Profiling signalling pathways of the receptor activator of NF-κB ligand-induced osteoclast formation in mouse monocyte cells, RAW264.7

Kim, M. H.; Kim, B. T.; Min, Yong Ki; Kim, S. H.

doi:10.1007/s00726-006-0461-4

Cited by 7 publications

(7 citation statements)

References 33 publications

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“…Our data additionally show that cathepsin B appears to promote the proteolysis seen during osteoclastogenesis. Previous studies have indicated negative effects of intracellular cathepsin B inhibitors on osteoclast activity and, more recently, formation, although the mechanisms by which cathepsin B promotes bone resorption have remained a mystery (51,54). In this study, we have identified a role for cathepsin B in osteoclast function through its modulation of myosin IIA levels, and subsequently, osteoclast formation.…”

Section: Discussionmentioning

confidence: 71%

Regulated Proteolysis of Nonmuscle Myosin IIA Stimulates Osteoclast Fusion

McMichael

Wysolmerski

Lee

2009

Journal of Biological Chemistry

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The nonmuscle myosin IIA heavy chain (Myh9) is strongly associated with adhesion structures of osteoclasts. In this study, we demonstrate that during osteoclastogenesis, myosin IIA heavy chain levels are temporarily suppressed, an event that stimulates the onset of cell fusion. This suppression is not mediated by changes in mRNA or translational levels but instead is due to a temporary increase in the rate of myosin IIA degradation. Intracellular activity of cathepsin B is significantly enhanced at the onset of osteoclast precursor fusion, and specific inhibition of its activity prevents myosin IIA degradation. Further, treatment of normal cells with cathepsin B inhibitors during the differentiation process reduces cell fusion and bone resorption capacity, whereas overexpression of cathepsin B enhances fusion. Ongoing suppression of the myosin IIA heavy chain via RNA interference results in formation of large osteoclasts with significantly increased numbers of nuclei, whereas overexpression of myosin IIA results in less osteoclast fusion. Increased multinucleation caused by myosin IIA suppression does not require RANKL. Further, knockdown of myosin IIA enhances cell spreading and lessens motility. These data taken together strongly suggest that base-line expression of nonmuscle myosin IIA inhibits osteoclast precursor fusion and that a temporary, cathepsin B-mediated decrease in myosin IIA levels triggers precursor fusion during osteoclastogenesis.The final stages of osteoclastogenesis involve fusion of differentiated precursors from the monocyte/macrophage lineage (1). Although the membrane structural components regulating preosteoclast fusion are not well understood, in recent years a number of candidate cell surface molecules have been implicated, including receptors CD44 (2, 3), CD47 and its ligand macrophage fusion receptor (also known as signal regulatory protein ␣) (4 -6), the purinergic receptor P2X 7 (7), and the disintegrin and metalloproteinase ADAM8 (8). A recently identified receptor, the dendritic cell-specific transmembrane protein, is essential for osteoclast fusion both in vitro and in vivo (9, 10). More recently, the d2 subunit of proton-translocating vacuolar proton-translocating ATPases, a membrane subunit isoform expressed predominantly in osteoclasts, similarly was demonstrated to be required for fusion in vitro and in vivo (11). However, elucidation of the mechanisms by which these molecules may mediate cell fusion has proved to be difficult.The mammalian class II myosin family consists of distinct isoforms expressed in skeletal, smooth, and cardiac muscle, as well as three nonmuscle forms designated IIA, IIB,. Although all class II molecules are composed of two heavy chains, two essential light chains, and two regulatory chains, their unique activities are a function of their particular heavy chain isoforms. Although the nonmuscle heavy chain isoforms share extensive structural homology, they have been shown to demonstrate distinct patterns of expression (15-18), enzyme kinetics and activ...

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Section: Discussionmentioning

confidence: 71%

Regulated Proteolysis of Nonmuscle Myosin IIA Stimulates Osteoclast Fusion

McMichael

Wysolmerski

Lee

2009

Journal of Biological Chemistry

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“…Cultures were maintained at 37°C in a 5% CO 2 humidified atmosphere and experiments were conducted on cells at approximately 80–90% confluence [19]. …”

Section: Methodsmentioning

confidence: 99%

5,7‐Dihydroxyflavone Analogues May Regulate Lipopolysaccharide‐Induced Inflammatory Responses by Suppressing IκBα‐Linked Akt and ERK5 Phosphorylation in RAW 264.7 Macrophages

Nishina

Shimizu

Koketsu

et al. 2017

Evidence-Based Complementary and Alternative Medicine

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We studied the anti-inflammatory activity of twelve 5,7-dihydroxyflavone analogues in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. We found that chrysin (1) and 4′-methoxytricetin (9) showed relatively significant anti-inflammatory activity and low cytotoxicity. Moreover, 1 and 9 recovered the expression levels of iNOS and COX2, as well as those of the intracellular inflammatory mediators IL-1β and IL-6, which were upregulated by LPS stimulation. In addition, 1 and 9 actively regulated the phosphorylation of IκBα, leading to the activation of NFκB. Phosphorylation of Akt and ERK5 (upstream of NFκB) by LPS stimulation was significantly regulated by 1 and 9, as well as by BIX 02189 and LY 294002, which are phosphorylation inhibitors of ERK5 and Akt, respectively. The results suggest that compounds 1 and 9 may suppress the levels of iNOS and COX2 by regulating phosphorylation of Akt, ERK5, and IκBα and thus NFκB-related signaling pathways, resulting in anti-inflammatory effects in the cells. Because 1 and 9 showed low cytotoxicity and regulated both PGE2 and NO production caused by inflammatory responses, they may hold promise as natural anti-inflammatory agents.

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“…Total protein in each fraction was determined using the Roti ® Quant assay kit (Roth, Karlsruhe, Germany). Fractions were analyzed by Western blot for the presence of the raft marker EGF receptor [4], [48], [50]–[52] (using anti-EGFR antibody, Biolegend, Diego, USA) as well as the plasma membrane marker transferrin receptor [8], [12], [48] (using anti-TFRC antibody, GeneTex, Irvine, USA). EGF receptor was found to be enriched in fraction 1 ( = membrane raft fraction), transferrin receptor was found to be enriched in fraction 7 ( = plasma membrane fraction).…”

Section: Methodsmentioning

confidence: 99%

Fatty Acid and Peptide Profiles in Plasma Membrane and Membrane Rafts of PUFA Supplemented RAW264.7 Macrophages

et al. 2011

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The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.

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Profiling signalling pathways of the receptor activator of NF-κB ligand-induced osteoclast formation in mouse monocyte cells, RAW264.7

Cited by 7 publications

References 33 publications

Regulated Proteolysis of Nonmuscle Myosin IIA Stimulates Osteoclast Fusion

Regulated Proteolysis of Nonmuscle Myosin IIA Stimulates Osteoclast Fusion

5,7‐Dihydroxyflavone Analogues May Regulate Lipopolysaccharide‐Induced Inflammatory Responses by Suppressing IκBα‐Linked Akt and ERK5 Phosphorylation in RAW 264.7 Macrophages

Fatty Acid and Peptide Profiles in Plasma Membrane and Membrane Rafts of PUFA Supplemented RAW264.7 Macrophages

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