Lnk Regulatory Functions in Hematopoietic Stem Cells

描述（由申请人提供）：造血干细胞（hsc）的体内平衡受复杂的信号通路和与微环境或干细胞生态位的相互作用调节。造血干细胞生物学的一个重要信号轴是血小板生成素（TPO）及其受体Mpl，通过Janus激酶（JAK2）发出信号。TPO-/-或Mpl-/-小鼠显示HSC自我更新减弱。相比之下，缺乏衔接蛋白link的小鼠的HSC数量增加了10倍，并且移植效果更好。我们发现HSC自我更新的link调控部分依赖于Mpl。为了支持这种遗传相互作用，我们发现Lnk直接与JAK2中磷酸化的酪氨酸结合，tpo介导的JAK2激活在Lnk-/- hsc中被增强。此外，Lnk-/-祖细胞（HPCs）比野生型（WT）更有效地归巢，并且Lnk-/-小鼠在髓外分布明显扩大，表明Lnk影响HSC与生态位的相互作用。一致地，我们发现link缺陷通过21个整合素增强HPC与纤维连接蛋白的粘附。因此，我们假设Lnk通过Mpl/JAK2控制HSC细胞周期动力学和存活，负向限制干细胞池，并通过调节整合素信号调节HSC与干细胞龛的相互作用。特异性目的1：研究Lnk在控制HSC细胞周期动力学、存活和衰竭中的作用。我们的目标是通过检测造血干细胞在发育过程中和应激后的细胞周期动力学、存活和静止状态，来研究在link -/- BM中扩大的干细胞池是如何实现的。此外，我们将使用串联BMTs研究link缺陷对HSC耗尽的影响。具体目标2：研究link在控制HSC归巢、粘附以及与HSC生态位相互作用中的作用。我们将扩展我们对祖细胞的初步研究，使用BMT试验来研究link缺陷是否会增强HSC的归巢/粘附。在分析依赖于link的HSC归巢/粘附机制时，我们将重点关注两种已知的主要途径：21整合素和基质衍生因子-1 （SDF-1）。此外，我们将测试link是否通过TPO/Mpl影响hsc中的整合素信号传导。具体目标3：研究link如何协调HSC自我更新、增殖/存活、归巢以及与生态位的相互作用。表面上不同的HSC性质是相互关联的。我们将研究整合素启动信号的扰动如何影响WT和link -/- HSC/PC的增殖、存活和植入。此外，我们将利用双光子/共聚焦显微镜在活体动物移植后研究Lnk在HSC归巢和分裂动力学中与体内微观解剖定位的关系。特异性目的4：在体内测试link - jak2相互作用在调节HSC功能中的作用。我们的遗传数据表明，在一定程度上，Lnk通过与JAK2最直接的关联来控制HSC的稳态。在这里，我们将在体内测试这一模型，通过产生两只“Knockin”小鼠，它们在Lnk和JAK2中发生点突变，从而破坏它们的相互作用。我们相信我们的研究将促进我们对干细胞稳态机制的理解，并促进干细胞治疗的临床应用。公共卫生相关性：在哺乳动物中，造血干细胞（hsc）在整个成年期负责每天产生数十亿成熟血细胞，异常的干细胞稳态机制与多种人类疾病有关，包括再生障碍性贫血和白血病。本文提出的研究旨在更好地理解内在信号分子Lnk如何调节干细胞的扩增、归巢和植入。我们相信我们的研究不仅将促进我们对正常干细胞调控机制的理解，而且还将为各种淋巴瘤和白血病的异常干细胞信号传导提供新的见解，并促进干细胞治疗的临床应用。

DESCRIPTION (provided by applicant): The homeostasis of hematopoietic stem cells (HSCs) is regulated by intricate signaling pathways and interactions with the microenvironment or stem cell niche. One important signaling axis for HSC biology is thrombopoietin (TPO) and its receptor Mpl, signaling through Janus Kinase (JAK2). TPO-/- or Mpl-/- mice show diminished HSC self-renewal. In contrast, mice lacking the adaptor protein Lnk harbor a >10-fold increase in HSC numbers and superior engraftment. We discovered that Lnk regulation of HSC self-renewal partially depends on Mpl. In support of this genetic interaction, we found that Lnk directly binds to phosphorylated tyrosines in JAK2, and TPO-mediated activation of JAK2 is potentiated in Lnk-/- HSCs. Furthermore, Lnk-/- progenitor cells (HPCs) home more efficiently than wild type (WT) controls, and Lnk-/- mice exhibit a marked expansion in extramedullary distribution, indicating Lnk affects HSC interaction with the niche. Consistently, we found that Lnk deficiency enhances HPC adhesion to fibronectin through 21 integrins. We thus hypothesize that Lnk negatively restricts stem cell pool by controlling HSC cell cycle kinetics and survival through Mpl/JAK2, and regulates HSC interaction with stem cell niches by modulating integrin signaling. Specific Aim 1: To investigate the role of Lnk in controlling HSC cell cycle kinetics, survival, and exhaustion. We aim to investigate how the expanded stem cell pool is achieved in Lnk-/- BM, by examining HSC cell cycle kinetics, survival and quiescence status of HSCs during development and after stress. In addition, we will study HSC exhaustion affected by Lnk deficiency using serial BMTs. Specific Aim 2: To investigate the role of Lnk in controlling HSC homing, adhesion, and interaction with HSC niches. We will extend our primary studies on progenitor cells to ask if Lnk deficiency enhances HSC homing/adhesion using BMT assays. In dissecting the mechanisms of Lnk-dependent HSC homing/adhesion, we will focus on two known major pathways: 21 integrin and stromal-derived factor-1 (SDF-1). Furthermore, we will test whether Lnk affects integrin signaling in HSCs through TPO/Mpl. Specific Aim 3: To investigate how Lnk coordinates HSC self-renewal, proliferation/survival, homing, and interaction with the niche. The ostensibly distinct HSC properties are inter-related. We will investigate how perturbation of integrin-initiated signaling will affect WT and Lnk-/- HSC/PC proliferation, survival, and engraftment. Furthermore, we will study the role of Lnk in HSC homing and division kinetics in relationship to micro-anatomic localization in vivo using two-photon/confocal microscopy on live animals upon transplanted. Specific Aim 4: To test Lnk-JAK2 interaction in regulating HSC functions in vivo. Our genetic data suggest Lnk functions in part through its direst association with JAK2 to control HSC homeostasis. Here we will test this model in vivo by generating two "Knockin" mice that bear point mutations in Lnk and JAK2 to disrupt their interaction. We believe our studies will advance our understanding of the mechanisms underlying stem cell homeostasis and facilitate clinical applications to stem cell therapy. PUBLIC HEALTH RELEVANCE: In mammals, hematopoietic stem cells (HSCs) are responsible for the daily production of billions of mature blood cells throughout adult life, and abnormal stem cell homeostatic mechanisms are associated with a variety of human disorders including aplastic anemia and leukemia. The research proposed here aims to achieve a better understanding of how an intrinsic signaling molecule, Lnk, regulates stem cell expansion, homing, and engraftment. We believe our studies will advance not only our understanding of the mechanisms underlying normal stem cell regulation, but also provide new insights into abnormal stem cell signaling in various lymphomas and leukemias, and facilitate clinical applications to stem cell therapy.