Revisiting asthma therapeutics: focus on WNT signal transduction

Revisiting asthma therapeutics

Koopmans, Tim; Gosens, Reinoud

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Drug Discovery Today

DOI:

10.1016/j.drudis.2017.09.001

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Koopmans, T., & Gosens, R. (2018). Revisiting asthma therapeutics: focus on WNT signal transduction.

Drug Discovery Today, 23(1), 49-62. https://doi.org/10.1016/j.drudis.2017.09.001

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Teaser

WNT

signalling

is

a

relevant,

yet

underappreciated

pathway

in

asthma.

Recent

insights

into

the

pathology

of

asthma

have

highlighted

this

pathway

as

a

potential

novel

therapeutic

point

of

intervention.

With

this

in

mind,

we

attempt

to

answer

the

question:

is

WNT

signalling

a

valid

target

for

asthma

therapy?

Revisiting

asthma

therapeutics:

focus

on

WNT

signal

transduction

Tim

Koopmans

1,2

and

Reinoud

Gosens

1,2

1_{DepartmentofMolecularPharmacology,UniversityofGroningen,TheNetherlands}

2_{GroningenResearchInstituteforAsthmaandCOPD(GRIAC),UniversityofGroningen,TheNetherlands}

Asthma

is

a

complex

disease

of

the

airways

that

develops

as

a

consequence

of

both

genetic

and

environmental

factors.

This

interaction

has

highlighted

genes

important

in

early

life,

particularly

those

that

control

lung

development,

such

as

the

Wingless/Integrase-1

(WNT)

signalling

pathway.

Although

aberrant

WNT

signalling

is

involved

with

an

array

of

human

conditions,

it

has

received

little

attention

within

the

context

of

asthma.

Yet

it

is

highly

relevant,

driving

events

involved

with

inflammation,

airway

remodelling,

and

airway

hyper-responsiveness

(AHR).

In

this

review,

we

revisit

asthma

therapeutics

by

examining

whether

WNT

signalling

is

a

valid

therapeutic

target

for

asthma.

Introduction

Asthmaisaheterogeneouschronicinflammatorydiseaseofthelargeandsmallairways.Overthe

pastcoupleofdecades,wehavecometoconsiderasthmanotasasinglediseaseentity,butrather

asacollectionofdifferentconditionswithoverlappingsymptomatology,butdiverseaetiologies

[1].Inmostpartsoftheworld,asthmaprevalenceiscontinuingtoincreaseorremainsstableand

isconsideredoneofthemostcommonchronicdisordersworldwide[2].Asthmaaffects

approxi-mately300millionpeopleacrosstheworldandisahugeburdenonhealthcareexpenditure[3,4].

AhallmarkfeatureofasthmaisAHR,definedastheexaggeratedbronchoconstrictionresponseto

specificandnonspecificstimuli.AHRresultsfromavariableandpersistentcomponent,drivenby

eitherchronicinflammationortheprogressivedevelopmentofstructuralchanges,respectively

[5].Structuralchanges,termed‘airwayremodelling’,encompassincreasedairwaysmoothmuscle

(ASM) mass,mucous gland hypertrophy, bronchial microvascular remodelling,subepithelial

fibrosis, and epithelial changes, including cell detachment and goblet cell hyperplasia [6].

Although the mortality rate hasreduced significantlyover the yearswith the regular useof

inhaledglucocorticosteroids,250000peoplestilldiefromasthmaannuallyandtheglobalimpact

ofasthmaremainshigh[7,8].Theprevalentmortalityandmorbidityisinpartbecauseofboth

pooradherence[9]and response to corticosteroidsinsevere asthmatics and asthmaticswho

smoke[10]and,insomecases,patientsexperiencenoclinicaleffectatall[11].Inaddition,the

effectsofcorticosteroidsonairwayremodellingremaincontroversial,andarerarelyclinically

Reviews FOUNDA TION REVIEW TimKoopmansisa postdoctoralresearcherat theComprehensive PneumologyCenteras partoftheHelmholtz ZentrumMfinchen, Germany.Whilehaving workedextensivelyonthe pathophysiologyofasthma

duringhisgraduateyears,withaspecialinterestin WNTsignalling,hiscurrentresearcheffortsare focusedonthepathologyofthesurfacemesothelium inthoracicandtrunkcavities,includingthelungs.In particular,heisinterestedinthestemcellcapacityof themesothelium,withinthecontextofhomeostasis, repair,andfibrosis.

ReinoudGosensis associateprofessorof translationalpharmacology attheFacultyofScience andEngineeringatthe UniversityofGroningen, TheNetherlands.Dr Gosens’currentresearch interestsarefocussedon

mechanismsthatregulatethestructuralremodelling andrepairoftheairwaysandparenchymainasthma andchronicobstructivepulmonarydisease.In particular,heisinterestedinthemechanismsand therapeuticvalueofmuscarinicreceptorand Wingless/Integrase-1(WNT)signalling.

Correspondingauthor:Gosens,R. (r.gosens@rug.nl)

significant for low doses [12–15]. Bronchial thermoplasty has

shownpromiseindecreasingsmoothmusclemassinsevere

asth-matics for up to at least 2 years [16], and is associated with

improvedqualityoflife,andreducedsymptomsandnumberof

exacerbations [17].However, the procedureis invasive and not

withoutcomplications[18]and,insomecases,iswithoutclinical

benefit [19]. Thus, there is a clear need for new therapies for

asthmathatovercometheshortcomingsofthosethatare

current-lyavailable.Inthisreview,wediscusstheevidencethatsupports

theinvolvementofWNTsignallinginasthmaandweevaluatethe

WNTpathwayasapotentialtherapeutictarget.

WNT

signalling

WNT signalling is an ancient pathway that dates back to the

earliestmetazoansthatstartedtodevelopapatternedbodyaxis,

andexpandeddramaticallyasanimalsevolvedintomorecomplex

organisms[20].Inmammals,thereare19differentWNTfamily

members.Theyarecriticallyinvolvedinregulatingembryogenesis

andcontroldiverseprocesseslaterinlife,includingcell

prolifera-tion, survival,migration,polarity, specificationofcell fate,and

self-renewalinstemcells[21].Itisofnosurprisethatperturbation

ofthelevelsofWNTligands,oralteredactivityofitsdownstream

effectors, results in developmental defects and contributes to

disease aetiology. Given the large diversity of WNT signalling

components, researchers have attempted to group individual

WNTproteinsintoclassesbasedontheirintrinsiccapabilitiesto

activatethetranscriptionalregulator

b-catenin

[22].Thisresulted

inWNTsbeingcategorisedaseithercanonical(b-catenin

depen-dent), or noncanonical(b-catenin independent). However, the

intrinsicpropertiesofWNTligandsonlycoverpartofthe story

and, in view of the increasing complexity of WNT signalling

networks,itseemsincongruoustorefertoindividualWNTsusing

thisnomenclature.Throughoutthisreview,weviewWNTswithin

the context of the pathway that they are part of and use the

terms ‘WNT/b-catenin’ and ‘b-catenin-independent’ signalling

accordingly.

WNTligandsaresecretedproteinsthatarecovalentlymodified

byglycosylationandpalmitoylationbeforeenteringthe

extracel-lularspace.Palmitoylationsrenderthemhydrophobicandtether

them to cell membranes or their cognatereceptors, known as

Frizzled (FZD) receptors.They signal in anauto- and paracrine

fashion,mostlythroughacell-boundmanner[23,24].Inthecase

of

b-catenin-dependent

signalling,oncesecretedfromtheirhost

cell, WNT ligands engage their cognate FZD receptorsand the

LRP5/6transmembraneco-receptor,inducingcomplexformation

betweenthetwo(Fig.1).Thisresultsinaconformationalchange

andenablesphosphorylationofthecytoplasmicLRPtail,which

inhibitsglycogensynthasekinase3(GSK-3)[25]andallows

bind-ingofthescaffoldproteinAxin.Conversely,whenWNTligands

are absent, Axin forms a complex together with adenomatous

polyposiscoli(APC)andtheconstitutivelyactiveserine-threonine

kinases Casein kinase (CK)-Ia and GSK-3. This so-called

‘destruction complex’ captures

b-catenin

and subjectsit to

se-quential phosphorylation at serine 45 by CK-Ia, followed by

phosphorylation atpositions41, 37,and 33byGSK-3 at theN

terminus, leadingto itsproteosomal degradation[26,27].WNT

pathway activation results in recruitment ofAxin to the

phos-phorylatedtailofLRP.Asaresult,thedestructioncomplex,while

remaining intact, becomes saturated with the phosphorylated

formof

b-catenin.

This results in newlysynthesised

b-catenin

accumulatingandtranslocatingtothenucleusindependentlyof

transporter receptors [28] to facilitate gene transcription [29].

Nuclear

b-catenin

governstranscriptional programsthrough

as-sociationwithanarrayoftranscriptionfactors,includingtheTcell

factor/Lymphoid enhancer-binding factor 1 (TCF/LEF1) family

[30].

The

b-catenin-independent

pathwaysaremorediverseintheir

intermediate effectorsand final biologicaloutcomes, including

orientationofcell division,planarcell polarity,andconvergent

extension,andcanincludebothtranscriptionaland

nontranscrip-tionalresponses in the cell (Fig. 1) [31]. The best-characterised

b-catenin-independent

WNTpathwaysaretheplanarcellpolarity

(PCP)pathwayandtheWNT/calciumpathway.ActivationofPCP

resultsindownstreameventsthatinvolveactivationofthesmall

GTPasesRac-1,RhoA,andJun-N-terminalkinase(JNK).Activation

oftheseeffectorscanleadtochangesincytoskeletalstructureor

cellpolarity,eitherdirectlyorthroughtranscriptionalactivation

[32].PCP signalling generally doesnot requirethe presence of

LRP5/6,butinsteadutilisestheco-receptorsRAR-relatedorphan

receptor (ROR), related to receptor tyrosine kinase (Ryk) and

tyrosine-proteinkinase-like7 (PTK7)[33].WNT/calcium

signal-ling involves the FZD-mediated activation of phospholipase C

(PLC), which stimulates the production of diacylglycerol and

inositol-1,4,5-triphosphate(Ins(1,4,5)P3)[34].Ins(1,4,5)P3triggers

calciumreleasefromintracellularstoresandsubsequentactivation

of calcium-dependent factors, such as calmodulin-dependent

kinaseII(CAMKII), calcineurin,andcertainisoformsofprotein

kinaseC(PKC).Theseinturnactonthetranscriptionalregulator

nuclear factor associated with T cells (NFAT) to promote gene

transcription.

Asthma

genetics

and

epigenetics

Indications

from

GWA

studies

Asthmafrequentlyexpressesitselfinearlylifeandhasasubstantial

heritablecomponent[35,36],indicatingastronggenetic

contri-butionto disease susceptibility.Furthermore, suboptimalfoetal

growth, maternal micronutrientdeficiencies(e.g., vitaminE or

vitaminD),andmaternalsmokingareassociatedwithimpaired

infant lungfunction and subsequent predispositionto develop

asthmalaterinlife[37–39],suggestingthatasthmadevelopsasa

consequence ofthe interactionof multipleenvironmental and

geneticfactors.Pre-orperinatalexposurescanalsodrive

remodel-linguponbirth.Forexample,maternalsmokingduringpregnancy

induces airway remodellingin mouse offspring [40],and these

changesareassociatedwith the differential expressionofWNT

pathwaygenesinneonates [41].This isinaccordance withthe

observationthat,inmanyasthmatics,airwayremodelling

devel-opsinearlylife,evenbeforeasthmaisofficiallydiagnosed[42–49].

Despitethelargenumberofstudiesaimedatidentifying

suscepti-bility loci, genome-wide associationstudies (GWAS) ofasthma

have onlyyielded a few targets asstrongasthma susceptibility

genes[50]thatonlyexplainasmallproportionofasthma

herita-bility,withlimitedabilityto predictoverall diseaserisk.GWA

studies are generally restricted to common single-nucleotide

polymorphisms (SNPs), but not rare or copy number variants,

and positive hits require exceedingly smallP valuesto declare

Reviews



FOUNDA

TION

significance,thusfilteringoutmanypotentialtrueassociations.In

addition,thestatisticalmodelsusedinGWASaresimplisticanddo

not take into account models of interactions, such as

gen-eenvironment,whichishighlyrelevantforasthma.Therefore,

acomplexdiseasesuchasasthmamightrequireamore

sophisti-catedapproach.Indeed,whenincorporatinggeneinterplay,WNT

signallingwas foundto stronglyassociate with asthma risk,in

particular FZD3 and FZD6 [51]. The importance of

genotype-specificresponsestoenvironmentalexposuressuggeststhatgenes

thatcontrollungdevelopmentareespeciallyrelevantforasthma

risk. Three large meta-analyses of GWAS from individuals of

Europeandecentwererecentlypublished,andidentified28loci

that were associatedwith lung function [52–55]. These studies

promptedthequestionwhetherthesamesetofgeneswere

impli-catedinchroniclungdisease,suchasasthmaorchronic

obstruc-tive pulmonary disease (COPD). Two follow-up meta-analyses

studieswereperformedbyasinglegrouptodeterminespecifically

whethertheidentifiedlocifromthesestudies,associatedwithlung

functioninthegeneralpopulation,alsodeterminedlungfunction

in individuals with asthma. They found that genetic variants

related to the gene encoding Family With Sequence Similarity

13MemberA(FAM13A)associatedwithbothlungfunction[52–

56]and asthma[57–59].Interestingly, FAM13Ahasalso

consis-tentlybeenlinkedwithCOPD[60–70],eveninthosewhohave

neversmoked[71].Importantly,FAM13Awasrecentlyfoundto

regulate

b-catenin

stability,highlightingWNTsignallingin

asth-ma[72].AlthoughthefunctionofFAM13Aremainstobefurther

investigated(Box1)[72,73],twosplicevariantshavebeen

identi-fiedinhumans[FAM13Aisoform1(longvariant)andisoform2

(shortvariant)[74]],expressedinmucosalcells,clubcells,airway

epithelial cells, alveolar cells, and alveolar macrophages [72].

Furtherevidencein supportofthisview hascomefromseveral

studies.In one study, offive selected WNT signallingpathway

genesthatweredifferentiallyexpressedinhumanfoetal

pseudo-glandular and canalicular-stage lungtissue samples,two genes,

encodingWNT-1-inducible-signalingpathwayprotein-1(WISP-1)

andWNTinhibitoryfactor-1(WIF-1),harbouredpolymorphisms

in childrendiagnosedwithmildtomoderatepersistent asthma

(Box1)[75].This waslater confirmedin asthmaticsofChinese

decent[76].

FIGURE1

Wingless/Integrase-1(WNT)signallingpathways.SimplifiedschemeshowingthemainWNTpathways.(a)WNT/

b

-cateninsignalling.Understeady-state conditionsandintheabsenceofWNTligands,glycogensynthasekinase3(GSK-3)phosphorylates

b

-catenin,whichtriggersitsdegradation.Inthepresenceof extracellularWNTligands,thedestructioncomplex[comprisingGSK-3,caseinkinase-I

a

(CK-I

a

),Axinandadenomatosispolyposiscoli(APC)]isrecruitedtothe WNT_–receptorcomplexandinactivated.Thissaturatesthedestructioncomplexandallowsnewlyformed

b

-catenintoaccumulateandtranslocatetothe nucleus,whereitactivatesthetranscriptionoftargetgenesunderthecontrolofTcellfactor(TCF),amongothers.(b)

b

-catenin-independentsignallingwith purple-andblue-labelledcomponentsdepictingplanarcellpolarity(PCP)andWNT/Ca2+_{signalling,respectively.PCPsignallingtriggersactivationofthesmall}

GTPasesRhoAandRac-1,whichinturnactivateRhokinase(ROCK)andJun-N-terminalkinase(JNK),leadingtoactinpolymerisation.Thispathwayisprominently involvedintheregulationofcellpolarity,cellmotility,andairwaysmoothmusclecontraction.TheWNT/Ca2+_{pathwayactivatesCa}2+_-and

calmodulin-dependentkinaseII(CamKII),proteinkinaseC(PKC),andcalcineurin(Cn).CalcineurinactivatesCa2+_{-sensitivetranscriptionfactors,includingnuclearfactorof}

activatedTcells(NFAT),whichregulatesthetranscriptionofgenescontrollingcellfateandcellmigration.Abbreviations:

b

-TrCP,beta-transducin repeat-containingE3ubiquitinproteinligase;DVL,Dishevelled;LRP5/6,low-densitylipoproteinreceptor-relatedprotein5/6;ub,ubiquitin.

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FOUNDA

TION

Indications

from

epigenetic

studies

GWASaloneisunabletoaddresswhetherSNPsareprotectiveor

whethertheyacceleratediseasedevelopment,orevenifthe

pre-dictedgeneisthekeygeneatthatGWASlocus.Thus,focussingon

epigeneticmarkersisahighlyvaluabletooltocomplementGWAS

data.Inonestudy,the

b-catenin-dependent

geneencoding

low-densitylipoproteinreceptor-related protein5 (LRP5),aswell as

WNT2, APC and several other WNT genes were differentially

methylatedspecificallyinbloodmonocytesofpatientswith

neu-trophilicasthma,butnoteosinophilicasthma[77].Anotherstudy

showedthat differentially methylatedregionscorresponding to

elevated expression of the CTNNB1 (encoding

b-catenin)

and

AXIN2 (a

b-catenin

target gene) genes in whole-bloodsamples

from children at the time of birth, were associated with the

increased riskofthe child developinglateorpersistent wheeze

laterinlife[78],whichincreasedwhenmotherswereexposedto

highlevelsofstress.Bycontrast,at4yearsofage,thisassociation

nolongerremained,suggestingthatearlyexposuresarecriticalin

diseasedevelopment.

Indications

from

lung

development

Theimportanceof

b-catenin

indrivinglungdevelopmental

path-ways has been demonstrated in numerous studies. Mice with

b-catenin

knocked outat embryonicday(E)14.5in pulmonary

epithelialcells(givingrisetoairwayandalveolarepithelialcells

afterbirth)developproximallungtubulesthatdifferentiate

nor-mally.However,lungsfailtoformperipheralairwaysandinstead

developintoproximaltubules,resultinginearlydeathafterbirth

[79].Bycontrast,overexpressionof

b-catenin

inCCSP-expressing

Claracells(whichstarttoexpressCCSPapproximatelyatE14.5)

perturbsepithelialcell differentiationandcausesgobletcell

hy-perplasiaandairspaceenlargement[80].Inaddition,constitutive

expression of stabilised

b-catenin

prevents differentiation into

secretory Claracells and terminallydifferentiated ciliated cells,

whichisaccompaniedbyacorrespondingincreaseinfunctionally

immatureepithelialcells[81].

b-catenin

isalsoimportantinthe

mesenchymal lineage. Mesenchymal deletion of

b-catenin

impairs the amplification, butnot differentiation,of

parabron-chialsmoothmuscleprogenitorcellsaswelldifferentiationinto

matureendothelialcells[82],andseveralWNTligands[83–85]are

essentialforsmoothmusclecelldevelopmentintheairways.

Animportant areaofstudywillbetofurthercharacterisethe

functionalsignificanceofgeneticvariantsassociatedwithWNT

signalling and asthma risk, where genetic and environmental

interactionsarekeyto furtheringourunderstandingofasthma.

Although large-scale GWA studies incorporating interactions

couldprovechallenging,a moreflexiblealternativetostudying

globaltranscriptionalandepigeneticresponsestokeyexposures

relevantforasthmacouldincludeinvivoandinvitromodels.Of

particularinteresthereisthe FAM13Alocus. HowFAM13A

reg-ulates

b-catenin

isanimportantquestiontoanswer,notonlyin

adultlife,butalsoduringlungdevelopment.Thiswillalsohelpus

understandhowdifferentSNPswithintheFAM13Aregionrelate

todifferentdiseases,suchasasthmaandCOPD,whichhaveboth

beenassociatedwithSNPslinkedtothFAM13A[60–70].

WNT

signalling

in

asthma:

evidence

from

animal

models

Animal models, although lacking the genetic background that

asthmaticindividualshave,nonethelessprovideavaluable tool

toobservehowdiseasedevelopmentmightoccur,andto

disen-tanglewhichfactors are a causeor determinantof the disease.

Allergic asthma in mice is typically modelled by exposure to

BOX1

Asthma

susceptibility

genes

Severalsusceptibilitygeneshavebeenassociatedwithasthma

risk,butforsomeitis notalwaysclearhowtheyaffectcell

behaviour.ThefunctionofFAM13Aisnotentirelyclear,butstudies

havesuggestedaroleinstabilisinglevelsof

b

-cateninthrough

interactionwithPP2A.InHEK293TandA549cells,FAM13Ais

phosphorylatedatSer322byAkt,whichincreasesitsbinding

affinitywith14-3-3,leadingtocytoplasmicsequestrationof

FAM13A[73].FAM13AboundtotheB56regulatorysubunitof

PP2AleadstodephosphorylationatSer322,andpromotes

nuclearlocalisation.FAM13AalsointeractswithAxin,butnot

GSK-3,andithasbeensuggestedthatFAM13Aregulates

post-translationalmodification(s)ofAxininthenucleus,leadingto

increasedAxinturnover,whichindirectlyincreases

b

-catenin

stability[73].Anotherstudyshowedthat,in16HBEcells,

overexpressionofFAM13Aresultedinincreasedphosphorylation

(Ser33and37,andThr41)andreducedlevelsof

b

-catenin[72].

Similarly,depletionofFAM13Aincreased

b

-cateninstabilityand

TOPFlashreporteractivity.Thesedifferentfindingswarrantfurther

investigation.

Inlightoftheseresults,itisworthnotingthatFAM13Acontainsa

putativenuclearexportsignal(NES)sequence[73],aswellasa

bipartitenuclearlocalisationsignal(NLS)andtwoshorterPat7

sequencemotifs,whichsuggestthenuclearpresenceandfunction

ofFAM13A[74].IsoformtwoisalsoassociatedwithaRhoGAP

domain,knowntoaffectRhofamilyGTPases[74].Belongingtothe

familyofsecretedmatricellularCCNproteins,WISP-1,alongwith

otherCCNfamilymembers,caninteractwithvariousreceptors,

includingLRPs,aspartofWNT/

b

-cateninsignalling[215].

However,itsprecisefunctionremainspoorlydescribed.WISP-1

caninteractwithintegrinsthroughseveralintegrinrecognition

sites[216–218].Thus,itcouldserveasamediatorofcell–matrix

adhesioninapleiotropic,cell-specificmanner,withpotential

distinctfunctionsdependingondifferentcellsurfacereceptorsin

differentcelltypes[219].Functionally,WISP-1hasbeenshownto

driveproliferativeandEMTresponsesinalveolarepithelialcells

andincreasethesynthesisofECMcomponentsinfibroblasts.

Antibody-mediatedinhibitionofWISP-1improvedlungfunctionin

thebleomycinmousemodelforpulmonaryfibrosis[220].Both

DKK-3andWIF-1aresecretednegativeregulatorsofWNTsignal

transduction[221].WIF-1candirectlybindandantagonisesome

WNTligands.Inaddition,itcontainsaheparinsulfate-bindingsite

(membrane-boundglycosaminoglycans,commonlycovalently

linkedtoheparinsulfateproteoglycans,thoughttomediate

localisationofWNTsnearthetargetcellsurface[222]),whichisnot

necessaryfor,butgreatlyfacilitates,WNTinhibition[221].

Generally,inhibitionofWIF-1exacerbatesWNT/

b

-catenin

signalling,anditsexpressioniscommonlysilencedinhumanlung

cancer[223,224].Bycontrast,DKKinhibitsWNTsignallingby

preventingWNTbindingwithLRP5/6[225].Interestingly,whereas

WNTligandstypicallybindtoonlyoneortwodistinctstructural

domainswithinLRP5/6,DKKbindsseveral,and,therefore,can

potentiallyantagonisedifferentWNTproteinssimultaneously

[226].SimilartoWIF-1,inhibitionofDKKgenerallyresultsin

activationofWNT/

b

-cateninsignalling,anditsdecreased

expressionisrelevantinlungcancer.Itsfunctionalsignificancein

relationtoasthmaisdescribedinmoredetailinthemaintext.

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ovalbumin(OVA)incombinationwithaluminiumhydroxideas

anadjuvanttofacilitatetheearly-phaseallergicresponseandskew

inflammatory events in favour of T-helper type 2 (Th2) cells.

Alternative allergens that are used include extracts of purified

proteinsfromhousedustmites,cockroaches,ragweed, orfungi

[86].Inaddition,occupationalasthmacanalsobemodelledandis

usually accomplished by exposure to di-isocyanates, the most

commonly identified cause of occupational asthma. Protocols

differ,but generallyinclude subcutaneousinjectionwith liquid

toluenedi-isocyanate(TDI)(sensitisation),followedbyinhalation

with TDI vapours (challenge). Although substantial differences

havebeennoted,manyfeaturesofdi-isocyanateasthmaare

simi-lartoatopicasthma,includingairwayinflammationcharacterised

by activated CD4+ _{T cells, eosinophils, and mast cells, airway}

remodelling,and increased levels ofinterleukin(IL)-4 and IL-5

[87].

Allergicasthmamodelshavefrequentlybeenassociatedwitha

change in WNT signalling, although the direction appears to

dependonthedurationoftheprotocolandrouteof

administra-tionoftheallergen.InacuteOVAmodels(upto3daysof

chal-lenge),

b-catenin

expressionisgenerallyreducedcomparedwith

controllungs[88–91],whereasinchronicOVAmodels(10weeks

or more),

b-catenin

expressionis generally higher [89,90]. For

occupationalasthmamodels,theresultsarelessclear.Balb/cmice

sensitised to TDI for 3 weeks and then challenged for 1 week

showedeitherreduced[83,92–95]ormildlyincreased[93]levels

oftotal

b-catenin,

concomitantwithincreasedlevelsofthe

non-phosphorylatedformof

b-catenin

[93,94].Alternative, but

less-frequentlyusedasthmamodelsarealsoassociatedwithchangesin

WNT/b-cateninsignalling.Miceexposedtoamixtureofbenzene,

toluene,xylene(collectivelycalledBTX),andformaldehyde(FA)

showed differential expression of several WNT-related miRNAs

[95], and Aspergillus fumigatus-exposed miceexhibited elevated

levelsofAxin-2intheASMandepitheliallayers[83].Theinitial

reductionin

b-catenin

activityintheacuteallergenmodelmight

reflectaphysiologicalresponsetoprotectthehostfromexcessive

amountsof

b-catenin.

Asovalbuminexposureincreasesovertime,

thisresponsemighteventuallylosegroundasairwayremodelling

startstodevelop,accompaniedbyincreasedactivationof

b-cate-nin.CTNNB1isapleiotropicgeneanditsactivationrequirestight

regulationtocoordinatecellbehaviour.Thistranslatesinto

tran-sientperiodsofactivation,wherebothactivationanddiminution

act in quick succession.As such, itis possible that CTNNB1 is

activatedinawavepatterninresponsetoallergens,andfailureto

detect differences in CTNNB 1expression could be a result of

‘missingthewave’.Inaddition,someofthemeasuredvariables

arenotrestrictedtoWNTsignalling.Forexample,inactivationof

GSK-3throughphosphorylationandthecorrespondingincrease

in

b-catenin

stabilityisachievedthroughWNT-independent

fac-tors,suchasPKB/Akt[96,97],phospholipaseC[98],orPKA[99].

Therefore,thesefindingsmightnotreflectWNT-pathway

activa-tion.Finally,WNTpathwayactivationmightnotalwaysbebest

determined by its expression. For example, studies with both

animal models[92,93] and biopsies from patients withasthma

[100]haveshowndecreasedexpressionofthemembrane-bound

protein E-cadherin,resulting in disruption of barrier function.

Thisobservedreductionwasparalleledbyadecreaseinjunctional

b-catenin,

which might become active as it diffuses into the

cytosol.Thesechangesaremaintained whenepithelialcellsare

isolatedandculturedinairliquidinterface(ALI),suggestingthat

theyareintrinsicinnature.

WNT

signalling

and

inflammation

in

asthma

Asthmaisprimarilyconsideredadiseaseassociatedwithactivation

of the adaptive immune response, most notably the Th2

cell-dependent promotionofimmunoglobulin(Ig)Eproduction and

recruitmentofmastcells.However,asthmaisalsocharacterisedby

innateimmuneresponsesthatinfluencetheactivationand

traf-ficking of dendritic cells (DCs), production of innate immune

cytokines,andprimingoflymphoidcells[101].Bothoftheseaxes

involveWNTsignalling(Fig.2).

Evidence

for

b

-catenin-independent

WNT

signalling

Evidencesuggests astronglinkbetween

b-catenin-independent

WNTsignallingandallergicinflammation.WNT-5Awasrecently

implicated in asthma in peripheral blood mononuclear cells

(PBMCs).PBMCsisolatedfromhealthyindividuals,treatedwith

eitherIL-4or IL-13for24h,and thenprocessedformicroarray

analyses,showedincreasedexpressionofWNT-5AforbothIL-4

and IL-13 [102]. Accordingly, WNT-5A expression could be

completely prevented by anti-IL-13 mAb. These findings were

extendedinanotherstudytowardspatientswithasthma,where

endobronchialbiopsiesfrompatientswithmild-to-moderate

asth-ma,stratifiedinto‘Th2-high’andTh2-low’subphenotypesonthe

basisofasignatureofthreeIL-13-induciblegenes,wereanalysed

bywhole-genomemicroarrayanalyses.Theauthorsreportedthat

multipleWNTgeneswerepositivelycorrelatedwiththeTh2-high

signature[103].Moreover,WNT5Awasfoundtobeincreasingly

expressedinPBMCsfromasthmaticsofKoreandecent[104].These

findingssuggestalinkbetween

b-catenin-independent

WNT

sig-nalling and Th2-high asthma, or possibly between WNTs and

allergy, whichisgenerallyconsidered tobeaTh2-predominant

response.Amorerecentpaperhassubstantiatedthisidea,inwhich

bronchialairwayepithelialbrushingswerescreenedfor

differen-tially expressedgenesand thencorrelatedtofractional exhaled

nitricoxide(FeNO)[105].Theauthorsthenusedk-means

cluster-ingtopartitionthesubsetofgenesthatcorrelatedwithFeNOinto

fivedifferentasthmaphenotypes,orsubjectclusters.Onecluster

wasenrichedwithWNTpathwaygenes,includingWIF1,WNT5B,

andDKK3.Ofnote,allofthepatientsinthisclusterwereatopic

and had a normal FeNO, butthe earliestage ofasthma onset,

longestdiseaseduration,andahighdiseaseseverityand

percent-ageofbronchoalveolarlavage(BAL)lymphocytes.Moreover,this

clustershowedelevatedlevelsoftumournecrosisfactor(TNF)-a

signalling,whichisknowntodriveexpressionofnoncanonical

WNTmediators[106].ASMcellsfrompatientswith

mild-to-mod-erateasthmahavealsobeenshowntocontainelevatedlevelsof

WNT-5AcomparedwithhealthyASM[107].Apartfromitsrolein

regulating bronchomotor tone, ASM is intimately involved

in modulating airway inflammation [108]. Collectively, these

results imply a role for

b-catenin-independent

WNT signalling

andinflammation,inparticularallergicresponses.

Evidence

for

WNT/

b

-catenin

signalling

In blood samples, polymorphisms within the promoter region

of CTNNB1 have been associated with either an increased or

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decreasedriskofdevelopingasthma,dependingonwhetherthese

variants increased or decreasedthe expressionof

b-catenin,

re-spectively [109]. Furthermore, endobronchial biopsies from

patients with mild-to-moderate asthma showed that WNT3A

andWNT10AassociatedwithTh2-highasthma[103].Insupport

ofthis,the

b-catenin

destructioneffectorgenesAxin1,APCand

GSK3b were all found to be decreased in PBMCs from Korean

patientswithasthma[104].

Collectively, theseresultssupport theview that bothaxesof

WNTsignallingareelevatedinasthmatictissuesandlinkto

Th2-specifc inflammation. These results are largely backed up by

mechanisticandtranslationalstudiesinanimalmodels,although

somediscrepanciesexist,whicharefurtheroutlinedbelow.

Evidence

from

mechanistic

studies

on

adaptive

immunity

WNT/b-cateninsignallingiscriticallyinvolvedinTcell

develop-ment inthethymus[110,111],primarilythroughinteractionof

b-catenin

with the transcription factor special AT-rich-binding

protein 1 (SATB1) [112], which was recently also shown to be

associated with mucous hypersecretion [113]. However, WNT/

b-catenin

hasalsobeenimplicatedintheTh2-mediatedresponse

thatoccursaftermaturationinthethymus,specificallywithinthe

contextofallergy.TransgenicmiceproducingWNT-1ina

tetra-cycline-based (tet-ON) manner, under control of the Clara cell

secretory protein(CCSP) promoter, specific forClara cells, and

subjectedtoOVAexposuretodriveallergicasthma-likechanges,

showed attenuated AHR,BAL eosinophilia, and a reductionin

mucusproduction[114].OverexpressedWNT-1hadnoeffecton

systemicsensitisation,asevidencedbyunchanged OVA-specific

IgE,IgG1,andIgG2blevelsinserum.Treatmentwiththe

nonse-lectiveGSK-3inhibitorlithiumchloridecouldmimictheseresults,

highlightingtheroleof

b-catenin

signallinginthisresponse.In

linewiththis,micewithahomozygoushypomorphicmutationat

the Dickkopf-1 (DKK-1) allele, in which DKK-1 expression is

reducedbyapproximately90%,showedamplifiedWNT/b-catenin

signalling,accompaniedbyreducedlevelsofneutrophils,

eosin-ophils, and CD4+ T cells in BAL fluid in response to allergen

challengewithhousedustmites[115].Inanotherstudy,

suppres-sionof DKK-1 by a neutralisingantibody, or administration of

WNT-3A,reducedneutrophiltraffickingduringacute

inflamma-tion[116].Moreover,inhibitionofDKK-1reducedtheproduction

of IL-4, IL-5, IL-10, and IL-13 in CD4+ T cells, and suppressed

interferon(IFN)-gexpressionunderTh1-cellpolarisation

condi-tions[112,117].WNT–10Bwasalso recentlyimplicated inTh2

activation[118].WNT-10isexpressedinairwayepitheliumaswell

FIGURE2

InvolvementofWingless/Integrase-1(WNT)signallinginasthmaticresponses.ReleaseofWNTligandsthatengageinWNT/

b

-cateninsignallinggenerally suppressesadaptiveimmuneresponsesatvariouslevels.Traffickingofinflammatorycellsintothealveolarspacebecauseofupregulationofadhesion molecules,proliferationofactivatedThelper2(Th2)cellsfollowingantigenicexposure,andexpressionofTh2cytokinesareallinhibiteduponactivationof WNT/

b

-cateninsignalling.Conversely,secretednegativeregulatorsofWNTsignalling[e.g.,Dickkopf-1(DKK-1)]canundothisinhibition.Suppressionof

b

-cateninalsosignallingattenuatesairwayremodelling,examplesincludingairwaysmoothmusclegrowthandsynthesisofextracellularmatrixproteins.

b

-catenin-independentWNTsignallingexertsdiverseeffectsthat,ingeneral,arepoorlydescribed.Examplesaremodulationofairwaysmoothmuscle contractionandactivationofinflammatoryresponses.Thereisalsoasubstantialamountofcross-regulationbetween

b

-catenin-independentWNTsignalling andotherpathways,suchastransforminggrowthfactor(TGF)-

b

signalling,whichcollectivelydrivesairwayremodelling.

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asinTcells.Full-bodyablationofWNT-10resultedinanincreased

Th2predominantinflammatoryresponseinanacutehousedust

mitemousemodel.BALfluideosinophilswereelevatedaswellas

whole-lunghomogenateexpressionofIL-4andIL-13and

infiltra-tionofantigen-specificeffectorcellsinthelungs,althoughthere

wasnodifferenceintheproportionofinfiltratedTcellswithinthe

lungsofWNT-10B–/–mice.However,amongtheinfiltratedcells

wasa highernumberofeffectorcells,characterisedasCD44high

CD62Llow,suggestingthatantigenexposureisarequisiteforthe

WNT-10B–/–statetotakeeffect[119].Inlinewiththis,sortedT

cellsfromWNT-10B–/–miceexposedtoIL-4todriveTh2

polarisa-tionexhibited increased GATA-3 and IL-4 expression. Ofnote,

these changes were absent under baseline conditions, and no

differenceswerefoundinexpressionofT-boxtranscriptionfactor,

(T-bet;expressedinCD4+_{TcellscommittedtoTh1Tcell}

devel-opment).Moreover,WNT-10B–/–_{TcellsexposedtoCD3/CD28to}

driveclonalTcellexpansionthroughligationoftheTcellreceptor

(TCR),showedincreasedproliferation.Otherimmunecellshave

alsobeenlinkedwithWNTsignalling,althoughnotallofthese

studies have been tested within the context of an asthma or

allergicinflammatorymodel.IsolatedDCsexposedtocurcumin,

anaturalsubstancethatincreases

b-catenin

activityinthesecells,

preventedupregulationoftheactivationmarkersCD40andCD68

induced by lipopolysaccharide (LPS).Curcumin also prevented

lymphocyteproliferationfollowingexposureto LPSin amixed

lymphocytereactionassay,andreducedOVA-induced

accumula-tionofinflammatorycellsintheBALfluidofmice[120].

Further-more,intestinalDCsdeficientin

b-catenin

arecompromisedin

theirabilitytoproduce retinaldehydedehydrogenases(RALDH)

[121],anenzymethatis partoftheconversion ofvitaminAto

retinoicacid.Failuretomount aRALDHresponse subsequently

shifts Th polarisation in favour of Th1 cells [122]. Although

retinoicacidproductionbyDCshasbeenconsideredtobelimited

togut-residentDCsonly,otherDCpopulationshaverecentlybeen

shown to also express RALDH, particularly lung-resident DCs,

which express RALDH-2 [123,124]. Survival of eosinophils has

alsobeenreportedtorequirethenuclearpresenceof

b-catenin,

whichcanbetriggeredvia IL-5ina WNT-independentmanner

[125].Moreover,eosinophilsfrompatientswithasthmacan

mod-ulate the WNT secretory profile of cultured ASM cells when

adhered to [126,127]. These changes can subsequently affect

howsmoothmusclecellsproliferateandmaintaintheir

extracel-lularmatrix(ECM)surroundings.Othercelltypes,suchasmast

cellsorBcells,havethusfarnotbeenresearchedinanasthmaticor

allergicsetting, although active WNT signallingis required for

theirproperdifferentiation[128,129].

Evidence

from

mechanistic

studies

on

innate

immunity

WNT/b-cateninsignallinghasalsobeendemonstratedtoregulate

innate immune responses, primarily through interaction with

nuclear factor kappa-light-chain-enhancer of activated B cells

(NF-kB).NF-kBisatranscriptionfactorthatdrivestheexpression

ofmultiplecytokines,chemokines,and celladhesionmolecules

thatareinvolvedinasthmapathophysiology.Itsactivationoccurs

mainly through ILs or TNF, or is elicited by the activation of

Toll-likereceptors(TLRs)duringabacterialorviralexacerbation.

The usefulness of targeting NF-kB in asthma has already been

demonstratedbytheefficacyofglucocorticosteroids,whichcanbe

contributedinparttotheinhibitionofNF-kB[130].

b-catenin

has

been shown to interact with both the p65 [131–133] and p50

[131,132,134–136]subunitofNF-kBinvariouscelltypes,

general-lyresultinginimpairedDNAbinding,transactivationactivity,and

targetgeneexpressionmediatedbyNF-kB.GSK-3isalsorequired

for NF-kB activation via degradation of

b-catenin

[137–139],

althoughdirectphosphorylationofNF-kBp65byGSK-3hasalso

been proposed[140,141].Interestingly,anotherlineofresearch

has proposed a dependency of NF-kB on

b-catenin.

Increased

b-catenin

signallingin alveolarepithelialcellsenhancedNF-kB

signallingandtranscriptionaloutputinvitro[142].Thenuclear

co-factorsCREB-bindingprotein(CBP)andE1Abindingproteinp300

(p300)havebeenshowntoberequiredfor

b-catenin

andNF-kB

interactions[143,144].Itwasrecentlyshownthat,inASM,

inhi-bition of the

b-catenin–CBP

interaction could amplify

NF-kB-mediated inflammation, whereas inhibition of the

b-catenin–

p300 interactioncould attenuateit (authors’unpublished

find-ings, 2017). Although detailed molecular events remain to be

determined,theseresultssuggestamolecularswitchthatdirectly

controls NF-kB output, requiring the presence of

b-catenin

(Fig.3).

The interconnectednatureofWNT/b-cateninsignallingwith

both adaptive and innate immune responses complicates the

interpretationofgenetic screeningstudiesthat haveimplicated

b-catenin

signallingin asthma.Inaddition, bothinflammatory

cascadesinteractwitheachother.Innateimmunemechanismsare

requiredforDCpriming[145]andcanamplifyaTh2responseto

inhaled ovalbumin[146,147].Furthermore, commercially

avail-ableOVAisknowntocontaintracesofLPS[148],whichfacilitate

theprimingofThcellstoinhaledOVA.ThedegreeofLPSexposure

in conjunction with OVA also determines the type of T cell

response that is elicited (e.g., Th1 versus Th2) [146,149–152].

NF-kBp50–/–_{micewerecompletelydevoidofairwayinflammation}

when challenged with inhaled allergen in a murine model of

asthma [153], and it hasbeen shown that NF-kBis critical for

Th2differentiationthroughexpressionofGATA-3[154].Tohelp

facilitate the drugdevelopment process,futureefforts aimed at

identifyingnewWNTtargetsshouldextricateinnatefrom

adap-tiveimmunity.

WNT

signalling

and

airway

remodelling

in

asthma

Evidence

for

b

-catenin-independent

WNT

signalling

WNT-5A isincreasinglyexpressedinASM cellsofpatients with

mild to moderate asthma compared with healthy individuals

[107].ArecentstudyimplicatedWNT-5AwithASMcontraction,

where WNT-5A acts via autocrine signalling to promote actin

polymerisationinASMcells[155].Theincreasedpresenceofactin

filamentsincreasedmaximumforcegenerationinASMcells

with-outaffectingsensitivitytohistamine.IncreasedactivityofWNT

pathway activation and modulation of the actin cytoskeletal

networkcould serveasanalternativemodeltoexplain AHRin

asthma[155].Inaddition,WNT-5Aisresponsibleforsomeofthe

actionsmediatedbytransforminggrowthfactor(TGF)-b.In

hu-manASM,fibronectinandcollagensynthesisfollowing

stimula-tion with TGF-b requires de novo synthesis of WNT-5A and

subsequent activation of TGF-b-activated kinase 1 (TAK1) and

specificity protein-1(SP-1) [107,156].Itwasfurther shown that

some of the effects of WNT-5A initiated by TGF-b require the

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release of actin-binding proteins following formation of actin

filaments. Ofparticular interesthereis myocardin-related

tran-scriptionfactorA,whichisreleaseduponWNTstimulationand

can drive expressionof TGF-b target genes[157].Individually,

WNT-5A is unable to achieve the effects mediated by TGF-b,

indicatingthatcooperativesignallingisarequisiteforthiseffect

[155].The effectsof TGF-b–WNT–MRTF-Aare also relevantfor

other aspects of airway remodelling. For example, MRTF-A is

criticallyinvolvedintheinductionofTGF-b-mediated

epithelial-–mesenchymal-transition (EMT) [158,159] and the epithelial–

myofibroblast transition [160]. Myofibroblasts area rich source

ofECMproteins,andMRTF-Aisanimportantmediatorof

myofi-broblastactivationandexpressionofECMproteins[161].

Inhibi-tionofmechanotransductionbyblockingtheRhoA–MRTF-Aaxis

attenuated experimental pulmonary fibrosis in mice [162]. In

asthma, cross-regulation between TGF-b and WNT signalling

could allow for the development of treatment strategies that

canovercometheshortcomingsofdrugsthattargetTGF-b

signal-ling more directly (which are associated with severe adverse

effects).Goingforward,itisessentialthatwestudythislevelof

integrationinmoredetail,becausethenatureofthiscrosstalkcan

beoverwhelminglycomplexandcontextdependent[163].Failure

torecognisethislevelofintegrationwillconfoundthe

develop-mentofeffectivetherapeuticinterventionsinacomplexdisease

suchasasthma.

Evidence

for

WNT/

b

-catenin

signalling

b-catenin

isacriticalregulatorofairwayremodelling,particularly

inASMandfibroblasts.Bothcellsrequireactive

b-catenin

signal-ling to promote cell growth [90,164] and production of ECM

proteins[165–167].Although the natureofWNT/b-catenin

ex-pressioninanimalmodelsforasthmaiscontroversial(seeabove),

targetingthis pathwaycould stillbebeneficial ina therapeutic

setting.OVA-exposedBalb/cmicetreatedwithsmallinterfering

(si)RNAtargetedagainst

b-catenin

showedconsiderablyreduced

parameters of airway remodelling. Both deposition of newly

synthesisedcollagenandexpressionofalphasmoothmuscleactin

(a-SMA)wereattenuatedfollowinginhibitionof

b-catenin

[89].

Similarly,inhibitionof the

b-catenin–CBP

interactionwiththe

small-moleculeICG-001wasabletopreventASMthicknessafter

repeatedOVAchallengeandshowedatrendtowardsadeclinein

peribronchialcollagen deposition[90].These resultshave been

FIGURE3

TargetingWingless/Integrase-1(WNT)signallinginasthma.WhenWNTpathwayinhibitionistobeachievedtotreatasthma,itisimportanttoconsiderthe differentlevelsofintervention,whichcanprofoundlyimpactthefinaloutcomeofthetreatment.Here,wecategorisetheselevelsinthreecompartmentsbased oncellulararchitecture:(i)theextracellularenvironment;(ii)thecytosolicenvironment;and(iii)thenuclearcompartment.Generally,designingcompoundsthat actupstreamcanresultinalackofspecificity,becauseoftheinterconnectednatureofcellsignallinganditsnumerousfeedbackloops.Inthiscase,potential off-targeteffectscanbeexpected.Atthesametime,thisapproachallowsforabroadertherapeuticreach,becauseupstreameffectors(e.g.,WNTligands)aremore likelytobesharedbydifferentcelltypescomparedwithdownstreameffectors.Italsopresentsamorediverseplatformfordrugdevelopment;becausecell permeabilityisnotrequired,smallmolecules,monoclonalantibodies,recombinantproteins,andreceptorconstructs(e.g.,fusionproteins)areallpartofthe drugrepertoire.Conversely,designingcompoundsthatactdownstreaminthecellallowsfortheinhibitionofspecificsignallingevents,thusminimising off-targeteffects.Maximumspecificitycanbeachievedbyinhibitingonlyaspecificsubsetofprotein–proteininteractions,forexampletheinteractionof

b

-catenin withCREB-bindingprotein(CBP),butnotE1Abindingproteinp300(p300).However,targetedtherapyinthenucleuspresentsmoredifficultiesinthedrug designingphase,becausecompoundshavetocrossseveralbarriersbeforereachingtheirdesignatedsite,requiringthemtobesolubleandcellpermeable,or encapsulatedbyadeliveryvehicle.Additionally,thedeliveryandretainmentofdrugsinsidethecellcanbehighlydependentonthepresenceandactivityof ABCtransportersthatareexpressedinthelungsandthatmightusepulmonarydrugsassubstrates.

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corroborated in a mouse model for occupational asthma [94].

Moreover, inhibitionof WISP-1 (an inducer ofWNT/b-catenin

signalling) by a neutralising antibody attenuatedOVA-induced

ASMthickeninginrats[168].

Current

therapies

and

WNT

signalling

AccordingtotheGlobalInitiativeforAsthma(GINA),keypoints

inasthmamanagementaretoachievegoodsymptomcontroland

tominimisefutureriskofexacerbations,fixedairflowlimitation,

and adverseeffectsoftreatment[169],highlightingour lackof

understandingofasthmaaetiologyandthefocusonsymptomatic

rather than curative treatment. To date, there have been no

clinicaltrialsforasthmainvolvingthe modulationoftheWNT

signallingpathway.Currenttherapyismainlybasedon

(combina-tionsof)inhaledcorticosteroids,

b

2-adrenergicreceptoragonists,

andleukotrieneinhibitors[169,170].Someofthese,mostnotably

glucocorticoids,havebeenreportedtoelicitsecondaryeffectson

WNTsignaltransduction,mainlyinoff-targettissues.

Mesenchy-malcellcommitmenttowardsosteoblasticdifferentiationto

pro-moteboneformationrequires endogenous glucocorticoidsthat

signalthrough WNT pathways downstream [171].In line with

this,osteoporosis,oneofthemostfrequentadverseeffectsof

long-termglucocorticoidtherapy[172],isaccompaniedbyinhibitionof

WNT/b-cateninsignallinginosteoblasts.Glucocorticoidsactivate

GSK-3[173],inhibitTCF/LEF[174],andincreasetheexpressionof

WNTpathwayinhibitors, suchasDKK-1 [175–177]and soluble

Frizzled-relatedprotein-1(sFRP-1)[178].GSK-3canalso

phosphor-ylate the glucocorticoid receptor (GR), which facilitates its

re-sponse to glucocorticoids [179–181]. It wouldbe interestingto

assess the effectsof glucocorticoids indifferent tissues that are

more relevant for asthma pathophysiology, and to evaluate

whetherpotentialeffectsonWNTsignallingactivationare

clini-callysignificant.Atthemoment,thereisnoevidencethatboth

short-actingandlong-acting

b

2-adrenergicreceptoragonistscan

modulateWNTsignallingin thelung.Onestudyaddressedthe

interactionoffenoterolwithWNTpathwaycomponentsin

hu-manbronchialrings[182],butthesefindingshavethusfarnot

beencorroboratedandrequireadditionalverification.Althoughit

hasbeenshownthatcysteinylleukotrienescanactivate

b-catenin

signalling [183,184], primarily in a WNT-independent manner

through activation of phosphatidylinositol 3-kinase (PI3K)

[185],therearenostudiesthathaveshownWNTpathway

modu-lation by any of the currently available cysteinyl

leukotriene-receptor antagonists (montelukast, zafirlukast, and pranlukast).

Thesameholdstrueformostotheravailabletreatmentstrategies,

examples beingIgE inhibitionwith omalizumabor cholinergic

pathwayinhibitionwithtiotropium.Ofnote,asthmatreatmentis

movingtowards personalisedmedicine and a focus onasthma

pheno- and endotypes [186–188]. Drug therapies previously

deemedineffectivehavegainedrenewedinterestinlightofthese

developments,oneexamplebeingbiologicstargetedagainstTh2

cytokines[189].Giventhe closeinvolvementofWNT pathway

components with innate and adaptive immunity, it would be

interesting to re-evaluate these drugs based on their potential

secondaryand/orindirecteffectsonWNTsignalling.Inaddition,

several other drugs that are currently under trial might prove

efficaciousintermsofWNTsignalmodulation.Forexample,drugs

thatinhibittheprostaglandin(PG)D2receptorsubtypeDP2[also

knownasthechemoattractanthomologousreceptorexpressedon

Th2cells(CRTh2)],importantinTh2andtype2innatelymphoid

cell(ILC2)function[190,191],butpossiblyalsoinairway

remo-delling[190],arenowinclinicaldevelopmentforasthma[192].It

is known that

b-catenin

functions downstream of the closely

relatedeicosanoidPGE2inacAMP-dependentmannerinseveral

malignant cell types[193,194], and itwould be worthwhile to

assesstheeffectsofCRTh2antagonistson

b-catenin

signalling.

Concluding

remarks

More than 30years afterthe discovery of whatis possibly the

oldestevolutionaryconservedpathwayinanimals,andextensive

researcheffortstocharacterisethisfundamentalpathway,

target-ing WNT signalling in a clinical setting is still in its infancy.

Despiteintensiveeffortstocharacterisethispathwayinadisease

setting, including asthma, unveiling a multitude of potential

therapeuticpointsofintervention,therehavebeensurprisingly

fewattemptstomodulateWNTpathwaycomponentsinclinical

trials.Thisisnotbecauseofalackofavailablereagentsthattarget

theWNTpathway[195],whichareincreasinglybeingdiscovered

and developed. Some of these compounds are currently being

tested in clinical trials, of whichmost are within the scope of

cancertreatment,butmostotherfieldshavesofarlaggedbehind,

includingasthma[196].Mostdrugstestedincurrenttrialstarget

extracellular modulatorsofWNTsignalling,including notonly

DKK-1, but also the WNT ligands themselves, as well as WNT

receptors [196]. This is surprising, considering the widespread

involvement of WNT signalling in almost every tissue within

the humanbody,and islikely tohave secondaryeffectsin

off-targettissues.Nonetheless,thesestudieswillprovidecriticalclues

tothesafetyprofileoftheseWNTmodulators,andwhetherthey

canbeefficaciousinatherapeutic(cancer)setting.Theywillalso

provideimportantinformationaboutwhetherfullinhibitionor

activation of WNT signalling isthe rightapproach for therapy

[197].Duringdrug-screeningapproaches,themostpotentdrugs

areusuallyselectedforandtestedinaclinicalsetting.However,

fullreductionofaberrantWNTsignallingmightnotnecessarilybe

the right approach. Given that WNT signalling is intricately

involved in tissue homeostasis, therapeutic targeting might

re-quireamoredelicateapproach,whereWNTpathwayactivation

needstobebroughtbackdowntonormallevels.Infact,thesafety

profile ofWNT modulatorscurrently in preclinicaland clinical

trials for cancer, including PRI-724 [198], LY209314 [199],

CWP232291[200],OMP-54F28[201],andOMP-18R5[202],show

thatmanyofthesecompoundssharesomeoftheiradverseeffects.

Althoughthesafetyresultsfromthesestudiesshowedgood

toler-abilityoverall,mostofthesecompoundsassociatedwith

symp-tomsofnausea,diarrhoea,andvomiting.Althoughtheseadverse

effects might raise concern over their general usability in the

clinic,limitingsystemicexposurebyrestrictingthereachofthese

drugs to the lungs through inhalation could largely overcome

theseissues.Nonetheless,theseresultshighlightthedifficultythat

residesin developingsafeand effectivetherapeuticcompounds

targetingthiscomplexpathway.

The increasinginterest in characterising asthma phenotypes

andendotypes,andtheemergingconceptthatasthmamighthave

a developmental basis, raises interesting thoughts in terms of

futuretherapy. Identifyingbiomarkersfor asthmadevelopment

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andsusceptibilityinearlylifecouldpavethe wayfortreatment

strategies that could alter disease progression entirely, possibly

evenhaltingorreversingit.Inlightofthegeneticand

develop-mentalaspectsofasthma,targetingtheWNTpathwaywouldbea

primarycandidateinthisregard.Ofparticularinterestwouldbe

WNT-associatedtherapiesthataffectairwayremodellinginearly

life,becauseremodellingmayalreadydevelopbeforetheonsetof

asthma symptoms. Targeting WNT signalling in patients who

alreadyhaveasthmamightalsobebeneficial.Ofspecialinterest

here are compounds that target the selective inhibition of the

interaction between

b-catenin

and co-factors, such as CBP or

p300,becausetheyonlydisruptasmallsubsetofco-factor

inter-actions,initiatingatranscriptionalprogramthatpotentially

inhi-bitsdiseaseparameters,whileleavingothersintact.Theyalsoact

significantlydownstreaminthecell,possiblypreventing

unwant-edsecondaryeffects.Toimprovethedevelopmentoftherapeutic

targetsdownstreamofWNTsignalling,itisessentialthatwelearn

more about the nuclear actions of

b-catenin,

because this is

currently an underappreciated topic. For example, CBP and

p300areparalogousgenesthatsharealargedegreeofstructural

similarity,yettheyareoftenascribedopposingroles[203].How

CBP and p300 can exert these seemingly bimodal functions

remainstobedetermined.Ithasbeensuggestedthatthe

interac-tionbetween

b-catenin

andCBPorp300resultsfromcompetition

[203],butthis modelseemstoomuch ofanoversimplification,

because CBP and p300 are known to facilitate transcriptional

output through a plethora of additional transcription factors.

Additionally, differential phosphorylation of CBP or p300

[203,204],oryettobediscoveredbindingpartners,couldgovern

selectivity and binding with regulatory components. Both the

abilityofCBPand/orp300tomodulatechromatinandacetylate

b-catenin

orotherproteinsthroughtheirhistone

acetyl-transfer-ase(HAT)domainwillbeanimportantfocusofstudy.Expanding

our knowledge ofthese architectural elements will further our

ability to design drug therapies that targeta selective rangeof

transcriptional events involved in disease, without interfering

withthecrucialroleofWNTsignallingintissuehomeostasis.Five

clinicaltrialsarecurrentlyunderway,allincancer,usingthe

small-moleculeinhibitor PRI-724(an enantiomer ofICG-001), which

selectivelytargetsthe

b-catenin–CBP

interaction,withnoeffect

onp300[205].

Type 2 inflammation can be efficiently suppressed in most

patientswithasthmawiththeregularuseofinhaled

glucocorti-costeroids.AlthoughTh2-highasthmaisgenerallya

corticoster-oid-responsive endotype [206–208], a notable subgroup of

patientswiththisendotypemaintainsymptomsandexperience

severeuncontrolledasthmadespiteregularuseofsteroids[209–

213].Novel drugtreatment ofthis group ofsteroid-insensitive

patients with severe asthma is warranted. Furthermore, some

Th2-high asthmatics require high doses of inhaled steroids or

oralsteroidsformaintenancetherapy,andthesepatientsarein

needofalternativestoavoidexcessiveadverseeffects.Theadvent

ofmorespecific inhibitors,suchasbiologicalstargetedagainst

type 2 inflammation, has raised hope that these drugs will

providesimilarbenefitstopatientswithasthma,whiledisplaying

feweradverseeffects.However,comparedwithglucocorticoids,

thesecompoundshaveamorelimitedeffectonairwayfunction

andasthma control,even when stratified fordifferent asthma

pheno-or endotypes. Thus, theyhave so farnot beenableto

replacesteroidtherapyandareadjunctiveatbest[214].In

addi-tion,glucocorticoidshavenonoticeableeffectonairway

remo-delling.Thisiswhereanti-WNTtherapycouldconferadditional

benefit,becauseofitscombinedeffectsonTh2immunity,airway

remodelling,andmusclebiology.Most trialsusing anti-DKK-1

antibody therapy in cancer are now complete, and a positive

outcome of these studies will be important in furthering our

understandingtowardsthedevelopmentofasthmatherapy.Over

thenextcoupleofyears,theseresultsandothersshouldshednew

lightonwhetherwecanusetheWNTpathwayasatherapeutic

targetinasthma.

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