Article

HFE downregulates iron uptake from transferrin and induces iron- regulatory protein activity in stably transfected cells

Department of Medicine, University Hospital of Heidelberg
Blood (Impact Factor: 10.43). 01/2000; 94(11):3915-21.
Source: PubMed

ABSTRACT Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular "labile iron pool." The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.

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    • "These molecules play a leading role in immune response; they present the antigens to the T cells to initiate the acquired (adaptive) immunity. The Hemochromatosis (Hfe) genes that influence the MHC I antigen transfer (Riedel et al., 1999), and the Lymphocyte activation gene3 (Lag3), responsible for limiting the pathogenic potential of CD4 + T cells (Bettini et al., 2011), both belong to MHC class II and are persistently suppressed after schistosoma infection. The production of Th2 cytokine IL-4 is important for inducing the granuloma formation and fibrosis, and IL-4 expression peaked at 4 weeks and 7 weeks pi in the spleen and liver, respectively (Burke et al., 2010a; Perry et al., 2011). "
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    • "A yellow co-labelling of the perinuclear region is seen with wild-type (Panels 2, 5) and mutant HFE (Panels 3, 6). levels (Roy et al., 1999, Riedel et al., 1999, Corsi et al., 1999, Ramalingam et al., 2000, Wang et al., 2002), reflecting iron deficiency. In our model, HFE-GFP over-expression had the opposite effect, illustrated by reduced expression of TfR. "
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    ABSTRACT: Hereditary hemochromatosis (HH) is a frequent recessive disorder of iron metabolism characterised by systemic iron overload. In Northern Europe, more than 90% of HH patients are homozygous for a mis-sense mutation (C282Y) in the HFE1 gene product. The HFE protein is the heavy chain of a MHC class I-related molecule and associates with beta2 microglobulin and the transferrin receptor. Its precise roles in iron metabolism and in the pathophysiology of HH are still unclear. In order to identify the cellular processing of HFE, an important step towards the understanding of the function of the protein, we stably over-expressed the wild type and mutated forms fused to the Green Fluorescent Protein in a melanocytic MHC class I expressing cell line, the Mel Juso cell line. In wild type and mutant clones, the fusion proteins were not detected at the cell surface but only in the cytoplasm. Their sub-cellular localisation was determined by co-labelling of cells with organite-specific antibodies and confocal microscopy. HFE-GFP followed initially HLA class I intracellular processing but co-localised with transferrin in early endosomes without recycling at the cell surface. The C282Y-GFP fusion protein followed a different folding pathway to exit endoplasmic reticulum. Over-expression of the wild-type protein lead to a decrease in diferric transferrin uptake. Our model will be of use in the elucidation of the functional interaction between intracellular HFE and iron transporters transferrin/transferrin receptor complexes and Slc11A2 (also named N-Ramp2 or DMT1) in different endosomal compartments.
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    ABSTRACT: Le cancer épithélial de l’ovaire est le cancer gynécologique le plus agressif avec le plus haut taux de mortalité. La croissance des cellules cancéreuses de l’ovaire est limitée par les nutriments de l’environnement, le fer étant un des éléments indispensables à leur prolifération. L’hémochromatose héréditaire est une maladie associée à une accumulation corporelle de fer. Cette maladie est liée à deux mutations majeures du gène HFE soit H63D et C282Y. Étant donnée l’influence de la protéine HFE sur l’entrée du fer dans la cellule, des mutations du gène HFE pourraient être associées à une croissance rapide des cellules cancéreuses. Des études de génotypage du gène HFE effectuées chez 526 patientes avec cancer épithélial de l’ovaire, ont révélées une fréquence allélique de la mutation C282Y significativement plus élevées chez les patientes avec tumeur ovarienne comparativement aux patientes du groupe contrôle (5.9% versus 1.3%, p = 0.02). De plus, le taux de survie des patientes avec mutations C282Y et tumeur ovarienne de G3, après 2 ans, est faible (20%) lorsque comparé à celui des patientes sans mutations (60%, p = 0.005). Une analyse de régression multivariée de Cox a démontrée un risque relatif de 3.1, suggérant que les patientes avec mutations C282Y ont 3 fois plus de chance d’avoir une faible survie (p=0.001). Également, des études de corrélation ont démontrées que les niveaux de ferritine du sérum étaient plus élevés chez les patientes avec grade avancé du cancer épithélial de l’ovaire (r = 0.445 et p= 0.00001), suggérant que ce paramètre pourrait servir comme marqueur tumoral. Afin de comprendre ces résultats, nous avons tout d’abord étudiés l’influence des mutations HFE sur les cellules cancéreuses. Pour ce faire, la lignée du cancer de l’ovaire TOV-112D, homozygote pour la mutation C282Y, a été transfectée avec les vecteurs HFEwt et HFEC282Y. Bien qu’aucune différence significative n’ait été trouvée en termes de TfR totaux, des analyses par FACS ont démontrées un phénotype de déficience de fer pour les clones stables HFEwt. In vitro, la restauration de la protéine HFE, dans la lignée TOV-112D du cancer de l’ovaire, n’influence pas la croissance cellulaire. Ensuite, nous avons étudiés l’influence des niveaux de fer sur la progression tumorale. Une expérience in vivo préliminaire a démontré une tendance à un volume tumoral supérieur dans un modèle de souris de surcharge de fer,HfeRag1-/-. De plus, les souris HfeRag1-/-, injectées avec la lignée du cancer de l’ovaire TOV-21G, ont montrées des niveaux significativement plus faibles de fer sérique comparativement à leur contrôle (fer sérique 40±7μM versus 27±6μM, p = 0.001). En conclusion, des études supplémentaires sont nécessaires afin de comprendre davantage le rôle des mutations HFE sur la progression tumorale. Notamment, les niveaux élevés de fer pourraient rendre les cellules tumorales résistantes aux traitements ou encore, augmenter la toxicité et ainsi, contribuer à un mauvais prognostique. Epithelial ovarian cancer is the most aggressive gynecological cancer with the highest mortality rates. Growth of the ovarian cancer cells is limited by nutrients in the environment; iron being one of the elements essential to their proliferation. Hereditary hemochromatosis is a disease associated with an accumulation of body iron, and is linked to two mutations of the HFE gene including C282Y and H63D. Given the influence of HFE protein on the entry of iron in the cell, mutations in the HFE gene may be associated with rapid growth of cancer cells. By genotyping the HFE gene of 526 patients with epithelial ovarian cancer, we have found that the allelic frequency of the C282Y mutation is significantly higher in patients with ovarian cancer compared to patients in the control group (5.9% versus 1.3% p = 0.02). Moreover, the 2-year survival rate, of patients with C282Y mutations and G3 ovarian tumor, is low (20%) when compared to patients without mutations (60%, p = 0.005). A multivariate survival analysis, using Cox’s regression model, also showed a hazard ratio value of 3.1,suggesting that patients with the C282Y mutation are 3 times more likely to have a poor survival (p =0.001). As well, correlation studies have demonstrated that serum ferritin levels were higher in patients with advanced grade of ovarian cancer (r = 0.445 and p = 0.00001), suggesting that this iron parameter could serve as a tumor marker for assessing the progression of ovarian cancer. In order to investigate these findings, we first studied the influence of HFE mutations on cancer cells. The ovarian cancer cell line TOV-112D, homozygous for the C282Y mutation, was transfected with the HFEwt and HFEC282Y vector, and although there were no differences in total TfR levels, FACS analysis demonstrated an iron deficient phenotype for the HFEwt stable clones (p<0.05). In vitro,restoration of the HFE protein, in the TOV-112D ovarian cancer cell line, does not influence cell growth. We then studied the influence of iron levels on tumor progression. A preliminary in vivo experiment has demonstrated a tendency to a higher tumor volume in a mouse model of iron overload,HfeRag1-/-. Furthermore, HfeRag1-/- mice that were injected with the ovarian cancer cell line TOV-21G showed significant lower serum iron levels compared to their controls (SI 40±7μM versus 27±6μM, p = 0.001). In conclusion, more studies are required to further understand the role of HFE mutations on tumor progression. Higher iron levels may confer tumor cells to be drug resistant or increase toxicity and thus, may contribute to a bad prognostic.
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