scholarly journals Quercetin Triggers Apoptosis of Lipopolysaccharide (LPS)-induced Osteoclasts and Inhibits Bone Resorption in RAW264.7 Cells

2012 ◽  
Vol 30 (1) ◽  
pp. 123-136 ◽  
Author(s):  
Chun Guo ◽  
Guo-qing Hou ◽  
Xue-dong Li ◽  
Xue Xia ◽  
Dong-xin Liu ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Raw264.7 Cells

scholarly journals Ferryl Hemoglobin Inhibits Osteoclastic Differentiation of Macrophages in Hemorrhaged Atherosclerotic Plaques

2020 ◽  
Vol 2020 ◽  
pp. 1-17
Author(s):  
Erzsébet Zavaczki ◽  
Tamás Gáll ◽  
Abolfazl Zarjou ◽  
Zoltán Hendrik ◽  
László Potor ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Carotid Arteries ◽  
Raw264.7 Cells ◽  
Calcium Deposition ◽  
Atherosclerotic Plaques ◽  
Nuclear Factor ◽  
Nuclear Factor Kappa ◽  
Receptor Activator ◽  
Osteoclastic Differentiation ◽  
Human Carotid

Intraplaque hemorrhage frequently occurs in atherosclerotic plaques resulting in cell-free hemoglobin, which is oxidized to ferryl hemoglobin (FHb) in the highly oxidative environment. Osteoclast-like cells (OLCs) derived from macrophages signify a counterbalance mechanism for calcium deposition in atherosclerosis. Our aim was to investigate whether oxidized hemoglobin alters osteoclast formation, thereby affecting calcium removal from mineralized atherosclerotic lesions. RANKL- (receptor activator of nuclear factor kappa-Β ligand-) induced osteoclastogenic differentiation and osteoclast activity of RAW264.7 cells were studied in response to oxidized hemoglobin via assessing bone resorption activity, expression of osteoclast-specific genes, and the activation of signalization pathways. OLCs in diseased human carotid arteries were assessed by immunohistochemistry. FHb, but not ferrohemoglobin, decreased bone resorption activity and inhibited osteoclast-specific gene expression (tartrate-resistant acid phosphatase, calcitonin receptor, and dendritic cell-specific transmembrane protein) induced by RANKL. In addition, FHb inhibited osteoclastogenic signaling pathways downstream of RANK (receptor activator of nuclear factor kappa-Β). It prevented the induction of TRAF6 (tumor necrosis factor (TNF) receptor-associated factor 6) and c-Fos, phosphorylation of p-38 and JNK (c-Jun N-terminal kinase), and nuclear translocation of NFκB (nuclear factor kappa-Β) and NFATc1 (nuclear factor of activated T-cells, cytoplasmic 1). These effects were independent of heme oxygenase-1 demonstrated by knocking down HO-1 gene in RAW264.7 cells and in mice. Importantly, FHb competed with RANK for RANKL binding suggesting possible mechanisms by which FHb impairs osteoclastic differentiation. In diseased human carotid arteries, OLCs were abundantly present in calcified plaques and colocalized with regions of calcium deposition, while the number of these cells were lower in hemorrhagic lesions exhibiting accumulation of FHb despite calcium deposition. We conclude that FHb inhibits RANKL-induced osteoclastic differentiation of macrophages and suggest that accumulation of FHb in a calcified area of atherosclerotic lesion with hemorrhage retards the formation of OLCs potentially impairing calcium resorption.

scholarly journals Osteoclastic Metabolism of 25(OH)-Vitamin D3: A Potential Mechanism for Optimization of Bone Resorption

Endocrinology ◽  
2010 ◽  
Vol 151 (10) ◽  
pp. 4613-4625 ◽  
Author(s):  
Masakazu Kogawa ◽  
David M. Findlay ◽  
Paul H. Anderson ◽  
Renee Ormsby ◽  
Cristina Vincent ◽  
...  
Keyword(s):  
T Cells ◽  
Bone Resorption ◽  
Mononuclear Cells ◽  
Osteoclast Differentiation ◽  
Raw264.7 Cells ◽  
Marker Genes ◽  
Null Mouse ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Mouse Splenocytes

The extrarenal synthesis of 1α,25 dihydroxyvitamin D3 (1,25D) has been demonstrated in a number of cell types including osteoblasts and cells of the monocyte/macrophage lineage. The skeleton appears responsive to serum levels of the 1,25D precursor, 25 hydroxyvitamin D3 (25D), in terms of bone mineralization parameters. The effect of metabolism of 25D into active 1,25D by osteoclast lineage cells is unknown. We found that CYP27B1 mRNA expression increased with exposure of human peripheral blood mononuclear cells (PBMCs) to macrophage colony-stimulating factor in the presence or absence of receptor activator of nuclear factor-κB ligand. Consistent with this, human osteoclast cultures incubated with 25D produced measurable quantities of 1,25D. Osteoclast formation from either mouse RAW264.7 cells or human PBMCs in the presence of physiological concentrations of 25D resulted in significant up-regulation of the key osteoclast transcription factor, nuclear factor of activated T cells-c1 in PBMCs and a number of key osteoclast marker genes in both models. The expression of the osteoblast coupling factor, ephrin-b2, was also increased in the presence of 25D. Levels of CYP27B1 and nuclear factor of activated T cells-1 mRNA correlated during osteoclastogenesis and also in a cohort of human bone samples. CYP27B1 short-hairpin RNA knockdown in RAW264.7 cells decreased their osteoclastogenic potential. 25D dose dependently reduced the resorptive capacity of PBMC-derived osteoclasts without compromising cell viability. 25D also reduced resorption by RAW264.7- and giant cell tumor-derived osteoclasts. Conversely, osteoclasts formed from vitamin D receptor-null mouse splenocytes had increased resorptive activity compared with wild-type cells. We conclude that 25D metabolism is an important intrinsic mechanism for optimizing osteoclast differentiation, ameliorating osteoclast activity, and potentially promoting the coupling of bone resorption to formation.

scholarly journals Undercarboxylated osteocalcin inhibits the early differentiation of osteoclast mediated by Gprc6a

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10898
Author(s):  
Hailong Wang ◽  
Jinqiao Li ◽  
Zihan Xu ◽  
Feng Wu ◽  
Hongyu Zhang ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Mononuclear Cells ◽  
Luciferase Activity ◽  
Immunofluorescent Staining ◽  
Raw264.7 Cells ◽  
Blue Staining ◽  
Marker Genes ◽  
Undercarboxylated Osteocalcin ◽  
Inhibitory Effects ◽  
Early Differentiation

Osteocalcin (OCN) was the most abundant noncollagen protein and considered as an endocrine factor. However, the functions of Undercarboxylated osteocalcin (ucOCN) on osteoclast and bone resorption are not well understood. In the present study, preosteoclast RAW264.7 cells and bone marrow mononuclear cells (BMMs) were treated with ucOCN purified from prokaryotic bacteria. Our results showed that ucOCN attenuated the proliferation of RAW264.7 cells with a concentration dependant manner by MTS assay. Scrape wounding assay revealed the decreased motility of RAW264.7 cells after ucOCN treatment. RT-qPCR results manifested the inhibitory effects of ucOCN on the expression of osteoclastic marker genes in RAW264.7 cells during inducing differentiation of RANKL. It was also observed that ucOCN inhibited the formation of multinucleated cells from RAW264.7 cells and BMMs detected by TRAP staining. The number and area of bone resorb pits were also decreased after treatment with ucOCN during their osteoclast induction by toluidine blue staining. The formation and integrity of the osteoclast actin ring were impaired by ucOCN by immunofluorescent staining. Time dependant treatment of ucOCN during osteoclastic induction demonstrated the inhibitory effects mainly occurred at the early stage of osteoclastogenesis. Signaling analysis of luciferase activity of the CRE or SRE reporter and ERK1/2 phosphorylation showed the selective inhibitor or siRNA of Gprc6a (a presumptive ucOCN receptor) could attenuate the promotion of ucOCN on CRE-luciferase activity. Taken together, we provided the first evidence that ucOCN had negative effects on the early differentiation and bone resorption of osteoclasts via Gprc6a.

SAT0022 CXCL7 PROMOTES OSTEOCLASTOGENESIS IN RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 941.1-941
Author(s):  
X. Wang ◽  
L. Sun ◽  
J. Zhao
Keyword(s):  
Rheumatoid Arthritis ◽  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Raw264.7 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bone Marrow Niche ◽  
Healthy Donors ◽  
Sat 1

Background:Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by bone destruction[1]. Chemokine signaling by skeletal cells or by other cells of the bone marrow niche regulates bone formation and resorption[2]. Recent studies have found that CXCL7 enhanced the osteoclast formation in mouse bone marrow cells[3, 4]. Whether CXCL7 plays a role in human osteoclastogenesis especially in RA patients remains unclear.Objectives:To examine the functional role of CXCL7 in the induction of osteoclastogenesis in RA.Methods:The level of CXCL7 in CD14+monocyte supernatant was assessed via enzyme-linked immunosorbent assay. Osteoclastogenesis of CD14+monocyte from RA patients and healthy donors were evaluated by TRAP staining and F-actin ring immunofluorescence. Bone resorption pit was observed by scanning electron microscopy. We performed quantitative reverse transcription polymerase chain reaction (RT-PCR) to detect changes in osteoclast markers. RAW264.7 macrophages were also used to investigate specific signaling pathway by which CXCL7 stimulated during osteoclast formation.Results:CXCL7 level in CD14+monocyte supernatant from RA patients (5690 ±627.05 pg/ml) was significantly higher than that in healthy controls (2301 ±535.52 pg/ml) (n=5, P<0.001). In the presence of M-CSF and RANKL, CXCL7 promoted osteoclast formation(Figure 1A and B) and increased bone resorption area(Figure 1C) of CD14+monocyte from healthy donors in the low concentration (10ng/ml) group (n=3, p < 0.05). While in high concentration of CXCL7 (50ng/ml) group, there were no significant changes in the number of osteoclasts. Transcription level of the osteoclast markers RANK, cathepsin K, and MMP-9 was significantly increased in the CXCL7 (10 ng/mL) group after 3 days in the presence of M-CSF and sRANKL (n=3, p < 0.05). When using CD14+ monocyte from RA patients, the optimal concentration of CXCL7 was 50ng/ml, which significantly increased the number of osteoclasts (Figure 2A and B)and bone resorption area (Figure 2C) (n=3, p < 0.01). Flow cytometry analysis revealed that a higher proportion of CD14+monocytes expressed CXCR2 from healthy donors than those from RA patients (n=6, p < 0.01). Consistent with the results obtained in CD14+monocytes, the effects of exogenous CXCL7 on osteoclast formation were also observed in RAW264.7 cells (p < 0.01). The addition of CXCL7 dramatically promoted phosphorylation ERK1/2 in RAW264.7 cells, but it did not affect the phosphorylation of P65.Conclusion:CXCL7 level in CD14+monocyte supernatant was higher in RA patients than that of healthy donors. CXCL7 promoted osteoclastogenesis in CD14+monocyte both from RA patients and healthy donors. CXCL7 could be a potential therapeutic target for bone destruction in RA.References:[1] McInnes, I.B. and G. Schett, The pathogenesis of rheumatoid arthritis. N Engl J Med, 2011. 365(23): p. 2205-19.[2] Brylka, L.J. and T. Schinke, Chemokines in Physiological and Pathological Bone Remodeling. Front Immunol, 2019. 10: p. 2182.[3] Nakao, K., et al., IGF2 modulates the microenvironment for osteoclastogenesis. Biochem Biophys Res Commun, 2009. 378(3): p. 462-6.[4] Goto, Y., et al., CXCR4(+) CD45(-) Cells are Niche Forming for Osteoclastogenesis via the SDF-1, CXCL7, and CX3CL1 Signaling Pathways in Bone Marrow. Stem Cells, 2016. 34(11): p. 2733-2743.Acknowledgments :We gratefully thank the Medical Research Center of Peking University Third Hospital for providing experimental equipment and technical support.Disclosure of Interests:None declared

Characterization of inorganic phosphate transport in osteoclast-like cells

2005 ◽  
Vol 288 (4) ◽  
pp. C921-C931 ◽  
Author(s):  
Mikiko Ito ◽  
Naoko Matsuka ◽  
Michiyo Izuka ◽  
Sakiko Haito ◽  
Yuko Sakai ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Transport System ◽  
Inorganic Phosphate ◽  
Phosphate Transport ◽  
Transport Systems ◽  
Raw264.7 Cells ◽  
Transport Activity ◽  
Phosphonoformic Acid ◽  
Carbonyl Cyanide ◽  
Bone Particles

Osteoclasts possess inorganic phosphate (Pi) transport systems to take up external Pi during bone resorption. In the present study, we characterized Pi transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-κB ligand (RANKL). In undifferentiated RAW264.7 cells, Pi transport into the cells was Na+ dependent, but after treatment with RANKL, Na+-independent Pi transport was significantly increased. In addition, compared with neutral pH, the activity of the Na+-independent Pi transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na+-independent system consisted of two components with Km of 0.35 mM and 7.5 mM. The inhibitors of Pi transport, phosphonoformic acid, and arsenate substantially decreased Pi transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K+ ionophore, valinomycin, significantly suppressed Pi transport activity. Analysis of BCECF fluorescence indicated that Pi transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K+ ion stimulated Pi transport, suggesting that membrane voltage is involved in the regulation of Pi transport activity. Finally, bone particles significantly increased Na+-independent Pi transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a Pi transport system with characteristics that are different from those of other Na+-dependent Pi transporters. We conclude that stimulation of Pi transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment.

IL-17A suppresses the expression of bone resorption-related proteinases and osteoclast differentiation via IL-17RA or IL-17RC receptors in RAW264.7 cells

Biochimie ◽  
2010 ◽  
Vol 92 (4) ◽  
pp. 398-404 ◽  
Author(s):  
Satoshi Kitami ◽  
Hideki Tanaka ◽  
Takayuki Kawato ◽  
Natsuko Tanabe ◽  
Tomoko Katono-Tani ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Osteoclast Differentiation ◽  
Raw264.7 Cells

scholarly journals Inhibition Effect of Zoledronate on the Osteoclast Differentiation of RAW264.7 Induced by Titanium Particles

2021 ◽  
Vol 2021 ◽  
pp. 1-7
Author(s):  
Wenhan Zhao ◽  
Zhusong Huang ◽  
Yu Lin ◽  
Jinfu Lan ◽  
Xi Gao
Keyword(s):  
Bone Resorption ◽  
Protein Expression ◽  
Osteoclast Differentiation ◽  
Raw264.7 Cells ◽  
Blue Staining ◽  
Toluidine Blue Staining ◽  
Mrna And Protein Expression ◽  
Polymerase Chain ◽  
Ti Particles

Objective. This study is aimed at studying the effect of zoledronate (ZOL) on the differentiation of osteoclast precursor RAW264.7 cells induced by titanium (Ti) particles and explores the possibility of preventing and treating periprosthetic osteoporosis using ZOL. Methods. RAW264.7 cells were cultured in vitro. Ti particles were prepared. The cell proliferation curve of RAW264.7 cells was plotted using the MTT assay to find the best concentration of ZOL for intervention. The cells were divided into three groups: control, Ti particles, and Ti particles+ZOL. The cell morphology was observed using tartaric acid–resistant acid phosphatase (TRAP) staining, and the activity of TRAP in cell supernatant was determined using the biochemical method. The number of bone resorption lacunae was detected using toluidine blue staining. The mRNA expression of RANK, NFATcl, CAII, and MMP-9 was detected using real-time polymerase chain reaction. The protein expression of RANK, NFATcl, and MMP-9 was detected using Western blot analysis. Results. Ti particles stimulated the differentiation of RAW264.7 cells into osteoclasts. They also increased the activity of TRAP, number of bone resorption lacunae, and mRNA and protein expression of RANK, NFATcl, and MMP-9. However, ZOL could suppress the effect of TI particles on the osteoclast differentiation of RAW264.7 cells. Conclusions. ZOL could effectively inhibit the differentiation of RAW264.7 cells into osteoclasts induced by Ti particles, decrease the activity of TRAP, reduce the number of bone resorption lacunae, and decrease the mRNA and protein expression of RANK, NFATcl, and MMP-9. Hence, it may be a promising candidate for preventing and treating periprosthetic osteoporosis after the artificial joint operation.

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