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

Journal of Cell Science 121, 3487-3495 (2008)
Published by The Company of Biologists 2008
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Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors

Dawid G. Nowak1,*, Jeanette Woolard1,*, Elianna Mohamed Amin2, Olga Konopatskaya1, Moin A. Saleem3, Amanda J. Churchill4, Michael R. Ladomery2 and Steven J. Harper1,{ddagger},§

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. Bates1,{ddagger},§


Figure 1
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  		Fig. 1. Exonic structure of the VEGF gene and identified splice variants of VEGF-A. (A) Full-length VEGF gene with two alternative exon 8 splice sites. (B) Two families of VEGF isoforms, VEGFxxx and VEGFxxxb.

 

Figure 2
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  		Fig. 2. Effect of IGF1 on expression of VEGFxxxb and VEGFxxx proteins. (A) Cell lysates from RPE cells that had been treated with increasing concentrations of IGF1 for 48 hours, and total VEGF and VEGFxxxb concentrations measured by ELISA. (B) Effect of TNF{alpha} on VEGF and VEGFxxxb levels in RPE cells. (C) VEGF and VEGFxxxb concentrations in podocytes treated with IGF1, *P<0.05, ***P<0.001 vs respective control, one-way ANOVA, Dunnett's test, n=3.

 

Figure 3
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  		Fig. 3. (A,B) Effect of IGF1 on expression of (A) VEGF165b and (B) VEGF165/VEGF165b in RPE cells. Cell lysates from RPE cells that had been treated with 100 nM IGF for 48 hours or left untreated were resolved by SDS-PAGE and immunoblotted using (A) monoclonal antibody against VEGF165b or (B) a pan-VEGF antibody. Blots were stripped and re-probed with anti-actin antibody. (C) Density of bands (mean ± s.e.m.) demonstrating a significant downregulation of VEGF165b (n=4) and upregulation of VEGF165 vs VEGF165b (n=5) in RPE cells (*P<0.05 compared with control, paired t-tests)

 

Figure 4
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  		Fig. 4. Effect of TGF1 on expression of VEGFxxxb and VEGFtotal in podocytes. (A) ELISA of podocyte cell lysate for VEGFxxxb and VEGFtotal treated with TGFβ1. *P<0.05, **P<0.01 vs respective control, one-way ANOVA, Dunnett's test, n=5. (B) Lysates of podocytes treated with TGF1 resolved by western blot using antibodies that detect VEGFxxxb. Densitometry of western blots show VEGF165b to be upregulated relative to control (n=3). (C) RT-PCR of cells exposed to 1 nM TGFβ1. The lower band, corresponding to VEGF165b is increased in intensity relative to the upper band (VEGF165). Densitometry (n=5) indicates a significant upregulation of VEGF165b relative to VEGF165 (**P<0.01, paired t-test).

 

Figure 5
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  		Fig. 5. Regulation of distal splice-site selection by p38 MAPK and p42/p44 MAPK, and Clk1 and Clk4. Podocytes were treated with the p38 MAPK inhibitor SB203580 (10 µM), the p42/p44 MAPK inhibitor PD98059 (10 µM) or the Clk/Sty inhibitor TG003 (1 µM) in the presence or absence of 1 nM TGF1. (A) mRNA from cells treated as above were subjected to RT-PCR using primers that detect both isoform families of VEGF, *P<0.05 compared with control, +P<0.05 compared with TGF alone. (B) Proteins were resolved by immunoblotting. (C) Quantitative ELISA for VEGFxxxb and VEGFtotal. TGF1-mediated upregulation of distal splicing was not seen in the presence of SB203580, DN-p38MAPK or TG003 but was present during PD98059 treatment. ***P<0.001 compared with vehicle, *P<0.05 compared with drug control. (D) TGFβ1 induces phosphorylation of p38 MAPK and p42/p44 MAPK in human podocytes was inhibited by SB203580 and PD98059, respectively.

 

Figure 6
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  		Fig. 6. Distribution of ESE consensus sequences in the C terminus of the VEGF gene. The SRp55 sites were associated with distal splicing whereas the SF2/ASF and SRp40 sites were more associated with the proximal splice sites.

 

Figure 7
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  		Fig. 7. Effect of overexpression of splicing factors on VEGF isoform production. (A) Retinal pigmented epithelial cells were transfected with splicing factors and distal splicing isoforms (VEGFxxxb) and total VEGF levels determined by SDS-PAGE and western blotting. (B) Densitometry of cell lysates showing expression of VEGF165b and total VEGF after transfection with splice factors. (C) The difference in VEGF expression between VEGFxxxb and total VEGF is shown relative to controls for splice factors. Solid line indicates an equal balance between the two sets of isoforms. Values below the line indicate anti-angiogenic balance, above a pro-angiogenic balance (*P<0.05 compared with untreated, ANOVA, Dunnett's test. n=3).

 

Figure 8
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  		Fig. 8. SRp55 regulates VEGF splice-site selection in exon 8. (A) RPE cells were transfected with SRp55 or shRNA targeting SRP55, and VEGF165b expression measured by immunoblotting. siRNA targeting SRp55 downregulated both SRp55 and VEGF165b. (B) Densitometry indicated that both shRNAs targeting SRp55 inhibited SRp55 expression (n=4). (C) shRNAs targeting SRp55 also inhibited VEGF165b expression (n=3, *P<0.05, **P<0.01 compared with control, ANOVA, Dunnett's test). (D) MS2-MBP assay for RNA-binding sites of HEK-cell nuclear extract. SRp55 was detected by immunoblotting in nuclear extract (NE) and when bound to a RNA construct containing the 35 nucleotides downstream of the stop codon for exon 8b (construct X), but not when this region was deleted (construct Y) or MS2 RNA (construct Z).

 

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