1 Dipartimento di Scienze e Tecnologie Avanzate, Università del Piemonte Orientale "Amedeo Avogadro," 15100 Alessandria; 2 Dipartimento di Biologia Sperimentale Ambientale ed Applicata, Università di Genova, 16132 Genoa; and 3 Dipartimento di Oncologia, Biologia e Genetica, Università di Genova, 16132 Genoa, Italy
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ABSTRACT |
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The mechanism of
lysosome activation by 17-estradiol has been studied in mussel blood
cells. Cell treatment with estradiol induced a sustained increase of
cytosolic free Ca2+ that was completely prevented by
preincubating the cells with the Ca2+ chelator BAPTA-AM.
Estradiol treatment was also followed by destabilization of the
lysosomal membranes, as detected in terms of the lysosomes' increased
permeability to neutral red. The effect of estradiol on lysosomes was
almost completely prevented by preincubation with the inhibitor of
cytosolic Ca2+-dependent PLA2
(cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), and
was significantly reduced by preincubation with BAPTA-AM. In contrast,
it was virtually unaffected by preincubation with the inhibitor of
Ca2+-independent PLA2,
(E)-6-(bromomethylene)tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (BEL). The Ca2+ ionophore A-23187 yielded similar effects
on [Ca2+]i and lysosomes. Exposure to
estradiol also resulted in cPLA2 translocation from cytosol
to membranes, lysosome enlargement, and increased protein degradation.
These results suggest that the destabilization of lysosomal membranes
following cell exposure to estradiol occurs mainly through a
Ca2+-dependent mechanism involving activation of
Ca2+-dependent PLA2. This mechanism promotes
lysosome fusion and catabolic activities and may mediate short-term
estradiol effects.
lysosome membrane stability; 17-estradiol; cytosolic
phospholipase A2; calcium signaling; AACOCF3; BEL
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INTRODUCTION |
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THE GONADAL STEROID
17-ESTRADIOL has been described mostly in terms of
its long-term effects related to genomic activation. However,
short-term nongenomic effects of the hormone have also been observed in
a variety of cell types. These effects are frequently associated with
increases in cytosolic free Ca2+ concentration
([Ca2+]i) (1, 3, 12, 26, 36, 44)
and may rely on the presence of estradiol receptors in the plasma
membrane (18, 41, 44). Because they may play a significant
role in the regulatory functions of the hormone, these short-term
effects warrant further study.
One short-term effect of estradiol that has not previously been studied
in association with other effects of the hormone is destabilization of
lysosomal membranes (29, 45, 51). Lysosomes are
membrane-bound organelles rich in acidic hydrolases that represent the
cellular site for bulk macromolecule degradation. Lysosomal activities
mediate several processes in cell feeding, homeostasis, and
antimicrobial defense, which involve lysosome fusion with endosomes and
(auto) phagosomes (22, 32, 43). Lysosomal membranes are
essential for the correct functioning of the lysosome, permitting
hydrolase compartmentalization, maintenance of an acidic internal
environment, and vacuole trafficking. Because of lysosomal sensitivity
to a variety of chemical agents and stressors, lysosomal alterations
are often used as a general biomarker of stress in environmental
biomonitoring (e.g., Refs. 10 and 48). Lysosomal membrane
destabilization involves increased fusion of lysosomes and phagosomes
and is frequently associated with cellular stress deriving from the
action of xenobiotics and pro-oxidant agents (16, 21, 27, 49,
52) or from pathological conditions (11, 14, 46).
Under extreme circumstances, normally latent acidic hydrolases may leak
into the cytoplasm, causing damage to cell components and eventually
leading to cell death (23, 50, 53). However, lysosomal
membrane destabilization can also occur independently from cellular
stress, such as during apoptosis (19, 34, 40) or
in the short-term response to 17-estradiol, as mentioned above.
This study is part of an attempt to address a broader question: is there a common explanation for the lysosomal (hyper)activations induced by stress conditions and by endogenous processes? To answer this question, the mechanisms responsible for lysosomal membrane destabilization and lysosome activation need to be further clarified. To reach this aim, we tried to understand the effect of estradiol on lysosomes by exploring possible links between the process of destabilization of the lysosomal membrane and other short-term effects of the hormone. We thus sought an estradiol-induced, Ca2+-dependent mechanism that leads to modification of the lysosomal membranes. Because both cytosolic and exogenous PLA2 have been found to cause lysosomal membrane destabilization (33) and lysosomal enzyme leakage (24), we focused on identifying a potential association between Ca2+-dependent cytosolic PLA2 and the effect of estradiol on lysosomal membranes.
Marine mussel blood cells served as our experimental model system.
These cells are lysosome-enriched elements involved in antipathogen
defense and have been widely used for investigations of lysosomes in
general (7, 37, 49, 52) and for the study of membrane
destabilizing effects produced by 17-estradiol in particular
(19). The presence of 17
-estradiol has been ascertained in mussels (39), and, in addition, steroid hormones seem
to play typical roles in the life cycle of these mollusks, for example, in gonad maturation during the reproductive period (38).
Estradiol treatments were used in combination with two selective inhibitors: the inhibitor of Ca2+-dependent PLA2, arachidonyl trifluoromethyl ketone (AACOCF3), and the inhibitor of Ca2+-independent PLA2, (E)-6-(bromomethylene)tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (BEL; Ref. 47). Digital imaging of fura 2-loaded cells was used to evaluate cytosolic free Ca2+ variations, whereas digital imaging of neutral red- stained cells served to evaluate lysosomal membrane destabilization. Translocation of Ca2+-dependent PLA2 from the cytosol to the membranes, an essential step in the enzyme activation pathway (8), was detected using cell fractionation and Western blotting. Because lysosomal membrane destabilization is correlated to an increase in protein degradation, we analyzed the catabolism of short-lived proteins, using cell labeling with [14C]valine, and structural protein catabolism, using actin staining with FITC-labeled phalloidin and confocal image analysis.
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MATERIALS AND METHODS |
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Chemicals
Bromoenol lactone (BEL), bovine serum albumin (BSA), digitonin, 17Solutions
Artificial sea water contained (in mM) 473 NaCl, 18 Na2SO4, 16 MgCl2, 6.2 KCl, 5 CaCl2, 1.5 NaHCO3, 0.045 NaF, 0.56 KBr, 0.32 H3BO3, and 0.048 SrCl2 · 6H2O, pH 8.0. Physiological saline contained (in mM) 20 HEPES, 436 NaCl, 53 MgSO4, 10 KCl, and 10 CaCl2, pH 7.3. Loading buffer contained (in mM) 30 HEPES, 0.5 sucrose, 2.5 MgCl2, 2.5 CaCl2, 125 NaCl, and 2.5 KCl, pH 7.3.Animals
Mussels (Mytilus galloprovincialis Lam.) with a shell length of 4-5 cm were obtained from a local farm (La Spezia, Italy) and acclimated for 3 days in an aquarium with aerated, recirculating, artificial seawater at 15°C.Blood Cell Collection
Mussel blood (1 ml) was withdrawn from the posterior adductor muscle by using a hypodermic syringe containing an equal volume of artificial seawater. The needle was then removed and the syringe content was put into a siliconized Eppendorf tube (Sigmacote).Evaluation of Lysosomal Membrane Stability and of Lysosomal Volume
Aliquots of 30 µl of blood cell suspension were placed on microscope slides coated with 2 µl of poly-L-lysine (1:10 in distilled water) to improve cell-to-substratum adhesion. Slides were incubated for 30 min in a light-proof humidity chamber at 15°C to allow the cells to settle. Excess solution was removed, and 30 µl of a 5% NR stock solution (20 mg/ml in DMSO) in artificial seawater were added. After 15 min, excess dye was washed out, 30 µl of artificial seawater were added, with or without 50 nM estradiol, and a coverslip was applied. Some slides were preincubated with 10 µM AACOCF3 or with 20 µM BEL for 10 min before estradiol treatment. At different times, slides were observed at room temperature under an Olympus IMT-2 inverted microscope equipped with a charge-coupled device CUE video camera, and images were recorded by a Dage MTI camera and digitized by the CUE2 imaging system (Galai Production, Israel). After image recording, the slides were returned to the humidity chamber. Different fields of each slide were recorded at different times.Digitized images allowed the evaluation of NR retention time within lysosomes. The optical density (OD) of 30 cells, randomly selected from each of four coverslips from different cell preparations, was measured first by tracing the cell contour and then recording the average OD within the selected area. An estimate of the ratio between the total cell volume and the overall volume of lysosomes was obtained by measuring whole cell areas and overall lysosomal areas in the 30 different cells selected as described above, using an OD threshold function in the CUE2 imaging system.
[Ca2+]i Measurements
Aliquots of 40 µl of blood suspension were settled on coverslips as described above, incubated with loading buffer containing 4 µM fura 2-AM for 30 min at 15°C, rinsed with buffer to remove extracellular dye, and bathed with 40 µl of physiological saline with or without estradiol. Immediately after exposure to estradiol or to control physiological saline, cells were observed at room temperature under an Olympus IMT-2 inverted microscope equipped with an IMT2-RFL fluorescence attachment (Olympus Optical, Germany) and with an MTI SIT 68 intensified camera (Oatencourt, England). Images were acquired every minute using the CUE2 RMS 4.0 imaging system (Galai Production, Israel). Background fluorescence was subtracted before analysis.Fura 2 calibration was achieved by the equation from Grynkiewicz et al.
(13)
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(1) |
Cell Fractionation and Western Blotting
Blood suspensions (see above) were settled in petri dishes at 15°C, and cells were then exposed to 50 nM estradiol for 30 min, with or without preincubation with 10 µM AACOCF3 for 10 min. Cells were then washed with artificial seawater, scraped in homogenization buffer (50 mM NaF, 0.2 mM Na orthovanadate, 130 mM NaCl, 10 mM phosphate buffer, pH 7.2, 5 µg/ml aprotinin, 5 µg/ml antipain, 5 µg/ml pepstatin A, 1 µg/ml chimostatin, and 1 µg/ml leupeptin; Sigma Chemical) and lysed by means of a tight glass/glass potter. Lysates were centrifuged at 100 g for 15 min using a Sorvall RC-5B (DuPont Instruments, Newtown, CT). The supernatant was centrifuged at 100,000 g for 1.5 h in a Beckman L5-50B Ultracentrifuge (Beckman Instruments, Fullerton, CA), and the ensuing supernatant and pellet were stored atProtein Degradation
Cell radiolabeling. Blood cells were settled onto slides and incubated in physiological saline with 1 µM DEVD-CHO, a caspase-3 inhibitor, 100 µM MG-132, a proteasome inhibitor, plus 1 µCi/ml [14C]valine, (specific activity 250 mCi/mmol), in a humidity chamber at 15°C for 2 h. The reaction was stopped by adding 2 mM of cold valine. Thereafter, cells were treated with 50 nM estradiol at 15°C for 2.5 h, and then 60-µl aliquots were spotted on 3 MM filter paper disks (Whatman, Whatman House, UK) moistened with 5% cold trichloroacetic acid solution (TCA) (diameter = 25 mm). Filters were then transferred to 5% cold TCA for 10 min, washed twice more with the same TCA before being dried, and counted for acid-soluble radioactivity with PicoFluor-40 (Packard, Camberra, AU) in a 1600 CA Tri-Carb scintillation analyzer (Packard). Radioactivity was expressed in cycles per minute per milligram of total protein.
Actin labeling. Cells were incubated with proteasome and caspase-3 inhibitors, as described above, and then treated with 50 nM estradiol at 15°C for 4 h. Thereafter, cells were washed with phosphate-buffered saline (PBS), fixed with 3% paraformaldehyde in PBS at room temperature for 10 min followed by 0.5% Triton X-100 in PBS for 1 min, washed twice with PBS, incubated with 0.1 mg/ml phalloidin-FITC label at room temperature for 30 min, and mounted in glycerol. Slides were observed under a Zeiss LSM 510 confocal microscope (Carl Zeiss, Thornwood, NY).
Statistics
Data were analyzed by the Systat 8.0 software (SPSS, Evanston, IL). ![]() |
RESULTS |
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[Ca2+]i Measurements and Lysosomal Membrane Stability Analysis
Loading of mussel blood cells with fura 2-AM and subsequent imaging with digital fluorescence microscopy revealed that exposure to 50 nM 17
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Lysosomal membrane destabilization was evaluated by staining cells with
the lysosomotropic dye NR. Transmission light microscopy and digital
image analysis showed no lysosome destaining in control cells within
1 h. By contrast, destaining of cells exposed to 50 nM estradiol
started at about 15 min and was highly significant after 1 h,
indicating dye leakage due to lysosomal membrane destabilization (Fig.
1B). The effect of estradiol on lysosomes was almost
entirely prevented by preincubation with AACOCF3, significantly lowered by preincubation with BAPTA-AM, and almost unaffected by preincubation with BEL (Fig. 1B). In addition, the use of fura 2 showed
that AACOCF3 did not produce any effect on cell Ca2+ and
did not prevent the Ca2+ rise induced by estradiol (Table
1).
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In another set of experiments, blood cells were exposed to the
Ca2+ ionophore A-23187 (20 µM), which yielded effects
quite similar to those induced by estradiol. By using fura 2 or NR in
different tests, A-23187 was shown to induce an increase in
[Ca2+]i and lysosomal membrane
destabilization (Fig. 2). Also,
preincubation with AACOCF3 significantly reduced the lysosome
destaining caused by A-23187 (Fig. 2B) without preventing
the Ca2+ rise induced by the ionophore (Table 1). Hence,
data deriving from the use of estradiol and the Ca2+
ionophore indicate that the destabilization of lysosomal membranes occurs mainly through a Ca2+-dependent mechanism, involving
the activation of Ca2+-dependent PLA2. However,
the failure of BAPTA to completely prevent the estradiol effect on
lysosomes suggests that a Ca2+-independent mechanism also
plays a minor role.
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Assessment of Ca2+-Dependent PLA2 Activation
The use of a goat polyclonal antibody on mussel blood cell homogenates permitted detection of a putative homolog of mammalian cPLA2, the Ca2+-dependent cytosolic PLA2. Western blotting after fractionation of mussel blood cell homogenates showed an increase of the cPLA homolog in the particulate fraction of cells exposed to estradiol, indicating that estradiol induces a translocation of Ca2+-dependent PLA2 from cytosol to membranes. Moreover, such an effect was significantly reduced by preincubation with AACOCF3 (Fig. 3).
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Assessment of Lysosomal Volume and Protein Degradation
Because lysosomal membrane destabilization generally leads to an increase in lysosomal volume and cell catabolic activities, we sought similar effects in estradiol-treated cells. In one experiment, transmission light digital imaging of NR-stained cells showed an increase of the ratio between the overall volume of lysosomes and the total cell volume after exposure to estradiol. In this case, too, preincubation with AACOCF3 successfully prevented the estradiol effect, whereas in the presence of BEL the effect of estradiol remained almost the same (Fig. 4A). Quite similar results were found after cell exposure to A-23187 (Fig. 4B).
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In another experiment, the degradation of long-lived protein was
evaluated by staining actin with fluorescein-labeled phalloidin. Quantitative analysis of confocal laser micrographs showed a sharp decrease of fluorescence in cells exposed to estradiol, indicating actin degradation (Fig. 5A).
However, the fluorescence decrease was significantly lowered by cell
preincubation with AACOCF3 (Fig. 5A).
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Finally, the ability of estradiol to induce an increase in protein turnover was investigated by cell labeling with the radioactive amino acid [14C]valine. The release of radioactive amino acid from labeled proteins was higher after exposure to estradiol, indicating an increase in protein breakdown (Fig. 5B), whereas AACOCF3 preincubation abolished such an estradiol effect (Fig. 5B). In these experiments, possible bias because of extralysosomal proteases was limited with the use of specific inhibitors. The use of caspase-3 protease inhibitor was essential for preventing the activation of caspase-3 because of lysosome labilization (15, 20), whereas the use of proteasome inhibitor decreased background noise.
Results from experiments using digital imaging and protein labeling consistently indicate that the lysosomal membrane destabilization induced by estradiol is strictly correlated to an increase of lysosomal volume and protein degradation.
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DISCUSSION |
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Our data have demonstrated that exposure of living mussel blood
cells to 17-estradiol causes a destabilization of lysosomal membranes, detected in terms of increased permeability to NR. Similar
effects of estradiol on lysosomes were pointed out in previous studies
(29, 51), but the present results indicate for the first
time a strict association between lysosomal membrane destabilization
and [Ca2+]i increase.
The estradiol-induced increase in [Ca2+]i can be correlated to different cell processes (36). Moreover, different studies showed that the increase in [Ca2+]i depends on Ca2+ entry (1, 36), Ca2+ release from intracellular stores (12), or both processes (4, 26). Although we did not establish the mechanism of [Ca2+]i elevation in mussel blood cells, we did show that it can be abolished by loading cells with the Ca2+ chelator BAPTA before estradiol stimulation, as previously shown in vascular endothelium by Goetz et al. (12). This evidence was essential for assessing the involvement of Ca2+ in the estradiol effect on lysosomes, because BAPTA also prevents most lysosomal membrane destabilization in estradiol-treated cells. Further confirmation arose from the use of the Ca2+ ionophore A-23187, showing that an increase in [Ca2+]i, similar to that caused by estradiol, is sufficient to produce a significant destabilizing effect on lysosomal membranes.
Having established the role of Ca2+ as a mediator of estradiol effects, several arguments pointed to Ca2+-dependent PLA2 as the potential connection between Ca2+ and lysosomes. Many cell types and organs express a cytosolic Ca2+-dependent PLA2 that selectively releases arachidonic acid from membrane phospholipids (17). PLA2 activity is responsible for eicosanoid synthesis, which is crucial to the initiation of the inflammatory response (2) and is activated by Ca2+-dependent translocation from the soluble to the membrane fraction of cells, allowing access to the arachidonoyl-containing phospholipid substrate (5, 42). The lipolytic activity of PLA2 can affect different membrane structures, possibly also including lysosomal membrane, as has been seen in snake venom PLA2 (33). Moreover, PLA2 plays an important role in blood phagocytic cells through the activation of NADPH oxidase (9).
In our experiments, BAPTA considerably reduced the effects of estradiol on lysosomes, whereas the inhibitor of Ca2+-dependent PLA2, AACOCF3, almost entirely blocked these effects without preventing an increase in [Ca2+]i. By contrast, the inhibitor of Ca2+-independent PLA2 (BEL) had no effect. These results indicate that Ca2+-dependent cytosolic PLA2 is the agent responsible for estradiol-induced lysosomal membrane destabilization. However, experiments with BAPTA showed that when Ca2+ is removed from the cytosol, estradiol is still capable of inducing a slight destaining of lysosomes, although the lysosome destaining induced by A-23187 was only partially abolished by AACOCF3. This suggests that either estradiol or Ca2+ can induce limited membrane destabilization on lysosomes independently from the Ca2+/PLA2 pathway. Overall, however, the data indicate that the Ca2+-dependent PLA2 pathway is the main mechanism by which estradiol produces its effect on lysosomes. A further confirmation of this mechanism comes from Western blotting experiments, which clearly showed cPLA2 translocation to the membranes induced by estradiol.
Membrane destabilization leads to fusion of membrane-bound organelles, in which a role is played by arachidonic acid (6). Accordingly, experimental evidence points to the involvement of PLA2 in lysosome/endosome fusion (25). Moreover, lysosomal membrane destabilization is known to be correlated both to enlargement of lysosomes (16), suggesting lysosome/endosome fusion, and to intracellular free Ca2+ elevation in fibroblasts (3). In addition, it has been shown that lysosomal fusion involves activation of different catabolic activities, particularly protein degradation (30). This scenario is consistent with the results of the present study. In our experimental system, the PLA2-dependent increase in lysosome volume following estradiol treatment is no doubt linked to increased fusion activities. Also, the increase in lysosome volume that was induced by estradiol or A-23187 was completely abolished by AACOCF3. This clearly indicates that lysosome fusion is totally dependent on PLA2 activation, in contrast to what happens in lysosome destaining during the NR assay. In addition, our data on degradation rates of both short- and long-lived proteins show that estradiol can induce metabolic activation of the lysosomal machinery, whereas the use of AACOCF3 confirmed the involvement of Ca2+-dependent PLA2 in this process.
In conclusion, we have identified a short-term pathway of estradiol, which consists in an activation of Ca2+-dependent PLA2 and leads to typical morphofunctional modifications of the lysosomal vacuolar system. Besides yielding a molecular explanation for endogenous lysosome activation, this study also offers a new perspective for the understanding of lysosomal alterations due to pathological conditions or the effects of xenobiotic compounds. Lysosomal membrane destabilization can be used as a biomarker of stress, and a variety of stressors causing lysosomal alterations, such as heavy metals and oxidants (28, 31, 49), are also known to affect cell Ca2+ homeostasis (35). Therefore, an involvement of Ca2+-dependent PLA2 could also be postulated in these cases. Hence, the mechanism described in the present study may be relevant to different fields of investigation, ranging from the understanding of cellular mechanisms of lysosome activation to the application of lysosome-based biomarkers in ecological risk assessment.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from Ministero dell' Università e della Ricerca Scientifica e Tecnologica (MURST). B. Marchi is the recipient of a MURST postdoctoral fellowship.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. Burlando, Dipartimento di Scienze e Tecnologie Avanzate, Università del Piemonte Orientale "Amedeo Avogadro," Corso Borsalino 54, 15100 Alessandria, Italy (E-mail: burlando{at}unipmn.it).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 24, 2002;10.1152/ajpcell.00429.2001
Received 6 September 2001; accepted in final form 16 July 2002.
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