Purchase this article with an account.
G. Chandrasekher, D. Sailaja, H.E. Bazan; Effect of Oxidative Stress on the Activation of Intracellular Signaling Cascades and Wound Healing of Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3839.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Reactive oxygen species have been shown to activate many intracellular protein kinases in several cell types. Generation of reactive oxygen species could occur due to chemical and environmental factors that damage corneal epithelium. In this study we investigated whether reactive oxygen species such as hydrogen peroxide (H2O2) can activate signal-transduction cascades in corneal epithelial cells and affect the wound-healing process. Methods: Rabbit corneal epithelial cells derived from primary cultures were treated with H2O2 (200 µM) for different times (0-4 hr). Cell lysates were subjected to SDS-PAGE and Western blot followed by immunoblotting using phospho-specific antibodies against protein kinase B (Akt), and mitogen-activated protein kinase superfamily members, p42/44 MAP kinase (Erk 1/2), stress-activated protein kinases, the p38 and c-jun N-terminal protein kinase (JNK). The effect of H2O2 (100-1000 µM) on 6-mm corneal epithelial wound closure was measured by image analysis of organ-cultured (24 hr) rabbit corneas. Apoptotic cells in cultures were stained with Hoechst reagent. Results: H2O2 induced the phosphorylation of all protein kinases examined by varying degrees in a time-dependent manner. Akt and Erk 1/2 were activated (3- and 6-fold respectively) within 10 min, after which the phosphorylation decreased slowly over 3-4 hr. Optimal increase (100%) in phosphorylation of p38 was observed within 60 min, and by 2 hr the activation reached basal levels. The phosphorylation of JNK, which peaked (3- to 4- fold) at 30 min, disappeared completely by 60 min. H2O2 (100-250 µM) caused about 15-20% inhibition in wound healing but did not induce significant apoptosis in cultured epithelial cells. However, when corneas were treated with 500-1000 µM H2O2 a significant delay (about 50-75% inhibition) in wound healing was observed. Apoptosis as well as necrosis occurred when corneal epithelial cell cultures were incubated with 1000 µM H2O2 for 24 hr. Conclusions: Low levels of oxygen radicals, if generated inside the corneal epithelial cells, may cause mitotic arrest through activation of stress kinases, leading to a moderate inhibition in wound healing but not apoptosis. Prolonged stimulation of Erk 1/2 and Akt induced by H2O2 could be a signaling response to maintain the normal cell metabolism. A significant delay in corneal epithelial wound healing at higher concentrations of H2O2 was due to apoptosis and necrosis.
This PDF is available to Subscribers Only