Identification of the Major Oxidative 3α-Hydroxysteroid Dehydrogenase in Human Prostate That Converts 5α-Androstane-3α,17β-diol to 5α-Dihydrotestosterone: A Potential Therapeutic Target for Androgen-Dependent Disease

Abstract

Androgen-dependent prostate diseases initially require 5alpha-dihydrotestosterone (DHT) for growth. The DHT product 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), is inactive at the androgen receptor (AR), but induces prostate growth, suggesting that an oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) exists. Candidate enzymes that posses 3alpha-HSD activity are type 3 3alpha-HSD (AKR1C2), 11-cis retinol dehydrogenase (RODH 5), L-3-hydroxyacyl coenzyme A dehydrogenase , RODH like 3alpha-HSD (RL-HSD), novel type of human microsomal 3alpha-HSD, and retinol dehydrogenase 4 (RODH 4). In mammalian transfection studies all enzymes except AKR1C2 oxidized 3alpha-diol back to DHT where RODH 5, RODH 4, and RL-HSD were the most efficient. AKR1C2 catalyzed the reduction of DHT to 3alpha-diol, suggesting that its role is to eliminate DHT. Steady-state kinetic parameters indicated that RODH 4 and RL-HSD were high-affinity, low-capacity enzymes whereas RODH 5 was a low-affinity, high-capacity enzyme. AR-dependent reporter gene assays showed that RL-HSD, RODH 5, and RODH 4 shifted the dose-response curve for 3alpha-diol a 100-fold, yielding EC(50) values of 2.5 x 10(-9) M, 1.5 x 10(-9) M, and 1.0 x 10(-9) M, respectively, when compared with the empty vector (EC(50) = 1.9 x 10(-7) M). Real-time RT-PCR indicated that L-3-hydroxyacyl coenzyme A dehydrogenase and RL-HSD were expressed more than 15-fold higher compared with the other candidate oxidative enzymes in human prostate and that RL-HSD and AR were colocalized in primary prostate stromal cells. The data show that the major oxidative 3alpha-HSD in normal human prostate is RL-HSD and may be a new therapeutic target for treating prostate diseases.

Full text

Identification of the Major Oxidative 3
␣
-
Hydroxysteroid Dehydrogenase in Human Prostate
That Converts 5
␣
-Androstane-3
␣
,17
␤
-diol to 5
␣
-
Dihydrotestosterone: A Potential Therapeutic Target
for Androgen-Dependent Disease
David R. Bauman, Stephan Steckelbroeck, Michelle V. Williams, Donna M. Peehl, and
Trevor M. Penning
Department of Pharmacology (D.R.B., S.S., M.V.W., T.M.P), University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104-6084; and Department of Urology (D.M.P.), Stanford
University School of Medicine, Stanford, California 94305
Androgen-dependent prostate diseases initially require
5
␣
-dihydrotestosterone (DHT) for growth. The DHT
product 5
␣
-androstane-3
␣
,17
␤
-diol (3
␣
-diol), is inac-
tive at the androgen receptor (AR), but induces prostate
growth, suggesting that an oxidative 3
␣
-hydroxys-
teroid dehydrogenase (HSD) exists. Candidate en-
zymes that posses 3
␣
-HSD activity are type 3 3
␣
-HSD
(AKR1C2), 11-cis retinol dehydrogenase (RODH 5), L-3-
hydroxyacyl coenzyme A dehydrogenase , RODH like
3
␣
-HSD (RL-HSD), novel type of human microsomal
3
␣
-HSD, and retinol dehydrogenase 4 (RODH 4). In
mammalian transfection studies all enzymes except
AKR1C2 oxidized 3
␣
-diol back to DHT where RODH 5,
RODH 4, and RL-HSD were the most efficient. AKR1C2
catalyzed the reduction of DHT to 3
␣
-diol, suggesting
that its role is to eliminate DHT. Steady-state kinetic
parameters indicated that RODH 4 and RL-HSD were
high-affinity, low-capacity enzymes whereas RODH 5
was a low-affinity, high-capacity enzyme. AR-depen-
dent reporter gene assays showed that RL-HSD, RODH
5, and RODH 4 shifted the dose-response curve for
3
␣
-diol a 100-fold, yielding EC
50
values of 2.5 ⴛ10
ⴚ9
M,
1.5 ⴛ10
ⴚ9
M, and 1.0 ⴛ10
ⴚ9
M, respectively, when
compared with the empty vector (EC
50
ⴝ1.9 ⴛ10
ⴚ7
M).
Real-time RT-PCR indicated that L-3-hydroxyacyl co-
enzyme A dehydrogenase and RL-HSD were ex-
pressed more than 15-fold higher compared with the
other candidate oxidative enzymes in human prostate
and that RL-HSD and AR were colocalized in primary
prostate stromal cells. The data show that the major
oxidative 3
␣
-HSD in normal human prostate is RL-HSD
and may be a new therapeutic target for treating pros-
tate diseases. (Molecular Endocrinology 20: 444–458,
2006)
ANDROGENS ARE ESSENTIAL for the develop-
ment and regulation of male sexual characteris-
tics (1–6). Androgens exert their action by binding to
the androgen receptor (AR), resulting in the trans-
activation of androgen-responsive genes (7, 8). Con-
sequently, androgen action is highly regulated, and its
dysregulation can result in androgen-dependent pros-
tate diseases, such as benign prostatic hyperplasia
(BPH) and prostate adenocarcinoma (CaP). BPH af-
fects approximately 50% of men by age 50, and its
incidence increases with age (2, 4). CaP is the second
leading cause of cancer-related deaths in men with
approximately 184,000 new cases a year and approx-
imately 32,000 related deaths a year (1, 9). Androgens
are essential for the development of the two diseases,
as prepubescent castrated male beagles never de-
velop BPH or CaP (10–12), and androgen ablation can
be a beneficial therapy in the treatment of these
diseases.
5
␣
-Dihydrotestosterone (DHT) is the most potent
androgen and is responsible for the growth, develop-
ment, and maintenance of the normal secretory func-
tion of the prostate (1, 2, 6, 13, 14). Within the prostate,
First Published Online September 22, 2005
Abbreviations: Adione, 5
␣
-Androstane-3,17-dione; AKR,
aldo-keto reductase; AKR1C2, type 3 3
␣
-HSD; androsterone,
3
␣
-hydroxy-5
␣
-androstan-17-one; AR, androgen receptor;
ARE, androgen response element; BPH, benign prostatic
hyperplasia; CaP, prostate adenocarcinoma; CAT, chloram-
phenicol acetyl transferase; CDT-FBS, charcoal/dextran
treated FBS; DHT, 5
␣
-dihydrotestosterone; 3
␣
-diol, 5
␣
-an-
drostane-3
␣
,17
␤
-diol; 3
␤
-diol, 5
␣
-androstane-3
␤
,17
␤
-diol;
ERAB, endoplasmic reticulum amyloid
␤
-peptide binding
protein/L-3-hydroxyacyl coenzyme A dehydrogenase/type
10 17
␤
-HSD; GAPDH, glyceraldehyde-3-phosphate dehy-
drogenase; HSD, hydroxysteroid dehydrogenase; IRES,
internal ribosome entry sequence; LC/MS, liquid chro-
matograpy/mass spectrometry; NT 3
␣
-HSD, novel type
of human microsomal 3
␣
-HSD; PBGD, porphobilinogen
deaminase; RL-HSD, RODH-like 3
␣
-HSD/human oxidative
3
␣
-HSD; RODH, retinol dehydrogenase; RODH 4, retinol de-
hydrogenase 4; RODH 5, 11-cis retinol dehydrogenase; SDR,
short-chain dehydrogenases and reductases; TLC, thin-layer
chromatography.
Molecular Endocrinology is published monthly by The
Endocrine Society (http://www.endo-society.org), the
foremost professional society serving the endocrine
community.
0888-8809/06/$15.00/0 Molecular Endocrinology 20(2):444–458
Printed in U.S.A. Copyright © 2006 by The Endocrine Society
doi: 10.1210/me.2005-0287
444
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

DHT is formed from the irreversible reduction of tes-
tosterone by type 2 5
␣
-reductase (13, 15, 16). Studies
in rat (17–19), dog (11, 20–23), and marsupials (24, 25)
show that DHT may also be formed from the inactive
androgen 5
␣
-androstane-3
␣
,17
␤
-diol (3
␣
-diol) by an
unknown oxidative 3
␣
-hydroxysteroid dehydroge-
nase(s) (HSD) (Fig. 1).
Regulating the levels of DHT by hormonal ablative
therapy is achieved either by surgical castration to
remove the testis and hence remove its precursor
testosterone or by directly blocking the intracrine for-
mation of DHT within the prostate by targeting type 2
5
␣
-reductase with the mechanism-based inactivator
finasteride. Surgical castration reduces the prostate
size by more than 80% and reduces prostate DHT
levels by 90% (26, 27). However, this approach has
undesired side effects that are associated with global
changes in androgen levels, e.g. osteoporosis (28).
Selective targeting of the type 2 5
␣
-reductase by fin-
asteride reduces both the volume and size of the pros-
tate by approximately 25% and decreases prostate
DHT levels by 80% with fewer side effects than cas-
tration (29–31). It is noteworthy that neither approach
completely attenuates DHT levels in the prostate, sug-
gesting that other sources of this potent androgen
exist.
Other sources of DHT include reduction of testos-
terone by type 1 5
␣
-reductase, which may be up-
regulated in the diseased prostate (32), and oxidation
of 3
␣
-diol, which can potently stimulate the growth of
prostate across species (19, 20, 23, 25). Because 3
␣
-
diol has a low affinity for the AR [dissociation constant
(K
d
)⫽10
⫺6
M[rsqb], it was concluded that 3
␣
-diol is
converted back to DHT by an unidentified oxidative
3
␣
-HSD. Furthermore, administration of 3
␣
-diol, but
not its epimer 5
␣
-androstane-3
␤
,17
␤
-diol (3
␤
-diol), re-
sulted in the induction of prostate growth in castrated
beagles (20, 23). Administration of [
3
H]3
␣
-diol in hu-
man males suggests a human oxidative 3
␣
-HSD exists
because approximately 65% and 50% of the radioac-
tivity was found to be [
3
H]DHT in the plasma and
prostate, respectively (33, 34). These data suggest
that the back reaction could be an important source of
DHT in humans and a potential therapeutic target for
treating prostate diseases.
In humans the candidate oxidative 3
␣
-HSDs are all
members of the short-chain dehydrogenase/reduc-
tase (SDR) family and include the following: 11-cis
retinol dehydrogenase (RODH 5) (35, 36), L-3-hy-
droxyacyl coenzyme A dehydrogenase/type 10 17
␤
-
HSD [endoplasmic reticulum amyloid
␤
-peptide bind-
ing protein (ERAB) (37, 38)], RODH like 3
␣
-HSD also
known as human oxidative 3
␣
-HSD (RL-HSD) (39),
novel type of human microsomal 3
␣
-HSD (NT 3
␣
-
HSD) (40), and retinol dehydrogenase 4 (RODH 4) (41,
42) (Table 1). Recently, aldo-keto reductase (AKR) 1C2
was shown to reduce DHT to 3
␣
-diol but was unable
to oxidize 3
␣
-diol back to DHT in transfected cells (9,
43). The ability of the other candidates to oxidize 3
␣
-
Fig. 1. Androgen Metabolism in Human Prostate
The formation of DHT from either the reduction of testosterone by type 2 5
␣
-reductase or by the oxidation of 3
␣
-diol (back
reaction) by an oxidative 3
␣
-HSD are shown. One source of circulating 3
␣
-diol is the hepatic metabolism of DHT by AKR1C4.
Inhibition of type 2 5
␣
-reductase with finasteride and the inhibition of the oxidative 3
␣
-HSD is also indicated as therapeutic targets
for prostate diseases. RED, Reductase; OX, oxidase.
Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate Mol Endocrinol, February 2006, 20(2):444–458 445
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

diol to produce sufficient DHT to activate AR-depen-
dent gene transcription has not been compared.
We report the identification of the major oxidative
3
␣
-HSD in human prostate as RL-HSD based on ac-
tivity assays, transfection studies, trans-activation of
the AR, and expression levels in human prostate.
Moreover, RL-HSD was shown to be colocalized with
the AR in primary prostate stromal cells, suggesting
that it can regulate androgen signaling in this cell type.
RESULTS
Experimental System to Monitor 3
␣
-Diol
Oxidation by 3
␣
-HSDs in Transfected Cells
The cDNAs that code for five candidate SDRs previ-
ously shown to convert 3
␣
-diol to DHT in transient
transfection studies were obtained by RT-PCR from
human liver RNA. The PCR products were subse-
quently cloned into either pcDNA3 or into pcDNA3-
LacZ to yield bicistronic constructs. Each construct
was transfected into either COS-1 cells or PC-3 cells,
and the ability of the transfected cells to convert 3
␣
-
diol to DHT was measured. COS-1 cells (44) were
selected as a null environment for androgen metabo-
lism, whereas PC-3 cells (45) were chosen because
they are a prototypic androgen-metabolizing prostate
cell line. The oxidative activity of the 3
␣
-HSDs to con-
vert 3
␣
-diol to DHT was determined using a double
transfection (pcDNA3–3
␣
-HSD and pCMV-
␤
-galacto-
sidase) or a single transfection of a bicistronic con-
struct (pcDNA3–3
␣
-HSD-LacZ).
At the commencement of these studies we deter-
mined that the steroid formed by the oxidation of
3
␣
-diol by the 3
␣
-HSDs was, in fact, DHT. In trans-
fection studies the product formed comigrated with an
authenticated DHT standard by thin-layer chromatog-
raphy (TLC) (Fig. 2). The reaction was replicated with
unlabeled 3
␣
-diol, and the resulting material was iso-
lated and identified by liquid chromatography/mass
spectrometry (LC/MS). Commercially available DHT
was also examined by LC/MS, and the reaction prod-
uct and the standard gave a single chromatographic
peak with a retention time of 15.42 min. The mass
spectrum for both species was identical and gave a
dominant molecular ion [M
⫹
NH
4
-H
2
O]
⫹
at m/z 290.44
(Fig. 2) where the predicted molecular ion m/z ⫽
291.44 [MH
⫹
].
Representative conversion of 5
␮
M[
3
H]3
␣
-diol to
DHT by COS-1 cells transiently transfected with
RODH 5 is also shown over time (0.25 h and 2 h) (Fig.
2). Because 3
␣
-diol has two functional substituents (a
3
␣
-hydroxy and a 17
␤
-hydroxy group), oxidation of
the 3
␣
-position was anticipated to yield DHT. Over
time, the initial product was converted to a second
product that comigrated with 5
␣
-androstane-3,17-di-
one (Adione). Oxidation of DHT by an endogenous
oxidative 17
␤
-HSD activity would be responsible for
this second product.
Oxidation of 3
␣
-Diol to DHT in Cells Transiently
Transfected with SDRs
The metabolism of 3
␣
-diol in COS-1- and PC-3-trans-
fected cells was compared using both low and high
substrate concentrations as previously reported (35,
43). The resulting activities were normalized either to
␤
-galactosidase by a double-transfection procedure,
whereby both the pcDNA3–3
␣
-HSD and pCMV-
␤
-ga-
lactosidase were cotransfected, or by a single trans-
fection of a bicistronic construct, whereby both the
3
␣
-HSD and
␤
-galactosidase were expressed under
the control of the same cytomegalovirus promoter.
This approach was taken because antibodies were
unattainable for all the enzymes used in the study. The
␤
-galactosidase values were used to normalize the
activity of the enzyme as a means to better control for
transfection efficiency. The variation of the
␤
-galacto-
sidase activity for the double transfection was as
Table 1. Candidate Oxidative 3
␣
-HSDs Assigned Names for this Study with Other Names with GenBank Accession Numbers
and Formal Gene Names
Name Assigned in this Study 100% Nucleotide Identity with GenBank Identification No. Formal Gene Name
AKR1C2 Aldo-keto reductase family 1, member C2 (BC063574) AKR1C2
Dihydrodiol dehydrogenase 2 (AB021654)
Bile acid binding protein (NM_205845)
Type 3 3
␣
-HSD (NM_001354)
ERAB 17
␤
-HSD type 10 (AF035555) HADH2
Short chain L-3-hydroxyacyl-CoA dehydrogenase (NM_004493) HADSC
Endoplasmic reticulum amyloid
␤
-peptide-binding protein (U96132)
RL-HSD Human oxidative 3
␣
-HSD (U89281) HSD17B6
Homo sapiens 3-hydroxysteroid epimerase (AF223225)
RODH-like 3
␣
-HSD
RODH 5 Human 11-cis retinol dehydrogenase (U43559) RDH5
Retinol dehydrogenase 5 (NM_002905)
NT 3
␣
-HSD Homo sapiens 3
␣
-HSD (AF343729) DHRS9
RODH 4 Homo sapiens sterol/retinol dehydrogenase (AF057034) RODH-4
Homo sapiens retinol dehydrogenase 16 (NM_003708)
446 Mol Endocrinol, February 2006, 20(2):444–458 Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

much as 2.5-fold; however the variation of
␤
-galacto-
sidase activity using the bicistronic construct was only
1.25-fold. Consequently, the bicistronic construct
gave a more reliable representation of transfection
efficiency, but at the cost of expression level. Both the
expression of the 3
␣
-HSD (as indicated by real-time
PCR) and the
␤
-galactosidase (as indicated by activity
measurements) were decreased when the bicistronic
construct was transfected. This is probably due to the
processing of the long mRNA and its subsequent
translation. Despite these differences, similar patterns
for DHT formation were seen in COS-1 and PC-3 cells
irrespective of the transfection protocol.
Transiently transfected cells were analyzed for the
formation of DHT using a low concentration (0.1
␮
M)of
3
␣
-diol, and the activity was normalized to
␤
-galacto-
sidase (Fig. 3). The formation of DHT was highest for
RODH 5, RODH 4, and RL-HSD for both the double-
transfection and the single-transfection procedures.
Due to the high endogenous 17
␤
-HSD activity of the
cells, DHT formed from 0.1
␮
M3
␣
-diol by the trans-
fected 3
␣
-HSDs was quickly converted to Adione.
Consequently, a more representative picture of the
3
␣
-HSD oxidase activity is obtained by combining the
DHT and Adione formed as a function of time (Fig. 3).
The metabolic profiles indicated that ERAB and NT
3
␣
-HSD were poor oxidases in comparison with the
other candidate enzymes because they were only able
to produce trace amounts of DHT when a high con-
centration (5
␮
M)of3
␣
-diol was incubated for longer
times (data not shown).
RL-HSD was also found to act as an epimerase
converting 3
␣
-diol first to DHT and then reducing DHT
back to 3
␤
-diol, as previously reported (46). However,
Fig. 2. Representative 3
␣
-Diol Metabolism in Transfected Cells and Validation of DHT as the Product
A, Androgen standards separated by TLC run three times using methylene chloride/ethyl ether [11:1 (vol/vol). B, Representative
analysis of metabolic profiles using the automatic TLC-linear analyzer. Standard [
3
H]3
␣
-diol (1); COS-1 cells transiently trans-
fected with RODH 5 using 5
␮
M[
3
H]-3
␣
-diol, 0.25 h (2) and 2 h (3); standard [
14
C]DHT (4). C, LC/MS/MS tandem mass
spectrometry of DHT (m/z ⫽290.44, [M⫹NH
4
-H
2
O]⫹, predicted MH⫹m/z ⫽291.44) and the unknown product predicted to be
DHT (m/z ⫽290.44, [M⫹NH
4
-H
2
O]⫹, predicted MH⫹m/z ⫽291.44)]. a, Androstanedione; b, DHT, c, androsterone, d, 3
␤
-diol;
e, 3
␣
-diol; f, polar metabolites.
Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate Mol Endocrinol, February 2006, 20(2):444–458 447
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

this activity was observed only when high substrate
concentrations (5
␮
M3
␣
-diol or 5
␮
MDHT) were incu-
bated and was not observed when a low substrate
concentration (0.1
␮
M3
␣
-diol) was used (data not
shown). The metabolism profiles indicated that three
candidates (RODH 5, RODH 4, and RL-HSD) could be
responsible for the conversion of physiological con-
centrations of 3
␣
-diol back to DHT in the human pros-
tate. Of all the enzymes studied, only AKR1C2 was
unable to convert 3
␣
-diol to DHT in vivo at all concen-
trations tested, and this confirmed previous findings
(9, 43).
Reduction of DHT to 3
␣
-Diol in Cells Transiently
Transfected with SDRs
The steroid specificity and preferred directionality of
the 3
␣
-HSDs was investigated by also determining
their ability to reduce 5
␮
MDHT in COS-1 and PC-3
cells. Normalized formation of 3
␣
-diol by transiently
transfected COS-1 and PC-3 cells is shown in Fig. 4.
Our results indicated that AKR1C2 acted as a robust
reductase as it catalyzed the reduction of DHT to
3
␣
-diol. Under these conditions the 3
␣
-HSDs were
unable to reduce DHT to 3
␣
-diol; however, substan-
tially lower 3
␣
-diol was present in the RODH 5-, NT
3
␣
-HSD-, RODH 4-, and RL-HSD-transfected cells as
compared with the no-transfected or the pcDNA3
(empty vector)-transfected controls. These differences
can be explained by other activities of these enzymes,
which are revealed when the high endogenous 17
␤
-
HSD activity is suppressed. It has been noted previ-
ously that high steroid concentrations will suppress
the 17
␤
-HSD activity in these recipient cells (43). For
example, at 5
␮
MDHT, transiently transfected NT 3
␣
-
HSD and RODH 4 exhibited oxidative 17
␤
-HSD activ-
ity whereby DHT and the 3
␣
-diol formed were now
converted to Adione and androsterone (3
␣
-hydroxy-
5
␣
-androstan-17-one), respectively. On the other
hand, transiently transfected RL-HSD exhibits reduc-
tive 3
␤
-HSD activity at the higher substrate concen-
trations and catalyzed the conversion of DHT to
3
␤
-diol. Although these additional activities were ob-
Fig. 3. 3
␣
-Diol Metabolism in PC-3 and COS-1 Cells
A, Normalized formation of DHT in PC-3 cells following the double-transfection protocol (pcDNA3–3
␣
-HSD plus pCMV-
␤
-
galactosidase) using 0.1
␮
M3
␣
-diol. B, Normalized formation of DHT and Adione in PC-3 cells using the double-transfection
protocol using 0.1
␮
M3
␣
-diol. C, Normalized formation of DHT in COS-1 cells by a single transfection of the bicistronic construct
(pcDNA3–3
␣
-HSD-Lac Z) using 0.1
␮
M3
␣
-diol. D, Normalized formation of both DHT and Adione in COS-1 cells by a
single-transfection protocol using 0.1
␮
M3
␣
-diol. Normalized activity is expressed as percent of total DHT or DHT ⫹Adione
formed divided by milliunits of
␤
-galactosidase with error bars representing the highest and lowest values with the average of three
independent experiments (see Materials and Methods).
␤
-Gal,
␤
-Galactosidase; NT, no transfection; NT 3
␣
-HSD, novel type
3
␣
-HSD.
448 Mol Endocrinol, February 2006, 20(2):444–458 Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

served (17
␤
-HSD oxidation and 3
␤
-HSD reduction),
they are minor in comparison with their 3
␣
-HSD oxi-
dative activity, because they only occur over a much
longer time frame. In contrast, RODH 5 is 3
␣
-HSD
specific because no other detectible activities were
observed. Thus, the decreased level of 3
␣
-diol ob-
served with RODH 5 was solely due to the ability of this
enzyme to oxidize the endogenously formed 3
␣
-diol
back to DHT (Fig. 4).
Steady-State Kinetic Analysis for the Conversion
of 3
␣
-Diol to DHT
Based on the transfection studies, RODH 5, RL-HSD,
and RODH 4 were all capable of converting 3
␣
-diol to
DHT. To identify the most efficient enzyme, the
steady-state kinetic parameters (V
maxapp
,K
m
, and
V
maxapp
/K
m
) were determined for the transfected en-
zymes using isolated membrane fractions. It was
found that RODH 4 and RL-HSD have similar kinetic
constants and were identified as being high-affinity
(submicromolar K
m
values), low-capacity enzymes
(low V
maxapp
values); however, RODH 5 was found to
be a low-affinity (micromolar K
m
value) and high-ca-
pacity enzyme (high V
maxapp
value) (Table 2). The
steady-state kinetic analysis indicated that RODH 4
and RL-HSD had much higher utilization ratios (7- to
16-fold greater) than RODH 5. Our steady-state kinetic
parameters for RODH 4, RL-HSD, and RODH 5 were
similar to those previously reported (36, 39, 42).
Trans-Activation of the AR by 3
␣
-Diol in Cells
Transfected with SDRs
The ability of the oxidative 3
␣
-HSDs to convert 3
␣
-diol
into sufficient amounts of DHT to trans-activate the AR
was assessed using a reporter gene assay. The chlor-
amphenicol acetyl transferase (CAT) reporter gene as-
say was performed using the appropriate controls to
ensure that the response was mediated through the
trans-activation of the AR. The specificity of the CAT
assay was determined by using a nontransfected con-
trol, a dimethylsulfoxide (DMSO) control, a no-steroid
control, an AR minus control, a p-tk-CAT [minus the
Fig. 4. DHT Metabolism in COS-1 and PC-3 Cells
Normalized formation of 3
␣
-diol in COS-1 (A) and PC-3 (B) cells following a double-transfection (pcDNA3 and pCMV-
␤
-
galactosidase) protocol using 5
␮
MDHT. Normalized activity is expressed as percent of total 3
␣
-diol formed divided by milliunits
of
␤
-galactosidase with error bars representing the highest and lowest values with the average of three independent experiments.
␤
-Gal,
␤
-Galactosidase; NT, no transfection; NT 3
␣
-HSD, novel type 3
␣
-HSD.
Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate Mol Endocrinol, February 2006, 20(2):444–458 449
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

androgen response element (ARE) tandem repeat]
control, and a pbasic-CAT (minus the tk promoter)
control, and the results are shown in Fig. 5.
Activation of the CAT reporter gene occurred only in
the presence of androgen when the AR and the
(ARE)
2
-tk-CAT constructs were cotransfected, indicat-
ing that the CAT assay was specific to monitor trans-
activation of the AR. Furthermore, the activation of
CAT by DHT, testosterone, and 3
␣
-diol was inhibited
with the AR antagonist flutamide. Consequently, the
CAT assay we developed could be used to determine
the ability of the oxidative 3
␣
-HSDs to modulate gene
transcription by trans-activation of the AR using 3
␣
-
diol as the substrate. Activation of the (ARE)
2
-tk-CAT
construct by the oxidative 3
␣
-HSDs using 3
␣
-diol is
shown in Fig. 5 and in Table 3. RODH 4, RODH 5,
RL-HSD, and NT 3
␣
-HSD were all able to mediate the
trans-activation of the reporter gene construct at low
levels of 3
␣
-diol yielding EC
50
values of 1.0 ⫻10
⫺9
M,
1.5 ⫻10
⫺9
M, 2.5 ⫻10
⫺9
M, and 5.5 ⫻10
⫺8
M,
respectively. In contrast, 3
␣
-diol gave an EC
50
value
for trans-activation of the AR equal to 1.9 ⫻10
⫺7
Min
pcDNA3 (empty vector)-transfected cells. Thus
RODH4, RODH 5, and RL-HSD each increased the
potency of 3
␣
-diol for the AR by greater than 100-fold.
From the metabolism data it is not surprising that
ERAB (EC
50
⫽2.1 ⫻10
⫺7
M) and AKR1C2 (EC
50
⫽
1.9 ⫻10
⫺7
M) were unable to trans-activate the re-
porter gene construct with 3
␣
-diol. Using the EC
50
values for 3
␣
-diol activation of the reporter gene, the
CAT reaction was subsequently inhibited with the AR
antagonist flutamide for pcDNA3, RL-HSD, RODH 5,
RODH 4 (Fig. 5), and NT-3
␣
-HSD (data not shown).
The inhibition of the CAT response by flutamide pro-
duced identical IC
50
values for all the constructs
tested, suggesting that the response was indeed AR
dependent and not construct dependent (pcDNA3 us-
ing 3
␣
-diol, IC
50
⫽3.5 ⫻10
⫺7
M; pcDNA3 using DHT,
IC
50
⫽3.5x10
⫺7
M; pcDNA3 using testosterone, IC
50
⫽4.6 ⫻10
⫺7
M; RL-HSD, IC
50
⫽3.6 ⫻10
⫺7
M; RODH
5, IC
50
⫽4.5 ⫻10
⫺7
M; RODH 4, IC
50
⫽2.8 ⫻10
⫺7
M;
NT 3
␣
-HSD, IC
50
⫽3.4 ⫻10
⫺7
M).
The activation of the (ARE)
2
-tk-CAT reporter by 3
␣
-
diol using transfected RODH 5, RL-HSD, and RODH 4
was compared with that observed with DHT, testos-
terone, and 3
␣
-diol (Table 3). The ability of RODH 5,
RL-HSD, and RODH 4 to activate the CAT reporter
using 3
␣
-diol gave a dose-response curve equivalent
to that observed with testosterone (EC
50
⫽2.5 ⫻10
⫺9
M), but less than the response observed with DHT
(EC
50
⫽6.8 ⫻10
⫺10
M). These differences can be
explained because at low concentrations (submicro-
molar), both 3
␣
-diol and DHT are quickly metabolized
to the inactive androgens, androsterone and andro-
stanedione, respectively, by a very high oxidative 17
␤
-
HSD activity. Taken together, RODH 5, RL-HSD, and
RODH 4 are able to alter gene transcription by oxidiz-
ing low levels of 3
␣
-diol to DHT when compared with
the pcDNA3 control.
Expression Levels of the SDRs in Human
Prostate and Cell Type Using Real-Time RT-PCR
The metabolism studies, kinetic parameters, and
trans-activation experiments indicate that three en-
zymes (RODH 5, RODH 4, and RL-HSD) may be re-
sponsible for the formation of DHT from 3
␣
-diol in the
human prostate. Consequently, a real-time PCR
method was developed to investigate the expression
levels of the 3
␣
-HSD oxidases in normal human pros-
tate. The development and specificity of the real-time
PCR assay included 1) the identification of primers
that amplified the desired gene, which was validated
by sequencing the PCR product; 2) placement of the
primers to cross over exon-intron boundaries to pre-
vent the nonspecific amplification of genomic DNA;
and 3) melting curves were analyzed at the end of each
run to ensure specificity between reactions. This RT-
PCR methods were linear (⬎r⫽0.995) over a dynamic
range (10
9
) as determined by plotting the log
10
fluo-
rescence intensity vs. the number of cycles and could
be used to determine variable expression levels of the
SDRs within the prostate. The expression levels of the
SDRs were determined using total RNA pooled from
32 Caucasian human prostates and normalized to the
high-abundance housekeeping gene glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) and the low
abundance PBGD, and similar patterns were ob-
served. The real-time PCR data indicated that all the
candidate oxidative 3
␣
-HSDs and the AR were ex-
pressed within whole prostate, but to varying levels
(Fig. 6). The expression patterns revealed that AR was
the highest expressed. Of the oxidative 3
␣
-HSDs,
ERAB and RL-HSD were expressed more than 15-fold
higher in comparison with the other candidates and
would therefore be the most likely candidates to be the
Table 2. Determination of the Steady-State Kinetic Parameters for 3
␣
-Diol Oxidation Catalyzed by 3
␣
-HSDs Using Isolated
Membrane Fractions from Transiently Transfected COS-1 Cells
Oxidative 3
␣
-HSD K
m
(
␮
M)
a
V
maxapp
(nmol/min/mg
protein)
a
V
maxapp
/K
m
(nmol/min/mg
protein)/(
␮
M)
a
RL-HSD 0.4 ⫾0.04 5.9 ⫾0.26 14.8
RODH 5 18.5 ⫾4.1 39.6 ⫾3.3 2.1
RODH 4 0.27 ⫾0.03 8.74 ⫾0.19 32.4
a
Kinetic parameters were determined from initial velocity measurements determined in triplicate, and the data were pooled from
two experiments.
450 Mol Endocrinol, February 2006, 20(2):444–458 Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

major oxidative 3
␣
-HSD in human prostate. However,
from the metabolism studies, ERAB was unable to
oxidize low levels of 3
␣
-diol and was unable to alter
gene transcription using the CAT assay. Conse-
quently, the major oxidative 3
␣
-HSD in normal human
prostate is identified as RL-HSD.
Although RL-HSD was expressed in prostate, its
abundance may be suppressed by examining the
whole gland. Subsequently, the expression levels of
the oxidative 3
␣
-HSDs were analyzed in cultured hu-
man prostate primary epithelial and stromal cells to
determine their cellular localization. Furthermore, be-
Fig. 5. Trans-Activation of the AR by 3
␣
-Diol in the Presence of Oxidative 3
␣
-HSDs
A, The specificity of the CAT assay was validated by comparing the no-transfected control with the positive control [pCMV-AR,
p-(ARE)
2
-tk-CAT, pCMV-
␤
-galactosidase and pcDNA3] and with all other possible combinations. All the systems tested included
a no-steroid and steroid (DHT; 1.0 ⫻10
⫺7
M) treatment. B, Activation of the (ARE)
2
-tk-CAT reporter gene by the AR in the presence
of cotransfected HSDs vs. the concentration of 3
␣
-diol was varied (10
⫺12
to 10
⫺6
M). Activation of the reporter gene is the average
of three independent experiments with the activity expressed as percent of CAT activity (fold activation of the CAT divided by the
maximal fold activation multiplied by 100%). C, Inhibition of the CAT response mediated by oxidative 3
␣
-HSDs and 3
␣
-diol using
flutamide. The concentration of 3
␣
-diol used was the EC
50
value in each case. The black triangle represents increasing flutamide
concentrations (0.01
␮
M, 0.1
␮
M, 0.3
␮
M,1
␮
M,3
␮
M, and 10
␮
M; not shown, 0.001
␮
Mflutamide) with the first value representing
no-flutamide treatment as indicated by a zero and the highest concentration (10
␮
M) shown. Inhibition of the reporter gene is the
average of three independent experiments with the activity expressed as percent of CAT activity (fold activation of the CAT divided
by the maximal fold activation multiplied by 100%). NT HSD, Novel-type 3
␣
-HSD.
Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate Mol Endocrinol, February 2006, 20(2):444–458 451
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

cause AR is prominently expressed in the stromal cells
of the prostate (47), it was important to determine
whether the SDRs are colocalized with the AR so that
they can regulate the androgen signal. Total RNA was
isolated from normal prostate primary epithelial (n ⫽
14) and primary stromal cells (n ⫽15), and 1
␮
g was
reverse transcribed. The results indicated that the en-
zymes displayed a cell type-specific distribution (Fig.
6). ERAB was most highly expressed in both cell types
in comparison with the other oxidative candidates with
an approximate 3-fold preference for the epithelial
cells, but it is a weak oxidase in comparison with the
other candidate enzymes. RL-HSD was expressed in
the stromal cells with an approximate 20-fold prefer-
ence. The AR was expressed in both epithelial and
stromal cells with an approximate 10-fold preference
for stromal cells as previously shown (47). The stromal
colocalization of RL-HSD and the AR indicated that
RL-HSD is positioned to regulate the trans-activation
of the AR. This is important because changes in the
expression levels of RL-HSD could lead to an increase
in activation of androgen-sensitive genes, due to the
stromal colocalization of the AR. RODH 5 was ex-
pressed equally in epithelial and stromal cells, but its
expression level was 5-fold lower in the stromal cells
as compared with RL-HSD. NT 3
␣
-HSD was ex-
pressed only in the epithelial cells; however, it poorly
metabolizes 3
␣
-diol and only weakly trans-activated
the AR with 3
␣
-diol. RODH 4 was very lowly expressed
in both epithelial and stromal cells, with a slight pref-
erence for the epithelial cells.
DISCUSSION
For more than 30 yr, animal experiments (rat, beagles,
and marsupials) have indicated that 3
␣
-diol will pro-
mote growth of the prostate, yet it has negligible af-
finity for the AR. It was assumed that the growth pro-
duced was accomplished by the oxidation of 3
␣
-diol
to DHT; however, the identity of the oxidative 3
␣
-HSD
has remained elusive. In the literature five SDRs have
been implicated by individual investigators as being
involved in this reaction, leading to confusion as to its
identity. In this study we have compared all candidate
oxidative enzymes in a single study and identified
RL-HSD as being the major oxidative 3
␣
-HSD in hu-
man prostate.
All the oxidative 3
␣
-HSD candidate enzymes were
able to oxidize 3
␣
-diol to DHT except for AKR1C2. The
3
␣
-diol metabolic profiles showed that three enzymes
(RODH 5, RODH 4, and RL-HSD) could be responsible
for the back reaction. In contrast, ERAB and NT 3
␣
-
HSD were only able to convert 3
␣
-diol to DHT at a high
concentration (5
␮
M) and over an extended time
course. These findings are supported by a steady-
state kinetic analysis of the transfected enzymes.
Thus, RL-HSD and RODH 4 had the highest utilization
ratios (V
maxapp
/K
m
). The metabolism studies also indi-
cated that RL-HSD was able to act as an epimerase by
converting 3
␣
-diol to DHT and then back to 3
␤
-diol,
but only at higher substrate concentrations (5
␮
M3
␣
-
diol or 5
␮
MDHT). Irrespective of this epimerase ac-
tivity, RL-HSD was able to regulate gene transcription
when low concentrations of 3
␣
-diol were incubated.
The ability of the oxidative 3
␣
-HSDs to reduce DHT
to 3
␣
-diol was also investigated. Our data confirmed
that AKR1C2 was the only enzyme that could reduce
DHT to 3
␣
-diol (9, 43). By contrast, in the RODH 5-,
RL-HSD-, RODH 4-, and NT 3
␣
-HSD-transfected cells
the amount of 3
␣
-diol formed was significantly lower;
this is due to the minor 17
␤
-HSD activity of NT 3
␣
-
HSD and RODH 4, which converts DHT to Adione, the
minor reductive 3
␤
-HSD activity of RL-HSD that con-
verts DHT to 3
␤
-diol, and the ability of RODH 5 to
oxidize 3
␣
-diol formed endogenously back to DHT.
We also investigated the ability of the oxidative 3
␣
-
HSDs to convert sufficient 3
␣
-diol to DHT to trans-
activate the AR. Using a reporter gene assay we found
that RODH 5, RL-HSD, and RODH 4 were able to shift
the dose-response curve of 3
␣
-diol to the left by 2
orders of magnitude as compared with the pcDNA3
control. This increased activation was blocked with
flutamide, indicating that this reaction was AR depen-
dent. The significant finding is that the oxidative 3
␣
-
HSDs altered gene transcription at the prereceptor
level by changing the concentration of active andro-
gen available to the AR.
The mRNA expression from normal human prostate
indicated that all the oxidative 3
␣
-HSD candidates
were expressed; however, RL-HSD was expressed
more than 15-fold as compared with the other two
remaining candidates (RODH 5 and RODH 4). Conse-
quently, the major oxidase was identified as RL-HSD
in normal human prostate. Furthermore, RL-HSD was
shown to be colocalized with the AR in primary pros-
tate stromal cells; thus RL-HSD could regulate andro-
gen-sensitive genes by oxidizing physiological con-
centrations of 3
␣
-diol back to DHT in this cell type.
Our data show that the major enzyme responsible
for the elimination of DHT in the prostate is likely
AKR1C2. Because the major oxidative 3
␣
-HSD re-
Table 3. EC
50
Values for Trans-Activation of the AR Using
a CAT Reporter Gene Assay
Steroid Construct EC
50
Value (M)
a
3
␣
-diol pcDNA3 1.9 ⫻10
⫺7
AKR1C2 1.9 ⫻10
⫺7
ERAB 2.1 ⫻10
⫺7
RL-HSD 2.6 ⫻10
⫺9
RODH 5 1.5 ⫻10
⫺9
NT 3
␣
-HSD 5.5 ⫻10
⫺8
RODH 4 1.0 ⫻10
⫺9
T pcDNA3 2.5 ⫻10
⫺9
DHT pcDNA3 6.8 ⫻10
⫺10
a
Activation of the (ARE)
2
-tk-CAT reporter gene by the oxi-
dative 3
␣
-HSDs in COS-1 cells are the average of three
independent experiments. T, Testosterone.
452 Mol Endocrinol, February 2006, 20(2):444–458 Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

sponsible for DHT formation from 3
␣
-diol is RL-HSD,
we have identified the HSD pair that modulates ligand
access to the AR in the human prostate (Fig. 7).
RL-HSD was identified as the major oxidative 3
␣
-
HSD in normal human prostate, but Northern analysis
indicates that RL-HSD is not ubiquitously expressed
(48). Because RODH 5, RL-HSD, or RODH 4 can form
DHT in transfected cells, the expression levels of these
enzymes need to be examined in other androgen-
dependent diseases, like acne or alopecia. Excess
DHT is implicated in acne and alopecia, and RODH 4
and RODH 5 may be important in regulating DHT
levels in the epidermis and sebaceous glands. For
example, Jurukovski et al. (41) showed that RODH 4
was expressed in the epidermis and Karlsson et al. (49)
showed that it was potently inhibited by 13-cis-retinoic
acid, a clinically relevant treatment for acne. The au-
thors suggested that the inhibition of acne by 13-cis-
retinoic acid could be due to the inhibition of RODH 4
and a local decrease of DHT where DHT increases the
differentiation and growth of sebaceous glands. New
approaches in the treatment of androgen-dependent
prostate diseases, acne, and/or alopecia may be to
inhibit RL-HSD, RODH 4, or RODH 5 by decreasing
the local production of DHT in these target tissues.
MATERIALS AND METHODS
Chemicals
The radioactive steroids were [4-
14
C]5
␣
-DHT (57.3 mCi/
␮
mol, PerkinElmer LLC, Norwalk, CT) and [9,11-
3
H(N)]5
␣
-
androstane-3
␣
,17
␤
-diol (40.0 Ci/
␮
mol, PerkinElmer LLC).
The following nonradioactive steroids used in the study were
all purchased from Steraloids (Wilton, NH): 3
␣
-diol, 3
␤
-diol,
androsterone, Adione, and DHT
Construction of the pcDNA3 Constructs and the
Bicistronic Constructs
The enzymes investigated were all cloned from human liver
total RNA (BD Bioscience, Palo Alto, CA). Total RNA (1
␮
g)
Fig. 6. Relative Expression of Oxidative 3
␣
-HSDs in Human Prostate and Cell Type as Determined by Real-Time RT-PCR
Normalized to PBDG
A, The expression of the oxidative HSDs in normal human prostate. Total RNA (1
␮
g) from 32 pooled human prostates was
reverse-transcribed to cDNA, and 12.5 ng of cDNA was added to each real-time PCR experiment that was performed in triplicate
with the mean shown. Data are normalized to the housekeeping gene PBGD and are represented as expressed femtograms of
each protein per ng of total cDNA. B, The expression of the oxidative HSDs in primary prostate epithelial and stromal cells show
a cell type-specific pattern. Total RNA (1
␮
g) from primary prostate epithelial (n ⫽14) and stromal (n ⫽15) cells was
reverse-transcribed to cDNA, and 50 ng of cDNA was added to each real-time PCR experiment that was performed in triplicate
with the median shown. NT HSD, Novel type 3
␣
-HSD.
Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate Mol Endocrinol, February 2006, 20(2):444–458 453
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

was reverse transcribed using GeneAmp RNA PCR Kit (Ap-
plied Biosystems, Foster City, CA) and the cDNA was ampli-
fied for each of the genes of interest using primers as previ-
ously reported [AKR1C2 (43, 51), ERAB (38), RL-HSD (50),
RODH 5 (35), NT 3
␣
-HSD (40), and RODH 4 (42)]. Each cDNA
was successfully subcloned into the mammalian expression
vector pcDNA3 using restriction sites previously reported (for
AKR1C2, KpnI and ApaI (43); for ERAB, BamHI and EcoRI
(38); for RL-HSD, EcoRI (50); for RODH, 5 BamHI and EcoRI
(35); for NT 3
␣
-HSD, EcoRI (40); and for RODH 4, EcoRI (42)],
and its identity was validated by dideoxy sequencing. The
bicistronic construct, pIRES (internal ribosome entry se-
quence)-Lac Z, was purchased from BCCM/LMBP (Ghent
University, Belgium), and subsequently ligated to pcDNA3 to
form a pcDNA3-pIRES-LacZ construct (pcDNA3-LacZ) using
EcoRI and XhoI. The construct was validated by dideoxy
sequencing, and after transfection the expression of
␤
-ga-
lactosidase activity was determined using the
␤
-galactosi-
dase enzyme assay system (Promega Corp., Madison, WI).
The 3
␣
-HSD enzymes were subcloned from pcDNA3 into the
bicistronic construct (pcDNA-3
␣
-HSD-LacZ) using the same
restriction enzymes that produced the respective pcDNA3–
3
␣
-HSD construct, and the final constructs were sequenced.
The constructs used in the reporter gene assays were
p-(ARE)
2
-tk-CAT, p-tk-CAT, and the pbasic-CAT (52); the
pCMV-
␤
-galactosidase was purchased from CLONTECH
Laboratories, Inc. (Palo Alto, CA).
Cell Culture
The cell lines (COS-1 and PC-3) were purchased and main-
tained according to the protocols provided by the American
Type Culture Collection (Manassas, VA). COS-1 cells were
maintained in DMEM (Invitrogen Corp., Grand Island, NY),
10% fetal bovine serum (FBS), 1% penicillin/streptomycin,
and 2% L-glutamine. COS-1 cells were plated in six-well
dishes at a density of 2.5 ⫻10
5
cells and were transfected
using FuGENE6 (Roche Diagnostics, Indianapolis, IN) with
0.7
␮
g pcDNA3–3
␣
-HSD, 0.7
␮
g pcDNA3, and 0.4
␮
g pCMV-
␤
-galactosidase. The plating condition for COS-1 cells was
held constant for transient transfection of the bicistronic con-
struct; however, 1.0
␮
g of plasmid (pcDNA3–3
␣
-HSD-LacZ)
was transfected. Approximately 3 h before metabolism stud-
ies, the medium was changed to DMEM (minus phenol red)
(Invitrogen Corp.), 1% charcoal/dextran-treated FBS (CDT-
FBS) (HyClone Laboratories, Inc., Logan, UT), 1% penicillin/
streptomycin, and 2% L-glutamine for COS-1 cells. PC-3
cells were maintained in FK-12 (Invitrogen Corp.), 10% FBS,
1% penicillin/streptomycin, and 1% L-glutamine. PC-3 cells
were plated in six-well plates at a density of 5.0 ⫻10
5
cells
and were transfected using FuGENE6 with 1.0
␮
g of con-
struct and 0.2
␮
gof
␤
-galactosidase. The plating condition
for COS-1 cells was held constant for transient transfection of
the bicistronic construct; however, 2.0
␮
g of plasmid was
transfected. Approximately 3 h before metabolism studies,
the medium was changed to RPMI (minus phenol red) (In-
vitrogen Corp.), 1% CDT-FBS, 1% penicillin/streptomycin,
and 1% L-glutamine for PC-3 cells.
Metabolism Studies Using Transiently Transfected
COS-1 and PC-3 Cells
The steroid stock concentrations used were determined by
weight and titrated by enzymatic conversion as previously
published (53). To study the metabolism of 0.1
␮
Mand 5
␮
M
3
␣
-diol or 5
␮
MDHT in transiently transfected COS-1 and
PC-3 cells, a mixture of radioactive and non-radioactive ste-
roid was used containing 2,200,000 cpm of [
3
H]5
␣
-andro-
stane-3
␣
,17
␤
-diol or 40,000 cpm of [
14
C]dihydrotestoster-
one. The organic soluble steroids were dried down under
nitrogen, redissolved in DMSO (Fisher Scientific, Pittsburgh,
PA), and added to the cells to give a final concentration of
0.25% DMSO, which had no effect on cell viability. Aliquots
(500
␮
l) of the culture media were removed over time and
extracted twice using 1 ml of water-saturated ethyl acetate
(⬎96% recovery). The ethyl acetate was evaporated to com-
plete dryness using a Sorvall Speed Vacuum and redissolved
in ethyl acetate-methanol-chloroform (1:0.5:0.5) (Fisher Sci-
entific) containing reference steroids as previously reported
(53). The dissolved steroids were plated on Whatman LK6D
Silica TLC plates (Fisher Scientific), prerun twice, and devel-
oped three times using methylene chloride-diethyl ether [11:1
(vol/vol)]. The TLC plates were analyzed with an automatic
TLC-linear analyzer (Bioscan Imaging Scanner System 200-
IBM with an AutoChanger 3000; Bioscan, Inc., Washington,
DC). The computer-aided software quantitatively determined
the radio signals emitted from the TLC plates and calculated
the percentages of each metabolic product vs. the total ra-
dioactivity. The positions of radioactive steroid signals on the
TLC plates were verified by staining reference standards and
Fig. 7. Regulation of the AR by 3
␣
-HSDs within the Prostate
The reduction of DHT by AKR1C2 and the oxidation of 3
␣
-diol by RL-3
␣
-HSD regulate ligand access to the AR. AKR1C2 is
highly expressed in human prostate cancer epithelial cells (43).
454 Mol Endocrinol, February 2006, 20(2):444–458 Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

quantified by scintillation counting as described previously
(53).
Validation that DHT Is the Product of the Enzymatic
Oxidation of 3
␣
-Diol
To validate that DHT was formed from 3
␣
-diol by the 3
␣
-HSD
oxidases, 5
␮
Munlabeled 3
␣
-diol was added to RODH
5-transiently transfected COS-1 cells as described earlier.
The reaction was terminated by removal of the medium after
4 h, a time by which more than 90% of 3
␣
-diol would be
converted to DHT. The medium (15 ml) was extracted three
times with 50 ml of ethyl acetate, concentrated by rotary
evaporation, and separated by TLC. Standards and a radio-
active control were run on the sides of the plate to determine
the location of DHT. The product was isolated, dissolved in
ethyl acetate, and separated over a silica column. DHT was
eluted using 10 ml of chloroform and ethyl acetate (1:1) and
condensed to 1 ml by rotary evaporation. The resulting liquid
was transferred into a small vial, dried under nitrogen, and
dissolved in 200
␮
l of acetonitrile. The product (predicted to
be DHT) was analyzed by LC/tandem mass spectrometry
using a Thermo Finnigan LCQ ion trap mass spectrometer
(Thermo Finnigan, San Jose, CA) equipped with an electro-
spray ionization source in positive ion mode. Operating con-
ditions were as follows: capillary temperature at 250 C. Ni-
trogen was used as the sheath (60 psi) and auxiliary (4
arbitrary units) gas to assist with nebulization. Full scanning
analyses were performed in the range of m/z 100 ⬃400.
Chromatography for LC/MS experiments using a gradient
system was performed using a Waters Alliance 2690 HPLC
system (Waters Corp., Milford, MA). An YMC C18 octadecyl-
silyl silicon-AQ column (250 ⫻2.0 mm inner diameter, 3
␮
m;
Waters, Milford, MA) was used with a flow rate of 0.15 ml/min.
Solvent A was 5 mMammonium acetate in water with 0.1%
formic acid, and solvent B was 5 mMammonium acetate in
acetonitrile with 0.1% formic acid. The linear gradient was as
follows: 50% B at 0 min, 50% B at 2 min, 90% B at 15 min,
90% B at 17 min, 50% B at 18 min, and 50% B at 25 min.
Formation of DHT Normalized to
␤
-Galactosidase
Cells (1.0 ⫻10
6
) were lysed using reporter lysis buffer (Pro-
mega Corp., Madison, WI) at the 2-h time point, and the
formation of DHT across the entire time course was normal-
ized to the coexpressed
␤
-galactosidase activity. The level of
␤
-galactosidase was detected spectrophotometrically fol-
lowing the manufacturer’s protocol (Promega). In the proto-
col, 2⫻reaction
␤
-galacosidase enzyme solution was made
using 200 mMsodium phosphate (pH 7.3) (Fisher Scientific),
2mMMgCl
2
, 100 mM
␤
-mercaptoethanol, and 4.4 mM
␱
-ni-
trophenyl-
␤
-D-galactopyranoside (Sigma-Aldrich Corp, St.
Louis, MO) as specified by Promega. The reaction was ter-
minated using 1 Msodium carbonate, and the formation of
the
␱
-nitro-phenolate anion at 420 nm was measured using
the molar extinction coefficient (E ⫽6900 M
⫺1
cm
⫺1
). Percent
conversion of steroid substrate was then normalized to mil-
liunits of
␤
-galactosidase.
Isolation of the Oxidative 3
␣
-HSDs from Transiently
Transfected COS-1 Cells
COS-1 cells were plated at 2.5 ⫻10
5
cells and transfected
using 2
␮
g of cDNA (pcDNA3–3
␣
-HSD). After 20 h the cells
were washed twice with PBS and harvested using a Tris-HCl-
sucrose buffer (pH 7.4) containing 50 mMTris-HCl, 250 mM
sucrose, 1 mMEDTA, and 1 mM
␤
-mercaptoenthanol. The
cells were sonicated using 4⫻10 bursts four times, and the
resulting supernatant was centrifuged at 800 ⫻gfor 10 min
at4Ctoremove the cellular debris. The supernatant was
removed and centrifuged at 100,000 ⫻gfor1hat4Cto
isolate the cytosolic and membrane fractions. After the high-
spin centrifugation the supernatant was removed, and glyc-
erol was added to the supernatant to a final concentration of
30% and stored as the cytosolic fraction. The membrane
pellet was washed in the Tris-HCl-sucrose buffer, resoni-
cated, resuspended, and centrifuged at 100,000 ⫻gfor an
additional hour at 4 C. The cytosolic fraction and this second
soluble fraction had no enzymatic activity with steroid sub-
strate. The membrane pellet was resuspended in the Tris-
HCl-sucrose buffer using a homogenizer, and glycerol was
added to a final concentration of 30% and stored at ⫺80 C
freezer. The total protein for the cytosolic and membrane
fractions was determined for each sample using the method
of Bradford (54).
Steady-State Kinetic Analysis of the Expressed
3
␣
-HSDs
The steady-state kinetic parameters for the 3
␣
-HSDs were
determined using the isolated cytosolic and membrane frac-
tions from transiently transfected COS-1 cells. The kinetic
analysis (final volume of 200
␮
l) was conducted at 37 C in the
Tris-HCl-sucrose buffer, pH 7.4 (final concentrations were 40
mMTris-HCl, 200 mMsucrose, 0.8 mMEDTA, and 0.8 mM
␤
-mercaptoenthanol), 10 mMMgCl
2
, 4% methanol, and a
combination of [
3
H]3
␣
-diol and unlabeled steroid to obtain
the final substrate concentration. Isolated cytosolic and
membrane fractions were added, and the reactions were
initiated by the addition of the oxidized cofactor nicotinamide
adenine dinucleotide to a final concentration equal to 1 mMas
previously determined (36, 39, 42). Reactions were termi-
nated using 1 ml of water-saturated ethyl acetate, extracted,
dried, and plated for TLC analysis as described earlier. This
discontinuous assay measures the formation of DHT from
3
␣
-diol vs. time. To obtain progress curves, time points were
collected to determine the initial velocity for each concentra-
tion tested by linear regression. Plots of velocity vs. substrate
concentration were hyperbolic and could be iteratively fit to
the Michaelis-Menten equation [v ⫽(V
max
* S)/(K
m
⫹S)] to
yield values for V
maxapp
,V
maxapp
/K
m
and K
m
, and their asso-
ciated SEs for the oxidation of 3
␣
-diol.
CAT Reporter Gene Assays
COS-1 cells were plated into six-well plates at a density of
3.5 ⫻10
⫺5
cells using DMEM (⫺phenol red), 1% CDT-FBS,
1% penicillin/streptomycin, and 2% L-glutamine. Twenty
hours after plating, the cells were transfected with FuGENE6
using 0.2
␮
g of pCMV-AR, 0.4
␮
g of (ARE)
2
-tk-CAT, 0.1
␮
gof
pCMV-
␤
-galactosidase, and 0.2
␮
g of pcDNA3 or the
pcDNA3–3
␣
-HSD of choice. Twenty-four hours after the
transfection, steroids were added to the individual wells.
Steroid concentrations included DHT (1 ⫻10
⫺12
to 1 ⫻10
⫺6
M), testosterone (1 ⫻10
⫺12
to 1 ⫻10
⫺6
M), and 3
␣
-diol (1 ⫻
10
⫺12
to1⫻10
⫺6
M). The specificity of the CAT assay was
determined by using the nontransfected control, the minus
AR control (i.e. p-(ARE)
2
-tk-CAT, pCMV-
␤
-galactosidase,
and pcDNA3); the tk-CAT promoter control (i.e. pCMV-AR,
p-tk-CAT, pCMV-
␤
-galactosidase, and pcDNA3); the pbasic-
CAT basic or no promoter control (i.e. pCMV-AR, pbasic-
CAT, pCMV-
␤
-galactosidase, and pcDNA3); and the DMSO
control (i.e. pCMV-AR, p-(ARE)
2
-tk-CAT, pCMV-
␤
-galactosi-
dase, and pcDNA3).
The CAT assay monitors the transfer of n-butyryl from
n-butyryl coenzyme A (Sigma-Aldrich Corp.) to D-threo-[di-
chloroacetyl-1,2–14C] chloramphenicol (60 mCi/mmol,
PerkinElmer LLC) whereby the products are separated by
TLC. The CAT reporter gene assay was performed using the
method described by Promega. Briefly, after 20 h of incuba-
tion with steroid, the cells were washed twice with PBS, and
200
␮
l of reporter lysis buffer was added to each well and
incubated at 37 C for 15 min. The cells were scraped, col-
Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate Mol Endocrinol, February 2006, 20(2):444–458 455
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

lected, and frozen at ⫺80 C until further processing. The cells
were thawed, lysed by vortexing for 15 sec, and centrifuged,
and the resulting supernatant was diluted with 800
␮
lof
reaction lysis buffer. The
␤
-galactosidase assay was per-
formed as described earlier, and the samples were normal-
ized to the no-steroid-treated cells using the
␤
-galactosidase
activity. The lysates were heated at 60 C for 10 min to
inactivate native cell deacetylases, as described by Promega,
and the CAT assay was subsequently performed at 37 C for
45 min. The reactions were terminated using 1 ml of water-
saturated ethyl acetate, extracted, dried, redissolved with
ethyl acetate, and plated for TLC analysis. The TLC plates
were prerun twice and developed using methylene chloride/
acetone [92.5:7.5 (vol/vol)]. The TLC plates were analyzed
using the TLC-linear analyzer as described previously. The
percentage of CAT activity was calculated and compared
with the no-steroid control to determine fold induction. The
EC
50
values were obtained by plotting the percent of CAT
activity (fold-activation/maximum activation * 100) vs. the
Log
10
concentration of steroid (M) using Grafit 5 [y ⫽
(Range)/(1 ⫹exp(slope factor * ln(abs(S/EC
50
)))) ⫹Back-
ground]. Inhibition of CAT activity was monitored using the
AR antagonist flutamide (Sigma-Aldrich Corp.) in these ex-
periments. Flutamide (0.001
␮
M–10
␮
M) and the test steroid
(EC
50
concentration) were combined, dried down under ni-
trogen, dissolved in DMSO, and added to the cells as a
cocktail.
Real-Time RT-PCR
Real-time RT-PCR determined the relative mRNA expression
levels of the oxidative 3
␣
-HSDs in human prostate tissue. The
development of the real-time PCR assay included the iden-
tification of specific primers to amplify only the desired gene
and placement of the primers to cross over an exon-intron
sequence to prevent the nonspecific amplification of
genomic DNA. Primer specificity was determined by sepa-
rating the PCR product on a 3% gel and by sequencing to
ensure only the amplification of the desired gene. Real-time
PCR was performed using a DNA Engine Opticon2 Continu-
ous Fluorescence Detector (MJ Research, Inc., Waltham,
MA), and each plate contained nine standards in duplicate
and four no-template controls. At the end of the PCR reac-
tion, melting curves were performed to ensure amplification
of the desired gene. The RT-PCR method was linear (⬎r⫽
0.995) over a dynamic range (10
9
) as determined by plotting
the log
10
fluorescence intensity vs. the amount of plasmid.
Total RNA pooled from 32 human Caucasian male prostates
was purchased from BD Bioscience (Palo Alto, CA) and 1
␮
g
of total RNA was reverse transcribed using the GeneAmp
RNA PCR Kit. The primer sequences for amplification of
ERAB, AR, and the housekeeping genes GAPDH and PBGD
were obtained from previous publications (55–57). The prim-
ers for RL-HSD are: forward, 5⬘-dGCT TTC TTT GTA GGA
GGC TAC TGT G-3⬘; reverse, 5⬘-dTCC TTA ATA TGC TTG
GGG GCT TCT-3⬘, giving a 187-bp product. Primers for
RODH 5 are: forward, 5⬘-dGAG GCC TTC TCT GAC AGC
CTG AG-3⬘; reverse, 5⬘-dCCA TAG TGG GCC TGT GTG
GCA-3⬘, giving a 169-bp product. NT 3
␣
-HSD primers are:
forward, 5⬘-dCCA AGT TGG GGA GAA AGG TCT-3⬘; reverse,
5⬘-dCAC TGG AGA CAT TAA TAA CTC TCC-3⬘, giving a
197-bp product. Primers for RODH 4 are: forward, 5⬘-dGAC
CGG TCC AGT CCA GAG GTC-3⬘; reverse, 5⬘-dTAG CGA
GTA CGG GGG TGG CAG-3⬘, giving a 160-bp product. The
conditions for the real-time PCR using SYBR Green (QIA-
GEN, Inc., Valencia, CA) were as follows: 95 C for 15 min
followed by 40 cycles of 94 C for 15 sec, X°C for 30 sec, and
72 C for 30 sec (where X ⫽58 C for GAPDH, PBGD, and AR;
60 C for RL-HSD, NT 3
␣
-HSD, and RODH 4; and 63 C for
ERAB and RODH 5). Full-length standards (2,500,000 fg ⫺
0.025 fg) were generated for ERAB, RL-HSD, RODH 5, NT
3
␣
-HSD, and RODH 4 from their appropriate cDNA plasmids
(pcDNA2-ERAB, pcDNA3-RL-HSD, pcDNA3-RODH 5,
pcDNA3-NT 3
␣
-HSD, and pcDNA3-RODH 4). PCR product
standards (2,500,000 fg ⫺0.25 fg) were generated for AR,
GAPDH, and PBGD by isolating the desired PCR product
after a PCR reaction using total human liver RNA. The prod-
uct was then isolated by gel purification and used as stan-
dards with correction factors for GADPH (3.30), PBGD (7.48),
and AR (11.79) due to the difference in molecular weight
between full-length and PCR product standards.
RT-PCR in Cultured Prostate Primary Epithelial and
Stromal Cells
The cultured primary prostate epithelial and stromal cells
were maintained as previously reported (58, 59). Total RNA (1
␮
g) from primary prostate epithelial (n ⫽14) and stromal (n ⫽
15) cells was reverse transcribed using GeneAmp RNA PCR
Kit, and 50 ng of cDNA was subsequently used to determine
the expression of the SDRs using the real-time PCR method
described above.
Acknowledgments
Received July 13, 2005. Accepted September 12, 2005.
Address all correspondence and requests for reprints to:
Trevor M. Penning, Department of Pharmacology, University
of Pennsylvania School of Medicine, 130C John Morgan
Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania
19104-6084. E-mail: penning@pharm.med.upenn.edu.
This work was supported by National Institutes of Health
(NIH) Grant R01 CA-90744 (to T.M.P.), and Department of
Defense (Army) Grant PC040420 (to D.M.P.). D.R.B. was sup-
ported, in part, by NIH Training Grant 1R25-CA-101871-D1.
REFERENCES
1. Handelsman DJ 2001 Androgen action and pharmaco-
logic uses. In: DeGroot LJ, Jameson JL, Burger H, eds.
Endocrinology. 4th ed. Philadelphia: W.B. Saunders Co;
2232–2242
2. Hayward SW, Cunha GR 2000 The prostate: develop-
ment and physiology. Radiol Clin North Am 38:1–14
3. Penning TM 1997 Molecular endocrinology of hydroxys-
teroid dehydrogenases. Endocr Rev 18:281–305
4. Weiss RE 1997 Management of prostate diseases. In:
Weiss RE, Fair WE, eds. Management of prostate dis-
eases. 2nd ed. Caddo, OK: Professional Communica-
tions, Inc.; 1–144
5. Wilson JD 1996 Role of dihydrotestosterone in androgen
action. Prostate Suppl 6:88–92
6. Geissler WM, Davis DL, Wu L, Bradshaw KD, Patel S,
Mendonca BB, Elliston KO, Wilson JD, Russell DW,
Andersson S 1994 Male pseudohermaphroditism caused
by mutations of testicular 17
␤
-hydroxysteroid dehydro-
genase 3. Nat Genet 7:34–39
7. McKenna NJ, O’Malley BW 2002 Combinatorial control
of gene expression by nuclear receptors and coregula-
tors. Cell 108:465–474
8. Carson-Jurica MA, Schrader WT, O’Malley BW 1990 Ste-
roid receptor family: structure and functions. Endocr Rev
11:201–220
9. Ji Q, Chang L, VanDenBerg D, Stanczyk FZ, Stolz A 2003
Selective reduction of AKR1C2 in prostate cancer and its
role in DHT metabolism. Prostate 54:275–289
10. Juniewicz PE, Berry SJ, Coffey DS, Strandberg JD, Ew-
ing LL 1994 The requirement of the testis in establishing
the sensitivity of the canine prostate to develop benign
prostatic hyperplasia. J Urol 152:996–1001
11. DeKlerk DP, Coffey DS, Ewing LL, McDermott IR, Reiner
WG, Robinson CH, Scott WW, Strandberg JD, Talalay P,
456 Mol Endocrinol, February 2006, 20(2):444–458 Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

Walsh PC, Wheaton LG, Zirkin BR 1979 Comparison of
spontaneous and experimentally induced canine pros-
tatic hyperplasia. J Clin Invest 64:842–849
12. Wilson JD 1987 Disorders of androgen action. Clin Res
35:1–12
13. Andersson S, Berman DM, Jenkins EP, Russell DW 1991
Deletion of steroid 5
␣
-reductase 2 gene in male
pseudohermaphroditism. Nature 354:159–161
14. Bruchovsky N, Wilson JD 1968 The intranuclear binding
of testosterone and 5
␣
-androstan-17-
␤
-ol-3-one by rat
prostate. J Biol Chem 243:5953–5960
15. Russell DW, Wilson JD 1994 Steroid 5
␣
-reductase: two
genes/two enzymes. Annu Rev Biochem 63:25–61
16. Andersson S, Bishop RW, Russell DW 1989 Expression
cloning and regulation of steroid 5
␣
-reductase, an en-
zyme essential for male sexual differentiation. J Biol
Chem 264:16249–16255
17. Orlowski J, Bird CE, Clark AF 1983 Androgen 5
␣
-reduc-
tase and 3
␣
-hydroxysteroid dehydrogenase activities in
ventral prostate epithelial and stromal cells from imma-
ture and mature rats. J Endocrinol 99:131–139
18. Kao LW, Lloret AP, Weisz J 1977 Metabolism in vitro of
dihydrotestosterone, 5
␣
-androstane 3
␣
,17
␤
-diol and its
3
␤
-epimer, three metabolites of testosterone, by three of
its target tissues, the anterior pituitary, the medial basal
hypothalamus and the seminiferous tubules. J Steroid
Biochem 8:1109–1115
19. Pilven A, Thieulant ML, Ducouret B, Samperez S, Jouan
P 1976 Rapid and intensive conversion of 5
␣
-andro-
stane-3
␣
,17
␤
-diol into 5
␣
-dihydrotestosterone in the
male rat anterior pituitary: in vivo and in vitro studies.
Steroids 28:349–358
20. Jacobi GH, Moore RJ, Wilson JD 1978 Studies on the
mechanism of 3
␣
-androstanediol-induced growth of the
dog prostate. Endocrinology 102:1748–1758
21. Jacobi GH, Moore RJ, Wilson JD 1977 Characterization
of the 3
␣
-hydroxysteroid dehydrogenase of dog pros-
tate. J Steroid Biochem 8:719–723
22. Jacobi GH, Wilson JD 1976 The formation of 5
␣
-andro-
stane-3
␣
,17
␤
-diol by dog prostate. Endocrinology 99:
602–610
23. Walsh PC, Wilson JD 1976 The induction of prostatic
hypertrophy in dog with androstanediol. J Clin Invest
57:1093–1097
24. Leihy MW, Shaw G, Wilson JD, Renfree MB 2001 Viril-
ization of the urogenital sinus of the tammar wallaby is
not unique to 5
␣
-androstane-3
␣
,17
␤
-diol. Mol Cell En-
docrinol 181:111–115
25. Shaw G, Renfree MB, Leihy MW, Shackleton CH, Roit-
man E, Wilson JD 2000 Prostate formation in a marsupial
is mediated by the testicular androgen 5
␣
-androstane-
3
␣
,17
␤
-diol. Proc Natl Acad Sci USA 97:12256–12259
26. Tieva A, Bergh A, Damber JE 2003 The clinical implica-
tions of the difference between castration, gonadotro-
phin releasing-hormone (GnRH) antagonists and agonist
treatment on the morphology and expression of GnRH
receptors in the rat ventral prostate. BJU Int 91:227–233
27. Geller J 1993 Basis for hormonal management of ad-
vanced prostate cancer. Cancer 71:1039–1045
28. Preston DM, Torrens JI, Harding P, Howard RS, Duncan
WE, McLeod DG 2002 Androgen deprivation in men with
prostate cancer is associated with an increased rate of
bone loss. Prostate Cancer Prostatic Dis 5:304–310
29. Uygur MC, Arik AI, Altug U, Erol D 1998 Effects of the
5
␣
-reductase inhibitor finasteride on serum levels of go-
nadal, adrenal, and hypophyseal hormones and its clin-
ical significance: a prospective clinical study. Steroids
63:208–213
30. McConnell JD, Bruskewitz R, Walsh P, Andriole G, Lieber
M, Holtgrewe HL, Albertsen P, Roehrborn CG, Nickel JC,
Wang DZ, Taylor AM, Waldstreicher J 1998 The effect of
finasteride on the risk of acute urinary retention and the
need for surgical treatment among men with benign
prostatic hyperplasia. Finasteride Long-Term Efficacy
and Safety Study Group. N Engl J Med 338:557–563
31. Andriole G, Bruchovsky N, Chung LW, Matsumoto AM,
Rittmaster R, Roehrborn C, Russell D, Tindall D 2004
Dihydrotestosterone and the prostate: the scientific ra-
tionale for 5
␣
-reductase inhibitors in the treatment of
benign prostatic hyperplasia. J Urol 172:1399–1403
32. Titus MA, Gregory CW, Ford III OH, Schell MJ, Maygar-
den SJ, Mohler JL 2005 Steroid 5
␣
-reductase isozymes
I and II in recurrent prostate cancer. Clin Cancer Res
11:4365–4371
33. Kinouchi T, Horton R 1974 3
␣
-Androstanediol kinetics in
man. J Clin Invest 54:646–653
34. Horst HJ, Dennis M, Kaufmann J, Voigt KD 1975 In vivo
uptake and metabolism of 3H-5
␣
-androstane-3
␣
,17
␤
-
diol and of 3H-5
␣
-androstane-3
␤
,17
␤
-diol by human
prostatic hypertrophy. Acta Endocrinol (Copenh) 79:
394–402
35. Huang XF, Luu-The V 2001 Characterization of the oxi-
dative 3
␣
-hydroxysteroid dehydrogenase activity of hu-
man recombinant 11-cis-retinol dehydrogenase. Bio-
chim Biophys Acta 1547:351–358
36. Wang J, Chai X, Eriksson U, Napoli JL 1999 Activity of
human 11-cis-retinol dehydrogenase (Rdh5) with ste-
roids and retinoids and expression of its mRNA in extra-
ocular human tissue. Biochem J 338:23–27
37. He XY, Merz G, Yang YZ, Pullakart R, Mehta P, Schulz H,
Yang SY 2000 Function of human brain short chain L-3-
hydroxyacyl coenzyme A dehydrogenase in androgen
metabolism. Biochim Biophys Acta 1484:267–277
38. He XY, Merz G, Mehta P, Schulz H, Yang SY 1999
Human brain short chain L-3-hydroxyacyl coenzyme A
dehydrogenase is a single-domain multifunctional en-
zyme. Characterization of a novel 17
␤
-hydroxysteroid
dehydrogenase. J Biol Chem 274:15014–15919
39. Biswas MG, Russell DW 1997 Expression cloning and
characterization of oxidative 17
␤
- and 3
␣
-hydroxysteroid
dehydrogenases from rat and human prostate. J Biol
Chem 272:15959–15966
40. Chetyrkin SV, Belyaeva OV, Gough WH, Kedishvili NY
2001 Characterization of a novel type of human micro-
somal 3
␣
-hydroxysteroid dehydrogenase: unique tissue
distribution and catalytic properties. J Biol Chem 276:
22278–22286
41. Jurukovski V, Markova NG, Karaman-Jurukovska N,
Randolph RK, Su J, Napoli JL, Simon M 1999 Cloning
and characterization of retinol dehydrogenase tran-
scripts expressed in human epidermal keratinocytes. Mol
Genet Metabol 67:62–73
42. Gough WH, VanOoteghem S, Sint T, Kedishvili NY 1998
cDNA cloning and characterization of a new human mi-
crosomal NAD
⫹
-dependent dehydrogenase that oxi-
dizes all-trans-retinol and 3
␣
-hydroxysteroids. J Biol
Chem 273:19778–19785
43. Rizner TL, Lin HK, Peehl DM, Steckelbroeck S, Bauman
DR, Penning TM 2003 Human type 3 3
␣
-hydroxysteroid
dehydrogenase (aldo-keto reductase 1C2) and androgen
metabolism in prostate cells. Endocrinology 144:
2922–2932
44. Gluzman Y 1981 SV40-transformed simian cells support
the replication of early SV40 mutants. Cell 23:175–182
45. Kaighn ME, Narayan KS, Ohnuki Y, Lechner JF, Jones
LW 1979 Establishment and characterization of a human
prostatic carcinoma cell line (PC-3). Invest Urol 17:16–23
46. Huang XF, Luu-The V 2000 Molecular characterization of
a first human 3(
␣
3
␤
)-hydroxysteroid epimerase. J Biol
Chem 275:29452–29457
47. El-Alfy M, Luu-The V, Huang XF, Berger L, Labrie F,
Pelletier G 1999 Localization of type 5 17
␤
-hydroxys-
teroid dehydrogenase, 3
␤
-hydroxysteroid dehydroge-
nase, and androgen receptor in the human prostate by in
situ hybridization and immunocytochemistry. Endocri-
nology 140:1481–1491
Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate Mol Endocrinol, February 2006, 20(2):444–458 457
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020

48. Ottander U, Poromaa IS, Bjurulf E, Skytt A, Backstrom T,
Olofsson JI 2005 Allopregnanolone and pregnanolone
are produced by the human corpus luteum. Mol Cell
Endocrinol 239:37–44
49. Karlsson T, Vahlquist A, Kedishvili N, Torma H 2003
13-cis-Retinoic acid competitively inhibits 3
␣
-hydroxys-
teroid oxidation by retinol dehydrogenase RoDH-4: a
mechanism for its anti-androgenic effects in sebaceous
glands? Biochem Biophys Res Commun 303:273–278
50. Chetyrkin SV, Hu J, Gough WH, Dumaual N, Kedishvili
NY 2001 Further characterization of human microsomal
3
␣
-hydroxysteroid dehydrogenase. Arch Biochem Bio-
phys 386:1–10
51. Lin HK, Jez JM, Schlegel BP, Peehl DM, Pachter JA,
Penning TM 1997 Expression and characterization of
recombinant type 2 3
␣
-hydroxysteroid dehydrogenase
(HSD) from human prostate: demonstration of bifunc-
tional 3
␣
/17
␤
-HSD activity and cellular distribution. Mol
Endocrinol 11:1971–1984
52. Hou YT, Lin HK, Penning TM 1998 Dexamethasone reg-
ulation of the rat 3
␣
-hydroxysteroid/dihydrodiol dehydro-
genase gene. Mol Pharmacol 53:459–466
53. Steckelbroeck S, Jin Y, Gopishetty S, Oyesanmi B, Pen-
ning TM 2004 Human cytosolic 3
␣
-hydroxysteroid dehy-
drogenases of the aldo-keto reductase superfamily dis-
play significant 3
␤
-hydroxysteroid dehydrogenase
activity: implications for steroid hormone metabolism
and action. J Biol Chem 279:10784–10795
54. Bradford MM 1976 A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal Biochem 72:
248–254
55. Steckelbroeck S, Watzka M, Reissinger A, Wegener-
Toper P, Bidlingmaier F, Bliesener N, Hans VH, Clus-
mann H, Ludwig M, Siekmann L, Klingmuller D 2003
Characterisation of estrogenic 17
␤
-hydroxysteroid dehy-
drogenase (17
␤
-HSD) activity in the human brain. J Ste-
roid Biochem Mol Biol 86:79–92
56. Steckelbroeck S, Watzka M, Lutjohann D, Makiola P,
Nassen A, Hans VH, Clusmann H, Reissinger A, Ludwig
M, Siekmann L, Klingmuller D 2002 Characterization of
the dehydroepiandrosterone (DHEA) metabolism via ox-
ysterol 7
␣
-hydroxylase and 17-ketosteroid reductase ac-
tivity in the human brain. J Neurochem 83:713–726
57. Sinha-Hikim I, Taylor WE, Gonzalez-Cadavid NF, Zheng
W, Bhasin S 2004 Androgen receptor in human skeletal
muscle and cultured muscle satellite cells: up-regulation
by androgen treatment. J Clin Endocrinol Metab 89:
5245–5255
58. Peehl DM 1992 Culture of human prostate epithelial cells.
In: Freshney RI, ed. Culture of epithelial cells. New York:
Wiley & Sons; 159–180
59. Peehl DM, Sellers RG 2000 Cultured stromal cells: an in
vitro model of prostatic mesenchymal biology. Prostate
45:115–123
Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost
professional society serving the endocrine community.
458 Mol Endocrinol, February 2006, 20(2):444–458 Bauman et al. • Major Oxidative 3
␣
-HSD in Human Prostate
Downloaded from https://academic.oup.com/mend/article-abstract/20/2/444/2741464 by guest on 04 August 2020