p47phox associates with the cytoskeleton through cortactin in human vascular smooth muscle cells: role in NAD(P)H oxidase regulation by angiotensin II.
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.


RESULTS
Electroporation of anti-p47phox antibody into VSMCs abrogated Ang II-mediated O2 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.
Touyz
R M
RM
Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Canada. touyzr@ircm.qc.ca
Yao
G
G
Quinn
M T
MT
Pagano
P J
PJ
Schiffrin
E L
EL
D000199:Actins / Q000378:metabolism; D000634:Aminoquinolines / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology; D051356:Cortactin; D003571:Cytochalasin B / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000098:blood supply; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013481:Superoxides / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000199:Actins; D000634:Aminoquinolines; C495868:CTTN protein, human; D051356:Cortactin; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D014662:Vasoconstrictor Agents; D013481:Superoxides; D000804:Angiotensin II; D003571:Cytochalasin B; C041715:6-anilino-5,8-quinolinedione; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases