, a novel anti-cancer compound isolated from the marine tunicate Aplidium albicans, exhibits anti-MM activity even in MM cells resistant to conventional chemotherapy and is currently in phase III clinical trials in Europe. However, the effects of Aplidin on bone cells and MM-induced bone disease are unknown. The goal of the current study was to examine the effect of Aplidin, alone or in combination with other anti-MM drugs, on myeloma cells and bone cells (osteocytes, osteoblasts, and osteoclasts) using in vitro approaches.Aplidin decreased the number of human JJN3 and murine 5TGM1 MM cells with an EC50~10nM, determined by MTT assay, due to decreased cell viability, assessed by Trypan blue exclusion, reaching a 20% increase in MM cell death after 48h. Treatment with glucocorticoids (GC) (10-5M) or Bortezomib (3nM) also decreased MM viability and enhanced the anti-MM activity of Aplidin by 1.6-fold and 2.5-fold, respectively. In addition, Aplidin at >10nM induced MLO-A5 osteocytic dead up to 15% and in combination with GC up to 50%; whereas, in contrast, Bortezomib did not affect osteocyte viability and partially prevented the increase in MLO-A5 cell death induced by Aplidin. Further, 1nM Aplidin increased OB-6 osteoblastic cell death up to 20% and decreased their matrix mineralization capacity, quantified by Alizarin red. These effects were enhanced when Aplidin was combined with GC, but partially prevented by combination of Aplidin with Bortezomib. Cells of the osteoclastic lineage exhibited higher sensitivity to Aplidin in comparison to MM cells or osteoblastic cells, and thus it was tested at lower concentrations. Specifically, 50 pM Aplidin decreased the number of hematopoietic precursors by 15-20%, as quantified by enumerating GM-CSF colonies in cultures established from murine bone marrow. Moreover, Aplidin, at concentrations as low as 0.01 pM, reduced osteoclast precursor commitment and differentiation by 10%, as quantified by enumerating the number of osteoclast precursors maintained in the presence of M-CSF for 3 days with or without Aplidin. In addition, Aplidin decreased by 40% the number of TRAP positive osteoclasts generated after 4 days in the presence of M-CSF and sRANKL; and blunted the rapid (5-15min) increased in ERK phosphorylation induced by Rankl, but did not affect RANKL-induced p38 phosphorylation, suggesting that Aplidin inhibited RANKL-induced differentiation of osteoclast precursors by preventing ERK activation. Lastly, to test the effects of Aplidin on osteoclasts function, mature osteoclasts generated in the presence of M-CSF/sRANKL were detached by trypsinization, replated on bovine dentin slides and exposed to Aplidin for 3 and 7 days. 10pM Aplidin reduced the number of mature osteoclasts by 60% and markedly decreased the number of resorption pits.In summary, Aplidin (1nM and up) decreases MM cell viability and GC or Borteozmib enhances its anti-MM activity. In addition, Aplidin (10nM and up) decreases osteocytic cell viability and this effect is exacerbated by GC but partially prevented by Bortezomib. Moreover, Aplidin (1nM and up) induces osteoblastic cell death and decreases their mineral production, and these effects are increased by GC, but partially prevented by Bortezomib. Furthermore, Aplidin (0.01pM and up) reduces osteoclast precursor commitment and differentiation, and inhibits mature osteoclast resorption activity. Taken together these findings suggest that combining Aplidin with proteosomal inhibitors may be a novel therapy for MM to enhance anti-tumor responses, decrease bone destruction by inhibiting bone resorption, and prevent potential side effects of Aplidin on cells of the osteoblastic lineage. DisclosuresDelgado-Calle:PharmaMar: Research Funding. Galmarini:PharmaMar: Employment. Roodman:PharmaMar: Research Funding. Bellido:PharmaMar: Research Funding.

Hypoxia inhibits RANKL-induced ferritinophagy and protects osteoclasts from ferroptosis

Ferroptosis is a new form of regulated cell death. Several studies have demonstrated that ferroptosis was involved in multiple diseases. However, the precise role of ferroptosis in osteoporosis remains unclear. Here, we demonstrated that ferroptosis was involved in osteoclasts over the course of RANKL-induced differentiation, and it was induced by iron-starvation response and ferrintinophagy. Mechanistically, under normoxia but not hypoxia, ferroptosis could be induced due to iron-starvation response (increased transferrin receptor 1, decreased ferritin) followed by RANKL stimulation, and this was attributed to the down-regulation of aconitase activity. We further investigated intracellular iron homeostasis and found that ferritinophagy, a process initiated by FTH-NCOA4 complex autophagosome degradation, was activated followed by RANKL stimulation under normoxia. Interestingly, these processes could not be observed under hypoxia. Moreover, we demonstrated that HIF-1α contributed to the decrease of ferritinophagy and autophagy flux under hypoxia. Additionally, HIF-1α impair autophagy flux via inhibition of autophagosome formation under hypoxia in BMDMs. In vivo study, we indicated that HIF-1α specific inhibitor 2ME2 prevent OVX bone loss. In conclusion, our study comprehensively investigated the role of ferroptosis in osteoclasts in vitro and in vivo, and innovatively suggested that targeting HIF-1α and ferritin thus inducing ferroptosis in osteoclasts could be an alternative in treatment of osteoporosis.

Bone Morphogenetic Protein-2 Promotes Osteoclasts-mediated Osteolysis via Smad1 and p65 Signaling Pathways.

An in vitro biological study. The aim of this study was to explore the role of bone morphogenetic protein-2 (BMP-2) in the regulation of osteoclast-mediated osteolysis, and the possible mechanism involving BMP-2 and nuclear factor-kappa B (NF-κB) signaling pathways. Recombinant human BMP-2 (rhBMP-2) has been approved as a therapeutic agent in spinal fusion and bone defect repair. However, its efficacy and clinical application are limited by associated complications including osteoclast-mediated bone resorption. The mechanism of BMP-2-induced osteolysis remains unknown. Bone marrow-derived macrophages (BMMs) were isolated from C57BL/6J mice and cultured with macrophage colony-stimulating factor (M-CSF) and receptor activator for nuclear factor-κB Ligand (RANKL) to induce osteoclast differentiation. An in vitro bone resorption assay was performed by co-culturing BMMs and bone slides. The expression of BMP canonical and NF-κB signaling factors and their interaction during signal transduction were quantitated by reverse transcription polymerase chain reaction, Western blot analysis, confocal microscopy, and co-immunoprecipitation. BMP-2 enhanced osteoclast-mediated bone resorption via inducing osteoclast differentiation in a concentration-dependent manner. In addition, a high concentration of BMP-2 significant upregulated phosphorylation of BMP signaling factors p-Smad1/5/8 and NF-κB downstream factor p65, and promoted the degeneration of IκBα. In addition, BMP-2 induced osteoclast differentiation through coupling between BMP receptor II and RANK. High concentrations of BMP-2 enhanced osteoclast-mediated bone resorption by promoting RANKL-induced pre-osteoclast differentiation, probably by mediating the cross-talk between BMP canonical and NF-κB signaling pathways.Level of Evidence: N/A.

Tungsten Increases Sex-Specific Osteoclast Differentiation in Murine Bone.

Tungsten is a naturally occurring metal that is increasingly used in industry and medical devices, and is labeled as an emerging environmental contaminant. Like many metals, tungsten accumulates in bone. Our previous data indicate that tungsten decreases differentiation of osteoblasts, bone-forming cells. Herein, we explored the impact of tungsten on osteoclast differentiation, which function in bone resorption. We observed significantly elevated osteoclast numbers in the trabecular bone of femurs following oral exposure to tungsten in male, but not female mice. In order to explore the mechanism(s) by which tungsten increases osteoclast number, we utilized in vitro murine primary and cell line pre-osteoclast models. Although tungsten did not alter the adhesion of osteoclasts to the extracellular matrix protein, vitronectin, we did observe that tungsten enhanced RANKL-induced differentiation into tartrate-resistant acid phosphatase (TRAP)-positive mononucleated osteoclasts. Importantly, tungsten alone had no effect on differentiation or on the number of multinucleated TRAP-positive osteoclasts. Enhanced RANKL-induced differentiation correlated with increased gene expression of differentiated osteoclast markers Nfatc1, Acp5, and Ctsk. Although tungsten did not alter the RANK surface receptor expression, it did modulate its downstream signaling. Co-exposure of tungsten and RANKL resulted in sustained positive p38 signaling. These findings demonstrate that tungsten enhances sex-specific osteoclast differentiation, and together with previous findings of decreased osteoblastogenesis, implicate tungsten as a modulator of bone homeostasis.

Linarin and its aglycone acacetin abrogate actin ring formation and focal contact to bone matrix of bone-resorbing osteoclasts through inhibition of αvβ3 integrin and core-linked CD44

BackgroundSince enhanced bone resorption due to osteoclast differentiation and activation cause skeletal diseases, there is a growing need in therapeutics for combating bone-resorbing osteoclasts. Botanical antioxidants are being increasingly investigated for their health-promoting effects on bone. Edible Cirsium setidens contains various polyphenols of linarin, pectolinarin, and apigenin with antioxidant and hepatoprotective effects. PurposeThis study aimed to determine whether linarin present in Cirsium setidens water extracts (CSE) and its aglycone acacetin inhibited osteoclastogenesis of RANKL-exposed RAW 264.7 murine macrophages for 5 days. MethodsThis study assessed the osteoprotective effects of CSE, linarin and acacetin on RANKL-induced differentiation and activation of osteoclasts by using MTT assay, TRAP staining, Western blot analysis, bone resorption assay actin ring staining, adhesion assay and immunocytochemical assay. This study explored the underlying mechanisms of their osteoprotection, and identified major components present in CSE by HPLC analysis. ResultsLinarin and pectolinarin were identified as major components of CSE. Nontoxic linarin and acacetin as well as CSE, but not pectolinarin attenuated the RANKL-induced macrophage differentiation into multinucleated osteoclasts, and curtailed osteoclastic bone resorption through reducing lacunar acidification and bone matrix degradation in the osteoclast-bone interface. Linarin and acacetin in CSE reduced the transmigration and focal contact of osteoclasts to bone matrix-mimicking RGD peptide. Such reduction was accomplished by inhibiting the induction of integrins, integrin-associated proteins of paxillin and gelsolin, cdc42 and CD44 involved in the formation of actin rings. The inhibition of integrin-mediated actin ring formation by linarin and acacetin entailed the disruption of TRAF6-c-Src-PI3K signaling of bone-resorbing osteoclasts. The functional inhibition of c-Src was involved in the loss of F-actin-enriched podosome core protein cortactin-mediated actin assembly due to linarin and acacetin. ConclusionThese observations demonstrate that CSE, linarin and acacetin were effective in retarding osteoclast function of focal adhesion to bone matrix and active bone resorption via inhibition of diffuse cloud-associated αvβ3 integrin and core-linked CD44.