title: Proteomic analysis identifies an NADPH oxidase 1 (Nox1)-mediated role for actin-related protein 2/3 complex subunit 2 (ARPC2) in promoting smooth muscle cell migration.
abstract: A variety of vascular pathologies, including hypertension, restenosis and atherosclerosis, are characterized by vascular smooth muscle cell (VSMC) hypertrophy and migration. NADPH oxidase 1 (Nox1) plays a pivotal role in these phenotypes via distinct downstream signaling. However, the mediators differentiating these distinct phenotypes and their precise role in vascular disease are still not clear. The present study was designed to identify novel targets of VSMC Nox1 signaling using 2D Differential In-Gel Electrophoresis and Mass Spectrometry (2D-DIGE/MS). VSMC treatment with scrambled (Scrmb) or Nox1 siRNA and incubation with the oxidant hydrogen peroxide (H2O2; 50 µM, 3 h) followed by 2D-DIGE/MS on cell lysates identified 10 target proteins. Among these proteins, actin-related protein 2/3 complex subunit 2 (ARPC2) with no previous link to Nox isozymes, H2O2, or other reactive oxygen species (ROS), was identified and postulated to play an intermediary role in VSMC migration. Western blot confirmed that Nox1 mediates H2O2-induced ARPC2 expression in VSMC. Treatment with a p38 MAPK inhibitor (SB203580) resulted in reduced ARPC2 expression in H2O2-treated VSMC. Additionally, wound-healing "scratch" assay confirmed that H2O2 stimulates VSMC migration via Nox1. Importantly, gene silencing of ARPC2 suppressed H2O2-stimulated VSMC migration. These results demonstrate for the first time that Nox1-mediated VSMC migration involves ARPC2 as a downstream signaling target.
authors.affiliation: Vascular Medicine Institute, 12th Floor BST, 200 Lothrop Street, University of Pittsburgh, PA 15261, USA. pagano@pitt.edu.
authors.forename: Imad
authors.initials: I
authors.lastname: Al Ghouleh
authors.forename: Andrés
authors.initials: A
authors.lastname: Rodríguez
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Pagano
authors.forename: Gábor
authors.initials: G
authors.lastname: Csányi
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D020543:Proteome; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; C000614712:NOX1 protein, rat