May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Anti-apoptotic Targets of Phosphatidylinositol-3 Kinase/Akt Signaling during HGF Promoted Survival of Corneal Epithelial Cells
Author Affiliations & Notes
  • A.H. Kakazu
    Ophthalmology, LSU Health Sciences Center, New Orleans, LA, United States
  • G. Chandrasekher
    Ophthalmology, LSU Health Sciences Center, New Orleans, LA, United States
  • H.E. Bazan
    Ophthalmology, LSU Health Sciences Center, New Orleans, LA, United States
  • Footnotes
    Commercial Relationships  A.H. Kakazu, None; G. Chandrasekher, None; H.E.P. Bazan, None.
  • Footnotes
    Support  NIH/NEI Ro1 EY06635
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1370. doi:
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      A.H. Kakazu, G. Chandrasekher, H.E. Bazan; Anti-apoptotic Targets of Phosphatidylinositol-3 Kinase/Akt Signaling during HGF Promoted Survival of Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1370.

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

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Abstract

Abstract: : Purpose: Cell proliferation and survival are critical stages during corneal epithelial regeneration after injury. Previously we showed that hepatocyte growth factor (HGF) protects corneal epithelial cell apotosis (induced by growth factor- deprived exhausted medium) through the activation of phosphatidylinositol-3 kinase (PI-3K) and Akt signaling ( ARVO Meeting 2002, Abs. No.1633). Akt targets multiple apoptosis regulating components such as Bad, which associates with Bcl-2 or Bcl-xl and IΚB, which interacts with NFΚB. Here we investigated the targets of Akt during HGF-promoted survival of corneal epithelial cells. Methods: Apoptosis was induced in corneal epithelial cells with staurosporin (10 ng/ml) or calcium ionophore, A23187 (0.5 µM). Presence of apoptotic cells in cultures was identified by staining with Hoechst reagent. Characteristic DNA fragmentation due to apoptosis was identified by DNA laddering in Agarose gel electrophoresis. Western immunoblotting using specific antibodies was used to identify Akt downstream proteins involved in apoptosis: Bcl-2 and Bcl-xl (anti-apoptotic), Bad and IΚB (pro-apoptotic), and their activated forms. Results: Staurosporin and A23187 induced apoptosis, and caused significant DNA fragmentation in corneal epithelial cells. Presence of HGF along with apoptotic agents greatly reduced the DNA breakdown and the number of Hoechst-stained epithelial cell nuclei in cultures. When PI-3K inhibitors wortmannin (200 nM) and LY294002 (10 µM) were present in the cultures, HGF could not overcome the DNA fragmentation produced by apoptotic agents. In addition Bcl-2, Bcl-xl, Bad, and IΚB were identified in corneal epithelial cells. However, Akt-activated forms of IΚB (phospho-IΚB) or Bad (phospho-Bad) were not detected in HGF-stimulated cells with the antibodies we used. Conclusions: Our studies demonstrate that HGF plays an important role in protecting corneal epithelial cells from apoptosis induced by several different mechanisms such as exhausted medium, staurosporin, and calcium ionophore. The PI-3K/Akt pathway is involved in all these cases. Absence of phosphorylated forms of Bad and IΚB, the well known targets of Akt (involved in apoptosis regulation) in our study suggests that, there could be other components through which Akt acts in HGF-promoted corneal epithelial cell survival. Differences in the profile and activation of components of the cell-death machinery could represent important points of regulation to restore a healthy epithelium after injury.

Keywords: signal transduction • apoptosis/cell death • growth factors/growth factor receptors 
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