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

Int J Mol Sci. 2013 Oct 11;14(10):20220-35. doi: 10.3390/ijms141020220.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actin-Related Protein 2-3 Complex / genetics
  • Actin-Related Protein 2-3 Complex / metabolism*
  • Animals
  • Cell Movement / drug effects
  • Cell Movement / genetics
  • Cell Movement / physiology*
  • Cells, Cultured
  • Hydrogen Peroxide / pharmacology
  • Muscle, Smooth, Vascular / drug effects
  • Muscle, Smooth, Vascular / metabolism*
  • Muscle, Smooth, Vascular / physiology
  • Myocytes, Smooth Muscle / drug effects
  • Myocytes, Smooth Muscle / metabolism*
  • Myocytes, Smooth Muscle / physiology
  • NADH, NADPH Oxidoreductases / genetics
  • NADH, NADPH Oxidoreductases / metabolism*
  • NADPH Oxidase 1
  • Proteome / genetics
  • Proteome / metabolism*
  • Proteomics / methods
  • Rats
  • Reactive Oxygen Species / metabolism
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Signal Transduction / physiology
  • Up-Regulation / drug effects
  • Up-Regulation / genetics

Substances

  • Actin-Related Protein 2-3 Complex
  • Proteome
  • Reactive Oxygen Species
  • Hydrogen Peroxide
  • NADH, NADPH Oxidoreductases
  • NADPH Oxidase 1
  • NOX1 protein, rat