LPS/IFN-γ-Induced RAW 264.7 Apoptosis is Regulated by Both Nitric Oxide–Dependent and –Independent Pathways Involving JNK and the Bcl-2 Family

Lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) stimulate macrophages to produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS) and activate stress signaling cascades including the c-jun-N-terminal kinase (JNK) pathway. These events trigger an apoptotic cascade that ultimately results in death. Since JNK regulates pro-apoptotic and anti-apoptotic Bcl-2 family members, the role of NO in LPS/IFNgamma-induced activation of JNK and its effects on the Bcl-2 family was examined in RAW 264.7 macrophage-like cells. Inhibition of JNK by siRNA verified a role for JNK in LPS/IFNgamma-induced apoptosis. Suppression of NO production by a pharmacologic agent, i.e., iNOS inhibitor L-NIL, altered the kinetics of JNK activation by LPS/IFNgamma. Examination of mitochondrial and nuclear compartments of RAW 264.7 cells demonstrated NO-dependent activation of mitochondrial JNK by LPS/IFNgamma, but NO-independent, cytokine-induced phosphorylation of Bim. NO did not affect phosphorylation, but did inhibit Bax phosphorylation. These results suggest a novel mechanism of LPS/IFNgamma-induced apoptosis in macrophages involving NO-independent phosphorylation of Bim and NO-dependent dephosphorylation of Bax.