Rac Activation Induces NADPH Oxidase Activity in Transgenic COS  Cells, and the Level of Superoxide Production Is Exchange Factor-dependent

Price, Marianne O.; Atkinson, Simon J.; Knaus, Ulla G.; Dinauer, Mary C.

doi:10.1074/jbc.m200061200

Cited by 110 publications

(112 citation statements)

References 76 publications

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“…Such cells expressed normal levels of MEF2C, featured regular sarcomeres, and thus differentiated normally into functional beating cardiac cells. Furthermore, day-to-day addition of 100 nM H 2 O 2 at early stage of differentiation (days 0 -7), at a concentration expected from RacV12 activity within differentiating cells (Price et al, 2002), to the EBs also severely impaired cardiac differentiation of ES cells, as shown by the lack of both transactivation of the MEF2C-dependent ␣-actin promoter and beating activity of EBs. On the other hand, the ROS scavenger, catalase added to the culture medium of EBs rescued MEF2C expression, myofibrillogenesis and in turn beating activity of RacV12 EBs.…”

Section: Discussionmentioning

confidence: 99%

A Dual Role of the GTPase Rac in Cardiac Differentiation of Stem Cells

Pucéat¹,

Travo²,

Quinn³

et al. 2003

MBoC

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The function of the GTPase Rac1, a molecular switch transducing intracellular signals from growth factors, in differentiation of a specific cell type during early embryogenesis has not been investigated. To address the question, we used embryonic stem (ES) cells differentiated into cardiomyocytes, a model that faithfully recapitulates early stages of cardiogenesis. Overexpression in ES cells of a constitutively active Rac (RacV12) but not of an active mutant (RacL61D38), which does not activate the NADPH oxydase generating ROS, prevented MEF2C expression and severely compromised cardiac cell differentiation. This resulted in poor expression of ventricular myosin light chain 2 (MLC2v) and its lack of insertion into sarcomeres. Thus ES-derived cardiomyocytes featured impaired myofibrillogenesis and contractility. Overexpression of MEF2C or addition of catalase in the culture medium rescued the phenotype of racV12 cells. In contrast, RacV12 specifically expressed in ES-derived ventricular cells improved the propensity of cardioblasts to differentiate into beating cardiomyocytes. This was attributed to both a facilitation of myofibrillogenesis and a prolongation in their proliferation. The dominant negative mutant RacN17 early or lately expressed in ES-derived cells prevented myofibrillogenesis and in turn beating of cardiomyocytes. We thus suggest a stage-dependent function of the GTPase during early embryogenesis.

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Section: Discussionmentioning

confidence: 99%

A Dual Role of the GTPase Rac in Cardiac Differentiation of Stem Cells

Pucéat¹,

Travo²,

Quinn³

et al. 2003

MBoC

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“…Proteins that regulate membrane fusion or fission include dynamin 2 [39], amphiphysin IIm [40], Arf6 [41,42], Rab5 [43,44], Rab11 [45], and PLA 2 [46]. Proteins that may affect the assembly and activation of the NADPH oxidase complex include Rac1, Rac2 [10], Vav1 [47], PAK1 [48], protein kinase C (PKC)␣ and PKCε [49,50], myristoylated alanine-rich C kinase substrate [49], and Akt [51]. Libraries of monoclonal antibodies and proteomic analyses have indicated that many more proteins, named or unnamed, are present on phagosomes [24,33].…”

Section: Proteins Downstream Of the Fcr Complexmentioning

confidence: 99%

The coordination of signaling during Fc receptor-mediated phagocytosis

Swanson

Hoppe

2004

Journal of Leukocyte Biology

274

234

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“…Esta línea celular había sido transfectada previamente de manera estable con los cADN de gp91 phox , p22 phox y p47 phox clonados en los vectores pEF-PGKpac, pEF-PGKneo y pEF-PGKhygro, respectivamente (40)(41)(42). Estas células se cultivaron en medio DMEM/10% suero bovino fetal (SBF) (Hyclone, Logan, UT) con suplemento de 50 unidades/ml de penicilina, 50 µg/ml de estreptomicina (Gibco BRLÒ Life Technologies), 0,2 mg/ml de higromicina (Invitrogen), 1,8 mg/ml de G418 y 1 µg/ml de puromicina (Sigma-Aldrich, St. Louis, MO).…”

Section: Transfección De La Línea Celular Cos91/22/47unclassified

“…Se mezclaron 3 x 10 6 células COS91/22/47 con 2 µg de los respectivos cADN clonados en pcADN3.1/zeo(+). Éstas se analizaron 21 horas después de la transfección como se menciona en los ensayos de Price et al (41,42).…”

Section: Transfección De La Línea Celular Cos91/22/47unclassified

“…Para esto se realizó un ensayo de cinética cuantitativa basada en la reducción del citocromo c, reacción que es inhibible por la superóxido dismutasa. El ensayo se realizó a 37°C con un lector de microplatos Thermomax (Molecular Devices, Inc., Sunnyvale, CA) como se ha descrito previamente (41,43). Brevemente, 2,5 x 10 5 células provenientes de cada una de las transfecciones se resuspendieron en 250 ul de PBSG (PBS más 0,5 mM de MgCl 2 , 0,9 mM de CaCl 2 y 7,5 mM de dextrosa) que contenía 75 µM de citocromo c. Las células se activaron por la adición de 100 µM de ácido araquidónico (AA).…”

Section: Cinética De Anión Superóxidounclassified

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Expresión y actividad de posibles polimorfismos provenientes de individuos normales en la proteína de 67 kd del sistema NADPH oxidasa utilizando el sistema COSphox.

Arias

Dinauer

Ding³

et al. 2004

biomedica

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El sistema NADPH oxidasa de las células fagocíticas cumple una función importante durante la respuesta antimicrobiana del organismo. La activación de este sistema está precedida por la translocación de las proteínas citosólicas p67 phox , p47 phox y p40 phox hacia la membrana para ponerse en contacto con el flavocitocromo b 558 , lo que induce la generación del anión superóxido, un precursor de agentes microbicidas oxidantes. El presente trabajo presenta un análisis funcional del sistema NADPH oxidasa basado en los hallazgos de polimorfismos encontrados en el gen de p67 phox de individuos sanos. Para esto se generaron mutaciones en el cADN que codifica la p67 phox y se expresaron en el sistema de células COS phox . Los datos obtenidos en el presente trabajo indican que los cambios Val 166 →Ile, Pro 329 →Ser y His 389 →Gln no generan alteraciones en el funcionamiento de la p67 phox cuando su función se analizó en el sistema transgénico basado en células COS-7. Por lo tanto, estos polimorfismos no generan ningún riesgo genético de producir deficiencias en la activación del sistema NADPH oxidasa. Además, se demuestra que el modelo de células COS phox representa un nuevo sistema celular, fácilmente transfectable que permite estudiar la función del sistema NADPH oxidasa de las células fagocíticas y sus particularidades genéticas. Finalmente, los hallazgos con estos polimorfismos nos permiten avanzar en el conocimiento sobre los mecanismos moleculares involucrados en la activación del sistema NADPH oxidasa células fagocíticas.Palabras clave: NADPH oxidasa, p67 phox , COS-phox cells, polimorfismos. Expression and activity of polymorphisms in the 67-kDa protein of the NADPH oxidase systemThe NADPH oxidase system plays a central role in the antimicrobial activity of phagocytes. This system is initiated by the translocation of cytosolic proteins p67 phox , p47 phox and p40 phox to be in close contact with membrane flavocytochrome b 558 . This event begins the electron transfer from cytosolic NADPH to molecular oxygen to produce superoxide anions. Herein, a functional analysis is presented of p67 phox polymorphisms identified from healthy humans. Mutations were generated in the p67 phox cDNA by site-directed mutagenesis and then transiently expressed in COS7 cells that also expressed gp91 phox , p22 phox , and p47 phox from stable transgenes. The changes Val166Ile, Pro329Ser and His389Gln correspond to possible polymorphisms identified in healthy individuals revealed a functional activity similar to COS phox cells transiently transfected with WT p67 phox ; therefore, these modifications are not associated with genetic deficiencies in NADPH oxidase. In conclusion, the COS phox system represents an easily transfectable model for analysis of NADPH oxidase function in intact cells. The analysis of mutant derivatives of p67 phox provides insight into molecular mechanisms by which this subunit regulates the NADPH oxidase.

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Rac Activation Induces NADPH Oxidase Activity in Transgenic COS Cells, and the Level of Superoxide Production Is Exchange Factor-dependent

Cited by 110 publications

References 76 publications

A Dual Role of the GTPase Rac in Cardiac Differentiation of Stem Cells

A Dual Role of the GTPase Rac in Cardiac Differentiation of Stem Cells

The coordination of signaling during Fc receptor-mediated phagocytosis

Expresión y actividad de posibles polimorfismos provenientes de individuos normales en la proteína de 67 kd del sistema NADPH oxidasa utilizando el sistema COSphox.

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