Molecular Analysis of the Erythropoietic Stress Response in vivo

描述（由申请人提供）：红细胞生成应激反应对于贫血、心肺疾病、化疗和骨髓移植的生存和恢复至关重要。它包括红细胞生成组织的急剧扩张，导致红细胞生成率增加。其主要介质是激素促红细胞生成素，其受体EpoR由红系祖细胞表达。应激过程中EpoR信号的分子靶点在很大程度上是未知的，它在应激过程中调节的祖细胞谱也没有完全确定。我们的长期目标是阐明红细胞生成应激反应的关键分子机制。一个主要的障碍是缺乏特异性的细胞表面标记物，在体内识别分化阶段特异性的红系祖细胞，因为它们处于应激状态。我们开发了一种利用细胞表面CD71和Ter119的流式细胞术，使我们能够直接识别造血组织中特定阶段的红母细胞。通过这项实验，我们发现epor激活的转录因子Stat5是早期红母细胞存活的关键调节因子。缺乏Stat5的小鼠贫血，应激反应不足。因此，我们的主要假设表明，早期红细胞存活直接调节红细胞生成率和应激反应。本研究的重点是死亡受体Fas及其配体FasL。通过流式细胞术CD71/Ter119检测，我们发现这两种基因在脾脏中约50%的早期红母细胞中表达。Fas表达与组织中早期红母细胞存活和频率呈负相关。此外，小鼠慢性应激或急性Epo给药可导致早期红母细胞表面Fas和FasL的下调。我们的具体目标是：(1)研究Fas和FasL在体内早期红母细胞中的功能。我们将在没有自身免疫性溶血的免疫缺陷背景下产生Fas和FasL突变小鼠，以检验早期红母细胞Fas负性调节基础红细胞生成的假设。(2)通过比较应激小鼠模型中新分离的早期红母细胞与对照小鼠分离的等效细胞，确定应激过程中红母细胞Fas和FasL下调的分子机制。3)我们将利用EpoR和红细胞特异性Stat5缺陷小鼠研究EpoR和Stat5信号在应激诱导的红母细胞Fas/ FasL下调中的作用。我们还将测试这些小鼠在Fas或fasl突变背景下繁殖时是否能够恢复缺乏的应激反应。相关性：为了从失血、贫血或骨髓移植和化疗等治疗过程中生存和恢复，我们的身体需要能够以比正常高得多的速度产生红细胞。这项工作旨在了解调节红细胞快速产生的机制。这可能有助于未来治疗贫血，包括与癌症相关的贫血和疲劳。

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.