Iron and Isocitrate Calibrate Erythropoietin Responsiveness of Erythroid Progenitors Via Aconitase Tuning of Protein Kinase C Activity.
Authors: Bullock Grant C. , Delehanty Lorrie L , Talbot Anne-Laure , Gonias Sara , Tong Wing-Hang , Rouault Tracey , Chruma Jason , Goldfarb Adam 
Published: October 2019

Abstract
Abstract 627
Our studies have focused on signaling alterations associated with the erythroid iron deprivation response, based on the hypothesis that a discrete pathway may calibrate erythropoietin (Epo) responsiveness according to iron availability. Previously we identified aconitase enzymatic activity as a critical target in the erythroid iron restriction response and provided evidence for a non-metabolic role for its product isocitrate in coupling iron and erythropoietin signaling. The striking ability of isocitrate to reverse the erythroid iron restriction response in vitro and in vivo raised the possibility for a novel therapeutic approach to Epo-resistant anemias. New data, presented below, further support a non-metabolic mechanism of action for isocitrate rescue of erythropoiesis and identify a specific signal transduction pathway mediating cross-talk among iron, aconitase/isocitrate, and erythropoietin signaling. Two approaches addressed the significance of isocitrate metabolism by isocitrate dehydrogenase (IDH) to yield NADH, NADPH, and αa-ketoglutarate. Firstly, direct measurement of cellular NADH, NADPH, and NADP showed no significant effects due to erythroid iron deprivation or isocitrate. Secondly, we synthesized a metabolically inactive enantiomer of isocitrate, L-(+)-isocitrate, which rescued erythroid viability under iron restriction with equivalent efficiency to the metabolically active D-(−)-isocitrate. L-(+)-isocitrate, also significantly rescued erythroid differentiation under iron restriction albeit somewhat less efficiently than D-(−)-isocitrate. Interestingly, high levels of L-(+)-isocitrate occur naturally in blackberries, which have been described as a folk remedy for anemia. Further evidence for non-metabolic signaling by isocitrate arose from identification of a specific signal transduction pathway dually regulated by iron and erythropoietin. In particular, erythroid deprivation of either iron or Epo induced hyperactivation of protein kinase Cαa (PKCαa), the relevance of which was supported by multiple findings. Firstly, the hyperactivation of PKCαa during erythroid iron deprivation preceded the onset of growth and differentiation defects. Secondly, PKC inhibitors significantly reversed both viability and differentiation defects associated with iron restriction. Thirdly, isocitrate reversed PKCαa hyperactivation in cells cultured with high Epo but failed to do so in cells cultured with low Epo, correlating with the previously described inability of isocitrate to reverse the iron restriction response in low Epo. Fourthly, we and others have demonstrated direct physical interaction between mitochondrial aconitase and active forms of PKCαa/β. To assess potential relevance of iron-sulfur clusters within aconitase, IRE binding assays were conducted on progenitors −/+ iron restriction −/+ isocitrate. The patterns of IRP1 and IRP2 activation suggested that aconitase iron-sulfur clusters in the erythroid lineage are uniquely sensitive to iron deprivation and that exogenous isocitrate may act to stabilize erythroid iron-sulfur clusters in the face of iron deprivation or stress. Taken together, our results suggest a role for aconitase and isocitrate in sensing the degree of erythroid iron restriction and transducing that signal to the Epo signaling pathway. A key target appears to be PKCαa. One possible mechanism involves aconitase acting as a highly dynamic scaffold that bridges iron metabolism and Epo signaling in early erythroid development. Isocitrate may act to stabilize the active site and prevent critical conformational changes during iron restriction. The resultant conformational stability may enable tonic suppression of PKC hyperactivation and thereby permit erythropoiesis in the face of iron deprivation. Novel compounds that stabilize the activity of aconitases may offer novel therapeutic avenues for Epo-refractory anemias.

Disclosures:
No relevant conflicts of interest to declare.