Regulating Hematopoietic Stem Cell Homeostasis through GSK-3

描述(申请人提供)：造血干细胞(HSC)维持自我更新和谱系承诺之间的平衡，这一调节的动态平衡机制还不是很清楚。糖原合成酶激酶-3(GSK-3)在造血过程中负向调节多种信号通路，包括Wnt信号通路和细胞因子，如血小板生成素(TPO)和红细胞生成素，通过JAK2信号通路激活STAT5、Akt和MAP激酶通路。我们发现GSK-3在控制HSC自我更新和分化之间的平衡中起着关键作用。抑制骨髓中的GSK3以ss-连环蛋白依赖的方式瞬时扩增表型HSCs和造血祖细胞(HPC)，这与Wnt信号转导的作用一致。然而，在长期的检测中，抑制GSK3通过激活mTOR逐渐耗尽HSCs。长期的HSC耗竭可以通过抑制mTOR来防止，而通过ss-catenin基因敲除来加剧。因此，GSK3同时调节HSC中的Wnt和mTOR信号，而对HSC的自我更新有相反的作用，我们发现联合抑制mTOR和GSK3可以导致HSC在体内持续的扩张。此外，通过联合GSK-3和mTOR抑制剂，我们能够在无细胞因子的培养液中培养来自小鼠或人类的功能性HSCs，然后在移植受者体内重建长期、多系的造血，为体外造血学研究提供了系统，并为扩大人类HSCs用于临床应用提供了一种潜在的方法。这个项目的总体目标是研究Wnt、mTOR和TPO/MPL/JAK2信号在GSK-3抑制反应中的各自作用，并确定GSK-3在造血系统中调节这些通路的作用。具体目标1将使用条件性基因敲除严格测试Gsk3a和Gsk3b在骨髓细胞中的独立和重叠功能。目的2探讨GSK-3下游的Wnt和mTOR通路在HSCs中的作用，并在体外条件下鉴定对GSK-3和mTOR抑制剂有反应的细胞群体。AIM 3将研究调节GSK-3和mTOR的上游通路，包括通过JAK2和Akt依赖的通路的细胞因子信号，并将具体区分Wnt和Akt信号通过GSK-3的各自作用。这些研究将确定GSK-3在正常造血中的功能(S)，并可能导致开发新的治疗方法来扩大造血障碍中的造血干细胞。

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSC) maintain a balance between self-renewal and lineage commitment, and the homeostatic mechanisms of this regulation are not well understood. Glycogen synthase kinase-3 (GSK-3) negatively regulates multiple signaling pathways in hematopoiesis, including Wnt signaling and cytokines such as Thrombopoietin (Tpo) and Erythropoietin, which signal through JAK2 to activate STAT5, Akt, and MAP kinase pathways. We have found GSK-3 plays a pivotal role in controlling the balance between HSC self-renewal and differentiation. Inhibition of Gsk3 in bone marrow transiently expands phenotypic HSCs and hematopoietic progenitor cells (HPCs) in a ss-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term assays, Gsk3 inhibition progressively depletes HSCs through activation of mTOR. Long-term HSC depletion is prevented by mTOR inhibition and exacerbated by ss-catenin knockout. Thus Gsk3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal, and we find that combined inhibition of mTOR and Gsk3 results in sustained HSC expansion in vivo. Furthermore, by combining GSK-3 and mTOR inhibitors, we are able to culture functional HSCs from mice or humans in cytokine- free medium and then reconstitute long-term, multilineage hematopoiesis in transplant recipients, providing a system for the ex vivo study of hematopoiesis and a potential method to expand human HSCs for clinical applications. The overall goal of this project is to examine the respective contributions of Wnt, mTOR, and Tpo/Mpl/JAK2 signaling in the response to GSK-3 inhibition and to define the role of GSK-3 in regulating these pathways within the hematopoietic system. Specific aim 1 will rigorously test both the independent and overlapping functions of Gsk3a and Gsk3b in bone marrow cells using conditional gene knockouts. Aim 2 will explore the role of Wnt and mTOR pathways downstream of GSK-3 in HSCs, and will identify the cell population that responds to GSK-3 and mTOR inhibitors under ex vivo conditions. Aim 3 will investigate the upstream pathways that regulate GSK-3 and mTOR, including cytokine signaling through JAK2 and Akt-dependent pathways, and will specifically distinguish the respective contributions of Wnt and Akt signaling through GSK-3. These studies will define the function(s) of GSK-3 in normal hematopoiesis and may lead to the development of novel therapeutic approaches to expand HSCs in hematopoietic disorders.