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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 10 3669-3672
Copyright © 2000 by The Endocrine Society

Original Studies

3ß-Hydroxysteroid Dehydrogenase/{Delta}5->4-Isomerase Activity Associated with the Human 17ß-Hydroxysteroid Dehydrogenase Type 2 Isoform1

Takashi Suzuki, Hironobu Sasano, Stefan Andersson and J. Ian Mason

Department of Pathology, Tohoku University School of Medicine (T.S., H.S.), Sendai 980-8575, Japan; Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center (S.A.), Dallas, Texas 75235; and Department of Reproductive and Developmental Sciences (Clinical Biochemistry), University of Edinburgh (J.I.M.), Edinburgh, United Kingdom EH3 9YW

Address all correspondence and requests for reprints to: Prof. J. Ian Mason, Department of Reproductive and Developmental Sciences (Clinical Biochemistry), University of Edinburgh, Royal Infirmary of Edinburgh, Lauriston Place, Edinburgh, United Kingdom EH3 9YW. E-mail: j.i.mason{at}ed.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The type 2 isoform of human 17ß-hydroxysteroid dehydrogenase (17ßHSD2) efficiently catalyzes the oxidative metabolism of androgens and estrogens, and it is expressed in a large series of human peripheral tissues. To obtain a better understanding of the regulation of local steroid biosynthesis and metabolism in human tissues, we have established a dual steroidogenic activity of the 17ßHSD2 enzyme after transfection of human 17ßHSD2-transfected human embryonic kidney (293) cells. After transient transfection, the metabolism of testosterone, pregnenolone, and dehydroepiandrosterone (DHEA) in intact transfected 293 cells was evaluated by TLC-based radiometric assays. 17ßHSD2-transfected cells converted 91% of testosterone (1 µmol/L) into androstenedione in a 2-h incubation period. In addition, pregnenolone (1 µmol/L) was converted to progesterone (18.5%), whereas DHEA (1 µmol/L) was metabolized to androstenedione (8.3% conversion) in a 15-h incubation period. The kinetics of the 3ß-hydroxysteroid dehydrogenase (3ßHSD) and 17ßHSD2 activities using cell homogenate protein of stably transfected 293 cells indicated that the catalytic efficiency (apparent catalytic efficiency = maximum velocity/Km) of this 3ßHSD activity is approximately 2000-fold (pregnenolone as substrate) or 3000-fold (DHEA as substrate) weaker than that of 17ßHSD2 activity. It is noteworthy, however, that the apparent catalytic efficiency of the HSD3B2 gene product is only approximately 50-fold higher than that of the 3ßHSD aspect of the 17ßHSD2 gene product. Pregnenolone or DHEA effectively inhibited 17ßHSD2 activity in a noncompetitive fashion. Furthermore, the potent 5{alpha}-reductase/3ßHSD inhibitor, 17ß-N,N-diethylcarbamoyl-4-methyl-4-aza-5{alpha}-androstane-3-one, inhibited neither 3ßHSD nor 17ßHSD2 activities. We conclude that human 17ßHSD2 enzyme exhibits 3ßHSD activity. Notwithstanding that this 3ßHSD activity is reduced compared to 17ßHSD oxidative activity, it may account for at least some of the reports of 3ßHSD activity found in human peripheral tissues that express notable amounts of the 17ßHSD2 isozyme as well as in individuals with severe classic 3ßHSD deficiency.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
17ß-HYDROXYSTEROID dehydrogenase (17ßHSD) enzymes, which catalyze the reversible reactions between estrone and estradiol and between androstenedione and testosterone, are believed to play essential roles in regulation of the tissue levels of bioactive steroids. In recent years various isoforms of 17ßHSD have been identified (1, 2, 3, 4), and it is revealed that 17ß-reduction and oxidation of androgens and estrogens are catalyzed by different isozymes. 17ßHSD type 1 catalyzes the 17ß-reduction of estrogens (1), type 3 is the most efficient in the reduction of testosterone to androstenedione (3), whereas 17ßHSD type 2 (17ßHSD2) is NAD+ dependent and efficiently catalyzes the oxidative metabolism of estrogens and androgens (2). 17ßHSD2 enzyme is expressed in a large series of human peripheral tissues, including the placenta, liver, pancreas, kidney, small intestine, and secretory endometrium (5), and it is believed to be a key enzyme in the transformation of bioactive steroids to inactive compounds locally.

The findings of recent investigations have demonstrated that some oxidoreductase enzymes show dual steroidogenic activity (6, 7, 8, 9). 17ßHSD2 was also reported to exhibit 20{alpha}-hydroxysteroid dehydrogenase (20{alpha}HSD) activity (2). To obtain a deeper understanding of the regulation of local steroid biosynthesis and metabolism in human tissues, it is important to examine such multiple steroidogenic activities of 17ßHSD enzymes in detail. In this study we have investigated the possibility that human 17ßHSD2 possesses other steroidogenic activities, using the recombinant enzyme expressed in nonsteroidogenic human embryonic kidney 293 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Vector and plasmid

The pCMV6 vector containing the full-length complementary DNA (cDNA) encoding the complete amino acid sequence for human 17ßHSD2 was used (2).

Enzymatic activity assay in intact cells

To determine enzymatic activity in intact transfected 293 cells, plasmids were transiently transfected into 293 cells plated in 24-well cell culture clusters (Costar, Cambridge, MA), using lipofectin reagent (Life Technologies, Inc., Gaithersburg, MD). Seventy-two hours after transfection, cells were directly incubated at 37 C with 0.5 mL medium containing 1 µmol/L [3H]testosterone, [3H]pregnenolone, or [3H]dehydroepiandrosterone ([3H]DHEA). As tracers, [1ß,2ß-N-3H]testosterone (42.5 Ci/mmol; NEN Life Science Products, Boston, MA), [7-3H]pregnenolone (23.5 Ci/mmol; NEN Life Science Products), and [1,2-N-3H]DHEA (48.7 Ci/mmol; NEN Life Science Products), respectively, were used (5 x 104 cpm/500 µL medium). The final concentration of ethanol in the medium was adjusted to be 1% (vol/vol). Mock transfections were carried out as appropriate controls.

Enzymatic activity assay in cell homogenates

To obtain further information of the enzymatic activities, we also performed in vitro examinations using homogenized transfected 293 cells. After cotransfection of the 17ßHSD2 plasmid with a pcDNA3.1 plasmid (Invitrogen, San Diego, CA) that contained the neomycin resistance gene using lipofectin reagent (Life Technologies, Inc.), selection of stable transfectants using G418 (Life Technologies, Inc.; 0.5 mg/mL) was achieved after at least three cell passages. Cells were cultured in Falcon’s 100 x 20-mm tissue culture dishes (Becton Dickinson and Co., Lincoln Park, NJ), harvested in the logarithmic growth phase, and rinsed with phosphate-buffered saline (pH 7.4), and the protein was collected with assay buffer (12 mmol/L Tris, 1 mmol/L ethylenediamine tetraacetate, and 0.25 mol/L sucrose) before homogenization. As negative controls, nontransfected 293 cells were used. The protein concentration was measured by the method of Lowry, using the Coomassie protein assay reagent (Pierce Chemical Co., Rockford, IL).

The cell homogenate protein was incubated at 37 C in 500 µL assay buffer containing the indicated concentration of 3H-labeled steroids (5 x 104 cpm/500 µL assay buffer) and NAD+ (1 mmol/L) as cofactor in the presence or the absence of the various unlabeled competitor steroid. Cell homogenates were also incubated in the presence of the 5{alpha}-reductase/3ßHSD inhibitor, 17ß-N,N-diethylcarbamoyl-4-methyl-4-aza-5{alpha}-androstane-3-one (4MA; 1 µmol/L). As a positive control of 3ßHSD activity, 293 cells stably transfected with cDNA for human 3ßHSD type 2 (3ßHSD2) (10) were also used (10).

Extraction, separation, and quantification of steroids

In each experiment after the indicated incubation period the enzymatic reaction was terminated by addition of methylene chloride (6 mL). The organic phase was concentrated under a steam of nitrogen and separated by TLC on silica gel 60 plates (Merck & Co., Inc., Darmstadt, Germany) in a chloroform/ethyl acetate (4:1, vol/vol) system. Then the rates of product formation were compared using an Imaging Scanner System 200 (Bioscan, Inc., Washington, DC). Apparent Km and maximum velocity (Vmax) values were calculated by Lineweaver-Burk analysis.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enzymatic activities of human 17ßHSD2 in intact transfected 293 cells

We first the analyzed enzymatic activity of human 17ßHSD2 in intact transfected 293 cells after TLC analysis. When we incubated 17ßHSD2-transfected cells with testosterone (1 µmol/L) as substrate, these cells demonstrated high 17ßHSD2 activity, i.e. in a typical experiment, conversion rates were 35.9%, 74.6%, 86.1%, and 90.7% of testosterone to androstenedione in 0.5-, 1-, 1.5-, and 2-h incubation periods, respectively. We also used mock-transfected cells as a negative control, in which only 2.1% conversion was detected in a 2-h incubation period. When 17ßHSD2-transfected cells were incubated with pregnenolone (1 µmol/L), 3ßHSD activity was detected, and the substrate was converted to progesterone in yields of 5.7% and 18.5% in 6- and 15-h incubation periods, respectively. There was no detectable progesterone formation in mock-transfected cells in a 15-h period of incubation (<0.1%). The identity of the radiolabeled progesterone product was confirmed by recrystallization studies to demonstrate a constant specific activity (data not shown). DHEA (1 µmol/L) was also converted to androstenedione in 6-h (3.5%) and 15-h (8.3%) incubation periods, respectively, whereas no conversion was observed in mock-transfected cells in a 15-h incubation period. To obtain further information about this 3ßHSD activity of 17ßHSD2 enzyme, we performed the following in vitro experiments using cell homogenates.

Kinetic properties of 17ßHSD2 and 3ßHSD activities of human 17ßHSD2 enzyme

First, we examined the kinetic properties of 17ßHSD2 and 3ßHSD activities of human 17ßHSD2 enzyme. As illustrated in Table 1Go, the apparent Vmax of the 3ßHSD activity was much lower than that of 17ßHSD2 activity [3.6 ± 1.8 pmol/min·mg protein (pregnenolone as substrate) or 3.8 ± 0.4 pmol/min·mg protein (DHEA as substrate) for 3ßHSD activity, and 4670 ± 640 pmol/min·mg protein for 17ßHSD2 activity]. The 3ßHSD apparent catalytic efficiency (apparent Vmax/apparent Km) was 2000- to 3000-fold weaker than that for 17ßHSD. As negative controls, we used homogenates of nontransfected 293 cells under the same conditions, but no activities were detected.


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  		Table 1. Kinetic properties of 17ßHSD2 and 3ßHSD activities of human 17ßHSD2 enzyme using homogenates of transfected 293 cells

 
Competition by 3ßHSD substrates of 17ßHSD2 activity

To analyze whether these activities have a similar catalytic site on human 17ßHSD2 protein, we next performed competition experiments for 17ßHSD2 activity, using 3ßHSD substrates. As illustrated in Fig. 1Go, when homogenate protein (10 µg) was incubated with radiolabeled testosterone (0.1 µmol/L), in the presence of various concentrations of nonradiolabeled competitors, pregnenolone and DHEA inhibited 17ßHSD2 activity with IC50 values of 4.5 µmol/L for pregnenolone, and 9.5 µmol/L for DHEA (the corresponding IC50 value after testosterone addition was 2.0 µmol/L).



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  		Figure 1. Lineweaver-Burk analysis of the competition by pregnenolone (A) and DHEA (B) of 17ßHSD2 activity in homogenates of human 17ßHSD2-transfected 293 cells using testosterone as substrate. Homogenate protein (10 µg) of 17ßHSD2-transfected cells was incubated for 15 min with 0.1, 0.3, 1, 3, 10, or 30 µmol/L [3H]testosterone and NAD+ (1 mmol/L) in the presence of 0, 0.1, 1, 10, or 100 µmol/L nonradiolabeled pregnenolone (B) or DHEA (C). Data represent the average of two experiments.

 
Lineweaver-Burk analysis of the competition by 3ßHSD substrates of 17ßHSD2 activity using testosterone as substrate are demonstrated in Fig. 1Go, A and B. Apparent Km (micromolar concentrations) and Vmax (nanomoles per min/mg protein) values at each inhibitor concentration were as follows: testosterone only (no inhibitor), 2.3 and 4.0; 0.1 µmol/L pregnenolone, 2.0 and 3.2; 1 µmol/L pregnenolone, 2.1 and 2.3; 10 µmol/L pregnenolone, 3.1 and 1.9; 100 µmol/L pregnenolone, 3.0 and 1.3; 0.1 µmol/L DHEA, 2.1 and 3.5; 1 µmol/L DHEA, 2.0 and 2.3; 10 µmol/L DHEA, 2.2 and 2.1; and 100 µmol/L DHEA, 2.6 and 1.2. These data indicate that the apparent Km value is constant, whereas the apparent Vmax value is decreased as the competitor steroid concentration is increased, suggesting that pregnenolone and DHEA, substrates of 3ßHSD, inhibit 17ßHSD2 activity in a noncompetitive manner.

Effects of 17ß-N,N-diethylcarbamoyl-4-methyl-4-aza-5{alpha}-androstane-3-one (4MA) on 3ßHSD activity of human 17ßHSD2 enzyme

4MA is known as a potent 3ßHSD inhibitor, especially inhibiting dehydrogenase activity (11). We examined the effect of 4MA on dual activities of the human 17ßHSD2 enzyme.

As shown in Fig. 2AGo, 4MA (1 µmol/L) strongly inhibited both 3ßHSD reactions of human 3ßHSD2-transfected 293 cells (P < 0.001). Nevertheless, 4MA (1 µmol/L) inhibited neither 3ßHSD (Fig. 2BGo) nor 17ßHSD2 (Fig. 2CGo) activity in 17ßHSD2-transfected cells.



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  		Figure 2. Effects of 4MA on 3ßHSD activity of human 3ßHSD2-transfected 293 cells (A) as well as human 17ßHSD2-transfected 293 cells (B and C). A, Homogenate protein (100 µg) of human 3ßHSD2-transfected cells was incubated for 3 h with [3H]pregnenolone or DHEA (1 µmol/L, respectively) and NAD+ (1 mmol/L) in the presence or the absence of 4MA (1 µmol/L). B, Homogenate protein (300 µg) of human 17ßHSD2-transfected cells was incubated for 8 h with [3H]pregnenolone or DHEA (1 µmol/L, respectively) and NAD+ (1 mmol/L) in the presence or the absence of 4MA (1 µmol/L). C, Homogenate protein (10 µg) of human 17ßHSD2 transfected cells was incubated for 15 min with [3H]testosterone (1 µmol/L) and NAD+ (1 mmol/L) in the presence or the absence of 4MA (1 µmol/L). Data represent the mean ± SD of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study demonstrates that 17ßHSD2 enzyme exhibits 3ßHSD activity in both intact cells and cell homogenate protein of transfected 293 cells. The oxidative pathway of 17ßHSD activity (estradiol to estrone, and testosterone to androstenedione) is detected in almost all human peripheral tissues (12). It has been demonstrated that 17ßHSD2 enzyme is expressed in a large series of human peripheral tissues, including the placenta, liver, pancreas, kidney, small intestine, and secretory endometrium, and it is regulated by progestins (5). In our study although 17ßHSD2-associated 3ßHSD activity is low compared to oxidative 17ßHSD2 activity, these findings support the idea that the 17ßHSD2 enzyme in various human peripheral tissues exhibits 3ßHSD activity.

It is well known that the adrenals, ovaries, and testes are the major source of androgens and estrogens in humans, but in addition, peripheral tissues can synthesize bioactive sex steroids locally (12, 13). 3ßHSD is a crucial step in the biosynthesis of hormonal steroids. 3ßHSD activity is found not only in classical steroidogenic tissues, but also in a large series of peripheral tissues, where it contributes to the local formation of bioactive sex steroids (13). Two types of human 3ßHSD genes have been characterized (14, 15). Human 3ßHSD type 1 (3ßHSD1) gene expression is detected in the placenta, skin, and mammary gland (15), whereas 3ßHSD2 gene is expressed in the adrenal and gonad (14). However, 3ßHSD gene expression in other human tissues is uncertain. Milewich et al. (16) reported that 3ßHSD activity is easily demonstrated widely among peripheral tissues of the human fetus, although 3ßHSD protein was not readily detected by Western analysis. Our present data may account for the some of the 3ßHSD activity found in some human peripheral tissues, and 17ßHSD2 enzyme may have a broad role in the regulation of local steroid biosynthesis as well as metabolism. Furthermore, our studies demonstrate that 3ß-hydroxysteroids, e.g., pregnenolone and DHEA, can alter 17ßHSD2 activity. Therefore, the 3ß-hydroxysteroids are possible modulators of 17ßHSD2 activity locally.

The amount of 17ßHSD2 messenger ribonucleic acid is extremely high in human placenta (2). Wu et al. (2) also reported that this enzyme possesses 20{alpha}HSD activity and suggested that it may be important in maintaining high progesterone levels during pregnancy. Our data are in agreement with such an interpretation, and this secondary 3ßHSD activity of 17ßHSD2 may have a role to maintain a high level of progesterone during pregnancy, in cooperation with the 3ßHSD1 enzyme and the 20{alpha}HSD activity of the 17ßHSD2 enzyme. This second activity of 17ßHSD2 may also be important in the formation of 3-ketosteroids in peripheral tissues of individuals with severe classic 3ßHSD deficiency (17).

The dual activity of certain steroid-specific oxidoreductatases has been reported using a preparation of purified enzymes, e.g., 3{alpha},20ßHSD from Streptomyces hydrogenans (6), 17ß,20{alpha}HSD from human placenta (7), and 3ß,20{alpha}HSD from fetal lamb blood (4). de Launoit et al. (9) demonstrated that recombinant rat 3ßHSD enzyme also exhibits androgenic 17ßHSD activity using transfected cells. As such secondary activity was suggested to be involved in the original active site in these previous experiments, it was postulated that some steroids can bind in opposite ways to the same enzyme site (8, 18). However, in our kinetic analysis, substrates of 3ßHSD inhibit 17ßHSD2 activity not in a competitive but in a noncompetitive fashion. These data indicate that the catalytic site of 3ßHSD activity is not closely involved with the 17ßHSD2 active site. In addition, we have shown that 4MA, which competitively and reversibly inhibits dehydrogenase activities of the human 3ßHSD gene products (11), does not inhibit the 3ßHSD activity of 17ßHSD2 protein, thus suggesting that this 3ßHSD activity does not have a typical 3ßHSD active site. To establish the characteristics of the 3ßHSD active site on human 17ßHSD2 protein in more detail, further detailed examination will be necessary.


   Footnotes
 
1 This work was supported in part by NIH Grant DK-52167 (to S.A.) and Grant G0000066 from the Medical Research Council (to J.I.M.). Back

Received October 19, 1999.

Revised June 30, 2000.

Accepted July 13, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Luu-The V, Labrie C, Simard J, et al. 1990 Structure of two in tandem human 17ß-hydroxysteroid dehydrogenase genes. Mol Endocrinol. 4:268–275.[Abstract/Free Full Text]
  2. Wu L, Einstein M, Geissler WM, Chan HK, Elliston KO, Andersson S. 1993 Expression cloning and characterization of human 17ß-hydroxysteroid dehydrogenase type 2, a microsomal enzyme possessing 20{alpha}-hydroxysteroid dehydrogenase activity. J Biol Chem. 268:12964–12969.[Abstract/Free Full Text]
  3. Geissler WM, Davis DL, Wu L, et al. 1994 Male psuedohermaphroditism caused by mutation of testicular 17ß-hydroxysteroid dehydrogenase 3. Nat Genet. 7:34–39.[CrossRef][Medline]
  4. Peltoketo H, Luu-The V, Simard J, Adamski J. 1999 17ß-Hydroxysteroid dehydrogenase (HSD)/17-ketosteroid reductase (KSR) family; nomenclature and main characteristics of the 17HSD/KSR enzymes. J Mol Endocrinol. 23:1–11.[Abstract]
  5. Casey ML, MacDonald PC, Andersson S. 1994 17ß-Hydroxysteroid dehydrogenase type 2: chromosomal assignment and progestin regulation of gene expression in human endometrium. J Clin Invest. 94:2135–2141.
  6. Strickler RC, Covey DF, Tobias B. 1980 Study of 3{alpha},20ß-hydroxysteroid dehydrogenase with an enzyme-generated affinity alkylator: dual enzyme activity at a single active site. Biochemistry. 19:4950–4954.[CrossRef][Medline]
  7. Thomas JL, LaRochelle MC, Asibey-Berko E, Strickler RC. 1985 Reactivation of human placental 17ß,20{alpha}-hydroxysteroid dehydrogenase affinity alkylated by estrone 3-(bromoacetate): topographic studies with 16{alpha}-(bromoacetoxy)estradiol 3-(methyl ether). Biochemistry. 24:5361–5363.[CrossRef][Medline]
  8. Chen Q, Rosik LO, Nancarrow CD, Sweet F. 1989 Fetal lamb 3ß,20{alpha}-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16{alpha}-(bromo[2'-14C]acetoxy)progesterone. Biochemistry. 28:8856–8863.[CrossRef][Medline]
  9. de Launoit Y, Simard J, Durocher F, Labrie F. 1992 Androgenic 17ß-hydroxysteroid dehydrogenase activity of expressed rat type 1 3ß-hydroxysteroid dehydrogenase/{Delta}5->4-isomerase. Endocrinology. 130:553–555.[Abstract/Free Full Text]
  10. McCartin S, Russell AJ, Fischer RA, et al. 2000 Phenotypic variability and origins of mutations in the gene encoding 3ß-hydroxysteroid dehydrogenase type II. J Mol Endocrinol. 24:75–82.[Abstract]
  11. Luu-The V, Takahashi M, de Launoit Y, Dumont M, Lachance Y, Labrie F. 1991 Evidence for distinct dehydrogenase and isomerase sites within a single 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein. Biochemistry. 30:8861–8865.[CrossRef][Medline]
  12. Marvel C, Rheaume E, Takahashi M, et al. 1992 Distribution of 17ß-hydroxysteroid dehydrogenase gene expression and activity in rat and human tissues. J Steroid Biochem Mol Biol. 41:597–603.[CrossRef][Medline]
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  14. Lachance Y, Luu-The V, Verreault H, et al. 1991 Structure of human type II 3ß-hydroxysteroid dehydrogenase/54-isomerase (3ß-HSD) gene: adrenal and gonadal specificity. DNA Cell Biol. 10:701–711.[Medline]
  15. Labrie F, Simard J, Luu-The V, et al. 1992 Structure and tissue-specific expression of 3ß-hydroxysteroid dehydrogenase/5-ene-4ene isomerase genes in human and rat classical and peripheral steroidogenic tissues. J Steroid Biochem Mol Biol. 41:421–435.[CrossRef][Medline]
  16. Milewich L, Shaw CE, Doody KM, et al. 1991 3ß-Hydroxysteroid dehydrogenase activity in glandular and extraglandular human fetal tissues. J Clin Endocrinol Metab. 73:1134–1140.[Abstract/Free Full Text]
  17. Moisan AM, Ricketts ML, Tardy V, et al. 1999 New insight into the molecular basis of 3ß-hydroxysteroid dehydrogenase deficiency: identification of eight mutations in the 3HSD3B2 gene in eleven patients from seven new families and comparison of the functional properties of twenty-five mutant enzymes. J Clin Endocrinol. Metab. 84:4410–4425.
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