Repression of transforming-growth-factor-beta-mediated transcription by nuclear factor kappaB.

Abstract

Activation of transforming growth factor-beta (TGF-beta) and activin receptors leads to phosphorylation of Sma- and Mad-related protein 2 (Smad2) and Smad3, which function as transcription factors to regulate gene expression. Smad7 is a regulatory protein which is able to inhibit TGF-beta and activin signalling in a negative-feedback loop, mediated by a direct regulation by Smad3 and Smad4 via a Smad-binding element (SBE) in the Smad7 promoter. Interestingly, we found that the Smad7 promoter was also regulated by nuclear factor kappaB (NF-kappaB), a transcription factor which plays an important role in inflammation and the immune response. Expression of NF-kappaB p65 subunit was able to inhibit the Smad7 promoter activity, and this inhibition could be reversed by co-expression of IkappaB, an inhibitor of NF-kappaB. In addition, the inhibitory activity of p65 was observed in a minimal promoter that contained only the Smad7 SBE and a TATA box, without any consensus NF-kappaB binding site. This inhibitory effect appeared to be common to other TGF-beta- and activin-responsive promoters, since p65 also inhibited the forkhead-activin-signal-transducer-2-mediated activation of a Xenopus Mix.2 promoter, as well as the Smad3-mediated activation of 3TP-lux which contains PMA-responsive elements and a plasminogen-activator-inhibitor-1 promoter. Activation of endogenous NF-kappaB by tumour necrosis factor-alpha (TNF-alpha) was also able to inhibit the Smad7 promoter in human embryonic kidney 293 cells. In human hepatoma HepG2 cells, TNF-alpha was able to inhibit TGF-beta- and activin-mediated transcriptional activation. Furthermore, overexpression of the transcription co-activator p300 could abrogate the inhibitory effect of NF-kappaB on the Smad7 promoter. Taken together, these data have indicated a novel mode of crosstalk between the Smad and the NF-kappaB signalling cascades at the transcriptional level by competing for a limiting pool of transcription co-activators.

Full text

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Biochem. J. (2000) 348, 591–596 (Printed in Great Britain)
591
Repression of transforming-growth-factor-β-mediated transcription by
nuclear factor κB
Raman P. NAGARAJAN*, Feifei CHEN*, Wei LI*, Eva VIG†, Maureen A. HARRINGTON†, Harikrishna NAKSHATRI†
and Yan CHEN*†1
*Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 West Walnut Street IB130, Indianapolis, IN 46202, U.S.A., and
†Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202, U.S.A.
Activation of transforming growth factor-β (TGF-β) and activin
receptors leads to phosphorylation of Sma- and Mad-related
protein 2 (Smad2) and Smad3, which function as transcription
factors to regulate gene expression. Smad7 is a regulatory protein
which is able to inhibit TGF-β and activin signalling in a
negative-feedback loop, mediated by a direct regulation by
Smad3 and Smad4 via a Smad-binding element (SBE) in
the Smad7 promoter. Interestingly, we found that the Smad7
promoter was also regulated by nuclear factor κB (NF-κB), a
transcription factor which plays an important role in inflam-
mation and the immune response. Expression of NF-κB p65
subunit was able to inhibit the Smad7 promoter activity, and this
inhibition could be reversed by co-expression of IκB, an inhibitor
ofNF-κB.Inaddition,theinhibitoryactivityofp65wasobserved
in a minimal promoter that contained only the Smad7 SBE and
a TATA box, without any consensus NF-κB binding site. This
inhibitory effect appeared to be common to other TGF-β- and
activin-responsive promoters, since p65 also inhibited the
INTRODUCTION
Sma- and Mad-related proteins (Smads) are a family of proteins
which mediate signalling for the transforming growth factor-β
(TGF-β) superfamily of cytokines, including TGF-β, activin,
bone morphogenetic proteins, and many others [1,2]. Structural
and functional characterization of Smad proteins has allowed
theircategorizationintothreegroups:pathway-specific,common
mediator and inhibitory. Smads 1–3, 5 and 8 are pathway-
specific Smads which are phosphorylated by the type I receptors
of the TGF-β superfamily [1,2]. Upon phosphorylation, the
pathway-specific Smads form hetero-oligomeric complexes with
Smad4, the common mediator Smad. These complexes then
migrate to the nucleus, and regulate gene transcription through
either direct DNA binding, via the MH1 domain of the Smad
proteins, or association with other specific transcription factors,
such as Xenopus FAST-1 (forkhead activin signal transducer-1)
or mouse FAST-2 [3–6]. The MH2 domain of Smad proteins
contains a transactivating activity that is mediated by interaction
with two closely related proteins, CREB-binding protein (CBP)
and p300, which function as transcriptional co-activators that
link specific transcription factors with the basal transcriptional
machinery[7–9].Ontheotherhand,Smad6andSmad7constitute
the third group of Smads, the inhibitory Smads. These two
Abbreviation used: CA-ALK, constitutively active activin-receptor-like kinase; CBP, CREB-binding protein; CMV, cytomegalovirus; FAST, forkhead
activin signal transducer; HEK-293 cells, human embryonic kidney 293 cells; NF-κB, nuclear factor κB; IκB, inhibitor of NF-κB; SBE, Smad binding
element; Smad, Sma- and Mad-related protein; TGF-β, transforming growth factor-β; TNF-α, tumour necrosis factor-α.
1To whom correspondence should be addressed (e-mail ychen3?iupui.edu).
forkhead-activin-signal-transducer-2-mediated activation of a
Xenopus Mix.2 promoter, as well as the Smad3-mediated acti-
vation of 3TP-lux which contains PMA-responsive elements and
a plasminogen-activator-inhibitor-1 promoter. Activation of
endogenous NF-κB by tumour necrosis factor-α (TNF-α) was
also able to inhibit the Smad7 promoter in human embryonic
kidney 293 cells. In human hepatoma HepG2 cells, TNF-α was
able to inhibit TGF-β- and activin-mediated transcriptional
activation. Furthermore, overexpression of the transcription co-
activator p300 could abrogate the inhibitory effect of NF-κB on
the Smad7 promoter. Taken together, these data have indicated
a novel mode of crosstalk between the Smad and the NF-κB
signalling cascades at the transcriptional level by competing for
a limiting pool of transcription co-activators.
Key words: activin, p300, signal transduction, Smad7, trans-
cription factor.
proteins inhibit TGF-β, activin and bone-morphogenetic-protein
signalling by interacting with the type I receptors of the TGF-β
superfamily, or competing for the complex formation between
pathway-specific Smads and Smad4 [1,2].
Nuclear factor κB (NF-κB) is a transcription factor which
controls expression of a wide range of genes [10,11]. It is mainly
involvedincellularfunctionsinimmunologicalandinflammatory
responses, as well as control of cell growth and apoptosis. For
example, NF-κB is involved in the cellular response initiated by
different proinflammatory cytokines, including interleukins and
tumour necrosis factor-α (TNF-α) [12]. In mammalian cells, NF-
κB exists in the cytoplasm as a homodimer or heterodimer
composed of variable subunits: p50, p52, p65(RelA), c-Rel, and
RelB; although the major form of NF-κB exists as a heterodimer
between p50 and p65 [10,11]. Three of these subunits, p65, RelB
and c-Rel, contain a C-terminal transactivation domain, and all
five subunits have an N-terminal Rel homology domain, which
mediates DNA binding, dimerization, and interaction with an
inhibitor of NF-κB (IκB). The interaction of NF-κB with IκB
masks a nuclear localization sequence of NF-κB, and this
interaction sequesters NF-κB in the cytoplasm. Activation of
NF-κB is initiated when IκB is phosphorylated and degraded in
response to inflammatory cytokines, such as TNF-α, UV radi-
ation,viralinfection,potentoxidants,orothersubstances[10,11].
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R. P. Nagarajan and others
These stimuli release NF-κB and enable it to translocate into the
nucleus, where it regulates transcription of target genes. Similar
to other transcription factors including Smads, the trans-
activating activity of NF-κB is mediated by an interaction with
the common transcription co-activators CBP and p300 which
contain an intrinsic acetyltransferase activity [13–15]. Upon
activation by associating with other DNA-binding transcription
factors, CBP?p300 bridge those transcription factors to the basic
transcriptionapparatus,altertheconformationofthechromatin,
and initiate stimulation of transcription.
TheTGF-βandNF-κBsignallingpathwaysmaycrosstalkwith
each other. For example, TNF-α and TGF-β1 can synergistically
enhance expression of type VII collagen gene that forms part of
the extracellular matrix [16]. On the other hand, treatment
of B-cell lymphomas with TGF-β1 can antagonize the activity of
NF-κB by elevating IκB transcription, leading to accelerated
apoptosis by attenuating the NF-κB-mediated repression of c-
myc transcription [17]. Conversely, NF-κB can block TGF-β1-
induced apoptosis of hepatocytes [18]. These observations in-
dicate that there exists an intrinsic interaction between these two
signalling pathways that might be dependent on the cellular
context, as well as the nature of gene expression.
We recently reported characterization of the mouse Smad7
promoter [19]. We have found that Smad7 transcription is
regulated by TGF-β signalling through direct binding of Smad3
andSmad4withaSmad-bindingelement(SBE)presentinSmad7
promoter [19]. This observation, in conjunction with recent
studies demonstrating an upregulation of Smad7 mRNA upon
TGF-β or activin treatment in the cells [20,21], suggests that
Smad7 participates in a negative-feedback loop which regulates
the length and intensity of the TGF-β and activin signalling.
Using the Smad7 promoter and other TGF-β-responsive
promoters as model systems, we investigated the functional
interaction between TGF-β and NF-κB signalling pathways at
the molecular level, and unveiled a novel crosstalk between them
at the transcriptional level.
MATERIALS AND METHODS
Cell culture and cell transfection
The human embryonic kidney 293 cells (HEK-293 cells) and the
human hepatoma HepG2 cells were cultured in Dulbecco’s
modified Eagle’s medium, containing 10% fetal bovine serum
supplemented with penicillin and streptomycin. Transient cell
transfection was performed by calcium phosphate method for
HEK-293cells,andDEAE-dextranmethodforhumanhepatoma
HepG2 cells [19].
Plasmids
The rat Smad3, the constitutively active activin-receptor-like
kinase TGF-β type I receptors (CA-ALK-5), the mouse FAST-
2, and the Smad7 promoter constructs have been described
previouslybyus [6,19].TogeneratetheminimalSmad7 promoter
containing the SBE of the Smad7 promoter, an E1B TATA box
(5?-TATATAAT-3?)inadenovirustype5[22]wasfirstengineered
intothepGL2-basicvector(Promega).Subsequently,twotandem
repeats of the SBE (5?-TCGACAGGGTGTCTAGACGGCC-
ACG-3?) of the Smad7 promoter were subcloned immediately 5?
to the TATA box. The human NF-κB p65 subunit, p50 subunit,
and IκB have been described previously [23,24]. The promoter
constructcontainingthreetandemrepeatsofHIVNF-κBbinding
sites (NF-κB3-luc) has been described previously [25,26]. The
Mix.2 AR3-lux containing three tandem repeats of activin-
responsive elements has been described in [6,27]. The 3TP-lux
promoter reporter containing three repeats of PMA-responsive
elements and a portion of plasminogen activator inhibitor-1
promoter was originally described by Wrana et al. [28].
Promoter assay
For the promoter assay, about 5?10? cells per well in six-well
plates were transfected with different combinations of plasmid
DNA. An empty vector pcDNA3 (Invitrogen) was used to make
up to 5 µg of total DNA for each transfection. The p cyto-
megalovirus(pCMV)-β-galactosidaseplasmidwasco-transfected
with other plasmids as an internal control for the transfection
efficiency. The cells were harvested at 48 h after transfection by
lysis with 400 µl of Nonidet P40 lysis buffer [29]. In TNF-α-
treated groups, cells were treated with TNF-α (2 or 20 ng?ml) for
16 h before harvest. A 20 µl aliquot of the lysate was used in the
β-galactosidase assay as reported previously [30], and 10µl of
the lysate was used in the luciferase assay with a Promega
luciferase assay kit, and counted for 10 s in a FB12 luminometer
(Zylux) with the data represented as the relative light unit per
second.
RESULTS AND DISCUSSION
Inhibition of Smad7 promoter by NF-κB
Our initial characterization with the mouse Smad7 promoter
indicated the presence of a putative NF-κB regulatory site at the
position of ?34bp to ?41bp, relative to the major transcription
initiation site [19]. To determine whether NF-κB indeed played a
role in regulating the transcription of the Smad7 gene, we tested
the effect of expression of NF-κB p65 subunit on the Smad7
promoter activity. We used three different promoter constructs
containing different lengths of the Smad7 promoter fused to a
luciferase reporter gene [19]. When transfected into HEK-293
cells, all three promoter constructs gave rise to a high level of
basal transactivating activity. As shown in Figure 1(A), the
largest reporter construct, spanning ?2.2 kb to ?112 bp of
theSmad7promoter,exhibitedthehighestlevelofbasalpromoter
activity. The second (?408 to ?112 bp) and the third (?408
to ?124 bp) promoters showed a little lower basal activity.
Interestingly, co-expression of p65 with each of these reporters
greatly reduced the basal transcription of the Smad7 promoter.
In p65 co-transfected cells, the basal luciferase activity was
reduced to approx. 10–15% of the control level for all three
Smad7 promoter constructs. This finding was somewhat un-
expected, because the putative NF-κB binding site in the Smad7
promoter was not covered by the third reporter that spans
?408 bp to ?124 bp of the promoter. Therefore these data not
only indicated that NF-κB was able to inhibit the basal Smad7
transcription, but also suggested that this inhibition was unlikely
to be caused by the interaction of NF-κB with the putative NF-
κB consensus site in the region of ?34 bp to ?41 bp.
In mammalian cells, the predominant form of NF-κB is
composed of a heterodimer of p65 and p50 [10,11]. When p50
alone was transfected in HEK-293 cells, it had no effect on the
basal transcription of Smad7 promoter (results not shown).
However, co-expression of p50 with p65 gave the similar level of
inhibition as p65 expression alone (Figure 1B). Because p65, but
not p50, contains the transactivating domain in the NF-κB
family of transcription factors, this finding suggested that the
inhibitory effect might be linked to the presence of the trans-
activating activity of NF-κB. In order to further test if the
inhibitory effect of NF-κB p65 on Smad7 promoter was specific,
we determined whether or not IκB was able to reverse the p65
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Nuclear factor-κB regulates transforming growth factor-β transcription
Figure 1 Repression of Smad7 promoter by NF-κB
(A) Effect of NF-κB p65 on different lengths of Smad7 promoter. HEK-293 cells were transiently
transfected with Smad7 promoter constructs (0.5 µg), pCMV-β-gal (0.5 µg) and p65 (2 µg)
as indicated. The absolute change of luciferase activity is shown as means?S.D. (B) IκB
reverses the repression effect of p65. HEK-293 cells were transiently transfected with Smad7
promoter ?408 to ?124 construct (0.5 µg), pCMV-β-gal (0.5 µg), p50 (1 µg), p65 (1 µg)
and IκB (2 µg) as indicated. The change of luciferase activity shown as means?S.D. was
derived by comparing to the value of the vector-transfected cells and normalized by β-
galactosidase activity.
effect. IκB is a specific inhibitor of NF-κB through masking of
the nuclear translocation site of NF-κB. When IκB was co-
expressed with p65, or p65 plus p50, it was able to completely
abrogate the inhibitory effect of NF-κB on the Smad7 promoter.
These data, therefore, provided further evidence that the in-
hibitory effect that we observed with Smad7 promoter was
specifically caused by p65.
Figure 2 Effect of p65 on multiple TGF-β-responsive promoters
(A) Repression of the SBE of the Smad7 promoter by p65. HEK-293 cells were transiently
transfected with pCMV-β-gal (0.5 µg), a control promoter containing only the TATA-box
(0.5 µg), a promoter containing two tandem repeats of the SBE and one TATA-box (0.5 µg),
p65 (1 µg), IκB (2 µg), Smad3 (1 µg), and the constitutively active ALK-5 (1 µg) as indicated.
The fold change of luciferase activity as compared to the vector-transfected cells (set to 1) is
shown as means?S.D after being normalized by β-galactosidase activity. (B) p65 inhibited
transcription of other TGF-β-responsive promoters. A similar experiment as above was
performed by transfection with AR3-lux (0.5 µg), 3TP-lux (0.5 µg) or the HIV NF-κB3-luc
reporter (0.5 µg) instead. The mouse FAST-2 (1 µg) was co-transfected with AR3-lux.
NF-κB represses the SBE of the Smad7 promoter
As we have shown before, Smad7 promoter contains a consensus
SBE that is able to associate with Smad3 and Smad4. To further
determine the regulatory role of NF-κB on the Smad7 promoter,
we generated a luciferase reporter that contained two tandem
repeats of this SBE and an E1B TATA box [22]. When the
control reporter only containing the TATA-box and the SBE-
containing reporter were transfected into HEK-293 cells, both of
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R. P. Nagarajan and others
them gave rise to a very low basal transactivation, as compared
with the longer Smad7 promoter constructs used in Figure 1.
However, expression of either CA-ALK-5 or Smad3 was able to
significantly stimulate the SBE-containing reporter, but not the
TATA-only reporter (Figure 2A). These data provided further
evidence that the regulatory function of TGF-β and activin
signalling through Smad proteins on Smad7 transcription was
mediated by this particular SBE present in the Smad7 promoter.
We next determined the effect of p65 on this shorter form of
Smad7 promoter. When p65 was co-transfected with the SBE-
containing reporter, it could completely reverse the stimulatory
effect of either CA-ALK-5 or Smad3 on the reporter (Figure 2A).
These data indicated that the inhibitory activity of p65 could
overcome the stimulatory effect of CA-ALK-5 or Smad3 on the
SBE of the Smad7 promoter. In addition, we also found that IκB
was able to reverse the p65 effect, further indicating that the
inhibitory effect of NF-κB on the SBE was mediated by p65. It
is noteworthy that IκB appeared to augment the stimulatory
effect of CA-ALK5 or Smad3 on the SBE transcription. This
might be caused by the possibility that the overexpressed IκB was
able to inhibit endogenous p65 and therefore relieve the basal
inhibitory effect of the endogenous p65 on the Smad-mediated
transcription.
NF-κB was able to inhibit other TGF-β-regulated promoters
Our experiments so far have indicated that NF-κB plays a unique
regulatory role on the Smad7 promoter. Is this regulatory
function only specific for Smad7 promoter, or common to other
promoters that are regulated by TGF-β signalling? To address
this issue, we tested two other promoters regulated by TGF-β
and activin signalling. AR3-lux is a luciferase reporter that
contains three tandem repeats of the activin-responsive elements
which are regulated by the FAST group of transcription factors
when complexed with Smad proteins following activation of
TGF-β or activin signalling [3,31]. Previous studies, as well as
our own observations, have indicated that a mouse FAST
homologue, FAST-2, was able to stimulate AR3-lux following
activationofTGF-βor activin signalling[5,6].3TP-luxisanother
reporter that has been commonly used as an indicator of TGF-
β or activin signalling. This reporter contains three tandem
repeats of PMA-responsive elements fused with the promoter of
plasminogen activator inhibitor-1 [28]. Numerous studies have
indicated that 3TP-lux could be activated by TGF-β or activin
signalling, possibly through binding of Smad proteins with a
CAGA motif in the plasminogen activator inhibitor-1 promoter
[32], or interaction of Smads with activator protein-1 that binds
PMA-responsive elements [33]. As shown in Figure 2(B), ex-
pression of Smad3 in HEK-293 cells was able to stimulate AR3-
lux in the presence of the mouse FAST-2. Similarly, Smad3 was
also able to elevate the transcription of 3TP-lux. However, the
stimulatory effect of Smad3 on both of these promoters was
largely abrogated by co-expression of p65. These data clearly
indicated that p65 has an inhibitory effect on different types of
promoters that are regulated by TGF-β or activin signalling. To
prove that p65 was indeed functionally expressed in our ex-
perimental system, we tested the effect of p65 on another reporter
construct that contains three tandem repeats of NF-κB binding
element present in the long terminal repeat of the HIV genome
[25,34]. As shown in Figure 2(B), p65 was able to strongly
stimulatethispromoter,indicating thatp65was indeed expressed
and functioning in these cells. Interestingly, Smad3 expression
appeared to further augment the stimulatory effect of p65 on this
promoter. This is consistent with a recent report demonstrating
that TGF-β is able to stimulate HIV enhancer through the NF-
κB binding element [35].
Inhibition of TGF-β-mediated transcription by TNF-α
NF-κB is mainly involved in cellular response to inflammatory
and immunological stimuli. One of the prototypic pathways in
which NF-κB plays a pivotal role is TNF-α signalling [12].
Activation of TNF receptors by TNF-α is followed by activation
of a whole array of signalling pathways, including activation of
NF-κB through phosphorylation and degradation of IκB. To see
if the regulatory role of NF-κB we observed here is of any
biological significance, we determined whether endogenous NF-
κB could mimic the inhibitory effect of ectopically expressed p65
following TNF-α treatment. To test this possibility, we expressed
Smad3 in HEK-293 cells, followed by treatment with different
amounts of TNF-α. We observed that TNF-α treatment could
significantly inhibit Smad3-induced stimulation of the Smad7
SBE reporter in a dose-dependent manner, and that this effect
could be reversed with IκB expression (Figure 3A). HEK-293
cells do express TNF-α receptors, and this TNF-α effect was
presumably due to the activation of endogenous NF-κB, since
this repression could be reversed by IκB. Taken together, these
results indicated that activation of endogenous NF-κB by bio-
logical stimuli may impose an inhibitory effect on the TGF-
β?activin-responsive Smad7 promoter in HEK-293 cells. It is
interesting to note here that the inhibitory effect of TNF-α on
Smad3 activation of the Smad7 promoter was unlikely due to an
inhibition of the Smad3 expression by p65. Western-blotting
analysis showed that the Smad3 protein level was higher in the
cells when it was co-expressed with p65 than in cells which were
transfected with Smad3 alone (results not shown). This was
probably due to the presence of the NF-κB binding site in the
CMV promoter that drove the ectopic expression of Smad3.
To further delineate the biological significance of our ob-
servation that p65 was able to inhibit TGF-β-mediated tran-
scription, we examined the effect of TNF-α treatment on TGF-
β- and activin-induced transcription of the AR3-lux reporter
derived from the Xenopus Mix.2 promoter in human hepatoma
HepG2 cells. This experiment excluded the use of exogenously
expressed p65 or Smad proteins in the experimental system. As
shown in Figure 3(B), TGF-β and activin treatment were able to
stimulate the AR3-lux transcription by approx. 140- and 20-fold
respectively. Furthermore, co-treatment with TNF-α was able to
inhibit the TGF-β- and activin-mediated transactivation. These
data would indicate that the TNF-α signalling pathway is able to
inhibit the transactivating activity of endogenous Smad proteins
following stimulation by TGF-β or activin.
Overexpression of p300 overcomes the inhibitory effect of p65
What is the molecular mechanism underlying the inhibitory
effect of NF-κB on TGF-β?activin-regulated promoters? Our
findings that p65 could inhibit different types of TGF-β or
activin-responsive promoters indicted that this effect is unlikely
due to the binding of p65 with a specific sequence element in
these promoters, since none of these promoters including the
Smad7 SBE, the FAST-binding activin-responsive element, and
the 3TP-lux, contain the consensus NF-κB binding element [36].
On the other hand, recent studies have indicated that the MH2
domain of Smad proteins was able to bind the general tran-
scription co-activators CBP and p300 at the C-terminal region to
mediate their transactivating activity [7–9]. Interestingly, NF-κB
is also able to bind the N-terminal and C-terminal regions of
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Nuclear factor-κB regulates transforming growth factor-β transcription
Figure 3TNF-α inhibited TGF-β-mediated transcription
(A) TNF-α inhibited SBE transcription of the Smad7 promoter. HEK-293 cells were transiently
transfected with the SBE-containing Smad7 minimal promoter (0.5 µg), pCMV-β-gal (0.5 µg),
Smad3 (1 µg) and IκB (2 µg) as indicated. The cells were treated with TNF-α 16 h before the
luciferase and β-galactosidase assays. The fold change of luciferase activity is shown as
means?S.D. after normalization with a β-galactosidase assay. (B) TNF-α inhibited TGF-β- and
activin-mediated transcription of AR3-lux derived from Xenopus Mix.2 promoter. Human
hepatoma HepG2 cells cells were transfected with AR3-lux (0.5 µg), mouse FAST-2 (1 µg) and
pCMV-β-gal (0.5 µg). The cells were treated with TGF-β1 (1 ng/ml), activin (10 ng/ml) or
TNF-α (20 ng/ml) for 16 h before luciferase assay. The fold change of luciferase activity
normalized by β-galactosidase assay is shown as means?S.D.
CBP?p300 [13–15]. There have been some observations dem-
onstrating that NF-κB is able to repress the transactivating
activity of other transcription factors through sequestration of a
limiting pool of CBP?p300. For example, NF-κB and p53
Figure 4
pression
Overexpression of p300 overcame the NF-κB-mediated re-
HEK-293 cells were transiently transfected with the SBE-containing Smad7 minimal promoter
(0.5 µg), pCMV-β-gal (0.5 µg), Smad3 (1 µg), p65 (1 µg) and different amounts of CBP or
p300 as indicated. The fold change of luciferase activity was shown is means?S.D after
normalization with β-galactosidase activity.
signalling pathways mutually repress each other’s transactivating
activity by competing for CBP?p300 [37]. NF-κB can also repress
STAT2-mediated HIV transcription through a similar mech-
anism [38].
To test if such a transcriptional sequestration of CBP?p300
may underlie the inhibitory effect of NF-κB on TGF-β?activin-
or Smad-mediated transcriptional activation, we determined the
effect of overexpression of CBP or p300 in our experimental
system. As shown in Figure 4, Smad3 expression was able to
stimulate the SBE-containing promoter, and the Smad3 effect
was inhibited by co-expressed p65. Both CBP and p300 were able
to enhance the stimulation of the promoter by Smad3, consistent
with previous reports that CBP and p300 are required for the
transactivating activity of Smad proteins [7–9]. Interestingly,
expression of p300 was indeed able to abrogate the inhibitory
effect of p65 on Smad3-activated Smad7 SBE activation in a
dose-dependent manner. CBP could also reverse part of the
inhibitory activity of p65, although to a lesser degree. Taken
together, these data indicated that p65 may inhibit the Smad-
mediated transcriptional control by competing for a limiting
pool of CBP?p300. When NF-κB is overexpressed or activated,
it may preferentially bind CBP or p300 and limit the amount of
these transcription co-activators available to Smad proteins that
are activated by TGF-β or activin signalling.
In conclusion, our results have indicated that the NF-κB-
mediated repression of gene expression via sequestration of
the common transcription co-activators may play a role in the
transcriptional regulation by Smad proteins. However, it is
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