Immunostaining study of cytokeratins in human hair follicle development,

Anais

Brasileiros

de

Dermatologia

www.anaisdedermatologia.org.br

INVESTIGATION

Immunostaining

study

of

cytokeratins

in

human

hair

follicle

development

夽,夽夽

Laura

Maria

Andrade

Silva

_,

_Ricardo

_Hsieh

_,

Silvia

Vanessa

Lourenc

¸o

_,

_Neusa

_Yuriko

_Sakai

_Valente

_,

_Geise

_Rezende

_Paiva

_,

Juliana

Dumet

Fernandes

a_{GraduatePrograminMedicineandHealth,FacultyofMedicineofBahia,UniversidadeFederaldaBahia,Salvador,BA,Brazil} b_{ServiceofDermatology,EscolaBahianadeMedicinaeSaúdePública,Salvador,BA,Brazil}

c_{ServiceofDermatology,HospitalSantaIzabel,Salvador,BA,Brazil}

d_{DepartmentofDermatology,FaculdadedeMedicina,UniversidadeFederaldaBahia,Salvador,BA,Brazil} e_{InstitutodeMedicinaTropicaldeSãoPaulo,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil}

f_{DepartmentofPathology,UniversidadeFederaldeAlfenas,Alfenas,MG,Brazil} g_{FaculdadedeOdontologia,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil}

h_{DepartmentofDermatology,FaculdadedeMedicina,HospitaldasClínicas,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil} i_{SectorofPathology,HospitalSantaIzabel,EscolaBahianadeMedicinaeSaúdePública,Salvador,BA,Brazil}

Received6June2019;accepted20September2019

Availableonline21March2020

KEYWORDS Hairfollicle; Hair-specific; Keratins; Molecular; Pathology Abstract

Background: The hair follicleis aunique structure, oneof themostdynamic structures in

mammalians,whichcanreproduceineverynewcycleallthemechanisminvolvedinitsfetal

development.Althoughalotofresearchhasbeenmadeaboutthehumanhairfolliclemuch

lesshasbeendiscoveredabouttheimportanceofthecytokeratins(CKs)initsdevelopment.

Objective: StudytheimmunohistochemicalpatternofepithelialCKsduringhumanhairfollicle development.

Methods: Weperformed an immunohistochemicalstudy using fresh post-mortemskin

biop-siesofhumanfetusesbetween4and25weeksofgestationalagetostudytheexpressionof

cytokeratins(CKs): CK1,CK10,CK13, CK14,CK16andCK20 duringhuman hairfolliclefetal

development.

Studylimitations: Restrospectivestudywithagoodnumberofmakersbutwithasmall popu-lation.

夽 _{Howtocitethisarticle:SilvaLMA,HsiehR,Lourenc¸oSV,ValenteNYS,PaivaGR,FernandesJD.Immunostainingstudyofcytokeratinsin} humanhairfollicledevelopment.AnBrasDermatol.2020;95:278---82.

夽夽_{StudyconductedattheInstitutodeMedicinaTropicaldeSãoPaulo,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil.} ∗_{Correspondingauthor.}

E-mail:contato@dralauraandrade.com(L.M.Silva).

https://doi.org/10.1016/j.abd.2019.09.028

0365-0596/©2020SociedadeBrasileiradeDermatologia.PublishedbyElsevierEspa˜na,S.L.U.ThisisanopenaccessarticleundertheCC BYlicense(http://creativecommons.org/licenses/by/4.0/).

Results/conclusion: Wefound that, theCKswere expressedinanintermediatetimeduring

folliculardevelopment.TheepithelialCKs(CK1,CK14,CK10,CK13)andtheepithelialCKswith

aproliferativecharactersuchasCK16wereexpressedfirst,asmarkersofcellularmaturation

andfollicularkeratinization.Atalaterphase,CK20wasexpressedinmoredevelopedprimitive

hairfolliclesaspreviouslydiscussedinliterature.

©2020SociedadeBrasileira deDermatologia.PublishedbyElsevierEspa˜na,S.L.U.Thisisan

openaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

Introduction

The hair follicle is in constant renewal, reproducing the sameembryonicmechanismsthatformeditfirstinthefetal period.1_{Thehairfolliclesdevelopatearlyphasesof}

follicu-lardevelopment,aroundthe9thand12thweekoriginating fromagerminativestratumoftheepidermis’sproliferation. Thekeratinocytesdifferentiateandformtheouterroot she-ath(ORS),thecompanionsheath,theinnerrootsheath,and thehairshaftitself.2_{Itsdevelopmentrespectsacranial}

cau-dalpatternofdevelopmentwiththefirsthairfolliclesbeing observedatfaceandeyebrow.2

Anextremelychallengingareaishowfollicular develop-mentanditsmaturationoccurs,folliculardevelopmentand itssubsequentdifferentiation involvesacomplexseriesof eventsthatgeneratecellmatrixremodeling,moleculessuch asproteoglycansandgrowthfactorsareimplicated inthis process.3---6

Cytokeratins(CKs)arethemainproteinsthatstructure epithelial cells, they provide shape,resistance and main-tenanceoftheintercellularcontacts;inthehairfolliclein additiontothestructuralfunction,theyaremarkersof folli-cularmaturationandcytodifferentiation.Theyaredivided intotwofamilies,Type1, acidkeratins andType 2,basic keratins.7_{Intotal,twenty-sixkeratinsareconsidered}

speci-fictohairfollicle,comparabletothetwenty-nineexpressed intheepithelium.8

ArangeofmutationsinCKsarerelatedtodiseasesthat affect hair structure and density in humans and animals. Diseasessuchashypotrichosisandmonilethrixhavealready been linked to specific mutations of some CKs.9---11 _Other

diseases that mayaffect thehair follicle aslichen planus pilarisshowanalteredpatternintheexpressionofcertain CKsthatcouldbeusedasearlymarkersforthisdisease.12,13

Thus,CKs maybetargets fordrugdevelopmentand diag-nosticmethods thatmay assistin the early diagnosis and treatmentofhairfolliclediseases.

Althoughthereisalreadyavastliteratureonhair folli-clesandepithelialCK,littleisknownabouttheexpressionof epithelialCKduringthedifferentiationofhumanhair folli-cles.ConsideringtheimportanceofCKinthedevelopment ofhair follicles andthescarcity ofinformationabout this subject,westudiedthepatternofepithelialCKexpression inthedevelopinghumanhairfollicle.

Methods

A retrospective study involving histological sections from hairyareasfromskinfragmentsofhumanembryonic/fetal derivatives from different gestational ages (4---25 weeks)

wereobtainedfromthearchivesof theDermatopathology Laboratory,Departmentof Dermatology, University ofSão Paulo.ThestudysubmittedtotheEthicsCommitteeofthis institution.CEPno.0172/08.Theanalyzedregionswerethe sameandstandardized inthegroups as: scalp,face, eye-brow,upperlimb,backandgenitalia.Themorphologiesof thestructureswerepreviouslyevaluatedinhematoxylinand eosinforbettervisualizationofthestructures.Inthese sec-tions,an immunohistochemical study wasperformed with thefollowing markers: CK1, CK10,CK13, CK14, CK16and CK20.

Immunohistochemicalprotocol

The 3␮m sectionswere dewaxed and diaphanized in two xylolbaths.Thespecimenswerethenrehydratedinethanol drop-offandimmersed in 10% ammonium hydroxide solu-tion.Recoveryoftheantigenicsitesoccurredinvariousways dependingontheprimaryantibodyused(Table1).

Afterwashing,thematerialwasincubatedin20volumes hydrogenperoxidesolution,andthen thespecimenswere immersedtwice in TRIS pH 7.4 solutionand subsequently incubatedwith0.5%BSA(Sigma)solution,2.5%SFB (Culti-lab)inTRISandincubatedwiththeprimaryserumdiluted in 1% BSA (Sigma) solution in TRIS. Subsequent procedu-res were always preceded by washes in the TRIS buffer. Afterincubationwiththeprimaryantibody,thespecimens wereincubatedwithEnVisionreagent(Dako).Forthe deve-lopment, the specimens were incubated in the solution containingDABagent(Sigma).Thehistologicalsectionswere then counterstained withCarazzi Hematoxylin and subse-quently dehydrated in upstream alcohols, diaphanized in three xylol baths, and mounted on Permount® _{resin for}

microscopicexamination.Negativecontrolswereperformed byincubatingspecimenswithnon-immuneserum.All immu-nohistochemicalreactionswereperformedinparallelwith adulttissuesasnegative/positivecontrols.

The resultsobtainedinthe immunohistochemical reac-tions were analyzed by two observers and qualitatively tabulated.Thespecimensthatshowedimmunostainingwere consideredpositive(+),thespecimensthatdidnotpresent immunoexpressionwereconsiderednegative(−).

Alltheresults wererecordedbytheAxioVision digital photomicrographsystem(ZeissandAxiophot).

Results

73specimensfromhairyareasofembryosorfetusesranging from4to25weeksofgestationalagewereevaluated.The followingmarkerswereevaluated:CK1,CK10,CK13,CK14,

Table1 Monoclonalantibodiesusedinstudyandprotocols.

Primaryantibody Clone Company Antigenicretrieval Dilution Incubation

CK1 34BB4 NovoCastra Microwave 1:25 ‘‘Overnight’’

CK14 AB-1 Neomakers Microwave 1:40 ‘‘Overnight’’

CK16 DE-K10 Dako Microwave 1:50 ‘‘Overnight’’

CK10 DE-K10 Dako Microwave 1:50 ‘‘Overnight’’

CK13 M7003 Dako Microwave 1:50 ‘‘Overnight’’

CK20 Ks20.8 Dako Microwave 1:50 ‘‘Overnight’’

Table 2 Resume of cytokeratins’s immunoexpression at

humanhairfollicledevelopment.

Marker Immunostannig percetual Firstgestational age immunoexpression CK1 25% 12weeks CK14 100% 9weeks CK10 55% 12weeks CK13 70% 12weeks CK16 61% 12weeks CK20 57% 14weeks

CK16andCK20,intheregionssuchas:scalp,face,eyebrow, upperlimb,backandgenitalsandtheresultscanbe

evalua-tedinTable2andFig.1.Weanalyzedallthecasesobtained

inthefilesoftheDermatopathologyLaboratoryreferringto hairyareasofeachmarker.Atotalof12CK1specimens,14 CK14specimens,9CK10specimens,10CK13specimens,21 CK16specimensandonly7CK20specimenswereobtained.

CK1,CK14,CK10andCK13

Immunostaining for CK1, CK10, CK13 wasnoted from the 12thweek gestationontheprimitivehair follicles andup tothe25th gestationalweekonthe mostfully developed follicles.25%,55%,70%ofthespecimensmarkedpositively forCK1,CK10andCK13respectively,inORS,intheterritory ofthefollicularostium,infundibulum,sebaceousglandand dermalpapilla.Themain markedregionsweretheeyelid, faceandgenital.ImmunostainingforCK14wasseenin pri-mitive follicles of 9week gestationembryos inthe upper limbandback,andeveninmoredevelopedfolliclesat 21 gestationalweeks.100%ofthecasesmarkedpositivelyfor CK14inORS.Allregionsanalyzedwerepositivelymarked.

CK16andCK20

Immunostainingfor CK16wasseen inprimitivefollicles of 12weekgestationembryosinthefacialregionandinmore developed 23week gestationalfollicles.61%of the speci-mensmarkedpositivelyforCK16atORS.Allregionsanalyzed

Figure1 Cytokeratin’simmunostaininginhumanhairfollicledevelopment.(A)CK1immunoexpressionat19weeksfetus.(B)

CK14immunoexpressionat12weeksfetus.(C)CK20immunoexpressionat21weeksfetus.(D)CK16immunoexpressionat12weeks

weremarkedpositively.ImmunostainingforCK20wasseen fromprimitivefolliclesof14weekgestationembryosinthe scalpregiontomore developed25 week gestational folli-cles. 57%fetuses marked forCK20 inthe territory ofORS anddermalpapilla.Allregionsanalyzedweremarked.

Discussion

ThisstudyevaluatedthepatternofepithelialCKexpression indevelopinghumanhairfollicle.Therearefew papersin theliteratureaboutthissubject.

Schirrenetal.werethepioneersshowingthatthe folli-cularcellsofthehumanfetalplacodesexpressedCK5,CK6, CK14,CK17andCK19,andthatsubsequentlywithfollicular developmentthepatternofexpressionofthoseCKchanges intheeachportionofthehairfollicle.14

Our data showed that between 9 and 14 gestational weeks, CK1, CK14, CK10, CK13, CK16 and CK20 became expressed.Onlytwoofourmarkerswereevaluatedby Schir-ren et al., CK14 and CK10, these CKs were found in the developing hair follicles, CK14 in the earliest phases still intheplacoidphaseandatlaterphases,CK10wasexpress in a slightly more differentiated hair follicle in the isth-musregioninORSatthesuprabasalcellsofinfundibulum along withCK14. Theresults ofthat workweresimilarto ours;theimmunostainingmaintainedthecranialpatternof distribution.

In our study,CK14 wasthe firstCK tobe expressedin embryos with less than 12 weeks of life, around the 9th gestationalweek.Initially,bothCK1andCK14were expres-sedin theregionontheisthmus.In2008, Lourenc¸oetal. whenanalyzedtheexpressionpatternofCKsinthe primi-tivefetal epidermis,showedCK1 expressionwasnotedin allepidermallayerswhileCK14wasnotedpredominatedin theperifollicularepidermalregion.15

Inourstudy,CK1,CK10,CK13andCK16wereexpressedat 12weeksofgestationalage.StudiesinvolvingCK1andCK14 at Shh signaling pathway have shown conflicting findings. WhenoccurstheoverexpressionofCK1atmicesin embryo-nicperiod,theywereunabletodevelophairfollicles.Inthe otherexperimentalmodel,whenoccursthesuperexpression ofCK14,afollicularhyperproliferationoccurred,inaddition tomultiplelesionssuchbasalcellcarcinomaintheanimals analyzed.16,17

It is reported in the literature that hyperproliferation ofepitheliallayerscausedbyoverexpressionofCK1during embryonicdevelopmentaltersthe abilityofbasal cellsto initiateasuitablecelldifferentiationprogramforhair folli-cleformation.16_{Thus,CK1andCK14playsanimportantrole}

in theformation ofthe fetalhair folliclethroughtheShh signaling cascade. The exact time, location,and concen-tration of expression of these Cks in this Shh signaling pathwayaffect thehairfolliclephenotypeinthe embryo-nicfetalperiod,butwithout changesinthepostnatalhair follicles.16

CK10isanepithelialCKclassicallyrelatedto Epidermoly-tic hyperkeratosis,eventual mutations may berelated to thisdiseaseorsimilarones,18_{inourworkitwasseenmarking}

thehairfolliclesinORS,aswellasCK1andCK14.

Inourresults,CK16wasexpressedinmoreproliferative regionsofhairfollicle,mainlyinthebulbregion.CK16,as

wellasCK6and CK17areproliferative andrepairing CKs, beingexpressedduringdiseasesandaroundhealingtissues. Corroboratingwithourfindings, itis described thatthese hyperproliferative CKs are expressed in the hair follicles atan intermediatestageof development,when epithelial differentiationalreadyexists.14

CK13isanepithelialCKalreadyusedasan immunotar-getagentfororal intraepithelialneoplasia,morerecently itsexpressionwithCK17hasbeenevaluatedasan immuno-targetforvulvarintraepithelialneoplasiaaswell.Ourwork evaluatedCK13 at the developing hair follicle and sawit markingfetalhairfollicles’sORSaswellastheother epithe-lialCKs.19

CK20isamarkerforMerkelcells.Asintheliterature,its immunoexpressionwasdetectedinhairfollicles atalater stageof development,fromthe 14th week ofgestational age.The literaturereportsthepresenceofMerkelcellsin theORSofhairfolliclesofrodentvibrissae,inhumansCK20 isreportedtomarkatthebasallayeroftheinfundibulum atprimitivefetalhair follicles,notmarkingatdeepareas of thehair follicles, includingthe bulb and the dermis.20

Inourwork,accordingtotheliterature,wefound expres-sionforCK20inthehairfolliclesalreadydevelopedaround the25thgestationalweekinthedermalpapillaandinthe ORS,butalsointhefolliclesintheintermediateperiodof developmentwith14gestationalweeks.

Conclusion

Hairfolliclemorphogenesis is arestructuring of epithelial tissueand mesenchymal tissue to forma completely dif-ferentiatedpilosebaceousunit.Wehave observedthatCK expressionpatternsduringhairfollicledevelopmentareas dynamicasseen by other authors in theepidermis.15 _CKs

begintheir expression in human hair follicles around the 9thgestationalweek,withCK14beingthefirsttobe identi-fiedandwiththehighestpercentageofimmunostaining.Our dataconfirmtheliteratureshowingthatCKsaremarkersof maturationofhair follicledevelopmentandareexpressed fromthe follicular placoid phase. Initially the expression ofthese CKs coincideswiththe CKs expressedin the pri-mitiveepithelium:CK1,CK14,CK10,CK13andproliferative CK:CK16.Aspreviouslyreportedintheliterature,CK20was expressedin more developed hair follicles fromthe 14th gestationalweek.Theresultswhentakentogetherindicate thatfromtheearlieststagesoffolliculardevelopmentthe hairfolliclesprogressivelyandcoordinatelyprepareto pro-tectthefetalskinforbirthandenvironmentalstimulation. Understandingof thesematuration steps is important for understandingthestructureofnormalhairfolliclesandtheir adultfunctionin healthanddisease.Ourwork hasserved tofillagapintheliteratureonapoorlystudiedtopic.We alsobelievethat byidentifyingpatternsof differentiation of CKs in hair follicles in humans, we can raise questi-onsandpromptinvestigations,andopenthepossibilityfor regenerativetherapies, identification of early biomarkers andpotential diagnostic targets for hair follicle diseases. However,furtherinvestigativestudiesareneededtobetter evaluatethedevelopmentandproteinexpressionofhuman hairfolliclesandtocollaborateinthediagnosisand treat-mentofdiseasesrelatedtothisstructure.

Financial

support

FUNADERM --- Fundo de Apoio à dermatologia Fundac¸ão de Apoio ao dermatologista (CAPES - Coordenac¸ão de Aperfeic¸oamentodePessoaldeNívelSuperior).

Authors’

contributions

LauraMaria Andrade Silva: Statistic analysis; approval of thefinalversionofthemanuscript;elaborationandwriting of themanuscript; obtaining, analysis,and interpretation ofthedata;effectiveparticipationinresearchorientation; intellectualparticipationinthepropaedeuticand/or thera-peuticconductof thestudied cases;criticalreviewofthe literature;criticalreviewofthemanuscript.

Ricardo Hsieh: Approval of the final version of the manuscript;conception and planning of the study; effec-tiveparticipationinresearchorientation;criticalreviewof themanuscript.

SilviaVanessaLourenc¸o:Conceptionandplanningofthe study;effectiveparticipationinresearchorientation; intel-lectualparticipationinthepropaedeuticand/ortherapeutic conductofthestudiedcases;criticalreviewofthe manus-cript.

Neusa Yuriko Sakai Valente: Approval of the final ver-sion of the manuscript; conception and planning of the study;effective participation in researchorientation; cri-ticalreviewofthemanuscript.

GeiseRezendePaiva:Approvalofthefinalversionofthe manuscript;obtaining, analysis, andinterpretation of the data;criticalreviewofthemanuscript.

JulianaDumetFernandes:Approvalofthefinalversionof themanuscript;conceptionandplanningofthestudy; ela-borationandwritingofthemanuscript;obtaining,analysis, andinterpretationofthedata;intellectualparticipationin thepropaedeuticand/ortherapeuticconductofthestudied cases;criticalreviewoftheliterature;criticalreviewofthe manuscript.

Conflicts

of

interest

Nonedeclared.

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