Shear-induced tyrosine phosphorylation in endothelial  cells requires Rac1-dependent production of ROS

Yeh, Li Hong; Park, Young J.; Hansalia, Riple; Ahmed, Syed Imraan; Deshpande, Shailesh; Goldschmidt‐Clermont, Pascal J.; Irani, Kaikobad; Alevriadou, B. Rita

doi:10.1152/ajpcell.1999.276.4.c838

Cited by 141 publications

(99 citation statements)

References 60 publications

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“…Rac proteins, in particular rac1, function similarly in nonphagocytic cells, 1,12 and such rac1-regulated ROS have been implicated in a variety of cellular processes including growth, migration, and transformation. 2,8,[12][13][14][15][16][17] We have recently shown that rac1-regulated ROS production also mediates apoptosis in response to I/R. 7 Hepatocyte growth factor (HGF), which was initially isolated as a potent mitogen for hepatocytes, is now known to be a broad-spectrum mitogen for a variety of cell types.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of hypoxia/reoxygenation-induced oxidative stress in HGF-stimulated antiapoptotic signaling: role of PI3-K and Akt kinase upon rac1

et al. 2003

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Rac1-regulated reactive oxygen species (ROS) production has been implicated in apoptosis. In contrast, pleiotropic protein kinase Akt protects against apoptosis. However, the pro-and antiapoptotic mechanisms of rac1 and Akt, respectively, and the intersection between these mechanisms are incompletely understood. In a model of oxidative stress and apoptosis induced by hypoxia/reoxygenation (H/R) in primary hepatocytes, activation of the PI3-K Akt axis by the prosurvival hepatocyte growth factor (HGF) inhibited H/Rstimulated rac1 activation and intracellular ROS production, and suppressed apoptosis. Suppression of PI3-K or Akt activity abrogated the inhibitory effect of HGF on rac1 activity and rac1-regulated oxidative stress. Furthermore, constitutive activation of Akt or PI3-K in the absence of HGF was sufficient to phosphorylate rac1, inhibit rac1 activation, and suppress rac1-regulated ROS production. These findings demonstrate that growth factor-stimulated activation of PI3-K-Akt is necessary and sufficient to suppress intracellular oxidative stress and apoptosis by inhibiting activation of proapoptotic, prooxidative rac1 GTPase.

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Section: Introductionmentioning

confidence: 99%

Inhibition of hypoxia/reoxygenation-induced oxidative stress in HGF-stimulated antiapoptotic signaling: role of PI3-K and Akt kinase upon rac1

et al. 2003

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“…However, the experimental setup is not readily applicable to adherent cell types, and the shear forces endemic to the procedure may induce phosphorylation changes that confound the responses to growth factor (10). Here, we present an alternative method for investigating early phosphorylation dynamics in adherent mammary epithelial cells on the timescale of 10 s. Increased temporal resolution, paired with a wide variety of treatment conditions, have provided insight into the mechanisms controlling activation of the EGFR signaling network that are obscured at later time points.…”

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confidence: 99%

Early signaling dynamics of the epidermal growth factor receptor

Reddy

Gajadhar

Swenson

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Despite extensive study of the EGF receptor (EGFR) signaling network, the immediate posttranslational changes that occur in response to growth factor stimulation remain poorly characterized; as a result, the biological mechanisms underlying signaling initiation remain obscured. To address this deficiency, we have used a mass spectrometry-based approach to measure system-wide phosphorylation changes throughout the network with 10-s resolution in the 80 s after stimulation in response to a range of eight growth factor concentrations. Significant changes were observed on proteins far downstream in the network as early as 10 s after stimulation, indicating a system capable of transmitting information quickly. Meanwhile, canonical members of the EGFR signaling network fall into clusters with distinct activation patterns. Src homology 2 domain containing transforming protein (Shc) and phosphoinositol 3-kinase (PI3K) phosphorylation levels increase rapidly, but equilibrate within 20 s, whereas proteins such as Grb2-associated binder-1 (Gab1) and SH2-containing tyrosine phosphatase (SHP2) show slower, sustained increases. Proximity ligation assays reveal that Shc and Gab1 phosphorylation patterns are representative of separate timescales for physical association with the receptor. Inhibition of phosphatases with vanadate reveals site-specific regulatory mechanisms and also uncovers primed activating components in the network, including Src family kinases, whose inhibition affects only a subset of proteins within the network. The results presented highlight the complexity of signaling initiation and provide a window into exploring mechanistic hypotheses about receptor tyrosine kinase (RTK) biology.signal transduction | tyrosine phosphorylation | epidermal growth factor receptor | mass spectrometry T he EGF receptor (EGFR) sits atop a complex signaling network that controls cell behavior in response to environmental cues. Cascades of posttranslational modifications initiated from EGFR, notably phosphorylation on tyrosine, serine, and threonine, influence protein-protein interactions and enzymatic activity to activate transcriptional programs that regulate proliferation, differentiation, and apoptosis (1). Mutation or overexpression of EGFR has been identified as an oncogenic driver for many tumor types, making it an attractive target for anticancer therapies (2).Building on decades of specific characterization using traditional biochemistry techniques, platforms such as protein microarrays and mass spectrometry have allowed systems biology to provide a comprehensive picture of intact and aberrant network behavior, which can be used to establish design criteria for therapeutic interventions (3). Manipulating components within the network experimentally and computationally has uncovered many features of the system that influence behaviors such as proliferation and survival (4-6). With many phenotypic responses occurring on the order of hours to days, most phosphorylation measurements have been on the timescale of minutes to...

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“…All flow studies with cultured ECs, as well as the reoxygenation (RO) phase of hypoxia (H)/RO studies, including work from our group (30,37,48,52,80,81), have been carried out at the atmospheric O 2 concentration (21% O 2 ), which gives rise to a partial pressure of O 2 (PO 2 ) of ϳ160 mmHg, above the levels in arterial blood (75-100 mmHg; 10 -13% O 2 ) and well above the levels in venous blood (35 mmHg; 5% O 2 ) or the levels to which ECs are exposed in tissue capillaries (mean capillary blood PO 2 in the heart is ϳ20 mmHg; 2.6% O 2 ) (72). Since it is known that 1-4% of O 2 reacting with the ETC is incompletely reduced to O 2 ⅐ Ϫ , and the O 2 ⅐ Ϫ formation rate by the ETC complexes III and I increases linearly with O 2 concentration (42,74), we hypothesized that, under flow, the relative "hyperoxic state" of 21% O 2 raises the O 2 concentration in EC mitochondria and enhances the formation of O 2 ⅐ Ϫ , which, via the reaction with shear-induced NO, increases the intramitochondrial ONOO Ϫ levels and potentiates the inhibition of respiration.…”

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confidence: 99%

Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite

Jones¹,

Han²,

Presley³

et al. 2008

American Journal of Physiology-Cell Physiology

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Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO Ϫ ) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the "hyperoxic state" of 21% O2, compared with more physiological O2 tensions (PO2), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O2 ⅐ Ϫ ) generation leading to ONOO Ϫ formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O2 consumption rates of bovine aortic ECs sheared (10 dyn/cm 2 , 30 min) at 5%, 10%, or 21% O2 or left static at 5% or 21% O2. Respiration was inhibited to a greater extent when ECs were sheared at 21% O2 than at lower PO2 or left static at different PO2. Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO Ϫ scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O2 ⅐ Ϫ production was higher in ECs sheared at 21% than at 5% O2, as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O2 was modestly increased compared with ECs sheared at lower PO2, suggesting that eNOS activity may be higher at 21% O2. Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O2 ⅐ Ϫ production, leads to enhanced ONOO Ϫ formation intramitochondrially and suppression of respiration. shear stress; endothelium; mitochondria; reactive oxygen species MITOCHONDRIA ARE THE SUBCELLULAR organelles for the production of cellular energy, but they also activate intracellular signaling pathways that modulate cell proliferation or promote cell cycle arrest and apoptosis by oxidative or nitrosative reactions. These reactions are regulated by the matrix concentration of nitric oxide (NO), which diffuses to the mitochondria from the cytosolic NO synthase (NOS) isoforms and is thought to be produced also locally by a mitochondrial NOS variant (11). In rat heart, skeletal muscle and liver mitochondria (15,57,58), isolated rat hearts (59), and whole animals (68), NO at physiological concentrations binds reversibly and in competition with oxygen (O 2 ) to the reduced binuclear center Cu B /a 3 of cytochrome-c oxidase (complex IV) of the electron transport chain (ETC) and inhibits mitochondrial O 2 uptake. At slightly higher concentrations, NO oxidizes ubiquinol of ubiquinolcytochrome c reductase (complex III) to increase unstable ubisemiquinone, which, by univalent electron transfer to O 2 , produces the reactive O 2 species (ROS) superoxide (O 2 ⅐ Ϫ ) (57, 58). O 2 ⅐ Ϫ generated from that location is released both into the matrix and intermembrane space (51), where it is dismutated to hydrogen peroxide (H 2 O 2 ) by manganese superoxide dismutase (MnSOD) and copper zinc SOD (CuZ...

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Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS

Cited by 141 publications

References 60 publications

Inhibition of hypoxia/reoxygenation-induced oxidative stress in HGF-stimulated antiapoptotic signaling: role of PI3-K and Akt kinase upon rac1

Inhibition of hypoxia/reoxygenation-induced oxidative stress in HGF-stimulated antiapoptotic signaling: role of PI3-K and Akt kinase upon rac1

Early signaling dynamics of the epidermal growth factor receptor

Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite

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