Dependence of EGF-Induced Increases in Corneal Epithelial Proliferation and Migration on GSK-3 Inactivation
ARMY AEROMEDICAL RESEARCH LAB FORT RUCKER AL
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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 Erk12 activation. Western blot analysis evaluated the phosphorylation status of Erk12 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 3H-thymidine incorporation and scratch wound healing assay, respectively. EGF induced increases in paxillin Ser-126 phosphorylation and cyclin D1 expression through transient Erk12 phosphorylation. However, preinhibition of glycogen synthase kinase GSK-3 activation with 20 M SB415286 prolonged and augmented this Erk12 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 M eliminated EGF-induced GSK-3 inhibition and Erk12 phosphorylation as well as increases in proliferation and migration. SB415286 or U0126 10 M suppression of Erk12 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. 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 Erk12 activation and alters the balance between EGFinduced increases in proliferation and migration. Therefore, these responses to EGF can be modulated through altering the feedback between these two pathways.
- Anatomy and Physiology
- Medicine and Medical Research