Both the Endoplasmic Reticulum and the Mitochondria Are Involved in Apoptosis of Erythroid Precursors in Low Grade Myelodysplastic Syndromes.

Cell death by apoptosis accounts for the ineffective erythropoiesis that characterizes low grade myelodysplastic syndromes (LG-MDS). We have shown that the death receptor Fas was over-expressed at the surface of LG-MDS erythroid precursors. In an ex vivo culture of bone-marrow CD34⁺ cells undergoing differentiation into red cells, we observed that apoptosis of LG-MDS erythroid precursors resulted from Fas-mediated activation of caspase-8. Both a Fas-Fc construct and the ectopic expression of a dominant negative mutant of the adaptor FADD (Fas-Associated Death Domain) could prevent the death of MDS erythroid precursors.

In this ex vivo culture system, apoptosis of erythroid precursors was associated with the release of cytochrome c from the mitochondria to the cytosol. To further address the mitochondria involvement in this apoptotic process, we ectopically expressed, by the use of a lentiviral vector, the anti-apoptotic protein Bcl-2 in MDS (n=20) and normal (n=10) CD34⁺ cells before inducing their erythroid differentiation. Bcl-2 expression delayed the erythroid differentiation of MDS as well as normal CD34⁺ cells. It also prevented phosphatidylserine exposure, mitochondrial membrane permeabilization, cytochrome c release, caspase-9 and -3 activation in LG-MDS erythroid precursors. Together with the cleavage of Bid, specifically observed in LG-MDS samples, these observations indicated that the Fas-mediated pathway that led to apoptosis of LG-MDS erythroid precursors was connected to the mitochondrial pathway to death.

We also addressed the role of the endoplasmic reticulum (ER) in this death pathway by using a lentiviral vector encoding an ER-targeted Bcl-2 construct, flag-Bcl2-Cb5. The corresponding protein co-localized with the ER protein calreticulin. ER-targeted Bcl-2 prevented apoptosis induced by thapsigargin, an inhibitor of endoplasmic/sarcoplasmic ATPases (SERCAs), without inhibiting apoptosis induced by lonidamine, a mitochondria targeting agent. Like wild-type Bcl-2, ER-targeted Bcl-2 inhibited phosphatidylserine exposure, mitochondrial membrane permeabilization, cytochrome c release, caspase-9 and caspase-3 activation that characterize spontaneous apoptosis of LG-MDS erythroid precursors in culture. Although ER calcium stores were increased in LG-MDS erythroid precursors, the release of calcium was not modulated by ER-targeted Bcl-2 in these cells. Altogether, our results indicate that, in LG-MDS erythroid precursors, spontaneous apoptosis starts at level of the death receptor Fas, then connects to the mitochondrial pathway that functions downstream of the ER through a calcium-independent and Bcl-2-regulated pathway.