Lysophosphatidic acid promoting corneal epithelial wound healing by transactivation of epidermal growth factor receptor.
OBJECTIVE
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.
Xu
Ke-Ping
KP
Department of Ophthalmology, Kresge Eye Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Yin
Jia
J
Yu
Fu-Shin X
FS
D000818:Animals; D018929:Cell Culture Techniques; D002465:Cell Movement; D003646:Debridement; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000293:injuries / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008246:Lysophospholipids / Q000494:pharmacology*; D051058:Oncogene Protein v-akt / Q000378:metabolism; D009924:Organ Culture Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D013552:Swine; D015533:Transcriptional Activation / Q000502:physiology*; D014945:Wound Healing / Q000187:drug effects*
C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008246:Lysophospholipids; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D051058:Oncogene Protein v-akt; D048049:Extracellular Signal-Regulated MAP Kinases; C032881:lysophosphatidic acid