Novel nuclear corticosteroid binding in rat small intestinal epithelia

Karen E. Sheppard1, Susanne Hourigan2, Kevin X. Z. Li1, and Zygmunt S. Krozowski1

1 Baker Medical Research Institute and the 2 Department of Pathology and Immunology, Monash University Medical School, Prahran, Victoria, Australia 3181


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 11beta -dehydrogenase activity, suggesting that binding may be to an 11beta -hydroxysteroid dehydrogenase (11beta HSD) isoform, although 11beta HSD1 is not present in small intestinal epithelia and 11beta 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 11beta HSD2 but is distinguishable from the latter by its distinct intracellular localization.

glucocorticoid receptor; mineralocorticoid receptor; 11beta -hydroxysteroid dehydrogenase; corticosterone


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

11beta -HYDROXYSTEROID DEHYDROGENASES (11beta 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, 11beta 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 11beta HSD isoforms have been cloned, 11beta HSD1 and 11beta HSD2 (1-3). 11beta 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, 11beta HSD1 catalyzes the reversible conversion of B to 11DHB (23), although in vivo 11beta 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 11beta HSD1 and 11beta HSD2, there is preliminary evidence for several other 11beta 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 11beta HSD2, 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 11beta HSD2 (31). The steroid specificity of this putative receptor distinguishes it from other steroid receptors and from 11beta HSD isoforms. We proposed that this putative receptor mediates glucocorticoid effects in cells expressing high levels of 11beta HSD2 activity. During the course of determining whether 11beta 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 11beta HSD2 and no 11beta 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 11beta HSD2 but is distinguishable from the latter by its distinct tissue and intracellular localization.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 11beta HSD2 (RAH23) polyclonal antibody (35), at a concentration of 1 µg/ml in the presence of 0.5% skim milk powder in PBS-T. The RAH23 antibody was raised against a 16-amino acid synthetic peptide corresponding to the last 16 amino acids of the nonconserved COOH terminus of the cloned rat 11beta HSD2. The filter was washed with PBS-T and then incubated at RT for 60 min with 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 (NEN, Boston, MA).

Cofactor analysis. To determine the cofactor dependence of the 11beta HSD 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Non-glucocorticoid receptor (GR), non-mineralocorticoid receptor (MR) binding of [3H]corticosterone (B) in small intestinal epithelial cells. Cells were incubated with [3H]B (25-30 nM) in the presence of 6 µM RU-28362 and 6 µM aldosterone to block binding to GR and MR. Nonspecific binding was determined in the presence of 6 µM unlabeled B. Following the incubation, nuclear 3H-labeled steroid binding was determined (A) and medium was extracted to assess conversion of [3H]B to [3H]11-dehydrocorticosterone (11DHB) (B). C: typical phosphorimage of the TLC profile of 3H-labeled steroids extracted from media at the end of incubation. Data are expressed as means ± SE; n = 6-12.

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-17beta 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, 11alpha -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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Fig. 2.   Competition for the non-GR, non-MR nuclear binding site. Jejunal epithelial cells were incubated with [3H]B (25-30 nM) and 6 µM of both aldosterone and RU-38486 to block binding to MR and GR with or without 6 µM competitor. Following the incubation, nuclear 3H-labeled steroid binding was determined. Binding data are expressed as a percent of total nuclear binding (means ± SE; n = 4-12). * P < 0.05 vs. total nuclear binding. CBX, carbenoxolone; DOC, deoxycorticosterone.


                              
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Table 1.   Steroids that did not markedly compete for the non-glucocorticoid receptor, non-mineralocorticoid receptor nuclear [3H]corticosterone binding site in jejunal epithelial cells

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 11alpha -hydroxyprogesterone for the binding site were compared in jejunal cells. Of the compounds tested, the rank order of potency was 11alpha -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 11beta -dehydrogenase activity was the same as that for competition for the small intestinal binding site.


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Fig. 3.   Competition for the small intestinal binding site (A) and inhibition of 11beta HSD (B). Jejunal epithelial cells were incubated with [3H]B (25-30 nM), with or without competitor, plus 6 µM of aldosterone and RU-38486 to block binding to MR and GR. Following the incubation, nuclear 3H-labeled steroid binding was determined and medium was extracted to assess conversion of [3H]B to [3H]11DHB. Binding data are expressed as a percent of total nuclear binding (fmol/µg DNA), and 11beta HSD activity is expressed as a percent of maximum conversion in the absence of added competitor. Data are expressed as means ± SE; n = 3-8.

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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Fig. 4.   Saturation and Scatchard analysis of [3H]B binding to the non-MR, non-GR nuclear binding site. Jejunal epithelial cells were incubated with [3H]B (7-300 nM) in the presence of 200-fold excess of RU-38486 and aldosterone. Nonspecific binding was determined in the presence of 200-fold excess unlabeled B. Shown is a typical saturation curve and Scatchard plot. The binding site concentration and the dissociation constant were 5 ± 1.8 fmol/µg DNA and 49 ± 7 nM, respectively, for 4 separate determinations.

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 11beta HSD isoform. We have previously demonstrated that 11beta HSD1 mRNA is not present in intestinal epithelial cells and that there is no 11-reductase activity (33). 11beta HSD2 mRNA is present in jejunal epithelial cells, although Western blot analysis failed to detect a 40-kDa 11beta HSD2 protein in jejunal homogenates (33) To determine whether 11beta HSD2 could be detected in nuclei from jejunal epithelial cells, subcellular fractions were isolated and subjected to Western blot analysis using the rat 11beta HSD2 antibody. As illustrated in Fig. 5, a 40-kDa protein corresponding to 11beta 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 11beta 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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Fig. 5.   Western blot analysis of relative levels of rat 11beta HSD2 in subcellular fractions of jejunal epithelial cells. Nuclei (N) were isolated from jejunal cell homogenate (H) on a discontinuous sucrose gradient as described for the binding assay. The microsome-enriched (M) pellet and cytosol (C) fractions were obtained by centrifuging the supernatant from the nuclei isolation procedure at 105,000 g for 60 min at 4°C. For jejunal fractions, 100 µg of protein was used; for kidney homogenate, 50 µg was used. The positions of the 40-kDa 11beta HSD2 protein and unidentified 38-kDa and 43-kDa proteins are indicated.

Characterization of 11beta HSD activity. To determine the cofactor preference of the 11beta 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 11beta HSD present was only an 11beta -dehydrogenase.

[3H]B and [3H]dexamethasone binding in subcellular fractions. The identical steroid specificity for [3H]B binding and inhibition of 11beta HSD activity in jejunum suggests that binding may be to an 11beta HSD isoform. To determine whether 11beta 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 11beta 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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Fig. 6.   Specific binding of [3H]B to the small intestinal binding site in nuclear and microsomal pellets. Epithelial cells were incubated with [3H]B, and binding to MR and GR was blocked by excess aldosterone and RU-38486. Nonspecific binding was determined in the presence of 6 µM carbenoxolone. Following the incubation, nuclear and microsomal pellets were isolated from jejunal and ileal cells, as described in the MATERIALS AND METHODS. Data are means ± SE; n = 3


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 11beta -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 11beta HSD isoform. In small intestinal epithelial cells, 11beta HSD2 but not 11beta HSD1 is present (33). The low level of 11beta 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. 11beta HSD2 shares the same enzyme characteristics, suggesting that the 11beta HSD activity in jejunal epithelial cells is probably due to the small amount of 11beta HSD2 that is present.

Western blot analysis of jejunal epithelia, using the RAH23 anti-rat 11beta HSD2 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 11beta 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 11beta HSD2 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 11beta HSD isoforms. Thus the small intestinal binding site is probably not an active 11beta HSD isoform, although we cannot rule out the possibility that it is an inactive isoform, immunologically distinct from 11beta HSD2 and 11beta HSD1. In support of this possibility is the presence of an inactive variant of 11beta HSD1 in ovine liver (41) and an inactive NH2 terminal truncated 11beta HSD1 in rat kidney, which has an intact substrate binding domain (17). The physiological role of an inactive 11beta HSD isoform is moot, although destabilization of enzyme dimerization leading to compromised 11beta 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-17beta 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 11beta 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 11beta HSD activity, and also between the activity of 11beta 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 11beta -dehydrogenase activity. In contrast, however, 11beta HSD2 did not colocalize intracellularly with binding. Furthermore, nuclear binding studies have failed to detect a nuclear binding site in cells transfected with 11beta HSD2, and in colonic epithelial cells that express high levels of 11beta 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 11beta HSD inhibition suggests that if the small intestinal binding site is not an inactive 11beta 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.


    ACKNOWLEDGEMENTS

Thanks to Rebecca Ridings for technical assistance and Professor J. W. Funder for helpful discussions.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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