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J Biol Chem, Vol. 274, Issue 47, 33190-33193, November 19, 1999
Promoter
Functions as a Nitric Oxide Response Element
From the Critical Care Medicine Department, Warren Grant
Magnuson Clinical Center, National Institutes of Health,
Bethesda, Maryland 20892
Regulation of gene transcription is an
incompletely understood function of nitric oxide (NO). Human leukocytes
produce increased amounts of tumor necrosis factor 
(TNF-) 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- promoter conveys NO responsiveness.
Human U937 cells were differentiated for TNF- production with
phorbol 12-myristate 13-acetate. NO donors and H89, an inhibitor of
cAMP-dependent protein kinase increased, while dibutyryl
cAMP (Bt2cAMP) decreased TNF- 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- 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 Bt2cAMP. Mutation of these inserted Sp1 sites prevented
this heterologous promoter from responding to NO, H89 and
Bt2cAMP. These results identify the Sp1 binding site as a
promoter motif that allows NO to control gene transcription.
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