Effects of mechanistically distinct NF-KB inhibitors on glial inducible nitric-oxide synthase expression

Nuclear factor (NF)-kappa B is all important regulator of inflammatory gene expression. Transcriptional regulation of Nos2, the inducible nitric-oxide synthase (iNOS) gene, is complex and not frilly understood, but appears to be regulated in part by NF-kappa B. To further understand the role of NF-kappa B in Nos2 expression, we compared three functionally distinct NF-kappa B inhibitors on NF-kappa B transactivation and iNOS induction by rat C6 glial cells. Cytokine-induced activation of a consensus NF-kappa B-reporter gene was concentration-dependently inhibited by BAY 11-7082, MG-132, and helenalin. The rank order of potency was MG-132 > helenalin > BAY 11-7082, with low concentrations of helenalin stimulating reporter gene activity. Cytokine-stimulated iNOS expression, measured by nitrite accumulation and ill Vitro L-citrulline production, was similarly reduced by exposing C6 cells to the NF-kappa B inhibitors. Surprisingly, activation of Nos2-reporter gene constructs containing the proximal 188 bp (containing one kappa B site) or proximal 94 bp (no kappa B site) of the rat promoter also was inhibited with the same rank order of potency. Interestingly, low concentrations of helenalin increased activity of both promoter constructs, while BAY 11-7082 poorly inhibited the 94-bp activity. This is the first report describing BAY 11-7082 and helenalin effects on NOS expression in astroglia. Given the reported mechanism of actions for these inhibitors, cytokine-induced glial NOS expression appears more sensitive to disruption of proteasome degradation and p65 function than modulation of I kappa B phosphorylation. These findings may foster the design of therapeutic agents aimed at NF-kappa B-associated pathways involved in neuroinflammation, especially NOS expression. (c) 2005 Elsevier Inc. All rights reserved.