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

Abstract

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 VEGF165, and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF165b. 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 VEGF165 and less VEGF165b. 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.

The transcription factor E2F1 and the SR protein SC35 control the ratio of pro-angiogenic versus antiangiogenic isoforms of vascular endothelial growth factor-A to inhibit neovascularization in vivo

“…An increasing amount of evidence now supports the idea that the balance between VEGF xxx (pro-angiogenic) and VEGF xxx b (antiangiogenic) splice variants has a crucial role in the control of tumour progression, as well as in the response of tumour cells to anti-VEGF therapies (Bates et al, 2002;Woolard et al, 2004;Rennel et al, 2008;Varey et al, 2008). Recently, it has been shown that VEGF splicing is controlled by the SR proteins ASF/SF2, SRp40 and SRp55 in primary epithelial cells (Nowak et al, 2008(Nowak et al, , 2010. To date, nothing is known about the factors that control the switch between VEGF xxx and VEGF xxx b splice variants in cancer cells.…”

“…As a whole, these data indicate that the balance between pro-(VEGF xxx ) and anti-(VEGF xxx b) angiogenic splice variants of VEGF-A has a critical role in both tumour progression and tumour cell response to antiangiogenic therapies. However, although there has been some investigation of splicing mechanisms in epithelial cells (Cohen et al, 2005;Nowak et al, 2008Nowak et al, , 2010, little is known about the proteins and signalling networks that control the splicing of VEGF-A pre-mRNA in cancer cells.…”

Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged

“…Inhibition of the p38 MAPK, activated by TGFb1 signaling, reversed the effect of TGFb1 on VEGFA splice site selection. The increase in VEGFA expression, as well as the splicing switch in favor of the canonical isoform in IGF1-treated cells, was inhibited when cells were treated simultaneously with inhibitors of protein kinase C (PKC) or the SR protein kinases SRPK1/2 (Nowak et al 2010).…”

Regulation of vascular endothelial growth factor in prostate cancer

“…The splice variants are differentially regulated (e.g., SRPK1 stimulates splicing to VEGFA 165 a, and Clk1/4 stimulates splicing to VEGFA 165 b) (Nowak et al 2008(Nowak et al , 2010 and are differentially regulated post-transcriptionally -for example, by T-cell intracellular antigen-1, an RNA-binding protein that differentially regulates translation and splicing of VEGF through activation by Ras (Hamdollah Zadeh et al 2015).…”