Abstract 18285: Poldip2, a Nox4 Interacting Protein, Regulates Vascular Smooth Muscle Cell Migration via RhoA-Dependent Focal Adhesion Turnover
NADPH oxidases, major sources of reactive oxygen species (ROS) in vascular cells, contribute to physiological as well as pathological signaling. The Nox4-type NADPH oxidase is highly expressed in all vascular cells. Polymerase delta interacting protein-2 (Poldip2) interacts with Nox4 and p22phox and positively regulates Nox4 function. Previous work implicates Nox4/Poldip2 in cytoskeletal dynamics and vascular smooth muscle cell (VSMC) migration, but the underlying regulatory mechanism is unclear. In the present study, we show that overexpression of Poldip2 (AdPoldip2) in VSMCs impairs migration in wound healing assays and significantly reduces the wound recovery (63±2.4 vs 38±2.1% in control vs AdPoldip2, respectively; n=16; p<0.05), with a characteristic phenotype of long lamellipodial extensions, suggesting impairment of focal adhesion turnover. In a Nocodazole-induced, microtubule-dependent focal adhesion dissolution and reformation assay, Poldip2 overexpression prevents focal adhesion turnover as measured by immunocytochemistry (ICC) for phospho-FAK (FAKpY397) and the focal adhesion protein paxillin. This effect is reversed by RhoA inhibition. Overexpression of Poldip2 also attenuates the decrease in RhoA activity observed at 30 minutes post-Nocodazole wash in control cells (74±11 vs 123±14 % in control vs AdPoldip2; n=4, p<0.05). Conversely, siPoldip2 and siNox4 treatment abolishes focal adhesion structures, as exhibited by the loss of FAKpY397 and Paxillin. This effect is reversed by constitutively active RhoA overexpression. Gene silencing of Poldip2 prevents the increase in RhoA activity after Nocodazole treatment (129±6 vs 69±7% in siControl vs siPoldip2, respectively; n=3; p<0.05). Interestingly, Poldip2 gene-trap (−/+) animals exhibit impaired neointima formation in a femoral artery wire injury model compared to their wild-type controls (neointima:media ratio 1.74±0.4 vs 0.66±.06 in wildtype vs Poldip2 gene-trap (−/+), respectively; n=3–4; p<0.05). These results clearly implicate Poldip2 in the migration of VSMC via RhoA-dependent focal adhesion turnover regulation. Our findings may thus identify a novel therapeutic target to control VSMC migration in atherosclerosis and restenosis.
- © 2010 by American Heart Association, Inc.