Suppression of GSK-3 activation prolongs Erk1/2 phosphorylation and augments EGF-induced migration in HCEC
Zheng Wang 1,
Hua Yang 1,
Fan Zhang 1,
Zan Pan 1,
Jose Capo-Aponte 2,
and
Peter S Reinach 3*
1 Biosciences, SUNY Optometry, New York, New York, United States
2 US Army Aeromedical Research Laboratory, Fort Rucker, Alabama, United States
3 Biosciences, SUNY Optometry, 33 w42nd St, New York, New York, 10036, United States
* To whom correspondence should be addressed. E-mail: preinach{at}gmail.com.
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Abstract |
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Purpose. To determine in human corneal epithelial cells (HCEC) if 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 as well as mitogen kinase protein phosphatase (MKP)-1. [3H]-thymidine incorporation and scratch wound assay determined proliferation and migration rates, 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-3 (GSK-3) activation with 20 µM 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 min sooner than without SB415286. In contrast, blocking PI3-K activation with LY294002 (50 µM) eliminated EGF-induced GSK-3 inhibition and Erk1/2 phosphorylation as well as increases in proliferation and migration. SB415286 or U0126 (10 µM) 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.
Key Words:
epidermal growth factor, extracellular regulated kinase, phosphoinositide3- kinase, glycogen synthase kinase3, migration, cyclinD1
