Regulation of vascular endothelial growth factor (VEGF) splicing from pro-angiogenic to anti-angiogenic isoforms: a novel therapeutic strategy for angiogenesis

Abstract

Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF(165), and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF(165)b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF(165) and less VEGF(165)b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.

FIGURE 1.

Inhibition of PKC by BIM1 prevents the down-regulation of VEGF_xxxb by IGF. A, exon structure of the VEGF pre-mRNA. Alternative splicing of exon 8 to either 8a or 8b results in use of proximal (PSS) or distal splice sites (DSS) resulting in shorter mRNA for distal splicing. Because the last stop codon is missing, the final six amino acid open reading frame is replaced by an identically sized open reading frame encoding six different amino acids. The primer position is shown by horizontal arrows. B–D, podocytes were treated with BIM1 (2.5 μm) alone or in combination with IGF-1 (100 nm). B, RT-PCR showed that BIM1 reduced the VEGF_xxx:VEGF_xxxb ratio at the RNA level. C, Western blot demonstrating that BIM1 inhibited the IGF-mediated down-regulation of VEGF_xxxb expression at the protein level. D, ELISA results confirming that BIM1 specifically attenuated the IGF-1-dependent down-regulation of VEGF_xxxb, but does not affect endogenous expression of VEGF_xxxb. **, p < 0.01 compared with untreated.

FIGURE 2.

Proximal splicing is activated by protein kinase C. A–C, treatment of podocytes with the PKC activator PMA reduced VEGF₁₆₅b expression, but increased expression of total VEGF as measured by Western blot (A) and ELISA (B). This results in a change of relative expression from 60% (anti-angiogenic) to just under 50% (angiogenic) (C).

FIGURE 3.

Inhibition of SPRK1/2 by SRPIN340 prevents the down-regulation of VEGF_xxxb by IGF. A–D, cells were treated with SRPIN340 (10 μm) alone or in combination with IGF-1 (100 nm). A, RT-PCR showed that SRPIN340 reduced the VEGF₁₆₅:VEGF₁₆₅b ratio at the RNA level. B, Western blot demonstrating that SRPIN340 inhibited the IGF-mediated down-regulation of VEGF_xxxb expression at the protein level. C, ELISA results confirming that SPRIN340 specifically attenuates the IGF-1-dependent down-regulation of VEGF_xxxb, but did not affect endogenous expression of VEGF_xxxb. D, ELISA of the protein extract shows that SRPK1 transfection reduces VEGF_xxxb expression, and total VEGF expression. SRPK2 reduces total expression, but did not affect VEGF_xxxb expression.

FIGURE 4.

Nuclear localization of ASF/SF2 is increased by IGF-1. A–F, cells were treated with vehicle or IGF in the presence or absence of SRPIN340 and stained for ASF/SF2 and counterstained with Hoechst. A, podocytes show expression of ASF/SF2 in the nucleus and in the cytoplasm. B, IGF induces nuclear localization of cytoplasmic ASF/SF2. C, SRPIN340 by itself does not affect localization of ASF/SF2. D, SRPIN340 inhibited this IGF-mediated localization. E, HEK cells also show cytoplasmic localization of ASF/SF2. F, in contrast, HeLa cells have nuclear ASF/SF2 localization. G, RT-PCR of mRNA from HEK cells shows VEGF₁₆₅b expression, but not in HeLa cells. MWM, molecular weight marker.

FIGURE 5.

ASF/SF2-1 binds a 35-nt region of VEGF pre-mRNA upstream of the proximal splice site of exon 8. A, constructs were generated containing fragments of the exon 7/exon 8 boundary, fused to a sequence encoding the stem loop structures recognized by the MBP-MS2-binding protein, which can bind maltose. These were transcribed in vitro. B, Western blot of HEK cell crude nuclear extract (NE) or NE incubated with mRNA constructs as above and run over a maltose column to isolate proteins that bind to the RNA constructs and probed with an ASF/SF2 antibody. Whereas mRNA containing the 5′ regions of the intron 7/exon 8 boundary contained ASF/SF2 immunoreactivity, RNA encoding the exon 8 region did not, identifying the binding site for ASF/SF2 in the intron 7/exon 8a boundary. C, immunoblot of HEK cell NE of cells treated as shown incubated with the RNA construct C and run over the MS2-MBP column. PMA activation increased binding, and this was blocked by SRPIN340 and phosphatase treatment. D, RNA immunoprecipitation of ASF/SF2 in cells expressing constructs with a mutated or deleted intron 7 sequence. The top shows RT-PCR of total cell extract (TCE) or immunoprecipitated RNA using a nonspecific mouse IgG (IgG), or using a mouse monoclonal antibody to ASF/SF2 using primers to detect the VEGF sequence. The bottom blot shows the same treatments subjected to GAPDH amplification. The wild-type sequence showed a stronger band in the ASF/SF2 IP, whereas the mutants showed no difference between mouse IgG and ASF/SF2.

FIGURE 6.

Neovascularization induced by hyperoxia is inhibited by a single dose of SRPK inhibitor, SRPIN340. A, low power fluorescence micrograph of FITC-labeled lectin staining of retinal whole mounts with areas of NV (white) and ischemic (orange) outlined. B, higher power view of a single retinal quadrant, with angiogenic areas highlighted by arrowheads. C, high power view of retinal angiogenic area showing sprouting endothelial cells. D, quantification of neovascular areas shows a small but significant inhibition by a single injection of 1 μl of 10 μm SRPIN340 1 day after removal from oxygen. E, ischemic area was also reduced in these mice. F, normal area was consequently increased. G, data shown as relative to control, uninjected contralateral eye. Neo, neovacular; isch, ischemia; Norm, normal.