title: NOX4-dependent Hydrogen peroxide promotes shear stress-induced SHP2 sulfenylation and eNOS activation.
abstract: Laminar shear stress (LSS) triggers signals that ultimately result in atheroprotection and vasodilatation. Early responses are related to the activation of specific signaling cascades. We investigated the participation of redox-mediated modifications and in particular the role of hydrogen peroxide (H2O2) in the sulfenylation of redox-sensitive phosphatases. Exposure of vascular endothelial cells to short periods of LSS (12 dyn/cm(2)) resulted in the generation of superoxide radical anion as detected by the formation of 2-hydroxyethidium by HPLC and its subsequent conversion to H2O2, which was corroborated by the increase in the fluorescence of the specific peroxide sensor HyPer. By using biotinylated dimedone we detected increased total protein sulfenylation in the bovine proteome, which was dependent on NADPH oxidase 4 (NOX4)-mediated generation of peroxide. Mass spectrometry analysis allowed us to identify the phosphatase SHP2 as a protein susceptible to sulfenylation under LSS. Given the dependence of FAK activity on SHP2 function, we explored the role of FAK under LSS conditions. FAK activation and subsequent endothelial NO synthase (eNOS) phosphorylation were promoted by LSS and both processes were dependent on NOX4, as demonstrated in lung endothelial cells isolated from NOX4-null mice. These results support the idea that LSS elicits redox-sensitive signal transduction responses involving NOX4-dependent generation of hydrogen peroxide, SHP2 sulfenylation, and ulterior FAK-mediated eNOS activation.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" CSIC-UAM, Campus Universidad Autónoma, E-28049 Madrid, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Sánchez-Gómez
authors.affiliation: Laboratory of Cardiovascular Proteomics, Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Calvo
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" CSIC-UAM, Campus Universidad Autónoma, E-28049 Madrid, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Bretón-Romero
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" CSIC-UAM, Campus Universidad Autónoma, E-28049 Madrid, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Fierro-Fernández
authors.affiliation: Cardiovascular Division, British Heart Foundation Centre of Research Excellence, King's College London, London SE5 9NU, UK.
authors.forename: Narayana
authors.initials: N
authors.lastname: Anilkumar
authors.affiliation: Cardiovascular Division, British Heart Foundation Centre of Research Excellence, King's College London, London SE5 9NU, UK.
authors.forename: Ajay M
authors.initials: AM
authors.lastname: Shah
authors.affiliation: Vascular Research Centre, Institute for Cardiovascular Physiology, Goethe University, 60590 Frankfurt am Main, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Schröder
authors.affiliation: Vascular Research Centre, Institute for Cardiovascular Physiology, Goethe University, 60590 Frankfurt am Main, Germany.
authors.forename: Ralf P
authors.initials: RP
authors.lastname: Brandes
authors.affiliation: Laboratory of Cardiovascular Proteomics, Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Vázquez
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" CSIC-UAM, Campus Universidad Autónoma, E-28049 Madrid, Spain. Electronic address: slamas@cbm.csic.es.
authors.forename: Santiago
authors.initials: S
authors.lastname: Lamas
mesh_terms: D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005260:Female; D005455:Fluorescent Antibody Technique; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D010766:Phosphorylation / Q000187:drug effects; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013314:Stress, Mechanical; D013434:Sulfenic Acids / Q000737:chemistry*; D013481:Superoxides
chemical_list: D016877:Oxidants; D013434:Sulfenic Acids; D013481:Superoxides; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; C516725:Ptpn11 protein, mouse
keywords: Endothelium; Free radicals; Hydrogen peroxide; Laminar shear stress; Redox signaling; Sulfenylation; Vasodilation