Poldip2, a novel regulator of Nox4 and cytoskeletal integrity in vascular smooth muscle cells.

RATIONALE: 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. METHODS AND 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.

Mechanism of hydrogen peroxide-induced cell cycle arrest in vascular smooth muscle.

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) can positively and negatively modulate vascular smooth muscle cell (VSMC) growth. To investigate these paradoxical effects of H(2)O(2), we examined its effect on apoptosis, cell cycle progression, and cell cycle proteins. High concentrations of H(2)O(2) (500 microM to 1 mM) induced apoptosis, whereas moderate concentrations (100 microM) caused cell cycle arrest in G1. H(2)O(2) (100 microM) blocked serum-stimulated cyclin-dependent kinase-2 (CDK2) activity, but not CDK4 activity, suggesting that cell cycle arrest occurred in part by inhibiting CDK2 activity. The serum-induced increase in cyclin A mRNA was also completely suppressed by H(2)O(2), whereas cyclin D1 mRNA was not affected. In addition, H(2)O(2) caused a dramatic increase in expression of the cell cycle inhibitor p21 mRNA (9.67 +/- 0.94-fold at 2 h) and protein (8.75 +/- 0.08-fold at 8 h), but no change in p27 protein. Finally, H(2)O(2 )transiently increased p53 protein levels (3.16 +/- 1.2-fold at 2 h). Thus, whereas high levels of H(2)O(2) induce apoptosis, moderate concentrations of H(2)O(2) coordinate a set of molecular events leading to arrest of VSMCs at the G1/S checkpoint of the cell cycle. These results provide insight into the mechanisms underlying positive and negative regulation of VSMC growth by H(2)O(2) in vascular disease.