NF-κB and AP-1 Activation by Nitric Oxide Attenuated Apoptotic Cell Death in RAW 264.7 Macrophages

Abstract

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 microM) or with lipopolysaccharide, interferon-gamma, and NG-monomethyl-L-arginine (LPS/IFN-gamma/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-gamma/NMMA prestimulation activated nuclear factor (NF)-kappaB and promoted Cox-2 expression. Cox-2 induction by low-dose GSNO demanded activation of both NF-kappaB and activator protein-1 (AP-1). NF-kappaB supershift analysis implied an active p50/p65 heterodimer, and a luciferase reporter construct, containing four copies of the NF-kappaB site derived from the murine Cox-2 promoter, confirmed NF-kappaB activation after NO addition. An NF-kappaB decoy approach abrogated not only Cox-2 expression after low-dose NO or after LPS/IFN-gamma/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-kappaB activation, a low-level NO-elicited Cox-2 response required activation of both NF-kappaB and AP-1.

Tyrosine‐nitration of caspase 3 and cytochrome c does not suppress apoptosis induction in squamous cell carcinoma cells

“…[35][36][37][38][39][40][41] In addition, ROS also play an important role in protein phosphorylation and activation of nuclear factor-kappa B (NF-B) and activating protein-1 (AP-1), which are both associated with apoptosis. 42,43 In OSC-4 cells used in the present study, the main apoptotic pathway seemed to be associated with cytochrome c release from the mitochondria. 6 ROS react with NO and highly reactive molecules are generated.…”

Mn‐SOD antisense upregulates in vivo apoptosis of squamous cell carcinoma cells by anticancer drugs and γ‐rays regulating expression of the BCL‐2 family proteins, COX‐2 and p21

“…The mechanism of the decrease of COX-2 expression is not clear, but high levels of NO have been reported to suppress COX-2 expression. 50 Therefore, estimations of the NO level in Mn-SOD antisense-transfected cells appears useful for the analysis of the decreased expression of COX-2.…”

Nitric oxide: a key regulator of myeloid inflammatory cell apoptosis

“…88 Agents such as LPS have been shown to delay apoptosis in this cell type through stimulation of NF-kB and subsequent caspase-1-dependent activation of IL-1b. 89 It has been demonstrated in several cell types that activation of NF-kB leads to the transcriptional upregulation of survival factors such as the immediate early gene, IEX-1L, inhibitor of apoptosis proteins (IAPs), and members of the antiapoptotic Bcl-2 gene family, including Mcl-1, Bfl1/A1, Bcl-XL and Nr13, potentially explaining the survival effects of NO which, at low concentrations, induces NF-kB activation in macrophage cell lines 90,91 and in human peripheral blood mononuclear cells. 92 Activation of NF-kB may occur through a cGMP-dependent mechanism, 93 and NO has been demonstrated to modulate the expression of several proteins affecting the activity of Bcl-2 family members through sGC activation.…”