Role of p47phoxin regulating Cdc42GAP, vimentin, and contraction in smooth muscle cells

Cdc42GAP (GTPase activating protein) has been shown to regulate smooth muscle contraction as well as cell motility, adhesion, proliferation, and apoptosis. We have recently shown that Cdc42GAP activity is suppressed in smooth muscle cells during contractile activation, which is reversed by inhibitors of reactive oxygen species (ROS). Because p47(phox), a regulatory subunit of NAD(P)H oxidase, has been implicated in smooth muscle signaling, we determined whether this subunit modulates Cdc42GAP activity in response to contractile stimulation. Transfection of smooth muscle cells with plasmids encoding short hairpin RNA (shRNA) against p47(phox), but not plasmids for luciferase shRNA, inhibited the expression of p47(phox). ROS production and the suppression of Cdc42GAP activity in response to stimulation with 5-hydroxytryptamine (5-HT) were attenuated in cells producing p47(phox) shRNA compared with cells producing luciferase shRNA. In contrast, the addition of hydrogen peroxide to p47(phox)-deficient cells suppressed the activity of Cdc42GAP. Furthermore, exposure to hydrogen peroxide led to a decrease in Cdc42GAP activity in an in vitro assay. Cdc42 activation, p21-activated kinase 1 (PAK1) phosphorylation at Thr-423 (an indication of PAK activation), and vimentin phosphorylation at Ser-56 in response to 5-HT activation were also attenuated in smooth muscle cells producing shRNA against p47(phox). The knockdown of p47(phox) inhibited smooth muscle contraction during stimulation with 5-HT but not hydrogen peroxide. These results suggest that the p47(phox) subunit of NAD(P)H oxidase may mediate the agonist-induced GAP suppression by controlling ROS generation in smooth muscle cells during agonist stimulation. p47(phox)-regulated GAP affects smooth muscle contraction likely through the Cdc42/PAK1/vimentin pathway.