scholarly journals Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 117
Author(s):  
Nadia Lampiasi ◽  
Roberta Russo ◽  
Igor Kireev ◽  
Olga Strelkova ◽  
Oxana Zhironkina ◽  
...  
Keyword(s):  
Gene Expression ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Protein Distribution ◽  
Cytoskeletal Organization ◽  
Receptor Activator ◽  
And Behavior ◽  
Nuclear Factor Κ B ◽  
Rankl Stimulation

The development of multi-nucleated cells is critical for osteoclasts (OCs) maturation and function. Our objective was to extend knowledge on osteoclastogenesis, focusing on pre-OC fusion timing and behavior. RAW 264.7 cells, which is a murine monocyte-macrophage cell line, provide a valuable and widely used tool for in vitro studies on osteoclastogenesis mechanisms. Cells were treated with the receptor activator of nuclear factor κ-B ligand (RANKL) for 1–4 days and effects on cell morphology, cytoskeletal organization, protein distribution, and OC-specific gene expression examined by TEM, immunofluorescence, and qPCR. Multinucleated cells began to appear at two days of Receptor Activator of Nuclear factor κ-B Ligand (RANKL) stimulation, increasing in number and size in the following days, associated with morphological and cytoskeletal organization changes. Interesting cellular extensions were observed in three days within cells labeled with wheat germ agglutinin (WGA)-Fluorescein isothiocyanate (FITC). The membrane, cytoplasmic, or nuclear distribution of RANK, TRAF6, p-p38, pERK1/2, and NFATc1, respectively, was related to OCs maturation timing. The gene expression for transcription factors regulating osteoclastogenesis (NFATc1, c-fos, RelA, MITF), molecules involved in RANKL-signaling transduction (TRAF6), cytoskeleton regulation (RhoA), fusion (DC-STAMP), migration (MMP9), and OC-specific enzymes (TRAP, CtsK), showed different trends related to OC differentiation timing. Our findings provide an integrated view on the morphological and molecular changes occurring during RANKL stimulation of RAW 264.7 cells, which are important to better understand the OCs’ maturation processes.

scholarly journals Role of nuclear factor-κ B and heme oxygenase-1 in the mechanism of action of an anti-inflammatory chalcone derivative in RAW 264.7 cells

2004 ◽  
Vol 142 (7) ◽  
pp. 1191-1199 ◽  
Author(s):  
María José Alcaraz ◽  
Ana María Vicente ◽  
Amparo Araico ◽  
José N Dominguez ◽  
María Carmen Terencio ◽  
...  
Keyword(s):  
Heme Oxygenase ◽  
Mechanism Of Action ◽  
Raw 264.7 Cells ◽  
Heme Oxygenase 1 ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Chalcone Derivative ◽  
Anti Inflammatory ◽  
Nuclear Factor Κ B

The Effect of Lunasin on Receptor Activator of Nuclear Factor Kappa-B Ligand−mediated Osteoclast Formation from RAW 264.7 Cells

2018 ◽  
Vol 44 (6) ◽  
pp. 997-999 ◽  
Author(s):  
Daisy Bachala ◽  
Nivine El-Refai ◽  
Edward Greenfield ◽  
Anita Aminoshariae ◽  
Andre Mickel
Keyword(s):  
Nuclear Factor Kappa B ◽  
Osteoclast Formation ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Nuclear Factor Kappa ◽  
Receptor Activator

scholarly journals (−)-Epigallocatechin-3-Gallate Decreases Osteoclastogenesis via Modulation of RANKL and Osteoprotegrin

Molecules ◽  
2019 ◽  
Vol 24 (1) ◽  
pp. 156 ◽  
Author(s):  
Shih-Tse Chen ◽  
Lin Kang ◽  
Chau-Zen Wang ◽  
Peng-Ju Huang ◽  
Hsuan-Ti Huang ◽  
...  
Keyword(s):  
Epigallocatechin Gallate ◽  
Secretory Protein ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Mineral Density ◽  
Protein Levels ◽  
Low Concentrations ◽  
Receptor Activator ◽  
Nuclear Factor Kb

Osteoporosis is the second most common epidemiologic disease in the aging population worldwide. Previous studies have found that frequent tea drinkers have higher bone mineral density and less hip fracture. We previously found that (−)-epigallocatechin gallate (EGCG) (20–100 µmol/L) significantly suppressed receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis and pit formation via inhibiting NF-κB transcriptional activity and nuclear transport of NF-κB in RAW 264.7 cells and murine primary bone marrow macrophage cells. The most important regulation in osteoclastogenesis is the receptor activator of nuclear factor-kB/RANKL/osteoprotegrin (RANK/RANKL/OPG) pathway. In this study, we used the coculture of RAW 264.7 cells and the feeder cells, ST2, to evaluate how EGCG regulated the RANK/RANKL/OPG pathway in RAW 264.7 cells and ST2 cells. We found EGCG decreased the RANKL/OPG ratio in both mRNA expression and secretory protein levels and eventually decreased osteoclastogenesis by TRAP (+) stain osteoclasts and TRAP activity at low concentrations—1 and 10 µmol/L—via the RANK/RANKL/OPG pathway. The effective concentration can be easily achieved in daily tea consumption. Taken together, our results implicate that EGCG could be an important nutrient in modulating bone resorption.

scholarly journals Inhibitory Effects of Astaxanthin on CML-HSA-Induced Inflammatory and RANKL-Induced Osteoclastogenic Gene Expression in RAW 264.7 Cells

Biomedicines ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
A. N. M. Mamun-Or-Rashid ◽  
Tanzima Tarannum Lucy ◽  
Masayuki Yagi ◽  
Yoshikazu Yonei
Keyword(s):  
Gene Expression ◽  
Raw 264.7 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Marker Genes ◽  
Tartrate Resistant Acid Phosphatase ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Inflammatory Gene Expression ◽  
Inflammatory Gene

Objective: Elevated levels of serum Nε-carboxymethyllysine (CML), a well-known advanced glycation end-product (AGE), were observed in patients with inflammation or osteoporosis. Astaxanthin was reported to possess anti-inflammatory and antioxidant effects. In the present study, we investigated the effects of commercially available dietary supplement AstaReal ACTR (ASR) capsule content as astaxanthin on CML-HSA-induced inflammatory and receptor activator of nuclear factor-kappa-Β ligand (RANKL)-induced osteoclastogenic gene expression. Methods: RAW 264.7 murine macrophage cells were stimulated with CML-HSA to trigger inflammatory gene expression and treated with either a vehicle control or varied concentrations of astaxanthin. Inflammatory gene expression was measured using an enzyme-linked immunosorbent assay (ELISA) or qPCR. We triggered osteoclastogenesis using RANKL, and osteoclastogenic gene expression was measured through tartrate-resistant acid phosphatase (TRAP) activity, staining, immunofluorescence, and qPCR analyses. Results: CML-HSA showed a stimulatory effect on inflammatory gene expression, and astaxanthin reduced the expression by at least two-fold. The levels of autoinflammatory gene expression were reduced by astaxanthin. The RANKL-induced osteoclastogenesis was significantly inhibited by astaxanthin, with reductions in the activation of nuclear factor-κB (NF-κB), the expression of NFATc1 (nuclear factor of activated T cells 1), multinucleated cell formation, and the expression of mature osteoclast marker genes. Conclusion: Astaxanthin has potential as a remedy for CML-HSA-induced inflammation and RANKL-induced excessive bone loss.

scholarly journals Febrile-range temperature modifies cytokine gene expression in LPS-stimulated macrophages by differentially modifying NF-κB recruitment to cytokine gene promoters

2010 ◽  
Vol 298 (1) ◽  
pp. C171-C181 ◽  
Author(s):  
Zachary A. Cooper ◽  
Arundhati Ghosh ◽  
Aditi Gupta ◽  
Tapan Maity ◽  
Ivor J. Benjamin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Receptor Activation ◽  
Cytokine Gene Expression ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Heat Shock Factor 1 ◽  
Gene Promoters ◽  
Cytokine Gene ◽  
Tnf Α

We previously showed that exposure to febrile-range temperatures (FRT, 39.5–40°C) reduces LPS-induced TNF-α expression, in part through the direct interaction of heat shock factor-1 (HSF1) with the TNF-α gene promoter. However, it is not known whether exposure to FRT also modifies more proximal LPS-induced signaling events. Using HSF1-null mice, we confirmed that HSF1 is required for FRT-induced repression of TNF-α in vitro by LPS-stimulated bone marrow-derived macrophages and in vivo in mice challenged intratracheally with LPS. Exposing LPS-stimulated RAW 264.7 mouse macrophages to FRT reduced TNF-α expression while increasing IL-1β expression despite the two genes sharing a common myeloid differentiation protein-88 (MyD88)-dependent pathway. Global activation of the three LPS-induced signaling intermediates that lead to cytokine gene expression, ERK and p38 MAPKs and NF-κB, was not affected by exposing RAW 264.7 cells to FRT as assessed by ERK and p38 phosphorylation and NF-κB in vitro DNA-binding activity and activation of a NF-κB-dependent synthetic promoter. However, chromatin immunoprecipitation (ChIP) analysis demonstrated that exposure to FRT reduced LPS-induced recruitment of NF-κB p65 to the TNF-α promoter while simultaneously increasing its recruitment to the IL-1β promoter. These data suggest that FRT exerts its effects on cytokine gene expression in a gene-specific manner through distal effects on promoter activation rather than proximal receptor activation and signal transduction.

cDNA microarray analysis of the gene expression of murine leukemia RAW 264.7 cells after exposure to propofol

2011 ◽  
Vol 28 (8) ◽  
pp. 471-478 ◽  
Author(s):  
Rick Sai-Chuen Wu ◽  
Kuo-Ching Liu ◽  
Nou-Ying Tang ◽  
Hsiung-Kwang Chung ◽  
Siu-Wan Ip ◽  
...  
Keyword(s):  
Gene Expression ◽  
Microarray Analysis ◽  
Cdna Microarray ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Cdna Microarray Analysis ◽  
Murine Leukemia

scholarly journals Anti-Inflammatory Effects of Lasia spinosa Leaf Extract in Lipopolysaccharide-Induced RAW 264.7 Macrophages

2020 ◽  
Vol 21 (10) ◽  
pp. 3439 ◽  
Author(s):  
Thanh Q. C. Nguyen ◽  
Tran Duy Binh ◽  
Tuan L. A. Pham ◽  
Yen D. H. Nguyen ◽  
Dai Thi Xuan Trang ◽  
...  
Keyword(s):  
Leaf Extract ◽  
Inflammatory Diseases ◽  
Raw 264.7 Cells ◽  
Inflammatory Factors ◽  
Heme Oxygenase 1 ◽  
Raw 264.7 ◽  
Related Factor ◽  
Nuclear Factor ◽  
Raw 264.7 Macrophages ◽  
Anti Inflammatory

Lasia spinosa (L.) Thwaites was used as a traditional medicine to treat many inflammatory diseases for centuries. However, its effects on the inflammatory response are not yet characterized. In this study, we investigated the anti-inflammatory activities of L. spinosa leaf extract in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. We found that ethanol extracts of L. spinosa leaves showed anti-oxidant activity due to the presence of high levels of polyphenolic compounds. Treatment with the leaf extract significantly repressed the production of inflammatory mediators such as nitric oxide and reactive oxygen species and the expression of pro-inflammatory cytokines in the LPS-stimulated RAW 264.7 cells. Moreover, L. spinosa leaf extract treatment prevented activation of the nuclear factor-kappa B pathway by inhibiting nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) degradation. Furthermore, the mitogen-activated kinase and phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathways were suppressed upon treatment with the leaf extract. In addition to suppressing inflammatory factors, the extract also activated the nuclear factor erythroid 2-related factor 2/heme-oxygenase-1 pathway. We propose that L. spinosa leaf extract has the potential as an effective therapeutic agent for alleviating oxidative stress and excessive inflammation.

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