REGULATION OF HEMATOPOIESIS BY THROMBOPOIETIN

本项目旨在了解细胞因子对巨核细胞和血小板产生的控制，长期目标是制定更好的临床策略来管理与癌症治疗和其他疾病相关的血小板减少症。最近的克隆TPO提供了一种具有刺激巨核细胞和血小板生成能力的分子。缺乏TPO或其受体c-Mpl的小鼠的血小板减少证明TPO是巨核细胞生成的主要生理调节剂，但有足够的TPO独立产生以维持止血。确定细胞因子对TPO独立过程的控制以及TPO与其他调节因子相互作用的机制是确定TPO和其他巨核细胞生成药物最有效临床应用的关键步骤。目前的建议旨在确定巨核细胞生成的已知调节因子的作用：IL-3、IL-6、IL-11、GM-CSF、LIF和SCF，在mpl缺陷小鼠的剩余稳态以及紧急巨核细胞和血小板生成中，尽管分析了缺乏这些调节因子或其受体的复合突变小鼠。初步结果表明，TPO信号在造血干细胞区室的调控中也起着关键作用。如果要将TPO用于患者，特别是白血病患者，就必须充分了解其对干细胞的作用。通过细胞分选策略分离的特定干细胞群体分析，在长期造血重建分析中将用于精确定义mpl缺陷小鼠和缺乏c-Mpl和其他干细胞调节因子（IL-6， LIF, Il-11， SCF, G-CSF）的复合突变体的干细胞缺乏症。从表达c-Mpl受体的正常小鼠中分离干细胞，并在c-Mpl位点的控制下产生表达绿色荧光蛋白的敲入小鼠，对其进行功能表征。对来自c-Mpl受体不同区域的信号所刺激的特定生物反应的理解，可能最终使TPO反应的治疗性操作的细胞内靶标的鉴定成为可能。将c-Mpl细胞质结构域突变纳入小鼠种系将用于确定TPO受体的不同细胞质区域是否参与体外增殖和分化，也介导这些反应在巨核细胞生成和干细胞的生理调节中。

DESCRIPTION: (Adapted from Applicant's Abstract) This project aims to understand the cytokine control of megakaryocytes and platelet production, with the long-term goal of devising better clinical strategies for the management of thrombocytopenias associated with cancer treatment and other disorders. The recent cloning of TPO has provided a molecule with potent capacity to stimulate megakaryocyte and platelet generation. The thrombocytopenia in mice lacking TPO or its receptor c-Mpl is evidence that TPO is the dominant physiologic regulator of megakaryocytopoiesis, but that sufficient TPO-independent production occurs to maintain hemostasis. Defining the cytokine control of the TPO-independent processes and the mechanisms by which TPO interacts with other regulators is a key step in determining the most effective clinical application of TPO and other megakaryocytopoietic agents. The current proposal aims to define the roles of known regulators of megakaryocytopoiesis: IL-3, IL-6, IL-11, GM-CSF, LIF, and SCF, in residual steady-state as well as emergency megakaryocyte and platelet production in Mpl-deficient mice, although analysis of compound mutant mice lacking these regulators or their receptors in addition of c-Mpl. Preliminary results suggest that TPO signaling also plays a critical role in regulation of the hemopoietic stem cell compartment. If TPO is to be administered to patients, particularly those with leukemia, its actions on stem cells must be fully understood. Analysis of specific stem cell populations, isolated by cell sorting strategies, in long term hemopoietic reconstitution assays will be used to precisely define the stem cell deficiency in Mpl-deficient mice and in compound mutants lacking c-Mpl and other stem cell regulators (IL-6, LIF, Il-11, SCF, G-CSF). Stem cells from normal mice that express the c-Mpl receptor will also be isolated and functionally characterized by generating knock-in mice expressing green fluorescent protein under the control of the c-mpl locus. An understanding of the specific biological responses stimulated by signals from different regions of the c-Mpl receptor may ultimately allow identification of intracellular targets for therapeutic manipulations of the TPO responses. Incorporation of c-Mpl cytoplasmic domain mutations into the mouse germline will be used to determine if the distinct cytoplasmic regions of the TPO receptor implicated in proliferation and differentiation in vitro, also mediate these responses in the physiological regulation of megakaryocytopoiesis and stem cells.