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. Author manuscript; available in PMC: 2017 Dec 6.
Published in final edited form as: Cell Rep. 2016 Dec 6;17(10):2532–2541. doi: 10.1016/j.celrep.2016.11.017

G-protein-coupled receptor-2-interacting protein-1 controls stalk cell fate by inhibiting Delta like 4-Notch1 signaling

Syamantak Majumder 1,#, GuoFu Zhu 2,#, Xiangbin Xu 3, Sharon Senchanthisai 1, Dongyang Jiang 2, Hao Liu 2, Chao Xue 1, Xiaoqun Wang 1, Heidi Coia 1, Zhaoqiang Cui 1, Elaine M Smolock 1, Richard T Libby 4, Bradford C Berk 1, Jinjiang Pang 1,2,*
PMCID: PMC5217468  NIHMSID: NIHMS831658  PMID: 27926858

SUMMARY

The spatiotemporal localization and expression of Dll4 are critical for sprouting angiogenesis. However, the related mechanisms are poorly understood. Here we show that G-protein-coupled receptor-kinase interacting protein-1 (GIT1) is a robust endogenous inhibitor of Dll4-Notch1 signaling that specifically controls stalk cell fate. GIT1 is highly expressed in stalk cells but not in tip cells. GIT1 deficiency remarkably enhances Dll4 expression and Notch1 signaling resulting in impaired retinal sprouting angiogenesis, which can be rescued by treatment with the Notch inhibitor, or Dll4 neutralizing antibody. Notch1 regulates Dll4 expression by binding to recombining binding protein suppressor of hairless (RBP-J, a transcriptional regulator of Notch) via a highly conserved ankyrin (ANK) repeat domain. We show that GIT1, which also contains an ANK domain, inhibits the Notch1-Dll4 signaling pathway by competing with Notch1 ANK domain for binding to RBP-J in stalk cells.

Graphical Abstract

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INTRODUCTION

Sprouting angiogenesis is required for new vessel formation during development, wound repair and tumor growth (Gerhardt et al., 2003; Trindade et al., 2008). An essential process for angiogenesis is endothelial cell (EC) sprouting by filopodia that sense and respond to vascular endothelial growth factor (VEGF) gradients (Gerhardt et al., 2003; Ruhrberg et al., 2002). Formation of a vascular plexus requires temporary spatial differentiation of EC into tip and stalk cells, a behavior that is tightly regulated by VEGF-Notch1-Dll4 signaling (Lobov et al., 2007; Phng and Gerhardt, 2009). The Notch family comprises highly conserved trans-membrane proteins, including ligands (Dll1, Dll4, Jag1) and receptors (Notch1-4). In the canonical Notch signaling pathway, upon ligand (Delta and Serrate/Jagged) binding, Notch is cleaved to generate the Notch intracellular domain (N-ICD), which translocates to the nucleus and binds to recombining binding protein suppressor of hairless (RBP-J), and subsequently regulates target gene expression. Notch signaling is essential for vasculature development in various vascular beds (Hrabe de Angelis et al., 1997; Krebs et al., 2000; Swiatek et al., 1994). Many aspects of the signaling pathway have been elucidated; however, the positive feedback regulation of Dll4-Notch signaling that controls tip and stalk cells behavior has never been described.

We recently showed that the major phenotype of GIT1 knock out (KO) mice was defective pulmonary vascular development (Pang et al., 2009). Our recent data also demonstrated that GIT1 is required for endothelial directional migration and tumor angiogenesis by mediating cortactin dependent actin remodeling and lamellipodia formation (Majumder et al., 2014). EC function in response to VEGF was impaired as demonstrated by decreased proliferation, tube formation and activation of multiple downstream signaling pathways (Pang et al., 2009). Interestingly, other vascular beds of GIT1-KO mice were normal except the retina. Both murine retina and lung vascular development occur postnatally, suggesting a unique role for GIT1 in postnatal angiogenesis (Hislop, 2005). However, the role of GIT1 in regulating sprouting angiogenesis, especially tip cell and stalk cell function, has not been explored. Here we demonstrated that GIT1 is required for regulating Dll4 positive feedback loop in stalk cells, thus maintaining the stalk cell phenotype, which is essential for sprouting angiogenesis.

EXPERIMENTAL PROCEDURES

Retina whole mount immunohistochemistry

Mouse pups were sacrificed at P5. Eyes were fixed in 4% paraformaldehyde (PFA) in PBS at 4°C overnight and washed in PBS. Retinas were dissected, permeabilized in PBS, 5% Normal goat serum (NGS), and 0.3% Triton X-100 at 4°C overnight. They were incubated in FITC (or TRITC)-conjugated isolectin B4 (Bandeiraea simplicifolia; L-2140; Sigma-Aldrich) 20 μg/ml in PBS at 4°C overnight. After washing and a brief post fixation in PFA, the retinas were either flat mounted or processed for multiple labeling. The following antibodies were used: Dll4 (1:100; Santa Cruz, sc-28915), GIT1 (1:200; Santa Cruz, sc-9657), VEGFR3 (1:80; R&D Systems, AF743), Tie2 (1:80; eBiosciences, 14-5987), ERG (1:150; Abcam, #ab92513), GFAP (1:500; Dako, Z0334), GFP 1:500 (Vector, A21311) and Alexa-546, Alexa-488 or Alexa-405 and Alexa-545 conjugated secondary antibodies (1:500, Molecular Probes). Flat mounted retinas were analyzed by fluorescence microscopy using an Olympus BX51 microscope equipped with a digital camera (Spot CCD camera) or by confocal laser scanning microscopy (Olympus confocal system, Fluoview). Images were processed using Adobe Photoshop® and analyzed by Image Pro Plus.

Immunoprecipitation and immunoblotting

For immunoblotting, freshly isolated P5 retina or cells were lysed in Cell Lysis Buffer (Cell Signaling Technology, #9803) and protein concentrations determined. Protein samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with appropriate primary antibodies. After incubating with fluorescence-conjugated secondary antibodies, immunoreactive proteins were visualized by an Odyssey infrared imaging system (LI-COR Biotechnology, Nebraska). Densitometric analysis of the blots was performed with Odyssey software (LI-COR Biotechnology). Results were normalized by arbitrarily setting the densitometry of control samples to 1.0. The antibodies used were GIT1 (Santa Cruz, sc-9657), Notch1 (EMD Millipore 07-220), Dll4 (Santa Cruz, sc-28915), VEGFR3 (eBiosciences, 16-5988-81), VE-cadherin (Santa Cruz, sc-6458), Flt-1 (Santa Cruz, sc-316), EphrinB2 (Santa Cruz, sc-1010), EphB4 (Santa Cruz, sc-5536), Hey1 (Millipore, AB5714) and RBP-J (Santa Cruz, sc-28713). IP was performed as published previously(Pang et al., 2008).

Cell fractionation

HUVECs were harvested using buffer A (250 mM Sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1mM EDTA, 1 mM EGTA) by incubation on ice for 20 min followed by shearing through a 25 G needle 10 times. The nuclear pellet was collected by centrifugation for 5 min. The supernatant contained membrane and cytoplasm fractions. The nuclear pellet was washed once with buffer A. The nuclear fraction was sheared through a 25 G needle 10 times and centrifuged again at 3000 rpm for 10 min. The nuclear pellet was re-suspended in the nuclear buffer (buffer A containing 10% glycerol and 0.1% SDS).

Luciferase reporter assay

HEK293 cells cultured in a 6-well plate were cotransfected with Hey1 luciferase reporter gene, pCMV-N1-ICD (generous gift from Dr. Eileen M. Redmond), CMV-β-galactosidase (β-gal) and GIT1-WT or GIT1 mutants using lipofectamine. After transfection for 18 h, cells were harvested. The luciferase activity in cell lysates was determined using the Luciferase Reporter Assay kit (Promega) and Wallac 1420 multilabel counter (PerkinElmer). β-gal activity was measured by ONPG assays and used to normalize for differences in transfection efficiency.

Statistical analysis

All values are expressed as mean ± SEM from three to eight samples. Data were assessed using the student’s t-test and p<0.05 was considered statistically significant. Power analysis was performed on line. (https://www.dssresearch.com/KnowledgeCenter/toolkitcalculators/statisticalpowercalculators.aspx). The data distribution was analyzed using SPSS software. Because the sample size is between three to eight, we used the Shapiro Wilks test.

RESULTS

Defective sprouting angiogenesis of global and endothelial specific GIT1-KO mice

We previously showed that the major phenotype of GIT1-KO mice is defective pulmonary vascular development due to impaired angiogenesis, implicating a critical role for GIT1 in angiogenesis (Pang et al., 2009). To define the role of GIT1 in sprouting angiogenesis, the retina vasculature at postnatal day 5 (P5) was visualized by isolectin B4 (IB4) staining. The retinas of GIT1-KO mice displayed decreases of 49.5±2.1% in vessel length (Fig. S1A–D, I), 26.4±1.4% in branch points (Fig. S1E–F, J), 37.3±1.8% in tip cells and 53.2±1.4% in filopodia extensions (Fig. S1G–H, K–L). Additional experiments using aortic ring (Fig. S1M–P) and human umbilical vascular EC (HUVEC) spheroid angiogenesis assay (Fig. S1Q–T) showed that GIT1 deficiency significantly inhibited sprouting angiogenesis ex vivo and in vitro, respectively. It is well established that astrocytes promote retinal vascular development by secreting VEGF (Watanabe and Raff, 1988). Thus, glial fibrillary acidic protein (GFAP, astrocyte marker) whole mount staining was performed on retina of GIT1-WT and KO mice at P5 and VEGF mRNA was also measured. The morphology and total number of astrocytes was comparable in GIT1-WT and KO mice (Fig. S1U) and there was no significant difference in VEGF mRNA expression between two groups (Fig. S1V). In addition, the artery and vein are very easily distinguished in GIT1-WT mice (vein has more branches compared to the artery, Fig. S1A). However, in GIT1-KO mice (Fig. S1B), no obvious vein or artery structures could be identified. Thus, we measured the artery marker (EphrinB2) and vein marker (EphB4) protein expression in retinas of GIT1-WT and GIT1-KO mice at P5 by western blot. We found a significant increase in EphrinB2 (2 fold) but no changes in EphB4 expression in retinas of gGIT1-KO mice (Fig. S1W–X).

To demonstrate the essential role of GIT1 in EC angiogenesis, we generated an EC specific GIT1-KO mice (ecGIT1-KO) using floxed Git1 mice and Tie2-Cre mice. We first confirmed the specificity of the Tie2-Cre activity in retina vascular ECs using Rosa26mT/mG; Tie2-Cre+ and Rosa26mT/mG; Tie2-Cre− mice at P5(Fig. S2A–D). Our data also demonstrated low expression of GIT1 in thymus and undetectable expression in spleen (Fig. S2E). Therefore, it is unlikely that ecGIT1-KO mouse generated using Tie2 Cre mouse has leakiness in hematopoietic cell. We found a similar retina phenotype in these ecGIT1-KO mice (Fig. S2I–R). These findings indicate that GIT1 is critical for sprouting angiogenesis.

GIT1 is expressed in stalk cells but not in tip cells

To define the specific role of GIT1 in tip cell and stalk cell function, GIT1 expression was measured in P1, P3 and P5 retinas by GIT1 immunostaining and costained with ETS-related gene (ERG, an EC specific nuclear marker). Surprisingly, there was no expression of GIT1 in tip cells (ERG positive cells with filopodia), but high expression in stalk cells (ERG positive cells without filopodia), locating in nuclei at P1 and P3 and in both nuclei and cytosol at P5 (Fig. 1A–B). These data suggest an important function of GIT1 in nuclei during retinal vasculature development. In addition, GIT1 is also expressed in veins but not in arteries (Fig. 1A, vein has more branches compared to the artery). It has been shown that Dll4 is highly expressed in tip cells (Hellstrom et al., 2007; Lobov et al., 2007; Jakobsson et al., 2010). To confirm these findings, we also performed co-staining of GIT1 and Dll4. GIT1 positive cells usually had no or very low Dll4 expression in the leading edge of retinal vasculature (Fig. 1C). These data imply an important role for GIT1 in stalk cells and artery-vein specification.

Fig. 1. GIT1 is expressed in stalk but not in tip cells and in veins but not in arteries.

Fig. 1

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A–B. Retinas from GIT1-WT mice were harvested at P1, P3 and P5. Retinas were processed and immunostained for GIT1 with counter staining the vessels with IB4-FITC or IB4-TRITC and ERG to locate the tip cells. Red boxes in B show the tip cells with filopodia structures, red arrow heads indicate stalk cells with GIT1 (red), white arrowheads indicate endothelial tip cells with no GIT1 (green) while green arrowheads indicate stalk cells with GIT1 (green) (n=4). GIT1 is also expressed in vein (v) but not in artery (a). C. Retinas from GIT1-WT and KO mice were harvested at P5. Retinas from WT mice were processed and immunostained for GIT1 with counter staining the tip cells with Dll4. Retinas from GIT1-KO mice were immunostained for GIT1 and IB4 to show the specificity of GIT1 antibody.

Absence of GIT1 increases Dll4 and Flt1 expression but inhibits VEGFR3 expression in vivo

Endothelial tip cell and filopodia numbers are tightly regulated by Dll4 (Hellstrom et al., 2007; Lobov et al., 2007; Suchting et al., 2007) and Dll4 is critical for artery specification (Duarte et al., 2004). Therefore, we detected Dll4 expression in P5 retina vasculature by immunohistochemistry. As reported, Dll4 was expressed in tip cells with sprouting filopodia in GIT1-WT mice (Fig. 2A). In contrast, in GIT1-KO mice, Dll4 expression was strikingly increased in both stalk and tip cells (Fig. 2A). Dll4 mRNA and protein expression in retinas of GIT1-KO mice were also significantly increased compared to GIT1-WT mice (Fig. 2B–D, 2.3±0.2 fold and 5.0±0.3 fold, respectively). We obtained similar results in ecGIT1-KO mice (Fig. S2S–Z). Dll4-Notch signaling strongly represses VEGF receptor 3 (VEGFR3) expression (Benedito et al., 2012). Consistently, we observed a 3.0±1.1 fold decrease of VEGFR3 protein expression in retinas of GIT1-KO mice (Fig. 2G–H). To confirm it, we performed VEGFR3 whole mount staining. VEGFR3 was highly expressed in the leading edge of retinal vasculature in GIT1-WT mice at P5. There was a 65% decrease of VEGFR3 in GIT1-KO mice (Fig. 2E–F).

Fig. 2. GIT1 is important for spatio-temporal expression of Dll4.

Fig. 2

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A. Whole mount Dll4 and IB4 staining of retinas of GIT1-WT and GIT1-KO mice at P5 (n=5). B–D. Retinas of GIT1-WT and GIT1-KO mice at P5 were isolated and expression of Dll4 was assayed by qRT-PCR (B) and western blot (C–D). Dll4 mRNA and protein expression were quantified (normalized to β-Actin or GAPDH). Student t-test, *P< 0.05 compared with control siRNA (mean ±SEM; n =3). E–F. Whole mount VEGF3 and IB4 staining of retinas of GIT1-WT and GIT1-KO mice at P5 (n=5, E). The fluorescent intensity of VEGF3 and IB4 was quantified (F). Student t-test, *P< 0.05 compared with GIT1-WT (mean ±SEM; n =5). G–H. Retinas of GIT1-WT and GIT1-KO mice at P5 were isolated and expression of VEGFR3 was assayed by western blot. VEGFR3 protein expression was quantified (normalized to Ve-Cadherin). Student t-test, *P< 0.05 compared with WT (mean ±SEM; n =3). I–K. Control and GIT1 siRNA were transfected for 48h in HUVECs. Dll4 and Hey1 mRNA and protein expression and Notch1 activation were detected by qRT-PCR (I) or western blot (J,K). Dll4 and Hey1 mRNA and protein expression and Notch1 activation were quantified (normalized to β-Actin or GAPDH or total Notch1). VEGFR3 protein expression (J,K) was quantified (normalized to GAPDH). Student t-test, *P< 0.05 compared with control siRNA group (mean ±SEM; n =3).

Deletion of GIT1 enhances Dll4-Notch signaling and downstream gene expression in vitro

Caolo et al showed a Dll4-Notch1-Dll4 positive feed loop in vitro (Caolo et al., 2010). Dll4 in a cell activates the Notch receptors and increases Dll4 expression in neighboring cells. Through these communications, Notch signaling is amplified or propagated (Fig. S3A). This positive feedback loop is remarkably enhanced in GIT1-KO mice, since increase of Dll4 in stalk cells affects both stalk and tip cell function. Based on these data, we hypothesized that GIT1 is important for stalk cell fate determination by inhibiting the Dll4-Notch1 feedback loop. To test this hypothesis, the effect of GIT1 on Dll4-Notch1 signaling was assayed in HUVECs. In the canonical Notch signaling pathway, upon ligand (Delta and Serrate/Jagged) binding, Notch1 is proteolytically cleaved by γ-secretase to generate the Notch intracellular domain (N1-ICD). N1-ICD translocates to the nucleus, where it binds to RBP-J and regulates gene expression (Gridley, 2007; Phng and Gerhardt, 2009; Roca and Adams, 2007). Since Dll4 is highly expressed in confluent ECs (Caolo et al., 2010), HUVECs were transfected with control non-silencing or GIT1 specific siRNA for 48h. Similar to the in vivo data, Dll4 mRNA and protein expression significantly increased upon GIT1 knockdown compared to control siRNA treated HUVECs (Fig. 2I; by 3.2±0.6 and 6.3±0.3 fold, respectively). Similar to Dll4 expression, the Notch1 and RBP-J target gene, Hairy/enhancer-of-split related with YRPW motif protein 1 (Hey1) was significantly increased after GIT1 siRNA treatment (mRNA and protein expression by 5.2±0.57 and 3.0±0.3 fold, Fig. 2J). We next studied the effect of GIT1 on basal Notch1 expression and cleavage. Notch1 was unchanged in GIT1 siRNA treated HUVECs. However, expression of N1-ICD in GIT1 siRNA treated HUVECs was significantly enhanced (1.7±0.4 fold; Fig. 2J–K). The increase of N1-ICD is consistent with the increased expression of Notch ligand Dll4 shown in Fig. 2J–K. Enhanced Dll4 in GIT1-KO mice significantly inhibited VEGFR3 expression by 10 fold (Fig. 2J–K), whereas it had no significant effect on VEGFR2 (Pang et al., 2009). This is consistent with result from the Adams’ group, which showed that Dll4 has a very weak inhibitory effect on VEGFR2 expression but substantial suppression on VEGFR3 (Benedito et al., 2012). In addition, it is reported that Dll4-Notch signaling enhances Fms-Related Tyrosine Kinase 1 (Flt-1, VEGFR-1) to reduce stalk cell response to VEGF (Harrington et al., 2008). Therefore, we also measured Flt-1 protein expression in GIT1 depleted EC and Flt-1 mRNA expression in retina of GIT1-WT and KO mice at P5. Flt-1 protein expression was 2 fold increased in GIT1 siRNA treated HUVEC compared to control siRNA treated cells. In vivo, Flt-1 mRNA expression was enhanced by 2.7 fold in retina of GIT1-KO mice (Fig. S3B–D). Other Notch ligands and receptors including Dll1, Jagged1, Notch2, Notch3 and Notch4 expression were not affected by depletion of GIT1 (data not shown).

Inhibition of Notch1 activation rescues defective sprouting angiogenesis of GIT1-KO mice

To prove a critical role for Dll4 dysregulation in the impaired angiogenesis of GIT1-KO mice, rescue experiments were performed using N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT, a γ-secretase inhibitor). To confirm that high Notch1 activation in GIT1 deficient mice leads to defective sprouting angiogenesis, we injected DAPT (50mg/kg body weight) subcutaneously in GIT1-WT and GIT1-KO mice at P3 and P4. Consistent with the findings of others (Benedito et al., 2012), DAPT treatment in WT mice increased sprouting angiogenesis (Fig. 3A–E), evident by increased branch points, tip cell number and filopodia numbers. DAPT treatment in GIT1-KO mice significantly rescued defective sprouting angiogenesis (Fig. 3A–E). To prove that the rescue effect of DAPT is mediated by inhibiting Dll4 positive feedback loop, we injected the ecGIT1-KO mice with DMSO or DAPT and detected the Dll4 protein expression in retinas of ecGIT1-KO after 24h. As we anticipated, DAPT administration significantly inhibited Dll4 protein expression by ~40% compared to DMSO treated mice (Fig. S3E–F). Moreover, to exclude the effect of DAPT on GIT1 expression, we detected GIT1 protein expression in HUVEC after DAPT treatment. GIT1 protein expression was not changed by DAPT treatment, whereas Dll4 protein expression was significantly decreased (Fig. S3G–H). Together, these data suggest that the rescue effect of DAPT on GIT1-KO mice is mediated by inhibition of Dll4.

Fig. 3. Inhibiting Notch signaling using DAPT or blocking excessive Dll4 in GIT1-KO mice rescued impaired angiogenesis.

Fig. 3

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A–E. DAPT (50mg/kg body weight) was subcutaneously injected in P3 and P4 mice. At P5, all the retinas were harvested and processed for IB4 staining. Images were acquired using Olympus confocal microscope IX81 and images were analyzed for vasculature length, tip cell number, branch points and filopodias. DMSO treatment served as control. Quantifications of vessel length (B), branch points (C), tip cell density (D) and filopodia extensions (E) of vessels. Student t-test, *P< 0.05 compared with WT mice treated with DMSO; # P< 0.05 compared with GIT1-KO mice treated with DAPT (mean ±SEM; n =6). F–J. IB4 staining of IgG or Dll4 neutralizing antibody treated retinas of P5 WT and GIT1-KO mice. IgG or Dll4 neutralizing antibodies were injected in P2 mice using a standardized intraorbital injection protocol. At P5, the retinas were harvested and processed for IB4 staining. Quantifications of vessel length (G), branch points (H), tip cell density (I) and filopodia extensions (J) of vessels. Red and yellow boxed areas in first panel of images are magnified and represented for branch points (middle panel) and filopodia analysis (third panel) respectively. (A–J) Red dots in the middle panel of images shows the branching points, green dots in the third panel of images indicates filopodia structures while blue line indicates the length covered by the leading edge. Student t-test, *P< 0.05 compared with WT mice treated with IgG; # P< 0.05 compared with GIT1-KO mice treated with Dll4 neutralizing antibody (mean ±SEM; n =6).

Dll4 neutralizing antibody recovers defective sprouting angiogenesis in GIT1-KO mice

To show a specific role of Dll4 in vivo, Dll4 neutralizing antibody or hamster IgG control were injected into the vitreous of the left and right eyes, respectively, of GIT1-WT and GIT1-KO mice at P2. The eyes were harvested at P5 and the retinal vasculature was visualized by IB4 staining. Similar to DAPT treatment, administration of Dll4 neutralizing antibody in GIT1-WT mice resulted in an increase of sprouting angiogenesis. Administration of Dll4 neutralizing antibody in GIT1-KO mice restored impaired retinal vasculature development (Fig. 3F–J). To further confirm that the rescue effect of Dll4 neutralizing antibody is mediated by inhibiting Dll4 positive feedback loop, we detected Notch target genes expression (Dll4, Hey1, Hey2, Hes1 and Hes2) in retinas of GIT1-KO mice after Dll4 neutralizing antibody injection for 24h. Dll4 and Hes2 expression was significantly decreased after injection compared to IgG treated GIT1-KO mice (Fig. S3I). Hey1, Hey2, Hes-1 expression was not affected, although there was a trend of decreasing Hey2 expression, which could be due to the specificity of Dll4 on Notch target genes in vivo and the time of the treatment. All these results demonstrate an essential role of Dll4 in angiogenesis of GIT1-KO mice.

ANK domain of GIT1 plays a critical role in GIT1 mediated Dll4-Notch signaling

Notch1 stimulates Dll4 expression by binding to RBP-J via a highly conserved ANK repeat domain (Tani et al., 2001). As GIT1 also contains an ANK domain, we hypothesized that GIT1 inhibits the Notch1-Dll4 feedback loop by competing with N1-ICD for binding to RBP-J. We first studied GIT1 subcellular localization. GIT1 localized to the nucleus and cytosol in HUVECs, as shown by confocal microscopy and cell fractionation (Fig. 4A–B). Importantly, GIT1 bound to RBP-J under basal conditions in HUVECs as shown by co-IP (Fig. 4C). Furthermore, ChIP assay using RBP-J antibody demonstrated binding of RBP-J to the promoter region of Hey1 was increased upon GIT1 depletion (Fig. 4D–E, Fig. S3J). To identify the specific domains of GIT1 required for the regulation of Notch signaling, we studied the effects of several GIT1 domain deletion mutants on expression of Hey1 induced by N1-ICD. As a model system, we transfected a Hey1-luciferase reporter construct together with N1-ICD and various GIT1 mutants. HEK293 cells were used since they express almost no GIT1 (Pang et al., 2008). In the absence of GIT1 (pcDNA group), N1-ICD induced high level transcriptional activation of Hey1. Transfection of GIT1-WT decreased Hey1 luciferase activity by 50% (Fig. 4F–G). Similarly, GIT1(1-420) and GIT1(1-635) significantly decreased Hey1 luciferase activity by 65% and 66%, respectively. In contrast, GIT1(420-770) and GIT1(250-770) had no significant effect. Based on these data, domain(s) present in GIT1(1-250) are critical for GIT1 mediated N1-ICD transcriptional effects. There are two known functional domains in GIT1(1-250): the ARF-GAP domain (1-124) and the ANK repeat domain (132-228) (Fig. 4F). The ANK domain contains a motif of 33 amino acid residues and was first identified in the sequence of Notch (Drosophila)(Breeden and Nasmyth, 1987). There are several highly conserved amino acids (TPLH) in the ANK domain of both GIT1 and Notch1, and these amino acids are essential for the helix-turn-helix conformation and protein-protein interactions (Fig. S4A) (Mosavi et al., 2004; Sedgwick and Smerdon, 1999). Although a recent sequence homology analysis demonstrated 3,608 proteins containing ANK repeat domains, protein-protein interactions dependent on ANK repeats are specific with limited numbers of binding partners (Mosavi et al., 2004). To determine whether GIT1 interacts with N1-ICD through the ANK repeat domain, GIT1-WT and N1-ICD were co-expressed in HEK293 cells. We found no association following GIT1 IP (Fig. S4B). It has also been established that N1-ICD interacts with RBP-J through two domains; the ANK repeats and the RBP-J associated module (Tani et al., 2001). To study the interaction between GIT1 and RBP-J, HEK293 cells were transfected with N1-ICD, GIT1-WT and two ANK repeat mutants; GIT1-ΔANK (missing AA132-228) and GIT1-ANK (expressing AA132-228). Similar to GIT1, GIT1-ANK localized to both the cytosol and nucleus (Fig. 4H). As anticipated, GIT1-WT and GIT1-ANK strongly associated with RBP-J, while GIT1-ΔANK associated weakly with RBP-J (Fig. 4I). Expression of GIT1-ANK significantly decreased the binding of N1-ICD with RBP-J (Fig. 4J). To confirm the effect of GIT1-ANK on Notch signaling, Hey1 expression was measured after transfection of GIT1-WT and GIT1-ANK mutants. GIT-WT and GIT1-ANK significantly decreased Hey1 promoter dependent luciferase activity by 42% and 58%, whereas GIT1-ΔANK had no significant effect (Fig. 4K). These data suggest that GIT1 associates with RBP-J to prevent RBP-J binding to N1-ICD, presumably by steric hindrance (Fig. 4L).

Fig. 4. GIT1 inhibits Dll4-Notch1 signaling by competing with N1-ICD binding to RBP-J through ANK repeat domain.

Fig. 4

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A–B. HUVECs were fixed and stained using GIT1 antibody (A). Cell fractionation was performed on HUVECs (n=3). Western blot was performed with GIT1, Histone H3 (nuclear marker) and tubulin (cytosol marker) antibodies. (B). C. The association of endogenous GIT1 and RBP-J in HUVECs was assayed by IP with RBP-J antibody and probed with GIT1 (n=3). D. ChIP assay was performed using RBP-J antibody to study RBP-J binding to Hey1 promoter region in GIT1 depleted HUVEC. E. The association of Hey1 promoter with RBP-J was quantified. (n=3, Student t-test, *P< 0.05 compared with control siRNA) F–G. GIT1 inhibits Notch signaling through GIT1(1-250). F. Functional domains of GIT1. G. HEK 293 cells were co-transfected with GIT1-WT or its mutants together with Hey1 luciferase reporter gene, N1-ICD and β-galactosidase (β-gal). After transfection for 18h, the luciferase and β-gal activities were measured (mean ±SE; n=4). (Student t-test, *P< 0.05 compared with N1-ICD+pcDNA group) H. HUVECs were transfected with GFP-GIT1-ANK plasmid. After transfection for 24h, GFP positive cells were detected and nuclei were stained by Hoechst (n=3). I. HEK293 cells were transfected with GFP-GIT1-WT, GFP-GIT1-ΔANK or GFP-GIT1-ANK for 24h. IP was performed with GFP antibody and probed for RBP-J to detect the interaction of RBP-J and GFP tagged GIT1 mutants. Western blot of total cell lysis (TCL) was performed to detect the successful expression of GFP-tagged GIT1-WT and GIT1 mutants. J. HEK293 cells were transfected with pcDNA+ N1-ICD or GFP-GIT1-ANK+ N1-ICD. The interaction of RBP-J and N1-ICD was assayed by IP with RBP-J antibody and probed for N1-ICD. Right panel shows expression of N1-ICD and GFP-GIT1-ANK in TCL. K. HEK 293 cells were cotransfected with GIT1-WT or its mutants together with Hey1 luciferase reporter gene, N1-ICD and β-gal. After transfection for 18h, luciferase and β-gal activities were detected (mean ±SEM; n=4). (Student t-test, *P< 0.05 compared with N1-ICD+pcDNA group). L. The mechanistic model of GIT1 regulation on Dll4 expression.

GIT1 gain-of-function in angiogenesis in vitro

To further determine the role of GIT1 in angiogenesis, we performed gain-of-function experiment in vitro. We transfected HUVECs with control siRNA or GIT1 siRNA in the presence of lenti-vector or lenti-GIT1. As anticipated, GIT1 depletion inhibited tube formation by 50% and increased Dll4 mRNA expression by about 12 fold compared to control siRNA treated cells (Fig. S4C–F). However, overexpression of GIT1 in GIT1 siRNA treated cells significantly restored tube formation and inhibited Dll4 expression by 84% (Fig. S4C–F) compared to GIT1 siRNA group. We further confirm the critical role of Dll4 in GIT1 mediated angiogenesis in vitro (Fig. S4G–H).

DISCUSSION

The major finding of this study is that GIT1 is mainly expressed in stalk cells and retinal veins. GIT1 is an inhibitor of Dll4-Notch1 positive feedback loop in stalk cells, thereby maintaining stalk cell phenotype and vein specification, which is essential for sprouting angiogenesis. GIT1 deficiency leads to increased Dll4 expression associated with decreased tip cell formation and sprouting angiogenesis. Because the GIT1(ANK) domain binds to RBP-J, as does the ANK repeat domain of N1-ICD, we propose that under normal physiological conditions, GIT1 regulates Notch signaling via controlling Dll4 expression by competing with N1-ICD in stalk cells (Fig. 4L, Fig. S4M). GIT1 regulation of Dll4 is particularly critical for angiogenesis, because Dll4 determines tip cell and stalk cell specification, both due to its trans function (Dll4 interacts with Notch1 of neighboring cell) and its cis function (Dll4 interacts with Notch1 in the same cell and inhibits Notch signaling) (Sprinzak et al., 2010).

The cross talk between Notch and VEGF signaling has been recognized and is controversial. Several groups demonstrate that Dll4 in tip cells activates Notch1 in stalk cells to specifically suppress VEGFR3 (but not VEGFR2) expression and prevent stalk cell sprouting (Benedito et al., 2012; Hellstrom et al., 2007; Lobov et al., 2007; Phng and Gerhardt, 2009). Consistently, we found that deletion of GIT1 inhibited VEGFR3 expression, while there was no effect on VEGFR2 expression (Pang et al., 2009). In addition, Dll4 treatment enhances Flt1 expression to reduce the cell response to VEGF (Harrington et al., 2008). Similarly, we observed increased Flt1 protein and mRNA expression in GIT1 depleted cells and GIT1-KO mice retinas. These results imply that decreased VEGFR3 and increased Flt1 expression are possibly responsible for the retinal vascular phenotypes of GIT1-KO mice. In contrast, Tosato’s group shows that Dll4-Notch signaling inhibits VEGFR2 expression (Williams et al., 2006). Angiogenesis is enhanced in EC specific VEGFR3-KO mice suggesting that VEGFR3 inhibits angiogenesis. These discrepancies are possibly due to the complexity of Notch transcriptional co-activators and co-repressors. The specific target genes responsible for the vascular phenotype of GIT1-KO mice need further investigation.

A major question in the field of sprouting angiogenesis is how stalk cells can remain highly proliferative despite a low level of VEGFR2 expression (Hellstrom et al., 2007; Jakobsson et al., 2010; Suchting et al., 2007). Previously our group and others showed that GIT1 is required for sustained VEGF stimulation of PLCγ and MEK1-ERK1/2, and modulated EC migration and proliferation (Pang et al., 2009; Yin et al., 2004; Za et al., 2006). Based on our current and previous findings, we show that GIT1 functions as the key regulator of stalk cell fate determination by two mechanisms: (i) GIT1 maintains low levels of Dll4 in stalk cells by specifically inhibiting the Dll4-Notch1-Dll4 feedback loop through competing with N1-ICD to bind to RBP-J using GIT1-ANK domain (Fig. 4L, Fig. S4M) and (ii) GIT1 promotes stalk cell proliferation via VEGF- PLCγ-ERK1/2 signaling (Pang et al., 2009) (Fig. S4M).

GIT1 is a scaffold protein, which mediates various signaling pathways by interacting with different partners through different domains. Based on the current study, we believe that the effects of GIT1 on Dll4-Notch and PLCγ-MEK1-ERK1/2 are regulated by different domains, since our previous data demonstrated that GIT1 interacts with PLCγ and MEK1-ERK1/2 through the Spa homology domain (Haendeler et al., 2003; Yin et al., 2004), while the ANK repeat domain is responsible for the Dll4-Notch pathway by interacting with RBP-J. However, due to the strong rescue effects of DAPT and Dll4 neutralizing antibody on retinal vascular development, it is possible that the effect of GIT1 on PLCγ is secondary to the disruption of Notch signaling. To investigate this possibility, we treated HUVECs with Dll4 at various time points. The phosphorylation of PLCγ was analyzed and no changes were observed (Fig. S4I), implying that the GIT1-PLCγ pathway is Notch independent. On the contrary, we also overexpressed GIT1-SHD (which is responsible for PLCγ activation) in HUVECs to measure Dll4 expression, we didn’t observe significant difference of Dll4 expression between vector and GIT1-SHD groups (Fig. S4J–K). All these data imply that different domains mediate different functions of GIT1.

Moreover, the role of GIT1 is also context dependent. For example, we recently published that GIT1 deletion in bone marrow cell decreases BMP signaling (Yin et al., 2014). However, we found no changes in BMP signaling in EC (data not shown). These data suggest that GIT1 function in signal transduction differs in various cell types, probably due to binding partners. The remarkable inhibitory effect of GIT1 on Dll4-Notch signaling suggests that the regulation of GIT1 itself is likely important in angiogenesis. Based on our and other group’s findings, we believe that GIT1 regulation is time and context dependent. GIT1 protein expression is robust in retinas (Fig. S2G–H) and lungs of GIT1-WT mice at P5 but significantly decreased at 3 months suggesting developmental regulation. In Fig. 1, we show that GIT1 is largely expressed in the leading edge of sprouting vessels (stalk cell) and veins in retina at P5, but not in tip cell. In addition, we found that GIT1, Dll4 and Notch1 protein expression is lower in human aortic EC (artery) compared to HUVEC (vein) (Fig. S2F). These data support the inhibitory role of GIT1 on Dll4 expression, since Dll4 is highly expressed in tip cell and artery to control tip cell fate and artery specification. In addition, we also observed GIT1 nuclei shuttling in vivo (Fig. 1B) and GIT1 nuclei location in vitro (Fig. 4A). These observations are consistent with its functional interaction with RBP-J, but the mechanism of GIT1 nuclear localization remains unknown. We propose that there is a specific repressor in tip cell and artery that can inhibit GIT1 expression during postnatal period. Premont’s group (Schmalzigaug et al., 2007) showed GIT1 expression is restricted to blood vessels (including EC and smooth muscle cells(SMC)) in adult mice. Similarly, we published previously that GIT1 is expressed in EC and SMC of carotid artery (mature vessel, 2–3 month old) and GIT1 protein expression is increased in intima after vessel injury. Mir-149 and Mir-491-5p regulate GIT1 expression in tumor cells (Chan et al., 2014). In vitro, Angiotensin II, EGF or FGF stimulation increases GIT1 protein expression in SMC (Pang et al., 2013). In the current study in HUVEC, we only found that GIT1 protein expression was only increased by EGF stimulation (Fig. S4L). In mature vessels, GIT1 is mainly expressed in cytosol, which is important for cell proliferation, migration and adhesion through ERK1/2 and PLCγ activation. The specific regulatory mechanism of GIT1 expression at different times, in different contexts and its nuclear localization need further investigation in future.

In summary, in this present study, we demonstrate that GIT1 is an endogenous inhibitor of the Dll4-Notch1 positive feedback loop that regulates angiogenesis by controlling stalk cell fate and vein specification. Due to the important role of Notch-Dll4 feedback loop in physiological and pathological angiogenesis, inhibitors of Notch1 or Dll4 have been proposed as therapeutic targets for angiogenesis related diseases (Lobov et al., 2007). Therefore, GIT1, or more specifically the ankyrin domain could be a promising therapeutic target in diseases with high expression of GIT1 (tumors) (Chan et al., 2014) or low expression of GIT1 (bronchopulmonary dysplasia). In future, investigating the therapeutic effects of specific mimic or blocking peptides based on the sequence of the GIT1 ANK domain on angiogenesis related diseases will be very informative and important.

Supplementary Material

1

Acknowledgments

This work was supported by a grant from NIH (HL122777-01) and a Scientist Development Grant from American Heart Association to Jinjiang Pang (0835626D). This work was also supported by grants from NIH to B.C.B. (HL63462) and an unrestricted grant to the Department of Ophthalmology from the Research to Prevent Blindness.

Footnotes

Authorship

JP, SM and GZ designed experiments, performed research, analyzed data, and wrote the paper. XX, SS, CX, XW, HC, ZC, DJ, HL performed research and analyzed data, RTL contributed vital new reagents and analytical tools, JP, SM, EMS and BCB wrote and edited the paper. We are grateful to Dr. Mark Sowden for editing the manuscript and preparing reagents.

Disclosures

The patent application of GIT1 ankyrin repeat domain acting as a therapeutic target in angiogenesis related diseases were filed (patent number: 61781832).

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