MECHANISMS REGULATING THE RESPONSE TO ANEMIC STRESS

New erythrocytes must be made constantly during a person's life because erythrocytes have, on average, a lifespan of 120 days. Erythrocytes carry oxygen to the body's tissues. Any decrease in erythrocyte number or their ability to carry oxygen results in anemia, which is a highly prevalent condition with multiple etiologies. Anemia can cause significant morbidity and mortality, and it can have significant impact on quality of life. The goal of our work is to understand the mechanisms by which the body responds to anemic or hypoxic stress. Previous work from our group has identified a novel stress response pathway that is induced by anemic stress, which functions to rapidly produce new erythrocytes to relieve tissue hypoxia. Ultimately, we hope to apply our knowledge of this system to design therapeutic interventions to treat anemia.Anemia is a condition that results in the impaired delivery of oxygen, which leads to tissue hypoxia. Because oxygen is required for survival, a physiological response to tissue hypoxia has evolved. One aspect of this response is the generation of new erythrocytes to deliver oxygen to hypoxic tissues. We have discovered that the erythroid response to anemic stress relies on the expansion and differentiation of a specialized population of stress erythroid progenitors. These progenitors are distinct from steady state erythroid progenitors and they develop outside of the bone marrow in a process referred to as extra-medullary erythropoiesis. In mice, this process occurs primarily in the spleen, and the fetal and adult liver. The signals that regulate stress erythropoiesis are different as well. Our data show that the expansion of stress erythroid progenitors requires bone morphogenetic protein 4 (BMP4), Hedgehog (HH), and growth and differentiation factor 15 (GDF15). These factors act on a specific population of erythroid progenitors that express stem cell markers. During the expansion stage, these progenitor cells are unable to differentiate into new erythrocytes. Following the expansion of the cells, erythropoietin (Epo) and tissue hypoxia act on macrophages in the microenvironment of the spleen to promote the differentiation of the stress erythroid progenitors. Once they acquire the ability to differentiate, the progenitor cells respond to BMP4, stem cell factor (SCF), Epo and hypoxia which promotes the proliferation and terminal differentiation of new erythrocytes.GDF15 regulates the hypoxia response: Our initial experiments showed that mice mutant for GDF15 (GDF15-/-) have no defects in steady state erythropoiesis. However, if challenged with anemic stress, the mice are unable to respond and fail to survive. Using this phenotype, we discovered that GDF15 signaling plays an important role in the expansion of progenitor cells early during the response to anemia. Previously, we showed that tissue hypoxia results in the up-regulation of BMP4 expression in the spleen and liver. Hypoxia mediates this response through the hypoxia-inducible transcription factor Hif2a. Under hypoxic conditions, Hif2a protein is stable where it forms a dimer with the Hifb subunit and activates hypoxia-inducible gene expression. In normoxia, Hif2a is unstable and is degraded by the ubiquitin-mediated protein degradation system. The VHL protein that is part of ubiquitin ligase complex that promotes the degradation of Hif2a. In GDF15-/- mice, BMP4 expression is not maintained in the spleen, and the lack of BMP4 contributes to the inability of these mice to produce new erythrocytes in response to anemia. GDF15-dependent signaling maintains BMP4 expression by repressing the expression of VHL, which leads to stabilization of Hif2a. Although this aspect of GDF15 plays an essential role in stress erythropoiesis, we know GDF15-dependent signaling has other targets because we still observe defects in stress erythropoiesis when we supplement BMP4 in the absence of GDF15.Stress erythropoiesis relies on interactions between progenitor cells and the microenvironment: Much of our published work has focused on the responses of progenitor cells to signals that drive their expansion and differentiation into new erythrocytes. Recent data from us and others has shown that this process can occur only through the interaction between progenitor cells and their microenvironment and, in particular, red pulp macrophages in the spleen. Papers from the Rivella and Frenette labs showed that removing macrophages by genetic ablation or chemical treatment impaired the response to anemic stress by impeding the proliferation and differentiation of stress erythroid progenitors. In my laboratory, we have shown that removal of macrophages from cultures of stress erythroid progenitors blocks their development. Macrophages express BMP4 and HH ligands that are known to drive the expansion and differentiation of stress erythroid progenitors, but in cultures where we add exogenous HH and BMP4, we cannot rescue cultures lacking macrophages.We have established a developmental pathway for the expansion and differentiation of stress erythroid progenitors. In addition, we have identified a number signals that regulate this process. This research project will focus on three questions: (1) How do BMP4-, HH- and GDF15-dependent signaling combine to promote the expansion of stress erythroid progenitors with stem cell-like properties? (2) What are the signals expressed by macrophages that regulate stress erythropoiesis? (3) What are the molecular mechanisms that define the switch from progenitor cells that are stem cell-like and proliferating to progenitors that are actively differentiating?