Steroid specificity of the putative DHB receptor: evidence
that the receptor is not 11
HSD
Karen E.
Sheppard1,
Karen
Khoo2,
Zygmunt S.
Krozowski1, and
Kevin X. Z.
Li1
1 Baker Medical Research
Institute, Melbourne, Victoria 8008; and
2 Department of Pathology and
Immunology, Monash University Medical School, Prahran, Victoria,
Australia 3181
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ABSTRACT |
Recently, we identified a novel putative nuclear
receptor in colonic crypt cells distinct from both mineralocorticoid
receptor and glucocorticoid receptor, with high affinity for
11-dehydrocorticosterone (11-DHB) (33). In the present study,
competitive nuclear binding assays demonstrated that this site has a
unique steroid binding specificity that distinguishes it from other
steroid receptors. Western blot analysis showed the presence of
11
-hydroxysteroid dehydrogenase-2
(11
HSD2) but not
11
HSD1 in colonic crypt cells and showed that
11
HSD2 was present
in the nuclear pellet. Differences in steroid specificity between the
putative DHB receptor and inhibition of 11
HSD activity indicate that
binding is not to the enzyme. Furthermore, modified Chinese hamster
ovary cells transfected with the
11
HSD2 gene express nuclear
11
HSD2 but not a nuclear DHB
binding site. In conclusion, these data support the existence of a
novel nuclear DHB receptor in rat colon that is distinct from the
classic steroid receptors and from both
11
HSD1 and
11
HSD2.
11-dehydrocorticosterone; 11
-hydroxysteroid dehydrogenase; glucocorticoid; mineralocorticoid; nuclear receptor; colon
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INTRODUCTION |
BOTH THE MINERALOCORTICOID receptor (MR) and the
glucocorticoid receptor (GR) have high affinity for the endogenous
glucocorticoid corticosterone (B) in rat and cortisol (F) in humans.
Corticosteroid responsiveness is modulated by 11
-hydroxysteroid
dehydrogenase (11
HSD), two isoforms of which have been cloned
(1-3). 11
HSD type 1 (11
HSD1) has micromolar
affinity for B and F and catalyzes the reversible conversion of these
steroids to their MR and GR inactive 11-keto metabolites,
11-dehydrocorticosterone (11-DHB) and cortisone (20). This enzyme has
been implicated in regulating the glucocorticoid response by increasing
and/or decreasing the local tissue concentration of endogenous
glucocorticoids (14, 16, 19, 39). 11
HSD type 2 (11
HSD2) has nanomolar
affinity for B and F and acts only as a dehydrogenase (3, 25). Given 11
HSD2 colocalization with MR
in sodium-transporting epithelia and the increase in sodium retention
when enzyme activity is compromised (38, 42), this enzyme is thought to
confer aldosterone specificity on MR (10, 13, 27).
Rat colonic epithelial cells express MR, GR, and
11
HSD2 (29, 33, 36, 45), and,
as expected, B binding to both MR and GR in these cells is decreased by
11
HSD activity (33). In addition to MR and GR, colonic crypt cells
also express a putative nuclear steroid receptor with high (~10 nM)
affinity for 11-DHB. We recently proposed that this receptor might
mediate glucocorticoid effects in cells expressing high levels of
11
HSD activity (33). In the present study, we have further
characterized this putative receptor by determining its steroid
specificity and clearly demonstrate that 11-DHB is not
binding to MR, GR, progesterone receptor (PR), androgen receptor (AR),
or estrogen receptor (ER). In addition, we demonstrate differences in
steroid specificity between the putative DHB receptor and 11
HSD
inhibition, indicating that binding is not to 11
HSD. Furthermore, we
show the presence of 11
HSD2 but
not 11
HSD1 in rat colonic crypt
cells and that cells transfected with
11
HSD2 express
11
HSD2 in nuclei but not a
nuclear DHB receptor.
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METHODS |
Colonic crypt isolation.
Colonic crypt cells were prepared from male Sprague-Dawley rats
weighing 180-220 g by a nonenzymatic technique (44). Briefly, colons were washed several times with ice-cold PBS (0.01 M) and then
incubated at 22°C (room temperature) in PBS containing 3 mM EDTA
and 0.5 mM dithiothreitol for 90 min. Crypts were removed by vigorous
shaking, pelleted by centrifugation (40 g for 5 min at 4°C), and then
resuspended in DMEM containing 25 mM HEPES (DMEM-HEPES). Previous
studies have demonstrated that this method results in the isolation of
single viable intact crypts free of stroma (43).
Modified Chinese hamster ovary cell culture and
transfection.
Modified Chinese hamster ovary cells (CHOP-C4) (15) were
grown on 100-mm plates in RPMI 1640 with 10% FCS, 2 mM glutamine, and
antibiotics. Rat 11
HSD1A cDNA
(1) and rat 11
HSD2 cDNA (17) in
the expression vector pcDNA1 (Invitrogen) were transfected into CHOP
cells by a modification of the DEAE-dextran method as previously
described (18). Enzyme activity and nuclear binding were measured in
cells 48-72 h after transfection.
Steroids, chromatography, and imaging.
Radioactive steroids were from Amersham (Buckinghamshire, UK), and
nonradioactive steroids were from Sigma (St. Louis, MO; B,
deoxycorticosterone, progesterone, estradiol, 5
-dihydrotestosterone, 11-DHB, 11
-hydroxyprogesterone, 5
-dihydrocorticosterone) or were
a gift from Roussel-Uclaf (Romainville, France; RU-28362 and RU-28318).
Carbenoxolone (CBX) was from Sigma. Ethyl acetate-extracted samples
spiked with nonradioactive B and 11-DHB were separated by TLC on silica
gel 60 F254 plates (Merck, Darmstadt, Germany) with 92% chloroform and
8% ethanol as the mobile phase. Silica gel plates containing the
TLC-separated radioactive steroids were exposed to a BAS-TR2040S
imaging screen (Fuji) for up to 2 days. 3H-labeled bands were then
visualized and quantified with a Fujix bioimaging analyzer (BAS1000
with Mac BAS, Fuji).
[3H]B and
11-[3H]DHB were
identified by the comigration of ultraviolet-visualized nonradioactive
B and 11-DHB, respectively.
Conditioned medium.
Conditioned medium was prepared by incubating
[3H]B (40 nM) with
colonic crypt cells for 3 h at room temperature. After incubation, cells were pelleted (40 g for 5 min at
4°C) and medium was removed. A sample of medium was extracted with
ethyl acetate, and the concentrations of
[3H]B and
11-[3H]DHB were
determined by TLC analysis and scintillation counting. Conditioned
medium contained 80%
[3H]DHB (32 nM) and
20% [3H]B (8 nM). For
the binding assay, conditioned medium was diluted with DMEM-HEPES to
achieve a final concentration of 16 nM
[3H]DHB and 4 nM
[3H]B.
Binding assay.
Colonic crypt cells or transfected CHOP cells were added to pregassed
(5% CO2-95%
O2) glass tubes containing
[3H]B (25-30 nM)
with or without 2-4 µM nonradioactive steroid. Nonspecific
binding was determined in the presence of 2 µM B and 2 µM 11-DHB.
Tubes were then covered in Parafilm and incubated at 22°C for 90 min. Where CBX was used, cells were preincubated for 20 min with 3 µM
CBX. To determine whether
11-[3H]DHB binds
directly to the DHB receptor, cells were incubated in conditioned
medium containing 16 nM
11-[3H]DHB and 4 nM
[3H]B in the presence
of 2 µM 11-DHB and 2 µM B. Low recovery of 11-[3H]DHB, partial
conversion of the
11-[3H]DHB to an
unidentified steroid during the purification procedure, and very high
nonspecific binding of purified
11-[3H]DHB
necessitated the use of conditioned medium. After incubation, a sample
of medium was ethyl acetate extracted, and the cells were washed three
times with 3 ml of ice-cold DMEM followed by centrifugation (200 g for 5 min at 4°C). Nuclei were
separated from cytoplasm as previously described (34). Briefly, washed cells were homogenized in 1.0 ml of cold lysis buffer [10%
(vol/vol) glycerol, 0.2% (vol/vol) Triton X-100, 10 mM KCl, and 50 mM
Tris, pH 7.4] containing 1.0 M sucrose, layered over 1.0 ml of
lysis buffer containing 1.4 M sucrose, and centrifuged for 20 min at 6,000 g, 4°C. The nuclear pellet
was then resuspended in 0.6 ml of 10% (vol/vol) glycerol and 50 mM
Tris (pH 7.4), and nuclei were collected on nitrocellulose filters
(0.45 µm pore size; Schleicher and Schuell, Dassel, Germany) under
vacuum. Filters were dried, DNA content was determined by the method of
Burton (8), and radioactivity was measured by liquid scintillation
spectrophotometry.
Western blot analysis.
Total tissue homogenates were prepared from 1 g of frozen rat tissue or
cells by homogenization in four volumes of homogenizing buffer (0.25 M
sucrose, 10 mM sodium phosphate, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4) in an Ika-Ultraturrex T25 homogenizer (Janke and
Kunkle, Stauten, Germany). Microsomal fractions were isolated from
homogenates by subsequent low-speed (1,500 g for 5 min at 4°C) and high-speed
(100,000 g for 60 min at 4°C)
centrifugation of supernatants. The remaining supernatant was cytosol.
Pelleted microsomes were resuspended in homogenizing buffer, frozen in liquid nitrogen, and kept at
70°C until required. Protein
concentration was determined by the Bradford method (5). For Western
blot analysis on subcellular fractions, nuclei were isolated as
described in Binding assay. The
microsome-enriched pellet and cytosol were obtained by centrifugation
of the supernatant from the nuclei isolation procedure at 105,000 g for 60 min at 4°C.
For Western blot analysis, proteins (50-100 µg) were separated
by 5-15% SDS-PAGE gradient gel electrophoresis under reducing conditions and then transferred to nitrocellulose filters (Scheicher and Schuell) for 2 h on ice. After nonspecific sites were blocked with
5% skim milk powder in PBS (pH 7.4) containing 0.1% Tween 20 (PBS-T),
the nitrocellulose blot was incubated overnight at 4°C with either
rabbit anti-rat HSD1 polyclonal
antibody (56-126) at 1:1,000 dilution (21) or the
immunopurified rabbit anti-rat HSD2 (RAH23) polyclonal antibody
(36) at a concentration of 1 µg/ml in the presence of 0.5% skim milk
powder in PBS-T. The filter was washed with PBS-T and then incubated at
room temperature for 60 min with a 1:5,000 dilution of goat anti-rabbit
IgG antibody conjugated to horseradish peroxidase. The blots were
washed in PBS-T for 60 min before the bands were visualized by the use
of a chemiluminescence kit (Du Pont-NEN, Boston, MA). When blots were
reprobed, the filter was stripped (62.5 mM
Tris · HCl, pH 6.8, 2% SDS, and 100 mM
2-mercaptoethanol) at 50°C for 30 min and then probed with RAH23
antibody at a concentration of 1 µg/ml and with the same procedure as
described above.
Inhibition of 11
HSD
activity.
Colonic crypt cells were added to pregassed (5%
CO2-95%
O2) glass tubes containing
[3H]B (15-20 nM)
with or without nonradioactive steroid. Medium was sampled for 20 min
at 5-min intervals and at 30 and 60 min. Steroids were ethyl acetate
extracted from medium and separated by TLC.
3H-labeled bands were then
visualized and quantified with a Fujix bioimaging analyzer, and
conversion of [3H]B to
11-[3H]DHB was
determined. The rate of conversion of
[3H]B to
11-[3H]DHB was linear
from 10 to 20 min in each experiment, and it was during this time
period that inhibition of enzyme activity was assessed. Data are
expressed as a percentage of enzyme activity in the absence of
inhibitor.
Data analysis.
Data were compared by one-way ANOVA followed by Fisher's protected
least significant differences test. Differences of
P < 0.05 were considered
significant. All data are expressed as means ± SE.
 |
RESULTS |
Steroid specificity of the putative DHB
receptor.
To determine whether 11-DHB was binding to one of the
already-characterized classic steroid receptors, colonic crypt cells were incubated with
[3H]B (25-30 nM)
in the presence or absence of 200-fold excess estradiol, 5
-dihydrotestosterone, or progesterone. Binding to MR and GR was
blocked by the addition of 2 µM RU-28318 and 2 µM RU-28362. Specific nuclear binding to the DHB receptor was 1.51 ± 0.18 fmol/µg DNA (n = 12), and
nonspecific binding as measured in the presence of an excess of both B
and 11-DHB was 0.34 ± 0.03 fmol/µg DNA (n = 12). As illustrated in Fig.
1A, none
of the steroids tested competed for nuclear binding, suggesting that
binding was not to ER, AR, or PR. TLC analysis of
[3H]steroids extracted
from medium showed that conversion of
[3H]B to
11-[3H]DHB was
63 ± 3% for control and that this was modestly decreased by 39 ± 5% by progesterone (Fig.
1B).

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Fig. 1.
Steroid specificity of 11-dehydrocorticosterone (DHB) receptor binding
and 11 -hydroxysteroid dehydrogenase (11 HSD) inhibition. Colonic
crypt cells were incubated with
[3H]corticosterone
([3H]B, 25-30 nM) ± 200-fold excess steroid. In addition, 2 µM RU-28318 and 2 µM
RU-28362 were added to block binding to mineralocorticoid receptor (MR)
and glucocorticoid receptor (GR). After incubation, nuclei were
isolated, nuclear 3H-labeled
steroid binding was determined, and medium was extracted to assess
conversion of [3H]B to
11-[3H]DHB. Binding
data (A) are expressed as
percentages of total nuclear binding (fmol/µg DNA), and 11 HSD
activity (B) is expressed as a
percentage of maximum conversion in absence of added competitor.
E2, estradiol; DHT,
dihydrotestosterone; prog, progesterone; B/DHB, combination of both B
and DHB (nonspecific binding). Data are means ± SE;
n = 4-12. * Significantly
different (P < 0.05) from total
nuclear binding (A, ) or
maximum conversion (B,
).
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To further analyze the specificity of the nuclear binding site
competition by unlabeled B, F and the 11-keto metabolites (11-DHB, cortisone) were compared. All compounds except cortisone significantly decreased nuclear binding (Fig.
2A).
Binding in the presence of excess B was 29 ± 3% of total nuclear
binding, which was not significantly different from nonspecific
binding; 11-DHB decreased binding to 41 ± 2%, and F decreased
binding to 72 ± 6%. Similarly, all steroids except cortisone
inhibited 11
HSD activity (Fig.
2B).

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Fig. 2.
Competition for DHB receptor and inhibition of 11 HSD by cortisol and
cortisone. Colonic crypt cells were incubated with
[3H]B (25-30 nM) ± 200-fold excess steroid and 2 µM of both RU-28318 and RU-28362
to block binding to MR and GR. After incubation, nuclear
3H-labeled steroid binding was
determined (A), and medium was
extracted to assess conversion of
[3H]B to
11-[3H]DHB
(B). Binding data are expressed as
percentages of total nuclear binding (fmol/µg DNA), and 11 HSD
activity is expressed as a percentage of maximum conversion in absence
of added competitor. Data are means ± SE;
n = 4-12. E, cortisone; F,
cortisol. * Significantly different
(P < 0.05) from total nuclear
binding (A, ) or maximum
conversion (B, ).
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To determine whether
11-[3H]DHB bound
directly to the DHB receptor or whether active conversion of
[3H]B to
11-[3H]DHB was a
requirement for binding, cells were incubated in conditioned medium
containing 16 nM
11-[3H]DHB and 4 nM
[3H]B, and binding to
MR and GR was blocked with 2 µM RU-28318 and RU-38486, respectively.
In addition, binding was assessed in the presence of either 21 nM or 4 nM [3H]B. Specific
nuclear binding occurred in the presence of conditioned medium and was
similar to binding in the presence of 21 nM
[3H]B (Fig.
3). There was no significant binding in the
presence of 4 nM
[3H]B, and CBX did not
alter binding of 3H-labeled
steroids in conditioned medium.

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Fig. 3.
Incubation of colonic crypt cells with conditioned medium. Colonic
crypt cells were incubated with either
[3H]B (21 and 4 nM)
or conditioned medium containing 16 nM
11-[3H]DHB and 4 nM
[3H]B ± 4 µM carbenoxolone (CBX). In addition, 2 µM RU-28318 and 2 µM
RU-28362 were added to block binding to MR and GR, and nonspecific
binding was determined in the presence of 200-fold excess 11-DHB and B. Data are averages of 2 separate determinations. , Total
binding.
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11
HSD expression in colonic crypt
cells.
To determine which isoform of 11
HSD was present in colonic crypt
cells, microsomal extracts were prepared from rat colonic crypts,
colon, liver, and kidney and then probed with antibodies to rat
11
HSD1 or rat
11
HSD2. As illustrated in Fig.
4, when probed with anti-rat
HSD1, extracts from rat
11
HSD1-transfected CHOP cells,
liver, kidney, and whole colon but not colonic crypt cells showed a
common signal at 34 kDa corresponding to the predicted size of rat
11
HSD1 (21). In addition, other
higher-molecular-weight proteins were detected in all tissues. Because
of the limited stock of the
11
HSD1 antibody, further
analysis and attempts to improve the
anti-HSD1 Western blot were not
feasible. The nitrocellulose blot was then stripped to remove rat
HSD1 primary antibody and reprobed
with antibodies to rat 11
HSD2.
Previous studies using this antibody (36) have demonstrated that rat
11
HSD2 migrates as a 40-kDa
protein. As illustrated in Fig. 4, kidney, whole colon, and colonic
crypt cell extracts contain
11
HSD2, whereas both liver and
11
HSD1-transfected CHOP cells
did not. The 34-kDa protein detected in liver and kidney presumably
reflects incomplete removal of the
anti-HSD1 primary antibody.

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Fig. 4.
Western blot analysis of relative levels of rat
11 HSD1 and
11 HSD2. Microsomal proteins
from liver (50 µg), kidney (50 µg), transfected modified Chinese
hamster ovary (CHOP) cells (50 µg), colon (100 µg), and colonic
crypt cells (100 µg) were separated by 5-15% SDS-PAGE gradient
gel electrophoresis under reducing conditions. Nitrocellulose blots
were initially probed with
anti-HSD1
(A) and then reprobed with
anti-HSD2
(B). Positions of 34-kDa
11 HSD1 protein and 40-kDa
11 HSD2 protein are indicated.
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Previous studies have demonstrated
11
HSD2 in nuclei of some (23,
35) but not all cells (24, 28). To determine the subcellular fraction
in which 11
HSD2 was present in
rat colonic crypt and
11
HSD2-transfected cells,
nuclei, microsome-enriched pellet, and cytosol were prepared as
described in METHODS. As illustrated
in Fig. 5,
11
HSD2 was present in all
subcellular fractions from both colonic crypt (Fig.
5A) and
11
HSD2-transfected cells (Fig.
5C). In addition to
11
HSD2, a protein >40 kDa was present in both nuclei and microsome-enriched pellet from colonic crypt
cells but not transfected cells. An apparent inconsistency in the
11
HSD2 Western blot analysis is
the absence of the higher-molecular-weight protein in microsomes
isolated from colonic crypt cells in Fig. 4. As described in
METHODS, the buffers and
centrifugation steps used to isolate nuclei and microsome-enriched
pellet (Fig. 5A) differ from those
used to isolate microsomes (Fig. 4). To further understand this
discrepancy, we analyzed the low-speed pellet (1,500 g), which would contain both nuclei
and microsomes, the microsomal pellet (high speed: 100,000 g), and the remaining cytosol. As
illustrated in Fig. 5B,
11
HSD2 was present in both the
low-speed and microsomal pellets but not in cytosol, whereas the
higher-molecular-weight band was only present in the low-speed pellet
(nuclei and microsomes). The presence of
11
HSD2 in cytosol in Fig.
5A but not in Fig. 5B probably reflects the
solubilization of the enzyme by detergent (0.2% Triton X-100) in the
lysis buffer used to isolate subcellular fractions in Fig.
5A.

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Fig. 5.
Western blot analysis of relative levels of rat
11 HSD2 in subcellular fractions
of colonic crypt and transfected CHOP cells.
A: nuclei (n) were isolated from
colonic crypt cells with use of a discontinuous sucrose gradient as
described in Binding assay.
Microsome-enriched (me) pellet and cytosol (c) fractions were obtained
by centrifugation of supernatant from nuclei isolation procedure at
105,000 g for 60 min at 4°C.
B: subsequent low- (1,500 g) and high-speed (100,000 g) centrifugation of colonic crypt
cells homogenized in homogenizing buffer resulted in pellets containing
microsomes (m) and both nuclei and microsomes (nm); remaining
supernatant was cytosol. Nuclei, microsome-enriched pellet, and cytosol
were obtained from 11 HSD2-
(C) and vector-transfected
(D) CHOP cells, using same method as
in A. Positions of 40-kDa
11 HSD2 protein and an
unidentified 43-kDa protein are indicated.
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Inhibitors of 11
HSD
activity.
To assess the steroid specificity of 11
HSD inhibition, colonic crypt
cells were incubated for 5-20 min with 10-20 nM
[3H]B and increasing
concentrations of steroid. Medium was sampled at 5-min intervals. The
rate of conversion of
[3H]B to
11-[3H]DHB was linear
from 10 to 20 min, and it was during this time period that inhibition
of enzyme activity was assessed. Of the compounds tested, the rank
order of potency was found to be 11
-hydroxyprogesterone > CBX > progesterone = 11-DHB > deoxycorticosterone in terms of inhibiting
11
HSD activity (Fig. 6). To test whether
these compounds competed for nuclear binding, colonic crypt cells were
incubated with [3H]B,
2 µM RU-28318, and 2 µM RU-28362 plus 4 µM competitor. As illustrated in Fig.
7A,
11
-hydroxyprogesterone decreased binding to nonspecific levels,
whereas CBX, deoxycorticosterone, and progesterone did not compete for
binding. In parallel with the 11
HSD inhibition dose-response study,
medium taken at the end of incubation showed 11
-hydroxyprogesterone > CBX > progesterone = deoxycorticosterone in terms of inhibiting
11
HSD activity. In addition, 5
-dihydrocorticosterone was potent
both as an inhibitor of 11
HSD activity and as a competitor for
nuclear binding (Fig. 7B).

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Fig. 6.
Dose-dependent inhibition of 11 HSD activity. Colonic crypt cells
were incubated for 10-20 min at room temperature with
[3H]B (15-20 nM) ± inhibitor. After incubation, a sample of medium was taken, and
conversion of [3H]B to
11-[3H]DHB was
determined. Rate of conversion of
[3H]B to
11-[3H]DHB was linear
from 10 to 20 min. Data are expressed as percentages of maximum
conversion in absence of added inhibitor (%control). Each point is
mean ± SE of 4-8 individual determinations. 11 -Hydroxyprog,
11 -hydroxyprogesterone; DOC, deoxycorticosterone.
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Fig. 7.
Competition for DHB receptor binding by competitive inhibitors of
11 HSD. Colonic crypt cells were incubated with
[3H]B (25-30 nM) ± 20-fold excess steroid and 2 µM of both RU-28318 and RU-28362
to block binding to MR and GR. After incubation, nuclear
3H-labeled steroid binding was
determined (A), and medium was
extracted to determine conversion of
[3H]B to
11-[3H]DHB
(B). Binding data are expressed as
percentages of total nuclear binding (fmol/µg DNA), and 11 HSD
activity is expressed as percentage of maximum conversion in absence of
added competitor. 11 OHProg, 11 -hydroxyprogesterone;
5 DiHydro-B, 5 -dihydrocorticosterone. Data are means ± SE;
n = 4-12. * Significantly
different (P < 0.05) from total
nuclear binding (A, ) or
maximum conversion (B, ).
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Transfection studies.
The differences between steroid specificity of 11
HSD inhibition and
the ability to displace nuclear binding suggest that the nuclear 11-DHB
binding site is not 11
HSD. To further rule out this possibility,
CHOP cells were transfected with rat
11
HSD2 or vector, and
3H-labeled steroid binding in the
nucleus was measured (Fig. 8). Nuclear
binding in vector-transfected CHOP cells was displaced to nonspecific
levels with excess RU-28362, indicating the presence of GR but not MR
in vector-transfected cells, and there was no significant conversion of
[3H]B to any
metabolite. When cells were transfected with rat
11
HSD2, 98% of B was converted
to 11-DHB, and there was no significant binding of
3H-labeled steroid in the nucleus,
suggesting that the 11-DHB site is not present and, in addition, that
11
HSD2 effectively inhibited binding to GR.

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Fig. 8.
3H-labeled steroid binding in
vector- and 11 HSD2-transfected
CHOP cells. CHOP cells were transfected with vector (pcDNA1) or rat
11 HSD2 gene. Transfected cells
were incubated with
[3H]B (20-25 nM) ± 200-fold excess RU-28362 or B. After incubation, total nuclear
3H-labeled steroid bound was
determined as described in METHODS,
and conversion of
[3H]B to
11-[3H]DHB was
measured in medium. Data are means ± SE;
n = 3-4. * Significantly
different (P < 0.05) from total
nuclear binding ( ).
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DISCUSSION |
Recently we identified a putative nuclear steroid receptor in colonic
crypt cells that bound 11-DHB with high affinity (33). The present
study demonstrates that the DHB receptor can directly bind
11-[3H]DHB and has a
unique steroid binding specificity that clearly distinguishes it from
other steroid receptors (MR, GR, AR, ER, PR). In addition, we show that
the competitive inhibitors of 11
HSD, CBX, progesterone, and
deoxycorticosterone (12, 22), do not compete for DHB receptor binding
and that there is no evidence of nuclear DHB receptor in
11
HSD2-transfected cells.
Collectively, these data are good evidence that the DHB receptor is a
novel steroid receptor and is not 11
HSD.
Previous studies have demonstrated
11
HSD2 in nuclei isolated from
human colonic cells (28, 35). In agreement with these studies, we can
demonstrate 11
HSD2 in nuclei
isolated from rat colonic crypt cells and
11
HSD2-transfected cells. In
addition, in colonic crypt nuclei, a second band of ~43 kDa is
detected by Western blot analysis. The absence of the 43-kDa protein in nuclei isolated from
11
HSD2-transfected cells
suggests that this protein is not a modified nuclear
11
HSD2; however, it may reflect cell-specific modification of the enzyme. Similarly, a protein ~4 kDa
greater than human 11
HSD2 has
been observed in Western blot analysis on human kidney homogenate (35).
In our previous study (33), we excluded the possibility that 11-DHB was
binding to 11
HSD on the basis that CBX, which is a competitive
inhibitor of 11
HSD (22), did not compete (33). A caveat on this
interpretation, however, was that CBX might not enter the nucleus. To
further explore the possibility that the DHB receptor is nuclear
localized 11
HSD, we assessed the ability of several known substrates
and competitive inhibitors of 11
HSD to compete for nuclear binding. In addition, we determined whether rat
11
HSD2-transfected CHOP cells
expressed the DHB receptor. In contrast to the enzyme inhibition data
in which deoxycorticosterone and progesterone were as potent as 11-DHB
in inhibiting 11
HSD activity, neither deoxycorticosterone nor
progesterone displaced nuclear binding, and, in agreement with previous
data, CBX was a potent inhibitor of enzyme activity but
not of binding (33). The discrepancy between enzyme inhibition and
binding to the DHB receptor is good evidence that these proteins are
distinct. In addition, the inability to detect a nuclear DHB receptor
in 11
HSD2-transfected cells
that express the enzyme in the nucleus further supports the distinction
between the putative DHB receptor and
11
HSD2.
The major circulating glucocorticoid in humans is cortisol, and
cortisone is the 11-keto metabolite produced by 11
HSD. When these
steroids were tested for their ability to compete for binding, cortisol
proved to be a weak competitor for the DHB receptor, and cortisone did
not significantly displace binding. Whether the putative DHB receptor
exists in human colonic crypt cells is yet to be determined. The
relatively low-affinity binding of cortisol to the DHB receptor
suggests that if this receptor is present in humans, it may have a
different steroid specificity and possibly a higher affinity for
cortisone and/or cortisol. It is also possible, given the
significant levels of circulating B in humans (~12 nM) (9, 40) and
the 10-fold increase observed in response to ACTH (40), that B
and/or 11-DHB may be the physiological ligand of the analogous
receptor in humans as well as rat.
Novel corticosteroid receptors have previously been described in
various rat tissues. The type III binding site detected in rat kidney
slices exhibits high affinity for B but does not bind aldosterone or
dexamethasone (11). This binding site is clearly different from the DHB
receptor in that progesterone and deoxycorticosterone compete for
binding. Studies by Naray-Fejes-Toth et al. (26) have suggested that
the type III binding site is 11
HSD, given the high correlation
between steroid specificity of binding and enzyme inhibition. In rat
colon and kidney, a novel glucocorticoid binder named the
corticosteroid IB receptor has also been described (4). It could be
distinguished from the classic GR by sedimentation coefficient and
non-cross-reactivity with GR antibodies. The steroid specificity of
this site, however, is identical to that of the classic GR, and thus it
is not the DHB receptor. A specific 11-DHB binding site in duck nasal
gland, a tissue important for salt balance, has previously been
demonstrated (31, 32). In the initial study (32), there were major
inconsistencies between B and 11-DHB binding characteristics, which
include the detection of 1,000-fold more binding sites when
[3H]B was used as
ligand compared with
11-[3H]DHB and the
observation that B was a better competitor than 11-DHB, although 11-DHB
had an apparent 100-fold higher affinity. These discrepancies and the
inability of excess aldosterone and 11-DHB to totally displace binding
indicate that more than one binding site was being detected. In a
follow-up study (31), using an ammonium sulfate-precipitated cytosolic
protein fraction, the same authors reported that 11-DHB did not compete
for binding, whereas B, aldosterone, and deoxycorticosterone did.
Interpretation of these studies is difficult because of the lack of
specific ligands to block binding to MR and GR and the presence of
11
HSD. In addition, the absence of receptor competition studies
makes it impossible to assess whether binding could be totally
accounted for by MR and/or GR, and the inability of 11-DHB to
displace binding in the ammonium sulfate-precipitated fraction may
indicate loss of the 11-DHB binding site during the process. Whether
the duck nasal 11-DHB binding protein is similar to the rat colonic DHB receptor thus requires further investigation.
Nuclear 11-DHB binding has also been reported in fetal mouse brain and
placental tissue (41). As in the present study and in contrast to the
duck nasal gland, excess 11-DHB was shown to totally displace binding.
These studies in fetal mouse predate the development of specific
ligands for corticosteroid receptors, so 11-DHB may have been
binding to MR and/or GR, although the low affinity of 11-DHB
for these receptors would suggest otherwise. In agreement with this
study, we can demonstrate specific 11-DHB binding in mouse colonic
crypt cells that is distinct from both MR and GR (Sheppard, unpublished
data).
The existence of a novel nuclear DHB receptor in mineralocorticoid
target cells is not unanticipated in that it may explain how B can
mediate glucocorticoid effects in
11
HSD2-expressing cells. Given
that cellular levels of 11-DHB will reflect circulating B except at the
highest levels of ACTH secretion, the receptor would therefore normally
respond to both diurnal and stress variations in corticosteroids. No
specific function has been ascribed to a novel DHB receptor, although
11-DHB has been shown to affect sodium transport in both toad bladder
and rat kidney (6, 7, 30, 37). The distinguishing characteristic of a
DHB receptor would clearly be an effect produced by 11-DHB but not by
dexamethasone or aldosterone. However, if the DHB receptor mediates
physiological glucocorticoid effects in
11
HSD2-expressing cells, this
distinction may not be easily tested experimentally when both GR and
the DHB receptor are present, as is the situation in colonic crypt
cells.
 |
ACKNOWLEDGEMENTS |
We thank Natalia Cordero and Rebecca Ridings for excellent
technical assistance and Dr. Jane Arthur and Prof. John W. Funder for
critical reading of the manuscript.
 |
FOOTNOTES |
We thank the National Health and Medical Research Council of Australia
for financial support.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: K. E. Sheppard, Baker Medical Research
Institute, PO Box 6492, Melbourne, Victoria, Australia 8008.
Received 17 February 1998; accepted in final form 20 April 1998.
 |
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