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 upst ream of exon 1 (5'-flanking region) and upstream of exon 2 (intron 1) regul ate its expression. Both promoter regions were cloned into pCBT-Basic plasm id 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 indu cibility when transfected into RAW 264.7 macrophages. To learn the regulato ry 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 crit ical, EMSA using an oligonucleotide probe encompassing the ICSBP binding si te showed that LPS treatment increased the formation of DNA binding complex . in addition, when supershift assays were performed, retardation of the pr otein-DNA complex was seen after the addition of anti-ICSBP Ab, For the act ivity 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 LP S treatment increased PU,1 binding activity. The addition of PU.1-specific Ab to LPS-treated nuclear extracts resulted in the formation of a supershif ted complex. Furthermore, cotransfection of ICSBP or PU,1 expression vector increased pi promoter activity or IL-18 expression, respectively. Taken to gether, these results indicate that ICSBP and PU,1 are critical elements fo r IL-18 gene expression.