Regulation of VEGF mRNA expression and protein secretion by TGF-β2 in human retinal pigment epithelial cells

VEGF secretion by the human retinal pigment epithelium (hRPE) plays an important role in retinal and choroidal neovascularization. In this study, transforming growth factor-β2 (TGF-β2)-induced vascular endothelial growth factor (VEGF) gene expression was investigated in hRPE cells. Treatment of hRPE cells with TGF-β2 for 24 and 48 h as compared to 8 h resulted in markedly increased VEGF secretion by fivefold and nine-fold, respectively. Induced VEGF mRNA peaked within 3 h of stimulation and remained above the basal at 36 h. Stimulation of VEGF expression by TGF-β2 was blocked by cycloheximide, suggesting that de novo protein synthesis is required. Induced VEGF production was strongly inhibited by anti-inflammatory agents, dexamethasone and cyclosporin A. Despite of the weak stimulation of VEGF expression by TNF-α or bFGF alone, co-administration of either of these two cytokines synergized the effect of TGF-β2 on VEGF mRNA expression and protein production. Quantitative RT-PCR revealed that the synergy was predominantly at the level of VEGF transcription. Moreover, TGF-β2-induced RPE VEGF secretion was significantly reduced by inhibitors of mitogen-activated protein (MAP) kinase (MEK) (U0126), p38 (SB202190), c-Jun NH2-terminal kinase (JNK), Sp600125, protein tyrosine kinase (PTK) (Genistein), and phosphatidylinositol 3-kinase (PI3K) (Ly294002). Induced VEGF expression was completely abrogated by inhibitors of protein kinase C (PKC) (Ro318220), nuclear factor-κB (NF-κB) [caffeic acid phenethyl ester (CAPE)], and reactive oxygen species (ROS) [N-acetyl-cysteine (Nac) and diphenyleneiodonium (DPI)]. These results suggest that MEK, p38, JNK, PI3K, and NF-κB as well as multiple essential signaling intermediates, including PKC, PTK and ROS, are involved in hRPE VEGF up regulation by TGF-β2.