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Y.–N. Huo, Y.–F. Yao, W.–Y. Qiu, Q. Pan, M.F. Lou; Association of Reactive Oxygen Species (ROS) With Epidermal Growth Factor (EGF)–Stimulated Corneal Epithelial Cell Proliferation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1609.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the role of reactive oxygen species (ROS) in EGF stimulated corneal epithelial cell proliferation.
Primary rabbit corneal epithelium cells were grown to confluence in SHEM medium with 5% FBS. The cells were gradually serum starved by first culturing in 0.5% FBS overnight and then serum–free medium for 30 min before use. Cells preloaded with fluorescent dye, 2’,7’–dichlorodihydrofluorescin diacetate (DCFHDA) were exposed to EGF (1 ng/ml) in the presence or absence of N–acetylcysteine (NAC, 30 mM, 30 min), an antioxidant, or mannitol (100 mM, 30 min), a free radical scavenger. The fluorescence produced by ROS–sensitive DCFH was captured by confocal microscopy (488 nm excitation and 522 nm emission). In a separate confocal study, EGF (1 mg/ml) was added to the cells pre–incubated in each of the following inhibitors: diphenyl–iodide (DPI, 5 µM) for NADPH oxidase; nordihydro– guaiaretic acid (NDGA, 10 µM) for lipoxygenase; rotenone (5 µM) for mitochondrial superoxide production. Cell proliferation was studied by stimulating cells with different concentrations of H2O2 (0–60 µM), or by EGF (1 ng/ml) with and without mannitol or DPI in media for 1 day, and evaluated by cell counting and MTT assay.
Confocal microscopy showed that EGF at 1 ng/ml strongly stimulated a transient increase of ROS–generated fluorescence in cultured rabbit corneal epithelial cells, starting at 5 min, peaking at 10 min, and gradually returning to basal level by 60 min. The EGF–activated fluorescence could be suppressed when cells were preloaded with NAC or mannitol. EGF–induced ROS production could be blocked by NDGA or DPI but not by rotenone. NDGA and DPI also exhibited synergistic inhibitory effect. EGF (1 ng/ml) stimulated cell proliferation 2–fold over the control (no EGF), but the mitogenic effect was suppressed by mannitol or DPI. H2O2 at the range of 1–20 µM stimulated cell growth in a dose–dependent manner, but at 40 µM or higher inhibited cell growth.
EGF stimulated intracellular ROS production enzymatically, and corroborated with cell proliferation. Exogenous H2O2 at physiological range also stimulated cell growth. This is a first report of ROS association with EGF mitogenic action in corneal epithelial cells.
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