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Laboratory of Molecular Hypertension, Baker Institute of Medical Research, Prahran, Victoria 3181, Australia
Address all correspondence and requests for reprints to: Paolo Ferrari, M.D., Laboratory of Molecular Hypertension, Baker Institute of Medical Research, Prahran, Victoria 3181, Australia.
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Abstract |
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In this study we compared both dehydrogenase and reductase characteristics of the cloned rat 11ßHSD1 and rat and human 11ßHSD2 for three different 11-hydroxysteroid substrates, cortisol (F), corticosterone (B), and dexamethasone (Dex), and the corresponding 11-keto metabolites, cortisone (E), 11-dehydrocorticosterone (A), and 11-dehydrodexamethasone (DH-Dex), respectively.
In cell homogenates expressing either the rat or the human 11ßHSD2, the relative potency for the dehydrogenase reaction was B > F > Dex. Although there was no reduction of A or E, DH-Dex was readily converted to Dex with an equilibrium far on the side of the 11-hydroxy metabolite. DH-Dex reduction in homogenates was inhibited by both glycyrrhetinic acid and carbenoxolone, with a 50% inhibition at 80 and 100 nM, respectively. In intact cells transfected with rat 11ßHSD1, the equilibrium was on the reductase side for all substrates. Dehydrogenation of B or F was more potent with rat 11ßHSD2 than with rat 11ßHSD1. There was no detectable 11ßHSD1 oxidation of Dex.
These data indicate that both the cloned human and rat 11ßHSD2 reduce DH-Dex and do this more readily than they oxidize Dex. Thus, 11ßHSD2 seems also to be a bidirectional enzyme, although no reduction of the physiological compounds A and E was observed.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When 11ßHSD2 activity is compromised, as occurs in licorice intoxication or in the congenital syndrome of apparent mineralocorticoid excess (AME), sodium retention, hypokalemia, and hypertension ensue, suggesting that F acts as a mineralocorticoid in these circumstances (17, 18). Recent studies have unequivocally established a link between 11ßHSD2 and AME by the demonstration of mutations in the HSD11B2 gene of AME patients (19, 20, 21, 22). Based on these observations, 11ßHSD2 is the most likely candidate to confer aldosterone selectivity on the mineralocorticoid receptor, whereas the role of 11ßHSD1 may include modulation of glucocorticoid bioavailability in glucocorticoid target tissues. High levels of 11ßHSD2 are also present in the human placenta (16), where it is thought to protect the fetus from high levels of maternal glucocorticoids, and there appears to be a correlation among placental enzyme activity, birth weight, and the development of hypertension latter in life (23, 24, 25).
The kinetics of 11ßHSD have been investigated largely in tissue homogenates of both rat liver, an organ in which message for 11ßHSD2 is undetectable (6, 9) and rat kidney, where both forms of 11ßHSD are present (11, 26). We recently established kinetics, concentration dependence, and saturability, as well as inhibition by other steroids, and described dexamethasone (Dex) dehydrogenation by the cloned human 11ßHSD2 (27). Oelkers et al. and Diedrich et al. recently reported oxoreduction of 11-dehydrodexamethasone (DH-Dex) by human kidney slices and human kidney microsomal preparations (28, 29). It is, however, unclear, whether DH-Dex reduction is catalyzed by 11ßHSD1 or 11ßHSD2 or by another, as yet unidentified 11ßHSD isoform. Thus, in the present study we used complementary DNAs coding for the rat 11ßHSD1, rat 11ßHSD2, and human 11ßHSD2 as well as mutated 11ßHSD2 plasmids derived from patients with AME to investigate DH-Dex oxidoreductase activity of the cloned 11ßHSD enzymes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture and preparation of homogenates
Modified Chinese hamster ovary cells (CHOP-C4) cells (30) were
cultured and transfected as previously reported (27). Experiments in
intact cells were performed 48 h after transfection in 35-mm
wells. CHOP cell homogenates were obtained 60 h after transfection
of cells. Confluent cells from 100-mm plates were homogenized and
stored at -70 C until use (27).
Analysis of enzyme activity in intact cells and homogenates
The 11ßHSD activity of rat or human 11ßHSD2 clones or that
of the rat 11ßHSD1 clone was determined by measuring the conversion
of radiolabeled 11-hydroxysteroid substrates F, B, and Dex to their
corresponding 11-keto metabolites E, A, and DH-Dex, respectively.
Similarly, 11ßHSD reductase activity was assessed by measuring the
conversion of radiolabeled 11-keto substrates to their respective
11-hydroxy metabolites.
Dehydrogenase and oxoreductase activities for F/E, B/A, and Dex/DH-Dex
were determined in intact cells after transfection of CHOP cells with 1
µg rat 11ßHSD2 or rat 11ßHSD1 plasmids. After 48 h, 2–5
nM of the corresponding 3H-labeled substrate
was incubated for 30–60 min with the cells in serum-free medium.
Culture medium was then extracted in 3 vol ethyl acetate. Steroid
metabolites were assayed by TLC as described below. Determination of
rat 11ßHSD2 enzyme kinetics for Dex was performed by incubating
transfected CHOP cells with substrate concentrations of 2
nM [3H]Dex and 400–12,800 nM
unlabeled Dex for 60 min. Determination of rat 11ßHSD2 or rat
11ßHSD1 kinetics for DH-Dex was performed by incubating transfected
CHOP cells with substrate concentrations of 2 nM
[3H]DH-Dex and 400–25,600 nM unlabeled
DH-Dex for 60 min. F dehydrogenation and DH-Dex oxoreduction were
measured in CHOP cells after transfection with 1 µg wild-type human
11ßHSD2 or the mutated AME plasmids R186C, L250P.L251S, R337C,
E356
-1nt, and R374X (31, 32). Cells transfected with mutated AME
plasmids were incubated with the substrate for 180 min.
To determine the amount of protein per well, cells from each dish were
removed and homogenized with homogenizing buffer, and the amount of
protein was determined colorimetrically by the Bradford method (33)
using Bio-Rad protein dye and calibration against standards of
-globulin. The amounts of product generated per dish and per min
were standardized for the amount of protein of the corresponding
dish.
11ßHSD2 activity in homogenates was determined by incubating 100 µg/ml CHOP cell homogenates at 37 C in a total volume of 500 µl assay buffer (0.25 M sucrose and 10 mM sodium phosphate, pH 7.4) containing 2 nM tritiated steroid and 500 µM cofactor (NAD or NADP for the dehydrogenase and NADH or NADPH for the reductase reaction). The reaction was terminated by the addition of 3 vol ethyl acetate. The inhibition of 11ßHSD2 reductase activity for DH-Dex by carbenoxolone (CBX) or glycyrrhetinic acid (GA) was assayed by incubating 50 µg transfected CHOP cell homogenates in 500 µl assay buffer at 37 C for 60 min with 2 nM [3H]DH-Dex in the presence of inhibitor concentrations between 10 nM and 10 µM. In all reactions, metabolism of substrate was always less than 25%.
For whole cell assays replicates were performed on separate plates, whereas assays on homogenates were conducted by homogenizing cells from one or more plates and performing the reaction in replicate tubes.
Extraction and quantitation of steroids
Ethyl acetate was used to extract steroids from culture medium
or assay buffer. The ethyl acetate was then transferred into a fresh
tube and evaporated under air. The dried steroids were resuspended in
100 µl ethanol containing unlabeled standards for visualization of
steroid migration under UV light. Separation of substrates and
metabolites was performed by TLC on plastic-backed silica gel plates
(Merck, Darmstadt, Germany). Redissolved steroids (10 µl) were
applied to the TLC plate, allowed to dry completely, and placed in an
equilibrated TLC tank with chloroform-ethanol (92:8) as solvent. Areas
corresponding to steroids were identified under UV light, cut out,
transferred to vials containing liquid scintillant, and counted in a
ß-counter.
Western blot analysis
Total proteins (100 µg) from CHOP cells transfected with rat
11ßHSD2 were separated by 5–15% gradient SDS-PAGE under reducing
conditions and transferred to nitrocellulose filters (Schleicher and
Schuell, Dassel, Germany) for 2 h on ice. After blocking
nonspecific sites on nitrocellulose, the blot was incubated overnight
at 4 C with 1 µg/ml immunopurified RAH23 polyclonal antibody (34)
diluted with 0.5% skim milk powder in PBS, pH 7.4, and 0.1% Tween-20.
The blots were then incubated at room temperature for 60 min with the
second antirabbit IgG labeled with peroxidase at a dilution of 1:1000
and washed in PBS-0.1% Tween-20 four times for 15 min each time before
revelation using a chemiluminescence kit (DuPont-New England Nuclear,
North Ryde, Australia) according to the manufacturer’s instructions.
For proteins from cells transfected with rat 11ßHSD1, rabbit
anti-HSD1 polyclonal antibody 56–126 (a gift from Dr. Carl Monder and
Dr. Brendan Waddell) was used at a dilution of 1:1000 (35).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). When dehydrogenase activity was examined in the
presence of Dex in intact mammalian cells the cloned 11ßHSD2 showed
marked conversion of Dex to DH-Dex. Dehydrogenase activity of 11ßHSD1
for Dex was not different from background. In intact cells transfected
with rat 11ßHSD1, the equilibrium was on the reductase side for all
substrates, whereas for 11ßHSD2 with F/E and B/A as substrates it was
on the oxidative side exclusively, but with DH-Dex/Dex it was on the
reductive side. Western blot analysis showed the presence of a similar
amount of immunoreactive 11ßHSD2 protein in the cells transfected
with 11ßHSD2 (Fig. 1b), so it was possible to compare the relative
activities of the 11ßHSD2 enzyme for the six different substrates.
The detection of equivalent amounts of immunoreactive 11ßHSD1 protein
in the cells transfected with 11ßHSD1 also allowed a direct
comparison of enzymatic activity (Fig. 1b). Assuming expression of
equal levels of both proteins, the dehydrogenase activity of 11ßHSD2
was consistently higher than that of 11ßHSD1, and the relative
reduction of DH-Dex with 11ßHSD2 was almost 90% higher than that
with 11ßHSD1.
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). In intact cells transfected with rat 11ßHSD2,
double reciprocal analysis of Dex oxidation showed that the
Km was 2.7 ± 0.4 µM, and the maximum
velocity (Vmax) was 26 ± 3 pmol/min·mg protein
(Fig. 2a), whereas for DH-Dex reduction, the Km was
1.9 ± 0.3 µM and the Vmax was 40
± 3 pmol/min·mg protein (Fig. 2b). The Km for DH-Dex for
rat 11ßHSD1 was 0.5 ± 0.2 µM, and the
Vmax was 12 ± 2 pmol/min·mg protein (Fig. 2b). We
have previously shown that 11ßHSD1 does not metabolize Dex (27). A
summary of the results comparing the bidirectional activities of the
two enzymes is shown in Table 1.
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). Rat and human 11ßHSD2 metabolized B, F, and Dex
with a hierarchy of B > F > Dex. Incubation of DH-Dex with
homogenates containing the 11ßHSD2 enzyme showed effective conversion
to Dex in the presence of NADH as cofactor. There was no NADP- or
NADPH-dependent 11ßHSD2 activity with any of the substrates (results
not shown). Neither the rat nor the human 11ßHSD2 enzymes showed
detectable reduction of E or A, with or without the addition of NADH or
NADPH as cofactor.
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. 11ßHSD2
inhibitory activity, calculated at 50% inhibition of conversion, was
observed at a concentration of 80 nM for glycyrrhetinic
acid and 100 nM for carbenoxolone (Fig. 4).
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shows the conversion of F and DH-Dex by the five
mutated enzymes. It can be seen that dehydrogenase activity was always
abolished in parallel with reductase activity, and that retention of
partial dehydrogenase activity (as in the R337C mutant) was also
reflected in the retention of partial oxoreductase activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In intact cells and in cell homogenates expressing either the rat or the human 11ßHSD2, the relative potency of the dehydrogenase reaction with various substrates was B > F > Dex. We recently reported Dex dehydrogenation by the cloned human 11ßHSD2 (27), a finding consistent with that of Brown et al. (36); Dex is not metabolized by human 11ßHSD1 (27). There was no reduction of A or E by either the rat or human 11ßHSD2 clones in either intact cells or homogenates using NADH or NADPH as cofactors, and this observation is consistent with previous findings (6, 10, 11, 12, 13). DH-Dex was, however, readily converted to Dex, and a consideration of kinetic parameters shows that the equilibrium for this reaction was far on the side of the 11-hydroxy metabolite. The 11ßHSD2-mediated DH-Dex reduction was exclusively NADH dependent, with no NADPH-dependent reductase activity detectable.
Probable explanations for the ability of 11ßHSD2 to reduce DH-Dex,
but not E or A, could be that either the reduction of the
9
-halogenated compound DH-Dex is catalyzed by a site of the
11ßHSD2 enzyme distinct from that driving the dehydrogenase reaction,
or alternatively, that the presence of the halogen group at the 9
position propels the reaction in the reductase direction only. Although
the existence of a distinct catalytic site cannot be ruled out, it
seems unlikely that this is the case for 11ßHSD2. First, most of the
enzymes that physiologically operate as reductases preferably use NADPH
as cofactor, whereas NAD is used primarily for the generation of NADH
(37). We were unable to show any reductase activity using 11ßHSD2
homogenates when NADPH was used as a cofactor with DH-Dex, E, or A.
Secondly, the similar pattern of relative activity of 11ßHSD2 mutants
derived from patients with AME for F and DH-Dex suggests that the point
mutations affected the oxidative and reductive enzymatic activities
similarly. Although this observation does not rule out distinct
catalytic sites, if DH-Dex reduction by mutant enzymes would have been
observed when no F oxidation was measured, it would have supported this
hypothesis.
The possibility that 9-halogenated steroid molecules are more
readily reduced than oxidized at the C-11 position deserves
consideration. 9-Halogenated 11-hydroxysteroids seem to be
metabolized by 11ßHSD enzymes in a different manner than the
nonhalogenated compounds. In a cell-free system, 9-fluorocortisol
(9FF), F, and aldosterone have the same affinity for the
mineralocorticoid receptor. In vivo, however, 9FF is 2-
to 400-fold more potent than F as a mineralocorticoid (28). The
explanation for the 9FF mineralocorticoid potency is thought to be
an impaired renal 11ß-oxidation of 9-halogenated
11-hydroxysteroids (28). Diedrich et al. addressed this
issue by studying the metabolism of 9-fluorinated steroids in human
kidney slices and microsomes (29). They found a distinct preference for
the reductase reaction for both Dex/DH-Dex and
9FF/9-flurocortisone. However, the kinetics for Dex
dehydrogenation and DH-Dex oxoreduction using human kidney preparations
showed a high affinity for both reactions.
Using NADH as a cofactor, Diedrich et al. reported reduction of E to F by human kidney microsomes (29). These results are in contrast with the present observations of the cloned 11ßHSD type I and II enzymes expressed in mammalian cells. The most likely explanation for this discrepancy is the existence of another, yet uncharacterized isoform of the 11ßHSD family. This isoform would convert DH-Dex to Dex more readily than the reverse reaction and would also convert E to F, using NADH as a cofactor. An alternative explanation might be that tissue-specific factors complex with the enzyme and facilitate bidirectional activity in mineralocorticoid target cells under some conditions. Further studies using stably transfected cell lines (38) from mineralocorticoid target cells are needed to address this issue.
When comparing rat 11ßHSD1 and 11ßHSD2 activities in intact transfected cells at equilibrium, 11ßHSD1 invariably favored the oxoreductase reaction with all substrates. In previous studies in the cell-free system oxoreductase activity was undetectable in preparations of purified hepatic 11ßHSD1 (39), or rate constants were equivalent to the dehydrogenase activity, but increased substantially with removal of NADP using the recombinant enzyme (7). There was no detectable Dex oxidation by 11ßHSD1, consistent with previous observations (27), and dehydrogenation of B or F appeared more potent with 11ßHSD2 than with 11ßHSD1. Our studies using competitors would appear to support this observation. As it seems that a permeability barrier slows the entry of GA into intact cells (40), we used homogenates to assess inhibition of 11ßHSD2 oxoreductase activity by GA or CBX. We found that the concentration of GA (80 nM) needed to inhibit half the oxoreductase activity in cell homogenates was comparable with the GA concentration required to inhibit half the cortisol metabolism of 11ßHSD2 (6), but was higher than previously found (10–20 nM) for 11ßHSD1 (7). These observations suggest that GA is a more potent inhibitor of 11ßHSD1 than 11ßHSD2.
Our results in the cell-free system as well as in intact cells show
that the oxidoreductase activity of 11ßHSD2 with 9-fluorinated
glucocorticoids is greater than that of the dehydrogenase. Thus, the
equilibrium of the reaction favors formation of the active synthetic
glucocorticoid. A similar conclusion was reach by others using
preparations of kidney slices (29). This observation would appear to
explain the mechanism by which synthetic glucocorticoids are
efficacious when administered to enhance fetal lung maturation, as the
human placenta expresses large amounts of 11ßHSD2 protein (16). These
data indicate that both the cloned human and rat 11ßHSD2 reduce
DH-Dex and do this more readily than they oxidase Dex. Thus, 11ßHSD2
seems also to be a bidirectional enzyme, although no reduction of the
physiological compounds A and E is observed.
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Acknowledgments |
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Footnotes |
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2 Recipient of a Foreign Research Grant from the Swiss National
Science Foundation.
Received November 25, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fluorocortisol: an explanation for its mineralocorticoid potency.
J Clin Endocrinol Metab 78:928–932[Abstract]
-fluorinated steroids in the human kidney. Endocr
Res 22:803–810[Medline]
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