Retinoid Metabolism Is Altered in Human and Mouse Cicatricial Alopecia

Transcription

1 See relted ommentry on pg 285 ORIGIL ARTICLE Retinoid Metolism Is Altered in Humn nd Mouse Citriil Alopei Helen B. Everts 1, Kthleen A. Silv 2,ShliseMontgomery 1, Liye Suo 1, Moni Menser 1, Amy S. Vlet 3, Lloyd E. King 3, Dvid E. Ong 4 ndjohnp.sunderg 2,3 C57BL/6 mie develop dermtitis nd srring lopei resemling humn itriil lopeis (CAs), prtiulrly the entrl entrifugl CA (CCCA) type. To evlute the role of retinoids in CA, the expression of retinoid metolism omponents were exmined in these mie with mild, moderte, or severe CA ompred with hir yle-mthed mie with no disese. Two feeding studies were onduted with dms fed either NIH 31 diet (study 1) or AIN93G diet (study 2). Adult mie were fed AIN93M diet with 4 (reommended), 28, or 56 IU vitmin Ag 1 diet. Feeding the AIN93M diet to dults inresed CA frequeny over NIH 31 fed mie. Inresed folliulr dystrophy ws seen in study 1 nd inresed derml srs in study 2 in mie fed the 28 IU diet. These results indite tht retinoid metolism is ltered in CA in C57BL/6J mie tht require preise levels of dietry vitmin A. Humn ptients with CCCA, pseudopelde (end-stge srring), nd ontrols with no lopei were lso studied. Mny retinoid metolism proteins were inresed in mild CCCA, ut were undetetle in pseudopelde. Studies to determine whether these dietry ltertions in retinoid metolism seen in C57BL/6J mie re lso involved in different types of humn CA re needed. Journl of Investigtive Dermtology (213) 133, ; doi:1.138/jid ; pulished online 25 Otoer 212 INTRODUCTION Vitmin A nd its derivtives (retinoids) re importnt for development nd mintenne of multiple epithelil tissues inluding skin, hir, nd seeous glnds, s severe, dverse effets re seen in vitmin A defiieny or exess (Zououlis, 26; Smith nd Thioutot, 28; Everts, 212). Vitmin A is stored in the liver nd extrhepti tissues s retinyl esters (O Byrne et l., 25). Retinol ound to retinol-inding protein 4 (RBP4) is the min irulting form of vitmin A (Blner et l., 22) nd mintined t onstnt lood level (Blomhoff et l., 1991). After mel, some retinyl esters enter nonhepti tissues from irulting hylomiron remnnts (Blner et l., 22). Retinoi id (RA) synthesis ours 1 Deprtment of Nutrition, The Ohio Stte University, Columus, Ohio, USA; 2 The Jkson Lortory, Br Hror, Mine, USA; 3 Deprtment of Mediine (Dermtology), Vnderilt University Medil Center, Nshville, Tennessee, USA nd 4 Deprtment of Biohemistry, Vnderilt University Medil Center, Nshville, Tennessee, USA Correspondene: Helen B. Everts, Deprtment of Humn Nutrition, The Ohio Stte University, Columus, Ohio 4321, USA. E-mil: everts.1@osu.edu Arevitions: ADH1, 3, nd 4, lohol dehydrogense 1, 3, nd 4; AIN, Amerin Institute of Nutrition; ALDH1A1, 2, nd 3, retinl dehydrogense 1, 2, nd 3; B6, C57BL/6J inred mouse strin; CA, itriil lopei; CCCA, entrl entrifugl CA; CRABP2, ellulr retinoi id-inding protein II; CYP26A1, B1, C1, ytohrome P45 26 fmily memers; DGAT1, diylglyerol yltrnsferse 1; DHRS9, dehydrogense redutse (SDR fmily) memer 9; IHC, immunohistohemistry; IR, immunoretivity; RA, retinoi id; RARA, B, nd G, retinoi id reeptor lph, et, nd gmm; RBP1, (formerly CRBP) ellulr retinol-inding protein 1 Reeived 8 Ferury 212; revised 13 August 212; epted 21 August 212; pulished online 25 Otoer 212 lolly in or ner ells tht utilize it. Preise sptil nd temporl levels of RA in skin re hieved y regulting key steps in ellulr vitmin A metolism: storge s retinyl esters, RA synthesis, nd RA degrdtion (Everts, 212). Retinol is trnsported into the ell through the protein lled stimulted y RA6 nd inds ellulr retinol-inding protein 1 (RBP1, k CRBP). This ound retinol is either esterified y leithin:retinol yltrnsferse for storge or reversily oxidized to retinl vi retinol dehydrogenses (dehydrogense redutse (SDR fmily) memer 9 (DHRS9), RDH1). Retinl is further oxidized to RA y retinl dehydrogenses 1 3 (ALDH1A1 3). RA then moves to the nuleus ound to ellulr RA-inding protein 2 (CRABP2), inds to RA reeptors lph, et, nd gmm (RARA, B, C) where it tivtes the trnsription of over 5 genes (Dong et l., 1999; Blmer nd Blomhoff, 22). Retinyl ester hydrolses onvert stored retinyl esters k to retinol when needed (Blomhoff et l., 1991; Go nd Simon, 25). When RBP1/CRBP is sturted or sent, retinol interts with different enzymes. Ayl CoA:diylglyerol yltrnsferse 1 (DGAT1) esterifies retinol for storge. Alopei with elerted telogen to ngen trnsition ws seen in sl epidermis nd outer root sheth speifi (Krt14 re) Dgt1 tm2fr null mie (Shih et l., 29). This inresed ngen indution nd lopei ws redued in Dgt1 tm2fr null mie y severely reduing dietry vitmin A intke, inditing tht lopei ws due to exess retinol nd RA. Free retinol n lso e oxidized y medium hin lohol dehydrogenses (ADH1, 3, nd 4; urrent gene nmes Adh1, 5, nd 7, Duester, 21). It hs een rgued y some (Kumr et l., & 213 The Soiety for Investigtive Dermtology 325

2 HB Everts et l. Retinoid Metolism in Citriil Alopei 212) nd refuted y others (Npoli, 212) tht exess retinol is oxidized to retinl y ADH1, nd then to RA y ALDH1A1. When given toxi retinol dose, mie tht lk Adh7 tm1gdu (lss IV, previously Adh4) hve deresed retinol oxidtion in the kidney (Deltour et l., 1999, ), ut not in the liver (Molotkov et l., 22).TheseAdh7 tm1gdu null mie re lso more sensitive to vitmin A defiieny. ADH1 nd ADH7 (lss IV) re present in skin (unpulished oservtion, Hselek et l., 1997), ut their roles in skin hve not een studied. RA is further metolized y ytohrome P45 26 fmily memers (CYP26A1, B1, C1) with the ssistne of ellulr RA-inding protein 1. Both Cyp261 nd Cyp261 messge levels re RA induile in oth orgnotypi ultures nd humn epidermis (Lorie et l., 29, ). Primry itriil lopeis (CAs) re srring hir loss diseses with permnent hir loss euse of destrution of folliles nd their stem ells y lymphoyti- nd neutrophilimedited inflmmtion (Bergfeld nd Elston, 23). Centrl entrifugl CA (CCCA) is progressive permnent lopei seen primrily in Afrin-Amerin women usully eginning in their mid-3s, in n nterior vertex nd mid-rown pttern. C57BL/6J (B6) mie develop CA with similr hrteristis s ptients with CCCA, inluding premture desqumtion of the inner root sheth nd folliulr dystrophy (Sperling nd Su, 1992; Sperling et l., 1994, 2; Sperling, 21; Sperling nd Cowper, 26; Sunderg et l., 211). In B6 mie, s the hir shfts twist, they penetrte through the outer root sheth nd indue n inflmmtory response. Studies in sei-mutnt mie (ABJ/Le-Sd1 -J /Sd1 -J nd DBA/1lJ-Sd1-2J / Sd1-2J ) lso suggest tht the inflmmtory response in CA follows hir shft penetrtion through the follile (Sunderg et l., 2). Other theories for the use of the vrious CAs (MElwee, 28) inlude: ltered seeous glnd funtion with redued seum (Stenn et l., 1999; Sunderg et l., 2; Al-Zid et l., 211) nd seeous glnd dut inflmmtion (Al-Zid et l., 211); redued peroxisome prolifertor tivted reeptor gmm nd lipid metolism (Krnik et l., 29); nd hir follile loss of immune privilege (Hrries et l., 29). Few studies hve exmined the role of diet in CA. Retinoid supplementtion improved CA in four ptients with low serum retinol levels (Klz, 1958). As result of this study nd the unique loliztion pttern of RA synthesis within the hir follile nd seeous glnd during the hir yle (Everts et l., 27), the expression of retinoid metolism proteins were exmined in the B6 mouse model of CA nd humn ptients with CA. The initil study reported tht ll sustrins of B6 mie hd polymorphism in Adh7, whih redued its funtion (Dolney et l., 21, ), nd only B6 sustrins with higher frequeny of disese hd higher levels of DHRS9 in telogen folliles (Sunderg et l., 211). As CA in B6 mie spred telogen folliles nd only lte ngen nd tgen folliles were ffeted, studies exmined retinoid metolism in ngen hir folliles. The urrent report identifies RA synthesis nd signling protein hnges in oth mouse nd humn CA, nd tht modifying vitmin A intke modifies CA severity in B6 mie. RESULTS RA synthesis, signling, nd degrdtion proteins were inresed in erly/mild CA, ut eme undetetle in severe disese The expression of retinoid synthesis omponents ws previously mesured in telogen folliles of C57BL/6 mouse sustrins (Sunderg et l., 211). The urrent study evluted ngen folliles, s it is the stge when B6 CA is most severe nd RA synthesis omponents peked (Everts et l., 27; Sunderg et l., 211). Biopsies of B6 mie tht spontneously developed CA in The Jkson Lortory prodution fility were evluted y immunohistohemistry (IHC) using ntiodies ginst RBP1, DHRS9, ALDH1A1, ALDH1A2, ALDH1A3, CRABP2, CYP26A1, RARA, nd RARB. These mie were grouped y severity of disese into no disese, mild, moderte, or severe disese with hir folliles in mid-lte ngen using stndrd stging riteri (Muller-Rover et l., 21). Biopsies from humn ptients with CCCA, end-stge CA (pseudopelde), nd ontrols with no lopei (tine pitis nd folliles djent to pilr ysts) were lso exmined. DHRS9 ws inresed in mild disese in mie nd humns with CCCA yet undetetle in severe disese (Figure 1, Supplementry Figures S1 nd S2 online). A similr pttern of expression ws lso seen for ALDH1A1, ALDH1A2, nd RARB in mie nd humns (Figure 2, Supplementry Figure S3 online, dt not shown). CYP26A1 showed the sme pttern s well, ut ws only tested in mie (Figure 2; Supplementry Figure S3 online). In moderte mouse CA, levels of these proteins were similr to ontrols (dt not shown). In res of derml srs in mie, DHRS9 nd ALDH1A2 were high, ut ALDH1A1 lolized only to the outer root sheth of telogen folliles nd ALDH1A3 ws not ltered (dt not shown). CRABP2 IR lso inresed in mild disese, ut remined high within dystrophi hir folliles in ll disese sttes (Figure 3). RARA loliztion hnged with more RARA-positive ells seen in the dermis, espeilly surrounding the ulge in mild, moderte, nd severe mouse disese (Figure 3). Dietry vitmin A ltered the frequeny nd severity of CA in B6 mie To test the hypothesis tht norml vitmin A metolism is one of the primry mehnisms using CA in B6 mie, 12- week-old femle B6 mie (n ¼ 9 1)werefedpurifieddiets ontining one of the three levels of vitmin A (Reserh Diets, New Brunswik, NJ). Diets ontined either the Amerin Institute of Nutrition (AIN; Reeves et l., 1993) reommended level (4 IU g 1 diet), the highest level found in ommeril diet used t The Jkson Lortory (high; 28 IU g diet), or twie the highest level (exess; 56 IU g 1 ). Mie fed exess vitmin A hd less lopei thn either of the other groups, nd ll of these mie hd signifintly fewer hir folliles in ngen, inditing tht folliles were in their norml telogen phse (Figure 4 nd ). Histologil nlysis showed tht more hir follile dystrophy ourred when hir folliles were in ngen or tgen with n inverse orreltion etween perent of hir follile dystrophy nd telogen folliles (Figure 4). Mie fed high vitmin A hd more folliulr dystrophy nd grnuloms (Figure 4d nd e). 326 Journl of Investigtive Dermtology (213), Volume 133

3 HB Everts et l. Retinoid Metolism in Citriil Alopei Control Mild Free retinol ADH7 Mod. Severe Retinol-CRBP DHRS9 Retinl Retinl ALDH1A1 ALDH1A1 ALDH1A2 ALDH1A3 Free retinoi id CL PM Retinoi id-crabp2 CYP26A1 Degrdtion Ative RARA RARB Ativte trnsription Figure 1. Immunoretivity of dehydrogense redutse (SDR fmily) memer 9 (DHRS9) inresed in C57BL/6J mie with mild dermtitis nd iopsies from humn ptients with entrl entrifugl itriil lopei (CCCA), ut ws redued in mie with severe disese nd iopsies from humn ptients with pseudopelde. Immunohistohemistry ws performed with n ntiody ginst DHRS9 in dorsl skin from how fed C57BL/6J inred mouse strin (B6) mie with no disese (, n ¼ 18), mild disese (, n ¼ 6), severe disese (e, n ¼ 38), iopsies from ptients with no lopei (, norml Cusin skin djent to pilr yst removed y exision n ¼ 2, tine pitus from Afrin-Amerin skin n ¼ 1), CCCA (d, n ¼ 14), or pseudopelde (f, n ¼ 3). nd f re from Cusin ptients, wheres d is from n AfrinAmerin ptient. Br ¼ 11 mm for min piture nd 1.1 mm for insets. A seond feeding study ws onduted to test the hypotheses tht signifintly lowering dietry vitmin A would redue the frequeny nd severity of CA. Typil mouse ommeril diets (how) ontin up to seven times the AIN reommended level of dietry vitmin A (4 IU g 1 diet, Reeves et l., 1993). The how diet fed to mie t The Jkson Lortory ws nlyzed nd ontined 6 28 IU vitmin A g 1 depending on the lot. This vriility is importnt s vitmin A is stored in the liver nd dms fed how during reeding produe pups with lrge mounts of vitmin A in their liver. Feeding dms vitmin A-defiient diet during reeding redued liver stores of vitmin A in their pups (Smith et l., 1987) nd redued lopei in Dgt1tm2Fr null mie (Shih et l., 29). To signifintly lower vitmin A levels in this study, dms were fed AIN93G diet for two genertions. This study lso evluted whether induing ngen y wx stripping would led to CA ut these hypotheses were inorret. Alopei ws onsistently high in ll the mie not wx stripped. However, wx stripping redued the Figure 2. Overview of retinoid metolism nd how these proteins hnged during the progression of itriil lopei. The left shows shemti of RA synthesis, degrdtion, nd signling pthwys in B6 mie. The right shows summry of IHC results in how fed B6 mie with no (ontrol), mild, moderte, or severe disese. Immunoretivity intensity rnges from drk to light; very strong (mroon), strong (red), moderte (hot pink), mild (pink), or undetetle (white). ALDH1A1, 2, nd 3, retinl dehydrogense 1, 2, nd 3; B6, C57BL/6J inred mouse strin; CL, ompnion lyer; CRABP2, ellulr retinoi id-inding protein II; CRBP, ellulr retinol-inding protein; CYP26A1, ytohrome P45 26 fmily memer A1; DHRS9, dehydrogense redutse (SDR fmily) memer 9; IHC, immunohistohemistry; Mod., moderte; PM, premedull; RA, retinoi id; RARA nd B, retinoi id reeptor lph nd et. frequeny of lopei in mie fed reommended nd high levels of vitmin A (Figure 4). The one wx-stripped mouse with lopei fed the reommended diet developed signifintly more ulers, derml srs, nd epiderml hyperplsi thn ny other mouse in the study. The only other mie with epiderml hyperplsi nd derml srs were the mie fed high vitmin A in study 2 (Figure 4f). They lso hd signifintly less folliulr dystrophy nd grnuloms thn mie fed the sme diet in study 1 (Figure 4d nd e, Po.5). Within study 2, mie fed high vitmin A hd signifintly lower numer of grnuloms thn mie fed reommended levels of vitmin A (Figure 4e, Po.5). This suggests tht these mie fed high vitmin A in study 2 hd previous out of severe disese. Liver retinoid nlysis reveled tht dms fed the 4 IU diet did not lower liver retinyl plmitte levels in their pups s predited (Figure 5, Smith et l., 1987). Both liver retinol nd retinyl plmitte inresed with dietry vitmin A s expeted (Figure 5 nd, Po.1). Liver retinol ws signifintly greter in the seond study (Figure 5, Po.1). In ddition, wx stripping signifintly redued liver retinyl plmitte levels (Figure 5, Po.1). Skin retinol inresed with dietry 327

4 HB Everts et l. Retinoid Metolism in Citriil Alopei Figure 3. CRABP2 nd retinoi id reeptor lph (RARA) levels were inresed in C57BL/6J mie with itriil lopei. Immunohistohemistry ws performed with ntiodies ginst CRABP2 (,, e) nd RARA (, d, f), in mie with no disese (, ), mild disese (, d), or severe disese (e, f). Br ¼ 11 mm for min piture nd 1.1 mm for insets. vitmin A in oth the studies (Figure 5, Po.5). In mie fed high vitmin A, skin retinol ws greter in study 1 thn in study 2. This trend ws seen when mie wx stripped nd not wx stripped were ompred seprtely, ut ws signifint when ll mie in study 2 fed high vitmin A were omined (Figure 5, Po.5). There were no signifint effets of pup or dm diets on skin retinyl ester levels (Figure 5d). Skin retinyl ester levels were signifintly redued in wx-stripped mie (Figure 5 nd d, Po.5). IHC nlysis ws performed to ompre results from these diet studies to those performed in NIH31 fed mie. RARB ws signifintly redued s the perentge of folliulr dystrophy inresed (Supplementry Figures S4, d nd S5 online). RARB ws lso low in derml srs (Supplementry Figures S4f nd S5 online). In ontrst, DHRS9 remined high in dystrophi hir folliles, ut dropped slightly in derml srs (Supplementry Figure S4,, e online). None of the mie in these diet ontrolled studies hd severe folliulr dystrophy s seen in NIH31 fed mie. DISCUSSION This study emphsizes tht the effets of n essentil nutrient, vitmin A, must e tightly regulted to prevent dverse effets euse of exess or defiieny. In this study, oth low nd high levels of dietry vitmin A given to B6 mie ffeted the development nd degree of folliulr srring, inditing tht preise ontrol of retinoid metolism is required for norml 328 Journl of Investigtive Dermtology (213), Volume 133 funtion of hir folliles. Initil studies lolized RA synthesis nd signling proteins in norml mouse skin (Everts et l., 24, 27). A susequent study found tht only B6 sustrins with higher frequeny of inflmed nd folliulr srring hd higher levels of DHRS9 in telogen folliles (Sunderg et l., 211), suggesting possile sis for their tendeny to develop CA. As CA ours primrily during mid-lte ngen, the urrent study nlyzed mid-lte ngen hir folliles nd found tht RA synthesis, degrdtion, nd inding proteins were ltered during the progression of CA in B6 mie. These proteins were inresed in mild disese nd redued in severe disese. A similr pttern of expression ws lso seen in iopsies from ptients with CCCA, lthough the full progression of disese ws not otined. This study hrterized hnges in retinoid metolism throughout the entire progression of CA in mouse model of primry humn CA. Others (Flowers et l., 211) found t only one time point Rp1 (Crp) nd Crp2 inresed in skin speifi (Krt14-re reominse) Sd1tm2Ntm-null mle B6 mie with mild CA. They lso reported tht severl other retinoid metolism genes were inresed in their mirorry nlyses, ut hnges in expression of these genes were not further vlidted t the messge or protein level. They lso found tht retinol, retinyl esters, nd RA levels were inresed in the skin of these null mie. Feeding vitmin A-defiient diet did not redue skin RA levels or hnge the phenotype, rguing tht the lk of Sd1 led to the inresed skin RA levels. In the urrent study, RA metolism ws ltered in the sene of Sd1 muttion nd signifintly reduing dietry vitmin A mde the disese worse. These urrent studies expnd on the oservtions of Flowers et l. (211) y showing tht s folliulr dystrophy inreses, the expression of proteins responsile for RA synthesis nd degrdtion egin to derese nd reh undetetle levels in severe CA. This oservtion ws further onfirmed y the redution of RARB, lssi RA trget gene, s folliulr dystrophy inresed in the two diet studies preeding the redution of DHRS9. In ddition, these ltertions in RA synthesis proteins were lso seen in iopsies from ptients with CA. It is unknown t this time whether ltered RA synthesis nd signling used the lopei or this ltertion in retinoid metolism is seondry to the disese. CYP26A1 lso lolized within the hir follile nd ws ltered during CA. CYP26A1 ws previously reported only in sl kertinoytes, erine swet glnds, nd seeous glnds (Heise et l., 26). CYP26A1 hs key role in mintining RA levels through feedk inhiitory loop (Reijntjes et l., 25). High levels of RA diretly indue CYP26A1 expression (Loudig et l., 2). Then CYP26A1 degrdes this exess RA. In ddition, when RA levels re low, CYP26A1 levels re lso low. In retinol dehydrogense 1 (Rdh1tm1.1Jln) null mie redution in CYP26A1 expression ompensted for the redued RA synthesis nd mintined norml RA levels (Zhng et l., 27). These oservtions re onsistent with the onurrent ltertions of CYP26A1 with DHRS9 in this study. Epigeneti silening my explin the expression results seen. Muttions in either CRABP2 or RARA resulted in

5 HB Everts et l. Retinoid Metolism in Citriil Alopei % Hir loss Study 1 Study 2 not wxed Study 2 wxed * # d % Folliulr dystrophy Study 1 Study 2 not wxed Study 2 wxed % Hir folliles in ngen Study 1 Study 2 not wxed Study 2 wxed e % Grnuloms Study 1 Study 2 not wxed Study 2 wxed % Hir follile dystrophy f % Derml srs % Telogen 12 Study 1 Study 2 not wxed Study 2 wxed Figure 4. Citril lopei ws ltered y dietry vitmin A nd wx stripping. The perentge of mie with hir loss () ws determined y visul inspetion in B6 mie fed 4 (open rs), 28 (losed rs), or 56 (hthed rs) IU vitmin A g 1 diet. H&E slides were then sored for % of hir folliles in ngen () nd telogen (), % of folliulr dystrophy (d), % grnuloms (e), nd % of derml srs (f). *Signifint effet of wx stripping s nlyzed y hi-squre (Po.5), # signifint diet effet in the wx-stripped mie (Po.5). Results with different letters re signifintly different from eh other in mie with hir loss (Po.5). () r ¼.86, Po.1. n ¼ 9 1 study 1, n ¼ 8 19 study 2. B6, C57BL/6J inred mouse strin; H&E, hemtoxylin nd eosin;, not pplile. hypermethyltion nd epigeneti silening of RARA trget genes suh s RARB, CRBP, nd CYP26A1 (Ren et l., 25; Corlzzoli et l., 29; Lursen et l., 212). As CRABP2 funtions to deliver RA to RARA (Dong et l., 1999) nd RARA represses trnsription in the sene of RA (Chen nd Evns, 1995; Horlein et l., 1995), the ove studies suggest tht ny defet resulting in redued RA inding to its reeptor ould use hypermethyltion nd gene silening of RARA trget genes. Suh defets would inlude redued levels of RA preursors retinol or retinyl esters, redued uptke of retinol or retinyl esters into the skin, redued RA synthesis, redued delivery of RA to RARA vi CRABP2, inresed RA degrdtion, or ny defet in RARA itself. It is possile tht the redution in RA synthesis enzymes, RARB, nd CYP26A1 in moderte nd severe CA, ws euse of this mehnism of errnt RA signling indued hypermethyltion. Alterntively, primry effet of inresed dietry vitmin A ould e euse of toxi effets during mild disese on speifi hir follile ells, leding to their deth or deresed iohemil funtion suh tht the levels of RA synthesis enzymes nd reeptors derese s CA disese progresses. The urrent studies indite the lne etween RA synthesis nd retinol degrdtion is ltered in mouse model of humn CA nd imply tht ltered retinoid metolism is involved in the pthogenesis of CA. Future studies will determine whether CA is euse of ltered retinoid metolism or is euse of loss of retinoid relted enzymes present in skin euse of srring indued y nother use. The urrent study implies tht preise levels of dietry vitmin A re required to ompenste for the defets in retinoid metolism found in sustrins of C57BL/6 mie. A polymorphism in Adh7 (lss IV, previously reported s Adh4) ws previously found in C57BL/6 mie tht redued its funtion (Dolney et l., 21, ; Sunderg et l., 211). C57BL/6 mie hve rginine12, wheres C3H/HeJ nd other strins of mie hve ysteine12. Humn ADH7 (lss IV) lso 329

6 HB Everts et l. Retinoid Metolism in Citriil Alopei Liver retinyl plmitte (nmol g 1 tissue) d Liver retinol (nmol/g tissue) 35 3 Study 1 Study 2 not wxed Study 2 wxed 25 * Dietry vitmin A (IU g 1 diet) 1, Skin retinol (nmol g 1 tissue) Skin retinyl esters (nmol g 1 tissue) 8, 6, 4, 2, Study 1 Study 2 not wxed Study 2 wxed 4 28 Dietry vitmin A (IU g 1 diet) Study 1 Study 2 not wxed Study 2 wxed 4 Study 1 Study 2 not wxed Study 2 wxed # ontins rginine12, similr to C57BL/6 mie (Kedishvili et l., 1995). Additionl polymorphisms in ADH7 (lss IV) were lso found in humns linked to lohol dependene, heroin ddition, nd redued risk of hed nd nek squmous ell rinom (Edenerg et l., 26; Levrn et l., 29; Wei et l., 21). Polymorphisms in ADH7 (lss IV) my predispose B6 mie nd humns to CA. Studies in trnsgeni mie suggest two roles for Adh7 (lss IV). * 28 Dietry vitmin A (IU g 1 diet) 4 28 Dietry vitmin A (IU g 1 diet) Figure 5. Dietry vitmin A ltered liver nd skin retinoids differently in the vrious studies. Liver retinol (), liver retinyl plmitte (), skin retinol (), nd skin retinyl esters (d) were nlyzed y HPLC in study 1 (open rs, n ¼ 9 1), study 2 with no wx stripping (losed rs, n ¼ 8 19), nd study 2 with wx stripping (hthed rs, n ¼ 1 16). *Signifint study effet (Po.1 in, nd Po.5 in ), # signifint effet of wx stripping (Po.5), results with different letters re signifintly different from eh other (Po.5) # Adh7 tm1gdu null mie hve redued fetl survivl when dms re defiient in vitmin A (Deltour et l., 1999). When these Adh7 tm1gdu null mie re stressed with exess retinol there ws redued retinol lerne in the kidney (Deltour et l., 1999, ), ut not the liver (Molotkov et l., 22). These dt imply tht redued Adh7 (lss IV) tivity inrese suseptiility to oth vitmin A defiieny nd toxiity in B6 mie nd some humns. This phenomenon my explin why more severe disese ws seen in the seond study when dms were fed lower vitmin A. The mie fed the AIN93G diet were lso slow to reed; the numer of litters nd the numer of pups per litter were deresed ompred with how fed mie. These pups ppered runted t first, ut ppered to th up their growth fter severl dys. In ddition, mny of the dm s first litters were stillorn. It is possile tht C57BL/6 mie require nrrower window of vitmin A for optiml funtion thn other strins of mie. In the urrent study, this optiml window is ove 4 nd elow 28 IU vitmin A g 1 of diet. Vritions in vitmin A levels in ommeril unpurified diets rnge from 6.2 to 28.7 IU in seven tests from two lortories, IU within the sme lot (Sunderg unpulished dt) implying mixing is not uniform. In suseptile strins, this vrition in vitmin A onentrtion in ommeril diets n hve profound impt on the frequeny of lopei nd dermtitis, nd explin the pprent rndomness of CA inidene in B6 mie in The Jkson Lortory fility. Humns with similr polymorphisms my lso require more preise levels of dietry vitmin A. Anedotl reports suggest redued lopei with retinyl plmitte supplementtion t roughly 3.5 times the reommended dily llowne (8, IU dy 1 ) in ptients with low serum vitmin A (Wilm Bergfeld, personl ommunition). Future studies re needed to determine the optiml dose of vitmin A in humns nd mie suseptile to CA. Retinoids re not the first tretment hoie in CA ptients, ut they do help some ptients nd re listed s seond or third line of tretment in some forms of CA (Hrries et l., 28). This vrile enefit of retinoid therpy is similr to the initil linil trils in ner (reviewed in, Freemntle et l., 23). It is now well estlished tht retinoid resistne ours in ner euse of epigeneti hypermethyltion of RARB nd other RA synthesis enzymes (Tng nd Guds, 211). Current studies omining low-dose retinoid tretment with methyl trnsferse inhiitors in mouse models of ner were more suessful (Tng et l., 29). A similr sitution of retinoid resistne my our in CA. If this is the se then this dul tretment strtegy my e worth testing for the tretment of CA. As high vitmin A worsened CA in this mouse study, this implies tht phrmologil doses of retinoids my hve een toxi. High vitmin A intke (12 IU g 1 diet) lso elerted lopei ret in grft-indued C3H/HeJ mie (Dunn et l., 212). Telogen effluvium ws lso seen in ptients treted with retinoids (Ruzik et l., 1992). Thus, etter tretment option my e to restore retinoid signling with methyl trnsferse inhiitors while mintining dequte dietry vitmin A levels. The ext dequte level of dietry vitmin A is likely to e very different in mie nd humns with different geneti kgrounds. 33 Journl of Investigtive Dermtology (213), Volume 133

7 HB Everts et l. Retinoid Metolism in Citriil Alopei In summry, this study suggests tht retinoid metolism is ltered in CA nd high dietry vitmin A my worsen the disese. Future studies re needed to determine whether the hnges oserved our in ll primry lymphoyti nd neutrophili forms of CA nd the optiml level of dietry vitmin A to prevent the ourrene nd severity of humn CA. In B6 mie studies, the diet should strt with times the reommended levels of vitmin A (6 12 IU g 1 diet in B6 mie). This optiml level mye different in different genotypes, therefore future studies should nlyze linkge etween Adh7 (lss IV) single-nuleotide polymorphisms with CA. As ll sustrins of B6 mie hve Adh7 polymorphisms, ut differ in disese frequeny, future studies should lso identify other geneti muttions in RA synthesis or signling genes. Epigeneti hnges in retinoid metolism in CA should lso e explored. In ddition, RARB expression levels my e useful iomrker for severity of CA in speifi sustrins of B6 mie nd types of humn CA. This study lso found tht the frequeny of this rre disese ould e inresed y diet so tht future studies n e onduted in B6 mie to define the pthogenesis of CA nd develop speifi tretments of humn CA. MATERIALS AND METHODS Mie The Jkson Lortory (Br Hror, ME) Institutionl Animl Cre nd Use Committee pproved ll proedures nd hir loss ws oserved weekly. Hemtoxylin nd eosin slides of iopsies were sored for speifi follile stge, folliulr dystrophy, grnuloms, epiderml hyperplsi, ulers, nd derml srs y ord ertified pthologist (JPS, Sunderg et l., 28). Initil expression study mie Mie were fed the NIH 31 diet with 6% ft; (LDiet 5K52, Purin Mills, St Louis, MO) throughout the study. There were 51 femle ontrols. Cses were divided into mild (n ¼ 6), moderte (n ¼ 15), severe (n ¼ 38), nd derml srs (26). Hir folliles were stged s ngen II III versus ngen IV tgen I, nd hir folliles in these preise stges were ompred etween the groups (Muller-Rover et l., 21). Diet study 1 mie Dms nd femle pups were fed the NIH 31 diet until 12 weeks of ge, until swithing to the AIN93 Mintenne (M) diet ontining 4(n ¼ 9), 28 (n ¼ 1), or 56 (n ¼ 1) IU vitmin A g 1 diet (Reserh Diets) for 4 months. Diet study 2 mie Dms were fed the AIN93 growth (G) diet throughout gesttion. Femle pups from these dms were mintined on this AIN93G diet during their growth phse nd when they eme pregnnt. Mie from this seond genertion of dms fed the AIN93G diet were used in this study. At 6 weeks of ge, these mie were fed the AIN93M diet ontining 4 (n ¼ 19), 28 (n ¼ 35), or 56 (n ¼ 22) IU vitmin A g 1 diet (Reserh Diets). At 16 weeks of ge, mie (n ¼ 1, 4 IU; n ¼ 16, 28IU; n ¼ 14, 56 IU) were depilted nd then euthnized 2 weeks lter. Humn smples Prffin loks of iopsies from 14 humn ptients with CCCA, 3 humn ptients with pseudopelde, nd 3 nonlopei dermtoses (tine pitus nd skin djent to pilr ysts) were otined with Institutionl Review Bord pprovl from rhives t Vnderilt University (Nshville, TN; Supplementry Tle S1 online). Proedures IHC ws performed s desried previously (Everts et l., 27). Antiodies were purhsed for CYP26A1 (Alph Dignosti Intl., Sn Antonio, TX), RARA, nd RARB (Snt Cruz Bioteh, Snt Cruz, CA). Other ntiodies used were produed nd vlidted in Dr Ong s lortory (Everts et l., 27). A linded IHC sore of RARB immunoretivity (IR) ws mesured using sle of 4 multiplied y the perentge of ells/mid-lte ngen hir folliles. Two linded reserhers sored humn smples for % of ells nd strength on 4 sle with ¼ no IR; 1 ¼ 1 25% ells, wek IR; 2 ¼ 26 5% ells, moderte IR; 3 ¼ 51 75% ells, intense IR; 4 ¼ 76 1% ells, very intense IR. The IHC level ws lulted s the sum of intensity plus % ells s previously vlidted (Shmonki et l., 26). Retinoids were extrted (Ppps et l., 1993) nd nlyzed y HPLC with modifition of Fleshmn et l., (212) using C mm, 3.5 mm olumn. The solvents were (A) 8:2 MeOH/.1% formi id nd (B) MtBE. In ll, 1 2 ml ofsmplein1%a ws injeted nd retinoids seprted with initil onditions of 1%A, liner grdient to 1%B over 13 minutes, held t 1%B for 3 minutes, then equilirted k to 1%A over 2 minutes. Spetr were nlyzed using photodiode rry (Agilent Tehnologies, Snt Clr, CA) t 325 nm. Stndrds were purhsed from Sigm (St Louis, MO). Chi-squred nlysis ws performed to determine differenes in hir loss using Prism 5 (Grph Pd; L Joll, CA). All other dt were nlyzed y nlysis of vrine, followed y Tukey post ho tests when pproprite using SPSS, v19 (IBM; Armonk, NY). See Supplementry Dt online for more detiled methods. CONFLICT OF INTEREST The uthors stte no onflit of interest. ACKNOWLEDGMENTS This work ws supported y grnts from the Ntionl Institutes of Helth (AR529 to HBE, AR5271 to JPS), the North Amerin Hir Reserh Soiety (HBE), the Citriil Alopei Reserh Foundtion (CARF to JPS), nd the Wuford Foundtion (HBE). SUPPLEMENTARY MATERIAL Supplementry mteril is linked to the online version of the pper t REFERENCES Al-Zid T, Vnderweil S, Zemowiz A et l. (211) Seeous glnd loss nd inflmmtion in srring lopei: potentil role in pthogenesis. JAm Ad Dermtol 65: Blmer JE, Blomhoff R (22) Gene expression regultion y retinoi id. J Lipid Res 43: Bergfeld WF, Elston DM (23) Citriil lopei. In: Olsen EA ed Disorders of Hir Growth: Dignosis nd Tretment. 2nd edn MGrw-Hill Medil Pulishing Division: New York,

8 HB Everts et l. Retinoid Metolism in Citriil Alopei Blner WS, Gmle MV, Vogel S et l. (22) Retinol-inding protein (RBP): essentil physiologi funtions. JNutr132:2979S Blomhoff R, Green MH, Green JB et l. (1991) Vitmin-A metolism new perspetives on sorption, trnsport, nd storge. Physiol Rev 71:951 9 Chen JD, Evns RM (1995) A trnsriptionl o-repressor tht interts with nuler hormone reeptors. Nture 377:454 7 Corlzzoli F, Rossetti S, Bistulfi G et l. (29) Derngement of ftor upstrem of RAR lph triggers the repression of pleiotropi epigeneti network. PLoS One 4:e435 Deltour L, Foglio MH, Duester G (1999) Impired retinol utiliztion in Adh4 lohol dehydrogense mutnt mie. Dev Genet 25:1 1 Deltour L, Foglio MH, Duester G (1999) Metoli defiienies in lohol dehydrogense Adh1, Adh3, ndadh4 null mutnt mie. Overlpping roles of Adh1 nd Adh4 in ethnol lerne nd metolism of retinol to retinoi id. JBiolChem274: Dolney DE, Szli G, Duester G et l. (21) Moleulr nlysis of geneti differenes mong inred mouse strins ontrolling tissue expression pttern of lohol dehydrogense 4. Gene 267: Dolney DE, Szli G, Felder MR (21) Differenes in hrge nd kineti properties of lohol dehydrogense 4 from C57BL/6 mie ompred to other inred strins re ssoited with ysteine12 to rginine12 sustitution. Biohem Genet 39:239 5 Dong D, Ruusk SE, Levinthl DJ et l. (1999) Distint roles for ellulr retinoi id-inding proteins I nd II in regulting signling y retinoi id. JBiolChem274: Duester G (21) Geneti dissetion of retinoid dehydrogenses. Chem-Biol Intert 13:469 8 Dunn FJ, Silv KA, Johnson C et l. (212) Endogenous retinoids in the pthogenesis of lopei ret. J Invest Dermtol (doi:1.138/jid ) Edenerg HJ, Xuei XL, Chen HJ et l. (26) Assoition of lohol dehydrogense genes with lohol dependene: omprehensive nlysis. Hum Mol Genet 15: Everts HB (212) Endogenous retinoids in the hir follile nd seeous glnd. Biohim Biophys At 1821:222 9 Everts HB, King LE Jr, Sunderg JP et l. (24) Hir yle-speifi immunololiztion of retinoi id synthesizing enzymes Aldh12 nd Aldh13 indite omplex regultion. J Invest Dermtol 123: Everts HB, Sunderg JP, King LE Jr. et l. (27) Immunololiztion of enzymes, inding proteins, nd reeptors suffiient for retinoi id synthesis nd signling during the hir yle. J Invest Dermtol 127: Fleshmn MK, Riedl KM, Novotny JA et l. (212) An LC/MS method for d8- et-rotene nd d4-retinyl esters: et-rotene sorption nd its onversion to vitmin A in humns. J Lipid Res 53:82 7 Flowers MT, Pton CM, O Byrne SM et l. (211) Metoli hnges in skin used y Sd1 defiieny: fous on retinol metolism. PLoS One 6:e19734 Freemntle SJ, Spinell MJ, Dmitrovsky E (23) Retinoids in ner therpy nd hemoprevention: promise meets resistne. Onogene 22: Go J, Simon M (25) Identifition of novel kertinoyte retinyl ester hydrolse s trnsylse nd lipse. J Invest Dermtol 124: Hrries MJ, Meyer KC, Pus R (29) Hir loss s result of utneous utoimmunity: frontiers in the immunopthogenesis of primry itriil lopei. Autoimmun Rev 8: Hrries MJ, Sinlir RD, MDonld-Hull S et l. (28) Mngement of primry itriil lopeis: options for tretment. Br J Dermtol 159:1 22 Hselek RJ, Ang HL, Duester G (1997) Clss IV lohol/retinol dehydrogense loliztion in epiderml sl lyer: potentil site of retinoi id synthesis during skin development. Dev Dyn 28: Heise R, Mey J, Neis MM et l. (26) Skin retinoid onentrtions re modulted y CYP26AI expression restrited to sl kertinoytes in norml humn skin nd differentited 3D skin models. J Invest Dermtol 126: Horlein AJ, Nr AM, Heinzel T et l. (1995) Lignd-independent repression y the thyroid-hormone reeptor-medited y nuler reeptor o-repressor. Nture 377: Klz F (1958) Citriil lopei nd vitmin A. AMA Arh Derm 78:74 3 Krnik P, Tekeste Z, MCormik TS et l. (29) Hir follile stem ell-speifi PPARg deletion uses srring lopei. J Invest Dermtol 129: Kedishvili NY, Bosron WF, Stone CL et l. (1995) Expression nd kineti hrteriztion of reominnt humn stoh lohol-dehydrogensetive-site mino-id-sequee explins sustrte speifiity ompred with liver isozymes. J Biol Chem 27: Kumr S, Sndell LL, Triner PA et l. (212) Alohol nd ldehyde dehydrogenses: retinoid metoli effets in mouse knokout models. Biohim Biophys At, Mol Cell Biol Lipids 1821: Lursen KB, Wong PM, Guds LJ (212) Epigeneti regultion y RAR lph mintins lignd-independent trnsriptionl tivity. Nulei Aids Res 4:12 15 Levrn O, Londono D, O Hr K et l. (29) Heroin ddition in Afrin Amerins: hypothesis-driven ssoition study. Genes Brin Behv 8:531 4 Lorie EP, Chmheu JC, Vhlquist A et l. (29) Both ll-trns retinoi id nd ytohrome P45 (CYP26) inhiitors ffet the expression of vitmin A metolizing enzymes nd retinoid iomrkers in orgnotypi epidermis. Arh Dermtol Res 31: Lorie EP, Cools M, Borgers M et l. (29) Topil tretment with CYP26 inhiitor tlrozole (R115866) dose dependently lters the expression of retinoid-regulted genes in norml humn epidermis. Br J Dermtol 16:26 36 Loudig O, Bihuk C, White J et l. (2) Cytohrome P45RAI(CYP26) promoter: distint omposite retinoi id response element underlies the omplex regultion of retinoi id metolism. Mol Endorinol 14: MElwee KJ (28) Etiology of itriil lopeis: si siene point of view. Dermtol Ther 21:212 2 Molotkov A, Fn X, Duester G (22) Exessive vitmin A toxiity in mie genetilly defiient in either lohol dehydrogense Adh1 or Adh3. Eur J Biohem 269: Muller-Rover S, Hndjiski B, vn der Veen C et l. (21) A omprehensive guide for the urte lssifition of murine hir folliles in distint hir yle stges. J Invest Dermtol 117:3 15 Npoli JL (212) Physiologil insights into ll-trns-retinoi id iosynthesis. Biohim Biophys At, Mol Cell Biol Lipids 1821: O Byrne SM, Wongsiriroj N, Liien J et l. (25) Retinoid sorption nd storge is impired in mie lking leithin: retinol yltrnsferse (LRAT). JBiolChem28: Ppps RS, Newomer ME, Ong DE (1993) Endogenous retinoids in rt epididyml tissue nd rt nd humn spermtozo. Biol Reprod 48: Reeves PG, Nielsen FH, Fhey GC Jr (1993) AIN-93 purified diets for lortory rodents: finl report of the Amerin Institute of Nutrition d ho writing ommittee on the reformultion of the AIN-76A rodent diet. JNutr123: Reijntjes S, Blenti A, Gle E et l. (25) The ontrol of morphogen signlling: regultion of the synthesis nd tolism of retinoi id in the developing emryo. Dev Biol 285: Ren MQ, Pozzi S, Bistulfi G et l. (25) Impired retinoi id (RA) signl leds to RAR et 2 epigeneti silening nd RA resistne. Mol Cell Biol 25: Ruzik T, Sommerurg C, Goerz G et l. (1992) Tretment of utneous lupus-erythemtosus with itretin nd hydroxyhloroquine. Br J Dermtol 127:513 8 Shmonki MI, Kligmn I, Shmonki JM et l. (26) Immunohistohemil expression of endometril L-seletin lignd is higher in donor egg reipients with emryoni implnttion. Fertil Steril 86: Shih MYS, Kne MA, Zhou P et l. (29) Retinol esterifition y DGAT1 is essentil for retinoid homeostsis in murine skin. JBiolChem284: Journl of Investigtive Dermtology (213), Volume 133

9 HB Everts et l. Retinoid Metolism in Citriil Alopei Smith KR, Thioutot DM (28) Seeous glnd lipids: friend or foe?. J Lipid Res 49: Smith SM, Levy NS, Hyes CE (1987) Impired immunity in vitmin A defiient mie. J Nutr 117: Sperling LC (21) Srring lopei nd the dermtopthologist. J Cutn Pthol 28: Sperling LC, Cowper SE (26) The histoplthology of primry itriil lopei. Semin Cutn Med Surg 25:41 5 Sperling LC, Su P (1992) The folliulr degenertion syndrome in lk ptients- hot om lopei revisited nd revised. Arh Dermtol 128:68 74 Sperling LC, Skelton HG, Smith KJ et l. (1994) Folliulr degenertion syndrome in men. Arh Dermtol 13:763 9 Sperling LC, Solomon AR, Whiting DA (2) A new look t srring lopei. Arh Dermtol 136: Stenn KS, Sunderg JP, Sperling LC (1999) Hir follile iology, the seeous glnd, nd srring lopeis. Arh Dermtol 135:973 4 Sunderg JP, Boggess D, Sunderg BA et l. (2) Asei-2J (Sd1 2J ): new llele nd model for srring lopei. Am J Pthol 156: Sunderg JP, Sunderg BA, Shofield P (28) Integrting mouse ntomy nd pthology ontologies into phenotyping dtse: Tools for dt pture nd trining. Mmm Genome 19:413 9 Sunderg JP, Tylor D, Lorh G et l. (211) Primry folliulr dystrophy with srring dermtitis in C57BL/6 mouse sustrins resemles entrl entrifugl itriil lopei in humns. Vet Pthol 48: Tng XH, Alert M, Sognmiglio T et l. (29) A D methyltrnsferse inhiitor nd ll-trns retinoi id redue orl vity rinogenesis indued y the rinogen 4-nitroquinoline 1-oxide. Cner Prevention Res 2:11 1 Tng XH, Guds LJ (211) Retinoids, retinoi id reeptors, nd ner. Ann Rev Pthol 6: Wei S, Liu ZS, Zho H et l. (21) A single nuleotide polymorphism in the lohol dehydrogense 7 gene (lnine to glyine sustitution t mino id 92) is ssoited with the risk of squmous ell rinom of the hed nd nek. Cner 116: Zhng M, Hu PR, Krois CR et l. (27) Altered vitmin A homeostsis nd inresed size nd diposity in the rdh1-null mouse. FASEB J 21: Zououlis CC (26) Isotretinoin revisited: pluripotent effets on humn seeous glnd ells. J Invest Dermtol 126: