STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide.
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-gamma-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.
Starzynski
Rafal Radoslaw
RR
Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, ul. Postepu 1, 05-552 Wolka Kosowska, Poland.
Gonçalves
Ana Sofia
AS
Muzeau
Françoise
F
Tyrolczyk
Zofia
Z
Smuda
Ewa
E
Drapier
Jean-Claude
JC
Beaumont
Carole
C
Lipinski
Pawel
P
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D035941:Iron Regulatory Protein 1 / Q000096:biosynthesis* / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism / Q000502:physiology; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D050799:STAT5 Transcription Factor / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors / Q000378:metabolism; D014162:Transfection
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D050799:STAT5 Transcription Factor; D016329:Sp1 Transcription Factor; D009569:Nitric Oxide; D007371:Interferon-gamma; D004247:DNA; D007501:Iron; D035941:Iron Regulatory Protein 1