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Endocrinology Vol. 144, No. 5 2084-2091
Copyright © 2003 by The Endocrine Society

17ß-Hydroxysteroid Dehydrogenase Type XI Localizes to Human Steroidogenic Cells

Zhonglin Chai, Phillip Brereton, Takashi Suzuki, Hironobu Sasano, Varuni Obeyesekere, Genevieve Escher, Richard Saffery, Peter Fuller, Carla Enriquez and Zygmunt Krozowski

Baker Heart Research Institute (Z.C., P.B., V.O., G.E., C.E., Z.K.), Melbourne, Victoria 8008, Australia; First Department of Surgery and Pathology, Tohoku University, School of Medicine, Sendai, Miyagi 980-8575, Japan (T.S., H.S.); and Murdoch Children’s Research Institute (R.S.), Melbourne 3052, Australia; and Prince Henry’s Institute (P.F.), Melbourne 3168, Australia

Address all correspondence and requests for reprints to: Zygmunt Krozowski, Ph.D., Baker Heart Research Institute, P.O. Box 6492, St. Kilda Road, Central Melbourne, Victoria 8008, Australia. E-mail: zygmunt.krozowski{at}baker.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We searched expressed sequence tag databases with conserved domains of the short-chain alcohol dehydrogenase superfamily and identified another isoform of 17ß-hydroxysteroid dehydrogenase, 17ßHSDXI. This enzyme converts 5{alpha}-androstane-3{alpha}, 17ß-diol to androsterone. The substrate has been implicated in supporting gestation and modulating {gamma}-aminobutyric acid receptor activity. 17ßHSDXI is colinear with human retinal short-chain dehydrogenase/reductase retSDR2, a protein with no known biological activity (accession no. AAF06939). Of the proteins with known function, 17ßHSDXI is most closely related to the retinol-metabolizing enzyme retSDR1, with which it has 30% identity. There is a polymorphic stretch of 15 adenosines in the 5' untranslated region of the cDNA sequence and a silent polymorphism at C719T. A 17ßHSDXI construct with a stretch of 20 adenosines was found to produce significantly more enzyme activity than constructs containing 15 or less adenosines (43% vs. 26%, P < 0.005). The C719T polymorphism is present in 15% of genomic DNA samples. Northern blot analysis showed high levels of 17ßHSDXI expression in the pancreas, kidney, liver, lung, adrenal, ovary, and heart. Immunohistochemical staining for 17ßHSDXI is strong in steroidogenic cells such as syncytiotrophoblasts, sebaceous gland, Leydig cells, and granulosa cells of the dominant follicle and corpus luteum. In the adrenal 17ßHSDXI, staining colocalized with the distribution of 17{alpha}-hydroxylase but was stronger in the mid to outer cortex. 17ßHSDXI was also found in the fetus and increased after birth. Liver parenchymal cells and epithelium of the endometrium and small intestine also stained. Regulation studies in mouse Y1 cells showed that cAMP down-regulates 17ßHSDXI enzymatic activity (40% vs. 32%, P < 0.05) and reduces gene expression to undetectable levels. All-trans-retinoic acid did not affect 17ßHSDXI expression or activity, but addition of the retinoid together with cAMP significantly decreased activity over cAMP alone (32% vs. 23%, P < 0.05). Cloning and sequencing of the 17ßHSDXI promoter identified the potential nuclear receptor steroidogenic factor-1 half-site TCCAAGGCCGG, and a cluster of three other potential steroidogenic factor-1 half-sites were found in the distal part of intron 1. Collectively, these results suggest a role for 17ßHSDXI in androgen metabolism during steroidogenesis and a possible role in nonsteroidogenic tissues including paracrine modulation of 5{alpha}-androstane-3{alpha}, 17ß-diol levels. 17ßHSDXI could act by metabolizing compounds that stimulate steroid synthesis and/or by generating metabolites that inhibit it.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEQUENCING OF THE human genome has resulted in the identification of a huge number of new genes of unknown function. Some insights into their biological roles can be inferred from knowledge of the evolution of proteins in superfamilies. The short-chain alcohol dehydrogenase (SCAD) superfamily is involved in the oxidoreduction and oxidation of secondary alcohol groups and ketones such as in the interconversion of cortisol and cortisone or estradiol and estrone. Members of this family are thought to have evolved from enzymes that metabolized sugars, and over time substrates have come to include polyols, organic acids, retinoids, vitamins, prostaglandins, and steroids. A handful of the 200 or so SCAD members are highly conserved, allowing insights into the nature of the substrate for newly identified proteins, but most proteins are less than 30% identical and have sequences that are uninformative with respect to biological function. To confound matters, dehydrogenases acting on the same substrate can be highly divergent.

The important biological role played by the SCAD superfamily is exemplified by the 11ß-hydroxysteroid dehydrogenase (11ßHSD), 11ßHSD1 and 11ßHSD2 (1). These enzymes play pivotal roles in modulating tissue levels of glucocorticoids and are thus excellent mediators of paracrine and autocrine actions. 11ßHSD1 activates glucocorticoids by converting cortisone into cortisol and effectively amplifies levels of this hormone in liver, adipose tissue, and brain (2). Deletion of this gene shows that it plays a role in lipogenesis and insulin sensitivity and may thus be potentially important in atherogenesis (3). Recently overexpression of 11ßHSD1 in mouse adipose tissue confirmed these observations and underlined the importance of local glucocorticoid activation in visceral obesity and the metabolic syndrome (4). In contrast, 11ßHSD2 inactivates glucocorticoids, endowing specificity on the mineralocorticoid receptor in sodium-transporting epithelia (5) and in the placenta protecting the fetus from high circulating levels of maternal glucocorticoids (6). The syndrome of apparent mineralocorticoid excess, in which patients exhibit excessive sodium retention and severely elevated blood pressure, is a result of mutations in this gene (7, 8). Similarly, various 17ß-hydroxysteroid dehydrogenases (17ßHSDs) regulate the levels of active androgens and estrogens in a tissue specific manner (9), and mutations in the 17ßHSD3 gene lead to male pseudohermaphroditism (10).

In previous studies a novel dehydrogenase was identified by searching expressed sequence-tagged databases translated in six frames for conserved motifs of the SCAD superfamily (11, 12). The protein has been previously denoted Pan1b or retSDR2 (13), but demonstration of 17ßHSD activity necessitated renaming it to 17ßHSDXI. Although it is present in the eye extensive studies in cell-free systems have been unable to demonstrate retinoid metabolizing activity (12). However, transfected CHOP cells rapidly metabolize androstane-3{alpha}, 17ß-diol, 5{alpha}-androstane, 7ß-diol [3{alpha}-Adiol] (13). In the present study, we describe polymorphic forms of the messenger RNA and its effects on enzyme activity and further characterize substrate specificity, tissue distribution, and regulation of 17ßHSDXI expression in steroidogenic cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All steroids and carbenoxolone were from Sigma (St. Louis, MO) or Steraloids (Newport, RI) with the exception of 3{alpha}-Adiol, which was from NEN Life Science Products (Boston, MA). CHOP-C4 cells were a gift from Dr. James Dennis (Samuel Lunenfield Institute, London, ONT). Silica gel thin-layer chromatography (TLC) plates with aluminum backing (HPTLC-Alufolien Kieselgel 60) were from E. Merck (Darmstadt, Germany).

Preparation of constructs of 17ßHSDXI with various lengths of poly adenosine (pA) in 5' untranslated regions (UTRs)
Human 17ßHSDXI cDNA was amplified by PCR from a pcDNA1 clone using SP6 reverse primer 5'-GATTTAGGTGACACTATAG-3' and a 17ßHSDXI-specific forward primer containing 5, 10, 15, or 20 adenosines at the 5' end (5'-(A)nCACACCAAAC-3' where n represents 5, 10, 15, or 20). The PCR products were cloned into pGEM-T EASY vector (Promega Corp., Madison, WI). The 17ßHSDXI inserts containing 5'pA of various lengths were excised using EcoRI and subcloned into the pcDNA1 expression vector. All constructs were confirmed by DNA sequencing. Several preparations of plasmid DNA were used throughout the study and all gave similar results.

Estimation of enzyme activity of 5'pA variants and competition studies
For estimation of enzyme activity of the 5'pA variants, CHOP cells were transfected with 3 µg construct DNA using DEAE-dextran (14). Transfection efficiency was checked by transfection with ß-galactosidase-expressing plasmid and was always around 75%. Tritiated 3{alpha}-Adiol (8 nM) in serum-free RPMI 1640 medium (Invitrogen, Carlsbad, CA) was added 48 h after transfection and incubated further for 18 h. Steroids were extracted from the medium and separated by TLC with standard steroids as previously described (13, 15). For competition studies, serum-free RPMI 1640 medium containing 8 nM tritium labeled 3{alpha}-Adiol and 100 µM cold steroid competitors was added to CHOP cells that were transfected with either wild-type 17ßHSDXI or empty vector control DNA and incubated for 18 h. The labeled 3{alpha}-Adiol and its converted products were separated by TLC and analyzed as previously described (13).

Immunoblotting of Myc-tagged human 17ßHSDXI
The HUP1 antibody does not recognize 17ßHSDXI on Western blots. Therefore, the human 17ßHSDXI fragment was subcloned into pCDNA3–2M vector in frame with the N-terminal double-Myc-epitope (16). Recombinant plasmid and empty vector were separately transfected to HeLa cells by electroporation and cells harvested 24 h later (16). Approximately 20 µg total cell lysate per lane were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. C-myc-tagged proteins were detected by anti-c-Myc monoclonal antibody 9E10 followed by incubation with antimouse IgG-horseradish peroxidase conjugate and chemiluminescence reagent (NEN Life Science Products). The same blot was then washed with PBS and reprobed with anti-{alpha}-tubulin monoclonal antibody (Sigma) to show equal loading.

Preparation of the HUP1 antibody
The HUP1 antibody was raised in rabbits against a chimeric peptide consisting of the first and last eight amino acids of 17ßHSDXI joined by a cysteine residue included for ease of coupling to affinity columns. The sequence was thus MKFLLDILCIGYKMKAQ. The peptide was conjugated to diptheria toxoid for injection into New Zealand White rabbits and coupled to a Biogel column for immunopurification as previously described for preparation of the HUH23 antibody (17).

Immunohistochemistry
Human tissue was obtained at autopsy and from surgical pathology files at Tohoku University Hospital (Sendai, Japan). The study was approved by the Ethics Committee of the Tohoku University School of Medicine. Specimens were fixed in 10% formalin and embedded in paraffin/wax. Immunohistochemical analysis was performed using the immunopurified HUP1 (Pan1b) antibody as previously described (13). Negative controls incorporated normal rabbit IgG instead of primary antibody. Antibody (2 µg) was preabsorbed with 5 µg peptide antigen at room temperature overnight and incubated with sections of human liver as an additional negative control.

Intracellular localization
A DNA fragment encoding human 17ßHSDXI amino acids 2–300 was amplified using Pfu DNA polymerase from a cloned full-length cDNA clone in pCDNA1. The PCR primers were: forward 5'-ACAAAGAATTCGAAATTTCTTCTGGACATCCTC-3' and reverse 5'-TCAGTCTCGAGAAAACTAGGTGCTTATTGCGCT 3'. An EcoRI site was introduced in the forward primer and XhoI in the reverse primer (italicized). The PCR fragment was cloned in frame to the pEGFP-C1 vector (CLONTECH Laboratories, Inc., Palo Alto, CA) and confirmed by sequencing. After overnight transfection to mouse Y1 cells cultured on round coverslips, the cells were fixed with 4% formaldehyde and permeabilized with 0.5% Triton X-100. Enhanced green fluorescent protein-tagged human 17ßHSDXI protein was examined for intracellular localization by immunofluorescent microscopy with nuclei stained with Hoechst 33342 (Sigma).

Effect of cAMP and all-trans-retinoic acid on 17ßHSDXI expression and progesterone production in mouse Y1 cells
Mouse adrenal Y1 cells were grown to confluence in 35-mm plates in DMEM/F12 medium (Invitrogen) with 10% fetal bovine serum (CSL, Parkville, Australia). Cells were washed with OPTI-MEM (Invitrogen) and incubated in OPTI-MEM containing 1 mM dibutyryl cAMP and/or 0.5 µM all-trans-retinoic acid (Sigma) for 24 h. The medium was harvested for progesterone assay using the Ultra Progesterone ELISA test kit (Neogen, Lexington, KY). The 17ßHSDXI activities in the intact cells were determined after 1 h of incubation with tritium-labeled 3-{alpha} Adiol. Total RNA was then extracted from the cells using an SV Total RNA isolation kit (Promega Corp.) to determine the 17ßHSDXI expression level by RT-PCR. First-strand cDNA primed with oligo-(deoxythymidine) was synthesized from approximately 0.8 µg total RNA using a first-strand reverse transcription (RT) kit (Invitrogen). One tenth of the RT reaction was used as the template in subsequent PCR to amplify 17ßHSDXI using primers 5'-CAGCATTGAGTCTCTTGTC-3' and 5'-GCACATTGACTTCGAAAGT-3'. A pair of mouse ß-actin primers (5'-GGCTACAGCTTCACCACCAC-3' and 5'-GCAGATGTGGATCAGCAAGC-3') were used to amplify actin mRNA from the RT samples as controls.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
17ßHSDXI cDNA is colinear with retSDR2
The full-length 17ßHSDXI cDNA was derived from a pregnant human uterus library after identification of a partial clone in expressed sequence tag (EST) databases with homology to the SCAD superfamily (Fig. 1AGo). The cDNA is 1407 bp in length and colinear with retSDR2, which was isolated from the retina (accession no. AF126780), but has shorter 5' and 3' UTRs. A consensus Kozak sequence was absent although the crucial G at -3 was present. The protein is 300 amino acids long and contains a potential N-linked glycosylation site at residue 228. Recently a clone with 98% identity to 17ßHSDXI at the protein level was isolated from an adenocarcinoma of the colon (accession no. BC008650). All three cDNAs contain an adenosine repeat in the 5' UTR (5'pA). A repeat of 14 adenosines is present in retSDR2, and there are 15 in 17ßHSDXI and the adenocarcinoma clone. A single nucleotide C719T silent polymorphism in the position of nt719 of retSDR2 is present in 17ßHSDXI and can be detected using the BsmA I enzyme that cuts in the position of the polymorphism with C (GTCTC) but not T (GTTTC). Sequence analysis of genomic DNA from 28 normal individuals showed that the T allele was present in 15% of cases. A 5'pA motif of 15 adenosines was present in all 52 samples of a normal population. In addition, retSDR2 contained two polyadenylation signal sequences (AATAAA) in the 3' UTR, and 17ßHSDXI as well as the colon adenocarcinoma clone used the first polyadenylation signal to add a polyA tail 12 nucleotides after the signal sequence (Fig. 1AGo). Western blot analysis was performed using an anti-c-myc antibody directed at 17ßHSDXI containing two N-terminal c-Myc epitopes (Fig. 1BGo). The results show that the enzyme migrates on SDS-PAGE gel with a molecular weight of approximately 32 kDA, which compares favorably with a calculated weight of 35,534 Da for the tagged protein.



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  		Figure 1. A, Molecular structure of 17ßHSDXI cDNA and comparison with retSDR2. Diagram of 17ßHSDXI cDNA is aligned with retSDR2. Nucleotide positions are indicated according to the cDNA sequence of retSDR2 (accession no. AF126780). The open reading frame (wide bar), UTRs (thin bar), and pA tail are shown. The 5'pA of 14-adenosine sequence [(A)14] in retSDR2 and 15 [(A)15] in 17ßHSDXI are shown. The silent polymorphism at position 719 is shown with C or T, and polyadenylation signals are represented by an oval-shaped symbol. B, Western blot analysis of the c-myc-tagged 17ßHSDXI protein. Western blotting was performed using anti-Myc monoclonal antibody (9E10) on a blot containing whole-cell lysates of HeLa cells transfected with empty vector DNA (lane 1) and Myc-17ßHSDXI construct DNA (lane 2). The same blot was reprobed with anti-{alpha}-tubulin monoclonal antibody to show equal loading.

 
17ßHSDXI belongs to the SCAD superfamily. Of proteins with known function, 17ßHSDXI is most closely related to the retinol dehydrogenase retSDR1, with which it is 30.3% identical, 22.9% identical with the cortisone reductase 11ßHSD1, and only 15.7% identical with 17ßHSD6, an enzyme with which it shares some substrate specificity (15). There exists a mouse ortholog 88% identical with the human sequence (accession no. AF304306). We also cloned and sequenced 2.2 kb of the 5' flanking region of the gene and identified the steroidogenic factor (SF)-1-binding site TCCAAGGCCGG 347 bp upstream from the translation initiation site (accession no. AY062939). A search of the Celera database identified another three SF-1-binding sites clustered in the distal region of intron 1.

17ßHSDXI metabolizes 3{alpha}-Adiol to androsterone
Screening of a range of radioactive steroids identified 3{alpha}-Adiol as the substrate with the highest rate of metabolism by 17ßHSDXI. The following tritiated steroids were not metabolized: androsterone, dihydrotestosterone, DHEA, testosterone, androstenedione, androstene-3ß, 17ß-diol, and estrone, but there was a small amount of conversion with estradiol (11). The two possible metabolites of 3{alpha}-Adiol are androsterone and dihydrotestosterone. Figure 2AGo shows that 3{alpha}-Adiol is converted to androsterone and proves that this enzyme is a 17ß-dehydrogenase. Further attempts to define kinetic constants showed nonsaturability with 3{alpha}-Adiol up to 100 µM (Fig. 2BGo). At this concentration DHEA inhibited metabolism of 3{alpha}-Adiol by 70%, but androsterone, 3ß-Adiol, and carbenoxolone did not affect activity of the enzyme. In other studies 50% inhibition of 3{alpha}-Adiol metabolism was achieved with 84 µM DHEA, 50 µM 13 cis-retinol, 31 µM all- trans-retinol, and 9 µM all-trans-retinal (data not shown), but we have been unable to demonstrate metabolism of these compounds by 17ßHSDXI overexpressed in transfected whole cells using HPLC analysis (13).



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  		Figure 2. A, Metabolism of 3{alpha}-Adiol to androsterone. Lane 1, [3H]-Androsterone standard; lane 2, [3H]-dihydrotestosterone standard; lane 3, [3H]-3{alpha}-Adiol incubated with CHOP cells transfected with p17ßHSDXI; lane 4, [3H]-3{alpha}-Adiol incubated with CHOP cells transfected with pcDNA1 vector. B, Competition by various compounds for [3H]-3{alpha}-Adiol metabolism in transfected CHOP cells. CHOP cells transfected with 17ßHSDXI expression plasmid (17ßHSDXI) or vector DNA (vector) were incubated with 8 nM [3H]-3{alpha}-Adiol and 100 µM competitors. Competitors are: no competitor (2, 3); 3{alpha}-Adiol (4, 5); dehydroepiandrosterone (6, 7); androsterone (8, 9); 3ß-Adiol (10, 11); and carbenoxolone (12, 13). Standards on the TLC plate are [3H]-3{alpha}-Adiol (S1) and [3H]-androsterone (S2).

 
The length of the 5'pA motif affects enzyme activity
EST database searches showed 10, 11, 13, 14, 15, 16, 17, and 20 adenosines in the 5'pA motif. Next we asked whether there might be a correlation of repeat length with 17ßHSDXI activity. We constructed 5'pA variants harboring 5–20 adenosine residues and transiently transfected CHOP cells and determined enzyme activity. Figure 3Go shows that there was no significant difference between wild type and constructs with 5, 10, or 15 adenosines. However, the construct with a 5'pA motif of 20 adenosines generated significantly more conversion than the next most active plasmid (43% ± 3.3% vs. 26% ± 3.6% for wild type, P < 0.005).



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  		Figure 3. Effect of length of the 5'pA motif on 17ßHSDXI activity. CHOP cells were transfected with p17ßHSDXI expression constructs containing 5'pA motifs of various adenosine length (5, 10, 15 and 20) as well as a wild-type p17ßHSDXI (15A motif) plasmid (WT) and empty vector DNA (V). The % conversion denotes metabolism of [3H]-3{alpha}-Adiol to [3H]-androsterone by the transfected CHOP cells after overnight incubation (n = 3, ± SEM). *, P < 0.02, compared with control.

 
Northern blot analysis shows a tissue-specific pattern of distribution
Northern blot hybridization showed a 17ßHSDXI mRNA transcript of about 2.1 kb (Fig. 4Go). High levels of 17ßHSDXI mRNA expression were seen in the pancreas, kidney, liver, lung, heart, small intestine, adrenal, and ovary, with significant levels also observed in peripheral blood lymphocytes. Lowest expression was seen in skeletal muscle, brain, stomach, thymus, prostate, and colon, although most tissues displayed some signal.



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  		Figure 4. Northern blot analysis of 17ßHSDXI expression in human tissues. Three commercial blots (CLONTECH Laboratories, Inc.) containing 2 µg pA+ RNA per lane were hybridized with full-length 17ßHSDXI cDNA probe. Blots were stripped and reprobed with a full-length ß-actin cDNA probe to check for loading of RNA. Lane 1, Pancreas; 2, kidney; 3, skeletal muscle; 4, liver; 5, lung; 6, placenta; 7, brain; 8, heart; 9, stomach; 10, small intestine; 11, thymus; 12; testis; 13, adrenal cortex; 14, thyroid; 15, adrenal medulla; 16, pancreas; 17, spleen; 18, thymus; 19, prostate; 20, testis; 21, ovary; 22, small intestine; 23, colon; and 24, blood leukocytes.

 
17ßHSDXI is present in steroidogenic cells
A striking cell-specific pattern of distribution was seen for 17ßHSDXI in human tissues. Immunostaining specific for 17ßHSDXI was evident in parenchymal cells of the liver but was absent from the central vein (Fig. 5aGo). Preimmune serum did not stain the liver (Fig. 5bGo) and immunopurified antibody preincubated with synthetic peptide also did not stain sections of human liver (results not shown). Staining was also seen in syncytiotrophoblasts of the placenta (Fig. 5cGo). In the ovary, only granulosa cells of the dominant follicle stained (Fig. 5dGo); this distribution differed from that of 17{alpha}-hydroxylase, which was restricted to the theca interna cells (Fig. 5eGo). The endometrium displayed glandular staining (Fig. 5fGo). In the functioning corpus luteum, luteinized granulosa cells stained for 17ßHSDXI (Fig. 5gGo), but 17{alpha}-hydroxylase was present in luteinized theca cells (Fig. 5hGo). In the testis immunostaining was present in Leydig cells (Fig. 5iGo). In the adult adrenal gland, 17ßHSDXI staining was present in the fasciculata/reticularis with higher levels evident in the outer cortex, but there was no staining in the medulla (Fig. 5jGo). Staining performed for 17{alpha}-hydroxylase in a serial section was coincident with the distribution of 17ßHSDXI but was of higher intensity toward the medulla (Fig. 5kGo). In a 1-d-old neonate, staining for 17ßHSDXI was present in the mid to outer cortex and was of lower intensity than in the adult (Fig. 5lGo). In the small intestine, surface epithelium was positive for 17ßHSDXI (Fig. 5mGo). The skin displayed staining only in sebaceous glands (Fig. 5nGo). Immunofluorescence studies localized 17ßHSDXI to the cytoplasm of the steroidogenic Y1 mouse cell line (Fig. 5oGo).



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  		Figure 5. Immunostaining of human tissues with the immunopurified HUP1 antibody. a, Liver; b, liver negative control; c, placenta; d, ovarian follicle; e, serial section of d stained with an antibody to 17{alpha}-hydroxylase (C17); f, uterus; g, corpus luteum; h, serial section of g stained with C17 antibody; i, testis; j, adult adrenal cortex; k, serial section of j stained with an antibody to C17; l, adrenal cortex from 1-d-old neonate; m, jejunum; n, sebaceous gland; o, immunofluorescence labeling of c-myc-tagged 17ßHSDXI (green) in transiently transfected mouse adrenal Y1 cells with the nucleus shown (blue).

 
Modulation of 17ßHSDXI during steroidogenesis
Because 17ßHSDXI staining localizes to steroidogenic cells, we sought to obtain evidence for a role of this enzyme in the steroid biosynthetic pathway. Mouse Y1 adrenocortical cells were treated with cAMP and all-trans-retinoic acid, two known stimulants of steroid synthesis. 3{alpha}-Adiol-metabolizing activity decreased significantly with cAMP treatment (40% vs. 32%, P < 0.05) but not with all-trans-retinoic acid alone (Fig. 6AGo). However, addition of the retinoic acid together with cAMP significantly decreased activity over cAMP alone (32% vs. 23%, P < 0.05). Message for 17ßHSDXI was reduced to undetectable levels by the addition of cAMP (Fig. 6BGo). In contrast, parallel measurements of progesterone production showed the expected 2- to 3-fold increases over control cells with all three treatments (Fig. 6CGo).



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  		Figure 6. Effect of cAMP and all-trans-retinoic acid treatment on 17ßHSDXI activity (A) and mRNA levels (B) in mouse Y1 cells. Lane 1, No-treatment control; lane 2, cAMP; lane 3, all-trans-retinoic acid; and lane 4, cAMP plus all-trans-retinoic acid. As a control for treatment efficacy, progesterone production was measured in the media of these cells with corresponding treatment (C; n = 3, ± SEM). *, P < 0.02, compared with no treatment control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have characterized an enzyme with 17ßHSD activity. This is the 11th isoform to be described. The protein belongs to the SCAD superfamily and retains conserved A-to-D domains with other members (18). The cDNA contains a polymorphic adenosine repeat in the 5'UTR, and other examples containing 10–20 adenosine repeats were found in EST databases. However, deviation from a 5'pA of 15 appears to be rare because we did not observe a single occurrence in a normal population of 52 subjects. We found higher levels of 17ßHSDXI activity when constructs containing 20 adenosines were expressed in CHOP cells. This effect may be mediated by poly-A-binding proteins that enhance binding of translation initiation factors and result in a higher efficiency of translation (19, 20). However, the structure of the human 5'UTR is not conserved in mouse, and no adenosine repeat exists in that species. The 5' flanking region of the human gene contains highly methylated CpG islands that bind to the methyl-CpG binding domain of the rat chromosomal protein MeCP2 (21). The gene was found at chromosome 4q21 by radiation hybrid mapping and sequencing by the human genome project.

17ßHSDXI is most active with 3{alpha}-Adiol as substrate in transfected CHOP cells. Currently we cannot rule out the possibility that 17ßHSDXI also has activity with other substrates, given that we observed endogenous 17-keto reductase activity in CHOP cells with a number of substrates during the course of these studies. However, further experiments with Y1, 3T3, and COS cells did not show enhanced 17ßHSDXI activity with the above-labeled substrates (results not shown). Nevertheless, the present data suggest that 17ßHSDXI participates in the pathway of androgen degradation whereby 3{alpha}-Adiol, which is directly produced by the metabolism of dihydrotestosterone, or indirectly from DHEA, is converted to androsterone. We were unable to demonstrate saturation of the enzyme with substrate, a property 17ßHSDXI shares with RoDH1 (22); previous studies show there is a considerable overlap in specificity between enzymes that metabolize 3{alpha}-Adiol and retinols (15, 23). 3{alpha}-Adiol is also metabolized to androsterone by 17ßHSD6, an enzyme that is 65% identical with the retinol dehydrogenase RoDH1, but CRAD2 exhibits 3{alpha},17ß hydroxysteroid and cis/trans-retinol catalytic activities, and RoDH1 converts 3{alpha}-Adiol back to dihydrotestosterone (DHT). This suggests that a pocket accommodating 3{alpha}-Adiol may also bind retinoids. Indeed, our studies show that high concentrations of retinoids can inhibit the metabolism of 3{alpha}-Adiol by 17ßHSDXI. However, using HPLC, we were unable to demonstrate metabolism by transfected whole cells of any retinoids. This finding is consistent with previous work employing membranes from insect cells (12). Carbenoxolone, a nonspecific inhibitor that blocks both 11ßHSDs and many 17ßHSDs, was unable to inhibit 17ßHSDXI activity.

Little is known about the biological actions of 3{alpha}-Adiol, although emerging evidence suggests that it is a hormone in its own right (24, 25, 26), and a few studies indicate an important role in parturition. Administration of 3{alpha}-Adiol increased live births in 5{alpha}-reductase deficient mice from 27% to over 90%. That this is not due to conversion to DHT is suggested by the observation that administration of DHT only partially restored levels. Furthermore, enzymes that lead to the production of 3{alpha}-Adiol are induced in the mouse uterus during the second half of gestation, leading to a peak in circulating 3{alpha}-Adiol-glucuronide levels during this time (27). In a subsequent study, it was shown that a failure to 5{alpha}-reduce androgens leads to excessive production of estrogens and thus to fetal mortality (28).

We also observed 17ßHSDXI immunostaining coincident with staining for 17{alpha}-hydroxylase, a marker of fasciculata/reticularis cells of the adrenal (29). This pattern of expression is consistent with a regulatory role for 17ßHSDXI in androgen synthesis/steroidogenesis. We suggest that 17ßHSDXI may not participate directly in the steroid biosynthetic pathway because two-dimensional TLC did not reveal metabolism of a pool of over a dozen steroids generated from H295R cells using labeled cholesterol, but 11ßHSD2 readily metabolized cortisol in this mixture (results not shown).

Although we observed evidence for 17ßHSDXI message in the adrenal medulla, we were unable to confirm the presence of the protein by immunostaining. This could be due to the nontranslation of this message or contamination of the medulla with cortex cells during tissue collection. 17ßHSDXI was also found in epithelia of the endometrium and small intestine. Androgens are thought to inhibit the growth of endometrial cells, suggesting that 17ßHSDXI may be important in lowering active androgen levels in this setting (30). Further evidence for a role of 17ßHSDXI in androgen action comes from our localization of this enzyme to the sebaceous gland in which previous studies have shown high 5{alpha}-reductase and 17ßHSD activity (31, 32). In addition, 17ßHSDXI is expressed at significant levels in lymphocytes in which recent evidence suggests metabolism of sex steroids (33). A large number of hydroxysteroid dehydrogenases have been described in the epithelium of the small intestine in which they may act on exogenous compounds to protect the organism from toxins of dietary origin. Airway epithelia also express significant amounts of the 17ßHSDXI in adult and fetal tissues (13).

The liver is clearly the organ with the highest amount of 17ßHSDXI and would most likely modulate plasma levels of 3{alpha}-Adiol, but paracrine effects of 17ßHSDXI are also important in organs such as the heart in which fibroblasts may produce 3{alpha}-Adiol (34). Interestingly, 3{alpha}-Adiol has been shown to act as a neurosteroid (25) and prevent changes induced by adrenalectomy in the brain (26, 35). Clinical studies suggest that it modulates central {gamma}-aminobutyric acid (GABA)ergic tone and that higher plasma concentrations could play a role in preventing anxiety (36). A decrease in cardiac 17ßHSDXI could result in higher local 3{alpha}-Adiol and changes in GABAergic tone in the heart. This may have deleterious effects given that stimulation of cardiac GABA receptors has been shown to enhance the cardiotoxicity of some agents (37).

Collectively, these results point to a role for 17ßHSDXI in androgen metabolism. Differences in cellular distribution and substrate specificity with other 17ßHSD isoforms suggest that this enzyme may also have an important role to play in modulating the paracrine actions of 3{alpha}-Adiol. Furthermore, in the present work, regulation studies showed an inverse relationship between steroid production in Y1 cells and 17ßHSDXI activity and gene expression. This observation, together with the finding of multiple potential SF-1-binding sites in the 17ßHSDXI gene, is consistent with a role in steroidogenesis (38). 17ßHSDXI could act by metabolizing compounds that stimulate steroid synthesis and/or by generating metabolites that inhibit it.


   Acknowledgments
 
The authors thank Michelle Cinel for sequencing, Benjamin Lamont for help with making the 5'pA constructs, Professor Jock Findlay for comments during the study, and Professor John Funder for insight and encouragement throughout this work.


   Footnotes
 
Abbreviations: 3{alpha}-Adiol, Androstane-3{alpha}, 17ß-diol, 5{alpha}-[9, 11-3H(N); 11ßHSD, 11ß-hydroxysteroid dehydrogenase; 17ßHSD, 17ß-hydroxysteroid dehydrogenase; DHT, dihydrotestosterone; EST, expressed sequence tag; GABA, {gamma}-aminobutyric acid; pA, poly adenosine; RT, reverse transcription; SCAD, short-chain alcohol dehydrogenase; SF, steroidogenic factor; TLC, thin-layer chromatography; UTR, untranslated region.

Received October 4, 2002.

Accepted for publication January 30, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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