Characterization, Expression, and Intmunohistochemical Localization of 3fi­ Hydroxysteroid Dehydrogenase/d5-d4 Isomerase in Human Skin

Martine Dumont, Van Luu-The, Eric Dupont, Georges Pelletier, and Fernand Labrie Medical Research Council Group in Molecular Endocrinology, CHUL Research Center and Laval University, Quebec, Canada

Three beta-hydroxysteroid dehydroge!lase/[..5-[..4 isomer­ entire coding region has been inserted in a pCMV expression ase (3,B-HSD) catalyses an obligatory step in the biosynthesis vector and transfected into human cervical carcinoma cells of all classes of hormonal steroids, namely, the oxidation/ (HeLa). The expressed enzyme efficiently catalyzes the isomerization of 3,B-hydroxy-5-ene steroids into the corre­ transformation of pregnenolone, DHEA, and dihydrotestos­ sponding 3-keto-4-ene steroids in gonadal as well as in pe­ terone into progesterone, 4-androstenedione, and 5a-an­ ripheral tissues. Because humans are unique with some drostane-3,B, 17,B-diol, respectively. Using the enzyme ex­ primates in having adrenals that secrete large amounts of the pressed in HeLa cells, we have shown cyproterone acetate, a steroid precursors dehydroepiandrosterone (DHEA) and its progestin used in the treatment of acne and hirsutism, as well sulfate (DHEA-S) and its exceptionally large volume makes as norgestrel and norethindrone, two steroids widely used as the skin an important site of steroid biosynthesis, we have oral contraceptives, to be relatively potent inhibitors, with Ki isolated and characterized eDNA clones encoding 3,B-hy­ values of 0.38 ,uM, 1.3,uM, and 1.2,uM, respectively. Im­ droxysteroid dehydrogenase/[..5-[..4 isomerase from a munohistochemical localization of 3,B-HSD, illustrated by human skin Agt11 library. The longest clone obtained con­ using an antibody raised against human placental 3,B-HSD, tains the entire coding sequence for type I 3,B-HSD (372 shows that the enzyme is localized in sebaceous glands. ] amino acids) as well as an additional 131 nucleotides in the Invest Dermato! 99:415 -421, 1992 5' -untranslated region. The insert of 1647 bp containing the

ecause the skin is rich in steroidogenic enzymes and is essential step in the biosynthesis of all steroids, namely, progester­ the largest organ in the human body, this tissue must be one, mineralocorticoids, glucocorticoids, androgens, and estrogens. considered as an important site of sex steroid forma­ Following purification of 3P-HSD from human placenta and de­ tion. In fact, the skin possesses all the enzymes required velopment of antibodies against the enzyme in rabbits [8,9], we have for the transformation of the steroid precursors of adre­ isolated and characterized a first cDNA species [6] and gene [7]. Bnal origin, namely, dehydroepiandrosterone (DHEA) and especially Recently, we have isolated a second 3P-HSD cDNA species that is its sulfate, DHEA-S, into active androgens and estrogens [1- 6]. the almost exclusive species expressed in the adrenals and gonads The first step in the conversion of DliEA and androst-5-ene- and was chronologically designated as human type II 3P-HSD [4] . 3p, 17P-diol (L15-diol) into 4-androstenedioJ1e and testosterone, re­ We have recently characterized the structure of the corresponding spectively, is catalyzed by 3p-hydroxystero id dehydrogenase/ L15- human type II 3P-HSD gene [10]. In the rat, three types of3P-HSD ~4 isomerase, hereafter called 3p-HSD. Because the same enzyme cDNA [11,12] have so far been characterized, and one bovine [13] also transforms C-21 steroids into the corresponding 4-ketoste­ and one macaque [14] eDNA clone have been described. roids, 3P-HSD is expressed, not only in classical steroidogenic tis­ In addition to the well-recognized importance of steroids in nor­ sues (adrenals, testes, ovaries, and placenta) but also in a large series mal skin physiology as well as in pathologic states, it is also quite of peripheral intracrine tissues [3,5,7]. In fact, 3P-HSD catalyzes an possible that steroids synthesized in the skin have some generalized effects in other organs and tissues. Considering the essential role of 3P-HSD in the biosynthesis of all steroids in the skin from the adrenal precursors DHEA-S and DHEA, we have cloned cDNA Manuscript received December 31, 1991; accepted for publication May 8, encoding 3P-HSD from a human skin eDNA library, inserted the 1992. cDNA in an expression vector, and expressed the functional eDNA This work was supported by the Medical Research Council of Canada, the Funds de la Recherche en Sante du Quebec, and Endorecherche, Inc. in HeLa cells. Immunohistochemical localization of 3P-HSD in Reprint requests to: Professor Fernand Labrie, MRC Group in Molecular human skin has also been performed by using an antiserum raised Endocrinology, CHUL Research Center, 2705 Laurier Boulevard, Quebec, against the purified human placental enzyme. Quebec G1V 4G2, Canada. Abbreviations: MATERIALS AND METHODS 3P-HSD: 3p-hydroxysteroid dehydrogenase/ t.5- L~.4 isomerase DHEA: dehydroepiandrosterone Materials All restriction enzymes and T4 DNA ligase were ob­ DHEA-S: DHEA-sulfate tained from Pharmacia Inc. (Canada), whereas [14C]DHEA and PCR: polymerase chain reaction (14C]androstenedione were from Dupont Inc. (Canada) HeLa cells

0022-202Xj92jSOS.00 Copyright © 1992 by The Society for Investigative Dermatology, Inc.

415 416 DUMONT ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

were obtained from the American Type Culture Collection (Rock­ purified by replating twice and grown in liquid culture. Phages ville, MD). Cyproterone acetate was obtained from Schering AG, were isolated by centrifugation for 1.5 h at 105,000 X g and DNA Germany, whereas Epostane was provided by Sterling-Winthrop was isolated by phenol extraction. EcoRI restriction fragments ob­ Research Institute, Rensslaer, New York and 4-MA was from tained from phage DNA were subcloned into Bluescript SK vector Merck Sharp & Dohme Research Laboratories, Rahway, NJ. (Stratagene, San Diego, CAl and plasmid DNA was prepared by alkaline lysis [16). Sequencing of double-stranded plasmid DNA Cloning and Sequence Determination A human skin Agt11 was performed according to the dideoxy chain termination method cDNA library obtained from Clontech Laboratories Inc. (Palo Alto, [17) using modified T7 DNA polymerase [18) (Sequenase Kit, CAl, was screened with human placental 3P-HSD cDNA (hp3P­ Unitcd States Biochemical Corp., Cleveland, Ohio). Oligonucleo­ HSD63) [6) labelcd with (a-32P)dCTP by the random primer tide primers were synthetized in our laboratory with a Biosearch method [15J as described [7J . Positive recombinant plaques were DNA synthetizer.

ACTCTTCTGTCC -1 20

hp hsk AGCTTTTAACAATCTAACTAATGCCCTCTCCAGGGTCACCCTAGGGTCACCCTAGAATCAGATCTGCTCCCCAGCATCTTCTGTTTCCTGGTGAGTGATTCCTGCTACTTTGGATGGCC -1

hp hsk ATG ACG GGC TGG AGC TGC CTT GTG ACA GGA GCA GGA GGG TTT CTG GGA CAG AGG ATC ATC CGC CTC TTG GTG AAG GAG AAG GAG CTG AAG 90 Met Thr Gly Trp Ser Cys Leu Val Thr Gly Ala Gly Gly Phe Leu Gly Gln Arg lle lle Arg Leu Leu Val Lys Glu Lys Glu Leu Lys 10 20 30

hp hsk GAG ATC AGG GTC TTG GAC AAG GCC TTC GGA CCA GAA TTG AGA GAG GAA TTT TCT AAA CTC CAG AAC AAG ACC AAG CTG ACA GTG CTG GAA 180 Glu lle Arg Val Leu Asp Lys Ala Phe Gly Pro Glu Leu Arg Glu Glu Phe Ser Lys Leu Gln Asn Lys Thr Lys Leu Thr Val Leu Glu 40 50 60 hp hsk GGA GAC ATT CTG GAT GAG CCA TTC CTG AAG AGA GCC TGC CAG GAC GTC TCG GTC ATC ATC CAC ACC GCC TGT ATC ATT GAT GTC TTC GGT 270 Gly Asp lle Leu Asp Glu Pro Phe Leu Ly s Arg Ala Cys Gln Asp Val Ser Va l lle lle His Th~ Ala Cys lle lle Asp Val Phe Gly 70 80 90

hp hsk GTC ACT CAC AGA GAG TCT ATC ATG AAT GTC AAT GTG AAA GGT ACC CAG CTC CTG TTA GAG GCC TGT GTC CAA GCT AGT GTG CCA GTC TTC J60 Val Thr His Arg Glu Ser lle Met As n Val Asn Val Lys Gly Thr Gln Leu Leu Leu Glu Ala Cys Val Gln Ala Ser Val Pro Val Phe 100 110 120

hp hsk ATC TAC ACC AGT AGC ATA GAG GTA GCC GGG CCC AAC TCC TAC AAG GAA ATC ATC CAG AAT GGC CAT GAA GAA GAG CCT CTG GAA AAC ACA 450 lle Tyr Thr Ser Ser lle Glu Val Ala Gly Pro Asn Ser Tyr Lys Glu lle lle Gln As n Gly His Glu Glu Glu Pro Leu Glu Asn Thr 130 140 1 50 hp hsk TGG CCC GCT CCA TAC CCA CAC AGC AAA AAG CTT GCT GAG AAG GCT GTA CTG GCG GCT AAC GGG TGG AAT CTG AAA AAC GGC GGC ACC CTG 540 Trp Pro Ala Pro Tyr Pro His Ser Lys Lys Leu Ala Glu Lys Ala Val Leu Ala Ala Asn Gly Trp Asn Leu Lys Asn Gly Gly Thr Leu 160 170 180 hp hsk TAC ACT TGT GCC TTA CGA CCC ATG TAT ATC TAT GGG GAA GGA AGC CGA TTC CTT TCT GCT AGT ATA AAC GAG GCC CTG AAC AAC AAT GGG 6JO Tyr Thr Cys Ala Leu Arg Pro Met Tyr lle Tyr Gly Glu Gly Ser Arg Phe Leu Ser Ala Ser lle Asn Glu Ala Leu Asn Asn Asn Gly 190 200 210

hp hsk ATC CTG TCA AGT GTT GGA AAG TTC TCC ACT GTT AAC CCA GTC TAT GTT GGC AAT GTG GCC TGG GCC CAC ATT CTG GCC TTG AGG GCC CTG 720 lle Leu Ser Ser Val Gly Lys Phe Ser Thr Val Asn Pro Val Tyr Val Gly Asn Val Ala Trp Ala His lle Leu Ala Leu Arg Ala Leu 220 2JO 240 hp hsk CAG GAC CCC AAG AAG GCC CCA AGC ATC CGA GGA CAG TTC TAC TAT ATC TCA GAT GAC ACG CCT CAC CAA AGC TAT GAT AAC CTT AAT TAC 810 Gln Asp Pro Ly s Lys Ala Pre Ser lle Arg Gly Gln Phe Tyr Tyr lle Ser Asp Asp Thr Pro Hi S Gln Ser Tyr Asp Asn Leu Asn Tyr 250 260 270 hp hsk ACC CTG AGC AAA GAG TTC GGC CTC CGC CTT GAT TCC AGA TGG AGC TTT CCT TTA TCC CTG ATG TAT TGG ATT GGC TTC CTG CTG GAA ATA 900 Thr Leu Ser Lys Glu Phe Gly Leu Arg Leu Asp Ser Arg Trp Ser Phe Pro Leu Ser Leu Met Tyr Trp lle Gly Phe Leu Leu Glu lle 280 290 JOO hp hsk GTG AGC TTC CTA CTC AGG CCA ATT TAC ACC TAT CGA CCG CCC TTC AAC CGC CAC ATA GTC ACA TTG TCA AAT AGC GTA TTC ACC TTC TCT 990 Val Ser Phe Leu Leu Arg Pro lle Tyr Thr Tyr Arg Pro Pro Phe As n Arg His lle Val Thr leU Ser As n Ser Val Phe Thr Phe Ser 310 J20 JJO Leu ~ ~- hsk TAT AAG AAG GCT CAG CGA GAT CTC GCG TAT AAG CCA CTC TAC AGC TGG GAG GAA GCC AAG CAG AAA ACG GTG GAG TGG GTT GGT TCC CTT 1080 Tyr Lys Lys Ala Gln Arg Asp Leu Ala Tyr Lys Pro Leu Tyr Ser Trp Glu Glu Ala Lys Gln LyS Thr Val Glu Trp Val Gly Ser Leu J40 J50 J60 Asn hp -A- hsk GTG GAC CGG CAC AAG GAG ACC CTG AAG TCC AAG ACT CAG TGA TTTAAGGATGACAGAGATGTGCATGTGGGTATTGTTAGGAGATGTCATCAAGCTCCACCCTCC 1185 Val Asp Arg His Lys Glu Thr Leu Lys Ser Lys Thr Gln End J70 hp ------______------______hsk TGGCCTCATACAGAAAGTGACAAGGGCACAAGCTCAGGTCCTGCTGCCTCCCTTTCATACAATGGCCAACTTATTGTATTCCTCATGTCATCAAAACCTGCGCAGTCATTGGCCCAACA 1J04 hp ------______------______hsk AGAAGGTTTCTGTCCTAATCATATACCAGAGGAAAGACCATGTGGTTTGCTGTTACCAAATCTCAGTAGCTGATTCTGAACAATTTAGGGACTCTTTTAACTTGAGGGTCGTTTTG ACT 142J hp ------______------______hsk ACTAGAGCTCCATTTCTACTCTTAAATGAGAAAGGATTTCCTTTCTTTTTAATCTTCCATTCCTTCACATAGTTTGATAAAAAGATCAATAAATGTTTGAATGTTAAAAAA 15J4

Figure 1. Comparison of the nucleotide sequences of human skin (hsk 3P-HSD17) (present data) and human placental (hp 3P-HSD63) eDNA. One-hundred thirty-one additional nucleotides are present in the 5' untranslated region of skin eDNA. The two nucleotide changes at amino acid positions 338 and 367 are indicated with the corresponding amino acids. VOL. 99, NO.4 OCTOBER 1992 HUMAN SKIN 3P-HSD 417

Construction ofExpression Vector and Transient Expression fragments were subcloned in the Bluescript KS vector and se­ iu Mammalian HeLa Cells A EcoRI-EcoRI fragment of human quenced in both directions. The recombinant plasmids were linear­ skin 3P-HSD (hsk3P-HSD) cDNA containing the entire coding ized with Not I and transcribed with [a_32 P] UTP (800 Ci/mmol) region, the 3'-untrans lated region and 131 nucleotides in the 5'­ (Amersham) to generate type I and type II specific cRNA probes of noncoding region was inserted downstream to the cytomegalovirus 315 nucleotides using T3 RNA [32P]labeled polymerase of the (CMV) promoter in the pCMV vector (kindly provided by Dr. transprobe T kit (Pharmacia LKB Biotechnology). Three to twenty Michael B. Mathews, Cold Spring Harbor Laboratories, NY). This micrograms of human total or poly(A)+ mRNA were hybridized construction was sequenced in both orientations and amplified in with 8 X 104 cpm, type I or type II cRNA probe at 35°C for 14 h. Escherichia coli DH5a competent cells. Recombinant plasmids Thereafter, RNA samples were diluted tenfold with 10 mM Tris­ (pCMV-hsk3PHSD) were prepared by the alkaline-lysis procedure HCI (pH 7.5), 300 mM NaCl, and 5 mM EDTA in the presence of [16] and purified by two cesium chloride-ethidium bromide den­ 40 mg/ml RNase-A and 250 U/ml RNase-Tl and incubated at sity-gradient centrifugations. Fifteen micrograms of vector were 37°C for 1 h. Samples were then treated with a final concentration used to trans feet human cervical carcinoma cells (He La) using the of 0.2 mg/ml proteinase-K and 0.5% sodium dodecyl sulfate for calcium-phosphate procedure [4,19]. Cells were plated initially at 15 min at 37°C, extracted, and purified as previously described 104 cells/cm2 in 75-cm2 Falcon culture flasks and grown in Dul­ [8,11]' RNase-resistant hybrids were analyzed by electrophoresis in becco's modified Eagle's medium containing 10% (v Iv) fetal bovine a denaturating polyacrylamide urea sequencing gel. Autoradiogra­ serum supplemented with 2 mM L-glutamine, 1 mM sodium pyru­ phy was performed at 80°C with two intensifying screens. vate, 100 IU penicillin/ml, and 100)1g streptomycin sulfate/ml. RESULTS Mock transfections were carried out with pCMV alone and the transfection efficiency was monitored by co-transfection of the Isolation and Characterization of 3P-HSD cDNA Clone pCMV-Hsk3P-HSD vector with the pXGH5 plasmid [20]. The from Human Skin A human skin cDNA library constructed in media were collected 24 h after transfection for the assay of GH Agtl1 was screened by using human placental3p-HSD eDNA clone secretion by RIA; cel ls were harvested by scraping with a rubber 3P-HSD63 as probe [6]. Five primary clones were isolated from policeman and suspended in 0.5 ml of buffer containing 50 mM about 400,000 plaques and their nucleotide sequence was deter­ potassium phosphate (pH 7.4), 20% glycerol, and 1 mM EDTA for mined [17] . The longest clone contains an open reading frame en­ enzymatic assay. coding 372 amino acids. The nucleotide sequence of the coding region of this cDNA is identical to that of human placental3p-HSD Enzymatic 3P-HSD Assay Transfected cells were incubated at previously reported [6] except for the substitution of two nucleo­ 37"C in 0.5 ml of 50 mM sodium phosphate buffer (pH 7.4) con­ tides, namely, the replacement of A by C at residue 367 (Fig 1) taining 20% glycerol, 1 mM EDTA, and 1 mM NAD+ for 1 h in changing Asn to Thr, whereas the replacement of C by T at residue the presence of the indicated concentrations of radio labeled steroid 338 conserves the Leu residue but deletes a BglII restriction site. substrate. The assay was performed essentially as described [7,8]. These two changes have also been observed in the 3P-HSD gene [7] Immediately after incubation, steroids were extracted twice from and have been attributed to polymorphism [22]. When compared the incubation medium with 2 ml of methylene chloride and the with the human placental 3P-HSD cDNA previously reported [6], organic phase was evaporated to dryness. The steroids were then separated by thin-layer chromatography on silica gel in the ben­ Zene-acetone (4: 1; v: v) system. Substrate and metabolite(s) were identified by comparison on each TLC plate with reference steroids and revealed by autoradiography. Radioactivity was measured by A liquid scintillation spectrometry. Immunoblot Analysis Immunoblotting was performed as de­ scribed [6] . Briefly, cells were Iyzed and electrophoresed on a 5- ~4- 15% sodium dodecyl sulfate polyacrylamide gel [21]. The gel was then transferred to nitrocellulose filters for 4 h at 60 V. Blots were treated three times for 30 min with 5% fat-free milk in phosphate­ DHEA- buffered saline (PBS) containing 0.1 % Nonidet P-40. Antiserum against 3P-HSD was added at dilution 1 : 2000 and incubated at4 °C for 18 h. Blots were then washed three times with PBS containing CONTROL TRANSF 5% fat-free milk and 0.1 % NP-40. After washing, (125I]-labeled goat antirabbit immunoglobulin was added at dilution 1 : 2000 and incubated for 4 h at 4 ° C. After three washes with the same buffer, B blots were exposed to Kodak XAR-5 films overnight at -80°C with intensifying screens. Immunohistochemistry Human skin from the mid-abdominal KDa region was fixed by immersion in Bouin's solution for 3 d, dehy­ drated in ethanol, and embedded in paraffin. Six-micron sections Were then cut and mounted onto glass slides. The sections were incubated overnight at 4°C with 3P-HSD antiserum [6,9] diluted 1: 500 in Tris-saline buffer (pH 7.6). The slides were then washed 42 - I!1 the same buffer and incubated at room temperature for 4 h with peroxidase-labeled goat anti-rabbit y-globulins (Hyclone, Logan, Dtah) diluted 1: 500. The peroxidase activity was revealed follow­ p T ing a 5-min incubation at room temperature in a medium contain­ c ing 0.05% diaminobenzidine and 0.01 % H 20 2. The sections were Figure 2. Expression of eDNA encoding human skin 3P-HSD in HeLa Ilot counterstained. cells. (A) Autoradiograph of thin-layer chromatography of the transforma­ tion of 1 ,uM [14c]DHEA by control mock-transfected cells (CONTROL) IUbonuclease Protection Assay Regions corresponding to nu­ and cells transfected with the pCMV-hsk3,BHSD expression vector. (B) cleo tides 688 - 907 for the human type I 3P-HSD and to nucleotides Immunoblot analysis of HeLa transfected cells. Lalle P, purified human 685-904 for the human type II 3P-HSD were obtained by amplifi­ placental 3P-HSD; ialle T, cells transfected with the pCMV-hsk3,BHSD cation using the polymerase chain reaction (PCR). The 220-bp vector; ialle C, mock-transfected cells. 418 DUMONT ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

TISSUE C S A P C S A P Table I. Kinetic Constants for Human Skin 3P-HSD Activity Transfected in HeLa Cells II PROBE I Vmax' Relative Specificity (nmole/min/mg (Vmax/Km) 320- Substrate Km·{!J.M) protein) (ml/min/mg) 300- 280- - DHEA 0.13 0.93 7.15 - Pregnenolone 0.33 2.43 7.23 260- DHT 2.1 14.12 6.72 240- , K.m and Vmax values were determined by Lineweaver-Burk plot analysis. Incubation 220- reactions were performed in duplicate as described in Materials a"d Methods with sub­ strate concentrations of 0.005, 0.01,0.025,0.050,0.1,0.25,0.5, 1,2.5,5, and 10 JIM. 200- NADH (1 mM) was used when DHT was the substrate.

180- mRNA, a ribonuclease assay was performed. This highly sensitive 160- assay permits the discrimination of a few base-pair mismatches that occur after annealing type I and type II 3P-HSD mRNA probes ~o .... the mRNA species extracted from human skin. As can be seen in Fig 140- 3, type I 3P-HSD mRNA is the exclusive mRNA species detectable in the skin and placenta, whereas in the adrenal, type II 3P-HSD mRNA was the only RNA species detected. These conclusions are 120- clearly supported by the presence of the expected full-length pr~­ tected fragment (220 nucleotides) when the type I cDNA probe IS used with skin and placental RNA as well as the occurrence of the expected small fragments of 98 and 64 nucleotides when the type II ,. cRNA probe was used. The opposite pattern was found when adre­ 100- • nal mRNA was studied, thus indicating the exclusive presence. of • type II 3P-HSD mRNA in the human adrenal. In agreement ~Ith our previous data [4], even with longer exposure, it was not pOSSible to detect type I 3P-HSD in human adrenal mRNA. . The homogenate of transfected cells was then used to determme 80- the affinity constant (Km) towards DHEA (C-19 steroid), Preg (C-21 steroid), and DHT (saturated steroid). As indicated in Table!, comparable relative specificities (Vmax/km) values of 7.15, 7.23, and 6.72 ml.mg/min were measured for pregnenolone, DHEA, and , dihydrotestosterone (DHT) transformation, respectively. HoW­ • ever, the Vmax calculated for DHT (14.12 nmoles/min/mg pro­ tein) is much higher than that of the unsaturated substrates DHEA/ • (0.95 nmoles/min/mg protein) and pregnenolone (2.4 nmoles Figure 3. Ribonuclease protection analysis of the distribution of human min/mg protein), whereas the Km was higher at 2.1 pM compare d types I and II 3ft-HSD mRNA in the human skin adrenal and placenta. with 0.13 pM and 0.33 pM for DHEA and pregnenolone, respec- Samples of total RNA from the human adrenal (10 Jig) (A), placenta (20 Jig) tively. . (P) , skin (20 I1g) (S), and yeast tRNA as control (20 I1g) (C) were hybridized as described in Materials and Methods with either probe (315 nucleotides). The In order to obtain more information about the properties of ~he longest protected fragment (220 nucleotides) corresponds to the homolo­ enzyme and the inhibition of its activity by steroids, we next studied. gous RNA species protected by the cRNA probe. Protected fra gments of98 the effect of cyproterone acetate, a steroid derivative used for the and 64 nucleotides correspond to predicted well recognized mismatches for treatment of hirsutism [23]' and norgestrel and norethindrone, rwO RNase A. Lalles corresponding to the adrenal and placenta were exposed to progestins widely used as contraceptives [24], as well as a Sa-reduc­ X-Ray film for 9 h, whereas those from skin and control were exposed for tase inhibitor, 4-MA [25] and a known inhibitor of 3P-HSD, Epos­ 9 d. tane [26]' on the activity of expressed human skin 3p-HSD. Dlxo plot analysis using two concentrations (0 .25 pM and 0.5 pM) °df DHEA as substrate shows Ki values of 0.12, 0.16, 0.38, 1.2, an 1.3 pM, respectively, for 4-MA, Epostane, cyproterone acetate, the present clone obtained from human skin contains an additional norethindrone, and norgestrel (Table II). . . 131 nucleotides in the 5' untranslated region (Fig 1), 35 of these As illustrated in Fig 4, a high level of3P-HSD immunoreactiVity nucleotides belonging to the first untranslated exon of the type I is seen in the sebaceous glands as well as sebaceous gland ducts, 3P-HSD gene [8]. Expression of Human Skin 3P-HSD in HeLa Cells As illus­ trated in Fig 2A, HeLa cells transfected with pCMV-hsk3PHSD Table n. Inhibition Constants of Steroid Derivatives on catalyze efficiently the transformation ofDHEA into androstenedi­ Human Skin 3P-HSD Transfected in HeLa Cells' one, whereas in mock-transfected cells, no enzymatic activity can be detected. As illustrated in Fig 2B, the 3P-HSD activity described ______S _te_ro_id______K_ I~{!J._M~) ____------above corresponds to the expression of a newly produced protein 4-MA 0.12 that migrates at the same position as purified human placental 3P­ Epostane 0.16 HSD, as revealed by immunoblot analysis using the antiserum raised Cyproterone acetate 0.38 against purified human placental 3p-HSD. Norethindrone 1.20 In order to determine with precision the relative level of expres­ ______N_ o_r~g~e_st_re_I______1_. 3_0______----- sion of the type I and type II 3P-HSD genes in human skin or the 'Inhibition experiments were performed at two fixed concentrations of D!{EA relative abundance of the two corresponding types of 3P-HSD (0.25 JIM and 0.5 JIM). K.i "alues were calculated by Dixon plot analysis. VOL. 99, NO.4 OCTOBER 1992 HUMAN SKIN 3P-HSD 419

Figure 4. Immunohistochemical localization of3P-HSO in human skin. Immunostaining for 3P-HSO can be seen in the cytoplasm of sebaceous gland (SG) and sebaceous duct (SO) cells. Magnification X 200.

whereas the surrounding tissue shows no significant immunoreac­ effect of increased sebaceous gland volume as well as being involved tivity. in the characteristics of lipid secretion by the sebaceous glands. It should also be mentioned that the metabolism of DHEA has been DISCUSSION found to be higher in the skin of hirsute compared to normal The present data show that the exclusive detectable 3P-HSD gene women [42]. expressed in human skin is type I with a coding sequence identical to The present observation of the immunocytochemical localization the gene exclusively expressed in the placenta [7]. When compared of 3P-HSD in sebaceous glands in the human skin is in agreement to the cDNA previously isolated from human placenta [7J, the with data obtained by histochemistry [1,47,48]. The localization of cDNA obtained from a human skin library contains, however, an 3fJ-HSD in sebaceous glands is also supported by the distribution of additional 131 nucleotides in the S'-noncoding region, thus coding enzymatic activity in different body sites of human skin during almost entirely the 5'-noncoding region, which contains 138 nu­ embryonic development [49] and in developed skin [32]. Microdis­ cleotides. sected sebaceous glands have also been shown to possess 3P-HSD The present data show that the Vmax of3P-HSD activity is much activity [32]' higher when DHT is used as substrate compared to the values mea­ It should be mentioned that more than 90% of 3P-HSD activity sured with pregnenolone and DHEA. This observation could be in forehead skin was localized in isolated sebaceous glands, although related to the fact that the transformation ofDHT requires only the some activity was also found in hair follicles, epidermis, and eccrine dehydrogenase activity, whereas transformation of DHEA and glands [32]. Three 3P-HSD activity was also found in the sebaceous pregnenolone requires both dehydrogenase and isomerase activities. glands from men with alopecia [50]. On the other hand, in axillary In fact, these two activities have been demonstrated to reside at two skin, most 3P-HSD was localized in sweat glands (both eccrine and distinct sites within the single 3P-HSD protein [27]. Km values for apocrine sweat glands are present in large numbers in axillary skin). DHEA and pregnenolone obtained with HeLa transfected cells The apocrine glands, like sebaceous glands, develop from the hair Were similar to the data obtained from HeLa cells transfected with follicle and form a much larger volume than sebaceous glands in the rat type I 3fJ-HSD [11]. pubic and axillary regions. Another argument supporting the role of Steroidogenic enzymes are well known to be present in human 3P-HSD in sebaceous gland activity is the finding that androst-5- skin. In fact, human skin has been shown to convert DHEA into ene-3p,17P-diol (i1S-diol) is a potent stimulator of sebum secretion 64-dione [28,29], which is then converted into testosterone [30- in the rat [51]. It should be mentioned that the conversion ofDHEA 32]. In addition, Sa-androstane-3fJ,17P-diol was a major metabolite into i14-steroids has also been observed in axillary apocrine glands when skin from back or scalp was incubated with [3H] testosterone [32] and in plucked hair follicles [51- 53]. [33] and i14-dione was found when skin was incubated with hair Both DHEA and i1S-diol require the action of3P-HSD as the first roots [34,35]. The physiologic importance of3fJ-HSD in the human step of enzymatic catalysis to form i14-dione and testosterone, re­ skin is clearly supported by the previous observation that D HEA can spectively. i14-dione is then converted into testosterone by the ac­ . stimulate sebaceous gland secretion in humans [2,36]. tion of 17p-hydroxysteroid dehydrogenase, whereas Sa-reductase At puberty, the increase in the secretion ofDHEA, especially the further transforms testosterone into DHT. The presence of 3fJ­ secretion of its sulfated form, DHEA-S [37], is associated with an HSD, 17P-HSD, and Sa-reductase in the sebaceous glands may increase in sebaceous gland size and sebum ~roduction [38,39], allow the sebaceous glands to develop fully in both boys and girls in Which frequently leads to problems of acne [40,41]. In fact, an utero and at puberty at the time of increased DHEA and DHEA-S elevation in skin 3P-HSD [42] and Sa-reductase [43,44] activities formation by the adrenals [54]. Three fJ-HSD is thus likely to playa has been described in hirsutism and acne. Because sebaceous gland major role during intrauterine life when the sebaceous glands de­ size has been found to be increased in hirsutism and acne [45,46] and velop to a state of maturity in both male and female fetuses, the 3fJ-HSD (present data) is apparently mainly localized in sebaceous androgens probably originating from the conversion ofDHEA and glands in the human, the observed elevation in 3P-HSD and andro­ DHEA-S by 3fJ-HSD [54]. gen formation in the skin of such patients [42] could be a cause or an In analogy with our recent studies that showed a close correlation 420 DUMONT ET , AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY between the levels of3jJ-HSD mRNA, protein, and enzymatic activ­ and sexual dimorphic expression of a liver-specific rat 3jJ-hydroxy­ ity as well as steroid production in bovine ovaries during the estrus steroid dehydrogenase/isomerase. Endocrinology 127:3237 - 3239, cycle [55) as well as in rat testis [5 6) and ovary [57) upon stimulation 1990 by hCG, changes in the level of 3jJ-HSD expression and activity 13. Zhao HF, Simard J, Labrie C, Breton N, Rheaume E, Luu-The V, could playa major role in androgen formation in sebaceous glands, Labrie F: Molecular cloning, eDNA structure and predicted amino thus controlling sebum secretion. This suggestion is well supported acid sequence of bovine 3jJ-hydroxy-5-ene steroid dehydrogenase/ 65-64 isomerase. FEBS Lett 259:153-157,1989 by the finding of a close correlation between 3jJ-HSD activity and in vivo sebum excretion rate in humans [58). 14. Simard J, Meiner MH, Breton N, Low KG, Zhao HF, Periman LM, Labrie F: Characterization of macaque 3jJ-hydroxy-5-ene sterOid Although the steroids produced in various components of the dehydrogenase/65-64 isomerase: structure and expression in ster­ skin are likely to have some systemic effects, it is most probable that Oidogenic and peripheral tissues in primate. Mol Cell Endocnnol the steroids synthesized in the skin from adrenal precursors exert 75:101-110,1991 major intracrine effects in the same cells where they are produced 15. Feinberg AP, Volgelstein B: A technique for radiolabeling DNA re­ [3). These steroids could also exert autocrine 9r paracrine effects on striction endonuclease fragments to high specific activity. Anal Bio­ neighboring cells. The availability of cDNA encoding the main chern 132:6 -13, 1983 steroidogenic enzymes expressed in the skin offers important tools 16. 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