1 Department of Physiology and Biophysics, University of California, Irvine, Irvine 92697; and 2 Department of Physiological Science, and 3 Brain Research Institute, University of California, Los Angeles, Los Angeles, California 90095-1761
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ABSTRACT |
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Chronic muscle inactivity with spinal cord
isolation (SI) decreases expression of slow type I myosin heavy chain
(MHC) while increasing expression of the faster MHC isoforms, primarily
IIx. The purpose of this study was to determine whether type I MHC downregulation in the soleus muscle of SI rats is regulated
transcriptionally and to identify cis-acting elements or
regions of the rat type I MHC gene promoter involved in this response.
One week of SI significantly decreased in vivo activity of the 3500-,
408-,
299-,
215-, and
171-bp type I MHC promoters. The activity
of all tested deletions of the type I MHC promoter, relative to the human skeletal
-actin promoter, were significantly reduced in the SI
soleus, except activity of the
171-bp promoter, which increased.
Mutation of the
e3 element (
214/
190 bp) in the
215- and
408-bp promoters and deletion of this element (
171-bp promoter) attenuated type I downregulation with SI. Gel mobility shift assays demonstrated a decrease in transcription enhancer factor-1 binding to
the
e3 element with SI, despite an increase in total binding to this
region. These results demonstrate that type I MHC downregulation with
SI is transcriptionally regulated and suggest that interactions between
transcription enhancer factor-1 and the
e3 element are likely
involved in this response.
e3 DNA regulatory element; transcription enhancer factor-1; spinal cord isolation; chronic muscle inactivity
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INTRODUCTION |
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CHRONIC DECREASES IN NEUROMUSCULAR activity strongly influence myosin heavy chain (MHC) expression in rodent skeletal muscle (for reviews see Refs. 27 and 36). Decreased neuromuscular activation and unloading, as occurs with spinal cord transection (37), and reduced loading or weight bearing, as induced by hindlimb suspension (simplified here as unloading) (14, 23), decrease the relative expression of slow type I MHC while increasing the relative expression of the faster MHC isoforms in the normally slow-twitch rat soleus muscle. Although these models decrease overall muscle activity and load, some residual neuromuscular activation remains, which could influence muscle gene expression. Specifically, MHC expression could be altered by any remaining activation-associated factors, despite the dramatic reduction in load and activity. Although denervation results in complete inactivity, it also removes non-activity-dependent neurotrophic factors, which may be involved in regulation of MHC plasticity (32, 33). Consequently, it has been difficult to develop a suitable model to study the effects of complete inactivity on MHC gene regulation while maintaining an intact motoneuron-muscle connection, i.e., without direct interruption of potential activity-independent neurotrophic factors.
Spinal cord isolation (SI) provides a unique model in which the hindlimb muscles are silenced for prolonged periods by functionally isolating the lumbar region of the spinal cord (6, 25, 28). This model eliminates supraspinal, infraspinal, and peripheral afferent input to the isolated cord segment while maintaining motoneuron-muscle connectivity. Thus SI provides a model that removes neuromuscular activation and loading, while the motoneuron continues to exert activity-independent neurotrophic effects on the inactive muscles (6, 28).
We recently reported that SI results in a time-dependent decrease in type I MHC in the rat soleus muscle, which is primarily offset by an increase in fast type IIx MHC (16). These MHC shifts were characterized by an early loss in type I MHC, as evidenced by the rapid muscle atrophy and concomitant decreases in muscle protein and MHC content over the first 8 days of SI. The loss of nearly half of the type I MHC protein in the soleus muscle after only 8 days of inactivity is the result of negative balance between protein synthesis and degradation. This imbalance may be due to alterations in transcriptional/translational and/or posttranslational processes. In a previous study (16), analysis of the time course of changes in type I MHC protein and mRNA expression in the soleus muscle of SI rats revealed that the changes at the mRNA level preceded changes at the protein level. Furthermore, over the complete SI time course of 4-90 days, there was a tight coupling between changes at the mRNA and protein levels, suggesting that pretranslational processes dominated MHC isoform shifts in the chronically inactive soleus muscle. However, the question remains as to whether these pretranslational processes are transcriptional; therefore, the present study further characterizes the molecular mechanisms mediating downregulation of type I MHC expression in inactive slow-twitch muscle. It is hypothesized that the molecular remodeling of the soleus muscle associated with SI is regulated in part at the transcriptional level via interaction of specific transcription factors with cis-acting regulatory elements located on muscle gene promoters. Furthermore, inactivity is likely associated with changes in the nuclear milieu that render it unfavorable for transcription of slow-twitch muscle genes.
Within the MHC gene family, slow type I MHC expression has been studied
most extensively in cardiac and skeletal muscle, and the gene has been
isolated from several mammalian species, including human (21,
30), rat (22), mouse (11, 42), and
rabbit (8). In vivo studies involving transgenic mice
(26) have provided evidence that type I MHC promoter
activity is muscle specific. Reporter activity was detected primarily
in cardiac and slow-twitch skeletal muscles. In contrast, no reporter
activity was detected in pure fast-twitch muscles (masseter) or
nonmuscle tissue of transgenic animals harboring the type I MHC
promoter fused to the chloramphenicol acetyltransferase reporter gene. Furthermore, in vivo studies using direct muscle gene injection demonstrated that the type I MHC promoter is 25-fold more active in the
slow-twitch soleus than in the fast-twitch tibialis anterior muscle
(9), which corresponds to endogenous gene expression. On
the basis of transient transfection assays in cardiac and muscle cell
culture (5, 7, 17, 18, 31, 38), several highly conserved
positive and negative cis-acting regulatory elements have
been identified within the most proximal region of the type I MHC
promoter [within 350 bp of the transcription start site (TSS)]. For
example, at least three positive elements that are necessary for
muscle-specific expression have been identified: e2 (
285/
269
bp), CACC box (
245/
233 bp), and
e3 (
210/
188 bp)
(38). A negative element,
e1 (
330/
300 bp), has also
been identified (5). In addition, the upstream distal
region (
3500/
2500 bp) was required for optimal promoter activity in
cardiac and slow-twitch skeletal muscles (9, 45).
Although several positive and negative regulatory elements have been
identified and found necessary for type I MHC promoter activity under
normal conditions, their role in modulating type I MHC gene expression
under altered physiological conditions is unclear. Studies have
implicated the e1 repressor region in type I MHC downregulation in
response to skeletal muscle unloading (9, 23), while all
three positive elements have been implicated in the adaptation to
skeletal muscle overloading (39, 43).
Although transcriptional regulation of the type I MHC gene has been demonstrated under altered loading states, regulation of this gene has never been studied under conditions of complete neuromuscular inactivity. It remains to be elucidated whether decreases in type I MHC expression with chronic inactivity in slow-twitch muscles are mediated transcriptionally and whether transcriptional downregulation would result from increased activity of a repressor element or decreased activity of an enhancer element. Consequently, SI provides an excellent model to begin to establish the functional significance of these positive and negative elements of the type I MHC promoter during periods of complete muscle inactivity. These findings will provide an excellent baseline with which to compare type I MHC regulation in other models of decreased muscle activity and/or neurotrophic influence such as hindlimb unloading and denervation.
The purpose of the present study, therefore, was to test the hypothesis
that downregulation of type I MHC expression in the soleus muscle of SI
rats is regulated at the transcriptional level using the in vivo direct
gene transfer approach. A second goal was to identify
cis-acting regulatory sequences on the rat type I MHC gene
promoter necessary for its downregulation in response to chronic
inactivity. We found that 1 wk of SI results in a significant decrease
in type I MHC promoter activity in the inactive rat soleus muscle and
that important cis-acting DNA regulatory elements mediating this response are contained within 215 bp of the proximal promoter. Within this fragment, the e3 element is likely involved in the type
I MHC downregulation, inasmuch as deletion and mutation of this element
attenuated the decrease in promoter activity associated with SI.
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METHODS |
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Experimental design and animal procedures.
The experiments were conducted over a period of ~1 yr and consisted
of repeating the following experimental design. For each tested
promoter construct, adult female Sprague-Dawley rats (145 ± 10 g) were randomly assigned to normal control (NC) and SI groups (n = 8 animals/group). Rats were anesthetized and
underwent surgery for intramuscular plasmid injection into left and
right soleus muscles, as described previously (9). A skin
incision was made to expose the soleus muscle, and 20 µl of
phosphate-buffered saline 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 a fixed amount (3.29 µg) of the human skeletal
-actin reference promoter construct (HSA2000-pRL). After plasmid injection, the rats in the SI group were subjected to complete spinal
cord transections at midthoracic and upper sacral levels, along with
bilateral deafferentation between the transection sites, as described
previously (10, 29). Additional rats were assigned to NC
(n = 12) and SI (n = 24) groups and
used to obtain soleus muscle for muscle nuclei extraction for use in
the DNA gel mobility shift assays (see below). The care and maintenance
of the SI rats have been described in detail previously (10,
29). All experiments were 7 days in duration. After 7 days, the
animals were euthanized, and the soleus muscle was quickly removed,
frozen in isopentane cooled by liquid nitrogen, and stored at
80°C
until subsequent analysis. This study was approved by the Animal Use
Committee at the University of California, Los Angeles, and followed
the animal care guidelines of the American Physiological Society.
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: 3500-,
408-,
299-,
215-, and
171-bp promoter
fragments, all extending to position +34 at the 3' end relative to the
TSS. These promoter constructs were the same as those used previously
by Wright et al. (44). The
408- and
215-bp type I MHC
promoter constructs with
e3 mutation (
408-bp
e2/
e3 mutant)
were also tested in the NC and SI soleus muscle. Mutations of the
target promoter sequence consisted of changing ACC to CGG in the
e3
element (17, 38) and GTG to TGT in the
e2 element (38) and were designed to disrupt specific transcription
factor binding sites (see Ref. 44 for details on
mutagenesis). The reference promoter consisted of the human skeletal
-actin gene extending from
2000 to +250 bp relative to the TSS,
which was linked to the Renilla luciferase (RLuc) reporter
gene in the pRL vector (Promega).
Reporter gene assay. Each soleus muscle was homogenized in 1 ml of ice-cold passive lysis buffer (Promega) supplemented with protease inhibitors [0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin], and the homogenate was centrifuged at 10,000 g for 10 min at 4°C. The supernatant was reserved to measure reporter gene activity using the Promega dual-luciferase assay kit, which is designed for sensitive detection of FLuc and RLuc activities within a single extract aliquot. Five microliters of supernatant were used in the assay, and light output was integrated over 10 s and expressed as relative light units (RLUs) using a luminometer (Analytical Luminescence). Background activity levels, based on measurements of noninjected tissue for both luciferases, were determined and subtracted from the activities measured in plasmid-injected tissue.
Rationale for use of skeletal -actin as a reference promoter.
In the present study, activity of the type I MHC promoter linked to an
FLuc reporter was tested in the SI soleus muscle using reporter gene
assays after direct muscle gene transfer. This approach is predicated
on the assumption that reporter gene expression (FLuc activity) is
directly proportional to the activity of the promoter linked to the
5'-upstream region. The method of gene transfer (in vivo or in cell
culture) is associated with variability in DNA uptake by the cells,
which makes it difficult to interpret changes in reporter gene
activity. Consequently, a reference promoter is coinjected with the
test promoter and used as an internal control for variable gene
transfer efficiency. The reference promoter drives expression of a
different reporter gene, which allows independent measurement of its
activity along with that of the test promoter. The reference gene
promoter should not interact with test promoter activity and should
ideally not change in response to experimental manipulations. Skeletal
-actin is a good reference promoter for studies utilizing muscle
gene promoters for the following reasons: 1) Skeletal
-actin and MHC are sarcomeric genes that are intrinsically active in
muscle cells; thus the transfection of exogenous promoters of these
genes into muscle cells is less likely to disturb the nuclear milieu
balance and cause competition for nuclear factors than transfection
with strong viral promoters (simian virus 40 and cytomegalovirus).
2) Skeletal
-actin and MHC are muscle-specific genes that
are not expressed in nonmuscle cells; thus the possibility of
confounding expression in surrounding nonmuscle cells is unlikely. 3) FLuc and RLuc reporter expression can be assayed in a
single aliquot with similar detection sensitivity and range (Promega dual-luciferase assay protocol). As evidence of the validity of
-actin for normalizing gene transfer efficiency in muscle, Giger et
al. (9) reported a strong correlation between FLuc and
RLuc activity after coinjection of type I MHC pGL3 and human
-actin pRL in the NC soleus.
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Plasmid uptake. Plasmid DNA uptake amounts can be used to normalize reporter activity without use of a reference plasmid; however, this approach does not account for possible alterations in posttranscriptional processes induced by the experimental manipulation. During the course of this study, it was clear that SI caused a dramatic decrease in test and reference promoter activities. Consequently, the possibility existed that plasmid uptake efficiency was less in SI muscles, which would cause this decrease in test and reference promoter activity. To gain insight into plasmid uptake in this model, tissue plasmid content at the time of analysis for reporter activity was determined using PCR.
Total tissue DNA (genomic and plasmid) was extracted from the resulting tissue pellet after centrifugation of the soleus homogenate in the reporter gene assay protocol described above. DNA extraction was performed using the Qiagen QIAamp DNA Mini Kit according to manufacturer's protocol for tissue extraction. DNA was eluted in 200 µl of nuclease-free water. Plasmid detection and quantification were based on PCR amplification. PCR primers to detect pGL3 plasmid were targeted specifically to the FLuc cDNA, and their sequence shares no identity with any of the rat genomic DNA sequence. The primers used to detect the pGL3 plasmid are CGGGCGCGGTCGGTAAAGT [5' pGL3 primer (1163-1181)] and AACAACGGCGGCGGGAAGT [3' pGL3 primer (1542-1524)], where the numbers in parentheses refer to the base position within the pGL3 basic sequence (GenBank accession no. U47295), and they generated a 380-bp PCR product. The FLuc cDNA gene is located from 88 to 1737 bp (Promega Technical Reference). Five microliters of the DNA template were amplified using PCR (final concentrations): 1× PCR buffer, 20 µM each dNTP, 1 µM primers, 2.8 mM MgCl2, and 0.75 U of Taq polymerase. Amplification was carried out in a thermocycler (Stratagene, La Jolla, CA) and was started with an initial denaturation step at 96°C for 3 min, followed by 1 min at 96°C, 45 s at 59°C, and 50 s at 72°C for 25 cycles and a final elongation step for 3 min at 72°C. Under these PCR conditions, the signal was in the linear range of a semilogarithmic plot of the yield vs. number of PCR cycles. Samples were run in duplicate with appropriate negative and positive controls. PCR products were separated on 2% agarose gels containing ethidium bromide. Pictures of the gels were obtained under ultraviolet light with Polaroid 55 film to obtain a negative and a positive gel image. The negatives were scanned (Molecular Dynamics personal densitometer), and band intensity was determined with the Image Quant 5.0 software package (Molecular Dynamics) using volume integration.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 SI soleus muscles (n = 6/group, each n consisted of a pool of 4 muscles for NC and
8 muscles for SI, ~200 mg/pool) and stored at 80°C.
Double-stranded oligonucleotides consist of 24 bp from the type I MHC
gene promoter spanning specific regulatory elements. The
e3
oligonucleotide sense strand sequence and
e3 mutant sense strand
sequence are given in Table 2. After strand annealing, double-stranded probes were end labeled with 32P using T4 polynucleotide kinase (Promega).
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Statistical analysis. Statistical analysis was performed with Graphpad Prism 3.0 statistical software. Values are means ± SE. An unpaired, two-tailed t-test was used to detect differences between NC and SI groups. For multiple comparisons, one-way ANOVA with Newman-Keuls post hoc tests were performed. P < 0.05 was accepted as the level of statistical significance.
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RESULTS |
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Body and muscle weights. Animals assigned to separate experiments were matched in initial body weight: 139 ± 1 and 140 ± 1 g for NC and SI groups, respectively. At the termination of the 7-day experiment, body weight increased to 166 ± 1 g in NC rats and decreased to 122 ± 1 g in SI rats. SI resulted in similar changes in body weight and muscle weight relative to NC across all experiments (Table 1). Average body weight was 26% less, absolute soleus muscle weight was 60% lower, and normalized soleus weight-to-body weight ratio was 46% lower in SI than in NC rats (Table 1). All these differences were highly significant (P < 0.0001).
Deletion analysis of the type I MHC promoter. The validity of the direct gene injection technique to study MHC regulation in intact skeletal muscle has been previously demonstrated in our laboratory (9). Transfected type I and IIb MHC promoter activities, based on reporter assays, correlated well with muscle fiber-type-specific expression of the corresponding endogenous genes.
In the NC soleus muscle, a "full-length"
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Mutational analysis of the type I promoter.
Mutation of the e3 element in the
215-bp type I promoter 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-twitch skeletal muscle (Fig.
3). Mutation of this element in the
215-bp promoter construct also attenuated type I MHC downregulation
in response to SI. Similar to the results observed with the
171-bp
promoter, activity of the
215-bp
e3 mutant promoter relative to
actin significantly increased in response to SI. Absolute FLuc activity
of the 215-bp
e3 mutant promoter was still decreased 65% in
response to SI (6,742 ± 1,990 to 2,274 ± 660 RLU); however,
this was smaller than the 96% decrease found with the 215-bp wild-type
promoter (29,360 ± 12,082 to 1,206 ± 550 RLU). Thus
decreased responsiveness to SI when the
e3 element is mutated
suggests that this element is involved in regulating transcriptional
downregulation of type I MHC (Fig. 3).
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Normalization of reporter activity to plasmid uptake.
As discussed in METHODS, an additional control for pGL3
plasmid uptake normalization was performed in selected experiments. In
contrast to actin promoter activity, which was significantly decreased
in response to SI (Fig. 2), plasmid uptake was slightly greater in the
soleus muscle of SI than NC rats (Table
3). Although normalization to actin
reduced reported responsiveness of the type I promoter to SI,
normalization to plasmid uptake resulted in type I downregulation,
which was more closely matched to the decrease in FLuc activity than to
the ratio of FLuc to RLuc activity. Specifically, although the
normalized type I MHC activity relative to actin decreased 92 and 39%
for the 3500- and
215-bp promoters, respectively, and increased
109% for the
171-bp promoter, the activities were decreased 99, 98, and 80% relative to plasmid uptake. These results clearly demonstrate
that significant downregulation of the type I MHC promoter in response
to SI was not due to reduced plasmid uptake but, rather, to SI-induced
transcriptional regulation.
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Activity of the type IIb MHC promoter.
Activity of a 1400-bp type IIb MHC promoter construct was
significantly increased in the soleus muscle of SI rats compared with
NC rats (Fig. 4). This upregulation is in
agreement with the shift in MHC expression in response to SI
(16) and demonstrates that the previously reported
increases in type IIb mRNA with SI are transcriptionally regulated.
Furthermore, upregulation of the type IIb promoter demonstrates that SI
does not cause a global decrease in transcription.
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Gel mobility shift assays.
The findings that 1) mutation of the e3 element in the
215- and
408-bp promoter fragments and 2) deletion of
this element (
171-bp fragment) decreased type I responsiveness to SI
strongly suggest that this element is involved in the SI response.
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DISCUSSION |
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Several models of chronic decreased activity, such as denervation,
hindlimb unloading, spinal cord transection, and SI have clearly
demonstrated the ability of skeletal muscles to adapt their MHC
phenotype to the imposed alterations in activity and load (for review
see Refs. 27 and 36). However, although variable amounts
of neuromuscular activity are known to impact muscle fibers in the
majority of these models, SI is unique, in that it provides a baseline
for complete inactivity while preserving potential neural, but
non-activity-related, factors. With the use of this model, the findings
reported here provide the first evidence that the complete absence of
neuromuscular activation and loading significantly decreases in vivo
transcription of the type I MHC gene in a slow-twitch muscle. In
addition, deletion and mutation analyses strongly suggest that the
e3 DNA regulatory element is involved in the type I MHC
downregulation associated with chronic inactivity. As evidenced by the
supershift assays, this downregulation with SI is potentially mediated
by decreased binding of TEF-1 to the
e3 element. Transcriptional regulation of the MHC genes with chronic inactivity was not confined to
the slow type I MHC gene, inasmuch as we also demonstrated a
significant transcriptional upregulation of the fast type IIb MHC gene.
Thus the previously observed time-dependent MHC shifts at the
steady-state mRNA and protein levels (i.e., decreases in slow type I
MHC and increases in the fast isoforms) (16) are initiated
at the level of transcription. Clearly, neuromuscular activation and
loading are essential factors in maintaining normal levels of type I
MHC transcription in a slow-twitch rat hindlimb muscle.
The significant depression in type I MHC promoter activity within 7 days of SI has a profound effect on muscle MHC protein expression, as evidenced by the rapid loss of muscle mass and protein/MHC content over the first 8 days of SI. We recently reported that the soleus muscle had lost nearly half of its type I MHC protein after only 8 days (16). As demonstrated by the present study, this rapid loss was primarily due to transcriptional regulation of the type I MHC gene, rather than posttranscriptional and/or translational regulation. With longer-duration SI, the rate of muscle atrophy and MHC protein loss was reduced, and the atrophied muscles then underwent additional remodeling of the myosin phenotype.
This is the first study to demonstrate that decreased type I MHC
expression with complete skeletal muscle inactivity is
transcriptionally regulated and likely involves the e3 regulatory
element. The enhancer core of the
e3 element has been characterized
in cardiac myocytes and is also referred to as the myocyte-specific CAT
(MCAT) cis-acting element (17, 19). Evidence
that
e3 is an enhancer element in the normal weight-bearing soleus
is demonstrated here by the significant decrease in
215- and
408-bp
type I promoter activity when the
e3 element is mutated (Fig. 3).
Members of the TEF-1 multigene family bind to this
e3 (MCAT) element
and activate transcription of muscle genes in cardiac myocytes such as
type I MHC (17, 18, 34, 40),
-MHC (12),
and skeletal
-actin (19). Recently, a TEF-1-related
protein, RTEF-1, was identified and found to be a target of
1-adrenergic signaling in hypertrophied cardiac myocytes
(40). Specifically, phosphorylation of a serine residue
accounted for a majority of the
1-adrenergic response.
Overexpression of TEF-1 activated a
215-bp type I MHC promoter, while
overexpression of RTEF-1 activated the
215- and
3300-bp type I
promoters (34). An intact MCAT element was necessary for
this transactivation of the type I promoter by TEF-1 and RTEF-1.
TEF-1 is also involved in regulating skeletal muscle genes, inasmuch as
3 days of stretch overload in the chicken anterior latissimus dorsi
muscle increased TEF-1 binding to the -actin promoter
(2). Our supershift assays clearly demonstrate that TEF-1
is also involved in type I MHC regulation in the rat soleus muscle and
is responsive to changes in neuromuscular activity (Fig. 5). A TEF-1
antibody supershifted protein complexes binding to the
e3
oligonucleotide in nuclear extracts from NC and SI soleus muscle.
Although total nuclear protein binding to the
e3 oligonucleotide
increased 85% with SI, the protein supershifted by the TEF-1 antibody
was only 50% of that observed in NC soleus muscle. Thus chronic
inactivity with SI decreased TEF-1 binding, in contrast to muscle
overload, which was previously shown to increase TEF-1 binding
(2). Consequently, one mechanism whereby SI could
downregulate type I MHC is a decrease in TEF-1 binding to the
e3
element, as evidenced by a significant reduction in downregulation when
this element was mutated or deleted. Taken together with previous work,
TEF-1 may upregulate muscle-specific genes in response to muscle
overload (2) while downregulating these genes in response
to chronic inactivity.
Because SI increased nuclear protein binding to the e3
oligonucleotide while decreasing the amount of TEF-1 binding, this suggests that the residual binding was composed of other transcription factors that increase in response to SI. One possibility is that SI may
increase other TEF-1-related factors (e.g., RTEF-1, DTEF-1, and ETF).
However, this is not likely, since this family of transcription factors
enhances transcription, while chronic inactivity downregulates type I
MHC expression. Another potential transcription factor that may
contribute to the increase in nuclear protein binding to the
e3
oligonucleotide is cAMP-responsive binding protein (CREB). The
e3
oligonucleotide probe used in the gel mobilty shift assays also
contains a putative CREB site (
213/
216 bp; Table 2) adjacent to the
TEF binding site (MCAT). Unphosphorylated CREB has been shown to
repress activity of the c-jun promoter (20);
consequently, deletion of this CREB site could remove another potential
regulatory site for decreasing type I MHC expression.
In our previous SI time-course study (16), we reported de novo expression of type IIb MHC mRNA after only 4 days of SI. These findings are supported by the present observation that 1 wk of SI significantly upregulates type IIb MHC promoter activity (Fig. 4). Interestingly, although short-term inactivity and/or unloading is sufficient to activate transcription of the IIb MHC gene, it is unable to induce expression of type IIb protein. For example, in hindlimb-unloaded rats, type IIb MHC was expressed at the mRNA, but not at the protein, level (14). Similarly, 60 days of SI (16), 30 days of denervation (13, 15), or 14 days of tetrodotoxin paralysis (24) did not induce de novo type IIb protein expression, despite increases in its mRNA. Taken together, these observations suggest a marked uncoupling between transcriptional and translational events in the upregulation of type IIb MHC in slow-twitch muscles during the initial stages of unloading. However, more prolonged inactivity (e.g., 90 days of SI) or decreased activity coupled with other factors such as thyroid hormone status have induced type IIb protein expression in the soleus muscle. For example, the combination of hindlimb unloading and triiodothyronine treatment resulted in increases in type IIb mRNA and protein in the rat soleus muscle (4, 14), which was transcriptionally regulated (4).
Collectively, we have used the direct gene transfer approach to
characterize the regulation of type I MHC gene expression in response
to chronic inactivity. In summary, our results demonstrate that chronic
inactivity and unloading of slow-twitch rat hindlimb muscles caused a
significant downregulation of type I MHC promoter activity. Deletion
and mutation analyses strongly suggest involvement of the e3 DNA
regulatory element in the type I MHC downregulation associated with SI.
Furthermore, decreased TEF-1 binding to the
e3 element likely
contributed to this type I MHC downregulation. Future studies should
investigate mechanisms whereby prolonged absence of activation and/or
loading alter the amount and binding of TEF-1 to the
e3 element and
how these interactions contribute to type I MHC downregulation with
different forms of reduced activity.
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ACKNOWLEDGEMENTS |
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The authors thank H. Zhong, J. Kim, and M. Herrera for animal care and surgery and A. Qin for plasmid preparation.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants NS-16333 (to V. R. Edgerton) and AR-30346 (to K. M. Baldwin).
Address for reprint requests and other correspondence: K. M. Baldwin, Dept. of Physiology and Biophysics, University of California, Irvine, 346-D Med Sci I, Irvine, CA 92697-4560 (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.00355.2001
Received 27 July 2001; accepted in final form 31 October 2001.
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