Inhibition of oxidant-induced barrier disruption and protein tyrosine phosphorylation in Caco-2 cell monolayers by epidermal growth factor

Abstract

The effect of epidermal growth factor (EGF) on the H 2O 2-induced increase in paracellular permeability in Caco-2 and T-84 cell monolayers was evaluated to examine the role of EGF in intestinal mucosal protection from oxidative stress. Oxidative stress was induced by exposing cell monolayers to H 2O 2 or a mixture of xanthine oxidase + xanthine (XO + X). Paracellular permeability was assessed by measuring transepithelial electrical resistance (TER), sodium chloride dilution potential, and unidirectional flux of [ 3H]mannitol. H 2O 2 (0.1 to 5.0 mM) reduced TER and dilution potential and increased mannitol flux. Administration of EGF delayed H 2O 2 and XO + X-induced changes in TER, dilution potential, and [ 3H]mannitol flux. This protective effect of apically or basally administered EGF was concentration-related, with A 50 (95% confidence limits) values of 2.1 (1.17 to 4.34) and 6.0 (4.37 to 8.34) n M, respectively. The EGF-mediated protection was prevented by treatment of cell monolayers with genistein (10 μM), a tyrosine kinase inhibitor. H 2O 2 and XO + X also induced tyrosine phosphorylation of a number of proteins in Caco-2 and T-84 cell monolayers. EGF treatment inhibited the oxidant-induced tyrosine phosphorylation of proteins, particularly those with a molecular mass of 110–220 kDa. Treatment of Caco-2 cells with anti-transforming growth factor-α antibodies potentiated the H 2O 2-induced changes in TER, dilution potential, and mannitol flux. These studies demonstrated that an EGF receptor-mediated mechanism delays oxidant-induced disruption of the epithelial barrier function, possibly by suppressing the oxidant-induced tyrosine phosphorylation of proteins.