NF-KAPPA-B REGULATES IL-1-BETA TRANSCRIPTION THROUGH A CONSENSUS NF-KAPPA-B BINDING-SITE AND A NONCONSENSUS CRE-LIKE SITE

In these studies, we show that NF-kappa B induces transcription from t he human pro-IL-1 beta (IL-1 beta) gene. A recombinant plasmid pIL-1(- 4000)-CAT, containing 4 kb of the IL-1 beta gene upstream regulatory s equence was transactivated by the p65 subunit of NF-kappa B or by trea tment of the cells with a combination of NF-kappa B inducers including LPS, PMA, and dibutyryl cyclic AMP (L+P+C) in U937 cells. Coexpressio n of p65 with L+P+C treatment led to a synergistic response, whereas c oexpression of the I kappa B alpha/MAD-3 protein, in place of p65, blo cked L+P+C induction. A series of 5' deletion mutants of the IL-1 beta promoter were used to define two p65 response regions: region located between -2800 to -2720 bp and region II located between -512 and -133 bp. Electrophoretic mobility shift assays confirmed that NF-kappa B-l ike proteins could bind to two consensus binding sites in region II. A site-specific mutation in only one of these NF-kappa B sites (-296/-2 86 bp) caused a specific loss of induction by p65 or L+P+C. A cyclic A MP response element (CRE) site (-2761/-2753 bp) in region I has been s hown previously to be critical for L+P+C induction. Mutation of the CR E in an enhancerless test plasmid containing two copies of region I bl ocked transactivation by p65. Likewise, coexpression of I kappa B alph a inhibited CRE-dependent L+P+C induction of the wild-type counterpart . These data show that NF-kappa B regulates a nonconsensus CRE site in addition to the consensus binding site at -296/-286 bp and suggest th at NF-kappa B may play multiple roles in the induction of IL-1 beta tr anscription.