Submitted June 22, 2006
Accepted October 5, 2006
Mad2 is required for optimal hematopoiesis: Mad2 associates with c-Kit in MO7e cells
Shigeki Ito, Charlie R Mantel, Myung-Kwan Han, Sunanda Basu, Seiji Fukuda, Scott Cooper, and Hal E. Broxmeyer*
Department of Microbiology & Immunology, Indiana University School of Medicine, Indianapolis, IN
Department of Microbiology & Immunology, Indiana University, School of Medicine, Indianapolis, IN
* Corresponding author; email: hbroxmey{at}iupui.edu.
Mitotic arrest deficiency 2 (Mad2) is a component of mitotic spindle checkpoint proteins and 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 CFU-GM did not manifest synergistic proliferation in response to SCF plus GM-CSF. The percent 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.
