Journal of Applied Physiology Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 102: 1483-1489, 2007. First published January 4, 2007; doi:10.1152/japplphysiol.01147.2006
8750-7587/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
102/4/1483    most recent
01147.2006v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Trenerry, M. K.
Right arrow Articles by Cameron-Smith, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Trenerry, M. K.
Right arrow Articles by Cameron-Smith, D.

STAT3 signaling is activated in human skeletal muscle following acute resistance exercise

Marissa K. Trenerry,1 Kate A. Carey,1 Alister C. Ward,2 and David Cameron-Smith1

1School of Exercise and Nutrition Science and 2School of Life and Environmental Sciences, Deakin University, Burwood, Victoria, Australia

Submitted 12 October 2006 ; accepted in final form 22 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
The transcription factor signal transducer and activator of transcription 3 (STAT3) has been identified as a mediator of cytokine signaling and implicated in hypertrophy; however, the importance of this pathway following resistance exercise in human skeletal muscle has not been investigated. In the present study, the phosphorylation and nuclear localization of STAT3, together with STAT3-regulated genes, were measured in the early recovery period following intense resistance exercise. Muscle biopsy samples from healthy subjects (7 males, 23.0 + 0.9 yr) were harvested before and again at 2, 4, and 24 h into recovery following a single bout of maximal leg extension exercise (3 sets, 12 repetitions). Rapid and transient activation of phosphorylated (tyrosine 705) STAT3 was observed at 2 h postexercise. STAT3 phosphorylation paralleled the transient localization of STAT3 to the nucleus, which also peaked at 2 h postexercise. Downstream transcriptional events regulated by STAT3 activation peaked at 2 h postexercise, including early responsive genes c-FOS (800-fold), JUNB (38-fold), and c-MYC (140-fold) at 2 h postexercise. A delayed peak in VEGF (4-fold) was measured 4 h postexercise. Finally, genes associated with modulating STAT3 signaling were also increased following exercise, including the negative regulator SOCS3 (60-fold). Thus, following a single bout of intense resistance exercise, a rapid phosphorylation and nuclear translocation of STAT3 are evident in human skeletal muscle. These data suggest that STAT3 signaling is an important common element and may contribute to the remodeling and adaptation of skeletal muscle following resistance exercise.

signal transducer and activator of transcription-3; inflammation; regeneration; hypertrophy

INTENSE MUSCULAR ACTIVITY, particularly eccentric muscular contractions, results in a rapid and localized muscle inflammatory response (5). In the minutes to hours of recovery, a diverse range of proinflammatory cytokines is released by the damaged muscle fibers, and resident neutrophils are rapidly mobilized and activated (30, 42). Typical of the actions of intense exercise is the marked increase in IL-6 (43), which signals via a heterodimer of IL-6 receptor (IL-6R) and glycoprotein 130 (gp130). gp130 is a common element of signal transduction events for a diverse range of cytokine receptors, including those for IL-11, IL-27, leukemia inhibitory factor (LIF), cardiotropin-1, ciliary neurotrophic factor, and oncostatin M (39), which all signal through a common receptor, gp130 (16). gp130 receptor binding activates the signal transducers and activators of transcription (STATs) family via the Janus family of tyrosine kinases (JAKs) (8, 18). Among the seven members of the STAT family (STAT1–4, 5A, 5B, and 6), STAT3 is activated by a wide spectrum of cytokines that signal via the cytokine type II receptor ligands (including IL-6, IL-10, and prolactin), as well as a diverse range of growth factors [including IGF-I, hepatocyte growth factor, epidermal growth factor, PDGF, and basic fibroblast growth factor (16)], ANG II, and mechanical stretch (Rho A) (20, 32, 41) The heterogeneity of signals that may mediate signaling via STAT3 activation suggests that STAT3 is a common and important element to the regenerative responses initiated in the recovery period after intense physical activity.

Activation and phosphorylation of STAT3 elicits dimerization and translocation to the nucleus where it participates in the regulation of many target genes. Subcellular localization of STAT3 in skeletal muscle has been identified in rats following muscle crush injury (21). However, the activation of the STAT3 signaling pathway in human skeletal muscle in response to a remodeling stimulus such as resistance exercise has not been investigated. Importantly, STAT3 activation may be a significant regulator of the transcriptional control of myofibers in the adaptation and recovery following resistance exercise. First, within proliferating satellite cells, the resident pluripotent stem cell population located within the muscle bed, STAT3 activation is evident (21, 36). Second, within many cell types, STAT3 is important in cell proliferation, differentiation, and survival by mediating the expression of the cell cycle regulators c-myc, cyclinD1 (23, 27), the antiapoptotic genes Bcl-2 and Bcl-XL (23, 27, 46), intermediate early response genes such as c-fos and junB (33), and the angiogenic factor vascular endothelial growth factor (VEGF) (7). These diverse actions have been described in cardiac myocytes, where STAT3 activation mediates cardiac hypertrophy and protection in response to cardiomyopathy induced by ischemia or drug treatment (24, 25).

Therefore, in the present study we investigated the activation of STAT3 signaling following an acute bout of heavy resistance exercise in healthy male volunteers. It was hypothesized that the STAT3 protein would be phosphorylated during the early recovery period following exercise and that this would be accompanied by the subsequent transcriptional activation of STAT3-responsive genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects.   Seven untrained but recreationally active male subjects volunteered to participate in this study. The mean (±SE) age, height, weight, and body mass index of the subjects were 23.0 ± 0.9 yr, 1.78 ± 0.04 m, 76.7 ± 4.5 kg, and 24.1 ± 0.1 kg/m2, respectively. Exclusion criteria included resistance training within the past 6 mo, medications, or a previous history of a diagnosed condition or illness that would endanger the subjects during strenuous resistance exercise. Informed written consent was obtained from each subject before participation in the study and after the nature, purpose, and risks of the study were explained. All experimental procedures were performed in accordance with the Helsinki declaration and were formally approved by the Deakin University Human Research Ethics Committee.

Familiarization.   Each subject completed a familiarization session on a Cybex NORM dynamometer (Cybex International) to become familiar with the execution of the exercise. The familiarization session involved assessment of isokinetic maximal voluntary contraction (MVC) during a knee extension-flexion exercise. MVC and peak torque (N·m) were determined at 60°/s over 12 maximal repetitions during concentric and eccentric phases. Subjects were instructed to maximally push up and contract and were verbally encouraged throughout the test. The familiarization session took place at least 7 days before the trial to prevent any residual effects of the strength test.

Experimental design.   For the 24 h preceding exercise, and the day of the trial, subjects consumed a standard diet (20% fat, 14% protein, and 66% carbohydrate) and abstained from alcohol, caffeine, tobacco, and additional exercise. On the morning of the trial, subjects presented to the laboratory in the fasted state. Following 30 min of supine resting, a muscle sample was collected from the vastus lateralis under local anesthesia (1% Xylocaine) by percutaneous needle biopsy technique (2) modified to include suction (11). Excised muscle tissue from each biopsy was immediately frozen and stored in liquid nitrogen for later analysis. Following this, subjects completed an acute bout of concentric and eccentric isokinetic leg extension exercise. All exercise was completed using the dominant limb on a Cybex NORM dynamometer at a constant speed of 60°/s. Subjects completed three sets of 12 repetitions of maximal single-legged knee extension exercise with 2 min of rest between each set. The system provided visual graphical feedback for both the subject and the investigator. Subjects were instructed to push up and contract maximally and were verbally encouraged to do so throughout each set. Further muscle samples were collected from the exercised leg at 2 and 4 h after the exercise. Subjects were provided with lunch (30 min following the 4 h muscle biopsy) and dinner (at 1800 for all subjects) on trial day. The following morning, subjects again reported to the laboratory in a fasted state for the collection of a final muscle sample 24 h postexercise. To minimize the potential for interference, serial biopsy samples were collected at least 2 cm from previous biopsy sites.

Protein extraction and Western blot analysis.   Tissue samples were homogenized in lysis buffer (20 mM Tris · HCl, 5 mM EDTA, 10 mM Na-pyrophosphate, 100 mM NaF, 2 mM Na3VO4, 1% Igepal, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 3 mM benzamidine, 1 mM PMSF) using a handheld homogenizer. The homogenate was rotated at 4°C for 1 h, then centrifuged at 13,000 rpm at 4°C for 10 min, and the supernatant was collected. Protein samples (50 µg) were denatured in sample buffer and separated by 8% SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane and blocked in 5% (wt/vol) BSA in Tris-buffered saline with 0.1% (vol/vol) Tween 20 (TBST) for 2 h at room temperature. Primary antibodies diluted in blocking buffer were applied and incubated overnight at 4°C. Membranes were subsequently washed with TBST and incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies before being washed again. Proteins were visualized by enhanced chemiluminescence (Western Lighting Chemiluminescence Reagent Plus, Perkin-Elmer, Boston, MA). The density of the bands was quantified using Kodak Imaging software, Kodak ID 3.5 (Perkin Elmer Life Sciences, Boston MA). The positive control for pSTAT3 was protein extracted from human vastus lateralis cells, treated with the cytokine IL-6 (M. K. Trenerry, unpublished observations). The positive control for phospho-STAT5 (pSTAT5) was protein extracted from human 293T cells and cotransfected with a hyperactive JAK2a and STAT5 for zebra fish (26). Total-STAT3 (no. 9132) and phospho-STAT3 (tyrosine 705) (no. 9131) antibodies were purchased from Cell Signaling Technology (Danvers, MA), total-STAT5 from BD Biosciences (no. 610191; San Jose, CA). and pSTAT5 (tyrosine 694) (no. 71–6900) from Zymed Laboratories (San Francisco, CA). Anti-mouse (no. 31430, Quantum Scientific) and anti-rabbit (no. AP342P, Chemicon) HRP-conjugated secondary antibodies were used.

Immunofluorescence.   A small section from each biopsy was dissected free of any connective tissue, blotted dry, and then mounted in TissueTek (ProSciTech) and frozen in isopentane cooled in liquid nitrogen. Tissue sections (10 µm) were fixed with 4% (wt/vol) paraformaldehyde in PBS for 10 min, permeated with 5% (vol/vol) Triton X-100 for 5 min, and blocked in 3% (wt/vol) BSA in PBS overnight at 4°C. Primary antibodies were diluted in blocking reagent [as follows: anti-pSTAT3 1:100 and anti-myosin heavy chain slow isoform (A4.840) 1:50 (Developmental Studies Hybridoma Bank)] and left overnight at 4°C. After four washes with PBS, sections were incubated at room temperature for 2 h with appropriate secondary antibody-coupled fluorophores, Alexa Fluor goat anti-mouse 488 (no. A21202 [GenBank] ) or donkey anti-rabbit 594 (no. A21207 [GenBank] ) (Molecular Probes). Nuclei were stained by incubating with the DNA binding dye bisbenzimide (Hoechst 33285) (Sigma, St. Louis, MO) for 10 min, followed by extensive washing. Immunostained sections were visualized with an Olympus IX70 fluorescent microscope, and digital images were collected using Spot RT slider camera and Magnifire Software (Olympus). Digitally captured images (x200 magnification) with four fields of view per muscle cross section (30 ± 1 fibers per field of view) were processed using ImageJ software (NIH Image). The total number of type I fibers was 12 ± 0 (per field of view) and type II fibers was 18 ± 2 (per field of view). Muscle nuclei were selected based on bisbenzamide staining (74 ± 4 per field of view).

RNA extraction and RT-PCR.   Total cellular RNA was extracted using a modification of the phenol-chloroform extraction and isopropanol precipitation protocol, using the ToTALLY RNA Kit (Ambion, Austin, TX). RNA quality and concentration were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). First-strand cDNA was generated from 0.5 µg total RNA using the AMV RT kit (Promega, Madison, WI). RT-PCR was performed using the GenAmp 7500 sequence detection system (Applied Biosystems, Foster City, CA). PCR was performed in duplicate with reaction volumes of 20 µl, containing SYBR/green 1 (Applied Biosystems), forward and reverse primers, and cDNA template (diluted 1:20). Data were analyzed using a comparative critical threshold (Ct) method where the amount of target normalized to the amount of endogenous control relative to control value is given by 2Formula (Applied Biosystems). The efficacy of GAPDH as endogenous controls was examined using the equation 2Formula. No changes in the expression of this gene were observed (data not shown), so it was considered an appropriate endogenous control for this study. Primers were designed using Primer Express software package version 3.0 (Applied Biosystems) from gene sequences obtained from GenBank (see Table 1 for details). Primers were designed spanning intron-exon boundaries to prevent amplification of the target region for any contaminating DNA. Primer sequence specificity was also confirmed using Basic Local Alignment Search Tool (BLAST). A melting point dissociation curve was generated by the PCR instrument for all PCR products to confirm the presence of a single amplified product.


View this table:
[in this window]
[in a new window]

  		Table 1. Details of primers used for real-time PCR analysis

 
Statistical analysis.   Statistical analysis was performed using GraphPad Prism 4.1 (GraphPad Software, San Diego, CA). Means were compared using one-way ANOVA, and any significant differences were analyzed using a Newman-Keuls multiple comparison test. Data are presented as means ± SE. A probability level of P < 0.05 was adopted throughout to determine statistical significance unless otherwise stated.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
STAT3 is phosphorylated in skeletal muscle following acute resistance exercise.   To examine STAT3 activation in skeletal muscle, STAT3 phosphorylation (pSTAT3) at tyrosine 705 was examined in vastus lateralis samples from human subjects following a single bout of resistance exercise. At rest, pSTAT3 was undetectable in skeletal muscle (Fig. 1A). By 2 h postexercise, pSTAT3 had increased significantly before returning to near resting levels by 4 h. To confirm that the increased phosphorylation observed was not due to an increase in STAT3 protein levels, the density of the pSTAT3 band was normalized against total STAT3 protein, which remained constant across the samples.


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 1. Activation of signal transducer and activator of transcription 3 (STAT3) in human skeletal muscle following acute resistance exercise. A: representative Western blots of protein extracted from samples taken at rest and 2, 4, or 24 h postexercise, with the inclusion of a positive control, probed with anti-phospho-STAT3 (Tyr705) (pSTAT3) or stripped and reprobed with anti-total STAT3 (tSTAT3). Phosphorylation of STAT3 increased significantly 2 h following an acute bout of exercise. The arrow indicates the pSTAT3 band at 86 kDa. The graph shows arbitrary units of pSTAT3 normalized to tSTAT3 representing the mean ± SE of 7 individuals. **Significantly different from resting value (P < 0.01). B: representative Western blots of the same samples as in A, with the inclusion of a positive control sample probed with anti-pSTAT5 (Tyr694). No phosphorylation of STAT5 was observed in skeletal muscle samples at any time point.

 
STAT5 phosphorylation is not detectable in skeletal muscle following exercise.   We next sought to determine if STAT3 is preferentially phosphorylated in muscle following exercise by examining the activation of another member of the STAT family, STAT5. Interestingly, while STAT5 protein was expressed in skeletal muscle (Fig. 1B), there was no detectable phosphorylation at any time point following exercise, although it was readily detectable in a positive control sample.

Phosphorylated STAT3 translocates to the nucleus postexercise.   Phosphorylated STAT3 is able to form homo- and heterodimers and translocates to the nucleus, where it can activate the expression of various target genes. To further explore STAT3 signaling events in skeletal muscle following exercise, the cellular localization of pSTAT3 protein was examined in muscle tissue sections collected at rest, as well as 2, 4, and 24 h postexercise. Immunohistochemical analysis revealed overwhelming translocation of pSTAT3 from within the muscle fibers to the nucleus 2 h after exercise (Fig. 2B), which again returned to resting levels by 4 h. Double immunostaining for pSTAT3 protein and the myosin heavy chain slow protein isoform showed that cytoplasmic pSTAT3 was preferentially localized to the type 2 (fast) fibers (Fig. 2A).


Figure 2
View larger version (96K):
[in this window]
[in a new window]

  		Fig. 2. Nuclear translocation of pSTAT3 following acute resistance exercise. Thin (10-µm) sections of skeletal muscle biopsy samples taken at rest (A) and 2 h (B), 4 h (C), or 24 h (D) following exercise were analyzed with anti-pSTAT3 antibodies (Tyr705) and anti-myosin heavy chain (slow isoform) [MHC(I)] to examine the cellular localization of activated STAT3 and fiber type distribution [pSTAT3 is shown in red using the Alexa Fluor 594 while MHC(I) is shown in green using the Alexa Fluor 488]. The degree of translocation was examined by comparing the percentage of pSTAT3-positive nuclei to total nuclei (E), which increased significantly 2 h postexercise. Bars represent means ± SE of 7 individuals. ***Significantly different from resting values (P < 0.001).

 
STAT3-responsive genes are transcriptionally activated following acute exercise.   We next sought to determine if the increased activation of STAT3 signaling resulted in a concomitant increase in the expression of potential downstream genes c-FOS, JUNB, VEGF, and c-MYC (Fig. 3). Interestingly, marked increases in expression were observed for all genes investigated, although differences in magnitude and timing were evident. Significant increases in the expression of the early response genes c-FOS and JUNB (800-fold and 38-fold, respectively) were observed at 2 h postexercise. This increased expression appeared to be largely transient, with a marked reduction evident by 4 and 24 h. Similarly, an increase in c-MYC expression peaked at 2 h postexercise (140-fold) before returning toward resting levels at 4 and 24 h. In comparison, VEGF mRNA expression increases were slightly delayed and of a smaller magnitude, with a fourfold increase observed at 4 h post-resistance exercise.


Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 3. Increased expression of STAT3 target genes following exercise. mRNA for c-FOS (A), JUNB (B), VEGF (C), and c-MYC (D) were examined in the skeletal muscle of young healthy males at rest or following an acute bout of resistance exercise. Values are arbitrary units normalized to the expression levels of the housekeeping gene GAPDH representing the mean ± SE for 7 individuals. Significantly different from resting levels: *P < 0.05, **P < 0.01.

 
Genes associated with STAT3 signaling are transiently upregulated following acute resistance exercise.   Last, we sought to investigate the expression of several genes implicated in the regulation of STAT3 signaling (Fig. 4). IL-6 and LIF, two cytokines that strongly activate STAT3, demonstrated marked transcriptional activation (85-fold and 120-fold, respectively) at 2 h following exercise. However, because of considerable between-subject variability, the change in LIF failed to reach statistical significance. The expression of IL-6R, through which IL-6 acts, was subsequently investigated. Although no significant changes were observed, a trend toward increased expression was evident at 4 h postexercise (8-fold, P = 0.08). Finally, we investigated the expression of suppressor of cytokine signaling 3 (SOCS3), a well-established negative regulator of STAT3 signaling. Interestingly, the expression of this gene was also markedly increased (60-fold) 2 h after exercise.


Figure 4
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 4. Altered expression of genes modulating STAT3 signaling following exercise. Expression of mRNA for IL-6 (A), IL-6 receptor (IL-6R; B), leukemia inhibitory factor (LIF; C), and suppressor of cytokine signaling 3 (SOCS3; D) were examined as described in Fig. 3. Significantly different from resting levels: **P < 0.01, ***P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGMENTS
 REFERENCES
 
Skeletal muscle remodeling in response to resistance exercise shares common features with many of the signaling events regulating the regenerative processes following muscle injury. Growth factors and cytokines are central mediators of repair and adaptation in skeletal muscle (42, 45), although the exact signaling pathways activated have yet to be fully elucidated. STAT3 signaling has been identified as a possible mediator of skeletal muscle regeneration (21), but the importance of this pathway in the adaptive response of human skeletal muscle to resistance exercise has not been investigated.

The results of the present study demonstrate that an acute bout of resistance exercise in young healthy males rapidly and transiently activates STAT3 signaling in human skeletal muscle. Phosphorylation of STAT3 at tyrosine 705 was undetectable in resting muscle but was markedly increased sevenfold within 2 h following a single session of strenuous leg extension exercise. Tyrosine phosphorylation of STAT3 at this site residue is essential for the formation of homo- or heterodimers, nuclear translocation, and subsequent STAT3-dependent increases in the transcriptional activity of responsive genes (9). Corresponding to the increased phosphorylation was the demonstration of increased nuclear accumulation of the phosphorylated STAT3 to the nucleus (10-fold) at 2 h postexercise. Interestingly, double labeling for myosin heavy chain I isoforms revealed preferential localization of cytoplasmic phosphorylated STAT3 to the fast (type II myosin heavy chain isoform) fibers. It is possible that greater STAT3 protein is resident within fast fibers or that a greater STAT3 activation is present within these fibers. Further analysis is then required to more adequately describe localization and mechanisms of activation of STAT3 following exercise.

To further examine the significance of STAT signaling in skeletal muscle, we also investigated the activation of another member of the STAT family, STAT5. This has been shown to be activated in skeletal muscle cells by growth hormone (12) and insulin (34), but its activation following resistance exercise has not been described. In contrast to STAT3, we observed no detectable level of STAT5 phosphorylation within skeletal muscle following exercise. While it is conceivable that STAT5 activation occurred outside the time points examined, these results more likely indicate differential activation of the STAT3 and STAT5 family members by acute exercise in human subjects.

The full spectrum of STAT3-regulated genes in skeletal muscle has not yet been characterized; however, potential targets of STAT3 have been known to include c-FOS and JUNB (13, 19), VEGF (14, 29), and c-MYC (3), among others. Having established activation and nuclear translocation of STAT3 in skeletal muscle following exercise, we next sought to determine if this activation was concomitant with increased transcription of STAT3 target genes. Of the genes investigated, the expressions of c-FOS, JUNB, and c-MYC were markedly but transiently upregulated within 2 h after the exercise stimulus, while the increase in VEGF expression peaked at 4 h. Increased expressions of members of the fos and jun gene family have previously been observed within minutes following an acute bout of cycling exercise (33). Fos and Jun proteins constitute the transcription factor complex AP-1, which is purported to regulate the transcriptional activity of genes that contribute to muscle adaptation (15). Increased mRNA levels of VEGF, an important regulator of angiogenesis, are evident in skeletal muscle during hypertrophy before increased capillarization is detected (10, 40). Increases in VEGF expression in human muscle following resistance exercise of a similar magnitude to that described in this study have been reported previously (6). c-MYC, a protooncogene, is a key factor regulating cell growth, transformation, and apoptosis induction in many tissues. In cardiac muscle, increased c-MYC mRNA is observed following exercise (17), while STAT3-induced c-MYC has been demonstrated to mediate cell cycle progression in skeletal muscle (22). However, the precise functions of each of these genes in muscle following resistance exercise are currently unknown. Equally, it cannot be categorically assumed that their upregulation following exercise was a direct result of increased STAT3 signaling and not a result of a myriad of other potential signaling pathways.

Last, we investigated the expression of several genes that positively or negatively regulate STAT3 signaling. While STAT3 is activated by many different ligands, the most widely studied activators in skeletal muscle are IL-6 and LIF. Both IL-6 and LIF mRNA increased markedly within 2 h of the completion of exercise although this increase was transient with levels having returned to their resting state by 4 h. It is generally well established that LIF and IL-6 are released from damaged and exercised muscle (31, 36, 38). LIF has also been established as an important mediator of skeletal muscle hypertrophy in response to increased loading (35). Activation of STAT3 signaling by IL-6 and LIF may contribute to the regeneration and remodeling of skeletal muscle. Conversely, we also demonstrated a marked upregulation in the expression of SOCS3, a negative regulator of STAT signaling, which draws a parallel with previous studies in rat skeletal muscle (37) . The SOCS3 gene is itself thought to be a direct target of STAT signaling (4, 28), suggesting that its transcriptional regulation forms part of a classical negative-feedback loop. Such tight control over the magnitude and length of STAT3 signaling is likely to have marked impacts on the physiological outcomes achieved. While the ultimate outcome of STAT3 signaling in this context is not clear, it is intriguing to speculate a role for this signaling pathway in the repair or remodeling of muscle following resistance exercise. Further support for this hypothesis is provided in cardiac tissue where STAT3 signaling may be critical for the development of cardiac hypertrophy (1, 44). Importantly, ongoing research is necessary to fully elucidate the intricacies of the STAT3 signaling pathway and its role in skeletal muscle remodeling.

Collectively, the results of this study describe the activation of the STAT3 signaling pathway in human skeletal muscle following an acute bout of resistance exercise. Although the significance of this signaling in skeletal muscle remains to be determined, the present study suggests that STAT3 signaling may contribute to the remodeling and adaptation of skeletal muscle following resistance exercise.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Dr Rowena Lewis for the STAT5 positive control sample, Dr. Andrew Garnham (Deakin University) for all muscle biopsy procedures, and the participants for volunteering in this study protocol.

The anti-myosin heavy chain slow isoform (A4.840) was obtained from the Developmental Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA).


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Cameron-Smith, School of Exercise and Nutrition Sciences, Deakin Univ., 221 Burwood Highway, Burwood, Victoria 3125, Australia (e-mail: david.cameron-smith{at}deakin.edu.au)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Ancey C, Menet E, Corbi P, Fredj S, Garcia M, Rucker-Martin C, Bescond J, Morel F, Wijdenes J, Lecron JC, Potreau D. Human cardiomyocyte hypertrophy induced in vitro by gp130 stimulation. Cardiovasc Res 59: 78–85, 2003.[Abstract/Free Full Text]
  2. Bergström J. Muscle electrolytes in man. Scand J Clin Lab Invest 14: 1–110, 1962.[ISI][Medline]
  3. Bowman T, Broome MA, Sinibaldi D, Wharton W, Pledger WJ, Sedivy JM, Irby R, Yeatman T, Courtneidge SA, Jove R. Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis. Proc Natl Acad Sci USA 98: 7319–7324, 2001.[Abstract/Free Full Text]
  4. Chen XP, Losman JA, Rothman P. SOCS proteins, regulators of intracellular signaling. Immunity 13: 287–290, 2000.[CrossRef][ISI][Medline]
  5. Chen YW, Hubal MJ, Hoffman EP, Thompson PD, Clarkson PM. Molecular responses of human muscle to eccentric exercise. J Appl Physiol 95: 2485–2494, 2003.[Abstract/Free Full Text]
  6. Coffey VG, Shield A, Canny BJ, Carey KA, Cameron-Smith D, Hawley JA. Interaction of contractile activity and training history on mRNA abundance in skeletal muscle from trained athletes. Am J Physiol Endocrinol Metab 290: E849–E855, 2006.[Abstract/Free Full Text]
  7. Cohen T, Nahari D, Cerem LW, Neufeld G, Levi BZ. Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem 271: 736–741, 1996.[Abstract/Free Full Text]
  8. Darnell JE Jr. STATs and gene regulation. Science 277: 1630–1635, 1997.[Abstract/Free Full Text]
  9. Decker T, Kovarik P. Transcription factor activity of STAT proteins: structural requirements and regulation by phosphorylation and interacting proteins. Cell Mol Life Sci 55: 1535–1546, 1999.[CrossRef][ISI][Medline]
  10. Degens H, Moore JA, Alway SE. Vascular endothelial growth factor, capillarization, and function of the rat plantaris muscle at the onset of hypertrophy. Jpn J Physiol 53: 181–191, 2003.[CrossRef][ISI][Medline]
  11. Evans W, Phinney S, Ivy J. Suction applied to a muscle biopsy maximises sample size. Med Sci Sports Exer 14: 101–102, 1982.[ISI][Medline]
  12. Frost RA, Nystrom GJ, Lang CH. Regulation of IGF-I mRNA and signal transducers and activators of transcription-3 and -5 (Stat-3 and -5) by GH in C2C12 myoblasts. Endocrinology 143: 492–503, 2002.[Abstract/Free Full Text]
  13. Fu AK, Fu WY, Ng AK, Chien WW, Ng YP, Wang JH, Ip NY. Cyclin-dependent kinase 5 phosphorylates signal transducer and activator of transcription 3 and regulates its transcriptional activity. Proc Natl Acad Sci USA 101: 6728–6733, 2004.[Abstract/Free Full Text]
  14. Funamoto M, Fujio Y, Kunisada K, Negoro S, Tone E, Osugi T, Hirota H, Izumi M, Yoshizaki K, Walsh K, Kishimoto T, Yamauchi-Takihara K. Signal transducer and activator of transcription 3 is required for glycoprotein 130-mediated induction of vascular endothelial growth factor in cardiac myocytes. J Biol Chem 275: 10561–10566, 2000.[Abstract/Free Full Text]
  15. Goswami SK, Shafiq S, Siddiqui MA. Modulation of MLC-2v gene expression by AP-1: complex regulatory role of Jun in cardiac myocytes. Mol Cell Biochem 217: 13–20, 2001.[CrossRef][ISI][Medline]
  16. Hirano T, Ishihara K, Hibi M. Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene 19: 2548–2556, 2000.[CrossRef][ISI][Medline]
  17. Iemitsu M, Maeda S, Jesmin S, Otsuki T, Kasuya Y, Miyauchi T. Activation pattern of MAPK signaling in the hearts of trained and untrained rats following a single bout of exercise. J Appl Physiol 101: 151–163, 2006.[Abstract/Free Full Text]
  18. Ihle JN. Cytokine receptor signalling. Nature 377: 591–594, 1995.[CrossRef][Medline]
  19. Joo A, Aburatani H, Morii E, Iba H, Yoshimura A. STAT3 and MITF cooperatively induce cellular transformation through upregulation of c-fos expression. Oncogene 23: 726–734, 2004.[CrossRef][ISI][Medline]
  20. Kakisis JD, Liapis CD, Sumpio BE. Effects of cyclic strain on vascular cells. Endothelium 11: 17–28, 2004.[CrossRef][ISI][Medline]
  21. Kami K, Senba E. In vivo activation of STAT3 signaling in satellite cells and myofibers in regenerating rat skeletal muscles. J Histochem Cytochem 50: 1579–1589, 2002.[Abstract/Free Full Text]
  22. Kataoka Y, Matsumura I, Ezoe S, Nakata S, Takigawa E, Sato Y, Kawasaki A, Yokota T, Nakajima K, Felsani A, Kanakura Y. Reciprocal inhibition between MyoD and STAT3 in the regulation of growth and differentiation of myoblasts. J Biol Chem 278: 44178–44187, 2003.[Abstract/Free Full Text]
  23. Kiuchi N, Nakajima K, Ichiba M, Fukada T, Narimatsu M, Mizuno K, Hibi M, Hirano T. STAT3 is required for the gp130-mediated full activation of the c-myc gene. J Exp Med 189: 63–73, 1999.[Abstract/Free Full Text]
  24. Kunisada K, Hirota H, Fujio Y, Matsui H, Tani Y, Yamauchi-Takihara K, Kishimoto T. Activation of JAK-STAT and MAP kinases by leukemia inhibitory factor through gp130 in cardiac myocytes. Circulation 94: 2626–2632, 1996.[Abstract/Free Full Text]
  25. Kunisada K, Negoro S, Tone E, Funamoto M, Osugi T, Yamada S, Okabe M, Kishimoto T, Yamauchi-Takihara K. Signal transducer and activator of transcription 3 in the heart transduces not only a hypertrophic signal but a protective signal against doxorubicin-induced cardiomyopathy. Proc Natl Acad Sci USA 97: 315–319, 2000.[Abstract/Free Full Text]
  26. Lewis RS, Stephenson SE, Ward AC. Constitutive activation of zebrafish Stat5 expands hematopoietic cell populations in vivo. Exp Hematol 34: 179–187, 2006.[CrossRef][ISI][Medline]
  27. Matsui T, Kinoshita T, Hirano T, Yokota T, Miyajima A. STAT3 down-regulates the expression of cyclin D during liver development. J Biol Chem 277: 36167–36173, 2002.[Abstract/Free Full Text]
  28. Naka T, Fujimtoto M, Kishimoto T. Negative regulation of cytokine signaling: STAT-induced STAT inhibitor. Trends Biochem Sci 24: 394–398, 1999.[CrossRef][ISI][Medline]
  29. Niu G, Wright KL, Huang M, Song L, Haura E, Turkson J, Zhang S, Wang T, Sinibaldi D, Coppola D, Heller R, Ellis LM, Karras J, Bromberg J, Pardoll D, Jove R, Yu H. Constitutive Stat3 activity upregulates VEGF expression and tumor angiogenesis. Oncogene 21: 2000–2008, 2002.[CrossRef][ISI][Medline]
  30. Peake J, Nosaka K, Suzuki K. Characterization of inflammatory responses to eccentric exercise in humans. Exerc Immunol Rev 11: 64–85, 2005.[ISI][Medline]
  31. Pedersen BK, Febbraio M. Muscle-derived interleukin-6—a possible link between skeletal muscle, adipose tissue, liver, and brain. Brain Behav Immun 19: 371–376, 2005.[CrossRef][ISI][Medline]
  32. Pelletier S, Duhamel F, Coulombe P, Popoff MR, Meloche S. Rho family GTPases are required for activation of Jak/STAT signaling by G protein-coupled receptors. Mol Cell Biol 23: 1316–1333, 2003.[Abstract/Free Full Text]
  33. Puntschart A, Wey E, Jostarndt K, Vogt M, Wittwer M, Widmer HR, Hoppeler H, Billeter R. Expression of fos and jun genes in human skeletal muscle after exercise. Am J Physiol Cell Physiol 274: C129–C137, 1998.[Abstract/Free Full Text]
  34. Sadowski CL, Choi TS, Le M, Wheeler TT, Wang LH, Sadowski HB. Insulin induction of SOCS-2 and SOCS-3 mRNA expression in C2C12 skeletal muscle cells is mediated by Stat5. J Biol Chem 276: 20703–20710, 2001.[Abstract/Free Full Text]
  35. Spangenburg EE, Booth FW. Leukemia inhibitory factor restores the hypertrophic response to increased loading in the LIF(–/–) mouse. Cytokine 34: 125–130, 2006.[CrossRef][ISI][Medline]
  36. Spangenburg EE, Booth FW. Multiple signaling pathways mediate LIF-induced skeletal muscle satellite cell proliferation. Am J Physiol Cell Physiol 283: C204–C211, 2002.[Abstract/Free Full Text]
  37. Spangenburg EE, Brown DA, Johnson MS, Moore RL. Exercise increases SOCS-3 expression in skeletal muscle: potential relationship to IL-6 expression. J Physiol 572: 839–848, 2006.[Abstract/Free Full Text]
  38. Steensberg A, Keller C, Starkie RL, Osada T, Febbraio MA, Pedersen BK. IL-6 and TNF-{alpha} expression in, and release from, contracting human skeletal muscle. Am J Physiol Endocrinol Metab 283: E1272–E1278, 2002.[Abstract/Free Full Text]
  39. Taga T, Kishimoto T. Gp130 and the interleukin-6 family of cytokines. Annu Rev Immunol 15: 797–819, 1997.[CrossRef][ISI][Medline]
  40. Takahashi A, Kureishi Y, Yang J, Luo Z, Guo K, Mukhopadhyay D, Ivashchenko Y, Branellec D, Walsh K. Myogenic Akt signaling regulates blood vessel recruitment during myofiber growth. Mol Cell Biol 22: 4803–4814, 2002.[Abstract/Free Full Text]
  41. Takeda K, Akira S. STAT family of transcription factors in cytokine-mediated biological responses. Cytokine Growth Factor Rev 11: 199–207, 2000.[CrossRef][ISI][Medline]
  42. Tidball JG. Inflammatory processes in muscle injury and repair. Am J Physiol Regul Integr Comp Physiol 288: R345–R353, 2005.[Abstract/Free Full Text]
  43. Tomiya A, Aizawa T, Nagatomi R, Sensui H, Kokubun S. Myofibers express IL-6 after eccentric exercise. Am J Sports Med 32: 503–508, 2004.[Abstract/Free Full Text]
  44. Uozumi H, Hiroi Y, Zou Y, Takimoto E, Toko H, Niu P, Shimoyama M, Yazaki Y, Nagai R, Komuro I. gp130 plays a critical role in pressure overload-induced cardiac hypertrophy. J Biol Chem 276: 23115–23119, 2001.[Abstract/Free Full Text]
  45. Vierck J, O'Reilly B, Hossner K, Antonio J, Byrne K, Bucci L, Dodson M. Satellite cell regulation following myotrauma caused by resistance exercise. Cell Biol Int 24: 263–272, 2000.[CrossRef][ISI][Medline]
  46. Zushi S, Shinomura Y, Kiyohara T, Miyazaki Y, Kondo S, Sugimachi M, Higashimoto Y, Kanayama S, Matsuzawa Y. STAT3 mediates the survival signal in oncogenic ras-transfected intestinal epithelial cells. Int J Cancer 78: 326–330, 1998.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 J. Physiol.Home page
O. H. Mortensen, K. Andersen, C. Fischer, A. R. Nielsen, S. Nielsen, T. Akerstrom, M.-b. Aastrom, R. Borup, and B. K. Pedersen
Calprotectin is released from human skeletal muscle tissue during exercise
J. Physiol., July 15, 2008; 586(14): 3551 - 3562.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
102/4/1483    most recent
01147.2006v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Trenerry, M. K.
Right arrow Articles by Cameron-Smith, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Trenerry, M. K.
Right arrow Articles by Cameron-Smith, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.