Abstract 19967: Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Michael K Delaney ◽  
Kyungho Kim ◽  
Brian Estevez ◽  
Aleksandra Stojanovic-Terpo ◽  
Bo Shen ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Platelet Activation ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Ros Generation ◽  
Ros Production ◽  
Oxygen Species ◽  
General Role ◽  
Glycoprotein Vi ◽  
Activation Pathways

Objective: Reactive oxygen species (ROS) generated from activated platelets is known to regulate platelet activation. However, it remains unclear whether and how different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs) play roles in different platelet activation pathways. Here we investigated the role of NOX1 and NOX2 in different platelet activation pathways using NOX1 and NOX2 knockout mice. Approach and Results: NOX1-/- platelets showed selective defects in G protein coupled receptor (GPCR)-mediated platelet activation induced by thrombin, protease-activated receptor 4 agonist peptide (PAR4AP) and thromboxane A2 analog U46619, but was not affected in platelet activation induced by collagen-related peptide (CRP), a glycoprotein VI (GPVI) agonist. In contrast, NOX2-/- platelets showed potent inhibition of CRP-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet aggregation and secretion. Consistently, production of reactive oxygen species (ROS) was inhibited in NOX1-/- platelets stimulated with thrombin, but not CRP, whereas NOX2-/- platelets showed reduced ROS generation induced by CRP or thrombin. Interestingly, laser-induced arterial thrombosis was impaired in NOX2-/- mice, and in thrombocytopenic mice transfused with NOX2-/- platelets, suggesting an important role for NOX2-dependent platelet ROS production in the laser-induced injury model of thrombosis. Conclusions: NOX1 and NOX2 play differential roles in different platelet activation pathways: NOX1 mediates GPCR-mediated ROS production and platelet activation, whereas NOX2 plays a general role in GPVI- and GPCR-induced ROS production and platelet activation in vitro , and in laser-induced thrombosis in vivo .

Abstract 54: Differential Regulation of NADPH-oxidases 1 and 2 and a Common Syk/PLC/Calcium-dependent ROS Signaling Pathway Mediating Platelet Activation

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Zheng Xu
Keyword(s):  
Reactive Oxygen Species ◽  
Platelet Aggregation ◽  
Signaling Pathway ◽  
Platelet Activation ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Differential Regulation ◽  
Ros Generation ◽  
Oxygen Species ◽  
Activation Pathways

Objective: Reactive oxygen species (ROS) generated from activated platelets is known to regulate platelet activation. This study investigates how different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs) mediates different platelet activation pathways. Approach and Results: ROS generation in different platelet activation pathways are mediated differentially by NOX1 and NOX2. NOX1 -/y platelets showed no defects in platelet aggregation and secretion induced by glycoprotein (GP) VI agonists, collagen-related peptide (CRP), but were partially defective in platelet aggregation and secretion induced by low doses of agonists of G protein coupled receptor (GPCR), thrombin, protease-activated receptor 4 agonist peptide (PAR4AP) and thromboxane A2 analog U46619. In contrast, NOX2 -/- platelets showed significantly defective platelet aggregation and secretion induced by CRP, and also showed partial inhibition of thrombin-induced platelet aggregation and secretion. Consistently, production of reactive oxygen species (ROS) was inhibited in NOX1 -/- platelets stimulated with thrombin, but not CRP, whereas NOX2 -/- platelets were defective ROS generation induced by CRP or thrombin. These differential effects of NOX1 and NOX2 is likely due to upstream differential regulation of these different enzymes, as thrombin-stimulated NOX1-/y platelets and CRP-stimulated NOX2-/- platelets similarly showed defective activation of tyrosine kinase Syk, its downstream target phospholipase Cγ (PLCγ) and calcium mobilization, which is mediated by PLC. Furthermore, mitogen-activated protein kinase pathways, which is another important platelet activation pathway was not significantly affected in either NOX1-/y or NOX2-/- platelets. Finally, NOX-/- platelets is defective in mediating arteriolar thrombosis in vivo, although minimally affected tail bleeding time. Conclusions: NOX1 and NOX2 play differential roles in different platelet activation pathways. The differential roles of these enzyme are due to differential upstream regulation. Both NOX isoforms mediates platelet activation via a common ROS-dependent activation Src-PLC-calcium signaling pathway.

scholarly journals Reactive Oxygen Species in Host Plant Are Required for an Early Defense Response against Attack of Stagonospora nodorum Berk. Necrotrophic Effectors SnTox

Plants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1586
Author(s):  
Svetlana Veselova ◽  
Tatyana Nuzhnaya ◽  
Guzel Burkhanova ◽  
Sergey Rumyantsev ◽  
Igor Maksimov
Keyword(s):  
Reactive Oxygen Species ◽  
Early Stage ◽  
Reactive Oxygen ◽  
Triticum Aestivum L ◽  
Redox Status ◽  
Stagonospora Nodorum ◽  
Ros Generation ◽  
Ros Production ◽  
Oxygen Species ◽  
Necrotrophic Effectors

Reactive oxygen species (ROS) play a central role in plant immune responses. The most important virulence factors of the Stagonospora nodorum Berk. are multiple fungal necrotrophic effectors (NEs) (SnTox) that affect the redox-status and cause necrosis and/or chlorosis in wheat lines possessing dominant susceptibility genes (Snn). However, the effect of NEs on ROS generation at the early stages of infection has not been studied. We studied the early stage of infection of various wheat genotypes with S nodorum isolates -Sn4VD, SnB, and Sn9MN, carrying a different set of NE genes. Our results indicate that all three NEs of SnToxA, SnTox1, SnTox3 significantly contributed to cause disease, and the virulence of the isolates depended on their differential expression in plants (Triticum aestivum L.). The Tsn1–SnToxA, Snn1–SnTox1and Snn3–SnTox3 interactions played an important role in inhibition ROS production at the initial stage of infection. The Snn3–SnTox3 inhibited ROS production in wheat by affecting NADPH-oxidases, peroxidases, superoxide dismutase and catalase. The Tsn1–SnToxA inhibited ROS production in wheat by affecting peroxidases and catalase. The Snn1–SnTox1 inhibited the production of ROS in wheat by mainly affecting a peroxidase. Collectively, these results show that the inverse gene-for gene interactions between effector of pathogen and product of host sensitivity gene suppress the host’s own PAMP-triggered immunity pathway, resulting in NE-triggered susceptibility (NETS). These results are fundamentally changing our understanding of the development of this economical important wheat disease.

scholarly journals CYP450 Mediates Reactive Oxygen Species Production in a Mouse Model of β-Thalassemia through an Increase in 20-HETE Activity

2021 ◽  
Vol 22 (3) ◽  
pp. 1106
Author(s):  
Rayan Bou-Fakhredin ◽  
Batoul Dia ◽  
Hilda E. Ghadieh ◽  
Stefano Rivella ◽  
Maria Domenica Cappellini ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Mouse Model ◽  
Cell Injury ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Ros Generation ◽  
Oxygen Species ◽  
Species Production ◽  
Different Sources ◽  
Dependent Pathway

Oxidative damage by reactive oxygen species (ROS) is one of the main contributors to cell injury and tissue damage in thalassemia patients. Recent studies suggest that ROS generation in non-transfusion-dependent (NTDT) patients occurs as a result of iron overload. Among the different sources of ROS, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes and cytochrome P450 (CYP450) have been proposed to be major contributors for oxidative stress in several diseases. However, the sources of ROS in patients with NTDT remain poorly understood. In this study, Hbbth3/+ mice, a mouse model for β-thalassemia, were used. These mice exhibit an unchanged or decreased expression of the major NOX isoforms, NOX1, NOX2 and NOX4, when compared to their C57BL/6 control littermates. However, a significant increase in the protein synthesis of CYP4A and CYP4F was observed in the Hbbth3/+ mice when compared to the C57BL/6 control mice. These changes were paralleled by an increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a CYP4A and CYP4F metabolite. Furthermore, these changes corroborate with onset of ROS production concomitant with liver injury. To our knowledge, this is the first report indicating that CYP450 4A and 4F-induced 20-HETE production mediates reactive oxygen species overgeneration in Hbbth3/+ mice through an NADPH-dependent pathway.

183 ADDITION OF DIPHENYLENEIODONIUM OR DEHYDROEPIANDROSTERONE TO CULTURE MEDIA INHIBITS REACTIVE OXYGEN SPECIES PRODUCTION DURING THE EARLY CLEAVAGE STAGES OF IN VITRO-PRODUCED PORCINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 208
Author(s):  
N. W. K. Karja ◽  
K. Kikuchi ◽  
M. Ozawa ◽  
M. Fahrudin ◽  
T. Somfai ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Nadph Oxidase ◽  
Culture Media ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Blastocyst Stage ◽  
Control Group ◽  
Ros Production ◽  
Oxygen Species

Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), an enzyme required to catalyze the oxidation of NADPH to NADP during the metabolism of glucose via the pentose phosphate pathway (PPP), was considered as contributing to intracellular reactive oxygen species (ROS) production. Production of superoxide anion and H2O2 via NADPH oxidase has been reported on a rabbit blastocyst surface (Manes and Lai 1995 J. Reprod. Fertil. 104, 69–75). The objective of this study was to examine the effects on in vitro development and intracellular ROS content after the addition of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, or dehydroepiandrosterone (DHEA), an inhibitor of glucose-6-phosphate dehydrogenase (G6PDH), to culture medium during the early embryonic development of in vitro-produced (IVP) porcine embryos. To confirm that these inhibitors lead to reduction in NADPH concentration in the embryo and hence likely to be inhibiting the PPP, a brilliant cresyl blue (BCB) test was performed on Day 2 (the day of insemination = Day 0) of culture. Porcine cumulus–oocyte complexes were matured and fertilized in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Prezumptive zygotes were then cultured in NCSU-37 supplemented with 5.5 mM glucose and DPI at concentrations of 0.5 or 1 nM or DHEA at concentrations of 10 or 100 �M (DPI-0.5, DPI-1, DHEA-10 and DHEA-100 groups, respectively) from Day 0 to Day 2 of culture. All of the embryos were cultured subsequently until Day 6 in NCSU-37 supplemented with only 5.5 mM glucose. Data were analyzed by ANOVA. On Day 6, the development to the blastocyst stage of embryos in DPI-0.5, DPI-1, DHEA-10, and DHEA-100 groups were 16.1, 17.6, 16.1, and 19.5%, respectively, which were not significantly different from that of the control group (17.5%) (n d 165 per group, 5 replicates). However, the mean cell number in blastocysts derived from DPI-1, DHEA-10, and DHEA-100 groups (40.8 � 2.3, 39.3 � 1.7, and 42.5 � 2.7, respectively) was significantly higher (P < 0.01) than those in the control (33.4 � 1.6) and DPI-0.5 (32.7 � 1.6) groups. At 20 min after an exposure to BCB, the percentage of BCB+ embryos in DPI-1, DHEA-10, and DHEA-100 groups (73.8, 79.9, and 77.8%, respectively) were significantly higher (P < 0.01) than those in the control and DPI-0.5 groups (42% and 53.9%, respectively) (n = 81-92 per group, 6 replicates), indicating that these two inhibitors effectively induce the reduction of NADPH concentration in the embryos. Moreover, the addition of DPI at 1 nM or DHEA at 10 or 100 �M significantly decreased the H2O2 content of Day 2 embryos as compared with control embryos (n = 48-53 per group, 7 replicates). These results suggest that the addition of either DPI or DHEA to the medium during the first 2 days of culture did not impair the development of the embryos to the blastocyst stage. Decrease of cellular ROS production in Day 2 embryos in this study is interpreted as a result of inhibition of the NADPH oxidase by DPI or of the G6PDH by DHEA.

scholarly journals Impact of CD14 on Reactive Oxygen Species Production from Human Leukocytes Primed byEscherichia coliLipopolysaccharides

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Dmitry S. Kabanov ◽  
Olga Yu. Vwedenskaya ◽  
Marina A. Fokina ◽  
Elena M. Morozova ◽  
Sergey V. Grachev ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Polymorphonuclear Neutrophils ◽  
Ros Generation ◽  
Ros Production ◽  
Oxygen Species ◽  
Human Pmns ◽  
Species Production ◽  
The Impact ◽  
Priming Activity

Lipopolysaccharides (LPS) from Gram-negative bacteria prime human polymorphonuclear neutrophils (PMNs) via multicomponent receptor cluster including CD14 and MD-2·TLR4 for the enhanced release of reactive oxygen species (ROS) were triggered by bacterial derived peptideN-formyl-methionyl-leucyl-phenylalanine (fMLP). In this study, we investigated the impact of CD14 on LPS-induced priming of human PMNs for fMLP-triggered ROS generation (respiratory or oxidative) burst. Monoclonal antibodies against human CD14 (mAbs) as well as isotype-matched IgG2a did not influence significantly fMLP-triggered ROS production from LPS-unprimed PMNs. Anti-CD14 mAbs (clone UCHM-1) attenuated LPS-induced priming of PMNs as it had been mirrored by fMLP-triggered decrease of ROS production. Similar priming activity of S-LPS or Re-LPS fromEscherichia colifor fMLP-triggered ROS release from PMNs was found. Obtained results suggest that glycosylphosphatidylinositol-anchored CD14 is the key player in LPS-induced PMN priming for fMLP-triggered ROS production. We believe that blockade of CD14 on the cell surface and clinical use of anti-CD14 mAbs or their Fab fragments may diminish the production of ROS and improve outcomes during cardiovascular diseases manifested by LPS-induced inflammation.

Reactive Oxygen Species Are Differentially Regulated in Chronic Myeloid Leukemia Versus Philadelphia Negative Myeloproliferative Neoplasms

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4215-4215
Author(s):  
Estelle Guerin ◽  
Francis Belloc ◽  
Gabriel Etienne ◽  
Pierre Duffau ◽  
Francois-Xavier Mahon ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Myeloproliferative Neoplasms ◽  
Reactive Oxygen ◽  
Ros Generation ◽  
Separate Analysis ◽  
Ros Production ◽  
Oxygen Species ◽  
Normal Cells

Abstract Deregulation of tyrosine-kinases is a characteristic of most Myeloproliferative Neoplasms (MPN); evolution from chronic phase to acute leukemia depends on the acquisition of additional mutations. Reactive Oxygen Species (ROS), the production of which is increased by tyrosine-kinase activation, can be responsible for additional mutations. The role of ROS in generating genetic aberrations has been mainly studied in BCR-ABL-positive cell lines. Little is known of ROS metabolism in primary cells from CML or Philadelphia-negative MPN (Ph-MPN). After informed consent, cells from blood or bone marrow were obtained from patients diagnosed with CML (12 bone marrow (BM), 8 peripheral blood (PB)), or Ph-MPN (4 Polycythemia Vera, 6 Essential Thrombocythemia, 3 Primary Myelofibroses, 2 atypical CML) and from healthy donors (bone marrow donors) or patients devoid of hematological disease undergoing thoracotomy. Cells were incubated with DCFDA, a fluorogenic marker of ROS production, labelled with an anti-CD45 antibody, stimulated with either the oxidant hydrogen peroxide (H2O2) or the PKC activator Phorbol Myristate Acetate (PMA), and analysed for ROS production by flow cytometry. CD45/SSC gating allowed separate analysis of granulocytes, monocytes or lymphocytes. The basal level of ROS was not higher in CML cells as compared to normal BM or PB leukocytes. It was even significantly lower in CML lymphocytes, either from the BM (2.35 Arbitrary Units vs 8.3 AU, p=5.5 10−5) or PB (2.47 AU vs 7.4 AU, p=3.10−5) and in CML granulocytes from peripheral blood (14 AU vs 45 AU, p =10 −5), but not bone marrow. The ROS levels of Ph-MPN cells were similar or slightly higher than control cells. Upon H2O2 stimulation however, ROS production increased significantly more in CML cells as compared to normal cells (6 fold increase), whatever the cell type (granulocytes, monocytes and lymphocytes) or their origin (PB or BM). In contrast, for Ph-MPN cells, H2O2-stimulated ROS production was close to that of normal cells, with only BM lymphocytes showing ROS generation four fold higher than control BM lymphocytes. After PMA stimulation, which yielded a more modest ROS production than H2O2, CML cells behaved similarly to normal cells, whereas ROS production was four fold higher in Ph-MPN cells, whatever their type and origin. In conclusion, ROS levels at the basal stage are not higher in MPN cells, whether they are Philadelphia positive or negative, as compared to normal cells. Various kinds of stimulation induce different patterns of response, CML cells being more sensitive to oxidants whereas Ph-MPN cells respond more to the cytokine-mimicking agent PMA. These results suggest that the mechanisms of ROS generation and thus of genetic instability are different in CML and Ph-MPN.

scholarly journals Wear Particles Promote Reactive Oxygen Species-Mediated Inflammation via the Nicotinamide Adenine Dinucleotide Phosphate Oxidase Pathway in Macrophages Surrounding Loosened Implants

2015 ◽  
Vol 35 (5) ◽  
pp. 1857-1867 ◽  
Author(s):  
Weishen Chen ◽  
Ziqing Li ◽  
Ying Guo ◽  
Yuhuan Zhou ◽  
Ziji Zhang ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Nicotinamide Adenine ◽  
Ros Generation ◽  
Wear Particles ◽  
Oxygen Species ◽  
Prosthesis Loosening ◽  
Ti Particles

Background/Aims: Prosthesis loosening is closely associated with chronic inflammatory cytokine secretion by macrophages, which are activated by wear particles or inflammatory stimulants such as lipopolysaccharide (LPS). Reactive oxygen species (ROS) are critical regulators of inflammation, but their enzymatic sources in response to wear particles and their effects on peri-implant LPS-tolerance remain unclear. Methods: Three ROS-related enzymes—nicotinamide adenine dinucleotide phosphate oxidase (NOX)-1 and -2 and catalase—were investigated in interface membrane tissues and in titanium (Ti) particle-stimulated macrophages in vitro. The generation of ROS and downstream inflammatory effects were measured with or without pre-incubation with apocynin, an NOX inhibitor. Results: Pre-exposure to Ti particles attenuated NF-κB activation in LPS-stimulated macrophages, indicating that wear particles suppress immune response, which may lead to chronic inflammation. NOX-1 and -2 were highly expressed in aseptically loosened interface membranes and in macrophages stimulated with Ti particles; the particles induced a moderate amount of ROS generation, NF-κB activation, and TNF-a secretion in macrophages, and these effects were suppressed by apocynin. Conclusion: Wear particles induce ROS generation through the NOX signaling pathway, resulting in persistent inflammation and delayed loosening. Thus, the suppression of NOX activity may be a useful strategy for preventing prosthesis loosening.

scholarly journals Bufalin Induces Reactive Oxygen Species Dependent Bax Translocation and Apoptosis in ASTC-a-1 Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Lei Sun ◽  
Tongsheng Chen ◽  
Xiaoping Wang ◽  
Yun Chen ◽  
Xunbin Wei
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Temporal Dynamics ◽  
Reactive Oxygen ◽  
Ros Generation ◽  
Ros Production ◽  
Oxygen Species ◽  
Induced Apoptosis

Bufalin has been shown to induce cancer cell death through apoptotic pathways. However, the molecular mechanisms are not well understood. In this study, we used the confocal fluorescence microscopy (CFM) to monitor the spatio-temporal dynamics of reactive oxygen species (ROS) production, Bax translocation and caspase-3 activation during bufalin-induced apoptosis in living human lung adenocarcinoma (ASTC-a-1) cells. Bufalin induced ROS production and apoptotic cell death, demonstrated by Hoechst 33258 staining as well as flow cytometry analysis. Bax redistributed from cytosol to mitochondria from 12 to 48 h after bufalin treatment in living cells expressed with green fluorescent protein Bax. Treatment with the antioxidantN-acetyl-cysteine (NAC), a ROS scavenger, inhibited ROS generation and Bax translocation and led to a significant protection against bufalin-induced apoptosis. Our results also revealed that bufalin induced a prominent increase of caspase-3 activation blocked potently by NAC. Taken together, bufalin induced ROS-mediated Bax translocation, mitochondrial permeability transition and caspase-3 activation, implying that bufalin induced apoptosis via ROS-dependent mitochondrial death pathway in ASTC-a-1 cells.

scholarly journals The impact of reactive oxygen species in the development of cardiometabolic disorders: a review

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Roland Akhigbe ◽  
Ayodeji Ajayi
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Acid Oxidation ◽  
Ros Generation ◽  
Metabolic Memory ◽  
Oxygen Species ◽  
Cardiometabolic Disorders ◽  
The Impact

AbstractOxidative stress, an alteration in the balance between reactive oxygen species (ROS) generation and antioxidant buffering capacity, has been implicated in the pathogenesis of cardiometabolic disorders (CMD). At physiological levels, ROS functions as signalling mediators, regulates various physiological functions such as the growth, proliferation, and migration endothelial cells (EC) and smooth muscle cells (SMC); formation and development of new blood vessels; EC and SMC regulated death; vascular tone; host defence; and genomic stability. However, at excessive levels, it causes a deviation in the redox state, mediates the development of CMD. Multiple mechanisms account for the rise in the production of free radicals in the heart. These include mitochondrial dysfunction and uncoupling, increased fatty acid oxidation, exaggerated activity of nicotinamide adenine dinucleotide phosphate oxidase (NOX), reduced antioxidant capacity, and cardiac metabolic memory. The purpose of this study is to discuss the link between oxidative stress and the aetiopathogenesis of CMD and highlight associated mechanisms. Oxidative stress plays a vital role in the development of obesity and dyslipidaemia, insulin resistance and diabetes, hypertension via various mechanisms associated with ROS-led inflammatory response and endothelial dysfunction.

A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation

Blood ◽  
2005 ◽  
Vol 106 (3) ◽  
pp. 852-859 ◽  
Author(s):  
Na Kyung Lee ◽  
Young Geum Choi ◽  
Ji Youn Baik ◽  
Song Yi Han ◽  
Dae-won Jeong ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Osteoclast Differentiation ◽  
Tumor Necrosis Factor Receptor ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Ros Production ◽  
Oxygen Species ◽  
Diphenylene Iodonium

Abstract Signaling by receptor activator of NF-κB (nuclear factor-κB) ligand (RANKL) is essential for differentiation of bone marrow monocyte-macrophage lineage (BMM) cells into osteoclasts. Here, we show RANKL stimulation of BMM cells transiently increased the intracellular level of reactive oxygen species (ROS) through a signaling cascade involving TNF (tumor necrosis factor) receptor-associated factor (TRAF) 6, Rac1, and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox) 1. A deficiency in TRAF6 or expression of a dominant-interfering mutant of TRAF6 blocks RANKL-mediated ROS production. Application of N-acetylcysteine (NAC) or blocking the activity of Nox, a protein leading to the formation of ROS, with diphenylene iodonium (DPI) inhibits the responses of BMM cells to RANKL, including ROS production, activation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and extracellular signal-regulated kinase (ERK), and osteoclast differentiation. Moreover, both RANKL-mediated ROS production and osteoclast differentiation were completely blocked in precursors depleted of Nox1 activity by RNA interference or by expressing a dominant-negative mutant of Rac1. Together, these results indicate that ROSs act as an intracellular signal mediator for osteoclast differentiation.

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