Regulation of vascular endothelial growth factor (VEGF) splicing from pro-angiogenic to anti-angiogenic isoforms: a novel therapeutic strategy for angiogenesis.
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.
Nowak
Dawid G
DG
Microvascular Research Laboratories, Bristol Heart Institute, Department of Physiology and Pharmacology, School of Veterinary Sciences, University of Bristol, Bristol BS2 8EJ, United Kingdom.
Amin
Elianna Mohamed
EM
Rennel
Emma S
ES
Hoareau-Aveilla
Coralie
C
Gammons
Melissa
M
Damodoran
Gopinath
G
Hagiwara
Masatoshi
M
Harper
Steven J
SJ
Woolard
Jeanette
J
Ladomery
Michael R
MR
Bates
David O
DO
D017398:Alternative Splicing; D020533:Angiogenesis Inhibitors / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D002461:Cell Line, Transformed; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D016601:RNA-Binding Proteins; D015861:Retinal Neovascularization / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D000068103:Serine-Arginine Splicing Factors; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
D020533:Angiogenesis Inhibitors; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C519530:insulin-like growth factor-1, mouse; C467480:vascular endothelial growth factor A, mouse; D000068103:Serine-Arginine Splicing Factors; D007334:Insulin-Like Growth Factor I; C087728:SRPK1 protein, human; C490215:Srpk1 protein, mouse; C484679:Srpk2 protein, mouse; D017346:Protein Serine-Threonine Kinases; C110522:SRPK2 protein, human