title: Regulation of VEGF mRNA expression and protein secretion by TGF-beta2 in human retinal pigment epithelial cells.
abstract: VEGF secretion by the human retinal pigment epithelium (hRPE) plays an important role in retinal and choroidal neovascularization. In this study, transforming growth factor-beta2 (TGF-beta2)-induced vascular endothelial growth factor (VEGF) gene expression was investigated in hRPE cells. Treatment of hRPE cells with TGF-beta2 for 24 and 48h as compared to 8h resulted in markedly increased VEGF secretion by fivefold and nine-fold, respectively. Induced VEGF mRNA peaked within 3h of stimulation and remained above the basal at 36h. Stimulation of VEGF expression by TGF-beta2 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-alpha or bFGF alone, co-administration of either of these two cytokines synergized the effect of TGF-beta2 on VEGF mRNA expression and protein production. Quantitative RT-PCR revealed that the synergy was predominantly at the level of VEGF transcription. Moreover, TGF-beta2-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-kappaB (NF-kappaB) [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-kappaB as well as multiple essential signaling intermediates, including PKC, PTK and ROS, are involved in hRPE VEGF up regulation by TGF-beta2.
authors.affiliation: Department of Ophthalmology, University of Michigan, Ann Arbor, MI 48105, USA.
authors.forename: Zong-Mei
authors.initials: ZM
authors.lastname: Bian
authors.forename: Susan G
authors.initials: SG
authors.lastname: Elner
authors.forename: Victor M
authors.initials: VM
authors.lastname: Elner
mesh_terms: D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004357:Drug Synergism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000893:Anti-Inflammatory Agents; D007166:Immunosuppressive Agents; D012333:RNA, Messenger; D011994:Recombinant Proteins; D053781:Transforming Growth Factor beta2; D014409:Tumor Necrosis Factor-alpha; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D003907:Dexamethasone; D016572:Cyclosporine