EGF Prevents Hydrogen Peroxide‐Induced Tyrosine Phosphorylation and Redistribution of Tight Junction and Adherens Junction Proteins in Human Colonic Mucosa

Abstract

Oxidative stress destabilizes intercellular junctions and promotes tumor metastasis. Previous studies demonstrated that H2O2 disrupts tight junctions (TJs) and adherens junctions (AJs) by a Tyr‐kinase‐dependent mechanism in Caco‐2 cells. In the present study, we evaluated the effect of H2O2 on TJs and AJs in human colonic mucosa. These studies were conducted according to the approved IRB protocol. Human colonic biopsies were incubated with 30 nM epidermal growth factor (EGF) 10 min prior to H2O2 (20 μM) for 1 hour. TJ and AJ integrity was evaluated by analyzing the distribution of occludin, ZO‐1, E‐cadherin and β‐catenin by immuofluorescence confocal microscopy. Triton‐insoluble and soluble fractions were immunoblotted for TJ and AJ proteins. Tyr‐phosphorylation of was determined by immunoprecipitation and immunoblot analysis. Results show that H2O2 induces a redistribution of occludin, ZO‐1, E‐cadherin and β‐catenin from the intercellular junctions into the intracellular compartments. H2O2 induced a significant reduction of these TJ and AJ proteins in the Triton‐insoluble fraction, while increasing the levels of these proteins in Triton‐soluble fraction. Tyr‐phosphorylation of TJ and AJ proteins was increased by H2O2. Pretreatment of tissues with EGF attenuated H2O2‐induced Tyr‐phosphorylation and redistribution of TJ and AJ proteins from the intercellular junctions and Triton‐insoluble factions. These results indicate that oxidative stress induces Tyr‐phosphorylation and redistribution of TJ and AJ proteins in human colonic mucosa and EGF prevents these effects of H2O2.