June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
NGF inhibited apoptosis through phosphorylation of FOXO3a in human corneal epithelial cells
Author Affiliations & Notes
  • Tingting Qian
    Ophthalmology, Eye & Ear, Nose, Throat Hosp, Shanghai, China
  • Jiaxu Hong
    Ophthalmology, Eye & Ear, Nose, Throat Hosp, Shanghai, China
  • JianJiang Xu
    Ophthalmology, Eye & Ear, Nose, Throat Hosp, Shanghai, China
  • Footnotes
    Commercial Relationships Tingting Qian, None; Jiaxu Hong, None; JianJiang Xu, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 993. doi:
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      Tingting Qian, Jiaxu Hong, JianJiang Xu; NGF inhibited apoptosis through phosphorylation of FOXO3a in human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):993.

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

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Abstract
 
Purpose
 

To investigate the possible mechanism by which NGF regulated the cell survival of human corneal epithelial cells.

 
Methods
 

Human corneal epithelial cells (HCECs) cultured in vitro were identified by immunostaining for cytokeratin12, a biomarker for corneal epithelium. Cells were stimulated with NGF at 25ng/ml for 1 hour. The cell apoptosis was analyzed by flow cytometry. Western blotting was applied to examine the expression levels of p140trkA, p-Akt, p-FOXO3a and Bim. Immunofluorescence was used to observe the distribution of p-FOXO3a in the cells. Then, PI3K/Akt pathway inhibitor LY294002 was treated to confirm the PI3K/Akt signaling pathway by which NGF regulated the apoptosis of human corneal epithelial cells. Furthermore, western blotting and RT-PCR was used to check the expression of pro-apoptotic FoxO target Bim. Gene reporter experiments were performed to examine the activity of Bim promoter, which was dependent on a FoxO binding site.

 
Results
 

HCECs expressed cytokeratin12. The results of flow cytometry analysis showed that the cell apoptosis was inhibited by NGF. P140trkA, as well as the PI3K/Akt cascade, was activated immediately cells were stimulated. Western blotting and immunofluorescence analysis revealed that he forkhead transcription factor FOXO3a, was phosphorylated and cytoplasm translocated by the regulation of PI3K/Akt signaling pathway in HCECs. Furthermore, both the mRNA and protein levels of Bim were decreased dramatically after NGF treatment. Gene reporter experiments demonstrated that in HCE cells inhibition of Bim promoter was dependent on a FoxO binding site.

 
Conclusions
 

These data suggested that NGF regulated HCECs survival via PI3K/Akt-regulated phosphorylation of FOXO3a in human corneal epithelial cells, and that the phosphorylation of FoxO3a was responsible for the transcriptional down-regulation of pro-apoptotic FoxO target Bim.

     
Keywords: 482 cornea: epithelium • 714 signal transduction • 426 apoptosis/cell death  
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