Zagariya A, Mungre S, Lovis R, Birrer M, Ness S, Thimmapaya B, Pope R
Department of Medicine, and Veterans Administration Lakeside Medical Center, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Mol Cell Biol. 1998 May;18(5):2815-24. doi: 10.1128/MCB.18.5.2815.
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.
肿瘤坏死因子α（TNFα）是一种关键的调节性细胞因子，其表达受多种细胞类型中一系列复杂刺激的控制。此前，我们发现富含单核细胞/巨噬细胞的核转录因子C/EBPβ在髓单核细胞中TNFα基因的调控中起重要作用。大量证据表明其他转录因子也参与其中。在此，我们分析了C/EBPβ与c-Jun之间的相互作用，c-Jun是普遍表达的AP-1复合物的一个组成部分。在佛波酯（PMA）处理的Jurkat T细胞中，该细胞不具有内源性C/EBPβ，单独的c-Jun表达对TNFα启动子活性影响相对较小。然而，C/EBPβ与c-Jun的组合具有协同作用，导致激活超过130倍。这种效应需要c-Jun的亮氨酸拉链和DNA结合结构域，但不需要其反式激活结构域，以及TNFα启动子中转录起始位点上游102/103 bp处的AP-1结合位点。为了确定C/EBPβ和c-Jun是否可能协同调节髓单核细胞中的细胞TNFα基因，我们检测了具有内源性C/EBPβ且稳定转染了野生型c-Jun或反式激活结构域缺失突变体（TAM-67）的U937细胞。在PMA加脂多糖刺激下，表达异位野生型c-Jun或TAM-67的U937细胞分泌的TNFα比对照细胞系多三倍以上。对表达TAM-67的U937细胞进行瞬时转染表明，TAM-67与-106/-99-bp AP-1结合位点的结合与内源性C/EBPβ协同激活-120 TNFα启动子报告基因。使用源自TNFα启动子的寡核苷酸进行的DNA结合分析表明，C/EBPβ和c-Jun在体外相互作用，且这种相互作用可能依赖于DNA。我们的数据表明，TNFα基因受普遍存在的AP-1复合物蛋白c-Jun与富含单核细胞/巨噬细胞的转录因子C/EBPβ相互作用的调控，这种相互作用有助于髓单核细胞中细胞TNFα基因的表达。这种相互作用的独特之处在于它不需要c-Jun的反式激活结构域，为TNFα基因的细胞类型特异性调控提供了新的见解。
