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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.

Hemopexin (Hx) is a plasma glycoprotein mainly expressed in liver and, less abundantly, in the central and peripheral nervous systems. Hx has a high binding affinity with heme and is considered to be a major transport vehicle of heme into the liver, thus preventing both heme-catalyzed oxidative damage and heme-bound iron loss. To determine the physiologic relevance of heme-Hx complex formation, Hx-deficient mice were generated by homologous recombination in embryonic stem (ES) cells. The Hx-deficient mice were viable and fertile. Their plasma iron level and blood parameters were comparable to those of control mice and they showed no evidence of tissue lesions caused by oxidative damage or abnormal iron deposits. Moreover, they were sensitive to acute hemolysis, as are wild-type mice. Nevertheless, Hx-null mice recovered more slowly after hemolysis and were seen to have more severe renal damage than controls. After hemolytic stimulus, Hx-deficient mice presented prolonged hemoglobinuria with a higher kidney iron load and higher lipid peroxidation than control mice. Moreover, Hx-null mice showed altered posthemolysis haptoglobin (Hp) turnover in as much as Hp persisted in the circulation after hemolytic stimulus. These data indicate that, although Hx is not crucial either for iron metabolism or as a protection against oxidative stress under physiologic conditions, it does play an important protective role after hemolytic processes.

Abstract 2090 Feline Leukemia Virus subgroup C Receptor 1 (FLVCR1) is a cell membrane heme exporter that contributes to maintain the balance between heme level and globin synthesis in erythroid precursors. Consistently, FLVCR1-null mice died in utero due to a failure of erythropoiesis1. We previously reported the identification of a mitochondrial isoform of FLVCR1, named FLVCR1b, that was able to support fetal murine erythroid differentiation in the absence of the cell membrane isoform (herein called FLVCR1a)2. The aim of this work was to investigate the role of FLVCR1b during erythroid differentiation. To this end, overexpression and silencing experiments were performed. FLVCR1b overexpression promotes in vitro erythroid differentiation of K562 cells, as indicated by the increased transcription of globin mRNA and a higher percentage of benzidine positive cells, compared to control cells. On the contrary, FLVCR1a-overexpressing K562 cells were not able to differentiate. Interestingly, shRNA-mediated down-regulation of FLVCR1a led to the elevation of the percentage of benzidine positive cells compared to controls that further increased upon the stimulation of erythroid differentiation using sodium butyrate. A decrease of the percentage of benzidine positive cells was observed in K562 cells lacking both FLVCR1 isoforms compared to FLVCR1a-deficient cells. Moreover these cells were not able to differentiate upon stimulation with sodium butyrate. These results suggest a fundamental role of FLVCR1b during erythroid differentiation in vitro, supporting previous data obtained in the mouse model2. To understand the molecular mechanisms in which FLVCR1b is involved, we investigate whether FLVCR1b could affect heme export from mitochondria. The overexpression of FLVCR1b in HeLa cells led to increased intracellular heme content together with a strong induction of the heme degrading enzyme heme oxygenase 1 (HO-1) mRNA. Inhibition of the heme biosynthetic pathway using succinylacetone, completely prevented intracellular accumulation of heme observed when FLVCR1b was overexpressed. So, increased heme biosynthesis rate is responsible for the elevation of heme content. Accordingly, HeLa cells overexpressing FLVCR1b showed an alteration of heme biosynthesis enzymes and transporters. On the contrary, the specific loss of FLVCR1b using siRNA causes heme accumulation in mitochondria and a subsequent block of heme biosynthesis. These data are consistent with a role of FLVCR1b as a mitochondrial heme exporter. Similar results were also obtained in K562 cells thus suggesting that loss of FLVCR1b reduces the availability of heme for haemoglobin synthesis, a process essential during erythroid differentiation. To assess whether mitochondrial heme accumulation due to the loss of FLVCR1b affect mitochondrial functionality, the mitochondrial Ca2+ response after agonist stimulation was monitored as a highly sensitive readout of mitochondrial state. It is well known that mitochondrial alterations cause defects in Ca2+ uptake by the organelle. The silencing of FLVCR1a and FVLCR1b in HeLa cells caused a significant reduction of Ca2+spike in the mitochondrial matrix evoked by agonist stimulation. These data suggested that when FLVCR1b is lost heme accumulates in mitochondria resulting in the alterations of mitochondrial functionality. All together these data indicate that the impairment of erythroid differentiation observed in the absence of FLVCR1b is due to the block of heme export from mitochondria and a consequent impairment of mitochondrial functionality, which is essential for cell survival. These results, linking heme biosynthesis pathway to mitochondrial Ca2+signaling, will have broad implications in cellular metabolism. Disclosures: No relevant conflicts of interest to declare.

Abstract 4243 Feline Leukemia Virus subgroup C Receptor (FLVCR) was originally identified and cloned as a cell-surface protein receptor for feline leukemia virus subgroup C, causing pure red blood cell aplasia in cats. Recent studies have demonstrated that FLVCR is a heme exporter which is essential for erythropoiesis. The heme efflux via FLVCR was shown to be essential for erythroid differentiation in K562 cells as well as in CD34+ precursors cells1. Moreover, Keel and co-authors have reported that Flvcr-null mice die in utero due to the failure of fetal erythropoiesis; also post-natal mice lacking FLVCR showed severe anemia. In addition to the erythroid defect, Flvcr-null embryos display defective growth and developmental anomalies2. We have identified an alternative transcription start site giving rise to a novel FLVCR isoform (FLVCRb). Flvcr-b transcript completely lacks the first exon of the canonical isoform (FLVCRa) and code for a putative 6 transmembrane domain containing protein ubiquitously expressed. In vitro over-expression of FLVCRa and FLVCRb showed that the two proteins display different subcellular localization. As expected, FLVCRa is localized at the cell membrane while FLVCRb is in the mitochondrial compartment. The mitochondrial localization of this novel isoform is further confirmed by the identification of a N-terminal mitochondrial sorting presequence. The mitochondrion is the site in which heme biosynthesis occurs. Although all the enzymatic reactions involved in heme synthesis are well characterized, how heme is exported to the cytosol is largely unknown. Because of FLVCRa is a heme exporter at the cell membrane, we hypothesized that FLVCRb could be the mitochondrial heme exporter. According to this hypothesis, FLVCRb expression increased following the stimulation of heme biosynthesis in vitro, in correlation with the increase in hemoglobin production. The ability of FLVCRb to bind and export heme out of the mitochondria is still under investigation. To gain insights into the specific roles of the two isoforms, we have generated Flvcr mutant mice different from those previously reported2. Keel and co-author generated a mouse model in which both FLVCRa and FLVCRb have been deleted. In our mouse model, FLVCRa has been specifically deleted while FLVCRb is still expressed (FLVCRa-null mice). Flvcr-a +/− mice were grossly normal, fertile and indistinguishable from their wild-type littermates. When Flvcr-a +/− mice were intercrossed, no Flvcr-a homozygous knock-out newborns were obtained. The analysis of the embryos from timed Flvcr-a +/− intercrosses showed that the Flvcr-a homozygous knock-out genotype was lethal between E14.5 and the birth. E13.5 Flvcr-a-null embryos showed multifocal and extended hemorrhages, visible in the limbs, head and throughout the body wall, as well as subcutaneous edema. Imcomplete vasculogenesis in the Flvcr-a-null embryos was observed at E11.5, a developmental stage in which hemorrhages were not still evident. This suggests that hemorrhages arise from a defect in the development of embryonic vasculature. Moreover, FLVCRa-null embryos showed skeletal abnormalities as demonstrated by Alcian blue-alizarin red staining. Skeletal malformations were evident in the limb where digits did not form properly and in the head where Meckel's cartilage was incomplete. It is interesting to note that this kind of malformations also occurs in Diamond Blackfan Anemia (DBA) patients. Surprisingly, flow cytometric analyses of E14.5 fetal liver cells double-stained for Ter119 (erythroid-specific antigen) and CD71 (transferrin receptor) showed normal erythropoiesis in Flvcr-a-null embryos, in opposition to what occurs in the previously reported Flvcr-null mice2. Taken together, these data demonstrated that FLVCRb is sufficient to support fetal erythropoiesis when the expression of FLVCRa is loss, likely exporting heme out of the mithocondrion for hemoglobin synthesis. Moreover, the loss of FLVCRa leads to incomplete vasculogenesis, hemorrhages and skeletal malformations highlighting new roles of FLVCRa in these processes. 1. Quigley JG et al. Identification of a human heme exporter that is essential for erythropoiesis. Cell 2004 2. Keel SB et al. A heme export protein is required for red blood cell differentiation and iron homeostasis. Science 2008. Disclosures: No relevant conflicts of interest to declare.

Abstract 4776 Introduction. A good indicator of the rate of cell ageing is the length of telomeres, the long tandem repeat sequences at the end of chromosomes. Telomere length (TL) in humans decreases at each somatic cell division, and undergoes progressive shortening with increasing age. Indeed, cell proliferation has been considered the primary factor responsible of TL shortening and ultimately of the cell ageing process. Exposure to DNA-damaging substances has been proposed as an alternative and relevant factor involved in the cell ageing process. In spite of the number of studies in both human and animal models, few information is available on the role of both cell proliferation and DNA damage in normal human stem cells. The present study addressed this issue by investigating the ageing process in cultured Bone-Marrow (BM) human Mesenchymal Stem Cells (MSCs). Aim of the study has been to investigate whether and how exposure to toxic agents may affect the rate of TL shortening in cultured MSCs. Methods. 1. MSCs were cultured from human mononuclear BM cells, obtained from normal volunteers or patients undergoing routine diagnostic procedures; cells were seeded in MEM-alpha medium containing heparin and 10% platelet lysates and fed at 3–4 day intervals, until they reached confluence; they were then detached with trypsin-EDTA solution and reseeded for the experimental tests. Cultured MSC were identified for positivity of CD105, CD90, CD29, CD44 and negativity for CD14, CD34 and CD45 by flow cytometry; 2. cultured MSC were assayed for response to DNA-damaging drugs between the second and the third passage in culture. Two chemotherapeutic drugs were employed to induce DNA damage: Doxorubicin (Doxo) and etoposide (Eto). Cells were incubated for 2 hours with decreasing doses of the tested drugs, and 10 nM Doxo and 500 ng/ml Eto were the highest doses of the drugs that were used without any distress on cell proliferation and cell viability. The 2-hour exposure to chemotherapy was repeated at 7 day intervals up to four times; 3. telomere was evaluate in MSCs before (day 0 or T0) and after exposure to chemotherapeutic drugs, i.e. at 7 – 14 –21 and 28 days of culture since drug exposure. TL was determined by the cytofluorimetric Flow-fish assay and by southern-blot analysis; 4. MSC were also cultured in differentiating medium in order to induce osteogenic and adipogenic differentiation, evaluated by Alizarin red and Oil red staining, respectively; 5. lastly, both ATM phosphorilation and telomerase activation were assessed, by Western blot analysis and the TRAP assay, respectively. Results. 1. as expected, TL progressively decreased after 7 days in culture in untreated (NT) MSC (88,8% compared to T0); TL was further shortened as long as the cells were maintained in culture; 2. a marked TL shortening occurred in MSC already at day 7 after exposure to sub-lethal doses of Doxo and Eto, with a mean of 81,7% and 79,6% compared to T0, respectively. Indeed, a minimum of 5 days in culture was required to identify the earliest signs of telomere loss. Again, further TL shortening was observed later on and the difference in TL between NT and chemotherapy-exposed cells progressively increased: compared to T0, TL resulted of 82 % and 74% in NT cells, whereas TL was 68% and 54% in Doxo-treated, and 70% and 62% in Eto-treated, at week 3 and 4, respectively; 3. a single 2-hr exposure to Doxo or Eto induced a TL loss compared to NT cells, that was maintained unchanged at long-term in culture; 4. when MSC were assayed in differentiation inducing medium, cells exposed to chemotherapy displayed enhanced adipogenic and osteogenic differentiation compared to NT cells; 5. ATM was strongly phosphorilated following exposure to Doxo and Eto compared to NT MSC; ATM phosphorilation preceded TL loss; by contrast, telomerase was unaffected by chemotherapy exposure. Conclusions. i. human cultured MSCs exposed to chemotherapeutic drugs offer a simple and reliable means to investigate the correlation between DNA damage and cell ageing; ii. telomere loss is clearly detectable in MSCs already after 7 days following exposure to Doxo and Eto and remains detectable at long-term in culture as a permanent signature of the previous DNA damage; iii. the in vitro observation suggests that not only hematopoietic cells but also stromal cells, including MSCs, may experience relevant DNA damages following in vivo treatments with chemotherapeutic drugs. Disclosures: No relevant conflicts of interest to declare.