Background: Recently, the list of intercellular communicators has been extended by extracellular vesicles, whose contents and membrane proteins are involved in signaling or in its modulation. In particular, it is known that exosomes, the most studied form of extracellular vesicles, can contain 20S proteasome subunits. It is also known that extracellular proteasomes exist in a vesicle-free form, and their activity has a physiological significance. In this work we investigated proteasomes and already activated apoptosomes that are secreted by adult human hematopoietic stem cells (aHSCs).
Methods: aHSCs were isolated from human bone marrow, cultivated in Serum-Free Expansion Medium (SFEM), supplemented with Flt3L, SCF, IL-3, IL-6 and TPO. The cultured cells were concentrated by positive selection for CD34+ antigen. The cells were permeabilized and apoptosis was induced with cytochrome C and dATP. Exosomes were purified from culture medium by centrifugation or using a special kit. The concentrated culture medium was fractionated on a gel filtration column. Caspase-3 activity was measured with (Z-DEVD)2-R110 substrate in the presence of bortezomib. Chymotrypsin-like proteasome activities were determined with Suc-LLYV-AMC and different proteasome inhibitors were used for the specific identification.
Results: Attempts to induce apoptosis in aHSCs were unsuccessful. Minor specific caspase activity was found in cell membranes and in exosomes but it was constitutive one and activation of apoptosome assembly by cytochrome C/dATP does not affect this in any way. At the same time, a notable caspase activity was found in SFEM freed from exosomes. Gel filtration of SFEM showed that this activity associated with components of a high molecular weight and is separated by gel filtration together with the proteasomes. It turned out that exosomes also contain proteosomal activity. This suggests that exosomes contain proteasomes and activated caspases. The presence of these active enzymes in the culture medium could be explained by the action of sphingomyelinase or phospholipases, which release them from exosomes.
Conclusion: Content of exosomes from aHSCs includes proteases, such as proteasomes and caspases, which could perform signaling and other functions.
