This article isprotected rights by All copyright. reserved. 10.1111/exd.13509 doi: lead todifferences version between this andthe ofPleasec Version Record. been and throughtypesetting, proofreadingwhich may thecopyediting, process, pagination This article undergone has for and buthas accepted been not review publication fullpeer effectors PPK mice naildystrophy, orallesion congenita(PC), a genodermat challenge derivedappendages and isinducibly The Abstract coulombe@umich Building,109 Zina Pi Biology, # 2 1 AbigailZieman mice in background by impacted modestly is phenotype null 16 The Articletype : Letter: MouseMutants with Absent Skin Phenotype DR. PIERRE ACOULOMBE (Orcid ID : 0000

Department Departmentof Biochemistry and Molecular Biology,Bloomberg School of Public Health, AcceptedAddress forcorrespondence: Pierre A.Coulombe, Ph.D., Dept. of Article AnnArbor,MI Johns Hopkins University,Baltimore, MD

typeIinterme

lesions developorallesions early after birth and PC UniversityMichiganof Medical School

( s e.g., .Dominantly

have

of

danger 1 Celland Developmental , 2

.edu. diate filament a marked and PierreCoulombeA. tcherPlace - associated molecularp

-

acting missense alleles in s and,s frequently,alterations in

dysregula osisinvolving

, keratin 16 AnnArbor, MI 48109

expressed inthe epidermis uponbarrier - tionof skin barrier related 0003 Biology

(K16) 1, 2 - 0680 debilitatingpalmoplantar keratoderma(PPK),

, # atterns), ,

3071 ,

- is - 2373) UniversityMichigan of Medical School, like PPKlesions as young adults. Thes KRT16 constitutive BiomedicalSciences Research , USA. , Tel:

andare preceded by

glands andhair.

arecausative pachyonychia for

lyexpressedectoderm in 734 Celland Developmental

- genetic strain strain genetic 615

and C57Bl/6 - 7509.E

ite this articleite this as innateimmunity

oxidative - compromising ;Krt16 - mail: stress stress - - / - e

This article isprotected rights by All copyright. reserved. Acceptedand effectorsof innateimmunity genes,including Danger showed lesionsthat the lesionsearly afterbirth andPC effects. consequences the associatedwith alterationsin epidermolytic forpatients. involve dram Focalnon (PPK) (PC) thatd psoriasis,(e.g. carcinoma) assuch the epidermis are Articlefollicles,nail, glands,oral papillae andetc.) isinducibly expressed i Introduction causingmutations in the observationlight in of variations the in clinical presentation ofindividuals harboring di phenotypeoccur as well lesionsPC of patients. Here we report that secondaryto hypoactive (2) ominantly ,nail dystrophy, oral lesions

Mice null for Keratin 16(K16), a typeIintermediate filament geneand , isconstitutively

, a , - e genodermatosis n

pidermolytic Of noteOf atic epidermalthickening and hy

specificcell types in ectoderm PPKin theabsence of otherlesions - acting missense alleles in ,s Krt16 imilaralleles in - PPKlesions in the Nrf2 Nrf2 signaling. KRT16 AssociatedMolecular Pa , originally , generated in the

experiencing footpad skinfootpad involve gross a misregulationof skin (1)

involvingpainful and debilitatingpalmoplantar keratoderma . - FVB Geneticlinkage analyse likePPK lesions as youngadults

(6)

and in

, strain background andare , which ,

KRT16 .

, , frequent

stress ( These K

allcomponents of -

RT16 derivedepithelial appendages (e.g., hair

occur atoccur

precededand caused by a state of oxidative perkeratosis can elicita presentation

molecular featuresare present in PPK e.g.,UV exposure, wounding) or ly, KRT16

tterns (DAMPs;also knownalarmins) as causativeare pachyonychia for congenita

characteristicof PC alterationsin pressurepoints in palmsand soles C57Bl/6 , albeit less, severely a so, significant

s and s expressed when stratifiedepithelia

are subject tomodifier gene(s)

and are DNA

the the background,develop oral glands and hair (5) C57Bl/6;Krt16

often sequencingdetermined . Follow . of

(4)

signficantly focalnon , , suggesting that - - up studiesup barrierrelated - /

- aswell disease

mouse -

painful s ease

(3) ,

- .

This article isprotected rights by All copyright. reserved. severe weaningand Altogether isdramatically reduced relatedK13 (Fig. 1D) and Immunostainingof (Fig. 1C), area or posterior the aspect of the dorsal tongueepithelium examination morethan what had been observedin the daybirth,after while ~ werenext conducted and theprogeny analyzed. backcrossesof Results backgroundon background purpose ofdefining the role of allele the in individuals PPK stress

AcceptedFVB; Article -

of dorsal the tonguereveals a mildlythickened,disorganized and occasionally cytolytic like lesionsin footpadskin secondaryto , The

Krt16 asthose

andDiscussion

theseobservations with PC Krt16 FVB (see postnatalat day5 (P5) +/+ the

express C57Bl/6;Krt16 mice (Fig 1B).Histologically mouse background.strain This studywas conducted for primary the previouslyreported for characteroforal lesions [ nullallele wastransferred to the 8 similar ] (6 )but providedopportunity an to assessthe impact of misregulation ofKeap1 with ,7 50% of50% these

) ionof in thedorsal tongueepithelium of .

Here,we describe a normal complementof oralpapilla its partnerits K4 (data notshown) tissuesections the +/ , , summarizedin Krt16 -

and wildtype of Krt16 C57Bl/6

mice survive adulthood to revealswhitish and raisedplaques (oralleukoplakia) in in HPV16 - / -

C57Bl/6; phenotype.

revealsthat in C57Bl/6 ; - Krt16 , examination , of sagittal cross sections of this

Nrf2 signaling FVB;Krt16 the phenotype the associated with the FVB -

inducedcarcinogenesis Suppl.Fig.

Athird of - Krt16 / -

mice.Intercrosses between

FVB mice arepresent in PPKlesions of strain FVB;

ex - - /

/ - strain backgroundvia -

mice pressionof the differentiation

mice

Krt16 (by17%; are FVB; FVB;Krt16 (7) 1 ,

showthatt .Several of the key featuresof unaltered,whereas thatof K17

(5) are similar to (data not shown) (Fig 1A) - Krt16 / -

mice,but in not .

[ 5 - / ] - - ). Macroscopic). /

- mice (Fig.1E).

, which , isslightly offspring dies within a in the h geneticstrain e

, survivalat

butnot as FVB six FVB;Krt16 , at P5 at , Krt16

FVB; sequential

null . Krt16 - +/ -

+/ -

This article isprotected rights by All copyright. reserved. activated ofform finding genesisof the Additionally, severalDAMPS including typepartnerII gene for basedantioxidant system, and of Keap1 the forDAMPs (danger misregulatedin footpad skinof C57Bl/6;Krt16 2 positive innate the for immune cell markerCD11b, suggesting an inflammatory infiltrate FVB;Krt16 phosphorylatedHistone H3 (Fig 2 quantitation startingat fourweeks 2 (Fig. paw ofskin similarsex bias PPK for alsoonset occurs in macroscopicand blinded measure oflesion severity,at 6 weeks of ag vs.0.37 5.04±0.41 weeks; Fig.see 2 mice developPPK comparedto I Accepted). Th Article (5) H eseobservations are qualitativelysimilar to thosemade when characterizing s Nextwe assessedsome of the key molecular readouts thathad been shown be to theAt two monthsmark post ),

.Male exhibit mice a slightly earlier onset of PPK relative to females (average 4.67± werecorroborated at the protein level suggestingimpaired +/+ FVB;Krt16 reportedin Fig.2F).Lesional footpad skinshow

the the mRNAtranscrip - epidermis,indicating a mild state of hyperproliferation WT like lesionson their pawsfront (Fig. 2 - / cornifiedenvelope, were -

mice

littermates ( IL - - 1β associated molecularpatterns; see (5) - / (datanot shown) - Krt1

mice exhibitthickened epidermis and modest hyperkeratosis (see Suppl(see Fig DefB4 - 6 positive nucleiin the basallayer of B

Suppl Table and asensitive indicatorof cell and tissuestress, and of

)and pea epidermaldifferentiation C57Bl/6;Krt16 , tlevels for S100A8 - birth,surviving D king at eightking weeks (Fig. 2 )and also exhibit a greaterPPK index, a also markedlyelevated2 (Fig. . 1 T ) , 1 . hese observations and ).

Sprr2d - Nrf2 system C57Bl/6;Krt16 A - , e.g., , / - llsurviving

mice.Emphasis was placed on mRNAlevels S100A9

and A

FVB;Krt16 bywestern blotting for ), as ), , SerpinB3a

(6) a~2.5 fold weremarkedly elevated 2 (Fig.

(7) we reported male and female ), keyeffectors of the glutathione - / s - . Transcriptlevels for

mice ( suggest decreas - / FVB;Krt16 -

mice , C

increase in which contributeto (Fig. 2G),(Fig. and cells 9 ;sex ).

e(Fig 2,Aand edstaining for still J that for for Histologically, the ). - specific So C57Bl/ - weigh less /

-

while qualitatively

S100A8 andthe FVB;Krt16 relative to me

of t 6;Krt16 Krt6a hese E -

/ - ).

J

, the mice (Fig. - / ). - A

-

This article isprotected rights by All copyright. reserved. advice Acknowledgments Materials andMethods mouse modelas a useful resource defineto determinants the involved individualswhose genome harbormutated findingsare thus consistent with thevariable penetranceof the clinical presentation in between l influe impactthe prese Krt16 crossis death the mice were Krt16 C57Bl/6;Krt16 similar, t e

Acceptedthality theseverityto of oraland Article Krt16 nce nullallele lossHPV16 for remainsundefined

and support Theauthors are grateful to The conclusion,In our findings of One the keyjustification of this set of experiments was toassess impact the of

indicativea of he alterationshe occurring in theskin barrierare less severein

C57Bl/6 - / theintensity of vir - ;HPV thus

methods usedin thisstudy - / -

. tg/+ bred to the mice

ntation of the

and

mice diedwithin weeksafter two birth . . Thiswork was supportedby grantAR044232 from the NationalInstitute

. tg/+

FVB strong - indu

and calls forsubstantive follow

t strains mayaccount for observationsthese FVB; uallya ced tumorigenesisced in

geneticinteraction betweenHPV16 the transgeneand the Krt16 HPV suggest PPK llofaspects the presentation, ranging perinatalfrom Dr.

nullpheno tg/+ Michelle Kerns - are described

lesions. mouse line

thatwhile strain ba genetic K RT type

16 Differences ininflammatory responses FVB

alleles (2 as described in mouse ata qualitative level

, under “Supplementa

Joseph Shen mouseskin (see (8)). -

up studiesup (Suppl Table - 4) ,and point to the (8) ,t

. and FVB;Krt16 Remarkab he ckgrounddoes not .

2

(

10,11 ) outcomeof this . Tara BiserTara

While l

Materials”. FVB; ) .

These These - ly,100% of / the the cause of Krt16 -

relative to , it , Krt16

for does

null +/ -

This article isprotected rights by All copyright. reserved. Kerns9. M Hakim,L, J. M., 8. 7. 6. 5. 4. 3. 2. 1. Re Conflictof Interest designed theexperiments Arthritis, of Musculoskeletal and Skin Diseases.

hallmark feature ofpachyonychia congeni Lessard C,J Coulombe KeratinPA. 16 Human molecular genetics1995: 4: 1875 underlyfocalnon ShamsherM NavsariaK, HA, Stevens HP, et al. Novelinmutations keratin 16 gen .J InvestigDermatol Symp Proc2005: 10: 3 LeachmanS A, Kaspar FleckmanRL, P, etClinicalal. and pathological featuresof pachyonychia congenita.Nature genetics19 McLeanH, Rugg EL, W LunnyetDP, al. Keratin and16 keratin17 mutationscause Biochem1998: 31: 173 intothe role of intermediate filamentsin specifying keratinocyte cytoarchitecture.Subcell McGowanCoulombeK, PA. Thewound repair Dermatol response ato Nrf2 inducerin a mouse model for pachyonychia congenita keratinocytes.Nat Genet2015: 47: 933 Coulombe,P. A. Keratin B. G., Guo,Y.,Han, J., Ong,S.,Zheng, Taube,J.M., W., Cihakova, D., Wan,F., Hobbs RP,DePianto, D. Jacob,J., J. T.,Han,M. Chung, C., B. M., Batazzi, A. S.,Poll, Clin Invest Oxidative anddysfunctional stress NRF2 underlie pachyonychia congenitaphenotypes. J KernsM Hakim,L, J.M., Lu, R. G.,Guo, Y.,Berroth, A.,Kaspar, R. L.,Coulombe, P. A. breach.Proc Natl Acad SciUSA2013: 110: 19537 Coulombe,P. A. Keratin regulates16 innate immunity in response toepidermal Lessard C,J Pina investigative dermatology132:2012: 1384

Accepted Articleferences Theauthors reportno conflict of interest.

(in press) 2016:126: 2356

- - epidermolyticpalmoplantar keratoderma (NEPPK)in two families. Paz,Rotty, S., J.D., Hickerson, R. P.,Kaspar, R. L.,Balmain, A., .

, interpreted , thedata - 204. - dependent regulation of and dependent regulation Aire ge Zieman,A - 2366.

., Lu,R.G. - - 938. nullmice develop ,a - taand relateddisorders. The Journalof 1881. - 1391.

95:9: 273 and wrotethe manuscript.

AZ performedall experiments. AZ ,Coulombe, P. A. -

associated keratins6, 16, and 17.

- 19542.

- 278.

neexpression in skin tumor Sexualdimorphism in - 17.

. J Invest

andP barrier Insights e AC This article isprotected rights by All copyright. reserved. (red)in FVB;Krt16 proliferation(measured by P thickness from8 old PPK of lesions severity basedpresenceon of waxy keratoderma on pawfront of macroscopiclesions (waxykeratoderma) were present. n=4 lesioninonset both sexes of junction. 4 controls.to female (left) F K17in sectionsof fe FVB;Krt16 determined byLog lesions.(A) Survival ofcurve Figure 1. toFigures Legends 11. 10. - igure 2.igure male

and8

Accepted Articleimpactsmodulation of the pathwayTLR4 by RONin tissue S.,Couto, Modrusan, Z., French, D., Cupp, Ashkenazi,J., A. Host genetic backgropund immunomodified Vetmice. Pathol 2012: 49:32 inbread laboratorymouse strains:Points to considerin phenotyping genetically

FVB;Krt16 Chaiuduri A, Wilson SellersR S, Clifford, C.B.,

FVB;Krt16 front pawfront skin from - Epi,epidermis; SC,stratum corneum. Scale bar, weeks

FVB;Krt16 FVB;Krt16

- / mice - (B and(B C

mice(both sexes)P5.at (C FVB;Krt16

mice.n . n old - week - / = fresh frozentongue tissue fromP5 - -

rank test. 3 mice/genotype. (H FVB;Krt16 lesions. - - ) H&E ) staining of

/ / - -

=2 mice developPPK lesions. (A mice have increased mortalitycompared to and developWT oral

- old / , , N.S.,Yang, B.,Paler Martinez Liu,A, J., Zhu, C., Bricker,N., -

- mice displayingmacroscopic appearance of PPK lesionscompared 4 mice/sex/genotype.

FVB;Krt16

8 -

- Histonestaining) H3 in FVB;Krt16 FVB;Krt16 (B)Pictures of macroscopictongue plaques thatoccur week - Treuting,P.M, Brayton, C. Immunologicalvariation between / -

male s Scale bar,

oldmale

mice front paws.front n=2 front pawfront skin sections -

)H&E staining of P5 tonguesto evaluate histologyof )Immunostaining for filaggrin(green) and k / mice -

mice.Mice werescored as lesional when evidence .

Dotted linecorresponds todermal/epidermal .n FVB;Krt16 50μm

(F =31 - ) Sex ) 43. - ) ) Images of6 36 mice/genotype. front paw front skin 8 from

- (Dand E male littermates. 3mice/genotype. (G - specific quantitationof epidermal

mice 50 μm. - . . (I relatinghistological changes in associate macrophages. - ) ) Immunostaining for 5 mice/sex. (E ) ) Immunostaining fo

- (D week )Quantitation of PPK

** denotes p<0.01 s

old male (right)and - weeksold male ) ) Quantitationof ) ) Quantitation s of s

6 eratin 5 - K week rCD11b in 13and

This article isprotected rights by All copyright. reserved. Accepted Article denotes p skin from μm.50 (green)and p65 (red) (J) Transcriptlevels Danger for Associated MolecularPatterns(DAMPs) in 8 < - 0.01as determined by weekold FVB;Krt16

in frontpaw ski

null unpaired2 micerelative n from8 - - tailedS weeksold male

to WT tudent’s

controls.* denotes p<0.05 and **

t

FVB;Krt16 test

on

dCq values.

mice . Scale bar, front pawfront

This article isprotected rights by All copyright. reserved. Accepted Article