The FASEB Journal • Research Communication
Modulation of ER stress and apoptosis by endoplasmic reticulum calcium leak via translocon during unfolded protein response: involvement of GRP78 Mehdi Hammadi,*,1,2 Agathe Oulidi,*,1 Florian Gackière,†,‡ Maria Katsogiannou,§,储,¶ Christian Slomianny,* Morad Roudbaraki,* Etienne Dewailly,* Philippe Delcourt,* Gilbert Lepage,* Sabine Lotteau,#,**,3 Sylvie Ducreux,#,**,3 Natalia Prevarskaya,*,4 and Fabien Van Coppenolle#,**,4,5 *Laboratoire de Physiologie Cellulaire (Cellular Physiology Laboratory), Institut National de la Santé et de la Recherche Médicale (INSERM) U1003, Université Lille I, Villeneuve d’Ascq, France; †Institut de Neurosciences de la Timone, Campus Santé Timone, ‡Unité Mixte de Recherche (UMR) 7289 Centre National de la Recherche Scientifique (CNRS), §INSERM U1068 (Molecular Oncology), and 储 CNRS UMR 7258, Aix-Marseille Université, Marseille, France;¶Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille (CRCM), Marseille, France; #Centre de Génétique et de Physiologie Moléculaires et Cellulaires, UMR CNRS 5534, Université Claude Bernard Lyon 1, Villeurbanne, France; and **Université de Lyon, Lyon, France The endoplasmic reticulum (ER) is involved in many cellular functions, including protein folding and Ca2ⴙ homeostasis. The ability of cells to respond to the ER stress is critical for cell survival, and disruption in such regulation can lead to apoptosis. ER stress is accompanied by alterations in Ca2ⴙ homeostasis, and the ER Ca2ⴙ store depletion by itself can induce ER stress and apoptosis. Despite that, the ER Ca2ⴙ leak channels activated in response to the ER stress remain poorly characterized. Here we demonstrate that ER Ca2ⴙ depletion during the ER stress occurs via translocon, the ER protein complex involved in translation. Numerous ER stress inducers stimulate the ER Ca2ⴙ leak that can be prevented by translocon inhibitor, anisomycin. Expression of GRP78, an ER stress marker, increased following treatment with puromycin (a translocon opener) and was suppressed by anisomycin, confirming a primary role of translocon in ER stress induction. Inhibition of ER store depletion by anisomycin significantly reduces apoptosis stimulated by the ER stress inducers. We suggest that translocon opening is physiologically modulated by GRP78, particularly during the ER stress. The ability to modulate the ER Ca2ⴙ permeability and subsequent ER stress can lead to development of a novel therapeutic approach.—Hammadi, M., Oulidi, A., Gackière, F., KatABSTRACT
Abbreviations: ANOVA, analysis of variance; ATF6, activating transcription factor 6; Bip, binding protein; BFA, brefeldin A; coIP, coimmunoprecipitation; DTT, dithiotreitol; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; GRP78, 78-kDa glucose-related peptide; IRE1␣, inositol-requiring enzyme 1␣; PERK, protein kinaselike endoplasmic reticulum kinase; RT-PCR, reverse transcription–polymerase chain reaction; SERCA, sarco-endoplasmic reticulum calcium ATPase; TG, thapsigargin; TLC, translocon; UPR, unfolded protein response 1600
sogiannou, M., Slomianny, C., Roudbaraki, M., Dewailly, E., Delcourt, P., Lepage, G., Lotteau, S., Ducreux, S., Prevarskaya, N., Van Coppenolle, F. Modulation of ER stress and apoptosis by endoplasmic reticulum calcium leak via translocon during unfolded protein response: involvement of GRP78. FASEB J. 27, 1600 –1609 (2013). www.fasebj.org Key Words: cancer 䡠 Bip Endoplasmic reticulum (ER) is the major cellular Ca2⫹ store where lumenal Ca2⫹ concentration ([Ca2⫹]ER) is balanced between active Ca2⫹ entry through sarco-endoplasmic reticulum calcium ATPase (SERCA) and passive Ca2⫹ leak. ER plays a crucial role in Ca2⫹ homeostasis and cell physiopathology. ER stress is a phenomenon that develops either due to accumulation of unfolded or misfolded proteins within the ER or a Ca2⫹ store depletion (for review, see refs. 1, 2). Accumulation of unfolded or misfolded 1
These authors contributed equally to this work. Current address: Laboratoire de Physiologie Cellulaire et Moléculaire, Université de Picardie Jules Verne, EA4667, 33 Rue Saint Leu, 80039 Amiens Cedex 1, France. 3 Current address: Laboratoire CarMeN, INSERM U1060CarMeN-“Equipe 5”, Université Claude Bernard Lyon 1, Facultés de Médecine Rockfeller et Charles Merieux LyonSud, 8 Ave. Rockefeller, 69373 Lyon Cedex 08, France. 4 These authors contributed equally to this work. 5 Correspondence and current address: Université Claude Bernard-Lyon 1, INSERM U1060-CarMeN-“Equipe 5”, 8 Ave. Rockefeller, 69373 Lyon Cedex 08, France. E-mail: fabien.
[email protected] doi: 10.1096/fj.12-218875 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 2
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proteins in the lumen of the ER induces unfolded protein response (UPR). During this cellular stress response related to the ER, specific signaling pathways are induced to cope with these proteins. ER stress is involved in an increased number of pathologies, such as cancers (3), diabetes (4), cardiac pathologies (5), and neurodegenerative diseases (6). It is suggested that Ca2⫹ plays an important role in ER stress and in UPR. Nevertheless, the molecular mechanisms of Ca2⫹ homeostasis during ER stress remain unknown. An important check point of ER stress relates to regulation of the ER Ca2⫹ homeostasis, determined by precise control of the permeability of ER Ca2⫹ leak channels, is so far not characterized. Knowledge of the modulation of ER Ca2⫹ permeability during UPR can improve understanding of Ca2⫹ involvement in UPR, the ER stress in both physiological and pathological conditions. In a previous study, we have shown that translocon (TLC), a protein complex involved in protein translocation during translation (7), is an ER Ca2⫹ leak channel (8, 9). The store depletion induced by puromycin, an antibiotic that maintains the TLC open, is able to activate Ca2⫹ entry through store-operated channels (9). In the same work, we have demonstrated that ER Ca2⫹ depletion by thapsigargin (TG; an inhibitor of SERCA pumps and an ER stress inducer) occurs mainly via TLC, which is also implicated in the retrotranslocation of unfolded proteins for their degradation by proteasome. This process is called ER-associated degradation (ERAD; for review, see ref. 10). In parallel to ER protein retrotranslocation through TLC, Ca2⫹ likely follows the same pathway. Thus, TLC appears to be a good candidate for the ER Ca2⫹ leak channel involved in Ca2⫹ homeostasis during ER stress. On the other hand, within the ER, certain chaperone proteins facilitate protein folding and, as such, prevent their aggregation. Among the best characterized chaperone proteins, the 78-kDa glucose-regulated protein (GRP78), also referred to as immunoglobulin binding protein (Bip), binds to unfolded proteins using its peptide-binding domain and possesses an ATPase domain for folding activity (11–13). It is known that GRP78 monitors ER protein activities. Under resting conditions, GRP78 binds to the luminal part of ER membrane proteins like inositolrequiring enzyme 1␣ (IRE1␣), protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6) to maintain them inactive. These proteins are involved in the UPR response pathway (for review, see ref. 3). GRP78 can also seal the pore of the TLC (14) and thus possibly reduces ER Ca2⫹ leak. On unfolded protein accumulation and during ER stress, GRP78 is released from IRE1␣, PERK, ATF6, and TLC, which then trigger the UPR response and probably enhance ER Ca2⫹ depletion. We show in this report that TLC is an ER Ca2⫹ leak channel involved in ER store depletion during ER stress. In addition, we suggest that this depletion can be regulated by GRP78 in control conditions and under the ER stress. In our cell model, ER stress induced ER CA2⫹ DEPLETION VIA TRANSLOCON DURING ER STRESS
apoptosis and GRP78 overexpression. These effects were decreased by maintaining TLC in a closed conformation by anisomycin, an inhibitor of the Ca2⫹ leak through TLC (8, 9, 15, 16). Thus, the decrease of ER Ca2⫹ leak via TLC inhibits apoptosis under ER stress conditions. The degree of Ca2⫹ leak via TLC is connected to ER stress and specifically to GRP78 expression. During ER stress, GRP78 overexpression maintains protein folding and also reduces the permeability of TLC to Ca2⫹. In summary, our data represent a new mechanism involved in the ER stress by controlling the ER Ca2⫹ content and subsequently contributing to the balance between cell survival and apoptosis during ER stress. MATERIALS AND METHODS Cell culture LNCaP cells from the American Type Culture Collection (Manassas, VA, USA) were cultured in RPMI 1640 medium (Life Technologies, Inc., Fontenay sous Bois, France) supplemented with 5 mM l-glutamine (Life Technologies), 10% fetal bovine serum (Seromed; Poly-Labo, Strasbourg, France), and 1% kanamycin (Life Technologies). Cells were routinely grown in 50-ml flasks (Nunc, Langenselbold, Germany) and kept at 37°C in a humidified incubator in an air/CO2 (95/5%) atmosphere. For Ca2⫹ imaging experiments, the cells were subcultured in Petri dishes (Nunc) and used after 3– 6 d. Ca2ⴙ imaging Intracellular Ca2⫹ concentration ([Ca2⫹]i) was measured using either ratiometric Fura-2 or Fluo-4 dyes. Fura-2 fluorescence was quantified according to the Grynkiewicz and Tsien formula (17). The following equation was used to determine the absolute values of [Ca2⫹]i in Fura-2 AM LNCaP loaded cells: [Ca2⫹]i ⫽ Kd(R ⫺ Rmin)(Rmax ⫺ R) (Sf2/Sb2), where Kd is a dissociation constant, R is the fluorescence ratio (F340/ F380), and Sf2/Sb2 is the ratio of proportionality coefficients of free Fura-2 and Ca2⫹-bound Fura-2 at 380 nm. We performed an in vivo calibration (with Fura-2 AM-loaded cells) to measure Rmax, Rmin, and Sf2/Sb2. Rmax was obtained by addition of 5 M ionomycin in a Ca2⫹-saturated medium (10 mM CaCl2). Rmin was obtained by addition of 5 M ionomycin in a Ca2⫹-free medium (10 mM EGTA). The extracellular solution contained 120 mM NaCl, 6 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 12 mM glucose. For Ca2⫹-free HBSS, CaCl2 was removed and EGTA (0.5 mM) added. LNCaP cells were grown on glass coverslips and loaded with 5 M of Fluo-4 AM for 25 min at 37°C or loaded with 5 M of Fura-2 AM for 45 min at 37°C. Ratio imaging measurements of Fluo-4 were made using a confocal microscope (LSM 510; Zeiss, Le Pecq, France). The microscope was equipped with a ⫻63 oil-immersion objective (NA⫽1.4). Fluo-4 was excited with the 488-nm line of an argon ion laser, and the emitted fluorescent light was measured at wavelengths ⬎505 nm. Images (512/512 pixels) were taken at 5-s intervals. Fluorescence of regions of interest was normalized to baseline fluorescence (F0[x]). Western blotting Subconfluent LNCaP cells were treated with an ice-cold lysis buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1601
10 mM MgCl2, 1 mM PMSF, 1% Nonidet P-40, and protease inhibitor cocktail from Sigma-Aldrich (l’Isle d’Abeau, France). The lysates were centrifuged at 15,000 g at 4°C for 20 min, mixed with a sample buffer containing 125 mM Tris-HCl (pH 6.8), 4% SDS, 5% -mercaptoethanol, 20% glycerol, and 0.01% bromophenol blue. Total protein samples were subjected to 8 –10% SDS-PAGE and transferred to a nitrocellulose membrane by semidry Western blotting (Bio-Rad Laboratories, Marnes-la-Coquette, France). The membrane was blocked in a 5% milk containing TNT buffer (Tris-HCl, pH 7.5; 140mM NaCl; and 0.05% Tween 20) overnight, then probed using specific rabbit polyclonal anti-GRP78 (1:200; Novocastra, Newcastle on Tyne, UK) and anti--actin (1:200; Lab Vision, Kalamazoo, MI, USA) or anti-calretinin (AbCam, Cambridge, UK) antibodies. -Actin or calretinin was used as internal control. The bands on the membrane were visualized using the enhanced chemiluminescence method (Pierce Biotechnologies Inc., Rockford, IL, USA).
Transfection GRP78 silencing was performed using siGRP78 (5=-GGAGCGCAUUGAUACUAGA-3=; Dharmacon, Illkirch, France) in LnCaP cells. Control siRNA (siLuc) experiments were performed by transfecting siRNA against luciferase (5=-CUUACGCUGAGUACUUCGA-3=; Dharmacon). Transfection was performed with jetSI ENDO (Polyplus Transfection, Illkirch, France). Protein knockdown was confirmed after 2 d using Western blot assay. Reagents and chemicals All chemicals were purchased from Sigma Aldrich except Fura-2-AM and Fluo 4-AM (Molecular Probes, Leiden, The Netherlands). Data analysis and statistics
Coimmunoprecipitation (coIP) Cells cultured at 80% of confluency were incubated for 18 h in control conditions, with 2 M puromycin or with 0.1 M TG alone or in combination with 0.2 M anisomycin. Cells were then harvested and homogenized in 600 l of an ice-cold lysis buffer (pH 7.4; 50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 1.1 mM EGTA, 1 mM PMSF, 1 mM NaVO4, 2 g/ml pepstatin, 2 g/ml aprotinin, 2 g/ml leupeptin, 5 g/ml trypsin inhibitor, and 0.5–1% Triton X-100), kept on ice for 1 h, then centrifuged at 10,000 g at 4°C for 10 min. Total protein (1 mg) was subjected to coIP overnight at 4°C with protein A- or protein G-Sepharose 4B beads (SigmaAldrich) preincubated at 4°C for 2 h with 1 g anti-sec61␣ antibody (Santa Cruz Biotechnology, Heidelberg, Germany). Proteins bound to the beads were washed 3 times with lysis buffer, then eluted by boiling the samples in Laemmli buffer, and analyzed by SDS-PAGE under reducing conditions and Western blotting, processed with anti-GRP78 antibody (1:200; Santa Cruz Biotechnology). Membrane was reblotted twice, first with anti-sec61␣ antibody (1:200; Santa Cruz Biotechnology), then with anti-SERCA2 (1:200; Santa Cruz Biotechnology). Apoptosis The level of apoptosis was estimated from a number of apoptotic bodies visualized by Hoechst staining. The detailed procedure has been previously described (18). The percentage of apoptotic cells was determined by counting ⱖ500 cells in random fields. All these experiments were performed in triplicate (3 dishes/condition). Reverse transcription–polymerase chain reaction (RT-PCR) RT-PCR was carried out as described previously (19). The 398-bp GRP78 (HSPA5) isoform amplicon (accession number NM_005347) was amplified with 5=-CTGGGTACATTTGATCTGACTGG-3= (forward), nt 1639 –1661, and 5=-GCATCCTGGTGGTTTCCAGCCAT-3= (reverse), nt 2036 –2013 (Eurogentec, Seraing, Belgium). The human 210-bp -actin amplicon (NM_001101) internal control was amplified with 5=-CAGAGCAAGAGAGGCATCCT-3= (forward), nt 248 –267, and 5=-GTTGAAGGTCTCAAACATGATC-3= (reverse), nt 457– 436. GRP78 mRNA expression was validated by gel density analysis by using human -actin mRNA as internal control. 1602
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Each experiment was repeated several times. Data were analyzed using Origin 8.0 (Microcal Software Inc., Northampton, MA, USA). Results are expressed as means ⫾ se. Differences between conditions were compared by 1-way analysis of variance (ANOVA). When a significant F value was obtained, means were compared using a Tukey’s test. Statistical significance was defined as a value of P ⬍ 0.05. For calcium experiments, N ⫽ number of independent experiments; n ⫽ biological replicates per experiment. The experiments (Western-blot, RT-PCR, apoptosis, and coIP) were performed in triplicate, using ⱖ3 flasks of cells per condition each time.
RESULTS TLC is one of the ER Ca2ⴙ leak channels involved in ER depletion during ER stress To deplete ER Ca2⫹ store and reveal ER Ca2⫹ leak, we used TG, a SERCA pump inhibitor. In LNCaP cells, TG (1 M) elicited a transient Ca2⫹ increase in control conditions and in a Ca2⫹-free medium (Fig. 1A). In line with previous experiments (9), the mean of the [Ca2⫹]i peak, in Fura-2 AM-loaded cells, was 265.3 ⫾ 7.35 nM (n⫽123). Anisomycin can be used to decrease the ER store depletion via the TLC aperture (8, 9, 15). Pretreatment of the cells with anisomycin (200 M, 1 h) lowered the above TG response by 45% (146.78⫾6.25 nM; n⫽89). These results clearly suggested that TLC is one of the major Ca2⫹ leak channels of the ER. We then hypothesized that decreasing the amount of opened TLC could slow down the speed of TG-induced store depletion. To illustrate this, we used increasing anisomycin concentrations (from 0.2 to 500 M) to pretreat cells for 25 min prior to TG perfusion and Ca2⫹ measurement. Anisomycin increased the time to peak (Fig. 1B) and consequently decreased the speed of ER Ca2⫹ depletion in a dose-dependent manner (control: 233.36⫾44.78 vs. 475.19⫾38 s with 500 M anisomycin, which represents a ⫹103.63% increase) in Fluo-4 AM loaded cells. In Fig. 1C, we used the amplitude of TG response in Fura-2 AM-loaded cells as a marker of ER Ca2⫹ content. ER stressor pretreatment
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Figure 1. Anisomycin and GRP78 inhibit Ca2⫹ release from the ER via TLC. A) Time course of TG (1 M) response in Fura-2 AM LNCaP-loaded cells in control conditions and after 1 h pretreatment with 200 M anisomycin. Anisomycin inhibits the ER Ca2⫹ leak occurring through the TLC from the lumen to the cytoplasm. B) Time to peak (means⫾se) of TG (1 M) action on cytosolic Ca2⫹ concentration. Cells were preincubated with several concentrations of anisomycin (from 0.2 to 500 M) for 25 min prior to TG perfusion. Increasing anisomycin concentrations reduced ER Ca2⫹ release induced by TG in Fluo-4 AM loaded cells. C) Cumulative data (means⫾se) for the amplitude of the ER Ca2⫹ release in Fura-2 AM-loaded cells after 1 M TG treatment of LNCaP cells. TG response was proportional to the ER Ca2⫹ concentration. Contrary to anisomycin alone (2 M; n⫽37), tunicamycin (5 g/ml; n⫽59), brefeldin A (BFA 50 M; n⫽42), or dithiotreitol (DTT 1 mM; n⫽37) significantly depleted the store after 3 h of treatment as compared to the control (n⫽43). At the same concentrations, anisomycin added to tunicamycin (n⫽55), to BFA (n⫽62), or to DTT (n⫽43) significantly prevented ER calcium depletion. aP⬍0.001, CTL vs. tunicamycin; bP⬍0.001, CTL vs. BFA; cP⬍0.001, CTL vs. DTT; dP⬍0.001, tunicamycin vs. tunicamycin ⫹ anisomycin; eP⬍0.001, BFA vs. BFA ⫹ anisomycin; fP⬍0.001, DTT vs. DTT ⫹ anisomycin. D) Expression levels of GRP78 and -actin in control conditions or 48 h after transfection with siRNA-GRP78 or siRNA-luciferase. E) Typical traces of TG response in Fura-2 AM loaded cells in control conditions or 48 h after transfection with siRNA-GRP78 or siRNA-luciferase. F) Cumulative data (means⫾se) illustrating the TG (1M) response in control conditions (n⫽100) or 48 h after transfection with siRNA-GRP78 (n⫽100) or siRNA-luciferase (n⫽51). **P ⬍ 0.001.
with tunicamycin (5 g/ml; N⫽7; n⫽59), brefeldin A (BFA; 50 M; N⫽6; n⫽42), or dithiotreitol (DTT; 1 mM; N⫽6; n⫽37) for 3 h significantly reduced TG response as compared to control conditions: 54.66 ⫾ 4.26, ⫺31.71 ⫾ 3.43, and ⫺29.69 ⫾ 4.27%, respectively. These results indicate that stressing the ER can induce its Ca2⫹ depletion. Tunicamycin, BFA, and DTT enhance protein unfolding and ER stress. TLC is also used to export unfolded proteins into the cytoplasm for their degradation by the proteasome (20). Therefore, TLC could be responsible for ER Ca2⫹ store depletion during ER stress. Used at 2 M, anisomycin alone slightly decreased TG response (⫺7.3⫾3.15%; N⫽8; n⫽37). Of importance, in the presence of both ER stressors and anisomycin, the TG response was not significantly different from the control conditions (tunicamycin and anisomycin: 8.32⫾2.15%; N⫽5; n⫽55; BFA and anisomycin: 8.9⫾4.43%; N⫽5; n⫽62; and DTT and anisomycin: 5.12⫾4.16%; N⫽5; n⫽43). Thus, ER stressors failed to deplete ER stores in presence of anisomycin. ER CA2⫹ DEPLETION VIA TRANSLOCON DURING ER STRESS
The luminal side of the aqueous TLC pore is sealed by GRP78 (14, 21, 22). We addressed the question of whether GRP78 inhibits ER Ca2⫹ leak when bound to TLC. To this end, LNCaP cells were transfected with 100 nM siRNAGRP78 or siRNA-luciferase as a control. As expected, GRP78 expression decreased after 2 d under siRNA-GRP78 conditions (Fig. 1D). GRP78 expression was similar either under siRNA-luciferase or in nontransfected conditions. Figure 1E, F illustrates the significant decrease in TG-induced Ca2⫹ release in siRNA-GRP78-transfected cells (40.38⫾4.12%; N⫽8; n⫽100) as compared with siRNA-luciferase-transfected cells (121.9⫾16.22%; N⫽7; n⫽51) or nontransfected cells (100⫾8.39%; N⫽8; n⫽71). Our data suggest a new role for GRP78, which could cork TLC to maintain [Ca2⫹]ER. Anisomycin decreases GRP78 expression at mRNA and protein levels during ER stress GRP78 is inducible by ER stress (11). To provoke an ER stress, LNCaP cells were treated with 2 M puromycin, 0.1 M TG, 5 g/ml tunicamycin, 1 mM DTT, or 50 M 1603
Figure 2. Anisomycin reduces GRP78 mRNA levels in control conditions and during ER stress: RT-PCR analysis of the GRP78 mRNA in LNCaP cells. A) GRP78 mRNA levels were evaluated using semiquantitative RT-PCR with or without 18 h treatments with ER stress inducers (puromycin, 2 M; TG, 0.1 M; tunicamycin, 5 g/ml; DTT, 1 mM, and BFA, 50 M) combined with anisomycin (0.2 M). Predicted size of the amplified product is 398 pb. -Actin was used as an internal standard. B) mRNA levels were analyzed by densitometric analysis and normalized by the -actin mRNA level. Experiments were performed in triplicate. a P⬍0.001, CTL vs. anisomycin; bP⬍0.001, CTL vs. puromycin; cP⬍0.001, CTL vs. TG; dP⬍0.001, CTL vs. tunicamycin; eP⬍0.001, CTL vs. BFA; fP⬍0.001, CTL vs. DTT; gP⬍0.001, puromycin vs. puromycin ⫹ anisomycin; hP⬍0.01, TG vs. TG ⫹ anisomycin; i P⬍0.001, tunicamycin vs. tunicamycin ⫹ anisomycin; jP⬍0.01, BFA vs. BFA ⫹ anisomycin; kP⬍0.05, DTT vs. DTT ⫹ anisomycin.
BFA for 18 h (Fig. 2). Contrary to the experiments of Fig. 1, we used here a lowered concentration of TG. Effectively, a high concentration of TG (1 M) allows visualization of an acute ER Ca2⫹ depletion (Fig. 1). Such treatment for 18 h is cytotoxic. Then, we employed 0.1 M of TG, which was effective to deplete ER stores and to induce ER stress in a longtime manner. At the level of transcript expression, each ER stress inducer significantly enhanced GRP78 expression compared to nonstressed conditions: with puromycin (⫹133⫾6%), TG (⫹202⫾8.8%), tunicamycin (⫹208⫾ 11%), BFA (⫹151⫾6%), or DTT (⫹104⫾4%) (Fig. 2A, B). At the concentration used, the GRP78 expression levels were higher with puromycin, TG, and tunicamycin as compared with DTT and BFA. When the previous treatments were combined with 0.2 M anisomycin, GRP78 mRNA levels were significantly decreased in nonstressed conditions (⫺50.2⫾4.1%) and when ER stress was induced by puromycin (⫺47.2⫾1.2%), TG (⫺16.1⫾2%), tunicamycin (⫺52.4⫾0.6%), BFA (⫺12.4⫾1.6%), or DTT (⫺8.38⫾3.8%) (Fig. 2B). Prior to Western blot analysis of GRP78, we verified the action of anisomycin on protein synthesis, using markers like -actin, Bcl-2, and calnexin; at 0.2 M, anisomycin had no effect (see Supplemental Data). Similarly, 2 M puromycin (also a translation inhibitor) did not reduce -actin synthesis. The same experimental conditions used for RT-PCR analysis of GRP78 mRNA level were carried out to measure GRP78 protein level by Western blot. The immunoblot showed detection of GRP78 (Fig. 3A). As expected, all ER stress inducers clearly increased cellular level of GRP78: with puromycin (⫹564⫾49%), TG (⫹2541⫾235%), tunicamycin (⫹1801⫾97%), BFA (⫹140⫾26%), or DTT (⫹154⫾5%). To determine the role of Ca2⫹ leak 1604
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and ER store depletion via TLC, anisomycin was added, combined with the ER stress inducers. Anisomycin treatment significantly decreased GRP78 synthesis in nonstressed conditions (⫺55.1⫾0.1%) and when ER stress was induced by puromycin (⫺83.3⫾2.8%), TG (⫺46.69⫾2.8%), tunicamycin (⫺68.52⫾2.33%), BFA (⫺32.5⫾2.1%), or DTT (⫺21.1⫾0.1%) (Fig. 3B). Collectively, these data implied that anisomycin decreases ER Ca2⫹ leak and attenuates ER stress. Involvement of GRP78 in apoptosis during ER stress LNCaP cells undergo apoptosis due to ER store depletion (18, 23). TG, like puromycin, induces Ca2⫹ release from the ER via TLC (Fig. 1). Apoptosis was evaluated using Hoechst staining after 18, 24, or 48 h of treatment with 0.1 M TG, or with 2 M puromycin alone or in combination with 0.2 M anisomycin. Anisomycin did not significantly increase apoptosis on its own as compared to the control (Fig. 4A, B). LNCaP cell apoptosis increased over time in the presence of TG, confirming our previous findings (18). The ER store depletion induced by SERCA pump inhibition led to apoptosis (18 h: 3.25⫾0.4%; 24 h: 12.85⫾1.25%; 48 h: 22.98⫾0.63%). Anisomycin (0.2 M) significantly reduced apoptosis when applied with TG after 24 and 48 h of treatment (7.01⫾0.35 and 15.12⫾ 0.55%, respectively). Similar results were observed with puromycin (Fig. 4B). Like TG, puromycin induced apoptosis after 18, 24, and 48 h of treatment (5.12⫾ 0.82, 13.55⫾2.1, and 37.01⫾1.18%, respectively). Anisomycin significantly reduced apoptosis after puromycin treatment for 24 and 48 h (7.2⫾1.08 and 22.73⫾1.98%, respectively). These findings indicate that the ER store depletion via TLC is central in the
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Figure 3. Anisomycin reduces GRP78 expression in control conditions and during ER stress: Western blot analysis of GRP78. A) GRP78 expression was evaluated using Western blot analysis in similar conditions as in Fig. 2. Predicted molecular weight of GRP78 is 78 kDa. Bottom: -actin (43 kDa) expression serves as control for total protein amount. B) Band intensity was determined by densitometry scanning and adjusted to actin levels. Experiments were performed in triplicate. aP⬍0.001, CTL vs. anisomycin; bP⬍0.001, CTL vs. puromycin; cP⬍0.001, CTL vs. TG; dP⬍0.001, CTL vs. tunicamycin; eP⬍0.001, CTL vs. BFA; f P⬍0.001, CTL vs. DTT; gP⬍0.001, puromycin vs. puromycin ⫹ anisomycin; hP⬍0.001, TG vs. TG ⫹ anisomycin; iP⬍0.001, tunicamycin vs. tunicamycin ⫹ anisomycin; jP⬍0.01, BFA vs. BFA ⫹ anisomycin; kP⬍0.05, DTT vs. DTT ⫹ anisomycin.
induction of apoptosis. The TLC inhibitor anisomycin can diminish cell death, pointing out the link between ER Ca2⫹ leak channel and cell death. Anisomycin treatment reduces association between sec61␣ and GRP78 in control conditions or after ER store depletion Sec61␣ is a pore protein of the TLC (14). As mentioned earlier, GRP78 seals the TLC pore under control conditions (14). We used a coIP experiment to test potential association and, consequently, regulation by GRP78
of the TLC opening during ER stress and anisomycin treatment. The immunoblot clearly revealed the 78kDa band of the GRP78 protein and its 72-kDa isoform, the 52-kDa band of sec61␣, the 100-kDa band of SERCA2 (LNCaP cells only express SERCA2 isoform), and the 43-kDa band of -actin (Fig. 5A). Cells were treated with anisomycin (0.2 M), puromycin (2 M), or TG (0.1 M) for 18 h (Fig. 5B). Anisomycin inhibited Ca2⫹ leak (Fig. 1), and diminished both GRP78 mRNA level and protein expression (Figs. 2 and 3, respectively). Anisomycin also significantly reduced interaction between sec61␣ and GRP78 in control condi-
Figure 4. Store depletion induced by TG or puromycin triggers apoptosis in LNCaP cells. A) Cumulative data (means⫾se) of temporal changes in apoptotic response of the LNCaP cells treated with TG (0.1 M) alone, or in association with anisomycin (0.2 M). B) Cumulative data (means⫾se) of temporal changes in apoptotic response induced by puromycin (2 M) with or without anisomycin (0.2 M). Experiments were performed in triplicate. *P ⬍ 0.01, **P ⬍ 0.001. ER CA2⫹ DEPLETION VIA TRANSLOCON DURING ER STRESS
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Figure 5. Anisomycin decreases interactions between sec61␣ (TLC) and GRP78/Bip. Interactions between sec61␣ and GRP78/Bip were assessed by coIP assay. Cells were treated for 18 h with anisomycin (0.2 M), puromycin (2 M), puromycin and anisomycin, TG (0.1 M), or TG and anisomycin. A) LNCaP extracts were immunoprecipitated by the anti-sec61␣ antibody and detected with the anti-GRP78 antibody, as well as anti-sec61␣ and anti-SERCA2. B) Input for each protein.
tions and during ER stress inflicted by puromycin or TG treatment (Fig. 5B). Therefore, with anisomycin, the cells did not overexpress GRP78 to stop ER Ca2⫹ leak through TLC either in controlled conditions or under ER stress.
DISCUSSION Perturbation of UPR could causally contribute to many diseases, including cancers (3), diabetes (24), cardiopathologies (5), and neurodegenerative disorders (6). Subsequently, there is a need for tools designed to modulate ER stress pathways and their interconnections within cellular homeostasis. Ca2⫹ plays an important role in the communication between organelles and the cytoplasm during ER stress (1, 2, 25, 26). ER Ca2⫹ plays an essential role in many physiological processes from cell proliferation to apoptosis (27, 28). Only few data are available on ER Ca2⫹ permeability under ER stress conditions. [Ca2⫹]ER is determined through a balance between Ca2⫹ entry mediated by SERCA pumps and Ca2⫹ release through Ca2⫹ leak channels. Chami et al. (29) have shown that the stress mediates induction of truncated isoforms of SERCA1 (S1Ts), which are responsible for Ca2⫹ leak. Nevertheless, LNCaP cells only express the SERCA2b isoform (23). We have demonstrated in previous works that TLC is one of the major ER Ca2⫹ leak channel in LNCaP cells (8, 9). We have also shown that TLC acts as a Ca2⫹ release channel in acinar pancreatic cells (15). Interestingly, a recent work has reported that calmodulin regulates TLC permeability to Ca2⫹ (30). In a previous study, we showed that ER Ca2⫹ depletion triggers apoptosis (18). Thus, we hypothesized that UPR and ER stress could lead to ER Ca2⫹ release via TLC and apoptosis if the ER remains depleted. To test this hypothesis, we have used anisomycin to reduce TLC permeability to Ca2⫹ (9). The time to peak of TGinduced Ca2⫹ release was decreased with anisomycin 1606
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treatment (Fig. 1). These results are in accordance with the fact that TLC is a major ER Ca2⫹ leak channel in LNCaP cells (9). We next undertook measurements of the ER Ca2⫹ content during UPR. Tunicamycin, BFA, and DTT are commonly used to trigger UPR (31, 32). ER stressors significantly decreased the content of the ER Ca2⫹ stores (Fig. 1), confirming the proposed hypothesis that decrease of ER luminal Ca2⫹ concentration is one of the early steps of the UPR. The ER Ca2⫹ leak channels involved in the ER stress remain poorly characterized. We pursued the suggestion that TLC might be a good potential candidate for involvement in this process. Anisomycin abolished ER stressor-induced ER depletion. These experiments confirm that TLC is one of the leak channels involved in ER store depletion during ER stress. Interestingly, TLC appears to be a common pathway used to perform unfolded protein retrotranslocation (33). UPR is associated with ERAD. The misfolded proteins are retrotranslocated through TLC to the cytoplasm to be degraded by the proteasome pathway (for review, see ref. 3). Thus, during UPR and the retrotranslocation of the misfolded proteins, an ER Ca2⫹ leak via TLC may occur simultaneously. Anisomycin, while ineffective in the accumulation of misfolded proteins, reduces the Ca2⫹ leak through TLC. Consequently, anisomycin prevents ER store depletion and ER stress. Chemical chaperones are used by the cells to restore ER function and ER homeostasis (11). During unfolding protein accumulation within the ER, as mentioned earlier, cells overexpress chaperone proteins, such as GRP78 (11), which acts as a stress sensor. Under control conditions, GRP78 binds to TLC (22) and to other ER proteins, such as protein kinases IRE1, PERK, and the transcription factor ATF6, to maintain them in an inactivated state (12). More precisely, GRP78 corks the luminal side of the TLC (14, 21, 22) and blocks ER Ca2⫹ leak. In this study, we measured a decrease in the
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ER Ca2⫹ concentration in siRNA-GRP78 transfected cells (Fig. 1). This ER Ca2⫹ depletion could not be inhibited by anisomycin (0.2 M) after 48-h treatments (data not shown). Our data are in accordance with the recent work of Schaüble et al. (34) showing a new role for GRP78: When bound to the luminal side of TLC, this chaperone protein acts as a plug to inhibit ER Ca2⫹ leak and to maintain [Ca2⫹]ER (34). Furthermore, to connect the Ca2⫹ permeability of TLC to ER stress and to the subsequent apoptosis, we assessed the effects of anisomycin pretreatment on the expression of GRP78 and its interactions with sec61␣ (a protein of the TLC pore). At the concentration used, anisomycin (like puromycin) did not affect -actin expression (see Supplemental Data), suggesting no effect on general protein translation. Tunicamycin, DTT, and BFA caused the accumulation of misfolded proteins in the ER and consequently induced a decrease in the [Ca2⫹]ER. We cannot exclude a capacitative calcium entry induced by ER store depletion during ER stress. Nevertheless, in each condition, the cytosolic resting Ca2⫹ concentration was similar, showing that secondary plasma mem-
brane Ca2⫹ entry would not interfere in our experimental conditions. Other ER stressors, like TG and puromycin, directly deplete the stores (Fig. 1). In each case, anisomycin reduced ER Ca2⫹ leak (Fig. 1) and decreased GRP78 expression at mRNA (Fig. 2) and protein levels (Fig. 3) in our cell model. Furthermore, in Fig. 5, we measured a decrease in the sec61/GRP78 interaction under anisomycin in control and in ER stress conditions. We speculated that it is due to a lowered GRP78 expression in parallel with a stable sec61␣ expression. This interaction is obviously affected by the amount of available GRP78. In nonstress conditions, GRP78 binds to TLC, IRE1␣, ATF6, and PERK and regulates their activities (Fig. 6). During UPR, GRP78 detaches from these ER membrane proteins and binds to misfolded proteins. In fact, there is a redistribution of GRP78 from its ER membrane partners to the lumen. It induces, in a second time, an overexpression of GRP78 to modulate ER stress. If the cells do not deal with accumulated misfolded proteins, a long term ER stress triggers apoptosis (for review, see ref. 3). In our experiments, apoptosis induced by ER
Figure 6. GRP78 modulates TLC opening and Ca2⫹ releasing during UPR response and ER stress. In basal conditions, GRP78 binds the UPR transducers (PERK, IRE1, and ATF6) into an inactive form and seals the TLC pore (top). During UPR response and ER stress, GRP78 is released in the lumen of the ER, inducing activation of UPR transducers to trigger apoptosis pathways. Loosening interactions between GRP78 and TLC allows Ca2⫹ release via the TLC pore from ER to cytoplasm and induces caspase 12-dependent apoptosis (bottom). ER stress conditions can be reproduced using puromycin. ER-stress-associated GRP78 overexpression, TLC-dependent Ca2⫹ release, and apoptosis can be reduced by anisomycin treatment. ER CA2⫹ DEPLETION VIA TRANSLOCON DURING ER STRESS
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store depletion was also inhibited by anisomycin (Fig. 4). In summary, this compound is not a chemical chaperone, but it inhibits ER Ca2⫹ leak through TLC and thus has similar effects to a GRP78 overexpression on [Ca2⫹]ER and ER stress and apoptosis. A mild ER stress promotes cell survival in part due to GRP78 overexpression (1–3). Conversely, a prolonged ER stress turns the balance toward cell death. It is evident that UPR and apoptosis are linked to Ca2⫹ signaling and particularly to ER store depletion (25, 26). Our results demonstrate that modulation of TLC permeability has a strong effect on apoptosis. As expected, the ER store depletion, either due to SERCA pump inhibition (with TG) or due to the increase in the Ca2⫹ leak through TLC (under puromycin treatment), provoked dramatic increase in apoptosis. Remarkably, the addition of anisomycin promoted cell survival. Taken together, our results indicate that in our cell model, ER store depletion mainly occurs via TLC during ER stress and induces apoptosis. To our knowledge, this study is the first to show that ER stress and apoptosis could be modulated using the ER Ca2⫹ leak pathway. Our findings could be helpful in cancer therapy, considering that highly elevated levels of GRP78 are correlated with cancer malignancy (35, 36). Reddy et al. (37) clearly demonstrated that drug resistance is associated with GRP78 overexpression in tumor cells. The researchers showed that GRP78 protects cells from apoptosis induced by the anticancer agent etoposide (inhibitor of topoisomerase II). Moreover, suppression of GRP78 increases breast cancer sensitivity to etoposide (38). Using MCF-7 (a breast cancer cell line), Fu et al. (39) showed that apoptosis-resistance to starvation is associated with GRP78 overexpression. In the case of chemoresistance associated with GRP78 overexpression, we hypothesize that the ER Ca2⫹ leak through TLC is reduced in a similar manner as in our experiments with anisomycin. Therefore, puromycin treatment (or equivalent) would enhance cancer cell sensitivity to chemotherapy and to apoptosis. In summary, this study is the first description of a new mechanism involving TLC as an ER Ca2⫹ leak channel in ER stress. We report 3 major findings. First, under UPR and ER stress conditions, TLC is one of the major ER Ca2⫹ leak channels involved in the pathway leading to ER store depletion and to cell death. Second, anisomycin inhibits ER Ca2⫹ depletion via TLC and also decreases ER stress and apoptosis under UPR conditions. Overexpressed GRP78/Bip during UPR and ER stress probably plugs TLC and minimizes ER store depletion. Finally, pharmacological tools that modulate ER Ca2⫹ leak from TLC could be helpful in the understanding and in the treatment of pathologies associated with deregulation of the ER stress. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Ligue Nationale Contre le Cancer, Association pour la Recherche contre le Cancer (ARC), and Ministère de l’Enseignement Supérieur et de la Recherche. The authors declare no conflicts of interest. 1608
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