Role of NIX in Erythroid Maturation

描述（由申请人提供）：总结：在本提案中，我们提供了证据，支持BCL 2相关蛋白在红系成熟中作用的新模型。我们发现，在终末分化过程中，促凋亡和抗凋亡BCL 2相关蛋白均上调。其中之一是促凋亡BH 3-only蛋白，NIX。为了了解它在发育中的作用，我们产生了有针对性地破坏Nix基因的小鼠。这些小鼠是可行的，但有轻度贫血和惊人的网织红细胞增多症。在第一个目标中，我们提出实验来表征这些小鼠中红系成熟的缺陷。我们建议检查红细胞存活，通过输血抑制红细胞生成，并用放血和苯肼应激小鼠。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，然后在它们成熟时通过电子显微镜检查它们的超微结构。我们提出的模型是，同时增加亲和抗凋亡BCL 2相关蛋白诱导自噬，这是必要的晚期成红细胞的重塑。在第二个目标中，我们提出了几个遗传实验来验证这一假设。我们建议繁殖Nix和Bcl-X小鼠，看看NIX是否负责在BCL-XL或细胞因子剥夺的条件下的细胞凋亡。我们建议繁殖Nix和Puma小鼠，看看这些仅含BH 3的蛋白质之间是否存在冗余。最后，我们建议繁殖Nix和条件Bcl-X小鼠，以确定BCL-XL是否在自噬中起作用。相关性：BCL-XL对红系细胞的发育至关重要。流行的模型是BCL-XL的主要功能是提供促红细胞生成素受体下游的存活信号。然而，有研究表明BCL-XL在红细胞分化后期发挥作用，超过了需要促红细胞生成素信号传导的点。现在，我们发现另一种高度调节的BCL 2相关蛋白在红细胞成熟后期起作用。总之，这些研究表明，BCL 2相关蛋白，包括BCL-XL，可能在红细胞分化中具有根本不同的作用。我们建议在Nix小鼠和其他BCL 2相关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.