Abstract
BackgroundErythroid development requires the action of erythropoietin (EPO) on committed progenitors to match red cell output to demand. In this process, iron acts as a critical cofactor, with iron deficiency blunting EPO-responsiveness of erythroid progenitors. Aconitase enzymes have recently been identified as possible signal integration elements that couple erythropoiesis with iron availability. In the current study, a regulatory role for aconitase during erythropoiesis was ascertained using a direct inhibitory strategy.Methodology/Principal FindingsIn C57BL/6 mice, infusion of an aconitase active-site inhibitor caused a hypoplastic anemia and suppressed responsiveness to hemolytic challenge. In a murine model of polycythemia vera, aconitase inhibition rapidly normalized red cell counts, but did not perturb other lineages. In primary erythroid progenitor cultures, aconitase inhibition impaired proliferation and maturation but had no effect on viability or ATP levels. This inhibition correlated with a blockade in EPO signal transmission specifically via ERK, with preservation of JAK2-STAT5 and Akt activation. Correspondingly, a physical interaction between ERK and mitochondrial aconitase was identified and found to be sensitive to aconitase inhibition.Conclusions/SignificanceDirect aconitase inhibition interferes with erythropoiesis in vivo and in vitro, confirming a lineage-selective regulatory role involving its enzymatic activity. This inhibition spares metabolic function but impedes EPO-induced ERK signaling and disturbs a newly identified ERK-aconitase physical interaction. We propose a model in which aconitase functions as a licensing factor in ERK-dependent proliferation and differentiation, thereby providing a regulatory input for iron in EPO-dependent erythropoiesis. Directly targeting aconitase may provide an alternative to phlebotomy in the treatment of polycythemia vera.
Highlights
Production of red blood cells, or erythropoiesis, is regulated by the cytokine erythropoietin (EPO) in conjunction with iron
The mean corpuscular hemoglobin concentration (MCHC) was unaffected by aconitase inhibition (Table 1), in contrast to the hypochromic anemias associated with defective heme biosynthesis
50 mM FA did not alter the mitochondrial membrane potential (MMP), as assessed by staining with the membrane permeant dye MitoTracker CMXRos [37]. In both early (GPA negative) and more mature (GPA positive) populations, the mean fluorescence intensity (MFI) of MitoTracker staining was unaffected by FA treatment (Fig. 4D). These results demonstrate that the degree of aconitase inhibition sufficient to impair erythropoiesis is below the threshold that disrupts cellular ATP levels, redox status, or MMP
Summary
Production of red blood cells, or erythropoiesis, is regulated by the cytokine erythropoietin (EPO) in conjunction with iron. In the context of an adequate iron supply, EPO promotes proliferation, differentiation, and survival of erythroid progenitors, beginning at the colony forming unit-erythroid (CFU-E) stage. I.e. diminished amounts of bio-available iron, results in diminished red cell production due to decreased erythroid proliferation and maturation [1]. Erythroid development requires the action of erythropoietin (EPO) on committed progenitors to match red cell output to demand. In this process, iron acts as a critical cofactor, with iron deficiency blunting EPO-responsiveness of erythroid progenitors. Aconitase enzymes have recently been identified as possible signal integration elements that couple erythropoiesis with iron availability. A regulatory role for aconitase during erythropoiesis was ascertained using a direct inhibitory strategy
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