Heme is required by all aerobic cells, yet toxic, so its intracellular concentration must be tightly regulated. We previously determined that the Feline Leukemia Virus, subgroup C, cell surface Receptor (FLVCR), is a heme export protein (
), and characterized the erythropoietic phenotype of inducible FLVCR null mice (Flvcrflox/flox;Mx-cre, ASH abstract 2006). Deleted mice develop a severe hyperchromic macrocytic anemia (HGB 3.8±0.2 g/dl, MCV 65.5±2.1 fL, MCH 22.7±0.6 pg vs. HGB 15.4±0.6, MCV 50.8±1.5, MCH 15.9±0.5). Morphologic, flow cytometric, and erythroid colony analyses of spleen and bone marrow cells showed a block in erythroid differentiation at the CFU-E/proerythroblast stage, a phenotype identical to that observed in cats with pure red cell aplasia (PRCA) due to retroviral inhibition of FLVCR. Mice transplanted with Flvcrflox/flox;Mx-cre marrow, and then treated with p(I)p(C) to delete Flvcr in hematopoietic cells, also developed PRCA. These studies show that FLVCR is required by CFU-E/proerythroblasts, likely to export excess heme and to ensure cell survival, and show that PRCA results from a lack of FLVCR in hematopoietic, and not microenvironmental cells. By western blot, FLVCR is highly expressed in tissues with high heme flux, like the placenta, uterus, duodenum, liver, and cultured macrophages, which suggests that it also has a role in heme-iron trafficking or in the prevention of heme toxicity in nonerythroid cells. To define its macrophage function, we exposed marrow-derived macrophages from deleted and control mice to ferric ammonium citrate (FAC) or IgG-coated red blood cells ± hepcidin and measured ferritin accumulation. Deleted and control macrophages exposed to FAC showed equivalent increases in ferritin which as predicted, increased further with hepcidin treatment. However, after exposure to opsonized red blood cells, deleted macrophages accumulated significantly more ferritin than controls (122.3±0.8 ng/mh protein vs. 67.3±1.3), which increased further with hepcidin treatment. Therefore, not all heme in macrophages is degraded to iron, but rather some traverses the cell intact via FLVCR. We next evaluated the role of FLVCR in iron homeostasis by examining all tissues in deleted mice. Within 5 weeks, deleted mice developed pronounced iron loading in hepatocytes and subsequently within duodenal enterocytes and splenic macrophages. By 7 months, there was swelling of hepatocytes lining bile canaliculi and bile stasis. In contrast, mice in which Flvcr was deleted only in hematopoietic cells showed no iron overload after 5–6 weeks. Liver hepcidin expression by quantitative RT-PCR was significantly increased in both deleted mice and in mice lacking FLVCR only in their hematopoietic cells. These data demonstrate that hepcidin expression alone does not account for the iron overload, and suggest that FLVCR exports heme from liver into bile, thus providing a mechanism for iron to exit the body. Since high hepcidin levels are in contrast to other iron loading anemias with ineffective erythropoiesis, the “erythropoietic regulator” of liver hepcidin expression must require cells more differentiated than proerythroblasts. Together, our work establishes that FLVCR is required for terminal red blood cell development and argues that systemic iron balance involves heme-iron trafficking via FLVCR, in addition to the well-described elemental iron pathways.
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