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首页> 外文学位 >Identification of Novel Regulators in Hematopoiesis: Roles for Gfer in Hematopoietic Stem Cell Proliferation and CaMKK2 in the Restriction of Granulopoiesis.
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Identification of Novel Regulators in Hematopoiesis: Roles for Gfer in Hematopoietic Stem Cell Proliferation and CaMKK2 in the Restriction of Granulopoiesis.

机译：造血干细胞中新型调节剂的鉴定：Gfer在造血干细胞增殖中的作用和CaMKK2在限制粒细胞生成中的作用。

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摘要

Hematopoiesis is the process in which billions of blood cells are produced on a daily basis, and is vital for sustaining life. This process is tightly regulated by a dynamic balance between hematopoietic stem cell (HSC) self-renewal and differentiation, and maintenance of this balance is of critical importance as dysregulation of HSCs can lead to hematopoietic deficiencies or malignancies such as leukemogenesis. While the signaling mechanisms that regulate HSC homeostasis and function are not well understood, our previous studies have identified a calcium/calmodulin (CaM)-dependent protein kinase, CaMKIV, that is intrinsically required for regulating normal proliferation and survival in HSCs. These findings suggest not only the importance of calcium-initiated pathways including CaMKIV-dependent signaling in hematopoietic cells, but also the potential for other calcium/CaM-dependent effector proteins such as other CaM-kinases to be involved in regulating HSCs and hematopoiesis.;The first major section of this dissertation work presented herein was based on the usage of RNA interference (RNAi) technology to specifically deplete HSCs of growth factor erv1-like (Gfer), a gene whose expression appeared to be absent in CaMKIV null HSCs based on comparative microarray analysis with wild-type HSCs, and seemed a potential target of CaMKIV. We showed that depletion of Gfer in HSCs compromised their in vivo engraftment potential and triggered a hyper-proliferative response that led to their exhaustion. We further assessed Gfer-depleted HSCs by using microscopy techniques and found that these cells possessed significantly reduced levels of the cyclin-dependent kinase inhibitor (CDKI) p27kip1. In contrast, ectopic over-expression of Gfer in HSCs resulted in significantly elevated total and nuclear p27kip1. We next performed immunoprecipitation-immunoblot analyses to determine whether alteration of Gfer levels would affect p27 kip1's binding with its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), that would subsequently lead to its ubiquitination, and determined that depletion of Gfer resulted in enhanced binding of p27kip1 to Jab1. Conversely, over-expression of Gfer resulted in its enhanced binding to Jab1 and inhibition of the Jab1-p27 kip1 interaction. Furthermore, normalization of p27kip1 in Gfer-KD HSCs rescued their in vitro proliferation deficits. These results provide evidence for a novel Gfer-Jab1-p27kip1 pathway present in HSCs that functions to restrict abnormal proliferation.;The second major section of this dissertation work describes our studies of a CaMKIV kinase, CaMKK2, and its role in HSCs and hematopoietic development. These studies were largely based on the usage of mice genetically ablated for the Camkk2 gene in the germline. Herein, we identified a role for CaMKK2 in the restriction of granulocytic fate commitment and differentiation of myeloid progenitor cells. We performed bone marrow transplantation studies and discovered that engraftment by Camkk2-/- donor cells resulted in the increased production of mature granulocytes in the bone marrow and peripheral blood. Similarly, we used fluorescence activated cell sorting (FACS) to determine that Camkk2-/- mice possessed elevated numbers of common myeloid progenitor cells, and exhibited an accelerated granulopoietic phenotype in the bone marrow. Expression of ectopic CaMKK2 in Camkk2-/- common myeloid progenitors was sufficient to rescue aberrant granulocyte differentiation, and when over-expressed in 32Dcl3 cells was also sufficient to impede granulocyte differentiation in a kinase activity-dependent manner. Collectively, our results reveal a novel role for CaMKK2 as an inhibitor of granulocytic fate commitment and differentiation in early myeloid progenitors.;While our original intent was to identify and link a downstream target and upstream kinase to CaMKIV in HSCs, our results ultimately did not suggest that Gfer or CaMKK2 function in the same pathway in HSCs as discussed in the following chapters. Nonetheless, our findings represent a considerable advance in identifying and characterizing the functions of two novel regulators, Gfer and CaMKK2, that are important for HSC proliferation and the commitment and early differentiation steps of granulopoiesis, respectively.
机译：造血过程是每天产生数十亿个血细胞的过程，对于维持生命至关重要。此过程受到造血干细胞（HSC）自我更新和分化之间动态平衡的严格调节，并且维持这种平衡至关重要，因为HSC的失调可导致造血缺陷或恶性肿瘤，例如白血病的发生。虽然尚不清楚调节HSC稳态和功能的信号传导机制，但我们先前的研究已经确定了钙/钙调蛋白（CaM）依赖性蛋白激酶CaMKIV，这是调节HSC正常增殖和存活的内在必需。这些发现不仅表明造血细胞中钙起始途径包括CaMKIV依赖性信号传导的重要性，而且还表明其他钙/ CaM依赖性效应蛋白（例如其他CaM激酶）参与调节HSC和造血作用的潜力。本文介绍的本论文的第一部分主要是基于RNA干扰（RNAi）技术的使用，目的是专门消耗生长因子erv1样（Gfer）的HSC，该基因的表达似乎在基于CaMKIV的HSC中不存在，与野生型HSC进行比较微阵列分析，似乎是CaMKIV的潜在目标。我们显示，HSC中Gfer的耗竭损害了它们的体内植入潜能，并引发了过度增殖反应，导致其疲惫。我们通过使用显微镜技术进一步评估了耗尽Gfer的HSC，发现这些细胞的细胞周期蛋白依赖性激酶抑制剂（CDKI）p27kip1水平显着降低。相比之下，HSC中异位表达Gfer导致总p27kipip1和核p27kip1明显升高。接下来，我们进行了免疫沉淀-免疫印迹分析，以确定Gfer水平的改变是否会影响p27 kip1与其抑制剂COP9信号体亚基jun激活域结合蛋白1（Jab1）的结合，从而导致其泛素化，并确定耗竭Gfer的作用导致p27kip1与Jab1的结合增强。相反，Gfer的过度表达导致其与Jab1的结合增强，并抑制了Jab1-p27 kip1的相互作用。此外，Gfer-KD HSC中p27kip1的正常化挽救了其体外增殖缺陷。这些结果提供了在HSC中存在的一种新的Gfer-Jab1-p27kip1途径的证据，该途径具有限制异常增殖的作用。 。这些研究主要是基于对种系中的Camkk2基因进行基因消融的小鼠的使用。在本文中，我们确定了CaMKK2在限制粒细胞命运和骨髓祖细胞分化中的作用。我们进行了骨髓移植研究，发现Camkk2-/-供体细胞的移植导致骨髓和外周血中成熟粒细胞的产生增加。同样，我们使用荧光激活细胞分选（FACS）来确定Camkk2-/-小鼠具有升高数量的普通髓样祖细胞，并在骨髓中表现出加速的粒细胞表型。在Camkk2-/-常见髓样祖细胞中异位CaMKK2的表达足以挽救异常的粒细胞分化，当在32Dcl3细胞中过表达时，也足以以激酶活性依赖性方式阻止粒细胞分化。总的来说，我们的研究结果揭示了CaMKK2在早期髓样祖细胞中作为粒细胞命运承诺和分化抑制剂的新作用。虽然我们的初衷是确定HSC中的下游靶标和上游激酶并将其与CaMKIV联系起来，但最终结果并未提示Gfer或CaMKK2在HSC的同一途径中起作用，如以下各章所述。但是，我们的发现代表了在鉴定和表征两种新型调节剂Gfer和CaMKK2的功能方面的重要进展，这两种调节剂分别对HSC增殖以及粒细胞生成和早期分化步骤很重要。

著录项

  • 作者

    Teng, Ellen Chao.;

  • 作者单位

    Duke University.;

  • 授予单位 Duke University.;
  • 学科 Biology Molecular.;Biology Cell.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 137 p.
  • 总页数 137
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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