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JAM-A Acts via C/EBP-α to Promote Claudin-5 Expression and Enhance Endothelial Barrier Function

Author(s): Kakogiannos, Nikolaos; Ferrari, Laura; Giampietro, Costanza; Scalise, Anna Agata; Maderna, Claudio; Ravà, Micol; Taddei, Andrea; Lampugnani, Maria Grazia; Pisati, Federica; Malinverno, Matteo; Martini, Emanuele; Costa, Ilaria; Lupia, Michela; Cavallaro, Ugo; Beznoussenko, Galina V.; Mironov, Alexander A.; Fernandes, Bethania; Rudini, Noemi; Dejana, Elisabetta; Giannotta, Monica

Publication Date: 2020-09

Permanent Link: https://doi.org/10.3929/ethz-b-000463163

Originally published in: Circulation Research 127(8), http://doi.org/10.1161/circresaha.120.316742

Rights / License: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International

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ORIGINAL RESEARCH JAM-A Acts via C/EBP-α to Promote Claudin-5 Expression and Enhance Endothelial Barrier Function Nikolaos Kakogiannos,* Laura Ferrari,* Costanza Giampietro, Anna Agata Scalise , Claudio Maderna , Micol Ravà, Andrea Taddei, Maria Grazia Lampugnani, Federica Pisati, Matteo Malinverno , Emanuele Martini, Ilaria Costa, Michela Lupia, Ugo Cavallaro, Galina V. Beznoussenko , Alexander A. Mironov , Bethania Fernandes , Noemi Rudini , Elisabetta Dejana , Monica Giannotta

RATIONALE: Intercellular tight junctions are crucial for correct regulation of the endothelial barrier. Their composition and integrity are affected in pathological contexts, such as inflammation and tumor growth. JAM-A (junctional adhesion molecule A) is a transmembrane component of tight junctions with a role in maintenance of endothelial barrier function, although how this is accomplished remains elusive. OBJECTIVE: We aimed to understand the molecular mechanisms through which JAM-A expression regulates tight junction organization to control endothelial permeability, with potential implications under pathological conditions. METHODS AND RESULTS: Genetic deletion of JAM-A in mice significantly increased vascular permeability. This was associated with significantly decreased expression of claudin-5 in the vasculature of various tissues, including brain and lung. We observed

Downloaded from http://ahajournals.org by on January 18, 2021 that C/EBP-α (CCAAT/enhancer-binding protein-α) can act as a transcription factor to trigger the expression of claudin-5 downstream of JAM-A, to thus enhance vascular barrier function. Accordingly, gain-of-function for C/EBP-α increased claudin-5 expression and decreased endothelial permeability, as measured by the passage of fluorescein isothiocyanate (FITC)-dextran through endothelial monolayers. Conversely, C/EBP-α loss-of-function showed the opposite effects of decreased claudin-5 levels and increased endothelial permeability. Mechanistically, JAM-A promoted C/EBP-α expression through suppression of β-catenin transcriptional activity, and also through activation of EPAC (exchange protein directly activated by cAMP). C/ EBP-α then directly binds the promoter of claudin-5 to thereby promote its transcription. Finally, JAM-A–C/EBP-α–mediated regulation of claudin-5 was lost in blood vessels from tissue biopsies from patients with glioblastoma and ovarian cancer. CONCLUSIONS: We describe here a novel role for the transcription factor C/EBP-α that is positively modulated by JAM-A, a component of tight junctions that acts through EPAC to up-regulate the expression of claudin-5, to thus decrease endothelial permeability. Overall, these data unravel a regulatory molecular pathway through which tight junctions limit vascular permeability. This will help in the identification of further therapeutic targets for diseases associated with endothelial barrier dysfunction. GRAPHIC ABSTRACT: A graphic abstract is available for this article.

Key Words: claudin-5 ◼ endothelium ◼ junctional adhesion molecule A ◼ tight junctions ◼ vascular permeability

Meet the First Author, see p 951

Correspondence to: Monica Giannotta, PhD, FIRC Institute of Molecular Oncology, Milan, Italy, Email [email protected] or Prof Elisabetta Dejana, FIRC Institute of Molecular Oncology, Milan, Italy, Email [email protected] *N.K. and L.F. contributed equally to this article. The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/CIRCRESAHA.120.316742. For Sources of Funding and Disclosures, see page 1071. © 2020 The Authors. Circulation Research is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made. Circulation Research is available at www.ahajournals.org/journal/res

1056 September 25, 2020 Circulation Research. 2020;127:1056–1073. DOI: 10.1161/CIRCRESAHA.120.316742 Original Research

11–14 1057 Moreover, we Moreover, 15 September 25, 2020 2–4 JAM-A is ubiquitously expressed and expressed ubiquitously is JAM-A 6–10 JAM-A, C/EBP-JAM-A, Function in Endothelial α, Claudin-5 Among the transmembrane proteins, the immu- 5 –claudin-5 pathway in pathological conditions, pathway in pathological α–claudin-5 expression in the absence of JAM-A. Finally, Finally, JAM-A. of absence the in expression α The functional integrity of the endothelial barrier functional The 1 he vascular barrier that separates the blood from he vascular barrier that separates the endothe- the of composed mainly is tissues the for maintenance of vascular is essential lium, which These proteins are potent activators of Rap-1, which, in proteins are potent activators of Rap-1, which, These Tight junctions are formed by different types of transmem- of types by different formed are junctions Tight It describes the dysregulation of the novel JAM-A–C/ the dysregulation It describes EBP- as cancer. such 13 have shown previously that JAM-A promotes activation andactivation promotes JAM-A that previously shown have Rap-1 (Ras- junctional localization of the small GTPase of EPAC related protein-1) through sustained expression protein directly activated by cAMP)-1(exchange and EPAC- 2. Recent studies have shown that ZO-1 can form and JAM-A a cooperative unit that activates junctional actomyosin, and thus induces endothelial barrier formation. noglobulin superfamily protein JAM-A (junctional adhesionnoglobulin superfamily protein JAM-A molecule A) has a key role in maintenance of endothelial function. barrier hemostasis. Defective barrier function through increased through function barrier Defective hemostasis. many of a common feature permeability is endothelial as inflammation and can- pathological processes, such cers. brane adhesion proteins (eg, occludin, claudins, junctional cingulin,(eg, intracellular proteins adhesion molecules) and afadin, ZO-1–3 these [zona occludens 1–3]). Collectively, solutes, and larger constitute a selective barrier to water, molecules. A detailed understanding of the molecular and cellular A detailed understanding vascular permeability is critical pathways that regulate for the tar- therapies and safe to the design of efficient a wide range of human diseases. geted treatment of circuit that involves JAM-A describe a signaling We regulates endothelial permeabil- and claudin-5 and show that the transcription We ity in vivo and in vitro. C/EBP-factor of claudin-5 expression the α induces endothe- thus restricting downstream of JAM-A, cascade is negatively signaling This lial permeability. C/ represses regulated by active β-catenin, which EBP- JAM-A–C/EBP-α–mediated regulation of claudin-5 glioblastoma is dysregulated in the ovarian cancer and aberrant tumor vasculature, implicating this circuit in of this pathway can help to discovery vessels. The associ- identify new therapeutic targets for diseases ated with vascular barrier dysfunction. is determined by the fine-tuned organization and activity is determined by the fine-tuned organization localized at of the adherens and tight junction complexes the intercellular contacts. regulates several processes, including cell migration, angio- genesis, and stem cell and leukocyte transmigration. • T

Novelty and SignificanceNovelty ′-O- 8-(4-chlorophenylthio)-2 methyladenosine-3′,5′-cyclic mono- ′-O-Me-cAMP)phosphate (8-pCPT-2 α protein- CCAAT/enhancer-binding cAMP response-element binding protein dominant negative enhanced green fluorescent protein exchange protein directly activated by exchange cAMP extracellular signal-regulated kinase extracellular freshly isolated lung endothelial cells forkhead box protein O glioblastoma multiforme immortalized lung endothelial cells junctional adhesion molecule A platelet/endothelial cell adhesion molecule-1 protein kinase A factor T-cell vascular endothelial cadherin zona occludens α

It defines the mechanism of action of JAM-A in the of of action It defines the mechanism in the regu- and, hence, control of claudin-5 expression lation of the vascular barrier. It identifies the transcription factor C/EBP-α (CCAAT/ - enhancer-binding protein-α) as a key player in control permeability. and endothelial ling claudin-5 expression Intercellular tight junctions are crucial for the regulation the regulation crucial for tight junctions are Intercellular function and the control of vas- of endothelial barrier are integrity and composition Their permeability. cular include which pathological contexts, in several affected inflammation and cancers. adhesion molecule A) is a trans- (junctional JAM-A of tight junctions with a role in membrane component barrier function. maintenance of endothelial proteins of the claudin family are key Besides JAM-A, an endo- Claudin-5 is junctions. tight components of that contributes to the selectiv- thelial-specific protein ity of paracellular barriers. Nonstandard Abbreviations and Acronyms Nonstandard Abbreviations 007 C/EBP- CREB DN eGFP EPAC • ERK What New Information Does This Article New Information Does This What Contribute? • fLECs FoxO GBM iLECs JAM-A PECAM-1 PKA TCF VE-cadherin WT wild-type ZO • • What Is Known? What • 2020;127:1056–1073. DOI: 10.1161/CIRCRESAHA.120.316742 Circulation Research. 2020;127:1056–1073. Kakogiannos et al et Kakogiannos

Downloaded from http://ahajournals.org by on January 18, 2021 Downloaded from http://ahajournals.org by on January 18, 2021 18, January on by http://ahajournals.org from Downloaded Original Research endothelial barrierisclaudin-5, the correctorganizationofcell-celljunctions. of cancers. EBP- reduces claudin-5expression. α naling, which in turn inhibits C/EBP- show that JAM-A deficiency promotes sion issuppressedbyactiveβ α activity dependsonEPAC, whereasC/EBP- α meability. We C/EBP- revealthatthecAMP-inducible downstream ofJAM-A, which decreasesendothelialper- transcription factorthattriggersclaudin-5expression EBP- mechanism thatisactivatedbycAMP. We identifyC/ claudin-5 expression throughanEPAC-dependent vivo andinvitro.We demonstratethatJAM-A induces ing circuitthatregulatesendothelialpermeabilityin din-5 expression remainstobeclarified. clau- that linksJAM-A expression andcAMP-induced 1058 the corresponding authorsonreasonablerequest. data thatsupport thefindingsofthisstudyare availablefrom Detailed Methodsareavailable intheDataSupplement.The METHODS turn, enhancesendothelialbarrierfunction, Kakogiannos etal the claudincompositionoftightjunctions. din-2 oroccludin,which suggestedthatJAM-A regulates ing claudin-10andclaudin-15,withnochanges inclau- JAM-A increasedthelevelsofpermeability-enhanc- in vitroandvivoepithelialmodels,theabsenceof between theexpression ofJAM-A andclaudins.Indeed, endothelial barrierdysfunction. further therapeutictargetsfor diseasesassociatedwith cial componentoftightjunctions.This willhelptoidentify enhancing theexpression ofclaudin-5,which isacru- tight junctions,JAM-A, restrictsvascularpermeabilityby molecular pathwaythroughwhich acomponentofthe maintenance ofvascularintegrityinhumans. the potentialrelevance of thissignaling pathway for andovariancancer, which indicates multiforme (GBM) downregulated inthefragilevasculatureofglioblastoma α, andclaudin-5areconcomitantly of JAM-A, C/EBP- PKA-independent pathways. PKA-independent (proteinkinaseA)-dependentand tion, throughPKA by cAMP, aknownregulatorofendothelialbarrierfunc- attention andremainstobebetterdefined. the molecularbasisofthisregulationhasreceivedlittle rier function. vessel permeability, andthusenhancedendothelialbar- ing oftightjunctions,andasaconsequence,decreased claudin family. Claudin-5expression promotestheseal- The mainstructuraldeterminantoftheparacellular It hasbeenreportedthatthelevelsofJAM-A, C/ In the present study, we define a novel signal- In summary, ourdatademonstrateanovelregulatory Claudin-5 expression inendothelialcellsisincreased α, andclaudin-5aredecreasedindifferent types α (CCAAT/enhancer-binding protein-α September 25,2020 28–31 20–22 Here,weobservedthattheexpression Apreviousreportsuggestedalink 24,25 -catenin. 19 However, thepathway which belongstothe transcription and 26,27 16–18 β -catenin sig- Indeed,we 23 to maintain tomaintain However, Circulation Research.2020;127:1056–1073. 10.1161/CIRCRESAHA.120.316742 DOI: ) asthe expres- tivity of paracellular barriers, that thesejunctionswerenotformedcorrectly. widening oftheparacellularspaces,which suggested JAM-A led to discontinuoustightjunctionstrands,with comparison withthecontrolJAM-A–WT, theabsenceof A–WT andJAM-A–nulliLECs. AsshowninFigure 1D, used transmissionelectronmicroscopytocompareJAM- JAM-A intriggeringclaudin-5expression. Data Supplement). in the through ID (Figure IB with small-interfering RNAs iLECs was also observed upon acute depletion of JAM-A ure 1A–1C). Downregulationofclaudin-5expression in immunoblotting and immunofluorescence staining (Fig- chain reaction analysis and also atthe protein level by were confirmedbyreal-timequantitativepolymerase the molecules werenotsignificantlyaffected (FigureIAin error test;foldchange >2),whereasadherensjunctions lation intheabsenceofJAM-A (P tions, Among theprominentlyaffected genesofthetightjunc- tight and adherens junctions. the organization of both ponents describedintheliteratureaskeymoleculesfor JAM-A–null mice.We examined theendothelialcom- endothelial cells(iLECs) ofwild-type(WT)miceand expression throughacomparisonofimmortalizedlung sion, we carried out Affymetrix analysis of gene junctions hasnotbeenfullyelucidated. limits permeability and maintains the integrity of tight ever, themolecularmechanism throughwhich JAM-A compared with WTcells.Moreover, reestablishment lial permeability wasincreasedintheabsence ofJAM-A the in and Western andIF blotting,respectively (FigureIE of claudin-5wereevaluated usingimmunofluorescence A iLECs andafterrestoringclaudin-5expression inJAM- across monolayersofJAM-A–WTand–null of fluoresceinisothiocyanate(FITC)–labeled dextran was determinedthroughmeasurementofthepassage permeability. To addressthispoint,invitropermeability absence ofJAM-A isresponsibleforincreasedvascular prompted ustopostulatethatitsdownregulationinthe tions. tant componentofendothelialandepithelialtightjunc- which controlsvascularpermeabilityalongwithRap-1. JAM-A deficiencyreducesthe expression ofclaudin-5, RESULTS migration ofinflammatoryandimmunecells. endothelial permeabilitytoplasmasolutesandfortrans- –null iLECs. The expression and junctional localization As claudin-5isthemajordeterminantinselec- To accuratelyexamine tightjunctionmorphology, we Overall, thesedatademonstrateapositiverolefor The differences in the expression levels of claudin-5 To identifytranscriptsregulatedbyJAM-A expres- Multiple studies have identified JAM-A as an impor- Data Supplement).AsshowninFigure 1E, endothe- Data Supplement). 9,32,33 claudin-5 showed greater significant downregu- JAM-A was shown to be required for control of α, Claudin-5inEndothelialFunction JAM-A, C/EBP- 20–22 <0.05, local-pooled the overall data 23,34 How- Original Research . –7 1059 ), JAM-A–WT vs ; JAM-A–WT vs JAM- –7 –3 ; claudin-5 ***P=4.17×10 –7 September 25, 2020 P=5.3×10 JAM-A, C/EBP-JAM-A, Function in Endothelial α, Claudin-5 ; JAM-A–WT vs JAM-A–null claudin-5 **P=4.3×10 –4 –null 007 ***P=5×10 ; JAM-A–WT vs JAM-A–null 007 –4 –null iLECs. Tubulin is shown as the loading control. Data is Tubulin and claudin-5 JAM-A–WT and JAM-A–null iLECs. B, Representative immunoblotting for JAM-A in (red) and claudin-5 (green) expression C, Representative confocal microscopy of JAM-A are representative of 3 independent experiments. PECAM-1 (platelet/endothelial cell adhesion molecule-1; white) is an endothelial junctional marker; DAPI JAM-A–WT and JAM-A–null iLECs. Scale bars: 20 µm. D, Representative (4′,6-diamidino-2-phenylindole; blue) stains nuclei. Data are representative of 3 independent experiments. Arrows indicate the tight junction strands of the endothelial cells. Scale transmission electron microscopy of JAM-A–WT and JAM-A–null iLECs. and JAM- of JAM-A–WT and JAM-A–null iLECs Permeability to fluorescein isothiocyanate (FITC)–dextran flux assay. bars: 200 nm. E, Tracer GFPA–null stably expressing JAM-A–null claudin-5) with treatment with (green fluorescent protein; JAM-A–WT and JAM-A–null) or claudin-5 ( ′-O-methyladenosine-3vehicle or 8-(4-chlorophenylthio)-2 ′-O-Me-cAMP;′,5′-cyclic monophosphate (8-pCPT-2 100 µmol/L), 007; to activate Rap-1. Data are means±SEM P values determined as representative of 3 independent experiments. of triplicates from a single experiment, followed by Dunnett T3 test for multiple comparisons. Comparison at 6 h: (overall **** ANOVA by Brown-Forsythe JAM-A–null ***P=3×10 2020;127:1056–1073. DOI: 10.1161/CIRCRESAHA.120.316742 Circulation Research. 2020;127:1056–1073. JAM-A (junctional adhesion molecule A) controls endothelial permeability through regulation of claudin-5 expression Figure 1. JAM-A (junctional adhesion molecule A) controls endothelial permeability through and Rap-1 (Ras-related protein-1) activation. from JAM-A–wild- cells (iLECs) in immortalized lung endothelial in JAM-A and claudin-5 gene expression A, Quantification of fold-differences reaction analysis. Data are means±SDtype (WT) and JAM-A–null mice following real-time quantitative polymerase chain from 3 independent t test. JAM-A–WT vs JAM-A–null: JAM-A ***P=9.54×10 P value determined by 2-sided unpaired Welch experiments. –null 007 *P=0.0117. P=0.25, not statistically significant [NS]; JAM-A–null vs JAM-A–null 007 A–null claudin-5 007, Kakogiannos et al et Kakogiannos

Downloaded from http://ahajournals.org by on January 18, 2021 Downloaded from http://ahajournals.org by on January 18, 2021 18, January on by http://ahajournals.org from Downloaded Original Research Rap-1 activation. claudin-5expression and through thecontrolofboth important regulatorofvascularpermeabilitythatacts (Figure 1E). 2′ methyladenosine-3′ EPAC-specific agonist 8-(4-chlorophenylthio)-2′ claudin-5 expression andactivatingRap-1usingthe of endothelialfunctionwasobtainedbyrestoringboth trol ofendothelialpermeability. Indeed,completerescue claudin-5–dependent and Rap-1–dependentcon- both expression induced by 8-chlorophenylthio-cAMP was expression inducedby 8-chlorophenylthio-cAMP ure 2A). Moreover, intheabsenceofJAM-A, claudin-5 (potentially EPAC-mediated) pathwaysiniLECs (Fig- expression and PKA-independent via PKA-dependent increases claudin-5 by 50%, which confirmed that cAMP claudin-5induction 8-chlorophenylthio-cAMP–mediated not affect thebasallevelsofclaudin-5, thisreduced did inhibitor H89 Although pretreatmentwiththePKA absence ofJAM-A, thiswaslower(1.5-fold;Figure 2A). 1/2, increased claudin-5 levels by 4-fold, whereas in the rophenylthio-cAMP, andEPAC- which activatesPKA expression. Treatment ofJAM-A–WTiLECs with8-chlo- analogsfortheirimpactonclaudin-5 permeable cAMP claudin-5 expression inendothelial cells. and EPAC-dependent pathways sustain sized that PKA- endothelial barrier. Based on this evidence, we hypothe- andEPAC-1/2, whichPKA act in concert to tighten the 1060 expression. inductionofclaudin-5 dependent andPKA-independent PKA- junctions inbrainendothelialcellsthroughboth has beenreportedtoenhancebarrierfunctionoftight involved intheregulationofclaudin-5expression. cAMP JAM-A, weanalyzedthedifferent factorsthatmightbe lie reducedexpression ofclaudin-5intheabsence To unravelthemolecularmechanisms thatunder- the AbsenceofJAM-A Through EPAC Signaling,Which IsImpaired in Increases Claudin-5Expression Cyclic AMP EPAC-2. Rap-1 throughsustainedexpression ofEPAC-1 and activation andjunctionlocalizationofthesmallGTPase intheDataSupplement). throughIG IE rier functiondownstreamofJAM-A (Figure 1E;Figure which indicatedthatclaudin-5controlsendothelialbar- showed 72% recoveryofthepermeabilityphenotype, of claudin-5expression intheseJAM-A–nulliLECs Kakogiannos etal tion. cell junctionsandenhancesendothelialbarrierfunc- Rap-1, which maintainsthecorrectorganizationofcell- -O-Me-cAMP; knownas007) inJAM-A–nulliLECs -O-Me-cAMP; Taken together, these data showthatJAM-A isan Therefore, we investigateddifferent membrane- Our previousstudyshowedthatJAM-A promotes 16–18 September 25,2020 Thus, wehypothesizedthatJAM-A regulates 13 These last 2 proteins are potent activators of 24,25,35 Moreover, the main cAMP targetsare Moreover, themaincAMP ,5′ -cyclic monophosphate(8-pCPT- Circulation Research.2020;127:1056–1073. 10.1161/CIRCRESAHA.120.316742 DOI: -O- tion and differentiation, and inflammatory responses. regulators ofcellularprocesses,includingcellprolifera- mediated acute depletion of JAM-A in iLECs (Figure IIG mediated acute depletionofJAM-A iniLECs (FigureIIG α expression wasdetected uponsmall-interfering RNA– Supplement). Moreover, significantreduction inC/EBP- inthe Data andIIF counterpart (Figure 2C;FigureIIE significantly reduced compared with the α levelsintheJAM-A–nullcellswere Nuclear C/EBP- EBP- JAM-A–WT and–nulliLECs intermsofC/ Supplement). intheData andIID downstream ofEPAC (FigureIIC suggests thatitisinvolvedintheregulationofclaudin-5 nificantly downregulatedintheabsenceof JAM-A, which identified (α expression ofEPAC-1/2. dependent claudin-5expression isaffected bythelow we hypothesizedthatinJAM-A–nulliLECs, EPAC- alone.Thus,claudin-5 expression iscontrolledbyPKA which suggeststhatthe completely abrogatedbyH89, EBP- JAM-A DeficiencyReducesExpression ofC/ the in andIID upregulated inJAM-A–nulliLECs (FigureIIC β the C/EBP- δ and C/EBP- γ reaction and immunoblotting revealed that C/EBP- WT andJAM-A–nulliLECs. Real-timepolymerasechain isoformsinJAM-A– sion levelsofthedifferent C/EBP andproteinexpres- JAM-A. First,wecomparedmRNA isoformsisaffected by expression ofspecificC/EBP of claudin-5expression, weinvestigatedwhetherthe As weshowedthatEPAC isinvolvedintheregulation andPKA. pendent ofCREB expression throughnonclassicalroutesthatareinde- canalsoregulate gene Evidence suggestedthatcAMP mechanism. lates claudin-5expression throughanEPAC-dependent intheDataSupplement). andIIB (Figure IIA dent on EPAC. activitiesthataredepen - shown tohavecAMP-inducible hasbeen of transcriptionfactorsknownasC/EBPs downstream effector ofPKA. response-element binding protein), a (cAMP CREB regulation ofclaudin-5,weanalyzedtheactivation Finally, toruleoutaroleforJAM-A inPKA-dependent ulates claudin-5expression throughEPAC (Figure 2B). the absenceofJAM-A, which confirmedthat JAM-A reg- ence ofJAM-A (by2-fold),whereasitfailedtodosoin levelsonlyinthepres- increased theclaudin-5mRNA ences in CREB phosphorylationatserineresidue133 ences inCREB JAM-A–WT and To furtherstrengthenthisevidence,wecompared Overall, these data demonstrate that JAM-A regu- α Data Supplement).Conversely, C/EBP- α nuclearlocalizationandtranscriptional activity. α , β isoforms, known as LIP andLAP, isoforms,knownasLIP were α, Claudin-5inEndothelialFunction JAM-A, C/EBP- 38,39 levelsremainedunchanged, whereas , γ JAM-A–null iLECs showed no differ- , To date, 6 C/EBP genes have beenTo date,6 C/EBP δ , ε , ζ ). These allfunctionasmaster 13 Indeed,treatmentwith007 37 Inthiscontext, agroup 36 Here,comparisonof JAM-A–WT wassig- 40 36

Original Research 1061 September 25, 2020 JAM-A, C/EBP-JAM-A, Function in Endothelial α, Claudin-5 2020;127:1056–1073. DOI: 10.1161/CIRCRESAHA.120.316742 Circulation Research. 2020;127:1056–1073. JAM-A (junctional adhesion molecule A) sustains claudin-5 expression through an EPAC (exchange protein directly (exchange EPAC Figure 2. JAM-A (junctional adhesion molecule A) sustains claudin-5 expression through an activated by cAMP)-dependent and C/EBP- protein-α) regulation. mechanism α (CCAAT/enhancer-binding –wild-type (WT) and JAM-A–null immortalized lung endothelial in JAM-A in claudin-5 gene expression A and B, Quantification of fold-differences analysis. Cells were treated with vehicle (A and B; fold- reaction (RT-qPCR) following real-time quantitative polymerase chain cells (iLECs) 1.0) and the specific difference, PKA (protein kinase A) inhibitor H89 (10 µmol/L), activator (Continued ) and the PKA and EPAC Kakogiannos et al et Kakogiannos

Downloaded from http://ahajournals.org by on January 18, 2021 Downloaded from http://ahajournals.org by on January 18, 2021 18, January on by http://ahajournals.org from Downloaded Original Research cldn-5: (overall****P=5.7×10 α: JAM-A–WTvs–null**P=3.2×10 3 independentexperiments. Pvaluedeterminedby2-sidedunpairedWelch ttest.C/EBP- α/lamin Bratioquantifiedbydensitometryscanand from expressed asfold changes. Dataaremeans±SD markers, respectively. Right,C/EBP- α inJAM-A–WTand JAM-A–null iLECs. Tubulinfor nuclear/cytoplasmdistributionofC/EBP- andlaminBareshownascytoplasmicnuclear (overall **P=9.2×10 ***P=6.4×10 +H89 **P=5.6×10 +H89 –WT fBECs: cldn-5: (overall****P=6.3×10 ANOVA followedbyDunnettT3testformultiplecomparisons.JAM-A–WTfBECs: from 3independentexperiments (n=12,JAM-A–WT;n=12,–nullmice;foreach experiment). PvaluesdeterminedbyBrown-Forsythe difference, 1.0),theselectiveEPAC EPAC Dataaremeans±SD andthespecific activator007 (10µmol/L). (100µmol/L), inhibitorESI-09 followingRT-qPCRpparg, cdh5expression analysis.Cellsweretreatedwithvehicle(fold- inJAM-A–WT(left)and –null (right)fBECs vehicle vs ESI-09 ****P=2.9×10 vehicle vsESI-09 1062 followed byDunnettT3testformultiplecomparisons.JAM-A–WT:(overall***P=10 from3independentexperiments. PvaluesdeterminedbyBrown-Forsythe B).Dataaremeans±SD 007; ANOVA 100 µmol/L; O-Me-cAMP; ′,5-cyclic monophosphate(8-pCPT-2 (A),orselectiveEPACcombination withH89 ′- activator8-(4-chlorophenylthio)-2′-O-methyladenosine-3 aloneandin (8-(4-chlorophenylthio)adenosineFigure 2Continued.CPT-cAMP 3 ′,5-cyclic monophosphatesodiumsalt;250µmol/L) csfr3, levelsofitsknowntargetgenes(ie,csf1r, ing mRNA EBP- in theDataSupplement).Consistentwiththis,whenC/ Kakogiannos etal vs JAM-A–null***P=2.9×10 ***P=1.2×10 from3independentexperiments. Pvaluesdeterminedby2-sidedunpairedWelchmeans±SD JAM-A–WTvs–null ttest.Claudin-5:fBEC, α/VE-cadherin ratiosquantifiedbydensitometryscanand expressed asfold changes. Dataare Right, Claudin-5/VE-cadherinandC/EBP- of JAM-A–WTand–nullmice.VE-cadherin(vascularendothelialcadherin)isshownasloadingcontrolforcellnumbers. andlungs(fLECs) α infreshlyisolatedendothelialcellsfrombrains(fBECs) Representative immunoblottingforJAM-A, claudin-5,andC/EBP- by 2-sidedunpairedWelch ttest.JAM-A–WTvs–null**P=8.3×10 4 sectionscontainingatleast10nuclei,expressed P valuedetermined asarbitraryunits(AU); n=3,JAM-A–WT;–null,means±SD. α meanfluorescenceintensityin α signalinlungvesselssectionsfromJAM-A–WTand–nullmiceasnuclearC/EBP- of nuclearC/EBP- α andVE-cadherin(right).Sectionswere4μmthick. Scalebars:20μm(n=3,JAM-A–WT;n=3,–null).G,Quantification filtered C/EBP- α (middle),formergedimagesof toisolatenuclearC/EBP- related gene)staining(nuclearmarker)wassegmentedwiththreshold 350-4.096 (ETS- α wasdetectedinthecytoplasmofperivascularcellslung,ERG sections fromJAM-A–WTand–nullmice.AsC/EBP- α (green)andVE-cadherin(red)inparaffin-embeddedlung 4 μmthick. Scalebars:20µm.F,RepresentativeconfocalmicroscopyforC/EBP- claudin-5 (green)inparaffin-embeddedlungsectionsfromJAM-A–WTand JAM-A–null mice(n=3,–WT;n=3,–null).Sectionswere A–WT: vehiclevs007 ***P=6×10 vehicle vs ESI-09 ***P=2.6×10 vehicle vsESI-09 *P=3.7×10 vs CPT-cAMP+H89 **P=2.4×10 vs CPT-cAMP+H89 2D). Densitometric analysis showed 30% trols (Figure 2D).Densitometricanalysisshowed30% C/EBP- JAM-A ledtosignificantdecreasesinclaudin-5and sistent withthepreviousinvitrodata,absenceof cells, epithelialfibroblasts,andastrocytes.Con- of othercelltypes,such aspericytes,smoothmuscle specific markers,withbarelydetectablecontamination rations showed strong enrichment of endothelial cell– fLEC andfreshlyisolatedbrainendothelialcellsprepa- the intheDataSupplement,both reported inFigureIIIA cell typesfromthebrainandlungswasmeasured.As The endothelialcellenrichment incomparisontoother polymerase chain reactionforcell-specificmarkers. We firsttestedthepreparationpurityusingreal-time obtained fromtheJAM-A–WTand–nullmice. cells [fLECs] andfreshlyisolatedbrainendothelialcells) different organs(ie,freshlyisolatedlungendothelial protein levelsinfreshlyisolatedendothelialcellsfrom α in vitro findings, we analyzed claudin-5 and C/EBP- Data Supplement). inthe A–null iLECs comparedwiththeWT(FigureIIH vehicle vs ESI-09 P=0.14,NS. vehicle vsESI-09 To determinethephysiologicalrelevanceofthese α transcriptionalactivitywasanalyzedbymonitor- id2, September 25,2020 α –2 pparg), proteinlevelscomparedwiththeWTcon- -4 -4 ; vehicle vs ESI-09 **P=5.8×10 ; vehiclevsESI-09 ), vehiclevs007 *P=1.1×10 ; fLEC, JAM-A–WTvs–null***P=5.4×10 –3 -3 ; CPT-cAMP vs CPT-cAMP+H89 **P=7×10 vsCPT-cAMP+H89 ; CPT-cAMP 41 =0.39, NS; vehicle vs ESI-09 *P=2.1×10 vehicle vsESI-09 ), vehiclevs007 NS; P=0.39, theseweresignificantlylowerinJAM- -4 -6 , Representative confocal microscopy for PECAM-1 (platelet/endothelialcelladhesionmolecule-1;red)and . E,RepresentativeconfocalmicroscopyforPECAM-1 ), vehiclevs007 *P=1.4×10 –4 –7 ; C/EBP-α:(overall***P=1.1×10 –3 –null fBECs: JAM-A–nullfBECs: P=0.13,NS. vehiclevs007 vehiclevsESI-09 ; cdh5:(overallP=0.12,NS), P=0.21,NS; –4 ; CPT-cAMP vs CPT-cAMP+H89 **P=10 vsCPT-cAMP+H89 ; CPT-cAMP ; JAM-A–null:vehiclevs007 C,leftRepresentativeimmunoblotting P=0.23,notstatisticallysignificant(NS). –2 ; vehicle vs ESI-09 **P=8.4×10 ; vehiclevsESI-09 –3 ; id2:(overall**P= 2.6×10 Circulation Research.2020;127:1056–1073. 10.1161/CIRCRESAHA.120.316742 DOI: –2 ; vehicle vs ESI-09 ***P=8.4×10 ; vehiclevsESI-09 -4 : fBEC, JAM-A–WT vs–null**P=1.5×10 α: fBEC, ; C/EBP- -4 –3 ), vehiclevs007 **P=1.4×10 ; JAM-A–null:(overall***P=2.2×10 -3 ), vehiclevs007 P=0.17, ***P=6×10 vehiclevsESI-09 NS; -3 . H,Quantificationoffold-differences forcldn-5,C/EBP-α,id2, –3 protein levelsofEPAC downstreameffectors inboth versely, and the EPAC reduced mRNA inhibitor ESI-09 intheDataSupplement).Con- (Figure 2H; FigureIIIC A, whereasitfailedtodosointheabsenceofJAM-A EBP-α andproteinlevelsofclaudin-5 C/ in themRNA EPAC-specific agonist007 ledtoasignificantincrease JAM-A–WT and–nullmice.Treatment withthe in culturedendothelialcellsfrombrainisolated we examined theimpactofitsactivationandinhibition Supplement). intheData cellular modelforfurtherstudies(FigureIIIB proteins, which suggeststhattheiLECs areasuitable levels of additional tight junction and adherens junction fLECs andiLECs, JAM-A expression didnotaffect the cadherin; Figure sion molecule-1)andVE-cadherin(vascularendothelial the levels (platelet/endothelial of PECAM-1 cell adhe- in bloodvesselsthatlacked JAM-A, withnochanges in α downregulation ofclaudin-5andC/EBP- fluorescence analysisofthelungsconfirmedsignificant lated brainendothelialcells(Figure 2D).Invivoimmuno- infreshlyiso- infLECs and60% were reducedby40% α and brain(Figure 2D),whereasC/EBP- lungs downregulation ofclaudin-5expression inboth –3 ; pparg:(overall***P=3.4×10 . B,Pvaluesdeterminedby2-sidedunpairedWelch ttest.JAM- Finally,EPAC toconfirm activityinthebrainvessels, –2 0.058, NS), vehicle vs007; cdh5:(overallP= 0.058, NS), P=0.74, NS; –4 ), H89 vs CPT-cAMP ****P=2×10 vsCPT-cAMP ), H89 anditstargetsonlyinthepresenceofJAM- –4 ; C/EBP-α:(overall***P=2.5×10 α, Claudin-5inEndothelialFunction JAM-A, C/EBP- –3 ; vehicle vs ESI-09 **P=1.9×10 ; vehiclevsESI-09 2E through 2G). Furthermore, in both –4 ), H89 vs CPT-cAMP *P=1.67×10 vsCPT-cAMP ), H89 -4 ), vehiclevs007 *P=1.1×10 -8 ), vehiclevs007 ****P=2.4×10 –5 ; H89 vs CPT-cAMP vsCPT-cAMP ; H89 -3 ; fLEC, JAM-A–WT -4 ), vehiclevs007 –3 proteinlevels ; id2:(overall –4 expression –3 ; pparg: . D,left –2 ; –2 ; H89 ; H89 –5 ; Original Research 1063 clau- September 25, 2020 JAM-A, C/EBP-JAM-A, Function in Endothelial α, Claudin-5 binding sites were identified within the within identified were sites binding α α sustains claudin-5 transcription by directly JAM-A–null C/EBP-α-WT in JAM-A–null cells ( –null iLECs infected with vectors to stably with vectors infected JAM-A–null iLECs ). This suggested that (Figure 4A). This JAM-A–null iLECs Conversely, the expression of C/EBP-the expression Conversely, α-DN in JAM- In chromatin immunoprecipitation assays, C/EBP- immunoprecipitation In chromatin α confirm these data, the sequences of the claudin-5 To demonstrate that the binding of C/EBP-To α to the –WT to compare JAM-A assays in vitro permeability and C/EBP- full-length express C/EBP--WT or α α-DN, or eGFP protein) as the green fluorescent (enhanced of C/ 3E, the expression shown in Figure control. As EBP- permeability compared with the α-WT) decreased the –null eGFP).control cells (JAM-A suggests that the This by the that is mediated expression increase in claudin-5 C/EBP-factor transcription of the tightening induces α C/EBP-in JAM-A–null expected, As junctions. the α- was not cells, the permeability defect WT–expressing as C/EBP-completely rescued, α failed to re-establish –WT con- to the level of the JAM-A claudin-5 expression shown (Figure 3C; Figure IIIFtrol cells, as previously in ). the Data Supplement the permeability compared with increased A–WT iLECs the control cells (JAM-A–WT eGFP), due to the down- regulation of claudin-5 (see Figure 3C; Figure IIIF in the the novel set of data confirms Data Supplement). This role of the transcriptional activity of C/EBP-α in the con- (Figure 3E). trol of endothelial barrier permeability JAM-A Promotes C/EBP-JAM-A Promotes α Binding to the and Induces Gene Claudin-5 Promoter Transcription then wanted to determine how C/EBP-We α acts on the As 3 putative claudin-5 gene to induce its expression. C/EBP- promoter region within −6.0 kb of the transcription din-5 promoter region within −6.0 kb of the gene is a start site, we hypothesized that the claudin-5 direct transcriptional target of C/EBP- in α (Figure IVA the Data Supplement). at the selected sites on the clau- binding was enriched din-5 promoter in the JAM-A–WT cells, compared with the C/EBP- is com- binding to its promoter and that this mechanism where C/EBP-promised in the absence of JAM-A, α is downregulated (Figure 4A) and less active. promoter were cloned upstream of the firefly luciferase gene; these comprised all 3 of the C/EBP-α binding site regions (ie, no. 1, no. 2, no. 3) or regions no. 1 and no. 2, or only region no. 3 (Figure IVB in the Data Supple- of these reporters the transfection ment). As expected, in the activities resulted in higher luciferase into iLECs (Figure 4B). presence of JAM-A a transcriptional active, functionally is promoter claudin-5 reporter assay was performed in HEK-293T cells, where with claudin-5 reporter constructs were cotransfected target . This inhibitory . This in both JAM-A–WT -WT was expressed, was expressed, -WT 42 -WT were comparable –null JAM-A–WT and JAM-A–null -DNIIID(Figure IIIEand in target genes at the mRNA level is a novel regulator of claudin-5 3C). When C/EBP- 3C). When α expression, which suggests a role for C/EBP- suggests which expression, α α-DN decreased claudin-5 mRNA and protein downregulation using small-interfering RNAs.α downregulation using small-interfering showed similar trends to that of to that of trends similar showed and pparg id2 -WT and C/EBP-and -WT α 4% (ie, 0.0417-fold) that of the WT counterpart, that of the WT counterpart, 4% (ie, 0.0417-fold) α α-DN were similar in ≈ –null iLECs. As shown in Figure 3C and 3D, As shown JAM-A–null iLECs. ). Taken together these data dem- Data Supplement). Taken Finally, we sought to determine whether the increase sought to determine whether we Finally, Similar data on claudin-5 protein expression and and expression claudin-5 protein data on Similar Thus, these data demonstrate that JAM-A sustains sustains that JAM-A data demonstrate these Thus, in EPAC-mediated claudin-5 induction in endothelial endothelial in induction claudin-5 in EPAC-mediated –null cells, where claudin-5 expression is maintained is maintained –null cells, where claudin-5 expression expression. induced by C/EBP-in claudin-5 expression α can reverse the higher endothelial permeability caused by this purpose, we performed For the absence of JAM-A. the onstrate that C/EBP-α although the levels of C/EBP-α claudin-5 (Figure 3D). regulation of C/EBP-α cells (Figure –null iLECs the absolute level of claudin-5 in JAM-A was in both cell types (Figure 3C, p42 isoform, highlighted in JAM- suggests an inhibitory mechanism in blue). This A low even in the presence of C/EBP-α Consistent with further below. is explored mechanism this, the mRNA levels of the selected C/EBP-α genes of C/ were obtained using transient overexpression EBP- C/EBP- and these were upregulated by full- whereas expression, length-C/EBP-(C/EBP-α fold-differences The α-WT). induced by C/EBP-in claudin-5 expression α and C/ EBP- 2020;127:1056–1073. DOI: 10.1161/CIRCRESAHA.120.316742 Circulation Research. 2020;127:1056–1073. To investigate the specific role of C/EBP- investigate the specific To α in the used several we expression, regulation of claudin-5 to modulate C/EBP-approaches and its α expression acute we induced First, factor. a transcription as activity C/EBP- the C/EBP- markedly reduced This α mRNA and pro- to 80% with the compared by ≈75% tein expression, of C/EBP- reduction control (Figure 3A and 3B). This α at expression led to significant decreases in claudin-5 both the mRNA with con- and protein levels, compared - trol cells (Figure 3A and 3B). Second, we stably overex pressed full-length C/EBP-α and a 30-kDa C/EBP-α dominant-negative (C/EBP-α-DN) JAM-A Triggers Claudin-5 Expression Expression Claudin-5 JAM-A Triggers C/EBP-Through α and Regulates Endothelial Permeability α cells. –null endothelial cells from cells from JAM-A–null endothelial JAM-A–WT and and ESI-09 007 neither of the treat- brain. As expected, the mRNAments affected 2H; of cdh5 (Figure levels Figure IIIC). the Data Supplement in C/EBP- Kakogiannos et al et Kakogiannos

Downloaded from http://ahajournals.org by on January 18, 2021 Downloaded from http://ahajournals.org by on January 18, 2021 18, January on by http://ahajournals.org from Downloaded Original Research α vsC/EBP- eGFP vs siRNA no.54**P vs siRNA C/EBP- siRNA no. 54***P siRNA 1064 ***P independent experiments. P EBP- α againstC/EBP- Ctrl[control])orwithsiRNAs (siRNA; small-interfering RNA A Figure 3.C/EBP- Kakogiannos etal EBP- α vsC/EBP- eGFP (overall **P not statistically significant (NS). D not statisticallysignificant(NS). determined byBrown-Forsythe ANOVA followedbyDunnettT3testfor multiplecomparisons.Comparison at6h:(overall****P=7.8×10 oftriplicatesfrom asingleexperiment, representative of3independentexperiments. P ascontrol.Dataaremeans±SEM andeGFP (p30), α isothiocyanate (FITC)–dextran kDa)inJAM-A–WTand–nulliLECs stably expressing (40 full-lengthC/EBP- Brown-Forsythe ANOVA followedbyDunnettT3test formultiplecomparisons.JAM-A–WT:(overall***P quantified bydensitometryscanningand from3independent expressed as fold experiments. P changes. Dataaremeans±SD α corresponding tofull-lengthC/EBP- (enhancedgreenfluorescent protein)ascontrol. highlightbands Vinculin isshownasloadingcontrol.Upper,Blueboxes andeGFP p30), (DN; α claudin-5 (lower)inJAM-A–wild-type (WT)andJAM-A–null iLECs stablyexpressing full-lengthC/EBP- α vsC/EBP- eGFP –null eGFP ****P vsJAM-A –null eGFP WT eGFP siRNA Ctrl vs siRNA no.55***P Ctrl vssiRNA siRNA α againstC/EBP- Ctrl;fold-difference, 1.0)andsiRNAs (siRNA expression iniLECs followingreal-timequantitativepolymerasechain reactionanalysis,asshowninA iLECs: 3 independentexperiments. P A determined byBrown-Forsythe ANOVA followed byDunnettT3testformultiplecomparisons.C/EBP-α α (control;fold-difference 1.0)orC/EBP- –null (right)iLECs, understableexpression ofeGFP , α left, RepresentativeimmunoblottingforC/EBP- = 5.8×10 α α id2: (overall***P /GAPDH and claudin-5/GAPDH ratiosquantifiedbydensitometryscanningand from3 expressed asfold changes. Dataaremeans±SD andclaudin-5/GAPDH /GAPDH -DN ***P -DN α September 25,2020 -DN null***P -DN = 2.9×10 -4 ), siRNA Ctrl vs siRNA no.54*P CtrlvssiRNA ), siRNA = 2.1×10 = 6.5×10 -WT **P -DN ****P -DN -DN ****P -DN -3 = α (CCAAT/enhancer-binding protein-α ) controlstheendothelialbarrierthroughclaudin-5expression. α vsC/EBP- ), eGFP 1.3×10 = =6.9×10 6.4×10 –4 ; -4 =2.6×10 pparg: (overall*P ; siRNA Ctrl vs siRNA no.55**P CtrlvssiRNA ; siRNA valuesdeterminedbyBrown-Forsythe ANOVA followedbyDunnettT3testformultiplecomparisons.C/EBP-α = = -3 ; siRNA Ctrl vs siRNA no.55***P CtrlvssiRNA ; siRNA 4.6×10 6.7×10 valuesdeterminedbyBrown-Forsythe ANOVA followed byDunnettT3testformultiplecomparisons.JAM-A–WT -4 =6.3×10 –4 α vsC/EBP- ), eGFP , Quantificationoffold-differences forid2, . –3 =1.3×10 -5 –6 α vsC/EBP- ; eGFP –null were loaded. Claudin-5/vinculin ratios μg JAM-A–nullwereloaded.Claudin-5/vinculinratios , 10μgJAM-A–WTand80 (p42isoform).Bottom ; . JAM-A–null: (overall**P JAM-A–null iLECs: id2: (overall****P -4 -WT *P . C = α , RepresentativeimmunoblottingforJAM-A (junctional adhesionmoleculeA),C/EBP- 1.5×10 –5 = ; 1×10 α vsJAM-A–WTC/EBP- JAM-A–WT eGFP =1.2×10 andclaudin-5inimmortalizedlungendothelialcells(iLECs) transfected withnontargeting -2 -2 -WT *P α vsC/EBP- ), eGFP , siRNA Ctrl vs siRNA no.55**P CtrlvssiRNA , siRNA Circulation Research.2020;127:1056–1073. 10.1161/CIRCRESAHA.120.316742 DOI: -DN ***P -DN –2 α vsC/EBP- ; eGFP =4.4×10 =1.1×10 =2.9×10 (no. 54, no. 55). Data are means±SD from3independentexperiments. P (no.54,no.55).Dataaremeans±SD = 5.2×10 = 3×10 -3 –2 . Claudin-5: (overall***P α vsC/EBP- ; eGFP -4 -3 . α vsC/EBP- ), eGFP –4 B pparg, andclaudin-5geneexpression inJAM-A–WT(left ) andJAM- -WT *P = ; , Quantificationoffold-differences forC/EBP-α cldn-5: (overall****P 3.9×10 (no. 54, no. 55). GAPDH isshownasloadingcontrol.Right , C/ (no.54,no.55).GAPDH -DN **P -DN =4.3×10 -5 α vsC/EBP- ), eGFP = 2.5×10 = 5.9×10 –2 -DN **P -DN -DN **P -DN = α vsC/EBP- ; eGFP -WT *P 5.1×10 α, Claudin-5inEndothelialFunction JAM-A, C/EBP- -3 α -WT orC/EBP- = . –3 2.8×10 Claudin-5: (overall****P . E = =1.7×10 . Cells were treated with nontargeting siRNA . CellsweretreatedwithnontargetingsiRNA , Quantification of permeability to fluorescein , Quantificationofpermeabilitytofluorescein 8.4×10 =2.8×10 -4 = : (overall***P ), siRNA Ctrl vs siRNA no.54**P CtrlvssiRNA ), siRNA 5.5×10 -6 α -WT (p42),C/EBP- α vsC/EBP- ), eGFP -WT ***P –3 –3 ; ; -2 pparg: (overall****P -4 –null vs JAM-A–null JAM-A–null eGFP α vsC/EBP- ; eGFP α vsC/EBP- ), eGFP -DN. Data are means±SD from Dataaremeans±SD -DN. -DN ****P -DN = =6.6×10 α -WT (p42),C/EBP- 2.7×10 = 3.9×10 = gene andclaudin-5gene values determined by valuesdeterminedby 5.9×10 -4 –4 ), siRNA Ctrl vs Ctrlvs ), siRNA ; eGFP vsC/ ; eGFP -WT ***P -dominant-negative -5 = ), siRNA Ctrl Ctrl ), siRNA ) and (upper)and 4.1×10 –5 -DN P -DN –7 ; -WT *P cldn-5: ), =3×10 = JAM-A– values values 2.9×10 -DN -DN : (overall : (overall =0.27, -6 values values ), ), =10 –4 ; -3 -2 ; ; Original Research 1065 September 25, 2020 . (Continued ) -2 JAM-A, C/EBP-JAM-A, Function in Endothelial α, Claudin-5 ; region no. 3 *P=2.9×10 -2 ; region no. 2 *P=4.5×10 -2 2020;127:1056–1073. DOI: 10.1161/CIRCRESAHA.120.316742 Circulation Research. 2020;127:1056–1073. JAM-A (junctional adhesion molecule A) promotes upregulation of claudin-5 expression via C/EBP-Figure 4. JAM-A (junctional adhesion molecule A) promotes upregulation of claudin-5 expression α (CCAAT/ β-catenin. is inhibited by active enhancer-binding protein-α), which chromatin immunoprecipitation analysis of C/EBP-A, Quantification of α binding to the claudin-5 promoter in JAM-A–wild-type (WT) and JAM- in the Data Supplement). DNA levels were normalized to the input. for details see Figure IVA A–null immortalized lung endothelial cells (iLECs; t P values determined by 2-sided unpaired Welch Data are means±SD as representative of 3 experiments. of triplicates from a single experiment, test. JAM-A–WT vs JAM-A–null: region no. 1 *P=3×10 Kakogiannos et al et Kakogiannos

Downloaded from http://ahajournals.org by on January 18, 2021 Downloaded from http://ahajournals.org by on January 18, 2021 18, January on by http://ahajournals.org from Downloaded Original Research remain to be defined. AU indicates arbitrary units; and EPAC,remain tobedefined. AU indicates arbitraryunits;and exchange proteindirectlyactivatedby cAMP. protein expression oractivity).The molecularmechanismsdashed arrows,regulationofdownstream targets(eg,mRNA, ofthisregulatorypathway EBP- =0.38, NS; TOP-FLASH, *P=1.9×10 TOP-FLASH, NS; P=0.38, A–null: FOP-FLASH from3independentexperiments. Pvaluesdeterminedby2-sidedunpaired Welchconstructs. Dataaremeans±SD ttest.JAM-A–WTandJAM- transcriptional activityofTCF (T-cell-factor)– β-catenin inJAM-A–WTand –null iLECs underexpression orFOP-FLASH ofTOP-FLASH ***P=3×10 α-DN vector vsC/EBP- lates 3 independentexperiments. Pvaluesdeterminedby2-sidedunpaired WelchP=8.6×10 **** vs TCF4-DN t test.C/EBP- α:JAM-A–WT:GFP from Dataaremeans±SD (green fluorescentprotein)or JAM-A–WT (left)and–null right) iLECs,TCF4-DN. underexpression ofGFP EBP- 3 JAM-A–WT 007 **P=1.5×10 **P=10 ANOVA followedbyDunnettT3test formultiplecomparisons.Regionno.1:(overall*** oftriplicatesfromasingleexperiment, representativeof3experiments.the input.Dataaremeans±SD PvaluesdeterminedbyBrown-Forsythe 007). levelswerenormalizedto DNA ′,5-cyclic monophosphate(8-pCPT-2′-O-Me-cAMP; or with8-(4-chlorophenylthio)-2′-O-methyladenosine-3 α bindingtotheclaudin-5promoterinJAM-A–WTand–nulliLECs treatedwithvehicleascontrol, immunoprecipitation analysisofC/EBP- 1066 region no.1,2,3****P=4.9×10 from asingleexperiment, representativeof3experiments. Pvaluesdeterminedby2-sidedunpairedWelch ttest.JAM-A–WTvs–null: (region no.1and2),oronlyregion33;fordetailsseeFigureIVBinthe regionno.1and2 α bindingsiteregionsoftheclaudin-5promoter(regionno.1,2,3),orboth luciferase reportersforall3C/EBP- Figure 4Continued.B,QuantificationoftranscriptionalreporterassaysinJAM-A–WTand–nulliLECs underoverexpression of α mRNA. sion ofC/EBP- of Wnt/β-catenin signaling suppresses the expres- Previous studieshavedemonstratedthatactivation α Inhibition Expression Through C/EBP- Signaling, Which InTurn ReducesClaudin-5 JAM-A DeficiencyIncreases β-Catenin This signalingpathwayispositivelyregulatedbyJAM-A. claudin-5 expression inresponsetoEPAC activation. din-5 promoterthroughJAM-A (Figure 4D). α bindingtotheclau- that EPAC isrequiredforC/EBP- absence ofJAM-A. These lastdatasupporttheconcept in thepresenceofJAM-A, withnoeffects seeninthe α bindingtotheclaudin-5promoteronly ened C/EBP- in thepresenceof007. Indeed,thistreatmentstrength- chromatin immunoprecipitationassayswereperformed α totheclaudin-5promoter, lates thebindingofC/EBP- EBP- C/EBP- promoter activity, andthiseffect wasreducedwhenthe α-WT induced claudin-5 Supplement). Here, C/EBP- respectively(FigureIVC intheData α-DN, or C/EBP- α-WT plasmids thatdrivetheoverexpression ofC/EBP- Kakogiannos etal vs JAM-A–null007 ***P=2.1×10 –WT: GFP vs TCF4-DN ***P=2.3×10 vs TCF4-DN claudin-5: JAM-A–WT:GFP =0.94, NS; cytoplasm:JAM-A –WT andJAM-A–null**P=3×10 P=0.94, NS; from3independentexperiments. Pvaluesdeterminedby2-sidedunpairedWelchmeans±SD ttest.Total lysate: JAM-A–WTand–null, respectively., phospho-β-cateninS552/total-ratiosquantifiedbydensitometryscanand expressed asfold Bottom changes. Dataare catenin S552(phosphor-β-catenin)inJAM-A–WTandJAM-A–nulliLECs. Tubulin andlaminBareshownascytoplasmicnuclearmarkers, (NS); regionno.3:(overall****P=4×10 (NS); no. 1,2,and3:(overall****P=1.8×10 representative of3experiments. PvaluesdeterminedbyBrown-Forsythe ANOVA followedbyDunnettT3testformultiplecomparisons.Region oftriplicatesfromasingleexperiment, as asindicated.Dataaremeans±SD α–dominant-negative (DN), α-WT, orC/EBP- full-length C/EBP- cellsoverexpressing claudin-5promoter-reporterplasmids(asinB)togetherwithemptyvectorascontrol,and reporter assaysinHEK-293T ; JAM-A–WTvs–null***P=4.8×10 α increases Overall, ourdatademonstratethatC/EBP- Finally, todeterminewhetherEPAC activationregu- -DN ****P=2.2×10 α-DN α-WT vsC/EBP- α-WT ***P=3.7×10 -DN; Figure 4C). α-DN; –3 β-catenin signalinginepithelialcells. . H,Schematic model ofJAM-A signalingintheregulationofvascular permeability. Full arrows,directeffects ondownstreamtargets; September 25,2020 α transcriptionalactivitywascompromised(C/ -4 -DN **P=1.3×10 α-DN ; emptyvectorvsC/EBP- -3 ; JAM-A–WTvs–null***P=8.7×10 -4 26,27 . E,Representativeimmunoblottingfortotallysate,cytoplasmandnuclear distributionoftotalandactiveβ- -4 -DN **P=8.1×10 α -DN α-WT vsC/EBP- ; C/EBP- JAM-A negativelyregu- -7 -6 ; regionno.2and3*P=1.6×10 ), JAM-A–WTvs007 **P=3.2×10 -4 -5 ; JAM-A–WTvs–null007 ***P=9.3×10 -7 ; regionno.2and3:(overall***P=7×10 α-WT ****P=3.2×10 ), emptyvectorvsC/EBP- –4 11 –null: GFP vs TCF4-DN ***P=7.1×10 vsTCF4-DN ; JAM-A–null:GFP Basedon Circulation Research.2020;127:1056–1073. 10.1161/CIRCRESAHA.120.316742 DOI: -2 . G,Quantificationoffold-differences ofC/EBP-αandclaudin-5geneexpression in -3 ; nucleus:JAM-A–WTand–null**P=4.9×10 -3 -DN ****P=10 α -DN α-WT vsC/EBP- ; C/EBP- -3 ; JAM-A–WTvs–null007 P=0.64,notstatisticallysignificant -2 ; regionno.3**P=1.9×10 EBP-α Indeed, Wnt-3a upregulatedaxin2,whileitinhibitedC/ β integration site family,MMTV member 3a), which stabilizes A EBP- TCF-mediated genetranscription. we canconcludeherethatJAM–A regulatesβ–catenin/ (Figure 4F; FigureIVG intheDataSupplement).Thus, that itwassignificantlyhigherintheabsenceof JAM-A a TCF (T-cell-factor) luciferase reporter, which showed expression ofitstargetgenesaxin2andccnd1using scriptional activity wasdetectedbymeasurementofthe Data Supplement).Consistentwiththis,β-catenintran- WT counterpart(Figure 4E;FigureIVEandIVFinthe levels of active nuclear absence of JAM-A, there was marked increase in the through IVFintheDataSupplement).Conversely, inthe JAM-A–null iLECs andfLECs (Figure 4E;FigureIVD phorylated atSer552wassimilarintheJAM-A–WTand Here, thetotalamountofactiveβ-cateninthatwasphos- localization andtranscriptionalsignalingofβ-catenin. JAM-A–WT and–nullcellsintermsofthenuclear α andclaudin-5expression. Therefore, wecompared enhances this evidence,wehypothesizedthatJAM-A depletion -catenin, –null iLECs weretreatedwithWnt-3a (wingless-type To determinewhetherβ -3 α ; regionno.3:(overall****P=9.2×10 at the mRNA level, compared with level,comparedwith andclaudin-5atthemRNA and claudin-5 regulation,JAM-A–WT and JAM- -3 P=2.1×10 ; JAM-A–WTvs–null***P=5.8×10 -4 43 α-WT **P=3.3×10 ), emptyvectorvsC/EBP- β-catenin signaling,tothusinhibitC/EBP- -4 totriggertheexpression ofitstargetgenes. ). Data are means±SD oftriplicates Data Supplement).aremeans±SD ; regionno.2:(overall**P=7.4×10 -6 -DN *P=3.2×10 α-DN ; emptyvectorvsC/EBP- α, Claudin-5inEndothelialFunction JAM-A, C/EBP- -4 ), JAM-A–WTvs007 **P=2.1×10 –4 -3 –null: GFP vs TCF4-DN vsTCF4-DN ; claudin-5:JAM-A–null:GFP . C,Quantificationoftranscriptional β-catenin, compared with the -catenin contributestoC/ -5 . D,Quantificationof chromatin -3 . F,Quantificationof -7 ), emptyvectorvsC/ -3 ), JAM-A–WTvs -4 ; JAM-A–WT -3 ; empty -2 ; C/ –6 ; - Original Research 1067 Of note, as claudin-5, and 48–50 mRNAs bloodin 5F, a through c; Fig- c; a through 5F, JAM-A, September 25, 2020 C/EBP-α we hypothesized that this is 46,47 has a tumor suppressor role in - the expres Hence, we evaluated 28 , and claudin-5, JAM-A, C/EBP-JAM-A, Function in Endothelial α, Claudin-5 Given the reduction in claudin-5 expression Given the reduction in claudin-5 expression 45 mRNA levels were significantly lower in GBM, Expression in Cancer Vasculature α Expression One of the characteristics of brain tumors is signifi- One of the characteristics staining in the tumor vasculature was lower compared vessels from tissue biopsies of 11 patients with grade IVgrade with patients 11 of biopsies tissue from vessels GBM., As illustrated in Figure 6A magnification. In the control littermate, the capillaries on littermate, the capillaries In the control magnification. of the electron microscopy cross-sections consecutive junction Here, a tight boxtomography examined. were channel) area (osmiophobic as a translucent appeared attached where they were plasma membranes, between 2 the dark to the cells), in side, relative (external other to each membrane (Figure plasma of the layers was pale channel ). This ure VIA in the Data Supplement consecutive serial tomography sec- observed in all of the or interruption, and the strands oftions without any break observed in close vicinity to each the tight junctions were VIA in the Data Supple- a through c; Figure other (Figure 5F, near an edematousment). In JAM-A–null capillaries located significantly more distant from each locus, the strands were the Data Supplement), and in the con- other (Figure VIA in was an interruption along the palesecutive sections, there 5E, d through f; Figure VIA in the (Figure strand channel pattern typically indicated that theData Supplement). This was impaired andbarrier function of these tight junctions leakage observed.that this break was responsible for the can impair the organization of the absence of JAM-A Thus, areas of edema,the tight junctions in correspondence with in the control of role of JAM-A supports the central which endothelial barrier function in vivo. related to JAM-A expression. Indeed, downregulation of Indeed, downregulation expression. related to JAM-A has been described for several solid tumors, such JAM-A as endometrial, pancreatic, and kidney tumors, and has been shown to correlate with tumor size and progression, as well as with poor prognostic outcome. sion of JAM-A, sion C/EBP-α Moreover, tissues. healthy patient-matched with compared and claudin-5 showed intense in the healthy tissue, JAM-A staining patterns at the endothelial cell-to-cell contacts, whereas in the GBM nor claudin-5 JAM-A vessels, neither were detectable (Figure 6B). In parallel, nuclear C/EBP- α with the vasculature of the healthy tissue (Figure 6C). in the GBM vasculature, JAM-A Positively Regulates Claudin-5 and C/ Regulates Claudin-5 and JAM-A Positively EBP- data presented aboveThe describe a novel molecular and clau- of JAM-A expression that links the mechanism of the din-5 and that is important for the maintenance 4H). integrity of the endothelial barrier (Figure as the can be explained cant peritumoral edema, which organization andresult of alterations to the tight junction claudin‑5. well as mediating JAM-A–positive regulation of claudin-5 well as mediating JAM-A–positive transcription, C/EBP-α solid tumors. different , JAM-A–null expression by expression -catenin signaling Given that claudin-5 19 negative TCF4 construct in JAM-A–WT construct in negative TCF4 –catenin transcriptional activity. C/EBP- activity. –catenin transcriptional α β compared with the control compared and claudin-5 5 is a key component of component key a is claudin‑5 Moreover, –null iLECs increased the mRNA increased JAM-A–null iLECs expression 10,23,44 C/EBP-α Furthermore, in the cortex of JAM-A–null mice, fluid in the cortex Furthermore, regulates the size-dependent paracellular pathway in regulates the size-dependent paracellular whether loss of we investigated the blood-brain barrier, endothelial permeability to the leads to increased JAM-A bioti- Da) lysine-reactive (443.43 weight molecular low nylation reagent sulpho–N-hydroxysuccinimide–biotin, brain parenchyma. from the intravascular space into the with JAM-A–WT littermates, Compared leakage of adult mice showed significantly increased in the cerebral sulpho–N-hydroxysuccinimide–biotin and midbrain (Figure 5A striatum, hypothalamus, cortex, with widespread the brain areas and 5B). Interestingly, pro- of claudin-5 downregulation also showed leakage is consistent with loss of barrier which tein expression, in the Data Supplement). function (Figure 5C; Figure VA barrier determine the size selectivity of the endothelial To molec- larger of extravasation examined we dysfunction, cadaverine (950 ular weight tracers in the brain, using leakage no while kDa). Interestingly, (10 dextran and Da) in the was detected, dextran weight high molecular of the the permeability to cadaverine was absence of JAM-A and the increased in the cortex but significantly, slightly, in the Data hypothalamus (Figure 5D; Figure VB and VC can signaling JAM-A that suggested Supplement). This regulate the paracellular passage of molecules into the brain in a claudin-5–like size-dependent manner. leakage from blood capillaries and edema were confirmed using 2-step transmission electron microscopy tomography. inter- with using serial sections samples, sectioned the We the first section (60 nm) was Initially, leaved thicknesses. and an area where under electron microscopy, examined was selected edema was evident in the absence of JAM-A examined 200-nm section was the first (Figure 5E). Then for the cross-section of the blood capillary located near the edematous area (Figure VIA in the Data Supplement). 2 serial tomography boxes were obtained at higherNext, 2020;127:1056–1073. DOI: 10.1161/CIRCRESAHA.120.316742 Circulation Research. 2020;127:1056–1073. in turn, sustains claudin-5 expression, which controls the which expression, in turn, sustains claudin-5 (Figure 4H). vascular permeability Multiple studies have demonstrated that JAM-A is demonstrated that JAM-A Multiple studies have regulation of epithelial barrier func- involved in positive tion. JAM-A Deficiency Increases Endothelial Increases JAM-A Deficiency in the Brain Microvasculature Permeability - using a dominant and of suppressing 4G). Taken together, these data demonstrate together, 4G). Taken cells (Figure positively regulates C/EBP-that JAM-A α ). in the Data Supplement cells (Figure IVH vehicle-treated β inhibition of endogenous In contrast, the tight junction strands, and it is highly expressed in expressed the tight junction strands, and it is highly permeability brain endothelial cells, where it decreases to small molecules (<800 Da). Kakogiannos et al et Kakogiannos

Downloaded from http://ahajournals.org by on January 18, 2021 Downloaded from http://ahajournals.org by on January 18, 2021 18, January on by http://ahajournals.org from Downloaded Original Research sections fromJAM-A –WT andJAM-A–nullmice.Scale bar: 20µm(n=3,JAM-A–WT;n=3, JAM-A–null). (Continued ) leakage(pseudocolor)inbrain (platelet/endothelial celladhesionmolecule-1; red),andstreptavidin,forsulpho-NHS-biotin (green), PECAM-1 1068 value determinedby2-sidedunpaired Welch ttest.JAM-A–WTvs JAM-A–null ***P=6.4×10 area/braintotalarea(n=4, JAM-A–WT; n=4,–nullmice).P asratioofsulpho-NHS-biotin sections, asshowninA.Dataaremeans±SD, leakageinJAM-A–WTand–nullmicebrain Scale bar:1mm(n=3,JAM-A–WT;n=3, JAM-A–null). B,Quantificationofsulpho-NHS-biotin brain sections(100μm)fromJAM-A –wild-type (WT)andJAM-A–nullmice.Redarrows(right)indicatefociofleakageindifferent brainregions. leakageinvibratome A, Representativeconfocalmicroscopy forstreptavidintodetectsulpho–N-hydroxysuccinimide–biotin (sulpho-NHS-biotin) decreased claudin-5. Figure 5.EndothelialpermeabilityisincreasedinvivobyJAM-A(junctional adhesionmoleculeA)deficiencyinareasof Kakogiannos etal September 25,2020 Circulation Research.2020;127:1056–1073. 10.1161/CIRCRESAHA.120.316742 DOI: -4 . α, Claudin-5inEndothelialFunction JAM-A, C/EBP- C, Representativeconfocalmicroscopy forclaudin-5 Original Research

, 54 20 that 1069 which which 28–30 39,40 indicate that indicate that 20 September 25, 2020 , and claudin-5 are decreased and different cancers, and different 55,56 Here, we now shown that JAM-A regu- Here, we now shown that JAM-A 13 JAM-A, C/EBP-JAM-A, Function in Endothelial α, Claudin-5 Interestingly, although increasing evidence has sug- has evidence increasing although Interestingly, We have shown here that EPAC activates C/EBP- here that EPAC have shown We α absence of have previously demonstrated that the We In conclusion, our data demonstrate a novel regulatory lates vascular permeability via the control of the strength oflates vascular permeability via the control highlights This tight junctions through claudin-5 expression. an important regulator of the vas- as a dual role for JAM-A, cular barrier properties to both solutes and circulating cells. underlying vascular leakage gested that the mechanisms organ-specialized vasculatures, might vary in different downstream of JAM-A in endothelial cells. However, the However, in endothelial cells. downstream of JAM-A be to of this regulation remain molecular mechanisms of C/EBP-modification Covalent further. explored by α might kinases protein EPAC-activated intermediate the cer- it has been demonstrated that In this respect, occur. tain C/EBP for ERK isoforms are substrates - (extracel kinase), ribosomallular signal-regulated and S6 kinase, resulting phos- kinases. The protein kinase C protein the activity of C/EBPs,phorylation enhances although C/EBP the different by specific proteins are regulated redundancy. without functional mechanisms activation of Rap-1, which impairs EPAC-mediated JAM-A of leukocytes andis required to restrict transmigration mesoangioblasts. the signaling pathway of JAM-A–EPAC–C/EBP-α we have identified here is conserved in diverse tissues,we have identified here is conserved underlines the possible broad- This as brain and lung. such the maintenancespectrum relevance of this signaling in its possible altera- of vascular integrity in physiology and reported that thetions in pathologies. Indeed, it has been C/EBP-levels of JAM-A, α in several diseases Therefore, our data and previous findings data and previous our Therefore, also implies a role for these molecules as tumor sup- also implies a role for these molecules the JAM-A in pressors. Our findings show that reduced human GBMpar- carcinoma vasculature and ovarian allels the downregulation of claudin-5 and C/EBP-α suggests a correlation between these players in which pathological situations in humans. a component of the tight junctions, loop through which restricts vascular permeability by enhancing the JAM-A, of claudin-5, a crucial functional component expression pathway should of the tight junctions themselves. This help to identify novel therapeutic targets for diseases associated with vascular barrier dysfunction. -catenin can repress the transcription of claudin-5 transcription of claudin-5 can repress the β-catenin through indirectly mechanisms: parallel different through of C/EBP-inhibition and directly through α expression; box (forkhead O1) and TCF. with FoxO1 interactions ; dextran P=0.56, not statistically significant (NS).; dextran E, Representative -4 β-catenin Claudin-5 Indeed, our 51 26,27,38,39 We describe here a novel role for We Here, we have shown that C/ β-catenin acts as a negative regu- 52 53 -catenin transcriptional activity, which, in turn, which, β-catenin transcriptional activity, were significantly lower in the ovarian cancer α were significantly binds directly to the claudin-5 promoter to induce binds directly to the claudin-5 promoter α The mechanism by which JAM-A activates C/EBP- JAM-A by which mechanism The α Finally, we extended this approach to high-grade to high-grade this approach we extended Finally, suggest that the dysregu- these data together, Taken Claudin-5 transcription can be controlled by various by various controlled be Claudin-5 transcription can EBP- - transcription of the claudin-5 gene, thus enhancing clau din-5 expression. in turn activates C/EBP- which requires EPAC, α. In this in contrast, context, lator of claudin-5 expression. Indeed, JAM-A deficiency Indeed, JAM-A lator of claudin-5 expression. increases of C/EBP-inhibits the expression α, thus reducing clau- din-5 transcription. A role for active β-catenin in suppres- of C/EBP-sion of the expression α is in agreement with cell types. in other previous reports is the major constituent of endothelial cell-to-cell tight is the major constituent of endothelial maintenance of junctions, and it has a critical role in the the vascular barrier. 2020;127:1056–1073. DOI: 10.1161/CIRCRESAHA.120.316742 Circulation Research. 2020;127:1056–1073. , Quantification of cadaverine and dextran leakage in JAM-A–WT and JAM-A–null mice brain sections. Data are Figure 5 Continued. D, Quantification of cadaverine and dextran means±SD, n=4, JAM-A–WT; n=5, JAM-A–null mice). P values determined by 2-sided measured as relative intensities (arbitrary units; AU; P=7.9×10 t test. JAM-A–WT vs JAM-A–null: cadaverine *** unpaired Welch DISCUSSION of deteriorating vascular integrity is One of the hallmarks occurs in pathologi- which increased vessel permeability, The as inflammation and tumor growth. such cal contexts the stabil- through vascular barrier is mainly maintained ity of the endothelial cell-to-cell junctions. serous ovarian cancer, where the expression of JAM-A, of JAM-A, where the expression cancer, serous ovarian and C/EBP-claudin-5, was evaluated α in blood vessels - by immunofluores tissue biopsies in patient-derived and claudin-5, JAM-A, levels of endothelial cence. The C/EBP- with the healthy ovarian controls (Fig- tissues, compared ure VIIA through VIIE). in the Data Supplement that involve the mechanisms lation of these molecular patho- has claudin-5 and JAM-A proteins junction tight logical implications. the transcription factor C/EBP-α that acts downstream junctions) tight cell-to-cell of component (a JAM-A of to promote the transcription of claudin-5, thus and EPAC in vivo and in vitro. strengthening the endothelial barrier JAM-A junctions, tight of component a as Therefore, of tight junc- contributes to the organization and function of claudin-5. tions through enhanced expression can be regulated signaling pathways, and its activity post-translationally. transmission electron microscopy of cerebral cortex from JAM-A–WT and JAM-A–null mice. Arrow denotes locus of edema. Scale bar: 1200 nm transmission electron microscopy of cerebral cortex from JAM-A–WT (n=3, JAM-A–WT; n=3, JAM-A–null). F, Representative 2-step transmission electron microscopy tomography of cerebral cortex showing a tight junction strand. White (a–c) and (d–f) are 3 consecutive serial tomography sections (a–c) and JAM-A–null mice (d–f). Parts between 2 plasma membranes; yellow arrow; interruption along arrows, tight junction that appears as the translucent area (osmiophobic channel) Scale bars (a–c): 80the pale strand channel. nm; (d–f) 90 nm (n=3, JAM-A–WT; n=3, JAM-A–null). LBC indicates lumen of brain capillary. data indicate that JAM-A deficiency increases deficiency that JAM-A indicate data in turn reduces and signaling, which nuclear localization C/EBP-of inhibition through expression claudin-5 α. Kakogiannos et al et Kakogiannos

Downloaded from http://ahajournals.org by on January 18, 2021 Downloaded from http://ahajournals.org by on January 18, 2021 18, January on by http://ahajournals.org from Downloaded Original Research EBP- (filteredC/ α andPECAM-1 formergedimagesoffiltered C/EBP- (S.ERG) related gene)staining wassegmentedwiththreshold 350-4.096 C/EBP- andpatient-matched healthytissue.Imagesarerepresentativeof11patients.Scalebars: 20µm. GBM (white),JAM-ARepresentative confocalmicroscopy forPECAM-1 (red),and claudin-5(green)inparaffin-embeddedtissuesections(4µm)from 1070 vspathological(P):JAM-A**P=6.8×10 by 2-sidedpairedttest.Non-pathological(NP) andpatient-matched healthy tissues.Pvaluesdetermined (platelet/endothelial celladhesionmolecule-1). The lineslink valuesbetweenGBM expression foreach followingreal-timequantitativepolymerase chain genewasnormalizedtoPECAM-1 reactionanalysis.mRNA (GBM) A, QuantificationofJAM-Aclaudin-5,andC/EBP-αexpression intissuebiopsiesfrom11patientswithgrade IVglioblastomamultiforme protein-α) expressioninglioblastomamultiforme. α (CCAAT/enhancer-binding Figure 6.JAM-A(junctionaladhesionmoleculeA)positivelyregulatesclaudin-5 andC/EBP- Kakogiannos etal PECAM-1). Yellowα PECAM-1). nucleitoexclude signalsbelonging tocirculatingcells.Scalebars: 20µm. dashedcirclesoutlineERG-positive m) from GBM and patient-matched healthy tissue. ERG (ETS- andpatient-matched healthytissue.ERG (red)inparaffin-embedded tissuesections(4μm)fromGBM α (green)andPECAM-1 September 25,2020 Circulation Research.2020;127:1056–1073. 10.1161/CIRCRESAHA.120.316742 DOI: -3 ; claudin-5***P=1.3×10 α, Claudin-5inEndothelialFunction JAM-A, C/EBP- C, Representativeconfocalmicroscopy for -4 ; C/EBP-α****P=2.1×10 -5 . 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REFERENCES Disclosures None. Supplemental Materials Major Resources Table Detailed Methods I and II Online Tables Online Figures I–VII Unedited Gels Full References Associazione Italiana per la Ricerca sul Cancro (AIRC;sul Ricerca per la Italiana Associazione study was supported by This Investigator Grants 18683 and 21320), the AIRC 5x1000 call Metastatic dis- ease: the key unmet need in oncology of the MYNERVA (My- 21267 project, no. AIRC), Council (proj- Venture the European Research eloid Neoplasms Research Networks BtRAINect EC-ERC-V-EPC, grant Initial Training contract 742922), - Founda the CARIPLO Lombarde) (Cassa di Risparmio delle Provincie 675619, tion (2014-1038, 2016-0461), the Italian Ministry of Health (Ricerca Finalizzata (GGP19202), Foundation IEO-CCMthe PE-2016-02362551),Telethon the (Istituto Europeo di Oncologia-Centro the Cardiologico Monzino) Foundation, and Council, and the Knut the Swedish Science Foundation, Umberto Veronesi Foundation. Alice Wallenberg Sources of Funding Sources We thank Amanda Oldani for image processing support and Fabrizio Orsenigo for for image processing support and Fabrizio thank Amanda Oldani We Ber- thank Christopher P. We claudin-5 promoter constructs. sharing luciferase patient informed consent Bello for collecting Lorenzo rie for editorial assistance, (IRB1299) procedure clinical the to according statistical for Binelli Giorgio and the lentiviral human for providing are indebted to Ruud D. Fontijn support. We for providing the retroviral C/EBP-claudin-5 constructs, and with Daniel Tenen designed the study; L. α constructs. M. Giannotta and E. Dejana conceived and and and A.A. Scalise performed the in vitro experiments M. Giannotta, Ferrari, Ravà and N. Kakogiannos analyzed the results with C. Giampietro; C. Maderna, M. and analyzed the results with M. Giannotta; performed the in vivo experiments Martini and I. Costa Pisati performed paraffin-embedded tissue sectioning; E. F. M. N. Kakogiannos, performed imaging analysis. M.G. Lampugnani, C. Giampietro, Lupia and U. discussions. M. contributed to scientific Malinverno, and A. Taddei cancer patients and Cavallaro provided tissue biopsies from healthy and ovarian and N. Rudini provided tis- analyzed the data with M. Giannotta; B. Fernandes multiforme and sue biopsies from healthy patients and patients with glioblastoma per- Mironov A.A. and Beznoussenko, G.V. Giannotta; M. with data the analyzed M. Giannotta and M.G. Lampugnani microscopy experiments; formed the electron revised by E. Dejana, C. Giampietro, and A. Taddei. was wrote the article, which has been approved by all of the authors. final form of the article The Acknowledgments Affiliations C.M., A.A.S., the FIRCFrom L.F., Milan, Italy (N.K., of Molecular Oncology, Institute Laborato- E.D., M.G.); EMPA, A.A.M., Swiss Federal I.C., G.V.B., M.G.L., M.M., E.M., Mechanics, Continuum Experimental Technologies, and Science Material ries for Experimental Oncology (M.R.) and Unit of Gynae- Dübendorf, Switzerland (C.G.); of Oncology IRCSS, (M.L., U.C.), European Institute cological Oncology Research - Mario Negri Institute for Phar United Kingdom (A.T.); Milan; Benevolent AI, London, SRL Milan (M.G.L.); Cogentech macological Research, Milan Benefit Corporation, Centre, Rozzano, Milan (B.F., Unit, Humanitas Clinical and Research Pathology (F.P.); Milanof Medicine, University of School and Haemato-Oncology, N.R.); Oncology Sweden (E.D.). Uppsala University, Genetics and Pathology, (E.D.); and Immunology, ARTICLE INFORMATIONARTICLE 10, 2020; accepted July 13, revision received July 10, 2020; Received February 2020. Kakogiannos et al et Kakogiannos

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