From the Departments of Biology and Kinesiology and Health Science, York University, Toronto, Ontario M3J 1P3, Canada
Received for publication, January 11, 2001
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ABSTRACT |
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Contractile activity induces adaptations in
the expression of genes encoding skeletal muscle mitochondrial
proteins; however, the putative signals responsible for these
adaptations remain unknown. We used electrical stimulation (5 Hz, 65 V)
of C2C12 muscle cells in culture to define some of the mechanisms
involved in contractile activity-induced changes in cytochrome
c gene expression. Chronic contractile activity (4 days, 3 h/day) augmented cytochrome c mRNA by 1.6-fold above
control cells. This was likely mediated by increases in transcriptional
activation, because cells transfected with full-length ( Elevations in skeletal muscle contractile activity are known
to induce large and rapid augmentations in the mRNA expression of
genes encoding mitochondrial proteins (1-4). These increases result
from a disruption in the equilibrium that exists between gene
transcription and mRNA stability during nonadaptive steady-state conditions. Elevations in the level of the mRNA encoding the
nuclear gene product cytochrome c in response to in
vivo contractile activity have been reported (5), and our recent
work has established that this adaptation is mediated via sequential,
time-dependent elevations in both of these processes (6).
This necessitates alterations in the expression of proteins involved in
regulating transcriptional activation and/or message stabilization
within the cytosol. However, the specific cellular events that occur during muscle contraction to initiate these adaptations remain largely
undefined. These events could include membrane depolarization, Ca2+ mobilization, cross-bridge cycling, and alterations in
energy metabolism. Each of these are known to initiate intracellular signaling cascades (7-11), which could ultimately alter rates of
transcription and/or mRNA stability. It is now established that
acute elevations in contractile activity can stimulate a number of
kinases involved in signal transduction, including mitogen-activated protein kinase, c-Jun N-terminal kinase, and p38 kinase
activities in skeletal muscle (4, 12, 13). However, the temporal
relationship between the onset of the putative signaling event
(i.e. kinase activity), transcription factor activation, and
the up-regulation of nuclear genes encoding mitochondrial proteins in
response to contractile activity remains unknown.
We have previously used cytochrome c as a model,
nuclear-encoded mitochondrial protein to define some of the adaptations
that occur in response to contractile activity and artificially
elevated muscle Ca2+ levels (6, 8). The cytochrome
c promoter contains multiple GC-rich regions that can serve
as binding sites for the transcription factor Sp1 (14-16). However,
the zinc finger transcription factor Egr-1 (17) may also interact with
these elements, because the consensus binding sequences are similar,
and it may be that Sp1 and Egr-1 can compete for similar, nonconsensus
sites (14). Furthermore, Sp1-mediated gene transcription is inhibited
by Egr-1 displacement of Sp1 from the promoter (18, 19). In addition, Egr-1 is rapidly induced in response to elevations in contractile activity (20, 21) as well as elevated intracellular Ca2+
levels (22). These findings suggest potential roles for both Sp1 and
Egr-1 in the regulation of cytochrome c expression during conditions of increased contractile activity. Thus, in the present study we adopted a contracting C2C12 murine skeletal muscle cell model
to define some of the events occurring during muscle contraction that
initiate the increase in cytochrome c expression and to
identify some of the transcription factors that are immediately
responsible for this increase.
Materials--
BDM,1
TTX, DNP, and ACT were purchased from Sigma.
[14C]Chloramphenicol, [ Cell Culture--
C2C12 murine skeletal muscle cells were
maintained at 37 °C in 5% CO2 on 100-mm gelatin-coated
plastic dishes containing Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin. Upon reaching 90% confluence, myoblast
differentiation was induced by switching to a lower serum medium
(Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated horse serum and 1% penicillin/streptomycin). Treatments were routinely carried out when the myotubes occupied 90-100% of the dish (~8-10 days).
Electrical Stimulation--
The design of the electrical
stimulation apparatus was derived by modifying existing methods
(23-25). Lids from plastic 100-mm culture dishes (Sarstedt, Montreal,
Canada) were fitted with two platinum wire electrodes such that 5-cm
lengths ran parallel to each other at opposite ends of the dish ~5 cm
apart. Electrodes were attached to the plates using miniature banana
plugs (Electrosonic, Toronto, Canada). Multiple plates were arranged in
parallel and connected to a stimulator unit, which reversed the
polarity of the output every second. Cells were stimulated at a
frequency of 5 Hz and an intensity of 65 V (1.2 V/cm2).
Stimulation was either performed acutely for 5, 15, 30, 60, or 240 min
or chronically for 4 days (3 h/day). Cell extracts obtained from the
chronic experiments were prepared 21 h after the last stimulation period.
Inhibitors--
To determine the event(s) of muscle
contraction responsible for activity-induced alterations in gene
expression, sarcomere shortening was inhibited at three different
levels 24 h prior to the onset of stimulation: 1) membrane
depolarization was inhibited by preventing the opening of voltage-gated
Na+ channels by treatment with 10 µM TTX, 2)
contraction was disrupted by pretreatment with the
membrane-permeable Ca2+ chelator BAPTA-AM (25 µM); this was done to prevent the increase in cytosolic
[Ca2+], which occurs subsequent to membrane
depolarization; and 3) contraction was inhibited by preventing
cross-bridge cycling while allowing membrane depolarization and
Ca2+ fluxes to occur by treatment with 1.5 mM
BDM. This concentration of BDM has been shown to have no effect on
Ca2+ transients in phenylepherine treated cardiac myocytes
(26) and appears to act by inhibiting myosin ATPase activity (27). Cells were electrically stimulated for 4 days during each treatment (3 h/day), and results from drug-treated cells were compared with quiescent cells treated with the corresponding vehicle (VEH). Finally,
mitochondrial ATP synthesis was inhibited by treatment of unstimulated
cells with 200 µM DNP to uncouple mitochondrial respiration. In this case, cells were treated for 9 h on two
consecutive days. Cells were harvested immediately after treatment on
the second day.
Steady-state mRNA Measurements--
Total RNA was isolated
from stimulated and unstimulated control cells as done previously (6)
and resuspended in diethyl pyrocarbonate-treated H2O.
Determination of the quality and subsequent size separation of total
RNA (15 µg for cytochrome c or 30 µg for Egr-1) was
achieved by electrophoresis using denaturing formaldehyde-1% agarose
gels, which were transferred and subsequently fixed to nylon membranes.
These membranes were then probed with radiolabeled cDNA probes
encoding cytochrome c, Egr-1, and 18 S rRNA as done previously (28). Stringent washes were performed at 55 °C for 15 min
in 0.1× SSC, 0.1% SDS followed by 15 min at 60 °C. Signals were
quantified by electronic autoradiography (Instantimager, Packard), and cytochrome c and Egr-1 mRNAs were
normalized to 18 S rRNA levels to correct for uneven loading.
Plasmids--
Plasmid constructs containing various lengths of
the cytochrome c promoters linked to a reporter gene
(pRC4CAT and pRC4LUC) were provided by Dr. R. Scarpulla (Northwestern
University, Chicago, IL) and Dr. F. Booth (University of Missouri,
Columbia, MO), respectively, and have been previously characterized
(16, 29). The pRC4CAT/ DNA Transfection and Expression Assays--
C2C12 myoblasts were
transfected with the appropriate cytochrome c
promoter/reporter construct (5 µg/100-mm dish) along with pRSV/ Measurement of mRNA Stability--
Differentiated myotubes
were either electrically stimulated for 4 days (3 h/day) or
left untreated for a similar time period. Immediately after the final
bout of stimulation, cells (stimulated and control) were treated with
either 10 µg/ml ACT to inhibit mRNA synthesis or an equivalent
volume of methanol, which served as the VEH. Myocytes were incubated
with ACT or VEH for 4, 24, or 48 h. At the appropriate time
points, cells were harvested, and total RNA was isolated. Equal amounts
of total RNA (15 µg) from ACT- and VEH-treated cells were subjected
to Northern blotting as described above. Blots were probed for
cytochrome c mRNA and uneven loading was corrected using
18 S rRNA. Cytochrome c mRNA degradation was assessed by
expressing the mRNA levels found at all time points as a percentage
of the t = 0 value.
Electromobility Shift Assays--
Nuclear proteins were isolated
from stimulated and control cells by scraping them from culture dishes
in ice-cold phosphate-buffered saline followed by centrifugation for
10 s in a microcentrifuge (4 °C). The supernatant was
discarded, and the pellet was resuspended in 400 µl of swelling
buffer (10 mM Hepes-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride).
Following a 10-min incubation on ice, cells were vortexed and pelleted
in a microcentrifuge (4 °C). The supernatant was discarded, and the
pellet was resuspended in 100 µl of resuspension buffer (20 mM Hepes-KOH, pH 7.9, 25% glycerol, 420 mM
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). Following a 20-min incubation on ice, cells were pelleted for 2 min in a microcentrifuge (4 °C). The supernatant was removed and used in electromobility shift assays following determination of
protein content using a Bradford assay (31). These nuclear extracts (25 µg) were incubated with 20 µg/ml poly(dI-dC), 50 µM
pyrophosphate, and 40,000 cpm of a [ Immunoblotting--
C2C12 muscle cells were rinsed in ice-cold
phosphate-buffered saline and subsequently scraped from culture dishes
in 200 µl of Laemmli buffer (62.5 mM Tris, 20% glycerol,
2% SDS, and 5% 2-mercaptoethanol). Equal amounts of nuclear extracts
(50 µg) were size-separated by electrophoresis on a 12%
SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose
membranes and probed with polyclonal antibodies for Egr-1, Sp1, or
cyclin D1 (1:500). Blots were then probed with the appropriate
horseradish peroxidase-conjugated secondary antibody (1:1000) and
visualized with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).
Cytochrome c Oxidase Enzyme Activity--
Cytochrome
c oxidase activity was measured as described previously
(2).
Statistical Analyses--
The data presented are the means ± S.E. Student's t test was used to evaluate the effects
of contractile activity, Sp1 overexpression, or DNP treatment on
mRNA levels and transcriptional activation. One-way analysis of
variance was used to evaluate the effects of TTX, BAPTA-AM, and BDM on
cytochrome c transcriptional activation in stimulated and
unstimulated control cells. In all cases individual differences were
determined using Tukey's post-hoc test and differences were considered
significant if p < 0.05.
Effectiveness of Stimulation of C2C12 Muscle Cells--
Using our
cell culture model, myotubes were observed to contract synchronously at
the correct frequency (5 Hz) using voltages as low as 35 V. Cells
appeared to reach maximal shortening at 55 V with no further increase
in contraction intensity apparent at voltages as high as 110 V. Thus,
cells in subsequent experiments were stimulated at an intensity of 65 V
(1.2 V/cm2). This voltage is much lower than that
previously used to stimulate cardiac myocytes in culture (32, 33) and
resulted in minimal myotube detachment following up to 8 h of
continuous stimulation.2 To
more objectively assess the effectiveness of the treatment, we
evaluated the acute effects of stimulation on Egr-1 mRNA levels, because Egr-1 mRNA is known to increase rapidly with contractile activity (20, 21). Increases (p < 0.05) in mRNA
were evident as early as 15 min after the onset of stimulation (Fig.
1). This increase was transient in
nature, reaching a maximum of 4.0-fold above that in unstimulated
control cells following 30 min of contractile activity and declining to
2.0-fold above control after 4 h of stimulation. This effect is
similar to that observed previously in vivo (20), and it
demonstrates the effectiveness of the myocyte electrical stimulation
model in rapidly altering muscle gene expression.
Electrical Stimulation Increases Cytochrome c mRNA Levels as a
Result of Transcriptional Mechanisms--
Cytochrome c was
chosen as a representative model of the effect of contractile activity
on the expression of nuclear genes encoding mitochondrial proteins.
Four days of electrical stimulation (3 h/day) resulted in an elevation
in cytochrome c mRNA to levels that were 1.6-fold above
those in unstimulated cells (Fig.
2A, p < 0.05). This adaptation occurred prior to changes in mitochondrial enzymes reflected by cytochrome c oxidase activity, which
was 157.3 ± 20.1 and 150.0 ± 2.7 nmol/min/mg
(n = 3) in control and stimulated cells, respectively.
To assess whether electrical stimulation affects cytochrome
c transcription, myocytes were transfected with
pRC4CAT/
To evaluate the possible contribution of contractile activity-induced
changes in mRNA stability to the increase in cytochrome c mRNA observed, we measured cytochrome c
mRNA decay in stimulated and unstimulated cells. Similar
degradation rates of cytochrome c mRNA were observed in
stimulated and control cells, which declined by 23.8 ± 4.5 and
30.1 ± 9.7%, respectively, after 48 h of transcriptional inhibition with ACT (Fig. 2C). Cytochrome c
mRNAs were very stable in these cells, exhibiting an extrapolated
half-life of ~89 h. These data suggest that the contractile
activity-induced increase in cytochrome c mRNA was due
to increases in gene transcription.
Sp1 Binding within the First Intron of the Cytochrome c Promoter Is
Increased by Contractile Activity--
Following 4 days of electrical
stimulation (3 h/day), there was a contractile activity-induced
2.4 ± 0.5-fold increase in Sp1 protein levels, whereas no effect
of electrical stimulation on Egr-1 protein levels was evident (Fig.
3A). Coincident with this
increase in Sp1 protein levels was a 1.8 ± 0.2-fold increase in
DNA binding within the first intron (+75 to +104 bp) of the cytochrome
c promoter (Fig. 3B, lane 2 versus
lane 3). This binding was prevented by incubation with
100-molar excess of a cold oligodeoxynucleotide containing a portion of
the first intron of the cytochrome c gene (lane
4). In addition, DNA binding was eliminated by preincubation with
a nonradiolabeled consensus Sp1 deoxyribonucleotide (lane 5), suggesting that Sp1 may be responsible for the
activity-induced activation of cytochrome c transcription.
In contrast, there was no significant effect of preincubation with a
cold oligonucleotide containing the consensus Egr-1 binding sequence
(lane 6). To further evaluate the effects of contractile
activity on Sp1 activation, extracts from stimulated and control cells
were incubated with a radiolabeled consensus Sp1 sequence (Fig.
3C, lanes 1-4). These analyses revealed a
stimulation-induced elevation in Sp1 DNA binding (lane 2 versus lane 3), which was completely eliminated by the addition of an excess of cold Sp1 consensus oligonucleotide (lane 4). In contrast, there was no effect of contractile activity on binding to an Egr-1 consensus oligonucleotide (lane 6 versus
lane 7). As expected, Egr-1 binding was not evident in the
presence of an excess of cold Egr-1 consensus oligonucleotides
(lane 8). Taken together, these results suggest that
electrical stimulation increases Sp1 binding within the first intron of
the cytochrome c gene and that Egr-1 does not bind within
this region.
Overexpression of Sp1 Increases Cytochrome c Transactivation and
Cytochrome c mRNA--
To further evaluate the role of Sp1 in
cytochrome c transcriptional activation, cells were
cotransfected with either empty vector or a vector directing the
overexpression of Sp1, along with the pRC4LUC/ Transcriptional Activation of Cytochrome c Is Proportional to
Mitochondrial ATP Synthesis--
To evaluate the event(s) occurring
during contractile activity that provide the putative signal
responsible for activity-induced cytochrome c
transactivation, muscle contraction was inhibited at various
subcellular levels. First, cells were transfected with the
To reduce the mitochondrial ATP synthesis rate below that found in
resting, noncontracting cells, we treated cells with DNP to inhibit
oxidative phosphorylation. Mitochondrial uncouplers such as DNP have
been used previously as energy stressors (34) that do not affect ATP
utilization (i.e. myosin ATPase activity) but that impair
ATP synthesis, reduce the mitochondrial transmembrane potential
( Contractile activity is a potent stimulus for the induction of
numerous cellular adaptations in skeletal muscle, including mitochondrial biogenesis (1, 4). This complex process involves the
coordinated expression of proteins encoded within the nucleus as well
as those encoded in mitochondrion, suggesting that a vital intracellular communication exists between these two organelles. It is
likely that mitochondrial assembly is regulated mainly by proteins
originating from the nucleus, because most of the factors required for
the expression of mitochondrial proteins are nuclear-encoded, and the
vast majority of the proteins found within the organelle are derived
from the nuclear genome. We have adopted the respiratory chain protein
cytochrome c as a model for the regulation of nuclear gene
expression in response to contractile activity. Recently, we
demonstrated that an elevation in contractile activity evokes time-dependent augmentations in cytochrome c
transcriptional activation in vivo (6). Increases in
cytochrome c mRNA stability were also partly responsible
for the contractile activity-induced increase in mRNA observed.
Here our goal was to utilize a cell culture model of contractile
activity to mimic the in vivo induction of cytochrome
c mRNA, while allowing for the definition of some of the
intracellular signals and transcription factors responsible for this
adaptation. Electrical stimulation of C2C12 cells in culture elevated
the contractile activity of differentiated myotubes, which was clearly
visible upon inspection under the microscope compared with unstimulated
control cells.
Contractile activity imposed for 4 days (3 h/day) induced an elevation
in cytochrome c mRNA. This was likely due to
transcriptional activation, as indicated by cytochrome c
promoter-reporter activity, as well as by the fact that no change in
cytochrome c mRNA stability was observed under these
conditions. Previous studies have shown that transcriptional activation
of cytochrome c can occur in response to elevated
intracellular Ca2+. This appears to be mediated by factors
that bind to elements within the minimal ( In contrast to Egr-1, it is known that Sp1 is widely involved in the
expression of a variety of mammalian genes involved in oxidative
phosphorylation (35, 36). In particular, Sp1 affects cytochrome
c expression by binding within the region between +83 and
+104 bp of the gene (16). Here we show that contractile activity
increases Sp1 protein levels and causes an elevation in DNA binding
within this region and to a consensus Sp1 oligonucleotide. In both
cases DNA binding is completely eliminated by preincubation with a
nonlabeled Sp1 oligonucleotide. Further, overexpression of Sp1 led to
an increase in cytochrome c transactivation and mRNA
levels. Thus, it appears that contractile activity in skeletal muscle
cells induces cytochrome c transcription via mechanisms that
involve Sp1, at least with respect to the activation of the To define more precisely the intracellular events associated with
muscle contraction that mediated this transcriptional response, muscle
contraction was systematically disrupted at three different levels.
Treatment of cells with TTX completely abolished muscle contraction by
preventing membrane depolarization, which should eliminate the
subsequent release of Ca2+ and actin-myosin interactions.
As expected, this prevented the activity-induced cytochrome
c transcriptional activation that was apparent in
vehicle-treated cells. Next, the role of Ca2+ in
activity-induced cytochrome c expression was evaluated using the membrane-permeable Ca2+ chelator BAPTA-AM. The
potential involvement of intracellular Ca2+ in
activity-induced cytochrome c expression was suggested
from observations that Ca2+ serves as a potent
intracellular second messenger for a variety of cellular adaptations
(11, 37, 38). In addition, we recently reported that a marked elevation
in cytochrome c transcriptional activation occurs following
the treatment of rat L6E9 muscle cells with the Ca2+
ionophore A23187 (8), an effect that is reproducible in the mouse C2C12
muscle cells used in the present study.2 Treatment with
BAPTA-AM should permit the transmission of membrane depolarization but
eliminate sarcoplasmic reticulum-mediated Ca2+ transients,
subsequent actin-myosin interactions, and the accelerated ATP turnover.
The complete inhibition of contractile activity that we observed during
these conditions was also associated with the abolition of the
simulation-induced cytochrome c transcriptional activation.
This suggests that membrane depolarization alone, leading to
voltage-sensitive activation of signaling cascades, which are known to
activate the transcription of numerous genes in excitable cells (see
Ref. 7 for review), cannot account for the observed increases in
cytochrome c transactivation. We then evaluated contractile
activity-induced increases in cytochrome c transactivation
in the presence and absence of BDM. This agent is known to eliminate
cross-bridge cycling and, therefore, the increased ATP turnover
associated with contractile activity, but it allows both membrane
depolarization and the increase in Ca2+ transients to
occur. We hypothesized that if Ca2+ was a primary
intracellular signal mediating the increase in muscle cytochrome
c transactivation (8), this transcriptional activation
should be evident even when muscle contraction was inhibited in
electrically stimulated cells by preventing cross-bridge cycling.
However, under these conditions no elevation in cytochrome c
transcription was evident. These data suggest that the increases in
Ca2+ invoked during the contractile activity conditions
employed were not a sufficient stimulus (i.e. either in
magnitude or duration) to induce the effect, as compared with treatment
with a calcium ionophore like A23187 (8). In contrast, our data support the idea that, during skeletal muscle contractile activity, alterations in the rate of mitochondrial ATP synthesis represent a more important signal for the induction mitochondrial biogenesis, as reflected by
cytochrome c expression. This is suggested by the three
levels of mitochondrial ATP synthesis induced in the present study,
using cells subject to contractile activity, unstimulated cells, as well as those in which oxidative phosphorylation is inhibited. Whether
this effect is mediated directly via effects on kinase or phosphatase
activities or indirectly via putative signaling molecules involved in
mitochondrial-to-nuclear interorganellar communication remains to be determined.
726 base
pairs) or minimal (
66 base pairs) cytochrome c
promoter/chloramphenicol acetyltransferase reporter constructs
demonstrated contractile activity-induced 1.5-1.7-fold increases in
the absence of contractile activity-induced increases in mRNA
stability. Transcriptional activation of the
726 promoter was
abolished when muscle contraction was inhibited at various subcellular
locations by pretreatment with either the Na+ channel
blocker tetrodotoxin, the intracellular Ca2+ chelator
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester, or the myosin ATPase inhibitor 2,3-butanedione monoxime. It was further reduced in unstimulated cells
when mitochondrial ATP synthesis was impaired using the uncoupler
2,4-dinitrophenol. Because the contractile activity-induced response
was evident within the minimal promoter, electromobility shift assays
performed within the first intron (+75 to +104 base pairs) containing
Sp1 sites revealed an elevated DNA binding in response to contractile
activity. This was paralleled by increases in Sp1 protein levels. Sp1
overexpression studies also led to increases in cytochrome
c transactivation and mRNA levels. These data suggest
that variations in the rate of mitochondrial ATP synthesis are
important in determining cytochrome c gene expression in
muscle cells and that this is mediated, in part, by Sp1-induced increases in cytochrome c transcription.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP,
[
-32P]ATP, nitrocellulose, and nylon membranes (Hybond
N) were obtained from Amersham Pharmacia Biotech. BAPTA-AM was
purchased from Calbiochem (San Diego, CA). Fetal bovine serum and horse
serum were obtained from Summit Biotechnologies (Fort Collins, CO)
and Life Technologies, Inc., respectively. Polyclonal antibodies
directed toward Egr-1, Sp1, and Cyclin D1 were from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA). The dual luciferase reporter
assay system was from Promega (Madison, WI). All other reagents were
purchased from Sigma and were of the highest grade available. The
oligodeoxynucleotides used for electromobility shift assays were 1)
Sp1, 5'-ATTCGATCGGGGCGGGGCGAGC-3' (Dalton
Chemicals, Toronto); 2) Egr-1,
5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3' (Santa Cruz
Biotechnology, Santa Cruz); and 3) a sequence composed of +75 to +104
of the first intron of the cytochrome c gene,
5'-GGGGACGCGGGGCGGGAAGAGGGCGAGGAG-3' (Dalton Chemicals, Toronto, Canada).
726 construct is composed of sequences from the
cytochrome c promoter from
726 bp upstream of the
transcription start site to position +115 within the first intron,
fused to a chloramphenicol acetyltransferase (CAT) reporter gene.
Experiments utilizing the minimal cytochrome c promoter
(pRC4CAT/
66 or pRC4LUC/
66) were also conducted, because this region
has been shown to be sufficient to confer cytochrome c
transcriptional activation in response to elevated levels of intracellular Ca2+ (8). Overexpression of wild type Sp1
driven by the cytomegalovirus promoter was achieved with a vector
provided by Dr. G. Suske (University of Marburg, Germany).
-Galactosidase activity, under the control of the Rous sarcoma virus
promoter or Renilla luciferase activity driven by the
cytomegalovirus promoter was used to correct for transfection efficiency.
-gal (5 µg/dish) or pCMV/RL (5 ng/dish) when they reached 70% confluence. Where applicable, wild type Sp1 or empty vector were
also cotransfected (5 µg/dish) in combination with cytochrome c promoter/reporter constructs. The total amount of DNA
added was maintained constant in all transfected cells. Transfections were done using a poly-L-ornithine method followed by a
Me2SO shock (30). Cells were then differentiated by
switching to a low serum medium. CAT and
-galactosidase activities
in stimulated and quiescent cells were measured as described previously
(8). Luciferase activities were measured in a EG & G Berthold (Lumat LB
9507) luminometer using the dual luciferase reporter assay system
according to the manufacturer's instructions.
-32P]ATP
end-labeled oligonucleotide (containing the sequence between +75 and
+104 bp of the cytochrome c gene) in a binding buffer (20 mM Tris, pH 7.6, 0.1 M MgCl2, 50 mM dithiothreitol, 1 mM spermidine, 1 mM EDTA) at room temperature for 20 min. To determine the
specificity of binding, competition assays were conducted by
preincubating extracts (20 min) with a 100 molar excess of cold
oligonucleotide before the addition of labeled oligonucleotide. The
oligonucleotides used in these competition assays were composed of 1)
+75 to +104 bp of the first intron of the cytochrome c gene,
2) the consensus Sp1 binding site, and 3) the consensus Egr-1 binding
site. Samples were run on a nondenaturing 4% acrylamide gel and were
electrophoresed for 3 h (200 V). The gel was subsequently fixed
for 15 min in acetic acid/methanol/H2O (10:30:60), dried,
and imaged utilizing an Instantimager (Packard).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of contractile activity on
Egr-1 mRNA levels in C2C12 myocytes. Cells were electrically
stimulated (5 Hz, 65 V) for 5, 15, 30, and 60 min and harvested
immediately following the cessation of stimulation. Total RNA was
extracted, and Northern blot analyses were carried out as described
under "Experimental Procedures." A typical Northern blot showing
the levels of Egr-1 mRNA in electrically stimulated cells
(S) compared with unstimulated control cells (C)
is shown (inset), and the results of repeated experiments
are depicted graphically. *, p < 0.05 versus control. Egr-1 mRNA levels were corrected for
uneven loading with 18 S rRNA and are expressed as percentages of the
levels observed in control cells. Values are the means ± S.E.
726 plasmid constructs, which contained the full-length cytochrome c promoter linked to a CAT reporter gene. CAT
assays revealed a 1.5-fold higher (p < 0.05)
cytochrome c transcriptional activation in stimulated cells
compared with unstimulated control cells (Fig. 2B), which
paralleled the stimulation-induced increase in cytochrome c
mRNA. Because it has been shown previously that the factors
involved in Ca2+-mediated increases in transcription act
within the minimal
66 bp cytochrome c promoter (8), we
transfected cells with plasmid constructs containing this region
(pRC4CAT/
66). Electrical stimulation of these cells elicited similar
increases (1.7-fold higher; p < 0.05) in CAT activity
as with the
726 construct, compared with unstimulated control cells
(Fig. 2B). This observation indicates the presence of a
contractile activity response element within this minimal
66-bp
promoter region.
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Fig. 2.
Effects of contractile activity on cytochrome
c mRNA, transcriptional activation, and mRNA
stability in skeletal muscle cells. A, C2C12 cells were
electrically stimulated (5 Hz, 65 V) for 4 days (3 h/day) or remained
quiescent for a similar time period. Total RNA extraction and Northern
blot analyses were carried out as described under "Experimental
Procedures." Autoradiograms demonstrating the levels of cytochrome
c mRNA (inset) in control and stimulated
cells were quantified by electronic autoradiography. *,
p < 0.05 versus control. B,
C2C12 myoblasts were transfected with 5 µg of either pRC4CAT/ 726 or
pRC4CAT/
66 along with 5 µg of pRSV/
-gal. Cells were then
electrically stimulated (5 Hz, 65 V) for 4 days (3 h/day) or remained
quiescent for a similar time period. CAT and
-galactosidase
activities were measured as described under "Experimental
Procedures." Autoradiograms of CAT assays (inset) from
control cells (C) and electrically stimulated cells
(S) were quantified by electronic autoradiography. *,
p < 0.05 versus control. Values are the
means ± S.E. of at least seven independent experiments.
C, degradation of cytochrome c mRNA in
stimulated and control cells was measured following treatment with 10 µg/ml ACT. RNA was isolated at 4, 24, and 48 h after the
addition of ACT, and cytochrome c mRNA levels were
measured and expressed as percentages of the t = 0 value. Values are the means ± S.E. of three or four
experiments.
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Fig. 3.
The effects of contractile activity on DNA
binding within the first intron of the cytochrome c
gene. A, C2C12 muscle cells were electrically
stimulated (3 h/day for 4 days), and nuclear proteins were isolated as
described under "Experimental Procedures." Nuclear extracts (50 µg) from stimulated (S) and control (C) cells
were separated by SDS-polyacrylamide gel electrophoresis and probed
with polyclonal antibodies for Sp1, Egr-1, or cyclin D1. B,
nuclear proteins (25 µg) from control (lane 2) and
stimulated cells (lanes 3-6) were incubated with a
32P-labeled oligonucleotide corresponding to +75 to +104 bp
of the first intron of the cytochrome c gene. Nuclear
extracts were also preincubated with nonradiolabeled oligonucleotides
corresponding to this region ( 66, lane 4), the
Sp1 consensus binding site (Sp, lane 5) or the Egr-1
consensus binding sequence (Egr, lane 6). C, cell
extracts (25 µg) from control (lanes 2 and 6)
and S (lanes 3, 4, 7, and
8) cells were incubated as in A with radiolabeled
Sp1 (lanes 1-4) or Egr-1 (lanes 5-8) consensus
oligonucleotides. Competition reactions were conducted with 100 molar
excess nonradiolabeled Sp1 (lane 4) or Egr-1 (lane
8) oligonucleotides. FP, free probe.
66 construct.
Luciferase activity controlled by this minimal promoter was ~3-fold
higher in Sp1 overexpressing cells (Fig.
4A; p < 0.05). This was accompanied by a modest but significant 25% increase
in cytochrome c mRNA levels (Fig. 4B; p < 0.05).
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Fig. 4.
Sp1 overexpression transactivates the
cytochrome c promoter and increases cytochrome
c mRNA levels. A, C2C12 myoblasts
were transfected with 5 µg of CMV-SP1 (Sp1) or an empty vector
(EV), 5 µg of pRC4LUC/ 66, and 5 ng of pCMV/RL. Firefly
and Renilla luciferase activities were measured as described
under "Experimental Procedures." Luciferase activities observed in
empty vector cells were set at 1.0, and activities measured in
Sp1-transfected cells were normalized to this value. Values are the
means ± S.E. of 10 separate experiments. *, p < 0.05 versus empty vector. B, C2C12 myoblasts were
transfected with 5 µg of CMV-SP1 (Sp1) or an empty vector
(EV). Total RNA extraction and Northern blot analyses were
performed as described under "Experimental Procedures." Cytochrome
c mRNA levels were determined by Northern blot analyses
and quantitated by electronic autoradiography. Equal loading was
verified by inspection of the ethidium bromide (EtBr)
stained gel (bottom panel).
726-bp
cytochrome c promoter and were treated 24 h prior to
the onset of stimulation with 1) TTX (10 µM) to prevent
membrane depolarization, 2) BAPTA-AM (25 µM) to prevent
the increase in [Ca2+]i associated with
Ca2+ release from the sarcoplasmic reticulum, or 3) BDM
(1.5 mM) to prevent cross-bridge cycling. Pretreatment of
control cells with TTX abolished muscle contraction entirely but had no
effect on basal levels of cytochrome c transcriptional
activation compared with VEH-treated cells (Fig.
5A). Contractile activity
induced a 1.8-fold activation (p < 0.05) of the
726-bp cytochrome c promoter in the presence of VEH. This
situation represents the condition in which the mitochondrial ATP
synthesis rate is the highest, because myosin ATPase is activated by
Ca2+, and the resulting production of free ADP is high and
capable of activating mitochondrial ATP regeneration through coupled
oxidative phosphorylation. This effect was completely prevented by TTX
treatment (Fig. 5A), as expected. BAPTA-AM (25 µM) was used to permit membrane depolarization while
preventing stimulation-induced muscle shortening via the chelation of
intracellular Ca2+. BAPTA-AM treatment resulted in a 37%
decrease (p < 0.05) in cytochrome c
transcriptional activation in unstimulated cells compared with
VEH-treated cells (Fig. 5B), suggesting the involvement of
Ca2+ in cytochrome c transcription, as described
recently (8). Myotubes subjected to electrical stimulation and VEH
treatment demonstrated a 1.5-fold increase (p < 0.05)
in cytochrome c transactivation above that in unstimulated
control cells. Stimulated cells pretreated with BAPTA-AM were
completely unable to contract and showed no elevation in cytochrome
c transcriptional activation above that found in C + VEH
cells (Fig. 5B). In this situation, mitochondrial ATP
synthesis is low and similar to unstimulated cells, because there is no
Ca2+ available to permit cross-bridge cycling and ATP
utilization. In addition, because BAPTA-AM prevented the increase in
Ca2+ associated with muscle contraction, and this occurred
coincident with a normalization of cytochrome c
transactivation, these data support a potential role for
Ca2+ in mediating the transcriptional response. We then
inhibited contractile activity at the level of cross-bridge cycling
with BDM to allow membrane depolarization and an increase in
[Ca2+]i to occur in response to electrical
stimulation, while preventing sarcomere shortening and maintaining ATP
turnover at a rate similar to unstimulated cells. BDM had no effect on
cytochrome c transcriptional activation in unstimulated
cells (Fig. 5C). Electrical stimulation of VEH-treated cell
resulted in a 1.4-fold elevation (p < 0.05) in
cytochrome c transcriptional activity, similar to the
results illustrated in Fig. 5 (A and B). However, coincident with the low ATP turnover and mitochondrial ATP synthesis rate, cytochrome c transcriptional activation was completely
prevented in the presence of BDM, despite the high levels of
Ca2+ available, expected in view of continued membrane
depolarization and Ca2+ release.
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Fig. 5.
The roles of membrane depolarization,
intracellular Ca2+ levels, and cross-bridge cycling in
activity-induced cytochrome c transcriptional
activation. A, C2C12 myoblasts were
transfected with 5 µg of pRC4CAT/ 726 and 5 µg of
pRSV/
-gal. Cells were treated with 10 µM TTX to
prevent opening of voltage-gated Na+ channels or treated
with vehicle (V) 24 h prior to the onset of
stimulation. Cells either remained quiescent (C) or were
electrically stimulated (S) for 4 days (5 Hz, 65 V, 3 h/day). CAT and
-galactosidase activities were measured as described
under "Experimental Procedures." CAT activities observed in control
and vehicle (C + V) cells were set at 100%, and all other
activities were expressed as percentages of this value. Values are the
means ± S.E. of six experiments. B, myoblasts were
transfected with 5 µg of pRC4CAT/
726. To prevent the
contraction-induced elevation in [Ca2+]i, cells
were treated with 25 µM BAPTA-AM or VEH 24 h prior
to the onset of stimulation. CAT activities were measured as in
A and expressed as percentages of the C + V
value. Values are the means ± S.E. of five separate experiments.
C, myoblasts were transfected with 5 µg of pRC4CAT/
726
and subsequently treated with 1.5 mM BDM, to prevent the
cross-bridge cycling, or VEH 24 h prior to the onset of
stimulation. CAT activities were measured as in A and
expressed as percentages of the C + V value. Values are the
means ± S.E. of five independent experiments. *,
p < 0.05 versus C + V.
), and lead to a reduction in ATP levels by about 50% (11).
Coincident with reduced mitochondrial ATP synthesis was a diminished
cytochrome c transactivation to ~45-50% of
Me2SO-treated cells (Fig. 6).
The addition of BDM did not further increase the effect of the
uncoupler on cytochrome c transactivation.
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Fig. 6.
Treatment of cells with DNP reduces
cytochrome c transcriptional activation in C2C12
myotubes. Myoblasts were transfected with 5 µg of pRC4CAT/ 726
and 5 ng of pCMV/RL. Cells were treated with 200 µM DNP,
1.5 mM BDM, alone or in combination, or dimethyl sulfoxide
vehicle (VEH) for two successive 9-h periods prior to
harvesting myotubes for CAT reporter assays. CAT and Renilla
luciferase activities were measured as described under "Experimental
Procedures." CAT activities observed in vehicle (DMSO)
cells were set at 100%, and all other activities were expressed as
percentages of this value. Values are the means ± S.E. of four
separate experiments. *, p < 0.05 versus
dimethyl sulfoxide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
66 to +115 bp) promoter
region of the cytochrome c gene (8). Our data indicate that
this region is also responsive to contractile activity. Within the
first intron of the cytochrome c gene are GC-rich regions
that may serve as binding sites for the zinc finger transcription
factors Sp1 and Egr-1 (14, 15). Indeed, Egr-1 mRNA levels were
elevated very rapidly after the onset of muscle contraction (Fig. 1),
although no changes in Egr-1 protein were evident after 4 days of
stimulation (Fig. 3A), emphasizing the transient nature of
Egr-1 gene expression, as reported previously (20). This lack of
increase evident at 4 days was reflected by the results of the DNA
binding assay, because no increases in Egr-1 DNA binding were evident
at that time.
66 bp
promoter. However, the effect of contractile activity on the
transactivation of the full (
726 bp) promoter is not likely entirely
due to Sp1 activity. Using cardiac myocytes, Xia et al. (37)
showed that a mutation of the Sp1 site within the first intron did not
abolish the transcriptional activation induced by cardiac pacing,
although the magnitude of transcriptional activation was reduced. In
particular, they showed that the NRF-1 and CRE sites were also
important for full contractile activity-induced transcriptional
activation of the gene, at least in cardiac cells. Our data compliment
those findings and uniquely illustrate the inducibility of Sp1-mediated
transcriptional activation in skeletal muscle cells in response to a
physiological stimulus.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. R. C. Scarpulla (Department of Cell and Molecular Biology, Northwestern University, Chicago, IL) and Dr. F. Booth (University of Missouri, Columbia, MO) for the donation of the cytochrome c promoter constructs and Dr. G. Suske (University of Marburg, Marburg, Germany) for the Sp1 expression vector.
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FOOTNOTES |
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* This work was supported by a grant from the Natural Sciences and Engineering Council of Canada.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: Dept. of Biology, York
University, North York, ON M3J 1P3, Canada. Tel.: 416-736-2100, Ext. 66640; Fax: 416-736-5698; E-mail: dhood@yorku.ca.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M100272200
2 M. K. Connor and D. A. Hood, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: BDM, 2,3-butanedione monoxime; ACT, actinomycin D; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; CAT, chloramphenicol acetyltransferase; DNP, 2,4-dinitrophenol; LUC, luciferase; TTX, tetrodotoxin; VEH, vehicle; bp, base pair(s).
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