Iron control of erythroid microtubule cytoskeleton as a potential target in treatment of iron-restricted anemia.

Anemias of chronic disease and inflammation (ACDI) result from restricted iron delivery to erythroid progenitors. The current studies reveal an organellar response in erythroid iron restriction consisting of disassembly of the microtubule cytoskeleton and associated Golgi disruption. Isocitrate supplementation, known to abrogate the erythroid iron restriction response, induces reassembly of microtubules and Golgi in iron deprived progenitors. Ferritin, based on proteomic profiles, regulation by iron and isocitrate, and putative interaction with microtubules, is assessed as a candidate mediator. Knockdown of ferritin heavy chain (FTH1) in iron replete progenitors induces microtubule collapse and erythropoietic blockade; conversely, enforced ferritin expression rescues erythroid differentiation under conditions of iron restriction. Fumarate, a known ferritin inducer, synergizes with isocitrate in reversing molecular and cellular defects of iron restriction and in oral remediation of murine anemia. These findings identify a cytoskeletal component of erythroid iron restriction and demonstrate potential for its therapeutic targeting in ACDI.

Iron modulation of erythropoiesis is associated with Scribble-mediated control of the erythropoietin receptor.

Iron-restricted human anemias are associated with the acquisition of marrow resistance to the hematopoietic cytokine erythropoietin (Epo). Regulation of Epo responsiveness by iron availability serves as the basis for intravenous iron therapy in anemias of chronic disease. Epo engagement of its receptor normally promotes survival, proliferation, and differentiation of erythroid progenitors. However, Epo resistance caused by iron restriction selectively impairs proliferation and differentiation while preserving viability. Our results reveal that iron restriction limits surface display of Epo receptor in primary progenitors and that mice with enforced surface retention of the receptor fail to develop anemia with iron deprivation. A mechanistic pathway is identified in which erythroid iron restriction down-regulates a receptor control element, Scribble, through the mediation of the iron-sensing transferrin receptor 2. Scribble deficiency reduces surface expression of Epo receptor but selectively retains survival signaling via Akt. This mechanism integrates nutrient sensing with receptor function to permit modulation of progenitor expansion without compromising survival.

A specialized pathway for erythroid iron delivery through lysosomal trafficking of transferrin receptor 2.

Erythroid progenitors are the largest consumers of iron in the human body. In these cells, a high flux of iron must reach the mitochondrial matrix to form sufficient heme to support hemoglobinization. Canonical erythroid iron trafficking occurs via the first transferrin receptor (TfR1)-mediated endocytosis of diferric-transferrin into recycling endosomes, where ferric iron is released, reduced, and exported to the cytosol via DMT1. However, mice lacking TfR1 or DMT1 demonstrate residual erythropoiesis, suggesting additional pathways for iron use. How iron moves from endosomes to mitochondria is incompletely understood, with both cytosolic chaperoning and "kiss and run" interorganelle transfer implicated. TfR2, in contrast to its paralog TfR1, has established roles in iron sensing, but not iron uptake. Recently, mice with marrow-selective TfR2 deficiency were found to exhibit microcytosis, suggesting TfR2 may also contribute to erythroid hemoglobinization. In this study, we identify alternative trafficking, in which TfR2 mediates lysosomal transferrin delivery. Imaging studies reveal an erythroid lineage-specific organelle arrangement consisting of a focal lysosomal cluster surrounded by a nest of mitochondria, with direct contacts between these 2 organelles. Erythroid TfR2 deficiency yields aberrant mitochondrial morphology, implicating TfR2-dependent transferrin trafficking in mitochondrial maintenance. Human TFR2 shares a lineage- and stage-specific expression pattern with MCOLN1, encoding a lysosomal iron channel, and MFN2, encoding a protein mediating organelle contacts. Functional studies reveal these latter factors to be involved in mitochondrial regulation and erythroid differentiation, with Mfn2 required for mitochondrial-lysosomal contacts. These findings identify a new pathway for erythroid iron trafficking involving TfR2-mediated lysosomal delivery followed by interorganelle transfer to mitochondria.

Calyculin A from Discodermia calyx is a dual action toxin that blocks calcium influx and inhibits protein Ser/Thr phosphatases.

Calyculin A (Caly A) is cell permeable toxin widely used in cell biology research as an inhibitor of type 1 and type 2A protein Ser/Thr phosphatases of the PPP family. Here we tested effects of low concentrations of Caly A on proliferation of human cancer and non-cancer cell lines. We found that long-term 0.3 nM Caly A prevented G1 to S phase cell cycle progression in human Hs-68 fibroblasts and ARPE19 epithelial cells, but not human breast cancer MDA-MB-468, MDA-MB-231 and MCF7 cells. These conditions produced no change in cyclin D1 levels or in the phosphorylation of endogenous proteins. However, acute application of 0.3 nM Caly A blocked serum-induced increase in intracellular calcium levels in Hs-68 fibroblasts, but not in MDA-MB-468 breast cancer cells. We propose that subnanomolar Caly A prevents cell cycle progression because it blocks calcium uptake by fibroblasts. This probably involves non-selective cation channels and cancer cell proliferation was not affected because calcium enters these cells by other channels. Our results suggest that calyculin A has dual actions and acts as a channel blocker, in addition to its well-established effects as a phosphatase inhibitor.