TSH is a novel neuroendocrine regulator of selected keratins in the human hair follicle

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 3 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). 4 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 5 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. 6 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 7 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. 8 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. 9 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 -