Molecular Analysis of the Erythropoietic Stress Response in vivo

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DESCRIPTION (provided by applicant): The erythropoietic stress response is essential for survival and recovery from anemias, cardio-pulmonary disease, chemotherapy and bone marrow transplantation. It consists of a dramatic expansion in erythropoietic tissue leading to increased erythropoietic rate. Its principal mediator is the hormone erythropoietin, whose receptor, EpoR, is expressed by erythroid progenitors. The molecular targets of EpoR signaling during stress are largely unknown, and the spectrum of progenitors it regulates during stress is not fully defined. Our long-term goal is to elucidate the molecular mechanisms critical to the erythropoietic stress response. A major obstacle had been the lack of specific cell-surface markers identifying differentiation stage-specific erythroid progenitors in vivo as they are undergoing stress. We have developed a flow cytometric assay utilizing cell-surface CD71 and Ter119 that allows us to recognize stage-specific erythroblasts directly in hematopoietic tissue. Using this assay, we found that the EpoR-activated transcription factor Stat5 is a key regulator of early erythroblast survival. Mice lacking Stat5 are anemic and have a deficient stress response. Therefore, our principal hypothesis suggests that early erythroblast survival directly regulates erythropoietic rate and the stress response. The present proposal focuses on the death receptor Fas and its ligand, FasL. Using the flow-cytometric CD71/Ter119 assay, we found that both are expressed by approximately 50% of early erythroblasts in spleen. Fas expression was inversely related to early erythroblast survival and frequency in tissue. Further, chronic stress in mice or acute Epo administration results in down-regulation of Fas and FasL from the early erythroblast cell surface. Our specific aims are: (1) Investigate Fas and FasL function in early erythroblasts in vivo. We will generate Fas and FasL mutant mice on an immune-deficient background free of autoimmune hemolysis, to test the hypothesis that early erythroblast Fas negatively regulates basal erythropoiesis. (2) Identify the molecular mechanisms responsible for down-regulation of erythroblast Fas and FasL during stress, by comparing early erythroblasts freshly isolated from mouse models of stress to equivalent cells isolated from control mice. 3) We will use EpoR and erythroid-specific Stat5-deficient mice to study the role of EpoR and Stat5 signaling in stress-induced erythroblast Fas/ FasL down-regulation. We will also test whether the deficient stress response of these mice is rescued when bred onto a Fas or FasL-mutant background. Relevance: In order to survive and recover from blood loss, anemia, or therapeutic procedures such as bone-marrow transplantation and chemotherapy, our bodies need to be able to generate red blood cells at a much higher rate than normal. The proposed work aims to understand mechanisms regulating the rapid production of red cells. This may contribute to future therapies of anemias, including anemia and fatigue associated with cancer.