Inhibition of protein geranylgeranylation causes a superinduction of nitric-oxide synthase-2 by interleukin-1β in vascular smooth muscle cells

Recently, we have designed farnesyltransferase and geranylgeranyltransferase I inhibitors (FTI-277 and GGTI-298) that selectively block protein farnesylation and geranylgeranylation, respectively. In this study, we describe the opposing effects of these inhibitors on interleukin-1β (IL-1β)-stimulated induction of nitric-oxide synthase-2 (NOS-2) in rat pulmonary artery smooth muscle cells (RPASMC) and rat hepatocytes. Pretreatment of cells with GGTI-298 caused a superinduction of NOS-2 by IL-1β. RPASMC treated with GGTI-298 (10 μM) prior to IL-1β (10 ng/ml) expressed levels of NOS-2 protein five times higher than those exposed to IL-1β alone. This superinduction of NOS-2 protein by pretreatment with GGTI-298 resulted in nitrite concentrations in the medium that were 5-fold higher at 10 ng/ml IL-1β and 10-fold higher at 1 ng/ml IL-1β. Furthermore, NOS-2 mRNA levels in RPASMC were also increased 6- and 14-fold (at 10 and 1 ng/ml IL-1β, respectively) when the cells were pretreated with GGTI-298. In contrast, treatment of cells with the inhibitor of protein farnesylation, FTI-277 (10 μM), blocked IL-1β-induced NOS-2 expression at mRNA and protein levels. Pretreatment with lovastatin, an inhibitor of protein prenylation, resulted in superinduction of NOS-2. This superinduction was reversed by geranylgeraniol, but not by farnesol, further confirming that inhibition of geranylgeranylation, not farnesylation, is responsible for enhanced NOS-2 expression. The results demonstrate that a farnesylated protein(s) mediates IL-1β induction of NOS-2, whereas a geranylgeranylated protein(s) represses this induction.