Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by known splicing and growth factors
J. Cell Sci. 121: 3487-3495 (2008) - PMID: 18843117
Authors: Nowak, D.G., Woolard, J., Amin, E.M., Konopatskaya, O., Saleem, M.A., Churchill, A.J., Ladomery, M.R., Harper, S.J., Bates, D.O.
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 VEGF isoforms (xxx is the number of amino acids), whereas distal splice-site selection (DSS) results in anti-angiogenic VEGFb 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 VEGFb formed the majority of VEGF isoforms (74%). IGF1, and TNFalpha treatment favoured PSS (increasing VEGF) whereas TGFbeta1 favoured DSS, increasing VEGFb 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 VEGFb (DSS) isoforms relative to VEGF. SRp55 knockdown reduced expression of VEGFb. 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.
Keywords: Alternative splicing,VEGF,angiogenesis