From the Department of Neurology and
Neuroscience, Weill Medical College of Cornell University, New York,
New York 10021, ¶ Department of Neurology, University of Miami
School of Medicine, Miami, Florida 33136, and
Department of
Psychiatry, Harvard Medical School and Laboratory for Oxidation Biology
Genetics and Aging Research Unit, Massachusetts General Hospital,
Charlestown, Massachusetts 02129
Received for publication, March 29, 2002, and in revised form, September 27, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Members of the BCL-2-related antiapoptotic
family of proteins have been shown previously to regulate ATP/ADP
exchange across the mitochondrial membranes and to prevent the loss of
coupled mitochondrial respiration during apoptosis. We have found that BCL-2/BCL-xL can also improve mitochondrial oxidative
phosphorylation in cells harboring pathogenic mutations in
mitochondrial tRNA genes. The effect of BCL-2 overexpression in mutated
cells was independent from apoptosis and was presumably associated with a modulation of adenine nucleotide exchange between mitochondria and
cytosol. These results suggest that BCL-2 can regulate respiratory functions in response to mitochondrial distress by regulating the
levels of adenine nucleotides.
The proto-oncogene BCL-2 prevents apoptosis and some
forms of cellular necrosis (1), although the exact mechanisms of its anti-cell death function are not known. BCL-2 was shown to localize to
multiple cell compartments, including the outer mitochondrial membrane
(2). A number of functions related to mitochondria have been proposed
for BCL-2, including the capacity to prevent loss of mitochondrial
membrane potential and opening of the mitochondrial permeability
transition pore (3), which might be responsible for the activation of
certain apoptotic pathways (4). In addition, BCL-xL,
another member of the BCL-2 antiapoptotic family of proteins, was shown
to regulate ATP/ADP exchange across the mitochondrial membranes and to
prevent the loss of coupled mitochondrial respiration in response to
proapoptotic stimuli (5, 6). It was proposed that the ability of the
BCL-2 family of proteins to maintain mitochondrial integrity after
proapoptotic stimuli might be related to maintaining the
voltage-dependent anion channel of the outer
mitochondrial membrane in an open configuration. This would prevent the
accumulation of anions, particularly ADP, ATP, and
creatine-PO4, in the mitochondrial intermembrane
space (5-7). Moreover, BCL-xL overexpression can stimulate
mitochondrial respiration in cultured cells independent of the
induction of apoptosis (8).
Mutations in mtDNA are associated with a heterogeneous group of
sporadic or maternally inherited metabolic disorders. Usually these
mutations cause impairment of oxidative phosphorylation (OXPHOS)1 with a reduction in
mitochondrial membrane potential and ATP synthesis (9).
We studied the effect of BCL-2 and BCL-xL overexpression on
mitochondrial and cytosolic ATP pools in human transmitochondrial cybrid lines containing normal mtDNA, mutated mtDNA causing defective respiratory chains, and cells lacking mtDNA ( BCL-2 and BCL-XL DNA Constructs, Cell Culture and
Transfection, and Generation of Hybrid Cell Lines--
Human
BCL-2 cDNA was cloned in the EcoRI site of
the eukaryotic expression vector pcDNA3 (Invitrogen). Human
BCL-XL (a gift from Lawrence Boise, University
of Miami) cloned in the expression vector pIRESneo
(Clontech) was obtained as described previously (8). The human osteosarcoma mtDNA-less cell line
(143B/206
143B/206
Hybrid cell lines were obtained by fusing 206 Measurements of ATPm and
ATPc--
WT, WTBCL-2, 143B,
143BBCLXL, 143B/206 Cell Death Studies--
Apoptotic cell death induced by
treatment with 0.5 µM staurosporine (STP) for 6 h
was assessed by using a cell death detection enzyme-linked
immunosorbent assay kit (Roche Molecular Biochemicals) on the basis of
the detection of nucleosome breakdown as described previously (8).
Biochemical Studies--
Oxygen consumption was measured in a
300-µl reaction chamber equipped with a Clark-type polarographic
electrode as described previously (17) with a few modifications. Cells
were trypsinized, counted in a Z1 automated cell counter
(Beckman-Coulter, Miami, FL), and resuspended at 1.5 × 106 cells in Dulbecco's modified Eagle's medium
containing no glucose and no fetal bovine serum and supplemented with 1 mM sodium pyruvate. 1 µM carbonyl cyanide
p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial
uncoupling agent, was added to a replicate sample to measure uncoupled
respiration. Respiration rates were determined as the rates of oxygen
consumption. Complex I-driven respiration was measured by polarography
with and without rotenone (1 µM) and expressed as
rotenone-sensitive oxygen consumption.
Oligomycin-sensitive ATP synthesis was measured on
digitonin-permeabilized cells using a luciferase-based kinetic assay as described previously (18) with some modifications. Cells were trypsinized and resuspended in the reaction buffer at 1 × 107/ml. Digitonin (50 µg/ml) was added to 160 µl of
cell suspension followed by incubation for 1 min at room temperature.
Cells were washed in 1 ml of reaction buffer, and the luminometric
measurement was performed as described (18) with and without the
addition of 10 µg/ml oligomycin.
Mitochondrial membrane potential was estimated using the membrane
potential-sensitive dye
5,5',6,6'-tetrachloro-1,1',3,3-'tetraethylbenzimidazole carbocyanide
iodide (JC-1; Molecular Probes, Eugene, OR) as described (8), except
that cells were grown in 6-well plates to ~80% confluence, and the
JC-1 staining was performed directly on attached cells. Fluorescence
was read at 535 (green monomer) and at 595 nm (orange aggregate) in an
HTS 7000 plus plate reader (PerkinElmer Life Sciences). Mitochondrial
membrane potential was expressed as the ratio of the absorbance at 595 nm to the absorbance at 535 nm.
Hydrogen peroxide levels were measured in cells by
2'-7'-dichlorofluorescein diacetate (H2DCFDA; Molecular
Probes) fluorescence as described elsewhere (19).
Analyses of Mitochondrial Translation Products--
Steady-state
levels of mitochondrial peptides were studied by Western blotting. 20 µg of total cellular protein was separated onto 15%
SDS-polyacrylamide gels and transferred to polyvinylidene difluoride
membrane (Bio-Rad). After transfer, membranes were incubated with
monoclonal antibodies against human cytochrome c oxidase
subunits I, II, and IV and the 39-kDa subunit of complex I (Molecular
Probes) at 4 °C for 12 h and subsequently incubated with
peroxidase-conjugated anti-mouse IgG for 1 h at room temperature. Bands were developed with Supersignal chemiluminescence substrate (Pierce) and quantified by densitometry using a Fluor-S analyzer (Bio-Rad).
BCL-2 Expression and mtDNA Genotype in Hybrid Cells--
The human
BCL-2 cDNA cloned in the eukaryotic expression vector
pcDNA3 was transfected into human osteosarcoma cells lacking mtDNA
(143B/206
206 Mitochondrial and Cytosolic ATP Levels in BCL-2- and
BCL-xL-Overexpressing Cells--
To measure ATP content,
cells were transfected transiently with pcDNA3 containing the ATP
reporter luciferase and engineered to be targeted to the mitochondrial
matrix or to the cytosol. Cytosolic luciferase or mitochondrial
luciferase-transfected cells were incubated in culture medium
containing pyruvate with or without glucose. Relative ATP content was
estimated by measuring luminescence after the addition of luciferin.
In untransfected cells containing normal mtDNA (WT cybrids and 143B
parental cells), the ratio between ATP content in the mitochondria
(ATPm) and in the cytosol (ATPc) was lower in
the presence of glucose than when pyruvate was the only substrate available. Overexpression of BCL-2 and BCL-xL significantly
increased the ATPm:ATPc ratio in the presence
of glucose but did not affect it in the presence of pyruvate alone
(Fig. 2). Therefore, when cells competent
for respiration were grown in pyruvate, medium mitochondrial ATP became
predominant over cytosolic ATP. However, overexpression of BCL-2 or
BCL-xL appeared to equalize the relative ATP concentrations
between the two compartments in cells grown in glucose medium (Fig. 2).
As expected, 206 BCL-2 Overexpression Improves Mitochondrial ATP Synthesis,
Respiration, and Mitochondrial Membrane Potential in Cybrids with
Mutated mtDNA--
Mitochondrial ATP synthesis was measured in
permeabilized cells with malate plus pyruvate as substrates. The rate
of ATP synthesis was ~10-fold lower in both MERRF MT and MELAS MT
cells compared with the respective WT lines. In MTBCL-2
cells, ATP synthesis was restored to ~40% of WT levels
(i.e. 4-fold increase; Fig.
3A). On the contrary, a
moderate reduction (~40-50%) of ATP synthesis was observed in
WTBCL-2 cells as compared with their untransfected
counterparts (Fig. 3A).
Mitochondrial respiration measured in MT cells (Fig. 3B) was
reduced to ~15% of WT. However, in MTBCL-2 cells it was
restored to ~60% of WT values (i.e. a 4-fold increase). The addition of the protonophore FCCP, which completely uncouples mitochondria, increased cell respiration 2-fold in WT cells and to a
lower degree in MT cells. The proportion by which respiration was
induced by FCCP was substantially unchanged in BCL-2-expressing cells,
suggesting that the increased respiration in intact cells was unlikely
to be caused by the uncoupling of mitochondria by BCL-2.
Mitochondrial membrane potential measured by the potentiometric
mitochondrial dye JC-1 in medium containing glucose and pyruvate was
significantly increased in both MTBCL-2 cell lines compared
with untransfected cells but was still lower than in WT cells (Fig.
3C).
The activity of respiratory chain complex I, measured as
rotenone-sensitive cell respiration (Fig. 3D), was severely
decreased in MERRF MT and to an even greater extent in MELAS MT. In
both MT cell lines, overexpression of BCL-2 partially restored complex I activity (~40% of WT levels).
To exclude the possibility that selection in medium lacking uridine
(during the production of the different cell lines used in this study)
might have favored the growth of spontaneous revertants, MERRF MT
cybrids were also directly transfected with pcDNA3-BCL-2 or
mock-transfected with pcDNA3 (neo) and selected in medium
containing G418 and high glucose and supplemented with uridine. In
addition, directly transfected cybrids served as controls for the extra dose of nuclear genes presumably present in the hybrids derived from
fusion of cybrids with 206 BCL-2 Overexpression Does Not Affect Free Radical Production in
Mutated Cells in Nonapoptotic Conditions--
It has been
suggested that members of the BCL-2 antiapoptotic protein
family can protect cells from the potentially deleterious effects of
mitochondrial reactive oxygen species production that follow
apoptotic stimuli (22, 23). Therefore, it could be hypothesized that if
mitochondrial tRNA mutations cause increased reactive oxygen species
production, BCL-2 overexpression could prevent damage to mitochondria
by scavenging reactive oxygen species. However, hydrogen peroxide
levels measured by H2DCFA dye in MT and WT cells
growing in normal conditions without apoptotic stimuli were not
increased in MT cells as compared with WT cells, and BCL-2
overexpression did not seem to affect hydrogen peroxide levels (not shown).
To verify whether the effect of BCL-2 overexpression on MT cells could
be mimicked by exogenous antioxidants, MERRF MT cells were grown for a
prolonged period of time in the presence of 10 mM
dihydrolipoic acid. This antioxidant agent has been shown to protect
PC12 rat cells from cell death induced by mitochondrial respiratory
chain inhibitors (24). Cells treated with dihydrolipoic acid for 1 or 2 weeks did not show any improvement in respiratory chain activities and
cell respiration (not shown), further suggesting that protection from
free radical damage is unlikely to play a major role in the biochemical
improvement induced by BCL-2 overexpression.
BCL-2 Overexpression Does Not Affect Respiratory Chain Subunit
Steady-state Levels--
The steady-state levels of respiratory chain
complex IV subunits I and II (cytochrome c oxidase I and
II), which are encoded by the mtDNA, were markedly decreased in MT
cells. BCL-2 overexpression did not modify the steady-state levels of
these subunits in MERRF and MELAS MT cells (not shown). The levels of
complex IV subunit IV (cytochrome c oxidase IV)
and complex I 39-kDa subunit (CO I 39-kDa) that are both encoded by the
nuclear DNA were similar in all cells and were unmodified by BCL-2
overexpression (not shown). This finding suggests that BCL-2 did not
exert its effects on mitochondrial respiration by increasing the levels
of mitochondrial enzymes, but rather by modulating their activities.
It was suggested that BCL-xL is able to regulate
mitochondrial membrane potential and ATP/ADP exchange across the
mitochondrial membranes in cells deprived of growth factors (5, 6).
BCL-2 has also been shown to promote H+ efflux from the
mitochondrial matrix into the intermembrane space of isolated
mitochondria as a mechanism to maintain membrane potential in response
to uncoupling agents (3). These observations suggest that the
antiapoptotic BCL-2 family of proteins might have a role in maintenance
of mitochondrial homeostasis that is not limited to their antiapoptotic
properties. These findings also seem to point to adenine nucleotide
translocation and transmembrane proton flux as potential critical sites
for the action of BCL-2 proteins.
In normal cells, ATP is generated both by glycolysis in the cytosol and
by OXPHOS in mitochondria, from which it is exported to the other
cellular compartments where most of it is used. We found that in
untransfected WT cells the ATPm:ATPc ratio was
higher in a medium without glucose (in which cells produce ATP mainly through oxidative phosphorylation) than when cells were grown with a
high concentration of glucose. BCL-2 and BCL-xL appeared to
increase the ATPm:ATPc ratio in glucose medium,
presumably by promoting the ATP exchange across the mitochondrial
membranes. Accordingly, we found that WT cells overexpressing
BCL-2/BCL-xL had a significantly reduced mitochondrial ATP
synthesis, possibly as a response to the increased
ATPm:ATPc ratios. In WT cells, which have a
normal respiratory chain, an increased
ATPm:ATPc ratio may have an inhibitory effect
on ATP synthase. On the contrary, we showed that BCL-2 expression
increased the ATPm:ATPc ratio in cells
harboring the MERRF mutation not only in glucose but also in the
oxidative substrate pyruvate, and it stimulated mitochondrial respiration and ATP synthesis.
It was suggested that ATP/ADP translocation is the most efficient
mechanism to maintain mitochondrial membrane potential in cells with a
defective respiratory chain, such as
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0 cells),
which have no functioning respiratory chain. We found that in both wild
type (WT) and mtDNA mutant (MT) cells BCL-2 and BCL-xL
overexpression tended to equalize the relative ATP content in the two
cell compartments, presumably promoting its exchange across the
mitochondrial membranes. We investigated the effect of BCL-2 and
BCL-xL expression in cells harboring pathogenic mutations
in mitochondrial tRNA-coding genes to assess whether modulation of
anion exchange and the protective effect on mitochondrial function
could improve defects of the mitochondrial respiratory chain caused by
mtDNA mutations. We showed that antiapoptotic BCL-2 family members
ameliorated the defective OXPHOS phenotype in three different cell
lines harboring distinct mitochondrial tRNA gene mutations. These data
raise the possibility that BCL-2 might have a more general protective
function against insulting agents on mitochondrial physiology that is
not necessarily limited to its antiapoptotic properties.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0 cells) was cultured in Dulbecco's
modified Eagle's medium containing high glucose (4.5 g/liter)
supplemented with 5% fetal bovine serum, 1 mM sodium
pyruvate, and 50 µg/ml uridine.
0 cells were transfected with pcDNA3-BCL-2
using FuGENE 6 (Roche Molecular Biochemicals) as described by the
manufacturer. G418 (GeneticinTM; Invitrogen)-resistant
clones were selected and screened for BCL-2 expression by Western
blotting using specific monoclonal antibodies against human BCL-2
(Santa Cruz Biotechnology Inc., Santa Cruz, CA). One
143B/206
0 clone with high BCL-2 expression
(206
0BCL-2) was chosen for mtDNA repopulation.
206
0 BCL-xL cells were obtained as described
previously (8).
0BCL-2 or
206
0 BCL-xL cells with previously described
mutated (MT) T8356C and WT myoclonus epilepsy with ragged red fibers
(MERRF) (10) and MT C3256T and WT mitochondrial encephalomyopathy,
lactic acidosis, and strokelike episodes (MELAS) (11) cybrids. Before
fusion, MERRF and MELAS cybrids were treated for 2 h with 1 µg/ml actinomycin D, a DNA transcription and replication inhibitor,
to limit the amount of nuclear DNA transferred from the cybrids to the
BCL hybrids. Actinomycin D-treated cells (1 × 106)
were washed three times in Dulbecco's modified Eagle's medium and
then fused to 1 × 106 206
0BCL-2 or
0206
0 BCL-xL cells as
described previously (12). Cells were selected in a medium containing
500 µg/ml G418 and lacking uridine. Surviving cells were pooled in
mass cultures. MERRF MT (8356) and MERRF MT (8344) (10) cells were also
directly transfected with pcDNA3 BCL-2 or with pcDNA3 empty
vector as described above. In this case, cells were selected in 500 µg/ml GeneticinTM, and individual colonies were isolated
for cell respiration and ATP synthesis analyses.
0,
206
0BCL-2, MERRF MT (8356), and MERRF
MTBCL-2 cells were transiently transfected as described
previously with pcDNA3 expressing the ATP reporter luciferase
targeted to the mitochondrial matrix or to the cytosol. Mitochondrial
luciferase was generated by appending to the N terminus of luciferase
the presequence of the mitochondrially targeted subunit VIII of
cytochrome c oxidase. This construct has been shown to be
efficiently imported into mitochondria (13-15). Cytosolic luciferase
was obtained by disrupting the natural C-terminal peroxisomal targeting
sequence with a leucine to valine amino acid substitution at position
550 (16). 48 h after transfection with cytosolic luciferase or
mitochondrial luciferase, aliquots of 2 × 105 cells
were resuspended in 1 ml of Dulbecco's modified Eagle's medium
containing 100 mM pyruvate with or without 4.5 mg/ml
glucose and incubated in a 24-well plate with gentle agitation at
37 °C in 5% CO2. After 1 h, cells were collected
by centrifugation and resuspended in 90 µl of 25 mM
Tricine, 150 mM NaCl, pH 7.4. 10 µl of 20 mM
luciferin (Promega Inc., Madison, WI) was added, and luminescence
was recorded in an Optocomp I luminometer (MGM Instruments, Hamden,
CT). To normalize for the variability of luciferase expression, the
luminescence values were expressed as a ratio to the "total potential
luminescence" measured on 2 × 105 aliquots of the
same cells lysed with a luciferase assay kit (Promega Inc.) containing
500 µM ATP.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0). G418-resistant clones were screened for
BCL-2 expression, and one high expressor (termed
206
0BCL-2) was selected. The 206
0BCL-2
line was then repopulated with exogenous WT mtDNA. Similarly, human
BCL-xL cDNA cloned in a pIRESneo vector was
transfected into 143B/206
0 or in parental osteosarcoma
143B cells, and G418-resistant BCL-xL overexpressing clones
were selected. The 206
0BCL-2 line was
repopulated with exogenous mtDNA from cybrid clones derived from a
patient harboring a C3256T transition in the
tRNALeu(UUR) gene and from a patient with a
T8356C transition in the tRNALys gene. The
C3256T mutation has been associated with a disease clinically
characterized by MELAS. The T8356C mutation causes a particular
type of encephalopathy known as MERRF. Because in both patients the
mutated mtDNA was heteroplasmic (i.e. MT and WT mtDNA
coexisted), it was possible to isolate homoplasmic cybrid clones
containing either exclusively MT or WT mtDNA.
0 cells are strictly auxotrophic for uridine. Thus,
hybrid cells harboring the mitochondrial genotypes described above and
expressing BCL-2 (termed MERRF MTBCL-2, MERRF
WTBCL-2, MELAS MTBCL-2, MELAS
WTBCL-2) were obtained by the fusion of
206
0BCL-2 cells with WT or MT MERRF and MELAS cybrids.
We note that in all the experiments involving hybrids, we deliberately
avoided isolating individual clones of repopulated cells and chose
instead to study pools of clones to minimize artifacts caused by
potential interclonal variability. As expected, all the newly produced
hybrid cell lines expressing BCL-2 contained either MT or WT mtDNA
(Fig. 1A). In an independent
set of experiments, 206
0 cells expressing
BCL-xL were fused with MERRF and MELAS MT cybrids. The
levels of BCL-2 in the expressor lines were ~10-20-fold higher than
the endogenous levels in untransfected cells (Fig. 1B). The antiapoptotic function of BCL-2 was tested by measuring nucleosome breakdown before and after STP induction. BCL-2-overexpressing cells
showed a marked decrease in the apoptotic response to STP (Fig.
1C). In addition, we observed that both untransfected MT cell lines displayed some degree of natural resistance against STP-induced apoptosis compared with their WT counterparts (Fig. 1C). It is possible that reduced mitochondrial ATP
synthesis and membrane potential in MT cells could lead to decreased
apoptotic response to STP as described previously in
143B/206
0 cells (8) and in Jurkat T cells depleted of
ATP by glucose deprivation combined with rotenone treatment (20).
View larger version (29K):
[in a new window]
Fig. 1.
mtDNA genotype and BCL-2 expression.
A, PCR-restriction fragment length polymorphism
analyses demonstrate that hybrid cells resulting from the fusion of
0BCL-2 cells with MERRF (top panel) and MELAS
(bottom panel) cybrids generated cell lines containing
either homoplasmic mutated or homoplasmic wild type mtDNA. Cybrid cells
are denoted with the prefix c. Hybrid cells are denoted with
the prefix h. U denotes uncut PCR products.
B, Western blot with anti-human BCL-2 antibodies showed a
10-20-fold increase in BCL-2 levels in the cell lines expressing the
recombinant BCL-2 compared with untransfected lines. C,
STP-induced nucleosome breakdown. The response to STP is expressed as
the -fold increase above pretreatment levels. BCL-2 expression induced
a clear decrease in STP-induced nucleosome breakdown in BCL-2 MERRF WT,
BCL-2 MERRF MT, and BCL-2 MELAS WT compared with untransfected cells.
STP-induced nucleosome breakdown was also decreased in MELAS MT cells
by BCL-2 overexpression. However, these cells had a lower tendency to
undergo apoptosis compared with other lines tested. Error
bars are the S.D. of three independent experiments.
0 cells, which are OXPHOS-incompetent,
were completely depleted of ATP in pyruvate medium (not shown). In
glucose medium, untransfected 206
0 cells had an equal
ATPm:ATPc ratio as BCL-2-overexpressing
206
0. MERRF MT cybrids transfected with BCL-2 also
showed an increased ATPm:ATPc ratio in the
presence of glucose compared with mock-transfected MERRF MT cybrids. In
MERRF MTBCL-2 cells the ATPm:ATPc
ratio was also increased when pyruvate was the only substrate in the
medium, probably reflecting an improved respiratory function (Fig. 2).
In fact, in pyruvate medium the absolute ATP contents in the two
compartments were ~10-fold higher in MERRF MT cells expressing BCL-2
than in mock transfected cells (not shown). These findings support the
concept that BCL-2 and BCL-xL may play a role in modulating
the exchange of ATP across the mitochondrial membranes, probably by
promoting its equilibration between the intramitochondrial and
extramitochondrial milieus. These results also suggest that BCL-2 may
improve respiratory functions in mtDNA mutant cells.
View larger version (19K):
[in a new window]
Fig. 2.
Cytosolic and mitochondrial ATP content in
cells. ATP-dependent luminescence was measured in
intact cells after the addition of 2 mM luciferin. 48 h after transfection with either cytosolic luciferase or mitochondrial
luciferase, cells were incubated for 1 h in medium containing
glucose and pyruvate or pyruvate alone. For each experiment,
luminescence was normalized by the "total potential luminescence"
obtained on lysates of aliquots of cells with the addition of 2 mM luciferin and 500 µM ATP. Bars
represent the ratios of the averages of ATPm and
ATPc luminescence values obtained in 20 replicate
measurements. Error bars represent 1 S.D.
View larger version (35K):
[in a new window]
Fig. 3.
ATP synthesis, cell respiration, respiratory
chain activities, and mitochondrial membrane potential. Data are
averages plus S.D. of three or more independent measurements.
p values of significant differences between untransfected
and BCL-2-overexpressing cells determined by unpaired Student's
t test are shown. A, oligomycin-sensitive ATP
synthesis measured in permeabilized cells using malate plus pyruvate as
substrates. ATP synthesis values are expressed as the percentages of
ATP synthesis in untransfected WT cells. B, cell respiration
with endogenous substrates in intact ( FCCP) and in uncoupled (+FCCP)
cells. Cell respiration is expressed as femtomoles of molecular oxygen
consumed in 1 min per cell. C, mitochondrial membrane
potential measurement using the potential-sensitive dye JC-1. Membrane
potential is expressed as the ratio of the two readings at 595 and 535 nm. D, complex I (NADH-coenzyme Q-reductase) activity was
measured by polarography in intact cells with and without 1 µM rotenone. Activities are expressed as
rotenone-sensitive oxygen consumption (nmol O2/min/mg
protein). Cybrid and hybrid cells are denoted as in Fig. 1.
0BCL-2 cells. MERRF
MTBCL-2 and WTBCL-2 as well as mock-transfected
clones were selected and mitochondrial respiration was measured. In
this case, we could not use pools of clones because many G418-resistant
cells might not express high levels of BCL-2. MERRF MTBCL-2
clones had higher respiration rates than mock-transfected clones: 2.9 ± 0.4 and 1.7 ± 0.8 fmol of O2/min/cell,
respectively (number of clones = 6; p < 0.01 (Fig. 3B)), confirming the results described previously on
MERRF MTBCL-2 hybrids created by fusion and selection in
medium containing G418 and lacking uridine. In addition, to further
confirm these findings, cybrids containing homoplasmic levels of a
different, more severe, tRNALys mutation at
nucleotide 8344 also associated with MERRF (21) were transfected with
pcDNA3-BCL-2 or mock-transfected with pcDNA3 (neo) and selected
in G418. MERRF 8344 MT clones overexpressing BCL-2 also showed improved
cell respiration and ATP synthesis. Oxygen consumption in BCL-2 and
mock-transfected MERRF 8344 MT clones was 1.2 ± 0.3 and 0.6 ± 0.3 fmol of O2/min/cell, respectively (number of BCL-2
expressing clones = 6; number of mock-transfected clones = 8;
p < 0.002 as determined by unpaired Student's
t test (Fig. 3B)). ATP synthesis was increased
5.5-fold in BCL-2 MERRF 8344 MT over the mock transfected (number of
clones = 5 in each group; p < 0.03 (not shown)).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0 cells (25). To
maintain membrane potential in respiration-deficient cells, ATP is
imported inside mitochondria and hydrolyzed by the F1-ATPase (Fig.
4). Maintenance of a mitochondrial
membrane potential, even if reduced, is necessary for cell survival,
because crucial functions such as mitochondrial protein import and
processing are membrane potential-dependent (26, 27). This
behavior could explain the differences between ATP production in wild
type and in mutant cells.
View larger version (22K):
[in a new window]
Fig. 4.
Model for BCL-2/BCL-xL
role in mitochondrial ATP/ADP translocation in mtDNA mutant
cells. In normal cells ATP is preferentially translocated outside
and ADP is translocated inside mitochondria. However, in cells with
defective mitochondrial respiration caused by reduced electron transfer
chain activity, ATP is translocated inside mitochondria and hydrolyzed
by the F1-ATPase to maintain a minimum membrane potential for cell
survival. Members of the antiapoptotic BCL-2 family of proteins might
modulate this mechanism, possibly by facilitating the entrance of
adenine nucleotides (ANT) through the
voltage-dependent anion channel (VDAC).
Cells harboring homoplasmic levels of mitochondrial tRNA mutations must
rely primarily on glycolysis, because their mitochondrial ATP synthesis
is extremely low. Therefore, it is likely that similar mechanisms to
those described in 0 cells, which involve ATP/ADP
exchange to maintain membrane potential, operate in mutated cells as
well. The events through which BCL-2 and BCL-xL facilitate
the exchange of creatine-PO4 and adenine nucleotides
between cytosol and mitochondria in response to perturbations of
cellular metabolism seem to occur at the level of the
voltage-dependent anion channel in the outer mitochondrial
membrane voltage-dependent anion channel (5). When
mitochondrial production of ATP becomes low, as in the initial phases
of apoptosis, or in cells that harbor mutations of the mtDNA,
BCL-xL and BCL-2 might promote the exchange of ATP and ADP
between mitochondria and cytosol. Mutant cells, which unlike
0 cells have some residual capability to maintain a
functioning respiratory chain, could benefit from the higher
mitochondrial membrane potential and higher intramitochondrial supplies
of adenine nucleotides. At this point it is unclear how BCL-2 improves
oxidative phosphorylation activity, but our results suggest that this
effect is associated with increased ATP flux into mitochondria. This increased flux could stimulate oxidative phosphorylation by leading to
the formation of ADP (through ATP-hydrolyzing reactions), which could
directly stimulate the respiratory chain. Alternatively, BCL-2 could
increase ATP-dependent functions of mitochondria containing mutated mtDNA, thereby increasing OXPHOS efficiency. This result could
be accomplished, for instance, by improved stability of certain
components of the mitochondrial respiratory chain such as cytochrome
c leading to more efficient electron transfer and OXPHOS.
Improved OXPHOS efficiency may then increase membrane potential and ATP synthesis.
In conclusion, we believe that antiapoptotic BCL-2-related proteins may
have a role in the homeostasis of mitochondrial metabolism not only in
response to apoptotic stimuli but also in a variety of conditions that
require mitochondrial adaptation to metabolic stresses.
BCL-2/BCL-xL can modulate the ATP gradient between
mitochondria and cytosol, and their increased expression may not only
protect cells during noxious stimuli but also improve energy metabolism within mitochondria with defective respiratory chain functions.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Muscular Dystrophy Association (to G. M. and C. T. M.), NINDS National Institutes of Health Grant NS02179 (to G. M.) and NCI National Institutes of Health Grant CA085700 (to C. T. M.).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.
§ To whom correspondence should be addressed: Weill Medical College of Cornell University, 525 E. 68th St., A-505, New York, NY 10021. Tel.: 212-746-4605; Fax: 212-746-4803; E-mail: gim2004@mail.med.cornell.edu.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M203080200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: OXPHOS, oxidative phosphorylation; MT, mutant; WT, wild type; MERRF, myoclonus epilepsy and ragged red fibers; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes; STP, staurosporine; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; ATPc, cytosolic ATP; ATPm, mitochondrial ATP.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Vaux, D. L., Cory, S., and Adams, J. M. (1988) Nature 335, 440-442[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Monaghan, P.,
Robertson, D.,
Amos, T. A.,
Dyer, M. J.,
Mason, D. Y.,
and Greaves, M. F.
(1992)
J. Histochem. Cytochem.
40,
1819-1825 |
3. | Shimizu, S., Eguchi, Y., Kamiike, W., Waguri, S., Uchiyama, Y., Matsuda, H., and Tsujimoto, Y. (1996) Oncogene 13, 21-29[Medline] [Order article via Infotrieve] |
4. | Susin, S. A., Zamzami, N., Castedo, M., Hirsch, T., Marchetti, P., Macho, A., Daugas, E., Geuskens, M., and Kroemer, G. (1996) J. Exp. Med. 184, 1331-1341[Abstract] |
5. |
Vander Heiden, M. G.,
Chandel, N. S., Li, X. X.,
Schumacker, P. T.,
Colombini, M.,
and Thompson, C. B.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4666-4671 |
6. | Vander Heiden, M. G., Chandel, N. S., Schumacker, P. T., and Thompson, C. B. (1999) Mol. Cell 3, 159-167[Medline] [Order article via Infotrieve] |
7. |
Vander Heiden, M. G., Li, X. X.,
Gottleib, E.,
Hill, R. B.,
Thompson, C. B.,
and Colombini, M.
(2001)
J. Biol. Chem.
276,
19414-19419 |
8. |
Dey, R.,
and Moraes, C. T.
(2000)
J. Biol. Chem.
275,
7087-7094 |
9. | DiMauro, S., Bonilla, E., Davidson, M., Hirano, M., and Schon, E. A. (1998) Biochim. Biophys. Acta 1366, 199-210[Medline] [Order article via Infotrieve] |
10. | Masucci, J. P., Davidson, M., Koga, Y., Schon, E. A., and King, M. P. (1995) Mol. Cell. Biol. 15, 2872-2881[Abstract] |
11. |
Hao, H.,
and Moraes, C. T.
(1996)
J. Biol. Chem.
271,
2347-2352 |
12. | King, M. P., and Attardi, G. (1989) Science 246, 500-503[Medline] [Order article via Infotrieve] |
13. |
Jouaville, L. S.,
Pinton, P.,
Bastianutto, C.,
Rutter, G. A.,
and Rizzuto, R.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
13807-13812 |
14. |
Kennedy, H. J.,
Pouli, A. E.,
Ainscow, E. K.,
Jouaville, L. S.,
Rizzuto, R.,
and Rutter, G. A.
(1999)
J. Biol. Chem.
274,
13281-13291 |
15. | Porcelli, A. M., Pinton, P., Ainscow, E. K., Chiesa, A., Rugolo, M., Rutter, G. A., and Rizzuto, R. (2001) Methods Cell Biol. 65, 353-380[Medline] [Order article via Infotrieve] |
16. | Gould, S. J., Keller, G. A., Hosken, N., Wilkinson, J., and Subramani, S. (1989) J. Cell Biol. 108, 1657-1664[Abstract] |
17. |
Barrientos, A.,
Kenyon, L.,
and Moraes, C. T.
(1998)
J. Biol. Chem.
273,
14210-14217 |
18. |
Manfredi, G.,
Gupta, N.,
Vazquez-Memije, M. E.,
Sadlock, J. E.,
Spinazzola, A., De,
Vivo, D. C.,
and Schon, E. A.
(1999)
J. Biol. Chem.
274,
9386-9391 |
19. |
Barrientos, A.,
and Moraes, C. T.
(1999)
J. Biol. Chem.
274,
16188-16197 |
20. | Single, B., Leist, M., and Nicotera, P. (2001) Exp. Cell Res. 262, 8-16[CrossRef][Medline] [Order article via Infotrieve] |
21. | Wallace, D. C., Zheng, X. X., Lott, M. T., Shoffner, J. M., Hodge, J. A., Kelley, R. I., Epstein, C. M., and Hopkins, L. C. (1988) Cell 55, 601-610[Medline] [Order article via Infotrieve] |
22. |
Gottlieb, E.,
Vander Heiden, M. G.,
and Thompson, C. B.
(2000)
Mol. Cell. Biol.
20,
5680-5689 |
23. |
Degli Esposti, M.,
Hatzinisiriou, I.,
McLennan, H.,
and Ralph, S.
(1999)
J. Biol. Chem.
274,
29831-29837 |
24. | Seaton, T. A., Cooper, J. M., and Schapira, A. H. (1997) Brain Res. 777, 110-118[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Buchet, K.,
and Godinot, C.
(1998)
J. Biol. Chem.
273,
22983-22989 |
26. | Suzuki, C. K., Rep, M., van Dijl, J. M., Suda, K., Grivell, L. A., and Schatz, G. (1997) Trends Biochem. Sci. 22, 118-123[CrossRef][Medline] [Order article via Infotrieve] |
27. | Neupert, W. (1997) Annu. Rev. Biochem. 66, 863-917[CrossRef][Medline] [Order article via Infotrieve] |