A New FLVCR Isoform Able to Support Erythropoiesis.

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.