Heme is critical to all aerobic cells both as the prosthetic group for a diverse number of hemoproteins and as a regulatory molecule in cellular processes. In addition, in erythroid cells, heme acts not only as structural component of hemoglobin but also initiates globin transcription and translation. However, because heme is highly reactive and directly toxic to cells, its intracellular level must be tightly controlled, particularly in cells with high heme requirements, like erythroid precursors. We previously determined that Feline Leukemia Virus, subgroup C, Receptor (FLVCR), is a heme export protein required for erythroid differentiation (Cell 885,757–66;2004 & Science 1206,825–8;2008). Conditional deletion of Flvcr in mice results in a severe hypoproliferative, macrocytic anemia with a block in proerythroblast maturation. These findings suggest that erythroid failure in mice lacking FLVCR is mediated by a transient excess of free heme at or around the proerythroblast stage, when heme synthesis exceeds its utilization and/or catabolism. However, we lacked proof to establish this cause and effect relationship. To test our hypothesis, we first measured intracellular free heme as sensed by the globin LCR. We adapted a luciferase assay system based on the transcriptional control of ß-globin expression by heme (J Biol Chem 279,5480–5487;2004). Specifically, we generated a lentiviral vector that contains a 838-bp fragment of the human ß-globin gene promoter and a 3-kb fragment of the LCR (which includes a heme-responsive MARE site) upstream of the firefly luciferase gene. We validated the assay's heme-specificity in a murine fetal liver erythroid culture and then transduced human CD34+ stem/progenitor cells and followed the expression of luciferase when erythroid differentiation was induced. Free heme was detected prior to Glycophorin A expression. We also determined (by q RT-PCR) that heme oygenase-1 (HMOX1), an inducible enzyme which catabolizes heme, is expressed in primary murine marrow cells and its expression decreases during terminal erythroid differentiation, potentially when ß-globin is available to bind heme. As Hmox1 expression is transcriptionally-upregulated by heme, these data suggest that heme excess occurs early in erythroid differentiation. To definitively determine whether excess heme causes the erythroid marrow failure in mice lacking FLVCR, we used a genetic approach to reduce heme synthesis. We bred Flvcrfloxflox;Mx-cre mice to mice with a recessive, severe loss of function mutation in the ferrochelatase gene, the final enzyme in the heme synthetic pathway. Fechm1Pas/m1Pas mice have a mild microcytic anemia compared to wild-type controls (Blood 109,811–818;2007, preliminary data: hemoglobin 13.4±0.6 vs. 16.2±0.8 g/dL, MCV 41.1± 0.9 vs. 46.5±1.5 fL, n=2 in each group, mean±SEM). Notably, in our first cohort of mice, the adult Flvcr-deleted mouse expressing mutant ferrochelatase had a near normal hemoglobin. This contrasted the severe anemia of the control Flvcr-deleted mice. Specifically, seven weeks after poly(I)poly(C) deletion of the floxed Flvcr allele, the hemoglobin in the Fechm1Pas/m1Pas;Flvcrfloxflox;Mx-cre mouse was 10.1 g/dL, while the hemoglobins in the Fechwildtype/wildtype;Flvcrfloxflox;Mx-cre and Fechwildtype/ m1Pas;Flvcrfloxflox;Mx-cre mice were 4.4 and 4.5 g/dL, respectively. Terminal erythroid differentiation was also corrected as assessed by flow cytometry (immunostained for CD44, Ter119, and CD71). Additional cohort studies are underway. As a second approach to reduce intracellular free heme in cells lacking FLVCR, we transduced Flvcrflox/flox;Mxcre bone marrow with a retroviral vector expressing Hmox-1, and transplanted the cells into irradiated recipients. After hematopoietic recovery, the mice were treated with poly(I)poly(C) to delete Flvcr specifically in engrafted cells. These studies are ongoing. Together, our preliminary data show that FLVCR protects proerythroblasts from excess intracellular heme and heme toxicity. This pathophysiology may be relevant to other models of ineffective or failed erythropoiesis where heme synthesis and globin expression are discordant.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.