1
Le e o he Edi o
TSH is a no el neu oendoc ine egula o o selec ed ke a ins in he human
hai ollicle
Yu al Ramo a,b,*, Guoyou Zhanga,c, Tamás Bí ód, E ika Lisz esd, Wol ang Funke, A ieh
Ingbe b, Lu z Langbein and Ral Pausa,g
aDepa men o De ma ology, Uni e si y o Lübeck, D-23538 Lübeck, Ge many
bDepa men o De ma ology, Hadassah - Heb ew Uni e si y Medical Cen e , Je usalem,
Is ael
cDepa men o Hand and Plas ic Su ge y, he Second A ilia ed Hospi al o Wenzhou
Medical College, Wenzhou, Zhejiang P o ince, China
dDepa men o Physiology, Uni e si y o Deb ecen, Medical and Heal h Science Cen e ,
Resea ch Cen e o Molecula Medicine, Deb ecen, Hunga y
eKlinik D . Koslowski, Munich, Ge many
Di ision o Skin Ca cinogenesis, Deu sches K ebs o schungszen um, Heidelbe g, Ge many
gSchool o T ansla ional Medicine, Uni e si y o Manches e , Manches e , UK
Wo d coun : 1099; Numbe o e e ences: 14; Numbe o ables: 1; Numbe o igu es: 2
This s udy was suppo ed in pa by a Mine a Fellowship o YR, and by g an s om
Manches e NIHSR Biomedical Resea ch Cen e o RP and om he Wilhelm Sande S i ung
o LL (2007.133.1.). Con lic o in e es : The au ho s ha e no con lic o in e es o decla e
*Co spending au ho : Depa men o De ma ology, Hadassah - Heb ew Uni e si y
Medical Cen e , Je usalem, Is ael, Tel.: +972 (0)2 677-7111, Fax: +972 (0)2 677-7299, e-mail
y[email p o ec ed]
2
Ke a ins and ke a in-associa ed p o eins (KAPs) cons i u e he majo s uc u al p o ein
componen s o he hai . Regula ion o hei exp ession is c i ical o p ope hai ollicle (HF)
s uc u e and unc ion [1]. The e o e, i is impo an o ully elucida e he con ols ha
egula e ke a in exp ession. Al hough i is accep ed ha he exp ession o selec ed ke a in
genes unde lies endoc ine con ols [1,2], ou unde s anding o he complex egula ion o
ke a in exp ession emains a he agmen a y. To be e elucida e his egula ion, human skin
and HF o gan cul u e o e an ins uc i e, physiologically ele an esea ch ool [1,3].
In pa icula , e y li le is known on he neu oendoc ine con ols o ke a in ansc ip ion. The
impo ance o he la e is highligh ed by he ecen disco e y ha he "pi ui a y" neu opep ide
ho mone, p olac in, which is also exp essed by human HFs, po en ly egula es he exp ession
o selec ed human ke a ins on he gene and p o ein le el [3]. Mo eo e , mic oa ay analyses
had p o ided i s clues ha hy oid s imula ing ho mone (TSH) and i s p oximal egula o in
he hypo halamic-pi ui a y- hy oid axis, hy o opin- eleasing ho mone (TRH), migh ope a e
as p e iously unsuspec ed modula o s o human hai ke a in and KAP gene ansc ip ion in
si u [4-6]. Also, we had ecen ly ound ha TSH up egula es ke a in K5 gene exp ession and
p o ein syn hesis as well as K14 ansc ip ion in human epide mis [4].
The e o e, we ha e asked whe he TSH ope a es as a no el neu oendoc ine egula o o
human ke a ins in si u, using mic odissec ed, o gan-cul u ed human scalp HFs as a
physiologically and clinically ele an assay sys ems [3-5]. This was complemen ed by
s udying he e ec o TSH on ke a in exp ession in cul u ed human ou e oo shea h (ORS)
ke a inocy es (KCs).
Anagen VI HFs we e isola ed om no mal on o empo al scalp skin ob ained a e w i en
in o med consen om h ee heal hy emales unde going ou ine ace-li su ge y, as
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p e iously desc ibed [4,5], adhe ing o Helsinki guidelines and unde a licence om he e hics
commi ee o he Uni e si y o Lübeck. HF mRNA ex ac s om one emale pa ien we e
subjec ed o quan i a i e eal ime PCR (qPCR) o selec ed hai ke a in genes a e 24 h s
ea men wi h TSH (100 mU ml-1) o ehicle. Fo immunohis ochemical analysis, isola ed
HFs om addi ional wo emale pa ien s we e o gan cul u ed o 6 days as desc ibed
p e iously [3,4], and exp ession o ke a ins K6, K14, K17, K31, K32, K85 and MSX-2 was
s udied wi h ou p e iously published basic immunohis ology p o ocols [3,4] (Table 1). Fo
epi helial ke a in qPCR expe imen s, human ORS KCs we e ob ained om an addi ional
emale pa ien , as p e iously desc ibed [3]. These we e ea ed o 24h wi h TSH (100 mU ml-
1) o ehicle.
Ou p e ious mic oa ay esul s had sugges ed ha TSH adminis a ion signi ican ly
down egula es exp ession o K31 [4], hus sugges ing a ole o TSH in he egula ion o hai
ke a ins. To u he explo e his mic oa ay lead, we pe o med qPCR on ca e ully selec ed
hai ke a ins ha a e exp essed in he hai sha -gene a ing HF epi helium, ep esen ing h ee
majo compa men s o he HF epi helium: he hai ma ix/p eco ex and cu icle (K35), he
hai co ex (K31) and he hai cu icle (K32) [2]. Pa icula emphasis was placed on s udying
KRT85 and KRT35 ansc ip ion, since bo h ke a ins a e he ea lies ones o be exp essed in
he p eco ical hai ma ix and ea ly cu icle [2].
These qPCR analyses demons a ed ha TSH down egula ed ansc ip ion o KRT31 and
KRT32 genes (Fig. 1A). In hese qPCR analyses, KRT35 ansc ip ion was la gely una ec ed
by TSH. Ins ead, ansc ip ion o i s ype II coun e pa ke a in gene, KRT85 [2], was
down egula ed (Fig. 1A).
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Immunohis ochemical s udies on TSH ea ed HFs con i med he down egula ion o he hai
ke a ins also a he p o ein le el (Fig. 2A-C): K31 immuno eac i i y, localized o he hai
co ex (Fig. 2A), K85 immuno eac i i y, which is exp essed in he hai co ex and in he
p eco ical hai ma ix (Fig. 2B) and K32 immuno eac i i y, which se es as a ma ke o he
hai cu icle (Fig. 2C), we e signi ican ly down egula ed ollowing TSH adminis a ion. Since
MSX2 se es as a majo ansc ip ion ac o ha egula es hai ke a in exp ession [7], analysis
o MSX2 exp ession was also pe o med. Indeed, TSH down egula ed MSX2 ansc ip ion in
human anagen HFs in si u (Fig. 1A). MSX2 immuno eac i i y was ound o co espond o he
exp ession pa e n in he mouse, he hai ma ix and co ex (Fig. 2D) [8]. In line wi h he
qPCR esul s, TSH down egula ed MSX2 immuno eac i i y (Fig. 2D).
Se e al epi helial ke a ins a e widely exp essed in he HF ORS, whe e hey a e hough o
play an impo an ole in main aining s uc u al in eg i y and homeos asis [1]. We ha e
p e iously con i med ha TSH has a egula o y e ec on K5 [4], which is p ominen ly
exp essed in he ORS [1]. We ha e he e o e u he explo ed his inding, by s udying he
e ec s o TSH on selec ed epi helial ke a ins which a e cons i u i ely exp essed in he ORS
[2]. HF ea men wi h TSH signi ican ly down egula ed K6, K14 and K17 immuno eac i i y
(Fig. 2E-G). In o de o con i m hese indings in isola ed HF KCs in si u, TSH ea ed ORS
KCs we e analyzed by qPCR. This showed a signi ican down egula ion o KRT6 and KRT17
ansc ip ion, bu no e ec on KRT14 ansc ip ion (Fig. 1B).
Exploi ing he human HF as a disco e y ool o ke a in esea ch, he cu en pilo s udy
iden i ies TSH as a no el neu oendoc ine egula o o ke a in exp ession in human skin. The
da a p o ided he e also sugges ha he hai sha abno mali ies seen in pa ien s wi h hype -
o hypo hy oidism [9] may esul no only om pe u ba ions in he hy oid ho mone blood
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le els, bu also om associa ed neu oendoc ine changes in TSH le els in he blood, o e en in
he skin [6,10].
The egula ion o hai ke a in gene exp ession is igh ly con olled by a complex mechanism
in ol ing se e al ups eam media o s. These include membe s o he Wn /β-ca enin pa hway
and o he TGF-β amily which con ol MSX2, FOXN1 and HOXC13 ac i i y [1,2,7,11,12].
Since MSX2 is a majo egula o o hai sha di e en ia ion, ou obse a ion ha TSH
educes MSX2 exp ession on he gene and p o ein le el encou ages one o pu sue he
hypo hesis ha TSH may ac a leas in pa ia inhibi ing MSX2 exp ession.
While KRT35 ansc ip ion was no inhibi ed by TSH, TSH down egula ed KRT85
exp ession. Such a unila e al ke a in egula ion has been documen ed be o e o he cu icle
ke a in K82 [13]. I is concei able ha he TSH-induced up egula ion o KAPs ansc ip ion
p e iously obse ed by mic oa ay analysis [4] may compensa e o he down egula ion o
one ype o ke a in, as an a emp o main ain s uc u al s abili y o he hai sha .
In summa y, ou pilo s udy e eals ha he exp ession o selec ed HF ke a ins unde lies
no el neu oendoc ine con ols, possibly as pa o he un olding hypo halamus-pi ui a y-
hy oid axis equi alen p esen in human skin [4-6,10]. As shown o p olac in [3] and TSH,
human HF o gan cul u e o e s an excellen esea ch ool o u he dissec ”no el”
neu oendoc ine in si u-con ols ha d i e ke a in exp ession in he human sys em unde
clinically ele an condi ions on he gene and p o ein le el.

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Acknowledgemen s
The excellen echnical assis ance o A. Becke and G. Scheel wi h HF o gan cul u e is
g a e ully app ecia ed. We hank E. Gáspá o he c i ical e iew o he manusc ip . This
s udy was suppo ed in pa by a Mine a Fellowship o YR, and by g an s om Manches e
NIHSR Biomedical Resea ch Cen e o RP and om he Wilhelm Sande S i ung o LL
(2007.133.1.).
Re e ences
1. Ramo Y, Paus R, Tiede S, e al. Endoc ine con ols o ke a in exp ession. Bioessays
2009;31:389-99.
2. Langbein L, Schweize J. Ke a ins o he human hai ollicle. In Re Cy ol
2005;243:1-78.
3. Ramo Y, Bí ó T, Tiede S, e al. P olac in - a no el neu oendoc ine egula o o human
ke a in exp ession in si u. FASEB J 2010;24:1768-79.
4. Bodó E, K omminga A, Bi ó T, e al. Human emale hai ollicles a e a di ec ,
nonclassical a ge o hy oid-s imula ing ho mone. J In es De ma ol
2009;129:1126-1139.
5. Gáspá E, Ha denbicke C, Bodó E, e al. Thy o opin eleasing ho mone (TRH): a
new playe in human hai -g ow h con ol. FASEB J 2010;24:393-403.
6. Bodó E, Kany B, Gáspá E, e al. Thy oid s imula ing ho mone (TSH), a no el, locally
p oduced modula o o human epide mal unc ions, is egula ed by hy o opin
eleasing ho mone and hy oid ho mones. Endoc inology 2010;151:1633-42.
7. Cai J, Lee J, Kopan R, e al. Gene ic in e plays be ween Msx2 and Foxn1 a e equi ed
o No ch1 exp ession and hai sha di e en ia ion. De Biol 2009;326:420-30.
8. Ma L, Liu J, Wu T, e al. ‘Cyclic alopecia’ in Msx2 mu an s: de ec s in hai cycling
and hai sha di e en ia ion. De elopmen 2003;130:379-89
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9. an Beek N, Bodó E, K omminga A, e al. Thy oid ho mones di ec ly al e human hai
ollicle unc ions: anagen p olonga ion and s imula ion o bo h hai ma ix
ke a inocy e p oli e a ion and hai pigmen a ion. J Clin Endoc inol Me ab
2008;93:4381-8.
10. Paus R. Explo ing he " hy oid-skin connec ion": concep s, ques ions, and clinical
ele ance. J In es De ma ol 2010;130:7-10.
11. Guo J, Rahman M, Cheng L, e al. Mo phogenesis and main enance o he 3D hymic
medulla and p e en ion o nude skin pheno ype equi e FoxN1 in p e- and pos -na al
K14 epi helium. J Mol Med 2010 No 26 [Epub ahead o p in ]
12. Po e CS, P ue ND, Ke n MJ, e al. The nude mu an gene Foxn1 is a HOXC13
egula o y a ge du ing hai ollicle and nail di e en ia ion. J I es De ma ol 2010
Dec 30 [Epub ahead o p in ]
13. Kiso M, Tanaka S, Saba R, e al. The dis up ion o Sox21-media ed hai sha cu icle
di e en ia ion causes cyclic alopecia in mice. P oc Na l Acad Sci USA
2009;106:9292-7.
14. Moll R, Di o M, Langbein L. The human ke a ins: biology and pa hology. His ochem
Cell Biol 2008;129:705-33.
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Figu e legends
Fig 1. A. Rela i e mRNA exp ession o KRT35, KRT31, KRT32, KRT85 and MSX2 genes
ollowing ea men wi h TSH (100 mU ml-1). This dose was selec ed since i is wi hin he
ange o cus oma ily employed TSH concen a ions in cell cul u e s udies, and because his
dose has been p e iously shown by mic oa ay analysis o egula e he exp ession o se e al
genes in o gan-cul u ed human hai ollicles [4]. Resul s ep esen iplica e de e mina ions o
samples. To al RNA was pooled om 20 HFs. B. Rela i e mRNA exp ession o KRT6,
KRT14 and KRT17 ollowing adminis a ion o TSH o ORS KCs in cul u e, ex ac ed om
HFs o an addi ional emale pa ien . *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; mean ± SEM. PCR
ampli ica ion was ca ied ou by using he TaqMan p ime s and p obes ( ecognizing he
ollowing human genes: Assay ID: Hs01699178_gH o KRT6, Assay ID: Hs00265033_m1
o KRT14, Assay ID: Hs00356958_m1 o KRT17, Assay ID: Hs00605539_m1 o KRT31,
Assay ID: Hs00605543_g1 o KRT32, Assay ID: Hs00605557_g1 o KRT35, Assay ID:
Hs00158558_m1 o KRT85, Assay ID: Hs00741177_m1 o MSX2) using he TaqMan
Uni e sal PCR Mas e Mix P o ocol (Applied Biosys ems). As in e nal con ols, ansc ip s o
glyce aldehyde 3-phospha e dehyd ogenase (GAPDH) we e de e mined (Assay ID:
Hs99999905_m1 o human GAPDH).
Fig. 2. TSH (100 mU ml-1) down egula es immuno eac i i y o K31 (A), K85 (B), K32 (C),
MSX-2 (D), K6 (E), K14 (F) and K17 (G) in mic odissec ed, o gan-cul u ed no mal, human
scalp skin HFs, a e 6 days o adminis a ion. Densi ome ic measu emen s o s aining
in ensi ies in de ined e e ence a eas (quan i a i e immunohis omo phome y) we e
pe o med using ImageJ so wa e (NIH, Be hesda, MD, USA; h p:// sbweb. nih.go /ij/) as
desc ibed p e iously [3,4]. Columns ep esen means±SEM ; n=15–18 HFs/g oup; cumula i e
esul s o wo di e en expe imen s. ***P<0.001.
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TABLE 1. P ima y an ibodies used
Name
Hos
Dilu ion
Posi i e con ol
Me hod
Sou ce
Clone
Ke a in 6
Mouse
1:10
Sup abasal laye s o he ORS; sup abasal laye s o
wounded skin [2,14]
Indi ec
IF
PROGEN, Heidelbe g, Ge many
Ks6.KA12
Ke a in
14
Mouse
1:50
Skin epide mis, basal laye ; basal and
sup abasal laye s o he ORS [2,14]
Indi ec
IF
Sigma-Ald ich, Tau ki chen,
Ge many
CKB1
Ke a in
17
Mouse
1:50
Sup abasal laye s o he ORS [14]
Indi ec
IF
PROGEN, Heidelbe g, Ge many
Ks17.E3
Ke a in
31
Guinea
Pig
1:7000
P eco ex egion [2]
Indi ec
IF
Lu z Langbein, DKFZ,
Heidelbe g, Ge many
hHa1
p o .1
Ke a in
32
Guinea
Pig
1:2000
Hai cu icle [10,14]
Indi ec
IF
Lu z Langbein, DKFZ,
Heidelbe g, Ge many
Ha2.1
Ke a in
85
Guinea
Pig
1:1000
Hai ma ix, co ex and cu icle [14]
Indi ec
IF
Lu z Langbein, DKFZ,
Heidelbe g, Ge many
hHb 5co.2
Msx-2
Goa
1:100
Hai ma ix and co ex [8]
Indi ec
IF
San ac uz, CA, USA
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