title: Binding of EBP50 to Nox organizing subunit p47phox is pivotal to cellular reactive species generation and altered vascular phenotype.
abstract: Despite numerous reports implicating NADPH oxidases (Nox) in the pathogenesis of many diseases, precise regulation of this family of professional reactive oxygen species (ROS) producers remains unclear. A unique member of this family, Nox1 oxidase, functions as either a canonical or hybrid system using Nox organizing subunit 1 (NoxO1) or p47(phox), respectively, the latter of which is functional in vascular smooth muscle cells (VSMC). In this manuscript, we identify critical requirement of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50; aka NHERF1) for Nox1 activation and downstream responses. Superoxide (O2 (•-)) production induced by angiotensin II (AngII) was absent in mouse EBP50 KO VSMC vs. WT. Moreover, ex vivo incubation of aortas with AngII showed a significant increase in O2 (•-) in WT but not EBP50 or Nox1 nulls. Similarly, lipopolysaccharide (LPS)-induced oxidative stress was attenuated in femoral arteries from EBP50 KO vs. WT. In silico analyses confirmed by confocal microscopy, immunoprecipitation, proximity ligation assay, FRET, and gain-/loss-of-function mutagenesis revealed binding of EBP50, via its PDZ domains, to a specific motif in p47(phox) Functional studies revealed AngII-induced hypertrophy was absent in EBP50 KOs, and in VSMC overexpressing EBP50, Nox1 gene silencing abolished VSMC hypertrophy. Finally, ex vivo measurement of lumen diameter in mouse resistance arteries exhibited attenuated AngII-induced vasoconstriction in EBP50 KO vs. WT. Taken together, our data identify EBP50 as a previously unidentified regulator of Nox1 and support that it promotes Nox1 activity by binding p47(phox) This interaction is pivotal for agonist-induced smooth muscle ROS, hypertrophy, and vasoconstriction and has implications for ROS-mediated physiological and pathophysiological processes.
authors.affiliation: Division of Cardiology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh School of Medicine Pittsburgh, PA 15260; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260;
authors.forename: Imad
authors.initials: I
authors.lastname: Al Ghouleh
authors.affiliation: Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh School of Medicine Pittsburgh, PA 15260; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260;
authors.forename: Daniel N
authors.initials: DN
authors.lastname: Meijles
authors.affiliation: Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh School of Medicine Pittsburgh, PA 15260; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260;
authors.forename: Stephanie
authors.identifier: 0000-0002-4288-4452
authors.initials: S
authors.lastname: Mutchler
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260;
authors.forename: Qiangmin
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh School of Medicine Pittsburgh, PA 15260; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260;
authors.forename: Sanghamitra
authors.initials: S
authors.lastname: Sahoo
authors.affiliation: Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh School of Medicine Pittsburgh, PA 15260; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260;
authors.forename: Anastasia
authors.initials: A
authors.lastname: Gorelova
authors.affiliation: Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh School of Medicine Pittsburgh, PA 15260; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260;
authors.forename: Jefferson
authors.initials: J
authors.lastname: Henrich Amaral
authors.affiliation: Heart Institute, University of São Paulo School of Medicine, Brazil;
authors.forename: Andrés I
authors.initials: AI
authors.lastname: Rodríguez
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260;
authors.forename: Tatyana
authors.initials: T
authors.lastname: Mamonova
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260; Brain Science and Engineering Institute, Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 41944, Korea;
authors.forename: Gyun Jee
authors.initials: GJ
authors.lastname: Song
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260;
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bisello
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260; Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260; Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Friedman
authors.affiliation: Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh School of Medicine Pittsburgh, PA 15260; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260;
authors.forename: M Eugenia
authors.initials: ME
authors.lastname: Cifuentes-Pagano
authors.affiliation: Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh School of Medicine Pittsburgh, PA 15260; Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260; pagano@pitt.edu.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Pagano
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000804:Angiotensin II / Q000008:administration & dosage / Q000009:adverse effects; D000818:Animals; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D005263:Femoral Artery / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D006801:Humans; D006984:Hypertrophy / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D008070:Lipopolysaccharides / Q000633:toxicity; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000378:metabolism / Q000473:pathology; D000074624:NADPH Oxidase 1 / Q000235:genetics* / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D014661:Vasoconstriction / Q000187:drug effects / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D008070:Lipopolysaccharides; C472287:Noxo1 protein, mouse; D010750:Phosphoproteins; D011506:Proteins; D017382:Reactive Oxygen Species; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D013481:Superoxides; D000804:Angiotensin II; D000074624:NADPH Oxidase 1; C000614711:NOX1 protein, mouse; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C518330:RUVBL2 protein, mouse
keywords: EBP50; NADPH oxidase; hypertrophy; smooth muscle; vascular tone