Mitotic Arrest Deficiency 2 (Mad2) Is Required for Proliferative Synergy of GM-CSF Plus SCF in Hematopoietic Progenitors Via Effects on Apoptosis and Is Mediated by Ligand-Dependent Association of Mad2 with SCF and GM-CSF Receptors.

Mitotic arrest deficiency 2 (Mad2) is a component of the mitotic spindle checkpoint complex and is essential for accurate chromosome segregation. We previously reported that Mad2 is involved in synergistic proliferation of hematopoietic progenitor cells in response to SCF combined with GM-CSF, but the underlying mechanism remains unclear. Recently, it was reported that Mad2 physically associates with common β chain (βc) of GM-CSF receptor. Because c-Kit constitutively binds to βc, we hypothesized that Mad2 protein physically associates with c-Kit receptor. We examined if Mad2 interacts with c-Kit using human growth factor-dependent cell line, MO7e, which expresses both c-Kit and GM-CSF receptors. Immunoprecipitation assays demonstrated interaction of c-Kit with Mad2. We then examined whether the interaction of Mad2 with c-Kit is cytokine dependent. Mad2 dissociates from c-Kit after stimulation with SCF plus GM-CSF, but not with SCF or GM-CSF alone. Additionally, because bone marrow hematopoietic progenitor cells from Mad2^+/− mice lack synergy in response to SCF plus GM-CSF, it is possible that interaction of Mad2 with c-Kit mediates their synergistic proliferative effects. To address this, we examined intracellular cytokine signaling and apoptosis in MO7e cells depleted of Mad2 by RNA interference. We observed no difference between control and Mad2-depleted cells in phosphorylation of Erk1/2 at Thr202/Tyr204 and Akt at Ser473 after synergistic stimulation with SCF plus GM-CSF. In contrast, the percent of apoptosis of Mad2-depleted cells was significantly higher than that of control (26.2±0.7% vs. 20.3±0.4%, P<0.01). This was compatible with the results of apoptosis assays using c-Kit⁺Lineage⁻ bone marrow cells from Mad2^+/− and wild type mice. These results suggest that Mad2 may be involved in synergistic proliferation of hematopoietic progenitor cells via regulation of apoptosis rather than early cytokine signaling. These effects are likely mediated through Mad2 interaction with c-Kit and the beta chain of the GM-CSF receptor.