INTRODUCTION

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Tumor necrosis factor alpha (TNF) contributes to the pathogenesis of many chronic inflammatory diseases, including rheumatoid arthritis, diabetes, hepatitis, and some causes of pulmonary inflammation and fibrosis (14, 16, 27, 29, 49, 53, 54). The controlled expression of TNF is critical during sepsis and adipocyte differentiation and in obesity (9, 19, 20, 22, 49). Although macrophages are the principal source of TNF secretion in conditions such as rheumatoid arthritis and sepsis (27, 49), the cytokine is produced by a wide variety of cells, including lymphocytes, adipocytes, mast cells, keratinocytes, and astrocytes.

A number of transcription factors contribute to the complex regulation of the TNF gene, and interactions between factors may vary depending on the cell type and the particular extracellular stimuli. The transcription factor C/EBP (also called NF-IL6, NF-M, LAP, IL6-DBP, AGP/EBP, and CRP2) (1) has been shown to be important in TNF gene activation in myelomonocytic cells (38). It binds to the TNF promoter at a site between 74 and 100 bp upstream of the transcription start site (38) and may also be important for the expression of TNF in other cell types, such as hepatocytes and adipocytes, which also express C/EBP.

Several types of evidence suggest that C/EBP works in concert with other transcription factors to regulate the TNF promoter in a cell-type-specific fashion. For example, the TNF promoter contains potential binding sites for several additional transcription factors, including AP-1, AP-2, NF-B, NFAT, Ets, SP-1, and cyclic AMP response element (CRE) (see Fig. 1) (13, 15, 25, 33, 38, 39, 48). The transcription factors AP-1, Ets, and NFAT have been shown to play important roles in the activation of the TNF gene (13, 15, 23, 33, 39, 48). In addition, C/EBP synergizes with a variety of transcription factors, including c-Jun, NF-B, Myb, and the glucocorticoid receptor, to regulate other genes (7, 17, 21, 23, 26, 32, 35, 42, 43). However, very little is known about interactions between C/EBP and other transcription factors in the cell-type-specific regulation of the TNF gene.

Here we describe interactions between C/EBP and the transcription factor AP-1 which affect the regulation of the TNF gene. We showed that c-Jun binds adjacent to C/EBP on the TNF promoter and synergistically activates the C/EBP-dependent expression of the TNF gene in phorbol myristate acetate (PMA)-treated Jurkat T cells. This cooperation required the DNA binding domain of c-Jun and an intact AP-1 binding site on the TNF promoter, suggesting that both C/EBP and c-Jun must bind in order to coactivate the gene. U937 cells stably overexpressing wild-type c-Jun secreted increased TNF. Interestingly, C/EBP also cooperated with the mutant form of c-Jun lacking the transcriptional transactivation domain, in both Jurkat T cells and U937 cells, suggesting that the cooperation between c-Jun and C/EBP is not just the additive effect of independent transcriptional activation domains.

	MATERIALS AND METHODS

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Plasmid vector constructs. The TNF promoter reporter constructs containing 615, 120, or 95 bp 5' of the transcription start site, linked to a luciferase gene, have been described elsewhere (13, 38), as have the expression vectors (cytomegalovirus encoding wild-type [CMV]-C/EBP) and dominant negative (CMV-5D229) versions of C/EBP (38). c-jun and c-jun mutant human cDNAs were cloned into the CMV vector plasmid and have been previously characterized (2, 6, 18, 36). Mutations had been generated by deletion of the leucine zipper (c-Jun-LZ), the DNA binding (c-Jun-DBD), and the transactivation (transactivation domain mutant TAM-67) domains (2, 6, 18, 36). Human c-fos was cloned into a pSV vector (2, 6). The pCDM8 (Invitrogen, San Diego, Calif.), pSV, and CMV vectors were used as controls. A promoter-reporter construct containing dual c-Jun binding sites from the interleukin-2 (IL-2) promoter [TRE(IL-2)-Luc] was used as a control (36). The p300- and CREB-binding protein (CBP)-expressing plasmids have been previously described (10, 12). Luciferase-expressing plasmids possessing the AP-1 and C/EBP binding sites of the TNF gene were constructed by using pT81-Luc, which possessed a weak tk promoter. Two plasmids, constructed by using oligonucleotides representing 115 to 74 bp of the TNF promoter, possess 0 (pT81TNF0bp) or 10 (pT81TNF10bp) irrelevant bp inserted between 98 and 97 bp. Since the two sites overlapped, 99 and 100 were included on both sides of the inserted base pairs. Plasmids were screened by restriction digestion, and the sequences were confirmed by DNA sequencing employing the dideoxynucleotide method.

Transfection and luciferase assay. Jurkat T cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, and penicillin-streptomycin (complete medium). Transfection of Jurkat T cells was performed by the DEAE-dextran method as previously described (38), keeping the total plasmid concentration constant (8 µg). Transfection of U937 cells was performed by electroporation (38), keeping the total DNA concentration at 16 µg/transfection. After transfection, cells were placed in complete medium for 4 to 6 h, and then PMA (10 ng/ml), alone or with lipopolysaccharide (LPS; 1 µg/ml), or ionomycin (0.5 µM) or control buffer, as indicated in the individual experiments, was added for an additional 12 h. Cells were harvested, washed, and lysed by freeze-thawing three times, and luciferase activities were determined on cell lysates as previously described (38), using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, Calif.). Promoter activities were expressed as relative light units (RLU), normalized for the total protein in each extract.

Oligonucleotides and electrophoretic mobility shift assay. Synthetic oligonucleotide probes were prepared which spanned the following regions of the TNF promoter: 100 to 74 (100/74) (29), 115 to 74 (115/74), and 115 to 98 (115/98) (Fig. 1). Oligonucleotides representing the C/EBP binding site of the IL-6 promoter and an AP-1 binding site from the collagenase promoter have been previously described (1, 3). Recombinant C/EBP was expressed in insect cells, using a baculovirus vector, as previously described (38). Recombinant c-Jun was obtained commercially (Promega Corp., Madison, Wis.), as were antibodies specific for C/EBP and c-Jun (Santa Cruz Inc., Santa Cruz, Calif.). Electrophoretic mobility shift assays were performed as described previously (38), using 1 ng of ³²P-labeled probe per reaction. DNA-binding complexes were analyzed by electrophoresis on a 4% nondenaturing polyacrylamide gel in buffer containing 67 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 33 mM sodium acetate. Gels were dried and exposed to radiographic film overnight at 80°C.

Cell lines. Jurkat and U937 cell lines were obtained from the American Type Culture Collection (Rockville, Md.) and maintained in complete medium as described above (38). U937 cell lines stably transfected with plasmids expressing a neomycin resistance gene alone or together with plasmids expressing the wild-type c-Jun or TAM-67 have been previously described and characterized (18, 45). These cell lines were maintained in medium containing 400 µg of G418 per ml.

TNF secretion and quantitation. U937 cell lines expressing the neomycin resistance gene alone or together with wild-type c-Jun or TAM-67 were plated at 0.5 × 10⁶/ml in complete medium. PMA (10 ng/ml), or PMA plus LPS (1 µg/ml), or control medium was added, and cells were incubated for 18 h. Supernatants were harvested and frozen at 20°C. TNF was measured by enzyme-linked immunosorbent assay using commercially available reagents as described previously (8).

	RESULTS

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C/EBP and c-Jun proteins synergistically activate the TNF gene. Our prior data (38) showed that C/EBP was important in activating a promoter reporter gene construct possessing as little as 120 bp of the TNF promoter (120 TNF-luciferase). However, this promoter reporter also possesses two functional AP-1 binding sites centered at 102/103 and 62 bp upstream from the transcription start site (Fig. 1). We used cotransfection assays in Jurkat T cells, which do not express endogenous C/EBP, under conditions in which c-Jun was not significantly activated (38, 44), in order to determine if there was an interaction between C/EBP and AP-1 complex transcription factors in regulation of the TNF promoter.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Sequence of the human TNF promoter region 123 bp upstream from the transcription start site (TSS). Previously described binding sites for C/EBP (100 to 74 bp), AP-1 (106 to 99 and 65 to 59 bp), AP-2 (36 to 28 bp), SP-1 (52 to 45 bp), Ets (116 to 112 bp), NFAT and NF-B (97 to 88 bp), and CRE (106 to 99) are indicated on the sequence representing the first 123 bp of the TNF promoter (13, 15, 25, 33, 38, 39, 48). The C/EBP binding site was defined by using the 32P-oligonucleotide employed in this study (38). The sites highlighted by the bold boxes are the focus of this study.

View larger version (26K): [in this window] [in a new window]   View larger version (59K): [in this window] [in a new window]   View larger version (44K): [in this window] [in a new window]   FIG. 2.   Both c-Jun and TAM-67 synergize with C/EBP to activate the TNF promoter. (A) Effects of C/EBP, c-Jun, and c-Jun mutants on PMA-induced TNF promoter activation. Jurkat T lymphocytes were transfected by the DEAE-dextran method with plasmid vectors expressing C/EBP, c-Jun, the c-Jun mutants c-Jun-LZ, c-Jun-DBD, and TAM-67, and the TNF promoter reporter construct (3 µg/transfection) containing 120 bp 5' of the transcription start site. The C/EBP plasmid was transfected at a suboptimal concentration (2 µg), while the plasmids expressing wild-type and mutant c-Jun and the CMV vector control were added at increasing concentrations (0.25, 0.5, 1, 2, and 3 µg, represented by the columns from left to right), with the total DNA added kept constant (8 µg). Luciferase activity was reported as RLU, corrected for the total protein in each lysate. Fold activation is expressed as a mean ± 1 SE for three or more experiments. (B) Effects of cotransfection of C/EBP with c-Jun or its mutants on PMA-induced activation of the TNF promoter. Jurkat T lymphocytes were transfected with plasmid vectors expressing C/EBP plus c-Jun or its mutants and the 120 TNF promoter reporter construct (3 µg). Various concentrations of control, c-Jun, or mutant c-Jun (0.25 to 3 µg) were added to C/EBP (2 µg). In addition, to more readily compare the results of different experiments, prior to analyses, the results of each experiment were normalized to the values obtained for C/EBP, which was defined as 100%. Data for wild-type c-Jun are the means ± 1 SE of six experiments, while those involving the mutants are the means of four experiments. Differences between C/EBP alone and with mutant or wild-type Jun were determined by t test for matched pairs. (C) Effects of TAM-67 and CMV vector control on c-Jun-induced activation of TRE(IL-2)-Luc promoter-reporter in PMA-stimulated Jurkat T cells. The TRE(IL-2)-Luc promoter reporter contains two copies of the c-Jun binding site of the IL-2 promoter. TRE(IL-2)-Luc (3 µg) was transfected with an optimal concentration of c-Jun (0.25 µg) and increasing concentrations of TAM-67 (0.25, 2, and 5 µg) or control CMV vector (0.25, 2, and 5 µg). The results presented are the means ± 1 SE of a single experiment that was representative of four experiments.

The c-Jun transactivation domain is not required for enhancement of C/EBP-induced activation of the TNF gene. Deletion mutants were used to identify the regions of c-Jun responsible for the synergistic activation of the TNF promoter. The TAM-67, c-Jun-DBD, and c-Jun-LZ domain deletion mutants of c-Jun alone resulted in little or no activation of the TNF promoter reporter in PMA-treated Jurkat T cells (Fig. 2A). In addition, no synergy was observed when c-Jun-LZ was coexpressed with C/EBP (Fig. 2B). These observations suggest that protein-protein interactions, most likely involving the c-Jun leucine zipper, were necessary for the synergistic activation of the TNF gene by C/EBP and c-Jun. To determine if c-Jun DNA binding was required, the c-Jun-DBD mutant, which is capable of dimerization but not DNA binding, was also used. No synergy with C/EBP was observed (Fig. 2B), suggesting that binding of c-Jun homodimers or formation of a complex of c-Jun and C/EBP was necessary for the observed synergistic activation. Unexpectedly, the ectopic expression of TAM-67, which has the c-Jun DNA binding and dimerization domains intact but lacks the transactivation domain, enhanced the activation of the TNF promoter by C/EBP (P < 0.02 at 0.25 and 0.5 µg) (Fig. 2B). The TAM-67 results suggest that the observed synergism of c-Jun with C/EBP was not due to additive effects of heterologous transactivation domains but more likely involved direct protein-protein interactions or cooperative DNA binding to the TNF promoter.

c-Jun must bind the TNF promoter to synergize with C/EBP. Prior studies have indicated that AP-1 binding sites centered 62 and 102/103 bp upstream of the transcription start site were involved in TNF gene activation (25, 39). The contribution of these sites for the enhancement of C/EBP-induced activation by c-Jun was examined. Since synergistic interactions between c-Jun and C/EBP were evident in assays using the 615 AP-1 mutant TNF promoter reporter, which possesses a 2-base substitution at 65 and 66 bp of the TNF promoter (data not presented), we reasoned that the AP-1 binding site at that position was not required for synergism with C/EBP. Therefore, we compared the 95 TNF-luciferase promoter reporter construct, which possesses the C/EBP binding site (38, 50) but lacks the AP-1 site centered at 103/102 bp (25, 33), to the 120 TNF promoter reporter, in which the AP-1 site is present. The initial cotransfection assays were performed at a limiting concentration of CMV-C/EBP (1 µg), which we had determined in preliminary studies more sensitively detected the potential synergistic effects of added c-Jun. The two promoter reporter constructs were comparably activated by cotransfection of 1 µg of C/EBP expression vector in PMA-treated Jurkat T cells (Fig. 3A). However, unlike the results observed with the 120 construct, no synergy between C/EBP and c-Jun was observed when the 95 TNF promoter was used. Additional experiments were performed with the 95 promoter reporter construct, using 2 µg of CMV-C/EBP and each of the c-Jun constructs (Fig. 3B). Again in contrast to the results observed with the 120 TNF promoter (Fig. 2B), cotransfection of TAM-67 with the 95 TNF promoter reporter had no effect on C/EBP-induced activation (Fig. 3B). Since the 95 TNF promoter reporter plasmid still possessed the AP-1 binding site centered 62 bp upstream of the transcription start site, the data confirm that this AP-1 site was not responsible for the synergistic enhancement of C/EBP-induced activation of the TNF promoter by c-Jun in PMA-treated Jurkat T cells. Thus, synergism between c-Jun and C/EBP required a c-Jun binding site in the promoter as well as expression of c-Jun proteins containing both the DNA binding and dimerization domains, strongly suggesting that DNA binding by c-Jun was essential.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   The AP-1 binding site is necessary for synergistic activation of the TNF promoter. (A) Comparison of 120 and 95 TNF promoter reporter constructs on C/EBP-plus-c-Jun activation in PMA-induced Jurkat T cells. Jurkat T lymphocytes were transfected with plasmid vectors expressing C/EBP (1 µg) and c-Jun (0.25 µg), separately or together, plus the 120 TNF or the 95 TNF promoter reporter (3 µg/transfection). The experiments were performed and analyzed as described for Fig. 2A. The results presented are the means ± 1 SE of three experiments. (B) Effects of cotransfection of c-Jun and c-Jun mutants on activation of the 95 TNF promoter reporter construct. Jurkat T cells were transfected with the 95 TNF-luciferase promoter reporter (3 µg), the CMV-C/EBP expression vector (2 µg), and various concentrations of wild-type or mutant c-Jun expression vectors (each at 0.25, 1, 3, and 5 µg/transfection). The experiments were performed, and cells were harvested and analyzed, as described for Fig. 2A. The results presented are the means ± 1 SE of two experiments.

The transactivation domain of c-Jun mediates suppression of C/EBP-induced activation in Jurkat T cells. Cotransfection of either wild-type c-Jun or the c-Jun-LZ mutant resulted in significant (P < 0.05 to 0.005 at 0.5 to 3 µg) suppression of the C/EBP-induced activation of both the 120 and the 95 TNF promoter reporters (Fig. 2B and 3B), suggesting that dimerization or protein-protein interactions mediated by the leucine zipper were not necessary for suppression. In contrast to the results with wild-type c-Jun, suppression was not observed at any concentration of TAM-67 with either the 120 or 95 TNF promoter reporter construct (Fig. 2B and 3B). These observations suggest that the c-Jun transactivation domain contributed to the inhibition of C/EBP-induced activation of the TNF promoter and that this repression was independent of the AP-1 binding site. The explanation for the lack of suppression observed with the c-Jun DNA binding mutant (Fig. 2B and 3B) remains unclear. The suppressive effect was not specific for c-Jun since c-Fos, at similar concentrations, also suppressed C/EBP-induced activation (data not shown).

Potential contribution of other transcription factors or coactivators to C/EBP-c-Jun-mediated TNF activation. Earlier studies demonstrated that NFAT was important in the activation of the TNF gene in T cells under certain conditions (15, 48). Jurkat T cells possess NFAT, which requires two signals, such as PMA plus ionomycin, for optimal activation. Since activation of the TNF gene by NFAT was inhibited by cyclosporin A (48), this inhibitor was used to determine if the TNF promoter expression induced in response to C/EBP plus PMA in Jurkat T cells was mediated by the activation of NFAT. Following transfection with CMV-C/EBP and stimulation with PMA, no inhibition of TNF promoter activation was observed by adding cyclosporin A (data not shown). In contrast, activation of the TNF promoter induced by PMA plus ionomycin, in the absence of C/EBP, was inhibited >90% by cyclosporin A, suggesting that cyclosporin A inhibited NFAT-induced TNF promoter activation, as previously described (15, 48).

Overexpression of c-Jun and TAM-67 was associated with increased TNF secretion in U937 cell lines. The results described above implicate c-Jun in the regulation of the TNF promoter but do not address whether similar mechanisms also regulate the chromosomal TNF gene. Since monocytes/macrophages are the principal source of TNF, we used the myelomonocytic cell line U937 to test whether ectopic expression of wild-type c-Jun or TAM-67 would affect the synthesis and secretion of TNF. U937 cells constitutively express C/EBP, and the concentration was increased by differentiation in response to PMA (31). Therefore, we examined TNF secretion in three independently derived stably transfected U937 cell lines overexpressing c-Jun and one line expressing TAM-67, each of which have been previously characterized (18, 45), as well as a similar line transfected only with the neomycin resistance gene. Western blots confirmed the increased wild-type c-Jun, both constitutively and following PMA-LPS treatment, in the c-jun-transfected lines compared to the control (data not shown). The TAM-67-transfected line expressed abundant TAM-67, and the wild-type c-Jun level was comparable to that in the neomycin-transfected control cell line (data not shown). Interestingly, no TNF was secreted by these lines constitutively or when the cells were differentiated with PMA alone (data not shown). However, following differentiation plus activation by LPS, TNF was secreted by each of the lines. TNF secretion was significantly increased (P < 0.02) in the lines overexpressing either wild-type c-Jun or TAM-67 compared to the control (Fig. 4), suggesting that wild-type and TAM-67 c-Jun proteins can enhance TNF protein production by myelomonocytic cells.

View larger version (51K): [in this window] [in a new window]   FIG. 4.   Overexpression of wild-type c-Jun or TAM-67 in U937 cells resulted in increased TNF secretion. U937 cell lines expressing the neomycin resistance gene alone (Neo) or together with wild-type c-Jun or with TAM-67 were cultured in complete medium at 0.5 × 106/ml. Three independent lines overexpressing c-Jun (c-Jun 1, 2, and 3) and one expressing TAM-67 (TAM-67) were used. Cells were incubated in medium alone or in medium containing PMA or PMA plus LPS. Little or no TNF was secreted when cells were cultured in medium alone or with PMA (data not shown). The data presented are the results obtained following incubation with PMA plus LPS. Supernatants were harvested at 18 h, and TNF was quantitated. The results presented are the means ± 1 SE of three independent experiments.

TAM-67-C/EBP interaction in U937 cells was associated with enhanced activation of the TNF promoter reporter. To determine if the AP-1 site centered 102/103 bp 5' of the transcription start site was involved in the enhanced activation observed in the U937 line expressing TAM-67, this line and the control U937 line were transfected with either the 120 or the 95 TNF-luciferase promoter reporters. The TAM-67 line, rather than a c-Jun-overexpressing line, was used to avoid potential activation by the wild-type c-Jun through the AP-1 site centered at 62 bp of the promoter. The 95 TNF construct does not possess the AP-1 site centered at 102/103 bp (Fig. 1) but does retain the ability to become activated by C/EBP (Fig. 3B). In the control neomycin-transfected U937 line, the 120 TNF promoter-reporter demonstrated no increased activation compared to the 95 TNF promoter reporter (Fig. 5A). In contrast, the 120 TNF construct demonstrated a fourfold activation compared to the 95 TNF construct in the U937 line stably expressing TAM-67 (Fig. 5A). This observation suggests that the enhanced activation of the TNF promoter in U937 cells overexpressing TAM-67 was mediated through the AP-1 site centered at 102/103 bp. This observation further documents that the 62 bp AP-1 site was not involved in the enhanced activation of the 120 TNF promoter reporter observed in the TAM-67 cells, similar to the results observed in the Jurkat T cells.

View larger version (32K): [in this window] [in a new window]   FIG. 5.   Effect of TAM-67 on the activation of the TNF promoter reporter in U937 cells. (A) The AP-1 binding site centered 102/103 bp 5' of the transcription start site contributes to the activation observed in TAM-67-expressing U937 cells. U937 cells stably transfected with TAM-67 were transiently transfected with either the 120 or the 95 TNF promoter reporter construct (3 µg). Sixteen hours later, the cells were harvested and RLU was determined and corrected for total protein in each lysate. The results presented are the means ± 1 SE of a transfection performed in duplicate and are representative of two experiments. (B) DN C/EBP inhibits activation of the 120 TNF promoter reporter in U937 cells stably expressing TAM-67. U937 cells stably transfected with TAM-67 were cotransfected with the 120 TNF promoter-reporter (3 µg) plus DN C/EBP (6 µg) or the CMV control vector (6 µg). Cells were harvested and analyzed as described above. The results presented represent the means ± 1 SE of an experiment performed in replicate and are representative of four experiments.

c-Jun affects the DNA binding of C/EBP to the TNF promoter. The transfection assays described above suggested that c-Jun must bind the TNF promoter to enhance C/EBP activity. To further characterize the interactions between c-Jun and C/EBP, in vitro DNA binding assays were performed with three different ³²P-labeled oligonucleotides derived from the TNF promoter. The 115/98 oligonucleotide contained only the crucial c-Jun binding site described above. The 100/74 oligonucleotide contained the C/EBP binding site but no AP-1 site, and the 115/74 oligonucleotide spanned both sites. C/EBP bound readily to the ³²P-labeled 100/74 bp oligonucleotide (Fig. 6A, lane 1). This binding has been shown to be specific since it was efficiently competed by adding an excess of either unlabeled 100/74 oligonucleotide or another C/EBP-binding oligonucleotide derived from the IL-6 promoter, and it was supershifted by monospecific antibody to C/EBP (38). Others have further narrowed the C/EBP binding site to 95 to 87 bp of the TNF promoter (50). The C/EBP binds as a broad band because it is synthesized as a mixture of three translation products of 45, 38, and 20 kDa, all of which bind DNA (1, 41). In contrast, c-Jun did not bind to this oligonucleotide (Fig. 6A, lanes 2 and 3). Addition of increasing concentrations of c-Jun resulted in reduced binding of C/EBP to the 100/74 oligonucleotide (Fig. 6A, lanes 4 and 5), similar to results reported for assay using a C/EBP-binding oligonucleotide from the IL-6 promoter (21). This inhibition was often not as complete as is apparent in Fig. 6A, although inhibition was reproducible at higher concentrations of c-Jun.

View larger version (56K): [in this window] [in a new window]   FIG. 6.   The effect of C/EBP and c-Jun interaction is DNA dependent. (A) Transcription factor c-Jun inhibits binding of C/EBP to the 100/74 but not the 115/74 oligonucleotide. TNF promoter oligonucleotides 100/74 (lanes 1 to 5) and 115/74 (lanes 6 to 10) were 32P labeled and used as probes in the gel shift assay. c-Jun (1 and 3 µg; lanes 2, 3, 7, and 8) and C/EBP (1.0 µl of baculovirus nuclear extract; lanes 1 and 7), alone or combined (lanes 4, 5, 9, and 10), were loaded on a 4% polyacrylamide gel and run under nonreducing conditions. (B) c-Jun and C/EBP form a complex binding to the 115/74 TNF promoter oligonucleotide. C/EBP (1 µl) and c-Jun (1 µg) were added alone (lanes 1 and 2, respectively) or together (lanes 3 to 6) to the 32P-labeled 115/74 oligonucleotide. Antibody to C/EBP (lane 4), control antibody (lane 5), or anti-c-Jun (lane 6) was added to the reaction mixture prior to loading on the gel. IgG, immunoglobulin G. (C) C/EBP alters the mobility of c-Jun bound to the 115/98 TNF promoter oligonucleotide. C/EBP (1 µl) and c-Jun (1 µg), alone (lanes 1 and 2) or together (lanes 3 to 5), were incubated with the 32P-labeled 115/98 oligonucleotide for 20 min at room temperature prior to running the gel. Antibodies to C/EBP and c-Jun (lanes 4 and 5) were incubated with the transcription factors for 20 min at room temperature prior to adding the radiolabeled oligonucleotide. (D) The proximity of AP-1 and C/EBP binding sites affects activation. A single copy of each of the AP-1 and C/EBP binding sites from the TNF promoter was inserted into pT81-Luc either unchanged (0 bp) or with 10 irrelevant oligonucleotides (10 bp) separating the two sites. The plasmids (5 µg) were transfected by electroporation into wild-type U937 cells, keeping the total concentration of plasmid constant (16 µg). After transfection the cells were treated with PMA and LPS as described in the text. Cells were harvested and analyzed as described in Materials and Methods. The parent plasmid was transfected in each experiment, and the results were subtracted prior to analysis. The results presented as the means ± 1 SE are representative of four independent experiments.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Our prior data indicated that C/EBP was capable of binding to the 100/74-bp region of the TNF promoter and of activating the TNF gene (38). Expression of DN C/EBP inhibited endogenous C/EBP in myelomonocytic cells, suppressing the activation of the TNF promoter (38). Since C/EBP is readily detected in the nuclei of monocytes not actively synthesizing TNF, activation of C/EBP and/or interaction with an additional transcription factor may be necessary for expression of the TNF gene. Our data demonstrate that c-Jun is capable of interacting with C/EBP to activate the TNF promoter in a unique manner that does not require the c-Jun transactivation domain.

Transient transfection of Jurkat T cells demonstrated that the macrophage-enriched nuclear transcription factor C/EBP and the ubiquitous c-Jun interacted, resulting in synergistic activation of the TNF promoter. These interactions involved the AP-1 and C/EBP binding sites, located 106/99 and 100/74 bp, respectively, 5' from the transcription start site of the TNF gene (Fig. 1). The data indicate that the enhanced activation was independent of the c-Jun transactivation domain. Both wild-type and TAM-67 c-Jun were capable of amplifying the activation observed with C/EBP, indicating that the enhancement by c-Jun did not result from the additive or synergistic effects of heterologous transactivation domains.

Studies were performed with U937 cell lines that constitutively express endogenous C/EBP to determine the potential relevance of the observations obtained with the Jurkat T cells. The overexpression of c-Jun and the expression of TAM-67 were both associated with enhanced secretion of TNF in differentiated cells stimulated with LPS, suggesting the possibility that the enhanced expression of the TNF gene in U937 cells was dependent on an interaction between c-Jun/TAM-67 and C/EBP. The results of the transient transfections in U937 cells expressing TAM-67 support a direct effect of TAM-67 and C/EBP in the activation of the TNF promoter in these cells. Alternatively, it is possible that c-Jun or TAM-67 affected differentiation or another mediator or transcription factor. Since c-Jun and TAM-67 affect differentiation differently, an effect on this process seems an unlikely explanation (18, 45). Although we cannot exclude the possibility that the effect on the TNF gene was not secondary to that of another mediator or transcription factor, this is this first characterization, to our knowledge, in which the wild-type and transactivation domain mutant c-Jun interacted similarly in the activation of a gene.

In Jurkat T cells, the synergistic effect required that c-Jun and TAM-67 bind to the AP-1 site centered 102/103 bp upstream of the transcription start site. Truncations that abolished the AP-1 binding site or mutations of c-Jun that did not bind DNA did not manifest the synergistic effect. Similarly, truncation of this AP-1 site resulted in diminished activation of the TNF promoter reporter in U937 cells expressing TAM-67. Our data indicate that the C/EBP transactivation domain was required for the enhanced activation of the TNF gene observed by the interaction between C/EBP and c-Jun/TAM-67, since DN C/EBP inhibited the activation of the TNF promoter reporter in TAM-67-expressing U937 cells. These observations, together with the proximity of the two binding sites, suggest that physical interactions between C/EBP and c-Jun/TAM-67, mediated by their leucine zippers (21), may contribute to the enhanced TNF activation.

The importance of the close proximity of these two binding sites is supported by the gel shift experiments. With the 115/74 oligonucleotide, both c-Jun and C/EBP bound preferentially to the same oligonucleotide and were supershifted by monospecific antibody against either protein. Previous studies documented the inhibition of binding by C/EBP to an IL-6 promoter oligonucleotide by c-Jun (21). Although similar inhibition of binding was noted with the shorter 100/74 TNF oligonucleotide that did not bind c-Jun, suppression of binding of C/EBP to the 115/74 oligonucleotide, capable of binding both C/EBP and c-Jun, was not observed. C/EBP binds weakly to the TNF promoter (38). Binding of c-Jun/TAM-67 at the neighboring cis site may facilitate the interaction of C/EBP and c-Jun that is mediated through their leucine zipper domains (21), stabilizing the C/EBP-DNA interaction and resulting in enhanced activation. Similar observations have been described for NF-B p65 interaction with c-Fos or c-Jun and C/EBP interaction with NF-B, despite lack of enhanced or cooperative binding to DNA in gel shift experiments (37, 42). Consistent with this possibility, insertion of 10 bp between the AP-1 and C/EBP binding sites of the TNF promoter resulted in diminished activation in PMA-LPS-treated U937 cells.

We did not find that interaction with NFAT, CBP, or p300 was responsible for the enhanced activation observed in this study. Consistent with the results observed with CBP and p300, E1A 12S demonstrated minimal or no effect on TNF promoter activity in PMA-treated Jurkat T cells and in LPS stimulated THP-1 myelomonocytic cells (28). If CBP or p300 were involved, inhibition by E1A might have been expected (4, 5, 28). Consistent with our data concerning NFAT, cyclosporin A did not inhibit TNF transcription in macrophages (34). Additionally, in studies not presented, we have observed that c-Jun also interacted with NF-B proteins to enhance TNF promoter activation. However, the transactivation domain of c-Jun was required, suggesting that the enhanced expression of TNF observed in U937 cells in response to TAM-67 was not due to interactions with NF-B. Additionally, the NF-B binding site (97 to 88 bp of the TNF gene) is partially removed in the 95 TNF promoter reporter construct, and NF-B fails to activate this promoter (data not shown).

The importance of the interactions between proteins binding to the 106/99 and 97/88 regions of the TNF promoter has been identified by others (48). ATF-2/Jun and NFATp, respectively, were identified as binding to these sites (48). Independent and noncooperative binding at these two sites was responsible for optimal activation of the TNF gene by anti-CD3 or ionomycin in Ar-5 T cells (48). Our observations concerning the interaction between c-Jun and C/EBP binding to these two regions were similar. We did not find evidence for cooperativity of binding of c-Jun and C/EBP to the 115/74 oligonucleotide or for the binding of heterodimers when the 115/98 and the 100/74 TNF oligonucleotides were independently used.

Our data suggest that the suppression observed with c-Jun in our cotransfection assays using the 120 TNF promoter reporter was due to squelching by the transactivation domain of c-Jun and not inhibition of binding to the promoter or the formation of heterodimers. The TAM-67 mutant, which lacks a transactivation domain, failed to suppress the C/EBP-induced activation of either the 120 or the 95 TNF promoter reporter in PMA-activated Jurkat T cells. This result suggests that the inhibition of C/EBP binding, as seen in the gel shift experiments using the 100/74 oligonucleotide (Fig. 6) and proposed as a mechanism of inhibition of activation of a C/EBP-driven promoter reporter (21), was not responsible for the inhibition of TNF promoter activation observed with wild-type c-Jun in Jurkat T cells in assays employing the 120 TNF promoter reporter. We cannot exclude the possibility that overexpression of c-Jun inhibited C/EBP binding to the 95 TNF promoter, since the effect of TAM-67 on C/EBP binding to the 100/74 oligonucleotide was not examined. Supporting the interpretation that interaction through the leucine zipper was not required for suppression, cotransfection of c-Jun-LZ also resulted in suppression of C/EBP-induced activation. The explanation for the lack of inhibition by the c-Jun-DBD is not clear, since wild-type c-Jun was suppressive of C/EBP-induced activation with the 95 TNF promoter-reporter, which lacked the AP-1 binding site centered at 102/103 bp. Our data do not support the consumption of CBP or p300 by c-Jun as a cause for the suppression, since their overexpression failed to reverse the inhibition. Suppression was not specific for c-Jun, however, since coexpression of c-Fos, which failed to enhance C/EBP-induced activation, also was suppressive. Since the CMV vector control did not suppress, inhibition was not due simply to promoter squelching. Suppression of TNF secretion was not observed in the U937 cell lines stably transfected with wild-type c-Jun. It is possible that this phenomenon does not occur in these cells or that the concentration of c-Jun was not high enough to allow this effect to be observed.

Although PMA was not required to activate the TNF promoter reporter constructs in U937 cells, the requirement for PMA in the Jurkat T cells deserves comment. PMA may be necessary to phosphorylate and activate C/EBP (24, 30, 46, 47, 51, 52). It is possible that the PMA induces an additional endogenous factor required for TNF activation. We did not find evidence to suggest that NFAT, CBP, or p300 was involved. Binding to the 115/74 TNF oligonucleotide was observed constitutively with nuclear extracts from Jurkat cells not treated with PMA (data not presented). This binding was not due to C/EBP and, although not further characterized, was greatly reduced following the addition of PMA, suggesting that PMA may inhibit a suppressive factor. For example, in pro-B-cell lines, CHOP was shown to bind C/EBP, inhibiting its ability to activate the Idl gene (40). As an additional mechanism, PMA may also increase the concentration of C/EBP, which was under the control of a CMV promoter. PMA resulted in a two- to fivefold increase in the expression of a CMV-luciferase promoter reporter in Jurkat T cells (data not presented). Activation of the TNF promoter was very sensitive to the concentration of C/EBP-expressing plasmid transfected, less than 5-fold at 1 µg and >40-fold at 2 µg. The ratio of the 20-kDa inhibitory and the 38- and 45-kDa stimulatory versions of C/EBP are important in the overall ability of C/EBP to become transcriptionally active within a cell. PMA may effect this ratio in favor of activation. This process is thought to be regulated at the level of mRNA translation. We have not been able to definitively ascribe the effects of PMA to any one of these mechanisms.

AP-1 and C/EBP are also known to cooperate in the regulation of other genes, including the chicken myelomonocytic growth factor (cGMF) gene and the human TSF-6 and collagenase-1 genes (11, 23, 32). An AP-1 binding site in the proximal promoter and a C/EBP