Post-induction, Stimulus-specific Regulation of Tumor Necrosis Factor mRNA Expression

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 kappa B-like DNA motifs in the region upstream of the TNF gene, some have proposed a direct role for NF-kappa B 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 kappa B-like motifs, kappa 3, which is bound by nuclear factor of activated T cell 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-kappa B activation ( using the proteasome inhibitor lactacystin and using cells lacking either functional NF-kappa B essential modulator, which is the I kappa B kinase regulatory subunit, or the Nemo gene itself). We find that TNF mRNA production in response to ionophore is NF-kappa B-independent, but inhibition of NF-kappa B 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-kappa B binding to the proximal promoter; rather, a stimulus- specific post-induction mechanism involving NF-kappa B, yet to be characterized, is involved in the maintenance of maximal TNF mRNA levels.