Transcriptional Upregulation of Nrf2-Dependent Phase II Detoxification Genes in the Involved Epidermis of Vitiligo Vulgaris

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1 ORIGIL ARTICLE Trnsriptionl Upregultion of Nrf-Depenent Phse II Detoxifition Genes in the Involve Epiermis of Vitiligo Vulgris Vivek T. Ntrjn 1, Arhn Singh, Avinsh A. Kumr, Pnkj Shrm 3, Hemnt K. Kr 3, Lurent rrot, Jen-Roh eunier, Krishnmurthy Ntrjn, Rjni Rni n Rjesh S. Gokhle 1,, Oxitive stress is wiely elieve to e ontriuting ftor in vitiligo pthogenesis. To explore mehnisms y whih epiermis respons to mounting oxitive stress, we investigte the involvement of phse II etoxifition genes in vitiligo. Phse II etoxifition pthwys hve reently een ientifie s eing import the regultion of epierml skin homeostsis. In this stuy we show tht the key trnsription ftor nuler ftor E relte ftor (Nrf) n the ownstrem genes D(P)H:quinone oxise-1 (), g-glutmyl ystine ligse tlyti suunit (), n g-glutmyl ystine ligse moifying suunit () re upregulte in the lesionl epierml skin of sujets with vitiligo vulgris. The ifferenes etween lesionl n nonlesionl skin were further investigte y stuying the inue expression of Nrf-epenent trnsripts in skin punh iopsies using urumin n sntlol. Surprisingly, nonlesionl skin showe inution of ll trnsripts while similr effet ws not oserve for the skin punhes from the lesionl skin. The use of urumin n sntlol on epierml ells showe tht kertinoytes were more suseptile to poptosis, wheres melnoytes inue phse II genes uner the sme onentrtions with negligile poptosis. Our stuies provie new insights into the role of phse II etoxifition pthwy in mintining skin homeostsis n sustining reox lne in vitiligo ptients. Journl of Investigtive Dermtology (1) 13, 71 79; oi:1.13/ji.1.1; pulishe online July 1 INTRODUCTION Vitiligo is epigmenting isorer of humn skin tht is hrterize y lolize loss of melnin from the lesionl epiermis (Lei et l., ; Spritz, ; Shllreuter et l., ). It is ler tht melnoytes the melnin-synthesizing ells of the skin re sustntilly erese in vitiligo lesions (Toin et l., ). Severl theories out the etiology of this omplex isorer hve een propose. These inlue geneti suseptiility (Spritz, ), utoimmunity (Le Poole n Luiten, ), impire melnoyte migrtion n/or prolifertion (Guthier et l., 3), n oxitive stress (Nmzi, 7; Shllreuter et l., ; Kovs et l., 9). 1 Institute of Genomis n Integrtive Biology (CSIR), Delhi, Ini; Ntionl Institute of Immunology, New Delhi, Ini; 3 Deprtment of Dermtology, Dr Rm nohr Lohi Hospitl, New Delhi, Ini; Deprtment of Sfety Reserh, Phototoxiity Unit, L OREAL Reserh, Aulny-Sous-Bois, rne; Shool of Life Sienes, Jwhrll Nehru University, New Delhi, Ini n Jwhrll Nehru Centre for Avne Sientifi Reserh, Bnglore, Ini Corresponene: Rjesh S. Gokhle, Institute of Genomis n Integrtive Biology, CSIR, Systems Biology Group, ll Ro, Delhi 117, Ini. E-mil: rsg@igi.res.in Rjni Rni, Ntionl Institute of Immunology, Arun Asf Ali rg, New Delhi 117, Ini. E-mil: rjni@nii.res.in Arevitions:, g-glutmyl ystine ligse tlyti suunit;, g-glutmyl ystine ligse moultory suunit;, heme oxygense-1; Nrf, nuler ftor E relte ftor ;, D(P)H:quinone oxise-1 Reeive Otoer 9; revise 9 April 1; epte y 1; pulishe online July 1 Although the ext mehnism is fr from ler, ttempts hve reently een me to evelop umultive theory to explin the origin of the isorer (Dell nn n Piro, ; Westerhof n Ishi, 7). In this ontext, ientifition of eregulte pthwys in vitiligo woul enle etter unerstning of the unerlying pthophysiology. Skin is the mjor trget of insult for ro spetrum of hemil pollutnts n UV rys (Brenner n Hering, ). These toxi ftors inue, iretly or iniretly, the proution of retive oxygen speies. Retive oxygen speies inlue singlet oxygen, superoxie nions, hyroxyl rils, n H O, ll of whih re short-live entities ontinuously generte t low levels uring the ourse of norml eroi metolism (Zou n Bnerjee, ). The inility of skin to regulte the levels of these speies results in oxitive stress. The rekown of this homeostsis efense mehnism in the skin n result in moifition of proteins, lipis, n D, whih in turn n ring out severl ermtologil iseses. To omt this ellulr mge, mmmls hve evolve elorte etoxifition mhinery ssoite with the phse I, II, n III lsses of enzymes. The phse II etoxifition enzymes re ruil, s they re iretly involve in etoxifition retions of oxitive stress response (Tirumli et l., ). The key trnsription ftor tht inues phse II gene expression is nuler ftor E relte ftor (Nrf), n it funtions y & 1 The Soiety for Investigtive Dermtology 71

2 VT Ntrjn et l. Homeostsis of Oxitive Stress in Vitiligo ining to the ntioxint response element prese the promoter of mny phse II genes (otohshi n Ymmoto, ; Kensler et l., 7). One of the import vivo regultory mehnisms involves formtion of the Nrf-Kep1 omplex, whih is known to regulte ellulr efense mehnisms suh s oxitive stress (Wkyshi et l., 3; Nguyen et l., 9). Reent stuies hve ientifie n importnt role for the Nrf pthwy in mintining skin homeostsis in epiermis (rrot et l., ). This pthwy opertes in oth melnoytes n kertinoytes, enling skin to pt to vrious environmentl stresses. Genetilly, Nrf promoter polymorphisms hve een ssoite with n inrese risk of eveloping vitiligo (Zhou et l., ; Gun et l., ). Altere ellulr loliztion of Nrf in lesionl vitiligo skin hs lso een reporte (Gun et l., ). Interestingly, vitiligo ptients seem to exhiit systemi s well s lolize oxitive stress (Pssi et l., 199; Rokos et l., ), n millimolr levels of H O re reporte to e prese their epiermis (Shllreuter, 1999; Shlf et l., ). Severl ntioxint proteins hve een shown to e ltere in ptient groups (Ines et l., ; Arin n Kuruts, ), n metoli hnges in the skin inue y H O hve lso een reporte (Shllreuter et l., ; Spener et l., 7). A linil stuy suggeste tht nrrow-n UVB-tivte pseuotlse ontrols H O levels n thus might e effetive for tretment of hilhoo vitiligo (Shllreuter et l., ). However, it is urrently not ler whether Nrf-regulte phse II etoxifition mhinery hs n importnt role in vitiligo etiopthogenesis. In this stuy, we exmine the integrity of the phse II etoxifition pthwy in the epiermis of vitiligo ptients. We foun tht the trnsript levels of Nrf s well s the ownstrem etoxifition genes D(P)H:quinone oxise (), g-glutmyl ystine ligse tlyti suunit (), n g-glutmyl ystine ligse moultory suunit () re upregulte in the lesionl epiermis ompre with the mthe nonlesionl skin. urthermore, we evlute the regultion of phse II genes in the punh iopsies from vitiligo ptients s well s in ulture melnoytes n kertinoytes using the eletrophili ompouns urumin n sntlol. Our stuy revele ritil role of the phse II etoxifition pthwy in vitiligo n suggests tht therpeuti intervention of this pthwy my e useful in mngement of the isorer. RESULTS Expression nlyses of phse II etoxifition pthwy genes To stuy the etoxifition pthwy in vitiligo, nonlesionl n lesionl skin iopsies were ollete from vitiligo vulgris ptients fter written informe onsent n institutionl ethil ommittee lerne h een otine. The levels of Nrf n phse II etoxifition gene trnsripts were ompre ross the lesionl n nonlesionl epiermis of vitiligo ptients, n this ws onsiere mesure of ellulr response to the previling oxitive stress. Totl R ws isolte from the lesionl n nonlesionl epiermis. Quntittive rel-time PCR nlysis ws rrie out to exmine the expression of heme oxygense-1 (),,, n trnsripts. Tqn proes use for the experiment were esigne t the exon intron ounry n hve een vlite to e mr speifi (rrot et l., ). Our preliminry nlysis showe tht -tin mr ws ifferentilly expresse in vitiligo s ompre with nonlesionl skin, n therefore 1S riosoml R (rr) ws use s the normliztion ontrol in uplex Tqn ssys. Quntittive PCR nlysis ws rrie out for eh smple in uplite. Replites tht iffere y n SD of X. were eliminte from the nlysis. The DC t vlue ws efine s the ifferene etween the C t vlue for the gene of interest n tht for the enogenous ontrol 1S rr. The DC t vlues of nonlesionl n lesionl skin for eh of the ptient smples were plotte s olumn stter plot (igure 1). ein DC t vlues were signifintly lower for the expression of,, n in vitiliginous skin s ompre with nonlesionl skin (P-vlue o.1, nn Whitney test). Interestingly, there ws no signifint ifferene in the mr levels etween nonlesionl n lesionl skin in this ohort of vitiligo vulgris ptients. Pire Stuent s t-test for the mthe smples of nonlesionl n lesionl skin lso showe signifint trens (P-vlue o.1), onfirming upregultion of etoxifition genes in the lesionl skin. To unerstn the signifine of this upregultion, we lso investigte the expression levels in norml n foreskin epiermis (igure 1 n ). The DC t vlues etween lesionl n nonlesionl smples n e reily ompre s these were otine from the sme sujets. However, the sme riterion nnot e pplie to the foreskin n norml skin smples. With our limite t, it is tempting to speulte tht the foreskin hs higher expression of phse II genes thn norml skin oes. We lulte the fol hnge in expression of eh mthe pir of nonlesionl n lesionl skin. The lesionl skin of most of the iniviuls h elevte expression of,, n genes (igure 1 f, Tle 1), wheres only 3 of iniviuls showe remrkle elevte expression of the gene (igure 1g). Although is n importnt element of the ntioxint response, its regultion hs een shown to e more omplex thn tht of other phse II genes (Bglole et l., ; Numt et l., 9). The n proteins intert to form the funtionl g-glutmyl ystine ligse enzyme, n, oringly, the elevte n levels in the lesionl skin ssume funtionl signifine. The t suggest efinite involvement of, s it ws upregulte in the lesionl skin in lmost ll sujets with fol hnge of.. Expression of Nrf in the lesionl n nonlesionl epiermis Given tht Nrf is the upstrem trnsription ftor regulting the inution of phse II genes upon oxitive stress, we exmine the levels of Nrf mr in skin punh smples from vitiligo ptients. The DC t vlues showe istintly higher expression of Nrf in the lesionl skin s ompre with the nonlesionl skin (igure ). The reltive Nrf levels vry etween two- n eightfol in ifferent smples (igure ). The trnsript of Nrf ws lso stuie in the 7 Journl of Investigtive Dermtology (1), Volume 13

3 VT Ntrjn et l. Homeostsis of Oxitive Stress in Vitiligo 3 ** ** ** ns Non-lesionl Lesionl Non-lesionl Lesionl Non-lesionl Lesionl Non-lesionl Lesionl 3 oreskin 3 Norml skin ol hnge (lesionl/non-lesionl) f ol hnge (lesionl/non-lesionl) VD1 VD1 HQ-1 HQ-1 VD1 VD VD VD3 VD VD VD VD1 VD VD VD3 VD VD VD e ol hnge (lesionl/non-lesionl) g ol hnge (lesionl/non-lesionl) 1 1 VD1 VD1 VD1 VD VD VD3 VD1 VD VD VD3 VD igure 1. Levels of phse II etoxifition gene trnsripts re ltere in the lesionl epiermis s ompre with the nonlesionl epiermis. () Rel-time PCR nlyses of,,, n rrie out using uplex ssys were normlize to 1S riosoml R (rr) n represente s olumn stter plot of DC t vlues for oth nonlesionl n lesionl skin. The horizontl line shows the mein (n ¼ ). **Signifint P-vlue (o.1), s lulte using the nn Whitney test. DC t vlues for phse II genes in () foreskin n () norml epiermis. ol hnge with respet to orresponing nonlesionl skin for eh vitiligo ptient is represente s r grph. (), (e), (f), n (g). ol hnge ws lulte using DDCt, where DDC t represents DC t of lesionl DC t of nonlesionl skin., g-glutmyl ystine ligse tlyti suunit ;, g-glutmyl ystine ligse moultory suunit ;, heme oxygense-1;, D(P)H:quinone oxise-1. VD VD VD VD VD norml skin n foreskin smples, s shown in igure. We further exmine the protein expression of Nrf in the epiermis using immunohistohemistry on the skin setions erive from the foreskin n the nonlesionl n lesionl skin (igure ). Interestingly, lthough the istriution of Nrf in the nonlesionl skin is uniform, the lesionl skin showe Nrf istriution in pthes. Effet of urumin n sntlol on humn melnoytes n kertinoytes Enhne levels of phse II genes inite isrupte reox homeostsis in the vitiligo epiermis. We eie to explore this further y investigting the effet of inue oxitive stress response in ulture melnoytes n kertinoytes. Two ommon skin-re ompouns urumin n sntlol were hosen for this stuy. Curumin possesses two phenoli rings onnete y two,-unsturte ronyl groups (igure 3). Sntlol onsists of iyli ring system ovlently linke to n liphti unsturte hin ontining hyroxyl funtionl group (igure 3). Both ompouns re eletrophili n similr to other known phse II gene inuers, inluing utylte hyroxynisol, tert-utylhyroquinone, green te polyphenol, ( )-epitehin-3-gllte, n isothioyntes suh s sulforphne (Rushmore et l., 199). Curumin hs previously een shown to tivte Nrf in severl ell types (Jeong et l., 9; Yng et l., 9). 73

4 VT Ntrjn et l. Homeostsis of Oxitive Stress in Vitiligo Tle 1. Detils of vitiligo vulgris ptients reruite for the stuy long with the het mp showing regultion of phse II etoxifition genes in lesionl s ompre with nonlesionl epiermis Levels of phse II gene trnsripts in vitiligenous epiermis wrt nonlesionl ID Age (yers) Sex Onset ge (yers) PUVA tretment 1 Involve re 1 1 Thigh VD1 No Shin VD Hn, elow 1 9 Yes Hn VD 7 Yes Leg Yes Leg VD 3 Nek 1 No Leg, feet No Leg 1 Yes Wrist VD3 1 orerm 1 Knee VD 9 Leg VD 1 Shin VD 1 No Ankle Arevition:, not ville. 1 History of psorlen n UVA light therpy. Log1 of fol ifferene etween involve n uninvolve regions plotte s het mp. Key to het mp Up Down Not regulte. 17. **. 17. Non-lesionl 1 μm Non-lesionl ol hnge (lesionl/non-lesionl) Lesionl 1. Nrf oreskin Norml VD VD VD VD1 VD1 VD3 VD VD Lesionl oreskin igure. Anlysis of nuler ftor E relte ftor (Nrf) mr levels in nonlesionl n lesionl epiermis. () DC t vlues for Nrf etermine y rel-time PCR in the nonlesionl n the lesionl epiermis of vitiligo vulgris ptients. **Signifint P-vlue (o.), s lulte using the nn Whitney test. () Chnges in the expression levels of Nrf etween the lesionl skin n the orresponing nonlesionl skin represente s fol hnge for eh vitiligo ptient. () DC t vlues for Nrf etermine y rel-time PCR in the foreskin n norml epiermis. () Immunohistohemil nlysis of Nrf levels in nonlesionl n lesionl skin setions n omprison with foreskin. Nrf ws stine rown using rit Nrf ntioy n evelope. Sle r ¼ 1 mm. 1 μm 1 μm 7 Journl of Investigtive Dermtology (1), Volume 13

5 VT Ntrjn et l. Homeostsis of Oxitive Stress in Vitiligo O O CH CH 3 HO OCH 3 Curumin OCH 3 OH H 3 C Sntlol HO e % Cell viility g % Cell viility 1 1 Kertinoytes Curumin (μ) Sntlol (μ) f % Cell viility h % Cell viility elnoytes Curumin (μ) Sntlol (μ) igure 3. Cell viility of melnoytes n kertinoytes upon urumin n sntlol tretment. Chemil strutures of () urumin n () sntlol. Brightfiel imges of ulture () kertinoytes n () melnoytes efore tretment; sle r ¼ 1 mm. Cell viility mesure olorimetrilly using the methylthizolyliphenyl-tetrzolium romie (TT) ssy t vrious onentrtions of urumin (e) in kertinoytes n (f) in melnoytes n of sntlol (g) in kertinoytes n (h) in melnoytes. Cells were trete with inite onentrtions of the ompouns for hours followe y the ition of TT for the lst 3 hours, n the purple formzn ws issolve in DSO. Kertinoyte n melnoyte ultures were estlishe from foreskins erive from helthy onors (igure 3 n ). TT (methylthizolyliphenyl-tetrzolium romie) ssys were performe to etermine the optiml onentrtion of the ompouns, n two onentrtions were hosen on the sis of ifferenes in the viility of these two ell types (igure 3e h). There re initions in the literture to suggest tht ells in the erly poptoti phse re vile n thus re lso positive in the TT ssy (Gomez-Lehon et l., ). Apoptosis ssys were performe on melnoytes n kertinoytes with 1. n m urumin n n 1 m sntlol inute for hours. To ssess the extent of ell eth inue y these two ompouns, ell viility ws etermine vi propiium ioie (PI) n nnexin V stining followe y flow ytometry. The perentge of ells unergoing poptosis (PI n nnexin V oule positive) ws oserve to e higher in kertinoytes thn in melnoytes (igure ). Even t low onentrtions of urumin (1. m) n sntlol ( m), pproximtely % of the kertinoytes were live, wheres t the sme onentrtions in melnoytes the extent of ell eth ws negligile (igure ). Although oth these ell types were epiermlly erive, the oserve responses to hemils were strikingly ifferent. To ssess the expression levels of Nrf n phse II etoxifition genes, ells t % onfluene were trete with the hosen onentrtions of urumin n sntlol for hours. The Nrf mr levels showe inue expression in melnoytes, n no signifint hnge oul e oserve in kertinoytes (igure ). or the etoxifition genes, showe inrese levels in kertinoytes on tretment with urumin n sntlol (igure ). In ontrst, melnoytes showe n inrese in ll fonsript levels 7

6 VT Ntrjn et l. Homeostsis of Oxitive Stress in Vitiligo Tretment % Apoptoti ells Kertinoytes (men ± S.E.) elnoytes (men ± S.E.) 19. ± ±. Curumin (1. μ) 7. ± ± 3.9 Curumin ( μ) 9.3 ± 1..9 ±. DSO ontrol 1. μ urumin μ urumin μ sntlol 1 μ sntlol Sntlol ( μ) Sntlol (1 μ).7 ± ± ± ±. Propiium ioie DSO ontrol Annexin V-ITC 1. μ urumin μ urumin μ sntlol 1 μ sntlol ol expression 3 1 Nrf Curumin 1. μ Curumin μ Sntlol μ Sntlol 1 μ Kertinoyte elnoyte ol expression Phse II genes in kertinoytes e ol expression Phse II genes in melnoytes Ctrl Snt μ Snt 1 m Ctrl Snt μ Snt 1 m Ctrl Snt μ Snt 1 m Ctrl Snt μ Snt 1 m Snt μ Snt 1 m Snt μ Snt 1 m Snt μ Snt 1 m Snt μ Snt 1 m igure. Curumin n sntlol lter nuler ftor E relte ftor (Nrf) n phse II etoxifition trnsripts in ulture humn melnoytes n kertinoytes n their effet on poptosis. Representtive flow ytometri ensity plots of urumin- n sntlol-trete () kertinoytes (top) n melnoytes (ottom) re given. In eh pnel the lower left qurnt represents unstine live ells, n the lower right n upper right qurnts show erly (nnexin V-positive ells) n lte (nnexin V propiium ioie (PI)-positive) poptoti ells, respetively. Upper left qurnt represents PI-positive neroti ells. The inset in the upper left qurnt shows the perentges of ells flling in eh qurnt. () Perentge of totl poptoti ells etete (men of sum of erly n lte poptoti ells of four smples eh) for inite onentrtions of urumin n sntlol. () Nrf levels mesure y rel-time PCR for eh onentrtion of urumin n sntlol in kertinoytes n melnoytes. Effet of urumin (1. n m) n sntlol ( n 1 m) on the levels of phse II etoxifition trnsripts in () kertinoytes n (e) melnoytes. Error rs represent men±se ross five inepenent ultures (n ¼ ). (igure e). It is tempting to speulte tht kertinoytes suum to mounting levels of oxitive stress euse of their inility to inue Nrf. elnoytes, y virtue of elevte levels of Nrf n lso the phse II trnsripts, resiste poptosis inue y urumin n sntlol. Effet of urumin n sntlol on lesionl n nonlesionl epiermis Stuies on ulture ells in the presene of urumin n sntlol hve shown tht oth Nrf n phse II etoxifition genes oul e inue ifferentilly in the two epiermis ell types. We therefore eie to investigte the overll oxitive homeostsis in epiermis using these two eletrophili regents. or this experiment we use orgn ulture of intt skin iopsies (Lteef et l., ). The iopsies were isinfete with 7% ethnol, rinse with ntiiotis, n ple in meium ontining urumin or sntlol for hours. These experiments were performe with 1. m urumin n m sntlol. Uner these onitions the expression of severl housekeeping genes ws minimlly perture. In the nonlesionl skin, oth urumin n sntlol use n inrese in the levels of ll four phse II trnsripts (igure ), lthough the hnge in seems to e mrginl. Interestingly, in the lesionl skin these ompouns i not show n nlogous inrese in the levels of trnsripts (igure ). It seems tht the elevte sl levels of phse II trnsripts re not further ltere y these ompouns. The foreskin lso i not show orresponing hnges in the trnsripts s oserve for the nonlesionl skin (igure ). Corroorting these oservtions, orresponing hnges in the Nrf levels were lso oserve in the nonlesionl skin (igure ). Immunohistohemil stining of melnoytes in the nonlesionl n lesionl skin shows the ifferenes in melnoyte numers for the two mthe skins (igure e n f). Our results thus strongly suggest tht the ellulr physiology of lesionl epiermis is inee perture in omprison with nonlesionl skin. 7 Journl of Investigtive Dermtology (1), Volume 13

7 VT Ntrjn et l. Homeostsis of Oxitive Stress in Vitiligo ol expression Lesionl skin C C lol C lo l l C ol o C n l ol e Nrf C C lol l C ol l C ol l ol oreskin C C lo l l C ol S umol C lol o C n l ol ol expression Non-lesionl skin ol expression Non-lesionl skin f Lesionl skin ol expression Curumin Sntlol μm Non-lesionl Vitiligo 1 μm oreskin igure. Effet of urumin n sntlol on whole epiermis. () Nonlesionl n () lesionl vitiligo skin iopsies n () foreskin were ulture in the presene of 1. m urumin n m sntlol for hours, n the levels of phse II etoxifition trnsripts were quntitte y rel-time PCR in the epiermis. Error rs represent men±se ross four iniviuls. Dotte line represents twofol upregultion threshol, whih is onsiere signifint. () ol hnges in the nuler ftor E relte ftor (Nrf) levels upon urumin n sntlol tretme nonlesionl n lesionl skin of vitiligo ptient n in foreskin. Immunohistohemistry using S-1 ntioy for eteting melnoytes in the (e) nonlesionl n (f) lesionl skin of the vitiligo ptient. Drk re stine ells re S1-positive melnoytes. Sle r ¼ 1 mm. DISCUSSION Oxitive stress hs signifint role in the inution of severl inflmmtory skin iseses (Bikers n Athr, ). Inrese oxitive stress hs lso een implite in the etiology of epigmente pthes in vitiligo (Pssi et l., 199; Shllreuter, 1999). The trnsription ftor Nrf is known to regulte the expression of network of ytoprotetive enzymes, resulting in protetion ginst toxiity fter exposure to oxitive stress or vrious eletrophili n oxitive hemils. In this stuy we esrie the trnsriptionl tivtion of Nrf s well s ownstrem phse II etoxifition genes from the lesionl regions. Comprtive nlysis etween lesionl n nonlesionl skin from the sme iniviuls ross severl sujets showe upregultion of,, n genes in lesionl skin. Interestingly, the expression of, nother phse II etoxifition response gene, is not ltere in the mjority of iniviuls. Inee, severl stuies hve reporte tht regultion my e more omplex (Zhng et l., ; Bglole et l., ). Aross ll sujets, the lesionl level of Nrf ws lso onsistently higher thn in the mthe nonlesions. Interestingly, uner sl onitions Nrf is oun to Kep1 in the ytoplsm, n the issoition of this omplex is ritil to the tivtion of Nrf. In ertin instnes, mr regultion of Nrf hs lso een emonstrte (Gong n Ceerum, ), n Nrf is propose to utoregulte its own expression through n ntioxint response element like element lote in the proximl region of its promoter, leing to persistent nuler umultion of Nrf n protrte inution of phse II genes (io et l., ). In this ontext, onsistent upregultion of Nrf mr n phse II etoxifition genes in lesionl skin gins relevne. We lso inepenently oserve tht the levels of Nrf mr n of the etoxifition genes seem to e higher in foreskin smples thn in norml skin, se on t from out five smples. Although the norml skin n foreskin smples were from unmthe iniviuls, the onsistent pttern of high Nrf expression n lk of inution of phse II genes suggests tht the foreskin my e uner high oxitive stress. Stuies using kertinoytes from foreskin hve shown n ugmente innte immune response owing to the presene of miroflor (Chung n Dle, ). This oul e reson for the inrese Nrf n phse II genes oserve in this skin type. or n, the mein levels in nonlesionl skin were seemingly higher thn in norml epiermis from iniviuls not suffering from vitiligo. Although the numer of norml smples nlyze ws smll, this pttern suggests tht the nonlesionl skin ws lso uner oxitive stress, lthough t lower level thn in the orresponing lesionl skin. Our results showe tht the nonlesionl skin ws le to tively respon to the eletrophili ompouns urumin n sntlol n inue lssi Nrf-epenent ntioxint response. The lesionl punh smples, however, i not 77

8 VT Ntrjn et l. Homeostsis of Oxitive Stress in Vitiligo respon in n nlogous mnner. We suggest tht this ws ue to the elevte levels of phse II genes in the lesionl skin. The effets of urumin n sntlol on kertinoytes n melnoytes in ulture highlight the ifferentil responses etween the two ell types. Wheres kertinoytes o not respon to the ompouns y elevting Nrf n phse II genes n eventully suum to poptosis, melnoytes upregulte the Nrf pthwy n re well protete. This suggests n integrte role of Nrf-epenent phse II genes in oxitive stress inue poptosis. Although the ells provie suitle moel for stuying skin homeostsis, the ler-ut ifferenes with the skin punh suggest omplex ehvior of this tissue. Thus, our stuies using skin punh iopsies provie n lterntive orgn moel n revele oxitive stress regultion in vitiligo. The roles of Nrf-epenent pthwys hve egun to emerge from vrious moel systems, rnging from pulmonry isorers to ner. The enefiil effet of enhne Nrf effetors seems to e the prime protetive funtion onferre y this pthwy on tissue homeostsis. Aprt from the protetive response, it is noteworthy tht the onsequenes of this inrese response of phse II genes oul e in sustining the epigmente lesions in vitiligo pthes. Although oxitive stress in lesionl n nonlesionl skin hs een reporte previously, our stuy emonstrtes the involvement of Nrf n phse II genes uring skin homeostsis in vitiligo. We lso showe tht nonlesionl pigmente skin from vitiligo ptients oes not unergo the sme level of oxitive stress. Thus, therpeuti intervention in this ltere oxitive stress pthwy my hve signifint relevne in the mngement n tretment of vitiligo. ATERIALS AND ETHODS Skin punhes use for the experiments were tken from ptients fter otining informe written onsent; the proeure is elorte in the Supplementry ethos online. The proeures followe were pprove y the institutionl humn ethis ommittee t the Ntionl Institute of Immunology n Dr Rm nohr t Lohi Hospitl, n were in greement with the Delrtion of Helsinki Priniples. Rel-time PCR for phse II etoxifition gene trnsripts Gene expression nlysis ws rrie out using the ABI Prism 7 rel-time PCR system (Applie Biosystems, oster City, CA). Retions (1 ml) were rrie out in replites using Tqn universl PCR mster mix (Applie Biosystems). A- n TARAlele Tqn proes use in the stuy were Hs79_m1 (), Hs7_m1 (), Hs9_m1 (), n Hs79_m1 (); normliztion ws rrie out using 1S rr proes lele with VIC n GB 31913E-713. or Nrf, the primer sequenes use were -GCTCATACTCTTT CCGTCGC-3 n -ATCATGATGGACTTGGAGC-3 n for 1S rr, -CGAAAGCATTTGCCAAGAAT-3 n -AGTCGGCATCG TTTATGGTC-3 were use. Rel time PCR ws rrie out using the SYBR Green (erments Intl, Ontrio, Cn) metho. All quntittive PCR retions were initite t 1C for minutes, 9 1C for 1 minutes, followe y yling onition of 9 1C for. minutes n 1C for 1 minute for yles. The t were nlyze using SDS 1. sequene-etetion softwre (Applie Biosystems), n C t vlues were otine. Dt nlysis or omprison, the C t metho (DC t vlue) ws use. C t vlues were normlize to their orresponing 1S rr levels to otin DC t vlues. The DC t vlues were lulte y sutrting the vlue of 1S R from tht of the gene of interest. The retions were performe in replites, n DC t vlues tht iffere y n SD of X. etween the replites were eliminte from further nlysis. Comprison of the mein DC t vlues ws rrie out using the nn Whitney test. ol hnge ws lulte using DDCt,whereDDC t represents DC t of vitiligo DC t of nonlesionl skin. Chnges in expression level were lulte s fol hnge in the level of trnsript etween nonlesionl n lesionl skin n normlize to 1S rr levels. Pire Stuent s t-test ws performe to stuy the signifine of the DC t vlue pirs. All sttistil nlyses performe in this stuy were rrie out using Grph-P Prism softwre (Sn Diego, CA). Apoptosis ssy At hours fter the foresi tretment, oth floting n herent ells were hrveste n stine with nnexin V ITC n PI s esrie y the mnufturer (Annexin V ITC etetion kit, Promokine, Heielerg, Germny). In rief, ml of nnexin V ITC n ml of PI were e to ells fter wshing n resuspension in ml of ining uffer. The mixture ws inute in the rk for minutes t room temperture. ITC n PI fluoresene ws mesure using BD LSR low ytometer (BD Biosienes, rnklin Lke, NJ). Approximtely 3, ells were nlyze for eh smple. All t nlysis ws performe using WinDI version.9 softwre (Joseph Totter, Sripps Reserh Institute, Sn Diego, CA). Immunohistohemistry for Nrf n S1 Immunohistohemistry ws performe using stnr protools. Briefly, skin iopsies were fixe with 1% formlin, ehyrte, n emee in prffin. Setions ( mm thik) were ple on polylysine-ote slies, eprffinize, hyrte, ntigen retrieve with 1 m itrte uffer ph., n loke with % norml got serum. Slies were stine with rit monolonl ntioies to Nrf (ilution 1:1; Epitomis, Burlingme, CA) n S1 (ilution 1:1; Dko Cytomtion, Glostrup, Denmrk) for minutes t room temperture. Slies were wshe n inute with lele polymer lkline phosphtse (Dko Cytomtion) for 3 minutes t room temperture. Retion ws evelope with fst re for Nrf stining n with fuhsin for S1 stining for minutes eh, n oth were ounterstine with hemtoxylin. CONLICT O INTEREST The uthors stte no onflit of interest. ACKNOWLEDGENTS RSG is the reipient of Swrnjynti ellowship from the Deprtment of Siene n Tehnology n is lso supporte y Tt Innovtive ellowship from the Deprtment of Biotehnology, Ini. This work ws supporte y grnts for the Progrm Support for skin pigmenttion n melnoyte-kertinoyte iology from the Deprtment of Biotehnology, Ini. AAK is Junior Reserh ellow of the Counil of Sientifi n Inustril Reserh, Ini. We thnk Trun Chopr for his initil help in ulturing melnoytes from the epiermis n Chnrim Shh for helping in IHC stining. 7 Journl of Investigtive Dermtology (1), Volume 13

9 VT Ntrjn et l. Homeostsis of Oxitive Stress in Vitiligo SUPPLEENTARY ATERIAL Supplementry mteril is linke to the online version of the pper t REERENCES Arin O, Kuruts EB () Oxitive stress in the loo of ptients with tive lolize vitiligo. At Dermtovenerol Alp Pnoni Arit 17:1 Bglole CJ, Sime PJ, Phipps RP () Cigrette smoke-inue expression of heme oxygense-1 in humn lung firolsts is regulte y intrellulr glutthione. Am J Physiol Lung Cell ol Physiol 9:L 3 Bikers DR, Athr () Oxitive stress in the pthogenesis of skin isese. J Invest Dermtol 1: 7 Brenner, Hering VJ () The protetive role of melnin ginst UV mge in humn skin. Photohem Photoiol :39 9 Chung WO, Dle BA () Innte immune response of orl n foreskin kertinoytes: utiliztion of ifferent signling pthwys y vrious teril speies. Infet Immun 7:3 Dell nn L, Piro () A review n new hypothesis for non-immunologil pthogeneti mehnisms in vitiligo. Pigment Cell Res 19: 11 Guthier Y, Crio Anre, Tie A (3) A ritil pprisl of vitiligo etiologi theories. 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() BRG1 interts with Nrf to seletively meite inution in response to oxitive stress. ol Cell Biol :79 Zhou N, Xu AE, Lu LJ et l. () [Assoition of single nuleotie polymorphisms of Nrf promoter region with suseptiility to vitiligo]. Zhonghu Yi Xue Z Zhi :99 7 Zou CG, Bnerjee R () Homoysteine n reox signling. Antioxi Reox Signl 7: