A Closer Look at Cellular Iron Metabolism in IRP2 Deficient Erythroblasts.

Developing red blood cells are the major consumers of body iron which is indispensable for the enormous production of heme for hemoglobin synthesis. The uptake of iron occurs via binding of iron-loaded transferrin to its cognate receptor (TfR). Thereafter the iron is shuttled to the mitochondria where it is incorporated into protoporphyrin IX to form heme. Excess iron is enclosed within the iron storage protein ferritin. Coordinated control between iron uptake and storage is mainly achieved by the post-transcriptional regulation of TfR1 and ferritin synthesis by the iron regulatory proteins IRP1 and IRP2. Recently, two groups independently created mice lacking either IRP1 or IRP2 and showed that only IRP2 deficient mice developed microcytic hypochromic anemia. Both groups observed a reduction in TfR1 protein expression levels in the developing red blood cells of IRP2 knockout animals and suggested that the decrease in receptor levels is responsible for the development of anemia.

For a more detailed analysis of how the loss of IRP2 expression influences iron metabolism and hemoglobinization during terminal erythroid differentiation, we isolated CFU-E-like erythroid cells from mouse fetal liver of wild type, IRP1 and IRP2 knock out animals. In vitro cultivation of these primary erythroid cells and their synchronous induction for differentiation allowed us to study their cellular iron metabolism at different time points. We analyzed the extent of hemoglobinization and cell size as well as the expression of ferritin and TfR1 during various stages of erythroid differentiation in IRP1, IRP2 and wild type cells. In agreement with the published phenotype of microcytic hypochromic anemia, only erythroblasts lacking IRP2 exhibited a reduction in hemoglobinization and showed a significant increase in ferritin protein levels before and after induction of differentiation. In contrast, TfR1 protein expression levels on the cell surface were significantly decreased in IRP2 deficient cells until 24h of differentiation, but converged with those of wild type cells at 48h of differentiation at the time point at which hemoglobinization is fully in progress. Moreover, measurement of 59Fe uptake and its cellular distribution showed that there is significantly more 59Fe located in cytosolic ferritin of IRP2 knock out cells at all time points compared to their wild type counterpart. In summary, these results suggest that not only the reduced expression of TfR1, but also the up-regulation of ferritin, play important roles in the development of anemic phenotype in IRP2 knock out mice.

This work was supported by the Canadian Institutes of Health Research and the Canadian Blood Services.