Heme Exporter Research Articles

Pathophysiology of Macrocytic Anemia in Diamond Blackfan Anemia and Del(5q) Myelodysplastic Syndrome

Twenty-five percent of Diamond Blackfan anemia (DBA) cases result from haploinsufficiency of RPS19 and ~30% from haploinsufficiencies of other ribosomal proteins. The macrocytic anemia of del(5q) MDS results from RPS14 haploinsufficiency. When tested, these mutations disrupt ribosome assembly, yet how this causes macrocytic anemia remains uncertain. The dominant hypothesis is that P53 pathway activation leads to cell apoptosis, however, why erythropoiesis is severely impacted, while other hematopoietic as well as non-hematopoietic cells function appropriately, is difficult to explain. These observations led us to predict that DBA and del(5q) MDS share a pathogenesis that reflects the rapid kinetics of red cell and hemoglobin production, and specifically that poor ribosome assembly leads to an inability to quickly translate mRNA to protein, delayed globin synthesis in early erythroid precursors, and excess heme. Since free heme is toxic, cell death ensues. To explore this, we studied marrow from DBA (n=3) and del(5q) MDS (n=6) patients in cultures that optimize erythroid differentiation and showed that (a) cell numbers significantly decrease between stages I (lin-CD71+CD36-GlyA-)(BFU-E/early CFU-E) and II, (lin-CD71+CD36+GlyA-)(CFU-E/early proerythroblasts; Li et al. Blood 2014;124:3636); and (b) the cells that survive this insult differentiate normally to stages III (lin-CD36+GlyA+) and IV (lin-CD36-GlyA+). Using marrow from the index DBA patient, a 28-year old transfusion-dependent female with inherited RPS19 haploinsufficiency, and (as available) marrow from other DBA and del(5q) MDS patients (n=2-7 patients per study), we demonstrated that heme synthesis (assessed by ALAS2mRNA and protein) initiates normally while globin production (α- and β-globin mRNA and protein) is slowed, and that stage I and II cells have excess heme. Next, to discern the cell-specific characteristics that allow a DBA or del(5q) MDS cell to survive and fully mature, we compared those cells able to advance to stages III and IV in patient and concurrent control cultures. Stage III-IV GlyA+ DBA cells expressed 2.4-fold less ALAS2 mRNAand 2-fold more FLVCR mRNA than GlyA+ control cells (p values<0.05). Since the levels of ALAS2 and FLVCR mRNA were similar in stage I DBA and control cells, the time when heme synthesis initiates, these data imply that DBA cells capable of down-regulating their heme synthesis (have less ALAS2) or content (have moreFLVCR and increased heme export) preferentially survive. Data from del(5q) MDS patients are similar - cells progressing to stages III and IV have 2.9-fold less ALAS2 mRNA (p<0.05) and 9.4-fold higher FLVCR mRNA (p<0.01) than control cells. That heme synthesis proceeds normally, while globin production initiates slowly, was confirmed by sequential sampling of marrow cultures at days 3, 7, 10, 13, and 17; and as expected, DBA and del(5q) MDS cells at day 7 had increased reactive oxygen species (ROS). Consistent with these observations, treatment with 10 μM succinylacetone, a specific inhibitor of heme synthesis, improved the erythroid cell output of DBA and del(5q) MDS cultures by 68-95% (p values<0.05), while the erythroid cell output of concurrent control cultures decreased by 4-13%. Cell death appears to result both from apoptosis (not shown) and ferroptosis, a ROS-induced process previously characterized in kidney epithelial cells and cancer cell lines (Dixon et al. Cell 2012;149:1060). Recent data showing P53 sensitizes cells to ferroptosis by impeding the transcription of SCL7A11, a cysteine-glutamate antiporter, leading to less cysteine uptake, less GSH synthesis, less GPX4, and less antioxidants (Jiang et al. Nature 2015;520:57) link our observations to work implicating P53 activation in DBA and del(5q) MDS anemia. In support of this connection, DBA marrow cells at culture day 10 have 57% less SLC7A11 mRNA than control cells in preliminary experiments. Together, our studies demonstrate that erythropoiesis fails when heme exceeds globin. The data also suggest that decreasing heme synthesis, either directly or by chelating iron, may improve these anemias. They further argue that lenalidomide (destabilizes P53) and therapies decreasing heme should be synergistic. Since diverse mutations could impede brisk protein transcription or translation, and hence brisk globin production, heme toxicity might also contribute to anemia in MDS patients without del(5q). DisclosuresNo relevant conflicts of interest to declare.

Coordinate expression of heme and globin is essential for effective erythropoiesis.

Erythropoiesis requires rapid and extensive hemoglobin production. Heme activates globin transcription and translation; therefore, heme synthesis must precede globin synthesis. As free heme is a potent inducer of oxidative damage, its levels within cellular compartments require stringent regulation. Mice lacking the heme exporter FLVCR1 have a severe macrocytic anemia; however, the mechanisms that underlie erythropoiesis dysfunction in these animals are unclear. Here, we determined that erythropoiesis failure occurs in these animals at the CFU-E/proerythroblast stage, a point at which the transferrin receptor (CD71) is upregulated, iron is imported, and heme is synthesized--before ample globin is produced. From the CFU-E/proerythroblast (CD71(+) Ter119(-) cells) stage onward, erythroid progenitors exhibited excess heme content, increased cytoplasmic ROS, and increased apoptosis. Reducing heme synthesis in FLVCR1-defient animals via genetic and biochemical approaches improved the anemia, implying that heme excess causes, and is not just associated with, the erythroid marrow failure. Expression of the cell surface FLVCR1 isoform, but not the mitochondrial FLVCR1 isoform, restored normal rbc production, demonstrating that cellular heme export is essential. Together, these studies provide insight into how heme is regulated to allow effective erythropoiesis, show that erythropoiesis fails when heme is excessive, and emphasize the importance of evaluating Ter119(-) erythroid cells when studying erythroid marrow failure in murine models.

Crucial Role of FLVCR1a in the Maintenance of Intestinal Heme Homeostasis.

The maintenance of heme homeostasis, mucosa cell renewal, and redox environment in the intestine is essential to permit digestion, absorption, cell proliferation, cell apoptosis, and immune response and to avoid the development of gut disorders. The feline leukemia virus, subgroup C, receptor 1a (FLVCR1a) is a heme exporter expressed in almost all cell types, including intestinal cells. This work investigates the role of FLVCR1a in the intestine, taking advantage of an intestine-specific conditional Flvcr1a-knockout mouse and of FLVCR1a-depleted Caco2 cells. The data show that FLVCR1a does not participate in the absorption of dietary heme, whereas it is involved in the export of de novo synthesized heme from intestinal cells. The loss of Flvcr1a is associated with a decrease of intestinal cell proliferation and with alterations in the peculiar homeostasis of proliferating cells, including the maintenance of their redox status. The involvement of FLVCR1a in these processes renders this exporter crucial for the survival of mice in a model of ulcerative colitis. These findings shed light on the role of heme export in the dietary heme absorption process and unravel a new role for heme export in the control of mucosal renewal and in proliferating cell redox status and metabolic activity, demonstrating a crucial role for FLVCR1a in maintaining intestinal homeostasis in both physiologic and pathologic situations. By exporting the excess of de novo synthesized heme from intestinal cells, FLVCR1a participates in the control of intestinal mucosa homeostasis.

Alteration of heme metabolism in a cellular model of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital pure red cell aplasia often associated with skeletal malformations. Mutations in ribosomal protein coding genes, mainly in RPS19, account for the majority of DBA cases. The molecular mechanisms underlying DBA pathogenesis are still not completely understood. Alternative spliced isoforms of FLVCR1 (feline leukemia virus subgroup C receptor 1) transcript coding for non-functional proteins have been reported in some DBA patients. Consistently, a phenotype very close to DBA has been described in animal models of FLVCR1 deficiency. FLVCR1 gene codes for two proteins: the plasma membrane heme exporter FLVCR1a and the mitochondrial heme exporter FLVCR1b. The coordinated expression of both FLVCR1 isoforms regulates an intracellular heme pool, necessary for proper expansion and differentiation of erythroid precursors. Here, we investigate the role of FLVCR1 isoforms in a cellular model of DBA. RPS19-downregulated TF1 cells show reduced FLVCR1a and FLVCR1b mRNA levels associated with heme overload. The downregulation of FLVCR1 isoforms affects cell cycle progression and apoptosis in differentiating K562 cells, a phenotype similar to DBA. Taken together, these data suggest that alteration of heme metabolism could play a role in the pathogenesis of DBA.

The heme exporter Flvcr1 regulates expansion and differentiation of committed erythroid progenitors by controlling intracellular heme accumulation.

Feline leukemia virus subgroup C receptor 1 (Flvcr1) encodes two heme exporters: FLVCR1a, which localizes to the plasma membrane, and FLVCR1b, which localizes to mitochondria. Here, we investigated the role of the two Flvcr1 isoforms during erythropoiesis. We showed that, in mice and zebrafish, Flvcr1a is required for the expansion of committed erythroid progenitors but cannot drive their terminal differentiation, while Flvcr1b contributes to the expansion phase and is required for differentiation. FLVCR1a-down-regulated K562 cells have defective proliferation, enhanced differentiation, and heme loading in the cytosol, while FLVCR1a/1b-deficient K562 cells show impairment in both proliferation and differentiation, and accumulate heme in mitochondria. These data support a model in which the coordinated expression of Flvcr1a and Flvcr1b contributes to control the size of the cytosolic heme pool required to sustain metabolic activity during the expansion of erythroid progenitors and to allow hemoglobinization during their terminal maturation. Consistently, reduction or increase of the cytosolic heme rescued the erythroid defects in zebrafish deficient in Flvcr1a or Flvcr1b, respectively. Thus, heme export represents a tightly regulated process that controls erythropoiesis.

Heme exporter FLVCR is required for T cell development and peripheral survival.

All aerobic cells and organisms must synthesize heme from the amino acid glycine and the tricarboxylic acid cycle intermediate succinyl CoA for incorporation into hemoproteins, such as the cytochromes needed for oxidative phosphorylation. Most studies on heme regulation have been done in erythroid cells or hepatocytes; however, much less is known about heme metabolism in other cell types. The feline leukemia virus subgroup C receptor (FLVCR) is a 12-transmembrane domain surface protein that exports heme from cells, and it was shown to be required for erythroid development. In this article, we show that deletion of Flvcr in murine hematopoietic precursors caused a complete block in αβ T cell development at the CD4(+)CD8(+) double-positive stage, although other lymphoid lineages were not affected. Moreover, FLVCR was required for the proliferation and survival of peripheral CD4(+) and CD8(+) T cells. These studies identify a novel and unexpected role for FLVCR, a major facilitator superfamily metabolite transporter, in T cell development and suggest that heme metabolism is particularly important in the T lineage.

Placental heme receptor LRP1 correlates with the heme exporter FLVCR1 and neonatal iron status

LDL receptor-related protein 1 (LRP1) is a transmembrane receptor highly expressed in human placenta. It was recently found to be the receptor for heme and its plasma-binding protein hemopexin (Hx) and is integral to systemic heme clearance. Little is known about systemic concentrations of Hx during pregnancy and whether maternal Hx and placental LRP1 contributes to fetal iron (Fe) homeostasis during pregnancy. We hypothesized that placental LRP1 would be upregulated in maternal/neonatal Fe insufficiency and would be related to maternal circulating Hx. Placental LRP1 expression was assessed in 57 pregnant adolescents (14-18 years) in relationship with maternal and cord blood Fe status indicators (hemoglobin (Hb), serum ferritin, transferrin receptor), the Fe regulatory hormone hepcidin and serum Hx. Hx at mid-gestation correlated positively with Hb at mid-gestation (r=0.35, P=0.02) and Hx at delivery correlated positively with cord hepcidin (r=0.37, P=0.005). Placental LRP1 protein expression was significantly higher in women who exhibited greater decreases in serum Hx from mid-gestation to term (r=0.28, P=0.04). Significant associations were also found between placental LRP1 protein with cord hepcidin (r=-0.29, P=0.03) and placental heme exporter feline leukemia virus C receptor 1 (r=0.34, P=0.03). Our data are consistent with a role for placental heme Fe utilization in supporting fetal Fe demands.

Control of Metazoan Heme Homeostasis by a Conserved Multidrug Resistance Protein

Several lines of evidence predict that specific pathways must exist in metazoans for the escorted movement of heme, an essential but cytotoxic iron-containing organic ring, within and between cells and tissues, but these pathways remain obscure. In Caenorhabditis elegans, embryonic development is inextricably dependent on both maternally derived heme and environmentally acquired heme. Here, we show that the multidrug resistance protein MRP-5/ABCC5 likely acts as a heme exporter, and targeted depletion of mrp-5 in the intestine causes embryonic lethality. Transient knockdown of mrp5 in zebrafish leads to morphological defects and failure to hemoglobinize red blood cells. MRP5 resides on the plasma membrane and endosomal compartments and regulates export of cytosolic heme. Together, our genetic studies in worms, yeast, zebrafish, and mammalian cells identify a conserved, physiological role for a multidrug resistance protein in regulating systemic heme homeostasis. We envision other MRP family members may play similar unanticipated physiological roles in animal development.

Hypoxia controls Flvcr1 gene expression in Caco2 cells through HIF2α and ETS1

The tissue-specific gene expression changes mediated by the hypoxia inducible factors (HIFs) allow the adaptation of cells to low oxygen tension and control several processes including erythropoiesis, angiogenesis and vasculogenesis. The Feline Leukemia Virus, subgroup C, Receptor 1 (Flvcr1) gene encodes for two isoforms, Flvcr1a and 1b, involved in the export of heme out of the cell and of mitochondria respectively. Studies in mouse models demonstrated a crucial role of Flvcr1 isoforms in erythropoiesis and during embryo development.Here, we showed the modulation of Flvcr1 gene expression in different tissues and cell lines in response to hypoxia. Chromatin immunoprecipitation analysis demonstrated that HIF2α and HIF-dependent transcription factor ETS1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1) bind at the region −318/+39 of the Flvcr1 promoter. Analysis of Caco2 cells in which HIF2α or ETS1 were silenced or overexpressed demonstrated that, both HIF2α and ETS1 are involved in the transcriptional regulation of Flvcr1a and that HIF2α is absolutely required for Flvcr1a induction upon hypoxia.The inclusion of the Flvcr1 gene in the group of HIF2α-responsive genes strengthens its role in hypoxia-stimulated processes like erythropoiesis, vasculogenesis and heme absorption.

Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

Background & AimsThe liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice.MethodsWe created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1afl/fl;alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured.ResultsFlvcr1afl/fl;alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1afl/fl;alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450.ConclusionsIn livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism.

Differential Activation of Staphylococcus aureus Heme Detoxification Machinery by Heme Analogues

The reactive nature of heme enables its use as an enzymatic cofactor while rendering excess heme toxic. The importance of heme detoxification machinery is highlighted by the presence of various types of these homeostatic systems in Gram-positive and Gram-negative microorganisms. A number of pathogens possess orthologs of the HssRS/HrtAB heme detoxification system, underscoring a potential role this system plays in the survival of bacteria in heme-rich environments such as the vertebrate host. In this work, we sought to determine the role of this system in protection against metalloporphyrin heme analogues identified by previous studies as antimicrobial agents. Our findings demonstrate that only toxic metalloporphyrins maximally activate expression of the Staphylococcus aureus heme detoxification system, suggesting that the sensing mechanism of HssRS might require a component of the associated toxicity rather than or in addition to the metalloporphyrin itself. We further establish that only a subset of toxic metalloporphyrins elicit the oxidative damage previously shown to be a significant component of heme toxicity whereas all toxic noniron metalloporphyrins inhibit bacterial respiration. Finally, we demonstrate that, despite the fact that toxic metalloporphyrin treatment induces expression of S. aureus heme detoxification machinery, the HrtAB heme export pump is unable to detoxify most of these molecules. The ineffectiveness of HrtAB against toxic heme analogues provides an explanation for their increased antimicrobial activity relative to heme. Additionally, these studies define the specificity of HssRS/HrtAB, which may provide future insight into the biochemical mechanisms of these systems.

Heme-Related Blood Disorders

Heme biosynthesis in erythroid cells is intended primarily for the formation of hemoglobin. As in every cell, this synthesis requires a multi-step pathway that involves eight enzymes including the erythroid-specific δ-aminolevulinate synthase (ALAS2, the first regulated enzyme that converts glycine and succinyl CoA into ALA) and the ubiquitous ferrochelatase (FECH, the final enzyme). Heme biosynthesis also requires membrane transporters that are necessary to translocate glycine, precursors of heme, and heme itself between the mitochondria and the cytosol. Defects in normal porphyrin and/or heme synthesis and transport cause four major erythroid inherited disorders, which may or may not be associated with dyserythropoiesis (e.g., sideroblastic, microcytic anemia and/or hemolytic anemia): "X-linked" sideroblastic anemia (XLSA) and X-linked dominant protoporphyria (XLDPP) are two different and opposing disorders but related to altered gene encoding ALAS2 only. Defective activity of this enzyme due to mutations in the ALAS2 gene causes the XLSA phenotype, including microcytic, hypochromic anemia with abundant ringed sideroblasts in the bone marrow. Vice versa, gain-of-function mutations of ALAS2 are responsible of the XLDPP characterized by predominant accumulation of the hydrophobic protoporphyrin (PPIX, the last heme precursor) in the erythrocytes without anemia or sideroblasts. Furthermore, the glycine transporter (SLC25A38) and Glutaredoxin 5 genes are reported to be involved in human non-syndromic sideroblastic anemia. Congenital erythropoietic porphyria (CEP) is the rarest autosomal recessive disorder due to a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. CEP leads to excessive synthesis and accumulation of type I isomers of porphyrins in the reticulocytes, followed by intravascular hemolysis and severe anemia. The ALAS2 gene may act as a modifier gene in CEP patients (Figueras J et al, Blood. 2011;118(6):1443-51). Erythropoietic protoporphyria (EPP) results from a partial deficiency of FECH and leads similarly to XLDPP, to deleterious accumulation of PPIX in erythroid cells. Most EPP patients present intrans to a FECH gene mutation an IVS3-48C hypomorphic allele due to a splice mutation. Abnormal spliced mRNA is degraded which contributes to the lowest FECH enzyme activity and allowed EPP phenotype expression. We have identified an antisense oligonucleotide (ASO) to redirect splicing from cryptic to physiological site and showed that the ASO-based therapy mediates normal splice rescue of FECH mRNA and reduction by 60 percent of PPIX overproduction in primary cultures of EPP erythroid progenitors. Therapeutic approaches to target both ALAS2 inhibition and heme-level reduction may be useful in other erythroid disorders such as thalassemia (where reduced heme biosynthesis was shown to improve the clinical phenotype) or the Diamond-Blackfan anemia (DBA). Indeed, in some DBA patients, an unusual mRNA splicing of heme exporter FLVCR has been found, reminiscent of Flvcr1-deficient mice that develop a DBA-like phenotype with erythroid heme accumulation. Thus, FLVCR may act as a modifier gene for DBA phenotypic variability. Recent advances in understanding the pathogenesis and molecular genetic heterogeneity of heme-related disorders have led to improved diagnosis and treatment. These advances include DNA-based diagnoses for all the porphyrias and some porphyrins and heme transporters, new understanding of the pathogenesis of the erythropoietic disorders, and new and experimental treatments such as chronic erythrocyte transfusions, bone marrow or hematopoietic stem cell transplants, and experimental pharmacologic chaperone and stem cell gene therapies for erythropoietic porphyrias. Disclosures:No relevant conflicts of interest to declare.

FLVCR1a But Not FLVCR1b Is Required For Effective Erythropoiesis In Adult Mice

There are two characterized isoforms of FLVCR. The long form, FLVCR1a, is a broadly expressed cell surface heme exporter (Cell 118: 757, 2004) which exports heme from the cytoplasm to an extracellular carrier protein (JBC 285: 28874, 2010). The short isoform, FLVCR1b, is transcribed off an internal promoter and localizes to the mitochondria where it exports heme out of mitochondria into the cytoplasm (JCI 112: 4569, 2012). Adult mice with an induced deletion of both isoforms of FLVCR develop severe macrocytic anemia (Science 319: 825, 2008), indicating that FLVCR is essential for erythropoiesis. Mice constitutively lacking both isoforms of FLVCR die midgestation with craniofacial and digit abnormalities and lack definitive erythropoiesis while mice lacking only FLVCR1a die midgestation with craniofacial and digit abnormalities yet appear to have intact erythropoiesis (Science 319: 825, 2008, JCI 112: 4569, 2012). These results suggest that FLVCR1b, and not FLVCR1a, is required for erythropoiesis.To definitively test whether FLVCR1a or FLVCR1b is sufficient for red cell development, we transplanted mice with marrow lacking both isoforms of FLVCR that had been transduced with either FLVCR1a or FLVCR1b along with a GFP marker. Both cohorts of mice had comparable marking frequency in granulocytes (31.1±16.4%, N=15, FLVCR1a vs 32.7±16.9%, N=8, FLVCR1b) at 4 weeks post-transplant. By 7 wks post-transplant, 71.3±19.7% of the RBC in mice that received FLVCR1a were derived from transduced cells, while only 1.0±0.8% of the RBC in mice that received FLVCR1b were derived from transduced cells. Mice that received FLVCR1a are healthy and have normal CBC parameters (WBC 8.34±4.7 k/ml, RBC 7.95±1.2 M/ml, HGB 12.5±1.7 g/dl, MCV 47.9±2.0 fl , PLT 879±387 k/ml) which persist 9 months later, while mice that received FLVCR1b are severely anemic (WBC 1.9±1.3 k/ml, RBC 1.5±0.6 M/ml, HGB 2.0±0.8 g/dl, MCV 36.5±0.8 fl , PLT 1442±1232 k/ml) and die by 8 weeks post-transplant. This demonstrates that only the FLVCR1a isoform is capable of reconstituting erythropoiesis in adult mice lacking both isoforms in hematopoietic cells. One possible way to reconcile these data with the reported role of FLVCR1b, would be if FLVCR1b were needed during fetal, but not adult, erythropoiesis.As mentioned above, adult mice with an induced deletion of FLVCR develop severe macrocytic anemia (HBG 6.5±2.2 g/dl, MCV 67.9±7.3 fL; vs controls 14.6±0.7, 44.7±3.6), in contrast, over expression of FLVCR1a results in mild hypochromic microcytic anemia (HBG 13.2±1.4 g/dl, MCV 41±5.2 fL; vs controls 15.4±0.5, 49.3±1.3). Because hypochromasia and microcytosis only result from heme or hemoglobin deficiency, FLVCR1a must export heme from differentiating erythroblasts in vivo. To confirm this, we sorted developing erythroblasts from FLVCR-deleted and control mice and measured heme content at each stage. Terminally differentiating erythroid precursors (populations I-IV, PNAS 106: 17413, 2009) from FLVCR-deficient mice have significantly more heme than those from control mice (I&II 359.6±99.9 pg/cell, III 702.2±302.2 pg/cell, IV 657.8±292.9 pg/cell, versus controls 131.4±65.2, 153.9±27.1, and 269.1±102.7 respectively, all p<0.05) and have significantly more apoptosis. To definitively demonstrate that heme toxicity causes proerythroblast apoptosis and macrocytic anemia, we are using existing mouse model systems to modulate impact heme synthesis or degradation and test whether they alter the effect of FLVCR deletion on erythropoiesis. The presence of the ferrochelatase mutation, Fechm1Pas/m1Pas, does not rescue FLVCR deficiency, most likely because the accumulation of toxic precursor products which are also substrates of FLVCR (JBC 285: 28874, 2010). We are currently evaluating whether over-expression of HO-1 or restricted expression of the transferrin receptor can mitigate the effect of FLVCR deletion. Disclosures:No relevant conflicts of interest to declare.

FLVCR is necessary for erythroid maturation, may contribute to platelet maturation, but is dispensable for normal hematopoietic stem cell function

AbstractHeme is a pleiotropic molecule that is important for oxygen and oxidative metabolism, most notably as the prosthetic group of hemoglobin and cytochromes. Because excess free intracellular heme is toxic, organisms have developed mechanisms to tightly regulate its concentration. One mechanism is through active heme export by the group C feline leukemia virus receptor (FLVCR). Previously, we have shown that FLVCR is necessary for embryonic and postnatal erythropoiesis. However, FLVCR is also expressed in numerous other tissues, including hematopoietic stem cells (HSCs). To explore a possible role for FLVCR in HSC function, we performed serial, competitive repopulation transplant experiments using FLVCR-deleted and control bone marrow cells, along with wild-type competitor cells. Loss of FLVCR did not impact HSC function under steady-state or myelotoxic stress conditions (such as arsenic or radiation exposure), nor did FLVCR deletion result in alterations in the various progenitor compartments. However, even when 95% of the donor bone marrow cells lacked FLVCR, all red cells in recipient mice were wild type. This is due to the increased apoptosis of FLVCR-deleted proerythroblasts. Also, remarkably, loss of FLVCR increased megakaryocyte ploidy. Together, these findings show FLVCR is redundant in stem cells but has critical and contrasting stage-specific roles in discrete hematopoietic lineages.

ATP-Binding Cassette B10 Regulates Early Steps of Heme Synthesis

Heme plays a critical role in gas exchange, mitochondrial energy production, and antioxidant defense in cardiovascular system. The mitochondrial transporter ATP-binding cassette (ABC) B10 has been suggested to export heme out of the mitochondria and is required for normal hemoglobinization of erythropoietic cells and protection against ischemia-reperfusion injury in the heart; however, its primary function has not been established. The aim of this study was to identify the function of ABCB10 in heme synthesis in cardiac cells. Knockdown of ABCB10 in cardiac myoblasts significantly reduced heme levels and the activities of heme-containing proteins, whereas supplementation with δ-aminolevulinic acid reversed these defects. Overexpression of mitochondrial δ-aminolevulinic acid synthase 2, the rate-limiting enzyme upstream of δ-aminolevulinic acid export, failed to restore heme levels in cells with ABCB10 downregulation. ABCB10 and heme levels were increased by hypoxia, and reversal of ABCB10 upregulation caused oxidative stress and cell death. Furthermore, ABCB10 knockdown in neonatal rat cardiomyocytes resulted in a significant delay of calcium removal from the cytoplasm, suggesting a relaxation defect. Finally, ABCB10 expression and heme levels were altered in failing human hearts and mice with ischemic cardiomyopathy. ABCB10 plays a critical role in heme synthesis pathway by facilitating δ-aminolevulinic acid production or export from the mitochondria. In contrast to previous reports, we show that ABCB10 is not a heme exporter and instead is required for the early mitochondrial steps of heme biosynthesis.

Placental expression of the heme scavenger receptor, LDL receptor‐related protein 1, is associated with expression of placental heme exporter, feline leukemia virus C receptor 1

LDL receptor‐related protein 1 (LRP 1) is the surface receptor for the plasma heme‐hemopexin complex and is involved in heme clearance following intravascular red cell destruction. Both LRP1 and the heme exporter feline leukemia virus C receptor 1 (FLVCR1) are highly expressed in the human placenta and may play a role in partitioning heme iron between the maternal and fetal compartments. To explore this possibility, protein expression of LRP1 (n=122) and FLVCR1 (n=62) were assessed in pregnant adolescents (13–18 y) in relation to plasma concentrations of the heme scavenger, hemopexin, and iron status indicators (hemoglobin, serum ferritin, transferrin receptor, and hepcidin) at mid‐gestation (~26 wks) and in maternal/cord blood at delivery. Placental LRP1 expression was positively associated with placental FLVCR1 (r=0.41, n=62, p&lt;0.01), maternal serum transferrin receptor at delivery (r=0.20, n=117, p=0.03), and cord hepcidin (r=0.22, n=113, p=0.02). Greater decreases in hemopexin across pregnancy were associated with higher placental LRP1 (r=0.33, n=55, p=0.01). Placental LRP1 is associated with placental FLVCR1 and a maternal index of intravascular hemolysis. Further studies are needed to elucidate functional roles of LRP1 in placental iron transport.Supported by: USDA 2008–0857Grant Funding Source: USDA

The mitochondrial heme exporter FLVCR1b mediates erythroid differentiation

Feline leukemia virus subgroup C receptor 1 (FLVCR1) is a cell membrane heme exporter that maintains the balance between heme levels and globin synthesis in erythroid precursors. It was previously shown that Flvcr1-null mice died in utero due to a failure of erythropoiesis. Here, we identify Flvcr1b, a mitochondrial Flvcr1 isoform that promotes heme efflux into the cytoplasm. Flvcr1b overexpression promoted heme synthesis and in vitro erythroid differentiation, whereas silencing of Flvcr1b caused mitochondrial heme accumulation and termination of erythroid differentiation. Furthermore, mice lacking the plasma membrane isoform (Flvcr1a) but expressing Flvcr1b had normal erythropoiesis, but exhibited hemorrhages, edema, and skeletal abnormalities. Thus, FLVCR1b regulates erythropoiesis by controlling mitochondrial heme efflux, whereas FLVCR1a expression is required to prevent hemorrhages and edema. The aberrant expression of Flvcr1 isoforms may play a role in the pathogenesis of disorders characterized by an imbalance between heme and globin synthesis.

Mitochondrial Heme Export Through FLVCR1b Controls Erythroid Differentiation.

AbstractAbstract 2090Feline 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.

Heme Excess Impairs the Maturation of Proerythroblasts From Patients with Diamond Blackfan Anemia (DBA) and 5q- Myelodysplastic Syndrome (MDS)

Abstract 514Free heme is toxic so heme and globin synthesis must be tightly coordinated. In early erythroid differentiation, CD71 (transferrin receptor) is upregulated, allowing iron influx and the initiation of heme synthesis (via an IRE in the 5' UTR of ALAS2 RNA). Heme then upregulates globin transcription and translation. Eventually heme and globin production synchronize to ensure efficient and robust hemoglobin assembly. Mice lacking FLVCR, the cytoplasmic heme exporter, have a severe macrocytic anemia and cell apoptosis and maturation arrest at the proerythroblast stage. This led us to hypothesize that early erythroid precursors must export heme to prevent heme toxicities (Science 319:825, 2008).To test this hypothesis using primary human marrow cells, we adapted the culture system of Giarratana, Blood 118:5071, 2011. At 10 days, >95% of cells are committed to erythropoiesis (CD71pos, linneg) and are at both early (predominantly) and later stages of differentiation, allowing sorting of maturing subsets (by CD36 and GlyA) for analyses of gene expression and heme content. We anticipated that: 1) Heme synthesis would intensify before globin synthesis; 2) Erythroid cells might lack heme oxygenases (HMOX1, HMOX2) which degrade heme and thus be especially vulnerable to heme toxicity; and 3) FLVCR would be high in early cells then decrease as cells mature. As expected, ALAS2 expression initiates in stage I (CD36negGlyAneg; 30±6×103 copies/5ngRNA, n=5) and there is 4.0±0.4 ng heme/104 cells. Both ALAS2 RNA and total heme content increase in stage II (CD36posGlyAneg) and are high and stable in stages III (CD36posGlyApos) and IV (CD36negGlyAneg)(65–73×104 and 16.4–16.7). In contrast, alpha and beta globin RNA are first upregulated in stage II (17 and 13×105 copies/5ngRNA) then increase 10–16 fold more in stages III-IV. Also as expected, HMOX1 RNA is barely detectable in stages I-III and present at only 30±4×102 copies/5ng RNA in stage IV; and HMOX2 RNA is low (22–30×102 copies/5ng RNA) in stages I and II, then decreases further, indicating minimal heme oxygenase activity occurs during erythroid differentiation. And as predicted, FLVCR RNA is high in stage I, highest in stage II, and declines in stages III and IV.We next explored abnormal erythropoiesis, including times when heme and globin synthesis may be discordant. DBA is a congenital macrocytic anemia. RPS19 is mutated in 25% of cases causing failed ribosome assembly; an additional ∼30% result from defects in 13 other ribosomal proteins. We hypothesized that in DBA, globin translation would initiate slowly, leading to an increased vulnerability of proerythroblasts to heme toxicity, as heme export via FLVCR may be insufficient to compensate for the added free heme load. Data from DBA patients support this. After 10 days of marrow culture, total cell numbers increase only 6–11 fold (n=3) compared to 21–28 fold for concurrent control studies (p<0.01). Heme synthesis initiates normally in DBA, as ALAS2 expression in stage I (20±1×103 copies/5ng RNA) is normal. However, the total heme content of stage I cells (a stage where globin is not yet available) is significantly higher in DBA patients (6.4±0.2 ng/104cells) than in controls (p<0.05) and by flow cytometry, differentiation appears blocked between stages I and II. Those relatively few DBA cells that reach stage II expand in number in stages III-IV, and interestingly this subset of cells that can mature has significantly less ALAS2 RNA (26–32×104 copies/5ng RNA stages III- IV, p<0.05) and more FLVCR RNA (p<0.10), thereby assuring a total heme content that is normal. There is no difference in HMOX1 or 2. An erythroid differentiation pattern similar to DBA was also observed in studies of two patients with 5q- MDS (acquired haploinsufficiency of RPS14), suggesting a shared pathophysiology of erythroid marrow failure. In both, escaping cells had significantly lower ALAS2 and ∼10-fold higher FLVCR RNA (p values <0.01). MDS patients with refractory anemia with ringed sideroblasts (RARS, n=1) and refractory cytopenia with multilineage dysplasia (n=1) were studied as well and did not show a DBA-like pattern; indeed, as anticipated, the heme content of cells from the RARS patient was significantly lower than in normal persons, p values<0.01. Together our results show that proerythroblasts are especially vulnerable to heme toxicity and argue that the macrocytic anemia of DBA, and perhaps 5q- MDS, results from excess free heme. Disclosures:No relevant conflicts of interest to declare.

FLVCR, a Heme Exporter, Is Required for Peripheral T Cell Survival.

AbstractAbstract 2162Heme is essential for every mammalian cell, however, free heme can induce free radical formation and cellular damage, therefore cells must carefully regulate heme levels. The feline leukemia virus subgroup C receptor (FLVCR) exports heme from cells. Conditional deletion of Flvcr was shown to cause progressive anemia in neonatal and adult mice (Science 319:825–8, 2008). Using a transplant model, we previously demonstrated that Flvcr-deleted thymocytes were blocked at the CD4+CD8+ double-positive (DP) stage (Blood [ASH Annual Meeting Abstracts] 114: 913, 2009). To characterize the temporal requirement for FLVCR in developing thymocytes, we crossed Flvcrflox/flox mice to thymocyte-specific cre recombinase strains: Lck-cre mice, which express cre in early CD4+CD8+ double-negative (DN) thymocytes, and CD4-cre mice, which turn on cre in late DN/early DP thymocytes. Flvcrflox/flox;Lck-cre mice had similar numbers of DN and DP thymocytes compared to controls, however, CD4+ and CD8+ single-positive (SP) thymocytes and peripheral T cells were nearly absent, similar to what we observed in our previous transplant model. In contrast, Flvcrflox/flox;CD4-cre mice had intact thymic development with normal numbers of SP, but there were few CD4+ and CD8+ T cells in the periphery. When we analyzed deletion efficiency of these T cells, CD8+ T cells showed only 50% Flvcr deletion and were nearly all CD44-high, implying that only incompletely-deleted CD8+ T cells survived and expanded. Taken together, these results show that FLVCR is required not only for T cell development beyond the DP stage, but also for the survival of mature T cells in the periphery.We next adoptively transferred thymocytes from Flvcrflox/flox;CD4-cre mice or controls into sub-lethally irradiated Rag1−/− mice. Normal SP thymocytes undergo homeostatic proliferation when transferred into an “empty” host. At day 12 and 20 post-adoptive transfer, few Flvcrflox/flox;CD4-cre CD4+ or CD8+ T cells were found, in contrast to mice that had received Flvcr+/flox;CD4-cre thymocytes. To determine whether Flvcr-deleted T cells failed to undergo homeostatic proliferation, we used carboxyfluorescein succinimidyl ester (CFSE) to label Flvcrflox/flox;CD4-cre or control thymocytes prior to adoptive transfer. At day 8, similar numbers of Flvcrflox/flox;CD4-cre and control T cell were found in the periphery and both had diluted CFSE equally, thus initial proliferation was not affected. However, by day 20, few Flvcr-deleted T cells were present compared to controls. Experiments are currently underway to understand how and why Flvcr-deleted T cells fail to persist long-term.The finding that FLVCR is required for T cell development and peripheral survival is intriguing because there is no known specific role for heme in T cell development or function. We carried out transcriptional profiling on sorted DP thymocytes from Rag1−/− mice transplanted with Flvcr-deleted or control bone marrow to determine whether FLVCR loss led to gene expression changes that might explain the block in T cell development. Surprisingly, there were few transcriptional changes, suggesting that FLVCR loss has an abrupt impact on T cell development late in the DP stage. This finding, together with the apparent normal development of Flvcr-deleted B lymphocytes and myeloid lineages, leads us to hypothesize that FLVCR plays a specific role in T cell development starting at the DP stage and persisting throughout T cell life. FLVCR is a member of the major facilitator superfamily of secondary active transporters. While FLVCR has been shown to export heme, it is not known whether it can import or export other small molecules or metabolites. We are now using a bioinformatics approach on published datasets to analyze metabolic gene expression during normal thymic development and in various mature T cell subsets to identify metabolic pathways that are specific for the DP-SP transition in thymocytes as well as in mature, peripheral T cells. We will then test whether these pathways are altered in Flvcr-deleted thymocytes and mature T cells. These studies may uncover a new role for heme in T cell metabolism, function, and survival, or a new non-heme role for FLVCR. Disclosures:No relevant conflicts of interest to declare.