STUDIES OF HEME-REGULATED eIF2a KINASE STRESS SIGNALING ON MATURATION OF MACROPHAGES AND ERYTHROBLASTIC ISLAND FORMATION IN IRON RESTRICTIVE ERYTHROPOIESIS

Iron is the most important metal for the human body. Different states of iron deficiency have long existed and remain very common in today’s population. The vast majority of cases of iron deficiency are acquired as a result from blood loss. Any condition in which dietary iron intake does not meet the body’s demands will result in iron deficiency. Iron and heme are both fundamental in hemoglobin synthesis and erythroid cell differentiation. In addition to act as a prosthetic group for hemoglobin, heme regulates the transcription of globin genes and controls the translational activity in erythroid precursors through modulation of the kinase activity of the eIF2α kinase HRI which is regulated by heme. HRI, the heme-regulated inhibitor of translation, was first discovered in reticulocytes under the conditions of iron and heme deficiencies. During heme deficiency conditions, in the erythroid precursors protein synthesis is inhibited by phosphorylation of the α-subunit of the eukaryotic initiation factor 2 (eIF2α) as the result of the activation of HRI. The role of HRI is to control that the amount of globin chains synthesized are not in excess of what can be utilized for hemoglobin tetramers depending of heme available. In the absence of HRI, during erythropoietic stress, heme-free globins precipitate causing severe demage of cells in iron-deficiency anemia. In addition to inhibiting protein synthesis, eIF2α phosphorylation increases the translation of specific mRNAs, most notably the activating transcription factor 4 (ATF4),a key factor during cell adaptation to stress. Chen laboratory published new important findings about the role of HRI in murine macrophage maturation. For the first time HRI was studied in a non-erythroid cellular line. HRI is expressed at a lower level in macrophages, but in HRI deficiency, macrophages did not develop the typical morphology and expressed less critical markers for the growth and maturation of macrophages compared to wt mice. Macrophages are responsible for iron recycling, taking iron from the hemoglobin of senescent red blood cells by phagocytosis and releasing it through the iron transporter ferroportin. During definitive erythropoiesis, erythroblasts are known to express a diverse array of adhesion molecules, these proteins arrange both erythroblast/erythroblast and erythroblast/macrophage interactions forming the erythroblastic islands (EI). These specialized niches within definitive mammalian erythroblasts have the task to facilitate proliferation and differentiation of erythroblasts. EIs can be described as a central macrophage surrounded by erythroblasts in different stages of maturation and are essential for the developing of erythroblasts that are going to enucleate. In this project we focused on the role of HRI pathway in the specific subset of macrophages that form EIs. We compared macrophages from Hri-/- mice, which lack HRI in all the tissues, with macrophages from an eAA mouse model, which are defective in eIF2α signaling specifically in the erythroid lineage to examine the differences between the two models under erythropoietic stress. We investigated the interactions of macrophages and erythroid precursors (EI) in the bone marrow and spleen of wild type, Hri-/-, and eAA mice using immunofluorescent microscopy to study the morphology of native EI. We also reconstituted EI by using macrophages from the bone marrow of wild type and mutant mice. We cultured the bone marrow and spleen derived macrophages (BMDMS and SDMS) from Hri-/-, eAA and Atf4-/- mutant mice. We were able to obtain more SDMS from iron deficient mice compared to iron sufficient mice starting with the same number of nucleated cells but there was much less BMDMS and SDMS from Atf4-/- mice and we validated these results with the colonies assay. EIs from Hri-/- mice showed impaired macrophages when compared to the EI from eAA and Wt mice. The Hri-/- macrophages were smaller with the cell extremities less expanded; in contrast, EIs from eAA mice were well differentiated and they formed large EI with many erythroblast precursors. This finding was validated with the reconstituted islands, using Wt macrophages reconstituted with Hri-/- erythroblasts.