By
From the Division of Nephrology, University Hospital of Berne, 3010 Berne, Switzerland
Endogenously released or exogenously administered glucocorticosteroids are relevant hormones for controlling inflammation. Only 11-hydroxy glucocorticosteroids, but not 11-keto
glucocorticosteroids, activate glucocorticoid receptors. Since we found that glomerular mesangial cells (GMC) express 11
-hydroxysteroid dehydrogenase 1 (11
-OHSD1), which interconverts 11-keto glucocorticosteroids into 11
-hydroxy glucocorticosteroids (cortisone/cortisol
shuttle), we explored whether 11
-OHSD1 determines the antiinflammatory effect of glucocorticosteroids. GMC exposed to interleukin (IL)-1
or tumor necrosis factor
(TNF-
) release group II phospholipase A2 (PLA2), a key enzyme producing inflammatory mediators.
11
-hydroxy glucocorticosteroids inhibited cytokine-induced transcription and release of
PLA2 through a glucocorticoid receptor-dependent mechanism. This inhibition was enhanced
by inhibiting 11
-OHSD1. Interestingly, 11-keto glucocorticosteroids decreased cytokine-induced PLA2 release as well, a finding abrogated by inhibiting 11
-OHSD1. Stimulating
GMC with IL-1
or TNF-
increased expression and reductase activity of 11
-OHSD1. Similarly, this IL-1
- and TNF-
-induced formation of active 11
-hydroxy glucocorticosteroids
from inert 11-keto glucocorticosteroids by the 11
-OHSD1 was shown in the Kiki cell line
that expresses the stably transfected bacterial
-galactosidase gene under the control of a glucocorticosteroids response element. Thus, we conclude that 11
-OHSD1 controls access of 11
-hydroxy glucocorticosteroids and 11-keto glucocorticosteroids to glucocorticoid receptors and
thus determines the anti-inflammatory effect of glucocorticosteroids. IL-1
and TNF-
upregulate specifically the reductase activity of 11
-OHSD1 and counterbalance by that mechanism
their own proinflammatory effect.
IL-1
Two isoenzymes accounting for 11 Supplies.
For cell culture and 11 Cell Cultures.
GMC were cultured from isolated rat (Sprague-Dawley) glomeruli (20). In brief, the cells were grown in RPMI-1640 supplemented with 10% FCS, penicillin (100,000 U/liter),
streptomycin (100,000 µg/liter), transferrin (5 mg/liter), insulin
(5 mg/liter), and sodium selenite (5 µg/liter). For the experiments, passages 20-30 were used. For 11 and TNF-
often act synergistically and cause a
wide array of in vitro and in vivo immune inflammatory
responses such as the secretion of phospholipase A2 (PLA2)1, a
key enzyme that releases arachidonic acid and therefore boosts prostaglandin production and secretion (1). This inflammatory reaction is regulated by 11
-hydroxy glucocorticosteroids; for instance, glucocorticoid deficiency increases,
whereas physiological and pharmacological doses of glucocorticosteroids suppress the enhanced expression of group
II PLA2 during inflammation (4). The biological activity
of glucocorticosteroids depends on their dose, metabolism,
local access to their cognate receptors, and on the responsiveness of the target cells (8, 9). Traditionally the 11-keto-
glucocorticosteroid molecules are believed to have hardly
any biological activity because of their negligible affinity to
glucocorticoid receptors. In the present investigation, we demonstrate that during inflammation, 11-keto steroids exhibit antiinflammatory properties. This effect is dependent on the
activity of the enzyme 11
-hydroxysteroid dehydrogenase
(11
-OHSD), which interconverts the 11-keto and the corresponding 11
-hydroxy glucocorticosteroids by the so-called
cortisone/cortisol shuttle (8, 10-15; Fig. 1).
Fig. 1.
Cortisone/cortisol shuttle. The endogenous hormones cortisol and corticosterone, as well as the pharmacologically used prednisolone, are biologically active 11-hydroxy glucocorticosteroids because they can
bind to the cognate receptor. The corresponding 11-keto glucocorticoids
cortisone, dehydrocorticosterone, and prednisone are unable to do so.
The enzyme 11
-OHSD1 converts 11-keto glucocorticosteroids to 11
-hydroxy glucocorticosteroids and vice versa, and thus regulates local intracellular access of the steroids to the receptors. 11
-OHSD activity can
be inhibited by glycyrrhetinic acid, a compound found in licorice and anise. Corticosterone and dehydrocorticosterone differ from cortisol and
cortisone because of the absence of a hydroxyl group at position C17,
whereas prednisolone and prednisone have an additional double bond in the
A ring.
[View Larger Version of this Image (11K GIF file)]
-OHSD activity
have been cloned and characterized: 11
-OHSD1 (11) is
dependent on the reduced form of nicotinamide adenine
dinucleotide phosphate [NADP(H)] and catalyses both the
oxidation and the reduction reactions, whereas 11
-OHSD2
requires nicotinamide adenine dinucleotide (NAD) as a cofactor and exhibits only oxidative activity (12). The biological role of 11
-OHSD2 is most likely to provide selective access of aldosterone to the mineralocorticoid receptor by
inactivating cortisol (8, 13). The absence of 11
-OHSD2 results in apparent mineralocorticoid excess with
hypertension and hypokalemia. Because specific inactivation
of cortisol is relevant only in distal tubular cells of the kidney, salivary glands, and colon (the target cells of aldosterone), 11
-OHSD2 is almost exclusively expressed in this
subset of cells. 11
-OHSD1, on the other hand, is expressed in a wide variety of tissues, but its function is still not clear. In this report, we studied the role of 11
-OHSD1 in
glomerular mesangial cells (GMC). These proinflammatory
cells were chosen because they play a pivotal role in certain
forms of glomerular diseases. During inflammation, these
cells release active substances such as enzymes, vasoactive endobiotics, extracellular matrix components, prostaglandins,
and cytokines such as IL-1
and TNF-
, which cause local
glomerular tissue damage (6, 16). In the present investigation, it is demonstrated that the activity of the 11
-OHSD1 determines the antiinflammatory effect of 11
-hydroxy glucocorticosteroids and that the proinflammatory
endobiotics IL-1
and TNF-
upregulate the reductase activity of 11
-OHSD1, and thus, these cytokines display a
dual mode of action in that they induce concomitantly inflammation and an antiinflammatory response.
-OHSD assay, corticosterone,
dehydrocorticosterone, glycyrrhetinic acid, transferrin, and insulin were obtained from Sigma Chemical Co. (Buchs, Switzerland), and NAD phosphate (NADP), NADPH, and NAD were
from Boehringer Mannheim (Rotkreuz, Switzerland). [1,2,6,7 3H]corticosterone with a specific activity of 83 Ci/ mM and
[3H]oleic acid (specific activity 10 Ci/mM) were purchased from Amersham Intl. (Buckinghamshire, U.K.). [3H]dehydrocorticosterone was prepared as already described (19). Bicinchonic acid
protein assay reagent was from Pierce Chemical Co. (Rockford,
IL). Triton X-100 and TLC plates (60 fluorescence indicator 254)
coated with silica gel were from Merck (Schweiz) AG (Dietilcon,
Switzerland). RPMI-1640, penicillin G (100,000 U/liter) and
streptomycin sulfate (100,000 µg/liter) were obtained from GIBCO
BRL (Basel, Switzerland); FCS was from Biological Industries (France). Tissue culture plates (24-well plates) were obtained from Becton Dickinson Labware (Basel, Switzerland), IL-1
and TNF-
were from Pharma Biotechnologie (Hannover, Germany), and forskolin was from Calbiochem-Novabiochem (Luzern,
Switzerland). RU 486 was a gift from Dr. B. Stadler (Institute of
Immunology, Berne, Switzerland). For reverse transcription and
polymerase chain reaction, deoxynucleotides (dNTPs), RNAse inhibitor, avian myeloblastosis virus reverse transcriptase, and BSA
were obtained from Boehringer Mannheim. Primers were ordered
from Microsynth (Balgach, Switzerland) and Thermus aquaticus
DNA polymerase from Perkin-Elmer Cetus Instrs. (Norwalk, CT).
The enhanced chemiluminescence detection kit was purchased
from Amersham Intl.
-OHSD assays, confluent
GMC cultures in 15-mm-diameter wells were incubated with
500 µl RPMI-1640 medium containing 10% FCS and increasing
concentrations of IL-1
, TNF-
, or both for 48 h in a CO2 incubator maintained at 37°C. The medium was removed and conversion by stimulated GMC of corticosterone to dehydrocorticosterone and dehydrocorticosterone to corticosterone was
analyzed in situ by incubating the cells for 4 h with 200 µl medium containing 5 nCi [3H]corticosterone or [3H]dehydrocorticosterone and 0.5 µM corticosterone or dehydrocorticosterone. After incubation, the medium was extracted with 200 µl ethyl acetate and TLC were performed as described below. Protein was
extracted from the cells by adding 200 µl 0.4 N NaOH at 37°C
for 1 h after neutralization with the same volume of 0.4 N HCl.
Specific activity was expressed as the percentage of conversion of
corticosterone to dehydrocorticosterone, and dehydrocorticosterone to corticosterone, respectively, per milligram of total protein
during 4 h. For PLA2 assays, confluent GMC cultures in 15-mm-
diameter wells were incubated with 500 µl RPMI-1640 medium
containing 10% FCS and 1 nM of IL-1
with and without indicated concentrations of corticosterone and/or glycyrrhetinic acid,
and forskolin or RU 486. 24 h later, the medium was removed,
centrifuged for 5 min at 4,000 rpm and used for PLA2 assay.
COS-1 cells were cultured and transfected with the cDNA of
11
-OHSD1 as previously described (19).
-galactosidase gene
(lacZ) under control of the mouse mammary tumor virus promoter. This promoter contains essential glucocorticoid response
elements. Thus, Kiki cells express
-galactosidase when exposed
to sufficient doses of 11
-hydroxy steroids. Kiki cells were cultured in DMEM supplemented with 10% FCS, 60 mg/liter kanamycin, and 30 mg/liter hygromycin B. For steroid activity assays,
the cells were incubated for 72 h with a combination of steroids
and cytokines, and then subjected to the in situ
-galactosidase
assay as described (21).
Assay for 11-OHSD1.
The assay was performed as previously described by Monder et al. (22). Oxidation or reduction at
C-11 was determined by measuring the rate of conversion of corticosterone to 11-dehydrocorticosterone in the presence of NADP
or dehydrocorticosterone to corticosterone in the presence of
NADPH. GMC were extracted with 10 mM Tris-HCl (pH 7.5),
5 mM EDTA (pH 8), 1% Triton X-100, 2 mM PMSF, and 100 µg total protein were used for the reaction. The assay was performed in 0.25 mM NADP or NADPH, 100 mM Tris (pH 8.3),
10 nCi [3H]corticosterone or [3H]dehydrocorticosterone, and 5 µM
corticosterone or dehydrocorticosterone. Samples were incubated
for 3 h at 37°C, reaction was stopped on ice, and steroids were
extracted with 500 µl ethyl acetate. The organic layer was separated by centrifugation at 13,000 rpm and evaporated under a
stream of nitrogen. The steroid residue was dissolved in 20 µl
methanol containing a mixture of 20 µg each of unlabeled corticosterone and dehydrocorticosterone. This was quantitatively
transferred to thin-layer plates and developed in chloroform-methanol (90:10 vol/vol). The spots corresponding to the steroids
were located under a UV lamp, cut out, transferred to scintillation vials, and counted in scintillation fluid in a Kontron (Zurich,
Switzerland) Betamatic fluid scintillation counter. Specific activity was expressed as nanomolar of product formed per microgram
protein per hour.
Assay for 11-OHSD2.
The assay was performed as previously described by Albitson et al. (12). Homogenization of cells
for measurement of 11
-OHSD2 activity was performed in homogenization buffer containing 250 mM sucrose and 10 mM
Tris-HCl (pH 7.5). Protein extract was incubated for 3 h at 37°C
with 1 mM NAD, 10 nM corticosterone, and 50 nCi [3H]corticosterone in 500 µl homogenization buffer. The subsequent steps
were the same as those described for 11
-OHSD1.
Assay for 17-OHSD.
The stimulation experiments were performed in parallel with those for 11
-OHSD1 measurements. 17
-OHSD activity was performed as already described (23) by measuring in situ the conversion of estradiol to estrone or estrone to
estradiol. GMC were incubated for 4 h with 200 µl medium
containing 5 nCi [3H]estradiol or [3H]estrone and 0.5 µM estradiol or estrone. After incubation, the medium was extracted with
1 vol diethyl ether and frozen. The unfrozen organic layer was
poured into a fresh tube, evaporated under a stream of nitrogen,
and TLC were performed in 4:1 dichloromethane/ethyl acetate,
using 20 µg unlabeled estradiol and estrone. The next steps were
the same as for 11
-OHSD1.
Assay for PLA2 Activity. [3H]oleic acid-labeled Escherichia coli were prepared as already described (24, 25). PLA2 was assayed using [3H]oleate-labeled, autoclaved E. coli as the substrate (26). The reaction mixture of 350 µl contained 100 mM Tris-HCl (pH 8.0), 5 mM Ca2+, 2.85 × 108 cells of autoclaved E. coli (corresponding to 10,000 cpm and 5.0 nm lipid phosphorus), and tissue acid extracts or supernatants of GMC. The amount of protein was chosen such that 6-15% hydrolysis of substrate was obtained when incubated at 37°C for 2 h. The reaction was stopped by adding 100 µl of 2N HCl. 100 µl of fatty acid-free BSA (100 µg/ml) was added, and the tubes were vortexed and centrifuged at 13,000 rpm for 5 min. An aliquot (140 µl) of the supernatant containing released [3H]oleic acid was mixed with scintillation cocktail and counted in a liquid scintillation counter.
Reverse Transcription of Messenger RNA and PCR.
Total RNA
was extracted from GMC after the guanidium thiocyanate method
(27). The RNA concentration was determined by measuring the
absorption at 260 nm and its quality was controlled by loading 1 µg
on a 1% formaldehyde gel. Reverse transcription was performed in 20 µl containing 50 mM Tris-HCl (pH 8.2), 6 mM MgCl2, 10 mM dithiothreitol, 100 mM NaCl, 200 µM dNTPs, 11 U of ribonuclease inhibitor RNAsin, 1 U avian myeloblastosis virus reverse
transcriptase, 10 pmol 3 primer of the corresponding cDNA position (852-873 for 11
-OHSD1, 1271-1295 for 11
-OHSD2,
335-359 for group I PLA2, 695-719 for group II PLA2, 980-
1004 for the internal standard glyceraldehydephosphate dehydrogenase [GAPDH]), and 2 µg total RNA (4, 11, 12, 24, 28). Initially, the 3
primer was incubated with total RNA for 5 min at
65°C and cooled at room temperature for 15 min. The remaining
reaction components were added and incubated for 1 h at 42°C.
Western Blot Analysis.
Electrophoresis was performed using a
12.5% polyacrylamide gel under reducing conditions. Prestained
protein standards were used as markers. Probes (25 µg of total
protein) were heated along with SDS sample buffer at 95°C for
15 min. After electrophoresis, the gel was equilibrated in transfer
buffer (20 mM Tris, 190 mM glycine, 20% methanol) for 10 min.
The transfer of protein to an Immobilon membrane (Millipore
Corp., Bedford, MA) was performed with a constant voltage of
60 V for 1 h on ice. The membrane was blocked in 5% BSA in
PBS for 2 h, washed with PBS, and incubated over night with a
rabbit polyclonal antibody for 11-OHSD1 (gift from Carl Monder, The Population Council, New York, NY). The Immobilon membrane was washed with PBS, saturated again for 2 h in 5%
BSA, and incubated for 1 h at room temperature with a goat
anti-rabbit IgG horseradish peroxidase conjugate (Bio Rad Labs.,
Hercules, CA). After washing, the detection was performed with
the enhanced chemiluminescence kit. The bands on the x-ray
film were scanned with a transmittance scanning densitometer
(Scanalytics, Billerica, MA).
To establish the presence of 11-OHSD1 and 11
-OHSD2, as well as PLA2 group I and group II, their messenger RNA (mRNA) levels were measured in GMC by
reverse transcribed PCR (RT-PCR) using specific primers.
The PCR products on the gel revealed signals for group II
PLA2 and 11
-OHSD1, but no signal for group I PLA2 (pancreatic), and only a very faint signal for 11
-OHSD2
(Fig. 2, top). These signals were also quantified by including
[
32P]-dCTP nucleotide during PCR (Fig. 2, bottom).
Oxidation of the hydroxyl group of corticosterone and
reduction of the keto group of dehydrocorticosterone by
11-OHSD1 were measured using NADP and NADPH as
cofactors, respectively, in Triton X-100 extracts of GMC.
The enzyme activity was plotted as a function of the
amount of protein (Fig. 3). A dose-dependent increase in the oxidation (Fig. 3, left) and reduction (Fig. 3, right) activity was observed. Glycyrrhetinic acid (GA), a known inhibitor of 11
-OHSD1 and 11
-OHSD2 (12, 29), completely inhibited oxidation and reduction activity of GMC
11
-OHSD1. The GMC extract had no 11
-OHSD2 oxidation activity with NAD as cofactor (data not shown), an
observation in line with the weak signal observed by RT-PCR. It has to be noted that 11
-OHSD2 has only oxidation and no reduction activity (12).
During glomerular inflammation, PLA2 levels rise
sharply in GMC after stimulation with IL-1, TNF-
, or
forskolin (30). Therefore, we studied the role of 11
-OHSD1 on the inhibitory effect of glucocorticoids on
PLA2. For that purpose, GA was used to modulate the activity of 11
-OHSD1. The level of group II PLA2 was first
assessed by RT-PCR (Fig. 4). Stimulation of GMC with IL-1
markedly enhanced the level of group II PLA2. The
rise was less pronounced in the presence of corticosterone
and was diminished further when GA was added. GA
alone, however, does not affect group II PLA2 expression
induced by IL-1
. The quantitatively assessed mRNA content of GAPDH in GMC was not affected in these different
experiments (data not shown).
In line with these observations, the enzymatic activity of
PLA2 increased upon stimulation with IL-1 or forskolin,
to be then decreased by the action of corticosterone and
corticosterone combined with GA (Fig. 5). Corticosterone
reduced the IL-1
-stimulated PLA2 enzyme activity by
60% and corticosterone in combination with GA diminished the activity by 90% (Fig. 5, top). GA alone did not alter the secretion of PLA2 induced by IL-1
(Fig. 5, top).
Similar results were obtained when forskolin, instead of IL-1
, was used for stimulaton of group II PLA2 activity
(Fig. 5, bottom).
To establish whether the inhibiton of group II PLA2 enzyme by corticosterone is mediated through glucocorticoid
receptors, the PLA2 activity was assayed in the presence of
the glucocorticoid antagonist RU 486. As shown in Fig. 6,
the effect of corticosterone was diminished by increasing
concentrations of RU 486, indicating that corticosterone
exerts its effect through glucocorticoid receptors.
Since it was shown previously that IL-1 increased corticosteroid levels, the hypothesis was tested whether IL-1
and TNF-
regulate the 11
-OHSD1 (33, 34). For that
purpose, GMC were incubated with TNF-
, IL-1
, or a
combination of both, and the mRNA content was quantified by RT-PCR for 11
-OHSD1 (Fig. 7, top). Both IL-1
and TNF-
only slightly enhanced the mRNA content in GMC. The combination of both, in contrast, yielded a
strong additive effect (Fig. 7, top). The increased mRNA
content of GMC was reflected by a sharp increase in the
amount of 11
-OHSD1 protein (Fig. 7, bottom). The cytokine-induced increase of protein levels was more marked
than the effect on steady-state mRNA levels, suggesting that the modulation may take place mainly at the translational level. When the specific activity of 11
-OHSD1 was
analyzed, no change was seen with respect to oxidation (results not shown). However, the reductive activity of 11
-OHSD1 increased as a function of the concentrations of
IL-1
and/or TNF-
(Fig. 8). The combination of TNF-
and IL-1
stimulated the reductive activity more than either of the cytokines added alone; this was shown when either increasing concentrations of TNF-
or IL-1
were
used, or when a fixed amount of TNF-
with an increasing amount of IL-1
were given to the cell cultures (Fig.
8). A similar pattern was observed when the specific activity (percentage of conversion of dehydrocorticosterone to
corticosterone per milligram protein per 4-h period) was
calculated (data not shown). To exclude that the increased
reductive activity after stimulation with cytokines was due
to an increased availability of cofactors, incubations of stimulated GMC with dehydrocorticosterone in the presence
of 5 mM NADPH were performed. The results from these
studies revealed that the increased reductase activity induced by cytokines was not attributable to a higher availability of NADPH (data not shown).
Besides IL-1 and TNF-
, other cytokines such as IL-3
and IL-6, and other stimulating agents including platelet-derived growth factor, PMA, and forskolin were added to
the GMC cultures. These agents exhibited neither an increase nor a decrease of the oxidative or reductive activity of
11
-OHSD1. To exclude a nonspecific effect of TNF-
and/or IL-1
on oxidoreductase activity in general, the activity of 17
-hydroxysteroid dehydrogenase was determined in GMC with and without the addition of TNF-
and IL-1
using estradiol and estrone as substrates. The activity of 17
-OHSD was not affected by these cytokines.
Thus, the enhancement of 11
-OHSD1 by IL-1
and
TNF-
is specific.
The biological relevance of the increased reductase activity of 11-OHSD1 after stimulation with IL-1
and/or
TNF-
was assessed first on the inhibition of PLA2 activity
in GMC. Unstimulated GMC or GMC treated with GA
alone or with the 11-keto-glucocorticosteroid dehydrocorticosterone alone displayed only background PLA2 activity (Fig. 9). Stimulation of GMC with IL-1
enhanced
PLA2 production at least fivefold. Dehydrocorticosterone
reduced the IL-1
-induced PLA2 activity. This effect was
abrogated with increasing concentrations of GA (Fig. 9). In
a second set of experiments, the relevance of the interconversion of biologically inactive 11-keto glycocorticosteroids into active 11
-hydroxy glucocorticosteroids was demonstrated in the Kiki cell line (21). We found that Kiki cells
contain high levels of 11
-OHSD1, but low levels of 11
-OHSD2 transcripts (data not shown). TNF-
and IL-1
induce the reductase activity of 11
-OHSD1 in these cells
(data not shown). Kiki cells express the bacterial
-galactosidase gene under the control of glucocorticoid-responsive elements of the mouse mammary tumor virus promoter.
This cell line therefore expresses
-galactosidase only when
biologically active 11
-hydroxy glucocorticosteroids are
present. The experiments in Fig. 10 demonstrate that although 11
-hydroxy glucocorticosteroids directly drive gene
expression, 11-keto glucocorticosteroids are unable to do
so unless the cells are concomitantly stimulated by IL-1
.
This effect is mediated by 11
-OHSD activity, because it
can be abolished with GA in a dose dependent fashion (Fig. 10 b). Furthermore, this effect is specific for 11-keto glucocorticosteroids as cortexolone, a steroid without a functional
group at position 11, displays an 11
-OHSD- and cytokine-independent constitutive activity, albeit weak (Fig. 10
a) The relevance of the conversion of cortisone to cortisol
was furthermore shown when these two endogenous glucocorticosteroids are present in the low physiological concentration range using Kiki cells (data not shown).
The present study revealed for the first time 11-OHSD
activity in GMC. The activity was attributable to 11
-OHSD1 as shown by measurement of mRNA, protein, and
activity using NADP as a cofactor. The absence of appreciable expression of 11
-OHSD2 in GMC is in line with
previous immunohistochemical data on kidney cortex (35,
36) showing only a weak staining for 11
-OHSD2 in the
visceral epithelial cells of the outer capillary loop of the
glomerulus.
The impact of 11-OHSD1 on glucocorticoid action
during inflammation was analyzed by studying the corticosterone-induced inhibition of group II PLA2 release in
GMC. As recently shown, GMC stimulated with IL-1
or
TNF-
enhance the eicosanoid formation concomittantly with an increased 14-kD group II PLA2 gene expression
and secretion (30, 37). A similar effect was observed
when GMC were stimulated with forskolin (37, 38). Forskolin and IL-1
stimulate PLA2 through two distinct
mechanisms: activation of adenyl cyclase and protein kinase
C (38). The enhanced expression of PLA2 in inflammation
is suppressed by glucocorticosteroids (39). In the present
study, the inhibitory effect of corticosterone on the release
of group II PLA2 stimulated by IL-1
or forskolin was enhanced by inhibiting the 11
-OHSD1 by GA. This effect
was most likely mediated through glucocorticoid receptors
as shown by abrogating the glucocorticoid inhibitory effect
with RU 486. When GA was added to the GMC, no direct inhibition of group II PLA2 transcription and release
was observed; the inhibitory effect of GA on PLA2 was
only detected in the presence of corticosterone. This indicates that the previously reported antiinflammatory (40) and antiallergic effects of GA (41), the active pharmacological component of licorice (Glycyrrhiza glabra), is attributable to an enhanced effect of endogenous glucocorticoids
as a consequence of the inhibition of 11
-OHSD.
GMC occupy a central position within the glomeruli (16). They are important for the regulation of glomerular filtration rate, and furthermore, GMC participate in the process of glomerular injury. For both processes, arachidonic acid-derived metabolites are relevant. Group II PLA2 plays a pivotal role for the arachidonic acid release. PLA2 is regulated by glucocorticosteroids, since glucocorticoid deficiency enhances and pharmacological doses of glucocorticosteroids suppress group II PLA2 transcription (4, 6, 16). This high sensitivity of group II PLA2 to exogenously prescribed or endogenously released steroid hormones underlines the relevance of glucocorticoids in health and disease states for prostaglandin release. The present study demonstrates that not only the concentrations of the glucocorticosteroids but, in addition, the intracellular disposition determines their antiinflammatory effect.
The addition of IL-1 and TNF-
enhanced the expression of 11
-OHSD1. The activity of the reductase, but not
that of the oxidase, of 11
-OHSD increased severalfold.
This observation has been made in two different cell lines.
The mechanism accounting for the enhanced reduction is a
specific phenomenon because the activity of another oxido-reductase, the 17
-hydroxysteroid dehydrogenase, was
not affected by the cytokines. The enhanced activity of
11
-OHSD1 cannot be explained by mere proliferation of
the GMC induced by these cytokines, since the specific activity of 11
-OHSD1 reductase increased and other GMC-stimulatory agents did not enhance the 11
-OHSD1 reductive capacity in GMC. The increased reductive capacity is
biologically relevant as evidenced first by the activation of a
glucocorticosteroid response element-dependent reporter
gene by biologically inert 11-keto steroids such as cortisone, dehydrocorticosterone, or prednisone, and by the inhibition of PLA2 through a glucocorticoid receptor-dependent pathway by dehydrocorticosterone.
The mechanism for the enhanced reductive but not oxidative activity, despite increased concentrations of 11-OHSD1 protein after stimulation with IL-1
and TNF-
,
is unknown. One possible mechanism is a different posttranslational modification. Such modifications exist for 11
-OHSD1, as shown by Agarwal et al. who incubated TK-143B human osteosarcoma cells transfected with the cDNA
of rat 11
-OHSD1 in the presence of the glycosylation inhibitor A1-tunicamycin and observed a 50% decrease in
dehydrogenase activity without affecting the reductase activity (15). Similarly, in the present investigation, a clear
decline in the oxidase activity, but a less pronounced decline in the reductase activity was seen when GMC were
incubated with A1-tunicamycin (data not shown). Thus, a
possible explanation of the altered oxidative/reductive ratio
induced by IL-1
and TNF-
, is a posttranslational modification. Nominally, however, we can not rule out the possibility that the relative increase in reductase activity relates
to the activation of a yet undiscovered and hypothetical
11
-OHSD3.
IL-1 and TNF-
often exert various synergistic in vitro
and in vivo effects including immune inflammatory responses
(1, 42, 43). In glomerular inflammation, increased expression of the inflammatory cytokines IL-1
and TNF-
has been observed (44). IL-1
and TNF-
induce,
among others, contraction, proliferation, expression of receptors, and metabolic effects in GMC. The present novel
observation, that IL-1
and TNF-
modulate the 11
-OHSD activity in such a way that the formation of active
11
-hydroxy glucocorticoids is favored, is a biologically
interesting phenomenon for the following reasons. First,
Besedowsky et al. and Sapolsky et al. presented evidence
for an interaction between IL-1
and glucocorticoids (33,
34). The injection of IL-1
activated the hypothalamo-pituitary-adrenal axis with an enhanced secretion of adrenocorticotropic hormone and corticosterone due to an IL-1- stimulated release of corticotropin-releasing factor (33).
This observation was interpreted to mean that during infections, cortisol or corticosterone appear in higher concentrations in serum to overcome the stress due to an infectious challenge. The present observation that IL-1
favors
the formation of biologically active glucocorticoids during
inflammatory reactions within the target cells, is in line
with the seminal observations of Besedovsky et al. and Sapolsky et al. (33, 34). Second, IL-1
and TNF-
are known
to induce apoptosis in some cells (50, 51). Similarly, 11
-hydroxy glucocorticosteroids induce apoptosis (52). Since TNF-
and IL-1
stimulate the intracellular formation of
active glucocorticosteroids, they enhance their own apoptoting potential. Third, inflammatory processes are limited
with respect to time in most situations. The mechanisms
accounting for such limitation are not completely understood. Glucocorticosteroids are powerful inhibitors of IL-1
and TNF-
secretion and of many of the inflammatory effects of these cytokines (53). IL-1
and TNF-
can
mediate the onset of inflammation and, by inducing the reductase activity of 11
-OHSD, enhance the GMC susceptibility to glucocorticoids. It is interesting to note that the
two processes are temporally distinct. It has been already
shown that PLA2 levels rise sharply as early as 12 h after exposure to proinflammatory cytokines (37). In contrast, we
have observed that a consistent increase in 11
-OHSD reductase activity is detectable only after 24 h. After 1, 4, 6, and 12 h, no significant increase in reductase activity was detected (data not shown). By this dual action, IL-1
and
TNF-
may counterbalance their proinflammatory effect,
and contribute to the ultimately necessary arrest of inflammation. A comparable dual effect of proinflammatory and
antiinflammatory actions has recently been described for
leukotriene B4 (57). Leukotriene B4 sustains and amplifies
inflammation, while concomitantly activating the peroxisome proliferator-activated receptor, which drives peroxisomal
-oxidative degradation of leukotriene B4 itself.
Address correspondence to Dr. Felix J. Frey, Division of Nephrology, Inselspital, 3010 Berne, Switzerland. Phone: 41-31-632-96-29; FAX: 41-31-632-94-44.
Received for publication 10 February 1997 and in revised form 7 May 1997.
1Abbreviations used in this paper: dCTP, desoxy CTP; GA, glycyrrhetinic acid; GAPDH, glyceraldehydephosphate dehydrogenase; GMC, glomerular mesangial cells; mRNA, messenger RNA; NAD, nicotinamide adenine dinucleotide; NADP, NAD phosphate; NADPH, reduced form of NADP; OHSD, hydroxysteroid dehydrogenase; PLA2, phospholipase A2; RT-PCR, reverse transcribed PCR.We thank Dr. M. Baggiolini (University of Berne, Berne, Switzerland), for critical discussion of the manuscript.
This work was supported by a grant from the Swiss National Foundation for Scientific Research (Nr 3200-040492.94).
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