Mol. Cells 2017; 40(4): 280-290
Published online March 28, 2017
https://doi.org/10.14348/molcells.2017.2320
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: kijang1@kbri.re.kr
Several lines of evidence suggest that endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of many neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Protein tyrosine phosphatase 1B (PTP1B) is known to regulate the ER stress signaling pathway, but its role in neuronal systems in terms of ER stress remains largely unknown. Here, we showed that rotenone-induced toxicity in human neuroblastoma cell lines and mouse primary cortical neurons was ameliorated by PTP1B inhibition. Moreover, the increase in the level of ER stress markers (eIF2α phosphorylation and PERK phosphorylation) induced by rotenone treatment was obviously suppressed by concomitant PTP1B inhibition. However, the rotenone-induced production of reactive oxygen species (ROS) was not affected by PTP1B inhibition, suggesting that the neuroprotective effect of the PTP1B inhibitor is not associated with ROS production. Moreover, we found that MG132-induced toxicity involving proteasome inhibition was also ameliorated by PTP1B inhibition in a human neuroblastoma cell line and mouse primary cortical neurons. Consistently, downregulation of the PTP1B homologue gene in
Keywords endoplasmic reticulum stress (ER Stress), MG132, reactive oxygen species (ROS), rotenone, ubiquitin proteasome system
The endoplasmic reticulum (ER) is a multifunctional continuous membrane system essential for the synthesis, folding and processing of secretory and transmembrane proteins in eukaryotic cells. Functional impairment of the ER causes accumulation of unfolded proteins in the ER lumen and leads to an evolutionarily conserved stress response called the unfolded protein response (Chung et al., 2015; Xu et al., 2005). Previous studies have indicated that prolonged ER stress is implicated in many human diseases including neurodegenerative diseases, atherosclerosis, type 2 diabetes and cancer (Ozcan and Tabas, 2012).
Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed enzyme anchored in the ER membrane (Popov, 2012). PTP1B plays a crucial role in ER homeostasis through dephosphorylating tyrosine kinase receptors and thereby regulating the intensity of their signaling cascades (Popov, 2012). Previous studies have indicated that ER stress elevated by a high-fat diet is attenuated by PTP1B deletion in mouse skeletal muscle (Panzhinskiy et al., 2013a). In the central nervous system, neuron-specific PTP1B deletion mice are hypersensitive to leptin and have reduced body weight (Prada et al., 2013). Moreover, PTP1B upregulation is observed in neuroinflammatory conditions. Rat spinal cord injury greatly increases the level of PTP1B in motor neurons, and brain injection of lipopolysaccharide (LPS, inflammation inducer) significantly elevates PTP1B expression (Song et al., 2016; Zhu et al., 2015). Collectively, this evidence suggests that PTP1B seems to play an important role in ER stress-mediated neuronal cell death, though the mechanism underlying its role in neuronal ER stress is mostly unknown.
To investigate the role of PTP1B in ER stress-mediated neuronal cell death, we treated the neuroblastoma cell line (SH-SY5Y) and mouse primary cortical neurons with ER stress inducers such as rotenone and MG132. Rotenone produces reactive oxygen species (ROS) through mitochondrial complex I inhibition (Nandipati and Litvan, 2016), and MG132 is a specific inhibitor of the 26S proteasome (Cui et al., 2013). Even though the two chemicals induce ER stress via different mechanisms, PTP1B inhibition mitigated both MG132 and rotenone-induced neuronal cell death. Furthermore, PTP1B inhibition decreased the level of ER stress markers such as phosphorylated eIF2 alpha and phosphorylated PERK. In addition, downregulation of the
Cell culture media and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific (USA). Rotenone (R8875), dimethyl sulfoxide, 2′,7′-dichlorofluorescein diacetate and tunicamycin (T7765) were purchased from Sigma-Aldrich (USA). The PTP1B (CAS-765317-72-4) inhibitor and MG132 were purchased from EMD Millipore (USA). Rabbit anti-phospho-eIF2a (Ser51) (catalog no. 3597), rabbit anti-eIF2a (catalog no. 9722) and HRP-conjugated anti-alpha-tubulin (catalog no. 9099) were obtained from Cell Signaling Technology. Rabbit anti-phospho-PERK (Thr981) (catalog no. sc32577) and rabbit anti-PTP1B (catalog no. sc14021) were purchased from Santa Cruz Biotechnology.
Human neuroblastoma cells, SH-SY5Y, were grown in DMEM with 10% fetal bovine serum (FBS) and anti-biotic (100 U/ml penicillin, 100 μg/ml streptomycin) solutions at 37°C in 5% CO2/95% air. SH-SY5Y cells were seeded in 96-well plates (1 × 105 cells/well). After 24 h, different treatments were performed. Cortical tissue from embryonic day 16 (E16) mouse brains was dissected out, incubated with 0.25% trypsin for 15 min at 37°C, and dissociated by mechanical trituration (Araki et al., 2000). The brains was removed and transferred to a 15 ml conical tube and washed twice with ice-cold HBSS (Gibco), and the cortex was separated and then incubated with 2 ml of pre-warmed papain (20 units/ml) (Worthington Biochemical Corporation) and DNase I (0.005%) for 30 min at 37°C in a humidified cell culture incubator supplied with 5% CO2. After incubation, cortical cells were centrifuged at 800 rpm for 10 min at room temperature. Dissociated cortical neurons were then plated in 48-well plates (2 × 105 cells/well) previously coated with 0.1% poly-D-lysine (Sigma-Aldrich), and grown in neurobasal media containing B27 supplement (Gibco), N2 supplement (Gibco), 2 mM glutamine (Gibco), and penicillin-streptomycin (Gibco). The culture media was changed initially after 5 days and then half-changed every 3 days, and cells were used after culture for 14–15 days.
Finally, the viability of the cells was determined by using the Cell Counting Kit-8 (CCK-8) assay, as previously described (Xu et al., 2012). CCK-8 is more sensitive than the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. For dose-response studies of rotenone toxicity, SH-SY5Y cells and primary cortical neurons were cultured with rotenone at 0–1000 μM and 0–20 μM concentrations for 24 h, respectively. Then, for dose-response studies of MG132 toxicity, SH-SY5Y cells and primary cortical neurons were cultured with MG132 at 0–20 μM and 0–1 μM concentrations for 24 h, respectively. Finally, for dose-response studies of tunicamycin toxicity, SH-SY5Y cells were cultured with tunicamycin at 0–20 μM concentrations for 24 h. Then, 10–20 μl CCK-8 (Enzo Life Sciences) solution was added to each well. Plates were incubated for an additional 2 h. The optical density of each well was measured using a microplate reader (Tecan) at a 450 nm wavelength. Cell viability was expressed as a percentage of that of the DMSO-treated cells. All experiments were performed in triplicate.
Flies (n < 100) from each experimental group were monitored for their survival along with aging. The rotenone, paraquat and MG132-induced toxicity survival assays were performed on regular food medium. Flies were maintained on standard cornmeal agar media at 24°C and transferred every day to a new vial containing food that was treated with chemicals for the chemical treatment groups. Next, a survival assay was performed on filter papers soaked with 450 μM MG132 and 5% sucrose at 30°C. Filters were changed daily. The non-chemical treatment groups were maintained in the same way except for the treatment with the chemicals. The percentage of flies that remained alive at the end of the experiment was calculated based on the starting number of flies for each treatment group. This experiment was repeated three times.
The levels of intracellular ROS generation were evaluated by DCFH-DA as previously described (Song et al., 2012). Briefly, after treatment, cells were washed with 1X PBS and loaded with DCFH-DA (final concentration 10 μM in colorless DMEM) for 30 min at 30°C. Fluorescence was measured by using a fluorescence spectrophotometer with excitation at 485 nm and emission at 530 nm.
The OCR was measured using a Seahorse XF24 analyzer according to the manufacturer’s instructions (Seahorse Bioscience Inc). Briefly, SH-SY5Y cells were plated at 1 × 105 cells per well in full growth medium (Hardie et al., 2017). After overnight attachment, the medium was washed and replaced with pre-warmed running medium (non-buffered DMEM supplemented with 4 mM L-glutamine, 25 mM D-glucose and 1 mM sodium pyruvate, pH 7.4) and incubated in a non-CO2 incubator at 37°C for 60 min. The OCR was further measured following injection of rotenone and the PTP1B inhibitor. After the OCR measurement, cells were lysed, and the protein content was estimated using a BCA Assay (Pierce). The OCR was plotted after normalizing by total protein.
After treatment, the cells were homogenized in RIPA buffer (Cell signaling Technology) with a phosphatase and protease inhibitor cocktail (Roche) and incubated at 4°C for 1 h. Cells were collected by centrifugation at 13,000 rpm for 30 min at 4°C. The supernatant was collected, and the protein concentration was determined using a BCA protein assay kit (Pierce). Next, denatured proteins were separated by Nu-PAGE 4–12% Bis-Tris Gels (Novex) and transferred to a poly-vinylidene difluoride (PVDF) membrane (Novex) at 20 V for 1 h. The blots were blocked for 1 h at room temperature in 5% skim milk. The membrane was then incubated overnight at 4°C with primary antibodies against phospho-eIF2a (Ser51), eIF2a, phospho-PERK (Thr981), PTP1B and α-tubulin. The membrane was incubated for 1 h with HRP-conjugated secondary antibodies, followed by detection with the ECL prime kit (Amersham Biosciences). Samples from three independent experiments were used in this analysis. The relative expression level was determined by using a Fusion-FX imaging system (Viber Lourmat).
Cell death was detected by using an FITC Annexin V apoptosis detection kit (BD Biosciences) and flow cytometry. In brief, cells were trypsinized and washed with chilled PBS twice, and the cell pellets were re-suspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) to create a cell suspension at a density of 1 × 106 cells/ml. Then, 3 μl of FITC-conjugated Annexin V was added to the suspension, which was incubated for 15 min at room temperature in the dark. Finally, 3 μl of propidium iodide was added, and flow cytometry was performed within 1 h using MoFlo Astrios cell sorter (Beckman Coulter).
Each experiment was performed at least three times, and the results were presented as the means ± standard deviations (SD) or means ± standard error (SEM). Comparison between groups was made by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test (GraphPad Prism Software) or analyzed by Student’s
To investigate the role of PTP1B in neuronal ER stress, we used rotenone-treated SH-SY5Y cells and a selective PTP1B inhibitor (CAS 765317-72-4) (Wiesmann et al., 2004). Rotenone is a widely used insecticide and is known to induce ER stress via ROS production (Pal et al., 2014). The cytotoxicity of the human neuroblastoma cell line (SH-SY5Y) was determined using a CCK-8 assay, and the percent cell viability was plotted. Treatment with rotenone (15 μM for 24 h) reduced cell viability by approximately 50% in SH-SY5Y cells (Fig. 1A). Pre-treatment with the PTP1B inhibitor at concentrations ranging from 5–20 μM reduced rotenone-induced cell death in a dose-dependent manner (Fig. 1A). Furthermore, PTP1B inhibition mitigated rotenone-induced toxicity in primary mouse cortical neurons (Fig. 1B, 53% compared to 68% in rotenone-treated primary cortical neuronal cells). The protective effect of the PTP1B inhibitor on rotenone-induced toxicity was also assessed by PI and Annexin V staining followed by flow cytometric analysis in SH-SY5Y cells. Annexin V and PI were used to determine the levels of apoptotic cell death (PI−/Annexin V+ or PI+/Annexin V+) or necrotic cell death (PI+/Annexin V−). Rotenone induced cell death in 25.79% of SH-SY5Y cells (PI−/Annexin V+ or PI+/Annexin V+: 20.94% and PI+/Annexin V−: 4.85%). In comparison, the percent cell death in SH-SY5Y cells treated with both rotenone and the PTP1B inhibitor was 8.84% (PI−/Annexin V+ or PI+/Annexin V+: 6.77% and PI+/Annexin V−: 2.07%). PTP1B inhibitor pretreatment decreased cell death by 16.95% in the rotenone-treated condition, whereas the PTP1B inhibitor alone without rotenone treatment did not affect neuronal viability (Fig. 1C). These data indicate that PTP1B inhibition protects neuronal cells from rotenone-induced cell death.
We next investigated the role of PTP1B in ROS-induced toxicity
We then wondered how PTP1B inhibition could suppress ROS-induced toxicity. Previous studies have reported that rotenone-induced ER stress increases the phosphorylation level of PERK (Thr981) and eIF2α (Ser51) (Chen et al., 2008). Phospho-PERK and phospho-eIF2α are well-known markers for ER stress. To investigate the role of the ER stress pathway in rotenone-treated neuronal cells, we performed an immunoblotting analysis with the antibodies against phospho-PERK (Thr981), phospho-eIF2α (Ser51), and total eIF2a and PTP1B from rotenone- and/or PTP1B inhibitor-treated SH-SY5Y cells. Interestingly, PTP1B inhibition significantly reduced the rotenone-induced increase in the phosphorylation of PERK-eIF2α (Fig. 2A). In contrast, the total eIF2a and PTP1B protein levels were not changed in rotenone- and PTP1B inhibitor-treated SH-SY5Y cells (Fig. 2A). We confirmed that the PTP1B inhibitor alone had no effect on the levels of phospho-PERK and phospho-eIF2α (Fig. 2B). These results indicated that neuronal ER stress could be mitigated by PTP1B inhibition.
Previous studies have shown that reduced mitochondrial complex I activity by rotenone treatment results in an increased production of ROS and thereby consequently induces ER stress-mediated neurotoxicity (Goswami et al., 2016). Therefore, rotenone-induced ROS production may be regulated by PTP1B.
To investigate this possibility, we measured intracellular ROS using the redox-sensitive fluorophore 2′,7′-dichloro-fluorescein diacetate (DCFH-DA). Non-fluorescent DCFH-DA is converted to the fluorescent molecule DCF by oxidation. We found that the level of DCF in SH-SY5Y cells treated with 15 μM rotenone was significantly increased in a time-dependent manner (Fig. 3A). ROS levels, when stimulated by 15 μM rotenone, peaked at 24 h and were 1.8-fold higher than in the DMSO treatment group. Next, SH-SY5Y cells were pre-incubated with the PTP1B inhibitor (20 μM) for 30 min and treated with 15 μM rotenone or 5 μM MG132 for 24 h. Pre-treatment with the PTP1B inhibitor for 30 min showed no significant reduction in the rotenone-induced intracellular ROS level. Treatment with the proteasome inhibitor, MG132, for 24 h showed no significant effect on ROS production compared to that observed in the DMSO treatment group (Fig. 3B).
For real-time analysis of mitochondrial oxidation, SH-SY5Y cells were analyzed using an XF-24 extracellular flux analyzer (Seahorse Bioscience Inc). In sequence, SH-SY5Y cells were treated with rotenone (5 μM) in the presence or absence of the PTP1B inhibitor (20 μM), and cellular oxygen consumption rate (OCR) was measured using the XF24 Seahorse Analyzer. As expected, rotenone significantly inhibited the OCR of SH-SY5Y cells; moreover, PTP1B inhibition did not result in any change in the reduced OCR through injection of rotenone (Fig. 3C). These data suggest that the neuroprotective effect of PTP1B inhibition does not involve ROS production.
We have shown that PTP1B inhibition could protect rote-none-induced neurotoxicity without reducing ROS production. Therefore, we hypothesized that PTP1B inhibition may primarily attenuate downstream processes of ROS such as ER stress. MG132 is a specific peptide-aldehyde inhibitor of the ubiquitin proteasome and known as an inducer of ER stress. Because unfolded and misfolded proteins are degraded by the proteasome system (Werner et al., 1996), MG132 treatment results in accumulation of misfolded-proteins in the ER, leading to ER stress (Nakajima et al., 2011).
We examined the level of ER stress markers in MG132-treated SH-SY5Y cells. Cells were treated with MG132 for different time periods and subjected to immunoblot analysis. MG132 treatment rapidly increased the level of the ER stress marker, phospho-eIF2α. Moreover, PTP1B inhibition significantly reduced the MG132-induced eIF2α phosphorylation (Fig. 4A). To elucidate the role of PTP1B in neuronal UPS impairment, we treated SH-SY5Y cells (Fig. 4B) and mouse primary cortical neurons (Fig. 4C) with MG132 and/or the PTP1B inhibitor. Cell viability was examined using cell counting kit-8. We found that within the 24-h treatment period, MG132 caused dramatic cell death, while the PTP1B inhibitor suppressed this MG132-induced cell death. To confirm the effect of the PTP1B inhibitor on MG132-induced neuronal cell death, SH-SY5Y cells were double-labeled for Annexin V and PI and then analyzed by flow cytometric analysis. MG132 induced cell death in 16.03% of SH-SY5Y cells (PI−/Annexin V+ or PI+/Annexin V+: 15.22% and PI+/Annexin V−: 0.81%). In comparison, the percent cell death in SH-SY5Y cells treated with both MG132 and the PTP1B inhibitor was 6.22% (PI−/Annexin V+ or PI+/Annexin V+: 5.03% and PI+/Annexin V−: 1.19%) (Fig. 4D).
To test the idea that therapeutic modulation of PTP1B may have an effect on toxicity induced by UPS impairment
Tunicamycin specifically blocks the initial step of glycoprotein biosynthesis in the ER (Oslowski and Urano, 2011). Moreover, a previous study showed that tunicamycin is induced in neuronal cell death and ER stress. Thus, we sought to confirm how the neuroprotective effect of PTP1B is regulated by other ER stress inducers in SH-SY5Y cells.
SH-SY5Y cells were treated with 5 μM of tunicamycin for 24 h and pre-treated with different dosages of the PTP1B inhibitor for 30 min. The cell viability was tested using the CCK-8 assay. The result of the CCK-8 assay indicated that PTP1B inhibition (20 μM) prevented cell death induced by tunicamycin (Fig. 5A, 30.8% compared to 40.9% in tunicamycin-treated SH-SY5Y cells). These data indicate that PTP1B plays an important role in various forms of ER stress-induced neuronal toxicity. Next, we also examined whether PTP1B inhibition influences tunicamycin-induced ER stress. PTP1B inhibition significantly reduced the tunicamycin-induced increase in the phosphorylation of eIF2α (Fig. 5B). These results suggest that PTP1B inhibition attenuates the toxicity of neuronal ER stress.
Oxidative stress and UPS impairment are common features of many neurodegenerative diseases including AD, PD and ALS. Both cause accumulation of misfolded and unfolded proteins in the ER lumen, and accumulation of these abnormal proteins induces ER stress. ER stress activates a stress-adaptive signaling process called the unfolded protein response (UPR) (Xiang et al., 2017). UPR activation is mediated via three ER stress sensors localized in the ER lumen: IRE1, ATF6 and PERK. Recently, many studies have reported that phosphorylated PERK and eIF2α are detected in postmortem brain and spinal cord tissue from AD, ALS, PD and frontotemporal dementia (FTD) patients (Smith and Mallucci, 2016). Moreover, pharmacological inhibition of the PERK pathway restored pathological features of ALS, Prion disease and FTD animal models (Kim et al., 2014; Moreno et al., 2013; Radford et al., 2015). Therefore, the PERK-eIF2α axis of the UPR might be a valuable therapeutic target for various neurodegenerative diseases.
PTP1B is a negative regulator of insulin signaling and is predominantly localized to the cytoplasmic surface of the ER (Popov, 2012). Recent studies have suggested that PTP1B is implicated in the regulation of ER stress signaling in cultured adipocytes and myotubes (Bettaieb et al., 2012; Panzhinskiy et al., 2013b). Interestingly, Song et al. (2016) discovered that inhibition of PTP1B has an anti-inflammatory effect in a mouse neuroinflammation model. Many reports have indicated that inflammatory responses are also implicated in ER stress and chronic metabolic diseases such as type 2 diabetes, obesity and insulin resistance (Hotamisligil, 2010; Zhang, 2010). Furthermore, Hakim et al. (2015) showed that chronic sleep fragmentation induces ER stress and PTP1B upregulation in hypothalamic neurons. Collectively, previous studies have suggested that PTP1B might modulate the toxic effect of neuronal ER stress.
To elucidate the relationship between neuronal ER stress and PTP1B, we examined whether neuronal toxicity induced by UPS impairment and ROS production were mitigated by PTP1B inhibition.
We showed that PTP1B inhibition attenuated oxidative stress- and UPS impairment-induced toxicity in
To the best of our knowledge, rotenone triggers massive ROS production through the inhibition of mitochondrial complex I, which finally results in ER stress and apoptotic cell death (Seoposengwe et al., 2013; Swarnkar et al., 2012). Therefore, PTP1B inhibition might mitigate rotenone-induced ROS production via improvement of mitochondrial function. Our study showed that significantly increased levels of ROS and mitochondria dysfunction were observed in rotenone-treated neuronal cells. However, the increase in ROS production and mitochondrial dysfunction induced by rotenone treatment were not altered by PTP1B inhibition. These data demonstrate that the protective effect of PTP1B inhibition seems to not be related to ROS production or mitochondrial activity.
We also tested the effect of the PTP1B inhibitor after inducing ER stress with tunicamycin. Tunicamycin inhibits the glycosylation of newly synthesized protein, and this biosynthetic step occurs within the ER. Consequently, impairment of protein glycosylation leads to the disruption of proper protein folding in the ER (Foufelle and Fromenty, 2016). We found that tunicamycin-induced cell death and eIF2α phosphorylation is also suppressed by PTP1B inhibition in SH-SY5Y cells. Taken together, these results suggest that PTP1B inhibition attenuates the toxicity of neuronal ER stress.
Possible neuroprotective mechanisms of PTP1B inhibition include BDNF/TrkB signaling. TrkB is a family of receptor tyrosine-kinases (RTKs) and a receptor of brain-derived neurotrophic factor (Vieira et al., 2017). Many studies have reported that the toxic effects of ER stress are significantly mitigated by the upregulation of BDNF in the brain and neuronal cells (Chen et al., 2007; Qiu et al., 2013; Shimoke et al., 2004; Wei et al., 2014; Zhu et al., 2004). Moreover, TrkB is a direct substrate of PTP1B; so, PTP1B negatively regulates TrkB activation via BDNF treatment (Ozek et al., 2014). Notably, deletion or inhibition of PTP1B potentiates BDNF signaling via enhanced TrkB phosphorylation in SH-SY5Y cells and the mouse hypothalamic brain region (Ozek et al., 2014). Therefore, PTP1B inhibition might mitigate the toxic consequences of neuronal ER stress via enhancing BDNF/TrkB signaling.
Another possible mechanism is the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. Nrf2 is a basic leucine-zipper transcription factor that plays an important role in mitigating proteotoxic stress (Pajares et al., 2017). Under normal conditions, the level of Nrf2 protein remains low because of its rapid turnover. In response to different stimuli, including accumulation of misfolded proteins, the Nrf2 protein is increased and translocated to the nucleus to promote the transcription of ARE-containing genes (Cui et al., 2016). Nrf2 activation upregulates key genes for the protein quality control system, and this gene upregulation promotes the degradation of misfolded proteins. Specifically, Nrf2 increases the expression level of several UPS subunits and protects cells from abnormal protein accumulation. Previous studies have indicated that Nrf2 activation is essential for transcriptional upregulation of the 26S proteasome subunit and improving proteasome activity in the liver and fibroblasts (Kapeta et al., 2010; Kwak et al., 2003a; 2003b; Pickering et al., 2012). Moreover, PTP1B deficiency enhances the nuclear accumulation of Nrf2 in acetaminophen-induced hepatotoxicity via the GSK3 beta/Src-Fyn pathway (Mobasher et al., 2013). In this study, we found that UPS impairment-induced toxicity is rescued by PTP1B inhibition. Therefore, PTP1B inhibition might mitigate neuronal ER stress by promoting the degradation of abnormal proteins through Nrf2 activation.
In conclusion, our study showed that PTP1B inhibition attenuates ER stress via mitochondria-independent mechanisms in neuronal cells. As a proof of principle that modulating PTP1B activity could be beneficial, a small molecule inhibitor of PTP1B significantly suppressed UPS impairment-induced toxicity
SH-SY5Y cells (A) and primary mouse cortical neuron (B) cells were pretreated with various concentrations of the PTP1B inhibitor for 30 min and then treated with rotenone for 24 h. The rotenone-induced increase in neuronal cell death was attenuated by PTP1B inhibition. The cell viability was then determined by CCK-8 assay, and the percentage of cell viability was plotted as the mean ± SD of the three different experiments
(A) SH-SY5Y cells were pretreated with the PTP1B inhibitor for 30 min and then treated with 15 μM rotenone for 12 h. The expression levels of p-PERK, eIF2α, p-eIF2α and PTP1B were determined with immunoblotting. Rotenone-induced p-PERK and p-eIF2α protein levels were decreased by PTP1B inhibition. (B) SH-SY5Y cells were pretreated with the PTP1B inhibitor for 30 min. The expression levels of p-PERK, eIF2α and p-eIF2α were determined with immunoblotting. There were no significant changes in p-PERK, p-eIF2α and eIF2α with PTP1B inhibitor treatment. The relative protein levels of p-PERK, eIF2α, p-eIF2α and PTP1B imaged by Fusion imaging systems were measured by densitometry and normalized to the expression of α-tubulin.
(A) Rotenone induces intracellular ROS production in SH-SY5Y cells. The indicated cells were treated with rotenone (15 μM) for 0–24 h, followed by ROS production using the dye DCFH-DA. The rotenone-induced ROS production was time-dependent. *
(A) SH-SY5Y cells were pretreated with the PTP1B inhibitor for 30 min and then treated with 5 μM MG132 for 24 h. The expression levels of eIF2α and p-eIF2α were determined by immunoblotting. The relative protein levels of eIF2α and p-eIF2α imaged by a Fusion imaging system were measured by densitometry and normalized to the expression of α-tubulin. SH-SY5Y cells (B) and primary mouse cortical neurons (C) were pretreated with various concentrations of the PTP1B inhibitor for 30 min and treated with MG132 for 24 h. The MG132-induced neuronal cell death was attenuated by PTP1B inhibition. Cell viability was then determined by CCK-8 assay, and the percent cell viability was plotted as the mean ± SD of the three different experiments (**
(A) SH-SY5Y cells were pretreated with various concentrations of the PTP1B inhibitor for 30 min and then treated with 5 μM tunicamycin for 24 h. The tunicamycin-induced increase in neuronal cell death was attenuated by PTP1B inhibition. The cell viability was then determined by CCK-8 assay, and the percentage of cell viability was plotted as the mean ± SD of the three different experiments
Mol. Cells 2017; 40(4): 280-290
Published online April 30, 2017 https://doi.org/10.14348/molcells.2017.2320
Copyright © The Korean Society for Molecular and Cellular Biology.
Yu-Mi Jeon1, Shinrye Lee1, Seyeon Kim1, Younghwi Kwon1, Kiyoung Kim2, Chang Geon Chung3, Seongsoo Lee4, Sung Bae Lee3, and Hyung-Jun Kim1,*
1Department of Neural Development and Disease, Korea Brain Research Institute (KBRI), Daegu 41068, Korea, 2Department of Medical Biotechnology, Soonchunhyang University, Asan 31538, Korea, 3Department of Brain & Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988, Korea, 4Gwangju Center, Korea Basic Science Institute (KBSI), Gwangju 61186, Korea
Correspondence to:*Correspondence: kijang1@kbri.re.kr
Several lines of evidence suggest that endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of many neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Protein tyrosine phosphatase 1B (PTP1B) is known to regulate the ER stress signaling pathway, but its role in neuronal systems in terms of ER stress remains largely unknown. Here, we showed that rotenone-induced toxicity in human neuroblastoma cell lines and mouse primary cortical neurons was ameliorated by PTP1B inhibition. Moreover, the increase in the level of ER stress markers (eIF2α phosphorylation and PERK phosphorylation) induced by rotenone treatment was obviously suppressed by concomitant PTP1B inhibition. However, the rotenone-induced production of reactive oxygen species (ROS) was not affected by PTP1B inhibition, suggesting that the neuroprotective effect of the PTP1B inhibitor is not associated with ROS production. Moreover, we found that MG132-induced toxicity involving proteasome inhibition was also ameliorated by PTP1B inhibition in a human neuroblastoma cell line and mouse primary cortical neurons. Consistently, downregulation of the PTP1B homologue gene in
Keywords: endoplasmic reticulum stress (ER Stress), MG132, reactive oxygen species (ROS), rotenone, ubiquitin proteasome system
The endoplasmic reticulum (ER) is a multifunctional continuous membrane system essential for the synthesis, folding and processing of secretory and transmembrane proteins in eukaryotic cells. Functional impairment of the ER causes accumulation of unfolded proteins in the ER lumen and leads to an evolutionarily conserved stress response called the unfolded protein response (Chung et al., 2015; Xu et al., 2005). Previous studies have indicated that prolonged ER stress is implicated in many human diseases including neurodegenerative diseases, atherosclerosis, type 2 diabetes and cancer (Ozcan and Tabas, 2012).
Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed enzyme anchored in the ER membrane (Popov, 2012). PTP1B plays a crucial role in ER homeostasis through dephosphorylating tyrosine kinase receptors and thereby regulating the intensity of their signaling cascades (Popov, 2012). Previous studies have indicated that ER stress elevated by a high-fat diet is attenuated by PTP1B deletion in mouse skeletal muscle (Panzhinskiy et al., 2013a). In the central nervous system, neuron-specific PTP1B deletion mice are hypersensitive to leptin and have reduced body weight (Prada et al., 2013). Moreover, PTP1B upregulation is observed in neuroinflammatory conditions. Rat spinal cord injury greatly increases the level of PTP1B in motor neurons, and brain injection of lipopolysaccharide (LPS, inflammation inducer) significantly elevates PTP1B expression (Song et al., 2016; Zhu et al., 2015). Collectively, this evidence suggests that PTP1B seems to play an important role in ER stress-mediated neuronal cell death, though the mechanism underlying its role in neuronal ER stress is mostly unknown.
To investigate the role of PTP1B in ER stress-mediated neuronal cell death, we treated the neuroblastoma cell line (SH-SY5Y) and mouse primary cortical neurons with ER stress inducers such as rotenone and MG132. Rotenone produces reactive oxygen species (ROS) through mitochondrial complex I inhibition (Nandipati and Litvan, 2016), and MG132 is a specific inhibitor of the 26S proteasome (Cui et al., 2013). Even though the two chemicals induce ER stress via different mechanisms, PTP1B inhibition mitigated both MG132 and rotenone-induced neuronal cell death. Furthermore, PTP1B inhibition decreased the level of ER stress markers such as phosphorylated eIF2 alpha and phosphorylated PERK. In addition, downregulation of the
Cell culture media and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific (USA). Rotenone (R8875), dimethyl sulfoxide, 2′,7′-dichlorofluorescein diacetate and tunicamycin (T7765) were purchased from Sigma-Aldrich (USA). The PTP1B (CAS-765317-72-4) inhibitor and MG132 were purchased from EMD Millipore (USA). Rabbit anti-phospho-eIF2a (Ser51) (catalog no. 3597), rabbit anti-eIF2a (catalog no. 9722) and HRP-conjugated anti-alpha-tubulin (catalog no. 9099) were obtained from Cell Signaling Technology. Rabbit anti-phospho-PERK (Thr981) (catalog no. sc32577) and rabbit anti-PTP1B (catalog no. sc14021) were purchased from Santa Cruz Biotechnology.
Human neuroblastoma cells, SH-SY5Y, were grown in DMEM with 10% fetal bovine serum (FBS) and anti-biotic (100 U/ml penicillin, 100 μg/ml streptomycin) solutions at 37°C in 5% CO2/95% air. SH-SY5Y cells were seeded in 96-well plates (1 × 105 cells/well). After 24 h, different treatments were performed. Cortical tissue from embryonic day 16 (E16) mouse brains was dissected out, incubated with 0.25% trypsin for 15 min at 37°C, and dissociated by mechanical trituration (Araki et al., 2000). The brains was removed and transferred to a 15 ml conical tube and washed twice with ice-cold HBSS (Gibco), and the cortex was separated and then incubated with 2 ml of pre-warmed papain (20 units/ml) (Worthington Biochemical Corporation) and DNase I (0.005%) for 30 min at 37°C in a humidified cell culture incubator supplied with 5% CO2. After incubation, cortical cells were centrifuged at 800 rpm for 10 min at room temperature. Dissociated cortical neurons were then plated in 48-well plates (2 × 105 cells/well) previously coated with 0.1% poly-D-lysine (Sigma-Aldrich), and grown in neurobasal media containing B27 supplement (Gibco), N2 supplement (Gibco), 2 mM glutamine (Gibco), and penicillin-streptomycin (Gibco). The culture media was changed initially after 5 days and then half-changed every 3 days, and cells were used after culture for 14–15 days.
Finally, the viability of the cells was determined by using the Cell Counting Kit-8 (CCK-8) assay, as previously described (Xu et al., 2012). CCK-8 is more sensitive than the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. For dose-response studies of rotenone toxicity, SH-SY5Y cells and primary cortical neurons were cultured with rotenone at 0–1000 μM and 0–20 μM concentrations for 24 h, respectively. Then, for dose-response studies of MG132 toxicity, SH-SY5Y cells and primary cortical neurons were cultured with MG132 at 0–20 μM and 0–1 μM concentrations for 24 h, respectively. Finally, for dose-response studies of tunicamycin toxicity, SH-SY5Y cells were cultured with tunicamycin at 0–20 μM concentrations for 24 h. Then, 10–20 μl CCK-8 (Enzo Life Sciences) solution was added to each well. Plates were incubated for an additional 2 h. The optical density of each well was measured using a microplate reader (Tecan) at a 450 nm wavelength. Cell viability was expressed as a percentage of that of the DMSO-treated cells. All experiments were performed in triplicate.
Flies (n < 100) from each experimental group were monitored for their survival along with aging. The rotenone, paraquat and MG132-induced toxicity survival assays were performed on regular food medium. Flies were maintained on standard cornmeal agar media at 24°C and transferred every day to a new vial containing food that was treated with chemicals for the chemical treatment groups. Next, a survival assay was performed on filter papers soaked with 450 μM MG132 and 5% sucrose at 30°C. Filters were changed daily. The non-chemical treatment groups were maintained in the same way except for the treatment with the chemicals. The percentage of flies that remained alive at the end of the experiment was calculated based on the starting number of flies for each treatment group. This experiment was repeated three times.
The levels of intracellular ROS generation were evaluated by DCFH-DA as previously described (Song et al., 2012). Briefly, after treatment, cells were washed with 1X PBS and loaded with DCFH-DA (final concentration 10 μM in colorless DMEM) for 30 min at 30°C. Fluorescence was measured by using a fluorescence spectrophotometer with excitation at 485 nm and emission at 530 nm.
The OCR was measured using a Seahorse XF24 analyzer according to the manufacturer’s instructions (Seahorse Bioscience Inc). Briefly, SH-SY5Y cells were plated at 1 × 105 cells per well in full growth medium (Hardie et al., 2017). After overnight attachment, the medium was washed and replaced with pre-warmed running medium (non-buffered DMEM supplemented with 4 mM L-glutamine, 25 mM D-glucose and 1 mM sodium pyruvate, pH 7.4) and incubated in a non-CO2 incubator at 37°C for 60 min. The OCR was further measured following injection of rotenone and the PTP1B inhibitor. After the OCR measurement, cells were lysed, and the protein content was estimated using a BCA Assay (Pierce). The OCR was plotted after normalizing by total protein.
After treatment, the cells were homogenized in RIPA buffer (Cell signaling Technology) with a phosphatase and protease inhibitor cocktail (Roche) and incubated at 4°C for 1 h. Cells were collected by centrifugation at 13,000 rpm for 30 min at 4°C. The supernatant was collected, and the protein concentration was determined using a BCA protein assay kit (Pierce). Next, denatured proteins were separated by Nu-PAGE 4–12% Bis-Tris Gels (Novex) and transferred to a poly-vinylidene difluoride (PVDF) membrane (Novex) at 20 V for 1 h. The blots were blocked for 1 h at room temperature in 5% skim milk. The membrane was then incubated overnight at 4°C with primary antibodies against phospho-eIF2a (Ser51), eIF2a, phospho-PERK (Thr981), PTP1B and α-tubulin. The membrane was incubated for 1 h with HRP-conjugated secondary antibodies, followed by detection with the ECL prime kit (Amersham Biosciences). Samples from three independent experiments were used in this analysis. The relative expression level was determined by using a Fusion-FX imaging system (Viber Lourmat).
Cell death was detected by using an FITC Annexin V apoptosis detection kit (BD Biosciences) and flow cytometry. In brief, cells were trypsinized and washed with chilled PBS twice, and the cell pellets were re-suspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) to create a cell suspension at a density of 1 × 106 cells/ml. Then, 3 μl of FITC-conjugated Annexin V was added to the suspension, which was incubated for 15 min at room temperature in the dark. Finally, 3 μl of propidium iodide was added, and flow cytometry was performed within 1 h using MoFlo Astrios cell sorter (Beckman Coulter).
Each experiment was performed at least three times, and the results were presented as the means ± standard deviations (SD) or means ± standard error (SEM). Comparison between groups was made by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test (GraphPad Prism Software) or analyzed by Student’s
To investigate the role of PTP1B in neuronal ER stress, we used rotenone-treated SH-SY5Y cells and a selective PTP1B inhibitor (CAS 765317-72-4) (Wiesmann et al., 2004). Rotenone is a widely used insecticide and is known to induce ER stress via ROS production (Pal et al., 2014). The cytotoxicity of the human neuroblastoma cell line (SH-SY5Y) was determined using a CCK-8 assay, and the percent cell viability was plotted. Treatment with rotenone (15 μM for 24 h) reduced cell viability by approximately 50% in SH-SY5Y cells (Fig. 1A). Pre-treatment with the PTP1B inhibitor at concentrations ranging from 5–20 μM reduced rotenone-induced cell death in a dose-dependent manner (Fig. 1A). Furthermore, PTP1B inhibition mitigated rotenone-induced toxicity in primary mouse cortical neurons (Fig. 1B, 53% compared to 68% in rotenone-treated primary cortical neuronal cells). The protective effect of the PTP1B inhibitor on rotenone-induced toxicity was also assessed by PI and Annexin V staining followed by flow cytometric analysis in SH-SY5Y cells. Annexin V and PI were used to determine the levels of apoptotic cell death (PI−/Annexin V+ or PI+/Annexin V+) or necrotic cell death (PI+/Annexin V−). Rotenone induced cell death in 25.79% of SH-SY5Y cells (PI−/Annexin V+ or PI+/Annexin V+: 20.94% and PI+/Annexin V−: 4.85%). In comparison, the percent cell death in SH-SY5Y cells treated with both rotenone and the PTP1B inhibitor was 8.84% (PI−/Annexin V+ or PI+/Annexin V+: 6.77% and PI+/Annexin V−: 2.07%). PTP1B inhibitor pretreatment decreased cell death by 16.95% in the rotenone-treated condition, whereas the PTP1B inhibitor alone without rotenone treatment did not affect neuronal viability (Fig. 1C). These data indicate that PTP1B inhibition protects neuronal cells from rotenone-induced cell death.
We next investigated the role of PTP1B in ROS-induced toxicity
We then wondered how PTP1B inhibition could suppress ROS-induced toxicity. Previous studies have reported that rotenone-induced ER stress increases the phosphorylation level of PERK (Thr981) and eIF2α (Ser51) (Chen et al., 2008). Phospho-PERK and phospho-eIF2α are well-known markers for ER stress. To investigate the role of the ER stress pathway in rotenone-treated neuronal cells, we performed an immunoblotting analysis with the antibodies against phospho-PERK (Thr981), phospho-eIF2α (Ser51), and total eIF2a and PTP1B from rotenone- and/or PTP1B inhibitor-treated SH-SY5Y cells. Interestingly, PTP1B inhibition significantly reduced the rotenone-induced increase in the phosphorylation of PERK-eIF2α (Fig. 2A). In contrast, the total eIF2a and PTP1B protein levels were not changed in rotenone- and PTP1B inhibitor-treated SH-SY5Y cells (Fig. 2A). We confirmed that the PTP1B inhibitor alone had no effect on the levels of phospho-PERK and phospho-eIF2α (Fig. 2B). These results indicated that neuronal ER stress could be mitigated by PTP1B inhibition.
Previous studies have shown that reduced mitochondrial complex I activity by rotenone treatment results in an increased production of ROS and thereby consequently induces ER stress-mediated neurotoxicity (Goswami et al., 2016). Therefore, rotenone-induced ROS production may be regulated by PTP1B.
To investigate this possibility, we measured intracellular ROS using the redox-sensitive fluorophore 2′,7′-dichloro-fluorescein diacetate (DCFH-DA). Non-fluorescent DCFH-DA is converted to the fluorescent molecule DCF by oxidation. We found that the level of DCF in SH-SY5Y cells treated with 15 μM rotenone was significantly increased in a time-dependent manner (Fig. 3A). ROS levels, when stimulated by 15 μM rotenone, peaked at 24 h and were 1.8-fold higher than in the DMSO treatment group. Next, SH-SY5Y cells were pre-incubated with the PTP1B inhibitor (20 μM) for 30 min and treated with 15 μM rotenone or 5 μM MG132 for 24 h. Pre-treatment with the PTP1B inhibitor for 30 min showed no significant reduction in the rotenone-induced intracellular ROS level. Treatment with the proteasome inhibitor, MG132, for 24 h showed no significant effect on ROS production compared to that observed in the DMSO treatment group (Fig. 3B).
For real-time analysis of mitochondrial oxidation, SH-SY5Y cells were analyzed using an XF-24 extracellular flux analyzer (Seahorse Bioscience Inc). In sequence, SH-SY5Y cells were treated with rotenone (5 μM) in the presence or absence of the PTP1B inhibitor (20 μM), and cellular oxygen consumption rate (OCR) was measured using the XF24 Seahorse Analyzer. As expected, rotenone significantly inhibited the OCR of SH-SY5Y cells; moreover, PTP1B inhibition did not result in any change in the reduced OCR through injection of rotenone (Fig. 3C). These data suggest that the neuroprotective effect of PTP1B inhibition does not involve ROS production.
We have shown that PTP1B inhibition could protect rote-none-induced neurotoxicity without reducing ROS production. Therefore, we hypothesized that PTP1B inhibition may primarily attenuate downstream processes of ROS such as ER stress. MG132 is a specific peptide-aldehyde inhibitor of the ubiquitin proteasome and known as an inducer of ER stress. Because unfolded and misfolded proteins are degraded by the proteasome system (Werner et al., 1996), MG132 treatment results in accumulation of misfolded-proteins in the ER, leading to ER stress (Nakajima et al., 2011).
We examined the level of ER stress markers in MG132-treated SH-SY5Y cells. Cells were treated with MG132 for different time periods and subjected to immunoblot analysis. MG132 treatment rapidly increased the level of the ER stress marker, phospho-eIF2α. Moreover, PTP1B inhibition significantly reduced the MG132-induced eIF2α phosphorylation (Fig. 4A). To elucidate the role of PTP1B in neuronal UPS impairment, we treated SH-SY5Y cells (Fig. 4B) and mouse primary cortical neurons (Fig. 4C) with MG132 and/or the PTP1B inhibitor. Cell viability was examined using cell counting kit-8. We found that within the 24-h treatment period, MG132 caused dramatic cell death, while the PTP1B inhibitor suppressed this MG132-induced cell death. To confirm the effect of the PTP1B inhibitor on MG132-induced neuronal cell death, SH-SY5Y cells were double-labeled for Annexin V and PI and then analyzed by flow cytometric analysis. MG132 induced cell death in 16.03% of SH-SY5Y cells (PI−/Annexin V+ or PI+/Annexin V+: 15.22% and PI+/Annexin V−: 0.81%). In comparison, the percent cell death in SH-SY5Y cells treated with both MG132 and the PTP1B inhibitor was 6.22% (PI−/Annexin V+ or PI+/Annexin V+: 5.03% and PI+/Annexin V−: 1.19%) (Fig. 4D).
To test the idea that therapeutic modulation of PTP1B may have an effect on toxicity induced by UPS impairment
Tunicamycin specifically blocks the initial step of glycoprotein biosynthesis in the ER (Oslowski and Urano, 2011). Moreover, a previous study showed that tunicamycin is induced in neuronal cell death and ER stress. Thus, we sought to confirm how the neuroprotective effect of PTP1B is regulated by other ER stress inducers in SH-SY5Y cells.
SH-SY5Y cells were treated with 5 μM of tunicamycin for 24 h and pre-treated with different dosages of the PTP1B inhibitor for 30 min. The cell viability was tested using the CCK-8 assay. The result of the CCK-8 assay indicated that PTP1B inhibition (20 μM) prevented cell death induced by tunicamycin (Fig. 5A, 30.8% compared to 40.9% in tunicamycin-treated SH-SY5Y cells). These data indicate that PTP1B plays an important role in various forms of ER stress-induced neuronal toxicity. Next, we also examined whether PTP1B inhibition influences tunicamycin-induced ER stress. PTP1B inhibition significantly reduced the tunicamycin-induced increase in the phosphorylation of eIF2α (Fig. 5B). These results suggest that PTP1B inhibition attenuates the toxicity of neuronal ER stress.
Oxidative stress and UPS impairment are common features of many neurodegenerative diseases including AD, PD and ALS. Both cause accumulation of misfolded and unfolded proteins in the ER lumen, and accumulation of these abnormal proteins induces ER stress. ER stress activates a stress-adaptive signaling process called the unfolded protein response (UPR) (Xiang et al., 2017). UPR activation is mediated via three ER stress sensors localized in the ER lumen: IRE1, ATF6 and PERK. Recently, many studies have reported that phosphorylated PERK and eIF2α are detected in postmortem brain and spinal cord tissue from AD, ALS, PD and frontotemporal dementia (FTD) patients (Smith and Mallucci, 2016). Moreover, pharmacological inhibition of the PERK pathway restored pathological features of ALS, Prion disease and FTD animal models (Kim et al., 2014; Moreno et al., 2013; Radford et al., 2015). Therefore, the PERK-eIF2α axis of the UPR might be a valuable therapeutic target for various neurodegenerative diseases.
PTP1B is a negative regulator of insulin signaling and is predominantly localized to the cytoplasmic surface of the ER (Popov, 2012). Recent studies have suggested that PTP1B is implicated in the regulation of ER stress signaling in cultured adipocytes and myotubes (Bettaieb et al., 2012; Panzhinskiy et al., 2013b). Interestingly, Song et al. (2016) discovered that inhibition of PTP1B has an anti-inflammatory effect in a mouse neuroinflammation model. Many reports have indicated that inflammatory responses are also implicated in ER stress and chronic metabolic diseases such as type 2 diabetes, obesity and insulin resistance (Hotamisligil, 2010; Zhang, 2010). Furthermore, Hakim et al. (2015) showed that chronic sleep fragmentation induces ER stress and PTP1B upregulation in hypothalamic neurons. Collectively, previous studies have suggested that PTP1B might modulate the toxic effect of neuronal ER stress.
To elucidate the relationship between neuronal ER stress and PTP1B, we examined whether neuronal toxicity induced by UPS impairment and ROS production were mitigated by PTP1B inhibition.
We showed that PTP1B inhibition attenuated oxidative stress- and UPS impairment-induced toxicity in
To the best of our knowledge, rotenone triggers massive ROS production through the inhibition of mitochondrial complex I, which finally results in ER stress and apoptotic cell death (Seoposengwe et al., 2013; Swarnkar et al., 2012). Therefore, PTP1B inhibition might mitigate rotenone-induced ROS production via improvement of mitochondrial function. Our study showed that significantly increased levels of ROS and mitochondria dysfunction were observed in rotenone-treated neuronal cells. However, the increase in ROS production and mitochondrial dysfunction induced by rotenone treatment were not altered by PTP1B inhibition. These data demonstrate that the protective effect of PTP1B inhibition seems to not be related to ROS production or mitochondrial activity.
We also tested the effect of the PTP1B inhibitor after inducing ER stress with tunicamycin. Tunicamycin inhibits the glycosylation of newly synthesized protein, and this biosynthetic step occurs within the ER. Consequently, impairment of protein glycosylation leads to the disruption of proper protein folding in the ER (Foufelle and Fromenty, 2016). We found that tunicamycin-induced cell death and eIF2α phosphorylation is also suppressed by PTP1B inhibition in SH-SY5Y cells. Taken together, these results suggest that PTP1B inhibition attenuates the toxicity of neuronal ER stress.
Possible neuroprotective mechanisms of PTP1B inhibition include BDNF/TrkB signaling. TrkB is a family of receptor tyrosine-kinases (RTKs) and a receptor of brain-derived neurotrophic factor (Vieira et al., 2017). Many studies have reported that the toxic effects of ER stress are significantly mitigated by the upregulation of BDNF in the brain and neuronal cells (Chen et al., 2007; Qiu et al., 2013; Shimoke et al., 2004; Wei et al., 2014; Zhu et al., 2004). Moreover, TrkB is a direct substrate of PTP1B; so, PTP1B negatively regulates TrkB activation via BDNF treatment (Ozek et al., 2014). Notably, deletion or inhibition of PTP1B potentiates BDNF signaling via enhanced TrkB phosphorylation in SH-SY5Y cells and the mouse hypothalamic brain region (Ozek et al., 2014). Therefore, PTP1B inhibition might mitigate the toxic consequences of neuronal ER stress via enhancing BDNF/TrkB signaling.
Another possible mechanism is the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. Nrf2 is a basic leucine-zipper transcription factor that plays an important role in mitigating proteotoxic stress (Pajares et al., 2017). Under normal conditions, the level of Nrf2 protein remains low because of its rapid turnover. In response to different stimuli, including accumulation of misfolded proteins, the Nrf2 protein is increased and translocated to the nucleus to promote the transcription of ARE-containing genes (Cui et al., 2016). Nrf2 activation upregulates key genes for the protein quality control system, and this gene upregulation promotes the degradation of misfolded proteins. Specifically, Nrf2 increases the expression level of several UPS subunits and protects cells from abnormal protein accumulation. Previous studies have indicated that Nrf2 activation is essential for transcriptional upregulation of the 26S proteasome subunit and improving proteasome activity in the liver and fibroblasts (Kapeta et al., 2010; Kwak et al., 2003a; 2003b; Pickering et al., 2012). Moreover, PTP1B deficiency enhances the nuclear accumulation of Nrf2 in acetaminophen-induced hepatotoxicity via the GSK3 beta/Src-Fyn pathway (Mobasher et al., 2013). In this study, we found that UPS impairment-induced toxicity is rescued by PTP1B inhibition. Therefore, PTP1B inhibition might mitigate neuronal ER stress by promoting the degradation of abnormal proteins through Nrf2 activation.
In conclusion, our study showed that PTP1B inhibition attenuates ER stress via mitochondria-independent mechanisms in neuronal cells. As a proof of principle that modulating PTP1B activity could be beneficial, a small molecule inhibitor of PTP1B significantly suppressed UPS impairment-induced toxicity
SH-SY5Y cells (A) and primary mouse cortical neuron (B) cells were pretreated with various concentrations of the PTP1B inhibitor for 30 min and then treated with rotenone for 24 h. The rotenone-induced increase in neuronal cell death was attenuated by PTP1B inhibition. The cell viability was then determined by CCK-8 assay, and the percentage of cell viability was plotted as the mean ± SD of the three different experiments
(A) SH-SY5Y cells were pretreated with the PTP1B inhibitor for 30 min and then treated with 15 μM rotenone for 12 h. The expression levels of p-PERK, eIF2α, p-eIF2α and PTP1B were determined with immunoblotting. Rotenone-induced p-PERK and p-eIF2α protein levels were decreased by PTP1B inhibition. (B) SH-SY5Y cells were pretreated with the PTP1B inhibitor for 30 min. The expression levels of p-PERK, eIF2α and p-eIF2α were determined with immunoblotting. There were no significant changes in p-PERK, p-eIF2α and eIF2α with PTP1B inhibitor treatment. The relative protein levels of p-PERK, eIF2α, p-eIF2α and PTP1B imaged by Fusion imaging systems were measured by densitometry and normalized to the expression of α-tubulin.
(A) Rotenone induces intracellular ROS production in SH-SY5Y cells. The indicated cells were treated with rotenone (15 μM) for 0–24 h, followed by ROS production using the dye DCFH-DA. The rotenone-induced ROS production was time-dependent. *
(A) SH-SY5Y cells were pretreated with the PTP1B inhibitor for 30 min and then treated with 5 μM MG132 for 24 h. The expression levels of eIF2α and p-eIF2α were determined by immunoblotting. The relative protein levels of eIF2α and p-eIF2α imaged by a Fusion imaging system were measured by densitometry and normalized to the expression of α-tubulin. SH-SY5Y cells (B) and primary mouse cortical neurons (C) were pretreated with various concentrations of the PTP1B inhibitor for 30 min and treated with MG132 for 24 h. The MG132-induced neuronal cell death was attenuated by PTP1B inhibition. Cell viability was then determined by CCK-8 assay, and the percent cell viability was plotted as the mean ± SD of the three different experiments (**
(A) SH-SY5Y cells were pretreated with various concentrations of the PTP1B inhibitor for 30 min and then treated with 5 μM tunicamycin for 24 h. The tunicamycin-induced increase in neuronal cell death was attenuated by PTP1B inhibition. The cell viability was then determined by CCK-8 assay, and the percentage of cell viability was plotted as the mean ± SD of the three different experiments
Jangham Jung, Issac Choi, Hyunju Ro, Tae-Lin Huh, Joonho Choe, and Myungchull Rhee
Mol. Cells 2020; 43(1): 76-85 https://doi.org/10.14348/molcells.2019.0210Taewook Nam, Jong Hyun Han, Sushil Devkota, and Han-Woong Lee
Mol. Cells 2017; 40(12): 897-905 https://doi.org/10.14348/molcells.2017.0226Ji I Baek, Dong-Won Seol, Ah-Reum Lee, Woo Sik Lee, Sook-Young Yoon, and Dong Ryul Lee
Mol. Cells 2017; 40(11): 871-879 https://doi.org/10.14348/molcells.2017.0184
SH-SY5Y cells (A) and primary mouse cortical neuron (B) cells were pretreated with various concentrations of the PTP1B inhibitor for 30 min and then treated with rotenone for 24 h. The rotenone-induced increase in neuronal cell death was attenuated by PTP1B inhibition. The cell viability was then determined by CCK-8 assay, and the percentage of cell viability was plotted as the mean ± SD of the three different experiments
(A) SH-SY5Y cells were pretreated with the PTP1B inhibitor for 30 min and then treated with 15 μM rotenone for 12 h. The expression levels of p-PERK, eIF2α, p-eIF2α and PTP1B were determined with immunoblotting. Rotenone-induced p-PERK and p-eIF2α protein levels were decreased by PTP1B inhibition. (B) SH-SY5Y cells were pretreated with the PTP1B inhibitor for 30 min. The expression levels of p-PERK, eIF2α and p-eIF2α were determined with immunoblotting. There were no significant changes in p-PERK, p-eIF2α and eIF2α with PTP1B inhibitor treatment. The relative protein levels of p-PERK, eIF2α, p-eIF2α and PTP1B imaged by Fusion imaging systems were measured by densitometry and normalized to the expression of α-tubulin.
(A) Rotenone induces intracellular ROS production in SH-SY5Y cells. The indicated cells were treated with rotenone (15 μM) for 0–24 h, followed by ROS production using the dye DCFH-DA. The rotenone-induced ROS production was time-dependent. *
(A) SH-SY5Y cells were pretreated with the PTP1B inhibitor for 30 min and then treated with 5 μM MG132 for 24 h. The expression levels of eIF2α and p-eIF2α were determined by immunoblotting. The relative protein levels of eIF2α and p-eIF2α imaged by a Fusion imaging system were measured by densitometry and normalized to the expression of α-tubulin. SH-SY5Y cells (B) and primary mouse cortical neurons (C) were pretreated with various concentrations of the PTP1B inhibitor for 30 min and treated with MG132 for 24 h. The MG132-induced neuronal cell death was attenuated by PTP1B inhibition. Cell viability was then determined by CCK-8 assay, and the percent cell viability was plotted as the mean ± SD of the three different experiments (**
(A) SH-SY5Y cells were pretreated with various concentrations of the PTP1B inhibitor for 30 min and then treated with 5 μM tunicamycin for 24 h. The tunicamycin-induced increase in neuronal cell death was attenuated by PTP1B inhibition. The cell viability was then determined by CCK-8 assay, and the percentage of cell viability was plotted as the mean ± SD of the three different experiments