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-κB 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-κB 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-κB induction in these cells.
- A549 cells
- lipopolysaccharide response
- regulation of inflammatory mediator expression
- nuclear factor-κB
the adenovirus E1Agene 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-κB (NF-κB) 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-β, IL-1β, 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 andIL-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-κB stands out as a central coordinating regulator (33). The active DNA-binding forms of NF-κB 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-κB family bear unique and overlapping functional activities, and combinations of these members form distinctive complexes that are characterized by their preference of certain κB sites and by their transactivation potentials (33). In fact, the Rel A homodimer has been reported to bind preferentially to the NF-κB binding sites of humanICAM-1 andIL-8 genes (14, 34). Under most circumstances, NF-κB complexes lie dormant in the cytoplasm of unstimulated cells, bound to an inhibitory protein, IκB (33). In response to extracellular signals such as TNF and phorbol 12-myristate 13-acetate (PMA), IκB is degraded, allowing NF-κB to translocate to the nucleus, where it binds tocis-acting κB sites in the regulatory region of NF-κB-responsive genes, includingICAM-1 andIL-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-8and 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.
METHODS AND MATERIALS
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 MN-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.
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 5E1A 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 5E1A 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-κB 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 [α-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 12,000g. 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 12,000g, 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-κB binding sequence of the immunoglobulin κ-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-κB, and C/EBP, respectively. For the human IL-8gene-specific NF-κB 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 [γ-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 theIL-8 gene-specific NF-κB 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-κB, antiserum (1–2 μg) against the Rel A or p50 subunit of NF-κB (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.
To test the effect of antioxidants on LPS-induced NF-κB activation, E4 cells were preincubated for 1 h in the presence or absence of 30 mM NAC (25) or 100 μM PDTC (30) before treatment with LPS for 2 h. Nuclear extracts were analyzed by EMSA using the NF-κB binding sequence of the immunoglobulin κ-chain gene, and these were compared with nuclear extracts from untreated cells or from cells treated with LPS alone where, as described above, excess unlabeled competitor oligonucleotide was added to the nuclear extract before the labeled probe.
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-κB 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-κB 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-κB, was calculated. The data from the E1A-positive and E1A-negative cells were pooled for each of the culture conditions and compared by at-test, with the assumption of unequal variances.
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-8gene 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.1 A). 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).
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.1 B). 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-κB binding activity in the nuclei of E1A transfectants but not in control cells.
When the EMSA using the oligonucleotide specifying the NF-κB binding sequence in the enhancer of the immunoglobulin κ-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.2 A). To facilitate their identification, the complexes are numberedI, II, andIII 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-κB 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. 2 A).Complex I, the slowest migrating of the three, was detected only at low levels and only whencomplexes II andIII 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-κB oligonucleotide, whereas an unrelated oligonucleotide specific for AP-1 binding did not affect the formation of these complexes (Fig.2 B). Bands designated NS were not affected by either competitor. Similar NF-κB-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).
Antisera against Rel A and p50, the major binding subunits of NF-κB, were used 1) to confirm that the complexes binding the immunoglobulin κ-chain NF-κB oligonucleotide were specific for this transcription factor and2) to identify the individual components within these complexes (Fig.3 A). In nuclear extracts from the E1A transfectant E4 treated with LPS antiserum against Rel A disrupted complex I and II formation to yield slower-migrating DNA-protein complexes, which are commonly referred to as supershifted bands. The same antiserum did not affect the formation of complex III. On the other hand, antiserum against p50 completely disruptedcomplex III and most ofcomplex II. Again, a supershifted band was present. Unrelated antiserum containing rabbit anti-mouse immunoglobulins did not affect the formation of the three complexes. When these antisera were applied to extracts from unstimulated E1A transfectants, no alterations in the pattern of bands could be detected (Fig. 3 A). Similar results were obtained using another transfectant, E20 (data not shown).
When E1A-positive E4 cells were pretreated with antioxidants before stimulation with LPS, only a small reduction in binding to the NF-κB binding sequence in the enhancer of the immunoglobulin κ-chain gene was found with 30 mM NAC and no reduction was found with 100 μM PDTC (Fig. 3 B). At the same time, complete abolition of specific binding was observed after competition with excess unlabeled NF-κB oligonucleotide, whereas the nonspecific competitor AP-1 had no effect (Fig.3 B).
LPS treatment induced IL-8 gene-specific NF-κB 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-κB consensus sequence, EMSA was performed using the labeled oligonucleotide, which included theIL-8 gene-specific NF-κB 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 ofcomplexes I andII in E1A transfectants, withcomplex I predominating. The same treatment did not affect the NF-κB 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 ofcomplexes I andII 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-κB 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).
As described above, confirmation of the specificity of the complexes formed and identification of the components making up these complexes induced in E1A transfectant E4 after LPS treatment were made by applying antisera against Rel A and p50 to the nuclear extracts (Fig.5). Antiserum against Rel A disrupted and supershifted complexes I andII, whereas antiserum against p50 disrupted only complex II. Unrelated antiserum did not affect the formation of these complexes. Similar results were observed when another E1A transfectant, E20, was tested (data not shown).
LPS treatment induced nuclear translocation of NF-κB in E1A transfectants but not in control cells.
The subcellular distribution of NF-κB was also examined by an indirect immunofluorescence assay with use of an antiserum against the Rel A subunit of NF-κB (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).
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.7 A). 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 and2) E20 and C4 (data not shown). This binding activity was competed out by excess unlabeled AP-1 oligonucleotide, whereas an unrelated NF-κB-specific oligonucleotide did not compete for the binding (Fig.7 B). 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).
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).
We previously reported that adenovirus 5 E1A upregulates the expression of ICAM-1 (11) andIL-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-κB activation that is associated with the upregulation of these two responsive genes in target epithelial cells.
In A549 cells, E1A-dependent upregulation ofIL-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-junand 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-κB, 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 andICAM-1 genes in pulmonary epithelial cells expressing adenovirus E1A. NF-κB, 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-κB activation appears to be essential for the transcription of humanICAM-1 andIL-8 genes (7, 19). Interestingly, recent studies have revealed that theIL-8 andICAM-1 genes are unique in terms of the nucleotide sequence of their NF-κB binding site and of the binding affinity of the different NF-κB subunits to these sites. The ICAM-1 NF-κB site 5′-TGGAAATTCC-3′ and the IL-8 NF-κB site 5′-TGGAATTTCC-3′ differ from the NF-κB 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 theIL-8 andICAM-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-κB complexes are induced on stimulation of pulmonary epithelial cells with LPS if adenovirus E1A is present. The two different NF-κB sites tested here are bound by these complexes in a selective fashion. For the prototypic immunoglobulin NF-κB sequence, EMSA and supershift assays revealed thatcomplex II mainly consists of a p50/Rel A heterodimer and complex IIIis another p50-containing complex, probably a p50 homodimer, although minor contributions from other members of the NF-κB 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-κB site located in theIL-8 gene promoter is consistent with other reports (13, 34). Inasmuch as these patterns of NF-κB 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-κB 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-κB (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-κB. Further evidence has been presented that the interactions between E1A and NF-κB 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-κB 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-κBcomplexes I andII 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-κB, 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-κB has bound to the regulatory sequence of theIL-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-κB 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-κB, 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 andICAM-1 genes (19, 36), and members of this transcription factor family are known to be affected byE1A 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-κB site in the regulatory region of theIL-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-κB 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-κB 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-κB 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.
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-κB immunofluorescent staining technique, and Dr. M. Liu for helpful comments on the manuscript.
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:).
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.
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- Copyright © 1999 the American Physiological Society