NF-kappa B and AP-1 activation by nitric oxide attenuated apoptotic cell death in RAW 264.7 macrophages

A toxic dose of the nitric oxide (NO) donor S-nitrosoglutathione (GSNO; 1 m M) promoted apoptotic cell death of RAW 264.7 macrophages, which was attenu ated by cellular preactivation with a nontoxic dose of GSNO (200 mu M) Or W ith lipopolysaccharide, interferon-gamma, and N-G-monomethyl-L-arginine (LP S/IFN-gamma/NMMA) for 15 h. Protection from apoptosis was achieved by expre ssion of cyclooxygenase-2 (Cox-2). Here we investigated the underlying mech anisms leading to Cox-2 expression. LPS/IFN-gamma/NMMA prestimulation activ ated nuclear factor (NF)-kappa B and promoted Cox-2 expression. Cox-2 induc tion by low-dose GSNO demanded activation of both NF-kappa B and activator protein-1 (AP-1). NF-kappa B supershift analysis implied an active p50/p65 heterodimer, and a luciferase reporter construct, containing four copies of the NF-kappa B site derived from the murine Cox-2 promoter, confirmed NF-k appa B activation after NO addition. An NF-kappa B decoy approach abrogated not only Cox-2 expression after low-dose NO or after LPS/IFN-gamma/NMMA bu t also inducible protection. The importance of AP-1 for Cox-2 expression an d cell. protection by low-level NO was substantiated by using the extracell ular signal-regulated kinase inhibitor PD98059, blocking NO-elicited Cox-2 expression, but leaving the cytokine signal unaltered. Transient transfecti on 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-kap pa B activation, a low-level NO-elicited Cox-2 response required activation of both NF-kappa B and AP-1.