Dependence of EGF-induced increases in corneal epithelial proliferation and migration on GSK-3 inactivation.
OBJECTIVE
This study was designed to determine in human corneal epithelial cells (HCEC) whether the balance between epidermal growth factor (EGF)-induced increases in proliferation and migration is dependent on the duration and magnitude of extracellular signal-regulated kinase (Erk)1/2 activation.


METHODS
Western blot analysis evaluated the phosphorylation status of Erk1/2 and phosphoinositide 3-kinase (PI3-K) along with cell cycle kinases, paxillin, and mitogen kinase protein phosphatase (MKP)-1. Proliferation and migration rates were determined by [(3)H]-thymidine incorporation and scratch wound healing assay, respectively.


RESULTS
EGF induced increases in paxillin Ser-126 phosphorylation and cyclin D1 expression through transient Erk1/2 phosphorylation. However, preinhibition of glycogen synthase kinase (GSK)-3 activation with 20 microM SB415286 prolonged and augmented this Erk1/2 response to EGF but decreased cyclin D1 expression, whereas p27Kip1 levels rose. In turn, the mitogenic response fell, whereas paxillin phosphorylation occurred 45 minutes sooner than without SB415286. In contrast, blocking PI3-K activation with LY294002 (50 microM) eliminated EGF-induced GSK-3 inhibition and Erk1/2 phosphorylation as well as increases in proliferation and migration. SB415286 or U0126 (10 microM) suppression of Erk1/2 phosphorylation blocked EGF-induced MKP-1 phosphorylation. Inhibition of EGF-induced increases in proliferation and migration by LY294002 was associated with sustained MKP-1 phosphorylation induced by GSK-3. Prolonging MKP-1 phosphorylation by LY294002 increased p27Kip1, whereas cyclin D1 levels fell.


CONCLUSIONS
GSK-3-induced MKP-1 phosphorylation mediates negative feedback control between EGF receptor-linked PI3-K and ERK signaling pathways. Inhibition of such control prolongs Erk1/2 activation and alters the balance between EGF-induced increases in proliferation and migration. Therefore, these responses to EGF can be modulated through altering the feedback between these two pathways.
Wang
Zheng
Z
State University of New York, State College of Optometry, Department of Biological Sciences, New York, New York 10036, USA.
Yang
Hua
H
Zhang
Fan
F
Pan
Zan
Z
Capó-Aponte
José
J
Reinach
Peter S
PS
D000627:Aminophenols / Q000494:pharmacology; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D054638:Dual Specificity Phosphatase 1 / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000502:physiology*; D019573:Epithelium, Corneal / Q000166:cytology*; D025461:Feedback, Physiological / Q000502:physiology*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D008301:Maleimides / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D051419:Paxillin / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D013997:Time Factors; D014945:Wound Healing / Q000502:physiology
C417520:3-(3-chloro-4-hydroxyphenylamino)-4-(4-nitrophenyl)-1H-pyrrole-2,5-dione; D000627:Aminophenols; D018797:Cell Cycle Proteins; D008301:Maleimides; C496061:PXN protein, human; D051419:Paxillin; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D038362:Glycogen Synthase Kinase 3; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1