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

Iron regulatory protein 1 (IRP1) is an RNA binding protein that posttranscr iptionally modulates the expression of mRNAs coding for proteins involved i n iron metabolism. It has long been held that its RNA binding activity is r egulated posttranslationally by the insertion/extrusion of a 4Fe-4S cluster , without changes in IRP1 levels. However, the question of a possible regul ation of the expression of this protein has remained open. In the present s tudy we analyzed the modulation of IRP1 expression in murine macrophages, W e showed that activation by IFN-gamma and/or lipopolysaccharide, which indu ces IRP1 RNA binding activity via nitric oxide (NO). results simultaneously in a reduction in IRP1 protein levels, as determined by Western blot analy ses. IRP1 expression decreased time-dependently to about 40% of control lev els 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 (N OS) inhibitor N-monomethyl-L-arginine. No changes in IRP1 levels could be d etected in stimulated peritoneal macrophages from NOS2 knockout (NOS2(-/-)) mice, unlike wild-type mice. Converse modulation of IRP1 RNA binding activ ity and IRP1 levels could be reproduced by exogenous NO and also was observ ed in nonmacrophage cells cocultured with NO-producing macrophages. We also analyzed IRP1 mRNA levels by Northern blotting and found a decrease in IRP 1 mRNA expression after stimulation with IFN-gamma plus lipopolysaccharide, which was abrogated in the presence of N-monomethyl-L-arginine. This is ev idence that IRP1 is regulated by a physiological stimulus other than posttr anslationally.