Fbxw7 Controls Angiogenesis by Regulating Endothelial Notch Activity

<div><p>Notch signaling controls fundamental aspects of angiogenic blood vessel growth including the selection of sprouting tip cells, endothelial proliferation and arterial differentiation. The E3 ubiquitin ligase Fbxw7 is part of the SCF protein complex responsible for the polyubiquitination and thereby proteasomal degradation of substrates such as Notch, c-Myc and c-Jun. Here, we show that Fbxw7 is a critical regulator of angiogenesis in the mouse retina and the zebrafish embryonic trunk, which we attribute to its role in the degradation of active Notch. Growth of retinal blood vessel was impaired and the Notch ligand Dll4, which is also a Notch target, upregulated in inducible and endothelial cell-specific <em>Fbxw7</em>^iECKO mutant mice. The stability of the cleaved and active Notch intracellular domain was increased after siRNA knockdown of the E3 ligase in cultured human endothelial cells. Injection of <em>fbxw7</em> morpholinos interfered with the sprouting of zebrafish intersegmental vessels (ISVs). Arguing strongly that Notch and not other Fbxw7 substrates are primarily responsible for these phenotypes, the genetic inactivation of Notch pathway components reversed the impaired ISV growth in the zebrafish embryo as well as sprouting and proliferation in the mouse retina. Our findings establish that Fbxw7 is a potent positive regulator of angiogenesis that limits the activity of Notch in the endothelium of the growing vasculature.</p> </div

Impaired ISV growth after knockdown of zebrafish <i>fbxw7</i> expression.

<p>Following injection with control morpholino (<i>control MO</i>) or fbxw7 transcription blocking morpholino (<i>fbxw7 ATG MO</i>), the fluorescent vasculature of <i>Tg(kdrl:EGFP)^s843</i> transgenic embryos was examined by live microscopy. Outgrowth of ISVs from the dorsal aorta (DA) and formation of the DLAV (as indicated in the left image) were impaired in <i>fbxw7</i> morphant embryos (arrowheads) at 32 hpf (A). Scale bar is 70 µm. Protein extracts from control and <i>fbxw7 ATG MO</i> embryos at 24 hpf and 32 hpf were analyzed by Western blotting (B). Note transient reduction of the Fbxw7 band (asterisk). Tubulin was used as loading control. Quantification of the number of ECs per ISV in control MO and <i>fbxw7 ATG MO</i> injected embryos at 32 hpf (based on 3 independent experiments) (C). Proliferation of endothelial cells at 32 hpf in ISVs of <i>Tg(fli1a:nEGFP)^y7</i> x <i>Tg(kdrl:HsHRAS-mCherry)^s896</i> double transgenic embryos injected with <i>control MO</i> or <i>Fbxw7 ATG MO</i> (D). Error bars indicate SEM. P value (*) in (C) is <0.05.</p

Targeting of <i>Fbxw7</i> induced the upregulation of Dll4 expression.

<p>Anti-Dll4 and Isolectin B4 (IsolB4)-stained retinal whole-mounts of <i>Fbxw7</i>^iECKO and littermate control retinas, as indicated (A, B). Arrowheads in (B) mark Dll4+ peripheral sprouts at the edge of the growing plexus, arrows indicate upregulated Dll4 in <i>Fbxw7</i>^iECKO retinal capillaries. Quantitative analysis (with Volocity 5; n = 3 for each group) of image data confirmed elevated Dll4 levels (number of pixels) in the mutant endothelium (C). Likewise, quantitative RT-PCR analysis showed reduced <i>Fbxw7</i> expression but upregulated <i>Dll4</i> transcript levels in P6 <i>Fbxw7</i>^iECKO lungs (D). Expression of the <i>Cdh5</i> gene was used for normalization. Error bars indicate SEM. P values are indicated as ** (<0.001) and * (p<0.05). Scale bars are 100 µm.</p

<i>Rbpj</i> inactivation reverses the impaired vascular growth of <i>Fbxw7</i> mutant mice.

<p>Whole-mount Isolectin B4 staining of the retinal vasculature in P6 control, <i>Fbxw7</i>^iECKO/<i>Rbpj</i>^+/iECKO (<i>Fbxw7</i> homozygous & <i>Rbpj</i> heterozygous EC-specific KO), <i>Fbxw7</i>^iECKO/<i>Rbpj</i>^iECKO (<i>Fbxw7</i> & <i>Rbpj</i> EC-specific double KO), and <i>Fbxw7</i>^+/iECKO/<i>Rbpj</i>^iECKO (<i>Fbxw7</i> heterozygous & <i>Rbpj</i> homozygous EC-specific KO), mice, as indicated. Inactivation of both <i>Rbpj</i> alleles enhanced EC proliferation and sprouting, and restored retinal angiogenesis in the <i>Fbxw7</i> mutant background. Scale bar represents 200 µm. Three independent experiments were performed with similar results.</p

Inducible targeting of the <i>Fbxw7</i> gene in retinal ECs.

<p>Confocal images showing the organization of the <i>Fbxw7</i>^iECKO mutant and littermate control retinal vasculature at postnatal day 6 (P6) after whole-mount Isolectin B4 staining (A and C). Postnatal, EC-specific loss-of-function mutants were either generated with the <i>Cdh5(PAC)-CreERT2</i> (A, B) or <i>Pdgfb-iCre</i> (C, D) transgenics. Radial extension, branchpoint number, sprouts, and vascular area (B and D) were quantitated (control, n = 7; <i>Fbxw7</i>^iECKO n = 6). Error bars indicate SEM. P value (***) <0.00001. Scale bar is 200 µm for all microscopic images.</p

Fbw7<i>β</i> is transcriptionally regulated by Hes5.

<p>(a) Schematic representation of the <i>Fbw7</i> genomic locus (adapted from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001586#pbio.1001586-Welcker2" target="_blank">[24]</a>). mRNA transcripts for the different <i>Fbw7</i> isoforms and Q-PCR primers used to detect <i>Fbw7</i> isoforms are depicted in the figure. (b) Q-PCR analysis of <i>Fbw7α</i>, <i>Fbw7β</i>, and <i>Fbw7</i>(exon5) in intestinal cells and NSCs isolated from <i>Fbw7^f/+</i> or <i>Fbw7^ΔG/+</i> mice and <i>Fbw7^f/+</i> or <i>Fbw^ΔN/+</i> NSCs, respectively (relative fold induction after normalizing to actin ± SEM, <i>n</i>≥3 for each genotype). (c) Q-PCR analysis of <i>Fbw7α</i> and <i>Fbw7β</i> in intestinal tumours isolated from <i>APC^min/+</i>; <i>Fbw7^f/+</i> or <i>APC^min/+</i>; <i>Fbw7^ΔG/+</i> mice (relative fold induction after normalizing to actin ± SEM, <i>n</i>≥3 for each genotype). (d) Schematic representation of <i>Fbw7α</i>, <i>Fbw7β</i>, and <i>Ngn3</i> promoter regions. Red boxes denote consensus N-box sites. Green arrows indicate primers used for ChIP. (e) ChIP was performed using HCT116 cells transfected with p-Dest-flag or p-Dest-Hes5-flag. Flag binding to the consensus sites in <i>Fbw7α</i>, <i>Fbw7β</i>, and <i>Ngn3</i> promoters was determined by Q-PCR. Data were represented as fold activation of percentage input versus the p-DEST-Flag samples. Red line denotes background-binding activity. (f) Schematic representation of the different <i>Fbw7β</i>-luciferase constructs generated. Red rectangles represent putative N-boxes. Crossed red rectangles represent mutated N-boxes. (g) HCT116 cells were transfected with <i>Fbw7β</i>–N1, <i>Fbw7β</i>–N2, <i>Fbw7β</i>–N1-mut, pGL3–<i>Fbw7β</i>–N2-mut together with p-Dest-Hes5-Flag overexpression vector or p-Dest-flag as a control. Data represent luciferase activity relative to <i>Fbw7β</i>–N1+pDest-flag transfected cells.</p

Fbw7 is haploinsufficient for Notch in the gut and NSCs.

<p>(a) H&E and AB/PAS staining in intestinal tissue from <i>Fbw7^f/+</i> or <i>Fbw7^ΔG/+</i> mice. (b) Quantification of goblet (AB-PAS+) cells in intestinal tissue from <i>Fbw7^f/+</i> or <i>Fbw7^ΔG/+</i> mice. (c) Western analysis of protein lysates from intestinal cells isolated from <i>Fbw7^f/+</i> or <i>Fbw7^ΔG/+</i> mice (numbers indicate the fold induction of NICD normalized to actin). (d) Q-PCR analysis of <i>Hes1, Hes5, c-Jun</i> and <i>c-Myc</i> transcripts in <i>Fbw7^ΔG/+</i> intestinal cells compared to <i>Fbw7^f/+</i> (relative fold induction after normalizing to actin ± SEM, <i>n</i>≥3 for each genotype). (e) Nestin staining of NSCs isolated from <i>Fbw7^f/+</i> or <i>Fbw7^ΔN/+</i> mice. (f) Quantification of Nestin+ cells in NSCs isolated from <i>Fbw7^f/+</i> or <i>Fbw7^ΔN/+</i> mice (percentage positive cells ± SEM, <i>n</i>≥20 for each genotype). (g) Western blot analysis of protein lysates from <i>Fbw7^f/+</i> or <i>Fbw7^ΔN/+</i> NSCs for NICD-1 (numbers indicate the fold induction of NICD normalized to actin). (h) Q-PCR analysis of <i>Hes1, Hes5, c-Jun</i> and <i>c-Myc</i> transcripts in <i>Fbw7^ΔN/+</i> NSCs compared to <i>Fbw7^f/+</i> NSCs (relative fold induction after normalizing to actin ± SEM, <i>n</i>≥3 for each genotype).</p

Impaired Notch signaling alleviates the <i>fbxw7</i> morphant phenotype in zebrafish.

<p>Offspring resulting from an intercross of heterozyogous <i>dll4^j16e1</i> mutants were injected with <i>fbxw7 ATG MO</i> and the ISV morphology was analyzed at 32 hpf. The fraction of embryos showing normal ISV growth (light grey) was highest in a <i>dll4</i> homozygous background. In contrast, <i>fbxw7</i> silencing led to defective ISV growth (black) in almost all <i>dll4</i> wild-type embryos. The ratio of ISV phenotypes within each genotype group was calculated (based on 3 independent experiments). Error bars reflect SEM. P value (*) is <0.05.</p

Fbxw7 controls endothelial Notch signaling.

<p>Protein extracts prepared from P6 control (lanes 1 & 3 from the left) and <i>Fbxw7</i>^iECKO lungs (lanes 2 & 4) showed increased amounts of active Notch1 (NICD-Val1744) and reduced VEGFR3 protein (A). VE-Cadherin (Cdh5) was used for normalization. Relative <i>Fbxw7</i> mRNA expression in HUVECs transfected with <i>siFbxw7</i> relative to <i>siControl</i> siRNAs (n = 3 per group) (B). Human <i>GAPDH</i> was used for normalization. VEGF-A stimulation (50 ng/ml for the indicated time periods) led to a transient accumulation of active Notch (NICD) in HUVECs transfected with <i>siControl</i>. NICD levels were upregulated after <i>Fbxw7</i> silencing (C). Western blot data (band intensity in pixels) was quantified with ImageJ; the graph shows average values from 3 independent experiments (D). Alpha-tubulin was used for normalization. Confocal images showing the pronounced reduction of VEGFR3 protein (green) in the endothelium (Isolectin B4; red) of the P6 <i>Fbxw7</i>^iECKO retina (E). Scale bar is 100 µm. Quantitative analysis (with Volocity 5; n = 3 for each group) of image data confirmed reduced VEGFR3 levels (number of pixels) in mutant ECs (F). Error bars (in D and F) indicate SEM. P values are indicated as ** (<0.001) and * (p<0.05).</p

Hes5 deletion rescues Fbw7 haploinsufficiency in gut and NSCs.

<p>(a) H&E and AB/PAS staining in the intestines of <i>Fbw7^+/+, Fbw7^ΔG/+</i> or <i>Fbw7^ΔG/+; Hes5</i>^−/− mice. (b) Quantification of goblet (AB-PAS+) cells in the intestines of <i>Fbw7^+/+, Fbw7^ΔG/+</i>, or <i>Fbw7^ΔG/+; Hes5</i>^−/− mice. (c) Q-PCR analysis of <i>Fbw7α</i>, <i>Fbw7β, Hes5</i>, and <i>Dll1</i> in intestinal cells isolated from <i>Fbw7^+/+, Fbw7^ΔG/+</i> or <i>Fbw7^ΔG/+; Hes5</i>^−/− mice. (d) Nestin, Map2, and DAPI staining on NSCs isolated from <i>Fbw7^+/+, Fbw7^ΔN/+</i>, or <i>Fbw7^ΔN/+; Hes5</i>^−/− mice, 3 d after differentiation and (e) quantification of Nestin+ and Map2+ cells from these NSCs. (f) Q-PCR analysis of <i>Fbw7α</i>, <i>Fbw7β, Hes5</i>, and <i>Dll1</i> from NSCs isolated from <i>Fbw7^+/+, Fbw7^ΔN/+</i> or <i>Fbw7^ΔN/+; Hes5</i>^−/− mice. (percentage positive cells ± SEM, <i>n</i>≥10 for each genotype; relative fold induction after normalizing to actin ± SEM, <i>n</i>≥3 for each genotype).</p