FLCN regulates transferrin receptor 1 transport and iron homeostasis.

Birt-Hogg-Dubé (BHD) syndrome is a multiorgan disorder caused by inactivation of the folliculin (FLCN) protein. Previously, we identified FLCN as a binding protein of Rab11A, a key regulator of the endocytic recycling pathway. This finding implies that the abnormal localization of specific proteins whose transport requires the FLCN-Rab11A complex may contribute to BHD. Here, we used human kidney-derived HEK293 cells as a model, and we report that FLCN promotes the binding of Rab11A with transferrin receptor 1 (TfR1), which is required for iron uptake through continuous trafficking between the cell surface and the cytoplasm. Loss of FLCN attenuated the Rab11A-TfR1 interaction, resulting in delayed recycling transport of TfR1. This delay caused an iron deficiency condition that induced hypoxia-inducible factor (HIF) activity, which was reversed by iron supplementation. In a Drosophila model of BHD syndrome, we further demonstrated that the phenotype of BHD mutant larvae was substantially rescued by an iron-rich diet. These findings reveal a conserved function of FLCN in iron metabolism and may help to elucidate the mechanisms driving BHD syndrome.

Parenteral vs. oral iron: influence on hepcidin signaling pathways through analysis of Hfe/Tfr2-null mice.

Treatment for iron deficiency anemia can involve iron supplementation via dietary or parenteral routes that result in different cellular iron distributions. The effect of the administered iron on the iron regulatory system and hepcidin in the liver has not been well studied. Hepcidin, the liver-expressed central iron-regulatory peptide, is itself regulated through the bone morphogenetic protein (BMP)/SMAD signaling pathway. Specifically, Bmp6 expression is upregulated in response to iron and induces hepcidin through phosphorylation of Smad1/5/8. The hemochromatosis-associated proteins Hfe and transferrin receptor 2 (Tfr2) are known upstream regulators of hepcidin, although their precise roles are still unclear. To investigate the mechanisms of this regulation and the roles of the Hfe and Tfr2, we subjected wild-type, Hfe(-/-), Tfr2(-/-), and Hfe(-/-)/Tfr2(-/-) mice to iron loading via dietary or parenteral routes. Systematic analysis demonstrated that Tfr2 is required for effective upregulation of Bmp6 in response to hepatocyte iron, but not nonparenchymal iron. Hfe is not required for Bmp6 upregulation, regardless of iron localization, but rather, is required for efficient downstream transmission of the regulatory signal. Our results demonstrate that Hfe and Tfr2 play separate roles in the regulatory responses to iron compartmentalized in different cell types and further elucidates the regulatory mechanisms controlling iron homeostasis.

Iron in Hodgkin's lymphoma.

Anemia is a frequent finding of Hodgkin's lymphoma (HL) diagnosis. It is usually mild, with hemoglobin levels between 10 and 12 g/dl; it is rarely (<10% of cases) a result of bone marrow infiltration; and it displays the characteristics of the anemia of chronic disease due to abnormalities in iron metabolism. The inflammatory cytokine IL-6 is frequently up-regulated in Hodgkin's lymphoma, and IL-6 levels are strongly associated with hepcidin, the main regulator of iron metabolism. Elevated hepcidin levels result in iron restriction and signs of anemia of chronic disease. In addition, the abundant microenvironment surrounding the neoplastic Hodgkin's and Reed-Sternberg cells may contribute to alterations in iron metabolism. Tumor-infiltrating macrophages can sequester iron using scavenger receptors. Iron-restricted anemia at HL diagnosis can be aggravated by intensive chemotherapy, and iron overload may become clinically relevant in heavily treated patients with relapsed or refractory disease undergoing high-dose chemotherapy and stem cell transplantation.

Neuroimmunomodulation and aging: a role for transferrin and the hypothalamus/thymus axis.

Advanced age is associated with an increased incidence of immune and degenerative disorders, mediated by metabolic changes, dysregulation of proinflammatory signals, and apoptosis. Concurrently, there is a progressive decline in self-recognition. Investigations on biologic functions of transferrin (Tf) other than iron transport showed that Tf has a profound cytoprotective (anti-apoptotic) effect on lympho-hematopoietic cells and the thymus, and interferes with stress-induced signals. Tf protects hepatocytes against Fas-induced cell death by reducing BID cleavage, inhibiting caspase-3 and -9 activation and up-regulating survival signals such as Bcl-xL. The involvement in the regulation of alloreactivity and apoptosis suggests that Tf participates in the maintenance of "self-identity" mechanisms, which are tightly linked to the capacity of the immune system to recognize and react against any noxious agent. Some of the disorders associated with aging are thought to be related to thymic involution, reflecting alterations in the interplay of neural, endocrine and immune factors. We established a murine model of thymic involution induced by stereotactically placed electrolytic lesions in the anterior hypothalamic area. The events observed in this model mimic those observed during senescence including thymus involution, i.e. enhanced glucocorticoid reaction to distress, and obesity. The described properties of Tf can be exploited to modify immune responses and provide cytoprotection against pro-apoptotic and cytotoxic signals when neuroimmunomodulatory mechanisms are impaired, as is the case with aging.

Brain transcriptome perturbations in the transferrin receptor 2 mutant mouse support the case for brain changes in iron loading disorders, including effects relating to long-term depression and long-term potentiation.

Iron abnormalities within the brain are associated with several rare but severe neurodegenerative conditions. There is growing evidence that more common systemic iron loading disorders such as hemochromatosis can also have important effects on the brain. To identify features that are common across different forms of hemochromatosis, we used microarray and real-time reverse transcription polymerase chain reaction (RT-PCR) to assess brain transcriptome profiles of transferrin receptor 2 mutant mice (Tfr2(mut)), a model of a rare type of hereditary hemochromatosis, relative to wildtype control mice. The results were compared with our previous findings in dietary iron-supplemented wildtype mice and Hfe(-/-) mice, a model of a common type of hereditary hemochromatosis. For transcripts showing significant changes relative to controls across all three models, there was perfect (100%) directional concordance (i.e. transcripts were increased in all models or decreased in all models). Comparison of the two models of hereditary hemochromatosis, which showed more pronounced changes than the dietary iron-supplemented mice, revealed numerous common molecular effects. Pathway analyses highlighted changes for genes relating to long-term depression (6.8-fold enrichment, p=5.4×10(-7)) and, to a lesser extent, long-term potentiation (3.7-fold enrichment, p=0.01), with generalized reductions in transcription of key genes from these pathways, which are involved in modulating synaptic strength and efficacy and are essential for memory and learning. The agreement across the models suggests the findings are robust and strengthens previous evidence that iron loading disorders affect the brain. Perturbations of brain phenomena such as long-term depression and long-term potentiation might partly explain neurologic symptoms reported for some hemochromatosis patients.

Silencing of RhoA nucleotide exchange factor, ARHGEF3, reveals its unexpected role in iron uptake.

Genomewide association meta-analysis studies have identified > 100 independent genetic loci associated with blood cell indices, including volume and count of platelets and erythrocytes. Although several of these loci encode known regulators of hematopoiesis, the mechanism by which most sequence variants exert their effect on blood cell formation remains elusive. An example is the Rho guanine nucleotide exchange factor, ARHGEF3, which was previously implicated by genomewide association meta-analysis studies in bone cell biology. Here, we report on the unexpected role of ARHGEF3 in regulation of iron uptake and erythroid cell maturation. Although early erythroid differentiation progressed normally, silencing of arhgef3 in Danio rerio resulted in microcytic and hypochromic anemia. This was rescued by intracellular supplementation of iron, showing that arhgef3-depleted erythroid cells are fully capable of hemoglobinization. Disruption of the arhgef3 target, RhoA, also produced severe anemia, which was, again, corrected by iron injection. Moreover, silencing of ARHGEF3 in erythromyeloblastoid cells K562 showed that the uptake of transferrin was severely impaired. Taken together, this is the first study to provide evidence for ARHGEF3 being a regulator of transferrin uptake in erythroid cells, through activation of RHOA.

Supplementation of endothelial cells with mitochondria-targeted antioxidants inhibit peroxide-induced mitochondrial iron uptake, oxidative damage, and apoptosis.

The mitochondria-targeted drugs mitoquinone (Mito-Q) and mitovitamin E (MitoVit-E) are a new class of antioxidants containing the triphenylphosphonium cation moiety that facilitates drug accumulation in mitochondria. In this study, Mito-Q (ubiquinone attached to a triphenylphosphonium cation) and MitoVit-E (vitamin E attached to a triphenylphosphonium cation) were used. The aim of this study was to test the hypothesis that mitochondria-targeted antioxidants inhibit peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC) through enhanced scavenging of mitochondrial reactive oxygen species, thereby blocking reactive oxygen species-induced transferrin receptor (TfR)-mediated iron uptake into mitochondria. Glucose/glucose oxidase-induced oxidative stress in BAECs was monitored by oxidation of dichlorodihydrofluorescein that was catalyzed by both intracellular H(2)O(2) and transferrin iron transported into cells. Pretreatment of BAECs with Mito-Q (1 microM) and MitoVit-E (1 microM) but not untargeted antioxidants (e.g. vitamin E) significantly abrogated H(2)O(2)- and lipid peroxide-induced 2',7'-dichlorofluorescein fluorescence and protein oxidation. Mitochondria-targeted antioxidants inhibit cytochrome c release, caspase-3 activation, and DNA fragmentation. Mito-Q and MitoVit-E inhibited H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, TfR overexpression, and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. We conclude that Mito-Q or MitoVit-E supplementation of endothelial cells mitigates peroxide-mediated oxidant stress and maintains proteasomal function, resulting in the overall inhibition of TfR-dependent iron uptake and apoptosis.

Iron and inflammatory bowel disease.

Both anaemia of iron deficiency and anaemia of chronic disease are frequently encountered in inflammatory bowel disease. Anaemia of iron deficiency is mostly due to inadequate intake or loss of iron. Anaemia of chronic disease probably results from decreased erythropoiesis, secondary to increased levels of proinflammatory cytokines, reactive oxygen metabolites and nitric oxide. Assessment of the iron status in a condition associated with inflammation, such as inflammatory bowel disease, is difficult. The combination of serum transferrin receptor with ferritin concentrations, however, allows a reliable assessment of the iron deficit. The best treatment for anaemia of chronic disease is the cure of the underlying disease. Erythropoietin reportedly may increase haemoglobin levels in some of these patients. The anaemia of iron deficiency is usually treated with oral iron supplements. Iron supplementation may lead to an increased inflammatory activity through the generation of reactive oxygen species. To date, data from studies in animal models of inflammatory bowel disease support the theoretical disadvantage of iron supplementation in this respect. The results, however, cannot easily be extrapolated to the human situation, because the amount of supplemented iron in these experiments was much higher than the dose used in patients with iron deficiency.

The transferrin receptor: role in health and disease.

The transferrin receptor is a membrane glycoprotein whose only clearly defined function is to mediate cellular uptake of iron from a plasma glycoprotein, transferrin. Iron uptake from transferrin involves the binding of transferrin to the transferrin receptor, internalization of transferrin within an endocytic vesicle by receptor-mediated endocytosis and the release of iron from the protein by a decrease in endosomal pH. With the exception of highly differentiated cells, transferrin receptors are probably expressed on all cells but their levels vary greatly. Transferrin receptors are highly expressed on immature erythroid cells, placental tissue, and rapidly dividing cells, both normal and malignant. In proliferating nonerythroid cells the expression of transferrin receptors is negatively regulated post-transcriptionally by intracellular iron through iron responsive elements (IREs) in the 3' untranslated region of transferrin receptor mRNA. IREs are recognized by specific cytoplasmic proteins (IRPs; iron regulatory proteins) that, in the absence of iron in the labile pool, bind to the IREs of transferrin receptor mRNA, preventing its degradation. On the other hand, the expansion of the labile iron pool leads to a rapid degradation of transferrin receptor mRNA that is not protected since IRPs are not bound to it. However, some cells and tissues with specific requirements for iron probably evolved mechanisms that can override the IRE/IRP-dependent control of transferrin receptor expression. Erythroid cells, which are the most avid consumers of iron in the organism, use a transcriptional mechanism to maintain very high transferrin receptor levels. Transcriptional regulation is also involved in the receptor expression during T and B lymphocyte activation. Macrophages are another example of a cell type that shows 'unorthodox' responses in terms of IRE/IRP paradigm since in these cells elevated iron levels increase (rather than decrease) transferrin receptor mRNA and protein levels. Erythroid cells contain the highest mass of the total organismal transferrin receptors which are released from reticulocytes during their maturation to erythrocytes. Hence, plasma contains small amounts of transferrin receptors which represent a soluble fragment of the extracellular receptor domain. Measurements of serum transferrin receptor concentrations are clinically useful since their levels correlate with the total mass of immature erythroid cells.

Effects of iron supplementation on iron uptake by differentiating cytotrophoblasts.

Cultured in Medium-199, cytotrophoblasts isolated from human placentae differentiate morphologically (e.g. syncytium formation) as well as biochemically (e.g. expression of transferrin receptors, TfRs) into syncytiotrophoblast-like structures. The highest TfR numbers are observed in cells cultured in iron-poor culture medium. Investigated were the implications of the variation in surface TfR numbers on the uptake of iron by cytotrophoblasts cultured in iron-poor Medium-199. Despite differences in TfR densities induced by culture time and iron availability, the initial rate of iron uptake did not change (80-100 pmol/mg protein/h). Homeostasis of iron uptake could be explained by adaptive changes in the rate constant for TfR endocytosis (kend), exocytosis (kexo) and TfR cycle times. In undifferentiated cells (cultured for 18 h) kend was 0.299 min-1. In differentiated cells (culture time 65 h, higher surface TfR densities), kend changed to 0.138 min-1. Culture for 65 h in diferric transferrin-enriched medium resulted in intermediate TfR densities together with an intermediate kend (0.210 min-1). Adaptive changes in the corresponding rate constant of exocytosis were less pronounced (0.192, 0.192 and 0.260 min-1 respectively). It is concluded that differentiating cytotrophoblasts regulate iron uptake by variation of both TfR numbers and the rate of receptor-mediated endocytosis and exocytosis.

Essential fatty acids and iron are involved at distinct stages of the proliferative cycle but not in the activation of human T cells.

In the absence of serum, optimal lymphocyte proliferation is obtained when cultures are supplemented with transferrin and an essential fatty acid (EFA). In order to study the effects of iron in conjunction with EFA on T-cell proliferation, we have utilized a chemically defined serum-free culture system to achieve better control of the variables involved. This system includes three different serum-free media (SFM) that differ in total iron content and source of iron: (i) transferrin-free medium containing a high concentration (500 microns) of a soluble iron salt in the form of ferric citrate (Fe-SFM); (ii) iron-saturated human transferrin (5 micrograms/ml) (T-SFM); and (iii) iron-free medium (SFM(-Fe)) without any apparent source of iron. None of these SFM supported proliferation of T cells stimulated by the combination of phorbol 12,13-dibutyrate/ionomycin or phytohemagglutinin. Restoration of the proliferative response was only observed following supplementation of the iron-containing media with linoleic acid (complexed to bovine serum albumin (LA/BSA)). In cultures containing LA/BSA, the addition of iron alone in the absence of transferrin (Fe-SFM) resulted in similar responses to the transferrin-containing medium (T-SFM). Low levels of RNA synthesis in mitogen-stimulated T cells could be demonstrated in the presence or absence of iron and the addition of LA/BSA resulted in marked enhancement of RNA synthesis, regardless of the availability of iron. Cell cycle analysis showed that 91-94% of the cells cultured in SFM were arrested in G0/G1. These cells could progress through the cell cycle following the addition of LA/BSA, but only in the iron-containing media. Unlike DNA or RNA synthesis, activation of T cells could be demonstrated in SFM with or without iron as shown by the normal induction of c-fos and early growth response gene mRNA, normal expression of IL2 and transferrin receptors, and normal IL2 production, despite the arrest of cells in G0/G1. These results suggest that although human T-cell growth is iron and EFA dependent, the early events of T-cell activation are both iron and EFA independent.

Induction of T cell activation by monoclonal antibodies specific for the transferrin receptor.

The effect of the monoclonal antibodies (mAb), FG 1/5, FG 1/6 and FG 2/12, specific for different epitopes of the transferrin receptor (TfR) on T cell activation was studied. mAb FG 1/6 but not FG 2/12 or FG 1/5 was able to induce T cell proliferation in presence of submitogenic doses of phorbol esters. The costimulatory effect of FG 1/6 was seen only with phorbol esters known to be activators of protein kinase C. This proliferation occurred at low concentration (0.5 micrograms/ml) of antibody, required the simultaneous presence of both stimuli, phorbol esters and FG 1/6, and was independent of the presence of accessory cells. Furthermore, FG 1/6 mAb was able to increase the rate of modulation of CD3 surface expression induced by phorbol esters. FG 1/6 induced interleukin (IL) 2 synthesis by normal and transformed T lymphocytes. In addition, anti-IL2 receptor antibodies inhibited FG 1/6 plus phorbol ester-induced proliferation. Our results indicate that FG 1/6 mAb may provide to the T cells complementary signals to protein kinase C and that this activation is mediated by the IL2/IL 2R pathway.