The Retinoid X Receptor Agonist 9-cis-Retinoic Acid and the 24-Hydroxylase Inhibitor Ketoconazole Increase Activity of 1,25-Dihydroxyvitamin D3 in Human Skin In Vivo
Sewon Kang, Xiao-Yan Li , Elizabeth A. Duell, and Jolm J. Voorhees Department of Dermatology, U niversity o f Micltigan M edical Center Ann Arbor. Michigan. U .S .A.
1,25-Dihydroxyvitamin D 3 [1,25(OHhD31 transacti 1,25(OH}zD3' however, caused a synergistic increase vates its target genes via the vitamin D receptor in 24-0Hase mRNA. Similarly, 1,25(OH)2D3 plus (VDR). VDR functions in physiology as a dimer ketoconazole increased 24-0Hase mRNA synergisti complexed with retinoid X receptor (RXR), whose cally. Ket oconazole inhibited ex vil10 1,25(OH}zD3- natural ligand is 9-cis-retinoic acid (9-c-RA). Inacti induced epidermal 24-0Hase activity. Thus, 24- vation of 1,25(OH}zD3 occurs through a cytochrome OHase mRNA induction is a sensitive reporter of P-450 24-hydroxylase (OHase). The promoter of the 1,25(OH}zD3 activity ill vivo; RXR bound to VDR is human 24-0Hase gene contains a 1,25(OH)2D 3-re- not a silent partner ill vivo, because 9-c-RA enhances sponsive enhancer element (VDRE). We have used 1,25(OHhDrliganded RXRJVDR stimulation of the this VDRE containing gene as an endogenous re VDRE containing 24-0Hase gene; ketoconazole in porter for vitamin D 3-mediated gene activation ;11 hibition of 24-0Hase enhances 1,25(OH}zD3 activity vivo. Normal adult human skin was keratomed after a by impeding its breakdown. Thus, the synergistic 2-d exposure to 1,25(OHhD3' 9-c-RA, all-tl'a1ls-RA, response of human skin to topical 1,25(OH}zD3 and/or and ketoconazole. 1,25(OH}zD3 caused a concentra 1,25(OHhD3 analogs plus RXR retinoids and/or ke tion-dependent increase in 24-0Hase mRNA expres toconazole may be exploited to give a desired biolog sion as determined by northern blot analysis. The ic/therapeutic response with less 1,25(OH}zD3, mini activity of epidermal 24-0Hase was also induced nlizing the potential calcemic risk from systemic by 1,25(OH}zD3. Compared with vehicle, neither of absorption of 1,25(OH}zD3' KCJI words: meta.bolistll. t he RA isonlers nor ketoconazole alone induced 24- ] lUllest Del'1lwtol 108:513-518, 1997 OHase mRNA. Addition of 9-c-RA or t-RA to
,2 5-0ihydroxyvitamin D J [1,25(OHh 0 31 is a bioactive hormone nuclear rccepto r supel'family. As such, it functi ons as a hormo ne critical in seven11 cellular and phys io logic li gand (ho rmone)-dependcllt transcriptio n f.1c to r, regulating the processes. In addition to its well-established importance activatio n o f vitamin O-responsive target genes. U nlike stero id in plasma calcium ho meostasis (D oyle et al. 1972; Holick receptors that fun ction as homodimers, the V OR appears to et ai, 1972). 1.25(O H)20 3 al so affects cell prolifera tio n function predominantly as a heterodimcr (Lemo n and Freedman, a1nd di ffe re ntiation (Colston CI ai , 1981; T anaka et ai, 1982; Smith el 1996). T he heterodimeri c parmer o f VDR is retinoid X recep tor ai, 1986) , as w e ll as T-cell acti vation (Mliller and Bendtzen, 1992; ( RXl~) whosc natural li gand is 9-cis-retino ic acid (9-c-RA) (Hey- B agor et ai , 1994). The seemingly dive rse e ffects of 1.25 (O H)20 3 1113n et ai, 1992; Levin 1'1 ai, 1992) . In addition to VD R , RXR are m ainly m ediated thro ugh its intracellular vitamin 0 receptor hetcrodimerizes w ith other nuclear reccpto rs including, but not (VDR). VDR is a member of the stero id/ retinoic acidlthyro id limited to. re tinoic acid receptors (RARs) and thyroid hormone receptors. Manuscript received September 9, 1996; revised November 13, 1996; M ost of o ur current understanding o f the m o lecular m echanism accepted for publicatio n January 3, 19'1 7. b y which VDR activates its target gcnes has been through studies in T lus work was presented in part at the annual mee ting of the Society of ;f( 1I;lro system s o r in artificial ceU-transfecti on m od els. T he inheren t lnvesri gative Dermatolot;y. W ashington. Districr o f Columbia. U .S.A .. limitati ons o f these ;f( ,,;fro system s to predict ;f( 11 ;110 situations, 1996. especiall y with regard to the ro le o fRXR and its li gand in vitamin R eprint requests to: Dr. Sewon Kant;. Dcpartmcllt o f Dermato logy. D si gnaling, arc clearl y dem onstrated by inconsistent findings U niversity ofMichit;an Medical Center. ·19 10 T aubman Center. Ann Arbo r. (M acD onald cl ai, 1993; Sasaki cl ai , 1995; Kane cl ai, 1996), w h.i ch M I 48109-0314. undcrscore the necessity to directl y in vesti gate the ;f( /1 ;/10 system o f Abbreviations: (O l-l h D.l' dihydrolCyv itamin D3; (O I-l )o D, . trihydroxyvi ra min D,; O I-l D,. hydrolCyvitamin D, or cholecalciferol; O l-la se, hyd rox interest. Skin is clearly a target o rgan for 1 ,25(O H hO,1 with its cells ylase; I-RA, all-fmlls-rctino ic acid ; 9-(-RA. 9-ci.<- rctinoic acid; VDR., constitutively expressing VDRs (Berger ef ai, 1988; Milde et ai, vitamin D receptor; RX R .. retinoid X recepto r; VDR.E. vitamin D respo n 1991). Furthermore, certain di seases. such as ichthyosis and psori sive element; It AR. retin o ic acid receptor. asis. respo nd to 1,25(O H)l D, and its re lated compounds (Krag-
0022-202X/97 IS I n.S!) • Copyri ght © 1997 by The Society for In vestigative Derlllatology. Inc.
513 9 514 KANG /i'1 ti l. T H E JOUR NAL OF INVESTIGATI VE DERMATOLOGY
balle, 1<)92) . PriOl' to o ur study, h o w e ve r, the re had been a lac k of nl. 19') I). e"('''pt that 36B4 rather than cycl ophilin was nsed as the control sp ecifi c m e asurable 1l1arke rs of vitamin D ac tivity in human skin ill probe. In tegrated autoradiographic intensity obtained by Ph osphorlll1ager I'iIJ() . This d e fi c ie n cy has limite d the study o f the e ffects and lo r th e 24-01-/ase gene was lirst di vid ed by that of the control gell e (3 6B4). The divisio n produc t" was then divided ag ain b y t'h ~ l t o f the contro l tre~ltlll e llt m o lecular si g naling o f these m o lecules in human sk in ;11 1';1'0. for relative cOl11pa risons. Beca use the nC[;ative ('o ntrol (vehicle) group 24-Hydro'-'Y lase (O Hase) is a cytochro m e P-4 50 e n z yl1l e tha t contained many sl11all nU111bers, including zero. which precillded the hydro x ylates 1,2S(O H h D at: the 24 p osition, resulting in a m e J require d CO lllptltatio n s (divisio n b y zero), data arc e xpressed as pe rcen t tabo li te. 1.24.25-trihydroxyvitilmin D:\ 1.1. 24,2 S(O H)}D }J. with a inducti on of thc positive control (1).05% I ,25 (0 1-l),D, ). which was consid m arke dly re duced ac ti v ity (Ho li ck ('/ ,Ii. J 9 73; O h yama and Okuda, e r ed Inaxil1lll1ll induc t-jo n . 1( 91). R,ecent c loning o f this gene hil S reveal e d that its natural Measurement of 24-0Hase Enzytne Ac tivity HUl11an skin trcared pro m o te r harbo rs vitamin D resp o n sive e le m ents (YD IU ) that are with vehiclc and 0.05% 1. 25(0 1-l ),D , lor 2 d under continuous occiusiolJ composed o f AGGTCA- like hexameric direct re p eats separate d b y w as kcrato nlc-biopsicd . T he biopsy specinlCl1 s we re incubated in trypsin 3-bp-a so calJ e d DR3 (Ohyama el ai, 1994; Zierold cl ai , 1995). (0 .1 % in solution consisting of 30 mM I-IE PES buffer, 10 111M glu cose. 3 mM This type (DR3) of e nhancer c le m e nt has also b een identified in KC I. 130 111M NaC I. and I 111M sodium phosphate. 1'1-1 7,2) ('or 30 min at o ste o calcin and calbindin D ~K gen es (Ke rne r £'1 ai , 1989; D arwish 35°C to separate thl' epidermis &om the dennis. Dispersed epidermal cell s and D eLuca, 1992) '\I1d appe ars to be the physiologic ally relevant were centrifuged at 2000 X .~ lo r 5 l11in . Thc pell eted cell s were resuspcnded YDRE. A lthoug h some reports of VDR- YDR h o modil1'1 e rs bind in 5 ~111 o f 0. 1% trypsin inhibitor and then centrifuged again at 2000 X g for ing to VDR.E exist (C arlbe rg 1'1 (/1 , 1993), prevailing evide n ce p o ints 5 111n1. T he cells wc re washed twice with 5 ml of the di ges ti on buffer to R X R - VDR h e tero dime r as the majo r complex tha t binds to the (wi thout trypsin). T he fin al cell pell et was resuspended in I 1111 of cnzy l11 e assay bull'c r (20 111M I-I Ei'ES bull'c r. 125 111M KC I. 2U u,M succin ate. 2 mM YDRE (Yu 1'1 ill. 199 1; Ke phart 1'( (fl , 19 96). Indeed. e ndogeno u s MgCI" I 111M di thi othreitol. and 1 111M ethylenediamine terraacetic acid. VDn.. in nucle ar extracts fro m human epide rmis and culture d pH 7.4 ). To 2U Jl.1 o{'res uspended cell s. I I.tM 25-1 ·' 1-1.1 0 I-I D.I ( I Jl.Ci) was human ke ratinocytes binds to its recognition si te (DR3) as a added to s,.art the reacti on. When lI sed . ketoconazole was added at a fi nal h e te ro dime r (RX H.. -VDR) and not as a homodime r (YDR-VDR) concentration of I X 10- " M. After a 30- l11in incubation at 35°C, th e (Li {'( (fl , 1(97) . In c ultured human k e ratinocytes, 'I ,2S(OHh D , has react'ion was stopped with rhe addition of 2.6 Inl of l11ethan ol:c hl oroform . b een reported to e nhan ce the expression of 24- 0 Hase mRNA 2: 1 (vol/vol). After adding 1.2 ml of chl oroforl11 and I 1111 of water, the (C h e n ('( aI, 1994). In this study. w e in vesti gated the regulation o f s;ll11plcs w ere vort'cx-lllixcd for 'I lniJl alld thell all owed to separate i_llto two the 2 4-0 J-lase g ene , which contains a natural VDrU in its pro layers. Thc aqueous la ye r was was hed twice with 1.2 1111 0 (' chl oroform . mote r. in human sk in ill 11;1'''. I-lerein w e repo rt that the 9-c-RA, W ith each wash. the chl oroform la ye r was pooled and then evaporated to li gand o fRXR. syn e rgistica ll y e nhances the abi li ty of J .25(O l-I), I), dryness . and the nlaterial wa s res lI spended in 200 Jl.1 of l11 e,.h anol. The rnaterial was fi lrered with Millipore SJI-IV 004 filters into high-performance to induce 2 4-0Hase ge n e transcripts, and furthe rmo re, a sin;ila;' liqnid chro/l)atogr;'phy vi,d s. After eva pora tion to dr)'ncss the material ,,,as can response b e ac hie ved by inhibiting the inactivatio n o f taken lip in a I'inal volume of 3(1 1.1, 1 f.o r hi gh- perto nnal1ce liquid chroma 1. 25(O H h D \ b y li se o f a 24- 0 Hase inhibitor. tograph y al1al ys is. MATErtlALS AND METI-I O DS High-Perforrrtance Liquid Chromatography Separation of Vita Materials 1.25 (0 I-l >O D ,. 25-hydroxyvital11in D , or cholecalciferol 125- min 0 , Metabolites The stnlldard used to idclltif), al1d quantitate 0 1-1])", 24.25-dihydroxyvitamin D, [24.25(01-l ),D, I. 25.26-dihydroxy vitamin D., metabolites were 25-01-11)" 24.25(0 I-l h l),. 1,25(O Hh D3' vita l11in Dol r25 .2() (0 1-l )2D, I. 1,24,25(0I-l h l), . 1,25.26-trih )'drox),v italll in 25.26(OH),I),. 1.24.25(O I-l) .,D,I' and 1. 25.26 (0 1-1) .,)).1' Vitamin D, me ta holi tes wcre separated by usin g a Hewlett-Packard I090M high-pcrfor DJ 11. 25.2('(0 1-l).,DJ I. and 9-(- I ~A were gifts (i'om Dr. Milan rt. Uskokovic and Dr. Peter Bo lI "g at I-I offmann La R oche (Nutley. NJ). AII -lm1lS-reti ll oic IlJaIlCC liquid c hrolllatography sysr C llt w ith a c he rn worksta t. io n. :l pilo t acid (I-R A) was purcha sed frol1l Sigma (S t. LOlli s. MO ). Ketoconazole wavelength of 264 nm, and a Sphcri sorb o n S I column of 4.6 X 200 nll11 . powder wa.' a gift fi'om Dr. Gccrt Cau wcnbergh at J ohnson and J ohnson An isocratic elution with ')0% solvent A (acetonitrile) and 10% solvent B (S killlllan. NJ) and Dr. Allnie I-I erelllans atJ'lII ssen I'ha,.,lIa ceuti c,,! (B eerse. 10 .0(15 M anll11 ol1ill1n acetate :a ceri<: acid. 100: I (v ol/vol),1 was achi eved Bel gilll1l ). Deoxycytidinc 5'-1a- ..12 Pltriphosplw tc and 25-I ' I-IJ O I-ID., (25- with a fl ow rate of U. 7 ml l l1,il1 at 26°C fo r 22 min . The elHlI ent fi'om thc 0 1-l1 26.27-lII clli yl-Jl-ll cholecalciferol) were purcha se d li'olll DuPo ll t-NEN colul11n Il owed directl y il1 to a Radio l11 ,ltic l11 0del 295A now through li quid (Boston. MA) and Alll ershalll Life Science (Arlington I-I eights. IL) . res pec sci ntiUatioll spcctro lllc tc r fo r quallfitatio n of triti uI11 in each o f the separated ti vel),. r1 lCtBho lircs . T reatment of Subjects and Procurement of Tissue Solu tions of Statistical Analysis Comparisons of mean levels of 24 -0 1-l ase m ftNA 1. 25 (0 1-1 )21), (O.()(l04 'YO •. 1I .lliI 2%. 0.0 I %. and 0. 05%) . 9-(-rtA (0 .1 %). I-RA a/\long treated sites were l11ad e with either the paired I' tes t or th e repeated ItlC:lSUrCS analysis o f vari:lll c t~ . All p values arc rw o-sided. SUllullary statisri cs (1) . 1' Yo ,). kctoconazole ('I %). 1.25(0I-l )o DJ pillS 9 - c- I ~A (0.002% and ll.'l %. respecti vely), 1. 25(OI-l ), D, plus I - I ~A (O. O() 2% and 0 .1 %. respecti vely). alld arc expressed as IllCOlns ± SEM . Data analysis w as pcrfo rlllcd w ith dlC use of the Mi chi gan Intera ctive Data All alys is Sys tem (M IDAS ). a sta tisti cal 1.25(01-I),Dol pillS ketoconazole (O. 002' Yo , and 1%. respeCli ve ly) were prepared in a vc hicl e consistillg of 95% ethanol alld propylelle glycol, 7:3 softw are p ac k~ gc developed at the Cel1ter to r Statisti cal Consulta tion and (vol/vol). Eac h solution also cOlltain ed the all ti-oxidant hlltylatl'd hydroxy I ~ese ar c h at the University of Mi chi gan . tolulcne (0 .5 IIl g per 1111 ). O ll e hundred micro li ters of the stll dy solutio", RESULTS were applied once to arcas 3 CI11 X 6 CI11 on buttock skin of norl1\al heal thy adu lts. Sites weI''' occlnded with Sa ran wrap and then covered with a 24-0Ha5e Gene Expression Is Induced by 1,25(OH)2D3 in tape-secllred li ght- proof dress in g fo r 2 d. Fo r treatl11 ent wi th ketoconazole Human Skin To dete rmine whether the 24-0Hase gene in plus 1.25(01-l ),D,I ' the stlld y sites were treated wjth the il1\ida zole for 3 h humnn skin can he regulate d ;11 l'iI,,, b y 1 ,25(OHh D • four diiferent befo re application of the cO l1lbina tion so lution. Treated areas were infil J con centrations o f th e hormone were applied once unde r o cclusion t rOl ted with I tXt li d oc:l ill c local an esthesia al1d biopsicd w irh a kcrfl to ll1 c (= 150 Jl.nl thi ck). J{ cra tO l11 ed specil1l ens were iml1l ediatel y fi'oze/1 in li qllid fo r 48 h . Northe rn blo t analysis re veale d a con centrati o n-depen nitrogt'n '\/ld stored at - 700 IIn tilllsed . All subjects gave inform ed written dent induction of the 24-0Hase m[~A le ve ls (Fig 1). [n vehicle CQ I1$cnt" ill a pro tocol th.n had received prior approval by the Ulliversity o f treated skin. the ml~A level was very low and often undetectable. Mic hi ga n Medi ca l Center In stiturio lwl R evie w Board. Fo r this reason , the data are expressed as fi'ac cions o f the level o bserved with O.05'X, 1,25(01-1)" 1),, the hig hest concentration Measurement of 24-0Hase mRNA Fo r ll1l!a sure l11 ent or 24-0 1-la se u sed. arbi traril y set at 1 00% (max imum induc tio n). As compared nrl ~N A . total ce lllllar RNA was ex tracted from kerato l11 e bi opsies obtained ilS dc'\crihcd :l bovc. The g ua nidiniull1 isothio natc-ccsiul11 chl o ri de proce with ve hic le treatme nt. the appli cation o f 0.002% I ,25(OH)2DJ dure was used as described (Eld er ('I al. 1990). RNA specics (20 Jl.g pe l' caused a small hut statisti call y sig nificant inc re ase in the mRNA " 'n'plc) were clectrophore ti call y size-fi 'acrionated on agarose gels and le ve l (p < (l.05; n = 5). transferred to d c ri vatized n ylo l1 111Cfllh r :lllcs . The blots were sequentia ll y hybridized again st .12 1'- labeled 24-01-la se cDNA and 3G B4 (Astrol1l ci nl. 24-0Hase Gene Expression by 1,25(OHhD3 Is Synergisti 199 1). DNA probes were prepared by randol11l,,;ming (B ochringer Mann cally Induced by 9-c-RA Afte r d e te rmining the dose-respo nse l1 e il11 . Indianapo lis. IN) and quantita ted by using a Phosphorlma ger (Mo c urve fo r 24-01-):l se mRNA induction by 1.2S(O H h D , applica leclli ar Dynamics. Sllllll )'va le. CAl as descrihed (Eld er ('1 al. 1990; Astro rn ('I ti o n , w e in vestig ate d whe the r the sllla ll increase in 24-0Hase VOL. 108 . NO.4 A I'IUL 1') 97 VITAM I N 1) SICNALING IN IIU MAN SK IN 1,\ ' 1'/1"0 515
* 100 -4Kb ~M (1) 0 80 24·0Hase t ..J~ Subject 1 Subject 2 100 l- Qj~ ~ ~ 60 ...... 3684 er .... ili O 50 E 0 ...J ~ Subject 1 Subject 2 03 9cRA 1,25(OH),D3 Induces I-Iu111an Epidermal 24- 0Hase Activity a nd Ketoconazole Inhibits the Induced Hydroxylase Activ F igure 2. 24-0Hasc gene expression in hUluan skin ;11 P;II() is ity To determine wh ether ketoco nazole C'ln indecd inhihit 24- increased synergistically by 1,25(OHhD, and 9-c-RA. Normali zed O I-Iase en zyme activity. we first studied the inducibility of thc 24 -0Hase mll...NA Icvels were detenllilled as described ill !\I(I(crinls (1/111 cytochrome P-4S0 enzyme activity in hUl1lan skin ;11 1';1''' by O.OS % Melhods. Error "ors, SEM (n = IS) . *1' < 0.05 /IS (I.00 2'X. 1. 25 (0I-lh D, : t p < 1.2S (OHhD occlusivc treatmcn t for 2 d. Epidermal cclls derived 0.05 vcrsus vehicle (opell bar). )) .1 ' 1. 25(01-1} ~ )) .1 (hatched bars). Fo r 3 from vehicle- and 1. 2S (OHh D ,-treated sites were assaycd tor 0.002% D, plu s 0. 1% 'J -r-ll...A (so li d bars) treatlllen .. , tbe bar height is ta ller than the addition of heights cau sed by trciltl\lent of each agelll alollc by th e transformation of 2S-0HD, to 24,2S(OH)2D ,. Compared to ve top (s tippled) segment (= synergisl\l). (Ill sel) Il...e sults from two rcpresellla hicle (25 ::!:: 15 pg pe r min per I11 g of protein). there was a I O-told rive subj ects. increase (p (l.OS; n = 5) in the fo rmation of 2.J., 2S (01-lh D , by 516 I N N T he concentration-dependent induction of 24-0Hase mRNA ><: ><: + + expression by 1 ,2s(OH)2D , indicates that in human skin ;11 lIillO, the ~ o~ '# 0 ~ ~ "'N N a" presence of only the VDR li gand is suffi cient to activate the gene. (30"'~ OUl (30 01.11 .r:.-0' 00.. '-0.c. . 00. . T he barely detectable 24- 0 Hase mRNA levels in no mlal (vehicle (!jMNt"lM (!jMNMM >Cl><:ClCl >Cl><:ClCl treated) skin and the J'Ob ust induction of tlus gene transcl;pt by 100 1- 24-0Hase 1,2s(OHhD) make this a potentially useful bioassay to test for cuM" 60 vitamin 0 activity of a compound in human Skill ;11 /1;110. T his >0 3684 appears analogous to cellular retino ic acid binding protein ~ ~ 50 «LO Subject 1 Subject 2 (CRABP)-II mRNA induction by topical retin o id in human skin. Z~ 40 Enhanced expression of thi s gene, w hose far upstream promoter 0:- contains a retinoic acid-responsive ele ment (Astrom CI (//. 1991), by E2 30 Ql C t an occlusive treatmen t with natural Or synthetic retino ids has > Ql .- () 20 predicted their retin o idal activity in human skin ;11 /1 ;110 (Grifriths CI -Ql ro .... (//, 1993; Elder ci (/ /, t 995). T he potential value of 24-0Hase gene -Ql 10 0:_Qla. expressio n as a measure of vitamin D activi ty in human skin, 0 however, would be greater than C RABP- II mIlNA induction for Vehicle .002% 1% .002% 03 .05% retinoids. T his is because, although retinoids typicall y give dose 03 Keto + 1% Keto 03 dependent responses clinicall y (i.e., erythema) and/or histologically (i.e., hyperplasia of the epidermis), 1,25(OH)2D) and its synthetic Figure 4_ 24-0Hase gene expression in human skin ;11 11;"0 by 1,25(OH)2D3 is increased synergistically with ketocollazole_ Nor analog calcipotl'i ene, which is cun'ently in clinical usc, do neither malized 24-01-lase mRNA levels were determined as described in Malaia/s (Kang and Voorhees, unpublished data; Kragball e, personal com a",1 Melhods. E n'Ol' hal'S, SEM (n = .15). "p < 0.05 "s 0.002')lu 1. 25(OH),03; munication). T hus, inductio n of 24- 0Hase mRNA provides a 'fP < 0.05 "CI'SIIS vehicle (open bars). 0 3' 1,25 (01-1) 20 3 (hatched bars); detectable 1,2s(OH)2D 3 target in the absence of any appreciable Keto. ketoconazole (so lid bars) . For (l.002% 0 3 plus 1% ketoconazole clinical or histologic activity in human ski n ;11 I' ; IJO. Furthermore, treatment. the bar height is taller than the addition of heights ca used by this enhanced expression of 24-0Hase mRNA by 1,25(OHhD3 treatment of each agent alone by the top (stippled bar) segment treatment correla tes w ith an in crease in 24-0 Hase enzyme activity. ( = synergism). (fllsel) Resul ts fi'om two representative subjects. T he abiJi ty of 9-c-RA to synerg is ticall y enhance the 24-0 Hase mRNA level induced by a low dose of 1 ,2s(OH hD) indicates that RXR is not a sil ent partner in RXR-VDR heterodimer complex 1,2s(OH)2D3 (260 ± 78 pg per min per mg of protein) (Fig 5). m ediated vitamin D sign alin g in human skin ;11 V;'1(' . T his ;11 lI;'m T his induced activity of24-0H ase by 1,25(OH)2D 3 treatment was finding is consistent w ith our ;11 1';lm data. In cell culture, human completely inhibited by addition ofketoconazole (1. X 10- " M) ex keratinocytes from normal adults transfected with a reporter gene lIillo (Fig 5). VD R E (DR.3)-lk-CAT (w here CAT is chlo ramphemicol acetyl DISCUSSION transferase) demo nstrated m ore than a doubling of chlorampheni col acetyl transferase activity when 9-c-RA was added to a low dose This study demonstrates that 24-0Hase gene expression in human of 1 ,2s(OH)2D3 (Li cl ai, t 997). T hi s chl oramphenicol acetyl trans epidermis is increased ;11 Il illO by topical appli cation of fe rase activity could be further in creased by overexpressing VDR t,2s(OH)2D 3' Furtherm ore, tlus stimulation is furthe r in creased and/ or RXR in the transfected keratinocytes, wluch strongly synergisticaLly by the presence of an RXR Li ga nd or a 24-0Hase i.ndi cates the direct con tri butio n of the receptors to the li gand inhibitor. mediated in ducibility. Although 9-c-RA is the o nl y naturalligalld ofRXR identified to date, it is also an agonist of l'lARs (Heyman cl ai , 1992; Allenby el (//, 1993). In addition, because 9-c-RA can isomerize to I-RA in human skin tissue (Duell cl (//, 1996), a contribution of RAR-VDR in vitamin D signaling after 9-r-RA. and 1 ,2s(OH )2D} co-treatment Vehicle ,.. cannot be excluded. T his is unlikely to be of m ajor significance in human ski.n , however, because in epidermal nuclear extract prep arations, we do not detect endogenous RAR-VDR heterodimers binding to VDREs (Li c/ (/ /, 1997). Furthermore, in o Ur ;11 I1;lro transfection system and in C57BL/6 adult Inice ill 11;110 , the use of RXR-specifi c ligand SR 11237 provided results that were compa rable w ith that of 9-c-RA, sy nergistically ellhancing 24- 0Hase .05% 03 mRNA induction by 1,25(OH)2D, (Li cl (//, 1997). The la ck of adequate toxicology data to assure safe use in humans prevented us ,.. from applying this synthetic compound to o ur normal volunteers (hence its appli cation to mice). Si milar to 9-c-RA, I-RA synerg istica Ll y enhanced 24-0Hase mRNA i.nduction with a low dose (0.002'10, ) of 1,2s(OH)2DJ' o 50 100 150 200 250 300 350 Compared to 1,2s(OH)2D} treatm ent alo ne, addition of I-RA increased the transcript level by 50%, and 9-c-RA did so by 76% 24,25 (OHh 03 Formation pglminlmg Protein (I' = 0.74 liS 50'1., in crease). The comparable magnitude of modest synergism caused by the two RA isomers with 1 ,25(OHhD, is not inconsisten t with R XR-VDR being the major heterodimer com F ig ure 5. 1,25(OH)2D3 stimulates 24-0Hase activity and kctocon pl ex in vitamin D signaling in human skin ;11 11 ; 110 . We have recently azoIc inhibits the stimulated 24-0Hase activity. Condi tions for ;11 reported that the sa me 0.1 "/t, solution of I-RA applied to human skin "ilm assay of epiderm al ce Us derived [rom vehicle- and 1.25(01-1)20 , pproximately 7 nM 9-c-RA in the viable epidemus treated sites and analytica l procedures are detailed in Materials am/ Mel h"ds. for 48 h yie lded a (Duell el (/ /, 1996). Dissociation constant (K,,) values reported for Emil' /,,//'s, SEM (11 = 5). ' 1' < 0.05 1'5 0.05% 1,25(0 1-l ),D, without kctocona zole. 0 3' 1.25(01-1 ),0, . Opell ba.r, vehicle trcatment; hatched bar. 9-c-RA binding to RXRs have ranged from 11 to 18 nM (Heyman 1,25(01-1 ),0 , treatment; solid bar. 1.25(01-1 ),0, treatment plus ketocon el (/ /, 1. 992; Levin cl (//, 1992; All enby c ( ai, 1993). T he Kd azoic (I X 10- 4 M). determination made with endogenous retino id receptors (RXR and VOL. 108, NO. 4 APRlL 1997 VITAMIN 0 SIGNALING IN H UMAN SKIN TN VIVO 517 RAR combined) in nuclear extracts from human epidemns, in 1,25(OH)2D3 required to produce a pharmacologic effect would winch the level ofRXR protein is 5-fold greater than that ofRAR minimize potential risk of hypercalcemia. protein, was 1.2 nM (Fisher et ai, 1994). Therefore, the 7nM 9-c-RA that forms after 0.1 % t-RA may be sufricient to activate skin RXRs. Indeed, although 0.1 % 9-c-RA treatment results in 260 11M Hfe thallk 511zall R chbille for her expe/tise ;11 applyillg the CO li/pOll lids alld 9-c-RA in stratum corneum-free epidermis (Duell e/ ai, 1996), no keratoll/illg of the treated areas, Ted A. Hall/i1toll for statisliw l allalysis, Lallra Vall significant difference in synergism with l,25(OHhD3 was detected Goor for th e i/lllsiratiolls, mid TOil, Kllczajda for secretarial ass is/alice. Hfe also between 9-c-RA and I-RA (Figs 2, 3) . Ihallk Li Qill for peifom,illg 1I0l1h em blot atl(llysis alld Dr. M .R . Hallssler for Although both t-RA and 9-c-RA augmented vitamin 0 signaling, prollidillg /lIl1l1all 24-0Hase eDNA. 5.K. is a recipiellt of Ih e D e,ma/ology 1,25(OH)20 3' at concentrations used in our study, did not increase FOlllldatioll , Gla;,"o Derll/atology C lill ;cal Career D ellelop,"e"t A ward. This work or decrease the abil.ity of t-RA or 9-c-RA to stimulate relevant was sllPported ill pat1 by the Babcock Elldo,vlI/elll allli the ) 0 /111.1011 alld )0/111.10 11 target genes. When the blots were stripped of 24-0Hase probes Co'poralioll. and reprobed with CRABP-U probes, we found, consistent with previous observations (Kang ct ai , 1995; Elder ct ai , 1996), that both t-RA and 9-c-RA markedly induced CRABP-II mRNA levels. REFERENCES Compared with a single treatment of /-RA or 9-c-RA, co-admin Allegretto EA. Shevdc N. Zou A. H o well SR . Boehm M F. H ollis BW. Pike JW: istration of each RA isomer with 1,25(OHhD3 did not influence Retinoid X receptor acts as a ho rmone receptor jll I, jlll) to induce it key metabolic the level of CRABP-II gene transcripts (data not shown). enzyme for 1.25-dihydroxyvital11in OJ. j Bioi C lIe", 270:23906-23909. 1995 AlI cnb), G. Bocqucl MT. Sau.nders M. Kazmer S. Speck Rosenberger M , Lovey A, In human skin ;11 1);110, neither 9-c-RA nor t-RA alone induced J. Kasmer 1', Grippo JF. Chambon P. l evin AA: Retinoic acid receptors and 24-0Hase mR.NA expression. Similar results were obtained from rctinoid X receptors: Interactions with cndogcnous rctinoic adds. Proc Nail Awd cultured human keratinocytes and mouse skin ;11 1) ;1)0 (Li e/ ai, Sci USA 90:30- 34. 1993 1997). By contrast, in mouse kidney systemic administration of Astrom A, Tavakkol A, Pettersson U. C romie M, Elder JT. Voorhees JJ: Molecular both natural and synthetic RXR ligands enhanced the expression of cloning of two human cellular retinoic acid-binding proteins (CltA13P). -' Bioi ctWI! 266: 17662-17666.1 99 1 the 24-0Hase gene without the presence of vitamin 0 (Allegretto 3 Bagot M, C harllc D, Lcscs MC . Pamphilc It, R.evuzJ: Immunosuppressive effects of et ai, 1995). This apparent tiss ue-specifi c gene regulation indicates 1.25-dihydroxyviranlin DJ and its analogue calcipo triol o n epidermal cell s. Dr J that, in addition to the receptors and their cognate I.igands, other Oem,.I,,{130: 424 -43 1, 1994 factors are involved in the process. The importance of relative ratios Berger U. \1(filson p. McClell and KA, Colston K, Hauss ler MR, Pike J W. Coombes ofVDR and RKR in determining the degree of ligand responsive RC: Immullocytochenlical detection of 1.25-dihydroxyv-iramin D receptors in no rmal human ti ss ue. ] e lill E"docri,,,,! J\t1ctab 67:607-61 3. "1 988 ness (Lemon and Frceman, 1996) and the identity of the RXR Boyle IT. Miravet l , Gray R W, Holick MF, De Lu ca HF: T he response oOntcstinal isoform in influencing VDR hetcrodimer transactivation of the calcium transport to 25-hydroxy and 1.25-dihydroxyvitamin 0 in nephrecto 24-0Hase genc by l,25(OHh03 and 9-c-RA (Kephart et ai, 1996) mized rats. E"doc"iu (lh~r:}1 90:605- 608. 1972 were recently demonstrated. It is also possible that as described for C arlberg C . Bcndik I. \Vyss A. Meier E. Struzenbecker lJ. Grippo JF. Hunziker W : Two nuclear signalling pathways for Vi(;1 l11ill D . Nature 361:657 -660. t 993 estrogen and thyroid hormone receptors (Chen and Evans, 1995; Chen JD. EV:l11s RM: A tra nscriptio nal co-repressor that interacts with nuclear H6r1ein c/ ai, 1995), co-activator and/or co-repressor proteins may hormone receptors. Natllre 377:454-457, 1995 be modulating VDR imd RXl~ activities. Therefore, tissue-re Chen Ml, Heinrich G . Ohya m:l Yl. O kuda K. Omdahl Jl. C hen TC. Holi ck MF: stricted expression of the receptors/rcceptor isoforms and/ or co Exprcssion of 25-hydroxyvitamin D.l -24-hydro :\,.... ylase m l"t..NA in cultured human keratinocytes. P,.,,( Soc EXf! Bioi Med 207:57-61. 1994 activators/ co-repressors may be responsible in part, for the differ Colston K. Colston MJ . Feldman 0: 1.25-d.ihydroxyvitamin OJ and malignant ential response to ligands observed in skin and kidney. melanoma: the presence of receptors and inhibito rs of cell growth in culture. Ketoconazole is a broad-spectrum P-450 inhibitor that has been Elldocrillo/"g), 108: 1 083-1 086. 198 1 DarWIsh !-1M . DeLu ca !-IF: Idcnti tic:ltion o f a t .25-dihydroxyvitamin D -respol1sivc used clinicall y in a variety of conditions ranging from fungal 3 clement in the 5' -flanking region of the rat calbindin D-9k gene. PI'(l( N atl Arml infections, seborrheic dermatitis, and psoriasis to prostatic carci Sci USA 89:603-607. 1992 noma (Farr e/ ai, 1985; Green et ai, 1987; Mahler e/ ai, 1993; DegrecfHJ. DeDoncker pI{.: C urrent therapy of dermatophytosis.} A lii Arml D(",,,w((I/ Degreefand OeOoncker, 1994). TopicaLly, ketoconazole has been 3 1:S25 - $30. 1994 safely used as a 2'Yo cream and shampoo formulations. W e found 1 % Duell EA, Astro m A. Griffiths CEM. Chambon 1'. Voorhees JJ: Human ski (( levels of rctinoic acid and cytochromc P-450-derived 4-hydro~.'yrctin o i c acid after topical ketoconazole to be cffective in syncrgistically increasing the 24- application of rctinoic 3(; id ill ' ); 11(1 compared [ 0 conccntrations required [ 0 OHase mR.NA level caused by a low dose of 1,25(OHhD3· The stimulate retinoic acid receptor-mediated transcription ill l1 ;tnJ. J CIi" I"" ('st marked inhibition of 1,25(OH)20 :I-induced human epidermal 24- 90:1269-1274. 1992 Ducll EA. Ka ll g S. Voorhees JJ : Retinoic acid isomers applied to hUI11 Horlcin Aj . Niiar AM, H e inzel T. Torch_in J. G loss 13 , Kurokawa R . R yan A, Kamci inilibits 1.25-dill ydroxyvitamill D 3-acriv:II'cd expressio n o f rJIC rat ostcocalcin Y. Soderstrom M, Glass C K, Rosenfeld MG: Ligand-independent repression by gene. Mol Cdl Bic,1 13:5907-5917, 1993 the thyroid hormone receptor I11 cdhtred by a nuclear receptor co-repressor. Mahler C , Vc rh elst J. Denis L: Kctocon3 zolc and li arozoic in the treatment of N aillre 377:397- 403. 1995 advanced prostatic cancer. C nllcer 7 1: I 068-1 073. 1993 Ka ne KF. Lang-man MjS, Willi:tl1ls GR: Antiprolifcrati ve responses o f two human Mjlde p. Hauser U, Sim on T, M;dl G. Enlst V. Hausslcr MR. • Frosch P. Itamerberg colon can cer cell lines to vita min D nrc differentially modified by 9-cis-rctinoic J EW : Expression of 1.25-dih ydroxyvil'ill1liJ1 D J receptors in nonnal and psoriatic acid . C allcer Res 56:623-632. 19% skin. J Ittl/ ( ~.'i ( D efllw(ol 97:230- 239. 199'1 Kang S. Duell EA. Fisher GJ . Datta SC. Wang ZQ. Reddy AP, Tavakkol A, Yi JY. Miill er K. Bendtzen K: Inhibitio n o rt11ll1lan T Iymphocytc pro lifc r:ltion aud cycokine Crjlliths CEM. Elder JT. Voorhees]]: Appli cation ofrctinoi to human skin ill II;"" production by 1.25-dihydroxyvitalllin D.\. DiU'c rel1tiai c fIccl'S on C D451'tA + and induces cpidcnl1al hyperplasia and cellular rcrinoid binding: pro teins charnctcristic CD45RO + cell s. A llioil/C/I/C//( il), 14:37-43. 1992 of rc tinoic acid by w itho ut measurable retinoid acid levels or irritation. J IlIItes t O h ya ll1~ V. O kuda K: Isolati on and ciwracterizfltion of;1 cytochrome P-4S0 from rat Ocrtlllllol 105:549-556. '1995 kidney m,itochOl ldria that c~ltaly zcs the 24-hydroxylnti oll of 25-hydroxyv'itamin J(:ln g: S, Duell EA. Kim I{J. Voorhecs J.J : Liarozolc inhibits Iluman e pidermal rctinoic D, . ./ Bioi C it el/C 266:8690-8695, 199 1 acid 4-hydroxylase activity and dilferentiaUy augments human skin responses to Ohyama Y. OZOIlO K, Uchida M. Shinki T , Ka to S. Suda V, Ya mamoro 0, Noshiro retinoic acid and retinol../ I",,,,sl Oemlnwl 107:183-187. 1996 M. Kato V: Idcll tific;ttioll of a vitamin responsive clement ill the 5'-Ranking Ke phart DO, Walfish PG. Dcluca H , Butt TR: R.etinoid X receptor isotype identity region of rh e rat 25-hydroxyvitalllin D . ~ 24-hydroxyl:l sc gene. J [J;jl! C i1 ctll eli.rects hurn;11I vitamin 0 receptor hcteroel imcr trallsaccivatio tl from the 24- 269:'10 545-10550, 1994 hydroxylase vitamin 0 response clcments in ycast. Mol Em/"c,.;" ,,1 1 0:408-419. Sasaki H . Harada H. I-Ianda Y, Mo rino H. Suzawa M. Shirnp E, KatslImata T. 1996 Masuhiro Y. Matsuda K. Ebihara K: Transcriptional activity of a Au orinated Kerner SA. Scott R.A, Pike JW: Scquence cle ments ill the human ostcocalcin gene vitamin 0 analog o n VOR-IlXR-mcdiatcd gene cxp,·cssion. Hiarlll!IIIisr,1' 3-.1: confer basa l activation tllld inducibl e respo nse to hormonal vi tamin DJ . Proc N flll Acml Sci USA 86:4455-4459. 1989 370- 377, 1995 KragbaUe K: Vitamin D, and skin diseases. A rcil OCl'lII alol Res 284:S30-S36, '1992 Smith EL, Walworth NO, H oli ck MF: E(fcc[ o f 1.25-dih ydroxyvit;ltllill D J on the Lemon DO. Freedman LP: Selective effccts of ligands on vitam.in 0 .1 rcccptor- and morphologic and biochemical di{fcrciltiluioll o f cultrcd human epidermal kcra rerino id X receptor-mcdiated gene activati on ill 1/ ;110. 1\1101 Cell 8io/1 6 :1 006 - '10 16. tinocytes grown in serum-free conditio ns. ) IIIIlest IJem",!ul 86:709 - 7 14, 1986 1996 Tanaka I-I. Abc E. Miya ura C: 1.25-DihydroxycholecHcrol and hUII1;lI1 mycloid Levin AA, Sturzcnbcckcr LJ . Ka zmer S. Bosakowski T, Huselton C, Allenby G, Speck le ukemia cell line (HL-60): the presence of cytosol receptor :mel induction of J. Kratzc.iscn C I. R osenberger M. lovey A, Grippo J F: 9-cis rctinoic acid diUc rcntiation. Bi"rit c/(c./ 204:713-1'19. 1982 stercoisome r binds and activates the l1u clear receptor RX R a. Nn!llre 355:359- Yu VC. Delscrt C. Andersen B. Holloway JM. Dcvary OV. Naar AM. Kim SY. Boutin 361, 1992 JM. Glass C K. Rosenfeld MG: fU(R heta: a corcglliator th"c enhances binding of Li XV, X ia o JI-I. Fcng X. Q in L. Voorhees JJ : Retinoid x rcceptor-specifi c li gands rc tino ic acid, thyroid hormo ne, and vit;lrl1in 0 receptors to their cogn:lfe response synergi stically upregul ates 1,25 di hydroxyvitamin Dr dcpendcnl' transcription ill clement. Cell 67: 125 1-1 266. 1991 epidermal keratinocytes ;" lJitro and ill I/i" O. J IIl11es! Demllltol '108:506-512. '1997 Z icrold C, Darwish HM, Deluca HF: Two vitamin D responsive elemcnts fUll ction in MacDonald I'N. Dowd DR. Nakajima S. Ga lli gan MA . Reeder MC , Hall ss ler CA. the rat '1. 25-dihyelroxyvitamin 0 24-hydroxylase prOI1lOl"cr. J lJicll ClwlII 270 : Ozato J{. Hauss le r Mil...: R etinoid X receptors stimulate and 9-cis retinoic acid 1675-1 678.1 995