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GROWTH, DIFFERENTIATION, AND APOPTOSIS
1School of Kinesiology and Health Science, York University, Toronto, Ontario, Canada; and Departments of 2Internal Medicine and 3Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas
Submitted 8 June 2005 ; accepted in final form 30 August 2005
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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satellite cell; myoblast; stem cell; transcriptional regulation; RGK protein
We previously undertook microarray analysis to define the global molecular events that characterize discrete stages of muscle regeneration (4). On the basis of these studies, we identified a Ras-related protein, termed Rad (Ras associated with diabetes), which was robustly expressed early (6–12 h) after skeletal muscle injury. Rad is the prototypical member of a novel class of 35- to 39-kDa Ras-related GTPases. Rad, as well as other members of this Ras-related GTPase protein subfamily (RGK family members Gem, Kir, Ges, and Rem1/2), possess a highly conserved 88-amino acid NH2-terminal extension and a 31-amino acid carboxy terminus that lacks the CAAX-like prenylation motif typical of other Ras proteins (20).
Rad was initially identified because its expression was upregulated in the skeletal muscle of some patients with type 2 diabetes mellitus (20), but it has since been documented to be dysregulated in patients with Duchenne muscular dystrophy (15) and in the amputated limbs of the newt (24). Rad is expressed most highly in the heart, lung, and skeletal muscle of humans and also has been demonstrated to exist in the C2C12 muscle cell line (1, 18, 20).
Although a number of biochemical studies characterizing Rad have been performed (11, 12, 30), the biological function of Rad was not fully understood until recently. Rad, as well as other members of the RGK protein subfamily, has been observed to play key roles in the regulation of intracellular Ca2+ dynamics and the remodeling of cell shape. In particular, Rad has been demonstrated to inhibit the activity of L-type Ca2+ channels by interacting with the 
-subunit (3) and to reduce Rho/RhoA kinase (ROK) signaling by inhibiting ROK-mediated phosphorylation (28). In the present study, we have defined the expression profile of Rad during mammalian development and muscle regeneration, and, for the first time, the key myogenic factors responsible for the transcriptional regulation of Rad within skeletal muscle have been identified.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Tissue preparations for in situ hybridization. Acute injury was induced in the gastrocnemius muscles of adult (2–4 mo old) wild-type mice using a 150-µl intramuscular injection of 10 µM cardiotoxin (Naja nigricollis; Calbiochem, La Jolla, CA) (6). The gastrocnemius muscles of these mice were harvested at 6 or 12 h or at 1, 2, 5, 7, or 14 days postinjury. To determine the expression of Rad in tissues undergoing chronic degeneration-regeneration cycles, we harvested the gastrocnemius muscles and diaphragms from adult mdx mice. To assess the spatiotemporal expression of Rad during development, embryos were harvested from timed C57BL/6 pregnant female mice at 8.5, 11.5, and 15.5 days postcoitum.
Embryos and tissues were fixed overnight in 4% paraformaldehyde/diethyl pyrocarbonate-PBS after administration of avertin-induced anesthesia and transcardiac perfusion. Paraformaldehyde-fixed tissue was paraffin embedded for rotary microtomy.
Cardiac injury. After they were under continuous anesthesia with 2.0% isoflurane, mice underwent a left thoracotomy/pericardiotomy to expose the left ventricle. To induce cardiac muscle damage, either the left anterior descending coronary artery was ligated with 8-0 Prolene sutures (ligation injury) or a frozen, 3-mm-diameter metal probe cooled to –190°C using liquid nitrogen immersion was applied to the left ventricular free wall between the left anterior descending coronary artery and the posterolateral branch for a period of 2–4 s (cryoinjury). The residual pneumothorax was evacuated to restore negative pressure, and buprenorphine (0.05 mg/kg body wt) was administered for postoperative pain control. For each of the protocols (ligation or cryoinjury), mice were euthanized and their hearts excised at day 0 (no injury) or at 1, 3, 7, or 14 days postinjury. Total RNA was harvested, and RT-PCR analysis was performed as described below.
Riboprobe synthesis.
An I.M.A.G.E. clone (no. 850142; I.M.A.G.E. Consortium) was verified using sequence analysis and prepared for in vitro transcription after restriction enzyme digestion and gel isolation. Linearized template (500 ng) was transcribed using either the T7 or SP6 RNA polymerase (Ambion, Austin, TX) with 7.0 µM [
-35S]UTP (1,000 Ci/mmol; Amersham, Piscataway, NJ) to produce the respective sense and antisense riboprobes. The Rad riboprobe was 
900 bp in length for sense and antisense probes. The probe was purified using MicroSpin G-50 columns (Amersham), analyzed for integrity, and stored at –80°C overnight.
In situ hybridization. In situ hybridization technique was performed as previously described (23). Briefly, 5-µm paraffin sections were cut, mounted on Vectabond-coated slides, dewaxed, permeabilized, acetylated, and hybridized at 70°C. Riboprobes were diluted in 50% formamide, 0.07 M NaCl, 20 mM Tris·HCl (pH 8.0), 5 nM EDTA (pH 8.0), 10 mM NaPO4 (pH 8.0), 10% dextran sulfate, 1x Denhardt's solution, and 0.5 mg/ml transfer RNA. After hybridization, the slides were rinsed with increasing stringency washes, treated with RNase A (2 µg/ml, 30 min at 37°C), dehydrated, and dipped in K5 nuclear emulsion gel (Polysciences, Warrington, PA). Autoradiographic exposure was undertaken for a 21-day period. The sections were counterstained with hematoxylin and examined using bright- and dark-field optics (23). In all cases, sections hybridized with the sense probe resulted in the absence of signal.
Northern blot analysis.
The Strip-EZ DNA probe synthesis kit (Ambion), Rad cDNA (50 ng), and 5 µl of [-32P]dATP (1,000 Ci/mmol; Amersham) were used to produce the cDNA probe. Adult tissue and embryo Northern blot analysis membranes (Ambion) were incubated with the Rad probe in ULTRAhyb solution (Ambion) at 42°C overnight. After being rinsed, hybridized membranes were exposed to radiographic film at –80°C for various lengths of time (12–48 h). A -actin probe was used as a control.
Cell culture. Primary myogenic progenitor cells were harvested from hindlimb skeletal muscle of wild-type neonatal (2 day old) mice (6, 7). For RT-PCR analysis, the mRNA was harvested from these cells after 5 days in culture.
C2C12 myogenic cells were grown as monolayers in DMEM supplemented with 20% FBS and antibiotics. This cell line was used for transient transfection of Rad promoter-reporter constructs and various myogenic transcription factors. For RT-PCR analysis, myotube differentiation was promoted by exposing 80% confluent myoblast cultures to differentiation medium (7).
Immunohistochemical analysis of regenerating skeletal muscle. Immunohistochemical detection for Rad and MyoD was undertaken on frozen sections of regenerating skeletal muscle (10 days postinjury). Sections were blocked with appropriate normal sera, and endogenous peroxidase activity was quenched by incubating sections in 0.6% hydrogen peroxide/methanol. Sections were incubated overnight at 4°C with the polyclonal rabbit anti-Rad antiserum (1:50 dilution; generous gift from R. Kahn) and detected using Texas red-conjugated secondary antibody (1:25 dilution). Coexpression of MyoD was performed by incubating the above sections with the MAb MyoD (1:500 dilution; BD Biosciences) for 1 h at room temperature and detected using a FITC-conjugated secondary antibody. All negative control sections (PBS substituted for primary antibody) had an absence of signal.
Construction of Rad promoter-reporter plasmid. A 2-kb 5' flanking region of the mouse Rad gene and a 5' deletion promoter fragment were generated by performing PCR from bacterial artificial chromosome clone RPCI-23. The 5' deletion of the 2-kb 5' flanking region generated a promoter fragment 708 bp (0.7 kb) in length. The PCR products were ligated into the PCR2.1 vector using the TA cloning kit (Invitrogen, Carlsbad, CA), digested from the PCR2.1 vector using restriction enzymes BamHI and XhoI, and ligated to the luciferase gene in the linearized pGL3Basic vector (XhoI and BglII; Promega, Madison, WI).
Transient transfection assays.
For transient DNA transfection, 2 x 105 C2C12 cells per well were added to six-well plates and allowed to adhere overnight. After 24 h, cells were transiently transfected with the Rad promoter-reporter vector and either empty vector (pCI-neo) or overexpression vectors for MyoD (0.5 µg), Myf-5 (0.5 µg), myocyte enhancer factor (MEF)2C (0.75 µg), or constitutively active calcineurin A (0.5 µg of pEMSV-MyoD, pEMSV-Myf-5, pCDNA-MEF2C, or pCI-Neo-CnA) using LipofectAMINE Plus lipid reagent and OptiMEM serum-free medium (Invitrogen) according to the manufacturer's protocols. Twenty-four hours after transfection, cells were rinsed and harvested using lysis buffer (Promega). Luciferase activity was measured in cell lysates using a luciferase assay system (Promega) according to the supplier's instructions. Transfection efficiencies were normalized by cotransfecting cells with pCMV-lacZ (0.1 µg) and measuring -galactosidase expression.
Each experiment was performed in triplicate, and a dose-response curve (using the 2-kb Rad promoter-reporter) was performed to determine the optimal DNA concentration for each of the MyoD, Myf-5, and MEF2C overexpression plasmids. The concentration of each overexpression plasmid that provided the greatest luciferase expression from the 2-kb promoter was used in subsequent studies.
Semiquantitative RT-PCR analyses. Total RNA was isolated with TriPure isolation reagent (Roche Applied Science, Basel, Switzerland) and was reverse transcribed with SuperScript II (Invitrogen) to cDNA. Semiquantitative RT-PCR (using 1:10 and 1:50 dilutions of cDNA) was performed under conditions in which the abundance of each amplified cDNA varied linearly with input RNA. Semiquantitative RT-PCR was performed using cDNA (1 µl) as a template for PCR in a 25-µl reaction volume, including 40 ng of each primer, 1.5 mM MgCl2, 0.2 mM 2-deoxynucleotide 5'-triphosphate, 2.5 µl of 10x Taq buffer, and 2.5 U of Taq polymerase (Invitrogen). Thermocycling conditions were 1) 96°C for 2 min, 2) 96°C for 15 s, 3) 62°C for 30 s, 4) 72°C for 30 s, and 5) 72°C for 10 min. Steps 2–4 were repeated for 22 cycles (18S rRNA) or 27 cycles (Rad). Primers for semiquantitative RT-PCR were Rad forward primer, 5'-AAGACGGACCTGAAGCAGAA-3', Rad reverse primer, 5'-CAAGGGCAGCTTTGAGAAAG-3'; and 18S rRNA forward primer, 5'-GGACCAGAGCGAAAGCATTTA-3', 18S rRNA reverse primer, 5'-TGCCAGAGTCTCGTTCGTTAT-3'.
Microscopy and photomicrography. Rad mRNA expression was visualized using a Leitz Laborlux-S microscope equipped with PlanApochromatic optics, a standard bright-field condenser, and a Mears low-magnification dark-field condenser. Photomicrographs were obtained with an Optronics VI-470 charge-coupled device camera and a Macintosh G3 Power computer using Scion Image 1.62 software.
Data analysis. Student's t-tests were performed to identify significant differences (P < 0.05) in data obtained from control and experimental samples. Data are presented as means ± SE.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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In situ hybridization analysis of regenerating skeletal muscle sections localized the expression of Rad to the periphery of the injured myofibers at early time points (<24 h) after chemically induced injury (Fig. 3, A, B, E, and F). These areas correspond to the location of the myogenic progenitor cells. On the basis of the expression of Rad within the primary myogenic progenitor cells using RT-PCR and the lack of expression within polymorphonuclear cells (infiltrating neutrophils; Fig. 3, C and D) or in other non-muscle cell types (Fig. 1), we suggest that the early expression of Rad during skeletal muscle regeneration is localized to the myogenic progenitor cell population. At 5 and 7 days after injury, Rad expression was observed in some newly regenerated myofibers as defined by their centrally located nuclei (Fig. 3, G and H). Because myogenic progenitor cells fuse together to form myofibers in response to extensive muscle damage, the expression of Rad within the newly regenerated myofibers at 5 and 7 days postinjury is consistent with our hypothesis that the early expression of Rad is localized to the myogenic progenitor cells. Immunohistochemical analysis of regenerating skeletal muscle (10 days postinjury; Fig. 3, I–K) demonstrated that Rad-positive cells coexpressed the myogenic factor MyoD, confirming that the Rad-positive cells observed within regenerating skeletal muscle were myogenic in nature. Myopathic mouse skeletal and diaphragmatic muscle from the mdx mouse also displayed focal regions of Rad expression consistent with the chronic cycles of degeneration and regeneration that characterize this mouse model (Fig. 4).
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The myogenic regulatory factor family of transcription factors bind to a canonical sequence (CANNTG), termed the E-box, on the promoter regions of key muscle-specific genes. The 2-kb promoter region of Rad contains two evolutionarily conserved E-box motifs (total of 4 E-boxes). Although truncation of the 2-kb Rad promoter (to create the 0.7-kb promoter construct) removed the two evolutionarily conserved E-boxes (Fig. 5A), the capacity of MyoD or Myf-5 to transactivate the Rad promoter was not reduced, indicating that the remaining E-box located within the 0.7-kb Rad promoter was capable of activating Rad transcription in response to MyoD and Myf-5 transcription factors (Fig. 5B).
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MyoD, Myf-5, or MEF2C overexpression in the COS-7 fibroblast cell line was ineffective in increasing Rad promoter-reporter luciferase expression. This suggests that factors within C2C12 muscle cells, but not in COS-7 cells, were necessary for these transcription factors to function effectively on the Rad promoter (data not shown).
There are two nuclear factor of activated T cell (NFAT) binding motifs located within the 2-kb Rad promoter with the proximal site demonstrating evolutionary conservation (Fig. 5A). Truncation of the 2-kb Rad promoter to generate the 0.7-kb promoter construct removed these NFAT binding motifs. Overexpression of activated calcineurin was capable of transactivating both the 2- and 0.7-kb Rad promoter constructs (Fig. 5C), although the increase observed in association with the 0.7-kb Rad promoter fragment was significantly lower than that observed in association with the 2-kb construct. Because calcineurin has the capacity to act through the transcription factors MyoD and MEF2, the effects of co-overexpressing activated calcineurin and these myogenic transcription factors in the presence of Rad promoter constructs was investigated. As shown in Fig. 5C, overexpression of activated calcineurin with any of the myogenic factors alone or in combination was capable of significantly increasing luciferase expression in the 2-kb Rad promoter construct above that observed with activated calcineurin alone, with no difference between any of the myogenic factors (MyoD, Myf-5, or MEF2C) regarding their capacity to increase Rad promoter activity in the presence of calcineurin. This increased promoter activity with overexpression of activated calcineurin and the myogenic factors was not observed upon truncation to the 0.7-kb Rad promoter construct (Fig. 5C), suggesting that the NFAT sites may be critical for the transcriptional regulation of Rad in response to the myogenic regulatory factors and MEF2.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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The present study has demonstrated that known myogenic transcription factors can increase the transcriptional activity of the Rad promoter and that this ability is significantly enhanced by activated calcineurin and the presence of a conserved NFAT binding motif within the Rad promoter. On the basis of the current findings, we propose that Rad might play a dual role in the regulation of the myogenic progenitor cell during skeletal muscle regeneration. The early, robust expression of Rad within the myogenic progenitor cell is consistent with the role of Rad in the inhibition of L-type Ca2+ channels and thereby the prevention of myoblast differentiation (3, 19), whereas the later expression of Rad within newly regenerated myofibers results in the downregulation of Rho/ROK signaling, which is necessary for myoblast fusion and subsequent terminal differentiation (14, 28)
Developmental expression of Rad is limited to muscle lineages.
As observed in the day E15.5 embryo, Rad expression is localized to the muscle lineages, with the heart, diaphragm, intercostals, tongue, esophageal smooth muscle, and back musculature all exhibiting high signal intensity (Fig. 1). The lack of expression within the nonmuscle lineages of the developing embryo is consistent with our finding that key muscle regulatory factors are involved in the transcriptional control of Rad expression. In contrast to the developing embryo and adult human skeletal muscle (18, 20), Rad demonstrates low-level expression in resting adult mouse skeletal muscle similar to that observed in the newt homolog of Rad (nRad), which displays 68% homology to the mouse and human Rad genes (24). Although Rad expression is low in resting skeletal muscle, this level is dramatically increased during skeletal muscle regeneration. We speculate that within skeletal muscle, Rad might play an analogous role during development and regeneration. This assumption is based on the observation that the events associated with development in which muscle precursor cells proliferate and fuse to form muscle fibers recapitulate, in part, events that occur during skeletal muscle regeneration (5).
Rad expression in regenerating myocardium. An increase in nRad expression has been observed in the regenerating newt heart after injury (24). Within normal adult mouse myocardium, the high level of Rad expression would suggest an important role for Rad in cardiomyocyte function. This hypothesis is supported by the finding that a nonsynonymous, single-nucleotide polymorphism within the Rad gene has been linked to congestive heart failure (8). Despite an increase in Rad expression after skeletal muscle injury (present study) or myocardial injury in the newt (24), neither ligation nor cryoinjury-induced myocardial damage produced a detectable increase in Rad expression in the present study. Interestingly, like mammalian myocardium, the cardiomyocytes of the adult newt ventricle are relatively quiescent; however, unlike its mammalian counterpart, removal of the apical ventricular tissue in the newt results in extensive DNA synthesis and mitosis in the vicinity of the wound, ultimately resulting in cardiac regeneration (16). On the basis of the currently proposed roles for Rad, we surmise that the presently unknown function of Rad within the mammalian cardiomyocyte may be distinct from its role within regenerating skeletal muscle.
Expression of Rad during skeletal muscle regeneration. The early expression of Rad after muscle damage in the present study corroborates the findings of Shimizu-Nishikawa et al. (24), who observed expression of nRad at 1 and 3 days after cold-induced injury in the newt. Similar to the expression of Rad in the myogenic progenitor cells of regenerating muscle in the present study, the expression of nRad in response to cryoinjury was due to infiltrating pericytes (which corresponded to mammalian myogenic progenitor cells).
Using cDNA microarrays, Noguchi et al. (15) previously demonstrated upregulation of Rad expression within the skeletal muscle of patients with Duchenne muscular dystrophy. Consistent with their results, we have demonstrated that dystrophic skeletal and diaphragmatic muscle from the mdx mouse displays discrete regions of Rad expression. The continued expression of Rad within the myopathic muscles of the mdx mouse (which demonstrates chronic cycles of degeneration and regeneration) suggests that Rad expression is necessary for skeletal muscle regeneration and that its upregulation in the present study is not an artifact of cardiotoxin injection.
Increased Rad expression in the myogenic progenitor cells after skeletal muscle damage (Fig. 3) is consistent with the role of Rad in the inhibition of L-type Ca2+ channel activity within myogenic progenitor cells (3). Porter et al. (19) found that inhibition of L-type Ca2+ channels resulted in a decrease in intracellular Ca2+ levels and inhibition of myoblast differentiation into mature myotubes. The attenuation of myoblast differentiation through increased Rad expression could allow for an increased proliferative potential. This hypothesis is supported by the findings reported in other studies in which stable expression of Rad within a breast cancer cell line resulted in increased cell cycle progression and tumorigenic potential (27) and in which the inhibition of the Rad family member Gem (60% homology to Rad) prevented the proliferation of 3T3 cells (9).
Although the expression of Rad was less pronounced on the basis of RT-PCR and microarray analyses during the later stages of regeneration, in situ hybridization analysis clearly revealed Rad expression within newly regenerated myofibers (Fig. 3). The reduced detection of Rad expression using microarray and RT-PCR analyses may be due in part to the diffuse time period in which newly regenerated myofibers were formed and to the time course of Rad expression, in which not all newly regenerated myofibers may express Rad at the time of muscle harvest. The observation of Rad expression within newly regenerated myofibers is supported by the finding of increased Rad expression within C2C12 myoblasts exposed to differentiation conditions (Fig. 2E).
The expression of Rad in newly regenerated myofibers and differentiating C2C12 myoblasts may seem paradoxical to its expression during the early phases of regeneration. However, in support of a role for Rad during myoblast fusion and differentiation, Rad has been shown to regulate the Rho/ROK signaling cascade negatively (28), allowing for nuclear translocation of the forkhead in human rhabdomyosarcoma transcription factor and ultimately myoblast fusion (14). Furthermore, Zhu et al. (30) demonstrated an interaction of Rad with the structural protein -tropomyosin, supporting its role in cell cytoskeletal organization and skeletal muscle differentiation.
Taken together, we propose that Rad might play a dual role within the myogenic progenitor cell population during skeletal muscle regeneration. Our hypothesis is that, initially, Rad prevents myogenic progenitor cell differentiation through inhibition of L-type Ca2+ channel activity. This function would be distinct from its role during the later stages of regeneration, when Rad would inhibit the Rho/ROK signaling cascade, thus allowing for myogenic progenitor cell fusion and ultimately terminal differentiation.
Transcriptional regulation of Rad. On the basis of the localization of Rad expression to the muscle lineage during development and regeneration, we investigated the capacity of myogenic factors known to be important in skeletal muscle regeneration to regulate the transcriptional activity of Rad.
The myogenic regulatory factors MyoD and Myf-5 bind a canonical binding motif known as an E-box and are associated with the regulation of the myogenic progenitor cell population in response to muscle damage (2, 21, 26). Although both MyoD and Myf-5 are important in the activation and proliferation of myogenic progenitor cells, they exhibit distinct expression profiles during skeletal muscle regeneration. On the basis of the microarray analysis results, MyoD displays a temporal expression pattern that parallels that of Rad, whereas Myf-5 is low during the early regenerative phase and is increased during the later stages in which myogenic progenitor cells undergo fusion to regenerate the myofiber population (4). These data suggest that MyoD may be involved in the early regulation of Rad transcription in response to injury, whereas Myf-5 may aid in the transcription of Rad during the later phases of muscle regeneration.
The MADS box (MCM1, agamous deficiens, and serum response factor) transcription factor family member MEF2 has been shown to play a key role in skeletal muscle differentiation (17). Preliminary studies conducted in our laboratory using MEF2 indicator mice (13, 29) confirmed a high level of MEF2 protein expression during the later stages of muscle regeneration in which newly regenerated myofibers were visible (5–10 days postinjury; data not shown). The late expression of MEF2 during skeletal muscle regeneration suggests that this transcription factor is involved in the regulation of Rad expression during myogenic progenitor cell differentiation.
In response to skeletal muscle injury, calcineurin protein expression is upregulated as early as 1 day postinjury and remains elevated for up to 14 days. In addition, the NFATc1 downstream target of calcineurin also displayed an increase in activated (dephosphorylated) protein levels from 1 to 6 days postinjury (22). Whether calcineurin or NFATc1 expression was increased before 1 day was not assessed in the study, however.
The coexpression of activated calcineurin with the other myogenic transcription factors resulted in Rad promoter activity levels that were far greater than expected as an additive effect. This robust increase in Rad promoter activity was dependent on the presence of the 2-kb Rad promoter. We suggest that this increase was dependent on the presence of a conserved NFAT binding motif within the 2-kb Rad promoter, although the reasons for this phenomenon have yet to be elucidated. Although it is conceivable that calcineurin increased the affinity of Myf-5 and MyoD for the evolutionarily conserved E-boxes that are removed in the 0.7-kb promoter construct, this hypothesis would not explain the results observed in association with coexpression of calcineurin and MEF2C.
Conclusion. In summary, we have demonstrated the expression pattern and transcriptional regulation of Rad, a prototypic member of the RGK family of novel Ras-related GTPases, during mammalian development and skeletal muscle regeneration. The expression pattern of Rad during skeletal muscle regeneration is consistent with its proposed role in attenuating myogenic progenitor cell fusion during the initial period after injury and its role in promoting terminal differentiation during the later stages of regeneration. We have also demonstrated that myogenic transcription factors (MEF2, MyoD, and Myf-5) that are expressed during skeletal muscle regeneration can increase the transcriptional activity of the Rad promoter and that this ability is significantly enhanced by the presence of activated calcineurin. Furthermore, this enhanced transcriptional activity appears to be dependent on the presence of a conserved NFAT binding motif within the Rad promoter. Together, these data define Rad as a novel factor within the skeletal muscle regenerative process. Future studies will aid in further elucidation of the role and regulation of this novel protein.
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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 T. J. Hawke, D. J. Atkinson, S. B. Kanatous, P. F. M. Van der Ven, S. C. Goetsch, and D. J. Garry Xin, an actin binding protein, is expressed within muscle satellite cells and newly regenerated skeletal muscle fibers Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1636 - C1644. [Abstract] [Full Text] [PDF]
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