Signaling pathways regulated by GSK3 in hematopoietic stem cell

造血干细胞（hematopoietic stem cells, hsc）具有两个不同的特征：自我更新的能力和向成熟血细胞的分化能力。调控HSC自我更新和分化的信号通路尚不清楚。糖原合成酶激酶3 （GSK3）是典型Wnt信号的负调节因子，也是造血生长因子信号通路的下游靶点，通过Jak/Stat和PI3激酶/Akt依赖通路发出信号，参与调节HSC活性。典型的Wnt信号与HSC自我更新有关，但该途径的作用仍存在争议。最近，PTEN-PI3K-mTOR通路已被证明在HSC维持中发挥重要作用。我们的假设是，GSK3是HSC中Wnt和PI3K下游的关键效应物，调节HSC群体。为了支持这一假设，我们的初步数据显示：1)锂和其他选择性GSK3抑制剂显著增加小鼠HSC的数量，2)RNAi介导的小鼠HSC中GSK3的缺失最初扩大了骨髓移植中小鼠HSC的数量，但长期抑制GSK3会耗尽HSC。3)抑制GSK3对hsc的扩增是通过2-catenin介导的，而hsc的消耗主要依赖于mTOR信号传导。这些观察结果支持了GSK3是造血重要调节因子的假设。在本提案中，我们将扩展我们的研究，探索GSK3磷酸化在造血干细胞中的功能作用。我们还将通过新构建的条件敲除小鼠造血细胞来研究Gsk3a与Gsk3b的异构体特异性功能。GSK3抑制对造血干细胞的非细胞自主作用也将通过在成骨细胞壁龛中条件敲除GSK3来探索。此外，我们将测试调节GSK3和mTOR的活性是否可以在离体培养中扩增hsc。将进行功能性筛选，以确定体外培养中造血干细胞自我更新的新调节因子。该项目的总体目标是研究Wnt和PTEN-PI3K-mTOR信号在GSK3抑制反应中的各自作用，并确定GSK3在造血系统中调节这些途径中的作用。从这些研究中获得的知识将为HSC自我更新和分化的调节机制提供新的见解，并可能改善造血疾病的治疗。1

Signaling pathways regulated by GSK3 in hematopoietic stem cells Abstract Hematopoietic stem cells (HSCs) possess two distinct features: the ability to self-renew and to differentiate into mature blood cells. The signaling pathways that regulate HSC self-renewal and differentiation are not well understood. Glycogen synthase kinase-3 (GSK3), a negative regulator of canonical Wnt signaling and a downstream target of hematopoietic growth factor signaling pathways that signal through Jak/Stat and PI3 kinase/Akt dependent pathways, has been implicated in the regulation of HSC activity. Canonical Wnt signaling has been implicated in HSC self-renewal, but the role of this pathway remains controversial. Recently, the PTEN-PI3K-mTOR pathway has been demonstrated to play an essential role in HSC maintenance. Our hypothesis is that GSK3 is a critical effector downstream of Wnt and PI3K within HSCs that regulates the HSC population. In support of this hypothesis, our preliminary data show that: 1) lithium and other selective GSK3 inhibitors significantly increase the number of HSCs in mice, 2) RNAi mediated depletion of Gsk3 in murine HSCs initially expands the HSC population in bone marrow transplants, but long-term inhibition of GSK3 exhausts HSC. 3) The expansion of HSCs by inhibition of GSK3 is mediated through 2-catenin while the depletion of HSCs is largely dependent on mTOR signaling. These observations support the hypothesis that GSK3 is an important regulator of hematopoiesis. In this proposal, we will extend our study to explore the functional role of GSK3 phosphorylation in HSCs. We will also address the isoform specific functions of Gsk3a vs. Gsk3b in hematopoietic cells with newly constructed conditional knockouts in mice. The non cell- autonomous effect of GSK3 inhibition on HSCs will also be explored with the conditional knockout of Gsk3 in the osteoblast niche. Furthermore, we will test whether modulating the activity of GSK3 and mTOR can expand HSCs in ex vivo culture. A functional screen to identify new regulators of HSCs self-renewal in ex vivo culture will be carried out. The overall goal of this project is to examine the respective contributions of Wnt and PTEN-PI3K-mTOR signaling in the response to GSK3 inhibition and to define the role of GSK3 in regulating these pathways within the hematopoietic system. The knowledge learned from these studies will provide new insights into the mechanisms of the regulation of HSC self-renewal and differentiation and may improve the therapy of hematopoietic disorders. 1