Baker Medical Research Institute, Melbourne, Victoria 8008, Australia
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ABSTRACT |
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To evaluate the potential roles that both
receptors and enzymes play in corticosteroid regulation of intestinal
function, we have determined glucocorticoid receptor (GR),
mineralocorticoid receptor (MR), and 11-hydroxysteroid dehydrogenase
(11
-HSD) expression in intestinal epithelial cells. GR and MR mRNA
and receptor binding were ubiquitously expressed in epithelial cells, with receptor levels higher in ileum and colon than jejunum and duodenum. RNase protection analysis showed that 11
-HSD1 was not expressed in intestinal epithelial cells, and enzyme activity studies
detected no 11-reductase activity. 11
-HSD2 mRNA and protein were
demonstrated in ileal and colonic epithelia; both MR and GR binding
increased when enzyme activity was inhibited with carbenoxolone. Duodenal and jejunal epithelial cells showed very little 11
-HSD2 mRNA and undetectable 11
-HSD2 protein; despite minor (<7%)
dehydrogenase activity in these cells, enzyme activity did not alter
binding of corticosterone to either MR or GR. These findings
demonstrate the ubiquitous but differential expression of MR and GR in
intestinal epithelia and that 11
-HSD2 modulates corticosteroid
binding to both MR and GR in ileum and proximal and distal colon but
not in duodenum or jejunum.
glucocorticoid receptor; mineralocorticoid receptor
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INTRODUCTION |
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GLUCOCORTICOID RECEPTORS (GR) and mineralocorticoid
receptors (MR) bind endogenous glucocorticoids with high affinity.
Intracellular availability of corticosterone (B) and cortisol (F) for
both GR and MR can be modulated by 11-hydroxysteroid dehydrogenase 1 (11
-HSD1) and 11
-hydroxysteroid dehydrogenase 2 (11
-HSD2). In
mineralocorticoid target cells, aldosterone specificity is conferred on
the nonselective MR by 11
-HSD2, which converts B and F to their
inactive 11-keto metabolites, 11-dehydrocorticosterone (11-DHB) and
cortisone (7, 9). In addition, 11
-HSD2 inhibits B binding to GR (20,
23). In tissues that express MR but not 11
-HSD2, MR bind endogenous
glucocorticoids and mediate glucocorticoid effects (5). When 11
-HSD2
activity is deficient (25, 29), mineralocorticoid activity is elevated,
presumably a result of endogenous glucocorticoids binding and
activating MR in mineralocorticoid target tissues.
Although 11-HSD2 acts as a dehydrogenase only for endogenous
glucocorticoids, 11
-HSD1 catalyzes the reversible conversion of B to
11-DHB. Inhibition of 11
-HSD1 in
GH3 cells has been shown to
potentiate the activity of B mediated via GR (26, 32), 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 (13, 16, 28). In addition
to 11
-HSD1 and 11
-HSD2, several other 11
-HSD isoforms have
been described but not cloned. They can be distinguished from
11
-HSD1 and 11
-HSD2 based on cofactor preference, affinity for
substrate, and whether they act as an oxidase, a reductase, or both
(10-12). The role these enzymes play in modulating corticosteroid
access to GR and MR is yet to be defined, although in rat Leydig cells
a novel 11
-HSD isoform appears to modulate the GR response (10).
In the present study we have evaluated GR, MR, and 11-HSD expression
and activity in duodenal, jejunal, ileal, and colonic epithelial cells
to define the role that these receptors may play in intestinal function
and that 11
-HSD isoforms may play in enhancing or limiting
glucocorticoid effects on rat small intestine.
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MATERIALS AND METHODS |
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Animals, intestinal dissection, and epithelial cell isolation. Adult male Sprague-Dawley rats weighing 180-220 g were used in all experiments. Animals were killed by decapitation, and intestinal sections were rapidly removed as follows: duodenum, 5 cm distal to the pylorus; jejunum, 20 cm distal from the duodenum; ileum, 20 cm proximal to the cecum; proximal colon, half the colon distal to the cecum; and distal colon, half the colon proximal to the rectum. Cells were prepared from intestinal sections by a nonenzymatic technique (31). Briefly, intestinal sections were washed with ice-cold PBS and then incubated at 22°C (room temperature) 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 (DMEM-HEPES). Previous studies have demonstrated that this method results in the isolation of viable intact cells essentially free of stroma (30).
RNA isolation and recombinant clones.
Total RNA was prepared from intestinal cells and tissues by the
guanidinium isothiocyanate method as previously described (4), and RNA
concentration was determined by absorbance at 260 nm. All RNA probes
were synthesized with the Promega transcription system (Promega) to a
specific activity of ~0.3 × 109 cpm/µg. The cDNA
templates used to generate
32P-labeled riboprobes were as
follows: for GR, a 294-bp Xba
I-BamH I rat GR cDNA fragment
corresponding to the 3' untranslated region; for MR, a 188-bp
EcoR
I-Stu I rat MR cDNA fragment
corresponding to nucleotides 3130-3318; for 11-HSD1, a 466-bp
EcoR
I-EcoR V fragment corresponding to
nucleotides 1-466; and for 11
-HSD2, a 628-bp fragment
corresponding to nucleotides 924-1552.
Solution hybridization/RNase protection assay.
A solution hybridization/RNase protection assay was used to quantify
mRNA levels of MR, GR, and 11-HSD enzymes. Total RNA (10-20
µg) was speed-vac dried and reconstituted in 25 µl of hybridization buffer (80% formamide, 40 mM PIPES, pH 6.7, 0.4 mM NaCl, and 1 mM
EDTA) containing 5 µl of
32P-labeled antisense RNA probe.
Samples were hybridized overnight at 45°C, followed by digestion
for 45 min at 37°C with 300 µl RNase buffer (300 mM NaCl, 10 mM
Tris, pH 7.5, and 5 mM EDTA) containing RNase T1 (400 units) for MR,
GR, and 11
-HSD2 mRNA measurements and both RNase A (40 µg/ml) and
RNase T1 (400 units) for 11
-HSD1. Protected hybrids were then
purified by proteinase K (10 mg/ml) digestion (37°C for 15 min) in
the presence of 1% SDS, followed by isopropanol precipitation.
Protected hybrids were reconstituted in diethyl pyrocarbonate-treated
H2O and separated on 5%
nondenaturing polyacrylamide gels.
32P-labeled hybrids were
visualized and quantified on a Fujix Bio-Imaging Analyzer (BAS1000 with
Mac BAS; Fuji). Because GR and MR riboprobes were uniformly labeled
with [32P]UTP but
differed in the number of bases protected (294 for GR and 188 for MR),
specific activity in molar terms (cpm/mol of riboprobe) differed
between GR and MR. To allow assessment of relative expression of MR and
GR mRNA, protected hybrids were corrected for specific activity
(cpm/mol).
Cytosol steroid receptor binding assay. Cells resuspended in TMD buffer (10 mM Tris, 100 mM NaMoO4, and 1 mM dithiothreitol, pH 7.4) were homogenized by hand (glass-Teflon, 4°C), and the homogenate was centrifuged (105,000 g for 40 min at 4°C) to yield cytosol. Cytosols (250 µl) were incubated (22°C for 90 min) with 150 µl of TMD buffer containing 25-30 nM of [3H]B with or without 6 µM RU-38486 or 6 µM aldosterone. Bound and free steroid were separated on dextran-coated charcoal as previously described (21). A sample of the cytosol was taken for protein determination by the Bradford method (2). Binding displaced by 6 µM RU-38486 was taken as specific GR binding, and binding displaced by aldosterone in the presence of RU-38486 was taken as specific MR binding.
Steroids and thin layer chromatography. Radioactive steroids were purchased from Amersham (Buckinghamshire, UK) and nonradioactive steroids (B and 11-DHB) from Sigma (St. Louis, MO), or were a gift from Roussel-Uclaf, Romainville, France (RU-38486). 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 separated radioactive steroids were exposed to a BAS-TR2040S imaging screen (Fuji) for up to 5 days. 3H-labeled steroids were then visualized and quantified by phosphorimage analysis. [3H]B and [3H]11-DHB were identified by the comigration of UV-visualized nonradioactive B and 11-DHB.
11-HSD activity.
Cells were added to pregassed (5%
CO2- 95%
O2) tubes containing
[3H]B (25 nM) or
[3H]11-DHB (20 nM) in
DMEM-HEPES. After 90-min incubation at 22°C, medium was removed,
and steroids were extracted with ethyl acetate and
separated by TLC. Chopped rat liver (~0.5-cm cube) was used as a
positive control for 11-reductase activity.
Whole cell steroid receptor binding assay. Intestinal cells were added to pregassed (5% CO2-95% O2) glass tubes containing 30-35 nM [3H]B with or without 6 µM nonradioactive RU-38486, aldosterone, or CBX. Tubes were then covered in Parafilm and incubated at 22°C for 90 min. After incubation, a sample of medium was extracted with ethyl acetate, 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). Washed cells were resuspended in TMD buffer and homogenized, and the homogenate was centrifuged (105,000 g for 60 min at 4°C) to yield cytosol. Protein-bound steroid was then separated from free steroid with dextran-coated charcoal (21), and a sample of cytosol was taken for protein determination (2). Binding displaced by 6 µM RU-38486 was taken as specific GR binding, and binding displaced by aldosterone in the presence of RU-38486 was taken as specific MR binding.
Western blot analysis.
Total tissue homogenates were prepared from 1 g of frozen rat tissue or
cells by homogenization in 4 vol of homogenizing buffer (0.25 M
sucrose, 10 mM sodium phosphate, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4). Protein concentration was determined by the Bradford
method (2). Proteins (50-100 µg) were separated by 5-15%
SDS-PAGE gradient gel electrophoresis under reducing conditions and
then were transferred to nitrocellulose filters (Schleicher & Schuell)
for 2 h at 4°C. 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
immunopurified rabbit anti-rat 11-HSD2 (RAH23) polyclonal antibody
(24) 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 with a
chemiluminescence kit (DuPont NEN, Boston, MA).
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.
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RESULTS |
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Expression of MR and GR mRNA in intestinal cells.
MR and GR mRNA were measured in epithelial cells isolated from the
duodenum, jejunum, ileum, proximal colon, and distal colon. Kidney RNA
was used as a positive control for both GR and MR mRNA, and yeast total
RNA was used as a negative control. Figure
1A shows
a typical phosphorimage after simultaneous solution hydridization/RNase protection analysis of GR and MR mRNA. GR mRNA expression was ubiquitous with no significant differences in levels of expression between different segments of intestine (Fig.
1B). Similarly, MR mRNA was
expressed in all intestinal segments but with significantly (P < 0.05) greater levels in cells
from ileum and colon than jejunum and duodenum; levels in colon
epithelial cells in turn were significantly higher than those in ileal
cells (Fig. 1C). In Fig. 1,
B and
C, the relative expression of GR and
MR was assessed by correcting values for riboprobe-specific activity
(cpm/mol). As illustrated in Fig. 1D,
MR mRNA expression was higher than GR mRNA in epithelial cells isolated
from all segments of the intestine.
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GR and MR binding in cytosol.
To confirm the presence of GR and MR protein in intestinal epithelial
cells, we performed cytosol binding assays on epithelial cell extracts.
As shown in Fig. 2, specific binding of
[3H]B to GR and MR
could be measured in epithelial cells from the different intestinal
segments. [3H]B
binding to GR was significantly greater in ileum and proximal colon
than in the other intestinal segments, reflecting the trend in GR mRNA
levels. MR binding correlates directly with the MR mRNA concentrations,
with MR binding significantly greater in ileal and colonic epithelial
cells than in cells from jejunum and duodenum. MR binding in colonic
cells was similarly significantly greater than in ileal cells. In
contrast to the MR-to-GR mRNA ratio, the MR-to-GR binding ratio was
<1 for all intestinal segments.
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Expression of 11-HSD in intestinal cells.
To determine whether 11
-HSD was expressed in intestinal epithelial
cells we measured both 11
-HSD1 and 11
-HSD2 mRNA in cells isolated
from the various intestinal segments. Figure
3A shows a
phosphorimage after solution hydridization/RNase protection analysis of
rat 11
-HSD1 mRNA. A faint band running at a higher position than
11
-HSD1 was seen in all samples, including the negative control
(yeast total RNA) and probably represents a small quantity of
undigested single-stranded riboprobe. There was no evidence for
11
-HSD1 mRNA expression in any of the gut epithelial cells. In
contrast, liver and the medulla/cortex of the kidney showed high levels
of expression, indicating that 11
-HSD1 mRNA could be detected in
tissue known to express this isoform. As previously shown (15),
multiple mRNA species are detected in kidney; the two major protected
bands in kidney cortex/medulla correspond to the predicted full-length
11
-HSD1 and the smaller band to a truncated nonfunctional form of
the enzyme. In renal papilla the major mRNA species is clearly smaller
than full-length 11
-HSD1.
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GR and MR binding in whole cells.
Because 11-HSD activity is absent from cytosol extracts (20), we
performed binding assays on whole cells in the presence or absence of
CBX to determine whether 11
-HSD regulates B access to GR and/or MR.
In accordance with the low levels of 11
-HSD activity in duodenum and
jejunum, [3H]B binding
to MR and GR was unaltered in the presence of CBX (data not shown).
[3H]B binding to GR
doubled in ileum, proximal colon, and distal colon when 11
-HSD was
inhibited. [3H]B
binding to MR in the presence of CBX increased approximately threefold
in ileum and distal colon and approximately fourfold in proximal colon
(Fig. 6). In media taken at the end of
incubation, 44% of 30 nM of
[3H]B was converted to
[3H]11-DHB in ileum
and 85% of 35 nM
[3H]B in proximal and
distal colon.
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DISCUSSION |
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11-HSD isoforms are known to regulate the response to endogenous
glucocorticoids by modulating the intracellular concentrations of B and
F. 11
-HSD2 acts as only a dehydrogenase for B and F; given its
colocalization with MR in sodium-transporting epithelia and the
increase in sodium retention when enzyme activity is compromised (27,
29), 11
-HSD2 is thought to confer aldosterone specificity on MR (7,
9, 17). 11
-HSD1, on the other hand, can act as both a dehydrogenase
and reductase and has been suggested as possibly modulating
glucocorticoid responses by either increasing or decreasing local
tissue concentrations of endogenous glucocorticoids (13, 14, 16, 28).
In the present study, we examined whether MR, GR, and 11
-HSD
isoforms were present in intestinal epithelial cells and whether
11
-HSD isoforms modulated endogenous glucocorticoid access to these receptors.
In duodenum and jejunum there is good evidence that glucocorticoids
regulate several aspects of electrolyte transport, whereas mineralocorticoids have minor or no effects (19). In agreement with
these functional studies is the relatively high expression of GR and
low expression of MR in duodenal and jejunal epithelial cells. The
cellular response to endogenous corticosteroids is dependent on many
factors, two of which are receptor concentration (1) and the presence
of 11-HSD (6, 8). The low level of MR in jejunal and duodenal cells
suggests that these receptors may not be capable of inducing a major
response. Furthermore, the minimal 11
-HSD2 activity plus the
observation that B access to MR or GR was not altered by CBX suggests
that, in vivo, B rather than aldosterone binds MR. Thus the MR in
duodenum and jejunum may resemble hippocampal MR that bind B in vivo
(22) and thus mediate physiological glucocorticoid actions (5).
Significant expression of MR, GR, and 11-HSD2 is present in
epithelial cells from ileum, proximal colon, and distal colon. When
11
-HSD activity is inhibited, an increase in B binding to both MR
and GR is observed. These data suggest that, in vivo, MR in these
intestinal segments bind aldosterone and that B binding to GR is also
modulated by enzyme activity. This is supported by functional studies
in which both mineralocorticoids and glucocorticoids have been shown to
regulate electrolyte transport in both ileum and colon (19). When
11
-HSD activity was inhibited in ileum and colon, B binding to MR
increased three- to fourfold, whereas, in contrast, binding to GR only
doubled, despite a threefold increase in
[3H]B in media (~10
nM to 30 nM) for ileal cells and a sevenfold increase in
[3H]B in media (~5
nM to 35 nM) for proximal and distal colonic cells. Given that MR in
the intestine and elsewhere have substantially higher affinity for B
than do GR, the consistently enhanced occupancy of MR compared with GR
when 11
-HSD2 is inhibited cannot be explained simply on the basis of
increased [3H]B
concentrations. A possible interpretation of these data is that
11
-HSD is in much closer association with MR than with GR. Whether
this is because of some epithelial cells expressing GR only or to an
intimate intracellular association of 11
-HSD2 with MR but not GR, so
that the local microconcentration of
[3H]B is lower for MR
than for GR, requires further investigation.
In all intestinal segments expression of MR mRNA is greater than that
of GR mRNA; however, GR binding was greater than MR binding. The
discrepancy between GR and MR mRNA and binding levels suggests that RNA
stability, protein stability, and/or translational efficiency may
differ in intestinal cells. Recently, a splice variant of MR mRNA was
described in rat colon that resulted in truncation of the
steroid-binding domain (33). Although receptor binding was not
performed on this truncated MR, aldosterone and B failed to increase
transactivation of a luciferase reporter system, suggesting that
neither steroid binds to the truncated MR. The riboprobe used in the
present study would measure both of these MR mRNA species, so that an
explanation for the discrepancy in the MR-to-GR mRNA and binding ratios
may reflect expression of truncated MR in intestinal epithelial cells.
Two GR isoforms have also been described, GR- and a nonhormone
binding form, GR-
. The GR binding assay only measures GR-
,
whereas the GR riboprobe used in the RNase protection assay is
complementary to a common region of both GR isoforms and thus would
detect both GR mRNA species. Previous studies have shown that GR-
is
expressed predominantly in epithelial cells (18), suggesting that this isoform may be present in intestinal epithelial cells. Although the
presence of the GR-
would add to the discrepancy between receptor
mRNA and binding levels, it may explain the discrepancy between the
relatively uniform expression of GR mRNA levels and the variation
between tissues in GR binding.
In addition to 11-HSD1 and 11
-HSD2, other 11
-HSD isoforms have
been described but not cloned. They can be distinguished from
11
-HSD1 and 11
-HSD2 based on cofactor preference, affinity for
substrate, and whether they act as an oxidase, a reductase, or both (3,
10-12). Western blot analysis with an antibody (RAH23) directed
against the last 16 amino acids in the nonconserved COOH terminus of
the cloned rat 11
-HSD2 protein (24) detected the 40-kDa 11
-HSD2
protein in ileum, colon, and kidney. In addition, this antibody
detected a protein of ~38 kDa in small intestinal epithelia but not
colonic epithelia or kidney. The insignificant amount of
11
-dehydrogenase activity and the absence of 11-reductase activity
in duodenal and jejunal epithelia argue against the presence of a novel
11
-HSD in these cells. If the immunoreactive 38-kDa protein is a
novel 11
-HSD then it is either nonfunctional or inactive under our
experimental conditions. Previous studies with the 11
-HSD2 antibody
RAH23 in Western blot analysis of various rat tissues have shown
multiple bands at 30 kDa, which correlated with levels of the 40-kDa
11
-HSD2 protein and therefore probably represent
NH2-terminal degradation of
11
-HSD2 (24). In the present study the 38-kDa protein was present in
the absence of detectable 11
-HSD2 and was consistently seen, so that
it is unlikely to be a degradation product of the enzyme.
In summary, we have demonstrated the ubiquitous but differential
expression of GR and MR in epithelial cells along the intestinal tract.
In duodenum and jejunum epithelia there is very little 11-dehydrogenase and no 11-reductase activity, suggesting that 11
-HSD isoforms do not modulate corticosteroid responsiveness in
these cells. In addition, MR expression is low, supporting functional
studies in which mineralocorticoids fail to regulate water and
electrolyte transport in these intestinal segments (19). In contrast,
ileum and colon epithelial cells express high levels of
11
-dehydrogenase activity that limits intracellular B availability to both GR and MR and would confer aldosterone specificity on MR in vivo.
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ACKNOWLEDGEMENTS |
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We thank Rebecca Ridings for technical assistance and Professor John Funder for constructive criticism.
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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.
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 and other correspondence: K. E. Sheppard, Baker Medical Research Institute, PO Box 6492, Melbourne, Victoria 8008, Australia (E-mail: karen.sheppard{at}baker.edu.au).
Received 8 March 1999; accepted in final form 14 May 1999.
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