Signaling pathways regulated by GSK3 in hematopoietic stem cell

造血干细胞中GSK 3调控的信号通路摘要造血干细胞具有自我更新和分化为成熟血细胞的能力。调控HSC自我更新和分化的信号通路还不清楚。糖原合成酶激酶-3（GSK 3）是经典Wnt信号传导的负调节剂，并且是通过Jak/Stat和PI 3激酶/Akt依赖性途径进行信号传导的造血生长因子信号传导途径的下游靶标，其已经涉及HSC活性的调节。经典的Wnt信号通路与HSC自我更新有关，但该通路的作用仍存在争议。最近，PTEN-PI 3 K-mTOR通路已被证明在HSC维持中起重要作用。我们的假设是GSK 3是HSC内Wnt和PI 3 K下游的关键效应子，其调节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GSK 3抑制剂显著增加小鼠中HSC的数量，2）RNAi介导的鼠HSC中GSK 3的消耗最初在骨髓移植中扩增HSC群体，但GSK 3的长期抑制耗尽HSC。3)通过抑制GSK 3的HSC扩增是通过2-连环蛋白介导的，而HSC的消耗在很大程度上依赖于mTOR信号传导。这些观察结果支持GSK 3是造血的重要调节剂的假设。在这个建议中，我们将扩展我们的研究，以探索GSK 3磷酸化在HSC中的功能作用。我们还将在小鼠中使用新构建的条件性敲除来解决Gsk 3a与Gsk 3b在造血细胞中的同种型特异性功能。GSK 3抑制对HSC的非细胞自主作用也将在成骨细胞龛中用GSK 3的条件性敲除来探索。此外，我们将测试调节GSK 3和mTOR的活性是否可以在离体培养中扩增HSC。将进行功能筛选以鉴定在离体培养中HSC自我更新的新调节剂。该项目的总体目标是检查Wnt和PTEN-PI 3 K-mTOR信号传导在对GSK 3抑制的响应中的各自贡献，并确定GSK 3在造血系统内调节这些通路中的作用。从这些研究中获得的知识将为HSC自我更新和分化的调控机制提供新的见解，并可能改善造血系统疾病的治疗。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