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K.H. Svoboda, P.B. Moessner; EGF and Type I Collagen Stimulate and AG1478 Inhibits Actin Reorganization in Whole Epithelial Sheets . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3811.
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© ARVO (1962-2015); The Authors (2016-present)
Whole embryonic corneal epithelial sheets can be isolated without the basal lamina. These sheets of cells respond to bombesin, lysophosphatidic acid (LPA) and extracellular matrix (ECM) molecules including fibronectin (FN), laminin and collagen (COL), by reorganizing the actin cortical mat (ACM). Purpose: As EGF and integrin mediated signaling stimulate many of the same secondary signal molecules we asked: do sheets of epithelia respond to EGF by increasing actin reorganization? In addition, we asked if we blocked the EGF receptor with a specific inhibitor, AG1478, would the actin still reorganize in the presence of EGF, COL, or the combination of EGF and COL? Methods: Epithelia were isolated without the basal lamina and incubated overnight with either control media or media containing EFG (10 ng/ml) in the presence or absence of AG1478 (30 nM). The following day, the epithelia were incubated in type I COL (100 µg/ml) for 60, 90 or 120 min. The tissues were rinsed, fixed and stained with Texas Red phalloidin. Whole tissues were morphologically analyzed using a Leica TSP SP2 microscope for actin reorganization. Epithelia were scored as +, +/-, or –actin cortical mat (ACM). Results: No treatment control tissues and epithelia treated with AG1478 incubated without EGF or COL were scored –ACM (90%). EGF treated epithelia reorganized the ACM in 80% of the epithelia scored, similar to COL (90%). Tissues treated with AG1478 and EGF or COL were either –ACM (60%) or +/- ACM (25%). Conclusions: Embryonic corneal epithelial cells respond to EGF by reorganizing the ACM. Blocking EGF decreased ACM reorganization by both the integrin mediated and EFG signal transduction pathways.
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