Purpose: To identify signal-transduction pathways induced by glial cell-derived neurotrophic factor (GDNF) in corneal epithelial cells and to characterize its effect on cell migration.
Methods: Expression of GDNF receptor (GFR) alpha-1 in human corneal epithelium was detected by RT-PCR and Western blot analysis. Expression and phosphorylation of Ret, activation of focal adhesion kinase (FAK) and mitogen-associated protein kinase (MAPK) signaling pathways, and phosphorylation of paxillin by GDNF were investigated by immunoprecipitation and Western blot analysis in primary human corneal epithelial cells and a corneal epithelial cell line. The tyrosine kinase inhibitor herbimycin A and Ras farnesyltransferase inhibitor manumycin were used to specifically inhibit GDNF-induced signaling pathways. In vitro wound-healing assays and modified Boyden chamber analysis were performed to investigate the effect of GDNF on epithelial cell migration.
Results: Expression of GFRalpha-1 was detected in normal and transformed human corneal epithelium. GDNF induced tyrosine phosphorylation of Ret. Furthermore, 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 presence of GDNF. Tyrosine phosphorylation of paxillin was also induced by GDNF. Migration of corneal epithelial cells was significantly stimulated by GDNF. 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 phosphorylation of c-Raf, MEK 1, Erk-1 and -2, and Elk 1, but not that of FAK.
Conclusions: Corneal epithelial cells express receptors specific for GDNF that are used by GDNF to induce intracellular signaling. FAK and MAPK pathways seem to be activated by GDNF to modulate gene transcription and cell migration. FAK seems to be an upstream regulator of the MAPK cascade for GDNF signal transduction. As an inducer of FAK-dependent corneal epithelial migration, GDNF may play an important role in corneal regeneration and wound healing.