Regulating Hematopoietic Stem Cell Homeostasis through GSK-3

描述（由申请人提供）：造血干细胞（HSC）在自我更新和谱系定型之间保持平衡，这种调节的稳态机制尚不清楚。 糖原合成酶激酶-3（GSK-3）负调节造血中的多种信号传导途径，包括Wnt信号传导和细胞因子如血小板生成素（Tpo）和促红细胞生成素，其通过JAK 2信号传导以激活STAT 5、Akt和MAP激酶途径。 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Gsk 3的抑制以β-连环蛋白依赖性方式瞬时扩增表型HSC和造血祖细胞（HPC），这与Wnt信号传导的作用一致。然而，在长期试验中，Gsk 3抑制通过mTOR的激活逐渐耗尽HSC。mTOR抑制可防止长期HSC耗竭，β-连环蛋白敲除可加重HSC耗竭。因此，Gsk 3调节HSC中的Wnt和mTOR信号传导，对HSC自我更新具有相反的作用，并且我们发现mTOR和Gsk 3的组合抑制导致体内持续的HSC扩增。此外，通过组合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HSC，然后在移植受体中重建长期、多谱系造血，从而提供用于造血的离体研究的系统和扩增人HSC用于临床应用的潜在方法。该项目的总体目标是检查Wnt、mTOR和Tpo/Mpl/JAK 2信号传导在响应GSK-3抑制中的各自贡献，并确定GSK-3在造血系统内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骨髓细胞中Gsk 3a和Gsk 3b的独立和重叠功能。目的2将探索GSK-3下游Wnt和mTOR通路在HSC中的作用，并将鉴定在离体条件下对GSK-3和mTOR抑制剂有反应的细胞群。目的3将研究调控GSK-3和mTOR的上游途径，包括通过JAK 2和Akt依赖性途径的细胞因子信号传导，并将具体区分Wnt和Akt信号传导通过GSK-3的各自贡献。这些研究将确定GSK-3在正常造血中的功能，并可能导致开发新的治疗方法来扩增造血疾病中的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.