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European Journal of Cell Biology
j ou rn a l h o m e p a g e :w w w . e l s e v i e r . c om / l o c a t e / e j c b
Research paper
Connexin43 high prostate cancer cells induce endothelial connexin43 up-regulation through the activation of intercellular
ERK1/2-dependent signaling axis
Katarzyna Piwowarczyk
a,1, Milena Paw
a,1, Damian Ryszawy
a,
Magdalena Rutkowska-Zapała
b, Zbigniew Madeja
a, Maciej Siedlar
b, Jarosław Czy ˙z
a,∗aDepartmentofCellBiology,FacultyofBiochemistry,BiophysicsandBiotechnology,JagiellonianUniversity,Kraków,Poland
bDepartmentofClinicalImmunology,InstituteofPaediatrics,FacultyofMedicine,JagiellonianUniversityMedicalCollege,Kraków,Poland
a r t i c l e i n f o
Articlehistory:
Received29December2016
Receivedinrevisedform24March2017 Accepted31March2017
Keywords:
Prostatecancer Cx43
Endothelialcells ERK1/2 Diapedesis
a b s t r a c t
Connexin(Cx)43regulatestheinvasivepotentialofprostatecancercellsandparticipatesintheirextrava- sation.ToaddresstheroleofendothelialCx43inthisprocess,weanalyzedCx43regulationinhuman umbilicalveinendothelialcellsintheproximityofCx43high(DU-145andMAT-LyLu)andCx43lowprostate cancercells(PC-3andAT-2).EndothelialCx43up-regulationwasobservedduringthediapedesisofDU- 145andMAT-LyLucells.ThisprocesswasattenuatedbytransientCx43silencingincancercellsandby chemicalinhibitionofERK1/2-dependentsignalinginendothelialcells.Cx43expressioninendothelial cellswasinsensitivetotheinhibitionofgapjunctionalintercellularcouplingbetweenCx43highprostate cancerandendothelialcellsby18␣-glycyrrhetinicacid.Instead,endothelialCx43up-regulationwascor- relatedwiththelocalcontractionofendothelialcellsandwiththeiractivationintheproximityofCx43high DU-145andMAT-LyLucells.Itwasalsosensitivetopro-inflammatoryfactorssecretedbyperipheral bloodmonocytes,suchasTNF␣.IncontrasttoCx43lowAT-2cells,Cx43lowPC-3cellsproducedangioac- tivefactorsthatlocallyactivatedtheendothelialcellsintheabsenceofendothelialCx43up-regulation.
Collectively,thesedatashowthatCx43lowandCx43highprostatecancercellscanadaptdiscrete,Cx43- independentandCx43-dependentstrategiesofdiapedesis.Ourobservationsidentifyanovelstrategy ofprostatecancercelldiapedesis,whichdependsontheactivationofintercellularCx43/ERK1/2/Cx43 signalingaxisattheinterfacesbetweenCx43highprostatecancerandendothelialcells.
©2017TheAuthors.PublishedbyElsevierGmbH.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Diapedesisisthefinalstepofcancercelldissemination.Itiniti- atestheformationofsecondarytumorsincolonizedorgans.During thediapedesis,circulatingcancercellsmigratethroughthecapil- larywallstowardsthesitesofprospectivemetastases.Duetothe barrierfunctionofcapillaryendothelium,cancercelldiapedesisis alimitingstepofthemetastaticcascade(Reymondetal.,2013;van Zijletal.,2011).Theabilityofcancercellstopenetrateendothelial continuumdependsontheirintrinsicproperties,suchasadhesive- ness,motility,nanomechanicalelasticityandproteolyticactivity
∗ Correspondingauthorat:DepartmentofCellBiology,FacultyofBiochemistry, BiophysicsandBiotechnology,JagiellonianUniversity,ul.Gronostajowa7,30-387, Cracow,Poland.
E-mailaddress:jarek.czyz@uj.edu.pl(J.Czy ˙z).
1 Theseauthorscontributedequallytothiswork.
(FriedlandAlexander,2011;FriedlandWolf,2010;Nauseefand Henry,2011).However,thedisruptionofendothelialcontinuum intheproximityofcancercellsisalsofacilitatedbythelocalacti- vationofendothelialcells,oftenmanifestedbytheinductionof endothelialcell motility(Piwowarczyket al.,2015).These pro- cessesareinducedbythechemical andphysical stimuli,which canbeexchangedbetweencancer,immuneandendothelialcells.
Humoralfactors,celladhesionreceptorsandconnexinsparticipate intheintercellularcommunicationloopsestablishedwithinsuch pre-metastaticnichesofcancercells(Reymondetal.,2013).
Connexinsareintegralmembraneproteinsthatspontaneously oligomerizeintohexamerichemichannels(connexons).Connexons incorporated into the membranes of adjacent cells form inter- cellular aqueous channels, which are often organized into the semi-crystallineclustersofgapjunctions(MaedaandTsukihara, 2011).Gapjunctionsmediatethedirectintercellularexchangeof ionsandsmallmetabolites(<1.5kDa)intheprocessofgapjunc-
http://dx.doi.org/10.1016/j.ejcb.2017.03.012
0171-9335/©2017TheAuthors.PublishedbyElsevierGmbH.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/
4.0/).
tionalintercellular coupling(GJIC;(Ek-Vitorinand Burt,2013)).
Singleconnexinmoleculescanalsoregulatetheintracellularsig- nalingindependentlyoftheirchannelfunction,whereasunpaired connexonslinkintracellularcompartmentswithextracellularenvi- ronment(Dbouketal.,2009;Vinkenetal.,2011).Thecomplexity of connexin functionsat thecellular level is paralleled by the multidirectionalinvolvementofconnexinsincancerdevelopment.
Etiopathology of cancer has long been attributed to the com- promised expression and function of tissue-specific connexins (KandouzandBatist,2010;Laird,2006;Leitheetal.,2006;Pointis etal.,2007).However,thereportsonthesuppressivefunctionof Cx43incancerpromotion weremore recentlyconfrontedwith theincreasingamount of datashowing that Cx43can increase theinvasivepotentialofcancercells.Nowadays, itis clearthat theGJIC-dependentandGJIC-independentfunctionsofconnexins (inparticularCx43)in cancerdevelopmentdependonitsstage (Czyz,2008).Stage-specificCx43functionsincarcinogenesisare manifestedbyCx43involvementinthemicroevolutionofinvasive sub-populationsofcancercells(Ryszawyetal.,2014)andinthe regulationoftheirmigration(Defamieetal.,2014).Cx43wasalso showntofacilitatethediapedesisofcancercells(Itoetal.,2000;
Pollmannetal.,2005;Tangetal.,2013).However,theinvolvement ofendothelialCx43intumorprogressionhasnotbeenaddressed sofar.
Stage-specific function of Cx43 has also been observed in prostate cancer (Czyz et al., 2012; Mol et al., 2007), which is a leading causeof cancer-related deathin developed countries (Siegel et al.,2016).Primary prostate tumors display relatively lowexpressionlevelsofconnexins(incl.Cx43)incomparisonto the“native”tissue.Ontheotherhand,elevatedCx43levelshave beenimplicatedinprostatecancerprogression(Czyzetal.,2012;
Lamicheetal.,2011).WehaverecentlyfoundthatCx43mediatesa GJIC-dependentandGJIC-independentintercellularsignalingthat locallyactivatesendothelialcellsandaugmentstheefficiencyof Cx43highprostatecancercelldiapedesis(Piwowarczyketal.,2015;
Ryszawyetal.,2014).Conceivably,partnerCx43moleculesbuilt inthemembranesofprostatecancerandendothelialcellsarenec- essarytoestablishCx43-dependentintercellularcommunication duringthediapedesis.ToaddresstheroleofendothelialCx43in thisprocess,weanalyzedCx43regulationinhumanumbilicalvein endothelialcellsincubatedintheproximityofCx43high(DU-145 andMAT-LyLu)andCx43low(PC-3andAT-2)prostatecancercells.
2. Materialsandmethods 2.1. Cellcultures
Humanumbilicalveinendothelialcells(HUVEC;obtainedfrom LifeTechnologiesCorporation)werecultured(upto6passages) in EBM medium supplemented with 10% FBS and supplement cocktail(rhEGF,bovinebrainextract,hydrocortisone,gentamicin, amphotericin-B;allfromLonza).HumanprostatecarcinomaDU- 145andPC-3cellswereroutinelycultivatedinDMEM-F12HAM medium(Sigma)supplementedwith10%FBSandantibiotics.Rat prostatecarcinomaMAT-LyLuandAT-2cellsweregrowninFBS- supplemented (10%) RPMI 1640 medium (Lonza),as described previously (Miekus et al., 2005). Co-culture experiments were performedonHUVEC monolayers between70 and 98% conflu- ence (as indicated in the text). Cancer cells were seeded into HUVECculturesatthedensityof1300cells/cm2 andco-cultures wereanalyzedinsupplementedEBMmedium.Whereindicated, conditionedmedia(CM),18␣-glycyrrhetinicacid(AGA), lithium chloride,TNF␣,PD98059orGM6001wereadministeredatthetime pointsandconcentration(s)indicatedinthetext.Prostatecancer cell-conditionedmediawereaspiratedfromprostatecancercell
cultures after24hof incubation,centrifuged, mixedwithfresh media(ratio3:5)andaddedtoHUVECculture.
2.2. Immunofluorescenceandcytofluorometry
Fortheimmunofluorescenceanalysis,cellswerefixedwith3.7%
formaldehydefor20minatroomtemperature(RT)andperme- abilisedfor5minwith0.1%TritonX-100in PBS.FortotalCx43 staining,cellswerealsofixedinMetOH/Acetone(7:3)solutionfor 10minin−20◦C.Fixedcellswereincubatedin3%BSAsolution for60minand withprimary antibodies(rabbitpolyclonal anti- VE-cadherinantibody(1:200;V1514;Sigma)orrabbitpolyclonal anti-Cx43antibody(1:200;C6219;Sigma))for1h.Then,thecells wereincubatedwithsecondary antibody(Alexa Fluor® 488-or AlexaFluor® 546-conjugatedgoatanti-rabbitantibodies(1:300 or1:600respectively;A11008andA11010;LifeTechnologies)or Alexa Fluor® 488-conjugated phalloidin (1u/slide; A12379; Life Technologies)andcounterstainedwithHoechst33358(0.5g/ml;
B2883; Sigma). Image acquisition was performed with a Leica DMI6000B microscope (DMI7000 version; Leica Microsystems, Wetzlar,Germany) equipped withthe Total Internal Reflection Fluorescence(TIRF)andNomarskiInterferenceContrast(DIC)mod- ules.
FreeconnexinsandconnexonsarelargelysensitivetoTriton- X100extraction,whereastheybecomeinsolubleinTriton-X-100 upon incorporation into mature gap junctions (Kelsey et al., 2014). Therefore, for the cytofluorometric estimation of total and detergent-insoluble Cx43 fraction, confluent HUVECs and HUVEC/DU-145 specimens were fixed/permeabilised with 3.7%
formaldehyde/0.1%TritonX-100orinMetOH/Acetone(7:3)solu- tion, respectively, and stained against Cx43/DNA as described above.For each specimen,thestacks of fluorescenceimagesof atleast16randomlychosenconfluentcultureregionswerecol- lectedinred(N2.1;excitation–BP515-560;emission–590LP)and bluechannel(A4;excitation–BP360/40;emission–BP470/40).In eachexperiment,thestackswereobtainedwiththesameexcita- tion/exposuresettings(excitation/cameragain/timeofexposition).
The Cx43Fluorescence Index (i.e.the ratioof Cx43- and DNA- specificfluorescenceintensity)wasestimatedforeachstackwith LasXsoftware(Leica)tonormalizeCx43contentagainstthenum- berofcells.Finally,theaveragedCx43FluorescenceIndex(FICx43) wascalculatedforeachspecimen(Pawetal.,2017).
2.3. qRT-PCR
TotalmRNAwasobtainedfromcellcultureswiththeRNeasy MiniKitPlus(Qiagen,Inc.)andsubsequentlyreverse-transcribed withHighCapacityReverseTrancriptionKit(AppliedBiosystems).
DetectionofCx43,VEGFandGAPDHmRNAlevelswasperformed byreal-timeRT-PCRassayusing7500FastSystem(AppliedBiosys- tems).ForthedetectionofspecificcDNAsTaqManGeneExpression Assaywas used including FAM-labeled probes: Hs00748445s1 (Cx43), HS00900055m1 (VEGF) and Hs99999905m1 (GAPDH) (AppliedBiosystems).GAPDHwasusedasareferencegene.Results areshownasaCtvalue.
2.4. Immunoblotandarrayanalyses
For immunoblot analyses, the cells were dissolved in lysis bufferandcellularproteins(20g/lane)wereappliedto10%or 15%SDS-polyacrylamidegels,followedbytransfertonitrocellu- losemembrane.Blotswereexposedtoprimaryrabbitpolyclonal anti-Cx43 (1:4000: C6219; Sigma), mouse monoclonal anti-␣- tubulin (1:2000; No. T9026; Sigma) IgG, rabbit monoclonal anti-phosphorylatedERK1/2(Thr202/Tyr204;1:000,9106;CellSig- naling) IgG and anti-ERK1/2 (1:1000,9107; Cell Signaling) IgG,
followed bytheapplication of theHRP-labeledsecondary anti- bodies(G21040andG21234;LifeTechnologies).HRPactivitywas detectedwithSuperSignalWestPicoSubstrate(Pierce,Rockford, IL;(Daniel-Wojciketal.,2008)),followedbybanddetectionwith PierceTMCN/DABSubstrateKit(ThermoScientific)andthechemi- luminescence imaging systems (MicroChemi; DNR Bio-Imaging Systems,Jerusalem,Israel).Bandintensitieswerecalculatedrel- ativetothecorrespondingcontrol(␣-tubulin)usingtheImageJgel analysistoolandsubjectedtostatisticalanalyses(Pawetal.,2017).
Angiogenesis-related protein expression was semi- quantitatively estimated with antibody array kit (Proteome ProfilerTMHumanAngiogenesisArrayKit,R&DSystems,ARY007) according tothe manufacturer’sprotocol. Sampleswere mixed withacocktail ofbiotinylatedantibodiesandthenappliedonto nitrocellulose membranes to allow their binding to cognate immobilizedcaptureantibodies.Complexesweredetectedwith streptavidin-HRP system. The signal produced at each spot in proportion totheamount of theanalyte boundwasregistered withtheMicroChemii imaging system(QuantityOnesoftware) anddotintensitieswerecalculatedusingtheImageJgelanalysis tool.
2.5. Transendothelialpenetrationassaysandcellmotilitytests
HUVECs wereseeded oncoverslips at2×104 cells/welland grown toconfluence for 72h. Prostatecancer cells (1300/cm2) were stained with CellTracker Orange CMRA (10M, C34551;
LifeTechnologies; 30min), washedin PBS, seededona HUVEC monolayersandincubatedfor6h.Thereafter,theco-cultureswere fixed/permeabilisedwith3.7%FA/0.1%Triton-X-100andstained againstactin/DNA(phalloidin-Alexa488/Hoechst33258).Alterna- tively, HUVEC monolayers were pre-incubated in the presence ofunstainedDU-145cellsfor6h.Thereafter,CellTrackerOrange CMRA-stainedDU-145cellswereseededonHUVECmonolayers.
Theco-cultureswereincubatedfor1and18h,fixed/permeabilised with 3.7% FA/0.1%/Triton X-100 and stained against actin/DNA (phalloidin-Alexa488/Hoechst33258).Then, thespecimens were microscopically assessed towards discontinuities of endothelial barrier in the proximity of CMRA+ cancer cells. At least 200 eventsin3independentexperimentsweretakentoquantifythe percentage ofcancercellscapableof disruptingtheendothelial continuumintheirproximity(transEndothelialPenetrationIndex –EPI;Piwowarczyketal.,2015).
The movement of HUVECs in control condition and in co- cultureswithprostatecancercellswasestimatedintheconditions ascertaining theirbasal motility (70%confluence; Piwowarczyk etal.,2015)withtheDMI6000Btime-lapsevidoemicroscopysys- temequippedwithatemperaturechamber(37◦C/5%CO2),IMC contrastopticsandCCDcamera.HUVECswereseededatdensity of500cells/cm2andculturedfor4daystoformislets(70%con- fluence).Onlythe“proximal”HUVECs,i.e.thecellsthathaddirect contactwithcancercellsattheonsetofregistration,wereanalyzed.
Thetrajectoriesofindividualcellswereconstructedfromaseriesof HUVECcentroidpositionsrecordedfor7hat300sintervalsinthe absence(control)andpresenceofcancercells(1300/cm2)usinga dry20×,NA-0.75objective.Thedatawerepooledandanalyzedto estimatetheaveragelengthofcelltrajectory;(Distance;m),the averagelengthofcelldisplacementi.e.thedistancefromthestart- ingpointdirectlytothecell’sfinalposition(Displacement;m), andtheaveragerateofcelldisplacement,i.e.thedistancefromthe startingpointdirectlytothecell’sfinalposition/timeofrecording (ARCD;m/h).Celltrajectoriesfromnolessthanthreeindepen- dentexperiments(numberofcells>50)werestatisticallyanalyzed (Korohodaetal.,2002).
2.6. Cx43silencingbysiRNA
DU-145cellswereseededatadensityof7×104/wellinto12- well plates in antibiotic-free DMEM-F12 HAM culture medium supplemented with 10% FBS. After 24h, the cells were trans- fectedwithMISSION®GJA1(EHU105621;114pmol;Sigma)using LipofectamineTM2000 (11668019; Invitrogen) according to the manufacturer’sprotocol.TheefficiencyofCx43silencingwasana- lyzedwithimmunoblottechnique(Piwowarczyketal.,2015).
2.7. Calceintransferassays
Calcein(LifeTechnologies;C3099)-loadeddonorcells(DU-145, PC-3)wereseededontothemonolayersofacceptorcells(HUVEC;
1:50)growninPetridishesasdescribedpreviously(Wybieralska et al., 2011).After1h, calceintransfer from donor toacceptor cells was evaluated using a Leica DMI6000B inverted fluores- cencemicroscope(Leica;excitation–BP470/40nM;emission–BP 525/50)equippedwithLasXsoftware.Heterologousgapjunctional couplingwasquantifiedasthepercentageofdonorcellssuccess- fullycoupledwithacceptormonolayer(couplingindex–ci).Adye transferfromatleast200donorcellspercoverslipwasanalyzedin3 independentexperiments(N=3)performedforeachexperimental condition.
2.8. Isolationofperipheralbloodmonocytes
PeripheralbloodmononuclearcellswereisolatedfromEDTA- treated blood of humanhealthy donors by the Ficoll/Isopaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation, accordingtotheapprovalofthelocalJagiellonianUniversityEthi- calCommittee(No.KBET/330/B/2012).Peripheralbloodmonocytes (PBMs)wereseparatedfrommononuclearcellsbycounter-flow centrifugalelutriationwiththeJE–6Belutriationsystemequipped witha5mlSandersonseparationchamber(Beckman-Coulter,Palo Alto,CA;Baranetal.,2009;Mytaretal.,2001)andsuspendedin RPMI1640culturemedium(PAALaboratories,Pasching,Germany) withgentamycin(25g/ml;Sigma).PurityofPBMswasover95%, asjudgedbystainingwithanti-CD14mAb(561383;BDBiosciences Pharmingen,SanDiego, CA) and flow cytometryanalysis (FAC- SCanto,BDBiosciencesImmunocytometrySystems,SanJose,CA).
Forendpointexperiments,PBMswereseededontheHUVECmono- layersandintoHUVEC/DU-145co-culturesatthedensityof104and 105cells/cm2.
2.9. Statisticalanalysis
Thestatisticalsignificanceofthedifferenceswastestedwith one-way ANOVAfollowedby post-hocTukey’s HSDorDunnett comparisonforvariableswithGaussianandnon-Gaussiandistri- bution,respectively.Data areexpressedasthemean±standard errorofthemean(SEM).Statisticalsignificancewassetat*p<0.05;
**p<0.01.
3. Results
3.1. DU-145prostatecancercellsinduceendothelialCx43 up-regulation
Toaddresstheinfluenceofprostatecancercellsonendothe- lialCx43 levels,we seededDU-145 cells onthemonolayers of humanumbilical veinendothelialcells(HUVECs;Fig.1).A local retractionofHUVECs wasobserved intheproximityofDU-145 cells.Itwasaccompaniedbythedisruptionofintercellularcon- tacts,thecytoskeletalrearrangements(incl.disappearanceofactin corticalbeltsinHUVECs (Fig.1A;c.f. Piwowarczyket al.,2015)
Fig.1.Cx43highDU-145prostatecancercellsinduceendothelialCx43up-regulation.(A)DU-145cells(markedwitharrows)wereseededontothemonolayerofHUVECs (98%confluence)atthedensityof1300cells/cm2andspecimenswerefixedafter6hwith3.7%FA,permeabilized,stainedforVE-cadherin(left)orF-actin(right)and counterstainedwithHoechst33258.(B)HUVECwereculturedintheabsence(control)orpresenceofDU-145cells(markedwitharrow).Cx43localizationandexpression levelswereestimatedatindicatedtimepointswithimmunostainingofMetOH/Acetone-fixedspecimens(left,cf.Fig.E1inSupplementarymaterial),qRT-PCR(upper right;thestatisticalsignificanceofthedifferencesinCx43mRNAlevelswastestedwithone-wayANOVAfollowedbypost-hocnon-parametricDunnettcomparison.
**p<0.01)andimmunoblotting(lowerright).ProteinlevelsinHUVEC/DU-145co-cultureswerecomparedwiththe“mock”control(HUVECsandDU-145celllysatesmixed atrelevantproportions);fordensitometricandstatisticalanalysesoftheblotsseeFig.E2AinSupplementarymaterial).(C)HUVECsco-culturedwithDU-145cellswere fixedwithMetOH/Acetone(7:3,−20◦C)orwith3.7%FAandpermeabilizedwithTritonX-100.Then,thespecimenswerestainedagainstCx43/DNAandthecytofluorometric quantificationofthelevelsoftotal/Triton-insolubleCx43fractionwasperformedatindicatedtimepoints.(D)PercentageofDU-145cellsthatdisruptednativeand“pre- activated”HUVECmonolayerat(transEndothelialPenetrationIndex;EPI)wasestimatedattheindicatedtimepoints.ThestatisticalsignificanceofthedifferencesinC andDwastestedwithone-wayANOVAfollowedbypost-hocTukey’sHSDcomparison(*p<0.05(Cytofluorometry)and**p<0.01(EPI)).Scalebars:40m.Allresultsare representativeofthreeindependentexperiments.NotethatDU-145celldiapedesisinvitroisaccompaniedbyendothelialCx43up-regulationatthemRNAandprotein levels.
andendothelialCx43up-regulationdetectedbyimmunofluores- cenceandRT-qPCR(Fig.1B;seeFig.E1inSupplementarymaterial).
SlightlyincreasedCx43mRNAlevelswereseeninHUVECsalready after3hoftheirco-culturewithDU-145cells.Theyreachedtheir maximum 3h thereafter and gradually decreased to the basal levels24hafterDU-145seeding.EndothelialCx43up-regulation was confirmed at the protein level by immunoblotting stud- ies(Fig.1B, seealsoFig.E2in Supplementarymaterial)and by cytofluorometricanalysesoftotal Cx43inMetOH/Acetone-fixed HUVECs (Fig. 1C). Concomitant increase of detergent-insoluble (formaldehyde-fixable)fractionofCx43illustratedtheincorpora- tionofnewlysynthesizedendothelialCx43intothefunctionalgap junctions.
ToestimatetheeffectofendothelialCx43up-regulationonthe efficiencyofDU-145diapedesis,wefurtheranalyzedthekineticsof thepenetrationthroughendotheliallayersbyDU-145cells.Only asmallportionofDU-145cells(ca.25%)wasabletodisruptthe
“native”(control)endotheliumwithin1hafterseedingasillus- tratedbythevaluesofendothelialpenetrationindex(EPI;Fig.1D).
TheaccelerationofthisprocesswasobservedwhenDU-145cells wereseededontheendotheliallayersthathadbeenpre-activated byDU-145cells for 6h. In suchconditions,endothelial discon- tinuitieswere observedin the proximity of ca. 75%of DU-145 cells1haftertheirseeding.Thesedata indicatedthat endothe-
lialCx43up-regulationmightaugmentHUVECresponsivenessto Cx43-mediatedsignalsfromcancercells.
3.2. Cx43-dependentintercellularsignalingaxisinduce endothelialCx43up-regulation
ToidentifythemechanismsofendothelialCx43up-regulation, wefurthercompared HUVECreactionstothesignals fromDU- 145cellsandPC-3cells.PC-3cells,whichoriginatefromprostate cancermetastasistobone,displayedconsiderablylowerCx43lev- elsthanbrainmetastasis-derivedDU-145cells(Fig.2A).Onlya slightandtransientincreaseofCx43-encodingmRNAlevelswas observedin PC-3/HUVECco-cultures(Fig.2B),whereas Cx43low PC-3cellsfailedtoinduceendothelialCx43up-regulationatthe proteinlevel.Furthermore,Cx43down-regulationinCx43highDU- 145cellsbyCx43siRNAstronglyaffectedthekineticsofendothelial Cx43up-regulation(Fig.2C).WedidnotobserveendothelialCx43 up-regulationafter6hofHUVEC/DU-145siRNAco-incubation(cf.
Fig.1B).Instead,increasedabundanceofCx43localizedatintercel- lularcontactswasobservedinendothelialcells1hand24hafter DU-145siRNAcellseeding.Finally,wecomparedHUVECreactions toratprostatecancerMAT-LyLuandAT-2cells.IncontrasttoAT-2 cells,MAT-LyLucellsdisplaythepredilectiontometastasizetolym- phaticnodesanglungs(Carteretal.,1989)andahighinvasiveness
Fig.2. ProstatecancercellsinduceendothelialCx43up-regulationinaCx43-dependentmanner.(A)HUVEC,PC-3andDU-145cellswerecultivatedtoconfluenceandCx43 expressionintheirpopulationswasestimatedwithimmmunoblotting(upperpanel;fordensitometricandstatisticalanalysesoftheblotsseeFig.E2BinSupplementary material)andimmunofluorescence(lowerpanel).(B)HUVECwereculturedintheabsence(control)orpresenceofPC-3cells.Cx43-encodingmRNAlevelswereanalyzedwith RT-qPCR(left;thestatisticalsignificanceofthedifferenceswastestedwithone-wayANOVAfollowedbyDunnettcomparison.**p<0.01).Cx43localizationandexpression levelsinco-cultureswereestimatedwithimmunoblotting(middle)andimmunofluorescence(right;PC-3cellsmarkedwitharrows).ProteinlevelsinHUVEC/PC-3co-cultures werecomparedwiththe“mock”control(HUVECsandPC-3celllysatesmixedatrelevantproportions);fordensitometricandstatisticalanalysesoftheblotsseeFig.E2C inSupplementarymaterial).(C)Cx43expressionwastransientlysilencedinDU-145cellsbyesiRNAGJA1(left;seeFig.E2DinSupplementarymaterial).Cx43expression levelsinHUVEC/DU-145siRNAco-cultureswereestimatedattheindicatedtime-pointswithimmunoblotting(middle;fordensitometricandstatisticalanalysesoftheblots seeFig.E2DinSupplementarymaterial)andwithimmunofluorescence(right;cf.Fig.1B;DU-145cellsaremarkedwitharrows).(D)HUVECswereculturedintheabsence (control)orpresenceofMAT-LyLu(left)andAT-2cells(right).Proteinlevelsinco-cultureswerecomparedwiththe“mock”control;fordensitometricandstatisticalanalyses oftheblotsseeFig.E2EinSupplementarymaterial).Scalebars:40m.Allresultsarerepresentativeofthreeindependentexperiments.NotethecorrelationbetweenCx43 expressionlevelsincancercellsandendothelialCx43up-regulation.
invitro(Miekusetal.,2005;Ryszawyetal.,2014).Cx43highMAT- LyLucellsinduced aprominentendothelialCx43up-regulation, whereaswedidnotobserveanyconsiderableeffectsofAT-2cells onCx43expressioninHUVECs(Fig.2D).Ourobservationsshow thatCx43incancercellscanaffecttheexpressionofendothelial Cx43.
3.3. ERK1/2participatesinGJIC-independentendothelialCx43 up-regulationbyprostatecancercells
Toidentify thesignalingpathways thatregulate endothelial Cx43 expression in the proximity of Cx43high prostate cancer cells,wefurtherinhibitedGJICbetweenDU-145andendothelial cells by 18␣-glycyrrhetinic acid(AGA; Fig. 3A). AGA treatment hadnoeffectonDU-145-inducedendothelialCx43up-regulation (Fig.3B).InconjunctionwiththeattenuatingeffectofCx43down- regulation in DU-145 cells on this process (see Fig. 2C), this resultindicatesthatDU-145cellsaffectendothelialCx43expres- sioninaCx43-dependent,GJIC-independentmanner.Furthermore, lithiumchloridewhich isanactivatorofGSK3-dependentsig- naling, did not affect Cx43 expression in HUVECs (Fig. E3 in Supplementary material).On the other hand,endothelial Cx43 up-regulationinHUVEC/DU-145co-cultureswasaccompaniedby ERK1/2phosphorylationinendothelialcells,whereastheinhibition of ERK1/2 phosphorylation by PD98059 attenuated Cx43 up- regulationinHUVECsasshownbyimmunoblottingassay(Fig.3C).
Immunofluorescenceanalysisofthetotal(MetOH/Acetone-fixed) and detergent-insoluble (FA/0.1% Triton-fixed) Cx43 levels in HUVECsco-culturedwithDU-145cellsconfirmedtheinvolvement ofERK1/2intheregulationofCx43synthesis.PD98059reducedthe total(incl.newlysynthesized)endothelialCx43levels,butexerted
alesspronouncedeffectondetergent-insolubleCx43fractionin HUVEC/DU-145co-cultures. Thisnotionis illustratedby a rela- tiveabundanceofCx43-positiveplaquesattheHUVECinterfaces inDU-145/PD98059-treatedcells(seeinsertsinFig.3D).Collec- tively,thesedatashowthattheintercellularCx43/ERK1/2signaling axisinducesendothelialCx43up-regulationinHUVEC/DU-145co- culturesinaGJIC-independentmanner.
3.4. Cx43up-regulationincreasesendothelialsusceptibilitytothe signalsfromprostatecancercells
Cancer cell diapedesis is facilitated by the local activation of endothelial cells, which can be illustrated by the induction of endothelial cell motility in the proximity of cancer cells (Piwowarczyketal.,2015).Therefore,tofurtherestimatewhether endothelialCx43up-regulationfacilitatesthelocalactivationof endothelialcellsduringprostatecancercelldiapedesis,weana- lyzedHUVECmotile behaviorin theproximityofDU-145cells.
RelativelyefficientdiapedesisofCx43highDU-145cells,illustrated byrelativelyhighEPIvalues,wassignificantlyattenuatedbytran- sientCx43silencing,whereasAGAhadnoeffectonthisparameter (Fig.4A).Acorrelationwasobservedbetweenthediapedesiseffi- ciencyofDU-145cellsandtheactivationofendotheliumintheir proximity,measuredbytheinductionofHUVECmotilityinsub- confluentco-cultures((Piwowarczyketal.,2015);Fig.4B,seeFig.
E4inSupplementarymaterial).In contrasttoAGA,Cx43down- regulation and ERK1/2 inhibition by PD98059 in DU-145 cells considerablydecreasedthemagnitudeofHUVECactivation.Acor- relationbetweenCx43expressionlevelsinprostatecancercells, theirdiapedesisefficiencyandHUVECactivationwasalsoobserved for Cx43high MAT-LyLuand Cx43low AT-2 cells. MAT-LyLucells
Fig.3.ERK1/2participatesinGJIC-independentendothelialCx43up-regulationinducedbyprostatecancercells.(A)Calcein-loadedPC-3andDU-145cellswereseededonto HUVECmonolayers(98%ofconfluence).TheeffectofAGA(100M)onGJICbetweenHUVECsandDU-145cellswasillustratedbyquantificationofcalceintransferassay (couplingindex,Ci,right;1h).Thestatisticalsignificanceofthedifferenceswastestedwithone-wayANOVAfollowedbypost-hocTukey’sHSDcomparison.**p<0.01.(B)DU- 145cellswereseededontoHUVECmonolayers(98%ofconfluence)andtheeffectofAGA(100M)onendothelialCx43up-regulationwasestimatedbyimmunoblotting(for densitometricandstatisticalanalysesoftheblotsseeFig.E2FinSupplementarymaterial).(C)HUVECwereculturedintheabsence(control)orpresenceofDU-145cellsand ofPD98059(20M)for6h.Cx43andERK1/2phosphorylationlevelswereestimatedbyimmunoblotting(fordensitometricandstatisticalanalysesoftheblotsseeFig.E2G inSupplementarymaterial).(D)HUVECswereculturedasinC.IntracellularCx43localizationwasestimatedwithimmunostainingofformaldehyde-andMetOH/Acetone- fixedspecimensafter6h.HighmagnificationinsertsshowCx43+gapjunctionalplaquesatcell-to-cellinterfaces.Scalebars:40m.Allresultsarerepresentativeofthree independentexperiments.NotethattheinhibitionofERK1/2attenuatesendothelialCx43up-regulation.
Fig.4. Cx43up-regulationincreasesendothelialsusceptibilitytothesignalsfromprostatecancercells.(A)DU-145,MAT-LyLu,AT-2andPC-3cells(markedwitharrows) wereseededontothemonolayerofHUVECs(98%confluence,left)atthedensityof1300cells/cm2.Specimenswerefixedafter6hwith3.7%FA,permeabilised,stainedfor F-actinandcounterstainedwithHoechst33258.Whereindicated,HUVECsweretreatedwithAGA(100M)1hbeforeDU-145seedingorDU-145cellsweresubjectedto Cx43down-regulation(bysiRNA).Transendothelialpenetrationindices(EPI)wereestimatedasin1D.Thestatisticalsignificanceofthedifferenceswastestedwithone-way ANOVAfollowedbypost-hocTukey’sHSDcomparison.**p<0.01.Scalebar–40m.(B)DU-145,PC-3,MAT-LyLuandAT-2cellswereseededontoHUVECsintheconditions ascertainingtheirbasalmotility(70%ofconfluence).Whereindicated,DU-145cellsweresubjectedtoCx43down-regulation(bysiRNA),orHUVECswerepretreatedwithAGA (100M)andPD98059(20M).HUVECmotilitywasvisualizedbyatime-lapsevideomicroscopy.Columnchartsshowaveragerateofcelldisplacement(ARCD)ofHUVECs (seedot-plotsinFig.E4inSupplementarymaterial).Thestatisticalsignificanceofthedifferenceswastestedwithone-wayANOVAfollowedbynon-parametricDunnett (HUVEC/DU–145+PD98059)orpost-hocTukey’sHSDcomparison.**p<0.01.Allresultsarerepresentativeofthreeindependentexperiments.NotethatCx43down-regulation inDU-145cellsattenuatestheirdiapedesisandendothelialactivation,whereasCx43lowPC-3cellsdisplayrelativelyhighinvasiveness.
morereadilypenetratedHUVECcontinuumandmoreefficiently activatedendothelial cells thanAT-2 cells. Inductionof HUVEC motilityintheproximityofCx43highprostatecancercellsconfirms that endothelialCx43up-regulationenhancesthesusceptibility ofHUVECstoCx43-mediatedsignalsfromcancercells.Ourdata alsoshowthatCx43high prostatecancercellscanadaptaCx43- dependentstrategyofdiapedesis.Ontheotherhand,Cx43lowPC-3 cells couldalsoefficiently penetrateendotheliallayers (Fig.4A) andactivateendothelialcellsintheirproximity(Fig.4B).Itindi- catesthatCx43deficiencymaybecompensatedbyothermediators ofintercellularcommunicationduringthediapedesisofprostate cancercells.
3.5. Angioactivefactorsevokeendothelialactivationinthe proximityofCx43lowPC-3cells
Toidentifythemechanismunderlyingrelativelyefficientdia- pedesis of Cx43low PC-3 cells, we further analyzed the effect ofprostatecancercells’secretome onHUVECproperties.Media conditionedbyDU-145,MAT-LyLuandAT-2cellshadnopromi- nenteffect onCx43levelsin HUVECs (Fig.5A).Neither didwe observeanyconsiderableeffectsofthesemediaonHUVECmotil- ity(Fig.5B).However,mediumconditionedbyCx43lowPC-3cells slightlyincreasedendothelial Cx43levelsand significantlyaug- mented HUVECmotility. Thiseffect wasnot dependentonthe activityofmetalloproteinases,whichwassimilarinPC-3andDU- 145secretome.InhibitionofmetalloproteinaseactivitybyGM6001 had no effect on the motility of HUVECs in the proximity of DU-145andPC-3cells (see Fig.E5in Supplementarymaterial).
However,increasedlevelsofmRNAencodingVEGFwereobserved inHUVECs/PC-3co-cultures(Fig.E6inSupplementarymaterial).
Furthermore,numerousangioactivefactors,includingEGF,VEGF, IL-1andaFGF,weredetectedinPC-3cellsbymicroarraytests (Fig.5C;seeFig.E7inSupplementarymaterial),whereasonlyET-1 wasfoundinrelativelyhighquantitiesinDU-145cells.Thesedata suggestthatangioactivefactorscanattenuateendothelialbarrier functionandfacilitatethediapedesisofCx43low prostatecancer cells.TheyindicatethatCx43lowprostatecancercellscanadapta Cx43-independentstrategyofdiapedesis.
3.6. Pro-inflammatorysignalingmodulatesthemagnitudeof endothelialCx43up-regulationintheproximityofCx43high DU-145cells
Angioactive factors exerted negligible effects onendothelial Cx43 up-regulation. Therefore, we asked whether this process can be modulated by inflammatory signals. Peripheral blood monocytes(PBMs)secretepro-inflammatorycytokinesupontheir activation bycancer cells (Zembala et al., 1994).Whenseeded onto themonolayerofHUVECs at low density(104/cm2)PBMs didnotaffectHUVECmotility(Fig.6A)butincreasedCx43expres- sioninHUVECs(Fig.6B).Interestingly,alesspronouncedeffecton Cx43expressioninHUVECswasseenwhenPBMswereseededon HUVECsatthehigherdensity(105/cm2).Correspondingeffectsof PBMswereobservedinHUVEC/DU-145co-cultures.Importantly, theeffectsofPBMsonendothelialCx43expressionweresensitive totheinhibitionofERK1/2signalingbyPD98059(Fig.6B).Because DU-145cellsand HUVECsinducedTNF␣secretioninPBMs(not shown),wefurtherestimatedtheeffectofTNF␣administrationon Cx43localizationinHUVECs.AslightinductionofCx43expression wasobservedinHUVECstreatedwithTNF␣.Ontheotherhand, TNF␣slightlydecreasedCx43levelsinHUVECsco-culturedwith DU-145cells,especiallytheabundanceofCx43attheintercellular contactsbetweenHUVECs(Fig.6C).ThesedataindicatethatTNF␣ isinvolvedintheregulationofendothelialCx43expression,how- everthisprocessistargetedbythecomplexnetworkofagonistic
andantagonisticsignalsduringthediapedesisofprostatecancer cells.
4. Discussion
Efficiencyofcancercellinvasionisregulatedbytheintrinsic invasivepropertiesofcancercellsandbythemicroenvironmen- talcuesthatcompromise thebarrier functionofcancerstroma.
Cx43hasbeenshowntoaugmenttheinvasivepotentialofcancer cellsinaGJIC-independentmannerviatheeffectoncellmotility, cytoskeletonarchitectureand mechanicalelasticity(Batesetal., 2007;Cronieretal.,2009;Lamicheetal.,2011;Olketal.,2009;
Omorietal.,2007).Inthemetastaticcascadeofprostatecancer, Cx43participatesinthediapedesisofcancercellsthroughtheGJIC- dependentandGJIC-independentactivationofadjacentendothelial cells(Piwowarczyketal.,2015;Ryszawyetal.,2014).Tillnow,the involvementofCx43inprostate cancerpromotionandprogres- sionhaspredominantlybeenconsideredintermsofitsfunction incancercells.Here, weshowthatCx43expressedbyprostate cancercellsmayup-regulateendothelialCx43levels,thuslocally sensitizingtheendotheliumtosignalsthatcompromiseitsbarrier functionduringprostatecancercellextravasation.ThisnovelGJIC- independentfunctionunderlinestheimportanceofCx43forthe metastaticcascadeofprostatecancer.
WehavepreviouslyshownthattheextravasatingDU-145cells activate endotheliumin their proximityin a GJIC-independent, Cx43-dependentmanner(Piwowarczyketal.,2015).Ourpresent data show a prominent endothelial Cx43 up-regulation in the proximity of Cx43high but not of Cx43low prostate cancercells.
The attenuation of this process was seen upon transient Cx43 down-regulationinDU-145cells,whereas chemicalGJICinhibi- tioninHUVEC/DU-145co-culturesdidnotaffectthemagnitudeof endothelialCx43up-regulation.Theseobservationsindicatethat Cx43inprostatecancercellsregulatesendothelialCx43expression inaGJIC-independentmanner.Numeroussignalingsystemshave beenimplicatedintheregulationofCx43expression(Oyamada etal.,2013).Manyofthemareanchoredto“gapjunctionalpro- teome”(Mroueetal.,2011).Inparticular,GSK3-activityhasbeen showntoregulateCx43expressionincardiomyocytes(Czyzetal., 2005),whereastheinvasivenessofprostatecancercellsdepends onafeedbackloopbetweenCx43expressionandTGF/Snail-1sig- nalingaxis(Ryszawyetal.,2014).Inourhands,DU-145-induced endothelialCx43up-regulationwasinsensitivetoGSK3activa- tionandwedidnotobserveSnail-1activationinHUVEC/DU-145 co-cultures(notshown).Instead,chemicalERK1/2inhibitorabol- ishedendothelialCx43up-regulationintheproximityofCx43high DU-145 cells. An involvement of ERK1/2 in the regulation of endothelialCx43expressionhaspreviouslybeenreported(Zhao etal.,2011).Whereasfurtherstudiesarenecessarytoidentifythe moleculesthatfunctionallylinkCx43inprostatecancercellsand ERK1/2activationin endothelialcells,ourdataidentifya novel intercellularCx43/ERK1/2/Cx43signalingaxis.ItcoordinatesCx43 expressionincancerandendothelialcells.
BiologicalsignificanceofendothelialCx43up-regulationisillus- tratedbythecorrelationofitsmagnitudewiththeefficiencyof DU-145 celldiapedesis and with themagnitude of endothelial activation.Previously,thecorrelationhasbeenshownbetweendis- turbanceofendothelialcontinuumbycancercells andthelocal activationofendothelialcellsintheproximityofcancercells(mea- sured byendothelialcellmotility insub-confluentco-cultures).
Here,weshowthatboth thetransientCx43down-regulationin DU-145cells andERK1/2inhibition attenuates theinductionof HUVECmotilityintheproximityofcancercells,whereasGJICinhi- bitionbyAGAhadnoeffectonthisparameter.Thus,Cx43may beimplicatedinintercellular transmissionofsignalsthat facili-
Fig.5.AngioactivefactorsevokeendothelialactivationintheproximityofCx43lowPC-3cells.(A)HUVECswereexposedtothemediaconditionedbyDU-145,PC-3,AT-2and MAT-LyLucellsfor24handtheireffectonCx43expressioninHUVECswasestimatedbyimmunoblotting(fordensitometricandstatisticalanalysesoftheblotsseeFig.E2Hin Supplementarymaterial).PhotomicrographsshowanexemplaryCx43staininginHUVECsincubatedinthepresenceofDU-145-conditionedmediafor6h.Scalebar:40m.
(B)MediaconditionedbyMAT-LyLu,AT-2,DU-145,andPC-3cellswereadministeredtoHUVECcultures.HUVECmotilitywasvisualizedbyatime-lapsevideomicroscopyin theconditionsascertainingtheirbasalmotility(70%confluence).Thestatisticalsignificanceofthedifferenceswastestedwithone-wayANOVAfollowedbynon-parametric Dunnettcomparison.**p<0.01.(C)DU-145andPC-3cellswereculturedinstandardconditionsfor24handtheexpressionofangioactiveproteinswassemi-quantitively estimatedwithantibodyarraykit(seeMethods).PlotsshowthedensitometricallyestimateddotintensitiesthatillustratetheproteinamountsinDU-145andPC-3cells.
Scalebar:40m.Allresultsarerepresentativeofthreeindependentexperiments.NoteawidespectrumofangioactivefactorssecretedbyPC-3,whichactivateHUVEC motilityintheabsenceofadistinctendothelialCx43up-regulation.
Fig.6.Pro-inflammatorysignalingmodulatesthemagnitudeofendothelialCx43up-regulationintheproximityofCx43highDU-145cells.(A)HUVECswereculturedin thepresenceofDU-145cellsand/orofperipheralbloodmonocytes(PBMs)for6handtheirmotilitywasanalyzedwithtime-lapsevideomicroscopy.Columnchartsshow averagerateofcelldisplacement(ARCD)ofHUVECs.Thestatisticalsignificanceofthedifferenceswastestedwithone-wayANOVAfollowedbypost-hocTukey’sHSD comparison.**p<0.01.(B)HUVECswereculturedinthepresenceofDU-145cellsand/orperipheralbloodmonocytes(PBMs)for6handCx43levelswereestimatedby immunoblottinginthepresenceorabsenceofPD98059(20M;fordensitometricandstatisticalanalysesoftheblotsseeFig.E2IinSupplementarymaterial).(C)HUVECs andHUVEC/DU-145co-cultureswereculturedinthepresenceofTNF␣for6h,fixedwithMetOH/Acetone,stainedagainstCx43andcounterstainedwithHoechst33258.
CytofluorometricapproachwasappliedtoquantifytheintracellularlevelsofCx43.HighmagnificationinsertsshowCx43+gapjunctionalplaquesatcell-to-cellinterfaces.
Thestatisticalsignificanceofthedifferenceswastestedwithone-wayANOVAfollowedbypost-hocTukey’sHSDcomparison.Differenceswithp>0.05wereregardedas significant.Scalebar:25m.Allresultsarerepresentativeofthreeindependentexperiments.Notethenon-linearPBMandTNF␣effectsonendothelialCx43up-regulation andtheirsensitivitytotheinhibitionofERK1/2.
tatescancercelldiapedesis throughthelocal,GJIC-independent activationofendothelialcells(Piwowarczyketal.,2015).Because Cx43-mediatedintercellularcommunicationdependsontheavail- abilityandcompatibilityofconnexinsexpressedbyadjacentcells (Kovaletal.,2014),endothelialCx43up-regulationmightfacilitate theinteractionsofendothelialCx43connexonswiththeircoun- terpartsonthesurfaceofcancercells.WhetherendothelialCx43 up-regulationincreasestheabundanceofendothelialCx43connex- onsintheapicalmembranesofendothelialcellsrequiresfurther study.However,thismechanismmayincreaseendothelialcellsus- ceptibilitytothesignalsgeneratedbyextravasatingcancercells, thushelpingthemtopenetrateendothelialbarriers(Lewalleetal., 1998;Piwowarczyketal.,2015).Notably,acorrespondingmecha- nismisinvolvedintheextravasationofimmunecells(Branesetal., 2002;Eugeninetal.,2003;Zahleretal.,2003).Collectively,ourdata demonstratethattheintercellularCx43/ERK1/2/Cx43axisisacti- vatedattheinterfacebetweenCx43highprostatecancercellsand endothelium.ThisaxisisanimportantplayerinCx43-dependent strategiesofprostatecancercelldiapedesis.
Whereas Cx43high prostate cancer cells can activate Cx43-dependent pathways during their extravasation, Cx43- independentsignalingbetweencancerandendothelialcellsmay account for the relatively efficient diapedesis of Cx43low cells.
Effective transendothelial migration of PC-3 cells was accom- panied by a strong activating effect of PC-3 cell-conditioned mediaonendothelialcellmotilityandbyarelativeabundanceof angioactivefactorsinPC-3incomparisontoDU145cells.Inturn, alowdiapedesisefficiencyofCx43low AT-2cellswascorrelated withtherelativelypooractivityofthefactorssecretedbythese cells.Apparently,theangioactivesecretomeofCx43low prostate cancercellscanefficientlyactivateendothelialcells,thushelping thecancercellstopenetrateendothelialbarriersintheabsenceof endothelialCx43up-regulation.Althoughthespecificmediators ofthesignalingbetweencancerand endothelialcells remainto beidentified,thesedataindicatethatparacrinesignalingbetween cancer and endothelial cells can facilitate prostate cancer cell diapedesisinaCx43-independentmanner(Reymondetal.,2013).
Humoralfactorssecretedbycirculatingcellscanalsoaffectthe magnitudeofendothelialCx43up-regulation.IncreasedCx43lev- elswereobservedinHUVECexposedtoangioactivePC-3secretome in conditionedmedia.Alsoa disturbedCx43expression, which wasobservedinHUVECsafteratransientCx43down-regulation inDU-145cells,suggeststhecontributionofhumoralfactorsto endothelialCx43up-regulation.Finally,DU-145-inducedendothe- lialCx43up-regulationwassensitivetopro-inflammatorysignals fromimmunecells.Cx43haspreviouslybeensuggestedtofacilitate theextravasationofpolymorphonuclearcells(Branesetal.,2002;
Soonetal.,2016;Zahleretal.,2003),whereasTNF␣regulatesCx43 expressioninimmuneandendothelialcells(Pfennigeretal.,2013;
vanRijenetal.,1998).Inourhands,peripheralbloodmonocytes (PBMs)andTNF␣(notshown)hadnoinducingeffectonendothe- lialcellmotilityintheproximityofDU-145cells.Instead,PBMs wereabletoincreasethemagnitudeofDU-145-inducedendothe- lialCx43up-regulationinamannerdependentonERK1/2signaling.
These datasuggest that discrete ERK1/2-dependentpathway(s) participateintheregulationofbothprocesses.Itisthusconceivable thatCx43/ERK1/2/Cx43axismayconstituteoneofthefunctional linksbetweencancercelldiapedesisandthelocalinflammation (Kwak etal., 2002;vanKempenand Jongsma,1999).However, non-linearity of dose-dependent effects of PBMs and TNF␣ on endothelialCx43expressionandthemotilityofendothelialcells illustratesthecomplexityoftheselinks.
Inconclusion,wedemonstratethatCx43expressedbyprostate cancercellsactsasaco-factorofERK1/2-dependentintercellular signalingaxis,whichregulatestheexpressionofCx43inendothe- lialcells.ThisnovelCx43functionmayparticipateintheselection
ofCx43highcellstothemetastaticfrontofprostatecancer.These observationsconfirmtheimportanceofCx43for themetastatic cascadeof prostatecancerand extendourknowledgeonCx43- dependentintercellularcommunicationnetworksinvolvedinthe diapedesisofprostatecancercells.SensitivityofCx43/ERK1/2/Cx43 signalingaxistoinflammatorysignalsillustratestherelationship betweenprostatecancercelldiapedesisand localinflammatory processes.Our dataalsoaddressthecontroversiesonCx43role inprostatecancerprogression(Czyzetal.,2012;Defamieetal., 2014; Naus and Laird, 2010) because they show that discrete lineagesof prostatecancercells (Marusyket al.,2012;Nguyen, 2011)mayadaptdifferentCx43-dependentandCx43-independent strategiesofdiapedesis.Angioactivesecretomeofprostatecancer cellscanefficientlycompromiseendothelialbarrierfunctioninthe absenceofCx43-mediatedcommunication.Consequently,itcan facilitatethediapedesisofCx43lowprostatecancercellsinaCx43- independentmanner.Alongwithtissue-specificityofendothelial andimmunecellproperties,thisdiversityofdiapedesisstrategies mayaccountfortissue-specifichomingofCx43high andCx43low prostatecancercellsduringthemetastaticcascade(Hart,1982;
Pientaetal.,2013).Therefore,diapedesisshouldberegardedas apotentialtargetfornewpalliativestrategiesofprostatecancer therapy (Piwowarczyket al., 2015). More thorough considera- tionofcooperativeCx43-dependentintercellularsignalingduring prostatecancercelldiapedesisshouldhelptoeffectivelyimple- mentthesestrategies.
Conflictofinterest
Theauthorsdeclarenoconflictofinterest.
Acknowledgements
Work supported by the Polish National Science Cen- tre (2015/17/B/NZ3/01040, 2011/01/B/NZ3/00004 and 2013/09/N/NZ3/00204).The Faculty ofBiochemistry, Biophysics and Biotechnologyof theJagiellonianUniversity isa partnerof theLeadingNationalResearchCenter(KNOW)supportedbythe MinistryofScienceandHigherEducation.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.ejcb.2017.03.012.
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