HRI Protects Erythroid Precursors in Iron Deficiency and in β-Thalassemia by Maintaining GATA-1 and Fog-1 Expressions.

HRI is the heme-regulated eIF2a kinase that controls the translation of mRNAs in erythroid precursors by phosphorylating the a-subunit of eukaryotic translational initiation factor 2 (eIF2a). HRI is essential for translational regulation of a and b globins and the survival of erythroid precursors in iron and heme deficiency. Furthermore, HRI modifies the severity of β-thalassemic mice that are devoid of the b-globin major gene. To gain insights into the molecular mechanisms by which HRI exert its protection during stress erythropoiesis, micrroarray gene profilings of erythroid precursors were carried out under the conditions of iron deficiency and HRI deficiency. HRI +/+ (Wt) and −/ − (Ko) mice were maintained in an iron deficient diet for 2 months and during gestation to induce iron deficiency in developing embryos. Under these conditions, both Wt and Ko fetal liver cells at E14.5 exhibited impaired erythroid differentiation, increased proliferation and elevated apoptosis, hallmarks of ineffective erythropoiesis. All these parameters were aggravated by HRI deficiency. Furthermore, examinations of embryonic blood smears and fetal liver cytospins revealed the presence of inclusions in reticulocytes of Ko-Fe mice (HRI^−/− in iron deficiency). These phenotypes of Ko-Fe in fetal definitive erythropoiesis were the same as those observed earlier in adult erythropoiesis. Hence, mRNAs from E14.5 fetal liver nucleated erythroid precursors were used for mouse expression arrays. In Wt-Fe, expression of 213 genes was altered more than 2-fold. The number of genes altered in Ko-Fe increased dramatically by 15-fold to 3,135. In contrast, the number of the genes changed in Ko+Fe was relatively small (85), consistent with the mild phenotype in Ko+Fe mice. Therefore, HRI is crucial in maintaining proper gene expression in iron deficient erythroid precursors. Large numbers of genes altered in Ko-Fe were involved in cell death, protein synthesis, cancer and the cell cycle, consistent with the phenotypes of elevated apoptosis and increased proliferation in Ko-Fe erythroid precursors. In addition, there were more known erythroid genes (29) and iron/heme homoeostasis genes (12) that were changed in Ko-Fe as compared to Wt-Fe. Most of these genes were targets of GATA-1. When gene profiling of Ko-Fe erythroid precursors was compared with that of G1E-Er4 cells upon induction of GATA-1 by estradiol, there were 892 overlapping genes affected in both cases. GATA-1 mRNA and protein levels in fetal liver cells were reduced in iron deficiency and in b-thalassemia, and were decreased further in HRI deficiencies. Fog-1 mRNA level was not significantly altered in iron deficiency or b-thlassemia alone, but were reduced significantly upon combined deficiencies with HRI. These results demonstrate for the first time the impairment of GATA-1 and Fog-1 expressions in iron deficiency and in b-thalassemia with deletion of the b-globin gene. Furthermore, in iron deficiency and b-thalassemia HRI is necessary to maintain both expressions of GATA-1 and Fog-1, which are essential for definitive erythropoiesis.