help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-1476
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Spangenburg, E. E.
Right arrow Articles by Booth, F. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Spangenburg, E. E.
Right arrow Articles by Booth, F. W.
Endocrinology Vol. 145, No. 4 2054-2063
Copyright © 2004 by The Endocrine Society

Insulin-Like Growth Factor-Induced Transcriptional Activity of the Skeletal {alpha}-Actin Gene Is Regulated by Signaling Mechanisms Linked to Voltage-Gated Calcium Channels during Myoblast Differentiation

Espen E. Spangenburg, Douglas K. Bowles and Frank W. Booth

Department of Biomedical Sciences (E.E.S., D.K.B., F.W.B.), Department of Medical Pharmacology and Physiology (F.W.B.), and Dalton Cardiovascular Research Center (D.K.B., F.W.B.), University of Missouri, Columbia, Missouri 65211

Address all correspondence and requests for reprints to: Frank W. Booth, Ph. D., University of Missouri, Department of Biomedical Sciences, E102 Veterinary Medical Building, 1600 East Rollins Road, Columbia, Missouri 65211. E-mail: boothf{at}missouri.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I activates signaling pathways that increase the expression of muscle-specific genes in differentiating myoblasts. Induction of skeletal {alpha}-actin expression occurs during differentiation through unknown mechanisms. The purpose of this investigation was to examine the mechanisms that IGF-I uses to induce skeletal {alpha}-actin gene expression in C2C12 myoblasts. IGF-I increased skeletal {alpha}-actin promoter activity by 107% compared with the control condition. Ni+ [T-type voltage-gated Ca2+ channel (VGCC) inhibitor] reduced basal-induced activation of the skeletal {alpha}-actin promoter by approximately 84%, and nifedipine (L-type VGCC inhibitor) inhibited IGF-I-induced activation of the skeletal {alpha}-actin promoter by 29–48%. IGF-I failed to increase skeletal {alpha}-actin promoter activity in differentiating dysgenic (lack functional L-type VGCC) myoblasts; 30 mM K+ and 30 mM K++IGF-I increased skeletal {alpha}-actin promoter activity by 162% and 76% compared with non-IGF-I or IGF-I-only conditions, respectively. IGF-I increased calcineurin activity, which was inhibited by cyclosporine A. Further, cyclosporine A inhibited K++IGF-I-induced activation of the skeletal {alpha}-actin promoter. Constitutively active calcineurin increased skeletal {alpha}-actin promoter activity by 154% and rescued the nifedipine-induced inhibition of L-type VGCC but failed to rescue the Ni+-inhibition of T-type VGCC. IGF-I-induced nuclear factor of activated T-cells transcriptional activity was not inhibited by nifedipine or Ni+. IGF-I failed to increase serum response factor transcriptional activity; however, serum response factor activity was reduced in the presence of Ni+. These data suggest that IGF-I-induced activation of the skeletal {alpha}-actin promoter is regulated by the L-type VGCC and calcineurin but independent of nuclear factor of activated T-cell transcriptional activity as C2C12 myoblasts differentiate into myotubes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SKELETAL MUSCLE SATELLITE cell/myoblast differentiation is a fundamental and multistep process that occurs during muscle development (1) and repair from muscle injury or insult (2). Differentiation induces maturation of the myoblasts/satellite cells and ultimately allows for the induction of adult specific gene expression, which includes various contractile and metabolic genes such as skeletal {alpha}-actin (3) and creatine kinase (4). The increased expression of these genes during differentiation is likely initially regulated at the transcriptional level, because nondifferentiated myoblasts fail to express any significant levels of adult skeletal muscle contractile gene mRNA or protein (5). The onset of differentiation necessitates the activation of a number of transcription factors to induce a muscle-specific gene program. The expression of one such transcription factor, myogenin, a member of the basic helix-loop-helix family, is necessary for myoblast differentiation (6) and increased contractile gene expression (7). Therefore, the understanding of mechanisms that regulate the activation of various transcription factors is fundamental to understanding how myoblast differentiation occurs in muscle.

Differentiation of skeletal muscle myoblasts is dependent upon a complex interplay of growth factors, ions, and transcription factors (8). The extracellular environment has a profound effect on the myoblast differentiation process by influencing signaling mechanisms that alter specific gene expression within the myoblast (9). For example, Shainberg et al. (10) demonstrated that myoblast differentiation was dependent upon the entry of extracellular calcium into the cell. Further, the induction of skeletal {alpha}-actin protein expression during myoblast differentiation is dependent upon extracellular calcium (8). Controversy exists in how the myoblast induces the entry of extracellular calcium into the sacroplasm, although it appears that the myoblast uses, either separately or in combination, T-type (11) or L-type (12) voltage-gated calcium channels (VGCC).

Another factor, IGF-I, has long been recognized as having the ability to stimulate the rate of myoblast differentiation (4) and influence myogenin gene expression (13, 14, 15). Unfortunately, at this time, it remains unclear how growth factors, like IGF-I, interact at the cell membrane to influence changes in gene expression that occur during differentiation. Previously, Semsarian et al. (16) demonstrated that exogenous IGF-I increased intracellular calcium levels by 77% and further activated the phosphatase activity of the calcium-sensitive phosphatase, calcineurin. In mature skeletal muscle fibers, Delbono et al. (17) have demonstrated L-type VGCC activation by IGF-I. Therefore, it is possible that growth factors, such as IGF-I, induce contractile gene expression in differentiating myoblasts by altering calcium levels through activation of various membrane-bound Ca2+ channels.

Very little is known about how IGF-I alters contractile gene expression, and most studies have focused on IGF-I’s effects on myogenin expression during differentiation (15). Therefore, we sought to delineate possible mechanisms that IGF-I may employ to induce skeletal {alpha}-actin gene expression. The purpose of this study was 3-fold: 1) to determine whether IGF-I induces activation of the skeletal {alpha}-actin promoter; 2) to determine whether activity of the membrane VGCC is necessary for activation of the skeletal {alpha}-actin promoter; and 3) to determine what cellular signaling mechanisms couple VGCC activation to skeletal {alpha}-actin promoter activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human IGF-I (Austral Biologicals, San Roman, CA) and factor of activated T-cells (NFAT)- and serum response factor (SRF)-sensor constructs (pGL3 luciferase construct) (Stratagene, La Jolla, CA) were purchased. Cyclosporine A (CsA) (calcineurin inhibitor), NiCl (T-type Ca2+channel inhibitor), and nifedipine (L-type Ca2+ channel inhibitor) were obtained from Sigma (St. Louis, MO). A 2.0-kb human skeletal {alpha}-actin promoter (pGL2 luciferase construct) [Dr. Steve J. Swoap, Williams College, Williamstown, MA) (18)], a constitutively active calcineurin (caCaN) construct (PM7 expression vector) (Dr. Grace K. Pavlath, Emory University, Atlanta, GA), and the dysgenic muscle cell line (Dr. Paul D. Allen, Harvard University, Boston, MA) (19) were all kind gifts. All other material was purchased from Fisher, unless otherwise stated.

Cell culture conditions
All cell culture experiments were performed using C2C12 mouse myoblasts (ATCC, Rockville, MD), which were maintained at a subconfluent density at 37 C in 10% CO2 in DMEM (Life Technologies, Rockville, MD) supplemented with 20% fetal bovine serum (Life Technologies) and 1% penicillin-streptomycin antibiotic (Life Technologies). Differentiation was induced by transferring the myoblasts to DMEM media containing 2% horse serum and 1% penicillin-streptomycin antibiotic. Dysgenic cells were maintained according to previously described methods of Rando and Blau (20).

Transient transfection of myoblasts
Transient transfections were performed with Lipofectamine PLUS reagent (Invitrogen, Carlsbad, CA) according to the manufacture’s directions and previously described methods (21, 22). Briefly, transfections were carried out in 24-well tissue culture plates (8–10 x 104 cells) with a total of 0.125 µg DNA per transfection in serum-free DMEM. All assays were preformed using equimolar ratios of DNA, and the total amount of DNA per transfection was adjusted using the pUC19 empty vector as previously described (22, 23). More specifically, when more than one vector (i.e. caCaN and NFAT luciferase or skeletal {alpha}-actin) was transfected, the total amount of DNA transfected remained at 0.125 µg; however, the ratio of reporter construct (0.065 µg) to signaling construct (0.065 µg) or empty plasmid (0.065 µg) was adjusted so that each condition for each experiment received equal amounts of DNA during transfection. After completion of the transfection, the culture media was changed to the described condition for each experiment. All solutions were changed every 24 h with each specific pharmacological agent, and IGF-I was added from sterilized stock solutions to the media before exposure to the cells. In all cases, the control-condition media was supplemented with the same volume amount of the specific dilution vehicle used for that specific agent. The duration of all the experiments was 72 h except for the experiment depicted in Fig. 1Go, in which the durations of time are described in the figure. In addition, basal- and IGF-I-induced differentiation had minimal effects [less than 500 relative light units (RLU)] on the luciferase activity of the empty pGL2 or pGL3 vectors (i.e. background activity) under any conditions (data not shown).



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 1. IGF-I increased transcriptional activity of the 2.0-kb human skeletal {alpha}-actin promoter in differentiating myoblasts. Subconfluent myoblasts were transfected with 0.125 µg of the 2.0-kb human skeletal {alpha}-actin promoter construct and induced to differentiate in differentiation media, with or without IGF-I, for varying durations of time. *, Statistically different from the 24- and 48-h control groups (P < 0.05). All activity is expressed as mean ± SE. Side panel: Representative example of formed myotubes after 72 h of differentiation with or without IGF-I; white arrows, formed myotubes. HS, Horse serum.

 
Cell lysis and luciferase measurements
Cell lysis and luciferase measurements were performed using the luciferase assay kit (Promega, Madison, WI), as previously described (22). Except for Fig. 1Go, all experiments were maintained in differentiation media for 72 h total, with the solutions renewed every 24 h. After 72 h, the cells were gently washed twice with PBS, cell lysis buffer was then added, and the plates were gently rocked for 15 min at 4 C. The homogenates were then transferred to a microcentrifuge tube and centrifuged at 13,000 rpm for 1 min. The supernatant was retained and stored at -80 C. All luciferase measurements were normalized to the total protein concentration of each well as previously described (21, 24). None of the conditions had any affect on the empty pGL2 plasmid, with IGF-I inducing less than 1000 RLU of activity from the empty plasmid compared with IGF-I inducing over 250,000 RLU on the skeletal {alpha}-actin plasmid. In addition, because IGF-I increases global protein synthesis in muscle (25), it was necessary to normalize all luciferase activity measures to the total protein content of each individual well. Therefore, we used the more conservative normalization method (i.e. total protein content) for all of our data. For all conditions, at least three independent experiments, each consisting of multiple wells, were performed.

Statistics
All values are means ± SE. All differences between groups were determined by using a one-way ANOVA. If the ANOVA revealed significant effects, Tukey’s post hoc test was applied. For all statistical tests, a 0.05 level was used as statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-I increases the transcriptional activity of the skeletal {alpha}-actin promoter in differentiating myoblasts
Initially, we determined whether IGF-I (250 ng/ml) increased the transcriptional activity of the human skeletal {alpha}-actin promoter in transiently transfected C2C12 myoblasts. After transfection, the myoblasts were subsequently induced to differentiate into myotubes with or without IGF-I for various durations. IGF-I significantly increased skeletal {alpha}-actin promoter activity after 48 h, compared with the control condition (Fig. 1Go). Skeletal {alpha}-actin promoter activity did not significantly increase in the control condition until after 72 h of differentiation, whereas IGF-I further enhanced actin promoter activity at the 72-h time point. Therefore, 72-h or greater incubation time points were selected for remaining experiments.

IGF-I enhances the skeletal {alpha}-actin promoter activity through the L-type Ca2+ VGCC
Using the same IGF-I concentration (250 ng/ml) as the current experiment, Semsarian et al. (16) found that IGF-I increased free intracellular Ca2+ levels in differentiating myoblasts by 76%, although the source of the increase in free Ca2+ was not readily distinguishable. However, other data have indicated that IGF-I can increase L-type VGCC channel activity and expression in skeletal muscle (26). Here we sought to determine whether increases skeletal {alpha}-actin gene transcription occurred through the L-type VGCC activation. Therefore, we simultaneously exposed control and IGF-I-stimulated differentiating myoblasts to varying concentrations of nifedipine, an L-type VGCC antagonist, for 72 h (Fig. 2AGo). Here, we demonstrate that, after 72 h, nifedipine (at known L-type VGCC inhibitory concentrations 0.1–100 µM) (27) prevented the IGF-I-induced activation of the skeletal {alpha}-actin promoter and maintained it at control levels. However, in non-IGF-I-stimulated differentiating myoblasts, only a very high concentration of nifedipine (100 µM) reduced activation levels of the skeletal {alpha}-actin promoter.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 2. L-type VGCC is necessary for IGF-I-induced stimulation of the 2.0-kb human skeletal {alpha}-actin promoter. Nifedipine (Nif) (0.1–100 µM) reduced IGF-I-stimulated 2.0-kb human skeletal {alpha}-actin promoter activities to control levels, but only 100 µM nifedipine reduced basal levels of promoter activation. Subconfluent myoblasts were transfected with 0.125 µg of the 2.0-kb human skeletal {alpha}-actin promoter construct and were induced to differentiate in differentiation media with (250 ng/ml) or without IGF-I and nifedipine for 72 h. #, Statistically different from the control group; *, different from the IGF-I without-nifedipine group (P < 0.05). All activity is expressed as mean ± SE. Side panel, Representative example of formed myotubes after 72 h of differentiation with 1 µM nifedipine in the presence or absence of IGF-I; white arrows, formed myotubes.

 
To further confirm the involvement of the L-type Ca2+ channel in IGF-I-induced stimulation of the skeletal {alpha}-actin promoter in differentiating myoblasts, a dysgenic myoblast cell line was employed. Dysgenic myoblasts were isolated from lethal muscular dysgenesis mice, which do not express the {alpha}1 subunit of the dihydropryridine (DHP) receptor (i.e. L-type VGCC) in skeletal muscle (28). Myotubes grown in culture from the dysgenic myoblasts lack both sarcolemmal ion charge movement associated with normal physiological voltage sensing (29) and the DHP-sensitive Ca2+ current (30). Interestingly, when stimulated, DHP receptors produce two different types of calcium components, slow and fast, that appear to have different physiological roles within the cell (31). Recent evidence indicates that the slow component is nifedipine sensitive, effects nucleoplasmic Ca2+ levels, and possibly gene transcription (31, 32), all of which is supportive of current findings. Here, dysgenic myoblasts were transiently transfected with the skeletal {alpha}-actin promoter and induced to differentiate with and without IGF-I. IGF-I failed to increase the activity of the skeletal {alpha}-actin promoter in dysgenic myoblasts above the level of the control as seen in wild-type myoblasts (data not shown), suggesting that L-type VGCC are required for IGF-I stimulation of {alpha}-actin gene transcription. However, the dysgenic cells did have high levels of basal, or non-IGF-I-induced, skeletal {alpha}-actin promoter activity, suggesting that the L-type VGCC is not necessary for basal levels of transcription, and would indicate the possible involvement of another VGCC. Additionally, the dysgenic cells were responsive to the exogenous IGF-I, because the total protein content of the IGF-I-stimulated wells was 43% greater than the control condition wells, indicating that the lack of a transcriptional response in the IGF-I condition is not due to a failure of the cells to respond to IGF-I.

To further confirm the involvement of the L-type VGCC, K+-induced depolarizations of myotubes were employed as previously described (31). Exposure of myotubes to high concentrations of K+ (30–50 mM) is known to induce membrane depolarization and increase intracellular Ca2+ levels through activation of L-type VGCC (31). K+-induced depolarization (30 and 45 mM) for 72 h (Fig. 3Go) increased skeletal {alpha}-actin promoter activity compared with the control condition, whereas the inclusion of IGF-I and K+ further significantly elevated promoter activity compared with the IGF-I-only condition. Thus, activation of the L-type VGCC, by two methods of stimulation (i.e. IGF-I and K+), increased the transcriptional activity of the skeletal {alpha}-actin promoter. Therefore, it appears that whereas the basal up-regulation of the skeletal {alpha}-actin gene expression during differentiation is not dependent upon L-type VGCC, activation of L-type VGCC, either by IGF-I or K+ depolarization, can increase skeletal {alpha}-actin gene expression.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 3. K+-induced depolarization of cultured myotubes increases the transcriptional activity of the skeletal {alpha}-actin promoter. Incubation of myotubes in media supplemented with increased levels of K+ (30–45 mM) increased skeletal {alpha}-actin promoter activities in the control and IGF-I conditions. Subconfluent myoblasts were transfected with 0.125 µg of the 2.0-kb human skeletal {alpha}-actin promoter construct and induced to differentiate in differentiation media with (250 ng/ml) or without IGF-I and with or without K+ for 72 h. *, Statistically different from the control group; #, different from the IGF-I without-K+ group (P < 0.05). All activity is expressed as mean ± SE.

 
Basal and IGF-I-stimulated skeletal {alpha}-actin promoter activity is dependent upon the T-type VGCC
Recent evidence indicates that T-type VGCC plays an integral role in human myoblast differentiation (11). Therefore, to determine whether the T-type VGCC could be contributing to the increase in skeletal {alpha}-actin promoter activity in differentiating myoblasts, the T-type VGCC inhibitor Ni2+ was used at various doses for 72 h (Fig. 4Go). At doses known to inhibit T-type VGCC (33), Ni+ (100 and 200 µM) abolished most basal activity of the skeletal {alpha}-actin promoter. In the IGF-I-stimulated cells, 50 µM Ni+ reduced skeletal {alpha}-actin promoter activity to control values, whereas 100 and 200 µM Ni+ abolished most of the promoter activity.



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 4. Inhibition of T-type VGCC with Ni+ reduces activation of the skeletal {alpha}-actin promoter under all conditions. Ni+ (100–200 µM) inhibited both basal- and IGF-I-stimulated skeletal {alpha}-actin promoter activities. Subconfluent myoblasts were transfected with 0.125 µg of the 2.0-kg human skeletal {alpha}-actin promoter construct and induced to differentiate in differentiation media in varying Ni+ concentrations with (250 ng/ml) or without IGF-I for 72 h. #, Statistically different from control group; *, different from the IGF-I without-Ni+ group (P < 0.05). All activity is expressed as mean ± SE. Side panel, Representative example of formed myotubes after 72 h of differentiation with or without IGF-I and 1 µM Ni+; white arrows, formed myotubes.

 
Calcineurin, but not NFAT, is a downstream regulator of the L-type VGCC of the skeletal {alpha}-actin promoter in differentiating myoblasts
The next purpose of the current study was to determine which signaling pathways are activated by the VGCC to increase the skeletal {alpha}-actin promoter activity in differentiating myoblasts. IGF-I has been shown to increase the activity of the Ca2+-mediated phosphatase, calcineurin, and induce NFAT nuclear translocation (16, 34). To confirm this, a reporter construct that contained four consensus NFAT transcription sites transactivating the luciferase reporter gene (NFAT-sensor construct) was transiently transfected into the myoblasts. After 72 h in differentiation media, IGF-I significantly increased NFAT transcriptional activity (Fig 5AGo). NFAT is a downstream target of calcineurin and has been implicated in the transcriptional activation of a number of muscle contractile genes (35, 36). Using CsA (5 µM), a known calcineurin inhibitor, IGF-I-induced activity of the NFAT reporter construct, was abolished, thereby implicating calcineurin in the activation of NFAT by IGF-I (Fig 5AGo). NFAT reporter gene activity served as an index of calcineurin activity as previously described (37, 38). The quantity 5 µM CsA was chosen because it completely abolished all IGF-I-induced NFAT transcriptional activity, whereas 1 µM only reduced IGF-I-induced transcriptional activity by 50% (data not shown). Further, 5 µM CsA has also been used in other investigations to inhibit calcineurin activity (34). To determine whether the VGCC-mediated activation of the skeletal {alpha}-actin gene occurred through activation of calcineurin and NFAT, we tested whether or not CsA inhibited activation of the skeletal {alpha}-actin promoter. Using the same concentration (5 µM) of CsA, which inhibited the IGF-I-induced activity of the NFAT reporter construct, it was found that simultaneous IGF-I- and K+-induced activation of the skeletal {alpha}-actin promoter activity was completely inhibited; whereas in the control condition, there was a minimal, but significant, inhibition of the promoter (Fig. 5BGo).



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 5. IGF-I increased NFAT and skeletal {alpha}-actin transcriptional activities through activation of a CsA-sensitive pathway. A, IGF-I stimulated luciferase activities transactivated by a 4x NFAT consensus-site promoter compared with the control condition. IGF-I-induced NFAT activity was inhibited after 72 h with 5 µM CsA in the media. Subconfluent myoblasts were transfected with 0.125 µg of the NFAT sensor promoter construct and induced to differentiate in differentiation media with (250 ng/ml) or without IGF-I in the presence or absence of CsA (5 µM) for 72 h. *, Statistically different from all groups (P < 0.05). All activity is expressed as mean ± SE. B, CsA (5 µM) prevented the activation of the skeletal {alpha}-actin promoter in differentiating myoblasts cultured in media supplemented with K+ in the presence (250 ng/ml) or absence of IGF-I. In the absence of IGF-I, means ± SE for control and CSA were 14559 ± 870 and 12939 ± 577, respectively (P = 0.0485). Subconfluent myoblasts were transfected with 0.125 µg of the 2.0-kb human skeletal {alpha}-actin promoter construct and induced to differentiate in differentiation media with or without CsA, IGF-I (250 ng/ml), and K+ for 72 h. *, Statistically different from the control without-K+ group; {dagger}, different from control with K+, but without CsA; #, different from the all groups (P < 0.05). All activity is expressed as mean ± SE.

 
The efficacy of a caCaN construct was verified by significantly increasing the activity of the NFAT-sensor construct, which was further enhanced by the inclusion of IGF-I (Fig. 6AGo). These data indicate that the caCaN was properly targeting specific substrates of the described calcineurin pathway (39). The caCaN was then cotransfected with the skeletal {alpha}-actin promoter, where caCaN induced a 154% increase in skeletal {alpha}-actin promoter activity compared with the control condition (Fig. 7BGo), implying that calcineurin may potentially regulate transcription of the skeletal {alpha}-actin gene during myoblast differentiation. Therefore, it was determined whether the caCaN construct could rescue inhibition of the skeletal {alpha}-actin promoter by nifedipine or Ni+. We found that caCaN prevented the inhibition of nifedipine in the presence of IGF-I on the skeletal {alpha}-actin promoter (Fig. 7AGo); however, caCaN surprisingly failed to rescue the Ni+-induced inhibition of the skeletal {alpha}-actin promoter under basal conditions (Fig 7BGo). Although these data suggest that IGF-I-induced activation of the skeletal {alpha}-actin promoter through the L-type VGCC is mediated by calcineurin, they also indicate that under basal conditions, calcineurin is not downstream of the T-type VGCC, and that another signaling pathway must contribute to basal activation of the promoter. Finally, NFAT transcriptional activation was determined under the conditions of myoblast differentiation during inhibition of L- or T-type VGCC. The NFAT-sensor construct was transiently transfected into the differentiating myoblasts, and its transcriptional activity was hypothesized to decrease in the presence of nifedipine and Ni+ with IGF-I. Surprisingly, nifedipine had no effect on NFAT transcriptional activity under basal- or IGF-I-induced conditions (Fig. 8Go). Further, Ni+ actually enhanced basal- and IGF-I-induced NFAT transcriptional activity (Fig. 8Go). These data suggest that calcineurin-mediated IGF-I-induced activation of the skeletal {alpha}-actin promoter through the L-type VGCC is via a transcription factor other than NFAT.



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 6. caCaN increases NFAT transcriptional activities in differentiating myoblasts. caCaN stimulated luciferase activities transactivated by a 4x NFAT consensus-site promoter compared with the control condition. Subconfluent myoblasts were cotransfected with 0.0625 µg of the NFAT sensor promoter construct and 0.0625 µg of the caCaN and then induced to differentiate in differentiation media with or without IGF-I for 72 h. *, Statistically different from the control group; #, different from the caCaN-only group (P < 0.05). All activity is expressed as mean ± SE.

 


View larger version (10K):
[in this window]
[in a new window]
  		FIG. 7. caCaN rescues skeletal {alpha}-actin transcriptional activity during L-type VGCC inhibition, but not T-type VGCC inhibition in differentiating myoblasts. A, caCaN recovered nifedipine-induced inhibition of the 2.0-kb human skeletal {alpha}-actin promoter activity compared with the nifedipine-IGF-I-only condition. Subconfluent myoblasts were cotransfected with 0.0625 µg of 2.0-kb human skeletal {alpha}-actin promoter construct and 0.0625 µg of caCaN and then induced to differentiate in differentiation media with IGF-I in the presence or absence of nifedipine (1 µM) for 72 h. *, Statistically different from IGF-I-only group; #, different from all groups (P < 0.05). All activity is expressed as mean ± SE. B, caCaN failed to recover Ni+-induced inhibition of 2.0-kb human skeletal {alpha}-actin promoter activity compared with the control and caCaN-only conditions. Subconfluent myoblasts were cotransfected with 0.0625 µg of the skeletal {alpha}-actin promoter construct and 0.0625 µg caCaN and then induced to differentiate in differentiation media with Ni+ (100 µM) for 72 h. *, Statistically different from control-only group; #, different from all groups (P < 0.05). All activity is expressed as mean ± SE.

 


View larger version (14K):
[in this window]
[in a new window]
  		FIG. 8. NFAT transcriptional activity is not inhibited by L-type or T-type VGCC inhibition in differentiating myoblasts. Neither basal- or IGF-I-stimulated luciferase activities transactivated by a 4x NFAT consensus-site promoter were inhibited by nifedipine (1 µM) or Ni+ (100 µm) exposure. Subconfluent myoblasts were transfected with 0.125 µg of the NFAT sensor promoter construct and then induced to differentiate in differentiation media with or without IGF-I and the designated VGCC inhibitors for 72 h. *, Statistically different from the control group; #, different from the control and IGF-I-only group (P < 0.05). All activity is expressed as mean ± SE.

 
Basal activation of the skeletal {alpha}-actin promoter may be mediated by SRF transcriptional activity
The necessity of two separate transcriptional pathways to drive the human skeletal {alpha}-actin promoter was initially suggested by Muscat et al. (23, 40), who found that the promoter contained two separate and distinct regulatory regions that interacted with different transactivating factors. SRF has been described by numerous reports to be a fundamental regulator of myoblast differentiation (41) and a transcriptional activator of the chicken skeletal {alpha}-actin gene (24, 42). Using a similar approach, as described above, we employed a SRF-sensor construct that contained four juxtaposed consensus serum response elements (SRE) able to transactivate a luciferase reporter construct when bound by SRF. First, we sought to determine whether SRE transcriptional activity was increased by IGF-I, as previously described for NFAT. Surprisingly, SRE transcriptional activity was decreased by IGF-I in differentiating myoblasts (Fig. 9Go). The SRE elements contained within this SRF-sensor construct are the same SRE elements in the c-FOS promoter (CCATATTAGG), which is indistinguishable from the CArG binding factor in the proximal element of the human skeletal {alpha}-actin promoter (43). These data suggest that IGF-I does not increase the activity of the skeletal {alpha}-actin promoter through subsequent activation of SRE alone.



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 9. SRE transcriptional activity is inhibited by IGF-I exposure in differentiating myoblasts. IGF-I (72 h) decreased luciferase activities transactivated by a 4x SRE consensus-site promoter in differentiating myoblasts. Subconfluent myoblasts were transfected with 0.125 µg of the SRE sensor promoter construct and then induced to differentiate in differentiation media with or without IGF-I. *, Statistically different from the control group (P < 0.05). All activity is expressed as mean ± SE.

 
Although IGF-I has been suggested to increase SRE transcriptional activity (24), this is the first measure of SRE-specific transcriptional activity in the presence of IGF-I. However, based upon prior published evidence (23, 24), it is very likely that SRF transcriptional activity is critical for the basal induction of the skeletal {alpha}-actin promoter in differentiating C2C12 myoblasts. Therefore, it was determined whether inhibition of the T- or L-type-VGCC had any affect on basal SRE transcriptional activity. Inhibition of T-type VGCC, with Ni+, reduced basal transcriptional activity of SRE (Fig. 10Go), whereas L-type VGCC inhibition had no effect (data not shown). These data indicate that basal regulation of the skeletal {alpha}-actin promoter through the T-type VGCC can be mediated by activation of SRE transcriptional activity.



View larger version (9K):
[in this window]
[in a new window]
  		FIG. 10. SRE transcriptional activity is inhibited by T-type VGCC inhibition in differentiating myoblasts. Ni+ (100 µM for 72 h) decreased luciferase activities transactivated by a 4x SRE consensus-site promoter in differentiating myoblasts. Subconfluent myoblasts were transfected with 0.125 µg of the SRE sensor promoter construct and then induced to differentiate in differentiation media. *, Statistically different from the control group (P < 0.05). All activity is expressed as mean ± SE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During myoblast differentiation, there is an up-regulation of contractile gene expression both in culture (5) and during development (3), which the environment can have a profound effect upon (9). For example, exogenous IGF-I accelerated the appearance and up-regulation of the skeletal {alpha}-actin mRNA during differentiation. The current study extends the earlier finding to show that during differentiation, IGF-I increases the promoter activity of the human skeletal {alpha}-actin gene through L-type VGCC and calcineurin signaling but not through downstream NFAT- or SRE-promoter activations. In addition, in the absence of IGF-I, the promoter activities of human skeletal {alpha}-actin and SRE approached zero when T-type VGCC were blocked, suggesting that, in contrast to IGF-I, basal levels of skeletal {alpha}-actin are signaled at least through the SRE.

IGF-I plays a fundamental role in myoblast differentiation during muscle development (9) and may influence muscle regeneration (44). The differentiation of cultured myoblasts requires extracellular Ca2+ (8, 10), and recent data indicate that VGCC may mobilize this extracellular Ca2+ (11, 12). IGF-I enhances the differentiation of cultured myoblasts (13), through activation of Ca2+-mediated mechanisms (16), which influences IGF-I’s potent effect on gene transcription (9). Further, IGF-I can increase L-type VGCC current, through phosphorylation of the L-type VGCC by protein kinase C (17). Insulin, which is structurally similar to IGF-I, can increase near-membrane free Ca2+ through activation of the L-type VGCC (45). The current study indicates that a downstream consequence of IGF-I activation of L-type VGCC in differentiating myoblasts is increased activation of the skeletal {alpha}-actin promoter, as evidenced by abolished IGF-I-induced promoter activation by inhibition or absence of L-type VGCC.

High extracellular K+ induces two types of Ca2+ signals, a fast- and slow-Ca2+ response, with each Ca2+ signal having a different suggested physiological role (31). Activation of the fast response is not abolished by nifedipine-induced inhibition of the L-type VGCC, and appears to be related to force production, whereas the slow response, which is inhibited by nifedipine blockage of the L-type VGCC, is thought to be more specific to signaling in the cell nucleus (46). Because our current observations show that an IGF-I-induced skeletal {alpha}-actin promoter activation is dependent upon a functional L-type VGCC, and others have reported that endogenous IGF-I increases during myoblast differentiation (47), the aforementioned information suggests that IGF-I induces transcriptional gene expression during myoblast differentiation through activation of L-type VGCC.

Porter et al. (12) reported that the L-type VGCC regulates the chicken skeletal {alpha}-actin promoter under non-IGF-I conditions or conditions analogous to the control group in this study. They found that 50 and 100 µM nifedipine, without IGF-I, reduced chick skeletal {alpha}-actin promoter activity in differentiating myoblasts, which confirms the current findings that 100 µM nifedipine reduced the activation levels in the control group. However, dosage levels of 100 µM nifedipine should be interpreted with caution because such concentrations of nifedipine have been suggested to be nonspecific to the L-type VGCC (27). In contrast, 10 µM nifedipine was reported to specifically abolish L-type VGCC activity in differentiated myoblasts (48), and 1–10 µM nifedipine was sufficient to reduce intercellular Ca2+ in C2C12 myotubes (12). Our data and Porter et al. (12) showed no inhibition of the skeletal {alpha}-actin promoter at these lower concentrations. Therefore, transcriptional regulation of the skeletal {alpha}-actin promoter under non-IGF-I-stimulated conditions is not likely regulated by the L-type VGCC and may be instead regulated by T-type VGCC activation.

Dysgenic myoblasts, which lack functional L-type VGCC but do express functional T-type VGCC (49), had activation of the skeletal {alpha}-actin promoter under control or nonexogenous IGF-I conditions (data not shown). In addition, dysgenic myoblasts differentiate and form normal myotubes (50). Finally, 100–200 µM Ni+ completely abolished skeletal {alpha}-actin promoter activity during differentiation, which suggests that the T-type VGCC is a significant contributor to skeletal {alpha}-actin gene transcription during myoblast differentiation. The involvement of T-type VGCC in myoblast differentiation is consistent with other reports (11). For example, T-type VGCC are expressed in embryonic and developing mouse skeletal muscle (51), with expression and function of the T-type VGCC declining 3 wk after birth (52). The {alpha}1H T-type VGCC is expressed in developing muscle, and a concentration of 100 µM Ni+ has been reported to block almost 90% of the {alpha}1H T-type current present in the developing muscle (51). Further, 200 µM Ni+ inhibits human myoblast fusion (11). Combined, these data suggest that the T-type VGCC is an important contributor to basal skeletal {alpha}-actin promoter activation during myoblast differentiation.

The rescue of nifedipine-induced inhibition of the IGF-I-induced activations of the L-type VGCC and skeletal {alpha}-actin promoter by a caCaN supports the notion that calcineurin is a downstream mediator of IGF-I-induced signaling through the L-type VGCC in differentiating myoblasts (Fig 7AGo). This interpretation is supported by the report indicating that activation of the L-type VGCC induces genomic signaling events (32). Consequently, it was determined whether IGF-I activation of the L-type VGCC resulted in activation of specific signaling mechanisms that may influence promoter activation of the skeletal {alpha}-actin gene. IGF-I increases free Ca2+ levels and activity of calcineurin, a calcium-sensitive phosphatase, in C2C12 myoblasts (16). Calcineurin affects gene transcription by removing phosphate groups from the serine/theronine residues on NFAT, allowing translocation of NFAT to the nucleus (39). IGF-I increased NFAT-driven promoter activity after 24 h (22) and continued to stimulate NFAT-driven promoter activity even after 72 h (Fig. 5AGo). IGF-induced NFAT activity is inhibited by the calcineurin inhibitor, CsA; and finally, Calcineurin induces transcriptional activity of various isoforms of the myosin heavy chain gene family in differentiating myoblasts (35, 36). These data suggest that IGF-I is, in fact, stimulating increased calcineurin activity to increase contractile gene expression in differentiating myoblasts.

Further, caCaN not only demonstrated that the skeletal {alpha}-actin promoter is a target of calcineurin in differentiating myoblasts (Fig. 6BGo), but also rescued the nifedipine-induced inhibition of the IGF-I-induced activation of the skeletal {alpha}-actin gene. These rescue data support the notion that calcineurin is a downstream mediator of IGF-I-induced signaling through the L-type VGCC in differentiating myoblasts.

Interestingly, the data imply that NFAT is not a downstream substrate of calcineurin to the skeletal {alpha}-actin promoter, because nifedipine failed to reduce NFAT transcriptional activity in the presence of IGF-I, whereas nifedipine or CsA both blocked skeletal {alpha}-actin promoter activity in the presence of IGF-I. Although unexpected, the finding that NFAT may not be the primary transcriptional factor between calcineurin and the skeletal {alpha}-actin promoter in differentiating myoblasts, it is not without precedence. More specifically, Friday et al. (8) found that calcineurin was required for myoblast differentiation, however did not use NFAT during differentiation. Recent data indicate that a complex interaction of MyoD and myocyte enhancer factor-2 (MEF2), a known calcineurin substrate, may be responsible for calcineurin-induced expression of myogenin (53). Interestingly, the distal element (-1300 to -626) of the human skeletal {alpha}-actin promoter, which, if deleted, induces a 10-fold decrease in activity in C2C12 myotubes, contains an A/T-rich-like element (54). MEF2-stimulated transcription is mediated by binding of MEF2 to the A/T rich element (55). Finally, Delling et al. (56) also found that IGF used calcineurin to induce slow myosin expression, but this mechanism was independent of NFAT. Our findings agree that calcineurin is responsible for inducing part of the differentiation process through a NFAT-independent signaling to the skeletal {alpha}-actin promoter.

SRF has been implicated in activation of the skeletal {alpha}-actin gene in differentiating myoblasts (24) and in regenerating skeletal muscle (44). The human skeletal {alpha}-actin promoter contains a CArG box (SRE) located in its proximal region (-153 to -87), which is essential to expression in C2C12 myotubes (23). Deletion of the entire proximal region reduces promoter activity in differentiating C2C12 myoblasts from 6.5-fold to 1.1-fold (40). Here we describe, for the first time, that, after 72 h of incubation, IGF-I inhibits SRE-driven reporter gene activity in differentiating myoblasts. Extrapolation of the latter observation to the endogenous skeletal {alpha}-actin promoter suggests that it is unlikely that the endogenous SRE promoter could be activated by IGF-I alone. However, SRE seems to play a clearer role under basal (non-IGF-I) conditions, because blockage of T-type VGCC with Ni+ reduced both SRE- and skeletal {alpha}-actin-driven promoter activities. Further, SRF transcriptional activity may be regulated in a Ca2+-dependent manner, potentially through a Ca2+/camodulin-dependent kinase (57). Thus, under the conditions employed here, SRE is an important regulatory element of basal skeletal {alpha}-actin promoter activity downstream of T-type VGCC.

In summary, the current findings delineate an IGF-I requirement for L-type VGCC to activate the human skeletal {alpha}-actin promoter in C2C12 myoblasts (Fig. 11Go). Intermediate IGF-I-induced signaling steps include calcineurin, but further IGF-I-downstream signaling is not via consensus NFAT- or SRE-activated transcription of a reporter gene (Fig. 11Go). On the other hand, basal activity of the skeletal {alpha}-actin promoter involves T-type VGCC-initiated activation. The requirement of VGCC for human skeletal {alpha}-actin promoter activity implies a role for Ca2+ regulation in basal- and IGF-I-regulated skeletal {alpha}-actin expression.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 11. Potential model for the role of basal- and IGF-I-stimulated increases in skeletal {alpha}-actin transcriptional activity in differentiating myoblasts. Basal-stimulated activity of the skeletal {alpha}-actin promoter is induced by SRF-mediated transcriptional activity that is up-regulated by T-type VGCC activity as determined from its activation of consensus SRE. One key CArG binding site (*) exists in the proximal binding element of the human skeletal {alpha}-actin promoter (23 ). The upstream signaling molecule activated by the T-type VGCC that is potentially inducing SRF activity remains unclear, but is not calcineurin. IGF-I-induced activity of the skeletal {alpha}-actin promoter is activated by calcineurin signaling, which is mediated by the L-type VGCC. Activation of the L-type VGCC by IGF-I appears to occur through increased activation of protein kinase C by IGF-I (17 ). However, the transcription factor by which calcineurin is mediating the promoter activity is not NFAT and is potentially another transcription factor. MEF2 may be the potential transcription factor, in that in the distal region of the skeletal {alpha}-actin promoter, there is an A/T rich element (*), which is a known binding site for MEF2 (55 ).

 


   Acknowledgments
 
The authors thank Drs. Shuichi Machida, Chris W. Ward, Grace K. Pavlath, Kurt G. Beam, and Chris L. Heaps for helpful and thoughtful discussions on various portions of these experiments. The authors also thank Dr. Steve Swoap (Williams College) for the kind gift of the 2.0-kb human skeletal {alpha}-actin promoter, Dr. Grace K. Pavlath (Emory University) for the kind gift of the caCaN, and Dr. Paul Allen (Harvard University) for the kind gift of the dysgenic cell line.


   Footnotes
 
This work was supported by NIH Grants AR48514 (to E.E.S., fellowship), AR19393 (to F.W.B.), and HL52940 (to D.K.B.).

Current address for E.E.S.: University of California, Exercise Biology Program, One Shields Avenue, Davis, California 95616. E-mail: eespangenburg{at}ucdavis.edu

Abbreviations: caCaN, Constitutively active calcineurin; CsA, cyclosporine A; DHP, dihydropryridine; MEF2, myocyte enhancer factor-2; NFAT, nuclear factor of activated T-cells; RLU, relative light units; SRE, serum response element(s); SRF, serum response factor; VGCC, voltage-gated Ca2+ channel(s).

Received October 31, 2003.

Accepted for publication December 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Seale P, Asakura A, Rudnicki MA 2001 The potential of muscle stem cells. Dev Cell 1:333–342[CrossRef][Medline]
  2. Schultz E, McCormick KM 1994 Skeletal muscle satellite cells. Rev Physiol Biochem Pharmacol 123:213–257[Medline]
  3. Wade R, Kedes L 1989 Developmental regulation of contractile protein genes. Annu Rev Physiol 51:179–188[CrossRef][Medline]
  4. Coolican SA, Samuel DS, Ewton DZ, McWade FJ, Florini JR 1997 The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways. J Biol Chem 272:6653–6662[Abstract/Free Full Text]
  5. Gunning P, Hardeman E, Wade R, Ponte P, Bains W, Blau HM, Kedes L 1987 Differential patterns of transcript accumulation during human myogenesis. Mol Cell Biol 7:4100–4114[Abstract/Free Full Text]
  6. Myer A, Wagner DS, Vivian JL, Olson EN, Klein WH 1997 Wild-type myoblasts rescue the ability of myogenin-null myoblasts to fuse in vivo. Dev Biol 185:127–138[CrossRef][Medline]
  7. Andres V, Walsh K 1996 Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis. J Cell Biol 132:657–666[Abstract/Free Full Text]
  8. Friday BB, Horsley V, Pavlath GK 2000 Calcineurin activity is required for the initiation of skeletal muscle differentiation. J Cell Biol 149:657–666[Abstract/Free Full Text]
  9. Florini JR, Ewton DZ, Coolican SA 1996 Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev 17:481–517[Abstract/Free Full Text]
  10. Shainberg A, Yagil G, Yaffe D 1969 Control of myogenesis in vitro by Ca2+ concentration in nutritional medium. Exp Cell Res 58:163–167[CrossRef][Medline]
  11. Bijlenga P, Liu JH, Espinos E, Haenggeli CA, Fischer-Lougheed J, Bader CR, Bernheim L 2000 T-type {alpha}1H Ca2+ channels are involved in Ca2+ signaling during terminal differentiation (fusion) of human myoblasts. Proc Natl Acad Sci USA 97:7627–7632[Abstract/Free Full Text]
  12. Porter Jr GA, Makuck RF, Rivkees SA 2002 Reduction in intracellular calcium levels inhibits myoblast differentiation. J Biol Chem 277:28942–28947[Abstract/Free Full Text]
  13. Florini JR, Ewton DZ, Roof SL 1991 Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression. Mol Endocrinol 5:718–724[Abstract/Free Full Text]
  14. Adi S, Cheng ZQ, Zhang PL, Wu NY, Mellon SH, Rosenthal SM 2000 Opposing early inhibitory and late stimulatory effects of insulin-like growth factor-I on myogenin gene transcription. J Cell Biochem 78:617–626[CrossRef][Medline]
  15. Tureckova J, Wilson EM, Cappalonga JL, Rotwein P 2001 Insulin-like growth factor-mediated muscle differentiation: collaboration between phosphatidylinositol 3-kinase-Akt-signaling pathways and myogenin. J Biol Chem 276:39264–39270[Abstract/Free Full Text]
  16. Semsarian C, Wu MJ, Ju YK, Marciniec T, Yeoh T, Allen DG, Harvey RP, Graham RM 1999 Skeletal muscle hypertrophy is mediated by a Ca2+-dependent calcineurin signalling pathway. Nature 400:576–581[CrossRef][Medline]
  17. Delbono O, Renganathan M, Messi ML 1997 Regulation of mouse skeletal muscle L-type Ca2+ channel by activation of the insulin-like growth factor-1 receptor. J Neurosci 17:6918–6928[Abstract/Free Full Text]
  18. Corin SJ, Levitt LK, O’Mahoney JV, Joya JE, Hardeman EC, Wade R 1995 Delineation of a slow-twitch-myofiber-specific transcriptional element by using in vivo somatic gene transfer. Proc Natl Acad Sci USA 92:6185–6189[Abstract/Free Full Text]
  19. Knudson CM, Chaudhari N, Sharp AH, Powell JA, Beam KG, Campbell KP 1989 Specific absence of the {alpha}1 subunit of the dihydropyridine receptor in mice with muscular dysgenesis. J Biol Chem 264:1345–1348[Abstract/Free Full Text]
  20. Rando T, Blau H 1994 Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol 125:1275–1287[Abstract/Free Full Text]
  21. Spangenburg EE, Booth FW 2002 Multiple signaling pathways mediate LIF-induced skeletal muscle satellite cell proliferation. Am J Physiol Cell Physiol 283:C204–C211
  22. Vyas DR, Spangenburg EE, Abraha TW, Childs TE, Booth FW 2002 GSK-3ß negatively regulates skeletal myotube hypertrophy. Am J Physiol Cell Physiol 283:C545–C551
  23. Muscat GE, Gustafson TA, Kedes L 1988 A common factor regulates skeletal and cardiac {alpha}-actin gene transcription in muscle. Mol Cell Biol 8:4120–4133[Abstract/Free Full Text]
  24. Wei L, Zhou W, Wang L, Schwartz RJ 2000 ß(1)-integrin and PI 3-kinase regulate RhoA-dependent activation of skeletal {alpha}-actin promoter in myoblasts. Am J Physiol Heart Circ Physiol 278:H1736–H1743
  25. Davis TA, Fiorotto ML, Burrin DG, Vann RC, Reeds PJ, Nguyen HV, Beckett PR, Bush JA 2002 Acute IGF-I infusion stimulates protein synthesis in skeletal muscle and other tissues of neonatal pigs. Am J Physiol Endocrinol Metab 283:E638–E647
  26. Wang ZM, Messi ML, Renganathan M, Delbono O 1999 Insulin-like growth factor-1 enhances rat skeletal muscle charge movement and L-type Ca2+ channel gene expression. J Physiol (Lond) 516:331–341[Abstract/Free Full Text]
  27. McDonald TF, Pelzer S, Trautwein W, Pelzer DJ 1994 Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol Rev 74:365–507[Free Full Text]
  28. Flucher BE, Phillips JL, Powell JA 1991 Dihydropyridine receptor {alpha}-subunits in normal and dysgenic muscle in vitro: expression of {alpha}1 is required for proper targeting and distribution of {alpha}2. J Cell Biol 115:1345–1356[Abstract/Free Full Text]
  29. Adams BA, Tanabe T, Mikami A, Numa S, Beam KG 1990 Intramembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs. Nature 346:569–572[CrossRef][Medline]
  30. Beam KG, Knudson CM, Powell JA 1986 A lethal mutation in mice eliminates the slow calcium current in skeletal muscle cells. Nature 320:168–170[CrossRef][Medline]
  31. Jaimovich E, Reyes R, Liberona JL, Powell JA 2000 IP(3) receptors, IP(3) transients, and nucleus-associated Ca(2+) signals in cultured skeletal muscle. Am J Physiol Cell Physiol 278:C998–C1010
  32. Powell JA, Carrasco MA, Adams DS, Drouet B, Rios J, Muller M, Estrada M, Jaimovich E 2001 IP(3) receptor function and localization in myotubes: an unexplored Ca(2+) signaling pathway in skeletal muscle. J Cell Sci 114:3673–3683
  33. Perez-Reyes E 2003 Molecular physiology of low-voltage-activated t-type calcium channels. Physiol Rev 83:117–161[Abstract/Free Full Text]
  34. Musaro A, McCullagh KJ, Naya FJ, Olson EN, Rosenthal N 1999 IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1. Nature 400:581–585[CrossRef][Medline]
  35. Allen DL, Sartorius CA, Sycuro LK, Leinwand LA 2001 Different pathways regulate expression of the skeletal myosin heavy chain genes. J Biol Chem 276:43524–43533[Abstract/Free Full Text]
  36. Allen DL, Leinwand LA 2002 Intracellular calcium and myosin isoform transitions. Calcineurin and calcium-calmodulin kinase pathways regulate preferential activation of the IIa myosin heavy chain promoter. J Biol Chem 277:45323–45330[Abstract/Free Full Text]
  37. Parsons SA, Wilkins BJ, Bueno OF, Molkentin JD 2003 Altered skeletal muscle phenotypes in calcineurin A{alpha} and Aß gene-targeted mice. Mol Cell Biol 23:4331–4343[Abstract/Free Full Text]
  38. Rusnak F, Mertz P 2000 Calcineurin: form and function. Physiol Rev 80:1483–1521[Abstract/Free Full Text]
  39. Rao A, Luo C, Hogan PG 1997 Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol 15:707–747[CrossRef][Medline]
  40. Muscat GE, Kedes L 1987 Multiple 5'-flanking regions of the human {alpha}-skeletal actin gene synergistically modulate muscle-specific expression. Mol Cell Biol 7:4089–4099[Abstract/Free Full Text]
  41. Wei L, Zhou W, Croissant JD, Johansen FE, Prywes R, Balasubramanyam A, Schwartz RJ 1998 RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation. J Biol Chem 273:30287–30294[Abstract/Free Full Text]
  42. Carson JA, Schwartz RJ, Booth FW 1996 SRF and TEF-1 control of chicken skeletal {alpha}-actin gene during slow-muscle hypertrophy. Am J Physiol 270:C1624–C1633
  43. Boxer LM, Prywes R, Roeder RG, Kedes L 1989 The sarcomeric actin CArG-binding factor is indistinguishable from the c-fos serum response factor. Mol Cell Biol 9:515–522[Abstract/Free Full Text]
  44. Marsh DR, Carson JA, Stewart LN, Booth FW 1998 Activation of the skeletal {alpha}-actin promoter during muscle regeneration. J Muscle Res Cell Motil 19:897–907[CrossRef][Medline]
  45. Bruton JD, Katz A, Westerblad H 1999 Insulin increases near-membrane but not global Ca2+ in isolated skeletal muscle. Proc Natl Acad Sci USA 96:3281–3286[Abstract/Free Full Text]
  46. Araya R, Liberona JL, Cardenas JC, Riveros N, Estrada M, Powell JA, Carrasco MA, Jaimovich E 2003 Dihydropyridine receptors as voltage sensors for a depolarization-evoked, IP3R-mediated, slow calcium signal in skeletal muscle cells. J Gen Physiol 121:3–16[Abstract/Free Full Text]
  47. Tollefsen SE, Lajara R, McCusker RH, Clemmons DR, Rotwein P 1989 Insulin-like growth factors (IGF) in muscle development. Expression of IGF-I, the IGF-I receptor, and an IGF binding protein during myoblast differentiation. J Biol Chem 264:13810–13817[Abstract/Free Full Text]
  48. Weigl LG, Hohenegger M, Kress HG 2000 Dihydropyridine-induced Ca2+ release from ryanodine-sensitive Ca2+ pools in human skeletal muscle cells. J Physiol (Lond) 525:461–469[Abstract/Free Full Text]
  49. Garcia J, Beam KG 1994 Calcium transients associated with the T type calcium current in myotubes. J Gen Physiol 104:1113–1128[Abstract/Free Full Text]
  50. Flucher BE, Phillips JL, Powell JA, Andrews SB, Daniels MP 1992 Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle, in vivo and in vitro. Dev Biol 150:266–280[CrossRef][Medline]
  51. Berthier C, Monteil A, Lory P, Strube C 2002 {alpha}(1H) mRNA in single skeletal muscle fibres accounts for T-type calcium current transient expression during fetal development in mice. J Physiol (Lond) 539:681–691[Abstract/Free Full Text]
  52. Beam KG, Knudson CM 1988 Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle. J Gen Physiol 91:799–815[Abstract/Free Full Text]
  53. Friday BB, Mitchell PO, Kegley KM, Pavlath GK 2003 Calcineurin initiates skeletal muscle differentiation by activating MEF2 and MyoD. Differentiation 71:217–227[CrossRef][Medline]
  54. Muscat GE, Perry S, Prentice H, Kedes L 1992 The human skeletal {alpha}-actin gene is regulated by a muscle-specific enhancer that binds three nuclear factors. Gene Expr 2:111–126[Medline]
  55. Black BL, Olson EN 1998 Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu Rev Cell Dev Biol 14:167–196[CrossRef][Medline]
  56. Delling U, Tureckova J, Lim HW, De Windt LJ, Rotwein P, Molkentin JD 2000 A calcineurin-NFATc3-dependent pathway regulates skeletal muscle differentiation and slow myosin heavy-chain expression. Mol Cell Biol 20:6600–6611[Abstract/Free Full Text]
  57. Davis FJ, Gupta M, Camoretti-Mercado B, Schwartz RJ, Gupta MP 2003 Calcium/calmodulin-dependent protein kinase activates serum response factor transcription activity by its dissociation from histone deacetylase, HDAC4: implications in cardiac muscle gene regulation during hypertrophy. J Biol Chem 278:20047–20058[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 DevelopmentHome page
S. Konig, A. Beguet, C. R. Bader, and L. Bernheim
The calcineurin pathway links hyperpolarization (Kir2.1)-induced Ca2+ signals to human myoblast differentiation and fusion
Development, August 15, 2006; 133(16): 3107 - 3114.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
E. E. Spangenburg, D. A. Brown, M. S. Johnson, and R. L. Moore
Exercise increases SOCS-3 expression in rat skeletal muscle: potential relationship to IL-6 expression
J. Physiol., May 1, 2006; 572(3): 839 - 848.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. E. Spangenburg
SOCS-3 Induces Myoblast Differentiation
J. Biol. Chem., March 18, 2005; 280(11): 10749 - 10758.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
Y.-C. Huang, R. G. Dennis, L. Larkin, and K. Baar
Rapid formation of functional muscle in vitro using fibrin gels
J Appl Physiol, February 1, 2005; 98(2): 706 - 713.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Spangenburg, E. E.
Right arrow Articles by Booth, F. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Spangenburg, E. E.
Right arrow Articles by Booth, F. W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals