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Melanocytes, melanin-synthesis, and related signalingpathway

yinjuan Wang

To cite this version:yinjuan Wang. Melanocytes, melanin-synthesis, and related signaling pathway. Dermatology. Uni-versité Bourgogne Franche-Comté, 2017. English. �NNT : 2017UBFCE005�. �tel-01823030�

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UNIVERSITE BOURGOGNE- FRANCHE COMTE ECOLE DOCTORALE « ENVIRONNEMENTS-SANTE »

Année 2017

THESE

Pour obtenir le grade de

Docteur de l’Université de Franche-Comté

Spécialité : Sciences de la Vie et de la Santé

Présentée et soutenue publiquement

Le 12 Sept 2017

Par

Yinjuan Wang

Melanocyte,melaninsynthesisandrelatedsignalingpathway

Thesis director: Professor Philippe Humbert

Li LI, Professor, University of Sichuan, China Reviewer

Xiuli WANG, Professor, University of Tongji, China Reviewer

Philippe HUMBERT, Professor, University of Bourgogne Franche-Comté, France Thesis Director

Li HE, Professor, University of Kunming Medical, China Examinator

Jean-Yves BERTHON, Director, Greentech Biotechnical Company, France Examinator

Céline VIENNET, Research engineer, University of Bourgogne Franche-Comté, France Examinator

2

Acknowledgments

ForProf.PhilippeHUMBERT

ThankyouforbelievingmeandgivingmethepossibilitytodomyPhDthesisinyourlab.

Thankyouforsupportingmesomuchchancestolearningandtraininginpast3-year.This

3-yearismeaningfulforcareerandmakemehavemoreconfidencetochoosethefuturelife

Ilove.

ForProf.LiHE

YouareoneofprofessorsIadmiremost.Youaremyprofessorbothinacademicandinlife.

Thankyouforyoualwaysbelievingmeandgivingmefreedomandspacetofinishmywork

individually.Yourencouragementandtrustsupportmestrongertofaceanydifficult.

ForCélineVIENNET

Thankyouforstayingwithmeinpast3-year,weexperiencedlotsofdifficultandchallenge

together.Youspenttoomuchspiritandtimeinconductingmythesisandhelpingme,always

bepatientandbepolite.Thanksalotforallyourhelpandwarmsmile.

ForMr.Jean-YvesBERTHON,

Thank you for being my co-director and providing me the precious chance to widemy

academicvisionandpresentmythesis inSPIM.Thankyouforthefinancialsupport from

GreentechBiotechologyCompany.

ForProf.LiLIandProf.Xiu-liWANG

Thankyouforbeingthereviewersofmythesis.Itismygreathonorthatyouwouldliketo

bethejuriesinmydefense,yourcomingwillsupportmemoreconfidencetodefenseand

finishmythesis.

3

ForProf.MURET,Sophie,Marion,andGwen

Thankyouforjoiningmythesisandgivingmeprecioussuggestions.ThankstoMarionfor

allthetechnicalsupport.

Forcolleagues，

ThankstoThomasforgivingmestatisticssupportinginmythesis,kindnessandalltheother

help.ThankstoFerialforgivingmeonemonthclinicaltrainingandfollowinghelpduring

work.ThankstoAdelineforhelpingmetofinishmyclinicalresearchandalltheotherhelp.

ThankstoRaneshaforprovidingmetheprecioussampleformyexperiment.Thankstoall

thehelpfromAgnès,Ahmed,Céline,Vanessa,Aurélie,Isabelle.

Formyfriends,

ThankstoYoussef,Vaheide,Som,Phaeng,Delphine,Victoria,Faty,thefriendshipfromallof

yougivemespiritandconfidencetoovercomethedifficulties.

Formyparents

Lastbutnotleast,Iwouldliketoexpressmysincerelygratefultomyparents.HowluckyI

amtohavemyopen-mindedparentswhoalwaysrespectmyidea,believeme,encourageme.

Thanksforunderstandingandsupportingmetoachievemydreaminpast3years.Thanks

forkeepinghealth.

4

Contents

Acknowledgments................................................................................................................................2

Contents...................................................................................................................................................4

ListofFigures.........................................................................................................................................8

ListofTables.......................................................................................................................................13

ListofAbbreviations........................................................................................................................14

GeneralIntroduction........................................................................................................................17

Abstract.................................................................................................................................................19

ChapterI...............................................................................................................................................22

LiteratureReview..............................................................................................................................22

I. Structureoftheskin...........................................................................................................................23

I.IEpidermis.........................................................................................................................................................24

I.IIDermis...............................................................................................................................................................25

I.IIISubcutaneoustissue...................................................................................................................................25

II. SkinPigmentation...............................................................................................................................25

II.IMelanocyteanditscharacteristics.......................................................................................................25

II.IIMelanogenesisandrelatedenzymesandmodulators................................................................26

II.IIIMainsignalingpathwaysregulatingmelanogenesisinmelanocyte....................................30

II.IVRoleofepidermalkeratinocytesinmelanogenesis....................................................................33

II.VRoleofdermalfibroblastsinmelanogenesis.................................................................................35

III. DKKsfamily...........................................................................................................................................38

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III.IStructure........................................................................................................................................................38

III.IIFunctions.......................................................................................................................................................39

IV.DKK1........................................................................................................................................................40

IV.IDKK1-LRP5/6complex..........................................................................................................................40

IV.IIDKK1skinexpression.............................................................................................................................40

IV.IIIDKK1effectsonkeratinocytes...........................................................................................................40

IV.IVDKK1effectsonmelanocytes..............................................................................................................44

IV.VRoleofDKK1invitiligo..........................................................................................................................45

IV.VIRoleofDKK1inmelanoma..................................................................................................................46

V. TGF-bfamily..........................................................................................................................................47

V.ITGF-bandrelatedsignalingpathway..............................................................................................47

V.IITGF-bsandfibroblasts...........................................................................................................................48

V.IIIp38-MAPKsignalingpathwayactivationbyTGF-b..................................................................49

VI. Conclusion.........................................................................................................................................50

Article1:Preciseroleofdermalfibroblastsonmelanocytepigmentation........................................50

ChapterII..............................................................................................................................................84

Developmentandvalidationofasimplemethodfortheextractionofhumanskin

melanocytes.........................................................................................................................................84

Article2:Developmentandvalidationofasimplemethodfortheextractionofhumanskin

melanocytes....................................................................................................................................................87

ChapterIII..........................................................................................................................................107

InvolvementofDickkopf-relatedprotein1inmelanogenesis:focusonsolarlentigo

lesion...................................................................................................................................................107

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I. AimsoftheStudy.....................................................................................................................108

II. Hypothesis.................................................................................................................................108

III. MethodsandMaterials.....................................................................................................111

III.I.PartI-Solarlentigobiopsies.................................................................................................................111

III.I.I.Collectionofskinbiopsies...............................................................................................................................111

III.I.II.Histology.................................................................................................................................................................111

III.I.II.IFixationandsectioning...........................................................................................................................111

III.I.II.IIMasson'strichromestainingforcollagenfibers...........................................................................111

III.I.II.IIIMasson'sFontanastainingformelanin...........................................................................................112

III.I.II.IV.Immunostainingforβ-catenin............................................................................................................112

III.I.III.Cellculture.............................................................................................................................................................112

III.I.III.I.Extractionoffibroblasts.........................................................................................................................112

III.I.III.II.Establishmentofskinfibroblastcultures......................................................................................112

III.I.IV.Enzyme-linkedimmunosorbentassay(ELISA)....................................................................................113

III.I.V.Biuretproteinassay..........................................................................................................................................113

III.I.VI.Real-timequantitativereversetranscription-PCR..............................................................................113

III.I.VII.ConditionedmediumfromFLandFNinculturedhumanmelanocytes...................................114

III.I.VIII.MTTassay............................................................................................................................................................115

III.I.IX.ImmunostainingforMITF.............................................................................................................................116

III.I.X.Melaninassay......................................................................................................................................................116

III.II.3Dbiologicalmodel.................................................................................................................................117

III.II.I.Fibroblasts-populatedtensecollagenlatticepreparation(FPCL)................................................117

III.II.II.Repeated-UVAirradiation.............................................................................................................................118

III.III.ELISA.............................................................................................................................................................119

III.IV.Statisticalanalysis....................................................................................................................................120

IV. Results....................................................................................................................................120

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IV.I.ComparativeanalysesofstaininginSLversusSNbiopsies...................................................120

IV.II.ExpressionandsecretionofDKK1fromFLandFL..................................................................121

IV.III.SecretionofTGF-β1fromFLandFL...............................................................................................123

IV.IV.EffectsofconditionedmediumfromFLandFNonnormalmelanocytecultures.......124

IV.IV.I.Melanocyteviability.........................................................................................................................................124

IV.IV.II.Melanocytecharacteristics............................................................................................................................126

IV.V.SecretionofDKK1afterirradiationofnormalfibroblastsembeddedin3Dcollagen

gel......................................................................................................................................................................................129

V. Discussion-Perspectives.......................................................................................................129

ListofPublications&Communications...................................................................................133

Publications.................................................................................................................................................134

Posters...........................................................................................................................................................135

Article3:Assessmentoftheefficacyofanewcomplexanti-sensitiveskincream.............138

References.........................................................................................................................................163

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ListofFigures

Figure1.Structureoftheskin(SourcefromPearsonEducation)...............................................23

Figure2.Structureofepidermis(SourcefromHumanAnatomyLibrary).............................24

Figure3.Fourstagesofmelanosome:stageI,premelanosome,sphericalformcontaining

dense spot and few filaments; stage II, premelanosome, ellipsoidal form containing

organized, structured fibrillar matrix; stage III, the beginning of the melanin

production;stageIV,beingfullofmelanin99...................................................................................27

Figure4.Melaninsynthesisinmelanocytes100.................................................................................29

Figure5.Canonicalandnon-canonicalWntsignalingpathways.a.CanonicalWnt/β-

catenin signaling pathway, the ternary complex of Wnt-Lrp5/6-Fzd interacts with

Axin/APC/GSK-3toactiveordegradetheaccumulationofβ-catenin.b.Non-canonical

Wnt/Ca2+signalingpathwaythroughinteractionofWntligandswithFzdreceptorscan

lead to an increase in intracellular calcium level, and involves activation of PLC

(phospholipase C). c. Non-canonicalWnt/PCP (Planar Cell Polarity) pathway, it’s is

characterizedbyanasymmetricdistributionofFzd,CELSR,PkandVANGL2,resulting

inthepolarizationofthecell16...............................................................................................................32

Figure6.Melanintransferfrommelanocytetokeratinocytebymelanosome.Different

formbetweenlightskinanddarkskin21...........................................................................................34

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Figure 7. Schematic representation of relationships between fibroblasts and

melanocytes.................................................................................................................................................37

Figure 8. The different structure of DKKs family. DKK1, 2 and 4 have similar gene

sequence;DKK3andDKKL1havesamehomologyofsgy-domain34....................................38

Figure9.DKK1andDKK2interactwithWnt/β-cateninsignalingpathway.(a)Wnt,Fz,

and LRP6 form a complex to active the signaling pathway by increasing β-catenin

accumulation; (b)DKK1bindingwithLRP6 prevents the formationof complexand

inhibitsthesignaling38...............................................................................................................................39

Figure10.TheeffectsofDKK1 frompalmoplantar fibroblastsonbothmelanocytes

and keratinocytes. DKK1 binds to LRP5/6 against Wnt signaling pathway,

downregulatesMITFinmelanocytes,andatthemeanwhile,downregulatesthetransfer

proteinPAR-2inkeratinocytesanddecreasesthemelanintakenbykeratinocytes.On

theotherhand,DKK1enhancesthethicknessoftheskinbyupregulatingkeratin9in

keratinocytes31..............................................................................................................................................42

Figure11.ReconstructedskinmodelwithrhDKK1treatment(left)showedsignificant

lesspigmentationthanthecontrolgroup(right)31..............................................................44

Figure12.TGF-bfamilysignalingbytypeIIandIreceptors,andsmadproteins56......48

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Figure 13. Hypothesis of the study: Structural damages, pro-inflammatory cytokines,

immune-modulatoryfactors(SASP)causedbyexternalstimulatedfactors(UVrays,air

pollution, environmental toxins) induce high secretion of TGF-b by fibroblasts and

inflammatorycells (Tcells,macrophages).Thisprocess inducesadownregulationof

DKK1geneexpressionandadecreaseofDKK1secretionbyfibroblasts.Lowexpression

andsecretionofDKK1leadstotheactivationofWnt/b-cateninsignalingpathway,and

resultsinincreasedmelaninsynthesisbymelanocytes..........................................................110

Figure14.TenseFPCLembeddedbyanylonmeshring...........................................................118

Figure15.Shematicrepresentationoftheirradiationprotocol..........................................119

Figure16.UVA-irradiationoftenseFPCL..........................................................................................119

Figure17.a)Melanin(HC,Masson-Fontanastaining)andb)β-catenin(IHC,DABstaining)

inSLandSNtissues.InSL,levelsofmelanin(a-1)andβ-catenin(b-1)↗comparedto

SN(a-2,b-2respectively).InSL,thebasallayerisdisrupted,causingmelaninreleasein

papillarydermis(a-2).MagnificationX20....................................................................................121

Figure18.RelativegeneexpressionofDKK1assessedbyreal-timequantitativeRT-PCR.

Toanalyzetherelativechangesingeneexpressionfromthereal-timePCRexperiments,

thecomparative2-△△Ctmethodwasused.The△CtvalueforFLandFNwascalculated

usingtheequation:Ct(DKK1)-Ct(reference,Abl).Thefoldchangeingeneexpression

ofDKK1wasfinallyobtainedfromtheformula2-△△Ct,wherethe△△Ctvaluewasthe

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differencebetween△Ct(FL)and△Ct(FN)values.Therelativegeneexpressionisset

to1 forFNsamples.DKK1expression↘ inFLcomparedtoFN.Themean±SDwas

determinedfrom9independentexperiments,eachperformedinduplicate.***p<0,001.

...........................................................................................................................................................................122

Figure19.DKK1secretion into themediumbyELISA.DKK1secretion isnormalized to

proteincontentasmeasuredusingBiuretassay.Themean±SDwasdeterminedfrom9

independentexperiments,eachperformedinduplicate.DKK1proteinlevels↘inthe

mediumofFLcomparedtoFNbutnosignificantdifferencewasdetectedbetweenboth

groups............................................................................................................................................................123

Figure20.TGF-β1secretionintothemediumbyELISA.TGF-β1secretionisnormalizedto

cellnumber.Themean±SDwasdetermined from9 independentexperiments,each

performedinduplicate.TGF-β1levels↗inthemediumofFLcomparedtoFN.**p<0,01.

...........................................................................................................................................................................124

Figure21.Phasecontrast imagesofmelanocytescultures ina)normalconditions,b)FL

conditionedmediumandc)FNconditionedmedium.Arrowpointstomelanin.Normal

epidermal melanocytes exhibit a typical dendritic morphology and produce more

melaninwithFL-conditionedmediumtreatment(15%v/v).MagnificationX20.......127

Figure22.Immunofluorescenceimagesofmelanocytesculturesina)normalconditions,b)

FLconditionedmediumandc)FNconditionedmedium.Normalepidermalmelanocytes

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exhibit a typical dendritic morphology and express MITF marker with FL or FN-

conditionedmediumtreatment(15%v/v).MagnificationX20...........................................128

Figure23.DKK1secretionintothemediumbyELISA.TGF-β1secretionisnormalizedto

cellnumber.Themean±SDwasdetermined from3 independentexperiments,each

performedinduplicate.DKK1levels↘inthemediumafterUVirradiation.***p<0,001.

...........................................................................................................................................................................129

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ListofTables

Table1.Comparisonofthreeclassicmelanocyteextractionmethods.........................................86

Table2.PrimersandTaqmanprobesusedforqRT-PCR................................................................114

Table3.Preparationofdifferentconcentrationsoffibroblast-conditionedmedium.........115

Table4.OpticaldensitymeasurementsforMTTassayofviabilityofmelanocytescultured

withdifferent concentrationsof fibroblast-conditionedmedium.Themean±SDwas

determined from 3 independent experiments, each performed 4 times. Fibroblasts-

conditionedmediumfrombothFLandFNinconcentrationrangingfrom0to15%v/v

showednocytotoxiceffectonmelanocytes..................................................................................125

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ListofAbbreviations

AMH:Anti-MüllerianHormone

APC:AdenomatousPolyposisColi

bFGF:basicFibroblastGrowthFactor

BMK1:BigMAPKinase1

BMPs:BoneMorphogeneticProteins

cAMP:cyclicAdenosineMonophosphate

CREB:cAMPResponsiveElementBindingprotein

DCT:DOPAChromeTautomerase

DHI:5,6-dihydroxyindole

DHICA:DHI-2-carboxylicAcid

DKK:Dickkopf

DMEM:Dulbecco’sModifiedEagle’sMedium

DMSO:DimethylSulfoxide

DOPA:L-3,4-dihydroxyphenylalanine

Dsh:Dishevelled

ERKs:ExtracellularSignal-regulatedKinases

FBS:FetalBovineSerum

FPCL:Fibroblast-populatedCollagenLattice

GDF:GrowthandDifferentiationFactors

Gsk-3b:GlycogenSynthaseKinase-3𝛽

HGF:HepatocyteGrowthFactor

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JNK/SAPK:c-junN-terminalkinases

KGF:KeratinocyteGgrowthFactor

LDLR:Low-densityLipoproteinReceptor

LEF:LymphoidEnhancer-bindingFactor

MAPK:Mitogen-activatedProteinKinase

MATP:MyoacitveTeradecaPeptide

MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide

MITF:Microphthalmia-associatedTranscriptionFactor

NT-3:Neurotrophin-3

NRG-1:Neuregulin-1

PAR-2:Protease-activatedReceptor-2

PBS:Phosphate-bufferedSaline

PI3K/Akt:phosphatidylinositol3′-kinase/Akt

PKA:ProteinKinaseA

P/S:Penicillin/Streptomycin

R-Smads:Receptor-activatedSmad

rhDKK1:recombinantHumanDKK1

ROS:ReactiveOxygenSpecies

Sema7a:Semaphorin7a

SCF:StemCellFactor

SFRP:SecretedFrizzled-relatedProteins

TGF-b：TransformingGrowthFactor-beta

TRP-1:Tyrosinase-relatedProtein-1

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TYR:Tyrosinase

WIf-1:WntInhibitoryFactor1

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GeneralIntroduction

Our skin reflects our general health and well-being. It plays the role of interface

between our body and the outside environment, and protective barrier against external

aggressors.Thereforemanypeoplepayspecialattentiontotheirskin.Amongskindiseases,

pigmentation disorders are now a very important area of research in cosmetology and

dermopharmacy. Melanin plays key roles in determining human skin pigmentation.

Melanocytes are cells that possess the unique capacity to synthesize melanin within

melanosomes. This thesis focuses on the study of skin pigmentation in different views:

melanocyte,melaninsynthesis,andrelatedsignalingpathway.

The first chapter introduces the process of melanogenesis and the factors that regulate

melanin production in melanocytes. Transcription factors specifically expressed by

melanocytes and keratinocytes-derived paracrine factors regulate the functions of

melanocytes.Today,thereisgrowingscientificevidencesuggestingthatfibroblastssecrete

factors that are involved in the regulation of skin pigmentation. Therefore, a literature

reviewwaswritten todescribe therecent findingsonmelanogenic factorssecreted from

fibroblasts.

The second chapter describes and validates an extraction method of human skin

melanocytesbeing simple, effectiveandapplicable to smaller skin samples, andavoiding

animal reagents. This method would be suitable for establishment of optimal primary

melanocyteculturesforclinicalapplicationandresearch.

Lastly, the third chapter explores the roleof fibroblast-derivedparacrine factorDKK1 in

melanogenesiswith a focus on solar lentigo lesion. An hypothesis proposes that TGF-β1

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mediatesdevelopmentofsolarlentigobyreducingDKK1expressioninfibroblaststhrough

thep38MAPkinasepathway,whichleadstoanactivationoftheWnt/b-cateninsignaling

cascade. The investigation is conducted with primary cultures of fibroblasts from solar

lentigo,anddermalequivalentmodelsexposedtoUVradiation.

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Abstract

Thisthesisfocusesonthestudyofskinpigmentationindifferentviews:melanocyte,

melaninsynthesis,andrelatedsignalingpathway.

The first part of the experimental work was to develop a new extraction method of

melanocytes. Cell extraction is an inevitable and critical step in the development and

productionofAdvancedTherapyMedicinalProducts (ATMP) as for the establishmentof

primarycellbank.Thetechniquesdescribedintheliteratureareusuallybasedontrypsin,

aloneorincombinationwithdispase.Suchenzymesareusedinordertoseparatedermis

from epidermis and subsequently provide a suspension of epidermal cells. The

implementation of these protocols is often operator-dependent, not suitable for small

samplesandrequiresanimalderivedproducts(notcompatiblewithclinicalapproach).The

objectiveofthisworkwastodefineandvalidateanepidermalcellextractionmethodbeing

the simplest, the most effective and applicable to smaller samples and avoiding animal

reagents.Anewproduct(TrypLE,Lifescience),animal-freeproduct,hasbeentestedonskin

biopsies.Theextractionefficiencywasjudgedonthefollowingcriteria:separationepidermis

/dermis,culturedmelanocytes,functionalityoftheextractedcells.Resultsshowedtheease

of separation between the dermis and epidermis on the one hand, and between the

epidermalcellsontheotherhand.Aminimumsizeofskinsamplewasdefinedtoallowthe

extraction of functional melanocytes. In conclusion, this optimal method opens new

perspectives for establishment optimal melanocyte cell lines suitable for cutaneous

pathophysiologyresearchandproductionofATMP.

20

Thesecondpartoftheexperimentalworkwastoinvestigatethemechanismunderlyingthe

lesion of solar lentigo by exploring the role of Dickkopf-related protein 1 (DKK1) in

hyperpigmentation. Recent studies reported that high DKK1 expression by dermal

fibroblastsiscloselyrelatedtohypopigmentedskin,aspalmoplantarskinandvitiligolesion.

DKK1encodedbyDKK1geneisanantagonisticinhibitoroftheWntsignalingpathway,by

isolating the LRP5/6 co-receptor and preventing the Frizzeled-Wnt-LRP5/6 complex

formation.Wnt/bcateninsignalingregulatesthetranscriptionofmelanocyte-specificgenes

likeMITF, a gene involved inmelanin synthesis.Wehypothesized that TGF-β1mediates

developmentofsolarlentigobyreducingDKK1expressioninfibroblaststhroughthep38

MAPkinasepathway,whichleadstoanactivationoftheWnt/b-cateninsignalingcascade.In

vitromodelsthatmimictheinvivoenvironmentwereused:fibroblastsisolatedfromsolar

lentigoandperi-lesionalbiopsies,andnormalfibroblastsembeddedin3Dcollagengeland

exposedtorepeateddosesofUVA.Q-PCRandELISAtechniquesshowedthatfibroblastsfrom

solar lentigo and fibroblasts irradiated to UVA, express low level of DKK1.

Immunohistochemicalstudiesrevealedastrongstainingofbcateninandmelanininsolar

lentigo skin, which indicates an activation of Wnt/b catenin signaling and melanocyte

function.Ourpreviousdatademonstratedthesenescent-likephenotypeoffibroblastsfrom

solar lentigo with particularly a high secretion of TGF-β1, suggesting its role in the

development of the lesion. Fibroblasts should respond tomany inflammatorymediators

releasedduringhyperpigmentarydisorderbyincreasingTGF-β1secretion.TGF-β1isknown

tosuppresstheexpressionofDKK1inap38-dependentmanner.Therefore,ourhypothesis

confirmedthatTGF-β1regulatesdevelopmentofsolarlentigobyreducingDKK1expression

21

infibroblaststhroughthep38MAPkinasepathway.Thisprocessmayresultinanuneven

distributionofactivemelanocytes,withareasofhyperpigmentationintheskin.

22

ChapterI

LiteratureReview

23

I. Structureoftheskin

Skinasoneofthelargestorganofhumanbodybecomesthemostimportantbarrierbetween

humanbodyandexternalenvironment.Theaverageareaof theskin is to1.8m2,and its

average sickness is to 1.2 mm. It is responsible of three main functions: protection

(mechanical trauma, UV radiation, microorganisms, infection), regulation (temperature,

fluid,immunity,synthesisofvitaminD)andsensation(pressure/pain,heat/cold)1.

Figure1.Structureoftheskin(SourcefromPearsonEducation)

24

Theskiniscomposedbythreemainlayers:theepidermis,thedermisandthesubcutaneous

tissue(Figure1).

I.IEpidermis

Theepidermisistheouterlayeroftheskinwhichisconsistedof5layers:stratumcorneum,

stratumlucidum,stratumgranulosum,stratumspinosum,stratumbasale.Itcontains95%

keratinocytes,melanocytes,Langerhanscells,Merkelcells.

Figure2.Structureofepidermis(SourcefromHumanAnatomyLibrary)

25

Keratinocytesoriginateandproliferatesfromdeepestlayeroftheepidermisupdatedtothe

surfacelayerstepbystep.Melanocytes,whichlocatedinbasallayerofepidermis,secrete

melanintoprotecttheskinfromUVradiation.Langerhanscell,areresponsibletotheskin

immunesystem.Merkelcellsdisplaythefeatureofsensoryreceptorcells(Figure2).

I.IIDermis

Thedermisisconnectedtotheepidermisthroughabasementmembrane.Itisdividedin2

layers:thesuperficialareaknownaspapillaryregion,anddeeperandthickerareaknownas

reticularregion.Itsskeletonstructureismainlyconstitutedbycollagenandelastinfibers,

extrafibrillarmatrix.Inaddition,therearealsohairfollicles,sweatglands,sebaceousglands,

lymphandbloodvessels.Fibroblastsaretheprincipaldermalcells.

I.IIISubcutaneoustissue

Subcutaneous tissue also known as hypodermis, consists of loose connective tissue, fat,

largerbloodvesselsandnerves.

II. SkinPigmentation

II.IMelanocyteanditscharacteristics

Melanocytesderivefromneural-crestcellslocatedonthebasallayeroftheepidermis,the

uvea of the eyes, inner ear, nervous system, heart, bones and so on. In epidermis, each

melanocyte connects with 30-40 associated keratinocytes to form a unit as “Epidermal

MelaninUnit”.Theproportionofmelanocytestokeratinocytesintheepidermalbasallayer

is1:10.1200melanocytespermm2areapproximatelycontainedinhumanskin.However,

26

the number of melanocytes in the skin of palms and soles are fivefold lower than

melanocytesnumberatotherpartoftheskin2.Italsodecreasesby10-20%perdecadesafter

30yearsold.Withsenescence,melanocytestrendtobelargerandmoredendritic,andto

havelesstyrosinaseactivity3.Melanocyteisnottheonlycellwhichcouldproducemelanin

inhumanbody,othercellssuchascellsofpigmentedepitheliumofretina,epitheliaofiris

andciliarybodyoftheeye,someneurons,adipocytescouldalsosynthesizemelanin4.

II.IIMelanogenesisandrelatedenzymesandmodulators

In melanocytes, melanin production takes place in cytoplasmic organelles named

melanosomes. Itexperiences fourstages tobemature:stage I,premelanosome,spherical

form containingdense spot and few filaments; stage II, premelanosome, ellipsoidal form

containing organized, structured fibrillar matrix; stage III, the beginning of the melanin

production;stageIV,beingfullofmelanin5(Figure3).

27

Figure 3. Four stages of melanosome: stage I, premelanosome, spherical form

containing dense spot and few filaments; stage II, premelanosome, ellipsoidal form

containingorganized,structuredfibrillarmatrix;stageIII,thebeginningofthemelanin

production;stageIV,beingfullofmelanin99.

28

Theprocessofmelanin formationstartswiththerate limitingenzyme, tyrosinase.

TyrosineishydroxylatedtoL-3,4-dihydroxyphenylalanine(DOPA)bytyrosinase(TYR),then

rapidlyoxidizedtoDOPAquinone.Withcysteineandglutathione,cysteinylDOPAisformed

thenoxidizedandpolymerizedtopheomelaninwhichpresentsyellow-redsolublemelanin.

Intheabsenceofcysteineandglutathione,DOPAquinoneiscyclizatedtoDOPAchrome,then

transferedto5,6-dihydroxyindole(DHI)whichisoxidizedtothedark-brown-blacknamed

as DHI-melanin. On the other way, DOPAchrome transformes to DHI-2-carboxylic acid

(DHICA)byDOPAchrometautomerase(TYRP2/DCT),thenTYRP1catalyzesDHICAtoform

lighter brown color DHOCA-melanin. Both DHICA-melanin and DHI-melanin belonge to

Eumelaninwhichtypeplaysmoreimportantroleintheskincoloranddiversityofethnic

groups. Comparing to pheomelanin, eumelanin ismore effective in photoprotectionwith

higherresistancetodegradation,andabilitytoneutralizereactiveoxygenspecies(ROS)4,6

(Figure4).

29

MITF(Microphthalmia-associatedtranscriptionfactor)actsasamasterregulatorof

melaninsynthesisinmelanocyte.Itregulatesmelanocytedifferentiation,proliferationand

survival by adjusting various genes of differentiation and cell-cycle. MITF itself is also

regulatedbymanyother transcription factors, includingPAX3, SOX9, SOX10,LEF-1/TCF,

CREB (cAMP responsive element binding protein), and DICER. There are three factors

affecting melanosome structure: Pmel17, MART-1 and GPNMB7,8. Enzymes and proteins

modulatemelaninsynthesisindistinctway:TYR,TYRP-1,DCT,BLOC-1(lysosome-related

Figure4.Melaninsynthesisinmelanocytes100

30

organelle complexe), OA1 (melanosomal G-protein-coupled receptor), P (membrane

transporters),andSLC45A2(implicatedinthecontrolofmelanosomeosmolarity)1.Some

proteinsinvolvemelanosometraffickingortransport:microtubules,F-actin,kinesin,dynein,

Rab27a,melanophilin,myosinVa,RILP5,9.

II.IIIMainsignalingpathwaysregulatingmelanogenesisinmelanocyte

a) MAPK/ERK (mitogen-activated protein kinases/extracellular signal-regulated

kinases) signaling is crucial to the proliferation and differentiation of melanocytes. The

kinases MEK and ERK in MAPK signaling pathway involve the activation of melanocyte

receptorsvialigandbindingtotheirextracellulardomain(eg,receptortyrosinekinasec-Kit).

Withbindingtotheirreceptors,ligandactivatescomplexmechanisms(Ras-Raf-MEK-ERK)

that lead to up-regulateMITF. Themutation ofMAPKs’ inhibitor such as BRAF affecting

MAPKstransductiongivesapotentialformelanoma10.TheMAPKsignalingpathwaybythe

synergistic action of factors derived from melanocytes and keratinocytes induces ERK

activation,whichtriggersmelanocyteproliferation11,12.

b) Wnt signaling pathway is involved in numerous developmental events during

embryogenesisandisessentialtokeeptissuehomeostasis.Italsoplayssignificantrolein

celldifferentiation.Thereare2typesofWntsignalingpathway:canonicalpathwayandnon-

canonicalpathway. Incanonical signalingpathway, the factorsconnectwithFrizzledand

WntcoreceptorLPR(Lowdensitylipoproteinreceptor-relatedprotein)5,6andregulate𝛽-

catenin by interactions with Axin/APC/GSK-3. In non-canonical signaling pathway, the

signal binding to Frizzled/LRP receptor transduces a signal to Ca2+or PCP (planar cell

31

polarity) without the participant of𝛽 -catenin. At the result, the accumulated𝛽 -catenin

interactswithTCF(Tcell factor)andregulates transcription.Thereare fiveextracellular

Wntantagonists:sFRP,WIF1,xenopuscerberus,DKKfamilyofsecretedproteins13,14(Figure

5).

Inmelanocytes,Wnt/β-cateninsignalingisanimportantpathwayinpigmentationprocess

andmelanocytesdifferentiation.ActivationofWnt/β-cateninsignalingoccursuponbinding

ofWnttofrizzledreceptorsandLRP5/6.Signalsaretransducedthroughtheinhibitionof

glycogensynthasekinase-3β(GSK-3β)activity,leadingtostabilizationandtransportofβ-

catenin into the nucleus that regulates transcription of MITF through interactions with

lymphoid enhancer-binding factor (LEF). Wnt signaling is modulated by secreted and

transmembraneWntinhibitorsandactivators13,15.

32

Figure5.Canonicalandnon-canonicalWntsignalingpathways.a.CanonicalWnt/𝛽-

catenin signaling pathway, the ternary complex of Wnt-Lrp5/6-Fzd interacts with

Axin/APC/GSK-3 to active or degrade the accumulation of𝛽 -catenin. b. Non-canonical

Wnt/Ca2+signalingpathwaythroughinteractionofWntligandswithFzdreceptorscanlead

toanincreaseinintracellularcalciumlevel,andinvolvesactivationofPLC(phospholipase

C). c. Non-canonicalWnt/PCP (Planar Cell Polarity) pathway, it’s is characterized by an

asymmetricdistributionofFzd,CELSR,PkandVANGL2,resultinginthepolarizationofthe

cell16.

c) cAMP/PKA(cyclicadenosinemonophosphate/proteinkinaseA) signalingcanalso

contributetoMITFexpression.Activationofsomemelanocytereceptorswiththeirligands

(eg, melanocortin receptorMCR-1) results in increased levels of intracellular cAMP and

33

activationofPKA.PKAphosphorylatesCREBwhichactsasatranscriptionfactorofMITF.It

has also been reported that activation of PKC (ProteinKinase C) can be associatedwith

cAMP-dependent pathway17. Various intrinsic and extrinsic factors affect melanogenesis

throughsignaltransductionpathways.Theyexerttheiractionsdirectlyonmelanocytesor

indirectlyviamediatorsproducedbysurroundingskincells12.

d) PI3K/Akt(phosphatidylinositol3′-kinase/Akt)signalingpathwayplaysacriticalrole

inproliferationandapoptosisofmelanocytethroughtheinhibitionofBAD(pro-apoptotic

memberoftheBcl-2family),aswellasadjustingcellcyclebyinactivatingGSK3andthen

activatingcyclinD1.ItalsocouldcooperatewithRAS-RAF-MEK-ERKpathwaytoeffecton

melanocyte activity. Because of the significant role of PI3K/Akt in melanoma by the

apoptosis resistance and cell proliferation, the utilization of its inhibitor was widely

developedintheclinicaltreatment18–20.

II.IVRoleofepidermalkeratinocytesinmelanogenesis

Melanosomeistheonlyorganellewhichcouldloadandtransmitmelanininthebasallayer

frommelanocytetokeratinocyte.Apartfromthetypeofmelanin,themelaninreleaseand

transferareanothersignificantfactorsinfluencingskincolor.Inlightskin,melanosomesare

released from melanocyte to keratinocyte in cluster form while in dark skin, they are

distributedindividually21(Figure6).

34

Inkeratinocytemembrane,a7-transmembraneG-protein-coupledreceptorknownas the

protease-activatedreceptor-2(PAR-2)controlsmelanosomeingestionandphagocytosisby

keratinocytes and exerts a regulatory role in skin pigmentation22,23. Moreover, PAR-2 is

inducedbyUV irradiationand inhibitionofPAR-2activation results in thepreventionof

UVB-inducedtanning.

Interactions between melanocytes and neighboring cells in the skin are important in

regulating skin color in humans. The proliferation, differentiation, melanogenesis, and

dendritogenesis of melanocytes in the epidermis are primarily regulated by paracrine

factorsderivedfromkeratinocytes.Keratinocyte-derivedfactorssuchasα-MSH,ACTH,NGF,

bFGF,ET-1,ET-2,ET-3,SCF,LIF,HGF,GMCSF,PGE2,andPGF2αbindtotheirspecificreceptors

Figure6.Melanintransferfrommelanocytetokeratinocytebymelanosome.Different

formbetweenlightskinanddarkskin21.

35

(e.g.,Mc1r,NGFR,FGFR-1,FGFR-2,ETBR,Kit,gp130,LIFRα,c-Met,GMCSFR,EP1,EP2,and

EP3)onthemembraneofmelanocytesandstimulatedifferentsignalingpathways,PKA,PKC,

andMAPK.

II.VRoleofdermalfibroblastsinmelanogenesis

Increasingevidencehasunderlinedthecontributionofdermalcomponentsintheregulation

ofpigmentation.Morerecently,dermalfibroblastsweredemonstratedtoexertaregulatory

roleonpigmentationthroughthesecretionofsolublefactors.

Factors secreted from fibroblasts bind to membrane receptors of melanocytes, active

differentsignalingpathwaysandmodulatemelanocytefunctionssuchasmelaninsynthesis.

Stemcell factor(SCF),hepatocytegrowthfactor(HGF),keratinocytegrowthfactor(KGF),

basic fibroblast growth factor (bFGF), neuregulin-1 (NRG-1), neurotrophin-3 (NT-3) and

semaphorin7a (Sema7a) target CREB promotor via MAPK signaling pathway and active

expressionof theMITF24–30.DKK1, an antagonist ofWnt/β-catenin signalingpathwayby

disruptingtheWnt-inducedFrizzle-Lrp5/6complex,preventstheWnt-inducedstabilization

of β-catenin and the binding to the LEF-1 transcription factor31. Secreted frizzle-related

protein (sFRP) activesWnt/β-catenin signaling pathway by binding to Frizzled receptor

whereasWntinhibitoryfactor-1inhibitsit32,33.WiththeadjustedpromotorofLEF-1,CREB

orPAX3,MITFcontrolstheactivityofTyrosinase,Tyrosinase-relatedprotein-1(TRP-1),

dopachrometautomerase (DCT)and therebymodulatesmelaninsynthesis.NRG-1,Nerve

growthfactor(NGF)andNT-3regulatePI3K/Aktsignalingpathway,andSema7aactivesFAK

andLIMKII,FAP-αandCCNwhichareassociatedwithmelanocyteproliferation,adhesion

andmigration5(Figure7).

36

Theroleofdermalfibroblastsinsolarlentigo(SL)lesionhasbeenrecentlydemonstrated.SL

is a common pigmentation disorder presented as aging spot on the exposure skin. It is

characterizedbyhyperpigmentedmacules,andthenumberofthespotsisrelatedtotheage

andskinphototype.ThehistologicalcharacterizationofSL isdefinedbyahighermelanin

depositioninthebasallayer,elongatedepidermalridgesandlargemelanosomalcomplexes.

Differentialgene-profilinganalysesbetweenSLandnormalskinbiopsiesrevealedthatSL

tissuesaremainlycomposedofactivatedmelanocytes34.Therearesomefibroblast-derived

paracrine factors such as HGF (Hepatocyte growth Factor), KGF (Keratinocyte Growth

Factor),SCF(StemCellfactor)involvedinSLlesionformation69,35.Immuno-staininganalyses

ofsomegrowth factorsandsecretedproteins in theupperdermisofSLbiopsiesstrongly

suggestthatdermalfibroblastscontributetofunctionallydysregulatingtheepidermalcells36.

Moreover, the treatmentwithKGFandKGFassociatedwithIL-1 induceshyperpigmented

lesionsinvivo37.

37

Figure7.Schematicrepresentationofrelationshipsbetweenfibroblastsandmelanocytes.

38

III. DKKsfamily

III.IStructure

Inmiceandhuman, thereweremultipleDKK(Dickkopf)genes.The familyofDKKgenes

includesDKK1,DKK2,DKK3,DKK4andDKKL1(Dickkopf-likeprotein1,alsocalledsoggy)

thatencodeDKKsecretedproteins.TheseDKKproteinsantagonizeWnt/𝛽-cateninsignaling

pathway by internalization withWnt coreceptor Lrp5 and 6, and by affinity ligands for

Kremen1and2transmembraneproteins.DKK1,DKK2andDKK4were identified tohave

similarsequence,4locatschromosome4/5/8/10paralogygroup,mappedto10q11,4q25

and8p11respectively.AtthemeanwhileDKK3andDKKL1showedthesamehomologyand

arelocatedonanotherparalogygroupmappedto11p15.338(Figure8).

Figure8.Thedifferent structureofDKKs family.DKK1,2and4have similargene

sequence;DKK3andDKKL1havesamehomologyofsgy-domain34.

39

III.IIFunctions

InthefunctionofeffectingonWntsignalingpathway,somedifferencesexist.DKK1,2and4

modulateWntsignalingpathwayasinhibitorswhilesometimesDKK2actsasWntactivator.

DKK3andsgydidnotshowevidencethattheyhavefunctioninWntsignalingpathwayand

theyareknownasdivergentmemberoftheDKKsfamily39.RelatedtoWnttrigger,thereare

severaldifferentpathwaysthatcouldbeeffectedsuchasWnt/𝛽-cateninsignalingpathway,

Wnt/Junkinase signalingpathway,Wnt/Ca2+Cascade.DKKsonly showeffect onWnt/𝛽-

cateninsignalingpathway38,40(Figure9).

DKKproteinshavedistinctpatternsofexpressioninadultandembryonictissuesandhavea

wide range of effects on tissue development andmorphogenesis41. They are involved in

cancer,Alzheimer’sdisease,rheumatoidarthritis,keloids…41,42

Figure9.DKK1andDKK2interactwithWnt/β-cateninsignalingpathway.(a)Wnt,Fz,

and LRP6 form a complex to active the signaling pathway by increasing β-catenin

accumulation;(b)DKK1bindingwithLRP6preventstheformationofcomplexandinhibits

thesignaling38.

40

IV. DKK1

IV.IDKK1-LRP5/6complex

DKK1,as the foundingmemberof the family,was identifiedasa secretedprotein that is

required for head formation duringXenopous embryogenesis. InWnt signaling pathway,

DKK1 is the only antagonist that interacts with co-receptors known as LRP5 and LRP6,

contrarytoWntinhibitoryfactor-1andsecretedfrizzled-relatedproteinwhichdirectlybind

with Wnt43. LRP5 and LRP6 are specific coreceptors for DKK1. DKK1 binding to LRP6

interrupts the complex withWnt-Fz and causes the accumulation of𝛽 -catenin, thereby

blockstheWnt/𝛽-cateninsignalingpathway.

ItexistsanothertypeofDKK1coreceptorsknownasKremen1andKremen2,asingle-pass

transmembraneproteins.

IV.IIDKK1skinexpression

IthasrecentlybedemonstratedthatDKK1secretedbyfibroblastsinthedermiselicitsthe

hypopigmentedphenotypeofpalmoplantarskinduetosuppressionofmelanocytefunction

andgrowthviatheregulationoftwoimportantsignalingfactors,MITFandβ-catenin.2The

levels of DKK1 in palmoplantar dermal fibroblasts (at mRNA and protein levels) are

physiologicallyhigher than thoseobserved innon-palmoplantardermal fibroblasts.Thus

DKK1 has a role on skin pigmentation and thickness by inducing the palmoplantar

phenotype.

IV.IIIDKK1effectsonkeratinocytes

In previous study, transfected-DKK1 keratinocytes showed a stimulation of growth and

41

densityandaninhibitionofmelaninuptaken(lowexpressionofPAR-2)44.Inaddition,the

thicknessofareconstructedskinmodelwithkeratinocytestreatedbyrhDKK1(recombinant

humanDKK1)wassignificantly thicker 31,44.Therefore,DKK1regulatespigmentationnot

only on melanin synthesis and melanocyte activity but also on melanin uptaken by

keratinocytes(Figure10).

42

Figure10.TheeffectsofDKK1frompalmoplantarfibroblastsonbothmelanocytes

andkeratinocytes.DKK1bindstoLRP5/6againstWntsignalingpathway,downregulates

MITFinmelanocytes,andatthemeanwhile,downregulatesthetransferproteinPAR-2in

keratinocytesanddecreasesthemelanintakenbykeratinocytes.Ontheotherhand,DKK1

enhancesthethicknessoftheskinbyupregulatingkeratin9inkeratinocytes31

43

In rhDKK1 treated-keratinocytes, upregulation of a-KLEIP and keratin 9 and

downregulationofTulp3wereobserved.Keratin9 isspecificallyexpressedinsuprabasal

palmoplantarepidermisandisonlyobservedinacrosyringiainnonpalmoplantarepidermis.

Highexpressionofkeratin9inrhDKK1treated-keratinocyteconfirmsthatDKK1undirectly

regulates the thicknessofpalmoplantar skin45.aKLEIP is aprotein that is related to cell

adhesion and cytokinesis. High expression of aKLEIP shows that DKK1 regulates

proliferationandcelldensityofkeratinocytesbyincreasingcelladhesionandcytokinesis31.

Tulp3 is a protein belong to Tubby-like protein family which involves cell apoptosis.

DownregulationofTulp3suggeststhatDKK1regulateskeratinocytenumberbydecreasing

cellapoptosis31.

44

IV.IVDKK1effectsonmelanocytes

Reconstructed skinmodel treated by rhDKK1 showed significant less pigmentation than

controlmodel44(Figure11).

ThedecreasedexpressionofDKK1hasaneffectonmelanocyteproliferationandfunctionin

theregulationoftherelatedproteinofmelanosomeandmelanin(TYR,DCT,andMART1).

Inmelanocyte,someWnt-relatedgenessuchasPKCb1,Krn1,andLRP6havehighresponse

toDKK1byupregulation.SomeothergenesencodingreceptorssuchasLDLR,GPR51,and

TNFRSF10A were responsive to DKK1 treatment39,46. LDLR has relationship with LRP6

known as the co-receptor of DKK147. GPR51 is related toMC1R known as a specifically

receptorinmelanocyte48.TNFRSF10Aactivesp53-independentapoptosis,whichindicates

thatDKK1hasclosedrelationshipwithmelanocyteapoptosis.Besides,aresearchhasalso

Figure11.ReconstructedskinmodelwithrhDKK1treatment(left)showed

significantlesspigmentationthanthecontrolgroup(right)31.

45

foundthatDKK1upregulatesGadd45bandinducesmelanocyteapoptosisbyp38mitogen-

activated protein kinase pathway49. These studies suggest that DKK1 is involved in the

inhibitionofmelanocytegrowth.

In terms ofmelanosomal proteins, DKK1plays a significant role directly or indirectly in

upregulation of some expression proteins such as SVF3B, caveolin, syntaxin 5A, and

melanophilin. DKK1 also could directly influences tyrosinase by upregulating the critical

proteinmyoacitveteradecapeptide(MATP)inmelanocyte.Thereby,wecouldalsosuggest

thatDKK1haseffectonmelanocytedifferentiation39.

DKK1alsoaffectstheHOXrelatedgenesthatplayaroleinthegrowthanddifferentiationof

melanocytes,byupregulatingacidfibroblastgrowthfactor-likeprotein50.

IV.VRoleofDKK1invitiligo

Vitiligoisahypopigmentationdisorderinducedbydysfunctionordeficiencyofmelanocytes.

Theetiologyofvitiligoisstillunclearanditcouldco-existwithotherautoimmunediseases.

Mostresearcheshaveconcentratedontheabnormalityofmelanocytesandkeratinocytes

ratherthantheabnormalityoffibroblasts.Animbalanceofkeratinocyte-derivedcytokines

andadysregulationofinteractionswiththeirrespectivereceptorhavebeendemonstrated.

Thereisonlyonestudy,basedonfifteenskinbiopsies,ontherelationshipbetweenDKK1

andvitiligo.The immunohistochemistrystainingrevealed that theexpressionofDKK1in

dermisofvitiligoissignificantlyhigherthaninnon-lesionaldermis.ThemRNAlevelofDKK1

in fibroblasts from vitiligo was also higher than fibroblast from non-lesion51. At the

46

meanwhile,theexpressionofPAR-2indermisofvitiligowassignificantlowerthaninnon-

lesion.ThesefindingsindicatethatDKK1invitiligonotonlydecreasesmelanocyteactivity

butalsomelaninuptakenbykeratinocyte.Inaddition,thelowerexpressionofb-cateninin

vitiligo epidermis compared to non-lesional part confirmed that DKK1 causes

hypopigmentationbyWnt/b-cateninsignalingpathwayaspreviousstudies51–55.

IV.VIRoleofDKK1inmelanoma

Melanomaisthemostserioustypeofhumanskincancerthatdevelopsfrommelanocytes

andoccursanywhereonthebodyexposedtodirectsunlight.Themainreasoniscausedby

excessiveUVexposure thatdamagesDNA inmelanocytes.Melanoma implicatesnotonly

malignantmelanocytes,butalsoaheterogeneousmixofgeneticallystablenon-cancercells,

including fibroblasts, endothelial and inflammatory cells. DKK1 was proved to be a

diagnostic/prognosticserumbiomarkerindifferenthumancancers56.Recently,DKK1was

foundtobelinkedtomelanomadevelopment.ThelevelofDKK1inbloodserumofmelanoma

patient is significantly higher than those in healthy control. In vitro, DKK1 inhibits the

invasion of melanoma cells40. In B16F10 melanoma induced by viral-mediated tumor

transfection, DKK1 was found to inhibit melanoma development by decreasing tumor

angiogenesisandvascularperfusion57.

47

V. TGF-bfamily

V.ITGF-bandrelatedsignalingpathway

TGF-bsuperfamilycomprisesnearly30growthanddifferentiationfactorsincludingBMPs

(Bone morphogenetic proteins), GDFs (Growth and differentiation factors), AMH (anti-

müllerianhormone),Activin,NodalandTGF-bs(Figure12).Thesignalingisconductedby

transmembranereceptorstypeIandtypeII.Inresponsetoligandbinding,typeIIreceptors

(BMPRII,ActRIIA,ActRIIB,TβRII (1-3)) catalyze thephosphorylationofType I receptors

(ALK1-7). Activated type I receptors phosphorylate downstream Smads (Smad1, Smad2,

Smad3,Smad5,andSmad8),activatingthemtotransducethesignaltothenucleus.Italso

exists non-smad signaling pathway in stem cells. TGF-b signaling pathway is involved in

embryonic development, wound healing, angiogenesis by adjusting cell proliferation,

differentiation,andapoptosis56,58.

48

Figure12.TGF-bfamilysignalingbytypeIIandIreceptors,andsmadproteins56.

V.IITGF-bsandfibroblasts

TGF-b,ononehand,isapotentinducerofECMaccumulation;ontheotherhand,itreduces

the turnover of ECM by inhibiting the expression of MMP-1 by dermal fibroblast59.

Accumulation of TGF-b is often found in cutaneous fibrosis diseases such as keloids,

hypertrophicscars,andscleroderma60–62.

AutocrineTGF-b/Smad signaling regulates gene expressionof contractility andmatrix in

dermalfibroblast.Smad7playsinhibitorroleintheformationofcollagenIanda-smooth

49

muscleactin63.TGF-b1ishighlyexpressedbothinmRNAandproteinlevelsinhypertrophic

andscartissuederivedfibroblastscomparedtonormalfibroblasts,indicatingthepotential

ofTGF-b1intheformationhypertrophicscar64,65.

V.IIIp38-MAPKsignalingpathwayactivationbyTGF-b

MAPKsignalingpathwaysareidentifiedbyfourdifferentgroupsincludingERKs,JNK/SAPK,

ERK/BMK1andP38,whichgeneratediversecellularresponsessuchascellproliferation,

differentiation, migration and apoptosis. p38-MAPK is activated by a variety of cellular

stresses including inflammatory cytokines, growth factors, UV… Activated p38-MAPK

phosphorylates and activates transcription factors which are involved in DNA damage

response,inflammation,proliferation,apoptosis,cellularregulation.

TGF-bregulatesviap38/MAPKsignalingpathwaytheexpressionofcollagenase-3(MMP-13)

ingingivalfibroblasts66.

TGF-b is akey regulatorof fibroblast activation thatdrives the synthesisof extracellular

matrixinwoundhealingandfibroticdiseases61,64,66,67.TheroleofWntsignalingpathwayin

fibrosisaswellasscarringmakesaconsideringaboutthecrosstalkbetweentwosignaling

pathways.Infibroticfibroblast,TGF-bstimulatescanonicalWntsignalinginap38/MAPK

mannerbydownregulatingDKK168.

50

VI. Conclusion

Review published in Journal of Dermatological Science

http://dx.doi.org/10.1016/j.jdermsci.2017.06.018

Article1:Preciseroleofdermalfibroblastsonmelanocytepigmentation

YinjuanWangM.D.a,CélineViennetPh.D.a*,SophieRobinPh.D.b,Jean-YvesBerthon

Ph.D.c,LiHeM.D.,Ph.D.d*,PhilippeHumbertM.D.,Ph.D.a,e

aEngineeringandCutaneousBiologyLaboratory,UMR1098,UniversityofBourgogneFranche-

Comté,Besançon,France; bBioexigenceS.A.R.L,Besançon,France; cGREENTECHSA,Biopôle

Clermont Limagne, Saint Beauzire, France; dDepartment of Dermatology, First Affiliated

Hospital of Kunming Medical University, Kunming, China; eDepartment of Dermatology,

UniversityHospital,Besançon,France

*Correspondingauthors:CélineViennet,LiHe

E-mailaddresses:celine.viennet@univ-fcomte.fr/drheli2662@126.com

Abstract

Dermalfibroblastsaretraditionallyrecognizedassynthesizing,remodelinganddepositing

collagen and extracellularmatrix, the structural framework for tissues, helping to bring

thicknessand firmness to theskin.However, theroleof fibroblastsonskinpigmentation

arouses concern recently. More is known about the interactions between epidermal

melanocytesandkeratinocytes.

This review highlights the importance of fibroblast-derived melanogenic paracrine

51

mediatorsintheregulationofmelanocyteactivities.Fibroblastsactonmelanocytesdirectly

and indirectly throughneighboring cells by secreting a largenumber of cytokines (SCF),

proteins(DKK1,sFRP,Sema7a,CCN,FAP-α)andgrowthfactors(KGF,HGF,bFGF,NT-3,NRG-

1, TGF-β) which bind to receptors and modulate intracellular signaling cascades

(MAPK/ERK,cAMP/PKA,Wnt/-catenin,PI3K/Akt)relatedtomelanocytefunctions.These

factorsinfluencethegrowth,thepigmentationofmelanocytesviatheexpressionofmelanin-

producing enzymes and melanosome transfer, as well as their dendricity, mobility and

adhesive properties. Thus, fibroblasts are implicated in both skin physiological and

pathologicalpigmentation.Inordertoinvestigatetheircontribution,variousinvitromodels

havebeendeveloped,basedoncellularsenescence.UVexposure,amajorfactorimplicated

inpigmentarydisorders,mayaffectthesecretorycrosstalkbetweendermalandepithelial

cells.Therefore,identificationoftheinteractionsbetweenfibroblastsandmelanocytescould

providenovelinsightsnotonlyforthedevelopmentofmelanogenicagentsintheclinicaland

cosmetic fields, but also for a better understanding of the melanocyte biology and

melanogenesisregulation.

Highlights

• Cutaneouspigmentationisregulatedbyacomplexdermal-epidermalnetwork.

• Fibroblastsinteractwithmelanocytesviathesynthesisofbiochemicalfactors

• Fibroblasts-derivedfactorsbindtomelanocytereceptorsandmodulateintracellular

melanogenicpathways.

• The importance of fibroblasts-derived factors is demonstratedby skinpigmentary

changes.

52

1.Introduction

Dermalfibroblastsaretraditionallyrecognizedassynthesizing,remodelinganddepositing

collagenandnon-collagenextracellularmatrix(ECM),thestructuralframeworkfortissues,

helpingtobringthicknessandfirmnesstotheskin.Theycommunicatewitheachotherand

neighboring cellsby secretinga largenumberof cytokines andgrowth factors, playinga

crucialroleinskinphysiology.Intermofpigmentation,fibroblastsexhibitagreatdynamic

in theepidermalmelanogenesis, andparticipateactively in thesignal cross-talkbetween

melanocytes and keratinocytes.Most of previous research studied the regulation of skin

pigmentationbyfocusingonbothmelanocyteswhichsynthesizemelanin,andneighboring

keratinocyteswhichreceiveanddistributethepigmentinupperlayersoftheskin.Interest

infibroblastshasincreasedinrecentyearsduetotheirabilitytosecretemelanogenicfactors.

Thisreviewoutlinestheroleofdermalfibroblastsinconstitutivepigmentationandinthe

developmentofpigmentarydisorders.

2.Correlationbetweenfibroblastandmelanocytepigmentation

Studiesincreasinglyelucidatedthesignificantroleoffibroblastinpigmentation.Photoaged

fibroblasts in reconstructed skin model stimulate pigmentation, including both melanin

production andmelanogenic gene expression, compared to unexposed fibroblasts[1]. In

addition, Salducci et al. observed an increase of melanocytes number in reconstructed

epidermis cultured with conditioned media of UVA-treated fibroblasts[2]. However,

fibroblasts limit thespontaneouspigmentationofmelanocytes in3D- reconstructedwith

cells frompatient of phototype I and II, suggesting a role of fibroblasts in the control of

pigmentation[3].Murineandhumanfibroblastsdidnotsecretethesamemelanogenicand

53

mitogenicmelanocyte factors, resulting in adifferent effect onpigmentation. It hasbeen

shown that fetal fibroblasts in reconstructed skin model induce dramatic increase of

pigmentation compared to adult fibroblast, resulting in the elevation of melanogenic

mediators fromfetal fibroblasts.Tsuchiyamaetal. foundthatmultilineage-differentiating

stress-enduringcells,distinctstemcellsamonghumanfibroblasts,couldbereprogrammed

intomelanocytes.TheseMuse-derivedmelanocytesresideinthebasallayerofepidermisin

3D-skinmodel,andacquiremelanocyticfunctions.Thistechniqueshouldpermittreatment

of vitiligo by autologous transplantation[4].Moreover, Yang et al. successfully converted

mouse and human fibroblasts to functional melanocytes by combination of several

transcriptional factors, Microphthalmia- associated transcription factor (MITF), paired

domain and homeodomain-containing transcription factor 3 (PAX3) and SRY-related

transcriptionfactor10(SOX10)[5].Theseinduced-melanocytesproducemelanosomesand

melanin,delivermelanintokeratinocytesin3D-skinmodeland,evengeneratepigmentation

invivo.Theymayprovideanewefficientwaytotreatmelanogenicdysfunctions.Therefore,

allthesefindingsconfirmanimportantcross-talkingbetweenfibroblastsandmelanocytes

in pigmentation. Specifically, these are fibroblast-derived secreted factors which are

involvedinthefibroblastinteractionswithmelanocytes.

3.Signalingpathwaysoffibroblast-derivedfactorsinmelanocytes

Asknown, thepigmentmelanin includingdifferent types,pheomelaninandeumelanin, is

producedbymelanocytesinacomplexprocesscalledmelanogenesis:melaninsynthesisin

melanocytes,melanin transport frommelanocytes to keratinocytes bymelanosome, and

melanin distribution in epidermis. All factors related to this process can affect melanin

54

synthesis, including structural proteins of melanosome (Pmel17, MART-1, GPNMB),

enzymes required for melanin synthesis (tyrosinase (TYR), tyrosinase-related protein-1

(TYRP-1) and dopachrome tautomerase (DCT)), and proteins necessary formelanosome

transport and distribution (Rab27A, myosin Va, Slac2-a/melanophilin). MITF plays a

significantroleinmelanogenesisandmelanocytesdifferentiation,dendricity,proliferation

andapoptosis. Specifically,MITF regulates theexpressionofmelanogenicenzymes (TYR,

TYRP-1andDCT),melanosomalmatrix(Pmel17,Rab27)andanti-apoptoticproteins(bcl-

2).Signalingpathwaysplayakeyroleinrelayingextracellularsignalsfromfactorbindingto

cellmembranereceptortocellnucleusviaacascadeofphosphorylationevents.Fourcrucial

intracellular signaling pathways regulate melanocyte functions, and three of them are

associatedwiththeexpressionandfunctionofMITF[6,7].

3.1MAPK/ERK

MAPK/ERK (mitogen-activated protein kinases/ extracellular signal-regulated kinases)

signaling is essential to the proliferation anddifferentiation ofmelanocytes. The kinases

MEKandERKinMAPKsignal transductionpathway involvetheactivationofmelanocyte

receptorsvia ligandbindingtotheirextracellulardomain(eg,receptortyrosinekinasec-

Kit)[8].With binding to their receptors, ligand activates complexmechanisms (Ras-Raf-

MEK-ERK)thatleadtoup-regulateMITF[9,10].AmutationinthegenethatencodestheRAF

kinaseBRAF leads to constitutive activation of downstream signaling in theMAP kinase

pathway[11,12].

3.2Wnt/β-catenin

55

Wnt/β-catenin signaling is another important pathway in pigmentation process and

melanocytes differentiation, also for melanocyte stem cell[13–15]. Activation of Wnt/β-

cateninsignalingoccursuponbindingofWnttofrizzledreceptorsandlipoproteinreceptor-

relatedprotein5and6(LRP5/6).Signalsaretransducedthroughtheinhibitionofglycogen

synthasekinase-3β(GSK-3β)activity,leadingtostabilizationandtransportofβ-catenininto

thenucleus,where itregulatestranscriptionofMITFthrough interactionswith lymphoid

enhancer-bindingfactor(LEF).Wntsignalingismodulatedbysecretedandtransmembrane

Wntinhibitorsandactivators[16].

3.3cAMP/PKA

cAMP/PKA (cyclic adenosine monophosphate/protein kinase A) signaling can also

contributetoMITFexpression.Activationofsomemelanocytereceptorswiththeirligands

(eg, melanocortin receptorMCR-1) results in increased levels of intracellular cAMP and

activation of PKA[17]. PKA phosphorylates cAMP responsive element binding protein

(CREB)whichactsasatranscriptionfactorofMITF.Ithasalsobeenreportedthatactivation

ofPKCcanbeassociatedwithcAMP-dependentpathway[18].Variousintrinsicandextrinsic

factors affect melanogenesis through this signal transduction pathway. They exert their

actionsdirectlyonmelanocytesorindirectlyviamediatorsproducedbysurroundingskin

cells[18,19].

3.4PI3K/Akt

PI3K/Akt (phosphatidylinositol 3′-kinase/Akt) signaling pathway plays a critical role in

melanocyteproliferationandapoptosis throughthecellcycleregulationwithGSK-3and

56

proteincyclinD1[20,21],andthecontroloftheproapoptoticproteinBAD[22,23].Itcould

alsocooperatewithRas-Raf-MEK-ERKsignalingcascadeonregulatingmelanocyteactivity.

4.Fibroblast-derivedfactorsinvolvedinmelanocyteactivity

Fibroblasts release melanogenic factors which act both directly and indirectly on

melanocytes. Numerousmediators secreted from fibroblasts play significant roles in the

processof skinpigmentation throughdifferent signalingpathways. Someare involved in

down-regulation(DKK1),modulation(sFRP)andinduction(KGF,NRG-1)ofpigmentation,

someareinvolvedininductionofproliferationandsurvival(SCF,bFGF,NT-3,Sema7a,TGF-

β,CNN,FAP-α),andsomeareuniversalcontributor(HGF)(Table.1).

4.1Inhibitingfactor

-DKK1

Dickkopf(DKK)familycomprises4members(DKK1-4),andencodessecretedproteinsthat

antagonize Wnt signaling by inhibiting Wnt coreceptors Lrp5 and 6. It takes part in

numerousprocessesasboneformation,Alzheimer’sdisease,eyedevelopmentandalsoskin

pigmentation. DKK1 is produced by fibroblasts in skin and its regulatory role in

melanogenesiswas firstly described in 2004. Yamaguchi et al. reported thatmelanocyte

densityinpalmoplantarhumanskinwasfivetimeslowerthanthatinnonpalmoplantarpart.

FibroblastsexpresshighlylevelsofDKK1mRNAonthepalmsandsoles,andhighlylevels

mRNAofDKK-3innonpalmoplantararea[24].Infurtherstudy,Yamaguchietal.provedthat

DKK1hasaninhibitoryeffectonMITFexpressionwhichresultsmainlyfromthedecreased

activity of GSK-3β and β-catenin[25]. In addition, DKK1 up-regulates the expression of

57

myoactivetetradecapeptide(MATP)whichreduceTYRactivity[26].Therefore,DKK1acts

on melanocytes by suppressing proliferation and melanin production. These combined

effectsexplainthelowerpigmentationobservedonthepalmsandsoles.

4.2Modulatingfactor

-sFRP

Secreted frizzle-related protein (sFRP) family consists of 5 secreted proteins in humans

(sFRP1-5) thatmodulateWnt signaling by bindingWnt proteins and Frizzled receptors.

Early studies found that sFRPbinding toWntprevented theactivationofWnt receptors,

leading to the initial classificationof sFRPs asWnt signaling inhibitors[27-28].However,

subsequentfindinghassuggestedthatsFRP2functionsasamelanogenicstimulatorthrough

Wnt/β-catenin signaling, but the precisemechanismneeds to be clarified[29]. Kimet al.

highlighted a certain paracrine role of fibroblast-derived sFRP2 in pigmentation. A co-

culture experiment with sFRP2 over/downexpressed fibroblasts demonstrated that

fibroblast-derivedsFRP2increasedpigmentationinnormalhumanmelanocytes.Thereby,

the termofWnt-signalingmodulator is preferentially attributed to sFRP.Wnt inhibitory

factor-1(WIF-1)belongingtosFRPfamilyisalsoanagonistofWntsignalingpathway.Itwas

shown that WIF-1 increases pigmentation in melanocytes co- cultured with WIF-1

overexpressedfibroblasts[30].

4.3Activatingfactors-KGF

The keratinocyte growth factor (KGF) derived from fibroblasts participates in

melanogenesis process by inducing melanosome transfer. Interleukin-1 α (IL-1α), an

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inflammatory mediator produced by keratinocytes after UVB exposure, stimulates

fibroblaststogenerateKGF.InsynergywithcAMP,transferrin,ET-1andbFGF,KGFincreases

differentiation,cellbodyexpansion,dendritesextensionandmelanosometransfer[31]. In

addition, KGF alone or in synergywith IL-1α and bFGF, inducesmelanin deposition and

elongatedreteridges[32].

-NGR-1

Neuregulin-1(NRG-1),anervegrowthfactorrelatedtothedifferentiationandmigrationof

neurons, is expressed differently among skin phototypes, and visibly increases skin

pigmentation[33]. A higher level of NRG-1 is expressed in 3D-skin equivalents included

fibroblastsfromtypeVI(darkskin).Inaddition,theamountandsizeofmelanocytes,aswell

asthicknessofdendritesareincreased[34].NRG-1bindsspecificallytoERBB3andERBB4

receptorsandactivatesPI3KandMAPKsignalingpathwaysinmelanocytes[35].

-SCF

Thecytokinestemcellfactor(SCF)issecretedconstitutivelybyfibroblasts.Thesolubleform

secreted from fibroblasts binds to the c-kit receptor of melanocytes and activates the

MAPK/ERK signaling pathway. SCF increases proliferation and differentiation of

melanocytes with or without factors produced by keratinocytes, as cAMP, ET-1 and

bFGF[36]. However the absence of SCF is correlated to a dysfunction of melanocyte

proliferation.ThesignalingSCF/c-kitisnecessarytotheviabilityofmelanocytes.Theuseof

anantibodyneutralizingc-kit(ACK2)inducesapoptosisofmurinemelanocytes[37].

-bFGF

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Thebasicfibroblastgrowthfactor(bFGF,FGF2),amemberofthefibroblastgrowthfactor

family,issynthesizedbyfibroblastsandactsinaparacrinemanneronmelanocytesviaits

transmembrane receptor FGFR2 and the intracellular signaling MAPK pathway. bFGF is

mitogenicandmelanogenicformelanocytes[38].

-NT-3

Neurotrophin-3 (NT-3) belongs to a family of nerve growth factors, synthesized by

fibroblasts.Thesefactorshavebeenextensivelystudiedfortheirroleinthedevelopmentof

neuronsandneuralcrest-derivedcellssuchasmelanocytes.NT-3canlinkeachTrkreceptor

includingTrk-A,Trk-BandTrk-C,butmainlyplaysabiologicalfunctionbybindingtoTrk-C.

It modulates intracellular signal transduction through MAPK and PI3K- Akt pathways,

regulatingmelanocytedifferentiationandsurvivalrespectively[39].

-Sema7a

Semaphorin7a(Sema7a)fromsemaphorinfamily,alargeclassofsecretedandmembrane

anchoredproteins that is involved innumerousbiologicalprocesses, stimulatesdendrite

outgrowth frommelanocytes. Sema7a is a paracrine and UV irradiation-inducible ligand

expressedbyfibroblasts[40].PlexinC1andβ1-integrinsreceptorsareligandsforSema7a,

and signaling by these receptors has opposing effects on Sema7a-induced dendrite

formation.Sema7ainducesfocalFAKandMAPKactivationviaβ1-integrin,andstimulates

melanocyte spreading and dendricity in human melanocytes. It regulates negatively

melanocytedendricityviathereceptorPlexinC1.

-TGF-β

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TheTransforminggrowthfactor-β(TGFβ)familyregulatesamultitudeofcellularprocesses,

including cell survival, proliferation and apoptosis[41]. It is secreted from various cells

includingfibroblasts.AfterbindingtoitstypeIandIIcellsurfacereceptors,TGF-βactivates

smadssignalingcascades.TGFβsignalinghasbeenshowntoexhibitarepressiveeffecton

bothmelanocytedifferentiationandmelanogenesisviadownregulationofMITFandPAX3,

and to influence quiescence of melanocyte stem cells[42–44]. It is interesting that both

TGFβ1 and TGFβ2 are upregulated by PAX3, and PAX3 itself is repressed by TGFβ1,

suggesting a negative feedback mechanism. On the other hand, TGFβ reduces CREB-

dependent transcriptionofMITFby repressionof PKA[45]. In termsof paracrine action,

TGF-βisapotentinhibitorofHGFsecretionfromfibroblasts.

-CCN

TheCCNfamilyisagroupofmultifunctionalsecretedproteinsdesignatedCCN1toCCN6.

CCN proteins regulate crucial biological processes by connecting cell surface and ECM.

Although they appear not to have specific high-affinity receptors, they signal through

integrins andproteoglycans. CCN2andCCN5aremostly expressed in thedermis[46,47].

CCN1isincreasinglyassociatedwithagegrowth[48].Aroleinskinpigmentationhasbeen

recently discovered. UV radiation upregulates CCN1-2 whereas CCN3-6 are

downregulated[49].

-FAP-α

Fibroblastactivationprotein-α(FAP-α),amemberofserineproteasefamily,isselectively

expressed in fibroblasts. It plays an important role on tumor spreading and is highly

61

expressedafterUVRtreatment[50].Thisprocesscouldbeupregulatedbyplateletderived

growthfactor-BB(PDGF-BB),TGF-β1,signalingproteinWnt5areleasedfrommelanocytes

andplasminogenactivatorfrommelanomacells[51,52].

-HGF

The hepatocyte growth factor (HGF, also known as scatter factor), highly expressed by

fibroblasts,bindstomelanocytereceptorc-METandtriggerstheMAPKandthePI3K-Akt

signaling pathways, modulating melanocyte proliferation, migration, and

melanogenesis[53,54]. The MAPK activates the ribosomal S6 kinase (RSK) family and

improves the phosphorylation of CREB protein. The secretion of HGF is stimulated by

keratinocytecytokines,IL-1αandtumornecrosisfactor-α(TNF-α)[55].

5.Fibroblastsinthedevelopmentofpigmentarydisorders

Alterationinmelaninproductionisthefundamentalchangeinpigmentarydisordersofthe

skin, which can be caused by defects of melanocytes, keratinocytes and fibroblasts.

Pigmentaryanomaliescanbeclassifiedaccordingwhetherthepigmentmelaninisincreased

(lentigo,melasma,café-au-laitmacules)ordecreased(vitiligo),withlocalizedorgeneralized

distribution(Table.2).

Theearlierscientiststhoughtthatkeratinocytesplaytheleadingrolesinpigmentation,and

fibroblastshavequitefewinfluenceonpigmentation.Forexample,membrane-boundSCF

derived from keratinocytes is more likely to increase the melanin production and

proliferation of melanocytes rather than soluble SCF derived from fibroblasts[56].

Fibroblastsareinvolvedinpathologicalpigmentation,byanalteredexpressionofvarious

62

factors. Levels of HGF, SCF and KGF in dermis of café-au-lait macules and freckles are

significantlyhigherthantheminnon-hyperpigmenteddermis[57].Thetanningresponseto

UVradiationexposureismediatedbyaspectrumoflocallyproducedcytokinesandgrowth

factors[58].Mutationsingenesencodingtheseregulatorsmodifytheirexpressionand/or

functionality, leading to altered signaling pathways, modified skin phenotypes, and

developmentofbenignlesionsortumors[59].Tobetterunderstandthedermalinfluenceon

skinpigmentation,thisreviewfocusesonsomecommonpigmentaryanomaliesthatinvolve

fibroblasts-derivedfactors.

5.1Melasma

Melasmaisacommonacquiredhyperpigmentationdisorder,occurredonforehead,cheeks

andmandible.ItaffectsappearanceandhashighlyincidenceinLatino,AsianandDark-skin

women.Thenumberofmelanocytesinmelasmalesionisincreasinghowevertheactivityof

melanin synthesis enhanced. The pathogenesis and etiology of melasma have not been

clearlyidentified,however,thepreviousresearchesidentifiedthehormonalfactors,family

history,sunexposureandcosmeticsasthefourmaintriggeringandaggravatingfactorsfor

melasmadevelopment[60].SomecytokinesinepidermissuchasSCF,PGE2,ET-1arehighly

expressed in epidermis of melasma lesion[61]. The paracrine linkage between dermal

fibroblasts and melanocytes also played an important role in the mechanism of

hyperpigmentation in melasma. UV-repeated radiations stimulate directly or indirectly,

throughkeratinocyte-derivedcytokines,thesecretionofsolubleSCFbydermalfibroblasts.

UVAradiationsinduceweakereffectonSCFsecretionthanthoseofUVB.Kangetal.found

thatSCFexpressioninthedermisofmelasmalesionissignificantlyincreased,itsreceptorc-

63

kitonmelanocytesisupregulatedindermisofmelasmaaswellbyimmunohistochemical

staining and RT-PCR[61]. Inmelasma, CCN proteins could be regulated by some factors

relatedtopigmentarydiseasessuchasFGF2,ET,estrogenanditsreceptors,progesterone

and its receptors. Fibroblasts extracted frommelasmaexpressmoreNGF-β compared to

fibroblastsfromperilesionalskin.Recently,researchersshowedthatexpressionofWIF-1is

significantly reduced in melasma lesion. Downregulation of WIF-1 in fibroblasts was

reportedtostimulatetyrosinaseexpressionandmelanosometransfer[62].

5.2SolarLentigo

Solar lentigo (SL) is a common pigmentation disorder presented as aging spot on the

exposureskin.Itischaracterizedbyhyperpigmentedmacules,andthenumberofthespots

isrelatedtotheageandskinphototype.ThehistologicalcharacterizationofSLisdefinedby

a higher melanin deposition in the basal layer, elongated epidermal ridges and large

melanosomalcomplexes.Differentialgene-profilinganalysesbetweenSLandnormalskin

biopsies revealed that SL tissues are mainly composed of activated melanocytes[63].

Overexpression ofHGF has been consistently proved in lentigo, and its receptor, c-MET,

highlyexpressedinmetastaticmelanoma[64].Therearesomefibroblast-derivedparacrine

factorssuchasHGF,KGF,SCFinvolvedinSLlesionformation[65].Immuno-staininganalyses

ofsomegrowthfactorsandsecretedproteinsintheupperdermisofSLbiopsiesstrongly

suggest that dermal fibroblasts contribute to functionally dysregulating the epidermal

cells[66].KGFsynergywithIL-1networkdescribedbeforeeffectwasprovedinsolarlentigo

aswell[67].KGFshowsalsoefficientlyabilitytopromotetheproductionandsecretionof

SCFinkeratinocytes[65].

64

5.3Vitiligo

Vitiligoisahypopigmentationdisorderinducedbydysfunctionordeficiencyofmelanocytes.

Theetiologyofvitiligoisstillunclear.Mostresearcheshaveconcentratedontheabnormality

ofmelanocytesandkeratinocytesratherthantheabnormalityoffibroblasts.Lowexpression

of SCF receptor c-kit in melanocyte at the edge of the lesion may indicate that

downregulationofSCF/c-kit/MAPKsignalingpathwaycontributestodysfunctionandloss

ofmelanocytes[56].ItwasreportedthatDKK1secretedfromfibroblastishighlyexpressed

invitiligolesionand,β-cateninandPAR-2expressioninmelanocyteissignificantlylowerin

vitiligo lesion compared to non-lesion skin[68-69]. The dysregulation of Wnt signaling

pathway in vitiligo lesion could prevent melanocyte differentiation. The occurrence of

vitiligocouldalsobeassociatedwithsomeimmunesystemdisorders[70]suchasthyroid

disease, it has effectively been proved that DKK1 could increase the expression level of

thyrotrophic embryonic factor and mitochondrial ribosomal proteins[71]. Furthermore,

attachment of melanocytes to collagen IV is mediated through collagen-receptor DDR1

(Discoidindomainreceptor1)whichisunderthecontrolofCCN3.ThedysfunctionofCCN3

andDDR1 interaction in vitiligo lesion causesweakly adhesion ofmelanocytes, and this

processinteractswithTGF-βandCCN2[72].

5.4Melanoma

Melanomaisthemostserioustypeofhumanskincancerthatdevelopsfrommelanocytes

andoccursanywhereonthebodyespeciallyinareasofthebodythatareexposedtodirect

sunlight. The main reason is caused by excessive UV exposure that damages DNA in

melanocytes. Melanoma implicates not only malignant melanocytes, but also a

65

heterogeneousmixofgeneticallystablenon-cancercells,includingfibroblasts,endothelial

and inflammatory cells. Infiltrated and surrounding stromal fibroblasts are recruited,

perpetually activated through paracrine factors released from melanoma cells and

transdifferentiatedintocancer-associatedfibroblasts(CAFs).OnemajorsourceofCAFsin

melanoma is resident normal skin fibroblasts. CAFs exhibit both morphological and

functional differences compared to normal fibroblasts. They acquire properties of

myofibroblasts, produce various growth factors, cytokines, and ECM proteins. Therefore

CAFsparticipateinthegrowthandinvasionofthetumorcellsbypromotingangiogenesis,

inflammation and metastasis[73]. However, the exact mechanisms of how normal skin

fibroblastsinteractwithmalignantmelanomacellsandsubsequentlytransformtoCAFs,are

poorlyunderstood[74].Melanomacellsanddermalfibroblastscommunicatethroughoutthe

tumorigenicprocess,includingexpressionofnumerouschemokines(IL-6,IL-8,CXCL1−3,

CXCL8,CCL5,CCL2...)andchemokine-receptors (CXCR4,CXCR2,CXCR3,CCR7andCCR10

and CXCL1...), and deregulation of signaling pathways (MAPK - PI3K/AKT, Wnt/β-

catenin)[75–77]. CAFs secretion of HGF results in activation of the c-MET receptor and

signaling pathways[78]. Expression of SCF, bFGF and KGF is involved in melanoma

genesis[79,80].UVexposureinducesupregulationofFAP-αinfibroblasts,andcontributes

tomigrationandinvasionofmelanomacells[52].HGFreceptor,c-MET,highlyexpressedin

metastaticmelanomapathwaymaintainsthemelanocytesurvivalbysuppressingthepro-

apoptoticBadprotein and increasing the anti-apoptoticBcl-xl[38].Nuclear Factor-kappa

(NF-kB), a transcription factor involved in the immune response, has been shown to be

upregulated inmelanoma throughderegulations inupstreamsignalingpathwayssuchas

Ras/Raf,PI3K/Akt.Therefore, targeting stromal fibroblasts and inhibiting stromalNF-kB

66

signaling could be a possible treatment formelanoma [35,50,81,82]. NT-3mediates cell

invasionandECMdegradationinmetastaticmelanoma[81].Additionally,developmentand

progressionofmelanomaareassociatedwithderegulationofWntsignalingandlossofDKK-

1 secretedby fibroblasts [83–85].DKK-1 isknown tobe regulatedby the canonicalWnt

pathway.Innormalmelanocytes,WntpathwayisactivatedbysecretionofWnt3a.However,

inmalignantmelanocytes,Wnt5awhich is an inhibitor of DKK-1, is expressed. It is also

suggestedthatphenotypicchangesofmelanocytesinmelanomatumorareassociatedwith

senescence,particularlyinthedermalcompartment.Thesecretionoffactorsfromsenescent

fibroblasts,suchasMMP-3,IL-6,TGF-,altersepithelialdifferentiation,promotesendothelial

cellmotilityandstimulatescancercellgrowthandtumorigenesis[86].Inaddition,senescent

fibroblasts accumulate during aging. Secreted sFRP of aged dermal fibroblasts drive

melanoma promoting both angiogenesis and metastasis[87]. Besides, loss of Sema7a

receptorPlexinC1mayinducemelanomainvasionandmetastasis,andthereforePlexinC1

isknownasapotentialtumorsuppressorformelanomaprogression[88].Downregulation

ofCCN3inmelanomacellscontributestotheirinvasivephenotype[89].

5.5Dermatofibroma

Dermatofibroma(DF),alsoknownasfibroushistiocytoma,isabenigncommoncutaneous

nodule of fibroblast-like cells with unknown etiology. Histologically, DF lesion is

characterizedbyahyperpigmentationintheoverlyingepidermiswithacanthosisandwith

an increased number of melanocytes. Proliferating fibroblast-like cells and histiocytes

accumulate in the dermis and are surrounded by mature collagen and by increased

capillaries. They secrete elevated gene and protein levels of SCF andHGF cytokines and

67

stimulate melanocytes located in the adjacent epidermis, which results in the

hyperpigmentationoftheoverlyingskin[50].ThealteredexpressionofSCFandHGFinDF

lesionscanbeassociatedwiththetumorcellproliferationandinductionofDF.SCForHGF

derivedfromthefibroblastictumormayfunctionasamitogenformelanocytes[90].SCFis

alsoknownasmastcellgrowthfactorandthisexplainswhyaccumulationanddegranulation

ofmastcellsareobservedwithmelanocyteactivationinDFlesions.However,furtherstudies

arerequiredtoidentifythefactorsthatstimulateexclusivelyinabundancethesecretionof

SCFandHGF.

6.Conclusion

Cutaneouspigmentationisregulatedbymelanogenicfactors,locallysynthesizedintheskin

bybothkeratinocytesandfibroblasts,orproducedbydistanttissuesandtransportedtothe

skin by the circulation. Many of those factors regulate constitutive and induced

pigmentation. In this review, the contribution of dermal fibroblasts to the regulation of

melanocyteactivities isdemonstrated.Fibroblasts interactwithmelanocytesdirectlyand

indirectlythroughkeratinocytes.Theyreleasenumerousbiochemicalfactorsthatmodulate

thepigmentedstatusofmelanocytesbyactivatingsignalingcascades,geneexpressionand

enzymeactivity.Levelsof fibroblast-derivedmelanogenicparacrinemediatorsdependon

intrinsicandextrinsicfactorssuchasskintype,genetic,sunexposition.Increasedresearches

on the importance role of fibroblasts in melanocyte function under physiological or

pathologicalconditionsprovidetheoryevidencesfortreatmentofpigmentarydisorders.

Financialsupport

68

ThisworkwasfinanciallysupportedbyProgramforInnovativeResearchTeaminUniversity

ofMinistryofEducationofChina(GrandNo.IRT13067)andTheFundofYunnanProvince

ChineseAcademyofSciencesCooperation(GrandeNo.2014IB008)(China)andGreentech

SA(France).

Conflictsofinterest

Theauthorshavenoconflictofinteresttodeclare.

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AuthorBiography

Yinjuan Wang is a Ph.D. candidate from the Laboratory of Engineering and Cutaneous

Biology, UMR 1098, Bourgogne Franche-Comte University, Besancon, France. Her thesis,

supervisedbyProfessorsPhilippeHumbertandHeLi,isconcernedwithskinpigmentation.

She receivedMedicalDoctor on2011andDermatologicalMaster on2014, fromSichuan

UniversityandKunmingMedicalUniversityofChina, respectively.Her research interests

involvemelaninsynthesisbymelanocytesanditspathways,inparticulartheepidemiology

andphysiopathologyofhyperpigmentedskinlesionsincludingmelasmaandlentigo.

82

Table.1.Effectsoffibroblast-derivedfactorsonmelanocytefunctions.

83

Table.2.Somecommonpigmentationdisordersinvolvingfibroblast-derivedfactors.

84

ChapterII

Developmentandvalidationofasimplemethodforthe

extractionofhumanskinmelanocytes

85

Theonlycellsthatproducemelaninaremelanocyteswhichareusefulforstudying

manypigmentarydisordersaswellasmechanismsregulatingmelanogenesis,andforcell

transplantationintreatmentofvitiligo.

Therearevariousmethodstoisolatemelanocytesfromskin,whicharegenerallybasedon

two-steps enzyme digestion (by dispase and trypsin) process (Table 1)76–78. Although

previouslydevelopedprotocolsaresatisfactoryintermsofcellyieldandviability,theyuse

enzymesofanimalorigin.Inthischapter,weintroducedasimpleandoptimizedmethodfor

extractionofmelanocytesfromlowsurfaceofhumanskinsample,whichshouldbeeffective

forfunctionalandmolecularstudiesandalsoformedicalapplicationsuchcellgrafting.We

compare the new protocol with a classic melanocyte extraction method. This work is

summarizedinthefollowingarticlesubmittedinCytotechnologyjournal.

86

Protocol Method77 Method78 Method76

Sterilization Iodine70%ethanolDPBSCa\Mg-free10μg/mlGentamicin

70%ethanolHBSS

IodineNormalSaline

Sizeofsample 1cmwidepieces(minimum1cm2)

5×5mm2pieces 0.5mmthickpieces

Solutionforseparatingdermisandepidermis

10mg/mldispaseIIinHank’ssolutionOvernight4℃（≥18h）

0.48mg/mldispaseII(BoehringerMannheim)0.1%BSAinPBSCa/Mg-free18-24h,4℃

0.25%dispase(Solarbio)Overnight,4℃

Solutionforgettingcellsuspension

Trypsin/EDTA10-15min,37℃

Trypsin/EDTA5min,37℃

Trypsin/EDTA5min,37℃

Termination RPMIMedium HBSS+Medium SerumCountingcellbeforeculture

Yes No Yes

Medium RPMI(Sigma)supplementwithTPA,CT,ET1,SCF

MCDB153(Sigma)supplementwithFBS,ChelatedFBS,L-Glutamine,CT,rh-bFGF,rET-3,rhSCF,Heparin

MC254(Gibco)1%HMGS2100U/ml，Penicillin，50μg/mlstreptomycin

Seedingnumber

15×104cellsperml AllcellsinT25cell-culturevessel

5x105cells/25cm²flask

Table1.Comparisonofthreeclassicmelanocyteextractionmethods

87

Article2:Developmentandvalidationofasimplemethodfortheextractionofhuman

skinmelanocytes

YinjuanWanga,CélineVienneta*,GwenaëlRolina,f,MarionTissota,PatriceMureta,Sophie

Robinb,Jean-YvesBerthonc,LiHed*,PhilippeHumberta,e

aEngineeringandCutaneousBiologyLaboratory,UMR1098,UniversityofBourgogneFranche-

Comté,Besançon,France

bBioexigenceS.A.R.L,Besançon,France

cGREENTECHSA,BiopôleClermontLimagne,SaintBeauzire,France

dDepartmentofDermatology,FirstAffiliatedHospitalofKunmingMedicalUniversity,Kunming,

China

eDepartmentofDermatology,UniversityHospital,Besançon,France

fClinicalInvestigationCenter,InsermCICB1431,UniversityHospital,Besançon,France

*Correspondingauthors:CélineViennet,LiHe

E-mailaddressforcorrespondance:celine.viennet@univ-fcomte.frordrheli2662@126.com

Acknowledgments: this work was financially supported by Program for Innovative

ResearchTeaminUniversityofMinistryofEducationofChina(GrandNo.IRT13067),the

FundofYunnanProvinceChineseAcademyofSciencesCooperation(GrandNo.2014IB008)

andGreentechSA(France).

Conflictofinterests:theauthorshavenoconflictofinteresttodeclare.

88

Abstract

Primarymelanocytesincultureareusefulmodelsforstudyingepidermalpigmentationand

efficacyofmelanogenic compounds,ordevelopingadvanced therapymedicinalproducts.

Cellextractionisaninevitableandcriticalstepintheestablishmentofcellcultures.Many

enzymatic methods for extracting and growing cells derived from human skin, such as

melanocytes, are described in literature. They are usually based on 2 enzymatic steps,

Trypsin in combination with Dispase, in order to separate dermis from epidermis and

subsequently provide a suspension of epidermal cells. The objective of thisworkwas to

develop and validate an extraction method of human skin melanocytes being simple,

effectiveandapplicable tosmallerskinsamples,andavoidinganimalreagents.TrypLETM

productwastestedonverylimitedsizeofhumanskin,equivalentofmultiple3-mmpunch

biopsies,andwascomparedtoTrypsin/Dispaseenzymes.Functionalityofextractedcells

was evaluated by analysis of proliferation, morphology and melanin production. In

comparisonwith Trypsin/Dispase incubationmethod, themain advantages of TrypLETM

incubationmethodwere theeasierof separationbetweendermisandepidermisand the

lower contamination by keratinocytes after extraction. Both protocols preserved

morphological and biological characteristics of melanocytes. The minimum size of skin

samplethatallowedtheextractionoffunctionalcellswas6x3-mmpunchbiopsies(e.g.,42

mm2)whatever themethod used. In conclusion, this newprocedure based onTrypLETM

incubationwouldbesuitableforestablishmentofoptimalprimarymelanocytesculturesfor

clinicalapplicationsandresearch.

89

Introduction

In human skin,melanocytes are specialized cells of the epidermis and hair follicles that

producethepigmentmelanin.Innormalepidermis,theyconstituteapproximately3to7%

of the cells, contactingwith severalneighboringkeratinocytesand forminganepidermal

melaninunit.Melanocytesarelocalizedinthebasallayeroftheepidermisatthejunctionof

thedermis,andtheirdendritesallowmelanintransfertokeratinocytes.Establishmentand

characterization of melanocyte cultures are important for understanding and treating

pigment-relateddisorders,ordevelopinginvitropigmentedskinmodels(Guerraetal.2003;

Duvaletal.2003;Yoonetal.2003;Vermaetal.2015;Kumaretal.2012).Althoughanumber

of cell lines originated fromhealthy and lesional skin are used for studying cellular and

molecularbiology, testing cosmetic and chemicalproducts, primary culture remains as a

more reliablemodel thatmimics in vivo skin. Severalmethods for the isolation and the

cultureofmelanocytesfromhumanskinhavebeendescribed(Hsuetal.2005;Godwinetal.

2014;Tangetal.2014;Nielsenetal.1984;Gilchrestetal.1984;Yaranietal.2013).Thefirst

human melanocyte culture from skin was published by Hu et al. (1957). Unlike basal

keratinocytes that are proliferative and capable of regenerating the epidermal layers,

melanocyte cultureshave lowproliferationpotential and short life span.Toobtainhigh-

densityprimaryculturesofmelanocytes,differentselectivemediaaresupplementedwith

mitogens as Phorbol 12-myristate 13-acetate (PMA), endothelins, growth factors (basic

fibroblastgrowthfactor,stemcellfactor,hepatocytegrowthfactorandnervegrowthfactor)

(Eisinger andMarko 1982; Halaban et al. 1987; Imokawa et al. 1998; Yada et al. 1991;

Peacocke et al. 1988). 5-fluorouracil (5-FU) is utilized to isolate pure populations of

melanocytes free from keratinocyte (Takuo et al. 1983). Isolation of melanocytes from

90

human skin explants is usually performed in a two-step protocol, dispase incubation to

separate the dermal and epidermal layers, following by trypsin treatment to release the

epidermalcells(Tangetal.2014).Althoughtrypsinisutilizedforavarietyofcellculture

applications,itisderivedfromanimalsandmaycontaintoxicsubstances.Thereforemostof

thetechniquesdescribedintheliteraturearealimitingfactorinusingcellsforclinic.

Thisworkdescribesanewmethodforisolationofmelanocytes,basedonone-stepenzyme

incubationusingtheTrypLETM,ananimalorigin-freeandrecombinantenzyme.Wereported

thenumberofcellsisolatedfromdifferentlimitedsizesofskintissueandcharacterizedthe

cellpopulation.Inaddition,wecomparedournovelprocedurewithanestablishedtechnique

(Godwinetal.2014),studyingthefunctionalityoftheextractedmelanocytes.

MaterialsandMethods

Collectionofskinbiopsies

Skinsampleswereobtainedfromdiscardedskinfollowingabdominoplastywithinformed

donor consent (5 patients, 43±17 years, skin types II-IV). The hypodermis layer was

removed.Subsequently,skinbiopsiesof3mmindiameterwererealizedandputfor10min

in phosphate-buffered saline (PBS) containing 100 IU/ml penicillin and 100 μg/ml

streptomycin (PS) (all from Pan-Biotech, Dutscher, France). In total, 45 biopsies were

collectedperskinand6distinctgroupswererealizedaccordingtothenumberofbiopsies

(5to10).

Dissociationofepidermisfromdermisandseparationofepidermalcells

Method I: simpleenzymaticdigestion (TrypLETM) (Figure.1)Biopsieswere incubated in

TrypLETMenzyme1X(GIBCOÒTrypLETMSelectTMCTSTM,ThermoFisherScientific,France)at

roomtemperatureovernight(17h).Duringthetreatment,theepidermallayerwaseasily

91

separated fromthedermis.After incubation,alldermiswerediscarded, thenmelanocyte

culture medium was added and epidermal cells were disaggregated mechanically by

repeatedpipettingupanddown.Mixtureswerefilteredthrough70μmcellstrainers,and

centrifugedfor5minat1,100rpm.Cellpelletswereresuspendedinmelanocytemedium

which consists ofM254 culturemedium supplementedwith humanmelanocytes growth

supplement (HMGS, Life Technologies, ThermoFisher Scientific, France), 100 IU/ ml

penicillinand100μg/mlstreptomycin.Epidermalcellswerecounted,placedinto25cm2

tissuecultureflasksatacelldensityof2to4x104cells/cm2,andkeptat37°Cinahumidified

atmospherewith5%CO2.

MethodII:double-enzymaticdigestion(dispaseandtrypsin)Acontrolgroupwassetupby

incubating biopsies in 10 mg/ml dispase II (GIBCOÒ, ThermoFisher Scientific, France)

overnight(17h)at4°C.Theepidermiswasseparatedfromthedermisusingfineforceps.

Isolatedepidermal layerswereput in0.05%trypsin/0.02%EDTAsolution (Pan-Biotech,

Dutscher,France)at37̊Cfor10min.

After incubation, melanocyte culture medium was added and epidermal cells were

disaggregatedmechanicallybyrepeatedpipettingupanddown.Thefollowingstepswere

similarasTrypLETMmethod.

Primarymelanocytecultures

Culture medium was firstly changed after 48 h, then every 3 days. When melanocytes

reached 70% confluency, they were harvested by 3 min treatment at 37°C with 0.05%

trypsin/0.02%EDTAsolution.Thecellsuspensionwasneutralizedwithfetalbovineserum

(Pan-Biotech,Dutscher,France)andcentrifugedfor5minat1,100rpm.Thecellpelletwas

resuspended in melanocyte medium. Viable cell counts were performed before seeding

92

melanocytesat16x103cells/cm2 intonewculture flask foranotherroundof incubation.

ImagesofcellcultureswereacquiredusinganOlympusIX50invertedmicroscope.

Melanocyteculturesatpassage2wereusedforthefollowingexperiments.

Melanocyteproliferationassay

Melanocyteswereseededatadensityof6x104cells/wellin12-wellplates.After1,2,3and

4 days of culture, cellswere harvested by trypsinization and counted using trypan blue

exclusion.

Measurementofmelanincontent

Melanocyteswere plated at 400000 cells on 25 cm2 flask and grown for 5 days to 70%

confluence.TheywerewashedwithPBS, collectedbyscrapingandpelleted. Intracellular

melaninwasextractedbysolubilizingfrozencellpelletsin1NNaOHcontaining10%DMSO

for 2 h at 90 °C. The samples were then centrifuged at 1100 rpm for 10 min and the

supernatantswere transferredto fresh tubes.Themelanincontent in thesesupernatants

wasmeasuredspectrophotometricallyatanabsorbanceof420nmagainstastandardcurve

ofknownconcentrationsofsyntheticmelanin(0-50μg/mL,Sigma-Aldrich,France).

TheproteinlevelincellpelletwasdeterminedusingtheBradfordassay.Theintracellular

melanincontentwasadjustedbytheamountofproteininthesamesample.

Immunocytochemistry

Melanocytesweregrownin8-wellchamberslides(NuncLab-TekChamberSlides,Dutscher,

France)atadensityof1×104cells/well.After24h,cellswerefixedin3%paraformaldehyde

in PBS, then rinsed in PBS and permeabilised for 15min in 0.1% Triton X-100 in PBS.

Nonspecificbindingsiteswereblockedatroomtemperaturebya10minincubationin1%

glycineinPBSfollowedbya1hincubationin3%bovineserumalbumin(BSA)solutionin

93

PBScontaining10%normalgoatserumand0.1%TritonX100.Cellswereincubatedat4°C

overnight with polyclonal rabbit anti-human microphthalmia-associated transcription

factor (MITF) at 1:1000 dilution, then 1 h at room temperature with goat anti-rabbit

antibodyconjugatedwithrhodaminediluted1:2000.Theyweredouble-stainedwithFITC-

conjugatedphalloidinfor15minatroomtemperature(allantibodiesfromSigma-Aldrich,

France).PBSwith1%BSAand0.1%TritonX-100wasusedforwashesbetweenincubations

andantibodydilutions.SamplesweremountedonglassslideswithFluoromountTMmedium

andobservedunderaZeissAxioskop40fluorescencemicroscope.

StatisticalAnalysis

Allvalueswereexpressedasmean±SE.Statisticalanalysesbetweenskinbiopsygroups

wereperformedusingANOVAwithSigmaStat.Differenceswereconsideredsignificantifthe

Pvaluewas<0.05.

Resultsanddiscussion

Primary cultures of human melanocytes are appropriate models to study the

pathomechanismsofpigmentation,thegeneticdisordersofmelanocytedevelopmentorthe

efficacy of melanogenic compounds. Unfortunately, the clinical size and the anatomical

locationofmelanocyticlesionsoftenpreventawideexcisionofskinand,thusmakedifficult

the establishment of melanocyte cultures. Many methods for extracting and growing

melanocytesaredescribed(Hsuetal.2005;Godwinetal.2014;Tangetal.2014;Nielsenet

al.1984;Gilchrestetal.1984;Yaranietal.2013).Theyareusuallybasedon2enzymatic

steps, dispase in combination with trypsin, to separate dermis from epidermis and

subsequently provide a suspension of epidermal cells. The objective of thisworkwas to

developandvalidateamelanocyteextractionmethodbeingthesimplest,themosteffective

94

andapplicabletosmallskinsamples.

Enzymaticdissociationofhumanskintissue

Melanocyteswereisolatedfromverylimitedsizeofhumanskin,equivalentofmultiple3-

mmpunchbiopsies(notexceeding70mm2intotalsurfacearea).Weusedone-stepenzyme

incubation with TrypLETM, an animal origin free and recombinant enzyme, for isolating

epidermis from dermis and release epidermal cells. We compared our method with a

standarddissociationprotocolwhichisperformedintwo-stepenzymeincubation.Asshown

inFigure.2,theincubationinTrypLETMresultedinatotalseparationoftheepidermisfrom

thedermis.Theincubationwithdispasewaslesseffectivewithapartialseparationofthe2

skinlayersontheoutsideedgeofbiopsies.Epidermalsheetswerepeeledfromthedermis

with forceps. Following TrypLETM treatment, the intact epidermal layer was easily

dissociatedmechanicallyintoacellsuspension.Withthestandardprotocol,thedigestionof

epidermisrequiredasecondenzymeincubationusingtrypsin,supportedbyamechanical

action.

Inaddition,contrarytothetraditionalmethod,theadvantageofourmethodistheabsence

of animal derived enzyme, a major condition for the production of advanced therapy

medicinal products. Kormos et al. (2016) described the use of the Mel-mix medium

supplementedwithhuman serum forobtainingpuremelanocyte cultures.Therefore, the

combinationofdifferent techniquesoffersvaluableopportunity inculturedmelanocytes-

basedtransplantationtherapies.

Growthpatternofextractedcells

As Normand and Karasek (1995) who focused on keratinocytes, endothelial cells and

fibroblasts,ourmethodestablishedmelanocyteculturesfromsmallamountoftissue.There

95

wasnosignificantdifferenceinnumberofcellextractedamonggroupsof6to10biopsies,

andwhatever themethodused (Figure. 3). Surfaceof skin tissue less thanor equal to5

biopsies(e.g.,35mm2)didn’tallowsurvivalandproliferationofextractedcells (datanot

shown).Extractedcellsattachedtothecultureflaskwithin48handmelanocytesinitiateda

dendritic appearance (Figure. 4). After 10 days of incubation, it was observed that

melanocytes were 40-50% confluent and displayed typical dendritic morphology. Cells

became highly proliferative. In case of TrypLETMmethod, we obtained a relatively pure

primarymelanocyteculture.Incontrast,primaryculturesobtainedfromdispaseandtrypsin

incubations were contaminated with keratinocytes, suggesting a significant increase of

adhesionandgrowthofextractedkeratinocytescomparedtoTrypLETM.Incubationfor17h

at4°CtoseparateepidermisfromdermiswithTrypLETMmethodgavebetterresultswith

less keratinocytes. However in subsequent passages these clusters of keratinocytes

disappeared, inducing the decrease of cell number between extraction day and end of

passage 1. Extracted cells reached 70% confluency around 14 days and they were

subculturedata1:2splitratioapproximatelyevery10to14days.Thereisnodifferencein

thecellyieldofearlypassagemelanocytesusingbothmethodsandfromskinsampleswith

morethan42andlessthan70mm2oftissue.

FunctionalityofmelanocytesPrimarymelanocytesinculturemighttobeoptimaltoexamine

invivopathomechanisms, toassessbiologicaleffectofcompoundsortotreatpigmentary

lesions. Previous work showed that non-enzymatic tissue incubation method for cell

isolationpreservesmolecularmarkers(Xinyanetal.2014).Prolongedpassagesofcultured

melanocytes tend to lose their pigmentation (Virador et al. 1999). Due to the limited

proliferativecapacityofprimarymelanocytesandthevariabilityofphenotypesgenerated,

96

it’s necessary to limitmelanocyte passage number. In the presentwork,we studied the

functionality of melanocytes cultured at passage 2. Several melanocytic markers can be

utilized to identify typically melanocytes, including MITF. MITF is a cell-type specific

regulator that is required for thedevelopmentandsurvivalofpigmentscells. Itactsasa

masterregulatorofmelaninproduction(Shietal.2016).Weanalyzedcellmorphologyand

moreprecisely thedynamic cytoskeletal networkby filamentous actin (F actin) labeling.

Resultsobtainedwereequivalentwhateverthenumberof3-mmpunchbiopsies.Onlythe

data for10x3-mmpunchbiopsiesareshown.Similargrowthratewereobservedamong

TrypLETM andDispase/Trypsinmethods (Figure. 5). Therewas also no difference in the

intracellular melanin content of melanocytes (Figure. 6). Immunocytochemical staining

confirmed that cells extracted by both methods positively expressed MITF and F actin

(Figure.7).Itwas concluded that TrypLETM andDispase/Trypsinmethods are of similar

efficacy for preserving cell biological characteristics. Melanocytes extracted with our

protocolandculturedwithclassicalconditionsexhibitednormalfunctionalitywithrespect

todendriticmorphology,pigmentproductionandgrowthkinetic.

Conclusion

Wereportinthisstudyanovelandsimpleextractionmethodofhumanskinmelanocytes

basedontheuseofTrypLETM.Thisprotocolprovidedpurepopulationsofmelanocyteafter

the extraction process and preserved cell morphology and functional characteristics. It

maximizedthenumberofmelanocytesobtainedfromsmallskinsurface(greaterthan42

mm2or6x3-mmpunchbiopsies),andthereforegeneratedsufficientcellstostartaprimary

culture.Inaddition,ourprocedurehadthesignificantadvantagetouseananimal-freecell

culturereagentandcouldbeapplicabletothedevelopmentofadvancedcelltherapyproduct

97

indicatedforthetreatmentofpigmentarydisorders.

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reconstructed human skin to evaluate UV-induced modifications and sunscreen

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vitro in the presence of phorbol ester and cholera toxin. Proc Natl Acad Sci USA

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melanocytesfromnewbornandadultepidermis.JInvestDermatol83:370-376

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DC(2014)Isolation,culture,andtransfectionofmelanocytes.CurrProtocCellBiol

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epidermisinthesurgicaltherapyofstablevitiligo.ArchDermatol139:1303-1310

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humanmelanocytes.InVitroCellDevBiol2347–52

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intheabsenceofphorbolesters.MethodsMolMed107:13-28

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adultepidermis.PloSOne6:e17197

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keratinocyte co-culture model for controls and vitiligo to assess regulators of

pigmentationandmelanocytes.IndianJDermatolVenereolLeprol78:599–604

- NielsenHI,DonP(1984)Cultureofnormaladulthumanmelanocytes.BrJDermatol

110:569–580

- NormandJ,KarasekMA(1995)Amethodfortheisolationandserialpropagationof

keratinocytes,endothelialcellsandfibroblastsfromasinglepunchbiopsyofhuman

skin.InvitroCellDevBiolAnimal31:447-455

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growth factor receptors on cultured humanmelanocytes. Proc Natl Acad Sci USA

85:5282–5286

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Fan.Biologicalcharacteristicsofmouseskinmelanocytes.Tissueandcell48:114-120

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forthestudyofhyperpigmentation.Cytotechnology66:891-8

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assessingregulatorsofpigmentation.AnalyticalBiochemistry270:207-219

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andproliferationinhumanmelanocytes.BiolChem266:18352–18357

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100

Figure.1Shematicdiagramfortheisolationofmelanocytesfromsmallamountofhuman

skin,viatheTrypLETMmethod.Theinitialseedingdensityforinitiatingtheprimaryculture

is2to4×104cellsper25cm2tissuecultureflask.Generally,onesuchflaskissetupfroman

initialyieldof4to10×105cellsisolatedfromabout42to70mm2pieceofskin.

101

Figure.2Epidermal-dermalseparationusingTrypLETMenzyme1X(A)anddispaseII(B).

After TrypLETM incubation, epidermis (arrow) was completely separated from dermis,

whereasafterdispasetreatment,itwasnotperfectlydetached.

102

Figure.3Skinepithelialcells isolatedfrommultiple3-mmpunchbiopsies.Comparisonof

numberofcellsusingTrypLETMandDispase/Trypsinmethods.Cellcountswereperformed

at the extraction day and the first passage (P1). Histograms are means ± SE of five

independentexperimentswithskinfromdifferentdonors.Whateverthemethodused,there

wasnosignificantdifferenceincellnumberfrom6biopsiesto10biopsies.

103

Figure.4Growthpatternofcellsextractedfrom10punchbiopsiesofskin,byTrypLETM(A,

C) and Dispase/Trypsin (B, C), at 2 days (A, B) and 10 days (C, D) after cell isolation.

Extracted cells attached to the culture flaskwithin 2 days andmelanocytes initiated a

dendriticappearance.After10daysofincubation,melanocytesdisplayedtypicaldendritic

morphology.WithTrypLETMmethod,relativelypuremelanocytecultureswereobtained,

whereas with Dispase/Trypsin method, melanocyte cultures were contaminated with

keratinocytes.Bar100µm

104

Figure.5GrowthrateofmelanocytesextractedbyTrypLEandDispase/Trypsinmethods.

Melanocytesatpassage2weredepositedin12-wellplatesatadensityof6x104cells/well.

Cellnumbersweremeasuredafter1,2,3and4daysofculture.Dataareexpressedasthe

means±SEof5independentexperimentscarriedoutinduplicate.Nodifferencewasfound.

105

Figure.6Intracellularmelanincontentnormalizedtotheamountofprotein.Melanocytesat

passage2weredepositedon25cm2flaskatadensityof4x105cells/flask.Melaninand

proteincontentsweremeasuredafter5daysofculture.Dataareexpressedasthemeans±

SEof5independentexperimentscarriedoutinduplicate.Nodifferencewasfoundbetween

TrypLEandDispase/Trypsinmethods.

106

Figure.7Immunostainingofcellsextractedfrom10punchbiopsiesofskin,byTrypLETM(A)

and Dispase/Trypsin (B), and cultured at passage 2. MITF (in red) was used as a

characteristic melanocyte marker while F actin (green) as a cell shape marker. Cells

extractedbybothmethodspositivelyexpressedMITFandFactin.Bar50µm

107

ChapterIII

InvolvementofDickkopf-relatedprotein1in

melanogenesis:focusonsolarlentigolesion

108

I. AimsoftheStudy

Recent studies reported that high DKK1 expression by dermal fibroblasts is closely

relatedtohypopigmentedskin,aspalmoplantarskinandvitiligolesion.DKK1encodedby

DKK1geneisanantagonisticinhibitoroftheWntsignalingpathway,byisolatingtheLRP5/6

co-receptorandpreventingtheFrizzeled-Wnt-LRP5/6complex formation.Wnt/bcatenin

signalingregulatesthetranscriptionofmelanocyte-specificgeneslikeMITF,ageneinvolved

inmelaninsynthesis.

The aims of the study were firstly, to determine the cellular level of DKK1 on

hyperpigmented skin including solar lentigo; secondly to conduct a comprehensive

investigationofthemechanismsunderlyingthislesion72.

Invitromodelsthatmimictheinvivoenvironmentwereused:fibroblastsisolatedfromsolar

lentigoandperi-lesionalbiopsies,andnormalfibroblastsembeddedin3Dcollagengeland

exposedtorepeateddosesofUVA.

II. Hypothesis

TGF-β1mediatesdevelopmentofsolarlentigobyreducingDKK1expressioninfibroblasts

through thep38-MAPkinasepathway,which leads toanactivationof theWnt/b-catenin

signalingcascade.Thisprocessmayresultinanunevendistributionofactivemelanocytes,

withareasofhyperpigmentationintheskin(Figure13).

109

110

Figure 13. Hypothesis of the study: Structural damages, pro-inflammatory cytokines,

immune-modulatory factors (SASP) caused by external stimulated factors (UV rays, air

pollution, environmental toxins) induce high secretion of TGF-b by fibroblasts and

inflammatorycells(Tcells,macrophages).ThisprocessinducesadownregulationofDKK1

gene expression and a decrease of DKK1 secretion by fibroblasts. Low expression and

secretionofDKK1leadstotheactivationofWnt/b-cateninsignalingpathway,andresultsin

increasedmelaninsynthesisbymelanocytes.

111

III. MethodsandMaterials

III.I.PartI-Solarlentigobiopsies

III.I.I.Collectionofskinbiopsies

Twobiopsiesof3-mmpunches(onefromsolarlentigolesionalskin(SL),onefromadjacent

normal skin (SN)) were obtained from nine female volunteers on the back of hands

(Caucasianfemales,67–89yearsofage)afterwritteninformedconsent(Universityhospital

ofBesançon,DepartmentofDermatology;Pr.PHumbert).

III.I.II.Histology

III.I.II.IFixationandsectioning

SLandSNbiopsieswerefixedinPFAsolution(3%inPBS),dehydratedthroughagraded

seriesofalcoholandembeddedinparaffin.Sections7µminthicknesswereobtainedusing

arotarymicrotome(Leitz1512;Leica).Theywereclearedinxylenerehydratedinsolutions

ofalcoholandbrieflywashedindistilledwater.

III.I.II.IIMasson'strichromestainingforcollagenfibers

ThesectionswerestainedinHarrishematoxylinsolution,rinsedindistilledwater,stained

with0.1%eosinsolution,treatedwith1%aceticacid-water,washedagainanddifferentiated

in1%phosphomolybdicacid.Finally,thesectionswerestainedwith0.5%lightgreenand

rinsedindistilledwaterbefore1%aceticacid-water.Thestainedsectionsweremounted

withFaramountaqueousmountingmedium,andobservedusingaZeissAxioskop40light

microscope.

112

III.I.II.IIIMasson'sFontanastainingformelanin

The sections were stained with Fontana-Masson kit following the manufacturer’s

instructions(Diapath,Italy).ThestainedsectionsweremountedwithFaramountaqueous

mountingmedium,andobservedusingaZeissAxioskop40lightmicroscope.

III.I.II.IV.Immunostainingforβ-catenin

Slideswereheatedat95 °C for20min in10 mM citratebuffer pH 6. Sampleswere then

incubatedovernightat4°Cwiththeprimaryantibodiesat1:50(MAB13291-SP,R&Dsystem,

USA).Subsequently,theywereincubatedfor1hwithanti-mouseHRP-DAB(CTS002,R&D

system,USA)followingmanufacturer’sprocedure.Thestainedsectionsweremountedwith

Faramount aqueous mounting medium, and observed using a Zeiss Axioskop 40 light

microscope.

III.I.III.Cellculture

III.I.III.I.Extractionoffibroblasts

All cell culture products were purchased from PAN Biotech, Germany. Primary human

fibroblasts from SL (FL) and from SN (FN) were obtained from the biopsies by explant

culture,andcellculturesweremaintainedinDulbecco’sModifiedEagle’sMedium(DMEM)

supplementedwith10%fetalbovineserum(FBS)and1%Penicillin/Streptomycin(P/S,100

units/mLpenicillin,100µg/mLstreptomycin)inahumidified5%CO2atmosphereat37℃.

III.I.III.II.Establishmentofskinfibroblastcultures

FLandFNweremaintaineduntilthemonolayerculturesreached80%confluencyandwere

passagedusing0.25%trypsin-EDTAsolution.Cellswereusedforexperimentsatpassages

2-7.FLandFSwereseededat400,000cellsin25cm2cultureflasks(F25)with3mLfull

DMEMmedium.

113

III.I.IV.Enzyme-linkedimmunosorbentassay(ELISA)

After3daysofculture,supernatantswerecollectedwith10%(v/v)anti-proteasesolution

(P1860,Sigma-Aldrich,France)andstoredat-80°C.ELISAanalyzesforDKK1(kitEHDKK1,

ThermoScientific,USA)andTGF-b1(kitBMS2065,Invitrogen,Australia)wereperformed

accordingtothemanufacturer’sinstructions.Duplicateanalyseswereperformedforeach

sample. Dkk1 concentration in the supernatantswas normalized to the concentration of

intracellular protein (which corresponds directly to the cell number) determined by the

Biuretmethod.TGF-b1concentrationinthesupernatantswasnormalizedtothecellnumber.

III.I.V.Biuretproteinassay

After3daysofculture,cellswerescrapped,lysedwithNaOH0.1NandmixedwithBiuret

reagent(theBiuretreaction(Cu2++e-→Cu+)usesbicinchoninicacidanditisbasedonthe

measurementoftheformationofpurple-coloredcomplexofbicinchoninicacidandCu+)73.

Mixtureswerecentrifugated,andabsorbanceofsupernatantswasdeterminedat620nm.

Bovineserumalbuminwasusedasstandard.Duplicateanalyseswereperformedforeach

sample.

III.I.VI.Real-timequantitativereversetranscription-PCR

After3daysofculture,cellpelletswerecollectedwith0.25%trypsin-EDTAsolution,lysed

inRLTbuffer(Qiagen,France)with1%b-mercaptoethanolandstoredat-80°C.TotalRNA

wasextractedusingtheRNeasyMiniKit(Qiagen,France)accordingtothemanufacturer’s

protocol. Next, cDNA was performed using the High Capacity RNA-to-cDNA kit

(Thermofisher, Applied Biosystems, France). TaqmanÒ Gene Expression Assay kits

(Thermofisher,AppliedBiosystems,France)wereusedtoassessDKK1(Hs00183740_m1)

andAbl(Abelson,Dm01843160_g1)geneexpressions.Ablwasusedashousekeepinggene.

114

ThePCRreactionmixturecomprised2μLofcDNAsample,10μLofTaqManuniversalPCR

mastermix,1μLofeachofTaqMangeneexpressionassayandRNase-freewater.Reactions

wereperformedandmonitoredusing theBioradCFX96Real-TimePCRSystem.Thermal

cyclingconditionswereinitiatedwith2minat50°C,followedby10minat95°C,andthen

40cyclesof95°Cfor10s(denaturation)and60°Cfor1min(annealing/extension).

Forquantification, geneexpression levelswerecalculatedby the comparativeCtmethod

(alsoknownasthe2-DDCt).Duplicatereactionspersamplewererun.

Table2.PrimersandTaqmanprobesusedforqRT-PCR

III.I.VII.ConditionedmediumfromFLandFNinculturedhumanmelanocytes

Primary melanocytes cultures were prepared from adult human abdominal skin.

Melanocytes(passage2-3)wereseededin24-wellplatesatadensityof40,000cells/mLand

culturedfor48hinmelanocyte medium which consists of M254 culture medium supplemented

with human melanocytes growth supplement (HMGS, Life Technologies, ThermoFisher

Scientific, France), 100 IU/mL penicillin and 100 µg/mL streptomycin. The fibroblast-

115

conditionedmediumwas obtainedby culturing FL andFS for4days at density 150,000

cells/well in full DMEM medium. It was then concentrated by lyophilization. Each

lyophilisatewas added to fullmediumM254 and themixturewas added onmelanocyte

cultures(Table3).

Table3.Preparationofdifferentconcentrationsoffibroblast-conditionedmedium

III.I.VIII.MTTassay

Melanocyteswereculturedin96-wellplatesataninitialdensityof5×103cellsperwellfor

24h,andthentreatedwithseveralconcentrationsoffibroblast-conditionedmedium.After

48 h, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 0,5 mg/mL,

Sigma-Aldrich,France)wasaddedtoeachwellandthecellswereincubatedat37°Cfor4h.

The medium was removed and dimethyl sulfoxide (DMSO) was added to dissolve the

Concentrationof

fibroblast-conditionedmedium

VolumeofmelanocytefullmediumM254

Volumeoflyophilisate(eachlyophilisateismixedin1.2mLoffullmediumM254)

0% 120µL 0µL

10% 108µL 12µL

15% 82µL 18µL

20% 96µL 24µL

25% 90µL 30µL

50% 60µL 60µL

75% 30µL 90µL

100% 0µL 120µL

116

formazancrystals.Theabsorbancewasmeasuredat571nmwithaspectrophotometer.The

experimentswereperformedinquadruplicate.

III.I.IX.ImmunostainingforMITF

MelanocyteswereculturedinLab-Tek®chamberataninitialdensityof10,000cellsperwell

for24h,andthentreatedwithfibroblast-conditionedmedium(15%v/v).After48h,cells

were fixed for15minwith3%PFA inPBS,washed inPBS andpermeabilizedwith cold

acetonefor10minat-20ºC.Afterwashing,nonspecificbindingsiteswereblockedby10

minincubationin1%glycineinPBSfollowedby1hincubationinPBScontaining3%BSA,

10%serumand0.1%TritonX100.Then,sampleswereincubatedovernightwithantiMITF

primaryantibody(rabbitAcManti-MITF,HPA003259,Sigma-Aldrich)diluted1:200inPBS

containing1%BSAand0.1%TritonX100.Afterwashing inPBSwith1%BSAand0.1%

Triton X100, the secondary antibody (goat anti-rabbit IgG-rhodamine or TRITC, 18772,

Sigma-Aldrich)diluted1:200inPBScontaining1%BSAand0.1%TritonX100,wasapplied

for1h.AfterwashinginPBS,sampleswereincubated15mininrhodamine-conjugatedFITC

(Fluorescein isothiocyanate, P5282, Sigma-Aldrich) for F-actin staining (2.5 µg/mL).

Samples were thenmounted in Dako fluorescent medium, observed under fluorescence

microscopy(Axioskop40,Zeiss).

III.I.X.Melaninassay

Melanocytes were cultured at 400,000 cells on 25 cm2 flask for 24 h, and treated with

fibroblast-conditionedmedium(15%v/v).After48h,theywerewashedwithPBS,collected

by scraping and pelleted. Intracellularmelanin was extracted by solubilizing frozen cell

pellets in 1 N NaOH containing 10% DMSO for 2 h at 90 °C. The samples were then

centrifugedat1100rpmfor10minandthesupernatantsweretransferredtofreshtubes.

117

Theintracellularmelanincontentinsupernatantswasmeasuredspectrophotometricallyat

anabsorbanceof420nmagainst a standardcurveofknownconcentrationsof synthetic

melanin (0-50µg/mL, Sigma-Aldrich, France).Experimentswereperformed induplicate.

Resultswereexpressedinµgmelanin/mL.

III.II.3Dbiologicalmodel

III.II.I.Fibroblasts-populatedtensecollagenlatticepreparation(FPCL)

Primary fibroblast cultures were prepared from adult human facial skin (passage 4-6).

Fibroblastswerebroughttoaconcentrationof8x105cells/mLinfullDMEMmedium.They

wereembeddedinthree-dimensionalhydratedcollagengels74,75.Eachcollagenlatticewas

preparedbymixing,inthefollowingorder:3mLof1.76XconcentratedDMEM(containing

0.5%NaHCO3,0.1NNaOH,15%FBS),1.5mLofrattailtypeIcollagensolution(2mg/mL,

InstitutdeBiotechnologiesJacquesBoy,France)and0.5mLoffibroblastsuspension.The

mixturewasquicklypouredintoa16X60-mmpetri-dishcontainingasterilizednylonmesh

ring(SefarNitex03-150/50,HeidenSwitzerland)(Figure14).Afterpolymerizationat37°C

for 1 h, 3 mL of full DMEM mediumwereadded.

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Figure14.TenseFPCLembeddedbyanylonmeshring

III.II.II.Repeated-UVAirradiation

After5daysofculture,collagengelswerewashedwithPBS(untilnophenol-redcolor)and

coveredwiththinlayerofPBSpriortoUVAirradiation(6J/cm2).TheUVAsourcewasabank

of8fluorescentblacklightlamps(F15T8,Sylvania,Danvers,MA,USA).Thepeakemission

wasat370nm,andwascheckedusingaUVradiometer IL-1700withUVA filter (Dexter

IndustrialGreen,Newburyport,MA,USA).UVAradiationprocesswasperformedthreetimes

at an interval of 48 h (Figures 15 and 16). After the last irradiation, collagen gelswere

cultivated for 24 h in full DMEMmedium. Control FPCL were kept in the same culture

conditionswithoutUVAexposure.Allexperimentalconditionsweremadeintriplicate.

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Figure15.Shematicrepresentationoftheirradiationprotocol

Figure16.UVA-irradiationoftenseFPCL.

III.III.ELISA

ELISAanalyzes forDkk1andTGF-b1 in the supernatants of tenseFPCLwereperformed

followingthesameprotocolsasdescribedbefore.

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III.IV.Statisticalanalysis

All quantitative data are expressed as means ± SD. Paired Student’s test (SPSS 24.0) was used to

compare groups. p < 0.05 was considered significant. (* p<0.05, ** p<0.01, *** p<0.001)

IV. Results

IV.I.ComparativeanalysesofstaininginSLversusSNbiopsies

Stainingtechniquessuchashistochemistry(HC)andimmunohistochemistry(IHC)provide

useful informationregarding theskinstructure, the localizationandrelativeamountofa

moleculeinskin.

SLandSNtissuesdisplayeddifferenthistological features.SLshoweda thinepidermis,a

disorganized basementmembrane and elongated rete ridges collapsing into the dermis.

Masson-Fontanastainingdemonstratedhighermelanindeposition intheepidermalbasal

layer of SL compared to SN. The disruption of epidermal basal layer in SL caused some

melaninreleaseintothedermis.β-cateninstainingpresentedmorepositivestaininginSL

comparedtoSN(Figure17).Altogether,histologicalanalysisvalidatedtheSLoriginofour

invitrofibroblasticmodel.

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a-1a-2

b-1b-2

IV.II.ExpressionandsecretionofDKK1fromFLandFL

Quantitativereal-timeRT-PCRwasperformedtoanalyzetheamountsofDKK1expression

in FL, normalized to the amounts expression in FN. As shown in Figure 18, the mRNA

encoding DKK1 was significantly down-expressed in FL. ELISA was used to measure

Figure17. a)Melanin (HC,Masson-Fontana staining) andb) β-catenin (IHC, DAB

staining) in SL and SN tissues. In SL, levels of melanin (a-1) and β-catenin (b-1) ↗

compared to SN (a-2, b-2 respectively). In SL, the basal layer is disrupted, causing

melaninreleaseinpapillarydermis(a-2).MagnificationX20.

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secretionofDKK1intothemediumoffibroblastcultures.AsshowninFigure19,secretion

ofDDK1inFLculturedecreasedbutnosignificantdifferencewasdetectedbetweenFLand

FN.

Figure18.RelativegeneexpressionofDKK1assessedbyreal-timequantitativeRT-PCR.

Toanalyzetherelativechangesingeneexpressionfromthereal-timePCRexperiments,the

comparative2-△△Ctmethodwasused.The△CtvalueforFLandFNwascalculatedusingthe

equation:Ct(DKK1)-Ct(reference,Abl).ThefoldchangeingeneexpressionofDKK1was

finallyobtainedfromtheformula2-△△Ct,wherethe△△Ctvaluewasthedifferencebetween

△Ct(FL)and△Ct(FN)values.Therelativegeneexpressionissetto1forFNsamples.DKK1

expression↘inFLcomparedtoFN.Themean±SDwasdeterminedfrom9 independent

experiments,eachperformedinduplicate.***p<0,001.

1

0,38

0

0.2

0.4

0.6

0.8

1

1.2

FN FL

DKK1

mRN

Arelativ

evalue

***

123

Figure19.DKK1secretion into themediumbyELISA.DKK1secretion isnormalized to

protein content asmeasuredusingBiuret assay.Themean± SDwasdetermined from9

independent experiments, each performed in duplicate. DKK1 protein levels ↘ in the

medium of FL compared to FN but no significant differencewas detected between both

groups.

IV.III.SecretionofTGF-β1fromFLandFL

ELISAwasusedtomeasuresecretionofTGF-β1intothemediumoffibroblastcultures.As

shown in Figure 23, secretion of TGF-β1 in FL cultureswas significantly higher than FN

cultures(Figure20).

0.06

0.03

0

0.02

0.04

0.06

0.08

0.1

FN FL

pgDKK

1/pgprotein

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Figure20.TGF-β1secretionintothemediumbyELISA.TGF-β1secretionisnormalizedto

cell number. The mean ± SD was determined from 9 independent experiments, each

performedinduplicate.TGF-β1levels↗inthemediumofFLcomparedtoFN.**p<0,01.

IV.IV.EffectsofconditionedmediumfromFLandFNonnormalmelanocytecultures

IV.IV.I.Melanocyteviability

Todeterminethecytotoxiceffectof fibroblasts-conditionedmedium,normalmelanocytes

were incubated with various concentrations of FL/FN-conditioned medium (v/v).

Cytotoxicity was rated in accordance with ISO-standard 10993-5 as non-cytotoxic (cell

viability higher than 75%), slightly cytotoxic (cell viability ranging from 50% to 75%),

moderatelycytotoxic(cellviabilityrangingfrom 25%to50%),andseverelycytotoxic(cell

viabilitylowerthan25%).AsshowninTable4,fibroblasts-conditionedmediumfromboth

FLandFN,atconcentrationlessthanorequalto15%v/v,showedrelativelynocytotoxic

effectonmelanocytes.

0.0009

0.0015

0

0.0005

0.001

0.0015

0.002

0.0025

FN FL

TGF-β1

pg/cell

**

125

FNConcentrationoflyophilizated

fibroblastmedium

Averageabsorbance

Standarddeviation Viability(%)

0% 0.61583 0.0848 100 10% 0.61623 0.1564 100.06 15% 0.52647 0.0766 85.49 20% 0.30455 0.0617 49.45 25% 0.38493 0.022 62.5 50% 0.28323 0.0429 45.99 75% 0.25637 0.0325 41.63 100% 0.2873 0.0485 46.65

FL Concentrationoflyophilizatedfibroblastmedium

Averageabsorbance

Standarddeviation Viability(%)

0% 0.46867 0.101 100 10% 0.42543 0.0626 90.78 15% 0.42123 0.533 89.88 20% 0.38343 0.322 81.81 25% 0.3582 0.082 76.43 50% 0.27873 0.0326 59.47 75% 0.27087 0.0353 57.8 100% 0.21635 0.0624 46.16

Table 4. OpticaldensitymeasurementsforMTTassayofviabilityofmelanocytescultured

with different concentrations of fibroblast-conditioned medium. The mean ± SD was

determined from 3 independent experiments, each performed 4 times. Fibroblasts-

conditionedmedium from both FL and FN in concentration ranging from 0 to 15% v/v

showednocytotoxiceffectonmelanocytes.

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IV.IV.II.Melanocytecharacteristics

In accordance with MTT results, the concentration of 15% v/v was chosen to evaluate

melanocytemorphologyandMITFexpression.

Following FL-conditioned medium treatment, melanocytes showed increased dendritic

morphology and intracellular melanin (Figure 21). These findings were supported by

stainingofMITFandF-actin,usedrespectivelyasacharacteristicmelanocytemarkeranda

cellshapemarker,revealingapositiveMITFexpressionandaclassicaldendriticmorphology

(Figure22).

127

Figure21.Phasecontrast imagesofmelanocytescultures ina)normalconditions,b)FL

conditioned medium and c) FN conditioned medium. Arrow points to melanin. Normal

epidermalmelanocytesexhibitatypicaldendriticmorphologyandproducemoremelanin

withFL-conditionedmediumtreatment(15%v/v).MagnificationX20.

128

Figure22.Immunofluorescenceimagesofmelanocytesculturesina)normalconditions,b)

FL conditionedmedium and c) FN conditionedmedium. Normal epidermalmelanocytes

exhibitatypicaldendriticmorphologyandexpressMITFmarkerwithFLorFN-conditioned

mediumtreatment(15%v/v).MagnificationX20.

129

IV.V.SecretionofDKK1afterirradiationofnormalfibroblastsembeddedin3Dcollagen

gel

Under6J/cm2repeatedUVA-treatment,facialfibroblastssecretedsignificantlylessDKK1

thanfibroblastwithoutUVAtreatment(Figure23).

Figure23.DKK1secretionintothemediumbyELISA.TGF-β1secretionisnormalizedto

cell number. The mean ± SD was determined from 3 independent experiments, each

performedinduplicate.DKK1levels↘inthemediumafterUVirradiation.***p<0,001.

V. Discussion-Perspectives

Theagingprocessinhumanskinresultsnotonlyinelasticityloss,dryandthinskinbut

also in benign lesion development such as solar lentigo (SL). Recent studies have

demonstratedthatfibroblastsareactivatedbyUVexposuretoreleasemelanogenicgrowth

factors (HGF, KGF, SCF) which act on melanocytes both directly and indirectly through

0.27

0.08

0

0.05

0.1

0.15

0.2

0.25

0.3

Non-UVA UVA

DKK1

pg/cell

***

130

keratinocytesand,thereforemaycontributetothehyperpigmentationofSL.Theprevious

research in our lab has identified cellular senescence-like features of fibroblasts derived

fromSL76.Otherfactorsareknowntobeinvolvedinskinpigmentation,suchasDKK1which

regulateshypopigmentationinvitiligoandpalmoplantarskin,andinhibitscellinvasionand

angiogenesisinmelanomadevelopment31,40,54,77,78.

ThisstudyinvestigatesanewpossibleroleofDKK1inhyperpigmentation,byusingsolar

lentigo(SL)andnormaladjacent(SN)skinbiopsiesfromthesamepatient,whichprovidesa

more precision evidence for the research. Our experimental data indicate a reduced

expression and secretion of DKK1 in fibroblasts from solar lentigo (FL) compared to

fibroblasts from normal adjacent skin (FN). As previous studies described, DKK1 is an

antagonisticinhibitoroftheWntsignalingpathway,byisolatingtheLRP5/6co-receptorand

preventing the Frizzeled-Wnt-LRP5/6 complex formation both in melanocyte and

keratinocyte31,77. The down-expression of DKK1 in FL compared to FN highlights the

potentialroleofDKK1inthehyperpigmentationprocess.ThisdecreaseofDKK1fromFL

maybecausedbydifferentendogenousandexogenousfactors,suchaspro-inflammatory

cytokines.

It was found that TGF-b1 stimulates canonical Wnt signaling in p38-MAPK by

downregulatingDKK1infibroticfibroblast.Weaimedtoexplorethehypothesisthathigh

levels of TGF-b1 may downregulate DKK1 by p38-MAPK signaling pathway in SL. We

reportedthatthesecretionofTGF-b1fromFLwashigherthanfromFN.

Tofurtherverifythehypothesis,wetriedtomimicinvitrotheenvironmentofsolarlentigo

andreproducetheskinphotoagingprocessunderpro-inflammatoryconditionsviaTGF-b1.

WehavechosenthemodelofUVA-irradiated3Dtensecollagengel. In3Dtensecollagen

131

lattices,fibroblastsundergomechanicalstress,differentiateinmyofibroblastsandsecrete

TGF-b1.Besides,UVA-irradiationwassetuptoreproduceUVexposurewhichisthemost

exogenousfactorinducingsolarlentigo36.ExperimentaldataindicatedthatUVAtreatment

couldstimulateTGF-b1secretionandinhibitDKK1expressionbyfibroblastsembeddedin

collagenmatrix.Toverifymorepreciselyourhypothesis,futureexperimentswillfocuson

theinhibitionofthep38MAPkinasepathway.

DKK1regulatesmelanocytefunctionviatheregulationoftwoimportantsignalingfactors,

MITFandβ-catenin.AdecreaseofDKK1expressioninducesanactivationofWnt/β-catenin

signalingassociatedwithanincreaseofmelaninandβ-catenin,whichwasconfirmedbyour

histologicalobservations.

In addition, undefined conditioned medium of fibroblast derived from SL can stimulate

melanin synthesis inmelanocytes. These results confirmed thatmelanogenic factors are

secretedbyfibroblasts.

ThereforeallthesedatarevealalinkbetweenTGF-β1,DKK1andtheWnt/βcateninpathway:

UVirradiationÞ↗TGF-β1Þ↘DKK1ÞupregulationofWnt/βcatenincascadeÞ↗MITF

Þ↗melaninÞSLlesion.

This process may result in an uneven distribution of active melanocytes, with areas of

hyperpigmentationintheskin.

In the future, it would be a good practice to do co-cultures of melanocyte and

keratinocyteinfibroblast-conditionedmediumtofurtherexploretheinfluenceofDKK1in

hyperpigmentation both in melanin synthesis in melanocyte and melanin taken by

keratinocyte.

132

Up-regulationofmelanocytefunctionscausedbyagingandUVirradiationisinvolvedinthe

development of solar lentigo lesions or other age-related hyperpigmentation disorders.

Cosmeticcompaniesusuallydevelopanti-photoagingproductsbyfocusingonmelanocyte

and keratinocyte functions. This study brings new approach to prevent and treat solar

lentigo lesions, by controlling the expression and secretion of several fibroblast-derived

solublefactors.

133

ListofPublications&Communications

134

Publications

• YJ.Wang, C. Viennet, S. Robin,M. Tissot, P.Muret, L.He, JY. Berthon, P.Humbert.

InvolvementofDickkopf-1inmelanogenesis:focusonsolarlentigo.(Inpreparation)

• YinjuanWang,Céline.Viennet,LiHe,PhilippeHumbert.Assessmentoftheefficacy

of a new complex anti-sensitive skin cream. (Original Article. Submitted to Journal of

CosmeticDermatology)

• YinjuanWang,CélineViennet,GwenaëlRolin,MarionTissot,PatriceMuret,Sophie

Robin, Jean-Yves Berthon, Li He, Philippe Humbert.Development and validation of a

simple method for the extraction of human skin melanocytes. (Original Article.

SubmittedtoCytotechnology)

• YJ. Wang, C. Viennet, S. Robin, JY. Berthon, L. He, P. Humbert. Precious role of

fibroblast on melanocyte pigmentation. Journal of Dermatology Science.

http://dx.doi.org/10.1016/j.jdermsci.2017.06.018

135

Posters

§ InvolvementofDickkopf-relatedprotein1inmelanogenesis:focusonsolar

lentigolesion

SkinPhysiologyInternationalMeeting,Vichy,France,2016

§ Developmentandvalidationofasimpleandcommonmethodforthe

extractionofepidermalcells

• CongrèsAnnuelDeRechercheDermatologique,Lille,France,2015

• EuropeanTissueRepairSociety,Copenhagen,Danmark,2016

136

137

138

Article3:Assessmentoftheefficacyofanewcomplexanti-sensitiveskincream

YinjuanWang, MD,1 Céline Viennet, PhD,1 Adeline Jeudy, PhD,2 Ferial Fanian, MD,

PhD,2LiHeMD,PhD,3*PhilippeHumbert,MD,PhD,1,2*

1Engineering and Cutaneous Biology Laboratory, UMR 1098, University of Bourgogne

Franche-Comté,Besançon,France

2Research and Studies Center on the Integument (CERT), Department of Dermatology,

UniversityHospital,Besançon,France

3Department of Dermatology, First Affiliated Hospital of Kunming Medical University,

Kunming,China

*Correspondingco-authors:PrPhilippeHumbertandPrLiHe

E-mail addresses for correspondence: philippe.humbert@univ-fcomte.fr or

drheli2662@126.com

Disclosureandconflictsofinterests:theauthorshavenoconflictofinteresttodeclare.

139

Abstract

Background Sensitive skin is frequently complaint in dermatology consultation with

cutaneous manifestations such as stinging, redness, dryness and burning sensation that

affectthequalityoflife.Itspathogenesisismainlyrelatedtodysfunctionofneurosensory,

skinbarrier,andalsoimmuneactivity.Thetreatmentisgenerallybasedoncontinuousand

topicaltherapybynon-irritatingcomplex.

Objective:Toevaluate theanti-sensitive functionof anewcomplex creamcomposedby

Yunnan Portulaca olreacea extracted, Prinsepia utilis oil, beta-glucan, and sodium

hyaluronateextractedfrommushroom.

MethodsArandomizeddouble-blindandself-controlstudywasconductedon20selected

volunteerswithsensitiveskin.Subjectsappliedthetestcreamtoonesideoftheface,andthe

controlcream(toleranceextremecream)totheothersideoftheface,twicedailyover28

days.Evaluationswereperformedatbaselineandat28days.Expertclinicalgradingoffacial

skinincludingdryness,roughness,desquamationanderythemawasassessed.Subjectself-

assessment questionnaires, digital photography and noninvasive bioinstrumentation of

hydration, transepidermalwater loss, lipid index, skin texture andwettabilitywere also

includedinthestudy.

ResultsProductswerewelltolerated.Forallparametersstudied,nosignificantdifference

wasobservedbetweentestandcontrolcreams.Resultsshowedthattestcreamprovideda

statisticallysignificantimprovementinclinicalgradingscoresfordryness,roughnessand

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erythemaat28dayscomparedtobaseline.Inaddition,statisticallysignificantimprovement

of skin hydration and texture parameters (eg, smoothness and roughness) were

demonstrated. Volunteers’questionnaire revealed self-perceived benefits consistent with

expertvisualgrading.

ConclusionThisstudyconfirmedtheeffectivenessandtoleranceofthenewcomplexcream

insubjectswithsensitiveskin.Thetestcreamcouldserveaasadailycaremoisturizerfor

face.

Keywords:skin,clinicaltrial,sensitive,hydration,cream

141

Introduction

Sensitive skin is a frequent complaint indermatology consultation. It isdefinedby some

clinicalmanifestations includingstinging, redness,burning,pruritus, tingling thathappen

mainly on the face but also on other areas such as scalp and hands.1 The potential

mechanisms of sensitive skin involve skin neurosensory dysfunction,2,3 neurogenic

inflammation,4epidermalbarrierdisruption(increasingtransepidermalwaterloss)5-7and

immunecellsactivity(transientreceptorpotentialchannels).Variousconditionsinducethe

symptomsofsensitiveskin,suchasenvironmentalfactors(e.g.UVradiation,heat,cold,hash

winds, pollution), lifestyle factors (e.g. cosmetic usage, diet and alcohol), and even

psychological factors (e.g. stress) underlying dermatological diseases as atopic

dermatitis.3,8,9Thediagnosisofsensitiveskincouldbeassistedbystingingtests,occlusion

tests,behind-the-kneetests,washingandexaggeratedimmersiontests,totheevaluationof

itching and quantitative sensory testing (QST).10 In Europe, approximately 39% of the

populationarebotheredbysensitiveskin,andfemaleskinisdeclaredmoresensitivethan

maleskin.ApparentlysensitiveandverysensitiveskinshowedhighincidenceinFranceand

Italy.11 In response to this prevalence, the effective treatment of sensitive skin becomes

challenging.Continuousandtropiccareplaysasignificantroleintreatmentofsensitiveskin

whether in daily care or in the combination of drug treatment.Mildness, UV protection,

hydrationarethefundamentaldemandingfortropicdailycareofsensitiveskin,andthere

areseveralcosmeticsandcosmeceuticalsforsensitiveskinaccordingtothisprinciple.

142

Inthisarticle,weevaluatedananti-sensitivemoisturizingtolerance-extremecreamthatis

an innovative formulationmainly composedbyChinaYunnanPortulacaolreaceaextract,

Prinsepia utilis oil, and beta-glucan, sodium hyaluronate extracted from mushroom.

Portulaca oleracea has anti-inflammatory and anti-allergy effects, it can relieve skin

irritationandenhanceskintolerance.12Prinsepiautilispromotesthesynthesisofceramide

bystratumcorneumcells,andthereforecaninducerepairoftheskinbarrier.13Mushroom

beta-glucanstimulatesproliferationofkeratinocytesand,thuspromoteshealingofdamaged

skin.14Weperformedamonocentric,double-blinded,controlledandrandomizedstudyina

panelof20volunteersusingclinicalandinstrumentalevaluations.

MaterialandMethods

Studydesign

Thisrandomizeddouble-blindcomparativestudywascarriedoutattheCERT(Researchand

StudyCenteronIntegument)locatedinthedepartmentofDermatology,UniversityHospital

of Besançon, France. The study protocol was approved by the ethics committee of the

institute.Thisstudywasconductedinaccordancewiththeethicalprinciplesderivedfrom

the Declaration of Helsinki with Good Clinical Practices (ICH E6) and the law of local

regulation clinical studies, and was considered as a non-interventional study (directive

2001/20/CE,decreedated27April2006).The finalprotocol and the relateddocuments

werereviewedbytheCPPEst2ofFranche-Comté,Besançon,aswellasANSM,Paris,France.

143

StudySubjects

20femalevolunteersaged37.1±10.6yearsoldwithFitzpatrickskinphototypesI–IVand

dryandsensitiveskin,participatedinthisstudyafterhavinggiventheirwritteninformed

consent.Theywereselectedbasedondermatologist’smedicalexamination,measurement

of skin hydration (facial hydration index < 60, Corneometerâ CM825, Courage-Khazaka

Electronic)andsensitivity(lacticacidstiningtest).Exclusioncriteriawerethepresenceof

any skin-related pathologies and abnormalities (eczema, psoriasis…), allergies,

hypersensitivity to the testedproduct, acuteand/or chronic inflammationor infectionof

facial skin, an exposure of sunlight or artificial UV rays within 15 days, pregnancy and

nursing.Subjectswereadvisedtoavoidtheapplicationofothersimilarproductduringthe

wholestudy.

Testproduct

The test product is an anti-sensitive moisturizing tolerance-extreme cream which is

manufacturedbyBeitainiBio-technologicalCo.,Ltd.(China).Atoleranceextremecreamwas

used as control. Both products were provided by Beitaini Bio-technological Co.,Ltd.

IngredientsoftestandcontrolcreamswerelistedinTable1.

Treatment

Volunteersservedastheirowncontrolswithonesideofthefacereceivingthetestcream

andtheothersideofthefacereceivingthecontrolcream.Evaluationswereperformedat

144

baseline(T0)and28days(T1)aftertwicedailyapplication.Volunteersstayedinacontrolled

room (temperature 22±2°C, humidity 50±10%) for at least 20 minutes before each

evaluation.Theywereevaluatedinthestandardskinsituation(lastfacewashingthenight

before visit, without any cosmetic, water and makeup application until the measures).

Measurements were recorded using a flexible transparent plastic mask. This allowed

measurements to be done in exactly the same place at each visit (Thermocell-Test®

professionalkit,Monaderm).

Evaluation

Clinicalscores

Visualclinicalscoreswererealizedincludingdryness,roughness,desquamation,erythema

witha5-pointscale(0=none,1=slight,2=mild,3=moderate,4=severe).

Self-assessment

Subjectscompletedaself-assessmentquestionnaireatT0andT1(Table2).

Photographies

AtT0 andT1, digital imageswere takenusing a standardizedphotography system (SSA,

Courage-KhazakaElectronic)connectedtoaCanonPowerShotA620camera. Allfrontaland

profilephotographswerestandardizedforlightandposition.

145

Bioinstrumentation

Transepidermalwaterloss(TEWL)

The skinbarrier functionof the stratum corneumwasmeasuredbyVapometer® (Delfin

Technologies).Thesensormonitorinclosedcylindricalchambermeasurestheincreasing

relative humidity (RH) of evaporated water from the skin covered by chamber. The

evaporation rate value was expressed in g /m²/ h. One assessment was performed on

externalsideofeachcheekbone.

Skinhydration

Skin hydration was determined on each cheekbone by the measurement of the electric

capacitance(i.e.thecapacityoftheintercellularwatertoconductelectrons)ofthestratum

corneum using a Corneometerâ CM825 (Courage-Khazaka Electronic). The mean value

(hydrateindex,HI)ofthreeassessmentswascalculated.

Lipidindex

Quantitativeanalysisof thesebumwasmeasuredon foreheadbyaSebumeter®SM815

(Courage-KhazakaElectronic).Valueswereexpressedinmg/cm2.Themeanvalueofthree

assessmentswascalculated.

146

Skintexture

Visioscanâ VC98 (Courage-Khazaka Electronic) was used for assessing skin texture and

roughness.15 It takes pictures of the skin surface under standardized homogenous ring-

shaped UVA illumination. Evaluation of images was carried out according texture

parameters: energy reflectingoverall skin condition (NRJ), skin smoothness (Sesm), skin

roughness(Ser),andarithmeticmeanroughness(R5).Themeanvalueofthreeacquisitions

oneachtemplewascalculated.

Skinsurfacewettability

Themeasurementof the contact angle formedbetween the skin surface andwater is an

indicatorofitslipophilicorhydrophilicproperty,thusthecontentinlipids.Thesystemis

composedofasurgicalmicroscope(WildM650Heerbrugg®,Leica)withamirrororiented

at45°totheskinsurface.Adropofdistilledwaterwasdepositedontheskinsurfaceand

thecontactanglewasmeasuredusingsoftwarebasedonthegeometricaldimensionsofthe

drop.16Themeanvalueofthreemeasuresonforeheadwascalculated.

Skincolor

TheMinolta400ChromaMeterisacolorimeterthatcombinesasource-filter-photodetector

andaunitthatdirectlycomputesthechromaticdimensionsofthecolour.Themeanvalueof

threemeasuresoneachcheekbonewascalculated.

147

Sensitivity

Stingingtestisnowacceptedasaroutinelytestandisusedfortherecruitmentofsubjects

with sensitive skin. Itmeasures the skin reactivity particularly in the face. It consists of

applyinga10%lacticacidatthenasolabialfoldcomparedwithphysiologicalserumapplied

simultaneously to the other side. Subjects assessed stinging based to a 4-point scale

(0=nothing,1=slighting tingling,2=moderate tingling,3= intense tingling)at10s,2.5min

and5min.

Statisticalanalysis

The statistical analysis was conducted using Statistica Version 7.1 and Graphpad Instat

Version3.06.DistributionofthedatawasassessedusingtheShapiro-Wilktest.Statistical

comparisonsofthedatawerecarriedoutwiththeAnovaorWilcoxontest(dependingofthe

normalityofthedistribution).Dataarepresentedasmean±standarderrorofmean(SEM).

Differenceswithp<0.05wereconsideredtobestatisticallysignificant.

Results

Test and control creams were well tolerated. For all parameters studied, no significant

differencewas observed between test and control creams. Only data for test cream are

shown.

Highresolutiondigitalimageswerecapturedforallsubjectsatbaselineandafter28

148

daysoftreatment.RepresentativeexampleofonesubjectisshowninFigure1.Visualgrading

analysis demonstrated that after test cream application, a significant improvement of

dryness, erythema, and roughness was observed (Figure 2). At T0, 85% of volunteers

exhibitedslightandmild skindrynesswhereasatT1after test cream treatment,90%of

volunteers had none and slight skin dryness (P<0.0001). 25% of volunteers showed

moderateskinroughnessbeforetreatment,whichpopulationdisappearedaftertreatment.

AtT1,roughnessscoresdecreasedwithonlynonetomildlevels(P=0.0107).AtT0,nearly

50%of volunteers exhibitedmild erythemawhereas atT1, thispercentagedecreased to

14%.Inadditionafter28daysoftreatment,10%and60%ofvolunteershadrespectivelyno

andslighterythema(P=0.0210).However,forskindesquamation,nosignificantdifference

wasnotedbetweenT0andT1.

Thehydrationindexofskinwasscoringfrom48.7±9.5atbaselineT0to57.2±10.6at

T1 after test creamapplication,which reflected a significant increaseof the skin surface

hydration(P=0.0017)(Figure3).

Skintextureparametersincludingroughness(Ser)andsmoothness(Sesm)showed

significantdecline(respectivelyP=0.0333andP=0.0111)aftertestcreamtreatment,andthe

textureparameterofenergyshowedsignificantincrease(P=0.0045)(Figure4).

However,nosignificanteffectwasobservedforskinbarrierfunction(TEWL),sebum

recovery(lipidindex),wettability,colorandstingingtest(datanotshown).

Resultsoftheself-assessmentquestionnaireshowedthattestcreamwassignificantly

149

favourable for 3 questions (Figure5). Subjects considered their facial skin less tightness

(p=0.0169), more hydrated (p=0.0004) and comfortable (p=0.0127) after test cream

application.

Questionnairerevealedself-perceivedbenefitsconsistentwithexpertvisualgrading.

Discussion

Thetreatmentofsensitiveskinischallengingandgenerallybasedoncontinuousandtopical

application of anti-sensitive moisturizing tolerance extreme product that improves skin

features associated with itching, stinging, dryness, tightness, burning or pain.10,17–19 To

maintainahealthyskin,itisrecommendedtohydrateandprotectit.Inthisstudy,ananti-

sensitivemoisturizingtolerance-extremecreamfromBeitainiBio-technologicalCo.,Ltd.in

China,wasfoundtobeaneffectivecosmeticcreamtoimproveskinhydrationandtexture.

Itseffectwassimilarasthecontrolcream.Theclinicalassessmentofqualityoffacialskin

usingvisualscoringsystem,theinstrumentalassessmentandtheself-assessmentbysubject

using questionnaire reported that after 28 days of twice daily application of test cream,

dryness,smoothness,roughnessanderythemaofskinweresignificantlyimproved.These

interesting skin benefits are due to the components of the innovative formulation. The

improvement of skin texture is likely contributedbyPrinsepia utilis oil, beta-glucan and

sodiumhyaluronate.

Prinsepiautilis isagenusoftrees intheRosaceaeandmainlydistributedinChina, India,

BangladeshandFarmosa. InChina,Prinsepiautilis is rich in theareaabove2000meters

150

altitudeofYunnanprovincewithpollution-free.Itskerneliscomposedofaround40%oil

whichcontainsamajorityofunsaturatedfattyacidandvitamin.Inpreviousstudy,Prinsepia

utilisoilenhancedthesynthesisofceramideincultureofkeratinocytes.Innudemicemodel,

its topical application during 7 days significantly decreased TEWL, increased epidermal

hydration and skin surface lipids.20,21 Therefore, Prinsepia utilis oil seems to be an ideal

activeingredientforrecoverytheskinbarrierinanti-sensitiveformulation.

Beta-glucansareglucosepolymers,extracted fromcellwallsofnumerous fungal species.

Theyarefoundtocontributetowoundhealing,depressingimmunesystem,andareeven

recommendedfortreatmentofatopicdermatitis.22–25Sodiumhyaluronateisthesodiumsalt

ofhyaluronicacidwhich isacomponentof thedermalextracellularmatrix. Itshydration

propertymakes it an excellentmoisturizing ingredient. ConcerningPortulacaoleracea, it

belongstoPortulacaceaefamilyandisdistributedinmainlywarm-climatearea.Thereare

many studies on its functions (anti-inflammatory,26,27 anti-oxydant,27 anti-ulcerogenic,28

anti-bacterial,29 anti-cancer,30 and inwound healing31,32). In addition, Portulaca oleracea

extracts could prevent the apoptosis of keratinocyte and fibroblast cultures and has an

assistant function in reversing the keratinocyte differentiation and skin barrier

dysfunction.33,34 In this study, theclinical scoringoferythemaafter test creamtreatment

firmlyprovedtheanti-sensitivefunctionofPortulacaoleraceaintheinnovativeformulation.

Conclusion

The combination of different actives in this new anti-sensitive moisturizing tolerance-

extreme cream improved skin hydration and texture after 28 days of treatment. This

151

complexcreamcouldgivefurtherassistancetothecontinuoustreatmentofsensitiveskin.

Acknowledgments

ThisworkwasfinanciallysupportedbyProgramforInnovativeResearchTeaminUniversity

ofMinistryofEducationofChina(GrandNo.IRT13067)andtheFundofYunnanProvince

ChineseAcademyofSciencesCooperation(GrandeNo.2014IB008)(China).

We would like to thank Céline Thiebaut, Aurélie Durai and Vanessa Ecarnot for their technical

help.

Conflictsofinterest

Theauthorshavenoconflictofinteresttodeclare.

152

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156

Table 1. List of ingredients of test cream

Test cream: Anti-sensitive moisturizing tolerance-extreme cream

Aqua, Glycerin, Pentylene glycol, butyrospermum parkII (shea butter), tridecyl,

trimellitate, dimethicone, glyceryl stearate, prinsepia utilis oil, sodium hyaluronate,

portulaca oleracea extract, beta-glucan, acrylates/c10-30 alkyl acrylate crosspolumer,

xanthan gum, butylene glycol, aminomethyl propano

Control cream: Tolerance extreme cream

Thermal spring water, glycerin, mineral oil (paraffinum liquidum), squalene, carthamus

tinctorius (Safflower), seed oil (carthamus tinctorius seed oil), cyclomethicone, glyceryl

stearate, sodium carbomer, titanium dioxide

157

Table 2. Self-assessment questionnaire designed for subjects

Does your skin reddens easily? (0 = never / 9 = very easy)

What is the intensity of your rash? (0 = no redness / 9 = very intense redness)

Is your skin reacts with emotions, stress? (0 = never / 9 = very often)

Is your skin reacts it with changes in temperature? (0 = never / 9 = very often)

Is your skin does presents persistent redness? (0 = never / 9 = very often)

Do you feel tingling? (0 = never / 9 = very often)

Do you feel tightness? (0 = never / 9 = very often)

Do you feel itchy? (0 = never / 9 = very often)

Do you feel your skin hydrated? (0 = not hydrated, dry skin / 9 = very hydrated)

Have you got desquamation? (0 = no desquamation / 9 = significant desquamation)

Do you feel the skin of your face comfortable? (0 = Very Comfortable / 9 = Uncomfortable)

Is your skin looks rough? (0 = no rough / 9 = very rough)

Is your skin soft? (0 = no soft / 9 = very soft)

158

Figure 1. Representative images of one subject before (T0) and after the 28 days treatment (T1)

with the test cream.

BEFORE

AFTER

159

Figure 2. Clinical scoring of a) dryness b) roughness c) desquamation d) erythema

An analogical scale (from 0 to 4) was used to score skin parameters before (T0) and after (T1) test

cream treatment.

*** indicates P < 0.0001, * indicates P < 0.05.

a) b)

c) d)

0

10

20

30

T0 T1

Dryness*** 4

3

2

1

00

10

20

30

T0 T1

Roughness* 4

3

2

1

0

0

5

10

15

20

25

T0 T1

Desquamation4

3

2

1

0 0

5

10

15

20

25

T0 T1

Erythema* 4

3

2

1

0

160

Figure 3. Effect of test cream treatment on skin hydration

T0: baseline; T1: after 28 days of treatment

** indicates P < 0.001.

01020304050607080

T0 T1

Hydrationindex**

161

Figure 4. Effect of test cream treatment on skin texture

T0: baseline; T1: after 28 days of treatment

** indicates P < 0.001, * indicates P < 0.05.

0.000.020.040.060.080.100.120.14

T0 T1

Texture:NRJ**

0.00.51.01.52.02.53.03.5

T0 T1

Texture:Ser*

0

10

20

30

40

50

T0 T1

Texture:Sesm*

0.00

0.02

0.04

0.06

0.08

0.10

T0 T1

Texture:R5

162

Figure 5. Significant results of self-assessment questionnaire

T0: baseline; T1: after 28 days of treatment

*** indicates P < 0.0001, ** indicates P < 0.001.

0

2

4

6

8

T0 T1

Do you feel your skin hydrated?

***

0

2

4

6

8

T0 T1

Do you feel the skin of your face comfortable?

**

02468

10

T0 T1

Do you feel tightness?

**

163

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