Interleukin 12 (IL-12) is a heterodimeric cytokine whose activity is critical for T-helper 1 responses. The gene for the IL-12 p40 subunit is expressed in macrophages following induction by bacterial products, and its expression is augmented by gamma interferon. In this study, we performed a functional analysis of the murine and human p40 promoters in the murine macrophage cell line RAW 264.7. Transcription from the murine p40 promoter was strongly induced by lipopolysaccharide and heat-killed Listeria monocytogenes (HKLM), but promoter activity was not enhanced by gamma interferon. Multiple cis-acting elements involved in activated transcription were identified through an extensive mutant analysis. The most critical element, whose activity is conserved in mice and humans, is located between positions ؊96 and ؊88 relative to the murine transcription start site. This element exhibits functional synergy with a previously described NF-B half-site which interacts with Rel proteins. DNase I footprinting and electrophoretic mobility shift assays demonstrated that C/EBP proteins interact with the critical element, but in nuclear extracts, cooperative binding of C/EBP and Rel proteins to their respective sites was not observed. Interestingly, promoter activity was induced by HKLM in the presence of cycloheximide, consistent with induction by posttranslational mechanisms. The results suggest that C/EBP and Rel proteins play important roles in the activation of IL-12 p40 transcription by bacteria. However, many complex interactions will need to be clarified to fully understand p40 regulation.

Interleukin (IL)-12 is a heterodimeric cytokine produced by macrophages in response to intracellular pathogens and provides an obligatory signal for the differentiation of T-helper-1 cells. We previously reported an analysis of the IL-12 p40 promoter in RAW264.7 macrophages. Multiple control elements were involved in activation of transcription by bacterial products. A critical control element, located between ؊96 and ؊88, interacts with C/EBP family members. In this study, using a strategy to demonstrate functional activity in a minimal promoter context, three novel cis-acting elements are found to have an important role in IL-12 p40 promoter activation by lipopolysaccharide. One of these elements is characterized in detail. Mutations from ؊79 to ؊74 in the murine IL-12 p40 promoter significantly reduce lipopolysaccharide-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 cooperativity between these elements. c-Jun and a mutant c-Jun molecule activate the IL-12 p40 promoter and synergistically activate the promoter when co-expressed with C/EBP␤. Finally, this region of the promoter is demonstrated to be a target for mitogen-activated protein kinase and tolllike receptor signaling pathways.