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K. Xu, F.–S.X. Yu; Lysophosphatidic Acid Promotes Corneal Epithelial Wound Healing via Transactivation of Epidermal Growth Factor Receptor . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5017.
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Growth factor like lipid mediator lysophosphatidic acid (LPA) has been shown to stimulate corneal epithelial cell migration and proliferation. The study sought to identify the underlying mechanisms for LPA to act like a growth factor in stimulating extracellular signal–regulated kinase (ERK) of the mitogen–activated protein kinase (MAPK) during corneal epithelial wound healing.
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 of tyrphostin AG1478 (an epidermal growth factor receptor, EGFR inhibitor), GM6001 (a matrix metalloproteinase inhibitor), or CRM197 (an HB–EGF antagonist) with or without LPA. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blotting with phosphor–tyrosine antibodies. Phosphorylation of ERK, AKT (a major substrate of phosphatidylinositol 3'–kinase) was analyzed by Western blotting with antibodies specific to phosphorylated proteins. Wound and LPA–induced shedding of heparin–binding EGF–like growth factor (HB–EGF) was assessed by measuring the release of alkaline phosphatase (AP) in stable THCE cell lines expressing HB–EGF with AP inserted in the heparin–binding site.
In both organ and cell culture models, LPA enhanced corneal epithelial wound healing. LPA–stimulated wound closure was inhibited by AG1478, GM6001, or CRM197. Consistent with the effects on epithelial migration, all inhibitors as well as Src kinase inhibitor (PP2) retarded LPA–induced EGFR and ERK activation in THCE cells. Unlike exogenously added HB–EGF, LPA stimulated moderate EGFR phosphorylation; the level was similar to that induced by wounding. However, LPA prolonged wound–induced EGFR signaling. The release of HB–EGF assessed by AP activity was shown to be significantly increased in response to wounding, LPA or their combination. Whereas Src kinase inhibitor PP2 and metalloproteinase inhibitor GM6001 significantly blocked the release of HB–EGF–AP shedding induced by LPA.
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 the ligands of G–protein–coupled receptors in response to pathophysiological challenge in human corneal epithelial cells.
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