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,

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.

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

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),

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

)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

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

) 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.

)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

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.
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