1 Baker Medical Research Institute and the 2 Department of Pathology and Immunology, Monash University Medical School, Prahran, Victoria, Australia 3181
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ABSTRACT |
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When small intestinal epithelial cells are incubated with
[3H]corticosterone, nuclear binding is displaced neither
by aldosterone nor RU-28362, suggesting that
[3H]corticosterone is binding to a site distinct from
mineralocorticoid receptor and glucocorticoid receptor. Saturation and
Scatchard analysis of nuclear [3H]corticosterone binding
demonstrate a single saturable binding site with a relatively low
affinity (49 nM) and high capacity (5 fmol/µg DNA). Competitive
binding assays indicate that this site has a unique steroid binding
specificity, which distinguishes it from other steroid receptors.
Steroid specificity of nuclear binding mirrors inhibition of the low
11-dehydrogenase activity, suggesting that binding may be to an
11
-hydroxysteroid dehydrogenase (11
HSD) isoform, although
11
HSD1 is not present in small intestinal epithelia and 11
HSD2
does not colocalize intracellularly with the binding site. In summary,
a nuclear [3H]corticosterone binding site is present in
small intestinal epithelia that is distinct from other steroid
receptors and shares steroid specificity characteristics with 11
HSD2
but is distinguishable from the latter by its distinct intracellular localization.
glucocorticoid receptor; mineralocorticoid receptor; 11-hydroxysteroid dehydrogenase; corticosterone
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INTRODUCTION |
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11-HYDROXYSTEROID
DEHYDROGENASES (11
HSDs) interconvert endogenous
glucocorticoids, corticosterone (B), and cortisol (F) to glucocorticoid
receptor (GR) and mineralocorticoid receptor (MR) inert 11-keto
metabolites, 11-dehydrocorticosterone (11DHB), and cortisone.
Consequently, 11
HSDs regulate intracellular availability of B and F
to both GR and MR and thus play a critical role in modulating
corticosteroid hormone action. Two 11
HSD isoforms have been cloned,
11
HSD1 and 11
HSD2 (1-3). 11
HSD2 operates as
an exclusive dehydrogenase for B and F, and given its colocalization with MR in sodium-transporting epithelia (15) and the
increase in sodium retention when enzyme activity is compromised
(24, 36, 38), this enzyme is
thought to confer aldosterone specificity on MR (7,
10, 27). In tissue homogenates, 11
HSD1
catalyzes the reversible conversion of B to 11DHB (23),
although in vivo 11
HSD1 is thought to act as a reductase and has
been suggested to potentiate glucocorticoid action by increasing the
local tissue concentration of endogenous glucocorticoids
(14, 21, 37). In addition to
11
HSD1 and 11
HSD2, there is preliminary evidence for several
other 11
HSD isoforms based on cofactor preference, affinity for
substrate, and whether they act as an oxidase, reductase, or both
(11-13, 22).
In cells expressing high levels of 11HSD2, B binding to both
MR and GR is compromised (29, 32). Therefore,
it is conceivable that there are cellular mechanisms that would
allow endogenous, albeit transformed, glucocorticoids to bind
receptor and thus mediate glucocorticoid effects. A putative steroid
receptor that binds 11DHB with high affinity (<10 nM) was recently
described in rat colonic epithelial cells that express high levels of
11
HSD2 (31). The steroid specificity of this putative
receptor distinguishes it from other steroid receptors and from
11
HSD isoforms. We proposed that this putative receptor mediates
glucocorticoid effects in cells expressing high levels of 11
HSD2
activity. During the course of determining whether 11
HSD2 and the
putative DHB receptor colocalize, we found specific B binding
to a site that was distinct from both MR and GR in rat jejunal and
duodenal epithelial cells that express very little 11
HSD2 and
no 11
HSD1 (33). In the present study, we have further
characterized B binding in rat small intestinal epithelia and show that
B is binding to a nuclear localized binding site that is distinct from
GR, MR, and the putative colonic DHB receptor and shares steroid
specificity characteristics with 11
HSD2 but is distinguishable from
the latter by its distinct tissue and intracellular localization.
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MATERIALS AND METHODS |
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Animals, intestinal dissection, and epithelial cell isolation. In all experiments, adult male Sprague-Dawley rats weighing 180-220 g were used. Sections of the intestine were taken as follows: duodenum, 5 cm distal to the pylorus; jejunum, 20 cm distal from the duodenum; and ileum, 20 cm proximal to the cecum. As previously described, a nonenzymatic technique was used to prepare epithelial cells (40). Briefly, intestinal sections were washed with ice-cold PBS (0.01 M) and then incubated at 22°C (RT) in PBS containing 3 mM EDTA and 0.5 mM dithiothreitol for 90 min. Cells 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. Previous studies have demonstrated that this method results in the isolation of single viable intact cells free of stroma (39).
Whole cell binding assay. Intestinal cells were added to pregassed (5% CO2-95% O2) glass tubes containing [3H]B (25-30 nM) with or without nonradioactive steroid or carbenoxolone. Nonspecific binding was determined in the presence of 200-fold excess B. Tubes were then covered in parafilm and incubated at 22°C for 90 min. Following incubation, a sample of medium was ethyl acetate extracted and cells were washed with 3 ml of ice-cold DMEM. 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 and 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 & Schuell, Dassel, Germany) under vacuum. Filters were dried, DNA content was determined (6), and radioactivity was measured by liquid scintillation spectrophotometry.
For binding in jejunal and ileal subcellular fractions, nuclei were isolated as described above. The microsomal-enriched pellet and cytosol were obtained by centrifuging the supernatant from the nuclei isolation procedure at 105,000 g for 60 min at 4°C. Radioactivity was measured by liquid scintillation spectrophotometry in the cytosol and microsomal-enriched pellet that had been homogenized in 10% (vol/vol) glycerol and 50 mM Tris (pH 7.4). Protein concentration was determined (5), as was DNA content as noted above.Steroids, chromatography, and imaging. Radioactive steroids were from Amersham (Little Chalfont, UK), and nonradioactive steroids (B and 11DHB) were from Sigma (St. Louis, MO) or a gift from Roussel-UCLAF (Romainville, France; RU-28362 and RU-38486). Carbenoxolone was from Sigma. Ethyl acetate-extracted samples spiked with nonradioactive B and 11DHB 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 5 days. 3H-labeled bands were then visualized and quantified with a Fujix Bio-Imaging Analyzer (BAS1000 with Mac BAS; Fuji). [3H]B and [3H]11DHB were identified by the comigration of ultraviolet-visualized nonradioactive B and 11DHB, respectively.
Western blot analysis. Kidney homogenates were prepared from 1 g of frozen tissue by homogenization in 4 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). For Western blot analysis on subcellular fractions, nuclei were isolated as described for binding assays with the addition of 100 µM leupeptin in all buffers. The microsomal-enriched pellets and cytosols were obtained by centrifuging supernatants from the nuclear isolation procedure at 105,000 g for 60 min at 4°C. Both nuclear and microsomal-enriched pellets were resuspended in homogenizing buffer, and protein concentration was determined (5).
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 & Schuell) for 2 h on ice. After blocking nonspecific sites 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 immunopurified rabbit anti-rat 11Cofactor analysis.
To determine the cofactor dependence of the 11HSD activity, jejunum
epithelial cells were homogenized with 4 volumes of homogenizing buffer
(10 mM sodium phosphate, 0.25 M sucrose, and 1 mM phenylmethylsulfonyl fluoride; pH 7.4). After homogenization, protein concentration was
determined (5). Homogenates (500 µg) were added to tubes containing 12 nM [3H]B and either NAD (500 µM) or NADP
(500 µM) then incubated at 37°C for 2 h. This experiment was
repeated eight times. At the end of incubation, samples were ethyl
acetate extracted.
Data analysis. Data were compared by one-way ANOVA followed by Fisher's protected least significant difference test. Differences of P < 0.05 were considered significant. All data are expressed as means ± SE.
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RESULTS |
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Non-GR, non-MR binding of [3H]B in small intestinal
epithelial cells.
In the initial study, we measured specific nuclear binding in
epithelial cells isolated from duodenum, jejunum, and ileum that had
been incubated with [3H]B in the presence of RU-28362 and
aldosterone to block binding to GR and MR. As illustrated in Fig.
1A, a non-MR, non-GR nuclear localized binding site was detected, with levels being significantly (P < 0.05) higher in jejunum than in duodenum.
3H-labeled steroids were extracted from the media taken at
the end of incubation to determine whether [3H]B was
metabolized. As illustrated in Fig. 1B, ileal cells
converted 18 ± 3% of [3H]B to
[3H]11DHB, but there was very little conversion in the
duodenum (5 ± 1%) and jejunum (6 ± 1%). In addition,
there was no evidence of other metabolites other than
[3H]11DHB (Fig. 1C).
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Steroid specificity.
We have previously described a novel putative corticosteroid receptor
(DHB receptor) in rat colonic epithelial cells (31). To
assess whether the small intestinal nuclear binding site was the DHB
receptor, we determined the steroid profile of the binding site.
Jejunal epithelial cells were incubated with [3H]B
(25-30 nM) in the presence or absence of 200-fold excess of various unlabeled steroids; for cortisol-17 acid a 1,000-fold excess
of steroid was used to compete for binding. In addition, 200-fold
excess of RU-38486 and aldosterone were added to block binding to GR
and MR. Illustrated in Fig. 2 are the
compounds that significantly (P < 0.05) displaced
nuclear [3H]B binding at the single dose tested, and
listed in Table 1 are the steroids that
did not markedly compete for nuclear binding. As for the putative DHB
receptor, the compounds B, 11DHB, 11
-hydroxyprogesterone, and
11-ketoprogesterone competed for nuclear binding. In contrast with the
colonic binder, carbenoxolone, progesterone and deoxycorticosterone also displaced binding, suggesting that the small intestinal binding site is distinct from DHB receptor.
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Dose-dependent competition studies.
To further analyze the specificity of the jejunal nuclear binding site,
dose-dependent competition by unlabeled B, 11DHB, carbenoxolone,
progesterone, deoxycorticosterone, 11-ketoprogesterone, and
11-hydroxyprogesterone for the binding site were compared in jejunal
cells. Of the compounds tested, the rank order of potency was
11
-hydroxyprogesterone > B = carbenoxolone > 11DHB = 11-ketoprogesterone > progesterone = deoxycorticosterone (Fig. 3A).
In addition, media were taken at the end of incubation, and conversion
of [3H]B to [3H]11DHB was assessed. As
illustrated in Fig. 3B, the rank order of potency of the
various steroids in inhibiting 11
-dehydrogenase activity was the
same as that for competition for the small intestinal binding site.
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Scatchard analysis.
On the basis of Scatchard analysis and relative binding studies on
cytosol from various tissues, the affinity of B for rat GR is 2-5
nM and for MR is 0.5-2 nM (18, 30). To
determine whether the small intestinal binding site had a similar high
affinity for B as does rat GR and MR, we performed saturation and
Scatchard analysis on [3H]B nuclear binding in jejunal
epithelial cells in the presence of excess RU-38486 and aldosterone to
block binding to GR and MR, respectively. As illustrated in Fig.
4, [3H]B yielded a
rectilinear Scatchard plot, consistent with a single class of binding
site. The dissociation constant and maximum binding were 49 ± 7 nM and 5.0 ± 1.8 fmol/µg DNA, respectively, of four separate
determinations. These data indicate that the binding site is of
relatively low affinity compared with rat GR and MR.
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Western blot.
The steroid competition profile together with the relatively low
affinity of [3H]B for the nuclear binding site suggests
that binding may be to an 11HSD isoform. We have previously
demonstrated that 11
HSD1 mRNA is not present in intestinal
epithelial cells and that there is no 11-reductase activity
(33). 11
HSD2 mRNA is present in jejunal epithelial
cells, although Western blot analysis failed to detect a 40-kDa
11
HSD2 protein in jejunal homogenates (33) To determine
whether 11
HSD2 could be detected in nuclei from jejunal epithelial
cells, subcellular fractions were isolated and subjected to Western
blot analysis using the rat 11
HSD2 antibody. As illustrated in Fig.
5, a 40-kDa protein corresponding to
11
HSD2 was detected in microsomal-enriched pellet but not in cytosol or nuclei and, as previously demonstrated, not in whole cell
homogenates (33). In addition to 11
HSD2, two other
proteins were found, one of ~43 kDa and the other 38 kDa. The
lower-molecular-weight protein was found in whole homogenate and
cytosol; this protein has previously been described in epithelial cells
isolated from the duodenum, jejunum, and ileum but not colon
(33). The higher-molecular-weight protein was found in
both microsomal-enriched pellets and nuclei and has been previously
observed in these subcellular fractions isolated from colonic crypt
cells (33).
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Characterization of 11HSD activity.
To determine the cofactor preference of the 11
HSD activity, 500 µg
of jejunal epithelial cell protein and 12 nM [3H]B were
incubated for 2 h at 37°C in the presence or absence of NAD or
NADP. In the absence of cofactor, 2.9 ± 0.8% of B was converted
to 11DHB; in the presence of 0.5 mM NAD, conversion significantly
increased to 5.6 ± 0.7%, whereas incubation with 0.5 mM NADP did
not increase conversion (3.1 ± 0.5%). Jejunal epithelial cells
incubated with 20 nM [3H]DHB for 2 h at 37°C
failed to convert [3H]11DHB to B (data not shown),
indicating that the 11
HSD present was only an 11
-dehydrogenase.
[3H]B and [3H]dexamethasone binding in
subcellular fractions.
The identical steroid specificity for [3H]B binding and
inhibition of 11HSD activity in jejunum suggests that binding may be
to an 11
HSD isoform. To determine whether 11
HSD2 colocalized intracellularly with the non-MR, non-GR binding site, jejunal cells
were incubated with [3H]B plus 6 µM RU-38486 and
aldosterone in the presence or absence of 6 µM carbenoxolone and
binding was determined in the subcellular fractions. As illustrated in
Fig. 6, and in contrast to 11
HSD2 localization (Fig. 5), binding of [3H]B was predominately
in nuclei, with little binding detected in the microsomal-enriched
fraction and none in cytosol (data not shown). In contrast, GR bound
[3H]dexamethasone in jejunal epithelial cells and binding
was found in both the nuclear (1.09 ± 0.03 fmol/µg DNA) and
microsomal-enriched (1.03 ± 0.09 fmol/µg DNA) fractions.
[3H]dexamethasone binding in cytosol was inconsistent and
was presumably compromised by the very high nonspecific binding in this
cellular fraction.
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DISCUSSION |
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The present series of studies describe a nuclear localized
[3H]B binding site in small intestinal epithelia of
relatively low-affinity, high-capacity, and broad steroid specificity
compared with the corticosteroid binding receptors MR and GR. The
affinity profile of this binding site distinguishes it from MR, GR, and
other classic steroid receptors (estrogen receptor, progesterone
receptor, and androgen receptor). The potency of various
steroids to inhibit the very low levels of 11-dehydrogenase activity
in small intestinal epithelia strikingly mirrors the steroid
specificity of nuclear binding, suggesting that binding may be to the
substrate binding site on an 11
HSD isoform. In small intestinal
epithelial cells, 11
HSD2 but not 11
HSD1 is present
(33). The low level of 11
HSD activity that is present
is NAD dependent, is unable to catalyze the reaction in the reductive
direction, and is inhibited by the endproduct 11DHB. 11
HSD2 shares
the same enzyme characteristics, suggesting that the 11
HSD activity
in jejunal epithelial cells is probably due to the small amount of
11
HSD2 that is present.
Western blot analysis of jejunal epithelia, using the RAH23 anti-rat
11HSD2 antibody, which is directed against the last 16 amino acids
of the nonconserved COOH terminus, detects two proteins in addition to
the classic rat 40-kDa 11
HSD2, one being larger than 40 kDa and the
other smaller. The lower-molecular- weight protein is found in whole
homogenate and cytosol and has previously been described in epithelial
cells isolated from the duodenum, jejunum, and ileum but not colon
(33). The higher-molecular-weight protein is found in
microsomal-enriched pellets and nuclei and has been previously observed
in similar subcellular fractions isolated from colonic crypt cells
(33). It is unlikely that the 43-kDa protein represents
the small intestinal binding site given that it is found in colonic
epithelial cells in which this binding site is not detected. The 38-kDa
protein is not found in nuclei, suggesting that it is also not the
binding site.
The intracellular localization of 11HSD2 immunoreactive proteins
suggests that they are not the small intestinal binding site, and from
the enzyme activity data there is no evidence for other active 11
HSD
isoforms. Thus the small intestinal binding site is probably not an
active 11
HSD isoform, although we cannot rule out the possibility
that it is an inactive isoform, immunologically distinct from 11
HSD2
and 11
HSD1. In support of this possibility is the presence of an
inactive variant of 11
HSD1 in ovine liver (41) and an
inactive NH2 terminal truncated 11
HSD1 in rat kidney, which has an intact substrate binding domain (17). The
physiological role of an inactive 11
HSD isoform is moot, although
destabilization of enzyme dimerization leading to compromised 11
HSD
activity has been suggested (28).
In addition to MR and GR, binding sites that have previously been
described that share some similarity in specificity to the small
intestinal binding are the kidney type III binding site (8), extravascular corticosteroid-binding globulin (CBG)
(19), and the putative DHB receptor
(31). The small intestinal binding site is probably not
extravascular CBG given that a 1,000-fold excess of cortisol-17
acid, a synthetic steroid that binds to CBG (20), competed
minimally for binding and that CBG is not found in nuclei
(19). Although the putative DHB receptor shares a similar
steroid profile (31), it can clearly be distinguished from
the small intestinal binding site by the inability of carbenoxolone, progesterone, and deoxycorticosterone to compete for binding. Naray-Fejes-Toth et al. (25, 26) suggested
that the kidney type III site was 11
HSD2. This was based on
colocalization of binding and enzyme activity in rabbit renal cortical
principal cells, a close correlation between binding specificity and
inhibition of 11
HSD activity, and also between the activity of
11
HSD and the number of B binding sites. Similarly, in the present
study there is a good correlation between binding specificity and
inhibition of the low level of 11
-dehydrogenase activity. In
contrast, however, 11
HSD2 did not colocalize intracellularly with
binding. Furthermore, nuclear binding studies have failed to detect a
nuclear binding site in cells transfected with 11
HSD2, and in
colonic epithelial cells that express high levels of 11
HSD2 the
small intestinal binding site is not present (31).
Similarities in steroid receptor structure with a conserved DNA binding domain, flanked by a variable NH2 terminal domain and COOH terminal ligand binding domain, has allowed the cloning and identification of receptors for many nuclear hormones as well as a myriad of orphan receptors for which physiological ligands are yet to be identified. Of the many orphan receptors that have been cloned, only three appear to be steroid responsive: constitutive androstane receptor (9), pregnane X receptor (PXR) (16), and steroid and xenobiotic receptor (SXR) (4), of which PXR and SXR are activated by corticosteroids. Both the mouse PXR and human SXR regulate expression of CYP3A genes, are expressed highly where these catabolic enzymes are abundant (liver and intestine), and respond to high concentrations of a diverse group of compounds, including xenobiotics and both synthetic and endogenous steroids. These receptors differ in terms of their DNA sequence, and transfection studies show that strong activators of one receptor are typically weak activators of the other. It has been suggested that PXR and SXR control detoxification and catabolism of steroids and xenobiotics by regulating cytochrome P-450 enzymes and that they may represent the same receptor, with the difference in pharmacology reflecting differences in ingested nutrients and xenobiotics between mouse and human (4). The binding site described in the present study is similar to PXR and SXR in its broad steroid specificity, nuclear localization, and expression in the small intestine. In contrast with these receptors is the 100-fold higher affinity for binding in the small intestine (~50 nM compared with 5 µM) and the restriction of steroid selectivity to C21 steroids. The rat equivalent of either SXR or PXR has not been cloned; whether the binding site in the small intestine is related to one or both of these receptors or the rat equivalent will require the sequence to be established.
The relatively low affinity and similarity in steroid specificity of
the small intestinal binding site with 11HSD inhibition suggests
that if the small intestinal binding site is not an inactive 11
HSD
isoform then it may be related to PXR and SXR as one of a novel branch
of the nuclear receptor family, in which the receptors are of low
affinity, high capacity, and regulate metabolism of a broad spectrum of
compounds. It has been proposed that the physiological function of
these receptors may then be to provide an intracellular environment
allowing cells to respond to circulating levels of endogenous hormones
while at the same time protecting them from ligands found in
ingested material.
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ACKNOWLEDGEMENTS |
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Thanks to Rebecca Ridings for technical assistance and Professor J. W. Funder for helpful discussions.
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FOOTNOTES |
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This work was supported by a block grant to the Baker Medical Research Institute from the National Health and Medical Research Council of Australia.
Address for reprint requests and other correspondence: K. E. Sheppard, Baker Medical Research Institute, P.O. Box 6492, St. Kilda Rd. Central, Melbourne, Victoria, Australia, 8008 (E-mail: karen.sheppard{at}baker.edu.au).
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.
Received 9 November 1999; accepted in final form 29 March 2000.
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