Regulation of IL-12 p40 Promoter Activity in Primary Human Monocytes:Roles of NF-#kappa#B,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

摘要

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/E8P, NF-#kappa#B, 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 GAT A core motif in unstimulated primary monocytes and RAW 264.7 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 in- ducible promoter activity in monocytes. Regulation of GAP-12 binding may thus modulate IL-12 p40 gene expression.