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Department of Physiology and Biophysics, University of California, Irvine, California 92697
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
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Previously, we have reported that the combination of hindlimb suspension (HS) and thyroid hormone [triiodothyronine (T3)] treatment induces the de novo expression of the fast IIb myosin heavy chain (MHC) gene in the soleus. Thus we tested the hypotheses that the induction of IIb MHC expression with HS + T3 is prevented with denervation and that this IIb induction is regulated by transcriptional processes. Adult female rats were subjected to 2 wk of combined HS + T3 in which one side of the lower leg was simultaneously denervated. HS + T3 caused decreased expression of the slow type I MHC and concomitant increases in both the fast type IIx and IIb MHC isoforms in the intact soleus muscle. Denervation prevented the endogenous expression of the IIb MHC gene at both the protein and mRNA levels. Although HS + T3 intervention was able to markedly increase the expression of the 2.6-kb IIb MHC promoter-reporter construct using direct gene transfer, this induction, however, was not inhibited by denervation. These findings collectively suggest that normal innervation is essential for inducing the unique expression of the IIb MHC in a slow muscle in response to HS + T3; however, in the denervated muscle, there is a discordance between the regulation of the endogenous IIb MHC gene relative to the exogenous IIb MHC promoter-reporter construct.
soleus; reverse transcriptase-polymerase chain reaction; hindlimb suspension; MyoD; direct gene transfer; transcription
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
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IN PREVIOUS STUDIES, we have made the novel observation that the combined treatments of unloading [hindlimb suspension (HS)] plus hyperthyroidism [triiodothyronine (T3)] induce the de novo expression of the fast type IIb myosin heavy chain (MHC) gene in a slow-twitch muscle (e.g., soleus) (2). This observation is unique in that the rodent soleus muscle does not express this gene under normal conditions or when either of the above interventions is imposed separately on this muscle. Interestingly, the HS + T3-treated soleus actually expressed such an abundance of the IIb MHC that it comprised as much as 30% of the MHC pool in many of the affected fibers, as well as contributed to the muscle's fast contractile properties as measured at the whole muscle level (2). In contrast to the above observations, denervation, in and of itself, has been reported either to markedly inhibit the developmentally induced transition for de novo expression of the IIb MHC in neonatal rats (1) or to repress the level of IIb MHC expression that is normally maintained in the adult state in a typical rodent fast-twitch muscle such as the plantaris and the tibialis anterior (8, 10); this process appears to be regulated at the pretranslational level (10).
To further examine the role of normal innervation in regulating the de
novo expression of the IIb MHC gene in the soleus muscle in response to
HS + T3, we tested the hypothesis that such expression 1) is inhibited by denervation and 2) is regulated at
the transcriptional level. Thus this study involved the use of a IIb
MHC promoter fragment [
2554/+13 bp] linked to a
firefly luciferase reporter gene in a plasmid vector to be tested in
normal control, HS + T3, and HS + T3 + denervated soleus, using in vivo direct gene transfer techniques. The
findings presented in this study support the above hypotheses and
further show that the upregulation of IIb MHC expression in soleus
muscle in response to HS + T3 is mediated by
transcriptional processes. A normal innervation was required for IIb
upregulation as studied at the protein and mRNA level. In contrast, the
nerve requirement for this IIb upregulation was not evident based on
reporter activity when driven by 2.6-kb IIb MHC promoter fragment.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
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Animal Care and Experimental Manipulations
Sixteen adult female Sprague-Dawley rats (weight ~250 g; Taconic Farms, Germantown, NY) were randomly assigned to either a normal control (NC, n = 8) or a HS plus thyroid hormone-treated (HS + T3, n = 8) group. The animals were housed individually in temperature- and light-controlled living quarters and were provided food and water ad libitum.Animals designated for suspension were treated with a noninvasive
tail-casting technique as described previously (9). The same group was
treated with daily intraperitoneal injections of T3 (150 µg · kg
1 · day1)
throughout the 14-day experimental period. This protocol has been shown
to 1) elevate plasma T3 levels, 2) induce
cardiac hypertrophy and MHC isoform shifts in striated muscle favoring
a fast phenotype, and 3) increase resting metabolic rate;
together, these three effects are the hallmarks of a hyperthyroid state
(8, 9, 13).
To accomplish denervation, animals in the HS + T3 group were anesthetized with ketamine-acepromazine (80:2 mg/kg). After an incision was made in the popliteal region of the left leg, the sciatic nerve was isolated, ligated in two places, and severed between the ligations so that 1 cm of nerve was removed to prevent regeneration. Denervation at this level eliminates innervation of both the anterior and posterior compartments of the lower leg while avoiding disruption of the structural integrity of the targeted muscles and their blood supply. After suturing, each animal was allowed to recover to full ambulation, at which time the combined suspension and T3-treatment procedures were initiated. A unilateral denervation approach was used to expose both the denervated and the innervated contralateral muscles to the same environmental conditions, i.e., T3 treatment and hindlimb unloading. The separate interventions of hindlimb unloading and of T3 treatment were not performed in the context of the present experiment because neither intervention by itself has been shown to cause a significant upregulation of the IIb MHC at the protein level in the soleus muscle (2, 9). Thus these additional groups would have added little insight relevant to the hypothesis presented in this experiment. Finally, a 2-wk period was selected because previous findings on this model suggest that upregulation of the IIb MHC, at both the mRNA and protein levels of expression, occurs in this time frame (2, 9). All the procedures reported above were approved by our institutional animal care and use committee.
In Vivo Gene Transfer
For promoter assays, plasmid DNA mixtures were directly injected into the soleus muscle of young rodents according to methods published previously (Refs. 4, 6, 15). Separate groups of NC (n = 4) and HS + T3-treated (n = 8) animals were used for the gene injection study; a left-leg denervation procedure was performed on the latter group. Each injection consisted of 10 µg of the test plasmid combined with an equimolar amount of a reference plasmid in 25 µl of sterile PBS. The test plasmid consisted of a mouse IIb MHC promoter fragment extending from2554 bp to +13 bp relative to
the transcription start site and linked to a firefly luciferase
reporter gene (pGL3 basic, Promega) and was designated as 2.6-kb IIb
MHC-Fluc. The reference plasmid consisted of the promoter sequence of
another sarcomeric protein, the human skeletal 
-actin (4), which was
linked to a Renilla luciferase reporter (pRL nul, Promega) and
designated as HSA2000-Rluc. Both promoter fragments were the kind gift
of Dr. Steve Swoap, Williams College, Williamstown, MA (15). The
reference plasmid was used to control for transfection efficiency
within a group and as a general marker for sarcomeric gene
transcription. After plasmid injection in the soleus muscle, the
sciatic nerve of the left leg of the HS + T3 group was cut,
and the rats were immediately subjected to 7 days of HS + T3 as described above, whereas the NC rats were allowed to
bear weight in standard vivarium cages. After 7 days of treatment,
animals were euthanized (see below), and the soleus muscles were
removed and quickly frozen and stored at 80°C. Each frozen
muscle was homogenized in 1 ml of ice-cold passive lysis buffer
(Promega) supplemented with protease inhibitors (0.2 mM AEBSF, 5 µg/ml aprotinin, 5 µg/ml leupetin), and the homogenate was
centrifuged at 10,000 g for 10 min in the cold. Five
microliters of the supernatant were immediately assayed for luciferase
activity with use of the Promega dual luciferase assay kit, which was
designed for sensitive detection of both firefly and Renilla
luciferase activities in a single extract aliquot. Light output from
each specific luciferase activity was measured for 10 s with an
analytic luminometer (Monolight 2010-C, Analytical Luminescence
Laboratory, Ann Arbor, MI). Background activity levels, based on
measurements in noninjected tissue for both luciferases, were
established and subtracted from the activities measured in the
DNA-injected tissue. Activities were reported as relative light units
per muscle per second. Firefly luciferase activity represents the IIb
MHC promoter activity, whereas the Renilla luciferase activity
represents that of the -actin promoter.
Tissue Processing and Biochemical Analyses
At the termination of the 2-wk HS + T3 experiment, the rats were killed by a lethal injection of Nembutal (50 mg/kg), immediately after which the heart, soleus, and plantaris muscles (both sides) were rapidly removed, trimmed of connective tissue, weighed, and stored at80°C until they were analyzed for MHC mRNA and protein content. Each soleus muscle was divided into two pieces. One piece was
homogenized in a solution that contained (in mmol/l) 250 sucrose, 100 KCl, 5 EDTA, and 10 Tris-HCl, pH 7.0. Total protein content was
determined by the Biuret method (7), and the homogenate protein was
adjusted to 1 mg/ml with a storage buffer containing 50% glycerol, 50 mmol/l Na4P2O7, 2.5 mmol/l EGTA,
and 0.5 mmol/l 
-mercaptoethanol (pH 8.8) and stored at
20°C until subsequent analysis for MHC protein determination.
The other piece of muscle was processed for total cellular RNA
extraction by homogenization in the TRI reagent (Molecular Research
Center, Cincinnati, OH) according to the company's protocol, which is
based on the method described by Chomczynski (3). Total RNA was
precipitated from the aqueous phase with isopropanol; after it was
washed with ethanol, it was dried and suspended in a small volume of
nuclease-free water. The RNA concentration was determined by using an
optical density at 260 nm equivalent to 40 µg/ml. The RNA samples
were stored frozen at 80°C until they were subsequently
analyzed by RT-PCR technology (see below).
Reverse Transcription
One microgram of total RNA was reverse transcribed for each muscle sample by using the SuperScript II RT from GIBCO BRL and a mix of oligo dT and random decamer primers (100 ng/reaction) according to the provided protocol. At the end of the RT reaction, the tubes were heated at 85°C for 5 min to stop the reaction and were stored frozen at80°C until they were used in the PCR reactions for MHC
isoforms as well as for MyoD mRNA analyses.
MHC Protein Analysis
Skeletal MHC protein isoforms were separated with an SDS-PAGE technique that is capable of separating both neonatal and embryonic MHCs in addition to the four adult MHCs typically expressed in rodent leg muscles. The separation technique for adult and neonatal/embryonic MHC isoforms was described in detail previously (1). Included in these analyses were Western blotting analytic techniques to identify the appearance of any detectable embryonic MHC isoform in the denervated muscle by using procedures described previously (1). The MHC protein isoform profile for each muscle was determined by laser scanning densitometry of the stained gel as described previously (1, 8, 9).MHC mRNA Analysis
Analyses of MHC mRNA isoforms used a modification of a RT-PCR technique designed to quantitate relative amounts of MHC mRNAs representing the various MHC isoforms, as described in detail previously (17).PCR primers and internal control fragment.
The 5' upstream primer was designed from a highly conserved
region in all known rat MHC genes located at ~500-550 bp
upstream from the stop codon. All seven MHC mRNA isoforms are identical in this region spanning ~35 nt. A 20-nt oligonucleotide was chosen of
the following common sequence: 5'-AGAAGGAGCAGGACACCAGC-3'. The 3' MHC isoform-specific oligonucleotides used in the PCR
reaction were basically the same as those used previously (17) except that the embryonic oligo sequence was as follows:
CCCTCAC
GAGGACATGC. A correction in the
sequence (underlined) was included based on the mRNA sequence published
in the GenBank (accession no. X04267). The internal fragment also was
modified to be used as a competitor for all known MHC isoforms. Through
the use of sequence information, the internal fragment was made shorter
and the new common primer sequence was ligated at the 5' end,
whereas the neonatal- and embryonic-specific antisense primers were
linked in tandem to its 3' end, which already contained type I,
IIa, IIx, and IIb primers (17).
PCR.
Each RT reaction was diluted 20-fold with nuclease-free water and was
mixed with an equal volume of control fragment at the appropriate
dilution (~1 amol/µl). Two microliters of this mixture were used
for 25-µl PCR reactions. The PCR reaction was carried out in the
presence of 2 mM MgCl2 by using standard PCR buffer (GIBCO), 0.2 mM dNTP, 1 µM primers, and 0.75 unit of DNA Taq
polymerase (GIBCO). Amplifications were carried out in a Stratagene
Robocycler with an initial denaturing step of 3 min at 95°C,
followed by 25 cycles of 45 s at 95°C, 50 s at 52°C, 50 s at
72°C, and a final step of 3 min at 72°C. The number of cycles
was optimized so that the amplified signal was still in the linear
range of the semilog plot of the yield expressed as a function of the
number of cycles. PCR products were separated on a 2% agarose gel by
electrophoresis, and signal quantification was done as reported
previously (17). A correction factor was calculated for each band to
normalize the intensity of the ethidium bromide staining to account for the different sizes of the control synthetic fragment (Table
1). In this method, each specific MHC mRNA
isoform expression is calculated relative to the total MHC mRNA pool.
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MyoD mRNA Analysis
A relative quantitative RT-PCR method using 18S as the internal standard (Ambion, Austin, TX) was applied to study MyoD mRNA expression in control and experimental muscles. 18S ribosomal RNA was coamplified in all the samples to serve as an internal standard and allowed correction for the starting amounts of total RNA.For the MyoD amplification, we used the following primers: 5'-CTACAGCGGCGACTCAGACG-3' (5' sense primer) and 5'-TTGGGGCCGGATGTAGGA-3' (3' antisense primer). The MyoD primers yielded a 563-bp product. For the 18S, we used the alternate 18S internal standards (Ambion), which yields a 324-bp product. The 18S competimers and primers were mixed at a 10-to-1 ratio, which allowed linear amplification of the 18S to the same range as the MyoD target mRNA (Quantum RNA, Ambion). One microliter of a 10-fold dilution of each RT reaction was used for the PCR amplification. The PCR reaction was carried out in the presence of 1.5 mM MgCl2 by using standard PCR buffer (GIBCO), 0.2 mM dNTP, 1 µM MyoD primers, 0.5 µM 18S primers mix, and 0.75 unit of DNA Taq polymerase (GIBCO) in 25 µl total volume. Amplifications were carried out in a Stratagene Robocycler with an initial denaturing step of 3 min at 96°C, followed by 25 cycles of 1 min at 96°C, 1 min at 59°C, 1 min at 72°C, and a final step of 3 min at 72°C. PCR products were separated on a 2% agarose gel by electrophoresis and stained with ethidium bromide; signal quantification was performed by laser scanning densitometry as reported previously (17). By this method, each MyoD signal was normalized to its corresponding 18S. Under these PCR conditions, both the MyoD and 18S product yield are still in the linear range of the semilog plot when the yield is expressed as a function of the number of cycles.
Statistical Analyses
All data are reported as means ± SE. Statistical differences between the NC, HS + T3, and HS + T3 + denervation groups were tested by using a one-way ANOVA procedure; when differences were detected for a given variable, a Newman-Keuls post hoc test was used. All statistical analyses were performed using a computer software package (Prism, Graphpad Software). Statistical significance was set at P < 0.05.| |
RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
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Body and Muscle Weights
The combination HS + T3 induced a 12% reduction in body weight (P < 0.05; Table 2), which is a typical adaptive response to the combined interventions (9). Heart weight and the heart weight-to-body weight ratio were significantly elevated by 35% in the treated group (Table 2), a finding that is also consistent with the action of T3 on this muscle type (13). Both the innervated soleus and plantaris muscles of the HS + T3 group underwent the typical atrophy response that has been routinely reported with this model (9). Denervation induced additional atrophy in the plantaris muscle but not in the soleus muscle (Table 2). These findings on the denervated muscle, as well as other findings in which denervation was interacted with thyroid deficiency (8), clearly suggest that fast muscle appears to be more sensitive to the denervation-induced atrophy response than slow muscle, especially when either the loading status or the thyroid state is altered in combination with denervation (8). Clearly, more information is needed to unravel this differential response between fast and slow skeletal muscle.
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Endogenous MHC Isoform Responses
The NC soleus expresses mainly two MHC isoforms, i.e., type I and IIa, which combine to account for more than 95% of the MHC protein pool. This is true at both the protein and mRNA levels (Figs. 1 and 2). Trace amounts of IIx (<3%) also were detected. In addition to types I, IIa, and IIx MHC, some IIb and embryonic MHC isoforms could be detected only at the mRNA level, accounting for ~1% and ~8% of the pool, respectively (Fig. 2). The expression of the embryonic mRNA is somewhat surprising given that its encoded MHC protein is seldom detected in normal adult slow muscle even with the use of more sensitive detection methods such as immunoblotting techniques (Fig. 3). This unique observation of a low level of embryonic MHC mRNA expression in a NC soleus was validated by using the more conventional Northern blot hybridization (Fig. 4B) and was also noted by others using RT-PCR methods (12).
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The combination of HS + T3 induced a significant remodeling of MHC expression by causing a decrease in the relative content of the type I MHC. This response was offset by significant increases in the relative content of both the fast type IIx and IIb MHCs (P < 0.05), whereas essentially no change in the relative content of the fast IIa MHC was produced. This pattern of response across the MHC pool is similar at both the protein and mRNA levels (Figs. 1 and 2), although the magnitude of shifts is much more significant at the mRNA level because the mRNA has a faster turnover rate compared with the MHC proteins. Also, the MHC isoform response to HS + T3 is similar to what was previously reported (2, 9).
When the variable of denervation was introduced in combination with the HS + T3 intervention, the de novo expression of the IIb MHC protein and induction of IIb MHC mRNA were repressed, whereas significant amounts of the IIx MHC continued to be expressed along with the type I and IIa MHCs (Figs. 1 and 2). In addition to blunting the induction of the IIb MHC mRNA expression, denervation was also associated with an induction of both embryonic (22%) and neonatal (3%) MHC mRNAs, which were not detected at the protein levels (Figs. 1-4). This observation was somewhat surprising especially for the embryonic MHC, which comprised as much as 22% of the total MHC mRNA pool even though it apparently was not translated (Fig. 1).
Interestingly, other reports (14) have shown detectable amounts of embryonic and neonatal MHC protein in denervated muscles using the same antibody (BF-45) used in this study. One explanation for this discrepancy is that the immunoblotting method we used (Fig. 3) is not as sensitive as the immunofluorescence method used by Schiaffino et al. (14). Alternatively, one cannot compare the denervated muscles in the two studies (this study and Ref. 14) because in this present study denervation was coupled to a hyperthyroid state, whereas in the study by Schiaffino et al. it was carried out in the euthyroid state, and this difference could possibly account for the difference in results. In fact, in a previous study (8), we were able to detect the embryonic MHC protein isoform expression in denervated soleus muscle under both hypothyroid and euthyroid states, but not under a hyperthyroid state, by using Coomassie blue-stained protein gels. Thus, based on our results, it is clear that there is a disproportionality between the amount of embryonic MHC mRNA vs. protein that is expressed. If embryonic MHC protein were expressed, it had to be at a very low level, i.e., below the limits of our detection method. Thus denervation, although essentially blunting the de novo expression of the IIb MHC in terms of both protein and mRNA, induced a unique profile at the mRNA level but not at the protein level. That is, denervation essentially mimics a pattern typically seen as a cross between undifferentiated fast and slow muscle during the early stages of neonatal development (1).
IIb MHC Promoter Activity
The 2.6-kb IIb MHC promoter fragment had a very low activity in the NC soleus based on total firefly luciferase reporter activity expressed and compared with the actin promoter activity (Fig. 5). This was not surprising because the former is normally expressed chiefly in fast-twitch muscles, whereas the latter is expressed in both fast- and slow-twitch skeletal muscles (4). HS + T3 caused an approximately four- to fivefold increase in the absolute activity of the 2.6-kb IIb MHC-Fluc promoter. Interestingly, this response to HS + T3 occurred even though the response of the HSA2000-Rluc promoter was markedly inhibited, thereby producing a marked increase (~40-fold) in the IIb-to-actin ratio (Fig. 5). When denervation was imposed in the context of the HS + T3 stimulus, the response of the 2.6-kb IIb MHC-Fluc remained elevated and was not different from that seen with HS + T3 (Fig. 5). Similarly, denervation in the context of HS + T3 did not alter the HSA2000-Rluc promoter activity any further than the inhibition due to HS + T3 alone. Thus these data suggest a discord between the response of the endogenous IIb MHC gene and the exogenous 2.6-kb IIb MHC promoter fragment under the unique conditions of denervation, and this response occurs when the 2.6-kb IIb MHC-Fluc promoter-reporter data are assessed independently of or in conjunction with the actin response.
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MyoD Expression
Analysis of the 2.6-kb IIb MHC promoter nucleotide sequence reveals that at least 14 E-box elements are located throughout the 2.6-kb promoter upstream of the transcription start site (16). The E-box element (CANNTG) is known to interact with proteins comprising the MyoD gene family (5, 11). Thus it was of interest to examine the response of MyoD mRNA expression in the present series of experiments. As shown in Fig. 5, MyoD mRNA signals were markedly elevated in the soleus muscle of rodents subjected to HS + T3, whereas only trace levels are detected in the normal soleus muscle. Given the fact that MyoD protein is abundantly expressed in fast muscles that highly express the type IIb MHC isoform (11), this factor could play a role in the adaptive process by impacting transcriptional activity of the IIb MHC in HS + T3 soleus muscle. If MyoD expression is a key factor in regulating IIb MHC transcriptional activity in the HS + T3 soleus, we would have expected a marked reduction in the MyoD signal in the denervated side. Surprisingly, however, we observed that the denervated HS + T3 soleus also expressed significant levels of MyoD mRNA. In fact, the levels were higher in the denervated side compared with HS + T3 alone (Fig. 6). These data suggest that the MyoD factor, in and of itself, is unlikely to be the primary factor in the regulation of endogenous IIb MHC expression in response to HS + T3, particularly in the context of the denervation response. Clearly, some other factor(s) must be involved in both the upregulation and repression of IIb MHC gene expression in our model system.
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Integrated Response
The collective data presented in this paper provide convincing evidence that normal muscle innervation is an essential factor in the de novo expression of the endogenous IIb MHC gene that occurs in the soleus muscle in response to the unique combination of hindlimb unloading and T3 treatment. This conclusion is based on the MHC protein and mRNA isoform expression data and confirms the first hypothesis. Using the in vivo gene transfer approach, we demonstrated that the activity of the exogenous 2.6-kb IIb MHC promoter fragment increases severalfold relative to control in response to the combined treatment (HS + T3). This increase correlated well with the endogenous IIb MHC expression in HS + T3 soleus and suggests that this de novo synthesis of IIb is due to transcriptional regulation. Also, this finding implies that the2554/+13 bp IIb
MHC promoter fragment contains cis regulatory elements that are
responsive to HS + T3 treatment, which, in turn, act to
enhance the promoter activity. Surprisingly, judging by reporter gene
activity level, denervation did not inhibit the increased
transcriptional activity of the exogenous 2.6-kb IIb MHC promoter
fragment the same way as the endogenous IIb MHC expression is
regulated. Thus a discordance between the regulation of the endogenous
IIb MHC mRNA expression vs. that of the transfected promoter fragment
activity was detected in the HS + T3 + denervated soleus.
This disagreement could be the result of at least two mechanisms:
1) the IIb repression by denervation is transcriptionally regulated but the "denervation response element" on the IIb
promoter is located outside the 2554/+13 bp IIb MHC promoter
fragment. That is, it is either further upstream of the 2554 bp
or downstream in the transcribed region of the gene; 2) the IIb
repression by denervation is at the posttranscriptional level and
involves processing and maturation of the primary IIb MHC transcript.
This latter possibility would have to be unique to the IIb MHC gene
involving specific regulatory protein-RNA interaction, given that it
was not operative for the reporter gene (Fluc). Presently, it is not certain which of the two mechanisms is occurring. To test either, a
more elaborate IIb MHC promoter gene sequence would have to be isolated
and tested in conjunction with the 2554/+13-bp promoter fragment. Of interest to the findings reported herein for the IIb MHC
promoter, we have recently reported that an enhancer sequence is
located in the upstream promoter region of the type I MHC promoter between 3500 and 2900 bp (18). Thus it is possible that a larger fragment of the IIb MHC promoter is needed to detect the regulation occurring during denervation.
On the other hand, one should not ignore the alternative possibility that the chromosomal context in which the endogenous IIb MHC gene exists may contain the required information for the full regulatory mechanism. It is known that the exogenous plasmid constructs are taken up and remain episomal. Although these plasmid constructs are exposed to the same transcription factors and nuclear milieu, some critical information linked to chromatin structure and interaction may be lacking. In conclusion, the exact mechanism by which denervation inhibits IIb MHC isoform expression in HS + T3 soleus remains to be elucidated.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-30346 and by National Space Biomedical Research Institute Grant NCC9-58.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: K. M. Baldwin, Dept. of Physiology and Biophysics, Univ. of California, Irvine, CA 92697 (E-mail: kmbaldwi{at}uci.edu).
Received 22 March 1999; accepted in final form 14 October 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
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