Regulation of IL-12 p40 promoter activity in primary human monocytes: roles of NF-kappaB, CCAAT/enhancer-binding protein beta, and PU.1 and identification of a novel repressor element (GA-12) that responds to IL-4 and prostaglandin E(2).
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-kappaB, and ETS were only found upon stimulation with LPS and IFN-gamma. 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 PGE(2). 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.
Becker
C
C
Laboratory of Immunology, First Medical Clinic, University of Mainz, Germany.
Wirtz
S
S
Ma
X
X
Blessing
M
M
Galle
P R
PR
Neurath
M F
MF
D000818:Animals; D001483:Base Sequence; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D018983:DNA Footprinting; D017931:DNA Primers / Q000235:genetics; D015232:Dinoprostone / Q000494:pharmacology; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics*; D015847:Interleukin-4 / Q000494:pharmacology; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D051379:Mice; D009000:Monocytes / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D000099232:Proto-Oncogene Protein Spi-1
D022782:CCAAT-Enhancer-Binding Protein-beta; D017931:DNA Primers; D015232:Dinoprostone; D018664:Interleukin-12; D015847:Interleukin-4; D018950:Lipopolysaccharide Receptors; D016328:NF-kappa B; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1