Essential role of Ca2+-dependent phospholipase A2 in estradiol-induced lysosome activation

Bruno Burlando1, Barbara Marchi2, Isabella Panfoli3, and Aldo Viarengo1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanism of lysosome activation by 17beta -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; 17beta -estradiol; cytosolic phospholipase A2; calcium signaling; AACOCF3; BEL


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE GONADAL STEROID 17beta -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 17beta -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 17beta -estradiol in particular (19). The presence of 17beta -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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals

Bromoenol lactone (BEL), bovine serum albumin (BSA), digitonin, 17beta -estradiol, neutral red (NR), FITC-labeled phalloidin, poly-L-lysine, and Sigmacote were from Sigma Chemical (St. Louis, MO). Fura 2 was from Molecular Probes (Eugene, OR). DEVD-CHO was from Biomol Research Laboratories (Plymouth Meeting, PA). AACOCF3, MG-132, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) were from CalBiochem (La Jolla, CA). [14C]valine was from NEN Life Science Products (Boston, MA). All other reagents were of analytical grade.

Solutions

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)
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB>(F − F<SUB>min</SUB>)/(F<SUB>max</SUB> − F)Sf<SUB>2</SUB>/Sb<SUB>2</SUB> (1)
where Kd = 135 nM, Fmax and Fmin are maximum and minimum fluorescence intensities, measured after cell treatment with 50 µM digitonin and 5 mM EGTA, respectively, and Sf2/Sb2 is the ratio between the excitation efficiencies of free probe and Ca2+-bound probe at 380 nm.

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 at -40°C until used. Protein determination was performed using the bicinchoninic protein assay (BCA; Pierce Chemical, Rockford, IL). Samples were electrophoresed under reducing conditions on 8.5% SDS-PAGE, and proteins were blotted to Hybond ECL filters (Amersham Pharmacia Biotech, Uppsala, Sweden) using a Hoefer TE blotting device (Hoefer Scientific Instruments, San Francisco, CA). Filters were incubated for 1 h at 4°C with goat polyclonal anti-cPLA2 (C-20; Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:100 in Tris-buffered saline containing Tween 20 (TBST)-BSA 3.5%, and then incubated for 1 h with horseradish peroxidase (HRP)-conjugated anti-goat IgG (Santa Cruz) diluted 1:500 in TBST-BSA 1%. Binding of antibodies was visualized by the enhanced chemiluminescence detection system (Roche, Basel, Switzerland), digitized by the Fluor-S Max gel analyzer (Bio-Rad Laboratories), and analyzed by Quantity One 4.2.1 software (Bio-Rad Laboratories).

Protein 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

[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 17beta -estradiol induced a significant increase in [Ca2+]i (Fig. 1A) that was completely prevented by cell loading with the Ca2+ chelator BAPTA-AM (15 µM for 15 min) before estradiol exposure (Fig. 1A).


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Fig. 1.   Effects of estradiol on the [Ca2+]i and lysosomes of mussel blood cells. A: free Ca2+ measurements in fura 2-loaded cells treated with 50 nM estradiol show a sustained Ca2+ rise starting from 10 min after exposure. Preincubation with the Ca2+ chelator BAPTA-AM (15 µM) for 15 min completely abolished the effect of estradiol on cytosolic Ca2+. Data are means ± SD of 8 Ca2+ measurements in different cells. B: digital imaging measurements of the optical density of cells 60 min after staining with the lysosomotropic dye neutral red (see MATERIALS AND METHODS). Data show significant destaining upon treatment with estradiol (50 nM, exposure starts at t = 0). Different preincubations before estradiol exposure show the following results: 1) almost complete destaining using 20 µM (E)-6-(bromomethylene)tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (BEL) for 10 min; 2) limited destaining using 15 µM BAPTA-AM for 15 min; and 3) nonsignificant destaining using 10 µM arachidonyl trifluoromethyl ketone (AACOCF3) for 10 min. Data are means ± SD (n = 30) expressed as a percentage of control optical density. Bars with different letters indicate significant differences according to the Bonferroni test (P < 0.01).

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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Table 1.   Variations of [Ca2+]i in mussel blood cells exposed to different compounds and in the presence or absence of AACOCF3

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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Fig. 2.   Effects of the Ca2+ ionophore A-23187 on [Ca2+]i and lysosome membrane stability. A: fura 2-loaded cells exposed to 20 µM A-23187 show sustained [Ca2+]i rise. B: cells stained with neutral red show significant destaining after treatment with A-23187 (exposure starts at t = 0), whereas the effect is partially prevented by preincubation with 10 µM AACOCF3 for 10 min. Data are expressed as described in Fig. 1.

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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Fig. 3.   Western blot analysis of cPLA2 in cytosolic and particulate fractions of mussel blood cells. Protein (50 µg) were solved on SDS-polyacrylamide gels, transferred to Hybond ECL membranes, and probed with goat polyclonal anti-cPLA2 (see MATERIALS AND METHODS). A: exposure to estradiol induces cPLA2 translocation to membranes, as shown by the increase of the cPLA2 band in the particulate fraction and by the parallel decrease in the cytosolic one with respect to control. Incubation with AACOCF3 before estradiol exposure produces a strong reduction of cPLA2 band increase in the particulate fraction. B: digital image quantification of band peak densities.

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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Fig. 4.   Effects of estradiol (A) and A-23187 (B) on the size of lysosomes. Digital imaging evaluation of the lysosome-to-cell volume ratio shows an increase in the overall lysosomal volume after 60 min of exposure to either 50 nM estradiol or 20 µM A-23187. The fraction of the cell volume pertaining to lysosomes varies from about 23-24% (t = 0 min) to about 36-38% (t = 60 min). The effects of both estradiol and A-23187 on lysosome volume are abolished by preincubation with AACOCF3 but not by preincubation with BEL. Estr, estradiol. Data are means ± SD (n = 30); * P < 0.01 in a t-test comparison between t = 0 and t = 60 min.

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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Fig. 5.   Effect of estradiol on protein degradation, as evaluated after cell labeling with phalloidin-FITC or [14C]valine. A: cell stained for actin using phalloidin-FITC and incubated with estradiol, with or without AACOCF3 preincubation, show different fluorescence intensities when observed under a confocal microscope. Treatment with 50 nM estradiol induces significant fluorescence decrease after 4.5 h with respect to control, whereas AACOCF3 preincubation partially prevents such a fluorescence decrease. Data are means ± SD (n = 46). All groups are significantly different from each other (P < 0.01) according to the Bonferroni t-test. Insets: confocal images of phalloidin-labeled cells after the different incubations (bar = 25 µm). B: cells labeled with [14C]valine show increased release of the radioactive amino acid after 2.5 h of treatment with estradiol. Preincubation with AACOCF3 is able to restore [14C]valine release to control values. Data are means ± SD (n = 6), expressed as a percentage of specific activity in control cells. * P < 0.05, Bonferroni test.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our data have demonstrated that exposure of living mussel blood cells to 17beta -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Audy, MC, Vacher P, and Duly B. 17beta -Estradiol stimulates a rapid Ca2+ influx in LNCaP human prostate cancer cells. Eur J Endocrinol 135: 367-373, 1996[ISI][Medline].

2.   Axelrod, J. Receptor-mediated activation of phospholipase A2 and arachidonic acid release in signal transduction. Biochem Soc Trans 18: 503-507, 1990[ISI][Medline].

3.   Bakker, AC, Webster P, Jacob WA, and Andrews NW. Homotypic fusion between aggregated lysosomes triggered by elevated [Ca2+]i in fibroblasts. J Cell Sci 110: 2227-2238, 1997[Abstract/Free Full Text].

4.   Benten, WP, Lieberherr M, Giese G, and Wunderlich F. Estradiol binding to cell surface raises cytosolic free calcium in T cells. FEBS Lett 422: 349-353, 1998[ISI][Medline].

5.   Bittova, L, Sumandea M, and Cho W. A structure-function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues. J Biol Chem 274: 9665-9672, 1999[Abstract/Free Full Text].

6.   Burger, KN, and Verkleij AJ. Membrane fusion. Experientia 46: 631-644, 1990[ISI][Medline].

7.   Cajaraville, MP, and Pal SG. Morphofunctional study of the haemocytes of the bivalve mollusc Mytilus galloprovincialis with emphasis on the endolysosomal compartment. Cell Struct Funct 20: 355-367, 1995[ISI][Medline].

8.   Clark, JD, Lin LL, Kriz RW, Ramesha CS, Sultzman LA, Lin AY, Milona N, and Knopf JL. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP. Cell 65: 1043-1051, 1991[ISI][Medline].

9.   Dana, R, Leto TL, Malech HL, and Levy R. Essential requirement of cytosolic phospholipase A2 for activation of the phagocyte NADPH oxidase. J Biol Chem 273: 441-445, 1998[Abstract/Free Full Text].

10.   Fernley, PW, Moore MN, Lowe DM, Donkin P, Evans S, and Gabrielescu E. Impact of the Sea Empress oil spill on lysosomal stability in mussel blood cells. Mar Environ Res 50: 451-455, 2000[ISI][Medline].

11.   Gabrielescu, E, Butur G, Nicolau N, Ciobanu A, and Nutu O. Histochemical investigation of myocardial proteases in heart anoxia, under protection with cardioplegic solution and protease inhibitors. Physiologie 26: 101-110, 1989[Medline].

12.   Goetz, RM, Thatte HS, Prabhakar P, Cho MR, Michel T, and Golan DE. Estradiol induces the calcium-dependent translocation of endothelial nitric oxide synthase. Proc Natl Acad Sci USA 96: 2788-2793, 1999[Abstract/Free Full Text].

13.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

14.   Harris, DC, Tay YC, Chen J, Chen L, and Nankivell BJ. Mechanisms of iron-induced proximal tubule injury in rat remnant kidney. Am J Physiol Renal Fluid Electrolyte Physiol 269: F218-F224, 1995[Abstract/Free Full Text].

15.   Ishisaka, R, Utsumi T, Yabuki M, Kanno T, Furuno T, Inoue M, and Utsumi K. Activation of caspase-3-like protease by digitonin-treated lysosomes. FEBS Lett 435: 233-236, 1998[ISI][Medline].

16.   Kohler, A. Lysosomal perturbations in fish liver as indicators for toxic effects of environmental pollution. Comp Biochem Physiol 100C: 123-127, 1991[ISI].

17.   Kramer, RM, and Sharp JD. Structure, function and regulation of Ca2+-sensitive cytosolic phospholipase A2 (cPLA2). FEBS Lett 410: 49-53, 1997[ISI][Medline].

18.   Levin, ER. Cellular functions of the plasma membrane estrogen receptor. Trends Endocrinol Metab 10: 374-377, 1999[ISI][Medline].

19.   Li, W, Yuan X, Nordgren G, Dalen H, Dubowchik GM, Firestone RA, and Brunk UT. Induction of cell death by the lysosomotropic detergent MSDH. FEBS Lett 470: 35-39, 2000[ISI][Medline].

20.   Lia, W, Yuana X, Nordgrena G, Dalenb H, Dubowchikc GM, Firestoned RA, and Brunka UT. Induction of cell death by the lysosomotropic detergent MSDH. FEBS Lett 470: 35-39, 2000[ISI][Medline].

21.   Lowe, DM, and Pipe RK. Contaminant induced lysosomal membrane damage in marine mussel digestive cells: an in vitro study. Aquat Toxicol (Amst) 30: 357-365, 1994.

22.   Luzio, JP, Rous BA, Bright NA, Pryor PR, Mullock BM, and Piper RC. Lysosome-endosome fusion and lysosome biogenesis. J Cell Sci 113: 1515-1524, 2000[Abstract/Free Full Text].

23.   Mak, IT, Misra HP, and Weglicki WB. Temporal relationship of free radical-induced lipid peroxidation and loss of latent enzyme activity in highly enriched hepatic lysosomes. J Biol Chem 258: 13733-13737, 1983[Abstract/Free Full Text].

24.   Marone, G, Fimiani B, Torella G, Poto S, Bianco P, and Condorelli M. Possible role of arachidonic acid and of phospholipase A2 in the control of lysosomal enzyme release from human polymorphonuclear leukocytes. J Clin Lab Immunol 12: 111-116, 1983[ISI][Medline].

25.   Mayorga, LS, Colombo MI, Lennartz M, Brown EJ, Rahman KH, Weiss R, Lennon PJ, and Stahl PD. Inhibition of endosome fusion by phospholipase A2 (PLA2) inhibitors points to a role for PLA2 in endocytosis. Proc Natl Acad Sci USA 90: 10255-10259, 1993[Abstract].

26.   Moini, H, Bilsel S, Bekdemir T, and Emerk K. 17beta -Estradiol increases intracellular free calcium concentrations of human vascular endothelial cells and modulates its responses to acetylcholine. Endothelium 5: 11-19, 1997[Medline].

27.   Moore, MN. Cytochemical demonstration of latency of lysosomal hydrolases in digestive cells of the common mussel, Mytilus edulis, and changes induced by thermal stress. Cell Tissue Res 175: 279-287, 1976[ISI][Medline].

28.   Moore, MN. Cellular responses to pollutants. Mar Pollut Bull 16: 134-139, 1985[ISI].

29.   Moore, MN, Lowe DM, and Fieth PEM Responses of lysosomes in the digestive cells of the common mussel, Mytilus edulis, to sex steroids and cortisol. Cell Tissue Res 188: 1-9, 1978[ISI].

30.   Moore, MN, and Viarengo A. Lysosomal membrane fragility and catabolism of cytosolic proteins: evidence for a direct relationship. Experientia 43: 320-323, 1987[ISI][Medline].

31.   Moore, MN, Widdows J, Cleary JJ, Pipe RK, Salkeld PN, Donkin P, Farrar SV, Evans SV, and Thomson PE. Responses of the mussel Mytilus edulis to copper and phenanthrene: interactive effects. Mar Environ Res 14: 167-183, 1984[ISI].

32.   Mortimore, GE, Miotto G, Venerando R, and Kadowaki M. Autophagy. In: Biology of the Lysosome. Subcellular Biochemistry, edited by Lloyd JB, and Mason RW.. New York, London: Plenum, 1996, vol. 27, p. 93-135.

33.   Mukherjee, AK, Ghosal SK, and Maity CR. Lysosomal membrane stabilization by alpha -tocopherol against the damaging action of Vipera russelli venom phospholipase A2. Cell Mol Life Sci 53: 152-155, 1997[ISI][Medline].

34.   Neuzil, J, Svensson I, Weber T, Weber C, and Brunch UT. alpha -Tocopherol succinate-induced apoptosis in Jurkat T cells involves caspase-3 activation, and both lysosomal and mitochondrial destabilization. FEBS Lett 445: 295-300, 1999[ISI][Medline].

35.   Orrenius, S, McConkey DJ, Bellomo G, and Nicotera P. Role of Ca2+ in toxic cell killing. Trends Pharmacol Sci 10: 281-285, 1989[ISI][Medline].

36.   Picotto, G, Massheimer V, and Boland R. Acute stimulation of intestinal cell calcium influx induced by 17beta -estradiol via the cAMP messenger system. Mol Cell Endocrinol 119: 129-134, 1996[ISI][Medline].

37.   Pipe, RK. Hydrolytic enzymes associated with the granular haemocytes of the marine mussel Mytilus edulis. Histochem J 22: 595-603, 1990[ISI][Medline].

38.   Reis-Henriques, MA, and Coimbra J. Variations in the levels of progesterone in Mytilus edulis during the annual reproductive cycle. Comp Biochem Physiol 95A: 343-348, 1990[ISI].

39.   Reis-Henriques, MA, Le Guellec D, Remy-Martin JP, and Adessi GL. Studies of endogenous steroids from the marine mollusc Mytilus edulis L. by gas chromatography and mass spectrometry. Comp Biochem Physiol 95B: 303-309, 1990.

40.   Roberg, K, and Ollinger K. Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes. Am J Pathol 152: 1151-1156, 1998[Abstract].

41.   Russell, KS, Haynes MP, Sinha D, Clerisme E, and Bender JR. Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling. Proc Natl Acad Sci USA 23: 5930-5935, 2000.

42.   Schievella, AR, Regier MK, Smith WL, and Lin LL. Calcium-mediated translocation of cytosolic phospholipase A2 to the nuclear envelope and endoplasmic reticulum. J Biol Chem 270: 30749-30754, 1995[Abstract/Free Full Text].

43.   Smythe, E. Endocytosis. In: Biology of the Lysosome. Subcellular Biochemistry, edited by Lloyd JB, and Mason RW.. New York, London: Plenum, 1996, vol. 27, p. 51-92.

44.   Stefano, GB, Cadet P, Breton C, Goumon Y, Prevot V, Dessaint JP, Beauvillain JC, Roumier AS, Welters I, and Salzet M. Estradiol-stimulated nitric oxide release in human granulocytes is dependent on intracellular calcium transients: evidence of a cell surface estrogen receptor. Blood 95: 3951-3958, 2000[Abstract/Free Full Text].

45.   Szego, CM. Lysosomal function in nucleocytoplasmic communication. In: Lysosomes in Biology and Pathology, edited by Dingle JT, and Dean R.. Amsterdam: Elsevier, 1975, vol. 4, p. 385-477.

46.   Tadevosian, IV, Karagezian KG, Gevorkian GA, and Batikian TB. Role of phospholipids in changes in lysosomal membrane stability in conditions of chronic alcoholic intoxication. Biull Eksp Biol Med 100: 553-554, 1985[Medline].

47.   Takuma, T, and Ichida T. Role of Ca2+-independent phospholipase A2 in exocytosis of amylase from parotid acinar cells. J Biochem (Tokyo) 121: 1018-1024, 1997[Abstract].

48.   Viarengo, A, Lafaurie M, Gabrielides GP, Fabbri R, Marro A, and Romeo M. Critical evaluation of an intercalibration exercise undertaken in the framework of the MED POL biomonitoring program. Mar Environ Res 49: 1-18, 2000[ISI][Medline].

49.   Viarengo, A, Marro A, Marchi B, and Burlando B. Single and combined effects of heavy metals and hormones on lysosomes of hemolymph cells from the mussel Mytilus galloprovincialis. Mar Biol (Berl) 137: 907-912, 2000.

50.   Wattiaux, R, and Wattiaux-De Coninck S. Lysosome pharmacology and toxicology. In: Biology of the Lysosome. Subcellular Biochemistry, edited by Lloyd JB, and Mason RW.. New York, London: Plenum, 1996, vol. 27, p. 387-409.

51.   Weissmann, G. The effects of steroids and drugs on lysosomes. In: Lysosomes in Biology and Pathology, edited by Dingle JT, and Fell HB.. Amsterdam: Elsevier, 1969, vol. 1, p. 276-295.

52.   Winston, GW, Moore MN, Kirchin MA, and Soverchia C. Production of reactive oxygen species by hemocytes from the marine mussel, Mytilus edulis: lysosomal localization and effect of xenobiotics. Comp Biochem Physiol 113C: 221-229, 1996.

53.   Zdolsek, J, Zhang H, Roberg K, and Brunk U. H2O2-mediated damage to lysosomal membranes of J-774 cells. Free Radic Res Commun 18: 71-85, 1993[ISI][Medline].


Am J Physiol Cell Physiol 283(5):C1461-C1468
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