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Transforming growth factor beta1 targets estrogen receptor signaling in bronchial epithelial cells

Material Information

Title:
Transforming growth factor beta1 targets estrogen receptor signaling in bronchial epithelial cells
Creator:
L. Cody Smith
Santiago Moreno
Lauren Robertson
Sarah Robinson
Kristal Gant
Andrew J. Bryant
Tara Sabo-Attwood
Publisher:
BMC, Respiratory Research
Publication Date:
Language:
English

Subjects

Subjects / Keywords:
Transforming growth factor beta ( fast )
Estrogen ( fast )
Estrogen receptor ( fast )
Lung ( fast )
Fibrosis ( fast )

Notes

Abstract:
Background: Sex differences in idiopathic pulmonary fibrosis (IPF) suggest a protective role for estrogen (E2); however, mechanistic studies in animal models have produced mixed results. Reports using cell lines have investigated molecular interactions between transforming growth factor beta1 (TGF-β1) and estrogen receptor (ESR) pathways in breast, prostate, and skin cells, but no such interactions have been described in human lung cells. To address this gap in the literature, we investigated a role for E2 in modulating TGF-β1-induced signaling mechanisms and identified novel pathways impacted by estrogen in bronchial epithelial cells. Methods: We investigated a role for E2 in modulating TGF-β1-induced epithelial to mesenchymal transition (EMT) in bronchial epithelial cells (BEAS-2Bs) and characterized the effect of TGF-β1 on ESR mRNA and protein expression in BEAS-2Bs. We also quantified mRNA expression of ESRs in lung tissue from individuals with IPF and identified potential downstream targets of E2 signaling in BEAS-2Bs using RNA-Seq and gene set enrichment analysis. Results: E2 negligibly modulated TGF-β1-induced EMT; however, we report the novel observation that TGF-β1 repressed ESR expression, most notably estrogen receptor alpha (ESR1). Results of the RNA-Seq analysis showed that TGF-β1 and E2 inversely modulated the expression of several genes involved in processes such as extracellular matrix (ECM) turnover, airway smooth muscle cell contraction, and calcium flux regulation. We also report that E2 specifically modulated the expression of genes involved in chromatin remodeling pathways and that this regulation was absent in the presence of TGF-β1. Conclusions: Collectively, these results suggest that E2 influences unexplored pathways that may be relevant to pulmonary disease and highlights potential roles for E2 in the lung that may contribute to sex-specific differences. Keywords: Transforming growth factor beta1, Estrogen, Estrogen receptor, Lung, Fibrosis
General Note:
Smith et al. Respiratory Research (2018) 19:160 https://doi.org/10.1186/s12931-018-0861-5; Pages 1-17

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University of Florida
Holding Location:
University of Florida
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© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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mods:abstract displayLabel Abstract Background: Sex differences in idiopathic pulmonary fibrosis (IPF) suggest a protective role for estrogen (E2);
however, mechanistic studies in animal models have produced mixed results. Reports using cell lines have
investigated molecular interactions between transforming growth factor beta1 (TGF-1) and estrogen receptor (ESR)
pathways in breast, prostate, and skin cells, but no such interactions have been described in human lung cells. To
address this gap in the literature, we investigated a role for E2 in modulating TGF-1-induced signaling
mechanisms and identified novel pathways impacted by estrogen in bronchial epithelial cells.
Methods: We investigated a role for E2 in modulating TGF-1-induced epithelial to mesenchymal transition (EMT)
in bronchial epithelial cells (BEAS-2Bs) and characterized the effect of TGF-1 on ESR mRNA and protein expression
in BEAS-2Bs. We also quantified mRNA expression of ESRs in lung tissue from individuals with IPF and identified
potential downstream targets of E2 signaling in BEAS-2Bs using RNA-Seq and gene set enrichment analysis.
Results: E2 negligibly modulated TGF-1-induced EMT; however, we report the novel observation that TGF-1
repressed ESR expression, most notably estrogen receptor alpha (ESR1). Results of the RNA-Seq analysis showed
that TGF-1 and E2 inversely modulated the expression of several genes involved in processes such as extracellular
matrix (ECM) turnover, airway smooth muscle cell contraction, and calcium flux regulation. We also report that E2
specifically modulated the expression of genes involved in chromatin remodeling pathways and that this regulation
was absent in the presence of TGF-1.
Conclusions: Collectively, these results suggest that E2 influences unexplored pathways that may be relevant to
pulmonary disease and highlights potential roles for E2 in the lung that may contribute to sex-specific differences.
Keywords: Transforming growth factor beta1, Estrogen, Estrogen receptor, Lung, Fibrosis
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Santiago Moreno
Lauren Robertson
Sarah Robinson
Kristal Gant
Andrew J. Bryant
Tara Sabo-Attwood
mods:note Smith et al. Respiratory Research (2018) 19:160
https://doi.org/10.1186/s12931-018-0861-5; Pages 1-17
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mods:topic Transforming growth factor beta
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Estrogen
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Estrogen receptor
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Lung
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Fibrosis
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RESEARCHOpenAccess Transforminggrowthfactorbeta1targetsestrogenreceptorsignalinginbronchialepithelialcellsL.CodySmith1,2,SantiagoMoreno2,LaurenRobertson2,3,SarahRobinson2,3,KristalGant2,3,AndrewJ.Bryant4andTaraSabo-Attwood2,3* AbstractBackground:Sexdifferencesinidiopathicpulmonaryfibrosis(IPF)suggestaprotectiveroleforestrogen(E2);however,mechanisticstudiesinanimalmodelshaveproducedmixedresults.Reportsusingcelllineshaveinvestigatedmolecularinteractionsbetweentransforminggrowthfactorbeta1(TGF-1)andestrogenreceptor(ESR)pathwaysinbreast,prostate,andskincells,butnosuchinteractionshavebeendescribedinhumanlungcells.Toaddressthisgapintheliterature,weinvestigatedaroleforE2inmodulatingTGF-1-inducedsignalingmechanismsandidentifiednovelpathwaysimpactedbyestrogeninbronchialepithelialcells.Methods:WeinvestigatedaroleforE2inmodulatingTGF-1-inducedepithelialtomesenchymaltransition(EMT)inbronchialepithelialcells(BEAS-2Bs)andcharacterizedtheeffectofTGF-1onESRmRNAandproteinexpressioninBEAS-2Bs.WealsoquantifiedmRNAexpressionofESRsinlungtissuefromindividualswithIPFandidentifiedpotentialdownstreamtargetsofE2signalinginBEAS-2BsusingRNA-Seqandgenesetenrichmentanalysis.Results:E2negligiblymodulatedTGF-1-inducedEMT;however,wereportthenovelobservationthatTGF-1repressedESRexpression,mostnotablyestrogenreceptoralpha(ESR1).ResultsoftheRNA-SeqanalysisshowedthatTGF-1andE2inverselymodulatedtheexpressionofseveralgenesinvolvedinprocessessuchasextracellularmatrix(ECM)turnover,airwaysmoothmusclecellcontraction,andcalciumfluxregulation.WealsoreportthatE2specificallymodulatedtheexpressionofgenesinvolvedinchromatinremodelingpathwaysandthatthisregulationwasabsentinthepresenceofTGF-1.Conclusions:Collectively,theseresultssuggestthatE2influencesunexploredpathwaysthatmayberelevanttopulmonarydiseaseandhighlightspotentialrolesforE2inthelungthatmaycontributetosex-specificdifferences.Keywords:Transforminggrowthfactorbeta1,Estrogen,Estrogenreceptor,Lung,FibrosisBackgroundEpidemiologicalstudieshaveassociatedsexwithidio-pathicpulmonaryfibrosis(IPF)wheremalesaremorenegativelyimpactedandfemaleshavebettersurvivalrates[1–5].Inaddition,astudybyourgroupfoundastatisticalinteractionbetweensexandIPFseveritybasedongeneexpressionindiseasedlungtissue[6].Basedontheseobservations,severalhypothesessuggestaroleforestrogensandandrogens.Sex-basedstudiesofpulmonaryfibrosisinsmallanimalmodelshaveproducedmixedresults.Severalreportssuggestthatestrogen(E2)isprotectiveandandrogensexacerbatefi-broticresponses.Forexample,bleomycincausedincreasedpulmonaryfibrosisinmalemicecomparedtofemales[7,8],andarecentstudyfoundthatitwasthepresenceoftheYchromosomeandnotnecessarilysexitselfthatpredisposedthelungtoincreasedbleomycin-inducedfibrosisinmaleandfemalemice[9].Conversely,astudyinratsindicatedthatfemalesexhibitedincreasedpulmonaryfibrosisinre-sponsetobleomycincomparedtomales[10].Importantly, *Correspondence:sabo@phhp.ufl.edu2CenterforEnvironmentalandHumanToxicology,UniversityofFlorida,Gainesville,FL,USA3DepartmentofEnvironmentalandGlobalHealth,CenterforEnvironmentalandHumanToxicology,UniversityofFlorida,Box110885,2187MowryRd,Gainesville,FL32611,USAFulllistofauthorinformationisavailableattheendofthearticle TheAuthor(s).2018OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.Smithetal.RespiratoryResearch (2018) 19:160 https://doi.org/10.1186/s12931-018-0861-5

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variableresponsestobleomycininmicemaybeduetodif-ferentialactivityofbleomycinhydrolasebetweenmaleandfemales[11].Conversely,E2wasprotectiveinovariecto-mizedmicewhereasignificantincreaseintotallungcolla-gencontentandairwaysubepithelialcollagendepositionwasobservedinovariectomizedmicewhichwasmitigatedbyE2administration[12].Currentreportshavenotprobedthemolecularmecha-nismsimpactedbyhormonesinthelungcontributingtosex-baseddifferencesinfibroticdiseaseexceptonestudywhichfoundincreasedTGF-1expressioninratfibroblastsexposedtoE2[10].RegulationofTGF-1byE2hasbeenextensivelycharacterizedinothermodelsystemsandtheef-fectsappeartobecontextual.Forexample,E2inhibitedTGF-1signalinginbreastcancercellsbyreducingtheexpressionofactivatorsofTGF-1[13]andbyincreasingdegradationofSMADproteins[14,15].Conversely,E2in-creasedTGF-1secretionindermalfibroblasts[16],andTGF-1levelswerereducedinthekidneysofdiabeticfe-malemicelackingestrogenreceptoralpha(ESR1)comparedtowildtypemicesuggestingpositiveregulationofTGF-1byE2[17].E2bindsandactivatesseveralreceptorsincludingthenu-cleartranscriptionfactorsestrogenreceptoralpha(ESR1),estrogenreceptorbeta(ESR2),andseveralvariantsthereof,andtheputativemembrane-boundG-proteincoupledestro-genreceptor1(GPER1).Studiesaimingtodecipherreceptor-specificeffectsonTGF-1signalinghavebeenlim-itedtonon-lungcellsandtissues(i.e.breast).Forexample,Stopeetal.foundthatESR1butnotESR2inhibitedTGF-1activationinbreastcancercells[18]whileothersfoundthatbothESR1andESR2suppressedTGF-1signalingbyasso-ciatingwithandactingasatranscriptionalcorepressorforSMAD3[19,20].OtherstudiesshowedaroleforGPER1inmediatingE2-dependentreductioninSMADproteinactiva-tioninbreastcancercells[21]andTGF-1-inducedextra-cellularmatrix(ECM)productioninhumanandratmesangialcells[22].GiventhepreponderanceofopposingactionsofE2onTGF-1ininvitrosystemsotherthanthelungandtheepidemiologicalevidencesuggestingamalesex-biasinIPF,wehypothesizedthatE2wouldinhibitTGF-1-inducedsignalinginlungepithelialcells.Totestthishypothesis,weinvestigatedtheimpactofE2onTGF-1-inducedepithelialtomesenchymaltransition(EMT),characterizedtheex-pressionofESRsinbronchialepithelialcellsandlungtissuefromindividualswithIPF,andperformedRNA-SeqanalysistoidentifytargetsofE2inbronchialepithelialcells.MethodsChemicalsRecombinanthumantransforminggrowthfactorbeta1(TGF-1)waspurchasedfromR&DSystems(>97%purity,CatalogNo.240-B002,Minneapolis,MN)and17-Estradiol(E2)waspurchasedfromSigma(98%purity,CatalogNo.E2758,SaintLouis,MO).HumanlungsamplesThehumanlungtissuesamplesusedinthisstudywereakindgiftfromDr.AndrewBryant.Theexplantedlungtis-suewasobtainedfromsubjectsundergoinglungtransplantforIPFandfromlungsrejectedfortransplantfromnormalcontrolspertheNationalInstitutesofHealthLungTissueResearchConsortium(protocolno.14-99-0011).Thisstudyconsistedoffifteenpatients,eightwithIPF(n=8)andsevencontrols(n=7)describedindetailinTable1.Adiag-nosisofIPFwasdeterminedbasedonATScriteria[23,24].Theprotocolforcollectionoflungtissuesamplesandsub-sequentstudieswereapprovedbytheinstitutionalreviewboardatVanderbiltUniversityandtheUniversityofFlorida[25].Immediatelyafterlungbiopsyorresection,aportionofthelungwasremovedandflash-frozeninliquidnitrogenordryiceandstoredatŠ80C.CellcultureHumanbronchialepithelialcells(BEAS-2Bs,CRL-9609™)werepurchasedfromATCCandculturedaccordingtomanufacturer’sspecifications.STRanalysisandmycoplasmacontaminationtestingwasnotperformed.Cellswerecul-turedinbronchialepithelialgrowthmedium(BEGM)con-sistingofbronchialepithelialbasalmedium(BEBM,LonzaCC-3171,Walkersville,MD)andtheBEGMSingleQuotKitSupplements&GrowthFactors(LonzaCC-4175)butex-changinggentamicinforPenicillin-Streptomycin-Neomycin(PSN)AntibioticMixture(Gibco15,640,ThermoFisherSci-entificInc.,Waltham,MA).CellswereculturedinT75Corning™U-ShapedCellCultureFlasks(Corning430,641U,FisherScientificCoLLC,Pittsburgh,PA)coatedwithamatrix(4.5mLper75cm2)consistingof0.01mg/mLfibro-nectin(AkronAK8350,City,State),0.03mg/mLbovinecollagen(GibcoA10644–01),and0.01mg/mLBSA(FisherBP1605)inBEBM.Cellsweresub-culturedupto11timesbeforeuseinexposurestudies.Allexposureswereper-formedinBEGMwithoutthesuppliedbovinepituitaryextract(BPE)aliquotbecauseitscompositionisnotdefined.Forthegeneexpressionexperiments,BEAS-2Bswereplatedat40,000cells/mLonmatrix-coated12-wellNunc™ Table1PatientdemographicdataCharacteristicsControlsPatientsMale/Female2/38/0Agea6414.7861.57.11SmokingStatusNever46Ever12FVC,%predicteda105.27.7942.914.39aMeanSDSmithetal.RespiratoryResearch (2018) 19:160 Page2of17

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Cell-Culture-TreatedMultidishes(ThermoFisherScientificInc.),allowedtoadhereovernight,andsubsequentlyexposedfor48htotheindicatedconcentrationsofTGF-1dissolvedin0.1%BSA(FisherBP1605)and4mMHClor10nME2dissolvedinDMSO(MediatechInc.MT25950CQC).DosesofTGF-1andE2werechosenbasedonefficacyinpreviousexperiments[26–30].Fortheproteinexpressionexperi-ments,BEAS-2Bswereplatedat60,000cells/mLonmatrix-coated6-wellCorningCostarcellcultureplates(Costar3516),allowedtoadhereovernight,andsubse-quentlyexposedfor48htotheindicatedconcentrationsofTGF-1inBEBMwithoutBPE.Allchemicalsolventcon-centrationsweremaintainedbelow0.1%.TotalRNAextractionandpurificationFrozenwholelungsampleswerepulverizedoverliquidnitrogenusingamortarandpestlethenmechanicallydis-ruptedinRNASTAT-60™Reagent(Tel-Test,Inc.Cs-502,Friendswood,TX)usingahandheldhomogenizer,andcelllysateswerecollectedinRNASTAT-60™Reagentandvor-texedtopromotelysis.RNAwasextractedpermanufac-turer’sspecificationsfollowedbyovernightprecipitationatŠ20Cusing100%molecularbiologygradeisopropanol(FisherBioReagents™BP26184)containing0.067%v/vGly-coBlue™Coprecipitant(AmbionAM9515).PrecipitatedRNAwaspelletedbycentrifugationat14000xgfor45minat4Candpurifiedbywashing2Xwith75%mo-lecularbiologygradeabsoluteethanol(FisherBioReagents™BP28184).RNApelletswerereconstitutedin15LRNAse-cure™(AmbionAM7010).RNAwasquantifiedusingaSynergy™H1platereader(BioTekInstrument,Inc.,Winoo-ski,VT)andRNAintegritywasanalyzedusingaBioanaly-zer2100instrument(AgilentTechnologies,SantaClara,CA).qPCRRNA(1–2g)wasDNase-treatedusingthePerfeCTaDNaseIKit(QuantaBioSciences95,150-01k,VWRInternationalLLC,Suwanee,GA)andsubsequentlyre-versetranscribedusingtheqScript™cDNASynthesisKit(QuantaBioSciences95,047).cDNAwasdiluted1:20inRNase-DNasefreewater.Each10LqPCRreactioncontained1SsoAdvanced™UniversalSYBRGreenSupermix(Bio-Rad172–5270,Hercules,CA),850nMforwardandreverseprimers,and3.3LofthecDNAdilution.PerMIQEguidelines[31],genespecificprimersandcyclingparametersaredisplayedinTable2.ESR2primerswerepurchasedfromBio-Rad(UniqueAssayID:qHsaCID0013184).EachqPCRreactionwasfollowedbymeltcurveanalysistoverifyprimerspecificity.CqvaluesweredeterminedbyregressionmethodusingtheCFXManager2.1softwareandquantifiedusingtherela-tiveCqmethod[32]ortheratiomethod[33]whenindicated.Inthecaseofnoamplification,aCqvalueof40wasapplied.Forthecellcultureexperiments,targetgeneexpressionwasnormalizedtoglyceraldehyde3-phosphatedehydrogenase(GAPDH)expressionandtoribosomalproteinS13(RPS13)expressioninthehumanlungtissuesamples.ProteinextractionandpurificationAfterexposure,mediawasremoved,andcellswerewashed3Xwithice-coldPBSandsubsequentlycollectedin200LRIPALysisandExtractionBuffer(PierceBio-technology89,900)containingPierce™ProteaseInhibitorTablets(PierceBiotechnology88,265)usingacellscraper.Thelysateswerepassed5Xthrougha25-gaugeneedleandincubatedonicefor30minwithintermittentmixing.Thereafter,thelysateswerecentrifugedat14000xgfor20minat4C,supernatantswereremoved,andtotalproteinquantifiedusingthePierceBCAProteinAssayKit(PierceBiotechnology23,225).WesternblotForSDS-PAGE,totalprotein(15g)wasdilutedinNovex™Tris-GlycineSDSSampleBuffer(2X)(Invitro-genLC2676)andloadedontoaNovex™WedgeWell™4–12%Tris-GlycineMiniGel(InvitrogenXP04120BOX).Electrophoresiswasperformedfor30minat225Vandelectrophoresedproteinsweresubsequentlytransferredtonitrocellulosemembranes(GVSLifeSciencesEP4HY00010)bysemi-drytransfermethodunder15Vfor30min.Blotswereblockedwith5%dehydratedmilkdissolvedinTBSTfor1hatroomtemperature,thenin-cubatedwithmousemonoclonalantibodyspecificforestrogenreceptor(anti-ESR1,SantaCruzBiotechnol-ogy,Inc.SC-514857,Dallas,TX)diluted1:100inblock-ingsolutionovernightat4C.Blotswerewashed3XinTBSTfor10minandincubatedwithHRP-linkedRabbitanti-MouseIgG(H+L)SecondaryAntibody(Pierce31,450,Rockford,IL)diluted1:4000inTBSTfor1hatroomtemperature.Blotswerewashed3XwithTBSTfor10minatroomtemperature,then1XwithTBSatroomtemperature.Thereafter,blotswereincubatedwith1:1solutionofClarity™WesternECLBlottingSubstrates(Bio-Rad170–5060,Hercules,CA)andimagedusingtheauto-exposureoptiononaBio-RadChemiDoc™MPsys-tem(Bio-Rad17,001,402).Afterprobingwithanti-ESR1,blotswereincubated2Xfor10minatroomtemperatureinmildstrippingbuffer(0.1MGlycine,20mMMgAce-tate,50mMKCl,pH2.2),thenwashed3XwithTBSTfor5minatroomtemperature.Toverifyantibodystrip-ping,theblotwasprobedwithHRP-linkedsecondaryantibodyandre-imagedasbefore.Afterverificationofanti-ESR1removal,theblotwasincubatedinblockingsolutionfor1hatroomtemperature,thenincubatedwithmousemonoclonalantibodyspecificfor-Actin(anti--Actin,SigmaA5441)diluted1:5000inblockingSmithetal.RespiratoryResearch (2018) 19:160 Page3of17

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solutionovernightat4C.Theblotwassubsequentlywashedasbefore,incubatedinHRP-linkedRabbitanti-MouseIgG(H+L)SecondaryAntibody(Pierce31,450)diluted1:5000inTBSTfor1hatroomtemperature,andimagedasbefore.DensitometrywasperformedinImageJ[34]usingtheGelAnalysismethodoutlinedintheImageJdocumentation.RNA-SeqlibrarypreparationandsequencingRNAlibraryconstructionwasperformedattheInterdis-ciplinaryCenterforBiotechnologyResearch(ICBR)GeneExpressionCore,UniversityofFlorida(UF).RNAconcen-trationwasdeterminedonQubit2.0Fluorometer(Ther-moFisher/Invitrogen,GrandIsland,NY),RNAqualitywasassessedusingtheAgilent2100Bioanalyzer.TotalRNAwith28S/18S>1andRNAintegritynumber(RIN)7wasusedforRNA-seqlibraryconstruction.TheRINsofalltotalRNAsampleswere9.7–10.2Lof1:2000dilutedExternalRNAControlsConsortium(ERCC)Spike-InMix(Ambion™4,456,740,ThermoFisherScientificInc.)wasaddedto100ngofhigh-qualitytotalRNAfollowedbymRNAisolationusingNEBNextPoly(A)mRNAMagneticIsolationmodule(NewEnglandBiolabsE7490,Ipswich,MA)andRNAlibraryconstructionwithNEBNextUltraRNALibraryPrepKitforIllumina(NewEnglandBiolabsE7530)accordingtothemanufacturer’suserguide.Specif-ically,100ngoftotalRNAtogetherwith2Lof1:2000dilutedERCCwasaddedtoextractedmRNAwith15LofNEBNextMagneticOligod(T)25andfragmentedinNEBNextFirstStrandSynthesisBufferbyheatingat94Cfor8min,followedbyfirst-strandcDNAsynthesisusingreversetranscriptaseandrandomprimers.SynthesisofdscDNAwasdoneusingthe2ndstrandmastermixpro-videdinthekit.Theresultingdouble-strandedcDNAwasend-repaired,dA-tailedandligatedwithNEBNextadap-tors.Finally,librarywasenrichedby13cyclesofamplifi-cation,andpurifiedbyMeg-BindRxnPurePlusbeads(OmegaBiotekM1386,Norcross,GA).Bar-codedlibrariesweresizedonthebioanalyzer,quantitatedbyQUBITandqPCR(KapaBiosystemsKK4824,Wilmington,MA).Seventeenindividuallibrarieswerepooledatequalmolar(20nM).Bar-codedcDNAwassequencedusingthe2100configurationin2lanesofaHiSeq3000instrument(Illumina,SanDiego,CA).Theyieldfortherunwasintheexpectedrange,thequalitywasgoodwithQ30>96.25%,andthepoolwaswell-balanced(intermsofnumberofreadspersamples).BioinformaticsShortreadsweretrimmedandfilteredtoremovelow-qualityreadsusingTrimmomaticversion0.36.QualitycontrolwasassessedusingtheFastQCtool,version0.11.4.ShortreadswerealignedtothetranscriptomeusingSTARversion2.5.2a.Transcriptquantificationanddifferentialana-lysiswasperformedusingRSEMversion1.2.31.Differentialanalysiswasperformedatthelevelofcodinggenes,alltran-script,andallsplicingisoforms.Codinggenes,transcripts,andsplicingisoformswereconsideredstatisticallysignificantifFDR-correctedp-value5%andfoldchange>1.5ineitherdirection.Clusteringanalysiswasperformedusingthe‘gplots’packageinRversion3.3.2(2016-10-31).Geneseten-richmentanalysiswasperformedusingPathwayStudioVersion11.4.0.8operatingontheResNetMammaliandatabase(Elsevier).Statisticallysignificantenrichment(p0.05)ofpredefinedgenesetswasdeterminedbyMann-WhitneyU-test.ThedatadiscussedinthispublicationhavebeendepositedinNCBI’sGeneExpres-sionOmnibus[35]andareaccessiblethroughGEOSeriesaccessionnumberGSE100574(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE100574).StatisticsNormalityofexperimentaldatawasdeterminedbyD’Agostino&Pearsonomnibusnormalitytest,Shapiro-Wilknormalitytest,orKSnormalitytestusing Table2PrimerinformationforqPCRGeneForward(5-3)Reverse(5-3)ProtocolEfficiencySourceGAPDHGAAGGTGAAGGTCGGAGTCGAAGATGGTGATGGGATTTC95C3m;95C10s,60C30s,4093.9%[92]ESR1CCACCAACCAGTGCACCATTGGTCTTTTCGTATCCCACCTTTC95C3m;95C10s,60C30s,40100.7%[93]ESR2ProprietaryProprietary95C3m;95C10s,60C30s,40102.9%Bio-RadGPERGCTCCCTGCAAGCAGTCTTTGAAGGTCTCCCCGAGAAAGC95C3m;95C10s,60C30s,4097.2%[94]SNAI1CCAGACCCACTCAGATGTCAAGGACTCTTGGTGCTTGTGGA95C3m;95C10s,58C30s,4097.2%[94]CDH1GAAAGCGGCTGATACTGACCCTCAGACTAGCAGCTTCGGA95C3m;95C10s,58C30s,40104.9%[94]ACTA2CATCATGCGTCTGGATCTGGGGACAATCTCACGCTCAGCA95C3m;95C10s,60C30s,4094.8%[95]MMP2TGTGTTCTTTGCAGGGAATGTCCAGAATTTGTCTCCAGCA95C3m;95C10s,58C30s,4093.6%[96]CTGFAATGCTGCGAGGAGTGGGTCGGCTCTAATCATAGTTGGGTCT95C3m;95C10s,60C30s,4096.4%[97]VIMGCGTGAAATGGAAGAGAACTGGTATCAACCAGAGGGAGTG95C3m;95C10s,56C10s,72C30S,40104.0%[30]MUC15CCATCGGCGACTTTATGACGTCTTCACTTTCTGGCATGGCT95C3m;95C10s,60C30s,4092.1%[94]Smithetal.RespiratoryResearch (2018) 19:160 Page4of17

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GraphPadPrismsoftware(Version5.01,GraphPadSoft-ware,Inc.).Dataweredeterminedtobenormalbypass-ingatleastonenormalitytest(p<0.05).ForqPCRandwesternblotanalyses,statisticallysignificantdifferences(p<0.05)inmeanfoldchangesbetweenexperimentalgroupsweredeterminedbyone-wayANOVAfollowedbyNewman-Keulsmultiplecomparisontestorunpaired,two-tailedttestwhenindicatedusingGraphPadPrismsoftware.Ifthedatawerenotnormal,statisticallysigni-ficantdifferencesweredeterminedbytwo-tailedMann-WhitneyUtest.ResultsTGF-1induceschangesconsistentwithEMTTobegininvestigations,weoptimizedawell-characterizedmodelofTGF-1-inducedEMTbasedonchangesingeneexpression.BEAS-2BswereexposedtoTGF-1(0.1,1,and5ng/mL)for48handmRNAexpressionofmolecu-larmarkersforEMTwereassayedbyqPCR.Resultsre-vealedadose-dependentresponsewhereexposureofcellsfor48hto1and5ng/mLTGF-1causedasignificantre-ductioninexpressionoftheepithelialcelltypemarker,E-cadherin(CDH1),comparedtocontrolcells(Fig.1a).Conversely,exposureofcellsto1and5ng/mLTGF-1causedasignificantincreaseinexpressionofthemesen-chymalcelltypemarkers,Vimentin(VIM),Snailfamilytranscriptionalrepressor1(SNAI1),N-cadherin(CDH2),andFibronectin(FN1)comparedtocontrolcells(Fig.1b,d-f).ExposureofcellstoTGF-1didnotaffectexpressionofthemyofibroblastcelltypemarker,alphasmoothmuscleactin(ACTA2)atanytesteddoses(Fig.1c).E2doesnotsignificantlyaffectTGF-1-inducedEMTAroleforE2inmodulatingTGF-1-inducedEMTwasassessedbyexposingBEAS-2Bcellsto5ng/mLTGF-1inthepresenceandabsenceof10nME2for48h.Thereafter,expressionofEMTmarkergeneswasassayedbyqPCR.Asexpected,5ng/mLTGF-1causedareduction(0.686-fold,p>0.05)inCDH1mRNAex-pression(Fig.2a).However,theadditionof10nME2didnotsignificantlyaffecttheTGF-1-inducedresponseorCDH1expressionindividually(Fig.2a).Exposureofcellsto5ng/mLTGF-1causedatrendofincreasedmRNAexpressionofthemesenchymalmarkers,VIM(p>0.05),SNAI1(p>0.05),CDH2,FN1(Fig.2b,d-f)andsimilartoCDH1,exposureofcellsto5ng/mLTGF-1inthepresenceof10nME2didnotresultinastatisticallysignificantdifferencefromcellsexposedtoTGF-1individually(Fig.2b,d-f).Asbefore,therewerenostatisticallysignificantdifferencesinexpressionofACTA2comparedtocontrolcellsinanyexposuregroup,howevertherewasa1.74-foldtrendofincreasedexpressionintheco-exposuregroup(Fig.2c).Statisti-callysignificantdifferencescouldnotbedeterminedbetweengroupsforCDH2andFN1becausethesedataweregeneratedfromonlytwoindependentexperiments.TGF-1reducesestrogenreceptormRNAandproteinexpressionNext,weinvestigatedwhetherestrogenreceptormRNAexpressionwaswasatargetforTGF-1.Thebaselineexpressionlevelsofestrogenreceptoralpha(ESR1),es-trogenreceptorbeta(ESR2),andg-proteincoupledes-trogenreceptor(GPER1)incontrolcellswereexpressedasaratiotoESR1basedonthemethoddescribedbyPfaffletal.[33]todeterminerelativebaselineexpressionlevels.TherelativeexpressionofeachreceptorsubtypewasGPER1>ESR1>ESR2(Fig.3a).ExposingBEAS-2BstoincreasingconcentrationsofTGF-1(0.1,1,and5ng/mL)for48hcauseda1.81-,3.11-,and2.87-fold(p<0.05)decreaseinESR1mRNAexpressioncomparedtocontrols(Fig.3b).SimilartrendswereobservedforESR2mRNAexpressioncomparedtocontrols(Fig.3c),anda1.44,1.72,and1.78-fold(p<0.05)decreaseinGPER1mRNAexpressioncomparedtocontrols(Fig.3d)wasobservedforthethreedoses,respectively.BEAS-2BswereexposedtoTGF-1(0.1,1,and5ng/mL)for48handESR1wasdetectedbywesternblottodetermineifTGF-1reducedESR1proteinlevels(Fig.4a).SignalintensitywasquantifiedbydensitometryusingImageJ(Fig.4b).SimilartothemRNAresults,TGF-1(0.1,1,and5ng/mL)causeda2.09-,2.77-,and3.76-foldsignificantdecreaseinESR1proteinlevels,re-spectively(Fig.4b).EstrogenreceptormRNAexpressionisreducedinlungsofpatientswithIPFWenextquestionedwhetherourinvitroresultstrans-latedinvivo.Toanswerthis,mRNAexpressionofESR1,ESR2,andGPER1wascomparedinlungtissuefromhealthycontrolstoindividualswithend-stageIPFgiventhatthosewithIPFtendtohavehigherTGF-1serumlevelscomparedtohealthycontrols[36].AqPCRana-lysisfoundthatESR1andGPER1mRNAexpressionwassignificantlyreducedinthelungsofpatientswithend-stageIPFcomparedtohealthycontrolswhiletherewasatrendofreducedexpressionofESR2intheformergroup(Fig.5a-c).TGF-1andE2exhibituniquetranscriptionalprofilesRNA-Seqwasperformedtoidentifytranscriptionaltar-getsofE2inbronchialepithelialcellsandcellularpro-cessesthatmaybeaffectedbytheobserveddown-regulationofESRexpressionbyTGF-1.Forthisexperiment,BEAS-2Bswereexposedtoeithervehiclecontrol,5ng/mLTGF-1for48h,10nME2for24h,orpre-exposedto5ng/mLTGF-1for24handsubse-quentlyco-exposedtoboth5ng/mLTGF-1and10nMSmithetal.RespiratoryResearch (2018) 19:160 Page5of17

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E2for24h(Fig.6a).Differentialexpressionanalysisre-sultedin2182codinggeneswithFDR-correctedp-value0.05andLog2(FoldChange)>|0.6|comparedtocon-trolsintheTGF-1group.Theexpressionof2119cod-inggeneswasalteredinthegroupco-exposedtoTGF-1andE2,and10inthegroupexposedtoE2.Insum,379,316,and6geneswerespecificallyalteredintheTGF-1,TGF-1+E2,andE2groups,respectively,while1798genesweredifferentiallyregulatedinboththeTGF-1andTGF-1+E2groups,and4werediffer-entiallyregulatedinallgroups(Fig.6b).Manyofthegenessignificantlyup-regulatedbyTGF-1,suchasCTGF,MMP2andVIM,arewell-knowntargetsofthispathway(Fig.7,Additionalfile1:TableS1).Othergenessignificantlyup-regulatedinalltreatmentgroupsin-cludedSproutyRTKsignalingantagonist4(SPRY4)and Fig.1TGF-1inducesEMT-likechangesinmRNAexpressioninBEAS-2Bcells.(a-f)BEAS-2BcellswereexposedtoTGF-1(0.1,1,and5ng/mL)for48handmRNAexpressionoftheepithelialcelltypemarker,E-cadherin(CDH1,a),themesenchymalcelltypemarkersVimentin(VIM,b),Snailfamilytranscriptionalrepressor1(SNAI1,d),Cadherin2(CDH2,e),andFibronectin(FN1,f),andthemyofibroblastcelltypemarker,Alphasmoothmuscleactin(ACTA2)wasmeasuredbyqPCR.TargetgeneexpressionwasnormalizedtoGAPDHmRNAexpressionandquantifiedasfoldchangetocontrolusingtherelativeCqmethod.DataaremeanSEMofthreeorfourindependentexperiments.Differentlettersindicatestatisticallysignificant(p<0.05)differencesbetweengroupsasdeterminedbyone-wayANOVAandNewman-KeulsmultiplecomparisontestSmithetal.RespiratoryResearch (2018) 19:160 Page6of17

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Dualspecificityphosphatase6(DUSP6),andsignifi-cantlydown-regulatedgenesincludedPotassiumvoltage-gatedchannelsubfamilyQmember1(KCNQ1)andRASproteinactivatorlike1(RASAL1)(Additionalfile1:TablesS1-S2,Table3).GenesthatwerespecificallyregulatedbyE2includedRetinolbindingprotein7[RBP7,Log2(FoldChange)=Š1.65]andChlorideintra-cellularchannel3[CLIC3,Log2(FoldChange)=Š0.73](Table3).AhierarchicalclusteringanalysisofgenesdifferentiallyregulatedinatleastoneexposuregroupshowedthattheexpressionprofilesoftheTGF-1andTGF-1+E2groupweremoresimilartoeachotherthantotheexpressionprofileofE2(Fig.6c).TheexpressionofgenesrelevanttopulmonaryfibrosisandthiscurrentworkwasvalidatedbyqPCRinaninde-pendentexperiment(Fig.7).Asexpected,exposuretoTGF-1causedasignificantreductioninESR1mRNAexpression(Fig.7a)andincreasedtheexpressionof Fig.2E2doesnotsignificantlyaffectTGF-1-inducedEMT.BEAS-2Bcellswereexposedto5ng/mLTGF-1inthepresenceorabsenceof10nME2for48h.(a-f)ExpressionofCDH1(a),VIM(b),ACTA2(c),SNAI1(d),CDH2(e),andFN1(f)mRNAwasmeasuredbyqPCR.TargetgeneexpressionwasnormalizedtoGAPDHmRNAexpressionandquantifiedasfoldchangetocontrolusingtherelativeCqmethod.DataaremeanSEMofthreeorfour(a-d)ortwo(e-f)independentexperiments.Differentlettersindicatestatisticallysignificant(p<0.05)differencesbetweengroupsasdeterminedbyone-wayANOVAandNewman-KeulsmultiplecomparisontestSmithetal.RespiratoryResearch (2018) 19:160 Page7of17

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knowntargetsofTGF-1suchasConnectivetissuegrowthfactor(CTGF,Fig.7b),VIM(Fig.7c),andMatrixmetalloproteinase2(MMP2,Fig.7d).ThepresenceofE2intheco-exposuregroupdidnothaveacleareffectontheexpressionofthesegenescomparedtotheTGF-1group.TGF-1andE2differentiallyregulategenesetsGenesetenrichmentanalysis(GSEA)[37]wasper-formedtoidentifystatisticalenrichmentintheRNA-SeqdataofcuratedpathwaysusingPathwayStudioVersion11.4.0.8(Elsevier).TheGSEAresultedindifferentialen-richmentofbiologicalfunctionanddiseasepathwaysamongtheexposuregroups.Asexpected,exposuretoTGF-1resultedinenrichmentofpathwayssuchasECMturnoverandskinfibrosis(Table4).ExposuretoTGF-1alsoresultedinstatisticalenrichmentofpath-waysincludingalveolarepithelialcelldysfunction,Ca2+fluxregulation,classicalandalternativecomplementpathways,andneutrophilchemotaxis(Table4).Inmostcases,similarenrichmentandmedianchangeswereob-servedintheco-exposuregroup(TGF-1+E2)andtheTGF-1onlyexposuregroupexceptforalveolarepithe-lialcelldysfunctionwhichwasnotstatisticallyenrichedintheco-exposuregroup(Table4).E2alsocausedenrichmentofclassicalandalternativecomplementpathways,airwaysmoothmusclecellcontraction,Ca2+fluxregulation,andECMturnover,however,themedianchangeofthelattertwopathwayswasinverse(down-regulated)comparedtothemedianchange Fig.3TGF-1down-regulatesESR1,ESR2,andGPER1mRNAexpressioninBEAS-2Bs.(a)RelativeexpressionofestrogenreceptorsubtypesincontrolcellswasGPER1>ESR1>ESR2.ESRgeneexpressionwasnormalizedtoGAPDHmRNAexpressionandcalculatedasaratiotoESR1mRNAexpression.(b-d)BEAS-2BcellswereexposedtoTGF-1(0.1,1,and5ng/mL)for48handexpressionofESR1(n=3;b),ESR2(n=2;c),andGPER1(n=3;d)mRNAwasmeasuredbyqPCR.TargetgeneexpressionwasnormalizedtoGAPDHmRNAexpressionandquantifiedasfoldchangetocontrolusingtherelativeCqmethod.DataaremeanSEManddifferentlettersindicatestatisticallysignificant(p<0.05)differencesbetweengroupsasdeterminedbyone-wayANOVAandNewman-KeulsmultiplecomparisontestSmithetal.RespiratoryResearch (2018) 19:160 Page8of17

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observedintheTGF-1andco-exposuregroups(Table4).TheECMpathwayispresentedgraphicallytohighlighttheinverseregulationofgenesinthepathwaybyTGF-1(Fig.8,top)andE2(Fig.8,bottom).E2specificallycausedstatisticalenrichmentofpathwaysincludinghistoneacetylationandphosphorylationpathways,nucleosome-remodelingfactor(NURF)inchromatinremodeling,andvasodilationactivation(Table4).DiscussionThisworkwasmotivatedbyevidencesuggestinghor-monesmayinfluencegeneregulationinthelungandcon-tributetosexdifferencesinpulmonarydiseasessuchasfibrosis.Usingawell-establishedmodellungepithelialcellline,weinvestigatedtheimpactofE2onTGF-1-inducedEMT.WereportthatalthoughTGF-1-inducedEMTwasnotsignificantlyaffectedbyE2,thismaybeduetothenovelobservationthatTGF-1repressedESRexpression,mostnotablyESR1.Weextendedthisobservationtoiden-tifynoveltargetsofE2byRNA-Seqthatmaybesuscep-tibletoTGF-1-inducedrepressionofESRssuchaschromatinremodelingprocessesandECMturnover.WefirstcharacterizedtherelativeexpressionlevelsoftheESRsinourmodelcellline.WefoundthatGPER1wasthemostabundantfollowedbyESR1whileESR2wasleastexpressed(Fig.3a).OurresultsaresimilartoastudybyStabileetal.thatfoundhigherexpressionofESR1thanESR2inhumanlungadenocarcinomasand Fig.4TGF-1down-regulatesESR1proteinexpression.BEAS-2BcellswereexposedtoTGF-1(0.1,1,and5ng/mL)for48handESR1proteinexpressionwasmeasuredbywesternblotfollowedbydensitometricanalysisinImageJ.aRepresentativewesternblot.bFoldchangeESR1proteinexpressionwasnormalizedtoBeta-actin(ACTB)andcalculatedasfoldchangetovehiclecontrol(0ng/mLTGF-1).DataaremeanSEMnormalizedarbitrarydensityunitsofduplicatemeasurementsperblotofthreeindependentexperiments.Differentlettersindicatestatisticallysignificant(p<0.05)differencesbetweengroupsasdeterminedbyone-wayANOVAandNewman-Keulsmultiplecomparisontest Fig.5EstrogenreceptormRNAexpressionisreducedinlungsofpatientswithsevereIPFcomparedtohealthycontrolsubjects.a-cGeneexpressionofESR1(a),ESR2(b),andGPER1(c)wasmeasuredinlungtissuefrompatientswithIPFandhealthycontrolsbyqPCR.TargetgeneexpressionwasnormalizedtoGAPDHmRNAexpressionandquantifiedasfoldchangetocontrolusingtherelativeCqmethod.Boxplotsrepresent5Â…95%confidenceintervalsandasterisks(*)indicatestatisticallysignificant(p<0.05)differencescomparedtocontrolsasdeterminedbytwo-tailedMann-WhitneyUtestSmithetal.RespiratoryResearch (2018) 19:160 Page9of17

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squamouscelllungtumorsalthoughadifferencebe-tweenESR1andESR2expressionwasnotevidentinnormallungcells[38].TheseresultsareincontrasttoastudybyMollerupetal.thatfoundthatESR2wasmoreabundantlyexpressedthanESR1[39]andanotherstudybyCouseetal.thatfoundgreaterexpressionofESR2inmouselung[40].Thediscrepanciesmaybearesultofvariabledetectionmethods[38–40].WeprobedaroleforE2inmodulatingTGF-1-inducedEMTbecausethisprocesshasbeenobservedinfibrosismodelsalthoughtherelativecontributiontofibrogenesisinhumansisheavilydebated[41–45].TGF-1causedasignifi-cantreductioninexpressionoftheepithelialcelltypemarkerandasignificantincreaseinexpressionofthemes-enchymalcelltypemarkers(Fig.1b,d-f)similartootherstudies[28–30].UnlikethestudybyDoermeretal.,wedidnotmeasureasignificantincreaseinexpres-sionofACTA2mRNA(Fig.1c)whichmaybearesultofthedurationofexposureasthisparticularmarkertendstobemorehighlyinducedatlatertime-points(5days)[29]andisindicativeoffurtherdifferentiationoffibroblastsintocontractilemyofi-broblasts[46].Usingthismodelsystem,wedeterminedwhetherE2af-fectedTGF-1-inducedEMT.AroleforE2ininhibitingEMTinhumanswassuggestedinastudywhichfoundthatreducedexpressionofESR1wasassociatedwithin-creasedexpressionofgenesinvolvedinEMTinendomet-rialcarcinomasamples[47].Further,EMTisatargetforsexhormonesincelltypessuchasbreastandprostatecancercellswhereE2signalingmaintainsanepithelialphenotypeandsuppressesEMT[48–51].Inouranalysis,exposuretoE2didnotsignificantlyaffectEMTmarkergeneexpressionindividuallynordiditimpactthenormalTGF-1response(Fig.2a-f).Co-exposuretoTGF-1andE2resultedinatrendofincreasedexpressionofVIMandACTA2comparedtoTGF-1alonewhichisconsistentwithonestudythatreportedE2promotedreversibleEMT-liketransitionandcollectivemotilityinbreastcan-cercells[52].E2maynotaffectTGF-1-inducedEMTduetodirectactionsofTGF-1onESRsthemselves.Interestingly,we Fig.6TGF-1andE2exhibitdistincttranscriptionalprofiles.aBEAS-2Bswereexposedto5ng/mLTGF-1and10nME2individuallyandincombination.Cellswereacclimatedfor24h,thengroups2and3wereexposedtoTGF-1.After24h,groups3and4wereexposedtoE2,andallsampleswerecollected24hthereafter.bVenndiagramhighlightingdistributionofdifferentiallyexpressedgenes[Log2(FoldChange)|0.6|andFDR-correctedp-value<0.05]amongthetreatmentgroups.cHeatmapshowingtheclusteringandrelativeexpressionlevels[Log2(FoldChange)comparedtocontrols]ofgenesthatweredifferentiallyexpressedinatleastonetreatmentgroup.Redcoloringindicatesup-regulationcomparedtocontrolsandgreencoloringindicatesdown-regulationcomparedtocontrols,(T,TGF-1;T+E,TGF-1+E2;E,E2)Smithetal.RespiratoryResearch (2018) 19:160 Page10of17

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Fig.7OrthogonalvalidationofRNA-Seqdata.a-dExpressionofselectgeneswasvalidatedbyqPCR;ESR1(a),Connectivetissuegrowthfactor(CTGF,b),VIM(c),andMatrixmetalloproteinase2(MMP2,d),inanidenticalandindependentexperiment.Barsrepresentexpression[Log2(FoldChange)]ofeachgeneintheRNA-Seqanalysis,andblackdotsrepresentexpression[Log2(FoldChange)]ineachsample(n=6)intheorthogonalexperimentasdeterminedbyqPCRrelativetovehiclecontrol(DMSO).TargetgeneexpressionasmeasuredbyqPCRwasnormalizedtoGAPDHmRNAexpressionandquantifiedasfoldchangetocontrolusingtherelativeCqmethod.Asterisks(*)indicatedifferentialexpressioncomparedtocontrols[Log2(FoldChange)|0.6|andFDR-correctedp-value<0.05]intheRNA-Seqanalysis,andpoundsigns(#)indicatestatisticallysignificant(p<0.05)differencescomparedtovehiclecontrolsintheqPCRdataasdeterminedbyone-wayANOVAandNewman-Keulsmultiplecomparisontest Table3GenesdifferentiallyregulatedbyE2ENSEMBLGeneIDGeneDescriptionLog2(FoldChange)p-valueENSG00000258588TRIM6-TRIM34TripartiteMotif-Containing6AndTripartiteMotif-Containing345.361.33E-06ENSG00000256966RP11-613M10.8AL513165.23.612.98E-02ENSG00000274944RP5-864K19.6AL139260.32.931.34E-14ENSG00000255439RP11-196G11.1AC135050.21.439.12E-03ENSG00000187678SPRY4sproutyRTKsignalingantagonist40.763.20E-05ENSG00000139318DUSP6dualspecificityphosphatase60.752.25E-08ENSG00000169583CLIC3chlorideintracellularchannel3Š0.731.40E-06ENSG00000053918KCNQ1potassiumvoltage-gatedchannelsubfamilyQmember1Š0.864.87E-03ENSG00000111344RASAL1RASproteinactivatorlike1Š1.341.79E-03ENSG00000162444RBP7retinolbindingprotein7Š1.653.08E-02Smithetal.RespiratoryResearch (2018) 19:160 Page11of17

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foundthatexposuretoTGF-1causedadose-dependentandsignificantreductioninESR1,ESR2,andGPER1mRNAexpression(Fig.3b-c).WeextendedthistoshowthatTGF-1-inducedrepressionofESR1persistedattheproteinlevel(Fig.4a-b).OtherstudieshaveshownthatTGF-1reducesESR1mRNAexpression[53,54]andESR1proteinexpression[54,55]inbreastepithelialcan-cercellsandESR2proteinexpressioninprostatecancercells[51],howeverthisisthefirststudytoshowthatTGF-1reducedESR2andGPER1mRNAexpressionandcertainlythefirsttoreportanyinteractionbetweenTGF-1andESRsinbronchialepithelialcells.Insupportofthisinteractionoccurringinvivo,weobservedreducedexpressionofESR1,ESR2,andGPER1inlungtissuefrompatientswithend-stageIPFcomparedtohealthycontrolsubjects(Fig.5a-c).Althoughwedonothavemeasure-mentsofTGF-1inoursamples,othershaveshownin-creasedserumTGF-1levelsinpatientswithIPFcomparedtohealthycontrols[36].TheseresultsshouldbecarefullyinterpretedgiventhesmallsamplesizeandtheabsenceofmechanisticinformationlinkingincreasedserumTGF-1levelstoESR1,ESR2,andGPER1mRNAexpression.Futurestudiesshouldinvestigatewhichsignal-ingmediatorsdownstreamofTGF-1,e.g.SMADs,CTGF,orSNAI1,amongothers,areresponsiblefortheobservedrepression.FewstudiestodatehaverevealedafunctionalroleforE2inlungcellsasmeasuredbygenesandpathwaysmodulateddownstream.UsingRNA-Seq,wesearchedforenrichedpathwaysinBEAS-2BsexposedtoE2andTGF-1individu-ally,andincombination,tobothidentifypointsofconver-genceofE2andTGF-1signalingandtohighlightnovelE2targetsthatmaybesusceptibletoTGF-1-inducedrepres-sionofESRs.SequencingdataindicatedgreaterregulationofgenesinresponsetoTGF-1exposureincomparisontoE2exposure,perhapsconsistentwiththewell-recognizedstrongpro-fibroticresponseassociatedwithTGF-1.Al-thoughsomegenesweredifferentiallyregulatedintheTGF-1orTGF-1+E2exposuregroups,mostweresharedsuggestingthatthepresenceofE2hadaminimaleffectontheTGF-1-inducedtranscriptome(Fig.5b)potentiallyduetoTGF-1-inducedrepressionofESRs.Weconfirmedtheexpressionofselectedgenesrelevanttothisworkand/orknowntobetargetsofTGF-1(Fig.6,ESR1,VIM,CTGF,MMP2)whichwasconsistentwithourpreviousresultsandthosereportedintheliterature[56–64].ExposuretoE2didnotinduceasrobustatranscrip-tionalresponseinBEAS-2BscomparedtoTGF-1.How-ever,weidentifiedstatisticallysignificantregulationof10genesbyE2(Table3)thathavenotbeenpreviouslyre-ported.Twogenesthatwerespecificallydown-regulatedbyE2includedChlorideintracellularchannel3(CLIC3)andRetinolbindingprotein7(RBP7)(Table3).CLIC3promotesmigrationandinvasionofcancercellsbyfacili-tatingthefunctionsofMT1-MMP(MMP14)[65,66].MT1-MMPisthemosthighlyexpressedMMPinIPFlungs[63]andmayprotectagainstPFbydegradingcolla-gen[67]andpromotinglungrepair[68].AnotherstudyindicatedthatMT1-MMPpromotedpulmonaryfibrosisbyactivatinglatentTGF-1[69].OurresultssuggestE2mayrepressMT1-MMPfunctionbydownregulatingCLIC3mRNAexpression.RBP7,alsoknownasCRABP4,isaretinolbindingproteinthoughttoplayanimportantroleinretinoluptake,storage,andmetabolism[70].RBP7hasbeenshowntobeup-regulatedinIPFlungtissue[71]andinwoundtissueinthenormalchickenchorioallantoicwoundmodel[72]althoughitsroleinfibrosisisunclear.RBP7ispositivelyregulatedbyE2inbreastcancercells[73]andmousemammarygland[74].Thediscrepancyinregulationinourstudymaybearesultofvariable Table4SignificantlyenrichedgenesetsNameTGF-1TGF-1+E2E2PathwayTypeMedianChangep-valueMedianChangep-valueMedianChangep-valueAirwaySmoothMuscleCellContractionDiseases1.144.72E-041.104.34E-03Š1.013.96E-02AlveolarEpithelialCellDysfunctionDiseases1.014.98E-02…………Ca2+FluxRegulationBiologicalFunction1.022.12E-071.061.40E-05Š1.005.48E-09ComplementAlternativePathwayBiologicalFunctionŠ1.633.17E-04Š1.492.64E-05Š1.111.36E-03ComplementClassicalPathwayBiologicalFunctionŠ1.342.27E-04Š1.493.09E-05Š1.154.87E-04ExtracellularMatrixTurnoverBiologicalFunction1.552.26E-081.533.98E-07Š1.046.86E-04HistoneAcetylationBiologicalFunction…………1.043.68E-02HistonePhosphorylationBiologicalFunction…………1.052.99E-05NeutrophilChemotaxisBiologicalFunction1.091.27E-031.077.25E-03……NURFinChromatinRemodelingBiologicalFunction…………1.062.48E-02SkinFibrosisDiseases1.072.15E-021.061.64E-02……VasodilationActivationBiologicalFunction…………Š1.003.34E-02(),notsignificantlyenrichedinspecifiedexposuregroupSmithetal.RespiratoryResearch (2018) 19:160 Page12of17

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Fig.8TGF-1andE2causedifferentialregulationofgenesinvolvedinextracellularmatrixturnover.AgenesetenrichmentanalysisusingPathwayStudioofgenesidentifiedbyRNA-Seqrevealedthatexposureto5ng/mLTGF-1(top)and10nME2(bottom)causedstatisticallysignificant(p<0.05)enrichmentoftheextracellularmatrixturnoverpathway.Grayboxesdenotecellularprocessesinvolvedintheextracellularmatrixturnoverpathway.Redproteinsindicateup-regulationandblueproteinsindicatedown-regulationasdeterminedbyRNA-SeqSmithetal.RespiratoryResearch (2018) 19:160 Page13of17

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exposuredynamicsasthestudybyCalvoetal.exposedmicetoonedoseofE2andsacrificedtheanimals3hlater[74]whileweexposedcellsinvitrofor24h.Nonetheless,E2appearstoregulateRBP7whichmayexhibitanunex-ploredeffectonfibrogenicsignaling.Asexpected,exposuretoTGF-1resultedinsignificantenrichmentoftheECMturnover(Fig.8a),alveolarepithe-lialcelldysfunction,andskinfibrosispathwaysasdeter-minedbyGSEA(Table4).ItiswellknownthatTGF-1isinvolvedinorganizationoftheECM[75]andneutrophilchemotaxis[76],andoneoftheprevailinghypothesesinIPFresearchisthatitisaresultofdysfunctionalbehaviorofalveolarepithelialcells[77].Inthiscase,skinfibrosisservesasasurrogateforpulmonaryfibrosisbecausetheunderlyingmechanismsaresimilarandlargelyregulatedbyTGF-1[78],andpulmonaryfibrosisdoesnotexistasacurated,predefinedpathwayinPathwayStudio.Inmostcases,pathwaysenrichedintheTGF-1individualexpos-uregroupwerealsoenrichedintheTGF-1+E2co-exposuregroup,andtheoveralldirectionalityasindi-catedbythemedianchangewassimilar.ThissuggeststhatE2hasalimitedeffectonTGF-1oncethepathwaysareinmotionand/orwasaresultofTGF-1-inducedre-pressionofESRsthusmirroringtheresultsseenatthegenelevel(Fig.3b-c).Interestingly,exposuretoE2resultedinspecificen-richmentofmultiplepathwaysinvolvedinepigeneticregulationofchromatinstructureandorganizationin-cludingHistoneAcetylation,HistonePhosphorylation,andNURFinChromatinRemodeling(Table4).WhileE2hasbeenshowntoregulatehistoneacetylationinA549cells[79],littleisknownaboutaroleforE2intranscriptionallyregulatingtheexpressionofgenesinvolvedinchromatinremodelinginthelung.Thisisimportantbecauseevidencefortheimportanceofepi-geneticsandchromatinorganizationinlungdiseaseisgrowing,particularlyinthecontextofpulmonaryfibrosis[80–85].Forexample,histonedeacetylasesareinvolvedinactivationoflungfibroblaststomyofibro-blasts[86]andaccumulationofECMcomponentsandEMTinthediabetickidney[87].Notably,exposuretoTGF-1individuallyandinthepresenceofE2didnotresultinenrichmentofchromatinremodelinggenesets.Thissuggeststhattheabsenceofenrichmentintheco-exposuregroup,despitethepresenceofE2,maybearesultofTGF-1-inducedrepressionofESRexpressionandnotthroughdirectregulationofgenesbyTGF-1.SimilartoTGF-1,exposuretoE2resultedinstatis-ticalenrichmentofgenesassociatedwithECMturnover,airwaysmoothmusclecellcontraction,andcalciumfluxregulationpathways(Table4).E2isknowntoinfluencetheECMintheuterusandvaginaltissues[88],inosteo-blasts[89],andintheskin[90].Interestingly,theoveralldirectionalityofthepathwayasindicatedbythemedianchange,wasopposite(negative)comparedtothedirec-tionalityofthepathwayintheTGF-1andTGF-1+E2groups(positive,Table4).ThisisconsistentwithastudythatfoundthatE2inhibitedTGF-1-inducedECMpro-ductioninhumanandratmesangialcellsthroughGPER1activation[22].OfnoteistherepressionofMMP14andMMP2asanotherstudyshowedthatE2decreasedMMP2-,MMP13-,andMMP14-mediatedtis-suematrixdestruction[91].TheseresultsareconsistentwiththesignificantreductionofCLIC3mRNAexpres-sionbyE2(Table3)whichisknowntoregulateMMP14[65,66].FuturestudiesshoulddelineatethepreciseroleofeachESRinregulatinggenesinvolvedinECMturnover.ConclusionsInconclusion,wewerenotabletodecipheraneffectofE2onTGF-1-inducedEMT,butwedoreportthenovelobser-vationthatTGF-1inhibitedESR1,ESR2,andGPER1mRNAexpressionandESR1proteinexpressioninBEAS-2Bs.WealsoreportthatE2specificallydown-regulatedtheexpressionofCLIC3andRBP7whichhavebeenassociatedwithpathogenicmechanismsofpul-monaryfibrosis.Wefurtherhighlightcellularpathwaysin-volvedinchromatinremodelingasnovelandspecifictargetsofE2inbronchialepithelialcellsandopposingac-tionsofTGF-1andE2signalingongenesinvolvedinECMturnover.AlthoughthesedatadonotexplicitlyindicateaprotectiveroleforE2inpulmonaryfibrosis,theseresultssuggestthatE2influencespathwaysrelevanttopulmonaryfibrosisandhighlightspotentialrolesforE2inthelungthatmaycontributetosex-specificdifferences.AdditionalfileAdditionalfile1:TableS1.Genesdifferentiallyexpressed(FDR-correctedp-value<0.05)inTGFB1groupcomparedtovehiclecontrolgroup.TableS2.Genesdifferentiallyexpressed(FDR-correctedp-value<0.05)inTGFB1+E2groupcomparedtovehiclecontrolgroup.(XLS479kb)AbbreviationsACTA2:Alphasmoothmuscleactin;ACTB:Beta-actin;BEAS-2Bs:Bronchialepithelialcells;CDH1:E-cadherin;CDH2:N-cadherin;CLIC3:Chlorideintracellularchannel3;CRABP4:Cellularretinoicacid-bindingprotein4;CTGF:Connectivetissuegrowthfactor;DMSO:Dimethylsulfoxide;DUSP6:Dualspecificityphosphatase6;E2:17-Estradiol;EMT:Epithelialtomesenchymaltransition;ERCC:ExternalRNAcontrolsconsortium;ESR:Estrogenreceptor;ESR1:Estrogenreceptoralpha;ESR2:Estrogenreceptorbeta;FN1:Fibronectin;FVC:Forcedvitalcapacity;FVC%:Percentpredictedforcedvitalcapacity;GAPDH:Glyceraldehydephosphatedehydrogenase;GPER1:G-proteincoupledestrogenreceptor;GSEA:Genesetenrichmentanalysis;IPF:Idiopathicpulmonaryfibrosis;KCNQ1:Potassiumvoltage-gatedchannelsubfamilyQmember1;MIQE:MinimuminformationforpublicationofqPCRexperiments;MMP14:Matrixmetalloproteinase14;MMP2:Matrixmetalloproteinase2;MT1-MMP:Matrixmetalloproteinase14;MT-MMP:Membrane-typematrixmetalloproteinase;NURF:Nucleosomeremodelingfactor;PDGF:Platelet-derivedgrowthfactor;RASAL1:RASproteinactivatorlike1;RBP7:Retinolbindingprotein7;RPS13:RibosomalproteinS13;SMAD3:SMADfamilymember3;SNAI1:SnailfamilytranscriptionalSmithetal.RespiratoryResearch (2018) 19:160 Page14of17

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repressor1;SPRY4:SproutyRTKsignalingantagonist4;TGF-1:Transforminggrowthfactorbeta1;UIP:Usualinterstitialpneumonia;VIM:VimentinAcknowledgementsTheauthorswouldliketothankYanpingZhangintheGeneExpression&GenotypingCoreoftheInterdisciplinaryCenterforBiotechnologyResearch(ICBR)forassistancewithRNA-Seqlibrarypreparation,DavidMoragaoftheNextGenDNASequencingcoreofUFICBRforassistancewithRNAsequencing,andAlbertoRivaoftheBioinformaticscoreofUFICBRforassistancewithbio-informaticsanalysisofRNA-Seqdata.TheauthorswouldalsoliketothankDavidDreierandAmandaBuergerforassistanceperformingclusteringanalysisandcreatingheatmaps.FundingThisworkwassupportedbyfundingfromNationalInstitutesofHealth(R01HL114907toT.S.A).ResearchreportedinthispublicationwassupportedbytheUFClinicalandTranslationalScienceInstitute,whichissupportedinpartbytheNIHNationalCenterforAdvancingTranslationalSciencesunderawardnumberUL1TR001427.ThecontentissolelytheresponsibilityoftheauthorsanddoesnotnecessarilyrepresenttheofficialviewsoftheNationalInstitutesofHealth.AvailabilityofdataandmaterialsThedatadiscussedinthispublicationhavebeendepositedinNCBIsGeneExpressionOmnibus(39)andareaccessiblethroughGEOSeriesaccessionnumberGSE100574(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE100574).Authors’contributionsLCSandTSAconceivedthestudyanddesignedtheexperiments.LCS,SM,LR,KG,andSRperformedtheexperiments.LCSandTSAinterpretedtheresults.LCSdraftedthemanuscriptandTSAedited.LCS,SM,LR,SR,AJB,andTSAapprovedthefinalversionofthemanuscript.EthicsapprovalandconsenttoparticipateThehumanlungtissuesamplesusedinthisstudywereakindgiftfromDr.AndrewBryant.Thedeidentified,explantedlungtissuewasobtainedfromsubjectsundergoinglungtransplantforIPFandfromlungsrejectedfortransplantfromnormalcontrolspertheNationalInstitutesofHealthLungTissueResearchConsortium(protocolno.14-99-0011).Theprotocolforcollectionoflungtissuesamples,andsubsequentstudies,wereapprovedbytheinstitutionalreviewboardatVanderbiltUniversityandtheUniversityofFlorida(30).ConsentforpublicationNotapplicable.CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.Publisher’sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.Authordetails1DepartmentofPhysiologicalSciences,UniversityofFlorida,Gainesville,FL,USA.2CenterforEnvironmentalandHumanToxicology,UniversityofFlorida,Gainesville,FL,USA.3DepartmentofEnvironmentalandGlobalHealth,CenterforEnvironmentalandHumanToxicology,UniversityofFlorida,Box110885,2187MowryRd,Gainesville,FL32611,USA.4DepartmentofMedicine,UniversityofFlorida,Gainesville,FL,USA.Received:4May2018Accepted:13August2018 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