Báo cáo khóa học: ICAM-1 expression is highly NF-jB-dependent in A549 cells No role for ERK and p38 MAPK docx

ICAM-1 expression is highly NF-jB-dependent in A549 cells
No role for ERK and p38 MAPK
Neil S. Holden
1
, Matthew C. Catley
2
, Lisa M. Cambridge
2
, Peter J. Barnes
2
and Robert Newton
1
1
Department of Biological Sciences, University of Warwick, Coventry, UK;
2
Thoracic Medicine, National Heart & Lung Institute,
Imperial College Faculty of Medicine, London, UK
The transcription factor nuclear factor jB(NF-jB) is an
activator of multiple cytokines, chemokines and adhesion
molecules, which are important in inflammatory diseases
such as asthma, and is consequently considered as an
attractive therapeutic target. In the present study, a con-
stitutively active dominant version of IjBa,IjBaDN, was
introduced into A549 pulmonary cells by adenovirus-
mediated delivery. The dominant IjB, but not a null viral
vector, prevented the induction of NF-jB-dependent
transcription by both tumor necrosis factor a (TNFa)and
interleukin-1b (IL-1b). Similarly, both TNFa and IL-1b
strongly induced mRNA and protein expression of inter-
cellular adhesion molecule (ICAM)-1 and in each case this
was prevented by adenovirus expressing the dominant IjB,

but not by the null virus, thereby establishing ICAM-1 as
an NF-jB-dependent gene. Numerous studies have sug-
gested key roles for the p38 and extracellular regulated
kinase (ERK) mitogen-activated protein kinase (MAPK)
cascades in the activation and transactivation of NF-jB.
We show here that SB203580, a selective inhibitor of the
p38 MAPK, and PD098059 and UO126, both selective
inhibitors of the ERK MAPK cascade, have no effect on
TNFa or IL-1b-induced translocation and DNA binding
of NF-jB. Furthermore, these inhibitors showed no
pharmacologically relevant effect on NF-jB-dependent
transcription nor was there any effect on expression of
ICAM-1. Taken together these data highlight the potential
use of inhibition of the NF-jB signalling pathway in
pulmonary inflammatory diseases and suggest that inhi-
bitors of the p38 and ERK MAPK pathways may be of
lesser effect.
Keywords:NF-jB; transactivation; ICAM-1; ERK; p38.
The transcription factor nuclear factor-jB(NF-jB) is a
central regulator of the immune system and promotes the
transcription of over 150 genes [1]. As many of these genes
are inflammatory, and include cytokines, chemokines,
adhesion molecules as well as other enzymes, such as
inducible nitric oxide synthase and cyclooxengenase-2,
NF-jB may provide an attractive target for therapeutic
intervention in inflammatory diseases [2]. NF-jBexistsas
a homo or heterodimer made up from subunits from the rel
family of proteins, which in vertebrates, comprises of p65
(RelA), p50/p105, p52/p100, c-Rel and RelB [3]. Within
resting cells, NF-jB is retained in the cytoplasm complexed

to an inhibitor protein from the IjB family [3]. Stimulation
with pro-inflammatory cytokines, such as tumour necrosis
factor a (TNFa) or interleukin 1b (IL-1b), activates the IjB
kinase (IKK) complex, which then phosphorylates the IjB
[3]. This leads to ubiquitination of the IjB, targeting it for
rapid degradation by the 26S proteosome [3]. Degradation
of IjB reveals a nuclear localization signal (NLS) allowing
NF-jB to interact with the nuclear import protein karyo-
pherin a2 [4]. This allows NF-jB to translocate into the
nucleus where it can bind to jB sequences in the promoters
of NF-jB-dependent genes to up-regulate transcription [3].
There are still many aspects of the NF-jB activation
pathway that have yet to be elucidated. For example, the
mechanisms that enhance the transactivation of NF-jB
once it is bound to its consensus sequence remain poorly
defined and numerous studies have suggested the involve-
ment of the p38 mitogen activated protein kinase (MAPK)
and the extracellular signal-regulated kinases (ERK) in the
activation or potentiation of NF-jB-dependent transcrip-
tion [5]. Thus, the selective p38 inhibitor, SB203580,
prevents NF-jB/p65-dependent, transactivation without
affecting NF-jB DNA binding [6,7]. Possible downstream
effects of the p38 MAPK include; phosphorylation of
histone H3 to enhance recruitment of NF-jB [8], phos-
phorylation of the TATA binding protein (TBP) to increase
transactivation of the DNA bound NF-jB [9], or phos-
phorylation of serine 276 of p65 or cAMP response element
binding protein (CREB) binding protein (CBP), events
Correspondence to R. Newton, Department of Biological Sciences,
University of Warwick, Coventry CV4 7AL, UK.

Fax: + 44 247 652 3701, Tel.: + 44 247 657 4187,
E-mail:
Abbreviations: CBP, CREB binding protein; ERK, extracellular
signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; ICAM-1, intercellular adhesion molecule 1; IKK, IjB
kinase; IL-1b, interleukin 1b; MAPK, MAPK/ERK kinase; MEK,
mitogen activated protein kinase; MOI, multiplicity of infection;
NF-jB, nuclear factor jB; TBP, TATA binding protein; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide;
TNFa, tumour necrosis factor a.
Note: A web site is available at: />molphys.asp
(Received 12 September 2003, revised 4 December 2003,
accepted 7 January 2004)
Eur. J. Biochem. 271, 785–791 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03982.x
which have both been shown to increase the ability of p65
to interact with CBP/p300 [10,11]. Similarly, inhibition of
the ERK pathway, with the MEK1 inhibitor, PD098059,
abrogated IKK activity and dominant negative constructs
of ERK prevented NF-jB DNA binding activity [12,13]. In
addition, the ERK pathway has been suggested to both
enhance DNA binding activity as well as to phosphorylate
downstream cofactors including CBP/p300 and positive
cofactor 1 to increase the transactivational potential of
NF-jB [10,14].
The pulmonary epithelium is the primary site of contact
with airborne allergens, irritants, pathogens and other
proinflammatory agents that trigger exacerbation in airway
diseases [15]. This epithelium is biosynthetically active and
acts as a source of multiple inflammatory cytokines,
chemokines, prostanoids and other mediators as well as

adhesion molecules such as intercellular adhesion molecule
1 (ICAM-1) [15]. In this context, ICAM-1 is particularly
important in inflammatory diseases such as asthma, where it
is not only responsible for the recruitment of inflammatory
cells from the blood to the airways, but also acts as a
receptor for many viruses that may exacerbate asthma [16].
Numerous studies suggest a role for NF-jBinICAM-1
expression, but an unequivocal demonstration in pulmon-
ary epithelial cells is currently lacking. As NF-jBis
considered to be a candidate for therapeutic intervention
in airway inflammation, we have investigated the role of
NF-jB in the induction of ICAM-1 expression by pro-
inflammatory cytokines. In addition, we have used NF-jB-
dependent transcriptional reporters and ICAM-1 expression
to address the role of p38 and ERK pathways in the
activation of NF-jB dependent transcription in pulmonary
epithelial cells.
Materials and methods
Cell culture, cytokines and drugs
A549 cells were grown to confluency in six-well plates as
described previously [17]. Cells were cultured overnight in
serum free media before changing to fresh serum free media
containing TNFa or IL-1b (both from R & D systems,
Abingdon, UK). SB203580, PD098059, and U0126 (all
from Calbiochem, Nottingham UK) were dissolved in
dimethylsulfoxide. Final concentrations of dimethylsulfox-
ide added to cells were < 0.1% and this had no effect on
any of the responses (data not shown).
NF-jB
luciferase

reporter cell lines
The NF-jB-dependent reporter cells, A549 6jBtkluc, were
grown as described previously [17]. These contain a stably
integrated plasmid with three tandem repeats of the
sequence 5¢-AGC TTA CAA GGG ACT TTC CGC
TGG GGA CTT TCC AGG GA-3¢, which has two copies
of the decameric NF-jB binding site (bold text) upstream of
a minimal thymidine kinase promoter driving a luciferase
gene. Confluent cells in 24-well plates were changed to
serum-free medium and treated as indicated before harvest-
ingat6hin1· reporter lysis buffer (Promega, South-
ampton, UK). Luminescence was measured using the
luciferase assay system (Promega).
Adenovirus infection
A549 cells were either infected with empty Ad5 expression
vector or a vector expressing an IjBa proteinwithan
N-terminal deletion (Ad5IjBaDN) [18]. This deletion
renders IjBa immune to signal-induced proteolysis and
generates a constitutive inhibitor of NF-jB. As previously
described, cells were infected at a multiplicity of infection
(MOI) of 10, a level which we have shown previously to
infect > 95% of cells, and then cultured for 24 h prior to
treatment with drugs or cytokines [19].
Cell viability assay
Cell viability was assessed by using the 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyl-tetrazolium bromide [thiazolyl blue
tetrazolium bromide (MTT)] assay (Sigma, Poole, UK)
according to the manufacturer’s instructions.
Semi-quantitative reverse transcription PCR
RNA isolation, reverse transcription, primers, PCR

conditions, and cycling parameters for glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) were as described
previously [17]. Primer pairs for ICAM-1 (Accession No:
BC015969) (5¢fi3¢) were (forward) CCG TGT ACT GGA
CTC CAG AA, (reverse) AGG TGT AGC TGC ATG
GCA TA. Cycling parameters for ICAM-1 were: 94 °C,
30 s; 58 °C, 30 s; 72 °C, 30 s. The number of amplification
cycles used was that necessary to achieve exponential
amplification where product formation was proportional
to starting cDNA and was established empirically [17].
Following amplification, PCR products (10 lL) were run
on 2.0% agarose gels stained with ethidium bromide. After
densitometric analysis using
TOTALLAB
software, version 1
(Nonlinear Dynamics), data were expressed as the ratio of
ICAM-1/GAPDH.
Electrophoretic mobility shift assay (EMSA)
Nuclear proteins were isolated 1 h after stimulation and the
consensus NF-jB(5¢-AGT TGA GGG GAC TTT CCC
AGG-3¢) probe (Promega) radioactively labelled as des-
cribed previously [20]. Specificity of binding was determined
by the prior addition of 100-fold excess unlabeled consensus
oligonucleotide. Reactions were separated on 7% native
acrylamide gels before vacuum drying and autoradiography.
Western blot analysis
Cells were harvested in 100 lLof10 m
M
Tris/HCl (pH 7.5),
0.15

M
NaCl, 1.5 m
M
MgCl
2
, 0.65% (v/v) NP-40, 0.5 m
M
phenylmethylsulfonyl fluoride, 0.5 m
M
dithiothreitol. Sam-
ples were run on 10% SDS polyacrylamide gels and
transferred to Hybond-ECL nitro-cellulose paper (Amer-
sham Pharmacia, Bucks, UK) using standard techniques.
Membranes were probed with mouse monoclonal anti-
human GAPDH Ig (#4699–9555) (Biogenesis Ltd, Poole,
UK) or mouse polyclonal anti-ICAM-1 (sc-8439) Ig (Santa
Cruz Biotechnology, CA, USA) at dilutions of 1 : 20 000
and 1 : 1000, respectively. Alternatively, membranes were
probed for phospho-hsp27 (#2401), or ERK 1/2 (#9100)
786 N. S. Holden et al. (Eur. J. Biochem. 271) Ó FEBS 2004
(both phospho and pan ERK 1/2) (New England BioLabs,
Hertfordshire, UK) at a dilution of 1 : 1000 and incubated
overnight at 4 °C. In all cases, proteins were visualized using
ECL (Amersham Pharmacia Biotech, Bucks, UK) accord-
ing to the manufacturer’s instructions.
Results
The establishment of
ICAM-1
as an NF-jB-dependent
gene

Western blot analysis revealed extremely low levels of
ICAM-1 protein in untreated cells, but following treatment
with either TNFa or IL-1b, ICAM-1 protein was rapidly
induced (Fig. 1). This was first apparent by 2 h poststim-
ulation and levels of ICAM-1 protein continued to rise over
the 18 h of the experiment. To examine the role of NF-jB
in the induction of ICAM-1, cells were infected with an
adenoviral vector that over-expresses IjBaDN, a dominant
inhibitor of NF-jB [18]. We have described previously the
effectiveness of this construct in repressing both NF-jB
DNA binding and transcriptional activity in A549 cells [19].
Both TNFa and IL-1b produced a robust increase in
reporter activity, measured 6 h poststimulation as des-
cribed previously [21]. In each case, this activity was reduced
to basal levels by the IjBaDN expressing virus (Fig. 2). The
null, or empty, viral vector showed no effect on induction
of reporter activity (Fig. 2). Likewise, there was no effect of
this virus on basal activation of the reporter (data not
shown) [19].
Having established that IjBDN over-expression is effect-
ive at preventing induction of NF-jB transcriptional
activity, this virus was used to test the role of NF-jBin
the induction of ICAM-1 expression. As in previous
experiments, unstimulated cells expressed very little
ICAM-1 protein and this was also true of ICAM-1 mRNA
(Fig. 3). Upon stimulation with either TNFa or IL-1b,
ICAM-1 mRNA and protein was dramatically increased.
Prior infection with the IjBaDN expressing adenovirus, but
not the null virus, totally prevented the expression of
ICAM-1 in response to stimulation by both TNFa,and

IL-1b. These data therefore establish that the induction of
ICAM-1 by both TNFa and IL-1b is highly NF-jB-
dependent in A549 cells.
Effect of SB203580, PD098059 and U0126 on NF-jB DNA
binding and NF-jB-dependent transcription
To examine the role of the p38 and the ERK MAPK
pathways, we examined the effects of the p38 MAPK
inhibitor, SB203580, the MEK1 inhibitor, PD098059 and
the MEK 1/2 inhibitor, U0126, on TNFa-andIL-1b-
induced NF-jB translocation and DNA binding. As
induction of NF-jB DNA binding was shown previously
to be maximal at 1 h poststimulation [21], the cells were
stimulated for 1 h with TNFa or IL-1b prior to analysis by
EMSA. Following each treatment, two NF-jB DNA
binding complexes were observed and these were both
removed by competition with the addition of 100-fold excess
of cold oligonucleotide (Fig. 4A). Densitometric analysis of
these complexes indicated a 10-fold increase in NF-jB
DNA binding when compared to unstimulated cells
(Fig. 4A, lower panels). In each case the prior addition of
SB203580, PD098059, or U0126 revealed no effect on the
induction of NF-jB DNA binding.
To investigate the potential role these MAPK cas-
cades on NF-jB-dependent transcription, 6jBtk cells
Fig. 1. Time course of ICAM-1 expression in TNFa and IL-1b stimu-
lated cells. Cells were either not stimulated (NS) or stimulated with
TNFa (10 ngÆmL
)1
)orIL-1b (1 ngÆmL
)1

) and harvested at 0, 2, 6 and
18 h post stimulation, prior to Western blot analysis of ICAM-1. A
representative blot is shown and optical densities (n ¼ 3) are plotted as
arbitrary units as means ± SEM.
Fig. 2. Validation of an adenovirus expressing a constitutively active
IjB. A549 6jBtk cells were either infected or not at a MOI of 10 with
either Ad5IjBDN or null virus as indicated. After changing to serum
free media, cells were either unstimulated or treated with TNFa
(10 ngÆmL
)1
)orIL-1b (1 ngÆmL
)1
) as indicated. Cells were harvested
after 6 h for luciferase activity determination. Data (n ¼ 4) are
expressed as percentage of stimulated cells and are plotted as means ±
SEM.
Ó FEBS 2004 The role of NF-jB and MAPK in ICAM-1 expression (Eur. J. Biochem. 271) 787
were stimulated with either TNFa or IL-1b in the presence
of absence of various concentrations of SB203580,
PD098059 or UO126. At concentrations from 0.01 and
1 l
M
these inhibitors showed little or no effect on luciferase
levels (Fig. 4B). However decreased luciferase activity was
observedwith10 l
M
SB203850 and this appeared to reach a
plateau by 100 l
M
. With PD098059 and UO126 a variable

effect was observed at 10 l
M
and by 100 l
M
amarked
repression was apparent (Fig. 4B). However, in each case,
these concentrations were 10 to 100-fold greater than the
reported K
i
values and so probably result from nonspecific
effects of these kinase inhibitors ([22] and refs therein). To
assess the effect of these compounds on cell viability, MTT
assays were performed on cells preincubated with a range
of concentrations of the inhibitors. With the exception of
U0126, which showed a 30% loss of cell viability at 100 l
M
,
MTT analysis revealed little or no effect of PD098059,
SB203580 or U0126 on cell viability at any of the
concentrations tested (0.01–100 l
M
; data not shown).
Effect of MEK and p38 MAPK inhibitors on ICAM-1
expression in A549 cells
As previous reports implicating MAPK pathways in NF-
jB-dependent transcription have suggested the involvement
of mechanisms that impact on the promoter architecture,
and this may not always be faithfully reproduced in a
reporter system, we examined the effect of SB203580 and
PD098059 on ICAM-1 expression as an endogenous

indicator of NF-jB transcriptional activity. As in previous
experiments, stimulation of A549 cells with TNFa or IL-1b
caused a considerable up regulation of ICAM-1 expression
at 6 h poststimulation compared to unstimulated cells
(Fig. 5). Pre-incubation of cells with a range of concentra-
tions of PD098059 or SB203580 showed little or no effect on
ICAM-1 expression in either TNFa or IL-1b stimulated
cells suggesting that neither the p38 MAPK nor the MEK1-
ERK pathways are involved in the expression of ICAM-1
in this system (Fig. 5).
Validation of the SB203580, PD098059 and U0126
in A549 cells
To validate the inhibitory action of SB203580, the phos-
phorylation of heat shock protein 27 (hsp27), a downstream
target of p38 MAPK was analysed [22]. Both TNFa and
IL-1b markedly induced hsp27 phosphorylation and this
was prevented by preincubation with SB203580 (Fig. 6).
Analysis of GAPDH expression confirmed equality of
loading. Following densitometric analysis, EC
50
values of
0.21 and 0.23 l
M
were calculated for inhibition of TNFa-
and IL-1b-stimulated hsp27 phosphorylation, respectively.
These values are consistent with the published IC
50
for
SB203580 (0.6 l
M

) indicating the inhibition of p38 at
pharmacologically relevant concentrations [23].
Similarly, the MEK inhibitors, PD098059 and U0126,
were validated by analysis of phosphorylation of ERKs 1
and 2, which lie downstream of MEK1 [24]. Both TNFa
and IL-1b upregulated phosphorylation of p42/44 ERKs
and preincubation with PD098059 abrogated this response.
Following densitometry, EC
50
values of 0.26 and 0.58 l
M
were calculated for TNFa-andIL-1b-stimulated cells,
respectively. These are consistent with published values for
MEK 1 inhibition (5 l
M
) [25]. As a loading and expression
control, the expression of total p42/44 was also examined.
Likewise, U0126 completely prevented the phosphorylation
of p42/44 with EC
50
values of 0.23 and 0.1 l
M
for TNFa
and IL-1b stimulated cells, respectively. This is again
consistent with the published IC
50
value for MEK1/2
inhibition (0.065 l
M
) suggesting that both PD098059 and

U0126 are functionally active in A549 cells [26].
Discussion
The adhesion molecule ICAM-1 has been shown by
numerous studies to be an important factor in many allergic
diseases such as asthma, where it not only plays a critical
role in airway inflammation and the development of hyper-
responsiveness [27], but also acts as a receptor for infection
by rhinoviral and respiratory syncytial viruses, which both
increase exacerbations in asthma [28,29]. The regulation of
ICAM-1 production is therefore a potential target for the
development of new therapeutics in asthma. Various studies
have implicated NF-jB in the transcriptional regulation of
ICAM-1 in a variety of cells [30–33], however, these studies
often rely on reporter systems, which may not faithfully
mimic the architecture of the endogenous promoter. In the
current study, we have over-expressed a dominant form of
Fig. 3. Effect of an adenovirus expressing a constitutively active IjBon
ICAM-1 expression. Cells were either infected or not at a MOI of 10
with either Ad5IjBDN or null virus as indicated. After changing to
serum free media, cells were either not stimulated (NS) or treated with
TNFa (10 ngÆmL
)1
)orIL-1b (1 ngÆmL
)1
) as indicated. Cells were
either harvested after 6 h for semiquantitative RT-PCR analysis or
after 24 h for Western blot analysis of both ICAM-1 and GAPDH
expression. Representative blots are shown, and data (n ¼ 6) were
normalized to GAPDH expression and are plotted as means ± SEM.
788 N. S. Holden et al. (Eur. J. Biochem. 271) Ó FEBS 2004

IjBa to unequivocally demonstrate that the induction of
ICAM-1 expression is NF-jB-dependent in TNFa-and
IL-1b-stimulated pulmonary epithelial A549 cells.
As noted above, both the p38 and ERK MAPK
cascades are variously implicated in the activation of NF-
jB. However, as many of these studies rely on transfection
analysis of reporter plasmids, which are prone to artefacts,
or on the effects of single high dose inhibitors, which may
lead to pharmacologically unrelated events, to define
functional roles for MAPK, they should be treated with
a degree off caution ([22] and references therein). Thus, in
contrast to the observation that dominant negative ERKs
completely prevented NF-jB DNA binding [12,13], the
present analysis showed no effect of either the MEK 1
inhibitor PD098059 or the MEK 1/2 inhibitor U0126 on
NF-jB translocation and DNA binding. As a similar result
was observed with the p38 MAPK inhibitor, SB203580,
these data indicate that neither the ERK nor p38 MAPK
pathways play a significant role in the activation of NF-jB
DNA binding in A549 cells. However, numerous studies
Fig. 4. Effect of PD098059, SB203580 and
U0126 on NF-jB DNA binding, and NF-jB-
dependent transcription. (A) Cells were pre-
incubated with PD098059, SB203580 or
U0126 (all at 10 l
M
) and then stimulated
with either TNFa (10 ngÆmL
)1
)orIL-1b

(1 ngÆmL
)1
).After1h,nuclearextractswere
prepared and analysed by EMSA. Represen-
tative blots are shown (n ¼ 4). XS indicates
the presence of a 100-fold excess of cold
NF-jB probe. Specific complexes, defined by
competition (XS), are indicated, and data,
expressed as a percentage of stimulated cells
are plotted as means ± SEM. (B) 6jBtk A549
cells were preincubated with various concen-
trations of PD098059, SB203580 or U0126
(0.01–100 l
M
) before stimulation with either
TNFa (10 ngÆmL
)1
)orIL-1b (1 ngÆmL
)1
).
Cells were harvested after 6 h for luciferase
activity determination. Data (n ¼ 5) was
expressed as percentage of stimulated cells and
are plotted as means ± SEM.
Fig. 5. Effect of p38 MAPK and MEK inhi-
bitors on ICAM-1 expression. Cells were pre-
incubated with various concentrations of
PD098059, SB203580 or U0126 (0.01–30 l
M
)

before stimulation with either TNFa
(10 ngÆmL
)1
)orIL-1b (1 ngÆmL
)1
). Cells were
harvested after 6 h for Western blot analysis
of ICAM-1 expression. Representative blots
are shown and data (n ¼ 2), expressed as
percentage of stimulated cells, are plotted as
means ± SEM.
Ó FEBS 2004 The role of NF-jB and MAPK in ICAM-1 expression (Eur. J. Biochem. 271) 789
have implicated roles for p38 and MEK 1/2 cascades in the
transactivation of NF-jB downstream of DNA binding
[5–7], through modulation of various components of the
basal transcriptional machinery such as TBP [9], or even
NF-jB/p65 itself [10,11]. In addition, p38-dependent
phosphorylation may lead to modification of histone
proteins causing changes in the chromatin structure of
NF-jB-dependent genes and allowing increased access of
the basal transcription machinery to the DNA [8]. More
recently, the p38 downstream kinase, mitogen- and stress-
activated protein kinase-1 (MSK-1) was shown to phos-
phorylate p65 at Ser276 and this was shown to be essential
for association with CBP/p300 and thus up-regulation of
NF-jB transactivation [10]. However, in the present study,
inhibitors of the p38 and ERK MAPK pathways were all
shown to be functionally active at the correct pharmaco-
logical concentrations, yet had no effect on the activation
of NF-jB-dependent transcription as determined by luci-

ferase reporter assay. However, as transcriptional reporters
may not mimic the true physiological architecture exhib-
ited by an endogenous promoter within cells, ICAM-1 was
also used as an endogenous NF-jB-dependent reporter.
However, this analysis confirmed the reporter data indica-
ting that neither the p38 nor the ERK MAPK pathways
play a major role in NF-jB-dependent transcription in
these cells. Finally, it should be noted that the use of the
p38 MAPK inhibitor, SB203580, only implicates the a and
b isofroms of p38, as SB203580 has little or no effect on
p38 c and d and it is possible that this could explain
discrepancies between over-expression and inhibitor based
studies [34].
In conclusion, the data presented here firmly demon-
strates that the adhesion molecule ICAM-1 is a highly
NF-jB-dependent gene in A549 pulmonary epithelial cells
and is therefore a useful endogenous reporter of NF-jB-
dependent transcription. Furthermore, despite the extensive
evidence documenting roles for the p38 and ERK MAPK
pathways in NF-jB-dependent transcription, we found that
highly selective inhibitors of these pathways had no effect on
activation of NF-jB DNA binding, NF-jB-dependent
transcription, or on the endogenous NF-jB-dependent gene
ICAM-1. Taken together, these data highlight the potential
utility of inhibiting the NF-jB signalling pathway in
pulmonary inflammatory diseases and suggest that inhibi-
tors of the p38 and ERK MAPK pathways may be of lesser
effect in this cell type.
Acknowledgements
N. S. H. and M. C. C. are MRC collaborative students supported by

Novartis Pharmaceuticals and Aventis Pharmaceuticals, respectively.
L. M. C. was funded by a grant from Novartis Pharmaceuticals.
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(10 ngÆmL
)1
)orIL-1b (1 ngÆmL
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). After 30 min, cells were harvested
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