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H. Bazan, J. He, N. Li; Epidermal Growth Factor (EGF) Stimulates Corneal Keratocyte Proliferation and Differentiation via PI-3 Kinase Activity. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1478.
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© ARVO (1962-2015); The Authors (2016-present)
Stromal keratocytes (SK) differentiate to myofibroblasts after they have been activated and are important in the development of a fibrotic, opaque appearance in the cornea. TGF-ß1 is important in this differentiation. We had reported (ARVO2006) that EGF, in conjunction with TGF-ß1 , increases the transformation of SK to myofibroblasts, but there is no information about the cellular and molecular mechanisms by which EGF exerts this action. Here, we investigated the involvement of the signaling pathways activated by the EGF receptor in the proliferation and differentiation of SK
: SK were treated with EGF, TGF-ß1 or EGF plus TGF-ß1 for 2 days to 1 week in the presence or absence of AG1478 (an inhibitor of EGF-receptors), LY294002 (a PI-3K inhibitor), PD98059 (a ERK1/2 inhibitor), and SB203580 (a p38 inhibitor). SK differentiation to myofibroblast was identified with anti-ADLH1 and α SMA antibodies. Cell proliferation was evaluated with anti-Ki-67 and PCNA antibodies. ECM components: keratan sulfate (KS), chondroitin sulfate (CS), fibronectin (FN), and thrombospondin-1(TSP-1), were assayed by immunochemistry and Western Blot. Cell migration was measured from images obtained under the microscope with MetaVue image software.
Addition of EGF (50ng/ml) to SK induced 100% transformation into proto-myofibroblasts and expression of α-SMA, CS, FN and TSP-1. TGF-ß1 (10ng/mL) for 1 week induced the cells to transform into proto-myofibroblasts (88%) and into differentiated-myofibroblasts (12%). However, TGF-ß1 plus EGF induced 90% of the cells to differentiated-myofibroblasts, with significant increase in the expression of CS, FN and TSP. EGF promoted cell proliferation and migration and TGF-ß1 increased migration. TGF-ß1 plus EGF also significantly promoted cell migration but had no significant effect on cell proliferation. AG1478 not only blocked the effect of EGF on SK proliferation but also prevented the effect of TGF-ß1 on SK differentiation. Similar inhibitory effects were observed in the presence of LY294002, but not in the presence of PD98059 and SB203580.
The data suggest that EGF through its receptor activation and PI-3K pathway promotes stromal wound healing by increasing SK proliferation and transformation to proto-myofibroblasts, amplifying the differentiation to myofibroblasts and the synthesis of ECM components such as FN and TSP-1, and synergistically enhancing cell migration induced by TGF-ß1.
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