The Proximal Promoter Region Is Essential for Lipopolysaccharide Induction and Cyclic AMP Inhibition of Mouse Tumor Necrosis Factor-α

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Abstract
Bacterial lipopolysaccharide (LPS) induces a rapid and transient increase in transcription of the tumor necrosis factor-α (TNF-α) gene in cells of monocyte/macrophage lineage. This study examines the role of potential regulatory elements within the proximal promoter region of the mouse TNF-α gene in LPS induction and cyclic AMP (cAMP)-mediated inhibition of TNF-α in RAW 264.7 murine macrophage-like cells. Transfection of proximal promoter chloramphenicol acetyltransferase (CAT) reporter constructs demonstrated that this region is LPS inducible in murine RAW 264.7 cells, with a 5.9-fold increase over nonstimulated transfectants. Site-specific mutations of the ETS, activated protein-1 (AP-1)/cAMP-responsive element (CRE)-like, or NF-κB-like motifs within this region caused a reduction in the LPS response by 52%, 46%, and 51%, respectively. LPS induction of the proximal promoter-CAT reporter construct was reduced by >40% by the addition of 8-bromo-cAMP (8-Br-cAMP). To determine the role of the proximal promoter region in the context of the entire TNF-α gene, we produced a hemagglutinin (HA)-tagged genomic TNF-α construct that contains a deletion of the proximal promoter region. Transfection of this construct into RAW 264.7 cells demonstrated a decrease in LPS-induced transcripts as well as a lack of response to cAMP. This suggested an essential role for this regulatory region in LPS-induced activation and cAMP inhibition of mouse TNF-α gene transcription in murine macrophages.

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