Caspase-3 Has Apoptotic and Non-Apoptotic Functions in Regulating the Fate of Hematopoietic Stem Cells.

The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Much emphasis has been placed on differentiation and proliferation in hematopoiesis, but little is known about control of the stem cell pool by mediators of apoptosis. We are particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitor, p21Cip1. p21 is known to regulate hematopoietic stem cell (HSC) cycling and we demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse HSC. Using mice engineered to be deficient in Caspase-3, we observed no difference from wild-type in blood counts. However, knock-out animals had reduced bone marrow cellularity and an increase in the stem cell enriched lin-kit+Sca-1+ CD34 low (LKS-CD34^lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions were minimally different from controls. However, under conditions of stress (gamma irradiation, heat shock or cytokine withdrawal), Caspase-3 deficient LKS cells demonstrated reduced sensitivity to apoptotic cell death as measured by TUNEL or Annexin-V assay. Primitive hematopoietic cells from these animals in vivo also had reduced BrdU incorporation reflecting decreased proliferation and, upon transplantation, demonstrated marked differentiation abnormalities. Caspase-3−/− marrow cells had significantly reduced ability to differentiate into lymphoid and myeloid lineages 12 weeks after transplantation. Similarly, WT bone marrow cells treated prior to transplantation with a selective Caspase-3 inhibitor, z-DEVD.fmk, displayed a significant disadvantage in peripheral blood reconstitution, compared with a mock control. These data indicate that Caspase-3 is involved in the regulation of apoptosis, differentiation and proliferation of HSC. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.