Differential protein expression profile in the hypothalamic GT1-7 cell line after exposure to anabolic

22 Pages·2017·5.92 MB·English

RESEARCHARTICLE
Differential protein expression profile in the
hypothalamic GT1-7 cell line after exposure to
anabolic androgenic steroids
FreddysonJ.Mart´ınez-Rivera1,JulianaPe´rez-Laspiur2,Mar´ıaE.Santiago-Gascot1,Abner
G.Alema´n-Reyes3,EmanuelGarc´ıa-Santiago4,YolandaRodr´ıguez-Pe´rez2,CristhianCalo-
Guadalupe4,IneliaOtero-Paga´n1,RoxsanaN.Ayala-Paga´n3,MagdielMart´ınez5,Yisel
M.Cantres-Rosario6,LoydaM.Mele´ndez2,6,JenniferL.Barreto-Estrada1*
a1111111111 1 DepartmentofAnatomyandNeurobiology,MedicalSciencesCampus,UniversityofPuertoRico,San
Juan,PuertoRico,UnitedStatesofAmerica,2 TranslationalProteomicsCenter-RCMI,MedicalSciences
a1111111111
Campus,UniversityofPuertoRico,SanJuan,PuertoRico,UnitedStatesofAmerica,3 Departmentof
a1111111111
Biology,UniversityofPuertoRico,R´ıoPiedrasCampus,SanJuan,PuertoRico,UnitedStatesofAmerica,
a1111111111 4 DepartmentofBiotechnology,UniversidaddelEste,Carolina,PuertoRico,UnitedStatesofAmerica,
a1111111111 5 DepartmentofPhysiologyandBiophysics,MedicalSciencesCampus,UniversityofPuertoRico,SanJuan,
PuertoRico,UnitedStatesofAmerica,6 DepartmentofMicrobiologyandMedicalZoology,MedicalSciences
Campus,UniversityofPuertoRico,SanJuan,PuertoRico,UnitedStatesofAmerica
*[email protected]
OPENACCESS
Citation:Mart´ınez-RiveraFJ,Pe´rez-LaspiurJ, Abstract
Santiago-GascotME,Alema´n-ReyesAG,Garc´ıa-
SantiagoE,Rodr´ıguez-Pe´rezY,etal.(2017)
Theabuseofanabolicandrogenicsteroids(AAS)hasbeenconsideredamajorpublichealth
Differentialproteinexpressionprofileinthe
problemduringdecades.SupraphysiologicaldosesofAASmayleadtoavarietyofneuro-
hypothalamicGT1-7celllineafterexposureto
anabolicandrogenicsteroids.PLoSONE12(7): endocrineproblems.Precisely,thehypothalamic-pituitary-gonadal(HPG)axisisoneofthe
e0180409.https://doi.org/10.1371/journal. bodysystemsthatismainlyinfluencedbysteroidalhormones.Fluctuationsofthehormonal
pone.0180409
milieuresultinalterationsofreproductivefunction,whicharemadethroughchangesin
Editor:HubertVaudry,UniversitedeRouen, hypothalamicneuronsexpressinggonadotropin-releasinghormone(GnRH).Infact,previ-
FRANCE
ousstudieshaveshownthatAASmodulatetheactivityoftheseneuronsthroughsteroid-
Received:July2,2016 sensitiveafferents.ToincreaseknowledgeaboutthecellularmechanismsinducedbyAAS
Accepted:June15,2017 inGnRHneurons,weperformedproteomicanalysesofthemurinehypothalamicGT1-7cell
lineafterexposureto17α-methyltestosterone(17α-meT;1μM).Thesecellsrepresenta
Published:July18,2017
goodmodelforstudyingregulatoryprocessesbecausetheyexhibitthetypicalcharacteris-
Copyright:©2017Mart´ınez-Riveraetal.Thisisan
ticsofGnRHneurons,andrespondtocompoundsthatmodulateGnRHinvivo.Two-dimen-
openaccessarticledistributedunderthetermsof
theCreativeCommonsAttributionLicense,which sionaldifferenceingelelectrophoresis(2D-DIGE)andmassspectrometryanalyses
permitsunrestricteduse,distribution,and identifiedatotalof17differentproteinsthatweresignificantlyaffectedbysupraphysiological
reproductioninanymedium,providedtheoriginal
levelsofAAS.Furthermore,pathwayanalysesshowedthatmodulatedproteinsweremainly
authorandsourcearecredited.
associatedtoglucosemetabolism,drugdetoxification,stressresponseandcellcycle.Vali-
DataAvailabilityStatement:Allrelevantdataare
dationofmanyoftheseproteins,suchasGSTM1,ERH,GAPDH,PEBP1andPDIA6,were
withinthepaperanditsSupportingInformation
files. confirmedbywesternblotting.WefurtherdemonstratedthatAASexposuredecreased
expressionofestrogenreceptorsandGnRH,whiletwoimportantsignalingpathwayproteins
Funding:ThisworkwasfundedbyRTRNSmall
GrantsProgram(U54MD008149)http://grants. p-ERK,andp-p38,weremodulated.Ourresultssuggestthatsteroidshavethecapacityto
rtrn.net/index.php/SMALLGRANTS20162017, directlyaffecttheneuroendocrinesystembymodulatingkeycellularprocessesforthecon-
toJLBE;NIH–NCRR(2P20RR016470)
trolofreproductivefunction.
http://www.nems.nih.gov/teams/Pages/NCRR.
aspx,toJLBE;NIGMS(8P20GM103475)
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ProteinprofileofhypothalamicGT1-7celllineafterAASexposure
https://www.nigms.nih.gov/Pages/default.aspxto Introduction
JLBE;MBRS-RISE(R25GM061838)http://mbrs-
Theanabolicandrogenicsteroids(AAS)aresyntheticderivativesoftestosterone,createdto
rise.rcm.upr.edu.toFJMR;MBRS-RISE
(R25GM066250)http://www.suagm.edu/une/ provideanabolicpotencyandlowandrogeniceffect[1].WhilemostusersabuseAASto
propuestas/rise/rise_index.html.toEGSandCCG; improvephysicalperformance,nearlyallofthemareunawareofthenumeroussideeffects
RCMITranslationalProteomicsCenterNIMHD thatcanbeassociatedwithandrogenmisuse[2].Cardiovasculardiseases,cancer,liver
(8G12MD007600),http://rcmi.rcm.upr.edu/
dysfunctionandpsychiatricdisordersaresomeofthosewell-documentedproblems
resreso/guidelines_rcmi.Thefundershadnorole
[2,3].Similarly,undertheinfluenceofAAS,thehypothalamic-pituitary-gonadal(HPG)
instudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthe axisisoneofthemostaffectedbodysystems[4,5].Theneuralcontrolofthisaxisresides
manuscript. withinthehypothalamus,whichischaracterizedbytheexpressionofhighlevelsofandro-
genandestrogenreceptors(ARandER)[6].Themedialpreopticarea(mPOA)isasteroid-
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. sensitivehypothalamicregion,thatispopulatedbyneuronsexpressinggonadotropin-
releasinghormone(GnRH)[7].Indeed,GnRHneuronsrespondtofluctuationsofgonadal
steroids,whichresultinneuroplasticitychangesleadingtopulsatilesecretionofthispep-
tide.Invivoandinvitrostudieshaveshownthatexposuretosteroidsisassociatedwith
alterationsonGnRHexpressionandsecretion.Forinstance,studiesinrodents[8],and
cells[9,10]showedthatexposuretoeitherandrogensorestrogensreducedGnRH
transcripts.
Todate,fewstudieshaveinvestigatedtheeffectofAASinGnRHneurons.Inthisregard,
electrophysiologicalstudieshaveshownthatAASmodulatetheactivityofthesecells[4,5].For
example,theAAS17α-methyltestosterone(17α-meT;7.5mg/kg),increasedpresynaptic
GABA receptor(GABA R)currentsofmPOAsteroid-sensitiveneurons,resultingininhibi-
A A
tionofGnRHcells[4].Similarly,femalemiceexposedto17α-meT(7.5mg/kg),displayeda
diestrous-likepatternactivityinGnRHneuronsthroughthesuppressionofpresynaptickis-
speptinexcitatoryinputsfromtheanteroventralperiventricularnucleus[5].Althoughthese
studiesdemonstratedneuroendocrinemodulationbyAAS,thereisstillalackofacomplete
proteinprofileonGnRHneurosecretorycellsafterAASexposure,whichhasthepotentialto
revealspecificalterationsinregulatoryprocessesofthereproductiveaxis.Inthisstudy,we
characterizedtheproteomicprofileofGnRHneuronsafterexposuretosupraphysiologicallev-
elsof17α-meT.
GiventhescatteredconcentrationofGnRHneurosecretorycellswithinthemPOA[11],
andthedifficultytoestablishaninvivoapproachtostudytheseneurons[9],weusedthe
murineimmortalizedcelllineofGnRH-secretinghypothalamicneurons(GT1-7)[12].Cer-
tainly,GT1-7cellshavebeenveryusefulinstudyingregulatoryprocessesbecausetheyexhibit
thetypicalcharacteristicsofGnRHneurons,andrespondtothesamecompoundsthatmodu-
lateGnRHsecretioninvivo[13–15].Moreover,evidencerevealsthatGT1-7cellsexpressAR
[16,17],ER[18,19],andreceptorsforGABA(GABA R)[20],cellularpropertiesthatconfer
A
responsivenesstoandrogenicandestrogeniccompounds.
Theuseofomicstechnologieshasgainedpopularitytouncovertheuseofanabolicagents
inhumansports,animalhusbandry[21],andaquaculture[22].Indeed,proteomicanalyses
havebeenappliedforthescreeningofsteroideffectsonbodytissuessuchasprostate[23],
gonads[22],breast[24],muscles[25]andbloodcomponents[26].Inthisstudy,weusedtwo-
dimensionaldifferenceingelelectrophoresis(2D-DIGE)incombinationwithmassspectrom-
etryandwesternblottingtoprofiletheproteomeofGnRHneurons.Wehypothesizedthat
17α-meTwillmodulatetheexpressionofproteinsassociatedwithneuroendocrineregulation,
synapticplasticity,andcellularstress;keybiologicalprocessesthatmightimpactreproductive
competenceandintegrity.
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ProteinprofileofhypothalamicGT1-7celllineafterAASexposure
Materialsandmethods
Cellcultureandreagents
GT1-7cellsweregrownaspreviouslydescribed[12].Inbrief,cellsweremaintainedinDMEM
(Mediatech,Manassas,VA)supplementedwith10%fetalbovineserum(FBS;Hyclone,Wal-
tham,MA)andpenicillin/streptomycin(Gibco,GrandIsland,NY).Cellsweregrownin25cm
cultureflasksandmaintainedinahumidifiedincubatorat37˚Cand5%CO .Eachculture
2
flaskrepresentsanindependentbiologicalreplicate.
Drug
A2D-DIGEexperimentwasperformedusingfourindependentbiologicalreplicatespertreat-
ment(Control:n=4;AAS:n=4).BeforeAASexposure,cellsweregrowninsteroid-free
serum(HycloneWaltham,MA)duringthelog-phasegrowth(70–80%confluency).Thereaf-
ter,four(4)samplesweretreatedwithasupraphysiologicaldoseoftheAAS,17α-methyltestos-
terone(17α-meT:1μM;Sigma,St.Louis,MO)for48haspreviouslydescribed[27].Control
samplesweretreatedwithvehicle(30%cyclodextrinin0.9%saline;Sigma,St.Louis,MO).
17α-meTwaschosenasthepresenceoftheC17methylgroupreducesitsaromatizationto
17β-estradiol[28],andinhibitsaromatization[29,30].Asthenormalleveloftestosteronein
maleserumis1X10−8M(0.01μM)[31,32],theregimenusedinourexperiment(1μMfor
48h)[27]reflectsachronicsupraphysiologicaldose.CellviabilityafterAASexposurewas
assessedbytrypanblueexclusionassayaspreviouslydescribedwithsomemodifications[33].
Caspaseactivity
Caspase3activitywasmeasuredusingtheCaspase-Glo13/7(PromegaCo.,Madison,WI),in
which50,000cells/wellwereinitiallyseededinaflatbottom96-wellplate.Cellswereincubated
withequalvolumesofthereagenttotheculturemediumfor1h,andaVarioskanFlashReader
(ThermoFisherScientific,Waltham,MA)wasusedtoassessrelativeluminescence.Caspase9
activitywasmeasuredusingtheCaspase-9Fluorometricassay(R&DSystems,Inc.Minneapo-
lis,MN).Eachsamplecontainedthecelllysate(100μgofproteinin50μL),50μLof2Xreac-
tionbuffer,and5μLofcaspase9fluorometricsubstrate(LEHD-AFC).Thereactionwas
incubatedfor1hat37˚Cinaflatbottom96-wellmicroplate.Fluorescencesignalwasindica-
tiveofcaspaseactivation,anditwasmeasuredonafluorescentplatereader(GeminiSpectra-
Max,MolecularDevices,CA,USA).
Proteomicanalyses
Proteinextractionandproteinquantification. Cellswereharvestedafter48hofAAS
exposure.LysisBuffer(7Murea,2Mthiourea,4%CHAPS;GEHealthcare,Pittsburgh,PA)
andacocktailofproteaseinhibitors(BioVision,Inc.,Milpitas,CA)wereaddedtothecellpel-
lets.Sampleswerecentrifugedat13,000gfor15minat4˚C.Supernatantscontainingthe
cellularproteinfractionswererecoveredandproteinextractswerepurifiedbyprecipitation,
usinga2-DClean-UpKit(GEHealthcare,Pittsburgh,PA).Afterprecipitation,proteinpellets
wereresuspendedincelllysisbuffer(30mMTris-HCLpH=8,7Murea,2Mthiourea,4%
CHAPS).Proteinconcentrationwasmeasuredusinga2-DQuantKit(GEHealthcare,Pitts-
burgh,PA).Kitswereusedassuggestedbymanufacturer’sinstructions.
Samplelabeling. Cy5andCy3labelingforanalyticalgels:Five(5)μgoftotalproteinfrom
GT1-7cellssamples(4controls,4AAS)werespeed-vacuumdriedandresuspendedincell
lysisbuffer.Sampleswerereducedbyincubationwith2nMTCEP(tris-[2-carboxyethyl]phos-
phoinehydrochloride;GEHealthcare,Pittsburgh,PA)at37˚Cfor1handthenlabeledwith4
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ProteinprofileofhypothalamicGT1-7celllineafterAASexposure
nMofCy5saturationdye(GEHealthcare,Pittsburgh,PA)for30minat37˚C.Onevolume
samplebuffer(7Murea,2Mthiourea,4%CHAPS),pH3-10NLIPGbuffer,and130mMDTT
wasaddedtothesamples.Aninternalstandardwaspreparedbypooling5μgtotalproteinof
eachsample,mixedtogether,andthenspeedvacuum-dried.Proteinpelletwasresuspended
withcelllysisbufferandreducedwith2nMTCEPper5μgproteinfor1hat37˚C.Thepro-
teinswerelabeledwith4nMofCy3saturationdyeper5μgproteinfor30minat37˚C.One
volumesamplebufferwasaddedtothesamples.Each5μgofsamplelabeledwithCy5was
mixedwith5μgofinternalstandardlabeledwithCy3.Rehydrationbuffer(7Murea,2Mthio-
urea,2%CHAPS),and60mMDTT,pH3-10NLIPGbufferwasaddedtoafinalvolumeof
450μl.
Cy3labelingforpreparativegels:Atotalof250μgofprotein(31.25μg/sample)fromsam-
plesrepresentedinanalyticalgelswerepooled,speedvacuum-dried,resuspendedin250μlof
celllysisbufferandtreatedwithTCEPfor1hat37˚C.Protein(250μg)waslabeledwithCy3
saturationdyefor30minat37˚C.Tostopthereaction,samplebufferwasaddedtoafinalvol-
umeof450μl.
2D-DIGE. FirstdimensionwascarriedoutwithanEttanIPGphorapparatus(GE
Healthcare,Pittsburgh,PA).Sampleswereloadedon24cmlongImmobilineDryStripsgels
withnon-linearimmobilizedpHgradient3–10byovernightrehydration.Eachgelcontained
theinternalstandardandoneofthesamples(analyticalgels,aftersaturationlabeling)or
pooledmaterialinthecaseofthepreparativegelstrip.Isoelectricfocusingwascarriedoutat
aconstanttemperatureof20˚Cwithatotalof78.5kVh.Beforetheseconddimension,sepa-
rationstripswereincubatedwithanequilibrationsolution(50mMTris-HCLpH=8.8,6M
urea,30%glycerol,2%SDS,0.01bromophenolbluecontaining100mMDTT)for15min.
Then,thestripswereloadedonthetopofpre-cast12%polyacrylamidegelsandfixedwith
0.5%agarose.TheseconddimensionwascarriedoutwithanEttanDALTtwelveElectropho-
resisSystem(GEHealthcare,Pittsburgh,PA)at20˚C.Currentwasheldconstantto12mA
pergelforanovernightperiod.Onthenextday,currentwasincreasedto20mApergeluntil
bromophenolbluemarktheendofthegel.Forvisualizationofproteinspots,signalswere
collectedatexcitationwavelengthforCy3andCy5labeledsampleat540and635nm,respec-
tively,usingEttanDIGEImager(GEHealthcare,Pittsburgh,PA).Gelswerescannedat
100μmresolutionandanalyzedusingDeCyder2-D6.5software(GEHealthcare,Pittsburgh,
PA).Usinga2mmdiametertip,spotsselectedforproteinidentificationafterDeCyderstatis-
ticalanalysis(P<0.05)werepickedfromthepreparativegelbyautomaticEttanSpotPicker
(GEHealthcare,Pittsburgh,PA).
In-geldigestion. Spotspickedfrom2D-DIGEpreparativegelwerewashedatRTwith
200μlof50%acetonitrileand50mMammoniumbicarbonatesolutionfor1h.Gelpieces
weredriedandthenincubatedwithtrypsinovernightdigestionat37˚C.Resultingpeptides
wereextractedusingamixtureof60%acetonitrileand0.1%trifluoroaceticacid(TFA).Sam-
plesweredriedandresuspendedin0.5%trifluoroaceticacid.Allsamplesweredesaltedand
purifiedusingC18ZipTips(EMDMillipore,Billerica,MA)accordingtomanufacturer’srec-
ommendationsandresuspendedin2%acetonitrilewith0.1%formicacidpriortoliquidchro-
matographytandemmassspectrometry(LC-MS/MS)analysis.
Massspectrometryandproteinidentification. Trypticpeptideswerereconstitutedin
40μLof0.1%TFA(v/v)inwater:acetonitrile(95:5)and4μLwasdirectlyloadedat4μL/min
for7minontoacustom-madetrapcolumn(100μmI.D.fusedsilicawithKasilfrit)containing
2cmof200Å,5μmMagicC18AQparticles(Bruker-Michrom,Auburn,CA).Peptideswere
elutedusingacustom-madeanalyticalcolumn(75μmI.D.fusedsilica)withgravity-pulledtip
andpackedwith25cm100Å,5μmMagicC18AQparticles(Bruker-Michrom,Auburn,CA).
Peptideswereelutedwithalineargradientfrom100%solventA(0.1%formicacid:acetonitrile
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ProteinprofileofhypothalamicGT1-7celllineafterAASexposure
[95:05])to35%solventB(acetonitrilecontaining0.1%formicacid)in30minat300nL/min,
usingaProxeonEasynanoLCsystemdirectlycoupledtoaLTQOrbitrapVelosmassspec-
trometer(ThermoScientific,Waltham,MA).Datawereacquiredusingadata-dependent
acquisitionroutineofacquiringonemassspectrumfromm/z350–2000intheOrbitrap(reso-
lution60,000)followedbytandemmassspectrometryscansintheLTQlineariontrapofthe
10mostabundantprecursorionsfoundinthemassspectrum.Chargestaterejectionofsingly
chargedionsanddynamicexclusionwasutilizedtominimizedataredundancyandmaximize
peptideidentification.Therawdatafileswereprocessedintopeaklistsfordatabasesearching.
DatabaseSearchingofallMS/MSsampleswasperformedusingMascot(MatrixScience,
London,UK;version2.3.02)andX!Tandem(TheGPM,thegpm.org;versionCYCLONE
[2010.12.01.1]).Chargestatedeconvolutionanddeisotopingwerenotperformed.X!Tandem
wassetuptosearchasubsetoftheSwissProt_012512databaseandMascotwassetuptosearch
theSwissProt_022212database(selectedforMus.,unknownversion,16531entries)assuming
thedigestionenzymetrypsin.MascotandX!Tandemweresearchedwithafragmentionmass
toleranceof0.50Daandaparentiontoleranceof15PPM.Pyro-glufromEofthen-terminus,
oxidationofmethionine,acetylationofthen-terminusandiodoacetamidederivativeofcyste-
inewerespecifiedinX!Tandemasvariablemodifications.Pyro-glufromEofthenterminus,
oxidationofmethionine,acetylationofthen-terminus,iodoacetamidederivativeofcysteine
andacrylamideadductofcysteinewerespecifiedinMascotasvariablemodifications.
Scaffold(versionScaffold_3.4.5,ProteomeSoftwareInc.,Portland,OR)wasusedtovali-
dateMS/MSbasedpeptideandproteinidentifications.Peptideidentificationswereaccepted
iftheycouldbeestablishedatgreaterthan80%probability,asspecifiedbythePeptide
Prophetalgorithm[34].Proteinidentificationswereacceptediftheycouldbeestablishedat
greaterthan90%probabilityandcontainedatleast2identifiedpeptides.Proteinprobabilities
wereassignedbytheProteinProphetalgorithm[35].Proteinsthatcontainedsimilarpeptides
andcouldnotbedifferentiatedbasedonMS/MSanalysisaloneweregroupedtosatisfythe
principlesofparsimony.
Networksandpathwaysanalysis
Uniprotdatabase(www.uniprot.org)wasusedtoidentifyproteinlocalizationandfunction.
Ingenuitypathwaysanalysis(IPA)knowledgebase(www.ingenuity.com)wasusedtoidentify
predominantinteractionnetworksandbiologicalfunctionsofdifferentiallyexpressedproteins
[36].Thissoftwareusescomputationalalgorithmstoidentifynetworksconsistingofproteins
ofinterestandtheirinteractionswithotherproteinsintheknowledgebase.Thenetwork
scores(negativelogofthePvalue)arecalculatedaccordingtothefitofthenetworktothe
focusproteins.Inaddition,IPAknowledgebaseidentifiesglobalfunctionandcanonicalpath-
waysoftheentiredatasetofproteins.Thesignificancevaluesforthecanonicalpathwaysare
calculatedbyFisher’sexacttestright-tailed.Specifically,thistestcomparesthenumberofpro-
teinsthatcontributeinagivenpathwayorprocess,relativetothetotalnumberofoccurrences
oftheseproteinsinallpathwayannotationsstoredintheIPAknowledgebase.
Validationofdifferentialproteinexpression
Samplepreparation. GT1-7cellsweregrownaspreviouslydescribedinproteomicexper-
iments.Cellswerewashedwithice-coldPBS,andlysedwithCelLyticMammalianLysis/
ExtractionReagent,supplementedwithSIGMAFAST™ProteaseInhibitorTablets(Sigma–
Aldrich,MO).Extractsweremaintainedwithconstantagitationfor30minat4˚Candthen
centrifugedfor20minat17,000g.Supernatantswerecollectedandusedtodeterminetotal
proteinconcentrationusingtheBradfordQuick-StartProteinAssay(Bio-RadLaboratories,
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ProteinprofileofhypothalamicGT1-7celllineafterAASexposure
Hercules,CA).Allprocedureswereperformedat4˚Candsampleswerestoredat-80˚Cfor
furtherproteinextractionandwesternblotting.
Westernblotting. Westernblottingwasperformedaspreviouslydescribed[37],with
somemodifications.Equalamountsofwholeproteinextractsweresuspendedin6xLaemmli
SampleBuffer,heated,loaded,andthenelectrophoresedonPre-CastedTGX-SDSgels(Bio-
Rad,CA).GelsweretransferredtonitrocellulosemembranesusingtheBio-RadTurboTrans-
Blotapparatus.Membraneswereblockedwith5%(w/v)non-fatmilkinTBST[25mMTris–
HCl,pH7.4,150mMNaCland0.1%(v/v)Tween-20]overnightat4˚Candtheappropriate
primaryantibodieswereaddedovernightat4˚C.MembraneswerethenwashedwithTBST
andprobedwiththecorrespondinghorseradishperoxidase-conjugated(HRP)secondaryanti-
bodiesat22˚Cfor1h.Blotswerevisualizedusinganenhancedchemiluminescencekit(Super-
SignalFemto,Pierce,IL)andimageswereobtainedusingaVersaDoc1000system(Bio-Rad,
CA).AllWesternblotswereperformedatleastintriplicatefromthreeindependentexperi-
ments,anddensitometryanalyseswerenormalizedtoβ-actinexpressionusingNIHImageJ
(v1.47d).DatafromWesternblotsispresentedasmean±standarderrorofthemean(S.E.M.).
Student’st-testanalyseswereemployedandstatisticalsignificancewasestablishedas
(cid:3)P(cid:20)0.05;(cid:3)(cid:3)P(cid:20)0.01.
Antibodies. Thefollowingprimaryantibodieswereused:mouseanti-ERH(1:1000),
mouseanti-PDIA6/ERP(1:1000),rabbitanti-ERα(1:1500),goatanti-AR(1:1000)andrabbit
anti-GnRH1(1:1000)fromSantaCruzBiotechnology(SCBT),Dallas,TX;rabbitanti-GSTM1
(1:2000)fromThermoScientific,Waltham,MA;rabbitanti-PEBP1/RKIP(1:1000),rabbit
anti-GAPDH(G3P)-HRPconjugated(1:3000),rabbitanti-p-ERK(1:10000),rabbitanti-ERK
(1:6000),rabbitanti-p-p38MAPK(Thr180/Tyr182)(1:10000),rabbitanti-AKT(pan)
(1:15000)andrabbitanti-β-actinHRPconjugated(1:3000)fromCellSignalingTechnology
(CST)Danvers,MA.
Results
ProteomicchangesinGT1-7cellsafterAAStreatment
ToinvestigatetheeffectofasupraphysiologicaldoseofAASintheproteomeofGT1-7cells,
weused2D-DIGEanalysesinextractsfromvehicleand17α-meTtreatedcells.Beforeexamin-
ingtheproteome,wefirstdemonstratedthat17α-meTat1μMdidnotaffectcellviabilityat48
h(Control:92.07±0.012;17α-meT:92.82±0.013;p=0.703,unpairedt-test).Likewise,there
werenoAAS-inducedapoptosisasindicatedbycaspaseactivityassays.Valuesofrelativelumi-
nescenceunitsforcaspase3were:Control:6.5X106±2.99X105;17α-meT:6.99X106±1.29
X105;t =1.527;p=0.177;unpairedt-test.Valuesofrelativefluorescenceunitsforcaspase9
6
were:Control:468.21±31.54;17α-meT:464.61±22.02;t =0.097;p=0.926,unpairedt-test.
5
Atotalof6,045proteinspotsweredetectedautomaticallyinthemaster2Dgelimage,of
which84proteinspotsweredifferentiallyexpressedatapvalue<0.05.Fromthose84spots,
only23weredistinguishableonthepreparativegelandpickedforproteinidentificationby
LC-MS/MS.Foreasyvisualization,whitearrowsinFig1Ashowthose23spotsfromarepre-
sentativeanalyticalgel.AfterDeCyderanalysis,spotpeaksweregenerated.Fig1Band1C
showexamplesofoverexpressed(spot2674)andunderexpressed(spot5747)protein-contain-
ingspots,respectively,duringvehicleorAAStreatment.
Tables1and2showoverexpressedandunderexpressedproteinspotsandtheircorrespon-
dentproteinsthatweredifferentiallyregulatedaftertreatmentwithAAS.Fromthose23recog-
nizedspots,17differentproteinswereidentified.Twotofourproteinsweredetectedper
proteinspotcollected.TopproteinhitswiththebestMascotscoreswereselectedforvalidation
ashavingmajorcontributiontoDIGEabundancedata.UniProtdatabase(www.uniprot.org)
PLOSONE|https://doi.org/10.1371/journal.pone.0180409 July18,2017 6/22
ProteinprofileofhypothalamicGT1-7celllineafterAASexposure
Fig1.Representativeimagesof2D-DIGEanalyticalgeland3Dviewofproteinspotsinvehicleand
AAS-treatedGT1-7cells.(A)Twenty-three(23)proteinspotswereidentifiedasdifferentiallyexpressedin
GT1-7cellsthatwereexposedtotheAAS,17α-meT(1μM).ProteinsampleswerelabeledwithCy5(red),
whereasthosefromtheinternalstandardwerelabeledwithCy3(green).Aftermerging,proteinspots
exhibitingnochangesappearyellowinDIGEimages,underexpressedproteinsappearingreen,and
overexpressedproteinsareinred.WhitenumbersandarrowscorrespondtotheidentifiedproteinsinTables
1and2.(B-C)Panelsdepicttheproteinspots’abundanceof(B)overexpressedand(C)underexpressed
proteinspotsanalyzedbyDeCydersoftware.Purplelinesencirclepeaksrepresentingproteinspotsfrom
AAS-treatedsamples(leftpanels)orcontrolvehicle(centerpanels).Proteinabundancewascalculatedfrom
normalizedspotvolume,standardizedagainstthein-gelstandardofeachgel(rightpanels).
https://doi.org/10.1371/journal.pone.0180409.g001
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ProteinprofileofhypothalamicGT1-7celllineafterAASexposure
Table1. OverexpressedproteinsinGT1-7cellsafterAASexposure.
Spot aAv. P-Value Gene Description/Name Accession bMW Peptides Location Function
Ratio no. kDa
4882 3.45 0.026 PPIA Peptidyl-prolylcis-trans P17742 18 9 Cytoplasm Proteinmodification
isomeraseA andbinding
2832 1.88 0.02 SUCB2 Succinyl-CoAligase[GDP- Q9Z2I8 47 4 Mitochondrion Metabolism(Krebs
forming]subunitbeta, cycle)
mitochondrial
3742 1.51 0.043 GSTM1 GlutathioneS-transferaseMu1 P10649 26 3 Cytoplasm Cellulardetoxification
3742 1.51 0.043 GSTM2 GlutathioneS-transferaseMu2 P15626 26 7 Cytoplasm Cellulardetoxification
2729 1.45 0.045 G3P Glyceraldehyde-3-phosphate P16858 36 10,12 Cytoplasm Metabolism
2674 1.21 0.035 (GAPDH) dehydrogenase (Glycolysis)
3925 1.42 0.047 PGAM1 Phosphoglyceratemutase1 Q9DBJ1 29 8,7 Cytoplasm Metabolism
3767 1.26 0.015 (carbohydrate
metabolism)
2002 1.42 0.045 STIP1 Stress-induced-phosphoprotein1 Q60864 63 20 Cytoplasm/Nucleus Stressresponse
2546 1.41 0.023 PDIA6 Proteindisulfide-isomeraseA6 Q922R8 48 11 Endoplasmic Proteinmodification
(ERP) reticulum/cell andbinding
membrane
1613 1.37 0.014 HSP90B HeatshockproteinHSP90-beta P11499 83 26,18 Cytoplasm Stressresponse
1804 1.23 0.047
1406 1.32 0.049 FLNA Filamin-A Q8BTM8 280 16 Cytoplasm Cellmotility
4371 1.32 0.031 COF1 Cofilin-1 P18760 18 5 Cytoplasm/cell Cellcycle/cellmotility
(CFL) membrane/nucleus
2585 1.27 0.013 ENOA Alpha-enolase P17182 47 14,21, Cytoplasm/cell Metabolism
2589 1.18 0.005 (ENO1) 23 membrane (carbohydrate
2622 1.18 0.009 metabolism)
aAverage(Av)ratiorepresentsthemeanfromfour(4)differentgelsforbothAAS-treatedandcontrolGT1-7cells.
bMolecularweight
https://doi.org/10.1371/journal.pone.0180409.t001
Table2. UnderexpressedproteinsinGT1-7cellsafterAASexposure.
Spot aAv. P-Value Gene Description/Name Accession bMW Peptides Location Function
Ratio no. kDa
5747 -1.39 0.031 ERH Enhancerofrudimentaryhomolog P84089 12 5 Cellmembrane Cellcycle/RNA
binding
1478 -1.50 0.0014 FLNA Filamin-A Q8BTM8 280 11 Cytoplasm Cellmotility
4713 -1.56 0.023 PPIA Peptidyl-prolylcis-transisomeraseA P17742 18 4 Cytoplasm Proteinmodification
andbinding
1260 -1.65 0.03 VINC Vinculin Q64727 117 21 Cytoplasm/cell Cellmotility
(VCL) membrane
5764 -2.07 0.031 CX6A1 Cytochromecoxidasesubunit6A1, P43024 12 4 Mitochondrion Metabolism/electron
mitochondrial transportchange
4213 -2.28 0.0021 VPS28 Vacuolarproteinsorting-associated Q9D1C8 25 3 Cellmembrane/ Proteintransport
protein28homolog Cytoplasm
4310 -2.32 0.039 PEBP1 Phosphatidylethanolamine-binding P70296 21 5 Cytoplasm Nucleotidebinding/
protein1 Proteaseinhibitor
aAverage(Av)ratiorepresentsthemeanfromfour(4)differentgelsforbothAAS-treatedandcontrolGT1-7cells.
bMolecularweight
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ProteinprofileofhypothalamicGT1-7celllineafterAASexposure
Fig2.CellularfunctionsandlocationsofdifferentiallyexpressedproteinsinGT1-7cellsafterAAS
exposure.(A)Atotalof8biologicalprocesseswiththeirrespectivecellularlocations(B)werecategorized
from17differentproteinsidentifiedbymassspectrometry.DatawereobtainedfromUniprotdatabase(http://
www.uniprot.org)andshownaspercentagesfromthetotalnumberofproteins.
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revealedthatdifferentiallyexpressedproteinsafterAAStreatmentarerelatedtocellular
metabolism,cellcycle,cellmotility,stressresponse,drugdetoxification,aswellasproteinand
nucleotideprocessingorbinding(Fig2A).Subcellularlocationandbiologicalfunction/pro-
cessanalysisrevealedthatthemajorityoftheidentifiedproteinswerelocalizedinthecyto-
plasm,cellmembraneandnucleus(Fig2B).
Overexpressedproteinsinthecategoryofglucosemetabolismwere:succinyl-CoAligase
[GDP-forming]subunitbeta,mitochondrial(SUCB2),glyceraldehyde-3-phosphatedehydro-
genase(GAPDH/G3P),phosphoglyceratemutase1(PGAM1),andalpha-enolase(ENOA).In
thissamecategory,cytochromecoxidasesubunit6A1,mitochondrial(CX6A1)wasunderex-
pressed.Inthecategoryofcellmotility,cofilin-1(COF1)andvinculin(VCL)werefoundover-
expressedandunderexpressed,respectively.Inproteinsassociatedtocellcycleregulation,
enhancerofrudimentaryhomolog(ERH)wasunderexpressed,whileCOF1wasalsoassoci-
atedwiththiscategory.Otheroverexpressedproteinsforthecellularstressresponse,were
stress-induced-phosphoprotein1(STIP1)andheatshockproteinHSP90-beta(HSP90B).Sim-
ilaroverexpressionpatternswereobtainedforglutathioneS-transferaseMu1&2(GSTM1&
2),twokeyproteinsassociatedwithdetoxificationprocesses.Ontheotherhand,overexpres-
sionofdisulfide-isomeraseA6(PDIA6),aproteinrelatedtoproteinmodificationandbinding,
wasalsoobserved.Inthecategoryoftransport-relatedproteins,vacuolarproteinsorting-asso-
ciatedprotein28homolog(VPS28)wasfoundunderexpressed.Finally,ERH,aproteinlinked
toRNAbindingandcellcycle,andphosphatidylethanolamine-bindingprotein1(PEBP1),a
regulatoryproteinofnucleotidebinding,werefoundunderexpressed.Sixproteins,FLNA,
HSP90B,ENOA,G3P,PGAMandPPIAwererepresentedinmultiplespotsandwerealso
emphasizedinTables1and2.Fromthe23identifiedproteins,onlytwoofthemwerefound
bothoverexpressedandunderexpressedindifferentspots.Theseproteinswerefilamin
(FLNA)andpeptidyl-prolylcis-transisomeraseA(PPIA),inthecategoriesofcellmotility
andproteinmodificationandbinding,respectively.
Pathwayanalysis
UsingtheIngenuity1PathwayAnalysissoftware(IPA),weprovidedinsightsintoAAS-
inducedproteinexpressionchangesintheGT1-7cellline.Thesenetworksarerankedby
scores,andarebasedonthenumberoffocusproteinsandthesizeofthenetwork.Scoresof10
orhigher(negativelogofthePvalue)havehighconfidence,avoidingrandomeffects[36].The
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ProteinprofileofhypothalamicGT1-7celllineafterAASexposure
Fig3.NetworkofproteinsmodulatedbyAASinGT1-7cells.IngenuityPathwayAnalysis(IPA)identified
amajorproteinnetworkassociatedwithdrugmetabolism,glutathionedepletion,proteinsynthesis,
immunologicaldiseaseandendocrinefunction.Redandgreensymbolsindicateoverexpressedand
underexpressedproteins,respectively.GSTM2isrepresentedasGSTM5intheIPAknowledgebase.Direct
interactionsarerepresentedassolidlines,whereasindirectinteractionsappearasdottedlines.
https://doi.org/10.1371/journal.pone.0180409.g003
pathwayanalysisidentifiedonlyonenetworkwithahighscoreof35,anditwasassociatedto
drugmetabolism,glutathionedepletion,proteinsynthesis,immunologicaldisease,andendo-
crinesystemdevelopmentandfunction.Thenetworkincorporated13focusproteinsoutof
the17identifiedproteinsmodulatedby17α-meT(Fig3).Thecannonicalsignalingpathway
showedthehighestsignificanceandnumberoffocusproteinsforglycolysis(3proteins),glu-
coneogenesis(3proteins),arylhydrocarbonreceptor(AHRsignaling;3proteins),glutathione
mediateddetoxification(2proteins)andNRF2-meditaitedoxidativestressresponse(3pro-
teins).OthersignalingpathwaysassociatedwithAAS-inducedproteinmodulationareshown
inTable3.
Confirmationofproteomicchangesbywesternblotting
InordertovalidateproteinsthatweredifferentiallyexpressedinGT1-7cellsbyAAS,weper-
formedwesternblottingoncelllysatesforseveralproteinsindifferentcategories.Wefound
thatallproteinstestedbyWesternblotswereexpressedinGT1-7cells.Thesewere:GSTM1,
GAPDH/G3P,ERH,PEBP1,andPDIA6.Thereafter,weperformedWesternblotstoGT1-7
celllysatesinordertodeterminetheexpressionoftheseproteinsaftersteroidtreatment.We
confirmedthatsimilartotheproteomicresults,treatmentwith17α-meTincreasedthe
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