SECRETORY INTERLEUKIN-1 RECEPTOR ANTAGONIST GENE-EXPRESSION REQUIRES BOTH A PU.1 AND A NOVEL COMPOSITE NF-KAPPA-B PU.1/GA-BINDING PROTEIN-BINDING SITE/

The human secretory interleukin-1 receptor antagonist (secretory IL-1R a) gene is controlled through three lipopolysaccharide (LPS)-responsiv e promoter elements, one of which was identified as an NF-kappa B bind ing site. Sequence analysis of the secretory IL-1Ra promoter identifie d a potential PU.1 binding site located between positions -80 and -90 on the complementary strand overlapping the NF-kappa B 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 ident ified as NF-KB, and the constitutive factors were identified as PU.1 a nd GA-binding protein. Site-directed mutagenesis of the -93 to -79 pro moter region demonstrated that mutation of either the NF-kappa B 5'-ha lf site or the PU.1/GA-binding protein half-site alone did not signifi cantly decrease LPS responsiveness. However, a mutation that disrupted the binding of all three factors resulted in a 50% decrease in LPS re sponsiveness. A second PU.1 binding site centered at -230 was identifi ed by gel shift and supershift assays. Mutation of the core GGAA regio n resulted ill a 50% decrease in LPS-responsive promoter activity. Mut ation of both the distal and proximal LPS response elements led to an almost complete loss of responsiveness. These data therefore suggest t hat the regulation of IL-1Ra gene expression is a complex event involv ing the interactions of three different transcription factors with a s ingle cis-acting element and that the two PU.1 binding sites are the m ajor response elements for LPS-induced IL-1Ra gene expression.