Glucocorticoids suppress tumor necrosis factor-α expression by human monocytic THP-1 cells by suppressing transactivation through adjacent NF-κb and c-Jun-activating transcription factor-2 binding sites in the promoter

Abstract

Glucocorticoid drugs suppress tumor necrosis factor-α (TNF-α) synthesis by activated monocyte/macrophages, contributing to an anti- inflammatory action in vivo. In lipopolysaccharide (LPS)-activated human monocytic THP-1 cells, glucocorticoids acted primarily on the TNF-α promoter to suppress a burst of transcriptional activity that occurred between 90 min and 3 h after LPS exposure. LPS increased nuclear c-Jun/ATF-2, NF-κB1/Rel- A, and Rel-A/C-Rel transcription factor complexes, which bound specifically to oligonucleotide sequences from the -106 to -88 base pair (bp) region of the promoter. The glucocorticoid, dexamethasone, suppressed nuclear binding activity of these complexes prior to and during the critical phase of TNF-α transcription. Site-directed mutagenesis in TNF-α promoter-luciferase reporter constructs showed that the adjacent c-Jun/ATF-2 (-106 to -99 bp) and NF-κB (-97 to -88 bp) binding sites each contributed to the LPS-stimulated expression. Mutating both sites largely prevented dexamethasone from suppressing TNF-α promoter-luciferase reporters. LPS exposure also increased nuclear Egr-1 and PU.1 abundance. The Egr-1/Sp1 (-172 to -161 bp) binding sites and the PU.1-binding Ets site (-116 to -110 bp) each contributed to the LPS-stimulated expression but not to glucocorticoid response. Dexamethasone suppressed the abundance of the c-Fos/c-Jun complex in THP-1 cell nuclei, but there was no direct evidence for c-Fos/c-Jun transactivation through sites in the -172 to -52 bp region. Small contributions to glucocorticoid response were attributable to promoter sequences outside the -172 to -88 bp region and to sequences in the TNF-α 3'-untranslated region. We conclude that glucocorticolds suppress LPS-stimulated secretion of TNF-α from human monocytic cells largely through antagonizing transactivation by c-Jun/ATF-2 and NF-κB complexes at binding sites in the -106 to -88 bp region of the TNF-α promoter.