Published online before print December 23, 2004, doi: 10.1161/01.ATV.0000154141.66879.98
© 2005 American Heart Association, Inc.
Vascular Biology |
p47phox Associates With the Cytoskeleton Through Cortactin in Human Vascular Smooth Muscle Cells
Role in NAD(P)H Oxidase Regulation by Angiotensin II
From Multidisciplinary Research Group on Hypertension (R.M.T., G.Y., E.L.S.), Clinical Research Institute of Montreal, University of Montreal, Canada; Department of Veterinary Molecular Biology (M.T.Q.), Montana State University, Bozeman; Hypertension and Vascular Research Division and Biostatistics Department (P.J.P.), Henry Ford Hospital, Detroit, Mich.
Correspondence to Rhian M Touyz, MD, PhD, Clinical Research Institute of Montreal, 110 Pine Ave West, Montreal, Quebec H2W 1R7, Canada. E-mail touyzr{at}ircm.qc.ca
Objective— We tested the hypothesis that p47phox associates with the actin cytoskeleton, enabling site-directed activation of NAD(P)H oxidase, and assessed whether these actions influence reactive oxygen species (ROS) generation and signaling by angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs) from human resistance and coronary arteries.
Methods and Results— Electroporation of anti-p47phox antibody into VSMCs abrogated Ang II-mediated O
2 generation, establishing the requirement for p47phox in this response. Immunfluorescence confocal microscopy demonstrated a cytosolic distribution of p47phox in basal conditions. After Ang II stimulation, p47phox rearranged in a linear fashion, colocalizing with F-actin. Co-immunoprecipitation studies confirmed an association between p47phox and actin and demonstrated an interaction with the actin-binding protein cortactin. Cytoskeletal disruption with cytochalasin prevented p47phox:actin interaction and attenuated ROS formation and p38MAP kinase and Akt phosphorylation by Ang II. Intracellular ROS generation in response to LY83583 (O2 generator) or exogenous H2O2 and Ang II-induced ERK1/2 activation were unaltered by cytochalasin.
Conclusions— The p47phox:actin interaction, through cortactin, plays an important role in Ang II-mediated site-directed assembly of functionally active NAD(P)H oxidase, ROS generation, and activation of redox-sensitive p38MAP kinase and Akt, but not ERK1/2. These findings demonstrate the importance of an intact actin-cytoskeleton in NAD(P)H oxidase regulation and redox signaling by Ang II in human VSMCs.
We demonstrate that p47phox:actin interaction, through cortactin, is involved in Ang II-mediated site-directed assembly of NAD(P)H oxidase, ROS generation, and activation of redox-sensitive p38MAPK and Akt, but not ERK1/2. These findings demonstrate the importance of an intact actin-cytoskeleton in NAD(P)H oxidase regulation and redox-signaling by Ang II in human VSMCs.
Key Words: reactive oxygen species • cytochalasin B • cortactin • LY83583 • hydrogen peroxide
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