Raymond T. Doty

A heme export protein is required for red blood cell differentiation and iron homeostasis

Hemoproteins are critical for the function and integrity of aerobic cells. However, free heme is toxic. Therefore, cells must balance heme synthesis with its use. We previously demonstrated that the feline leukemia virus, subgroup C, receptor (FLVCR) exports cytoplasmic heme. Here, we show that FLVCR-null mice lack definitive erythropoiesis, have craniofacial and limb deformities resembling those of patients with Diamond-Blackfan anemia, and die in midgestation. Mice with FLVCR that is deleted neonatally develop a severe macrocytic anemia with proerythroblast maturation arrest, which suggests that erythroid precursors export excess heme to ensure survival. We further demonstrate that FLVCR mediates heme export from macrophages that ingest senescent red cells and regulates hepatic iron. Thus, the trafficking of heme, and not just elemental iron, facilitates erythropoiesis and systemic iron balance.

Kinetics and Specificity of Feline Leukemia Virus Subgroup C Receptor (FLVCR) Export Function and Its Dependence on Hemopexin

The feline leukemia virus subgroup C receptor (FLVCR) is a heme export protein that is required for proerythroblast survival and facilitates macrophage heme iron recycling. However, its mechanism of heme export and substrate specificity are uncharacterized. Using [55Fe]heme and the fluorescent heme analog zinc mesoporphyrin, we investigated whether export by FLVCR depends on the availability and avidity of extracellular heme-binding proteins. Export was 100-fold more efficient when the medium contained hemopexin (Kd < 1 pm) compared with albumin (Kd = 5 nm) at the same concentration and was not detectable when the medium lacked heme-binding proteins. Besides heme, FLVCR could export other cyclic planar porphyrins, such as protoporphyrin IX and coproporphyrin. However, FLVCR has a narrow substrate range because unconjugated bilirubin, the primary breakdown product of heme, was not transported. As neither protoporphyrin IX nor coproporphyrin export improved with extracellular hemopexin (versus albumin), our observations further suggest that hemopexin, an abundant protein with a serum concentration (6.7–25 μm) equivalent to that of the iron transport protein transferrin (22–31 μm), by accepting heme from FLVCR and targeting it to the liver, might regulate macrophage heme export and heme iron recycling in vivo. Final studies show that hemopexin directly interacts with FLVCR, which also helps explain why FLVCR, in contrast to some major facilitator superfamily members, does not function as a bidirectional gradient-dependent transporter. Together, these data argue that hemopexin has a role in assuring systemic iron balance during homeostasis in addition to its established role as a scavenger during internal bleeding or hemolysis.

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.

Evidence that the expression of transferrin receptor 1 on erythroid marrow cells mediates hepcidin suppression in the liver.

Hepcidin is the key regulator of iron absorption and recycling, and its expression is suppressed by red blood cell production. When erythropoiesis is expanded, hepcidin expression decreases. To gain insight into the stage of erythroid differentiation at which the regulation might originate, we measured serum hepcidin levels in archived pure red cell aplasia samples from patients whose block in erythroid differentiation was well defined by hematopoietic colony assays and marrow morphologic review. Hepcidin values are high or high normal in pure red cell aplasia patients in whom erythropoiesis is inhibited prior to the proerythroblast stage, but are suppressed in patients with excess proerythroblasts and few later erythroid cells. These data suggest that the suppressive effect of erythropoietic activity on hepcidin expression can arise from proerythroblasts, the stage at which transferrin receptor 1 expression peaks, prompting the hypothesis that transferrin receptor 1 expression on erythroid precursors is a proximal mediator of the erythroid regulator of hepcidin expression. Our characterization of erythropoiesis, iron status, and hepcidin expression in mice with global or hematopoietic cell-specific haploinsufficiency of transferrin receptor 1 provides initial supporting data for this model. The regulation appears independent of erythroferrone and growth differentiation factor 15, supporting the concept that several mechanisms signal iron need in response to an expanded erythron.

An all-feline retroviral packaging system for transduction of human cells

Abstract The subgroup C feline leukemia virus (FeLV-C) receptor FLVCR is a widely expressed 12-transmembrane domain transporter that exports cytoplasmic heme and is a promising target for retrovirus-mediated gene delivery. Previous studies demonstrated that FeLV-C pseudotype vectors were more efficient at targeting human hematopoietic stem cells than those pseudotyped with gibbon ape leukemia virus (GALV), and thus we developed an all FeLV-C-based packaging system, termed CatPac. CatPac is helper-virus free and can produce higher titer vectors than existing gammaretroviral packaging systems, including systems mixing Moloney murine leukemia virus (MoMLV) Gag-Pol and FeLV-C Env proteins. The vectors can be readily concentrated (>30-fold), refrozen (three to five times), and held on ice (>2 days) with little loss of titer. Furthermore, we demonstrate that CatPac pseudotype vectors efficiently target early CD34(+)CD38(-) stem/progenitor cells, monocytic and erythroid progenitors, activated T cells, mature macrophages, and cancer cell lines, suggesting utility for human cell and cell line transduction and possibly gene therapy.

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

Heme 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.

Feline leukemia virus integrase and capsid packaging functions do not change the insertion profile of standard Moloney retroviral vectors

Adverse events linked to perturbations of cellular genes by vector insertion reported in gene therapy trials and animal models have prompted attempts to better understand the mechanisms directing viral vector integration. The integration profiles of vectors based on MLV, ASLV, SIV and HIV have all been shown to be non-random, and novel vectors with a safer integration pattern have been sought. Recently, we developed a producer cell line called CatPac that packages standard MoMLV vectors with feline leukemia virus (FeLV) gag, pol and env gene products. We now report the integration profile of this vector, asking if the FeLV integrase and capsid proteins could modify the MoMLV integration profile, potentially resulting in a less genotoxic pattern. We transduced rhesus macaque CD34+ hematopoietic progenitor cells with CatPac or standard MoMLV vectors, and determined their integration profile by LAM-PCR. We obtained 184 and 175 unique integration sites (ISs) respectively for CatPac and standard MoMLV vectors, and these were compared with 10 000 in silico-generated random IS. The integration profile for CatPac vector was similar to MoMLV and equally non-random, with a propensity for integration near transcription start sites and in highly dense gene regions. We found an IS for CatPac vector localized 715 nucleotides upstream of LMO-2, the gene involved in the acute lymphoblastic leukemia developed by X-SCID patients treated by gene therapy using MoMLV vectors. In conclusion, we found that replacement of MoMLV env, gag and pol gene products with FeLV did not alter the basic integration profile. Thus, there appears to be no safety advantage for this packaging system. However, considering the stability and efficacy of CatPac vectors, further development is warranted, using potentially safer vector backbones, for instance those with a SIN configuration.