May 2008
Volume 49, Issue 13
ARVO Annual Meeting Abstract  |   May 2008
Dependence of EGF-Induced Increases in Corneal Epithelial Cell Migration on Paxillin Phosphorylation
Author Affiliations & Notes
  • Z. Wang
    Biological Sciences, SUNY College of Optometry, New York, New York
  • P. Reinach
    Biological Sciences, SUNY College of Optometry, New York, New York
  • Footnotes
    Commercial Relationships  Z. Wang, None; P. Reinach, None.
  • Footnotes
    Support  EY04795
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3619. doi:
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      Z. Wang, P. Reinach; Dependence of EGF-Induced Increases in Corneal Epithelial Cell Migration on Paxillin Phosphorylation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3619. doi:

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Paxillin is a scaffolding protein that is a component of focal adhesion complexes whose phosphorylation status modulates cell migration rate. Increases in its status resulting from EGF exposure are dependent on sequential PI3-K-induced Akt activation followed by glycogen synthase kinase-3 (GSK-3 α/β) serine 21/9 phosphorylation (i.e inhibition). The associations were determined between such rises and paxillin serine 126 phosphorylation as well as human corneal epithelial cell migration.

Methods: : Scratch wound assay was performed on SV40-immortalized human corneal epithelial cells (HCEC) in the absence and presence of mediators that alter GSK-3 α/β phosphorylation status. Phosphorylation status changes of GSK-3 α/β (i.e. serine 21/9) and paxillin (i.e. serine126) were determined with Western blot analysis. Time dependent Erk1/2 activation was characterized along with changes in p27 and cyclin D-1 expression levels. [3H] thymidine incorporation measurements assessed cell proliferation.

Results: : EGF (5 ng/ml) mediated cell migration stimulation was associated with GSK-3 α/β inhibition. GSK-3 α/β inhibition prolonged time dependent increases in EGF-induced Erk1/2 phosphorylation. Despite such prolongation, the mitogenic response to EGF was lessened, which was associated with rises in p27 expression levels and declines in cyclin D-1 expresion. PDBu (1 uM) phosphorylated GSK-3 α/β whereas LY294002 (20 uM) fully blocked EGF-induced GSK-3 α/β paxillin serine 126 phosphorylation. Paxillin phosphorylation was maximal following GSK-3 α/β inhibition by either EGF or SB415286 (10 uM) whereas UO126 (10 uM) preincubation blocked this increase in phosphorylation. Wound closure rates were maximal with either EGF alone or EGF in combination with SB415286. Similarly, SB415286 alone induced wound closure was faster than that in the control. There was a correspondence between either EGF or SB415286-induced increases in GSK-3 α/β, paxillin serine126 phosphorylation and wound closure rate.

Conclusions: : EGF-induced stimulation of cell migration is associated with increases in GSK-3 α/β inhibition and Erk1/2-induced serine 126 phosphorylation of paxillin. Since GSK-3 α/β inhibition suppressed the mitogenic response to EGF and prolonged transient Erk1/2 phosphorylation, GSK-3 α/β mediates through protein phosphatase activation crosstalk control of Erk1/2 phosphorylation status. Therefore, this mitogenic response is dependent on the time course of transient changes in Erk1/2 phosphorylation status.

Keywords: cornea: epithelium • proliferation • cell survival 

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