Date Published: April 4, 2019
Publisher: Public Library of Science
Author(s): Jyoti Naik, Chi M. Hau, Lysbeth ten Bloemendaal, Kam S. Mok, Najat Hajji, Ann M. Wehman, Sander Meisner, Vanesa Muncan, Nanne J. Paauw, H. E. de Vries, Rienk Nieuwland, Coen C. Paulusma, Piter J. Bosma, Ramin M. Hakami.
Extracellular vesicles (EVs) released by cells have a role in intercellular communication to regulate a wide range of biological processes. Two types of EVs can be recognized. Exosomes, which are released from multi-vesicular bodies upon fusion with the plasma membrane, and ectosomes, which directly bud from the plasma membrane. How cells regulate the quantity of EV release is largely unknown. One of the initiating events in vesicle biogenesis is the regulated transport of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes. This process is catalyzed by P4-ATPases. The role of these phospholipid transporters in intracellular vesicle transport has been established in lower eukaryotes and is slowly emerging in mammalian cells. In Caenorhabditis elegans (C. elegans), deficiency of the P4-ATPase member TAT-5 resulted in enhanced EV shedding, indicating a role in the regulation of EV release. In this study, we investigated whether the mammalian ortholog of TAT-5, ATP9A, has a similar function in mammalian cells. We show that knockdown of ATP9A expression in human hepatoma cells resulted in a significant increase in EV release that was independent of caspase-3 activation. Pharmacological blocking of exosome release in ATP9A knockdown cells did significantly reduce the total number of EVs. Our data support a role for ATP9A in the regulation of exosome release from human cells.
Extracellular vesicles (EVs) are carriers of a wide range of signaling molecules, including proteins, messenger- and micro-RNAs, that regulate a wide range of (patho)physiological processes, including blood coagulation, angiogenesis, detoxification and immune responses [1–4]. For instance, cancer cells use EVs to dictate their microenvironment to promote their proliferation and survival . In addition, EVs are used by cells to selectively externalize proteins, such as the transferrin receptor during the maturation of reticulocytes . Furthermore, drug transport by extracellular vesicles underlies multidrug resistance in cancer cells and to dispose of active caspase-3 thereby preventing apoptosis [7, 8]. Two classes of EVs (sizes ranging from 50–1000 nm) can be distinguished, i.e. exosomes and ectosomes, which differ in their route of secretion [9, 10]. Exosomes are released by fusion of multivesicular endosomes (MVEs) with the plasma membrane, whereas ectosomes are formed by direct outward budding of the plasma membrane .
Given the mild increase in PS externalization in ATP9A depleted cells, we studied the phospholipid flippase activity of ATP9A. ATP9Aflag was over-expressed in Chinese Hamster Ovary-derived UPS-1 cells, which are knockout cells for the hamster P4-ATPase ATP11C . Similar to HepG2 cells, surface biotinylation of UPS-ATP9Aflag cells showed that 10–15% of the ATP9Aflag pool localized to the plasma membrane in hamster cells (Fig 7A). Internalization of NBD-labeled phospholipid analogs NBD-PS and NBD-PE was studied in UPS-ATP9Aflag cells (Fig 7B–7D). ATP11Cflag over-expressing UPS-1 cells were included as a positive control for NBD-PS and NBD-PE internalization, as ATP11C is known to have flippase activity towards these two NBD-labeled phospholipids . As expected, the internalization of NBD-PS and NBD-PE was significantly and time-dependently increased in ATP11C-overexpressing cells compared to control cells; however, ATP9Aflag cells showed no enhanced uptake of these phospholipid analogs (Fig 7B and 7C). These data indicate that ATP9Aflag does not show detectable flippase activity towards the tested NBD-labeled phospholipids.
Here we show that the P4-ATPase ATP9A plays an important role in the release of EVs from human cells. Depletion of ATP9A from cell lines of different origin, including HepG2, MCF-7 and THP-1, resulted in enhanced EV release, suggesting an universal role of ATP9A in this process. EVs play crucial roles in intercellular communication by delivering proteins, DNA, miRNA and mRNAs that regulate a wide range of processes, including blood clotting, angiogenesis, detoxification and immune responses. This requires tight regulation of EV release, however, the mechanisms hereof are poorly understood. Our data suggest that ATP9A inhibits the secretion of exosomes, as inhibition of neutral sphingomyelinase with GW4869 resulted in a normalization of EVs release in ATP9A-depleted HepG2 cells.