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STIM2 degradation
Accepted Manuscript Title: Inhibition of Store-Operated Calcium Entry by Sub-lethal Levels of Proteasome Inhibition is Associated with STIM1/STIM2 Degradation Author: Xiu-li Kuang Yimei Liu Yuhua Chang Jing Zhou He Zhang Yiping Li Jia Qu Shengzhou Wu PII: DOI: Reference:
S0143-4160(16)30003-3 http://dx.doi.org/doi:10.1016/j.ceca.2016.01.007 YCECA 1727
To appear in:
Cell Calcium
Received date: Revised date: Accepted date:
4-11-2015 21-1-2016 22-1-2016
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Inhibition of Store-Operated Calcium Entry by Sub-lethal Levels of Proteasome Inhibition is Associated with STIM1/STIM2 Degradation
*Xiu-li Kuang a,b, *Yimei Liu a,b, Yuhua Chang a,b, Jing Zhou a,b , He Zhang a,b, Yiping Li c, Jia Qu a,b，Shengzhou Wu a,b *Xiu-li Kuang and Yimei Liu contributed to the work equally a School
of Optometry and Ophthalmology and the Eye Hospital, Wenzhou Medical
University;
b
Cultivation Base and Key Laboratory of Vision Science, Ministry of Health
and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou Medical University. 270 Xueyuan Road, Wenzhou, Zhejiang, 325003, P.R.China c
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, P.R. China
Correspondence should be directed to: Dr. Shengzhou Wu, Ph.D, M.D School of Optometry and Ophthalmology Wenzhou Medical University Email: [email protected] or [email protected] Phone: (86)-577-88067974 Fax：(86)-577-88067934
Graphical abstract
Highlights:
Proteasome inhibition repressed SOCE. Proteasome inhibition decreased STIM1 and STIM2 protein levels. Proteasome inhibition activated autophagy. Rapamycin decreased vs. Bafilomycin increased STIM1/STIM2.
Abstract Dysfunction of the ubiquitin-proteasome system (UPS) and calcium homeostasis has been implicated in the neurodegeneration of Alzheimer’s and Parkinson’s diseases. The cytosolic calcium concentration is maintained by store-operated calcium entry (SOCE), which is repressed by Alzheimer’s disease-associated mutants, such as mutant presenilins. We hypothesized that inhibition of UPS impacts SOCE. This study
showed that pretreatment with sub-lethal levels of proteasome inhibitors, including MG132 and clasto-lactacystin-β-lactone (LA), reduced SOCE after depletion of endoplasmic reticulum calcium in rat neurons. With the same treatment, MG-132 and LA reduced the protein levels of stromal interaction molecule 1and 2 (STIM1/2), but not the levels of Orai1 and canonical transient receptor potential channel 1 (TRPC1). STIM1 or STIM2 protein was mobilized to lysosome by MG-132/LA treatment as observed under an immunofluorescence confocal laser microscope. In the neurons, MG-132 and LA degraded p62/SQSTM1, promoted autophagy, converted LC3I to LC3II, and promoted co-localization of LC3 and lysosomes. Rapamycin, which enhances autophagy, reduced STIM1/2 protein levels, whereas bafilomycin, which inhibits autophagy, increased their protein levels. The protein levels of STIM1/2 and the amplitude of SOCE were decreased in SH-SY5Y with decreased protein level of proteasome subunit beta type-5 induced by shRNA. We conclude that sub-lethal levels of proteasome inhibition reduce SOCE and promote autophagy-mediated degradation of STIM1/2. UPS inhibition, a common finding in neurodegenerative diseases, interferes with calcium homeostasis via repression of SOCE.
Abbreviations: AD, Alzheimer’ disease; PD, Parkinson’s disease; SOCC, storeoperated calcium channel; SOCE, store-operated calcium entry; UPS, ubiquitinproteasome system; ER, endoplasmic reticulum; STIM, stromal interaction molecule; TRPC, canonical transient receptor potential channel; PSMB5, proteasome beta subunit type-5; MG-132, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal LA, clastolactacystin-β-lactone; LC3, microtubule-associated protein light chain; P62/ SQSTM1, sequestosome1; PS, presenilin.
Key words: Stromal interaction molecule, ubiquitin-proteasome system, PSMB5, Orai1, store-operated calcium channel, presenilin, low calcium, ER stress, neurodegeneration
1. Introduction The ubiquitin-proteasome system (UPS) is involved in protein degradation, regulating cellular events, such as the cell cycle, apoptosis, and signaling [1, 2]. The inhibition of UPS may cause neuronal death and degeneration seen in Alzheimer’s[3], Parkinson’s [4], and Huntington’s diseases[5]. The correlation between inhibition of UPS and neurodegeneration may be a result of oxidative stress[6], calcium dyshomeostasis [7, 8], and endoplasmic reticulum stress [9] with proteasome inhibition. However, sub-lethal levels of proteasome inhibition may play a protective role [10-13]. Conversely, sub-lethal levels of proteasome inhibition can cause harmful effects; for example, by disturbing the ER quality-control system, such as the calnexin/calreticulin cycle [14], which plays a role in protein-folding and ER-associated degradation. In contrast with UPS-mediated protein degradation, macro-autophagy (hereafter referred to as autophagy) depends on lysosomes, and it targets long-lived proteins and organelles. Autophagy begins with the formation of a phagophore and autophagosome from subcellular structures, which engulf unused proteins and damaged organelles. Autophagosomes fuse with lysosomes to form autolysosomes, in which lysosomal hydrolases degrade the contents [12, 15]. Proteasome degradation occurs in the inner core of the 20S proteasome complex, in which β-subunits form the two inner rings and α-subunits form the two outer rings[16]. Eukaryotic proteasome complex has substrate specificity, with the β1, β2, and β5 subunits corresponding to peptidyl-glutamyl peptide-
hydrolyzing function and trypsin- and chymotrypsin-like degradation. Chymotrypsin-like degradation, which is specific for the proteasome complex [17], occurs at the PSMB5 subunit in the mammalian proteasome complex. The two intracellular protein degradation systems (autophagy and proteasomes) also interact with each other, with the inhibition of autophagy enhancing or inhibiting proteasomes [18, 19], and the inhibition of proteasomes enhancing autophagy [12, 20, 21]. Store-operated calcium entry (SOCE), also called capacitative calcium entry, maintains calcium homeostasis in non-excitable cells and is induced by depletion of endoplasmic reticulum (ER) calcium [22]. Depletion of ER calcium redistributes STIM1 localized on ER membrane to the proximity of the plasma membrane. STIM1 is an ER membrane-anchored protein with E2F in the lumen that senses ER calcium levels [23, 24]. At the plasma membrane-ER junction, STIM1 oligomerizes with Orai1 or canonical transient receptor potential channel 1 (TRPC1), thereby allowing calcium to enter [25]. STIM1 activates Orai1 in response to relatively greater reduction in ER calcium, whereas STIM2, a homologue of STIM1, maintains basal cytosolic and ER calcium levels. STIM2 activates Orai1 in response to relatively smaller reduction in ER calcium [26, 27]. Although SOCE was discovered three decades ago, the importance of SOCE in neurons was only recently assessed [28, 29] because of its much smaller amplitude than that of voltage-dependent calcium channel (VGCC). Presenilin1and 2, which are family Alzheimer disease (FAD)-associated mutants, reduce SOCE [30-32] and interfere with homeostasis of ER calcium, which is an early event in neurodegeneration [33]. It is
unclear whether and how proteasome inhibition, which is commonly seen in Alzheimer’s and Parkinson’s diseases, impacts SOCE.
2. Materials and Methods Materials The following reagents were used: carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; clasto-lactacystine-β-lactone; adenosine-triphosphate; DL-dithiothreitol; ethylene glycol tetraacetic acid (EGTA); ethylenediaminetetraacetic acid (EDTA); glycerine; N-succinylLeu-Leu-Val-Tyr-7-amido-4-methylcoumain; 7-amino-4-methylcoumarin (Sigma, St. Louis, MO); LI-COR IRDye® 800CW secondary antibody ( LI-COR, Lincoln, NE); fura2/AM, and Neurobasal/B27 (Molecular Probes/Invitrogen, Carlsbad, CA); full-range rainbow molecular-weight markers (GE Healthcare, Piscataway, NJ); CL-XPosure film (Thermo Scientific Branch, Shanghai, P. R. China); Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL); protease inhibitor cocktail (Calbiochem, San Diego, CA); psmb5 shRNA lentivirus transfer plasmid carrying GFP fluorescence (Origene, Rockville, MD); Lyso Tracker; bafilomycin (Invitrogen, MA, USA); rapamycin (LC laboratories, MA, USA); antibodies for TRPC1 and Orai1 (Abcam, Cambridge, MA); and antibodies for LC3,STIM1, STIM2, PSMB5, GAPDH ( Cell Signaling Technology, Danvers, MA).
Animals All experiments were conducted in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and EU Directive 2010/63/EU for animal experiments. The study protocol was approved by the Wenzhou Medical University Committee on the Use and Care of Animals.
Methods Primary cortex neuronal culture The primary cultures were prepared from the cortical neurons of E18 SpragueDawley rats. The cultures were maintained in Neurobasal/B27 for eight to ten days for use in experiments. The culture procedures have been described [34] .
Cell proliferation assays The survival assays of neurons were examined with the MTS assay, as described [11].
In vitro proteasome activity assay The in vitro proteasome assays were performed as described[14]. Chymotrypsinlike activity, which is mostly specific to proteasomes, was assayed either in primary neuronal cultures or in SH-SY5Y cultures.
Western blot analysis
Cultured neurons were washed with phosphate-buffered saline (PBS) and harvested in the lysis buffer. The lysis buffer was composed of 460 mM Tris-HCl, pH 7.4; 138
mM
NaCl;
1
mM
EDT;
2.5
mM
NaF;
2.5
mM
Na3VO4;
1
mM
phenylmethanesulfonylfluoride; 1 mM dithiothreitol, supplemented with 0.1% Nonidet P40; and 1 X protease / 1 X phosphatase inhibitor cocktail (Sigma, St. Louis, MO). The nitrocellulose membranes, carrying the proteins transferred from SDS-PAGE, were incubated with individual primary antibody STIM1 (1:500) / STIM2 (1:500) / Orai1 (1:500) / TRPC1 (1:500) / PSMB5 (1:1000) overnight at 4 °C. The membranes were washed with PBS and incubated with an infrared fluorescent dye-labeled secondary antibody, LI-COR IRDye® 800CW (1:5000), at room temperature for one hour. Images of the membrane were taken with LI-COR Odyssey imager 9120, and the band optical densities were obtained with ImageJ.
GAPDH blotting (1:1000) was used as the
internal loading reference.
Ratiometric calcium analysis A ratiometric calcium-imaging system was used to acquire the data. This system was composed of a high-speed monochromator (DeltaRAMTM from Photon Technology International, PTI), an EMCCD camera (AndorTM) , and an inverted microscope (Nikon Eclipse Ti). The cultures were loaded with Fura-2 by incubation with Fura-2 AM (2 M) and pluronic F-127 (0.05%) at 37 0 C for 45 minutes with the loading buffer (144 mM NaCl, 10 mM glucose, 10 mM Hepes, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 10 M glycine, pH,7.2). The buffer was washed out and replaced with the dye-free loading buffer for another 45 minutes and switched to calcium-free media (144 mM NaCl, 10
mM glucose, 10 mM Hepes, 5 mM KCl, 1 mM MgCl2, 10 M glycine, and 2 mM EGTA, pH,7.2). After the baseline [ Ca 2+]i was measured, thapsigargin (5 M) was added to the cultures and [Ca 2+] i was collected for five minutes. Thapsigargin was washed out, and the regular buffer containing calcium (144 mM NaCl, 10 mM glucose, 10 mM Hepes, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 10 M glycine, pH,7.2) was added to the cultures. The [Ca 2+]i was collected until it reached a steady state.
Immunofluorescent confocal laser microscope Cortex neurons from the E18 Sprague-Dawley rats were cultured on polyethyleneimine-coated coverslips in 6-well plates. After seven days, the neurons were treated with MG-132, LA, or sham. The procedure for staining LC3 has been described [12]. The STIM1/STIM2 distribution in cellular organelles was determined by incubating the rat neurons with lysotracker, a lysosome reporter dye, for 90 minutes at 37 oC. After washing with PBS, the neurons were fixed in 4% paraformaldehyde for five to ten minutes. The neurons were washed with PBS again, blocked with 10% goat sera for 45 minutes, and incubated with STIM1 (1:80) or STIM2 (1:30) antibody in PBS containing 0.1% Triton-X100 for two hours. The neurons were washed and incubated with FITCconjugated second antibody (1:3000) in PBS with 0.1% Triton-X100 for 45 minutes. The nuclei were stained with DAPI for three minutes, washed, and observed under a Zeiss LSM 710 confocal microscope system (Carl Zeiss, Germany). The images were taken under oil-immersion lens (X 63) and processed with Zen Le (Carl Zeiss, Germany).
Establishment of stable PSMB5 knockdown SH-SY5Y We evaluated the knockdown effects of various types of shRNA. SH-SY5Y (<20 passages) were cultured in DMEM/F12 with 10% fetal bovine serum until 70% confluency in 96-well plates. Before transfection, the culture media were changed to serum-free media and transfected with plasmids by Lipofectamine® 2000 (Invitrogen). The plasmids carry gene fragments expressing PSMB5 or scramble shRNA, EGFP, and puromycin selection marker (Origene). Four shRNA (A, B, C, D) were transfected individually or in combination with SH-SY5Y, and the maximal knockdown effect of the combination of A and B was noted. The cultured SH-SY5Y was transfected with A and B in serum-free media for five hours and switched to regular culture media supplemented with penicillin (100 unit/mL) and streptomycin (100 µg/mL) (PS) for another 48 hours. The transfected SH-SY5Y was gently trypsinized, suspended, and selected with fluorescence-activated cell sorting (FACS), based on GFP fluorescence. SH-SY5Y with GFP from FACS was cultured in the regular medium supplemented with PS and gentamycin (10 µg/mL) for a week. Puromycin (1µg/mL) was added to the culture to exclude SH-SY5Y discharging the transfected plasmids, and the selected cultures were used for experiments.
Statistical analysis
The significance in each experiment was tested by one-way ANOVA and Bonferroni post hoc test if not mentioned; otherwise by Student’s t-test. Differences were considered statistically significant if p<0.05.
3. Results 3.1. Pretreatment with proteasome inhibitors reduces the store-operated calcium entry in rat primary neuronal cultures The cellular events in the early stages of neuronal death from proteasome inhibitors may explain the process of neurodegeneration associated with UPS dysfunction. Therefore, we used doses of inhibitors that suppressed proteasome activity but did not compromise neuronal survival. At 1 µM, MG-132/LA significantly decreased proteasome activity but did not cause death at 12 hours (Figs.1A,B). However, death occurred when the treatment was extended to 36 hours (not shown). We used a maximal dose of 1 µM and maximal treatment time of 12 hours when assessing the regulation of SOCE. Under these conditions, the amplitudes of SOCE were significantly decreased after thapsigargin (Tg) treatment (5 µM). For example, the ratio differences before and after addition of calciumin either in MG-132- or LA-treated cultures, which were representative of SOCE, were much smaller than those with the sham-washed cultures (Fig.1C,D). Interestingly, the application of MG-132 or LA alone had no effect on basal calcium influx (data not shown), which suggests that modulation of SOCE by proteasome inhibitors is dependent on the depletion of ER calcium and activation of SOCE. In the treated cultures, release of calcium from the ER after thapsigargin decreased dramatically compared with that in the sham-washed cultures. This
decrease reflects a reduction in the thapsigargin-releasable ER calcium pool (Figs. 1C), which is consistent with our previous observations [7].
3.2. Proteasome inhibitors decrease the levels of STIM1 and STIM2 but not of Orai1 or TRPC1 SOCE is activated by the dynamic assembly of STIM/Orai or STIM/TRPC after depletion of ER calcium [25]. In the Orai family, Orai1 is more likely to contribute to SOCE than is Orai2 or Orai3 [26]. Similarly, TRPC1 is more consistently induced by internal calcium depletion than is TRPC3, TRPC4, or TRPC7 [25]. Therefore, we aimed to determine whether MG-132 or LA treatment changed the levels of STIM1, STIM2, Orai1, or TRPC1. Both MG-132 and LA, at 1µM for 12 hours, reduced STIM1 to ~74% of sham-washed control values (Figs, 2, P<0.05). The reduction of STIM2 was more sensitive than STIM1 to MG-132/LA treatment. For example, at 0.3 µM for 12 hours, either treatment reduced STIM2 significantly, whereas at 1 µM, MG-132 reduced STIM2 to 62%, and LA reduced it to 41% of that in sham-washed control (Fig. 2, p<0.05). Conversely, MG-132 and LA did not change the level of Orai1/TRPC1 (Figs, 2A, C). We monitored the distribution of STIM1 before and after MG-132/LA treatment by labeling lysosomes (Fig. 3, 2nd column) and STIM1 (Fig. 3, 3rd column). The staining of STIM1 was diffuse in sham-washed neurons (Fig. 3, 1st row of 3rd column) but clustered around the nuclei in treated neurons (Fig. 3, 2nd and 3rd rows of 3rd column). A significantly higher percentage of stained STIM1 co-localized with lysosomes (Fig. 3, 2nd and 3rd rows of 4th column) in those with treatments than in those without treatment (Fig. 3, 1st row of 4th column). This co-localization suggests that STIM1 was mobilized to the
lysosomes by MG-132 and LA. Increased co-localization between STIM2 staining and lysosome was seen as well (Supplemental Fig1).
3.3. Proteasome inhibitors induce autophagy in primary rat cortical cultures Previous studies indicate that proteasome inhibition induces autophagy [12, 20, 21]. We measured the level of p62, an autophagy substrate. LA, at 0.3 or 1 µM, and MG132, at 1µM, significantly decreased p62 compared with sham-washed control (Figs, 4A and C). At higher concentrations, the levels of p62 recovered (not shown). This biphasic pattern of change with proteasome inhibitors was consistent with our observation in retinal pigmented epithelial cells [12], except that the recovery in neuronal cultures occurred at higher doses. Additionally, MG-132 and LA increased the conversion of LC3I to LC3II (Figs, 4A and C). Autophagy is a multiple-step and dynamic process. Increased autophagosome accumulation such as increased LC3II conversion reflects the status either at blockade of autophagy or autophagy flux [35]. To differentiate the increased LC3II conversion either due to autophagosome accumulation or to autophagy flux, we labeled lysosomes with the lysotracker (Fig.5, column2) and LC3 (Fig.5. column3); the result indicated significantly higher amount of merging puncta in treated neurons (Fig.5, column4). This observation suggests that MG-132 and LA increased autophagy flux in the cultures.
3.4. Autophagy enhancer or inhibitor modulates STIM1/STIM2 Previous studies suggest that MG-132 and LA degrade ST1M1/2 by inducing autophagy. Rapamycin, an autophagy enhancer, at 200 nM for 16 hours, reduced
STIM1 and STIM2 protein levels to 58% and 52% of sham-washed control values, respectively (Fig.6). Bafilomycin, an autophagy inhibitor, at 100 nM for 12 hours, increased the STIM1 and STIM2 levels (Fig.6). This finding supports the hypothesis that MG-132/LA treatment degrades STIM1/STIM2 by activating autophagy.
3.5. PSMB5 knockdown reduces the protein level of STIM1/STIM2 and the amplitude of SOCE Previous studies indicate that proteasome inhibitors are associated with several undesirable effects. We knocked down the proteasome subunit beta type-5 (PSMB5), which possesses chymotrypsin-like degradation activity, in the SH-SY5Y cell line with transfection of vectors expressing PSMB5 shRNA. We screened the transfected cells with FACS based on GFP fluorescence and multiplied the transfected clones to separate the non-transfected ones. Transfection with the PSMB5 shRNA vectors knocked PSMB5 down to ~63% (Figs, 7C,D) and reduced the proteasome activity to ~61% of sham-washed control values (Fig. 7B). In cell lines with the knockdown level of PSMB5, p62 level was reduced, suggesting that the autophagy pathway is activated (Supplemental Fig.2). In SH-SY5Y with PSMB5 knockdown (sh-PSMB5), the protein levels of STIM1and STIM2 were decreased to ~68% and ~43.5%, respectively, of the SH-SY5Y with normal level of PSMB5 (sh-scr) (Figs, 7C, D). In contrast, the levels of TRPC1 and Orai1 were not changed. These findings are consistent with the results of experiments conducted in primary neuronal cultures, as described above. The amplitudes of SOCE were also significantly reduced after ER calcium depletion in sh-scr compared with those in sh-PSMB5 cells (Fig. 7E).
4. Discussion This study showed that proteasome inhibition reduced SOCE in primary rat neuronal cultures, a finding which is consistent with prior observations in primary murine neuronal cultures [7]. Presenilin1 and presenilin2 have a similar effect on SOCE [30, 32]. We confirmed the inhibition of SOCE in SH-SY5Ywith decreased proteasome activity arising from knocking down PSMB5. The reduced SOCE was associated with lower STIM1 or STIM2 levels. This reduction was mediated by the autophagy pathway, which coincides with previous observation, viz. autophagy-deficient T cells express more STIM1 [36]. A previous study showed that pretreatment with MG-132, at 10 µM for one hour, potentiates the SOCE induced by depletion of ER calcium in HEK293 cells [37]. This observation is contradictory to ours. The apparent discrepancy can be explained by the difference in treatments used: we used MG-132 or LA at 1 µM for 12 hours, which is a chronic, low dose compared with the acute, high dose used in the previous study. We ruled out the non-specific effects of proteasome inhibitors by confirming the negative regulation of SOCE subsequent to depletion of ER calcium in PSMB5 knockdown SH-SY5Y. The chemical and genetic manipulations modulating proteasome activity in our study suggest that sub-lethal levels of proteasome inhibitors suppress, rather than potentiate, SOCE. Proteasome inhibition by chemical treatment or genetic manipulation reduced the amplitude of SOCE and levels of STIM1 and STIM2. Previous studies showed that STIM2 maintains basal and ER luminal free calcium, whereas STIM1 activates Orai1 in
rat neurons at relatively greater reduction of ER calcium [26, 27]. In this study, STIM1 was reduced by ~26%, which differs significantly from the ~90% decrease in SOCE with MG-132/LA treatments. Therefore, a part of SOCE inhibition is independent of the reduction of STIM1. MG-132 and LA reduce ER luminal free calcium concentration [7, 14] and calreticulin protein level [14]. A previous study indicated that calreticulin plays a role in maintaining the ER calcium reservoir [38] but not ER luminal free calcium [39]. In this study, we showed that MG-132 and LA reduced STIM2. It is possible that reduced STIM2 levels account for the reduction of ER luminal free calcium with MG-132 and LA. An increase or decrease in calcium is associated with neuronal death [40-43]. Calcium overload leads to oxidative and nitrosative stresses, resulting in DNA and protein damage [44]. Reduced calcium levels lead to calcium redistribution in cellular compartments; for example, proteasome inhibitors induce calcium depletion from endoplasmic reticulum, resulting in ER stress and dysfunctional ER quality-control [7, 14]. However, the temporal relationship between high and low calcium levels during neuronal death is unclear. We found that sub-lethal level of proteasome inhibition reduces SOCE, suggesting that calcium deficiency is an early event in UPS inhibitionassociated neurodegeneration seen in Alzheimer’s and Parkinson’s diseases. We conclude that sub-lethal levels of proteasome inhibitors reduce SOCE and promote autophagy-mediated degradation of STIM1/2. UPS inhibition, a common finding in neurodegenerative diseases, interferes with calcium homeostasis via
repression of SOCE. The findings of this work may contribute to understanding the pathogenesis of diseases such as Alzheimer’s and Parkinson’s diseases.
Conflict of interests The authors have found none to disclose.
Acknowledgments The work was supported by National Natural Science Foundation of China (81371027), Chinese Ministry of Education (20133321120002), and by funding (QTJ10 001) from Wenzhou Medical University to Dr. Shengzhou Wu, and also supported by Wenzhou Scientific Bureau (Y20150155) to Dr. He Zhang. The funding resources have no roles in study design, data collection, interpretation, manuscript writing, and decision to submission.
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Figure1. Pretreatment with proteasome inhibitors reduces the store-operated calcium entry in primary rat neuronal cultures. A. The cortical neuronal cultures were treated with MG-132 or LA for 12 hours and subjected to chymotrypsin-like proteasome activity assay. The values from treated conditions were normalized to those of sham-washed control (100% of proteasome activity). B. MTS assay was used to measure cell viability in neuronal cultures under the same treatment conditions as in A. The values from sham-washed control were set as 100%, and survival values in treated conditions were normalized to the sham-washed control values. The results shown are a mean (+ SEM) of experiments conducted thrice in quadruplicate cultures. *,#p<0.05 vs. control. C. The cortical neuronal cultures were treated with1 µM MG-132 or LA for 12 hours and used for ratiometric calcium measurement. The neurons were loaded with Fura-2 by incubation with Fura-2 AM, the culture media were changed to calcium-free buffer, and the baseline calcium ratios were acquired for five minutes. The cultures were exposed to thapsigargin (5 M) for five minutes, and a regular buffer containing 1.8 mM calcium was added. The ratios were acquired for another five minutes. Calcium ratios were acquired every 30 seconds and reported as mean (+ SEM) from 25-57 neurons at each timepoint. *,# p< 0.05 indicated that calcium levels without treatment rose immediately after thapsigargin, which differed significantly from those of MG-132 (red line)/LA (blue line)–treated cultures. D. The differences in calcium ratios before and after addition of calcium-containing buffer from C were calculated. The number was averaged from 25-57 neurons. * p<0.05 vs. control.
Figure 2. Proteasome inhibitors reduce the levels of STIM1and STIM2, but not of Orai1 or TRPC1. After 7-9 days in culture, rat primary cortical neurons were exposed to MG-132 (A) or LA (C) for 12 hours at concentrations ranging from 100 nM to 1 μM. Lysates containing equal amounts of protein were subjected to western blot analysis of STIM1 (1:500), STIM2 (1:500),Orai1 (1:500), and TRPC1 (1:500). The ratios of optical densities were normalized to the sham-washed control value, set as 100%. The ratios of STIM1/GAPDH,STIM2/GAPDH, Orai1/GAPDH, and TRPC1/GAPDH were quantified from at least three independent experiments (B, D). The blots shown are typical of the determinations. *, # p<0.05 vs. non-treatment.
Figure 3. Proteasome inhibitors mobilize STIM1 to lysosomes. A. After 7-9 days in culture, rat primary cortical neurons were exposed to MG-132 or LA at 1 µM for 12 hours and labeled with fluorescence dye. The images, taken with a confocal laser microscope, are typical of five non-contiguous fields in each dish from triplicate experiments. Blue, DAPI-labeled nuclei; red, lyso tracker-labeled lysosome; green, STIM1-labeled puncta. The merged images are shown in the rightmost column, and the orange-stained cells indicate STIM1 staining, co-localized with lysosome. Scale bar, 20 µM. B, The STIM1-positive puncta overlying labeled lysosomes for MG-132, or LA treatment, and sham condition were manually counted with ImageJ plug-in counter; the numbers were averaged from 25 cells and quantified. * p<0.05 vs. control.
Figure 4. Proteasome inhibitors induce p62 degradation and LC3 lipidation. The neurons at 7-9 days in culture were exposed to MG-132, LA, or sham-washed control for 12 hours. The lysates were subjected to immunoblotting with p62 (1:1000) and LC3 (1:1000) (A, C). GAPDH was used as an internal reference. The blots shown are typical of experiments performed thrice. The optical densities were quantified in B and D. *p<0.05 vs. control.
Figure 5. Proteasome inhibitors increase autophagy flux. A.The rat primary cortical neurons at 7-9 days in culture were exposed to MG-132 or LA at 1 µM for 12 hours and labeled with fluorescence. The images taken with a confocal laser microscope are typical of the images from five non-contiguous fields in each dish from triplicate experiments. Blue, DAPI-labeled nuclei; red, lyso tracker-labeled lysosome; green, LC3 staining. The merged images are shown in the rightmost column, and the orangestained cells indicate the co-localization between LC3 and lysosome. Scale bar, 20 µM. B. The co-localization puncta between LC3 and lysosome for MG-132, LA, and sham conditions were averaged from 25 cells and quantified. * p<0.05 vs. control.
Figure 6. Rapamycin reduces and bafilomycin increases STIM1/STIM2 levels. The neurons at 7-9 days in culture were exposed to rapamycin, bafilomycin, or shamwashed control for 12 hours. The lysates were subjected to immunoblotting with p62 (1:1000), STIM1(1:500), and STIM2 (1:500). GAPDH was used as an internal reference. The blots shown are typical of experiments performed thrice; the optical densities were quantified in B and D. *p<0.05 vs. control.
Figure 7. Reduction of STIM1, STIM2, and SOCE in PSMB5 knockdown SH-SY5Y cells. A. SH-SY5Y cells were stably transfected with a vector expressing RNA of a scrambled sequence (scr) or vectors expressing PSMB5 shRNA (A and B). The cells were screened and multiplied. The SH-SY5Y cells with either vector expressing GFP were detected in a fluorescence (FL) image of the AB clone. A phase contrast (PH) image is shown for comparison. B. Non-denatured lysates containing equal amounts of protein from the SH-SY5Y stably transfected with scr vector (scr) or PSMB5 shRNA vector (shPSMB5) were subjected to chymotrypsin degradation assay. The average results of experiments performed thrice are shown. Student’s t-test was used; *p<0.05 vs. scr. C. Denatured lysates containing equal amounts of protein were subjected to western blot analysis of TRPC1 (1:500), STIM1 (1:500), Orai1 (1:500), and PSMB5 (1:1000). The blots are typical of experiments performed thrice. D.The ratios of STIM1/GAPDH, Orai1/GAPDH, TRPC1/GAPDH, and PSMB5/GAPDH were quantified from experiments performed thrice. *,#p<0.05 vs scr. E. The analyses of SOCE showed linear trends averaged from 32 cells either for scr or shPSMB5. *p<0.05 indicated significant difference in the amount of calcium released from ER induced by thapsigargin between in scr and in shPSMB5. ++p<0.05 indicated significant difference in the amplitude of calcium influx subsequent to thapsigargin-induced ER calcium depletion between in scr and in shPSMB5.
S0143-4160(16)30003-3 http://dx.doi.org/doi:10.1016/j.ceca.2016.01.007 YCECA 1727
To appear in:
Cell Calcium
Received date: Revised date: Accepted date:
4-11-2015 21-1-2016 22-1-2016
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Inhibition of Store-Operated Calcium Entry by Sub-lethal Levels of Proteasome Inhibition is Associated with STIM1/STIM2 Degradation
*Xiu-li Kuang a,b, *Yimei Liu a,b, Yuhua Chang a,b, Jing Zhou a,b , He Zhang a,b, Yiping Li c, Jia Qu a,b，Shengzhou Wu a,b *Xiu-li Kuang and Yimei Liu contributed to the work equally a School
of Optometry and Ophthalmology and the Eye Hospital, Wenzhou Medical
University;
b
Cultivation Base and Key Laboratory of Vision Science, Ministry of Health
and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou Medical University. 270 Xueyuan Road, Wenzhou, Zhejiang, 325003, P.R.China c
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, P.R. China
Correspondence should be directed to: Dr. Shengzhou Wu, Ph.D, M.D School of Optometry and Ophthalmology Wenzhou Medical University Email: [email protected] or [email protected] Phone: (86)-577-88067974 Fax：(86)-577-88067934
Graphical abstract
Highlights:
Proteasome inhibition repressed SOCE. Proteasome inhibition decreased STIM1 and STIM2 protein levels. Proteasome inhibition activated autophagy. Rapamycin decreased vs. Bafilomycin increased STIM1/STIM2.
Abstract Dysfunction of the ubiquitin-proteasome system (UPS) and calcium homeostasis has been implicated in the neurodegeneration of Alzheimer’s and Parkinson’s diseases. The cytosolic calcium concentration is maintained by store-operated calcium entry (SOCE), which is repressed by Alzheimer’s disease-associated mutants, such as mutant presenilins. We hypothesized that inhibition of UPS impacts SOCE. This study
showed that pretreatment with sub-lethal levels of proteasome inhibitors, including MG132 and clasto-lactacystin-β-lactone (LA), reduced SOCE after depletion of endoplasmic reticulum calcium in rat neurons. With the same treatment, MG-132 and LA reduced the protein levels of stromal interaction molecule 1and 2 (STIM1/2), but not the levels of Orai1 and canonical transient receptor potential channel 1 (TRPC1). STIM1 or STIM2 protein was mobilized to lysosome by MG-132/LA treatment as observed under an immunofluorescence confocal laser microscope. In the neurons, MG-132 and LA degraded p62/SQSTM1, promoted autophagy, converted LC3I to LC3II, and promoted co-localization of LC3 and lysosomes. Rapamycin, which enhances autophagy, reduced STIM1/2 protein levels, whereas bafilomycin, which inhibits autophagy, increased their protein levels. The protein levels of STIM1/2 and the amplitude of SOCE were decreased in SH-SY5Y with decreased protein level of proteasome subunit beta type-5 induced by shRNA. We conclude that sub-lethal levels of proteasome inhibition reduce SOCE and promote autophagy-mediated degradation of STIM1/2. UPS inhibition, a common finding in neurodegenerative diseases, interferes with calcium homeostasis via repression of SOCE.
Abbreviations: AD, Alzheimer’ disease; PD, Parkinson’s disease; SOCC, storeoperated calcium channel; SOCE, store-operated calcium entry; UPS, ubiquitinproteasome system; ER, endoplasmic reticulum; STIM, stromal interaction molecule; TRPC, canonical transient receptor potential channel; PSMB5, proteasome beta subunit type-5; MG-132, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal LA, clastolactacystin-β-lactone; LC3, microtubule-associated protein light chain; P62/ SQSTM1, sequestosome1; PS, presenilin.
Key words: Stromal interaction molecule, ubiquitin-proteasome system, PSMB5, Orai1, store-operated calcium channel, presenilin, low calcium, ER stress, neurodegeneration
1. Introduction The ubiquitin-proteasome system (UPS) is involved in protein degradation, regulating cellular events, such as the cell cycle, apoptosis, and signaling [1, 2]. The inhibition of UPS may cause neuronal death and degeneration seen in Alzheimer’s[3], Parkinson’s [4], and Huntington’s diseases[5]. The correlation between inhibition of UPS and neurodegeneration may be a result of oxidative stress[6], calcium dyshomeostasis [7, 8], and endoplasmic reticulum stress [9] with proteasome inhibition. However, sub-lethal levels of proteasome inhibition may play a protective role [10-13]. Conversely, sub-lethal levels of proteasome inhibition can cause harmful effects; for example, by disturbing the ER quality-control system, such as the calnexin/calreticulin cycle [14], which plays a role in protein-folding and ER-associated degradation. In contrast with UPS-mediated protein degradation, macro-autophagy (hereafter referred to as autophagy) depends on lysosomes, and it targets long-lived proteins and organelles. Autophagy begins with the formation of a phagophore and autophagosome from subcellular structures, which engulf unused proteins and damaged organelles. Autophagosomes fuse with lysosomes to form autolysosomes, in which lysosomal hydrolases degrade the contents [12, 15]. Proteasome degradation occurs in the inner core of the 20S proteasome complex, in which β-subunits form the two inner rings and α-subunits form the two outer rings[16]. Eukaryotic proteasome complex has substrate specificity, with the β1, β2, and β5 subunits corresponding to peptidyl-glutamyl peptide-
hydrolyzing function and trypsin- and chymotrypsin-like degradation. Chymotrypsin-like degradation, which is specific for the proteasome complex [17], occurs at the PSMB5 subunit in the mammalian proteasome complex. The two intracellular protein degradation systems (autophagy and proteasomes) also interact with each other, with the inhibition of autophagy enhancing or inhibiting proteasomes [18, 19], and the inhibition of proteasomes enhancing autophagy [12, 20, 21]. Store-operated calcium entry (SOCE), also called capacitative calcium entry, maintains calcium homeostasis in non-excitable cells and is induced by depletion of endoplasmic reticulum (ER) calcium [22]. Depletion of ER calcium redistributes STIM1 localized on ER membrane to the proximity of the plasma membrane. STIM1 is an ER membrane-anchored protein with E2F in the lumen that senses ER calcium levels [23, 24]. At the plasma membrane-ER junction, STIM1 oligomerizes with Orai1 or canonical transient receptor potential channel 1 (TRPC1), thereby allowing calcium to enter [25]. STIM1 activates Orai1 in response to relatively greater reduction in ER calcium, whereas STIM2, a homologue of STIM1, maintains basal cytosolic and ER calcium levels. STIM2 activates Orai1 in response to relatively smaller reduction in ER calcium [26, 27]. Although SOCE was discovered three decades ago, the importance of SOCE in neurons was only recently assessed [28, 29] because of its much smaller amplitude than that of voltage-dependent calcium channel (VGCC). Presenilin1and 2, which are family Alzheimer disease (FAD)-associated mutants, reduce SOCE [30-32] and interfere with homeostasis of ER calcium, which is an early event in neurodegeneration [33]. It is
unclear whether and how proteasome inhibition, which is commonly seen in Alzheimer’s and Parkinson’s diseases, impacts SOCE.
2. Materials and Methods Materials The following reagents were used: carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; clasto-lactacystine-β-lactone; adenosine-triphosphate; DL-dithiothreitol; ethylene glycol tetraacetic acid (EGTA); ethylenediaminetetraacetic acid (EDTA); glycerine; N-succinylLeu-Leu-Val-Tyr-7-amido-4-methylcoumain; 7-amino-4-methylcoumarin (Sigma, St. Louis, MO); LI-COR IRDye® 800CW secondary antibody ( LI-COR, Lincoln, NE); fura2/AM, and Neurobasal/B27 (Molecular Probes/Invitrogen, Carlsbad, CA); full-range rainbow molecular-weight markers (GE Healthcare, Piscataway, NJ); CL-XPosure film (Thermo Scientific Branch, Shanghai, P. R. China); Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL); protease inhibitor cocktail (Calbiochem, San Diego, CA); psmb5 shRNA lentivirus transfer plasmid carrying GFP fluorescence (Origene, Rockville, MD); Lyso Tracker; bafilomycin (Invitrogen, MA, USA); rapamycin (LC laboratories, MA, USA); antibodies for TRPC1 and Orai1 (Abcam, Cambridge, MA); and antibodies for LC3,STIM1, STIM2, PSMB5, GAPDH ( Cell Signaling Technology, Danvers, MA).
Animals All experiments were conducted in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and EU Directive 2010/63/EU for animal experiments. The study protocol was approved by the Wenzhou Medical University Committee on the Use and Care of Animals.
Methods Primary cortex neuronal culture The primary cultures were prepared from the cortical neurons of E18 SpragueDawley rats. The cultures were maintained in Neurobasal/B27 for eight to ten days for use in experiments. The culture procedures have been described [34] .
Cell proliferation assays The survival assays of neurons were examined with the MTS assay, as described [11].
In vitro proteasome activity assay The in vitro proteasome assays were performed as described[14]. Chymotrypsinlike activity, which is mostly specific to proteasomes, was assayed either in primary neuronal cultures or in SH-SY5Y cultures.
Western blot analysis
Cultured neurons were washed with phosphate-buffered saline (PBS) and harvested in the lysis buffer. The lysis buffer was composed of 460 mM Tris-HCl, pH 7.4; 138
mM
NaCl;
1
mM
EDT;
2.5
mM
NaF;
2.5
mM
Na3VO4;
1
mM
phenylmethanesulfonylfluoride; 1 mM dithiothreitol, supplemented with 0.1% Nonidet P40; and 1 X protease / 1 X phosphatase inhibitor cocktail (Sigma, St. Louis, MO). The nitrocellulose membranes, carrying the proteins transferred from SDS-PAGE, were incubated with individual primary antibody STIM1 (1:500) / STIM2 (1:500) / Orai1 (1:500) / TRPC1 (1:500) / PSMB5 (1:1000) overnight at 4 °C. The membranes were washed with PBS and incubated with an infrared fluorescent dye-labeled secondary antibody, LI-COR IRDye® 800CW (1:5000), at room temperature for one hour. Images of the membrane were taken with LI-COR Odyssey imager 9120, and the band optical densities were obtained with ImageJ.
GAPDH blotting (1:1000) was used as the
internal loading reference.
Ratiometric calcium analysis A ratiometric calcium-imaging system was used to acquire the data. This system was composed of a high-speed monochromator (DeltaRAMTM from Photon Technology International, PTI), an EMCCD camera (AndorTM) , and an inverted microscope (Nikon Eclipse Ti). The cultures were loaded with Fura-2 by incubation with Fura-2 AM (2 M) and pluronic F-127 (0.05%) at 37 0 C for 45 minutes with the loading buffer (144 mM NaCl, 10 mM glucose, 10 mM Hepes, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 10 M glycine, pH,7.2). The buffer was washed out and replaced with the dye-free loading buffer for another 45 minutes and switched to calcium-free media (144 mM NaCl, 10
mM glucose, 10 mM Hepes, 5 mM KCl, 1 mM MgCl2, 10 M glycine, and 2 mM EGTA, pH,7.2). After the baseline [ Ca 2+]i was measured, thapsigargin (5 M) was added to the cultures and [Ca 2+] i was collected for five minutes. Thapsigargin was washed out, and the regular buffer containing calcium (144 mM NaCl, 10 mM glucose, 10 mM Hepes, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 10 M glycine, pH,7.2) was added to the cultures. The [Ca 2+]i was collected until it reached a steady state.
Immunofluorescent confocal laser microscope Cortex neurons from the E18 Sprague-Dawley rats were cultured on polyethyleneimine-coated coverslips in 6-well plates. After seven days, the neurons were treated with MG-132, LA, or sham. The procedure for staining LC3 has been described [12]. The STIM1/STIM2 distribution in cellular organelles was determined by incubating the rat neurons with lysotracker, a lysosome reporter dye, for 90 minutes at 37 oC. After washing with PBS, the neurons were fixed in 4% paraformaldehyde for five to ten minutes. The neurons were washed with PBS again, blocked with 10% goat sera for 45 minutes, and incubated with STIM1 (1:80) or STIM2 (1:30) antibody in PBS containing 0.1% Triton-X100 for two hours. The neurons were washed and incubated with FITCconjugated second antibody (1:3000) in PBS with 0.1% Triton-X100 for 45 minutes. The nuclei were stained with DAPI for three minutes, washed, and observed under a Zeiss LSM 710 confocal microscope system (Carl Zeiss, Germany). The images were taken under oil-immersion lens (X 63) and processed with Zen Le (Carl Zeiss, Germany).
Establishment of stable PSMB5 knockdown SH-SY5Y We evaluated the knockdown effects of various types of shRNA. SH-SY5Y (<20 passages) were cultured in DMEM/F12 with 10% fetal bovine serum until 70% confluency in 96-well plates. Before transfection, the culture media were changed to serum-free media and transfected with plasmids by Lipofectamine® 2000 (Invitrogen). The plasmids carry gene fragments expressing PSMB5 or scramble shRNA, EGFP, and puromycin selection marker (Origene). Four shRNA (A, B, C, D) were transfected individually or in combination with SH-SY5Y, and the maximal knockdown effect of the combination of A and B was noted. The cultured SH-SY5Y was transfected with A and B in serum-free media for five hours and switched to regular culture media supplemented with penicillin (100 unit/mL) and streptomycin (100 µg/mL) (PS) for another 48 hours. The transfected SH-SY5Y was gently trypsinized, suspended, and selected with fluorescence-activated cell sorting (FACS), based on GFP fluorescence. SH-SY5Y with GFP from FACS was cultured in the regular medium supplemented with PS and gentamycin (10 µg/mL) for a week. Puromycin (1µg/mL) was added to the culture to exclude SH-SY5Y discharging the transfected plasmids, and the selected cultures were used for experiments.
Statistical analysis
The significance in each experiment was tested by one-way ANOVA and Bonferroni post hoc test if not mentioned; otherwise by Student’s t-test. Differences were considered statistically significant if p<0.05.
3. Results 3.1. Pretreatment with proteasome inhibitors reduces the store-operated calcium entry in rat primary neuronal cultures The cellular events in the early stages of neuronal death from proteasome inhibitors may explain the process of neurodegeneration associated with UPS dysfunction. Therefore, we used doses of inhibitors that suppressed proteasome activity but did not compromise neuronal survival. At 1 µM, MG-132/LA significantly decreased proteasome activity but did not cause death at 12 hours (Figs.1A,B). However, death occurred when the treatment was extended to 36 hours (not shown). We used a maximal dose of 1 µM and maximal treatment time of 12 hours when assessing the regulation of SOCE. Under these conditions, the amplitudes of SOCE were significantly decreased after thapsigargin (Tg) treatment (5 µM). For example, the ratio differences before and after addition of calciumin either in MG-132- or LA-treated cultures, which were representative of SOCE, were much smaller than those with the sham-washed cultures (Fig.1C,D). Interestingly, the application of MG-132 or LA alone had no effect on basal calcium influx (data not shown), which suggests that modulation of SOCE by proteasome inhibitors is dependent on the depletion of ER calcium and activation of SOCE. In the treated cultures, release of calcium from the ER after thapsigargin decreased dramatically compared with that in the sham-washed cultures. This
decrease reflects a reduction in the thapsigargin-releasable ER calcium pool (Figs. 1C), which is consistent with our previous observations [7].
3.2. Proteasome inhibitors decrease the levels of STIM1 and STIM2 but not of Orai1 or TRPC1 SOCE is activated by the dynamic assembly of STIM/Orai or STIM/TRPC after depletion of ER calcium [25]. In the Orai family, Orai1 is more likely to contribute to SOCE than is Orai2 or Orai3 [26]. Similarly, TRPC1 is more consistently induced by internal calcium depletion than is TRPC3, TRPC4, or TRPC7 [25]. Therefore, we aimed to determine whether MG-132 or LA treatment changed the levels of STIM1, STIM2, Orai1, or TRPC1. Both MG-132 and LA, at 1µM for 12 hours, reduced STIM1 to ~74% of sham-washed control values (Figs, 2, P<0.05). The reduction of STIM2 was more sensitive than STIM1 to MG-132/LA treatment. For example, at 0.3 µM for 12 hours, either treatment reduced STIM2 significantly, whereas at 1 µM, MG-132 reduced STIM2 to 62%, and LA reduced it to 41% of that in sham-washed control (Fig. 2, p<0.05). Conversely, MG-132 and LA did not change the level of Orai1/TRPC1 (Figs, 2A, C). We monitored the distribution of STIM1 before and after MG-132/LA treatment by labeling lysosomes (Fig. 3, 2nd column) and STIM1 (Fig. 3, 3rd column). The staining of STIM1 was diffuse in sham-washed neurons (Fig. 3, 1st row of 3rd column) but clustered around the nuclei in treated neurons (Fig. 3, 2nd and 3rd rows of 3rd column). A significantly higher percentage of stained STIM1 co-localized with lysosomes (Fig. 3, 2nd and 3rd rows of 4th column) in those with treatments than in those without treatment (Fig. 3, 1st row of 4th column). This co-localization suggests that STIM1 was mobilized to the
lysosomes by MG-132 and LA. Increased co-localization between STIM2 staining and lysosome was seen as well (Supplemental Fig1).
3.3. Proteasome inhibitors induce autophagy in primary rat cortical cultures Previous studies indicate that proteasome inhibition induces autophagy [12, 20, 21]. We measured the level of p62, an autophagy substrate. LA, at 0.3 or 1 µM, and MG132, at 1µM, significantly decreased p62 compared with sham-washed control (Figs, 4A and C). At higher concentrations, the levels of p62 recovered (not shown). This biphasic pattern of change with proteasome inhibitors was consistent with our observation in retinal pigmented epithelial cells [12], except that the recovery in neuronal cultures occurred at higher doses. Additionally, MG-132 and LA increased the conversion of LC3I to LC3II (Figs, 4A and C). Autophagy is a multiple-step and dynamic process. Increased autophagosome accumulation such as increased LC3II conversion reflects the status either at blockade of autophagy or autophagy flux [35]. To differentiate the increased LC3II conversion either due to autophagosome accumulation or to autophagy flux, we labeled lysosomes with the lysotracker (Fig.5, column2) and LC3 (Fig.5. column3); the result indicated significantly higher amount of merging puncta in treated neurons (Fig.5, column4). This observation suggests that MG-132 and LA increased autophagy flux in the cultures.
3.4. Autophagy enhancer or inhibitor modulates STIM1/STIM2 Previous studies suggest that MG-132 and LA degrade ST1M1/2 by inducing autophagy. Rapamycin, an autophagy enhancer, at 200 nM for 16 hours, reduced
STIM1 and STIM2 protein levels to 58% and 52% of sham-washed control values, respectively (Fig.6). Bafilomycin, an autophagy inhibitor, at 100 nM for 12 hours, increased the STIM1 and STIM2 levels (Fig.6). This finding supports the hypothesis that MG-132/LA treatment degrades STIM1/STIM2 by activating autophagy.
3.5. PSMB5 knockdown reduces the protein level of STIM1/STIM2 and the amplitude of SOCE Previous studies indicate that proteasome inhibitors are associated with several undesirable effects. We knocked down the proteasome subunit beta type-5 (PSMB5), which possesses chymotrypsin-like degradation activity, in the SH-SY5Y cell line with transfection of vectors expressing PSMB5 shRNA. We screened the transfected cells with FACS based on GFP fluorescence and multiplied the transfected clones to separate the non-transfected ones. Transfection with the PSMB5 shRNA vectors knocked PSMB5 down to ~63% (Figs, 7C,D) and reduced the proteasome activity to ~61% of sham-washed control values (Fig. 7B). In cell lines with the knockdown level of PSMB5, p62 level was reduced, suggesting that the autophagy pathway is activated (Supplemental Fig.2). In SH-SY5Y with PSMB5 knockdown (sh-PSMB5), the protein levels of STIM1and STIM2 were decreased to ~68% and ~43.5%, respectively, of the SH-SY5Y with normal level of PSMB5 (sh-scr) (Figs, 7C, D). In contrast, the levels of TRPC1 and Orai1 were not changed. These findings are consistent with the results of experiments conducted in primary neuronal cultures, as described above. The amplitudes of SOCE were also significantly reduced after ER calcium depletion in sh-scr compared with those in sh-PSMB5 cells (Fig. 7E).
4. Discussion This study showed that proteasome inhibition reduced SOCE in primary rat neuronal cultures, a finding which is consistent with prior observations in primary murine neuronal cultures [7]. Presenilin1 and presenilin2 have a similar effect on SOCE [30, 32]. We confirmed the inhibition of SOCE in SH-SY5Ywith decreased proteasome activity arising from knocking down PSMB5. The reduced SOCE was associated with lower STIM1 or STIM2 levels. This reduction was mediated by the autophagy pathway, which coincides with previous observation, viz. autophagy-deficient T cells express more STIM1 [36]. A previous study showed that pretreatment with MG-132, at 10 µM for one hour, potentiates the SOCE induced by depletion of ER calcium in HEK293 cells [37]. This observation is contradictory to ours. The apparent discrepancy can be explained by the difference in treatments used: we used MG-132 or LA at 1 µM for 12 hours, which is a chronic, low dose compared with the acute, high dose used in the previous study. We ruled out the non-specific effects of proteasome inhibitors by confirming the negative regulation of SOCE subsequent to depletion of ER calcium in PSMB5 knockdown SH-SY5Y. The chemical and genetic manipulations modulating proteasome activity in our study suggest that sub-lethal levels of proteasome inhibitors suppress, rather than potentiate, SOCE. Proteasome inhibition by chemical treatment or genetic manipulation reduced the amplitude of SOCE and levels of STIM1 and STIM2. Previous studies showed that STIM2 maintains basal and ER luminal free calcium, whereas STIM1 activates Orai1 in
rat neurons at relatively greater reduction of ER calcium [26, 27]. In this study, STIM1 was reduced by ~26%, which differs significantly from the ~90% decrease in SOCE with MG-132/LA treatments. Therefore, a part of SOCE inhibition is independent of the reduction of STIM1. MG-132 and LA reduce ER luminal free calcium concentration [7, 14] and calreticulin protein level [14]. A previous study indicated that calreticulin plays a role in maintaining the ER calcium reservoir [38] but not ER luminal free calcium [39]. In this study, we showed that MG-132 and LA reduced STIM2. It is possible that reduced STIM2 levels account for the reduction of ER luminal free calcium with MG-132 and LA. An increase or decrease in calcium is associated with neuronal death [40-43]. Calcium overload leads to oxidative and nitrosative stresses, resulting in DNA and protein damage [44]. Reduced calcium levels lead to calcium redistribution in cellular compartments; for example, proteasome inhibitors induce calcium depletion from endoplasmic reticulum, resulting in ER stress and dysfunctional ER quality-control [7, 14]. However, the temporal relationship between high and low calcium levels during neuronal death is unclear. We found that sub-lethal level of proteasome inhibition reduces SOCE, suggesting that calcium deficiency is an early event in UPS inhibitionassociated neurodegeneration seen in Alzheimer’s and Parkinson’s diseases. We conclude that sub-lethal levels of proteasome inhibitors reduce SOCE and promote autophagy-mediated degradation of STIM1/2. UPS inhibition, a common finding in neurodegenerative diseases, interferes with calcium homeostasis via
repression of SOCE. The findings of this work may contribute to understanding the pathogenesis of diseases such as Alzheimer’s and Parkinson’s diseases.
Conflict of interests The authors have found none to disclose.
Acknowledgments The work was supported by National Natural Science Foundation of China (81371027), Chinese Ministry of Education (20133321120002), and by funding (QTJ10 001) from Wenzhou Medical University to Dr. Shengzhou Wu, and also supported by Wenzhou Scientific Bureau (Y20150155) to Dr. He Zhang. The funding resources have no roles in study design, data collection, interpretation, manuscript writing, and decision to submission.
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Figure1. Pretreatment with proteasome inhibitors reduces the store-operated calcium entry in primary rat neuronal cultures. A. The cortical neuronal cultures were treated with MG-132 or LA for 12 hours and subjected to chymotrypsin-like proteasome activity assay. The values from treated conditions were normalized to those of sham-washed control (100% of proteasome activity). B. MTS assay was used to measure cell viability in neuronal cultures under the same treatment conditions as in A. The values from sham-washed control were set as 100%, and survival values in treated conditions were normalized to the sham-washed control values. The results shown are a mean (+ SEM) of experiments conducted thrice in quadruplicate cultures. *,#p<0.05 vs. control. C. The cortical neuronal cultures were treated with1 µM MG-132 or LA for 12 hours and used for ratiometric calcium measurement. The neurons were loaded with Fura-2 by incubation with Fura-2 AM, the culture media were changed to calcium-free buffer, and the baseline calcium ratios were acquired for five minutes. The cultures were exposed to thapsigargin (5 M) for five minutes, and a regular buffer containing 1.8 mM calcium was added. The ratios were acquired for another five minutes. Calcium ratios were acquired every 30 seconds and reported as mean (+ SEM) from 25-57 neurons at each timepoint. *,# p< 0.05 indicated that calcium levels without treatment rose immediately after thapsigargin, which differed significantly from those of MG-132 (red line)/LA (blue line)–treated cultures. D. The differences in calcium ratios before and after addition of calcium-containing buffer from C were calculated. The number was averaged from 25-57 neurons. * p<0.05 vs. control.
Figure 2. Proteasome inhibitors reduce the levels of STIM1and STIM2, but not of Orai1 or TRPC1. After 7-9 days in culture, rat primary cortical neurons were exposed to MG-132 (A) or LA (C) for 12 hours at concentrations ranging from 100 nM to 1 μM. Lysates containing equal amounts of protein were subjected to western blot analysis of STIM1 (1:500), STIM2 (1:500),Orai1 (1:500), and TRPC1 (1:500). The ratios of optical densities were normalized to the sham-washed control value, set as 100%. The ratios of STIM1/GAPDH,STIM2/GAPDH, Orai1/GAPDH, and TRPC1/GAPDH were quantified from at least three independent experiments (B, D). The blots shown are typical of the determinations. *, # p<0.05 vs. non-treatment.
Figure 3. Proteasome inhibitors mobilize STIM1 to lysosomes. A. After 7-9 days in culture, rat primary cortical neurons were exposed to MG-132 or LA at 1 µM for 12 hours and labeled with fluorescence dye. The images, taken with a confocal laser microscope, are typical of five non-contiguous fields in each dish from triplicate experiments. Blue, DAPI-labeled nuclei; red, lyso tracker-labeled lysosome; green, STIM1-labeled puncta. The merged images are shown in the rightmost column, and the orange-stained cells indicate STIM1 staining, co-localized with lysosome. Scale bar, 20 µM. B, The STIM1-positive puncta overlying labeled lysosomes for MG-132, or LA treatment, and sham condition were manually counted with ImageJ plug-in counter; the numbers were averaged from 25 cells and quantified. * p<0.05 vs. control.
Figure 4. Proteasome inhibitors induce p62 degradation and LC3 lipidation. The neurons at 7-9 days in culture were exposed to MG-132, LA, or sham-washed control for 12 hours. The lysates were subjected to immunoblotting with p62 (1:1000) and LC3 (1:1000) (A, C). GAPDH was used as an internal reference. The blots shown are typical of experiments performed thrice. The optical densities were quantified in B and D. *p<0.05 vs. control.
Figure 5. Proteasome inhibitors increase autophagy flux. A.The rat primary cortical neurons at 7-9 days in culture were exposed to MG-132 or LA at 1 µM for 12 hours and labeled with fluorescence. The images taken with a confocal laser microscope are typical of the images from five non-contiguous fields in each dish from triplicate experiments. Blue, DAPI-labeled nuclei; red, lyso tracker-labeled lysosome; green, LC3 staining. The merged images are shown in the rightmost column, and the orangestained cells indicate the co-localization between LC3 and lysosome. Scale bar, 20 µM. B. The co-localization puncta between LC3 and lysosome for MG-132, LA, and sham conditions were averaged from 25 cells and quantified. * p<0.05 vs. control.
Figure 6. Rapamycin reduces and bafilomycin increases STIM1/STIM2 levels. The neurons at 7-9 days in culture were exposed to rapamycin, bafilomycin, or shamwashed control for 12 hours. The lysates were subjected to immunoblotting with p62 (1:1000), STIM1(1:500), and STIM2 (1:500). GAPDH was used as an internal reference. The blots shown are typical of experiments performed thrice; the optical densities were quantified in B and D. *p<0.05 vs. control.
Figure 7. Reduction of STIM1, STIM2, and SOCE in PSMB5 knockdown SH-SY5Y cells. A. SH-SY5Y cells were stably transfected with a vector expressing RNA of a scrambled sequence (scr) or vectors expressing PSMB5 shRNA (A and B). The cells were screened and multiplied. The SH-SY5Y cells with either vector expressing GFP were detected in a fluorescence (FL) image of the AB clone. A phase contrast (PH) image is shown for comparison. B. Non-denatured lysates containing equal amounts of protein from the SH-SY5Y stably transfected with scr vector (scr) or PSMB5 shRNA vector (shPSMB5) were subjected to chymotrypsin degradation assay. The average results of experiments performed thrice are shown. Student’s t-test was used; *p<0.05 vs. scr. C. Denatured lysates containing equal amounts of protein were subjected to western blot analysis of TRPC1 (1:500), STIM1 (1:500), Orai1 (1:500), and PSMB5 (1:1000). The blots are typical of experiments performed thrice. D.The ratios of STIM1/GAPDH, Orai1/GAPDH, TRPC1/GAPDH, and PSMB5/GAPDH were quantified from experiments performed thrice. *,#p<0.05 vs scr. E. The analyses of SOCE showed linear trends averaged from 32 cells either for scr or shPSMB5. *p<0.05 indicated significant difference in the amount of calcium released from ER induced by thapsigargin between in scr and in shPSMB5. ++p<0.05 indicated significant difference in the amplitude of calcium influx subsequent to thapsigargin-induced ER calcium depletion between in scr and in shPSMB5.