(Received for publication, May 21, 1997, and in revised form, June 23, 1997)
From the Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Novum F60, S-141 86 Huddinge, Sweden
Glucocorticoids inhibit NF-B signaling by
interfering with the NF-
B transcription factor RelA. Previous
studies have identified the DNA-binding domain (DBD) in the
glucocorticoid receptor (GR) as the major region responsible for this
repressive activity. Using GR mutants with chimeric DBDs the repressive
function was found to be located in the C-terminal zinc finger. As
predicted from these results the mineralocorticoid receptor that
contains a C-terminal zinc finger identical to that of the GR was also able to repress RelA-dependent transcription. Mutation of a
conserved arginine or a lysine in the second zinc finger of the GR DBD
(Arg-488 or Lys-490 in the rat GR) abolished the ability of GR to
inhibit RelA activity. In contrast, C-terminal zinc finger GR mutants with mutations in the dimerization box or mutations necessary for full
transcriptional GR activity were still able to repress RelA-dependent transcription. In addition, we found that
the steroid analog ZK98299 known to induce GR transrepression of AP-1
had no inhibitory effect on RelA activity. In summary, these results demonstrate that the inhibition of NF-
B by glucocorticoids involves two critical amino acids in the C-terminal zinc finger of the GR.
Furthermore, the results from the use of mineralocorticoid receptor and
anti-glucocorticoids suggest that the mechanisms for GR-mediated
repression of NF-
B and AP-1 are different.
Glucocorticoid hormones regulate many different biological
processes via a specific intracellular receptor, the glucocorticoid receptor (GR),1 which is
present in most cell types. The GR is a member of the superfamily of
nuclear receptors, which all contain three main functional domains (1,
2). After binding of hormone to the C-terminal ligand binding domain
(LBD) and dissociation of heat shock proteins, the GR homodimerizes and
interacts with specific DNA sequences termed glucocorticoid response
elements (GREs) through its central DNA binding domain (DBD). The
transcriptional activity of GR is mainly dependent on the 1 domain
localized in the N-terminal part of the protein (3, 4). The highly
conserved DBD contains two zinc fingers and in each of them a zinc ion
is tetrahedrally coordinating four cysteine residues. One function of
the DBD is to discriminate between different response elements, thus
determining target genes to be activated (5, 6). This function is
achieved by a few amino acids localized in the C-terminal part of the
N-terminal zinc finger, the so-called the P box. A second subdomain
termed the D box in the N-terminal knuckle of the C-terminal zinc
finger has been shown to harbor amino acid residues important for
homodimerization (7). DNA binding of the ligand-activated GR results in
an increased rate of formation of transcriptionally competent
pre-initiation complexes. This is thought to be achieved by
protein-protein interactions between the receptors and different
components of the transcriptional machinery (8, 9). Besides the more
well studied transcriptional activation process, the GR can repress
transcription via different mechanisms (10). The GR has been shown to
bind to overlapping DNA response elements for other transcription
factors leading to repression (11-13). These GR binding elements have
been termed negative GREs. Inhibition of gene expression by
glucocorticoids can also occur in the absence of GR DNA binding. The
most well studied system for this is the repression by the GR of genes
activated by the AP-1 transcription factor complex. In this case there
is evidence for a direct physical association between the proteins present in AP-1 and the GR (14-16). Although direct binding of the GR
to DNA is not necessary, the DBD has in some cases been shown to be
essential for this interaction (15). Furthermore, the composition of
the AP-1 complex determines whether the GR will cause a positive or a
negative effect on AP-1 controlled transcription. In addition to the
ability of GR to interfere with AP-1 controlled transcription, we and
other (17-20) have shown that the GR also can repress NF-
B
signaling.
NF-B is an inducible transcription factor complex that plays an
essential role in the inflammatory and immune responses (21). It is
activated by a diverse range of signals including the pro-inflammatory cytokines tumor necrosis factor-
and interleukin-1 as well as phorbol esters, physical or oxidative stress, and bacterial and viral
proteins. In most cells the NF-
B is composed of a heterodimer between RelA (p65) and NFKB1 (p50), where the RelA protein is responsible for the transactivation potential. In the non-activated state the NF-
B is sequestered in the cytoplasm through the
interaction with the inhibitory protein I
B. During activation, the
I
B protein becomes phosphorylated and degrades allowing NF-
B to
translocate to the nucleus where it binds to specific DNA elements and
subsequently regulates genes involved in inflammation and immune
responses (22-24).
Glucocorticoids have potent immunosuppressive effects and are commonly
used in the clinic to suppress different immunological and inflammatory
responses. Different molecular mechanisms have been suggested to be
involved in this process including inhibition of AP-1 and NF-B (25).
In an earlier report (19) using transient transfections of GR and
NF-
B responsive reporter genes, we have shown a mutual
transcriptional inactivation between the GR and the NF-
B protein
RelA. Direct DNA binding of GR is not required for the NF-
B
repression to occur, since a GR, in which the P box had been mutated so
that it no longer recognized a GRE, still was able to repress. Instead
we showed that the GR and the RelA can directly or indirectly interact
with each other in vitro and mutually interfere with
transcriptional activity. Using deletion mutants and chimeric
receptors, we demonstrated that the GR DBD is the major GR domain
responsible for repression of RelA activity. In addition, an
alternative mechanism for glucocorticoid repression of NF-
B activity
has been suggested, which involves induction of I
B
by GR leading
to the sequestering of NF-
B in the cytoplasm (26, 27).
In this report we investigated the subdomain and critical amino acids
in the GR DBD involved in glucocorticoid repression of the NF-B
protein RelA. In addition, we investigated the role of GR dimerization
and transactivation for the repression of RelA activity. Finally, we
analyzed the ability of glucocorticoid antagonists to cause repression
of NF-
B activity.
Deep Vent® DNA polymerase and T4 DNA ligase were from New England Biolabs. Media, antibiotics, fetal bovine serum, and Lipofectin® were purchased from Life Technologies, Inc. Dexamethasone and aldosterone was obtained from Sigma. The monoclonal antibody number 7 against rat GR has previously been described (28). RU486 was obtained from Roussel-UCLAF (Romainville, France) and ZK98299 was from Schering, Berlin, Germany.
Reporter and Expression PlasmidsThe luciferase reporter
plasmids 3xNF-B(IC)tk-LUC and (GRE)2tk-LUC (29, 30) and
the expression plasmid RcCMV-RelA (19) have been described previously.
GR mutants with chimeric DBDs were constructed using the
PCR-ligation-PCR protocol (31). For this purpose the DBDs of GR and
thyroid receptor
(TR
) were separated into three parts as
follows: the N-terminal zinc finger, the linker region between the two
fingers, and the C-terminal zinc finger, and termed ggg or ttt,
respectively (the first, second, and third lowercase letter
representing the N-terminal zinc finger, linker region, and C-terminal
zinc finger region of GR (g) or TR
(t), respectively). The
expression plasmids GRnx (32) and GTG (33) in this report
named GgggG and GtttG, respectively, were used for the amplification of
the different DBD regions. The first PCR reaction contained 1 ng of
plasmid DNA, 250 µM each dNTP, 1 µM
primers, 1 × reaction buffer, and 1 unit of Deep Vent® DNA
polymerase. The PCR program contained a 30-s denaturation step at
94 °C, 30 s annealing time at 56 °C, and a 30-s extension at
72 °C, for 25 cycles. Plasmid DNA templates and pairs of primers were as follows: PCR-1, GgggG, 5
-AAGCCCCAGCATGAGACCAGAT-3
(primer A)
and 5
-GCAGCCTTCACACGTGATA-CAG-3
; PCR-2, GgggG, primer A and 5
-ACATAGGTAATTGTGCTGTCCTTCC-3
; PCR-3, GgggG,
5
-GCTGGAAGGAATGATTGCATC-3
and 5
-ACTCCTGTAGTGGCCTGCTGAA-3
(primer
B); PCR-4, GtttG, 5
-AAGGGTTTCTTTAGAAGGACCATTC-3
and
5
-TACCAGGATTTTCAGAG-GTTTC-3
; PCR-5, GtttG,
5
-AAATATGAAGGAAAATGTGTCATAGACA-3
and primer B; PCR-6, GtttG, primer A
and 5
-ACAGGAATAGGATGGATGGAGATT-3
. After phosphorylation, 5 µl of
PCR-1, -2, and -3 were mixed with 5 µl of PCR-4, -5, and -6, respectively, and ligated with 400 units of T4 DNA ligase for 15 min at
room temperature. The resulting fragments were amplified in a second
PCR reaction using primers A and B and under the same conditions as for
the first PCR reaction. The PCR products were digested with
NotI and XhoI and inserted into the
GRnx expression plasmid instead of the wild type GR DBD to
create the chimeric GR/TR
-DBD mutants GgttG, GggtG, and GttgG. The
GgtgG mutant was constructed by using the GgttG expression plasmid as
template DNA instead of the GgggG in the PCR-6. The rat GR mutants
R488Q, K490E, and N491A were created by digesting the corresponding
yeast expression vectors (34) with NcoI and PstI,
and the isolated GR fragments containing the mutation were subcloned
into the corresponding sites of the mammalian rat GR expression vector
6RGR (35) after removing the wild type sequence. All constructs were
verified by sequencing.
Green monkey COS-1 cells
were grown in 1:1 mixture of Dulbecco's modified Eagle's medium and
Ham's F-12 medium supplemented with 7.5% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 5% CO2. Cells were
plated in 24-well multidishes at a density of 3 × 104
cells/well 24 h before transfection. Cells were transiently
transfected using Lipofectin® according to the recommendations of the
manufacturer. In transrepression experiments, cells were co-transfected
with 200 ng of 3xNF-B(IC)tk-LUC reporter plasmid, 2.5 ng of RelA
expression plasmid, and 25 ng of wild type or mutant GR expression
plasmid. In the transactivation experiments, cells were co-transfected with 200 ng of (GRE)2tk-LUC and 25 ng of wild type or
mutant GR expression plasmid. Following overnight exposure of the cells to the DNA/lipid mixture, fresh medium was added, and cells were incubated in the absence or presence of 1 µM
dexamethasone for 24 h. Cells were lysed and luciferase activity
was determined.
COS-1 cells were plated in 10-cm cell culture dishes and transfected with 1 µg of expression plasmids for wild type or mutant GR as described above. Cells were pelleted, resuspended in 200 µl of ETG buffer (1 mM EDTA, 10 mM Tris-HCl, pH 7.4, 10% (v/v) glycerol) containing 0.4 M KCl, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 1 mM dithiothreitol and homogenized with a micro-Dounce homogenizer by 20 strokes. The homogenates were centrifuged at 265,000 × g for 40 min at 4 °C. Protein concentrations were determined, and supernatants were mixed with 1 volume of 2 × SDS buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromphenol blue, 20% (v/v) glycerol), and 10 µg of total protein was separated by 10% SDS-polyacrylamide gel electrophoresis and electroblotted to a C-Extra Hybond membrane (Amersham Corp.). Immunodetection was carried out using the enhanced chemiluminescence detection kit from Amersham Corp. after incubation with the monoclonal antibody number 7 (28) followed by secondary horseradish peroxidase-labeled anti-mouse antibody (Amersham Corp.) according to the suggested protocols.
We and others (19, 20)
have previously shown that the DBD of the GR is of crucial importance
for the ability of GR to interfere with the NF-B-mediated response,
since deletion of the GR DBD or replacement of the GR DBD with the
corresponding TR
DBD abolished repression. To further determine if a
particular region in the GR DBD is responsible for the functional
interference with NF-
B activity, GR mutants were created in which
individual parts of the DBD were replaced by the corresponding regions
of the TR
DBD. For this purpose the GR DBD was divided into three parts, the N-terminal zinc finger, the linker region, and the C-terminal zinc finger, respectively (Fig.
1A). The ability of the
different GR mutants to repress NF-
B was tested on RelA-mediated transactivation of a luciferase reporter gene controlled by three NF-
B sites from the human intercellular adhesion molecule-1 promoter and a minimal thymidine kinase promoter (3xNF-
B(IC)tk-LUC) in COS-1
cells. Exchanging the whole GR DBD with the DBD from TR
(GtttG)
resulted in a 70-80% loss of repressive activity in comparison to the
wild type GR (GgggG) (Fig. 1B). Exchanging the C-terminal zinc finger alone (GggtG) or the linker region together with the C-terminal zinc finger (GgttG) with the corresponding region of TR
DBD destroyed the ability of the GR to inhibit RelA-mediated transactivation to a similar degree as for GtttG (Fig. 1B).
In contrast, exchanging the linker region alone (GgtgG) did not affect the repressive activity of GR. This suggested that this region was
dispensable and that the C-terminal zinc finger was critical for the
repression to occur. The importance of the C-terminal zinc finger was
confirmed with the GttgG mutant, since most of the repressive activity
was maintained when the C-terminal zinc finger alone was from the GR
(Fig. 1B). These results demonstrate that the contribution
of the GR DBD to the functional interference between GR and RelA is
localized in the C-terminal zinc finger of the GR.
Since the amino acid sequences of the C-terminal zinc finger of the GR and the mineralocorticoid receptor (MR) are identical (36), this would suggest that the MR is able to repress RelA-mediated transactivation. Indeed, in transfection experiments performed as above, the MR activated by 10 nM aldosterone repressed RelA-mediated transactivation as efficiently as the GR (data not shown). This also shows that the four amino acids outside the second zinc finger that differ between the MR and GR DBDs are not critical for the repressive capacity.
An Intact GR Dimerization Box Is Not Required for Repression of RelA-mediated TransactivationA major function for the C-terminal
zinc finger in the GR DBD is to contribute to receptor
homodimerization, a prerequisite for the receptor to bind DNA and
transactivate efficiently (7, 37). This function is achieved by the D
box region which is localized in the N-terminal knuckle of the
C-terminal zinc finger. Since dimerization and NF-B repression
functions are localized in the same zinc finger of the GR, we tested if
GR dimerization is a prerequisite for repression of Rel A
transactivation. For this purpose we used a GR mutant (D4X) in which
three amino acids out of five in the D box have been mutated (Fig.
2A) (38). Transfection experiments with the D4X mutant demonstrated that mutations in the D
box did not impair the ability of GR to repress RelA transcriptional activity (Fig. 2B). This shows that receptor dimerization is
not a prerequisite for GR-mediated repression of RelA. As previously shown, the D4X mutant harbored no significant transcriptional activity
(Fig. 2C).
Identification of GR Amino Acids Involved in Repression of RelA-mediated Transactivation
Previous studies have shown that in
addition to GR, the estrogen receptor, progestin receptor (PR), and the
androgen receptor (AR) also repress NF-B activity (39-41). In
addition, as shown in this study, the MR also has this capacity. In
contrast, the TR
, the retinoic acid receptor
isoform (RAR
),
and the ecdysone receptor are unable to repress RelA-mediated
transactivation (19).2 An
amino acid sequence comparison of the C-terminal zinc fingers of
repressive and non-repressive receptors revealed that the arginine and
the lysine in position 488 and 490 in the rat GR are conserved only in
the repressing receptors, suggesting an important role for these amino
acids in the repressive activity (Table
I). To test the importance of these amino
acids for GR-mediated repression of RelA activity, we exchanged these
amino acids in the rat GR to a glutamine and a glutamic acid,
respectively (R488Q and K490E, respectively), and tested the ability of
the mutated GRs for their ability to repress RelA transcriptional
activity in COS-1 cells. Transfection experiments showed that both GR
mutants, R488Q and K490E, had lost almost all their repressive activity
as compared with the wild type GR (Fig. 2B). In contrast,
the GR mutants N491A and LS7 with substitutions of amino acids that are
not conserved among the repressive receptors (Fig. 2A, Table
I) had retained their ability to repress RelA activity. None of these
C-terminal zinc finger GR mutants except the N491A mutant could
transactivate the (GRE)2tk-LUC reporter gene. The N491A
mutant retained approximately 50% transcriptional activity as compared
with the wild type GR (Fig. 2C). The inability of the
R488Q and K490E mutant to repress the RelA activity was not due
to poor expression of the receptor proteins, since Western blot
analysis of the transfected cells showed that the expression
levels of the mutated receptors were the same as for the wild type GR
(Fig. 2D). These results demonstrate that the arginine and
the lysine residues in positions 488 and 490 in the rat GR
(corresponding to positions 469 and 471 in the human GR) are critical
for GR-mediated inhibition of RelA-dependent transactivation.
|
The steroid analogs RU486 and ZK98299 are
antagonists of GR transactivation but are able to stimulate GR
transrepression of AP-1 activity (38). Furthermore, we and others (19,
20, 40) have previously shown that RU486 can also act as a partial agonist for GR- and PR-mediated repression of NF-B activity. We
tested if the steroid analog ZK98299 could inhibit NF-
B activity in
analogy to RU486. As shown in Fig.
3A, 10 nM RU486
repressed RelA activity to a level that was approximately 40% the
repression obtained with 10 nM dexamethasone. In contrast,
no repression was observed with 10 or 100 nM ZK98299. This
was not due to the lack of biological activity of ZK98299, since it
could inhibit GR transactivation as efficiently as RU486 (Fig.
3B). These results suggest that RU486 and ZK98299 induce GR
to states with different competence to repress RelA activity.
The recent discovery of an inhibitory cross-talk between the
NF-B and GR signaling pathways has provided one molecular mechanism by which glucocorticoids exert their potent anti-inflammatory effects
(17, 19, 20). The inhibition of NF-
B activity by estrogen receptor,
PR, and AR has also been reported (39-42). Here we show that the MR
also has the ability to repress RelA activity in a co-transfection
assay. Thus, all steroid receptors are able to repress NF-
B in
contrast to nuclear receptors from the RAR/TR subfamily, suggesting
that a distinct structural determinant within the steroid receptor
subfamily is responsible for the repression of NF-
B. The importance
of the DBD in these receptors for the repressive activity has been
demonstrated in several previous studies, where it was shown that
deletion or replacement of the whole DBD resulted in the loss of the
repressive activity (19, 20, 39-41). To identify which subdomain in
the GR DBD is responsible for the repression of RelA activity, we have
used GR mutants in which various parts of the GR DBD have been replaced
with the corresponding regions of the non-repressive TR
DBD. Our
results demonstrate that most of the repression of NF-
B activity
could be attributed to the C-terminal zinc finger of the GR DBD. This localizes a new function to this finger, which previously has been
known mainly to harbor functions important for dimerization and
transactivation (7, 37). Analysis of two C-terminal zinc finger GR
mutants with substitutions of the arginine (amino acids 488 in the rat
GR) and lysine (amino acids 490 in the rat GR) to a glutamine and a
glutamic acid, respectively, confirmed the importance of this finger
and identified two critical basic residues for repression of NF-
B
activity. No particular function has previously been attributed to the
arginine residue 488 (corresponding to amino acid 469 in the human GR)
with regard to dimerization or interaction with DNA, since substitution
of this residue to a glutamine did not impair DNA binding (34). The
lysine residue 490 (corresponding to amino acid 471 in the human GR),
on the other hand, is involved in making contact with the phosphate
backbone (43). Interestingly, these two residues are conserved only in the C-terminal zinc finger of steroid receptors, consistent with the
observation that only members of this subfamily of nuclear receptors
seem to be able to repress NF-
B. Mutation of either of these two
amino acids resulted in a significantly decreased transcriptional
activity of GR (this study and Ref. 34). However, no correlation
between transcriptional activation by GR and transrepression of RelA
activity exists, since the two GR mutants D4X and LS7, which lack most
of their transcriptional activity, were fully active with regard to
repression of RelA activity. This is also in line with previous data
showing that GR mutants with a deletion of the major transactivation
domain
1 or a substitution of the P box by that of the TR
were
still repressive (19, 20). Thus, glucocorticoid induction of the
NF-
B inhibitor I
B
which previously has been suggested as a
mechanism controlling NF-
B inhibition by glucocorticoids cannot
explain why transcriptionally deficient GR mutants can still repress
NF-
B (26, 27). In addition, it has been shown in monocytic U937
cells that inhibition of ICAM-1 gene expression by glucocorticoids
occurs in the absence of protein synthesis (44). More recently, several
studies also reported that NF-
B could be inhibited in osteoblast
U2-OS cells (39), alveolar epithelium-like A549/8 cells (45), kidney
epithelial NRK-52E cells (46), and aortic endothelial BAEC cells (47) in the absence of I
B
induction. Collectively, these data argue for a general mechanism for glucocorticoid inhibition of NF-
B which
involves GR transrepression via protein-protein interaction between
non-DNA binding GR and NF-
B transcription factors as initially
suggested (17, 19, 20). However, induction of I
B
may play a more
significant role in some specific cell types such as lymphocytes
(47).
Several results reported here together with previous observations
indicate that the mechanisms by which GR inhibits NF-B and AP-1
signaling pathways are different. Most notable is that MR, as reported
in this study, is an efficient inhibitor of NF-
B, whereas it is
known to be a very weak repressor of AP-1 activity (48, 49). In
contrast, RAR
is unable to repress NF-
B activity but is able to
inhibit AP-1 (19, 50). These differences may be related to the fact
that although the GR DBD is critical for repression of both NF-
B and
AP-1, different parts of the DBD contribute to this effect. This is
supported by the data from Heck et al. (38) who demonstrated
that point mutations within the N-terminal zinc finger of GR could
severely impair AP-1 repression, whereas our results indicate that this
zinc finger does not play a significant role in the inhibition of
NF-
B activity. In addition, previous data showed that GR mutants
with deletion of the N-terminal domain could efficiently inhibit
NF-
B but not AP-1 (15, 19, 20). It has recently been suggested that
the mechanism by which GR represses AP-1 involves competition between
GR and AP-1 for limiting amounts of the co-activator CBP/p300 (51).
Although, as recently reported, a physical interaction between CBP/p300 and RelA also occurs (52, 53), no evidence has yet been reported that
GR inhibits NF-
B by competing for the co-activator CBP/p300. However, if this occurs, the mechanism is likely to be different from
that of the GR and AP-1 cross-talk, since some receptors such as RAR
for instance can interact with CBP/p300 and inhibit AP-1 but have no
repressive activity on NF-
B (51). Finally, by using glucocorticoid
antagonists, we also found that ZK98299 is unable to repress NF-
B
activity, whereas it has previously been shown that the ZK98299·GR
complex is an efficient inhibitor of AP-1 activity (38). This shows
that ligands can exhibit a selectivity with regard to GR
transrepression, probably as a consequence of different receptor
conformations required for inhibiting various signaling pathways.
In conclusion, we have identified two critical residues within the
C-terminal zinc finger of the GR DBD that are critical for repression
of NF-B activity. This finding identifies a new function for the GR
C-terminal zinc finger. Furthermore, using C-terminal zinc finger GR
mutants and antagonists, we further characterized the mechanism by
which GR represses NF-
B, and we obtained evidence that the
mechanisms by which GR inhibits NF-
B and AP-1 have different
features. This knowledge could be very useful in the search for new GR
ligands with selective activity for GR transrepression of different
signaling pathways. Such ligands could be a useful tool for basic
research regarding mechanisms of glucocorticoid action and possibly in
clinical use with less side effects.
We thank Drs. A. C. B. Cato, K. R. Yamamoto, and R. M. Evans for providing some of the mutant receptors.