RESUMO
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.
Assuntos
Processamento Alternativo , Inibidores da Angiogênese/biossíntese , RNA Mensageiro/biossíntese , Neovascularização Retiniana/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Inibidores da Angiogênese/genética , Animais , Linhagem Celular Transformada , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Neovascularização Retiniana/genética , Neovascularização Retiniana/terapia , Fatores de Processamento de Serina-Arginina , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Vascular endothelial growth factor A (VEGFA; hereafter referred to as VEGF) is a key regulator of physiological and pathological angiogenesis. Two families of VEGF isoforms are generated by alternate splice-site selection in the terminal exon. Proximal splice-site selection (PSS) in exon 8 results in pro-angiogenic VEGFxxx isoforms (xxx is the number of amino acids), whereas distal splice-site selection (DSS) results in anti-angiogenic VEGFxxxb isoforms. To investigate control of PSS and DSS, we investigated the regulation of isoform expression by extracellular growth factor administration and intracellular splicing factors. In primary epithelial cells VEGFxxxb formed the majority of VEGF isoforms (74%). IGF1, and TNFalpha treatment favoured PSS (increasing VEGFxxx) whereas TGFbeta1 favoured DSS, increasing VEGFxxxb levels. TGFbeta1 induced DSS selection was prevented by inhibition of p38 MAPK and the Clk/sty (CDC-like kinase, CLK1) splicing factor kinase family, but not ERK1/2. Clk phosphorylates SR protein splicing factors ASF/SF2, SRp40 and SRp55. To determine whether SR splicing factors alter VEGF splicing, they were overexpressed in epithelial cells, and VEGF isoform production assessed. ASF/SF2, and SRp40 both favoured PSS, whereas SRp55 upregulated VEGFxxxb (DSS) isoforms relative to VEGFxxx. SRp55 knockdown reduced expression of VEGF165b. Moreover, SRp55 bound to a 35 nucleotide region of the 3'UTR immediately downstream of the stop codon in exon 8b. These results identify regulation of splicing by growth and splice factors as a key event in determining the relative pro-versus anti-angiogenic expression of VEGF isoforms, and suggest that p38 MAPK-Clk/sty kinases are responsible for the TGFbeta1-induced DSS selection, and identify SRp55 as a key regulatory splice factor.