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.

. Quantitation of transferrin receptor expression and localization in erythroid progenitors subjected to iron restriction and isocitrate treatment. histogram plots of TfR1 levels (total, surface, intracellular) and distribution (intracellular/surface ratio, co-localization with lysosome), with mean and median signal intensity. Cells were cultured, stained and analyzed as in Supplementary Fig. 3, with each analysis using data acquired from >19,000 cells. Graphs depict relative mean values from 4 independent experiments ± SEM; *, **P = 0.017, 0.008, one-way ANOVA with Tukey post hoc test. Abbreviations: IC (isocitrate), BF (bright field), rel (relative), MFI (mean fluorescence intensity), Coloc (colocalization), NS (not significant).
Cell fumarate quantitation. Metabolite quantitation occurred as described 3 . ~5 × 10 7 cells washed twice in ice-cold PBS were suspended in ice-cold distilled H2O and then supplemented with an equal volume of cold (−20°C) absolute methanol. After vortexing briefly, samples were frozen in a dry ice bath for 30 minutes and then thawed in a wet ice bath for 10 minutes. After centrifugation (18,000 × g, 10 minutes), the supernatant was collected, and the pellet underwent repeat extraction with 50% v/v cold (−20°C) methanol, followed by pooling of first and second supernatants. These extracts were then evaporated to dryness, and re-dissolved in 150 μl of distilled H2O. After briefly heating to 90°C, samples were cooled on ice, centrifuged (18,000 × g, 10 minutes), and then supernatants were harvested for desiccation by Speed-Vac.
For NMR spectroscopy, the extracts were dissolved in 300 µl D2O with 1.5 mM (trimethylsilyl-2,2,3,3-tetradeuteropropioninc acid (TSP) and placed in 3 mm NMR tubes points, and an exponential line broadening of 0.1 Hz was employed, followed by Fourier transformation. Phase and baseline-corrected spectra were referenced to the TSP peak at 0.00 ppm. Metabolite identification and quantification employed Chenomx NMR Suite (version 5.1; Chenomx Inc., Edmonton, Canada).

Analysis of iron-related parameters in ACDI mice. Mice underwent ACDI induction
and treatment as in Fig. 7b and c. Two days after the last oral gavage, mice were anesthetized with isoflurane and euthanized by cervical dislocation. Immediately after, blood was collected by heart puncture for serum studies and portal veins were perfused with 10 ml Hanks' Balanced Salt Solution (HBBS) containing 0.5 mM EGTA, followed by a 5 ml HBSS flush.
For serum studies, blood samples underwent clotting at room temperature for 30 minutes followed by a microfuge spin at 3000 g at room temperature for 15 minutes.
Serum was collected and stored in aliquots at -20°C. For measurements, samples were thawed and diluted (1:2 for IL-6 and 1:10 for hepcidin) in buffers provided by the kit manufacturers. IL-6 was quantified using the IL-6 Mouse Uncoated ELISA Kit (Invitrogen,