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F. Zhang, H. Yang, P. Gjorstrup, P. Reinach; Cell Signaling Pathways Underlying Resolvin Induced Increases in Human Corneal Epithelial Cell Migration: Effects Of RX-10001 (RvE1) and RX-10008. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3492.
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Resolvins are lipid mediators generated through the oxidation of omega-3 fatty acids: eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). While there is evidence that resolvin analogues hasten human corneal epithelial cell (HCEC) wound healing through increase in cell migration, the signaling pathways underlying this response require additional clarification. Accordingly, we determined in HCEC if wound closure stimulation by resolvins is mediated through phosphoinositide 3-K (PI3-K), p38 mitogen activated protein kinase (MAPK) signaling activation and changes in paxillin phosphorylation status.
A scratch wound assay was used to delineate the effects of the endogenous resolvin RvE1 (RX-10001) and the analog RX-10008 on wound closure rates of SV40-immortalized HCEC. Cells were seeded onto 6-well plates and grown to 80% confluence. After overnight exposure to serum-free medium, the cultures were wounded using a scrapper. Floating cells were removed and the culture was replenished with medium in the absence or presence of drug. The responses to 10 ng/ml EGF were used as a positive control. Hydroxyurea (2.5 mM) was added to reduce cell proliferation. Wound closure rates were monitored using an inverted microscope and photographing the wound area at 5 and 24 h. Sigma scan evaluated the extent of wound closure. To resolve the individual roles of PI3-K and p38 MAPK signaling on wound closure, the effects of the PI3-K inhibitor, LY294002, and the p38 inhibitor, BIRB 796 were determined on this response. Western Blot analyses determined RvE1 and RX-10008-induced changes in the phosphorylation status of p38 and paxillin (Tyr 118).
RvE1 and RX-10008 dose-dependently increased HCEC migratory rates to levels comparable to those induced by EGF. At 24 h, the rates normalized to the untreated controls increased at 0.1 µM up to 1.68 ± 0.11 and 1.82 ± 0.12 (n=11), respectively. These increases were blocked by exposure to either 0.5 µM BIRB 796 or 1 µM LY294002. Both RvE1 and RX-10008 induced time dependent increases in p38 and paxillin (Tyr 118) phosphorylation for up to 60 min. Preincubation with the epidermal growth factor receptor inhibitor, AG1478 (1 µM), blocked the resolvin-induced increases in migration and phosphorylation of p38 and paxillin (Tyr 118).
Resolvins enhanced HCEC wound closure by stimulating their migratory activity following EGFR transactivation. This response is dependent on PI3-K and p38 linked signaling. PI3-K activation leads to paxillin (Tyr 118) phosphorylation.
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