STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide

Abstract

RNA-binding activity of IRP1 (iron regulatory protein 1) is regulated by the insertion/extrusion of a [4Fe-4S] cluster into/from the IRP1 molecule. NO (nitic oxide), whose ability to activate IRP1 by removing its [4Fe-4S] cluster is well known, has also been shown to down-regulate expression of the IRP1 gene. In the present study, we examine whether this regulation occurs at the transcriptional level. Analysis of the mouse IRP1 promoter sequence revealed two conserved putative binding sites for transcription factor(s) regulated by NO and/or changes in intracellular iron level: Sp1 (promoter-selective transcription factor 1) and MTF1 (metal transcription factor 1), plus GAS (interferon-γ -activated sequence), a binding site for STAT (signal transducer and activator of transcription) proteins. In order to define the functional activity of these sequences, reporter constructs were generated through the insertion of overlapping fragments of the mouse IRP1 promoter upstream of the luciferase gene. Transient expression assays following transfection of HuH7 cells with these plasmids revealed that while both the Sp1 and GAS sequences are involved in basal transcriptional activity of the IRP1 promoter, the role of the latter is predominant. Analysis of protein binding to these sequences in EMSAs (electrophoretic mobility-shift assays) using nuclear extracts from mouse RAW 264.7 macrophages stimulated to synthesize NO showed a significant decrease in the formation of Sp1-DNA and STAT-DNA complexes, compared with controls. We have also demonstrated that the GAS sequence is involved in NO-dependent down-regulation of IRP1 transcription. Further analysis revealed that levels of STAT5a and STAT5b in the nucleus and cytosol of NO-producing macrophages are substantially lower than in control cells. These findings provide evidence that STAT5 proteins play a role in NO-mediated down-regulation of IRP1 gene expression.

Iron Regulatory Proteins: From Molecular Mechanisms to Drug Development

“…8). When iron availability changes, IRP1 protein levels remain nearly constant, and the same occurs under other conditions known to alter IRP1 binding activity, although a STAT5-dependent (195) decrease in IRP1 protein levels has been reported in nitric oxide-treated cells (148). IRE binding is also controlled by the oxidation-reduction state of the cysteine residues involved in cluster coordination (79,80), which is why it was initially proposed that a reversible switch between a cluster-containing holoprotein and a cluster-deficient apoprotein allows aconitase=IRP1 to sense iron levels constantly and adapt them to cell requirements without any changes in protein levels.…”

High D‐glucose reduces SLC29A1 promoter activity and adenosine transport involving specific protein 1 in human umbilical vein endothelium

“…Several studies report that NO modulates transcription factors including Sp1 (Wang et al, 2002; Jeong et al, 2004; Starzynski et al, 2006) and NF‐κB (Park et al, 1997; Park and Wei, 2003). Equally, PKC activators increased Sp1 protein abundance in calf pulmonary artery endothelial cells (Tanaka et al, 2000) and insulin‐increased expression of PKC‐δ is regulated by PKC‐α via a mechanism requiring Sp1 activation in skeletal muscle L6 cells (Horovitz‐Fried and Sampson, 2007).…”

“…Activity of the zinc finger promoter‐selective transcription factor specific protein 1 (Sp1) is modulated by NO (Wang et al, 1999; Bogdan, 2001; Sellak et al, 2002; Starzynski et al, 2006), MEK/ERKs (Fiuza et al, 2003), and PKC (Tanaka et al, 2000; Horovitz‐Fried and Sampson, 2007), altering gene expression in several cell types (Bogdan, 2001; Jeong et al, 2004; Chu and Ferro, 2005; Paradkar and Roth, 2006; Starzynski et al, 2006). SLC29A1 promoter activity of the sequence upstream −795 bp from ATG translation start codon is repressed in a NO‐dependent manner in primary cultures of HUVEC (Farías et al, 2006; Puebla et al, 2007).…”

Glucocorticoid Causes Iron Accumulation in Liver by Up-Regulating Expression of Iron Regulatory Protein 1 Gene Through GR and STAT5

“…It has been described that STAT5 -/-mouse embryos often suffer from severe microcytic anemia, a disease mostly associated with iron deficiency and characterized by smallsized red blood cells [12], suggesting that STAT5 might be involved in iron metabolism. Besides, mouse IRP1 promoter has been shown to have a binding site for STAT5 protein, which participates in NO-mediated down-regulation of IRP1 expression [13]. Thus, it appears that STAT5 may play an essential role in the regulation of iron metabolism and IRP1 gene expression.…”