Lnk Regulatory Functions in Hematopoietic Stem Cells

造血干细胞中的 Lnk 调节功能

• 批准号: 8293214
• 负责人: Wei Tong
• 金额: $ 40.71万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-30 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8293214
• 关键词: Ablation; Adaptor Signaling Protein; Address; Adhesions; Adult; Affect; Anatomy; Animals; Aplastic Anemia; Binding; Binding Sites; Biological Assay; Blood; Blood Cells; Bone Marrow; Bone Marrow Transplantation; Breeding; CXCR4 gene; Cell Communication; Cell Count; Cell Cycle; Cell Cycle Kinetics; Cell Surface Receptors; Cell Survival; Cell physiology; Chemicals; Confocal Microscopy; Cytokine Signaling; Data; Development; Disease; Dominant-Negative Mutation; Dyes; Engineering; Engraftment; Equilibrium; Event; Exhibits; Extracellular Matrix; Extramedullary; Family; Fetal Liver; Fibronectins; Fluorouracil; Focal Adhesion Kinase 1; Frequencies; Genetic; Genetically Modified Animals; Guanosine Triphosphate Phosphohydrolases; Health; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hepatocyte; Home environment; Homeostasis; Homing; Human; Image; Integrin Signaling Pathway; Integrins; JAK2 gene; Janus kinase; Kinetics; Label; Life; Ligands; Location; Lymphocyte; Maintenance; Mammals; Maps; Mediating; Microscopy; Mus; Mutate; Mutation; Non-Malignant; Osteoblasts; PTK2 gene; Pathway interactions; Phenotype; Play; Point Mutation; Production; Property; Regulation; Research; Resistance; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Specificity; Stem Cell Development; Stem cells; Stress; Structure; System; Testing; Thrombopoietin; Transplantation; Tyrosine; Ursidae Family; Wild Type Mouse; base; cancer therapy; chemokine; clinical application; cytokine; exhaustion; in vivo; in vivo Model; inhibitor/antagonist; insight; leukemia; leukemia/lymphoma; mutant; neutralizing antibody; postnatal; premature; receptor; retroviral transduction; rho; self-renewal; src Homology Region 2 Domain; stem cell biology; stem cell division; stem cell niche; stem cell therapy; two-photon

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.

描述(申请人提供)：造血干细胞(HSCs)的动态平衡由复杂的信号通路和与微环境或干细胞生态位的相互作用来调节。HSC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 Kinase(JAK2)进行信号转导。TPO-/-或MPL-/-小鼠的HSC自我更新能力减弱。相比之下，缺乏适配蛋白LNK的小鼠的HSC数量增加了10倍，植入能力更强。我们发现LNK对HSC自我更新的调节部分依赖于MPL。为了支持这种遗传相互作用，我们发现LNK直接与JAK2中的磷酸化酪氨酸结合，并且在LNK-/-HSCs中TPO介导的JAK2的激活被增强。此外，LNK-/-祖细胞(HPC)比野生型(WT)对照组更有效地回家，并且LNK-/-小鼠在髓外分布上显示出明显的扩张，表明LNK影响HSC与小生境的相互作用。我们一致地发现，LNK缺乏通过21种整合素增强HPC与纤维连接蛋白的黏附。因此，我们假设LNK通过MPL/JAK2控制HSC细胞周期动力学和存活来负面限制干细胞库，并通过调节整合素信号来调节HSC与干细胞利基的相互作用。具体目的1：研究LNK在控制HSC细胞周期动力学、存活和衰竭中的作用。我们的目的是通过检测HSC细胞周期动力学，以及HSC在发育和应激后的存活和静止状态，来研究LNK-/-BM是如何实现扩大的干细胞库的。此外，我们还将使用系列BMT研究LNK缺乏对HSC耗竭的影响。具体目的2：研究LNK在控制HSC归巢、黏附和与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，利用骨髓移植检测LNK缺乏是否会增强HSC的归巢/黏附。在剖析LNK依赖的HSC归巢/黏附机制时，我们将重点介绍两条已知的主要途径：21整合素和基质衍生因子-1(SDF-1)。此外，我们将测试LNK是否通过TPO/MPL影响HSCs中的整合素信号转导。具体目标3：研究LNK如何协调HSC的自我更新、增殖/存活、归巢以及与生态位的相互作用。表面上不同的HSC特性是相互关联的。我们将研究整合素启动信号的扰动如何影响WT和LNK-/-HSC/PC的增殖、存活和植入。此外，我们将在活体动物移植后，利用双光子/共聚焦显微镜研究LNK在HSC归巢和分裂动力学中的作用以及与体内显微解剖定位的关系。具体目的4：检测体内LNK-JAK2相互作用对HSC功能的调节作用。我们的遗传数据表明，LNK的部分功能是通过与JAK2的最直接联系来控制HSC的动态平衡。在这里，我们将通过产生两只携带LNK和JAK2点突变的“Knockin”小鼠来在体内测试这一模型，以破坏它们之间的相互作用。我们相信，我们的研究将促进我们对干细胞动态平衡机制的理解，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(HSCs)负责在成年一生中每天产生数十亿个成熟血细胞，而异常的干细胞稳态机制与包括再生障碍性贫血和白血病在内的各种人类疾病有关。这里提出的这项研究旨在更好地了解内在信号分子LNK如何调控干细胞的扩张、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节机制的理解，还将为各种淋巴瘤和白血病中异常的干细胞信号提供新的见解，并促进干细胞治疗的临床应用。