Interdependence of thyroglobulin processing and thyroid hormone export in the mouse thyroid gland

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Year: 2017

Interdependence of thyroglobulin processing and thyroid hormone export in

the mouse thyroid gland

Weber, Jonas ; McInnes, Joseph ; Kizilirmak, Cise ; Rehders, Maren ; Qatato, Maria ; Wirth, Eva K ;

Schweizer, Ulrich ; Verrey, François ; Heuer, Heike ; Brix, Klaudia

Abstract: Thyroid hormone (TH) target cells need to adopt mechanisms to maintain suﬀicient levels of

TH to ensure regular functions. This includes thyroid epithelial cells, which generate TH in addition to

being TH-responsive. However, the cellular and molecular pathways underlying thyroid auto-regulation

are insuﬀiciently understood. In order to investigate whether thyroglobulin processing and TH export

are sensed by thyrocytes, we inactivated thyroglobulin-processing cathepsins and TH-exporting

monocar-boxylate transporters (Mct) in the mouse. The states of thyroglobulin storage and its protease-mediated

processing and degradation were related to the levels of TH transporter molecules by immunoblotting

and immunofluorescence microscopy. Thyroid epithelial cells of cathepsin-deficient mice showed increased

Mct8 protein levels at the basolateral plasma membrane domains when compared to wild type controls.

While the protein amounts of the thyroglobulin-degrading cathepsin D remained largely unaffected by

Mct8 or Mct10 single-deficiencies, a significant increase in the amounts of the thyroglobulin-processing

cathepsins B and L was detectable in particular in Mct8/Mct10 double deficiency. In addition, it was

observed that larger endo-lysosomes containing cathepsins B, D, and L were typical for Mct8- and/or

Mct10-deficient mouse thyroid epithelial cells. These data support the notion of a crosstalk between

TH transporters and thyroglobulin-processing proteases in thyroid epithelial cells. We conclude that a

defect in exporting thyroxine from thyroid follicles feeds back positively on its cathepsin-mediated

prote-olytic liberation from the precursor thyroglobulin, thereby adding to the development of auto-thyrotoxic

states in Mct8 and/or Mct10 deficiencies. The data suggest TH sensing molecules within thyrocytes that

contribute to thyroid auto-regulation.

DOI: https://doi.org/10.1016/j.ejcb.2017.02.002

Posted at the Zurich Open Repository and Archive, University of Zurich

ZORA URL: https://doi.org/10.5167/uzh-139734

Journal Article

Published Version

The following work is licensed under a Creative Commons: Attribution-NonCommercial-NoDerivatives

4.0 International (CC BY-NC-ND 4.0) License.

Originally published at:

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thyroglobu-lin processing and thyroid hormone export in the mouse thyroid gland. European Journal of Cell Biology,

96(5):440-456.

DOI: https://doi.org/10.1016/j.ejcb.2017.02.002

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ContentslistsavailableatScienceDirect

European

Journal

of

Cell

Biology

j ou rn a l h o m epa g e :w w w . e l s e v i e r . c o m / l o c a t e / e j c b

Research

paper

Interdependence

of

thyroglobulin

processing

and

thyroid

hormone

export

in

the

mouse

thyroid

gland

Jonas

Weber

a

_,

_Joseph

_McInnes

a,1

_,

_Cise

_Kizilirmak

a

_,

_Maren

_Rehders

a

_,

_Maria

_Qatato

a

_,

Eva

K. Wirth

b

_,

_Ulrich

_Schweizer

c

_,

_Francois

_Verrey

d

_,

_Heike

_Heuer

e

_,

_Klaudia

_Brix

a,∗ a_Jacobs_University_Bremen,_Department_of_Life_Sciences_and_Chemistry,_Campus_Ring_1,_D-28759_Bremen,_Germany

b_{Charité-Universitätsmedizin}_Berlin,_Institut_für_{Experimentelle}_{Endokrinologie,}_{Augustenburger}_Platz_1,_D-13353_Berlin,_Germany c_Universität_Bonn,_Institut_für_Biochemie_und_{Molekularbiologie,}_Nußallee_11,_D-53115_Bonn,_Germany

d_Universität_Zürich,_{Physiologisches}_Institut,_{Winterthurerstrasse}_190,_CH-8057_Zürich,_Switzerland

e_IUF_–_Leibniz_Institut_für_{umweltmedizinische}_Forschung,_Auf’m_Hennekamp_50,_D-40225_Düsseldorf,_Germany

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22December2016

Receivedinrevisedform8February2017 Accepted9February2017 Key-words: Cathepsins Endo-lysosomes Mct8 Mct10 Thyroidauto-regulation Thyroidstates Thyroxine

Translocationacrossmembranes

a

b

s

t

r

a

c

t

Thyroidhormone(TH)targetcellsneedtoadoptmechanismstomaintainsufficientlevelsofTHto

ensureregularfunctions.Thisincludesthyroidepithelialcells,whichgenerateTHinadditiontobeing

TH-responsive.However,thecellularandmolecularpathwaysunderlyingthyroidauto-regulationare

insufficientlyunderstood.InordertoinvestigatewhetherthyroglobulinprocessingandTHexportare

sensedbythyrocytes,weinactivatedthyroglobulin-processingcathepsinsandTH-exporting

monocar-boxylatetransporters(Mct)inthemouse.Thestatesofthyroglobulinstorageanditsprotease-mediated

processinganddegradationwererelatedtothelevelsofTHtransportermoleculesbyimmunoblottingand

immunofluorescencemicroscopy.Thyroidepithelialcellsofcathepsin-deficientmiceshowedincreased

Mct8proteinlevelsatthebasolateralplasmamembranedomainswhencomparedtowildtypecontrols.

Whiletheproteinamountsofthethyroglobulin-degradingcathepsinDremainedlargelyunaffectedby

Mct8orMct10single-deficiencies,asignificantincreaseintheamountsofthethyroglobulin-processing

cathepsinsBandLwasdetectableinparticularinMct8/Mct10doubledeficiency.Inaddition,itwas

observedthatlargerendo-lysosomescontainingcathepsinsB,D,andLweretypicalforMct8-and/or

Mct10-deficientmousethyroidepithelialcells.Thesedatasupportthenotionofacrosstalkbetween

THtransportersandthyroglobulin-processingproteasesinthyroidepithelialcells.Weconcludethata

defectinexportingthyroxinefromthyroidfolliclesfeedsbackpositivelyonitscathepsin-mediated

prote-olyticliberationfromtheprecursorthyroglobulin,therebyaddingtothedevelopmentofauto-thyrotoxic

statesinMct8and/orMct10deficiencies.ThedatasuggestTHsensingmoleculeswithinthyrocytesthat

contributetothyroidauto-regulation.

BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Thyroiddysfunctionscausedbyalteredthyroidhormone(TH)2 generationordisturbedTHactionareconsideredtobeamongthe mostfrequentendocrinedisorders(Brixetal.,2011;Führeretal.,

∗ Correspondingauthor.

E-mailaddress:[email protected](K.Brix).

1 _Present_address:_VIB_Center_for_Brain_and_Disease_Research_and_KU_Leuven

DepartmentforHumanGenetics,3000Leuven,Belgium.

2 _{Abbreviations:}_CMF-PBS,_calcium-_and_{magnesium-free}_phosphate_buffered

saline;Cy3,indocarbocyanine;HPT,hypothalamus-pituitary-thyroid;Mct, mono-carboxylatetransporters;PTC,papillarythyroidcarcinoma;Tg,thyroglobulin;TH, thyroidhormones;TRH,thyrotropin-releasinghormone;TSH,thyroidstimulating hormone;WT,wildtype.

2015).THareofcrucialimportancefortheproperfunctioningof nearlyeveryorgan,includingthethyroidglanditself.Hence,allTH targetcellsneedtoadoptmechanismstomaintainsufficientlevels ofTHtoensureregularfunctions.Sincethethyroidglanditselfnot onlyproducesTHbutalsorespondstoTHlevelsinaself-sustaining manner,auto-protectionofthethyroidglandisvitalnotonlyfor thyrocytesbutalsoforallotherTH-dependentcells.Properthyroid statesofanorganismarecentrallyregulatedbythe hypothalamus-pituitary-thyroid(HPT)–axis,wherebylowserumTHlevelstrigger hypothalamicthyrotropin-releasinghormone(TRH)releasewhich thenpromotesreleaseofthyroidstimulatinghormone(TSH)from thepituitary(Fliersetal.,2014),therebytriggeringTHgeneration inthethyroidgland,andTHreleaseintotheblood.

Tofulfillandmaintainitstasks,thethyroidglandiscomposed offunctionalunits,theso-calledthyroidfollicles,thatarebuiltby http://dx.doi.org/10.1016/j.ejcb.2017.02.002

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Table1

SpecificfunctionsofdifferentcathepsinsexpressedinthethyroidregardingtheirprocessingofTg. extracellularTg solubilisationfrom cross-linked storageforms extracellularTg processing intracellular limitedTg processing (endosomal) intracellularTg degradation (lysosomal) directT4liberation fromTg exhaustiveTg degradationforTg utilization (endo-lysosomalTH liberation) References

CathepsinB x x x x involvedtomajor

extent

Brixetal.,1996;Dunn etal.,1996;Tepeletal., 2000;Friedrichsetal., 2003;Jordansetal., 2009

CathepsinD x involvedtominor

extent

Brixetal.,1996;Dunn etal.,1996;Friedrichs etal.,2003

CathepsinK x x x Tepeletal.,2000;

Friedrichsetal.,2003; Jordansetal.,2009

CathepsinL x x x x involvedtomajor

extent

Brixetal.,1996;Dunn etal.,1996;Tepeletal., 2000;Friedrichsetal., 2003;Jordansetal., 2009

Fig.1. Cathepsin-mediatedsolubilization,processing,anddegradationofTg.

SchematicdrawingsummarizingthesequentialstepsofTgprocessinganddegradationbyendo-lysosomalcathepsins(Brixetal.,1996;Tepeletal.,2000;Brixetal.,2001; Linkeetal.,2002a;Friedrichsetal.,2003;Jordansetal.,2009;Dauthetal.,2011).CathepsinsBandLinvolveinextracellularTgsolubilizationfromitscovalentlycross-linked storageforms,whilecathepsinKisinstrumentalinTgutilizationforT4liberation.Uponre-internalizationofintactandpartiallydegradedTg,itisfurtherprocessedand degradedbyasparticandcysteinecathepsinswithinendo-lysosomesofthyrocytes.

amonolayerofthyroidepithelialcells(Fujita,1988;Nilssonand Fagman,2013).ThemacromolecularTHprecursorthyroglobulin (Tg)is storedin covalently cross-linkedformin the extracellu-larthyroidfolliclelumen(Herzogetal.,1992;Berndorferetal., 1996;Kleinetal.,2000;Saber-Lichtenbergetal.,2000).Covalently cross-linkedTgisprocessedforsolubilizationanddegradationby proteases,whicharesecretedinaTSH-regulatedfashionintothe extra-cellular folliclelumenupon a demand of TH inthe body periphery(forreviews,seeBrixetal.,2001;Dauthetal.,2011). Thus,solubilizationofTgfromitscovalentlycross-linkedstorage formsisenabledthroughlimited,extracellularproteinprocessing facilitatedbycathepsinsBandL,whileTHliberationfromTgis mediatedbycathepsinsKandL,andalsoinitiatedinthethyroid folliclelumen(Brixetal.,1996;Linkeetal.,2002a;Friedrichsetal., 2003;Tepeletal.,2000).Subsequently, partiallydegradedTgis internalizedandreachesendo-lysosomalcompartments,whereT3

andT4liberationiscompletedforTgutilizationthroughexhaustive

proteolysismediatedbyavarietyofhydrolyticenzymesincluding asparticandcysteinecathepsinsB,D,K,L,andS(Brixetal.,1996; Tepeletal.,2000;Linkeetal.,2002a;Friedrichsetal.,2003;Jordans etal.,2009).CathepsinKisspecialamongtheTg-processing pro-teasesasitisabletoliberateT4directlyfromTg(Tepeletal.,2000).

Inbriefsummary,thyroidfunctionsareenabledbysequential pro-teolyticprocessingofTg(Table1,Fig.1),resultingintheliberation ofT4and,toalowerextent,T3.

Considering that thyroid epithelial cells need to release TH throughtransmembranetranslocationintothebloodcirculation, andkeepinginmindthatthyrocytesareTHtargetcellsthemselves, aregulatorymechanismmustexistthatdetermineshowmuchof theT4 liberated fromTgis eitherretainedin thethyrocytesor

transportedbackintothesame.Weproposethatintra-thyroidal THtransporterslikethemonocarboxylatetransportermolecules Mct8andMct10mayactassuchsensorsof(i)theextentof pro-teolyticTHliberation,(ii)theamountsofTHreleasedfromthyroid follicles,and(iii)thelevelsofre-uptakeofcirculatingTHbackinto thyroidfollicles.

Mice with a global Mct8-deficiency (Mct8 knock-out mice) exhibitcentralTH-resistance,i.e.TRHlevelsaredramatically ele-vateddespite highlyincreasedserum T3 levels,whereas serum

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Table2

Thyroidfunctionparameters,peripheralandintra-thyroidalthyroidstates,THgenerationandTHreleaseinMct8/10-deficientmice.

Mct8−/y _Mct10−/− _Mct8−/y_/Mct10−/− _References

Thyroidfunctionparameters,HPTaxis

• serumT4 ↓ = = Dumitrescuetal.,2006;Trajkovicetal.,2007;DiCosmoetal.,2010;

Liaoetal.,2011;Wirthetal.,2011;Trajkovic-Arsicetal.,2010b; Mulleretal.,2014

• serumT3 ↑↑↑ = ↑↑↑ Dumitrescuetal.,2006;Trajkovicetal.,2007;DiCosmoetal.,2010;

Liaoetal.,2011;Wirthetal.,2011;Trajkovic-Arsicetal.,2010b; Mulleretal.,2014

• serumTSH,orTSH mRNA(pituitary)

↑ = ↑ Dumitrescuetal.,2006;Trajkovicetal.,2007;Trajkovic-Arsicetal., 2010b;Liaoetal.,2011;Wirthetal.,2011;Mulleretal.,2014

• TRHmRNA (hypothalamus)

↑↑ = = Trajkovicetal.,2007;Trajkovic-Arsicetal.,2010b;Mulleretal.,2014

peripheralDio1activity (liver,kidney)

↑ = ↑↑ Dumitrescuetal.,2006;Trajkovicetal.,2007;Liaoetal.,2011; Trajkovic-Arsicetal.,2010a;Trajkovic-Arsicetal.,2010b;Mulleretal., 2014

intra-thyroidalthyroidstates • intra-thyroidalTH • (non-Tg-boundand

Tg-bound)

↑↑ = ↑↑↑ DiCosmoetal.,2010;Trajkovic-Arsicetal.,2010b;Liaoetal.,2011; Mulleretal.,2014

• intra-thyroidalDio1 activity

= DiCosmoetal.,2010;Wirthetal.,2011;Mulleretal.,2014

• auto-thyrotoxicstate,i.e. papillarystructureswith nuclearchanges;or alteredtumourmarker likeGalectin-3

↑↑ = ↑↑↑ Wirthetal.,2011;Mulleretal.,2014

• thyroidfollicleand/or luminalareas

↑ = ↑↑ DiCosmoetal.,2010;Trajkovic-Arsicetal.,2010b;Wirthetal.,2011; Mulleretal.,2014

• thyroidepithelial extensions

↑ DiCosmoetal.,2010;Wirthetal.,2011

THgeneration • Nis(sodiumiodide

symporter),Ttf1 (transthyretin)

↑notsignifi-cant = ↑↑ DiCosmoetal.,2010;Mulleretal.,2014

• Tshr(TSHreceptor),Tpo (thyroidperoxidase),Tg (thyroglobulin),Dehal1 (dehalogenase1)

= = ↑↑ DiCosmoetal.,2010;Trajkovic-Arsicetal.,2010b;Mulleretal.,2014

THreleasefromthyroidfollicles • TSH-stimulatedT4

release

↓ DiCosmoetal.,2010;Trajkovic-Arsicetal.,2010b

• TSH-stimulatedT3 release ↑ Trajkovic-Arsicetal.,2010b • Mct10,Lat1,Lat2(TH transporters) = DiCosmoetal.,2010

2014).Interestingly,micedeficientinbothMct8andMct10 pro-teins(Mulleretal.,2014)showedapartialrescueoftheunusual serumTHstatusobservedinMct8-deficientanimals,whereasthe intra-thyroidalauto-thyrotoxicstateswereevenmorepronounced thaninMct8 deficiencyalone.Therefore, bothMct8 andMct10 appeartoberesponsiblefor THexportfromthyrocytes(Muller etal.,2014).

Theastonishingchangesintissue-specificTHstatesinmicewith THtransporter-deficienciesindicatethatTHlevelsareregulated inanorgan-specific manner,andnot entirelygovernedby sys-temicfactorssuchasregulationthroughtheHPTaxis.Withregard tothethyroidglanditself,suchorgan-specificmechanismsmust compriseofintra-thyroidalself-regulatorypathwaystomanageits thyroidstate.Inthisstudy,pathwaysofthyroidauto-regulation werestudiedinMct8-and/orMct10-deficientmalemicein

com-parisontowildtype(WT)controls.Inaddition,Ctsk−/−_male_mice

were used, because they provide a suitable animal model for analyzingintra-thyroidalpathwaysofthyroidauto-regulation,in particularwithregardtoTgproteolysis(Dauthetal.,2011).Serum TH levelsare not altered in cathepsin Kdeficiency, and hence, TSHlevelsarelikelynotenhancedinthismousemodel,despite developinganenlargedthyroidglandandfeaturingchangesin Tg-processingabilitiesinthatcathepsinLisup-regulated(Friedrichs etal.,2003).ThismeansthatCtsk−/−_mice_show_signs_of_thyroid

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2. Materialsandmethods 2.1. Animals

All studies were performed with 10–14 weeks and 20–28 weeksoldmaleCtsk−/−_,_Mct8−/y_,_Mct10−/−_,_Mct8−/y_/Mct10−/− _or

WT C57Bl/6J mice. The mice were back-crossed to a congenic C57Bl/6JbackgroundintheanimalfacilityofJacobsUniversity Bre-men,Germany,licencedunderSfAFGJSAz.513-30-00/2-15-32and 522-27-11/3-1,05-A20andA21.Thegenerationandgenotyping proceduresofmousestrainslackingMct8(Trajkovicetal.,2007; Wirthetal.,2009),Mct10(Mariottaetal.,2012),Mct8andMct10 (Muller etal.,2014), orCtsk(Saftig etal.,1998)weredescribed previously.Ctsb−/−_,_Ctsk−/−_,_Ctsl−/−_,_and_Ctsb−/−_/Ctsk−/−_male_mice

werebredand housedaspreviouslydescribed(Friedrichsetal., 2003).Ingeneral,micewerehousedunderstandardconditions, witha12h/12hlight/darkcyclewithlightsoutat07:00p.m.and adlibitumwaterandfood.

2.2. Tissuesamplingandsectioning

MiceweredeeplyanesthetizedbyCO2inhalationorbyusingthe

inhalation-anestheticIsoFlou(AbbottGmbH&Co.KG, Wiesbaden-Delkenheim,Germany)beforeintra-peritonealinjectionof100␮l of10%ketamine(CEVASanteAnimale,Düsseldorf,Germany).The abdominal and thoracic cavities wereopenedand the abdomi-nalaortawascut. Perfusion wascarriedout through theheart with0.9%NaClsupplementedwith200IUheparin(Braun Mel-sungenAG,Melsungen,Germany).Thethyroidwasimmediately removed,whilestill attachedtothetrachea,either snap-frozen inliquidnitrogen,orincubatedin4%PFAin200mMHEPES,pH 7.4,and leftovernightat4◦_C. _Then,_{cryo-preservation} _was

car-riedoutbyembeddinginTissueFreezingMedium(Jung,through LeicaMicrosystems,Wetzlar,Germany)overnight,beforetissue wasfrozen in thegasphase of liquid nitrogenand stored at -80◦_C_until_sectioning._Thyroid_samples_were_then_mounted_on_the

sectioningtableauofthecryotome,andsectionsof5␮mwere pre-paredat−20◦_C_(Leica_{Microsystems).}

2.3. Indirectimmunofluorescence

Embedding material was washed out of the tissue sections withPBSpriortothestainingprocedure.Blockingwasperformed with3%bovineserumalbumin(AlbuminFractionV,Roth, Karl-sruhe, Germany) for 60min at 37◦_C. _Samples _were _incubated

overnightwithspecificantibodiesdilutedin0.1%BSAin calcium-andmagnesium-freePBS(CMF-PBS)at4◦_C_in_a_moisturized

cham-ber.Specificantibodieswererabbitanti-Mct8(HPA003353;1in 250ATLASAntibodies,Stockholm,Sweden),sheepanti-cathepsin L (06-0008; 1 in 20, RD antibodies through Merck-Millipore, Darmstadt,Germany),goatanti-cathepsinB(GT15047;1in200, Neuromics, through Acris, Himmelstadt, Germany), sheep anti-cathepsinD(06-0003; 1in50, Bio-Ass,Diessen, Germany),and rabbitanti-bovineTg(Brixetal.,1998).Secondaryantibodiesand counter-stainswereincubatedfor60minat37◦_C._As_secondary

antibodies,indocarbocyanine-(Cy3)orAlexa488-conjugatedgoat anti-rabbitIgG,donkeyanti-sheepand rabbitanti-goatIgG,and AlexaFluor546coupledtodonkeyanti-sheepwereused.Draq5TM

(DR05500, 1 in 500, BioStatus, Leicestershire, UK) wasused as counter-staintovisualizenuclearDNA.

2.4. Imageacquisitionofmousethyroidtissueanddensitometry ConfocalimagesweretakenusingaZeissLSM510METAlaser scanningmicroscopeequippedwithArgonandHelium-Neonlasers (CarlZeissJenaGmbH,Jena,Germany).Opticalsectionsweretaken with a pinhole opening of 1 Airy unit and at a resolution of

1024×1024pixelsor2048×2048,usingLSM5software(version 3.2;CarlZeissGmbH).Forquantitationofcysteinecathepsinand Mct8levels,thyroidtissuefromatleastthreedifferentanimalswas analyzedsuchthatimagesfromtissuesectionswereobtainedat thesamedetectorgain andpinholesettings.Thenumberof fol-liclesanalyzedisgivenintherespectivefigurelegends.Byusing CellProfilersoftware,signalsofimmunostainingintensitieswere quantifiedandnormalizedtothenumberofcells by enumerat-ingthecorrespondingDraq5TM_-positive_nuclei₍_Kamentsky_et_al., 2011).

2.5. Proteinextraction

Thyroidtissue sampleswere homogenizedand lysedin ice-coldPBScontaining0.5%TritonX-100,1mMEDTA,andprotease inhibitors(0.2␮g/mlaprotinin,Sigma-Aldrich,1153;EDTA;10␮M E-64, Enzo Life Science, Farmingdale, NY, USA, ALX 260007-M005;1␮MpepstatinA,Sigma-Aldrich,77170).Afterclearingat 10,000×gand4◦_C_for₁₀_min,_the_lysates_were_stored_at₋₂₀◦_C._The

proteincontentofallsampleswasdeterminedusingtheNeuhoff assay(Neuhoffetal.,1979)asdescribedbefore(Arampatzidouetal., 2012).

2.6. Gelelectrophoresisandimmunoblotting

Tissue lysates were normalized to equal amounts of pro-tein,heatedat95◦_C_in_sample_buffer_and_separated_on_self-cast

12.5% acrylamide gels or horizontal SDS Gradient 8–18 Excel-Gelgels(GEHealthcare,Uppsala,Sweden).Insomeexperiments, proteinsoftissuelysateswereseparatedundernon-reducing con-ditions.Gelswereeitherstainedfortotalproteinbysilverstaining (HeukeshovenandDernick,1988),orseparatedproteinswere fur-thertransferred onto a nitrocellulosemembrane by a semi-dry blottingprocedure(Kyhse-Andersen,1984).

Immunodetectionwasperformedwithprimaryantibodies rab-bitanti-ß-tubulin(ab6046,1in500,Abcam),goatanti-cathepsinB (GT15047;1in1000,NeuromicsthroughAcris,Herford,Germany), rabbitanti-cathepsinD(IM16;1in80,CalbiochemthroughMerck Millipore),goatanti-cathepsinL(GT15049;1in1000,Neuromics through Acris),rabbit anti-Mct8(HPA003353; 1in 2500ATLAS Antibodies),andrabbitanti-bovine Tg(W.S.3,Brixetal.,1998) appliedfor1.5h(see,Friedrichsetal.,2003).Therespective sec-ondaryantibodies,HRP-conjugatedgoatanti-rabbitIgG(H+Lsp.) andrabbitanti-goatIgG(H+Lsp.)wereappliedfor1hatroom tem-perature.VisualizationwasperformedbychemiluminescenceECL WesternBlottingsubstrateontoXPosureTM_film_(Pierce_through

ThermoScientific,Schwerte,Germany),whichwerescannedusing a transmittedlightscannerdevice.Densitometryanalyseswere performedbyusingImageJasdescribedelsewhere(TanandNg, 2008;Gassmannetal.,2009).Sampleswerenormalizedtoequal amountsofproteinandbandswerenormalizedtoWTcontrolsor toß-tubulinloadingifnotstatedotherwise.

2.7. Statisticalanalyses

Levelsofstatisticalsignificanceofnon-pairedobservationswere calculatedbytwo-tailedStudent’st-testorone-wayANOVAwith Dunnett’scorrectionformultiple comparisonsusingMicrosoft® Excel® 2016(MicrosoftCorp.)orGraphPadPrismTM _(GraphPad

SoftwareInc.,SanDiego,CA,USA). 3. Results

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tissueof Mct8-and Mct10-double-deficientmice(Mulleret al., 2014)can,in principle,beexplainedby theinabilityof thyroid folliclestoproperlyreleaseTHintothebloodstreamdueto lack-ingMct8-and/orMct10-mediatedtransportacrossthebasolateral plasmamembranedomainofthyroidepithelialcells,leadingtoTH accumulationwithinthesecells.Inaddition,reducedT4levelsin

thebloodstreamstimulateTSHproduction,inprinciple,possibly affectingTgsolubilizationandutilizationbymeansofitsextra-and intracellularproteolysismediatedbythecysteinecathepsins.Thus, alteredTgturnoveranddegradationmayfurthercontributetothe enhancedlevelsof THdetectableinthyrocytesofMct8-and/or Mct10-deficientmice.Therefore,thestatusofTgcross-linkingand degradationwascorrelatedtotheamountsofTg-processing pro-teasesinthyroidtissueofTHtransporter-deficientmice,andvice versa,theexpressionandlocalizationoftheTHtransporterMct8 wasdeterminedinthyroidtissueofcathepsin-deficientmice. 3.1. AlteredthyroglobulinstatusinMct8-and/orMct10-deficient mousethyroidtissue

TheTgstatusofMct8and/orMct10-deficientthyroidglandswas analyzedmorphologicallybystainingthyroidtissuesectionswith antibodiesdirectedagainstTg.LuminalTgdepositionsare hetero-geneousbutshowthetypicalmultilayeredappearance(Berndorfer etal.,1996;Friedrichsetal.,2003)(Fig.2).Regionsrepresenting sol-ubilizedTgcanbeidentifiedbyhigherimmuno-stainingintensities, becausetheseTgformsaremoreeasilyaccessible,whereas com-pact,covalentlycross-linkedformsofTgarestainedlessintense bytheantibodies(Friedrichsetal.,2003).Whilecovalently cross-linkedformsofTgareprevalentinthecentreoffolliclelumina,the solubleandmoreeasilyaccessibleTgformsaretypicallydetected nexttotheapicalplasmamembrane(Fig.2,panelsAandB,WT;cf. panelC).ThedifferencesinappearanceofluminalTg-stainingthus dependontheaccessibilityofTgfortheantibodies,andweused thateffecttoquantifytheproportionoffolliclescontainingtightly compactedTg(Fig.2C,a,black),multilayeredTg(Fig.2C,b,grey) andhighlysolubilized,multilayeredTgapicallywithdense, com-pactTginthecentreofthatfollicle(Fig.2C,c,lightgrey;diamonds). Manualcountingrevealedthattherewasatrendofadecline infolliclecountswithtightlycompactedTginMct8−/y_/Mct10−/−

mice(60.8±5.7%)comparedtotheWT(87.4±2.5%),whileno sig-nificantdifferenceswereobservedinMct10−/−_mice_(72.0_±_11.3%)

andinMct8−/y_mice_(69.4_±_7.7%)₍_Fig.₂_,_bar_charts_beneath_panel

B).Additionally,follicleswithperipheralregionsofwell-accessible TgwerenotdetectedinWT(0.3%)orMct10−/−_mice_(0.0%),_but_4.3%

ofallfolliclesinMct8-deficientmicecontainedthoseTg appear-anceswhichwereevenmorepronouncedinMct8/Mct10-deficient animals(9.0%)(Fig.2,barchartsbeneathpanelB).Thus,asdeduced fromantibodyaccessibility,Tgstatesarealteredwithregardtothe well-accessibleTgappearancesintheperi-cellularregionatthe apexofthyrocytes,inparticularinMct8−/y_and_in_Mct8−/y_/Mct10−/−

mice,indicatingmoresolubilizedTgthaninWTanimals(Fig.2, panelsAandB,cf.Mct8−/y_and_Mct8−/y_/Mct10−/−_with_WT).

3.2. ThyroglobulindegradationisenhancedinMct8-and/or Mct10-deficientmice

TofurtherinvestigatetheeffectsofTHtransporterdeficiencyon thestatusoftheTHprecursorTg,weanalyzedwholethyroidlysates preparedfromyoung(<2.5month,seeFig.3)andadult(5–8month, seeFig.4)mice.

Immunoblots revealed an increase of soluble Tg fragments (<330kDa)inyoungMct8−/y_/Mct10−/−_mice_compared_to_all_other

genotypes(Fig.3AandB),andlessabundantmultimericformsofTg insilver-stainedSDS-gelsofthesamegenotype(Fig.3C),indicating enhancedsolubilizationofTginMct8-andMct10-double-deficient

animals.Thepresence ofmore Tgfragments pointstowardsan enhancedutilizationoftheTHprecursorTgbythyrocytesupon deficienciesin Mct8and Mct10.Sincea decreaseofmono- and dimericTgformsinMct8−/y_/Mct10−/−_mice_in_comparison_to

Mct8-deficientmicewasdetectedunderreducingconditions(Fig.3D), andatthesametime,thesolubledimericformofTgwasincreased whenthyroidslysateswereseparatedundernon-reducing condi-tions(Fig.3C),itmustbeassumedthatTgcross-linkingisalteredin thyroidfolliclesofyoungMct8-andMct10-double-deficientmice. Toconfirm thisinterpretation,adult animalswereanalyzed. InaccordancewiththeTgstatusobservedinyounganimals(see

Fig. 3), significantly enhanced amounts of Tg fragments were detectedinbothMct8-andMct8/Mct10-deficientadultmicewhen the protein lysates were separated under reducing conditions (Fig.4A).However,itwasfoundthattheeffectofincreaseddimeric TginMct8/Mct10-deficiencyof younganimals wasnot present anymore when thyroid lysates of adult animalswere analyzed undernon-reducingconditions(Figs.4B,cf.3C).Moreover,Tg mul-timerizationandtheamountsofdimericTgwereobservedtooccur tocomparableextents inthyroidtissue lysatesofallgenotypes analyzedundernon-reducingconditions(Fig.4B),whileless di-andmonomericTgwasobservedinMct8-and/orMct10-deficient thyroidtissuelysatesincomparisontoWTwheninspectedunder reducingconditions(Fig.4C).

Thus, gross degradation of Tg is enhanced in Mct8- and/or Mct10-deficientmiceatallages,whilecross-linkingforTg stor-ageisaffectedprimarilyinMct8−/y_/Mct10−/−_mice_at_an_age_of_up

to2.5months,butbyfarlessprominentlyalteredin5–8monthold mice.Theseresultsindicatethatintra-thyroidalregulationofTg turnoveraffectsenzymesinvolvedincross-linkingofTg(forreview, seeBrixetal.,2001)toalowerdegreethantheproteasesinvolved inextra-andintracellularprocessingofTgforitssolubilizationand utilization,eventuallyresultinginTHliberation.

3.3. Tg-processingproteasesaredifferentiallyaffectedbyMct8 and/orMct10deficiencies

Toinvestigatewhethertheobserved alterationsin Tgstatus mightbeduetoalteredlevelsofproteolyticenzymesweanalyzed theproteinlevelsofthecathepsinsB,D,andLinthyroidtissueof youngandadultanimals.Inyoungmice(<2.5month),proteinlevels ofcathepsinBweresignificantlyincreasedin Mct8/Mct10-double-deficientanimals,only(Fig.5).Inadultmice(5–8months),the proteinamountsofcathepsinsBandLinthyroidlysateswere sig-nificantlyincreasedinMct8-and/orMct10-deficiencies(Fig.6,top andbottompanels),whereascathepsinDproteinlevelswere sig-nificantlyincreasedinMct8/Mct10-double-deficientanimals,only (middlepanel)whencomparedtotheWT.

Sincethelocalization ofproteases,andthereforetheir possi-bleaccesstothesubstrateTg,definestheirimpactonmaintaining thyroidfunction,we usedimmunohistochemistrytoinvestigate whethertheintra-follicularandsubcellularlocalizationpatterns ofthecathepsinsB,D,andLchangedinTHtransporterdeficiency. Thyroidtissuecryo-sectionsfromWTorMct8/10-deficient ani-malswere immunolabeledwithantibodies againstcathepsin B. Inallgenotypesinspected,astainingpatternoflargelyendocytic cathepsin B, and, in few follicles,staining in thefolliclelumen aswellasattheapicalplasmamembranedomainofthyrocytes was observed (Fig. 7A–D). Moreover, the appearance of thyro-cyteswithenlargedcathepsinBpositivevesiclesinMct8−/y _was

detected(Fig.7G,arrows), which waseven moreprominent in Mct8−/y_/Mct10−/− _mice ₍_Fig. ₇_H, _arrows). _However, _the _overall

cathepsinBsignalintensitywasincreasedonlyslightlyinMct8−/y

andMct8−/y_/Mct10−/−_mice_when_compared_to_the_WT₍_Fig.₇_J).

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Fig.2. AssessmentofTgstatesinthyroidfolliclesofMct8/Mct10-deficientmice.

Confocalfluorescenceimagesofcryo-sectionsofthyroidglandsfromadultWTorTHtransporter-deficientmice(5–8monthsold)oftheindicatedgenotypesimmunolabeled withantibodiesagainstTg.TgwasdetectedinmultilayeredforminthefolliclelumenofWTmicewithincreasedlabellinginproximitytotheapicalplasmamembrane domainofthyrocytes,thusindicatingsolubleformsofTg(A,asterisks).AbundanceoffollicleswithoutprominentlabellingofsolubleTg(C,a)wasdecreasedin Mct8-andMct8/Mct10-doubledeficientanimals(AandB).Likewise,theproportionoffollicleswithintenselyimmuno-labeledsolubleTg(C,b)isincreasedinMct8-deficient animalsandevenmoreincreasedinMct8/Mct10-doubledeficientanimals.Interestingly,4.3%and9.0%ofallanalyzedfolliclesofMct8orMct8/Mct10-deficientanimals, respectively,featuredhighlysolubilizedTg(C,c)intheluminalperipherybutadensemassofcovalentlycross-linkedTginthecentralregionoflargefollicles(B,diamond). A:representative,overviewimagesusedforquantification,n=250,72,190,and127follicles,respectively.B:close-upimagesdisplayingfollicleswithdifferentformsofTg. BarchartsinBandC:black(a),percentageoffollicleswithoutlabeledsolubleTg;grey(b),percentageoffollicleswithlabeledsolubleTg;lightgrey(c),percentageoffollicles withhighlysolubilizedTggivenasmeans.Scalebars:100␮m.

signalintensityofcathepsinDstainingwaselevatedinthyroid tis-sueofMct10−/−_,_Mct8−/y_and_Mct8−/y_/Mct10−/−_mice₍_Fig.₈_B_and_F,

CandG,DandH,respectively)whencomparedtotheWT. Interest-ingly,cathepsinDpositivevesiclesinMct8-and/orMct10-deficient miceappeared largerthan those of therespectiveWT controls andinsomecases,vesicleswithprominentlyenhanceddiameters werefound(Fig.8F–H,cf.E,dashedarrows).Astrikingdifference betweenMct8−/y_and_Mct8−/y_/Mct10−/− _mice_was_the_occurrence

ofcathepsinDstainingatthebasalsurfaceofthyrocytes,which couldeitherbedue tocathepsin Dstainingin endothelialcells (Fig.8G,asterisks),secretionofcathepsinDatthebasalpole,and itsre-associationwiththebasolateralplasmamembranedomains ofthyrocytes(arrows),orboth.

InWTanimals,cathepsinLislocalisedinvesicularstructures, butinfewfollicles,associationwiththeapicalplasmamembrane domain of thyrocytes was also observed (Fig. 9A). In

Mct10-deficientmice,thelocalizationofcathepsinLdidnotchangewhen comparedtotheWTcontrol,howeveraslightincreaseofsignal intensitywasmeasuredindicatingelevatedproteinexpression lev-els(Fig.9BandH).InMct8−/y_and_Mct8−/y_/Mct10−/−_mice,_cathepsin

Lwasdetectedintracellularlywithinvesicularstructuresthatwere muchlargerthanthoseinWTtissue,insomecasesreaching diam-eters comparableof thenucleioftherespectivecells, i.e. upto 6␮m(Fig.9CandD,EandF,E’andF’,arrows).Additionally, local-izationofcathepsinLattheapicalplasmamembranedomainof Mct8−/y_/Mct10−/− _thyrocytes_was_increased_and_luminal_staining

nexttotheapicalplasmamembranedomainwasmuchmore abun-dantthanintheWT(Fig.9F,cf.E,arrows).

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Fig.3.Thyroglobulindegradationpatternandcross-linkagein<2.5monthsoldmice.

WholethyroidlysatesofWTmiceormicedeficientinMct10,Mct8and/orMct10wereseparatedonhorizontalSDS-gels,silver-stainedortransferredtonitrocellulosefor subsequentincubationoftheblotswithantibodiesagainstTgasindicated,andcomparedtotheWT.(A)Shownisarepresentativeimmunoblotwiththeindicatedgenotypes. (B)LowmolecularmassfragmentsofTgarerepresentativeofdegradationproductswhichwererevealedbydensitometrytobesignificantlyincreasedinMct8/Mct10-deficient animals.(C)Undernon-reducingconditions,theamountsofthesolubleformofTgareincreasedinMct8/Mct10-deficientanimals,whereasthemultimericTgformswereless abundantcomparedtotheWT,Mct10-andMct8-deficientanimals.Mct8-deficientmicewereobservedtohavemoretotalTgamountsincludingthemultimericform.Upon reducingsampleswithDTTbeforeSDS-PAGE,thepersistenceofdimericTgwasreducedinMct8/Mct10-deficientanimals(E),thusindicatingalteredsolubilizationofTg, whereasMct8-deficientanimalsfeaturedincreaseddimericandmonomericTgwhencomparedtotheWT.Animalsanalyzed:n=5,3,5,and5,respectively.InB,means±SD aregiven;*,P<0.05;**,P<0.005.

levelswereenhancedinthyroidtissueofallgenotypes,andmost prominentlyinMct8−/y_/Mct10−/−_mice,_which_were_further

charac-terizedbyenhancedamountsofcathepsinD.Thus,thedatapointto mostsignificantlyenhancedamountsofendo-lysosomalcathepsin LinparticularinMct8−/y_and_Mct8−/y_/Mct10−/−_mouse_thyrocytes,

i.e.intheanimalmodelsfeaturingthemostsevereintra-thyroidal thyrotoxicstates(Mulleretal.,2014).ConsideringcathepsinL’s main function in Tg processing, the enhanced amounts of this protease in Mct8−/y_/Mct10−/− _thyroid _tissue _explain _the_largely

enhanced extent of Tg degradation observed in Mct8/Mct10-deficientmice(Figs.3and4),andsuggestthatT4accumulatesin

thyrocytesoftheseanimals.Thus,theenhancedamountsof cathep-sinL,whichisalsovitalforT4liberationfromTg,inthyroidsofmice

lackingtheTH-transportingMct8,whichismainlyresponsiblefor T4releasefromthyroidfollicles,iscounterintuitive.

3.4. AlackoftheT4-liberatingcysteinecathepsinKisassociated

withenhancedamountsofthemainT4-exportingtransporter

Mct8

Thecysteine cathepsins Kand L together arecritical for TH liberation(seeTable1;Friedrichsetal.,2003).CathepsinK,in par-ticular,mediatesdirectliberationofT4fromTg(Tepeletal.,2000).

AlossofcathepsinKcanbecompensatedbyothercathepsins;this notionwasderivedfromanalysesofmiceshortlyafter establish-ingtheCtsk−/−_model,_i.e._when_the_animals_were_still_on_a_mixed

129Ola:C57BL/6Jbackground(Friedrichsetal.,2003).However,in thisstudy,weusedcongenicanimalsontheC57BL6/Jbackground. Immunoblottingrevealed thatthelossof cathepsinKledtoan increaseof cathepsinLexpression,whereas proteinamountsof cathepsinsBandDwerenot,oronlymarginallyelevatedinCtsk−/−

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Fig.4. Thyroglobulindegradationpatternandcross-linkagein5–8monthsoldmice.

WholethyroidlysatesofmicedeficientinMct10,Mct8and/orMct10wereseparatedonverticalorhorizontalSDS-gels,silver-stainedortransferredtonitrocellulose forsubsequentincubationoftheblotswithantibodiesagainstTg,asindicated,andcomparedtotheWT.(A)Displayedisarepresentativeimmunoblotresultandthe correspondingdensitometrydatashowingincreasedamountsofsmallerTgfragments,withmolecularmassesbelow330kDa,inMct8-and/orMct10-deficientmiceas comparedtotheWTmice.(B)Whenseparatingwholethyroidlysatesinnon-reducingconditions,thebandofthedimericTgwascomparableinallgenotypesinvestigated, howeverthemultimericformsofTgwerelessabundantinMct8-andMct8/Mct10-deficientmice.(C)ReducingsampleswithDTTbeforeSDS-PAGEyieldedindecreased intensityofbandsrepresentingthemono-anddimericTginMct8-and/orMct10-deficientanimals,howevermoststrikinginMct8-andMct8/Mct10-deficientmice,which mayindicateincreasedsolubilizationandproteolyticdegradationofTgbycysteinecathepsins.Animalsanalyzed:n=2–3.InA,means±SDaregiven;*,P<0.05.

previousfindings(Friedrichsetal.,2003),andfurthervalidatedthe importanceofcathepsinL-mediatedTgprocessing.

LossofbothcathepsinsKandLleadstodecreasedT4levelsinthe

blood;however,lossofcathepsinKalonedoesnotaffectserumTH levels(Friedrichsetal.,2003).Whileendo-lysosomalTg process-ingiscompensatedforinCtsk−/−_mice_by_cathepsin_L,_the_function

ofcathepsinKinextracellularT4liberationdirectlyfromTgisstill

lacking,leadingustowonderhownormalserumT4levelsare

main-tainedinCtsk−/−_mice._We_reasoned_that_one_possible_mechanism

explainingnormalserumTHlevelsmaybeanupregulationofTH transporters,andtothisextentweinvestigatedMct8expressionin thyroidsofCtsk−/−_mice.

Thesignaldetectedafterincubationofcryo-sectionswith anti-bodiesagainstMct8(Fig.11AandB)wasquantified,anda3-fold increaseinthyroidtissueofCtsk−/−_animals_was_found₍_Fig.₁₁_F).

Likewise,inimmunoblots,Mct8proteinamountswerefoundto beincreasedinthyroidtissuelysatesofcathepsinK-deficientover WTanimals(Fig.11GandH).However,thelocalizationofMct8at thebasolateralplasmamembranedomainofthethyrocytesdidnot changeinCtsk−/−_animals₍_Fig.₁₁_D_and_E).

As described above, alterations in the levels of TH trans-porters feedback on the gross degradation of Tg mediated by endo-lysosomalproteases(seeFigs.3and4).Therefore,we fur-therinvestigatedwhetherthereversesituation,namelyalossof theTg-degradingcathepsinsBandL,isaccompaniedbydifferent expression of the TH transporter Mct8. In mice with single-deficiencies in the cathepsins B or L, or in cathepsin B- and

K-double-deficientanimals, similarobservations weremade,as such,thatMct8stainingintensitieswereincreasedoverWT,but its localization at thebasolateral plasma membrane domainof thyrocytesdidnotchange(Fig.12).However,anti-Mct8-positive punctawereadditionallyobservedinthecytoplasmofthyrocytes ofcathepsinB-,K-,orL-single-andcathepsinB/K-double-deficient mice(Fig.12,arrows).

Thus,thenotionofenhancedamountsoftheT4-exportingMct8

inconditionsofTg-processingcathepsinslackingfrommouse thy-roidepithelialcells(thisstudy)iscounterintuitive,asitmostlikely meansthatthethyroidglanditselfbecomeshypothyroid,especially intheabsenceoftheT4-liberatingcathepsinK(Tepeletal.,2000; Friedrichsetal.,2003),inordertomaintainproperserumlevelsof TH.ThebloodserummustthereforebeviewedasasinkforfreeT4

(HeuerandVisser,2013). 4. Discussion

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Fig.5.ProteinlevelsofcathepsinB,D,andLinthyroidsofTHtransporter-deficientyoungmice.

Wholethyroidlysatesofyoungmice(lessthan2.5monthsold)wereseparatedonhorizontalSDS-gels,transferredtonitrocelluloseandincubatedwithantibodiesagainst therespectiveproteins(leftpanels).DensitometryoftheimmunolabeledbandsrevealedthatcathepsinB,D,andLlevelswereelevatedsignificantlyinMct8/Mct10-deficient animals,howeverthyroidlysatesofMct8-orMct10-deficientanimalsdidnotexhibitelevatedproteinamountswhencomparedtotheWT(rightpanels).Animalsanalyzed: n=6,3,6and10,respectively.HC=heavychain;SC=singlechain;pro=proform.Dataaregivenasmeans±SD;***,P<0.001.

Fig.6.ProteinlevelsofcathepsinB,D,andLinthyroidsofTHtransporter-deficientadultmice.

Wholethyroidlysatesofadultmice(5–8monthsold)wereseparatedonhorizontalSDS-gels,transferredtonitrocelluloseandincubatedwithantibodiesagainstcathepsinsB, DorL(leftpanels).DensitometryoftheindicatedbandsrevealedthatcathepsinDproteinlevelswereelevatedsignificantlyinMct8/Mct10-deficientmice,whereascathepsin BandLlevelswereincreasedinallinvestigatedgenotypeswhencomparedtoWT(rightpanels).Animalsanalyzed:n=3pergenotype.HC=heavychain;SC=singlechain; pro=proform.Dataaregivenasmeans±SD;*,P<0.05;**,P<0.005.

TH-resistantMct8-andMct8/Mct10-deficientmice(Trajkovicetal., 2007;Mulleretal.,2014).Inthisstudy,weproposethatTH trans-porters likeMct8 and Mct10 collectivelymeet the demands of facilitatingthereleaseofTHintothebloodinordertoproperly supportperipheralTHtargetorgans,whilealsoinvolvingin

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cys-Fig.7. ExpressionandlocalizationofcathepsinBinthyroidglandsfromTHtransporter-deficientmice.

CathepsinB(white,red)wasmainlylocalisedinvesiclesofthyrocytesofWT(A)andMct10-(B)Mct8-(C),andMct8/Mct10-deficient(D)animalsbutalsoassociatedwith theapicalplasmamembranedomainandwithinthelumenofthyroidfollicles(EtoH,asterisks).ThyroidtissuetakenfromcathepsinB-deficientanimalsservedascontrols forantibodyspecificity,whichwasconfirmedbylackofprimaryantibodyreactivity(I).Vesicularstaining(arrows)ofcathepsinBismorepronouncedandsignalintensities (J)areslightlyincreasedinboth,Mct8-(G)orMct8/Mct10-deficient(H)mousethyroidswhencomparedtoWT(E).Representativeimagesof3animals(5–8monthsold) pergenotypearedisplayed.J:normalizedsignalintensitypercellgivenasmeans±SD;n=12,8,8,and12,respectively.Nucleiwerecounter-stainedwithDraq5TM_(blue)._L

denotesfolliclelumen.Scalebars:50␮m(A–D,I)and25␮m(E–H).

teinecathepsins,whilethereversesituation,cathepsindeficiency, results in up-regulated T4-exporting capabilities of thyrocytes,

sinceMct8ispresentinenhancedamounts.Although counterintu-itive,thisnotioncontributestoexplanations,whyMct8deficiency leadstointra-thyroidalhyperthyroidstatesandtotheunusual thy-roidstatesinthebloodcirculation(seebelow;MüllerandHeuer, 2014).Instarkcontrast,singledeficienciesinthecathepsinsB,K, andLfeatureeu-oronlyverymildlyhypothyroidstates(Friedrichs etal.,2003).CathepsinKandLdouble-deficiency,only,resultsin hypothyroidisminmice(Friedrichsetal.,2003).

4.1. ThyroidfunctionismaintainedbyTHreleasefromthyroid follicleswhichisenabledbyprotease-mediatedTHliberationfrom Tg

Mct8 knock-out mice exhibit an unusual pattern of altered TH serum levels with low T4 and highly increased T3 levels

(Dumitrescuetal.,2006;Trajkovicetal.,2007;Wirthetal.,2011). AnimalslackingbothMct8andMct10exhibitpartiallynormalized serumT4levels,despiteTSHlevelselevatedoverWTtoanextent

comparabletoMct8-deficientmice,buttheelevatedT3serum

lev-elstypicalforMct8-deficientmicearemaintained(Mulleretal., 2014).Thereasonfor thesehighlyunusualserumTHstatesare consideredtoresideindecreasedT4releasefromthyroidfollicles

duetodysfunctionalMct8-facilitatedexportacrossthebasolateral plasmamembranedomainofthyrocytes(Mulleretal.,2014).Inthe sameanimals,increasedconversionofT4toT3mediatedby

deiod-inase1(Dio1)inliverandkidneyexplainstheabnormallyhighT3

serumlevels.Inaddition,webelievethatthethyroidglandofMct8 knock-outmicereleasesmoreT3duetointactMct10-facilitated transport,whichcontributesfurthertotheunusuallyhighT3/T4

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Fig.8.ExpressionandlocalizationofcathepsinDinthyroidglandsfromTHtransporter-deficientmice.

CathepsinDwaslocalisedinvesiclesdistributedevenlythroughoutthecytoplasminthyrocytesofWT(A),Mct10-(B),Mct8-(C),andMct8/Mct10-deficient(D)mice. Enlargedanti-cathepsinD-positivevesicleswherefoundinMct10-(F),Mct8-(G),andMct8/Mct10-deficient(H)mice(dashed,emptyarrows)whencomparedtotheWT(E). InMct8-deficientmice,cathepsinDstainingwasobservedatthebasalsurfaceofthyrocytes(G,arrows),orattheplasmamembranedomainofendothelialcells(G,asterisks). Dottedlines(E-H)representoutlinesoffollicleepithelia.Representativeimagesof3animals(5–8monthsold)pergenotypearedisplayed.Ldenotesfolliclelumen.Scale bars:50␮m(A–D)and10␮m(E–H).

theTHtransporter-deficientmice.Indeed,itislikelythat Mct8-deficient thyrocytesare stimulated more strongly than thyroid folliclecellsofMct8/Mct10-doubledeficientmice,becauseTRH lev-elsarehigherinMct8-deficientanimals(Mulleretal.,2014).Thus, intra-thyroidalTHproductionandTHliberationfromTgismost likely de-regulateddifferently in Mct8−/y _and _Mct8−/y_/Mct10−/−

animals.Therefore,weinvestigatedtheTgstatusandthe molec-ularpathwaysofTgutilizationforTHliberationbyanalyzingthe keyplayersinvolvedinproteolyticprocessinganddegradationof Tg,thecathepsins(seeTable1andFig.1).

ThedatacollectedinthisstudybyinspectingtheTgstatusof THtransporter-deficientmice(seeFigs.2–4),suggeststhatbothTg solubilizationfromcovalentlycross-linkedstorageformsby extra-cellularTgprocessing,anditsutilizationforTHliberationby extra-andintracellularmeansofproteolyticTgdegradationareenhanced inMct8-and/orMct10-deficiencies.Therefore,wepostulatedthat Tg-processingenzymesmaybedifferentlyexpressedandlocalised inthyroidtissueofMct8-and/orMct10-deficientmice.

Secretionof cathepsin B at the apical pole of thyrocytes is increaseduponacuteTSHstimulation,i.e.1–2hafterTSH stimu-lation,whereas enhancedbiosynthesisofcathepsinBwilloccur only during long-term chronic TSH stimulation (Linke et al., 2002a,b).Accordingly,becausetheelevatedTSHlevelsin Mct8-orMct8/Mct10animalsarechronic(DiCosmoetal.,2010;Muller etal., 2014), we expectedincreasedprotein levelsof cathepsin B,which wereindeedobservedbyimmunoblotting(seeFig.6). Moreover,theintra-follicularlocalizationofcathepsinBdiffered fromWT,inparticularinMct8-andMct8/Mct10-deficientmice, suchthattheproteasewasalmostexclusivelypresentinenlarged endo-lysosomesofthethyroidepithelialcells(seeFig.7).

Anotherimportantproteinthatisinvolvedinendo-lysosomalTg degradation,butlesssoinTHliberationfromTg,istheaspartic pro-teasecathepsinD(Dunnetal.,1991;Jordansetal.,2009).Inmouse thyrocytes,cathepsinDisusuallylocalisedinvesiclesdistributed throughout thecytoplasm and accumulatingin theperinuclear region,i.e.inlysosomesofthyrocytes(Friedrichsetal.,2003).In

thisstudy,cathepsin Dwasfoundinimmenselyenlarged endo-lysosomal vesicles of Mct8- and Mct8-/Mct10-deficient mouse thyrocytes(seeFig.8),indicatingenhancedabilitiesofthesefollicle cellstocompletelydegradeTgbyintracellularmeansoflysosomal proteolysis.Moreover,cathepsinDwasfoundtobesecretedfrom thyrocytesofMct8-deficientmiceatthebasolateralpole.This find-ingisinterestingwhenconsideringtherolethatcathepsinDplays incancer(Brixetal.,2008;Brixetal.,2015).Mct8-deficientmice developpapillarythyroidcarcinoma(PTC)atolderage,possibly duetoalossofauto-regulativecontrolofthyrocyteproliferation (Wirthetal.,2011).However,basolaterallysecretedcathepsinD mayfurthercontributetotheprogressionofPTCdevelopmentas itcan,inprinciple,enhanceextracellularmatrixdegradation,and therebyfacilitatecancerprogression.

CathepsinLplaysavitalroleinthyrocytefunction,anditwas showntobeimportantforsurvivalofthyrocytes,sincethe respec-tiveknock-outmodelsshowedluminalinclusionsofdeadcellsand deadcellremnants(Friedrichsetal.,2003).Hereweshowthatthe availabilityofcathepsinLintheextracellularfolliclelumenismore pronouncedinMct8/Mct10-deficientmicethaninMct8−/y _mice

(seeFig.9),suggestingenhancedTgsolubilisationforsubsequent endocyticuptake.Inaddition,thisenzymewithacriticalfunction inmaintainingproperserumlevelsofT4(Friedrichsetal.,2003)

wasenhancedinproteinamounts,inparticular,inMct8andMct10 deficientmousethyrocytes.

Hence,theproteinlevelsoftheTg-processingproteases corre-latewiththelackofTHtransporterproteinslikeMct8and/orMct10. Theseobservationsmostlikelycontributetoexplainingwhyamong theTHtransporter-lackinggenotypesinspectedinthisstudy, espe-ciallytheMct8−/y_/Mct10−/−_mouse_model_features_the_most_severe

auto-thyrotoxicstates(Mulleretal.,2014).In reverse,themain T4-exportingtransportermoleculeMct8isup-regulatedinCtsk−/−

mice,aTgprocessingenzymethatisabletodirectlyliberateT4

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Fig.9. ExpressionandlocalizationofcathepsinLinthyroidglandsfromthyroidhormonetransporterdeficientanimals.

CathepsinLwasdetectedinvesicularstructuresinallgenotypes(A,B,C,andD),exceptintissuefromcathepsinL-deficientmice(G),whichservedascontrolforantibody specificity.FewfolliclesofWT(A)andMct10-deficient(B)animalsshowedapicalandluminalcathepsinLstaining,howeverintensitiesofcathepsinLlabelling(H)were increasedinMct8and/orMct10-deficientmice(BandC)whencomparedtoWTanimals(A).Notetheincreasedluminal,apicalandvesicularstainingpatternsandintensities ofcathepsinL(green)inthyrocytesofMct8/Mct10-deficientanimals(F)comparedtotheWT(E),andintherespectivehighermagnifications(E’andF’).Representative imagesof3animals(5–8monthsold)pergenotypearedisplayed.Nucleiwerecounter-stainedwithDraq5TM_(blue)._H:_normalized_signal_intensity_per_cell;_mean_+/−_SEM;

n=1193,1422,1309,and1638,respectively.Ldenotesfolliclelumen.Scalebars:50␮m(A–D,G)and10␮m(E,F).*,P<0.05.

2003),whichalsofeatureenhancedMct8atthebasolateralplasma membranedomainsofthyrocytes(thisstudy).

Such correlations between TH-liberating proteases and TH transportersaresomewhatcounterintuitive,andindicatealossof THsensingcapabilitiesofthyrocytesuponfunctionallossofeither key-playerenablingthyroidglandfunctions(seeFig.13).

4.2. Thyroidauto-regulationtoleratesminorchangesinthyroid statesbyredundancy,butaself-destructiveviciouscycleis switchedonwhenthekey-playerscathepsinLandMct8are affectedbydisease

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Fig.10.ProteinlevelsofcathepsinsB,D,andLinthyroidsofcathepsinK-deficientmice.

WholethyroidlysateswereseparatedonhorizontalSDS-gels,transferredtonitrocelluloseandincubatedwithantibodiesagainsttherespectiveproteins.(A)Immunoblots ofcathepsinsB,D,andLwerequantifiedbydensitometry(B).CathepsinLlevelswereelevatedsignificantlyinCtsk-deficientanimals.Animalsanalyzed:n=3pergenotype. HC=heavychain;SC=singlechain.Dataaregivenasmeans±SD;*,P<0.05.

ofTHtransporters(asinCtsb−/−_,_Ctsk−/−_,_Ctsl−/−_,_or_Ctsb−/−_/Ctsk−/−

mice),orbyenhancingcathepsin-mediatedTg-processing(asin Ctsk−/− _mice), _or _both, _without _the _necessity _of _{TSH-triggered}

classicalregulation(seeFig.13).Suchauto-regulatorypathways, drivenexclusivelybyintra-thyroidalmeans,mightbebasedupon thyrocyte-specificmeasuresofTH-sensing.Thus, withinthyroid follicles,thethyroidepithelialcellsdetecttheamountsofTH lib-erated,andintegratethesewiththoseTHamountsexportedfrom thefolliclesforglobalsupply(Fig.13,WT).Suchapathwayof TH-sensingwithinthyroidepithelialcellsofindividualfolliclesmost likelyrequirestheinvolvementofTHtransportersactingat differ-entcellularmembranes(Fig.13).Interestingly,althoughdifferent TH transporterproteins areexpressed in mousethyrocytes (Di Cosmoetal.,2010),compensatoryup-regulationofTHtransporters inMct8-deficientanimalshasnotbeenfound(DiCosmoetal.,2010; Wirthetal.,2011).Incontrast,enhancedTgdegradationand uti-lizationtakesplacewhenMct8andMct10arelacking(thisstudy). Eventually,thisleadstoincreasedintra-thyroidalTHlevels,which shouldfeedbacktothethyrocytetostoptheelevationofTH produc-tionatonepoint.Ironically,andagainstalllogics,sucharegulatory feedbackdoesnothappeninMct8/Mct10-deficientanimals.

Insummary,theresultsofthisstudyprovideamolecular mech-anisticexplanationforthealteredthyroidfunctionalstatesofmice withMct8-and/orMct10-deficienciesinthatenhancedTg degra-dationbycathepsinsBandL(actingextra-andintracellularlyonTg) andbyintra-lysosomallyactingcathepsinDoccurs,althoughthisis counterintuitiveandlikelyworsenstheintra-follicularthyrotoxic states.

At first glance, up-regulation of Mct8 in thyroid glands of animals lacking the Tg-processing cathepsin proteases (Figs. 11 and 12) makes more sense as the export of T4 from

the thyroid follicles would be facilitated in suchconditions of enhancedMct8atthebasolateralplasmamembraneofthyrocytes.

However,againcounterintuitively,thethyroidglanditselfmight becomehypothyroid,atleasttransiently,attheexpenseof main-tainingproperTHsupplytotheperipheralorgans,inparticular,in Ctsk−/−_mice₍_Friedrichs_et_al.,₂₀₀₃_)._Within_the_thyroid_follicles_of

Ctsk−/−_mice,_such_states_of_too_little_TH_liberation_due_to_lacking

cathepsin K-mediated Tg-processing by extra-and intracellular meansofproteolysisareobviouslysensedandcounter-actedsuch thattheyeventuallyleadtoacompensatoryup-regulationofthe amountsofcathepsinL(Friedrichsetal.,2003).

4.3. Perspectives

In conclusion, we propose that the intra-thyroidal TH sens-ingmechanismspresentinCtsb−/−_,_Ctsk−/−_,_Ctsl−/−_,_Mct8−/y_,_and

Mct8−/y_/Mct10−/−_mice_are_enabled_such_that_different_TH

trans-porters are present in specific subcellular locations of thyroid epithelial cells, thereby selectively monitoring individual steps ofthyroglobulinprocessingandthus,thecellularTHstatus(see

Fig.13).Hence,futurestudiesmustelucidatethemolecularnature oftheseproposedintra-thyroidalT4-sensingmechanismsbeyond

Mct8,inordertounravel,howT4-sensingbyMct8and/orMct10at

thebasolateralplasmamembranedomainismechanistically con-nectedtoregulationoftranslationandtraffickingofTg-processing proteaseswithinindividualfollicles,andhowpreciselythe associ-ationwithendo-lysosomalfunctionsismaintained.Wepropose aninvolvement oftheTH transporterLat2, which ispresent at theendo-lysosomalmembraneofthyrocytesinWTmice(McInnes etal.,2013;Weberetal.,2015).

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Fig.11.Mct8expressionlevelsandlocalizationinthyroidtissueofcathepsinK-deficientmice.

SignalintensitieswerequantifiedinmicrographsofcryosectionslabeledwithantibodiesagainstMct8(A,B).TissueofMct8-deficientanimalsservedtocontrolforstaining specificityoftheprimaryantibody(C).NotethesignificantincreaseofMct8incathepsinK-deficientmicewhencomparedtotheWT(F).However,thelocalisationofMct8 (green)atthebasolateralplasmamembranedomain(DandE)didnotchangeincathepsinK-deficiency.WholethyroidlysateswereseparatedonhorizontalSDS-gels, transferredtonitrocelluloseforsubsequentincubationoftheblotswithantibodiesagainstMct8(G)andthequantificationbydensitometryrevealedelevatedMct8levels inCtsk-deficientmice(H).F:animalsanalyzedwere8and9,respectively;dataaregivenasmeans±SD;n=41and49follicles,respectively.H:animalsanalyzedn=6,and 6,respectively.Scalebars:40␮m(A–C)and10mm(DandE).Nucleiwerecounter-stainedwithDraq5TM(blue).***,P<0.001.

andTgbiosynthesis(Brixetal.,2001).Thus,itwillbeimportantin futurestudiestodiscriminatebetweenTSH-dependentregulation andthyroidauto-regulationthatisindependentofthesystemic HPT-axis.Inthisregard,wearecurrentlyanalyzingknock-out mod-elsfeaturingthyroid-specificMct8-deficiency.

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Fig.12.Mct8expressionlevelsandlocalizationinthyroidtissueofWTandcathepsinB,K,L,andB/K-deficientmice.

ConfocalfluorescencemicrographsofcryosectionslabeledwithantibodiesagainstMct8(green)revealedenhancedstainingintensityincathepsin-deficientmice(B–E)when comparedtotheWT(A).However,thelocalizationofMct8atthebasolateralplasmamembranedomaindidnotchangeincathepsin-deficiencies.Thyroidof cathepsin-deficientmicewerecharacterizedbyanti-Mct8-positivepuncta(arrows)whichweremostabundantinCtsl−/−_(D)_and_Ctsb−/−_/Ctsk−/−_(E)_mice._Scale_bars:₁₀_␮m._Nuclei

werecounter-stainedwithDraq5TM_(blue).

Fig.13.TheinfluenceofcathepsinKandTHtransporterdeficiencyonliberationofTHandtheirreleasefromthyroidfollicles.

Tgissynthesisedandsecretedattheapicalplasmamembraneofthyrocytes(WT,left)tobestoredincovalentlycross-linkedforminthelumenofthyroidfollicles.Upon TSHstimulation,cysteinecathepsinsaresecreteduponretrogradetrafficking,andcathepsinsBandLareinvolvedinsolubilizationofTg,whereascathepsinKisinvolvedin directT4liberationinthefolliclelumen(WT,upperportion,andlowerpanel),whichmaybetransportedacrosstheapicalplasmamembranedomainbyanasyetunknown

T4transporter(cyan).Then,TgandpartiallydegradedTgmoleculesareendocytosedfordeliverytoearlyandlateendosomesaswellaslysosomes,whereT4andT3canbe

liberatedbytheintracellularproteolyticactionofcysteinecathepsinsbeforebeingtransportedacrosstheendo-lysosomalmembranesintothecytosol(darkpink).T4may

thenbedeiodinatedandthusactivatedtoT3,orT4isreleasedfromthethyrocytesbytransportacrossthebasolateralplasmamembranedomain(chartreuse).

MicedeficientincathepsinKexhibitnormalT4serumlevels,whileMct8deficiencyresultsinreducedT4andslightlyelevatedserumTSHconcentrations.TSHinfluencesthe

increaseofTHproductionandlikewise,expressionofcathepsinsiselevateduponlong-termTSHstimulation(Linkeetal.,2002b).MicewithdeficienciesinMct8andMct10 exhibitsimilarTSHlevelsasMct8-deficientmicebutdifferentserumT4levels.UponlossofcathepsinK(Ctsk−/−),weobservedup-regulatedcathepsinLexpressionand

activity(Friedrichsetal.,2003;redarrows)andenhancedexpressionoftheTHtransporterMct8atthebasolateralplasmamembranedomainofthethyrocytes(thisstudy; greenarrows),suggestingT4releasefromthyroidfolliclestobefavoredoverself-sustainingregulation,therebyrenderingthethyroidglandhypothyroid.Uponlossofboth

Mct8andMct10(Mct8−/y_/Mct10−/−_),_we_observed_altered_{cross-linking}_and_{multimerization}_of_Tg_which_may_have_led_to_enhanced_utilization_of_Tg_for_TH_liberation_(red

arrows)andtherebyincreasingtheintra-thyroidalTHlevels(Mulleretal.,2014).WespeculatethatthelossofMct8(greencrosses)andMct10fromthebasolateralplasma membranedomainofthyrocytesledtoalossofsensingcapabilityfortheamountofliberatedTHbycathepsins,whichyieldedinincreasedprocessinganddegradationof Tgdespitetoxicintra-thyroidalTHconcentrations,therebyhoweveralsocontributingtothenormalserumT4levelsinMct8/Mct10-deficientmice.

releasefromthyrocytes,beforeTgiseventuallystoredin cross-linkedform in the thyroid folliclelumen.Both, Tgfolding and Tgcross-linkingarechaperonedandmediatedbyprotein disul-fideisomerase(PDI;Berndorferetal.,1996;MuresanandArvan,

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theTgmoleculefurtherfeaturesthepresenceofdifferentinternal homologydomains(forreview,seeBrixetal.,2001),someofwhich containtheaforementionedCXXC-motifs ofthioredoxins,while othersarecharacterizedbyCWCV-motifsthatareindicativeofthe so-calledthyropins,i.e.smallproteinsorpeptidesactingascysteine peptidaseinhibitors(Lenarcicetal.,1997).Clearly,futurestudies areneededinmicetoexplorethesignificanceofTgcross-linking andthepossibleinterconnectionswithitsdegradation,sinceboth arepotentiallysubjecttointra-thyroidalauto-regulation,i.e.byTg itself(RoussetandMornex,1991;Brixetal.,2001).

Funding

ThisworkwassupportedbytheDeutsche Forschungsgemein-schaft,DFG,grantBR1308/11-1and11-2toK.B.,WI3768/1-1to E.K.W.,SCHW914/3-1toU.S.,andHE3418/7-1and7-2toH.H.in theframeworkofthepriorityprogramSPP1629/1and/2‘Thyroid TransAct’.

Disclosurestatement

Theauthorsdeclarethatnoconflictofinterestexists. Acknowledgements

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