Fas-Dependent Apoptosis in Early MDS Erythroid Precursors Involves Endoplasmic Reticulum.

The anemia that characterizes most early myelodysplastic syndromes (MDS) was proposed to involve a deregulation in cell death pathways leading to excessive apoptosis of bone marrow erythroid precursors. Pathways leading to this excess in MDS erythroid precursors have been partially depicted in ex vivo liquid cultures of patients CD34⁺ bone marrow cells induced to differentiate into red cells in the presence of various cytokines. For example, we have identified the Fas-dependent activation of caspase-8 as a key initiating event. In order to further understand the mechanisms of MDS erythroid precursor death, we explored the role of the endoplasmic reticulum (ER) in this process. We first observed that Fas-dependent activation of caspase-8 in these cells induced the cleavage of BAP-31, an ER protein that is associated to Bcl-2 at the ER surface and was demonstrated to be a caspase-8 substrate. We also detected a proteolysis of caspase-4, which was proposed to play a role in ER-mediated apoptosis. To further explore the role of the ER, we constructed a lentivirus expressing a Bcl-2 mutant targeted to the ER membrane. The specific expression of Bcl-2 at the ER level prevented BAP-31 and caspase-4 cleavage induced by Fas engagement at the surface of MDS erythroid precursors and inhibited Fas-dependent apoptosis. Interestingly, ER-targeted Bcl-2 also inhibited mitochondrial membrane permeabilization (MMP) and cytochrome c release in MDS erythroid precursors undergoing spontaneous or Fas-induced apoptosis. These data argued for a role of the ER in MDS erythroid precursor apoptosis, upstream of the mitochondria. MDS erythroid precursors also demonstrated elevated ER Ca²⁺ stores when compared to normal erythroid precursors cultured in the same conditions. Ca²⁺ chelation with BAPTA or treatment with pharmacologic Ca²⁺ inhibitors such as nicardipine prevented the spontaneous apoptosis of MDS erythroid precursors. Altogether, these data suggest that the ER is involved in the spontaneous apoptosis of MDS erythroid precursors, downstream of Fas and upstream of the mitochondria, through mechanisms that can be inhibited by Bcl-2 and that involve Ca²⁺ stores.