A Prominent Role for Sp1 During Lipopolysaccharide- Mediated Induction of the IL-10 Promoter in Macrophages

Abstract

IL-10 is an antiinflammatory cytokine secreted by activated macrophages and Th2 cells. IL-10 secretion promotes the down-regulation of proinflammatory cytokine synthesis and the development of Th2 responses. In macrophages, proinflammatory cytokines appear to be induced by similar mechanisms, but the IL-10 induction mechanisms have not been examined. We have analyzed the murine IL-10 promoter in the RAW264.7 macrophage line activated with LPS. A comprehensive mutant analysis revealed only one element upstream of the core promoter that was essential for promoter induction. A refined mutant analysis localized this element to nucleotides −89 to −78, and gel shift experiments revealed that it represents a nonconsensus binding site for Sp1. The functional relevance of Sp1 was supported by the high affinity of the interaction, the close correlation between the nucleotides required for Sp1 binding and promoter function, and the ability of an Sp1 consensus sequence to substitute for the −89/−78 promoter sequence. Evidence that Sp1 may be a target of signaling pathways involved in IL-10 induction was provided by the exclusive requirement for the Sp1 binding site, by the ability of the Sp1 site to confer induction to a heterologous promoter, and by the delineation of an Sp1 domain that can mediate induction. No relevant contribution from Rel, C/EBP (CCAAT/enhancer-binding protein), or AP-1 binding sites, which regulate most proinflammatory cytokine promoters, was observed. Together, these results demonstrate that IL-10 gene regulation is distinct from the regulation of proinflammatory cytokine genes, and suggest that Sp1 may be a central mediator of IL-10 induction.

Sp1 binds to the G allele of the−1087 polymorphism in the IL-10 promoter and promotes IL-10 mRNA transcription and protein production

“…LPS-induced activation of Sp1 expression is involved in the regulation of the transcription of the human IL-10 gene [34][35][36][37] and one Sp1-binding site was identified in the region immediately upstream of À571 in the IL-10 promoter. 38 LPS activates the three mitogen-activated protein kinase (MAPK9) pathways ERK, JNK and p38 and, thereby, transcription factors that are involved in gene regulation.…”

Regulation of cytokine production during phagocytosis of apoptotic cells

“…However, the kinetics of its induction differs from those of the proinflammatory mediators [63,68,97]. Recent molecular analyses of the murine IL-10 promoter show that IL-10 transcription in macrophage cell types can be regulated by constitutive and ubiquitous transcription factors such as Sp1 and Sp3, suggesting that IL-10 may be produced at low levels constitutively to maintain certain level of control over "baseline" inflammation [98,99]. Another study provided evidence that post-transcriptional regulation of IL-10 gene expression through sequences in the 3'-untranslated region of the IL-10 mRNA contributes to its overall production as well [100,101].…”

STAT‐1‐mediated repression of monocyte interleukin‐10 gene expression in vivo

“…The successful host response to invading pathogens is a careful balance, and while an excessive pro-inflammatory response has negative consequences for the host, overabundant IL-10 can compromise inflammation designed to clear invading pathogens [17]. In vitro, two transcription factors designated SP1 and SP3 have been implicated as important for the regulation of IL-10, suggesting that IL-10 mRNA may be constitutively produced under the influence of SP1/SP3 transcriptional elements and largely regulated by post-transcriptional control mechanisms such as 3 0 mRNA instability sequences present in the IL-10 mRNA transcript [18][19][20]. In addition to these observations, further regulation of IL-10 was suggested by in vitro work that observed decreases in IL-10 production following removal of a putative STAT-3-binding site at position -120 in the IL-10 promoter [21].…”