Hamp1 mRNA and plasma hepcidin levels are influenced by sex and strain but do not predict tissue iron levels in inbred mice

Iron homeostasis is tightly regulated, and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex- and strain-dependent differences in mRNA and serum levels of hepcidin and reported conflicting data on the relationship between hepcidin ( Hamp1) mRNA levels and iron status. Our aim was to clarify the relationships between strain, sex, and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, Hamp1 mRNA, liver iron, and plasma diferric transferrin levels were measured in 14 inbred strains on a control diet; and second, Hamp1 mRNA and plasma hepcidin levels in both sexes and iron levels in the heart, kidneys, liver, pancreas, and spleen in males were measured in nine inbred/recombinant inbred strains raised on an iron-sufficient or high-iron diet. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). However, liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice fed iron-sufficient or high-iron diets, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. We also measured plasma erythroferrone, performed RNA-sequencing analysis of liver samples from six inbred strains fed the iron-sufficient, low-iron, or high-iron diets, and explored differences in gene expression between the strains with the highest and lowest hepcidin levels. NEW & NOTEWORTHY Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). Liver iron and diferric transferrin levels are not predictors of Hamp1 mRNA levels in mice, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males.

The Oxidative Function of Diferric Transferrin

There is evidence for an unexpected role of diferric transferrin as a terminal oxidase for the transplasma membrane oxidation of cytosolic NADH. In the original studies which showed the reduction of iron in transferrin by the plasma membranes NADH oxidase, the possible role of the reduction on iron uptake was emphasized. The rapid reoxidation of transferrin iron under aerobic conditions precludes a role for surface reduction at neutral pH for release of iron for uptake at the plasma membrane. The stimulation of cytosolic NADH oxidation by diferric transferrin indicates that the transferrin can act as a terminal oxidase for the transplasma membrane NADH oxidase or can bind to a site which activates the oxidase. Since plasma membrane NADH oxidases clearly play a role in cell signaling, the relation of ferric transferrin stimulation of NADH oxidase to cell control should be considered, especially in relation to the growth promotion by transferrin not related to iron uptake. The oxidase can also contribute to control of cytosolic NAD concentration, and thereby can activate sirtuins for control of ageing and growth.

Further Evidence for the “Kiss and Run” Hypothesis of Iron Delivery to Mitochondria in Erythroid Cells,

Abstract 3178 Delivery of iron (Fe) to most cells occurs following the binding of diferric transferrin (Tf) to its cognate receptors on the cell membrane. The Tf-receptor complexes are then internalized via endocytosis, and iron is released from Tf by a process involving endosomal acidification and reduction by Steap3. Iron is then transported across the endosomal membrane by the divalent metal transporter, DMT1. Unfortunately, the post-endosomal path of iron within cells remains elusive or is, at best, controversial. It has been commonly accepted that a low molecular weight intermediate chaperones iron in transit from endosomes to mitochondria and other sites of utilization; however, this much sought iron binding intermediate has never been identified. In erythroid cells, more than 90% of iron has to enter mitochondria where ferrochelatase, the final enzyme in the heme biosynthetic pathway that inserts Fe2+ into protoporphyrin IX, resides. Indeed, strong evidence exists for specific targeting of Fe toward mitochondria in developing red blood cells in which iron acquired from Tf continues to flow into mitochondria even when the synthesis of protoporphyrin IX is suppressed. Based on this, we have formulated the hypothesis that, in erythroid cells, a transient mitochondrion-endosome interaction is involved in Fe translocation to its final destination and have collected experimental support for this proposition (Zhang et al. Blood 105:368, 2005; Sheftel et al. Blood 110: 125, 2007). We have previously shown, using 3D live confocal imaging, that the iron delivery pathway in reticulocytes involves a transient interaction of endosomes with mitochondria. Moreover, we have demonstrated the interaction of these organelles by a novel method exploiting flow cytometry to analyze reticulocyte lysates labeled with Alexa Green Transferrin (AGTf) and MitoTracker Deep Red (MTDR). By using this new technique of flow subcytometry, we identified a double-labeled population representing endosomes interacting with mitochondria. The dynamic nature of this interaction was shown by chase experiments in which a time-dependent decrease of the double-labeled population was observed when reticulocytes were washed and re-incubated with unlabeled Fe2-Tf. Furthermore, we have shown that the iron status of endosomes governs the efficacy of endosome-mediated iron delivery to mitochondria. Experiments with heme, which feedback inhibited the release of iron from Tf in endosomes, slows the generation of the double-labeled population. Additionally, treatment of cells with heme in chase experiments retarded the dissociation of endosomes from mitochondria. In this study, we provide further evidence for an interaction between mitochondria with endosomes. Fluorescently-labeled mitochondria isolated from mouse reticulocytes with MTDR and AGTf were analyzed using 2D confocal microscopy. Results from these experiments confirmed that mitochondria indeed come in physical contact with endosomes. In addition, we have used different constructs of fluorescently labeled, recombinant human Tf, which either remain permanently bound to iron (recombinant diferric-transferrin; rTf) or cannot bind to iron (recombinant apotransferrin; rapoTf), in flow subcytometry studies. As expected, these studies showed that reticulocytes incubated with MTDR and rapoTf failed to produce a double-labeled population in uptake experiments. Interestingly, when reticulocyte lysates were incubated with MTDR and rTf, compared to controls using wild type human Tf, the size of the double-labeled population was decreased. This suggests that failure of iron release from rTf may interfere with the process of endocytosis or endosomal trafficking. Disclosures: No relevant conflicts of interest to declare.

In silico QTL mapping of basal liver iron levels in inbred mouse strains

Both iron deficiency and iron excess are detrimental in many organisms, and previous studies in both mice and humans suggest that genetic variation may influence iron status in mammals. However, these genetic factors are not well defined. To address this issue, we measured basal liver iron levels in 18 inbred strains of mice of both sexes on a defined iron diet and found ∼4-fold variation in liver iron in males (lowest 153 μg/g, highest 661 μg/g) and ∼3-fold variation in females (lowest 222 μg/g, highest 658 μg/g). We carried out a genome-wide association mapping to identify haplotypes underlying differences in liver iron and three other related traits (copper and zinc liver levels, and plasma diferric transferrin levels) in a subset of 14 inbred strains for which genotype information was available. We identified two putative quantitative trait loci (QTL) that contain genes with a known role in iron metabolism: Eif2ak1 and Igf2r. We also identified four putative QTL that reside in previously identified iron-related QTL and 22 novel putative QTL. The most promising putative QTL include a 0.22 Mb region on Chromosome 7 and a 0.32 Mb region on Chromosome 11 that both contain only one candidate gene, Adam12 and Gria1, respectively. Identified putative QTL are good candidates for further refinement and subsequent functional studies.

Comparison of 3 Tfr2-deficient murine models suggests distinct functions for Tfr2-α and Tfr2-β isoforms in different tissues

Transferrin receptor 2 (TFR2) is a transmembrane protein that is mutated in hemochromatosis type 3. The TFR2 gene is transcribed in 2 main isoforms: the full-length (α) and a shorter form (β). α-Tfr2 is the sensor of diferric transferrin, implicated in the modulation of hepcidin, the main regulator of iron homeostasis. The function of the putative β-Tfr2 protein is unknown. We have developed a new mouse model (KI) lacking β-Tfr2 compared with Tfr2 knockout mice (KO). Adult Tfr2 KO mice show liver iron overload and inadequate hepcidin levels relative to body iron stores, even though they increase Bmp6 production. KI mice have normal transferrin saturation, liver iron concentration, hepcidin and Bmp6 levels but show a transient anemia at young age and severe spleen iron accumulation in adult animals. Fpn1 is strikingly decreased in the spleen of these animals. These findings and the expression of β-Tfr2 in wild-type mice spleen suggest a role for β-Tfr2 in Fpn1 transcriptional control. Selective inactivation of liver α-Tfr2 in KI mice (LCKO-KI) returned the phenotype to liver iron overload. Our results strengthen the function of hepatic α-Tfr2 in hepcidin activation, suggest a role for extrahepatic Tfr2 and indicate that β-Tfr2 may specifically control spleen iron efflux.

Diferric Transferrin-Sensed Hepcidin Upregulation in Human Hepatoma-Derived Cell Line Is Differentially Controlled in Each Isoform 20, 22, 25; Confirmation by a Novel Simultaneous Quantification by Liquid Chromatography/ Tandem Mass Spectrometry.

Abstract 4046 Poster Board III-981 Introduction and aim Hepcidin is a key molecule of body iron metabolism, and the expression at mRNA level is thought to be upregulated by iron loading. As the mature processed form of human hepcidin is known to have 3 isoforms, hepcidin -20, -22, and -25, and hepcidin -25 is thought to be the major isoform active in iron metabolism. However, the physiological roles of other isoforms are poorly understood. Concerning the study on the regulatory mechanism on hepcidin expression, most studies have been only performed at the transcriptional level because of the difficulty of quantification of hepcidin in cell culture media; therefore, the experiments in vitro would be valuable. We therefore developed a sensitive new method for measuring hepcidin that can simultaneously measure the isoforms in culture media, and studied the expression patterns of isoforms at mature protein level in various human hepatoma-derived cell lines with and without diferric transferrin. Methods Quantification of human hepcidin -20, -22, -25 was performed using liquid chromatography (LC) - tandem mass spectrometry (MS) which we newly developed. Selected reaction monitoring (SRM) transitions and the collision energies were settled for each isoform respectively. Quantification of hepcidin isoforms in culture medium of 13 strains of hepatoma-derived cell lines was performed. Various stimulants for hepcidin expression, such as interleukin-6, diferric transferrin and etc, were also used for investigating the response patterns of hepcidin isoforms. Results Upon optimization of SRM conditions, the most intense precursor ions were selected in each mass spectrum to detect hepcidin isoforms. Product ions were selected to maximize sensitivity and selectivity. Despite using culture media including 10% FBS as matrix, isoform peaks were not interfered with by a blank matrix, indicating the method has good selectivity. Calibration curves were constructed over the range 2-1,000 ng/mL, and linearity of the calibration curves by weighted (1/x2) linear regression was excellent (correlation coefficient: r=0.9974 for hepcidin-20, r=0.9937 for hepcidin-22, r=0.9950 for hepcidin-25). Accuracies for back-corrected concentrations were 99.7-122.1% for hepcidin-20, 102.6-132.5% for hepcidin-22, and 99.1-141.2% for hepcidin-25. These results indicate that the method is adequate for quantifying hepcidin isoforms in culture media. We also found that substantial difference of hepcidin isoforms' expression patterns among human hepatoma-derived cell lines, and the patterns were divided into 5 groups. Response patterns for various stimulants were also different among those groups. Especially, human diferric transferrin upregulates hepcidin-20 and -22 in WRL68 cells, and hepcidin-22 in Hep3B, HuH-2, HuH-4, and HuH-6 cells; this should be the first report that human diferric transferrin upregulates hepcidin isoforms other than hepcidin-25 in human hepatocyte-derived cells. Conclusions We have devised a novel method for simultaneous quantification of hepcidin isoforms in culture media. Although most previous studies only observe the changes of hepcidin expression at mRNA level, our method revealed heterogeneous expressions of hepcidin isoforms and hepcidin upregulation by human diferric transferrin in human hepatocyte-derived cells at the peptide level. The fact of hepcidin isoforms' upregulation by human diferric transferrin in human hepatocyte-derived cells might be the clue to elucidate the mechanism for iron sensor in human body. We believe that this novel quantification method can contribute to further progress, especially in vitro research on the regulation of hepcidin expression. Disclosures: No relevant conflicts of interest to declare.

Characterization of the Interaction Between Diferric Transferrin and Transferrin Receptor 2 by Functional Assays and Atomic Force Microscopy (AFM).

[Introduction and aim] Transferrin receptor 2 (TfR2) is a homologue of the classical transferrin receptor 1 (TfR1), and TfR2 has two isoforms, α and β. Like TfR1, TfR2α is a type II membrane protein expressing mainly in the hepatocytes on their cell surface, but the β form lacks intracellular and transmembrane portions and therefore is likely to be an intracellular protein. Although the main physiological functions of these two isoforms are still not fully understood, TfR2α binds diferric transferrin (Tf), and is therefore thought likely to be involved in cellular iron metabolism. In fact, mutations of TfR2 cause hemochromatosis, implying that the function of TfR2 might be a regulation of iron metabolism. The aim of the present study was to investigate the interaction of TfR2α with Tf by functional assays and atomic force microscopy (AFM), which would be a powerful tool for investigation of the interaction between ligand and receptor on living cell surface. [Methods] To investigate the functional properties of TfR2α, we established the stable clone that expresses TfR2α protein with FLAG-tagging from transferrin receptor-deficient Chinese hamster ovary (CHO) cells (TRVb). That clone was applied for the 125I –labeled Tf (125I –Tf) binding study at 4 oC. The cells were applied for Tf and iron uptake study using 125I –Tf and 59Fe –loaded Tf at 37 oC. AFM is the method that can investigate the unbinding force between receptor and ligand at single molecule level when their binding is physically detached, and we applied this method for determine the interaction between Tf and TfR2α using the transiently transfected human hepatome-derived HLF cells with TfR2α-expression vector. [Results] The association constant for binding of 125I-Tf to TfR2α was calculated to be 5.6 x 106 M−1 from non-linear least squares curve fitting to a saturable binding isotherm, which is much lower than that of TfR1. Although CHO cells showed a receptor-independent non-specific association with Tf at 37 oC, we observed cell-associated Tf persisting after acid-washing in TfR2α overexpressing cells, We also confirmied the existence of internalized Tf into cells via TfR2α. Overexpressed TfR2α protein was also shown to mediate iron uptake although its rate of iron donation is slower than TfR1. The interaction between Tf and TfR2α was also confirmed by AFM, but the unbinding force was different from that between Tf and TfR1. This report might be the first that showed the binding and unbinding properties between Tf and TfR2α by AFM. [Discussion and conclusions] TfR2α binds to Tf although its affinity for Tf is low, and TfR2α possesses iron donating ability to the cells when TfR2α is over-expressed in our functional assays. AFM study also showed the binding between Tf and TfR2α on cell surface, but the properties of unbinding between them were different from those between Tf and TfR1. In conclusion, TfRα binds to Tf at cell surface and functions as iron donator to the cells like TfR1, but there should be the difference in the properties of binding with Tf between TfR1 and TfR2α, implying that the difference makes them having different physiological role in living organisms.