Feedback control of Wnt signaling based on ultrastable histidine cluster co-aggregation between Naked/NKD and Axin.

Feedback control is a universal feature of cell signaling pathways. Naked/NKD is a widely conserved feedback regulator of Wnt signaling which controls animal development and tissue homeostasis. Naked/NKD destabilizes Dishevelled, which assembles Wnt signalosomes to inhibit the β-catenin destruction complex via recruitment of Axin. Here, we discover that the molecular mechanism underlying Naked/NKD function relies on its assembly into ultra-stable decameric core aggregates via its conserved C-terminal histidine cluster (HisC). HisC aggregation is facilitated by Dishevelled and depends on accumulation of Naked/NKD during prolonged Wnt stimulation. Naked/NKD HisC cores co-aggregate with a conserved histidine cluster within Axin, to destabilize it along with Dishevelled, possibly via the autophagy receptor p62, which binds to HisC aggregates. Consistent with this, attenuated Wnt responses are observed in CRISPR-engineered flies and human epithelial cells whose Naked/NKD HisC has been deleted. Thus, HisC aggregation by Naked/NKD provides context-dependent feedback control of prolonged Wnt responses

Regulation of Dishevelled DEP domain swapping by conserved phosphorylation sites.

Wnt signals bind to Frizzled receptors to trigger canonical and noncanonical signaling responses that control cell fates during animal development and tissue homeostasis. All Wnt signals are relayed by the hub protein Dishevelled. During canonical (β-catenin-dependent) signaling, Dishevelled assembles signalosomes via dynamic head-to-tail polymerization of its Dishevelled and Axin (DIX) domain, which are cross-linked by its Dishevelled, Egl-10, and Pleckstrin (DEP) domain through a conformational switch from monomer to domain-swapped dimer. The domain-swapped conformation of DEP masks the site through which Dishevelled binds to Frizzled, implying that DEP domain swapping results in the detachment of Dishevelled from Frizzled. This would be incompatible with noncanonical Wnt signaling, which relies on long-term association between Dishevelled and Frizzled. It is therefore likely that DEP domain swapping is differentially regulated during canonical and noncanonical Wnt signaling. Here, we use NMR spectroscopy and cell-based assays to uncover intermolecular contacts in the DEP dimer that are essential for its stability and for Dishevelled function in relaying canonical Wnt signals. These contacts are mediated by an intrinsically structured sequence spanning a conserved phosphorylation site upstream of the DEP domain that serves to clamp down the swapped N-terminal α-helix onto the structural core of a reciprocal DEP molecule in the domain-swapped configuration. Mutations of this phosphorylation site and its cognate surface on the reciprocal DEP core attenuate DEP-dependent dimerization of Dishevelled and its canonical signaling activity in cells without impeding its binding to Frizzled. We propose that phosphorylation of this crucial residue could be employed to switch off canonical Wnt signaling

SRPK1 inhibition modulates VEGF splicing to reduce pathological neovascularisation in a rat model of Retinopathy of Prematurity

PURPOSE: We tested the hypothesis that recombinant human VEGF-A165b and the serine arginine protein kinase (SRPK) inhibitor, SRPIN340, which controls splicing of the VEGF-A pre-mRNA, prevent neovascularization in a rodent model of retinopathy of prematurity (ROP). METHODS: In the 50/10 oxygen-induced retinopathy (50/10 OIR) model that exposes newborn rats to repeated cycles of 24 hours of 50% oxygen alternating with 24 hours of 10% oxygen, pups received intraocular injections of SRPIN340, vehicle, VEGF165b, anti-VEGF antibody, or saline. Whole mounts of retinas were prepared for isolectin immunohistochemistry, and preretinal or intravitreal neovascularization (PRNV) determined by clock hour analysis. RESULTS: The anti-VEGF antibody (P < 0.04), rhVEGF165b (P < 0.001), and SRPIN340 (P < 0.05) significantly reduced PRNV compared with control eyes. SRPIN340 reduced the expression of proangiogenic VEGF165 without affecting VEGF165b expression. CONCLUSIONS: These results suggest that splicing regulation through selective downregulation of proangiogenic VEGF isoforms (via SRPK1 inhibition) or competitive inhibition of VEGF signaling by rhVEGF165b has the potential to be an effective alternative to potential cyto- and neurotoxic anti-VEGF agents in the treatment of pathological neovascularization in the eye

The carboxyl terminus of VEGF-A is a potential target for anti-angiogenic therapy

Anti-VEGF-A therapy has become a mainstay of treatment for ocular neovascularisation and in cancer; however, their effectiveness is not universal, in some cases only benefiting a minority of patients. Anti-VEGF-A therapies bind and block both pro-angiogenic VEGF-A(xxx) and the partial agonist VEGF-A(xxx)b isoforms, but their anti-angiogenic benefit only comes about from targeting the pro-angiogenic isoforms. Therefore, antibodies that exclusively target the pro-angiogenic isoforms may be more effective. To determine whether C-terminal-targeted antibodies could inhibit angiogenesis, we generated a polyclonal antibody to the last nine amino acids of VEGF-A(165) and tested it in vitro and in vivo. The exon8a polyclonal antibody (Exon8apab) did not bind VEGF-A(165)b even at greater than 100-fold excess concentration, and dose dependently inhibited VEGF-A(165) induced endothelial migration in vitro at concentrations similar to the VEGF-A antibody fragment ranibizumab. Exon8apab can inhibit tumour growth of LS174t cells implanted in vivo and blood vessel growth in the eye in models of age-related macular degeneration, with equal efficacy to non-selective anti-VEGF-A antibodies. It also showed that it was the VEGF-A(xxx) levels specifically that were upregulated in plasma from patients with proliferative diabetic retinopathy. These results suggest that VEGF-A(165)-specific antibodies can be therapeutically useful

Detection of VEGF-A_xxxb Isoforms in Human Tissues

Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls

qRT-PCR using protocols shown in figure 2D and E can detect changes in splicing induced by splicing factor knockdown.

<p>A. C_tmax-C_t for cDNA extracted from prostate cancer (PC3) cells with lentiviral knockdown of SRPK1 or scrambled. B. Amount of VEGF calculated from standard curves in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068399#pone-0068399-g002" target="_blank">Figure 2</a>. C. Amount of VEGF-A₁₆₅b identified by Exon 8b primers (VEGF-A₁₆₅b) or that calculated from mispriming of VEGF-A₁₆₅a. D. Proportion of VEGF that is VEGF-A₁₆₅a or VEGF-A₁₆₅b in control and knockdown cells. Values are Mean±SEM (n = 2). 3E. qPCR for VEGF-A₁₆₅a on commercially available cDNAs from 2 different companies (open bars) or cDNA reverse transcribed from freshly extracted human kidney RNA (solid bar). 3F qPCR for VEGF-A₁₆₅b on commercially available cDNAs from 2 different companies (open bars) or cDNA reverse transcribed from freshly extracted human kidney RNA (solid bar).</p

Isoform specific PCR requires positive controls to ensure specificity.

<p>A. Sequence of the VEGF 3′ exon sequence. (i) Exon 7 (red) contains the same last three nucleotides (underlined) as the last three nucleotides of exon 8a (blue, underlined), requiring specific PCR primers that extend into exon 7 (arrow). (ii) mispriming (VEGF-A₁₆₅a -specific primers priming on VEGF-A₁₆₅b, and VEGF-A₁₆₅b -specific primers priming on VEGF-A₁₆₅a) can occur both ways round if the conditions are not tested. B. Published control PCR gels demonstrating specificity of primer conditions. The original description of VEGF-A₁₆₅b describing conditions at which VEGF-A₁₆₅b is not misprimed in the presence of 100ng VEGF-A₁₆₅a (lane highlighted by arrow), but still able to amplify 0.1ng VEGF-A₁₆₅b. C. Annealing temperature dependence of the specificity of the isoform specific primers. Only at >62°C is specificity resolved. D. qPCR using VEGF-A₁₆₅a specific primers on VEGF-A₁₆₅a and VEGF-A₁₆₅b plasmid E. qPCR using VEGF-A₁₆₅b specific primers on VEGF-A₁₆₅a and VEGF-A₁₆₅b plasmid.</p