TUMOR-NECROSIS-FACTOR-ALPHA GENE-REGULATION - ENHANCEMENT OF C EBP-BETA-INDUCED ACTIVATION BY C-JUN/

Tumor necrosis factor alpha (TNF alpha) is a key regulatory cytokine w hose expression is controlled by a complex set of stimuli in a variety of cell types. Previously, we found that the monocyte/macrophage-enri ched nuclear transcription factor C/EBP beta played an important role in the regulation of the TNF alpha gene in myelomonocytic cells. Abund ant evidence suggests that other transcription factors participate as well. Here we have analyzed interactions between C/EBP beta and c-Jun, a component of the ubiquitously expressed AP-1 complex. In phorbol my ristate acetate (PMA)-treated Jurkat T cells, which did not possess en dogenous C/EBP beta, expression of c-Jun by itself had relatively litt le effect on TNF alpha promoter activity. However, the combination of C/EBP beta and c-Jun was synergistic, resulting in greater than 130-fo ld activation. This effect required both the leucine zipper and DNA bi nding domains, but not the transactivation domain, of c-Jun, plus the AP-1 binding site centered 102/103 bp upstream of the transcription st art site in the TNF alpha promoter. To determine if C/EBP beta and c-J un might cooperate to regulate the cellular TNF alpha gene in myelomon ocytic cells, U937 cells that possess endogenous C/EBP beta and were s tably transfected with either wild-type c-Jun or the transactivation d omain deletion mutant (TAM-67) were examined. U937 cells expressing ec topic wild type c-Jun or TAM-67 secreted over threefold more TNF alpha than the control line in response to PMA plus lipopolysaccharide. Tra nsient transfection of the U937 cells expressing TAM-67 suggested that TAM-67 binding to the -106/-99-bp AP-1 binding site cooperated with e ndogenous C/EBP beta in the activation of the -120 TNF alpha promoter- reporter. DNA binding assays using oligonucleotides derived from the T NF alpha promoter suggested that C/EBP beta and c-Jun interact in vitr o and that the interaction may be DNA dependent. Our data demonstrate that the TNF alpha gene is regulated by the interaction of the ubiquit ous AP-1 complex protein c-Jun and the monocyte/macrophage-enriched tr anscription factor C/EBP beta 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 tra nsactivation domain, providing new insight into the fell-type-specific regulation of the TNF alpha gene.