Regulation of Vascular Endothelial Growth Factor (VEGF) Splicing from Pro-angiogenic to Anti-angiogenic Isoforms

• DOI: 10.1074/jbc.m109.074930
• Publication Date: 2009-11-12T01:18:05Z
• Authors: Dawid G. Nowak; Elianna Mohamed Amin; Emma S. Rennel; Coralie Hoareau-A...; Melissa Gammons; Gopinath Damodoran; Masatoshi Hagiwara; Steven J. Harper; Jeanette Woolard; Michael R. Ladomery; David O. Bates
• Keywords: Vascular Endothelial Growth Factor A; 0301 basic medicine; 570; Messenger; 610; Angiogenesis Inhibitors; Protein Serine-Threonine Kinases; Retinal Neovascularization; Cell Line; Mice; 03 medical and health sciences; Animals; Humans; Protein Isoforms; RNA, Messenger; Enzyme Inhibitors; Insulin-Like Growth Factor I; Cell Line, Transformed; Serine-Arginine Splicing Factors; Animal; Nuclear Proteins; RNA-Binding Proteins; Protein-Serine-Threonine Kinases; 3. Good health; Alternative Splicing; Disease Models, Animal; Transformed; Disease Models; RNA

Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF(165), and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF(165)b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF(165) and less VEGF(165)b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.