BIOSYNTHESIS OF NITRIC-OXIDE ACTIVATES IRON REGULATORY FACTOR IN MACROPHAGES

Biosynthesis of nitric oxide (NO) from L-arginine modulates activity o f iron-dependent enzymes, including mitochondrial aconitase, an [Fe-S] protein. We examined the effect of NO on the activity of iron regulat ory factor (IRF), a cytoplasmic protein which modulates both ferritin mRNA translation and transferrin receptor mRNA stability by binding to specific mRNA sequences called iron responsive elements (IREs). Murin e macrophages were activated with interferon-gamma and lipopolysacchar ide to induce NO synthase activity and cultured in the presence or abs ence of N(G)-substituted analogues Of L-arginine which served as selec tive inhibitors of NO synthesis. Measurement of the nitrite concentrat ion in the culture medium was taken as an index of NO production. Mito chondria-free cytosols were then prepared and aconitase activity as we ll as IRE binding activity assessed in parallel. Inhibition of enzymat ic activity and induction of IRE binding activity were correlated and depended on NO synthesis after IFN-gamma and/or LPS stimulation. Authe ntic NO gas as well as the NO-generating compound 3-morpholinosydnonim ine (SIN-1) also conversely modulated aconitase and IRE binding activi ties of purified recombinant IRF. These results provide evidence that endogenously produced NO may modulate the post-transcriptional regulat ion of genes involved in iron homeostasis and support the hypothesis t hat the [Fe-S] cluster of IRF mediates iron-dependent regulation.