Purpose: To investigate the effect of epidermal growth factor (EGF) on lipoxin A4 (LXA4) synthesis and how it regulates corneal epithelial wound healing through mitogen-activated kinases, extracellular regulated kinase (ERK) 1/2, and p38.
Methods: Rabbit corneal epithelial (RCE) cells were stimulated with EGF or LXA4 at different times. In some experiments, cells were pretreated with 12/15-lipoxygenase (12/15-LOX) inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC), ERK1/2 inhibitor PD98059, or p38 inhibitor SB203580. For wound-healing experiments, corneas from rabbits and 12/15-LOX (ALOX-15)-deficient mice were injured by epithelial removal and maintained in organ culture in the presence of EGF or LXA4 with or without inhibitors. Epithelial cell proliferation was assayed by immunofluorescence with Ki67 and cell counting. Scrape migration assays were performed in 6-well plates. LXA4 synthesis was analyzed by liquid chromatography-tandem mass spectrometry analysis.
Results: EGF activated ERK1/2 and p38 in RCE cells in a sustained manner. EGF activation was partially inhibited by CDC. EGF and LXA4 increased corneal epithelial wound closure. ERK1/2 inhibition with PD98059 or p38 with SB203580 blocked the effect of LXA4 on wound healing. ALOX-15 corneas displayed inhibition of epithelial wound closure promoted by EGF, whereas LXA4 stimulation induced similar wound closure in wild-type and knockout mice. EGF-stimulated LXA4 synthesis in RCE cells was inhibited by CDC or the EGF receptor antagonist AG1478.
Conclusions: These results demonstrate that EGF-stimulated epithelial wound healing is partially mediated through a 12/15-LOX-LXA4 pathway, and activation of ERK1/2 and p38 is required for LXA4 action.