Involvement of nuclear factor-κB (NF-κB) signaling in the expression of inducible nitric oxide synthase (iNOS) gene in rat C6 glioma cells

It has been demonstrated from studies using NF-κB inhibitors that NF-κB may be involved in the iNOS induction stimulated by cytokines and/or lipopolysaccharide (LPS) in various cell types and tissues. However, the actions of the inhibitors are less selective and highly cytotoxic. We constructed stable clones of C6 cells transfected with two types of IκBα mutant genes (IκBα(ss → AA); Ser-32/36 to Ala-32/36, IκBα (KK → RR); Lys-21/22 to Arg-21/22). IκBα(SS → AA) strongly inhibited (1) LPS-, IL-1β-, and TNF-α-induced nuclear translocation and DNA binding of NF-κB to the κB site; and (2) iNOS induction stimulated by LPS or IL-1β plus IFN-γ. These results indicate that NF-κB plays a critical role in cytokines and/or LPS-induced iNOS induction. Surprisingly, similar to the endogenous IκBα, IκBα(KK → RR) was degraded by various stimuli, and proteasome inhibitors blocked this event. These results suggest that another Lys residue(s), other than Lys-21/22, may be required for the ligand-induced IκBα degradation by the ubiquitin-proteasome pathway. (C) 2000 Academic Press.