Involvement of endoplasmic reticulum stress in β-cell apoptosis: a proteomic approach in INS-1E cells. - PDF

Involvement of endoplasmic reticulum stress in β-cell apoptosis: a proteomic approach in INS-1E cells. D Hertog W. 1, Maris M. 1, Ferreira G.B. 1, Lage K. 2, Hansen D.A. 2, Cardozo A.K. 3,Workman C.T.

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Involvement of endoplasmic reticulum stress in β-cell apoptosis: a proteomic approach in INS-1E cells. D Hertog W. 1, Maris M. 1, Ferreira G.B. 1, Lage K. 2, Hansen D.A. 2, Cardozo A.K. 3,Workman C.T. 2, Moreau Y. 4, Eizirik D.L. 3, Waelkens E. 5,6, Overbergh L. 1 and Mathieu C. 1. Affiliations: 1 Laboratory for Experimental Medicine and Endocrinology (LEGENDO), University Hospital Gasthuisberg, Catholic University of Leuven, Herestraat 49, box 902, B-3000 Leuven, Belgium 2 Centre for Biological Sequence Analysis, BioCentrum-DTU, Technical University of Denmark, Building 208, DK-2800 Lyngby, Denmark 3 Laboratory for Experimental Medicine, Université Libre de Bruxelles (U.L.B.), route de Lennik 808, box CP618, B-1070 Brussel, Belgium 4 jesat-sdc, Department of Electrical Engineering, Catholic University of Leuven, Kasteelpark Arenberg 10, box 2446, B-3001 Heverlee, Belgium 5 ProMeta, University Hospital Gasthuisberg, Catholic University of Leuven, Herestraat 49, box 901, B-3000 Leuven, Belgium 6 Laboratory of Biochemistry, University Hospital Gasthuisberg, Catholic University of Leuven, Herestraat 49, box 901, B-3000 Leuven, Belgium Corresponding author: Lut Overbergh, PhD, LEGENDO, UZ-Gasthuisberg, Onderwijs en Navorsing, Herestraat 49, bus 902, B-3000 Leuven, Belgium. Tel: ; Fax: Running title: Involvement of endoplasmic reticulum stress in β-cell apoptosis Abbreviations: ATF6: activating transcription factor 6 CHOP: CCAAT/enhancer binding protein CPA: cyclopiazonic acid EF2: elongation factor 2 ER: endoplasmic reticulum hnrpk: heterogeneous nuclear ribonucleoprotein K HPRT: hypoxanthine-guanine phosphoribosyltransferase HSPA5: 78 kda glucose-regulated protein HSPA8: heat shock 70 kda protein 8 HYOU1: hypoxia up-regulated protein 1 IFN-γ: interferon gamma IL-1β: interleukin-1 beta IRE1a: inositol-requiring ER-to-nucleus signal kinase 1 a PCSK2: prohormone convertase 2 PDIA6: protein disulfide-isomerase A6 PERK: protein kinase RNA-dependent-like ER kinase UPR: unfolded protein response Summary Exposure of insulin secreting β-cells to inflammatory cytokines or nutrients, factors involved in the pathogenesis of type 1 and type 2 diabetes, leads to endoplasmic reticulum (ER) stress, β-cell dysfunction and eventually apoptotic β-cell death. RNA studies suggest that ER stress is a major contributor to β-cell dysfunction and death. The aim of this study was to analyze the protein changes induced by ER stress, in order to gain insight on the exact role of ER stress in β-cell damage. For this purpose, we have exposed insulin-producing rat INS-1E cells in vitro to the reversible ER stress inducer cyclopiazonic acid (CPA) at two different concentrations (6.25µM and 25µM), representing low and high ER stress levels. Protein changes were evaluated using 2D- DIGE, followed by MALDI-TOF/TOF at three different time points (6h, 12h and 24h). In INS-1E cells, 25µM CPA led to massive apoptosis, accompanied by a near complete protein translation shut-down, indicating a higher sensitivity of INS-1E cells to CPAinduced ER stress compared to other endocrine cells (rat adrenal medulla derived PC12). The lower concentration of CPA (6.25µM) led to adaptation of ER stress. Identification of the differential expressed proteins in the two conditions identified a clear pattern of defense pathways, with posttranslational modifications playing a crucial role. Moreover, many discrepancies with RNA findings were observed, suggesting that this latter technique may not be optimal for analyzing effects of ER stress, as many effects will only appear posttranscriptionally. We conclude that insulin secreting INS-1E cells are excessively sensitive to pure ER stress and that studying posttranscriptional and posttranslational modifications are of major importance to understand the impact of ER stress on β-cell function and survival. Introduction Pancreatic β-cells are responsible for synthesizing, processing and secreting the peptide hormone insulin into the circulation, thus maintaining normal blood glucose levels, a situation which is imbalanced in both type 1 and type 2 diabetic patients. Because of this metabolic role focused on insulin processing, β-cells have a highly developed endoplasmic reticulum (ER). Proper function of the ER is essential for β-cell survival and perturbation of its function induces cellular damage, eventually resulting in apoptosis. Various conditions, like inhibition of protein glycosylation, impaired formation of disulfide bonds and calcium depletion from the ER lumen can disturb ER function. Accumulation of unfolded or misfolded proteins in the organelle is met by a selfprotective mechanism against ER stress, the unfolded protein response (UPR). This is mediated through the action of three pathways, namely protein kinase RNA-dependentlike ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring ER-to-nucleus signal kinase 1 a (IRE1a). The UPR is a protective pathway which decreases ER protein levels and restores ER function by different mechanisms, such as 1) attenuation of protein translation through activation of the PERK eukaryotic translation initiation factor 2alpha (eif2alpha pathway); 2) up-regulation of ER chaperones through activation of the ATF6 X-box binding protein 1 (XBP1) pathway; 3) degradation of misfolded proteins through activation of the ER degradation pathway (ERAD); and 4) halting new protein production via activation of IRE1 (1, 2). In case of prolonged or excessive ER stress, the different steps activated by the UPR response fail to restore ER homeostasis and apoptotic pathways are triggered. Cyclopiazonic acid (CPA) is produced by Penicillium cyclopium as a secondary metabolite and is a highly selective and reversible inhibitor of the sarco-endoplasmic reticulum pump (SERCA-pump), thereby depleting the ER from Ca 2+. CPA is thus used as a well established model for the induction of pure ER stress. Also cytokines are known to cause ER stress, at least in part, through inhibition of the same SERCA pump, via activation of inos and nitric oxide production (3) and JNK activation (4), as well as through activation of inositol 1,4,5-trisphosphate receptor type 2 (IPTR2) (5), both leading to a depletion of intracellular Ca 2+ stores. We choose the well established rat cell line INS-1E (6), which is considered an accepted in vivo model for the study of β-cells. Taking into account the high amount of cells necessary for proteomic analysis, using a cell line opens the possibility to investigate different experimental conditions, i.e. different time points combined with different concentrations of CPA. In the present study we now investigate the global changes induced by CPA at the protein level. The results point to two different mechanisms. INS-1E cells under high ER stress quickly and massively go in apoptosis. We observe major defects in insulin processing, suggesting that not only insulin mrna expression is decreased upon ER stress, but also the conversion of pro-insulin to insulin. Also, marked decreases in different ER chaperones are observed, a phenomenon that is not observed at mrna level. This indicates that important differences at mrna and protein or even post-translational level are taking place in the process of ER-stress mediated β-cell death. As these INS-1E cells are extremely sensitive to ER stress as compared to other secretory cell types like e.g. the rat pheochromocytoma cell line PC12, we want to assess the effect of a lower dose of CPA (6.25µM) as a model for low ER stress, where the molecular mechanisms behind ER stress might be more visible. Under low ER stress, the INS-1E cells are able to adapt, as deducted at the protein level by the transient downregulation or inactivation of many proteins involved in cell fate, followed at later time points by upregulation or activation. Experimental procedures Cell culture conditions INS-1E cells, a kind gift from Prof. C. Wollheim (Centre Medical Universitaire, Geneva, Switzerland), were cultured in RPMI 1640 (Cambrex, Belgium) supplemented with 10 mmol/l HEPES, 10% v/v heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 1 mmol/l sodium pyruvate, and 50 µmol/l β-mercaptoethanol. Cells used for experiments ranged from passage 62 until 66 (6). The presently used INS-1E cells have a well-preserved insulin release in response to glucose (6) and respond to cytokines and CPA in a similar way as primary β-cells (7). PC-12 cells were cultured in F-12K Nutrient Mixture Kaighn s modification medium supplemented with 15% horse serum, 2.5% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. Cells or whole islets from rat (isolation see below) were treated with CPA (Sigma, Bornem, Belgium) dissolved in DMSO and used at a final concentration of 3.1µM; 6.25µM; 12.5µM or 25µM. Control cells were treated with DMSO alone. For cytokine treatment 10 units/ml recombinant human IL-1β (a kind gift from Dr. C. W. Reinolds, National Cancer Institute, National Institutes of Health, Bethesda, MD) and 500 units/ml recombinant rat IFN- (R&D Systems, Minneapolis, MN) were used. Rat islet isolation Wistar rats were purchased from xxx. Islets from young rats (1 week of age) were isolated as described previously (8). Cell death analysis For the INS-1E cells, the percentage of living, apoptotic and necrotic cells was assessed by cell counting. INS-1E cells were cultured in 96-well plates (8000 cells/well). After 6, 12h and 24h of exposure cells were incubated for 15 min with propidium iodide (10 µg/ml)(invitrogen, Merelbeke, Belgium) and Hoechst HO 342 (20 µg/ml)( Invitrogen, Merelbeke, Belgium). A minimum of 500 cells was counted in each experimental condition by two researchers, one of them unaware of the sample identity, on an inverted fluorescent Ti-E microscope (Nikon, Brussels, Belgium). For the rat islets, the percentage of living and death cells was assessed by cell counting. Wistar rat islets were cultured in 96-well plates (15 to 20 islets /well). After 24h of exposure to 25µM CPA or control condition, islets were incubated for 15 min with propidium iodide (10 µg/ml) and Hoechst HO 342 (20 µg/ml). Rat islets were counted in each experimental condition by two researchers, one of them unaware of the sample identity, on an inverted fluorescent Ti-E microscope (Nikon, Brussels, Belgium). Real-time RT-PCR Total RNA was extracted from 5 x 10 5 cells using the High Pure RNA Isolation Kit (Roche, Vilvoorde, Belgium) and 0.5µg was reverse transcribed using 100U Superscript II RT (Life Technologies, Merelbeke, Belgium) at 42 C for 80min, in the presence of 5µM oligodt 16. Real time PCR was performed for CHOP10, HSPA5, HYOU1, PDIA6 and HPRT using TaqGold (Eurogentec, Belgium), TaqMan probes or Syber-Green, and the MyiQ system (Bio-Rad, Nazareth, Belgium) as described previously (9). The following primers were designed making use of the software program Primer3 (10): CHOP10-FW: 5 -TCTCATCCCCAGGAAACGAA-3 ; CHOP-RV: 5 -ATCTGGAGA- GCGAGGGCTTT-3 ; and CHOP-TP: 5 -ACCCTGCGTCCCTAGCTTGGCTG-3. INS2-FW: 5 - GCTGGCCCTGCTCATCCT-3. INS2-RV: 5 - CCACCAAGTGAGAACCACAAAG-3. HPRT-FW: 5 -TTATCAGACT- GAAGAGCTACTGTAATGATC-3 ; HPRT-RV: 5 -TTA- CCAGTGTCAATTATATCTTCAACAATC-3 ; and HPRT-TP 5 -TGAGAGATCATC- TCCACCAATAACTTTTATGTCCC-3. HYOU1-FW: 5 -CACATGGCACAGATTG- AAGG-3 ; and HYOU1-RV: 5 -CAGGCACTCGATCAAACAAA-3. PDIA6-FW: 5 - GCAGCAAGTGCACTGAAAGA-3 ; and PDIA6-RV: 5 -GGAAATCCCTGGACAC- CATAC-3. HSPA5-FW: 5 -ACCTATTCCTGCGTCGGTGT-3 ; HSPA5-RV: 5 - AGGAGTGAAGGCCACATACGA-3 and HSPA5-TP: 5 - AAGAACGGCCGCGTGGAGATCAT-3. Normalization was performed using the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT), for which no regulation by the treatment itself was observed. Quantification was based on the Ct method. Where necessary, a correction for differences in efficiency between target and housekeeping gene was performed using the Pfaffl method (11). 2D-DIGE analysis 8.5x10 6 INS-1E cells were incubated during 6, 12 or 24h with 6.25µM CPA or, with 25µM CPA during 6 or 12h. Control cells were treated with DMSO alone. Quadruplicate experiments were performed, originating from 4 independent experiments. Sample collection and processing was performed as described earlier (5). Protein samples were separated in the first dimension using two ph ranges (ph4-7 and ph6-9) on 24cm strips (GE Healthcare, Diegem, Belgium). First and second dimension were performed as described earlier (5). Spot digestion and protein identification by MALDI-TOF/TOF analysis For spot picking, two preparative gels for each ph range were run (350µg protein lysate each). First and second dimension runs were performed as described (5), except that Cy Dye-labeling was omitted. Glass plates were pre-treated with BindSilane and two reference markers were applied to enable automatic picking. The gels were post-stained using Krypton (Pierce, Rockford, USA). Matching with the analytical gels was performed using the BVA module of the DeCyder V6.5 software. A pick list was generated and exported into the Spot Picker V1.20 software which controls the Ettan Spot Picker (GE Healthcare). Spot digestion and peptide purification was performed as previously reported (5). MS/MS analyses were performed on a 4800 MALDI TOF/TOF (Applied Biosystems, CA, USA). The instrument was calibrated with the Applied Biosystems Calibration Mixture 1. Measurements were taken in the positive ion mode between 900 and 3000 m/z. Sequences were automatically acquired by scanning first in MS mode and selecting the 15 most intense ions for MS/MS using an exclusion list of peaks arising from tryptic auto-digestion. Air was used as the collision gas, while the collision energy was adapted automatically. Data interpretation was carried out using the GPS Explorer software (V3.6) and database searching was carried out using the Mascot program (version ). Since all experiments were performed on rat INS-1E cells, MS/MS searches were conducted in the following databases with the taxonomy set on Rattus: NCBI (3446 sequences), MSDB (20748 sequences) and SwissProt (5769 sequences). MS/MS tolerance for precursor and fragment ions was set between 0.2 Da and 1 Da depending on the sample, methionine oxidation as variable modification and carbamidomethylation of cysteine as fixed modification. As enzyme, trypsin was selected and a maximum of one missed cleavage was allowed. Using these parameters the probability-based MOWSE scores greater than the given cut-off value for MS/MS fragmentation data were taken as significant (p 0.05). Interactome network analysis Network analysis was performed as reported in previous work (5) based on (12, 13). Western blotting 2.5x10 6 INS-1E cells were cultured as described above. The cells were lysed in 150mM NaCl, 1mM CaCl 2, 1mM MgCl 2, 10mM NaF, 1% NP-40, 1mM Na 3 VO 4, 1mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Complete Protease Inhibitor, Roche Diagnostics, Basel, Switzerland). Protein concentrations were determined using the BCA Protein Assay Reagent Kit (Perbio Science, Aalst, Belgium). 10µg cell lysate was denatured using NuPage Sample Buffer (4X), NuPage 10X Reducing Agent and heated for 10min at 70 C, before loading on a 4-12% NuPage Bis- Tris gel. Proteins were transferred to an Amersham Hybond LFP Low-fluorescent PVDF membrane (GE Healthcare). Protein electrophoresis and electroblotting were performed following the instructions of the manufacturer. Equal loading and transfer efficiency was tested by staining for β-actin. Previously blocked membranes were incubated overnight at 4 C with one of the following antibodies: anti-chop (1/350, Santa Cruz Biotechnology, Santa Cruz, USA), anti-actin (1/12000, Sigma, Bornem, Belgium), anti-hyou1 (1/5000, Abcam, Cambridge, UK), anti-hspa5 (1/500, Santa Cruz Biotechnology, Santa Cruz, USA), anti-pdia6 (1/5000, Abcam, Cambridge, UK). Imaging and quantification of the blots was done using the ECL-plex (GE Healthcare) goat-anti-mouse Cy2 and goat-antirabbit Cy5 secondary antibodies and subsequent scanning of the blots on the Typhoon scanner. Image analysis and quantification of the bands was performed using ImageQuant TL (v2003). ELISA Insulin release was measured upon stimulation with low (3mM) or high (20mM) glucose concentration. After incubation, INS-1E cells were washed twice with pre-warmed glucose-free Krebs-Ringer HEPES bicarbonate (KRHB) (134mM NaCl, 4.7mM KCl, 1.2mM NaH 2 PO 4.H 2 O, 1.2mM MgSO 4.7H 2 O, 1mM CaCl 2.2H 2 O, 5mM NaHCO 3, 10mM HEPES, 0.5% BSA) and equilibrated for 30min at room temperature. The solution was replaced with KRHB with either 3mM or 20mM glucose and incubated for 1h at 37 C. Insulin content in the supernatant was determined using the rat insulin ELISA kit from Mercodia (Uppsala, Sweden), according to the manufacturer s guidelines. Statistical analysis For 2D-DIGE experiments the one-way ANOVA and the paired Student s t-test was used. To analyze differences in protein levels, the Decyder V6.5 software was used, and a p-value of less than or equal to 0.05 was considered statistically significant. For all other tests the paired Student s t-test was used and p 0.05 was considered statistically significant. Results Effect of CPA on apoptosis susceptibility of INS-1E cells, compared to whole islets and PC12 cells. To investigate the susceptibility of INS-1E cells to CPA-induced apoptosis, cells were cultured for 6h, 12h or 24h in the presence of different concentrations of CPA, ranging from 3.1µM to 25µM. For the lowest concentration of 3.1µM CPA, no apoptosis was induced at any of the time points investigated. When using higher concentrations of 6.25µM and 12.5µM CPA, the percentage of apoptotic cells increased in a dose dependent manner. Using the highest concentration of 25µM CPA, the proportion of apoptotic cells increased from 2.8±1.1% in control condition to 10.3±1.7% (p 0.05) after 6h of exposure, increasing to 22.9±0.9% (p 0.001) after 12h, resulting in a massive induction of apoptosis after 24h treatment (39.8±4.5% apoptotic cells versus 1.9±1.0% in control condition, p 0.001) (Fig. 1a). No significant increases in the level of necrosis were observed in any of the conditions analyzed (data not shown). This increase in apoptosis was paralleled by a massive increase in transcript levels for the pro-apoptotic transcription factor CHOP. No increase in CHOP mrna levels was observed using 3.1 or 6.25µM CPA. Using the higher concentrations of 12.5 or 25µM, a marked induction of CHOP mrna levels was observed, and this already from 6h onwards (17.5-, 4.8-, and 3.5-fold for 12.5µM and 49.8-, and 23.1-fold for 25µM; p 0.05 compared to control for all conditions) (Fig. 1b). This was paralleled by an increase in CHOP protein levels, as measured upon 25µM CPA stimulation (8.8-fold after 6h (p 0.05, n=3) and 7.3 fold after 12h (p 0.01, n=3) (Fig. 2) (14). These findings are in agreement with our previous findings, confirming that CPA specifically induces apoptosis, and not necrosis, in INS-1E cells. The susceptibility of INS-1E cells was compared to CPA-induced apoptosis in whole rat islets. Here, using 25µM CPA, an increase in apoptosis was observed after 24h (from 17.8±1.4% apoptosis in control to 36.4±1.3% apoptosis in islets treated with CPA, p 0;001, n=3). The susceptibility of INS-1E cells was compared to CPA-induced apoptosis in PC12 cells. Here, using the same concentration of CPA (25µM), only a minor increase in apoptosis after 24h was induced (2.7±2.1% to 7.4±3.1%, p 0.05). This was indicative for a higher susceptibility of INS-1E cells to ER-stress induced apoptosis as compared to PC12 cells. Effects of high concentrations of CPA (25µM) on INS-1E cells Alterations in proteomic profile: 2D-DIGE analysis. Differential proteomic profiles of INS-1E cells were determined after exposure to 25µM CPA, and this af
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