September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Downstream signal pathway of epidermal growth factor receptor on the in vitro corneal wound healing model
Author Affiliations & Notes
  • Masataka Ito
    Developmental Anatomy, National Defense Med College, Tokorozawa, Japan
  • Yoko Karasawa
    Ophthalmology, National Defense Med College, Tokorozawa, Japan
  • Masaru Takeuchi
    Ophthalmology, National Defense Med College, Tokorozawa, Japan
  • Footnotes
    Commercial Relationships   Masataka Ito, None; Yoko Karasawa, None; Masaru Takeuchi, None
  • Footnotes
    Support  Grants‐in‐aid for Scientific Research #26462673
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1277. doi:
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      Masataka Ito, Yoko Karasawa, Masaru Takeuchi; Downstream signal pathway of epidermal growth factor receptor on the in vitro corneal wound healing model. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1277.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : We have previously reported that, in the in vitro corneal wound healing model using mouse corneal epithelial cell line, epidermal growth factor (EGF) promoted re-epithelialization of denuded area, and that the re-epithelialization did not depend on cell proliferation. The purpose of this study was to investigate which downstream signal pathway, MAP kinase pathway or phosphatidyl inositol 3 (PI3) kinase pathway, is involved in this re-epithelialization.

Methods : Corneal epithelial cell line TKE-2 was cultured and multilayered cultures were scratch-wounded with P10 pipette tip. Either EGF receptor kinase inhibitor AG1478 (2.5μM), MAP kinase kinase inhibitor PD08059 (20μM), PI3 kinase inhibitor LY294002 (50μM), or Wortmannin (5μM) was added to the culture and decrease of denuded area was evaluated. ImageJ software was used for the measurement of the area. Cell proliferation was evaluated by BrdU uptake and cell death by colorimetric analyses of lactate dehydrogenase in proliferating cell cultures.

Results : When EGF receptor kinase inhibitor AG1478 was added, re-epithelialization was completely inhibited, while inhibition of re-epithelialization by MAP kinase kinase inhibitor PD08059 was mild. Similar to AG1478, two PI3 kinase inhibitors LY294002 and Wortmannin inhibited re-epithelialization completely. Under the administration of these inhibitors to proliferating cells, BrdU uptake was suppressed compared with control, while levels of cell death were not significantly different.

Conclusions : For the re-epithelialization of denuded area, PI3 kinase pathway is the main pathway of the downstream of EGF receptor.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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