Steroid specificity of the putative DHB receptor: evidence that the receptor is not 11beta 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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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 11beta -hydroxysteroid dehydrogenase-2 (11beta HSD2) but not 11beta HSD1 in colonic crypt cells and showed that 11beta HSD2 was present in the nuclear pellet. Differences in steroid specificity between the putative DHB receptor and inhibition of 11beta HSD activity indicate that binding is not to the enzyme. Furthermore, modified Chinese hamster ovary cells transfected with the 11beta HSD2 gene express nuclear 11beta 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 11beta HSD1 and 11beta HSD2.

11-dehydrocorticosterone; 11beta -hydroxysteroid dehydrogenase; glucocorticoid; mineralocorticoid; nuclear receptor; colon

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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 11beta -hydroxysteroid dehydrogenase (11beta HSD), two isoforms of which have been cloned (1-3). 11beta HSD type 1 (11beta 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). 11beta HSD type 2 (11beta HSD2) has nanomolar affinity for B and F and acts only as a dehydrogenase (3, 25). Given 11beta 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 11beta HSD2 (29, 33, 36, 45), and, as expected, B binding to both MR and GR in these cells is decreased by 11beta 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 11beta 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 11beta HSD inhibition, indicating that binding is not to 11beta HSD. Furthermore, we show the presence of 11beta HSD2 but not 11beta HSD1 in rat colonic crypt cells and that cells transfected with 11beta HSD2 express 11beta HSD2 in nuclei but not a nuclear DHB receptor.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 11beta HSD1A cDNA (1) and rat 11beta 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, 5alpha -dihydrotestosterone, 11-DHB, 11alpha -hydroxyprogesterone, 5alpha -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 11beta 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
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Abstract
Introduction
Methods
Results
Discussion
References

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, 5alpha -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 11beta -hydroxysteroid dehydrogenase (11beta 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 11beta 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, -).

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 11beta HSD activity (Fig. 2B).


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Fig. 2.   Competition for DHB receptor and inhibition of 11beta 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 11beta 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, -).

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.

11beta HSD expression in colonic crypt cells. To determine which isoform of 11beta 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 11beta HSD1 or rat 11beta HSD2. As illustrated in Fig. 4, when probed with anti-rat HSD1, extracts from rat 11beta 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 11beta HSD1 (21). In addition, other higher-molecular-weight proteins were detected in all tissues. Because of the limited stock of the 11beta 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 11beta HSD2. Previous studies using this antibody (36) have demonstrated that rat 11beta HSD2 migrates as a 40-kDa protein. As illustrated in Fig. 4, kidney, whole colon, and colonic crypt cell extracts contain 11beta HSD2, whereas both liver and 11beta 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 11beta HSD1 and 11beta 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 11beta HSD1 protein and 40-kDa 11beta HSD2 protein are indicated.

Previous studies have demonstrated 11beta HSD2 in nuclei of some (23, 35) but not all cells (24, 28). To determine the subcellular fraction in which 11beta HSD2 was present in rat colonic crypt and 11beta HSD2-transfected cells, nuclei, microsome-enriched pellet, and cytosol were prepared as described in METHODS. As illustrated in Fig. 5, 11beta HSD2 was present in all subcellular fractions from both colonic crypt (Fig. 5A) and 11beta HSD2-transfected cells (Fig. 5C). In addition to 11beta 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 11beta 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, 11beta 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 11beta 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 11beta 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 11beta HSD2- (C) and vector-transfected (D) CHOP cells, using same method as in A. Positions of 40-kDa 11beta HSD2 protein and an unidentified 43-kDa protein are indicated.

Inhibitors of 11beta HSD activity. To assess the steroid specificity of 11beta 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 11alpha -hydroxyprogesterone > CBX > progesterone = 11-DHB > deoxycorticosterone in terms of inhibiting 11beta 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, 11alpha -hydroxyprogesterone decreased binding to nonspecific levels, whereas CBX, deoxycorticosterone, and progesterone did not compete for binding. In parallel with the 11beta HSD inhibition dose-response study, medium taken at the end of incubation showed 11alpha -hydroxyprogesterone > CBX > progesterone = deoxycorticosterone in terms of inhibiting 11beta HSD activity. In addition, 5alpha -dihydrocorticosterone was potent both as an inhibitor of 11beta HSD activity and as a competitor for nuclear binding (Fig. 7B).


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Fig. 6.   Dose-dependent inhibition of 11beta 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. 11alpha -Hydroxyprog, 11alpha -hydroxyprogesterone; DOC, deoxycorticosterone.


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Fig. 7.   Competition for DHB receptor binding by competitive inhibitors of 11beta 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 11beta HSD activity is expressed as percentage of maximum conversion in absence of added competitor. 11alpha OHProg, 11alpha -hydroxyprogesterone; 5alpha DiHydro-B, 5alpha -dihydrocorticosterone. Data are means ± SE; n = 4-12. * Significantly different (P < 0.05) from total nuclear binding (A, -) or maximum conversion (B, -).

Transfection studies. The differences between steroid specificity of 11beta HSD inhibition and the ability to displace nuclear binding suggest that the nuclear 11-DHB binding site is not 11beta HSD. To further rule out this possibility, CHOP cells were transfected with rat 11beta 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 11beta 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 11beta HSD2 effectively inhibited binding to GR.


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Fig. 8.   3H-labeled steroid binding in vector- and 11beta HSD2-transfected CHOP cells. CHOP cells were transfected with vector (pcDNA1) or rat 11beta 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 (-).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 11beta 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 11beta HSD2-transfected cells. Collectively, these data are good evidence that the DHB receptor is a novel steroid receptor and is not 11beta HSD.

Previous studies have demonstrated 11beta HSD2 in nuclei isolated from human colonic cells (28, 35). In agreement with these studies, we can demonstrate 11beta HSD2 in nuclei isolated from rat colonic crypt cells and 11beta 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 11beta HSD2-transfected cells suggests that this protein is not a modified nuclear 11beta HSD2; however, it may reflect cell-specific modification of the enzyme. Similarly, a protein ~4 kDa greater than human 11beta 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 11beta HSD on the basis that CBX, which is a competitive inhibitor of 11beta 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 11beta HSD, we assessed the ability of several known substrates and competitive inhibitors of 11beta HSD to compete for nuclear binding. In addition, we determined whether rat 11beta 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 11beta 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 11beta HSD2-transfected cells that express the enzyme in the nucleus further supports the distinction between the putative DHB receptor and 11beta HSD2.

The major circulating glucocorticoid in humans is cortisol, and cortisone is the 11-keto metabolite produced by 11beta 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 11beta 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 11beta 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 11beta 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 11beta 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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 275(1):E124-E131
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