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Vol. 10, Issue 2, 361-372, February 1999
B and AP-1 Activation by Nitric Oxide Attenuated Apoptotic
Cell Death in RAW 264.7 Macrophages
University of Erlangen-Nürnberg, Faculty of Medicine,
Department of Medicine IV-Experimental Division, Erlangen, Germany
A toxic dose of the nitric oxide (NO) donor
S-nitrosoglutathione (GSNO; 1 mM) promoted
apoptotic cell death of RAW 264.7 macrophages, which was attenuated by
cellular preactivation with a nontoxic dose of GSNO (200 µM) or with
lipopolysaccharide, interferon-
, and
NG-monomethyl-L-arginine
(LPS/IFN-/NMMA) for 15 h. Protection from apoptosis was
achieved by expression of cyclooxygenase-2 (Cox-2). Here we
investigated the underlying mechanisms leading to Cox-2 expression.
LPS/IFN-/NMMA prestimulation activated nuclear factor (NF)-
B and
promoted Cox-2 expression. Cox-2 induction by low-dose GSNO demanded
activation of both NF-B and activator protein-1 (AP-1).
NF-B supershift analysis implied an active p50/p65 heterodimer, and
a luciferase reporter construct, containing four copies of the NF-B
site derived from the murine Cox-2 promoter, confirmed NF-B
activation after NO addition. An NF-B decoy approach abrogated not
only Cox-2 expression after low-dose NO or after LPS/IFN-/NMMA but
also inducible protection. The importance of AP-1 for Cox-2 expression
and cell protection by low-level NO was substantiated by using the
extracellular signal-regulated kinase inhibitor PD98059, blocking
NO-elicited Cox-2 expression, but leaving the cytokine signal
unaltered. Transient transfection of a dominant-negative c-Jun
mutant further attenuated Cox-2 expression by low-level NO. Whereas
cytokine-mediated Cox-2 induction relies on NF-B activation, a
low-level NO-elicited Cox-2 response required activation of both
NF-B and AP-1.
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