Caspase Activated Rock-1 Allows Erythroblasts Enucleation Independently of Cytokine Signaling

Abstract

Stem Cell Factor (SCF) and Erythopoietin (Epo) are strictly required for preventing apoptosis and stimulating proliferation, allowing differentiation of erythroid precursors from CFU-E until the polychromatophilic stage. In contrast, terminal maturation generating erythrocytes occurs independently of cytokine signaling by a mechanism poorly understood. Terminal differentiation is characterized by a sequence of morphological changes including a progressive decrease in cell size and nucleus with chromatin condensation and disappearance of organelles that require transient caspase activation. These events are followed by nuclei extrusion as a consequence of plasma membrane and cytoskeleton reorganization. Two serine/threonine protein kinases, Rock-1 and Rock-2, the main Rho effectors, are key regulators of the actomyosin-based contractility. To determine the role of the Rho/Rock pathway in the terminal stage of erythropoiesis, we used an in vitro model of erythroid differentiation based on human hematopoietic cells derived from cord blood. We showed that in early step, SCF stimulates Rho/Rock pathway that is involved in erythroblasts proliferation and cytokinesis until the basophilic stage. Thereafter, Rock-I is activated independently of Rho signaling by caspase-3-mediated cleavage. To investigate the role of Rock during erythroid terminal differentiation, we used a Rock inhibitor (Y-27632). At day 8 of the CD36+ control culture, 55% of cells exhibited a mature phenotype (30% polychromatic, and 25% orthochromatic erythroblasts). By contrast, in the Y-27632 treated culture, there were no more than 23% mature cells (22% polychromatic and 1% orthochromatic erythroblasts) and 47% exhibited apoptotic features. Most of these apoptotic cells exhibited mature morphology suggesting that apoptosis occurred during the late step of maturation at the time of caspase activation. The percentage of enucleated cells was low in these culture conditions, thus, to determine whether or not Rock was also involved in the enucleation process, we used a coculture on MS5 stromal layer system (Giarratana, Nat Biotechnol 2005). At day 18, the rate of enucleated cells was 30% in the control culture whereas it was two-fold decreased in the Y-27632 treated culture and many of mature cells were instead dying by apoptosis. This finding suggested that Rock inhibition could be also involved in the enucleation process. By using shRNA technique, we demonstrated that Rock-1 is involved in terminal maturation and enucleation through phosphorylation of the light chain of Myosin II (MLC2). Therefore, in this differentiation system, caspase activation allows final maturation independently of cytokine signaling through kinase cleavage and activation.