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.
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Research Article|
November 14 2006
STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide
Rafal Radoslaw Starzynski;
Rafal Radoslaw Starzynski
*Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, ul. Postepu 1, 05-552 Wolka Kosowska, Poland
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Ana Sofia Gonçalves;
Ana Sofia Gonçalves
†INSERM U773, Centre de Recherche Biomédical Bichat Beaujon CRB3, Faculté Xavier Bichat, BP416, 16 Rue Henri Huchard, 75870 Paris cedex 18, France
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Françoise Muzeau;
Françoise Muzeau
†INSERM U773, Centre de Recherche Biomédical Bichat Beaujon CRB3, Faculté Xavier Bichat, BP416, 16 Rue Henri Huchard, 75870 Paris cedex 18, France
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Zofia Tyrolczyk;
Zofia Tyrolczyk
*Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, ul. Postepu 1, 05-552 Wolka Kosowska, Poland
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Ewa Smuda;
Ewa Smuda
*Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, ul. Postepu 1, 05-552 Wolka Kosowska, Poland
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Jean-Claude Drapier;
Jean-Claude Drapier
‡CNRS, Institut de Chimie des Substances Naturelles, 91190 Gif-sur-Yvette, France
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Carole Beaumont;
Carole Beaumont
†INSERM U773, Centre de Recherche Biomédical Bichat Beaujon CRB3, Faculté Xavier Bichat, BP416, 16 Rue Henri Huchard, 75870 Paris cedex 18, France
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Pawel Lipinski
Pawel Lipinski
1
*Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, ul. Postepu 1, 05-552 Wolka Kosowska, Poland
1To whom correspondence should be addressed (email P.Lipinski@ighz.pl).
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Publisher: Portland Press Ltd
Received:
April 26 2006
Revision Received:
July 11 2006
Accepted:
August 04 2006
Accepted Manuscript online:
August 04 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 400 (2): 367–375.
Article history
Received:
April 26 2006
Revision Received:
July 11 2006
Accepted:
August 04 2006
Accepted Manuscript online:
August 04 2006
Citation
Rafal Radoslaw Starzynski, Ana Sofia Gonçalves, Françoise Muzeau, Zofia Tyrolczyk, Ewa Smuda, Jean-Claude Drapier, Carole Beaumont, Pawel Lipinski; STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide. Biochem J 1 December 2006; 400 (2): 367–375. doi: https://doi.org/10.1042/BJ20060623
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