CCAAT/enhancer binding protein is involved in the expression of the tumour necrosis factor gene in human monocytes

Within the human TNF promoter we have identified two sites at positions -189 and -101 that show C/EBP specific binding of nuclear proteins from cells of the human monocytic line Mono Mac 6. Supershift analysis with anti C/EBP antibodies revealed that the complexes formed consist of both C/EBP alpha and C/EBP beta. When studying reporter constructs with a 5'-deletion series of the TNF promoter in cotransfection experiments with a C/EBP beta expression plasmid, a construct with the -1064 TNF fragment gave 26-fold transactivation, the -630 fragment showed 23-fold transactivation and the -107 fragment (containing the -101 C/EBP binding motif) still gave 16-fold transactivation. Mutagenesis of the -101 site in the -630 construct resulted in a reduction of C/EBP driven transactivation from 26-fold to 7-fold. Finally, when Mono Mac 6 cells were transfected with these constructs, stimulation by LPS induced a 19-fold transactivation in the -630 wild type construct, while the -630 construct carrying the -101 mutation was transactivated only 4-fold. Hence, the data indicate that the -101 C/EBP motif is crucial for TNF gene expression in human monocytes.