Biosynthesis of nitric oxide (NO) from L‐arginine modulates activity of iron‐dependent enzymes, including mitochondrial acontiase, an [Fe‐S] protein. We examined the effect of NO on the activity of iron regulatory 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). Murine macrophages were activated with interferon‐gamma and lipopolysaccharide to induce NO synthase activity and cultured in the presence or absence of NG‐substituted analogues of L‐arginine which served as selective inhibitors of NO synthesis. Measurement of the nitrite concentration in the culture medium was taken as an index of NO production. Mitochondria‐free cytosols were then prepared and aconitase activity as well as IRE binding activity and induction of IRE binding activity were correlated and depended on NO synthesis after IFN‐gamma and/or LPS stimulation. Authentic NO gas as well as the NO‐generating compound 3‐morpholinosydnonimine (SIN‐1) also conversely modulated aconitase and IRE binding activities of purified recombinant IRF. These results provide evidence that endogenously produced NO may modulate the post‐transcriptional regulation of genes involved in iron homeostasis and support the hypothesis that the [Fe‐S] cluster of IRF mediates iron‐dependent regulation.

P.Kaldy and E.Menotti contributed equally to this workPost-transcriptional regulation of mRNA translation and stability in iron metabolism involves the interaction between the trans-acting cytoplasmic iron regulatory proteins (IRP-1 and IRP-2) and cis-acting iron-responsive elements (IREs) in mRNA 5Ј-or 3Ј-untranslated regions. IRP-1 can adopt two conformations: one with a [4Fe-4S]-cluster, unable to bind IREs, which functions as a cytoplasmic aconitase; one lacking this cluster, which accumulates in iron-deprived cells and binds mRNA firmly. We investigated which surfaces of IRP-1 interact with IREs. Surface areas were predicted on the basis of the crystallized porcine mitochondrial aconitase structure. We selected nine sequences absent or different in mitochondrial and Escherichia coli aconitases, both being devoid of RNAbinding properties. Mutations in two regions of domain 4 of IRP-1 lowered the affinity for a wild-type IRE up to 7-fold in vitro, whereas the aconitase activity, a control for structural integrity, was not affected. Scatchard plot analysis with mutant IREs indicated that domain 4 is involved in the binding specificity. This conclusion was confirmed with hybrid proteins in which IRP-1 surface loops were grafted into IRP-2. The results indicate that arginines 728 and 732 contact the IRE bulge, whereas region 685-689 is necessary for recognition of the IRE loop.

It has recently been proposed that cellular iron homeostasis in mammalian cells is regulated at the post-transcriptional level by the reciprocal control of transferrin receptor and fenitin mRNA expression via an iron-regulatory factor. This iron-regulatory factor has been shown to be a cytoplasmic aconitase which can bind to iron-responsive elements in the corresponding mRNAs with greater or lesser affinity as a function of the iron status of the cell. In the present study, we show that in vivo the affinity of iron-regulatory factor for iron-responsive elements in liver reflects the long-term iron status of the tissue in animal models for iron overloading and iron deficiency, when combined with altered transferrin saturation and serum iron levels. In contrast hepatic iron overload achieved without altering such haematopoeitic indices, had a less pronounced effect. In both spleen and heart, the affinities of iron-regulatory factor changed in parallel with both altered iron status and haematological markers. In brain and duodenum, there were no consistent changes in ironregulatory-factor activity with iron loading or depletion. Iron-regulatory-factor activity in kidney responded in an as yet unexplained manner.Iron is an essential element for the growth, differentiation and well-being of most living organisms [l]. It is therefore of paramount importance to understand at the molecular, cellular and whole-organism level how iron homeostasis is regulated. Cellular iron homeostasis can be defined as the mechanisms whereby the intracellular concentration of the metal ion is maintained at a level adequate for the cell's requirements, but not sufficiently high to cause toxic effects. This is achieved by regulating the uptake of the metal ion in concert with its intracellular utilisation, storage and eventual externalization. It has been suggested that the expression of several key proteins of iron metabolism is regulated by intracellular iron levels [2-61. The post-transcriptional regulation of the iron-storage protein ferritin, the erythroid 5-aminolaevulinate synthase (AlS) and the transfenin receptor operate at the level of mRNA. All H-chain and L-chain ferritin mRNAs [3, 7, binds tightly to the IRE of transferrin-receptor mRNA, thereby ensuring its protection against nuclease digestion [20] and translation of the transferrin receptor, thus enhancing the uptake of iron by the cell. The same iron-depleted form of IRF binds strongly to the femtin and A1S mRNAs and thus blocks the biosynthesis of ferritin erythroid AlS. When cells are replete in iron the IRF binds poorly to femtin and erythroid AIS mRNAs ensuring their expression, and dissociates from the transfenin-receptor mRNA ensuring that it will be degraded by cellular nucleases. Thus, the model implies a coordinate regulation at the level of translation of mRNAs for ferritin and erythroid AlS, on the one hand, and transferrin receptor on the other.The IRF has been clearly shown to be a cytosolic aconitase which, in its active 4Fe-4S form, has a turnover number s...

Activation pathways inducing the expression of the interferon (IFN)-gamma gene in a cytotoxic T lymphocyte (CTL) clone were studied for their effects on transcription and on mRNA stability. IFN-gamma was secreted by the CTL clone in response to the Ca2+ ionophore ionomycin when used in conjunction with either protein kinase C (PKC)-activating phorbol 12-myristate 13-acetate (PMA) or with agents increasing cAMP, including prostaglandin E2. We describe that ionomycin induced IFN-gamma gene transcription, which was totally inhibited in the presence of cyclosporin A (CSA), an immunosuppressant forming a calcineurin-inhibiting complex with cyclophilin. Ionomycin did not, however, permit accumulation of IFN-gamma mRNA. Activation of PKC by PMA or of cAMP-dependent protein kinase through increase in cAMP had no transcription-inducing effect, either alone or in conjunction with ionomycin, as measured in run on assays of the IFN-gamma gene. When transcription of the IFN-gamma gene, initiated in the presence of ionomycin and an agent increasing intracellular cAMP, was inhibited by CSA in the absence of PKC or cAMP-dependent protein kinase activation, the IFN-gamma mRNA was rapidly degraded (half-life = 30 min). When either PKC was activated or intracellular cAMP was increased at the time of inhibition with CSA, a stabilizing effect was observed on IFN-gamma mRNA, which led to an increase in secreted IFN-gamma. These effects were selective, they did not affect the rate of transcription of the actin gene, nor the accumulation of actin mRNA. These results show that (i) post-transcriptional events can be critical for IFN-gamma expression in activated lymphocytes, and (ii) specific stabilization of IFN-gamma mRNA can be mediated by activation of two different protein kinases involved in T cell activation.