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. 2011 Feb 1;2011(2):273-278.
doi: 10.1039/C0SC00297F.

Borrelidin Modulates the Alternative Splicing of VEGF in Favour of Anti-Angiogenic Isoforms

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Borrelidin Modulates the Alternative Splicing of VEGF in Favour of Anti-Angiogenic Isoforms

Jeanette Woolard et al. Chem Sci. .
Free PMC article

Abstract

The polyketide natural product borrelidin 1 is a potent inhibitor of angiogenesis and spontaneous metastasis. Affinity biopanning of a phage display library of colon tumor cell cDNAs identified the tandem WW domains of spliceosome-associated protein formin binding protein 21 (FBP21) as a novel molecular target of borrelidin, suggesting that borrelidin may act as a modulator of alternative splicing. In support of this idea, 1, and its more selective analog 2, bound to purified recombinant WW domains of FBP21. They also altered the ratio of vascular endothelial growth factor (VEGF) isoforms in retinal pigmented endothelial (RPE) cells in favour of anti-angiogenic isoforms. Transfection of RPE cells with FBP21 altered the ratio in favour of pro-angiogenic VEGF isoforms, an effect inhibited by 2. These data implicate FBP21 in the regulation of alternative splicing and suggest the potential of borrelidin analogs as tools to deconvolute key steps of spliceosome function.

Figures

Fig. 1
Fig. 1
Structures of borrelidin, analogs and chemical probes.
Fig. 2
Fig. 2
(a) Four serial rounds of biopanning resulted in the progressive enrichment of two phage products of approx. 450 and 600 bp respectively (highlighted by arrows) as judged by PCR of total phage after each round. The final round was eluted using either SDS (5a) or E. coli (5b), and resulted in differential enrichment of these two bands (see right hand image for clarity); (b) PCR analysis of single colony isolation of phage from the final round of biopanning (representatives of rounds 5a&b are shown); (c) Sequence analysis of the phage PCR products identified a common insert containing a translated 83 amino acid section (red) of FBP21 including its tandem WW domains (underlined) - the translated sequence for a single clone BV14F is shown alongside that for FBP21 (NM_007187).
Fig. 3
Fig. 3
Comparison of the effect of 0.5 and 5 μM 1 and 2 on total VEGF (a) and VEGFxxxb (b) concentrations in RPE cells measured by ELISA, *p<0.05, **p<0.01, Dunnett’s compared with vehicle, n=3; Insert in b) shows PCR for VEGF165b in control cells, vehicle treated and cells treated with 2 (c) RPE cells treated with increasing concentrations of 2 for 24 h; VEGFxxxb and total VEGF concentrations measured by ELISA, **p<0.05, Dunnett’s, **p<0.01, n=3; (d) Effect of IGF-1 and 2 treatment on VEGFxxx protein levels; RPE cells were treated with increasing concentrations of 2 and 1μM IGF-1 for 24 h; total VEGF levels measured by ELISA, p=0.0113, one way ANOVA, **p<0.01, Dunnett’s compared with IGF alone, n=3.
Fig. 4
Fig. 4
Enhanced FBP21 expression mitigates the effect of 2 on alternative splicing of VEGF in RPE cells. RPE cells were transfected with either control vector or FBP21 expression vector. (a) ELISA for VEGFxxxb using biotinylated VEGFxxxb specific detection antibody, *p<0.05 compared with control, Paired t test, n=3; (b) Transfected cells were then treated with 0.5μM 2 for 72 h and protein assayed by ELISA; results are expressed relative to vehicle (0.1% DMSO) treated cells, P=0.05 Mann Whitney U test, n=4.

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