From the Departments of Biology and Kinesiology and Health Science, York University, Toronto, Ontario M3J 1P3, Canada
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ABSTRACT |
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Mitochondrial biogenesis can occur rapidly in
mammalian skeletal muscle subjected to a variety of physiological
conditions. However, the intracellular signal(s) involved in regulating
this process remain unknown. Using nuclearly encoded cytochrome
c, we show that its expression in muscle cells is increased
by changes in cytosolic Ca2+ using the ionophore A23187.
Treatment of myotubes with A23187 increased cytochrome c
mRNA expression up to 1.7-fold. Transfection experiments
using promoter-chloramphenicol acetyltransferase constructs revealed that this increase could be transcriptionally mediated since
A23187 increased chloramphenicol acetyltransferase activity by
2.5-fold. This increase was not changed by KN62, an inhibitor of
Ca2+/calmodulin-dependent kinases II and IV,
and it was not modified by overexpression of protein kinase A and cAMP
response element-binding protein, demonstrating that the A23187 effect
was not mediated through
Ca2+/calmodulin-dependent kinase- or protein
kinase A-dependent pathways. However, treatment of myotubes
with staurosporine or 12-O-tetradecanoylphorbol-13-acetate reduced the effect of A23187 on cytochrome c
transactivation by 40-50%. Coexpression of the
Ca2+-sensitive protein kinase C isoforms Ionized calcium (Ca2+) acts as a second messenger in a
variety of biological processes, including gene expression (1, 2), cell
cycle regulation, (3) and cell death (4). Since Ca2+ cannot
be metabolized like other second messengers, cells tightly regulate
intracellular Ca2+ concentration through a set of
specialized molecules by sequestering Ca2+ into
intracellular compartments. In skeletal muscle, depolarization causes
the release of Ca2+ from the sarcoplasmic reticulum and a
subsequent increase in cytosolic Ca2+ concentration. This
rise triggers muscle contraction (5) and promotes mitochondrial
metabolism (6, 7). However, it is not known to what extent
Ca2+ is involved in the phenotypic adaptations observed in
skeletal muscle in response to an altered functional demand, such as
chronic contractile activity.
Mitochondrial biogenesis is one of the striking responses observed in
skeletal muscle cells in response to a variety of physiological conditions (see Refs. 8-10 for reviews). The synthesis of
mitochondrial proteins involves the expression of genes originating
from two distinct genetic compartments. Thirteen proteins are derived
from the mitochondrial genome, whereas the remaining gene products are
transcribed from the nuclear genome, translated, and subsequently imported into the mitochondria. In skeletal muscle, chronic contractile activity is known to markedly increase the mRNA levels of several nuclear and mitochondrial genes encoding mitochondrial proteins (11-14). Since contractile activity necessarily involves the
mobilization of Ca2+ from intracellular stores (5) and
since Ca2+ is an important signaling ion in muscle and
other tissues (15), we hypothesized that Ca2+ could play a
role as an intracellular signal leading to mitochondrial biogenesis in
skeletal muscle. Several arguments support this possibility. First,
changes in internal Ca2+ concentration in skeletal muscle
cells using the Ca2+ ionophore A23187 are known to induce
alterations in gene expression (16, 17). Second, a
Ca2+-regulated, calcineurin-dependent pathway
has been implicated in determining skeletal muscle phenotype (18).
Third, the treatment of myotubes in culture with the Ca2+
ionophore A23187 has been shown to increase the mitochondrial enzyme
activities of citrate synthase, malate dehydrogenase, and fumarate
hydratase (16, 19). Fourth, myotubes cultured in low Ca2+
medium possess very low levels of mitochondrial citrate synthase (20).
Fifth, DNA sequencing and footprinting analyses within the rat
cytochrome c promoter have demonstrated the existence of
multiple binding sites, including two cAMP response elements (CRE)1 (21, 22).
Phosphorylation of the cAMP response element-binding protein (CREB) on
Ser133 by calcium/calmodulin-dependent protein
kinase (CaM kinase) IV and its binding to the CRE are known to enhance
the transcriptional activation of specific genes (23, 24). Finally,
electrical stimulation of skeletal muscle cells has been shown to
induce the translocation of the Ca2+-dependent
protein kinase C (PKC) from the cytosolic to the plasma membrane
fraction (25) and to cause an increase in the activity of PKC in the
nucleus (26).
Thus, to define more precisely the potential role of Ca2+
in skeletal muscle mitochondrial biogenesis, we began with a systematic study of the effect of the Ca2+ ionophore A23187 on
cytochrome c gene expression in skeletal muscle. We report
here, using the L6E9 muscle cell line, that the treatment of myotubes
with A23187 increases cytochrome c gene expression through a
PKC-dependent pathway that depends, in part, on the
activation of MAPK.
Materials--
A23187, KN62, TPA, and staurosporine were
purchased from Sigma. They were prepared as stock solutions in
Me2SO at concentrations of 0.25 mM, 2 mM, 0.25 mM, and 1 µM,
respectively. PD98059 was obtained from Calbiochem and prepared as a 25 mM solution in Me2SO. [14C]Chloramphenicol, [ Cell Culture--
L6E9 myoblasts were cultured at 37 °C and
5% CO2 in air on 100-mm gelatin-coated plastic dishes
containing Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum and 1% penicillin/streptomycin. At ~70%
confluence, cells were switched to a lower serum differentiation medium
(Dulbecco's modified Eagle's medium supplemented with 5%
heat-inactivated horse serum and 1% penicillin/streptomycin).
Treatments began when the plates contained 85-90% myotubes (4-6 days later).
Dose-response and Time Course Experiments--
All experiments
were done with differentiated cells (myotubes) and were matched with
vehicle-treated controls. The dose-dependent effects of
A23187 on cytochrome c mRNA were evaluated at final concentrations ranging between 0.25 and 1 µM. Total RNA
was extracted 72 h later as described below. In time course
experiments, myotubes were incubated with 1 µM A23187 for
24, 48, or 72 h prior to the RNA extraction.
Total RNA Isolation, Northern Blotting, and Radiolabeled Probes--
To isolate total RNA, cells were harvested and centrifuged at
4 °C for 5 min. The pellet was resuspended with 1 ml of lysis solution consisting of 1 volume of solution containing 4 M
guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% Sarkosyl, and 0.1 M Plasmids--
Plasmid constructs used for cytochrome
c mRNA expression (pRC4) and promoter analysis (pRC4CAT)
were generously provided by Dr. Richard Scarpulla (Department of Cell
and Molecular Biology, Northwestern University, Chicago, IL) (20, 28).
The pRC4CAT/ DNA Transfections and Expression Assays--
L6E9 myoblasts at
60% confluence were transfected with the appropriate cytochrome
c promoter-reporter construct (10 µg/100-mm dish).
pRSV/
To prepare cell extracts for CAT and Immunoblotting--
To prepare whole cell extracts for MAPK
immunoblotting, myotubes were rinsed with cold phosphate-buffered
saline and scraped in 200 µl of modified Laemmli buffer (20%
glycerol, 2% SDS, and 5% Statistics--
The data are presented as means ± S.E.
One-way analyses of variance were used to evaluate the effect of A23187
incubation time and concentration on cytochrome c mRNA
levels. Individual mean differences were assessed with Scheffé's
post-hoc test. Two-way analyses of variance were employed to examine
the effects of A23187 incubation time and cotransfection of plasmids on
cytochrome c transactivation. Unpaired t tests
were used for evaluating the effects of PD98059, staurosporine, and TPA
on CAT activity compared with A23187 alone and to evaluate the effect
of PKC isoforms on CAT activity. An Calcium-dependent Regulation of Cytochrome c Gene
Expression--
Northern blot analyses were first used to determine
the effect of A23187 treatment on cytochrome c mRNA
levels. Concentrations of A23187 ranging from 0.25 to 1.0 µM elicited a progressive increase in cytochrome
c mRNA levels by up to 1.7-fold (p < 0.05) (Fig. 1A). Higher
concentrations did not further enhance the response (data not shown).
The effect of A23187 on cytochrome c mRNA levels was
also time-dependent (Fig. 1B). By 48 and 72 h of exposure to the ionophore, mRNA levels were increased by 1.6- and 1.7-fold (p < 0.05), respectively. This effect was
abolished when myotubes were preincubated with the extracellular
Ca2+-chelating agent EGTA (Fig. 1A). Taken
together, these data show a calcium-dependent increase in
cytochrome c mRNA levels and indicate that A23187 exerts
its effect by increasing the intracellular calcium concentration from
the extracellular Ca2+ pool.
Preliminary work suggested that the A23187 effect on cytochrome
c mRNA was not mediated by a change in mRNA
stability.2 Thus, to
determine whether the effect of A23187 on cytochrome c
mRNA levels was mediated via transcriptional activation, L6E9 myoblasts were transfected with pRC4CAT/ Ca2+-mediated Cytochrome c Transcriptional Activation
Is Not Mediated via the CRE--
Next, we investigated the potential
role of cis-elements present in the cytochrome c
promoter in response to A23187, particularly the role of the two
Ca2+/cAMP response elements. One CRE (CRE-1) is located
To specifically investigate the role of CREB in cytochrome c
gene expression, cotransfection experiments were carried out in the
presence and absence of CREB and PKA expression plasmids. A23187 alone
produced a 3-fold increase (p < 0.05) in CAT activity. Transfection of myotubes with pRSV/CREB elicited 7.2 ± 1.3-fold (n = 5) increases in CREB protein levels (Fig.
3A, inset).
Cotransfection with CREB or PKA alone or in combination did not further
enhance the typical response elicited by A23187 (Fig. 3A).
These results show that cytochrome c gene expression in
skeletal muscle cells does not appear to involve a PKA-mediated
phosphorylation of CREB.
Ca2+-mediated Cytochrome c Transcriptional Activation
Is Not Mediated via a CaM Kinase-dependent Pathway--
To
examine the possibility that the Ca2+-mediated effect on
cytochrome c gene expression occurs through a CaM
kinase-dependent pathway, KN62, a potent inhibitor of both
CaM kinases II and IV, was used. Inhibition of CaM kinases was
ineffective in abolishing the A23187-induced increase
(p < 0.05) in cytochrome c transcriptional activation, suggesting that neither CaM kinase II nor CaM kinase IV
mediates the intracellular pathway triggered by A23187 (Fig. 3B).
Ca2+-mediated Cytochrome c Transcriptional Activation
Is Mediated via a PKC-dependent Pathway--
Next, we
examined the possibility that the induction of cytochrome c
gene expression by A23187 was mediated through a
PKC-dependent pathway. In these experiments, A23187 induced
a 2.6-fold increase in cytochrome c transactivation. In the
presence of staurosporine, a nonspecific inhibitor of PKC, cytochrome
c transactivation by A23187 was still evident
(p < 0.05), but was markedly reduced to a 1.4-fold
effect, significantly less than that observed in the absence of
staurosporine (Fig. 3B). To more specifically inhibit PKC,
myotubes were preincubated for 24 h with the phorbol ester TPA.
Long-term treatment with TPA has been shown to down-regulate PKC
expression in skeletal muscle (26) and in L6 cells (36), thus providing
a useful tool to test the involvement of PKC in the signaling pathway.
TPA treatment reduced the effect of A23187 on cytochrome c
transactivation from 2.6- to 1.6-fold (40%) (Fig. 3C),
indicating that the effect of A23187 is partly mediated by a
PKC-dependent pathway. Therefore, we also evaluated the
role of specific PKC isoforms in determining this effect.
Cotransfection experiments were performed using pRC4CAT/ Ca2+-mediated Cytochrome c Transcriptional Activation
Involves the Activation of MEK and MAPK--
Since various lines of
evidence (37) indicate that PKC isoforms activate the ERK1 and ERK2
MAPKs, we examined the possible activation of a MAPK cascade in
response to A23187 treatment. Myotubes that were preincubated for 30 min with the MEK inhibitor PD98059 (38) demonstrated a 38% decrease
(p < 0.05) in Ca2+-induced cytochrome
c gene transactivation (Fig. 4B). Immunoblot analyses using a phospho-specific MAPK antibody showed that the effect
of A23187 was a relatively early event since a transient 4.5 ± 0.7-fold (n = 4) activation of ERK1 (p42 MAPK) and ERK2 (p44 MAPK) occurred with a peak at 2 h (Fig. 4C). This
activation was completely prevented by preincubation with the MEK
inhibitor PD98059. The stimulatory effect of A23187 was still present
at 24 h, but was undetectable at 48 h after treatment with
A23187 (data not shown). This transient pattern observed could also not be explained by differences in gel loading since both Ponceau staining
and immunostaining of the same blot with a p44 antibody to illustrate
total p44 levels revealed equal loading in all lanes (data not shown).
It is well known that oscillations in cytosolic Ca2+
concentration play a fundamental role in the contraction and relaxation of striated muscles. It is also established, mainly from work in other
tissue types, that Ca2+ is active in signaling alterations
in gene expression (1, 2, 15). Recent investigations have documented
that this occurs in skeletal myofibers (18) and that artificially
induced increases in Ca2+ concentration lead to muscle
phenotypic adaptations (16). These investigations are physiologically
relevant since sustained elevations in internal Ca2+ occur
during contractile activity (39), a situation in which marked
alterations in muscle phenotype have been documented (40). In view of
earlier work that demonstrated that mitochondrial enzyme activities
could be modified by the presence or absence of Ca2+ (19,
20), we hypothesized that modifications in Ca2+
concentration could represent a putative signal leading to the enhanced
expression of nuclear genes encoding mitochondrial proteins, thereby
enhancing organelle biogenesis. To begin addressing this question, we
have specifically studied the behavior of cytochrome c, a
nuclearly encoded mitochondrial protein of the respiratory chain, in
response to a model of augmented cellular Ca2+
concentration using the Ca2+ ionophore A23187. Increases in
intracellular Ca2+ concentration of 3-10-fold are
typically obtained 10 min after the addition of the ionophore,
apparently lasting up to 16 days following addition of the drug (16).
This treatment has previously been used to document changes in
acetylcholine receptor (17), acetylcholinesterase (41), and myosin
isoform (16) gene expression. Here, we show for the first time that an
increase in intracellular Ca2+ concentration brought about
by A23187 can increase the transcriptional activation of a nuclear gene
encoding a mitochondrial protein (i.e. cytochrome
c). This effect may be applicable to other nuclearly encoded
genes since we have observed a similar finding using the cytochrome
c oxidase subunit Vb
promoter.3
We were surprised to find that the augmentation of cytochrome
c transcriptional activation was independent of the
involvement of the two CRE sites present in the cytochrome c
promoter as well as the activation of the CaM
kinase-dependent pathway since a CREB/CRE pathway of
cytochrome c transcriptional activation was previously found
in Balb/c/3T3 cells (22). However, our observations are consistent with
those of others using cardiac myocytes, in which the CRE appears to
interact with c-Jun rather than CREB (42). The difference between these
results and those previously reported in Balb/c/3T3 cells (22) suggests
that cytochrome c gene expression is regulated via different
transduction pathways in a variety of cell types. This appears to be
true even within striated muscles since we have previously shown
differences in the regulation of cytochrome c gene
expression between heart and skeletal muscle during development and in
response to variations in thyroid status (43).
In agreement with previous reports using other cell types (21, 29), we
show that the DNA sequence of the cytochrome c promoter
encompassed between positions PKC exists as a family of 11 homologous serine/threonine kinases, of
which four isoforms ( In vitro, PKC and
II, but not the Ca2+-insensitive
isoform, exaggerated the A23187-mediated response. The short-term
effect of A23187 was mediated in part by mitogen-activated protein
kinase (extracellular signal-regulated kinases 1 and 2) since its
activation peaked 2 h after A23187 treatment, and cytochrome c transactivation was reduced by PD98089, a
mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase inhibitor. These results demonstrate the existence of a
Ca2+-sensitive, protein kinase C-dependent
pathway involved in cytochrome c expression and implicate
Ca2+ as a signal in the up-regulation of nuclear genes
encoding mitochondrial proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP, nylon
membrane (Hybond N), and the ECL immunoblot detection reagents were
obtained from Amersham Pharmacia Biotech (Mississauga, Ontario,
Canada). Thin-layer chromatography plates were purchased from Fisher
Scientific (Unionville, Ontario). The CREB antibody was purchased from
Santa Cruz Biotechnology, whereas the MAPK antibody was from New
England Biolabs Inc. (Beverly, MA). Other cell culture and molecular
biology reagents were purchased from Sigma or Life Technologies, Inc.
(Burlington, Ontario) and were of the highest grade available.
-mercaptoethanol; 1 volume of
phenol; and 0.1 volume of 2 M sodium acetate, pH 4.0. The
cell lysate was then mixed with 0.2 ml of chloroform/isoamyl alcohol
(24:1, v/v) and centrifuged at 4 °C for 15 min. The upper phase was
removed and added to 0.5 ml of isopropyl alcohol to precipitate the
RNA. Total RNA was centrifuged for 10 min at 4 °C, washed with 1 ml
of 75% ethanol, dried in a vacuum desiccator, and finally resuspended
in 30-50 µl of diethyl pyrocarbonate-treated water. Total RNA (10 µg) was separated on a denaturing formaldehyde-containing 1% agarose
gel and transferred overnight to a nylon membrane. The quality of the
total RNA isolated in this fashion was high as indicated by 1) an
~2-fold greater intensity of the 28 S rRNA band, compared with 18 S
rRNA, on ethidium bromide-stained gels; 2) high
A260/A280 ratios; and 3)
a lack of visible RNA degradation or DNA contamination. Membranes were
hybridized with random primer-labeled cDNA probes encoding
cytochrome c and 18 S rRNA, as done previously (27, 28).
Final stringent washes of the blots were done in 0.1× SSC and 0.1%
SDS for 15 min at 55 °C followed by 15 min at 60 °C. Signals were
quantified by electronic autoradiography (Packard Instrument Co.), and
specific cytochrome c mRNA levels were corrected for
differences in loading using the 18 S rRNA signal.
726 construct contains sequences of the rat
cytochrome c gene extending from position
726 upstream of
the start site of transcription fused to the chloramphenicol
acetyltransferase (CAT) reporter gene. Truncated cytochrome
c promoter constructs (pRC4CAT/
326 and pRC4CAT/
66) were
also used. pRSV/
-gal was used to direct the synthesis of
-galactosidase, whereas pRSV/CREB (provided by Dr. R. Gaynor,
University of Texas Southwestern, Dallas, TX) and pC
EV (provided by
Dr. G. S. McKnight, University of Washington, Seattle, WA) were
used for the overexpression of CREB (30) and the catalytic subunit of
PKA (31), respectively. Expression vectors encoding PKC isoforms
,
II, and
were originally described by Ono et
al. (32) and were obtained from Dr. J. McDermott (Department of
Kinesiology, York University, Toronto, Canada).
-gal (2 µg/dish) was used as an internal control to assess
and correct for variations in transfection efficiency. However, no
differences in
-galactosidase activity were found in A23187- and
vehicle-treated cells. Where indicated, 5 µg of pRSV/CREB, pC
EV
(PKA), PKC
, PKC
II, or PKC
were cotransfected. Total DNA concentration was maintained constant in transfection experiments by adding pGEM-4Blue (Promega) or the specific empty vector
as a carrier plasmid. Transfections were done using the poly-L-ornithine method followed by a Me2SO
shock (33, 34). Cells were then switched to low serum medium to allow
differentiation. Where indicated, myotubes were preincubated for
24 h with KN62, staurosporine, or TPA prior to A23187 treatment.
Some cells were treated with PD98059 (12.5 µM) for
0.5 h or with EGTA (1 mM) for 5 h prior to A23187 treatment.
-galactosidase expression
assays, myotubes were scraped and centrifuged at 4 °C for 5 min. The
cell pellet was resuspended in 100 µl of 0.25 M Tris, pH
7.9, and then subjected to three freeze-thaw cycles in an ethanol/dry ice slurry. Cell debris was centrifuged at 4 °C for 5 min. The resulting supernatant was removed and used directly in the assays. CAT
activity was determined using 10-20 µl of cell extract in the
presence of 1 mM acetyl-CoA and 0.23 µCi of
[14C]chloramphenicol in a total volume of 40 µl. The
mixture was incubated at 37 °C for 1.5 h, extracted with ethyl
acetate, and dried in a vacuum desiccator. Acetylated and
non-acetylated forms of [14C]chloramphenicol were
resuspended in 30 µl of ethyl acetate and separated by thin-layer
chromatography for 30 min at room temperature with chloroform/methanol
(95:5, v/v) as the mobile phase. The percent conversion of
[14C]chloramphenicol to its acetylated products was
quantified by electronic autoradiography. Results were corrected for
transfection efficiency using
-galactosidase activity of the
cotransfected plasmid.
-Galactosidase activity was measured using
10-30 µl of cell extract in a reaction mixture consisting of 60 mM Na2HPO4, 40 mM
NaH2PO4, 1 mM MgCl2, 50 mM
-mercaptoethanol, and 0.66 mM o-nitrophenyl-
-D-galactopyranoside. After
incubation for 2.5 h at 37 °C, the reaction was stopped by the
addition of 1 M NaCO3, and the reaction product
absorbance was read at 420 nm.
-mercaptoethanol in 62.5 mM
Tris-HCl, pH 6.8) containing protease inhibitors. Cells were sonicated
(3 × 10-s pulses) on ice, heat-denatured at 95 °C for 5 min,
and spun briefly to pellet insoluble material. Extracts (100 µg) were
electrophoresed on SDS-10% polyacrylamide gels and transferred to
nylon membranes by electrotransfer (35). The extracts used to detect
CREB were the same as those used in the CAT assay, with the addition of
1 volume of lysis buffer (10% glycerol, 2.3% SDS, 5%
-mercaptoethanol, and 62.5 mM Tris-HCl, pH 6.8). The
extracts were heat-denatured for 5 min, and 30 µg of protein/lane
were subjected to electrophoresis on SDS-10% polyacrylamide gels
followed by transfer to nylon membranes. Primary antibody incubations
using dilutions of 1:1000 (CREB) and 1:250 (MAPK) were carried out at
4 °C overnight. Signals were detected by the use of an anti-rabbit
antibody coupled to horseradish peroxidase and enhanced
chemiluminescence according to the manufacturer's instructions
(Amersham Pharmacia Biotech).
level of 0.05 was used to
indicate statistical significance.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effects of A23187 and EGTA on cytochrome
c mRNA levels in L6E9 myotubes. A,
myotubes were treated with various concentrations of A23187 for 72 h prior to harvesting myotubes for Northern blot analysis. For EGTA
experiments, cells were preincubated for 5 h with 1 mM
EGTA before the addition of A23187. Total RNA extraction and Northern
blot analyses were performed as described under "Experimental
Procedures." An image from a Northern blot showing the effect of
A23187 on cytochrome c (Cyto c) mRNA levels
is presented in the upper panel. Quantification of the
effects of A23187 and EGTA on cytochrome c mRNA levels
in L6E9 myotubes is presented in the lower panel (*,
p < 0.05 versus 0 µM A23187).
B, myotubes were treated with either the vehicle ( ) or 1 µM A23187 (+) for 0, 24, 48, and 72 h. The
cytochrome c mRNA levels were determined by Northern
blot analysis (upper panel) and quantitated by electronic
autoradiography (lower panel; *, p < 0.05 versus 0 h). Cytochrome c mRNA levels
were corrected for variations in loading with 18 S rRNA levels and are
expressed graphically as a percent of the level obtained in control
(C) myotubes. Values are means ± S.E. of at least
three independent experiments.
726, allowed to
differentiate, and incubated with 1 µM A23187 or vehicle.
Analysis of variance revealed significant main effects of time and
A23187 on the response. After 24 and 48 h of treatment, the
activity of the CAT reporter gene was significantly increased by 2.3- and 3.0-fold, respectively (Fig.
2A), indicating that A23187
increases cytochrome c mRNA levels through
transcriptional activation. In addition, as previously shown with
cytochrome c mRNA, preincubation of the myotubes with 1 mM EGTA abolished the A23187-dependent increase
in transcriptional activation, indicating a requirement for
extracellular Ca2+ (Fig. 2B).
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Fig. 2.
Effect of A23187 on cytochrome c
transcriptional activation. A, L6E9 myoblasts
were transfected with 10 µg of pRC4CAT/ 726 and 2 µg of
pRSV/
-gal plasmid. Cells were then treated for 48 h with either
the vehicle alone (
) or 1 µM A23187 (+). CAT and
-galactosidase activities were determined in control and
A23187-treated cells as described under "Experimental Procedures."
Autoradiograms (right panel) were quantified by electronic
autoradiography (*, p < 0.05 versus without
A23187). B, cells were transfected with 10 µg of the
different pRC4CAT promoter constructs along with 2 µg of
pRSV/
-gal. Myotubes were then treated for 48 h with the vehicle
alone (
) or 1 µM A23187 (+). For EGTA experiments,
myotubes were preincubated for 5 h in the presence of 1 mM EGTA. CAT activity were normalized for transfection
efficiency using
-galactosidase activity. Data are means ± S.E. of three to five independent transfection experiments (*,
p < 0.05 versus without A23187).
281 base pairs upstream of the transcription start site, whereas
another CRE (CRE-2) is found at position
119 (22). Analysis of
variance indicated that significant main effects of A23187 and promoter construct existed, but that the effect of A23187 depended on the construct used. A23187 increased cytochrome c
transactivation mediated by the
726 promoter construct by 2.4-fold
(p < 0.05). Elimination of 400 base pairs downstream
of position
726 using pRC4CAT/
326 did not modify the magnitude of
the transcriptional activation induced by A23187 (Fig. 2B),
as expected on the basis of the locations of the CREs. Surprisingly,
myotubes transfected with pRC4CAT/
66 showed a greater relative
response (3.9-fold) to A23187 treatment compared with cells transfected
with pRC4CAT/
726 or pRC4CAT/
326, despite drastic reductions in
absolute CAT activity. These experiments verify that the DNA sequence
encompassed between positions
326 and
66 is necessary for a high
constitutive level of cytochrome c gene expression in L6E9
cells and that A23187-induced transcriptional activation does not seem
to involve the activation of the CRE sites.
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Fig. 3.
Effects of CREB and PKA cotransfections,
inhibition of CaM kinase, and inhibition of PKC on cytochrome
c transcriptional activation. A, 5 µg of pRSV/CREB and 5 µg of pC EV (plasmids expressing CREB and
the catalytic subunit of PKA, respectively) were cotransfected into
L6E9 myoblasts along with 10 µg of pRC4CAT/
326 and 2 µg of
pRSV/
-gal. Plasmid DNA concentration was adjusted with carrier
plasmid. Myotubes were then treated for 48 h with either the
vehicle alone (
) or 1 µM A23187 (+). Cell extracts and
CAT and
-galactosidase activity assays were performed as described
under "Experimental Procedures." The normalized CAT activity
obtained with pRC4CAT/
326 in the absence of transactivating plasmids
was defined as 1.0. Values are means ± S.E. of three to seven
independent experiments (*, p < 0.05 versus
without A23187). Inset, typical immunoblot of CREB
expression in the presence of empty vector (E) or pRSV/CREB
(C). B, myoblasts were transfected with 10 µg
of pRC4CAT/
326 and 2 µg of pRSV/
-gal expression vector. Once
differentiated, the myotubes were preincubated with various
concentrations of KN62 for 24 h prior to treatment for 48 h
with 1 µM A23187 (+) or vehicle (
). CAT and
-galactosidase activities were then determined. Data were
derived from three to nine independent experiments (*,
p < 0.05 versus without A23187).
C, transfections of myoblasts were performed with 10 µg of
pRC4CAT/
326 and 2 µg of pRSV/
-gal expression vector. Once
differentiated, the myotubes were preincubated with the indicated
concentrations of staurosporine or TPA for 24 h before treatment
for 48 h with either vehicle (
) or with 1 µM
A23187 (+). The activity obtained with pRC4CAT/
326 in the absence of
PKC inhibitors was defined as 1.0. Values are taken from three to five
independent experiments (*, p < 0.05 versus
without A23187; ¶, p < 0.05 versus
with A23187, no additions).
326 along
with either the Ca2+-sensitive PKC
or
PKC
II isoform or the Ca2+-insensitive PKC
isoform. Transcriptional activation by A23187 was significantly
(p < 0.05) enhanced in the presence of the
and
II isoforms compared with the empty vector pTB, but not
in the presence of the Ca2+-insensitive PKC
isoform
(Fig. 4A). These experiments
confirmed the involvement of Ca2+-sensitive PKC isoforms in
the response triggered by A23187.
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Fig. 4.
Role of PKC isoforms and MEK inhibition in
cytochrome c transcriptional activation and effect of
A23187 on ERK1 and ERK2 activities. A, effect of PKC
isoform overexpression on cytochrome c transcriptional
activation. L6E9 myoblasts were cotransfected with 10 µg of
pRC4CAT/ 326 and 5 µg of PKC expression vectors or empty vector
(pTB). Following transfection, the myoblasts were induced to
differentiate by switching to low serum conditions until the myotubes
reached 85-90% confluence. The myotubes were then treated with A23187
(0.75 µM) or vehicle (dimethyl sulfoxide
(DMSO)) for 48 h. CAT assays were normalized with
-galactosidase activity (*, p < 0.05 versus the empty vector pTB). B, role of MEK in
A23187-mediated cytochrome c transactivation. L6E9 myoblasts
were transfected with 10 µg of pRC4CAT/
326. Following
differentiation, the myotubes were treated with PD98059 (PD;
12.5 µM) or vehicle 30 min prior to the addition of
A23187 (A; 0.75 µM) or vehicle (dimethyl
sulfoxide (D)) for 48 h. CAT assays were normalized
with
-galactosidase activity (*, p < 0.05 versus without PD98059). C, time course of ERK1
(p42) and ERK2 (p44) MAPK activation in response to A23187. L6E9
myotubes were treated with A23187 (A; 0.75 µM)
or vehicle (dimethyl sulfoxide (D)) for the indicated time
points or were not treated (NT). Some cells were also
treated with PD98059 (PD; 12.5 µM) for 30 min
prior to the addition of the ionophore. Whole cell lysates were
prepared for immunoblot analyses as described under "Experimental
Procedures" using a phospho-specific ERK antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
326 and
66 is necessary for a high
constitutive level of cytochrome c gene expression in L6E9
muscle cells. Despite this, the minimal cytochrome c
promoter (pRC4CAT/
66) still possessed a high degree of
Ca2+ responsiveness. The Ca2+-responsive
element downstream of position
66 is currently under investigation.
Although there are no apparent AP-1 or CRE sites in this region, the
response may be related to the presence of CCAAT box-like sequences at
positions +23 and +48 of the first intron. This sequence has previously
been shown to confer A23187-mediated transcriptional activation of the
grp78 promoter (44). It does not appear that Sp1 by itself is involved
since preliminary investigations have shown that Sp1 overexpression
actually reduces transcriptional activation of the full-length
cytochrome c promoter and that restoration of
transcriptional activation by A23187 is independent of
Sp1.2
,
I,
II, and
)
are Ca2+-dependent (45). Here, we show that the
effect of the ionophore on cytochrome c transactivation is
dramatically reduced by PKC inhibitors, indicating that a
Ca2+-sensitive, PKC-dependent pathway is
involved in L6E9 cells. This conclusion is further fortified by the
fact that cells cotransfected with the Ca2+-sensitive
PKC
and PKC
II isoforms illustrate a greater response to the Ca2+ ionophore than cells cotransfected with the
Ca2+-insensitive PKC
isoform. Since it was reported that
the only detectable Ca2+-sensitive isoform in L6 cells
under non-transfected conditions is PKC
(36) and that this isoform
is known to be down-regulated by prolonged treatment with TPA (36),
these data suggest that the Ca2+-mediated effect on
cytochrome c gene expression occurs via PKC
. However, it
appears that other regulatory influences exist as well since ~25% of
the response triggered by A23187 was not abolished by TPA treatment,
but was eliminated by the nonspecific PKC inhibitor staurosporine. The
broader spectrum of action of staurosporine likely inhibited other
kinases involved in parallel signal transduction pathway(s) that could
have been activated by the ionophore. Such a candidate may be c-Jun
N-terminal kinase since recent work using contractile activity to
increase cytosolic Ca2+ levels in cardiac myocytes (46) and
skeletal muscle (47) have demonstrated its activation.
has been shown to phosphorylate c-Raf (48),
and c-Raf is known to phosphorylate and activate MEK directly (49),
thus leading to the activation of the ERK1 and ERK2 MAPKs. The facts
that the A23187-induced increase in cytochrome c
transactivation was enhanced by PKC
, that it was reduced by
treatment of the cells with the MEK inhibitor PD98059, and that A23187
induced an increase in ERK1 and ERK2 activities with a time course
consistent with their involvement in downstream cytochrome c
transactivation strongly suggest that this pathway is active in
Ca2+-mediated cytochrome c expression. Thus, the
following sequence of events is supported by our data: 1) A23187
mobilizes Ca2+ from the extracellular pool to increase its
intracellular concentration, which 2) triggers the activation of a
PKC/MEK/MAPK-dependent pathway leading to 3) the
transactivation of cytochrome c. This is the likely cause of
the subsequent increase in cytochrome c mRNA observed. Further work will define the requirement for de novo protein
synthesis as well as the Ca2+-responsive element involved
and whether an increase in intracellular Ca2+ levels can
act as a general signal mediating an increase in the expression of a
variety of nuclear genes encoding mitochondrial proteins.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. C. Scarpulla for the donation of the cytochrome c promoter constructs and Dr. J. McDermott for the provision of L6E9 cells.
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FOOTNOTES |
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* This work was supported in part by a grant from the National Science 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.
Recipient of a postdoctoral fellowship from the Région
Rhônes-Alpes (France). Present address: Lab. de Physiologie,
Faculté de Médicine, 15 rue A. Paré, 42023 Saint-Etienne Cedex 2, France.
§ To whom correspondence should be addressed: Dept. of Biology, York University, Toronto, Ontario M3J 1P3, Canada. Tel.: 416-736-2100 (ext. 66640); Fax: 416-736-5698; E-mail: dhood{at}yorku.ca.
2 M. Di Carlo and D. A. Hood, unpublished observations.
3 P. Kumar and D. A. Hood, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CRE, cAMP response element; CREB, CRE-binding protein; CaM kinase, calcium/calmodulin-dependent protein kinase; PKC, protein kinase C; PKA, protein kinase A; TPA, 12-O-tetradecanoylphorbol-13-acetate; CAT, chloramphenicol acetyltransferase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase.
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