Characterization of an activation protein-1-binding site in the murine interleukin-12 p40 promoter - Demonstration of novel functional elements by a reductionist approach

Interleukin (IL)-12 is a heterodimeric cytokine produced by macrophages in response to intracellular pathogens and provides an obligatory signal for t he differentiation of T-helper-l cells. We previously reported an analysis of the IL-12 p40 promoter in RAW264.7 macrophages, Multiple control element s were involved in activation of transcription by bacterial products. A cri tical control element, located between -96 and -88, interacts with C/EBP fa mily members. In this study, using a strategy to demonstrate functional act ivity in a minimal promoter context, three novel cis-acting elements are fo und to have an important role in IL-12 p40 promoter activation by lipopolys accharide. One of these elements is characterized in detail. Mutations from -79 to -74 in the murine IL-12 p40 promoter significantly reduce lipopolys accharide-induced promoter activity. Electrophoretic mobility shift assays demonstrate binding of AP-1 family members to this region. Spacing between the C/EBP and AP-1 site is important for promoter activation, suggesting co operativity between these elements. c-Jun and a mutant c-Jun molecule activ ate the IL-12 p40 promoter and synergistically activate the promoter when c o-expressed with C/EBP beta. Finally, this region of the promoter is demons trated to be a target for mitogen-activated protein kinase and toll-like re ceptor signaling pathways.