Mitochondrial Ferritin Expression Research Articles

Mitochondrial ferritin expression in human macrophages is facilitated by thrombin-mediated cleavage under hypoxia.

Ferritins are iron storage proteins, which maintain cellular iron homeostasis. Among these proteins, the ferritin heavy chain is well characterized, but the regulatory principles of mitochondrial ferritin (FTMT) remain elusive. FTMT appears to be cleaved from a 27 kDa to a 22 kDa form. In human macrophages, FTMT increased under hypoxia in a hypoxia-inducible factor 2-dependent manner. Occurrence of FTMT resulted from cleavage by thrombin, which was supplied by serum. Inhibition of thrombin as well as serum removal decreased FTMT, while supplementation of thrombin under serum-deprived conditions restored its expression. Besides hypoxia, thrombin facilitated FTMT expression after treatment with the ferroptosis inducer RSL3 and the pro-inflammatory stimulus lipopolysaccharide. This study provides insights into the regulation of FTMT under hypoxia and identifies thrombin as a FTMT maturation-associated peptidase.

Mitochondrial Ferritin: Its Role in Physiological and Pathological Conditions.

In 2001, a new type of human ferritin was identified by searching for homologous sequences to H-ferritin in the human genome. After the demonstration that this ferritin is located specifically in the mitochondrion, it was called mitochondrial ferritin. Studies on the properties of this new type of ferritin have been limited by its very high homology with the cytosolic H-ferritin, which is expressed at higher levels in cells. This great similarity made it difficult to obtain specific antibodies against the mitochondrial ferritin devoid of cross-reactivity with cytosolic ferritin. Thus, the knowledge of the physiological role of mitochondrial ferritin is still incomplete despite 20 years of research. In this review, we summarize the literature on mitochondrial ferritin expression regulation and its physical and biochemical properties, with particular attention paid to the differences with cytosolic ferritin and its role in physiological condition. Until now, there has been no evidence that the alteration of the mitochondrial ferritin gene is causative of any disorder; however, the identified association of the mitochondrial ferritin with some disorders is discussed.

Aberrant mitochondrial iron distribution and maturation arrest characterize early erythroid precursors in low-risk myelodysplastic syndromes

Early erythroblasts from patients with refractory anemia (RA) and RA with ringed sideroblasts (RARS) show constitutive mitochondrial release of cytochrome c. Moreover, mature erythroblasts in RARS, but not in RA, display aberrant accumulation of mitochondrial ferritin (MtF). We analyzed cytochrome c release, MtF expression, and gene expression during erythroid differentiation in bone marrow cells from myelodysplastic syndrome (MDS) patients and healthy controls. Whereas none or few cultured erythroid cells from healthy individuals and RA patients expressed MtF, those from RARS patients showed MtF expression at an early stage, when cells were CD34+ and without morphologic signs of erythroid differentiation. The proportion of RARS erythroblasts that were MtF+ increased further upon in vitro maturation. Moreover, a significant overexpression of mRNA encoding cytochrome c, and proapoptotic Bid and Bax, was seen in freshly isolated cells from MDS patients. Genes involved in erythroid differentiation were also dysregulated in MDS cells. Importantly, GATA-1 expression increased during normal erythroid maturation, but remained low in MDS cultures, indicating a block of erythroid maturation at the transcriptional level. In conclusion, aberrant MtF expression in RARS erythroblasts occurs at a very early stage of erythroid differentiation and is paralleled by an up-regulation of genes involved in this process.

Iron loss triggers mitophagy through induction of mitochondrial ferritin.

Mitochondrial quality is controlled by the selective removal of damaged mitochondria through mitophagy. Mitophagy impairment is associated with aging and many pathological conditions. An iron loss induced by iron chelator triggers mitophagy by a yet unknown mechanism. This type of mitophagy may have therapeutic potential, since iron chelators are clinically used. Here, we aimed to clarify the mechanisms by which iron loss induces mitophagy. Deferiprone, an iron chelator, treatment resulted in the increased expression of mitochondrial ferritin (FTMT) and the localization of FTMT precursor on the mitochondrial outer membrane. Specific protein 1 and its regulator hypoxia-inducible factor 1α were necessary for deferiprone-induced increase in FTMT. FTMT specifically interacted with nuclear receptor coactivator 4, an autophagic cargo receptor. Deferiprone-induced mitophagy occurred selectively for depolarized mitochondria. Additionally, deferiprone suppressed the development of hepatocellular carcinoma (HCC) in mice by inducing mitophagy. Silencing FTMT abrogated deferiprone-induced mitophagy and suppression of HCC. These results demonstrate the mechanisms by which iron loss induces mitophagy and provide a rationale for targeting mitophagic activation as a therapeutic strategy.

Mitochondrial ferritin expression in erythroid cells from patients with alpha-thalassaemia

ABSTRACTBackground: Patients with thalassaemia who received regular transfusions had increased iron accumulation, leading to iron overload, which was associated with oxidative stress. Mitochondrial ferritin, encoded by the FTMT gene is an iron-storage protein in the mitochondria. The aim of this work was to investigate the expression levels of FTMT in the reticulocytes of patients with alpha-thalassaemia who were regularly transfused and rarely transfused compared with healthy controls and to evaluate the relationships of the levels of FTMT mRNA with malondialdehyde (MDA) and ferritin in these patients.Methods: The levels of FTMT mRNA in the reticulocytes of patients (30 regularly transfused and 30 rarely transfused) and 30 healthy individuals were assessed by quantitative reverse transcription-polymerase chain reaction. The levels of ferritin and MDA were analysed by ELISA and by a thiobarbituric acid reactive substance assay, respectively.Results: The levels of FTMT mRNA, ferritin and MDA in both groups of patients were significantly increased compared with those in the healthy controls. In addition, the levels of FTMT mRNA, ferritin and MDA in the regularly transfused patients were significantly higher than those in the rarely transfused patients. Furthermore, the relative expression levels of FTMT in patients correlated with those of MDA and ferritin.Conclusion: These results suggest that the elevation of expression levels of FTMT in the reticulocytes of patients with alpha-thalassaemia may be associated with iron loading and oxidative stress.

Establishment of a cell model of X-linked sideroblastic anemia using genome editing

ALAS2 gene mutations cause X-linked sideroblastic anemia. The presence of ring sideroblasts in a patient's bone marrow is the hallmark of sideroblastic anemia, but the precise mechanisms underlying sideroblast formation are largely unknown. Using a genome-editing system, a mutation was introduced in the erythroid-specific enhancer of the ALAS2 gene in HUDEP2 cells, which were derived from human umbilical stem cells and can produce erythrocytes. The established cell line, termed HA2low, expressed less ALAS2 mRNA than did wild-type cells, even after erythroid differentiation. Although the mRNA expression of α-globin, β-globin, and the mitochondrial iron importer mitoferrin-1 was induced similarly in wild-type and HA2low cells, hemoglobinization of differentiated cells was limited in HA2low cells compared with wild-type cells. Importantly, Prussian blue staining revealed that approximately one-third of differentiated HA2low cells exhibited intracellular iron deposition and these cells looked like ring sideroblasts. Electron microscopy confirmed that the mitochondria in HA2low cells contained high-density deposits that might contain iron. Ring sideroblastic cells appeared among HA2low cells only after differentiation, whereas the induced expression of mitochondrial ferritin was observed in both cell types during differentiation. These results suggest that the induction of mitochondrial ferritin expression might be essential for, but not the primary cause of, ring sideroblast formation. Our results also suggest that the insufficient supply of protoporphyrin IX due to ALAS2 deficiency in combination with increased iron import into mitochondria during erythroid differentiation results in the formation of ring sideroblasts. Furthermore, HA2low cells are a useful tool for characterizing ring sideroblasts in vitro.

Upregulation of mitochondrial ferritin by proinflammatory cytokines: implications for a role in Alzheimer's disease.

Studies have shown an increased expression of mitochondrial ferritin (FtMt) and an antioxidant role for the protein in the brains of Alzheimer's disease (AD) patients. However, little information is available concerning the role of FtMt in other AD pathologies, including inflammation and amyloidogenesis. Therefore, we investigated the regulation and function of FtMt in inflammation and amyloidogenesis. FtMt protein expression was increased by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin 6 (IL-6), whereas FtMt mRNA levels were increased by TNF-α but not by IL-1β or IL-6 in IMR-32 cells. The transcription factor nuclear factor-κB (NF-κB) inhibitor, Bay 11-7082, suppressed this TNF-α-induced FtMt expression. FtMt overexpression increased NF-κB activity and translocation of p65 into the nucleus in HEK293 cells. Conversely, knockdown of FtMt attenuated TNF-α-induced NF-κB activity. Overexpression of FtMt inhibited TNF-α-induced apoptosis in the cell culture. FtMt overexpression reduced iron-mediated expression of amyloid-β protein precursor and decreased NF-κB-dependent increases in β- and γ-secretase, leading to decreased amyloid-β production. Our data provide new insights into the mechanism underlying the regulation of FtMt expression by proinflammatory cytokines and indicate further roles for FtMt in AD.

Expression and localization of mitochondrial ferritin mRNA in Alzheimer's disease cerebral cortex.

Mitochondrial ferritin (MtF) has been identified as a novel ferritin encoded by an intron-lacking gene with specific mitochondrial localization located on chromosome 5q23.1. MtF has been associated with neurodegenerative disorders such as Friedreich ataxia and restless leg syndrome. However, little information is available about MtF in Alzheimer's disease (AD). In this study, therefore, we investigated the expression and localization of MtF messenger RNA (mRNA) in the cerebral cortex of AD and control cases using real-time polymerase chain reaction (PCR) as well as in situ hybridization histochemistry. We also examined protein expression using western-blot assay. In addition, we used in vitro methods to further explore the effect of oxidative stress and β-amyloid peptide (Aβ) on MtF expression. To do this we examined MtF mRNA and protein expression changes in the human neuroblastoma cell line, IMR-32, after treatment with Aβ, H2O2, or both. The neuroprotective effect of MtF on oxidative stress induced by H2O2 was measured by MTT assay. The in situ hybridization studies revealed that MtF mRNA was detected mainly in neurons to a lesser degree in glial cells in the cerebral cortex. The staining intensity and the number of positive cells were increased in the cerebral cortex of AD patients. Real-time PCR and western-blot confirmed that MtF expression levels in the cerebral cortex were significantly higher in AD cases than that in control cases at both the mRNA and the protein level. Cell culture experiments demonstrated that the expression of both MtF mRNA and protein were increased by treatment with H2O2 or a combination of Aβ and H2O2, but not with Aβ alone. Finally, MtF expression showed a significant neuroprotective effect against H2O2-induced oxidative stress (p<0.05). The present study suggests that MtF is involved in the pathology of AD and may play a neuroprotective role against oxidative stress.

Over-expression of mitochondrial ferritin affects the JAK2/STAT5 pathway in K562 cells and causes mitochondrial iron accumulation

Mitochondrial ferritin is a nuclear encoded iron-storage protein localized in mitochondria. It has anti-oxidant properties related to its ferroxidase activity, and it is able to sequester iron avidly into the organelle. The protein has a tissue-specific pattern of expression and is also highly expressed in sideroblasts of patients affected by hereditary sideroblastic anemia and by refractory anemia with ringed sideroblasts. The present study examined whether mitochondrial ferritin has a role in the pathogenesis of these diseases. We analyzed the effect of mitochondrial ferritin over-expression on the JAK2/STAT5 pathway, on iron metabolism and on heme synthesis in erythroleukemic cell lines. Furthermore its effect on apoptosis was evaluated on human erythroid progenitors. Data revealed that a high level of mitochondrial ferritin reduced reactive oxygen species and Stat5 phosphorylation while promoting mitochondrial iron loading and cytosolic iron starvation. The decline of Stat5 phosphorylation induced a decrease of the level of anti-apoptotic Bcl-xL transcript compared to that in control cells; however, transferrin receptor 1 transcript increased due to the activation of the iron responsive element/iron regulatory protein machinery. Also, high expression of mitochondrial ferritin increased apoptosis, limited heme synthesis and promoted the formation of Perls-positive granules, identified by electron microscopy as iron granules in mitochondria. Our results provide evidence suggesting that Stat5-dependent transcriptional regulation is displaced by strong cytosolic iron starvation status induced by mitochondrial ferritin. The protein interferes with JAK2/STAT5 pathways and with the mechanism of mitochondrial iron accumulation.

The Effects of Mitochondrial Ferritin Expression in Normal and Sideroblastic Erythropoiesis.

Abstract 736In erythroid cells from patients with refractory anemia with ringed sideroblasts (RARS), the expression of mitochondrial ferritin (MtF) - encoded by the nuclear FTMT gene located on chromosome 5q21.3 - occurs at a very early stage of differentiation and is closely related to the sideroblastic phenotype [Blood. 2003 Mar 1;101(5):1996-2000]. On the other hand, in extra-hematopoietic tissues MtF expression is associated with high cellular metabolic activity and oxygen consumption, suggesting a possible role of this protein in protecting mitochondria from iron-dependent oxidative damage. It is known that reactive oxygen species (ROS) act as second messengers in the JAK/STAT pathway, which is involved in the regulation of erythropoiesis, and that ROS scavengers inhibit STAT5 phosphorylation. It was the aim of this study to investigate the influence of experimentally induced MtF over-expression on erythroid differentiation in normal hematopoietic progenitors, and in addition to define the relationship between MtF expression and erythroid maturation, apoptosis, and JAK-STAT pathway activation in RARS. A liquid culture model was adopted to expand erythroid progenitors from CD34+ cells in the presence of IL-3, IL-6, stem cell factor and Epo [Blood. 2005 Jul 1;106(1):247-53]. To study the effect of MtF induction in normal hematopoiesis, CD34+ cells from 5 healthy donors were transduced using lentivirus carrying cDNA of FTMT downstream to the ubiquitous PGK promoter (transduction efficiency equal to 30-40%), and then cultured for 21 days. To assess the effect of MtF expression in myelodysplastic syndromes (MDS), CD34+ cells from 24 patients with RARS, as well as cells from 20 patients with refractory anemia (RA) and from 8 healthy donors (as control groups) were cultured. Cytospins were performed for MtF, H-ferritin (HF) and L-ferritin (LF) immunocytochemical analysis, and samples of cultured cells were removed at various days of culture for biological studies. Lentivirus-mediated FTMT transduction of normal CD34+ progenitors did not inhibit cell growth nor prevent the differentiation of erythroid progenitors. By flow cytometry analysis, MtF-positive erythroid progenitors showed significantly reduced levels of HF and increased expression of transferrin receptor (CD71) compared with MtF-negative progenitors (P = .004 and P = .01, respectively). In this model, induction of MtF resulted in increased cellular apoptosis (median number of apoptotic cells by TUNEL assay at day 21 equal to 83% in MtF-positive cells vs 18% in MtF-negative cells, P < .001). A significantly lower proliferation rate and higher apoptotic index were observed in cultures from patients with RARS and RA with respect to healthy controls. FTMT mRNA was detected by RT-PCR in CD34+ progenitor cells from patients with RARS, while protein expression was observed only from day 4 of culture, with a significant increase in the percentage of MtF-positive cells during culture (median value from 5% at day 4 to 24% at day 21, P = .002). No MtF expression was observed in RA patients. In MtF-positive cells from RARS patients, an inverse relationship between MtF and HF cellular content, and a direct relationship between MtF and CD71 expression were observed. In erythroid progenitors from RARS patients, the apoptotic rate was higher in MtF-positive than in MtF-negative cells (median number of apoptotic cells equal to 17% vs 6%, P < .001). Finally, we analyzed by flow cytometry the relationship between MtF expression and STAT5 phosphorylation in cultured cells following Epo stimulation. Preliminary data from three RARS patients showed that p-STAT5 expression was lower in MtF-positive than in MtF-negative cells (P = .02). In conclusion, experimental overexpression of MtF in normal erythroid progenitors may reduce mitochondrial iron availability thus inducing apoptosis. In erythroid progenitors from RARS patients, the pathological expression of MtF is associated with increased apoptosis of immature red cells (ineffective erythropoiesis), which may be at least in part determined by reduced activation of the JAK-STAT pathway in response to Epo. Disclosures:No relevant conflicts of interest to declare.

Granulocyte JAK2 (V617F) Mutation Status in Myeloid Neoplasms with Ringed Sideroblasts.

The most common type of acquired sideroblastic anemia is the myelodysplastic syndrome (MDS) defined as refractory anemia with ringed sideroblasts (RARS). We have previously demonstrated that mitochondrial iron accumulation in this condition is in the form of mitochondrial ferritin (MtF). A gain-of-function mutation of JAK2 is found in most patients with chronic myeloproliferative disorders. A high frequency of this mutation has been also reported in RARS associated with marked thrombocytosis (RARS-T), a provisional entity characterized by marked increase in platelet count, hypercellular marrow with increased megakaryocytes, and ringed sideroblasts. In this study, we investigated the granulocyte JAK2 (V617F) mutation status in 73 patients receiving a diagnosis of myeloid malignancy with ringed sideroblasts at the Division of Hematology, University of Pavia Medical School & IRCCS Policlinico San Matteo Pavia, Italy between 2001 and 2006. According to the WHO classification of the myeloid neoplasms, 23 patients had RARS, 17 had refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), 16 had refractory anemia with blasts excess, four had MDS with isolated del(5q), and 13 fulfilled the criteria for RARS-T. JAK2 (V617F) mutation status was analyzed on peripheral blood granulocytes through a quantitative real time PCR-based allelic discrimination assay. We compared clinical and biological features of patients with RARS-T with those of patients with refractory anemia or cytopenia with ringed sideroblasts, and found that RARS-T patients had higher neutrophil and platelet counts, lower frequency of cytogenetic abnormalities and higher incidence of the JAK2 (V617F) mutation (P values ranging from <.001 to .02). JAK2 (V617F) was detected in six out of 63 evaluable cases (9.5%), one being diagnosed as MDS with isolated del(5q) and five as RARS-T, resulting in an incidence of mutation in the latter group of 45%. The proportion of mutant alleles ranged between 2.8% and 18.4%, values commonly observed by us in essential thrombocythemia [Blood. 2006 May 1;107(9):3676–82]. Focusing the analysis on RARS-T, a significantly higher hemoglobin level at diagnosis was found in mutated (median value 11.2 g/dL, range 10.1–15.4) compared with non mutated patients (median value 9 g/dL, range 6–9.9) (P=.009). JAK2-positive patients also showed a significantly lower percentage of ringed sideroblasts in the bone marrow (P=.01), and an increased marrow reticulin fibrosis (P=.03). We then evaluated the clonality of hematopoiesis in female patients through analysis of X-chromosome inactivation patterns (XCIPs) in circulating granulocytes and bone marrow CD34-positive cells. Twenty-one out of 23 informative female patients with ringed sideroblasts (91%), as well as 5/6 RARS-T (83%) displayed a clonal pattern of X-chromosome inactivation. These observations suggest that refractory anemia with ringed sideroblasts with marked thrombocytosis is a clonal stem cell disorder significantly associated with the JAK2 (V617F) mutation. This disorder shows both dysplastic and proliferative features, the presence of the mutation being associated with a predominant myeloproliferative phenotype. The recognition of a heterogeneous genetic background in myeloid neoplasms with ringed sideroblasts suggests that different mechanisms might be involved in the induction of mitochondrial ferritin expression in these disorders.

In vivo tumor growth is inhibited by cytosolic iron deprivation caused by the expression of mitochondrial ferritin

AbstractMitochondrial ferritin (MtFt) is a mitochondrial iron-storage protein whose function and regulation is largely unknown. Our previous results have shown that MtFt overexpression markedly affects intracellular iron homeostasis in mammalian cells. Using tumor xenografts, we examined the effects of MtFt overexpression on tumor iron metabolism and growth. The expression of MtFt dramatically reduced implanted tumor growth in nude mice. Mitochondrial iron deposition in MtFt-expressing tumors was directly observed by transmission electron microscopy. A cytosolic iron starvation phenotype in MtFt-expressing tumors was revealed by increased RNA-binding activity of iron regulatory proteins, and concomitantly both an increase in transferrin receptor levels and a decrease in cytosolic ferritin. MtFt overexpression also led to decreases in total cellular heme content and heme oxygenase-1 levels. In addition, elevated MtFt in tumors was also associated with a decrease in total aconitase activity and lower frataxin protein level. In conclusion, our study shows that high MtFt levels can significantly affect tumor iron homeostasis by shunting iron into mitochondria; iron scarcity resulted in partially deficient heme and iron-sulfur cluster synthesis. It is likely that deprivation of iron in the cytosol is the cause for the significant inhibition of xenograft tumor growth.

Flow cytometry evaluation of erythroid dysplasia in patients with myelodysplastic syndrome

Erythroid dysplasia is the pathologic hallmark of myelodysplastic syndromes (MDS). To develop a quantitative flow-cytometry approach to its evaluation, we analyzed the expression of CD71, CD105, cytosolic H-ferritin (HF), cytosolic L-ferritin (LF) and mitochondrial ferritin (MtF) in erythroblasts from 104 MDS patients, 69 pathologic control patients and 19 healthy subjects. Six-parameter, 4-color flow cytometry was employed, and data were expressed as mean fluorescence intensity. Compared with pathologic and healthy controls, MDS patients had higher expression of HF (P < 0.001) and CD105 (P < 0.001), and lower expression of CD71 (P < 0.001). MtF was specifically detected in MDS with ringed sideroblasts, and there was a close relationship between its expression and Prussian blue staining (r = 0.89, P < 0.001). In vitro cultures of myelodysplastic hematopoietic progenitors showed that both HF and MtF were expressed at a very early stage of erythroid differentiation, and that MtF expression is specifically related to mitochondrial iron loading. A classification function based on expression levels of HF, CD71 and CD105 allowed us to correctly classify > 95% of MDS patients. This flow-cytometry approach provides an accurate quantitative evaluation of erythroid dysplasia and allows a reliable diagnosis of sideroblastic anemia, and may therefore be a useful tool in the work-up of patients with MDS.

Mitochondrial Ferritin Expression and Clonality of Hematopoiesis in Patients with Refractory Anemia with Ringed Sideroblasts.

Sideroblastic anemias are a heterogeneous group of disorders that have in common the presence of erythroblasts with iron-loaded mitochondria defined as ringed sideroblasts. We have previously demonstrated that mitochondrial iron deposition in these disorders is in the form of mitochondrial ferritin (MtF), suggesting that this latter may be a specific marker of sideroblastic anemia (Cazzola et al, Blood 2003; 101:1996–2000). The most common type of acquired sideroblastic anemia is the myelodysplastic syndrome (MDS) defined as refractory anemia with ringed sideroblasts, which is generally associated with a relatively benign clinical course. In the present work, we studied the relationship between MtF expression and clonality of hematopoiesis in 55 consecutive female patients with low-risk MDS, including 20 cases with ringed sideroblasts and 35 cases without ringed sideroblasts. The expression of MtF, as well as that of cytosolic ferritin (H and L subunits) and of transferrin receptor (CD71), was evaluated by flow cytometry in bone marrow erythroid cells; in selected cases, these immunophenotypic investigations were also performed on liquid cultures of purified CD34-positive cells. X-chromosome inactivation patterns (XCIPs) were assessed in peripheral blood granulocytes and in bone marrow CD34-positive cells by analysis of both DNA methylation at the HUMARA and PGK loci and of IDS gene expression. Within informative females, 11 out of 12 patients with ringed sideroblasts displayed clonal XCIPs in granulocytes; by contrast, only 9 out of 22 patients without ringed sideroblasts displayed clonal XCIPs. Purified CD34-positive cells showed clonal XCIPs in 6 out of 7 patients with ringed sideroblasts but had polyclonal XCIPs in 4 out of 5 individuals without ringed sideroblasts. Flow cytometry evaluation of bone marrow erythroid cells showed that MtF expression was restricted to MDS patients with ringed sideroblasts. A close positive relationship was found between MtF and CD71 expression (r=.49, P=.001); this association may reflect the cytosolic iron deprivation induced by MtF overexpression. Analysis of cultured erythroid progenitor cells showed that MtF was detectable at a very early stage of differentiation from CD34-positive cells. Addition of erythropoietin to the culture system sustained the appearance of a polyclonal erythroid population in 2 out of 4 patients with ringed sideroblasts and clonal CD34-positive cells. These observations suggest that refractory anemia with ringed sideroblasts is a truly clonal stem cell disorder, while more than 50% of patients with refractory anemia without ringed sideroblasts have evidence of polyclonal hematopoiesis. The clonal pattern of CD34-positive cells and the early appearance of MtF during erythroid differentiation suggest that - despite be benign natural history of refractory anemia with ringed sideroblasts - the initial pathogenetic event in this condition occurs in multipotent stem cells. Although the mechanisms responsible for overexpression of MtF are still unclear, flow cytometry evaluation of this protein is a useful diagnostic tool that also provides helpful prognostic information.

Effects of Mitochondrial Ferritin Expression on Tumor Iron Metabolism and Tumor Growth in Nude Mice Xenografts.

Mitochondrial ferritin (MtFt) is a mitochondrial iron storage protein, whose function and regulation is largely unknown. Our previous results have shown that MtFt markedly affects intracellular iron distribution and homeostasis in mammalian cells (Blood 105: 2161–2167, 2005). Using tumor xenografts, we examined the effects of expression MtFt on tumor iron metabolism and growth. H1299 parental or MtFt overexpressing cells were implanted into nude mice. As compared to control tumor xenografts, the expression of MtFt dramatically reduced the implanted tumor growth. A cytosolic iron starvation phenotype in MtFt expressing tumors was revealed by increased RNA-binding activity of iron regulatory proteins (IRPs) and, concomitantly, both an increase in transferrin receptor levels and a decrease in cytosolic ferritin. MtFt overexpression also led to a decrease in both total cellular heme content and heme oxygenase-1 levels. In addition, the expression of MtFt in tumors was associated with a decrease in aconitase activity and lower frataxin protein levels. Mitochondrial iron deposition in MtFt expressing tumors was directly observed by transmission electron microscopy. The pattern of iron accumulation in MtFt overexpressing tumor cells is remarkably similar to that observed in the mitochondria of sideroblastic anemia patients. In conclusion, our study shows that MtFt expression significantly affected tumor iron homeostasis by shunting iron into mitochondria; iron scarcity resulted in partial defects in heme and iron-sulfur cluster syntheses. It is likely that deprivation of iron in the cytosol is the cause of the significant inhibition of xenograft tumor growth.

Flow Cytometry Evaluation of Erythroid Dysplasia in Patients with Myelodysplastic Syndrome.

In the last few years, several studies have evaluated the clinical utility of flow cytometry immunophenotyping in myelodysplastic syndromes (MDS). We developed a flow cytometry approach for the evaluation of marrow dysplasia in MDS using discriminant analysis to generate erythroid and myeloid classification functions (Hematol J 2004; vol 5 S2: pag S91). However, a major problem in the immunophenotypic analysis of MDS is evaluation of erythroid dysplasia because of the limited availability of specific antibodies (Ab). In order to define new tools, we prospectively analyzed the expression of mitochondrial ferritin (MtF) and H ferritin subunit (HF) in bone marrow cells from 69 consecutive patients with MDS and 40 pathologic controls. Six-parameter, 4-color flow cytometry was performed with an EPIX XL flow cytometer (Beckmann Coulter, Fullerton, CA). Erythroid bone marrow cells were analyzed for the expression of HF using a monoclonal mouse Ab (rH02), expression of MtF using a polyclonal mouse Ab, and expression of Glycophorin A, CD71 and CD105 using monoclonal Abs (Instrumentation Laboratory, Milan, Italy). Data on HF and MtF expression obtained by flow cytometry were compared with those obtained on bone marrow smears by an immunocytochemical method (LSAB kit, Dakopatts, Glostrup, Denmark). Discriminant function analysis was performed to identify erythroid dysplasia, with the aim of distinguishing MDS without ringed sideroblasts (RS), MDS with RS, and controls. A significant correlation was found between immunocytochemically and cytometrically detected HF expression (r=.46, P=.016). Overall, the expression of HF was higher in MDS than in controls (P<.001), and was positively correlated with the degree of morphological erythroid dysplasia (r=.56, P<.001). A negative correlation was noticed between HF and CD71 expression (r=−.53, P<.001). Despite its mitochondrial localization, we were able to detect MtF by flow cytometry. Positive correlations were found between flow cytometry detection of MtF and both Prussian blue staining (r=.81, P<.001) and immunocytochemical data (r=.65, P<.001). MtF expression was significantly higher in MDS with RS than in MDS without RS or in controls (P<.001). Finally, compared with controls, MDS patients had a higher percentage of erythroblasts (P<.001), lower expression of CD71 (P<.001) and a higher expression of CD105 (P<.001). A classification function based on HF, CD71 and CD105 expression was defined with the aim of distinguishing MDS from controls:Y=12.44573*(HFX-mean)-2.7698*(CD71X-mean)+13.33752*(CD105X-mean)-4.38426.A patient will be classified as having erythroid dysplasia if the Y value obtained is greater than zero, or as not having dysplasia if the value is lower than zero. This function was integrated with the immunophenotypic evaluation of MtF (Xmean) in order to differentiate between MDS with and without RS. This function allowed us to classify 96% of MDS patients correctly with no false positive cases. Two MDS patients were incorrectly assigned to the RS subgroup by cytometric evaluation of MtF. Based on these findings, we conclude that flow cytometry analysis of MtF and HF expression is reliable and reproducible. Analysis of the expression of these antigens, together with CD71 and CD105, provide an accurate evaluation of erythroid marrow dysplasia and could be usefully included in the work-up of patients with MDS.

The expression of human mitochondrial ferritin rescues respiratory function in frataxin-deficient yeast.

Mitochondrial ferritin (MtF) is structurally and functionally similar to the cytosolic ferritins, molecules designed to store and detoxify cellular iron. MtF expression in human and mouse is restricted to the testis and few tissues, and it is abundant in the erythroblasts of patients with sideroblastic anemia, where it is thought to protect the mitochondria from the damage caused by iron loading. Mitochondria iron overload occurs also in cells deficient in frataxin, a mitochondrial protein involved in iron handling and implicated in Friedreich ataxia. We expressed human MtF in frataxin-deficient yeast cells, a well-characterized model of mitochondrial iron overload and oxidative damage. The human MtF precursor was efficiently imported by yeast mitochondria and processed to functional ferritin that actively sequestered iron in the organelle. MtF expression rescued the respiratory deficiency caused by the loss of frataxin protecting the activity of iron-sulfur enzymes and enabling frataxin-deficient cells to grow on non-fermentable carbon sources. Furthermore, MtF expression prevented the development of mitochondrial iron overload, preserved mitochondrial DNA integrity and increased cell resistance to H2O2. The data show that MtF can substitute for most frataxin functions in yeast, suggesting that frataxin is directly involved in mitochondrial iron-binding and detoxification.

Mitochondrial ferritin

A novel ferritin type specifically targeted to mitochondria has been recently found in human and mouse. It is structurally and functionally similar to the cytosolic ferritins, well-characterized molecules found in most living systems which are designed to store and detoxify cellular iron. Cytosolic ferritins in mammals are ubiquitous while mitochondrial ferritin expression is restricted mainly to the testis, neuronal cells and islets of Langherans. In addition, it is abundant in the iron-loaded mitochondria of erythroblasts of patients with sideroblastic anaemia. The characterization of recombinant and transfected mitochondrial ferritin indicated that this protein has a role in protecting mitochondria from iron-induced damage. These data suggest that it is an interesting tool to study the iron metabolism in this organelle. In addition, it may be useful for the diagnosis of myelodysplastic syndromes and in protecting mitochondria from the toxic effects of excess iron.

Mitochondrial ferritin expression in erythroid cells from patients with sideroblastic anemia

The sideroblastic anemias are characterized by ring sideroblasts, that is, red cell precursors with mitochondrial iron accumulation. We therefore studied the expression of mitochondrial ferritin (MtF) in these conditions. Erythroid cells from 13 patients with refractory anemia with ring sideroblasts (RARS) and 3 patients with X-linked sideroblastic anemia (XLSA) were analyzed for the distribution of cytoplasmic H ferritin (HF) and MtF using immunocytochemical methods. We also studied 11 healthy controls, 5 patients with refractory anemia without ring sideroblasts (RA), and 7 patients with RA with excess of blasts (RAEB). About one fourth of normal immature red cells, mostly proerythroblasts and basophilic erythroblasts, showed diffuse cytoplasmic positivity for HF, but very few were positive for MtF (0%-10%). Similar patterns were found in anemic patients without ring sideroblasts. In contrast, many erythroblasts from patients with sideroblastic anemia (82%-90% in XLSA and 36%-84% in RARS) were positive for MtF, which regularly appeared as granules ringing the nucleus. Double immunocytochemical staining confirmed the different cellular distribution of HF and MtF. There was a highly significant relationship between the percentage of MtF(+) erythroblasts and that of ring sideroblasts (Spearman R = 0.90; P <.0001). Reverse transcription-polymerase chain reaction studies demonstrated the presence of MtF mRNA in circulating reticulocytes of 2 patients with XLSA but not in controls. These findings suggest that most of the iron deposited in perinuclear mitochondria of ring sideroblasts is present in the form of MtF and that this latter might be a specific marker of sideroblastic anemia.

Mitochondrial Ferritin: A New Player in Iron Metabolism

ABSTRACT Mitochondrial ferritin (MtF) is a novel H-type ferritin encoded by an intronless gene on chromosome 5q23.1. The protein is synthesized as a precursor of about 30 kDa that is targeted to mitochondria by a leader sequence of 60 amino acids. This leader is proteolytically removed inside the mitochondria and the resulting 22 kDa subunit forms typical ferritin shells. These shells have ferroxidase activity and are therefore likely to sequester potentially harmful free iron. However, this may be a limited function since MtF has a very restricted tissue expression. High amounts are found in testis but only very low levels are found in iron storage organs. The levels of MtF appear to correlate more with mitochondrial abundance than with iron metabolism. MtF does not seem to be an obligatory intermediate in transfer of free iron to heme and other iron compounds in mitochondria. However, its level increases dramatically in sideroblastic anemia when heme synthesis is disrupted. This increased synthesis does not appear to involve the classical translational control since MtF mRNA lacks an apparent iron response element. In transfected HeLa cells added iron is incorporated as quickly into MtF as into cytosolic ferritin. In addition, increased levels of MtF cause a redistribution of iron from cytosol to mitochondria and this effect is enhanced by iron chelation. Thus high levels of MtF result in an iron deficient phenotype in cytosol with decreased expression of ferritin and increased expression of transferrin receptor. This avidity for iron may explain why MtF levels are maintained at low levels in most normal cells. The regulation of MtF expression and possible therapeutic applications of MtF in neurological disorders involving increased iron deposition are topics for future research.