TRANSCRIPTIONAL ACTIVATION OF THE HUMAN TNF-ALPHA PROMOTER BY SUPERANTIGEN IN HUMAN MONOCYTIC CELLS - ROLE OF NF-KAPPA-B

We have studied the transcriptional activation of the human TNF-alpha gene by the superantigen staphylococcal enterotoxin A (SEA) in the hum an premonocytic cell line THP-1. Nuclear proteins from SEA-stimulated THP-1 cells bound strongly to kappa 3, the most proximal of three puta tive NF-kappa B binding sites (kappa 1-kappa 3) found in the 5' regula tory region of the TNF-alpha gene, but only weakly to kappa 1,the most distal of the NF-kappa B binding sites, and showed no binding to kapp a 2. The mobility of the kappa 3-nucleoprotein complex was identical t o that of complexes formed between nuclear proteins and the consensus NF-kappa B sequence. Moreover, both 5' and 3' mutants of kappa 3 were unable to displace kappa 3 binding, suggesting that the kappa 3 bindin g complex induced by SEA has the characteristics of NF-kappa B. Studie s using Abs directed against the NF-kappa B subunits p50 and p65 sugge sted that both p50 and p65 bind to the kappa 3 sequence. Reporter gene assays showed that deletion of kappa 3 (-99 to -89 bp) and point muta tion of the three 5' guanine bases in the kappa 3 sequence reduced the inducibility of the TNF-alpha promoter by SEA and LPS. These results indicate that superantigen induces NF-kappa B in human monocytic cells and suggest that binding of NF-kappa B to the kappa 3 site of the TNF -alpha promoter plays an important role in the transcriptional activat ion of the TNF-alpha gene by superantigen.