Department of Physiology and Biophysics, University of California, Irvine, California 92697
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ABSTRACT |
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Denervation (DEN) of rat soleus is
associated with a decreased expression of slow type I myosin heavy
chain (MHC) and an increased expression of the faster MHC isoforms. The
molecular mechanisms behind these shifts remain unclear. We first
investigated endogenous transcriptional activity of the type I MHC gene
in normal and denervated soleus muscles via pre-mRNA analysis. Our
results suggest that the type I MHC gene is regulated via
transcriptional processes in the denervated soleus. Deletion and
mutational analysis of the rat type I MHC promoter was then used to
identify cis elements or regions of the promoter involved in this
response. DEN significantly decreased in vivo activity of the 3,500,
2,500,
914,
408,
299, and
215 bp type I MHC promoters,
relative to the
-skeletal actin promoter. In contrast, normalized
171 promoter activity was unchanged. Mutation of the
e3 element
(
214/
190) in the
215 promoter and deletion of this element (
171
promoter) blunted type I downregulation with DEN. In contrast,
e3
mutation in the
408 promoters was not effective in attenuating the
DEN response, suggesting the existence of additional DEN-responsive
sites between
408 and
215. Western blotting and gel mobility
supershift assays demonstrated decreased expression and DNA binding of
transcription enhancer factor 1 (TEF-1) with DEN, suggesting that this
decrease may contribute to type I MHC downregulation in denervated muscle.
slow muscle; transcriptional regulation; pre-mRNA; e3 DNA
regulatory element; denervation
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INTRODUCTION |
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THE IMPORTANCE OF NEURAL ACTIVATION and loading state in the determination and maintenance of myosin heavy chain (MHC) expression in rodent skeletal muscle has been clearly demonstrated in models such as cross-reinnervation, electrical stimulation, spinal cord isolation (SI), and denervation (DEN; for reviews, see Refs. 37 and 43). When neuromuscular activation is absent or decreased as occurs with spinal cord transection (44), SI (20), or DEN (19), there is a rapid loss of muscle mass associated with a specific reduction in the relative expression of slow type I myosin heavy chain (MHC) at the expense of the faster MHC isoforms in slow muscles such as the soleus. Although similarities exist in the phenotypic shift in a muscle deprived of either neural activation and/or loading, the molecular mechanisms leading to these transformations may be different and specific to the imposed perturbation. For example, SI is unique in that there is virtually no residual activation or loading of the hindlimb muscles, but the motoneuron-muscle connections remain intact (35, 38). Thus, although both SI and DEN muscles are inactive, the models differ significantly due to the presence (SI) or absence (DEN) of an intact motoneuron-muscle connection and the associated neurotrophic influences. Consequently, the affected muscle is exposed to different conditions that potentially lead to significant divergence in the gene expression regulatory pathways between the two models. For example, several studies have shown that neurotrophic factors may influence gene expression independent of electrical activity (6, 9, 18, 25, 29, 50). Therefore, even though both DEN and SI decrease type I MHC, the molecular mechanisms mediating the shifts with DEN are likely unique in the absence of motoneuron-muscle connections.
Previous studies have demonstrated significant decreases in type I MHC expression at both the mRNA and protein levels in the rat soleus in response to DEN (19, 21, 31). However, no studies to date have investigated whether these changes are initiated at the transcriptional or posttranscriptional level. Gene expression can be regulated at several cellular levels, i.e., transcriptional, posttranscriptional/pretranslational, translational, and posttranslational levels. One may gain understanding as to the site of regulation by examining the relationship among expression levels of protein, mRNA, and pre-mRNA. A previous study reported that DEN induces a time-dependent decrease in type I MHC expression at both the mRNA and protein level (19), suggesting that this downregulation is largely pretranslational. Furthermore, this pretranslational control could be mediated via transcriptional or posttranscriptional processes. Assessment of the transcriptional activity of endogenous genes is commonly determined with nuclear run-on assays. However, because of the difficulties in obtaining skeletal muscle nuclei, and perhaps the inconvenience of the run-on assay (using large amounts of radioisotopes), very few studies have reported nuclear run-on results for muscle-specific gene transcription (12, 17). Alternatively, analysis of pre-mRNA expression can be used to assess a gene's transcriptional activity (5, 33, 34). To our knowledge, this approach has not been applied to study MHC gene transcription. In this study, we have utilized this approach to investigate transcriptional regulation of the type I MHC gene in denervated rat soleus. We found that type I MHC pre-mRNA expression decreased in the denervated soleus, suggesting transcriptional regulation of the gene in this model. Therefore, we used in vivo gene transfer and promoter-reporter system analyses to characterize promoter function and delineate cis-regulatory elements involved in this response.
Slow type I MHC expression has been studied extensively in both cardiac
and skeletal muscle, and the gene has been isolated from several
mammalian species, including human (27, 39), rat
(28), mouse (13, 48), and rabbit
(8). Transgenic mice have been utilized to demonstrate
that type I MHC promoter activity in muscles is fiber-type specific.
Type I MHC reporter activity was detected primarily in cardiac
ventricles and slow skeletal muscles, whereas activity was not detected
in pure fast muscles (tibialis anterior) or nonmuscle tissue (liver)
(36). Furthermore, in vivo rat studies using direct muscle
gene injection demonstrated 25-fold greater activity of the type I MHC
promoter in the slow soleus compared with the fast tibialis anterior
(TA) (11). Transient transfection assays in cardiac and
muscle cell culture (4, 7, 22, 23, 40, 45) have identified
several highly conserved positive and negative cis regulatory elements within the most proximal region of the type I MHC promoter [within 350 base pairs (bp) of the transcription start site]. For example, at
least three positive elements that are necessary for muscle-specific expression have been identified: e2 (
285/
269), CACC Box
(
245/
233), and
e3 (
210/
188) (45). A negative
element,
e1, (
330/
300) has also been identified
(4). Furthermore, the upstream distal region
(
3,500/
2,500) was found to be required for maximal promoter activity in both cardiac and slow skeletal muscles (11,
53).
Studies have implicated the e1 repressor region in type I MHC
downregulation in response to skeletal muscle unloading (11, 30). All three positive elements have been implicated in the adaptation to skeletal muscle overloading (46, 49), and
the
e3 element has been implicated in the overload-induced type I MHC upregulation in the rat plantaris (10). Recently, we
reported that the
e3 element is involved in the type I MHC
downregulation associated with SI (20). It remains to be
determined whether type I MHC promoter activity is also downregulated
in response to DEN and the proximal regulatory elements potentially
involved in this response. In particular, the role of
e3 is of
interest on the basis of its role in type I MHC downregulation in a
different model of muscle inactivity, SI (20).
e3 element-mediated regulation of type I MHC gene expression has
been shown to involve transcription enhancer factor 1 (TEF-1) binding
to this element (22, 23, 41, 47). In the SI model, the
involvement of the
e3 element in type I MHC downregulation was
associated with decreased TEF-1 expression and DNA binding. The present
study also supports the finding that type I MHC gene downregulation in
inactive muscles involves changes in the expression and binding of
TEF-1 to the
e3 element. In contrast, stretch-overload-induced muscle hypertrophy was associated with a significant increase in TEF-1
binding to the
-actin promoter (2). Whereas increased expression and DNA binding of TEF-1 may be associated with muscle hypertrophy, a reduction in TEF-1 may contribute to muscle atrophy. One
potential model of muscle gene regulation is that TEF-1 could upregulate muscle contractile genes in response to muscle overload (2) while downregulating these genes in response to
chronic inactivity.
Consequently, through deletion and mutational analysis of the type I MHC promoter, this is the first study to establish the functional significance of regulatory elements in the type I MHC promoter in a denervated muscle. Understanding the molecular mechanisms underlying MHC phenotype shifts in response to DEN has clinical importance not only for peripheral nerve injury but also aging, which is also associated with selective muscle fiber denervation. This research can ultimately help identify ways to maintain muscle phenotype in a denervated muscle that will eventually be reinnervated.
The purpose of the present study, therefore, was to test the hypothesis
that downregulation of type I MHC expression in the denervated rat
soleus is regulated at the transcriptional level, using pre-mRNA
expression as a marker of gene transcriptional activity. Our goal was
to characterize type I MHC gene promoter activity in the denervated
soleus muscle and to identify cis-acting regulatory sequences necessary
for its downregulation in response to DEN. We report that 1 wk of DEN
was associated with a significant decrease in type I MHC endogenous
gene transcription. Promoter analyses demonstrated that important cis
DNA regulatory elements mediating this response are contained within
215 bp upstream of the transcription start site. Within this fragment,
the e3 element is likely to be involved in the type I
downregulation, because both deletion and mutation of this element
reduced the responsiveness of the type I MHC promoter to DEN.
Furthermore,
e3 regulation likely involves decreased TEF-1
expression and binding to this element.
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MATERIALS AND METHODS |
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Experimental design and animal procedures.
These experiments were conducted over a period of ~1 yr and consisted
of repeating the following experimental design. Ten adult female
Sprague-Dawley rats (96 ± 1.0 g) were used for each tested
promoter construct. Rats were anesthetized and underwent surgery for
intramuscular plasmid injection into both left and right soleus muscles
as described previously (11). A skin incision was made to
expose the soleus, and 20 µl of phosphate-buffered saline (PBS)
containing plasmid DNA mixtures were injected into the muscles using a
29-gauge needle attached to a 0.5-ml insulin syringe. Plasmid mixtures
consisted of the rat type I MHC test promoter (molar equivalent to 10 µg of 3.5 kb type I MHC-pGL3 promoter construct) and of a fixed
amount (3.29 µg) of the human skeletal
-actin reference promoter
construct (HSA2000-pRL). Approximately 15 min after plasmid injection
and while anesthetized, the rats underwent unilateral hindlimb muscle
DEN by removal of ~2 mm of the sciatic nerve in the midthigh region.
The contralateral leg served as a control based on our previous results
showing that soleus MHC composition was similar between a normal
control and the contralateral muscle when studied 4 wk after DEN
(13).
Plasmid constructs.
The test promoter consisted of the rat type I MHC gene promoter linked
to the firefly luciferase (Fluc) reporter gene in the pGL3 vector
(Promega). A series of deletions of the type I MHC promoter were tested
in this study as follows: 3,500,
2,500,
914,
408,
299,
215,
and
171 bp promoter fragments all extending to position +34 at the
3'-end relative to the transcription start site (TSS). These promoter
constructs were the same as those used previously by this laboratory
(10, 20, 52). The
408 and
215 type I MHC promoter
constructs with
e3 mutation, the
408
e2/
e3 mutant, and
408
e2/
e3/GATA mutant were also tested in the normal control (NC) and
DEN soleus. Mutations of the target promoter sequence consisted
of changing ACC to CGG in the
e3 element (22,
45), and GTG to TGT in the
e2 element (45), GATAT to
ACTCG in the GATA element, and CCCACCC to
CCCAAAG in the C-rich region and were designed to disrupt
specific transcription factor binding sites (see Ref. 52
for details on mutagenesis).
Rationale for the use of skeletal -actin as a reference
promoter.
To correct for variation in gene transfer efficiency in the direct
injection technique, a reference promoter linked to a different reporter gene is coinjected with the test plasmid. Typically, a plasmid
containing a viral promoter, such as the CMV (cyto megalo virus) or
SV40 (simian virus) promoter, is selected as the reference vector
because the viral promoter is ubiquitously and constitutively expressed
in transfected cells. The activity of the reference promoter is used as
an independent internal control for the variability of gene transfer
efficiency. However, in our hands, these viral promoters subcloned into
a renilla luciferase expression vector (CMV-Rluc, SV40-Rluc; Promega)
proved unsatisfactory as control vectors. As reported previously by our
group (11), CMV-Rluc activity was persistently variable
and often significantly lower than the test promoter-Fluc (
3,500 type
I MHC-Fluc), thus a poor reflection of transfection efficiency.
Therefore, we chose to use a promoter fragment of the human skeletal
-actin (32) extending from
2,000 to +250 bp relative
to TSS, which was linked to the RLuc reporter gene in the pRL vector
(Promega). Both MHC and skeletal
-actin are integral
sarcomeric muscle proteins but, unlike the MHC, skeletal
-actin is
the only actin isoform expressed in adult skeletal muscle and,
therefore, is not fiber type specific. Any change in
-actin
expression reflects global changes in general transcription activity of
sarcomeric proteins due to either atrophic or hypertrophic stimuli.
Unlike a viral promoter, the
-actin promoter will not be expressed
in surrounding nonmuscle tissue, which compromises its function in the
normalization of plasmid uptake in muscle cells. Previously, we have
shown (11) that the level of Fluc activity (type I
MHC-Fluc) correlated well to the level of Rluc (
-actin-Rluc)
activity in control soleus muscles, demonstrating the effectiveness of
-actin reference plasmid as a control for transfection efficiency.
On the basis of reporter activity generated in this study, a strong
relationship between Fluc and Rluc activity is observed in control
muscles (Fig. 1A).
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Reporter gene assays.
For the luciferase assay, each soleus was homogenized in 1 ml of
ice-cold passive lysis buffer (Promega) supplemented with protease
inhibitors (0.2 mM AEBSF, 5 µg/ml aprotinin, and 5 µg/ml leupeptin), and 200 µl were transferred into a tube containing 600 µl of TRI reagent LS (Molecular Research Center), mixed immediately, and stored at 80°C for subsequent RNA extraction. The remaining homogenate was centrifuged at 10,000 g for 10 min at 4°C.
The supernatant was reserved for the luciferase assay using the Promega dual luciferase assay kit, which is designed for sensitive detection of
both firefly and renilla luciferase activities within a single extract
aliquot. Reporter activity was assayed using 5 µl of supernatant, and
light output was integrated over 10 s using a Monolight 2010-C luminometer (Analytical Luminescence Laboratories). Background activity
levels, based on measurements in noninjected tissue for both
luciferases, were determined and subtracted from the activities measured in the tissues injected with the test plasmid.
Type I MHC mRNA and pre-mRNA analyses.
For the first two experiments, total RNA was extracted from 200 µl of
the luciferase homogenate using TRI reagent LS according to the
manufacturer's protocol. After extraction, the RNA pellet was
suspended in nuclease-free water and immediately treated with DNase
according to supplier's (Promega) recommendation to remove any trace
of genomic DNA contamination. At the end of DNase treatment, the RNA
samples were re-extracted with TRI-reagent LS, and the RNA pellet was
suspended in nuclease-free water. RNA concentration was determined by
ultraviolet absorption at 260 nm. RNA samples were stored at 80°C
for future analysis with reverse transcription-polymerase chain
reaction (RT-PCR).
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Gel mobility shift assays.
Skeletal muscle nuclei were isolated and extracted according to the
method described previously by Blough et al. (1). Nuclear extract was prepared from NC and DEN soleus muscles (n = 6/group, each "n" consisted of a pool of four muscles
for control and six muscles for DEN, ~200 mg/pool) and stored at
80°C. Double-stranded oligonucleotides consisting of 24 bp from the
type I MHC gene promoter spanned the
e3 regulatory elements.
(10, 20, 30). After strand annealing, double-stranded
probes were end-labeled with 32P using T4 polynucleotide
kinase (Promega).
Western blotting. Approximately 10 µg of soleus nuclear proteins were subjected to SDS-PAGE according to standard protocol (26) and electrophoretically transferred to a PVDF membrane (Immobilon-P) using 10% methanol, 25 mM Tris, and 193 mM glycine, pH 8.3. The membrane was reacted overnight with a TEF-1 monoclonal antibody (BD Bioscience) at 1:500, and after washing and reaction with the secondary antibody, the signal was detected using enhanced chemiluminescence (ECL Plus Kit, Amersham) according to the manufacturer's protocols. Signal intensity was determined by laser scanning densitometry (Molecular Dynamics/ImageQuant). Antibody reaction resulted in a specific ~53-kDa band matching the band obtained from a positive control A431 cell lysate (provided by BD Bioscience) and a band from HeLa nuclear extract (Santa Cruz). A duplicate membrane blot of the same samples was reacted with preimmune mouse serum and a secondary antibody under similar conditions as the TEF-1 blot, and signal was used as negative control to insure the specificity of TEF-1 antibody reaction.
Statistical analysis. Statistical analysis was performed with GraphPad Prism 3.0 statistical software. Values are means ± SE. Differences between mean type I MHC promoter activity in NC and DEN soleus was determined by a paired, two-tailed t-test. P < 0.05 was accepted as the level of statistical significance. Differences among mean type I MHC promoter activity of the deletion constructs in either NC or DEN soleus were determined by a one-way ANOVA with Newman-Keuls post hoc comparisons.
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RESULTS |
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Body and muscle weights. The average initial body weight at the time of unilateral denervation and gene transfer was 96 ± 0.7 g and increased to 133 ± 0.8 g after 7 days. Mean soleus weight was 58 ± 0.7 mg and 34 ± 0.8 mg for the NC and DEN soleus, respectively. In all conducted experiments, whether to measure promoter activity or to obtain muscle nuclei, 7 days of DEN was associated with similar muscle atrophy. The average muscle loss in the denervated soleus ranged from 35 to 46% (overall average was 42%, P < 0.01).
Endogenous type I MHC gene mRNA and pre-mRNA expression.
Seven days of DEN was associated with a 36% reduction in pre-mRNA
(P < 0.05) and a 56% decrease in mRNA expression
(P < 0.05) (Fig. 2).
Although the mature mRNA changes appear more pronounced than the
pre-mRNA, these changes are not significantly different (P = 0.2). These results demonstrate that
transcriptional processes primarily regulate the reduction in type I
MHC mRNA expression in denervated muscles.
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Deletion analysis of the type I MHC promoter.
In the normal control soleus, the longest promoter fragment (3,500
type I MHC) demonstrated the greatest activity, confirming the
existence of positive regulatory elements in the distal upstream region
of the promoter (Fig. 3). In addition to
the
3,500 type I MHC promoter, six shorter promoter fragments were
tested for their responsiveness to DEN. All of the deletion fragments
tested (
2,500,
914,
408,
299,
215, and
171) were active in
NC soleus, but to a lesser degree than the
3,500 promoter fragment.
In response to DEN,
3,500 type I MHC promoter activity was reduced by
77% relative to control. The responsiveness to DEN was similar in all
deletion fragments down to
215 (Fig. 3), with the decrease ranging
from 59 to 76% relative to activity in normal control muscle (Fig. 3,
inset).
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Mutational analysis of the type I promoter.
Mutation of the e3 element in the
215 type I promoter
significantly decreased activity in the NC soleus, confirming the role
of the
e3 element as a positive regulatory element in normal expression of the type I MHC gene in slow skeletal muscle (Fig. 4). Mutation of this element blunted the
promoter downregulation in response to DEN (Fig. 4). Thus decreased
responsiveness to DEN when the
e3 element is mutated suggests that
this element may confer responsiveness of the type I MHC promoter to
DEN.
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Activity of the type IIb MHC promoter.
Denervation of slow muscle is associated with an increase in fast MHC
isoform expression (19, 21, 31). A type IIb MHC-pGL3 promoter-reporter construct was injected into NC and DEN soleus to
examine the promoter response of a gene that is upregulated in response
to DEN. Our results show that the activity of a 1,400/+13 type IIb
MHC promoter construct was significantly increased in the soleus of DEN
rats compared with NC (Fig. 6). This
upregulation of the type IIb promoter demonstrates that DEN does not
cause a global decrease in the transfected promoter activities.
Furthermore, it shows that the IIb promoter response to DEN is in the
same direction as the endogenous IIb MHC gene.
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Gel mobility shift assays.
The findings that mutation of the e3 element in the
215 promoter
fragment and that deletion of this element (
171 fragment) decreased
type I responsiveness to DEN suggest that this element is involved in
the response to DEN. Thus gel mobility shift assays were used to
examine interactions of the
e3 element with nuclear proteins
extracted from NC and DEN soleus (Fig.
7A). Although DNA-protein
interactions were resolved into several complexes on the gel, only one
complex, based on competition with different DNA probes, was specific
to the
e3 probe (labeled Sp. C in Fig. 7A). This specific
band (Sp. C) was competed out by 100-fold molar excess of unlabeled
e3 probe. In contrast, an unlabeled
e3 mutant probe and an
unrelated sequence (GATA) were not able to effectively compete out
binding to the radiolabeled
e3 probe when incubated at 100-fold
molar excess. Densitometric analysis determined that there was a
significant 3.6-fold increase in binding with the DEN nuclear extracts
compared with NC. Further, the increase was not due to differences in
nuclear protein concentration between the groups, because binding to
probes containing other cis DNA elements (GATA or NFAT) was not
different between the two groups (data not shown).
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DISCUSSION |
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Motor innervation and muscle activation are critical factors
influencing muscle gene expression. Previous denervation studies at
both the mRNA and protein levels have demonstrated that the muscle
shifts its MHC phenotype when the motoneuron-muscle connection is
severed (19, 21, 31). Similar to other models of muscle inactivity, the MHC remodeling that occurs with DEN is characterized by
decreased expression of slow type I MHC and increased expression of the
faster MHC isoforms (19, 21, 31). The findings reported herein provide the first evidence that the previously reported decrease
in type I MHC protein and mRNA in denervated slow muscles is mediated
largely via decreased transcriptional activity of the endogenous type I
MHC gene based on pre-mRNA and mRNA analyses of type I MHC gene
expression. Furthermore, the findings reported herein demonstrate that
the activity of an exogenous type I MHC promoter fragment, when studied
via reporter gene assays 7 days after direct gene transfer in vivo, is
also downregulated in denervated soleus similar to the endogenous gene.
This downregulation in reporter gene activity was observed for both the
type I MHC and -skeletal actin promoters; however, reporter activity
increased severalfold when driven by a fast type IIb MHC promoter
fragment (Fig. 6). This latter observation validates the specificity of the system and negates the possibility of a generalized decrease in
reporter activity in the denervated muscle. A systematic functional analysis of the type I MHC promoter via serial deletions of the 5'
flanking region demonstrates that promoter responsiveness to DEN is
contained within 215 bp upstream of the transcriptional start site
(Fig. 3). Mutation analysis of important cis-regulatory elements
partially implicates the
e3 DNA regulatory element (45) in the response to DEN (Figs. 4 and 5).
Likely, both the neurotrophic effects and activation-associated factors
present with an intact motoneuron-muscle connection are essential for
maintenance of slow type I MHC expression in a slow rat hindlimb
muscle. We recently reported that SI is associated with type I MHC
promoter downregulation in the rat soleus (20). SI is
unique in providing a baseline for complete inactivity while maintaining potential activity-independent neural influences. In
contrast, the motoneuron-muscle connection is severed with DEN, thus
eliminating both activity-dependent and -independent neural influences
on the muscle. Despite possible differences between these two models of
inactivity, the e3 element was involved in type I MHC downregulation
in both models. Although the
e3 element is likely a critical
activity/load response element, there are some subtle differences in
type I MHC transcriptional regulation when the motoneuron-muscle
connection is maintained. With SI, the
e3 element was necessary and
sufficient for transcriptional downregulation of the type I MHC gene
promoter (20). Specifically, mutation of this element in
two different promoter fragment lengths (
215 and
408) prevented
type I MHC downregulation. In contrast, although mutation of the
e3
element in the
215 fragment blunted type I MHC downregulation in
response to DEN, mutation of this element in the
408 fragment did not
prevent type I MHC downregulation. The differing
408 promoter
response between DEN and SI with the
e3 mutation suggests that in SI
muscles the
e3 element was the primary mechanism whereby type I MHC
promoter activity was blunted. In contrast, severing the
motoneuron-muscle connection with DEN increased potential pathways for
the muscle to downregulate type I MHC gene expression. Despite the
mutation of four important DNA regulatory elements, the
408 type I
MHC promoter fragment was still responsive to DEN (Fig. 5). This
responsiveness of the triple mutant (
e2,
e3, and GATA) was
similar to the
408 WT but was slightly attenuated (P = 0.06 ANOVA) when the C-rich mutation was added to the previous three
elements (Fig. 5, inset).
These results point to the complex regulation of the type I MHC
promoter in denervated muscle. The e3 element is important for
conferring responsiveness to DEN in the
215 promoter; however, this
same element's regulatory capacity is overridden by other influences
in the longer 408 promoter fragment. It is puzzling that simultaneous
mutations of the
e3 with other known upstream regulatory elements in
the
408 fragment did not abolish the responsiveness to DEN observed
with
215
e3 mutant. This suggests that other untested elements
located between 408 and 215 bp upstream of TSS may play a role in type
I MHC downregulation in the absence of neural activation and trophic
factors. These elements could involve the
e1 repressor element
(4) or other unknown elements.
The present study demonstrates that decreased type I MHC expression
with skeletal muscle inactivity, independent of motoneuron-muscle connectivity, is transcriptionally regulated and involves the e3
regulatory element. Studies in cardiac myocytes have characterized the
enhancer core of the
e3 element, also referred to as the myocyte-specific CAT (M-CAT) cis element (22, 24). We have demonstrated that
e3 is a positive element in the normal
weight-bearing soleus muscle, as evidenced by the significant decrease
in
215 and
408 type I promoter activity when the
e3 element is
mutated (Figs. 4 and 5). Members of the TEF-1 multigene family bind to this
e3 (M-CAT) element and activate transcription of muscle genes
in cardiac myocytes such as type I MHC (22, 23, 41, 47),
MHC (14), and skeletal
-actin (24). For
example, TEF-1 overexpression and activation of a
215 type I MHC
promoter in cardiac myocytes required an intact M-CAT element
(41).
Studies in both cardiac and skeletal muscle suggest TEF-1 involvement
in muscle contractile gene regulation. Recently, a TEF-1-related protein, RTEF-1, was implicated in cardiac myocyte hypertrophy (47). Skeletal muscle hypertrophy is also associated with
significantly increased TEF-1 binding to -actin promoter
(2). Although increased expression and DNA binding of
TEF-1 may be associated with muscle hypertrophy, a reduction in TEF-1
expression and binding may contribute to muscle atrophy. Our supershift
assays demonstrate that TEF-1 is part of the nuclear protein complex
bound to the
e3 element (Fig. 7). A TEF-1 antibody supershifted
protein complexes binding to the
e3 oligonucleotide in nuclear
extracts from both NC and DEN soleus. Although total nuclear protein
binding to the
e3 oligonucleotide increased almost fourfold with
DEN, protein supershifted by the TEF-1 antibody was only 50% of that
observed in NC soleus. These results, taken together with our previous
finding in the soleus of SI rats (20), demonstrate that
two different models of skeletal muscle inactivity decrease TEF-1
binding to the type I MHC promoter. This decreased binding to the
e3
probe in the GMSA resulted from decreased expression of TEF-1 protein
as demonstrated by Western blot analysis (Fig. 7B).
Consequently, one mechanism by which muscle inactivity could
downregulate type I MHC is via decreased TEF-1 expression, thus
decreasing activation of the
e3 element of the type I MHC promoter.
This possibility is supported by the significant reduction in promoter
responsiveness when
e3 element was mutated or deleted in the shorter
promoter fragment. Potentially, TEF-1 could upregulate muscle
contractile genes in response to muscle overload (2) while
downregulating these genes in response to chronic inactivity.
Because DEN significantly increased nuclear protein binding to the
e3 oligonucleotide while decreasing the amount of TEF-1 binding, the
residual binding was likely composed of other yet to be determined
transcription factors that increase in response to DEN. Another
possibility is that DEN causes changes in the availability of
promoter-specific cofactors. For example, TEF-1 and a transcription
factor designated as Max associate to exert a positive cooperative
effect for gene regulation (14). Consequently, residual
binding to the
e3 element may include factors such as Max; however,
without TEF-1, gene activation is blunted.
In NC soleus, IIb MHC mRNA and protein expression are below detection limits. After 7 days of DEN, we detected de novo expression of type IIb MHC mRNA up to 6 ± 2% of the total MHC mRNA pool as determined by RT-PCR (unreported data). Testing the IIb MHC promoter in the denervated soleus showed that its activity increases several fold, mimicking the regulation of the endogenous gene (Fig. 6). This observation further validates gene injection as an approach to study regulation of promoter activity. However, although short-term inactivity and/or unloading is sufficient to activate IIb MHC promoter activity and significantly increase endogenous mRNA, such inactivity is unable to induce expression of IIb protein. For example, in hindlimb-unloaded rats, IIb mRNA was detected with no changes at the protein level (16). Similarly, 60 days of SI, 30 days of DEN (15, 19), or 14 days of tetrodotoxin paralysis (31) did not induce de novo IIb protein expression. Taken together, these observations suggest that in a slow muscle there is a marked uncoupling between transcriptional and translational events in the upregulation of IIb MHC. However, more prolonged inactivity (e.g., 90 days of SI) or decreased activity coupled with other factors such as thyroid hormone status has induced IIb protein expression in the soleus. For example, hindlimb unloading coupled with triiodothyronine treatment increased both IIb mRNA and protein in the rat soleus (3, 16).
In summary, using a novel approach, we demonstrated that type I MHC
downregulation in the denervated soleus involves transcriptional processes. Using the direct gene transfer approach, we have
characterized important regulatory elements in the type I MHC gene
promoter that mediate downregulation in response to DEN. Deletion and
mutation analyses suggest involvement of the e3 DNA regulatory
element in the type I MHC downregulation; however, other potential
mechanisms exist with the addition of upstream regulatory elements.
Supershift assays demonstrated that one mechanism contributing to type
I MHC downregulation with DEN is decreased TEF-1 DNA binding,
potentially leading to a decreased activation of the
e3 positive
regulatory element on the type I MHC promoter (Fig. 7). Mutation of the
e3 element mutation in the
215 promoter fragment rendered it
unresponsive to DEN (Fig. 4). Furthermore, the
171 promoter, which
lacks the
e3 element, was also unresponsive to DEN (Fig. 4).
However, strong involvement of the
e3 element was dependent on
promoter length as the addition of upstream regulatory elements (
408
promoter) altered the role of
e3. Consequently, future studies to
further delineate the role of
e3 in conferring this responsiveness
could ligate a single or double 24bp-
e3 element upstream of the
171-bp promoter or the 215
e3 mutant promoter to test whether
responsiveness to DEN is restored. Furthermore, utilization of
different models of reduced muscle activity will be useful in
establishing the distinct roles of neurotrophic factors, neural
activation, and loading on muscle contractile gene expression.
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ACKNOWLEDGEMENTS |
---|
We thank Sam A. McCue and Ming Zeng for technical assistance.
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FOOTNOTES |
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This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases AR-30346.
Present address of K.A. Huey: Arizona State University, Dept. of Kinesiology, Physical Education East 107-B, Tempe, AZ 85287.
Address for reprint requests and other correspondence: K. M. Baldwin, Univ. of California, Irvine, Physiology and Biophysics, 346-D Med Sci I, Irvine, CA 92697-4560 (E-mail: kmbaldwin{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.
First published November 20, 2002;10.1152/ajpcell.00389.2002
Received 27 August 2002; accepted in final form 14 November 2002.
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