Heme-Regulated eIF2α Kinase Coordinates Translational Repression of eIF2αP and mTORC1 Signaling during Iron Deficiency to Mitigate Ineffective Erythropoiesis

Iron deficiency anemia (IDA) is a prevalent disease, yet molecular mechanisms by which iron and heme regulate erythropoiesis are not well understood. Heme-regulated eIF2α kinase (HRI) is a hemoprotein highly expressed in erythroid precursors. HRI senses intracellular heme concentrations to ensure that globin synthesis does not exceed the amount of heme available for hemoglobin production. In heme deficiency, HRI is activated and phosphorylates eIF2α, which inhibits the translation of globin mRNAs as well as enhances selectively the translation of activating transcription factor 4 (ATF4) mRNA to induce transcription of stress response genes. Previously, we reported that HRI is also necessary to promote erythroid differentiation during iron deficiency (ID). To further interrogate the molecular mechanism of HRI-eIF2αP-ATF4 signaling in erythropoiesis, we generated a novel mouse model (eAA mice) that lacks of eIF2α phosphorylation (eIF2αP) by substituting serine 51 residue with alanine specifically in the erythroid lineage. eAA mice did not display significant erythroid phenotypes during normal adult steady-state erythropoiesis. However, eAA mice developed ineffective erythropoiesis with splenomegaly and macrocytic hyperchromic anemia in ID, similar to Hri^-/- mice. In contrast, iron deficient Atf4^-/-mice developed more severe anemia, and were microcytic hypochromic. Thus, regulation of cell size and hemoglobin content of RBCs by HRI is mediated through eIF2αP and not ATF4. Consistent with the regulation of globin translation by eIF2αP, globin inclusions were present in reticulocytes of eAA mice, but not Atf4^-/- mice. Both eAA and Atf4^-/-mice exhibited inhibition of terminal erythroid differentiation starting at basophilic erythroblasts in ID. The ROS levels in RBCs and reticulocytes were elevated, demonstrating that HRI-eIF2αP-ATF4 signaling contributes significantly to mitigation of oxidative stress in late stage erythroid cells. Our results demonstrate for the first time that both eIF2αP and ATF4 are necessary for iron-restricted erythropoiesis.

mTORC1 (mechanistic target of rapamycin complex 1) signaling in blood cells was recently reported to be down regulated in ID. Furthermore, constitutively activated mTORC1 signaling in the hematopoietic lineage results in macrocytic hyperchromic anemia with splenomegaly (similar to our ID Hri^-/-and eAA mice) whereas inactivation of mTORC1 signaling results in microcytic hyperchromic anemia, characteristics of IDA in Wt mice (Knight et al. eLife 2014). We therefore investigated whether HRI stress signaling was necessary to down-regulate mTORC1 in ID. mTORC1 signaling increases protein synthesis by phosphorylating eIF4E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K), which phosphorylates S6. We found that mTORC1 signaling activities as measured by pS6 and p4E-BP1, were higher in iron deficient Hri^-/-, eAA and Atf4^-/-erythroid precursors in comparison to Wt cells. Treatment of iron deficient Hri^-/-, eAA and Atf4^-/-mice with rapamycin, an mTORC1 inhibitor, increased RBC counts and hemoglobin content in the blood, improved erythroid differentiation and reduced splenomegaly significantly. However, globin inclusions remained visible in Hri^-/- and eAA reticulocytes, indicating a non-redundant role of HRI-eIF2αP in inhibiting globin translation. Dietary iron repletion of the iron deficient mice rapidly and completely reversed the anemia in 10 days by promoting erythroid differentiation and decreasing HRI and mTORC1 signaling in Hri^-/-, eAA and Atf4^-/-mice. Furthermore, the elevated serum erythropoietin (EPO) levels in our ID anemic mice were decreased significantly upon rapamycin treatment or iron repletion. Together, our results demonstrate that HRI is indispensible for both the activation of the eIF2αP-ATF4 signaling and the repression of mTORC1 signaling during ID to circumvent ineffective erythropoiesis and macrocytic anemia. Our findings also indicate that HRI stress signaling could serves as a feedback mechanism for EPO signaling by inhibiting mTORC1. This study provides molecular insights into the role of heme and translation in the regulation of erythropoiesis and may provide novel targets for the treatment of hemoglobinopathies.