Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors

Abstract

Summary 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 TNFα treatment favoured PSS (increasing VEGFxxx) whereas TGFβ1 favoured DSS, increasing VEGFxxxb levels. TGFβ1 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 TGFβ1-induced DSS selection, and identify SRp55 as a key regulatory splice factor.

Deficient angiogenesis in redox-dead Cys17Ser PKARIα knock-in mice

“…This unexpected phenomenon suggests VEGF may mediate both pro-, as well as anti-angiogenic signalling. This is consistent with evidence that splice variants of VEGF can have opposing effects on vessel growth 31,32 . In this scenario, PKA in its oxidized form induces pro-angiogenic signalling that overrides the possible concomitant anti-angiogenic effects of VEGF to have a net increase in vessel growth.…”

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.…”

VEGF-A121a binding to Neuropilins – A concept revisited

“…1, 2), while the ‘xxxb’ isoforms, containing the exon 8b-encoded sequence, cannot. 8-10 All pro-angiogenic VEGF-A isoforms retain the exon 8a-encoded sequence. However, while the exon 8a-encoded sequence appears to be necessary for Neuropilin binding, experiments had not directly clarified whether it is sufficient; VEGF-A121a, for example, has been shown in some studies to bind Neuropilins and in other studies not to bind.…”