LPS-induced expression of the human IL-1 receptor antagonist gene is controlled by multiple interacting promoter elements.
Expression of the IL-1 receptor antagonist (IL-1ra) can be induced by treatment of monocytes and macrophages with LPS. We have previously demonstrated that the most proximal 294 bp of the human IL-1ra promoter are sufficient for full basal activity and LPS responsiveness. In the present study, we demonstrate the presence of one inhibitory and three positive-acting LPS response elements (LRE) within this proximal 294-bp IL-1ra promoter fragment. By both 5'-deletional analysis and heterologous promoter studies, an element between -294 and -250 was found to mask the LPS response. By 5'-deletional analysis and heterologous promoter experiments, two positive-acting LRE were identified between -250 and -200 (LRE3) and -200 and -148 (LRE2) which exhibited cooperativity in that neither element alone was active. Furthermore, LRE2 also cooperated with a more proximal site between -148 and -31 (LRE1), which also was not active alone. LRE1 was identified as an NF-kappa B-binding site. Site-directed mutagenesis of this site, located between -93 and -84, resulted in a > 50% decrease in the LPS responsiveness of the 294-bp promoter. By electrophoretic mobility shift assays, with or without specific antisera to members of the rel/NF-kappa B family, the complex binding to LRE1 was shown to contain primarily NF-kappa B1/p50 and lesser amounts of RelA/p65. These results indicate that the net activation of the human IL-1ra promoter in response to LPS involves the functional interaction of at least four cis-acting DNA elements within the proximal 294 bp.
Smith
M F
MF
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Eidlen
D
D
Arend
W P
WP
Gutierrez-Hartmann
A
A
D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000502:physiology; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D012795:Sialoglycoproteins / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012795:Sialoglycoproteins; D014157:Transcription Factors