NOX4 (NADPH Oxidase 4) and Poldip2 (Polymerase -Interacting Protein 2) Induce Filamentous Actin Oxidation and Promote Its Interaction With Vinculin During Integrin-Mediated Cell Adhesion

Objective Actin cytoskeleton assembly and organization, as a result of focal adhesion (FA) formation during cell adhesion, are dependent on reactive oxygen species and the cellular redox environment. Poldip2 (polymerase -interacting protein 2), a novel regulator of NOX4 (NADPH oxidase 4), plays a significant role in reactive oxygen species production and cytoskeletal remodeling. Thus, we hypothesized that endogenous reactive oxygen species derived from Poldip2/NOX4 contribute to redox regulation of actin and cytoskeleton assembly during integrin-mediated cell adhesion. Approach and Results Using vascular smooth muscle cells, we verified that hydrogen peroxide (H2O2) levels increase during integrin-mediated cell attachment as a result of activation of NOX4. Filamentous actin (F-actin) was oxidized by sulfenylation during cell attachment, with a peak at 3 hours (0.800.04 versus 0.08 +/- 0.13 arbitrary units at time zero), which was enhanced by overexpression of Poldip2. Depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2O2 with catalase, inhibited F-actin oxidation by 78 +/- 26%, 99 +/- 1%, and 98 +/- 1%, respectively. To determine the consequence of F-actin oxidation, we examined the binding of F-actin to vinculin, a protein involved in FA complexes that regulates FA maturation. Vinculin binding during cell adhesion as well as migration capacity were inhibited after transfection with actin containing 2 oxidation-resistant point mutations (C272A and C374A). Silencing of Poldip2 or NOX4 also impaired actin-vinculin interaction, which disturbed maturation of FAs and inhibited cell migration. Conclusions These results suggest that integrin engagement during cell attachment activates Poldip2/Nox4 to oxidize actin, which modulates FA assembly.