Characterization of an Activation Protein-1-binding Site in the Murine Interleukin-12 p40 Promoter

Abstract

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.

Inhibition of Interleukin-12 p40 Transcription and NF-κB Activation by Nitric Oxide in Murine Macrophages and Dendritic Cells

“…LPS signals in macrophages and dendritic cells through TLR4. In our previous study, dominant negative signal transduction molecules downstream of TLRs (MyD88, IRAK, and TRAF6) inhibited LPSinduced activity of the IL-12 p40 promoter in RAW 264.7 cells (18). In the present study, NO inhibited IRAK activity and disrupted the interaction of IRAK with TRAF6 in activated macrophages.…”

“…Labeled probes were purified with Nuctrap purification columns (Roche Applied Science). Electrophoretic mobility shift assays were performed as described previously (18), using 10 5 cpm probe and 5 g of nuclear extracts per reaction. Supershift experiments were performed as described previously (9) by using 2 l of antibodies to c-Rel, NF-B p50, and NF-B p65 (Santa Cruz Biotechnology).…”

Morphine Withdrawal Inhibits IL-12 Induction in a Macrophage Cell Line through a Mechanism That Involves cAMP

“…We further show that Rp cAMP, a cAMP antagonist, significantly reversed morphine-induced inhibition in a concentration-dependent manner. It is also well documented that cAMP and cAMP-elevating agents are known to decrease the production of IL-12 (11,19,27). Taken together, it can be speculated that it is the ability of morphine withdrawal to increase the intracellular concentrations of cAMP, which ultimately decreases IL-12 production possibly via a NF-B pathway.…”

“…The production of IL-12p40 in both macrophages and dendritic cells is transcriptionally regulated by key transcription factors which include AP-1, PU.1, C/EBP, and NF-B (22)(23)(24)(25)(26)(27). Recent studies (21) show that NF-B activation is essential for LPS-induced IL-12 production and mutation of the IL-12 promoter at the NF-B site significantly reduced IL-12 transcription (21).…”

Toward an Understanding of the Gene-Specific and Global Logic of Inducible Gene Transcription

“…Electrophoretic mobility shift assays, DNase I footprinting, and other assays could then be used to identify transcription factors that bind the important elements. Using a functional assay in which macrophage cell lines were transfected with plasmids containing the Il12b promoter fused to a luciferase or chloramphenicol acetyltransferase gene, followed by lipopolysaccharide (LPS) stimulation of the transfected cells, putative binding sites for NF-kB, C/EBP, AP-1, and NFAT were identified (Plevy et al 1997;Zhu et al 2001Zhu et al , 2003. Subsequent studies led to the identification of an Il12b enhancer located 10 kb upstream of the transcription start site that also appears to contribute to Il12b induction (Zhou et al 2007).…”