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. 2001 Dec 3;20(23):6909-18.
doi: 10.1093/emboj/20.23.6909.

Heme-regulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency

A P Han  1 , C Yu, L Lu, Y Fujiwara, C Browne, G Chin, M Fleming, P Leboulch, S H Orkin, J J Chen
Affiliations

Heme-regulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency

A P Han et al. EMBO J. .

Abstract

Although the physiological role of tissue-specific translational control of gene expression in mammals has long been suspected on the basis of biochemical studies, direct evidence has been lacking. Here, we report on the targeted disruption of the gene encoding the heme-regulated eIF2alpha kinase (HRI) in mice. We establish that HRI, which is expressed predominantly in erythroid cells, regulates the synthesis of both alpha- and beta-globins in red blood cell (RBC) precursors by inhibiting the general translation initiation factor eIF2. This inhibition occurs when the intracellular concentration of heme declines, thereby preventing the synthesis of globin peptides in excess of heme. In iron-deficient HRI(-/-) mice, globins devoid of heme aggregated within the RBC and its precursors, resulting in a hyperchromic, normocytic anemia with decreased RBC counts, compensatory erythroid hyperplasia and accelerated apoptosis in bone marrow and spleen. Thus, HRI is a physiological regulator of gene expression and cell survival in the erythroid lineage.

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Figures

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Fig. 1. Targeted disruption of the HRI gene. (A) HRI wild-type locus (top), targeting construct (middle) and targeted homologous recombination at the HRI locus before and after Cre-mediated excision of the neomycin resistance gene (bottom). Exons were marked by the filled rectangles and labeled with Roman numerals. Abbreviations used for restriction enzymes: N, NdeI; B, BssHII; E, EcoRV; Bg, BglII. (B) Genotyping of the targeted disrupted mice by PCR. The primers P1 and P2 were used for amplification of the HRI+/+ DNA of 625 bp, whereas primers P1 and P3 were used for the amplification of HRI–/– DNA of ∼1000 bp. (C) HRI mRNA. HRI mRNA was determined by RT–PCR from RNA in E19.5 fetal livers of HRI +/+ and –/– embryos. (D) HRI protein and eIF2α kinase activity. The levels of HRI protein in the lysates of 1 × 106 reticulocytes of HRI +/+, +/– and –/– mice were examined by western blot analysis (top). The eIF2α kinase activity of HRI in reticulocyte lysates was determined using in vitro protein kinase assays followed by western blot analysis using antibody specific to the phosphorylated eIF2α (bottom).
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Fig. 2. Characterization of the HRI–/– reticulocytes. (A) Protein synthesis and eIF2α phosphorylation in intact reticulocytes. Protein synthesis was carried out by labeling HRI +/+ or –/– reticulocytes (2 × 108 reticulocytes/ml) with [35S]methionine for 90 min. Samples (3 × 105 reticulocytes) were taken every 15 min to be analyzed for rate of protein synthesis (top, autoradiogram), eIF2α phosphorylation (middle, western) and total eIF2α (bottom, western). (B) Polysome profiles of HRI +/+ and –/– reticulocytes. Polysomal profiles were obtained with 5 × 107 reticulocytes. Numbers of ribosomes in the polysomes were labeled. (C) Northern blot analysis of β-globin mRNA. Total RNAs from HRI +/+ and –/– reticulocytes (0.5, 1 and 5 µg) were analyzed. (D) Loss of heme-dependent globin synthesis in HRI–/– reticulocytes. HRI+/+ and HRI–/– reticulocytes were incubated with [35S]methionine for 90 min in the presence of hemin (H) or cycloheximide (CHX). C, untreated control reticulocytes. Samples were taken every 30 min to be analyzed for globin synthesis.
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Fig. 3. Hematological analysis of HRI +/+ and –/– mice in iron deficiency. The RBC number, Hb (total hemoglobin) and the RBC indices, MCV and MCH were obtained from tail vein blood. Time courses of these changes from day 17 to 84 are shown. Four to six mice were used for each group. Wt + Fe, Wt mice in a normal diet; Wt – Fe, Wt mice in a low iron diet; Ko + Fe, knockout mice in a normal diet; Ko – Fe, knockout mice in a low iron diet. The differences in these red cell indices are statistically significant for a low iron diet (p <0.001 for all these four parameters).
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Fig. 4. Erythroid hyperplasia and increased apoptosis of HRI–/– erythroid precursors in iron deficiency. (A) Erythroid hyperplasia in the spleens of HRI–/– mice. The tissue sections of the spleens of Wt and HRI–/– mice both on normal and iron-deficient diets for 43 days were stained with hematoxylin and eosin. The arrows indicate the expanded red pulp. (B) Increased Ter-119+ cells in HRI–/– spleens. Single-cell suspensions from the spleens of HRI +/+ and –/– mice were gated with Ter-119 monoclonal antibody. (C) Increased apoptosis of erythroid progenitor cells in HRI–/– spleens. The Ter-119+ cells were further analyzed for their AnV and 7AAD stainings to gate the living cells (lower left quadrant), apoptotic cells (upper left quadrant) and dead cells (lower and upper right quadrant). The number in each quadrant represents the percentage of total cells.
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Fig. 5. Precipitation of globins in RBCs of HRI–/– mice in iron deficiency. (A) Wright–Giemsa-stained peripheral blood smears. Peripheral blood smears were prepared from HRI +/+ and –/– mice maintained on an iron-deficient diet for 33 days. (B) Heinz body staining. Blood from mice at day 92 after receiving a low iron diet was collected and stained with crystal violet for the presence of Heinz bodies. (C) Precipitation of globins in the blood cells of HRI–/– mice. Precipitated proteins from the blood of Wt and HRI–/– mice at day 67 after receiving a low iron diet were collected by centrifugation and separated by 15% SDS–PAGE. Proteins were analyzed by Coomassie Blue staining (upper two panels) and by western blot analysis using anti-mouse hemoglobin antibody (lower two panels). P2, pellets of 2000 g centrifugation; P100, pellets of 100 000 g centrifugation.
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Fig. 6. Electron microscopic examination of the inclusions in the reticulocytes of HRI–/– mice in iron deficiency. Blood from mice of all four groups at day 59 after receiving low iron diet was collected and processed for electron microscopy. The inclusion, indicated by an arrow, in a reticulocyte of HRI–/– mice is shown in (A) at a magnification of 30 000 ×. Inclusions were not observed in the blood samples of other groups. Higher magnification (150 000 ×) of the boxed area is shown in (B). The long arrow indicates the border of the inclusions and the shorter arrow indicates the membrane of a mitochondrion.
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Fig. 7. Decreased survival of HRI–/– mice in phenylhydrazine-induced hemolysis. Both Wt and HRI–/– mice were injected with phenylhydrazine at the dosages indicated at days 0, 1 and 3. The mice were observed daily. (A) The time courses of survival of the iron-deficient Wt and HRI–/– mice at a dosage of 50 mg/kg. n = 12 for Wt mice; n = 11 for HRI–/– mice. (B) The percentage survival at different dosages at day 6 after initial phenylhydrazine injection. Six mice of each genotype were used for each dosage.
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Fig. 8. Models of the role of HRI during erythroid differentiation. (A) Regulation of α- and β-globin synthesis by HRI and heme. (B) Altered hematological response of HRI–/– mice to iron deficiency.
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