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.
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.
Smith
M F
MF
Department of Medicine, Division of Gastroenterology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA. mfs3k@virginia.edu
Carl
V S
VS
Lodie
T
T
Fenton
M J
MJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051268:GA-Binding Protein Transcription Factor; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012795:Sialoglycoproteins / Q000096:biosynthesis / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D000099232:Proto-Oncogene Protein Spi-1
D004268:DNA-Binding Proteins; D051268:GA-Binding Protein Transcription Factor; D053590:Interleukin 1 Receptor Antagonist Protein; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C507763:IL1RN protein, human; C507764:Il1rn protein, mouse