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Section on Molecular Endocrinology, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Dr. M. L. Dufau, National Institutes of Health, Building 49, Room 6A-36, 49 Convent Road, Bethesda, Maryland 20892-4510. E-mail dufau{at}helix.nih.gov
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Introduction |
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5-androstene-3ß,17ß-diol. Seven distinct 17ßHSD
isoenzymes have been cloned and characterized from various tissues and
are designated types I, II, III, IV (revieved in Refs. 2, 3, 4); V (5); VI
(6); and VII (7). Types I-IV, VI, and VII are members of the short
chain alcohol dehydrogenase super family (2, 3, 4, 6, 7). However, type V
belongs to the aldo-keto reductase family (5). The 17ßHSD isoenzymes
differ in their tissue/cellular localization, substrate specificities,
cofactor requirements, and preference for oxidation or reduction
reactions. Three of the isoforms, types I, III, and VII, catalyze the
reductive reaction, whereas the other four preferentially catalyze the
oxidation reaction. The type III isoform was isolated from the human and mouse testes (8, 9). The human type III enzyme appears to be testis specific (8), whereas the mouse type III 17ßHSD, in addition to its strong expression in the testis, is weakly expressed in the ovary, uterus, seminal vesicle, and prostate, as determined by PCR amplification (9). The human type III enzyme is predominantly associated with the production of testosterone by testicular Leydig cells. Mutations of the human testicular type III 17ßHSD that involve either spliced junction abnormalities or single base substitutions result in nonexpression or severely compromised enzyme activity, respectively, and cause male pseudohermaphroditism (8, 10, 11). In the rat Leydig cell, 17ßHSD activity is under ATP control, and intracellular glucose plays an important role in the regulation of the enzyme. The contribution of the glycolytic pathway to meet optimal provision of functional ATP for 17ßHSD activity has been recently demonstrated (12). However, hormonal regulation of 17ßHSD type III activity and its gene expression in the gonads have not been investigated. Gonadotropins exhibit dual control of testicular Leydig cell function (reviewed in Ref. 13). Low doses of LH/hCG maintain LH/hCG receptors and steroidogenic enzymes in the up-regulated state, whereas high doses cause receptor down-regulation and desensitization of steroidogenic enzymes. Previous studies have demonstrated a blockade at the site of conversion of progesterone to androgens and a consequent reduction of testosterone production that is partly attributable to a decrease of CYP17 (17-hydroxylase/lyase) expression (14, 15). More recently, we have demonstrated rapid gonadotropin-induced negative transcriptional control of types I and II 3ß-hydroxysteroid dehydrogenase genes in rat Leydig cells (16). However, the participation of 17ßHSD in this desensitizing process remains to be elucidated.
To further understand the functional requirements of the enzyme, the mechanism of its activation, and molecular regulation, we have cloned the 17ßHSD type III enzyme from rat Leydig cells in this study. The rat type III enzyme encodes a single 1.4-kb messenger RNA (mRNA) species with high similarity to the human and mouse type III isoform. A glucose requirement for 17ßHSD optimal activity was observed in cells transiently transfected with 17ßHSD complementary DNA (cDNA). Moreover, 17ßHSD was down-regulated in vivo by hCG in a dose- and a time-dependent manner. These studies provide a basis for further investigation of the metabolic and molecular regulation of this enzyme.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal treatment and Leydig cell preparation
Adult male rats (200–250 g; Charles River Laboratories, Inc., Wilmington, MA) were housed in pathogen-free, temperature-
and light-controlled conditions (20 C; alternating 14-h light, 10-h
dark cycles). All animal studies were approved by the NICHHD animal and
care and use committee (protocols 97–039 and 97–041). The male adult
rats were given single sc injections of various doses of Pregnyl
(Organon, West Orange, NJ): 0.1, 1, and 25 IU, equivalent
to 0.01, 0.1, and 2.5 µg purified hCG, in 100 µl of Dulbecco’s
PBS. hCG international units (IU) are equivalent to hCG USP (United
States Pharmacopea unit). Control animals were injected with vehicle
alone. Animals were killed by asphyxiation with CO2 and
decapitated 24 h after hCG treatment unless otherwise indicated.
Leydig cells were prepared by collagenase dispersion and purified by
centrifugal elutriation, as previously described (17). These cells were
immediately frozen on dry ice and maintained at -70 C until extraction
of RNA. In vivo ovarian luteinization and down-regulation of
LH receptor (LHR) and LHR mRNA was induced by sequential PMSG/hCG
treatment of 21-day-old female rats (Sprague Dawley, Charles River Laboratories, Inc., Wilmington, MA) following established
protocols (22). The animals received a sc injection of 50 IU PMSG, a
preparation rich in FSH activity that produces follicular maturation,
followed 65 h later by injection of 25 IU hCG
(superovulation/luteinization). Six days later, the animals
received a second injection of 25 IU hCG (LHR down-regulation,
steroidogenic desensitization). Animals were killed, and their ovaries
were removed, rapidly frozen, and preserved at -70 C until RNA
extraction.
Ribonuclease (RNase) protection analysis
The RNase protection assay was performed by established
methodology (23). The 17ßHSD complementary RNA (cRNA) probes were
generated by PCR followed by subcloning. Primers F2 (5-ATGTTGTACTTA
TCAGCCGGACAC-3) and R5
(5-AAATGTGCACACAAAAGCCTTGGAAGCTGAGTA-3) were used to
amplify the fragment complementary to the coding region from 230–633
nucleotides relative to the translational start site of 17ßHSD.
Primers of 5-GCTACAGCTTCACCAC CACA-3 and 5-GGTCTTTACGGATGTCAACG-3,
located at exons 4 and 5, respectively, of the rat ß-actin gene (24),
were employed to amplify the ß-actin fragment used as an internal
control for the RNase protection assay. The fragments were cloned into
pGEM vector (Promega Corp., Madison, WI) and verified by
sequencing. The constructs were linearized by the digestion with
SpeI. The cRNA probes were produced by in vitro
transcription with RNA polymerase T7 (Life Technologies)
following the manufacturer’s protocol and labeled by
[32P]UTP (800 Ci/mmol; ICN Biomedicals, Inc., Costa Mesa, CA). Ten micrograms of total RNA samples were
applied for hybridization. Ribonuclease T1 (Life Technologies) and ribonuclease A (Amersham,
Arlington Heights, IL) were used for the digestion of unhybridized RNA
and cRNA probe. The protected fragments were resolved on a 6%
sequencing gel, dried, and autoradiographed for visual evaluation only.
Bands were quantified by phosphorimage analysis using PhosphorImager
Scanner model Storm 860 from Molecular Dynamics, Inc.
(Sunnyvale, CA).
Northern blot and mRNA half-life analysis
mRNA was extracted from rat Leydig cells and from various male
and female rat tissues using the Invitrogen isolation method (Carlsbad,
CA). The mRNA samples (10 µg for each lane) were resolved on 1%
agarose gels and transferred onto a Gene Screen membrane (Biotechnology
System NEN Research Products, Boston, MA). The fixed membrane was
prehybridized with 0.5 M phosphate buffer (pH 7.0)
containing 0.1 mM EDTA, 0.5% BSA, and 7% SDS at 50 C for
3 h and hybridized with a 32P-labeled PCR-generated
403-bp fragment (230–633 bp) at 50 C overnight. Thereafter, the
membrane was washed twice in 2 x SSC (standard saline
citrate)-0.1% SDS at 50 C for 15 min each and twice in 0.5 x
SSC-0.1% SDS for 10 min each. In experiments investigating the
half-life of the 17ßHSD mRNA, cells were incubated with 10 µg/ml
actinomycin D for 0, 1, 3, 6, and 10 h in medium 199/0.1% BSA.
Ten micrograms of mRNA samples were resolved as described above.
Hybridization was recorded by radioautography and quantified by
phosphorimage analysis. The half-life of mRNA was calculated by linear
regression analysis.
The expression of 17ßHSD type III in the ovary was followed by RT-PCR analysis using total RNA of ovaries at different times after treatment of animals with two doses of hCG (2.5 µg). RNA (1 µg) was reverse transcribed using random hexamers and Superscript II RNase H reverse transcriptase. Paired primers corresponding to nucleotide positions 61–81 and 311–331 of 17ßHSD cDNA were used to amplify the 270-bp fragment. Also, primers corresponding to exons 4 and 5 of ß-actin (24) were used to amplify a 290-bp fragment for use as an internal control. PCR products were analyzed in 3% agarose gels.
Nuclear run-off assay
Nuclei were isolated from batches of 50 x 106
rat Leydig cells from rats injected with or without 2.5 µg hCG were
prepared as previously described and suspended in 50 mM
Tris-HCl (pH 8.3), 40% glycerol, and 5 mM
MgCl2 (16). Nuclear RNA was labeled by in vitro
transcription as previously described (25). Two hundred microliters of
Leydig cell nuclear extracts (30 x 106 nuclei) were
incubated with 20 µl [
-32P]UTP (3000 Ci/mmol; 10
mCi/ml; from DuPont NEN) and 200 µl buffer containing 10
mM Tris-HCl (pH 8); 5 mM MgCl2; 0.3
M KCl; and 1 mM each of unlabeled ATP, CTP, and
GTP for 30 min at 30 C. After deoxyribonuclease I and proteinase K
treatments, the labeled transcripts were extracted by
phenol-chloroform-isoamyl alcohol (25:24:1) and subsequently
subjected to chromatography on G-25 columns to remove free nucleotides.
The linearized complementary DNA fragments of 17ßHSD type III,
ß-actin, and pGEM vector were immobilized to the nitrocellulose
membranes using a slot blot apparatus. The labeled RNA (3 x
106 cpm) were then hybridized to the membranes in 2 ml
hybridization buffer (10 mM
N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic
acid, 10 mM EDTA, 0.2% SDS, and 0.3 M
NaCl) for 20 h at 60 C. The membranes were washed with 2 x
SSC and exposed to Kodak x-ray film (Eastman Kodak Co.,
Rochester, NY) at -70 C with intensifier screens. The densities of the
bands were quantified by phosphorimage analysis.
Transient expression of rat 17ßHSD type III in mammalian
cells
Expression studies were performed in COS-1 and 293 cells
(American Type Tissue Collection, Manassas, VA). The cells were plated
into six-well plates in DMEM supplemented with 10% FBS, penicillin
(100 IU/ml), and streptomycin (100 IU/ml) and were transfected at
approximately 70–80% confluence using DOTAP
(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl
ammonium methyl sulfate) following the manufacturer’s procedure
(Boehringer Mannheim, Indianapolis, IN). Briefly, empty
vector (pBK) or pBK-17HSD (2 µg/well each) were incubated for 15 min
with 15 µg/well DOTAP in 140 µl HBS (20 mM
HEPES containing 150 mM NaCl, pH 7.4). After
incubation, the reaction mixture was added to wells containing 2 ml
OPTI-MEM I (Life Technologies, Inc.). Transfection
was carried out for 5 h; subsequently, OPTI-MEM I was replaced by
fresh DMEM, and cells were further cultured for 48 h.
Measurement of 17ßHSD activity
Both reductive and oxidative activities of 17ßHSD were
measured essentially as previously described (4, 12) in Leydig cells
and in transiently transfected COS-1 and 293 cells with 17ßHSD cDNA
(see above). Enzyme activity assays were performed by addition to cell
suspensions (Leydig cells) and cell cultures (COS-1 and 293 cells) of 2
µM 14C-labeled substrate in the presence of 5
µM of the respective unlabeled substrates:
androstenedione (53.86 mCi/mmol), testosterone (mCi/mmol),
dehydroepiandrosterone (54 mCi/mmol), estrone (57 mCi/mmol), and
estradiol (54 mCi/mmol), purchased from DuPont NEN
(Boston, MA). Forty-eight hours after transfection, cell cultures were
washed three times with serum and glucose-free DMEM-0.1% BSA. Enzyme
activity was determined in 1 ml DMEM-0.1% BSA in the presence or
absence of 5.8 mM glucose. The Leydig cells were first
incubated with 100 µg/ml aminoglutethimide (Sigma Chemical Co., St. Louis, MO) for 15 min to block steroid metabolism early
(cholesterol side-chain cleavage) and distally (aromatase) in the
pathway (26). This was followed by incubation with
14C-labeled androstenedione (2 µM) and
unlabeled substrate (5 µM) in HEPES buffer (5
mM; pH 7.4) containing 4 mM KCl, 140
mM NaCl, 1 mM MgCl2, 2.5
mM CaCl2, 5.8 mM glucose, and 0.1%
BSA with the addition of substrate(s) (see above). After incubation for
the designated times, the media were collected, and steroids were
extracted with ethyl acetate and separated on silica-coated TLC plates
with chloroform and ethyl acetate (3:1, vol/vol) as previously
described (12). Plates were exposed to x-ray films for visual record
only. The radioactivities corresponding to unconverted steroid
substrates and products in the TLC plate were quantified by
phosphorimage analysis.
The LHR binding studies included in Fig. 5
are taken from our recent
report (16) and are presented for comparison with the changes observed
in 17ßHSD gene expression. The materials obtained for analysis were
derived from the same experiments. LH receptor binding presented in
Fig. 4 was performed as previously described (16).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; GenBank accession no. AF035156).
The sequence identity between the rat type III 17ßHSD and
previously cloned human and mouse type III 17ßHSD is shown in Fig. 1B. Pairwise comparison of rat type III to human and mouse displays an
overall 75% and 85% amino acid similarity, respectively (Fig. 1B).
The encoded protein contains a region of homology to the short chain
alcohol dehydrogenase super family (amino acids 174–225) with the
conserved YXXXK structure (amino acids 194) found in all members of
short chain alcohol dehydrogenase/reductase family. The N-terminal of
17ßHSD contains a putative secretory signal sequence of 20 amino
acids. Two putative transmembrane helixes are predicted at amino acid
position 2–21 close to the N-terminus, and at amino acid
position 255–277 close to the C-terminus. The primary structure
also shows the presence of four potential N-linked
glycosylation sites (amino acids 153, 178, 236, and 302), four
potential PKC sites (amino acids 170, 237, 251, and 287), three casein
kinase II sites (amino acids 104, 142, and 243), and at least three
potential sites for phosphorylation by cAMP-dependent protein kinase
and cGMP-dependent protein kinase (27) at amino acids 61, 256, and 296.
A consensus NADPH binding motif (GXXXGXG) is located at amino acids
51–57 (PC Gene PROSITE program).
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). The molecular size of the mRNA is
slightly higher than the 1.1-kb 17ßHSD cDNA isolated from the rat
Leydig cell library (Fig. 2A). This difference may be due to the
presence of additional sequences at the 5'- and/or 3'-end in the mRNA.
Only using RT-PCR amplification, a minor band of 17ßHSD mRNA could be
detected in rat ovaries at 9 and 24 h after PMSG administration
and after hCG treatment in rats with superovulated/luteinized ovaries
(Fig. 2B).
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). The time-course study revealed
significant 17ßHSD activity within 15 min of substrate addition in
the presence of glucose in the medium compared with that in cells
incubated in the absence of glucose or cells transfected with vector
only. A further increase in activity was observed in the presence of
glucose at the 60 min point and reached a plateau at 120 min. In the
absence of glucose, the enzyme activity was undetectable at 15 and 30
min and increased at 60 and 240 min to 29% and 65%, respectively, of
the activity observed in the presence of glucose. Overall, the 17ßHSD
activity in COS-1 cells was significantly increased by the addition of
glucose in the medium throughout the time examined (P
< 0.001; Fig. 3).
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). The rat type III isozyme
catalyzed the reductive reaction almost exclusively with substrate
preference for androstenedione over dihydroepiandrosterone in both cell
types employed. Estrogen failed to serve as a substrate for the cloned
17ßHSD enzyme.
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). A small, but significant, inhibition
of 17ßHSD mRNA levels was observed even at low doses of hCG (by 30%;
P < 0.007 at 0.01 µg hCG; P < 0.003
at 0.1 µg hCG), and marked down-regulation was induced by the high
dose of hCG (2.5 µg; by 
80%; P < 0.001).
Temporal analysis of 17ßHSD gene expression using the high dose hCG
(2.5 µg) showed no significant changes in 17ßHSD mRNA levels at 1
and 4 h (P > 0.05), whereas these were
significantly inhibited at 12 and 24 h (P <
0.001) and recovered to control levels on day 9 post-hCG treatment
(Fig. 5B). It is important to note that
the significant reduction of 17ßHSD gene expression (Fig. 5B) at
12 h preceded the major down-regulation of LH/hCG receptors
observed at 24 h (Fig. 5A). This result suggests that regulation
of 17ßHSD gene transcription is not related to the loss of
hormone-binding sites. Furthermore, the 17ßHSD enzyme activity was
inhibited at 12 h by 30% (P < 0.001; Fig. 6), and a further reduction was observed
at 24 h (to 25% of controls; P < 0.001).
Although major reductions in 17ßHSD mRNA levels were observed at 12
and 24 h, the enzyme activity was only moderately reduced at
12 h, whereas a marked decrease was observed at 24 h (Fig. 5).
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). To determine whether the
gonadotropin-induced decline in steady state levels of 17ßHSD mRNA
was due to increased mRNA degradation, mRNA stability was evaluated
after the addition of actinomycin D to control and hCG-treated groups.
Significant reduction of 17ßHSD gene expression by hCG was observed
at all times after actinomycin D treatment (P <
0.001). The rates of degradation were similar for the two groups, with
half-lives of 3.8 and 3.5 h for the control and hCG-treated
groups, respectively (Fig. 7A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The highest expression of rat 17ßHSD type III mRNA was observed in
the Leydig cells, in contrast to the ovary, where it could only be
detected by RT-PCR amplification. This is consistent with a previous
report that the human type III 17ßHSD was only detected in the testis
(8). However, the mouse type III form detected by RT-PCR was reported
to be ubiquitously expressed in gonadal and nongonadal tissues, with
highest expression in the testis (9). The low level of mRNA expression
observed in murine nongonadal tissues indicates their potential
capacity for conversion of 17-ketosteroids to 17-hydroxysteroids.
The apparent difference in tissue distribution patterns between species
is in part attributable to the level of sensitivity of the analysis
employed (Northern blot vs. RT-PCR amplification). In the
gonads (ovary vs. testis), the catalytic activity of
17ßHSD is dependent on the presence of the specific isoform and the
level of its expression. Previous expression studies (8, 30, 31) have
shown that the various isoforms will catalyze the conversion of both
C19 and C18 steroids with different affinities.
The type I isoform displays high affinity for the reduction of
C18 steroids, whereas the type III enzyme reduces
C19 steroids with high affinity. In this study, the rat
type III isozyme was mainly responsible for the synthesis of
testosterone. This enzyme prefers androstenedione (C19) as
a substrate, and its failure to convert C18 steroids
(e.g. estrone to estradiol or vice versa)
demonstrated that the cloned rat type III 17ßHSD possesses catalytic
activity only for androgen formation (Table 1). The findings of the
present study, which have demonstrated that the main function of the
rat type III enzyme is the testicular production of testosterone, and
those of previous reports (8, 30, 31), indicating that the type I
isoform is expressed mainly in the ovary and catalyzes the conversion
of estrone to estradiol, are consistent with the low expression of the
type III enzyme found in the ovary.
When transiently expressed in COS-1 cells, type III 17ßHSD displayed catalytic activity that was markedly stimulated by the presence of glucose in the medium. This finding indicated that the cloned enzyme had similar requirements to the endogenous Leydig cell enzyme, where the integrity of the glucose transport system and the viability of the glycolytic pathway for ATP generation are necessary for optimal 17ßHSD activity at the final step of androgen pathway (12). However, no such requirement was observed for other enzymes of the steroidogenic pathway (12). Thus, further investigation with the cloned enzyme should reveal the extent to which posttranslational events, such as phosphorylation or ATP binding, are of relevance to the control of 17ßHSD activity in the Leydig cell.
Hormonal regulation of 17ßHSD gene expression in rat Leydig cells
became evident during our investigation of hCG action on 17ßHSD mRNA
expression and activity as components of steroidogenic desensitization.
These studies demonstrated a gonadotropin-induced steroidogenic block
at the level of conversion of androstenedione to testosterone by
17ßHSD in rat Leydig cells, in addition to the impairment of other
steroidogenic enzymes, including 3ßHSD (16) and
17-hydroxylase/lyase (14, 15). In intact animals, treatment with hCG
inhibited 17ßHSD type III gene expression in a dose-dependent manner.
This reduction was observed even with low doses of the hormone 24
h after treatment (0.01 and 0.1 µg/hCG; by 40%). This is in contrast
with the lack of effect of low doses of hormone on 3ß-hydroxysteroid
dehydrogenase type II gene expression (16). The high dose of hCG (2.5
µg) reduced 17ßHSD mRNA levels significantly after 12 h, and a
further decrease was observed at 24 h followed by recovery over
several days. The reduction at 12 h preceded the major decrease in
LH receptors induced by the hormone, which occurred at 24 h,
indicating that the former change was not related to receptor
down-regulation. Moreover, with the lower doses of the hormone (0.01
and 0.1 µg/hCG) the changes in 17ßHSD mRNA levels were not
accompanied by changes on the LHR, further confirming the lack of
dependence between these parameters. The hormone-induced reduction of
17ßHSD gene expression resulted in decreased 17ßHSD activity and
probably contributed to the in vitro decrease in
testosterone responses to hCG in rat Leydig cells (13). Although major
reductions in 17ßHSD mRNA levels (by 60%) were observed at 12
h, the activity of the enzyme was only modestly reduced (by 30%).
However, at 24 h the major decrease in mRNA (by 80%) was
consistent with the changes in enzyme activity. Overall, the changes
observed in 17ßHSD mRNA and enzyme activity were noted at later times
than those in 3ßHSD (12 vs. 4 h).
These studies have shown that hCG treatment decreased the incorporation
of [-32P]UTP into 17ßHSD transcripts in total
nuclear RNA and had no effect on the apparent half-life of the enzyme,
indicating that the loss of 17ßHSD type III gene expression results
from an inhibitory action of hCG at the transcriptional level. It is
possible that the consequences of desensitization by hCG include
changes in transcriptional regulation of the steroidogenic enzymes
through a common control mechanism. The promoter domain and 5'-flanking
region of 17ßHSD type III gene and the mechanisms of its regulation
have not been determined. However, the control of this enzyme could be
exerted directly or indirectly by the actions of steroid products or
second messengers elicited by the hormonal stimulus.
In conclusion, the present study describes the cloning and characterization of the rat type III 17ßHSD. Furthermore, we have demonstrated that the Leydig cell enzyme is down-regulated at the transcriptional level by gonadotropin. The cloning of the rat type III 17ßHSD will permit further elucidation of the structural requirements that influence the enzymatic activity of the gene product.
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Footnotes |
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Received January 22, 1999.
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-hydroxysteroid
dehydrogenases from rat and human prostate. J Biol Chem 272:15959–15966This article has been cited by other articles:
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, NADPH binding motif. TM1 and -2,
Putative transmembrane region. *, Potential
N-glycosylation site. •, Potential protein kinase C.
, Casein kinase II. 
, Sites for cAMP- and cGMP-dependent protein
kinases.
