Fibroblast-to-myofibroblast switch is mediated by NAD(P)H oxidase generated reactive oxygen species.

0.71 MB·English

by  Alili, Lirija

Biosci.Rep.(2014)/34/art:e00089/doi10.1042/BSR20130091
Fibroblast-to-myofibroblast switch is mediated by
NAD(P)H oxidase generated reactive oxygen
species
LirijaALILI*1,MarenSACK*,KatharinaPUSCHMANN*andPeterBRENNEISEN*
*InstituteofBiochemistryandMolecularBiologyI,MedicalFaculty,Heinrich-Heine-University,Du¨sseldorf,Germany
Synopsis
Tumour–stromainteractionisaprerequisitefortumourprogressioninskincancer.Hereby,acriticalstepinstromal
function is the transition of tumour-associated fibroblasts to MFs (myofibroblasts) by growth factors, for example
TGFβ (transforminggrowthfactorbeta().Inthisstudy,thequestionwasaddressedofwhetherfibroblast-associated
NAD(P)Hoxidase(NADH/NADPHoxidase),knowntobeactivatedbyTGFβ1,isinvolvedinthefibroblast-to-MFswitch.
g The up-regulation of αSMA (alpha smooth muscle actin), a biomarker for MFs, is mediated by a TGFβ1-dependent
r
o increase in the intracellular level of ROS (reactive oxygen species). This report demonstrates two novel aspects of
. theTGFβ1signallingcascade,namelythegenerationofROSduetoabiphasicNAD(P)HoxidaseactivityandaROS-
p
dependent downstream activation of p38 leading to a transition of dermal fibroblasts to MFs that can be inhibited
e
by the selective NAD(P)H oxidase inhibitor apocynin. These data suggest that inhibition of NAD(P)H oxidase activity
r
i preventsthefibroblast-to-MFswitchandmaybeimportantforchemopreventionincontextofa‘stromaltherapy’which
c
wasdescribedearlier.
s
o Keywords: MAPK,myofibroblast,NAD(P)Hoxidase,reactiveoxygenspecies,TGFβ1,tumour–stromainteraction.
i
b
Citethisarticleas:Alili,L.,Sack,M.,Puschmann,K.andBrenneisen,P.(2014)Fibroblast-to-myofibroblastswitchismediatedby
.
w NAD(P)Hoxidasegeneratedreactiveoxygenspecies.Biosci.Rep.34(1),art:e00089.doi:10.1042/BSR20130091
w
w
(cid:9)
INTRODUCTION esis,awidevarietyofdifferentcytokinesandgrowthfactorsare

expressed in tumour–stroma interaction, which stimulate intra-
s
cellular signal transduction pathways resulting in angiogenesis
t
r Tumourprogressionischaracterizedbythelocalaccumulation and tumour growth as well as migration during tumour inva-
o
of extracellular matrix components and connective tissue cells sion. Among the autocrine and paracrine acting growth factors
p
surrounding the tumour cluster, a phenomenon called tumour– involved in molecular processes of tumour–stroma interaction,
e
stromainteraction[1,2].Disturbanceinstroma,composedofin- transforming growth factor-beta1 (TGFβ1), a 25kDa homodi-
R
flammatorycells,smallvessels,fibroblasticandmyofibroblastic mericprotein,playsapivotalrole[2,3,9,10].

e cells,constitutesthedesmoplasticreaction,suggestedtobees- Aparacrineeffectoftumourcell-derivedTGFβ1onthedown-
c sentialinthedevelopmentoftheinvasionprocess[3]. regulationofgapjunctionalintercellularcommunicationbetween
n
Thecompositionofreactivestroma,providingstructuraland stromalfibroblastswasshownearlier,dependentonthegenera-
e
vascularsupportfortumourgrowth,resemblesthatofgranulation tionofROS(reactiveoxygenspecies)[11,12].Inlinewiththis,
i
c tissue, and MFs (myofibroblasts) play a critical role in driving TGFβ1inducedanincreaseinH O (hydrogenperoxide)levels
2 2
s boththestromalreactionofphysiologicalwoundrepair[4,5]and inhumanlungfibroblasts,whichwasabrogatedbyaninhibitorof
o
of invasive tumours [6]. The MF has acquired the capacity to NAD(P)Hoxidase(NADH/NADPHoxidase)[13].Furthermore,
Bi expressthebiomarkersαSMA(alphasmoothmuscleactin)and aTGFβ1-triggeredactivationofNAD(P)Hoxidaseinitiatedap-
theFN(fibronectin)splicevariantED-AFN[7,8].Incarcinogen- optosisoffetalrathepatocytes[14].Tumourcell-derivedTGFβ1
.A..b..b..r..e..v..i.a..t..i.o..n..s..:...A..P..-.1..,..a..c..t.i.v..a..t.i.n..g...p..r.o..t.e..i.n..-.1..;...C..C..D...,..c..h..a..r.g..e..-.c.o..u..p...le..d....d..e..v.i.c..e..;..C...M...,..c..o..n..d..i.t.i.o..n..e..d...m...e..d..i.a..;..D...C..F.,...2..(cid:2).,.7..(cid:2)..-d..i.c..h..l.o..r.o..fl..u..o..r.e..s..c..e..i.n..;..D...M...E..M...,..D...u..l.b..e..c..c.o..’.s...m...o..d..i.fi..e..d...E..a..g..l.e..’.s...m...e..d..i.u..m...;..D...N..P.,...........
2,4-dinitrophenyl;FN,fibronectin;GPx,glutathioneperoxidase;H2O2,hydrogenperoxide;H2DCF-DA,2(cid:2),7(cid:2)-dichlorodihydrofluoresceindiacetate;HDF,humandermalfibroblasts;HPRT1,
hypoxanthineguaninephosphoribosyltransferase;HRP,horseradishperoxidase;MAPK,mitogen-activatedproteinkinase;MF,myofibroblast;NAD(P)Hoxidase,NADH/NADPHoxidase;
NGS,normalgoatserum;O2.−,superoxide,ROS,reactiveoxygenspecies;rTGFβ1,recombinanttransforminggrowthfactor-beta1;SCL-1,squamouscellcarcinomaline;αSMA,alpha
smoothmuscleactin.
1 Towhomcorrespondenceshouldbeaddressed(email:[email protected]).
(cid:2)c 2014TheAuthor(s) ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicence(CC-BY)(http://creativecommons.org/licenses/by/3.0/) 7
whichpermitsunrestricteduse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.
L.Aliliandothers
increasedthegenerationofmyofibroblasticcells,whichpromote Table1 SequencesofprimersforRT–PCR
the invasion of tumour cells in an in-vitro 3D model [15] and Genes Primer(5(cid:2)→3(cid:2))
which can be prevented by redox-active nanoparticles (stromal
HPRT1 Forward:ATTCTTTGCTGACCTGCTGGATT
therapy)[16].
Reverse:CTTAGGCTTTGTATTTTGCTTTTC
However,thecomponentsoftheTGFβ1/ROS-initiateddown-
αSMA Forward:CTGTTCCAGCCATCCTTCAT
streamsignallingpathwaysresultinginαSMAexpressionhave
Reverse:TCATGATGCTGCTGTTGTAGGTGGT
notbeensufficientlyidentified.Here,wedemonstratetwonovel
findings in TGFβ1-initiated αSMA expression. First of all, p67phox Forward:CGAGGGAACCAGCTGATAGA
TGFβ1 initiates two activity peaks of the NAD(P)H oxidase. Reverse:CATGGGAACACTGAGCTTCA
Secondly,thesecondactivitypeakaccompaniedbyasignificant NOX4 Forward:GAAGCCCATTTGAGGAGTCA
expression of the regulatory subunit p67phox, is responsible for Reverse:GGGTCCACAGCAGAAAACTC
the ROS-dependent increase in stress-activated kinase expres-
sion/activation(especiallyp38)thatisinvolvedinαSMAinduc-
ginally derived from the face of a 74-year-old woman [18]
tion.Interestingly,theNAD(P)Hoxidaseinhibitorapocyninal-
(generously provided by Professor Dr Norbert Fusenig, DKFZ
mostcompletelyabrogatedTGFβ1-mediatedαSMAexpression,
Heidelberg,Germany)weremaintainedinDMEMsupplemented
whereasthexanthinoxidaseinhibitorallopurinol,forexample,
withglutamine(2mM),penicillin(400units/ml),streptomycin
has no effect. These data give a novel insight into the ROS- (50μg/ml) and 10% (v/v) FCS in a humidified atmosphere of
dependentsignallingleadingtoMFgenerationandopenupnew 5%(v/v)CO and95%(v/v)airat37◦C.MFsweregenerated
2
possibilitiesforchemopreventionincontextofastromaltherapy. bytreatmentofHDFswithrTGFβ1for48hinHDFconditioned
medium(CMHDF)[15].
MATERIALS AND METHODS
Preparationofconditionedmedia(CM)
CMwasobtainedfromSCL-1cells(CMSCL)andHDF(CMHDF).
Cell culture media [DMEM (Dulbecco’s modified Eagle’s SCL-1 cells at an initial density of 1×106 cells were grown
medium)]werepurchasedfromInvitrogenGmbHandthedefined tosubconfluence(∼70%confluence)and1,5×106HDFcellsto
fetalcalfserum(FCSgold)wasfromPAALaboratories(Linz, confluencein175cm2cultureflaskstogetidenticalcellnumbers.
Austria).Allchemicalsincludingproteaseaswellasphosphatase Theserumcontainingmediumwasremoved,andafterwashing
inhibitorcocktail1and2wereobtainedfromSigmaorMerck threetimesinPBSthecellswereincubated forfurther48hin
Biosciencesunlessotherwisestated.Theproteinassaykit(Bio- 15mlserum-freeDMEMbeforecollectionoftheCM.
RadDC,detergentcompatible)wasfromBio-RadLaboratories
GmbH (Mu¨nchen, Germany). Apocynin was delivered by Cal-
biochem.Theenhancedchemoluminescencesystem(SuperSig- RNAisolationandquantitativereal-timeRT–PCR
nalWestPicoMaximumSensitivitySubstrate)wassuppliedby TotalRNAwasisolatedandtranscribedintocDNAasdescribed.
Pierce. The Oxyblot Protein Oxidation Detection kit was from mRNAlevelswereanalysedbyRT–PCReitherbyusingaTher-
Millipore.ThedyeH DCF-DA(2(cid:2),7(cid:2)-dichlorodihydrofluorescein mocycler(Biometra)asdescribedin[19]orintheLightCycler
2
diacetate)wassuppliedfromMoBiTec.PCRprimersweresyn- system(Roche).Real-timeRT–PCRwasperformedwith40ng
thesizedbyInvitrogen.ReagentsforSDS–PAGEwerefromRoth. cDNAinglasscapillariescontainingLightCyclerFastStartDNA
Monoclonal mouse antibody raised against human αSMA and MasterSYBRGreenIReactionMix(Roche),2mMMgCl and
2
αTubulin was supplied by Sigma. Polyclonal rabbit antibody 1μMofprimers.QuantificationofthePCRampliconswasper-
raised against human phospho P38 was supplied by New Eng- formedusingtheLightCyclerSoftware.HPRT1(hypoxanthine
land Biolabs. The following secondary antibodies were used: guanine phosphoribosyltransferase) was used as internal nor-
polyclonal HRP (horseradish peroxidase) – conjugated rabbit malizationcontrol[20].Sequencesofprimerpairsaregivenin
anti-mouse IgG antibody (DAKO), goat anti-rabbit immuno- Table1.
globulin G antibodies were from Dianova and Alexa Fluor
488-coupled goat anti-mouse IgG antibody (H+L) (MoBiTec
GmbH). rTGFβ1 (recombinant human TGFβ1) was delivered MeasurementofintracellularROS
byR&DSystems. TheintracellularROSlevelwasmeasuredusingthefluorescent
dyeH DCF-DA,whichisdiffusibleintocellsandtherehydro-
2
lysedtothenon-fluorescentderivativeH DCF[12].Inthepres-
2
Cellculture enceofperoxides,H DCFisconvertedintothehighlyfluorescent
2
HDF(humandermalfibroblasts)wereestablishedbyoutgrowth DCF(2(cid:2),7(cid:2)-dichlorofluorescein).Forassays,subconfluentfibro-
from foreskin biopsies of healthy human donors aged from 3– blastmonolayercultureswereloadedwith20μMH DCF-DAin
2
6years. Cells were used in passages 2–11, corresponding to PSGbuffer(100mMKH PO4,10mMNaCl,and5mMglucose;
2
cumulative population doubling levels of 3–23 [17]. Dermal pH7.4)for15mininthedark.Afterwashingthreetimeswith
fibroblasts and the SCL-1 (squamous cell carcinoma line), ori- PSGbuffer,theloadedcellsweresubjectedto10ngrTGFβ1/ml
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8 (cid:2)c 2014TheAuthor(s) ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicence(CC-BY)(http://creativecommons.org/licenses/by/3.0/)
whichpermitsunrestricteduse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.
Fibroblast-to-myofibroblastswitch:roleofNAD(P)Hoxidase
PSG.ROSgenerationwasdetectedasaresultoftheoxidation 10minat4◦C.AfterwashingwithPBS,non-specificbindingof
of H DCF and the fluorescence (excitation 488nm; emission antibodieswasblockedwith3%(v/v)NGS(normalgoatserum)
2
515–540nm),giveninarbitraryunits,wasfollowedwithaZeiss inTBSTcontaining0.3%(v/v)TritonX-100atroomtemperature
axiovert fluorescent microscope with a CCD (charge-coupled (20◦C).CellswereincubatedwithmonoclonalαSMAantibody
device)camera(ORCAII,Hamamatsu)for20min. diluted1:1000in1%(v/v)NGS/TBSTovernightat4◦C.After
washing the cells were incubated with an Alexa 488-coupled
goatanti-mouseIgG(1/1000dilutedinTBST)for1hatroom
DeterminationofextracellularH O
2 2 temperature.ForDAPIstaining,cellswereincubatedfor10min
An extracellular concentration of H O was quantified by am-
2 2 atroomtemperaturewith1:500dilutedDAPIsolution(Sigma,
perometricdeterminationusingtheApollo4000(Worldprecision
stocksolution0.5mg/10mlH O)inMcIlvaine’sbuffer(100mM
Instruments)withtheH O sensorISO-HPO-2sensortipaccord- 2
2 2 citricacid,200mMNa HPO ;pH7.2).Afterwashingandem-
ingtothemanufacturer’sinstruction.Briefly,acalibrationcurve 2 4
bedding,imagesweretakenwithaZeissAxiovertfluorescence
was generated by the injection of different amounts of H O to
2 2 microscopewithaCCDcamera.
thecalibrationsolution(PBS).Plottingofthechangesincurrent
(pA) against the changes in concentration (nM) creates a cal-
ibrationcurve.FormeasuringextracellularH O concentration,
2 2
50μlofcellsupernatantswereinjectedtothecalibrationsolution SDS–PAGEandWesternblotting
andcurrentchangeswererecorded.Although,thesensitivityof SDS–PAGEwasperformedaccordingtothestandardprotocols
thesensordoesnotchangesignificantlywithininthetemperat- publishedelsewhere[21],withminormodifications.Briefly,cells
urerangeof20–37◦C,allmeasurementsandgenerationofthe werelysedafterincubationwithrTGFβ1(10ng/ml)in1%(w/v)
calibrationcurveweredoneat37◦C. SDSwith1:1000proteaseinhibitorcocktail(Sigma).Aftersonic-
ation,theproteinconcentrationwasdeterminedbyusingamodi-
fiedLowrymethod(Bio-RadDC).4xSDS–PAGEsamplebuffer
MeasurementofNAD(P)Hoxidaseactivity
[1.5MTris–HCl(pH6.8),6ml20%SDS,30mlglycerol,15ml
NAD(P)H oxidase has the E.C. number 1.6.1.3 reflecting one
β-mercaptoethanol and 1.8mg bromophenol blue] was added,
enzyme that uses both NADH and/or NADPH oxidase as sub-
andafterheating,thesamples(10–30μgtotalprotein/lane)were
strateandO aselectronacceptor.ThereforethetermNAD(P)H
2 appliedto8–15%(w/v)SDS–PAGE.Afterelectroblottingonto
oxidaseisoftenused–andweliketodoitaswell–torepresent
PVDFmembrane(GEHealthcare),immunodetectionwascarried
bothpossiblesubstrates.However,astheK valueforNADPHis
m outusingan1:1000dilutionofprimaryantibodies(mousemono-
lowerthanforNADH,theendogenousNAD(P)Hoxidasewould
clonalantiαSMAandα-tubulinorrabbitmonoclonalantiphos-
preferNADPH.
phop38),1:20000dilutionofanti-mouse/rabbitantibodyconjug-
Nevertheless, many publications used NADH as substrate
atedtoHRP).Antigen–antibodycomplexeswerevisualizedby
for the cell-free activity measurements as we did as well (see
anenhancedchemiluminescencesystem.α-tubulinorCoomassie
Figure 4A). Here, we like to discriminate between ‘NADH
BrilliantBluestainingwasusedasinternalcontrolforequalload-
oxidaseactivity’and‘NAD(P)Hoxidase’(meaningtheenzyme
ing.Molecularsizesofthebandswerecalculatedbycomparison
ingeneral).
withaprestainedproteinmarker(Fermentas,St.Leon-Rot).For
In this paper, the NADH oxidase activity of the NAD(P)H
quantificationofthebands,thedevelopedfilmswerescannedby
oxidase was measured. Fibroblasts were grown to 70% con-
animageanalysissystemandanalysedwiththeImageJsoftware.
fluence and washed with prewarmed HBSS (Hank’s buffered
salt solution). After 15min incubation with 10ng rTGFβ1/ml
ormocktreatmentinserum-freemedium,cellswereexposedto
250μMNADH/HBSSorNADPH/HBSSfor1min.Therateof Determinationofoxidized(carbonylated)proteins
NADH/NADPHconsumptionwasmeasuredasdecreaseinab- HDFweregrowntosubconfluenceontissueculturedishes.After
sorbanceat340nmusingaspectrophotometer(Ultrospec1000, removalofserum-containingmedium,cellswereculturedinthe
Pharmacia Biotech). The extinction coefficient for calculation serum-freemediumandeithermock-treatedortreatedfor24h
of the concentration of consumed NADH/NAD(P)H was 6.22 with TGFβ1 (10ng/ml). As a positive control, the cells were
mM−1 cm−1.FormeasurementsofthespecificNAD(P)Hoxi- treatedwithH O (1mM)for1h.Thereafter,cellswerelysed
2 2
daseactivity,hereintherateofNADHconsumptioninhibitable and carbonyl groups of oxidized proteins were detected with
by apocynin, a specific NAD(P)H oxidase inhibitor was used the OxyBlotTM Protein Oxidation Detection Kit, following the
as described earlier [13]. Data were expressed in nmol NADH manufacturer’s protocol. Briefly, the protein concentration was
consumptionmin−1mg−1protein. determinedbyusingamodifiedLowrymethod(Bio-RadDC).
Roughly,5μgofthecelllysateswereincubatedwithDNP(2,4-
dinitrophenyl)hydrazinetoformtheDNPhydrazonederivatives.
Immunocytochemistry LabelledproteinswereseparatedbySDS–PAGEandimmunos-
HDFmonolayerculturesweregrowninDMEMplus10%(v/v) tained using rabbit anti-DNP antiserum (1:500) and goat anti-
FCSoncoverslipsin3.5cmdiametertissueculturedishesbefore rabbitIgGconjugatedtoHRP(1:2000).Blotsweredevelopedby
use. Cells were washed with PBS and fixed with methanol for enhancedchemiluminescence.
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(cid:2)c 2014TheAuthor(s) ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicence(CC-BY)(http://creativecommons.org/licenses/by/3.0/) 9
whichpermitsunrestricteduse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.
L.Aliliandothers
Figure1 TGFβ1-mediatedtransitionoffibroblaststoMFs
SubconfluentHDFwereeithermock-treated(CMHDF),treatedwithrTGFβ1(10ng/ml)for48h(CMHDF,TGF)andinCMof
squamous carcinoma cells SCL-1 (CMSCL−1). (A) The amount of αSMA protein was immunostained for αSMA and (B)
determinedbyWesternblotanalysis.Thedensitometricvaluesrepresentthefoldincreaseovercontrol,whichwassetat
1.0.Thedatarepresentmeans+−S.E.M.ofthreeindependentexperiments.CM,conditionedmedium.
Statisticalanalysis Pharmacological approaches using ROS level-modulating
Means were calculated from at least three independent exper- substancessuchasselenite,butylatedhydroxytolueneandthevit-
iments, and error bars represent standard error of the mean aminE-derivateTroloxclearlydemonstratedaTGFβ1-mediated
(S.E.M.).Analysis of statistical significance was performed by generation of ROS [12,15], which was prevented by that anti-
Student’s t test or ANOVA with *P<0.05, **P<0.01 and oxidants.SeleniteandtheGPx(glutathioneperoxidase)mimic
***P<0.001asthelevelsofsignificance. ebselen inhibited the TGFβ1 initiated αSMA expression deal-
ing with GPx to play a major role in that context [15]. An in-
volvementofTGFβ1-initiatedhigherROSlevelmediatingdown-
regulationofgapjunctionalintercellularcommunication[12]and
RESULTS expressionofαSMA[15]indermalfibroblastswasdemonstrated
aswellasaTGFβ1-dependentactivationofNADHoxidasein
lungfibroblast[24].ToaddressthequestionofwhetherNAD(P)H
Recombinant-andtumourcell-derivedTGFβ1 oxidasealoneorothermajorO .−/H O sourcessuchasxanthine
2 2 2
inducefibroblast-to-MFtransition oxidaseplayaroleinthetransitionoffibroblaststoMFs,HDF
Immunocytochemistry studies show a significant increase in were exposed to rTGFβ1in the presence and absence of non-
αSMA staining after treatment with rTGFβ1 (CMHDF,rTGFβ1) toxic concentration of allopurinol (10μM), apocynin (1mM)
comparedwithmock-treatedcells(CMHDF).Furthermore,asig- andDPI(5μM).Theeffectofthexanthineoxidaseinhibitor,al-
nificant amount of active TGFβ1-containing CM of SCL-1 tu- lopurinol,ontheexpressionofαSMAwasexamined.Atadose
mour cells (CMSCL) [12] resulted in formation of MFs as well thathasbeenpreviouslyreportedtoinhibitthexanthineoxidase,
(Figure1A).ThestainingrevealstheorganizationofαSMAin allopurinol did not affect the αSMA expression (Figure 2A).
stressfibres,amorphologicalpropertyofMFs. Therefore,αSMAexpressionisindependentonxanthineoxidase.
In order to evaluate an x-fold increase in TGFβ1-triggered AsignificantincreaseintheαSMAproteinamountwasmeas-
expression of αSMA (CMHDF,rTGFβ1; CMSCL) in compar- ured at 48h on treatment with rTGFβ1 compared with mock-
ison with mock-treatment (CMHDF), subconfluent HDF were treatedcontrols.Thisincreasewasnearlycompletelyabolished
treatedwithrecombinant-andtumourcell-derivedTGFβ1.Treat- bypreincubationofthecellswiththeNAD(P)Hoxidaseinhib-
ment with both rTGFβ1 and CMSCL resulted in an about 9- itorsapocynin(1mM)orDPI(5μM)(Figure2B).Apocynin,a
fold and 4-fold increase of αSMA expression, respectively methoxy-substitutedcatecholandusedasselectiveinhibitorof
(Figure 1B). As CMSCL and rTGFβ1 show the similar results, NAD(P)Hoxidase,inhibitsNAD(P)Hoxidasebyimpedingthe
rTGFβ1wasusedforthefurtherexperiments. assemblyofp47phox andp67phox subunitswithinthemembrane-
associatedNAD(P)Hoxidasecomplex[25].Newly,apocyninwas
showntohaveahighcapacityasascavengerofH O inaddition
2 2
Effectofallopurinol,apocyninandDPIonTGFβ1 toitsfunctionasNOXinhibitor[26].ApocyninandDPIalone
inducedαSMAexpression hadnoeffectonαSMAexpressioncomparedwithmock-treated
ThegrowthfactorTGFβ1wasshowntobeinvolvedinproduction controls(datanotshown).AstheinhibitionofTGFβ1-mediated
ofROS,especiallyO .− (superoxide)andH O [12,15,22,23]. αSMAexpressionbyapocyninissignificant,furtherexperiments
2 2 2
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10 (cid:2)c 2014TheAuthor(s) ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicence(CC-BY)(http://creativecommons.org/licenses/by/3.0/)
whichpermitsunrestricteduse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.
Fibroblast-to-myofibroblastswitch:roleofNAD(P)Hoxidase
Figure2 TGFβ1-mediatedexpressionofαSMA
(A)SubconfluentHDFswereeithermock-treatedorpretreatedfor24hwithallopurinol(10μM)beforeadditionofrTGFβ1
(10ng/ml).TGFβ1andtheallopurinolwerepresentforanadditional48h.ThelevelofαSMAproteinwasdeterminedby
Westernblot.α-tubulinwasusedasloadingcontrol.Threeindependentexperimentswereperformed.(B)HDFmonolayer
cultureswereculturedinCMHDFcontainingapocynin(1mM)for1horDPI(5μM)for24hbeforetreatmentwithTGFβ1
(10ng/ml)forfurther48h.ThelevelofαSMAproteinwasdeterminedbyWesternblot.α-tubulinwasusedasloading
control. The experiments were performed in triplicate. (C) Subconfluent HDF were preincubated for 1h with apocynin
(1mM) in serum-free medium and then TGFβ1 (10ng/ml) treated for 24h. Steady-state mRNA levels of αSMA were
analysedbyreal-timeRT-PCR.Dataaregivenasmeansofthreeindependentexperiments+−S.E.M.
focus onNAD(P)H oxidase andits downstream signallingres- activationofNAD(P)Hoxidaseandisaffectedbyapocynin.H O
2 2
ultinginMFformation. treatmentofcells,preincubatedwiththeapocyninandTGFβ1,
Tostudytheeffectofapocyninonlevelsofsteady-statemRNA resultedinasignificantincreaseinDCFfluorescence(Figure3A).
ofαSMAinHDF,real-timeRT–PCRwasperformed.The‘house- ApotentialextracelluarincreaseinH O shouldbemeasured
2 2
keeping’geneHPRTwasusedasinternalcontrol.TGFβ1caused byanamperometricapproach,whichishighlysensitiveforthe
a 20+−2-fold increase in αSMA steady-state mRNA levels at determination of extracellular H2O2. TGFβ1 exposure resulted
24h after the treatment compared with mock-treated controls. inasignificantincreaseinH O generation(Figure3B).At24h
2 2
Preincubation with apocynin (1mM) completely abolished the after treatment of HDF cells with TGFβ1, the H O level was
2 2
TGFβ1-mediated increase in the steady-state mRNA level of 2-foldhighercomparedwithmock-treatedcontrols.Asthepro-
αSMA(Figure2C).ThesedatacorrelatedwiththeαSMApro- ductionofH O byTGFβ1needsaO .−source[27],theeffectof
2 2 2
teinamount(Figure2B). apocyninontheproductionofH O wasmeasured.Preincubation
2 2
offibroblastsfor1hwithapocyninpriortorTGFβ1treatment
down-regulatedtheTGFβ1-mediatedH O generation.However,
2 2
ModulationofROSgenerationandprotein it is evident that TGFβ1 exposure results in a solid generation
oxidationbyapocynin ofH O .Interestingly,theincubationwithapocyninaloneinhib-
2 2
To test a direct effect of apocynin on ROS production in the ited the H O generation compared with mock-treated control
2 2
fibroblasts,theROSgenerationwasassessedbothintracellularly (Figure3B).
andextracellularly. Another,moreindirectapproachtomeasuretheintracellular
IncubationwiththegrowthfactorTGFβ1resultedinasigni- generation of ROS, the occurrence of carbonylated proteins, a
ficant2-foldincreaseindichlorofluorescein(DCF)fluorescence biomarkerforintracellularoxidativestress,wasinvestigated.For
whichwasmaintainedoverthestudiedtimerange.Anon-toxic that,HDFweretreatedwithTGFβ1andthecarbonylatedpro-
concentrationof1mMH O ,usedasacontrol,furtherincreased teinsverified.Alowamountofcarbonylatedproteinswasdetec-
2 2
theintracellularROSlevel.PreincubationofHDFswithanon- tedinmock-treatedcells,whereastheamountwassignificantly
toxic concentration of the specific NAD(P)H oxidase inhibitor increased in H O – and TGFβ1-treated cells compared with
2 2
apocynin(1mM)(Figure3A)priortoTGFβ1stimulationpre- mock-treatedcells(Figure3C).Treatmentwithapocyninsignific-
ventedthegrowthfactor-initiatedincreaseintheROSlevel,in- antlyloweredtheTGFβ1-mediatedproteinoxidation.H O was
2 2
dicatingthatgenerationofelevatedROSlevelsisdownstreamof usedaspositivecontrol.Eventhoughtheoccurrenceofprotein
..........................................................................................................................................................................................................................................................................................................................................................................
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L.Aliliandothers
Figure3 ApocynininhibitstheROSproductionandtheoxidativedamageinHDF
(A)SubconfluentHDFswerepreincubatedwithapocynin(1mM)for1h(closedcircles)beforetreatmentwithrTGFβ1or
H2O2(1mM)fortheindicatedtime.IncreaseofDCFfluorescencewasfollowedover20minversusmock-treatedcontrols
(open circle). The experiments were performed in triplicate. Arrows indicate addition of rTGFβ1 or H2O2.(B) H2O2was
detectedbyamperometricdetermination.Thedatarepresentthemean+−S.E.M.ofthreeindependentexperiments.(C)
HDFcellswereexposedtorTGFβ1for24horpreincubatedwithapocynin(1mM)beforeoxidizedproteinsweredetected
byWesternblotanalysisviaderivatizationwithDNPhydrazine.H2O2 wasusedaspositivecontrol.Threeindependent
experimentswereperformed.
carbonylsisproofforoxidativestress,themeasurementofthose Therefore,thecytosolicsubunitp67phox andNOX4mRNAwas
carbonylsisratherageneralmeasureofanalterationofthecel- estimated by RT–PCR. cDNA integrity was checked simultan-
lularredoxstatus. eouslybyamplificationofthehousekeepinggeneHPRT1.The
expressionofNOX4andp67phox wereup-regulatedinTGFβ1-
TGFβ1stimulatesarapidincreaseintheNAD(P)H treated cells after 8h (Figure 4B). These data confirm the pre-
oxidaseactivity viously shown NADH consumption peak after 8h of TGFβ1
The rates of NADH consumption by control and TGFβ1- treatment(Figure4A).
stimulated cells were determined at various time points over a Even though the increase of p67phox and NOX4 mRNA
24h period. TPA (12-O-tetradecanoylphorbol-13-acetate) was levels (Figure 4B) and the high activity of NAD(P)H oxidase
used as a positive control. As shown in Figure 4(A), the (Figure4A)bothat8hdealwiththeimportanceofthat8hpeak
rate of NADH consumption in TGFβ1-treated cells resulted in incontextofαSMAexpression,itcouldnotbeexcludedthatthe
two peaks. After 10min the NADH consumption in TGFβ1- firstpeakat10minpost-treatment(Figure4A)affectstheαSMA
treated cells was 2-fold higher than that of ct (control cells), expressionaswell.
with no measurable increase at 1 and 4h. A second peak of Thereforedifferentincubationperiodswithapocyninshould
NADHconsumptionwasdetectedat8hwitha7-foldincrease solvetheproblem.HDFwereexposedtorTGFβ1inthepresence
ofNADHconsumptioncomparedtoct(P<0.05).Thetreatment (+)andabsence(−)ofapocynin(1mM).Asignificantincrease
withTGFβ1ledtoagradualdecreasetobaseline(undetectable intheαSMAproteinamountwasmeasured48hupontreatment
levels)by24h. with rTGFβ1 compared with mock-treated controls. Apocynin
In most cell types, the members of the NOX family are the treatment (+) over the total time period of 48h after TGFβ1
sourcefortheoccurrenceofROS,namelysuperoxide.NAD(P)H incubation completely abolished the αSMA signal. The incub-
oxidase consists of membrane-associated and cytosolic sub- ation with apocynin starting 4 and 8h after TGFβ1 treatment
units.TherearefivehumanNAD(P)Hoxidases,namelyNOX1 showedamarkedloweringofαSMAexpressionaswell.How-
to NOX5 and several cytosolic and regulatory subunits, e.g. ever, apocynin treatment starting 16h after TGFβ1 incubation
p67phox[28]. It is described, that NOX4 is involved in TGFβ1- didnotaffecttheαSMAexpression(Figure4C).Furthermore,
mediated differentiation of human cardiac fibroblasts to MFs apocyninincubationduringthefirsthourafterTGFβ1treatment
[29].Inthisstudy,wecheckedwhetherrTGFβ1isinvolvedin also showed no inhibitory effect on αSMA expression. Thus,
expressionofgenesencodingcomponentsofNAD(P)Hoxidases. the first NAD(P)H oxidase peak seems to play a rather minor
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Fibroblast-to-myofibroblastswitch:roleofNAD(P)Hoxidase
Figure4 rTGFβ1activatestheNADHoxidaseindermalfibroblasts
(A) Rates of NADH consumption by ct and time course of the rates from HDF following rTGFβ1 (10ng/ml) treatment.
TPAwasusedasapositivecontrol.Inpresenceof250μMNADH,subconfluentHDFwereeithermock-treatedortreated
with rTGFβ1. The consumption of NADH was measured spectrophotometrically. data represent the mean+−S.E.M. (B)
SubconfluentHDFwerepreincubatedfor1hwithapocynin(1mM)intheserum-freemediumandthenrTGFβ1(10ng/ml)
treated for various time points. p67phox and NOX4 mRNA expression were analysed by RT–PCR. HPRT1 was used as
housekeepinggene.Threeindependentexperimentswereperformed.(C)SubconfluentHDFswereeithermock-treated,
treatedwithrTGFβ1(10ng/ml)for48horincubatedwithapocyninfor1horstarting4,8and16hafterrTGFβ1treatment.
ThelevelofαSMAproteinwasdeterminedbyWesternblot.CoomassieBrilliantbluestainingwasusedasloadingcontrol.
Threeindependentexperimentswereperformed.
roleinαSMAsignalling.Insummary,theNAD(P)Hoxidaseis kinase) activation U0126 had no effect on αSMA expression
essential for αSMA signalling in a time period of 4–8h after (Figure5A).Asthespecificp38-inhibitorS202190hadthemost
TGFβ1-stimulation. inhibitory effect on αSMA protein level, the effect on αSMA
mRNAexpressionwastested.Inthefollowing,thefocuswason
EffectsofMAPK(mitogen-activatedprotein thep38kinaseandthechronologicalinvolvementofp38inthe
kinases)onMFformation αSMA signalling. Preincubation with non-toxic concentrations
In fibroblasts of adventitia from vascular cells, ROS generated of the p38 MAPK inhibitor significantly (P<0.001) counter-
byNAD(P)Hoxidase,activatedtheMAPKandfinallythefibro- actedtheTGFβ1-initiatedtranscriptionofαSMAmRNA(Fig-
blasts differentiated to MFs [30]. To check the importance of ure5B),indicatingacrucialroleofp38kinaseinthesignalling
MAPKduringthetransitionprocessofHDF,HDFwereexposed pathway,whichresultsinαSMAexpressionandMFformation.
torTGFβ1inthepresenceandabsenceofnon-toxicconcentra- In the following, a time course analysis after stimulation with
tionofU0126,SP600125andSB202190(10μM).Mock-treated rTGFβ1 was performed. The cells were co-incubated with the
ctshowedabasalαSMAexpression.Asignificantincreaseinthe specificp38inhibitorSB202190fordifferenttimeperiods.Mock-
αSMAproteinamountwasmeasuredat48hupontreatmentwith treated ct showed a basal αSMA expression. After stimulation
TGFβ1comparedwithmock-treatedcontrols.Inourstudy,JNK withrTGFβ1theαSMAproteinamountincreased.TheαSMA
(c-Jun N-terminal kinase) inhibitor SP600125 and p38 MAPK protein level of cells co-incubated either with rTGFβ1 and SB
inhibitorSB202190significantly(P<0.001)attenuatedTGFβ1- 202190overthetotaltimeperiodorwithrTGFβ1andp38in-
inducedαSMAexpressionwhenaddedtothecultures1hprior hibitor starting 4 and 8h after rTGFβ1 treatment was nearly
toTGFβ1.TheinhibitorofERK(extracellular-signal-regulated completelyabolished.Bycontrast,incubationwiththeinhibitor
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L.Aliliandothers
Figure5 Involvementofp38kinaseinTGFβ1/ROS-dependentexpressionofαSMA
(A)SubconfluentHDFswerepreincubatedwithMAPKinhibitorsU0126,SP600125orSB202190beforetreatmentwith
rTGFβ1.ExpressionofαSMAwasdetectedbyWesternblots.Thedensitometricanalysisdescribesproteinexpressionas
foldincreaseovercontrol,whichwassetat1.0.Thedatarepresentthemean+−S.E.M.ofthreeindependentexperiments.
(B) Subconfluent HDF were preincubated for 1h with SB202190 (10μM) in the serum-free medium and then rTGFβ1
(10ng/ml)treatedfor24h.αSMAmRNAlevelswereanalysedbyreal-timeRT-PCR.Dataaregivenasmeansofthree
independentexperiments+−S.E.M.(C)SubconfluentHDFswereeithermock-treated,treatedwithrTGFβ1(10ng/ml)for
48horincubatedwithSB202190for48horstarting4,8and16hafterrTGFβ1treatment.ThelevelofαSMAprotein
wasdeterminedbyWesternblot.α-tubulinwasusedasloadingcontrol.Threeindependentexperimentswereperformed.
(D)SubconfluentHDFswereeithermock-treatedorpretreatedfor1hwithapocynin(1mM)beforeadditionofrTGFβ1
(10ng/ml). TGFβ1 and apocynin were present for an additional 12h. Anisomycin (0.5μg/ml) was used as technical
controlandincubatedfor20min.Thelevelofphospho-p38MAPKproteinwasdeterminedbyWesternblot.α-tubulinwas
usedasloadingcontrol.Twoindependentexperimentswereperformed.
starting16hafterrTGFβ1stimulationshowedonlyaslightbut DISCUSSION
notsignificantinhibitoryeffectonαSMAexpression,keepingin
mindtheα-tubulinloadingcontrol(Figure5C).Herein,wehave
shownthatapocyninaswellasthep38inhibitorSB202190in- The first crucial step in tumour invasion and metastasis is the
hibittheTGFβ1-mediatedαSMAexpressionandconsequently movement of cancer cells into the tumour-surrounding tissue.
theMFformation.TGFβ1generatesROSbecauseofNAD(P)H Recentdatabroughtprominencetothehypothesisofarolefor
oxidase,whichactivatesp38andfurtherstimulatesαSMAsig- tumour stromal environment as a leading player, and not just
nalling. The link between the NAD(P)H oxidase and p38 was a supporting additional, in the progression of carcinomas, the
investigated using apocynin. Incubation with Anisomycin for most common form of human cancer. Fibroblasts have a more
20min showed a distinct signal, the mock-treated fibroblasts profoundinfluenceonthedevelopmentandprogressionofcar-
showed a weak activation of p38. Treatment with rTGFβ1 for cinomasthanpreviouslyappreciated[1,2].Inthatcontext,MFs
12h induced a significant p38 phosphorylation. Preincubation and cancer cells are known to exchange proteolytic enzymes,
withapocynin(1mM)nearlycompletelyabolishedtheincrease cytokines and growth factors, which promote proliferation and
oftheactivatedp38(Figure5D). survivalaswellasinvasionofthetumour[31].Inthisstudy,we
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14 (cid:2)c 2014TheAuthor(s) ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicence(CC-BY)(http://creativecommons.org/licenses/by/3.0/)
whichpermitsunrestricteduse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.
Fibroblast-to-myofibroblastswitch:roleofNAD(P)Hoxidase
Figure6 SchemeofTGFβ1-mediatedsignalling
Tumourcellsreleasegrowthfactors,e.g.TGFβ1,whichgeneratesROSduetoNAD(P)Hoxidase,whichisresponsiblefor
thedownstreamsignallingresultinginROS-triggeredactivationofthestresskinasep38andexpressionofαSMA.Both
canbeinhibitedbythespecificNAD(P)Hoxidaseinhibitorapocynin.
haveshowninvitrothattheNAD(P)Hoxidaseisresponsiblefor ing signalling as well as transcription factors can be modified
thedownstreamsignallingresultinginROStriggeredactivation by TGFβ1 induced ROS [37,40]. As several cellular mechan-
ofthestresskinasep38andexpressionofαSMAafterTGFβ1 ismsresultintheproductionofROS[41,42],weaskedforthe
treatment(Figure6). sourceofROS.Somestudiesshowthegrowthfactor-/cytokine-
Fibroblastscanbeactivatedbyvariousgrowthfactorsandcy- dependent generation of ROS during physiological signalling
tokines,whicharesecretedbytumourcells[15,32,33],andshow duetomembrane-boundenzymecomplexes,e.g.theNAD(P)H
thenmyofibroblasticdifferentiation[34,35].Thereisadynamic oxidasethatgenerateO .−/H O [43–45].TGFβ1isknownto
2 2 2
interactionbetweenmalignantcellsandstromaltissue,whichis activateNAD(P)Hoxidase,resultinginthegenerationofROS,
mediated by cell–cell- and cell–matrix communiacations [36]. whichpromotescarcinogenesis[46].AsTGFβ1isknowntoin-
Inourstudy,themesenchymal–mesenchymaltransitionwasin- duce the formation of MFs [3], we addressed the question of
ducedbyrTGFβ1atconcentrationsrangingfrom5to10ng/ml. whetherNAD(P)Hoxidasealoneorothersourcessuchasxanth-
Recently, similar concentrations of rTGFβ1 were shown to be ineoxidasehaveasynergisticeffectinTGFβ1-initiatedandROS-
sufficienttoincreaseαSMAproteinlevelsinratproximaltubu- dependentfibroblast-to-MFtransition.Sofar,itwasknown,that
lar epithelial cell line NRK52E [37] as well as in human fetal TGFβ1treatmentincreasestheNAD(P)Hoxidaseactivityinlung
lungfibroblasts[38].Moreover,thesecellsshowedsignificantly fibroblasts[13].Thenon-phagocyticNAD(P)Hoxidaseproduces
higheramountsofαSMAproteinat24–48hafterTGFβ1treat- primarilysuperoxideanions(O .−)onthecytosolicsideofthe
2
ment,whichcorrespondstoourstudy.Herein,theincubationof cellmembrane[47],whichmaybesubsequentlydismutatedto
HDFwithsupernatantsofsquamoustumourcellsresultedina H O [40,48].
2 2
significantincreaseinαSMAexpressionaswell.Theloweref- Inourstudy,thecommonflavoproteininhibitorDPIandthe
fectofthetumourcellsupernatantsonαSMAexpressionisdue selectiveNAD(P)Hoxidaseinhibitorapocynin[49]completely
to the lower concentration of active TGFβ1in the supernatant prevented both the TGFβ1-mediated expression of αSMA and
comparedwiththeactivityofrTGFβ1[12]. thegenerationofROS,excludingmitochondriaorxanthineoxi-
ThesupernatantofSCL-1tumourcellsandTGFβ1triggered dasetobeinvolvedintheROS-dependenttransitionoffibroblasts
a rapid increase in intracellular ROS levels in HDF leading to toMFs.Thus,itislikelytoassumethattheNAD(P)Hoxidaseis
animpairedgapjunctionalintercellularcommunication[11],a theonlycellularsourceforthemeasuredROSinthatcontext.The
prerequisitefortumourprogression[39].Cellularstructuresdur- rateofNAD(P)HconsumptionbyTGFβ1-treatedcellsresulted
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whichpermitsunrestricteduse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.
L.Aliliandothers
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7 Kunz-Schughart,L.A.andKnuechel,R.(2002)Tumor-associated
MFs[13,40].Incontrast,weshowthattherapidactivationofthe
fibroblasts(partII):functionalimpactontumortissue.Histol.
NAD(P)HoxidaseisnotpartoftheTGFβ1-mediatedsignalling
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and phosphorylation-dependent activation of AP-1 (activating 11Stuhlmann,D.,Ale-Agha,N.,Reinehr,R.,Steinbrenner,H.,Ramos,
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AUTHORCONTRIBUTIONS
transforminggrowthfactorbeta1.J.Biol.Chem.270,
Lirija Alili analysed data and wrote the paper. Maren Sack and 30334–30338
Katharina Puschmann designed and conducted research. Peter 14Herrera,B.,Murillo,M.M.,Alvarez-Barrientos,A.,Beltran,J.,
Brenneisen contributed to the design of research and did the fi- Fernandez,M.andFabregat,I.(2004)Sourceofearlyreactive
nalapprovaloftheversiontobepublished. oxygenspeciesintheapoptosisinducedbytransforminggrowth
factor-betainfetalrathepatocytes.FreeRadic.Biol.Med.36,
16–26
ACKNOWLEDGMENTS 15Cat,B.,Stuhlmann,D.,Steinbrenner,H.,Alili,L.,Holtkotter,O.,
ThisworkispartofthePh.D.thesisofK.P.attheHeinrich-Heine- Sies,H.andBrenneisen,P.(2006)Enhancementoftumorinvasion
dependsontransdifferentiationofskinfibroblastsmediatedby
UniversityofDu¨sseldorf.WethankC.Wyrichforexcellenttechnical
reactiveoxygenspecies.J.CellSci.119,2727–2738
assistance.
16Alili,L.,Sack,M.,Karakoti,A.S.,Teuber,S.,Puschmann,K.,Hirst,
S.M.,Reilly,C.M.,Zanger,K.,Stahl,W.,Das,S.etal.(2011)
FUNDING Combinedcytotoxicandanti-invasivepropertiesofredox-active
nanoparticlesintumor–stromainteractions.Biomaterials32,
Thisresearchreceivednospecificgrantfromanyfundingagency
2918–29
inthepublic,commercialornot-for-profitsectors.
17Bayreuther,K.,Francz,P.I.,Gogol,J.andKontermann,K.(1992)
Terminaldifferentiation,aging,apoptosis,andspontaneous
transformationinfibroblaststemcellsystemsinvivoandinvitro.
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whichpermitsunrestricteduse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.