NFkappaB 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 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-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 infection. Binding of Sp1/Sp3 and NF-kappaB (p50/p65) was confirmed by EMSA. Further studies showed that three copies of Sp1 elements or NF-kappaB elements are not sufficient to confer M. 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 partial increase in promoter activity when cotransfected with the TLR2 promoter with one of the Sp1 sites mutated. Sp1 and NF-kappaB 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-kappaB as well as Sp1 in regulating TLR2 transcription.
Wang
T
T
Department of Microbiology and Molecular Virology, Ohio State University, Columbus, OH 43210, USA.
Lafuse
W P
WP
Zwilling
B S
BS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D004330:Drosophila / Q000378:metabolism; D029721:Drosophila Proteins; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000276:immunology* / Q000382:microbiology; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009162:Mycobacterium avium / Q000502:physiology*; D016328:NF-kappa B / Q000235:genetics / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D020218:Response Elements; D016329:Sp1 Transcription Factor / Q000502:physiology*; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors; D015533:Transcriptional Activation; D014162:Transfection
D029721:Drosophila Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D016329:Sp1 Transcription Factor; D051195:Toll-Like Receptor 2; D051193:Toll-Like Receptors; D008156:Luciferases