Cloning and characterization of lipopolysaccharide-induced tumor necrosis factor α factor promoter

Abstract

We have recently identified lipopolysaccharide tumor-induced tumor necrosis factor α factor (LITAF) as a novel transcription factor controlling necrosis factor (TNF)-α expression in the human monocytic cell line, THP-1. To characterize the human (h) LITAF promoter, we isolated a 1.2-kb DNA fragment and followed this by a screening of human genomic DNA with a hLITAF cDNA probe. A 34-bp sequence domain located from nucleotides -74 to -43 in the hLITAF promoter exhibited the highest basal reporter gene activity; however, the activity was not elevated by lipopolysaccharide (LPS) stimulation. The sequence domain included a consensus sequence for hepatocyte nuclear factor (HNF)-3α, regulating the transcription of many kinds of genes. Interestingly, the DNA sequence position between -542 and -538 in the hLITAF promoter contained the CTCCC motif, which has been reported to act as a specific binding site for hLITAF protein. Electrophoretic mobility shift assays demonstrated that LPS induced the binding of THP-1 nuclear factors to a 22 bp probe containing the CTCCC motif. In addition, hLITAF mRNA and nuclear hLITAF protein increased significantly in the THP-1 cells immediately after LPS stimulation. These results suggest that the consensus sequence for HNF-3α, or a nuclear binding protein to the CTCCC motif, may play an important role in regulating LPS-dependent LITAF transcription. © 2006 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.