Molecular Analysis of the Erythropoietic Stress Response in vivo

红细胞生成应激反应对于贫血、心肺功能衰竭和其他疾病的生存和恢复至关重要。 疾病、化疗和骨髓移植。它包括一个戏剧性的扩张， 红细胞生成组织，导致红细胞生成率增加。它的主要介质是激素 促红细胞生成素，其受体EpoR由红系祖细胞表达。EpoR的分子靶点 在应激过程中的信号传导在很大程度上是未知的，并且在应激过程中它调节的祖细胞谱也不是 完全定义。我们的长期目标是阐明红细胞生成应激的分子机制 反应一个主要障碍是缺乏识别分化的特异性细胞表面标记物 阶段特异性红系祖细胞在体内，因为他们正在经历压力。我们已经形成了一个流程- 利用细胞表面CD 71和Ter 119的细胞计数分析，使我们能够识别阶段特异性 直接在造血组织中的成红细胞。使用这种分析，我们发现EpoR激活的 转录因子StatS是早期成红细胞存活的关键调节因子。缺乏StatS的小鼠贫血， 都有应激反应不足因此，我们的主要假设表明，早期成红细胞存活 直接调节红细胞生成率和应激反应。目前的建议集中在死亡- Fas受体及其配体Fast。使用流式细胞术CD 71/Ter 119检测，我们发现两者都是 由脾脏中约50%的早期成红细胞表达。Fas的表达与 早期成红细胞存活率和组织中的频率。此外，小鼠的慢性应激或急性Epo 施用导致来自早期成红细胞细胞表面的Fas和FasL的下调。我们的具体 目的：（1）研究Fas和FasL在早期成红细胞中的功能。我们将产生Fas和FasL 突变小鼠在免疫缺陷背景下无自身免疫性溶血，以检验假设， 早期成红细胞Fas负调节基础红细胞生成。(2)识别分子机制 通过比较早期成红细胞， 从小鼠应激模型新鲜分离的细胞与从对照小鼠分离的等同细胞。3)我们将使用 EpoR+/-和红系特异性StatS缺陷小鼠，以研究EpoR和StatS信号传导在应激中的作用。 诱导成红细胞Fas/ FasL下调。我们还将测试是否有缺陷的压力反应， 当在Fas或FasL突变体背景下繁殖时，这些小鼠被拯救。相关性：为了生存 从失血、贫血或骨髓移植等治疗过程中恢复过来， 化疗时，我们的身体需要能够以比正常高得多的速度生成红细胞。 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，包括贫血和与癌症相关的疲劳。

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 erythropoeitic 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 hematopoeitic tissue. Using this assay, we found that the EpoR-activated transcription factor StatS is a key regulator of early erythroblast survival. Mice lacking StatS 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, Fast. 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 result 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 StatS-deficient mice to study the role of EpoR and StatS 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.