3 NF-KAPPA-B SITES IN THE I-KAPPA-B-ALPHA PROMOTER ARE REQUIRED FOR INDUCTION OF GENE-EXPRESSION BY TNF-ALPHA

NF-kappa B was first identified as a postive regulator which bound to a 10 bp sequence in the first intron of the lgk light chain gene. Furt her characterization of this transcription factor has revealed that NF -kappa B is kept from binding to its consensus sequence by its inhibit or, IkB-alpha, which retains NF-kappa B in the cytoplasm. Upon receivi ng various extra- and intracellular signals, I kappa B-alpha is rapidl y degraded and NF-kappa B is induced to translocate into the nucleus. This process precedes the rapid induction of I kappa B-alpha mRNA and protein. To understand how I kappa B-alpha is replenished, we have clo ned and sequenced the 5' flanking region of the I kappa B-alpha gene a nd have identified the transcription start site and three NF-kappa B s ites in this region. Further characterization of these NF-KB sites sho w that they have different affinities for three specific protein compl exes which we identify here to consist of various members of the Rel f amily. In transient assays, cotransfection with a p65 expression vecto r is able to activate an I kappa B-alpha promoter-CAT reporter constru ct and all three NF-kappa B sites are required for full activation of the I kappa B-alpha gene following stimulation with TNF-alpha. Our dat a confirm a transcriptional autoregulatory loop involved in maintainin g appropriate NF-kappa B and I kappa B-alpha levels in the cell.