A Regulatory Network Between Notch and AKT Signaling Pathways Differentially Controls Megakaryocyte Development from Hematopoietic Stem or Committed Progenitor Cells.
Blood(2009)
摘要
Abstract Abstract 384 The Notch signaling pathway is implicated in a broad range of developmental processes, including cell fate decisions. However, the molecular basis for its role at the different steps of stem cell lineage commitment to a specific lineage is unclear. During hematopoiesis, the Notch signaling pathway is known to play an important role in T cell lineage development. Recently, we demonstrated that the Notch signaling pathway is also a positive regulator of megakaryocyte lineage specification from hematopoietic stem cells (HSC). The importance of a tight regulation of this latter role is highlighted by the aberrant activation of the canonical Notch pathway transcription factor RBPJ by OTT-MAL, a fusion oncogene specifically associated with infant acute megakaryoblastic leukemia (AMKL). Here, we report a crosstalk between the Notch and PI3K/AKT pathways that provides new insights into the mechanism through which Notch signaling pathway regulates HSC differentiation into the erythro-megakaryocytic lineages. First, we observed that cells expressing a constitutively active Notch mutant had an increased level of phosphorylation of AKT compared to controls, indicating an association between Notch and AKT pathway activation. Using a Notch-GFP reporter mouse line, we confirmed that phosphorylation of AKT was increased in wild-type bone marrow cells upon physiological Notch stimulation (i.e. GFP+ cells) compared to control cells (i.e. GFP- cells) in vivo. Next, we assessed whether PI3K/AKT activation could replace or mimic Notch signaling during megakaryocyte development by transducing Lineage-Sca-1+cKit+ (LSK) cells or committed common myeloid progenitors (CMP) with a constitutively activated myristoylated AKT (myrAKT) mutant, followed by plating with or without Notch pathway stimulation on OP9-DL1 stroma or OP9 control stroma, respectively. MyrAKT-expressing LSK cells did not efficiently give rise to CD41+ megakaryocytic cells in the absence of Notch pathway stimulation, whereas myrAKT-expressing CMP showed partial rescue of development of megakaryocytes. Conversely, expression of a kinase-dead AKT mutant resulted in a pronounced reduction in megakaryocyte development from CMP, but had only a modest effect on LSK differentiation. Similar results were obtained with a chemical inhibitor of the AKT pathway. These results indicate that PI3K/AKT activation acts as an essential effector of the Notch pathway and can mimic Notch stimulation in CMP, whereas Notch-induced megakaryopoiesis from LSK cells is largely independent of the status of the PI3K/AKT pathway. To investigate the role of PI3K-AKT pathway on megakaryocyte development in vivo, we used FoxO1/3/4-deficient and PTEN-deficient mice, and observed that both mouse lines had significantly increased megakaryopoiesis compared to control animals both in vivo and ex vivo after culture on OP9-DL1 stroma. Importantly, FoxO1/3/4-deficient progenitors had upregulation of Nrarp and Hes1, two Notch pathway targets, and chromatin immunoprecipitation assays revealed the presence of FoxO factors at the Hes1 promoter, indicating a feedback control of the PI3K/AKT pathway on Notch pathway activation. Taken together, these data demonstrate a complex regulatory network between the Notch and PI3K/AKT pathways during megakaryopoiesis. In addition, our results annotate developmental mechanisms in the hematopoietic system that enable a decision to be made either at the hematopoietic stem cell or the committed progenitor level to commit to the megakaryocyte lineage, supporting the existence of two distinct developmental pathways. Disclosures: Gilliland: Merck: Employment.
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