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

It has been demonstrated from studies using NF-kappa B inhibitors that NF-k appa B may be involved in the iNOS induction stimulated by cytokines and/or lipopolysaccharide (LPS) in various cell types and tissues. However, the a ctions of the inhibitors are less selective and highly cytotoxic, We constr ucted stable clones of C6 cells transfected with two types of I kappa B alp ha mutant genes (I kappa B alpha(SS-->AA); Ser-32/36 to Ala-32/36, I kappa B alpha(KK-->RR);Lys-21/22 to Arg-21/22). I kappa B alpha(SS-->AA) strongly inhibited (1) LPS-, IL-1 beta-, and TNF-alpha-induced nuclear translocatio n and DNA binding of NF-kappa B to the kappa B site; and (2) iNOS induction stimulated by LPS or IL-1 beta plus IFN-gamma, These results indicate that NF-kappa B plays a critical role in cytokines and/or LPS-induced iNOS indu ction. Surprisingly, similar to the endogenous I kappa B alpha, I kappa B a lpha(KK-->RR) was degraded by various stimuli, and proteasome inhibitors bl ocked this event. These results suggest that another Lys residue(s), other than Lys-21/22, may be required for the ligand-induced I kappa B alpha degr adation by the ubiquitin-proteasome pathway. (C) 2000 Academic Press.