Upstream Stimulatory Factor Represses the Induction of
Carnitine Palmitoyltransferase-I
Expression by PGC-1*
Meredith L.
Moore,
Edwards A.
Park
, and
Jeanie B.
McMillin§
From the Department of Pathology and Laboratory Medicine, The
University of Texas Medical School at Houston, UT-Houston Health
Science Center and the Graduate School of Biomedical Sciences, The
Texas Medical Center, Houston, Texas 77030 and the
Department of Pharmacology, The University of Tennessee,
Memphis, Tennessee 38163
Received for publication, October 13, 2002, and in revised form, February 20, 2003
 |
ABSTRACT |
Transcriptional regulation of carnitine
palmitoyltransferase-1
(CPT-1
) is coordinated with contractile
gene expression through cardiac-enriched transcription factors, GATA4
and SRF. Metabolic modulation of CPT-1
promoter activity
has been described with the stimulation of gene expression by oleate
that is mediated through the peroxisome proliferator-activated receptor
(PPAR) pathway. The coactivator, peroxisomal proliferator-activated
receptor
coactivator (PGC-1), enhances gene expression through
interactions with nuclear hormone receptors and the myocyte enhancer
factor 2 (MEF2) family. PGC-1 and MEF2A synergistically activate
CPT-1
promoter activity. This stimulation is enhanced by
mutation of the E-box sequences that flank the MEF2A binding site.
These elements bind the upstream stimulatory factors (USF1 and USF2),
which activate transcription in CV-1 fibroblasts. However,
overexpression of the USF proteins in myocytes depresses CPT-1
activity and significantly reduces MEF2A and PGC-1 synergy.
Co-immunoprecipitation studies demonstrate that PGC-1 and USF2 proteins
can physically interact. Our studies demonstrate that PGC-1 stimulates
CPT-1
gene expression through MEF2A. USF proteins
have a novel role in repressing the expression of the
CPT-1
gene and modulating the induction by the
coactivator, PGC
1.
 |
INTRODUCTION |
Carnitine palmitoyltransferase-1
(CPT-1)1 is located on the
outer mitochondrial membrane and functions in the transport of fatty
acids into the matrix for
-oxidation. The
isoform (CPT-1
) is
primarily expressed in skeletal and cardiac muscle and white adipose
tissue (1). The
isoform of CPT-1 (CPT-1
) predominates in the
remaining tissues and exhibits different kinetics from the muscle
isoform. Early evidence suggests that CPT-1
is transcriptionally up-regulated in response to electrical stimulation and consequent hypertrophic growth (2). The cardiac-enriched factors GATA4 and serum
response factor (SRF) are synergistic activators of CPT-1
expression
(3). Because metabolic substrate preference shifts with hypertrophy and
failure, recent studies have focused on the identification of
transcription factors capable of transmitting nutritional as well as
pathological messages into changes in gene transcription.
Fatty-acid induction of gene expression through peroxisome proliferator
activator receptor-
(PPAR
) binding to the muscle-specific form of
CPT-1
has been extensively studied (4, 5). However, the rat
CPT-1
gene is only modestly induced by physiological levels of fatty acids (6), and CPT-1
message is increased 2-fold in
rodent models of fasting and diabetes where circulating fatty acids are
elevated (7). The PPAR
-mediated regulation of several
genes is enhanced by the PPAR
coactivator-1 (PGC-1). PGC-1 is highly
expressed in metabolically active tissues including brown fat, skeletal
muscle, and heart (8). PGC-1 has been implicated in mitochondrial
biogenesis in the heart and increased mitochondrial respiration in
brown fat (8). PGC-1 is a coactivator for many factors in the nuclear
hormone receptor family including PPAR
, the glucocorticoid receptor,
the thyroid hormone receptor, and several orphan receptors (9-12).
These combinatorial interactions upregulate the expression of fatty
acid oxidation, oxidative phosphorylation, and tricarboxylic acid cycle
enzymes as well as uncoupling proteins in response to cold, fasting, or
exercise (8). PGC-1 also physically interacts with myocyte enhancer
factor 2C (MEF2C) to upregulate GLUT4 expression and glucose uptake in
L6 cells that were overexpressing PGC-1 (13). Associations with
negative binding partners have been proposed but no specific proteins
have yet been identified (14).
Upstream stimulatory factor (USF) is a member of the basic
helix-loop-helix leucine zipper family and preferentially binds to the
E-box consensus CANNTG with CG as interior nucleotides (15). In the
heart, USF proteins regulate the expression of energy transfer and
contractile-responsive sarcomeric genes (16). We have shown that USF
heterodimers bind to two E-box sites within the CPT-1
promoter. However, the regulatory significance of USF in the context of
this gene is unknown (3). Although USF is expressed ubiquitously, the
relative amount of USF proteins varies with tissue type (17).
Alterations in USF protein stoichiometry may be involved in regulating
different sets of genes. While a high degree of amino acid conservation
characterizes their dimerization and DNA binding sequences, their
extreme divergence in N-terminal amino acid sequences could direct
selective contacts for a variety of transcription factors (17).
Here, we have examined the regulation of the CPT-1
gene by PGC-1 and USF proteins. We found that PGC-1 stimulates
CPT-1
expression through interactions with MEF2A. Furthermore, the
induction by PGC-1 and MEF2A is inhibited by USF. We demonstrate that
PCG-1 and USF proteins can physically interact. Our results demonstrate that PCG-1 is a powerful stimulator of a key regulatory protein in long
chain fatty acid oxidation in the heart and suggest a novel role for
USF in modulating PGC-1 action on the CPT-1
gene.
 |
EXPERIMENTAL PROCEDURES |
Plasmids and Luciferase Constructs--
The p-391/+80 rat
CPT-1
luciferase construct has been described (McMillin et
al., Ref. 18). Generation of site mutations within the
391/+80
fragment was performed with the QuickChange Mutagenesis kit
(Stratagene, La Jolla, CA) as reported (3). All promoter constructs and
their correct insertions were confirmed by DNA sequencing.
Cell Culture and Transfections--
Isolation of neonatal rat
cardiac myocytes was performed as previously described (19). Cells were
plated in 6-well plates (Primeria, Fisher, Pittsburgh, PA) at a cell
density of 6 × 105 cells/well. Dulbecco's Modified
Eagle's Medium was supplemented with 1% penicillin/streptomycin
(Invitrogen) and 10% calf serum (Hyclone Laboratories, Inc.,
Logan, UT). Cultures were maintained at 37 °C in an atmosphere of
95% air and 5% CO2 for 36 h before transfection.
CV-1 fibroblast cells (ATCC no. CCL-70) were trypsinized 24 h
before transfection and plated to 85% confluence. Both cell types were
transfected with LipofectAMINE PLUS Reagent (Invitrogen) in serum-free
medium for 3 h. The medium was then replaced with serum-containing
culture medium for an additional 48 h. Transfections included 1.0 µg of wild-type or mutated CPT-1
391/+80 firefly luciferase
reporter gene construct and 0.25 µg of pRL CMV-Renilla luciferase construct to control for transfection efficiency.
Co-transfections included various combinations of the following
CMV-driven expression vectors: USF1, USF2, USF2
B or USF2
N (from
Dr. M. Sawadogo), and pSV-PGC-1 (from Dr. B. Spiegelman). Total DNA
concentrations were kept constant with the corresponding empty vectors.
Final protein concentrations were determined with the BCA protein assay reagent kit (Pierce, Rockford, IL), and luciferase activities were
measured with the Promega Dual Luciferase kit (Madison, WI). Promoter
activity is expressed as Renilla-corrected firefly
luciferase/total protein.
Electrophoretic Mobility Shift Assays--
Nuclear extracts from
primary rat neonatal myocytes were prepared as described (20).
Double-stranded DNA probes that contained sequences from the rat
CPT-1
promoter were synthesized by Operon Technologies,
Inc. (Alameda, CA) as follows: Ebox-315
ctagcaggctacacagctgactcctggg (underlined sequences from
Ebox-315 were mutated to TagcCT); Ebox-252 ctagcaccatgctcacgtgagaccctcg (underlined sequences from
Ebox-252 were mutated to cGcgCA). EMSA reaction mixtures included 2-5
µg of nuclear extract, 25 mM Hepes, 100 mM
KCl, 0.1% Nonidet P40 (v/v), 1 mM dithiothreitol, 5%
glycerol, and 50 ng of polydeoxyinosine:deoxycytosine (poly(dIdC)) as a
nonspecific competitor in a 20-µl reaction volume. After a 10-min
room temperature incubation, 0.3 ng of radiolabeled probe was added,
and the reaction was allowed to incubate for an additional 20 min.
Antibody (SantaCruz Biotechnology, Santa Cruz, CA) or 25-100-fold
molar excess of cold probe was added during the first incubation when
included. Protein-DNA complexes were separated on a 4% non-denaturing
polyacrylamide gel at room temperature.
Western Blotting--
Transfected myocytes or CV-1 cells were
harvested in 200 µl of sample buffer (1.5% SDS, 60 mM
Tris-HCl, 11% glycerol in phosphate-buffered saline), sonicated, and
boiled. 10 µg of protein were separated on a 10% polyacrylamide
ready gel (Bio-Rad Laboratories, Hercules, CA) and transferred to
polyvinylidene difluoride membrane (PerkinElmer Life Science Products,
Boston, MA). Immunoblots were performed with a USF2 polyclonal primary
antibody (Santa Cruz Biotechnology) diluted 1:3000 or a PGC-1 antibody
(Calbiochem, San Diego, CA) diluted 1:1000. Reactive proteins were
detected with the appropriate secondary antibody and chemi-luminescence
reagents (PerkinElmer Life Science Products).
Immunoprecipitation--
Rat neonatal myocytes were cultured in
10-cm plates, washed with 1× phosphate-buffered saline, and harvested
in 0.75 ml of modified RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.5% deoxycholate,
1 mM phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, and protease inhibitors) for 10 min at
80°. Cell lysates were cleared with centrifugation, and the
supernatant was precleared with protein A beads for 2 h. 500 µl
of precleared extract was incubated with 2 µl of appropriate antibody
for 6 h. Protein A beads were added for an additional overnight
incubation under identical conditions. Beads were washed in modified
RIPA buffer, diluted in Lammeli buffer, boiled for 5 min, and analyzed by Western blot as described above. Where indicated, cell extracts were replaced with 10 µg of purified GST fusion proteins and 15 µl
of in vitro transcription translation (TNT)
product programmed per manufacturer's instructions (Promega, Madison
WI), and diluted in 500 µl of modified RIPA buffer.
GST Pull-down Assays--
GST and GST-PGC-1
fusion proteins
were prepared as described previously (21).
[35S]methionine-labeled USF-1 and USF-2 were expressed
using a linked transcription/translation kit (PROTEINscript II from
Ambion) (22). GST-PGC-1
proteins bound to Sepharose-4B were
incubated with 35S-labeled proteins in 20 mM
Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and
a protease inhibitor mixture (Sigma). The Sepharose-4B-bound GST
proteins were washed four times with the buffer used for binding. Bound
proteins were eluted in Laemmli buffer and resolved by SDS-PAGE. The
35S-labeled proteins were visualized by storage phosphor autoradiography.
 |
RESULTS |
Our first experiments examined the relative affinity of nuclear
protein binding to the E-boxes in the CPT-1 promoter. We
have previously shown that USF1 and 2 bind to the E-box sequences from the CPT-1
promoter as heterodimers (3). The consensus
E-box element
252 robustly binds USF proteins (Fig.
1, lane 2). The E-box element
315 requires additional nuclear extract from cardiac myocytes to
produce a similar band (Fig. 1, compare lane 2, 2-µl nuclear extract versus lane 8, 5-µl nuclear
extract). To examine further the relative binding strengths of the two
E-box regions, increasing concentrations of the unlabeled alternate DNA
sequence were added to the gel shift binding reaction. Residual USF
binding persists on element
252 even in the presence of 100-fold
molar excess of the unlabeled
315 sequence (Fig. 1, lanes
3-5). However, a 25-fold excess of
252 competitor is sufficient
to compete all protein binding to the E-box
315 (Fig. 1, lanes
9-11). Consistent with its consensus sequence, E-box
252 is the
stronger USF binding site. However, E-box-315 also binds USF with lower
affinity.

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Fig. 1.
USF1 and USF2 proteins preferentially bind to
E-box element 252. USF binding to CPT-1 promoter
sequences was characterized by gel mobility shift assay. Oligomers for
the two E-box elements were 32P-end-labeled and incubated
with nuclear extract (2 µl for E-Box-252; 5 µl for E-Box-315) from
rat neonatal cardiac myocytes as described (see "Experimental
Procedures"). Unlabeled double-stranded oligomers were used as
competitors and were added in excess of the labeled probe as is
indicated above each lane. The unlabeled mutant oligomer contains a
mutation in the USF site and was added in up to 100-fold excess.
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To define a regulatory role for USF proteins with the
CPT-1
gene, both USF1 and USF2 were transfected into CV-1
fibroblasts with the CPT-1
luciferase reporter construct. A model of
binding sites in the CPT-1
promoter is shown in Fig.
2, top panel. Expression of
the CPT-1
promoter increased 15.8 ± 1.5-fold by USF
overexpression (Fig. 2). Co-transfection of a vector with the
N-terminal activation domain of USF2 deleted (Fig. 2,
USF
N) significantly reduced USF1/USF2 up-regulation of
the CPT-1
construct. Removal of the DNA binding/dimerization domain
of USF2 (Fig. 2, USF
B) diminished promoter activation by
6.5 ± 0.9-fold (p < 0.0001). A weaker functional
inhibition by USF
N compared with USF
B may be, in part, due to
reduced expression of the former construct in CV-1 cells (Fig. 2,
inset). Therefore, USF proteins can activate CPT-1
in
CV-1 fibroblasts and requires intact protein structure to regulate gene
expression.

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Fig. 2.
Domain deletions within the USF2 protein
abrogate function in CV-1 cells. Top, diagram of the
CPT-1 promoter is shown and the key regulatory elements
identified. Early passage CV-1 fibroblasts were co-transfected with 1 µg each 391/+80 wild-type reporter, USF1, USF2, USF2 N, or
USF2 B as indicated. Cells were harvested, and luciferase activity
measured and normalized as described. Bars represent the
mean ± S.E. for triplicate measurements from three independent
experiments. Inset, 10 µg of protein from CV-1 cells
transfected with USF2, USF2 N, or USF2 B was separated on 10%
ready gels and analyzed by Western blot with specific USF2 antibodies.
*, p < 0.0005; §, p < 10 6.
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Since cell-type dependent functions have been reported for USF1 and
USF2 (23, 24), we wished to determine whether similar effects of USF-1
on CPT-1
would be observed in cardiac myocytes. We transfected out
CPT-1
-luciferase into cardiac myocytes with the USF expression
vectors. In cardiac myocytes, mutation of E-box
315 increases
promoter activity 2.2 ± 0.2-fold (p < 0.01),
suggesting that USF proteins function to suppress CPT-1
gene expression in this cell type. Consistent with this idea,
overexpression of USF1 and USF2 in cardiac myocytes reduces CPT-1
luciferase expression to less than 40% of basal activity (Fig.
3). Substitution of a DNA
binding-deficient mutant USF2 for the wild-type factor does not affect
USF2-mediated suppression. However, truncation of the N-terminal domain
returns luciferase values to baseline, suggesting that the suppressor
activity of USF2 may be localized in this region (Fig. 3). The finding
of opposite effects of USF overexpression in CV-1 fibroblasts
versus cardiac myocytes suggests that USF-mediated repression of CPT-1
reporter gene expression requires USF
interactions with myocyte-enriched factors.

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Fig. 3.
Overexpression of USF proteins inhibits
activity of the CPT-1 promoter construct.
Myocytes were co-transfected as described with 1 µg of 391/+80
reporter gene construct. Combinations of 1.0 µg of USF1 and either
1.0 µg of USF2 (wild-type) or USF2 B or USF2 N (1 µg each) were
overexpressed, as indicated below the x axis. Total DNA concentrations
were kept constant with the empty vector pSG5. Cells were harvested
after 48 h in serum containing Dulbecco's modified Eagle's
medium. Luciferase was measured and corrected for Renilla
expression and protein content. Bars represent mean ± S.E. for triplicate measurements from three separate passages. *,
p < 10 5.
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PPAR
and RXR
can bind to the fatty acid response element (FARE)
(also called PPRE) of CPT-1
, and these nuclear receptors can interact with PGC-1 (25, 26). In CPT-1
, the PPRE is flanked by
the two E-box motifs, and this region also contains the
myocyte-specific (MEF2) site that binds MEF2A and MEF2C (3). To
identify potential cell-specific interactions, we first investigated
the effects of USF on the PGC-1 regulation of the CPT-1
promoter in neonatal cardiac myocytes. Transfection of PGC-1-stimulated
reporter activity in cardiac myocytes in a dose-dependent
manner (Fig. 4A).
Overexpression of USF proteins in the myocyte culture completely
blunted the PGC-1 induction of CPT-1
and returned expression to
basal levels (Fig. 4B). To determine the interactions by
which USF interferes with PGC-1 function in cardiac myocytes, we
investigated this regulatory motif for myocyte-specific proteins,
e.g. MEF-2, that are known to recruit the latter cofactor
(13).

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Fig. 4.
USF expression blunts PGC-1 up-regulation of
the CPT-1 promoter construct.
A, myocytes were co-transfected as described with 1 µg of
391/+80 reporter gene construct and increasing concentrations of
PGC-1 expression vector, as indicated. B, myocytes were
co-transfected with 1 µg of the 391/+80 CPT-1 construct and 0.5 µg each of USF1, USF2, and PGC-1 as indicated. Total DNA
concentrations were kept constant with the appropriate empty vector.
Cells were harvested after 48 h in serum containing Dulbecco's
modified Eagle's medium. Luciferase was measured and corrected for
Renilla expression and protein content. Bars
represent mean ± S.E. for triplicate measurements from 3 separate
passages. *, p < 0.0001compared to PGC-1 alone.
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Endogenous levels of muscle-specific transcription factors often
interfere with transfection studies in myocytes or are already expressed at saturating concentrations. Therefore, to determine the
magnitude of MEF2A and PGC-1 regulation of CPT-1
, these expression vectors were co-transfected into a cardiac-null fibroblast cell line,
CV-1. Here, MEF2A synergistically and significantly co-activates PGC-1
induction of CPT-1
reporter gene expression by greater than 40-fold (Fig. 5). Because USF
prevented PGC-1 activation of CPT-1
luciferase expression in cardiac
myocytes (Fig. 4), we asked whether the bHLH factor could also
interfere with PGC-1/MEF2A synergy. Addition of USF1 and 2 expression
vectors abolished synergy and decreased PGC-1/MEF2A activation by
greater than 60% (Fig. 5). Substitution of either serum response
factor (SRF), or the cardiac-enriched factor, Nkx2.5, for USF does not
affect PGC-1/MEF2A synergy (Table I). To
determine if USF proteins might be interfering with the ability of
MEF2A to stimulate CPT-1
, we cotransfected CPT-1
-Luc
with expression vectors for USF proteins and MEF2A. USF1/2 alone
stimulated
391/+80 CPT-1
-Luc 4.3 ± 0.6-fold, while MEF2A
stimulated 1.7 ± 0.15-fold. When both USF and MEF2A were
transfected, the CPT-1
-Luc vector was stimulated 6.1 ± 0.65-fold. These data indicate that USF represses CPT-1
promoter activity by blocking synergistic interactions between PGC-1
and the muscle-specific transcription factor, MEF2A.

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Fig. 5.
PGC-1 and MEF2A synergistic activation of
CPT-1 is sensitive to USF1/2
overexpression. Subconfluent CV-1 cells were transfected as
described with 1 µg of reporter, 0.5 µg PGC-1, 0.5 µg MEF2A, and
0.25 µg each USF1 and USF2, as indicated. Total DNA concentrations
were kept constant at 2.5 µg with the appropriate empty vector. Cells
were maintained in serum-containing media for 48 h, harvested, and
assayed as described. Bars represent triplicate measurements from three
independent experiments presented as mean ± S.E. The significance
of comparisons indicated as an asterisk (p < 10 6) compared with PGC-1 alone, and § (p < 10 6) compared with PGC-1 plus MEF2A.
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Table I
Inhibition of PGC-1/MEF2A synergy is specific for USF
CV-1 fibroblasts were co-transfected with 1.0 µg of luciferase
reporter, 0.5 µg of PGC-1, 0.5 of µg MEF2A and either 0.1 µg of
SRF, 0.5 µg of Nkx2.5, or 0.25 µg each USF1 and USF2. USF
inhibition studies were also performed on the double E-box mutant
promoter (DEM). Data are expressed as percent reduction in the
magnitude of synergistic activation compared to empty vector controls.
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To confirm that E-box-binding proteins could alter PGC-1/MEF2A
interactions, reporter constructs with mutations in either or both
E-box elements were transfected with the synergistic combination into
CV-1 cells. Individual E-box mutations in either E-box
315 or E-box
252 increased synergy to 69 ± 4- and 82 ± 7-fold,
respectively (versus 43 ± 3-fold for wild type, Fig.
6). The double E-box mutant showed an
even greater response to PGC-1/MEF2A, i.e. 117 ± 8-fold induction (Fig. 6). PGC-1/MEF2A synergistic activation of the double E-box mutant construct remained sensitive to USF overexpression (Table I), suggesting that direct DNA binding was not essential for USF
modulation of PGC-1/MEF2A interactions. Therefore, based on these data,
USF appears to function by regulating MEF2 recruitment of cofactors
like PGC-1.

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Fig. 6.
PGC-1/MEF2A synergy is enhanced by E-box
sequence mutation. Early passage CV-1 cells were transfected with
the synergistic combination 0.5 µg of PGC-1 and 0.5 µg of MEF2A
with 1.0 µg of either wild-type or E-box mutant reporter construct as
indicated (DEM, double E-box mutant). Cells were harvested
after 48 h in serum containing Dulbecco's modified Eagle's
medium, and luciferase was measured and corrected for both
Renilla expression and protein content. Bars represent
mean ± S.E. for at least three experiments performed in
triplicate, and p values are compared with synergy on the
wild-type promoter.
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Our next experiments were performed to determine if USF proteins could
interact with PGC-1. Co-immunoprecipitation experiments with anti-USF2
IP of neonatal rat cardiac myocyte extract revealed PGC-1 interactions
following immunoblots with anti-PGC-1 antibodies (Fig.
7A, IP). Anti-USF2
IP of neonatal rat cardiac myocyte extract revealed PGC-1 interactions
following immunoblot with anti-PGC-1 antibodies (Fig. 7A,
IB). PGC-1 protein was also detected in control anti-MEF2
immunoprecipitates but not anti-actin precipitates (data not shown). To
confirm USF2/PGC-1 interaction, anti-GST immunoprecipitation with
PGC-1-GST fusion protein and USF2 TNT products also
revealed the presence of USF2 protein (Fig. 7A). IP
reactions with unprogrammed TNT products did not pull-down USF2 protein
(data not shown). We also conducted GST-PGC-1 pull-down experiments
using bacterially expressed GST-PGC-1 and
[35S]methionine-labeled USF-1 or USF-2. As is shown in
Fig. 7B, the full-length PGC-1 or PGC-1 vectors containing
either amino acids 1-400 or 1-170 were able to pull-down USF-1 and
USF-2. GST alone did not interact with either isoform of USF. These
data indicate that USF proteins can interact with peptides within the
first 170 amino acids of PGC-1.

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Fig. 7.
PGC-1 and USF2 physically interact.
A, co-immunoprecipitation (IP) with MEF2 and USF
antibodies was performed with protein extracts from cultured, neonatal
rat cardiac myocyte as described under "Experimental Procedures."
Antibodies used for Western immunoblot analysis (IB) are
listed on the left hand side of the gel. In the GST lane,
PGC-1 fused to GST was incubated with GST antibodies and USF2 TNT
reaction products. Proteins bound to Sepharose beads were released by
boiling and analyzed by Western blot. Input lane contained 5-10% of
the total protein used for the immunoprecipitation. Nonspecific bands
detected by the PGC-1 antibody in the input lane were removed during
the washing steps and are not present in the final IP lanes.
B, GST-PGC-1 was prepared as described under "Experimental
Procedures." [35S]USF-1 or [35S]USF-2
were incubated with GST-PGC proteins of varying lengths in in
vitro pull-downs. The proteins were subjected to electrophoresis
in SDS-acrylamide gels and visualized by autoradiography.
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 |
DISCUSSION |
Fatty acid oxidation accounts for the majority of energy
utilization in the normal, beating heart and fatty acid is the dominant fuel used by the diabetic heart. Since CPT-1
regulates fatty acid
entry into
-oxidation, this enzyme has a prominent role in
modulating changes in fuel selection. Long term adaptive responses extend beyond localized adjustments in enzyme activity from changes in
the malonyl-CoA pool. Metabolic up-regulation of CPT-1
gene expression by fatty acids occurs through the
ligand-dependent activation of PPAR
binding to a PPRE
motif in the promoter region of human (5) and rat CPT-1
(6). The
stimulation of genes encoding proteins involved in cardiac fatty acid
transport and metabolism by long chain fatty acids is well documented
(5, 27). Consistent with these observations, the rat
CPT-1
promoter is only moderately increased in neonatal
cardiac myocytes exposed to physiological concentrations of oleate (6).
Cotransfection of PPAR
and PGC-1 into NIH3T3 cells stimulated
luciferase genes driven by the promoters of the human
CPT-1
or mouse MCAD genes (10). Here we show
that CPT-1
gene expression is activated in cardiac
myocytes in a concentration-dependent manner by
overexpression of PGC-1.
Our data indicate that PGC-1 will induce CPT-1
through interactions
with MEF2C. Since the MEF2A and PPAR
binding sites are adjacent in
the rat CPT-1
promoter, we believe that both factors will
recruit PCG-1 to the CPT-1
promoter and induce high
expression of CPT-1
in the heart. In fact, PPAR
and MEF2C
interact with different regions of PGC-1 allowing for the possibility
that PGC-1 could physically interact with both proteins simultaneously.
MEF2C interacts with amino acids in the 400-570 region of PGC-1 (8), while PPAR
interacts with the LXXLL peptide motif at
amino acids 142-146 of PGC-1 (10). The MEF2C and PPAR
binding sites
in the CPT-1
promoter are flanked by E-boxes that bind
USF proteins. Our results indicate that the USF proteins can disrupt
the synergistic stimulation between MEF2C and PGC-1. The ability of USF
to interfere with the transcriptional cooperation of PGC-1 and MEF2C
may stem from the specific architecture of the CPT-1
promoter. Further studies are needed to determine if the inhibition of
PGC-1 transactivation by USF proteins is a general phenomenon or is
mediated in a gene-specific and tissue-specific manner.
The up-regulation of metabolic genes critical to mitochondrial energy
production in the heart is controlled by factors binding to conserved
regulatory motifs found in heart and striated muscle, including the
GATA and MEF2 elements and the CArG and E-boxes. These motifs, present
in the
myosin heavy chain gene (28), troponin I (29),
actin
(30), muscle creatine kinase (31), and the
Na+-Ca2+ exchanger (32), are also conserved in
the muscle-specific mitochondrial isoforms of cytochrome oxidase (33),
mitochondrial creatine kinase (34), and CPT-1
(18). These genomic
sequences have been proposed to be necessary in specifying expression
of genes involved in high energy phosphate production and energy
channeling to the unique oxidative requirements of cardiac muscle
(34).
In contrast to the normal working heart, carbohydrate utilization
increases at the expense of lipids during cardiac hypertrophy. Hypertrophied hearts exhibit reduced levels of PPAR
expression with
a down-regulation of fatty acid oxidation genes (35). Therefore, maladaptive reliance on glycolytic pathways in heart failure and concomitant down-regulation of CPT-1
suggest that glucose-responsive transcription factors may play a role in appositional changes in fatty
acid and glucose metabolism genes.
USF binding to E-box sites in cardiac-specific promoters has been
proposed to be a component of protein complexes that coordinately control the expression of myosin light-chain 2 and
myosin heavy chain genes (36, 37). The role of internal and flanking E-box nucleotide sequences in the context of the cardiac TnI gene
demonstrates that the specific nucleotide composition is important to
the regulation of bHLH-mediated gene expression (29). Likewise, the
majority of genes in which the promoter contains USF-binding E-boxes
are not regulated by glucose (38). Thus, the context of the E-box and
the protein composition of the USF-binding complex are important in
eliciting a transcriptional response. USF is ubiquitously expressed (17), so tissue-specific factors should play an important role in
determining the ultimate regulatory consequences of USF activity. We
have demonstrated that the action of USF on CPT-1
reporter gene expression is dependent on cell-specific protein
interactions, where USF up-regulates CPT-1
in fibroblasts and
down-regulates CPT-1
in cardiac myocytes. PGC-1-mediated activation
of CPT-1
is abolished by USF in cardiac myocytes as well as in
fibroblasts when the cardiac context for the MEF2 element adjacent to
E-252 on the CPT-1 gene is invoked by overexpression of the
cardiac-specific factor, MEF2A.
PGC-1 is most widely recognized for its role in co-activating nuclear
hormone receptors so that the downstream targets, while varied, are
genes involved in thermogenesis, energy production, and mitochondrial
biogenesis (8, 25, 39, 40). The inducible nature of PGC-1 expression
provides the cell with a mechanism for stimulation of metabolism in
response to stress (40). PGC-1-mediated up-regulation of GLUT4 responds
to an increased need for metabolic substrate to prevent self-catabolism
(13). This proposition could also be extended to include a possible
role for the up-regulation of fatty acid import (27).
USF binding to E-boxes is required for transcriptional activation of a
variety of cardiac genes including contractile proteins as well as for
the activity of the
B-crystallin enhancer (41). Alteration in
MAPK/ERK signaling pathways is a molecular mechanism by which factor
phosphorylation, e.g. PPAR
(35), may coordinate and
modulate these various signaling pathways during hypertrophic growth.
Phosphorylation of p38 enhances PGC-1 co-activation of PPAR
(42, 43)
and promotes transduction of cytokine signals to PGC-1 regulated genes
(44). During contractile stimulation, USF1 is phosphorylated resulting
in enhanced DNA binding and increased
myosin heavy chain promoter
activity (45). Application of a phosphorylation-dependent
regulatory scheme to PGC-1/USF interactions would identify another
transcription factor interaction responsible for cross-talk among
cardiac gene families. MAPK activity releases PGC-1 from the inhibitory
association of a PGC-1/repressor complex associated with the L2/L3
region of PGC-1 (14) and promotes co-activation of
PGC-1-dependent promoters. If PGC-1/USF association is
regulated in a similar manner, this
phosphorylation-dependent mechanism could potentially
integrate PGC-1 stimulated fatty acid metabolism with increased
contractile gene expression. Alternatively, the MEF2 binding site
flanked by E-box elements within the promoters of contractile genes
(46-48) could also recruit modulatory USF proteins.
In summary, the current data suggests that PGC-1 will promote high
levels of CPT-1
gene expression in the heart through
interactions with PPAR
and MEF2C. USF proteins can modulate the
inductive effects of MEF2C and PGC-1. USF2 and PGC-1 physically
interact, defining a new mechanism for the upstream stimulatory factors in the coordination of fatty acid oxidation genes. The potential exists
for phosphorylation control to integrate stress kinase signaling with
gene expression through these transcription factor interactions. Given
the altered substrate preference and increased p38 activity in
pathologic hypertrophy, this regulatory mechanism may play a
significant role in the pleitrophic genetic response to disease.
 |
ACKNOWLEDGEMENTS |
We thank Chad Jones for assistance with the
cardiac myocyte preparations and Dr. S. Song for the GST pull-down
assays. We also thank Dr. Michelle Sawadogo for USF expression vectors
and Dr. Bruce Spiegelman for the PGC-1 expression vector.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant RO1 HL38863, the Juvenile Diabetes Research Foundation (JDRF), and the American Heart Association (AHA).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 Pathology and
Laboratory Medicine, The University of Texas Medical School at Houston,
6431 Fannin, Houston, TX 77030. Tel.: 713-500-5335; Fax: 703-500-0730;
E-mail: Jeanie.B.McMillin@uth.tmc.edu.
Published, JBC Papers in Press, February 28, 2003, DOI 10.1074/jbc.M210486200
 |
ABBREVIATIONS |
The abbreviations used are:
CPT-1, carnitine
palmitoyltransferase-1;
SRF, serum response factor;
PPAR, peroxisome
proliferator activator receptor;
PGC-1, PPAR
coactivator-1;
MEF2, myocyte enhancer factor 2;
USF, upstream stimulatory factor;
L-PK, liver-type pyruvate kinase;
MAPK, mitogen-activated protein kinase;
TNT, transcription/translation;
GST, glutathione
S-transferase;
RIPA, radioimmune precipitation assay
buffer;
CMV, cytomegalovirus;
ERK, extracellular-regulated
kinase.
 |
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