1 Pulmonary Research Laboratory, University of British Columbia, Vancouver, British Columbia, Canada V6Z 1Y6; and 2 Third Department of Internal Medicine, University of Tokyo, Tokyo 113, Japan
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ABSTRACT |
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Adenovirus E1A DNA and proteins are detected in
lung epithelial cells of patients with chronic obstructive pulmonary
disease. In investigating E1A regulation of inflammatory mediator
expression in human lung epithelial cells, we found increased
intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 expression
after lipopolysaccharide (LPS) stimulation of A549 cells stably
transfected with adenovirus 5 E1A. We now show that E1A-dependent
induction of interleukin-8 expression is specific to LPS, superinduced
by cycloheximide, and not observed after tumor necrosis factor or phorbol 12-myristate 13-acetate stimulation. Electrophoretic mobility shift assays revealed that tumor necrosis factor or phorbol
12-myristate 13-acetate induced nuclear factor-B binding complexes
of Rel A and p50 in E1A and control transfectants, whereas LPS was
effective only in E1A transfectants. Similarly, LPS-induced nuclear
translocation of nuclear factor-
B was observed only in E1A
transfectants. CCAAT-enhancer binding protein binding was undetected
and activator protein-1 binding was unaffected by LPS in either cell
type, whereas basal mRNA levels of
c-jun were unchanged by E1A. We
conclude that E1A enhances the expression of these inflammatory
mediator genes by modulating events specific to LPS-triggered nuclear
factor-
B induction in these cells.
A549 cells; lipopolysaccharide response; regulation of inflammatory
mediator expression; nuclear factor-B
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INTRODUCTION |
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THE ADENOVIRUS E1A
gene is the first viral gene expressed during infection by
this virus and encodes nuclear phosphoproteins that regulate the
expression of a selected set of viral and cellular genes (31). Inasmuch
as E1A itself does not bind to DNA to effect this regulation, it must
do so by interacting with endogenous host transcription factors. E1A
proteins interact with specific members of the family of the activator
protein-1 (AP-1) and of the AP-1-related cellular activating
transcription factors (35), possibly by binding directly to these
transcription factors (15). E1A can also interact with the
retinoblastoma gene product and with p300, which in turn regulate the
activity of the transcription factors E2F and cAMP-responsive element
binding protein, respectively (1, 16). More recently, indirect
regulation of the activity of the transcription factor nuclear
factor-B (NF-
B) by E1A has been reported (4, 8, 24, 32). In this
manner, the interaction of adenovirus E1A with these host proteins
plays a pivotal role in altering the functional state of the host cell.
Studies from our laboratory showed that greater copy numbers of the E1A
DNA from group C adenovirus are found in the lungs of smokers with
chronic obstructive pulmonary disease than in smokers without airway
obstruction (17) and that E1A proteins are detected in lung epithelial
cells of these patients (3). These observations led to a working
hypothesis that chronic expression of E1A might amplify the cigarette
smoke-induced inflammatory process in the lungs of smokers with chronic
obstructive pulmonary disease. To examine this possibility in vitro, we
introduced the adenovirus 5 E1A gene
into A549 human lung epithelial cells and isolated stable transfectants
producing E1A proteins (11). In previous reports, we demonstrated that
the presence of E1A induces intercellular adhesion molecule-1 (ICAM-1)
and interleukin (IL)-8 expression in these lung epithelial cells after
their stimulation with lipopolysaccharide (LPS) (11, 12). This effect
of E1A on the induction by LPS of other inflammatory mediators,
monocyte chemotactic and activating factor, transforming growth
factor-, IL-1
, IL-6, granulocyte-macrophage colony-stimulating
factor, and granulocyte colony-stimulating factor, could not be
demonstrated (12). These findings suggest that the mechanisms
underlying control of the ICAM-1 and
IL-8 genes by E1A in lung epithelial cells are different from those controlling the expression of the other cytokines.
The accumulation of inflammatory cells at sites of chronic inflammation
is influenced by the expression of adhesion receptors and the action of
a variety of cytokines. These mediators are induced by proinflammatory
stimuli such as IL-1, tumor necrosis factor (TNF), and endotoxin, and
their production is ultimately controlled at the level of gene
transcription by the binding of transcription factors to specific sites
in the regulatory region of responsive genes. Among the various
transcription factors that contribute to the induction of these genes,
NF-B stands out as a central coordinating regulator (33). The active
DNA-binding forms of NF-
B transcription factors are dimeric
complexes of subunits, the most abundant of which are Rel A
(p65) and p50. Rel A, p50, and other members of the NF-
B family bear
unique and overlapping functional activities, and combinations of these members form distinctive complexes that are characterized by their preference of certain
B sites and by their transactivation
potentials (33). In fact, the Rel A homodimer has been reported to bind preferentially to the NF-
B binding sites of human
ICAM-1 and IL-8 genes (14, 34). Under most
circumstances, NF-
B complexes lie dormant in the cytoplasm of
unstimulated cells, bound to an inhibitory protein, I
B (33). In
response to extracellular signals such as TNF and phorbol 12-myristate
13-acetate (PMA), I
B is degraded, allowing NF-
B to translocate to
the nucleus, where it binds to
cis-acting
B sites in the
regulatory region of NF-
B-responsive genes, including
ICAM-1 and
IL-8. This process, which does not require de novo protein synthesis, allows a rapid and efficient transcriptional activation of these target genes.
The regulatory regions of human IL-8 and ICAM-1 genes also have putative AP-1 (19, 36) and CCAAT- enhancer binding protein (C/EBP) binding sites (7, 19). AP-1 is another dimeric transcription factor, and its activity is rapidly induced in response to a vast array of extracellular stimuli, including PMA. This response is regulated partly by the synthesis of Jun and Fos proteins, which are major components of AP-1 complexes, and partly by the posttranslational modification of these preexisting and newly synthesized proteins (10).
The purpose of the present study was twofold. First, to investigate the regulation of IL-8 expression in A549 cells in response to LPS stimulation in more detail, we determined whether the E1A effect on this gene was restricted solely to activation by LPS and whether it required de novo protein synthesis. Second, to link this regulation by E1A to transcriptional activation, we identified transcription factors that were activated on LPS stimulation of E1A-transfected A549 cells.
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METHODS AND MATERIALS |
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Reagents.
LPS (Escherichia coli 0111:B4, Sigma,
St. Louis, MO) was dissolved in sterile distilled water at 10 mg/ml.
Recombinant human TNF (Calbiochem, La Jolla, CA) was reconstituted in
1% BSA in PBS (Oxoid, Basingstoke, UK) at 5 × 105 U/ml. PMA (Sigma) was prepared
at 10 mM in DMSO. These reagents were stored at 70°C and
diluted to the appropriate concentrations before use. Cycloheximide
(CHX; Sigma) was dissolved in the cell culture medium at 10 mg/ml and
diluted before use. Antioxidants, 0.5 M
N-acetyl-L-cysteine
(NAC; Sigma) dissolved in MEM (GIBCO BRL, Gaithersburg, MD) and
adjusted to pH 7.4 with NaOH before filter sterilization and
filter-sterilized 1 M pyrrolidine dithiocarbamate (PDTC; Sigma), were
prepared immediately before use.
Cell culture.
A549 cells (American Type Culture Collection, Manassas, VA), a human
lung epithelial cell line originally derived from a patient with
bronchioloalveolar carcinoma, were transfected with the adenovirus 5 E1A gene (11). E1A transfectants
analyzed in this study, E4, E11, and E20, are three independent clones
of A549 cells stably transfected with a plasmid carrying adenovirus 5 E1A gene driven by its own promoter,
and the control transfectants tested here, C4 and C8, are two
independent clones of A549 cells transfected with the control plasmid
lacking the E1A gene (11). Northern and Western blotting and immunocytochemistry showed that all E1A transfectants produced relatively high levels of E1A mRNA and E1A
proteins (11). HeLa cells (American Type Culture Collection) were used
as a control cell line to identify the binding activity of the
transcription factors NF-B and AP-1 because the components of their
binding complexes induced in HeLa cells are well characterized (23,
35). A549 cells, HeLa cells, and all the transfectants were grown in
MEM supplemented with 10% fetal bovine serum (HyClone, Logan, UT). In
all the experiments presented here, 10% fetal bovine serum was
included in the medium. The transfectants were maintained under
constant selection with 280 µg/ml of active G-418 (GIBCO BRL) and
were not used beyond passage 13 to
avoid the generation of variants.
cDNA probes used for Northern blot analysis. A 0.5-kb EcoR I fragment from the plasmid carrying the human IL-8 cDNA (a generous gift from Dr. K. Matsushima) has been previously described (12). The murine c-jun probe, which cross-hybridizes with human c-jun mRNA (27), was obtained from American Type Culture Collection. As described previously (11), the rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, a 1.2-kb Pst I fragment that cross-hybridizes with human GAPDH mRNA, was used as an internal control for RNA loading.
Northern blot analysis.
Cells, both E1A transfectants and control cells, which had been grown
in 75-cm2 cell culture flasks,
were exposed to medium alone or to medium with 10 µg/ml LPS, 100 U/ml
TNF, or 100 ng/ml PMA for 4 h. The concentrations of LPS, TNF, and PMA
were based on the dose effective in inducing ICAM-1 and IL-8 expression
in the E1A-expressing A549 cells in previous studies (11, 12); at these
concentrations, cytotoxic effects, as measured by the
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma) assay (18), were not observed. In experiments to
determine the effect of a protein synthesis inhibitor, both cell types
were incubated for 4 h with or without 10 µg/ml CHX in the absence or
presence of 10 µg/ml LPS. Total RNA was isolated with the single-step
acid guanidinium thiocyanate method. This RNA, at 15 µg/lane, was
separated on a formaldehyde-1% agarose gel, transferred to a nylon
membrane (Hybond N, Amersham, Oakville, ON, Canada), and fixed to the
membrane by exposure to ultraviolet radiation. The amount, quality, and
size of the RNA were assessed before their transfer to the membrane by
monitoring 18S and 28S rRNA bands stained with ethidium bromide.
Membranes were prehybridized and then hybridized for 20 h with each of
the DNA probes, which were labeled with
[-32P]dCTP
(Amersham) by using the random primer-labeling technique. The membranes
were washed three times in 0.3 M NaCl-0.03 M sodium citrate, pH 7.0, with 0.1% SDS for 5 min at room temperature and twice in 0.03 M NaCl-3
mM sodium citrate, pH 7.0, with 0.1% SDS for 15 min at 65°C before autoradiography.
Nuclear extract preparation.
E1A transfectants, control cells, and HeLa cells that had been grown in
75-cm2 cell culture flasks were
incubated for 2 h in medium alone or in medium with 10 µg/ml LPS, 100 U/ml TNF, 100 ng/ml PMA, or 10 µg/ml CHX. In a time-course study, E1A
transfectants were incubated with 10 µg/ml LPS for 0, 0.5, 1, 2, and
4 h. After incubation, nuclear extracts were prepared on the basis of
the methods described previously (2). The cells were rinsed and scraped
in 1 ml of ice-cold solution of 0.05 M Tris · HCl in
0.15 M NaCl, pH 7.6, transferred to microcentrifuge tubes, and spun for
18 s at 12,000 g. All centrifugation
was done in the cold room at 6°C. Cell pellets were resuspended in
400 µl of 10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM
MgCl2, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, 0.1 mM EDTA, and 50 µg/ml leupeptin
(Sigma) and incubated on ice for 15 min to allow swelling. Then 25 µl
of 10% Nonidet P-40 were added, and the cells were vortexed vigorously
for 15 s before centrifugation for 30 s at 12,000 g. The pelleted nuclei were resuspended in 50 µl of 50 mM HEPES, pH 7.8, 50 mM KCl, 300 mM NaCl,
0.1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride,
10% glycerol, and 50 µg/ml leupeptin. After gentle mixing for 20 min
at 4°C, the tubes were spun for 5 min at 12,000 g, and the supernatant containing
nuclear proteins was immediately stored at 70°C until the
time of assay. Protein concentrations were determined using a Bradford
protein assay kit (Bio-Rad Laboratories, Hercules, CA ).
Electrophoretic mobility shift assay..
Double-stranded oligonucleotides used as DNA probes in the
electrophoretic mobility shift assay (EMSA) were 5'-CGC TTG
G CCG GAA-3' (Promega,
Madison, WI) for AP-1, 5'-AGT TGA
C AGG C-3' (Promega)
for the NF-
B binding sequence of the immunoglobulin
-chain gene,
and 5'-TGC AGA
T CTG
CA-3' (Santa Cruz Biotechnology, Santa Cruz, CA) for C/EBP. The
underlined sequences are binding motifs for AP-1, NF-
B, and C/EBP,
respectively. For the human IL-8
gene-specific NF-
B binding site, two complementary single-stranded
oligonucleotides with the sequence
5'-
TCT G-3'
from nucleotides
83 to
68, relative to the transcription start site of the IL-8 gene (19)
(GenBank accession number M28130), and its complement were prepared by
the University of Calgary Core DNA Synthesis Service. Each
double-stranded oligonucleotide was end labeled with
[
-32P]ATP (6,000 Ci/mmol) with T4 polynucleotide kinase (GIBCO BRL), and the labeled DNA
was purified on Bio-Spin 6 chromatography columns (Bio-Rad). The two
single-stranded oligonucleotides containing the
IL-8 gene-specific NF-
B binding
site were labeled as described above in a single reaction, heated at
65°C for 10 min before the complementary strands were allowed to
anneal at room temperature, and then purified on the Bio-Spin 6 column.
An aliquot of each nuclear extract (2-4.5 µl) corresponding to
10 µg of protein was incubated on ice for 15 min with four volumes of
binding buffer (10 mM Tris · HCl, pH 7.5, 50 mM NaCl,
1 mM EDTA, 1 mM dithiothreitol, and 4% glycerol) and 1.5 µg of
poly(dI-dC) (Boehringer Mannheim). For competition assays, excess
unlabeled double-stranded oligonucleotide (3.5 pmol) of sequence
identical with or unrelated to that of the labeled probe was added to
the nuclear extract. For supershift assays of NF-
B, antiserum
(1-2 µg) against the Rel A or p50 subunit of NF-
B (Santa Cruz
Biotechnology) or control rabbit antiserum (2 µg) against unrelated
protein (rabbit anti-mouse immunoglobulin, Dako, Glostrup, Denmark) was
added to the nuclear extract and incubated for 30 min on ice. Finally,
50,000 counts/min of the labeled probe, equivalent to 0.06-0.20
pmol of the oligonucleotide, were added, with further incubation for 20 min at room temperature to allow formation of protein-DNA complexes.
The complexes were separated at 12 V/cm on a nondenaturing 6%
polyacrylamide gel with an acrylamide-to-bis-acrylamide ratio of 37.5:1
in 22 mM Tris-borate and 0.5 mM EDTA. Gels were dried under vacuum and autoradiographed.
Immunofluorescent staining.
E1A transfectants, control transfectants, or A549 cells were grown on
coverslips and incubated for 2.5 h in medium alone or in medium with 10 µg/ml LPS or 100 U/ml TNF. Indirect immunofluorescent staining with
anti-NF-B antibody was performed as described previously (29).
Briefly, cells were fixed in 4.5% paraformaldehyde for 10 min,
permeabilized with 0.5% Triton X-100 in PBS for 10 min, and
preincubated with 5% normal goat serum for 20 min at room temperature.
The cells were incubated with rabbit antiserum against the Rel A
subunit of NF-
B or control serum for 45 min at 37°C, then washed
three times with PBS. The cells were then incubated with fluorescein
isothiocyanate-conjugated goat anti-rabbit whole IgG antibody (Cappel
Laboratories, Durham, NC) for 20 min at 37°C, then washed with PBS.
Two hundred cells were counted, and the percentage of cells with
nuclear staining, indicating nuclear translocation of NF-
B, was
calculated. The data from the E1A-positive and E1A-negative cells were
pooled for each of the culture conditions and compared by a
t-test, with the assumption of unequal variances.
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RESULTS |
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E1A-dependent induction of IL-8 gene expression is specific to LPS
and not observed after TNF or PMA stimulation.
When expression levels of the IL-8
gene in cells grown in medium alone were determined by Northern blot
analysis, we found that the E1A transfectant E20 and the control
transfectant C8 expressed trace amounts of this mRNA (Fig.
1A).
After treatment with LPS, E1A transfectants, but not control cells,
expressed increased levels of IL-8 mRNA, whereas TNF and PMA induced
IL-8 expression in both. At the same time, GAPDH mRNA levels were
similar in both cell types and remained unchanged after treatment with the three stimuli. Similar results were obtained after testing another
E1A transfectant, E4, and control parental A549 cells (data not shown).
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LPS-mediated IL-8 gene expression in E1A-producing cells is superinduced by CHX. To determine whether the induction of IL-8 mRNA by LPS requires de novo protein synthesis, the combined effect of LPS and CHX was examined in the E1A transfectant E11 and the control transfectant C8 (Fig. 1B). Without LPS or CHX, little or no IL-8 mRNA was detected in either cell type, whereas LPS alone induced this mRNA in the E1A transfectant but not in the controls. With CHX alone, IL-8 mRNA was induced in both cell types, with a greater effect in E1A transfectants. In the latter case, CHX was more effective than LPS. The combination of CHX and LPS amplified the expression of the IL-8 mRNA in both cell types to levels greater than that observed with either agent alone. Again, levels of IL-8 mRNA were higher in E1A transfectants than in controls. Similar results were obtained after testing of another pair consisting of E1A transfectant, E4, and control parental A549 cells (data not shown).
LPS treatment induced NF-B binding activity in the
nuclei of E1A transfectants but not in control cells.
When the EMSA using the oligonucleotide specifying the NF-
B binding
sequence in the enhancer of the immunoglobulin
-chain gene was
applied to nuclear extracts from cells grown in medium alone, specific
DNA-protein complexes were detected at low levels in the E1A
transfectant E4 and in A549 cell controls (Fig.
2A). To
facilitate their identification, the complexes are numbered I, II, and
III in the order of their migration
through the gel from the slowest to the fastest. In E1A transfectants,
LPS treatment increased the levels of complexes
I and II, although
predominantly of complex II, whereas
this treatment did not change the NF-
B binding activity in the
controls. Similar results were obtained after testing two other pairs
of E1A and control transfectants: 1)
E11 and C8 and 2) E20 and C4 (data
not shown). The increase in the level of complex
II in E1A transfectants was detected 30 min after LPS
treatment and reached the maximum level 2-4 h after stimulation
(data not shown). In E1A and control cells, TNF and PMA stimulation
resulted in a markedly increased level of complex II and, to a lesser degree, of complex
III (Fig. 2A).
Complex I, the slowest migrating of
the three, was detected only at low levels and only when
complexes II and
III were induced. In nuclear extracts
from an E1A transfectant, E4, treated with LPS, all three complexes
were competed out by a 30- to 60-fold excess of unlabeled NF-
B
oligonucleotide, whereas an unrelated oligonucleotide specific for AP-1
binding did not affect the formation of these complexes (Fig.
2B). Bands designated NS were not
affected by either competitor. Similar NF-
B-specific complexes were
induced in control and E1A transfectants by CHX, although more weakly
than that observed above, and also in HeLa cells after TNF and PMA
stimulation (data not shown).
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LPS treatment induced IL-8 gene-specific NF-B
binding activity consisting of Rel A and p50 in the nuclei of E1A
transfectants but not in control cells.
Similar to the above experiments, which used the prototypic NF-
B
consensus sequence, EMSA was performed using the labeled oligonucleotide, which included the
IL-8 gene-specific NF-
B sequence (Fig. 4). As described above, basal levels
of binding activity were undetectable or low in E1A transfectant E4 and
control A549 cells. LPS treatment resulted in increased levels of
complexes I and
II in E1A transfectants, with
complex I predominating. The same
treatment did not affect the NF-
B binding activity in control cells.
Similar results were obtained after testing two other pairs of E1A and
control transfectants: 1) E11 and C8
and 2) E20 and C4 (data not shown).
TNF and PMA stimulation increased the levels of
complexes I and
II in E1A and control cells (Fig. 4).
In comparison, induction of both complexes in these cells by CHX was
not as strong (data not presented). These two complexes were competed
out by excess unlabeled IL-8-specific NF-
B oligonucleotide and were unaffected by an unrelated oligonucleotide specific for AP-1 binding, whereas the band labeled NS was not affected by either competitor (data
not shown).
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LPS treatment induced nuclear translocation of NF-B
in E1A transfectants but not in control cells.
The subcellular distribution of NF-
B was also examined by an
indirect immunofluorescence assay with use of an antiserum against the
Rel A subunit of NF-
B (Table 1, Fig.
6). In the resting state, Rel A was mainly
confined to the cytoplasm in E1A transfectants and control cells. After
LPS stimulation, Rel A staining shifted to the nucleus of E1A
transfectants, whereas it remained in the cytoplasm after the same
treatment of control cells. In contrast, TNF induced increased nuclear
staining for Rel A in both cell types (Table 1). The overall
immunofluorescence intensity of Rel A staining was similar in
E1A-positive and E1A-negative transfectants and, although more
difficult to assess accurately because of differences in cell
morphology, also in the parent A549 cells. Nuclear staining with
control serum was always <10% (data not shown).
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AP-1 or C/EBP binding activities are not upregulated after LPS
stimulation in either cell type.
When basal levels of AP-1 binding activity in the cells were tested by
EMSA, the E1A transfectant E4 and control A549 cells demonstrated AP-1
binding activity (Fig.
7A). PMA
treatment appreciably increased this activity. In contrast, after LPS
and TNF treatment, no obvious increase was observed. Similar results
were obtained after testing of two other pairs of E1A and control
transfectants: 1) E11 and C8 and
2) E20 and C4 (data not shown). This
binding activity was competed out by excess unlabeled AP-1
oligonucleotide, whereas an unrelated NF-B-specific oligonucleotide
did not compete for the binding (Fig.
7B). The same specific complex was
detected in the nuclear extracts of HeLa cells and was, again, further induced by PMA stimulation (data not shown). EMSA using a labeled C/EBP
oligonucleotide did not show any TNF- or LPS-inducible bands in A549
cells or the E1A transfectant E4 (data not shown).
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Expression of c-jun was not changed in either cell type.
Because c-jun, a major component of
AP-1, was reported to be constitutively elevated at the level of
transcription in several cell lines in the presence of E1A (35), basal
levels of the mRNA of this transcription factor and levels in the
presence of CHX were measured by Northern blot analysis (Fig.
8). Although c-jun expression was superinduced by
CHX treatment in both cell types, no appreciable difference between the
E1A transfectant E11 and the control C8 cells was observed before or
after CHX treatment. At the same time, CHX did not affect the level of
the GAPDH mRNA. Similar results were obtained after another E1A
transfectant, E4, and control parental A549 cells were tested (data not
shown).
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DISCUSSION |
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We previously reported that adenovirus 5 E1A upregulates the expression
of ICAM-1 (11) and
IL-8 (12) genes after LPS stimulation of A549 pulmonary epithelial cells. In the present study, we
characterized this novel effect of the virus E1A on these inflammatory
genes and found that it potentiates the LPS-mediated NF-B activation that is associated with the upregulation of these two responsive genes
in target epithelial cells.
In A549 cells, E1A-dependent upregulation of
IL-8 gene expression was observed only
after LPS stimulation and not after stimulation with other inducers,
e.g., TNF and PMA. This implies that a specific process involving LPS
recognition or one of the many possible downstream processes emanating
from the LPS-mediated activation is affected by E1A. Because the
LPS-mediated increase in IL-8 mRNA was not reduced but was further
elevated by a potent protein synthesis inhibitor, CHX, de novo protein
synthesis is not required for gene induction through this pathway.
Superinduction by CHX is characteristic of short-lived mRNAs, such as
the immediate-early genes, e.g., c-jun
and c-fos (5), and is explained by the
inhibition of the translation of proteins, i.e., those responsible for
mRNA degradation (28) or for repression of transcriptional activation (22). Support for the first of these possibilities, in the case of
IL-8, comes from the observation that CHX markedly increased the
stability of this mRNA in pulmonary epithelial cells (21). Moreover,
our result that costimulation with LPS and CHX markedly enhanced IL-8
mRNA induction by CHX alone suggests that LPS and CHX act
synergistically by affecting a common control element in the IL-8
induction pathway, e.g., the transcription factor NF-B, as shown by
the results of our gel shift assays, as well as by affecting other
parts of the process of IL-8 mRNA expression discussed above.
E1A proteins localize in the nuclei of target cells where they interact
with a variety of cellular transcription factors that regulate the
expression of certain cellular genes (1, 4, 8, 15, 16, 23, 24, 31, 32,
35). In this manner, E1A could affect transcription factors, which, in
response to LPS stimulation, bind to the promoter-enhancer regions of
inflammatory genes. To investigate this possibility, we analyzed the
binding activity of transcription factors responsible for the
regulation of IL-8 and
ICAM-1 genes in pulmonary epithelial
cells expressing adenovirus E1A. NF-B, a transcription factor
induced by a variety of signals, including bacterial LPS (20, 33), has
been implicated in the increased expression of numerous genes involved
in inflammatory and immune responses. NF-
B activation appears to be
essential for the transcription of human
ICAM-1 and
IL-8 genes (7, 19). Interestingly,
recent studies have revealed that the
IL-8 and ICAM-1 genes are unique in terms of
the nucleotide sequence of their NF-
B binding site and of the
binding affinity of the different NF-
B subunits to these sites. The
ICAM-1 NF-
B site 5'-TGGAAATTCC-3' and the IL-8 NF-
B
site 5'-TGGAATTTCC-3' differ from the NF-
B consensus
sequence 5'-GGGRNNYYCC-3' at the 5'-most nucleotide where the conserved guanine residue is replaced by a thymine residue (14, 34). This substitution appears to weaken the binding affinity of
the p50 subunit to these variant sites (13), and instead Rel
A-containing complexes are preferentially bound to them (14, 34).
Whether these distinctive features of the
IL-8 and
ICAM-1 genes could be related to our
observation that, among the inflammatory mediators studied, only these
two were upregulated by LPS and only in cells expressing adenovirus E1A
(11, 12) was of interest to us.
Our results demonstrate that NF-B complexes are induced on
stimulation of pulmonary epithelial cells with LPS if adenovirus E1A is
present. The two different NF-
B sites tested here are bound by these
complexes in a selective fashion. For the prototypic immunoglobulin
NF-
B sequence, EMSA and supershift assays revealed that
complex II mainly consists of a
p50/Rel A heterodimer and complex III
is another p50-containing complex, probably a p50 homodimer, although
minor contributions from other members of the NF-
B family, such as
p52, c-Rel, and Rel B, cannot be ruled out (33). The minor band
representing complex I contains Rel A
subunits. In contrast, the IL-8-specific oligonucleotide was bound
mainly by complex I, a Rel A
homodimer, and complex II, a Rel A/p50
heterodimer. Here, no binding of the p50 homodimer was found. This
preferential binding of the Rel A-containing complexes, but not of the
p50 homodimer, to the variant NF-
B site located in the
IL-8 gene promoter is consistent with
other reports (13, 34). Inasmuch as these patterns of NF-
B complexes
are identical to those found on stimulation of these cells by TNF or
PMA, in the presence or absence of adenovirus E1A, this specificity of binding is most likely dictated by the DNA sequence of the binding site
and not by the inflammatory stimuli or the presence of the viral
protein. However, inasmuch as neither set of complexes was induced by
LPS in the absence of E1A, the viral protein must be present for LPS to
be effective.
Of the many investigations into the regulation of host cell
transcription by adenovirus E1A, some have documented the activation of
NF-B in the presence of these viral proteins (23, 24, 32). In Jurkat
cells, this activation was strictly due to the protein product of 289 amino acid residues from the 13S E1A mRNA and only observed after the
induction of NF-
B (24). In contrast to these findings, the
repression of the activity of this transcription factor by E1A, usually
by the protein product of 243 amino acid residues from the 12S E1A
mRNA, has also been reported (4, 8, 24). In none of these studies is
the activation or repression associated with the direct binding of E1A
to NF-
B. Further evidence has been presented that the interactions
between E1A and NF-
B are mediated by the coactivators CBP, the
cAMP-responsive element binding (CREB) binding protein, and the related
protein p300 (4, 24). These results are in keeping with the current
concept that CBP/p300 can interact with a wide variety of factors that
regulate transcription, including DNA binding proteins, the basal
transcriptional machinery, and viral proteins such as E1A, and, in
turn, some of these can bind to each other (reviewed in Ref. 9). The
vast array of signals that bombard a cell could be integrated in this fashion and result in the formation of these specific multiprotein complexes bound to DNA that ultimately controls the activity of a gene.
Within this schema, E1A could exert its influence, whether it be
activating or repressing, through its direct interaction with different
components of this multiprotein complex.
The results of our present studies that adenovirus E1A affects the
induction of NF-B by LPS in A549 cells come from the
immunofluorescence studies that revealed that Rel A translocated to the
nucleus in E1A transfectants, but not in control cells, in response to
LPS stimulation. These results demonstrating the translocation of the
Rel A subunit, which is essential for the formation of the NF-
B
complexes I and
II detected by our EMSA studies,
strongly suggest that, in this particular case, E1A exerts its
influence before the level of the formation of the transcriptional
complex, at an extranuclear site further upstream, perhaps during LPS
recognition or within the LPS signaling pathway. E1A is, however, a
nuclear protein, so it is unlikely that these viral proteins interact directly with the cell membrane or with intracytoplasmic processes. It
is more likely that E1A alters the expression of other genes, the
products of which affect the LPS recognition process or parts of the
ensuing signal transduction pathway. Inasmuch as CD14 was not detected
on the transfectants (11) and because LPS receptors other than CD14 and
the LPS signaling pathway are poorly delineated in nonmacrophage
lineages (20), a novel LPS signaling mechanism must be present in the
E1A-transfected A549 cells. These results on the induction of NF-
B,
however, do not exclude the possibility that E1A might also be involved
in enhancing the activity of this transcription factor, as described
above, once NF-
B has bound to the regulatory sequence of the
IL-8 gene.
Because reactive oxygen species have been documented to act as second
messengers in intracellular signaling of LPS (26, 30) and because
antioxidants are known to block NF-B activation (30), we
investigated the possibility that the regulation of inflammatory
mediator expression by adenovirus E1A in A549 cells could involve
oxygen free radicals. Our results, however, showed that pretreatment of
E1A-transfected cells with NAC only slightly inhibited the LPS-induced
activation of NF-
B, whereas no inhibition was found with the
otherwise potent blocker of LPS stimulation, PDTC (30). These findings
suggest that the LPS signaling pathway affected by adenovirus E1A in
A549 cells is primarily one other than that where reactive oxygen
species are the intermediates and support the presence of a novel LPS
signaling pathway in these E1A-transfected cells.
Putative AP-1 transcription factor binding sites are also located in the regulatory regions of IL-8 and ICAM-1 genes (19, 36), and members of this transcription factor family are known to be affected by E1A gene products (35). E1A represses AP-1-mediated transactivation of the human collagenase gene, whereas its presence increases the basal mRNA levels of the c-jun gene, which encodes a major component of the AP-1 complexes (35). In our present system, neither AP-1 binding activity nor c-jun mRNA levels were altered by E1A. Because the basal levels of AP-1 activity are relatively high in the parental A549 cells, this intrinsic activity might overshadow any effect of the adenovirus E1A in these cells.
A member of the C/EBP family binds to its motif, which is adjacent to
the NF-B site in the regulatory region of the
IL-8 gene (19, 34). In the present
study, however, the binding complexes of C/EBP were barely detected in
nuclei of control cells or E1A transfectants, most likely because of
the relatively weak binding of this transcription factor (34). Also,
the juxtaposition of the C/EBP binding motif to the NF-
B site might
be necessary to allow the interaction of the two transcription factors
before effective C/EBP binding occurs.
Many researchers have reported that primary cultured airway epithelial
cells as well as A549 cells are hyporesponsive or nonresponsive to LPS
(reviewed in Ref. 12). Our results demonstrated that adenovirus E1A
proteins augment LPS responsiveness of human pulmonary epithelial
cells. These cells are a natural target of the virus and of inhaled
dust particles contaminated with endotoxin. The mechanism of this
response is activation of NF-B and the subsequent upregulation of a
specific set of inflammatory genes. This implies a possible cooperative
amplification of the host inflammatory response by viral proteins and a
bacterial product known to contaminate respirable dust particles and
suggests that one of the ways in which latent adenoviral infection
contributes to chronic airway inflammation is through E1A-dependent
NF-
B activation. However, inasmuch as A549 cells are a carcinoma
cell line and as such may differ from epithelial cells in the lung, our
results must be confirmed in primary human pulmonary epithelial cells,
bronchial epithelial cells (6), or type II alveolar epithelial cells before their relevance to human disease can be established.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. K. Matsushima for the generous gift of the human
interleukin-8 cDNA plasmid, Dr. S. Sakurada for expert advice on the
nuclear factor-B immunofluorescent staining technique, and Dr. M. Liu for helpful comments on the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by the National Centres of Excellence for Respiratory Health and by the British Columbia Lung Association.
N. Keicho is a recipient of a Canadian Cystic Fibrosis Foundation fellowship, and S. Hayashi is a recipient of a British Columbia Lung Association scholarship.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Hayashi, Pulmonary Research Laboratory, University of British Columbia, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6 (E-mail: shayashi{at}prl.pulmonary.ubc.ca).
Received 8 December 1998; accepted in final form 16 April 1999.
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