Blocking VEGF signaling augments interleukin-8 secretion via MEK/ERK/1/2 axis in human retinal pigment epithelial cells

AIM: To identify proangiogenic factors engaged in neovascular age-related macular degeneration(AMD) except vascular endothelial growth factor(VEGF) from human retinal pigment epithelial(h RPE) cells and investigate the underlying mechanisms.METHODS: VEGF receptor 2(VEGFR2) in ARPE-19 cells was depleted by si RNA transfection or overexpressed through adenovirus infection. The m RNA and the protein levels of interleukin-8(IL-8) in ARPE-19 cells were measured by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. The protein levels of AKT, p-AKT, MEK, p-MEK, ERK1/2, p-ERK1/2, JNK, p-JNK, p38 and p-p38 were detected by Western blotting. A selective chemical inhibitor, LY3214996, was employed to inhibit phosphorylation of ERK1/2. Cell viability was determined by MTT assay.RESULTS: Knockdown of VEGFR2 in ARPE-19 cells robustly augmented IL-8 production at both the m RNA and the protein levels. Silencing VEGFR2 substantially enhanced phosphorylation of MEK and ERK1/2 while exerted no effects on phosphorylation of AKT, JNK and p38. Inhibiting ERK1/2 phosphorylation by LY3214996 reversed changes in VEGFR2 knockdown-induced IL-8 upregulation at the m RNA and the protein levels with no effects on cell viability. VEGFR2 overexpression significantly reduced IL-8 generation at the m RNA and the protein levels.CONCLUSION: Blockade of VEGF signaling augments IL-8 secretion via MEK/ERK1/2 axis and overactivation of VEGF pathway decreases IL-8 production in h RPE cells. Upregulated IL-8 expression after VEGF signaling inhibition in h RPE cells may be responsible for being incompletely responsive to anti-VEGF remedy in neovascular AMD, and IL-8 may serve as an alternative therapeutic target for neovascular AMD.