Decision letter: CCN1 interlinks integrin and hippo pathway to autoregulate tip cell activity

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract CCN1 (CYR61) stimulates active angiogenesis in various tumours, although the mechanism is largely unknown. Here, we report that CCN1 is a key regulator of endothelial tip cell activity in angiogenesis. Microvessel networks and directional vascular cell migration patterns were deformed in ccn1-knockdown zebrafish embryos. CCN1 activated VEGFR2 and downstream MAPK/PI3K signalling pathways, YAP/TAZ, as well as Rho effector mDia1 to enhance tip cell activity and CCN1 itself. VEGFR2 interacted with integrin αvβ3 through CCN1. Integrin αvβ3 inhibitor repressed tip cell number and sprouting in postnatal retinas from endothelial cell-specific Ccn1 transgenic mice, and allograft tumours in Ccn1 transgenic mice showed hyperactive vascular sprouting. Cancer patients with high CCN1 expression have poor survival outcomes and positive correlation with ITGAV and ITGB3 and high YAP/WWTR1. Thus, our data underscore the positive feedback regulation of tip cells by CCN1 through integrin αvβ3/VEGFR2 and increased YAP/TAZ activity, suggesting a promising therapeutic intervention for pathological angiogenesis. https://doi.org/10.7554/eLife.46012.001 Introduction Angiogenesis involves extensive remodelling of the extracellular matrix (ECM) and endothelial cells (ECs) (Stupack and Cheresh, 2002). The ECM is important for dynamic and multifunctional regulation of cell behaviours and can modulate the bioavailability and activity of growth factors, cytokines, and extracellular enzymes (Aszódi et al., 2006). In addition, ECM proteins directly interact with cell surface receptors to activate signal transduction (Bornstein and Sage, 2002). Matricellular proteins are ECM proteins that do not contribute directly to the formation of these structures in vertebrates but are involved in cell–matrix interactions and other diverse cellular functions (Bornstein, 1995). Such proteins include thrombospondin-1, osteonectin, and members of the CYR61/CTGF/NOV (CCN) family of proteins (Lau and Lam, 1999). Within the CCN protein family, cysteine-rich angiogenic inducer 61 (CYR61, also known as CCN1) is a secreted protein that is a target of the YAP/TAZ Hippo signalling molecules (Zhang et al., 2011; Yu et al., 2012). CCN1 has been reported to mediate a variety of cellular processes, including adhesion, chemotaxis stimulation, survival, and angiogenesis, in a cell type-dependent manner (Grzeszkiewicz et al., 2002; Lin et al., 2004). CCN1 also regulates the expression and activities of angiogenic factors, such as vascular endothelial growth factor (VEGF), in fibroblasts and osteoblasts (Chen et al., 2001; Ivkovic et al., 2003; Athanasopoulos et al., 2007; Dean et al., 2007). It is required for re-endothelialisation and wound repair in contexts where angiogenesis is indispensable (Athanasopoulos et al., 2007; Bär et al., 2010). The angiogenic behaviour of CCN1 has been attributed to its binding to integrin αvβ3, a major integrin expressed in ECs (Leu et al., 2002). Ccn1-null mice suffer embryonic lethality due to insufficient placental and embryonic blood vessel integrity (Babic et al., 1998), and Ccn1-knockdown in mouse embryos causes atrioventricular valve and septal defects due to apoptosis in the cushion tissue and reduction in gelatinase activity (Mo and Lau, 2006); this phenotype overlaps with VEGF-knockout phenotypes in the cardiovascular system (Dor et al., 2001). However, CCN1 is not expressed much in ECs but is rather expressed in other parenchymal or mesenchymal cells (Su et al., 2004), and thus, an EC-specific knockout strategy is likely not suitable for identification of the role of CCN1 in sprouting angiogenesis. YAP/TAZ are overexpressed in various cancer tissues and have been identified as prognostic markers (Wang et al., 2013; Han et al., 2014). Immunohistochemistry (IHC) of tumours has revealed prominent expression and nuclear localization of YAP/TAZ in correlation with malignant features and poor patient outcomes (Zanconato et al., 2016). CCN1 is a well-known transcriptional target of YAP/TAZ (Zhao et al., 2008), is secreted in cancer cells, and induces extensive tumour angiogenesis, underscoring its paracrine angiogenic effect (Harris et al., 2012; Maity et al., 2014). Thus, targeting abnormally activated YAP/TAZ is a promising strategy for the suppression of tumour progression, metastasis, and cancer relapse (Corvaisier et al., 2016; Warren et al., 2018). In addition, activation of YAP/TAZ by VEGF, a known angiogenic factor, facilitates expression of CCN1 (Wang et al., 2017). The presence of YAP in embryonic retinal vessels, along with reduced retinal vascular sprouting and decreased numbers of vascular branches upon EC-specific deletion of embryonic YAP/TAZ, has further emphasised the importance of YAP/TAZ in vascular development (Choi and Kwon, 2015; Sakabe et al., 2017). Three types of functionally different ECs participate in the angiogenic process: tip cells, stalk cells, and phalanx cells (Eilken and Adams, 2010). All of these are engaged in the processes of vascular maturation and the maintenance of vascular integrity, thereby optimising blood flow, tissue perfusion, and oxygenation (Eilken and Adams, 2010). Tip cells are characterised by their position at the very tops of angiogenic sprouts and have extensive filopodial protrusions directed toward angiogenic attractants. Tip cells have a specific molecular signature, characterised by the expression of vascular endothelial growth factor receptor 2 (VEGFR2), VEGFR3, and DLL4. It has been reported that the VEGF gradient is important in the selection and induction of endothelial tip cells. Binding of VEGFR2 induces a signalling cascade that enables the activation of Notch-Delta signalling via DLL4 expression in ECs, converting them into tip cells; however, mechanism of sustained tip cell activity other than VEGF-mediated signalling has not yet been elucidated. Here, we report that CCN1 plays crucial role as an auto-inducer of tip cell fate that stimulate angiogenesis through the interplay of YAP/TAZ signalling with the integration of integrin αvβ3-VEGFR2, suggesting a promising approach for the treatment of pathological angiogenesis facilitated by extensive stimulation of tip cells. Results CCN1 promotes sprouting angiogenesis in zebrafish Secreted CCN1 is reported to facilitate EC migration and tumour angiogenesis via a paracrine effect (Harris et al., 2012; Maity et al., 2014), and YAP, an upstream regulator of CCN1, is expressed in the developing front of mouse retinal vessels (Chintala et al., 2015). Thus, to examine the precise mechanistic involvement of CCN1 in vascular formation, we designed two kinds of morpholino (MO) to target the transcription start site (ATG MO) or intron 1/exon 2 boundary of the ccn1 gene (Splicing MO) (Figure 1A) and observed vascular development in TG (flk1:EGFP) embryos. MO-mediated knockdown of ccn1 caused the formation of small heads, oedema, and bent trunk regions (Figure 1B). In TG (flk1:EGFP) embryos, control MO injection resulted in the formation of T-shaped intersegmental vessels (ISVs) and dorsolateral anastomose vessels (DLAVs) at 40 hr post-fertilisation (hpf) (I′ and II′ in Figure 1B), whereas the vasculature in the head and trunk regions of ccn1 morphants was abnormally sprouted and disconnected (III′, IV′, V′, and VI′ in Figure 1B). ccn1 morphants lost the T-shaped morphology previously displayed at the DLAV and ISV connexions (IV′ and VI′ in Figure 1B). When we observed more precisely in ISVs, in control animals, frontal cells from the DA migrated along a left-ascending path to the parachordal vessel (PAV) and then along a right-ascending path to the DLAV (I and II, arrows in Figure 1B); conversely, in two kinds of ccn1 morphants, these cells took a right and then left-ascending or bifurcating path to DLAV or not migrating from DAV (III-VI, arrows in Figure 1C, Figure 1D), and disconnected or malformed DLAV (III-VI arrowheads in Figure 1C, Figure 1D) were significantly increased at both 32 and 40 hpf. However, injection of sense RNA of ccn1 significantly rescued the vascular malformations and altered phenotypes induced by ccn1 morpholinos (Figure 1C VII and VIII), suggesting that CCN1 is an essential factor for the vascular development in zebrafish. In addition, ccn1 morphants demonstrated ectopic expression of flk-1, which was expressed not in vascular ECs but across the entire anterior–posterior body axis (Figure 1E). Aortic vessels disappeared and changed into venous types as detected by in situ expression of Notch-1b (aortic marker) and Flt-4 (venous marker) (Figure 1—figure supplement 1). Therefore, the results suggest that CCN1 is essential for the normal migration and sprouting as well as the identity of ECs in zebrafish vascular development. Figure 1 with 1 supplement see all Download asset Open asset CCN1 knockdown induces defects in vessel migration and sprouting as well as identity of endothelial cells in zebrafish vascular development. (A) Scheme showing ccn1 gene exon and intron. Morpholinos (MO) binding to ccn1 ATG starting site (ATG MO) and intron 1/exon two boundary region (Splicing MO) denoted as red lines were synthesised and injected into one-cell-stage zebrafish embryos. (B) Lateral view of Tg(Flk;EGFP) embryos in head and trunk regions of control (I–II for bright field, I′–II′ for dark field) and ccn1 morphants (MO) (III–VI for bright field, III′–VI′ for dark field) at 40 hpf. Arrows indicates sprouting vessels. Arrows indicate dorsolateral anastomose vessels (DLAV), intersegmental vessels (ISV), parachordal vessel (PAV), dorsal aorta (DA) and pericardial vein (PCV). The white square was enlarged and placed next to it. Scale bar = 25 µm (C) Anterior regions in trunk vessels were magnified at 32 and 40 hpf, in control, ccn1 MO-treated (III-VI) and ccn1 RNA rescued (VII-VIII) embryos. Arrows indicate misdirected or disconnected vessels and arrowheads do disconnected or abnormal DLAV. Scale bar = 50 µm. (D) Number of intersomitic vessel (ISV) defect were counted in each embryo and graphed. (F) Whole mount in situ hybridization was performed with control and ccn1 MO embryos at 24 hpf and 32 hpf with zFlk1 antisense probe. Purple colour shows expression of zFlk1 mRNA expression. Arrows indicates ISVs. Scale bar = 100 µm. https://doi.org/10.7554/eLife.46012.002 CCN1 promotes endothelial sprouting activity in angiogenesis CCN1 is expressed during active angiogenic stages in postnatal retina tip cells, and its expression decreases upon EC maturation (Chintala et al., 2015). However, the above experiment raised the question of why CCN1 deletion increases sprouting and whether CCN1 can stimulate or inhibit tip cell activity. We used recombinant CCN1 protein to mimic the secreted protein ex vivo and performed a mouse aortic ring assay, as well as an EC spheroid sprouting assay in vitro, using VEGF-A (VEGF) as a positive control. CCN1 induced intensive vascular sprouts from aortic explants and significantly increased tip cell populations to the same degree as VEGF, as determined by tip cell markers, DLL4 and CD34 (Siemerink et al., 2012) as well as an EC marker, CD31 (Figure 2A). CCN1 also significantly increased sprouting branches from spheroids, as determined by both lengths and numbers of tip cells after immunostained with DLL4 and CD34 antibodies (Figure 2B). Filopodial formation, which is an important marker of tip cells in migrating cells, was also detected in vitro by immunostaining with phalloidin. In human ECs, filopodia markedly increased upon exposure to CCN1 (Figure 2C). To investigate the angiogenic activity of CCN1, we performed in vitro angiogenesis, tube formation, and EC migration assays. CCN1 markedly increased capillary tube formation (Figure 2D) and promoted EC migration in the wound migration assay (Figure 2E, Videos 1 and 2). Because tip and stalk cell activities are increased during active angiogenesis (Eilken and Adams, 2010), we checked whether the expression of marker genes for tip and stalk cells changed following CCN1 treatment in human umbilical vein ECs (HUVECs). Expression levels of tip cell marker genes like VEGFR2, DLL4 (Hellström et al., 2007), and SOX17 (Lee et al., 2014) were elevated, but those of stalk cell marker genes like JAG1, ROBO4, and NOTCH1 were not changed by CCN1, nor by VEGF (Figure 2F). Protein levels of VEGFR2, DLL4, and SOX17 were also increased by CCN1 (Figure 2G). To confirm these results, DLL4 promoter-driven luciferase (DLL4-Luc) reporter activity was observed. DLL4-Luc reporter activity was significantly increased by CCN1, as by VEGF (Figure 2H). These results clearly suggest that CCN1 induces tip cell activity and identity in sprouting during angiogenesis. Figure 2 Download asset Open asset CCN1 stimulates tip cell activity in ECs. (A) Mouse aorta isolated and cut at 6 weeks old, planted on Matrigel-coated plate and incubated for 7 days in presence or absence of CCN1 (10 ng/mL) or VEGF (10 ng/mL). Expression of CD31, DLL4, and CD34 were visualised with respective antibodies by the immunofluorescent assay under the confocal microscope. Tip cells expressing CD31+/DLL4+/CD34+ cells were counted and graphed. Three independent experiments were performed (n = 3/group). *p<0.05 vs control. Scale bar = 100 µm. (B) HUVEC spheroids were cultured in presence or absence of CCN1 (10 ng/mL) or VEGF (10 ng/mL) for 48 hr. Expression of CD31, DLL4, and CD34 were visualised with respective antibodies by the immunofluorescent assay under the confocal microscope. Length of sprout and tip cells expressing CD31+/DLL4+/CD34+ cells were counted and graphed (n = 3/group). **p<0.01 vs Control. Scale bar = 100 µm. (C) Filopodia number per field was counted in HUVEC cells treated with PBS (Control) or CCN1 (10 ng/mL) for 30 min and imaged using confocal microscope after staining with vinculin (red) and phalloidin (green). *p<0.0001 vs. Control. Scale bar = 25 µm. (D) Branching points of tubular networks were counted in HUVECs cultured in the presence or absence of CCN1 or VEGF for 48 hr (n = 3/group). Magnification = 40 × . *p<0.05 vs. control. (E) Confluent HUVECs were wounded and cultured in the presence or absence of CCN1 or VEGF for 6 hr. Migrating cells from wound line (dotted line) were counted (n = 3/group). Magnification = 40 × . *p<0.05 vs. control. (F) HUVECs were treated with CCN1 (10 ng/mL) or VEGF (10 ng/mL) for 24 hr, and total RNA was collected for real-time qRT-PCR analysis. Expression of tip cell marker genes (SOX17, DLL4, VEGFR2) and stalk cell marker genes (JAG1, NOTCH1, ROBO4) was analyzed. *p<0.05 or **p<0.01 vs. Control. (G) Western blotting analysis of VEGFR2, DLL4, and SOX17 protein levels in the same samples as used in F. β-actin was used as an internal control. Fold increase was indicated as a number below each band. (H) Luciferase assays in HUVECs transfected with DLL4-Luc reporter vector and treated with CCN1 or VEGF (10 ng/mL) for 24 hr. Luciferase activity was normalised to Renilla luminescence. *p<0.001 vs. Control. https://doi.org/10.7554/eLife.46012.004 Figure 2—source data 1 Source data for Figure 2G. https://doi.org/10.7554/eLife.46012.005 Download elife-46012-fig2-data1-v2.xlsx Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg CCN1 promoted EC migration. https://doi.org/10.7554/eLife.46012.006 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg CCN1 promoted EC migration. https://doi.org/10.7554/eLife.46012.007 CCN1-induced YAP/TAZ activation via VEGFR2 signalling is critical for tip cell activity YAP/TAZ have critical roles in vascular sprouting (Kim et al., 2017; Park and Kwon, 2018), and their activation promotes tip cell activity and expression of target genes, such as Ccn1 (Sakabe et al., 2017). Thus, the probable involvement of VEGFR2 signalling in the regulation of YAP/TAZ was investigated in CCN1-treated ECs. YAP and TAZ translocation to the nucleus was significantly upregulated by treatment with CCN1 for 1 hr in a dose-dependent manner (Figure 3A). Phosphorylation of YAP/TAZ at S127/89, respectively, leads to cytosolic degradation, thus inhibiting their nuclear localization. In growth factor-mediated YAP signalling via the MAPK and PI3K nexus, large tumour suppressor kinase 1 (LATS1), a tumour suppressor that phosphorylates YAP/TAZ, is repressed through reduced phosphorylation, thus enhancing the nuclear localization of YAP (Gumbiner and Kim, 2014; Kim and Gumbiner, 2015). We found that levels of phosphorylated LATS were decreased maximally at 1 hr and those of phosphorylated YAP/TAZ were decreased from 15 min, maximally at 1 hr by CCN1 in HUVECs (Figure 3B), indicating that CCN1 activates YAP/TAZ in ECs at the early time after stimulation. Figure 3 with 1 supplement see all Download asset Open asset CCN1-VEGFR2-MAPK, PI3K-YAP/TAZ signalling involved in CCN1-mediated tip cell activation. (A) Nuclear localization of YAP/TAZ in HUVECs treated with CCN1 at 5, 10, or 50 ng/mL for 1 hr. (B) Western blotting of LATS, YAP, and TAZ and their phosphorylated forms in HUVECs treated with CCN1 (10 ng/mL) for indicated times. Quantitation for phosphorylated protein level was normalised by each protein and graphed shown in right panel. *p<0.05 vs. Control. (C) Western blotting of phosphorylated VEGFR2 at tyrosine (Y)−1054/1059 and Y1175 in HUVECs treated with VEGF (10 ng/mL) or CCN1 (10 ng/mL) for 5, 10, 15, 30, or 60 min. β–actin was used as a loading control. (D) Western blotting of VEGFR2 phosphorylated at Y1054/1059 and Y1175 in HUVECs treated with VEGF (10 ng/mL) or CCN1 (0, 1, 5, or 10 ng/mL) for 30 min. β–actin was used as a loading control. (E) Western blotting of VEGFR2 phosphorylated at Y1054/1059 and Y1175 in HUVECs transfected with CCN1 siRNA. β–actin was used as a loading control. (F) Western blotting of phosphorylated or natural forms of p-ERK, p38 MAPK, and p85 PI3K in HUVECs were treated with VEGF or CCN1 (10 ng/mL) for 15 min. (G, H) DLL4 mRNA (G) and protein (H) levels in EA.hy926 cells treated with PBS (Control) or CCN1 (10 ng/mL) alone or together with MAPK kinase inhibitors PD98059, SP600125, or SB203580 (20 mM) or PI3K inhibitor LY294002 (20 mM) for 24 hr. *p<0.005 vs. Control, #p<0.0001 vs. CCN1. (I) Filopodia number per field was quantified after staining with anti-vinculin antibody (red) and phalloidin (green) in EA.hy926 cells treated with PBS (Control) or CCN1 (10 ng/mL) alone or together with PD98059 (20 mM), SP600125 (20 mM), SB203580 (20 mM), or LY294002 (20 mM) for 30 min. Scale bar = 10 µm. *p<0.0001 vs. Control, #p<0.0001 vs. CCN1. (J) Western blotting of indicated proteins in EA.hy926 cells treated with PBS (Control) or CCN1 (10 ng/mL) alone or together with PD98059 (20 mM), SB203580 (20 mM), or LY294002 (20 mM) for 1 hr. (K) CCN1 mRNA expression determined by qRT-PCR of total RNA obtained from the same groups as in G but treated with CCN1 for 24 hr. *p<0.005 vs. Control, #p<0.005 vs. CCN1. (L) of DLL4, and CCN1 proteins in HUVECs treated with CCN1 (10 ng/mL) for 24 hr in the presence or absence of 100 for 24 hr. Fold were under each protein in and Figure data 1 Source data for Figure Download VEGFR2 signalling is to YAP/TAZ activation in angiogenesis (Wang et al., 2017). VEGFR2 activation is a key in the fate of an EC as a tip cell et al., Thus, we investigated whether VEGFR2 is activated by CCN1 in ECs. CCN1 induced the of VEGFR2 from 10 min at time of YAP/TAZ activation 1 hr (Figure and in a dose-dependent manner (Figure VEGFR2 activation after stimulation (Figure supplement 1A) et al., was repressed upon CCN1 knockdown (Figure suggesting that CCN1 activates To confirm whether CCN1 is to activate VEGFR2 in the absence of VEGF, we transfected HUVECs with and found that CCN1 activated VEGFR2 of VEGF (Figure supplement 1B). of CCN1 with repressed the mRNA expression of the tip cells markers, DLL4 and VEGFR2 (Figure 2 and Figure supplement as well as the migration and sprouting (Figure supplement 1D), indicating that endothelial CCN1 is important for EC tip cell activity. The of Y1054/1059 in the VEGFR2 kinase kinase activity, and that of Y1175 binding to the p85 of PI3K and to which is involved in migration as well as et al., 2006). has been reported that activation induces downstream to inhibit LATS and activate YAP/TAZ et al., 2018). Therefore, we checked that extracellular kinase p38 MAPK, and p85 all downstream of Y1175 phosphorylation, were activated by CCN1 (Figure to confirm whether CCN1-mediated tip cell activity involves the MAPK and PI3K signalling we used inhibitors for p38 MAPK MAPK and PI3K treatment with all inhibitors SP600125, DLL4 mRNA and protein levels were repressed (Figure and To confirm these results, filopodial formation, as determined by phalloidin was reduced in ECs to CCN1 together with the inhibitors (Figure CCN1-mediated increases in the of and further its probable involvement in VEGFR2 to that of VEGF et al., et al., (Figure supplement 1E). Thus, these results suggest that CCN1 induces tip cell activation through the and signalling in ECs. we determined whether the CCN1-induced MAPK and PI3K signalling are involved in the activation of The of and YAP/TAZ was decreased by CCN1, but the PD98059, and LY294002 inhibitors to control thus the of phosphorylated in ECs, in YAP/TAZ activation by CCN1 (Figure In support of these expression of YAP/TAZ target gene CCN1 was also significantly increased by CCN1 treatment and repressed by PD98059, and LY294002 (Figure suggesting the involvement of MAPK and PI3K as well as the expression of CCN1 via the activation of YAP/TAZ in ECs. inhibitor of YAP/TAZ, VEGFR2, CCN1, and DLL4 expression induced by CCN1, the involvement of YAP/TAZ in tip cell activity (Figure These results suggest that CCN1 activates the to facilitate the of CCN1, further tip cell activity in ECs. mDia1 is involved in CCN1-induced filopodial formation and YAP/TAZ activation in ECs of and the of is a crucial in filopodial formation, and a family is essential for Rho filopodial formation et al., 2003; and 2012). Therefore, we investigated whether mDia1 is involved in CCN1-induced filopodial formation in ECs. We found that CCN1 increased mRNA expression by (Figure and also of increased filopodial numbers to that in CCN1-treated HUVECs (Figure However, decreased CCN1-induced filopodial formation (Figure suggesting that mDia1 is essential for CCN1-induced filopodial formation in ECs. the small Rho is critical for EC by endothelial and filopodial formation by et al., and tip cell migration (Sakabe et al., we levels of activated The active of by and analysis as previously (Wang et al., was increased by CCN1 treatment and but not the with CCN1 (Figure induced by active (Sakabe et al., was also detected in CCN1-treated and cells but not in cells (Figure suggesting that mDia1 is required for CCN1-induced activation in tip cell we further identified whether CCN1-induced mDia1 activation is for the YAP/TAZ activation. Nuclear localization of YAP/TAZ was induced by of as well as CCN1 but not by the (Figure indicating that mDia1 is for YAP/TAZ nuclear translocation in CCN1-induced tip cell Figure Download asset Open asset essential for CCN1-induced activation in tip cell (A) HUVECs were treated with PBS (Control) or CCN1 (10 ng/mL) for 24 hr, and total RNA was used for the of mRNA expression by *p<0.001 vs. Control. (B) with and HUVECs were treated with CCN1 and immunostained with anti-vinculin antibody (red) and phalloidin (green) to Scale bar = 10 µm. HUVECs transfected with and were treated with CCN1 (10 ng/mL) for 30 min, and active was by blotting analysis (C) and visualised by Fold were under each protein band. (E) HUVECs were transfected with and cells for hr were treated with CCN1 10 for 1 hr and detected YAP/TAZ by Scale bar = 100 µm. Figure data 1 Source data for Figure Download Integrin αvβ3 is a key of induced by CCN1 involved in various are induced by CCN1, and are also reported to activate VEGFR2 and its downstream signalling (Chen and Lau, αvβ3 and are involved in EC migration induced by CCN1 (Chen and Lau, and are the major receptors on ECs. the of ECs to to in the presence of CCN1 was by the of antibody at min (Figure supplement αvβ3 may be the major receptor of CCN1 to ECs et al., We also found that integrin was not involved in the of to ECs (Figure supplement To confirm whether integrin αvβ3 is essential for the induction of tip cells by CCN1, we checked whether CCN1 regulates integrin expression. CCN1, as well as VEGF increased integrin expression to blotting and assays (Figure supplement suggesting regulation of CCN1 angiogenic signalling through integrin to into the molecular involvement of integrin αvβ3 in CCN1-induced tip cell activity, we investigated whether integrin αvβ3 with VEGFR2 via CCN1. and showed interactions VEGFR2 and integrin αvβ3, as well as their with CCN1 upon CCN1 treatment for 24 hr. However, cell exposure to the αvβ3 inhibitor the interactions of VEGFR2 with integrin αvβ3, CCN1 with integrin αvβ3, and CCN1 with VEGFR2 (Figure In addition, in situ assay also clearly revealed increased interactions αvβ3/VEGFR2 in ECs to that in (Figure and Figure supplement CCN1-induced VEGFR2 downstream kinase signalling activity was by (Figure We also determined whether integrin αvβ3 is essential for YAP/TAZ activation by CCN1. with rescued the decreased of YAP/TAZ by CCN1 (Figure the same increased YAP/TAZ nuclear localization in the CCN1-treated was by (Figure by repressed DLL4-Luc activity and expression of tip cell marker genes DLL4 and SOX17 (Figure and These results suggest that CCN1-mediated tip cell activation is by YAP/TAZ through the integrin αvβ3/VEGFR2 signalling Figure with 1 supplement see all Download asset Open

Topic: Connective Tissue Growth Factor Research
Subfield: Molecular Biology
Field: Biochemistry, Genetics and Molecular Biology
Domain: Life Sciences