MULTIPLE CONTROL ELEMENTS MEDIATE ACTIVATION OF THE MURINE AND HUMAN INTERLEUKIN-12 P40 PROMOTERS - EVIDENCE OF FUNCTIONAL SYNERGY BETWEEN C EBP AND REL PROTEINS/

Interleukin 12 (IL-12) is a heterodimeric cytokine whose activity is c ritical for T-helper 1 responses. The gene for the IL-12 p40 subunit i s 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 hea t-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 a nalysis. The most critical element, whose activity is conserved in mic e and humans, is located between positions -96 and -88 relative to the murine transcription start site. This element exhibits functional syn ergy with a previously described NF-kappa B half-site which interacts with Rel proteins, DNase I footprinting and electrophoretic mobility s hift assays demonstrated that C/EBP proteins interact with the critica l element, but in nuclear extracts, cooperative binding of C/EBP and R el proteins to their respective sites was not observed. Interestingly, promoter activity was induced by HKLM in the presence of cycloheximid e, consistent with induction by posttranslational mechanisms. The resu lts suggest that C/EBP and Rel proteins play important roles in the ac tivation of IL-12 p40 transcription by bacteria. However, many complex interactions will need to be clarified to fully understand p40 regula tion.