Iron control of erythroid development by a novel aconitase-associated regulatory pathway

Abstract

Human red cell differentiation requires the action of erythropoietin on committed progenitor cells. In iron deficiency, committed erythroid progenitors lose responsiveness to erythropoietin, resulting in hypoplastic anemia. To address the basis for iron regulation of erythropoiesis, we established primary hematopoietic cultures with transferrin saturation levels that restricted erythropoiesis but permitted granulopoiesis and megakaryopoiesis. Experiments in this system identified as a critical regulatory element the aconitases, multifunctional iron-sulfur cluster proteins that metabolize citrate to isocitrate. Iron restriction suppressed mitochondrial and cytosolic aconitase activity in erythroid but not granulocytic or megakaryocytic progenitors. An active site aconitase inhibitor, fluorocitrate, blocked erythroid differentiation in a manner similar to iron deprivation. Exogenous isocitrate abrogated the erythroid iron restriction response in vitro and reversed anemia progression in irondeprived mice. The mechanism for aconitase regulation of erythropoiesis most probably involves both production of metabolic intermediates and modulation of erythropoietin signaling. One relevant signaling pathway appeared to involve protein kinase C␣/␤, or possibly protein kinase C␦, whose activities were regulated by iron, isocitrate, and erythropoietin. IntroductionRed cell production results from erythropoietin (Epo)-driven survival, proliferation, and maturation of committed bone marrow progenitor cells. This process critically depends on cellular uptake of adequate bioavailable iron, provided in the form of diferric transferrin. Compromise in iron uptake or intracellular trafficking results in iron-restricted erythropoiesis, characterized by diminished marrow responsiveness to Epo. 1 Epo acts during an early phase of erythroid development, between late (erythroid burst-forming unit) and early pronormoblast stages, before the initiation of hemoglobin synthesis. [1][2][3] Therefore, iron restriction serves as a checkpoint to restrain Epo-driven progenitor expansion in the face of limited iron stores. It has been documented in zebrafish, in which defects in intracellular iron utilization block early erythroid differentiation, 4 and in mammals, in which dietary iron deficiency impairs the transition from erythroid colony-forming unit to pronormoblast. 5 In the clinical setting, iron-restricted erythropoiesis underlies many of the anemias that are refractory to Epo treatment (eg, anemias of chronic renal disease and inflammation). 1 The lineage-and stage-selective nature of the erythroid iron restriction response has suggested involvement of a specialized signaling pathway distinct from the iron depletion response that occurs in a wide variety of cell types treated with chelators. Supporting this notion, chelator-induced iron depletion in MCF-7 cells causes cell-cycle arrest in G 1 phase followed by apoptosis, 6 neither of which is seen in iron-restricted erythropoiesis in vivo. 2,6,7 Thus, to identify mechanisms applicable to ...

Timing and Determinants of Erythropoietin Deficiency in Chronic Kidney Disease

“…EPO reduces circulating hepcidin in humans (27) and directly affects the intestinal handling of iron in rat models (28). Inversely, absolute iron deficiency is associated with increased EPO levels and early blockage of erythroid differentiation (29). Transferrin receptor 2, recently described as a sensor of body iron levels, may mediate EPO change with absolute iron deficiency (30).…”

Mechanistic and Therapeutic Studies Related to Anemia of Chronic Disease and Inflammation

“…Secondly, fluoracetate, which produces fluorocitrate within mitochondria, inhibited erythropoiesis much more efficiently than fluorocitrate, which accumulates primarily in the cytosol [27]. Thirdly, shRNA knockdown of mitochondrial aconitase, but not of cytosolic aconitase caused inhibition of erythroid differentiation [28].…”

“…Isocitrate treatment abrogates the erythroid iron restriction response in cell culture and animal models of iron deprivation [28]. Because the erythroid iron restriction response may also contribute to ACDI [53], we determined the effects of isocitrate administration in a rat arthritis model that faithfully recapitulates human ACDI [43,44].…”

“…Figure 1 PKCα/β hyperactivation [28]. Therefore, understanding the mechanisms underlying the erythroid iron restriction response and developing therapies to target this response are critically important.…”

“…High levels of aconitase activity are specifically required for erythropoiesis such that in vitro enzymatic inhibition blunts cellular responsiveness to Epo, and in vivo inhibition causes anemia [27,28]. Figure 1 PKCα/β hyperactivation [28].…”