Ito Shigeki, Mantel Charlie R, Han Myung-Kwan, Basu Sunanda, Fukuda Seiji, Cooper Scott, Broxmeyer Hal E
Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis 46202, USA.
Blood. 2007 Mar 1;109(5):1923-30. doi: 10.1182/blood-2006-06-030841. Epub 2006 Oct 12.
Mitotic arrest deficiency 2 (Mad2) is a component of mitotic spindle checkpoint proteins and is essential for accurate chromosome segregation. We investigated a role for Mad2 in hematopoiesis using Mad2-haploinsufficient (Mad2+/-) mice. Mad2+/- bone marrow (BM) and spleen manifested decreased absolute numbers and cycling status of immature, but not mature, hematopoietic progenitor cells. Mad2+/- BM granulocyte-macrophage colony-forming units (CFU-GMs) did not manifest synergistic proliferation in response to stem cell factor (SCF) plus GM-CSF. The percentage of annexin V+ cells was higher in Mad2+/- than Mad2+/+c-Kit+lin- BM after culture with SCF and GM-CSF. However, no significant difference in phosphorylation of extracellular signal-related kinase (Erk1/2) at Thr202/Tyr204 and Akt at Ser473 between Mad2+/- and Mad2+/+BM c-Kit+lin- cells was observed. Immunoprecipitation assays performed in human MO7e cells demonstrated physical association of c-Kit with Mad2. Moreover, stimulation with SCF plus GM-CSF led to dissociation of Mad2 from c-Kit. Confocal microscopy demonstrated that Mad2 colocalized with c-Kit in the cytoplasm of MO7e cells. These results suggest that Mad2 is involved in synergistic growth of immature hematopoietic progenitor cells in response to SCF plus GM-CSF, effects that may be mediated via physical association of Mad2 with c-Kit.
有丝分裂阻滞缺陷蛋白2（Mad2）是有丝分裂纺锤体检查点蛋白的一个组成部分，对准确的染色体分离至关重要。我们使用Mad2单倍体不足（Mad2+/-）小鼠研究了Mad2在造血过程中的作用。Mad2+/-骨髓（BM）和脾脏中未成熟造血祖细胞的绝对数量和增殖状态降低，但成熟造血祖细胞无此现象。Mad2+/- BM粒-巨噬细胞集落形成单位（CFU-GM）对干细胞因子（SCF）加粒细胞-巨噬细胞集落刺激因子（GM-CSF）无协同增殖反应。用SCF和GM-CSF培养后，Mad2+/-的膜联蛋白V+细胞百分比高于Mad2+/+ c-Kit+lin- BM。然而，未观察到Mad2+/-和Mad2+/+ BM c-Kit+lin-细胞在苏氨酸202/酪氨酸204处的细胞外信号调节激酶（Erk1/2）和丝氨酸473处的Akt磷酸化有显著差异。在人MO7e细胞中进行的免疫沉淀试验表明c-Kit与Mad2存在物理关联。此外，用SCF加GM-CSF刺激导致Mad2与c-Kit解离。共聚焦显微镜显示Mad2与c-Kit在MO7e细胞的细胞质中共定位。这些结果表明，Mad2参与了未成熟造血祖细胞对SCF加GM-CSF的协同生长，其作用可能是通过Mad2与c-Kit的物理关联介导的。
