Role of apoptotic and anti-apoptotic mechanisms in erythroblast differentiation

The present study focuses on deciphering the differentiation mechanisms of erythroid cells. In particular, the involvement of apoptotic mechanisms in erythroblast differentiation and enucleation was examined. A model system of erythroid differentiation was established. The primary culture of human erythroid cells fully recapitulated in vivo erythropoiesis as shown by cell morphology and cell surface markers. In addition this culture system was amenable for manipulation. Using this culture system, caspase activation was found to be associated with and required for erythroblast differentiation. In vitro erythropoiesis was blocked by the pan-caspase inhibitor zVAD.fmk as indicated by the accumulation of early progenitors with concomitant drop in the number of late erythroblasts. We also found that caspase-3 and caspase-9 were specifically activated during erythroid differentiation. DNA fragmentation has emerged as a second apoptotic mechanism involved in erythropoesis. DNA degradation occurs at late stages of differentiation as assessed by the detection of TUNELpositive erythroblasts in cord blood, bone marrow and erythroid culture. The mechanism of DNA degradation was cell-autonomous and resulted in production of high molecular DNA fragments with approximate length of about 50 kb. During these studies we made the rather unexpected observation that late stage erythroblast did not undergo apoptosis when treated with apoptosis inducers. This observation suggested to us that mature erythroblasts were therefore protected from cell death. We next examined the mechanisms involved in erythroblast protection. The levels of cytochrome c, a potent inducer engaging the mitochondrial pathway in apoptosis, were shown to significantly decrease with the differentiation. In addition, the levels of anti-apoptotic protein Bcl-xL increased, reaching maxima just before enucleation. Altogether these mechanisms might contribute to the protection from apoptosis in mature erythroblasts, described here for the first time.