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.