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First published online October 8, 2008
doi: 10.1242/10.1242/jcs.016410

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

Dawid G. Nowak^1,*, Jeanette Woolard^1,*, Elianna Mohamed Amin², Olga Konopatskaya¹, Moin A. Saleem³, Amanda J. Churchill⁴, Michael R. Ladomery² and Steven J. Harper^1,,

¹ Microvascular Research Laboratories, Bristol Heart Institute, Department of Physiology and Pharmacology, School of Veterinary Sciences, University of Bristol, Southwell Street, Bristol BS2 8EJ, UK
² Centre for Research in Biomedicine, Faculty of Health and Life Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK
³ Department of Clinical Sciences at North Bristol (Academic Renal Unit), Paul O'Gorman Lifeline Centre, Southmead Hospital, Bristol BS10 5NB, UK
⁴ Department of Ophthalmology, Bristol Eye Hospital, University of Bristol, Lower Maudlin Street, Bristol BS1 2LX, UK

David O. Bates^1,,

Author for correspondence (e-mail: s.harper{at}bris.ac.uk)

Author for correspondence (e-mail: Dave.Bates{at}bristol.ac.uk)

Accepted 7 July 2008

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_xxx isoforms (xxx is the number of amino acids), whereas distal splice-site selection (DSS) results in anti-angiogenic VEGF_xxxb 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 VEGF_xxxb formed the majority of VEGF isoforms (74%). IGF1, and TNF treatment favoured PSS (increasing VEGF_xxx) whereas TGFβ1 favoured DSS, increasing VEGF_xxxb 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 VEGF_xxxb (DSS) isoforms relative to VEGF_xxx. 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.

Key words: VEGF, VEGF₁₆₅b, splicing, VEGF_xxxb, SRp55, TGFβ1, IGF1, Clk1/sty (CLK1), CLK4