Secretory interleukin-1 receptor antagonist gene expression requires both a PU.1 and a novel composite NF-kappaB/PU.1/ GA-binding protein binding site

• Published in: The Journal of biological chemistry Vol. 273; no. 37; p. 24272
• Main Authors: Smith, Jr, M F, Carl, V S, Lodie, T, Fenton, M J
• Format: Journal Article
• Language: English
• Published: United States 11.09.1998
• Subjects: Animals; Base Sequence; Binding Sites; Cell Line; Cell Nucleus - metabolism; DNA-Binding Proteins - metabolism; GA-Binding Protein Transcription Factor; Humans; Interleukin 1 Receptor Antagonist Protein; Lipopolysaccharides - pharmacology; Macrophages; Mice; Mutagenesis, Site-Directed; NF-kappa B - metabolism; Nuclear Proteins - metabolism; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Proto-Oncogene Proteins - metabolism; Recombinant Proteins - biosynthesis; Sialoglycoproteins - biosynthesis; Sialoglycoproteins - genetics; Trans-Activators - metabolism; Transcription Factors - metabolism; Transfection
• ISSN: 0021-9258

The human secretory interleukin-1 receptor antagonist (secretory IL-1Ra) gene is controlled through three lipopolysaccharide (LPS)-responsive promoter elements, one of which was identified as an NF-kappaB binding site. Sequence analysis of the secretory IL-1Ra promoter identified a potential PU.1 binding site located between positions -80 and -90 on the complementary strand overlapping the NF-kappaB site. Gel shift analysis using this potential binding site with nuclear extracts from RAW 264.7 macrophages demonstrated the formation of three complexes, one LPS-inducible and two constitutive. The inducible factor was identified as NF-kappaB, and the constitutive factors were identified as PU.1 and GA-binding protein. Site-directed mutagenesis of the -93 to -79 promoter region demonstrated that mutation of either the NF-kappaB 5'-half site or the PU.1/GA-binding protein half-site alone did not significantly decrease LPS responsiveness. However, a mutation that disrupted the binding of all three factors resulted in a 50% decrease in LPS responsiveness. A second PU.1 binding site centered at -230 was identified by gel shift and supershift assays. Mutation of the core GGAA region resulted in a 50% decrease in LPS-responsive promoter activity. Mutation of both the distal and proximal LPS response elements led to an almost complete loss of responsiveness. These data therefore suggest that the regulation of IL-1Ra gene expression is a complex event involving the interactions of three different transcription factors with a single cis-acting element and that the two PU.1 binding sites are the major response elements for LPS-induced IL-1Ra gene expression.