PCBP1 and NCOA4 regulate erythroid iron storage and heme biosynthesis
Moon-Suhn Ryu, … , Minoo Shakoury-Elizeh, Caroline C. Philpott
Moon-Suhn Ryu, … , Minoo Shakoury-Elizeh, Caroline C. Philpott
Published May 1, 2017; First published April 4, 2017
Citation Information: J Clin Invest. 2017;127(5):1786-1797. https://doi.org/10.1172/JCI90519.
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Research Article Hematology

PCBP1 and NCOA4 regulate erythroid iron storage and heme biosynthesis

Abstract

Developing erythrocytes take up exceptionally large amounts of iron, which must be transferred to mitochondria for incorporation into heme. This massive iron flux must be precisely controlled to permit the coordinated synthesis of heme and hemoglobin while avoiding the toxic effects of chemically reactive iron. In cultured animal cells, iron chaperones poly rC–binding protein 1 (PCBP1) and PCBP2 deliver iron to ferritin, the sole cytosolic iron storage protein, and nuclear receptor coactivator 4 (NCOA4) mediates the autophagic turnover of ferritin. The roles of PCBP, ferritin, and NCOA4 in erythroid development remain unclear. Here, we show that PCBP1, NCOA4, and ferritin are critical for murine red cell development. Using a cultured cell model of erythroid differentiation, depletion of PCBP1 or NCOA4 impaired iron trafficking through ferritin, which resulted in reduced heme synthesis, reduced hemoglobin formation, and perturbation of erythroid regulatory systems. Mice lacking Pcbp1 exhibited microcytic anemia and activation of compensatory erythropoiesis via the regulators erythropoietin and erythroferrone. Ex vivo differentiation of erythroid precursors from Pcbp1-deficient mice confirmed defects in ferritin iron flux and heme synthesis. These studies demonstrate the importance of ferritin for the vectorial transfer of imported iron to mitochondria in developing red cells and of PCBP1 and NCOA4 in mediating iron flux through ferritin.

Authors

Moon-Suhn Ryu, Deliang Zhang, Olga Protchenko, Minoo Shakoury-Elizeh, Caroline C. Philpott

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Figure 1

PCBP1 and NCOA4 activities are differentially regulated during rbc maturation in G1E-ER4 cells.

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PCBP1 and NCOA4 activities are differentially regulated during rbc matur...
Cells were treated with β-estradiol (β-Est), EPO, and 55Fe2-Tf for 48 hours. (A) Accumulation of 55Fe in ferritin precedes incorporation into Hb in developing G1E-ER4 cells. Cells harvested at the indicated times were imaged as cell pellets (top) and analyzed by native gel electrophoresis and phosphorimaging. (B) Quantitation of 55Fe accumulation in cells, heme extracts, ferritin, and Hb. Iron levels at 8 hours and 24 hours of development are expressed as percentage of final levels (48 hours). (C) Temporal protein expression patterns of PCBPs and NCOA4 during erythroid maturation. Cells treated as in A were analyzed by immunoblotting. X indicates nonspecific protein. (D) Developmental regulation of physical interactions between PCBP1 or NCOA4 and ferritin in differentiating erythroid progenitors. Cells treated as in A were analyzed by quantitative IP of ferritin and immunoblotting. PCBP2 was not detectable in any ferritin precipitates. Ferritin-bound PCBP1 and NCOA4 were quantified and normalized to α-tubulin in post-IP supernatant. Bulk rabbit IgG included as negative control for nonspecific interactions. n = 3 independent experiments. *P < 0.05; **P < 0.01, repeated-measures ANOVA with Bonferroni’s post-test.