Canonical Wnt Signaling Induces Vascular Endothelial Dysfunction via p66Shc-Regulated Reactive Oxygen Species

OBJECTIVE—Reactive oxygen species regulate canonical Wnt signaling. However, the role of the redox regulatory protein p66 in the canonical Wnt pathway is not known. We investigated whether p66 is essential for canonical Wnt signaling in the endothelium and determined whether the canonical Wnt pathway induces vascular endothelial dysfunction via p66-mediated oxidative stress. APPROACH AND RESULTS—The canonical Wnt ligand Wnt3a induced phosphorylation (activation) of p66 in endothelial cells. Wnt3a-stimulated dephosphorylation of β-catenin, and β-catenin–dependent transcription, was inhibited by knockdown of p66. Exogenous H2O2-induced β-catenin dephosphorylation was also mediated by p66. Moreover, p66 overexpression dephosphorylated β-catenin and increased β-catenin–dependent transcription, independent of Wnt3a ligand. P66-induced β-catenin dephosphorylation was inhibited by antioxidants N-acetyl cysteine and catalase. Wnt3a upregulated endothelial NADPH oxidase-4, and β-catenin dephosphorylation was suppressed by knocking down NADPH oxidase-4 and by antioxidants. Wnt3a increased H2O2 levels in endothelial cells and impaired endothelium-dependent vasorelaxation in mouse aortas, both of which were rescued by p66 knockdown. P66 knockdown also inhibited adhesion of monocytes to Wnt3a-stimulated endothelial cells. Furthermore, constitutively active β-catenin expression in the endothelium increased vascular reactive oxygen species and impaired endothelium-dependent vasorelaxation. In vivo, high-fat diet feeding–induced endothelial dysfunction in mice was associated with increased endothelial Wnt3a, dephosphorylated β-catenin, and phosphorylated p66. High-fat diet–induced dephosphorylation of endothelial β-catenin was diminished in mice in which p66 was knocked down. CONCLUSIONS—p66 plays a vital part in canonical Wnt signaling in the endothelium and mediates Wnt3a-stimulated endothelial oxidative stress and dysfunction