Báo cáo khoa học: Inhibition of the NF-jB transcriptional activity by protein kinase A pot

Inhibition of the NF-jB transcriptional activity by protein kinase A
Naoko Takahashi, Toshifumi Tetsuka, Hiroaki Uranishi and Takashi Okamoto
Department of Molecular Genetics, Nagoya City University Medical School, Japan
The cAMP-dependent protein kinase (PKA) signaling
pathway plays a major role in a number of pathophysio-
logical conditions. However, there have been conflicting
evidences regarding the action of cAMP/PKA on nuclear
factor- jB(NF-jB). In this study, we have explored the effect
of cAMP/PKA on NF-jB activity and determined its
molecular mechanism. PKA activating agents or expression
of the catalytic subunit of PKA (PKAc) inhibited the NF-jB-
dependent reporter gene expression induced by tumor nec-
rosis factor a (TNFa). PKA activators affected neither IjBa
phosphorylation, IjBa degradation, nor the NF-jB/DNA
binding. Expression of PKAc inhibited the transactivation
potential of Gal4-p65 (286–551) suggesting that the inhibi-
tory action of PKA is through the C-terminal transactivation
domain of p65 but not by phosphorylation of the consensus
PKA recognition site containing serine at position 276.
Overexpression of coactivators, CBP (CREB-binding pro-
tein) and p300, failed to reverse the PKA-mediated inhibition
of p65 transactivation. Thus, the inhibitory action of the
cAMP/PKA pathway on the transcriptional activity of
NF-jB appears to be exhibited by modifying the C-terminal
transactivation domain of p65, either directly or indirectly.
Keywords:NF-jB; PKA; cAMP; signal transduction.
Nuclear factor jB (NF-jB) is an inducible cellular tran-
scription factor that regulates a wide variety of cellular and
viral genes including several cytokines, cell adhesion mole-
cules and human immunodeficiency virus (HIV) [1–4].
Members of the NF-jB family in mammalian cells include

the proto-oncogene c-Rel, Rel A (p65), Rel B, NF-kB1
(p50/105), and NF-kB2 (p52/p100)
1
. These proteins share a
conserved 300 amino acids region known as the Rel
homology domain, which is responsible for DNA binding,
dimerization, and nuclear translocation [1–4]. In most cells,
Rel family members form hetero- and homo-dimers with
distinct specificities in various combinations. A common
feature of the regulation of the NF-jB family is their
sequestration in the cytoplasm as inactive complexes with a
class of inhibitory molecules known as IjBs [1–4]. Treat-
ment of cells with a variety of inducers such as phorbol ester,
interleukin-1b (IL-1b) and tumor necrosis factor a (TNFa)
results in phosphorylation, ubiquitination and degradation
of the IjB proteins [3,5,6]. The degradation of IjB proteins
exposes the nuclear localization sequence in the remaining
NF-jB dimers, leading to nuclear translocation and subse-
quent binding of NF-jB to the DNA cis-regulatory element
of target genes [1–4]. In addition to the nuclear translocation
and DNA-binding of NF-jB, its transcriptional activity is
regulated by coactivators, CBP (CREB-binding protein)
and p300, that associate with the C-terminal transactivation
domain of p65. It has been demonstrated that these
coactivators physically interact with p65 and promote its
transcriptional activity [7,8].
On the other hand, elevation of intracellular cAMP
induces the expression of numerous genes through the
protein kinase A (PKA)-mediated phosphorylation of
transcription factors, including the cAMP response ele-

ment binding protein (CREB) [9]. Interestingly, cAMP
inhibits the induction of a distinct set of NF-jB regulated
genes. For example, cAMP inhibits IL-2 expression in
activated T-lymphocytes. In monocytes, pharmacological
agents that elevate intracellular cAMP, such as pentoxif-
ylline, iloprost, dibutyryl cAMP (db-cAMP), forskolin
(FSK), and isomethylxanthine, inhibit the TNFa produc-
tion and tissue factor expression that are also under the
control of NF-jB [10–14]. In endothelial cells, elevation of
cAMP inhibits the induction of E-selectin and VCAM-1
mediated by IL-1b or TNFa [15,16]. Thus, elevation of
intracellular cAMP and/or activation of PKA appear to
modulate the transcriptional activity of NF-jBina
negative way.
In this report, we examined whether cAMP and PKA
modulates NF-jB activity and found that the cAMP/PKA
signaling pathway inhibits the NF-jB activity by acting on
the transactivation domain of p65. Possible mechanisms for
this inhibitory action are discussed.
EXPERIMENTAL PROCEDURES
Reagents
Forskolin (FSK), 8-bromo-cAMP (Br-cAMP), dibutyryl
cAMP (db-cAMP) were purchased from Wako (Osaka,
Japan). Recombinant human TNFa and recombinant
human IL-1b were purchased from Roche (Tokyo,
Japan).
Correspondence to T. Okamoto, Department of Molecular Genetics,
Nagoya City University Medical School, 1 Kawasumi,
Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.
Fax: + 81 52 859 1235, Tel.: + 81 52 853 8205,

E-mail:
Abbreviations:NF-jB,nuclearfactorjB; PKA, cAMP-dependent
protein kinase; PKAc, catalytic subunit of PKA; FSK, forskolin;
db-cAMP, dibutyryl cAMP; Br-cAMP, 8-Bromo-cAMP; CREB,
cAMP response element binding protein; CBP, CREB binding pro-
tein; TNFa, tumor necrosis factor a;IL-1b, interleukin-1b;C/EPBb,
CCAAT/enhancer-binding protein b.
(Received 20 May 2002, revised 9 July 2002, accepted 29 July 2002)
Eur. J. Biochem. 269, 4559–4565 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03157.x
Cell cultures
Jurkat T-lymphocytes were maintained in RPMI1640
medium supplemented with 10% fetal bovine serum. HeLa
and human embryonic kidney 293 cells were maintained in
Dulbecco’s modified Eagle’s medium supplemented with
10% fetal bovine serum. Primary culture of rat mesangial
cells were prepared as described previously [17], and
maintained in RPMI1640 medium supplemented with
15% fetal bovine serum and 0.15 IUÆmL
)1
insulin.
Plasmids
Mammalian expression vectors, pCMV-p300, pRSV-CBP,
and pRSV-CREB were generous gifts from Drs David
Livingston (Dana-Farber Cancer Institute), Shunsuke Ishii
(Riken) and Masatoshi Hagiwara (Tokyo Medical and
Dental University), respectively. The constructions of
luciferase reporter 4 jB-luc, pCMV-p65, pGal4-p65
(1–551), and pGal4-p65 (286–551) expression plasmids were
described previously [18]. The pGal4-CREB (1–280),
pCRE-luc containing four tandem repeats of the CREB-

binding sites, pGal4-luc containing five tandem repeats of
Gal4-binding sites, and the mammalian expression vector
pCMV-PKAc encoding the PKA catalytic subunit were
obtained from Stratagene (La Jolla, CA).
Transfection conditions and luciferase assay
Transient transfection of Jurkat T-lymphocytes was
accomplished by using 5 lL of SuperFect transfection
reagent (Qiagen) per ml of culture medium and a total of
1 lg of plasmid DNA. Blank control plasmids
pcDNA3.1(–) and pUC19 were used to equalize the
amount of DNA for each transfection. SuperFect-DNA
complexes were allowed to form for 10 min at room
temperature in serum-free medium before being added to
the cells. Cells were incubated with the complex for 24 h,
stimulated with 10 ngÆmL
)1
of human recombinant TNFa
for an additional 24 h and harvested. Gene expression was
measured by luciferase activity as previously described [18].
Luciferase assays were performed and relative light units
were determined with an AutoLumat LB9507 luminometer
(EG & G Berthold, Japan). Transfection efficiency was
monitored by Renilla luciferase activity with pRL-TK
plasmid containing TK promoter as an internal control. All
luciferase activities shown in transient transfection assays
are corrected by the internal control activity of Renilla
luciferase by pRL-TK.
Electrophoretic mobility shift assay (EMSA)
Jurkat T-lymphocytes were pretreated with FSK (50 l
M

)or
vehicle (DMSO) for 30 min, and then stimulated with
TNFa (10 ngÆmL
)1
) for 30 min. Nuclear extracts were
prepared as previously described [19–21]. The double
stranded oligonucleotide probe for NF-jB was synthesized
and endlabeled by [c-
32
P]-ATP. The jB sequence was taken
from the human immunodeficiency virus long-terminal
repeat (HIV-LTR). The jB sequence used was forward
(5¢-TTTCTAGGGACTTTCCGCCTGGGGACTTTCCAG-3¢)
and complement (5¢-TTTCTGGAAAGTCCCCAGGCG
GAAAGTC CCTAG-3¢). The EMSA was performed as
previously described [19–21]. Nuclear extracts were incuba-
ted in 10 lL EMSA buffer containing the radiolabeled jB
oligonucleotide probe. The samples were analyzed by 6%
nondenaturing PAGE.
Western blot analysis
Jurkat T-lymphocytes were pretreated with or without FSK
(50 l
M
) for 30 min and then stimulated with TNFa
(10 ngÆmL
)1
)orIL-1b (10 ngÆmL
)1
). After stimulation,
cells were washed twice with ice-cold NaCl/Pi and lysed in

TOTEX buffer (20 m
M
Hepes/KOH, pH 7.9, 350 m
M
NaCl, 20% glycerol, 1% NP-40, 1 m
M
MgCl
2
,0.5m
M
EDTA, 0.1 m
M
EGTA, 1 m
M
dithiothreitol, 20 m
M
2-gly-
cerophosphate, 0.1 m
M
Na
3
VO
4
,1m
M
phenylmethane-
sulfonyl fluoride, and 2.5 lgÆmL
)1
each of aprotinin,
leupeptin and pepstatin). Equal amounts of samples

(20 lg of total protein) were subjected to 10% SDS/PAGE
and transferred onto a nitrocellulose membrane (Hybond-
C; Amersham Pharmacia Biotech). The membranes were
probed with anti-(IjBa) serum, anti-(IjBa-P) serum (New
England Bio Laboratories, Beverly, MA) and proteins were
visualized by enhanced chemiluminescence (Amersham Life
Science Inc., Cleveland, OH).
RESULTS
cAMP/PKA inhibits TNFa-induced NF-jB activation
To examine the effect of cAMP/PKA on TNFa-induced
NF-jB activity, Jurkat T-lymphocytes were treated with
PKA activating agents, FSK, db-cAMP and Br-cAMP.
Stimulation with these agents decreased TNFa-induced
NF-jB reporter gene expression in a dose dependent
manner (Fig. 1A–C). To test whether these inhibitory
effects of PKA activating agents are not limited for 4 kB-
luc, we examined the effects of FSK and db-cAMP on HIV-
LTR-luc which contains two NF-jB binding sites. As
shown in Fig. 1D, FSK and db-cAMP inhibited the TNFa-
induced HIV-LTR activation. To further test whether this
inhibitory effect of cAMP was mediated by PKA, we
transfected the catalytic subunit of PKA (PKAc) into Jurkat
and HeLa cells along with the jB-dependent luciferase
reporter. Transfection of PKAc led to a dramatic decrease
in the TNFa-induced expression from the jB-reporter gene
in both cells (Fig. 1E and F). These data indicate that
elevation of intracellular cAMP and subsequent activation
of PKA lead to the inhibition of NF-jB activity.
To further examine the effect of PKA on NF-jB activity,
we cotransfected Jurkat T-lymphocytes and HeLa cells with

plasmids expressing PKAc and p65 subunit of NF-jB,and
measured the level of NF-jB-dependent luciferase gene
expression. Transfection of p65 alone resulted in high levels
of luciferase expression, whereas cotransfection of p65 with
PKAc led to a dramatic decrease in the expression of the
reporter gene in both cells (Fig. 2A and B) supporting an
idea that PKA generally inhibits NF-jB-mediated tran-
scription. One of the major nuclear PKA substrates is the
transcription factor CREB (cAMP response element-bind-
ing protein) which binds to the cAMP response element
(CRE) in cAMP-inducible genes [9]. The phosphorylation
of CREB by PKA results in increased transactivation.
To examine whether the inhibitory effect of PKAc on
4560 N. Takahashi et al.(Eur. J. Biochem. 269) Ó FEBS 2002
transcription factor is specific for NF-jB, we cotransfected
the reporter plasmid pCRE-luc with PKAc and measured
CRE-dependent luciferase activity in Jurkat T-lymphocytes.
As expected, overexpression of PKAc led to an increase in
the CRE-dependent gene expression (Fig. 2C).
PKA activation does not affect IjB degradation
nor DNA-binding activity of NF-jB
In the unstimulated cells, NF-jB is sequestrated in the
cytoplasm as inactive complexes with IjBs, which are
phosphorylated and degraded upon stimulation of cells with
inducers of NF-jB,suchasTNFa and IL-1b [1–4]. As
Neumann et al. indicated that elevation of cAMP reduces
NF-jB activity, possibly by stabilizing IjBa [22], we
investigated whether the activation of PKA could inhibit
IjBa degradation. Jurkat T-lymphocytes were stimulated
with TNFa (20 ngÆmL

)1
) in the presence or absence of FSK
(50 l
M
), and IjBa were detected by immunoblotting. As
demonstrated in Fig. 3A, there was no difference in IjBa
degradation in the presence or absence of FSK. Similar
observation was obtained with HeLa cells (data not shown).
In addition, in primary culture of rat glomerular mesangial
cells, FSK did not affect the IL-1b-induced IjBa phospho-
rylation and subsequent degradation (Fig. 3B and C). These
data indicate that PKA activation by FSK does not affect
TNFa-orIL-1b-induced IjBa degradation in most cells. To
confirm that PKA activation does not modify DNA-
binding activity of NF-jB, we performed EMSA using
nuclear extract prepared from Jurkat cells. FSK did not
inhibit TNFa-induced DNA-binding of NF-jB (Fig. 3D).
Thus, it is likely that the inhibitory effect of PKA on NF-jB
is not due to the inhibition of DNA-binding activity of
NF-jB.
PKA impairs transactivation potential of p65
p65 protein has a putative consensus PKA recognition site
(RRPS) located approximately 25 amino acids N-terminal
from the nuclear localizing signal (NLS) in the Rel
homology domain [1–4]. (Fig. 4A). Zhong et al. reported
that the phosphorylation of this serine residue (at 276 amino
acid position) could modulate the transcriptional activity of
p65 [23]. Thus we have examined whether the phosphory-
lation of p65 at Ser276 is involved in the mechanism by
which PKA inhibits NF-jB activation. In Fig. 4B, we

examined the transcriptional activity of pGal4-p65 (1–551),
containing the full-length p65, and pGal4-p65 (286–551),
containing the transactivation domain of p65 but lacking
the Rel homology domain and the RRPS site (Ser276) of
p65, in response to PKAc overexpression. When we
cotransfected Jurkat T-lymphocytes with pCMV-PKAc
and either pGal4-p65 (1–551) or pGal4-p65 (286–551) and
measured the Gal4-dependent luciferase reporter gene
expression, PKAc inhibited the Gal4-dependent luciferase
activity induced by either pGal4-p65 (1–551) or pGal4-p65
Fig. 1. The effect of PKA activating agents and
PKAc on NF-jB-dependent transcription
inducedbyTNFa. (A–C) Suppression of
NF-jB-dependent gene expression (4 jB-luc)
in Jurkat cells by cAMP elevating agents
including FSK (A), db-cAMP (B), and
Br-cAMP (C). (D) Inhibition of TNFa-
induced HIV-LTR-luc activation in Jurkat
cells by FSK and db-cAMP. (E and F) Inhi-
bition of TNFa-induced NF-jB-dependent
gene expression (4 jB-luc) in Jurkat (E) and
HeLa cells (F). Cells were transfected with
100 ng of 4 jB-luc or HIV-LTR-luc reporter
plasmid and/or 150 ng of pCMV-PKAc
expression plasmid. After 24 h of transfection,
cells were stimulated with 10 ngÆmL
)1
of
TNFa for 24 h in the presence or absence of
the PKA activating agent. Cells were then

harvested and subjected to luciferase assay.
The data are presented as the fold-increase in
luciferase activities relative to control trans-
fection (no TNF stimulation). Values are the
mean of two independent transfections (A–C)
or the mean ± SE of three independent
transfections (D–F). Experiments were repea-
ted at least three times with the same results.
Ó FEBS 2002 PKA inhibits NF-jB transactivation (Eur. J. Biochem. 269) 4561
(286–551) almost to a similar extent (Fig. 4B). These results
excluded the possibility that the activation of PKA inhibits
NF-jB by direct phosphorylation of the consensus PKA
recognition site at Ser276 of p65 but suggested that PKA
inhibits NF-jB by acting at the C-terminal transactivation
domain of p65 subunit. These results also excluded the
possibility that PKAc directly affected the NF-jB DNA
binding. In contrast, PKAc augmented the transcriptional
activity of Gal4-CREB (1–280) containing the CREB
transactivation domain and the possible PKA-mediated
phosphorylation site (Fig. 4C). Thus, the inhibitory effect of
PKAc on transcription appeared to be specific for NF-jB
subunit p65.
CREB and coactivator CBP/p300 are not involved in the
PKA-mediated inhibition of NF-jB
To examine if CREB, a well-known nuclear substrate of
PKA, is involved in the PKA-mediated inhibition of p65
activity, Jurkat cells were transfected with pRSV-CREB in
addition to pCMV-p65 and 4 jB-luc (Fig. 5A). However,
CREB did not affect the transcriptional activity of p65. As
both CREB and p65 use coactivators CBP/p300 [7,8], the

limiting amount of CBP/p300 might account for the PKA-
mediated inhibition of p65 transactivation. To test this
hypothesis, pRSV-CBP expressing CBP was cotransfected
with pCMV-PKAc and pCMV-p65. However, overexpres-
sion of CBP did not reverse the PKA-mediated inhibition of
p65 transactivation (Fig. 5B). Similar results were obtained
with p300 overexpression (data not shown). These obser-
vations indicate that PKA-mediated inhibition of p65
transactivation is not likely through the sequestration of
CBP/p300.
DISCUSSION
In this study we have observed reproducibly that the cAMP/
PKA signaling pathway inhibits transcriptional activity of
NF-jB. Although a previous report by others [22] suggested
that PKA inhibited NF-jB activation by stabilizing IjB,
PKA activating agents such as FSK did not significantly
affect the TNFa-induced IjBa degradation or DNA-
binding activity of NF-jB (Fig. 3). These findings clearly
indicated that PKA inhibits NF-jB activation at a step
downstream of IjB degradation. Sequestration of CBP/
p300 coactivators by CREB is not likely because CBP/p300
overexpression did not abolish the inhibitory effect of PKA
on NF-jB(Fig.5B).
2
In this study, we have demonstrated
the evidence that the C-terminal transactivation domain of
p65 is responsible for the inhibition of NF-jBmediatedby
the cAMP/PKA signaling (Fig. 4).
Fig. 2. Inhibition of NF-jB (p65)-dependent gene expression by PKAc.
Jurkat (A) or HeLa (B) cells were transfected with 100 ng of 4 jB-luc,

50 ng of pCMV-p65 and/or 150 ng of pCMV-PKAc. (C) Activation of
CREB-dependent gene expression by PKAc. Jurkat cells were trans-
fected with 100 ng of pCRE-luc and various amounts of pCMV-
PKAc. The data are presented as the fold-increase in luciferase activ-
ities relative to control transfection. Values are the mean ± SE of
three independent transfections.
Fig. 3. Forskolin does not affect IjBa degradation or DNA-binding
activity of NF-jB. (A) Jurkat cells were pretreated with 50 l
M
of FSK
for 30 min, and then stimulated with 10 ngÆmL
)1
of TNFa for indi-
catedtimeperiods.CellswereharvestedandsubjectedtoWesternblot
analysis. (B) The time course of IL-1b-induced IkBa degradation and
phosphorylation in rat glomerular mesangial cells. Mesangial cells
were stimulated with IL-1b (10 ngÆmL
)1
) for indicated time periods.
Cells were harvested and subjected to Western blot analysis with anti-
IjBa antibody or the antibody against phosphorylated IjBa at Ser32
(IjBa-P). (C) Effect of FSK on the IL-1b-induced IjBa degradation
and phosphorylation in rat mesangial cells. Cells were pretreated with
50 l
M
ofFSKfor30min,andthenstimulatedwithIL-1b
(10 ngÆmL
)1
) for 5 and 15 min. Cells were harvested and total cell
lysates were subjected to Western blot analysis. (D) Effect of FSK on

the NF-jB DNA-binding activity. Jurkat cells were pretreated with or
without 50 l
M
FSK for 30 min, and then simulated with TNFa
(10 ngÆmL
)1
) for 30 min. Cells were harvested and nuclear extracts
were subjected for EMSA analysis with the jB probe.
4562 N. Takahashi et al.(Eur. J. Biochem. 269) Ó FEBS 2002
There have been conflicting evidences regarding the
action of PKA on the NF-jB activity. Although the
majority of reports [10–14] including this study, demonstra-
ted the inhibitory actions of PKA on the NF-jB dependent
gene expression, some reports such as Zhong et al. [23,24]
showed that PKAc, associated with the cytosolic NF-jB/
IjB complex, enhanced NF-jB-dependent gene expres-
sion by transient luciferase reporter assay. This marked
difference may be explained by use of distinct reporter
plasmids. It is noteworthy that several commonly used
reporter plasmids contain CRE sites that mediate induction
of reporter gene expression in cells stimulated with cAMP-
increasing agents [25]. It is also possible that NF-jB-
dependent gene expression may be enhanced or inhibited
depending on the level of PKA enzyme activity.
Induction of C/EBPb
3
(CCAAT/enhancer-binding pro-
tein b) by cAMP/PKA might be involved in cAMP/PKA-
mediated inhibition of NF-jB activity. In our preliminary
experiments, cAMP elevating agents induced C/EBPb and

formed a complex with NF-jB, and overexpression of
C/EBPb inhibited the transactivation potential of p65,
Gal4-p65 (1–551) and Gal4-p65 (286–551) (data not
shown). A number of reports suggest a physiological role
of C/EBPb in mediating the PKA signaling. For instance,
PKA stimulates C/EBPb transcription in hepatocytes,
neuronal cells and in fibroblasts [26]. In addition, C/EBPb
is induced through PKA signaling pathway upon stimula-
tion with human chorionic gonadotropin in rat granulosa
Fig. 5. Effects of CREB and CBP on transcriptional activity of p65. (A)
Effect of CREB on the p65-mediated transcription. Jurkat cells were
transfected with 4 jB-luc reporter plasmid (100 ng), pCMV-p65 and/
or pRSV-CREBs. After 24 h of transfection, cell lysates were harves-
ted and the luciferase assay was performed. (B) Effect of CBP over-
expression on the PKAc-mediated inhibition of p65 activity. Jurkat
cells were transfected with 4 jB-luc, pCMV-p65 (50 ng), pCMV-
PKAc (150 ng), and various amounts of pRSV-CBP (50, 150, 500 ng).
The relative luciferase activity as compared with control (4 jB-luc
reporter plasmid alone) is shown. Values are the mean ± SE of three
independent transfections.
Fig. 4. Inhibition of NF-jB activity by PKAc through the C-terminal
transactivation domain of p65. (A) Functional configuration of p65
protein. Location of rel-homology domain (RHD), putative PKA
phosphorylation site (Ser276), and two transactivation domains (TA1
and TA2) are indicated. (B) Inhibition of p65 transcriptional activity
by PKAc. In order to evaluate the effect of PKAc in Jurkat cells, either
pGal4-p65 (1–551), encoding a fusion protein of Gal4 DNA-binding
domain and the full-length p65 (amino acids 1–551), or pGal4-p65
(286–551), that lacking the RHD and the putative PKA phosphory-
lation site was cotransfected with pCMV-PKAc and the gene expres-

sion from the pGal4-luc reporter plasmid (100 ng) was assessed. (C)
Activation of CREB activity by PKAc. Jurkat cells were transfected
with Gal4-CREB (1–280), pCMV-PKAc, and pGal4-luc reporter
plasmid. Data are presented as percentage inhibition (B) or percentage
activation (C) relative to control transfection without pCMV-PKAc.
Values are the means of two independent transfections. Similar results
were obtained repeatedly.
Ó FEBS 2002 PKA inhibits NF-jB transactivation (Eur. J. Biochem. 269) 4563
cells and in rat Leydig cells [27,28]. C/EBPb can physically
interact with NF-jB [29], and form a repressor protein
complex with NF-jB and estrogen receptor in the context of
interleukin-6 promoter [30]. These findings collectively
suggest that C/EBPb may mediate the inhibitory effects of
cAMP/PKA on NF-jB activity by interacting with p65 at
least in part. However, the effect of C/EBPb on NF-jB-
dependent transcription may be context-dependent because
in some promoters such as IL-6 and IL-8, in which NF-jB
binding site and C/EBP binding site are very closely located,
C/EBPb synergistically activates the NF-jB-dependent
transcription [31]. Thus, further studies are needed to clarify
theroleofC/EBPb in controlling the transcriptional activity
of NF-jB.
The C-terminal transactivation domain of p65 has been
shown to be phosphorylated at Ser529 and Ser536 by
various upstream stimuli [32–35]. Thus, we cannot exclude a
possibility that PKA may inhibit NF-jB by modulating the
phosphorylation status of p65 with its C-terminal transac-
tivation domain, either directly or indirectly. It is possible
that PKA inhibits NF-jB in a multiplicative way including
stabilization of inhibitor IjB, induction of a repressor

complex such as C/EBPb which interacts with NF-jB, and
modulation of the phosphorylation status of the transacti-
vation domain of p65.
ACKNOWLEDGEMENT
N. T and T. T. contributed equally to this work. This work was
supported in part by grants in aid from the Ministry of Health, Labor
and Welfare, the Ministry of Education, Culture, Sports, Science and
Technology, and the Japanese Health Sciences Foundation. We thank
Drs Shunsuke Ishii, Masatoshi Hagiwara, and David Livingston for
plasmids.
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