Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells.
OBJECTIVE
Reactive oxygen species (ROS) that act as signaling molecules in vascular smooth muscle cells (VSMC) and contribute to growth, hypertrophy, and migration in atherogenesis are produced by multi-subunit NAD(P)H oxidases. Nox1 and Nox4, two homologues to the phagocytic NAD(P)H subunit gp91phox, both generate ROS in VSMC but differ in their response to growth factors. We hypothesize that the opposing functions of Nox1 and Nox4 are reflected in their differential subcellular locations.


RESULTS
We used immunofluorescence to visualize the NAD(P)H subunits Nox1, Nox4, and p22phox in cultured rat and human VSMC. Optical sectioning using confocal microscopy showed that Nox1 is co-localized with caveolin in punctate patches on the surface and along the cellular margins, whereas Nox4 is co-localized with vinculin in focal adhesions. These immunocytochemical distributions are supported by membrane fractionation experiments. Interestingly, p22phox, a membrane subunit that interacts with the Nox proteins, is found in surface labeling and in focal adhesions in patterns similar to Nox1 and Nox4, respectively.


CONCLUSIONS
The differential roles of Nox1 and Nox4 in VSMC may be correlated with their differential compartmentalization in specific signaling domains in the membrane and focal adhesions.
Hilenski
Lula L
LL
Division of Cardiology, Emory University School of Medicine, Atlanta, GA 30322, USA. lhilens@emory.edu
Clempus
Roza E
RE
Quinn
Mark T
MT
Lambeth
J David
JD
Griendling
Kathy K
KK
D000818:Animals; D021941:Caveolae / Q000201:enzymology*; D051242:Caveolin 1; D022461:Caveolins / Q000032:analysis; D002455:Cell Division; D002458:Cell Fractionation; D002478:Cells, Cultured / Q000201:enzymology / Q000648:ultrastructure; D016922:Cellular Senescence; D003599:Cytoskeleton / Q000378:metabolism; D022001:Focal Adhesions / Q000201:enzymology*; D006801:Humans; D046911:Macromolecular Substances; D008297:Male; D026901:Membrane Transport Proteins / Q000032:analysis; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000201:enzymology* / Q000648:ultrastructure; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis* / Q000502:physiology; D009252:NADPH Dehydrogenase / Q000032:analysis; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000032:analysis* / Q000502:physiology; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000032:analysis; D021122:Protein Subunits; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D016596:Vinculin / Q000032:analysis
C495540:CAV1 protein, human; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D021122:Protein Subunits; D017382:Reactive Oxygen Species; D016596:Vinculin; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C402799:NOX1 protein, human; C000614712:NOX1 protein, rat; C498910:NOX4 protein, human; C498911:Nox4 protein, rat; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase