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