Exosome Release Is Regulated by a Calcium-dependent Mechanism in K562 Cells*

Ariel Savina {ddagger}, Marcelo Furlán {ddagger}, Michel Vidal  § and Maria I. Colombo {ddagger} ||

From the {ddagger}Laboratorio de Biología Celular y Molecular-Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), 5500 Mendoza, Argentina and §Unité Mixte de Recherche 5539, Université Montpellier II, Montpellier 34095, France

Received for publication, February 17, 2003 , and in revised form, March 13, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Multivesicular bodies (MVBs)1 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 (1). The released vesicles, termed exosomes (for a review see Refs. 2 and 3), 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 (4, 5, 6). 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 (7, 8). Exosomes from cytotoxic T cells and B lymphocytes may be involved in targeting molecules for cell death (9) or antigen presentation (10, 11).

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 (12). 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 (13), 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 (14, 15, 16).

A rise in intracellular Ca2+ concentration, a universal intracellular signal (for a review see Refs. 17 and 18), is necessary to induce regulated secretion in most cell types (reviewed in Refs.19 and 20). 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 (21). 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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—RPMI cell culture medium and fetal calf serum were obtained from Invitrogen. EGTA-acetoxymethyl ester (AM), BAPTA-AM, and A23187 [GenBank] 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 x 106 cells) cultured over 7–15 h. The culture media were collected on ice, centrifuged at 800 x g for 10 min to sediment the cells, and then centrifuged at 12,000 x g for 30 min to remove the cellular debris. Exosomes were separated from the supernatant by centrifugation at 100,000 x 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 (12). Acetylcholinesterase activity was assayed following a previously described procedure (22). 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 (23). 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 x 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Monensin Induces the Formation of Large MVBs and Stimulates Exosome Secretion—K562 cells are human erythroleukemic cells that secrete exosomes (24), 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 (25, 12). 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 (12, 26). 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.



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FIG. 1.
Monensin generates large MBVs and stimulates exosome release in K562 cells. A, K562 cells were labeled with the fluorescent lipid analog N-Rh-PE and incubated for 3 h at 37 ° to label MBVs in the absence or presence of 7 µM of MON. Cells were mounted on coverslips and immediately analyzed by fluorescence microscopy. PhC, phase contrast image. Bar, 2 µm. B, a confocal image showing several MVBs with the characteristic internal vesicles. Bar, 2 µm. Inset, a higher magnification of the MVB depicted in panel B (arrow) in a different confocal plane to specifically show the internal vesicles. C, Western blot of samples of the exosomal fraction (see "Experimental Procedures") harvested from cells incubated in the presence of different concentrations of MON and probed with antibodies against the heat shock protein Hsc70. The corresponding bands were detected using an enhanced chemiluminescence detection kit, quantitated by densitometry, and expressed as arbitrary units. Data are representative of at least three independent experiments. D, cells were incubated in the presence of different concentrations of MON, and exosomes were collected from the culture media. The AChE activity was assayed on the exosome fractions by standard procedures as described under "Experimental Procedures." Data represent the mean ± S.E. of three independent experiments.

 

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) (27). 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 (12). 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 (14, 15, 16). 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).



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FIG. 2.
Monensin stimulates exosome release by a calcium-dependent mechanism. A, cells were loaded with Fura-2/AM, and the intracellular Ca2+ concentration was measured as described under "Experimental Procedures." Shown are changes in Ca2+ concentration elicited by 7 µM MON with or without the previous addition of 30 µM BAPTA-AM. Data are representative of at least three independent experiments. B, cells were loaded with Fura-2 and incubated with MON as in panel A. The intracellular Ca2+ concentration was measured over the 2-h period tested. Data are representative of at least three independent experiments. C, cells were incubated for 7 h in the presence of 30 µM BAPTA-AM (B-AM), 1.5 mM EGTA, 7 µM MON, or the combination of 30 µM BAPTA-AM/7 µM monensin (Mon + B-AM) or 1.5 mM EGTA/7 µM monensin (Mon + EGTA). The secreted exosomes were collected and quantitated by measuring the AChE activity. Asterisk, significantly different from the control, p < 0.05. Fragmented diamond, significantly different from the MON-treated cells, p < 0.05.

 

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 [GenBank] . 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).



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FIG. 3.
Intracellular calcium rise induces exosome release. K562 cells were incubated for 7 h in the presence of 7 µM MON (Mon), 1 µM A23187 [GenBank] (Ion), or 7 µM monensin/1 µM A23187 [GenBank] (Mon + Ion) (panel A) and with 1 µM A23187 [GenBank] (Ion), 1 µM A23187 [GenBank] /30 µM BAPTA-AM (Ion + B-AM), or 1 µM A23187 [GenBank] /1.5 mM EGTA (Ion + EGTA) (panel B). The secreted exosomes were collected and quantitated by measuring the AChE activity. Asterisk, significantly different from the control, p < 0.01. Fragmented diamond, significantly different from the A23187 [GenBank] -treated cells, p < 0.05.

 

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 (28). 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 (29). Previous work has shown that these compounds may also discharge intracellular Ca2+ pools from acidic compartments (30, 31). 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.



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FIG. 4.
Agents that alter the pH of intracellular compartments induce exosome release by a calcium-dependent mechanism. Panel A: cells were incubated for 7 h in the presence of 10 µM chloroquine (Chlor), 7 µM monensin (Mon), or 7 µM monensin/10 µM chloroquine (Chlor + Mon) (panel A) and with 10 µM chloroquine (Chlor), 10 µM bafilomycin A1 (Baf), 10 µM chloroquine/1.5 mM EGTA (Chlor +EGTA), or bafilomycin/1.5 mM EGTA (Baf +EGTA) (panel B). The secreted exosomes were collected and quantitated by measuring the AChE activity. Asterisk, significantly different from the control, p < 0.01. Fragmented diamond, significantly different from the Chlor- or Baf-treated cells, p < 0.01.

 

Visualizing a MVB Calcium Pool by Fluo3-AM Imaging— Numerous reports indicate the existence of several intracellular Ca2+ pools (32); for a review see Refs. 33 and 34. 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 (35). 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.



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FIG. 5.
The large MBVs generated by monensin or chloroquine treatment accumulate intralumenal calcium, and their production is inhibited by calcium chelators. K562 cells were loaded with 15 µM Fluo3-AM for 1 h at 37 °C. Cells were then washed twice with PBS buffer and labeled with the fluorescent lipid analog N-Rh-PE and incubated for 3 h at 37 ° in the presence of 7 µM MON, 7 µM MON/30 µM BAPTA, 10 µM chloroquine (CHLOR), or 10 µM chloroquine/30 µM BAPTA-AM. Samples were protected from light during the whole experimental period. Cells were mounted on coverslips and immediately analyzed by fluorescence microscopy. Left panels, N-Rh-PE (red); middle panels, Fluo3-AM (green); right panels, merged image.

 

Calcium Levels Regulated by IP3 Receptors 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 (36), 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.



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FIG. 6.
Calcium from internal stores regulates exosome release. Cells were incubated for 7 h in the presence of 7 µM MON (Mon), 1 µM thapsigargin (Tg), 1 µM thapsigargin/7 µM monensin (Tg + Mon) or 1 µM thapsigargin/1.5 mM EGTA (Tg + EGTA) and with 7 µM MON (Mon), 100 µM 2-APB, or 7 µM MON/100 µM 2-APB (Mon + 2-APB). The secreted exosomes were collected and quantitated by measuring the AChE activity. Asterisk, significantly different from the control; p < 0.01. Fragmented diamond, significantly different from the thapsigargin-treated cells, p < 0.01 (panel A), or 2-APB-treated, p < 0.05 (panel C). B and D, K562 cells were loaded with 15 µM Fluo3-AM for 1 h at 37 °C. Cells were then washed twice with PBS buffer and labeled with the fluorescent lipid analog N-Rh-PE and incubated for 3 h at 37 °C in the presence of 1 µM thapsigargin (TG), 7 µM MON/1 µM thapsigargin (TG MON), 100 µM 2-APB, or 7 µM MON/100 µM 2-APB (2-APB MON). Cells were mounted on coverslips and immediately analyzed by fluorescence microscopy. Left panels, N-Rh-PE (red); middle panels, Fluo3-AM (green); right panels, merged images.

 

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. 37 and 38). 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 (39). 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.



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FIG. 7.
Amiloride inhibits exosome release and the formation of the large MBVs. A: K562 cells were incubated for 7 h in the presence of 7 µM MON (Mon), 15 nM amiloride (Amil), or 7 µM MON/15 nM amiloride (Mon + Amil). The secreted exosomes were collected and quantitated by measuring the acetylcholinesterase activity. Asterisk, significantly different from the control, p < 0.01. Fragmented diamond, significantly different from the MON-treated cells, p < 0.01. B, cells were loaded with 15 µM Fluo3-AM for 1 h at 37 °C. Cells were then washed twice with PBS buffer, labeled with the fluorescent lipid analog N-Rh-PE, and incubated for 3 h at 37 °C in the presence of 15 nM amiloride or 15 nM amiloride/7 µM MON. Samples were protected from light during the whole experimental period. Cells were mounted on coverslips and immediately analyzed by fluorescence microscopy. Left panels, N-Rh-PE (red); middle panels, Fluo3-AM (green); right panels, merged images.

 

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 that binding of Tf to its receptor increases intracellular Ca2+ concentration (40), Tf might be a candidate for regulating exosome secretion. Therefore, we first assessed whether Tf increases intracellular Ca2+ in K562 cells. For this purpose, cells were loaded with Fura-2/AM as described above, and 20 µg/ml human Tf was added to the incubation media. As described previously for other cell types (40), Tf induced an increase in Ca2+ that was sustained for the whole 60-min period studied (Fig. 8A).



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FIG. 8.
Transferrin stimulates exosome release in a calcium-dependent manner. A, K562 cells were loaded with Fura-2/AM, and the intracellular Ca2+ concentration was measured as described under "Experimental Procedures." Shown are the changes in Ca2+ concentration elicited by 20 µg/ml of human Tf. B, cells were incubated overnight in the presence of 20 µg/ml human Tf or the combination of 30 µM BAPTA-AM/20 µg/ml Tf (Tf + B-AM), or 1.5 mM EGTA/20 µg/ml of Tf (Tf + EGTA), or 100 µM 2-APB/20 µg/ml Tf (Tf + 2-APB). The secreted exosomes were collected and quantitated by measuring the acetylcholinesterase activity. Asterisk, significantly different from the control, p < 0.05. Fragmented diamond, significantly different from the Tf-treated cells, p < 0.05.

 

We next addressed whether adding Tf to the culture media modifies exosome secretion. Cells were incubated for 12 h in the presence of 20 µg/ml human Tf, and exosomes were collected from the incubation media as described above. As shown in Fig. 8B, Tf stimulated exosome release, an effect that was hampered by EGTA or BAPTA-AM, indicating that was a Ca2+-dependent process. Furthermore, the Tf-stimulated exosome release was also inhibited by 2-APB, suggesting that IP3-sensitive Ca2+ channels participate in this process. This is in agreement with a recent observation that the addition of apotransferrin to cultured oligodendroglial cells increased the levels of IP3 (41).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study we have shown that the ionophore MON induces the formation of large MVBs and stimulates the release of the internal vesicles called exosomes. It has been shown that MON induces catecholamine secretion from adrenal chromaffin cells (14, 42). The release of regulated secretory granules is known to be Ca2+-dependent. Here, we present evidence that the MON-stimulated exosome secretion in K562 cells is indeed a calcium-dependent event. The application of MON generated a marked elevation of Ca2+ that was dependent on both an extracellular source and intracellular Ca2+ stores. The role for Ca2+ on exosome release was confirmed by the use of the Ca2+ ionophore A23187 [GenBank] . Similar results were obtained with agents known to alter the pH of vacuolar compartments such as chloroquine and bafilomycin, an inhibitor of the proton pump. Both compounds stimulated exosome release via a Ca2+-dependent mechanism, because the effect was abrogated by BAPTA-AM. We have evidence that chloroquine elevated intracellular Ca2+ in a similar manner as MON. These data are in agreement with previous observations indicating that chloroquine causes a substantial Ca2+ release in the parasite Plasmodium chabaudi (30). Similarly, it has been shown that bafilomycin alters cytosolic Ca2+ by discharging intracellular Ca2+ pools in lizard red blood cells (31). Therefore, taken together, our results clearly indicate that Ca2+ is a key participant in the exosome release process. We believe that this is a relevant observation because, depending on their origin, exosomes can play roles in different physiological processes (8). For example, activated platelets release exosomes at sites of vascular injury where they may have a signaling/adhesion function (7). Antigen-presenting cells also secrete exosomes that carry peptide-loaded MHC molecules functioning as intercellular vehicles for antigenic material. Therefore, our observation that Ca2+ regulates exosome release suggests that a signal transduction mechanism is likely involved in the activation of exosome-carrying cells to release these small vesicles at the proper site.

The use of MON allowed us to assess that Ca2+ plays an important role in exosome secretion. It has been proposed that MON acts on acidic intracellular organelles such as endosomes and lysosomes, exchanging H+ for Na+ and causing swelling of these vesicles by passive water influx (44). Therefore, it would be possible that a similar mechanism might be involved in the generation of the large MVBs formed after MON treatment. However, our results indicate that the formation of the enlarged endosomes is a calcium-dependent event, because this process was completely abrogated by the Ca2+ chelator BAPTA-AM. It is tempting to speculate that the formation of these gigantic endosomes is not only due to swelling of the vesicles but requires also the contribution of membranes from other sources, implying fusion among vesicular compartments. It is becoming evident that many intracellular transport events depend on Ca2+ (45) and, thus, it is likely that Ca2+ might be required for the fusion events involved in the generation of the enlarged MVBs. Experiments are underway to address this possibility.

MON, as a Na+ ionophore, transports a molecule of Na+ inside the cells per molecule of H+ transported to the extracellular media. The increase in intracellular Na+ activates the Na+/Ca2+ exchanger in a reverse mode, leading to an initial increase in cytosolic Ca2+ (16). Our data are in agreement with a requirement for an early entrance of Na+, because the MON-mediated effects were completely abrogated by amiloride, an inhibitor of the H+/Na+ and Na+/Ca2+ exchangers. Our data are also compatible with the idea that Na+ entry mediated by MON could activate the production of IP3, which in turn releases Ca2+ from intracellular stores. Subsequently, the emptiness of these stores might trigger the opening of store-operated Ca2+ channels (SOC) at the plasma membrane that induces a sustained Ca2+ rise consistent with the requirement for extracellular Ca2+ in our system. Data presented in this report indicate that IP3-dependent channels are involved in MON-stimulated exosome release, indicating that a transient Ca2+ rise mediated by the stimulation of IP3 receptors is critical for this event. However, the formation of the enlarged MVBs was only partially inhibited and not completely abrogated by inhibition of the IP3 receptors, suggesting that an additional mechanism might be involved in the generation of these large endosomes.

Interestingly, we have observed that the large MVBs generated by MON-treatment are filled with Ca2+. It is likely that the MON-stimulated antiport activity might be exerted primarily at the plasma membrane, but it is also possible that a similar effect occurs on intracellular membranes. Indeed, it has been shown that MON selectively induces the secretion of azurophil granule contents by directly affecting the granule membrane (46). Swanson and co-workers (28) have reported that lysosomes contain high concentration of Ca2+ and therefore could function as an intracellular Ca2+ source. These authors have shown that changes in lysosomal pH resulted in the movement of vacuolar Ca2+ out of lysosomes into the cytoplasm, possible via pH-dependent calcium channels or pumps. Our results indicate that, even though a thapsigargin-sensitive Ca2+ pump is involved in the release of exosomes, inhibition of this pump does not block either the formation of the gigantic MVBs or the accumulation of Ca2+ inside the large membranous structures. Therefore, it is likely that another type of Ca2+ pump, present in intracellular compartments, might be responsible for filling the enlarged endosomes. PMR1, a Ca2+ ATPase present in yeast Golgi (47) similar to the plasma membrane Ca2+ ATPase (PMCA), is thapsigargin-insensitive. A Ca2+ ATPase in the Golgi of mammary tissue (48) with characteristics slightly different from the PMCA and the endoplasmic reticulum Ca2+ ATPase (SERCA) has also been identified. This mammary Golgi secretory pathway Ca+2-ATPase (SPCA) seems to be a homologue to the yeast PMR1 and is believed to be important for the function of the secretory pathway. Interestingly, we have recently shown that MVBs in K562 cells are formed, at least in part, by membrane influx from Golgi compartments (12). Furthermore, it has been shown that endocytic vesicles from reticulocytes possess a Ca2+ ATPase that pumps Ca2+ into the lumen of the vesicles (49). Therefore, it would not be unprecedented that a thapsigargin-insensitive Ca2+ pump is involved in the formation of the enlarged MVBs and the accumulation of the lumenal Ca2+.

An interesting possibility is that the Ca2+ present inside the MVBs is playing a critical role in the exosome secretory process. A role for intravesicular Ca2+ in several secretory and intracellular fusion events has been proposed. Fusion of early endosomes seems to require the release of lumenal Ca2+ for fusion to occur (50). Also, in insulin-secreting cells it has been shown that Ca2+ depletion from granules inhibits exocytosis (21). Indeed, the presence of IP3 receptors on insulin and somatostatin secretory granules has been demonstrated, suggesting that these organelles represent a readily mobilizable IP3-regulated Ca2+ pool during the secretory process (52). As we mentioned above, it is known that MON disrupts proton gradients across the membranes, allowing the release of intra-vacuolar Ca2+. Therefore, it is possible that the localized release of Ca2+ from the MVBs at the docking site may play an active role in the fusion complex/pore formation. Even though our results clearly indicate that Ca2+ is a critical participant in the MON-stimulated exosome secretion, further experiments are required to elucidate whether the MVB luminal Ca2+ participates in this process.

Finally, we have presented data indicating that Tf increased intracellular calcium in K562 cells and stimulated exosome release in a Ca2+-dependent manner, suggesting that the secretion of exosomes is under physiological control. Our results are consistent with previous observations that the binding of transferrin to its receptor raises intracellular Ca2+ and stimulates receptor recycling in L2C cells (40). Also, transferrin recycling was stimulated by Ca2+ in bovine chromaffin cells (53). Our results suggest that a component of the vesicular transport machinery involved in exosome secretion is regulated by Ca2+. Proteins such as synaptotagmins and calmodulin have been implicated in vesicular transport events where they function as Ca2+ sensors (54). Therefore, Tf, by increasing intracellular Ca2+, may modulate some critical components of the transport/fusion machinery. Interestingly, in a recent paper it has been shown that the binding of apotransferrin to its receptor increased, via a calcium-calmodulin-dependent kinase, the levels of tubulin and actin, proteins known to be involved in vesicular trafficking (41). In conclusion, the TfR seems to function as a signal transduction molecule that modulates not only its own recycling but also its shedding via the exosome pathway, and Ca2+ is one of the components of this signaling pathway triggered by Tf binding. It is important to mention that exosomes released to circulation from maturing red cells are the principal source of the soluble, circulating, truncated TfR (24). Soluble TfR levels 8-fold greater than normal have been reported in hemolytic anemias (51). Even though the physiological role of the soluble truncated TfR (43) has not been established yet, our observation that Tf stimulates exosome release and, as a consequence, the availability of soluble TfR, led us to speculate that this is a mechanism to regulate the free levels of circulating Tf.


    FOOTNOTES
 
* This work was partly supported by Consejo Nacional de Investigaciones Científicas y Técnicas Grant PIP 0695/98), Agencia Nacional de Promoción Científica y Tecnológica Grant PICT99 1-6058, and grants from Consejo de Investigaciones de la Universidad Nacional de Cuyo (to M. I. C.), and the Association pour la Recherche sur le Cancer to (to M. V.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Recipient of Programa de Cooperación Argentino-Francesa de Formación para la Investigación Científica y Tecnológica (SECYT-ECOS)-Sud Joint Grant A98B04. Back

|| To whom correspondence should be addressed: María Isabel Colombo, Instituto de Histología y Embriología (IHEM), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Casilla de Correo 56, Mendoza, Argentina. Fax: 54-261-4494117; E-mail: mcolombo{at}fmed2.uncu.edu.ar.

1 The 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. Back


    ACKNOWLEDGMENTS
 
We thank Drs. Sebastian Amigorena and Luis Mayorga for critical reading of this manuscript. We also thank Silvina Pelozo for technical assistance.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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