Regulation of IL-12 p40 Promoter Activity in Primary Human Monocytes: Roles of NF-κB, CCAAT/Enhancer-Binding Protein β, and PU.1 and Identification of a Novel Repressor Element (GA-12) That Responds to IL-4 and Prostaglandin E2

Abstract

Appropriate regulation of IL-12 expression is critical for cell-mediated immune responses. In the present study, we have analyzed the regulation of IL-12 p40 promoter activity in primary human monocytes in vivo. Accordingly, we analyzed the p40 promoter by in vivo footprinting in resting and activated primary human blood CD14+ monocytes. Interestingly, footprints at binding sites for trans-activating proteins such as C/EBP, NF-κB, and ETS were only found upon stimulation with LPS and IFN-γ. In contrast, a footprint over a purine-rich sequence at −155, termed GA-12 (GATA sequence in the IL-12 promoter), was observed in resting, but not activated, cells. Further characterization of this site revealed specific complex formation at a protected GATA core motif in unstimulated primary monocytes and RAW264.7 macrophages. Mutagenesis within the GA-12 sequence caused a significant up-regulation of inducible IL-12 p40 promoter activity in both transient and stable transfection systems, suggesting a repressor function of this site. Furthermore, binding activity of the GA-12 binding protein GAP-12 was increased by treatment with two potent inhibitors of IL-12 expression, IL-4 and PGE2. Finally, we observed that IL-4-mediated repression of IL-12 p40 promoter activity is critically dependent on an intact GA-12 sequence. In summary, our data underline the complex regulation of the human IL-12 p40 promoter and identify GA-12 as a potent, novel repressor element that mediates IL-4-dependent suppression of inducible promoter activity in monocytes. Regulation of GAP-12 binding may thus modulate IL-12 p40 gene expression.

Dexamethasone Inhibits IL-12p40 Production in Lipopolysaccharide-Stimulated Human Monocytic Cells by Down-Regulating the Activity of c-Jun N-Terminal Kinase, the Activation Protein-1, and NF-κB Transcription Factors

“…Conversely, mutagenesis of the AP-1 site alone in this IL-12p40 promoter construct failed to abrogate luciferase activity (data not shown). These results are contrary to the observations provided by Becker et al (25) whereby mutations of NF-B, Ets, and C/EBP sites reduced IL-12p40 promoter activity even though the AP-1 site was left intact. Although the reasons for this discrepancy are not clear, it appears that IL-12p40 gene transcription may be subject to differential regulation depending on the cell type and the nature of the external stimuli used.…”

“…In contrast, Ma et al (22,23) demonstrated the role of the Ets-2 transcription factor in stimulation of human IL-12p40 (hIL-12p40) promoter in IFN-␥-and LPS-stimulated murine RAW264 monocytic cells. Recently, several other studies have reported the involvement of IFN-␥ regulatory factors, NF-B, Ets-2, and PU.1 transcription factors in IL12p40 regulation (25,27).…”

Interferon Regulatory Factor-8 Is Indispensable for the Expression of Promyelocytic Leukemia and the Formation of Nuclear Bodies in Myeloid Cells

“…The interaction between IRF-8 and IRF-1, both of which are IFN-␥-inducible, together with PU.1, which is an hematopoietic specific factor expressed constitutively in these cells, is a paradigm for the expression of many genes in mature macrophages like the phagocytic oxidase subunits (p67 and gp91) (44), IL-1␤ (45), ISG15 (32), Nramp1 (31), , and IL-12 (47,48). This complex is therefore characteristic of enhanceosomes of many IFN-␥ induced genes in mature macrophages.…”

MAPK-activated Protein Kinase 2 Differentially Regulates Plasmodium falciparum Glycosylphosphatidylinositol-induced Production of Tumor Necrosis Factor-α and Interleukin-12 in Macrophages

“…GAP-12, to the GATA sequence (GA-12) in the IL-12p40 promoter has been shown to repress IL-12p40 promoter activity (37,38). To determine the role of GAP-12 in GPI-induced IL-12 regulation, EMSA was performed using a DNA probe containing the GATA sequence (GA-12) of the IL-12p40 promoter.…”