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ARTICLE A novel role of transient receptor potential mucolipin1 (TRPML1) in protecting against imidazole-induced cytotoxicity Zhenxing Liu, Shuan Zhao, Shuaishuai Wu, Jingyou Zhang, Zunyang Nie, and Shenming Zeng
Abstract: Lysosomotropic amines cause serious side effects such as cytoplasmic vacuolation and cell death. TRPML1 (also known as mucolipin1), a member of the transient receptor potential (TRP) protein family, may regulate fusion/fission of vesicles along the endocytic pathway and some aspects of lysosomal ion homeostasis. Nevertheless, it is still unknown whether TRPML1 is involved in death of mammalian cells induced by lysosomotropic agents. In this study, imidazole was used as a model to investigate the role of TRPML1 in the cytotoxicity of lysosomotropic agents. Overexpression of wild-type TRPML1 inhibited imidazole-induced vacuole formation and cell death in human endometrial adenocarcinoma (HEC-1B) cells. In contrast, siRNAmediated TRPML1 knockdown increased the cell death induced by imidazole. Bafilomycin A1 raises the pH of acidic organelles and therefore suppresses accumulation of weak bases in them. Similarly, lysosomal pH was raised in TRPML1-overexpressing cells; therefore, we inferred that TRPML1 protected against imidazole toxicity by regulating the pH of acidic organelles. We concluded that TRPML1 had a novel role in protecting against lysosomotropic amine toxicity. Key words: imidazole, TRPML1, cytoplasmic vacuolation, cell death, lysosomal pH. Résumé : Les amines lysosomotropes provoquent des effets secondaires sérieux comme la vacuolisation cytoplasmique et la mort cellulaire. TRPML1 (aussi connu sous le nom de mucolipine 1), un membre de la famille des TRP (transient receptor potential), peut réguler les vésicules de fusion/fission le long de la voie endocytique et certains aspects de l’homéostasie des ions des lysosomes. On ignore cependant si TRPML1 est impliqué dans la mort des cellules de mammifères induite par des agents lysosomotropes. Dans cette étude, l’imidazole a été utilisé comme modèle afin d’examiner le rôle de TRPML1 dans la cytotoxicité d’agents lysosomotropes. La surexpression de TRPML1 sauvage inhibait la formation de vacuoles et la mort cellulaire induites par l’imidazole chez des cellules d’adénocarcinome de l’endomètre humain (HEC-1B). Au contraire, l’inactivation de TRPML1 par un pARNi augmentait la mort cellulaire induite par l’imidazole. La bafilomycine A1 accroit le pH des organites acides et supprime ainsi l’accumulation de bases faibles. De la même façon, le pH des lysosomes était accru chez les cellules qui surexprimaient TRPML1 ; ainsi les auteurs ont supposé que TRPML1 protégeait les cellules de la toxicité de l’imidazole en régulant le pH des organites acides. Les auteurs concluent que TRPML1 exerce un nouveau rôle en protégeant les cellules de la toxicité des amines lysosomotropes. [Traduit par la Rédaction] Mots-clés : imidazole, TRPML1, vacuolisation cytoplasmique, mort cellulaire, pH du lysosome.
Introduction Imidazole is an important pharmacophore in drug discovery. Its derivatives have diverse pharmacological activities, including anti-inflammatory (Labanauskas et al. 2000), histamine-H3 antagonist (Grassmann et al. 2002), antioxidant (Can-Eke et al. 1998), gastroprotective (Sevak et al. 2002), and anticancer (Krezel 1998) properties. However, imidazole can also have serious side effects, as high concentrations cause cell death (Iguchi et al. 2002). As a weak base, imidazole enters acidic spaces and increases the pH of acidic organelles (Poole and Ohkuma 1981). Accumulation of protonated weak bases in acidic organelles induces osmotic swelling and cytoplasmic vacuolation (Ohkuma and Poole 1981). Thus, permeabilization of lysosomal membranes and the subsequent release of hydrolases have been considered the mechanism of lysosomotropic compounds toxicity (Boya and Kroemer 2008). TRPML1 (also mucolipin1), a member of the transient receptor potential (TRP) protein family, primarily resides in membranes of late endosomes and lysosomes (Hersh et al. 2002; Kiselyov et al. 2005;
LaPlante et al. 2002; Vergarajauregui and Puertollano 2006) and functions as an inwardly rectifying cation channel potentiated by low pH (Xu et al. 2007; Zhang et al. 2009). Mutations in human TRPML1 cause mucolipidosis type IV (MLIV), a severe neurodegenerative disease associated with defective lysosomal biogenesis and trafficking (Bassi et al. 2000; Sun et al. 2000). TRPML1 may regulate fusion/fission of vesicles along the endocytic pathway (Cheng et al. 2010; LaPlante et al. 2004, 2006; Pryor et al. 2006). In addition, it also has crucial roles in lysosomal ion homeostasis, including modulating pH (Soyombo et al. 2006) and concentrations of iron (Dong et al. 2008, 2010a) and zinc (Eichelsdoerfer et al. 2010). However, whether TRPML1 is involved in cell death induced by lysosomotropic amines in mammalian cells has apparently not been reported. In this study, we used overexpression or knockdown to investigate whether TRPML1 was involved in imidazole-induced cytoplasmic vacuolation and cell death, in an attempt to gain further insights on the novel role of TRPML1 in protecting against lysosomotropic amine toxicity.
Received 31 March 2014. Revision received 14 May 2014. Accepted 19 May 2014. Z. Liu,* S. Zhao,* S. Wu, Z. Nie, and S. Zeng. Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P.R. China. J. Zhang. Reproduction and Breeding Research Center, Animal Husbandry and Veterinary 9 Research Institute, Heilongjiang Academy of Agricultural and Reclamation Science, Harbin 150038, P.R. China. Corresponding author: Shenming Zeng (e-mail: [email protected]). *Zhenxing Liu and Shuan Zhao contributed equally to this work. Biochem. Cell Biol. 92: 279–286 (2014) dx.doi.org/10.1139/bcb-2014-0044
Published at www.nrcresearchpress.com/bcb on 28 May 2014.
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Fig. 1. Induction of cell death by imidazole in HEC-1B cells. (A) Effect of imidazole on cell viability. Cells were treated with indicated concentrations of imidazole for 12 h or 100 mmol/L imidazole for various intervals. Cell viabilities were determined by MTT assay and expressed as percentages of nontreated cells. The results from 3 biological replicates were presented as the means ± SEM (*P < 0.05, **P < 0.01). (B) Activation of caspase-3 in imidazole-treated cells. Cells treated with or without 100 mmol/L imidazole for indicated intervals were subjected to Western blotting analysis using an antibody against cleaved-caspase-3 (c-cas3). -Actin was used as a loading control.
Fig. 2. Imidazole induced cell vacuolization and decreased the protein level of TRPML1 in HEC-1B cells. (A) Vacuoles formation in imidazole-treated cells. Cells with or without 50 mmol/L imidazole treatment for 2 h were observed (phase-contrast microscopy, 400× magnification). For neutral red dye uptake, cells were incubated with 0.05% neutral red dye at room temperature for 5 min, washed, and observed with light microscopy. The distribution of acidic organelles was examined by AO staining and visualized with various channels using confocal microscopy. C: control group; T: treatment group; Baf A1: bafilomycin A1; NR: neutral red; AO: acridine orange. Scale bar = 20 m. (B) Effect of imidazole on expression of TRPML1 protein. Cells were treated with or without 50 mmol/L imidazole for indicated intervals, and Western blotting was used to analyze the abundance of TRPML1 protein using anti-TRPML1 C-terminal antibody. -Actin was used as a loading control. Comparison of the intensities of Western blotting were analyzed and represented as means ± SEM of 3 independent experiments. *P < 0.05.
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Fig. 3. Overexpression of wild-type TRPML1 inhibited imidazole-induced vacuoles formation. (A–C) The HEC-1B cells were transfected with pEGFP-TRPML1 vectors and cultured in complete medium with or without 50 mmol/L imidazole for 2 h. The fluorescent signals of TRPML1-GFP (ML1-GFP) were sequentially acquired by fluorescence microscopy (400× magnification). Imidazole failed to induce cytoplasmic vacuolization in cells expressing ML1-GFP (arrows). (D–F) Cells transfected with pEGFP vectors were treated 2 h with imidazole. In contrast, GFP alone did not alter imidazole-induced vacuolization in cells (arrowheads). Images were merged using ImageJ software. Scale bar = 20 m.
Materials and methods Cell culture and reagents A human endometrial adenocarcinoma cell line (HEC-1B) was purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). The HEC-1B cells were grown in DMEM-F12 medium supplemented with 100 IU/mL penicillin, 100 g/mL streptomycin sulfate, and 10% fetal bovine serum, and maintained at 37 °C in a humidified incubator containing 5% CO2. Cell culture medium DMEM-F12, fetal bovine serum, and phosphate buffered saline (PBS) were purchased from Invitrogen (Carlsbad, Calif., USA). The anti-caspase-3 antibody and anti-TRPML1 antibody (C-terminal) were obtained from Cell Signaling Technology (Boston, Mass., USA) and Sigma-Aldrich (St Louis, Mo., USA) respectively. The anti--Actin antibody was purchased from Abmart (Shanghai, China). All other chemicals were obtained from SigmaAldrich, unless otherwise indicated. Imidazole treatment and cell viability The HEC-1B cells were seeded in 96-well plates (density, 5 × 103 cells/well). The following day, cells were treated with 100 mmol/L imidazole for various intervals, or for 12 h with various concentrations of imidazole. Cell viabilities were determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and expressed as percentages of non-treated cells. After exposure of cells to imidazole, culture media were changed by serum-free culture media. Then, MTT dissolved in PBS was added to each well for 4 h at 37 °C. After this interval, the culture media containing MTT were discarded and DMSO was added to each well, dissolving the precipitate. The optical density was measured at 570 nm spectral wavelength using a microplate reader (Bio-Rad, Hercules, Calif., USA). Neutral red and acridine orange staining The HEC-1B cells were treated with 50 mmol/L imidazole for 2 h and then examined for vacuolization under an inverted light microscope with 400× magnification (Olympus, Tokyo, Japan). The
neutral red uptake of intracellular vacuoles was performed as described in Cover et al. (1991). Cells on coverslips were loaded with the pH-sensitive fluorescent dye acridine orange (AO, 5 g/mL in PBS) at room temperature for 10 min. After three washes in PBS, cells were directly observed and photographed by laser scanning confocal microscopy (400× magnification; Nikon, Tokyo, Japan). Reverse transcription and quantitative PCR (qPCR) Total RNA was isolated from cells using TRIzol reagent (Invitrogen) and RNA yield quantified using an Eppendorf BioPhotometer (Eppendorf, Hamburg, Germany). The cDNA was synthesized using the First-Strand Synthesis System (Thermo Scientific, Waltham, Mass., USA) with 2 L of oligo (dT) priming. qPCR was performed using SYBR Green PCR Master Mix (TOYOBO, Tokyo, Japan) according to the manufacturer’s protocol. The primers used for qPCR were: 5=-ACACAGTGGGCGGATCCCCA-3= (sense) and 5=-TGCCGCCAC ATGAACCCCAC-3= (antisense) for TRPML1; 5=-AACGGATTTGGTCGT ATTG-3= (sense) and 5=-GGAAGATGGTGATGGGATT-3= (antisense) for GAPDH. Reactions were run on the 7300 Real Time System (Applied Biosystems, Carlsbad, Calif., USA). All experimental samples were normalized to GAPDH endogenous control. TRPML1 expression constructs and cell transfection The full-length sequence corresponding to human TRPML1 coding region was amplified by PCR, using the above cDNA as template. The PCR was performed with the following primers: 5=-TTCTCGAGATGACAGCCCCGGCGGGT-3= (sense) and 5=-TTACCG GTGGATTCACCAGCAGCGAATG-3= (antisense). Amplified products were subcloned into the multicloning site of the expression vector pEGFP-N1 (Clontech, Palo Alto, Calif., USA) with XhoI and AgeI restriction enzymes (New England Biolabs, Ipswich, Mass., USA). Insert orientation and polymerase fidelity were verified by restriction enzyme mapping and DNA sequencing. The HEC-1B cells were transfected in 35 mm dishes with 1 g of plasmid DNA using Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer’s protocols. Cells were analyzed after transfection Published by NRC Research Press
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24 h under a fluorescence microscopy (400× magnification; Olympus, Tokyo, Japan). LysoTracker Red staining The HEC-1B cells on coverslips were incubated with 50 nmol/L LysoTracker Red (Invitrogen) in growth medium for 1 h at 37 °C. Nuclei were stained by incubating cells with 2 mol/L Hoechst 33342 for 5 min. When labeling was complete, we removed the labeling solution and washed cells twice in PBS. Cells were observed and imaged under a fluorescence microscopy (400× magnification, Olympus). The fluorescence intensity of LysoTracker Red was detected using ImageJ software (National Institutes of Health, Bethesda, Md., USA). Statistical analysis of relative average fluorescence intensity value was performed between GFP group and ML1-GFP group. RNA interference All reagents for the RNA interference experiment were obtained from Invitrogen, including siRNA targeting the human TRPML1, negative control siRNA duplex, and BLOCK-iT transfection kit. The HEC-1B cells were plated in 24-well dishes and allowed to grow to 30%−50% confluence. Negative control siRNA (NC) and TRPML1-specific siRNA (ML1siRNA) were transfected into cells by using Lipofectamine 2000, according to the manufacturer’s protocols. The BLOCK-iT fluorescent oligo was used as an indicator of transfection efficiency. TRPML1 knockdown was confirmed after transfection 48 h by quantitative real-time RT-PCR and Western blotting analysis.
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Fig. 4. Overexpression of wild-type TRPML1 inhibited imidazoleinduced cell death. (A) Effect of TRPML1 overexpression on cell viability after imidazole treatment. The HEC-1B cells transfected with pEGFP-TRPML1 or pEGFP vectors were cultured in complete medium with or without 100 mmol/L imidazole for indicated intervals. Cell viabilities were determined by MTT assay and expressed as percentages of nontreated cells. The results from 3 independent experiments were presented as the means ± SEM (*P < 0.05). (B) Effect of TRPML1 overexpression on activation of caspase-3 after imidazole treatment. Cells transfected with pEGFPTRPML1 or pEGFP vectors were treated with or without 100 mmol/L imidazole for indicated intervals. Corresponding changes in caspase-3 were monitored by Western blotting with an anti-caspase-3 antibody, and -Actin was used as loading control. The intensities of Western blotting were analyzed and represented as means ± SEM of 3 independent experiments. *P < 0.05.
Western blotting analysis Protein concentrations were determined using a BCA assay. Equal amounts of protein (20 g) were electrophoresed on 12% SDS-PAGE gels for each experimental sample. Proteins were transferred to nitrocellulose membrane (Millipore, Billerica, Mass., USA) and blocked in 5% nonfat dry milk at 37 °C for 1 h. The following primary antibodies were used: anti-TRPML1 antibody (C-terminal) at 1:1000 dilution, anti-caspase-3 antibody at 1:500 dilution, and anti--Actin antibody at 1:2000 dilution. The membrane was incubated with primary antibodies overnight at 4 °C. The HRPconjugated goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China) were used at 1:5000 dilution. Immunodetection was performed with the enhanced chemiluminescence system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Band densities were measured using ImageJ software. Statistical analysis Statistical significance was analyzed using a one-tailed, unpaired Student’s t-test with P < 0.05 considered significant. Data were presented as mean ± SEM of at least 3 independent experiments.
Results Induction of cell death by imidazole Imidazole reduced viability of HEC-1B cells in a dose- and timedependent manner (Fig. 1A). To determine whether or not imidazoleinduced cell death resulted from caspase-dependent apoptosis, cleaved-caspase-3 (c-cas3) protein levels were investigated after imidazole treatment. Based on Western blotting analysis, the c-cas3 protein levels in the extract of HEC-1B cells increased in a time-dependent manner after imidazole treatment (Fig. 1B). Imidazole induced cell vacuolization and decreased the protein level of TRPML1 in HEC-1B cells Imidazole (10−100 mmol/L) induced the vacuole formation, which occurred within 1−2 h and lasted 24 h in HEC-1B cells. The most representative vacuolation was observed in cells treated with 50 mmol/L imidazole for 2 h (Fig. 2A). These large vacuoles were stained by neutral red or acridine orange (indicating that
imidazole treatment increased the size of acidic organelles). Consistent with these data, bafilomycin A1 (Baf A1), a specific inhibitor of the vacuolar type H+-ATPase, inhibited formation of vacuoles. Moreover, TRPML1 protein level decreased 2 h after imidazole treatment (Fig. 2B), indicating imidazole might have affected TRPML1 protein degradation. Overexpression of wild-type TRPML1 inhibited imidazoleinduced vacuole formation and cell death In HEC-1B cells treated with imidazole for 2 h (starting 24 h after transfection), imidazole failed to induce cytoplasmic vacuole formation in cells expressing TRPML1-GFP (Fig. 3A−3C, arrows). In contrast, transient transfection of GFP alone did not alter imidazole-induced vacuolization in HEC-1B cells (Fig. 3D−3F, arrowheads). Moreover, in cells exposed to 100 mmol/L imidazole, the viability of TRPML1 overexpressing cells was significantly higher (⬃20%) than the viability of GFP controls under the same regimen (Fig. 4A). The level of activated Published by NRC Research Press
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Fig. 5. LysoTracker Red staining in cells expressing TRPML1-GFP fusion protein. (A) The HEC-1B cells were pre-treated with DMSO (control) or 50 nmol/L bafilomycin A1 (Baf A1) for 45 min. The distribution of lysosomes was examined by LysoTracker Red staining and visualized with various channels using fluorescence microscopy (400× magnification). Hoechst staining was used to identify nuclei. (B) Cells transfected with pEGFP-TRPML1 or pEGFP vectors were stained with LysoTracker Red. Fluorescent signals of LysoTracker Red were sequentially acquired by fluorescence microscopy. Fluorescent image illustrated loss of LysoTracker Red staining in cells expressing ML1-GFP (arrows), whereas GFP control had no loss of staining. Scale bar = 20 m. A total of 50 cells from GFP group or ML1-GFP group were randomly selected, and red fluorescence intensity values were detected by ImageJ software. The statistical significance of relative average fluorescence intensity value was analyzed using Student’s t-test (n = 3, **P < 0.01).
caspase-3 also was significantly lower in the TRPML1 overexpressing cells than in the GFP controls after imidazole treatment 4−8 h (Fig. 4B). Overexpression of wild-type TRPML1 raised the pH of acidic organelles Following pretreatment with bafilomycin A1, loss of LysoTracker Red staining was observed in HEC-1B cells, indicating the increased luminal pH of acidic compartments blocked LysoTracker Red from entering lysosomes (Fig. 5A). Similarly, loss of LysoTracker Red staining was also observed in cells expressing TRPML1-GFP (Fig. 5B, arrows), whereas no remarkable loss of staining was detected in GFP control. The relative fluorescent intensity value of LysoTracker Red was significantly decreased in TRPML1-GFP group compared to GFP control. Therefore, overexpression of wild-type TRPML1 increased the pH of acidic organelles. siRNA-mediated knockdown of TRPML1 increased imidazole toxicity in HEC-1B cells The siRNA-mediated knockdown significantly decreased TRPML1 mRNA and protein levels after transfection 48 h in HEC-1B cells (Fig. 6A and 6B). At 100 mmol/L imidazole, the viability of TRPML1 knockdown cells decreased significantly compared to viability of control cells under the same treatment (Fig. 6C). These results indicated that loss of TRPML1 increased imidazole toxicity.
Discussion In this study, overexpression of lysosomal ion channel TRPML1 inhibited imidazole-induced vacuole formation and cell death in
HEC-1B cells. Conversely, TRPML1 knockdown cells were more vulnerable to imidazole-induced cytotoxicity than control cells. Imidazole, a lysosomotropic weak base, causes serious toxicity by accumulating in lysosomes. Prominent side effects of imidazole are vacuole formation (Ohkuma and Poole 1981) and cell death (Iguchi et al. 2002). Lysosomotropic compounds enter acidic organelles and become protonated; accumulation of protonated weak bases in the acidic organelles induces osmotic swelling of these organelles and permeabilization of their membranes (Ohkuma and Poole 1981; Poole and Ohkuma 1981). Increased permeabilization initiates release of lysosomal enzymes (e.g., cathepsins) into the cytoplasm (Boya and Kroemer 2008) and induces apoptosis, either by directly activating caspase-11 (Schotte et al. 1998) or by indirectly activating caspase-3 through mitochondrial membrane permeabilization (Boya et al. 2003). Similarly, in the present study, imidazole rapidly stimulated cell vacuolization (due to enlarged acidic compartments) and resulted in caspase-3 activation in HEC-1B cells. Moreover, when the pH of the cell culture medium was increased from 7.4 to 7.7 (similar to what occurred in imidazole-containing medium), there was no cell vacuolation or cell death (data not shown). Furthermore, bafilomycin A1, which raises the pH of acidic organelles and prevents accumulation of weak bases, did not cause cell death, but rather inhibited imidazole-induced cytotoxicity. Therefore, we concluded that osmotic swelling and release of lysosomal enzymes, rather than an increase in pH, was the primary cause of imidazole-induced cytotoxicity. Cell vacuolation has been assumed to be a hybrid of late endosomes and lysosomes (Molinari et al. 1997; Papini et al. 1997). TRPML1 (also called mucolipin1), a member of the transient recepPublished by NRC Research Press
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Fig. 6. siRNA-mediated knockdown of TRPML1 increased imidazole cytotoxicity. (A) The HEC-1B cells were transfected with negative control siRNA (NC) or TRPML1 siRNA (ML1siRNA) for 48 h. The levels of TRPML1 mRNA were measured by qRT-PCR and normalized to GAPDH control. Data were analyzed and expressed as means ± SEM of 3 independent experiments. **P < 0.01. (B) The corresponding changes in TRPML1 and caspase3 protein levels were monitored by Western blotting analysis. -Actin was used as a loading control. (C) Cells transfected with negative control or TRPML1 siRNA for 48 h were cultured in complete medium with or without 100 mmol/L imidazole for indicated intervals. Cell viabilities were measured by MTT assay and expressed as percentages of nontreated cells. Data were presented as means ± SEM of 3 independent experiments (*P < 0.05).
tor potential (TRP) protein family, primarily localizes in late endosomes and lysosomes (Dong et al. 2008; Pryor et al. 2006) and may regulate fusion/fission of vesicles in the endocytic pathway (Cheng et al. 2010). In the present study, there was no obvious change in mRNA expression of TRPML1 gene (data not shown); however, TRPML1 protein level decreased after imidazole treatment. Therefore, it was unclear how imidazole decreased TRPML1 protein. The function of TRPML1 is regulated by lysosomal pH and ion homeostasis (Cheng et al. 2010), perhaps imidazole-induced changes in the intralysosomal environment accelerated TRPML1 degradation. However, further studies are needed to elucidate the mechanism. The level of juxtaorganellar Ca2+ plays a central role in regulating membrane trafficking (Hay 2007; Luzio et al. 2007a; Pryor et al. 2000). However, ion channels responsible for intralysosomal Ca2+ release have remained elusive. TRPML1 is a candidate endolysosomal Ca2+ channel (Cheng et al. 2010). In this study, overexpression (Supplementary data, Fig. S11) was used to determine whether TRPML1 was involved in imidazole-induced vacuole formation. It was noteworthy that overexpression of wild-type TRPML1 did not accelerate formation of vacuoles in imidazole-treated cells, but rather inhibited imidazole-induced vacuolization and cell death. There are several potential explanations. Firstly, alteration in TRPML1 function could have changed lysosomal pH homeostasis. It has been reported that TRPML1 can function as a H+ channel,
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and the increased lysosomal acidification in TRPML1−/− cells was likely caused by loss of a TRPML1-mediated H+ leak (Soyombo et al. 2006). If TRPML1 functions as a H+ leak channel to prevent lysosomal over-acidification, overexpression of TRPML1 will result in the alkalization of endolysosomes, which would prevent protonation of imidazole in the luminal space and subsequent osmotic swelling of acidic organelles. Secondly, the endolysosomal Ca2+ gradient is maintained by an unidentified H+-Ca2+ exchanger at the expense of the H+ gradient (Dong et al. 2010b; Luzio et al. 2007b). Overexpression of TRPML1 channel may impair intraluminal H+ homeostasis and prevent accumulation of protonated imidazole in acidic organelles, which would inhibit osmotic swelling of these organelles. LysoTracker Red, a fluorescent weak base that accumulates in acidic spaces, was used to verify the change in lysosomal pH induced by TRPML1. Pretreatment with bafilomycin A1 resulted in loss of LysoTracker Red staining in HEC-1B cells. Similarly, loss of staining also was observed in cells overexpressing TRPML1. Therefore, we inferred that TRPML1 raised the luminal pH of acidic compartments. However, more studies are needed to clarify how TRPML1 changes lysosomal pH homeostasis. Although we observed an inhibition of imidazole-induced cell death both at cell viability and caspase-3 cleavage, the overexpression of TRPML1 did not completely abolish imidazole-caused cell death (⬃20%) in HEC-1B cells. Thus, there were other effects of imidazole that TRPML1 could not prevent. The mechanisms by which
Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/bcb-2014-0044. Published by NRC Research Press
Liu et al.
imidazole induces HEC-1B cells death still require further investigation. Moreover, cells from patients with mucolipidosis type IV (MLIV), associated with mutations in gene coding for ion channel TRPML1, are highly sensitive to chloroquine, a weak base (Goldin et al. 1999). In this study, down-regulation of TRPML1 increased imidazole-induced cell death in HEC-1B cells. Perhaps a TRPML1 knockdown (KD) reduced lysosomal pH in these cells, although the permeability of TRPML1 to H+ remains controversial. Consistent with this hypothesis, Soyombo et al. (2006) demonstrated that the lysosomes of TRPML1−/− cells were more acidic than those of wild-type cells and that TRPML1 regulated lysosomal pH. Increased lysosomal acidification in TRPML1 KD cells would cause more accumulation of imidazole in the acidic organelles, which would have accelerated osmotic swelling of these organelles and release of lysosomal enzymes, resulting in cell death. In addition, in our study, a 17 kDa activated caspase-3 band was detected 48 h after TRPML1 KD. It was recently reported that acute TRPML1 KD increased apoptosis mediated by cytoplasmic cathepsin B (CatB) and Bax activity (Colletti et al. 2012). Perhaps imidazole increased CatB translocation into cytoplasm and cell death through CatB-mediated apoptosis after TRPML1 KD. Regardless, TRPML1 KD cells were more sensitive to imidazole toxicity. On the basis of the present study, TRPML1 deficiency should be considered a risk factor for the potential use of a weak base for treatment of lysosomal storage diseases, e.g., mucolipidosis type IV (MLIV). In conclusion, TRPML1 prevented accumulation of imidazole in acidic organelles by changing lysosomal pH and inhibited imidazole-induced vacuolization in HEC-1B cells. Imidazole-caused cell death was partly inhibited by TRPML1, whereas TRPML1 deficiency increased imidazole-induced toxicity in cells. Therefore we inferred that TRPML1 had a novel role in protecting against lysosomotropic amines toxicity. These findings should promote further study of mechanisms for clearance of lysosomal amines, with implications for novel therapeutic strategies in human membrane trafficking defects or some cancers.
Acknowledgements We thank Dr. John P. Kastelic of the University of Calgary for useful discussion and suggestions on article preparation. This work was supported by the National Science & Technology Pillar Program during the 12th Five-year Plan Period (2012BAI32B05).
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ARTICLE A novel role of transient receptor potential mucolipin1 (TRPML1) in protecting against imidazole-induced cytotoxicity Zhenxing Liu, Shuan Zhao, Shuaishuai Wu, Jingyou Zhang, Zunyang Nie, and Shenming Zeng
Abstract: Lysosomotropic amines cause serious side effects such as cytoplasmic vacuolation and cell death. TRPML1 (also known as mucolipin1), a member of the transient receptor potential (TRP) protein family, may regulate fusion/fission of vesicles along the endocytic pathway and some aspects of lysosomal ion homeostasis. Nevertheless, it is still unknown whether TRPML1 is involved in death of mammalian cells induced by lysosomotropic agents. In this study, imidazole was used as a model to investigate the role of TRPML1 in the cytotoxicity of lysosomotropic agents. Overexpression of wild-type TRPML1 inhibited imidazole-induced vacuole formation and cell death in human endometrial adenocarcinoma (HEC-1B) cells. In contrast, siRNAmediated TRPML1 knockdown increased the cell death induced by imidazole. Bafilomycin A1 raises the pH of acidic organelles and therefore suppresses accumulation of weak bases in them. Similarly, lysosomal pH was raised in TRPML1-overexpressing cells; therefore, we inferred that TRPML1 protected against imidazole toxicity by regulating the pH of acidic organelles. We concluded that TRPML1 had a novel role in protecting against lysosomotropic amine toxicity. Key words: imidazole, TRPML1, cytoplasmic vacuolation, cell death, lysosomal pH. Résumé : Les amines lysosomotropes provoquent des effets secondaires sérieux comme la vacuolisation cytoplasmique et la mort cellulaire. TRPML1 (aussi connu sous le nom de mucolipine 1), un membre de la famille des TRP (transient receptor potential), peut réguler les vésicules de fusion/fission le long de la voie endocytique et certains aspects de l’homéostasie des ions des lysosomes. On ignore cependant si TRPML1 est impliqué dans la mort des cellules de mammifères induite par des agents lysosomotropes. Dans cette étude, l’imidazole a été utilisé comme modèle afin d’examiner le rôle de TRPML1 dans la cytotoxicité d’agents lysosomotropes. La surexpression de TRPML1 sauvage inhibait la formation de vacuoles et la mort cellulaire induites par l’imidazole chez des cellules d’adénocarcinome de l’endomètre humain (HEC-1B). Au contraire, l’inactivation de TRPML1 par un pARNi augmentait la mort cellulaire induite par l’imidazole. La bafilomycine A1 accroit le pH des organites acides et supprime ainsi l’accumulation de bases faibles. De la même façon, le pH des lysosomes était accru chez les cellules qui surexprimaient TRPML1 ; ainsi les auteurs ont supposé que TRPML1 protégeait les cellules de la toxicité de l’imidazole en régulant le pH des organites acides. Les auteurs concluent que TRPML1 exerce un nouveau rôle en protégeant les cellules de la toxicité des amines lysosomotropes. [Traduit par la Rédaction] Mots-clés : imidazole, TRPML1, vacuolisation cytoplasmique, mort cellulaire, pH du lysosome.
Introduction Imidazole is an important pharmacophore in drug discovery. Its derivatives have diverse pharmacological activities, including anti-inflammatory (Labanauskas et al. 2000), histamine-H3 antagonist (Grassmann et al. 2002), antioxidant (Can-Eke et al. 1998), gastroprotective (Sevak et al. 2002), and anticancer (Krezel 1998) properties. However, imidazole can also have serious side effects, as high concentrations cause cell death (Iguchi et al. 2002). As a weak base, imidazole enters acidic spaces and increases the pH of acidic organelles (Poole and Ohkuma 1981). Accumulation of protonated weak bases in acidic organelles induces osmotic swelling and cytoplasmic vacuolation (Ohkuma and Poole 1981). Thus, permeabilization of lysosomal membranes and the subsequent release of hydrolases have been considered the mechanism of lysosomotropic compounds toxicity (Boya and Kroemer 2008). TRPML1 (also mucolipin1), a member of the transient receptor potential (TRP) protein family, primarily resides in membranes of late endosomes and lysosomes (Hersh et al. 2002; Kiselyov et al. 2005;
LaPlante et al. 2002; Vergarajauregui and Puertollano 2006) and functions as an inwardly rectifying cation channel potentiated by low pH (Xu et al. 2007; Zhang et al. 2009). Mutations in human TRPML1 cause mucolipidosis type IV (MLIV), a severe neurodegenerative disease associated with defective lysosomal biogenesis and trafficking (Bassi et al. 2000; Sun et al. 2000). TRPML1 may regulate fusion/fission of vesicles along the endocytic pathway (Cheng et al. 2010; LaPlante et al. 2004, 2006; Pryor et al. 2006). In addition, it also has crucial roles in lysosomal ion homeostasis, including modulating pH (Soyombo et al. 2006) and concentrations of iron (Dong et al. 2008, 2010a) and zinc (Eichelsdoerfer et al. 2010). However, whether TRPML1 is involved in cell death induced by lysosomotropic amines in mammalian cells has apparently not been reported. In this study, we used overexpression or knockdown to investigate whether TRPML1 was involved in imidazole-induced cytoplasmic vacuolation and cell death, in an attempt to gain further insights on the novel role of TRPML1 in protecting against lysosomotropic amine toxicity.
Received 31 March 2014. Revision received 14 May 2014. Accepted 19 May 2014. Z. Liu,* S. Zhao,* S. Wu, Z. Nie, and S. Zeng. Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P.R. China. J. Zhang. Reproduction and Breeding Research Center, Animal Husbandry and Veterinary 9 Research Institute, Heilongjiang Academy of Agricultural and Reclamation Science, Harbin 150038, P.R. China. Corresponding author: Shenming Zeng (e-mail: [email protected]). *Zhenxing Liu and Shuan Zhao contributed equally to this work. Biochem. Cell Biol. 92: 279–286 (2014) dx.doi.org/10.1139/bcb-2014-0044
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Fig. 1. Induction of cell death by imidazole in HEC-1B cells. (A) Effect of imidazole on cell viability. Cells were treated with indicated concentrations of imidazole for 12 h or 100 mmol/L imidazole for various intervals. Cell viabilities were determined by MTT assay and expressed as percentages of nontreated cells. The results from 3 biological replicates were presented as the means ± SEM (*P < 0.05, **P < 0.01). (B) Activation of caspase-3 in imidazole-treated cells. Cells treated with or without 100 mmol/L imidazole for indicated intervals were subjected to Western blotting analysis using an antibody against cleaved-caspase-3 (c-cas3). -Actin was used as a loading control.
Fig. 2. Imidazole induced cell vacuolization and decreased the protein level of TRPML1 in HEC-1B cells. (A) Vacuoles formation in imidazole-treated cells. Cells with or without 50 mmol/L imidazole treatment for 2 h were observed (phase-contrast microscopy, 400× magnification). For neutral red dye uptake, cells were incubated with 0.05% neutral red dye at room temperature for 5 min, washed, and observed with light microscopy. The distribution of acidic organelles was examined by AO staining and visualized with various channels using confocal microscopy. C: control group; T: treatment group; Baf A1: bafilomycin A1; NR: neutral red; AO: acridine orange. Scale bar = 20 m. (B) Effect of imidazole on expression of TRPML1 protein. Cells were treated with or without 50 mmol/L imidazole for indicated intervals, and Western blotting was used to analyze the abundance of TRPML1 protein using anti-TRPML1 C-terminal antibody. -Actin was used as a loading control. Comparison of the intensities of Western blotting were analyzed and represented as means ± SEM of 3 independent experiments. *P < 0.05.
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Fig. 3. Overexpression of wild-type TRPML1 inhibited imidazole-induced vacuoles formation. (A–C) The HEC-1B cells were transfected with pEGFP-TRPML1 vectors and cultured in complete medium with or without 50 mmol/L imidazole for 2 h. The fluorescent signals of TRPML1-GFP (ML1-GFP) were sequentially acquired by fluorescence microscopy (400× magnification). Imidazole failed to induce cytoplasmic vacuolization in cells expressing ML1-GFP (arrows). (D–F) Cells transfected with pEGFP vectors were treated 2 h with imidazole. In contrast, GFP alone did not alter imidazole-induced vacuolization in cells (arrowheads). Images were merged using ImageJ software. Scale bar = 20 m.
Materials and methods Cell culture and reagents A human endometrial adenocarcinoma cell line (HEC-1B) was purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). The HEC-1B cells were grown in DMEM-F12 medium supplemented with 100 IU/mL penicillin, 100 g/mL streptomycin sulfate, and 10% fetal bovine serum, and maintained at 37 °C in a humidified incubator containing 5% CO2. Cell culture medium DMEM-F12, fetal bovine serum, and phosphate buffered saline (PBS) were purchased from Invitrogen (Carlsbad, Calif., USA). The anti-caspase-3 antibody and anti-TRPML1 antibody (C-terminal) were obtained from Cell Signaling Technology (Boston, Mass., USA) and Sigma-Aldrich (St Louis, Mo., USA) respectively. The anti--Actin antibody was purchased from Abmart (Shanghai, China). All other chemicals were obtained from SigmaAldrich, unless otherwise indicated. Imidazole treatment and cell viability The HEC-1B cells were seeded in 96-well plates (density, 5 × 103 cells/well). The following day, cells were treated with 100 mmol/L imidazole for various intervals, or for 12 h with various concentrations of imidazole. Cell viabilities were determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and expressed as percentages of non-treated cells. After exposure of cells to imidazole, culture media were changed by serum-free culture media. Then, MTT dissolved in PBS was added to each well for 4 h at 37 °C. After this interval, the culture media containing MTT were discarded and DMSO was added to each well, dissolving the precipitate. The optical density was measured at 570 nm spectral wavelength using a microplate reader (Bio-Rad, Hercules, Calif., USA). Neutral red and acridine orange staining The HEC-1B cells were treated with 50 mmol/L imidazole for 2 h and then examined for vacuolization under an inverted light microscope with 400× magnification (Olympus, Tokyo, Japan). The
neutral red uptake of intracellular vacuoles was performed as described in Cover et al. (1991). Cells on coverslips were loaded with the pH-sensitive fluorescent dye acridine orange (AO, 5 g/mL in PBS) at room temperature for 10 min. After three washes in PBS, cells were directly observed and photographed by laser scanning confocal microscopy (400× magnification; Nikon, Tokyo, Japan). Reverse transcription and quantitative PCR (qPCR) Total RNA was isolated from cells using TRIzol reagent (Invitrogen) and RNA yield quantified using an Eppendorf BioPhotometer (Eppendorf, Hamburg, Germany). The cDNA was synthesized using the First-Strand Synthesis System (Thermo Scientific, Waltham, Mass., USA) with 2 L of oligo (dT) priming. qPCR was performed using SYBR Green PCR Master Mix (TOYOBO, Tokyo, Japan) according to the manufacturer’s protocol. The primers used for qPCR were: 5=-ACACAGTGGGCGGATCCCCA-3= (sense) and 5=-TGCCGCCAC ATGAACCCCAC-3= (antisense) for TRPML1; 5=-AACGGATTTGGTCGT ATTG-3= (sense) and 5=-GGAAGATGGTGATGGGATT-3= (antisense) for GAPDH. Reactions were run on the 7300 Real Time System (Applied Biosystems, Carlsbad, Calif., USA). All experimental samples were normalized to GAPDH endogenous control. TRPML1 expression constructs and cell transfection The full-length sequence corresponding to human TRPML1 coding region was amplified by PCR, using the above cDNA as template. The PCR was performed with the following primers: 5=-TTCTCGAGATGACAGCCCCGGCGGGT-3= (sense) and 5=-TTACCG GTGGATTCACCAGCAGCGAATG-3= (antisense). Amplified products were subcloned into the multicloning site of the expression vector pEGFP-N1 (Clontech, Palo Alto, Calif., USA) with XhoI and AgeI restriction enzymes (New England Biolabs, Ipswich, Mass., USA). Insert orientation and polymerase fidelity were verified by restriction enzyme mapping and DNA sequencing. The HEC-1B cells were transfected in 35 mm dishes with 1 g of plasmid DNA using Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer’s protocols. Cells were analyzed after transfection Published by NRC Research Press
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24 h under a fluorescence microscopy (400× magnification; Olympus, Tokyo, Japan). LysoTracker Red staining The HEC-1B cells on coverslips were incubated with 50 nmol/L LysoTracker Red (Invitrogen) in growth medium for 1 h at 37 °C. Nuclei were stained by incubating cells with 2 mol/L Hoechst 33342 for 5 min. When labeling was complete, we removed the labeling solution and washed cells twice in PBS. Cells were observed and imaged under a fluorescence microscopy (400× magnification, Olympus). The fluorescence intensity of LysoTracker Red was detected using ImageJ software (National Institutes of Health, Bethesda, Md., USA). Statistical analysis of relative average fluorescence intensity value was performed between GFP group and ML1-GFP group. RNA interference All reagents for the RNA interference experiment were obtained from Invitrogen, including siRNA targeting the human TRPML1, negative control siRNA duplex, and BLOCK-iT transfection kit. The HEC-1B cells were plated in 24-well dishes and allowed to grow to 30%−50% confluence. Negative control siRNA (NC) and TRPML1-specific siRNA (ML1siRNA) were transfected into cells by using Lipofectamine 2000, according to the manufacturer’s protocols. The BLOCK-iT fluorescent oligo was used as an indicator of transfection efficiency. TRPML1 knockdown was confirmed after transfection 48 h by quantitative real-time RT-PCR and Western blotting analysis.
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Fig. 4. Overexpression of wild-type TRPML1 inhibited imidazoleinduced cell death. (A) Effect of TRPML1 overexpression on cell viability after imidazole treatment. The HEC-1B cells transfected with pEGFP-TRPML1 or pEGFP vectors were cultured in complete medium with or without 100 mmol/L imidazole for indicated intervals. Cell viabilities were determined by MTT assay and expressed as percentages of nontreated cells. The results from 3 independent experiments were presented as the means ± SEM (*P < 0.05). (B) Effect of TRPML1 overexpression on activation of caspase-3 after imidazole treatment. Cells transfected with pEGFPTRPML1 or pEGFP vectors were treated with or without 100 mmol/L imidazole for indicated intervals. Corresponding changes in caspase-3 were monitored by Western blotting with an anti-caspase-3 antibody, and -Actin was used as loading control. The intensities of Western blotting were analyzed and represented as means ± SEM of 3 independent experiments. *P < 0.05.
Western blotting analysis Protein concentrations were determined using a BCA assay. Equal amounts of protein (20 g) were electrophoresed on 12% SDS-PAGE gels for each experimental sample. Proteins were transferred to nitrocellulose membrane (Millipore, Billerica, Mass., USA) and blocked in 5% nonfat dry milk at 37 °C for 1 h. The following primary antibodies were used: anti-TRPML1 antibody (C-terminal) at 1:1000 dilution, anti-caspase-3 antibody at 1:500 dilution, and anti--Actin antibody at 1:2000 dilution. The membrane was incubated with primary antibodies overnight at 4 °C. The HRPconjugated goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China) were used at 1:5000 dilution. Immunodetection was performed with the enhanced chemiluminescence system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Band densities were measured using ImageJ software. Statistical analysis Statistical significance was analyzed using a one-tailed, unpaired Student’s t-test with P < 0.05 considered significant. Data were presented as mean ± SEM of at least 3 independent experiments.
Results Induction of cell death by imidazole Imidazole reduced viability of HEC-1B cells in a dose- and timedependent manner (Fig. 1A). To determine whether or not imidazoleinduced cell death resulted from caspase-dependent apoptosis, cleaved-caspase-3 (c-cas3) protein levels were investigated after imidazole treatment. Based on Western blotting analysis, the c-cas3 protein levels in the extract of HEC-1B cells increased in a time-dependent manner after imidazole treatment (Fig. 1B). Imidazole induced cell vacuolization and decreased the protein level of TRPML1 in HEC-1B cells Imidazole (10−100 mmol/L) induced the vacuole formation, which occurred within 1−2 h and lasted 24 h in HEC-1B cells. The most representative vacuolation was observed in cells treated with 50 mmol/L imidazole for 2 h (Fig. 2A). These large vacuoles were stained by neutral red or acridine orange (indicating that
imidazole treatment increased the size of acidic organelles). Consistent with these data, bafilomycin A1 (Baf A1), a specific inhibitor of the vacuolar type H+-ATPase, inhibited formation of vacuoles. Moreover, TRPML1 protein level decreased 2 h after imidazole treatment (Fig. 2B), indicating imidazole might have affected TRPML1 protein degradation. Overexpression of wild-type TRPML1 inhibited imidazoleinduced vacuole formation and cell death In HEC-1B cells treated with imidazole for 2 h (starting 24 h after transfection), imidazole failed to induce cytoplasmic vacuole formation in cells expressing TRPML1-GFP (Fig. 3A−3C, arrows). In contrast, transient transfection of GFP alone did not alter imidazole-induced vacuolization in HEC-1B cells (Fig. 3D−3F, arrowheads). Moreover, in cells exposed to 100 mmol/L imidazole, the viability of TRPML1 overexpressing cells was significantly higher (⬃20%) than the viability of GFP controls under the same regimen (Fig. 4A). The level of activated Published by NRC Research Press
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Fig. 5. LysoTracker Red staining in cells expressing TRPML1-GFP fusion protein. (A) The HEC-1B cells were pre-treated with DMSO (control) or 50 nmol/L bafilomycin A1 (Baf A1) for 45 min. The distribution of lysosomes was examined by LysoTracker Red staining and visualized with various channels using fluorescence microscopy (400× magnification). Hoechst staining was used to identify nuclei. (B) Cells transfected with pEGFP-TRPML1 or pEGFP vectors were stained with LysoTracker Red. Fluorescent signals of LysoTracker Red were sequentially acquired by fluorescence microscopy. Fluorescent image illustrated loss of LysoTracker Red staining in cells expressing ML1-GFP (arrows), whereas GFP control had no loss of staining. Scale bar = 20 m. A total of 50 cells from GFP group or ML1-GFP group were randomly selected, and red fluorescence intensity values were detected by ImageJ software. The statistical significance of relative average fluorescence intensity value was analyzed using Student’s t-test (n = 3, **P < 0.01).
caspase-3 also was significantly lower in the TRPML1 overexpressing cells than in the GFP controls after imidazole treatment 4−8 h (Fig. 4B). Overexpression of wild-type TRPML1 raised the pH of acidic organelles Following pretreatment with bafilomycin A1, loss of LysoTracker Red staining was observed in HEC-1B cells, indicating the increased luminal pH of acidic compartments blocked LysoTracker Red from entering lysosomes (Fig. 5A). Similarly, loss of LysoTracker Red staining was also observed in cells expressing TRPML1-GFP (Fig. 5B, arrows), whereas no remarkable loss of staining was detected in GFP control. The relative fluorescent intensity value of LysoTracker Red was significantly decreased in TRPML1-GFP group compared to GFP control. Therefore, overexpression of wild-type TRPML1 increased the pH of acidic organelles. siRNA-mediated knockdown of TRPML1 increased imidazole toxicity in HEC-1B cells The siRNA-mediated knockdown significantly decreased TRPML1 mRNA and protein levels after transfection 48 h in HEC-1B cells (Fig. 6A and 6B). At 100 mmol/L imidazole, the viability of TRPML1 knockdown cells decreased significantly compared to viability of control cells under the same treatment (Fig. 6C). These results indicated that loss of TRPML1 increased imidazole toxicity.
Discussion In this study, overexpression of lysosomal ion channel TRPML1 inhibited imidazole-induced vacuole formation and cell death in
HEC-1B cells. Conversely, TRPML1 knockdown cells were more vulnerable to imidazole-induced cytotoxicity than control cells. Imidazole, a lysosomotropic weak base, causes serious toxicity by accumulating in lysosomes. Prominent side effects of imidazole are vacuole formation (Ohkuma and Poole 1981) and cell death (Iguchi et al. 2002). Lysosomotropic compounds enter acidic organelles and become protonated; accumulation of protonated weak bases in the acidic organelles induces osmotic swelling of these organelles and permeabilization of their membranes (Ohkuma and Poole 1981; Poole and Ohkuma 1981). Increased permeabilization initiates release of lysosomal enzymes (e.g., cathepsins) into the cytoplasm (Boya and Kroemer 2008) and induces apoptosis, either by directly activating caspase-11 (Schotte et al. 1998) or by indirectly activating caspase-3 through mitochondrial membrane permeabilization (Boya et al. 2003). Similarly, in the present study, imidazole rapidly stimulated cell vacuolization (due to enlarged acidic compartments) and resulted in caspase-3 activation in HEC-1B cells. Moreover, when the pH of the cell culture medium was increased from 7.4 to 7.7 (similar to what occurred in imidazole-containing medium), there was no cell vacuolation or cell death (data not shown). Furthermore, bafilomycin A1, which raises the pH of acidic organelles and prevents accumulation of weak bases, did not cause cell death, but rather inhibited imidazole-induced cytotoxicity. Therefore, we concluded that osmotic swelling and release of lysosomal enzymes, rather than an increase in pH, was the primary cause of imidazole-induced cytotoxicity. Cell vacuolation has been assumed to be a hybrid of late endosomes and lysosomes (Molinari et al. 1997; Papini et al. 1997). TRPML1 (also called mucolipin1), a member of the transient recepPublished by NRC Research Press
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Fig. 6. siRNA-mediated knockdown of TRPML1 increased imidazole cytotoxicity. (A) The HEC-1B cells were transfected with negative control siRNA (NC) or TRPML1 siRNA (ML1siRNA) for 48 h. The levels of TRPML1 mRNA were measured by qRT-PCR and normalized to GAPDH control. Data were analyzed and expressed as means ± SEM of 3 independent experiments. **P < 0.01. (B) The corresponding changes in TRPML1 and caspase3 protein levels were monitored by Western blotting analysis. -Actin was used as a loading control. (C) Cells transfected with negative control or TRPML1 siRNA for 48 h were cultured in complete medium with or without 100 mmol/L imidazole for indicated intervals. Cell viabilities were measured by MTT assay and expressed as percentages of nontreated cells. Data were presented as means ± SEM of 3 independent experiments (*P < 0.05).
tor potential (TRP) protein family, primarily localizes in late endosomes and lysosomes (Dong et al. 2008; Pryor et al. 2006) and may regulate fusion/fission of vesicles in the endocytic pathway (Cheng et al. 2010). In the present study, there was no obvious change in mRNA expression of TRPML1 gene (data not shown); however, TRPML1 protein level decreased after imidazole treatment. Therefore, it was unclear how imidazole decreased TRPML1 protein. The function of TRPML1 is regulated by lysosomal pH and ion homeostasis (Cheng et al. 2010), perhaps imidazole-induced changes in the intralysosomal environment accelerated TRPML1 degradation. However, further studies are needed to elucidate the mechanism. The level of juxtaorganellar Ca2+ plays a central role in regulating membrane trafficking (Hay 2007; Luzio et al. 2007a; Pryor et al. 2000). However, ion channels responsible for intralysosomal Ca2+ release have remained elusive. TRPML1 is a candidate endolysosomal Ca2+ channel (Cheng et al. 2010). In this study, overexpression (Supplementary data, Fig. S11) was used to determine whether TRPML1 was involved in imidazole-induced vacuole formation. It was noteworthy that overexpression of wild-type TRPML1 did not accelerate formation of vacuoles in imidazole-treated cells, but rather inhibited imidazole-induced vacuolization and cell death. There are several potential explanations. Firstly, alteration in TRPML1 function could have changed lysosomal pH homeostasis. It has been reported that TRPML1 can function as a H+ channel,
1
and the increased lysosomal acidification in TRPML1−/− cells was likely caused by loss of a TRPML1-mediated H+ leak (Soyombo et al. 2006). If TRPML1 functions as a H+ leak channel to prevent lysosomal over-acidification, overexpression of TRPML1 will result in the alkalization of endolysosomes, which would prevent protonation of imidazole in the luminal space and subsequent osmotic swelling of acidic organelles. Secondly, the endolysosomal Ca2+ gradient is maintained by an unidentified H+-Ca2+ exchanger at the expense of the H+ gradient (Dong et al. 2010b; Luzio et al. 2007b). Overexpression of TRPML1 channel may impair intraluminal H+ homeostasis and prevent accumulation of protonated imidazole in acidic organelles, which would inhibit osmotic swelling of these organelles. LysoTracker Red, a fluorescent weak base that accumulates in acidic spaces, was used to verify the change in lysosomal pH induced by TRPML1. Pretreatment with bafilomycin A1 resulted in loss of LysoTracker Red staining in HEC-1B cells. Similarly, loss of staining also was observed in cells overexpressing TRPML1. Therefore, we inferred that TRPML1 raised the luminal pH of acidic compartments. However, more studies are needed to clarify how TRPML1 changes lysosomal pH homeostasis. Although we observed an inhibition of imidazole-induced cell death both at cell viability and caspase-3 cleavage, the overexpression of TRPML1 did not completely abolish imidazole-caused cell death (⬃20%) in HEC-1B cells. Thus, there were other effects of imidazole that TRPML1 could not prevent. The mechanisms by which
Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/bcb-2014-0044. Published by NRC Research Press
Liu et al.
imidazole induces HEC-1B cells death still require further investigation. Moreover, cells from patients with mucolipidosis type IV (MLIV), associated with mutations in gene coding for ion channel TRPML1, are highly sensitive to chloroquine, a weak base (Goldin et al. 1999). In this study, down-regulation of TRPML1 increased imidazole-induced cell death in HEC-1B cells. Perhaps a TRPML1 knockdown (KD) reduced lysosomal pH in these cells, although the permeability of TRPML1 to H+ remains controversial. Consistent with this hypothesis, Soyombo et al. (2006) demonstrated that the lysosomes of TRPML1−/− cells were more acidic than those of wild-type cells and that TRPML1 regulated lysosomal pH. Increased lysosomal acidification in TRPML1 KD cells would cause more accumulation of imidazole in the acidic organelles, which would have accelerated osmotic swelling of these organelles and release of lysosomal enzymes, resulting in cell death. In addition, in our study, a 17 kDa activated caspase-3 band was detected 48 h after TRPML1 KD. It was recently reported that acute TRPML1 KD increased apoptosis mediated by cytoplasmic cathepsin B (CatB) and Bax activity (Colletti et al. 2012). Perhaps imidazole increased CatB translocation into cytoplasm and cell death through CatB-mediated apoptosis after TRPML1 KD. Regardless, TRPML1 KD cells were more sensitive to imidazole toxicity. On the basis of the present study, TRPML1 deficiency should be considered a risk factor for the potential use of a weak base for treatment of lysosomal storage diseases, e.g., mucolipidosis type IV (MLIV). In conclusion, TRPML1 prevented accumulation of imidazole in acidic organelles by changing lysosomal pH and inhibited imidazole-induced vacuolization in HEC-1B cells. Imidazole-caused cell death was partly inhibited by TRPML1, whereas TRPML1 deficiency increased imidazole-induced toxicity in cells. Therefore we inferred that TRPML1 had a novel role in protecting against lysosomotropic amines toxicity. These findings should promote further study of mechanisms for clearance of lysosomal amines, with implications for novel therapeutic strategies in human membrane trafficking defects or some cancers.
Acknowledgements We thank Dr. John P. Kastelic of the University of Calgary for useful discussion and suggestions on article preparation. This work was supported by the National Science & Technology Pillar Program during the 12th Five-year Plan Period (2012BAI32B05).
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