RESEARCH ARTICLE

The Robo4–TRAF7 complex suppresses endothelialhyperpermeability in inflammationKeisuke Shirakura1, Ryosuke Ishiba1, Taito Kashio1, Risa Funatsu1, Toru Tanaka1, So-ichiro Fukada1,Kenji Ishimoto1, Nobumasa Hino1, Masuo Kondoh1, Yukio Ago1, Yasushi Fujio1, Kiichiro Yano2,Takefumi Doi1,*, William C. Aird2 and Yoshiaki Okada1,*

ABSTRACTRoundabout guidance receptor 4 (Robo4) is an endothelial cell-specific receptor that stabilizes the vasculature in pathologicalangiogenesis. Although Robo4 has been shown to suppressvascular hyperpermeability induced by vascular endothelial growthfactor (VEGF) in angiogenesis, the role of Robo4 in inflammation ispoorly understood. In this study, we investigated the role of Robo4 invascular hyperpermeability during inflammation. Endotoxemiamodels using Robo4−/− mice showed increased mortality andvascular leakage. In endothelial cells, Robo4 suppressed tumornecrosis factor α (TNFα)-induced hyperpermeability by stabilizingVE-cadherin at cell junctions, and deletion assays revealed that theC-terminus of Robo4 was involved in this suppression. Throughbinding and localization assays, we demonstrated that in endothelialcells, Robo4 binds to TNF receptor-associated factor 7 (TRAF7)through interaction with the C-terminus of Robo4. Gain- and loss-of-function studies of TRAF7 with or without Robo4 expression showedthat TRAF7 is required for Robo4-mediated suppression ofhyperpermeability. Taken together, our results demonstrate that theRobo4–TRAF7 complex is a novel negative regulator of inflammatoryhyperpermeability. We propose this complex as a potential futuretarget for protection against inflammatory diseases.

KEYWORDS: Endothelial permeability, Inflammation, TRAF7, Robo4

INTRODUCTIONBlood vessels function as a barrier to separate the blood from organtissues. Endothelial cells (ECs) on the inner surface of thevasculature play essential roles in the maintenance and regulationof this barrier function (Mehta andMalik, 2006;Weber et al., 2007).Endothelial permeability is regulated by various molecules such asvascular endothelial growth factor (VEGF), lipopolysaccharide(LPS) and tumor necrosis factor α (TNFα). These factors alter thecytoskeleton and cell adhesion molecules in ECs and regulateangiogenesis and immune cell trafficking to maintain vascularhomeostasis. However, excessive inflammatory cytokines ininflammatory diseases induce extreme vascular permeability andlethal symptoms such as septic shock and pulmonary edema(Madge and Pober, 2001; Zhu et al., 2012).

Roundabout guidance receptor 4 (also known as Roundabouthomolog 4, Robo4) is an endothelial-specific receptor that islocalized in both the plasma membrane and the cytoplasm(Huminiecki et al., 2002; Okada et al., 2014, 2008, 2007; Parket al., 2003; Sheldon et al., 2009; Zhang et al., 2016). Previousstudies using Robo4-knockout mice have shown that Robo4 is notessential for developmental vasculogenesis and angiogenesis, butsuppresses pathological angiogenesis (Jones et al., 2008; Kochet al., 2011; Zhang et al., 2016). In pathological angiogenesis,Robo4 and its binding proteins suppress VEGF-induced vascularpermeability by attenuating VEGF signaling. The N-terminaldomain of Robo4 interacts with Unc-5 netrin receptor B (Unc5B)and Slit guidance ligand 2 (Slit2). The binding of Robo4 to thetransmembrane receptor Unc5B induces Unc5B downstreamsignaling, which results in suppression of VEGF receptor 2(VEGFR2, also known as KDR) phosphorylation, and of vascularhyperpermeability (Koch et al., 2011; Suchting et al., 2005; Zhanget al., 2016). The binding of Robo4 to Slit2 also suppresses VEGF-induced cell migration by modulating the functions of Robo1 andGTPases, including Rac1 and Cdc42 (Enomoto et al., 2016; Joneset al., 2008; Kaur et al., 2006; Sheldon et al., 2009). Further, theC-terminal domain of Robo4 regulates cell migration by interactingwith the focal adhesion-associated protein paxillin and thecytoskeleton-regulating protein enabled homolog (Enah) andWiskott-Aldrich syndrome protein (Jones et al., 2009; Park et al.,2003; Sheldon et al., 2009).

Although the relationship between Robo4 andVEGF signaling hasbeen well studied, Robo4 functions in inflammation are not wellunderstood. A previous study suggested a potential role of Robo4 ininflammation by showing that Slit2 suppresses vascular permeabilityin mouse sepsis and influenza infectionmodels (London et al., 2010).This study provided an important insight that Robo4 could be apotential therapeutic target in infectious and inflammatory diseasesrelated to vascular hyperpermeability. However, the study wasperformed following administration of exogenous Slit2, which hasbeen shown to regulate endothelial cell (EC) function in a Robo4-independent pathway (Rama et al., 2015). Thus, to clearly elucidatethe physiological roles of Robo4 in inflammation, Robo4 needs to beevaluated in inflammatory models without exogenous Slit2.

In this study, we analyzed Robo4 functions in inflammation usingin vivo and in vitro inflammatory models. We successfullydemonstrated that Robo4 decreased vascular leakage and improvedsurvival in endotoxemic mice. Moreover, Robo4 suppressedendothelial hyperpermeability in inflammation through cooperationwith a newly identified Robo4-binding protein, TNF receptor-associated factor 7 (TRAF7). Our findings indicate a new regulatorymechanism for inflammatory hyperpermeability via the Robo4–TRAF7 complex and suggest that this complex could be used as atherapeutic target for the treatment of inflammatory diseases.Received 7 May 2018; Accepted 28 November 2018

1Graduate School of Pharmaceutical Sciences, Osaka University, Osaka,565-0781, Japan. 2The Center for Vascular Biology Research and Division ofMolecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston,MA 02215, USA.

*Authors for correspondence (okadabos@phs.osaka-u.ac.jp;doi@phs.osaka-u.ac.jp)

K.S., 0000-0002-9487-9495; Y.O., 0000-0002-2962-244X

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RESULTSRobo4 depletion enhanced vascular permeability inendotoxemic miceTo analyze the function of Robo4, Robo4−/− mice were generated byreplacing exons 2 and 3 with a neomycin resistance cassette (Fig. 1A).Generation of Robo4−/− mice was confirmed using PCR genotyping(Fig. 1B). Robo4−/−mice showed no obvious pathological phenotypeandwere able to breed. In endotoxemiamodel mice,Robo4 deficiencywas associated with significantly lower survival compared withRobo4+/+ mice (Fig. 1C). To investigate the mechanism modulatingthis lower survival rate accompanying Robo4 deficiency, the vascularpermeability of Robo4−/− and Robo4+/+ mice was analyzed withEvans blue dye (Fig. 1D). Compared with Robo4+/+ mice, Robo4−/−

mice injected with PBS showed increased extravasation of the dye inorgans. A significant increase was observed in the lungs, in whichRobo4 is highly expressed (Okada et al., 2007; Park et al., 2003).

Similarly, Robo4−/− mice injected with LPS showed increasedextravasation of the dye compared to Robo4+/+ mice injected withLPS. In particular, significant increases in extravasation wereobserved in the heart, lungs and small intestine. Taken together,these results indicate that Robo4 depletion decreases the survivalrates of mice with endotoxemia, possibly by increasing vascularpermeability in the heart, lungs and small intestine. This suggeststhat Robo4 suppresses inflammatory mediator-induced vascularhyperpermeability.

Robo4 attenuated endothelial hyperpermeability induced byTNFαTo investigate whether Robo4 regulated endothelialhyperpermeability induced by inflammatory mediators, weemployed an in vitro model using human umbilical vein endothelialcells (HUVECs) and TNFα, which is known to induce vascular

Fig. 1. Depletion of Robo4 decreased survival and enhanced vascular leakage in endotoxemia model mice. (A) Schematic representation of thewild-type Robo4 allele, the targeting vector, and the targeted Robo4 allele. Shaded boxes indicate exons 1–12 of the Robo4 gene. Neor, FRP, HSV-TK promoter,and DT-A denote neomycin-resistance, short flippase recognition target, Herpes simplex virus-thymidine kinase and diphtheria toxin genes, respectively.(B) Genomic PCR analysis of Robo4+/+ (+/+), Robo4+/− (+/−), and Robo4−/− (−/−) mice. DNA fragments amplified from the wild-type and targeted Robo4 alleleswere detected. (C) Survival curves for Robo4+/+ (n=10), Robo4+/− (n=10), and Robo4−/− (n=10) littermates injected intraperitoneally with LPS (16.5 mg/kg).P-values were determined by Mantel–Cox’s tests (versus Robo4+/+). (D) Vascular leakage in Robo4+/+ and Robo4−/− mice in the endotoxemia model. Micewere intraperitoneally injected with LPS or PBS. Six hours later, the mice were intravenously injected with Evans blue dye, and extravasated dye was quantifiedby measuring the OD620. Data are expressed as mean±s.e.m. (n=6). *P<0.05, **P<0.01 by ANOVA followed by Tukey–Kramer’s test for vascular leakage.

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hyperpermeability in sepsis (McKenzie andRidley, 2007;Mong et al.,2007; Qiu et al., 2011). We first examined the effects of Robo4knockdown on TNFα-induced hyperpermeability. HUVECmonolayers transfected with control siRNA (siCont) or siRNAagainst Robo4 (siRobo4) were treated with TNFα, and endothelialpermeability was analyzed using measuring transendothelial electricalresistance (TEER; Fig. 2A). TEER was observed to increaseimmediately after TNFα treatment in HUVECs transfected withboth siCont and siRobo4. This increase could be caused by TNFα-induced upregulation of cAMP and S1P, as suggested in previousreports (Pober et al., 1993; Xia et al., 1998). Transfection withsiRobo4 induced significantly lower TEER than siCont. In addition,transfection with siRobo4 did not affect EC viability in the WST-8assay (Fig. S1A). These results indicate that Robo4 knockdownincreases endothelial permeability without affecting cell viability.We next investigated the effects of Robo4 overexpression on

endothelial permeability. HUVEC monolayers infected withadenoviral vectors encoding AcGFP as a control (Ad-Cont) orRobo4 (Ad-Robo4) were stimulated with TNFα, and TEER wasmeasured (Fig. 2B; Fig. S2A). Ad-Robo4 significantly suppressedthe decrease in TEER induced by TNFα compared with Ad-Cont,indicating that Robo4 overexpression suppressed endothelialpermeability. In addition, Ad-Robo4 completely cancelled theenhanced hyperpermeability mediated by siRobo4 (Fig. 2C). Takentogether, these results indicate that Robo4 suppresses TNFα-induced endothelial hyperpermeability.

Robo4 increased expression of VE-cadherin localized at ECjunctionsTo investigate the mechanisms through which Robo4 suppressedTNFα-induced hyperpermeability, the subcellular localization andexpression levels of the adherens junction protein VE-cadherin(also known as CDH5) were analyzed. VE-cadherin is normallylocalized at junctions between ECs, but redistributed upon TNFαstimulation to increase permeability (Nwariaku et al., 2002). Onobservation of immunofluorescence staining using HUVECstransfected with siCont, VE-cadherin was shown to be located atjunctions, and TNFα treatment led to redistribution of VE-cadherin from junctions (Fig. 2D). Transfection with siRobo4decreased VE-cadherin localization at junctions between ECs,both in HUVECs treated with or without TNFα. In addition,treatment with siRobo4 did not alter total and cell surface levels ofVE-cadherin in HUVECs, but slightly decreased the totalVE-cadherin level in HUVECs treated with TNFα (Fig. 2F;Fig. S3). In contrast, transfection with Ad-Robo4 increased theamount of VE-cadherin localized at junctions and suppressedTNFα-induced redistribution of VE-cadherin in HUVECscompared with cells treated with Ad-Cont (Fig. 2E). These resultsindicate that Robo4 suppresses endothelial hyperpermeability bystabilizing VE-cadherin at junctions.Since destabilization of VE-cadherin has been reported to induce

extravasation of immune cells (Corada et al., 1999; Gotsch et al.,1997), we investigated whether Robo4 regulates immune celltrafficking. Transfection with siRobo4 significantly increasedtransmigration of monocytic U937 cells through a HUVEC layertreated with TNFα (Fig. 2G). In contrast, siRobo4 transfection led todecreased expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin by decreasing the TNFα-induced nuclearlocalization of the NF-κB p65 (also known as RELA) and p50(also known as NFKB1) subunits (Fig. S4). These results suggestthat Robo4 suppresses extravasation of immune cells by stabilizingVE-cadherin at junctions without inhibiting the NF-κB pathway.

The C-terminal domain of Robo4 regulates endothelialpermeabilityTo investigate the essential domain of Robo4 for the inhibition ofhyperpermeability and redistribution of VE-cadherin, we preparedRobo4 mutants lacking N- or C-terminal domains (Fig. 5A;Fig. S2B,C). Adenoviral expression of the Robo4 mutant lackingthe N-terminal domain (ΔN28–447), as well as wild-type Robo4,inhibited TNFα-induced hyperpermeability and redistribution ofVE-cadherin (Fig. 3A,C). In contrast, both of the Robo4 mutantslacking the C-terminal domain (ΔC785–1007 and ΔC570–1007)failed to inhibit hyperpermeability and redistribution of VE-cadherin (Fig. 3B,D). Taken together, these results indicate thatthe C-terminal, but not the N-terminal, domain of Robo4 is essentialfor inhibition of TNFα-induced endothelial hyperpermeability andredistribution of VE-cadherin.

TRAF7 is a novel Robo4-interacting protein in ECsTo investigate how Robo4 regulates endothelial permeability via itsC-terminal domain, Robo4-interacting proteins were purified fromHUVECs infected with Ad-Cont or Ad-Robo4-FLAG by means ofimmunoprecipitation using anti-FLAG antibodies. The precipitatedproteins were enzymatically digested, and the resulting peptideswere analyzed using mass spectrometry. These data identifiedTRAF7 as a novel Robo4-binding protein (Fig. 4A). The interactionbetween Robo4 and TRAF7 was confirmed through co-immunoprecipitation using COS-7 cells (Fig. 4B). Taken together,these results indicated that Robo4 interacts with TRAF7 in ECs.

The C-terminal domain of Robo4 interacts with TRAF7 andmodulates its localizationTo identify the Robo4 domain that interacts with TRAF7, co-immunoprecipitation assays were performed with Robo4 mutants(Fig. 5A,B). Robo4 mutants lacking the N-terminal domain (ΔN28–228 and ΔN28–447) showed similar or strong binding to TRAF7compared with wild-type Robo4. In contrast, Robo4 mutantslacking the C-terminal domain (ΔC785–1007, ΔC711–1007, andΔC570–1007) showed weaker binding to TRAF7 than wild-typeRobo4; ΔC570–1007 completely lost its binding affinity forTRAF7. These results indicate that Robo4 interacts with TRAF7via its C-terminal domain.

We next investigated the effects of the interaction between Robo4and TRAF7 on the subcellular localization of each protein inHUVECs using immunofluorescence staining (Fig. 5C). Robo4–FLAG was localized in the cytoplasm, particularly around thenucleus. Additional expression of TRAF7–myc did not alter thislocalization. In contrast, the additional expression of Robo4–FLAGshifted localization of TRAF7–myc from cytoplasm to theperinuclear region (Fig. 5D) and caused TRAF7–myc tocolocalize with Robo4–FLAG (Fig. 5C). This Robo4-mediatedalteration of TRAF7 localization was not observed with Robo4mutants lacking the C-terminal domain. Similar results wereobtained in HUVECs treated with TNFα (Fig. S5). Takentogether, these findings indicate that Robo4 interacts with TRAF7via the C-terminal domain and regulates TRAF7 localization,further suggesting that Robo4 modulates TRAF7 function byregulating its localization.

TRAF7 suppressed TNFα-induced hyperpermeabilityand VE-cadherin redistributionTo investigate TRAF7 functions in ECs, we analyzed the effects ofTRAF7 knockdown on endothelial permeability using siRNAagainst TRAF7 (siTRAF7). siTRAF7 transfection resulted in a

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significantly lower TEER in HUVECs than siCont transfection,both before and after TNFα stimulation (Fig. 6A). In contrast,infection with an adenoviral vector encoding TRAF–myc

(Ad-TRAF7-myc) suppressed the TNFα-induced decrease inTEER (Fig. 6B). In addition, treatment with siTRAF7, unlikewith siRobo4, reduced cell viability in the WST-8 assay (Fig. S1B).

Fig. 2. Robo4 suppressed TNFα-induced endothelial hyperpermeability by modulating VE-cadherin localization. (A–C) Effects of Robo4 down- andupregulation on endothelial hyperpermeability induced by TNFα. HUVECswere transfected with siRNA (siCont or siRobo4) (A) or infected with adenoviral vectors(Ad-Cont or Ad-Robo4) (B), treated with TNFα, and used for measurement of transendothelial electric resistance (TEER). Data are expressed as mean±s.e.m.(n=6 or 8). *P<0.05, **P<0.01 by two-sided Student’s t-tests. (C) HUVECs were treated with siRNA (siCont or siRobo4) and adenoviral vectors (Ad-Contor Ad-Robo4), stimulatedwith TNFα, and used for measurement of TEER. Data are expressed asmean±s.e.m. (n=6). *P<0.05, **P<0.01 versus siCont+Ad-Cont,by two-sided Dunnett’s test. (D,E) VE-cadherin (red) localization in siRNA- or adenoviral vector-treated HUVECs stimulated with or without TNFα for 24 h.DAPI, blue. The images are representative of three independent experiments. Scale bars: 25 µm. Graphs show the mean±s.e.m. fluorescence intensity ratio ofnon-junctional to junctional VE-cadherin (n≥50). *P<0.05, **P<0.01 by Kruskal–Wallis test. (F) VE-cadherin expression in siRNA-transfected HUVECstreated with or without TNFα for 24 h. Graph shows mean±s.e.m. relative immunoblot band intensity of VE-cadherin, GAPDH and Robo4 (n=3). *P<0.05 byTukey–Kramer test. (G) Effects of Robo4 on TNFα-induced transmigration of monocytic U937 cells. Mean±s.e.m. number of monocytic U937 cells transmigratingthrough HUVECs transfected with siRNA and stimulated with TNFα (n=10). **P<0.01 by Mann–Whitney U-test.

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These results indicate that TRAF7 suppresses TNFα-inducedendothelial hyperpermeability and that it is important for ECviability.

We next analyzed whether TRAF7 affects the localization and theprotein expression of VE-cadherin. Through immunofluorescencestaining, we observed that siTRAF7 decreased VE-cadherin

Fig. 3. See next page for legend.

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localization at cell junctions and induced intercellular gaps betweenHUVECs before and after TNFα stimulation (Fig. 6C). In contrast,Ad-TRAF7-myc treatment increased VE-cadherin localization atjunctions, particularly in HUVECs treated with TNFα (Fig. 6D). Inaddition, siTRAF7 transfection reduced protein expression of VE-cadherin in HUVECs treated with or without TNFα (Fig. 6E). Theseresults indicate that TRAF7 stabilizes VE-cadherin expressionand its localization at junctions, and suppresses endothelialhyperpermeability. Furthermore, siTRAF7 transfection decreasedexpression of ICAM-1 and E-selectin by decreasing TNFα-inducednuclear localization of the NF-κB p65 and p50 subunits(Fig. S4). This suggested that TRAF7 suppresses endothelialhyperpermeability without inhibiting NF-κB pathway.

Robo4 regulates endothelial permeability by modulatingTRAF7 functionThe functional analyses of Robo4 and TRAF7 indicated that bothproteins similarly suppress endothelial permeability, suggesting thatthis function stems from the Robo4–TRAF7 complex. To determinewhich of these factors is a major regulator of endothelial permeability,we investigated the effects of Robo4 overexpression on TNFα-inducedhyperpermeability with or without TRAF7 knockdown in HUVECs(Fig. 6F). The Ad-Robo4-mediated suppression of the TNFα-induceddecrease in TEER was observed after transfection with siCont but notwith siTRAF7. This indicates that TRAF7 is essential for Robo4function.We next investigated the effects of TRAF7 overexpression on

hyperpermeability with or without Robo4 knockdown. Interestingly,the Ad-TRAF7-myc-mediated suppression of the TNFα-induceddecrease in TEERwas observed after transfectionwith both siCont andsiRobo4 (Fig. 6G). This suggests that Robo4 is not essential forTRAF7 function. Taken together, these results suggest that TRAF7 is amajor regulator of endothelial permeability and that Robo4 functionsas a modulator for enhancing TRAF7 function.

DISCUSSIONIn the present study, we demonstrated that Robo4 suppresses vascularhyperpermeability and improves the survival of endotoxemic micewithout administration of exogenous Slit2. We further identifiedTRAF7 as a novel binding protein of the Robo4 C-terminal domainand demonstrated that Robo4 suppresses TNFα-inducedhyperpermeability by modulating TRAF7 function, therebyregulating VE-cadherin localization and protein expression. Thus,our findings support a novel Robo4-mediated regulatory mechanismof endothelial hyperpermeability in inflammation (Fig. 7).

In a Miles assay, we employed the endotoxemia model usingRobo4-knockout mice and observed a significant increase invascular leakage in the heart, lung and small intestine. Since weand others have previously reported that Robo4 is highly expressedin lung and heart vasculature (Okada et al., 2007, 2008; Park et al.,2003), Robo4 depletion in these organs may strongly affect vascularpermeability. This also supports an organ-specific function forRobo4 in inflammation, and vascular heterogeneity dependent onRobo4. Based on our Miles assay results, we speculated that Robo4suppresses some inflammatory signaling that induces vascularpermeability. Since TNFα, one of the major inflammatorymediators, is known to be expressed at the early stage ofendotoxemia and to induce vascular leakage (Ashkenazi et al.,1991; Norman et al., 1996; Tracey et al., 1987; Walsh et al., 1992),we focused on TNFα and successfully demonstrated suppression ofTNFα signaling by the Robo4–TRAF7 complex. However, it is stillpossible that Robo4 regulates hyperpermeability and inflammatoryresponses induced by other inflammatory mediators, as we haveshown previously (Shirakura et al., 2018).

Previous reports have demonstrated that Robo4 regulates VEGF-induced vascular permeability through the N-terminal domain via

Fig. 3. The C-terminal domain of Robo4 was essential for suppressingTNFα-induced endothelial hyperpermeability. (A,B) Effects of Robo4lacking the N-terminal (A) or C-terminal (B) domains on endothelialhyperpermeability induced by TNFα. HUVECs were infected with adenoviralvectors (Ad-Cont, Ad-Robo4-FLAG, Ad-ΔN28–447, Ad-ΔC785–1007, and Ad-ΔC570–1007), treated with TNFα, and used for TEER measurement. Data areexpressed as mean±s.e.m. (n=8 or 10). *P<0.05, **P<0.01 versus Robo4-FLAG byANOVA followed by two-sided Dunnett’s test. (C,D) Effect of wild-typeandmutant Robo4 lacking the N-terminal (C) or C-terminal (D) domains on VE-cadherin (red) localization in HUVECs treated with or without TNFα for 24 h.DAPI, blue. The images are representative of four independent experiments.Scale bars: 25 µm. Graphs show the mean±s.e.m. fluorescence intensity ratioof non-junctional to junctional VE-cadherin (n≥50). *P<0.05; **P<0.01; N.S.,not significant versus Ad-Cont+TNFα by Kruskal–Wallis test.

Fig. 4. Identification of the novel Robo4-binding protein TRAF7. (A) Peptides derived from the Robo4-binding protein TRAF7 identified using FLAGimmunoprecipitation. The 15 identified TRAF7 (NCBI Reference Sequence: NP_115647.2) peptides specifically included in the Robo4–FLAG sample are shownin gray. (B) Immunoblot results following a co-immunoprecipitation assay using COS-7 cells transfected with Robo4–FLAG and TRAF7–myc.

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two mechanisms. In the Robo4-Slit2 pathway, Slit2 binds to theN-terminal domain of Robo4 and induces downstreamsignaling, which suppresses VEGF-induced hyperpermeability inpathological angiogenesis (Jones et al., 2008). In the Robo4-Unc5Bpathway, the N-terminal domain of Robo4 binds to the netrinreceptor Unc5B and suppresses VEGF receptor 2 activation andvascular permeability (Koch et al., 2011; Zhang et al., 2016). Incontrast, the Robo4–TRAF7 complex, identified in this study,represents a new model of inflammatory endothelial permeabilityregulation. In this model, the C-terminal domain of Robo4, but notthe N-terminal domain, is necessary for the suppression ofendothelial hyperpermeability in inflammation.The C-terminal domain of Robo4 was found to bind TRAF7.

TRAF7 belongs to the TRAF family of proteins (TRAF1–TRAF7),which regulate inflammation and apoptosis (Bouwmeester et al.,2004; Xu et al., 2004; Yoshida et al., 2005; Zotti et al., 2011).Unlike other TRAF family members, TRAF7 does not contain aTRAF-C domain, which interacts with TNF receptors, but insteadcontains a uniqueWD40 domain (Xie, 2013). Although TRAF7 has

been shown to interact with mitogen-activated protein kinase kinasekinase 3 (MAP3K3), modulate its activity, and regulate TNFαsignaling in cell line-based assays (Bouwmeester et al., 2004; Xuet al., 2004), TRAF7 function in ECs is unclear. In this study, for thefirst time, we demonstrate that TRAF7 regulates endothelialpermeability in inflammation. Surprisingly, our results indicatethat TRAF7 is essential for Robo4-mediated suppression ofhyperpermeability, but not vice versa, suggesting that Robo4functions as an upstream modulator of TRAF7 to regulateendothelial permeability in ECs.

One possible mechanism through which Robo4 modulatesTRAF7 is via regulation of TRAF7 localization. Robo4 has beenshown to be localized in the cell membrane and cytoplasm, and toshuttle between these compartments (Sheldon et al., 2009; Zhanget al., 2016). Consistent with previous reports, we observed Robo4in the perinuclear region, whereas TRAF7 was observed incytoplasm. Co-expression of Robo4 and TRAF7 altered thelocalization of TRAF7 but not of Robo4, and Robo4 and TRAF7almost completely colocalized. Interestingly, Robo4 mutants

Fig. 5. Robo4modulated the subcellular localization of TRAF7 via theC-terminal domain. (A) Schematic illustration of FLAG-tagged wild-type and truncatedRobo4. Membrane localization of signal sequence (SS), Ig-like domain (Ig), fibronectin type III domain (FNIII), transmembrane domain (TM) and conservedcytoplasmic motifs (CC0 and CC2) are indicated. (B) Immunoblot results following co-immunoprecipitation assays in COS-7 cells transfected with TRAF7–mycand Robo4 mutants lacking the C-terminal domain (ΔC785–1007, ΔC711–1007, and ΔC570–1007) or N-terminal domain (ΔN28–447 and ΔN28–228).Graph shows mean±s.e.m. relative band intensity of immunoprecipitated TRAF7 (n=3). *P<0.05, **P<0.01 versus Robo4–FLAG by ANOVA, followed byDunnett’s test. (C) TRAF7 localization under co-expression with wild-type or mutant Robo4 in HUVECs infected with adenoviral vectors to express Robo4–FLAG(red) or its deletion mutants (ΔC785–1007 and ΔC570–1007) with or without TRAF7–myc (green). DAPI, blue. Scale bars: 25 µm. (D) Cytoplasmic distribution ofTRAF7 quantified as mean±s.e.m. relative TRAF7 fluorescence intensity versus distance from the nucleus (n=50).

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Fig. 6. See next page for legend.

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lacking the C-terminal domain, which did not suppresspermeability, also did not alter TRAF7 localization. Theseresults suggest that Robo4 modulates TRAF7 localization andfunction by interacting with TRAF7 via the Robo4 C-terminaldomain. Interestingly, we have previously reported that TNFαinduces Robo4 expression via NF-κB signaling. This suggests thatRobo4 induced by TNFα increases the formation of Robo–TRAF7complex molecules in ECs and suppresses hyperpermeability, thusserving as a negative feedback mechanism. To test this hypothesis,further detailed study to elucidate the localization and interactionof endogenous Robo4 and TRAF7 would be needed.In conclusion, our study successfully demonstrated that

Robo4 suppresses inflammatory endothelial hyperpermeabilityvia the Robo4 C-terminal domain and its binding protein TRAF7.The Robo4–TRAF7 complex could thus serve as a noveltherapeutic target in inflammatory diseases related to vascularhyperpermeability.

MATERIALS AND METHODSGeneration of Robo4−/− miceThe targeting vector was prepared through BAC recombination-mediatedgenetic engineering using the Escherichia coli strain EL350 (Lee et al.,2001). Briefly, the mouse Robo4 gene (extending from base pair −2560 toexon 11) was inserted into the vector. A neomycin resistance cassetteflanked by two loxP sites was inserted between exons 1 and 2 of theresulting plasmid, and the cassette was then removed using Cre recombinasein EL350. Another neomycin resistance cassette flanked by a loxP site andtwo FRT sites was inserted between exons 3 and 4 of the resulting plasmid,and exons 2 and 3, flanked by loxP sites, were removed using Crerecombinase. The resulting targeting vector was transfected into embryonicstem cells derived from 129/Sv mice using electroporation, and successfullytransfected cells were selected through resistance to G418 selectionantibiotic. Surviving embryonic stem colonies were picked and processedfor genomic DNA extraction and Southern blot analysis. Correctly targetedembryonic stem clones were used to generate Robo4−/− mice. Theestablished mice were backcrossed with C57BL/6N mice for more than10 generations. Genotyping polymerase chain reaction (PCR) wasperformed using mouse tail DNA and specific primers (Table S1).

Studies for survival and permeability using a mouseendotoxemia modelFor analysis of survival, Robo4−/−, Robo4+/−, and Robo4+/+ malelittermates (8–10 weeks old) were used to establish an endotoxemiamodel, as previously described (Yano et al., 2006). Survival was assessedover 96 h in Robo4−/− and Robo4+/+ mice that received an intraperitonealinjection of LPS (16.5 mg/kg body weight; Sigma-Aldrich). Forpermeability assays, Robo4−/− and Robo4+/+ littermates (8–12 weeks old)were intraperitoneally injected with LPS (16.5 mg/kg body weight). Sixhours later, the mice were intravenously administered 100 µl of 1% Evansblue dye in phosphate-buffered saline (PBS). One hour later, the mice wereperfused with PBS containing 2 mM ethylenediaminetetraacetic acid(EDTA) during anesthesia with isoflurane, and organs were harvested.Evans blue dye was eluted by mincing and incubating the organs informamide for 2 days. The eluted dye was quantified by measuring theoptical density at 620 nm. All the animal studies were approved by the ethicscommittee of Osaka University.

Cell cultureHuman umbilical vein endothelial cells (HUVECs; Lonza) were cultured inEGM-2-MV medium (Lonza). Human embryonic kidney (HEK293) cellsand African green monkey SV40-transfected kidney fibroblasts (COS-7cells; ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modifiedEagle’s medium (Nacalai Tesque) supplemented with 10% fetal bovineserum (FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin. MonocyticU937 cells were cultured in RPMI 1640 (Sigma-Aldrich) containing 10%FBS and 2% penicillin-streptomycin. All cells were cultured at 37°C in anatmosphere containing 5% CO2. All cells were free of mycoplasmacontamination.

Small interfering RNA (siRNA)-mediated gene knockdownsiRNA against Robo4 (SI03066896) and its control (AllStars NegativeControl) were purchased from Qiagen. siRNA against TRAF7 (10620319)and its control (Stealth RNAi siRNA Negative Control, Med GC) werepurchased from Invitrogen. Each siRNA was transfected usingLipofectamine RNAiMAX (Invitrogen).

Preparation of expression vectors and adenoviral vectorsThe DNA fragments encoding Robo4 and TRAF7 with or without peptidetags (FLAG and Myc) were amplified by PCR using HUVEC cDNA andspecific primers (Table S1) and inserted into pcDNA3 (Invitrogen) or theadenoviral shuttle vector pHMEF5 (Kawabata et al., 2005). The DNAfragments for Robo4 deletionmutants were prepared by PCR from pHMEF5-Robo4-FLAG using specific primers (Table S1). Each shuttle vector wasdigested, and the expression cassette was purified and inserted into theparental adenoviral vector pAdHM4. The resulting plasmids were linearized

Fig. 6. Robo4 suppressed TNFα-induced endothelial hyperpermeabilityin a TRAF7-dependent manner. (A,B) Effects of TRAF7 down- andupregulation on endothelial hyperpermeability induced by TNFα. HUVECswere transfected with siRNA (siCont or siTRAF7) (A) or infected withadenoviral vectors (Ad-Cont or Ad-TRAF7-myc) (B), treated with TNFα, andused for TEER measurement. Data are expressed as mean±s.e.m. (n=8).*P<0.05; **P<0.01 versus Robo4-FLAG by two-sided Student’s t-tests.(C,D) VE-cadherin (red) localization in siRNA-treated (C) or adenoviral vector-treated (D) HUVECs stimulated with or without TNFα for 24 h. DAPI, blue. Theimages are representative of three independent experiments. Scale bars:25 µm. Graphs show the mean±s.e.m. fluorescence intensity ratio of non-junctional to junctional VE-cadherin (n≥50). *P<0.05, **P<0.01 by Kruskal–Wallis test. (E) VE-cadherin, GAPDH, and Robo4 expression in siRNA-transfected HUVECs treated with or without TNFα for 24 h. Graph showsmean±s.e.m. relative VE-cadherin immunoblot band intensity (n=4). *P<0.05;**P<0.01 by Tukey-Kramer’s test. (F) Effects of TRAF7 downregulation onRobo4-mediated suppression of hyperpermeability. HUVECswere transfectedwith siRNA (siCont or siTRAF7) and infected with adenoviral vectors (Ad-Contor Ad-Robo4), treated with TNFα, and used for TEER measurement.(G) Effects of Robo4 downregulation on TRAF7-mediated suppression ofhyperpermeability. HUVECs were transfected with siRNA (siCont or siRobo4)and infected with adenoviral vectors (Ad-Cont or Ad-TRAF7), treated withTNFα, and used for TEER measurement. (F,G) Data are expressed asmean±s.e.m. (n=8). *P<0.05, **P<0.01 versus siCont/Ad-Cont by ANOVA,followed by Dunnett’s test.

Fig. 7. Schematic illustration of the Robo4–TRAF7 pathway suppressingTNFα-induced endothelial hyperpermeability. The C-terminal domain ofRobo4 interacts with TRAF7 at perinuclear region. The Robo4–TRAF7complex suppresses TNFα-induced vascular hyperpermeability by inhibitingVE-cadherin redistribution in a Robo4 ligand-independent manner.

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and transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen).Amplified adenoviral vectors were purified by centrifugation on a CsCl2gradient and quantified as described previously (Maizel et al., 1968).

Measurement of TEER in HUVECsHUVECs (4×104 cells) were seeded onto cell culture inserts with pore sizeof 0.4 µm (BD Falcon), treated with siRNAs (2.5 pmol) and/or adenoviralvectors (3000–10,000 virus particles/cell), and incubated for 48 h. TNFα(400 ng;Wako Pure Chemicals) was added to the upper chamber, and TEERwas measured using a Millicell ERS-2 Voltohmmeter (Merck Millipore).The TEER value was calculated by the following formula: (resistance ofexperimental wells−resistance of blank wells)×0.32 (the membrane area ofthe cell culture insert), according to the manufacturer’s instructions.

Western blottingHUVECs (8×105 cells) were transfected with siRNA (50 pmol) or adenoviralvectors (3000–10,000 virus particles/cell) and incubated for 48 h. The resultingcells were treated with TNFα for 24 h, and total cell lysates were extracted. Forthe detection of NF-κB, HUVECs were treated with TNFα for 30 or 60 min,and nuclear extract was prepared using the Qproteome Cell Compartment Kit(Qiagen). Western blotting was performed with the lysates and antibodiesagainst Robo4 (N-17, Santa Cruz Biotechnology, 1:100; or AF2366, R&Dsystems, 1:500), TRAF7 (H-300, Santa Cruz Biotechnology, 1:2000), FLAG(F1804, Sigma-Aldrich, 1:1000), VE-Cadherin (F-8, Santa CruzBiotechnology, 1:2000), Myc (9B11, Cell Signaling Technology, 1:1000),NF-κBp65 (C-20: Santa Cruz Biotechnology, 1:2000), NF-κBp50 (H-119,Santa Cruz Biotechnology, 1:2000), LaminB2 (LN43, Abcam, 1:2000) andGAPDH (MAB374, Merck Millipore, 1:10,000) and secondary antibodiesconjugated to horseradish peroxidase (Jackson ImmunoResearch LaboratoriesInc., PA). The resulting data were quantified by measuring intensities of thebands using ImageJ/Fiji software (Schindelin et al., 2012).

Flow cytometryHUVECs (8×105 cells) were transfected with siRNA (50 pmol) andincubated for 48 h. The resulting cells were treated with TNFα for 24 h, andharvested using Hanks’ Balanced Salt Solution containing 5 mM EDTA.The cells were incubated with R-Phycoerythrin (PE)-conjugated mouseanti-human VE-cadherin (16B1, Thermo Fisher Scientific, 1:40) orPE-conjugated mouse IgG1 (12-4714-81, Thermo Fisher Scientific, 1:40)for 30 min on ice and then washed with PBS containing 2% FBS.Fluorescence intensity of the cells was analyzed using a flow cytometer(FACSCalibur, Becton-Dickinson).

Transmigration assayHUVECs (4×104 cells) were seeded onto the FluoroBlok insert with poresize of 8.0 µm (Corning Life Sciences), treated with siRNAs (2.5 pmol),incubated for 48 h, and treated with TNFα (200 ng) for 24 h. U937 cells(1×106 cells) labeled with Cellstain Calcein AM solution (Dojindo) wereadded onto the HUVECs and incubated for 15 min. The transmigrated U937cells were counted using a BZ-X700 fluorescence microscope and BZ-Xanalyzer software (KEYENCE).

Real-time RT-PCRHUVECs (2×105 cells) were transfected with siRNA (25 pmol), incubatedfor 48 h and treated with TNFα (80 ng) for 30 min. Total RNA from cellswas prepared using the RNeasy Mini Kit (Qiagen) and reverse-transcribedwith Superscript VILO Master Mix (Invitrogen). Real-time PCR wasperformed using the cDNA, specific primers (Table S1), and QuantiTectSYBR Green PCR Kit (Qiagen). Copy numbers were calculated from thestandard curve prepared using known amounts of plasmids including targetsequences. The expression levels of Robo4, ICAM-1, and E-selectin (Sele)were normalized against the GAPDH level.

Identification of Robo4-binding proteinsHUVECs were infected with adenoviral vectors to express Robo4–FLAG or Ac green fluorescent protein (AcGFP) and incubated for36 h. The resulting cells were suspended in 1 ml lysis buffer (50 mMTris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium

deoxycholate, 1 mM EDTA, and Roche protease inhibitor cocktail).The resulting cell lysates were immunoprecipitated and eluted withthe FLAG Immunoprecipitation Kit (Sigma-Aldrich). The precipitatedproteins were digested with trypsin and analyzed by means of liquidchromatography tandem mass spectrometry (LC-MS/MS) using theUltimate 3000 HPLC/UPLC system and Q Exactive spectrometer(Thermo Fisher Scientific). The resulting data were analyzedusing Mascot Software (Matrix Science) to identify the peptidesspecifically included in the Robo4–FLAG sample but not in theAcGFP sample.

Co-immunoprecipitation assayCOS-7 cells (3×106 cells) were transfected with 3 μg of each expressionvector using Lipofectamine 2000 and cultured for 24 h. The resulting cellswere lysed in 1 ml lysis buffer and used for immunoprecipitation with theFLAG Immunoprecipitation Kit. The precipitated proteins were analyzedusing western blotting. The data were quantified by measuring the intensityof the bands using ImageJ/Fiji software.

Immunofluorescence stainingHUVECs (2×105 cells) were seeded on gelatin-coated coverslips, treatedwith adenoviral vectors (3000–10,000 virus particles/cell) or siRNAs(12.5 pmol), and incubated for 48 h. The resulting cells were treated withor without TNFα for 24 h and fixed with 4% paraformaldehyde. Forstaining of Robo4–FLAG and TRAF7–myc, the cells were permeabilizedwith PBS containing 0.5% sodium dodecyl sulfate and 4 mMdithiothreitol, and blocked with PBS containing 1% bovine serumalbumin. For the staining of VE-cadherin, the cells were permeabilizedwith PBS containing 0.3% Triton X-100 and blocked with PBS containing1% bovine serum albumin. The resulting blocked cells were incubated withantibodies against FLAG (F1804, Sigma-Aldrich, 1:1000), TRAF7 (H-300, Santa Cruz Biotechnology, 1:1000), or VE-cadherin (F-8, Santa CruzBiotechnology, 1:50), followed by incubation with secondary antibodiesconjugated with Alexa Fluor 488 or Alexa Fluor 555 (Life Technology).The coverslips were mounted with VECTASHIELD Mounting Mediumwith DAPI (Vector Laboratories) and analyzed using a BZ-X700fluorescence microscope (KEYENCE). Fluorescence intensity in thewhole cell and the non-junctional region was measured using ImageJ/Fijisoftware, and the ratio of non-junctional VE-cadherin was calculated.TRAF7 localization was also analyzed using ImageJ/Fiji by measuringfluorescence intensity of TRAF7 within various regions that were markedby progressively increasing by 1 pixel. The relative intensity of TRAF7was calculated by dividing the intensity in each region with that of thewhole cell.

WST-8 assayHUVECs (4×104 cells) were transfected with siRNAs (2.5 pmol) andincubated for 48 h. Cell viability was analyzed using the Cell Counting Kit-8 (Dojindo). Briefly, the cells transfected with siRNAs were incubated inmedium containing 10% WST-8 reagent for 1 h, after which their opticaldensity was measured at 450 nm.

Statistical analysisData are expressed as the mean±standard error (s.e.m.). No statisticalmethods were used to determine the sample size before experiments.Animals were selected for experiments based on certain criteria established:their genotypes, proper age, and sex. No randomization and blinding wereused. Normality and variance were tested by the Shapiro–Wilk test and theBrown–Forsythe test, respectively. For samples with a normal distributionand equal variance, P-values were calculated by Student’s t-test andANOVA followed by Tukey–Kramer test or Dunnett’s test. For sampleswith non-normal distribution and equal variance, P-values were calculatedby the Kruskal–Wallis test. The statistical significance of differences in themeans was determined by the tests stated in the figure legends. P-values of<0.05 were considered to be statistically significant.

AcknowledgementsWe thank Dr Hiroyuki Mizuguchi and Yurie Nakano for excellent assistance.

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Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: K.S., W.C.A., Y.O.; Methodology: K.S., R.I., T.K., R.F., T.T., S.F.,K.I., N.H., M.K., Y.A., Y.F., K.Y., T.D., W.C.A., Y.O.; Investigation: K.S., R.I., T.K.,R.F., T.T., S.F., K.I., N.H., K.Y., T.D., W.C.A., Y.O.; Writing - original draft: K.S.,W.C.A., Y.O.; Writing - review & editing: K.S., K.I., N.H., M.K., Y.A., Y.F., T.D.,Y.O.; Supervision: W.C.A., Y.O.; Project administration: W.C.A., Y.O.; Fundingacquisition: W.C.A., Y.O.

FundingThis work was supported by grants to Y.O., including Japan Society for thePromotion of Science KAKENHI [JP25670056, JP26293014 and JP17K19487],Japan Agency for Medical Research and Development [grants 18cm0106310h0003(P-CREATE) and 18am0101084j0002], and SENSHIN Medical ResearchFoundation.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.220228.supplemental

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