Lipopolysaccharide Induction of the Tumor Necrosis Factor-α Promoter in Human Monocytic Cells

Biosynthesis of tumor necrosis factor-α (TNF-α) is predominantly by cells of the monocytic lineage. This study examined the role of various cis-acting regulatory elements in the lipopolysaccharide (LPS) induction of the human TNF-α promoter in cells of monocytic lineage. Functional analysis of monocytic THP-1 cells transfected with plasmids containing various lengths of TNF-α promoter localized enhancer elements in a region (−182 to −37 base pairs (bp)) that were required for optimal transcription of the TNF-α gene in response to LPS. Two regions were identified: region I (−182 to −162 bp) contained an overlapping Sp1/Egr-1 site, and region II (−119 to −88) contained CRE and NF-κB (designated κB3) sites. In unstimulated THP-1, CRE-binding protein and, to a lesser extent, c-Jun complexes were found to bind to the CRE site. LPS stimulation increased the binding of c-Jun-containing complexes. In addition, LPS stimulation induced the binding of cognate nuclear factors to the Egr-1 and κB3 sites, which were identified as Egr-1 and p50/p65, respectively. The CRE and κB3 sites in region II together conferred strong LPS responsiveness to a heterologous promoter, whereas individually they failed to provide transcriptional activation. Furthermore, increasing the spacing between the CRE and the κB3 sites completely abolished LPS induction, suggesting a cooperative interaction between c-Jun complexes and p50/p65. These studies indicate that maximal LPS induction of the TNF-α promoter is mediated by concerted participation of at least two separatecis-acting regulatory elements.