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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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 1–4) (5, 6, 7, 8, 9) and six
rodent 17HSDs (types 1–6) 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 1–4
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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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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). 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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). 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 18–28%. 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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, 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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). 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 0.5–1.5% 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+.
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). The 4.6-kb mRNA
was very abundantly expressed in the ovaries of pregnant (14–20 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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). Neither 17HSD
nor 20HSD activity was observed when E2, A-dione, T,
20-OHP, or P were used as substrates.
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), 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 1–5, 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 2–2.5 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
1021–1003 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 9–27 and
from 1021–1003 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 2–24 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 2–24 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 33–849 in the cDNA, Fig. 2) or r17HSD7/PRAP cDNA
[nucleotides 9–897 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 351–370 and from 849–831 in m17HSD7 cDNA and nucleotides from 54–75 and from 912–849 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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