Androgens Attenuate Vitamin D Production Induced by UVB Irradiation of the Skin of Male Mice by an Enzymatic Mechanism

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1 ORIGINAL ARTICLE Androgens Attenute Vitmin D Production Induced y UVB Irrdition of the Skin of Mle Mice y n Enzymtic Mechnism Yingen Xue 1, Lee Ying 2, Ronld L. Horst 3, Gordon Wtson 2 nd Dvid Goltzmn 1 Cutneous exposure to UVB irrdition is n importnt source of vitmin D. Here, we exmined sex-specific differences in cutneous vitmin D production in mice. Both mle nd femle mice on vitmin D deficient diet mnifested vitmin D deficiency, with minerl normlities, secondry hyperprthyroidism, nd osteomlci. UVB irrdition significntly incresed vitmin D levels in the skin of femle mice nd normlized serum 25-hydroxyvitmin D 3 nd 1,25-dihydroxyvitmin D 3 levels, s well s minerl nd skeletl normlities. However, in mle mice, the vitmin D response to UVB ws ttenuted nd minerl nd skeletl normlities were not normlized. The vitmin D precursor, 7-dehydrocholesterol (7DHC), ws significntly lower in the skin of mle thn femle mice. This reduction ws due to locl ndrogen ction in the skin s demonstrted y cstrtion studies nd skin-specific ndrogen receptor deletion in mle mice, oth of which reversed the mle phenotype. Locl ndrogen regultion in the skin of the CYP11A1 gene, which encodes crucil enzyme tht metolizes cholesterol, 7DHC, nd vitmin D, ppered to contriute to the gender differences in UVB-induced vitmin D production nd to its reversl of vitmin D deficiency. Sex-specific, enzymticlly regulted differences in cutneous production of vitmin D my therefore e of importnce to ensure vitmin D sufficiency. Journl of Investigtive Dermtology (215) 135, ; doi:1.138/jid ; pulished online 2 August 215 INTRODUCTION Vitmin D is otined y mmmls either from dietry sources or y its production s choleclciferol (vitmin D 3 ) in skin exposed to sunlight. In cutneous production, exposure of the skin to UV light in the wvelengths nm (UVB) permits UVB photons to penetrte the epidermis nd y physicochemicl process strting with sorption of UVB energy y the B ring trnsforms 7-dehydrocholesterol(7DHC)to previtmin D 3, which is then isomerized to vitmin D 3. Photochemicl conversion of previtmin D 3 to lumisterol nd tchysterol is the mjor fctor tht prevents vitmin D intoxiction fter single prolonged exposure to the sun (Holick et l., 1981), nd photoproducts of sun-induced photoisomeriztion of vitmin D 3 hve lso een descried nd identified s suprsterol I, 1 Deprtment of Medicine, Clcium Reserch Lortory nd the Centre for Trnsltionl Biology, McGill University Helth Centre Montrel, Montrel, Queec, Cnd; 2 Children s Hospitl Oklnd Reserch Institute, Oklnd, Cliforni, USA nd 3 Hertlnd Assys LLC South Loop Drive, Suite 44, Ames, Iow, USA Correspondence: Dvid Goltzmn, Deprtment of Medicine, Clcium Reserch Lortory nd the Centre for Trnsltionl Biology, McGill University Helth Centre, Glen Site, 11 Decrie Blvd, Room EM1.322, Montrel, Queec H4A3J1, Cnd. E-mil: dvid.goltzmn@mcgill.c Arevitions: 7DHC, 7-dehydrocholesterol; DHCR7, 7DHC reductse; VDd, vitmin D deficient Received 2 April 215; revised 1 June 215; ccepted 3 June 215; ccepted rticle preview online 27 July 215; pulished online 2 August 215 suprsterol II, nd 5,6-trnsvitmin D 3 (We et l., 1989). 7DHC in ddition to serving s vitmin D 3 skin precursor cn lterntively serve s precursor of cholesterol, rection ctlyzed y the enzyme 7DHC reductse (DHCR7). Vitmin D 3 cn then exit the skin nd e converted, in the liver, to the most undnt circulting metolite, 25-hydroxyvitmin D 3 [25(OH)D 3 ], which cn then e converted, vi the ction of the 25-hydroxyvitmin D 1-lph hydroxylse enzyme, CYP27B1, to the ctive form 1,25-dihydroxyvitmin D 3 [1,25(OH) 2 D 3 ]. The 1,25(OH) 2 D 3 cn lso stimulte the degrding enzyme 24 hydroxylse, CYP24A1, thus fcilitting its clernce. 7DHC cn lso e metolized y cholesterol side chin clevge enzyme coded y cytochrome P45scc (CYP11A1) through hydroxyltion t C22 followed y C2 clevge of the side chin nd susequent metolism of the 7-dehydropregnenolone produced y existing steroidogenic enzymes (Slominski et l., 24, 29, 212, 213). CYP11A1 cn lso convert cholesterol to pregnenolone (Miller nd Bose, 211) nd cn metolize vitmin D y sequentil hydroxyltions strting t C2 (Guryev et l., 23; Slominski et l., 25, 212, 214; Tongko-On et l., 215). In contrst, 25(OH)D 3 is not sustrte for CYP11A1 (Slominski et l., 25). The CYP11A1 pthwy therefore ppers to e of mjor significnce in the skin (Slominski et l., 214). The cutneous production of vitmin D hs trditionlly een n extremely importnt source of vitmin D, which my, however, e limited y the intensity nd durtion of UV skin 215 The Society for Investigtive Dermtology

2 exposure (Greene-Finestone et l., 211). Thus, UV efficcy my e limited y fctors such s degree nd durtion of skin exposure, skin type in view of the fct tht melnin pigment in humn skin competes for nd sors the UVB photons responsile for 7DHC photolysis, time of dy of skin exposure nd seson of the yer during which exposure occurs, s well s geogrphic ltitude where the skin exposure occurs. However, reltively little is known out the regultion of cutneous vitmin D synthesis in vitmin D deficient (VDd) sttes, when UVB exposure is not limiting. Interestingly, genome-wide ssocition study exmining gene vrints ssocited with vitmin D deficiency, s defined y low serum 25(OH)D 3, identified severl loci, one of which is the DHCR7 locus, which would e determinnt of 7DHC levels in the skin (Wng et l., 25). In the present study, we exmined the cpcity of UVB skin exposure to reverse dietry vitmin D deficiency, which hd induced secondry hyperprthyroidism nd osteomlci in mle nd femle mice. We oserved differences in the responses of femle nd mle mice tht pper to e ndrogen sed nd enzymticlly regulted. RESULTS UVB irrdition incresed serum 25(OH)D 3 in gender-dependent mnner C57BL/6 mice were fed VDd diet or control diet from 3 weeks of ge nd irrdited with UV light for 1 week t 8 weeks of ge s per n cute exposure protocol (Supplementry Figure S1 online). Serum 25(OH)D 3 ws significntly decresed fter 6 weeks of VDd diet tretment in oth femle nd mle mice. After UVB irrdition, serum 25 (OH)D 3 incresed much more mrkedly in femle mice on VDd diet (8.62 ±.77 nmol l 1 vs ± 8.9 nmol l 1, VDd vs. VDd+UVB) thn in mle mice on VDd diet (9. ±.71 nmol l 1 vs ± 1.46 nmol l 1, VDd vs. VDd+UVB; Figure 1). Vitmin D 3 levels in the skin were not significntly different in femle or mle mice on control diet or on VDd diet; however, UVB irrdition incresed skin vitmin D 3 levels much more mrkedly in femle thn in mle mice (Figure 1). UVB irrdition meliortes vitmin D 3 deficiency in femle ut not in mle mice To investigte whether UVB irrdition meliortes vitmin D 3 deficiency, 3-week-old femle nd mle C57BL/6 mice were fed control diet or VDd diet for 8 weeks nd were irrdited with UVB t 5 weeks of ge s per chronic exposure protocol (Supplementry Figure S1 online). Serum 25(OH)D 3 decresed y 82% nd 77% in VDd diet treted femle nd mle mice, respectively; UVB irrdition significntly incresed serum 25(OH)D 3 levels from ±.53 nmol l 1 to ± 5.26 nmol l 1 in VDd diet treted femle mice ut only slightly incresed serum 25(OH)D 3 from ± 1.67 nmol l 1 to ± 1.22 nmol l 1 in VDd diet treted mle mice (Figure 2). Chnges in serum 1,25(OH) 2 D 3 levels generlly prlleled chnges in serum 25(OH)D 3 levels in femle nd mle mice on VDd diet nd fter UVB irrdition. Thus, serum 1,25(OH) 2 D 3 levels were similrly reduced on VDd diet (25. ± 1.4 pmol l 1 in femle mice nd 22.4 ± 2.24 pmol l 1 in mle mice); however, 1,25(OH) 2 D 3 levels were significntly higher in femle thn in mle mice fter UVB irrdition ( ± 9.94 pmol l 1 vs ± 4.4 pmol l 1, femle vs. mle; Figure 2). Both femle nd mle mice developed hypoclcemi, hypophosphtemi, nd secondry hyperprthyroidism of similr mgnitude fter 8 weeks of VDd diet tretment. UVB irrdition normlized serum clcium nd phosphorus in VDd diet treted femle mice ut not in VDd diet treted mle mice (Figure 2c nd d). After UVB irrdition, serum prthyroid hormone levels fell significntly into the norml rnge in VDd diet treted femle mice. In contrst, lthough prthyroid hormone levels fell in VDd diet treted mle mice fter UVB irrdition, the levels were not normlized (Figure 2e). UVB irrdition reversed the VDd diet-induced decreses in BMD nd BMC nd skeletl normlities in femle mice ut not in mle mice After tretment with VDd diet, BMD ws decresed y 17% nd 9%, nd BMC ws decresed y 18% nd 13% in femle mice nd mle mice, respectively. BMD nd BMC were normlized fter UVB irrdition of VDd diet treted femle mice ut not fter UVB irrdition of VDd diet treted mle mice (Supplementry Figure S2 nd S2 online). Associted with the minerl nd hormonl normlities induced y the VDd diet, osteoid volumes, osteolst numers, nd osteoclst numers were incresed in oth femle nd mle mice. These prmeters were normlized in VDd diet treted femle mice fter UVB irrdition. Although fter UVB irrdition osteoid volumes, osteolst numers, nd osteoclst numers were improved in VDd diet treted mle mice, these prmeters remined persistently higher thn in mle mice on control diet (Supplementry Figure S2c, S2d nd S2e online). Androgen locks the increse in 25(OH)D 3 fter UVB irrdition of the skin To exmine whether estrogen or ndrogen hs role in the production of vitmin D 3 in UVB-irrdited skin, we ovriectomized femle mice nd orchidectomized (cstrted) mle mice t 4 weeks of ge. Five-week-old mice were then treted with VDd diet for 6 weeks. At 1 weeks of ge, mice were irrdited with UVB for 1 week (Supplementry Figure S1c online). The results showed tht the ltion of estrogen did not ffect the response of femle mice to UVB irrdition (Figure 3 nd c). However, ndrogen depletion significntly incresed the response of mle mice to UVB irrdition such tht the increses were similr to those oserved in femles. Furthermore, fter UVB irrdition, vitmin D 3 levels in the skin of cstrted mice on VDd diet were sixfold higher thn the skin vitmin D 3 levels in shm-operted mle mice on VDd diet (45.86 ± ng g 1 vs ± ng g 1, cstrted vs. shm mice, respectively; Figure 3d). As well, fter UVB irrdition, serum 25(OH)D 3 in cstrted mice on VDd diet ws normlized nd ws threefold higher thn in shm-operted mle mice on VDd diet (97.4 ± 4.28 nmol l 1 vs. 3 ± 1.47 nmol l 1 cstrted vs. shm mice, respectively; Figure 3) Journl of Investigtive Dermtology (215), Volume 135

3 Serum 25(OH)D 3 (nmol l 1 ) Vitmin D 3 in skin (ng g 1 ) Femle Femle Control diet VDd VDd+UVB Mle Mle Figure 1. UVB irrdition incresed serum 25-hydroxyvitmin D 3 [25(OH) D 3 ] differently in femles nd mles. Three-week-old C57BL/6 mice were fed vitmin D deficient (VDd) diet or control diet for 6 weeks. The dorsl skin of mice ws shved nd exposed to UVB s per n cute exposure protocol. () Serum 25(OH)D 3 levels nd () Vitmin D 3 levels in the skin. Dt re expressed s men ± SEM (n = 6). Po.5 compred with control diet in mice of the sme sex. Po.5 compred with mice of the sme sex fed VDd diet. Po.5 compred with UVB-irrdited femle mice fed VDd diet. Androgen receptor skin-specific knockout mice showed incresed vitmin D production fter UVB irrdition of the skin of mle mice To exmine the locl role of ndrogens in UVB-induced vitmin D production in the skin, we generted ndrogen receptor skin-specific knockout mice (skin-ar KO) y crossing mice expressing the cre recominse under the control of the kertin- 14 promoter, with mice expressing floxed ndrogen receptor. PCR results demonstrted strong expression of 44 p excised AR nd confirming deletion of the skin AR (Figure 4), lthough fint residul levels of floxed AR nd (952 p) were oserved. The very low levels of residul floxed AR likely indicte tht recomintion efficiency ws slightly less thn 1%, or genomic DNA from non-kertinocyte tissues ws dmixed in the skin smple. In the wild-type littermtes, fter UVB irrdition, serum 25(OH)D 3 incresed y twofold in the VDd diet treted mles compred with non-irrdited VDd diet treted mles (33.31 ± 1.53 nmol l 1 vs ±.47 nmol l 1,VDd+UVB vs. VDd). In AR-skin KO mice, fter UVB irrdition, serum 25(OH) D 3 incresed y sixfold in the VDd diet treted mles compred with non-irrdited VDd diet treted mles (69.69 ± 4.43 nmoll 1 vs ± 1.3 nmol l 1,VDd+UVB vs. VDd; Figure 4). UVB irrdition incresed vitmin D 3 in the skin of VDd diet treted wild-type mle littermtes from ± 1.55 ng g 1 to ± 7.62 ng g 1, wheres in VDd diet treted AR-skin KO mice UVB irrdition incresed vitmin D 3 from ± 1.97 ng g 1 to ± ng g 1 (Figure 4c). Androgen decreses 7DHC levels through the ndrogen receptor in the skin To exmine the effect of ndrogen on cholesterol nd 7DHC levels in the skin, we mesured cholesterol levels in the skin Serum 25(OH)D 3 (nmol l 1 ) Femle Control diet VDd VDd+UVB Mle Serum 1,25(OH) 2 D 3 (pmol l 1 ) Femle c d e Femle Mle Femle Mle Femle Serum clcium (mg dl 1 ) Serum phosphorus (mm) Serum PTH (pg ml 1 ) Mle Mle Figure 2. UVB irrdition meliortes vitmin D 3 deficiency nd normlizes vitmin D deficient (VDd) diet induced hypoclcemi, hypophosphtemi, nd secondry hyperprthyroidism in femle mice ut not in mle mice. Three-week-old C57BL/6 mice were fed VDd diet or control diet for 8 weeks. At 5 weeks of ge, the dorsl skin of mice ws shved nd exposed to UV light twice weekly for 6 weeks s per chronic exposure protocol. Serum 25(OH)D 3 (), 1,25(OH) 2 D 3 (), clcium (c), phosphorous (d), nd prthyroid hormone (e) were mesured. Dt re expressed s men ± SEM (n = 6 per group). Po.5 compred with mice of the sme sex fed control diet. Po.5 compred with mice of the sme sex fed VDd diet. Po.5 compred with UVB-irrdited femle mice fed VDd diet

4 Serum 25(OH)D 3 (nmol l 1 ) c Vitmin D 3 level in skin (ng g 1 ) Shm Shm Control diet VDd VDd+UVB OVX OVX Serum 25(OH)D 3 (nmol l 1 ) d Vitmin D 3 level in skin (ng g 1 ) Shm Shm Cstrtion Cstrtion Figure 3. Androgen locks the production of 25(OH)D 3 induced y UVB irrdition of the skin. At 4 weeks of ge, femle mice were ovriectomized, nd mle mice were orchidectomized (cstrted); shm-operted mice were used s controls. One week lter, mice were fed vitmin D deficient (VDd) diet or control diet for 6 weeks. At 1 weeks of ge, the dorsl skin of mice ws shved nd exposed to UVB once dily for 7 dys (2.5 kj m 2 per dy). Serum 25(OH)D 3 levels nd vitmin D 3 levels in the skin were mesured in shm-operted (shm) nd OVX femle mice ( nd c) nd in shm-operted nd cstrted mle mice ( nd d). Dt re expressed s men ± SEM (n = 6). Po.5 compred with mice of the sme sex fed control diet. Po.5 compred with mice of the sme sex fed VDd diet. Po.5 compred with UVB-irrdited shm-operted mice fed VDd diet. nd 7DHC levels in the skin nd serum. The results showed no difference in skin cholesterol levels etween mle nd femle mice on control diet or VDd diet (Figure 5). This is consistent with previous studies tht hve shown tht vitmin D sttus does not lter cutneous cholesterol synthesis (Feingold et l., 1987). There were lso no differences in serum 7DHC levels etween mle nd femle mice fed control diet (Figure 5). Although there ws trend towrd n incresed 7DHC levels in the skin in oth femle nd mle mice on VDd diet, the results did not rech sttisticl significnce. However, 7DHC levels in the skin were significntly higher in femle mice thn in mle mice fed either control diet or VDd diet. Furthermore, cstrtion or conditionl deletion of the AR in the skin of mle mice mrkedly incresed skin 7DHC levels (Figure 5c nd d). Androgens influence gene expression of enzymes involved in 7DHC metolism in the skin We then exmined gene expression of 3-hydroxy-3- methylglutryl coenzyme A reductse (HMGCR), the key enzyme in cholesterol synthesis, DHCR7, which converts 7DHC to cholesterol, nd CYP111, which metolizes 7DHC to 7-dehydropregnenolone. This ws performed in 3- week-old mle nd femle mice, cstrted mle mice (5-week old), nd 3-week-old mle mice with skin-ar KO tht were fed VDd diet for 6 weeks. We found no difference of HMGCR mrna levels etween mle nd femle mice nd cstrted mle mice nd mle mice with skin-ar KO (Figure 6). DHCR7 mrna levels were significntly higher in femles, mle mice with skin-ar KO, nd cstrted mles fed VDd diet compred with mle mice fed VDd diet, which would not ccount for the higher 7DHC in femles, mle mice with skin-ar KO, nd cstrted mles fed VDd diet compred with mle mice fed VDd diet (Figure 6). In contrst, CYP111 mrna levels were significntly lower in femles, cstrted mles, nd mles with skin-ar KO fed VDd diet compred with mle mice fed VDd diet; this would pper to ccount for the lower 7DHC in mle mice (Figure 6c). DISCUSSION Our results demonstrte tht, in C57BL/6 VDd mle mice reltive to VDd femle mice, equivlent exposure of the skin to UVB irrdition results in lower skin vitmin D 3 levels, lower serum 25(OH)D 3, nd lower serum 1,25(OH) 2 D 3 levels. This is consistent with the study of Gormn et l. 212 who lso found, using VDd BALB/c mice, tht UVB significntly incresed serum 25(OH)D 3 levels in femles ut not in mles. In ddition, in our study, ndrogen receptor skin specific knockout mice nd wild-type littermtes were on mixed genetic ckground nd hd similr reltive responses to UVB s did cstrted C57BL/6 mle mice reltive to shm-operted C57BL/6 mice. Consequently, our oservtions do not pper to e specific to one strin of mice. The lower concentrtion of ctive vitmin D metolite in the VDd mle mice reltive to the VDd femle mice trnslted 3128 Journl of Investigtive Dermtology (215), Volume 135

5 Serum 25(OH)D 3 (nmol l 1 ) c Vitmin D 3 level in skin (ng g 1 ) WT WT Control diet VDd VDd+UVB Floxed AR WT AR Excised AR KRT-cre Skin-AR-KO into reduced cpcity to reverse secondry hyperprthyroidism nd osteomlci. Interestingly, UVB irrdition of the skin ws reported to e effective in correcting vitmin D deficiency nd suppressing secondry hyperprthyroidism in femle psychogeritric ptients, ut n equivlent study in mles hs not een reported (Chel et l., 1998). The lower skin vitmin D 3 levels in the UVB-irrdited VDd mle mice were ssocited with lower levels of its skin precursor, 7DHC. Reduction in estrogen levels y oophorectomy of femle mice did not result in mle phenotype, i.e., Skin-AR-KO Figure 4. The deletion of ndrogen receptor in the skin increses serum 25 (OH)D 3 nd vitmin D 3 in the skin induced y UVB irrdition. Three-weekold ndrogen receptor skin-specific knockout mice (skin-ar-ko) nd wildtype (WT) littermtes were fed vitmin D deficient (VDd) diet or control diet for 6 weeks. The dorsl skin of mice ws shved nd exposed to UVB once dy in the lst week s descried in the Mterils nd Methods section. (). PCR of the skin from skin-ar KO mice, from floxed AR mice, nd from WT mle littermtes is shown. Lnes 1, 2, nd 5 show the PCR results from the skin-ar KO mice (X ARflox Y; KRT-cre), Lnes 3, 4, nd 6 show the PCR results from the AR floxed mice (X ARflox Y), nd lne 7 shows the PCR results from WT mle littermte. Floxed AR, WT AR, nd excised AR products were 952, 855, nd 44 p, respectively. () Serum 25(OH)D 3 levels nd (c) vitmin D 3 levels in the skin. Dt re expressed s men ± SEM (n = 5). Po.5 compred with mice on control diet. Po.5 compred with mice fed VDd diet. Po.5 compred with UVB-irrdited WT mle littermtes on VDd diet. Cholesterol in skin (µg g 1 ) Femle control diet c 7DHC in the skin (ng g 1 ) 6, 5, 4, 3, 2, 1, Femle VDd 3, 2, 1, Femle control diet Mle control diet Femle VDd Mle VDd reduced levels of skin 7DHC or skin vitmin D 3 production fter UVB irrdition. In contrst, removl of ndrogens y cstrtion of mle mice did increse skin 7DHC nd skin vitmin D 3 production fter UVB irrdition, thus recpitulting the femle phenotype. Previous reports (Esvelt et l., 1978) showed tht the levels of vitmin D production fter exposure to UVB of mle rt skin in vitro ppered similr to wht we found in femle mice fter exposure to UVB in vivo. Asno ndrogen ws dded to the in vitro mlertskins,theseresults my e nlogous to our results in cstrted mles nd therefore my e consistent with our findings. We lso found increses in responses to UVB irrdition in mles tht pproximted the levels in femles fter conditionl deletion of the ndrogen receptor in kertinocytes in vivo,demonstrting the locl cutneous regultion of ndrogens in modifying the skin7dhcndvitmind 3 production fter UVB irrdition. We then exmined gene expression of enzymes ltering 7DHC synthesis nd metolism, i.e., HMGCR one of the key enzymes in 7DHC iosynthesis; DHCR7, which converts 7DHC to cholesterol; nd CYP111, which metolizes 7DHC to 7-dehydropregnenolone nd metolizes vitmin D to numer of side chin metolites (Slominski et l., 214). In our Serum 7DHC (ng ml 1 ) Femle control diet d 7DHC in skin (ng g 1 ) Mle control diet Mle control diet Mle VDd P<.5 P<.5 Cstrted mle VDd AR skin specific KO VDd Figure 5. Androgen decreses 7-dehydrocholesterol (7DHC) level in the skin. () Cholesterol levels in the skin. () Serum 7DHC levels. (c) 7DHC in the skin of femle mice fed control diet or vitmin D deficient (VDd) diet. (d) 7DHC levels in the skin of mle mice fed control diet or VDd diet nd in the skin of cstrted mice nd AR skin specific KO mice fed VDd diet

6 DHCR7 mrna level c reltive to GAPDH HMGCR mrna level reltive to GAPDH CYP11A1 mrna level reltive to GAPDH Mle Femle Cstrted mle Skin-AR KO mle Figure 6. Androgen decreses 7DHC reductse (DHCR7) nd increses cyp111 gene expression. Three-week-old mle, femle, skin-ar KO mice, or 5-week-old cstrted mle mice were fed VDd diet for 6 weeks. The totl mrna ws extrcted from the shved dorsl skin of mice for rel-time PCR nlysis s descried in the Mterils nd Methods section. HMGCR (), DHCR7 (), nd CYP11A1 (c) mrna levels were exmined using rel-time PCR nlysis nd normlized to glycerldehyde-3-phosphte dehydrogense (GAPDH) mrna levels. Dt re expressed s men ± SEM (n = 5). Po.5 compred with mle mice. study, we found no difference in HMGCR mrna levels etween mles nd femles. DHCR7 mrna levels were higher in femle thn mle mice nd were incresed in cstrted mle mice nd mle mice with skin-ar-specific knockout. Our studies indicte therefore tht gender-specific differences in the levels of 7DHC re not cused y ltertions in gene expression of HMGCR or of DHCR7. In contrst, gene expression of CYP111 ws lower in femle mice nd ws lso reduced in cstrted mles nd mles with skin-ar KO compred with control mle mice. Our findings re supported y previous study in which O'Shughnessy et l. (22) reported tht testiculr feminized mice with loss function of the ndrogen receptor nd cryptochidized mice hd decresed CYP111 mrna compred with norml mice. Furthermore, Hzr R et l. (213) reported tht trnsgenic mice with the ndrogen receptor overexpressed in Sertoli cells displyed upregulted CYP111 gene expression, nd Wu et l. (211) reported tht testosterone stimulted CYP11A1 gene expression in grnulos cells. Therefore, the reduced expression of CYP111 in femle, cstrted mle, nd AR-skin-specific KO mle mice in our studies is consistent with ndrogen regultion of this enzyme nd would pper to contriute to the higher levels of 7DHC nd vitmin D 3 production fter UVB irrdition in these mice. The mechnism wherey ndrogen regultes CYP111 gene expression requires further study; however, the present studies demonstrte n enzymtic locus of the regultion of cutneous vitmin D production. It is possile tht differences in susceptiility to UVBinduced vitmin D production etween mles nd femles underlie other cutneous phenomen tht hve een descried. Thus, it hs een reported (Thoms-Ahner et l., 27) tht mle mice developed more tumors nd more dvnced tumors compred with femle mice fter chronic exposure to equl doses of UVB. This ws found to e ssocited with the extent of oxidtive DNA dmge nd ntioxidnt cpcities. Kertinocytes express CYP27B1, llowing locl ctivtion of 25(OH)D 3 nd utocrine nd prcrine effects of 1,25(OH) 2 D 3. It is therefore possile tht reduced production of vitmin D in the mle mice fter UVB irrdition nd susequently of 1,25(OH) 2 D 3 my contriute to n ltered redox sttus, which my e influenced y the ctive form of vitmin D (Dixon et l., 213; Song et l., 213). Our study hs severl limittions. In view of the difficulty of isolting the epidermis from mouse skin, our mrna dt nd chemicl nlyses were performed on whole skin rther thn the epidermis, nd it would hve een preferle to perform these studies purely on the epidermis. We lso used mouse model where hir covering likely restricts the cpcity of UVB to optimlly stimulte vitmin D in the skin. Nevertheless, numerous studies hve used mouse models with shved skin to exmine the physiology of vitmin D fter UV exposure (Gormn et l., 212; Wng et l., 215). Finlly, there is very little evidence in the existing literture tht there re ny significnt sex differences in humns in response to the production of vitmin D from sun exposure. In study on trditionlly living popultions in Est Afric (Luxwold et l., 212), 25(OH)D levels were studied in Msi group composed of 57% women nd Hdze group composed of 84% men. The 25(OH)D levels in the two groups were 119. ± 26. nmol l 1 nd 19. ± 28 nmol l 1, respectively, nd, if one excluded 25(OH)D 2 (presumly derived exclusively from dietry sources) the 25(OH)D 3 would e 119 nmol l 1 in the predominntly femle group (Msi) nd 13.9 nmol l 1 in the predominntly mle group (Hdze), suggesting the presence of higher levels in the predominntly femle group. Mles nd femles were included in oth smples, however, nd the smple sizes were very smll nd would preclude significnt conclusions. A pper y Hddd 313 Journl of Investigtive Dermtology (215), Volume 135

7 nd Chyu (1971) reported sustntil elevtions of 25(OH)D levels in lifegurds; however, no reference ws mde s to the sex of the lifegurds precluding comprtive informtion on 25(OH)D in response to sunlight. In study tht exmined 25 (OH)D levels in lrge dtse (Kroll et l., 215), 8% of men were elow 3 ng ml 1 nd 7% of women were elow this level, which therefore represents only smll difference. In lrge representtive smple of dolescents living in Northern Irelnd (54 55 N), girls were found to hve higher levels of 25(OH)D 3 during summertime (Hill et l., 28), nd, in popultion-sed study of Norwegin dolescents living t 69 N (Oerg et l., 214), significnt difference in 25(OH)D 3 levels ws found etween oys nd girls, with higher rte of deficiency mong oys. Although the differences in the Norwegin study were ttriuted in prt to differences in sun seeking ehvior etween oys nd girls, it is possile tht incresed sensitivity of girls to UVB exposure might hve ugmented vitmin D 3 levels nd contriuted to lower vitmin D deficiency. Nevertheless, cross-sectionl oservtionl studies my e confounded y vrile dietry intke of vitmin D, nd fctors such s mlsorption tht my influence such intke, nd not solely reflect the influence of UV exposure. No humn studies hve to dte compred the cpcity to increse vitmin D from sun exposure in VDd mles nd femles, nd in popultions where dietry sources of vitmin D nd vitmin D supplements re limited this informtion in humns might e of importnce. In summry, our studies hve found sex-specific differences in cutneous production of vitmin D in mice, nd mechnism involving ndrogen regultion of CYP111 gene expression is implicted. If this oservtion cn e confirmed in humns, sex-specific strtegies might need to e considered to ensure vitmin D sufficiency in popultions where vitmin D insufficiency is endemic. MATERIALS AND METHODS Animls All niml experiments were reviewed nd pproved y the McGill University Animl Cre nd Use Committee. Mice were housed individully, exposed to n UV light-free 12-hour light drk cycle environment (Cler UV Tue Gurds, Pegsus Assocites) nd wter d liitum. The mice used in this study re descried in the Supplementry Mterils nd Methods online. UVB exposure Mice were shved on the dorsl region using electric clippers one dy efore UVB tretment. UVB-treted mice were irrdited, t distnce of 4 cm from the UV source, with nk of four unfiltered FS2T12 fluorescent sunlmps (Solrc Systems, Brrie, Ontrio, Cnd) emitting rod nd of UVR from 28 to 32 nm. The rdition output ws mesured efore ech tretment using UVX rdiometer equipped with 32-nm sensor (UVP). Approximtely 6% of the output ws in the UVB rnge (29 32 nm; Becklund et l., 21). Mice were individully irrdited in six-comprtment ox to prevent mice from sheltering ech other from the UVR. To prevent possile differences of UVB output in the different chmers, mice were rotted through the different chmers during the UVB tretment period. For cute UVB irrdition, 3-week-old wild-type or AR-skin KO mice or wild-type littermtes were fed VDd diet or control diet with vitmin D dded t 2.2 IU g 1 (control diet; Hrln Tekln, Mdison, WI) for 6 weeks. One week efore euthnsi, mice were shved nd irrdited for 13 minutes once dily (2.5 kj m 2 per dy), for period of 7 dys. An cute UVB irrdition protocol ws lso used for ovriectomized femle or cstrted mle mice nd the corresponding shm-operted controls. Thus, VDd diet ws egun t 5 weeks of ge (1 week fter surgery) nd continued for 6 weeks. UVB irrdition ws then dministered once dy, s per the protocol ove, in the finl week. For chronic UVB irrdition, 3-week-old mice were put on VDd diet or control diet for 8 weeks. Mice were irrdited t distnce of 4 cm from the UV source for 13 minutes (2.5 kj m 2 ) twice weekly from 5 weeks of ge for period of 6 weeks (Supplementry Figure S1 online). Serum nlysis Serum clcium nd phosphorus levels were determined y QuntiChrom clcium or phosphorus ssy kits (BioAssy Systems, Hywrd, CA), respectively. Serum 1,25(OH) 2 D 3 nd 25(OH)D 3 were mesured y ELISA (ImmunoDignostic Systems, Fountin Hills, AZ). Serum intct mouse prthyroid hormone ws mesured y n ELISA (Immunotopics, Sn Clemente, CA). Mss spectrometry nlysis Dorsl skin (1 2 g) nd serum (1 μl) smples were collected for totl cholesterol, 7DHC, nd vitmin D 3 mesurement. Both GC/MS nd LC-MS/MS pltforms were employed. GC/MS ws used for nlyses of totl cholesterol in the skin, nd LC-MS/MS ws employed for nlyses of 7DHC in the skin nd serum nd vitmin D 3 in the skin. The detiled description of the mss spectrometry is included in the Supplementry Mterils nd Methods online. Rel-Time PCR A RNesy firous tissue kit (QIAGEN, Vlenci, CA) ws used to extrct totl RNA from skin smples. Totl RNA ws reverse trnscried into complementry DNA (cdna) s previously descried (Xue nd Fleet, 29). HMGCR, DHCR7, nd CYP11A1 mrna (Hzr et l., 213) levels in the skin were exmined using reltime PCR. The mouse glycerldehyde-3-phosphte dehydrogense (GAPDH) gene ws used s housekeeping gene (Xue nd Fleet, 29). Bone phenotyping (one minerl density nd histomorphometry) nd sttisticl nlyses re descried in the Supplementry Mterils nd Methods online. CONFLICT OF INTEREST RLH is the owner nd director of Hertlnd Assys LLC, Ames, Iow, USA. The remining uthors stte no conflict of interest. ACKNOWLEDGMENTS We thnk Dr Guido Verhoeven nd Dr Krel De Gendt (Ktholieke Universiteit Leuven, Belgium) nd Dr Irin Agoulnik (Florid Interntionl University, Mimi, FL, USA) for shring nd providing ndrogen receptor floxed mice. This study ws supported y grnt from the Cndin Institutes for Helth Reserch (CIHR) to Dr Goltzmn. SUPPLEMENTARY MATERIAL Supplementry mteril is linked to the online version of the pper t

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