Primate Monocytes - CD14, CD16 - Ziegler-Heitbrock

Post-induction, stimulus-specific regulation of tumor necrosis factor mRNA expression

Abstract

The tumor necrosis factor (TNF) gene is activated by multiple extracellular signals in a stimulus- and cell type-specific fashion. Based on the presence of kappaB-like DNA motifs in the region upstream of the TNF gene, some have proposed a direct role for NF-kappaB in lipopolysaccharide (LPS)-induced TNF gene transcription in cells of the monocyte/macrophage lineage. However, we have previously demonstrated a general and critical role for a minimal TNF promoter region bearing only one of the kappaB-like motifs, kappa3, which is bound by nuclear factor of activated T cells (NFAT) proteins in lymphocytes and fibroblasts in response to multiple stimuli and Ets proteins in LPS-stimulated macrophages. Here, in an effort to resolve these contrasting findings, we used a combination of site-directed mutagenesis of the TNF promoter, quantitative DNase I footprinting, and analysis of endogenous TNF mRNA production in response to multiple stimuli under conditions that inhibit NF-kappaB activation (using the proteasome inhibitor lactacystin and using cells lacking either functional NEMO, which is the IkappaB kinase regulatory subunit, or the Nemo gene itself). We find that TNF mRNA production in response to ionophore is NF-kappaB-independent, but inhibition of NF-kappaB activation attenuates virus- and LPS-induced TNF mRNA levels after initial induction. We conclude that induction of TNF gene transcription by virus or LPS does not depend upon NF-kappaB binding to the proximal promoter; rather, a stimulus-specific post-induction mechanism involving NF-kappaB, yet to be characterized, is involved in the maintenance of maximal TNF mRNA levels.