NFκB and Sp1 Elements Are Necessary for Maximal Transcription of Toll-like Receptor 2 Induced byMycobacterium avium

Abstract

We have previously reported that Toll-like receptor (TLR) 2 mRNA was induced after infection with Mycobacterium avium. To investigate the molecular basis of TLR2 expression in macrophages, we cloned and analyzed the murine putative 5′-proximal promoter. Transient transfection of a 326-bp region from nucleotides −294-+32 relative to the first transcription start site was sufficient to induce maximal luciferase activity at the basal level and after infection with M. avium in J774A.1 cells. Sequence analysis showed that the region lacked a TATA box but contained two typical stimulating factor (Sp) 1 sites, two NF-κB sites, one IFN-regulatory factor site and one AP-1 site. Site-directed mutagenesis revealed that the NF-κB and Sp1 sites but not the IFN-regulatory factor site or the AP-1 site contributed to the basal level and the induction of luciferase activity during M. avium infection. Binding of Sp1/Sp3 and NF-κB (p50/p65) was confirmed by EMSA. Further studies showed that three copies of Sp1 elements or NF-κB elements are not sufficient to confer M. avium induction on a heterologous promoter. By contrast, overexpression of NF-κB p65 caused a strong increase in transcription from an intact TLR2 promoter, whereas it caused only a partial increase in promoter activity when cotransfected with the TLR2 promoter with one of the Sp1 sites mutated. Sp1 and NF-κB were the minimum mammalian transcription factors required for effective TLR2 transcriptional activity when transfected into Drosophila Schneider cells. Together, these data provide genetic and biochemical evidence for NF-κB as well as Sp1 in regulating TLR2 transcription.

The Toll-Like Receptor 5 Stimulus Bacterial Flagellin Induces Maturation and Chemokine Production in Human Dendritic Cells

“…For example, the human TLR2 gene is constitutively expressed in monocytes and is not inducible by microbial pathogens. In contrast, the mouse TLR2 gene, which has NF-B and STAT5 transcription factor binding sites in its proximal promoter, is rapidly induced by microbial stimuli (57). The human TLR3 gene is constitutively and selectively expressed in myeloid DCs, whereas the mouse TLR3 gene is inducible and expressed in macrophages.…”

“…54) clarified the species-specific variations of TLR expression in mice and humans. Recent data from several groups suggest that constitutive and inducible TLR expression in different cell types and in different species are controlled by transcriptional regulation (55)(56)(57). Moreover, gene regulatory elements found in the proximal promoters of TLR genes control cell-type specificity and inducible TLR expression in mice and humans.…”

Ligand-induced differential cross-regulation of Toll-like receptors 2, 4 and 5 in intestinal epithelial cells

“…3A and 5B), suggesting a common signalling pathway. Such a pathway may include the central TLR regulator NF-B, which has been shown to play an essential role in the activation of the mouse TLR2 promoter (Musikacharoen et al, 2001;Wang et al, 2001). The mouse TLR4 promoter does not contain NF-B sites but carries several other elements involved in positive (AP-1, Ets, PU.1) and negative (GATA-1 and Oct-1) regulation of TLR4 transcription (Pedchenko et al, 2005;Roger et al, 2005).…”

A Dominant Role of Toll-Like Receptor 4 in the Signaling of Apoptosis in Bacteria-Faced Macrophages

“…Because it has been shown that expression of the tlr2 gene is up-regulated after NF-B activation (41,42), we wondered whether the low rate of apoptosis in TLR2-stimulated and MG-132-pretreated cells could necessarily result from the prevention of TLR2 up-regulation by MG-132-mediated NF-B inhibition. Thus, we stimulated peritoneal mouse macrophages prepared from C3H/HeN mice with mouse TNF-␣ (40 ng/ml) for 3 or 16 h, which significantly increases TLR2 expression (43)(44)(45), before treatment with MG-132 and P3CSK4.…”