Title: Exosome Release Is Regulated by a Calcium-dependent Mechanism in K562 Cells
Abstract: Multivesicular bodies (MVBs) are endocytic structures that contain small vesicles formed by the budding of an endosomal membrane into the lumen of the compartment. Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles termed exosomes. K562 cells are a hematopoietic cell line that releases exosomes. The application of monensin (MON) generated large MVBs that were labeled with a fluorescent lipid. Exosome release was markedly enhanced by MON treatment, a Na+/H+ exchanger that induces changes in intracellular calcium (Ca2+). To explore the possibility that the effect of MON on exosome release was caused via an increase in Ca2+, we have used a calcium ionophore and a chelator of intracellular Ca2+. Our results indicate that increasing intracellular Ca2+ stimulates exosome secretion. Furthermore, MON-stimulated exosome release was completely eliminated by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), implying a requirement for Ca2+ in this process. We have observed that the large MVBs generated in the presence of MON accumulated Ca2+ as determined by labeling with Fluo3-AM, suggesting that intralumenal Ca2+ might play a critical role in the secretory process. Interestingly, our results indicate that transferrin (Tf) stimulated exosome release in a Ca2+-dependent manner, suggesting that Tf might be a physiological stimulus for exosome release in K562 cells. Multivesicular bodies (MVBs) are endocytic structures that contain small vesicles formed by the budding of an endosomal membrane into the lumen of the compartment. Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles termed exosomes. K562 cells are a hematopoietic cell line that releases exosomes. The application of monensin (MON) generated large MVBs that were labeled with a fluorescent lipid. Exosome release was markedly enhanced by MON treatment, a Na+/H+ exchanger that induces changes in intracellular calcium (Ca2+). To explore the possibility that the effect of MON on exosome release was caused via an increase in Ca2+, we have used a calcium ionophore and a chelator of intracellular Ca2+. Our results indicate that increasing intracellular Ca2+ stimulates exosome secretion. Furthermore, MON-stimulated exosome release was completely eliminated by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), implying a requirement for Ca2+ in this process. We have observed that the large MVBs generated in the presence of MON accumulated Ca2+ as determined by labeling with Fluo3-AM, suggesting that intralumenal Ca2+ might play a critical role in the secretory process. Interestingly, our results indicate that transferrin (Tf) stimulated exosome release in a Ca2+-dependent manner, suggesting that Tf might be a physiological stimulus for exosome release in K562 cells. Multivesicular bodies (MVBs) 1The abbreviations used are: MVBs, multivesicular bodies; MON, monensin; Tf, transferrin; TfR, Tf receptor; AM, acetoxymethyl ester; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 2-APB, 2-aminoethoxy-diphenylborate; N-Rh-Pe, N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine; PBS, phosphate-buffered saline; PM, plasma membrane, AchE, acetylcholinesterase; TG, thapsigargin; IP3, inositol 1,4,5-triphosphate. are endocytic organelles that contain small internal vesicles generated from inward budding of the limiting membrane. In antigen-presenting cells, the fusion of these MVBs with the plasma membrane leads to the release of internal vesicles into the extracellular space (1Clayton A. Court J. Navabi H. Adams M. Mason M.D. Hobot J.A. Newman G.R. Jasani B. J. Immunol. Methods. 2001; 247: 163-174Crossref PubMed Scopus (425) Google Scholar). The released vesicles, termed exosomes (for a review see Refs. 2Stoorvogel W. Kleijmeer M.J. Geuze H.J. Raposo G. Traffic. 2002; 3: 321-330Crossref PubMed Scopus (659) Google Scholar and 3Théry C. Zitvogel L. Amigorena S. Nat. Immunol. 2002; 2: 569-579Crossref Scopus (3835) Google Scholar), were initially described in reticulocyte maturation, where their function was to discard plasma membrane proteins that were no longer necessary, such as the transferrin receptor (4Johnstone R.M. Mathew A. Mason A.B. Teng K. J. Cell. Physiol. 1991; 147: 27-33Crossref PubMed Scopus (214) Google Scholar, 5Harding C. Heuser J. Stahl P. J. Cell Biol. 1983; 97: 329-339Crossref PubMed Scopus (1140) Google Scholar, 6Harding C. Heuser J. Stahl P. Eur. J. Cell Biol. 1984; 35: 256-263PubMed Google Scholar). Although other plasma membrane proteins (e.g. acetylcholinesterase) are secreted via exosomes, these small vesicles are devoid of both cytosolic proteins and proteins associated with other intracellular organelles, indicating that only a select group of macromolecules is shed via this pathway. Exosomes are also secreted by other cell types such as activated platelets, which may function in signaling/adhesion, thus having a role at sites of vascular injury (7Heijnen H.F. Schiel A.E. Fijnheer R. Geuze H.J. Sixma J.J. Blood. 1999; 94: 3791-3799Crossref PubMed Google Scholar, 8Denzer K. Kleijmeer M.J. Heijnen H.F. Stoorvogel W. Geuze H.J. J. Cell Sci. 2000; 113: 3365-3374Crossref PubMed Google Scholar). Exosomes from cytotoxic T cells and B lymphocytes may be involved in targeting molecules for cell death (9Peters P.J. Geuze H.J. Van der Donk H.A. Slot J.W. Griffith J.M. Stam N.J. Clevers H.C. Borst J. Eur. J. Immunol. 1989; 19: 1469-1475Crossref PubMed Scopus (190) Google Scholar) or antigen presentation (10Raposo G. Nijman H.W. Stoorvogel W. Liejendekker R. Harding C.V. Melief C.J. Geuze H.J. J. Exp. Med. 1996; 183: 1161-1172Crossref PubMed Scopus (2517) Google Scholar, 11Zitvogel L. Regnault A. Lozier A. Wolfers J. Flament C. Tenza D. Ricciardi-Castagnoli P. Raposo G. Amigorena S. Nat. Med. 1998; 4: 594-600Crossref PubMed Scopus (1704) Google Scholar). Despite the diverse extracellular functions that are carried out by exosomes, very little is known about the molecular machinery involved in either the formation of the MVBs or in the exosome secretory process. We have recently shown that in K562 cells, a human erythroleukemia cell line, overexpression of Rab11 regulates the exosome pathway (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). Interestingly, treatment of green fluorescent protein-Rab11-transfected cells with the ionophore monensin (MON) generated large MVBs decorated with Rab11 and labeled with a fluorescent lipid that accumulates in exosomes. MON, a membrane-permeable Na+ ionophore that mediates an antiporter activity exchanging Na+ ions with H+ ions (13Pressman B.C. Ann. Rev. Biochem. 1976; 45: 501-530Crossref PubMed Scopus (1457) Google Scholar), acts on acidic intracellular organelles such as endosomes and lysosomes, causing swelling of these vesicles. MON is also known to induce Ca2+ entry by reversed activity of the Na+/Ca2+ exchanger (14Nassar-Gentina V. Rojas E. Luxoro M. Cell Calcium. 1994; 16: 475-480Crossref PubMed Scopus (13) Google Scholar, 15Dömötör E. Abbott N.J. Adam-Vizi V. J. Physiol. 1999; 515: 147-155Crossref PubMed Scopus (45) Google Scholar, 16Wang X.D. Kiang J.G. Scheibel L.W. Smallridge R.C. J. Investig. Med. 1999; 47: 388-396PubMed Google Scholar). A rise in intracellular Ca2+ concentration, a universal intracellular signal (for a review see Refs. 17Cullen P.J. Lockyer P.J. Nat. Rev. Mol. Cell Biol. 2002; 3: 339-348Crossref PubMed Scopus (304) Google Scholar and 18Carafoli E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1115-1122Crossref PubMed Scopus (678) Google Scholar), is necessary to induce regulated secretion in most cell types (reviewed in Refs.19Gerber S.H. Sudhof T.C. Diabetes. 2002; 51: S3-S11Crossref PubMed Google Scholar and 20Wasle B. Edwardson J.M. Cell. Signal. 2002; 14: 191-197Crossref PubMed Scopus (49) Google Scholar). During regulated exocytosis, the membrane of a secretory vesicle fuses with the plasma membrane in a tightly controlled Ca2+-triggered reaction. In endocrine cells, secretory granules contain large amounts of Ca2+ ions, and it has been suggested that the high intragranular Ca2+ concentration is needed to sustain optimal exocytosis (21Scheenen W.J. Wollheim C.B. Pozzan T. Fasolato C. J. Biol. Chem. 1998; 273: 19002-19008Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Because MON generates large MVBs in K562 cells, the aim of the present study was to determine whether MON affects exosome release and establish whether Ca2+ is involved in this process. Our results indicate that both MON treatment and a rise in intracellular Ca2+ markedly stimulate exosome secretion. Furthermore, the MON-stimulated exosome release was a Ca2+-dependent process. Interestingly, we have also observed that MON induced the accumulation of Ca2+ in the enlarged MVBs, suggesting that intravesicular Ca2+ might be involved in the secretory step. To determine whether a physiological signal might regulate the Ca2+-dependent exosome release, cells were incubated with transferrin (Tf). Our results indicate that Tf stimulates exosome release in a Ca2+-dependent manner. Materials—RPMI cell culture medium and fetal calf serum were obtained from Invitrogen. EGTA-acetoxymethyl ester (AM), BAPTA-AM, and A23187 were purchased from Molecular Probes (Eugene, OR). Fura2-AM, Fluo3-AM, xestopongin C, cyclopiazonic acid, and 2-aminoethoxy-diphenylborate (2-APB) were from Calbiochem. N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (N-Rh-PE) was obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Acetylthiocholine and 5,5′-dithiobis(2-nitrobenzoic acid) were obtained from Sigma. Bodipy-TR ceramide and Hoechst 33342 were from Molecular Probes. All other chemicals were purchased from Sigma or ICN Biochemicals, Inc. (Aurora, OH). Cell Culture—K562, a human erythroleukemia cell line, was grown in RPMI supplemented with 10% fetal calf serum, streptomycin (50 μg/ml), and penicillin (50 units/ml). Exosome Isolation—Exosomes were collected from 10 ml of K562 media (15–20 × 106 cells) cultured over 7–15 h. The culture media were collected on ice, centrifuged at 800 × g for 10 min to sediment the cells, and then centrifuged at 12,000 × g for 30 min to remove the cellular debris. Exosomes were separated from the supernatant by centrifugation at 100,000 × g for 2 h. The exosome pellet was washed once in a large volume of PBS and resuspended in 100 μl of PBS (exosome fraction). Quantitation of Released Exosomes—The amount of released exosomes was quantitated by measuring the activity of acetylcholinesterase, an enzyme that is specifically directed to these vesicles (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). Acetylcholinesterase activity was assayed following a previously described procedure (22Ho I.K. Ellman G.L. J. Neurochem. 1969; 16: 1505-1513Crossref PubMed Scopus (59) Google Scholar). Briefly, 25 μl of the exosome fraction were suspended in 100 μl of phosphate buffer and incubated with 1.25 mm acetylthiocholine and 0.1 mm 5,5′-dithiobis(2-nitrobenzoic acid) in a final volume of 1 ml. The incubation was carried out in cuvettes at 37 °C, and the change in absorbance at 412 nm was followed continuously. The data represent the enzymatic activity at 20 min of incubation. As an independent assay, exosomes were quantitated by determining the levels of the protein Hsc70 by Western blot. Samples of the exosomal fraction (15 μl) were solubilized in reducing SDS loading buffer, incubated for 5 min at 95 °C, run on 10% polyacrylamide gels, and transferred to an Immobilon (Millipore) membrane. The membranes were blocked for 1h in Blotto (5% nonfat milk, 0.1% Tween 20, and PBS) and subsequently washed twice with PBS with 0.1% Tween 20 or Tris-buffered saline with 0.1% Tween 20. Membranes were incubated with primary antibodies and peroxidase-conjugated secondary antibodies. The corresponding bands were detected using an enhanced chemiluminescence detection kit (Pierce) and quantitated by densitometry. Labeling MVBs with the Fluorescent Lipid N-Rh-PE and Fluo3-AM for Imaging Calcium—The fluorescent phospholipid analog N-Rh-PE was inserted into the plasma membrane as described previously (23Willem J. ter Beest M. Scherphof G. Hoekstra D. Eur. J. Cell Biol. 1990; 53: 173-184PubMed Google Scholar). Briefly, an appropriate amount of the lipid, stored in chloroform/methanol (2:1), was dried under nitrogen and subsequently solubilized in absolute ethanol. This ethanolic solution was injected with a Hamilton syringe into serum-free RPMI (<1%, v/v) while vigorously vortexing. The mixture was then added to the cells, which were incubated for 60 min at 4 °C. After this incubation period, the medium was removed, and the cells were extensively washed with cold PBS to remove excess unbound lipids. After the addition of complete RPMI medium and Fluo3-AM (15 μm), labeled cells were cultured for 2–3 h under conditions as described and washed twice with ice-cold PBS. Cells were mounted on coverslips and immediately analyzed by fluorescence microscopy. In some experiments, the cells were preloaded with Fluo3-AM by incubating for 60 min at 37 °C before labeling with the fluorescent lipid. No major differences were observed between these experimental procedures. Fluorescence Microscopy—K562 cells were analyzed using an inverted microscope (Nikon Eclipse TE 300, Japan) equipped with the following filter systems: excitation filter 450–490 nm, barrier filter 515 nm to visualize Fluo3-AM; and excitation filter 510–560 nm, barrier filter 590 nm to localize N-Rh-PE. Images were captured with a CCD camera (Orca I, Hamamatsu) and processed using the program Meta-Morph 4.5 (Universal Images Corporation). Some images were obtained with a Nikon Confocal C1 and processed with the EZ-C1 program. Measurement of Intracellular Calcium Concentration—Cells were incubated in the presence of 10 μm Fura2-AM for 60 min at 37 °C. They were washed to remove the extracellular dye and resuspended in complete RPMI medium containing 1 × 106 cells/ml. Fura2-AM loaded cells were protected from light. Experiments were completed within 2 h. Changes in fluorescence after the addition of 7 μm MON or by adding 30 μm BAPTA-AM before MON were analyzed in a Hitachi F-2000 fluorescence spectrophotometer. Monensin Induces the Formation of Large MVBs and Stimulates Exosome Secretion—K562 cells are human erythroleukemic cells that secrete exosomes (24Johnstone R.M. J. Cell. Physiol. 1996; 168: 333-345Crossref PubMed Scopus (30) Google Scholar), the small internal vesicles released into the extracellular media by fusion of MVBs with the plasma membrane (PM). It has been shown by electron microscopy that treatment of K562 cells with the ionophore MON causes the formation of dilated MVBs (25Stein B.S. Bensch K.G. Sussman H.H. J. Biol. Chem. 1984; 259: 14762-14772Abstract Full Text PDF PubMed Google Scholar, 12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). However, the mechanism by which these large MVBs are formed and the effect of MON on exosome release have not been explored. To get insights into these issues, MVBs in K562 cells were labeled with the fluorescent lipid analog N-Rh-PE. Sucrose gradient analysis and immunoisolation experiments have demonstrated that this lipid is efficiently internalized via endocytosis and targeted to the MVBs. Indeed, N-Rh-PE accumulates in exosomes that are eventually secreted into the extracellular medium (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar, 26Vidal M. Mangeat P. Hoekstra D. J. Cell Sci. 1997; 110: 1867-1877Crossref PubMed Google Scholar). The lipid N-Rh-PE was first bound to the PM at 4 °C, and cells were washed and subsequently incubated at 37 °C for 3 h in the absence or the presence of 7 μm MON. As shown in Fig. 1A, MON treatment caused the formation of large MVBs labeled by the fluorescent lipid. In Fig. 1B a confocal image of the MVBs formed is shown, with the internal vesicles labeled with the fluorescent lipid clearly depicted. Because fusion of the MVBs with the PM results in the release of exosomes, we tested the effect of MON on the release of exosomes from K562 cells. Exosomes are enriched in proteins such as the transferrin receptor (TfR), Hsc70, and acetylcholinesterase (AChE) (27Johnstone R.M. Bianchini A. Teng K. Blood. 1989; 74: 1844-1851Crossref PubMed Google Scholar). Therefore, exosomes were quantitated in the exosomal fraction by measuring the activity of AChE (see “Experimental Procedures”). Also, the amount of Hsc70 and TfR was determined by Western blot as described previously (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). Exosomes were harvested from the extracellular media after 7-h incubations with different concentrations of MON and quantitated by determining the levels of the proteins Hsc70 (Fig. 1C) and TfR (not shown) by Western blot. As shown in Fig. 1C, MON induced a marked increase in exosome release in a concentration-dependent manner. A similar increase was observed by measuring, in the exosomal fraction, the activity of AChE (Fig. 1D), which was maximal at 10 μm MON. At higher MON concentrations some alterations in cell viability were observed as assessed by trypan blue exclusion, for which reason a 7 μm concentration of MON was used in the rest of the experiments. At this concentration, cells were also assayed for apoptosis by staining the nucleus with Hoechst 33342 (Molecular Probes). No morphological evidence of apoptotic nuclei was observed (data not shown). In some experiments, the amount of exosomes released was also quantified by assaying the fluorescent lipid N-Rh-PE. As mentioned above, this lipid accumulates in intracellular vesicles that are ultimately secreted into the extracellular medium as exosomes. As expected, MON also increased the release of exosomes labeled with the fluorescent lipid (data not shown). Taken together the results indicate that MON not only generates large MVBs but also increases the secretion of the internal vesicles termed exosomes. A Calcium-dependent Mechanism Is Involved in the Monensin-stimulated Exosome Release—It has been shown that MON, a Na+ ionophore, can increase cytosolic Ca2+ by reversing the Na+/Ca2+ exchange mechanism (14Nassar-Gentina V. Rojas E. Luxoro M. Cell Calcium. 1994; 16: 475-480Crossref PubMed Scopus (13) Google Scholar, 15Dömötör E. Abbott N.J. Adam-Vizi V. J. Physiol. 1999; 515: 147-155Crossref PubMed Scopus (45) Google Scholar, 16Wang X.D. Kiang J.G. Scheibel L.W. Smallridge R.C. J. Investig. Med. 1999; 47: 388-396PubMed Google Scholar). Therefore, to assess whether in our system the enhanced exosome release induced by MON was due to an increase in intracellular Ca2+, we first measured whether MON could modify the intracellular Ca2+ concentration in K562 cells. For this purpose, cells were loaded for 1 h at 37 °C with 10 μm Fura-2/AM. Subsequently, the intracellular Ca2+ concentration was measured by spectrofluorometry for different periods of time after the addition of MON. Fig. 2A shows that there was an initial Ca2+ peak and a subsequent marked rise in intracellular Ca2+ that was sustained over the 2-h period tested (Fig. 2B). The MON-induced Ca2+ rise was abolished by the previous addition of the intracellular Ca2+ chelator BAPTA-AM (Fig. 2A). Interestingly, in the presence of the extracellular Ca2+ chelator EGTA, MON induced the initial rise, which was likely due to Ca2+ release from intracellular stores. However, no sustained increase was observed, indicating that the latter is a result of Ca2+ influx from the extracellular environment (data not shown). The results suggest that the increase in exosome release might be due to a Ca2+-dependent mechanism. To test this hypothesis, we assessed whether the MON effect on exosome release could also be prevented by Ca2+ chelators. To chelate the Ca2+ present in the extracellular media, cells were incubated for several hours in the presence of 1.5 mm EGTA. Under these conditions, the free Ca2+ concentration was less than 10 nm as calculated with the Sliders program (see “Experimental Procedures”). BAPTA-AM was used to chelate intracellular Ca2+, because this is a membrane-permeable agent that efficiently chelates Ca2+. The released exosomes were collected from the media and quantitated by measuring AChE activity as indicated under “Experimental Procedures.” As shown in Fig. 2C, both EGTA and BAPTA-AM decreased, although slightly, the basal release of exosomes. Moreover, the MON-dependent increase was completely abrogated by the Ca2+ chelators, and no additive effects were observed when both chelators were added together (data not shown). The result clearly indicates that Ca2+ from the extracellular media and also from intracellular stores is required for the MON-induced exosome secretion. Ca2+ involvement in exosome release was evaluated using the Ca2+ ionophore A23187. As shown in Fig. 3A, incubation with the Ca2+ ionophore stimulated exosome secretion to a similar extent as MON. No additive effects were observed when both agents were added together. As expected, the secretory effect of the Ca2+ ionophore was inhibited by the chelators EGTA or BAPTA-AM (Fig. 3B). It is known that MON acts on acidic compartments by altering the proton gradient across vesicle membranes, resulting in a Ca2+ movement into the cytosol (28Christensen K. Myers J. Swanson J. J. Cell Sci. 2001; 115: 599-607Crossref Google Scholar). We tested the effect of two agents known to alter the pH of vacuolar compartments, the weak base chloroquine and the vacuolar proton pump inhibitor bafilomycin A1 (29Mousavi S.A. Kjeken R. Berg T.O. Seglen P.O. Berg T. Brech A. Biochim. Biophys. Acta. 2001; 1510: 243-257Crossref PubMed Scopus (57) Google Scholar). Previous work has shown that these compounds may also discharge intracellular Ca2+ pools from acidic compartments (30Passos A.P. Garcia C.R. Biochem. Biophys. Res. Commun. 1998; 245: 155-160Crossref PubMed Scopus (68) Google Scholar, 31Beraldo F.H. Sartorello R. Lanari R.D. Garcia C.R. Cell Calcium. 2001; 29: 439-445Crossref PubMed Scopus (16) Google Scholar). We have evidence that chloroquine elevated intracellular Ca2+ in a similar manner to MON (not shown). As shown in Fig. 4A, chloroquine stimulated the release of exosomes, although to a lesser degree than MON. Non-additive effects were observed by the addition of chloroquine together with MON. Bafilomycin also increased the release of exosomes (Fig. 4B). Both chloroquine and bafilomycin effects were abrogated by clamping extracellular Ca2+ with the chelator EGTA, indicating that these compounds indeed act via a calcium-dependent mechanism. Visualizing a MVB Calcium Pool by Fluo3-AM Imaging— Numerous reports indicate the existence of several intracellular Ca2+ pools (32Martinez J.R. Willis S. Puente S. Wells J. Helmke R. Zhang G.H. Biochem. J. 1996; 320: 627-634Crossref PubMed Scopus (27) Google Scholar); for a review see Refs. 33Verkhratsky A. Shmigol A. Cell Calcium. 1996; 19: 1-14Crossref PubMed Scopus (244) Google Scholar and 34Rizzuto R. Curr. Opin. Neurobiol. 2001; 11: 306-311Crossref PubMed Scopus (107) Google Scholar. Fluo3-AM is a membrane-permeant compound that accumulates in the cytoplasm where cytosolic esterases clip the AM groups, rendering the fluorescent probe membrane impermeable. However, it has been shown that, when used at higher concentrations, part of this indicator is capable of accumulating also in intracellular compartments and can be used as an indicator for intracellular Ca2+ stores (35Takahashi A. Camacho P. Lechleiter J.D. Herman B. Physiol. Rev. 1999; 79: 1089-1125Crossref PubMed Scopus (623) Google Scholar). Cells were incubated with Fluo3-AM for 1 h at 37 °C to visualize the calcium-containing compartments. MVBs were labeled with the fluorescent lipid N-Rh-PE as mentioned above, and the cells were subsequently incubated with the indicated agents for 3 h at 37 °C. As shown in Fig. 5, the large MVBs induced by MON treatment were clearly labeled by Fluo3-AM, indicating that Ca2+ accumulates in these intracellular compartments. Similarly, Ca2+ was also present in the large MVBs formed by chloroquine treatment. The presence of BAPTA-AM depleted the MVBs calcium pool in both conditions. Strikingly, the size of the MVBs was markedly reduced, indicating that a calcium-dependent mechanism is involved in the development of the gigantic MVBs formed by MON or chloroquine treatment. Calcium Levels Regulated by IP3Receptors and a Thapsigargin-sensitive Ca2+Pump Are Involved in the Release of Exosome—It is well established that thapsigargin (TG) causes a rapid inhibition of the calcium-ATPase pump present in the membranes of the endoplasmic reticulum (36Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (3010) Google Scholar), followed by a fast Ca2+ leak from other Ca2+ stores as well as influx from the extracellular media. This leads to a rapid and pronounced increase in the concentration of cytosolic-free calcium. As expected, treatment of K562 wells with this inhibitor stimulated exosome secretion in a similar manner as MON, and this effect was also blocked by EGTA (Fig. 6A). These findings confirmed a role for Ca2+ in the exosome secretory pathway and the participation of a TG-sensitive Ca2+ pump in the process. Because TG stimulated exosome release, we were interested in knowing whether the large MVBs developed by MON were also formed by treatment with TG. As shown in Fig. 6B, TG neither generated large MVBs nor impaired the formation of the MON-induced gigantic structures that were filled with calcium. This suggests that an increase in cytosolic Ca2+ is not by itself enough to generate the enlarged MVBs, despite being sufficient to stimulate exosome secretion. The phosphoinositide signaling cascade plays a prominent role in the mobilization of Ca2+ from intracellular stores (for a review see Refs. 37Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2272) Google Scholar and 38Balla T. Curr. Pharm. Des. 2001; 7: 475-507Crossref PubMed Scopus (48) Google Scholar). The receptors for the second messenger, inositol 1,4,5-trisphosphate (IP3), constitute a family of Ca2+ channels responsible for the mobilization of intracellular Ca2+ stores. The increase in the levels of IP3 result in the opening of Ca2+ channels present in the endoplasmic reticulum and the subsequent release of Ca2+ into the cytosol (39Taylor C.W. Richardson A. Pharmacol. Ther. 1991; 51: 97-137Crossref PubMed Scopus (122) Google Scholar). To test whether this type of channel might be involved in the calcium-induced exosome release, cells were incubated with 2-APB, a membrane-permeable inhibitor of IP3-induced Ca2+ release. Fig. 6C shows that 2-APB inhibited monensin-stimulated exosome release. Similar results were obtained with xestopongin, a potent blocker of IP3 receptors (data not shown), indicating that a transient Ca2+ rise mediated by the stimulation of an IP3 receptor is critical for the MON-stimulated exosome release. However, the formation of the large Ca2+-rich MVBs induced by MON was not completely abrogated by 2-APB (Fig. 6D), although there was a decrease in the size of the MVBs compared with the vesicles generated by MON in the absence of 2-APB. Vesicle area in the MON-treated cells was 436 ± 30 (relative units), whereas the addition of 2-APB reduced the size to 182 ± 16 (n = 50 vesicles counted). This suggests that the release of Ca2+ via the IP3-sensitive Ca2+ channels contributes, at least in part, to the generation of the enlarged MVBs. The Formation of the Gigantic MVBs Filled with Calcium Are Completely Blocked by Amiloride—The results presented here indicate that Ca2+ is absolutely required for generation of the enlarged MVBs, because these structures are not formed in the presence of BAPTA-AM (see above). However, even though IP3-sensitive Ca2+ channels seem to participate in the process, the development of the large MVBs was only partly decreased by specific modulators. This implies that another type of Ca2+ channel (see “Discussion”) is involved in the MON-dependent Ca2+ rise and the accumulation of Ca2+ in the MVBs. It is known that the rapid sodium influx initiated by MON increases cytosolic Ca2+ by reversing the Na+/Ca2+ exchange mechanism. Therefore, because the activity of a Na+/Ca2+ exchanger seems to be critical for the MON effect, we tested dimethyl amiloride, an inhibitor of the H+/Na+ and Na+/Ca2+ exchangers. As shown in Fig. 7A, amiloride decreased the basal exosome release and also completely inhibited the MON-stimulated secretion of exosomes. As expected, the formation of the gigantic Ca2+-filled MVBs generated by MON-treatment was completely abrogated by amiloride. In contrast, the process was not affected by verapamil, an inhibitor of a voltage-dependent Ca2+ channel (data not shown). These results are consistent with the idea that activation of a Na+/Ca2+ exchanger is a prerequisite for the Ca2+ rise in the cytoplasm that leads to exosome secretion and the formation of the enlarged MVBs filled with Ca2+ generated by MON. Transferrin Stimulates Exosome Release in a Calcium-dependent Manner—Taken together the results discussed above clearly indicate that Ca2+ is a key participant in the exosome release process. Therefore, we were interested in addressing whether a physiological stimulus might also enhance exosome secretion in a Ca2+-dependent manner. As mentioned previously, K562 is a human erythroleukemia cell line that presents high levels of TfR, and, because it has been shown th