Endoplasmic reticulum stress induced apoptosis and caspase activation is mediated through mitochondria during megakaryocyte differentiation

Abstract

Megakaryocytopoiesis involves the process of the development of hematopoietic stem cells into megakaryocytes (MKs), which are the specialized cells responsible for the production of blood platelets. Platelets are one of the crucial factors for hemostasis and thrombosis. In terminally differentiated MKs, many molecular process such as caspase activation and a massive cytoskeletal rearrangement drive the formation of cytoplasmic extensions called proplatelets. These cytoplasmic extensions packed with granules and organelles are then released from the bone marrow into the blood circulation as platelets. Classically, caspase activation is associated with apoptosis and recent reports suggest their involvement in cell differentiation and maturation. There is no clear evidence about the stimulus for caspase activation during megakaryocyte development. In the current study, we attempted to understand the importance of endoplasmic reticulum stress in the caspase activation during megakaryocyte maturation. We used human megakaryoblstic cell line (Dami cells) as an experimental model. We used PMA (Phorbol 12-myristate 13 acetate) to induce megakaryocytic differentiation to understand the involvement of ER stress and caspase activation during MK maturation. Further, we used Thapsigargin, a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) as a positive control to induce ER stress. We observed larger and adherent cells with the increased expression of megakaryocytic markers (CD41 and CD61) and UPR markers in PMA or Thapsigargin treated cells as compared to control. Also, Thapsigargin treatment induced increased caspase activity and PARP cleavage. The increased expression of megakaryocyte maturation markers alongside with ER stress and caspase activation suggests the importance of ER stress in caspase activation during MK maturation.