Kappa B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor alpha gene in primary macrophages.
We have explored the cis-acting elements necessary for the LPS-mediated activation of the mouse TNF-alpha 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 kappa B enhancer. Constructs containing two or more copies of one of the kappa B 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-kappa B and/or related proteins are involved in the LPS-induced transcriptional activation of the TNF-alpha gene, and that factors interacting with the Y box can additionally modulate the activity of the gene in macrophages.
Shakhov
A N
AN
Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland.
Collart
M A
MA
Vassalli
P
P
Nedospasov
S A
SA
Jongeneel
C V
CV
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D002478:Cells, Cultured; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005796:Genes / Q000187:drug effects*; D005803:Genes, Immunoglobulin; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D007145:Immunoglobulin kappa-Chains; D008070:Lipopolysaccharides; D015345:Oligonucleotide Probes; D014409:Tumor Necrosis Factor-alpha