Lysophosphatidic acid promoting corneal epithelial wound healing by transactivation of epidermal growth factor receptor

PURPOSE. To identify the underlying mechanisms by which lipid mediator lysophosphatidic acid ( LPA) acts as a growth factor in stimulating extracellular signal-regulated kinase ( ERK) and phosphatidylinositol 3'-kinase ( PI3K) during corneal epithelial wound healing. METHODS. Epithelial debridement wounds in cultured porcine corneas and scratch wounds in an epithelial monolayer of SV40-immortalized human corneal epithelial ( THCE) cells were allowed to heal in the presence or absence of an epidermal growth factor receptor ( EGFR) inhibitor ( tyrphostin AG1478), a matrix metalloproteinase inhibitor ( GM6001), or a heparin-binding EGF-like growth factor ( HB-EGF) antagonist ( CRM197) with or without LPA. EGFR activation was analyzed by immunoprecipitation using EGFR antibodies and Western blotting with phosphotyrosine antibodies. Phosphorylation of ERK and AKT ( a major substrate of PI3K) was analyzed by Western blotting with antibodies specific to the phosphorylated proteins. Wound- and LPA-induced shedding of HB-EGF was assessed by measuring the release of alkaline phosphatase ( AP) in a stable THCE cell line that expressed HB-EGF with AP inserted in the heparin-binding site. RESULTS. In organ and cell culture models, LPA enhanced corneal epithelial wound healing. LPA-stimulated and spontaneous wound closure was attenuated by AG1478, GM6001, or CRM197. Consistent with the effects on epithelial migration, these inhibitors, as well as the Src kinase inhibitor ( PP2), retarded LPA-induced activation of EGFR and its downstream effectors ERK and AKT in THCE cells. Unlike exogenously added HB-EGF, LPA stimulated moderate EGFR phosphorylation; the level of phosphorylated EGFR was similar to that induced by wounding. However, LPA appeared to prolong wound-induced EGFR signaling. The release of HB-EGF assessed by AP activity increased significantly in response to wounding, LPA, or both, and the release of HB-EGF-AP induced by LPA was inhibited by PP2 and GM6001. CONCLUSIONS. LPA accelerates corneal epithelial wound healing through its ability to induce autocrine HB-EGF signaling. Transactivation of EGFR by LPA represents a convergent signaling pathway accessible to stimuli such as growth factors and ligands of G-protein-coupled receptors in response to pathophysiological challenge in human corneal epithelial cells.