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The high iron demand associated with enhanced erythropoiesis during high-altitude hypoxia leads to skeletal muscle iron mobilization and decrease in myoglobin protein levels. To investigate the effect of enhanced erythropoiesis on systemic and muscle iron metabolism under nonhypoxic conditions, 8 healthy volunteers were treated with recombinant erythropoietin (rhEpo) for 1 month. As expected, the treatment efficiently increased erythropoiesis and stimulated bone marrow iron use. It was also associated with a prompt and considerable decrease in urinary hepcidin and a slight transient increase in GDF-15. The increased iron use and reduced hepcidin levels suggested increased iron mobilization, but the treatment was associated with increased muscle iron and L ferritin levels. The muscle expression of transferrin receptor and ferroportin was up-regulated by rhEpo administration, whereas no appreciable change in myoglobin levels was observed, which suggests unaltered muscle oxygen homeostasis. In conclusion, under rhEpo stimulation, the changes in the expression of muscle iron proteins indicate the occurrence of skeletal muscle iron accumulation despite the remarkable hepcidin suppression that may be mediated by several factors, such as rhEpo or decreased transferrin saturation or both.

Iron is essential for oxygen transport because it is incorporated in the heme of the oxygen-binding proteins hemoglobin and myoglobin. An interaction between iron homeostasis and oxygen regulation is further suggested during hypoxia, in which hemoglobin and myoglobin syntheses have been reported to increase. This study gives new insights into the changes in iron content and iron-oxygen interactions during enhanced erythropoiesis by simultaneously analyzing blood and muscle samples in humans exposed to 7 to 9 days of high altitude hypoxia (HA). HA up-regulates iron acquisition by erythroid cells, mobilizes body iron, and increases hemoglobin concentration. However, contrary to our hypothesis that muscle iron proteins and myoglobin would also be up-regulated during HA, this study shows that HA lowers myoglobin expression by 35% and down-regulates iron-related proteins in skeletal muscle, as evidenced by decreases in L-ferritin (43%), transferrin receptor (TfR; 50%), and total iron content (37%). This parallel decrease in L-ferritin and TfR in HA occurs independently of increased hypoxia-inducible factor 1 (HIF-1) mRNA levels and unchanged binding activity of iron regulatory proteins, but concurrently with increased ferroportin mRNA levels, suggesting enhanced iron export. Thus, in HA, the elevated iron requirement associated with enhanced erythropoiesis presumably elicits iron mobilization and myoglobin down-modulation, suggesting an altered muscle oxygen homeostasis.

We describe the use of small interfering RNAs (siRNAs) to down-regulate H- and L-ferritin levels in HeLa cells. siRNAs repressed H- and L-ferritin expression to about 20% to 25% of the background level in both stable and transient transfections. HeLa cells transfected with H- and L-ferritin cDNAs were analyzed in parallel to compare the effects of ferritin up- and down-regulation. We found that large modifications of L-ferritin levels did not affect iron availability in HeLa cells but positively affected cell proliferation rate in an iron-independent manner. The transient down-regulation of H-ferritin modified cellular iron availability and resistance to oxidative damage, as expected. In contrast, the stable suppression of H-ferritin in HeLa cell clones transfected with siRNAs did not increase cellular iron availability but made cells less resistant to iron supplementation and chelation. The results indicate that L-ferritin has no direct effects on cellular iron homeostasis in HeLa cells, while it has new, iron-unrelated functions. In addition, they suggest that H-ferritin function is to act as an iron buffer.

Ferritin, the iron-storing molecule, is made by the assembly of various proportions of 2 different H and L subunits into a 24-mer protein shell. These heteropolymers have distinct physicochemical properties, owing to the ferroxidase activity of the H subunit, which is necessary for iron uptake by the ferritin molecule, and the ability of the L subunit to facilitate iron core formation inside the protein shell. It has previously been shown that H ferritin is indispensable for normal development, since inactivation of the H ferritin gene by homologous recombination in mice is lethal at an early stage during embryonic development. Here the phenotypic analysis of the mice heterozygous for the H ferritin gene (Fth+/− mice) is reported, and differences in gene regulation between the 2 subunits are shown. The heterozygousFth+/− mice were healthy and fertile and did not present any apparent abnormalities. Although they had iron-overloaded spleens at the adult stage, this is identical to what is observed in normal Fth+/+ mice. However, these heterozygous mice had slightly elevated tissue L ferritin content and 7- to 10-fold more L ferritin in the serum than normal mice, but their serum iron remained unchanged. H ferritin synthesis from the remaining allele was not up-regulated. This probably results from subtle changes in the intracellular labile iron pool, which would stimulate L ferritin but not H ferritin synthesis. These results raise the possibility that reduced H ferritin expression might be responsible for unexplained human cases of hyperferritinemia in the absence of iron overload where the hereditary hyperferritinemia-cataract syndrome has been excluded.

Abstract 903 OBJECTIVE: To test the phenotypic cure we have discovered for SCD and malaria in vivo in mice and in vitro in human red blood cell precursors from SCD patients in two-phase cultures. BACKGROUND: Gene regulation of developmental hemoglobin switching provides phenotypic cures for beta thalassemias, sickle cell disease, and malaria since it is known that reactivation of fetal globin expression alleviates all these disorders. We discovered a protein that regulates this developmental switch. Ferritin heavy chain (FtH) represses adult β-globin and activates γ(fetal)-globin gene expression in embryonic/K562 erythroid cells (Broyles et al., PNAS98: 9145, 2001; US Patents #7,517,669, #7,718,669, & #2009/0232783 A1; EU Patent # EP1354032B1; Australia patent #2002217964), leading us to propose FtH as a therapeutic agent. METHODS: Normal C57BL/6 mice, transgenics carrying the complete human beta-globin gene cluster (Beta-YAC Tg), and transgenic mice that express human FtH in definitive erythroid cells have been used under an IACUC-approved protocol to show the safety and efficacy of human FtH and our phytotherapeutic in vivo. Erythroid precursor cells from pediatric SCD patients obtained under an IRB-approved protocol and cultured in a two-phase system allow direct testing of an FtH expression plasmid, FtH protein, and our phytotherapeutic for manipulating Hb phenotypes. Hemoglobins were identified and quantified by HPLC; fetal Hb was confirmed by immunofluorescence. RESULTS AND DISCUSSION: Human FtH transgenic mice, in which the FtH gene is driven by a truncated b-promoter lacking the FtH-binding motif, express human FtH in definitive erythroid cells which results in repression of bMajor-globin but not bMinor-globin. Thus, these TgFtH mice are born with a reduced ratio of bMajor/bMinor globins, resulting in α-globin chain excess and a mild b-thalassemia. In applying this therapy to humans, thalassemia would not be expected since FtH also activates g (fetal)-globin, preventing the chain imbalance. Recent results with normal mice and Beta-YacTg mice show that FtH protein and our phytotherapeutic are both well tolerated in vivo. We have used numbers of target cells (found in the FtH transgenics) as an initial screen for determining an effective dosing regimens. Initial results indicate that both therapeutics are altering the Hb phenotype as predicted. With cultured erythroid precursor cells from the pediatric SCD patients, our results show a complete switch from HbS to HbF with each mode of delivery of FtH or the phytotherapeutic. These results were replicated 15 times using cells from 9 SCD donors, as well as in erythroid precursors from normal donors. Human erythroid precursor cells have surface receptors specific for FtH. Immunofluorescence showed that human FtH is taken into the precursor cells in culture; and confocal microscopy confirmed nuclear localization of exogenously applied FtH. These results suggest that the purified protein and/or the phytotherapeutic can be directly delivered without gene therapy. Therefore, these methods of generating a phenotypic cure for malaria, beta-thal, and SCD should be inexpensive to deliver in vivo. Supported in part by donations to The Sickle Cell Cure Foundation and by a Grand Challenges in Global Health grant from the Bill & Melinda Gates Foundation. Disclosures: No relevant conflicts of interest to declare.

Mitochondrial ferritin (FtMt) is an iron-sequestering protein that specifically localizes into mitochondria and whose physiological role is only partly characterized. It was first identified in humans and subsequently described in mice, Drosophila, and plant. FtMt is a 24-homo-polymeric shell protein, with structure and function similar to those of cytosolic H-ferritin. Different from cytosolic ferritins, FtMt does not show ubiquitous distribution, and its expression is not iron-dependent. Studies on mice revealed that the protein has a tight tissue-specific expression pattern; it is preferentially expressed in cells characterized by high-energy consumption, like spermatozoa, neurons, and cardiomyocytes, while it is not present in iron storage tissues. In humans, the protein is also highly expressed in sideroblasts of patients affected by sideroblastic anemia (SA), and data on patients with myelodysplastic syndromes has provided evidence that FtMt represents a specific marker of SA. In refractory anemia with ringed sideroblasts, FtMt expression occurred in CD34+ cells before any morphological sign of maturation and iron accumulation appeared. This argues for a complex mechanism of gene regulation that remains to be elucidated. Previous studies on HeLa and yeast cells showed that FtMt expression affected cellular iron homeostasis, driving iron into mitochondria and inducing cytosolic iron starvation; however, FtMt is also able to reduce mitochondrial iron accumulation and improve iron-sulphur cluster (Fe/S) enzymes activity. Thus, it remains to be established under which conditions the presence of FtMt could be either helpful or detrimental to the cells. To clarify the physiological role of human FtMt, we tested its properties on a HeLa-tTa inducible clone. Expression of FtMt in cells was associated with a protective effect against oxidative damage promoted by hydrogen peroxide or Antimycin A insults, and against oxidative stress occurring under increased mitochondrial respiratory metabolism. The FtMt expression reduced the Reactive Oxygen Species (ROS) level and preserved ATP production and mitochondrial Fe/S enzymes activity, with a consequent positive effect on cell viability. FtMt reduced cytosolic and mitochondrial labile iron pools during glycolytic cellular growth, where FtMt was slowly degraded. In cells grown in glucose-free media, FtMt was degraded faster, determining iron redistribution among cellular compartments, which promoted mitochondrial enzymes activity without affecting cytosolic iron status. However, even under enhanced respiratory condition, FtMt maintained ROS buffering properties, indicating that the primary function of FtMt consists of the control of ROS formation through the regulation of mitochondrial iron availability. This FtMt protective effect might be particularly important in iron-overloaded mitochondria, as in SA. We produced FtMt-lentivirus vectors to transduce human CD34+ cells, and preliminary results indicate that they can be effectively employed to clarify the role of FtMt in pathophysiology of SA.

We have found that ferritin heavy chain (FtH), an antioxidant/stress response/iron-storage protein, localizes to the nucleus in K562 cells and represses the human adult beta-globin promoter in transient assays in primate cells (Broyles et al., PNAS98: 9145, 2001). Since other work indicates FtH is also a gene activator of fetal-globin genes, we hypothesize that FtH is a long-sought developmental hemoglobin (Hb) switching factor and that delivery of FtH to human adult erythroid cell precursors will reverse the phenotype to HbF, offering a phenotypic cure for sickle cell disease (SCD). Chromatin immunoprecipitation (ChIP) assays, antisense treatments, and an FtH transgenic mouse have confirmed that FtH is a globin gene regulatory protein in vivo. With erythroid precursor cells from pediatric SCD patients, under an IRB-approved protocol, we have used a two-phase culture system for in vitro maturation of erythroid cells in the presence of FtH, delivered to the cells as pure protein, as an expression plasmid, or as a priority inducer compound that activates the endogenous FtH gene. HPLC with a PolyCAT A column was used to separate and quantify human Hbs. With each mode of delivery, FtH stimulated a complete switch from HbS to HbF. This result was repeatable in multiple experiments using erythroid precursor cells from three different SCD donors. Fluorescently-labeled recombinant human FtH protein was taken into red cell precursors in culture, suggesting that the purified protein can be directly delivered without gene therapy. This method of producing a phenotypic cure in SCD patients should be easy and inexpensive to deliver in vivo.