Regulating Hematopoietic Stem Cell Homeostasis through GSK-3

描述（由申请人提供）：造血干细胞（HSC）在自我更新和谱系承诺之间保持平衡，这种调节的稳态机制尚不清楚。糖原合成酶激酶3 （GSK-3）负调控造血过程中的多种信号通路，包括Wnt信号通路和通过JAK2信号激活STAT5、Akt和MAP激酶通路的血小板生成素（Thrombopoietin, Tpo）和红细胞生成素（Erythropoietin）等细胞因子。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。骨髓中Gsk3的抑制以依赖于ss-catenin的方式瞬时扩增表型hsc和造血祖细胞（HPCs），这与Wnt信号传导的作用一致。然而，在长期实验中，Gsk3抑制通过激活mTOR逐渐消耗hsc。mTOR抑制可阻止长期的HSC消耗，而ss-catenin敲除则会加重。因此，Gsk3同时调节HSC中的Wnt和mTOR信号，对HSC自我更新具有相反的作用，我们发现mTOR和Gsk3的联合抑制导致体内持续的HSC扩增。此外，通过结合GSK-3和mTOR抑制剂，我们能够在无细胞因子的培养基中培养来自小鼠或人的功能性造血干细胞，然后在移植受体中重建长期的多系造血，为体外造血研究提供了一个系统，并为临床应用扩展人类造血干细胞提供了一种潜在的方法。该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途径中的作用。Specific aim 1将使用条件基因敲除技术严格测试Gsk3a和Gsk3b在骨髓细胞中的独立和重叠功能。Aim 2将探索hsc中GSK-3下游的Wnt和mTOR通路的作用，并将确定在离体条件下对GSK-3和mTOR抑制剂有反应的细胞群。Aim 3将研究调节GSK-3和mTOR的上游通路，包括通过JAK2和Akt依赖的细胞因子信号通路，并将具体区分Wnt和Akt通过GSK-3信号通路各自的贡献。这些研究将确定GSK-3在正常造血中的功能，并可能导致新的治疗方法的发展，以扩大造血障碍中的hsc。

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.