Departments of * Medicinal Chemistry, Environmental and Occupational Health Sciences, and
Pathology, University of Washington, Seattle, Washington 98195
1 To whom correspondence should be addressed at Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle WA 981957610. Fax: (206) 685-3252. E-mail: hankiat{at}u.washington.edu.
Received March 17, 2005; accepted May 9, 2005
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ABSTRACT |
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Key Words: tetrafluoroethylcysteine; mitochondrial dysfunction; Nrf2; oxidative stress; ER stress.
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INTRODUCTION |
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Previous investigations have characterized a predominant mitochondrial role in progression of injury involving translocation of pro-apoptotic cytosolic BAX to the mitochondrial outer membrane as a relatively early event in TFEC toxicity (Ho et al., 2005). Subsequent membrane permeability transition (MPT) and cytochrome c release have also been shown to be reversible with either BCL-xL overexpression or bongkekric acid administration (Ho et al., 2005
; James et al., 2002
). Collectively, these studies indicate that TFEC is a unique intramitochondrial toxicant, which acts to disrupt bioenergetics at precisely defined sites. Nonetheless, clear evidence linking the binding of intramitochondrial proteins to the final outcome of TFEC-induced cytotoxicity is still lacking. We have previously reported extensive biochemical characterizations of TFEC-induced damage in vitro utilizing the differentiated TAMH mouse cell line (Ho et al., 2005
; James et al., 2002
; Pierce et al., 2002
; Wu et al., 1994
). For example, the covalent modification of aconitase and
KGDH protein targets in vitro attenuates their enzymatic activities and adequately models the deficits observed in vivo (Ho et al., 2005
; James et al., 2002
; Pierce et al., 2002
; Wu et al., 1994
). The relationship(s) between reduced protein function and subsequent mitochondrial damage and cell death have yet to be elucidated. The signaling pathways to and from the mitochondria leading to progression of cellular injury also lacks adequate characterization. Therefore, the specific aim of this study was to use global gene expression-profiling to identify unknown cellular and biochemical responses to TFEC treatment in the TAMH cell line that may be involved in pathways progressing to cytotoxicity.
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MATERIALS AND METHODS |
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RNA isolation.
Cells were grown to confluence in 150 cm2 tissue culture dishes in quadruplicate for each sample and treated with 200 µM TFEC for 2, 4, and 6 h. At the end of respective treatments, cells were harvested by scraping with rubber policemen. The resultant cell pellets were spun down and washed once with Dulbecco's PBS (Gibco). Immediately, 1 ml of Trizol reagent (Gibco) per 107 cells was added for cell lysis. After vortexing, the lysate was passed through 22G needles 10 times to ensure complete lysis. Quickly, 0.2 ml of chloroform was added to every 1 ml of cell lysate and vortexed vigorously for 15 s (1-ml aliquots of lysates were measured into microcentrifuge tubes). The tubes were left to stand for 23 min before spinning at 10,000 rpm for 15 min at 4°C. Gently, 0.5 ml of aqueous phase was transferred to a fresh tube, and an equivolume of 70% ethanol was added. This resulting mix was loaded onto an RNeasy column (Qiagen, Valencia, CA), and purified total RNAs were extracted according to the manufacturer's protocol.
Microarray analysis procedures.
Gene expression analyses were performed using the Amersham, "Codelink" 10K mouse array (Amersham Biosciences, Piscataway, NJ) according to manufacturer's protocols. Briefly, total RNA from each sample was quantified before first and second strand cDNA synthesis. The resulting double-stranded cDNA was purified with a QIAquick spin column (Qiagen). After drying the cDNA in a SpeedVac concentrator, cRNA was synthesized by in vitro transcription and purified using the RNeasy kit. The quality of the cRNA was evaluated using an Agilent, 2100 Bioanalyzer (Agilent, Palo Alto, CA), and only those with A260:A280 ratio of 1.82.1 were used for subsequent microarray analysis. Each 10 µg of cRNA sample was hybridized onto Codelink microarray slides and incubated for 18 h at 37°C. At the end of incubation, the arrays were washed with 0.75x TNT buffer (0.1 M TrisHCl pH 7.6, 0.15 M NaCl, 0.05% Tween-20) at 46°C for 1 h and incubated with streptavidin-Alexa 647 (Molecular Probes, Eugene, OR) working solution at 25°C for 30 min to label the fluorogenic probe. The arrays were scanned with an Axon GenePix 4000B fluorescent scanner and the GenePix Pro imaging software (Axon Instruments, Foster City, CA). Fluorescent intensity of each spot in the image was determined using ImaGeneTM 5 (Biodiscovery, Marina del Rey, CA) for spot finding and analysis.
Real time RT-PCR.
Fluorogenic 5' nuclease assays (TaqMan) were carried out using an ABI Prism 7700 Sequence detection system (Applied Biosystems, Foster City, CA). The thermal cycling condition comprised an initial denaturation step at 95°C for 10 min, followed by 40 cycles at 95°C for 20 s and 62°C for 60 s. The gene-specific sequences of the primer pairs and probes used in the assays are as follows: GCLc (U85498): forward primer, ATGTGGACACCCGATGCAGTATT; reverse primer, TGTCTTGCTTGTAGTCAGGATGGTTT; probe, CCTAAAGCTAATTAAGAAGAGAGC. GCLm (NM_008129): forward primer, GCCACCAGATTTGACTGCCTTT; reverse primer, CAGGGATGCTTTCTTGAAGAGCTT; probe, TCTGAGGCAAGTTTCCA. GSTA3 (NM_010356): forward primer, AGGAACAAACCAGGAACCGTTACTT; reverse primer, CAGCGCTCCTCAGCCTGTT; probe, TCTTCAACACCTTTTCAAAGG. GSTA2 (NM_008182): forward primer, GTATTATGTCCCCCAGACCAAAGAG; reverse primer, CTGTTGCCCACAAGGTAGTCTTGT. GAPDH (NM_008084
[GenBank]
): forward primer, TCCTGCACCACCAACTGCTT; reverse primer, GAGGGGCCATCCACAGTCTT; probe, CACTCATGACCACAGTCCATGCCATCAC. GSTA2 was analyzed by SYBR green instead of TaqMan, and no probe was needed.
Isolation of cytosolic/nuclear fractions.
Nuclear and cytosolic fractions were isolated with slight modifications to the protocol described previously (Buckley et al., 2003). Briefly, the harvested cells were pelleted and resuspended in 475 µl of cytosolic extraction buffer (10 mM Tris-base, 60 mM KCl, 1 mM EDTA, 1 mM DTT, protease inhibitor cocktail) and kept on ice for 10 min. Subsequently, 25 µl of 10% v/v Igepal CA-630 was added to the cell suspension and mixed with gentle pipetting for 1015 s. This mixture was spun at 12,000 x g for 5 min at 4°C. The resultant supernatant was removed as the cytosolic fraction. The pellet, which contained the nuclear-enriched fraction, was then resuspended again in nuclear extraction buffer (20 mM Tris-base, 400 mM NaCl, 1.5 mM MgCl2, 1.5 mM EDTA, 1 mM DTT, 25% v/v glycerol, protease inhibitor cocktail). Independent verification of the relative purity of subcellular fractions was by immunoblot (as described below).
Immunoblot procedures.
All the fractions collected were assayed for protein concentration using the BCA protein assay kit (Pierce Chemical Co., Rockford, IL). Each 3050 µg of sample proteins were resolved by denaturing electrophoresis, SDSPAGE (Mini-PROTEAN II; Bio-Rad Laboratories, Hercules, CA) and transferred to nitrocellulose membrane for 1 h at 15 V using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad). Immunodetection was by chemiluminescence (SuperSignal ULTRA; Pierce, Rockford, IL) using specific antibodies diluted in PBS with 0.05% v/v Tween 20 and 5% w/v powdered milk. Anti-Nrf2 (1:1,000), anti-Nrf1, anti-Gadd153, anti-Gadd34, and anti-Histone-H1 were from Santa Cruz Biotechnology (San Diego CA), anti-HO-1 from Stressgen (Victoria, BC, Canada), and anti-GAPDH was developed in-house (Dietze et al., 1997). Secondary anti-mouse and anti-rabbit horseradish peroxidase conjugated secondary antibodies (Pierce) were used at 1:20,000 dilution. All primary antibodies were used at 1:2,000 dilution unless otherwise stated. Densitometric analyses were performed on selected immunoblots using Bio-Rad ChemiDoc and the Quantity One Version 4.3.0 program (Bio-Rad).
Immunocytochemistry.
Cells were grown on 4-well chambered slides (Labtek II, Nalgen, Naperville, IL). Cultures were dosed with 200 µM TFEC for 04 h. After treatment, medium was aspirated, and cells were washed twice with Hank's BSS and fixed with 3.7% (v/v) paraformaldehyde (EMS, Ft. Washington, PA) in Hank's BSS for 20 min at room temperature. Ice-cold acetone was added for 5 min, and nonspecific binding was blocked by soaking the chambers overnight in PBS with 10% FBS at 4°C. Immunostaining was with anti-Nrf2 and anti-rabbit fluorescein isothiocyanate (FITC) conjugated IgG (Molecular Probes) in the presence of saponin (0.2% w/v) to enhance antibody accessibility. Nuclear staining was performed by incubating 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) at 0.5 µg/ml in PBS for 5 min. Cells were washed extensively with PBS before being mounted with Fluoromount G (Southern Biotechnologies, Birmingham, AL) and examined using Nikon Eclipse fluorescence microscope (Nikon, Melville, NY) with 40x lenses. Images were then processed with Q-Imaging software (Burnaby, BC, Canada).
Spectrofluorometric analyses of hydrogen peroxide formation and free cytosolic calcium.
Cells were grown on 6-well dishes and treated with TFEC as indicated. Cells were then incubated with either 10 µM dihydro-dichlorofluorescein-diacetate (H2DCFDA) for measuring intracellular hydrogen peroxide, or 2 µM Indo-1 AM for measuring intracellular calcium. Cells were rinsed, harvested, and resuspended in 1 ml Hanks' BSS. Fluorescence detection was by SLM-Aminco 8100 spectrofluorometer (Spectronic Instruments, Rochester, NY), monitoring excitation 468 nm, emission 528 nm for H2DCFDA. Indo-1 detection and calcium quantification required a ratiometric analysis of [(excitation 320 nm, emission 405 nm) / (excitation 355 nm, emission 475 nm)].
Viability assay by MTT.
Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability assay according to protocol as previously described (Ho et al., 2005). Experiments investigating the impact of antioxidants on TFEC toxicity were with Trolox (1 mM) and t-butylhydroperoxide as a positive control.
ATP depletion assay.
Intracellular ATP level was measured by its activity using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI) according to the manufacturer's protocol. Briefly, cells were treated with 250 µM TFEC for 08 h on 96-well plates. After incubation, an equivolume of the luminescent substrate and lysis buffer mix from the assay kit was added. The mixture was transferred to an opaque 96-well plate, and luminescence was read and analyzed with PlateLumino (Phenix, Hayward CA).
Flow cytometry.
Flow cytometry methods were adopted as previously described (Botta et al., 2004). Briefly, cells were grown on 12-well dishes and treated for the time periods indicated. All cells were harvested, resuspended, and incubated in nonylacridine orange (NAO, Molecular Probes; 2 µM; excitation 488 nm, emission 530 nm), as a measure of mitochondrial cardiolipin content (Molecular Probes), with monochlorobimane (4 µM; excitation 351362 nm, emission 450 nm) for reduced glutatione (GSH) content, or with hydroethidine for superoxide (Molecular Probes; 5 µM; excitation 488 nm, emission 590 nm). Diethylmaleate (DEM) was used as a positive control for GSH depletion (125 µM; 4 h). After staining for 30 min at 37°C in the dark, the cells were examined by flow cytometry (Epics Elite, Beckman-Coulter Corp, Miami, FL) for the intensity of green fluorescence. The PMTs were gated for live cells using propidium iodide (2 µM), added just before acquisition. NAD(P)H redox status was also monitored concurrently with a UV-excited blue autofluorescence (excitation 351362 nm, emission 450 nm). Data from at least 5,000 cells were collected in list mode and processed with MPlus Software (Phoenix Flow Systems, San Diego, CA).
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RESULTS |
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To further determine the importance of intracellular ATP to subsequent cell signaling, TAMH cells were preincubated in glucose-free or high-glucose (4,500 mg/l) medium before subjecting to cytotoxic concentrations of TFEC (250 µM) for an additional 24 h. Our results show that cells survived significantly better in the presence of high-glucose medium (Fig. 5B), and this was associated with the maintenance of cellular ATP content (approx. 4-fold higher) by glucose supplementation (Fig. 5C).
Induction of ER Stress Response Genes
Activation of Nrf2 in the absence of oxidative stress suggests involvement of another signaling pathway known to occur at the level of the ER (Cullinan et al., 2003; Liu et al., 2005
). Our microarray analyses have indicated a strong link to ER stress with an early and pronounced upregulation of a number of ER-stress response genes (Table 3). This list includes four ER resident proteins (Gadd153, Gadd45, Gadd34, Atf3) as well as cytosolic proteins that have been well-established to be induced in response to ER stress (i.e., Ndr1). At least one of these genes was shown here to be upregulated at the protein level (Gadd153), while Gadd34 did not show any increase in protein level despite significant transcriptional changes (Fig. 6 and Table 3). Western blot analyses for both Hsp70i and Atf3 showed that Atf3 is strongly upregulated temporally from 4 to 8 h after the initiation of TFEC treatment, whereas Hsp70i levels remained high even at 20 h (Fig. 7).
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DISCUSSION |
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Nrf2 is an important stress-responsive transcription factor of the "cap-and-collar" ß-leucine zipper family, especially during oxidative stress (Nguyen et al., 2003). It regulates the expression of a number of Phase II enzymes (e.g., NQO1, GSTs) and antioxidant proteins (e.g., GCL, HO-1, thioredoxin). This process is driven by the association of Nrf2 to the ARE consensus sequence (5'-TGACnnnGCA-3') on the promoter region of these genes (Itoh et al., 2004
; Jaiswal, 2004
; Lee and Johnson, 2004
; Numazawa and Yoshida, 2004
). Recent reports continue to identify new downstream effector genes for Nrf2, including thioredoxin reductase and MafG (Katsuoka et al., 2005
; Sakurai et al., 2005
).
At present, the mechanisms of Nrf2 upstream activation are not completely understood. A few independent, yet not mutually exclusive, theories have emerged in this respect. Nrf2 has been shown to be constitutively expressed and localized in the cytosol and maintained in a repressed state by complexing with the actin-associating protein, Keap1. This heterodimerization confines most of Nrf2 to the cytoskeleton and away from the nucleus. Keap1 has a cysteine-rich surface which is subject to oxidation in cases of oxidative and nitrosative stress. This apparently results in global conformational changes to Keap1 thereby, leading to the liberation of Nrf2. The monomeric Nrf2 is then available to translocate to the nucleus. In this manner, Keap1 acts as a redox-sensor that upregulates ARE antioxidant responses through Nrf2 (Itoh et al., 2003, 2004
; Kang et al., 2004
; Levonen et al., 2004
; Zhang and Hannink, 2003
).
Nrf2 activation also has been shown to be mediated through phosphorylation of Nrf2 by mitogen-activated protein kinases (MAPKs), protein kinase C (atypical isoform), and phosphoinositol-3-kinase (PI3K) (Nakaso et al., 2003; Nguyen et al., 2003
; Numazawa et al., 2003
; Yu et al., 2000
). Further upstream kinases may also play a role in these events. In addition, it has been proposed that Nrf2 can be activated through a redox-independent pathway. This involves a prior ER stress that induces an ER-specific protein kinase, termed PKR-like endoplasmic reticular kinase (PERK), which can directly phosphorylate Nrf2 (Cullinan and Diehl, 2004
; Cullinan et al., 2003
).
From the studies reported here, TFEC can be seen to induce some Phase II enzymes and antioxidant-responsive genes in TAMH cells, and this is likely a consequence of early Nrf2 induction. This phenomenon has not been previously reported for TFEC or other halogenated aliphatics. To establish mechanistic links to the activation of Nrf2, we examined numerous indicators of cellular oxidative stress, but the overall lack of any significant changes in these parameters suggests that classical oxidative stress does not contribute significantly to TFEC-mediated cytotoxicity in TAMH cells. These findings are consistent with previously reported in vivo findings (Groves et al., 1991). While this does not rule out a role for Keap1 in the regulation of Nrf2 translocation, it does imply that redox changes related to GSH status or reactive oxygen species (ROS) production are not pivotal in the activation pathway. In future studies it will be of interest to examine the possibility of Keap1 alterations following transient, low-level oxidative stress using highly sensitive mass spectrometric techniques.
One of the earliest effects of TFEC observed was a rapid depletion of intracellular ATP. TFEC-mediated modification and inhibition of mitochondrial aconitase and KGDH activities (both important enzymes in the TCA cycle) might result in a localized intramitochondrial oxidative stress leading to the rapid inhibition of ATP productionan important determinant of the commitment to necrosis or apoptosis (Leist et al., 1997
, 1999
). Intracellular ATP concentrations are critical for cell viability, and marked ATP depletion (1525% of control) has been hypothesized to switch the cell death mechanism from apoptosis to necrosis (Lieberthal et al., 1998
). In fact, our previous studies with TFEC have shown that, despite mitochondrial changes supporting apoptosis (e.g., cytochrome c release), activation of pro-apoptotic caspases does not occur. Rather, energy-independent cysteine proteases like calpains replace caspases as the major enzymes catalyzing proteolysis (Ho et al., 2005
). The present work provides direct evidence for an immediate loss of ATP that is consistent with the decay of early apoptotic signals into a secondary necrosis. More importantly, we have also demonstrated that replenishment of ATP by glucose supplementation significantly restored cell viability. Even though depletion of ATP in glucose supplemented cells was still significant, it appears that a higher basal level was sufficient to "cushion" some of the damage produced by TFEC. These data confirm the importance of low intracellular ATP levels on critical downstream energy-dependent processes.
The lack of clear evidence for a generalized cellular oxidative stress, coupled to significant ATP depletion, implicates an alternative pathway for the activation of Nrf2 in this cell death model. One possibility is an ER stressmediated Nrf2 induction. An ER resident protein kinase, PERK, is known to directly phosphorylate Nrf2 and trigger dissociation from Keap1 without the involvement of ROS (Cullinan et al., 2003). PERK itself is strongly activated as part of the unfolded protein response (UPR) and has been shown to be critical for cell survival during ER stress (Cullinan and Diehl, 2004
). A recent study has also reported that ER stressstimulated HO-1 induction occurs through Nrf2 binding to ARE, consistent with our findings (Liu et al., 2005
). Furthermore, regulation of calcium homeostasis appears to be important during Nrf2 activation (Lee et al., 2003
), and recently, a selective calmodulin/CaMK inhibitor, KN93, was shown to block Nrf2 activation of ARE gene induction in HepG2 cells treated with diallyl trisulfide (Chen et al., 2004
).
The role of ER stress in TFEC-induced cytotoxicity is not well studied, but there are scattered reports which suggest that this might be a significant and under-appreciated phenomenon. For example, a role for calpains in renal cell death following TFEC treatment has been demonstrated (Schnellmann and Williams, 1998), in agreement with our previous work using TAMH cells (Ho et al., 2005
). Of particular significance, calpain activation was blocked by overexpression of anti-apoptotic BCL-xL (Ho et al., 2005
). This study also provides additional evidence for ER stress coupled with induction of specific ER stress proteins in TFEC-induced cytotoxicity. Particularly, Atf3 is recognized as an important stress-responsive ER-bound transcription factor that has been shown to induce Gadd153 (Wolfgang et al., 1997
). Since covalent binding precedes all other events, these findings suggest that ER stress occurs as a result of some prior mitochondrial dysfunction. Because a strong link has already been made between ATP depletion and calcium release from the ER (Harriman et al., 2002
), we have hypothesized that interruption of mitochondrial function causes a rapid depletion of intracellular ATP by TFEC, which leads to ER calcium release and an unfolded protein response (UPR), which is an energy-dependent process (Fig. 8). It is also possible that ATP depletion directly inhibits Ca-ATPase such that calcium is released from ER stores. Our ongoing efforts will include detecting activation of PERK, as well as other calcium-dependent signaling pathways, in Nrf2 phosphorylation and activation.
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ACKNOWLEDGMENTS |
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