Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), ethylene glycol tetra acetic acid (EGTA), hydrogen peroxide solution (H₂O₂), d-mannitol, and lipopolysaccharides were purchased from Sigma-Aldrich, USA. Bafilomycin A1 and GPN were from Santa Cruz Biotechnology, while BAPTA-AM, cyclosporin A, FK506, ionomycin, nicotinic acid adenine dinucleotide phosphate (NAADP), trans-Ned-19 (Ned-19), and U18666A were from Tocris Biosciences, USA. Thapsigargin (TG) was bought from Almone Labs, USA, while LysoTracker Red DND-99 Dye and Rhod dextran were from Invitrogen, USA. Antibodies used for immunoblotting and immunostaining were as follows: anti-mouse lysosome-associated membrane protein-1 (LAMP-1; sc-20011, Santa Cruz Biotechnology, USA), anti-rabbit cathepsin D (2284S, Cell Signaling, USA), anti-rabbit caspase-1 (2225S, Cell Signaling, USA), anti-rabbit TFEB (37785S, Cell Signaling, USA), anti-rabbit histone H3 (D1H2) (4499S, Cell Signaling, USA), anti-rabbit Integrin β1 (4706S, Cell Signaling, USA), anti-rabbit GAPDH (2118S, Cell Signaling, USA), and anti-rabbit α/β-tubulin (2148S, Cell Signaling, USA).

U937 (ATCC, USA) and THP-1 (InvivoGen, USA) monocytic cell lines were grown in RPMI 1640 (Gibco, USA) supplemented with 10% FBS, 2 mM l-glutamine, and 100 U/mL of penicillin and streptomycin. In HG experiments, before HG stimulation, the cells were cultured in RPMI 1640 with 5.5 mM glucose for 48 h, and then were changed to 10, 20, or 30 mM glucose RPMI 1640 for indicated time points. 30 mM mannitol was used as an osmotic control. For the experiments using chemical inhibitors, Cs A, FK506, and U18666A were pre-treated for 24 h, while TG was pre-treated for 45 min. EGTA and BAPTA-AM were treated in the presence of HG stimulation. For the immunoblotting experiments measuring TFEB translocation by calcium inducers, GPN, H₂O₂, ionomycin, NAADP, and TG were stimulated for 20 min. The supernatants from U937 and THP-1 cells were collected for the detection of human IL-1β levels by ELISA (eBioscience, USA).

Cells were transiently transfected with TFEB siRNA (100 nmol/L; Ambion, USA), by using Lipofectamine^® RNAiMAX Transfection reagent (Gibco, USA). The protocol was synthesized according to the manufacturer’s protocol. GAPDH siRNA was used as a control (40 nmol/L; Ambion, USA). Transfection efficiency was >70% assessed by BLOCK-iT™ Alexa Fluor^® Red Fluorescent Control (Ambion, USA) and western blotting. Cells were transfected with siRNA for 24 h before experiments.

The intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in single cells as previously described (27). Cells were loaded with Fluo-4 AM (2 µM; Molecular Probes, USA) in Tyrode solution containing 136.5 mM NaCl, 5.4 mM KCl, 0.53 mM MgCl₂, 1.8 mM CaCl₂, 0.33 mM NaH₂PO₄, 5.5 mM glucose, and 5.5 mM HEPES (pH 7.4, adjusted with NaOH) for 30 min at 37°C. Fluo-4 fluorescence intensity (494 nm excitation; 506 nm emission) was sampled at 5 s intervals using a Cell^® system (MT20, Olympus, USA). To enable comparisons between cells, the maximal change in fluorescence intensity was measured before and after GPN (400 µM), NAADP (1 µM), Baf A1 (500 nM), or TG (1 µM) was added.

The lysosomal Ca²⁺ concentration was measured as previously described (28). For lysosomal Ca²⁺ measurements, the cells were incubated with Rhod dextran (25 mg/ml) for 12 h after treatment as indicated in results, while for all cytosolic Ca²⁺ measurements, the cells were incubated with Fluo-4 (2 µM) for 30 min. The median fluorescence intensity (MFI) was determined using a FACS Canto flow cytometer (BD Biosciences, USA), and the data were analyzed using FlowJo software (Tree Star, USA).

Total protein was extracted with ice-cold lysis buffer, the nuclear/cytosolic proteins were extracted by using the Nuclear and Cytoplasmic Extraction Kit (Pierce, USA), and the PM/cytosolic proteins were extracted by using the Mem-PER™ Plus Membrane Protein Extraction Kit (Pierce, USA). The protein concentrations of the lysates were measured by the bicinchoninic acid kit (Pierce, USA). 40 µg proteins were used and separated by 10% SDS-PAGE gels and were transferred onto the nitrocellulose membranes. Membranes were incubated with primary antibodies (1/1,000 dilution) overnight at 4°C, and secondary antibodies (1/1,000 dilution) for 1 h at room temperature, and the immunoblots were developed by enhanced chemiluminescence (GE Healthcare Life Sciences, USA) with a ChemiDoc™ MP System (Bio-Rad Laboratories, USA). GAPDH, β-actin, α/β-Tubulin, Histone H3, and Integrin β1 were used as housekeeping controls.

Total RNA was extracted using RNeasy Mini Kit (Qiagen, USA), and cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). cDNA was quantified using Taqman assays by ViiA 7 Real-Time PCR System (Applied Biosystems, USA). The Taqman probes (Applied Biosystems, USA) used were as follows: TFEB (Hs00292981_m1), Cathepsin B (Hs00947433_m1), Cathepsin D (Hs00157205_m1), LAMP-1 (Hs00174766_m1), IL-1β (Hs00174097_m1), and β-actin (4326315E). β-Actin was used as an endogenous control. All gene expressions were calculated using the ΔΔCt method and were normalized to control.

For cells labeling with lysotracker, the cells were incubated with LysoTracker DND-99 Dye (250 nM) for 45 min at 37°C after treatment as indicated in results. The MFI was determined using a FACS Canto flow cytometer (BD Biosciences, USA), and the data were analyzed using FlowJo software (Tree Star, USA).

The LAMP-1 level on the PM was measured as previously described (29). After treatment as indicated in results, the intact cells were incubated with LAMP-1 antibody overnight at 4°C and then fixed with 4% paraformaldehyde solution (PFA; Santa Cruz Biotechnology, USA). After fixation, the cells were incubated with secondary antibody (1/400 dilution). The MFI was determined using a FACS Canto flow cytometer (BD Biosciences, USA), and the data were analyzed using FlowJo software (Tree Star, USA).

β-Hexosaminidase secretion was measured as previously described (25). After treatment, 200 ml supernatants of the cells were equilibrated in 1 mM EGTA-Ca²⁺-free buffer for 3 h and then mixed with 200 ml of 1 mM 4-methylumbelliferyl N-acetyl-β-d-glucosaminide (Sigma-Aldrich, USA) in 0.1 M citrate buffer (0.05 M citric acid, 0.05 M sodium citrate, pH 4.5, Sigma-Aldrich, USA) for 1 h at 37°C. The reaction was stopped with 400 ml 0.1 M sodium carbonate buffer (Sigma-Aldrich, USA), and the absorbance was measured at 405 nm. To determine the total cellular content of β-hexosaminidase, the cells were lysed with 1% (v/v) Triton X-100, and 10 µl of the cell extracts were used for the enzyme activity reaction. The percentage of β-hexosaminidase release was calculated from the enzyme activity of the supernatants and lysates.

Cells were extracted with 200 µl of chilled Cell Lysis Buffer following the manufacturer’s instruction. Cathepsin D activity was measured by using a flourimetric assay kit (Abcam, USA) and was normalized to control.

After treatment as indicated in results, the cells were fixed with 4% PFA for 15 min, followed by permeabilization with 0.1% Triton X-100 for 5 min, and were blocked in 20% goat serum (Cell Signaling, USA) for 30 min. Next, the cells were incubated with TFEB antibody (1/50 dilution) overnight at 4°C, and stained with secondary antibody (1/400 dilution) for 1 h and DAPI for 10 min. For the acquisition of the images, at least six images were taken per well of the 96-well plate by IN Cell Analyzer 2000 (GE Healthcare, USA), and quantitative analysis was performed with ImageJ software.

The cells were seeded onto confocal dishes (SPL Life Sciences, Korea) and were treated with indicated conditions as described. The cells were fixed with 4% PFA for 15 min, blocked in 20% goat serum (Cell Signaling, USA) for 30 min, and incubated with primary antibodies (1/50 dilution) overnight at 4°C, and then secondary antibodies (1/400 dilution) for 1 h. Images were captured with a confocal microscope (LEICA TCS SP8, Leica Microsystems, Germany), and quantitative analysis was performed with the ImageJ software.

All data were expressed as mean ± SEM and were analyzed by GraphPad Prism 5.0 (GraphPad, USA). Significant differences were determined by one-way ANOVA followed by a Dunnett’s test. P < 0.05 was considered as significant. Sample size (n) represented the number of independent experiments.

Impaired lysosomal Ca²⁺ homeostasis could lead to lysosomal dysfunction (30), and chronic exposure of HG to macrophages was demonstrated to induce the inhibition of lysosomal function (31); however, whether lysosomal Ca²⁺ homeostasis was altered under HG condition is still unclear. To examine the role of lysosomes in hyperglycemic environment in human monocytic cells, we first measured Ca²⁺ release from the lysosomes. GPN is a cathepsin C substrate that was reported to induce lysosomal Ca²⁺ release in monocytes (25). In Fluo-4-loaded U937 cells, treatment with HG (10, 20, 30 mM glucose for 48 h) or 30 mM glucose for 24, 48, or 72 h significantly reduced GPN-evoked Ca²⁺ release (Figures 1A–C), compared to low glucose (LG; 5.5 mM glucose) and 30 mM mannitol (Ma). Ma was used to as an osmotic control. Since 30 mM glucose treatment for 48 h, but not Ma, induced approximately 85% reduction of GPN-evoked Ca²⁺ release in U937 cells; therefore, it was regarded as our HG model in this study. Moreover, we also used another human monocytic cell line, THP-1, to confirm this observation. Similarly, we also observed that there was a reduction of GPN-evoked Ca²⁺ release under HG condition in THP-1 cells (Figure 1D), suggesting that HG might influence lysosomal Ca²⁺ homeostasis in human monocytic cells. In THP-1 cells, pre-treatment with U18666A, a drug that was used to deplete lysosomal Ca²⁺ store, significantly blocked GPN-evoked Ca²⁺ release (Figure 1D), this confirmed that GPN-evoked Ca²⁺ release was from the lysosomes in human monocytic cells.

To further examine the role of Ca²⁺ homeostasis under HG condition in human monocytic cells, we used NAADP, a Ca²⁺-mobilizing secondary messenger that was known to release Ca²⁺ from the acidic endo-lysosomal vesicles (32), and bafilomycin A1, an inhibitor of the vacuolar-ATPase to induce lysosomal Ca²⁺ release. Figures 2A,B showed that NAADP- and bafilomycin A1-evoked Ca²⁺ release were significantly reduced under HG in U937 cells. By contrast, we also measured Ca²⁺ release from the ER and mitochondria under HG condition. The cells were treated with TG to release ER Ca²⁺, or with CCCP, a mitochondrial uncoupler to release mitochondria Ca²⁺. No differences in Ca²⁺ release from the ER or mitochondria were observed between LG-, Ma-, and HG-treated U937 cells (Figures 2C,D). Taken together, this suggested that HG induced a disruption of Ca²⁺ homeostasis within lysosomes, but was dispensable for ER and mitochondria Ca²⁺ in human monocytic cells.

To determine the relationship between Ca²⁺ homeostasis and lysosomes, we measured cytosolic and lysosomal Ca²⁺ levels directly with Fluo-4 and Rhod-dextran, respectively, as previously described (28). Bafilomycin A1 was reported to increase the pH level of lysosomes that increased cytosolic Ca²⁺ concentration by reducing lysosomal Ca²⁺ level (33). In agreement with that, after 60 min treatment with bafilomycin A1, an increase in Fluo-4 MFI and a decrease in Rhod-dextran MFI were observed in U937 cells (Figure 3A). This further confirmed the change in cytosolic and lysosomal Ca²⁺ levels with Fluo-4 and Rhod-dextran by bafilomycin A1. Next, we examined whether HG affected Ca²⁺ homeostasis in monocytic cells, we observed a decrease in lysosomal Ca²⁺ level with Rhod-dextran and elevation in cytosolic Ca²⁺ level with Fluo-4 under HG (30 mM; 24, 48, 72 h) in U937 cells, this strongly suggested that HG decreased lysosomal Ca²⁺ concentration and affected cytosolic Ca²⁺ homeostasis (Figure 3B). Besides, we also determined whether HG influenced lysosomal function in human monocytic cells. LysoTracker dye was used to label lysosomes in live cells (34). By using flow cytometry, we observed a significant decrease in LysoTracker staining under HG for 72 h, but not for 24 h and 48 h in U937 cells (Figure 3C), suggesting that HG for 48 h caused a defect in lysosomal Ca²⁺ store, and HG for up to 72 h could inhibit lysosomal function in human monocytic cells. Taken together, our results suggested that HG induced the loss of lysosomes, affected lysosomal and cytosolic Ca²⁺ homeostasis in human monocytic cells.

Previous studies have suggested that Ca²⁺ signals was involved in lysosomal exocytosis-mediated IL-1β secretion in response to multiple stimuli (15, 25, 35, 36), whether HG disturbed Ca²⁺ homeostasis to promote lysosomal exocytosis is still unknown. To examine lysosomal exocytosis, we stained surface LAMP-1, a marker of the lysosomal exocytosis process (37). Figure 4A showed that HG (10, 20, and 30 mM glucose for 48 h) induced LAMP-1 translocation from cytosol to the PM in a dose-dependent manner in U937 cells. Similarly, we observed that treatment with HG (30 mM glucose) for 24, 48, and 72 h significantly increased surface LAMP-1 level by flow cytometry, where it reached maximum at 48 h in U937 cells (Figure 4B), suggesting that HG induced an active movement of lysosomes toward the PM. Moreover, we also examined the effects of different Ca²⁺ chelators and blockers on the surface LAMP-1 level under HG. In U937 cells, buffering of [Ca²⁺]_i by BAPTA significantly inhibited HG-induced surface LAMP-1 level (Figure 4C). Besides, we observed that the depletion of lysosomal Ca²⁺ store by U18666A also blocked this effect (Figure 4C). Similar results were obtained in THP-1 cells (Figure 4C). By contrast, EGTA did not affect the LAMP-1 level (Figure 4C), suggesting that HG rapidly triggered intracellular Ca²⁺ signals, which contributed to lysosomal exocytosis in human monocytic cells. Furthermore, HG-triggered translocation of LAMP-1 was accompanied by the lysosomal hydrolase, including cathepsin D (Figure 4D). We found that HG induced the maturation and activity of cathepsin D with maximal effects occurring at 48 h in U937 cells, whereas pre-treatment with U18666A could block these effects (Figures 4D–G). Similarly, Ned-19, an inhibitor of NAADP that blocks NAADP-induced Ca²⁺ mobilization from the lysosomes, also inhibited HG-induced cathepsin D activity in U937 cells (Figure 4G). This indicated that lysosomal Ca²⁺ signals was involved in HG-induced lysosomal exocytosis.

We then investigated whether IL-1β was accompanied by lysosomal exocytosis. The intracellular distribution of IL-1β and cathepsin D was examined under HG in U937 cells by confocal microscopy. We found out that IL-1β was co-localized with cathepsin D under HG, whereas this effect was abolished by the removal of Ca²⁺ with BAPTA plus EGTA (Figure 5A). Moreover, during HG (30 mM glucose) stimulation, IL-1β maturation and release were also abolished by buffering of [Ca²⁺]_i with BAPTA and the removal of extracellular Ca²⁺ with EGTA in U937 cells (Figure 5B). Meanwhile, we also examined the effect of Ca²⁺ chelators and agents on IL-1β secretion under HG condition in U937 and THP-1 cells. As expected, BAPTA significantly reduced IL-1β secretion by HG (Figure 5C). To further investigate whether lysosomal Ca²⁺ release participated in IL-1β secretion by HG, we used three antagonists, U18666A, Ned-19, and bafilomycin A1. Figure 5C showed that U18666A, Ned-19, and bafilomycin A1 markedly blocked HG-induced IL-1β secretion in U937 and THP-1 cells. Taken together, these results indicated that HG altered lysosomal Ca²⁺ homeostasis, which resulted in an increase in [Ca²⁺]_i and surface LAMP-1 level, facilitation in lysosomal exocytosis, lysosomal cathepsin D maturation and activity, and IL-1β release in human monocytic cells.

The activation of TFEB was reported to regulate lysosomal exocytosis by raising [Ca²⁺]_i (22); therefore, we examined whether it was also involved in HG stimulation. Immunoblotting results showed that HG increased TFEB translocation to the nucleus in a dose-dependent manner in U937 cells (Figure 6A). In addition to the nuclear translocation of TFEB, we also observed that HG upregulated TFEB mRNA in U937 cells (Figure 6B), indicating that HG did not only induce TFEB activation, but could also increase its mRNA expression. Notably, we found that the depletion of internal Ca²⁺ stores by ionomycin (28), or U18666A significantly reduced HG-induced nuclear translocation of TFEB (Figure 6C). By contrast, the depletion of ER Ca²⁺ store by TG had no effect on it (Figure 6C), suggesting that Ca²⁺ release from the lysosomes, but not from the ER, mediated the activation of TFEB under HG. Conversely, short and acute exposure to ionomycin, GPN, NAADP, or TG, that triggered internal Ca²⁺ release, could significantly induce nuclear translocation of TFEB in U937 cells (Figure 6D). However, H₂O₂ stimulation, which was reported to regulate monocytic function via extracellular Ca²⁺ influx (38), did not induce nuclear translocation of TFEB (Figure 6D). Therefore, our results supported that lysosmal Ca²⁺ signals played a key role in the regulation of TFEB translocation during HG condition in human monocytic cells.

We next investigated whether TFEB could regulate lysosomal exocytosis in U937 cells. Lysosomal Ca²⁺ response induced by GPN was proposed to be responsible for lysosomal exocytosis in human monocytes (25). Here, we measured the release of the lysosomal marker enzyme, β-hexosaminidase, to examine lysosome exocytosis. Our results demonstrated that GPN-induced β-hexosaminidase release in a time-dependent manner, and it was inhibited by TFEB siRNA and BAPTA in U937 cells (Figure 7A); this suggested that intracellular Ca²⁺ signals was involved in GPN-induced lysosomal exocytosis through TFEB pathway. The efficiency of the knockdown was shown by immunoblotting (Figure 7B). Moreover, we also found that HG-induced surface LAMP-1 level was reduced by TFEB siRNA (Figure 7C). Next, to further determine whether TFEB could control lysosomal exocytosis through its target genes, we measured the mRNA levels of TFEB target genes that were previously linked to lysosomal exocytosis, including LAMP-1, cathepsin B, and cathepsin D (15, 29, 39). We demonstrated that the mRNA expressions of LAMP-1, cathepsin B, and cathepsin D were upregulated under HG in U937 cells (Figure 7D). As expected, these effects were abolished by TFEB siRNA (Figure 7D), suggesting that TFEB directly controlled lysosomal exocytosis under HG condition. In addition, previous study demonstrated that calcineurin interacted with TFEB and modulated its activation (24). We observed that calcineurin inhibitors, cyclosporin A and FK506, significantly inhibited HG-induced IL-1β secretion in U937 cells (Figure 7E); so this further confirmed that HG induced lysosomal exocytosis-mediated IL-β secretion via calcineurin/TFEB pathway. Besides, our results also found that HG-mediated upregulation of IL-1β mRNA level and its maturation were suppressed by TFEB siRNA in U937 cells (Figures 7F,G). By contrast, TFEB siRNA did not induce HG-induced caspase-1 cleavage (p20) (Figure 7H). Taken together, this further suggested that TFEB/calcineurin pathway was responsible for HG-induced IL-1β release via regulation of synthesis of pro-IL-1β and lysosomal exocytosis, but independent of caspase-1 activation in human monocytic cells.