Role of Survivin in Development of Megakaryocytes and Erythroid Cells

描述（由申请人提供）：尽管红系细胞和巨核细胞源自共同的祖细胞并共享许多必需的转录因子，但它们的终末成熟遵循非常不同的路径：红系细胞经历细胞周期退出和去核，而巨核细胞继续通过细胞周期进展，但跳过有丝分裂的晚期阶段成为多倍体细胞。在我们的努力，以确定参与这一过程的基因，我们发现，生存素，细胞凋亡抑制剂（IAP）家族的成员，也发挥了重要作用，在胞质分裂，红细胞与巨核细胞发育过程中的差异表达。红系细胞在其整个成熟过程中表达生存素，直至分化的终末正染阶段。与此相反，纯化的小鼠巨核细胞表达的生存素mRNA水平比红系细胞低近4倍，并且没有检测到蛋白质。为了研究生存素是否参与造血祖细胞的分化和/或存活，我们用携带人生存素cDNA的逆转录病毒或对照逆转录病毒感染原代小鼠骨髓细胞，然后在液体培养和集落形成试验中诱导红系和巨核细胞分化。这些研究表明，生存素的过度表达拮抗巨核细胞的发展，但不影响终末分化的红细胞。与此相反，生存素mRNA的50%的减少，所造成的一个杂合子的生存素的损失，阻止红细胞，但不是巨核细胞，在体外的发展。因此，我们的初步数据支持持续生存素在红系细胞中的表达和巨核细胞中的表达水平显着降低的生理作用。基于这些发现，我们假设，持续生存素的表达是需要分化和/或红系细胞的生存，而其减少是必不可少的巨核细胞的终末成熟。在这个应用中，我们建议调查如何生存素有助于红系细胞和巨核细胞的发育。具体而言，我们计划1）通过小鼠中的条件基因靶向来确定红细胞和巨核细胞发育中对存活素的需求，2）通过分析具有降低的存活素表达的培养红系细胞的表型和通过鉴定红系细胞中的存活素蛋白复合物来表征存活素参与红系细胞分化的机制，3）通过分析在巨核细胞和红系细胞中异位表达生存素的转基因小鼠，研究生存素持续表达抑制巨核细胞多倍化和成熟的原因。生存素如何有助于造血的更详细的评估将有助于我们了解细胞周期调控和细胞凋亡在正常血细胞发育中的作用，并可能揭示红系细胞和巨核细胞从共同的前体细胞产生的机制。

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.