In Vitro and in Vivo Abrogation of An Erythroid Iron Restriction Developmental Checkpoint by Isocitrate through a Novel Erythropoietin- Coupled Signaling Mechanism

Erythropoietin (Epo) signaling drives normal erythropoiesis by promoting the survival, proliferation and maturation of committed erythroid progenitor cells. Epo acts at an early stage in erythroid development, prior to the initiation of hemoglobin synthesis. Under conditions of iron restriction, erythroid progenitors become refractory to Epo, resulting in hypoplastic anemia. The resulting iron restriction checkpoint protects iron stores from depletion by preventing Epo-driven erythroid expansion and inappropriate iron utilization for hemoglobin synthesis. In addition to diminished body stores, defects in iron uptake or intracellular trafficking can also activate this checkpoint and contribute to Epo-refractory anemias, e.g. sideroblastic anemias. Our previous work using primary human hematopoietic cultures had implicated aconitase enzymes, which interconvert citrate and isocitrate, as critical regulators of the erythroid iron-restriction checkpoint. In those studies, supplying cells with isocitrate had completely abrogated the block in erythroid development caused by iron restriction. In the current studies, we examine the mechanism for isocitrate rescue of erythropoiesis in iron deprived human progenitors and determine the in vivo effects of isocitrate administration in mice with iron deficiency anemia. Initial experiments addressed whether the activity of isocitrate was due to its catabolism to yield ATP and succinyl CoA, a precursor of heme. Several independent findings argued against such a metabolic mechanism. Firstly, erythroid progenitors showed no changes in cellular [ATP] with iron deprivation −/+ isocitrate. Secondly, a bioactive analog of alpha-ketoglutarate, TaKG, failed to rescue erythropoiesis under iron deprivation. Thirdly, isocitrate promoted erythroid differentiation in progenitors with blockade in mitochondrial biogenesis, induced by chloramphenicol. In these last studies, isocitrate reversed chloramphenicol inhibition of glycophorin A and globin expression; exogenous hemin by contrast reversed only the inhibition of globin expression. The combination of isocitrate and hemin, however, showed strong synergy in the rescue of growth and globin expression in cholaramphenicol treated progenitors. Subsequent experiments tested the hypothesis that isocitrate functions as a second messenger in erythroid development. Accordingly, iron deprived erythroid progenitors exposed to a range of Epo levels (0.05–20 U/ml) underwent isocitrate treatment. Remarkably, isocitrate showed no rescue of iron deprived erythroid cultures with 0.05 U/ml Epo, a dose that still promotes erythroid differentiation in high iron cultures. Partial rescue was obtained with 0.2 U/ml Epo, and complete rescue with > 4.5 U/ml. Previous studies have suggested that Epo-mediated calcium signaling shows a strong dosage dependency, requiring relatively high doses of Epo to activate calcium influx. In human CD34+ progenitors exposed to 4.5 U/ml Epo for either 5 hours or 3 days, iron deprivation induced a drop in steady state intracellular calcium levels. Inclusion of isocitrate in the medium restored the intracellular calcium to the levels seen in the iron-replete cultures. Finally, the in vivo activity of isocitrate was assessed in a murine model of iron deficiency anemia. Strikingly, intraperitoneal injections of isocitrate (200 mg/kg/day for 5 days) significantly augmented the red cell counts in C57BL/6 weanlings subjected to a low iron diet: mean RBC of 9.3 ± 0.35 × 10e12 cells/liter for the isocitrate group compared to an average RBC of 7.4 ± 0.51 × 10e12 cells/liter for the saline control group (P=0.012, N = 6 for each group). Interestingly, this increase accompanied a parallel decrease in red cell mean corpuscular hemoglobin concentration. Taken together, our results suggest a role for isocitrate as a second messenger coupled to Epo signaling and potentially involved in intracellular calcium regulation. In vivo administration abrogates the iron restriction checkpoint on red cell production, as in ex vivo studies, but cannot drive hemoglobin synthesis in the face of limited iron stores. The ability of isocitrate to collaborate with hemin in overriding erythroid mitochondrial defects offers novel therapeutic avenues for sideroblastic anemias.