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.
Subject(s)
Erythrocytes/cytology , Erythrocytes/enzymology , Gene Expression Regulation, Enzymologic , Iron Deficiencies , Protein Biosynthesis , eIF-2 Kinase/metabolism , eIF-2 Kinase/physiology , Animals , Apoptosis , Blotting, Northern , Blotting, Western , Cell Lineage , Cell Separation , Cell Survival , Cloning, Molecular , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Eukaryotic Initiation Factor-2/metabolism , Flow Cytometry , Gene Library , Genotype , Heme/biosynthesis , Iron/metabolism , Mice , Microscopy, Electron , Models, Biological , Phosphorylation , Polyribosomes/metabolism , Protein Binding , Protein Structure, Tertiary , Protoporphyrins/biosynthesis , Reticulocytes/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stress, Physiological , Time FactorsABSTRACT
Cytoplasmic stresses, including heat shock, osmotic stress, and oxidative stress, cause rapid inhibition of protein synthesis in cells through phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) by eIF2alpha kinases. We have investigated the role of heme-regulated inhibitor (HRI), a heme-regulated eIF2alpha kinase, in stress responses of erythroid cells. We have demonstrated that HRI in reticulocytes and fetal liver nucleated erythroid progenitors is activated by oxidative stress induced by arsenite, heat shock, and osmotic stress but not by endoplasmic reticulum stress or nutrient starvation. While autophosphorylation is essential for the activation of HRI, the phosphorylation status of HRI activated by different stresses is different. The contributions of HRI in various stress responses were assessed with the aid of HRI-null reticulocytes and fetal liver erythroid cells. HRI is the only eIF2alpha kinase activated by arsenite in erythroid cells, since HRI-null cells do not induce eIF2alpha phosphorylation upon arsenite treatment. HRI is also the major eIF2alpha kinase responsible for the increased eIF2alpha phosphorylation upon heat shock in erythroid cells. Activation of HRI by these stresses is independent of heme and requires the presence of intact cells. Both hsp90 and hsc70 are necessary for all stress-induced HRI activation. However, reactive oxygen species are involved only in HRI activation by arsenite. Our results provide evidence for a novel function of HRI in stress responses other than heme deficiency.