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

Amin, Elianna Mohamed; Nowak, Dawid G.; Woolard, Jeanette; Konopatskaya, Olga; Saleem, Moin A.; Ladomery, Michael; Churchill, Amanda J.; Harper, Steven J.; Bates, David O.

Authors

Elianna Mohamed Amin

Dawid G. Nowak

Jeanette Woolard

Olga Konopatskaya

Moin A. Saleem

Amanda J. Churchill

Steven J. Harper

David O. Bates



Abstract

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 proangiogenic 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 CIk/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.

Journal Article Type Article
Publication Date Oct 15, 2008
Journal Journal of Cell Science
Print ISSN 0021-9533
Publisher Company of Biologists
Peer Reviewed Peer Reviewed
Volume 121
Issue 20
Pages 3487-3495
DOI https://doi.org/10.1242/jcs.016410
Keywords VEGF, VEGF165b, splicing, VEGFxxxb, SRp55, TGFβ1, IGF1, Clk1/sty (CLK1), CLK4
Public URL https://uwe-repository.worktribe.com/output/1020686
Publisher URL http://dx.doi.org/10.1242/jcs.016410