Secretory Interleukin-1 Receptor Antagonist Gene Expression Requires both a PU.1 and a Novel Composite NF-κB/PU.1/ GA-binding Protein Binding Site

Abstract

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-B 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-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 identified as NF-B, 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-B 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.Interleukin 1 is an important proinflammatory cytokine produced by a wide variety of cell types and has been implicated in the pathogenesis of acute and chronic inflammatory or autoimmune diseases (1). The discovery of a naturally occurring IL-1 receptor antagonist (IL-1Ra) 1 has suggested a means through which the pathologic effects of IL-1 can be modulated (2, 3). The human IL-1Ra gene encodes two closely related proteins that act as specific antagonists of the binding of IL-1␣ and IL-1␤ to both the type I and type II IL-1 receptors (4, 5). Despite being members of the IL-1 gene family (6) and binding to the same cell surface receptors with approximately equal affinities as IL-1␣ and IL-1␤ (7, 8), the IL-1Ra proteins exert no agonist activity.IL-1Ra was originally described as a 22-26-kDa secreted product of monocytes (9) but is now known to also exist as an 18-kDa intracellular molecule originally found in the cytoplasm of epithelial cells (10). These two isoforms of IL-1Ra, termed sIL-1Ra and icIL-1Ra for the secreted and intracellular variants, respectively, are produced by alternative splicing of two distinct first exons. The first exon for icIL-1Ra is located approximately 9.6 kilobase pairs upstream of the first exon for sIL-1Ra (11) and splices into an internal site with the sequences encoding for the sIL-1Ra signal peptide. Thus, icIL1Ra contains no secretory leader peptide and remai...

Identification of a Novel IL-1 Cytokine Family Member in Teleost Fish

“…Analysis for regulatory elements within the 5Ј flanking region revealed a number of potentially important transcription factors involved in controlling the expression of this gene. However, in contrast to the IL-1␤ promoter (trout and mammals) (40) and IL-1ra promoter (63,64), no NF-B sites were discovered, consistent with the rather slow induction seen, with NF-B being a rapidly activated transcription factor induced via pattern recognition receptor recognition of pathogen-derived molecules (8). IL-1ra exists as secreted (sIL-1ra) and intracellular (icIL-1ra) molecules, driven by two independent promoters, with alternative splicing of the exon containing the signal peptide generating the two forms (65).…”

“…IL-1ra exists as secreted (sIL-1ra) and intracellular (icIL-1ra) molecules, driven by two independent promoters, with alternative splicing of the exon containing the signal peptide generating the two forms (65). The sIL-1ra promoter also requires PU.1 and GABP for LPS responsiveness (63), whereas the icIL-1ra promoter requires NF-IL-6 in addition to NF-B (64), with AP-1 driving constitutive expression (66). Multiple NF-IL-6 and AP-1 sites were identifiable in the nIL-1F promoter region, although in contrast to the icIL-1ra promoter there was a TATA box, also present in the sIL-1ra promoter (65), suggesting conventional transcriptional initiation of the nIL-1F gene.…”

NF-κB Down-regulates Expression of the B-lymphoma Marker CD10 through a miR-155/PU.1 Pathway

“…2B). Although the core sequence GAGGAA is known to be required for efficient PU.1 binding, bases outside of this core motif can also affect proper PU.1 binding (48,49). Mutation of the PU-1 binding site only partially reduced PU.1-induced activation of the CD10 promoter (Fig.…”

Transcriptional Regulation of Intracellular IL-1 Receptor Antagonist Gene by IL-1α in Primary Mouse Keratinocytes

“…We identified p65 as the transcription factor binding to the NK-B site on the icIL-1Ra promoter in keratinocytes treated with IL-1␣. Smith et al (32) demonstrated that the NF-B site is the regulatory sequence responsible for the induction of sIL-1Ra by LPS. Supershift experiments showed that in keratinocytes the binding complex contains the p65/p65 homodimer.…”

“…Twenty micrograms of RNA were separated on a formaldehyde-containing 1% agarose gel, transferred onto nylon membranes (Micron Separations, Westboro, MA), and UV cross-linked to the membrane using a Stratalinker (Stratagene, La Jolla, CA). cDNA for the C/EBPs and GAPDH were radiolabeled with [␣- 32 P]dCTP using the Decaprime II random prime kit (Ambion, Austin, TX). The blot was prehybridized (5ϫ SSC phosphate/EDTA (SSPE), 5ϫ Denhardt's solution, 150 g/ml denatured salmon sperm DNA, 0.5% SDS, and 10% dextran sulfate) for 3 h and hybridized for 20 h at 68°C.…”