Mad2 (Mitotic Arrest Deficiency 2) Is Essential for the Synergistic Proliferation Induced by Stem Cell Factor Combined with GM-CSF in Hematopoietic Progenitor Cells, Effects Reflecting the Association of c-kit with Mad2.

Mitotic spindle checkpoint protein, Mad2, is required for proper functioning of the mitotic checkpoint which ensures correct chromosome segregation during cell division. Homozygous Mad2 gene deletion is embryonic-lethal. Mad2 interacts with mitosis-associated molecules such as Mad1 and anaphase promoting complex/cyclosome to ensure proper cell cycle progression. Recently, Mad2 was shown to physically associate with the common beta chain of the GM-CSF receptor which raises the possibility that Mad2 may also be involved in cytokine signaling and regulation of mitosis in hematopoietic progenitor cells. To investigate this, we studied hematopoiesis and cytokine signaling in Mad2-haploinsufficient (+/−) mutant mice (M2MT). Colony formation by granulocyte macrophage progenitor cells (CFU-GM) from bone marrow of wild type (WT) mice is synergistically stimulated in vitro by the combination of stem cell factor (SCF) and GM-CSF. We found that bone marrow CFU-GM from M2MT mice are deficient in the synergistic proliferative/colony formation response in vitro to stimulation with the combination of GM-CSF plus SCF. In contrast, there was no difference in stimulation of CFU-GM formation in response to the individual cytokines, GM-CSF or SCF alone, nor a difference in response to pokeweed mitogen mouse spleen cell conditioned medium between M2MT and WT mice. Because there was no difference in the frequency of c-kit+Sca-1+Lin- (KSL) cells nor a difference in the intensity of c-kit surface expression on KSL cells from wild type and M2MT mice, we considered whether the suppression of the SCF/GM-CSF synergy response was due to a difference in intracellular growth-factor receptor signaling pathways. We found that the kinetics of Erk1/2 phosphorylation signaling differ in M2MT Lin- cells compared to WT Lin- cells and that the duration of Erk1/2 phosphorylation in M2MT cells was at least one half of that in WT Lin- cells. On the other hand, we found no difference in the kinetics of Akt phosphorylation between WT and M2MT Lin- cells suggesting a specificity of involvement of the MAP-kinase pathways. To understand how Mad2 plays a role in SCF/GM-CSF synergy, we tested the physical interaction between Mad2 and c-kit in primary Lin- mouse bone marrow cells. Primary Lin- bone marrow cells from WT mice were expanded in liquid culture with SCF and thrombopoietin for 5 days. We found that Mad2 physically associated with c-kit as indicated by co-immunoprecipitation. These results suggest that Mad2 is required for the SCF/GM-CSF proliferative-synergy response in primary Lin- mouse bone marrow cells and that Mad2 is involved in growth-factor signaling pathways, such as the MAP-kinase cascade, in addition to spindle checkpoint function in primary hematopoietic cells. These effects are likely mediated through Mad2 interaction with c-kit and the beta chain of the GM-CSF receptor.