Molecular Analysis of the Erythropoietic Stress Response in vivo

红系应激反应对心肺性贫血患者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 红系组织导致红系造血率增加。它的主要媒介是荷尔蒙 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 应激期间的信号转导在很大程度上是未知的，它在应激期间调节的前体细胞的光谱也不是很清楚。 完全定义好了。我们的长期目标是阐明红血球应激的关键分子机制。 回应。一个主要的障碍是缺乏识别分化的特定细胞表面标记。 体内特定阶段的红系祖细胞，因为他们正在经历压力。我们开发了一个流程- 利用细胞表面CD71和Ter119的细胞学分析，使我们能够识别特定的阶段 红血球直接在造血组织中。利用这一检测方法，我们发现EPOR激活 转录因子STATS是早期红细胞存活的关键调节因子。缺乏统计数据的小鼠会贫血 有不足的压力反应。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。目前的建议侧重于死亡- Fas受体及其配体Fast。使用流式细胞仪CD71/Ter119检测，我们发现两者都是 约50%的早期红细胞在脾中表达。Fas的表达与 早期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促红细胞生成素 给药可下调早期红细胞表面Fas和FasL的表达。我们的特定 目的：(1)研究体内早期红细胞Fas和FasL的功能。我们将生成Fas和FasL 在没有自身免疫溶血的免疫缺陷背景下的突变小鼠，以检验这一假设 早期红细胞Fas负性调节基础红细胞生成。(2)明确分子机制 通过比较早期红细胞，负责在应激过程中下调红细胞Fas和FasL 从应激模型小鼠新鲜分离的细胞与从对照小鼠分离的等量细胞。3)我们将使用 EPOR+/-和红系特异性STATS缺陷小鼠研究EPOR和STATS信号在应激中的作用 诱导红细胞Fas/FasL表达下调。我们还将测试是否有缺陷的应激反应 当这些小鼠被培育到Fas或FasL突变背景上时，它们就获救了。相关性：为了生存 并从失血、贫血或治疗程序中恢复，如骨髓移植和 化疗后，我们的身体需要能够以比正常速度高得多的速度产生红细胞。 这项拟议的工作旨在了解调节红细胞快速产生的机制。今年5月 有助于未来的贫血治疗，包括与癌症相关的贫血和疲劳。

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.