Down-regulation of iron regulatory protein 1 gene expression by nitric oxide

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Abstract
Iron regulatory protein 1 (IRP1) is an RNA binding protein that posttranscriptionally modulates the expression of mRNAs coding for proteins involved in iron metabolism. It has long been held that its RNA binding activity is regulated posttranslationally by the insertion/extrusion of a 4Fe-4S cluster, without changes in IRP1 levels. However, the question of a possible regulation of the expression of this protein has remained open. In the present study we analyzed the modulation of IRP1 expression in murine macrophages. We showed that activation by IFN-γ and/or lipopolysaccharide, which induces IRP1 RNA binding activity via nitric oxide (NO), results simultaneously in a reduction in IRP1 protein levels, as determined by Western blot analyses. IRP1 expression decreased time-dependently to about 40% of control levels after 16 h. Down-regulation of IRP1 protein levels was correlated with the amount of NO produced and was partially abolished by the NO synthase (NOS) inhibitorN-monomethyl-l-arginine. No changes in IRP1 levels could be detected in stimulated peritoneal macrophages from NOS2 knockout (NOS2−/−) mice, unlike wild-type mice. Converse modulation of IRP1 RNA binding activity and IRP1 levels could be reproduced by exogenous NO and also was observed in nonmacrophage cells cocultured with NO-producing macrophages. We also analyzed IRP1 mRNA levels by Northern blotting and found a decrease in IRP1 mRNA expression after stimulation with IFN-γ plus lipopolysaccharide, which was abrogated in the presence ofN-monomethyl-l-arginine. This is evidence that IRP1 is regulated by a physiological stimulus other than posttranslationally.