S‐nitrosylation of β‐catenin and p‐120 catenin: a novel regulatory mechanism in endothelial hyperpermeability

Abstract

Platelet‐activating factor (PAF) increases endothelial permeability (hyperpermeability) via endothelial NOS (eNOS), which stimulates cGMP production to enhance permeability. We explored the complementary hypothesis that PAF‐stimulated hyperpermeability involves eNOS‐derived NO induced S‐nitrosylation of adherens junction proteins. We measured PAF‐stimulated S‐nitrosylation of β‐catenin and p120‐catenin (p120) in 3 cell lines: ECV‐eNOSGFP, EAhy926 and CVEC. S‐nitrosylation reduced expression of β‐catenin and p120 at the adherens junction and enhanced permeability. To ascertain the significance of eNOS location, we used ECV‐304 cells transfected with constructs that target eNOS to the cytosol (G2AeNOSGFP) and plasma membrane (CAAXeNOSGFP). We detected S‐nitrosylation of β‐catenin and p120 only in ECV‐GFPeNOS‐G2A, i.e., in cells with cytosolic eNOS. PAF‐stimulated S‐nitrosylation decreased the association between β‐catenin and p‐120. PAF significantly decreased the association between β‐catenin and p‐120 only in ECV‐GFPeNOS‐G2A. Inhibitors of NO production and of S‐nitrosylation blocked PAF‐induced S‐nitrosylation and hyperpermeability. Our results demonstrate that agonist‐induced S‐nitrosylation contributes to junctional membrane protein changes that enhance endothelial permeability.