Research Article
4015
TNFa-induced lysosomal membrane permeability is
downstream of MOMP and triggered by caspasemediated NDUFS1 cleavage and ROS formation
Jisen Huai1,2,*, F-Nora Vögtle3,4, Lars Jöckel1,5,6, Yunbo Li2, Thomas Kiefer1,6, Jean-Ehrland Ricci7,8,9 and
Christoph Borner1,4,5,*
1
Institute of Molecular Medicine and Cell Research (ZBMZ), Albert Ludwigs University Freiburg, Stefan Meier Strasse 17, D-79104 Freiburg,
Germany
2
Institute of Microanatomy and Neurobiology, University Hospital Mainz, Langenbeckstrasse 1, D-55131 Mainz, Germany
3
Institute of Biochemistry and Molecular Biology (ZBMZ), Albert Ludwigs University Freiburg, Stefan Meier Strasse 17, D-79104 Freiburg, Germany
4
Centre for Biological Signaling Studies (BIOSS), Schänzlestrasse 18, Albert Ludwigs University Freiburg, D-79104 Freiburg, Germany
5
Spemann Graduate School of Biology and Medicine (SGBM), Albertstrasse 19a, Albert Ludwigs University Freiburg, D-79104 Freiburg, Germany
6
Faculty of Biology, Schänzlestrasse 1, Albert Ludwigs University Freiburg, D-79104 Freiburg, Germany
7
Centre Hospitalier Universitaire de Nice, Département d’Anesthésie Réanimation, F-06204 Cedex 3 Nice, France
8
Inserm, U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), Équipe ‘Contrôle Métabolique Des Morts Cellulaires’, Nice, F-06204 Cedex 3,
France
9
Université de Nice-Sophia-Antipolis, Faculté de Médecine, F-06204 Cedex 3 Nice, France
*Authors for correspondence ([email protected]; [email protected])
Journal of Cell Science
Accepted 30 May 2013
Journal of Cell Science 126, 4015–4025
ß 2013. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.129999
Summary
When NF-kB activation or protein synthesis is inhibited, tumor necrosis factor alpha (TNFa) can induce apoptosis through Bax- and
Bak-mediated mitochondrial outer membrane permeabilization (MOMP) leading to caspase-3 activation. Additionally, previous studies
have implicated lysosomal membrane permeability (LMP) and formation of reactive oxygen species (ROS) as early steps of TNFainduced apoptosis. However, how these two events connect to MOMP and caspase-3 activation has been largely debated. Here, we
present the novel finding that LMP induced by the addition of TNFa plus cycloheximide (CHX), the release of lysosomal cathepsins and
ROS formation do not occur upstream but downstream of MOMP and require the caspase-3-mediated cleavage of the p75 NDUFS1
subunit of respiratory complex I. Both a caspase non-cleavable p75 mutant and the mitochondrially localized antioxidant MitoQ prevent
LMP mediated by TNFa plus CHX and partially interfere with apoptosis induction. Moreover, LMP is completely blocked in cells
deficient in both Bax and Bak, Apaf-1, caspase-9 or both caspase-3 and -7. Thus, after MOMP, active caspase-3 exerts a feedback action
on complex I to produce ROS. ROS then provoke LMP, cathepsin release and further caspase activation to amplify TNFa apoptosis
signaling.
Key words: Apoptosis, TNFa, LMP, ROS, Caspase, MOMP, Bax, Bak, Apoptosome, MitoQ
Introduction
TNFa is a pleiotropic inflammatory cytokine, which was
discovered decades ago (Carswell et al., 1975; Pennica et al.,
1984). Since then, it has been shown to exert many functions in
various physiological and pathological processes. In particular, its
implication in the survival and death of normal and cancerous cells
has been widely investigated. For most mammalian cells, TNFa is
non-cytotoxic, but triggers, after ligation with TNF-receptor 1 at
the membrane, a TRADD–TRAF2–RIP1–c-IAP1/2 complex
(complex I)-dependent signaling pathway that ends up in the
activation of the transcription factor NF-kB (Walczak, 2011;
Micheau and Tschopp, 2003; Andrew, 2004; Wang et al., 2008).
NF-kB in turn induces genes for cell survival (such as those
encoding Bcl-xL, c-IAPs, c-FLIP, etc.) and pro-inflammatory
mediators (TNFa itself, COX-2, iNOS, etc.). Upon c-IAPs
inhibition by so called Smac mimetics, low expression of the
caspase-8 inhibitor c-FLIP, or protein synthesis inhibition by
cycloheximide (CHX), complex I dissociates from the membrane
and forms in the cytosol a second complex (complex II) which now
includes caspase-8 and its activator FADD to cleave the BH3-only
protein Bid to tBid (Micheau and Tschopp, 2003; Andrew, 2004;
Wang et al., 2008). tBid activates mitochondrial outer membrane
permeabilization (MOMP) via Bax/Bak (i.e. Bax and Bak)
activation leading to the release of cytochrome c, the formation
of the Apaf-1–caspase-9 apoptosome and the processing and
activation of effector caspases-3/7 (i.e. caspase-3 and -7), the main
executioners of apoptotic programmed cell death (Kroemer et al.,
2007). Recently, another type of TNFa-induced cell death, called
programmed necrosis or necroptosis has been discovered when
caspases are inhibited by the pan-caspase inhibitor Z-VAD-fmk.
This pathway includes RIP1 and other novel signaling components
(RIP3, MLK, etc.) whose functions are only now being unveiled
(Degterev and Yuan, 2008, He et al., 2009; Zhang et al., 2009).
A characteristic feature of TNFa-induced apoptotic and
necroptotic cell death is the production of reactive oxygen
species (ROS) such as superoxide anions (O22?) and hydrogen
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Journal of Cell Science 126 (17)
peroxide. The intracellular source of these ROS, their link to cell
death signaling components and their exact role in the effector
phase of apoptosis or necroptosis have been largely debated (Van
Herreweghe et al., 2010; Taujimoto et al., 1986; Meier et al.,
1989; Yazdanpanah et al., 2009; Kim et al., 2010; Woo et al.,
2000). While in macrophages and neutrophils TNFa triggers
ROS production as a quick oxidative burst required for an early
innate immune response, in fibroblasts and other nonphagocytic
cells this production occurs continuously over a time period of
hours (Van Herreweghe et al., 2010; Taujimoto et al., 1986;
Meier et al., 1989; Yazdanpanah et al., 2009). This suggests that
phagocytic and nonphagocytic cells may exploit distinct
molecular machineries for ROS generation. In innate immune
cells TNFa is known to stimulate the activity of the plasma
membrane-residing Nox2/gp91 phox subunit of NADPH oxidase
which produces superoxides within seconds in order to kill
invasive microorganisms (Yazdanpanah et al., 2009; Lambeth,
2004). Interestingly, after treating with TNFa, mouse fibroblasts
can also produce ROS via NADPH oxidase. In this case, ROS
generation depends on a signaling complex composed of
TRADD, RIP1, Nox1 and the small GTPase Rac1 (Lin et al.,
2004). Other reports further implicate calcium mobilization,
activation of the Rac-cPLA2-LTB4 cascade and activation of acid
sphingomyelinases in this process (Taujimoto et al., 1986;
Yazdanpanah et al., 2009; Woo et al., 2000; Shoji et al., 1995).
Due to the role of RIP1, it was suggested that this pathway of
ROS production is crucial for necrotic cell death. However, the
other important component RIP3, which forms a pro-necrotic
complex with RIP1 after phosphorylation (Cho et al., 2009), did
not activate NADPH oxidase but enzymes controlling glycolytic
flux and glutaminolysis, thereby causing ROS formation via
mitochondrial complex I (Zhang et la., 2009; Vandenabeele et al.,
2010). Hence for necroptosis, and probably also for apoptosis,
ROS may be rather produced by mitochondria, preferentially at
the ubisemiquinone site (Schulze et al., 1993). To trace
mitochondrial ROS, the Murphy group succeeded to generate
synthetic compounds, called MitoSOXTM and MitoQ, which after
removal of the targeting sequence by mitochondrial esterases, are
stably inserted into the inner mitochondrial membrane and act as
ROS detection probe and antioxidant, respectively (James et al.,
2005; Smith et al., 2011).
Mitochondrial ROS production has been closely linked to a fall
in the mitochondrial membrane potential. Although this process
is seen in all apoptotic cells, including when cells die in response
to TNFa, it is not an early event of cell death signaling but rather
a consequence of Bax/Bak-mediated MOMP. Ricci et al.
convincingly showed that caspase-3, which is activated after
MOMP, cytochrome c release and apoptosome formation, feeds
back on complex I of the respiratory chain by cleaving the p75
NDUFS1 subunit of this complex and thereby provoking a fall in
the mitochondrial membrane potential and ROS production
(Ricci et al., 2004). However, this feedback loop has not yet been
investigated for TNFa. By contrast, a recent study using HEK
293, HeLa and cervix carcinoma cells reported that TNFainduced mitochondrial ROS production was mediated by the
recruitment of the survival factor Bcl-xL to a mitochondrial
protein called Romo1, which then resulted in a reduction of the
mitochondrial membrane potential (Kim et al., 2010). Other
studies claimed that MOMP itself triggered ROS production
(Kirkland et al., 2002; Kirkland and Franklin, 2003; Kirkland and
Franklin, 2007; Jiang et al., 2008). Finally, complex III of the
respiratory chain was found to be involved in mitochondrial ROS
formation induced by Bak in vascular endothelial cells (Childs
et al., 2008). Thus, it is still unclear at which step in the apoptotic
pathway ROS are produced and how they impact on the cell
death machinery.
One widely discussed mode of action of ROS in the cell death
pathway is their destabilizing effect on the lysosomal membrane
leading to lysosomal membrane permeability (LMP) of various
extent. Certain ROS species such as hydrogen peroxides can
easily penetrate the organelle and be converted into highly
reactive hydroxyl radicals due to the iron content of lysosomes
(Fenton reaction). These radicals are known to effectively
damage membranes by lipid peroxidation. While a full lysis of
lysosomes has been associated with necrosis, a partial, more
selective lysosomal membrane perforation is supposed to assist
apoptosis by releasing cathepsins (Boya and Kroemer, 2008). An
oxidative burst elicited by interferon-c was also reported to
induce LMP and cathepsin release (Kowanko and Ferrante,
1987). Cathepsins are cysteine, serine or aspartyl proteases,
which, once in the cytosol can impinge on the apoptotic
machinery by either directly processing and activating caspases
or cleaving Bid to tBid (Boya and Kroemer, 2008; Blomgran
et al., 2007; Guicciardi et al., 2000; Guicciardi et al., 2005;
Droga-Mazovec et al., 2008). The problem with this pathway is
that it was previously claimed to be an early event of apoptosis
occurring before MOMP and effector caspase-3/7 activation and
prior to ROS production (Werneburg et al., 2002; Werneburg
et al., 2004; Gyrd-Hansen et al., 2006). We, however, recently
published that for a variety of apoptotic stimuli, LMP is not an
early requiring, but a late amplifying event of apoptosis induction
because it strictly depends on Bax/Bak (i.e. MOMP), apoptosome
formation and caspase-3/7 activation (Oberle et al., 2010). The
link from caspase-3/7 to LMP has however not been sorted out
yet. Moreover, our study did not include TNFa, which has so far
been the golden standard stimulus for implying LMP as an early
apoptotic trigger (Guicciardi et al., 2000; Guicciardi et al., 2005;
Droga-Mazovec et al., 2008; Werneburg et al., 2002; Werneburg
et al., 2004).
Here we show that also for TNFa, LMP is a late amplifying
process downstream of Bax/Bak, the apoptosome and caspase-3/
7. In addition, we uncovered the connection between caspase-3/7
activation and LMP. As previously shown by Ricci et al., 2004,
caspase-3 cleaves the p75 subunit of complex I and thereby
produces ROS, in particular O22?, which then damage the
lysosomal membrane leading to LMP.
Results
TNFa/CHX-triggered ROS are O22? of mitochondria origin
TNFa is only able to trigger apoptosis of mammalian cells when
the expression of NF-kB induced survival genes is blocked by
transcriptional or translational inhibition (Walczak, 2011; Micheau
and Tschopp, 2003; Andrew, 2004; Wang et al., 2008). We
therefore used in our study a combination of TNFa and CHX
(designated TNFa/CHX, T/C in figures) to treat mouse embryo
fibroblasts (MEFs). We first monitored the origin and amount of
ROS generated by this treatment. For that purpose, we used
dihydroethidium (DHE) and 29,79-dichlorodihydrofluorescein
(DCDHF) to estimate O22? and H2O2/nitric oxide (NO) by
FACS analysis and fluorescence microscopy detection,
respectively. After exposure to 10 ng/ml TNFa and 1 mg/ml
CHX for 4 h MEFs were strongly stained with DHE (Fig. 1A,B),
Journal of Cell Science
TNFa-induced LMP requires ROS downstream of MOMP
4017
Fig. 1. TNFa/CHX-induced ROS production involves O22? and is blocked by the mitochondrially localized antioxidant MitoQ and general caspase
inhibition. (A) MitoSOXTM or DHE fluorescence microscopy of MEFs (magnification 4006) treated with a combination of 10 ng/ml TNFa and 1 mg/ml CHX, or
0.5 mM rotenone in the presence or absence of the antioxidant MitoQ (5 mM) or the general caspase inhibitor Z-VAD.fmk (100 mM) for 4 h. Note that TNFa/CHX
triggers O22? production (red fluorescence) specifically in mitochondria (detected by the mitochondrially targeted MitoSOXTM ROS sensor) and that this is
blocked by the mitochondrial antioxidant MitoQ and by caspase inhibition (therefore implying caspases in ROS production). Rotenone, which inhibits the transfer
of electrons from iron-sulfur centers in complex I to ubiquinone, serves as a positive control of mitochondrial ROS production detected by MitoSOXTM and
blocked by MitoQ. Only a low level of ROS are produced in untreated cells. (B) FACS histogram of DHE fluorescence (FL-3) showing that 50% of the cells
produce O22? after treatment with 10 ng/ml TNFa and 1 mg/ml CHX for 4 h. This increased ROS production is blocked by MitoQ and is not seen in untreated
cells (control). (C) ScanR cytometry of MEFs (magnification 40 6) treated with 0.5 mM rotenone for 4 h. Note that after this time, about the same percentage of
cells (46%) are positive for mitochondrial O22? production/staining as with TNFa/CHX treatment (see B, 50%).
but not with DCDHF (data not shown) indicating that this
treatment majorly induced the production of O22?. To detect the
source of O22? production, we incubated the cells with the
mitochondrial red fluorescent ROS sensor MitoSOXTM. As shown
in Fig. 1A,B, TNFa/CHX-induced O22? were effectively
produced in mitochondria, and this effect was strongly inhibited
by a pretreatment with the mitochondria-specific antioxidant
MitoQ. This compound mimics the endogenous mitochondrial
antioxidant coenzyme Q10 (James et al., 2005; Smith et al., 2011)
thereby markedly scavenging the ROS produced at the
ubisemiquinone site. As a positive control for ROS production at
the same site, we used rotenone (Fig. 1A,C). Interestingly, the
amount of ROS/O22? produced by the treatment of MEFs with
0.5 mM rotenone was approximately the same (46% by ScanR
cytometry, Fig. 1C) as that generated by TNFa/CHX for the same
time period (50%, FACS analysis, Fig. 1B). Again the rotenoneinduced ROS production was completely ablated by MitoQ
(Fig. 1A) confirming that the ROS induced by TNFa/CHX is of
mitochondrial origin. It should be noted that DHE can be
intracellularly oxidized to ethidium (ETH) by ROS and then
stains DNA. Indeed, both DHE and MitoSOXTM (which is a DHE
analog) show a clear DNA and other intracellular (mitochondrial)
staining after cellular treatment with TNFa/CHX or rotenone
(Fig. 1A).
TNFa/CHX-triggered ROS production is dependent on the
Bax/Bak–apoptosome–caspase gateway and the caspase3-mediated cleavage of the p75 NDUFS1 subunit of
complex I of the respiratory chain
To elucidate if ROS production induced by TNFa/CHX is an
early event occurring before or simultaneously with MOMP or
rather a late event downstream of MOMP, we determined the
amount of O22? generation by DHE FACS analysis in WT, Bax/
Bak2/2 [double knockout (KO) in both Bax and Bak], Apaf12/2, caspase-92/2 and caspase-3/72/2 (double KO in both
caspase-3 and -7) MEFs. As shown in Fig. 2A, the ROS
production observed after 4 h of TNFa/CHX treatment (see
Fig. 1B) was completely ablated in all the KO cell lines, although
Apaf-12/2 MEFs retained a slightly higher ROS level above
background (which was however not significant). These data
clearly show that ROS is generated downstream of MOMP and
caspase-3/7 in response to TNFa/CHX. In order to exclude
possible respiratory chain deficiencies in Bax/Bak2/2 MEFs, we
measured oxygen consumption of intact cells. As shown in
supplementary material Fig. S1A,C, Bax/Bak2/2 cells did not
show any respiration deficit as they even consumed 55617%
more oxygen than WT cells. After treating with TNFa/CHX, WT
cells expectedly respired less but the Bax/Bak2/2 counterparts
still consumed oxygen at a higher rate (supplementary material
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(Fig. 2A). In the presence of the complex I inhibitor
rotenone, MEFs did hardly respire indicating that we indeed
measured the respiration of mitochondria (supplementary
material Fig. S1A).
Journal of Cell Science
Fig. S1A,C). This is consistent with the finding that the Bax/
Bak-deficient cells were protected from TNFa/CHX-induced
apoptosis (Fig. 6A) and cytochrome c release (MOMP)
(Kroemer et al., 2007) and did not produce any ROS
Fig. 2. TNFa/CHX-induced ROS production occurs downstream of Bax/Bak and the apoptosome, and involves the caspase-3-dependent cleavage of the
p75 NDUFS1 subunit of complex I. (A) Quantitative FACS analysis of DHE fluorescence measuring the amount of O22? produced in WT, Bax/Bak2/2, Apaf12/2, caspase-92/2 or caspase-3/72/2 MEFs exposed to 10 ng/ml TNFa and 1 mg/ml CHX (grey bars) or medium only (black bars) for 4 h. Note that O22?
production depends on Bax/Bak and the Apaf-1–caspase-9 apoptosome, as well as caspase-3/7. The data are the means6s.d. of at least three independent
experiments; P,0.001 between WT and KO cells. (B) FACS histogram of DHE fluorescence (FL-3) of WT HeLa cells expressing the WT p75 NDUFS1 subunit
of complex I or its D255A mutant, which cannot be cleaved by caspase-3, treated either with 10 ng/ml TNFa and 1 mg/ml CHX (T/C) for 4 h or 1 mg/ml
tunicamycin (Tuni) or 100 mM etoposide (Etop) for 24 h. Gray histograms represent untreated cells. Note that O22? production induced by T/C, but not induced by
tunicamycin or etoposide is less in HeLa cells expressing the p75D255A mutant, indicating that caspase-3-mediated cleavage of p75 is required for T/C-induced
ROS production. (C) Western blot analysis showing the levels of mitochondrial full-length (FL) p75 and caspase-3 cleaved (CL) p28 NDUFS1 or full-length
p17 NDUFB6 subunits of complex I or cytosolic cytochrome c (Cyto C) in p75WT or mutant p75D255A HeLa cells treated with 10 ng/ml TNFa and 1 mg/ml
CHX or 1 mg/ml CHX alone for 0–4 h, or 1 mg/ml tunicamyin (Tuni) or 100 mM etoposide (Etop) for 0–24 h. Note that although p75 is cleaved into p28 after
2–4 h of T/C treatment, the levels of p75 in response to CHX, Tuni or Etop, as well as those of the p75D255A mutant, stay the same. Cytochrome c appears in the
cytosol and diminishes in the mitochondria after 2 h of T/C treatment or 14 h of Tuni or Etop treatment in both p75WT and p75D255A cells, although the kinetics
were a bit slower for T/C-treated p75D255A cells. The loss of cytochrome c in the cytosol after 4 h of T/C treatment is due to secondary necrosis, releasing
cytochrome into the medium. Anti-actin and anti-COX-IV western blots serve a control for equal cytosolic and mitochondrial protein loading, respectively.
Molecular masses are indicated in kDa on the right side of the blots.
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Journal of Cell Science
TNFa-induced LMP requires ROS downstream of MOMP
Fig. 3. TNFa/CHX-triggered LMP and cathepsin B release from lysosomes depend on the Bax/Bak–apoptosomal pathway, the caspase-3-mediated
cleavage of the p75 NDUFS1 subunit of complex I and consequent ROS production. (A) Quantitative FACS measurement of lysosomal AO staining (red
fluorescence) and (B) fluorometric determination of cytosolic cathepsin B/L activities (Z-FRase) in WT, Bax/Bak2/2, Apaf-12/2, caspase-92/2 or caspase-3/72/2
MEFs treated (gray bars) or not (black bars) with T/C for 4 h. Note that WT cells exhibit a lack of retention of lysosomal AO staining and increased cytosolic
cathepsin B/L activities in response to T/C, which reflects induction of LMP. Both changes do not occur at all in the KO cell lines, indicating that LMP and
cathepsin B/L release is dependent on Bax/Bak, the caspase-9–Apaf-1 apoptosome and caspase-3/7. (C) The diminished AO staining in WT MEFs exposed to T/C
(dark gray bars) is almost completely ablated by the co-treatment with 5 mM of the mitochondrial antioxidant MitoQ (light gray bars), indicating that T/C-induced
LMP depends on mitochondrial ROS production. Treatment of these cells with 100 mM etoposide (Etop) for 4 h also induces LMP which is partially inhibited by
MitoQ, whereas treatment with 1 mg/ml tunicamycin (Tuni) only slightly affects LMP. The data presented in A–C are the means6s.d. of at least three
experiments. P-values were as follows: (A,B) P,0.001, n56 between WT and KO MEFs; (C) P,0.001 (T/C), 0.005 (Etop) and 0.01 (Tuni) between untreated
and treated cells, P,0.001 (T/C), P,0.004 (Etop) and P,0.01 (Tuni) between 6 Mito Q cells, n56. (D) FACS dot-plot analysis of AO staining of MEFs treated
with T/C in the presence or absence of 100 mM of the general antioxidant N-acetylcysteine (NAC) or 5 mM of the mitochondrially targeted antioxidant MitoQ for
4 h (left panels) or of p75WT and p75D255A mutant HeLa cells treated with T/C for the same time period (right panels). T/C induces a loss of the red fluorescence
of AO in lysosomes of MEFs (FL-3, red, y-axis) (59.4% less staining) concomitant with a slight gain of green fluorescence of AO in the cytosol (FL-1, green,
x-axis) indicating that AO is released into the cytosol due to LMP. Both processes are reduced in cells treated with MitoQ and NAC (only 18.2% and 19.4%
less FL-3 red staining, respectively), implicating ROS in the LMP process. Similarly, AO fluorescence decreases in the lysosomes of T/C-treated p75WT HeLa
cells (from 94.7 to 49%), but not in the p75D255A mutant counterparts (red staining stays at 82.5%) showing that LMP depends on the caspase-3-mediated cleavage
of p75 at D255. For unknown reasons, the green fluorescence of released AO in the cytosol does not increase but instead decreases. (E) Cathepsin B western
blot of the cytosolic and postmitochondrial membrane (containing lysosomes) fractions of p75WT HeLa cells treated with 10 ng/ml TNFa and 1 mg/ml CHX
in the presence (+) or absence (2) of 5 mM MitoQ for 0–4 h. Note that CTB release into the cytosol is blocked by the MitoQ antioxidant, indicating that it required
mitochondrially generated ROS. (F) The same western blot analysis as in E but showing p75WT and p75D255A cells treated with T/C for up to 4 h. Whereas, in
WT cells, CTB levels increase in the cytosol and decrease in the lysosomal fraction, this does not occur in the p75D255A cells, suggesting that caspase-3-mediated
cleavage at D255A of p75 is required for cathepsin B release from lysosomes to the cytosol. Anti-actin and anti-Lamp-1 western blotting serves as a control
for equal loading of cytosolic and lysosomal proteins. Molecular masses are indicated in kDa on the right side of the blots.
Journal of Cell Science
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After knowing that ROS production was downstream of
MOMP we wanted to know how it was caused. The most likely
mediator was caspase-3 because it was previously shown to feed
back on mitochondria by cleaving the p75 NDUFS1 subunit of
complex I, thereby triggering a fall in the mitochondrial
membrane potential and ROS production (Ricci et al., 2004).
We first treated WT MEFs with the general caspase inhibitor ZVAD-fmk and found that TNFa/CHX-induced mitochondrial
O22? production measured by DHE and MitoSOXTM
fluorescence was entirely inhibited (Fig. 1A). We then used
HeLa cells expressing either WT p75 or a mutant version that
cannot be cleaved by caspase-3 any more (p75D255A). As shown
in Fig. 2B, while p75WT HeLa cells produced significant amounts
of O22? in response to TNFa/CHX, this was not the case with the
caspase non-cleavable p75 mutant cells. The p75WT, but not the
mutant protein, was indeed cleaved concomitantly with the
release of cytochrome c from mitochondria into the cytosol
whereas another subunit of complex I, p17 NDUFB6 remained
intact (Fig. 2C, left panel). Importantly, the kinetics of
cytochrome c release was only slightly delayed in p75D255A as
compared to p75WT cells indicating that caspase-3-mediated
cleavage of p75 did not majorly affect MOMP, but a process
downstream of it (ROS production) (Fig. 2C, left panel). To
determine whether this caspase-3-mediated feedback effect for
ROS production was specific for TNFa/CHX, we performed the
same DHE FACS analysis with p75WT and p75D255A HeLa cells
treated with the genotoxic agent etoposide or the ER stress
stimulus tunicamycin, which are also known to induce MOMP
and ROS production (Kroemer et al., 2007). Indeed, both stimuli
triggered ROS production but no difference was detected
between p75WT and p75D255A mutant cells (Fig. 2B).
Moreover, the p75 protein was not proteolyzed despite
effective cytochrome c release (Fig. 2C, right panel). These
data indicate that TNFa/CHX specifically provokes ROS
generation via the caspase-3-mediated cleavage of p75 of
complex I which supports the finding above that ROS
production originates from a mitochondrial source.
p75 cleavage and mitochondrial ROS mediate TNFa/CHXinduced LMP downstream of MOMP, and lysosomes can
be spontaneously permeabilized by oxidative damage
Like other apoptotic stimuli TNFa is able to induce LMP.
However based on previous studies, it has been postulated that
LMP is an early event occurring before MOMP, i.e. right after
TNF-R1 stimulation (Boya and Kroemer, 2008; Blomgran et al.,
2007; Guicciardi et al., 2000; Guicciardi et al., 2005; DrogaMazovec et al., 2008; Werneburg et al., 2002; Werneburg et al.,
2004; Gyrd-Hansen et al., 2006). Surprisingly, the acid test,
namely the role of Bax/Bak and the apoptosome in TNFainduced LMP has not been performed yet. We therefore looked at
TNFa/CHX-triggered LMP in WT, Bax/Bak2/2, Apaf-12/2,
caspase-92/2 and caspase-3/72/2 MEFs by two means, the
decrease of lysosomal red fluorescent acridine orange (AO)
staining and the release of cathepsin B and -L (cathepsin B/L,
CTB/L) activity and/or protein into the cytosol. As shown in
Fig. 3A,B, the TNFa/CHX-induced diminishment of AO staining
and the concomitant appearance of cathepsin B/L activity (ZFRase) in the cytosol were both entirely prevented in all KO
MEFs indicating that LMP triggered by TNFa/CHX clearly
occurred downstream of MOMP. To identify if mitochondrial
ROS production via caspase-3-mediated p75 cleavage was the
mediator of this LMP, we quantified diminished AO staining and
cathepsin B protein release in WT MEFs co-treated with TNFa/
CHX and either MitoQ or the general antioxidant Nacetylcysteine (NAC) and in TNFa/CHX-treated p75WT and
p75D255A HeLa cells. For comparison, we again exposed MEFs to
etoposide and tunicamycin. While LMP was markedly (although
not completely) inhibited by both MitoQ and NAC when MEFs
were treated with TNFa/CHX, much less LMP inhibition was
detected by MitoQ in the case of etoposide and tunicamycin
(Fig. 3C,D). As discussed below, the partial dependence of LMP
on ROS in the case of these two drugs could be due to ROS
production independent of p75 cleavage. Moreover, the release of
cathepsin B induced by TNFa/CHX was significantly diminished
by MitoQ (Fig. 3E). In addition, while only 49% of p75WT HeLa
cells retained lysosomal AO staining (Fig. 3D), and cathepsin B
largely appeared in the cytosol (Fig. 3F) after 4 h of TNFa/CHX
treatment, 82.5% of the p75D225A mutant cells exhibited AO
staining (Fig. 3D) and no cytosolic cathepsin B release was
observed in these cells (Fig. 3F). Consistent with these data, a
cellular treatment with rotenone, which provokes mitochondrial
ROS production (see Fig. 1C), also effectively triggered LMP
(Fig. 5A). Thus, our results show for the first time that TNFa/
CHX-induced LMP is mediated via caspase-3-triggered p75
cleavage and mitochondrial ROS production.
To validate this mechanism, we needed to show that ROS
production was prior to LMP. For that purpose, we performed a
kinetic FACS analysis of TNFa/CHX-treated HeLa cells, using
AO (Fig. 4A) and DHE (Fig. 4B) stainings for LMP and ROS
measurements, respectively. Since AO exhibits both green and
red fluorescence and DHE stains red, the data could not be
depicted on a single FACS dot-plot. But by comparing the extent
of LMP and ROS at each hour post-treatment we could show that
ROS production peaked at 2 h (Fig. 4B, black bars) when only
20% of the cells had undergone LMP (Fig. 4A). This confirms
that mitochondrial ROS production in response to TNFa/CHX
occurs before the induction of LMP.
To get an idea if it is principally possible that ROS can
provoke LMP in vitro, we incubated a post-mitochondrial
fraction containing lysosomes with a buffer containing no
antioxidants (oxidizing) or reducing agents for up to 60 min
and then measured the amount of cathepsin B/L activity (ZFRase) remaining in the lysosomes. As shown in Fig. 5B, left
panel, the lysosomal Z-FRase activity gradually decreased with
time of incubation, and this was markedly inhibited by the
addition of NAC or glutathione (GSH) to the buffer system
(Fig. 5B, right panel). Simultaneously, the AO staining, which
was diminished in these lysosomes after 60 min buffer incubation
also recovered by the presence of the two antioxidants (Fig. 5B,
right panel). We could therefore demonstrate that ROS is able to
effectively mediate LMP in vitro.
TNFa/CHX-induced apoptosis is partially prevented by the
mitochondria-specific antioxidant MitoQ and in cells
expressing the p75D255A mutant
We finally needed to show that the caspase-3-mediated p75
cleavage and mitochondrial O22? production had any
physiological significance for TNFa/CHX-induced apoptosis, at
least as an amplification mechanism. As reported before, this
apoptosis was entirely prevented in Bax/Bak2/2, Apaf-12/2,
caspase-92/2 and caspase-3/72/2 MEFs (Fig. 6A) confirming
that in these cells TNFa/CHX used the intrinsic mitochondrial
Journal of Cell Science
TNFa-induced LMP requires ROS downstream of MOMP
4021
Fig. 4. TNFa/CHX triggers ROS production prior to
LMP. (A) FACS dot-plot analysis of AO staining of HeLa
cells treated with 10 ng/ml TNFa and 1 mg/ml CHX (T/C)
for 0–6 h. T/C induces a loss of the red fluorescence of AO
in lysosomes (FL-3, Red, y-axis) indicating that AO is
released into the cytosol owing to LMP. For unknown
reasons, the green fluorescence of released AO does not
increase but instead decreases in these cells. (B) The same
cells as in A were analyzed in parallel for ROS production
by DHE FACS analysis. Note that O22? production is
already maximal after 2 h of T/C treatment and stays at the
same level up to 5 h (black bars). LMP (i.e. a drop in red
fluorescence), however, only starts at 2 h and further
increases until 6 h, confirming that ROS production occurs
before LMP. The data represent the means6s.d. of at least
three experiments, P,0.05 between untreated and 1 h T/Ctreated cells, P,0.01 between 1 h with 2 h T/C-treated
cells, n56.
pathway for apoptosis induction. The mitochondria-specific
antioxidant MitoQ (Fig. 6B) or the presence of the caspase-3
non-cleavable p75D225A mutant (Fig. 6C) also partially protected
the cells from TNFa/CHX-induced apoptosis. By contrast NAC
was unable to interfere with this type of apoptosis (Fig. 6B)
despite the fact that it blocked LMP (Fig. 3D), most likely
because this antioxidant was slightly cytotoxic on its own (data
not shown). For unknown reasons untreated p75D225A cells
respired less effectively than their WT counterparts
(supplementary material Fig. S1B,C). Like WT MEFs, p75WT
cells consumed less oxygen upon TNFa/CHX treatment.
p75D225A cells showed a slightly improved respiration under
these conditions confirming that their respiratory chain was less
impaired and they produced less ROS than p75WT cells (Fig. 2B).
However, the p75D225A cells were not entirely protected
against TNFa/CHX-induced apoptosis since the p75/ROS
pathway is not expected to be an initiating, requiring event
leading to MOMP but a downstream amplifying process to
further enhance apoptosis through LMP, cathepsin release and
additional caspase activation. Indeed, the released cathespins
seemed to contribute to this amplification loop as both cathepsin
B/L2/2 MEFs as well as MEFs pre-treated with the cysteine
protease inhibitor E64d showed a similar diminishment of TNFa/
CHX-induced apoptosis as p75D225A HeLa cells (Fig. 6C).
Discussion
TNFa, the founding member of the TNF-superfamily, mediates
both necroptotic and apoptotic cell death (Walczak, 2011;
Micheau and Tschopp, 2003; Andrew, 2004; Wang et al., 2008;
Kroemer et al., 2007; Degterev and Yuan, 2008, He et al., 2009;
Zhang et al., 2009). Although many investigations have been
conducted over the last few years the detailed mechanisms of
these death signaling pathways are still not completely known.
This particularly concerns our understanding about where ROS
are produced intracellularly, how ROS and LMP are
mechanistically linked and how both processes contribute to
TNFa-induced apoptosis (Boya and Kroemer, 2008; Shen and
Pervaiz, 2006; Zdolsek et al., 1993; Brunk et al., 1995). Our
study here bridges these missing gaps. We identified that TNFa/
CHX-induced LMP is not an apoptosis initiating, but amplifying
process downstream of MOMP, and it is triggered by
mitochondrial ROS generated as the result of a caspase-3mediated cleavage of the p75 NDUFS1 subunit of complex I. A
similar finding was obtained in two different cellular systems,
MEFs and HeLa cells, although molecular tools for manipulating
p75 levels were not available for MEFs, and HeLa cells were less
sensitive to TNFa/CHX-induced apoptosis due to the expression
of HPV E6 and E7 genes (Garnett et al., 2006; Filippova et al.,
2002).
The mitochondrial production of ROS by TNFa has been
shown in previous reports, and it was also suggested that these
ROS somehow contribute to the apoptotic or necrotic process
(Kim et al., 2010; Lin et al., 2004; Shoji et la., 1995; Schulze et
la., 1993; Shen and Pervaiz, 2006). However, since other
intracellular sources of ROS exist, such as the plasma
membrane residing NADPH oxidase complex (Yazdanpanah
et al., 2009; Woo et al., 2000; Lambeth, 2004), we had to make
sure that the majority of ROS implicated in TNFa-induced death
signaling is of mitochondrial origin. Indeed, the mitochondria
specific antioxidant MitoQ, but not the general antioxidant NAC,
partially protected fibroblasts from TNFa/CHX-induced
apoptosis. Moreover, DHE and MitoSOXTM stainings were
completely abrogated by MitoQ and in Bax/Bak2/2 MEFs
indicating that the major source of ROS production induced by
TNFa/CHX is indeed the mitochondria. We further showed that
rotenone which interferes with complex I, triggered similar
ROS production as TNFa/CHX, and this was also blocked by
MitoQ. Finally, the fact that HeLa cells expressing the caspase
Journal of Cell Science
4022
Journal of Cell Science 126 (17)
Fig. 5. Rotenone triggers a similar extent of LMP to that induced by TNFa/CHX, and isolated lysosomes can be permeabilized by a process that is prevented
by the antioxidants NAC and GSH. (A) WT MEFs were treated with 0.5 mM rotenone for 4 h followed by staining with AO, as described in the Materials and
Methods. AO fluorescence in lysosomes was then quantified by ScanR cytometry (magnification 406). Although 96% of the untreated cells showed lysosomal AO
staining, only 33% of the cells retained this staining after rotenone treatment, indicating that most of the AO was not kept in lysosomes due to LMP to a similar extent
as observed upon TNFa/CHX treatment (see Fig. 3A, WT). (B) The post-mitochondrial fraction (containing lysosomes) was extracted from cells loaded with 5 mM
AO, washed and resuspended in 16 AT buffer, as described in the Materials and Methods. The fractions were then incubated at 37˚C with or without 1 mM
N-acetylcysteine (NAC) or 1 mM glutathione (GSH) for up to 60 min, followed by centrifugation and measurement of cathepsin B/L activity (Z-FRase activity,
measured as relative fluorescence units, RFU) (left panel) or AO fluorescence (right panel) in the membrane (lysosomal) fraction. Note that cathepsin activity
decreased in the lysosomes by simply incubating them at 37 ˚C. This decrease, as well as the reduction in AO staining, were reversed by NAC or GSH, suggesting that
LMP is mediated by ROS. In the right panel, the remaining Z-FRase activity and AO staining after 60 min of incubation (without antioxidant addition) was
taken as 100% (blue column, boxed). The data in B are the means6s.d. of at least three experiments. P,0.001 comparing 30 and 60 min to 0 min (left panel), and
comparing NAC and GSH to no addition (right panel), n54.
non-cleavable p75D255A mutant did not produce any ROS in
response to TNFa/CHX confirms that complex I is one major
source of this ROS generation. It has been reported that ROS can
be a direct consequence of Bax/Bak-mediated MOMP (Kirkland
et al., 2002; Kirkland and Franklin, 2003; Kirkland and Franklin,
2007; Jiang et al., 2008, Childs et al., 2008), because of the loss
of cytochrome c from the respiratory chain complex III/IV and
the fall in the mitochondrial membrane potential. This might be
the case for apoptotic stimuli such as etoposide or tunicamycin,
which did not provoke p75 cleavage despite effective cytochrome
c release, and did not depend on p75 for ROS production and
apoptosis. As with TNFa/CHX, the kinetics of cytochrome c
release induced by these two drugs were also similar between
p75WT and p75D255A cells. However, the situation is clearly
different for TNFa/CHX. Although it similarly triggers
cytochrome c release and ROS production as other apoptotic
stimuli, the latter event is entirely blocked in Apaf-12/2, caspase92/2 or caspase-3/72/2 MEFs. This reinforces the notion that in
response to TNFa/CHX ROS is generated downstream of
MOMP, e.g. by a caspase-3-mediated cleavage of p75, as has
previously been shown by Ricci et al., 2004. In their report they
also clearly revealed that the fall in the mitochondrial membrane
potential occurs after cytochrome c release in a caspase-3/p75
cleavage dependent manner (Ricci et al., 2004). More studies are
required to determine if other apoptotic stimuli (for example
other members of the TNF-superfamily such as FasL or TRAIL)
use the same mechanism to amplify apoptosis via LMP.
Although the major ROS species, which we measured in our
system were O22? (as evidenced by DHE or MitoSOX staining),
we cannot exclude that they were further converted into hydrogen
peroxide (H2O2) by mitochondrial superoxide dismutases. In fact,
in contrast to O22?, H2O2 is more cell permeable and may be the
ROS species that leave mitochondria and enter lysosomes where
they react with iron to produce the highly reactive hydroxyl
radicals (NOH) (Fenton reaction) which then induce LMP via lipid
peroxidation. Indeed the role of intralysosomal iron in oxidantinduced cell death has been previously reported (Yu et al., 2003).
Moreover, we could show here that incubating a postmitochondrial cellular fraction containing lysosomes with an
oxidizing buffer triggered LMP in vitro over time. The reason
why we did not detect major H2O2 production by DCDHF
staining might have been due to the use of CHX, which was
required for TNFa to effectively induce apoptosis (Walczak,
2011; Micheau and Tschopp, 2003; Andrew, 2004; Wang et al.,
Journal of Cell Science
TNFa-induced LMP requires ROS downstream of MOMP
4023
Fig. 6. TNFa/CHX-induced apoptosis depends on the Bax/Bakapoptosomal pathway and is amplified by the caspase-3mediated cleavage of the p75 NDUFS1 subunit of complex I,
mitochondrial ROS production and cathepsin B/L activities.
(A) Quantitative annexin-V/PI FACS analysis of WT, Bax/Bak2/2,
caspase-92/2, Apaf-12/2 or caspase-3/72/2 MEFs treated (gray
bars) or not (black bars) with T/C for 4 h showing that the T/Cinduced reduction of the number of annexin-V/PI-negative
(surviving) cells is dependent on the Bax/Bak–apoptosomal
pathway. (B) Dot-plot analysis of annexin-V (FL-1-green)/PI (FL-3red) stained WT MEFs treated or not (control) with T/C in the
presence or absence of 100 mM NAC or 5 mM MitoQ for 4 h. Note
that the number of apoptotic (lower right quadrant, 20.6%) and
secondary necrotic (upper right quadrant, 18.3%) in response to T/C
is largely not affected by NAC (25.8% and 13.6%, respectively), but
markedly decreased by MitoQ (9.0% and 7.2%, respectively),
indicating that this cell death is partially mediated via mitochondrial,
not general ROS production. (C) Quantitative annexin-V FACS
analysis of p75WT and p75D255A HeLa cells, as well as WT and
cathepsin-B/L-deficient MEFs treated or not with TNFa/CHX (or
E64d) for 4 h. The number of dying cells (apoptotic and necrotic)
upon T/C treatment is significantly reduced in the presence of the
caspase non-cleavable p75D255A mutant, indicating that cleavage of
p75 at its D255 site is not only crucial for ROS production (see
Fig. 2B) and LMP (Fig. 3D,E) but also amplifies T/C-induced cell
death. Cathepsin B and L appear to amplify T/C-induced cell death
in a similar way. The data in A and C represent the means6s.d. of at
least three experiments. For A, P,0.001 when comparing WT with
mutant cells; for C, P50.0004 between p75WT and p75D255A cells,
P50.0008 between WT and CTB/L2/2 MEFs and P50.001
between untreated and E64d-treated WT MEFs, n55.
2008), but which also is known to downregulate the
mitochondrial superoxide dismutase and ferritin heavy chain
(Sasazuki et al., 2004; Pham et al., 2004). This may explain why
ROS levels reached such high levels within a short time period
(2 h) of the TNFa/CHX treatment (Fig. 4B). These levels were
already at the maximum before a significant LMP was observed
(Fig. 4A) reinforcing that notion that LMP is caused by ROS.
The surprising finding of our study was that TNFa-induced
LMP turned out to be a process downstream of MOMP. Many
previous studies proposed an early induction of LMP by this
cytokine, for example via the activation of acid or neutral (FAN)
sphingomyelinases, cathepsin B or caspase-8 (Boya and
Kroemer, 2008; Blomgran et al., 2007; Guicciardi et al., 2000;
Guicciardi et al., 2005; Droga-Mazovec et al., 2008; Werneburg
et al., 2002; Werneburg et al., 2004; Gyrd-Hansen et al., 2006;
Ullio et al., 2012). How the lysosomal membrane is damaged by
these molecules has however not been unveiled so far, although a
direct perforation by Bax or Bak (Werneburg et al., 2007, Castino
et al., 2009; Kagedal et al., 2005; Feldstein et al., 2006) or the
implication of ROS (without showing from where they in fact
originated) were proposed (Zdolsek et al., 1993; Brunk et al.,
1995; Roberg and Öllinger, 1998; Brunk and Svensson, 1999;
Terman et al., 2006). LMP results in the release of cathepsins,
which then either cleave Bid to tBid, process caspases or degrade
Bcl-2-like survival factors to induce MOMP (Boya and Kroemer,
2008; Blomgran et al., 2007; Guicciardi et al., 2000; Guicciardi
et al., 2005; Droga-Mazovec et al., 2008). These signaling
pathways, and therefore LMP, were supposed to be initiating,
requiring events for TNFa-induced apoptosis. Astonishingly,
none of the previous studies verified if LMP was dependent on
MOMP, i.e. if it was blocked in Bax/Bak2/2 cells. Our study now
reveals for the first time that TNFa/CHX-induced LMP, as
determined by a diminished red fluorescent staining of the
lysosomotropic dye AO and the release of cathepsin B/L activity
and cathepsin B protein into the cytosol, is entirely dependent on
Bax/Bak, the apoptosome and caspase-3/7. Since ROS
production is also dependent on this pathway, and that LMP is
inhibited by MitoQ treatment or in p75D225A HeLa cells, the
mechanistic link between TNFa and LMP is mitochondrially
generated ROS. Thus, rather than being an initiating process,
LMP occurs as an amplification loop downstream of MOMP.
Such a mechanism explains why inhibiting ROS generation or
performing the experiments in p75D225A HeLa cells did not
entirely eliminate TNFa/CHX-induced apoptosis, because cell
death could still proceed at a low level without the amplification
loop. This adds TNFa to the growing list of apoptotic stimuli,
which we have previously shown to trigger LMP downstream of
MOMP (Oberle et al., 2010). We therefore propose that in most,
if not all cases, LMP and the release of cathepsins are not
required for apoptosis induction but rather amplify the process to
make sure that cell death occurs effectively, rapidly and
irreversibly.
Materials and Methods
Cell lines and cell growth conditions
3T9 MEFs from wild type (WT) C57BL/6 mice or mice deficient of Apaf-1,
caspase-9 or both Bax and Bak (Bax/Bak2/2) were obtained from Andreas
Strasser, WEHI, Melbourne. SV40 T antigen transformed MEFs deficient of both
caspase-3 and -7 (caspase-3/72/2) were kindly provided by Richard Flavell, Yale,
USA. 3T9 transformed MEFs deficient for both cathepsin B and L (CTB/L2/2) are
described in Oberle et al., 2010. HeLa cells stably expressing the wild-type (WT)
p75 NDUFS1 subunit of respiratory chain complex I or its caspase-3 non-cleavable
4024
Journal of Cell Science 126 (17)
mutant p75D255A are described in Ricci et al., 2004. All cells were cultured in
DMEM supplemented with 10% FCS, 100 units/ml penicillin and 100 mg/ml
streptomycin at 37 ˚C/5% CO2.
Apoptosis assay
For TNFa/CHX-induced cell death, fibroblasts were preincubated or not with
5 mM MitoQ or 30 mM E64d (Biomol, Germany) for 24 h and then treated with
10 ng/ml of TNFa in combination with 1 mg/ml CHX for 4 h. For etoposide or
tunicamycin-induced cell death, fibroblasts were treated with 100 mM etoposide or
1 mg/ml tunicamycin with or without 5 mM MitoQ for 24 h. Apoptotic cells were
determined by flow cytometry after staining with His–GFP–Annexin-V/propidium
iodide (PI) as described in Egger et al., 2003.
Cellular fractionation
Cellular fractionation was carried out using the Cell Fractionation Kit Standard
from Abcam which retains mitochondria intact. Briefly, cells were collected,
washed once with buffer A and centrifuged. The pellet was resuspended in buffer
A to 6.66106 cells/ml, an equal volume of buffer B was added and then the cells
were incubated for 7 min on a rotator at room temperature and centrifuged
sequentially at 5000 g and 10,000 g at 4 ˚C for 1 min each. The supernatant was
collected as cytosolic fraction. The cytosol-depleted pellet was resuspended in
buffer A and mixed with buffer C containing detergent and then incubated for
10 min on a rotator at room temperature. After two sequential centrifugations as
above the supernatant was collected as mitochondrial fraction. To get the
lysosomes the cytosolic fraction was further centrifuged at 100,000 g, 1 h. The
pellet represented the lysosomal fraction.
Journal of Cell Science
Cathespsin B/L activity assay
Cathepsin B (CTB) and cathepsin L (CTL) specific activities of cytosolic and
heavy membrane fractions were measured in a buffer containing 200 mM NaOAc,
1 mM EDTA, 0.1% Brij 35, 0.1% Triton X-100 at pH 6.7. Briefly, 5 ml of
cytosolic or heavy membrane fraction (25 mg protein) was mixed with 90 ml assay
buffer and 5 ml of the cathepsin B substrate Z-FR-AMC (25 mM) in each well of a
black Nunc 96 MicroWellTM plate (Nunc, Denmark) and incubated at 37 ˚C for 1 h
before measuring fluorescence at excitation 355 nm and emission 460 nm in a
Fluoroskan Ascent (Thermo Labsystem).
LMP assays
Cells were exposed or not to apoptotic stimuli and LMP was detected by flow
cytometry (FL1:FL3) after staining with 5 mM AO in 16Hank’s balanced salt
solution (HBSS) with Ca2+/Mg2+ (0.137 M NaCl, 5.4 mM KCl, 0.25 mM
Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 4.2 mM
NaHCO3) for 10 min at 37 ˚C, or alternatively by ScanR cytometry (Olympus)
after in situ staining and mounting. To measure LMP induced by ROS in vitro, a
post-mitochondrial fraction was extracted from MEFs loaded with 5 mM AO,
washed twice and then incubated with or without 1 mM N-acetylcysteine (NAC)
or 1 mM glutathione (GSH) at 37 ˚C for up to 1 h. Afterwards the postmitochondrial fraction was centrifuged as described above to separate cytosol from
membranes, and AO fluorescence was measured at excitation 485 nm and
emission 538 nm. In addition, the distribution of cathepsin B/L activities between
the membrane and cytosolic fraction was determined by the Z-FR-AMC assay as
described above.
ROS measurement
After treatment with various pro-apoptotic stimuli in the presence or absence of
5 mM MitoQ or 100 mM of the general caspase inhibitor Z-VAD.fmk (Promega)
the MEFs were incubated for 10 min in 16HBSS with Ca2+/Mg2+ buffer
containing 2 mM dihydroethidium (DHE) or 1 mM 29,79-dichlorofluorescein
(CM-H2DCFDA) or 1 mM MitoSOXTM red mitochondrial superoxide indicator
(Invitrogen). After two washes the cells were either subjected to FACS analysis
(for DHE and CM-H2DCFDA) or fluorescence microscopy (Zeiss) or ScanR
fluorescence cytometry (Olympus) (for MitoSOXTM). As positive control for ROS
production, cells were incubated with 0.5 mM rotenone for 2 or 4 h.
Cellular respiration assay
The experiment was carried out using high-resolution respirometry (Oxygraph 2k
Oroboros Instruments) according to the manufacturer’s instruction. Oxygen
consumption was measured in intact cells pretreated or not with TNFa/CHX for
2 h. Chamber A and B were filled with 2 ml of complete DMEM and compensated
for at least 5 min to stabilize temperature and oxygen. 26106 in 50 ml DMEM
were injected into each chamber and oxygen consumption was measured as
described in Kuznetsov et al., 2008. As a control for mitochondria-specific
respiration the cells were treated with 0.5 mM rotenone for 2 h prior to harvesting.
Western blot analysis
Cytosolic or heavy membrane fractions (containing all other organelles except the
nuclei) were collected as described above and the membrane fraction was lysed in
RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X100, 1% n-octylglucoside,
0.1% SDS, 1 mM EDTA, 150 mM NaCl) containing proteinase inhibitors
(16protease inhibitors cocktail, Roche) for 30 min on ice. 20 mg of cytosolic or
membrane proteins were subjected to SDS-PAGE followed by western blotting to
nitrocellulose (NC) membranes according to standard protocol. Immunodetection
was performed by incubating the NC membranes with the first antibody in PBS
containing 0.05% Tween-20 for 4 h followed by a horse radish peroxidase (HRP)conjugated secondary antibody for 1 h. The following first and secondary
antibodies were used: goat polyclonal anti-cathepsin B (R&D systems), rabbit
polyclonal anti-cytochrome c and anti-COX IV (3E11) (Cell Signalling), mouse
polyclonal anti-NDUFS1 (ab52690), rabbit polyclonal anti-NDUFB6 (ab103531),
rat monoclonal anti-Lamp-1 (1D4B) (Abcam), mouse monoclonal anti-b-actin
(Pierce), and HRP-conjugated donkey anti-goat, donkey anti-rabbit and goat antimouse antibodies (Dianova). Signals were visualized by enhanced
chemiluminescence (ECL) (Pierce).
Statistical analysis
All quantitative data are shown as means6standard deviation (s.d.) and the
statistical significance was determined by two-tailed Student’s t-test.
Acknowledgements
We thank Michael P. Murphy, MRC, Cambridge, United Kingdom
for MitoQ, Andreas Strasser, WEHI, Melbourne, Australia for the
Bax/Bak2/2, Apaf-12/2 and caspase-92/2 3T9 MEFs and Richard
Flavell, University of Yale, CT, USA, for the SV40 transformed
caspase-3/72/2 cells. We are grateful to Robert Pick, Walter Brendel
Center, Munich, Germany for the technical assistance with the
ScanR and Thomas Reinheckel, Freiburg, for helping with the
cathepsin assays. The authors declare that they have no conflict of
interest.
Author contributions
C.B. and J.H. conceived the project. J.H., Y.L., L.J., T.K. and F-N.V.
carried out the experiments. J.E.R. provided the p75WT and
p75D255A HeLa cells. C.B. and J.H. prepared the figures and
wrote the manuscript.
Funding
This work was supported by the Jose Carreras Foundation, Germany
(DJCS) [grant number R 06/09 to T.K. and C.B.]; the Excellence
Initiative, Germany, Spemann Graduate School of Biology and
Medicine (GSC-4) (to C.B. and L.J.); and by l’Agence Nationale de
la Recherche [grant number ANR-09-JCJC-0003 to J.E.R.]. J.H. was
supported by core funding from the Institute of Molecular Medicine,
F-N.V from the Institute of Biochemistry and Molecular Biology,
University of Freiburg, Germany. Y.L. received a FTN fellowship of
the Uniklinik Mainz, Germany.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.129999/-/DC1
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Supplementary Fig. S1. Oxygen consumption in intact WT and Bax/Bak-/- MEFs
or p75WT and p75D255A HeLa cells treated or not with TNFα/CHX
Cellular respiration assay carried out for up to 30 min in intact, untreated or
TNFα/CHX (T/C)-treated (2 h) WT or Bax/Bak-/- MEFs (A) or p75WT and p75D255A
HeLa cells (B) in the high-resolution respirometer Oxygraph 2k (Oroboros
Instruments) according to manufacturer´s instruction. At some points 1 mM NADH
and succinate were added but this did not further stimulate oxygen consumption
because we measured intact cells. To make sure that oxygen was consumed in
mitochondria, 0.5 µM rotenone was added 2 h prior to the measurement. Oxygen
concentration is depicted in blue (nmol/ml), and oxygen flow per cell in red
(pmol/(s*ml). Arrows mark the beginning of the respiration measurements when 2 x
106 cells were injected into the Oxygraph). (C) Cells were either untreated or treated
with TNFα/CHX for 2 h before respiration measurements. The consumption rate was
calculated over a period of 5 min. WT MEFs and p75 WT HeLa cells were taken as
100%, respectively. Note that Bax/Bak-/- respired faster than WT MEFs. By contrast,
the respiration of p75D255A was slower than that of p75WT HeLa cells. T/C treatment
diminished respiration in both WT cells, but Bax/Bak-/- MEFs and p75D255A HeLa
cells retained their oxygen consumption rates. As expected rotenone drastically
diminishes respiration. Data from WT and Bax/Bak-/- cells represent the means of at
least three independent experiments ± SD, p < 0.001 between Bax/Bak-/- and WT
MEFs (untreated and treated) and between T/C treated and untreated WT MEFs (left
panel); p = 0.005 between p75D255A and p75WT HeLa (untreated and treated) and p <
0.001 between T/C treated and untreated p75WT cells (right panel), n=5.
1 O2 conc. (nmol/ml)
O2 conc. (nmol/ml)
O2 conc. (nmol/ml)
O2 conc. (nmol/ml)
O2 conc. (nmol/ml)
O2 conc. (nmol/ml)
WT
+ rotenone
O2 flow per cell
(pmol/s*ml)
Bax/Bak-/+ T/C
O2 flow per cell
(pmol/s*ml)
WT
+ T/C
O2 flow per cell
(pmol/s*ml)
p75WT
+ T/C
p75D255A
+ T/C
+
p75WT
rotenone
O2 flow per cell
(pmol/s*ml)
D255A
p75
p75D255A
O2 flow per cell
(pmol/s*ml)
Bax/Bak-/-
O2 flow per cell
(pmol/s*ml)
p75WT
O2 flow per cell
(pmol/s*ml)
B
O2 flow per cell
(pmol/s*ml)
O2 flow per cell
(pmol/s*ml)
cell injection
O2 conc. (nmol/ml) O2 conc. (nmol/ml)
WT
O2 flow per cell
(pmol/s*ml)
O2 conc. (nmol/ml) OO2 2conc.
conc.(nmol/ml)
(nmol/ml)
A
cell injection
Figure S1
Relative oxygen consumption (%)
Relative oxygen consumption (%)
p < 0.001
p < 0.001
p < 0.001
+
TNFα/CHX
*+,!ɲ-./0'
Relative oxygen consumption (%)
Relative oxygen consumption (%)
.'
C
p = 0.005
p = 0.005
p < 0.001
+ TNFα/CHX
*+,!ɲ-./0'
Figure S1
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