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 the 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 sequence was transactivated by the p65 subunit of NF-kappa B or by treatment of the cells with a combination of NF-kappa B inducers including LPS, PMA, and dibutyryl cyclic AMP (L+P+C) in U937 cells. Coexpression of p65 with L+P+C treatment led to a synergistic response, whereas coexpression of the I kappa B alpha/MAD-3 protein, in place of p65, blocked 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 I located between -2800 to -2720 bp and region II located between -512 and -133 bp. Electrophoretic mobility shift assays confirmed that NF-kappa B-like 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/-286 bp) caused a specific loss of induction by p65 or L+P+C. A cyclic AMP response element (CRE) site (-2761/-2753 bp) in region I has been shown previously to be critical for L+P+C induction. Mutation of the CRE in an enhancerless test plasmid containing two copies of region I blocked transactivation by p65. Likewise, coexpression of I kappa B alpha 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 that NF-kappa B may play multiple roles in the induction of IL-1 beta transcription.
Department of Molecular Genetic, Glaxo Research Institute, Inc., Research Triangle Park, NC 27709.
J P
JP
Cogswell
M M
MM
Godlevski
G B
GB
Wisely
W C
WC
Clay
L M
LM
Leesnitzer
J P
JP
Ways
J G
JG
Gray
D001483:Base Sequence; D001665:Binding Sites; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; D003994:Bucladesine; D013755:Tetradecanoylphorbol Acetate