Specific Signaling Cascades Involved in Cell Spreading During Healing of Micro-Wounded Gastric Epithelial Monolayers

Mechanisms that specifically modulate cell spreading and/or cell migration following epithelial wounding are poorly understood. Using micro-wounded human gastric epithelial monolayers, we show herein that EGF and TGF alpha maximally increase spreading of epithelial sheets under a cell proliferation-independent mechanism. Treatment of confluent HGE-17 cells with the phosphatidylinositol 3-kinase inhibitor, LY294002, and the epidermal growth factor receptor inhibitor, PD 153035, strongly reduced basal and TGF alpha-stimulated cell spreading. While pharmacological inhibition of pp60src-kinase activity also attenuated basal epithelial spreading, addition of the mTOR/p70S6K inhibitor rapamycin or a specific siRNA targeting ILK sequence did not affect the kinetic rates of wound closure. Epithelial wound healing was initiated by actin purse-string contraction followed by lamellae formation. Conversely, disruption of actin and tubulin stability with cytochalasin D and nocodazole, respectively, inhibited epithelial sheet spreading. Finally, antibodies directed against the alpha 3 integrin subunit, but not against the alpha 6 or alpha 2 subunits, attenuated epithelial sheet spreading as well as lamellae formation. In conclusion, the current investigation establishes that EGF/TGF alpha and the alpha 3 beta 1 integrin, pp60c-src, EGFR and PI3K pathways are mainly associated with the cell spreading of the restitution process during healing of human gastric epithelial wounds. J. Cell. Biochem. 105: 1240-1249, 2008. (c) 2008 Wiley-Liss, Inc.