Pro-angiogenic effect of IFNgamma is dependent on the PI3K/mTOR/translational pathway in human retinal pigmented epithelial cells.
OBJECTIVE
To investigate the molecular signaling pathway of Interferon gamma (IFNgamma) contributing to angiogenesis in retinal pigmented epithelial (RPE) cells and the role of Phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) in this process.


METHODS
Human adult and fetal RPE cells were used in this study. Real-time polymerase chain reaction was used to detect human vascular endothelial growth factor (VEGF) mRNA expression. Thiazolyl blue tetrazolium bromide (MTT) assay was used to detect cell viability. VEGF expression from cell supernatant was measured using enzyme-linked immunosorbent assay (ELISA). Small interfering RNA (SiRNA) of signal transducers and activators of transcription 1 (stat1) and protein kinases B (akt) were transfected into ARPE-19 cells to directly study the roles of these molecules in VEGF expression. Sodium dodecyl sulfate PAGE (SDS-PAGE) and western blot analysis were used to detect the expression of signaling molecules.


RESULTS
IFNgamma promoted human VEGF expression in both adult and fetal RPE cells. The PI-3K/Akt/mTOR/p70 S6 kinase pathway is required for IFNgamma-induced VEGF expression in retinal cells. The mTOR inhibitor, rapamycin, along with the SiRNA targeted to akt and the PI3K inhibitor, LY294002, decreased hVEGF secretion from RPE cells. Moreover, IFNgamma-induced hVEGF expression was not affected by SiRNA targeted to Stat1, implying that the classic Jak-Stat1 pathway of IFNgamma may not be involved in this process.


CONCLUSIONS
We provide evidence that IFNgamma induces VEGF expression in human retinal pigment epithelial cells. Our work emphasizes that the activation of the PI-3K/mTOR/translational pathway is important for IFNgamma-mediated VEGF expression in RPE cells. By elucidating molecular signaling involved in this process, our findings provide further mechanistic insight into the successful clinical application of rapamycin therapy for choroidal neovascularization in age-related macular degeneration (AMD) and uveitis.
Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Baoying
B
Liu
Lisa
L
Faia
Mengjun
M
Hu
Robert B
RB
Nussenblatt
D002460:Cell Line; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D050794:STAT1 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D007371:Interferon-gamma; D047908:Intracellular Signaling Peptides and Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; D058570:TOR Serine-Threonine Kinases; D042461:Vascular Endothelial Growth Factor A; C494079:STAT1 protein, human; C546842:MTOR protein, human