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From the Department of Ophthalmology, University of Heidelberg Medical School, Germany.
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) 
-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 GFR-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.
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