IRON REGULATES NITRIC-OXIDE SYNTHASE ACTIVITY BY CONTROLLING NUCLEAR TRANSCRIPTION

Recently, it was reported that nitric oxide (NO) directly controls int racellular iron metabolism by activating iron regulatory protein (IRP) , a cytoplasmic protein that regulates ferritin translation. To determ ine whether intracellular iron levels themselves affect NO synthase (N OS), we studied the effect of iron on cytokine-inducible NOS activity and mRNA expression in the murine macrophage cell line J774A.1. We sho w here that NOS activity is decreased by about 50% in homogenates obta ined from cells treated with interferon gamma plus lipopolysaccharide (IFN-gamma/LPS) in the presence of 50 mu M ferric iron [Fe(3(+))] as c ompared with extracts from cells treated with IFN-gamma/LPS alone. Con versely, addition of the iron chelator desferrioxamine (100 mu M) at t he time of stimulation with IFN-gamma/LPS increases NOS activity up to 2.5-fold in J774 cells. These effects of changing the cellular iron s tate cannot be attributed to a general alteration of the IFN-gamma/LPS signal, since IFN-gamma/LPS-mediated major histocompatibility complex class II antigen expression is unaffected. Furthermore, neither was t he intracellular availability of the NOS cofactor tetrahydrobiopterin altered by treatment with Fe(3(+)) or desferrioxamine, nor do these co mpounds interfere with the activity of the hemoprotein NOS in vitro. W e demonstrate that the mRNA levels for NOS are profoundly increased by treatment with desferrioxamine and reduced by Fe(3(+)). The half-life of NOS mRNA appeared not to be significantly altered by administratio n of ferric ion, and NOS mRNA stability was only slightly prolonged by desferrioxamine treatment. Nuclear run-off experiments demonstrate th at nuclear transcription of cytokine-inducible NOS mRNA is strongly in creased by desferrioxamine whereas it is decreased by Fe(3 C). Thus, t his transcriptional response appears to account quantitatively for the changes in enzyme activity. Our results suggest the existence of a re gulatory loop between iron metabolism and the NO/NOS pathway.