NF kappa B and SP1 elements are necessary for maximal transcription of toll-like receptor 2 induced by Mycobacterium avium

We have previously reported that Toll-like receptor (TLR) 2 mRNA was induce d after infection with Mycobacterium avium. To investigate the molecular ba sis of TLR2 expression in macrophages, we cloned and analyzed the murine pu tative 5'-proximal promoter. Transient transfection of a 326-bp region from nucleotides -294-+32 relative to the first transcription start site was su fficient to induce maximal luciferase activity at the basal level and after infection with M. avium in J774A.1 cells. Sequence analysis showed that th e region lacked a TATA box but contained two typical stimulating factor (Sp ) 1 sites, two NF-kappaB sites, one IFN-regulatory factor site and one AP-1 site. Site-directed mutagenesis revealed that the NF-kappaB 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 infect ion. Binding of Sp1/Sp3 and NF-kappaB (p50/p65) was confirmed by EMSA. Furt her studies showed that three copies of Sp1 elements or NF-kappaB elements are not sufficient to confer Al. avium induction on a heterologous promoter . By contrast, overexpression of NF-kappaB p65 caused a strong increase in transcription from an intact TLR2 promoter, whereas it caused only a partia l increase in promoter activity when cotransfected with the TLR2 promoter w ith one of the Spl sites mutated. Sp1 and NF-kappaB were the minimum mammal ian transcription factors required for effective TLR2 transcriptional activ ity when transfected into Drosophila Schneider cells. Together, these data provide genetic and biochemical evidence for NF-kappaB as well as Sp1 in re gulating TLR2 transcription.