April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Extracellular ATP Stimulates Corneal Epithelial Cell Motility through Pyk2-mediated Activation of the EGF Receptor
Author Affiliations & Notes
  • Jes K. Klarlund
    Ophthal & Vis Sci Res Ctr, Univ of Pittsburgh Eye & Ear Inst, Pittsburgh, Pennsylvania
  • Ethan R. Block
    Ophthal & Vis Sci Res Ctr, Univ of Pittsburgh Eye & Ear Inst, Pittsburgh, Pennsylvania
  • Footnotes
    Commercial Relationships  Jes K. Klarlund, None; Ethan R. Block, None
  • Footnotes
    Support  This work was supported by the National Institutes of Health National Eye Institute grants EY-08098 and EY-017271, Research to Prevent Blindness, and The Eye and Ear Foundation (Pittsburgh, PA).
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 1943. doi:
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      Jes K. Klarlund, Ethan R. Block; Extracellular ATP Stimulates Corneal Epithelial Cell Motility through Pyk2-mediated Activation of the EGF Receptor. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1943.

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Abstract

Purpose: : Extracellular ATP is an important inducer of motility in corneal epithelial cells, and it is known to act through the src family of kinases (SFK). Here we test the hypothesis that Pyk2, a member of the focal adhesion kinase family, is the immediate activator of the SFKs, and that Pyk2 is activated through phospholipase D2 signaling by extracellular ATP.

Methods: : The activation states of Pyk2 and the SFKs were monitored by immunoblotting with phospho-specific antibodies. Pyk2-SFKs interaction was monitored by co-immunoprecipitation assays. Pyk2 and PLD2 activities were modulated by reducing expression with siRNAs and dominant negative constructs. Cell motility was assayed in human corneal limbal epithelial (HCLE) cells by introducing gaps in the cell layers.

Results: : Pyk2 was rapidly and potently activated by treating corneal epithelial cells with ATP, and Pyk2 interaction with SFKs was increased after ATP stimulation. Disruption of Pyk2 signaling either by siRNA or by expression of a dominant-negative mutant of Pyk2 led to inhibition of ATP-induced activation of the SFKs and the EGF receptor. Phospholipase D2 was shown to be upstream of Pyk2 activation by the ability of its reaction product, phosphatidic acid, to stimulate Pyk2 and by the inhibition of ATP-induced Pyk2 activation by reducing phospholipase D2 expression with siRNA. Reducing Pyk2 activity by siRNA inhibited healing of wounds in sheets of HCLE cells, and this could be rescued by addition of EGF to the cultures.

Conclusions: : Extracellular ATP induces activation of the SFK through Pyk2 signaling, and PLD2 is an upstream regulator of Pyk2. In conjunction with other data, a detailed picture of the intracellular signals that couple ATP stimulation to the critical EGF receptor activation is now emerging.

Keywords: cornea: basic science • cornea: epithelium • signal transduction 
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