Roles of IFN consensus sequence binding protein and PU.1 in regulating IL-18 gene expression

IL-18 is expressed from a variety of cell types. Two promoters located upstream of exon 1 (5'-flanking region) and upstream of exon 2 (intron 1) regulate its expression. Both promoter regions were cloned into pCBT-Basic plasmid to yield p1-2686 for the 5'-flanking promoter and p2-2.3 for the intron 1 promoter. Both promoters show ed basal constitutive activity and LPS inducibility when transfected into RAW 264.7 macrophages. To learn the regulatory elements of both promoters. 5'-serial deletion and site-directed mutants were prepared. For the activity of the p1-2686 promoter, the IFN consensus sequence binding protein (ICSBP) binding site between -39 and -22 was critical, EMSA using an oligonucleotide probe encompassing the ICSBP binding site showed that LPS treatment increased the formation of DNA binding complex. in addition, when supershift assays were performed, retardation of the protein-DNA complex was seen after the addition of anti-ICSBP Ab, For the activity of the p2-2.3 promoter, the PU,I binding site between -31 and -13 was important. EMSA using a PU.1-specific oligonucleotide demonstrated that LPS treatment increased PU,1 binding activity. The addition of PU.1-specific Ab to LPS-treated nuclear extracts resulted in the formation of a supershifted complex. Furthermore, cotransfection of ICSBP or PU,1 expression vector increased pi promoter activity or IL-18 expression, respectively. Taken together, these results indicate that ICSBP and PU,1 are critical elements for IL-18 gene expression.