Functional interaction of regulatory factors with the
Pgc-1{alpha} promoter in response to exercise by in
vivo imaging
Takayuki Akimoto, Ping Li and Zhen Yan
Am J Physiol Cell Physiol 295:288-292, 2008. First published Apr 23, 2008;
doi:10.1152/ajpcell.00104.2008
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Am J Physiol Cell Physiol 295: C288 –C292, 2008.
First published April 23, 2008; doi:10.1152/ajpcell.00104.2008.
Report
Functional interaction of regulatory factors with the Pgc-1␣ promoter
in response to exercise by in vivo imaging
Takayuki Akimoto, Ping Li, and Zhen Yan
Department of Medicine, Duke University Medical Center, Durham, North Carolina
Submitted 19 February 2008; accepted in final form 17 April 2008
⫺222) sequences in the promoter that converge biological
signals for the transcriptional control of the gene (6, 12). We
previously established a novel optical bioluminescence imaging system in living mice and showed that double mutation of
the two MEF2 sites or single mutation of the CRE site resulted
in loss of Pgc-1␣ promoter responsiveness to motor nerve
stimulation in skeletal muscle (2). These findings support the
notion that exercise-induced Pgc-1␣ transcription is mediated
by regulatory factors that interact with these cis elements on
the Pgc-1␣ promoter. The functional interaction between regulatory factors and Pgc-1␣ promoter in response to increased
neuromuscular activities remains to be elucidated in vivo.
MATERIALS AND METHODS
EXTENSIVE RESEARCH IN SIGNALING and molecular mechanisms of
skeletal muscle plasticity suggests that peroxisome proliferator-activated receptor-␥ coactivator-1␣ (Pgc-1␣), a versatile
transcriptional coactivator (15), plays a pivotal role in exerciseinduced genetic reprogramming in skeletal muscle (1, 3, 10,
17, 27). Although Pgc-1␣ activity could be controlled by
posttranslational modification (8, 16), there is clear evidence
that the Pgc-1␣ gene is transcriptionally activated in response
to exercise, which could play an important role in skeletal
muscle adaptation. Elucidating the signal transduction that
leads to transcriptional regulation of the Pgc-1␣ gene will,
therefore, likely provide valuable insights into the fundamental
mechanisms of skeletal muscle plasticity and help in development of effective therapeutics for chronic diseases that are
related to skeletal muscle contractile and metabolic functions.
A great challenge to exercise scientists is that exercise training
cannot be faithfully recapitulated in experimental models in
vitro. An imaging system with real-time imaging of gene
regulation in living animals will significantly facilitate this
important research area.
The mouse Pgc-1␣ gene, similar to its mammalian homologs, contains conserved myocyte enhancer factor 2 (MEF2;
at ⫺2901 and ⫺1539) and cAMP response element (CRE; at
Animals. Male C57BL/6J mice (7– 8 wk old) were obtained commercially (Jackson Laboratory) and housed in temperature-controlled
(21°C) quarters with a 12:12-h light-dark cycle and water and chow
(Purina) provided ad libitum. All experimental protocols were approved by the Duke University Institutional Animal Care and Use
Committee.
Plasmid DNA constructs. Pgc-1␣L is a construct containing a
3.1-kb 5⬘-flanking region of the mouse Pgc-1␣ gene upstream of the
firefly luciferase coding sequence (6), and mutations of each of the
MEF2 binding sites, ⌬MEF2(⫺2901) and ⌬MEF2(⫺1539), were
generated by site-directed mutagenesis based on the sequence information described previously (6). Pgc-1␣L with CRE site mutation,
⌬CRE(⫺222), has been used previously (2). Plasmid HDAC5(S2A)
encodes FLAG-tagged histone deacetylase 5 (HDAC5) with mutations of serine sites at 259 and 498 to nonphosphorylatable alanine
sites (31). Similarly, HDAC4(S3A) encodes FLAG-tagged HDAC4
with serine-to-alanine mutations at residues 246, 467, and 632 (38).
These mutations render the mutant HDAC proteins resistant to upstream signals (31, 38). Plasmid PKD(K/W) encodes a catalytically
inactive mutant of protein kinase D (PKD) with a hemagglutinin (HA)
tag (31). The dominant-negative ATF2⌬N encodes a FLAG-tagged,
truncated activating transcription factor 2 (ATF2) with NH2-terminal
deletion from amino acid 1 to 137 (30). pCI-neo is a mammalian
expression vector without coding region (Promega) used as a negative
control for the mutant/deletion expression vectors described above.
pEGFP-N1 was purchased from Invitrogen. All plasmid DNAs were
purified using the Endo-Free Plasmid Mega kit (Qiagene) and dissolved at a concentration of 2.5 mg/ml in 0.9% NaCl solution.
Electric pulse-mediated gene transfer. Electric pulse-mediated
gene transfer was modified from previous studies (2, 9, 25). Briefly,
under pentobarbital sodium anesthesia (50 mg/kg ip), both tibialis
anterior (TA) muscles were injected with a mixture of DNA (15 ␮g of
Pgc-1␣L and 50 ␮g of plasmid DNA encoding a mutant/deletion form
of regulatory factor) by use of a 0.5-ml syringe with a 28-gauge needle
at a rate of ⬍0.015 ml/min. Eight electric pulses (100 ms, 1 Hz, 100
V) were delivered immediately to the injected TA muscle using a
Address for reprint requests and other correspondence: Z. Yan, 4321
Medical Park Dr., Suite 200, Duke Univ. Independence Park Facility, Durham,
NC 27704 (e-mail: zhen.yan@duke.edu).
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.
signal transduction; transcriptional control; reporter gene; optical
biolunminescence imaging; electric pulse-mediated gene transfer
C288
0363-6143/08 $8.00 Copyright © 2008 the American Physiological Society
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Akimoto T, Li P, Yan Z. Functional interaction of regulatory
factors with the Pgc-1␣ promoter in response to exercise by in vivo
imaging. Am J Physiol Cell Physiol 295: C288 –C292, 2008. First
published April 23, 2008; doi:10.1152/ajpcell.00104.2008.—Realtime optical bioluminescence imaging is a powerful tool for studies of
gene regulation in living animals. To elucidate exercise-induced
signaling/transcriptional control of the peroxisome proliferator-activated receptor-␥ coactivator-1␣ (Pgc-1␣) gene in skeletal muscle, we
combined this technology with electric pulse-mediated gene transfer
to cotransfect the Pgc-1␣ reporter gene with plasmid DNA encoding
mutant/deletion forms of putative regulatory factors and, thereby,
assess the responsiveness of the promoter to skeletal muscle contraction. We show that each of the myocyte enhancer factor 2 sites on the
Pgc-1␣ promoter is required for contractile activity-induced Pgc-1␣
transcription. The responsiveness of the Pgc-1␣ promoter to contractile activity could be completely blocked by overexpression of the
dominant-negative form of activating transcription factor 2 (ATF2),
the signaling-resistant form of histone deacetylase (HDAC) 5
(HDAC5), or protein kinase D (PKD), but not by HDAC4. These
findings provide in vivo evidence for functional interactions between
PKD/HDAC5 and ATF2 regulatory factors and the Pgc-1␣ gene in
adult skeletal muscle.
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CONTROL OF Pgc-1␣ PROMOTER BY REGULATORY FACTORS IN VIVO
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square-pulse stimulator (model S88K, Grass Telefactor) through a
two-needle array (model 533, BTX) placed on the medial and lateral
sides of the TA muscle, so that the electrical field was perpendicular
to the long axis of the myofibers. Mice were allowed to recover for 10
days before electrode implantation, nerve stimulation, and imaging
analysis.
Motor nerve stimulation and optical bioluminescence imaging.
Under anesthesia, electrodes were implanted, and motor nerve stimulation was initiated within 30 min and continued for 2 h. In vivo
bioluminescence images were acquired and analyzed as described
previously (2). The total flux (in photons 䡠 s⫺1 䡠 cm⫺2 䡠 steradian) within
the region of interest was measured. The ratio of the total flux from the
stimulated muscle to that from the contralateral control muscle was
determined before and after motor nerve stimulation.
Western immunoblot analysis. TA muscles were homogenized and
analyzed by immunoblot as described elsewhere (32). Antibodies used
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Fig. 1. Peroxisome proliferator-activated receptor-␥ coactivator-1␣ (Pgc-1␣)
promoter activity is dependent on each of the myocytic enhancer factor 2
(MEF2) and cAMP response element (CRE) sites. After transfection of both
tibialis anterior (TA) muscles by Pgc-1␣L or its derivative mutant constructs,
the motor nerve of one of the TA muscles was stimulated for bioluminescence
imaging analysis. A: schematic presentation of plasmid DNAs. Pgc-1␣ promoter (3.1 kb) is presented as a solid line with two MEF2 sites and one CRE
site. Crosses denote site-directed mutagenesis as described elsewhere (6, 12).
B: in vivo imaging of luciferase activity in mouse TA muscles from Pgc-1␣L
and its mutant derivatives ⌬MEF2(⫺2901), ⌬MEF2(⫺1539), and ⌬CRE(⫺222).
Imaging analysis was performed 10 days after gene transfer. Pseudocolors
overlaid on the image indicate intensity of luminescent signals from luciferase
reporter gene activity. Animals’ right TA muscles were stimulated (S), and
their left TA muscles were sham-operated without stimulation and used as
reference control (C). C: quantification of luciferase activity in TA muscles.
Dashed horizontal line denotes basal level before stimulation. Values are
means ⫾ SE; n ⫽ 6 – 8. **P ⬍ 0.01 vs. poststimulation values.
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Fig. 2. Efficient gene expression following electric pulse-mediated gene transfer. Plasmid DNA [65 ␮g of pEGFP-N1 or 15 ␮g of Pgc-1␣L and 50 ␮g of
plasmid DNA encoding an empty control vector (pCI-neo) or a mutant/deletion
form of regulatory factor] was transfected into TA muscles, which were
visualized by epifluorescent microscopy (A) and subjected to homogenization
and immunobloting analysis (B). A: TA muscle transfected with pEGFP-N1
was harvested and sectioned for phase contrast (Phase) and fluorescent microscopic examination for expression of green fluorescent protein (GFP). There
were ⬃60% GFP-positive myofibers. Red lines outline boundary of muscle
sections. *, Myofibers that are negative for GFP signals. Scale bars, 250 and 50
␮m for ⫻4 and ⫻40 images, respectively. B: expression of ATF2⌬N,
HDAC5(S/A), HDAC4(S/A), and PKD(K/W) could be easily detected in
transfected TA muscles by immunoblot using anti-FLAG or anti-hemagglutinin (HA) antibodies for tagged proteins and anti-␣-tubulin antibodies for
protein loading. ATF2, activating transcription factor 2; HDAC, histone
deacetylase; PKD, protein kinase D.
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CONTROL OF Pgc-1␣ PROMOTER BY REGULATORY FACTORS IN VIVO
Fig. 3. Contractile activity-dependent functional interaction between Pgc-1␣
promoter and upstream regulatory factors. A: in vivo imaging of luciferase
activity in mouse TA muscles from Pgc-1␣L cotransfected with an empty
control vector (pCI-neo), ATF2⌬N, HDAC5(S/A), HDAC4(S/A), or PKD(K/W)
before (Pre) and 2 h after (Post) low-frequency (10-Hz) nerve stimulation (2 h).
Animals’ right TA muscles were stimulated (S), and their left TA muscles were
sham-operated without stimulation and used as reference control (C).
B: quantitative analysis of luciferase activity from Pgc-1␣L. Dashed horizontal
line denotes basal level before stimulation. Values are means ⫾ SE. *P ⬍ 0.05;
**P ⬍ 0.01 vs. poststimulation values.
were ANTI-FLAG polyclonal antibody (F-7425, Sigma), anti-HA
antibody (Roche), and anti-␣-tubulin antibody (Sigma).
Statistics. Luciferase activity in the stimulated TA muscle was
divided by that in the contralateral control muscle. Values before and
after motor nerve stimulation were compared by paired Student’s
t-test, with P ⬍ 0.05 considered statistically significant. Values are
means ⫾ SE.
RESULTS AND DISCUSSION
To further define the functional role of the cis elements on
the Pgc-1␣ promoter, we performed site-directed mutagenesis
for each of the MEF2 sites individually and confirmed that they
are equally important for the transcriptional activation of the
AJP-Cell Physiol • VOL
Fig. 4. Model for exercise-induced transcription of the Pgc-1␣ gene in skeletal
muscle. Increased neuromuscular activities elicit signals leading to activation
of the p38 MAPK pathway and transcriptional upregulation of the Pgc-1␣ gene
through ATF2 (1, 34) and MEF2 (11, 37). Activation of the p38 MAPK
pathway can also promote Pgc-1␣ transcription by inhibiting p160 and derepressing PGC-1␣ protein function (8), exerting a positive regulation of Pgc-1␣
gene transcription through MEF2 (12, 24). In parallel, exercise also promotes
Pgc-1␣ transcription through PKD/HDAC5-mediated derepression of MEF2
function (6). These signaling-transcription coupling cascades integrate contractile and metabolic cues from neuromuscular activity to control expression
of the Pgc-1␣ gene in skeletal muscle adaptation. Regulatory factors/cis
elements, the functional role of which has been tested and confirmed in the
present study, are shown as gray boxes with solid lines. Other relevant
regulatory factors that were not investigated in the present study are shown as
open boxes with dashed lines.
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Pgc-1␣ gene in skeletal muscle in vivo (Fig. 1). These findings
raised the possibility that exercise-induced Pgc-1␣ transcriptional activation is fully dependent on functional interactions of
regulatory factors with the MEF2 and the CRE cis elements on
the Pgc-1␣ promoter.
To address this issue, we chose to employ a loss-of-function
approach by cotransfecting mutant/deletion forms of the putative regulatory factors in adult skeletal muscle with the Pgc1␣L reporter gene. This approach, analogous to cotransfection
approaches used extensively in cultured cells, had not been
possible in a living animal until recent improvement of the gene
transfer technique in adult skeletal muscle (9, 25). We first checked
the efficiency of in vivo cotransfection to ensure possible
dominant-negative effect of the transgenes. Gene transfer of a
plasmid DNA containing the enhanced green fluorescent protein transfected ⱖ60% of the myofibers in mouse TA muscles
(Fig. 2A), which maintained normal morphology following the
recovery (10 days). We then cotransfected TA muscles with
plasmid DNAs containing Pgc-1␣L reporter gene (3.1-kb
mouse Pgc-1␣ promoter driving firefly luciferase) and an
empty control vector (pCI-neo) or plasmid DNA encoding
epitope-tagged mutant/deletion forms of putative regulatory
proteins. Consistent with efficient gene transfer, transgene
products, but not the empty control vector, could be readily
detected by immunoblot analysis (Fig. 2B). These results
demonstrated high feasibility for the in vivo approach and
prompted us to proceed with the investigation of functional
interactions of the putative regulatory factors with the MEF2
and CRE cis elements on the Pgc-1␣ promoter.
MEF2 activity has long been implicated in muscle plasticity
(35, 36), and association of MEF2 with class II HDACs, such
as HDAC4 and HDAC5, results in repression of the MEF2
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CONTROL OF Pgc-1␣ PROMOTER BY REGULATORY FACTORS IN VIVO
AJP-Cell Physiol • VOL
tion of the p38 MAPK pathway or overexpression of a dominant-negative form of ATF2 blocks the transcriptional activation of the Pgc-1␣ gene in skeletal muscles in culture (1, 34).
However, the functional role of ATF2 in transcriptional control
of the Pgc-1␣ gene in vivo has not been confirmed in skeletal
muscle in a physiological model of exercise. Here, we showed
that overexpression of a truncated, dominant-negative form of
ATF2 (30) completely blocked contractile activity-induced
Pgc-1␣ reporter gene activity, providing direct evidence that
ATF2 plays an essential role in the transcriptional control of
the Pgc-1␣ gene in skeletal muscle (Fig. 3).
Interestingly, the PKD/HDAC5/MEF2 and p38/ATF2 signal
transductions have been linked to the stress and the calcium
signals in striated muscles (7, 23, 31, 34). The functional
interactions of these two regulatory modules with the Pgc-1␣
promoter observed in the present study are consistent with the
notion that multiple cellular signals converge to control a
powerful regulatory factor, such as Pgc-1␣, in defining skeletal
muscle phenotype (Fig. 4). The multiple signal/transcription
control system would not only allow for more sophisticated
fine tuning of the adaptive functions, but it would also ensure
the validity of the signals to avoid unnecessary dysregulation
of the system. Future research should focus on dissection of
each one of the physiological signals in skeletal muscle adaptation. The analytic system employed in the present study by
combining efficient gene transfer of regulatory factors with
biochemiluminescence imaging of reporter gene is an exciting
step toward a complete elucidation of all the signaling and
transcription mechanisms of skeletal muscle adaptation and
could be applicable to other organ systems and disease models.
ACKNOWLEDGMENTS
We thank E. N. Olson and R. Bassel-Duby for kind gifts of Pgc-1␣L and
HDAC5(S2A) and PKD(K/W) DNA constructs, T. P. Yao for providing
HDAC4(S3A) construct, and G. Thiel for providing ATF2⌬N construct. We
thank Mei Zhang for excellent technical assistance.
Present address of T. Akimoto: Division of Biomedical Materials and
Systems, Center for Disease Biology and Integrative Medicine, Graduate
School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo
113-0033, Japan.
GRANTS
This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-050429 to Z. Yan.
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