CELL-TYPE-SPECIFIC REGULATION OF THE HUMAN TUMOR-NECROSIS-FACTOR-ALPHA GENE IN B-CELLS AND T-CELLS BY NFATP, AND ATF-2 JUN/

The human tumor necrosis factor alpha (TNF-alpha) gene is one of the e arliest genes transcribed after the stimulation of a B cell through it s antigen receptor or via the CD40 pathway. In both cases, induction o f TNF-alpha gene transcription can be blocked by the immunosuppressant s cyclosporin A and FK506, which suggested a role for the NFAT family of proteins in the regulation of the gene in B cells, Furthermore, in T cells, two molecules of NFATp bind to the TNF-alpha promoter element kappa 3 in association with ATF-2 and Jun proteins bound to an immedi ately adjacent cyclic AMP response element (CRE) site. Here, using the murine B-cell lymphoma cell line A20, we show that the TNF-alpha gene is regulated in a cell-type-specific manner. In A20 B cells, the TNF- alpha gene is not regulated by NFATp bound to the kappa 3 element. Ins tead, ATF-2 and Jun proteins bind to tile composite kappa 3/CRE site a nd NFATp binds to a newly identified second NFAT site centered at -76 nucleotides relative to the TNF-alpha transcription start site. This n ew site plays a critical role in the calcium-mediated, cyclosporin A-s ensitive induction of TNF-alpha in both A20 B cells and Ar-5 T cells. Consistent with these results, quantitative DNase footprinting of the TNF-alpha promoter using increasing amounts of recombinant NFATp demon strated that the -76 site binds to NFATp with a higher affinity than t he kappa 3 site. Two other previously unrecognized NFATp-binding sites in the proximal TNF-alpha promoter were also identified by this analy sis. Thus, through the differential use of the same promoter element, the composite kappa 3/CRE site, the TNF-alpha gene is regulated in a c ell-type-specific manner in response to the same extracellular signal.