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

Abstract

Biosynthesis of tumor necrosis factor-␣ (TNF-␣) 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-␣ promoter in cells of monocytic lineage. Functional analysis of monocytic THP-1 cells transfected with plasmids containing various lengths of TNF-␣ promoter localized enhancer elements in a region (؊182 to ؊37 base pairs (bp)) that were required for optimal transcription of the TNF-␣ 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-B (designated B3) 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 B3 sites, which were identified as Egr-1 and p50/p65, respectively. The CRE and B3 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 B3 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-␣ promoter is mediated by concerted participation of at least two separate cis-acting regulatory elements.

TNF-α Gene Expression in Macrophages: Regulation by NF-κB Is Independent of c-Jun or C/EBPβ

“…As early as 20 min following stimulation with LPS, NF-B p65/p50 heterodimers bound to the B1 and B3 binding sites of the proximal TNF-␣ promoter, consistent with earlier studies (7,8). The current study extends these observations by directly documenting the functional relevance of these heterodimers, because expression of NF-B p65 and p50 resulted in greater than additive, or synergistic, activation of the TNF-␣ promoter.…”

“…These observations suggest that both B1 and B3 binding sites contribute to the greater than additive activation of the TNF-␣ promoter by NF-B p65 plus p50. Supporting the importance of B1, mutation of this site reduced LPS-induced activation (7,8). Increased promoter activity was observed following transient transfection with the increasing concentrations of the p50 expression plasmid (ratios of 1:3 for both, and 1:4 for the Ϫ120-bp promoter).…”

“…Oligonucleotide probes spanning the following regions of the TNF-␣ promoter were employed: Ϫ100 to Ϫ74 bp (Ϫ100/Ϫ74), possessing the B3 (Ϫ98 to Ϫ89) and the C/EBP␤ binding sites; Ϫ115 to Ϫ98 bp (Ϫ115/Ϫ98), possessing the AP-1 binding site; Ϫ598 to Ϫ589 bp (Ϫ598/Ϫ589) representing the B1 binding site; and Ϫ217 to Ϫ200 bp (Ϫ217/Ϫ200) representing the B2 binding site (7,8,26). In addition, oligonucleotides representing the C/EBP␤ binding site of the IL-6 promoter and an AP-1 binding site from the collagenase promoter have been previously described (22).…”

Anticancer agent CHS 828 suppresses nuclear factor‐κB activity in cancer cells through downregulation of IKK activity

“…The THP-1 cells were transiently transfected during the log phase of growth using a DEAE-dextran transfection procedure (modified from Yao et al 16 ). Briefly, 3.5 ϫ 10 7 cells were resuspended in transfection medium (RPMI-1640 supplemented with 2 mM L-glutamine) and incubated with 5 g of construct DNA, 0.5 g pRL-TK vector plus 500 g of DEAE-dextran (Sigma-Aldrich, Vallensbaek Strand, Denmark).…”

Silencing of TNF‐α receptors coordinately suppresses TNF‐α expression through NF‐κB activation blockade in THP‐1 macrophage

“…Exposure of macrophages to endotoxins such as lipopolysaccharides (LPS) stimulates TNF-a secretion and some of the effects of LPS are executed predominantly by the production of TNF-a [3,4]. Nuclear factor-jB (NF-jB) acts as a key transcription factor in the synthesis of TNF-a.…”