The adenovirus (Ad) early gene product 13S transactivates the tumor necrosis factor (TNF)-α promoter in inflammatory cells. We examined both the subdomains of E1A and the upstream TNF promoter elements involved. In both Jurkat and U-937 cells, zinc finger or carboxyl region mutation of Ad E1A 13S conserved region 3 resulted in a significant loss of transactivation of the TNF promoter (≥69%). For both cell types there was a TNF-negative regulatory element in the −242 to −199 region and a positive regulatory element between −199 and −118. In contrast, an upstream positive regulatory element was detected in different regions in both cell types. In U-937 cells the positive regulatory unit was between −600 and −576, whereas in Jurkat cells it was between −576 and −242. The U-937 upstream element was dependent on a site previously designated epsilon in cooperation with an adjacent nuclear factor-κB-2a site. Therefore, transactivation of the TNF promoter by Ad 13S in lymphocyte and monocyte cell types involves similar subdomains of the E1A protein, but cell-specific TNF promoter elements.
- human gene expression/regulation
- Jurkat cells
- U-937 cells
- virus replication
- zinc finger protein
- third conserved region
adenoviral infection causes pneumonia and disseminated disease in immunocompromised and nonimmunocompromised hosts (1, 7, 33). Chronic sequelae of inflammation also occur, including bronchiectasis, bronchiolitis obliterans, and hyperlucent lung syndrome (12, 17,30-32). In addition, persistence of adenovirus or its proteins has been suggested as a risk factor for the development of steroid-resistant childhood asthma and chronic obstructive lung disease in some patients (21, 22).
Expression of adenovirus early genes may be important in mediation of inflammation by adenovirus, as infection in animal models with nonreplicating virus results in inflammation that is quantitatively similar to active infection (10, 11, 25). Tumor necrosis factor (TNF)-α is elevated in lung homogenates of these animals, suggesting that induction of this cytokine is important in the inflammatory response to adenovirus (11). TNF infusion causes inflammation in many animals (2, 35, 37). Furthermore, TNF appears to mediate many other forms of lung injury triggered by a variety of inciting agents (1, 6, 18).
We previously demonstrated that one of the gene products of the adenovirus early gene region E1A 13S transactivates the TNF gene in inflammatory cells (23). In that study, the third conserved region (CR3) of 13S appeared to be responsible for this effect, as transactivation was not seen with the 12S protein lacking this region. There are two subdomains of this CR3, a zinc finger and carboxyl region domain. Both of these subdomains are important for transactivation of downstream promoter elements of adenovirus (40). The first objective of this current study was to determine whether these subdomains are also important for transactivation of the TNF promoter by adenovirus in inflammatory cells.
The TNF promoter contains consensus sequences for many transcription factor binding sites (8, 14, 27). The importance of individual sites varies depending on the stimulus and cell type used (13, 27). We also sought to determine which regions of the TNF promoter are important for transactivation of the TNF promoter by adenovirus.
Our findings demonstrate that 1) both subdomains of E1A CR3 are necessary for transactivation of the TNF promoter by adenovirus and2) discrete and cell-type specific regions of the TNF promoter are important for these effects.
MATERIALS AND METHODS
Jurkat (ATCC TIB 152) and U-937 (ATCC CRL-1593.2) cell lines, obtained from American Type Culture Collection (Rockville, MD), were used for these studies, as adenoviruses infect both lymphocytes and monocyte/macrophages (5, 19). The Jurkat line was derived from a cell line established from a patient with T-cell leukemia. This derived line produces interleukin (IL)-2 and interferon-γ on stimulation and is CD2+, CD3+, CD4−, and CD8−. The U-937 line was derived from a patient with acute histiocytic lymphoma, has Fc and C3 receptors, produces lysozymes, and is phagocytic. Both cell types were maintained in suspension cell culture in RPMI 1640 medium containing 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 2 mM l-glutamine, and 80 μg/ml gentamicin.
The plasmid (p) SKE1AWT codes for the wild-type (WT) E1A 13S 289R protein under control of the native adenovirus promoter. pSKC171S and pSKS185N contain mutations resulting in single amino acid substitutions in the carboxyl (S185N) and zinc finger (C171S) subdomains of the CR3. pSKC171S/S185N encodes a double mutant of CR3. pSKT178S contains a mutation causing a single amino acid substitution in the region between the zinc finger and carboxyl region subdomains. All of these plasmids were gifts of Dr. Robert Ricciardi (Wistar Institute, Philadelphia, PA) (40). The parent control plasmid Bluescript II SK− (pBIISK−) was obtained from Stratagene (La Jolla, CA). The TNF promoter-CAT reporter constructs contain serial 5′ deletions of the human TNF promoter (−600, −576, −242, −118, −80, −52) upstream of the protein coding region of bacterial chloramphenicol acetyltransferase and were kindly given by Dr. A. Goldfeld (14). The human immunodeficiency virus (HIV)-1 long terminal repeat (LTR) promoter-CAT construct pHIV-1, which contains the HIV-1 LTR −450 to +180 upstream of CAT, was kindly given by Dr. B. M. Peterlin (34). This construct was used to determine whether transactivation by adenovirus E1A was specific in the cells tested. HIV-1 LTR is not transactivated by E1A 13S in Jurkat, THP-1, or HeLa cells (28,38). The thymidine kinase-β-galactosidase plasmid (pTK-β-gal) contains the herpes simplex virus thymidine kinase promoter linked to the β-gal protein-coding gene and was constructed from the pSV-β-gal (Promega) and pMC1neo poly-A (Stratagene). It was used to confirm similar transfection efficiencies between separate experiments (40). The plasmids used to determine whether TNF protein production is enhanced by E1A were pE1A-WT, p13S-WT, and p12S-WT, which were kindly given by Dr. Elizabeth Moran (24). These plasmids express either all the native proteins from the E1A region, WT E1A 13S 289R, or WT E1A 12S 243R, respectively. The parent vector for these constructs pUC18 was used as a negative control (GIBCO-BRL, Rockville, MD).
The TNF promoter/CAT construct p3xNFκBTNFCAT contains a triplicate NF-κB-2a site, and the construct p24bpTNFCAT contains the −600- to −576TNF promoter sequence. These plasmids were constructed by creating multiple nucleotide changes between the multicloning site of the parent pUC12 vector and the region immediately upstream of the TNF promoter element in −52TNFCAT using double-stranded DNA, site-directed mutagenesis (Stratagene). −52TNFCAT was chosen from among the 5′-deletions that were not significantly transactivated by E1A, because it contains the least number of residual transcription factor binding sites, thereby avoiding potential cooperative interactions between any residual sites and the created epsilon (ε), NF-κB-2a, or −600- to −576TNF promoter sequences. p24bpTNFCAT was subsequently used to generate a set of point mutations and base insertions. The construct pε-mutant (mt) 24bpTNFCAT contains three sequential point mutations to the ε-site. pκB2a-mt24bpTNFCAT contains three point mutations to the NF-κB-2a site. Construct pε-κB2a-mt24bpTNFCAT was derived from pε-mt24bpTNFCAT and contains the point mutations present in both pε-mt24bpTNFCAT and pκB2a-mt24bpTNFCAT. pε+6κB2a24bpTNFCAT contains a six-base insertion between the WT-ε and NF-κB-2a sites. The DNA sequences of all plasmids were confirmed by automated DNA sequencing (ABI Systems, Foster City, CA).
Transfection of both cell types was performed by electroporation. Cells were resuspended at a concentration of 2.5 × 107cells/ml (Jurkat cells) or 1.0 × 107 cell/ml (U-937 cells) in the same medium used for culture (see Cell culture). Plasmid DNA was added to 0.4 ml of this cell suspension in 0.4-cm electroporation cuvettes; the cells were gently mixed and were then electroporated at settings of 950 μF and 0.29 V. The electroporated cells were then transferred to 10-cm culture dishes with 10 ml of culture medium. Viability of the cells after electroporation was 35–53% as determined by trypan blue exclusion. For each transfection, additional cells were transfected in triplicate with 10 μg of pTK-β for later β-gal assays. After 48 h of culture, cells were collected for CAT and β-gal assays. E1A protein expression by transfected cells was confirmed by Western analysis as previously described (23).
CAT assays were performed as described using butyrylated coenzyme A (Sigma, St. Louis, MO) (29). Cell extract protein was measured by the method of Bradford (4), and equal amounts of protein were used for assays. The 14C-labeled chloramphenicol (NEN/Perkin Elmer, Boston, MA) was separated into unbutyrylated and butyrylated derivatives by phase extraction using mixed xylene (Sigma), and the butyrylated derivatives were quantified by liquid scintillation. CAT activity was interpolated from a standard curve derived from dilutions of purified chloramphenicol acetyltransferase. The assay was linear over about three orders of magnitude of CAT, and all assays were performed in the linear range of the assay. The results were adjusted for β-gal activity from cells transfected separately with pTK-β to control for variations in transfection efficiency.
TNF-α protein determination.
U-937 cells were transfected with 40 μg of pE1A-WT, p13S-WT, p12S-WT, or pUC18 as described. After 48 h of incubation, 106viable cells/ml were transferred to 12-well plates in fresh medium and stimulated with 1, 10, or 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma) or were mock stimulated with dimethyl sulfoxide (DMSO). After incubation for an additional 24 h, the media supernatants were collected and analyzed in triplicate for TNF-α protein by a sandwich enzyme-linked immunosorbent assay that has a sensitivity of ≤15 pg TNF-α/ml (R&D Systems, Minneapolis, MN). The interassay variability of a TNF-α control standard was ± 3.8% SD.
β-Gal assays were performed by a chemiluminescent assay (Tropix, Bedford, MA). The assay is sensitive to 2 fg of β-gal and linear over approximately five orders of magnitude.
The data are expressed as the means ± SE. Statistical significance was determined by either ANOVA with Ryan-Einot-Gabriel-Welsch multiple-range post hoc analysis or two-tailed t-test with Bonferroni correction for multiple comparisons as appropriate. Significance was considered asP < 0.05 (41).
Effect of CR3 mutation of adenovirus E1A 13S on transactivation of the TNF promoter in Jurkat and U-937 cells.
To evaluate the effect of mutation of specific regions of the adenovirus E1A 13S CR3 on transactivation of the TNF promoter, we cotransfected Jurkat cells and U-937 cells with E1A CR3 mutant plasmids (Fig.1 A) and the −600TNF promoter-CAT construct plasmid.
In Jurkat cells, WT E1A 13S resulted in a sevenfold increase in TNF promoter activity over cells transfected with pBIISK−, as assessed by CAT activity. Substitution of serine for cysteine in the E1A CR3 zinc finger subdomain (C171S) resulted in a significant 9.6-fold loss of TNF transactivation relative to WT E1A 13S (10.41 ± 7.61% relative CAT activity, P < 0.01; Fig.1 B). Furthermore, substitution of asparagine for serine in the carboxyl region (S185N) resulted in a similarly significant, 20-fold loss of activity (4.76 ± 3.49%, P < 0.01). Importantly, substitution at both sites (C171S/S185N) also resulted in a significant 10-fold loss of activity (9.26 ± 2.38%, P < 0.01). Transactivation using this double mutant was similar to that seen with the single mutants (P = 0.24), suggesting that mutations in one subdomain did not unmask a negative regulatory site in the other.
In contrast to the above findings, substitution of serine for threonine in the interdomain region of CR3 (T178S) resulted in only a 15% loss of activity compared with WT E1A (85.07 ± 2.29%, Fig.1 B). Transactivation using this mutant was significantly different from that seen with all of the subdomain mutants (P < 0.05).
In U-937 cells, WT E1A resulted in an 11-fold increase in TNF promoter activity as assessed by CAT activity (Fig. 1 C). The effect of mutations in the CR3 subdomains on transactivation of the TNF promoter in these cells was similar to that seen in Jurkat cells. Mutation of the zinc finger subdomain (C171S) resulted in a significant, fourfold loss of TNF transactivation compared with WT E1A 13S (25.2 ± 2.10%, P < 0.05). Mutation of the carboxyl region (S185N) also resulted in a significant 3.2-fold reduction in activity (31.2 ± 5.65%, P < 0.05). Similarly, mutation of both regions (C171S/S185N) led to a significant 3.6-fold decrease in TNF promoter activity (27.8 ± 4.48%,P < 0.05). As in the Jurkat cells, transactivation of the TNF promoter by E1A was not affected by a mutation in the interdomain region of CR3 (T178S, 124 ± 23.16%). These results suggest that transactivation of the TNF promoter by adenovirus E1A 13S involves the zinc finger- and carboxyl-specific subdomains of the CR3 and that these regions have activity in both cell types.
Transactivation by adenovirus E1A 13S appears not to be due to nonspecific gene activation in unstimulated Jurkat and U-937 cells, as no transactivation of the HIV-1 LTR was seen by either WT or mutant adenovirus E1A 13S (Fig. 1, B and C).
Effect of 5′-TNF promoter deletion on transactivation by WT E1A 13S in Jurkat and U-937 cells.
Mutation of specific subdomains of adenovirus E1A 13S had similar effects on transactivation of the TNF promoter in both Jurkat and U-937 cell lines. Therefore, we subsequently used 5′-TNF promoter deletions to determine whether similar regions of the TNF promoter were activated by E1A in both cell types (Fig.2 A). By comparing the relative activities of adjacent promoter deletions, we could infer the presence of either positive or negative regulatory elements.
For Jurkat cells the 5′-TNF promoter deletions resulted in three levels of transactivation by E1A 13S relative to the activity of −600TNFCAT. TNF activity was significantly diminished, not changed, or significantly augmented (Fig. 2 B). Diminished TNF promoter transactivation was seen with −242TNF (22.2 ± 7.3%,P < 0.01), −118TNF (18.0 ± 5.9%,P < 0.01), −80TNF (20.5 ± 2.0%,P < 0.01), and −52TNF (19.7 ± 1.8%,P < 0.01). The TNF promoter activity of −576TNF (90.7 ± 8.1%) was nearly equivalent to −600TNF. Augmented TNF promoter activity was seen with −199TNF (192.2 ± 18.5%,P < 0.01).
The presence of a positive regulatory element in Jurkat cells was indicated by the significant decrease in TNF promoter activity seen between −576TNF and −242TNF (68.5% difference, P < 0.01). Likewise, a positive regulatory element was seen between −199 and −118, as evidenced by the significant loss of TNF activity (174.2% difference, P < 0.01). Deletion of the promoter region between −242 and −199 resulted in a significant increase in transactivation by E1S 13S (170% difference,P < 0.01), indicating the presence of a negative regulatory element (Fig. 2 B).
In U-937 cells, the 5′-TNF promoter deletions displayed two levels of E1A 13S transactivation relative to −600TNF (Fig. 2 C). The TNF promoter activity was either significantly diminished or significantly augmented. Significantly reduced TNF promoter transactivation occurred with −576TNF (9.5 ± 5.6%,P < 0.01), −242TNF (9.3 ± 4.5%,P < 0.01), −118TNF (6.4 ± 6.3%,P < 0.01), −80TNF (38.3 ± 15.5%,P < 0.01), and −52TNF (45.2 ± 6.2%,P < 0.01). Significantly enhanced TNF promoter transactivation was seen with −199TNF (223.8 ± 33.3%,P < 0.01; Fig. 2 C).
Positive regulatory elements in the TNF promoter were localized between −600 and −576 in transfected U-937 cells, as the shorter construct displayed a significant decrease in the TNF promoter activity (90.5% difference, P < 0.01). Similarly, deletion between −199 and −118 resulted in a significant loss of transactivation (217.4% difference, P < 0.01), indicating the presence of another positive regulatory element. A negative regulatory element was indicated between −242 and −199, as the shorter construct displayed a significant increase in activity (214.5% difference,P < 0.01).
For U-937 cells the regions of the TNF promoter important in E1A transactivation were different from the regions identified for Jurkat cells. In U-937 cells a positive regulatory region was localized to the −600 to −576 region (Fig. 2 C), whereas in Jurkat cells no such regulatory element was seen in this region. In contrast, for Jurkat cells a positive regulatory element was localized to the −576 to −242 region (Fig. 2 B), but in U-937 cells no such element was seen in this region. Therefore, the location of the upstream positive regulatory element differs in the two cell types studied.
Cell-specific activation of the distal TNF promoter region.
The 24-bp region between −600 and −576 contains two transcription factor binding sites. NF-κB-2a has been shown to bind NF-κB proteins, as has a second site know as ε, both in response to lipopolysaccharide (LPS) stimulation. The ε-site also binds a non-NF-κB protein constitutively (36). We first sought to confirm the cell-specific activity of the 24-bp segment in response to E1A transactivation and to determine whether this activity was due to the NF-κB-2a site. We performed transient transfections in Jurkat and U-937 cells with the pSKE1AWT expression vector or the control plasmid along with the proximal deletion −52 or the −52 proximal deletion to which an NF-κB-2a triplet or the 24-bp region had been added. Data are expressed as CAT activity relative to that seen with cells transfected with pSKE1AWT and the −600TNF promoter construct.
In Jurkat cells, addition of the 24-bp region upstream of the −52TNF promoter did not restore transactivation to levels seen with the −600TNF construct (P < 0.05, Fig.3 A). Neither the NF-κB triplet (25.8 ± 9.6%) nor the 24-bp insert (17.8 ± 7.4%) conferred activity that was different from the −52TNF promoter deletion (26.6 ± 12.7%).
In contrast in U-937 cells, addition of the 24-bp region upstream of the −52TNF promoter resulted in transactivation levels similar to that seen with the −600TNF construct (79.9 ± 31.3%). This activity appeared to be greater than that seen with the NF-κB triplet (27.2 ± 8.1%), although this did not reach statistical significance. Transactivation of the 24-bp TNF construct was different from that seen with the −52TNF proximal deletion (11.9 ± 2.8%,P < 0.05; Fig. 3 B). These findings confirm the importance of the −600 to −576 region in E1A transactivation of the TNF promoter in U-937 cells and this region's lack of activity in Jurkat cells.
Effect of CR3 mutation of adenovirus E1A 13S on transactivation of the −600- to −576TNF promoter.
To determine whether transactivation of the 24-bp region occurred through similar subdomains of adenovirus E1A 13S CR3 as for transactivation of the entire promoter, U-937 cells were cotransfected with the 24-bp TNF construct and either the WT or CR3 mutant adenovirus E1A 13S expression vectors (Fig. 4). These results mirror those obtained with the intact −600TNFCAT (Fig. 1 C). CAT activity is expressed as a percentage of that seen in cells transfected with pSKE1AWT and p24bpTNFCAT.
WT E1A 13S enhanced CAT activity from the 24-bp TNFCAT construct 18-fold over the control vector pBIISK− (Fig. 4). Mutation of the zinc finger (C171S, 12.6 ± 3.2%) or carboxyl region (S185N, 40.7 ± 18.4%) of E1A CR3 or both regions (C171S/S185N, 4.9 ± 4.0%) resulted in a significant loss of activity compared with that induced by WT E1A 13S (P < 0.05). In contrast, mutation of the interdomain region (T178S, 81.5 ± 14.8%) resulted in a minimal loss of activity. These results suggest that transactivation of the −600- to −576TNF promoter region required both the zinc finger and carboxyl subdomains of adenovirus E1A CR3.
Localization of the TNF promoter upstream positive regulatory element in U-937 cells.
To determine the active site for transactivation in the −600- to −576TNF promoter region, we introduced mutations in the ε-site, the NF-κB-2a site, or both, and cotransfected the constructs along with the WT E1A 13S expression vector or control plasmid. CAT activity was expressed as a percentage of the 24-bp TNF construct, which contains no mutations (Fig. 5 A).
Mutation of the ε-site (ε-mt24bpTNF, 9.4 ± 1.8%) resulted in a significant 10.6-fold loss of activity (P < 0.01; Fig. 5 B). Mutation of the adjacent NF-κB-2a site (κB2a-mt24bpTNF, 46.8 ± 13.5%) resulted in a significant but lesser 7.4-fold loss of activity (P < 0.01). Combining the mutations in both regions (ε+κB2a-mt24bpTNF, 12.79 ± 6.30%, P < 0.01) resulted in no additional loss of activity over that seen with the mutation of the ε-site alone. To determine whether the ε-site functioned cooperatively with the NF-κB-2a site, we introduced a 6-bp sequence between the ε- and the NF-κB-2a site. This promoter construct (ε+6 κB2a24bpTNF, 5.23 ± 2.06%) resulted in significant 19-fold loss of activity (P < 0.01; Fig. 5 B).
These findings show that both the ε-site and, to a lesser extent, the NF-κB-2a site are responsible for U-937-specific E1A transactivation in this region and that these two adjacent sites act in cooperation to confer TNF promoter activity in U-937 cells.
TNF protein induction by adenovirus E1A.
To determine whether induction of the TNF promoter by E1A under control of the native adenovirus promoter resulted in induction of TNF protein production, we transfected U-937 cells with plasmids expressing all proteins from the E1A region (pE1A-WT), the 13S protein (p13S-WT), or the 12S protein (p12S-WT). Transfection with the control vector pUC18 was used as a negative control. Cells were also stimulated with increasing concentration of PMA or were left unstimulated.
E1A 13S expression by unstimulated U-937 cells resulted in at least a 2.2-fold increase in TNF protein production over unstimulated control cells (33 ± 8 vs. <15 pg/ml; Fig.6). TNF protein production from U-937 cells expressing E1A 13S and stimulated with 10 ng/ml PMA displayed a significant 2.3-fold increase compared with identically treated control cells (456 ± 30 vs. 198 ± 21 pg/ml, P < 0.05). Similarly, at 100 ng/ml PMA, a significant 2.8-fold increase in TNF protein was induced by cells expressing E1A 13S compared with identically stimulated control cells (598 ± 43 vs. 210 ± 23 pg/ml, P < 0.05). Additional experiments confirmed this effect is specific for E1A 13S, as transfection with plasmids expressing all the E1A proteins (WT E1A) or the E1A 12S (WT E1A 12S) protein did not enhance TNF protein production, even in the presence of PMA (Fig. 6). These results confirm that the presence of E1A 13S in mitogen-stimulated, monocyte/macrophage-like cells significantly enhances TNF protein production.
Stimulation of TNF-α by adenovirus early proteins expressed during replicative infection or latency may well play a role in chronic inflammation due to adenovirus. The current results confirm and extend previous data demonstrating upregulation of the TNF gene by adenovirus E1A 13S in inflammatory cells. (23) The importance of the specific subdomains in the CR3 necessary for transactivation of the TNF promoter by adenovirus is demonstrated. Localizing the sites within the E1A 13S CR3 responsible for transactivation suggests which of several mechanisms are responsible for the findings demonstrated.
There are three conserved regions in the adenovirus 13S 289R protein: CR1, CR2, and CR3 (20). Only CR1 and CR2 are also present in the shorter 12S 246R protein. We previously demonstrated that transactivation of the TNF promoter occurs only with the CR3-containing 13S protein and not with the 12S protein, which contains only CR1 and CR2 (23). In the present study, we confirm the importance of the E1A 13S CR3 in TNF promoter transactivation by demonstrating loss of this activity with deletion in either or both of two putative subdomains of the CR3.
Transcriptional activation by adenovirus E1A 13S CR3 appears to occur indirectly by binding of the subdomains to general and specific transcription factors. The zinc finger subdomain [amino acid (aa) 147–177] interacts with the general transcription factor IID through binding to TATA-binding protein (TBP) and TBP-associated factors, whereas the carboxyl region (aa 180–188) binds to cellular or specific transcription factors (9). E1A thus acts as a type of coactivator by linking cell specific transcription factors and the basal transcriptional complex. In this way E1A alters transcription rates without binding to DNA specifically in vitro.
As the target of E1A-mediated transactivation, the TNF promoter includes consensus sequences for many transcription factors, including activator protein (AP)-1, AP-2, and cAMP response elements (CRE) (16, 26, 27). Also, there are multiple sites for NF-κB (13). It appears that the importance of these sites varies with the cell type and stimulus used. Rhoades and colleagues (26), evaluating promoter transactivation by E1A in U-937 cells and a gibbon T-lymphocyte line, concluded that CRE were important for this activity in T lymphocytes, whereas AP-1 and AP-2 sites were also important in U-937 cells according to mutational analysis of these sites. The authors ascribed no importance to TNF promoter sequences upstream of −120 or to NF-κB sites in either cell type, despite the demonstration of a strong regulatory element between −615 and −120. They also did not perform any mutations of putative NF-κB sites, and although they used AP-1, AP-2, and CRE expression vectors to confirm their findings, they did not use any method to increase NF-κB expression and evaluate this effect on E1A transactivation. With regard to this region, Udalova and colleagues (36), using a variety of different methods and mutational analysis in the −600TNF promoter region, implicated multiple NF-κB sites, including an NF-κB-like site and a site termed ε as important in stimulation of the TNF promoter by LPS in Mono Mac 6 cells. Our findings confirm that these sites, particularly ε and NF-κB-2a, are important for transactivation of the TNF promoter by E1A in the monocyte/macrophage-like U-937 cells.
It is important that the sites in this region in E1A transactivation of TNF is cell type dependent. Comparing data from the Jurkat and U-937 cells, it appears that the NF-κB-2a and ε containing −600 to −576 region is not active in Jurkat cells.
Previous studies in our laboratory using E1A in a dextran-transfected monocyte/macrophage THP-1 cell line showed minimal induction of TNF mRNA but did not demonstrate induction of TNF protein (23). In the study by Rhoades et al. (26), E1A controlled by the stronger Rous sarcoma virus promoter was shown to induce IL-8 protein. In the data presented here, we demonstrate that E1A controlled by its native adenovirus promoter enhances TNF protein production from electroporated U-937 cells, at least in the presence of PMA. This effect was not due to enhancement of E1A expression by PMA, as Western analysis for E1A from PMA-stimulated E1A-transfected U-937 cells did not show enhanced E1A protein expression (not shown). Enhancement of TNF production by PMA in E1A-transfected cells is likely related to NF-κB activation, perhaps in association with AP-1, because modest induction of NF-κB binding occurs in the presence of E1A, and PMA enhances NF-κB and AP-1 binding activity in monocyte/macrophage cells (3, 15). Our findings using the various E1A region constructs in TNF protein production experiments may provide insight into the mechanisms of inflammation due to adenovirus infection and subsequent expression of the native E1A proteins. The WT E1A construct expresses all transcripts from the E1A region, whereas the WT 12S construct expresses 12S but not 13S E1A. We did not find TNF induction with either of these constructs, whereas expression of 13S E1A enhanced TNF protein production. This is not surprising, as, with expression of 12S or the other E1A proteins, CR1 and CR2, which have variable effects on DNA expression, would have more influence on the overall results. These findings may relate to how E1A expression stimulates inflammation in animal models. E1A gene expression has been shown to enhance lung inflammation in vivo in animal models in the presence of a cofactor such as cigarette smoke (39). Our data suggest that this inflammation is likely accompanied by or associated with specifically enhanced E1A 13S expression, as expression of other E1A transcripts failed to induce cytokine promoter activation in our model. We recognize that this would have to be confirmed by measurements of specific transcripts or use of E1A 13S protein-specific expression vectors in these animal models, which has not been done.
In conclusion, we demonstrate a mechanism whereby an adenovirus protein can interact with specific promoter elements of the cytokine gene promoter. It appears that this occurs by interaction of specific conserved subdomains of the adenovirus E1A 13S protein with varying factors that bind to discrete but diverse regions of the TNF promoter that vary according to cell type.
We thank Andrea Vincent for assistance with the statistical analysis and Drs. Gary Kinasewitz and Mark Coggeshall for review of the manuscript.
This work was supported by National Heart, Lung, and Blood Institute Grant K08-HL-03106 and an American Lung Association Research grant.
Address for reprint requests and other correspondence: J. P. Metcalf, Oklahoma Univ. Health Sciences Center, Research Park, Bldg. #1, 800 N. Research Pkwy, Rm. 425, Oklahoma City, OK 73104 (E-mail:).
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. Section 1734 solely to indicate this fact.
April 26, 2002;10.1152/ajplung.00342.2001
- Copyright © 2002 the American Physiological Society