Afadin downregulation by helicobacter pylori Induces epithelial to mesenchymal transition in gastric cells

micb-09-02712 No embe 7, 2018 Time: 21:31 # 1 ORIGINAL RESEARCH published: 09 No embe 2018 doi: 10.3389/ micb.2018.02712 Edi ed by: Alain Pie e Gobe , Vande bil Uni e si y Medical Cen e , Uni ed S a es Re iewed by: Lydia E. W oblewski, Vande bil Uni e si y Medical Cen e , Uni ed S a es Yoshimi Takai, Kobe Uni e si y G adua e School o Medicine, Japan *Co espondence: Ceu Figuei edo [email p o ec ed] †These au ho s ha e sha ed co-au ho ship Special y sec ion: This a icle was submi ed o In ec ious Diseases, a sec ion o he jou nal F on ie s in Mic obiology Recei ed: 04 Augus 2018 Accep ed: 23 Oc obe 2018 Published: 09 No embe 2018 Ci a ion: Ma ques MS, Melo J, Ca adas B, Mendes N, Pe ei a L, Ca nei o F, Figuei edo C and Lei e M (2018) A adin Down egula ion by Helicobac e pylo i Induces Epi helial o Mesenchymal T ansi ion in Gas ic Cells. F on . Mic obiol. 9:2712. doi: 10.3389/ micb.2018.02712 A adin Down egula ion by Helicobac e pylo i Induces Epi helial o Mesenchymal T ansi ion in Gas ic Cells Miguel Sa dinha Ma ques1,2,3, Joana Melo1,2,4, B uno Ca adas1,2,4, Nuno Mendes1,2, Luísa Pe ei a1,2,3, Fá ima Ca nei o1,2,3,5, Ceu Figuei edo1,2,3*†and Ma ina Lei e1,2,3† 1i3S – Ins i u o de In es igação e Ino ação em Saúde, Uni e sidade do Po o, Po o, Po ugal, 2Ipa imup – Ins i u e o Molecula Pa hology and Immunology, Uni e si y o Po o, Po o, Po ugal, 3Depa men o Pa hology, Facul y o Medicine, Uni e si y o Po o, Po o, Po ugal, 4Ins i u o de Ciências Biomédicas Abel Salaza , Uni e si y o Po o, Po o, Po ugal, 5Depa men o Pa hology, Cen o Hospi ala São João, Po o, Po ugal A adin is a cy oplasmic p o ein o he adhe ens junc ions, which egula es he o ma ion and s abiliza ion o bo h he adhe ens and he igh junc ions. Abe an exp ession o A adin has been shown in cance and i s loss has been associa ed wi h epi helial- o- mesenchymal ansi ion (EMT). EMT is cha ac e ized by he change om an epi helial o a mesenchymal pheno ype, wi h modi ica ions on he exp ession o adhesion molecules and acquisi ion o a mig a o y and in asi e cell beha io . While i is known ha Helicobac e pylo i dis up s he igh and he adhe ens junc ions and induces EMT, he e ec o he bac e ia on A adin is s ill unknown. The aim o his s udy was o disclose he e ec o H. pylo i on A adin and i s impac in he induc ion o an EMT pheno ype in gas ic cells. Using wo di e en cell lines, we obse ed ha H. pylo i in ec ion dec eased A adin p o ein le els, independen ly o CagA, T4SS, and VacA i ulence ac o s. H. pylo i in ec ion o cell lines ecapi ula ed se e al EMT ea u es, displacing and down egula ing mul iple p o eins om cell–cell junc ions, and inc easing he exp ession o ZEB1, Vimen in, Slug, N-cadhe in, and Snail. Silencing o A adin by RNAi p omo ed delocaliza ion o junc ional p o eins om he cell–cell con ac s, inc eased pa acellula pe meabili y, and dec eased ansepi helial elec ical esis ance, all compa ible wi h impai ed junc ional in eg i y. A adin silencing also led o inc eased exp ession o he EMT ma ke Snail, and o he o ma ion o ac in s ess ibe s, oge he wi h inc eased cell mo ili y and in asion. Finally, and in line wi h ou in i o da a, he gas ic mucosa o indi iduals in ec ed wi h H. pylo i showed dec ease/loss o A adin memb ane s aining a cell–cell con ac s signi ican ly mo e equen ly han unin ec ed indi iduals. In conclusion, A adin is down egula ed by H. pylo i in ec ion in i o and in i o, and i s down egula ion leads o he eme gence o EMT and o he acquisi ion o an agg essi e pheno ype in gas ic cells, which can con ibu e o gas ic ca cinogenesis. Keywo ds: Helicobac e pylo i, A adin, epi helial o mesenchymal ansi ion, cell–cell junc ion dis up ion, gas ic cance F on ie s in Mic obiology | www. on ie sin.o g 1No embe 2018 | Volume 9 | A icle 2712 micb-09-02712 No embe 7, 2018 Time: 21:31 # 2 Ma ques e al. H. pylo i, A adin and EMT INTRODUCTION Helicobac e pylo i is he mos p e alen ch onic in ec ion wo ldwide, wi h almos hal o he human popula ion being in ec ed by his bac e ium (Zamani e al., 2018). All indi iduals in ec ed wi h H. pylo i de elop ch onic in lamma ion o he gas ic mucosa, which in some cases may p og ess h ough a cascade o al e a ions ha culmina e in gas ic cance (Polk and Peek, 2010). In ac , H. pylo i is ega ded as he majo isk ac o o gas ic cance de elopmen , and has been conside ed as a class I ca cinogen by he Wo ld Heal h O ganiza ion (IARC, 1994, 2011). Gas ic mucosal in lamma ion and he de elopmen o mo e se e e clinical ou comes o H. pylo i in ec ion ha e been a ibu ed o a ia ion o i ulence ac o s be ween di e en s ains. Among hem, he ype 4 sec e ion sys em (T4SS)- ansloca ed CagA oncop o ein and he VacA cy o oxin a e he bes ecognized, and in ec ion wi h s ains ha bo ing he mos pa hogenic a ian s o hese ac o s a e associa ed wi h g ea e in ensi ies o gas ic in lamma ion, and wi h inc eased isk o de eloping gas ic p emalignan lesions, and gas ic cance (A he on e al., 1995;Figuei edo e al., 2002;Gonzalez e al., 2011). In he s omach, H. pylo i can be ound in he mucus and in close con ac wi h he epi helium, wi h a opism o cell– cell junc ions (Tan e al., 2009;Bugay so a e al., 2017). This p oximi y o H. pylo i o in e cellula con ac s, leads o dis up ion o he epi helial apical junc ional complex (AJC), which includes he igh junc ions (TJs) and he adhe ens junc ions (AJs) (Amie a e al., 2003;W oblewski e al., 2009, 2015;Hoy e al., 2010). The TJs con ibu e o he egula ion o epi helial pa acellula pe meabili y and o main enance o cell pola i y, and a e cons i u ed by ansmemb ane p o eins, such as occludin, claudins, and junc ional adhesion molecules (JAMs), and by cy oplasmic-associa ed p o eins, like zonula occludens 1 (ZO- 1) (Zihni e al., 2016). The AJs a e loca ed below he TJs, unc ion mainly in cell–cell adhesion, and a e composed by he E-cadhe in-ca enins and by he nec in-A adin complexes (Takai e al., 2008a;Zihni e al., 2016). A adin (AFDN, AF6 o MLLT4) is an ac in-binding p o ein ha associa es wi h nec ins a AJs, and ansien ly wi h ZO-1, and ha egula e he o ma ion and s abiliza ion o he junc ional complexes (Ikeda e al., 1999;Zhadano e al., 1999;Yokoyama e al., 2001;Fukuha a e al., 2002;Lo ge and Moelling, 2006; Takai e al., 2008b). A g owing body o e idence sugges s ha A adin is in ol ed in ca cinogenesis. In addi ion o epo s o loss o A adin exp ession in epi helial-de i ed b eas , colon, and panc eas umo s (Le essie e al., 2007;Sun e al., 2014;Xu e al., 2015), i s down egula ion led o inc eased cell in asion in i o and o accele a ed umo g ow h in mice (Fou nie e al., 2011). Fu he mo e, A adin was shown o be a nega i e egula o o he epi helial- o-mesenchymal ansi ion (EMT) ma ke Snail in panc ea ic cance (Xu e al., 2015). Epi helial- o-mesenchymal ansi ion desc ibes he di e en ia ion o epi helial cells in o mesenchymal cells, and is an impo an p ocess du ing emb yogenesis, o gan de elopmen , issue egene a ion, and cance p og ession (Kallu i and Weinbe g, 2009). EMT is cha ac e ized by loss o he AJC, whe e junc ional p o eins a e deg aded o delocalized, he co ical ac in cy oskele on is eo ganized wi h he o ma ion o lamellipodia and ilopodia, and he e is ep ession o cy oke a in in e media e ilamen s and exp ession o imen in ilamen s (Lamouille e al., 2014). Accompanying hese mo phological changes, he e is ep og amming o gene exp ession h ough ac i a ion o he mesenchymal pheno ype egula o s, such as Snail, Slug, and zinc- inge E-box-binding homeobox 1 (ZEB1) ansc ip ion ac o s, concomi an ly wi h down egula ion o epi helial ma ke s (Lamouille e al., 2014). Along wi h he changes in exp ession and localiza ion o p o eins o he AJC, H. pylo i in ec ion is able o inc ease cell in asi e p ope ies (Oli ei a e al., 2006;Cos a e al., 2016), and o ac i a e se e al signaling pa hways ha induce an EMT pheno ype in he in ec ed cells (Sai o e al., 2010;Yin e al., 2010; Baud e al., 2013;Bessede e al., 2014;Lee e al., 2014;Yu e al., 2014;W oblewski e al., 2015). The e o e, we aimed o de e mine he e ec o H. pylo i in ec ion on A adin and i s impac in he induc ion o an EMT pheno ype in gas ic cells. MATERIALS AND METHODS Cell Cul u e and T ans ec ions The human gas ic cance cell lines MKN74 (a kind gi om Ca la Oli ei a, Uni e si y o Po o) and NCI-N87 (ATCCR CRL- 5822TM), we e cul u ed in RPMI 1640 (Biowes ), supplemen ed wi h 10% e al bo ine se um (HyCloneTM, GE Heal hca e Li e Sciences) and wi h 100 U-100 µg/mL penicillin-s ep omycin sul a e (GibcoR ), a 37◦C, unde a 5% CO2humidi ied a mosphe e. Cell ans ec ions we e pe o med using he Lipo ec amineR  2000 ans ec ion eagen (In i ogenTM Li e Technologies), acco ding o he manu ac u e ’s p o ocol. An siRNA silencing A adin exp ession (sc-43007, San a C uz) and a nega i e con ol siRNA (All S a s Nega i e Con ol siRNA; QIAGEN, Ge many), we e used a a inal concen a ion o 75 nM in se um- and an ibio ic- ee Op i-MEM medium (In i ogen). The e iciency o ans ec ion was e alua ed by Wes e n blo . Bac e ial S ains and G ow h Condi ions H. pylo i s ain 26695 (ATCC 700392, cagPAI+, acA s1/m1) was ob ained om ATCC (LGC S anda ds, Uni ed Kingdom), and H. pylo i s ain 60190 (ATCC 49503 cagPAI+, acA s1/m1) and i s espec i e 60190CagA−, 60190CagE−, and 60190VacA−mu an s we e a kind gi om P o esso John A he on (Uni e si y o No ingham). S ains we e cul u ed in T yp icaseTM Soy Aga wi h 5% Sheep Blood (TSAII; Bec on, Dickinson and Company) a 37◦C unde mic oae ophilic a mosphe e (GENbox mic oae ; bioMé ieux S.A.) o 48 h, as p e iously desc ibed (Cos a e al., 2016). Fo he p oduc ion o condi ioned media, bac e ia we e g own in F12 medium (Gibco) supplemen ed wi h 1x choles e ol (Gibco) unde mic oae ophilic condi ions a 37◦C wi h cons an o a ion (150 pm) o e 24 h. Subsequen ly, bac e ial suspensions we e cen i uged a F on ie s in Mic obiology | www. on ie sin.o g 2No embe 2018 | Volume 9 | A icle 2712 micb-09-02712 No embe 7, 2018 Time: 21:31 # 3 Ma ques e al. H. pylo i, A adin and EMT 15,000 ×g, o 15 min, and he supe na an s we e il e ed h ough a 0.2-µm s e ile il e , and concen a ed by ul a il a ion using a 10 kDa po e size Amicon (EMD Millipo e). The esul ing concen a ed bac e ial condi ioned media was used as media o gas ic cell cul u es in compa ison wi h F12 medium plus 1x choles e ol alone wi hou bac e ia, as con ol. In ec ion o Gas ic Cells Gas ic cell lines we e g own in an ibio ic- ee medium a 100% con luence o 5 days in 6-well pla es (TPPR Plas ic P oduc s AG). Medium changes we e ca ied ou e e y day. Fo in ec ion expe imen s, bac e ia g own o 48 h we e collec ed in phospha e bu e saline (PBS, pH 7.4) and added o gas ic cell monolaye s, a a mul iplici y o in ec ion (MOI) o 100 bac e ia pe cell. Co- cul u es we e main ained o 16 h a 37◦C, unde a 5% CO2 humidi ied a mosphe e. Unin ec ed con ol cell cul u es we e p ocessed simila ly, wi h he addi ion o PBS ins ead o bac e ia. Fo s udies wi h bac e ial condi ioned media, F12 medium supplemen ed wi h 1x choles e ol (Gibco) alone o a e 24 h o bac e ial g ow h was used as cul u e medium. Wes e n Blo and An ibodies Cells we e collec ed and lysed in 1% T i on X-100, 1% NP- 40 in PBS, pH 7.4, con aining a cock ail o inhibi o s o p o eases (Roche Applied Science, Mannheim, Ge many) and o phospha ases (Sigma-Ald ich). A e supe na an eco e y, he p o ein concen a ion was assessed using he Bio-Rad p o ein assay ki (Bio-Rad), acco ding o he manu ac u e ’s p o ocol. Iden ical amoun s o p o ein we e subjec ed o SDS-PAGE and ans e ed on o ni ocellulose memb anes (Ame sham). Memb anes we e blocked wi h 5% a - ee milk o 1 h, a oom empe a u e, incuba ed o e nigh wi h p ima y an ibodies a 4◦C, and incuba ed wi h seconda y an ibodies o 1 h, a oom empe a u e. An ibodies we e as ollows: an i-AF6 (610732) and an i-β-Ca enin (610153) we e om BD T ansduc ion Labo a o iesTM; an i-Vimen in (5741), an i-N-Cadhe in (13116), an i-Snail (3879), an i-Slug (9585), an i-TCF8/ZEB1 (3396), and an i-E-Cadhe in (3195) we e om Cell Signaling Technology; an i-GAPDH (sc-47724) was om San a C uz Bio echnology; an i-E-cad HECD1 (13-1700), an i-ZO-1 (61-7300), and an i- Occludin (71-1500) we e om The mo Fishe Scien i ic; an i- αTubulin (T9026) was om Sigma-Ald ich; and ho se adish pe oxidase-conjuga ed seconda y an i- abbi (NA934) and an i- mouse (NA931) we e om GE Heal hca e Li e Sciences. T answell Ma igel In asion Assay Ma igel-coa ed 24-well in asion inse s wi h 8 µm po es (Co ningTM, BD Biosciences) we e used o he in i o in asion assay. Upon hyd a ion wi h RPMI medium, cells we e seeded a a densi y o 5 ×104cells in RPMI wi h 10% FBS, on op o he Ma igel in he uppe side o he inse ; he bo om side was illed wi h cul u e medium. Fo in ec ion expe imen s, H. pylo i was added a a MOI o 100. A e 24 h incuba ion, non-in ading cells we e emo ed wi h a co on swab, he emaining cells we e ixed in me hanol o 10 min on ice, and he il e was moun ed wi h Vec ashieldR wi h DAPI (Vec o Labo a o ies). The whole il e was coun ed, using a 20×magni ica ion. Immuno luo escence Cells we e g own on glass co e slips (Ma ien eld, Ge many), ixed wi h 4% pa a o maldehyde o ice-cold me hanol. Upon pe meabiliza ion and blocking wi h 5% goa se um, 0.3% T i on X-100 in PBS, o 1 h, a oom empe a u e, p ima y an ibody was added o 2 h a oom empe a u e. Co e slips we e hen washed se e al imes wi h PBS pH 7.4, be o e incuba ion wi h he espec i e Alexa Fluo -conjuga ed seconda y an ibodies (In i ogen). Co e slips we e moun ed wi h Vec ashieldR wi h DAPI, and luo escence was moni o ed in a Zeiss Axio Image Z1 Apo ome mic oscope. T ansepi helial Elec ical Resis ance (TER) and Fluo escein Iso hiocyana e (FITC)-Dex an Pe meabili y Assay Cells we e seeded a 100% con luence in 6.5 mm T answellR  wi h 3.0 µm Po e Polyes e Memb ane Inse (Co ning). In eg i y o he epi helial monolaye was de e mined using a Millicell ERS Vol ohmme e (Millipo e). P ocedu es o elec ode equilib a ion and decon amina ion we e pe o med acco ding o he manu ac u e ’s ins uc ions. T ansepi helial elec ical esis ance (TER) was measu ed e e y 24 h, o six consecu i e days. Fo in ec ion expe imen s, H. pylo i was added a MOI o 100, and TER was measu ed e e y 24 h, o wo consecu i e days. To a oid empe a u e in luence, pla es we e allowed o each oom empe a u e o 15 min, and medium was changed a e each measu emen . TER alues we e calcula ed as ohms/cm2. On he six h day pos A adin silencing, and he second day pos in ec ion pa acellula pe meabili y was assessed by measu ing he pe meabili y o he cell monolaye o FITC- dex an (FD4, Sigma). A e measu ing he TER, he medium was changed in he inne and bo om chambe , and allowed o equilib a e o a leas 1 h. One mg/mL o FITC-dex an was added o he inne chambe and incuba ed a 37◦C o 1 h. A e incuba ion, luo escence was measu ed a an exci a ion o 485 nm and emission o 544 nm, on a Syne gy Mx mic opla e eade . The amoun o di used dex an was de e mined using calib a ion cu es es ablished wi h he s ock solu ion. Medium wi hou FITC-dex an was used as blank. Single Cell Mo ili y Fo e alua ion o single cell mo ili y, 12-well pla es (TPPR  Plas ic P oduc s AG) we e coa ed wi h 5 µg/mL o Fib onec in (Bioch om), o e nigh a 4◦C. Pla es we e washed wi h s e ile PBS pH 7.4, and 1.5 ×103cells we e added o each well, allowing hem o adhe e o 16 h. Adhe en cells we e labeled wi h 5µg/mL o Vyb an R CM-Dil Cell-Labeling Solu ion (The mo Fishe Scien i ic) o e 20 min, ollowed by h ee washes wi h wa med RPMI wi hou phenol ed media. Ten di e en a eas we e selec ed pe well, and li e images we e cap u ed e e y 15 min on a Leica DMI 6000 ime-lapse mic oscope (Leica Mic osys ems, We zla , Ge many). The mig a o y pa hs and eloci y o each cell we e analyzed wi h he Manual T acking plugin unc ion a ailable a he Fiji so wa e package1. 1h p://imagej.ne / F on ie s in Mic obiology | www. on ie sin.o g 3No embe 2018 | Volume 9 | A icle 2712 micb-09-02712 No embe 7, 2018 Time: 21:31 # 4 Ma ques e al. H. pylo i, A adin and EMT AFDN Gene Exp ession in No mal H. pylo i-In ec ed and Non-in ec ed S omachs RNA-seq eads sequenced in s omach samples om 181 indi iduals wi hou disease we e ob ained om he Geno ype- Tissue Exp ession (GTEx) da abase (Conso ium, 2015). Non- human unmapped eads we e aligned agains a da ase o 197 bac e ial whole genomes collec ed om NCBI, comp ising species iden i ied in he gas oin es inal ac by he Human Mic obiome P ojec and addi ional species appea ing in disease condi ions (Tu nbaugh e al., 2007). Bac e ial quan i ica ion and no maliza ion we e es ima ed using he QmihR pipeline (Ca adas e al., 2017). A sample was conside ed as in ec ed by H. pylo i when coun s o his species eached a log2cu o alue o 5. mRNA exp ession da a o he AFDN gene, in eads pe kilobase pe million (RPKM), was ex ac ed om GTEx elease 6. Pa ien Ma e ials and His opa hology Fo y- wo o malin- ixed and pa a in-embedded gas ic issue specimens we e e ospec i ely e ie ed om he Depa men o Pa hology o Cen o Hospi ala S. João (CHSJ; Supplemen a y Table 1), om pa ien s unde going ba ia ic su ge y ha had specimens ob ained o his opa hologic examina ion (mean age [±SD], 40.4 ±12.6 yea s; emale o male a io, 6:1). Samples ha e been used e ospec i ely, and ha e been delinked and uniden i ied om hei dono s. The s udy was app o ed by he e hics commi ee o CHSJ. Specimens we e s ained wi h hema oxylin/eosin and modi ied Giemsa, he la e o H. pylo i de ec ion. His ological assessmen e alua ed he ollowing pa ame e s: H. pylo i in ec ion, ch onic in lamma ion, polymo phonuclea ac i i y, glandula a ophy, and in es inal me aplasia. His ological slides we e examined by an expe ienced pa hologis , blinded o he clinical in o ma ion o he pa ien s. Immunohis ochemis y Immunohis ochemis y was pe o med in a LabVision Au os aine 480S (The mo Scien i ic). B ie ly, slides we e depa a inized in xylene and hyd a ed h ough passages in 100%, 95%, 70% e hanol, and insed in wa e . An igen unmasking was pe o med o 35 min using 1x an igen e ie al solu ion om he Enzo Poly iewR Plus HRP-DAB (an i-Rabbi ) ki . Slides we e cooled o 20 min and washed wi h TBS-Tween-20, 0.01%. Endogenous pe oxidase ac i i y was blocked o 10 min, and slides we e washed and placed in he Au os aine . A e blocking o endogenous pe oxidase ac i i y, slides we e incuba ed wi h An ibody blocke /diluen (p o ided in he ki ) o 10 min o p e en unspeci ic binding, ollowed by 1 h incuba ion wi h p ima y polyclonal an ibody An i-AFDN (HPA030213, Sigma), 1:1250 dilu ed in an ibody diluen (The mo Scien i ic, Lab ision). To p e en non- speci ic binding, slides we e again incuba ed wi h an ibody blocke /diluen o 5 min. A e wash, he Poly iew Plus HRP (an i-Rabbi ) was added and incuba ed o 1 h. The eac ion was e ealed wi h 3,30-diaminobenzidine (DAB) 5 min. Coun e s ain was pe o med wi h HIGHDEFR hema oxylin (ENZO). RNA Ex ac ion and Quan i ica ion o A adin Exp ession by Quan i a i e Real-Time PCR To al RNA was ex ac ed using mi VanaTM miRNA Isola ion Ki (The mo Fishe Scien i ic) ollowing manu ac u e ’s ins uc ions om unin ec ed and 24 h-in ec ed MKN74 cells. RNA was e e sed- ansc ibed using M-MuLV Re e se T ansc ip ase and andom-hexame s (NZYTech). Quan i ica ion o A adin exp ession was pe o med by quan i a i e eal- ime PCR (RT- qPCR) using he TaqMan Gene Exp ession Assays (Applied Biosys ems), Hs00984486_m1 o A adin and Hs99999905_m1 o GAPDH, as endogenous con ol gene, on an ABI P ism 7000 Sequence De ec ion Sys em (Applied Biosys ems), in ou independen expe imen s. Da a we e analyzed by he compa a i e 2(−11CT)me hod (Li ak and Schmi gen, 2001) and Wilcoxon signed- ank es applied. S a is ical Analysis The unpai ed S uden ’s - es was used o compa isons be ween wo independen g oups, and he one-way ANOVA wi h pos hoc Tukey’s es was used o compa isons be ween h ee independen g oups. K uskal–Wallis es was applied o GTEx da a ha compa ed AFDN mRNA exp ession be ween in ec ed and non- in ec ed samples. Wilcoxon signed- ank es was applied o quan i a i e eal- ime PCR analysis. The ela ionship be ween he p esence o H. pylo i and al e a ions o he immunohis ochemical pa e n o A adin was assessed using he Fishe ’s exac es . Da a analysis was pe o med using G aphPad P ism Ve sion 6.0. S a is ically signi icance was se a P≤0.05 (∗∗∗∗p≤0.0001, ∗∗∗p≤0.001, ∗∗p≤0.01, ∗p≤0.05). Da a in g aphs ep esen mean ±s anda d e o o he mean (SEM) o a leas h ee expe imen s. RESULTS H. pylo i In ec ion Dec eases A adin Exp ession Al e a ions o se e al TJ and AJ p o eins induced by H. pylo i in ec ion ha e been epo ed (Amie a e al., 2003;Hoy e al., 2010), bu he in luence o H. pylo i on A adin is so a unknown. To in es iga e he e ec o H. pylo i on A adin, MKN74 and NCI-N87 cell lines ha es ablish compe en junc ional complexes, we e in ec ed wi h s ain 26695 o 24 h. Immuno luo escence analysis showed A adin p esen bo h a he memb ane and in he nucleus in unin ec ed condi ions. Upon in ec ion wi h H. pylo i he e was a signi ican dec ease in A adin s aining in bo h cell lines, and a bo h memb ane and nucleus subcellula localiza ions, as de e mined by imaging quan i ica ion (Figu e 1A). Wi hin he nucleus, a signi ican dec ease in he numbe o A adin nuclea do s was also obse ed in in ec ed cells compa ed o unin ec ed gas ic cells (Supplemen a y Figu e 1A). F on ie s in Mic obiology | www. on ie sin.o g 4No embe 2018 | Volume 9 | A icle 2712 micb-09-02712 No embe 7, 2018 Time: 21:31 # 5 Ma ques e al. H. pylo i, A adin and EMT FIGURE 1 | Impac o H. pylo i in ec ion on A adin exp ession. MKN74 and NCI-N87 gas ic cell lines we e le un ea ed o in ec ed wi h H. pylo i 26695 (A,B) o in ec ed wi h H. pylo i 60190, o wi h he espec i e CagA-, CagE-and VacA-mu an s (C), o 24 h a a MOI o 100. A adin de ec ion by immuno luo escence ( ed) and quan i ica ion o he luo escence in ensi y a he memb ane and he nucleus (A), and by Wes e n blo and i s quan i ica ion, using GAPDH o ubulin as loading con ols (B,C). Scale ba , 10 µm. Da a co espond o he mean alue ±SEM and a e ep esen a i e o a leas h ee independen expe imen s. S a is ical signi icance was e alua ed wi h he S uden ’s - es o wi h one-way ANOVA. A adin has wo majo iso o ms, he l-A adin iso o m ha con ains an F-ac in-binding domain, and he less exp essed s-A adin iso o m ha lacks he F-ac in-binding domain (Buche e al., 2007). Wes e n blo analysis wi h an an ibody ha ecognizes he wo A adin iso o ms e ealed he p esence in bo h cell lines o a highly exp essed 220 kDa p o ein, co esponding o l-A adin, and ain bands abo e he 150 kDa ma ke , co esponding o s-A adin. Silencing o A adin by siRNA con i med he loss o he wo iso o ms (Supplemen a y Figu e 1B). Upon in ec ion wi h H. pylo i 26695, he e was a signi ican dec ease o he p o ein le els o A adin (Figu e 1B), wi hou changes in he mRNA exp ession le els (Supplemen a y Figu e 1C). To e alua e he impac o he majo bac e ia i ulence ac o s on H. pylo i-media ed A adin down egula ion, cells we e in ec ed wi h H. pylo i s ain 60190 and i s espec i e CagA−, CagE− ( equi ed o a unc ional T4SS), and VacA−mu an s. As p e iously obse ed in in ec ions wi h s ain 26695, H. pylo i 60190 was also able o signi ican ly educe A adin p o ein le els in he wo cell lines (Figu e 1C). In ec ions wi h all o he H. pylo i mu an s ains also led o signi ican dec eases in A adin exp ession meaning ha loss o A adin is independen o CagA, VacA, and any ac o deli e ed ia he T4SS (Figu e 1C). To de e mine i sec e ed soluble bac e ial ac o s o iable bac e ia a e in ol ed in loss o A adin, ei he condi ioned medium o H. pylo i liquid cul u es o hea -killed H. pylo i we e incuba ed wi h epi helial cells. The esul s e ealed ha loss o A adin p o ein exp ession was dependen on he p esence o iable bac e ia bu ha was no igge ed by soluble bac e ial ac o s sec e ed by H. pylo i (Supplemen a y Figu e 1D). H. pylo i Induces an EMT Pheno ype in Gas ic Cells Epi helial- o-mesenchymal ansi ion is cha ac e ized by he ansi ion om an epi helial o a mesenchymal pheno ype, wi h he modi ica ion o he adhesion molecules exp essed by he cell, allowing i o adop a mig a o y and in asi e beha io (Nie o e al., 2016). I has been shown ha H. pylo i is able o modi y se e al cell–cell adhesion molecules (Fedwick e al., 2005;Fio en ino e al., 2013;Schmid e al., 2016; Teg meye e al., 2017), o induce an elonga ion in he cell shape (Moese e al., 2004;Bou zac e al., 2007), o inc ease cell in asion (Oli ei a e al., 2006;Cos a e al., 2016), and o up- egula e EMT gene exp ession (Yin e al., 2010;Baud e al., 2013). H. pylo i was also shown o al e Snail localiza ion in F on ie s in Mic obiology | www. on ie sin.o g 5No embe 2018 | Volume 9 | A icle 2712 micb-09-02712 No embe 7, 2018 Time: 21:31 # 6 Ma ques e al. H. pylo i, A adin and EMT bo h MKN28 cells, which o m unc ional TJs, and in human gas ic specimens (W oblewski e al., 2015). S ill, he g ea majo i y o hese esul s we e ob ained using gas ic cance cell lines ha ha e de icien AJs due o mu a ions in he E-cadhe in-encoding gene, as is he case o AGS cells. The e o e, we wan ed o know i H. pylo i could ecapi ula e some o hese p ope ies in ou gas ic cell line models ha es ablish p o icien AJs. E alua ion o he e ec s o H. pylo i on cell monolaye s, showed ha a e 24 h o in ec ion, he e was a dec ease in he exp ession and/o memb ane displacemen o E-cadhe in, occludin, and ZO-1 (Figu e 2A). Concu en ly, H. pylo i in ec ion signi ican ly educed E-cadhe in, β-ca enin, occludin, and ZO-1 p o ein le els, bo h in MKN74 and NCI-N87 cell lines (Figu e 2B). Fu he mo e, H. pylo i in ec ion signi ican ly inc eased he p o ein le els o ZEB1, Vimen in, Slug, N-cadhe in, and Snail in bo h cell lines (Figu e 2C). These esul s con i m ha in addi ion o A adin, H. pylo i in ec ion is able o al e junc ional complexes, dampening epi helial cell p ope ies. These esul s u he show ha H. pylo i-media ed inc ease in he exp ession o EMT-associa ed ma ke s also occu s in cell lines ha es ablish AJs. FIGURE 2 | H. pylo i induces an EMT pheno ype in epi helial gas ic cells. MKN74 and NCI-N87 gas ic cell lines we e le un ea ed o in ec ed wi h H. pylo i 26695 o a pe iod o 24 h a a MOI o 100. (A) Immuno luo escence o E-cadhe in, occludin, and ZO-1. Scale ba , 10 µm. (B) Wes e n blo and espec i e quan i ica ions o he le els o apical junc ional complex p o eins E-cadhe in, β-ca enin, ZO-1, and occludin. Tubulin was used as loading con ol. (C) Wes e n blo and espec i e quan i ica ions o he le els o mesenchymal ma ke s Snail, N-cadhe in, ZEB1, Slug, and Vimen in. GAPDH was used as loading con ol. Da a co espond o he mean alue ±SEM and a e ep esen a i e o h ee independen expe imen s. S a is ical signi icance was e alua ed wi h he S uden ’s - es . F on ie s in Mic obiology | www. on ie sin.o g 6No embe 2018 | Volume 9 | A icle 2712 micb-09-02712 No embe 7, 2018 Time: 21:31 # 7 Ma ques e al. H. pylo i, A adin and EMT A adin Down egula ion Delocalizes AJ and TJ P o eins, P omo es he Fo ma ion o Ac in S ess Fibe s, and Up egula es he EMT Ma ke Snail To mimic he e ec o H. pylo i in ec ion on A adin down egula ion and o e alua e he e ec o he loss o A adin in gas ic cells, he nex expe imen s we e pe o med wi h cells ha we e ansien ly ans ec ed wi h a siRNA o A adin o wi h a non-silencing siRNA con ol. E icien knockdown was achie ed in MKN74 (87%) and in NCI-N87 (54%) cells. Since A adin is an AJC p o ein, we i s e alua ed he e ec o A adin down egula ion on he localiza ion o AJs and TJs p o eins in MKN74 cells. In cells ans ec ed wi h he non- silencing siRNA, he AJs p o eins E-cadhe in and β-ca enin, and he TJs p o eins occludin and ZO-1 p e e en ially localized a he cell memb ane. In con as , in cells whe e A adin was silenced, he e was an al e a ion o he memb ane localiza ion o E-cadhe in, β-ca enin, occludin, and ZO-1 (Figu e 3A), wi hou changes in he espec i e p o ein exp ession le els (Figu e 4A). Al e a ions in he cellula localiza ion o hese p o eins in cells whe e A adin was silenced we e accompanied by a s iking change in he epi helial cell mo phology, wi h loss o he ac in bel , o ma ion o ac in s ess ibe s, and o ma ion o lamellipodia and ilopodia (Figu e 3A). In e es ingly, a si es whe e A adin was no e icien ly silenced by he siRNA, cells e ained he polyhed al epi helial mo phology wi h igh cell- o-cell con ac s, con as ing wi h si es whe e A adin exp ession was ully abolished by siRNA, and in which cell–cell adhesion was los and he AJC p o eins we e disloca ed o he cy osol (Figu e 3A). Quan i ica ion o A adin luo escence in ensi y a he memb ane and he nucleus in bo h A adin silenced and non- silenced cells is depic ed in Figu e 3B. A signi ican dec ease in A adin exp ession was obse ed in bo h localiza ions in A adin silenced cells, ein o cing he speci ici y o he A adin siRNA and an ibody. FIGURE 3 | A adin down egula ion displaces apical junc ional complex p o eins and p omo es he o ma ion o ac in s ess ibe s. Double immuno luo escence o A adin ( ed) wi h AJs p o eins E-cadhe in and β-ca enin (g een), and wi h TJs p o eins ZO-1 and occludin (g een) in MKN74 cells ans ec ed wi h a non-silencing siRNA (siNS) o wi h a siRNA o A adin (siAFDN). Immuno luo escence o ac in (g een) and o Snail ( ed) a e also shown. Nuclei we e coun e s ained wi h DAPI. Scale ba , 10 µm. Whi e a ows ep esen cells wi h A adin no e icien ly silenced by he siRNA and ha e ain he epi helial mo phology; Yellow a ows, lamellipodia; cyan a ows, ilopodia (A). Quan i ica ion o A adin luo escence in ensi y in MKN74 cells in bo h memb ane and he nucleus upon ea men wi h non-silencing siRNA o wi h a siRNA o A adin (B). F on ie s in Mic obiology | www. on ie sin.o g 7No embe 2018 | Volume 9 | A icle 2712 micb-09-02712 No embe 7, 2018 Time: 21:31 # 8 Ma ques e al. H. pylo i, A adin and EMT FIGURE 4 | A adin down egula ion does no al e he exp ession o apical junc ional complex p o eins, bu up egula es he EMT ma ke Snail. Wes e n blo analyses o MKN74 and NCI-N87 cells, cells ans ec ed wi h a non-silencing siRNA (siNS), o wi h a siRNA o A adin (siAFDN), e alua ing (A) apical junc ional complex p o eins E-cadhe in, β-ca enin, ZO-1, and occludin, and (B) mesenchymal ma ke s Snail, N-cadhe in, ZEB1, Slug, and Vimen in. GAPDH was used as loading con ol. Numbe s below he bands co espond o quan i ica ions. Da a a e ep esen a i e o h ee independen expe imen s. S a is ical signi icance was e alua ed wi h he S uden ’s - es . Ha ing shown ha A adin down egula ion al e s he s uc u e o gas ic cell–cell junc ions and cell mo phology, and knowing ha A adin nega i ely egula es he ansc ip ion o Snail o ac i a e EMT in panc ea ic cance cells (Xu e al., 2015), we sough o in es iga e he in ol emen o A adin in he EMT pheno ype in he gas ic con ex . In bo h MKN74 and NCI-N87 cell lines, silencing o A adin signi ican ly inc eased he le els o he EMT-associa ed ansc ip ion ac o Snail (Figu es 3A, 4B), bu no o he EMT ma ke s Slug, ZEB1, Vimen in, and N-Cadhe in (Figu e 4B). O e all, hese esul s a e sugges i e ha A adin down egula ion is implica ed in he eme gence o he EMT pheno ype in he gas ic se ing. A adin Down egula ion Al e s he TJ Ba ie Func ion and Inc eases Cell Mo ili y and In asion Ha ing shown ha A adin silencing esul ed in delocaliza ion o TJ and AJ p o eins om he memb ane o he cy oplasm, we nex add essed he unc ional consequences o A adin down egula ion in e ms o TJ in eg i y, cell mo ili y/mig a ion, and in asion. To e alua e TJ in eg i y, cells we e seeded in answells and allowed o each 100% con luence, a e which TER was measu ed o e six consecu i e days, allowing cells o pola ize. Cells whe e A adin was down egula ed had signi ican ly lowe elec ical esis ance in compa ison o cells ans ec ed wi h he non-silencing siRNA o o un ea ed cells, an e ec ha s a ed o be obse ed om day 3 (Figu e 5A). Pe meabili y assays we e pe o med on day 6, by measu ing he pe meabili y o he gas ic monolaye s o he high molecula weigh 4 kDa dex an. Monolaye s es ablished wi h cells whe e A adin exp ession was silenced we e signi ican ly mo e pe meable han con ol cell monolaye s (Figu e 5B). Simila esul s ega ding TER and pe meabili y o FITC-4 kDa Dex an we e ound o MKN74 (Figu es 5A,B) and NCI-N87 gas ic cells (Supplemen a y Figu es 2A,B) upon in ec ion wi h H. pylo i s ain 26695. To analyze whe he he e was a di ec link be ween loss o A adin and cell mo ili y/mig a ion, cells we e seeded a low densi y, s ained wi h he lipophilic dye CM-Dil, and acked o 24 h wi h ime-lapse mic oscopy. Analysis o he ime-lapse mic oscopy ideos e ealed ha , e en when small g oups o cells we e seeded, cells wi h down egula ion o A adin we e unable o main ain cell–cell adhesion, as opposed o un ea ed cells, o cells ans ec ed wi h he non-silencing siRNA (Figu e 5C). Coo dina es o each indi idual cell pa h o e he 24 h pe iod we e ep esen ed in wo-dimensional g aphs (Figu e 5D). Silencing o A adin signi ican ly inc eased he mig a ion eloci y (Figu e 5E) and he dis ance (Figu e 5F) co e ed by each indi idual cell, compa ed o non-silenced o o un ea ed con ol cells. No signi ican di e ences on di ec ionali y we e obse ed, wi h each cell displaying a andom mig a ion pa e n. We nex add essed he in ol emen o A adin in gas ic cell in asion in Ma igel in asion assays. Silencing o A adin esul ed in a wo old inc ease in he cell in asi e capaci y compa ed o con ol cells ans ec ed wi h he non-silencing siRNA (Figu e 5G). Inc eased cell in asion was also obse ed upon in ec ion wi h H. pylo i s ain 26695 in bo h MKN74 cells (Figu e 5H) and NCI-N87 cells (Supplemen a y Figu e 2C). O e all, hese esul s sugges ha A adin plays a ole in gas ic epi helial in eg i y by main aining he s uc u e and unc ions o F on ie s in Mic obiology | www. on ie sin.o g 8No embe 2018 | Volume 9 | A icle 2712 micb-09-02712 No embe 7, 2018 Time: 21:31 # 9 Ma ques e al. H. pylo i, A adin and EMT FIGURE 5 | A adin down egula ion al e s he TJ ba ie unc ion and inc eases cell mo ili y and in asion. (A) T ansepi helial elec ical esis ance (TER) measu emen s o MKN74 gas ic cells du ing a pe iod o 6 days pos A adin silencing (siAFDN) in compa ison wi h non-silenced (siNS) and wi h un ea ed cells, and du ing 2 days a e H. pylo i in ec ion in compa ison wi h unin ec ed cells. (B) Cell monolaye pe meabili y o 4 kDa FITC-Dex an on day 6 pos A adin silencing and on day 2 a e H. pylo i in ec ion. (C–F) Single cell mo ili y analysis: (C) Time-lapse mic oscopy pho os o cells du ing he 24 h pe iod. (D) Rep esen a i e g aphs o he X and Y coo dina es (in pixels, px) o each cell pa h, on a ib onec in coa ed su ace. (E) Cell eloci y in µm/minu e and (F) o al dis ance co e ed by and indi idual cell in µm. (G,H) Cell in asion capaci y on Ma igel-coa ed answells o MKN74 cells ans ec ed wi h siNS con ol o ans ec ed wi h siAFDN (G), and o MKN74 cells in ec ed o no wi h H. pylo i 26695 (H). Da a a e p esen ed as mean ±SEM. S a is ical signi icance was e alua ed wi h he one- and wo-way ANOVA and wi h S uden ’s - es . he TJs and AJs, namely p ese ing he epi helial ba ie and cell– cell adhesion, and supp essing cell mo ili y and in asion. H. pylo i In ec ion A ec s he Exp ession and Localiza ion o A adin in he Human Gas ic Mucosa To de e mine he impac o H. pylo i in ec ion on A adin in i o, we i s e alua ed AFDN gene exp ession using he da a a ailable a he GTEx da abase. Da a was e ie ed om gas ic samples o 181 indi iduals wi hou disease, and 67 indi iduals (37%) we e iden i ied o be in ec ed wi h H. pylo i. No s a is ically signi ican di e ences (p= 0.30) we e obse ed be ween AFDN gene exp ession le els in unin ec ed and H. pylo i-in ec ed pa ien s (Figu e 6A). The exp ession o A adin was also e alua ed by immunohis ochemis y in a se ies o 42 issue specimens ha ha e been cha ac e ized ega ding he his opa hological ea u es o he gas ic mucosa and he p esence o F on ie s in Mic obiology | www. on ie sin.o g 9No embe 2018 | Volume 9 | A icle 2712