Cross-Talk between Nuclear Factor-
B and the Steroid Hormone Receptors: Mechanisms of Mutual Antagonism
Lorraine I. McKay and
John A. Cidlowski
Laboratory of Signal Transduction National Institutes of
Environmental Health Sciences National Institutes of Health
Research Triangle Park, North Carolina 27709
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ABSTRACT
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Nuclear factor
B (NF-
B) is an inducible
transcription factor that positively regulates the expression of
proimmune and proinflammatory genes, while glucocorticoids are potent
suppressors of immune and inflammatory responses. NF-
B and the
glucocorticoid receptor (GR) physically interact, resulting in
repression of NF-
B transactivation. In transient cotransfection
experiments, we demonstrate a dose-dependent, mutual antagonism between
NF-
B and GR. Functional dissection of the NF-
B p50 and p65
subunits and deletion mutants of GR indicate that the GR antagonism is
specific to the p65 subunit of NF-
B heterodimer, whereas multiple
domains of GR are essential to repress p65-mediated transactivation.
Despite its repression of GR transactivation, p65 failed to block the
transrepressive GR homologous down-regulation function. We also
demonstrate that negative interactions between p65 and GR are not
selective for GR, but also occur between NF-
B and androgen,
progesterone B, and estrogen receptors. However, although each of these
members of the steroid hormone receptor family is repressed by NF-
B,
only GR effectively inhibits p65 transactivation. Further, in
cotransfections using a chimeric estrogen-GR, the presence of the GR
DNA-binding domain is insufficient to confer mutual antagonism to the
p65-estrogen receptor interaction. Selectivity of p65 repression for
each steroid receptor is demonstrated by I
B rescue from
NF-
B-mediated inhibition. Together these data suggest that NF-
B
p65 physically interacts with multiple steroid hormone receptors, and
this interaction is sufficient to transrepress each steroid receptor.
Further, the NF-
B status of a cell has the potential to
significantly alter multiple steroid signaling pathways within that
cell.
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INTRODUCTION
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Nuclear factor
B (NF-
B) is a widely expressed, inducible
transcription factor of particular importance to cells of the immune
system. Originally identified as an enhancer binding protein for the Ig
-light chain gene in B cells (1), NF-
B is now known to positively
regulate the expression of many genes involved in mammalian immune and
inflammatory responses, including cytokines, cell adhesion molecules,
complement factors, and a variety of immunoreceptors (for a detailed
list of NF-
B-regulated immune and inflammatory genes, see Ref.2).
The NF-
B transcription factor is a heterodimeric protein that
comprises the p50 and p65 (Rel A) subunits. These subunits are proteins
of the Rel family of transcriptional activators. Members of the Rel
family share a conserved 300-amino acid Rel homology domain responsible
for DNA binding, dimerization, and nuclear localization. While
transcriptionally active homodimers of both p50 and p65 can form, the
p50/65 heterodimer is preferentially formed in most cell types (3).
In the absence of stimulatory signals, the NF-
B heterodimer is
retained in the cytoplasm by its physical association with an
inhibitory phosphoprotein, I
B. Multiple forms of I
B have been
identified (4). Two of these forms, I
B
and I
Bß, have been
shown to modulate the function of the NF-
B heterodimer, and these
two I
Bs are phosphorylated in response to different extracellular
stimuli (4, 5). Recent studies indicate that the catalytic subunit of
protein kinase A (PKAC) is associated with the
NF-
B/I
B
complex (6). In this p50/p65/I
B
/PKAC
tetrameric configuration, I
B
renders PKAC inactive
and masks the nuclear localization signal on NF-
B. A variety of
extracellular stimulatory signals, such as cytokines, viruses, and
oxidative stressors (2), can activate kinases that phosphorylate I
B.
[A cytokine-activated I
B kinase termed IKK was recently isolated
and identified as the key regulatory kinase for I
B
(5).]
Phosphorylation at serines 32 and 36 targets I
B
for
ubiquitination and subsequent rapid proteolysis via a
proteasome-mediated pathway (7, 8, 9, 10), resulting in the release of
NF-
B/PKAC. The now active PKAC subunit
dissociates and phosphorylates the p65 subunit of NF-
B.
Phosphorylated NF-
B then translocates to the cell nucleus, where it
binds to target sequences in the chromatin and activates specific gene
subsets, particularly those important to immune and inflammatory
function (4).
Glucocorticoids are steroid hormones whose effects are mediated by the
glucocorticoid receptor (GR), a member of the steroid/thyroid/retinoid
receptor superfamily of nuclear receptors. GR binds glucocorticoid in
the cytoplasm, which results in the receptor undergoing a
conformational change. The ligand-activated receptor then
translocates to the nucleus, where it functions as a modulator of gene
transcription, activating the transcription of specific sets of genes
while repressing the expression of others. Synthetic glucocorticoids
that act via these pathways are widely employed as clinical
immunosuppressive/antiinflammatory agents. However, the widespread use
of these steroid hormones in the treatment of a broad range of
conditions including chronic asthma (11, 12), rheumatoid arthritis
(13), systemic lupus erythematosus (11), and tissue/organ
transplantation (14) is based on empirical evidence of their efficacy,
while surprisingly little is understood concerning the mechanisms by
which glucocorticoids suppress immune function. Recent studies indicate
that NF-
B and GR physically interact, resulting in a mutual
transcriptional antagonism (4, 15, 16). We have shown that the GR
DNA-binding domain (DBD) is required for this interaction. GR is also
reported to increase the expression of the NF-
B-inhibitory subunit,
I
B
(17). These findings suggest that an additional mechanism of
glucocorticoid- induced repression of cytokine transcription involves
GR-mediated inhibition of NF-
B transactivation. To clarify the
mechanism(s) by which GR and NF-
B interactions cause the antagonism
of both transcription factors, we examined the specificity of the
interaction between GR and NF-
B. We show that the observed
dose-dependent mutual antagonism is mediated by the p65, but not the
p50, subunit of NF-
B. Further, we identify multiple regions of the
GR as essential for mutual antagonism with p65.
Interestingly, despite the potent repressive effect of NF-
B on
GR-mediated transactivation, we show for the first time that NF-
B
p65 is incapable of interfering with homologous transcriptional
down-regulation of the GR. Since these results indicate that NF-
B
does not prevent all nuclear effects of GR, it is unlikely that the
mechanism of p65 repression of GR involves sequestration of GR in the
cytoplasm of the cell.
Our data demonstrate that p65 inhibition of GR transactivation is not
specific to GR, but occurs with the structurally and functionally
related androgen, progesterone B, and estrogen receptors (AR, PRB, and
ER) as well. Interestingly, these data also indicate that the
mechanisms of mutual transcriptional antagonism are more specific.
While all steroid receptors examined here were repressed by p65,
presumably through direct physical interaction, only the GR, which has
an immune suppressive function, effectively antagonized NF-
B
transactivation. Examination of the repressive effects of a chimeric ER
with a GR DBD provided further new evidence that the DBD of GR, while
required for negative interaction with NF-
B, is not sufficient for
mutual antagonism. Taken together, these data indicate that a simple
physical interaction between activated NF-
B and the steroid
receptors is sufficient to repress steroid receptor-mediated
transactivation, but the potent immunosuppressive effects of
glucocorticoids rely on a more specific, multidomain interaction
between p65 and GR to repress NF-
B transactivation.
We also provide new data, which indicate that overexpression of
I
B
blocks p65-mediated transrepression of GRs, ARs, PRBs, and
ERs. This ability of I
B
to rescue p65-repressed steroid receptor
function demonstrates the specificity of the p65-steroid receptor
interaction. Further, it suggests that NF-
B p65 interferes with
steroid hormone receptor-mediated signaling only in its
transcriptionally active state.
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RESULTS
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Coexpression of NF-
B and GR Results in Mutual Transcriptional
Inhibition
Previous studies have demonstrated that NF-
B and the GR
mutually repress each others transactivation function (15, 18). To
further characterize the interaction between these two transcription
factors, we transiently cotransfected COS-1 cells with NF-
B, GR, and
chloramphenicol acetyl transferase (CAT) reporter. Since the p65
subunit of NF-
B has been shown to be the transcriptionally active
and inducible subunit in most cell types (Refs. 3 and 19 and our
unpublished observations), we employed the p65 subunit expression
vector for these studies. The data in Fig. 1
demonstrate that when a constant amount
of p65 plasmid DNA is cotransfected with increasing amounts of GR
plasmid DNA (Fig. 1A
), p65-mediated transactivation of the
NF-
B-responsive 3XMHCCAT reporter is inhibited by ligand-activated
GR in a dose-dependent manner. Similarly, dexamethasone-mediated GR
transactivation of the glucocorticoid-responsive GRECAT reporter is
dose-dependently inhibited by p65 (Fig. 1C
). Western analyses indicate
that the observed repression of p65 and GR transactivation function is
not associated with an alteration of p65 (Fig. 1B
) or GR (Fig. 1D
)
protein expression in these cell cultures.

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Figure 1. GR and NF- B Interactions Result in Mutual
Transcriptional Antagonism
COS-1 cells were transiently transfected with 12 µg total DNA,
including 5 µg of the appropriate CAT reporter construct (3XMHCCAT or
GRECAT), the p65 subunit of NF- B (pCMVp65), hGR (pYChGR), and empty
expression vector (pCMV5) to maintain a constant 12 µg DNA per
transfection. After transfection, the appropriate cultures were treated
with 10-7 M dexamethasone, and all cultures
were incubated at 37 C in 5% CO2 humidified air for
18 h before harvest. Cell extracts were assayed for CAT activity.
CAT activities per µg protein are expressed as percent control
activity (control = p65, 0 µg GR, and no dexamethasone for panel
A; GR, 0 µg p65, and no dexamethasone for panel C) for plotting. For
Western blots, cells were transfected as for the corresponding CAT
assays, and 100 µg protein from whole cell extracts were loaded per
lane and then detected as described in Materials and
Methods. A, p65 transactivation vs. amount of
transfected GR. Cells were transfected with 0.1 µg pCMVp65, 5 µg
3XMHCCAT. B, p65 protein levels corresponding to 0, 1, 2.5, and 5 µg
cotransfected GR. C, GR transactivation vs. amount of
transfected p65. Cells were transfected with 5 µg pYChGR, 5 µg
GRECAT. D, GR protein levels corresponding to 0, 0.5, 1, and 2.5 µg
cotransfected p65. The CAT data represent the mean ±
SEM of three independent experiments. Western blots shown
are representative examples of three independent experiments.
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To eliminate the possibility that the observed transrepression of GR by
p65 might be specific to the minimal promoter reporter construct
GRECAT, we also performed the GR transactivation assay using two other
GR-responsive reporter constructs with more complex promoter regions:
GRE2CAT (data not shown) and MMTVCAT (Fig. 2
). The same dose-dependent repression of
GR transactivation by p65 was observed regardless of which reporter
construct was employed. To confirm that the observed reduction in CAT
activity was not due to some direct inhibition of the reporter gene
product by GR or NF-
B, we performed control experiments using a
constitutively active thymidine kinase-CAT reporter, PBL2CAT, which is
not responsive to NF-
B or GR (data not shown). Increasing amounts of
either transcription factor had no effect on the level of CAT activity
in these samples.

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Figure 2. NF- B p65 Represses GR Transactivation of the
MMTVCAT Reporter
GR transactivation of the complex-promoter construct MMTVCAT was
determined in the presence of increasing amounts of p65. Transfections
and CAT assays were performed as in Fig. 1 , using 5 µg MMTVCAT, 5
µg pCYhGR, 02.5 µg p65, and pCMV5 for a constant 12 µg per
transfection. Data are presented as fold induction over control
transactivation (control = 0 µg p65, no dexamethasone) and
represent the mean ± SEM of three independent
experiments.
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The p65 Subunit of NF
B Is Required for Inhibition of GR
Transactivation
Having established that the p65 subunit of NF-
B negatively
interacts with the GR, we wished to examine whether the p50 subunit of
the NF-
B heterodimer could also antagonize GR transactivation. As
Fig. 3
demonstrates, p65 homodimers (Fig. 3A
), as well as equimolar amounts of cotransfected p65 and 50 (Fig. 3B
), are capable of repressing GR transactivation. (Since p65/p50
heterodimers are preferentially formed when both p65 and p50 subunits
are present (19), the effect in Fig. 2B
is presumably mediated by the
heterodimer.) In contrast, p50 homodimers have no effect on GR-mediated
transcription, regardless of the amount of p50 transfected (Fig. 3C
).
This indicates that negative regulation of GR by the NF-
B
heterodimer is mediated solely through the p65 subunit. Since the p50
subunit of NF-
B is expressed at similar levels to p65, but not
transcriptionally active in this cotransfection system (as assessed by
Western analysis and CAT assay, data not shown), these data also
suggest that the mechanisms of reciprocal transcriptional repression of
NF-
B by GR are mediated solely through the p65 subunit.
The Mutual Antagonism of GR and NF-
B Is Mediated by Multiple
Domains of the GR
To identify regions of the GR that might be involved in the mutual
transrepression with NF-
B, a series of GR deletion mutants were
assessed for their ability to repress and be repressed by NF-
B p65.
Each of these mutant GRs has been previously demonstrated to be
efficiently expressed and capable of hormone-mediated nuclear
translocation in transfected COS-1 cells (with the exception of the
I550 steroid-binding domain mutant, which is localized to the nucleus
both in the presence and absence of hormone) (20).
In terms of GRs ability to inhibit p65 transactivation, three
separate regions of the receptor appear to play a role (Fig. 4A
). As previously demonstrated (15, 21),
the DNA binding domain (DBD) of GR is essential to this repressive
function (see
428490). More specifically, ablation of either of
the two zinc finger domains (
420451,
450487) of this region
completely releases the inhibition of NF-
B. The mutant GR I550
demonstrates the importance of the steroid-binding domain (SBD) to p65
transrepression. This receptor, which lacks the SBD, is incapable of
NF-
B transrepression despite its nuclear localization (20) and the
presence of an intact DBD.

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Figure 4. Multiple Domains of GR Are Necessary for Mutual
Antagonism with the p65 Subunit of NF- B
Deletion mutants of hGR were assessed both for their ability to repress
p65 transactivation and for the ability of p65 to repress their ligand-
dependent transactivation function. Cotransfections contained 5 µg of
the appropriate reporter construct, 0.1 µg of the p65 construct, and
5 µg of GR mutant construct per assay. CAT activities were determined
as described in Fig. 1 . and plotted as percent of control CAT activity.
A, Effect of GR on p65 transactivation. Control = p65
transactivation, no GR or dexamethasone (data not shown). B, Effect of
p65 on GR transactivation. Control = wild-type GR transactivation,
no dexamethasone or p65. C, Schematic diagram of GR deletion mutants
used in A and B.
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Interestingly, the GR amino-terminal mutant
9385 was also
incapable of transrepression. However, GR
77262, which contains a
smaller deletion of the transactivation domain nested within the region
missing from
9385, transrepresses p65. One possible
interpretation of this result is that a small region of the GR present
in
77262, but missing from
9385, functions as a third site
on the receptor involved in repression of p65 transactivation. An
alternative interpretation is that the deletion from 9385 of the GR
is sufficiently adjacent to the DBD of GR to affect the function of
that region. As we have shown, a functional DBD is essential for
transrepression of NF-
B p65 by GR. The current experiments do not
enable us to distinguish between these alternative interpretations.
These data demonstrate that multiple regions of the GR are involved in
the repression of p65 transactivation. In contrast, inhibition of
GR-mediated transcription by p65 is a more general phenomenon. As shown
in Fig. 4B
, any mutant that is transcriptionally active can have that
activity repressed by p65. Of particular interest is the ability of p65
to repress the function of GR I550. This receptor mutant, because it
lacks a steroid-dependent component, is nuclear and constitutively
transcriptionally active. That p65 inhibits the function of this mutant
GR suggests that the mechanism of p65 action involves something other
than a simple sequestering of GR in the cytoplasm, where it cannot
interact with chromatin.
p65 Does Not Inhibit Homologous Down-Regulation of the GR
While widely known to be a transcriptional activator, GR also
functions as a negative modulator of gene transcription. A special case
of negative regulation of gene expression by GR is the homologous
down-regulation of the GR, both at the message and protein levels, via
direct binding of intragenic elements within the GR DNA (22). Given the
generally repressive effects of NF-
B p65 on steroid hormone receptor
transactivation function, we sought to determine whether NF-
B also
negatively impacted this transrepressive function of GR. To address
this previously unexamined question, we determined whether
cotransfected p65 could block the dexamethasone-mediated
down-regulation of both GR mRNA and protein by GR in transiently
transfected COS cells. Figure 5
shows
that cotransfection of p65, at a dose that caused almost complete
repression of GR transactivation (see Fig. 1C
, 2
.5 µg p65), cannot
prevent GR-mediated repression of GR message (Fig. 5A
) or GR protein
(Fig. 5B
) expression. The inability of p65 to block this nuclear effect
of GR indicates that the mechanism by which NF-
B represses GR does
not involve cytoplasmic sequestration of the GR by activated NF-
B.
It also argues against the hypothesis that NF-
B interaction with GR
completely blocks GR DNA binding.

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Figure 5. NF- B p65 Does Not Inhibit Homologous
Down-Regulation of the GR
T-75 flasks of COS-1 cells were transiently cotransfected with a total
of 35 µg DNA, containing 25 µg pCYhGR and 10 µg of either pCMVp65
of pCMV5 backbone vector. After an overnight incubation, cells were
treated with 10-7 M dexamethasone as
appropriate and incubated for an additional 24 h before harvest.
For Northern blots, 30 µg total RNA were loaded per lane. For Western
blots, 25 µg of protein from whole-cell extracts were loaded per
lane. Blots shown are representative of at least three independent
experiments. A, GR mRNA. B, GR protein from a parallel cotransfection
experiment to that shown in panel A.
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p65 Antagonizes Multiple Members of the Steroid Hormone Receptor
Family, but Not All These Receptors Reciprocally Repress p65
The difference between p65 inhibition of GR and the reciprocal
inhibition of p65 by GR was intriguing and prompted us to question the
specificity of the p65 interaction with other closely related steroid
hormone receptors. Figures 6
and 7
address the interaction of p65 and
human (h) ARs, PRBs, and ERs. (The A form of PR was found to be
transcriptionally inactive in our system and, therefore, was not
examined in detail.) Figure 6A
demonstrates that AR inhibits p65 transactivation in a dose-dependent
manner. This effect is similar to the GR-mediated inhibition of p65
observed in Fig. 1A
, but is much weaker, requiring 2.55 µg of
transfected plasmid hAR to achieve the same 50% reduction in maximal
transactivation observed with 1 µg of plasmid GR. PRB and ER,
however, have little or no inhibitory effect on p65
transactivation.

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Figure 6. AR, but Not PR or ER, Can Repress Transactivation
by NF- B p65
Transient cotransfection assays were performed as in Fig. 1A , using
varying amounts of human AR, PRB, or ER expression vector in place of
hGR. Data show p65 transactivation of 3XMHCCAT in the presence of
increasing amounts of ligand-activated steroid receptor. A, AR ±
10-6 M R1881 B, PR ± 10-7
M progesterone. C, ER ± 10-6
M estradiol.
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Figure 7. NF- B p65 Represses Ligand-Dependent
Transactivation by AR, PR, and ER
Transient cotransfection assays were performed as in Fig. 1B , using 5
µg human AR, PRB, or ER expression with 5 µg of the corresponding
CAT reporter construct in place of hGR and GRECAT. Data show the
ligand-dependent steroid receptor transactivation with increasing
amounts of p65 (02.5 µg). A, AR transactivation. Ligand =
10-6 M R1881, reporter construct =
MMTVCAT. B, PR B transactivation. Ligand = 10-7
M progesterone, reporter construct = MMTVCAT. C, ER
transactivation. Ligand = 10-6 M
estradiol, reporter construct = vit-tk-CAT.
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While the steroid receptors clearly differ in their ability to repress
p65 transactivation (Fig. 6
), no differences are observed in terms of
their repression by p65. As depicted in Fig. 7
, p65 strongly and dose-dependently
transrepresses each of the steroid hormone receptors tested, much like
it represses GR function. (Control Western blots, not shown, confirm
that p65 has no effect on the levels of AR, ER, or PR expression in
this cotransfection system). These data suggest that a similar physical
interaction underlies the repression in each case. While this
interaction between p65 and steroid hormone receptors is sufficient to
inhibit the function of each receptor, it is apparently insufficient
for reciprocal repression of p65. Taken together, the data in Figs. 6
and 7
support the conclusions in Fig. 4
: the mechanism of mutual
antagonism between p65 and GR is highly specific, whereas the one-way
antagonism of all steroid receptors by p65 is of a more general
nature.
The DBD of GR Cannot, by Itself, Confer Reciprocal Transrepression
to the p65-ER Interaction
We have established that the DBD of GR is essential to mutual
functional antagonism with p65. In addition, regions of the SBD and
possibly the amino terminus of GR are involved in this specific
inhibitory interaction (Fig. 4A
). These observations led us to consider
whether the DBD of GR is responsible for the specific interaction with
p65, which results in mutual antagonism, while the SBD and/or
amino-terminal regions of the receptor are simply necessary to
stabilize the interaction.
To address this issue, we employed a chimeric ER/GR construct. The
construct expresses a hER that has a GR DBD substituted for its own.
The chimeric receptor binds and is activated by estradiol but
recognizes and binds a glucocorticoid-response element in the
chromatin. If the DBD of the GR were the only region of the receptor
responsible for mutual antagonism with p65, an intact chimeric ER/GR
would be able to suppress p65 transactivation in our system. Figure 8
demonstrates that this is not the case.
ER/GR exhibits the same pattern of repression as ER, not GR.
Specifically, p65 represses ER/GR transactivation of the GRE2CAT
reporter (Fig. 8A
), but ER/GR has no inhibitory effect on p65
transactivation (Fig. 8B
).

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Figure 8. Substitution of the DBD of ER with That of GR Does
Not Make ER-p65 Transrepression Reciprocal
ER-GR, an ER construct with a GR DNA binding domain, was cotransfected
into COS-1 cells with p65 and the appropriate reporter constructs to
assess both steroid receptor and p65 transactivation. Transfections and
CAT assays were performed as described in Fig. 1A , Ligand-activated
ER-GR transactivation in the presence of increasing amounts of p65
expression vector; 10-6 M estradiol
(E2) was used as ligand, and the CAT activity was
determined with a GRE2CAT reporter construct. B, p65 transactivation in
the presence of increasing amounts of ER-GR expression vector.
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These data indicate that other regions of the GR, in addition to the
DBD, are required for specific interactions with p65 and do not act
simply as stabilizers of the NF-
B p65-receptor interaction.
I
B
Blocks p65-Mediated Repression Transactivation by Multiple
Steroid Receptors
Although our (unpublished) observation that transcription of
constitutively expressed PBL2CAT reporter was unaffected in our
cotransfection system argued against general transcriptional
repression, we sought to verify the specificity of p65 as the mediator
of steroid receptor transrepression. We approached this question by
exploiting the interaction of NF-
B with the inhibitory rel subunit
I
B
. Increased expression of this inhibitory NF-
B subunit
causes an increased binding of nuclear NF-
B and attenuates NF-
B
transactivation by sequestering the transcription factor in the cell
cytoplasm. Glucocorticoids have also been shown to modulate NF-
B
function indirectly by increasing expression of I
B
(17). We
sought to determine whether overexpression of I
B
could interfere
with NF-
B-mediated repression of steroid-dependent GR
transactivation in this cotransfection system. Figure 9A
indicates that p65-mediated repression
of GR transactivation of the GRE2CAT reporter is completely blocked by
cotransfection of an equal amount of I
B
. Interestingly,
steroid-mediated GR transactivation function is actually considerably
enhanced compared with control in the presence of transfected I
B
(Fig. 9A
, I
B, I
B/p65). Since I
B has no effect on the reporter
construct in the absence of steroid receptor (data not shown), we
interpret this to be the result of I
B
sequestration of the
endogenous NF-
B present in COS-1 cells and subsequent release of the
basal GR inhibition by NF-
B.

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Figure 9. I B Blocks p65 Inhibition of Steroid Hormone
Receptor Action
Ligand-activated steroid hormone receptor transactivation was assessed
in the presence of cotransfected NF- B p65, I B, or both I B and
p65. In each experiment, 3 µg each of the appropriate steroid hormone
receptor expression vector (for GR, AR, PRB, or ER) and 2 µg of the
appropriate reporter construct were cotransfected into COS-1 cells.
pCMVp65 (3 µg) and pCMVI B (3 µg) were cotransfected as
indicated in each figure. Transfected DNA was held constant at 12 µg
by the addition of pCMV5 backbone vector DNA. Cells were treated with
ligand (as in Figs. 1 and 6 ) then harvested for CAT assay. Results
represent the mean ± SEM of three experiments and are
presented as percent of control transactivation. (Control =
ligand-dependent transactivation in the absence of p65 or I B,
represented by the first bar of each panel, and set to
100%). A, Dexamethasone-activated GR transactivation of GRE2CAT. B,
R1881-activated AR transactivation of MMTVCAT. C,
Progesterone-activated PRB transactivation of MMTVCAT. D, Estradiol
activated ER transactivation of vit-tk-CAT.
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The ability of I
B
to block the p65 repression of other steroid
hormone receptors was also examined. Figure 9
, B-D, shows that p65
repression of ARs, PRBs, and ERs, respectively, is similarly blocked by
overexpression of I
B
. However, some differences in efficacy were
observed with the different receptors. For GRs, PRs, and ERs, I
B
completely restored the receptor transactivation function in the
presence of p65, while for AR, I
B
could only restore
approximately 50% of control transactivation. It is also intriguing
that I
B
overexpression in the absence of p65 resulted in an
approximate 5-fold enhancement of steroid-dependent PR
transactivation and 3-fold enhancement of GR transactivation, but
no enhancement of AR or ER transactivation. These data suggest that
I
B
and its induction by GR may play an important role in
enhancing and sustaining the immunosuppressive effects of
glucocorticoids: ligand-activated GR simultaneously represses NF-
B
transactivation and enhances I
B
expression. I
B
then further
represses p65 and enhances GR function, possibly resulting in a
feed-forward mechanism of GR repression of NF-
B.
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DISCUSSION
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Negative cross-talk between NF-
B and the GR has recently gained
attention as a potentially important general mechanism by which
glucocorticoids exert their potent antiinflammatory and
immunosuppressive actions (15, 16, 21). Because the processes that
underlie glucocorticoid-mediated immunosuppression are of clinical
importance, but are as yet poorly understood, the work we present here
focuses on the mutual antagonism between NF-
B and the steroid
hormone receptors that transduce endocrine signals. In particular, we
begin to examine the mechanisms that underlie the negative cross-talk
between these transcription factors.
We have shown that the transrepression of NF-
B by GR is both
reciprocal and dose-dependent. Further, we show for the first time that
this mutual antagonism is mediated solely through the p65 subunit of
NF-
B. The data presented here support the possibility of two
mechanisms by which this mutual antagonism might occur. We propose that
one mechanism of mutual repression involves direct physical
interactions between p65 and GR and that this interaction prevents both
transcription factors from binding to their DNA-responsive elements and
activating transcription. The idea that this dose-dependent pattern of
mutual inhibition is due to a physical interaction between NF-
B and
the GR is supported by work from both our own and other laboratories
(15, 16, 21). Specifically, these studies have demonstrated by
coimmunoprecipitation that p65 and the GR physically associate with
each other. In addition, electrophoretic mobility shift assays (15, 16)
show that GR can inhibit the binding of p65 to its response element. A
recent report demonstrating coimmunoprecipitation of the progesterone
receptor and p65 (18) lends further support to the idea of a direct
steroid receptor-p65 interaction as the basis of our observed
transrepression.
A second potential mechanism by which GR and NF-
B might function as
mutual antagonists is competition between these two transcription
factors for a common transcriptional cofactor(s). The dose-dependent
nature of the observed antagonism, as well as the attenuating effect of
I
B
on p65 transrepression of each steroid receptor, demonstrated
here for the first time, would also be consistent with such a
mechanism. In addition, the fact that p65 cannot interfere with
homologous down-regulation of the GR, a process that involves GR
binding to intragenic elements of GR but is independent of the promoter
region of the GR gene, suggests that p65 interactions with GR do not
completely block GR binding to DNA.
We evaluated the effect of increasing amounts of p65 on transactivation
by PRBs, ARs, and ERs. These data prove that each of these steroid
hormone receptors is dose-dependently inhibited by p65 in a manner
similar to the inhibition of GR by p65. From this observation, we
conclude that a similar direct physical interaction or competition for
cofactors underlies the transrepression in each case. Surprisingly,
although this interaction between NF-
B and the steroid receptors
represses receptor-mediated transactivation in all cases, it is
insufficient for the reciprocal inhibition of p65 function. While for
GR and AR mutual antagonism with p65 was observed, PRB and ER had no
effect on p65 activation. These findings contradict a recently
published study that suggests that PRB can repress p65 (21). Although
that study did not present data to experimentally document this
repression, our own work indicates only a very small repression of p65
at very high levels of transfected PRB plasmid. Also, in contrast to
our current findings, recent publications provide evidence for ER
effects on p65 function (23, 24, 25); however, these findings appear to be
dependent on the presence of cell-type/tissue-specific cooperating
factors. The existence of cell type-specific differences in steroid
receptor/p65 interactions is consistent with the hypothesis that
cofactor competition is involved in NF-
B/steroid receptor
antagonism.
The observation that, in our experimental system, some steroid
receptors are capable of mutual antagonism with NF-
B, while others
cannot reciprocate, fits well with the conclusions from our experiments
with deletion mutants of the GR. Taken together, Fig. 4
, A and B, shows
that the mutual antagonism of p65 and GR is complex and requires
multiple domains of the GR. In contrast, the one-way inhibition of
active GR by p65 is more general, occurring regardless of what region
of GR has been deleted. These data indicate that all transcriptionally
active deletion mutants of the GR tested are transrepressed by p65.
However, we were able to identify at least two, and possibly three,
regions of the GR that play a role in the transrepression of p65. We
interpret these data to mean that the mechanisms of transrepression of
p65 and NF-
B are distinct. While both inhibitory functions require
physical interactions between the transcription factors, inhibition of
p65 requires the presence of multiple domains of the GR, including the
DBD, the SBD, and possibly small regions of the amino terminus. No
specific regions of the GR necessary for its repression by p65 could be
definitively identified.
Further evidence supporting the importance of multiple domains of the
GR in p65 transrepression came from our examination of the chimeric ER.
The DBD of GR has been identified both here and in previous studies
(15, 21) as an essential region of the receptor for the antagonism of
p65. In addition, DBD of GR has been identified as the region through
which cross-talk with another transcription factor, AP-1, occurs (26).
For this reason, we speculated as to whether the DBD of GR was the only
specific region of the receptor for p65-GR cross-talk, while the SBD
and amino-terminal regions of the receptor were merely stabilizers of
the physical interaction. If this were true, then replacement of the
DBD of ER with one from GR should confer mutual antagonism to the ER
interaction with p65. The data presented here indicate that this is not
the case. Chimeric ER does not have the ability to repress p65 despite
the presence of a GR DBD, indicating that specificity lies in multiple
regions of the GR.
Glucocorticoids are known to negatively impact many aspects of immune
and inflammatory response. They have been shown to repress the
expression of interleukin 6, CINC/gro, and other cytokines that cause
inflammation (16, 27). They also suppress the cytokine-induced
expression of NOS II, an important mediator of macrophage activity and
other immune and inflammatory responses (28). This GR-mediated
inhibition of cytokines occurs despite the lack of any identifiable
glucocorticoid-responsive elements in the promoter regions of these
genes. Glucocorticoids have also been shown to interfere with immune
function by altering the distribution pattern of lymphocytes (28) and
decreasing the number of lymphocytes by both inhibiting cell growth and
inducing apoptosis (Ref. 30 and unpublished observations). GR is also
known to positively regulate the expression of I
B, a powerful
inhibitor of NF-
B transactivation function (17). The functions of
NF-
B in the immune system, on the other hand, include the
up-regulation of cytokine expression and the regulation of cell
adhesion molecules that are involved in lymphocyte infiltration during
inflammation (2, 15, 27, 28). The ability of glucocorticoids to
suppress immune function is closely related to the ability of GR to
inhibit NF-
B transcriptional activity.
We maintain that the direct physical association of NF-
B and GR is
one important mechanism of NF-
B repression in immune cells, and
consequently, a key factor in suppression of immune and inflammatory
response. We further propose that additional mechanisms of NF-
B-GR
antagonism may also contribute to modulation of the immune response.
Specifically, our data are also consistent with a mechanism of
antagonism involving competition for common transcriptional cofactors.
Our observations concerning the specific nature of NF-
B antagonism
by activated GR speak to the physiological importance of this
mechanism. We have shown that NF-
B is a ubiquitously expressed
transcription factor that interacts with a variety of steroid hormone
receptors. Inhibition of NF-
B by any activated steroid hormone
receptor with which it interacts would reduce the value of GR as a
specific inhibitor of immune response. Instead, we demonstrate that
multiple regions of GR are required for this inhibition, ensuring that
immune suppression occurs in those cells in which glucocorticoids, GR,
and NF-
B are all present. Other steroid hormone receptors, such as
estrogen or progesterone receptors, while under the conditions
evaluated here are not specific repressors of NF-
B, may specifically
interact with NF-
B in a cell type-specific manner due to the
presence of specific transcriptional cofactors in that cell. The
identity of cofactors that might modulate the interactions between
NF-
B and the steroid hormone receptors remains to be elucidated and
is likely to become a prime focus of future investigation.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
COS-1 (African Green Monkey kidney) cells were maintained at 37
C in a 5% CO2 humidified atmosphere in DMEM with high
glucose (DMEM-H), supplemented with 2 mM glutamine, 10%
FCS/calf serum (CS) 1:1 (Irvine Scientific, Santa Ana, CA), 100 IU/ml
penicillin, and 100 µg/ml streptomycin. Cells were passaged every
34 days. One hour before each transfection experiment, culture media
were changed to a supplemented 3% FCS/CS formulation and maintained in
this media for the duration of transfection.
Steroids
R1881 (methyltrienolone) was obtained from NEN Research Products
(Boston, MA). Progesterone (4-pregnen-3, 20-dione), dexamethasone
(1, 4-pregnadien-9
-fluoro-16
-methyl-11ß, 17, 21-triol-3,
20-dione), and 17ß- estradiol (1, 3, 5(10)-estratrien-3,17-ß-diol)
were purchased from Steraloids, Inc. (Wilton, NH).
Recombinant Expression Vectors
Vectors containing the human transcription factor NF-
B
subunits p65 (pCMVp65) and p50 (pCMVp50) in a pCMV4T backbone, the
inhibitory
B subunit I
B in the pCMV4T backbone (I
B
), as
well as the NF-
B reporter 3XMHCCAT, were obtained from Dr. A.
S. Baldwin, University of North Carolina (Chapel Hill, NC). Detailed
descriptions for these constructs are available elsewhere (15, 31, 32).
3XMHCCAT has three copies of a major histocompatibility complex class I
NF-
B site cloned upstream of a minimal promoter CAT expression
vector (15). The GR expression vector pCYGR contains hGR cDNA cloned
into the pCMV5 plasmid. For determination of GR transactivation, GRECAT
and GRE2CAT reporter constructs were employed. These constructs are
described in detail elsewhere (33). Briefly, GRECAT and GRE2CAT contain
one or two copies, respectively, of the GRE sequence from the tyrosine
aminotransferase gene and an adenoviral TATA box upstream from the
chloramphenicol acetyl transferase gene. hAR cloned into the CMV3
expression vector was obtained from Dr. M. McPhaul, University of Texas
Southwestern Medical Center (Dallas, TX) (34). hPRB in a pRST7 (RSV
promoter) expression vector backbone was a gift from Dr. D. McDonnell,
Duke University (Durham, NC) (35). For both hAR and hPRB
transactivation studies the reporter construct pGMCS, which contains
mouse mammary tumor virus promoter sequences upstream of the CAT gene,
was used. This reporter was obtained from Dr. K. Yamamoto, University
of California (San Francisco, CA) (36). The hER cloned into a pKCR2
backbone (HEO), an ER/GR cassette (ER/GR) construct containing the hER
with an hGR DNA binding domain, and the vit-tk-CAT (estrogen
responsive) reporter construct were obtained from Dr. P. Chambon,
INSERM (Paris, France) (37, 38). Expression vectors containing deletion
mutants of the hGR (
428490,
420451,
450487,
77262,
9385, and I550) were obtained from Dr. R. Evans (Salk Institute,
San Diego, CA) and are described elsewhere (39, 40, 41).
Transient Transfections
COS-1 cells were transfected with vector DNA constructs using a
standard calcium phosphate method (42) or by SuperFect (Qiagen,
Inc. Santa Clarita, CA) cationic transfection reagent. The cells were
exposed to calcium phosphate-DNA of SuperFect-DNA precipitates for
4 h. Calcium phosphate- transfected cells were then subjected to
osmotic shock for 30 sec in a 15% glycerol/3% FCS/CS supplemented
DMEM-H solution. Fresh 10% FCS/CS supplemented media were applied to
all cells, and steroid hormone (10-7 M
dexamethasone or progesterone, or 10-6 M R1881
or estradiol) was added to the media as appropriate. After
transfection, cells were incubated in the presence or absence of
hormone for 1820 h before harvest in ice-cold PBS (1x PBS).
CAT Assays
Transcriptional activities were determined by standard CAT assay
(42). Briefly, 1820 h after transfection, monolayer cells were
harvested by scraping from culture dish into cold PBS. Cells were then
pelleted, resuspended in 0.25 M Tris buffer (pH 8), and
lysed by tip sonication. Cellular debris was pelleted at 14,000 x
g, and the supernatant was incubated at 65 C for 10 min to
inactivate endogenous inhibitors of CAT. Activated cell extracts were
assayed for protein content by the method of Bradford (43), using a
commercially available reagent kit (Bio-Rad, Hercules, CA). Volumes of
extract containing known amounts of total protein from 1150 µg were
then incubated for 18 h with 14C-labeled
chloramphenicol (NEN Research Products) in the presence of 1
mM acetyl coenzyme A (Boehringer Mannheim, Indianapolis,
IN). Reactions were stopped by ethyl acetate extraction, and CAT
activities were determined by separation of acetylated products from
substrate by TLC on silica gel 60 plates in 1:19 methanol-chloroform.
Acetylated product formed was quantitated by liquid scintillation
counting. CAT activities are measured as percent substrate converted to
acetylated products. Average percent acetylation in triplicate control
samples is set equal to 1, and all other values are presented in terms
of fold increase over average control.
Isolation of Total RNA and Northern Hybridization
Total RNA was isolated from transiently transfected COS-1 cells
with TRIzol Reagent (GIBCO, Grand Island, NY), and RNA integrity was
assessed by visual inspection of ribosomal RNA bands after agarose gel
electrophoresis and ethidium bromide staining.
For Northern hybridization, RNA samples were denatured by treatment in
glyoxal/dimethylsulfoxide and electrophoresed on a 1% agarose/10
mM NaHPO4 (pH 7) gel. After transfer to
0.2-µm Biotrans nylon membrane (ICN, Irvine, CA) and UV cross-link,
RNA was hybridized at 65 C in 50% formamide with an antisense
riboprobe synthesized from the pT3/T7 hGR cDNA vector as previously
described (22).
Western Blot Analyses
Transiently transfected COS-1 cells were incubated in the
absence or presence of hormone as for CAT assay. Cells were then washed
in ice-cold PBS, then harvested by scraping with a rubber spatula into
cold PBS containing a protease inhibitor cocktail (containing
phenylmethylsulfonyl fluoride, pepstatin, aprotinin, leupeptin,
antipain) at manufacturer-specified concentrations (Boehringer
Mannheim, Indianapolis, IN). Cells were homogenized on ice using three
10-sec bursts with a Tekmar Tissuemizer (Cincinnati, OH) with cooling
for 30 sec between bursts, after which the whole-cell lysates were
cleared by ultracentrifugation at 45,000 x g for 45
min. Resulting supernatant cell extracts were mixed with 6x Fairbanks
gel loading buffer (6 mM EDTA, 6% SDS, 30% sucrose, 60
µg/ml Pyronin Y, 36 mg/ml dithiothreitol), boiled for 4 min, and then
electrophoresed on 7.5% polyacrylamide gels with 3% polyacrylamide
stack gel. Electrophoretically separated proteins were electroblotted
onto nitrocellulose filters. Quality of transfer and consistency of
loading were visually assessed using Ponceau S staining. Filters were
blocked in 10 mM Tris/15 mM NaCl, pH 7.4 buffer
(CTT) containing 10% nonfat dry milk and 0.1% Tween-20.
Primary antibody incubations were performed in CTT buffer plus 1%
nonfat dry milk for 1 h at room temperature. [Primary antibodies:
GR = antibody 57, epitope-specific rabbit polyclonal antibody,
described previously (44); p65= NF-
B p65 (A) rabbit polyclonal
antibody (Santa Cruz Biotechnology, Santa Cruz, CA); p50 =
anti-human NF-KB p50 rabbit monospecific antibody (Rockland,
Gilbertsville, PA).] Secondary antibody incubations and subsequent
chemiluminescent detection with the ECL detection kit (Amersham,
Buckinghamshire, U.K.) were performed in accordance with kit
recommendations.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. John A. Cidlowski, National Institutes of Environmental Health Sciences, P.O. Box 12233, MD E202, Research Triangle Park, North Carolina 27709.
Received for publication August 28, 1997.
Accepted for publication October 3, 1997.
 |
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