Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors.
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.
Nowak
Dawid G
DG
Microvascular Research Laboratories, Bristol Heart Institute, Department of Physiology and Pharmacology, School of Veterinary Sciences, University of Bristol, Southwell Street, Bristol BS2 8EJ, UK.
Woolard
Jeanette
J
Amin
Elianna Mohamed
EM
Konopatskaya
Olga
O
Saleem
Moin A
MA
Churchill
Amanda J
AJ
Ladomery
Michael R
MR
Harper
Steven J
SJ
Bates
David O
DO
D017398:Alternative Splicing / Q000187:drug effects / Q000502:physiology*; D020533:Angiogenesis Inhibitors / Q000096:biosynthesis*; D002478:Cells, Cultured; D005091:Exons / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D009389:Neovascularization, Pathologic / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000502:physiology; D010766:Phosphorylation / Q000502:physiology; D020033:Protein Isoforms / Q000096:biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D022821:RNA Splice Sites / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis*
D020533:Angiogenesis Inhibitors; D020033:Protein Isoforms; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D007334:Insulin-Like Growth Factor I; C066780:Clk dual-specificity kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases