The proximal promoter region is essential for lipopolysaccharide induction and cyclic AMP inhibition of mouse tumor necrosis factor-alpha.
Bacterial lipopolysaccharide (LPS) induces a rapid and transient increase in transcription of the tumor necrosis factor-alpha (TNF-alpha) 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-alpha gene in LPS induction and cyclic AMP (cAMP)-mediated inhibition of TNF-alpha 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-kappaB-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-alpha gene, we produced a hemagglutinin (HA)-tagged genomic TNF-alpha 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-alpha gene transcription in murine macrophages.
O'Donnell
Phyllis M
PM
SUNY Upstate Medical University, Department of Microbiology/Immunology, Syracuse, NY 13210, USA.
Taffet
Steven M
SM
D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D000242:Cyclic AMP / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D005809:Genes, Regulator; D017930:Genes, Reporter; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase