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Biocenter Oulu and World Health Organization Collaborating Centre for Research on Reproductive Health (T.J.P., R.M.K., M.H.P., P.V.I., J.P.J.M., R.K.V., P.T.V.) and the Department of Anatomy (J.T.L.), University of Oulu, FIN-90220 Oulu, Finland; the Hauptman-Woodward Medical Research Institute, Inc., and Roswell Park Cancer Institute (D.G.), Buffalo, New York 14203; and the Department of Biosciences, Division of Biochemistry (P.T.V.), FIN-00014 University of Helsinki, Finland
Address all correspondence and requests for reprints to: Prof. Pirkko Vihko, Biocenter Oulu and World Health Organization Collaborating Centre for Research on Reproductive Health, University of Oulu, Kajaanintie 50, FIN-90220 Oulu, Finland. E-mail: pvihko{at}whoccr.oulu.fi
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Abstract |
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-dihydroprogesterone. The oxidation of
estradiol brought about by human 17HSD type 2 was effectively inhibited
by several other steroidal compounds, such as 2-hydroxyestradiol,
5ß-androstan-3,17ß-diol, 5-androstan-3,17ß-diol, and
5-androstan-3ß,17ß-diol. The broad substrate specificity of
human 17HSD type 2 together with its predominant oxidative activity and
intracellular location, as observed in this study, indicate the
physiological role of the enzyme to be primarily an inactivator of
highly active 17ß-hydroxysteroids. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydroprogesterone (20-P) to progesterone (P) (3). The
expression of human 17HSD type 2 messenger RNA (mRNA) has been detected
in a large variety of tissues. Its 1.5-kb mRNA is strongly expressed in
the endometrium, placenta, liver, and small intestine, and in smaller
amounts in the pancreas, colon, kidney, and prostate (8, 9, 10). 17HSD
type 2 mRNA has also been found to be present in human breast,
endometrial and prostate cancer cell lines (8, 9). The broad tissue
distribution together with the predominant oxidative activity of 17HSD
type 2 suggest that the enzyme may play an essential role in the
inactivation of highly active 17ß-hydroxysteroids from the blood
circulation, thus diminishing sex hormone action in target tissues. Although there are several reports concerning 17HSD type 2 mRNA and the protein translated in vitro, studies involving purified type 2 protein have not been reported. The primary structure of human 17HSD type 2 possesses two putative signal sequences and a putative transmembrane region consisting of 30 hydrophobic amino acids close to the N-terminus (3). The enzyme is, therefore, thought to be associated with the endoplasm reticulum (ER). In the present study, precise intracellular localization of human 17HSD type 2 was determined using a double immunofluorescence labeling technique. The substrate specificity of the enzyme was also analyzed in some detail. In addition, by studying the structural elements needed for both membrane interaction and enzyme activity, we were able to characterize the biochemical properties of an active truncated form of human 17HSD type 2 that lacks strong association within the membrane.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) by PCR, intact
cDNA was used as a template. All synthesized PCR products were then
cloned into the NotI and SalI sites of the
pK503–9 vector (11, 12), which is a FLAG sequence containing transfer
vector of the Bac-to-Bac Expression System (Life Technologies, Grand Island, NY).
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, resulted in a modified pcDNA3 vector
that allowed the cloning of NotI-SalI human 17HSD
type 2 cDNA fragments into the NotI-XhoI sites of
the vector to obtain amino-terminal FLAG fusion proteins. All of the
expression constructs generated were confirmed by sequencing (13).
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Production of the truncated N-29 17HSD type 2 enzyme was scaled up to a 30-liter bioreactor (Biostat UD 30, B. Braun International, Melsungen, Germany). The optimal level of expression was reached at about 72 h postinfection, whereafter the Sf9 cells were harvested by centrifugation at 1,000 x g for 10 min and stored at -70 C. To purify the fusion protein, harvested cells were suspended in buffer A [50 mM Tris-HCl buffer (pH 7.4), 150 mM NaCl, 0.5 mM phenylmethylsulfonylfluoride, 0.02% NaN3, 20% glycerol, 14 mM 2-mercaptoethanol, and 0.5% n-octylglucoside] and then disrupted in an ice bath by sonication (four times, 20 sec each time, 0.5-min intervals). The protein was then immunoprecipitated from the 100,000 x g fraction of the cell lysate using an anti-FLAG M1 affinity chromatography column (Eastman Kodak Co., New Haven, CT), according to the manufacturer’s instructions. The fractions containing the highest 17HSD type 2 concentrations, detected by SDS-PAGE, were pooled, concentrated, and loaded onto a Superose 12 gel filtration column (1.0 x 30 cm; 0.2 ml/min) connected to a fast protein liquid chromatography system (Pharmacia Biotech, Uppsala, Sweden). N-29 17HSD type 2 protein was eluted with 50 mM potassium phosphate buffer (pH 7.4) containing 150 mM KCl, 0.02% NaN3, 20% glycerol, and 14 mM 2-mercaptoethanol. The pure protein peak was collected and stored at -70 C.
The protein concentration of the purified N-29 17HSD type 2 was measured by the method of Bradford et al. (16). The purified N-29 protein was further characterized by 11% SDS-PAGE carried out on a Mini-PROTEAN II apparatus (Bio-Rad Laboratories, Inc., Richmond, CA). The proteins were visualized in the electrophoresis gels by 0.1% Coomassie blue staining.
Measurement of 17HSD type 2 activity in vitro and in cultured
cells
The activity of 17HSD was measured in vitro as
previously described by Puranen et al. (12, 28) with minor
modifications. Protein samples diluted in 10 mM potassium
phosphate, pH 7.5, containing 0.01% BSA and 14 mM
2-mercaptoethanol were mixed with 3H-labeled
E2, estrone (E1), T, androstenedione
(A-dione), DHT, 5-androstenediol (A-diol), dehydroepiandrosterone
(DHEA), 20-P, or P to a final concentration of 0.5–4
µmol/liter. The reactions were initiated by adding
NAD+/NADH (1.0 µmol/liter) as a cofactor, followed by
incubation for 1 min at 37 C. After incubation, the reactions were
stopped by immediate freezing in an ethanol-dry ice bath, and the
steroids were extracted into diethyl ether-ethyl acetate (9:1).
Substrates and reaction products were separated using an
acetonitrile-water (48:52, vol/vol) solution as a mobile phase in a
Symmetry C18 reverse phase chromatography column (3.9
x 150 mm) connected to a HPLC system (Waters Corp.,
Milford, MA), as previously described (15, 17). Km and
kcat values of 17HSD type 2 were calculated using a GraFit
program (Erithacus Software Ltd., Staines, UK). The kinetic values
shown represent the average ± SD of at least three
independent experiments carried out in triplicate. One micromole of
product formed per min was defined as representing 1 U enzyme
activity.
For comparing the catalytic properties of the truncated 17HSD type 2 enzymes with those of the wild-type enzyme, activity measurements in cultured human embryonic kidney 293 cells were carried out by plating the cells at a density of 1.2 x 106 cells/10-cm diameter petri dish in DMEM (Life Technologies) supplemented with 10% FCS. The cells were allowed to attach overnight and were then transfected with the intact and truncated forms of 17HSD type 2 cDNA constructs (5 µg DNA/1.2 x 106 cells) using a lipofection-based transfection method (DOTAP, Boehringer Mannheim, Mannheim, Germany). Transfection was carried out for 18 h, 10 ml fresh medium were added, and incubation was continued for an additional 48 h. Transfected cells were then plated into 6-well plates at a density of 200,000 cells/well and allowed to attach for 24 h. Activity measurements of the intact and truncated human 17HSD type 2 enzymes were performed by applying 2 ml serum-free medium containing 0.2 µM 3H-labeled E2 (0.4 nmol/well) to the cells. Oxidative 17HSD activity, from E2 to E1, was measured at four different time points (0.5–4 h). After incubation, the media were collected, frozen in dry ice, and kept at -20 C before extracting and separating the steroids (see above).
For analyzing the substrate specificity of the wild-type 17HSD type 2
enzyme, full-length cDNA was transiently expressed in 293 cells under
the cytomegalovirus promoter using pCMV6 vector (9). With
E2, E1, T, A-dione, DHT,
A-diol, DHEA, 20-P, and P as substrates, 17HSD activity
was measured using a 0.2-µM concentration of the
3H-labeled substrate at three different time points (1–4
h). Furthermore, the substrate specificities of the intact human 17HSD
type 2 expressed in 293 cells and the purified recombinant N-29 17HSD
type 2 were analyzed by studying the ability of several steroidal
molecules (Steraloids, Inc., Wilton, NY) to compete with 0.2 and 0.5
µM [3H]E2,
respectively. Steroidal compounds were added at final concentrations of
1 and 10 µmol/liter, using an incubation time of 2 h in the
experiments of the intact 17HSD type 2 enzyme and 1 min in the assays
of recombinant N-29 17HSD type 2 enzyme. The amount of converted
E2 in the experiments of the intact 17HSD type 2
was analyzed in triplicate samples in at least three independent
experiments and in the assays of recombinant N-29 17HSD type 2 enzyme
in duplicate samples in two or three independent experiments.
Immunoblotting
The amounts of wild-type and truncated forms of human 17HSD type
2 protein expressed in 293 cells were detected by immunoblotting. The
proteins from cell homogenates were separated by SDS-PAGE
(Mini-PROTEANII, Bio-Rad Laboratories, Inc.). The proteins
were then transferred to an Immobilon-P polyvinylidene fluoride
membrane (Millipore Corp., Bedford, MA) and immunostained
using the enhanced chemiluminescence detection system
(Amersham, Aylesbury, UK). Monoclonal M5 antibody raised
against the sequence of Met-FLAG peptide (Eastman Kodak Co., Rochester, NY) was used as primary antibody.
The immunoblotting of the recombinant N-29 17HSD type 2 fractions during the purification process was carried out as described above. The immunostaining was performed with the mouse monoclonal M2 antibody raised against FLAG-peptide (Eastman Kodak Co.) and the ProtoBlot AP system (Promega Corp., Madison, WI).
Immunocytochemistry
For subcellular localization of wild-type human 17HSD type 2 by
indirect immunofluorescence, NIH-3T3 cells were seeded in a 6-cm
diameter plate, 0.25 x 106 cells/plate. After
overnight incubation, the cells were transiently transfected with 17HSD
type 2 cDNA-pcDNA3 plasmid (10 µg DNA/
0.5 x 106
cells) using SuperFect transfection reagent (Qiagen,
Hilden, Germany), according to the manufacturer’s instructions. The
transfected NIH-3T3 cells were then grown for 48 h before plating
them at a density of 2.0 x 104 cells/chamber of an
eight-chamber slide (Nalge Nunc International, Naperville,
IL). The cells were allowed to attach overnight before staining.
For double immunofluorescence labeling, the cells were briefly washed with PBS and fixed for 5 min in 100% methanol and for 2 min in 100% acetone at -20 C. The cells were washed with PBS and then permeabilized in 1% Triton X-100-PBS solution for 2 min. After blocking the nonspecific binding sites with 50% goat serum for 4 h at room temperature, the cells were incubated with anti-FLAG M5 monoclonal antibody (27 µg/ml) for 3 h. To analyze whether the fusion protein was colocalized with ER membranes, the cells were further incubated with either 1:50 diluted polyclonal antisera specific for the carboxyl-terminus of rat Grp78 (BiP) or for the C-terminal region of canine calnexin (StressGen Biotechnologies Corp., Victoria, Canada) at 4 C overnight. The cells were then washed with PBS and incubated for 2 h at room temperature, first with rhodamine-labeled donkey antirabbit IgG antibodies (20 µg/ml) and then with fluorescein-labeled goat antimouse IgG antibodies (20 µg/ml). The cells were then washed with PBS and mounted on slides with Glycergel mounting medium (DAKO Corp., Glostrup, Denmark). The double immunofluorescence labeled cells were viewed using a confocal laser scanning microscope (CLSM, revision 4.0, Leica Corp. Lasertechnic, Heidelberg, Germany) as described previously (18, 19). The instrument is built around a Nikon Optiphot-2 epifluorescence microscope (Nikon, Tokyo, Japan).
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Results |
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). In similar conditions, the truncated
N-49 enzyme catalyzed 51.4% conversion of E2,
indicating that deletion of the first 49 N-terminal amino acids did not
affect the catalytic properties of the enzyme. Corresponding
conversions brought about by the truncated type 2 enzymes lacking the
first 29 (N-29) or last 6 carboxyl-terminal (C-6) amino acids were
29.4% and 26.5%, respectively. Thus, these enzymes catalyzed the
oxidative reaction of E2 at an approximately
0.6-fold lower rate than that found for the intact enzyme. However,
deleting the first 80 amino-terminal residues (N-80) or the first 49
amino-terminal residues simultaneously with the last 17 amino acids
from the C-terminus (N-49/C-17) resulted in totally inactive enzymes
(Fig. 3). The C-17 truncated form converted 1.3% of
E2 in 2 h (Fig. 3), also indicating complete
loss of activity. Conversions of E2 to
E1 for the intact and truncated 17HSD type 2
enzymes were calculated according to the amount of protein expressed in
transfected 293 cells measured by Western blotting (data not shown).
The solubility of the catalytically active truncated (N-29, N-49, and
C-6) 17HSD type 2 enzymes was then analyzed using several ionic and
nonionic detergents. In optimal conditions, 50% of the N-29 truncated
type 2 enzyme could be solubilized in the presence of 0.5%
n-octylglucoside detergent.
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), very close to that calculated (41.1
kDa). The catalytic properties of purified N-29 type 2 enzyme were
characterized in vitro (Table 1). The Km values were
similar when using E2, T, 20-P, DHT, and
A-diol as substrates (Table 1), indicating broad substrate specificity
of the enzyme. The catalytic efficiencies
(kcat/Km) indicated that the enzyme
catalyzed the oxidative reactions of estrogens and androgens 10-fold
more efficiently compared with the values measured for the
corresponding reductive reactions in vitro (Table 1).
Accordingly, the results of activity measurements performed in cultured
293 cells transiently transfected with intact 17HSD type 2 showed that
the type 2 enzyme possesses exclusive oxidative activity by equally
well converting E2, T, DHT, and A-diol to their
inactive counterparts (Fig. 5). However,
the enzyme catalyzed the oxidative reaction from 20-P to P
approximately 2-fold less efficiently. This is in line with results
obtained after activity measurement in vitro, which
indicated that the catalytic efficiency of the purified N-29 enzyme
with regard to oxidation of 20-P was 16-fold lower compared with the
corresponding estrogenic reaction (Table 1). We next carried out
extensive analysis to characterize the substrate specificity of human
17HSD type 2, using a series of steroidal compounds that were tested
for their ability to compete with E2 as a
substrate in 293 cells transfected with intact human enzyme and in
assays with the purified recombinant N-29 17HSD type 2 (Table 2). Strong inhibition of type 2 enzyme
activity was measured when using 2-hydroxyestradiol,
5ß-androstan-3,17ß-diol, 5-androstan-3,17ß-diol, and
5-androstan-3ß,17ß-diol as competitive molecules (Table 2).
These compounds inhibited the conversion of E2 to
E1 to around the same degree as did T, A-diol,
and 20-P (Table 2), which are known to possess high specificity
toward 17HSD type 2 (Fig. 5). In addition, 17-estradiol, ethynyl
estradiol, 17ß-dihydroequilin, and coumestrol had significant
inhibitory effects on the oxidation of E2
catalyzed by the human type 2 enzyme. In contrast, no such inhibition
was observed for glucocorticoids or progestins, except for 20-P
(Table 2).
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, A–C). The results were identical regardless of which of the two
ER-specific antisera (BiP or calnexin) were used for the experiments.
Interestingly, colocalization was observed all over the cytoplasm,
although the most intense staining pattern was found in ER located next
to the cell nuclei. To control the specificity of the immunoreactions,
primary antibodies were used to stain nontransfected NIH-3T3 cells
(Fig. 6, D–F), or they were omitted from some experiments (data not
shown). In both cases, no visible staining pattern was observed,
suggesting that the primary antibodies recognize their targets
specifically.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As antibodies to 17HSD type 2 were not available, cDNA constructs encoding a FLAG-tagged full-length human protein were transiently expressed in NIH-3T3 cells to allow localization of the enzyme by immunocytochemical techniques. The present results reveal that recombinant N-FLAG/17HSD type 2 is colocalized with Grp78 (BiP), a soluble protein present in the lumen of the ER (23, 24). This indicates that the human 17HSD type 2 protein, when expressed in NIH-3T3 cells, is localized in the ER, and that the N-terminal lysine-rich signal peptide is not cleaved out during the insertion process. This, together with the poor solubility of the enzyme, further suggest that human 17HSD type 2 is a transmembrane protein in the endoplasmic reticulum. Our finding is consistent with previous results reported by Wu et al. (3), who showed that expression of the enzyme in vitro was stimulated by microsomes, and that the translated protein cofractionated with the membrane pellet. However, the exact subcellular localization of the enzyme has remained unclear. The intracellular ER location of human 17HSD type 2 allows the enzyme to rapidly and efficiently inactivate high activity 17ß-hydroxysteroids before they are able to occupy nuclear steroid receptors.
To avoid the difficulties in purification associated with intact 17HSD
type 2, we produced a truncated form of the enzyme lacking the first 29
amino-terminal residues. Activity measurements of the purified N-29
enzyme carried out in vitro showed that it possesses high
specificity toward C18- and C19-steroids, but
it is also able to catalyze, although at a lower reaction rate,
interconversion between C20-steroids. Similarly, the intact
type 2 enzyme catalyzes the oxidation of E2, T,
A-diol, DHT, and 20-P in cultured cells. In addition to the broad
substrate specificity, both truncated and intact 17HSD type 2 enzymes
possess predominant oxidative activities, converting highly active
17ß-hydroxysteroids into less potent ketosteroids. The results
obtained in the activity measurements indicate identical catalytic
properties of the truncated and intact forms of 17HSD type 2, and thus
confirm the suitability of the constructed N-29 enzyme for
structure-function analyses. Furthermore, the purification protocol
described here results in a highly homogeneous protein, allowing us to
scale up protein production, which is needed for extensive
crystallization studies. As the structure of human 17HSD type 1
according to x-ray crystallography has been recently resolved (25, 26, 27),
elucidation of the three-dimensional structure of the type 2 protein
would be useful to study more closely the structural features of the
enzymes that allow them to use different steroids as substrates and to
catalyze opposite reactions.
Based on the present findings, human 17HSD type 2 shows a wider
specificity for androgenic substrates than for estrogens or progestins.
Our findings and those of others (3, 8, 28) indicate that by
inactivating the most active prostate-specific androgens, T, DHT, and
A-diol, 17HSD type 2 could play a central role in protecting prostatic
cells from excessive androgen action. Furthermore, the present results
reveal the effective inhibition of oxidation of
E2 by several other androgens present in the
prostate and urine, such as 5ß-androstan-3,17ß-diol,
5-androstan-3,17ß-diol, and 5-androstan-3ß,17ß-diol.
This suggests that the enzyme could also diminish androgen action in
the prostate by metabolizing intermediary steroids as well. The
metabolic pathways of these compounds could also be physiologically
important in epithelial cells of the gastrointestinal and urinary
tracts, where strong 17HSD type 2 mRNA expression has been detected
(29). The results showed that 17ß-dihydroequilin has a significant
inhibitory effect on the activity of 17HSD type 2 when
E2 was used as a substrate. This compound is a
metabolite of equilin, which is commonly used for estrogen replacement
therapy and prevention of osteoporosis in postmenopausal women (30).
Similarly, a contraceptive compound, ethynyl estradiol, is a potent
substrate for 17HSD type 2. The data therefore indicate that the enzyme
is capable of metabolizing several orally administered steroidal
compounds possessing a tetracyclic ring and a hydroxy group at position
C17, at the epithelium of the intestine. This, in turn, is
likely to be important with regard to the pharmacology of steroidal
drugs. Human 17HSD type 2 can also use 2-hydroxyestradiol as a
substrate, further suggesting that in addition to
E2, the enzyme could catabolize catechol
estrogens in the liver. The recent finding that 17HSD type 2 mRNA is
expressed in liver hepatocytes in both male and female mice (29)
suggests that the enzyme has a role in the general hepatic inactivation
of steroids. In conclusion, the predominant oxidative activity and
broad substrate specificity of human 17HSD type 2 observed in this
study indicate that this enzyme is primarily involved in diminishing
the stimulatory effects of highly active 17ß-hydroxysteroids, even in
tissues not classified as traditional sex steroid target tissues.
However, there is one exception, in that human 17HSD type 2 catalyzes
the oxidation of 20-P to a biologically more potent form (P),
as also observed previously (3). Substrate preference of human 17HSD
type 2 for several distinct steroidal molecules suggests easy
accessibility and open conformation of the active site. The activity
measurements showed that the recombinant N-29 17HSD type 2 has
preserved the broad substrate specificity during the purification
process. Based on the present findings, the purified N-29 truncated
form of human 17HSD type 2 could be used for resolving the structure of
the enzyme by x-ray crystallography, which would further allow detailed
characterization of the structural motifs and amino acids related to
the substrate specificity of the enzyme.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Physiology, University of Turku,
Kiinamyllyntie 10, FIN-20520 Turku, Finland.
Received October 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxysteroid
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