κB-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor α gene in primary macrophages

We have explored the cis-acting elements necessary for the LPS-mediated activation of the mouse TNF-α promoter by transfecting a set of 5' deletion mutants linked to the CAT reporter gene into primary bone marrow-derived macrophages. A major drop in inducibility by LPS was seen upon deletion of a region mapping between nt -655 and nt -451. Gel retardation assays revealed that LPS induced the appearance in this region of several specific DNA-protein complexes mapping to sequence motifs with strong homology to the κB enhancer. Constructs containing two or more copies of one of the κB enhancer motifs linked to a heterologous promoter were inducible by LPS. Additional deletion of a region between nt -301 and nt -241, which contains a MHC class II-like 'Y box' and formed a Y box-specific complex with a protein whose concentration was increased by LPS, caused a nearly complete loss of inducibility by LPS. We speculate that NF-κB and/or related proteins are involved in the LPS-induced transcriptional activation of the TNF-α gene, and that factors interacting with the Y box can additionally modulate the activity of the gene in macrophages.