Glial cell-derived neurotrophic factor (GDNF)-induced migration and signaltransduction in corneal epithelial cells

PURPOSE. To identify signal-transduction pathways induced by glial cell-der ived neurotrophic factor (GDNF) in corneal epithelial cells and to characte rize its effect on cell migration. METHODS. Expression of GDNF receptor (GFR) alpha -1 in human corneal epithe lium was detected by RT-PCR and Western blot analysis. Expression and phosp horylation of Ret, activation of focal adhesion kinase (FAK) and mitogen-as sociated protein kinase (MAPK) signaling pathways, and phosphorylation of p axillin by GDNF were investigated by immunoprecipitation and Western blot a nalysis in primary human corneal epithelial cells and a corneal epithelial cell line. The tyrosine kinase inhibitor herbimycin A and Ras farnesyltrans ferase inhibitor manumycin were used to specifically inhibit GDNF-induced s ignaling pathways. In vitro wound-healing assays and modified Boyden chambe r analysis were performed to investigate the effect of GDNF on epithelial c ell migration. RESULTS. Expression of GFR alpha -1 was detected in normal and transformed human corneal epithelium. GDNF induced tyrosine phosphorylation of Ret. Fur thermore, tyrosine phosphorylation of FAK and phosphotyrosine kinase (Pyk) 2; serine phosphorylation of c-Raf, MEK1, and Elk 1; and tyrosine-threonine phosphorylation of Erk-1 and -2 were time-dependently activated in the pre sence of GDNF. Tyrosine phosphorylation of paxillin was also induced by GDN F. Migration of corneal epithelial cells was significantly stimulated by GD NF. Herbimycin A strongly inhibited the activation of Ret, FAK, c-Raf, and Erk-1 and -2; the phosphorylation of paxillin; and corneal epithelial cell migration. More specifically, the Ras inhibitor manumycin inhibited phospho rylation of c-Raf, MEK 1, Erk l and -2, and Elk-1, but not that of FAK. CONCLUSIONS. Corneal epithelial cells express receptors specific for GDNF t hat are used by GDNF to induce intracellular signaling. FAK and MAPK pathwa ys seem to be activated by GDNF to modulate gene transcription and cell mig ration. FAK seems to be an upstream regulator of the MAPK cascade for GDNF signal transduction. As an inducer of FAK-dependent corneal epithelial migr ation, GDNF may play an important role in corneal regeneration and wound he aling.