Role of BNIP3 in TNF-induced cell death
— TNF upregulates BNIP3 expression
Saeid Ghavami
a,b,c, Mehdi Eshraghi
a, Kamran Kadkhoda
a, Mark M. Mutawe
b,c, Subbareddy Maddika
a,1,
Graham H. Bay
a, Sebastian Wesselborg
e, Andrew J. Halayko
b,c, Thomas Klonisch
d, Marek Los
f,g,⁎
a
Manitoba Institute of Cell Biology, and Department of Biochemistry and Medical Genetics, University of Manitoba, Canada
b
Department of Physiology, University of Manitoba, Canada
c
Manitoba Institute of Child's Health, University of Manitoba, Canada
d
Department of Human Anatomy and Cell Sciences, and Manitoba Institute of Child Health, Winnipeg, Canada
eDepartment of Internal Medicine I, University of Tübingen, Tübingen, Germany fBioApplications Enterprises, Winnipeg, Manitoba, Canada
g
Interfaculty Institute for Biochemistry, University of Tübingen, D-72076 Tübingen, Germany
a b s t r a c t
a r t i c l e i n f o
Article history:
Received 17 September 2008
Received in revised form 8 December 2008 Accepted 5 January 2009
Available online 15 January 2009 Keywords: Caspase Cathepsin Flow cytometry Lysosome ROS
Tumor necrosis factor alpha (TNF) is a cytokine that induces dependent (apoptotic) and caspase-independent (necrosis-like) cell death in different cells. We used the murinefibrosarcoma cell line model L929 and a stable L929 transfectant over-expressing a mutated dominant-negative form of BNIP3 lacking the C-terminal transmembrane (TM) domain (L929-ΔTM-BNIP3) to test if TNF-induced cell death involved pro-apoptotic Bcl2 protein BNIP3. Treatment of cells with TNF in the absence of actinomycin D caused a rapid fall in the mitochondrial membrane potential (ΔΨm) and a prompt increase in reactive oxygen species (ROS)
production, which was significantly less pronounced in L929-ΔTM-BNIP3. TNF did not cause the mitochondrial release of apoptosis inducing factor (AIF) and Endonuclease G (Endo-G) but provoked the release of cytochrome c, Smac/Diablo, and Omi/HtrA2 at similar levels in both L929 and in L929-ΔTM-BNIP3 cells. We observed TNF-associated increase in the expression of BNIP3 in L929 that was mediated by nitric oxide and significantly inhibited by nitric oxide synthase inhibitor N5
-(methylamidino)-L-ornithine acetate. In L929, lysosomal swelling and activation were markedly increased as compared to L929-ΔTM-BNIP3 and could be inhibited by treatment with inhibitors to vacuolar H+-ATPase and cathepsins−B/−L. Together, these data indicate that TNF-induced cell
death involves BNIP3, ROS production, and activation of the lysosomal death pathway.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Apoptosis and necrosis are two classical forms of cell death but a mixed death mode that shares elements of both characteristics may also occur. In multicellular organisms, apoptosis can be triggered by multiple events, including cancer therapeutics, and its purpose is to
eliminate damaged or undesirable cell[1–3]. By contrast, necrosis is a largely uncontrolled cell death and can result from exposure to extremely noxious stimuli [4]. Apoptosis and necrosis are distin-guished by morphological and biochemical criteria. Morphological characteristics of apoptosis include condensed, fragmented cell nuclei, a reduction in cell volume and cell membrane blebbing. Typical morphological characteristics of necrosis include pronounced swel-ling of the mitochondria and cell membrane, which leads to cell rupture. Biochemical characteristics unique to apoptosis include an increase in outer-mitochondrial membrane permeability, inter-nucleosomal fragmentation of chromosomal DNA, and exposure of phosphatidylserine on the outer surface of the cell[3,5].
Originally identified as a factor causing hemorrhagic necrosis in established tumors[6,7], the cytokine tumor necrosis factor alpha (TNF) has since been shown to play a crucial role in the pathogenesis of acute and chronic inflammatory diseases [8,9]. TNF can induce apoptotic (caspase-dependent) or necrotic (caspase-independent) cell death in vitro, depending on the cell type used[10–12]. Inflammatory responses include components of both TNF-induced apoptosis and necrosis but in the presence of the pan-caspase/apoptosis inhibitor and enhancer of necrosis, zVAD shows increased TNF toxicity in vivo Abbreviations: TNF, tumor necrosis factor alpha; AIF, apoptosis inducing factor;
Endo-G, Endonuclease G; tBid, truncated bid; BNIP3, Bcl2/E1B 19kD interacting protein; TM, trans-membrane; PTP, permeability transition pore; HIF-1α, hypoxia inducing factor-alpha-1; NO, nitric oxide; ROS, reactive oxygen species; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; PARP-1, poly(ADP-ribose)polymer-ase-1; DAF-2DA, di-aminofluorescein-2 diacetate; GAPDH, glyceraldehyde-3-phos-phate dehydrogenase; HSP60, heat shock protein 60; HDAC1, histone deacytalase 1; HtrA2, high-temperature requirement A2; Smac, second mitochondrial activator of caspases; DIABLO, direct inhibitor of apoptosis binding protein of low PI; LNMMA, N5
-(methylamidino)-L-ornithine acetate salt, JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide; DHR-123, dihydrorhodamine-123; ΔΨm,
mito-chondrial membrane potential change
⁎ Corresponding author. BioApplications Enterprises, Winnipeg, MB, R2V 2N6, Canada. Tel./fax: +1 204 334 5192.
E-mail address:mjelos@gmail.com(M. Los).
1
Current address: Department of Therapeutic Radiology, Yale School Of Medicine, USA. 0167-4889/$– see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2009.01.002
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Biochimica et Biophysica Acta
[13]. The different effects induced by TNF are mediated via two cell surface TNF receptors known as high affinity TNF-R1 (55 kD) and low affinity TNF-R2 (75 kD)[14]. TNF binding to its receptors activate a cascade of biochemical reactions including caspase activation, activa-tion of protein kinases[15], and the generation of ROS[16].
Upon binding to its high-affinity receptor, TNF-family members trigger a cascade of events, which lead to the recruitment of adaptor proteins and the activation of caspase-8. The binding of CD95 to its receptor activates caspase-8. Activated caspase-8 starts a mitochon-dria-independent caspase cascade directly relaying on the activation of caspase-3. Caspase-8 may also cleave and hyper-activate the pro-apoptotic Bid molecule. Truncated bid (tBid) translocates to mito-chondria and activates the mitomito-chondrial death pathway [17,18]. Thus, mitochondria may serve as a signal amplifier leading to the activation of caspase-9, -6, -8 and -3 [19]. BNIP3 (Bcl2/E1B 19kD interacting protein) over-expression induces an atypical, mixed caspase-independent form of cell death. BNIP3 was discovered in a yeast two-hybrid screen as the protein that interacts with adenovirus E1B 19K, which is homologous to Bcl2[20]. All Bcl2 family proteins possess at least one of four Bcl2 homology domains (BH1 to BH4) which determine the ability of these proteins to induce or inhibit apoptosis[21]. BNIP3 belongs to the BH3-only subfamily and has a C-terminal trans-membrane (TM) domain [22]. Over-expression of BNIP3 leads to the opening of the mitochondrial permeability transition pore (PTP), thereby abolishing the proton electrochemical gradient. This activates a chain of events culminating in chromatin condensation and DNA fragmentation[23,24]. Although chromatin condensation is an established marker of apoptosis, it has been proposed that BNIP3 induces a novel necrosis-like form of cell death. BNIP3-induced cell death is independent of caspases and the nuclear translocation of AIF, a mitochondrialflavoprotein. Furthermore, the release of cytochrome c from mitochondria is not involved in BNIP3-mediated cell death[24]. Thus, BNIP3-mediated cell death resembles TNF-induced cell demise that combines hallmarks of necrosis and apoptosis, in L929 cells[12].
Here we report that TNF-triggered cell death induced in the absence of actinomycin D (commonly used accelerator of TNF-induced cell death) is partially inhibited by the dominant negative mutant of BNIP3, L929-ΔTM-BNIP3, which lacks the C-terminal BNIP3 domain essential for insertion of BNIP3 into the mitochondrial membrane and execution of apoptotic function. TNF induced both a HIF-1α independent but NO-dependent increase of BNIP3 expression, and transfer of this Bcl2-family member from the nucleus to mitochondria. TNF-triggered cell death involved ROS-production and the activation of the lysosomal pathway. The protective effect ofΔTM-BNIP3 was associated with decreased mitochondrial ROS production.
2. Materials and methods 2.1. Reagents
Cell culture media were purchased from Sigma (Oakville, ON, Canada) or Gibco (Canada). Phosphate buffered saline (PBS; pH = 7.4), N5-(methylamidino)-L-ornithine acetate salt (L-NMMA), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and murine anti-human BNIP3 were from Sigma. Cell culture plastics ware obtained from Nunc Co. (Canada). Anti-human/murine/rabbit BNIP3, rabbit anti-human/murine/rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), murine anti-human/murine/rat tubulin, rabbit anti-human/murine/rat Omi/HtrA2, murine anti-human/ murine/rat cytochrome c, rabbit anti-human/murine/rat HIF-1α, and goat anti-human/murine/rat Endo-G were obtained from Santa Cruz Biotechnologies (USA). 5,5 ′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1), and DAF-2DA were obtained from Invitrogen Molecular Probes (Canada). Murine anti-human/murine cleaved caspase-3 (Asp175), was obtained from R&D,
Canada. Rabbit anti-human/murine cleaved PARP-1 were purchased from Cell Signaling, Canada. Caspase-Glo®-8, and -9 assay systems were obtained from Promega, Canada.
2.2. Cell culture
Murinefibroblast cell lines L929 and stable transfectant of L929 with a dominant negative mutant of BNIP3, L929-ΔTM-BNIP3, were cultured in DMEM supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100μg/ml streptomycin. Cells were incubated at 37 °C in a humidified CO2 incubator with 5% CO2 and maintained under logarithmic growth conditions.
2.3. Development of L929-ΔTM-BNIP3 cells
L929 cells were grown in 100 mm dishes. After reaching 80% confluence, cells were collected (106 cells) and transfected with pCDNA3-BNIP3-ΔTM (5 μg) using electroporation. After 48 h trans-fected cells were treated with selection media containing Geneticin (G418) (1000 μg/ml) and kept in selection media for 3 months to produce cells stably over-expressΔTM-BNIP3 protein. The expression ofΔTM-BNIP3 protein was controlled by Western blot.
2.4. MTT cytotoxicity assay
Colorimetric MTT assay was employed to evaluate the cytotoxicity effect of TNF on L929 and L929-ΔTM-BNIP3 cell lines[25]. Briefly, cells (1.5 × 104 cells/ml) were grown for 24 h in 96-well culture plates containing 200μl of culture medium. Defined concentrations of TNF were added and incubated for different time intervals, as shown in the figures, followed by the incubation with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide. The percentage of cell viability was calculated using the equation: [mean optical density (OD) of treated cells / mean OD of control cells] × 100.
2.5. Measurement of apoptosis byflow cytometry
Apoptosis was measured using the Nicoletti method [26,27]. Briefly, cells grown in 12 well plates were treated with S100A8/A9 (100μg/ml) for the indicated time periods. After scrapping, cells were pelleted by centrifugation at 800 g for 5 min, washed once with PBS, and resuspended in hypotonic propidium iodide (PI) lysis buffer (1% sodium citrate, 0.1% Triton X-100, 0.5 mg/ml RNase A, 40 μg/ml propidium iodide). Cell nuclei were incubated for 30 min at 30 °C and the nuclei were subsequently analyzed byflow cytometry. Nuclei to the left of the G1 peak containing hypo-diploid DNA were considered to be apoptotic.
2.6. ROS measurement usingflow cytometry
L929 and L929-ΔTM-BNIP3 cells were treated with TNF with indicated concentrations for different time points in 6 well plates. Following treatment, cells were incubated in 1 μM dihydro-rhodamine-123 (DHR-123; 1μM) in DMSO (final concentration 0.1% v/v). Incubation with DHR-123 was for 30 min at 37 °C. To prevent light accelerated oxidation, samples were maintained in the dark prior to and during analysis. Fluorescence within dye-loaded cells was analyzed byflow cytometry (FACS Calibur; Becton Dickinson, Mississauga, ON, Canada. Forflow cytometry, the excitation wave-length for rhodamine-123fluorescence was 488 nm and emission at 530 nm (FL-1). Signals were processed using a logarithmic amplifier andfluorescence distributions were plotted on a 4-decade logarith-mic scale. 10,000 events were counted and, as a semi-quantitative indicator for ROS levels, the geo-mean and mean-linearfluorescence values were calculated using Cell Quest Software (Becton Dickinson, Mississauga, ON, Canada).
2.7. Measurement of mitochondrial membrane potential
To measure theΨmof cells, thefluorescent probe JC1 (5,5,6,6 -tetrachloro-1,1,3,3 -tetraethylbenzimidazole carbocyanide iodide) was used. JC-1 exists as a monomer at low values of Ψm (green fluorescence), while it forms aggregates at high Ψm(redfluorescence). Thus, mitochondria with normalΨmconcentrate JC-1 into aggregates (redfluorescence), but with de-energized or depolarized Ψm, JC-1 forms monomers (greenfluorescence). Briefly, L929 and L929-ΔTM-BNIP3 (5 × 105cells/well were treated with TNF (0.2, 0.8 and 1 ng/ml) for 8 and 12 h, washed in PBS (pH = 7.4), and incubated for 15 min at 37 °C with 2.5μg/ml JC-1. Cells were pelleted at 400 g for 5 min in room temperature, washed in PBS, and analyzed byflow cytometry. The analyzer threshold was adjusted on the forward light scatter channel to exclude most of the sub-cellular debris. When excited simultaneously by a 488 nm argon-ion laser source, JC-1 monomers and JC-1-aggregates were separated into theflow cytometer FL1 and FL2 channels, respectively. Meanfluorescence intensity values for FL1 and FL2 expressed as relative linear fluorescence channels were obtained for at least 15,000 events in all experiments. The decrease in Ψm was determined employing a 3-D plot with FL2, FL1, and cell counts depicted on the x/y/z axis, respectively[28].
2.8. Cell fractionation
Cytosolic, mitochondrialand and nuclear fractions were generated using a digitonin-based subcellular fractionation technique as described previously [29,30]. Briefly, 107 cells were harvested by centrifugation at 800 g, washed in PBS pH 7.2, and re-pelletted. Cells were digitonin-permeabilized for 5 min on ice at a density of 3 × 107/ ml in cytosolic extraction buffer (250 mM sucrose, 70 mM KCl, 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4pH 7.2, 100μM PMSF,10 μg/ml leupeptin, 2μg/ml aprotinin, containing 200 μg/ml digitonin). Plasma membrane permeabilization of cells was confirmed by staining in a 0.2% trypan blue solution. Cells were then centrifuged at 1000 g for 5 min at 4 °C. The supernatants (cytosolic and mitochondria fractions) were saved and the pellets solubilized in the same volume of nuclear lysis buffer, followed by pelletting at 12,500 g for 10 min at 4 °C. The mitochondria was separated from cytosolic fraction pelletting 13,000 g and the pellets solubilized in equal amount of mitochondrial lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X-100, 0.3% NP-40, 100μM PMSF, 10 μg/ml leupeptin, 2 μg/ ml aprotinin). For the detection of proteins, equal amount of fractions protein were supplemented with 5 × SDS-PAGE loading buffer, subjected to standard 12% SDS-PAGE and transferred to nitrocellulose membranes.
2.9. Immunoblotting
Upon treatment of cells with TNF (1 ng/ml) for indicated time points, BNIP3, cytochrome c, and caspase-3 were detected by immunoblotting in total cell lysates and fractionated cell compart-ments, respectively. Briefly, harvested cells were washed once with cold PBS and cells were re-suspended for 20 min on ice in lysis buffer: 20 mM Tris–HCl (pH 7.5), 0.5% Nonidet P-40, 0.5 mM PMSF, and 0.5% protease inhibitor cocktail (Sigma). Then, experimental procedures were followed exactly as described previously[28].
2.10. Quantitative PCR measurement of BNIP3 mRNA
Cells were treated with TNF (1 ng/ml), TNF solvent (PBS) and medium (control). Total cellular RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. 1μg of total RNA was reverse transcribed by using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's recommendations. mRNA expression of murine BNIP3 in L929 cells
was quantified on a Applied Biosystems 7500 Real-Time PCR System instrument using Power SYBR Green PCR Master Mix (Applied Biosystems)[31]. A dissociation curve was generated at the end of each PCR cycle to verify that a single product was amplified. The relative expression levels of the BNIP3 gene was calculated as fold difference = 2(− Ct), where Ct is threshold cycle normalized to 18S internal control. Oligonucleotide primers were as follows for BNIP3, 5 ′-GCTCCCAGACACCACAAGAT-3′ (forward) and 5′-TGAGAGTAGCTG-TGCGCTTC-3′ (reverse), and for 18S: 5′-CGCCGCTAGAGGTGAAATTC-3′ (forward) and 5′-TTGGCAAATGCTTTCGCTC-3′ (reverse).
2.11. Nitric oxide measurement using DAF-2DA
Recently, a newfluorometric method based on di-aminofluorescein-2 diacetate (DAF-di-aminofluorescein-2 DA) has been developed for the direct detection of NO production. The DAF-2DA penetrates the cell membrane and is then hydrolysed by cytosolic esterases, producing DAF which traps NO to produce the green-fluorescence signal fluorescein. The high sensitivity of DAF and its broad concentration response range make this system a highly sensitive tool to detect even low amounts of NO produced by the constitutive enzymes[32]. Briefly, DAF-2DA stock solution was diluted (2.5μM) in pre-warmed HBSS buffer (Hank's Balanced Salt solution with 20 mM Hepes buffer with Ca2+and Mg2+(pH = 7.4) and added to the cells treated with TNF (0–24 h, 1 ng/ml) and control cells (treated with medium and TNF solvent, PBS). The cells were incubated with DAF-2DA for 40 min and then washed with HBSS one time to remove excess probe and incubated in HBSS for more 30 min to allow complete de-esterification of the intracellular diacetates. Fluorescence excitation and emission were calculated in 495 and 515 nm, respectively. 2.12. Immunocytochemistry and confocal imaging
Cells were grown overnight on coverslips and then treated with TNF for 12 h. Cells were washed in PBS,fixed in 4% paraformaldehyde in PBS at room temperature, and permeabilized with 0.25% Triton X-100 in PBS. To detect BNIP3, AIF, Endo-G, Omi/HtrA2, and Smac/Diablo translocation, cells were incubated with anti-BNIP3 murine IgG (Sigma; diluted 1:200) anti-AIF murine IgG (Santa Cruz Biotechnol-ogy; diluted 1:500), anti-Endo-G rabbit IgG (Biovision, 1:50 dilution), anti-Omi/HtrA2 rabbit IgG, and anti-Smac/Diablo rabbit IgG (Santa Cruz Biotechnology; both at 1:150). Slides were washed three times with PBS and incubated with Cy5-conjugated secondary antibody (1:1000; BNIP3, AIF, and Endo-G) or FITC-conjugated secondary antibody (1:50; BNIP3, Omi/HtrA2 and Smac/Diablo). To visualize nuclei, cells were stained with DAPI (10μg/ml). The mitochondria were stained with Mitotracker Red CMXRos (Molecular Probes; 200 nM) in DMEM culture medium for 15 min prior tofixation. The cell-permeant MitoTracker probes contained a mildly thiol-reactive chloromethyl moiety that appeared to be responsible for keeping the dye associated with the mitochondria after fixation. To label mitochondria, cells were incubated with sub-micromolar concentra-tions of a MitoTracker probe, which passively diffused across the plasma membrane and accumulated in active mitochondria. Once the mitochondria were labeled, cells were treated with an aldehyde-based fixative to allow further processing of the sample. Because most of the MitoTracker probes were retained after permeabilization with detergents or organic solvents, the sample continued to exhibit the fluorescent staining pattern characteristic of live cells during subsequent processing steps (e.g., immunocytochemistry, in situ hybridization, or electron microscopy)[26]. Fluorescent images were analyzed using an Olympus-IX81 multi-laser confocal microscope. 2.13. Caspase activity assays
Luminometric assays Caspase-Glo®-8, -9 (Promega, Canada, Nepean, ON) were used to measure the proteolytic activity of Caspase-8
(IETD-ase) and -9 (LEHD-(IETD-ase). The assays were performed according to manufactures instruction. Briefly, cells cultured in 96-well plate (15000 cells/well), were treated with indicated concentrations of TNF for different time points. The freshly prepared caspase reagents (100μl per well) contained either z-LETD-Luciferin alone or z-LEHD-Luciferin and whole protein cell lysate extract buffer. For each experiment, negative control cells (treated cells in medium without caspase reagent and reagent blank containing medium only) were included. The measure-ments were then performed exactly as described previously[28]. 2.14. Statistical analysis
The results were expressed as means ± SD and statistical differ-ences were evaluated by one-way and two-way ANOVA followed by Tukey's post-hoc test, using the software package SPSS 11.05 and Graph Pad Prism 4.0, Pb0.05 was considered significant.
3. Results
3.1. TNF induced cell death in L929 cells
L929 cells were treated with different concentrations of TNF in different time points and TNF cytotoxic effect was measured using MTT assay. In each concentration/time point cell viability was accessed comparing to corresponding control (cells treated with TNF solvent, PBS). To avoid experimental artifacts, all experiments were performed without the transcriptional inhibitor actinomycin D. Our results showed that TNF induced significant cell death in all concentration/ time point in L929 cells (Pb0.001) (Fig. 1A). To confirm that the observed form of cell death was apoptosis, the experiments were independently repeated using an apoptosis-specific flow cytometric method (Nicoletti), which detects apoptosis-typical hypodiploid nuclei. TNF-treated cells showed apoptotic cell death (Fig. 1B). It was previously shown that TNF-induced apoptosis caused PARP-1 cleavage
[12]. In our experimental model we also show that TNF induced PARP-1 cleavage in L929 cells, thus, providing additional confirmation of apoptosis induction by TNF (Fig. 1C).
3.2. TNF induced time and concentration-dependent BNIP3 expression in L929 cells
We investigated the effect of TNF treatment on the regulation of BNIP3 expression. Time-kinetics experiments indicate that BNIP3 protein expression (Fig. 2A, B) and BNIP3-mRNA content as assayed by quantitative PCR (Fig. 2C), were increased in L929 cells treated with TNF. BNIP3 protein expression also showed dependence on TNF concentration. Thus, higher concentrations of TNF induced higher expression of BNIP3 (Fig. 2D). Therefore for the further experiments we have been mostly using the higher (1 ng/ml) TNF concentration. 3.3. Dominant-negative mutant of BNIP3 partially inhibited TNF toxicity and TNF-induced BNIP3 mitochondrial translocation without affecting cytochrome c release and caspase-9 activation
To study the role of BNIP3 in TNF cytotoxicity, we compared TNF-triggered changes between L929 and a stable transfectants with the dominant-negative mutant of BNIP3 that lacks the trans-membrane domain, which is critical for its association with mitochondria (L929-ΔTM-BNIP3). Due to the strong toxicity of BNIP3 even in transient transfection experiments, we were unable to provide data on cells even transiently overexpressing BNIP3. MTT assay revealed that L929-ΔTM-BNIP3 cells were significantly more resistant towards TNF as compared to the parental L929 cell line (Pb0.01) (Fig. 3A). To avoid experimental artifacts, all experiments were performed without the transcriptional inhibitor actinomycin D. During the initiation of cell death BNIP3 can associate with mitochondria and interact with Bcl2
Fig. 1. TNF induced cell death in L929 cells. (A) L929 cells were treated with different concentrations of TNF for 0–24 h and cell viability was assessed by MTT assay. For each TNF concentration the treated cells were compared with control cells which had been treated with the TNF solvent PBS. Results are expressed as percentage of corresponding control and represent the means ± SD of three independent experiments. TNF significantly induced cell death in L929 cells in different concentration and time points (Pb0.001). (B) DNA histogram of L929 cells treated with TNF. The Nicoletti method was used for sample processing, typical DNA-histogram s are shown. M2 (statistical marker) has been placed to mark sub-diploid DNA. TNF- treatment (24 h, 1 ng/ml) induced apoptosis only a fraction of cells. G1 and G2 peaks are clearly visualized and a sub-diploid peak corresponding to apoptotic cells was clearly visible to the left from both peaks that represent normal cells. (C) L929 cells were treated with TNF (1 ng/ml) for indicated times, then total cell extracts were harvested, resolved by SDS-PAGE, and cleaved PARP-1 was detected by Western blot. Protein loading was controlled with GAPDH, (parallel experiment performed with the same protein extracts).
and Bcl-XL [23,33,34]. The predicted molecular weight of BNIP3 is 21.5 kD. However, in SDS-PAGE it migrates as a monomer of 30 kD and a homodimer of∼60 kD. In some models, homodimerization appears to be a feature of mitochondrial localization [24] and TM-domain mutants of BNIP3 including BNIP3Δ179–185 and point mutations at L179 and G180 fail to homodimerize, as does a C-terminal deletion mutant (BNIP3Δ184–194)[34]. Furthermore, exclusive mitochondrial localization for BNIP3 was only shown for some tissues (i.e. muscle), but not for others (i.e. the brain)[24]. Interestingly, the BH3 domain of BNIP3 does not appear to be essential for BNIP3-dependent cell death induction[34]. We have investigated the intracellular BNIP3 distribu-tion upon TNF treatment in L929 and L929-ΔTM-BNIP3 cells. In untreated L929 cells, BNIP3 was mainly localized in nuclei (Fig. 3B), whereas in L929-ΔTM-BNIP3 cells negligible amounts of nuclear BNIP3 were observed (Fig. 3B). In both L929 and L929-ΔTM-BNIP3 cells, TNF-treatment resulted in an increased presence of BNIP3 in the mitochondrial fraction, while BNIP3 content decreased in the nuclear fraction in L929 (Fig. 3B). The data obtained by cell fractionation were confirmed by immunohistochemistry followed by confocal micro-scopy (Fig. 3C, D).
TNF-induced apoptosis and necrosis both involve mitochondrial participation. Interaction of TNF with its TNF receptor-1 ultimately leads to the activation of caspase-8 as well as activation of the mitochondrial, caspase-9 dependent death pathway. We investigated caspase activation upon TNF treatment in L929 and L929-ΔTM-BNIP3 cells. TNF-triggered mitochondrial cytochrome c release, and the activation of caspase-9. The release of cytochrome c from mitochon-dria and the extent of caspase-9 were similar in L929 and L929- ΔTM-BNIP3 (Fig. 3E, F). This indicated that the TNF-mediated activation of the mitochondrial death pathway did not require the presence of fully-functional BNIP3. Furthermore, similar levels of caspase-3 activation were detected in L929 and L929-ΔTM-BNIP3 cells (Fig. 3G), again indicating that the caspase cascade was not affected by BNIP3 mutation.
3.4. TNF-mediated increase in BNIP3 expression involves NO-synthase We investigated the effect of TNF treatment on the regulation of BNIP3 expression. BNIP3 protein expression (Fig. 2A, B) and mRNA (Fig. 2C), was increased in L929 cells treated with TNF. As HIF-1α is one of the transcription factors regulating BNIP3 expression[24,34,35], we investigated if HIF-1α played role in TNF-induced increase of BNIP3 expression. TNF failed to induce HIF-1α expression, thus excluding HIF-1α transcriptional activity as a cause of the TNF-mediated BNIP3 upregulation (data not shown). It was shown recently that BNIP3 expression could be affected by nitric oxide[36,37]. Endogenously produced or exogenously added NO activates the Bnip3 promoter and induces expression of BNIP3 protein in RAW264.7 macrophages under normoxic conditions [36]. TNF significantly induces NO and NO2− production in L929 cells (Pb0.01) (Fig. 4A). These cells express both constitutive and a TNF-inducible nitric oxide synthase (NO-synthase)
[38,39]. Treatment with the competitive nitric oxide synthase (NOS) inhibitor (L-NMMA) prior to TNF stimulation significantly inhibited TNF-induced cell death and upregulation of 30 kD BNIP3-expression and cell death in L929 cells (Fig. 4B, C). In the presence of L-NMMA, there was a significant increase in 60 kD BNIP3 but 30 kD BNIP3 significantly decreased (Pb0.05) (Fig. 4C, D). The NOS inhibitor attenuated the overall increase of BNIP3 content in TNF-treated cells and shifted the equilibrium towards the 60 kD BNIP3 form. We have also investigated if L-NMMA could prevent BNIP3 translocation to mitochondria. Cells were pre and then co-treated with L-NMMA and TNF (1 ng/ml). L-NMMA could not prevent BNIP3 translocation to mitochondria (data was not show).
3.5. Dominant-negative BNIP3 mutant protects from TNF-triggered mitochondrial depolarization and ROS generation
The change in Ψm is a common cellular event during TNF-induced cell death[40–42]. TNF treatment induced changes inΨm Fig. 2. TNF induced time and concentration dependent BNIP3 expression in L929 cells. (A) Western blot detection of BNIP3 expression in L929 cells treated with 1 ng/ml TNF for 0, 8, 16 and 24 h. Tubulin was included as loading control. 30 and 60 kD signals of BNIP3 were significantly increased after 8 and 16 h. In control samples “C”, the cells were treated with the TNF solvent PBS. The Western blot signals are representative of three independent experiments. (B) Quantification of BNIP3 expression in TNF treated. Western blots of experiments performed as described in (A) were quantified as indicated in the method section (C) TNF induced an increase in BNIP3 mRNA expression. L929 cells were treated with TNF for indicated times and BNIP3 mRNA was measured using qPCR. TNF induced a significant increase in BNIP3 mRNA (Pb0.05). The data represent triplicates of three independent experiments. (D) Western blot of BNIP3 expression in L929 cells treated with 0.75 and 1 ng/ml TNF for 12 h. BNIP3 expression was TNF-concentration dependent. Tubulin was included as loading control. The Western blot data is representative for of three independent experiments.
Fig. 3. The expression of BNIP3 dominant negative mutantΔTM-BNIP3 partially protects L929 against TNF-induced cell death via prevention of BNIP3 translocation to mitochondria, without significantly affecting in cytochrome c release and caspase-9, and -3 activation. (A) Effect of TNF on L929 and L929-ΔTM-BNIP3 cells. Cells were treated with different concentrations of TNF for 0–24 h and cell viability was assessed by MTT assay. For each TNF concentration the treated cells were compared with control cells which had been treated with the TNF solvent PBS. Results are expressed as percentage of corresponding control and represent the means ± SD of three independent experiments. There is significant difference between parental L929 and L929-ΔTM-BNIP3 cell in TNF cytotoxicity (Pb0.01). (B) TNF induced mitochondrial translocation of BNIP3. BNIP3 presence in mitochondrial, and nuclear fractions of L929 and L929-ΔTM-BNIP3 cells treated with TNF (1 ng/ml). BNIP3 signals were detected by Western blot. BNIP3 was mainly located in the nucleus in L929 cells and translocated to mitochondria when cells were treated with TNF. In L929-ΔTM-BNIP3 cells, TNF induced less pronounced BNIP3 translocation to mitochondria. Protein loading was controlled with HDAC1 (nuclear fraction), and HSP60 (mitochondrial fraction). The data is representative for three independent experiments. (C, D) Intracellular localization of BNIP3 in (C) L929 and (D) L929-ΔTM-BNIP3 cells was determined by confocal microscopy. Cells were either left untreated or treated with TNF (1 ng/ml) for 12 h. Cells were immunostained with an anti-BNIP3 antibody followed by a FITC conjugated secondary antibody (green), and DAPI (nuclear DNA counterstain, blue). TNF-treated cells showed mitochondrial BNIP3 translocation which was less pronounced in L929-ΔTM-BNIP3 cells. The anti-BNIP3 antibodies used were unable to differentiate between wild type and mutated BNIP3. The magnification was 1000×. Representative micrographs are shown for all samples. (E) Cytochrome c detection by Western blot in mitochondrial and cytosolic fractions of L929 and L929-ΔTM-BNIP3 cells treated with TNF (1 ng/ml) for indicated time. Protein loading was controlled with GAPDH (cytosolic fraction) and HSP60 (mitochondrial fraction). The data represent representative blots of three independent experiments. (F) Caspase-9 activity in L929 and L929-ΔTM-BNIP3 cells treated with TNF (1 ng/ml) was measured by a Caspase-Glo®
luminometric assay. The caspase activity is presented as“fold increase” in comparison to the control of each time point. The data represent triplicates of three independent experiments. There was no significant difference in caspase-9 activity between the two cell lines (PN0.05). (G) L929 and L929-ΔTM-BNIP3 cells were treated with TNF (1 ng/ml) for indicated time periods, then total cell extracts were harvested, resolved by SDS-PAGE, and active subunits of caspase-3 were detected by Western blot. Protein loading was controlled with GAPDH, (parallel experiment performed with the same protein extracts). GAPDH represented for L929 cells. The data represent triplicates of three independent experiments.
in both wild type L929 and ΔTM-BNIP3-over-expressing cells (Fig. 5A). These changes were dose and time dependent (data not shown) and revealed L929 that exhibited higher changes inΨmthan L929-ΔTM-BNIP3 transfectants (Fig. 5A). Employing the ROS-sensitive fluorogenic marker DHR-123, we investigated ROS generation by flow cytometry in untreated and treated (TNF at 1 ng/ml) L929 and L929-ΔTM-BNIP3 cells. When compared with wild type L929 cells, L929-ΔTM-BNIP3 transfectants produced significantly less ROS (Fig. 5B, C) (Pb0.05). N-acetycysteine (NAC) (a clinically-applied anti-oxidant) interferes with increased ROS generation and TNF-induced death in L929 cells[25]. In our experimental system, NAC pre- and co-treatment partially prevented TNF-induced cell death in L929 cells (Fig. 5D).
3.6. TNF treatment does not induce EndoG and AIF release from mitochondria in L929 cells
AIF is a phylogenetically conserved flavoprotein essential for apoptosis during embryonic development [43]. It is a redox-active NADH-oxidase and, upon apoptosis induction, translocates from mitochondria to the nucleus to induce chromatin condensation[44].
Endo-G is a mitochondrial nuclease encoded by a nuclear gene. Once released from mitochondria into the cytosol, Endo-G trans-locates to the nucleus where it causes oligo-nucleosomal DNA fragmentation even in the presence of caspase inhibitors [45]. In mammalian cells, Endo-G cooperates with exonuclease and DNase-I to facilitate DNA processing[46]. We did not observe the release of these molecules from mitochondria upon TNF treatment (Fig. 6A, B). Thus, AIF and Endo-G do not appear to be components of TNF-triggered cell death pathway in L929 cells in our experimental system. Similar data has also been observed for L929 ΔTM-BNIP3 (not shown).
3.7. TNF-triggered mitochondrial release of Smac/Diablo and Omi/HtrA2 is unaffected byΔTM-BNIP3
To further examine the effect of TNF on mitochondria, we monitored the TNF-triggered release of Smac/Diablo and Omi/HtrA2 from mitochondria. Murine Smac and its human ortholog Diablo are proteolytically processed in mitochondria and released from the intermembrane mitochondrial space upon triggering apoptosis[47]. The mammalian serine protease Omi, also known as high-Fig. 3(continued).
temperature requirement A2 (HtrA2), is released from the mito-chondria to the cytosol during apoptosis where it contributes both to caspase-dependent and caspase-independent apoptosis [48]. Both Smac/Diablo and Omi/HtrA2 bind and antagonize the actions of inhibitors of apoptosis (IAPs)[49]. L929 cells were treated with TNF (1 ng/ml, 12 h) and the subcellular location of Smac/Diablo and Omi/HtrA2 was examined by confocal imaging (Fig. 6C, D). Immunoreactive Smac/Diablo and Omi/HtrA2 were present in mitochondria of cells treated with medium alone (Fig. 6C, D, control panel). Upon TNF-treatment, these proteins were released from the mitochondria to the cytosol (Fig. 6C, D) indicating that these proteins are involved in TNF-induced cell death. Over-expression of ΔTM-BNIP3 did not significantly inhibit the TNF-mediated release of Smac/Diablo and Omi/HtrA2 (Fig. 6C, D). This might be explained by the TNF-induced increase in wild-type BNIP3 expression, which may compensate for the presence of dominant negative mutant ΔTM-BNIP3.
3.8. Dominant-negative BNIP3 mutant counteracts the activation of lysosomal death pathway by TNF
Lysosomes are important organelles for the control of caspase-independent cell death. In the event of cell death, lysosomal acid hydrolases of the cathepsin (cath) family frequently translocate from lysosomes into the cytosol. In some experimental systems cath-B and cath-D are rate limiting for death induced by interferon-γ, TNF, p53, or pro-oxidants[50]. The vacuolar H+-ATPase inhibitor bafilomycin A1 (Baf A1; 0.05μM), the irreversible inhibitor of Cath-B and cath-L zFF-fmk (100μM), and the cath-B inhibitor CA-074-Me (10 μM), partially inhibited TNF-induced cell death in L929 and L929-ΔTM-BNIP3 cells (Fig. 7A–C) (Pb0.05). Staining of cells with the acidophilic lysosomal probe LTR revealed that TNF caused an increase in lysosomal volume in L929 cells (Fig. 7D), which was absent in L929-ΔTM-BNIP3 cells (Fig. 7E). Pre-treatment with bafilomycin A1, zFF-fmk, CA-074-Me, or antioxidant N-acetyl-L-cysteine (5 mM) abolished the differences in Fig. 4. TNF induces generation of reactive nitrogen species that are involved in TNF cytotoxicity and BNIP3 expression in L929 cells. (A) L929 cells were treated with TNF (1 ng/ml) for indicated time and NO production was measured using DAF-2A. TNF induced a significant increase in NO production (Pb0.05). The data represent triplicates of three independent experiments. (B) NO-synthase inhibitor interfered with TNF-induced cell death. Pre-treatment of L929 cells for 1 h with L-NMMA (500, 2500, 5000 nM) reduced TNF cytotoxicity (TNF: 1 ng/ml for 24 h) as measured by MTT assay. Data represent values from quadruplicates of four independent experiments (Pb0.05). Cells treated with TNF solvent PBS served as control. (C) Western blot analysis of BNIP3 expression in L929 cells upon pre-treatment with L-NMMA (500 nM for 1 h), with treatment with 1 ng/ml TNF for indicated time (right panel). Left panel, that represents the treatment with TNF alone served as a control. GAPDH was included as loading control. In each sets of data, cells treated with TNF solvent PBS and L-NMMA (500 nM and pre-treated for 1 h) served as an internal control“C”. (D) Quantification of BNIP3 expression in TNF treated cells pretreated with L-NMMA. NO synthase inhibitor shifted the 30 kD/60 kD BNIP3 ratio toward the 60 kD BNIP3 form, whereas total BNIP3 expression remained unchanged. Data were adjusted to the“C” control signal and represent triplicates of three independent experiments.
Fig. 5. The over-expression of dominant-negative form of BNIP3 decreased TNF-induced ROS production andΔψm. (A)ΔTM-BNIP3 over-expression partly protected from
TNF-triggeredΔψmas shown by cytofluorimetric analysis of ψmin L929 (upper panel) and a derivative that over-expressesΔTM-BNIP3 (lower panel). Cells were treated for 12 h with
medium alone (left diagrams), or with TNF (1 ng/ml). TNF treatment triggered marked changes in mitochondrial membrane potential that were partially abolished inΔTM-BNIP3 over-expressing cells. A representative experiment of four is shown. (B, C) In L929 (B) TNF (1 ng/ml) triggered marked ROS production, whereas in (C) L929-ΔTM-BNIP3 cells TNF treatment elicited only minimal (∼4–15%) induction of ROS (Pb0.05). Cells were treated with TNF for the indicated time periods and ROS was measured using dihydrorhodamine-123. Experiments were repeated four times and mean ROS values are shown. (D) Broad-spectrum anti-oxidant, NAC, partially inhibited TNF induced cell death. L929 cells were pre-treated with indicated concentrations of NAC for 3 h and than co-pre-treated with TNF (1 ng/ml) and indicated concentration of NAC for 24 h. NAC partially inhibited TNF induced cell death (Pb0.05). The data represents triplicates of three independent experiments.
lysosomal volume between L929 and L929-ΔTM-BNIP3 cells in response to TNF treatment (data not shown). Thus, the higher levels of ROS observed in L929 as compared to L929-ΔTM-BNIP3 cells appeared to contribute to lysosomal damage in TNF-treated L929 cells and this may involve the release of cathepsins into the cytoplasm
[51,52]. 4. Discussion
In the present study, we have investigated the role of the pro-apoptotic Bcl2-family member BNIP3 in TNF-induced cell death. We employed the rodent fibrosarcoma cell line L929, which has been widely used to study the mechanisms of TNF cytotoxicity. TNF induces cell death in L929, which may resemble necrosis or apoptosis[50,53]. BNIP3 is a pro-death member of the Bcl2-family of apoptotic proteins
[54]. Its mechanism of action is not fully understood. It is accepted that, at least in part, it exerts its activity by inserting into the mitochondrial membrane. This leads to the opening of the mitochon-drial permeability pore with resultant membrane depolarization and
subsequent ROS generation[35]. This rapid and profound mitochon-drial dysfunction does not involve the release of mitochonmitochon-drial cytochrome c, caspase activation, or the nuclear translocation of AIF
[24]. The resulting cell death is accompanied by an increase in plasma membrane permeability[34]. To address the role of BNIP3 in TNF-induced cell death, we employed wild-type BNIP3 and a deletion mutant of BNIP3 which lacks the transmembrane domain ( ΔTM-BNIP3) known to be important in BNIP3-induced cell death[55]. TNF-stimulated cell death in L929 was BNIP3-dependent and the presence ofΔTM-BNIP3 resulted in significantly higher TNF-resistance. BNIP3-mediated cell death is independent of caspase activation and the release of cytochrome c from mitochondria[24]. Likewise, caspase-3 and -9 activity and cytochrome c release were similar in the presence of wild type BNIP3 and ΔTM-BNIP3 in L929. Thus, TNF triggered caspase-independent cell death, which was partially inhibited by ΔTM-BNIP3. Some Bcl2-like proteins contain a single C-terminal TM helix to facilitate their membrane targeting[56]. Upon induction of cell death, BNIP3 integrates into the mitochondrial outer membrane with the N-terminus (residues 1–162) pointing into the cytoplasm and
Fig. 6. TNF did not affect the mitochondrial release of caspase-independent apoptosis inducing proteins (AIF, Endo-G) andΔTM-BNIP3 over-expression did not affect the mitochondrial release of caspase-dependent apoptosis inducing proteins (Smac/DIABLO, Omi/HtrA2). (A, B) TNF-treatment (in the absence of actinomycin D) did not trigger the release of pro-apoptotic mitochondrial proteins AIF and Endo-G from L929. Cellular localization of AIF (A) and Endo-G (B) were determined by confocal microscopy. L929 cells were treated with TNF (1 ng/ml) for 12 h prior to immunostaining with anti-AIF and anti-Endo-G antibodies followed by detection with Cy-5-conjugated secondary antibody (magenta). Cells were counterstained with DAPI (blue) and mitotracker (red) to visualize nuclei and mitochondria. (C, D) TNF-treatment (in the absence of actinomycin D) triggered the release of pro-apoptotic mitochondrial proteins Omi/HtrA2 and Smac/Diablo in L929 and L929-ΔTM-BNIP3 cells. ΔTM-BNIP3 over-expression did not prevent the release of pro-pro-apoptotic mitochondrial proteins Omi/HtrA2 and Smac/Diablo. Cellular localization of Omi/HtrA2 (C) and Smac/Diablo (D) in L929 and L929-ΔTM-BNIP3 cells was probed by confocal microscopy. Cells were treated with 1 ng/ml TNF for 12 h prior to immunostaining with anti-Omi/HtrA2 and anti-Smac/Diablo antibodies, followed by detection with FITC-conjugated secondary antibody (green). Cells were counterstained with DAPI (blue) and mitotracker (red). In control experiments, cells were treated with TNF solvent PBS. The magnification was 1000 ×. Representative micrographs are shown for all samples.
the C terminus (163–185) projecting inwards[24]. Delineating the molecular mechanisms of BNIP3 actions is very important. It has been found that both the pro-apoptotic activity of this protein and its interaction with other members of the Bcl2 family is sensitive to the presence of its TM segment[57,58]. The functional importance of TM segment was also confirmed by the fact that the enforced expression of a BNIP3 mutant lacking this domain partially blocks
hypoxia-induced cell death[58]. The results of our study also confirm the importance of TM domain in BNIP3 induced cell death.
Reactive nitrogen intermediates, including NO, NO2−and NO3−, are involved in TNF-mediated cytolysis[38]but the mechanism remained unknown. Here we show that NO intermediates can promote the transcriptional upregulation of BNIP3. This BNIP3 gene activation is abolished after treatment with the nitrogen oxide synthase inhibitor Fig. 7.ΔTM-BNIP3 over-expression counteracts TNF-triggered activation of lysosomal cell death pathway. Pretreatment for 1 h with the inhibitors of lysosomal death pathway (A) Baf A1 (0.05μM), (B) z-FF-fmk (100 μM), and (C) CA-074-Me (20 μM) reduced TNF-cytotoxicity (TNF at 1 ng/ml for 24 h) in L929 but not in L929-ΔTM-BNIP3. L929-ΔTM-BNIP3 cells were partly protected by the expression of the dominant-negative mutant of BNIP3. TNF cytotoxicity was assessed by MTT assay. Data represent average values from quadruplicates of four independent experiments. In control experiment, cells were either treated with TNF solvent (PBS) or with the inhibitors alone at the same concentration as used in the experiments. (D, E) TNF triggered swelling of lysosomes in L929, but not in L929-ΔTM-BNIP3. L929 (D) and L929-ΔTM-BNIP3 cells (E) were treated with TNF (1 ng/ml for 24 h) in the absence of actinomycin D and stained with the acidophilic lysosomal probe LysoTracker Red (LTR). TNF caused an increase in volume and frequency of cytoplasmic granule staining with LTR which was partially inhibited byΔTM-BNIP3 over-expression (E). Nuclei were visualized by DAPI (blue). In control experiment, cells were treated with PBS. The magnification was 1000×. Representative micrographs are shown for all samples.
(L-NMMA) in L929. Thus, we concluded that nitric oxide participates in TNF-induced cytotoxicity by the upregulation of the expression of BNIP3. The increased level and cellular translocation of BNIP3 from the nucleus to mitochondria may then trigger mitochondrial dysfunction frequently observed upon TNF-treatment. L929 cells over-expressing ΔTM-BNIP3 were partially protected from TNF-induced changes to Ψmand this coincided with higher residualΨmin L929-ΔTM-BNIP3 than observed in parental L929 cells. Thus, our finding provided further evidence for a role of TNF and BNIP3 in mitochondrial malfunction as part of the TNF-induced cell death.
Mitchell's chemi-osmotic theory[59]identified ΔΨmas the driving force for mitochondrial synthesis of ATP, an essential component for cell viability and the execution of apoptosis.Ψmvary depending on the physiological status of cells and is increased in activated cells and tumor cells[60,61]. This suggests that higherΨm, as observed with L929-ΔTM-BNIP3 cells, may contribute to increased resistance against apoptosis. TNF treatment causes a rapid and significant ATP depletion
[12,55,62]. The TNF-induced mitochondrial translocation of BNIP3 and the subsequent loss of mitochondrial membrane potential may, in part, explain this effect on cellular ATP levels and stresses the importance of the TM of BNIP3 for its pro-apoptotic function. By contrast,ΔTM-BNIP3 over-expression partially attenuated the effect of TNF onΔΨmsuggesting that this deletion variant may act as a partial antagonist of BNIP3.
Apoptosis involves the synchronized release [63] with similar kinetics[64]of cytochrome c, Smac/Diablo, and Omi/HtrA2. This was also observed in TNF-treated L929 and L929-ΔTM-BNIP3. These proteins reside in the mitochondrial inter-membrane space whereas AIF is anchored in the inner membrane[65], and Endo-G likely mainly localized in the matrix[42]. This would explain the lack of AIF and Endo-G in TNF-induced cell death in our experimental model. Insertion of BNIP3 into the mitochondrial outer membrane and changes inΨmmay participate in the release of Smac/Diablo and Omi/ HtrA2 during TNF-induced cell death. Indirect evidence in support of this interpretation came from L929 over-expressing ΔTM-BNIP3. When treated with TNF, these cells showed similar levels of release for cytochrome c, Smac/Diablo, and Omi/HtrA2 as in L929, emphasiz-ing the functional importance of the BNIP3 TM domain, and the inability ofΔTM-BNIP3 to interfere with this release.
Over-expression of ΔTM-BNIP3 counteracted lysosomal damage during TNF-mediated cell death. Loss of lysosomal integrity with subsequent activation of cell death cascades has been implicated in apoptosis during oxidative stress[59,66], growth factor starvation, Fas activation, α-tocopheryl-succinate-mediated apoptosis in Jurkat T-cells[60,61], 6-hydroxydopamine-associated death of cultured micro-glia[67], apoptosis induced by the synthetic retinoid CD437 in human leukemia HL-60 cells[68], TNF- and bile acid-mediated hepatocyte apoptosis[69,70], and p53-mediated apoptosis of M1-t-p53 myeloid leukemic cells[71]. Recently, TNF-cytotoxicity was shown to associate with the permeabilization of lysosomes, release of cath-B into the cytosol [72], and subsequent cath-B-induced activation of the mitochondrial death pathway [69,73]. TNF-mediated apoptosis is markedly attenuated by alkalinizing acidic vesicles, employing cath-B inhibitors, or in cath-B knockout mice[73,74]. Furthermore, cath-B deficient mice are resistant to TNF-mediated liver injury[75]. We show here that pre-treatment of L929 cells with the vacuolar H+ -ATPase inhibitor bafilomycin A1 and the inhibitors of cath-B/cath-L zFF-fmk and CA-074-Me attenuates the enhanced TNF-mediated cell death in L929. Moreover, TNF-induced enlargement of lysosomal volume was inhibited byΔTM-BNIP3 over-expression and coincided with decreased levels of ROS in L929-ΔTM-BNIP3 cells. Alterations in mitochondrial structure and function, as observed with TNF-treated L929, and subsequent ROS formation seem to be an important step in TNF cytotoxicity[75]. Inhibition of ROS by pre-treatment with NAC abolished the increase in lysosomal volume in TNF-exposed L929. Thus, BNIP3 appears to be involved in the activation of the lysosomal
pathway in TNF-mediated cell death. In part, the actions of BNIP3 are mediated indirectly via increased production of ROS as a result of ΔΨm. ROS act as locally active messenger and signal mitochondrial damage to lysosomes which respond by swelling and cathepsin release, thereby, executing cell death[76].
In conclusion, we show that BNIP3 plays an important role in TNF-induced cell death. TNF TNF-induced an NO-mediated increase in BNIP3 expression and the translocation of BNIP3 to mitochondria, which resulted in perturbed mitochondrial function and increased ROS production. Both effects were antagonized by over-expression of ΔTM-BNIP3 and consequently ΔTM-BNIP3 protected against lysoso-mal activation in TNF-treated L929 cells. Our data provide a functional basis and link between BNIP3, a mediator of atypical cell death, and TNF, known for decades to induce a mixed necro-apoptotic form of cell demise. We used TNF in the absence of the transcriptional inhibitor actinomycin D, which is frequently employed among TNF-researchers. This allowed for the detection of TNF-induced changes in gene expression, which let to our discovery of TNF-induced and NO-mediated upregulation of BNIP3 as another mechanism contributing to TNF-induced cell death.
Acknowledgements
S.G. thankfully acknowledges fellowships from MHRC and CCMF, MICH, NTPAA, and CLA/GSK/CIHR. M.E. is thankful for fellowships from CCMF and MICH. G.H.B. is thankful for the support through the BSc-Med program of the Univ. Manitoba. M.L. thankfully acknowl-edges support through CFI-Canada Research Chair program, MHRC-, CIHR, and MICH-founded programs. S.W. acknowledges support from DFG (We 1801/2-4, GRK1302, SFB 685); the German Federal Ministry of Education, Science, Research and Technology (Hep-Net); the IZKF Tübingen (IZKF; Fö. 01KS9602); the Wilhelm Sander-Stiftung (2004.099.1); and the Landesforschungsschwerpunktprogramm of the Ministry of Science, Research and Arts of the Land Baden-Wuerttemberg.
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