Formation Of Multinuclear Osteoclasts Research Articles

Effect of Chitosan Degradation Products, Glucosamine and Chitosan Oligosaccharide, on Osteoclastic Differentiation.

Chitosan, a natural cationic polysaccharide derived from crustaceans and shellfish shells, is known for its advantageous biological properties, including biodegradability, biocompatibility, and antibacterial activity. Chitosan and its composite materials are studied for their potential for bone tissue repair. However, the effects of chitosan degradation products, glucosamine (GlcN) and chitosan oligosaccharide (COS), on osteoclasts remain unclear. If these chitosan degradation products promote osteoclastic differentiation, careful consideration is required for the use of chitosan and related materials in bone repair applications. Here, we assessed the effects of high (500 μg/mL) and low (0.5 μg/mL) concentrations of GlcN and COS on osteoclastic differentiation in human peripheral blood mononuclear cells (PBMCs) and murine macrophage-like RAW264 cells. A tartrate-resistant acid phosphatase (TRAP) enzyme activity assay, TRAP staining, and actin staining were used to assess osteoclastic differentiation. High concentrations of GlcN and COS, but not low concentrations, suppressed macrophage colony-stimulating factor (M-CSF)- and RANKL-dependent increases in TRAP enzyme activity, TRAP-positive multinuclear osteoclast formation, and actin ring formation in PBMCs without cytotoxicity. Similar effects were observed in the RANKL-dependent osteoclastic differentiation of RAW264 cells. In conclusion, chitosan degradation products do not possess osteoclast-inducing properties, suggesting that chitosan and its composite materials can be safely used for bone tissue repair.

Amakusamine from a Psammocinia sp. Sponge: Isolation, Synthesis, and SAR Study on the Inhibition of RANKL-Induced Formation of Multinuclear Osteoclasts.

A simple methylenedioxy dibromoindole alkaloid, amakusamine (1), was isolated from a marine sponge of the genus Psammocinia, and its structure was determined from spectroscopic data, time-dependent density-functional theory calculations, and synthesis. Compound 1 inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinuclear osteoclasts with an IC50 value of 10.5 μM in RAW264 cells. The structure-activity relationship of 1 was also investigated with synthetic derivatives.

Taichunins E-T, Isopimarane Diterpenes and a 20-nor-Isopimarane, from Aspergillus taichungensis (IBT 19404): Structures and Inhibitory Effects on RANKL-Induced Formation of Multinuclear Osteoclasts.

Fifteen new isopimarane-type diterpenes, taichunins E-S (1-15), and a new 20-nor-isopimarane, taichunin T (16), together with four known compounds were isolated from Aspergillus taichungensis (IBT 19404). The structures of these new compounds were determined by NMR and mass spectroscopy, and their absolute configurations were analyzed by NOESY and TDDFT calculations of ECD spectra. Taichunins G, K, and N (3, 7, and 10) completely inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinuclear osteoclasts in RAW264 cells at 5 μM, with 3 showing 92% inhibition at a concentration of 0.2 μM.

Deficiency of Omentin-1 leads to delayed fracture healing through excessive inflammation and reduced CD31hiEmcnhi vessels

Fracture healing is a complicated process affected by many factors, such as inflammatory responses and angiogenesis. Omentin-1 is an adipokine with anti-inflammatory properties, but whether omentin-1 affects the fracture healing process is still unknown. Here, by using global omentin-1 knockout (omentin-1−/−) mice, we demonstrated that omentin-1 deficiency resulted in delayed fracture healing in mice, accompanied by increased inflammation and osteoclast formation, and decreased production of platelet-derived growth factor-BB (PDGF-BB) and osteogenesis-promoting vessels that are strongly positive for CD31 and Endomucin (CD31hiEmcnhi) in the fracture area. In vitro, omentin-1 treatment suppressed the ability of the tumor necrosis factor-α (TNF-α)-activated macrophages to stimulate multi-nuclear osteoclast formation, resulting in a significant increase in the generation of mono-nuclear preosteoclasts and PDGF-BB, a pro-angiogenic protein that is abundantly secreted by preosteoclasts. PDGF-BB significantly augmented endothelial cell proliferation, tube formation and migration, whereas direct treatment with omentin-1 did not induce obvious effects on angiogenesis activities of endothelial cells. Our study suggests a positive role of omentin-1 in fracture healing, which may be associated with the inhibition of inflammation and stimulation of preosteoclast PDGF-BB-mediated promotion of CD31hiEmcnhi vessel formation.

OC‐STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up‐regulation of permissive fusogen CD9

Cell fusion-mediated formation of multinuclear osteoclasts (OCs) plays a key role in bone resorption. It is reported that 2 unique OC-specific fusogens [ i.e., OC-stimulatory transmembrane protein (OC-STAMP) and dendritic cell-specific transmembrane protein (DC-STAMP)], and permissive fusogen CD9, are involved in OC fusion. In contrast to DC-STAMP-knockout (KO) mice, which show the osteopetrotic phenotype, OC-STAMP-KO mice show no difference in systemic bone mineral density. Nonetheless, according to the ligature-induced periodontitis model, significantly lower level of bone resorption was found in OC-STAMP-KO mice compared to WT mice. Anti-OC-STAMP-neutralizing mAb down-modulated in vitro: 1) the emergence of large multinuclear tartrate-resistant acid phosphatase-positive cells, 2) pit formation, and 3) mRNA and protein expression of CD9, but not DC-STAMP, in receptor activator of NF-κB ligand (RANKL)-stimulated OC precursor cells (OCps). While anti-DC-STAMP-mAb also down-regulated RANKL-induced osteoclastogenesis in vitro, it had no effect on CD9 expression. In our mouse model, systemic administration of anti-OC-STAMP-mAb suppressed the expression of CD9 mRNA, but not DC-STAMP mRNA, in periodontal tissue, along with diminished alveolar bone loss and reduced emergence of CD9+ OCps and tartrate-resistant acid phosphatase-positive multinuclear OCs. The present study demonstrated that OC-STAMP partners CD9 to promote periodontal bone destruction by up-regulation of fusion during osteoclastogenesis, suggesting that anti-OC-STAMP-mAb may lead to the development of a novel therapeutic regimen for periodontitis.-Ishii, T., Ruiz-Torruella, M., Ikeda, A., Shindo, S., Movila, A., Mawardi, H., Albassam, A., Kayal, R. A., Al-Dharrab, A. A., Egashira, K., Wisitrasameewong, W., Yamamoto, K., Mira, A. I., Sueishi, K., Han, X., Taubman, M. A., Miyamoto, T., Kawai, T. OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9.

Isolation of Aaptic Acid from the Marine Sponge Aaptos lobata and Inhibitory Effect of Aaptamines on RANKL-Induced Formation of Multinuclear Osteoclasts

Isolation of Aaptic Acid from the Marine Sponge Aaptos lobata and Inhibitory Effect of Aaptamines on RANKL-Induced Formation of Multinuclear Osteoclasts

Enantioselective inhibitory abilities of enantiomers of notoamides against RANKL-induced formation of multinuclear osteoclasts

The marine-derived Aspergillus protuberus MF297-2 and the terrestrial A. amoenus NRRL 35600 produce enantiomeric prenylated indole alkaloids. Investigation of biological activities of the natural and synthetic derivatives revealed that (−)-enantiomers of notoamides A and B, 6-epi-notoamide T, and stephacidin A inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)–induced osteoclastogenic differentiation of murine RAW264 cells more strongly than their respective (+)-enantiomers. Among them, (−)-6-epi-notoamide T was the most potent inhibitor with an IC50 value of 1.7μM.

Hydroxychloroquine affects bone resorption both in vitro and in vivo.

We recently showed that patients with primary Sjögren syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favorable effects on BMD. The aim of the study was to evaluate whether HCQ modulates osteoclast function. Osteoclasts were cultured from PBMC-sorted monocytes for 14 days and treated with different HCQ doses (controls 1 and 5 μg/ml). TRAP staining and resorption assays were performed to evaluate osteoclast differentiation and activity, respectively. Staining with an acidification marker (acridine orange) was performed to evaluate intracellular pH at multiple timepoints. Additionally, a fluorescent cholesterol uptake assay was performed to evaluate cholesterol trafficking. Serum bone resorption marker β-CTx was evaluated in rheumatoid arthritis patients. HCQ inhibits the formation of multinuclear osteoclasts and leads to decreased bone resorption. Continuous HCQ treatment significantly decreases intracellular pH and significantly enhanced cholesterol uptake in mature osteoclasts along with increased expression of the lowdensity lipoprotein receptor. Serum β-CTx was significantly decreased after 6 months of HCQ treatment. In agreement with our clinical data, we demonstrate that HCQ suppresses bone resorption in vitro and decreases the resorption marker β-CTx in vivo. We also showed that HCQ decreases the intracellular pH in mature osteoclasts and stimulates cholesterol uptake, suggesting that HCQ induces osteoclastic lysosomal membrane permeabilization (LMP) leading to decreased resorption without changes in apoptosis. We hypothesize that skeletal health of patients with increased risk of osteoporosis and fractures may benefit from HCQ by preventing BMD loss.

Ceylonins A-F, Spongian Diterpene Derivatives That Inhibit RANKL-Induced Formation of Multinuclear Osteoclasts, from the Marine Sponge Spongia ceylonensis.

Six new spongian diterpene derivatives, ceylonins A-F (1-6), were isolated from the Indonesian marine sponge Spongia ceylonensis along with spongia-13(16),14-dien-19-oic acid (7). They contained three additional carbons in ring D to supply an ether-bridged bicyclic ring system. Their structures were elucidated by analyzing NMR spectroscopic data and calculated ECD spectra in comparison to experimental ECD spectra. The bicyclic ring system may be derived from the major metabolite 7 and a C3 unit (an acrylic acid equivalent) through an intermolecular Diels-Alder reaction, which was experimentally supported by the formation of 1-6 from 7 and acrylic acid. The inhibitory effects of the isolated compounds on the RANKL-induced formation of multinuclear osteoclasts in RAW264 macrophages were examined.

Follistatin-like 1 promotes osteoclast formation via RANKL-mediated NF-κB activation and M-CSF-induced precursor proliferation

Follistatin-like 1 (FSTL1) functions as a pivotal modulator of inflammation and is implicated in many inflammatory diseases such as rheumatoid arthritis. Here, we report that FSTL1 is strongly upregulated and secreted during osteoclast differentiation of bone marrow-derived macrophages (BMMs) and that FSTL1 positively regulates osteoclast formation induced by RANKL and M-CSF. The overexpression of FSTL1 or treatment with recombinant FSTL1 (rFSTL1) in BMMs enhances the formation of multinuclear osteoclasts and the induction of c-Fos and NFATc1, transcription factors important for osteoclastogenesis. Conversely, knockdown of FSTL1 using a small hairpin RNA suppresses osteoclast formation and the expression of these transcription factors. While FSTL1 does not affect RANKL-stimulated activation of p38 MAPK, phosphorylation of IκBα, JNK, and ERK were increased by overexpression or addition of rFSTL1. Furthermore, rFSTL1 increased RANKL-induced NF-κB transcriptional activity in a dose-dependent manner. In addition to its role in osteoclastogenesis, FSTL1 promotes proliferation of osteoclast precursors by increasing M-CSF-induced ERK activation, which in turn leads to accelerated osteoclast formation. Together, our findings demonstrate that FSTL1 is a secreted osteoclastogenic factor that plays a critical role in osteoclast formation via the NF-κB and MAPKs signaling pathways.

Lipocalin-2 inhibits osteoclast formation by suppressing the proliferation and differentiation of osteoclast lineage cells

Lipocalin-2 (LCN2) is a member of the lipocalin superfamily and plays a critical role in the regulation of various physiological processes, such as inflammation and obesity. In this study, we report that LCN2 negatively modulates the proliferation and differentiation of osteoclast precursors, resulting in impaired osteoclast formation. The overexpression of LCN2 in bone marrow-derived macrophages or the addition of recombinant LCN2 protein inhibits the formation of multinuclear osteoclasts. LCN2 suppresses macrophage colony-stimulating factor (M-CSF)-induced proliferation of osteoclast precursor cells without affecting their apoptotic cell death. Interestingly, LCN2 decreases the expression of the M-CSF receptor, c-Fms, and subsequently blocks its downstream signaling cascades. In addition, LCN2 inhibits RANKL-induced osteoclast differentiation and attenuates the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important modulators in osteoclastogenesis. Mechanistically, LCN2 inhibits NF-κB signaling pathways, as demonstrated by the suppression of IκBα phosphorylation, nuclear translocation of p65, and NF-κB transcriptional activity. Thus, LCN2 is an anti-osteoclastogenic molecule that exerts its effects by retarding the proliferation and differentiation of osteoclast lineage cells.

A High Content Screening Assay for Evaluation of Biomaterial‐Mediated Cell Fusion Processes

SummaryBiomaterials are of increasing importance in regenerative medicine and entail delivery systems in somatic cell therapies, matrices for tissue engineering or tissue regeneration. The evaluation of biomaterial induced biological effects remains a key issue in clinical application. Cell‐based assays for potential cytotoxic and immunological responses have been developed but are often inadequate to address cell‐type specific responses to biomaterials. To quantitatively monitor attachment, survival, proliferation and fusion‐controlled differentiation of osteoclasts (bone resorbing cells), a High Content Screening (HCS) assay has been developed based on osteoclast differentiation of the murine monocytic cell line RAW 264.7. This assay was applied to investigate the influence of degradation products of polymers from gelatin and lysine diisocyanate, which display tailorable mechanical properties and have potential as biomaterials. The data show that the degradation products inhibit formation of multinuclear osteoclasts and suggest a potential support of bone regeneration by suppression of bone resorption.

Halenaquinone inhibits RANKL-induced osteoclastogenesis

Halenaquinone was isolated from the marine sponge Petrosia alfiani as an inhibitor of osteoclastogenic differentiation of murine RAW264 cells. It inhibited the RANKL (receptor activator of nuclear factor-κB ligand)-induced upregulation of TRAP (tartrate-resistant acid phosphatase) activity as well as the formation of multinuclear osteoclasts. In addition, halenaquinone substantially suppressed RANKL-induced IκB degradation and Akt phosphorylation. Thus, these results suggest that halenaquinone inhibits RANKL-induced osteoclastogenesis at least by suppressing the NF-κB and Akt signaling pathways.

Osteoclastic resorptive capacity is suppressed in patients receiving hyperbaric oxygen therapy

Background and purpose — Hypoxia, necrosis, and bone loss are hallmarks of many skeletal diseases. Hyperbaric oxygen therapy (HBO) is often used as an adjunctive therapy in these cases. However the in vivo effect of HBO on osteoclast formation has not been fully established. We therefore carried out a longitudinal study to examine the effect of HBO on osteoclast formation and bone resorptive capacity in patients who were referred to the Plymouth Hyperbaric Medical Centre.Methods — Osteoclast precursors were isolated from peripheral blood prior to and following 10 and 25 daily hyperbaric treatments (100% O2 at 2.4 atmospheres absolute ATA for 90 min) to determine osteoclast formation and resorptive capacity. The expression of key regulators of osteoclast differentiation RANK, Dc-STAMP, and NFATc1 was also assessed by quantitative real-time PCR.Results — HBO reduced the ability of precursors to form osteoclasts and reduced bone resorption in a treatment-dependent manner. The initial suppressive effect of HBO was more pronounced on mononuclear osteoclast formation than on multinuclear osteoclast formation, and this was accompanied by reduction in the expression of key regulators of osteoclast formation, RANK and Dc-STAMP.Interpretation — This study shows for the first time that in vivo, HBO suppresses the ability of monocytic precursors to form resorptive osteoclasts.

Kruppel-like factor 4 expression in osteoblasts represses osteoblast-dependent osteoclast maturation.

Kruppel-like factor 4 (KLF4) is a zinc-finger-type transcription factor with a restricted expression pattern during skeletal development. We have previously shown that KLF4 represses osteoblast mineralization concomitant with a down-regulation in the expression of a number of osteoblastic genes, both in vivo and in vitro. In addition to the cell-autonomous effects of KLF4 in osteoblasts, transgenic osteoblastic-KLF4 mice show severe defects in osteoclast maturation. Wild-type bone-marrow-derived macrophages co-cultured with KLF4-expressing osteoblasts exhibit reduced formation of multinuclear osteoclasts as compared with control cultures overexpressing green fluorescent protein. Significantly, the transduction of Runx2, a master regulator of osteoblastogenesis, together with KLF4 into osteoblasts restores the reduction in osteoclastogenesis induced by KLF4 alone. Various extracellular matrix molecules are down-regulated by KLF4 overexpression but this down-regulation can be partially restored by the co-transduction of Runx2. These results suggest that osteoblastic-KLF4 affects osteoclast maturation by regulating cell-matrix interactions and reinforce the importance of the regional down-regulation of KLF4 expression in the subset of osteoblasts for normal skeletal modeling and remodeling.

SLAT negatively regulates RANKL-induced osteoclast differentiation.

RANKL induces the formation of osteoclasts, which are responsible for bone resorption. Herein, we investigated the role of SWAP-70-like adapter of T cells (SLAT) in RANKL-induced osteoclastogenesis. Expression levels of SLAT were reduced during RANKL-induced osteoclastogenesis. Overexpression of SLAT in BMMs inhibited TRAP-positive multinuclear osteoclast formation and attenuated the expression of NFATc1, which is an important modulator in osteoclastogenesis. Furthermore, silencing of SLAT by RNA interference enhanced osteoclast formation as well as NFATc1 expression. In addition, SLAT was involved in RANKL-induced JNK activation in osteoclasts. Taken together, our data suggest that SLAT acts as a negative modulator of RANKL-induced osteoclastogenesis.

Early growth response 2 negatively modulates osteoclast differentiation through upregulation of Id helix–loop–helix proteins

Early growth response 2 (Egr2) is a zinc finger transcription factor that acts as an important modulator of various physiological processes. In this study, we show that Egr2 negatively regulates receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. The overexpression of Egr2 in bone marrow-derived macrophages (BMMs) suppresses the formation of multinuclear osteoclasts and the expression of osteoclastogenic markers, including nuclear factor of activated T cells c1 (NFATc1). On the other hand, Egr2 overexpression does not impact the phagocytic activity of osteoclast precursors or the expression of macrophage-specific markers in the presence of the osteoclastogenic stimuli, RANKL and M-CSF. We further demonstrate that Egr2 induces the expression of the inhibitors of differentiation/DNA binding (Ids) helix–loop–helix (HLH) transcription factors, which are important repressors in RANKL-mediated osteoclastogenesis. Egr2 transactivates the Id2 promoter and increases its recruitment to the Id2 promoter region. In addition, Egr2-dependent induction of Id2 promoter activity, and its binding to the Id2 promoter is abrogated by the overexpression of the Egr2 repressor, NGFI-A binding protein 2 (Nab2). Accordingly, coexpression with Nab2 restores Egr2-mediated suppression of osteoclast differentiation. Furthermore, knockdown of Egr2 using shRNA enhances osteoclastogenesis and decreases Id2 gene expression. Ectopic expression of Id2 reverses the phenotype mediated by Egr2 silencing. Taken together, our results identify Egr2 as an important modulator of RANKL-induced osteoclast differentiation and provide the link between RANKL, Egr2 and Id proteins in osteoclast-lineage cells.

The Influence of Interleukin-32γ on Osteoclastogenesis with a Focus on Fusion-Related Genes

We previously reported that interleukin-32 gamma (IL-32γ) has a direct effect on osteoclast differentiation and activation in vitro in the context of receptor activator of NF-κB ligand (RANKL) co-stimulation. However, the stage of osteoclast differentiation at which IL-32γ exerts its effect was not determined. Here, we demonstrated that IL-32γ plays an important role in the fusion of preosteoclasts to yield multinuclear osteoclasts, particularly large osteoclasts. The synergistic effect of IL-32γ on RANKL-induced formation of multinuclear osteoclasts was readily apparent when cells were treated with IL-32γ at the fusion stage. In addition, we demonstrated that IL-32γ induced the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), and NFATc1 inactivation by cyclosporine treatment attenuated the effect of IL-32γ. These results indicate that IL-32γ is a potential mediator of osteoclast fusion, likely through up-regulation of NFATc1 and DC-STAMP.

Effect of pioglitazone on receptor activator of nuclear factor-kappa B expression in osteoclast from RAW264.7 cells

To study the effect of pioglitazone, a synthetic peroxisome proliferator-activated receptor γ (PPARγ) agonist, on the RANKL-mediated osteoclastogenesis of osteoclast precursor cells, and to explore the function and mechanism of PPARγ in the osteoclast differentiation. Pioglitazone treatment of RAW264.7 murine macrophages were compared with those of simply cultured control and RANKL-mediated control. Accordingly, the RANKL-mediated cells were cultured with 30 ng/ml RANKL, then induced into significant multinuclear osteoclast formation. And pioglitazone treated cells were exposed to 10 µmol/L pioglitazone during the process of osteoclast differentiation under RANKL. The number of mature osteoclasts was calculated and the mRNA levels of RANK analyzed by real-time quantitative PCR. Pioglitazone significantly inhibited osteoclastogenesis of osteoclast precursor cells, the number of mature osteoclasts of pioglitazone treated group was (176 ± 58)/cm(2) and significantly less than the mature cells of RANKL induced group which number was (322 ± 74)/cm(2) (P < 0.01); and pioglitazone also significantly inhibited the mRNA expression of RANK, a typical differentiated factor of osteoclast, the number of the mRNA expression of RANK of pioglitazone treated group was 2.16 ± 0.74 and significantly less than the number of RANKL induced group (4.94 ± 0.39, P < 0.01). PPARγ agonist inhibited the differentiation of RAW264.7 towards osteoclast. It might be due to the suppression of RANK gene expression in the process of osteoclast differentiation.

THIAZOLIDINEDIONES INHIBIT TNF-α-MEDIATED OSTEOCLAST DIFFERENTIATION OF RAW264.7 MACROPHAGES AND MOUSE BONE MARROW CELLS THROUGH DOWNREGULATION OF NFATc1

TNF-alpha plays critical roles in bone-resorbing diseases, such as rheumatoid arthritis. Recent evidence suggests that thiazolidinediones (TZDs), which are peroxisome proliferator-activated receptor gamma agonists, have anti-inflammatory effects. The aim of this study was to evaluate the effect of TZDs on the TNF-alpha-mediated osteoclastogenesis of osteoclast precursor cells. TNF-alpha treatment of RAW264.7 murine macrophages or mouse bone marrow cells induced significant multinuclear osteoclast formation, and these differentiated osteoclast cells possessed bone-resorbing activity. The TZD drugs, rosiglitazone and pioglitazone, significantly inhibited TNF-alpha-induced osteoclast differentiation from both cell types and subsequent bone resorption. Reverse transcriptase-polymerase chain reaction, reporter gene assays, and Western blot results revealed that TZD treatment significantly suppressed NFATc1 expression. Moreover, GW9662 (a peroxisome proliferator-activated receptor gamma antagonist) prevented the inhibitory effect of TZDs on NFATc1 expression and osteoclast differentiation. In summary, our results demonstrate that TZDs inhibit TNF-alpha-mediated osteoclast differentiation by downregulation of NFATc1 expression. This observation increases the therapeutic applications of TZDs in inflammatory bone-resorbing diseases.