Hydrogen peroxide inhibits colonic epithelial ion transport by MAP kinase and PI3‐kinase (PI3K) independently of activated epidermal growth factor receptor (EGFr)

Abstract

Reactive oxygen species contribute to epithelial damage and dysfunction in inflammatory bowel disease (IBD). We have shown that hydrogen peroxide (H2O2) inhibits intestinal Ca2+-dependent ion transport by PI3K-and MAP kinase-dependent mechanisms, while also activating the EGFr. Here, we studied whether EGFr regulates H2O2-mediated inhibition of ion transport in colonic epithelial cells. Protein phosphorylation was measured in T84 colonic epithelial monolayers by Western blot, and ion transport was measured in Ussing chambers. H2O2 (500 μM) increased EGFr tyrosine phosphorylation and its association with the catalytic p110β subunit of PI3-K in T84 cells. However, neither an EGFr kinase inhibitor, tyrphostin AG1478 (1 μM), nor a neutralizing anti-EGFr antibody that blocks the EGFr ligand binding domain (5 μg/ml), had any significant effect on H2O2-induced activation of ERK or the PI3-K target, Akt1. Both AG1478 and anti-EGFr inhibited H2O2-induced EGFr activation indicating that H2O2 can activate EGFr via EGFr ligand release. Tyrphostin AG1478 also had no effect on H2O2 inhibition of CCh-stimulated Cl- secretion across T84 monolayers. In summary, H2O2 activates EGFr in colonic epithelial cells, probably via the release of an EGFr ligand, but this effect does not account for inhibition of Ca2+-dependent Cl− secretion by H2O2. Because we have previously shown that EGFr activated by other means can lead to inhibition of Cl− secretion, our data imply selective outcomes of EGFr signaling depending on its mode of activation. Supported by the Crohn's and Colitis Foundation of America and NIH.