Role of Survivin in Development of Megakaryocytes and Erythroid Cells

描述（由申请人提供）：虽然红系细胞和巨核细胞来源于一个共同的祖细胞，并共享许多必需的转录因子，但它们的最终成熟遵循非常不同的途径：红系细胞经历细胞周期退出和去核，而巨核细胞继续在细胞周期中前进，但跳过有丝分裂的后期成为多倍体细胞。在我们努力鉴定参与这一过程的基因时，我们发现凋亡抑制剂（IAP）家族的成员survivin在细胞分裂中也起着重要作用，在红细胞和巨核细胞发育过程中表达差异。红细胞在其成熟过程中表达survivin，直至分化的终末正色期。相比之下，纯化的小鼠巨核细胞表达的survivin mRNA水平比红系细胞低近4倍，且无可检测到的蛋白。为了研究survivin是否参与造血祖细胞的分化和存活，我们用携带人survivin cDNA的逆转录病毒或对照逆转录病毒感染小鼠原代骨髓细胞，然后在液体培养和集落形成实验中诱导红细胞和巨核细胞分化。这些研究表明，survivin的过表达可拮抗巨核细胞的发育，但不影响红细胞的终末分化。相反，由survivin杂合缺失引起的survivin mRNA减少50%，在体外阻断了红细胞的发育，而不是巨核细胞的发育。因此，我们的初步数据支持红细胞中持续的survivin表达和巨核细胞中显著降低的表达水平的生理作用。基于这些发现，我们假设持续的survivin表达是红系细胞分化和/或存活所必需的，而其减少是巨核细胞最终成熟所必需的。在这个应用中，我们建议研究survivin如何促进红细胞和巨核细胞的发育。具体来说，我们计划1)通过条件基因靶向确定小鼠红细胞和巨核细胞发育对survivin的需求，2)通过分析培养的survivin表达降低的红细胞的表型和鉴定红细胞中survivin蛋白复合物来表征survivin参与红细胞分化的机制。3)通过分析巨核细胞和红细胞中异位表达survivin的转基因小鼠，探讨为何持续表达survivin会抑制巨核细胞的多倍体化和成熟。对survivin如何促进造血的更详细的评估将有助于我们理解细胞周期调节和细胞凋亡在正常血细胞发育中的作用，并可能揭示红细胞和巨核细胞从共同前体产生的机制。

DESCRIPTION (provided by applicant): Although erythroid cells and megakaryocytes derive from a common progenitor and share many essential transcription factors, their terminal maturation follows very different paths: erythroid cells undergo cell cycle exit and enucleation while megakaryocytes continue to progress through the cell cycle, but skip late stages of mitosis to become polyploid cells. In our efforts to identify genes that participate in this process, we discovered that survivin, a member of the inhibitor of apoptosis (IAP) family that also plays an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express survivin throughout their maturation, up through the terminal orthochromatic stage of differentiation. In contrast, purified murine megakaryocytes express nearly 4-fold lower levels of survivin mRNA compared to erythroid cells and no detectable protein. To investigate whether survivin is involved in the differentiation and/or survival of hematopoietic progenitors, we infected primary mouse bone marrow cells with retroviruses harboring the human survivin cDNA or control retrovirus, and then induced erythroid and megakaryocyte differentiation in both liquid culture and colony-forming assays. These studies revealed that overexpression of survivin antagonized megakaryocyte development, but did not affect the terminal differentiation of red blood cells. In contrast, a 50% reduction in survivin mRNA, caused by a heterozygous loss of survivin, blocked erythroid, but not megakaryocyte, development in vitro. Thus, our preliminary data support a physiologic role for persistent survivin expression in erythroid cells and a significantly reduced level of expression in megakaryocytes. Based on these findings, we hypothesize that persistent survivin expression is required for differentiation and/or survival of erythroid cells, while its reduction is essential for terminal maturation of megakaryocytes. In this application, we propose to investigate how survivin contributes to erythroid cell and megakaryocyte development. Specifically, we plan to 1) Determine the requirement for survivin in red cell and megakaryocyte development by conditional gene targeting in mice, 2) Characterize the mechanism by which survivin participates in erythroid cell differentiation by analyzing the phenotype of cultured erythroid cells with reduced survivin expression and by identifying survivin protein complexes in erythroid cells, and 3) Investigate why persistent survivin expression is inhibitory to megakaryocyte polyploidization and maturation by analysis of transgenic mice that ectopically express survivin in megakaryocytes and erythroid cells. A more detailed assessment of how survivin contributes to hematopoiesis will aid in our understanding of the role of cell cycle regulation and apoptosis in normal blood cell development, and may shed light into the mechanism by which erythroid cells and megakaryocytes arise from a common precursor.