Tumor necrosis factor alpha gene regulation: enhancement of C/EBPbeta-induced activation by c-Jun

Abstract

Tumor necrosis factor alpha (TNF alpha) is a key regulatory cytokine whose expression is controlled by a complex set of stimuli in a variety of cell types. Previously, we found that the monocyte/macrophage-enriched nuclear transcription factor C/EBPbeta played an important role in the regulation of the TNF alpha gene in myelomonocytic cells. Abundant evidence suggests that other transcription factors participate as well. Here we have analyzed interactions between C/EBPbeta and c-Jun, a component of the ubiquitously expressed AP-1 complex. In phorbol myristate acetate (PMA)-treated Jurkat T cells, which did not possess endogenous C/EBPbeta, expression of c-Jun by itself had relatively little effect on TNF alpha promoter activity. However, the combination of C/EBPbeta and c-Jun was synergistic, resulting in greater than 130-fold activation. This effect required both the leucine zipper and DNA binding domains, but not the transactivation domain, of c-Jun, plus the AP-1 binding site centered 102/103 bp upstream of the transcription start site in the TNF alpha promoter. To determine if C/EBPbeta and c-Jun might cooperate to regulate the cellular TNF alpha gene in myelomonocytic cells, U937 cells that possess endogenous C/EBPbeta and were stably transfected with either wild-type c-Jun or the transactivation domain deletion mutant (TAM-67) were examined. U937 cells expressing ectopic wild-type c-Jun or TAM-67 secreted over threefold more TNF alpha than the control line in response to PMA plus lipopolysaccharide. Transient transfection of the U937 cells expressing TAM-67 suggested that TAM-67 binding to the -106/-99-bp AP-1 binding site cooperated with endogenous C/EBPbeta in the activation of the -120 TNF alpha promoter-reporter. DNA binding assays using oligonucleotides derived from the TNF alpha promoter suggested that C/EBPbeta and c-Jun interact in vitro and that the interaction may be DNA dependent. Our data demonstrate that the TNF alpha gene is regulated by the interaction of the ubiquitous AP-1 complex protein c-Jun and the monocyte/macrophage-enriched transcription factor C/EBPbeta and that this interaction contributes to the expression of the cellular TNF alpha gene in myelomonocytic cells. This interaction was unique in that it did not require the c-Jun transactivation domain, providing new insight into the cell-type-specific regulation of the TNF alpha gene.

FIG. 1

Sequence of the human TNFα promoter region −123 bp upstream from the transcription start site (TSS). Previously described binding sites for C/EBPβ (−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-κB (−97 to −88 bp), and CRE (−106 to −99) are indicated on the sequence representing the first 123 bp of the TNFα promoter (13, 15, 25, 33, 38, 39, 48). The C/EBPβ 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.

FIG. 2

Both c-Jun and TAM-67 synergize with C/EBPβ to activate the TNFα promoter. (A) Effects of C/EBPβ, c-Jun, and c-Jun mutants on PMA-induced TNFα promoter activation. Jurkat T lymphocytes were transfected by the DEAE-dextran method with plasmid vectors expressing C/EBPβ, c-Jun, the c-Jun mutants c-Jun-LZ, c-Jun-DBD, and TAM-67, and the TNFα promoter reporter construct (3 μg/transfection) containing 120 bp 5′ of the transcription start site. The C/EBPβ 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/EBPβ with c-Jun or its mutants on PMA-induced activation of the TNFα promoter. Jurkat T lymphocytes were transfected with plasmid vectors expressing C/EBPβ plus c-Jun or its mutants and the −120 TNFα promoter reporter construct (3 μg). Various concentrations of control, c-Jun, or mutant c-Jun (0.25 to 3 μg) were added to C/EBPβ (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/EBPβ, 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/EBPβ 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.

FIG. 2

Both c-Jun and TAM-67 synergize with C/EBPβ to activate the TNFα promoter. (A) Effects of C/EBPβ, c-Jun, and c-Jun mutants on PMA-induced TNFα promoter activation. Jurkat T lymphocytes were transfected by the DEAE-dextran method with plasmid vectors expressing C/EBPβ, c-Jun, the c-Jun mutants c-Jun-LZ, c-Jun-DBD, and TAM-67, and the TNFα promoter reporter construct (3 μg/transfection) containing 120 bp 5′ of the transcription start site. The C/EBPβ 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/EBPβ with c-Jun or its mutants on PMA-induced activation of the TNFα promoter. Jurkat T lymphocytes were transfected with plasmid vectors expressing C/EBPβ plus c-Jun or its mutants and the −120 TNFα promoter reporter construct (3 μg). Various concentrations of control, c-Jun, or mutant c-Jun (0.25 to 3 μg) were added to C/EBPβ (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/EBPβ, 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/EBPβ 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.

FIG. 2

Both c-Jun and TAM-67 synergize with C/EBPβ to activate the TNFα promoter. (A) Effects of C/EBPβ, c-Jun, and c-Jun mutants on PMA-induced TNFα promoter activation. Jurkat T lymphocytes were transfected by the DEAE-dextran method with plasmid vectors expressing C/EBPβ, c-Jun, the c-Jun mutants c-Jun-LZ, c-Jun-DBD, and TAM-67, and the TNFα promoter reporter construct (3 μg/transfection) containing 120 bp 5′ of the transcription start site. The C/EBPβ 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/EBPβ with c-Jun or its mutants on PMA-induced activation of the TNFα promoter. Jurkat T lymphocytes were transfected with plasmid vectors expressing C/EBPβ plus c-Jun or its mutants and the −120 TNFα promoter reporter construct (3 μg). Various concentrations of control, c-Jun, or mutant c-Jun (0.25 to 3 μg) were added to C/EBPβ (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/EBPβ, 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/EBPβ 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.

FIG. 3

The AP-1 binding site is necessary for synergistic activation of the TNFα promoter. (A) Comparison of −120 and −95 TNFα promoter reporter constructs on C/EBPβ-plus-c-Jun activation in PMA-induced Jurkat T cells. Jurkat T lymphocytes were transfected with plasmid vectors expressing C/EBPβ (1 μg) and c-Jun (0.25 μg), separately or together, plus the −120 TNFα or the −95 TNFα 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 TNFα promoter reporter construct. Jurkat T cells were transfected with the −95 TNFα-luciferase promoter reporter (3 μg), the CMV-C/EBPβ 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.

FIG. 4

Overexpression of wild-type c-Jun or TAM-67 in U937 cells resulted in increased TNFα 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 TNFα 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 TNFα was quantitated. The results presented are the means ± 1 SE of three independent experiments.

FIG. 5

Effect of TAM-67 on the activation of the TNFα 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 TNFα 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/EBPβ inhibits activation of the −120 TNFα promoter reporter in U937 cells stably expressing TAM-67. U937 cells stably transfected with TAM-67 were cotransfected with the −120 TNFα promoter-reporter (3 μg) plus DN C/EBPβ (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.

FIG. 6

The effect of C/EBPβ and c-Jun interaction is DNA dependent. (A) Transcription factor c-Jun inhibits binding of C/EBPβ to the −100/−74 but not the −115/−74 oligonucleotide. TNFα 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/EBPβ (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/EBPβ form a complex binding to the −115/−74 TNFα promoter oligonucleotide. C/EBPβ (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/EBPβ (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/EBPβ alters the mobility of c-Jun bound to the −115/−98 TNFα promoter oligonucleotide. C/EBPβ (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/EBPβ 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/EBPβ binding sites affects activation. A single copy of each of the AP-1 and C/EBPβ binding sites from the TNFα 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.