Role of NIX in Erythroid Maturation

描述(由申请人提供)：总结：在这项提案中，我们提出了支持bcl2相关蛋白在红系成熟中作用的新模型的证据。我们发现，在终末分化过程中，促凋亡和抗凋亡的bcl2相关蛋白均上调。其中之一是促凋亡的BH3-Only蛋白NIX。为了了解它在发育中的作用，我们培育了靶向干扰Nix基因的小鼠。这些小鼠是存活的，但有轻微的贫血和显著的网织红细胞增多症。在第一个目标中，我们提出了实验来描述这些小鼠的红系成熟缺陷。我们建议检测红细胞存活率，通过高输血抑制红细胞生成，并用放血和苯肼刺激小鼠。我们还建议在细胞水平上通过分离新生网织红细胞来检查成熟缺陷，然后在成熟时用电子显微镜检查它们的超微结构。我们提出的模型是，促凋亡和抗凋亡的bcl2相关蛋白的同时增加诱导了自噬，自噬是晚期红细胞重塑所必需的。在第二个目标中，我们提出了几个基因实验来验证这一假设。我们建议培育NIX和BclX小鼠，以了解NIX在缺乏bclxl或在细胞因子剥夺的条件下是否与细胞凋亡有关。我们建议培育NIX和彪马小鼠，看看这些仅有BH3的蛋白质之间是否存在冗余。最后，我们建议培育NIX和条件性的Bcl-X小鼠，以确定bcl-xl是否在自噬中起作用。相关性：bc1-xl对红系细胞的发育是必不可少的。目前流行的模型认为bclxl的主要功能是在促红细胞生成素受体下游提供生存信号。然而，有研究表明，bcl-xl在红系分化后期发挥作用，超过了需要促红细胞生成素信号的程度。现在，我们发现另一种高度受调控的BCL2相关蛋白在红系成熟后期起作用。总之，这些研究表明，bcl2相关蛋白，包括bclxl，在红系分化中可能具有根本不同的作用。我们建议在用Nix小鼠和其他与bcl2相关的小鼠品系的实验中探索这种可能性。

DESCRIPTION (provided by applicant): Summary: In this proposal we present evidence, which supports a new model for the role of BCL2-related proteins in erythroid maturation. We show that both pro- and anti-apoptotic BCL2-related proteins are upregulated during terminal differentiation. One of these is the pro-apoptotic BH3-only protein, NIX. To see its role in development, we generated mice with targeted disruption of the Nix gene. These mice are viable, but have mild anemia and striking reticulocytosis. In the first aim, we propose experiments to characterize the defect in erythroid maturation in these mice. We propose to examine erythrocyte survival, to suppress erythropoiesis through hypertransfusion, and to stress the mice with phlebotomy and phenylhydrazine. We also propose to examine the maturation defect at the cellular level by isolating nascent reticulocytes, then examining their ultrastructure by electron microscopy as they mature. The model we propose is that the simultaneous increase in pro- and anti-apoptotic BCL2-related proteins induces autophagy, which is necessary for the remodeling of late erythroblasts. In the second aim, we propose several genetic experiments to test this hypothesis. We propose to breed Nix and Bcl-X mice to see if NIX is responsible for apoptosis in the absence of BCL-XL or under conditions of cytokine deprivation. We propose to breed Nix and Puma mice, to see if there is redundancy between these BH3-only proteins. Finally, we propose to breed Nix and conditional Bcl-X mice to if BCL-XL has a role in autophagy. Relevance: BCL-XL is essential for the development of erythroid cells. The prevailing model is that the primary function of BCL-XL is to provide a survival signal downstream of the erythropoietin receptor. There are studies, however, which suggest that BCL-XL functions late in erythroid differentiation, beyond the point where erythropoietin signaling is required. Now we show that another highly-regulated BCL2-related protein has role in late erythroid maturation. Together, these studies suggest that BCL2-related proteins, including BCL-XL, may have a fundamentally different role in erythroid differentiation. We propose to explore that possibility in experiments with Nix mice and other BCL2-related mouse strains.