Attenuation of the Lysosomal Death Pathway by Lysosomal Cholesterol Accumulation
2011; Elsevier BV; Volume: 178; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2010.10.030
ISSN1525-2191
AutoresHanna Appelqvist, Cathrine Nilsson, Brett Garner, Andrew J. Brown, Katarina Kågedal, Karin Öllinger,
Tópico(s)Autophagy in Disease and Therapy
ResumoIn the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability. In the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability. Lysosomes are organelles involved in macromolecule turnover and, consequently, contain numerous hydrolytic enzymes, including the cathepsin family of proteases. Cathepsins are synthesized as inactive proenzymes, which are transported to the lysosomes and converted to active proteases inside these organelles. In addition to their involvement in protein degradation, cathepsins also have several other functions, including promotion of apoptotic cell death.1Boya P. Kroemer G. Lysosomal membrane permeabilization in cell death.Oncogene. 2008; 27: 6434-6451Crossref PubMed Scopus (1016) Google Scholar Release from the lysosomes into the cytosol is critical for participation of cathepsins in apoptosis. Lysosomotropic detergents, which selectively target the lysosomal membrane, are compounds that remain predominantly unprotonated and inert in the cytosol, but they become protonated, accumulate, and acquire detergent properties in the acidic interior of lysosomes.2Firestone R.A. Pisano J.M. Bonney R.J. Lysosomotropic agents.1. Synthesis and cytotoxic action of lysosomotropic detergents. J Med Chem. 1979; 22: 1130-1133Google Scholar Low concentrations of lysosomotropic detergents cause a limited translocation of lysosomal contents leading to apoptosis, whereas higher concentrations result in massive lysosomal release and subsequent necrotic cell death.3Li W. Yuan X. Nordgren G. Dalen H. Dubowchik G.M. Firestone R.A. Brunk U.T. Induction of cell death by the lysosomotropic detergent MSDH.FEBS Lett. 2000; 470: 35-39Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar Lysosomal membrane permeabilization (LMP) and subsequent release of cathepsins is an early event during apoptosis induced by a number of stimuli, such as oxidative stress, death receptor ligation, and DNA-damaging drugs.1Boya P. Kroemer G. Lysosomal membrane permeabilization in cell death.Oncogene. 2008; 27: 6434-6451Crossref PubMed Scopus (1016) Google Scholar Because the presence of cathepsins in the cytosol is sufficient to trigger apoptosis,4Roberg K. Kågedal K. Öllinger K. Microinjection of cathepsin D induces caspase-dependent apoptosis in fibroblasts.Am J Pathol. 2002; 161: 89-96Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar the permeability of the lysosomal membrane is tightly regulated to prevent accidental cell death. Increasing evidence suggests that several proteins, including antiapoptotic Bcl-2 proteins and heat shock proteins (Hsps), act as safeguards of lysosomal membrane integrity.5Johansson A.C. Appelqvist H. Nilsson C. Kågedal K. Roberg K. Öllinger K. Regulation of apoptosis-associated lysosomal membrane permeabilization.Apoptosis. 2010; 15: 527-540Crossref PubMed Scopus (339) Google ScholarAs an essential component of cellular membranes, cholesterol is critical for many cellular functions, including signal transduction and membrane trafficking. The majority of cellular cholesterol normally resides in the plasma membrane.6Mesmin B. Maxfield F.R. Intracellular sterol dynamics.Biochim Biophys Acta. 2009; 1791: 636-645Crossref PubMed Scopus (196) Google Scholar However, cells from patients with Niemann-Pick disease type C (NPC) are notable exceptions, in that a large amount of cholesterol is found in lysosomes.7Sokol J. Blanchette-Mackie J. Kruth H.S. Dwyer N.K. Amende L.M. Butler J.D. Robinson E. Patel S. Brady R.O. Comly M.E. et al.Type C Niemann-Pick disease Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol.J Biol Chem. 1988; 263: 3411-3417Abstract Full Text PDF PubMed Google Scholar NPC is an inherited lysosomal storage disorder that is characterized by a failure in cholesterol trafficking that leads to accumulation of unesterified cholesterol and sphingolipids in the endo-lysosomal system.8Liscum L. Faust J.R. The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one.J Biol Chem. 1989; 264: 11796-11806Abstract Full Text PDF PubMed Google Scholar This disease is caused by mutations in the genes coding for the proteins NPC1 and NPC2, which are crucial for transport of cholesterol from the lysosomal compartment to the endoplasmic reticulum for esterification and redistribution to other cellular compartments.6Mesmin B. Maxfield F.R. Intracellular sterol dynamics.Biochim Biophys Acta. 2009; 1791: 636-645Crossref PubMed Scopus (196) Google Scholar U18666A [3-β-(2-[diethylamino]ethoxy)-androst-5-en-17-one, monohydrochloride] is an amphiphilic drug known to inhibit cholesterol transport8Liscum L. Faust J.R. The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one.J Biol Chem. 1989; 264: 11796-11806Abstract Full Text PDF PubMed Google Scholar and, therefore, commonly used to mimic NPC defects in cell culture models. U18666A treatment results in lysosomal cholesterol accumulation,8Liscum L. Faust J.R. The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one.J Biol Chem. 1989; 264: 11796-11806Abstract Full Text PDF PubMed Google Scholar which is associated with induction of apoptosis in the primary cortical neurons of mice.9Cheung N.S. Koh C.H. Bay B.H. Qi R.Z. Choy M.S. Li Q.T. Wong K.P. Whiteman M. Chronic exposure to U18666A induces apoptosis in cultured murine cortical neurons.Biochem Biophys Res Commun. 2004; 315: 408-417Crossref PubMed Scopus (36) Google Scholar Cholesterol reinforces lysosomal membrane stability in cell-free experiments. Addition of cholesterol to isolated lysosomes reduces permeability,10Fouchier F. Mego J.L. Dang J. Simon C. Thyroid lysosomes: the stability of the lysosomal membrane.Eur J Cell Biol. 1983; 30: 272-278PubMed Google Scholar whereas a reduced lysosomal membrane cholesterol level is associated with increased permeability to protons and potassium ions that, in turn, causes an osmotic imbalance and destabilization of the lysosomal membrane.11Jadot M. Andrianaivo F. Dubois F. Wattiaux R. Effects of methylcyclodextrin on lysosomes.Eur J Biochem. 2001; 268: 1392-1399Crossref PubMed Scopus (32) Google Scholar The aim of this study was to investigate whether lysosomal accumulation of cholesterol alters lysosomal membrane permeability and cellular sensitivity to induction of apoptosis.Materials and MethodsAntibodiesThe following antibodies were used: rabbit anti-cathepsin D (01-12-030104; Athens Research and Technology, Athens, GA); mouse anti-LBPA (anti–lysobisphosphatidic acid; Z-SLBPA; Echelon Biosciences, Salt Lake City, UT); mouse anti-LAMP-2 (anti–lysosomal-associated membrane protein 2; 9840-01; Southern Biotech, Birmingham, AL); rabbit anti-LC3B (NB600-1384; Novus Biologicals, Littleton, CO); goat anti-LDH (anti–polyclonal lactate dehydrogenase; 20-LG22; Fitzgerald Industries International, Concord, MA); mouse anti-GAPDH (anti–glyceraldehyde 3-phosphate dehydrogenase; GTX78213, GeneTex, Irvine, CA); and rabbit anti–Mcl-1(sc-958), rabbit anti–Bcl-XL (sc-7195), mouse anti–Bcl-2 (sc-509), and mouse anti-Hsp70 (sc-24), all from Santa Cruz Biotechnology (Santa Cruz, CA).Cells and Culture ConditionsHuman foreskin fibroblasts (AG01518; passages 12–24; Coriell Institute for Medical Research, Camden, NJ) were cultured in Eagle's minimum essential medium supplemented with 2 mmol/L glutamine, 50 IU/ml penicillin-G, 50 μg/ml streptomycin, and 10% fetal bovine serum (all from GIBCO, Paisley, UK). Cells were incubated in humidified air with 5% CO2 at 37°C and were subcultured once a week. Before experiments, cells were trypsinized and seeded to reach 80% confluence at apoptosis induction. Cells were pretreated with 0.5 μg/ml U18666A (Sigma-Aldrich, St. Louis, MO) in complete cell culture medium for the indicated durations. For selected experiments, 25-hydroxycholesterol (25-HC; 1 μg/ml; Sigma-Aldrich) was added simultaneously with U18666A. At the times indicated, cells were pretreated with 3-methyladenine (3-MA; 5 mmol/L, 1 hour; Sigma-Aldrich). Apoptosis was induced by exposing cells to the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride [MSDH; 15 μmol/L; kindly provided by Gene M. Dubowchik (Bristol-Myers Squibb, Wallingford, CT)], the protein kinase inhibitor staurosporine (STS; 0.1 μmol/L; Sigma-Aldrich), or the anticancer drug cisplatin (40 μg/ml; Meda, Solna, Sweden). Apoptosis-inducing agents were added in serum-free medium and, for pretreated cultures, in the presence of U18666A.Chinese hamster ovary 2-2 (CHO 2-2) cells are mutated from CHO-K112Dahl N.K. Reed K.L. Daunais M.A. Faust J.R. Liscum L. Isolation and characterization of Chinese hamster ovary cells defective in the intracellular metabolism of low density lipoprotein-derived cholesterol.J Biol Chem. 1992; 267: 4889-4896Abstract Full Text PDF PubMed Google Scholar and represent a classic NPC phenotype.13Dahl N.K. Daunais M.A. Liscum L. A second complementation class of cholesterol transport mutants with a variant Niemann-Pick type C phenotype.J Lipid Res. 1994; 35: 1839-1849Abstract Full Text PDF PubMed Google Scholar The Npc1 gene of the CHO 2-2 cells contains an insertion resulting in a frame shift and early termination of NPC1 translation, creating a nonfunctional protein.14Wojtanik K.M. Liscum L. The transport of low density lipoprotein-derived cholesterol to the plasma membrane is defective in NPC1 cells.J Biol Chem. 2003; 278: 14850-14856Crossref PubMed Scopus (125) Google Scholar CHO cells were cultured in 1:1 Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture supplemented with 2 mmol/L glutamine, 50 IU/ml penicillin-G, 50 μg/ml streptomycin, and 10% fetal bovine serum (all from GIBCO). Cells were incubated in humidified air with 5% CO2 at 37°C and were subcultured twice a week. Before experiments, CHO-K1 and CHO 2-2 were trypsinized and seeded 1 day before experiments to reach 80% confluence at the time of exposure to STS (0.5 μmol/L) or MSDH (50 μmol/L).Measurement of Viability and ApoptosisViability of cell cultures was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay (Calbiochem, San Diego, CA). Cells were incubated with 0.25 mg/ml MTT for 2 hours at 37°C. The MTT solution was then removed and the formazan product dissolved in dimethyl sulfoxide. The absorbance was measured at 550 nm. Caspase-3–like activity was analyzed using the substrate Ac-DEVD-AMC (Becton, Dickinson and Company, Franklin Lakes, NJ) according to the manufacturer's instructions. Fluorescence was correlated to protein content.Cholesterol MeasurementCholesterol content of fibroblasts was measured in cell lysates using the Amplex Red Cholesterol Assay Kit (Invitrogen, Paisley, UK), as described by the manufacturer. Cholesterol content in CHO cells was determined by reversed-phase, high-performance liquid chromatography using an established method.15Fedorow H. Pickford R. Hook J.M. Double K.L. Halliday G.M. Gerlach M. Riederer P. Garner B. Dolichol is the major lipid component of human substantia nigra neuromelanin.J Neurochem. 2005; 92: 990-995Crossref PubMed Scopus (54) Google Scholar Cells were lysed in 0.2 mol/L NaOH and then mixed with methanol. Hexane was added, and the samples were centrifuged at 3000 × g for 15 minutes (4°C). The hexane phase was collected and dried down in a SpeedVac centrifuge (Thermo Electron Corporation, Waltham, MA) and the lipids resuspended in 100 μl of isopropanol. Cholesterol content was expressed relative to cellular protein content.ImmunocytochemistryFor staining of mitochondria, cells were incubated with MitoTracker Red CMXRos (200 nmol/L, 30 minutes, 37°C; Invitrogen). Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline for 20 minutes at 4°C. For visualization of cathepsin D, this was followed by methanol fixation (20 minutes, −20°C). After incubation in 0.1% saponin and 5% fetal bovine serum (20 minutes, room temperature), cells were incubated with primary antibodies overnight at 4°C. Cells were rinsed then incubated (1 hour, room temperature) with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to AlexaFluor 488 or AlexaFluor 594 (Invitrogen). To visualize unesterified cholesterol, cells were stained with filipin (125 μg/ml; Sigma-Aldrich) for 1 hour at room temperature. Coverslips were washed and mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Cells were examined using a Nikon Eclipse E600 laser scanning confocal microscope (Nikon, Tokyo, Japan) together with the EZC1 3.7 software (Nikon Instruments, Melville, NY) or a Nikon Eclipse TE2000U microscope (Nikon) with Bio-Rad Radiance 2100 MP confocal system (Carl Zeiss, Jena, Germany). The fluorescence intensity was quantified using ImageJ software 1.42q (National Institutes of Health, Bethesda, MD). For each treatment, 10 images (5 each from two separate experiments) were analyzed. The lysosomal area was selected (from images capturing LAMP-2–stained lysosomes), and the filipin intensity within these areas was analyzed. The mean fluorescence value from each image was recorded, and the overall mean was calculated (n = 10). The intracellular bis(monoacylglycero)phosphate (BMP) fluorescence was analyzed similarly.Flow Cytometric Determination of LysoTracker FluorescenceCells were stained with 50 nmol/L LysoTracker Green DND-26 (Invitrogen) for 5 minutes at 37°C and detached by trypsinization. LysoTracker Green was excited using a 488-nm argon laser, and the resulting fluorescence was detected in the FL1 channel using a 530 ± 28 nm filter. We collected 10,000 cells, and data were analyzed using CellQuest software (Becton, Dickinson and Company).Extraction of CytosolCytosol was extracted using the cholesterol-solubilizing agent digitonin (Sigma-Aldrich). When used at low concentrations, digitonin permeabilizes the plasma membrane, which has high cholesterol content, but leaves intracellular membranes with lower cholesterol content intact. Cell culture dishes were incubated for 12 minutes, rocking on ice, in extraction buffer [250 mmol/L sucrose, 20 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), and 1 mmol/L ethylene glycol tetraacetic acid (EGTA)] containing the indicated concentrations of digitonin before the extraction buffer was withdrawn. The release of cytosolic content was measured as lactate dehydrogenase (LDH) activity. LDH catalyzes the conversion of NADH to NAD+ and can be monitored as decreased absorbance of NADH. Samples were added to 1 ml of 0.1 mol/L potassium phosphate buffer (pH 7.5), 90 μmol/L NADH, and 790 μmol/L pyruvate at 37°C, and the reaction was followed for 150 seconds in a spectrophotometer at a wavelength of 340 nm. Disruption of the lysosomal membrane was analyzed by measuring the activity of the lysosomal enzyme N-acetyl-β-glucosaminidase (NAG) in the isolated fraction. Samples were incubated at 37°C for 40 minutes in 125 μl of 0.2 mol/L citrate buffer containing 0.8 mmol/L 4-methylumbelliferyl-2-acetamido-2-deoxy-β-d-glucopyranoside (NAG substrate; Sigma-Aldrich). The amount of the fluorescent product was determined at λex 356 nm and λem 444 nm. Proteins in the cytosolic fraction were precipitated by addition of trichloroacetic acid (final concentration 5%). After a 10-minute incubation on ice, proteins were pelleted by centrifugation at 20800 × g for 15 minutes. The pellets were resuspended in lysis buffer (see below) containing 6 mol/L urea to dissolve the precipitated proteins and neutralized by the addition of 3 μl 1 mol/L sodium hydroxide. The isolated fraction was analyzed by immunoblot technique.Immunoblot AnalysisCell pellets were lysed in 63 mmol/L Tris-HCl, 10% glycerol, and 2% SDS, and protein determination was performed. Bromophenol blue (0.05%) and dithiothreitol (50 mmol/L) were added before the samples were heated to 95°C. Proteins were separated by SDS polyacrylamide gel electrophoresis (200 V, 60 mA/gel) and blotted onto a nitrocellulose membrane (100 V, 250 mA, 1 hour). The membrane was blocked in 5% dry milk in Tris-buffered saline with polysorbate 20 (Tween 20) and probed with a primary antibody (diluted in 0.1% dry milk in Tris-buffered saline with polysorbate 20) overnight at 4°C. The membranes were washed and probed with horseradish peroxidase–conjugated goat anti-rabbit antibodies, goat anti-mouse antibodies (P0448, P0447; Dako, Glostrup, Denmark), or bovine anti-goat antibodies (sc-2350; Santa Cruz Biotechnology) for 1 hour at room temperature. After washing, immunodetection of the bound antibodies was performed using Western Blotting Luminol Reagent (Santa Cruz Biotechnology). Equal protein loading was verified by analyzing the expression of GAPDH (for whole cell extract) and LDH (cytosolic extract). Reprobing of membranes was performed after stripping in 1% Tween, 0.1% SDS, and 0.2 mol/L glycine, pH 2.2 (55°C, 1 hour) or blocking in the presence of 1% sodium azide.Statistical AnalysisResults are presented as mean and standard deviations. Data were statistically evaluated using a nonparametric Kruskal-Wallis test for multiple comparisons. Correlation analysis was performed using the Spearman test. P values ≤0.05 are considered to be significant and marked with an asterisk in figures. Experiments were routinely performed at least three times.ResultsU18666A Treatment Results in Cholesterol Accumulation and Lysosomal Alterations but Does Not Affect Cell ViabilityTreatment of human fibroblasts with 0.5 μg/ml U18666A induced a time-dependent increase in total cholesterol content that was significant after 24 hours and increased further after 48 hours of treatment (Figure 1A). Unesterified cholesterol can be specifically labeled using the antibiotic filipin.16Gimpl G. Gehrig-Burger K. Cholesterol reporter molecules.Biosci Rep. 2007; 27: 335-358Crossref PubMed Scopus (89) Google Scholar In untreated cells, we observed pale and diffuse filipin staining, whereas cells treated with U18666A revealed a punctate, perinuclear filipin staining pattern (Figure 1B) that colocalized with the lysosomal marker LAMP-2. No colocalization of filipin staining and mitochondria was detected (results not shown). U18666A treatment alone did not influence the viability of human fibroblasts as assessed by MTT assay and caspase-3 activity measurements (Figure 1, C and D). U18666A treatment caused an approximately 12-fold elevation of LysoTracker Green fluorescence as analyzed by flow cytometry (Figure 1, E and F). This increase of the lysosomal compartment was associated with a time-dependent increase in the expression of the lysosomal protein LAMP-2 and the proforms of cathepsin D (Figure 1G). By immunostaining and quantification of fluorescence, U18666A-treated cells were shown to have increased levels of the phospholipid BMP, also known as lysobisphosphatidic acid (LBPA), which is found exclusively in the endo-lysosomal system (Figure 1H). Together, these results show that U18666A induces an up-regulation of the lysosomal system.Fibroblasts with High Cholesterol Content Are Protected from Apoptosis Induced by MSDHTo investigate whether lysosomal cholesterol overload could alter the cellular sensitivity to apoptosis, fibroblasts were pretreated with U18666A before exposure to the lysosomotropic detergent MSDH. Microscopic examination and viability analysis revealed that U18666A-treated cells were less susceptible to cell death induced by MSDH (Figure 2, A and B). Accordingly, U18666A prevented MSDH-induced caspase-3 activation (Figure 2C). When cells were exposed to MSDH for 24 hours and then transferred to standard culture conditions, there was 100% survival of U18666A-pretreated cells compared with 17% in non-pretreated cells, indicating that cell death signaling is not only delayed but prevented. No apoptosis protection was noted when cholesterol accumulation was omitted and U18666A only was added at the time of MSDH-exposure (results not shown), excluding a direct effect of U18666A on the apoptotic response.Figure 2Fibroblasts with high cholesterol content are protected from apoptosis induced by MSDH. Human fibroblasts were pretreated with U18666A (0.5 μg/ml, 48 hours) to induce lysosomal cholesterol accumulation before exposure to the lysosomotropic detergent MSDH (15 μmol/L). A: Phase contrast images of cells exposed to MSDH for 24 hours. Scale bar = 20 μm. B: Viability of cell cultures after MSDH exposure as assessed by the MTT assay (n = 4). Viability is expressed as percentage of untreated cultures. C: Caspase-3–like activity after MSDH treatment determined using the fluorescent substrate Ac-DEVD-AMC and correlated to total protein content (n = 4). Asterisks represent statistically higher caspase-3–like activity in non-pretreated cells compared with U18666A-pretreated cells. D: Filipin staining of unesterified cholesterol in untreated cells, cells pretreated with U18666A, and cells pretreated with U18666A plus 25-HC (1 μg/ml, 48 hours). Scale bar = 20 μm. E: Viability of cultures pretreated with U18666A with or without 25-HC for 48 hours then exposed to MSDH (24 hours). Results are presented as mean values with error bars representing SD. Significant difference at *P ≤ 0.05.View Large Image Figure ViewerDownload (PPT)To verify the importance of cholesterol for the cytoprotective effect, U18666A-induced cholesterol accumulation was reverted by exogenously supplied 25-HC (Figure 2D). Simultaneous treatment of cells with U18666A and 25-HC totally abolished the protective effect of U18666A on MSDH-induced apoptosis (Figure 2E).Fibroblasts with High Cholesterol Content Are Less Sensitive to Apoptosis Induced by Staurosporine and CisplatinTo investigate if the protective effect of U18666A was specific for apoptosis induced by a lysosomotropic agent or a more general phenomenon, cells were exposed to the protein kinase inhibitor STS and cisplatin, two established apoptosis inducers. Earlier we reported that exposure of human fibroblasts to STS results in an early release of cathepsins from lysosomes with subsequent activation of the mitochondrial pathway to apoptosis.17Johansson A.C. Steen H. Öllinger K. Roberg K. Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine.Cell Death Differ. 2003; 10: 1253-1259Crossref PubMed Scopus (181) Google Scholar Cisplatin, clinically used to treat various types of cancer, has been shown to induce apoptosis associated with LMP when added to cell cultures.18Nilsson C. Roberg K. Grafstrom R.C. Ollinger K. Intrinsic differences in cisplatin sensitivity of head and neck cancer cell lines: correlation to lysosomal pH.Head Neck. 2010; 32: 1185-1194Crossref PubMed Scopus (20) Google Scholar The results show that pretreatment with U18666A protected cells from both STS-induced apoptosis and cisplatin-induced cell death (Figure 3, A–C), indicating that the protective effect of cholesterol accumulation is not limited to insults caused by lysosomotropic detergents directly targeting the lysosomal membrane.Figure 3Fibroblasts with high cholesterol content are less sensitive to apoptosis induced by STS and cisplatin. Human fibroblasts were pretreated with U18666A (0.5 μg/ml, 48 hours) to induce lysosomal cholesterol accumulation before exposure to STS (0.1 μmol/L, 24 hours) or cisplatin (40 μg/ml; 48 hours). A: Phase contrast images of cells exposed to STS and cisplatin. Scale bar = 20 μm. Viability and caspase-3–like activity of cell cultures after STS (B) or cisplatin (C) exposure (n = 4–8). Viability was assessed by the MTT assay and is expressed as percentage of untreated cultures. Caspase-3–like activity was determined using the fluorescent substrate Ac-DEVD-AMC and correlated to total protein content (n = 4). Results are presented as mean values with error bars representing SD. Significant difference at *P ≤ 0.05.View Large Image Figure ViewerDownload (PPT)Augmented Lysosomal Cholesterol Content Results in Increased Lysosomal StabilityNext, we investigated the effect of cholesterol accumulation on lysosomal stability. Double staining of cathepsin D and the lysosomal marker LAMP-2 showed a punctate lysosomal pattern in control cells (Figure 4A). After MSDH exposure, cathepsin D was partly found in the cytosol of non-pretreated cells, but it was still found colocalized with LAMP-2 in cells pretreated with U18666A. Due to difficulties in quantifying cathepsin D translocation using this method, lysosomal release was also estimated by extraction of the cytosolic fraction using digitonin, a detergent that selectively extracts cholesterol. Because the plasma membrane contains more cholesterol than membranes of intracellular organelles, an optimal concentration of digitonin solubilizes the plasma membrane but leaves organelles intact.19Zuurendonk P.F. Tager J.M. Rapid separation of particulate components and soluble cytoplasm of isolated rat-liver cells.Biochim Biophys Acta. 1974; 333: 393-399Crossref PubMed Scopus (262) Google Scholar As presented in Figure 4B, release of cytosolic content, assessed by measuring the activity of the cytosolic enzyme LDH, was similar in control and U18666A-pretreated cells. The release of lysosomal constituents, as determined by measurement of NAG activity, was affected by U18666A (Figure 4B). U18666A-treated cells had increased digitonin sensitivity, consistent with cholesterol accumulation in the lysosomal membrane. By using 20 μg/ml digitonin, the plasma membrane was permeabilized, but the lysosomal membrane was intact both in control and U18666A-treated cells. Thus, this concentration of digitonin was used to study the release of lysosomal content. By immunoblotting of extracted cytosol, we demonstrated that the MSDH-induced release of cathepsin D was reduced in U18666A-pretreated cells (Figure 4C).Figure 4Augmented lysosomal cholesterol content results in increased lysosomal stability. Normal human fibroblasts were pretreated with U18666A (0.5 μg/ml, 48 hours) before exposure to MSDH (15 μmol/L). A: Immunocytochemical staining of cathepsin D and LAMP-2 after 2 hours of MSDH exposure. Scale bar = 10 μm. B: Evaluation of plasma membrane sensitivity (measured as LDH activi
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