Otto K G, Broudy V C, Lin N L, Parganas E, Luthi J N, Drachman J G, Ihle J N, Blau C A
Department of Medicine, Division of Hematology, University of Washington, Seattle WA , USA.
Blood. 2001 Oct 1;98(7):2077-83. doi: 10.1182/blood.v98.7.2077.
Cellular trafficking of growth factor receptors, including cross-talk among receptors at the cell surface, may be important for signal transduction in normal hematopoietic cells. To test this idea, the signaling domain of Mpl (the thrombopoietin receptor) was targeted to the plasma membrane, or to the cytoplasm of murine marrow cells, and the ability of the cells to proliferate and differentiate in response to Mpl dimerized at the plasma membrane or free in the cytoplasm was assessed. Constructs encoding the signaling domain of Mpl linked to an FK506 binding protein domain (to permit dimerization by the membrane-permeable ligand AP20187) with or without a myristylation sequence (to target the receptor to the plasma membrane) and a hemagglutinin epitope tag were generated and introduced into murine marrow cells using a murine stem cell virus (MSCV)-based retroviral vector. Both populations of transduced marrow cells proliferated in Iscoves modified Dulbecco medium-10% FCS-100 nM AP20187 without exogenous growth factors for more than 100 days and achieved greater than a 10(7)-fold expansion of cells by day 50 (n = 4 transductions). Growth was dimerizer dependent, and myeloid, erythroid, and megakaryocytic progenitors were generated. Activation of Mpl either at the plasma membrane or in the cytoplasm allowed for the terminal maturation of transduced progenitor cells. Introduction of membrane-targeted or cytoplasmic Mpl into fetal liver cells from homozygous JAK2 knock-out mice or wild-type littermates demonstrated that both forms of Mpl require JAK2 for signaling. These data show that the activation of Mpl independent of its normal plasma membrane location can support production of the full range of normal hematopoietic progenitor cells in vitro.
生长因子受体的细胞运输，包括细胞表面受体之间的相互作用，对于正常造血细胞中的信号转导可能很重要。为了验证这一观点，将Mpl（血小板生成素受体）的信号结构域靶向鼠骨髓细胞的质膜或细胞质，并评估细胞在质膜上二聚化的Mpl或细胞质中游离的Mpl刺激下增殖和分化的能力。构建了编码与FK506结合蛋白结构域相连的Mpl信号结构域（以允许通过膜通透性配体AP20187进行二聚化）的构建体，该构建体带有或不带有肉豆蔻酰化序列（将受体靶向质膜）以及血凝素表位标签，并使用基于鼠干细胞病毒（MSCV）的逆转录病毒载体将其导入鼠骨髓细胞。在没有外源性生长因子的情况下，两种转导的骨髓细胞群体在Iscove改良的Dulbecco培养基-10%胎牛血清-100 nM AP20187中增殖超过100天，到第50天时细胞扩增超过10^7倍（n = 4次转导）。生长依赖于二聚体形成剂，并且产生了髓系、红系和巨核系祖细胞。在质膜或细胞质中激活Mpl可使转导的祖细胞终末成熟。将靶向膜或细胞质的Mpl引入纯合JAK2基因敲除小鼠或野生型同窝小鼠的胎肝细胞中，结果表明两种形式的Mpl信号传导都需要JAK2。这些数据表明，Mpl的激活不依赖于其正常的质膜定位，在体外能够支持产生全套正常造血祖细胞。
