April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Environment Stress-induced H2AX Phosphorylation through an Alternative Signaling Pathway in Human Corneal Epithelial Cells
Author Affiliations & Notes
  • Ling Wang
    Department of Medicine, HMC, David Geffen School of Medicine, UCLA, Torrance, California
  • Luo Lu
    Department of Medicine, HMC, David Geffen School of Medicine, UCLA, Torrance, California
  • Footnotes
    Commercial Relationships  Ling Wang, None; Luo Lu, None
  • Footnotes
    Support  NIH-EY18343
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 1945. doi:
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      Ling Wang, Luo Lu; Environment Stress-induced H2AX Phosphorylation through an Alternative Signaling Pathway in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1945.

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Abstract

Purpose: : Environment stresses, such as UV irradiation, hypoxia, and hyperosmotic pressure, play important roles in stimulating cell proliferation, migration and apoptosis through activation of signaling pathways to affect corneal epithelial wound healing. The purpose of this study is to investigate a novel/alternative signaling pathway that mediates environmental stress-induced phosphorylation of histone variant H2AX (γ-H2AX) that controls DNA repair process in human corneal epithelial (HCE) cells.

Methods: : HCE cells were cultured in DMEM/F12 medium containing 10% FBS and 5 µg/ml insulin at 37°C with 5% CO2. UV irradiation, hypoxia, and hyperosmotic stress were deployed by exposed cells to UV irradiation (40 µJ/cm2), hypoxia (1% O2, 5%CO2, 94% N2), and hyperosmotic (600 mM) stresses. Western analysis was performed to detect expression of targeting proteins. Immunoprecipitation and kinase assays were employed to measure UV irradiation, hypoxia, and hyperosmotic stress-induced cellular changes.

Results: : We found that Polo-like kinase 3 (Plk3) plays a functional role in the signaling pathway to mediate stress-induced cellular responses including phosphorylation of H2AX. Our results demonstrated in HCE cells: 1) stresses induced H2AX phosphorylation, which is correlated to Plk3 activation; 2) stress-activatedPlk3 could directly phosphorylate H2AXat the site of Ser-139; 3) knockdown of Plk3 by using specific siRNA diminished stress-induced H2AX phosphorylation; and 4) the effect of caffeine on stress-induced Plk3 activation and H2AX phosphorylation were also determined in HCE cells.

Conclusions: : Plk3 is a stress-induced protein kinase and plays an important role in environment stress-induced phosphorylation of H2AX to regulate the DNA repair process in human corneal epithelial cells.

Keywords: cornea: epithelium • gene/expression • apoptosis/cell death 
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