PCBP1 and NCOA4 regulate erythroid iron storage and heme biosynthesis
Moon-Suhn Ryu, Deliang Zhang, Olga Protchenko, Minoo Shakoury-Elizeh, Caroline C. Philpott
Moon-Suhn Ryu, Deliang Zhang, Olga Protchenko, Minoo Shakoury-Elizeh, Caroline C. Philpott
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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 2

Depletion of PCBP1 or NCOA4 impairs iron flux through ferritin and heme biosynthesis in maturing erythroid progenitors.

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Depletion of PCBP1 or NCOA4 impairs iron flux through ferritin and heme ...
G1E-ER4 cells were subjected to 2 sequential treatments with a nontargeting control siRNA or with siRNA against PCBP1 or NCOA4, then treated with β-estradiol and 55Fe2-Tf for 24 hours. (A) Reduced hemoglobinization of PCBP1- and NCOA4-deficient cells. Representative cell pellets after treatment are shown. Con, control. (B) Altered levels of 55Fe in ferritin and Hb in PCBP1- and NCOA4-depleted cells. Lysates from cells treated as in A were analyzed by native gel electrophoresis and phosphorimaging. Quantitation of 55Fe in ferritin and Hb is shown at right (n = 3). (C) Reduced heme synthesis in PCBP1- and NCOA4-depleted cells. Levels of 55Fe in heme extracts and whole cells were determined by scintillation counting (n = 3). (D) Effects of PCBP1 and NCOA4 depletion on indicators of cellular iron balance. Cells treated as in A were analyzed by immunoblotting for ferritin and IRP2. X indicates nonspecific protein. IRP2 quantitation shown at right (n = 6). (E) Effects of PCBP1 and NCOA4 depletion on indicators of heme levels. Cells were analyzed by immunoblotting for BACH1 and β-globin; quantitation at right (n = 6). (F) Repression of Hba-a1/2 (α-globin) transcripts in PCBP1- and NCOA4-depleted cells. Cells treated as in A were analyzed by real-time PCR (RT-PCR) (n = 5). Protein and transcript abundance were normalized to β-actin and are shown as relative to control siRNA-treated cells. *P < 0.05; **P < 0.01; ***P < 0.001, repeated-measures ANOVA with Dunnett’s test for multiple comparisons with control.