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Background Macrophages of the reticuloendothelial system play a key role in recycling iron from hemoglobin of senescent or damaged erythrocytes. Heme oxygenase 1 degrades the heme moiety and releases inorganic iron that is stored in ferritin or exported to the plasma via the iron export protein ferroportin. In the plasma, iron binds to transferrin and is made available for de novo red cell synthesis. The aim of this study was to gain insight into the regulatory mechanisms that control the transcriptional response of iron export protein ferroportin to hemoglobin in macrophages. Design and Methods Iron export protein ferroportin mRNA expression was analyzed in RAW264.7 mouse macrophages in response to hemoglobin, heme, ferric ammonium citrate or protoporphyrin treatment or to siRNA mediated knockdown or overexpression of Btb And Cnc Homology 1 or nuclear accumulation of Nuclear Factor Erythroid 2-like. Iron export protein ferroportin promoter activity was analyzed using reporter constructs that contain specific truncations of the iron export protein ferroportin promoter or mutations in a newly identified MARE/ARE element. Results We show that iron export protein ferroportin is transcriptionally co-regulated with heme oxygenase 1 by heme, a degradation product of hemoglobin. The protoporphyrin ring of heme is sufficient to increase iron export protein ferroportin transcriptional activity while the iron released from the heme moiety controls iron export protein ferroportin translation involving the IRE in the 5′untranslated region. Transcription of iron export protein ferroportin is inhibited by Btb and Cnc Homology 1 and activated by Nuclear Factor Erythroid 2-like involving a MARE/ARE element located at position −7007/−7016 of the iron export protein ferroportin promoter. Conclusions This finding suggests that heme controls a macrophage iron recycling regulon involving Btb and Cnc Homology 1 and Nuclear Factor Erythroid 2-like to assure the coordinated degradation of heme by heme oxygenase 1, iron storage and detoxification by ferritin, and iron export by iron export protein ferroportin.

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 1080 Poster Board I-102 Feline Leukemia Virus, subgroup C, Receptor (FLVCR) was originally identified and cloned as a cell-surface protein receptor for feline leukaemic virus, subgroup C causing pure red blood cell aplasia in cats. Recent studies have demonstrated that FLVCR is a heme exporter 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 recently reported that Flvcr-null mice die in utero due to the failure of fetal erythropoiesis2. We have recently identified a novel FLVCR isoform, called FLVCRb, coding for a putative 7 transmembrane domain-containing protein. Both FLVCRb and the canonical FLVCR (described herein as FLVCRa) were expressed in all tissues: FLVCRa mRNA level was significantly higher than FLVCRb in liver, kidney, colon and duodenum, whereas the opposite was observed in heart and skeletal muscle. In bone marrow, spleen and brain, FLVCRa and FLVCRb mRNA levels were comparable. FLVCRa and FLVCRb display different sub-cellular distribution when over-expressed in vitro. As expected, FLVCRa is localized at the cell membrane while FLVCRb is expressed in intracellular compartment suggesting different functions of the two proteins in heme metabolism. 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 were deleted. In our mouse model, FLVCRa has been specifically deleted and FLVCRb is still expressed (Flvcr-a-null mice). Flvcr-a heterozygous 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, and the analysis of the embryos from timed Flvcr-a +/- intercrosses showed that the Flvcr-a-/- genotype was lethal between E14.5-E16.5. Flvcr-a-null embryos showed multifocal and extended hemorrhages, visible in the limbs, head and throughout the body wall, as well as subcutaneous edema. Alcian blue-alizarin red staining demonstrated skeletal abnormalities in limbs and head similar to that observed in Diamond-Blakfan anemia (DBA) patients. Interestingly, flow cytometric analyses of E14.5 fetal liver cells double-stained for Ter119 (erythroid-specific antigen) and CD71 (transferrin receptor) show normal erythropoiesis in Flvcr-a-null embryos, opposite to the previously reported Flvcr-null mice2. Taken together, these data demonstrated that FLVCRb is sufficient to support fetal erythropoiesis, but not to prevent endothelial ruptures responsible for hemorrhages, thus suggesting that FLVCRa is needed for detoxifying heme excess at these sites. The involvement of the two isoforms in the pathogenesis of DBA is currently under investigation. Quigley JG, Yang Z, Worthington MT, Phillips JD, Sabo KM, Sabath DE, Berg CL, Sassa S, Wood BL, Abkowitz JL. Identification of a human heme exporter that is essential for erythropoiesis. Cell 2004;118:757-66. Keel SB, Doty RT, Yang Z, Quigley JG, Chen J, Knoblaugh S, Kingsley PD, De Domenico I, Vaughn MB, Kaplan J, Palis J, Abkowitz JL. A heme export protein is required for red blood cell differentiation and iron homeostasis. Science 2008;319:825-8. Disclosures No relevant conflicts of interest to declare.