STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide
| Autor: | Jean-Claude Drapier, Ana Sofia Gonçalves, Françoise Muzeau, Ewa Smuda, Paweł Lipiński, Rafał R. Starzyński, Zofia Tyrolczyk, Carole Beaumont |
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| Přispěvatelé: | Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC) |
| Rok vydání: | 2006 |
| Předmět: |
Lipopolysaccharides
Sp1 Transcription Factor Iron Response element CAAT box Down-Regulation Biology Nitric Oxide Transfection Biochemistry Cell Line Interferon-gamma Mice 03 medical and health sciences Upstream activating sequence Cytosol Sp3 transcription factor STAT5 Transcription Factor Animals Humans Iron Regulatory Protein 1 RNA Messenger Promoter Regions Genetic Molecular Biology Transcription factor 030304 developmental biology Cell Nucleus 0303 health sciences Sp1 transcription factor Binding Sites General transcription factor [CHIM.ORGA]Chemical Sciences/Organic chemistry Macrophages 030302 biochemistry & molecular biology Promoter DNA Cell Biology Molecular biology Research Article |
| Zdroj: | Biochemical Journal Biochemical Journal, Portland Press, 2006, 400(Part2), pp.367-375. ⟨10.1042/BJ20060623⟩ |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj20060623 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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