ri nohla cells endothelial IL-1 brain in Cldn5 of downregulation Ato o orsodne([email protected]) correspondence for *Author Tampa, Florida, South of USA. University 33612, Medicine, FL of College Morsani Surgery, eiie nvriyo ot lrd,Tma L362 USA. 33612, FL Tampa, Florida, South of University Medicine, o-uceMc srqie for required is Mlck Non-muscle ARTICLE RESEARCH ß 1840 2014 January 12 Accepted 2013; October 11 Received restrictive most The the (BBB). 1 barrier be blood–brain the to termed is considered cerebral and the endothelium of Malik, is endothelium and The is 1999). microcirculation (Mehta Curry, permeability tree and endothelial Michel vascular 2006; completely of the than throughout degree rather heterogeneous the permeable although selectively restrictive, is vascular be endothelium the of to of component function considered barrier crucial The homeostasis. a tissue is and function barrier Endothelial INTRODUCTION Claudin-5, kinase, IL-1 chain FoxO1, light myosin Non-muscle WORDS: KEY nmMlck. on by dependent caused repression from Cldn5 isolated that identified b BMVECs we nmMlck, primary for using null mice importantly, Most Cldn5. of of translocation nuclear repression transcriptional on depends that by mediated manner in a in dysfunction that BMVECs IL-1 hypothesis the barrier modulates tested we nmMlck nmMlck-dependent factors, these IL-1 mediate Considering epithelia. and to hyperpermeability, shown endothelial light in myosin molecules involved Non-muscle the signaling by encoded the translocation. (nmMlck, kinase and chain nuclear (FoxO1), their O1 regulating microvascular protein of factors brain box transcription regulation the forkhead to maintaining Transcriptional cell–cell attributed been integrity. endothelial for has Cldn5 (BMVEC) at crucial found cell are protein endothelial junction that tight contacts Claudin-5 a unclear. is remain IL-1 (Cldn5) mechanisms that the of but known phenotype hyperpermeability is hallmark a It is neuroinflammation. dysfunction cytokine (BBB) pro-inflammatory barrier the Blood–brain of levels the interleukin-1 in elevation Aberrant ABSTRACT Jr Beard, S. Richard ihIL-1 with ioa .Malinin L. Nikolay eateto oeua hraooyadPyilg,MraiCleeof College Morsani Physiology, and Pharmacology Molecular of Department ctnnadFx1i IL-1 in FoxO1 and -catenin 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,14–83doi:10.1242/jcs.144550 1840–1853 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. b b b nue are yfnto ocmtnl ihthe with concomitantly dysfunction barrier induced , b b ctnnadFx1 efudta raigBMVECs treating that found We FoxO1. and -catenin (IL-1 ctnn lo–ri are,Neuroinflammation barrier, Blood–brain -catenin, b otiue onuonlmaoydiseases. neuroinflammatory to contributes ) 1 itrChatterjee Victor , b b 1 ctnnadFx1adterepression the and FoxO1 and -catenin mdae oneuaino ln in Cldn5 of downregulation -mediated ic .Haines J. Ricci , b mdae are yfnto was dysfunction barrier -mediated Mylk b ee sakyregulator key a is gene) ietyidcsBBB induces directly 1 2 yogJ Cha J. Byeong , ei .Wu Y. Kevin , 2 b eatetof Department b ctnnand -catenin a been has 1 ao .Reynolds J. Jason , b 1 b akH Wu H. Mack , mdae are yfnto in dysfunction barrier -mediated ctnn n FoxO1-dependent and -catenin- nlmaoymdae B irpini fcuilimportance. crucial of of is mechanisms disruption al., the BBB et understanding inflammatory-mediated Winter encephalitis Hence, 2005; 2008). as sclerosis, Davis, Zlokovic, and 2004; such multiple (Hawkins disease stroke, diseases, Alzheimer and injury, neuroinflammatory brain and/or several traumatic onset thus the of and in BBB, implicated progression the been of routes has flux hyperpermeability, Dysfunction transcellular solute BBB 2010). by Miller, occur most 1999; to forcing (Banks, endothelium cerebral diffusion, the the of paracellular across contacts restrict (EC–EC) cell–cell BBB endothelial developed highly vdnefo nmlsuishv upre oefrIL-1 of for role lines a supported Several have studies diseases. animal from neuroinflammatory evidence in dysfunction BBB (IL)-1 interleukin PKC endothelia th microvascular reported recently have We 2003). IL-1 recombinant injecting (2) 2007); al., al., et et Chakraborty 2004; Simi al., 2010; et (Basu BB neuroinflammation of with models concomitantly elevated neuro during dysfunction ncnrs oteeiec nfvro oefrIL-1 for role a of favor in evidence barrier epithelial the 1990). gut to al., et and contrast (Ligumsky 2008) In disease al., bowel et inflammatory in (Ganter dysfunction injury lung acute endo in microvascular lung as dysfu such causing in implicated been also IL-1 of effect 1995). by reinforced al., also et is Yamasaki IL-1 2005; for evidence al., Furthermore, et (Lazovic hype neuroinflammation BBB attenuate receptor, IL-1 IL-1 (3) and 1991); (Blamire hyperpermeability BBB n eeoyi otcsa CE oncin,adaelinked are and and (Ocln) connections, homotypic EC–EC both occludin at form of that contacts proteins family heterotypic properties (Cldn) the 2008). and claudin al., of the ‘barrier’ et of (Liebner consist members the BBB junctions the forming as Tight such and endothelia, the specialized sealing cell–cell gaps for commonly and to important morphogenesis are paracellular addition are vascular junctions tight in in whereas role junctions and, remodeling, important cells an Adherens play endothelial adhesion, 2012). all adherens in al., as 2004; expressed Dejana, known in et detail junctions in McEwen (reviewed distinct junctions tight two and junctions of largely consists rel the regarding information dis several in permeability vascular brateeaini h eeso h r-nlmaoycytokine pro-inflammatory the of levels the in elevation Aberrant h oeua opsto fE–Ccnat scmlx but complex, is contacts EC–EC of composition molecular The h dpnetbrirdsucin(io ta. 02.IL-1 2012). al., et (Rigor dysfunction barrier -dependent 2 b n aa .Yuan Y. Sarah and nteidcino B yfnto Dde tal., et (Didier dysfunction BBB of induction the in b 1 b tpai .Davis M. Stephanie , a enipiae sasgiiatmdao of mediator significant a as implicated been has eetratgnss rgntcdlto fthe of deletion genetic or antagonists, receptor nvitro in nlmain 1 IL-1 (1) inflammation: el BVC)wt IL-1 with (BMVECs) cells l tdmlclrmechanisms. molecular ated xeiet htdmntaeadirect a demonstrate that experiments b hla el(ME)permeability (LMVEC) cell thelial eitn B hyperpermeability BBB mediating ae,teei eaiepuiyof paucity relative a is there eases, yepreblt nanimal in hyperpermeability B ta. 00 ugirloe al., et Quagliarello 2000; al., et preblt hti nue by induced is that rpermeability ttetn rmr ua brain human primary treating at cino te isebarriers, tissue other of nction 1, * 1 onE Elliott E. John , b ocnrtosare concentrations b b b -mediated b induces induces nBBB in b has 1 ,

Journal of Cell Science eosrtdtesgiiac fnMc nmediating in nmMlck of have Rigor, significance animals and modified unique the (Yuan genetically a endothelium using demonstrated represents mature Experiments kDa), in 2010). 210 expressed or (nmMlck, Mlck1, isoform cells. endothelial Mlck in expressed non-muscle are Mlck2) and (Mlck1 vrxrsino cni el htarayepesCldns express yet already the strands, that Of 2013). cells junction Daneman, and (Sohet in tight properties normal barrier Ocln strengthens of exhibit of maturation mice overexpression already-existing Ocln-knockout unknown, and strengthen is because to The BBB development only the junctions, 2005). serve of might Davis, junctions tight protein tight and occludens the in (Hawkins zonula Ocln and of ZO-2) by role and cytoskeleton precise (ZO-1 actin the proteins to intracellularly ARTICLE RESEARCH ftefu ao sfrsecddb the by encoded isoforms activity. to contractile major during promote four known actin and the with myosin is (MLC) Of of chains interaction that regulatory the is Mlck kinase myosin permeability. endothelial phosphorylate of calcium/calmodulin-dependent regulator crucial a in a as dysfunction act to BBB potential for targets investigate important conditions. therapeutic be neuroinflammatory to could determining information and Such for pathway. this dysfunction, of IL-1 regulators in barrier present to is valuable BMVEC be mechanism would this it stimuli, whether pathologic determine to BBB, response the (McColl in the gene mice at in of role injury expression the brain Considering 2008). Cldn5 ischemic al., et mild of after downregulation IL-1 al., particularly that et to demonstrated (Jang been injury (Beard has leads lung it hyperhomocysteinemia addition, acute In acrolein-induced in 2011). to and 2011) response as al., in this such et reversed that be stimuli, emerging can the is pathologic regulation Cldn5 Evidence in for for stimuli. mainly mechanism endothelium external explored mature mechanisms in to been to responding opposed is other have as endothelium junctions developing and FoxO1 the FoxO1 tight This of reducing whereas upregulation Akt, localization. junctions, active nuclear constitutively adherens by phosphorylated at (VE)-cadherin confluence, and reach of (FoxO1) factor transcription transcription TCF/LEF-dependent box inhibits forkhead cells, the endothelial the subconfluent with of In localizes 2008). repression al., of transcriptional et (Taddei accumulation resulting nuclear the the and preventing thus entirely contact, whereby discovered, not was b development still Cldn5 endothelial during of Cldn5 are of mechanisms conditions the models pathologic However, animal understood. 2008). in 2011; in al., al., regulation et et permeability (Beard McColl is neurodegeneration BBB and expression associated neuroinflammation Cldn5 increased been al., has of et downregulation with degree (Daneman Cldn5 ‘tightness’ the Furthermore, junction 2010). and with brain, correlated only positively the both is and the expression in (Hawkins its in is pan-endothelial, endothelium highest is in Cldn5 Cldn5 expressed Although found 2005). junctions, exclusively are Davis, is tight isoforms that endothelial isoform Cldn and the several in epithelial 2003). expressed al., are though et 12 Wolburg and 2008; Even al., 5 et 3, (Liebner 1, endothelium Cldns vascular only isoforms, Cldn 24 rnlcto and translocation ctnni eusee tahrn ucin pnEC–EC upon junctions adherens at sequestered is -catenin eety ehns for mechanism a Recently, ysnlgtcankns Ml rMc)hsbe shown been has Mlck) or (Mylk kinase chain light Myosin b b ctnni eusee yvsua endothelial vascular by sequestered is -catenin ctnnmdae ersino the of repression -catenin-mediated b ctnndpnetrglto of regulation -catenin-dependent b ctnnatvto,nuclear activation, -catenin Cldn5 Mylk hncells When . Cldn5 b b ee two gene, -mediated b b signaling -catenin -catenin Cldn5 gene nohla otatlt n yepreblt ne pro- under ( hyperpermeability nmMlck-isoform-specific knockout Importantly, and conditions. inflammatory contractility endothelial 02,TRrsossbgnt rpi oe n time- and dose- IL- with a treatment in of hours drop 6 first to 1 the within began al., manner et responses dependent (Rigor 2A,B). models (Fig. TER endothelial other system 2012), in reports (ECIS) with sensing Consistent electric an impedance to using IL-1 current cells cell–substrate with electrical to hours confluence bEnd.3 resistance 24 post adhesive for days growing cell–cell them 5 treating by for then mature model, and to them our allowing in confluence, (TER) experiments, IL- aberrant resistance of with associated this set is extend 1 that underlying first dysfunction mechanisms we barrier this the cells), BMVEC characterize In further (bEnd.3 and 2012). observation BMVECs al., immortalized et using Rigor 2003; irpino h B MCl ta. 08.Furthermore, 2008). al., et (McColl BBB neuroinflammation-induced the in of target IL-1 disruption molecular that predominant suggesting kDa), 2D). (66.4 bEnd.3 (Fig. FITC–albumin hyperpermeability of size-selective to mediates permeability kDa), IL-1 not the (4.4 TRITC–dextran that but in and fluorescein found increase sodium to sodium we monolayers significant molecules, to a sized induced monolayer differently the comparing of IL-1 permeability ( the fluorescein in increase yepreblt nbanmcoaclrendothelial microvascular brain in hyperpermeability IL-1 RESULTS esrdtepreblt ofiin ( of coefficient molecules permeability we to the assays, monolayer does permeability measured transwell the TER using Therefore, of current. sizes. permeability different electric the to indicate only not but resistance), paracellular hours 24 least at for persisted stimulation. and after hours 6 beyond continued TER BMVECs cultured treating that shown have IL-1 others with and We monolayers cell oneuaino ln n yfnto fteBMVEC the the of involves IL-1 of repression that dysfunction and manner modulates translocation and a in nmMlck Cldn5 barrier study, of that this in has downregulation 2011), hypothesized al., nmMlck et (Sun Because dissociating we junction in adherens investigated. the involved at been be cadherin not to shown has activated been BBB and IL-1 in Yuan the and nmMlck in of of (reviewed role ROS the documented the 2010), well Rigor, thrombin, and proinflammatory been has histamine, junctions to neutrophils) cell–cell (including response endothelial shock agents hyperpermeability of endotoxic mediating in opening in paracellular nmMlck survival of the role improved the both Although and 2007). al., atherosclerosis 2011) et 2007), (Ranaivo al., al., attenuated et et (Reynoso an burns (Sun severe (Wainwright display 2003), injury al., lung mice et acute to these response inflammatory yet vascular defects, cardiovascular 4hus n oprdterslswt hs funtreated of those with IL-1 results with the Treatment compared monolayers. IL-1 and with treated hours, were 24 that monolayers bEnd.3 in molecules b b hr sagoigbd feiec hwn htCd5i the is Cldn5 that showing evidence of body growing a is There E esrste‘ihns’o elcl dein(i.e. adhesion cell–cell of ‘tightness’ the measures TER utemr,w dniidta IL-1 that identified we Furthermore, . b b inln.Frt eaaye rnedteilelectrical transendothelial analyzed we First, signaling. nue ln oneuainadsize-selective and downregulation Cldn5 induces mdae E hne Fg C.Adtoal,when Additionally, 2C). (Fig. changes TER -mediated ora fCl cec 21)17 8015 doi:10.1242/jcs.144550 1840–1853 127, (2014) Science Cell of Journal b nmmlck nue nohla are yfnto Dde tal., et (Didier dysfunction barrier endothelial induces , . D)ta a nesl orltdwt the with correlated inversely was that kDa) 0.4 2 / 2 iedmntaeapeoyedvi of devoid phenotype a demonstrate mice ) Cldn5 b b ctnnatvto,nuclear activation, -catenin asdatime-dependent a caused P b b S mdae erae in decreases -mediated ee(i.1). (Fig. gene mdae dysfunction -mediated o ifrnl sized differently for ) b ctnnfo VE- from -catenin b hl measuring while b -mediated 1841 b for b b

Journal of Cell Science EERHARTICLE RESEARCH 1842 2003). al., in localization et and (Nitta expression postpartum Cldn5 hours the examined several we of die Therefore, ‘loosening’ and size-selective BBB exhibit mice Cldn5-deficient mechanisms. IL-1 established in previously involved indicate be lines might solid (nmMlck) whereas kinase of chain mechanisms, translocation light nuclear myosin the non-muscle promotes which through pathways dysfunction. signaling barrier of Diagram 1. Fig. ehpteieta mlkmdltsIL-1 modulates nmMlck that hypothesize We b ctnnadterpeso fteCd5gn.Qeto ak n ahdlnsidct nnw signaling unknown indicate lines dashed and marks Question gene. Cldn5 the of repression the and -catenin b mdae oneuaino ln n yfnto fteBVCbriri anrthat manner a in barrier BMVEC the of dysfunction and Cldn5 of downregulation -mediated epnet IL-1 to response uniaieR-C.Cnoa irsoyrvae ht in that, revealed microscopy Confocal and assays RT-PCR. (ICW) quantitative western in-cell analysis, immunofluorescence ora fCl cec 21)17 8015 doi:10.1242/jcs.144550 1840–1853 127, (2014) Science Cell of Journal b ramn yuigwsenbotn,confocal blotting, western using by treatment o analysis. hoc ofiins( coefficients permeability determine to were performed assays permeability transwell then IL-1 vehicle or with control treated were Monolayers (D) fluorescein. sodium to permeability the of IL-1 Time-course (C) ng/ml). 200 IL-1 with bEnd.3 monolayers of points treatment time the indicated during the at relative decline of TER analysis Statistical (B) indicates s.e.m. shading and arrow) by (indicated t mean * the as presented are enrssac omlzdto normalized resistance mean or PBS) in IL-1 BSA (0.1% control treated vehicle monolayers with of tracings ECIS (A) permeability. size- monolayer increasing selective while TER in decrease IL- with 1 cells) (bEnd.3 cells microvascular endothelial brain Treating 2. Fig. 6. D;Soe’radius Stokes’ kDa; (66.4 radius Stokes’ kDa; (4.4 TRITC–dextran radius Stokes’ kDa; (0.376 fluorescein sodium molecules: 5 P b , ;oewyAOAwt unt’ post Dunnett’s with ANOVA one-way 0; nue ie n dose-dependent and time- a induces b b .5cmae ihvhcecnrlor control vehicle with compared 0.05 mdae nraei monolayer in increase -mediated 20n/l.Taig niaethe indicate Tracings ng/ml). (200 < . m n FITC–albumin and nm) 1.4 P b S o h following the for ) 10n/l o 4hours, 24 for ng/ml) (100 b b ete 0 or 100 (either mdae BMVEC -mediated < < .8n) Data nm). 3.48 6 .5nm), 0.45 s.e.m. t 5 0

Journal of Cell Science yae ofre infcn euto nCd5lvl in levels whole-cell Cldn5 of in reduction analysis significant IL-1 Cldn5 to blot IL-1 a response However, confirmed with western lysates treated significantly Additionally, panels). was monolayers panels). junctions upper in tight at reduced 3A, localization (Fig. and expression junction ZO-1 tight to the localized with protein was colocalized strongly protein and Cldn5 junctions monolayers, EC–EC cell bEnd.3 control ARTICLE RESEARCH esuh oaayeteefc fIL-1 of Therefore, effect 2008). the analyze al., to et sought (Taddei we translocation nuclear prevent its sequesters to prevent VE-cadherin FoxO1) and Akt) (pT24 translocation, nuclear (pT308 24 its activated threonine Akt at of active FoxO1 constitutively presence phosphorylates endothelia, the brain by upon mature dependent modulated is be b the which can in region process stimuli repressor a a pathologic to FoxO1 of to the binding of the response regulation the in that gene identified have reports Previous and IL-1 FoxO1 by regulation Transcriptional IL-1 with correlated downregulation Cldn5 whether IL-1 of time-course the fbt oO and FoxO1 both of of activation reached IL-1 expression of in hours 6 decrease after this time- significance a and statistical in decreased manner, Cldn5 was total expression dependent quantify Cldn5 to 3C). assays (Fig. ICW expression used we dysfunction, barrier iepitps-ramn ihIL-1 in decrease with post-treatment time-point IL-1 with or post-treatment Cldn5 hours synthesis 24 to whether protein 1.5 altered from discern points of to result degradation, the order was In downregulation hyperpermeability. IL-1 and with correlated was decrease nlss edtrie htteewsa nraei FoxO1 in IL-1 of increase result an a as was immunofluorescent 4C,E) there (Fig. confocal accumulation that nuclear determined and we by fractionation Furthermore, analysis, 4A,B). nuclear (Fig. levels IL-1 FoxO1 using with pT24 and treatment Akt pT308 1.5-hour a that their whereas TRITC–dextran, unchanged. and remained Cldn5-deficient FITC–albumin to treating permeability fluorescein Interestingly, sodium IL-1 molecules. with monolayers permeability small paracellular the reduce to to cell– BMVECs at in needed to junctions is not cell a Cldn5 but 0.4- that TRITC–dextran, suggesting to FITC–albumin, showed 4.4-kDa monolayer 66.4-kDa and the fluorescein of monolayers sodium permeability kDa the Cldn5-deficient in increase Indeed, significant IL-1 without 3E,F). or (Fig. with monolayers controls fluorescein, Cldn5-deficient scrambled and sodium between FITC–albumin for and coefficients TRITC–dextran, permeability the compared Therefore, correlative. used largely gene we remains targeted data this a association downregulation, IL-1 temporal of strong a result is a between the there Although was IL-1 was event. to regulatory downregulation response there Cldn5 in changes that protein that Cldn5 of revealed onset in analysis decrease significant RT-PCR Quantitative ctnnt tblz hsrpeso Tde ta. 08.In 2008). al., et (Taddei repression this stabilize to -catenin ee yuigwsenbotn n C sas eidentified we assays, ICW and blotting western using by Here, b idcdgn ersinof repression gene -induced Cldn5 Cldn5 Cldn5 b ctnn n h ula rnlcto n binding and translocation nuclear the and -catenin, seii iN ordc h eeso ln,and Cldn5, of levels the reduce to siRNA -specific b b mdae are yfnto n Cldn5 and dysfunction barrier -mediated ramn o 4hus(i.3) odetermine To 3B). (Fig. hours 24 for treatment RAlvl 15hours). (1.5 levels mRNA b RAlvl eeasse tvrostime- various at assessed were levels mRNA ctnnt the to -catenin b b Cldn5 mdae oneuaino ln,and Cldn5, of downregulation -mediated ute nrae hi emaiiyto permeability their increased further RAlvl htpeee the preceded that levels mRNA b mdae are dysfunction barrier -mediated Cldn5 Cldn5 b b b twihw eetda detected we which at nAtFx1signaling, Akt–FoxO1 on eitdadces in decrease a mediated erso tteearliest the at repressor b ctnnmediates -catenin b b Fg A lower 3A, (Fig. Cldn5 ramn.This treatment. b b b suggesting , b b ctnnto -catenin b Fg 3D). (Fig. treatment. promoter, treatment -induced Cldn5 netnleihlu M ta. 05.Hwvr h oeof role in the IL-1 nmMlck However, in through 2005). or barrier al., BMVECs in gut et nmMlck the (Ma epithelium of intestinal dysfunction induce to b 1 putative the with and of FoxO1 whether determine level to assays the (ChIP) in IL-1 to decrease response a and translocation, were model genes our only in altered The significantly significance. were statistical that reach not treated did cells bEnd.3 it expression in genes target same IL-1 with the of array RT-PCR an ( protein vesicle-associated plasmalemmal in increase iaei rndcn r-nlmaoyo hyperpermeability IL-1 Furthermore, or endothelium. the pro-inflammatory in transducing signals important in an as nmMlck kinase implicated have evidence of lines nmMlck Several on dependent is dysfunction The Fg F n are yfnto t2 or Fg 6G). (Fig. hours 24 at both IL-1 by mediated that of dysfunction repression suggest involves downregulation results barrier these and Collectively, 6F) (Fig. either of of quantitative downregulation knockdown and the or Furthermore, 6A,C) FoxO1 (Fig. results. PCR qualitative 6B,D) both (Fig. by determined as ciei h rncitoa euaino agtgns(i.4D). 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(Fig. -catenin ctnnatvto lctdb IL-1 by elicited activation -catenin b ora fCl cec 21)17 8015 doi:10.1242/jcs.144550 1840–1853 127, (2014) Science Cell of Journal ctnn(ln E) sdtrie ywsenbo analysis blot western by determined as 8E7), (clone -catenin b b Cldn5 Fg D.Atog hr a enices in increase mean a was there Although 5D). (Fig. Cdkn1a Cldn3 ctnn(i.6)atnae ohteIL-1 the both attenuated 6E) (Fig. -catenin b b Cldn5 b xrsinwr eotd–Wtmdae nuclear Wnt-mediated – reported were expression epromdcrmtnimmunoprecipitation chromatin performed we , b ctnnwt Ecdei fe . or of hours 1.5 after VE-cadherin with -catenin xrsinadadces nteepeso of expression the in decrease a and expression ctnnws nta,ol soitdwt an with associated only instead, was, -catenin ctnnadFoxO1. and -catenin and Cldn5 b erso nrsos otetetwt IL- with treatment to response in repressor Pdk4 ctnnsaiie VE-cadherin-mediated stabilizes -catenin RAa . or fe treatment after hours 1.5 at mRNA eosrtdta h oO that FoxO1 the that demonstrated ) b nrae h ula localization nuclear the increased b b mdae yfnto fthe of dysfunction -mediated mdae oneuainof downregulation -mediated Cldn5 b Plvap b ctnnwt adherens with -catenin b ctnnt h nucleus, the to -catenin b Fg A,arsl that result a 5A), (Fig. mdae barrier -mediated b rncito htis that transcription .Teeoe eran we Therefore, ). b b b b ctnnfo the from -catenin Cldn5 ctnnassociate -catenin mdae Cldn5 -mediated b ohFx1and FoxO1 both , Cldn5 b Cldn5 a enshown been has ctnnnuclear -catenin ramn was treatment RAin mRNA b and repressor, b b -induced -catenin -catenin Cldn1 Ocln 1843 b . -

Journal of Cell Science sdt eemn h iecus o h IL-1 the for time-course the determine to used uniiaino ln ncdw htwsotie ytetetwt agtdsRA( siRNA targeted with treatment by obtained was that knockdown Cldn5 of quantification of IL-1 with downregulation treatment that demonstrated lysates cell IL-1 in however, Cldn5 ZO-1; IL-1 for for with blotting observed treatment were 24-hour changes a localization after or (ZO-1) 20 expression occludens-1 bars: zonula and Scale Cldn5 control. proteins vehicle junction tight the of analysis immunofluorescence EERHARTICLE RESEARCH 1844 IL-1 3. Fig. NV ihDnets(,)adTkys()ps o analysis. hoc post (F) Tukey’s and (C,D) Dunnett’s with ANOVA emaiiyo h ooae osdu loeci n RT–eta,weesFT–lui emaiiyrmie nhne.Dt r presented are Data unchanged. remained IL-1 permeability with FITC–albumin monolayers whereas Cldn5-deficient TRITC–dextran, treating and Moreover, fluorescein FITC–albumin. sodium mean to to not monolayer but the of TRITC–dextran, permeability and fluorescein sodium to monolayer ramn o 4hus n eecmae ihtoeof ( those monolayers with BMVEC compared Cldn5-deficient were in and measured hours, were 24 FITC-albumin for and treatment TRITC-dextran fluorescein, sodium for 6 ...* s.e.m. b erae ln xrsini Ed3clsi anrcnitn ihIL-1 with consistent manner a in cells bEnd.3 in expression Cldn5 decreases P , Cldn5 .5cmae ihtevhcecnrl the control, vehicle the with compared 0.05 RAta rcddteIL-1 the preceded that mRNA m .Cd5adZ- r ooaie tcl–elcnat nvhcecnrltetdcls nIL-1 In cells. vehicle-control-treated in contacts cell–cell at colocalized are ZO-1 and Cldn5 m. b mdae eraei ln rti xrsin D eltm C eut eosrt htIL-1 that demonstrate results PCR Real-time (D) expression. protein Cldn5 in decrease -mediated b mdae eraei ln rti xrsin E etr ltigpoiigcnimto and confirmation providing blotting Western (E) expression. protein Cldn5 in decrease -mediated siScr t 5 ooaes iia oIL-1 to Similar monolayers. iepitor time-point 0 b 10n/l induced ng/ml) (100 b nue infcn euto nCd5epeso tcl–elcnat.()Western (B) contacts. cell–cell at expression Cldn5 in reduction significant a induced siScr ; # siCldn5 P , ora fCl cec 21)17 8015 doi:10.1242/jcs.144550 1840–1853 127, (2014) Science Cell of Journal b .5cmae with compared 0.05 , ramn,Cd5dfcec nrae h emaiiyo the of permeability the increased deficiency Cldn5 treatment, -oddces nttlCd5poenepeso.()IW were ICWs (C) expression. protein Cldn5 total in decrease 2-fold rsrmldsRA( siRNA scrambled or ) b mdae are dysfunction. barrier -mediated siCldn5 siCldn5 siScr ln;Student’s alone; iho ihu IL-1 without or with ) .()Preblt ofiins( coefficients Permeability (F) ). b tetdcls oobvious no cells, -treated b 10n/l,cmae with compared ng/ml), (100 A Confocal (A) b ute nrae the increased further t ts rone-way or -test b 10ng/ml) (100 b induced sthe as P S )

Journal of Cell Science ciainocre meitl n esse o or after hours 2 for time- persisted the a and in by immediately IL-1 occurred (Y464) that indicated activation Given 464 7A). as (Fig. tyrosine manner minutes), dependent IL-1 of 15 phosphorylation with (within increased nmMlck BMVECs activated Treating cells. eemndteefc fIL-1 of effect of the role determined the investigated IL-1 we the S1), in nmMlck Fig. material (supplementary EERHARTICLE RESEARCH mlki h ntblt fahrn ucin,a ela the as of accumulation well nuclear IL-1 the as after that junctions, hypothesized instability adherens junction of of adherens role instability observed the the Considering in understood. nmMlck poorly is barrier endothelial sn rmr MEsfrom IL-1 to BMVECs response primary in activity using nmMlck of result the fe sltn n utrn rmr MEs efirst we BMVECs, primary culturing and isolating After b mdae oneuaino Cldn5. of downregulation -mediated b nmmlck nnMc ciaini these in activation nmMlck on +/+ b and 10n/l acutely ng/ml) (100 b mdae nmMlck -mediated b ctnnmgtbe might -catenin nmmlck b b hrfr,by Therefore, . ramn,we treatment, 2 / 2 mice rti xrsin upeetr aeilFg 2 nBMVECs both in total from S2) changing Fig. isolated (without material FoxO1 supplementary pT24 expression; and protein Akt IL-1 pT308 7B). of (Fig. levels the BMVECs in IL-1 pathway assess Akt–FoxO1 to the used the were in ICWs nmMlck of above. role the and determine to FoxO1- aimed next we treatment, b IL-1 that revealed active for IL-1 blotting western for However, required not is nmMlck that hs sltdfrom isolated those mlkat ptemof upstream acts nmMlck mlkdfcec teutdIL-1 attenuated deficiency nmMlck cuuain u o oO ula cuuain(i.7D,E). (Fig. accumulation nuclear FoxO1 not but accumulation, ctnni MEsioae from isolated BMVECs in -catenin ora fCl cec 21)17 8015 doi:10.1242/jcs.144550 1840–1853 127, (2014) Science Cell of Journal b ctnndpnetpoessta eediscussed were that processes -catenin-dependent b nmmlck nrae h ai factive of ratio the increased nmmlck +/+ 2 b cuuaino oO nrsos oIL- to response in FoxO1 of accumulation nuclear increased an revealed analysis immunofluorescence Confocal (E) cl as 20 bars: staining. Scale nuclear without images in nuclei of location the represent Arrowheads (a,b). control vehicle with treated 1 eil oto;Student’s control; vehicle h mean the Cdkn1a targets, transcriptional FoxO1 known of two upregulation showed results PCR Real- time (D) fractionation. nuclear the of determine efficacy to used control was loading Gapdh a and as used was A/C Lamin IL-1 to response in of FoxO1 accumulation nuclear the in increase significant a demonstrated lysates of nuclear analysis blot Western (C) FoxO1. and Akt of IL-1 dephosphorylation quantify mediated to used were ICWs (B) T24. on FoxO1 of phosphorylation decreased to led also IL-1 treatment Akt, of inactivation with T308. Consistent of phosphorylation in decrease a indicated by as Akt, inactivated hours) 1.5 for IL-1 that demonstrate blots expression. gene of regulation FoxO1-dependent and accumulation nuclear in FoxO1 resulting inactivation, Akt with i.4 IL-1 4. Fig. / b ctnnatvto.A expected, As activation. -catenin 2 and ramn cd oprdwt cells with compared (c,d) treatment ie(i.7) ugsigthat suggesting 7C), (Fig. mice b nmmlck and b b -mediated 6 nmmlck mdae k inactivation. Akt -mediated ctnnadtotal and -catenin b ...* s.e.m. b mdae nciainof inactivation -mediated Pdk4 A ersnaiewestern Representative (A) ramn sassociated is treatment m m. 2 aaaepeetdas presented are Data . / P +/+ 2 , b .5cmae with compared 0.05 b ie suggesting mice, ie u o in not but mice, ctnnnuclear -catenin ctnnt total to -catenin t b -test. b treatment. b 10ng/ml (100 decreased b -catenin b 1845 - b

Journal of Cell Science saswr eetdi MEs(i.7) lhuhtreatment Although nmmlck 7F). IL-1 (Fig. BMVECs with in repeated were assays h ulio IL-1 of nuclei the EERHARTICLE RESEARCH 1846 of repression on signaling transcriptional nmMlck of FoxO1-dependent impact the determine To ersieatvte fboth of activities repressive IL-1 the that to FoxO1 of binding fe . or ftetetadteIL-1 the Western hours. 24 and for treatment treatment after IL-1 observed is of that by dysfunction hours 1.5 induced mechanisms after molecular nmMlck-dependent ncliain ecnimdtecneto ewe the between connection the confirmed we culmination, In +/+ b b idcdnMc inln lestetranscriptional the alters signaling nmMlck -induced and e onceracmlto fFx1i both in FoxO1 of accumulation nuclear to led nmmlck b -treated 2 / 2 Cldn5 MEs h bec of absence the BMVECs, nmmlck b ctnnadFoxO1. and -catenin erso.Teerslssuggest results These repressor. 2 / 2 MEspeetdthe prevented BMVECs b mdae barrier -mediated b Cldn5 ctnn and -catenin- b ctnnin -catenin ChIP , b nmmlck eraei E htpritdfra es 4hus However, hours. 24 least at for persisted IL-1 that with TER treated in decrease were that IL-1 to monolayers response of BMVECs pattern and same bEnd.3 TER the both showed shown), basal IL-1 not higher (data without BMVECs had than monolayers or levels bEnd.3 with average, cultured on Although, were that BMVECs 10n/lfr2 or)dmntae htBVC from BMVECs that demonstrated hours) 24 nmmlck for ng/ml (100 esrmnswr bandfrom obtained were measurements rescued BMVECs in deficiency IL-1 nmMlck However, cells. bEnd.3 ltigfrCd5i yae bandfrom obtained lysates in Cldn5 for blotting b mdae ln oneuain(i.8) TER 8A). (Fig. downregulation Cldn5 -mediated +/+ 2 ora fCl cec 21)17 8015 doi:10.1242/jcs.144550 1840–1853 127, (2014) Science Cell of Journal / 2 iersoddt IL-1 to responded mice MEsta eeclue iho ihu IL-1 without or with cultured were that BMVECs ulisann.Saebr:20 bars: Scale without staining. images nuclei in nuclei of location the represent nuclei Arrowheads with staining). b staining, nuclei (a without control vehicle with staining) nuclei compared with d staining, nuclei epnet IL-1 to response cuuainof accumulation nuclear increased demonstrates immunocytochemistry Confocal (E) ihvhcecnrl Student’s control; vehicle with * control. vehicle ( levels mRNA mean IL-1 represent with treated were that Plvap i.4,a hs xeiet were experiments these as in 4C, shown Fig. controls the are of Gapdh duplicates and A/C Lamin showing blots control nuclear The of fractionation. efficacy the determine used to was Gapdh a and as control, used loading was A/C Lamin IL-1 treatment. to response in catenin of accumulation nuclear in significant increase a showed the fraction of nuclear analysis blot Western (C) mean the represent Data IL-1 after immunolabeling active in an increase demonstrated 8E7) (clone active catenin for blotting Western hours). (B) 1.5 ng/ml, (100 treatment IL-1 after (VE-cad) cadherin of ( amount the in a decrease was there that demonstrated experiments immunoprecipitation of localization of pool active the in increase an complex, VE-cadherin– the Cldn3 for determined were mRNA levels (D) blots. western and nuclear extracts same the on performed i.5 ramn fbn. cells IL-1 bEnd.3 with of Treatment 5. Fig. 6 b ct htpeiiae ihVE- with precipitated that -cat) ...,sona ecnaeof percentage a as shown s.e.m.), nmmlck b , and b Cldn5 ctnn n nuclear and -catenin, nasmlrfsinto fashion similar a in b Axin2 b ed ouculn of uncoupling to leads +/+ b , .Namely, ipae gradual a displayed Cldn12 b b b P versus -catenin. nmmlck rmbn. cells bEnd.3 from ramn cwithout (c treatment ctnnin -catenin , b b -catenin -catenin .5compared 0.05 , Ocln b nmmlck 6 b nmmlck +/+ b Fg 8B). (Fig. A Co- (A) b s.e.m. b treatment. Cldn1 , Data . b -catenin Tjp1 t b versus m -test. - m. , , 2 +/+ b / 2 b -

Journal of Cell Science oprdwt control, with compared eut ugs htnMc srqie o IL-1 for required is nmMlck that suggest results IL-1 restored also h coi xrsino active of expression ectopic the of activation the that evidence 1 EERHARTICLE RESEARCH nti td,w eotnwyietfe oeua details molecular IL-1 aberrant identified of newly effects report the of we some study, underlying this In DISCUSSION IL-1 in abolished expression nmMlck ectopic of completely the Furthermore, dysfunction. BMVECs barrier mediated in deficiency nmMlck IL-1 6. Fig. oneuaino ln nBMVECs. in Cldn5 of downregulation sda eaiecnrl o o-pcfcDApeiiain AC ulttv C eut rmCI sas BD uniaieR-C eut fr results RT-PCR Quantitative (B,D) assays. of ChIP downregulation from induced results against PCR siRNA targeted Qualitative using (A,C) knockdown precipitation. DNA non-specific for assays. controls negative as used IL-1 with hPsmlswr sae yuigPRwt rmr o h putative the for primers with PCR using by assayed were samples ChIP b mdae are yfnto Fg C.Cnitn ihthe with Consistent 8C). (Fig. dysfunction barrier -mediated D b q(rpretg fttl o the for total) of percentage (or Cq 10n/l rvhcecnrl on hoai a muorcpttd(hP ihat-oO,anti- anti-FoxO1, with (ChIP) immunoprecipitated was chromatin bound control, vehicle or ng/ml) (100 b eurstercuteto oO and FoxO1 of recruitment the requires nmmlck b mdae are yfnto Fg D.These 8D). (Fig. dysfunction barrier -mediated # P 2 , / Cldn5 2 .5cmae ihsSrtetdwt IL-1 with treated siScr with compared 0.05 MEsrvre h teuto fIL- of attenuation the reverted BMVECs RAa . or fe ramn F n are yfnto t2 or G.Dt ersn h mean the represent Data (G). hours 24 at dysfunction barrier and (F) treatment after hours 1.5 at mRNA b b ctnni eedn nnmMlck, on dependent is -catenin ctnnin -catenin b ctnn(si -catenin Cldn5 erso a acltdb omlzn oipt E etr ltigcnimto n uniiaino gene of quantification and confirmation blotting Western (E) input. to normalizing by calculated was repressor nmmlck b ct,Fx1(ioO)o cabe oto sSr.Fx1and FoxO1 (siScr). control scrambled or (siFoxO1) FoxO1 -cat), b ctnnt the to -catenin b 2 / inln in signaling 2 b -mediated BMVECs b Student’s ; Cldn5 Cldn5 b - erso ein hra rmr o the for primers whereas region, repressor erso ordc ln expression. Cldn5 reduce to repressor oO nrsos oIL-1 to response of translocation in nuclear the FoxO1 that Furthermore, indicate dysfunction. data barrier our BMVEC and downregulation in that, IL-1 demonstrate model, we our First, endothelium. microvascular brain icvrdta mlki iaeta sesnilfor essential is that kinase a is IL-1 nmMlck transducing that discovered Cldn5 in IL-1 decrease the that concomitant determining a After with expression. monolayers, BMVEC rncito rmthe from and FoxO1- transcription by of mediated downregulation are by preceded was Cldn5 expression protein Cldn5 in t -test. RA edtrie htIL-1 that determined we mRNA, ora fCl cec 21)17 8015 doi:10.1242/jcs.144550 1840–1853 127, (2014) Science Cell of Journal b b tmlto,wihutmtl ed oCldn5 to leads ultimately which stimulation, nue ieslciehpremaiiyin hyperpermeability size-selective induced Cldn5 b ctnno g stp-oto antibodies. isotype-control IgG or -catenin b b b ctnndpnetrpeso of repression -catenin-dependent ctnnkokonatnae IL-1 attenuated knockdown -catenin ee otipraty we importantly, Most gene. Cldn5 . or fe raigbn. cells bEnd.3 treating after hours 1.5 sarsl findependent of result a is b oigsqec CS were (CDS) sequence coding idcdCd5changes Cldn5 -induced b mdae decrease -mediated 6 ...* s.e.m. b ctnnand -catenin mChIP om P , 1847 0.05 b -

Journal of Cell Science eiinyatnae IL-1 attenuated deficiency EERHARTICLE RESEARCH 1848 of putative upstream acts the nmMlck to that demonstrate bind we proteins Moreover, both nucleus, the in when converge that mechanisms trafficking intracellular for required is nmMlck 7. Fig. cuuaino oO nboth in FoxO1 of accumulation ( otntby u oksosta ako nmMlck-induced of However, lack a accumulation. that shows nuclear work our FoxO1 notably, most and in involved not inactivation is yet Akt translocation, nuclear and activation catenin aha nBVC,b sn hshseii nioisaantT0 nAt(T0 k)adT4o oO p2 oO) nioisaantttlAtand Akt total against Antibodies FoxO1). IL-1 (pT24 (C) FoxO1 signals. on phosphospecific IL-1 T24 normalize assess to and used to Akt) were (pT308 used measurements Akt were the on active and ICWs T308 co-labeling, (B) for against manner. used antibodies time-dependent were phosphospecific FoxO1 a using in by Y464 BMVECs, of in pathway phosphorylation increased by indicated as aei ula rnlcto ssfiin oargt IL-1 abrogate to sufficient is translocation nuclear catenin h idn fFx1t the to FoxO1 of binding the from el ihVG nue eae n rlne 64 hours) (6–48 al. prolonged et and sustained delayed Cohen longer endothelial a induced artery 2010). a VEGF initial pulmonary (Bates, with and bovine cells an hours treating minutes many that – few demonstrated for response a lasting For just response hyperpermeability 2004). lasting Dejana, biphasic to response known and a is (VEGF) Bazzoni factor induce beds growth 1999; molecules endothelial vascular of vascular Curry, different example, size in and and barriers charge endothelial (Michel the across of (onset timing move terms their that in of and terms duration) in differ and stimuli various to responses repression. Cldn5 induced nmmlck urn vdneidctsta nohla hyperpermeability endothelial that indicates evidence Current nmmlck b ctnn(yohshrltda GSK3 at (hypophosphorylated -catenin +/+ n mlkhmzgu ncot( knockout homozygous nmMlck and ) 2 / 2 ie * mice. P , b mdae ula cuuainof accumulation nuclear -mediated .5cmae ihcnrl DE ossetwt h nciainadatvto inl nAt oO and FoxO1 Akt, on signals activation and inactivation the with Consistent (D,E) control. with compared 0.05 Cldn5 nmmlck b ctnnadFx1ocpnyo the of occupancy FoxO1 and -catenin erso.Dt ersn h mean the represent Data repressor. +/+ and b nmmlck ie e3 n h4)t total to Thr41) and Ser37 sites nmmlck 2 Cldn5 / 2 2 MEs h bec of absence the BMVECs, / 2 ie A raigBVC ihIL-1 with BMVECs Treating (A) mice. ) b ctnn u o ula cuuaino oO.()Atog IL-1 Although (F) FoxO1. of accumulation nuclear not but -catenin, repressor. 6 ...* s.e.m. b b b - - - Cldn5 b P ctnni MEsioae from isolated BMVECs in -catenin ln oso h B Ntae l,20) hydsoee htthe that discovered They 2003). al., Cldn5-null et (Nitta assays Using BBB permeability the of effect BBB. on BBB loss the hyperpermeability determine Cldn5 of the to masses series molecular with different in a of tracers performed Cldn5 with consistent al. of et also Nitta loss mice, the are by al., data mediated et IL-1 Puhlmann Our 2008; that al., 2005). note et derived (Ganter non-brain of monolayers in cell is hyperpermeability endothelial albumin it induce However, to shown molecules. small to longer hyperpermeability induce monolayer increase only that and responses sustained factors permeability-inducing with IL-1 12– consistent Our for 1989). for al., et lasts (Royall data cells endothelial aortic which bovine in hyperpermeability, hours 48 prolonged induce also nue yepreblt osltswt oeua aisthat radius molecular than larger a thrombin was with with treatment solutes and to hyperpermeability minutes), induced (5–90 transient more found be was hyperpermeability to thrombin-induced studies, same than the smaller radius molecular molecules of a coefficient with permeability the increased only that response , repressor. .5cmae ihalohrgop;Student’s groups; other all with compared 0.05 b ctnni h uliof nuclei the in -catenin ora fCl cec 21)17 8015 doi:10.1242/jcs.144550 1840–1853 127, (2014) Science Cell of Journal rmr MEswr sltdfo otcso wild-type of cortices from isolated were BMVECs Primary b b , 10n/l ctl wti 5mnts ciae nmMlck, activated minutes) 15 (within acutely ng/ml) (100 mdae ME yfnto em obe to seems dysfunction BMVEC -mediated 0A 60 ˚ te yoie,sc sTNF- as such cytokines, Other . nmmlck b 10n/l . or)icesdtertoof ratio the increased hours) 1.5 ng/ml, (100 nmmlck b 2 mdae nciaino h Akt–FoxO1 the of inactivation -mediated / 2 MEstetdwt IL-1 with treated BMVECs , +/+ 0A 60 ie u o ntoeisolated those in not but mice, ˚ b Chne l,19) In 1999). al., et (Cohen t -test. ramn e onuclear to led treatment b ctnn nmMlck -catenin, a b n IL-1 and b a been has prevented a ,

Journal of Cell Science rmBVC,adta hsls screae ihIL-1 with correlated is loss this that determined. and be IL-1 BMVECs, to that yet process from report this has that we pathogenesis role Here, inflammatory The 2012). in al., plays it et short tagging (Mandel membrane, relatively degradation the a in has for polyubiquitylated (Shen Cldn5 be that thought can reported and been previously half-life has than It tight 2008). shorter in al., proteins much et of is turnover strands the dynamic that junction and found photobleaching have fluidic after analysis highly recovery (FRAP) fluorescence a using have Studies to nature. discovered been recently IL-1 but albumin. kDa, that to induced 0.8–4.4 not BMVECs of of in molecules in to similar, extravasation hyperpermeability levels size-selective Cldn5 are demonstrate in data reduction siRNA-mediated to Our albumin. failed endogenous analysis histological EERHARTICLE RESEARCH eeso lsahmcsen as eraei Cldn5 the in involving decrease mechanism a a of through cause translocation nuclear BMVECs, homocysteine elevated in gene that plasma known expression the is of explain It would observed. levels was that that mechanism response a regulatory determine to sought IL-1 of onset of IL-1 downregulation that the identify triggers we Furthermore, hyperpermeability. mediated of than molecules to permeable less more was mice Cldn5-deficient of BBB ih ucin,rte hnbigmrl ttcbrir,have barriers, static merely being than rather junctions, Tight , .–0kacmae ihwl-yemc,and mice, wild-type with compared kDa 0.8–10 b mdae oso ln rti.Acrigy we Accordingly, protein. Cldn5 of loss -mediated b ctnnadtebnigo hsprotein this of binding the and -catenin b eitstels fCd5protein Cldn5 of loss the mediates Cldn5 RA hc rcdsthe precedes which mRNA, b -or b b - osof IL-1 the loss that discovered we Indeed, BMVECs. in Cldn5 nciaino k n eutn ula rnlcto fFoxO1; of of translocation nuclear translocation and activation nuclear the resulting (2) and Akt the (1) of with: associated inactivation was concomitantly and FoxO1, and catenin spatial the explore to us led they point, examine of end regulation directly affected not an did as studies Cldn5 these Although 2007). Weksler, of translocation the subsequent of the b disassembly and the complexes induce junction to adherens cells endothelial to cardiac response mouse IL- Furthermore, in 2011). LMVECs al., 1 et in (Jang reported injury lung been acrolein-induced also has putative mechanism the to idn fbt oO and FoxO1 both of binding nrae are ihns Lenre l,20;Poiel tal., and et expression Paolinelli 2008; Cldn3 al., in et increase (Liebner tightness an barrier by increased accompanied was that Gsk-3 the regulating recently of colleagues inhibitors BMVECs and in of during Dejana treatment 2009). that role al., reported et essential signaling (Daneman a an BBB described the play al. Wnt Wnt– et for to Daneman role BBB. of the known of development well downstream also transcription is catenin b ctnnt h ulu Brir ta. 08 abeiand Barbieri 2008; al., et (Barbieri nucleus the to -catenin nadto oisrl nsaiiigteahrn junction, adherens the stabilizing in role its to addition In a ensont c ncnucinwt oac mk in smoke tobacco with conjunction in act to shown been has ora fCl cec 21)17 8015 doi:10.1242/jcs.144550 1840–1853 127, (2014) Science Cell of Journal Cldn5 b b ctnndrn IL-1 during -catenin RAi MEswsdpneto both on dependent was BMVECs in mRNA ctnnsgaigi rvn h eeomn of development the driving in signaling -catenin Cldn5 b eutdin resulted erso Bade l,21) This 2011). al., et (Beard repressor b ctnnt the to -catenin oprdwt l te groups; Student’s other all with compared IL-1 restores also BMVECs of active expression ectopic nmMlck, on of that evidence activation the with Consistent (D) of IL-1 attenuation the reverts BMVECs IL-1 attenuates deficiency nmMlck 8. Fig. xrsino mlkin nmMlck of Ectopic expression (C) cells. bEnd.3 to IL-1 to respond ersn h mean the represent Data dysfunction. barrier mediated from BMVECs that demonstrated ECIS experiments (B) nmMlck. on dependent is downregulation IL-1 Cldn5 that mediated revealed hours) 24 ml, IL-1 with from treated lysates BMVECs in Cldn5 for blotting downregulation. Cldn5 and barrier endothelial brain the of dysfunction b ctnnnceraccumulation nuclear -catenin b b mdae oneuainof downregulation -mediated nvitro in mdae are dysfunction. barrier -mediated b ctnnin -catenin t -test. b Cldn5 b ihete n3 or Wnt3a either with ctnni dependent is -catenin nmmlck ctnn n 3 the (3) and -catenin; b b -mediated nasmlrfashion similar a in A Western (A) nmmlck 6 repressor. ...* s.e.m. +/+ b nmmlck b -mediated 2 b mice 10ng/ (100 / - 2 P , 1849 b 0.05 - 2 / 2 b b - -

Journal of Cell Science rnlcto nbrirloeigo are iheig we tightening, barrier IL-1 of or from effects loosening mRNA barrier conflicting evaluated on the translocation Given 2013). ARTICLE RESEARCH 1850 niiino eiinyi eotdt ecebt.Ti has This both. or rescue Although signaling MLC. phosphorylates than to other nmMlck proteins nmMlck regulatory reported that hypothesis and is a endothelium, prompted deficiency in or contractility inhibition actinomyosin simultaneously occur in to with junctions reported junctions adherens often of is uncoupling conditions intercellular inflammatory counteracts The motor 2010). widened Rigor, that cytoskeletal and of producing force (Yuan the role of adhesion, contractile the activation cell–cell produces past, the which the mediate to machinery, attributed to In been ability 2010). has its hyperpermeability al., endothelial et in nmMlck (Shen agents increasing inducing mediates is r that in kinase There dysfunction convergent barrier a endothelial 2010). as serves al., nmMlck that et evidence (Shen inflammatory- permeability modulating in mediated nmMlck for role a describing reports with concomitant nmMlck-dependent simultaneous IL-1 the pathways. activity. is FoxO1 signaling pathway increased such and other Akt one of of that inactivation inactivation indicate (differing studies or depend specific Our might activation and the context contexts) on pathologic is versus developmental activity in transcriptional of that accumulation conclude catenin we dysfunction, nuclear barrier with the association in increase catenin stimuli pathologic that in Ocln increase an with Wnt3a-mediated IL-1 that al., demonstrate et (Liebner we treatment Wnt3a Namely, from result 2008). to reported that than a with indicated associated results profile Our expression those Wnt3a. different with with results treated the BMVECs compared for and reported genes, junction tight for array novsteatvto of activation the involves 1 nucleus. the to translocated that catalyzing be so mediate can kinase, protein it the serine/threonine to possibility stabilizing thereby a the and shown phosphorylation as support catenin serves data been Our nmMlck have models. 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Journal of Cell Science etakJnta vrtetfrhseclettcnclassac nthe in assistance technical excellent his for Overstreet Jonathan thank We Acknowledgements mean the as presented are data The Statistics ARTICLE RESEARCH 1852 Jr C., R. Schaeffer, and M. J. Carbajal, W., A. Cohen, A. Basu, and M. Gupta, K., D. Kaushik, S., Chakraborty, and H. V. E. Perry, R., N. S. Sibson, B., Rajagopalan, Bearden, C., D. Anthony, and M., A. Blamire, J. J. Reynolds, Jr, S., R. Beard, B. B. Weksler, and E. Tremoli, L., Ruggiero, S., S. Barbieri, B. B. Weksler, and S. S. Barbieri, A. W. Banks, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.144550/-/DC1 online available material Supplementary material Supplementary months. 12 after release Research for Health and PMC of HL096640 in Institutes HL070752, Deposited National GM097270, HL061507]. the numbers by [grant part Grants in Project supported was work This Funding was manuscript S.Y.Y. The and authors. R.J.H. listed R.S.B.J., all by by were prepared Experiments S.Y.Y. interpreted and and R.S.B.J. executed by designed, designed and conceived was study This contributions Author interests. competing no declare authors The interests Competing technical excellent for images. staff for microscopy the Laboratory obtaining and Weitz in Imaging Muma assistance Cell Lisa and Health Microscopy USF Advanced the thank also We laboratory. eaa . reio .adLmunn,M G. M. Lampugnani, and F. Orsenigo, E., Dejana, E. Dejana, J. C. Gottardi, and L. R. Daugherty, A. B. Barres, and A. Kaushal, D., J. Cahoy, A. D., B. Agalliu, L., Barres, Zhou, and R., J. Daneman, C. Kuo, F., Kuhnert, L., Zhou, D., Agalliu, R., Daneman, E. Dejana, and G. Bazzoni, W. S. O. Levison, D. and Bates, K. J. Krady, A., Basu, eecmae yaayi fvrac ihTkyo unt othoc post Dunnett or Pair-wise Tukey with experiments). variance of independent analyses; analysis Student’s three by using compared least by were made at were from comparisons assays cell iir . oeo .A,Cre A., I. Romero, N., Didier, inln ttehato eta evu ytmpathology. system nervous central 1615-1631. of heart the at signaling rat the study. in resonance volume magnetic blood a cerebral brain: and coefficient, diffusion apparent permeability, P. Styles, junctions. tight by and barrier adherens blood-brain of 118 the regulation of receptor-dependent NMDA permeability increases Hyperhomocysteinemia endothelium vascular vivo. in in and expression trafficking vitro cyclooxygenase-2 of in induction beta-catenin and permeability increased in alter resulting to interleukin-1beta neurodegenerative and delivery drug pathogenesis, disorders. microbial for implications aodo A. Mabondzo, elBiol. Cell for functions. signaling resource and cells. adhesion new endothelial a brain of transcriptome: function barrier and ONE development non-CNS, blood-brain the not mouse understanding but The CNS, (2010). for required is signaling angiogenesis. barrier Wnt/beta-catenin endothelial (2009). small-pore and beta-catenin of dysfunction. phosphorylation tyrosine homeostasis. vascular in role and organization permeability. neuroinflammation. of regulator Biol. Vasc. Thromb. VE-cadherin/beta- modulates Arterioscler. interleukin-1beta plus of smoke tobacco dissociation by activity PTEN Suppressing ucin n Ecdei ntecnrlo aclrpermeability. vascular of control the in 121 VE-cadherin and junctions 2007-2014. , 2115-2122. , 5 e13741. , a 20) nohla elcl ucin:hpytogether. happy junctions: cell-cell Endothelial (2004). 5 .Neurovirol. J. 261-270. , a e t0.05 at set was 20) nelui-bt idcdcagsi lo-ri barrier blood-brain in changes -induced Interleukin-1beta (2000). m .Physiol. J. Am. adoac Res. Cardiovasc. 19) hsooyadptooyo h lo–ri barrier: blood–brain the of pathology and Physiology (1999). 21) aclredteilgot atr n vascular and factors growth endothelial Vascular (2010). rc al cd c.USA Sci. Acad. Natl. Proc. 20) erto fitrekn1eab srctsmediates astrocytes by interleukin-1beta of Secretion (2003). AE J. FASEB 5 538-555. , 20) nohla elt-eljntos molecular junctions: cell-to-cell Endothelial (2004). 277 priori a 21 mnn . ikusn . rsi .and J. Grassi, A., Wijkhuisen, C., ´minon, 87 .Nuoc.Res. Neurosci. J. 1831-1843. , H2038-H2049. , 28 262-271. , hsooy(Bethesda) Physiology 20) hsh-euaino Beta-catenin of Phospho-regulation (2007). .Neurosci. J. 732-738. , 6 20) oac mk oprtswith cooperates smoke Tobacco (2007). aei opee nendothelium. in complexes catenin ...( s.e.m. o ttsia significance. statistical for 106 641-646. , n 20) nelui-:amaster a Interleukin-1: (2004). hso.Rev. Physiol. t stenme faiasor animals of number the is 20 ts,adgopws data group-wise and -test, 20) h oeo adherens of role The (2008). 78 8153-8159. , 151-156. , 19) EFstimulates VEGF (1999). 21) Inflammasome (2010). 22 .Nuoc.Res. Neurosci. J. 303-309. , 84 869-901. , a.Rv Mol. Rev. Nat. .Cl Sci. Cell J. (2011). (2008). Blood PLoS 88 , cwn .E,Ecbr .E n otri .J. C. Gottardi, and E. D. Escobar, E., A. McEwen, col .W,Rtwl,N .adAln .M. S. Allan, and J. N. Rothwell, W., B. McColl, ens,R,Pri,R . rsi,J . ans .A,Wto,K D., K. Watson, A., D. Daines, W., J. Breslin, M., R. Perrin, C., R., Loichot, Reynoso, P., Ohlmann, R., Wangensteen, N., Carusio, R., H. Ranaivo, a .Y,Bii,M . e . erm .adSi,H M. H. Said, and A. Pedram, D., Ye, A., M. Boivin, Bourguignon, Y., Y., T. Ma, K. Kwong, Y., Wen, A., Matin, W., Xia, K., Makino, Y., S. Lin, D. Rachmilewitz, and F. Karmeli, L., P. Simon, M., Ligumsky, J., C. Czupalla, A., Taddei, J., Babbage, T., Bangsow, M., Corada, S., Liebner, adl . aen,T,Vloih . ehv . ls-amr .and L. Glass-Marmor, M., Merhav, A., Volkowich, T., Paperna, I., Mandel, hn . io,R . iet,C . u .H n un .Y. S. Yuan, and H. M. Wu, D., C. Pivetti, R., R. Rigor, R. Q., J. Shen, Turner, and R. C. Weber, S. L., Matalon, K., Shen, M. Cunningham, S., J. Beckman, L., R. Berkow, A., J. Y. Royall, S. Yuan, and P. O. Litovka, Jr, S., R. Beard, R., R. Rigor, M. W. Scheld, and Jr J., W. Long, B., Wispelwey, J., V. Quagliarello, and M. E. Turner, M., N. Carroll, M., J. Farma, M., D. Weinreich, M., Puhlmann, J., C. Czupalla, C., Artus, K., Devraj, L., Ferrarini, M. M., Furuse, Corada, N., R., Hashimoto, Paolinelli, H., Sasaki, Y., Seo, S., Gotoh, M., Hata, T., Nitta, S. D. Miller, E. F. Curry, and C. C. Michel, aoi,J,Bs,A,Ln .W,Rtsen .P,Kay .K,Sih .B and B. M. Smith, K., J. Krady, P., R. Rothstein, W., H. Lin, A., Basu, J., H., Lazovic, Pope-Varsalona, S., Liu, A., K. Brant, K., Bein, J., V. Concel, S., A. Jang, A. Kikuchi, and I. K. Nakayama, C., Tanji, S., Hino, et,D n ai,A B. A. Malik, and D. Mehta, atr .T,Ru,J,Myzw,B,Hwr,M,Fak .A,S,G., Su, A., P. T. Davis, J. and T. Frank, B. Hawkins, M., Howard, B., Miyazawa, J., Roux, T., M. Ganter, B., G. Mills, H., Nika, J., Meisenhelder, Y., Xia, Y., Zheng, D., Hawke, D., Fang, deesjunction. adherens experimental after disruption mice. junction tight in cerebrovascular stroke of kinetics the alters ih hi iae(LK20 nmcoaclrhpremaiiydrn severe during hyperpermeability microvascular burns. S. in Yuan, (MLCK-210) reduced and kinase chain H. of light M. Wu, consequence M., D. a Watterson, as al. mice. et shock MLCK210-null M. in D. endotoxic stress Watterson, against nitrosative J., Haiech, Protection I., Lobysheva, (2007). 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