From the Vascular Research Division, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Transcriptional coactivators may function as
nuclear integrators by coordinating diverse signaling events. Here we
show that the p65 (RelA) component of nuclear factor- Nuclear factor- Activation of NF- In contrast to NF- The divergent roles played by NF- Cells and Transfections--
SaoS2 and COS-7 were obtained from
the American Type Culture Collection (ATCC). ECV-304 cells were
obtained from the European Collection of Animal Cell Cultures (ECACC,
Salisbury, Wiltshire, United Kingdom). The cells were cultivated in
Dulbecco's modified Eagle's medium from Life Technologies, Inc.
supplemented with 10% fetal calf serum, 2 mM
L-glutamine, and antibiotics. Cells were grown on
10-cm2 dishes and cultured at 37 °C in a 5%
CO2 incubator. The COS cells were cotransfected with
Transfected cells were treated with 100 units/ml TNF Western Blot--
Aliquots from the cotransfection studies were
saved prior to the freeze/thaw step in the CAT assay harvesting
procedure. Cells were lysed in 5 × cell lysis buffer (Promega,
Madison, WI) for 20 min on ice. Samples were centrifuged briefly, and
equivalent amounts of protein were resuspended in SDS sample buffer,
boiled for 2 min, and analyzed on 10% SDS-polyacrylamide gels.
Proteins were electrophoretically transferred to nitrocellulose
(Schleicher and Schuell), the membranes blocked with 5% non-fat dry
milk in TBST buffer (containing 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.5% Tween 20), and incubated with either p65 or
p53 (Rockland, Gilbertsville, PA and PharMingen, San Diego, CA,
respectively) antisera for 16 h at 4 °C. Blots were washed
three times with TBST buffer, incubated for 1 h with a secondary
antibody conjugated to horseradish peroxidase (Amersham Pharmacia
Biotech), and then washed three times in TBST. The antigen antibody
interactions were visualized by incubation with ECL chemiluminescence
reagent (Amersham Pharmacia Biotech). Blots were exposed to x-ray film
for 10 s to 10 min.
p65 and p53 Mutually Repress Each Other's Transcription--
If
CBP functions as a signal integrator for the NF-
To determine whether p53 could alter NF- CBP Rescues p53 Inhibited p65-dependent
Transactivation--
One possibility suggested by the preceding
findings is that the formation of complexes between either p65 or p53
and specific coactivators would reduce the amount of coactivator
available for transcriptional activation. If competition for limiting
amounts of CBP accounts for the inhibitory effect of p53, then
increased levels of the coactivators should restore, or rescue,
p65-dependent gene expression. Indeed, the inhibitory
effect of p65 on p53-dependent gene expression was
completely abolished by cotransfection of a vector expressing CBP (Fig.
1B, lane 6). In a similar manner, the suppression
of p53 on p65-dependent gene expression was significantly decreased by CBP (Fig. 1C, lanes 10-13). Control
studies demonstrated that CBP overexpression did not alter levels of
either p53 (Fig. 1, B and C, insets)
or p65 (Fig. 1C, inset). Additionally,
overexpression of an irrelevant transcriptional activator, or mutated
forms of CBP, did not result in rescue (data not shown). Collectively, these functional studies demonstrate that CBP is limiting for both p65-
and p53-dependent transactivation and suggest that CBP can
rescue the mutually repressive interaction between the two activators.
TNF-activated NF- NF-
From these studies we suggest that cross-talk between the p53 and
NF-
This control process may be important in determining the fate of cells
in which both NF-
Key to understanding this level regulation is determining how CBP
can simultaneously integrate the functions of these diverse transcriptional activators. The context in which the transcription factors are positioned in the various target gene promoters may determine their ability to compete for the coactivators and
synergistically activate transcription (37). Additionally,
CBP-associated proteins, such as p/CAF, may generate a series of
distinct coactivator complexes (38). These assemblies could be used
differentially by specific groups of transcription factors, limiting
cross-talk between signaling pathways.
B (NF-
B) and
p53 mutually repress each other's ability to activate transcription. Additionally, tumor necrosis factor-activated NF-
B is inhibited by
UV light-induced p53. Both p65 and p53 depend upon the coactivator CREB-binding protein (CBP) for maximal activity. Increased levels of
the coactivator relieve p53-mediated repression of NF-
B activity and
p65-mediated repression of p53-dependent gene expression. Nuclear competition for limiting amounts of CBP provides a novel mechanism for altering the balance between the expression of
NF-
B-dependent proliferation or survival genes and
p53-dependent genes involved in cell cycle arrest and apoptosis.
INTRODUCTION
Top
Abstract
Introduction
References
B
(NF-
B)1 is an inducible
transcription factor that plays an essential role in the regulation of
gene expression in response to inflammatory stimuli (1). It is composed
of members of the Rel family (p50, p52, p65 (RelA), c-Rel, and RelB), which share a region of homology known as the Rel homology domain capable of directing DNA binding and mediating dimerization. In most
cells, NF-
B is found in an inactive form in the cytoplasm bound to
an inhibitory protein, I
B. In response to multiple activating signals, the inhibitor is degraded by the ubiquitin-proteasome complex,
and NF-
B translocates to the nucleus and induces gene expression.
NF-
B components can interact with other DNA binding proteins, as
well as with a series of non-DNA-binding coactivator proteins. Among
these interactions, the p65 component of NF-
B, like a variety of
signal-dependent transcriptional activators, can associate
with CREB-binding protein (CBP) or its structural homolog p300 (2,
3).
B is associated with resistance to programmed cell
death (4-10). Mice with a targeted mutation in the p65 component of
NF-
B die before birth with extensive liver cell apoptosis (11).
Fibroblasts derived from these mice show increased apoptosis following
TNF
stimulation, an effect that can be reversed by overexpression of
p65 (4). Inhibition of NF-
B activation increases cell death in
response to multiple stimuli (7). Moreover, inhibition of
constitutively active NF-
B in lymphoid cell lines causes apoptosis
(8). One mechanism by which NF-
B inhibits cell death is to induce
the expression of genes that promote resistance to apoptosis. These
anti-apoptotic gene include A20 (12), the immediate early response
gene IEX-1L (13), as well as TRAF1 (TNFR-associated factor 1), TRAF2,
and the inhibitor-of-apoptosis (IAP) proteins c-IAP and c-IAP2 (14).
Thus NF-
B can activate a set of genes that function cooperatively to
suppress apoptosis.
B's role in promoting cell survival, the p53 tumor
suppressor gene plays an important role in cell cycle arrest or
apoptosis in response to various types of stress (15, 16). p53
functions as a transcriptional activator by binding to specific DNA
sequence elements (17) and interacting with coactivators, such as CBP
(18-21). These interactions appear to be necessary for p53 to function
as a transcription factor. p53 increases expression of multiple genes,
including the cyclin-dependent kinase inhibitor
p21/WAF1/Cip and murine double minute (mdm2) (15, 16, 22,
23). Increased expression of the p21 gene by p53 correlates with cell
cycle control by inducing G1 arrest or apoptosis (24). The
ability of p53 to induce cell cycle arrest or apoptosis is closely
regulated under normal conditions (22, 23). Some oncogenes and stress
signals regulate p53 activity through MDM2. This negative regulator of
p53 functions in two ways: it binds to the activation domain of p53 and
inhibits its ability to stimulate transcription, and MDM2 mediates the
degradation of p53. In addition to having a role as a transcriptional
activator, p53 represses the expression of multiple genes that lack p53
binding sites (25). In contrast to activation by p53, no common
consensus DNA binding site has been identified which correlates with
the transcriptional repression. The biological consequences of
p53-mediated repression of gene expression are not fully understood.
B and p53 suggest that there might
be mechanisms that integrate the activities of these regulatory
factors. Some of this control may be provided by the common dependence
of both NF-
B and p53-dependent gene expression on
limiting levels of transcriptional coactivators. Since transactivation by both p53 and p65 involves CBP, we investigated the role of this
coactivator in the p53 and NF-
B signaling pathways. We find that
mutual transrepression of these diverse signaling systems results, at
least in part, from competition for a limiting amount of this versatile
transcriptional coactivator. Interactions between the p65 and p53
signaling pathways mediated by CBP may be an important aspect of
regulating the diverse changes in gene expression associated with cell survival.
EXPERIMENTAL PROCEDURES
578
E-selectin promoter-CAT (26) or a p21-promoter-luciferase reporter
construct (p21-luciferase), along with expression vectors for human p53
(pC53-SN3), p65 (pcDNA p65), MDM2 (pCHDMIA), or CBP
(pRc/RSV-mCBP-HA), as described in the figure legends. Whole cell
extracts were prepared from the transfected cells and CAT or luciferase
activity determined as described previously (2, 27).
(Endogene
Sciences) for 30 min and then exposed to increasing amounts of
ultraviolet light (10, 20, and 40 J/m2) using a
Stratalinker (Stratagene) at 254 nm UV. Cells were harvested 4 h
after exposure to UV light and whole cell extracts prepared.
RESULTS AND DISCUSSION
B and p53 pathways,
there might be mutual transcriptional interference between these two
signal-dependent activators. To determine whether the p65
component of NF-
B alters p53 function, a p53 reporter plasmid
containing the p21 promoter, a known p53 target gene associated with
arrest of the cell cycle, was cotransfected with a fixed amount of p53
and increasing amounts of a p65 expression plasmid. As expected, p53
strongly activates the p21 promoter (Fig.
1A, lanes 2 and
3). Cotransfection of p65 resulted in a
dose-dependent suppression of this reporter plasmid in
SaoS2 cells (Fig. 1A, lanes 5-8) or in COS cells
(Fig. 1B, lanes 3-5). p65-mediated suppression
was also seen with a Bax promoter-reporter, a gene that is associated
with the induction of apoptosis, as well as an artificial promoter
containing only multiple p53 binding sites (data not shown). In
contrast to the p53-dependent genes, no repression of a
Gal4-dependent promoter-reporter gene was seen (data not shown). Control studies also demonstrate that overexpressed p65 did not
decrease production of p53 from the corresponding expression construct
(Fig. 1, A and B, insets).
View larger version (16K):
[in a new window]
Fig. 1.
p65 and p53 mutually repress each other's
transcription in a CBP-dependent manner. A,
p65 represses p53-dependent activation of a
p21-promoter-reporter. Saos2 cells were cotransfected with 3 µg of a
p21 promoter-luciferase reporter, increasing amounts (10 and 20 ng) of
p53 alone (lanes 2 and 3), or 20 ng of p53 with
increasing amounts (0.1, 0.2, 0.5, 1.0 µg) of a p65 expression vector
(lanes 5-8). B, p65 repression of p53-activated
gene expression is CBP-dependent. COS cells were
transfected with 3 µg of a p21 promoter-luciferase reporter
construct, 20 ng of p53 alone (lane 2), or with increasing
amounts (0.1, 0.5, or 1.0 µg) of a p65 expression vector, in the
absence (lanes 3-5), or presence, of 10 µg of a CBP
expression vector (lane 6). C, p53 repression of
p65-dependent gene expression is CBP-dependent.
COS cells were cotransfected with 2 µg of an E-selectin
promoter-CAT reporter construct ( 578-CAT), 100 ng of a p65 expression
vector alone (lane 2), or with increasing amounts (5, 10, 20, or 35 ng) of a p53 expression vector (lanes 3-6), in
the presence or absence of increasing amounts (0.5 and 1.0 µg) of an
MDM2 expression vector (lanes 7-9) or increasing amounts
(3, 5, 8, or 10 µg) of a CBP expression vector (lanes
10-13). The total concentration of DNA was adjusted to 10 µg/6-cm tissue culture dish with empty pCR3 expression vector.
Forty-eight hours post-transfection, luciferase or CAT activities in
cell extracts were measured as described previously. Portions of the
cellular extracts from A-C were analyzed for p65 and p53 by
Western blot analysis (insets). Data presented are
representative of at least three independent transfections.
B-dependent gene
expression, similar studies were done with a
NF-
B-dependent E-selectin promoter-reporter construct
(2, 28), a fixed amount of p65, and increasing amounts of p53. As
predicted, p65 results in an induction of the
NF-
B-dependent reporter gene (Fig. 1C,
lane 2). Increasing amounts of p53 resulted in a
dose-dependent suppression of p65-mediated transactivation
(Fig. 1C, lanes 3-6). This effect was reversed
by MDM2 (Fig. 1C, lanes 7-9), an inhibitor of
p53 transactivation. Control studies demonstrated that overexpression of MDM2 did not alter levels of p65 (Fig. 1C,
insets), although in some experiments it modestly increased
p65-dependent gene expression (Fig. 1C,
lanes 7-9). MDM2 does not physically interact with p65 (data not shown). The presence of p53 had no effect on nuclear accumulation of NF-
B or DNA binding activity, and it did not physically interact with p65 (data not shown). Additionally, p53 did
not increase expression of an inhibitor of NF-
B, I
B-
(data not
shown). Thus p65-mediated transcriptional activation is repressed by
p53, and p53-dependent gene expression is repressed by p65.
B Is Inhibited by UV Light-induced
p53--
The mutual transrepression of p53- and
p65-dependent gene expression described above was observed
with over expressed activators and might not reflect the situation with
authentic levels of these transcription factors. To address this
important issue, we determined whether TNF
-activated NF-
B was
capable of inhibiting endogenous p53-mediated gene expression.
Endothelial cells transfected with an E-selectin promoter-reporter were
treated with TNF
and exposed to increasing amounts of UV
irradiation. As expected, TNF
activated expression of the E-selectin
promoter-reporter construct (Fig. 2A, lane 2). UV
illumination resulted in a dose-dependent suppression of
this activity (Fig. 2A, lanes 3-5).
Cotransfection of an MDM2 expression plasmid, while significantly
inhibiting a p53 transcriptional response (Fig.
3 and data not shown), increased
expression from the NF-
B-dependent reporter gene (Fig.
2A, lane 6). This suggests that the
transcriptional activating capacity of endogenous p53 is required for
the suppression of the E-selectin promoter-reporter construct. The
inhibitory effect of endogenous p53 on NF-
B-dependent gene expression was completely abolished by cotransfection of a vector
expressing CBP (Fig. 2A, lane 7). In control
studies, levels of
-galactosidase activity were measured in parallel
with the activity of the reporter gene. In contrast to the results with
the NF-
B-dependent reporter, neither the UV treatment
nor cotransfection with either MDM2 or CBP altered the level of
-galactosidase activity (Fig. 2B). These results are
consistent with the possibility that the coactivator rescues endogenous
p53-suppressed NF-
B-dependent gene expression.
View larger version (14K):
[in a new window]
Fig. 2.
TNF -activated
NF-
B activity is suppressed by UV
light-induced p53. A, UV irradiation suppresses
TNF
-activated NF-
B activity. ECV-304 endothelial cells were
cotransfected with 2 µg of an E-selectin promoter-CAT reporter
construct (
578-CAT) and 0.25 µg of a
-galactosidase expression
vector, either alone (lane 1) or in the presence of 1 µg
of an MDM2 expression vector (lane 6) or 1 µg of a CBP
expression vector (lane 7). Transfected cells were treated
with 100 units/ml TNF
for 30 min (lane 2) and then some
of the cells were exposed to increasing amounts of ultraviolet light
(10, 20, and 40 J/m2) (lanes 3-5). Cells were
harvested 4 h after exposure to UV light. B, UV
irradiation does not alter expression of
-galactosidase activity.
Portions of the cellular extracts described above were analyzed for
-galactosidase activity. Data presented in A and
B are representative of at least three independent
transfections.
View larger version (16K):
[in a new window]
Fig. 3.
UV-induced NF- B
activity is suppressed following irradiation. A, UV
exposure induces expression of a p53-dependent reporter
construct. ECV-304 cells were cotransfected with 2 µg of a
p21-promoter-luciferase construct in the presence or absence of 1 µg
of an MDM2 expression vector. Treated cells were exposed to 40 J/m2 UV ultraviolet light and cells were harvested at the
times indicated following stimulation. B, NF-
B activity
induced by UV is stimulated by MDM2. ECV-304 cells were transfected
with 2 µg of an E-selectin promoter-CAT reporter construct
(
578-CAT) in the presence or absence of 1 µg of an MDM2 expression
vector. Transfected cells were treated with UV as described above
and harvested at the times indicated. The results shown are
representative of three separate experiments.
B Activity Is Suppressed following UV Stimulation--
The
findings described above predict that if endogenous NF-
B and p53 are
induced simultaneously they would inhibit each other's transcriptional
activity. Because UV irradiation activates both transcription factors
(29), we used it as a stimulus to examine the effect of the signaling
pathways on each other. UV exposure strongly induces expression of a
p21 promoter-reporter construct (Fig. 3A) in a
time-dependent manner. In parallel, UV also activated expression of an E-selectin promoter-reporter gene (Fig.
3B). Cotransfection of an MDM2 expression plasmid blocked
the p53 transcriptional response (Fig. 3A), while increasing
expression of the NF-
B-dependent reporter plasmid (Fig.
3B). These results are consistent with the proposal that the
transcriptional activity of endogenous NF-
B is regulated by p53.
B signaling cascades is mediated by CBP. These findings are
consistent with observations that the levels of p300 are limiting relative to those of p65 (30) and that developmental processes are
sensitive to the overall gene dosage of either CBP or its homolog, p300
(31). The common dependence of both p65 and p53 on CBP/p300 suggests
that the coactivators may integrate multiple signaling pathways that
converge on these transcription factors. For example, p53 inhibits
AP-1-dependent transcription, and overexpression of p300
abolished the ability of p53 to inhibit AP-1 activity (18). Similar
interplay was observed between p53 and E2F through the coactivator p300
(32). NF-
B-dependent gene expression is also regulated
by coactivator competition. The p65 component of NF-
B and the
glucocorticoid receptor mutually repress each other's ability to
activate transcription (33). Increased levels of CBP relieve the
inhibition of glucocorticoid-mediated repression of NF-
B activity
and the NF-
B mediated repression of GR activity (27). The results
described here indicate that NF-
B and p53 mutually repress each
other's transcription, not only in vitro, but also in
intact cells, and suggest that these signaling systems converge on the
coactivators. Collectively, these studies are consistent with proposals
that coactivator sequestration can function as a selection mechanism to
determine patterns of gene expression (34).
B and p53 are activated. In settings where NF-
B
is acting as a survival factor, NF-
B would induce several genes that
promote resistance to apoptosis and entry into cell cycle. Indirectly,
through coactivator competition, NF-
B would also suppress p53's
ability to stimulate genes involved in cell death, such as Bax (35),
which might block the survival effect. In the context of cell death,
p53 would activate expression of genes involved in apoptosis and
suppress the survival signals generated by
NF-
B-dependent gene expression. This control process could be dysregulated during formation of tumors (36).
![]() |
ACKNOWLEDGEMENTS |
---|
The wild-type p53 expression vector and a p21 promoter-luciferase reporter construct were kindly provided by Dr. B. Vogelstein (The Johns Hopkins University). The human MDM2 expression vector was provided by Dr. A. Levine (Princeton University), a CBP expression vector was provided by R. Goodman (Oregon Health Science Center), and a Bax promoter-reporter construct was provided by J. Reed (La Jolla Cancer Research Foundation).
![]() |
FOOTNOTES |
---|
* This work was supported by Research Grants HL 35716, HL45462, and PO1 HL 36028 from the National Institutes of Health (to T. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pathology,
Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-732-5990; Fax: 617-278-6990; E-mail: tcollins{at}bustoff.bwh.harvard.edu.
The abbreviations used are:
NF-B, nuclear
factor-
B; CAT, chloramphenicol acetyltransferase; CBP, CREB-binding
protein; CREB, cAMP-response element-binding protein; TNF, tumor
necrosis factor.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|