Department of Physiology and Biophysics, University of California, Irvine, California 92697
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ABSTRACT |
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Functional overload (OL)
of the rat plantaris muscle by the removal of synergistic muscles
induces a shift in the myosin heavy chain (MHC) isoform expression
profile from the fast isoforms toward the slow type I, or, -MHC
isoform. Different length rat
-MHC promoters were linked to a
firefly luciferase reporter gene and injected in control and OL
plantaris muscles. Reporter activities of
3,500,
914,
408, and
215 bp promoters increased in response to 1 wk of OL. The smallest
171 bp promoter was not responsive to OL. Mutation analyses of
putative regulatory elements within the
171 and
408 bp region were
performed. The
408 bp promoters containing mutations of the
e1,
distal muscle CAT (MCAT;
e2), CACC, or A/T-rich (GATA), were still
responsive to OL. Only the proximal MCAT (
e3) mutation abolished the
OL response. Gel mobility shift assays revealed a significantly higher
level of complex formation of the
e3 probe with nuclear protein from
OL plantaris compared with control plantaris. These results suggest
that the
e3 site functions as a putative OL-responsive element in
the rat
-MHC gene promoter.
gel mobility shift assay; plantaris muscle; direct gene transfer; dual luciferase; -myosin heavy chain
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INTRODUCTION |
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SKELETAL MUSCLE
FIBERS have the potential to adapt their phenotypic properties to
meet different functional demands. The -myosin heavy chain (MHC)
isoform is typically expressed in "slow" muscles, which are
characterized by a relatively slow speed of shortening but exhibit
fatigue resistance and thus are able to maintain contractile activity
for long periods of time. They typically function as weight-bearing and
postural muscles. In the soleus, a slow muscle in the hindlimb, ~90%
of fibers express the
-form of MHC (13, 16). The
relationship between weight-bearing activity and the expression of
-MHC in soleus muscle is demonstrated by removing the factor of load
in experimental animal models. This is achieved by subjecting the
animal to either the microgravity environment of spaceflight
(14) or through the ground-based model of hindlimb suspension (4) such that the leg extensor muscles bear
little or no weight. When these manipulations are imposed, the
expression of
-MHC is significantly reduced along with a shift
toward increased expression of faster MHC isoforms (4,
14).
At the other extreme, functional overload induces the expression of
-MHC in the plantaris, a fast-twitch ankle extensor muscle that
normally expresses the fast MHC isoforms, IIb and IIx, which account
for >80% of the MHC pool (28). In this model, the
overload stress is induced by the removal of synergistic muscles along with normal weight-bearing activity, which collectively causes compensatory hypertrophy of muscle fibers (1, 26).
Adaptation to the overload stimulus also prompts a shift in the
fiber-type profile toward a slow fiber-type pattern, characterized by a
lower myofibrillar ATPase activity, reduced glycogenolytic enzyme
activities, decreased expression of fast MHC isoforms IIx and IIb, and
increased expression of slow myosin light chain isoforms and slower MHC isoforms
(type I) and IIa (2, 28, 30, 34).
We are interested in determining the molecular mechanisms responsible
for the increase in the slow, -MHC gene expression in overloaded
plantaris. It is believed that the phenotypic adaptations are mediated
in some way by the interaction of transcriptional regulatory proteins
with specific cis elements within the
-MHC gene's
promoter sequence. It may be that fast and slow fibers express a
different pool of transcriptional regulatory proteins, or transcription
factors, that interact with the cis elements that are
present only in the genes of "fast" or slow isoforms. Thus we
tested the hypothesis that the upregulation of
-MHC expression in
response to overload is regulated through specific elements of the
-MHC promoter.
The proximal region of the -MHC promoter has been shown to be
required for muscle-specific expression in cardiac and skeletal cell
culture (7, 29) and transgenic mice models (20,
34). Several highly conserved positive and negative regulatory
elements within the proximal region (
408 bp) promoter have been
identified (7, 20, 29, 34). A putative repressor element,
e1 (
321/
297), appears to play a distinct role in
-MHC
expression in skeletal and cardiac muscle cells (7, 9, 10)
and has been implicated in conferring the decreased expression of
-MHC that occurs in response to soleus unloading (23).
It has been suggested that four positive elements [
e2 or muscle CAT
(MCAT)-binding site (
285/
269); an A/T-rich site (
267/
259); the
CCAC box (
245/
233); and the
e3 element (
214/
190)] are
essential for muscle-specific gene activation (20, 29,
34). In fact, the region containing these positive motifs
appears to be required for the increased expression of the human
-MHC gene after plantaris overload in transgenic mice
(34).
Unlike previous studies (20, 30, 32), the present report
involves a comprehensive mutation analysis of all of the putative regulatory elements in the rat -MHC proximal promoter to resolve their role in the load responsiveness of the
-MHC gene in vivo. Herein, we describe a direct gene transfer approach whereby deleted or
mutated promoters are linked to a firefly luciferase reporter gene and
injected directly in rat plantaris muscles. After injection, the
synergistic gastrocnemius and soleus muscles were removed from the left
leg to induce plantaris overload, and the right leg was left intact to
act as the control muscle. After 7 days, the plantaris muscles were
harvested, and luciferase activity was measured. We have successfully
used this gene transfer technique to examine the relevant promoter
sequences for maintaining high levels of
-MHC activity in normal
slow-twitch soleus muscles and after unloading (11).
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METHODS |
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Animal Procedures
Female Sprague-Dawley rats weighing 110-125 g were anesthetized (10 mg ketamine and/or 20 mg acepromazine/100 g) for all aseptic surgical/injection procedures.DNA injection.
A skin incision was made to expose the muscle of interest. PBS (20 µl) containing a mixture of two supercoiled DNA plasmids [-MHC
test plasmid (molar equivalent to 10 µg of
3,500 bp MHC-Fluc) and a
control skeletal
-actin-Rluc reporter plasmid (molar equivalent to 5 µg of
3,500 bp MHC-Fluc)] was injected in the muscles using a
29-gauge needle attached to a 0.5-ml insulin syringe.
Electroporation. To improve plasmid uptake by the muscle, electropermeabilization was induced on the muscles immediately after plasmid injection. This procedure, termed electroporation, was performed according to the method described by Mir et al. (24). Two gold-plated stainless steel electrodes set 4 mm apart were placed in contact with the muscle at the injection site. Using a Grass electrical stimulator, four pulses (200 V/cm, lasting 50 ms each, 1 Hz) were delivered, and then four additional pulses of opposite polarity were applied.
Unilateral functional overload.
Plantaris overload was performed via removal of the synergistic
muscles, gastrocnemius, and soleus in one leg only, as described in
detail previously (2). The contralateral leg remained
intact, and the contralateral plantaris represented the control. The
overload surgical procedure was carried out immediately after plasmid
injections/electroporation. All experiments were for a duration of 7 days. After 7 days, plantaris muscle tissues were excised after
pentobarbital sodium (100 mg/kg) anesthesia and were quick-frozen and
stored at 80°C. All animals in the study were allowed food and
water ad libitum, and all procedures were approved by the institutional
animal care and use committee.
Reporter Plasmid Constructs and Expression Assays
The plasmids
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Frozen muscle tissues were homogenized in ice-cold passive lysis buffer from Promega, using a glass homogenizer. The homogenate was centrifuged at 10,000 g for 10 min at 4°C. The supernatant was reserved for the luciferase activity assay using Promega's dual luciferase assay kit, which is designed for sensitive detection of both firefly and renilla luciferase activities in a single extract aliquot. Activities were measured as total light output (as measured by a Monolight 2010-C luminometer) per muscle per second and were expressed as relative light units. Background levels, based on luciferase activities of noninjected tissue, were subtracted from the activities of test samples. These experiments were predicated on the assumption that the level of luciferase activity is proportional to the degree of promoter activity.
Justification for the Use of Skeletal -Actin as a
Reference Promoter
Ideally, the reference promoter activity should not change in response
to experimental manipulation. However, as a sarcomeric muscle protein,
we expect -actin to be upregulated in response to the hypertrophic
stimuli of plantaris overload. Indeed, we know that
-actin promoter
is responsive to functional overload in chicken anterior latissimus
dorsi muscles (6). Yet, because the overload procedure by
synergist ablation is the same in all experiments, we expect the degree
of overload to be equivalent in each subject as demonstrated by the
similar level of plantaris enlargement (see RESULTS).
Therefore, although the activity of
-actin promoter increases in
response to overload, the degree of increase should remain relatively
constant from one experiment to another. Indeed, in the present paper,
the increase of
-actin activity was not significantly different
among the experiments, with an average increase of 135% above control.
The consistency of the
-actin promoter activity validates its
function as a reference vector and thus allows comparisons between the
following test promoters: 1) different length promoter
fragments and 2) wild-type vs. mutated sequences.
As an additional control, plasmid uptake can be determined by directly
measuring the amount of plasmid DNA in the tissue using PCR. One set of
experiments was sampled using this method to assess whether plasmid
uptake was different in overloaded and normal control plantaris. No
difference was detected in either the firefly luciferase test plasmid
(-MHC-pGL3) or the reference
-actin renilla luciferase plasmid
between normal control and overloaded groups. Given this fact,
normalizing the firefly luciferase activity to plasmid DNA would
exaggerate the
-MHC response to overload because it would not
reflect the overall increase in global transcriptional and
posttranscriptional activity that likely occurs with hypertrophic stimuli. Therefore, we feel that dividing the
-MHC promoter-driven firefly activity by
-actin-driven renilla activity is a better conceived method of normalization because it factors out this generalized increase in transcriptional activity. Thus any increase in
the
-MHC-Fluc/
-actin-Rluc reporter activity in response to overload is a result of the specific increase in
-MHC isoform promoter activity.
Gel Mobility Shift Assay
Nuclear extraction of skeletal muscle tissue.
Nuclear protein was extracted from skeletal muscle according to the
method described by Blough et al. (3). Briefly, frozen plantaris muscle tissue (400-700 mg) was homogenized in 35 ml of
buffer 1 [10 mM HEPES (pH 7.5), 10 mM MgCl2, 5 mM KCl, 0.1 mM EDTA (pH 8.0), 0.1% Triton X-100, 0.2 mM
phenylmethylsulfonyl fluoride (PMSF), 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, and 1 mM dithiothreitol (DTT)]. Homogenates were
centrifuged for 5 min at 3,000 g at 4°C. The pellets were
resuspended in 500-1,000 µl of buffer 2 [20 mM HEPES
(pH 7.5), 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA (pH 8.0),
25% glycerol, 0.2 mM PMSF, 2.5 µg/ml aprotinin, 2.5 µg/ml
leupeptin, and 0.5 mM DTT]. The suspension was incubated on ice with
intermittent mixing for 30 min and was centrifuged for 5 min at 4,000 g at 4°C. The supernatant was transferred to an Amicon
2-ml Centricon filter unit (YM-10; Millipore, Bedford, MA). An equal
volume of binding buffer [20 mM HEPES (pH 7.9), 40 mM KCl, 2 mM
MgCl2, 10% glycerol, 0.2 mM PMSF, 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, and 0.5 mM DTT] was added to the filter unit and centrifuged for 30 min at 4,500 g at 4°C. Another
500-1,000 µl were added and centrifuged again for 3 min;
centrifugation was repeated until the concentrate achieved the desired
volume (1 ml). The protein concentration of the nuclear extract was
determined using the Bio-Rad protein assay with BSA as a standard.
Nuclear extract samples were stored at 80°C.
Gel mobility shift assay.
Gel mobility shift assays (GMSAs) were used to examine and
quantitate binding of nuclear extract protein to the e3
cis-regulatory element of the rat type I MHC gene promoter
(29). This approach allows one to detect changes in
transacting factor content and/or changes in the pattern of shifted
bands resulting from binding of one or more factors. The
e3
oligonucleotide sequences were purchased from GIBCO, and the sense
strand was of the following sequence:
-MHC
e3:
5'-CATGCCATACCACAACAATGACGC-3' (bases that were mutated are
underlined). After strand annealing, the double-stranded probe was end
labeled with [
-32P]ATP (6,000 Ci/mmol) using T4
polynucleotide kinase (Promega). For each binding reaction, 20-µg
nuclear extract were preincubated for 10 min at room temperature with
225 ng poly(dI-dC) homopolymer, which was used as a nonspecific
competitor, in a binding buffer containing 40 mM KCl, 1 mM DTT, 1 mM
EDTA, 1 mM MgCl2, 7.5% glycerol, 0.05% BSA, and 20 mM
HEPES (pH 7.9) in a total volume of 20 µl. For competition studies,
the preincubation was carried out in the presence of 150× molar excess
of either cold
e3 wild type (specific), a mutated cold
e3, or an
A/T-rich oligonucleotide (nonspecific). At the end of the
preincubation, 100,000 counts/min of labeled
e3 were added and
incubated for 30 min at room temperature. At the end of the reaction, 2 µl loading buffer (20% ficoll, 0.2% bromphenol blue, and 0.2%
xylene cyanol) were added, and the reaction mixtures were loaded on a
6% polyacrylamide gel, which was preelectrophoresed at 20 mA/gel for
2 h. Electrophoresis was carried out in 0.5× Tris-borate-EDTA
buffer at constant current (30 mA) at room temperature for 3 h. After electrophoresis, the gels were dried and visualized by
phosphorimaging (Molecular Dynamics, Sunnyvale, CA). The intensity of
the shifted bands was quantified by scanning densitometry (Molecular Dynamics) using volume integration on the shifted bands (Image Quant;
Molecular Dynamics).
Statistical Analysis
Statistical analysis was performed using the Graphpad Prism 2.0 statistical software package. Values are means ± SE. Differences among groups were determined by ANOVA followed by the Newman-Keuls posttest. Differences between the means of two experimental groups were assessed by an unpaired, two-tailed t-test. P < 0.05 was taken as the level of statistical significance. ![]() |
RESULTS |
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Body/Muscle Weights
Compensatory growth of the plantaris muscle of the left leg was observed after 1 wk of unilateral functional overload. The contralateral right leg remained intact, and the plantaris muscle of this leg served as the normal control muscle in the experiments. Previously, we have demonstrated that the unilateral functional overload model does not alter MHC gene expression in the contralateral control (unpublished observations). The average body weight of female Sprague-Dawley rats after 1 wk of overload was 147.1 ± 0.96 g (n = 100). Muscle wet weight of normal control plantaris muscle was 138 ± 1.6 mg (muscle wt/body wt = 0.94 ± 0.01 mg/g). Overloaded plantaris muscle was 316 ± 4.9 mg (muscle wt/body wt = 2.16 ± 0.03 mg/g), which corresponds to a significant 129% increase in muscle wet weight (Students t-test, P < 0.0001). Thus the overload stimulus provides a robust stimulus of muscle enlargement in these young female rats.Deletion Analysis of the -MHC Promoter in Normal Control
Plantaris Muscle
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Surprisingly, with the exception of the 171 construct, deletion
analyses of the parent promoter had no significant effect on activity
levels in normal control plantaris. This is worth noting because, in
soleus muscle, the activity is very responsive to deletion mutations
(Fig. 1B and Ref. 11). Also in soleus muscle,
-MHC-Fluc reporter activity was highest using the
3500 promoter,
whereas in plantaris the smallest deletion fragment,
171, showed the
highest level of activity. The very different pattern of promoter
activity in response to deletion analysis between the soleus and
plantaris muscles suggests that distinct mechanisms are functioning in
the regulation of this gene in these two different muscles of
contrasting fiber type.
Deletion Analysis in Overloaded Plantaris
Deletion analysis revealed that the
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Mutation Analysis of 408
-MHC Promoter
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A 408-A/T-rich mutant was tested in overloaded and normal control
plantaris muscles (Fig. 4). Evidence
supporting the A/T-rich mutant (or GATA element) as a load-responsive
element stems from a report by Vyas et al. (33) that
demonstrated increased binding of the A/T-rich element by nuclear
extracts from overloaded plantaris compared with normal control
plantaris.
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Compared with the 408 wild-type promoter, the A/T-rich mutant
exhibited a large increase in reporter activity in both normal control
and overloaded muscle, a pattern that suggests that a negative/repressor element may have been altered. However, despite this
upregulation compared with the wild type, the mutant was still highly
responsive to overload, with the reporter activity increasing up to
160% over control activity levels (Fig. 4B).
The last element to be tested that could be responsible for the
diminished activity of the triple mutant was the e3 site. The
408
e3 mutation eliminated the overload response, indicating that the
e3 element may be a critical site by which the
-MHC gene is
transcriptionally activated in plantaris muscle after overload (Fig.
4). Initially, the potential role of the
e3 element in the overload
response was not expected, given that 1) this element is
located within the
215 promoter fragment, and 2) the majority of the regulation appears to be taking place upstream of the
215 deletion fragment (within the
408 fragment). However, as seen
in the deletion analysis (Fig. 2A), although the
215 promoter exhibited an overload response, the response was significantly smaller than that of the
408 construct. In contrast, the
171 construct, which lacks the
e3 element, did not respond to the overload stimulus. Of all the
408 mutations tested in overloaded plantaris, only the triple mutant (i.e..
e2,
e3, and A/T rich) and the
e3 single mutant were significantly different than the response of the
408 wild type.
Gel Mobility Shift Analysis
Further evidence supporting a role for the
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DISCUSSION |
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There is a relationship between the functional characteristics of
a muscle and the types of MHC isoforms that are expressed in the muscle
fibers. In the hindlimb, for example, the chronically weight-bearing
soleus muscle expresses primarily the slow, -MHC isoform
(15), whereas its fast-twitch synergist, the plantaris muscle, expresses a predominance of the fast MHC isoforms, IIx and IIb
(14). Muscle fibers have the unique ability to undergo changes in MHC phenotype as an adaptive response to altered loading states; however, the regulatory mechanisms involved in these phenotype transitions are largely unknown. Our interest lies in further understanding how
-MHC isoform gene expression is regulated in fast
and slow muscles in response to various loading conditions. Deletion
analysis revealed that
-MHC promoter activity responds uniquely in
slow-twitch soleus compared with the fast-twitch plantaris muscle,
suggesting that distinct mechanisms are involved in the regulation of
this gene in these two different muscle types. Previously, we have
noted that, in soleus muscle (11) and cardiac muscle (35), the largest level of
-MHC promoter activity
occurs in the parent promoter (
3500), and promoter activity decreases
with successive deletions. Of further interest, herein we report that, in the normal control plantaris muscle, a muscle that normally does not
abundantly express the
-MHC gene (28), no such promoter length dependence was demonstrated (Fig. 1). The only difference in
activity was seen with the smallest deletion construct (
171), which
unexpectedly had the highest level of activity. In fact, aside from the
A/T-rich mutant, even when specific regulatory elements were mutated,
activity levels in normal control plantaris were not changed. It is
conceivable that a pool of inhibitory transcription factors bind the
-MHC promoter to maintain low levels of expression that are typical
of normal fast-twitch plantaris, and only after the binding sites are
eliminated by severe deletion are the inhibitory factors removed.
Support for this concept stems from the findings of Lamph et al.
(21), which suggest that the cAMP-responsive
element-binding protein (CREB) in an unphosphorylated state can act as
a repressor of transcriptional activity of the c-jun
promoter. The sequence between
215 and
171 contains, among others,
a putative CREB binding site. Perhaps CREB may be acting as a repressor
in the
-MHC promoter in normal plantaris muscle. Consequently, with
the creation of the
171 construct, this "repressor" is removed.
Mechanical overload of the plantaris results in marked increases in
expression of -MHC isoform mRNA and protein (28) and an
increase in
-MHC promoter activity (Fig. 2). However, the mechanism
by which loading stimuli affect
-MHC gene activation is unclear.
Based on the findings by Vyas et al. (33), we tested a
mutant
408-A/T-rich rat promoter in our gene transfer model to
determine if an intact A/T-rich element was required for the overload
response. Despite an upregulation in activity with the mutant
408-A/T-rich promoter, the overload response was similar to that of
the wild type. Therefore, factors binding to the A/T-rich element do
not appear to be relevant in conferring the overload-stimulated activity of the rodent
-MHC promoter. Perhaps our results differ from Vyas et al. (33) because their GMSA experiments used
the human
-MHC A/T-rich sequence, which contains about 10 extra base pairs that flank the 5'-A/T-rich region, whereas we used a rat sequence
that did not contain these flanking base pairs.
In the present study, a triple mutant (e2,
e3, and A/T rich) and
the
e3 single mutant were the only mutants that had a significantly
lower overload response than the
408 wild-type construct.
Interestingly, only the
e3 single mutant completely abolished the
overload response. One curious finding was that the activity of the
triple mutant in overloaded plantaris was higher than the
e3 mutant
alone, even though
e3 was one of the three mutations (see Figs. 3
and 4). Perhaps the reason for this unexpected result is that one of
the other mutations within the triple mutant was the A/T-rich element,
which exhibited a large upregulation in activity when it alone was
mutated. In other words, the A/T-rich mutation may have compensated for
the loss of activity brought about by the
e3 mutation.
To our knowledge, we are the first to identify the e3 site as a
putative overload-responsive element in the rat
-MHC gene promoter.
We base this conclusion on three findings. First, in the normal control
and overloaded plantaris gene transfer studies, the
171 construct,
which lacks the
e3 element, did not respond to the overload stimulus
(Fig. 2A). Second, the overload response was completely
abolished when 3 bp of the
e3 element were mutated (Fig.
4A). Finally, in the GMSA experiment (Fig. 5) a significant increase in the level of specific binding of the
e3 probe was detected with the extracts from overloaded plantaris muscles compared with normal control plantaris extracts. However, this conclusion conflicts with a transgenic mouse plantaris overload study using a
mouse
-MHC promoter-CAT reporter transgene that harbored
simultaneous mutations of
e2, CACC, and
e3 elements
(30). That report found that, despite the mutations,
reporter activity increased in response to 8 wk of bilateral plantaris
overload similar to that of the wild type. These findings may differ
between the two studies because of the different model systems
employed. Not only were different species and different mutated
sequences used, but we used a direct gene transfer technique to
introduce the promoter-reporter construct, whereas the other
investigators used a transgenic mouse model. Moreover, the time period
of the overload stimulus differed greatly between the two studies. Our
1-wk model explored the regulatory processes that are involved during
the adaptive phase of the overload response. In contrast, their 8-wk
study (30) assessed the promoter activity at the
steady-state level of the overload stimulus. It is possible that the
mechanisms regulating promoter activity are different during the steady
state compared with the adaptive phase such that, after such a long
period of overload perturbation, the involvement of other elements may
override the
e3 mutation and induce an increase in promoter activity.
In the GMSA experiment (Fig. 5), mutant competition suggests that the
protein(s) comprising complexes A and B bind at the site including the
3 bp ACC of the MCAT sequence. The MCAT motif has been shown to be
required for muscle-specific expression in genes of sarcomeric
proteins, such as -skeletal actin (19),
-MHC
(12), cardiac tropin T (22), and
-MHC
(9). Therefore, our results indicate that the
overload stimulus triggers an as yet unknown signaling pathway whereby
transcription factor(s) bind the MCAT core of the
e3 element, thus
initiating the transactivation of the
-MHC promoter.
Although we have not yet examined which transcription factors may be
binding the MCAT motif of e3 in our skeletal muscle overload model,
evidence from cardiomyocyte culture studies indicate that the
transcriptional enhancer factor (TEF) 1 (or MCAT binding factor; see
Ref. 8) may be involved. In fact, two members of the TEF
family, TEF1 and TEF-related (TEFR) 1, have been shown to regulate
transactivation of the
-MHC and other muscle genes such as
-MHC
(12) and skeletal
-actin (19) through an
MCAT site. For example, sequential mutation analysis in GMSA assays determined that both phenylephrine and protein kinase C (PKC)-
stimulation of cardiomyocytes activated the
215
-MHC promoter through the MCAT core of the
e3 element (18) and the
protein factor binding the
e3 sequence immunoreacted with a TEF1
antibody (17). Stewart et al. (27), on the
other hand, concluded that, although basal levels of
-MHC activity
require an intact MCAT for TEF1 and TEFR1 activation, the pathway
whereby phenylephrine potentiates
215
-MHC expression does not
involve the TEF family of factors or the MCAT box. The MCAT-TEF1-TEFR1
signaling pathway may also play a role in the regulation of skeletal
-actin, another fetal gene activated in response to
1-adrenergic stimulation. For example, Karns et al.
(19) maintain that PKC-phosphorylated TEF1 binds to an
MCAT site within the skeletal
-actin proximal promoter, whereas
Ueyma et al. (31) contend that it is the mitogen-activated protein kinase phosphorylation of serine-322 in TEFR1, a site not
present in TEF1, that transactivates the skeletal
-actin promoter.
Stewart et al. (27) noted that
1-adrenergic
stimulation of skeletal
-actin is via TEFR1, yet not through an MCAT
site. Given the differences of opinion about the role of TEF1/TEFR1 factors and signaling pathways in
-MHC and skeletal
-actin gene regulation in cardiomyocyte cultures, both TEF1 and TEFR1 factors need
to be tested in our model.
More relevant to our in vivo model are findings by Carson et al.
(5, 6) that indicate a role of TEF1 in the regulation of
the skeletal -actin gene in hypertrophied skeletal muscle. With the
use of a stretch-overload model of the chicken anterior latissimus
dorsi muscle, skeletal
-actin promoter activity increased significantly in response to stretch overload (6). Also, 3 days of stretch increased TEF-1 binding to the skeletal
-actin promoter, as demonstrated by GMSA. These results support the hypothesis that the TEF1 family of transcription factors may be involved in
conferring the overload-stimulated increase in
-MHC gene activity in
skeletal muscle.
Finally, it is important to note that we cannot completely disregard
the fact that other elements upstream of e3/
215 may play some role
in our model given the fact that the
408 construct has a
significantly higher level of overload responsiveness than that of the
215 promoter. In conclusion, the major findings derived from this
study indicate that the proximal region (
408) of the rat
-MHC
promoter is essential for maximal promoter activity in response to
plantaris overload, and an intact MCAT sequence within the
e3
element is required for the overload responsiveness.
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ACKNOWLEDGEMENTS |
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This work was performed during the tenure of a fellowship for J. M. Giger from the American Heart Association, Western States Affiliate, and was supported by National Institute of Arthritis and Musculoskeletal Diseases Grant AR-30346.
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. M. Baldwin, Dept. of Physiology and Biophysics, Univ. of California, Irvine, Irvine, CA 92697 (E-mail: kmbaldwi{at}uci.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.
10.1152/ajpcell.00444.2001
Received 17 September 2001; accepted in final form 31 October 2001.
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