Expression Cloning of a Novel Estrogenic Mouse 17ß-Hydroxysteroid Dehydrogenase/ 17-Ketosteroid Reductase (m17HSD7), Previously Described as a Prolactin Receptor-Associated Protein (PRAP) in Rat
Pasi Nokelainen,
Hellevi Peltoketo,
Reijo Vihko and
Pirkko Vihko
Biocenter Oulu and World Health Organization Collaborating Centre
for Research on Reproductive Health University of Oulu
FIN-90220 Oulu, Finland
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ABSTRACT
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17ß-Hydroxysteroid dehydrogenases/17-ketosteroid
reductases (17HSDs) modulate the biological activity of certain
estrogens and androgens by catalyzing reductase or dehydrogenase
reactions between 17-keto- and 17ß-hydroxysteroids. In the present
study, we demonstrate expression cloning of a novel type of 17HSD,
chronologically named 17HSD type 7, from the HC11 cell line derived
from mouse mammary gland. The cloned cDNA, 1.7 kb in size, encodes a
protein of 334 amino acids with a calculated molecular mass of 37,317
Da. The primary structure contains segments characteristic of enzymes
belonging to the short-chain dehydrogenase/reductase superfamily.
Strikingly, mouse 17HSD type 7 (m17HSD7) shows 89% identity with a
recently cloned rat protein called PRL receptor-associated protein
(PRAP). The function of PRAP has not yet been demonstrated.
The enzymatic characteristics of m17HSD7 and RT-PCR-cloned rat PRAP
(rPRAP) were analyzed in cultured HEK-293 cells, where both of the
enzymes efficiently catalyzed conversion of estrone
(E1) to estradiol (E2).
With other substrates tested no detectable 17HSD or
20
-hydroxysteroid dehydrogenase activities were found. Kinetic
parameters for m17HSD7 further indicate that E1
is a preferred substrate for this enzyme. Relative catalytic
efficiencies (Vmax/Km
values) for E1 and E2
are 244 and 48, respectively. As it is the case with rPRAP, m17HSD7 is
most abundantly expressed in the ovaries of pregnant animals. Further
studies show that the rat enzyme is primarily expressed in the middle
and second half of pregnancy, in parallel with
E2 secretion from the corpus luteum. The mRNA
for m17HSD7 is also apparent in the placenta, and a slight signal for
m17HSD7 is found in the ovaries of adult nonpregnant mice, in the
mammary gland, liver, kidney, and testis.
Altogether, because of their similar primary structures, enzymatic
characteristics, and the tissue distribution of m17HSD7 and rPRAP, we
suggest that rPRAP is rat 17HSD type 7. Furthermore, the results
indicate that 17HSD7 is an enzyme of E2
biosynthesis, which is predominantly expressed in the corpus luteum of
the pregnant animal.
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INTRODUCTION
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17ß-Hydroxysteroid dehydrogenases (17HSDs) catalyze the
conversion of low-activity 17-ketosteroids [e.g. estrone
(E1), androstenedione (A-dione), and 5
-androstanedione]
to their more potent 17ß-hydroxysteroid forms [estradiol
(E2), testosterone (T), and dihydrotestosterone] and vice
versa. Reductive 17HSDs are essential for the biosynthesis of
E2 and testosterone in the gonads and, in addition, they
modulate the activity of these steroids in certain extragonadal tissues
of several species, especially primates. Oxidative 17HSDs are widely
expressed and are suggested to play a role in the inactivation of
17ß-hydroxysteroids shifted to the tissues from the gastrointestinal
tract, blood circulation, and amniotic fluid (Refs. 1, 2, 3, 4 and references
therein). Four different human 17HSDs (types 14) (5, 6, 7, 8, 9) and six
rodent 17HSDs (types 16) have been cloned to date (10, 11, 12, 13, 14, 15, 16, 17, 18). In
addition to their distinct primary structures, they are dissimilarly
distributed, they differ in substrate specificity, and consequently
they have apparently separate physiological functions. 17HSD types 14
and 6 belong to the short-chain dehydrogenase/reductase (SDR) protein
family, whereas the type 5 enzyme belongs to the aldoketoreductase
family.
Up to now, estrogens have mostly been found to be modulated by
17HSD type 1 (17HSD1) and type 2 (17HSD2). 17HSD1 is predominantly
expressed in ovarian granulosa cells (10, 19, 20, 21, 22) and in the
syncytiotrophoblasts of human placenta (23, 24), being essential for
E2 biosynthesis. The human type 1 enzyme is also expressed
in certain estrogen target tissues such as breast (25) and endometrium
(26), where it converts circulating E1 to E2.
While human 17HSD1 primarily catalyzes reactions between phenolic
steroids, i.e. estrogens (27), rodent 17HSD1 enzymes are
also able to catalyze androgens efficiently (10, 28). In contrast to
type 1, the oxidative 17HSD2 decreases the biological activity of
estrogens and androgens and may thus protect tissues from excessive
hormone action (3, 4, 7, 29). The type 2 enzyme is particularly
expressed in human and rodent placenta, liver, kidney, and small
intestine (7, 11, 16, 29). It is the predominant 17HSD in human
endometrium (30), and it is also expressed in human prostate, at least
in neoplastic tissue (31).
The function of other 17HSDs in estrogen biosynthesis and
metabolism is less well understood. 17HSD type 3 (17HSD3) is
indispensable for testicular T biosynthesis and is thus crucial in male
sexual differentiation and reproduction, although it also reduces
E1 to E2 (8). 17HSD type 4 (17HSD4) is part of
peroxisomal multifunctional enzyme II, whose role in steroid metabolism
appears to be minor compared with the other activities of the enzyme
(9, 17, 32, 33). 17HSD type 5 (17HSD5), in turn, which shows oxidative
17HSD activity toward androgens, E2, and xenobiotics, is
mainly expressed in the liver and kidney, and the physiological role of
the enzyme has remained open (14). Finally, the recently cloned 17HSD
type 6 (17HSD6) takes part in an inactivation path of
dihydrotestosterone and is most abundantly expressed in the prostate
and liver (18).
In the present study we demonstrate cloning of a novel 17HSD
which we suggest to be involved in E2 biosynthesis. The
main source of E2 in cycling humans and rodents is the
ovary. E2 is synthesized in the granulosa cells of
developing follicles from theca cell-derived androgens, by P450
aromatase (P450arom), and 17HSD1 (15, 19, 20, 21, 34). After ovulation, the
follicles luteinize and turn to corpora lutea, which secrete
progesterone (P), E2, and peptide hormones, for example.
When impregnation occurs, the rat corpus luteum (CL) further develops
as a result of PRL stimulation being essential for maintaining the
pregnancy (35). During human pregnancy the placenta develops as a major
source of E2, whereas in rodents E2 is produced
in the ovaries from ovarian and placental precursors. Throughout
gestation, some rodent follicles mature and express 17HSD1 (36), but
from midpregnancy until parturition E2 is secreted mainly
from the CL (37). 17HSD1, however, has not been found in the CL of
either cycling or pregnant rats (15, 36), in contrast to human
granulosa-luteal cells (19, 20, 22), suggesting that, at least in
rodents, another enzyme is responsible for E2 biosynthesis
in the CL. We have now cloned a novel type of 17HSD from a cell line
originating from mouse mammary gland epithelial cells, but which is
most abundantly expressed in the ovary of the pregnant mouse, and which
efficiently catalyzes the reaction from E1 to
E2. The enzyme, which has chronologically been named 17HSD
type 7 (17HSD7), shows great identity to PRAP, recently cloned from rat
ovary (38).
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RESULTS
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Expression Cloning of m17HSD7
When screening different cell lines for 17HSD activity, we found
that the HC11 cell line originating from the epithelial cells of the
mammary gland of a pregnant mouse (39) possessed strong estrogenic
17HSD activity. E1 was effectively converted to
E2 in the cultured cells but, in contrast to the activity
of m17HSD1, the observed activity poorly catalyzed A-dione to T (Fig. 1
). HC11 cells were not able to catalyze
oxidative 17HSD reactions either. Probes for mouse 17HSD1 (m17HSD1) as
well as those for mouse 17HSD type 2 and 5, human 17HSD3, and rat
17HSD4 did not recognize any mRNAs in HC11 cell samples (data not
shown). The cell line was therefore assumed to express an unidentified
type of 17HSD enzyme. An expression cDNA library was prepared from
poly(A)+-enriched RNA of the HC11 cell line, and a total of
600,000 independent clones from the library were screened by monitoring
their capability to convert E1 to E2.

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Figure 1. Characterization of 17HSD Activity in Cultured HC11
Cells
The cells were seeded into six-well plates at a density of 230,000
cells per well and allowed to grow for 2 days. 17HSD activity
measurements were then carried out in the cultured cells using
estrogens (E1, E2) and androgens (A-dione, T)
as substrates (100 nM) in the culture media. After
incubation, the media were collected and the amount of product was
measured.
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Of the 243 plasmid pools, each containing DNA from 2500 individual
colonies, two gave a positive signal, i.e. possessed 17HSD
activity. These two plasmid pools were subdivided into smaller pools
until two independent cDNA samples (m17HSD7.1 and m17HSD7.2) were
obtained and sequenced. The two clones were found to be identical
except that the m17HSD7.1 clone had a 42 bp longer 5'-region than the
m17HSD7.2 clone and, in addition, the m17HSD7.2 clone had a G-to-T
substitution at position 1414, found to be in the noncoding region
(Fig. 2
). The m17HSD7.1 clone had an open
reading frame of 1005 bp, a 5'-region of 63 bp, and a 3'-region of 670
bp followed by a polyadenylate tract. The open reading frame encoded a
peptide of 334 amino acids with a predicted molecular mass of 37,317
Da, and the protein was named m17HSD7. A stop codon situated in the
frame upstream of the ATG triplet further indicated that the m17HSD7.1
clone contained the whole coding region. A putative polyadenylation
signal (AATTAAA) was located at position 1703 in the 3'-noncoding
region of the clone (Fig. 2
).

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Figure 2. Nucleotide Sequence and Deduced Amino Acid Sequence
of m17HSD7
Stop codons are marked with a box, and potential
polyadenylation signal is underlined. The
nucleotide sequence data reported in this paper will appear in the
EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under
accession number Y15733.
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Comparison of the predicted amino acid sequence of m17HSD7 with
the sequences in the protein databases revealed a significant 89%
identity between m17HSD7 and a protein called PRAP recently cloned from
the rat (38) (Fig. 3
). Analysis of the
amino acid sequence of m17HSD7 against the PROSITE database, allowing
one mismatch, further showed that the protein belonged to the SDR
superfamily. It contained the typical glycine pattern
(Gly9-Xaa-Xaa-Xaa-Gly13-Xaa-Gly15
in m17HSD7) and the three amino acid residues (Ser180,
Tyr193 and Lys197) (Fig. 3
) that are involved
in the catalytic reaction and are characteristic of SDR enzymes (40, 41). Identity of the predicted amino acid sequence to other mouse 17HSD
enzymes was 1828%. In addition, several putative posttranslational
modification sites were found in m17HSD7: four N-glycosylation sites at
positions 37, 127, 178, and 229, three casein kinase II phosphorylation
sites at positions 118, 125, and 180, and two protein kinase C
phosphorylation sites at positions 170 and 195 (Fig. 3
).

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Figure 3. Comparison of the Amino Acid Sequences of m17HSD7
(m7) and r17HSD7/PRAP (r7)
The glycine pattern and the conserved amino acids recognized as the SDR
consensus sequence in the PROSITE database, in both enzymes, are marked
with white and gray boxes, respectively.
Putative sites for N-linked glycosylation (#), casein kinase II
phosphorylation (- - -), and protein kinase C phosphorylation
(+++) are marked above the sequences. These consensus
sequences are present in both proteins, excluding the N-linked
glycosylation site at position 127, which is present only in m17HSD7. A
hydrophobic region suggested to be a membrane-spanning domain in
r17HSD7/PRAP (38 ) (underlined) is also included in
m17HSD7, according to a hydropathy plot (data not shown).
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Characterization of the Enzymatic Properties of m17HSD7
To characterize the enzymatic activities of the cloned
m17HSD7, the cDNA was expressed under a cytomegalovirus (CMV)-promoter
in human embryonic kidney 293 (HEK-293) cells. As shown in Fig. 4A
, m17HSD7 catalyzed only a reductive
reaction from E1 to E2. No significant
17HSD or 20
-hydroxysteroid dehydrogenase (20HSD) activity was
observed when E2, A-dione, T, P, or
20
-hydroxyprogesterone (20-OHP) were added to the reaction at
concentrations of 100 nM (Fig. 4A
) or 1 nM
(data not shown). To compare the efficacies of m17HSD1 and m17HSD7, the
cDNAs encoding them were separately transfected into HEK-293 cells. The
comparison indicated that the 17HSD activity of the type 7 enzyme was
relevant, i.e. of same magnitude as that of 17HSD1, even
though it was not as great as that of the type 1 enzyme in the
conditions used (Fig. 4B
). As also described previously, m17HSD1
catalyzes reactions from E1 to E2 and from
A-dione to T (Fig. 4B
and Ref. 10), while m17HSD7 was able to catalyze
only the reduction of E1 (Fig. 4
).

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Figure 4. Characterization of 17HSD Activity of m17HSD7 in
Cultured HEK-293 Cells after Transient Transfection with m17HSD7 cDNA
(A) and Comparison with m17HSD1 (B)
Cells grown in six-well plates (300,000 cells per well) were
transfected with 0.5 µg of m17HSD7.1-cDNA3.1 plasmids per well (A),
0.5 µg of m17HSD7.1-cDNA3.1 plasmids (B) or m17HSD1-CMV6 plasmids per
well. 17HSD activity measurements were carried out in cultured cells
using estrogens (E1, E2), androgens (A-dione,
T), or progestins (20-OHP, P) as substrates (100 nM) in the
culture media. After incubation periods, the media were collected and
the amount of product was measured. The background 17HSD activity
observed was subtracted from the results. The experiments were repeated
twice and typical reaction curves and columns are shown.
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To further characterize the enzymatic properties of the novel
17HSD, HEK-293 cells were transiently transfected with m17HSD7 cDNA and
then homogenized in phosphate buffer containing 20% glycerol and
0.08% nonionic detergent (Big Chap), which were found to stabilize the
labile enzyme (data not shown). Kinetic parameters (Km and
Vmax) were determined for E1 and E2
(Table 1
). The Vmax values
for E1 and E2 did not differ considerably, but
the Km values, and consequently the catalytic efficiencies
(Vmax/Km), clearly showed that of these two
E1 is the preferred substrate for the enzyme. While 49% of
E1 (1 µM) was converted to E2 in
20 min, the 17HSD activity of the homogenate toward A-dione, T,
dehydroepiandrosterone (DHEA), and androst-5-ene-3ß,17ß-diol
(A-diol) (0.5 µM) was 0.51.5% in 2 h and,
therefore, kinetic constants could not be determined for these
steroids. In similar conditions, conversions between 20-OHP and P were
less than 2.5%. Finally, in the conditions used, the conversion of
E1 to E2 was approximately 40 times faster when
the phosphorylated form of cofactor, NADPH, was used as when NADH was
added to the reaction. Likewise, the rate of reaction from
E2 to E1 was 7-fold greater with
NADP+ than with NAD+.
Tissue Distribution of m17HSD7
In Northern blot analysis, the m17HSD7 cDNA probe recognized mRNAs
of 4.6, 3.6, 1.7, and 1.2 kb in size, of which the 4.6-kb transcript
was the dominant form (Fig. 5A
). The 4.6-kb mRNA
was very abundantly expressed in the ovaries of pregnant (1420 day)
mice (Fig. 5
, A and B). This mRNA was also expressed in the placenta
and was detectable in the mammary gland, liver, kidney, testis, the
HC11 cell line, and in the ovaries of nonpregnant mice. Since the major
mRNA recognized (4.6 kb) and the cDNA cloned (1.7 kb) differed so
remarkably in size, and also because 28S ribosomal RNA is approximately
4.7 kb in size, the authenticity of the weak signals detected was
confirmed by RT-PCR. A specific fragment of 0.5 kb was amplified from
all the tissues tested, including a barely detectable amplification
signal from the spleen and uterine samples, and excluding the
pancreatic sample, where the RNA was found to be degraded (Fig. 5C
).
The quality of the total RNA used for RT-PCR was tested by checking the
integrity of ribosomal bands in agarose-formaldehyde gel (data not
shown) and also by performing RT-PCR with mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (Fig. 5C
).

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Figure 5. Tissue Distribution of m17HSD7 Analyzed by Northern
Blot (A and B) and RT-PCR (C) Analyses
A, Six micrograms of poly(A)+-enriched RNA extracted from
adult female mouse tissues, from testis, and from the HC11 cell line
(10 µg) were subjected to Northern blotting. Ovaries were collected
from pregnant mice at gestation days 1420, mammary glands were from
gestation days 15 and 20, and placentae were from day 15. Note the
variable exposure times. B, Expression of the enzyme in the ovaries of
normal cycling (control) and pregnant mice (1420 days) was analyzed
in a separate experiment using 20 µg total RNA. The exposure time was
16 h. The sizes of the mRNA transcripts were determined using a commercial
RNA ladder, and the amount of RNA loaded was controlled by
hybridization of the filter with a rat GAPDH cDNA probe
(bottom; exposure time, 20 h). (C) RT-PCR was
carried out using 125 ng of the same total RNA preparation as used for
the isolation of poly(A)+-enriched RNAs for the Northern
blot analysis (panel A). Primers for m17HSD7 and for mouse GAPDH were
used. Eighty percent of each PCR product was run in a agarose gel,
blotted, and analyzed by hybridization with m17HSD7 or rat GAPDH
probes. Cont., Negative control samples containing sterile
H2O instead of RNA template.
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Characterization of the Enzymatic Nature of rPRAP Suggested to be
Rat 17HSD Type 7, and the Expression Pattern of the Enzyme in the Ovary
during Gestation
rPRAP was cloned by RT-PCR from RNA isolated from the ovaries of
pregnant rats, using the sequence described (38). The rPRAP cDNA was
then subcloned to CMV6-vectors and transfected to HEK-293 cells. In a
identical manner to the mouse type 7 enzyme, the rat enzyme catalyzed a
reductive reaction from E1 to E2 in the
cultured cells (Fig. 6
). Neither 17HSD
nor 20HSD activity was observed when E2, A-dione, T,
20-OHP, or P were used as substrates.

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Figure 6. Characterization of the Activity of Transiently
Transfected r17HSD7/PRAP in Cultured HEK-293 Cells
Cells grown in six-well plates (300,000 cells per well) were
transfected with 1 µg of r17HSD7/PRAP-CMV6 plasmid per well.
Estrogens (E1, E2), androgens (A-dione, T), or
progestins (20-OHP, P) were added to the culture media at a
concentration of 100 nM to determine 17HSD and 20HSD
activities. After incubation, the media were collected and the amount
of product was measured. Conversion of E1 to E2
was measured after 2, 4, 8, and 16 h, while other conversions were
measured only after 20 h. The background activities observed were
subtracted from the results. The experiments were repeated and typical
conversions are shown.
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m17HSD7 mRNA was most abundant in ovarian samples from pregnant mice
(Fig. 5
), and rPRAP has also been shown to be mostly expressed during
pregnancy (42). Expression of the enzyme was therefore followed in more
detail in rat ovaries during pregnancy. Northern blot analysis revealed
two distinct PRAP mRNAs, 1.8 and 4.3 kb in size, of which the
longer species is dominant (Fig. 7
),
similar to m17HSD7 mRNAs. After 40 h exposure the 4.3-kb mRNA
could be detected in samples taken throughout the first part of
pregnancy, on days 15, but the expression of PRAP was remarkably
up-regulated at day 8. Expression of the 4.3-kb mRNA continued at a
high level until close to parturition. On day 21 its expression was
again barely detectable, after a long (40 h) exposure.

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Figure 7. Expression of 17HSD7/PRAP in the Ovaries of
Pregnant Rats
Thirty micrograms of total RNA from the ovaries of pregnant rats on
days 1, 3, 5, 8, 12, 15, 18, and 21 were analyzed in Northern blot
analysis. The upper and middle panels
represent the same film after two different exposure times. The sizes
of the mRNA transcripts were determined using a commercial RNA ladder,
and the amount of RNA loaded was controlled by hybridization with rat
GAPDH cDNA (bottom).
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DISCUSSION
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In addition to being crucial for gonadal sex steroid synthesis,
17HSD activity has been found ubiquitously in human and rodent tissues
(43, 44). Within the last few years, the 17HSD family has rapidly
expanded to six different types, although not all of them have been
linked primarily to steroidogenesis. In the present work we describe
the expression cloning of a novel type of 17HSD, chronologically named
17HSD7. m17HSD7 is an enzyme of 334 amino acids and it belongs to the
SDR protein family, as do 17HSD types 14 and 6. The identity of
m17HSD7 with other mouse 17HSD enzymes is between 1828%, which is in
the range characteristic of SDR members (40). Like 17HSD1, the type 7
enzyme contains the three critical amino acid residues,
Ser180, Tyr193, and Lys197, of the
catalytic triad (28, 41, 45) that lie on the segment recognized as an
SDR signal in the PROSITE database. The primary structure also includes
the glycine pattern that forms part of the ß
ß
Rossmann fold involved in the binding of a nucleotide cofactor
(40).
While the identity between m17HSD7 and other 17HSDs is less than 30%,
the cloned enzyme shares 89% identity with a rat protein called PRAP
(38). rPRAP also contains the SDR consensus sequence, including the
three highly conserved amino acid residues, and the glycine pattern
(see Fig. 3
), which are needed for 17HSD activity in other 17HSD
enzymes (45). In the present work we further demonstrate the 17HSD
activity of rPRAP, hereafter called r17HSD7/PRAP, thus confirming the
identity between 17HSD7 and PRAP. r17HSD7/PRAP is a microsomal
phosphoprotein that has been shown to be associated with a short form
of PRL receptor (PRL-R) (38) and, like r17HSD7/PRAP, m17HSD7 contains
several putative N-linked glycosylation and phosphorylation sites.
Lability of m17HSD7, which can be decreased with a nonionic detergent,
and a putative transmembrane domain of the enzyme similar to one in
r17HSD7/PRAP (38) also point to the membrane-associated character of
the mouse type 7 enzyme.
Characterization of 17HSD activities of m17HSD7 and r17HSD7/PRAP in
cultured HEK-293 cells showed that in in vivo conditions
both enzymes exclusively catalyze the reduction of E1 to
E2. Thus the rodent type 7 enzymes resemble human 17HSD1,
but differ from rodent type 1 enzymes, which also catalyze androgens
efficiently (10, 28). In addition, the activity of mouse and rat 17HSD7
enzymes corresponds to the activity originally found in the HC11 cell
line. Comparison of the activities of m17HSD1 and m17HSD7 further
indicates that the type 1 and 7 enzymes catalyze E1 to
E2 to the same degree and thus a noteworthy role for the
type 7 enzyme in E2 biosynthesis can be presented. Cell
homogenate enriched with 17HSD7 is also able to catalyze, to some
extent, the reverse reaction from E2 to E1 in
the presence of excess NADP+. As in the case of human
17HSD1 (28), the direction of the reaction may be reversed by an excess
of cofactor in vitro. Km and
Vmax/Km values and particularly the results
obtained in vivo, however, show that the type 7 enzyme
evidently prefers E1 over E2 as a substrate.
Hence, 17HSD7 belongs to the group of reductive enzymes, as do types 1
and 3, while the other isoenzymes, 2, 4, 5, and 6, favor 17ß-hydroxy
substrates. As suggested to be typical of reductive enzymes (46),
m17HSD7 also utilizes the phosphorylated forms of cofactor, NADPH and
NADP+, more efficiently than the nonphosphorylated
forms.
Northern blot analysis of the mouse type 7 enzyme revealed a remarkable
difference between the size of the cDNA (1.7 kb) and the major mRNA
(4.6 kb). The 1.7-kb mRNA is barely detectable as compared with the
4.6-kb transcript. This is also the case with r17HSD7/PRAP cDNA (1.8 kb
in size), which recognizes two to three mRNAs of 1.8 kb, 4.3 kb, and
5.5 kb (present study and Ref. 47). In rat tissues, the mRNAs longer
than 4 kb are also the dominant forms. A possible explanation for the
difference in lengths of the cDNAs and major mRNAs is an exceptionally
long 5'-noncoding sequence, or a 3'-noncoding region including more
than one polyadenylation signal. Thorough studies, most likely
structural analyses of the HSD17B7 genes, are needed to solve the size
differences. Nevertheless, the results of functional studies and an
in-frame stop codon in the 5'-noncoding area confirm that the cDNA
contains the whole coding area, and correspondence of results of
Northern blotting and RT-PCRs indicates that the mRNA, 4.6 kb in size,
is the major m17HSD7 transcript.
In agreement with the results published by Duan and co-workers (47),
our results show that 17HSD7/PRAP is most abundantly expressed in the
ovaries of pregnant rodents, even though the 17HSD7/PRAP mRNA and
protein is also detectable in ovarian samples from nonpregnant animals
(present study and Ref. 42). 17HSD7/PRAP is expressed in the ovaries
throughout pregnancy but it is particularly strongly up-regulated
around day 8 of pregnancy, and this is sustained at least until day 18.
Data of Parmer and co-workers (42) and Duan and co-workers (47)
confirmed that the expression of 17HSD7/PRAP occurs in the CL. During
rodent pregnancy, biosynthesis of E2 especially takes place
in the CL, even though some follicles continue to mature and the
granulosa cells are therefore also secreting E2. In the
first half of rodent pregnancy, steroidogenesis in the CL is stimulated
by PRL and LH, and E2 is synthesized from ovarian androgen
precursors (Ref. 35 and references therein). LH action on the CL of the
pregnant rat is known to be mediated by LH stimulation of
E2 biosynthesis. E2, together with decidual
luteotropin (DLt), further maintains P production. This is in line with
the data showing that 17HSD7/PRAP is tightly regulated by
E2 and that removal of the effect of tropic hormones on the
CL leads to disappearance of r17HSD7/PRAP (47).
At midpregnancy, remarkable endocrinological changes take place, such
as down-regulation of LH and pituitary PRL, and a luteal-placental
shift as a result of which the placenta starts production of the
androgen precursors needed for increased E2 synthesis in
the ovaries. E2 then acts in an autocrine and intracrine
manner together with placental lactogen (PL), causing differentiation
of the CL, characterized by hypertrophy of luteal cells,
vascularization of the CL, and stimulation of steroidogenesis
[synthesis of progesterone in particular (35)]. High expression of
17HSD7/PRAP from day 8 onward and during the second half of pregnancy
coincides first with LH-induced E2 production from the CL
and then enhanced A-dione secretion from the placenta, the expression
of P450arom in the CL (48), and elevated serum E2
concentrations (37). Altogether, our results, in combination with those
reported by Duan and co-workers (38, 47), strongly suggest
that 17HSD7/PRAP, not 17HSD1, is the enzyme required for E2
biosynthesis in the CL.
We demonstrate here a steroidogenic function for 17HSD7/PRAP. Moreover,
the enzyme has been shown to be associated with the short form of
PRL-R, and a potential role for 17HSD7/PRAP in PRL signaling has been
much discussed (38, 47). Both long and short forms of PRL-R are
expressed in the CL throughout rodent pregnancy (49). While the longer
form of the receptor mediates the PRL signal via the JAK2/Stat5 system,
the function of the short form is known only superficially (50, 51).
PRL or PRL-like hormones, DLt and PL, and E2 act together
on the CL throughout rodent pregnancy. Furthermore, PRL and PRL-like
hormones maintain high concentrations of estrogen receptor in the CL
(Ref. 35 and references therein), and 17HSD7/PRAP is up-regulated not
only by its product E2, but also by PRL (47). It will thus
be of great interest to further investigate the cross-talk between E2-
and PRL-signaling systems in the CL and the possible role of
17HSD7/PRAP. One question of interest is the association between
17HSD7/PRAP and the cell membrane (38) in terms of its steroidogenic
activity and possible role in PRL signaling.
In addition to the ovaries, 17HSD7 was found to be expressed in mouse
placenta, and weak signals for m17HSD7 were also detected in the
mammary gland, liver, kidney, and testis of the mouse. Moreover,
m17HSD7-PCR-amplicons were detected in samples from the uterus, brain,
and small intestine. r17HSD7/PRAP mRNAs have been detected only in the
ovaries (47), which may be due to species-specific differences or, more
likely, differences in sensitivity of the methods used in recognition
of the mRNAs. The physiological significance, if any, of low expression
of 17HSD7 in the mouse tissues remains open. Transcripts of
m17HSD3, for example, have also been detected in several male and
female tissues, in addition to its major expression in the testis, when
a very sensitive method, RT-PCR, has been used (12). The presence of
17HSD7 in HC11 cells and low but detectable concentrations of 17HSD7
mRNA in samples from the mammary glands of pregnant mice suggest,
however, that 17HSD7 is expressed in the mouse mammary gland, at least
during pregnancy. 17HSD7 might therefore catalyze circulating
E1 to E2 locally in the mammary gland, in a
similar manner as 17HSD1 has been suggested to do in human breast
epithelial cells (1, 52). Interestingly, the mammary gland is also a
well known target of both E2 and PRL action.
The expression of 17HSD7 in mouse placenta introduces new issues in
placental steroidogenesis. In contrast to the human placenta, rodent
placentae have been assumed to be unable to synthesize E2
as a result of their lack of P450arom (53) and 17HSD1 (10, 11). The
mouse placenta is rich in 17HSD2 (4, 11), but Blomquist and co-workers
(54) have shown that it may also express multiple forms of 17HSDs.
17HSD7 might thus catalyze circulating E1 to E2
in the placenta for local needs, while the oxidative type 2 enzyme is
suggested to prevent the transfer of active 17ß-hydroxy forms of sex
steroids between the fetus and the maternal circulation (4).
In summary, a novel type of 17HSD has been cloned from the HC11 cell
line. 17HSD7, also known as PRAP, is primarily expressed in the ovaries
of pregnant rodents. Based on the enzymatic character of 17HSD7/PRAP
and its expression pattern, which shows developmental regulation in the
ovary during gestation, we suggest that 17HSD7/PRAP is responsible for
the final step in the biosynthesis of E2 in the CL.
 |
MATERIALS AND METHODS
|
---|
Chemicals and Reagents
[A-dione, A-diol, DHEA, E2, E1,
20-OHP, P, and T were purchased from Steraloids, Inc. (Wilton, NH).
[1,2,6,7-3H]A-dione (110 Ci/mmol),
[2,4,6,7-3H]E2 (81 Ci/mmol),
[2,4,6,7-3H]E1 (99 Ci/mmol),
[1,2,6,7,16,17-3H]P (97 Ci/mmol),
[1,2,6,7-3H]T (105 Ci/mmol), and
[
32P]deoxy-CTP (3000 Ci/mmol) were obtained from
Amersham Life Science (Little Chalfont, Buckinghamshire, UK).
[1,2-3H(N)]A-diol (56 Ci/mmol),
[1,2,6,7-3H(N)]DHEA (92 Ci/mmol), and
[1,2-3H(N)]20-OHP (52 Ci/mmol) were from DuPont-New
England Nuclear Life Science (Boston, MA). The transfection reagent,
N-[1-(2,
3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethylsulfate
(DOTAP) was a product of Boehringer Mannheim GmbH (Mannheim, Germany).
Other reagents not mentioned in the text were purchased from Boehringer
Mannheim GmbH, Sigma Chemical Co. (St. Louis, MO), or Merck AG
(Darmstadt, Germany) and were of the highest purity grade
available.
Expression Cloning
For the construction of a cDNA library,
poly(A)+-enriched RNA was isolated using FastTrack 2.0 kit
(Invitrogen, San Diego, CA) from the HC11 cell line, derived from the
epithelial cells of the mammary gland of a pregnant mouse (39). The
cDNA library was generated in cDNA3.1 plasmids containing a
cytomegalovirus promoter and transformed to Escherichia coli
TOP10F' cells by Invitrogen. The cDNA library obtained was then divided
into pools of 2500 colonies on LB-ampicillin (50 µg/ml) plates.
Colonies from each pool were collected and combined, and the plasmid
fractions were isolated using a Qiagen Midi plasmid purification kit
(Hilden, Germany).
The cDNA library was screened based on the method described by Wu
et al. (7). Briefly, 5 µg of plasmid DNA were transfected
from each pool together with 0.5 µg of pAdVAntage plasmid (Promega,
Madison, WI) into HEK-293 cells (300,000 cells per well of a six-well
plate) using DOTAP lipofection reagent. After transfection (18 h) the
cells were allowed to grow for 30 h, and the media were then
replaced with medium containing 100 nM unlabeled
E1 + 200,000 cpm of 3H-labeled E1/ml. After
additional incubation for 18 h, the media were collected, frozen
in dry ice, and kept at -20 C until the steroids were extracted and
analyzed as previously described (45).
Altogether, 243 pools, each containing cDNAs from 2500 colonies,
were screened. Transfection of two plasmid pools resulted in the
conversion of E1 to E2, which was 22.5 times
higher than the conversion obtained in mock-transfected cells. The DNA
from the two positive pools was transformed into Epicurian
coli XL1-Blue MRF' supercompetent cells (Stratagene, La Jolla, CA)
to generate 20 pools of approximately 1000 transformants from both
original positive pools. Plasmid DNA samples isolated from these 40
pools were then transfected into HEK-293 cells, and conversion of
E1 to E2 was again analyzed. One positive pool
from both original lines was further subfractionated into 20 pools of
100 transformants and analyzed. At the final stage, a positive DNA pool
(containing 100 cDNA clones) was transformed into the supercompetent
cells, and 100 single colonies were picked up for plasmid isolation on
a miniprep scale to identify a pure positive clone. The two enriched
clones originating from the two independent positive pools were named
m17HSD7.1 and m17HSD7.2. The nucleotide sequence of the cDNAs was
determined from both their strands using an automatic DNA sequencer
(ABI Prism 377 DNA Sequencer, Perkin-Elmer, Foster City, CA). Fasta and
PROSITE searches for the sequences, homology comparisons, and
determination of the hydropathicity profile were carried out with a GCG
package, version 8.1 (Genetics Computer Group, Madison, WI).
Isolation of RNA and Cloning of r17HSD7/PRAP cDNA
Total and poly(A)+-enriched RNAs were extracted
using standard methods (55, 56). r17HSD7/PRAP cDNA was derived from
total RNA isolated from the ovaries of pregnant rats (12 day) using
SuperScriptII RT (GIBCO BRL, Gaithersburg, MD). The primer needed for
generation of the antisense strand corresponded to nucleotides
10211003 in the PRAP cDNA (38) and the RT reaction was carried out at
42 C for 50 min. The RNA strand was next degraded by ribonuclease H
(RNase H) (Pharmacia Biotech, Uppsala, Sweden) at 37 C for 15 min.
r17HSD7/PRAP cDNA was then amplified with Pyrococcus
furiosus polymerase (Stratagene) using primers containing the
EcoRI site and corresponding to nucleotides from 927 and
from 10211003 in the PRAP cDNA. The PCR consisted of denaturation at
94 C for 1 min, annealing for 1 min, and extension at 72 C for 2 min,
the total number of cycles being 35. In the first five cycles, the
annealing temperature was 57 C, and in the rest of the cycles 62 C. The
PCR product was digested with EcoRI, cloned into CMV6 and
Bluescript KS+ plasmids, and sequenced as mentioned
above.
Measurement of 17HSD and 20HSD Activities in Cultured Cells and
in Vitro
HC11 cells were seeded into six-well plates at a density of
230,000 cells per well. After 48 h, the media were replaced with
medium containing 100 nM unlabeled substrate
(E1, E2, A-dione, or T) + 200,000 cpm of
3H-labeled substrate per ml. After additional incubations
from 224 h, the media were collected, frozen in dry ice, and
maintained at -20 C. Steroids were extracted and separated from each
other, and the conversion of substrate to product was determined as
previously described (45).
17HSD/20HSD activity measurements after transient transfections with
m17HSD1, m17HSD7, and r17HSD7/PRAP cDNAs under CMV-promoter were
carried out in cultured HEK-293 cells. The cells were plated into
six-well plates at a density of 300,000 cells per well 1 day before
transfection. One half or 1 µg of plasmids was transfected using
DOTAP. After transfection (18 h), the cells were allowed to grow for
30 h, and the medium was then replaced with one containing 100
nM or 1 nM unlabeled substrate + 200,000 cpm of
3H-labeled substrate/ml. After incubations from 224 h,
the media were collected and stored, and steroids were analyzed as
above.
For activity measurements of m17HSD7 in vitro, HEK-293 cells
were transfected with m17HSD7.1-cDNA3.1 plasmids, after which they were
collected and lysed by sonication in 10 mM potassium
phosphate buffer, pH 8, containing 1 mM EDTA, 20%
glycerol, 0.08%
N,N-bis-(3-D-gluconamidopropyl)cholamide
(Big Chap, Calbiochem-Novabiochem International, San Diego, Ca), 0.05%
BSA, 0.02% NaN3, and PMSF, 87 ng/ml. Cell debris was
removed by centrifugation at 1500 x g for 10 min, and
the supernatant was stored at -70 C. For determination of
Km and Vmax values, cell extracts were diluted
in 10 mM potassium phosphate buffer, pH 8, containing 1
mM EDTA and 0.05% BSA, and the samples were then mixed
with a substrate (0.1, 1, and 5 µM E1, and
3.3, 5, and 10 µM E2). When the conversion
rates for A-dione, T, A-diol, DHEA, 20-OHP, and P were measured, a 0.5
µM substrate concentration was used. The enzymatic
reactions were started by adding a cofactor (NADP+/NADPH,
Boehringer Mannheim) to a final concentration of 1 mM, and
the samples were incubated for an appropriate time at 37 C. The
reactions were stopped by freezing the reaction mixture quickly in an
ethanol-dry ice bath. Steroids were extracted and separated from each
other, and the amount of substrate formed per min and per total amount
of protein was calculated. Kinetic parameters (Km and
Vmax) were calculated by using a GraFit-program (Erithacus
Software Ltd., Staines, UK). The program fits data to the
Michaelis-Menten equation using nonlinear regression analysis. The
values presented represent the average ± SD of three
independent experiments.
Northern Blotting and RT-PCR Analyses
Total or poly(A)+-enriched RNAs were subjected to
electrophoresis in 1% (wt/vol) agarose-formaldehyde gel (56), which
was blotted with a positively charged nylon membrane (Boehringer
Mannheim). The fixed membranes were hybridized with a
32P-labeled fragment of either m17HSD7 cDNA (corresponding
to nucleotides 33849 in the cDNA, Fig. 2
) or r17HSD7/PRAP cDNA
[nucleotides 9897 in the cDNA (38)]. Hybridization was performed at
42 C in 5 x NaCl/Pi,/EDTA-buffer (0.75 M
NaCl, 50 mM NaH2PO4, 5
mM EDTA, pH 7.0), containing 50% formamide, 1% BSA, 1%
Ficoll 400, 1% polyvinyl pyrrolidone, 0.5% SDS, and 100 µg salmon
sperm DNA/ml. To control the amount of RNA applied to the gels, the
membranes were also hybridized with a rat GAPDH cDNA probe.
RT-PCR analysis was run as previously described (10) using a
Thermostable recombinant Thermus thermophilus RNA RT-PCR kit
(Perkin Elmer-Roche Molecular Systems, Inc., Branchburg, NJ). The
primers corresponded to nucleotides from 351370 and from 849831 in
m17HSD7 cDNA and nucleotides from 5475 and from 912849 in mouse
GAPDH cDNA (57). After PCR, 80 µl aliquots of the PCR mixtures were
subjected to electrophoresis in 1.5% agarose gel and blotted with a
nylon membrane. Hybridization of the membrane was performed similarly
to Northern blot analysis, with m17HSD7 or rat GAPDH cDNA probes.
 |
ACKNOWLEDGMENTS
|
---|
We thank Ms. Helmi Konola and Mrs. Eeva Holopainen for their
skillful technical assistance. We are also grateful to Dr. Bernd Groner
for providing the HC11 cell line and Dr. Lateef Akinola for preparing
the filters containing RNA samples from rat tissues.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Pirkko Vihko, Professor, Biocenter Oulu and WHO Collaborating Centre for Research on Reproductive Health, University of Oulu, FIN-90220, Oulu, Finland.
This work was supported by the Research Council for Health of the
Academy of Finland (project no. 3314 and 40990). The World Health
Organization Collaborating Centre for Research on Reproductive Health
is supported by the Ministries of Education, Social Affairs and Health,
and Foreign Affairs, Finland.
Received for publication January 30, 1998.
Revision received March 19, 1998.
Accepted for publication March 19, 1998.
 |
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