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B (Nuclear Factor-B) and Glucocorticoid Receptor Physical Interactions and Antagonism
Molecular Endocrinology Group Laboratory of Signal Transduction National Institute of Environmental Health Sciences National Institutes of Health Research Triangle Park, North Carolina 27709-2233
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B (NF-B) and the
glucocorticoid receptor (GR) are transcription factors with opposing
actions in the modulation of immune/inflammatory responses. NF-B
induces the expression of proinflammatory genes, while GR suppresses
immune function in part by suppressing expression of the same genes.
Previously, we demonstrated that physiological antagonism between
NF-B and GR is due to a mutual transcriptional antagonism that
requires the p65 subunit of NF-B and multiple domains of GR
(1). To elucidate the mechanism(s) of NF-B p65 and GR
transcriptional antagonism, we analyzed the interactions of wild-type
p65 and p65 RHD (rel homology domain, a dominant negative mutant of p65
which lacks a transactivation domain) with GR. We show that p65RHD
blocks p65-mediated transactivation, yet does not block the repression
of GR transactivation by p65, indicating that transcriptional activity
by p65 is not required to repress GR function. Both p65 and p65 RHD
physically interact with GR, but only intact p65 represses GR-mediated
signaling, implicating the p65 transactivation domain in the
transcriptional repression of GR. To further characterize p65-GR
interactions, we examined the role of the transcriptional co-integrator
CREB binding protein (CBP) in their mutual antagonism. GR-mediated
repression of p65 transactivation and p65-mediated repression of GR
transactivation, as well as the physical interaction between NF-B
and GR, are enhanced by CBP. GR bound to the antagonist RU 486,
although transcriptionally inactive, retains the ability to repress p65
transactivation. However, CBP does not physically interact with
antagonist-bound GR and does not enhance its repressive effect on p65.
These data suggest that CBP functions as an integrator of p65/GR
physical interaction, rather than as a limiting cofactor for which p65
and GR compete. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B (NF-B) and glucocorticoid-mediated signal
transduction cascades.
NF-B is a widely expressed transcription factor that positively
regulates the expression of genes involved in immune responses and
inflammation, including cytokines and cell adhesion molecules (for a
detailed list of NF-B-regulated immune and inflammatory genes,
see Ref. 2). NF-B transcription factors are dimers whose subunits
comprise the Rel family of transcriptional activators. Rel proteins
share a conserved, 300-amino acid Rel homology domain that contains the
DNA binding, dimerization, and nuclear localization functions of
NF-B. The preferentially formed NF-B transcription factor in most
cell types is the p50/p65 (Rel A) heterodimer (3). Unactivated NF-B
is retained in the cytoplasm by the inhibitory protein IB. Multiple
forms of IB have been identified (4), and two of these forms
(IB
and IBß) have been shown to modulate the function of the
NF-B p65/p50 heterodimer. When a stimulus such as a proinflammatory
cytokine or an oxidative stressor activates the NF-B signaling
pathway, IB is phosphorylated (5) and consequently targeted for
ubiquitination (6, 7, 8) and removal via the proteosome. IB and
IBß are phosphorylated in response to different extracellular
stimuli (4, 9). Degradation of IB allows NF-B to migrate to the
nucleus (10, 11). There are two different but related ser/thr kinases,
IKK-1 and IKK-2, which are generally held responsible for the inducible
phosphorylation of IB (9, 12, 13, 14).
Activation of NF-B also involves phosphorylation of the p65 subunit
by the catalytic subunit of protein kinase A (PKAc). Dissociation of
IB allows PKAc to phosphorylate p65 on serine 276, dramatically
increasing the transcriptional activity of NF-B (15). Recent studies
show that this PKA-mediated phosphorylation of p65 is required for
recruitment of the transcriptional cofactor CREB-binding protein
(CBP)/p300 by NF-B p65 (16). In the nucleus, activated NF-B binds
to consensus sequences in the chromatin and activates specific
gene subsets, particularly those important to immune and
inflammatory function (4).
Glucocorticoid effects are mediated primarily by the -form of the
glucocorticoid receptor (GR or simply GR), a member of the
steroid/thyroid/retinoid receptor superfamily of nuclear receptors. GR
binds glucocorticoid in the cytoplasm, resulting in receptor
activation. Activation of GR involves a conformational change,
dissociation from cytoplasmic regulatory proteins, and increased
receptor phosphorylation. Ligand-activated receptor 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. Endogenous and exogenous glucocorticoids,
presumably acting via the GR, are potent
immunosuppressive/antiinflammatory agents. However, the widespread
clinical use of these steroid hormones to treat a broad range of
inflammatory disorders including chronic asthma (17, 18), rheumatoid
arthritis (19), and systemic lupus erythematosus (17) has been based
largely on the empirical evidence of their efficacy, while until
recently, little was understood concerning the mechanisms by which
glucocorticoids suppress the inflammatory response. Recent attention,
however, has turned to the opposing roles that NF-B and GR play in
regulating the immune system as an important mechanism for
glucocorticoid suppression of immunity and inflammation. The mechanisms
by which NF-B and GR antagonize each other’s function is now the
subject of intensive study (Refs. 1, 20, 21, 22, 23, 24, 25, 26 ; reviewed in Ref. 2).
Several studies have demonstrated that NF-B and GR physically
interact (4, 27, 28), which presumably mediates their functional
antagonism. We have also demonstrated that GR and NF-B are mutual
transcriptional antagonists. This mutual transcriptional antagonism
requires the p65 subunit of NF-B and multiple domains of GR. We have
now expanded our examination of the mechanisms by which NF-B/GR
mutual antagonism occurs. Using functional dissection of the p65
subunit of NF-B, we demonstrate that the transactivation domain of
p65 is required to repress GR function but not for physical interaction
between NF-B and GR. Thus, physical interaction alone is
insufficient to drive p65/GR antagonism. We show that CBP enhances the
physical interaction of GR and p65 and plays a role in their mutual
transcriptional antagonism. Based on these data, we propose a model for
NF-B/GR antagonism that involves a physical stabilization mediated
by CBP.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B heterodimer inhibits GR transactivation function (1, 24). To
determine whether specific subdomains of p65 could be identified as
essential for the observed repression of GR function, we performed a
functional dissection of p65 using a C-terminal truncation of p65,
termed p65 RHD (Rel homology domain), in transient cotransfection/CAT
reporter assays as described in Fig. 1
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p65 RHD (completely described in Ref. 29 and diagrammed in Fig. 1A)
contains the highly conserved RHD found in all members of the Rel
family of transcriptional activators (reviewed in Refs. 2, 4),
including sequences that drive the nuclear localization of p65, DNA
binding, and homo-/heterodimerization. However, it lacks the C-terminal
transactivation domains that are present in wild-type p65 and is
therefore transcriptionally inactive. Figure 1B demonstrates the
transcriptional inactivity of p65RHD as compared with wild-type p65
(control Western blots, not shown, confirm that the truncated form of
p65 is efficiently expressed, at levels comparable to intact p65, in
our transfection system). These data also indicate that p65RHD is a
dominant negative regulator of p65-mediated transactivation in our
model system as in others. Anrather et al. (29) have
demonstrated that this dominant negative function of p65RHD is probably
due to p65 RHD homodimers competing for NF-B binding sites in the
DNA. Although other possibilities for the mechanism of dominant
negative activity cannot be ruled out, p65 RHD does not interfere with
the expression of the p65 protein (data not shown).
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. In contrast
to wild-type p65, p65RHD is unable to block GR-mediated
transactivation, indicating that the transactivation domain of p65
contains a function that mediates the repression of GR by p65.
Interestingly, when the truncated and full-length constructs of p65 are
coexpressed, p65RHD does not block the ability of p65 to repress GR
function, despite the fact that under these same conditions, p65RHD is
a dominant negative for p65 transactivation function (Fig. 1B). These
data suggest that, while the transactivation domain of p65 is required
to block the transactivating function of GR, transcriptional activity
by p65 is not essential.
Physical Interaction of GR and p65 Is Independent of the p65
Transactivation Domain
To further understand the role of p65/GR physical interactions in
the repression of GR transactivation by NF-B, we assessed the
ability of GR to physically interact with both intact p65 and the
truncated p65RHD (which lacks the LXXLL protein-protein interaction
motif found in the C terminus of intact p65) in an in vitro
coimmunoprecipitation assay. Confirming previous observations that p65
and GR physically interact (24, 27, 28),
35S-labeled in vitro translated GR
weakly coimmunoprecipitates with antisera recognizing p65.
Surprisingly, GR coimmunoprecipitates with p65 RHD more efficiently
than it does with intact p65. Similar results (not shown) were obtained
when 35S-labeled p65 or p65RHD were pulled down
in immune complexes with unlabeled GR using antisera recognizing GR.
These data indicate that the LXXLL motif is not necessary for the
physical interaction of p65 and GR in vitro. Taken together,
the data from Figs. 1 and 2 demonstrate
that a physical interaction alone is insufficient to explain the
antagonism of GR by p65 and suggests that a more complex function
mediates mutual p65-GR transcriptional antagonism.
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B, the potential role of transcriptional cofactors as mediators
of the antagonism was considered. The transcriptional cointegrator
molecule CBP has been reported to bind, and in some cases to
coactivate, both GR (30, 31) and NF-B p65 (32, 33). We considered
that CBP might modulate p65-GR antagonism by serving as a limiting
cofactor required by both NF-B and GR. Therefore, using transient
cotransfection/CAT reporter assays, we examined whether
overexpression of CBP influences p65/GR antagonism. [A similar
approach has recently been reported by Sheppard et al. (30).
However, the ratios of transfected components in that manuscript
differed significantly from those presented here.] Western blot
analyses showed that COS-1 cells have some detectable endogenous CBP,
and that CBP is overexpressed under the conditions used for the
transactivation assay (Fig. 3A, top
panel, 3-fold induction over mock transfected cells). These
analyses also demonstrate that CBP cotransfection has little effect on
the expression of p65 and significantly influences neither the
expression nor the characteristic ligand-induced homologous
down-regulation of GR (Fig. 3A, center and lower panels).
Transactivation of an NF-B responsive reporter by low levels of
cotransfected p65, shown in Fig. 3B, is coactivated by coexpressed CBP.
Surprisingly, coexpression of CBP under these submaximal
transactivation conditions significantly enhances the repressive effect
of GR on p65 signaling (Fig. 3C). Specifically, coexpression of CBP
allowed full repression of p65 transactivation with only 100 ng of
transfected GR, while in the absence of CBP, 250 ng of GR are required
for full repression. When larger amounts of p65 are transfected (Fig. 3D) the p65 signaling is not enhanced by overexpression of CBP,
presumably because there is already a maximal transcriptional response.
However, even under these conditions, GR-mediated repression of p65 is
enhanced by coexpressed CBP. Dexamethasone has been shown to increase
expression of the NF-B inhibitory subunit IB in some cell
types (21). We therefore considered whether the observed repression of
p65 by GR might be due, in part, to induction of IB levels, an
effect that could be stimulated by overexpression of CBP. Western
analyses of the cotransfected cells 6 and 18 h after addition of
dexamethasone indicated that neither GR activation nor overexpression
of CBP alters IB levels in these cells (18 h, shown in Fig. 3C).
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. When
assessed under these conditions, GR transactivation is significantly
enhanced by coexpression of CBP. Although CBP coactivates GR
transcriptional activity under these conditions, GR function is not
fully rescued from p65-mediated repression by CBP (Fig. 4B). Rather,
the partial repression of GR by p65 (Fig. 4B, fourth set of
bars) is significantly enhanced to full repression by coexpressed
CBP. Figure 4C shows that, under maximal stimulation (i.e.
10-7 M
dexamethasone), GR transactivation of the GRE2CAT reporter is not
enhanced by overexpressed CBP, but CBP slightly enhances the repressive
effect of p65 on GR function. We attribute the lack of GR coactivation
by overexpressed CBP to the fact that the saturating dexamethasone
concentration fully activated the GR in our transactivation assay,
leaving no room for transcriptional enhancement by CBP.
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and 4A–C support the idea that
CBP is an important modulator of NF-B/GR interactions and
antagonism. However, since overexpressed CBP enhances NF-B/GR
antagonism rather than rescuing their transcriptional activation, these
data argue that CBP does not function as a limiting factor for which
these transcription factors compete, as previously proposed (30). Since
CBP enhanced the mutual antagonism of p65 and GR, we also considered
the possibility that CBP might facilitate the ability of p65 RHD to
repress GR signaling. As shown in Fig. 4D, overexpression of CBP
enables p65 RHD to partially repress GR transactivation. However,
although this repression is statistically significant, it is very weak
when compared with the repressive effect of the wild-type p65.
Together, the results of these functional studies indicate that CBP is
an integrator of GR/NFB antagonism.
CBP Enhances the Physical Interaction and Function of p65 with
Agonist-Bound, but Not Antagonist-Bound, GR
To further elucidate the mechanism(s) by which CBP enhances p65-GR
antagonism, we examined the role of CBP in p65-GR physical interaction
by in vitro coimmunoprecipitation assay. Addition of
in vitro translated CBP enhanced the physical interaction of
p65 with GR bound to the glucocorticoid agonist dexamethasone (Fig. 5A), suggesting that CBP enhances p65-GR
transcriptional antagonism by facilitating their proper physical
interaction. When GR was treated with the glucocorticoid antagonist RU
486 (Fig. 5B), GR/p65 interactions in the absence of transfected CBP
were similar to those seen for agonist bound GR. However, addition of
CBP had no enhancing effect on the physical interaction of
antagonist-bound GR and p65. These data suggested that antagonist-bound
GR possesses a conformation sufficiently similar to the agonist-bound
GR to allow physical interaction with p65, yet sufficiently different
to block the GR interaction with CBP. To test this hypothesis, we
assessed the ability of CBP to coimmunoprecipitate GR in the presence
of dexamethasone as compared with RU 486. Figure 5C shows that
dexamethasone-bound GR precipitates in immune complexes with CBP,
whereas RU 486-bound GR does not coimmunoprecipitate with CBP (similar
results were seen when an antibody against unlabeled GR was used to
coimmunoprecipitate labeled CBP, data not shown).
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, with the exception that RU 486 replaced
dexamethasone as the ligand for GR. The results, shown in Fig. 6, confirm that RU 486-bound GR is
capable of repressing p65-mediated transactivation (the level of
repression is 
50% of that seen with dexamethasone-bound GR, Figs. 6A and 3B), but CBP coexpression does not enhance the repressive effect
of antagonist-bound GR.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B and GR,
transcription factors with prominent and opposing roles in the
regulation of inflammation, interact to regulate these physiological
processes has become a major focus of research over the past
decade.
We have focused on understanding the nature of the mutual
transcriptional antagonism that occurs between NF-B and GR, the
mechanism of which is understood to involve a physical interaction
between the p65 subunit of NF-B and multiple domains of GR (1, 20, 24, 27, 28). The data presented here more clearly define the mechanisms
of interaction between p65 and GR that result in their mutual
antagonism, both by identifying the transactivation domain of p65 as an
essential element for transrepression of GR and by identifying a
potential role for transcriptional cofactors such as CBP as mediators
of the NF-B/GR physiological antagonism.
We demonstrate that the p65 transactivation domain is required to block
GR transactivation (Fig. 1C), but not for efficient physical
interactions between GR and p65 (Fig. 2). These data argue that, while
physical interactions between NF-B and GR probably mediate their
mutual antagonism, some function beyond the physical interaction must
occur to elicit efficient transrepression. Therefore, we considered the
role of the transcriptional cointegrator molecule CBP as a mediator of
the observed mutual antagonism. CBP is known to interact with a wide
variety of basal transcription factors, inducible transcription factors
(including NF-B and GR), and cofactors, serving in some instances as
a coactivator and in others as a central "integrator/adapter" that
assembles multiple transcription regulatory proteins together on DNA
(30, 31, 32, 33, 34, 35, 36). Given the apparent dual nature of CBP function, we considered
two possible ways that CBP might modulate the interactions of p65 and
GR: first, as a limiting, mutually required transcriptional cofactor
for which GR and p65 compete [a mechanism of action previously
described for CBP as a modulator of GR/AP-1 antagonism (31)] or
second, as an adapter which brings p65 and GR into a specific physical
association on the DNA. A recently published study by Sheppard et
al. (30), using in vitro transactivation assays similar
to those employed here, concludes that CBP is indeed a limiting
cofactor for p65 and GR. The data shown here also indicate a role for
CBP in p65-GR antagonism but do not support the same proposed mechanism
of action. Given that our experiments showed overexpression of CBP to
enhance both the mutual antagonism of p65 and GR (Figs. 3 and 4) and
the physical interaction between these two transcription factors (Fig. 5A), our data clearly argue against a competition mechanism and support
a mechanism whereby CBP integrates p65 and GR on the DNA. Another
possible mechanism for CBP involvement in NF-B/GR antagonism is
hinted at in studies by Lopez et al. (37) concerning the
role of CBP and other coactivators in the cross-talk between estrogen
(ER) and thyroid hormone (TR) receptors. These investigators also found
that overexpression of the coactivators CBP and SRC-1A exacerbated,
rather than abolished, nuclear receptor negative cross-talk.
Interestingly, unpublished data from our laboratory also suggest a role
for SRC-1A in mediating NF-B/GR interactions.
Some important differences exist between the CBP overexpression studies
shown here and those of Sheppard et al., which may explain
the lack of agreement concerning the role of CBP in p65-GR
interactions. For example, we used an NF-B reporter
construct containing three copies of the major
histocompatibility complex (MHC) class I NF-B site (27),
which results in robust transactivation in response to p65 even in the
absence of cotransfected CBP; in contrast, those investigators used a
reporter construct containing a portion of the E-selectin promoter,
which apparently requires the presence of CBP, SRC-1, or other
coactivator to strongly activate transcription. While the GR reporter
construct and cell type we used for GR transactivation studies did not
differ from those reported by Sheppard et al., CBP or SRC-1
was required for strong transactivation by GR under their experimental
conditions; under the conditions reported here, however, GR exhibits a
strong ligand-dependent transactivation function in the absence of
cotransfected coactivators. Perhaps most importantly, we find that at
submaximal dexamethasone concentrations we get significant coactivation
of GR transactivation function with a 10-fold excess of transfected CBP
DNA, while Sheppard et al. demonstrate coactivation using as
much as 1000-fold excesses of CBP DNA.
Previous work from our laboratory showed that RU-486 (antagonist)-bound
GR, which is localized in the nucleus and binds DNA but is
transcriptionally inactive, represses p65-mediated transactivation
(27). However, as confirmed in Fig. 6, the repression of p65
transactivation by antagonist-bound GR is only about 50% as efficient
as that observed with agonist-bound GR. These data led us to
hypothesize that antagonist-bound GR attains a conformation that allows
it to physically interact with both DNA and p65 in a manner similar to
agonist-bound GR, allowing partial repression of p65 transactivation,
yet differs in its interactions with CBP, therefore lacking the
efficiency of repression observed with the agonist-bound conformation.
Figure 6 shows that, unlike the situation for dexamethasone-bound GR
(Figs. 3B and 6A), CBP does not enhance the repression of p65
transactivation by antagonist-bound GR. Taken together with the data in
Fig. 5, B and C, showing that 1) RU-486-bound GR coimmunoprecipitates
with p65, 2) the coimmunoprecipitation is not enhanced in the presence
of CBP, and 3) this antagonist-bound form of GR does not
co-immunoprecipitate with CBP whereas the agonist-bound form of GR
does, these data make a strong case for the validity of our
hypothesis.
As diagrammed in Fig. 7, we propose a
working model for the mechanisms of NF-B/GR interactions and
transcriptional antagonism that might explain, in part, their
physiological antagonism. Figure 7A represents two alternative models
for the role of CBP in p65/GR interaction. As shown, activated NF-B
and GR physically interact, and both transcription factors are capable
of binding to CBP. These two transcription factors may compete for
binding to available CBP, a mechanism that we term the "mutually
exclusive" model. Alternatively, as we propose, CBP might bind to
both factors simultaneously, resulting in the ternary complex
diagrammed in the bottom panel and termed the "integrative
model." This schematic may help to explain the apparent
inconsistencies in the observed role of CBP in p65-GR antagonism
between our studies and those of Sheppard et al. (30), since
vast overexpression of CBP would greatly increase the likelihood of
having each transcription factor bound to a separate molecule of CBP,
in effect driving the integrated complex of NF-B, GR, and CBP to the
separate entities diagrammed in the mutually exclusive model. Figure 7B
summarizes the p65- and GR-mediated signaling pathways and the proposed
role of CBP both as a coactivator and as an integrator of
transcription factor cross-talk. Transactivation of a
glucocorticoid-responsive gene is mediated by binding of activated GR
homodimer to glucocorticoidresponsive elements in the promoter.
The DNA-bound GR then interacts, directly, and indirectly via CBP, with
the general transcriptional machinery to activate transcription.
Similarly, transactivation of an NF-B-responsive gene is
mediated by binding of activated NF-B p65/p50 heterodimer to
B-sites in the gene promoter and subsequent interaction of this
DNA-bound heterodimer with CBP and the general transcriptional
machinery. The simultaneous activation of GR and NF-B results in
both factors being present in the same nucleus. We propose that the
interaction of these factors in the nucleus causes mutual
transcriptional antagonism because it results in a transcription
factor/cofactor complex that holds both GR and NF-B in a
conformation that is incapable of forming correct contacts with the
general transcriptional machinery and/or other transcriptional
cofactors. Since p65 and GR interact both directly and through the
integrator CBP (reviewed in Ref. 2), the theorized function of CBP in
this model is to enhance or stabilize the interaction that occurs
between p65 and GR. The finding that antagonist-bound GR does not
interact with CBP, yet partially represses p65-mediated transcription,
suggests, however, that CBP integration is not the sole mediator of
p65-GR interaction. It is likely that direct transcription factor
interaction or the actions of other transcriptional cofactors also
contribute to the observed mutual antagonism.
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B/GR interactions and suggest that the relative abundance of
p65, GR, and CBP may be an important factor in the transcriptional
outcome of these interactions. For transcriptional
activation of the GR- and NF-B responsive reporter genes examined in
this report, the result of NF-B interacting with GR is mutual
transcriptional antagonism. Future study into the mechanisms of
NF-B/GR interaction may also reveal other outcomes of this
transcription factor cross-talk, which are modulated by additional,
cell type-specific transcriptional cofactors. However, mutual
antagonism, mediated by the action of CBP and probably other
transcriptional cofactors [e.g. reports by Sheppard
et al. (30), Lopez et al. (37), and our
unpublished results suggest a role for SRC-1A in NF-B/GR antagonism
as well], has been well studied and is very likely the predominant
outcome of NF-B/GR interactions in immune cells in which both these
signaling pathways are activated. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Steroids
Dexamethasone (1,4-pregnadien-9-fluoro-16-methyl-11ß,
17, 21-triol-3, 20-dione) was purchased from Steraloids, Inc. (Wilton,
NH). RU 38486 (RU 486) was obtained from Roussel Uclaf (Hoescht Marion Roussel, Inc., Kansas City, MO).
Recombinant Expression Vectors
Human transcription factor NF-B p65 subunit (pCMVp65) in a
pCMV4T backbone and 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 (27, 38, 39). 3XMHCCAT has three copies of a major histocompatibility
complex class I NF-B site cloned upstream of a minimal promoter CAT
(chloramphenicol acetyl transferase) expression vector (27). The GR
expression vector pCYGR contains human GR cDNA cloned into the pCMV5
plasmid. For determination of GR transactivation, the GRE2CAT reporter
was used. This construct, described in detail elsewhere (40) contains
two copies of the GRE sequence from the tyrosine aminotransferase gene
and an adenoviral TATA box upstream from the CAT gene. p65 RHD was
obtained from Dr. H. Winkler (Sandoz Center for Immunobiology, Harvard
Medical School, Boston, MA). This truncated human p65 construct,
generated by PCR from the parent pCMV4Tp65 vector, was completely
described by Anrather et al. (29). The murine CBP expression
construct pcDNA3-CBP-FLAG2X, obtained from Dr.
M. G. Rosenfeld (University of California, San Diego, La Jolla,
CA) was described by Kamei et al. (31).
Transient Transfections
COS-1 cells were transfected with vector DNA constructs using
SuperFect (QIAGEN, Inc. Santa Clarita, CA) cationic
transfection reagent after a 2-h incubation in medium stripped of
endogenous steroids. SuperFect-DNA precipitates were applied to cells
for 1.5 h, after which the transfection solution was replaced with
fresh supplemented, charcoal-stripped growth medium. Where
appropriate, 10-7 M dexamethasone or
10-6 M RU 486 was added to the
medium after transfection, and cells were allowed to grow in the
presence or absence of hormone for 18–20 h before harvest for CAT
assay. Parallel transfections to assess protein expression by Western
blot were processed as described previously (1).
CAT Assays
Transcriptional activities were determined by standard CAT assay
(41). Briefly, 18–20 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 (42), using a
commercially available reagent kit (Bio-Rad Laboratories, Inc. Hercules, CA). Volumes of extract containing known amounts
of total protein from 1–150 µg were then incubated for 18 h
with 14C-radiolabeled chloramphenicol (NEN Life Science Products, Boston, MA) in the presence of 1
mM acetyl-CoA (Roche Molecular Biochemicals, 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 detected by
exposing silica plates to an imaging plate and quantitated using
PhosphorImager technology (Molecular Dynamics, Inc.,
Sunnyvale, CA). CAT activities are measured as percent substrate
converted to acetylated products. Average percent acetylation in
control samples is set equal to 1, and all other values are presented
in terms of percent average control transactivation (acetylation).
Variations in fold induction of dexamethasone samples over control
samples between GR transactivation experiments are attributable to
fluctuations in absolute levels of background GR transactivation/CAT
activity, which we have observed to vary somewhat with batch of serum
and cell passage number. In all cases, the data represent the mean
± SEM of at least three independent
experiments.
In Vitro Coimmunoprecipitation Assays
Proteins to be assessed were in vitro translated
(IVT) using the TnT Reticulocyte Lysate System from Promega Corp. (Madison, WI). When a labeled product was desired,
Tran35S-label 70%
L-methionine (ICN Biochemicals, Inc., Irvine, CA) was included with the programmed lysate.
In vitro translated GR was treated with
10-7 M dexamethasone or
10-6 M RU 486 for 2 h
at 0 C and then activated by incubation at room temperature for 30 min.
IVT products were incubated together in cross-link buffer (20
mM HEPES, 50 mM KCl, 2.5
mM MgCl2 and 1
mM dithiothreitol) at room temperature and then
cross-linked with 2.5 mM DSP (dithiobis
succinmidyl propionate, Pierce Chemical Co., Rockford,
IL). Cross-link reagent was quenched with 0.1 M
ethanolamine, and samples were then diluted in IP buffer (10
mM Tris, pH 7.4, 150 mM
NaCl, 2 mM EDTA, 0.5% Na+
deoxycholate, 0.5% NP40, 0.1 M ethanolamine) for
subsequent immunoprecipitation. A protease inhibitor cocktail
(containing phenylmethylsulfonyl fluoride, pefabloc, pepstatin,
aprotinin, leupeptin, antipain) at manufacturer-specified
concentrations (Roche Molecular Biochemicals) was included
in all buffers. Reactions were precleared for 20 min at room
temperature with normal rabbit serum and protein A sepharose to reduce
nonspecific binding to the sepharose beads. Antisera (described below)
to target proteins and protein A sepharose were then added to the
cleared reactions, and samples were rotated at 4 C overnight. Sepharose
was pelleted at 14,000 x g for 1 min and then washed
extensively in cold IP buffer. Pellets were resuspended in Laemmli
sample buffer boiled for 5 min to dissociate immune complexes from the
sepharose. Samples were subjected to SDS-PAGE and autoradiography to
detect the amount of labeled protein that was coimmunoprecipitated with
the unlabeled target protein. Amount of labeled protein
immunoprecipitated was quantitated (in pixels) by densitometry using
NIH Image software and was expressed as percent above background
(amount pulled down with nonspecific serum, i.e. preimmune
or normal rabbit serum, present). For each coimmunoprecipitation shown,
a converse control experiment was also performed where the opposite
component of the complex was radiolabeled and pulled down with antisera
against the unlabeled component. These data, not shown, were used to
confirm that precipitation results were not peculiar to one antiserum
but could be reproduced with antibodies against any component of the
immune complex.
Antibodies
To immunoprecipitate GR, a 1:1 mixture of antibodies 57 and 59
[epitope-specific rabbit polyclonal antibodies, described previously
(43)] were used. For p65 immunoprecipitation, a 1:1 mixture of NF-B
p65 (A) and NF-B p65 (C) rabbit polyclonal antibodies that
recognize, respectively, the amino- and carboxy-terminal regions of
p65, (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was
used. To immunoprecipitate CBP, anti-CBP CT, a rabbit polyclonal IgG
raised against the C terminus of murine CBP, was used (Upstate Biotechnology, Inc. Lake Placid, NY). For detection of proteins
by Western blot (some data not shown), antibody 57, NF-B p65 (N),
anti- IB (Santa Cruz Biotechnology, Inc.), and
anti-CBP CT were used.
Statistical Analyses
All data indicated as significant were analyzed using a
t test at the 0.05 confidence level. Western blot data were
analyzed for significance by performing densitometry using NIH Image
and performing a t test on the pixel data from each
lane.
Other
All other reagents were obtained from Sigma (St.
Louis, MO).
|
FOOTNOTES |
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Received for publication October 22, 1999. Revision received April 20, 2000. Accepted for publication May 1, 2000.
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=
significantly enhanced p65-mediated repression of GR) C,
Transactivation of the reporter GRE2CAT by GR
(10-7 M dex).
Cotransfection of CBP (black bars) weakly enhances
p65-mediated antagonism of ligand-dependent GR transactivation. D,
Transactivation of GRE2CAT by GR (10-7
M dex). CBP (third bar) has no
significant effect on GR transactivation. p65 represses transactivation
by 87% (fourth bar), while coexpression of CBP with p65
increases this repression to 94% (fifth bar). p65RHD does
not significantly repress GR-mediated transactivation (sixth
bar), while coexpression of CBP with p65RHD (seventh
bar) represses GR function by 28%.
