Mouse genetic background impacts both on iron and non-iron metals parameters and on their relationships

International audienceIron is reported to interact with other metals. In addition, it has been shown that genetic background may impact iron metabolism. Our objective was to characterize, in mice of three genetic backgrounds, the links between iron and several non-iron metals. Thirty normal mice (C57BL/6, Balb/c and DBA/2; n = 10 for each group), fed with the same diet, were studied. Quantification of iron, zinc, cobalt, copper, manganese, magnesium and rubidium was performed by ICP/MS in plasma, erythrocytes, liver and spleen. Transferrin saturation was determined. Hepatic hepcidin1 mRNA level was evaluated by quantitative RT-PCR. As previously reported, iron parameters were modulated by genetic background with significantly higher values for plasma iron parameters and liver iron concentration in DBA/2 and Balb/c strains. Hepatic hepcidin1 mRNA level was lower in DBA/2 mice. No iron parameter was correlated with hepcidin1 mRNA levels. Principal component analysis of the data obtained for non-iron metals indicated that metals parameters stratified the mice according to their genetic background. Plasma and tissue metals parameters that are dependent or independent of genetic background were identified. Moreover, relationships were found between plasma and tissue content of iron and some other metals parameters. Our data: (i) confirms the impact of the genetic background on iron parameters, (ii) shows that genetic background may also play a role in the metabolism of non-iron metals, (iii) identifies links between iron and other metals parameters which may have implications in the understanding and, potentially, the modulation of iron metabolis

Etude de la répression transcriptionnelle des gènes des interferons-A murins (rôles du facteur à homéodomaine Pitx1)

L'étude de la régulation transcriptionnelle des différents membres de la famille multigénique des interférons-A (IFN-A) constitue un bon modèle pour comprendre les mécanismes d'activation et de répression d'un gène eucaryote. Certains de ces gènes sont hautement inductibles par les virus alors que d'autres ne le sont pas. Dans les cellules L929 murines après infection par le virus NDV, l'INF-A4 est hautement inductible tandis que l'IFN-A11 est faiblement inductible. L'expression différentielle des gènes des IFN-A murins s'explique par des substitutions dans les éléments proximaux activateurs de réponse aux virus (VRE), mais également par la présence dans la partie distale du promoteur IFN-A11 d'un domaine de régulation négative. Le même élément est présent dans le promoteur de l'IFN-A4 hautement inductible cependant, l'effet silenceur du DNRE est rendu inopérant par la présence d'un domaine antisilenceur central situé entre l'élément distal DNRE et l'élément proximal activateur VRE. L'élément DNRE est capable de fixer les protéines High Mobility Group I (Y) qui ne semblent pas être impliquées dans l'effet répresseur et une protéine qui pourrait être responsable de l'activité silenceur. Le clonage d'expression "in vivo" chez "Saccharomyces cerevisiae" (clonage simple-hybride) a permis d'isoler une protéine à homéodomaine : Pitx1 (Pituitary homeo box 1). Nous avons montré que Pitx1 joue un rôle important sur la répression transcriptionnelle des gènes IFN-A et qu'elle est impliquée dans l'expression différentielle de ces gènes. Nous avons recherché le mécanisme de répression de ce facteur par l'étude de sa structure et de sa fonction dans le cadre de la régulation transcriptionnelle des gènes IFN-A après induction virale. Cette étude a permis de mettre en évidence de nouveaux domaines fonctionnels de la protéine Pitx1 impliqués dans la trans-répression et dans l'interaction avec de nouveaux partenaires de ce facteur présents sur les éléments VRE-A.The regulation of the expression of type I interferon (IFN-A and IFN-B) multigene family is a model for understanding the positive and negative transcriptional mechanisms of eukaryotic gene regulation. The interferon (IFN)-A4 gene is highly inducible upon virus infection, whereas the IFN-A11 is not. In mouse L929 cells, the weak response of this promoter to viral induction is due to a combination of both point mutations in the virus responsive element (VRE) and the presence of negatively regulating sequences surrounding the VRE that can negatively modulate the transcriptional response to viral induction. This minimal 20-mer distal negative regularoty element (DNRE) in both promoters is necessarey and sufficient for the silencing and a region in the highly inducible IFN-A4 promoter located between the silencer and the virus responsive element overrides the silencer activity. (...)PARIS5-BU Saints-Pères (751062109) / SudocSudocFranceF

GATA-4 transcription factor regulates hepatic hepcidin expression.

International audienceHepcidin, an hormone mainly synthesized by hepatocytes and secreted in plasma, controls iron bioavailability. Thus, by inducing the internalization of the iron exporter ferroportin, it regulates iron release from macrophages, enterocytes and hepatocytes towards plasma. Abnormal levels of hepcidin expression alter plasma iron parameters and lead to iron metabolism disorders. To understand the mechanisms controlling hepcidin gene expression is therefore an important goal. We identified a potential GATA binding site within the human hepcidin promoter. Indeed, in hepatic HepG2 cells, luciferase experiments demonstrated that mutation of this GATA binding site impaired the hepcidin promoter transcriptional activity in basal condition. Gel retardation experiment showed that GATA-4 could bind this site. Cotransfection of GATA-4 expression vector with an hepcidin promoter reporter construct enhanced hepcidin promoter transcriptional activity. Furthermore, modulation of GATA-4 mRNA expression using specific siRNAs downregulated endogenous hepcidin gene expression. Finally, we found that mutation of the GATA binding site impaired the interleukin-6 induction of hepcidin gene expression, but did not prevent the bone morphogenetic protein-6 response. In conclusion, our findings: i) indicate that GATA-4 may participate to the control of hepcidin expression, and ii) suggest that alteration of its expression could contribute to the development of iron-related disorders

A new mutation in the hepcidin promoter impairs its BMP response and contributes to a severe phenotype in HFE related hemochromatosis

Bone morphogenetic protein (BMP) signaling activates transcription of the master iron regulator hepcidin in the liver. This study shows that a heterozygous mutation in the BMP-responsive element of the hepcidin gene promoter is associated with massive iron overload in a patient homozygous for the common HFE mutation, suggesting a new molecular mechanism of iron overload

Valproic Acid Induces Hepcidin Expression

International audienceMeeting Abstract: 14

The POU Transcription Factor Oct-1 Represses Virus-Induced Interferon A Gene Expression

Alpha interferon (IFN-α) and IFN-β are able to interfere with viral infection. They exert a vast array of biologic functions, including growth arrest, cell differentiation, and immune system regulation. This regulation extends from innate immunity to cellular and humoral adaptive immune responses. A strict control of expression is needed to prevent detrimental effects of unregulated IFN. Multiple IFN-A subtypes are coordinately induced in human and mouse cells infected by virus and exhibit differences in expression of their individual mRNAs. We demonstrated that the weakly expressed IFN-A11 gene is negatively regulated after viral infection, due to a distal negative regulatory element, binding homeoprotein pituitary homeobox 1 (Pitx1). Here we show that the POU protein Oct-1 binds in vitro and in vivo to the IFN-A11 promoter and represses IFN-A expression upon interferon regulatory factor overexpression. Furthermore, we show that Oct-1-deficient MEFs exhibit increased in vivo IFN-A gene expression and increased antiviral activity. Finally, the IFN-A expression pattern is modified in Oct-1-deficient MEFs. The broad representation of effective and potent octamer-like sequences within IFN-A promoters suggests an important role for Oct-1 in IFN-A regulation

Molecular diagnosis of genetic iron-overload disorders.

International audienceGenetic iron overload has long been confined to the picture of classical hemochromatosis related to the HFE C282Y mutation (type 1 hemochromatosis). C282Y homozygosity affects approximately three people out of 1000 of the Caucasian population, representing one of the most frequent genetic predispositions. It has, however, rapidly become clear that the HFE C282Y mutation is not the sole culprit in genetic iron overload. Several novel mutations in HFE and other genes have been discovered and related to various entities, which are now known as types 2, 3 and 4 hemochromatosis. These diseases are far less frequent than the classical type 1 hemochromatosis but, by contrast, are not limited to the Caucasian population. Molecular diagnosis obviously plays a key role in the diagnostic strategy. In the future, it will undoubtedly enable not only identification of new diagnostic markers, but also provide potential molecular targets for pathophysiologically based innovative therapeutic approaches