Insights on systemic and cellular iron homeostasis: hepcidin responses to oral and parenteral iron loading and an alternative mechanism for ferritin mRNA translation

Iron is vital for all living organisms, but due to its ability to readily accept or donate electrons, it is also potentially toxic. Finely tuned mechanisms have evolved to control iron homeostasis at the systemic and cellular level. The peptide hormone hepcidin controls systemic iron homeostasis by binding to and degrading the iron exporter, ferroportin, leading to decreased iron efflux from duodenal enterocytes, macrophages and hepatocytes into the blood stream. Cellular iron homeostasis is regulated by the IRE / IRP system, which controls the levels of proteins involved in iron uptake, utilization, export and storage in a coordinated manner. Excess intracellular iron is stored and detoxified in ferritin. In this work, we study how an excess of iron is managed at the systemic and cellular level. In chapter II, we hypothesize that dietary and parenteral iron loading have differential effects on body iron status and hepcidin expression in mice. We perform time – course experiments and compare the effects of dietary and parenteral iron loading on circulating and tissue iron parameters. We show that dietary iron overload exceeds the capacity of hepcidin to lower body iron levels, and parenteral iron loading elicits a delayed hepcidin response. We provide evidence that circulating holo – transferrin and hepatocytic iron are the sole iron signals for hepcidin activation. In chapter III, we examine how an excess of iron is managed at the cellular level. We hypothesize that ferritin might benefit from an alternative, IRES - dependent mechanism of translation. We inhibit global or ferritin - specific cap – dependent translation initiation and challenge the cells with iron. We show that ferritin by – passes both the global and the specific inhibition of translation. We then test for the presence of an IRES in the 5'UTR of ferritin mRNA and further validate this sequence.Le fer est vital pour tous les organismes vivants, cependant étant donné son habilité à donner ou accepter des électrons facilement, il a aussi le potentiel d'être toxique. Des mécanismes très précis ont évolué pour contrôler l'homéostasie du fer aux niveaux systémique et cellulaire. L'hormone peptidique, hepcidine, contrôle l'homéostasie du fer au niveau systémique par la dégradation de la ferroportine, l'exportateur cellulaire du fer. En conséquence, l'efflux du fer des entérocytes, des macrophages et des hépatocytes vers la circulation diminue. Au niveau cellulaire, le système IRE / IRP contrôle, d'une manière coordonnée, les niveaux des protéines impliquées dans l'acquisition, l'utilisation, l'exportation et le stockage du fer. L'excès de fer est stocké dans la ferritine. Dans ce travail, nous examinons comment l'excès de fer est géré aux niveaux systémique et cellulaire. Dans le chapitre II, nous émettons l'hypothèse que les surcharges orale et parentérale en fer ont des effets différents sur l'homéostasie systémique du fer et sur l'expression de l'hepcidine chez les souris. Nous comparons les effets des surcharges orale et parentérale en fer aux niveaux circulatoire et tissulaire. Nous démontrons que la surcharge orale en fer excède la capacité hypoferrémique de l'hepcidine alors que la surcharge parentérale en fer induit une réponse retardée de l'hepcidine. Nous apportons aussi la preuve que la holo – transferrine circulatoire et le fer hépatocytaire sont les signaux uniques de l'activation ferrique de l'hepcidine. Dans le chapitre III, nous examinons comment l'excès de fer est géré au niveau cellulaire. Nous émettons l'hypothèse que la ferritine bénéficie d'un mécanisme alternatif de traduction, dépendant d'une séquence IRES. Nous inhibons l'initiation de la traduction dépendante de la coiffe 5' globalement, ou spécifiquement pour la ferritine, et traitons les cellules avec une source de fer. Nous démontrons que la ferritine surpasse le blocage global ou spécifique de la traduction dépendante de la coiffe 5'. Nous testons la présence d'une séquence IRES dans l'extrémité 5' de l'ARNm et par la suite nous la validons

Iron-Dependent Regulation of Hepcidin in Hjv−/− Mice: Evidence That Hemojuvelin Is Dispensable for Sensing Body Iron Levels

<div><p>Hemojuvelin (Hjv) is a bone morphogenetic protein (BMP) co-receptor involved in the control of systemic iron homeostasis. Functional inactivation of Hjv leads to severe iron overload in humans and mice due to marked suppression of the iron-regulatory hormone hepcidin. To investigate the role of Hjv in body iron sensing, Hjv−/− mice and isogenic wild type controls were placed on a moderately low, a standard or a high iron diet for four weeks. Hjv−/− mice developed systemic iron overload under all regimens. Transferrin (Tf) was highly saturated regardless of the dietary iron content, while liver iron deposition was proportional to it. Hepcidin mRNA expression responded to fluctuations in dietary iron intake, despite the absence of Hjv. Nevertheless, iron-dependent upregulation of hepcidin was more than an order of magnitude lower compared to that seen in wild type controls. Likewise, iron signaling via the BMP/Smad pathway was preserved but substantially attenuated. These findings suggest that Hjv is not required for sensing of body iron levels and merely functions as an enhancer for iron signaling to hepcidin.</p></div

Effects of dietary iron manipulations on hepatic and splenic iron content.

<p>Livers and spleens from the Hjv−/− and wild type mice described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085530#pone-0085530-g001" target="_blank">Fig. 1</a> were used for histological detection of iron by staining with Perls’ Prussian blue, and for tissue iron quantification by the ferrozine assay. (A) Visualization of ferric deposits in representative liver sections (magnification: 10×). (B) Quantification of non-heme hepatic iron. (C) Visualization of ferric deposits in representative spleen sections (original magnification: 10×). (D) Quantification of non-heme splenic iron. Data in (B) and (D) are presented as the mean ± SEM. The p values were calculated by using one-way ANOVA with Bonferroni post-test correction. Detailed statistical analysis is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085530#pone.0085530.s006" target="_blank">Table S1</a>.</p

Residual iron-dependent regulation of hepatic hepcidin mRNA expression in Hjv−/− mice.

<p>RNA was extracted from tissues of the Hjv−/− and wild type mice described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085530#pone-0085530-g001" target="_blank">Fig. 1</a> and used for qPCR analysis. (A) Expression of hepatic hepcidin mRNA. (B) Expression of splenic hepcidin mRNA. Note that absolute hepcidin mRNA levels in the spleen are >100 times lower than in the liver. Data are presented as the mean ± SEM. The p values were calculated by using one-way ANOVA with Bonferroni post-test correction. Detailed statistical analysis is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085530#pone.0085530.s006" target="_blank">Table S1</a>.</p

Serum and liver iron indices in wild type and Hjv−/− mice of 129S6/SvEvTac or C57BL/6 genetic background (n = 10 male C57BL/6 mice for each genotype; n = 5 male 129S6/SvEvTac mice for each genotype).

<p>p<0.05;</p><p>p<0.01;</p><p>p<0.001 vs 129S6/SvEvTac mice of the same genotype (Student’s t test).</p><p>All differences among wild type and Hjv−/− mice of the same strain are statistically significant (p values not shown).</p

Hjv−/− mice exhibit elevated serum iron indices independently of dietary iron intake.

<p>Ten-week old male Hjv−/− and wild type mice (n = 10 for each group) in C57BL/6 background were placed on diets with variable iron content (low: 75–100 ppm; normal: 225 ppm; high: 225 ppm plus 2% carbonyl iron). After four weeks the animals were sacrificed and sera were analyzed for iron (A), transferrin saturation (B), and ferritin (C). Data are presented as the mean ± SEM. Statistical analysis is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085530#pone.0085530.s006" target="_blank">Table S1</a>.</p

List of primers used for qPCR or genotyping.

<p>List of primers used for qPCR or genotyping.</p