Aconitase: An Iron Sensing Regulator of Mitochondrial Oxidative Metabolism and Erythropoiesis

Anemia is a global health problem that decreases quality of life for billions of people. Previous studies have concluded that there is an iron-regulated checkpoint in erythropoiesis that suppresses red blood cell production. This results in anemia and the conservation of iron for use in other vital processes. We have shown that the aconitase enzymes are key to this pathway and that inhibition of aconitase enzyme activity by iron restriction or pharmacologic inhibitors blocks erythropoiesis in primary human hematopoietic progenitor cells (HPCs). Mitochondrial aconitase (ACO2) functions as an isomerase within the tricarboxylic acid (TCA) cycle to convert citrate into isocitrate, which contributes to ATP and heme synthesis. During iron restriction, there is a significant decrease of intracellular isocitrate with only a slight increase in intracellular citrate. A corresponding increase in ATP-citrate lyase activity suggests that excess citrate is shunted into acetyl-CoA production. The addition of exogenous isocitrate to iron-deprived HPCs abrogates the block in erythropoiesis and protects iron-deprived mice and chronically-inflamed rats from anemia. These results suggest that ACO2 regulates mitochondrial metabolism and erythropoiesis. Recent unpublished data shows that ACO2 inhibition by iron deprivation or by treatment with an ACO2 inhibitor decreases mitochondrial respiratory rates (RR) and increases mitochondrial reactive oxygen species (mito-ROS). Isocitrate normalizes RR and mito-ROS and restores erythropoiesis. Importantly, disruption of ROS generation with a variety of anti-oxidants blocks erythropoiesis, while surprisingly, treatment of iron restricted HPCs with oxidant generators or ROS promotes erythropoiesis. These data inform our overarchinghypothesis that iron-restriction inhibits ACO2, thereby inhibiting mitochondrial metabolism, resulting in the loss of a mitochondrial ROS signal that is required for erythropoiesis. We have recently extended these studies to ACO2 knock down (ACO2-KD) K562 cell lines which provide more material for biochemical assessment of mitochondrial function. New pilot data shows that a 70% decrease in ACO2 expression significantly reduces the induction of erythroid specific genes during hydroxyurea or hemin treatment. This confirms that ACO2 plays a direct role in erythropoiesis. Extracellular flux (XF, Seahorse Bioscience) experiments show a decreased RR in ACO2-KD cells. Mitochondrial complex activity assays show no differences in complex IV or citrate synthase activity between control and ACO2-KD cell lines. These studies also demonstrate that the shRNA-ACO2 lentiviral constructs are effectively targeting ACO2 and can be used in our HPC model system. We now have evidence in two different human cell culture models of erythropoiesis that mitochondrial aconitase is an iron-sensing regulator of both mitochondrial respiration and erythropoiesis. Our long term goals are to identify novel therapeutic targets in this iron dependent metabolic regulatory pathway that enhance or suppress erythropoiesis and have potential clinical application in the treatment of anemia or polycythemia. We are also investigating the role of the mitochondrion in the differentiation of other hematopoietic cell lineages.