Journal of Cell Science ocnui aeiga ucin,anra oaiaino Jpoen,adnra Jsrcueadbrirfnto.Mcora analysis CGN Microarray between function. expression barrier and gene structure in TJ changes normal significant and show proteins, not TJ of does localization cells normal intestinal a of junctions, at labeling cingulin no CGN nlsssosatoodices ntelvl fcadn2poeni h udnmadtekde fCGN of kidney the and duodenum the in protein claudin-2 of levels the in increase twofold a shows analysis nuyo h oo ydxrnsdu uft lct nitnusal epne nCGN in responses undistinguishable elicits sulfate sodium dextran by colon the of injury xrsino lui-,adtemcslrsos oaueijr nteduodenum. the the control that in networks injury signaling in acute embedded to is response and TJ, mucosal of the function barrier and and claudin-2, structure of the for expression dispensable is cingulin level organism oto fcadn2epeso n epneto the response in and role expression a claudin-2 plays of and control structure, function junction barrier tight epithelial and for dispensable is Cingulin Article Research luis aiyo rtista comprises that permeability size-selective proteins and charge- proteins. tissue-specific of the cytoplasmic regulate family and a transmembrane Claudins, of network al., complex et (Simon syndrome wasting magnesium and 1999). 1999), of (Schmitz pathogenesis al., disease the bowel et in inflammatory barrier including implicated normal diseases, been human the has be of epithelia disruption of must function The compartments where maintained. barrier and intestine, extracellular generated the The and distinct 2009). kidney major of compositionally the al., physiology the including in et organs, importance critical epithelial McCrea of is 2008b; expression TJ of gene function al., and Van differentiation, et roles 2006; cell important (Guillemot al., signaling, play TJ of the et addition, control (Shin maintaining In in 2006). permeability cells for Anderson, and epithelial and the Itallie of tissues, for polarity epithelial responsible apicobasal of primarily function are barrier apicolateral adherens TJ and characteristic (TJ) junctions. junctions a tight comprising display complex, junctional cells epithelial Vertebrate Introduction words: Key knockout CGN generated gene we regulates level, and organism (TJ), the junctions at tight CGN vertebrate of of function region the cytoplasmic investigate the To to cells. localized cultured (CGN is in which signaling protein, RhoA and kDa expression 140 a is (CGN) Cingulin Summary 10.1242/jcs.101261 doi: 5005–5014 125, ß Science Cell of Journal 2012 July 30 Accepted ( correspondence for *Author 4 3 2 1 Guillemot Laurent injury mucosa duodenal muoitceity muoloecne lcrnmcocp n emaiiyasy feihla ise fCGN of tissues epithelial of assays permeability and microscopy electron immunofluorescence, Immunohistochemistry, inlJond Lionel nttt fPtooy H60 oan,Switzerland Italy Locarno, Padova, CH-6601 35100 Pathology, Padova, Italy of of Padova, Institute University 35100 Gastroenterology, Padova, of of Department University Histology, of and Department Biology, Molecular of Department 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. are n inln ucin fT r rhsrtdb a by orchestrated are TJ of functions signaling and Barrier +/+ 2 / 2 itrae.Frhroe CGN Furthermore, littermates. ieb oooosrcmiain CGN recombination. homologous by mice ) igln lui-,Mue ih ucin,Z-,Ocui,RhoA Occludin, ZO-1, junctions, Tight Mouse, Claudin-2, Cingulin, 1 asm Bongiovanni Massimo , [email protected] 1 anSchneider Yann , 5 eateto elBooy nvriyo eea H11 eea Switzerland Geneva, CH-1211 Geneva, of University Biology, Cell of Department ) 2 / 2 ieso neaebtdrsos oteucrgncato fcsemn,weesacute whereas cysteamine, of action ulcerogenic the to response exacerbated an show mice 1 4 al Brun Paola , n adaCiti Sandra and , 5isoforms, 25 2 / 2 ieaeval n etl,adaebr tteepce edla ratios. mendelian expected the at born are and fertile, and viable are mice 2 gai Castagliuolo Ignazio , 1,5, * oan(o)(odnnie l,19) G neat with interacts CGN globular 1999). central comprising al., a subunit et and (Cordenonsi each the tail), (rod) dimer, in and domain 1989). localized a al., (head et specifically Citi as domains 1988; is al., exists et which (Citi CGN TJ protein, the underlying kDa cytoplasm 140 a is McCrea GTPases 2008b; 2011). al., family al., et et Rho Guillemot Citi the 2009; 2006; the al., al., of et to et and regulation Shin contribute transcription in the of (reviewed which to regulation proteins and the translation, contains to cytoplasmic polarity, also The of 2008b). establishment TJ al., regulating of et Anderson, thus Guillemot and region 2000; (Mitic cytoskeleton, Turner, function the 1998; barrier they and to and architecture proteins, proteins junction membrane membrane TJ a other form link and they critical since claudins function, of for barrier are TJ scaffold of TJ The control 2002). of the al., region in cytoplasmic et importance Amasheh the 2001; in that al., increases complexes cells et protein and (Furuse in it epithelia, expressed lack ectopically ‘leaky’ normally when of cations, to typical Anderson, permeability and is Itallie Van Claudin-2 2000; Furuse, 2006). and (Tsukita ions to TJ of igln(G;fo h Latin the from (CGN; Cingulin 2 / 2 2 2 / 2 ail Pizzuti Daniela , n CGN and n CGN and +/+ +/+ ie hra immunoblotting whereas mice, cingere ie ecnld hta the at that conclude We mice. 2 2 ig Martines Diego , ofr etaround) belt a form to / 2 a hlclcoiled-coil -helical ie oprdto compared mice, 2 / 2 ieshow mice 5005 3 , Journal of Cell Science raim ytreigCNi ie n nlzn the analyzing and mice, in CGN CGN of targeting 2009). phenotype al., by et Citi organism, 2006; cells Citi, MDCK and cingulin Guillemot in 2005; and proliferation al., cell GEF-H1, et increased (Aijaz activator in RhoA results the through depletion cells, with addition, epithelial In interaction cultured claudin-2 in systems. its controls activity model RhoA that culture Significantly, controls network cell CGN signaling increased different 2009). a suggesting in of in 2006), expression al., part Citi, resulted is and CGN et also (Guillemot that cells claudin-2 Citi occludin MDCK of expression 2004; in and depletion al., claudin-7 cingulin claudin-6, et protein in claudin-2, TJ (Guillemot large and resulted namely factors, a transcription targeting of genes, including proteins, expression genes, CGN of the TJ number However, in other changes function. of differentiation-dependent barrier localization and CGN, ultrastructure, and of al., devoid normal were et homologous CGN TJ (EB), of had (Guillemot bodies by differentiation embryoid domain by into targeted cells obtained ES head was cells epithelial the locus In the 2004). delete CGN clarify to the To (ES) recombination, 2002). which stem embryonic al., in CGN generated et previously cells for we D’Atri required CGN, ZO-1 1999; of is with function al., interaction CGN its et of via (Cordenonsi domain primarily junctions, head D’Atri to 1999; The al., recruitment et 2002). (Cordenonsi al., myosin and et actin with ZO-3, as and ZO-2 well ZO-1, as including proteins, TJ cytoplasmic several 5006 vdneta G sdsesbefrT are ucinand function barrier TJ for tissues dispensable epithelial in is organization CGN that evidence nkde n udnm n ntersos oauemucosal acute to response the duodenum. in the in and injury duodenum, expression claudin-2 and control kidney that in networks signaling in involved eew netgt h hsooia oeo G nawhole a in CGN of role physiological the investigate we Here ora fCl cec 2 (21) 125 Science Cell of Journal 2 / 2 ie u eut rvd conclusive provide results Our mice. nvivo in n niaeta G is CGN that indicate and , 2 / 2 feo n h e aste rmrst oae ohwti and within both located sets Primer cassette. deletion Neo the the in and resulting 1 allele), exon mutated of 1A, (Fig. sites and LoxP first in third the mice allele, between select occurred to recombination Cre genotyped, Cre-mediated (KO) was which the progeny the expressing WT the and knockout mice to strain, mated C57BL/6 to was mutated offspring mosaic mated resulting The were the recombinase. mice mouse obtain F1 F1 heterozygous heterozygote To the produce cell to generation. strain, stem C57BL/6 were embryonic chimeras to and mated Positive blastocysts, C57BL/6 of into 1B). by injected clones (Fig. were from determined clones cells isolated was The DNA stem genomic vector cingulin. embryonic of targeting of analysis the blot recruitment Southern domain, of junctional head integration globular for Neo the correct required of the most is flanking for which the codes 1, contains and 1 Exon ATG, exon vector). putative targeting of 1A, downstream (Fig. one cassette sites, resistance two LoxP three and locus CGN upstream the within insert to designed was usd h eee einwr sdfrgntpn Fg 1C). 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LoxP head first cingulin long the mouse between 334-residue recombination the mediated the of comprising of 1–279 residues arm, most for right (e.g. codes sequence kb 1 protein 2.4 Exon 4. a and and 3 sites, 2, LoxP exons by flanked and 2 and ( LoxP a containing arm Kpn left kb vector targeting 7.6 The a diagrams. comprises the probe) Neo the 5 (for (for below and above probes) indicated are digestion by generated fragments Apa 5 the of position h idtp lee n . bfamn ntetree lee u to due allele, of targeted analysis the in an fragment of kb insertion 7.2 the a and allele, wild-type the are (EI, Exons sites diagrams. Restriction the numbers. below boxed indicated by are indicated genotyping for used d) c, ( mutated the and allele, (T) targeted the vector, targeting recombination. homologous by locus gene ( the Targeting 1. Fig. A 2 ceai igasrpeetn h idtp lee() the (+), allele wild-type the representing diagrams Schematic ) ;K, I; ie,anoyi eitnecset NO lndbteneos1 exons between cloned (NEO) cassette resistance neomycin a site), I / , 2 7k fgnmcsqec.TeCNtreigvector targeting CGN The sequence. genomic of kb 27 iewr ona h xetdmneinratios mendelian expected the at born were mice Kpn Eco Apa ;Xb, I; Idgse eoi N ihteNopoerslsi a in results probe Neo the with DNA genomic RI-digested -ietdgnmcDAwt h 3 the with DNA genomic I-digested 9 n 3 and Eco Xba 9 Ist lnigtefrtLx ie otenblot Southern site. 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(D) sections KO ultrathin in of KO detected micrograph and was electron (A) abnormality WT No from mice. duodenum (B) of section histological Eosin-stained opooial nitnusal rmteT fCGN of TJ the from indistinguishable morphologically / 6 (26) (%) 165 mice of No. +/ +/+ Genotype i.2 omleihla itlg n J n odtcal cingulin detectable no CGN and of TJ, intestine and the in histology immunolabeling epithelial Normal 2. Fig. CGN of tissue the in TJ typical of duodenum presence of apical the showed sections microscopy thin electron transmission of the by analysis in Ultrastructural crypts, 2A,B). example observed and was (Fig. epithelium villi intestinal For the of of organization morphology abnormalities. normal normal a any and a ileum, detect the and not duodenum did and skin, Fg CD.Imnhsohmclaayi ftedoeu of duodenum the of analysis CGN Immunohistochemical 2C,D). (Fig. 2 al .Gntp fofpn rmmtn fheterozygous of mating from offsping of Genotype 1. Table / 2 2 m AB,20n CD,30 (C,D), nm 200 (A,B), m +/+ iesoe h oaiaino G ln h apical the along CGN of localization the showed mice G mice CGN m (E,F). m 6 (26) (48) 164 298 2 / 2 2 mice. / 2 E , F Immunohistochemical ) ( ie hc were which mice, A , C B , eaoyi-and Hematoxylin- ) D Transmission ) +/+ mice hte hne nteepeso eeso hs Jproteins TJ these of levels expression CGN in the investigate occur To in 2006). changes Citi, and whether (Guillemot cells MDCK depleted ucinlrgoso oaie pteilcls(i.2;and 2E; (Fig. CGN cells from duodenum epithelial of sections contrast, polarized In inset). of magnified regions junctional xrsino aygns nldn nrae ntemRNA the in increases including genes, the in many changes in of in CGN resulted of expression bodies inactivation embryoid targeted mouse the mice differentiated that KO cingulin showed in we genes Previously, protein TJ of Expression proteins from labeling examined TJ we CGN that tissue different junctional epithelial any CGN and of No cells in S1). CGN detected carried was Fig. were against material liver antibodies the (supplementary and using colon and the tissues out kidney, epithelial the of including corresponding sections organs, and in of Immunohistochemistry inset). analysis signal magnified immunofluorescence CGN and 2F; detectable (Fig. regions no showed littermates bevdcagsi rti eeso pcfcT rtisin previously proteins we TJ levels, specific of transcript levels in protein CGN in changes changes to observed addition CGN In of the kidney in and increased duodenum is expression protein Claudin-2 CGN and TJ of tissues, of couples group intestinal selected in a and genes of expression kidney protein the liver, qRT-PCR by we from examined Next, 3A). RNA (Fig. noise isolated gene over background in random involving change above significant comparison statistically expression, no a was isolated there In RNA genes, cells. on 46,600 analysis epithelial microarray CGN intestinal a of out from tissues test carried in first To occur we 2006). expression mice, of gene Citi, in levels and changes threefold) mRNA (Guillemot whether increased threefold) (about in kidney (about in resulted claudin-2 occludin depletion cells CGN (MDCK) Furthermore, and epithelial 2004). twofold), al., fourfold) (about et (Guillemot claudin-6 (about 20-fold), (about claudin-7 claudin-2 of levels lui-,ocui,Z-,adcadn5wsidsigihbein CGN indistinguishable was from claudin-5 tissues and ZO-1, occludin, claudin-7, Gilmte l,20;GilmtadCt,2006). not Citi, models does and cell Guillemot CGN in 2004; cultured al., TJ, of using et of observations lack (Guillemot composition previous the molecular with and that agreement structure and the alter CGN, cell-cell detectably of with tissues devoid epithelial demonstrate in junctions observations results approach these targeting together, our that Taken S1). Fig. material oigfrZ-,cadn2 lui-,cadn7adoccludin, and mRNA claudin-7 of claudin-6, CGN levels claudin-2, between the ZO-3, not in for did differences coding we In significant analysis, WT. microarray statistically of the percent find of as results levels expressed the the were with and sample of agreement 100%, KO expression as the taken of in was mRNA levels sample of WT measured the the in pair, mRNA each each In 3B–D). (Fig. xrsin(i.3–) h eeso h aegnsin mRNA genes of same levels were the examined shown). altered not we of (data that show unchanged ileum) levels also colon, not (spleen, The did tissues additional 3B–D). also (Fig. Paschoud 2002; 2011), expression al., ZO-1, et al., and (D’Atri cingulin Paracingulin et with expression. complex not a of form was the levels that increase in claudin-7, the samples, variability of and tissues, heterogeneity and to claudin-6 KO due in probably for significant, higher statistically example were for levels mRNA cases, some in 2 2 / / 2 2 ie hra h oaiaino aaigln claudin-1, paracingulin, of localization the whereas mice, mrodbde Gilmte l,20)adCGN- and 2004) al., et (Guillemot bodies embryoid 2 +/+ / 2 n CGN and ie yae eepeae rmkde,liver, kidney, from prepared were lysates mice, +/+ iglnkoku nmc 5007 mice in knockout Cingulin n CGN and 2 / 2 itrae Fg BD.Although 3B–D). (Fig. littermates 2 2 / 2 / +/+ 2 mice itrae (supplementary littermates n CGN and 2 / 2 littermates 2 2 / / 2 2 Journal of Cell Science o oa rti otn,uigat-uui niois(i.4A). (Fig. antibodies anti-tubulin were using content, normalized conditions carefully protein were total lysis lysates for and Optimal tissue, each colon). intestine for the established and of segments ileum different three (duodenum, from cells epithelial and 5008 ora fCl cec 2 (21) 125 Science Cell of Journal ifrne nmN eeswr ttsial infcn ( significant statistically were littermates. levels bars) mRNA on and (gray based in (C), KO percentage differences kidney average and the (B), bars) represent liver (white values in WT The proteins, from TJ tissues WT indicated of (D) value the intestinal the for taking 100%) and as qRT-PCR, tissue by ( (determined events. levels triplicate random mRNA using as relative 2.0 analyzed, considered were below be that changes can transcripts fold of these detect number Suite the to considering Genomics able Therefore, Partek not samples. the are these using we Moreover, analysis that 1.6). power shows probes change since two fold significant, only Tnfsrf22, not found and are we 1.2, analysis change (FDR) fold decrease) rate (Gm9934, or discovery (increase false change by fold However Frs3, a B930014J03Rik, with Atxn10, Cpa1) Gcnt2, A930023F12Rik, Taf3, Pogz, Epb4.1, Pias3, CGN eta ierpeettectf iiso .-ad5fl hnei expression. a in with change probes 5-fold and 367 1.5- found of We limits cutoff the represent comparing line transcripts, central mouse ( 46,664 KO the in of transcripts units) arbitrary in (expressed 4 D G oyetd nlstso CGN of lysates in the polypeptide detected CGN kDa antibodies 140 anti-CGN with analysis Immunoblotting netnltat(i.4) osgiiatcagsi their CGN in in detected changes the were significant expression along No of expression 4A). levels barely (Fig. increasing relative at tract a of expressed suggesting intestinal samples, gradient were ileum and rostro-caudal duodenum proteins data). in unpublished levels ZO-1 (2) S.C., cells detectable and CGN-depleted and rod–tail 2004), (L.G., of the 2006) Paracingulin phenotype (3) Citi, al., the and and rescue gives (Guillemot 2008), active et not cells Citi, is does epithelial and Guillemot domain CGN in (Paschoud domain where phenotype 1999; rod–tail no 2), the (Fig. al., of junctions form overexpression et truncated to this (1) target (Cordenonsi since not irrelevant, functionally does is this but irpe h G ee(i.4) rnae protein of lysates truncated in A detected 4A). was (Fig. domains, gene rod-tail CGN successfully the CGN approach containing targeting the our disrupted that demonstrating mice, i.3 xrsino Jpoengnsi h ie,kde,adintestine and kidney, liver, the in genes protein CGN TJ of of Expression 3. Fig. 4C). (Fig. tissues eeicesdb bu wfl,ol nkde n duodenum and expression kidney in of CGN only levels twofold, from claudin-2 about by contrast, increased In were 4A). (Fig. occludin CGN el,adtelvl fatv hAwr o significantly not were CGN RhoA from active lysates of comparing levels when MDCK of the different lysates to and compared tissues, active cells, mouse of of lysates amounts in low RhoA showed Immunoblotting assay. pulldown Fg AB.Teicesdepeso ftecadn2poenin protein claudin-2 the of expression increased The 4A,B). (Fig. 06,w esrdtelvl fatv hAi h inyand kidney the CGN in RhoA from active of levels duodenum of the Citi, measured and labeling we (Guillemot activation 2006), no RhoA increased on or dependent claudin was weak other gave and as but polypeptides. attempted, claudin-7, specific claudin-2, different was claudin-6, with of subcellular isoforms lysates against tissue distribution, mouse labeling sections antibodies of tissue Immunoblotting tissue of of 5). analysis (Fig. of immunocytochemical intensity detectable by pattern any determined and in the result localization, not in did tissues changes duodenum and kidney ic nMC el h peuaino lui- expression claudin-2 of upregulation the cells MDCK in Since +/+ 2 2 / / 2 2 2 mice. / 2 os ise ntson e uleo ta. 2004), al., et Guillemot see shown, (not tissues mouse itrae,smlryt htosre o O3and ZO-3 for observed what to similarly littermates, iei o infcnl ifrn rmteepeso in expression the from different significantly not is mice 2 ( / 2 A o-ltaayi ftenraie xrsinlevels expression normalized the of analysis Dot-plot ) y ie hncmae oCGN to compared when mice, ai)adW ( WT and -axis) 2 P / 2 -value n CGN and x ai)smls h ie aallt the to parallel lines The samples. -axis) , .5 nldn 2(ln Poldip3, (Plin, 12 including 0.05, B–D +/+ itgassoigthe showing Histograms ) itrae,uiga using littermates, +/+ n 5 u o CGN not but , ar.Nn fthe of None pairs. 6 +/+ P +/+ , n CGN and 0.05). littermates . +/+ 2. 2 2 and / / 2 2 Journal of Cell Science CGN o infcnl ifrn nCGN in different significantly not ise,uigatbde gis ifrn Jpoen seMtrasadMtos.Teiae hwrpeettv xmlso lt rmtoCGN two from blots of examples representative show images The Methods). and Materials (see proteins TJ different against antibodies using tissues, CGN fedtxnadssei lo eeso TNF- of levels blood systemic and endotoxin of CGN splmnaymtra i.SBC hwdn significant no cytokines showed inflammatory S2B,C) CGN between intestinal Fig. difference and 6C) of material (Fig. loss levels (supplementary weight (DSS), of (Okayasu of pattern diarrhea sulfate the measurement and of sodium Analysis loss 1990). dextran weight al., et colitis, with acute mice induces different injury, which the a inflammatory in acute challenged result to mucosa we would intestinal CGN the of of sensitivity lack the whether determine CGN Zisge l,20;Wbre l,20) enx xmndthe examined CGN next we in 2008), function al., barrier et Weber intestinal disease 2007; al., bowel et inflammatory to (Zeissig in linked been permeability has intestinal expression increased claudin-2 CGN of in levels sulfate increased sodium Since dextran to response parameters and inflammatory permeability, intestinal Normal CGN of duodenum and CGN kidney and the in protein claudin-2 of levels Increased 4. Fig. el eeue sapstv oto o h ulonasy ihttladatvtdRo ape hc eerni h aegladepsdfrtesame the for exposed and gel same the in run were which samples RhoA activated samples. and duodenum total with and assay, kidney pulldown corresponding the the for as control time positive of a length as used were cells 05.Mcslpeaain rmete h udnmo the or duodenum CGN the either of from al., colon et preparations (Brun Mucosal blood systemic 2005). and portal in cytokines inflammatory esrd ssoni i.6,n ttsial significant statistically tissues no comparing when 6A, detectable CGN Fig. was from resistance in was in resistance shown difference electrical As transepithelial measured. and chambers, Ussing eai tlaeclssiuae yLS intestinal LPS, by IL-1 of stimulated expression intestinal TNF- cells and and in activity, collagen-I stellate myeloperoxidase and fibronectin intestinal of hepatic and levels hepatic including of markers inflammation, additional Similarly, 6B). (Fig. 2 2 2 2 / / / 2 2 2 / 2 n CGN and +/+ n CGN and yae.( lysates. a itrae splmnaymtra i.SA.To S2A). Fig. material (supplementary littermates itrae.Euvln rti odnswr nlzdadnraie by normalized and analyzed were loadings protein Equivalent littermates. +/+ eentsgiiatydfeetbtenCGN between different significantly not were n CGN and +/+ B +/+ +/+ itga hwn eiqatttv esrmn ftedfeecsi xrsino lui- nkde n udnmtsusfrom tissues duodenum and kidney in claudin-2 of expression in differences the of measurement semi-quantitative a showing Histogram ) itraepis( pairs littermate ie eemndb muoltaayi fatv hAioae yGTpldw ihGTroei,adttlRo ees MDCK levels. RhoA total and GST–rhotekin, with pulldown GST by isolated RhoA active of analysis immunoblot by determined mice, n CGN and 2 +/+ / 2 n CGN and ie(i.6) otlbodlevels blood Portal 6A). (Fig. mice 2 / 2 n 5 itrae eemutdin mounted were littermates ) h ifrne r ttsial infcn (** significant statistically are differences The 8). 2 +/+ / 2 2 / n CGN and 2 ie n h eesof levels the and mice, littermates. 2 / 2 2 littermates / 2 a mice +/+ were and b nlmaoyrsos.Cnieignra uoa samples mucosa, normal CGN from Considering response. inflammatory hwducrlsos hra ieoto h wleCGN twelve the of out five whereas lesions, ulcer showed ( higher significantly was seMtrasadMtos.Teucridxo CGN of index ulcer The mouse and Methods). each for and ulcers, index Materials and ulcer of the (see calculate ulcer number to with dissected The used together and were parameters, prevalence, These 1986). was presence score). (ulcer the al., severity mice their assess et on to cysteamine-treated substances Cahill examined, different of 1978; of (Szabo, effect chemically duodenum the to pathogenesis study agent ulcer and used ulcers, widely induce a hydrochloride, cysteamine fcysteamine of udnlmcs.Spriiladde oae lesshowed ulcers CGN located in deep changes histopathological the and similar of Superficial analysis histopathological mucosa. ulcer a duodenal out cysteamine-induced carried to first response we formation, the influence could CGN CGN the lesions. of and no all showed severity mice number, example, the For in increase ulcers. an of to prevalence due 7A), (Fig. mice CGN more expression targets observed. claudin-2 which was increased injury where acute duodenum, of the model specifically disease a used next We CGN ihncoi feihlu n toa el,amild ulcer the a tissue of cells, severity granulation CGN greater of stromal in the that absence and induction suggesting and epithelium 7B,C), (Fig. infiltrate, of mononuclear necrosis with 2 otyt nesadtemcaimtruhwihtelc of lack the which through mechanism the understand to try To / 2 b 2 tblnsann.Nt htfl-eghcnui sudtcal in undetectable is cingulin full-length that Note staining. -tubulin mice. / 2 P , ieaemr estv oteucrgncaction ulcerogenic the to sensitive more are mice .0) ( 0.005). ( +/+ A muoltaayi fpoeni yae rmteindicated the from lysates in protein of analysis Immunoblot ) n CGN and C hAatvt nlstso inyaddoeu of duodenum and kidney of lysates in activity RhoA ) +/+ 2 / iglnkoku nmc 5009 mice in knockout Cingulin 2 n CGN and 2 P / iewsntdet different a to due not was mice 2 , iedslydasmlrdge of degree similar a displayed mice 0.0005, 2 / 2 n iewr hlegdwith challenged were mice 5 2 hnta fCGN of that than 12) +/+ n CGN and 2 2 2 / / / 2 2 2 2 / 2 mice, mice mice +/+ +/+ Journal of Cell Science CGN eklbln sdtce neihla el iigtevli(arrowheads) 50 villi bar: the Scale and lining (arrows), 2001). villi cells al., the epithelial et of in (Rahner base detected the is at claudin-2 zone labeling for proliferative labeling weak crypt loop strong the Henle’s duodenum, in of the detected limb In is descending 2002). thin al., the et in (Kiuchi-Saishin and (arrows) tubules proximal in muoitceia oaiaino lui- ntekde cortex kidney the in ( claudin-2 of localization Immunohistochemical n udnmfo nrae ie n hr a osignificant no was CGN there colon, between and differences the mice, untreated assessed from also duodenum and was Proliferation activity. proliferating nti td erpr h eeainadphenotypic and protein generation TJ the cytoplasmic the report CGN lacking (CGN). we mice cingulin of study characterization this In Discussion ipa eetbehsooia bomlte nmjrepithelial major in abnormalities histological detectable display n CGN and of mucosa intact the CGN within from Next, cells duodenum shown). the epithelial (not the ratio whether crypt/villus asked normal we a of and morphology crypts similar and with granulocytes, villi propria, of lamina absence the complete in almost infiltrate cell plasma and lymphocytic CGN in claudin-2 of of duodenum localization and subcellular kidney and distribution the tissue The 5. Fig. 5010 n te eetbehsooia rmcocpccagsin changes macroscopic or or CGN histological hyperplastic from of detectable tissues absence other the with any consistent S3), Fig. material rlfrtn el a lgtyhge ntedoeu of duodenum the in of higher percentage slightly the Although was cycle. 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al., not al., et was Paschoud et overlapping 2011; expression CGN al., (Ohnishi paracingulin partially et cells Although Paschoud has 2012). 2008a; cultured al., in and is et One Guillemot CGN that CGN, cingulin. with protein to of functions a related lack (CGNL1/JACOP), the structurally paracingulin for is and proteins compensate candidate redundant that may is cells mechanisms MDCK and bodies embryoid eoi N ape rmnorssatE elcoe ( clones cell ES neo-resistant from CGN samples of DNA Genomic generation and RT-PCR, and mice blotting Southern by Genotyping the with and (TV-CGN-3LoxP) LoxP vector a targeting the cloning lacking The pBluescriptSK(-), by a 2004). from al., obtained isolated were et was locus (Guillemot (CGN) cingulin library mouse 129SvJ the of fragments vector DNA targeting Genomic cingulin the of Construction Methods and Materials of not pathophysiological are administration different CGN rates the after of proliferation response in or no different implicated that conditions mechanistically showing suggest results, normal cells, cysteamine, intestinal Our under of rates 2008). proliferation either the Dignass, in al., difference and et significant (Moore Sturm cells and Repair epithelial tract, 1989; of intestinal 1983). differentiation the al., in and process wounds proliferation a et superficial restitution, Boesby reseals epithelial rapidly 1979; that include al., injury after et mechanisms (Szabo histamine gastrin as local such and hormones, enzymes, and mucosal neurotransmitters, of systemic and modulation and including hypersecretion, mechanisms, acid complex Cysteamine multiple, studies. through future ulcers by this induces in investigated claudin-2 be of should role potential phenotype, the and hypotheses, These itself. 5012 ilb neetn oaayetepeoyeo CGNL1 of phenotype the analyze to interesting be will h hsooia oe fteerltdpoen nawhole a in proteins related these of organism. roles physiological the n obeCGN double and hAatvto hntp fCGN of phenotype activation RhoA protein junctional a pathogenesis. of ulcer in the knockout knowledge formation that our ulcer influences to evidence is reported This the selected to first cysteamine. in mucosa of duodenal expression the injury claudin-2 of ulcerogenic response regulating the TJ, in and of organs, role function epithelial and a structure plays the for and dispensable is cingulin level otenbo nlss sn 3 using analysis, blot Southern sn rmr 5 primers using the of upstream 5 The Switzerland). 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P t 2 ewe .5 n .4;A(absent): A 0.045; and 0.050 between ts n diinlseso filtering of steps additional and -test 20 9 ˚ ) n tie ih cingulin with: stained and C), -CAGGGTGTTATAAGCAAT- 2 / 2 iewr backcrossed were mice m )wr re onto dried were m) 9 frad;(d):(forward); 9 9 - - Journal of Cell Science 5m T,1%gyeo.Tsuswr ooeie naduc homogenizer dounce a in homogenized were 4 Tissues X- glycerol. at cocktail Triton 10% inhibitor 1% DTT, protease mM NaCl PMSF, 65 mM mM 100 1 (1 azide, 7.4, sodium mix pH 0.03% Hepes, glycerol, mM 10% 25 100, colon): ileum, (duodenum, U ( DA 0m oimotoaaae MPS,1 rtnX10 1% X-100, mM Triton 10 (1 1% mix fluoride, PMSF, cocktail sodium inhibitor mM protease 2 mM Tris-HCl, DTT, 100 orthovanadate, mM mM 50 pyrophosphate, 10 sodium (kidney): SDS, mM sodium A 10 buffer mM and EDTA, lysis 100 solubilization, selected: protein 7.4, TJ were pH maximise buffers to tested following were the protocols and buffers lysis hmclyidcddoea les(zb,17;Chl ta. 96 were 1986) al., (175 et hydrochloride cysteamine Cahill of 1978; administration oral (Szabo, single a ulcers by produced duodenal induced induction Chemically ulcer of model Cysteamine (12–16 pairs age-matched of colon and CGN duodenum of weeks) from inflammation preparations and function Mucosal barrier intestinal of Measurement MgCl buffer mM MLB 10 ml NP-40, 0.5 1% 7.5, in pH lysed Hepes, segment, mM intestinal 25 the NaCl, mM off (150 scraped was mucosa The activity RhoA of Measurement h lcrclrssac ( resistance electrical the rti odnswr dutdt normalize to adjusted 1:200), revelation. were sc-179, chemiluminescent loadings and Cruz 32-5600; antibodies, Protein (Santa (Invitrogen secondary RhoA HRP-labeled claudin-2 1:500), by Dr 1:200), followed 33-1500, from (Invitrogen London, serum, occludin College, (rabbit 1:250), ZO-3 University 1:500; 39- 1:2000), Matter, Invitrogen 61-73000, 3644-01, K. 1:10,000, (Invitrogen (Invitrogen and 20896 ZO-1 hr, 1:500), (rabbit cingulin 1 8900, paracingulin for antibodies: 1:2500), milk) skimmed following 37-4300 5% Invitrogen the Tween-20, 0.1% with 7.5, pH incubated Tris-HCl mM 50 NaCl, hr). 2 on oguahoeSpaoeBbas oioaeteatv hAfraction RhoA active the isolate 2006). to Citi, beads, and equivalent (Guillemot glutathione–Sepharose4B (RBD) and domain to levels, GST–rhotekin/Rho-binding with RhoA bound incubated total were lysates normalize of to amounts immunoblotting and SDS-PAGE le ne ihsadr eito a lte o ahgenotype. each for plotted was deviation standard with index ulcer ites h udnmwsdsetdfrmcocpceaiainand and of 0 examination counted, signs 1978): after was (Szabo, macroscopic showed score ulcers hr severity they for 9 duodenal 2 a given ulcer, if of dissected within was number times, ulcer sacrificed was each The earlier analysis. were duodenum at to histopathological experiment, Mice or The the genotype cysteamine, of bias. distress. mice completion The of experimenter period. until 175 administration hr ulcer experimenter 9 any of typical the a induce to dose within eliminate to unknown mice, the required was WT selected dose in mice effective mice mortality minimum without WT the lesions on as preliminary curve weight) In status. (mouse dose-effect health undistinguishable a had and experiments, room, facility animal same ciiyadpoifamtr rti ees(TNF- levels protein pro-inflammatory TNF- and systemic experimenter of activity measurement the bias. by to experimenter and unknown any sections, was eliminate mice to assay the experiment, ELISA the of an using of genotype measured completion The were until 2007). vein al., portal et the from (Brun collected sera in levels oCGN to cr,and score, 0 lcrl 5m a,1m oimotoaaae . MPS,1 PMSF, mM 0.1 orthovanadate, sodium mM 4 1 at cocktail) NaF, inhibitor protease mM Roche 25 glycerol, 10% etiue t4 at centrifuged etiue 0mna 400rm t4 at rpm, 14,000 at min 10 centrifuged fW.Dt eeepesda eno ecnaevalues. percentage percent of as mean expressed as were mice expressed WT KO were of of Data values values WT. and couple, of 100%, each as For taken software. were were QuantityOne littermates littermates Bio-Rad WT/KO the of pairs with from analyzed autoradiograms levels, protein of evaluation a eemndb R-C)o irnci,clae yeI monocyte I, type collagen was LPS fibronectin, liver by of from expression isolated qRT-PCR) mRNA of (HSCs) by fold-induction cells determined of stellate measurement (as hepatic by of determined Activation 2007). ees xeiet n rtcl nmc eeatoie yteUiest of University the by experimentation. authorized animal were for mice Committee or on Ethics IL-1 examination, Padova protocols body of measurement and histological Animal and Experiments activity, for Colon MPO days. levels. paraformaldehyde daily. of evaluation 6 recorded in for were frozen, for fixed blood snap either fecal (DSS) of were sulfate presence samples and sodium consistency dextran stool 2007). weight, al., w/vol et 3% Brun 2005; containing al., et (Brun (MCP-1) 1 protein chemoattractant n I 5 5 o muoltig ircluoeset eebokdi B-ik(5 mM (150 TBS-milk in blocked were sheets nitrocellulose immunoblotting, For nlmainwsasse itlgclyo eaoyi-adEosin-stained and Hematoxylin- on histologically assessed was Inflammation ct netnlijr n nlmainwsidcdb diitrn water administering by induced was inflammation and injury intestinal Acute 2 sn h olwn oml VgladVgl 1997): Vogel, and (Vogel formula following the using 12) ˚ U ,icbtdfr6 i t4 at min 60 for incubated C, N 6 + +/+ 5 oh) yi ufrC(ie) 0m rsHl H95 %SDS, 2% 9.5, pH Tris-HCl, mM 50 (liver): C buffer lysis Roche); , U epucr 3 ulcer, deep S U n CGN and + P U sucrpeaec pretg faiaswt les.Teaverage The ulcers). with animals of (percentage prevalence ulcer is P +/+ 6 ˚ A 200rm3 i,B 200rm1 i,C 500rpm 45,000 C: min, 15 rpm 12,000 B: min, 30 rpm 12,000 (A: C 10 n CGN and 2 2 1 / where , 2 5 itrae ae1–6wes hthdbe erdi the in reared been had that weeks) 12–16 (age littermates R efrto.Ucridxwsmaue o ahmouse each for measured was index Ulcer perforation. ; 2 V / 2 /cm U itrae eemutdi sigcabr,and chambers, Ussing in mounted were littermates I 2 sucrindex, ulcer is a esrd(rne l,20) Endotoxin 2007). al., et (Brun measured was ) ˚ ol o ape nlssbfe ) and B), buffer lysis in samples for (only C ˚ ,icbtdfr1 i t4 at min 15 for incubated C, ˚ .Tespraat eeaaye by analyzed were supernatants The C. b tblncnet o quantitative For content. -tubulin a U ees ylprxds (MPO) myeloperoxidase levels, N sucrnumber, ulcer is a 6 5 n IL-1 and oh) yi ufrB buffer lysis Roche); , oucr 1 ulcer, no b 2 ˚ MEDTA, mM 1 , Bu tal., et (Brun ) hkn,and shaking, C b 5 U n TNF- and superficial S sulcer is m g/g), m g/g 6 a uleo,L n ii S. Citi, and L. Guillemot, uue . uue . aai .adTuia S. Tsukita, and H. Sasaki, K., Furuse, S. M., Furuse, Citi, and F. Nadalutti, F., D’Atri, P. Pulimeno, and J. Stutz, Y., Schneider, D., Spadaro, S., Citi, odnni . ’ti . amr . ar,D . edikJns . hr,D. Shore, J., Kendrick-Jones, A., D. Parry, E., Hammar, F., D’Atri, M., and Cordenonsi, L. Jond, F., Robertis, De F., B. Timolati, Geiger, P., Pulimeno, and S., J. Paschoud, S., Kendrick-Jones, Citi, H., Sabanay, S., J. Citi, Kendrick-Jones, and B. Geiger, R., Jakes, H., Sabanay, S., Citi, ail .C,Glahr .T n zb,S. Szabo, and T. G. Gallagher, C., M. Cahill, rn . atgioo . iLo . ua . izn,M,Palu M., Pinzani, A., Buda, V., Leo, Di I., Castagliuolo, P., Brun, Palu M., Pinzani, I., Castagliuolo, P., Brun, uleo,L,Pshu,S,Plmn,P,Fgi,A n ii S. Citi, and A. Foglia, P., Pulimeno, S., Paschoud, L., S. Guillemot, Citi, and A. Foglia, L., Jond, S., Paschoud, L., Guillemot, J. Spencer, and R. Mendez-Diaz, K., W. Man, S., Boesby, upeetr aeilaalbeoln at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.101261/-/DC1 online available material Supplementary 2009HLNNRL_002 of I.C.]. number Ministry [grant to Italian (MIUR) the Research from and [grant and University (ESCMID) P.B.]; Diseases to Society Infectious AZMAT03031159 European number and 3100-064089, the Microbiology of S.C.]; Clinical 3100-052903, Section to of 31003A116763 numbers the National and Geneva; Swiss [grant 31003A103637 the of from Foundation grants State Geneva; the Science of University by the of supported Biology was work This materials. and reagents of Funding gifts generous their cited for colleagues Schaad to text, Genetics’) also the in Thanks Olivier in statistics. ‘Frontiers and and Program analysis NCCR microarray analysis, with for the help of RT-PCR for Platform and and Stutz (Genomics and Lunghi Jeff construction immunohistochemistry and Lara Kaister plasmid with Marq, Christian assistance microscopy, Lin electron for Nathalie Henchoz Gindre, Philippe Patrizia thank We Acknowledgements when Student’s significant using statistically data considered parametric were Results for two-tailed). groups between means drawn as expressed were were data quantitative All analysis Statistical uleo,L,Hma,E,Kitr . iz . ale . od . ae,C., Bauer, L., Jond, D., Caille, J., Ritz, C., Kaister, E., Hammar, L., Guillemot, Scho H., A. 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