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

Alla V Tsytsykova,James V Falvo,Marc Schmidt-Supprian,Gilles Courtois,Dimitris Thanos,Anne E Goldfeld

doi:10.1074/jbc.m611418200

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 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-kappaB activation (using the proteasome inhibitor lactacystin and using cells lacking either functional NF-kappaB essential modulator, 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.

Highlights

After the identification of NF-␬B as an inducible DNA-binding factor [5, 6], four DNA motifs resembling NF-␬B-binding sites were described in the promoter of the murine tumor necrosis factor (TNF) gene [7, 8], eventually designated ␬B1, ␬B2, ␬B2a, and ␬B3 [9, 10], and three in the promoter of the human TNF gene, ␬1, ␬2, and ␬3 [11] (Fig. 1)
It has been reported that point mutations of these sites tate 13-acetate; IKK, I␬B kinase; MEF, murine embryonic fibroblast; TLR4, toll-like receptor 4; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; Btk, Bruton’s tyrosine kinase; IRF, interferon regulatory factor; P ϩ I, phorbol ester plus ionophore
We have previously demonstrated that the ␬3 site, which lies within the proximal human TNF promoter region (Fig. 1), behaves as a composite element in conjunction with an adjacent cAMP-response element site and is a functional binding site for an NFATp dimer [2, 21,22,23,24]

Summary

Introduction

After the identification of NF-␬B as an inducible DNA-binding factor [5, 6], four DNA motifs resembling NF-␬B-binding sites were described in the promoter of the murine TNF gene [7, 8], eventually designated ␬B1, ␬B2, ␬B2a, and ␬B3 [9, 10], and three in the promoter of the human TNF gene, ␬1, ␬2, and ␬3 [11] (Fig. 1). In an effort to resolve the basic discrepancy between the lack of function of upstream ␬B-like motifs in the TNF promoter and the apparent influence of NF-␬B upon LPS-mediated TNF transcription in the studies described above, we examined the effects of altering the interaction of NF-␬B with the TNF promoter and the time dependence of NF-␬B inhibition upon TNF gene expression in human and murine cells in response to multiple extracellular stimuli.

Results

Conclusion