Poldip2, a novel regulator of Nox4 and cytoskeletal integrity in vascular smooth muscle cells.
BACKGROUND
NADPH oxidases (Noxes) regulate vascular physiology and contribute to the pathogenesis of vascular disease. In vascular smooth muscle cells (VSMCs), the interactions of individual Nox homologs with regulatory proteins are poorly defined.


OBJECTIVE
The objective of this study was to identify novel NADPH oxidase regulatory proteins.


RESULTS
Using a yeast 2-hybrid screen, we identified a novel p22phox binding partner, Poldip2, and demonstrated that it associates with p22phox, NADPH oxidase (Nox)1, and Nox4 and colocalizes with p22phox at sites of Nox4 localization. Poldip2 increases Nox4 enzymatic activity by 3-fold and positively regulates basal reactive oxygen species production in VSMCs (O2(.-): 86.3+/-15.6% increase; H2O2: 40.7+/-4.5% increase). Overexpression of Poldip2 activates Rho (180.2+/-24.8% increase), strengthens focal adhesions, and increases stress fiber formation. These phenotypic changes are blocked by dominant negative Rho. In contrast, depletion of either Poldip2 or Nox4 results in a loss of these structures, which is rescued by adding back active Rho. Cell migration, which requires dynamic cytoskeletal remodeling, is impaired by either excess (70.1+/-14.7% decrease) or insufficient Poldip2 (63.5+/-5.9% decrease).


CONCLUSIONS
These results suggest that Poldip2 associates with p22phox to activate Nox4, leading to regulation of focal adhesion turnover and VSMC migration, thus linking reactive oxygen species production and cytoskeletal remodeling. Poldip2 may be a novel therapeutic target for vascular pathologies with a significant VSMC migratory component, such as restenosis and atherosclerosis.
Lyle
Alicia N
AN
Emory University, Division of Cardiology, 319 WMB, 1639 Pierce Dr, Atlanta, GA 30322, USA.
Deshpande
Nita N
NN
Taniyama
Yoshihiro
Y
Seidel-Rogol
Bonnie
B
Pounkova
Lily
L
Du
Pingfeng
P
Papaharalambus
Christopher
C
Lassègue
Bernard
B
Griendling
Kathy K
KK
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; C533897:PDIP38 protein, rat; C473592:POLDIP2 protein, human; C000593573:Poldip2 protein, rat; D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C498911:Nox4 protein, rat; C086986:CYBA protein, human; C515768:Cyba protein, rat; D020742:rhoA GTP-Binding Protein