A Sp1 binding site of the tumor necrosis factor alpha promoter functions as a nitric oxide response element.
Regulation of gene transcription is an incompletely understood function of nitric oxide (NO). Human leukocytes produce increased amounts of tumor necrosis factor alpha (TNF-alpha) in response to NO. This effect is associated with decreases in intracellular cAMP, suggesting that NO might regulate gene transcription through promoter sequences sensitive to cAMP such as cAMP response elements (CRE) and Sp1 binding sites. Here we report that a Sp1 binding site in the TNF-alpha promoter conveys NO responsiveness. Human U937 cells were differentiated for TNF-alpha production with phorbol 12-myristate 13-acetate. NO donors and H89, an inhibitor of cAMP-dependent protein kinase increased, while dibutyryl cAMP (Bt(2)cAMP) decreased TNF-alpha promoter activity. Deletion or mutation of the proximal Sp1 site, but not the CRE site, abolished the activating effects of NO donors and H89. Further, NO- and H89-mediated increases in TNF-alpha promoter activity were associated with decreased Sp1 binding. The insertion of Sp1 sites into a minimal cytomegalovirus promoter conferred NO responsiveness, an effect blocked by Bt(2)cAMP. Mutation of these inserted Sp1 sites prevented this heterologous promoter from responding to NO, H89 and Bt(2)cAMP. These results identify the Sp1 binding site as a promoter motif that allows NO to control gene transcription.
Wang
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Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
Wang
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Wesley
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Danner
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D001483:Base Sequence; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004789:Enzyme Activation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism* / Q000502:physiology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000502:physiology; D020298:U937 Cells
D017931:DNA Primers; D016329:Sp1 Transcription Factor; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D004247:DNA; D017868:Cyclic AMP-Dependent Protein Kinases