Phospholipase C-γ inhibition prevents EGF protection of intestinal cytoskeleton and barrier against oxidants

Abstract

Loss of intestinal barrier integrity is associated with oxidative inflammatory GI disorders including inflammatory bowel disease. Using monolayers of human intestinal epithelial (Caco-2) cells, we recently reported that epidermal growth factor (EGF) protects barrier integrity against oxidants by stabilizing the microtubule cytoskeleton, but the mechanism downstream of the EGF receptor (EGFR) is not established. We hypothesized that phospholipase C (PLC)-γ is required. Caco-2 monolayers were exposed to oxidant (H2O2) with or without pretreatment with EGF or specific inhibitors of EGFR tyrosine kinase (AG-1478, tyrphostin 25) or of PLC (L-108, U-73122). Other Caco-2 cells were stably transfected with a dominant negative fragment for PLC-γ (PLCz) to inhibit PLC-γ activation. Doses of EGF that enhanced PLC activity also protected monolayers against oxidant-induced tubulin disassembly, disruption of the microtubule cytoskeleton, and barrier leakiness as assessed by radioimmunoassay, quantitative Western blots, high-resolution laser confocal microscopy, and fluorometry, respectively. Pretreatment with either type of inhibitor abolished EGF protection. Transfected cells also lost EGF protection and showed reduced PLC-γ phosphorylation and activity. We conclude that EGF protection requires PLC-γ signaling and that PLC-γ may be a useful therapeutic target.