From the Laboratory of Biochemistry I, In the present study we show that
N-acetylsphingosine (C2-ceramide),
N-hexanoylsphingosine (C6-ceramide), and, to a
much lesser extent, C2-dihydroceramide induce cytochrome
c (cyto c) release from isolated rat liver
mitochondria. Ceramide-induced cyto c release is prevented
by preincubation of mitochondria with a low concentration (40 nM) of Bcl-2. The release takes place when cyto c is oxidized but not when it is reduced. Upon cyto
c loss, mitochondrial oxygen consumption, mitochondrial
transmembrane potential ( Recently, the importance of ceramide in cell metabolism has been
broadly investigated. It is now evident that ceramide is involved as a
second messenger in what has become known as the sphingomyelin cycle
(1), apoptosis, and differentiation in many cell types (2). The
mechanisms by which ceramide mediates apoptosis have not yet been fully
addressed, however, it is known that mitochondria are targets of
ceramide. Thus, direct inhibition of complex III of the mitochondrial
respiratory chain by ceramide (3), ceramide-induced generation of
reactive oxygen species in intact mitochondria (4) and in cells (5, 6),
and ceramide-induced cell death via disruption of mitochondrial
functions (7) are lines of evidence of the strong influence of ceramide
on mitochondria.
Cytochrome c (cyto
c)1 plays a dual
role in cell homeostasis. As a part of the respiratory chain, it is
needed for cell life, and as one of the triggers of apoptosis, it is
needed for cell death. It is now well accepted that many apoptogenic
factors induce cell death via mitochondrial cyto c release
(8). The released cytochrome switches on the death machinery, for
example, by activation of caspases (9, 10). The anti-apoptoic protein,
Bcl-2, was shown to prevent apoptosis both upstream (8) and downstream (11) of cyto c release.
In the present study we show that 1) C2-ceramide
(N-acetylsphingosine), C6-ceramide
(N-hexanoylsphingosine), and, to a much lesser extent, DHC
(C2-dihydroceramide) release cyto c from
isolated mitochondria, 2) ceramide-induced cyto c release
occurs when cyto c is oxidized but not when it is reduced,
3) this release is prevented by Bcl-2, 4) cyto c release
causes a decrease in mitochondrial oxygen consumption, transmembrane
potential ( C2- and C6-ceramide were obtained from
Alexis Biochemicals (Läufelfingen, Switzerland), DHC from
Calbiochem (Luzern, Switzerland), horse heart cytochrome c
from Sigma, mouse monoclonal cyto c antibody from RDI
(Flanders, NJ), anti-mouse Ig and horseradish peroxidase from Amersham
and His6-human Bcl-2 from Novartis (Basel, Switzerland). Ceramide stock solutions were prepared at a 500 times concentration in
ethanol (containing 1% Me2SO) and kept at Mitochondrial Preparation--
Isolation of rat liver
mitochondria was performed by differential centrifugation as described
(12). The protein content of mitochondria and the mitochondrial
supernatants were determined by the Biuret method with bovine serum
albumin as standard.
Detection of Cytochrome c Release--
Freshly isolated
mitochondria (10 mg protein/ml) were incubated at room temperature in
0.1 M HEPES buffer, pH 7.0, containing aprotinin, pepstatin
A, and leupeptin (1.5 µg/ml each). To investigate the effect of
ceramide when cyto c is oxidized, mitochondrial respiratory
chain complex III was blocked by 50 nM antimycin A (AA),
and after 1 min, ceramide (20 µM) or the vehicle was
added. Mitochondria were incubated for 2 min and then energized with 1 mM ascorbate (Asc) plus 0.4 mM
tetramethyl-1,4-phenylenediamine (TMPD) (Asc/TMPD). The effect of
ceramide on mitochondria when cyto c is reduced was studied
by addition of ceramide 1 min after Asc/TMPD. Bcl-2 (40 nM)
was added 5 min before AA. After 10 min of incubation at room
temperature, mitochondria were spun at 12,000 × g for
10 min at 4 °C, and the resulting supernatant was spun at
100,000 × g for 15 min at 4 °C. The supernatant of
the second centrifugation was used for the detection of cyto
c either spectrophotometrically or by gel electrophoresis.
Spectrophotometric measurements were done in a Varian Cray
spectrophotometer. As the blank sample, 10 mg of mitochondrial protein
was diluted in 1 ml of the buffer, mixed gently, and spun immediately
in 2 steps as mentioned above, and the supernatant was considered as
the blank. The absorption of the supernatants of mitochondria incubated
with ceramide or the vehicle was then recorded before and after
reduction with sodium dithionite. The concentration of cyto
c was calculated using an extinction coefficient of 19.0 mM
For studying the possible influence of Analysis of Mitochondrial Functions--
For studying
mitochondrial functions, 1 mg of mitochondrial protein/ml was incubated
in 0.1 M HEPES buffer, pH 7.0, with the following compounds
present where appropriate: 10-20 µM ceramide, 50 nM AA to block complex III, Asc/TMPD to reduce cyto
c, 1 mM KCN to block complex IV, 1 mM ATP as the substrate of ATPase, 1 µM
carbonyl cyanide m-chlorophenylhydrazone to uncouple
mitochondria, and 1.7 µg/ml oligomycin to block ATPase. Ceramide was
added when cyto c was oxidized, i.e. after AA, or
when it was reduced, i.e., after Asc/TMPD. Bcl-2 was added 5 min prior to the above mentioned compounds.
Oxygen consumption was measured at room temperature with a Clark-type
oxygen electrode (Yellow Spring Instruments, Yellow Spring, OH) under
continuous stirring. Mitochondrial transmembrane potential was measured
in an Aminco DW-2A spectrophotometer at 511-533 nm in the presence of
10 µM safranin as described (14). Mitochondrial
Ca2+ uptake and release was measured in 0.1 M
HEPES buffer, pH 7.0, containing 10 µM CaCl2
(10 nmol of Ca2+/mg of mitochondrial protein) at 685-675
nm in the presence of 50 µM Arsenazo III as described
(15).
Assessment of Cytochrome c Reduction--
The optical density of
a 10 µM oxidized horse heart cyto c solution
in the presence of 10 µM ceramide or the vehicle was
recorded between 380 and 600 nm at 1-nm intervals with a Varian Cray
spectrophotometer. Oxidized cyto c was then reduced stepwise
by the repeated addition of 2 µM ascorbate (taken from a
2 mM freshly prepared stock solution) to the cuvette. After
each reduction step, the optical density was again recorded. The
recorded optical density of the oxidized cyto c in each
wavelength was subtracted from the corresponding one of the reduced
cyto c and plotted against the wavelengths.
Ceramide-induced mitochondrial cyto c release was
determined by SDS-PAGE (Fig.
1A), by Western blotting (Fig.
1B), and spectrophotometrically (Fig. 1C). In
these experiments, ceramide was added when cyto c was mainly
oxidized, i.e. after blocking the complex III. Addition of
ceramide to mitochondria when cyto c was mainly reduced,
i.e., in the presence of Asc/TMPD, did not increase the
released cyto c, compared to the control (Fig.
1D). Preincubation of mitochondria with 40 nM
Bcl-2 (4 pmol of Bcl-2/mg of mitochondrial protein) fully prevented the
release of cyto c induced by ceramide (see Fig.
1B). When equal volumes (20 µl) of the supernatants of
mitochondria incubated with ceramides were separated by SDS-PAGE, an
increase in the total protein amount released into the supernatant was detected (Fig. 1A). When equal amounts (20 µg) of the
released proteins were analyzed by Western blot, a specific increase in the cyto c was found (Fig. 1B).
To investigate the consequences of cyto c release on
mitochondrial functions, we measured mitochondrial oxygen consumption,
ABSTRACT
Top
Abstract
Introduction
References
), and Ca2+ retention are
diminished. Incubation with Bcl-2 prevents, and addition of cyto
c reverses the alteration of these mitochondrial functions.
In ATP-energized mitochondria, ceramides do not alter
, neither
when cyto c is oxidized nor when it is reduced, ruling out
a nonspecific disturbance by ceramides of mitochondrial membrane integrity. Furthermore, ceramides decrease the reducibility of cyto
c. We conclude that the apoptogenic properties of ceramides are in part mediated via their interaction with mitochondrial cyto
c followed by its release and that the redox state of cyto c influences its detachment by ceramide from the inner
mitochondrial membrane.
INTRODUCTION
Top
Abstract
Introduction
References
), and Ca2+ retention, all of which are
prevented by preincubation of mitochondria with Bcl-2 and reversed by
addition of cyto c, and 5) ceramide interacts with cyto
c and changes its reducibility.
EXPERIMENTAL PROCEDURES
20 °C. The
vehicle always served as control.
1 cm
1 for reduced cyto
c at 550-540 nm (13) and is expressed as the percentage of
the control. For gel analysis, 20 µl of the supernatant of
mitochondria incubated with ceramide or the vehicle was separated by
15% SDS-PAGE and stained with Coomassie Blue. For Western blotting, 20 µg of protein of supernatants were separated by 15% SDS-PAGE, blotted onto a nitrocellulose membrane, probed by a monoclonal mouse
anti-cyto c antibody, and developed by enhanced chemiluminescence.
on mitochondrial cyto
c release,
was fully abrogated by incubation of
mitochondria with 5 µM rotenone, 50 nM AA,
and 1 µM carbonyl cyanide
m-chlorophenylhydrazone. The absence of
was verified
as explained below. After 10 min, mitochondria were spun as described
above, and the supernatant was tested for the presence of cyto
c by Western blotting.
RESULTS
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Fig. 1.
Ceramide-induced cyto c
release from rat liver mitochondria. Release was assayed by
Coomassie Blue staining (A), Western blotting
(B), and spectrophotometry (C). In these
experiments, ceramide was added when cyto c was oxidized. In
D (Coomassie Blue staining), ceramide was added when cyto
c was reduced. Mitochondria were treated with vehicle
(Cont), C2-ceramide (C2),
C6-ceramide (C6), or DHC (for details, see under
"Experimental Procedures"). The left lane of
A and D shows 5 µg of pure cyto c
(arrowhead).
, and Ca2+ homeostasis. Fig.
2A shows that addition of
ceramide to mitochondria when cyto c was oxidized decreased
the oxygen consumption supported by Asc/TMPD. Conversely, ceramide
added to mitochondria when cyto c was reduced did not change
the oxygen consumption (Fig. 2B). The decreased oxygen
consumption caused by ceramide was prevented by preincubation of
mitochondria with 40 nM Bcl-2 (Fig. 2C) and was
reversed by the addition of 200 nM exogenous cyto
c (200 pmol of cyto c/mg of mitochondrial
protein) (Fig. 2D).
View larger version (27K):
[in a new window]
Fig. 2.
. Mitochondrial oxygen consumption in the
presence of ceramide. 10 µM ceramide was added
when cyto c was oxidized (A) or reduced
(B). C, as for A except that
mitochondria were preincubated for 5 min with 40 nM Bcl-2.
D, as A except that 200 nM cyto
c was added at the point marked by the asterisk.
Mitochondria were treated with vehicle (Cont),
C2-ceramide (C2), C6-ceramide
(C6), or DHC (for details, see under "Experimental
Procedures"). , addition of AA;
, addition of Asc/TMPD.
A decrease in is considered important when cells commit suicide
(16). Fig. 3A shows that
addition of ceramide to mitochondria when cyto c was
oxidized caused a decrease in
. This figure also shows that
addition of 200 nM exogenous cyto c resulted in a full gain of
. Incubation of mitochondria with 40 nM Bcl-2 prevented the loss of
caused by ceramide
(not shown). Fig. 3B shows that ceramide did not alter
when cyto c was mainly reduced. When
was built
up as a consequence of ATP hydrolysis instead of respiration, ceramide
did not change
, neither when cyto c was oxidized nor
when it was reduced (not shown).
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Mitochondria are important calcium buffers in eukaryotic cells, and mitochondrial calcium release is involved in apoptosis (17). Fig. 4 shows that C6-ceramide, added to mitochondria when cyto c was mainly oxidized, caused a decrease in Ca2+ retention by mitochondria, in a Bcl-2 sensitive manner. The same results were obtained with C2-ceramide and, to a minor extent, with DHC (not shown). Addition of ceramide to mitochondria when cyto c was reduced did not change the mitochondrial Ca2+ homeostasis (not shown).
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Reduction of cyto c can be followed photometrically. Fig.
5 shows that in the presence of
C2-ceramide, the reduction of cyto c by Asc was
hampered, as evidenced by the smaller increase in the optical densities
of the - and
-regions. Again, C2-ceramide was
most effective, followed by C6-ceramide and DHC (not
shown).
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DISCUSSION |
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The present study shows that ceramide induces cyto c
release from isolated mitochondria, an event strongly influenced by the redox state of cyto c, and that incubation of mitochondria
with Bcl-2 prevents the cyto c release. Release of cyto
c decreases mitochondrial oxygen consumption, , and
the Ca2+ buffering capacity of mitochondria, all of which
are reversed by addition of exogenous cyto c. For all
parameters measured, the observed rank order of potency is
C2 > C6 > DHC. This study also provides
evidence for a possible direct interaction of ceramide and cyto
c.
There is growing evidence that mitochondria are involved in apoptosis
(16). The release of cyto c from mitochondria triggers apoptosis (18), and decreases during apoptosis (16, 19-21). Accordingly, prevention of cyto c release (22) as well as
stabilization of
(23) prevent apoptosis. Furthermore,
mitochondria carry the pro- and antiapoptotic proteins, cyto
c and Bcl-2. The presence of procaspase-3 in mitochondria
was also shown recently (24).
Several mediators, pathways, and factors are involved in apoptosis (25, 26). Among them, ceramide has been shown to directly target mitochondria. Zhang et al. (27) showed that in Molt-4 leukemic cells, 6 h of incubation with C6-ceramide increased the cytosolic concentration of cyto c, which was preventable by overexpression of Bcl-2. In a study by Amarante-Mendes et al. (28) 6 h of incubation with C2-ceramide caused the cytosolic accumulation of cyto c in control but not in Bcr-Abl-overexpressing HL-60 cells. In the present study, we show that ceramide directly causes the release of cyto c from isolated mitochondria and accordingly hypothesize that cyto c is the prime mitochondrial target of ceramide. The fact that incubation of mitochondria with a low concentration of Bcl-2, used in this study, prevents ceramide-induced cyto c release and its consequences suggests a specific function of the oncoprotein in ceramide-mediated apoptotic signals.
To investigate a possible direct interaction of ceramide with cyto c, we primed Sepharose columns with the biologically active D-C2-ceramide or biologically inactive L-C2-ceramide.2 We observed that D-C2-ceramide but not L-C2-ceramide columns selectively retained cyto c and that the retained protein could be eluted with D-C2-ceramide. We also observed that reduced cyto c had a much lower affinity to D-C2-ceramide column, compared to the oxidized cyto c. Because of that observation together with the fact that ceramide affects the reducibility of cyto c (Fig. 5), we hypothesize that ceramide may directly interact with cyto c, with a higher affinity for the oxidized protein, and that this interaction changes the physicochemical properties of cyto c, leading to its rejection from mitochondria. The fact that cyto c has multiple lipid binding sites and that the lipid-bound cyto c shows a lower affinity for attachment to the artificial membranes (29) strengthens this hypothesis.
Under our experimental conditions, prevention of mitochondrial cyto
c reduction is paralleled by the disappearance of . To
distinguish which of these two is decisive for ceramide-induced cyto
c release, we investigated by Western blot analysis whether cyto c is released because of
collapse. We found that
the absence of
, achieved by blocking respiration and uncoupling
of mitochondria, does not result in cyto c release,
indicating that binding of cyto c to the outer side of the
inner mitochondrial membrane does not require
. These results
also indicate that the release of cyto c by ceramide is not
a consequence of a nonspecific solubilization of mitochondrial
membranes due to the lipophilicity of ceramide, but rather is a
specific event.
The release of other mitochondrial proteins apart from cyto c is also increased upon treatment with ceramide (Fig. 1A). We argue that this apparently nonspecific protein release by ceramide is due to a general weakening of mitochondria upon cyto c release. Fig. 1D supports this argument by showing that when ceramide does not cause cyto c loss, other mitochondrial proteins are also not released. This notion, together with the fact that addition of exogenous cyto c reverses all the altered mitochondrial functions, leads us to conclude that mitochondrial cyto c is a prime target for ceramide. This conclusion does not rule out other possibilities, for example, the modification of Bcl-2 by ceramide.
In contrast to the finding that binding of cyto c to the
inner mitochondrial membrane is not a function of , the formation and the maintenance of
is dependent on the presence of a
functional cyto c (cf. Fig. 3A).
Disruption of
was shown to be involved in ceramide-induced
apoptosis (30). According to our study, cyto c has a
critical role in
formation, in that stabilization of cyto
c stabilizes
. Bcl-2 is located at the outer
mitochondrial membrane (31) and has been shown to stabilize
(23). It is known that Bcl-xL, another oncoprotein with mitochondrial
location, binds cyto c (32). Based on the present results
and the close proximity of Bcl-2 to mitochondrial cyto c, it
may be speculated that Bcl-2 stabilizes
by preventing cyto
c loss.
Nonspecific solute transport across the inner mitochondrial membrane,
operated via a Bcl-2-sensitive megachannel (the "permeability transition pore") is considered to be the reason for the collapse of
and many other features of apoptosis (reviewed in Ref. 33). It
was also reported that operation of such a pore causes cyto c release, and thus it was concluded that cyto c
release occurs downstream to pore formation and
collapse (34).
Because addition of cyto c reverses the ceramide-induced
decrease in mitochondrial oxygen consumption,
, and
Ca2+-maintaining capacity (see Figs. 2-4), we argue that
ceramide-induced apoptosis is not mediated via opening a nonspecific
megachannel or pore. The fact that ceramide does not affect
when
it is supported by ATP gives further support to this argument.
It was reported by Gudz et al. (3) that ceramide decreases
the activity of complex III of the mitochondrial respiratory chain, as
deduced from the measurement of mitochondrial oxygen consumption
supported by Asc/TMPD. Cyto c shuttles electrons between complex III and IV, and oxygen is consumed at the level of complex IV.
Figs. 2D and 3A show that the addition of cyto
c to mitochondria recovered the decreased respiration and
the reduced . Therefore, we conclude that parts of the
ceramide-induced reduction of complex III activity is due to cyto
c loss.
Garcia-Ruiz et al. (4) showed that ceramide induces superoxide radical formation in isolated rat liver mitochondria. From the elegant recent study by Cai and Jones (35), it is evident that superoxide formation by mitochondria is a consequence, and not the cause, of cyto c loss. Release of cyto c from mitochondria produces a gap in the hierarchically arranged mitochondrial respiratory complexes III and IV, and therefore a site for electron leakage. Present finding explains the ceramide-induced superoxide formation, reported by Garcia-Ruiz et al. (4), and supports the hypothesis that cyto c is the prime mitochondrial site of action of ceramide.
We reported (23) that TNF- added to cells induces a drop in
that is accompanied by increased reactive oxygen species formation in
mitochondria and that both events are prevented in Bcl-2-overexpressing
cells. It is known that de novo ceramide synthesis is part
of the transmembrane signaling transduction mechanism of TNF-
receptor (36-40). It was also reported that stimulation of TNF-
receptors causes apoptotic cell death by releasing mitochondrial cyto
c (41). The present study makes the link: after de
novo ceramide synthesis upon TNF-
receptor stimulation,
mitochondrial cyto c is released,
is decreased, and
reactive oxygen species formation is favored. Accordingly, overexpression of Bcl-2 prevents the apoptogenic properties of TNF-
(37) by prevention of ceramide-induced cyto c release.
It was reported by Hampton et al. (42) that cyto
c does not necessarily need to be reduced to activate
caspases. The investigators acknowledged, however, that they were
unable to keep cyto c oxidized in the presence of cytosolic
extracts. In the present study, we show that oxidized cyto c
is released by ceramide (Fig. 1, A-C) and that ceramide
decreases the reducibility of oxidized cyto c (Fig. 5).
Regarding the fact that both ceramide and cyto c are strong
mediators of caspase-induced apoptosis (43), the possible interaction
of ceramide and cyto c may thus amplify the progression of
the apoptotic cascade.
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ACKNOWLEDGEMENTS |
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We thank Dr. Wolfgang Blodig for very useful discussions, Dr. Theresa Visarius for critically reading the manuscript, Novartis Pharma AG for providing Bcl-2, and Dr. Kaspar H. Winterhalter for his interest and support. D- and L-ceramide Sepharose affinity chromatography columns were a generous gift of Dr. Joseph Brunner, Federal Institute of Technology (Zurich, Switzerland).
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Parts of this work were presented in the Biochemical Society meeting, Sheffield, United Kingdom, July 1998.
Financially supported by Swiss National Research Foundation Grant
31-45821.95.
§ Financially supported by Leica and Avina Foundation (Switzerland).
To whom correspondence should be addressed. Tel.:
41-1-632-31-36; Fax: 41-1-632-11-21; E-mail:
richter{at}bc.biol.ethz.ch.
2 P. Ghafourifar, C. Richter, and J. Brunner, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
cyto c, cytochrome c;
AA, antimycin A;
Asc, ascorbate;
TMPD, tetramethyl-1,4-phenylenediamine;
C2-ceramide, N-acetylsphingosine;
C6-ceramide, N-hexanoylsphingosine;
DHC, C2-dihydroceramide;
, mitochondrial transmembrane potential;
PAGE, polyacrylamide gel
electrophoresis.
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REFERENCES |
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