Synthetic Glucocorticoids That Dissociate Transactivation and AP-1 Transrepression Exhibit Antiinflammatory Activity in Vivo
Béatrice M. Vayssière1,
Sonia Dupont2,
Agnès Choquart,
Francis Petit,
Teresa Garcia,
Christian Marchandeau,
Hinrich Gronemeyer and
Michèle Resche-Rigon
Roussel UCLAF (B.M.V., S.D., A.C., F.P., T.G., C.M., M.R.-R.)
93235 Romainville Cedex, France
Institut de
Génétique et de Biologie Moléculaire et Cellulaire
(H.G.) IGBMC-BP. 163 67404 Illkirch Cedex, C.U. de Strasbourg,
France
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ABSTRACT
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Some of the most potent antiinflammatory and
immunosuppressive agents are synthetic glucocorticoids. However, major
side effects severely limit their therapeutic use. The development of
improved glucocorticoid-based drugs will require the separation of
beneficial from deleterious effects. One possibility toward this goal
is to try to dissociate two main activities of glucocorticoids,
i.e. transactivation and transrepression. Screening of a
library of compounds using transactivation and AP-1 transrepression
models in transiently transfected cells identified dissociated
glucocorticoids, which exert strong AP-1 inhibition but little or no
transactivation. Importantly, despite high ligand binding affinity, the
prototypic dissociated compound, RU24858, acted as a weak agonist and
did not efficiently antagonize dexamethasone-induced transcription in
transfected cells. Similar results were obtained in hepatic HTC
cells for the transactivation of the endogenous tyrosine amino
transferase gene (TAT), which encodes one of the enzymes involved in
the glucocorticoid-dependent stimulation of neoglucogenesis. To
investigate whether dissociated glucocorticoids retained the
antiinflammatory and immunosuppressive potential of classic
glucocorticoids, several in vitro and in vivo
models were used. Indeed, secretion of the proinflammatory lymphokine
interleukin-1ß was severely inhibited by dissociated glucocorticoids
in human monocytic THP 1 cells. Moreover, in two in vivo
models, these compounds exerted an antiinflammatory and
immunosuppressive activity as potent as that of the classic
glucocorticoid prednisolone. These results may lead to an improvement
of antiinflammatory and immunosuppressive therapies and provide a novel
concept for drug discovery.
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INTRODUCTION
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The glucocorticoid receptor (GR) is a member of the superfamily of
nuclear receptors which, upon binding to their cognate ligands, act as
positive and negative regulators of target gene transcription. In the
case of positive regulation, they transactivate through
cis-acting palindromic glucocorticoid response elements
(GREs), located in the promoter region of responsive genes (for reviews
see Refs. 1, 2, 3, 4, 5).
Apart from acting as transactivators, several nuclear receptors,
including GR, are able to negatively regulate transcription. For GR,
two distinct mechanisms have been described. One implies a competition
between GR and other transcription factors for binding to their cognate
DNA elements (6, 7). The second, generally referred to as
transrepression, is much less well understood. It refers to the
original observation that transactivation by AP-1 was impaired in the
presence of glucocorticoids. Reciprocally, glucocorticoid action was
inhibited by AP-1 (Refs. 8, 9, 10, 11, 12 ; reviewed in Ref. 13). Although evidence
for direct interaction between GR and AP-1 was provided from in
vitro studies indicating that GR and AP-1 mutually interfere with
each others DNA- binding ability (8, 9, 12), genomic footprinting
experiments demonstrated that in the presence of glucocorticoids, AP-1
remains bound to the collagenase promoter in vivo (14).
Together with the observation of cell- and promoter-specific
transrepression, these results suggested that AP-1 and GR interact
rather indirectly, possibly through other transcriptional factors (Ref.
15 ; reviewed in Ref. 13). Mutational studies have demonstrated that
both GR DNA- and ligand-binding domains (LBDs) are required for AP-1
transrepression (9, 10, 11, 12, 15, 16). Furthermore, by mutating individual
amino acids of the DNA-binding domain of GR, it could be demonstrated
that transactivation and transrepression are two separable functions
(17). A similar conclusion was drawn for retinoic acid receptors since
certain synthetic retinoids elicited AP-1 inhibition without
significantly transactivating cognate reporter genes (18, 19, 20).
Glucocorticoids can also repress members of the NF-
B-Rel
transcription factor family (21, 22, 23). In addition to a cross-coupling
mechanism of inhibition between NF-
B and the GR, induction of
transcription of the gene encoding I
B
has also been recently
demonstrated in lymphocytes and monocytes (24, 25). However, induction
of I
B
expression by glucocorticoids seems to be cell
type-restricted since it cannot account for inhibition of NF-
B
activity in endothelial cells (26).
Glucocorticoids are highly potent antiinflammatory and
immunosuppressive agents, due to their pleiotropic effects on the
expression/activity of multiple immunomodulators and their ability to
induce apoptosis in lymphocytes (Ref. 27 and references therein).
However, their therapeutic use is limited by severe side effects,
especially during long-term treatment (see Discussion).
Whether the dissociation of glucocorticoid-dependent transactivation
and transrepression may provide the possibility to separate (some of
the) negative side effects from the beneficial antiinflammatory action
of classic glucocorticoids is still not clear. Therefore, we have
systematically compared the transactivation and transrepression
abilities of various glucocorticoid agonists and antagonists and
screened a series of synthetic compounds to detect those exhibiting
dissociated characteristics (i.e. mainly transactivating or
AP-1 transrepressing). Here we report the identification of a novel
class of synthetic glucocorticoids that inhibit transcription of the
collagenase promoter in transfected Hela cells, while only weakly
activating GRE-based reporter genes. Moreover, we show that these
ligands are potent inhibitors of interleukin-1ß (IL-1ß) secretion
in activated monocytes, while they are unable to induce significant
tyrosine amino transferase (TAT) activity in rat HTC hepatoma cells.
Finally, we provide evidence that these dissociated glucocorticoids act
as antiinflammatory and immunosuppressive drugs in vivo.
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RESULTS
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Identification of Dissociated Glucocorticoids
More than 200 synthetic glucocorticoids, including several
clinically important GR ligands as reference compounds, were subjected
to a transient transfection-based screening system to compare their
potencies for GR-mediated transactivation and AP-1 transrepression
(for details see Materials and Methods). Briefly, ligand
dose-response curves were established for transactivation and AP-1
transrepression by the endogenous HeLa cell GR from the stimulation of
the GRE5-tk-CAT, and the inhibition of the c-Jun-activated
collagenase promoter-CAT reporter genes, respectively. As expected,
dexamethasone (Dex) and prednisolone were found to be both strong
transactivators and strong AP-1 inhibitors (Fig. 1
); we refer to this
class of glucocorticoids as "symmetrical" compounds. All tested
glucocorticoids currently used in medical therapies as antiinflammatory
agents were found to belong to this class (data available upon
request). Each compound was classified relative to the maximal
transactivation and AP-1 transrepression obtained with Dex (100%) and
characterized by its relative activation and repression activities at a
given concentration (Table
I, and data not shown).

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Figure 1. Effect of Glucocorticoids on GRE-tk-CAT
Transactivation and AP-1 Transrepression in HeLa Cells
Transactivation assays (A). Hela cells were cotransfected with the
GRE5-tk-CAT reporter (1 µg), the polyIIßGal internal
control vector (1 µg), and 3 µg of the empty vector Bluescript KS
and treated with the various compounds as indicated. CAT activity is
relative to the ß-galactosidase activity originating from the
internal control as described in Materials and Methods.
Activation is given relative to the maximal GRE5-tk-CAT
activity obtained with 10-6 M Dex (100%).
Transrepression assays (B). For AP-1 transrepression, cells were
transfected with the Coll(-517/+63)CAT reporter (3 µg), together
with pSVc-Jun (250 ng), polyIIßGal (1 µg), and 1 µg Bluescript
KS. The cells were treated with the GR ligands as indicated. The
Coll(-517/+63)CAT reporter was only weakly active in absence of
pSVc-Jun; cotransfection of 250 ng pSVc-Jun resulted in a 20-fold
induction of transcription (data not shown). Repression of the
Coll(-517/+63)CAT reporter in the presence of 10-7
M Dex was taken as -100%. Positive (transactivation) and
negative (AP-1 transrepression) effects of the glucocorticoids are
represented as positive and negative values, respectively. The results
presented are means ± SEM of at least three independent
experiments.
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Three of the analogs tested were not, or only weakly, able to confer
transactivation ability on GR, whereas its AP-1 transrepression
activity was fully retained or only marginally reduced in the presence
of these glucocorticoids (hereafter collectively referred to as
"dissociated" glucocorticoids). Their structures are presented in
Fig. 2
. For example, 1 µM RU24858
activated transcription from the GRE5-tk-CAT reporter only
25% as efficiently as the same concentration of Dex (Fig. 1A
), but was similarly potent (85%) in repressing
AP-1-induced transcription from the collagenase promoter (Fig. 1B
).
Note that the IC50 of RU24858 for AP-1 transrepression was
20 nM, close to that of prednisolone (Fig. 1B
). Two other
compounds, RU24782 and RU40066, exhibited similar dissociated
transactivation and AP-1 transrepression characteristics (see Table 1
and Fig. 1
; note that the results given in Table 1
correspond to 100 nM of the indicated ligand). In
vitro competition assays with GR-bound [3H]RU28362,
a pure glucocorticoid agonist (28), or [3H]Dex revealed
that RU24858 and RU24782 bound to GR with a similar relative affinity
as Dex, while RU40066 displayed a 3.7-fold lower affinity (Table 1
and
data not shown). In view of their low agonistic potential and high
affinity to GR, we expected the three dissociated glucocorticoids to
act as efficient antagonists of Dex-induced transactivation. Indeed,
RU40066, at 100 and 1000 nM, efficiently antagonized both
10 nM Dex-induced GRE-tk-CAT (transient transfection) and
TAT (non-transfected HTC cells) activities (Fig. 3
).
Surprisingly, however, a 100-fold excess of RU24858 was unable to
antagonize the same Dex-induced activities (Fig. 3
), although in
vitro it exhibited an even higher affinity to GR than Dex (Table 1
). In these experiments RU24782 was able to partially antagonize the
Dex-induced activities. As in vitro binding assays, RU24858,
RU24782, and RU40066 were shown to compete with
[3H]RU28362; we also measured transcriptional activation
of the GRE-tk-CAT reporter and induction of the TAT activity in Hela
and HTC cells, respectively, exposed to 5 or 10 nM RU28362
with increasing amounts of the dissociated compounds. Again, RU40066
was able to antagonize RU28362-induced transactivation and RU24782
displayed partial antagonist activity, whereas a 100- or 500-fold
excess of RU24858 was unable to antagonize RU28362-induced activity
(data not shown). Thus, apparently dissociated glucocorticoids with a
prevalent potency for AP-1 repression can be designed which are nearly
devoid of both agonistic and antagonistic activities.

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Figure 2. Structure of the Dissociated Glucocorticoids
Analogs
The structure of Dex is also shown for comparison.
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Figure 3. The Dissociated Glucocorticoid RU24858 Does Not
Antagonize Dex-Induced Transactivation in Intact Cells
A, HeLa cells, transfected with the GRE5-tk-CAT reporter
(and the internal control, see Materials and Methods),
were exposed to Dex alone or 10 nM Dex with increasing
amounts of the indicated dissociated glucocorticoids. The normalized
CAT activity induced by the endogenous HeLa cell GR in the presence of
1 µM Dex was taken as 100% induction. B, Analysis of
tyrosine amino transferase (TAT) activity (see Materials and
Methods for details) originating from transactivation of the
endogenous TAT gene by the endogenous GR in HTC cells under identical
conditions as in panel A. The enzymatic activity measured in
non-ligand-treated control cells was subtracted, and the results were
expressed relative to the maximal activity measured in cells treated
with 1 µM Dex (100%). The results shown are the
means ± SEM of at least three independent
experiments.
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Divergent Potencies of Dissociated Glucocorticoids in TAT Induction
and Inhibition of IL-1ß Secretion
Transcription of the TAT gene, one of the enzymes involved in the
glucocorticoid-dependent stimulation of neoglucogenesis, is induced by
glucocorticoids in vitro and in vivo, due to the
presence of GREs in the TAT gene promoter (29). To assess the activity
of the various dissociated glucocorticoids identified as described
above with an endogenous GR target gene, rat HTC cells were treated
with the synthetic glucocorticoids, and the resulting TAT activity was
measured. A 10-fold induction was observed in cells treated with Dex at
1 µM (100% in Fig. 4A
; and data not
shown). Prednisolone (1 µM) induced TAT to 93% of the
stimulation observed with 1 µM Dex (Fig. 4A
).
Importantly, only a very weak induction of TAT activity was observed in
HTC cells treated with the different dissociated glucocorticoids, in
keeping with the transactivation results obtained in Hela cells
transiently transfected with GRE5-tk-CAT (Figs. 1A
and 4A
).
For example, at 1 µM, RU24858 and RU24782 induced TAT
activity to 11% and 17%, respectively, of the stimulation seen with 1
µM Dex. RU40066, like the antagonist RU486, did not
significantly induce TAT activity (Fig. 4A
).

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Figure 4. Dissociated Glucocorticoids Are Weak
Transactivators of Tyrosine Amino Transferase (TAT) in Cultured Liver
Cells but Potent Inhibitors of LPS-Induced IL-1ß Secretion in the
Human Monocytic Cell Line THP 1
A, HTC cells were treated with 1 µM of either Dex (DEX),
prednisolone (PRED), RU24782, RU24858, RU40066, or RU38486 (RU486). TAT
activities were determined as described in Materials and
Methods and expressed relative to the activity seen with 1
µM Dex (100%). Note that half-maximal activation of TAT
induction was observed at Dex and prednisolone concentrations, which
resulted also in half-maximal stimulation of transactivation from the
GRE5-tk-CAT reporter gene in transfected Hela cells,
i.e. 9 nM, and 40 nM,
respectively (see Fig. 1 ). B, THP 1 cells, activated with 5 µg/ml
LPS, were treated for 18 h with 1 µM of the
indicated compounds. IL-1ß was measured in 50 µl of the culture
supernatant, using the ELISA kit from R&D Systems. IL1ß level was
undetectable in untreated control cell supernatants. LPS-treated cells
secreted 13 ng/ml of IL-1ß. Inhibition of IL-1ß secretion in the
presence of 1 µM Dex was taken as -100%. The results
presented are means ± SEM of at least three
independent experiments.
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IL-1ß plays a central role in the regulation of inflammatory
responses (for review see Ref. 30 and refs therein). Its induction is
repressed by glucocorticoids at both the transcriptional and
posttranscriptional level (31, 32, 33). To investigate whether dissociated
glucocorticoids would also exibit IL-1ß-repressing activity, their
effect on IL-1ß secretion was measured in THP1 cells, a monocytic
cell line that responds to lipopolysaccharide (LPS) stimulation by
secreting high amounts of IL-ß and tumor necrosis factor-
(TNF
). Dex, which at 1 µM inhibited 75% of
LPS-induced IL-1ß secretion (not shown), was taken as the reference
compound (-100% in Fig. 4B
). Note that the same IC50
values (2 nM) were observed for the Dex-dependent
inhibition of LPS-induced IL-1ß secretion from THP1 cells and the
c-Jun-induced collagenase promoter activity in transiently transfected
Hela cells (Fig. 1
, and data not shown). At 1 µM, the
dissociated glucocorticoids RU24782 and RU24858 inhibited IL-1ß
expression 70% and 95%, respectively, of the maximum inhibition
obtained with Dex (Fig. 4B
). RU40066 was inactive at doses up to 10
nM and displayed moderate activity (65%) at high
concentrations only, with an IC50 of 0.3 µM
(Fig. 4B
, and data not shown). As was the case for Dex, the
IC50 values for inhibition of IL-1ß secretion by
prednisolone, RU24858, and RU24782 were very close to those determined
for AP-1 transrepression in transiently transfected Hela cells (the
following IC50 values were obtained: prednisolone, 6
nM; RU24858, 30 nM; RU24782, <20
nM; compare with Fig. 1B
). It is noteworthy that similar
results were obtained with the dissociated glucocorticoids for the
inhibition of LPS-actived IL-1ß secretion from peripheral blood
mononuclear cells (data not shown).
Dissociated Glucocorticoids Display Antiinflammatory and Thymolytic
Activity in Vivo
Glucocorticoids are major tools in the therapy of inflammatory
disorders. Encouraged by the observation that expression of IL-1ß, an
immunomodulator at the top of the inflammatory cascade, was affected by
dissociated glucocorticoids, we investigated whether these compounds
could also exibit antiinflammatory activity in vivo. Two
classic animal models were selected. In the "cotton pellet
granuloma" test (34) the compounds were given orally to rats using
Dex and prednisolone as reference compounds. RU24858 and RU24782
displayed a similar antiinflammatory activity as shown by the
inhibition of granuloma formation; the corresponding ED50
values were estimated to 7 mg/kg and 5 mg/kg, respectively (Table 2
).
Dexamethasone, prednisolone, RU24858, and RU24782 inhibited granuloma
formation up to 80%. Prednisolone exhibited maximal efficiency above 5
mg/kg, whereas the activity of RU24782 in this assay plateaued off
above doses of 10 mg/kg (Fig. 5
). Although these
compounds were 50 to 70 times less active than Dex, their activities
were very similar to that of prednisolone (ED50 of 2.5
mg/kg; Table 2
); in this test system RU40066 was
inactive at the dose of 10 mg/kg, but was not tested at higher
doses.

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Figure 5. Dissociated Glucocorticoids Display in
Vivo Antiinflammatory Activity in the Cotton Pellet Granuloma
Model
Two cotton pellets (10 mg each) were inserted subcutaneously into the
upper dorsal area of female Wistar rats. Test compounds were
administered orally, once a day, for 4 days. Eight animals were
included for each treatment. Twenty-four hours after the last
administration the pellet dry weight was determined (see
Materials and Methods). The activities of the various
synthetic glucocorticoids were expressed as percent inhibition of
granuloma weight increase. **, P < 0.01.
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The "croton oil-induced ear edema" model (35) was the second
test selected to study the antiinflammatory activity of the dissociated
glucocorticoids in vivo and was performed in mice. In this
model, the antiinflammatory activity of compounds is determined by
their ability to prevent local inflammation induced by croton oil.
Topically applied RU24858 and RU24782 displayed very strong
antiinflammatory activities with ED50 values of 2 µg/ear
and 10 µg/ear, respectively. Note that RU24858 was only 2-fold less
efficient than Dex and 2-fold more efficient than prednisolone (Table 2
). RU40066 displayed a significant, albeit low activity when applied
topically (Table 2
).
Notably, RU486, which displayed some transrepression activity with the
Coll-CAT (but not TRE-tk-CAT; Table 1
) reporter, was completely
inactive in the cotton pellet granuloma and croton oil-induced ear
edema models at concentrations up to 100 mg/kg (Table 2
). Finally, two
further animal models, the "mouse zymosan paw edema" (36) and
"rat carrageenin paw edema" (37), confirmed that the
antiinflammatory potency of RU24858 was, albeit lower than that of Dex,
similar to that of prednisolone (data not shown).
As the immunosuppressive activity of classic glucocorticoids is linked
to their ability to induce lymphocyte apoptosis, we measured thymolytic
activity of dissociated glucocorticoids in the same rats that were used
for the cotton-pellet granuloma test. The doses at which a reduction of
thymus weights could be observed were generally lower than those needed
for inhibiting granuloma formation (Table 2
). RU24782 and RU24858
displayed the same activity (ED50 estimated to 2.5 mg/kg)
which was, as in the granuloma test, near to that of prednisolone (1.6
mg/kg). Doses of 5 mg/kg of RU24858 or RU24782 induced a thymus weight
reduction of 81% and 77%, respectively, compared with control
animals. Notably, even at 1 mg/kg, these glucocorticoids exhibited
significant activity (25% reduction of thymus weight,
P < 0.05), whereas they were inactive at these doses
in the granuloma test (data not shown). As expected, Dex was highly
active in the thymolysis test (with an ED50 below 0.05
mg/kg) since at this lowest dose tested, the decrease of thymus weight
was 60% (data not shown). As previously observed in the cotton-pellet
granuloma, RU40066 was inactive at the dose tested (10 mg/kg). Taken
together, in several in vivo animal models, the dissociated
glucocorticoids RU24858 and RU24782, which are weak activators of
positively regulated GR target genes, displayed consistently a strong
antiinflammatory action that was similar, or even superior, to
that of the classic antiinflammatory glucocorticoid prednisolone.
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DISCUSSION
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A Novel Class of Dissociated Glucocorticoids
Three classes of glucocorticoids have been distinguished by their
ability to induce/inhibit certain functions of GR: agonists, and type I
and type II antagonists (2). Agonists may induce the activation
function AF-2 in the ligand-binding domain (LBD) with varying
efficiencies (38), while in the presence of type I antagonists, such as
RU486 (39, 40, 41), GR AF-2 will be inactive (38, 42). Type I antagonists
do not impair the activity of the activation function AF-1, located in
the N-terminal region A/B of GR (42, 43) and may act as cell-specific
agonists, since it has been shown in the case of the estrogen receptor
that its AF-1 acts in a cell-specific manner in the presence of the
type I antagonist hydroxytamoxifen (44). In contrast, type II
antiglucocorticoids, such as RU43044, are defined by their ability to
impair DNA binding of GR and, thus, do not induce any significant
transcriptional activity of GR target genes (43).
With the aim of better understanding how glucocorticoids enable GR to
transrepress AP-1 activity (see Introduction for references), we have
compared AP-1 inhibition in the presence of the three above types of
ligands and, moreover, screened a library of glucocortcoids to find
ligands that would inhibit AP-1 activity without significantly inducing
agonistic activity (termed "dissociated" glucocorticoids).
Confirming the original data of Jonat et al. (9), we
observed that in transfected HeLa cells the type I antiglucocorticoid
RU38486 was unable to repress AP-1-induced transactivation of a
TRE5-tk-CAT reporter (Table 1
), and, indeed, could reverse
the effect of the agonist Dex (data not shown). With a collagenase
promoter-based reporter, some RU38486-dependent AP-1 repression was
observed but the antagonist still reversed the repression induced by 10
nM Dex (Fig. 6
). No transrepressing ability
was conferred onto GR in the presence of the type II antagonist RU43044
with any of the two AP-1 reporters (Table 1
). Note that in the above
cases transrepression was via the endogenous GR; transiently expressed
exogenous GR can apparently further increase the AP-1 inhibition (17)
but leads to nonphysiological GR concentrations. In conclusion, neither
type I nor type II antiglucocorticoids have the characteristics of
dissociated glucocorticoids and, thus, have only limited promise for
use as antiinflammatory agents (see below).

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Figure 6. The Classic Antiglucocorticoid RU38486 Induces Only
Weak AP-1 Transrepression with the Endogenous Hela Cell GR and
Antagonizes the Effect of High Dex Concentrations
Hela cells were transfected as described in the legend to Fig. 1B . AP-1
transrepression of c-Jun-induced Coll(-517/+63)CAT transcription was
measured after treating the cells with increasing concentrations of Dex
or RU38486 (RU486) alone, or with 10 nM Dex and increasing
concentrations of RU486.
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Screening of a library of glucocorticoids yielded three molecules
(RU24782, RU24858, and RU40066) that exhibited the dissociated
characteristics. All of them exerted residual agonistic activity (9%
to 35% of that of Dex) but induced 58% to 83% of the anti-AP-1
activity seen with Dex (Table 1
). Note in this context that
prednisolone, widely used as antiinflammatory glucocorticoid, showed a
similar anti-AP-1 activity in transfected cells (Table 1
). The most
interesting of these compounds is RU24858, since it exemplifies a novel
class of glucocorticoids: despite a binding affinity that is even
higher than that of Dex, RU24858 does not significantly antagonize
Dex-induced transcription (Fig. 3
). However, in in vitro
binding assays RU24858 does efficiently compete with radiolabeled Dex
(and other glucocorticoid agonists) for binding to the GR. This implies
that, in contrast to classic glucocorticoids, the binding affinity of
RU24858 to transcriptionally engaged GR in vivo is lower
than that measured in vitro. Importantly, however, this
lower in vivo binding affinity does not significantly
decrease the ability of the GR-RU24858 complex to repress AP-1
activity. We conclude from these data that AP-1 repression involves
very specific ligand-induced structural alterations of GR.
How can RU24858 bind GR with different affinities in
vitro and in vivo? Transcriptional
interference/squelching studies have suggested that transcriptional
intermediary factors (TIFs, also termed mediators, coactivators,
bridging factors) mediate the ligand-dependent activation function AF-2
of steroid receptors to the transcriptional machinery (45, 46, 47).
Putative TIFs have been recently isolated and characterized on the
basis of their ligand-dependent interaction with several nuclear
receptors (48, 49, 50, 51, 52, 53, 54). We propose that the interaction between the LBD and
TIF(s) (or any other factor that interacts with the GR LBD in
vivo) may differentially affect the binding of Dex and RU24858,
resulting in a decreased affinity of RU24858, but not Dex, to the
GR-TIF complex. However, noncomplexed GRs, or a set of GR complexes
distinct from those involved in transactivation, may bind RU24858 with
high affinity and give rise to the strong AP-1 inhibition observed in
the presence of this compound. Note that there is a precedent for an
altered affinity of glucocorticoids upon GR complex formation, since
different affinities have been reported for the GR and the GR-hsp90
complex (55, 56).
Taken together, the above results suggest that we have identified a
novel class of glucocorticoids that are neither efficient agonists nor
antagonists in vivo, but can very efficiently induce
GR-dependent AP-1 repression.
In Contrast to Classic Antiglucocorticoids, Dissociated
Glucocorticoids Are Strong Inhibitors of LPS-Induced IL-1ß
Secretion
Glucocorticoids are powerful antiinflammatory agents, most likely
due to their ability to block the expression of multiple cytokines.
Inhibition of cytokine expression has been reported to occur at the
transcriptional level for IL-1 to IL-6, IL-8, TNF
,
colony-stimulating factor (CSF)-1/macrophage (M)-CSF, granulocyte
macrophage (GM)-CSF, and
-interferon (IL-1 expression is blocked at
other levels as well; TNF and GM-CSF expression may be blocked through
degradation of their mRNAs; for reviews see Refs. 27, 57, 58, 59, 60 , and
references therein). Most of these genes require transcription factors,
such as AP-1, NF-
B, or NF-AT, for their expression, suggesting that
glucocorticoids exert their antiinflammatory functions by negatively
interfering with the activity of (some of) these factors. Dissociated
glucocorticoids, such as RU24858, were as potent inhibitors of IL-1ß
secretion as prednisolone in LPS-stimulated cultured human monocytes,
whereas classic antiglucocorticoids with no (RU43044) or
promoter-dependent AP-1-transrepression activities (RU486)
inhibited IL-1ß secretion only weakly (RU486) or not at all (RU43044)
in this assay (Fig. 4
and data not shown).
In Vivo Dissociated Glucocorticoids Are
Antiinflammatory Agents as Potent as Prednisolone
To investigate whether dissociated glucocorticoids could be
active as antiinflammatory drugs in vivo, we studied two
established animal models, the cotton pellet granuloma model, in which
the glucocorticoid is given orally to rats, and the croton oil-induced
ear edema model, in which the drug is applied topically to mice (see
Materials and Methods for details and references).
Importantly, in these in vivo models, RU24858 was as active
as prednisolone (Fig. 5
and Table 2
). In the croton oil-induced ear
edema model, which relies on phorbol ester-induced skin inflammation,
RU24858 was even 2-fold more active than prednisolone, and RU24782 was
about half as active as prednisolone. It is unclear why RU40066 was
inactive; at present, no data on the metabolism and stability in
vivo of this compound are available. Importantly, in keeping with
their inability to inhibit IL-1ß secretion, the classic antagonists
RU486 and RU43044 were completely inactive in these in vivo
tests (Table 2
and data not shown).
The immunosuppressant potential of glucocorticoids relates to
their ability to induce T cell apoptosis and to inhibit cytokine gene
expression. These activities are therapeutically exploited in organ
transplantation and in treatment of leukemias. We compared, in the
cotton pellet granuloma model, the extent of thymolysis induced by
classic (anti)glucocorticoids and the novel class of dissociated
glucocorticoids. Again we observed a high activity of RU24858 and
RU24782, similar to that of prednisolone, while antiglucocorticoids
were inactive (Table 2
and data not shown).
Dissociated Glucocorticoids May be Novel Tools to Improve
Antiinflammatory Glucocorticoid Therapy
We describe here a novel class of dissociated glucocorticoids,
distinct from the various previously reported agonists and antagonists.
These compounds efficiently transrepress AP-1 activity, while only
marginally activating glucocorticoid target genes. The dissociated
glucocorticoids have maintained the antiinflammatory and, according to
our as yet limiting data, also the thymolytic capacity of classical
glucocorticoid agonists. Moreover, its prototypic member RU24858 is as
active, or, depending on the system, even more active, than
prednisolone in standard in vivo models.
Hypothalamic-pituitary-adrenal axis insufficiency, osteoporosis,
diabetes, steroid myopathy, and infectious and neuropsychiatric
complications limit the therapeutic use of classic glucocorticoid
agonists (61). At least some of these complications are likely to
derive from the agonistic activities of the classic glucocorticoids,
and it has not escaped our attention that our glucocorticoid analogs,
provided that the dissociation of transactivation and transrepression
is maintained in target organs, have the potential to constitute a new
class of antiinflammatory glucocorticoids with significantly reduced
side effects. Studies are under way to confirm this hypothesis.
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MATERIALS AND METHODS
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Plasmids
GRE5-tk-CAT, TRE5-tk-CAT reporters, and
pSVc-Jun have been described (15, 62). The polyIIßGal used as
internal control was constructed by inserting the
HindIII-BamHI fragment of pCH110 (Pharmacia,
Piscataway, NJ) containing the coding sequence of the ß-galactosidase
gene into the HindIII-BglII sites of
pG4MpolyII (63). The plasmid Coll(-517/+63)CAT was a kind gift of C.
Wasylyk.
Cell Culture and Transfection
Hela cells were cultured in DMEM supplemented with 10% FCS and
grown at 37 C and 5% CO2. Transient transfection assays
for transactivation or AP-1 transrepression were carried out using the
calcium phosphate coprecipitation procedure. Briefly, 4 x
105 cells were seeded 24 h before transfection in
six-well plates containing DMEM supplemented with 5%
charcoal/dextran-treated serum. For transactivation assays, HeLa cells
were transfected with 1 µg GRE5-tk-CAT reporter, 1 µg
polyIIßGal, and 3 µg Bluescript KS (Stratagene, La Jolla, CA). For
transrepression assays, HeLa cells were transfected with 3 µg of the
Coll(-517/+63)CAT reporter, 1 µg polyIIßGal, 250 ng pSVc-Jun
expression plasmid, and 1 µg Bluescript KS. Sixteen hours later,
cells were rinsed with Earles Balanced Salt Solution (EBSS), the
medium was replaced, and the hormones were added as indicated. After
24 h, cells were washed with ice-cold EBSS and resuspended in 250
µl of lysis buffer provided with the chloramphenicol
acetyltransferase enzyme-linked immunosorbent assay (CAT ELISA) kit
(Boehringer Mannheim, Indianapolis, IN). The amount of CAT enzyme was
quantified according to the manufacturers instructions. Results were
normalized according to the ß-galactosidase activity originating from
cotransfected polyIIßGal and plotted relative to the transactivation
seen with 1 µM Dex (=100%) or relative to the AP-1
transrepression exerted by 1 µM Dex (=-100%).
SEMs were calculated from a minimum of three independent
assays per compound and per concentration.
Determination of Relative Binding Affinities
Relative binding affinities were determined by incubating HeLa
cell cytosol for 24 h at 0 C with either [3H]RU28362
(28) or [3H]Dex with or without different concentrations
of competitor steroids. Bound and free ligands were separated by the
dextran-coated charcol method (38). The relative binding affinity of
Dex was taken as reference (100%). Similar results were obtained with
crude SF9 cell extracts containing high levels of human GR expressed
from recombinant baculoviruses.
TAT Assay
HTC cells were cultured in DMEM supplemented with 10% FCS and
kept at 37 C and 5% CO2. For steroid treatment
106 cells per well were seeded into six-well plates. After
24 h, at confluency, the medium was replaced by DMEM without
serum, and hormones were added for 18 h. TAT activity was modified
from Ref. 64 to be measured in 96-well plates. The endogenous enzymatic
activity measured in control cells was subtracted, and the results were
expressed relative to the maximal activity measured in cells treated
with 10-6 M Dex.
Assay of IL-1ß Secretion
The monocytic cell line THP1 (ATCC: TIB202) was cultured in RPMI
1640 supplemented with 10% FCS and grown at 37 C and 5%
CO2. IL-1ß secretion was induced by treating the cells
with 5 µg/ml LPS (Sigma, St. Louis, MO); glucocorticoids were added
simultaneously. After 18 h, supernatants were collected and
IL-1ß was quantitated using the human IL-1ß ELISA kit from R&D
Systems (Minneapolis, MN).
Antiinflammatory Activity in the Cotton-Pellet Granuloma Test
The cotton-pellet granuloma assay was performed as described
previously (34). Two cotton pellets (10 mg each) were inserted
subcutaneously into the upper dorsal area of female Wistar rats (weight
range 90100 g, Iffa Credo, France). Test compounds were administered
orally, once a day, for 4 days. Twenty-four hours after the last
treatment, the animal was killed and the pellet, along with the
surrounding granuloma, was carefully dissected from the animal and its
dry weight was determined. For this, granuloma and pellet were heated
at 60 C overnight. By subtracting the initial weight of the pellet, the
dry weight of the granuloma was determined. The activities of the
various synthetic glucocorticoids were expressed as ED50
values (dose causing a reduction of granuloma weight by 50%).
Thymolytic Activity
The thymolytic activity of the synthetic compounds was
determined by weighing the thymus of the rats treated in the granuloma
test. The ED50 values correspond to the glucocorticoid
doses inducing a 50% decrease in thymus weights.
Topical Antiinflammatory Effect on the Croton Oil-Induced Ear
Edema
This test was carried out as described previously (35) on groups
of eight male OF1 mice weighing 1822 g (Iffa Credo, LArbresle,
France). The edema was induced on one ear by the application of a
solution of croton oil (2% vol/vol) in pyridine-water-ether 4:1:14.6
(by volume). Animals were killed 6 h later, and the ears were
removed and weighed. Edema was determined from the difference in weight
between the irritant-treated and the contralateral ear. The compounds
to be tested were dissolved in the croton oil solution and topically
applied on the ear. ED50 values correspond to the doses
reducing the control edema by 50%.
 |
ACKNOWLEDGMENTS
|
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We thank Dr. Michel Pons (Montpellier, France) for the kind gift
of HTC cells. We are grateful to the staff members of the endocrinology
department, and especially to Dr. Martine Gaillard and Dr. André
Ulmann for their help and support. Dr. F. Fassy is acknowledged for her
help and advice in setting up the TAT assay. We are grateful to O.
Krebs for her contribution in the set-up of the CAT ELISA assay.
Evelyne Cérède and Françoise Bouchoux are
acknowledged for their essential contribution in binding experiments.
Finally, we wish to thank Dr. Neerja Bhatnagar for compound synthesis
and Elisa Francesconi and Françoise Perrier for their excellent
technical assistance.
 |
FOOTNOTES
|
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Address requests for reprints to: Michele Resche-Rigon, ROUSSEL UCLAF, 102 route de Noisy, 93235 Romainville Cedex France.
1 Present address: INSERM U 248, Institut Curie, 26 rue dUlm, 75231
Paris Cedex 05. 
2 S. Dupont is currently working at Institut de Génétique
et de Biologie Moléculaire et Cellulaire. 
Received for publication November 27, 1996.
Revision received May 14, 1997.
Accepted for publication May 19, 1997.
 |
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