From the Departments of Medicine and
§ Cell Biology & Physiology, Washington University School of
Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Nuclear respiratory factor (NRF)-1 appears to be
important for the expression of several respiratory genes, but there is
no direct evidence that NRF-1 transduces a physiological signal into the production of an enzyme critical for mitochondrial biogenesis. We
generated HeLa cells containing plasmids allowing doxycycline-inducible expression of uncoupling protein (UCP)-1. In the absence of
doxycycline, UCP-1 mRNA and protein were undetectable. In the
presence of doxycycline, UCP-1 was expressed and oxygen consumption
doubled. This rise in oxygen consumption was associated with an
increase in NRF-1 mRNA. It was also associated with an increase in
NRF-1 protein binding activity as determined by electrophoretic
mobility shift assay using a functional NRF-1 binding site from the
Tissues adapt to an increased need for energy by increasing
mitochondria, resulting in an enhanced capacity to generate ATP by
oxidative phosphorylation. Examples of this phenomenon include the
adaptation of skeletal muscle to exercise (1, 2) and the adaptation of
liver to thyrotoxicosis (3, 4). Respiration, the transfer of electrons
from fuels to oxygen, provides most of the energy for mammalian cells.
This electron transfer is accomplished by respiratory cytochromes at
the mitochondrial inner membrane that contain heme (5).
Increasing respiratory capacity requires an increase in mitochondrial
size and number. Mitochondria have their own genome, a 16.5-kilobase
circular strand of DNA encoding only 13 of the more than 100 proteins
necessary for electron transfer to oxygen (5). The remaining proteins
necessary for oxidative phosphorylation (including ALA synthase), the
enzymes for fatty acid oxidation and the tricarboxylic acid cycle, and
the factors regulating mitochondrial DNA transcription and replication
are products of nuclear genes. Mitochondrial biogenesis thus depends on
the coordination of nuclear and mitochondrial events.
Nuclear respiratory factor (NRF)-1 may be responsible for this
coordination. NRF-1, a transcription factor encoded by nuclear DNA
(human chromosome 7, Ref. 7), was first identified by Evans and
Scarpulla (8) as an activator of cytochrome c gene
transcription. Functional binding sites for NRF-1 were subsequently
described in several nuclear genes critical for mitochondrial
biogenesis, including ALA synthase (9) and human mitochondrial
transcription factor A (TFAM) (10). Disruption of the Tfam
gene in mice abolishes oxidative phosphorylation and prevents
mitochondrial biogenesis (11).
There is no direct evidence that NRF-1 transduces physiological signals
to mitochondria. We addressed the question of whether an increase in
cellular metabolism prompts an increase in mitochondrial biogenesis
through NRF-1. HeLa cells, the source for the initial purification of
the NRF-1 protein (12), were engineered to inducibly express uncoupling
protein (UCP)-1, and ALA synthase protein was assayed as a marker for
stimulation of the assembly of mitochondrial respiratory complexes. The
results suggest that NRF-1 is a critical component of the
energy-sensing mechanism in mammalian cells.
Cloning of the Mouse UCP-1 cDNA--
Mouse UCP-1 was cloned
by RT-PCR using brown adipose tissue from cold-induced mice as the
source of mRNA. Adult C57BL/6J mice were placed at 4 °C for
6 h (13). Interscapular brown adipose tissue was harvested, total
RNA was prepared by equilibrium centrifugation in cesium chloride (14),
and poly(A)+ RNA was isolated using reagents in kit form
(QIAGEN, Valencia, CA). RT-PCR was performed using avian myeloblastosis
virus reverse transcriptase (TitanTM, Roche Molecular
Biochemicals). Primers were based on a mouse UCP-1 sequence
(nucleotides 184-207 and 1184-1207; GenBankTM accession
no. U63419). The upstream primer was 5'-TGA GTC CTT GAA TTC
TTG CAC TCA-3' (with underlined bases indicating substitutions generating an EcoRI site), and the downstream primer was
5'-GTC TCC CAG TCT AGA AGC CCA ATG-3' (with underlined
bases generating an XbaI site). Experiments with mice were
approved by the Animal Studies Committee at Washington University.
Engineering HeLa Cells for Inducible Expression of
UCP-1--
Regulated expression of UCP-1 was achieved using a
doxycycline-inducible gene expression system (Tet-On,
CLONTECH, Palo Alto, CA) originally described by
Gossen et al. (15). UCP-1 cDNA generated by RT-PCR was
directionally cloned into the EcoRI/XbaI sites of the plasmid pTRE and sequenced in entirety. The product, pUCP-1 (Fig.
1), was cotransfected with a plasmid
carrying a hygromycin resistance cassette (the ratio of pUCP-1 to the
hygromycin resistance plasmid was 20:1) into HeLa cells stably
transfected with the pTet-On plasmid (Fig. 1) by calcium phosphate
precipitation. Stable transfectants were identified by selection with
hygromycin at a concentration of 200 µg/ml. Three independent cell
lines stably transfected with mouse UCP-1 (as verified by RT-PCR in the
presence of doxycycline) were used for these experiments, and each
showed the same results.
HeLa Cell Culture--
After expansion, aliquots of stably
transfected UCP-1 HeLa cells were frozen until needed for specific
experiments. Cells were treated with trypsin-EDTA, washed, resuspended
in cell freezing medium (10% Me2SO, 50% fetal bovine
serum, and 40% culture medium (described below)) at 4 °C, kept at
Oxygen Consumption--
Oxygen consumption of UCP-1 stably
transfected HeLa cells treated with doxycycline and mouse brown fat was
measured using a model 5300 oxygen monitor (YSI Inc.). In preliminary
experiments, oxygen uptake was shown to be linearly dependent on input
cellular protein. After 6 h in the presence (+Dox) or absence
(
For measurement of oxygen consumption in brown fat, three adult
C57BL/6J mice were placed at 4 °C for 4.5 h followed by
isolation of interscapular brown fat in the following medium: 20 mM potassium phosphate (pH 7.4), 20 mM
potassium chloride, 1.6 mM EDTA, 5 mM magnesium
chloride, 1 mM sodium malate, 10 mM sodium
pyruvate, 123 mM sucrose, and 2 mM Tris (pH
7.4). Brown fat was also isolated from three littermates kept at room
temperature. Oxygen consumption was measured as described above.
Antibodies and Western Blotting--
Rabbit anti-mouse UCP-1
antiserum (recognizing the C terminus of mouse and rat UCP-1) was
purchased from Alpha Diagnostic International (San Antonio, TX).
Western blotting was performed using SDS-polyacrylamide gel
electrophoresis with transfer to Immobilon-P as described previously
(16), with the exception that chemiluminescence was used to generate
autoradiographs. The anti-UCP-1 primary antiserum and the secondary
antibody were used at a dilution of 1:3000.
Custom antiserum to ALA synthase was generated by Alpha Diagnostic
International. Rabbits were immunized with a 19-residue synthetic
peptide containing a C-terminal ALA synthase sequence conserved across
species: SER EKA YFS GMS KMV SAQ A. The antiserum was strongly reactive
in an enzyme-linked immunosorbent assay based on the injected peptide
and detected a protein of the predicted size for ALA synthase (70 kDa,
see Fig. 6) on Western blots. For ALA synthase Western blotting, the
primary antibody was used at a dilution of 1:2500, and all experiments
were performed using input protein within the linear response range of
the assay as verified by densitometric scanning.
NRF-1 Antisense Oligonucleotides--
18-mer antisense
oligonucleotides were designed to hybridize in the region of the human
NRF-1 translation initiation codon. Positive results were achieved
using the following antisense phosphorothioate-modified oligonucleotide
(complementary to human NRF-1 nucleotides 108-125, GenBankTM accession no. L22454): 5' CCT CCA TGA AGT TCT ACA
3'. The control "scrambled" phosphorothioate-modified
oligonucleotide (with the same base composition as the antisense
oligonucleotide) was 5' CAT GTA CGC AAC TCT ACT 3'.
HeLa cells containing pUCP-1 were transfected with antisense or
scrambled oligonucleotides using cationic liposomes (LipofectAMINE, Life Technologies, Inc.) by a modification of the protocol described by
Quaggin et al. (17). 30 µg of liposomes were complexed
with antisense or scrambled oligonucleotide and then added to 5 ml of
prewarmed culture medium containing 2 µg/ml doxycycline to yield a
final oligonucleotide concentration of 1 µM.
For antisense experiments, cells were fed culture medium containing 2 µg/ml doxycycline to initiate transcription of the UCP-1 gene. After
20 min, the medium was replaced with medium containing 2 µg/ml
doxycycline and either NRF-1 antisense or scrambled oligonucleotide complexed with cationic liposomes. 16 h later, cells were
harvested, extracts were prepared, and ALA synthase protein was assayed
by Western blotting.
Detection of UCP-1,
For all assays, HeLa total RNA was subjected to first strand synthesis
using avian myeloblastosis virus reverse transcriptase at 50 °C for
30 min. For multiplex amplification of UCP-1 and Electrophoretic Mobility Shift Assay for NRF-1
Protein--
Oligonucleotides A and B, containing a functional NRF-1
binding site in the ALA synthase promoter (9), were synthesized as
follows (with the recognition sequence for NRF-1 underlined): Oligo A,
5' G GCC GCT GCGCATGCGC TGT G 3'; Oligo B, 5' CCC ACA GCGCATGCGC AGC GG 3'. Control oligonucleotides were
synthesized as follows (with the mutated regions underlined): Oligo C,
5' G GCC GCT GAAAATGAAA TGT G 3'; Oligo D, 5' CCC ACA
TTTCATTTTC AGC GG. Probes were labeled using Klenow in
reaction mixtures containing dGTP and [
Nuclear extracts were prepared by the method of Dignam et
al. (19) as modified by Towler et al. (20). Binding
assays were performed in a total volume of 20 µl with 10 µg of
bovine serum albumin and 1 µg of sonicated salmon sperm DNA.
Preliminary experiments were performed to determine the range of input
nuclear protein that generated a linear response for the gel shift
signal on autoradiographs. Quantitative binding experiments were
performed under conditions of probe excess. After 20 min at room
temperature, binding reactions were loaded on prerun 4-15% native
gradient gels. For supershifting, reactions included 1 µl of
undiluted goat anti-NRF-1 antiserum (a gift from Richard Scarpulla,
Northwestern University) or 1 µl of undiluted control antiserum
(rabbit anti-ALA synthase antiserum, generated as described above).
Overexpression of NRF-1 in HeLa Cells--
The human NRF-1
cDNA (GenBankTM accession no. L22454) sequence between
nucleotides 37 and 1680, encompassing the coding region, was subcloned
into pCI-neo (Promega, Madison, WI), a neomycin-selectable mammalian
expression vector containing the CMV promoter. The resulting plasmid,
pNRF-1, was sequenced and then transfected into HeLa cells by calcium
phosphate precipitation. Parallel cells were subjected to the
transfection protocol in the absence of pNRF-1 (mock transfection).
24 h after transfection, pNRF-1 plates were fed medium containing
G418 at a concentration of 400 µg/ml. Resistant clones were isolated
and expanded over the next several weeks whereas mock-transfected cells
were passaged in parallel.
After expansion, cell lysates were subjected to Western blotting using
goat anti-NRF-1 antiserum as the primary antibody at a dilution of
1:3000. Once NRF-1-positive clones were identified, ALA synthase
Western blotting was performed as described above.
HeLa cells were isolated that contained both pTet-On (Fig.
1A), directing constitutive expression of a transcriptional
activator that binds to a tetracycline-responsive element (TRE) in the
presence of doxycycline, and pUCP-1 (Fig. 1B), which
contains a TRE. In the absence of doxycycline, control human -aminolevulinate (ALA) synthase promoter. Respiratory uncoupling
also caused a time-dependent increase in protein levels of
ALA synthase, an early marker for mitochondrial biogenesis. ALA
synthase induction by respiratory uncoupling was prevented by
transfecting cells with an oligonucleotide antisense to the region of
the NRF-1 initiation codon; a scrambled oligonucleotide with the same
base composition had no effect. Respiratory uncoupling increases oxygen
consumption and lowers energy reserves. In HeLa cells, uncoupling also
increases ALA synthase, an enzyme critical for mitochondrial
respiration, but only if translatable mRNA for NRF-1 is available.
These data suggest that the transcription factor NRF-1 plays a key role
in cellular adaptation to energy demands by translating physiological signals into an increased capacity for generating energy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Aminolevulinate (ALA)1
synthase is rate-limiting for the synthesis of heme (6). ALA synthase
catalyzes the reaction of succinyl-CoA with glycine to form ALA, a
precursor for protoporphyrin IX. Protoporphyrin combines with iron to
form heme, essential for electron transfer and energy generation.
Therefore, the induction of ALA synthase expression is critical to
effect an increase in cellular respiratory capacity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
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Fig. 1.
Strategy for engineering HeLa cells for
inducible expression of UCP-1. HeLa cells were isolated that were
stably transfected with the plasmids depicted in A and
B. pTet-On contains a CMV promoter driving constitutive
expression of a fusion protein consisting of a reverse tetracycline
repressor (rTetR) and the herpes virus VP16 activation
domain. pUCP-1 contains a TRE upstream of the minimal (min)
immediate early CMV promoter. The mouse UCP-1 cDNA, generated by
RT-PCR using brown fat RNA from cold-induced mice, was subcloned at
EcoRI/XbaI sites. In the presence of the
antibiotic doxycycline (C, +Dox), rTetR/VP16
binds to the TRE and drives UCP-1 transcription. ATG and
TAA indicate the relative positions of the start and stop
codons, respectively, in the UCP-1 cDNA.
20 °C for 2 h and
80 °C overnight, and then transferred
to liquid nitrogen. Aliquots were thawed rapidly at 37 °C and plated
in culture medium (Dulbecco's modified Eagle's medium with 10% fetal
bovine serum, 2 mM glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin). Medium was changed every 3-4 days. To
induce UCP-1 expression, cells were fed fresh culture medium, and
24 h later the medium was replaced with culture medium containing
either 2 µg/ml doxycycline (+Dox) or an equal volume of carrier
(
Dox).
Dox) of 2 µg/ml doxycycline, cells were trypsinized, washed,
resuspended in prewarmed culture medium, and placed in magnetically
stirred sample chambers containing Clarke-type polarographic oxygen
probes. Oxygen consumption was continuously monitored for 15 min for
each sample, and data were expressed by normalizing to input cell protein.
-Actin, NRF-1, and GAPDH Messages by
RT-PCR--
For UCP-1, primers were based on nucleotides 395-970
(GenBankTM accession no. U63419): upstream, 5' ATA AAG GTG
TCC TAG GGA CCA TCA 3'; downstream, 5' ACA GCT TGG TAC GCT TGG ATA CTG
3'. For
-actin, primers were based on nucleotides 81-394
(GenBankTM accession no. X00351): upstream, 5' TCC GGC
ATG TGC AAG GCC GGC TTC 3'; downstream, 5' TTC TCG CGG TTG GCC TTG GGG
TTC 3'. For NRF-1 mRNA, primers were based on nucleotides 131-694
(GenBankTM accession no. L22454): upstream, 5' GGA GTG ACC
CAA ACC GAA CAT ATG 3'; downstream, 5' TCC GTC GAT GGT GAG AGG CGG CAG
3'. For GAPDH, primers were based on nucleotides 241-522
(GenBankTM accession no. M17701): upstream, 5' CCC ATC ACC
ATC TTC CAG GAG CG 3'; downstream, 5' GTC ATG GAT GAC CTT GGC CAG GG
3'.
-actin, samples
were immediately subjected to amplification for 33 cycles using an
annealing temperature of 60 °C, and products were separated by
agarose gel electrophoresis. For multiplex amplification of NRF-1 and
GAPDH, messages were analyzed semiquantitatively, essentially as
described by Zhou et al. (18). After first strand synthesis,
reaction mixtures were diluted 1-16-fold. Fresh PCR reagents were
added to each tube, and amplification using an annealing temperature of
60 °C was carried out for 33 cycles after preliminary experiments
showed these conditions to be optimal for detecting linear,
dilution-dependent amplification signals.
-32P]dCTP.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin
message was detected in pTet-On/pUCP-1 HeLa cells, but there was no
expression of UCP-1 mRNA in multiplex RT-PCR assays of total RNA
(Fig. 2A,
Dox).
After treatment with 2 µg/ml doxycycline for 6 h, the same cells
expressed UCP-1 mRNA (Fig. 2A, +Dox). Protein
levels mirrored mRNA expression. The 32-kDa UCP-1 protein was
undetectable in extracts from cells cultured in the absence of
doxycycline but present when parallel cultures of the same cells were
treated with 2 µg/ml doxycycline for 6 h (Fig.
2B).
View larger version (42K):
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Fig. 2.
Mouse UCP-1 expression is detected only in
the presence of doxycycline. HeLa cells containing the plasmids
depicted in Fig. 1 were treated with 2 µg/ml doxycycline
(+Dox) or carrier ( Dox). 6 h later, cells
were isolated and processed for preparation of either total RNA
(A) or postnuclear cell protein (B). For
A, RNA was subjected to multiplex RT-PCR using primers
specific for human
-actin and mouse UCP-1. For B, cell
extracts were subjected to Western blotting using rabbit anti-mouse
UCP-1 antiserum known to recognize the C terminus of the UCP-1
protein.
Induction of UCP-1 expression by doxycycline increased oxygen
consumption (Fig. 3A). The
maximum effect was observed at 6 h. At this time point, HeLa cells
carrying pUCP-1 utilized twice as much oxygen as untreated cells
(8.37 ± 1.19 versus 4.37 ± 0.36 nmol/min/mg
protein, mean ± S.E., p = 0.0007, 4 experiments). Doxycycline treatment of HeLa cells carrying only pTet-On (Fig. 3A, first two bars from the left) had
no effect on oxygen consumption.
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To compare the physiological properties of HeLa cells to brown fat, mice were exposed to cold (4 °C for 4.5 h) followed by isolation of brown fat and determination of its oxygen consumption and UCP-1 protein content. Cold exposure increased oxygen consumption by 52% and UCP-1 expression by 62% in brown fat (Fig. 3B). Oxygen consumption in doxycycline-treated HeLa cells was similar to that of brown fat (54% or 8.37 densitometry units for HeLa +Dox versus 15.55 units for 24 °C brown fat). However, UCP-1 expression was proportionally lower in HeLa cells. In a typical experiment using equal amounts of protein, UCP-1 protein was 10.7 densitometry units in HeLa +Dox compared with 49.6 units in 24 °C brown fat and 80.1 units in 4 °C brown fat.
Like messages for many transcription factors, NRF-1 mRNA was not
abundant. NRF-1 mRNA was assayed semiquantitatively as described under "Experimental Procedures." cDNA synthesized from HeLa RNA was serially diluted and then subjected to PCR under conditions within
the linear response range of the assay. As shown in Fig. 4, the NRF-1 mRNA was higher in the
setting of UCP-1 induction for 16 h (+Dox), as compared
with cells not expressing UCP-1 (Dox), whereas there was
no effect of uncoupling on GAPDH mRNA levels. This is best seen by
comparing lanes 4 and 9, which contain products from template that was diluted 16-fold.
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The RT-PCR signals from Fig. 4A are graphically displayed in
Fig. 4B. For NRF-1, there is a 2.4-fold difference
(p = 0.0046) between the slopes of the +Dox
line (r = 0.9857) and
Dox
line (r =
0.9843). The same results were
seen in three experiments.
NRF-1 protein was undetectable by Western blotting. However, NRF-1
protein binding (Fig. 5) was detected by
electrophoretic mobility shift assay using an oligonucleotide
containing a functional NRF-1 binding site from the ALA synthase
promoter and HeLa nuclear extracts. The NRF-1-specific band is
indicated by the asterisk in Fig. 5A. This band
was absent from lanes containing probe but no extract (lane
1). It also did not appear in gel shift assays performed using
negative control oligonucleotides containing a mutated NRF-1 binding
site (data not shown, see "Experimental Procedures" for the
sequence of the negative control oligonucleotides). The band was
supershifted to the level of the arrow in panel A when reactions included an NRF-1 antibody (lane 4) but not
when they were performed in the presence of an antibody to ALA
synthase (lane 3).
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NRF-1 gel shift activity was greater when respiration was uncoupled by
doxycycline (Fig. 5B). The asterisk in Fig.
5B indicates the position of the same band identified by the
asterisk in Fig. 5A. The NRF-1 gel shift was more
intense in the presence of doxycycline (Fig. 5B, lane
2) as compared with Dox cells (lane 1), and the signal was essentially abolished by including a 10-fold molar excess of
unlabeled probe (lane 3). Radiographic density comparisons were made using data from experiments conducted under conditions of
probe excess and using protein concentrations within the linear response range of the assay. Fig. 5C shows a quantitative
analysis of the NRF-1 gel shift; doxycycline increased the signal by
83% (p = 0.0047). Similar results were seen in three
additional experiments.
Respiratory uncoupling also induced ALA synthase expression (Fig.
6). Treatment with doxycycline for
16 h increased the mass of the 70-kDa ALA synthase protein as
assayed by Western blotting (Fig. 6A). The induction of ALA
synthase was time-dependent; an intermediate signal was
detected after 6 h of doxycycline treatment (Fig. 6B).
At 16 h of doxycycline treatment (Fig. 6B, double
asterisk), ALA synthase protein was increased 2.2-fold
(p = 0.0005 by two-tail t test).
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The increase in ALA synthase associated with uncoupling was prevented by transfecting cells with an oligonucleotide antisense to the NRF-1 message. After 16 h of doxycycline treatment, NRF-1 antisense-treated cells did not show an increase in ALA synthase protein (Fig. 6B, second bar from the right), but an induction did occur in cells transfected with a scrambled (same base composition as the NRF-1 antisense oligonucleotide in a different sequence) oligonucleotide (Fig. 6B, asterisk, p = 0.0176 for antisense versus scrambled by two-tail t test).
The relevant comparisons of Fig. 6B remained significant
when analyzed by analysis of variance and the Tukey-Kramer multiple comparisons test. In cells treated with doxycycline for 16 h
(double asterisk), ALA synthase expression was greater than
in Dox cells at 16 h (p < 0.001) and 0 h
(p < 0.001). In cells treated with doxycycline and the
scrambled oligonucleotide (asterisk), ALA synthase
expression was greater than in cells treated with the NRF-1 antisense
oligonucleotide (p < 0.05),
Dox cells at 16 h (p < 0.01), and
Dox cells at 0 h
(p < 0.01).
To address the issue of whether the observed increase in ALA synthase
is due entirely to an increase in NRF-1, HeLa cells overexpressing
NRF-1 were generated (Fig. 7). Cells were
stably transfected with pNRF-1 (containing human NRF-1 cDNA driven
by the CMV promoter) and compared with mock-transfected cells. The 68-kDa NRF-1 protein was not detected in three independent
mock-transfected HeLa cell isolates but was easily detected in three
independent cell lines stably transfected with pNRF-1 (Fig. 7,
arrows in inset). ALA synthase protein levels
were higher in the cells overexpressing NRF-1 (Fig. 7,
p = 0.028). However, the magnitude of the ALA synthase increase (43%) in the setting of high level NRF-1 expression was less
than that observed after UCP-1 expression (see Fig. 6), which was
associated with low level induction of NRF-1 expression.
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DISCUSSION |
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Endurance exercise elevates skeletal muscle respiratory capacity by increasing mitochondrial size and number (1), but the mechanisms that translate an exercise-derived physiological signal into mitochondrial biogenesis are unknown. The current work establishes a system suitable for dissecting the link between energy demands and mitochondrial adaptations to those demands.
Expression of UCP-1 is a suitable mimic of exercise and the stimulation of mitochondrial proliferation. By moving protons from outside to inside the inner mitochondrial membrane without ATP synthesis (21), uncoupling proteins generate heat, decrease energy stores, and increase oxygen consumption, which are classic exercise responses. Exercise causes mitochondrial proliferation in muscle. An unexplained human syndrome of acquired respiratory uncoupling is associated with mitochondrial proliferation in muscle (22). The mitochondrial proliferation that occurs in brown adipose tissue after cold exposure is associated with an increase in UCP-1 and mitochondrial uncoupling (13).
Our results show that inducible expression of UCP-1 results in a 2-fold increase in oxygen consumption (Fig. 3), NRF-1 message (Fig. 4), NRF-1 binding activity (Fig. 5), and ALA synthase protein (Fig. 6A). The induction of ALA synthase protein is prevented by an oligonucleotide antisense to the NRF-1 message (Fig. 6B). These data suggest that increased energy consumption increases expression of the NRF-1 gene, which increases ALA synthase expression by interacting with the NRF-1 binding site in the ALA synthase promoter. Functional binding sites for NRF-1 are also found in the promoters for other genes critical for mitochondrial biogenesis, including human cytochrome c (23), ATP synthase (24), cytochrome oxidase (12), and mitochondrial transcription factor A (10). This observation is consistent with a role for NRF-1 in coordinating energy-related increases in mitochondrial biogenesis.
NRF-1 is not exclusively responsible for the changes in ALA synthase protein observed after UCP-1 expression. High level overexpression of NRF-1 alone increased ALA synthase protein to a lesser degree (Fig. 7) than expression of UCP-1 (Fig. 6). These results suggest that additional transcription factors are necessary to achieve maximal induction of ALA synthase expression or that UCP-1 expression is associated with posttranscriptional mechanisms promoting the accumulation of ALA synthase mass. However, there was no UCP-1-mediated increase in ALA synthase when NRF-1 translation was inhibited using an antisense oligonucleotide. Taken together, these data suggest that NRF-1 is necessary for the induction of ALA synthase expression but not sufficient for maximal expression.
We specifically chose ALA synthase as a marker for activation of mitochondrial function for three reasons. First, exercise is known to increase ALA synthase expression in skeletal muscle (25, 26). Second, ALA synthase, with a half-life estimated at 0.5-2 h (27, 28), has a more rapid turnover rate than other major proteins important for respiration. Even if one assumes a protein half-life of 2 h, 16 h of UCP-1 induction represents 8 half-lives, more than sufficient time for ALA synthase protein mass to reflect a physiological signal. Third, the ALA synthase promoter contains a functional NRF-1 binding site (9).
NRF-1 is a transcription factor belonging to a small family of regulatory proteins important for neuromuscular development in Drosophila and sea urchins (29, 30). These proteins share a conserved N-terminal DNA binding domain; phosphorylation of this domain in NRF-1 promotes DNA binding (31). Because exercise stimulates the mitogen-activated protein kinase pathway in skeletal muscle (32) and functional NRF-1 binding sites are found in the promoters of respiratory genes, it is reasonable to implicate NRF-1 in cellular adaptation to energy demands. Electrical stimulation of neonatal cardiac myocytes increases NRF-1 mRNA and increases NRF-1 binding to the cytochrome c promoter (33). An acute bout of exercise transiently increases NRF-1 expression in rat muscle (34). However, no direct data link a physiological signal and induction of a mitochondrial protein through NRF-1. Our work provides that link. Uncoupling, an exercise mimic, induces NRF-1 expression and ALA synthase protein but only when translatable NRF-1 mRNA is available.
The specific signal upstream of NRF-1 that is triggered by UCP-1 expression is unknown. Calcium is a prime candidate. In other cell systems, mitochondrial uncoupling is known to increase intracellular calcium (35). Calcium-regulated phosphorylation pathways have recently been shown to affect fiber-type specific gene expression, in part through the MEF2 family of transcription factors (36). MEF2C (37) and NRF-1 (31) phosphorylation are strikingly similar, raising the possibility that exercise-induced calcium fluxes alter the activities of these transcription factors through a common mediator.
Our data provide initial evidence that NRF-1 transduces energy-related
signals to mitochondria. Future studies will address how NRF-1 senses
the energy state of the cell, whether muscle cells respond similarly to
uncoupling, and whether NRF-1 alone is sufficient to promote
mitochondrial proliferation in the skeletal muscle of animals.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health (AG00425, HL58427, and DK53198), the Washington University Diabetes Research and Training Center (DK20579) and General Clinical Research Center (RR00036), and an established investigatorship from the American Heart Association.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: Washington University School of Medicine, 660 South Euclid Ave., Box 8046, St. Louis, MO 63110. E-mail: semenkov{at}im.wustl.edu.
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ABBREVIATIONS |
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The abbreviations used are:
ALA, -aminolevulinate;
UCP, uncoupling protein;
NRF, nuclear respiratory
factor;
RT-PCR, reverse transcriptase-polymerase chain reaction;
Dox, doxycycline;
GAPDH, glyceraldehyde-phosphate dehydrogenase;
CMV, cytomegalovirus;
TRE, tetracycline-responsive element.
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REFERENCES |
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