EGF stimulates lipoxin A4 synthesis and modulates repair in corneal epithelial cells through ERK and p38 activation.

The cornea is the most powerful refracting tissue in the eye, with a structure that allows for preservation of the delicate visual axis. Sustained inflammation can alter the wound-healing process, leading to visual impairment and, in the most severe cases, to blindness. The corneal epithelium, the most outer layer of the tissue, is frequently exposed to infection and injury. Thus, it is important to maintain an adequate host defense while actively suppressing inflammatory and immunogenic responses.1 It is therefore imperative to understand the functions of pro- and anti-inflammatory signals and their roles in corneal wound healing. Lipids play an important role in the complex inflammatory processes that occur after corneal injury.2,3 Once generated, pro- and anti-inflammatory lipids activate signaling pathways involved in injury and repair.4 Previous studies show that arachidonic acid (AA) is released and serves as a substrate for lipoxygenase (LOX) enzymes, resulting in more than a 700% increase in lipoxygenase products in the epithelium after injury to rabbit corneas.5–7 12/15-Lipoxygenases (12/15-LOX) are members of the LOX family that oxygenate free polyenoic fatty acids.8 The primary oxidation products are reduced by glutathione peroxidases to corresponding hydroxyl derivatives or are metabolized into secondary oxidized lipids such as leukotriens, lipoxins, and hepoxillins, which act as lipid mediators.9–11 The main lipoxygenase metabolites of AA formed in the cornea are 12-(S)-hydroxyl/peroxyeicosatetraenoic acid [12(S)-HETE] or 15(S)-HETE, depending on the species,12,13 and lipoxin A4 (LXA4).14 These metabolites may act as secondary messengers in promoting epithelial wound healing. Previous work shows that inhibition of LOX delays the rate of corneal epithelial repair.15 A more recent study on 3T6 fibroblast cultures shows that 12(S)-HETE is enantioselective in DNA synthesis, protein synthesis, and cell growth.16 Other studies show that LXA4 exhibits potent anti-inflammatory properties in in vitro and in vivo models of acute or aberrant inflammation by inhibiting neutrophils (PMN)17 and lymphocyte activation18 and by significantly increasing re-epithelization in corneal wounds.14 However, the biochemical mechanism involved in these processes is unknown. Epidermal growth factor (EGF) is expressed in tears, the corneal epithelium, and keratocytes, and it exerts autocrine and paracrine functions in corneal epithelium, thus stimulating migration and proliferation. The concentration of EGF in tears rapidly increases after epithelial wounding.19 mRNA and its product (EGF) increase up to 3 days after injury,20 demonstrating that there is a rapid pool released from tears followed by an increase in synthesis of this growth factor. EGF mRNA is also expressed in the epithelium and keratocytes, and there is a marked increase in EGF expression in keratocytes after epithelial scrape wounds.21 These changes in EGF expression demonstrate the importance of this growth factor in epithelial wound healing. In a variety of cells, including corneal epithelial cells, EGF binds to its receptor (EGFR) and activates the extracellular signal-regulated kinases 1/2- mitogen-activated protein kinase (ERK1/2-MAPK) cascade.22 Once this cascade is activated, ERK1/2 are imported into the nucleus, where they phosphorylate specific transcription factors involved in cell proliferation.23 Another member of the MAPK kinases, p38, is also activated, and after injury cross-talk occurs between these two MAPKs.24,25 While ERK1/2 are involved mainly in proliferation, p38 is activated during cell migration.24,25 Previously we have shown that after rabbit corneal injury, growth factors such as EGF activate cytosolic phospholipase A2 (cPLA2), leading to the formation of AA. This fatty acid is further converted by a 12-LOX to 12(S)-HETE, which plays a role in proliferation of corneal epithelial cells.26 In human corneal epithelial cells, EGF stimulates the rapid synthesis of 15(S)-HETE, the main LOX in the human cornea.27 In the present study, we investigated whether EGF induces the synthesis of LXA4 in corneal epithelial cells. We also studied the signaling mechanism implicated in corneal wound healing by EGF-mediated synthesis of LXA4.