(Received for publication, February 27, 1997, and in revised form, April 18, 1997)
From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9046
Intracellular levels of active steroid hormones
are determined by their relative rates of synthesis and breakdown. In
the case of the potent androgen dihydrotestosterone, synthesis from the
precursor testosterone is mediated by steroid 5-reductase, whereas
breakdown to the inactive androgens 5
-androstane-3
,17
-diol (3
-adiol), and androsterone is mediated by reductive
3
-hydroxysteroid dehydrogenases (3
-HSD) and oxidative
17
-hydroxysteroid dehydrogenases (17
-HSD), respectively. We
report the isolation by expression cloning of a cDNA encoding a
17
-HSD6 isozyme that oxidizes 3
-adiol to androsterone. 17
-HSD6
is a member of the short chain dehydrogenase/reductase family and
shares 65% sequence identity with retinol dehydrogenase 1 (RoDH1),
which catalyzes the oxidation of retinol to retinal. Expression of rat
and human RoDH cDNAs in mammalian cells is associated with the
oxidative conversion of 3
-adiol to dihydrotestosterone. Thus,
17
-HSD6 and RoDH play opposing roles in androgen action; 17
-HSD6
inactivates 3
-adiol by conversion to androsterone and RoDH activates
3
-adiol by conversion to dihydrotestosterone. The synthesis of
an active steroid hormone by back conversion of an inactive metabolite
represents a potentially important mechanism by which the steady state
level of a transcriptional effector can be regulated.
The formation and growth of the prostate as well as other male
reproductive organs is dependent on androgens, a class of
C19 steroids that act through the androgen receptor to
regulate gene expression. The two predominant androgens in this regard
are testosterone,1 which is formed in the
testis from androstenedione by the action of a reductive
17-hydroxysteroid dehydrogenase (17
-HSD3) enzyme (1), and
dihydrotestosterone, which is formed from testosterone by steroid
5
-reductase isozymes and is the active androgen in the prostate (2).
5
-Reduction of testosterone is an irreversible reaction. Androgen
action is terminated when these steroids are converted to metabolites
that have little or no affinity for the androgen receptor. A major
catabolic pathway involves the sequential conversion of
dihydrotestosterone to 5
-androstane-3
,17
-diol (3
-adiol) and
thereafter to androsterone (3). Both of these reactions are potentially
reversible (see Fig. 1).
Under normal conditions, an equilibrium exists between androgen
synthesis and breakdown and a steady state level of active hormone is
maintained in target tissues. An imbalance between input and output can
lead to androgen excess and contribute to certain disease states,
especially benign prostatic hyperplasia and prostate cancer. Two
mechanisms are postulated to produce excess dihydrotestosterone in the
prostate. In one, an elevated expression of steroid 5-reductase
leads to increased synthesis of the hormone. In the other, a decrease
in the breakdown of dihydrotestosterone occurs as a consequence of
either reduced expression of a catabolic enzyme or by a change in the
direction of the reaction. The latter mechanism is illustrated by the
differential metabolism of 3
-adiol (Fig. 1). If a reductive
3
-hydroxysteroid dehydrogenase (3
-HSD) activity predominates in
the prostate, then dihydrotestosterone is converted into 3
-adiol and
thereafter into androsterone, which is readily glucuronidated (4) and
excreted from the tissue. However, if an oxidative 3
-HSD activity
predominates, then 3
-adiol arising within the tissue or from
exogenous sources is converted back into the active androgen
dihydrotestosterone (Fig. 1).
Evidence supporting that androgen excess can be caused by an increase
in steroid 5-reductase activity has been obtained from enzyme
measurements and immunohistochemical analysis of benign prostatic
hyperplastic and prostate cancer tissue (5, 6). The levels of steroid
5
-reductase activity and protein are increased in these tissues.
Dihydrotestosterone can also be synthesized from 3
-adiol. When
radiolabeled 3
-adiol is administered to castrated and functionally
hepatectomized rats, it is converted into dihydrotestosterone and to a
lesser extent into androsterone in the ventral prostate (7). The
mechanism responsible for this conversion has not been elucidated, in
part due to a paucity of information regarding androgen catabolizing
enzymes in the prostate. Two closely related reductive 3
-HSD
isozymes are expressed in the human prostate (8), and an oxidative
17
-HSD type 2 isozyme and mRNA are present in benign and
malignant human prostate tissue (9) but the roles of these enzymes in
the metabolism of dihydrotestosterone are not known.
To gain further insight into androgen catabolism in the prostate, we
took an expression cloning approach to identify gene products capable
of metabolizing 3-adiol. These experiments identified an oxidative
17
-HSD isozyme that converts 3
-adiol to androsterone and whose
mRNA is expressed in the rat ventral prostate and liver. The
encoded protein, referred to as 17
-HSD6, shares extensive sequence
homology with retinol dehydrogenase 1 (RoDH1) (10), which led us to
test the hypothesis that RoDH1 might also metabolize steroid hormones.
Expression studies with rat and human RoDH cDNAs show that RoDH can
also serve as an oxidative 3
-HSD that efficiently catalyzes the back
reaction of 3
-adiol to dihydrotestosterone.
Oligo(dT)-primed cDNA was
synthesized from rat ventral prostate poly(A)+-enriched RNA
(5 µg) using a SuperScript cDNA synthesis kit (Life Technologies,
Inc., Bethesda, MD). cDNAs were ligated to SalI adaptors, size-fractionated by gel filtration, and unidirectionally ligated into SalI-NotI digested pCMV6 vector
(11). A 20-µl ligation mixture was divided into 0.1-µl aliquots,
which were individually transformed into 100 µl of MAX Efficiency
DH5 competent cells (Life Technologies, Inc.), yielding pools of
5,000 independent recombinants per transformation. The transformation
mixture (1 ml) was diluted by the addition of 3 ml of Luria-Bertani
medium supplemented with ampicillin (100 µg/ml). After an overnight
incubation at 37 °C, plasmid DNA was isolated from the bacterial
cells by column purification (Promega Mini-Prep, Madison, WI) and
transfected into mammalian cells.
Pools of cDNAs were transfected into human embryonic kidney 293 cells (ATCC CRL number 1573) by the calcium phosphate method using an
MBS Transfection kit (Stratagene, La Jolla, CA). The 293 cells were
grown in monolayer at 37 °C in an atmosphere of 8-9%
CO2 and maintained in medium A (Dulbecco's modified
Eagle's medium containing 100 units/ml of penicillin and 100 µg/ml
streptomycin sulfate and supplemented with 10% (v/v) fetal calf
serum). On day 0 of a transfection experiment, 700,000 cells were
plated in medium A per 50-mm dish. On day 2, cells were cotransfected with 4.5 µg of plasmid cDNA pool and 0.5 µg of pVA1, a plasmid encoding adenovirus VA1 RNA, which enhances expression of transfected cDNAs via a translational mechanism (12). After the transfection procedure, which involved a 3-h incubation at 35 °C in a 3%
CO2 atmosphere, the cells were washed once with
phosphate-buffered saline and then placed under 3 ml of medium A. At
16 h, medium A was replaced with medium A containing 50 nM [3H]5-androstane-3
,17
-diol
(3
-adiol, specific activity 49.0 Ci/mmol), and the cells were
incubated at 37 °C for an additional 3 h. Thereafter, 1 ml of
medium was removed from each dish and subjected to extraction with 7 volumes of methylene chloride. Separation of steroids was performed as
outlined below.
In an initial screen of 36 pools of 5,000 cDNAs each, 13 pools
showed conversion of 3-adiol into androsterone over background levels obtained in mock-transfected cells. The DNAs from one of several
positive pools (number 12, chosen at random), were retransformed into
Escherichia coli DH5
cells to generate pools of
approximately 1,000 independent transformants each. These plasmid DNAs
were transfected into 293 cells to identify 6 positive pools. Further subfractionation into pools of 200, 50, 10, and individual
cDNA-containing plasmids yielded a final positive clone.
For assay of enzyme activity in transfected
cell lysates, cells were harvested in ice-cold phosphate-buffered
saline from dishes 30 h post-transfection using a rubber policeman
pelleted by centrifugation, and either frozen in liquid N2
for storage at 80 °C, or lysed and assayed immediately. Cell
pellets were homogenized in 10 mM potassium phosphate, pH
7.0, 150 mM KCl, 1 mM EDTA with three short
pulses of a Brinkmann Polytron. Enzyme assays were performed in 0.1 M sodium phosphate buffer at pH 7.5. Incubations were
performed at 37 °C without shaking for 5-15 min and contained a
final cofactor concentration of 1 mM. The concentration of
protein in cell extracts was determined by the method of Bradford using
a kit (Bio-Rad Protein Assay) and bovine serum albumin as a standard.
Assay mixtures (500 µl) were extracted with 14 volumes of methylene
chloride. Following isolation and evaporation of the organic phase,
steroids were dissolved in 35 µl of chloroform/methanol (2:1),
spotted on thin layer chromatography plates (LK5D Silica Gel, Whatman
Lab Sales) and resolved by development in chloroform/ethyl acetate
(3:1). Reaction products were visualized by exposure to PhosphorImaging
plates and analyzed on a BAS 1000 PhosphorImager (Fuji, Stamford,
CT).
Kinetic constants for steroid substrates and cofactors were measured by
conventional Lineweaver-Burk analyses. All assays were repeated in
triplicate using cell lysates from different transfection experiments.
Ten concentrations of substrate between 0.01 and 5 µM
were used for each steroid. For the determination of apparent cofactor
Km values, five concentrations between 1 and 16 µM were used for NAD+ and five concentrations
between 1 and 10 mM were used for NADP+, both
with [3H]3-adiol at 2.0 µM.
13-cis-Retinoic acid (13-cis-RA) and
9-cis-retinoic acid (9-cis-RA) were dissolved in
ethanol to a final concentration of 10 mM and stored under
nitrogen at 20 °C in amber vials. Enzyme assay solutions described
above were supplemented with these solutions to achieve final retinoid
concentrations between 0.001 and 100 µM and incubations
were performed under low light conditions. Inhibition constants
(apparent Ki values) were obtained by Dixon plot
analysis of transfected cell lysate activity in the presence of
inhibitory concentrations of 13-cis-RA and
9-cis-RA with two different [3H]3
-adiol
substrate concentrations. (15 and 50 nM).
A directional rat liver cDNA
library was constructed in LambdaZiplox (Life Technologies, Inc.) using
a superscript cDNA synthesis kit (Life Technologies, Inc.).
Oligo(dT)-primed cDNA was prepared from 5 µg of mRNA, cDNAs
were unidirectionally ligated into the LambdaZiplox vector and packaged
with a MaxPlax Lambda Packaging extract (Epicentre Technologies,
Madison, WI). This library was screened with a probe generated by
radiolabeling with random hexamers (Mega Prime Labeling kit, Amersham)
from the 17-HSD6 cDNA shown in Fig. 3. Out of 100,000 recombinants screened, 18 hybridization positive plaques were
identified, two of which were purified through additional rounds of
screening, and subjected to DNA sequence analysis using SP6 and T7
primers. Another 100,000 recombinants were screened with a
3
-untranslated region probe to produce an additional seven
hybridization positive plaques, of which four were analyzed by DNA
sequencing.
Human RoDH cDNAs were isolated from a prostate cDNA library prepared as described above. Approximately 105 recombinants were screened with a radiolabeled probe corresponding to nucleotides 1-966 of the rat RoDH1 cDNA (10). After hybridization in 50% formamide at 42 °C, filters were washed under reduced stringency conditions (0.3 M NaCl, 0.03 M sodium citrate, 0.2% (w/v) SDS at 55 °C for 1 h). Two identical hybridization positives were isolated and subjected to DNA sequence analysis.
RNA BlottingTotal cellular RNA was isolated by guanidine
thiocyanate extraction using RNA STAT 60 (TEL-TEST "B", Inc.,
Friendswood, TX). Poly(A)+-enriched RNA was isolated
with a mRNA Purification Kit (Pharmacia Biotech Inc.). RNA was
size-fractionated by electrophoresis through a 1.4% (w/v) agarose gel,
transferred to nylon filters (Biotrans, ICN, Costa Mesa, CA) by
capillary blotting, and hybridized and washed as described (13). Human
Multiple Tissue RNA blots were purchased from
CLONTECH (Palo Alto, CA). All RNA blots were probed with 3-untranslated region probes, which were generated by
amplification via the polymerase chain reaction, gel purification, and
random hexamer radiolabeling. The individual probes were: 17
-HSD6,
nt 1246-1521 of Fig. 3 (GenBank accession number [GenBank]); rat RoDH1,
nt 1252-1347 (GenBank accession number [GenBank]) described in Ref. 10;
and human RoDH, nt 1226-1513 (GenBank accession number [GenBank]), this
study.
The effects of androgens on
the expression of 17-HSD6 mRNA in the rat ventral prostate were
determined in normal (N), castrate (C), and androgen-supplemented
castrates (C+T) as described (14). Briefly, groups of sexually mature
Sprague-Dawley rats (Harlan, Inc., Indianapolis, IN) (n = 4-7) were either castrated via a scrotal route or sham operated on day
0 of an experiment. On days 7-10, the N and C groups received daily
injections (0.2 ml) of vehicle (sesame oil) alone, and the C+T group
received 0.2-ml injections containing 2 mg of testosterone acetate.
Animals were killed on the morning of day 10, ventral prostates were
dissected free of connective tissue and fat, and total RNA was isolated by extraction with acidified phenol/guanidinium isothyocyanate. Hybridization signals were quantified by densitometry, normalized to
the level of
-actin mRNA, and expressed as a percentage of signal measured in sham operated animals.
An expression cloning approach in mammalian cells was used to
identify rat prostatic gene products that metabolize 3-adiol. A
cDNA library comprised of approximately one million independent clones was first prepared in a pCMV vector using rat ventral prostate mRNA as a template. Transformants from the library were divided into pools of 5,000 colonies, and plasmid DNA prepared from each pool
was transfected into cultured embryonic kidney 293 cells. After 16-20
h, [3H]3
-adiol was added to the cell medium at a
concentration of 50 nM and the incubation was continued for
an additional 3 h. Thereafter steroids were extracted from the
medium and separated by thin layer chromatography on silica gel plates.
An initial screen of 36 pools revealed several that contained a
cDNA encoding an enzyme that converted 3
-adiol into androsterone
(Fig. 2A). No pools were identified that
contained an activity capable of converting 3
-adiol to
dihydrotestosterone. One of the pools (number 12) that contained a
3
-adiol
androsterone activity was subdivided into progressively
smaller pools by transfection screening until a single cDNA that
encoded this activity was identified (Fig. 2, B-F).
The nucleotide sequence of the rat cDNA insert harbored by the
active clone is shown in Fig. 3. Analysis of the DNA
sequence revealed a 5-untranslated region of 231 nucleotides (nt), a
potential coding region of 981 nt that could specify a protein of 327 amino acids, and a 3
-untranslated region of 318 nt followed by a
polyadenylate tract. An in-frame stop codon was located 15 nucleotides
upstream of the methionine codon that initiated the longest
translational reading frame. The sequence around this methionine codon
demonstrated a poor match (7/13 nt) with the Kozak consensus
(GCCGCC(A/G)CCAUGG, Ref. 15). A second methionine codon
within a similar context (7/13 nt matches) was located at amino acid 11 of the deduced protein sequence (Fig. 3). This arrangement of
methionine codons and their contexts' precluded an unambiguous
identification of the true amino terminus and complicated calculation
of the molecular weight of the encoded protein. If the first methionine
marks the amino terminus, then the predicted molecular weight of the
enzyme is 37,100, whereas if the second methionine fills this role then the molecular weight is 36,100.
The reaction catalyzed by the cDNA-encoded enzyme involves the
oxidation of an alcohol substituent at carbon 17 of the 3-adiol substrate to a 17-oxo group in the androsterone product (Fig. 1).
Enzymes that catalyze this reaction are referred to as
17
-hydroxysteroid dehydrogenases (17
-HSD). Five 17
-HSD enzymes
have previously been identified (17
-HSD1 through 17
-HSD5, see
"Discussion") and for this reason, the protein encoded by the
cDNA was termed 17
-HSD6.
The kinetic constants that describe the conversion of 3-adiol to
androsterone by rat 17
-HSD6 were determined in cell lysates prepared
from transfected 293 cells. An apparent Km of 0.1 µM and a Vmax of 2.5 nmol/min/mg
cell lysate were measured for this enzyme (Table I). The
expressed enzyme exhibited lower affinity and activity with
dihydrotestosterone, testosterone, and estradiol. Additional
experiments revealed that the 17
-HSD6 enzyme possessed a weak
oxidative 3
-HSD activity, converting androsterone into
5
-androstanedione with an apparent Km of 0.2 µM and Vmax of 0.1 nmol/min/mg
cell lysate protein. No oxidative 3
-HSD activity against 3
-adiol
was detected in these experiments. The 17
-HSD6 enzyme preferred
NAD+ (apparent Km = 3 µM)
over NADP+ (apparent Km = 1 mM) as a cofactor when 3
-adiol was used as substrate. A
comparison of the relative efficiencies (determined as
Vmax/Km) of the reactions
catalyzed by 17
-HSD6 showed that the enzyme was most active as an
oxidative 17
-hydroxysteroid dehydrogenase against 3
-adiol (Table
I).
|
The tissue distribution of rat 17-HSD6 was assessed by RNA blotting.
Poly(A+)-enriched RNA was prepared from 10 tissues,
transferred to a nylon filter, and probed with a radiolabeled DNA
fragment. The probe was derived from the 3
-untranslated region of the
rat 17
-HSD6 cDNA to reduce the possibility of
cross-hybridization to homologous mRNAs (see below). A single
mRNA of approximately 1.5-1.6 kb was detected in three tissues,
including the ventral prostate, liver, and kidney (Fig.
4A). The levels of this mRNA were highest
in the ventral prostate and liver, and much lower in the kidney. The
abundance of 17
-HSD6 mRNA in the ventral prostate agrees well
with the large number of positive pools identified in the initial
expression cloning experiments (Fig. 2A) and the size of the
rat mRNA detected by blotting (1.5-1.6 kb) is similar to that of
the isolated cDNA (1.53 kb, Fig. 3).
To determine if circulating androgens influenced the expression of
17-HSD6 in the ventral prostate, the level of this mRNA was
compared in normal (lane 1), castrated (lane 2),
and castrated plus androgen-supplemented (lane 3) rats (Fig.
4B). Castration was associated with a substantial decrease
of 17
-HSD6 mRNA (to 13% of normal) in the ventral prostate.
Administration of androgens to castrates for a 3-day period increased
the level of 17
-HSD6 mRNA to 56% of normal levels. The amount
of a control mRNA (
-actin) remained unchanged (right
panel, Fig. 4B). These marked changes in 17
-HSD6
mRNA in response to androgen depletion and repletion could mean
that the hormone is required for expression of 17
-HSD6 in the
ventral prostate. Alternatively, the reduction in 17
-HSD6 mRNA
levels after castration may be a secondary effect resulting from the
marked decrease in the number of epithelial cells that occurs upon
androgen withdrawal in this gland (16).
A comparison of the predicted protein sequence of rat 17-HSD6 to
those in the data bases indicated that the enzyme shared sequence
identity with numerous members of the short chain
dehydrogenase/reductase family. Among these family members, the most
closely related protein was rat RoDH1, an enzyme that catalyzes the
conversion of retinol to retinal (10). An alignment of rat 17
-HSD6
and RoDH1 protein sequences is shown in Fig. 5 and
revealed that the two proteins share 65% sequence identity over their
entire lengths. The shared sequence identity began at the second
methionine of the 17
-HSD6 sequence (see Fig. 3), which suggested
that the second and not the first methionine may be the true initiator
methionine codon. A cDNA clone encoding a human RoDH protein was
subsequently isolated as described under "Experimental Procedures,"
and a comparison of the deduced protein sequences of the rat
17
-HSD6, rat RoDH1, and human RoDH enzymes again revealed extensive
sequence identities (Fig. 5).
The sequence conservation between 17-HSD6, a steroid metabolizing
enzyme, and RoDH, a retinoid metabolizing enzyme, raised the question
of whether RoDH might act on certain steroid substrates. To test this
idea, expression vectors containing either the rat or human RoDH
cDNAs were constructed and transfected into cultured 293 cells. A
vector containing a rat 17
-HSD6 cDNA served as a positive
control in these experiments. Cell lysates were prepared from
transfected cells, incubated with different radiolabeled steroid
substrates, and the metabolism of these compounds was followed by thin
layer chromatography. Recombinant rat and human RoDH actively catalyzed
the oxidation of 3
-adiol to dihydrotestosterone (Fig.
6A), indicating that these enzymes were
potent oxidative 3
-HSD (Fig. 1). For example, when 1 µg of cell
lysate containing either recombinant human or rat RoDH was incubated
with 3
-adiol, approximately half of the substrate was converted into
a product (Fig. 6A, lanes 8 and 10) with a
mobility identical to that of dihydrotestosterone standards (Fig.
6A, lanes 1 and 12). Increasing the amount of
cell lysate containing human RoDH resulted in more conversion of
substrate to product (lanes 4-7). NAD+ was the
preferred cofactor in these experiments (apparent Km = 8-15 µM for rat and human isozymes) versus
NADP+ (apparent Km = 7-10
mM).
The rat 17-HSD6 enzyme initially converts 3
-adiol to
androsterone, whereas the RoDH enzymes convert 3
-adiol to
dihydrotestosterone. Androsterone and dihydrotestosterone migrate with
almost identical Rf values on silica gel plates
(Fig. 6A), which makes unambiguous identification of the two
compounds difficult. To resolve this issue, an experiment identical to
that shown in Fig. 6A was carried out except that the
steroids were chromatographed on aluminum oxide plates. Chromatography
on this separation medium readily resolved androsterone and
dihydrotestosterone (Fig. 6B), and confirmed the initial
findings of oxidative 17
-hydroxysteroid dehydrogenase activity
associated with 17
-HSD6 and of oxidative 3
-HSD activity
associated with rat and human RoDH.
Several additional steroid products arising from the actions of rat and
human RoDH were visualized in the experiments of Fig. 6, suggesting
that like 17-HSD6, the RoDH enzymes were multifunctional. The
identities of the various products, their origins, and the kinetic
characteristics of each reaction were determined by incubating cell
lysates with appropriate steroid substrates. The results indicated that
under the assay conditions employed, both rat and human RoDH enzymes
were most efficient at converting 3
-adiol to dihydrotestosterone,
but the human RoDH also possessed weak 3
-hydroxysteroid
dehydrogenase activity and both enzymes manifest oxidative
17
-hydroxysteroid dehydrogenase activities when presented with
appropriate substrates (Table I).
We next tested the ability of endogenous retinoids to act as inhibitors
of the metabolism of 3-adiol by 17
-HSD6 and RoDH. Using assay
conditions described under "Experimental Procedures," 13-cis-retinoic acid competitively inhibited 17
-HSD6
(apparent Ki = 4 µM), rat RoDH1
(Ki = 12 µM), and human RoDH
(Ki = 30 µM) enzymes. In contrast,
9-cis-retinoic acid was an excellent competitive inhibitor
of 17
-HSD6 (Ki = 0.4 µM) but was
essentially inactive (Ki > 100 µM) against the rat and human RoDH enzymes. The ability of endogenous retinoids to inhibit RoDH at these concentrations is of potential pharmacological importance but the physiological significance of this
inhibition is unknown since intracellular retinoid concentrations are
not thought to exceed the low nanomolar range.
The finding that rat and human RoDH were capable of producing
dihydrotestosterone from 3-adiol suggested that these enzymes might
play a physiologically important role in androgen target tissues. To
assess this potential role, RNA extracted from human and rat tissues
was subjected to blot hybridization using probes derived from the
3
-untranslated regions of the respective cDNAs. The RoDH1 mRNA
was detected in the liver of the rat (Fig.
7A). In contrast, a human RoDH mRNA of
approximately 1.6 kb was detected in the liver and in lesser amounts in
spleen, testis, and prostate (Fig. 7B). A human mRNA of
a different size (approximately 0.8 kb) was detected at a moderate
level in placenta. Whether this mRNA represents another gene
product or arises from RNA processing of human RoDH transcripts was not
determined. No cross-hybridizing mRNAs were detected when the
filters containing human tissue RNAs were probed at low stringency with
radiolabeled cDNA fragments derived from the 3
-untranslated region
of the rat 17
-HSD6 clone or when these filters were probed at high
stringency with near full-length rat 17
-HSD6 cDNA fragments
(data not shown).
We report isolation of a cDNA encoding a rat
17-hydroxysteroid dehydrogenase from ventral prostate. The
17
-HSD type 6 isozyme prefers to catalyze the oxidation of androgen
substrates, particularly that of 3
-adiol to androsterone. The
mRNA encoding this isozyme is present at high levels in rat liver
and ventral prostate where the encoded protein presumably plays a
largely catabolic role in degrading androgens. The 17
-HSD6 is a
member of the short chain dehydrogenase/reductase family and shares
65% sequence identity with RoDH1. The RoDH mRNA is expressed at
high levels in the rat and human liver and at lower levels in human
spleen, testis, and prostate. Rat and human RoDH enzymes expressed in
cultured cells actively convert 3
-adiol to dihydrotestosterone and
thus exhibit strong oxidative 3
-HSD activities. The tissue
distributions and preferred reaction directions of these enzymes
suggest that RoDH may play an important anabolic role in androgen
metabolism.
Six different enzymes that catalyze oxidation and/or reduction
reactions at carbon 17 of steroid substrates have been isolated. Each
of these 17-HSD isozymes demonstrates a favored substrate and
reaction direction, and they often have unique tissue distributions (17, 18). For example, the type 1 isozyme preferentially reduces estradiol to estrone and is abundant in the ovary and placenta (19).
The 17
-HSD type 2 isozyme catalyzes the oxidation of androgens and
is present in the endometrium and placenta (20, 21), whereas the type 3 isozyme is exclusively a reductive enzyme of the testis (1). The type 4 isozyme shows an oxidative preference with steroid substrates and the
type 5 a reductive preference (17, 18). Although the different
17
-HSD isozymes act at the same carbon of a steroid substrate,
pairwise comparisons between individual isozymes reveal less than 25%
sequence identity, which suggests that their catalytic activity was
acquired by convergent evolution (17, 18). This diversity in primary
sequence is also characteristic of the 17
-HSD type 6 isozyme, which
shares 28% sequence identity with the type 2 isozyme and lesser
identities with the other five isozymes.
The 17-HSD type 6 isozyme reported here appears to be largely an
oxidative enzyme that is active against both androgens and estrogens
when expressed in 293 cells (Table I). The mRNA encoding this
isozyme is abundant in the rat liver and ventral prostate (Fig. 4).
These properties predict that the physiological role of 17
-HSD6 is
to inactivate androgens and estrogens in tissues and thereby to limit
the extent of signaling activity mediated by these steroid hormones.
Inactivation may be particularly important in the androgen-sensitive
prostate where 3
-adiol can be converted to dihydrotestosterone,
which in turn can stimulate growth of the gland. The presence of the
17
-HSD6 enzyme may thus exert a protective effect against
unregulated benign or malignant prostate growth (22) by preventing the
buildup of 3
-adiol and hence the synthesis of dihydrotestosterone
via the back reaction.
The 17-HSD6 isozyme shares signature sequence motifs with members of
the short chain dehydrogenase/reductase family. Enzymes in this large
family (>50 members, Ref. 23) are both soluble (3
/20
-hydroxysteroid dehydrogenase) and membrane bound (RoDH, Ref. 24). A hydropathy analysis of the 17
-HSD6 sequence did not
reveal extended stretches of hydrophobic amino acids resembling classical transmembrane domains. However, the fact that 17
-HSD6 shares extensive sequence identity with the RoDH1 enzyme suggests that
it may also be associated with the membrane compartment of the cell.
Members of the short chain dehydrogenase/reductase class share
conserved primary and tertiary sequences and catalyze diverse biochemical reactions with different stereochemistries (23). For
example, the 3/20
-hydroxysteroid dehydrogenase enzyme will reduce
both the 3-oxo and 20-oxo groups of progestagens (8) and the
17
-HSD2 enzyme will oxidize both 17- and 20
-alcohol substituents
of steroids (17, 18). The 17
-HSD6 is also ambidextrous in its
catalytic activities, oxidizing both carbon 17 and carbon 3 alcohol
groups on certain steroid substrates (Table I). An analysis of the
kinetic constants associated with these two reactions suggests that
oxidation at carbon 17 takes place more readily than does oxidation at
carbon 3 (Table I), which may suggest that the latter reaction only
occurs in transfected cells at nonphysiological substrate and/or enzyme
concentrations.
The high degree of sequence identity between rat 17-HSD6 and rat
RoDH1 led us to test whether RoDH1 was active against steroid substrates. This enzyme has been extensively characterized as a
membrane-bound retinol dehydrogenase that participates in the conversion of retinol to retinal (10). Efficient oxidation of retinoids
by RoDH1 is dependent on their prior association with the cellular
retinol-binding protein (24). RoDH1 preferentially utilizes
NADP+ as a cofactor with retinoid substrates (10), and like
17
-HSD6, exists in at least three isozymic forms (10, 25, 26). The observation reported here that rat RoDH1 and a human RoDH are oxidative
3
-HSDs with nanomolar affinities for 3
-adiol is important for
several reasons. First, it confirms a hypothesis put forth in two
recent reviews that RoDH enzymes might be active against both retinoids
and steroids (27, 28). Second, although several reductive 3
-HSD
isozymes have been isolated (reviewed in Ref. 8), an oxidative isozyme
is a novelty. Third, and most importantly, RoDH isozymes may contribute
to the synthesis of dihydrotestosterone, a powerful androgen that
exerts both endocrine and autocrine effects in target tissues.
The rat RodH1 isozyme has been extensively characterized at both the
biochemical (24) and molecular (10) levels. More recently, two
additional rat RoDH isozymes, termed RoDH2 and RoDH3, have been defined
at the molecular level (25, 26). The human RoDH cDNA isolated here
shares 62% amino acid sequence identity with the rat RoDH1 isozyme and
61 and 63% identities with the RoDH2 and RoDH3 isozymes, respectively.
We thus cannot state with certainty which of the three rat RoDH
isozymes is orthologous to the human protein encoded by the cDNA
characterized here. Additional studies on human RoDH cDNAs and
proteins will be required to determine the existence of isozymes and
their relationships to the rat proteins. Despite this uncertainty in
nomenclature, however, the current studies demonstrate that a known rat
RoDH isozyme (type 1) and a highly related human enzyme have potent
oxidative 3-HSD activities.
A majority of dihydrotestosterone is thought to be synthesized from
testosterone by the actions of steroid 5-reductase isozymes (2), and
consistent with this biosynthetic origin, mutations that impair either
steroid 5
-reductase type 1 or type 2 cause androgen insufficiency
syndromes in males and females (29, 30). Nevertheless, several findings
suggest that the so-called back reaction, in which 3
-adiol is
converted into dihydrotestosterone (Fig. 1), may also serve as a
physiologically important source of this androgen. Thus, 3
-adiol is
a potent stimulator of in vivo prostate growth in the dog
(31) and rat (7, 14, 32) and growth of the rat prostate in organ
culture is also stimulated by this steroid (33). The identity of the
RoDH isozyme(s) that catalyze this reaction in the rat ventral prostate
remains to be determined, however, using reduced stringency
hybridization conditions and coding region probes, RoDH transcripts
were detected in this tissue and 13-cis-retinoic acid
inhibited oxidative 3
-HSD enzyme activity in prostate extracts (data
not shown).
Finally, inhibition of the major human prostatic steroid 5-reductase
isozyme decreases the concentration of dihydrotestosterone within
the tissue to 90% of normal (34), but no further, suggesting that
another biosynthetic source of this hormone exists. The observation that the mRNA for a RoDH isozyme is present in the human prostate (Fig. 7B) provides one possible explanation for this
alternate biosynthetic source and confirms an earlier report of RoDH
activity in prostatic microsomes (35). It will be of interest in the future to correlate the expression of this RoDH in diseased and normal
prostate in an effort to understand the role of the enzyme in
androgen-regulated growth of the gland and in prostatic retinoid metabolism.
We thank Helen Hobbs and Jean Wilson for advice and critical reading of the manuscript, Joe Goldstein for comments, Charles Landrum for filters containing prostate RNAs, Stefan Andersson for discussions, Eric Lund for advice concerning steroids, and Lisa Beatty and Constance Martinelli for instruction in tissue culture.