Wnt/{beta}-catenin signaling activates growth-control genes during overload-induced skeletal muscle hypertrophy

Dustin D. Armstrong1,2 and Karyn A. Esser1,2

1University of Illinois Chicago, School of Kinesiology, Chicago, Illinois; and 2University of Kentucky, Department of Physiology, Lexington, Kentucky

Submitted 3 March 2005 ; accepted in final form 8 May 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{beta}-Catenin is a transcriptional activator shown to regulate the embryonic, postnatal, and oncogenic growth of many tissues. In most research to date, {beta}-catenin activation has been the unique downstream function of the Wnt signaling pathway. However, in the heart, a Wnt-independent mechanism involving Akt-mediated phosphorylation of glycogen synthase kinase (GSK)-3{beta} was recently shown to activate {beta}-catenin and regulate cardiomyocyte growth. In this study, results have identified the activation of the Wnt/{beta}-catenin pathway during hypertrophy of mechanically overloaded skeletal muscle. Significant increases in {beta}-catenin were determined during skeletal muscle hypertrophy. In addition, the Wnt receptor, mFrizzled (mFzd)-1, the signaling mediator disheveled-1, and the transcriptional co-activator, lymphocyte enhancement factor (Lef)-1, are all increased during hypertrophy of the overloaded mouse plantaris muscle. Experiments also determined an increased association between GSK-3{beta} and the inhibitory frequently rearranged in advanced T cell-1 protein with no increase in GSK-3{beta} phosphorylation (Ser9). Finally, skeletal muscle overload resulted in increased nuclear {beta}-catenin/Lef-1 expression and induction of the transcriptional targets c-Myc, cyclin D1, and paired-like homeodomain transcription factor 2. Thus this study provides the first evidence that the Wnt signaling pathway induces {beta}-catenin/Lef-1 activation of growth-control genes during overload induced skeletal muscle hypertrophy.

lymphocyte enhancement factor-1; glycogen synthase kinase-3{beta}; paired like homeodomain transcription factor-2; c-Myc


THE WNT PATHWAY has been shown to be critical for skeletal muscle development (12, 51, 52). Wnt1 and Wnt3 ligands, secreted by the dorsal neural tube, induce myogenesis during embryonic growth and Wnt activation is both necessary and sufficient to induce myogenesis in P19 embryonic carcinoma cells (12, 21, 35). In addition to its role in skeletal muscle development, Wnt signaling is associated with neuromuscular junction formation, the activation of stem cells during regeneration, the regulation of muscle fiber type, and the prevention of lipid accumulation with aging (3, 29, 47, 53). However, a role for Wnt signaling in the regulation of adult skeletal muscle size has not been tested.

The classic identifier of Wnt signaling is the accumulation and thus activation of {beta}-catenin function (32, 59). {beta}-Catenin is a multifunctional protein that can act in the cytoplasm to link cadherins to the actin cytoskeleton or enter the nucleus and function as a transcription factor (7, 33, 48). In the absence of Wnt signaling, free {beta}-catenin is phosphorylated by glycogen synthase kinase-3{beta} (GSK-3{beta}) and rapidly targeted for proteosomal degradation (2, 14, 20, 26, 27). On Wnt activation, Wnt ligands bind Frizzled (Fzd) and low-density lipoprotein receptor-related protein-5 or -6 coreceptors, stimulate phosphorylation of the protein disheveled (Dvl), and thereby inhibit {beta}-catenin degradation (23, 25, 34). Whereas the exact mechanism is unknown, Dvl phosphorylation/activation is thought to block GSK-3{beta} activity by sequestering GSK-3{beta} via the inhibitory protein frequently rearranged in advanced T cell lymphomas (Frat) (6, 26, 54, 59).

Alternatively, GSK-3{beta} inhibition via phosphorylation at the Ser9 site can increase {beta}-catenin accumulation independent of Wnt signaling (13, 16, 49). Several signaling pathways have been shown to induce GSK-3{beta} phosphorylation in other cell types (22, 60, 61). However, studies of GSK-3{beta} phosphorylation in skeletal muscle have focused on the activation of the phosphatidylinositol 3-kinase/Akt pathway via insulin and/or insulin-like growth factor-1 (IGF-1) (42, 44, 57). Interestingly, both IGF-1 signaling and Akt activation have been linked to skeletal muscle hypertrophy, whereas GSK-3{beta} activity has been shown to be a negative regulator of this process (9, 16, 42). In support of this model, Akt-mediated GSK-3{beta} phosporylation was shown to activate {beta}-catenin during cardiomyocyte hypertrophy (15).

Activated {beta}-catenin is associated with cellular growth through its well-characterized function as a transcription factor (32, 33). In the nucleus, {beta}-catenin can form a transcriptional complex with T cell factor/lymphocyte-enhancement factor-1 (Lef-1) family members and subsequently promote expression of target genes (33, 40). Two important transcriptional targets of active {beta}-catenin in developmental and oncogenic growth are c-Myc and cyclin D1 (17, 50). Besides direct transcriptional activation, {beta}-catenin can induce c-Myc and cyclin D1 indirectly through paired-like homeodomain transcription factor2 (Pitx2) activation. Pitx2 has been shown to directly activate c-Myc and cyclin D1 in skeletal muscle cells exposed to Wnt/{beta}-catenin stimulation in vitro (5). Both c-Myc and cyclin D1 are thought to function as cell cycle regulators but recent evidence suggests these proteins can regulate cell size (18, 31, 36, 41). For example, overexpression of c-Myc has been shown to be sufficient to induce hypertrophy in both postmitotic cardiac myocytes and hepatocytes in vivo (24, 62).

The overall goal of this study was to examine the regulation of {beta}-catenin during overload-induced skeletal muscle hypertrophy. The hypotheses tested were 1) {beta}-catenin will be activated during overload-induced skeletal muscle hypertrophy, 2) increased {beta}-catenin levels will result from phosphorylation-induced GSK-3{beta} inhibition and not via Wnt signaling, and 3) {beta}-catenin activation will correspond to an induction of known target genes associated with growth control. The results of this study demonstrate that {beta}-catenin is activated and its transcriptional targets are induced during skeletal muscle hypertrophy. The Wnt pathway is implicated as the predominant mediator of {beta}-catenin function during skeletal muscle growth because 1) several markers of Wnt pathway activation are identified following synergist ablation and 2) the {beta}-catenin accumulation is detected at a time in which levels of unphosphorylated GSK-3{beta} (active GSK-3{beta}) are significantly increased.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Induction of skeletal muscle hypertrophy. All experimental procedures performed in this study were approved by the University of Illinois at Chicago Institutional Animal Care and Use Committee. Animals were housed in temperature- and humidity-controlled holding facilities with lights on at 7 AM and off at 7 PM, and had access to food and water ad libitum. Bilateral synergist ablation of the plantaris muscle was performed on 10- to 12-wk-old male C57BL6 mice (Jackson Laboratories, Bar Harbor, ME) as previously described (19, 55). After anesthesia was induced with 80 mg/kg ketamine and 10 mg/kg xylazine, the gastrocnemius and soleus muscles were exposed by a longitudinal incision through the skin and fascia and removed in their entirety, whereas the plantaris muscle was left intact. Incisions were closed with the use of 6-0 silk sutures, and the mice were returned to their cages and resumed normal locomotor activity. At 7 or 14 days after surgery, overloaded and control plantaris muscles from nontreated mice were collected, quickly weighed, frozen in liquid nitrogen, and stored at –80°C until analysis.

Preparation of protein samples. For the analyses of protein expression, whole cell extracts were prepared by homogenizing muscles from control and overloaded mice by a polytron in 750 µl or 1 ml of buffer, respectively, containing 20 mmol/l Tris (pH 7.5), 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 10 g/l Nonidet P-40, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l {beta}-glycerolphosphate, 1 mmol/l sodium orthovanadate, 1 mg/l leupeptin, and 1 mmol/l phenylmethylsulfonyl fluoride. For the analyses of nuclear proteins, extracts were prepared using the nuclear protein isolation kit (NE-PER, Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. Briefly, control and overloaded muscles were homogenized in 750 µl or 1 ml of cytoplasmic extraction reagent and centrifuged at 5,000 rpm for 6 min. The supernatant, consisting of free cytosolic protein, was collected and the remaining pellet was treated with 150 or 200 µl of nuclear extraction reagent. The samples were then centrifuged at 14,000 rpm for 10 min and the supernatant was collected for nuclear proteins. Protein concentrations were determined using the DC protein assay (Bio-Rad Laboratories, Hercules, CA). To verify that the NE-PER kit isolated nuclear proteins without the presence of cytoplasmic proteins, cytoplasmic and nuclear protein isolates were run out on SDS page gels, stained with Coomassie blue, and examined for the presence or absence of the 205 KDa form of myosin heavy chain. As predicted, myosin expression was only detected in the cytoplasmic protein fraction (data not shown).

Western blot analysis. SDS-PAGE was performed on 7.5% acrylamide gels and proteins were transferred to polyvinylidene difluoride membranes. For these experiments, 40 µg of total or nuclear protein were analyzed with antibodies against {beta}-catenin (no. c-2206; Sigma, St. Louis, MO) 1:2,000, Lef-1 (no. L-3275, Sigma) 1:1,000, GSK-3{beta} (no. 610202, BD Transduction Laboratories, San Diego, CA) 1:1,000, phosphorylated (Ser9) GSK-3{beta} (no. 9336S, Cell Signaling, Beverly, MA) 1:1,000, Dvl-1 (no. 06-939, Cell Signaling) 1:1,000, Frat1 (no. ab2533-100, Abcam, Cambridge, MA) 1:1,000, c-Myc (no. sc-788, Santa Cruz Biotechnology, Santa Cruz, CA) 1:500, cyclin D1 (no. sc-718, Santa Cruz) 1:500, Pitx2 (no. sc-8748, Santa Cruz) 1:500, Wnt-10b (no. sc-25524, Santa Cruz) 1:1,000, and mFzd-1 (no. AF1120, R&D Systems, Minneapolis, MN) 1:1,000. Peroxidase-conjugated anti-rabbit (no. PI-1000), mouse (no. PI-2000), and goat (no. PI-9500) secondary antibodies were used (Vector Laboratories, Burlingame, CA) 1:5,000 and blots were visualized with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ). Densitometric measurements were performed on a FluorS Max Imager using QuantityOne Software (Bio-Rad Laboratories). Representative images and denstitometric values were made from analysis of individual muscle samples (n ≥ 6 per group).

Coimmunoprecipitation assays. Coimmunoprecipitation studies were performed using the Immunoprecipitation Starting Kit (Amersham). Briefly, 300 µg of total protein was precleared with a 50% mixture of A/G beads and incubated with a 1:100 dilution of {beta}-catenin or Frat1 antibodies at 4°C with rotation overnight, followed by an additional 1 h treatment with A/G beads. The beads were isolated by centrifugation, washed three times, and boiled in 2x loading buffer containing 20% glycerol, 100 nM Tris, 4% SDS, 0.017% bromophenol blue, and 0.25 M DTT for 10 min. Samples were loaded on SDS-PAGE gels for Western blot analysis as previously described.

Immunohistochemistry. Plantaris muscles were dissected from control or 7-day-old ablated mice, placed in tissue freezing medium (Fisher Scientific, Hanover Park, IL), frozen in isopentane cooled with dry ice, and stored at –80°C. Cross-sections (10 µM thick) were cut at the muscle midbelly and mounted on Superfrost/Plus slides (Fisher Scientific) and allowed to air dry for 60 min. Sections were fixed in acetone for 5 min, washed with 50 mM Tris-buffered saline (TBS) pH 7.6 for 5 min and blocked in 5% fetal bovine serum in TBS for 30 min. Excess blocking reagent was removed and replaced with c-Myc primary antibody, 1:100 in TBS overnight. The sections were next washed 3 x 10 min in TBS containing 0.1% Triton X-100 and treated with a rhodamine-conjugated anti-rabbit secondary antibody (no. 211–025-109, Jackson ImmunoResearch Laboratories, West Grove, PA) 1:200 in TBS for 40 min. The sections were washed in the TBS-Triton X-100 mixture and then costained with an anti-dystrophin goat polyclonal antibody (no. sc-7461, Santa Cruz) 1:100 in TBS for 1 h. Sections were washed again before being stained with a FITC-conjugated anti-goat secondary antibody (no. 705-095-003, Jackson) 1:200 in TBS for 40 min. Finally, sections were washed in TBS and then mounted with coverslips with the use of VectaMount (Vector Laboratories). Immunoflourescent images were captured on a Kodak DC290 camera taken with a Nikon Diaphot 200 microscope.

RNA isolation and analysis. Total RNA was extracted by homogenizing muscles in TRIzol reagent (Invitrogen, Carlsbad, CA) following the protocol provided and RNA was obtained and suspended in diethyl pyrocarbonate-treated water. cDNA was synthesized from 0.5 µg total RNA using SuperScript II (Invitrogen) and random hexamer primers. Semiquantitative PCR amplification was performed in a 50 µl reaction buffer containing: 1 µl Taq DNA polymerase, 5 µl 10x buffer with 15 mM MgCl2, and 200 µM each of dNTPs (Promega, Madison, WI) along with 0.1 µg of cDNA and 0.2 mM of primers directed against c-Myc (i) forward 5'-TGCGACGAGGAAGAGAATTT and (ii) reverse 5'-GAATCGGACGAGGTACAGGA that yields a 599-bp product and 18S rRNA (i) forward 5'-AAACGGCTACCACATCCAAG and reverse 5'-ccctcttaatcatggcctca that yields a 481 bp product. PCR was performed using an Epgradient S Mastercycler (Eppendorf, Hamburg, Germany) under the following conditions: denaturation for 30 s at 94°C, annealing for 30 s at 58°C, and extension for 45 s at 72°C. PCR products were resolved by running 15 µl of the PCR reaction products on a 1% agrose and 45 mM Tris-borate-1 mM EDTA) gel containing 3 µg/ml ethidium bromide and visualized under UV light. Images represent analysis of individual muscle samples (n = 6 per group).

Statistical analysis. Statistical analysis for these studies was performed using a paired Student's t-test or ANOVA for multiple groups. All data are expressed as means ± SE. Differences between groups were considered statistically significant if P ≤ 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{beta}-Catenin is activated during overload-induced skeletal muscle hypertrophy. Synergist ablation was used to induce mechanical overload of the mouse plantaris muscle. Consistent with previous studies, 7 days of overload produced a 67% increase in plantaris wet weight, whereas 14 days of overload resulted in a 139% increase in plantaris mass (P < 0.0001; Fig. 1). No differences in protein concentration (µg protein/mg muscle) were found among any of the groups, which indicate that changes in mass reflect total protein accumulation (data not shown). For the remaining experiments, muscles were studied at the 7-day time point because 1) mass is significantly elevated and 2) the muscle is in an active growth phase.



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Fig. 1. Plantaris muscle mass increases after synergist ablation. A 67% increase in muscle weight is seen after 7 days of overload, whereas 14 days of compensatory hypertrophy resulted in a 139% increase in plantaris mass, 23 ± 1 vs. 39 ± 2 vs. 57 ± 2 (mg). Values represent means ± SE (P < 0.0001). Open star, significantly greater than day 0. Solid star, significantly greater than day 7.

 
To examine the role of {beta}-catenin in skeletal muscle hypertrophy, {beta}-catenin levels were compared in control and overloaded plantaris muscle. Levels of total {beta}-catenin protein increased 301% (P = 0.0004) after 7 days of mechanical overload (Fig. 2A). To gain insight into potential functions, nuclear protein fractions were examined for {beta}-catenin expression. Muscle overload resulted in a 434% increase (P = 0.0001) in {beta}-catenin protein found in the nuclear enriched fraction (Fig. 2B). This large increase in nuclear protein accumulation suggests {beta}-catenin is acting as a transcription factor during skeletal muscle hypertrophy.



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Fig. 2. {beta}-Catenin is activated during overload-induced hypertrophy. A: total {beta}-catenin protein is expressed at a level {approx}3-fold higher in overloaded muscle, 100% ± 19 vs. 401% ± 33 (*P = 0.0004). B: nuclear {beta}-catenin levels increased {approx}4-fold with overload, 100 ± 25% vs. 534 ± 65% (*P = 0.0001). C: the transcriptional co-activator lymphocyte enhancement factor-1 (Lef-1) and complex formation between {beta}-catenin and Lef-1 is induced with 7 days of overload. Nuclear protein fractions and coimmunoprecipitation (IP) experiments were performed as described in MATERIALS AND METHODS. Values represent mean optical density expressed as a percentage of control ± SE.

 
Reports (5, 8, 40, 45) have established that {beta}-catenin dimerizes in the nucleus with the DNA binding protein Lef-1 to function as a transcriptional activator in skeletal muscle cells. Lef-1 protein levels were undetectable in nuclear lysates (40 µg) from control plantaris muscles but were strongly induced after 7 days of muscle overload (Fig. 2C). To test for a functional interaction between {beta}-catenin and Lef-1, coimmunoprecipitation studies were performed with nuclear extracts. Results of these experiments confirmed that a {beta}-catenin/Lef-1 transcriptional complex is not detectable in control lysates but is formed during overload-induced hypertrophy (Fig. 2C).

{beta}-Catenin activation does not require increased GSK-3{beta} phosphorylation. Numerous studies (28, 61) indicate that {beta}-catenin protein levels are regulated by targeted degradation after GSK-3{beta}-mediated phosphorylation. To determine the role of GSK-3{beta} in {beta}-catenin activation, the amount of total and phosphorylated GSK-3{beta}, at the Ser9 site, was examined in control and overloaded muscle samples. Total GSK-3{beta} protein levels increased 46% (P < 0.02) with 7 days of muscle overload, whereas the amount of phosphorylated GSK-3{beta} did not significantly change (Fig. 3). These results show that during skeletal muscle growth, {beta}-catenin protein levels are significantly increased, whereas there is also a significant increase in the level of unphosphorylated GSK-3{beta} protein.



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Fig. 3. Skeletal muscle overload is associated with an induction in unphosphorylated glycogen synthase kinase-3{beta} (GSK-3{beta}) protein. Total GSK-3{beta} protein levels increased 46% with overload, 100% ± 11 vs. 146% ± 13 (*P = 0.02), whereas the amount of GSK-3{beta} phosphorylated (p) at Ser9 did not significantly change, 100% ± 19 vs. 104% ± 17 (P = 0.86). Values represent mean optical density expressed as a percentage of control ± SE.

 
{beta}-Catenin activation corresponds with increased expression of Wnt signaling components. Adult skeletal muscle expresses several different Wnt and Fzd isoforms (38, 43). However, Wnt10b and mFzd-1 have previously been detected in murine skeletal muscle and are known to function in Wnt/{beta}-catenin signaling (10, 43, 53). Therefore, to examine whether these components of Wnt signaling change after mechanical overload, levels of Wnt10b and mFzd-1 were examined in whole cell extracts from control and overloaded muscle. Wnt-10b protein expression did not significantly change with overload but levels of mFzd-1 increased 71% (P = 0.006) (Fig. 4A).



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Fig. 4. Skeletal muscle overload is associated with increased expression of frizzled-1 (mFzd)-1, disheveled-1 (Dvl-1), and GSK-3{beta} frequently rearranged in advanced T cell lymphoma (Frat1) complex formation. A: Wnt-10b protein levels showed no significant change, 100% ± 9 vs. 109% ± 11 (P = 0.55), but mFzd-1 protein levels increased 71% with overload, 100% ± 17 vs. 171% ± 8 (*P = 0.006). B: total Dvl-1 protein levels increased 88% with overload, 100% ± 6 vs. 188 ± 12 (*P = 0.002). C: total Frat1 levels did not significantly change, 100% ± 10 vs. 99% ± 9 (P = 0.95), but GSK-3{beta} bound to Frat1, whereas not detectable in control muscle, was significantly expressed in overloaded muscle samples. Values represent mean optical density expressed as a percentage of control ± SE.

 
Dvl proteins have been shown to function downstream of Wnt binding to its receptor (Fzd) in the inactivation of the {beta}-catenin degradation complex (25, 34). Of the three mouse Dvl genes (Dvl-1, -2, and -3), Dvl-1 is the most well characterized (25). Dvl-1 is expressed in skeletal muscle cells and is known to participate in Wnt-induced {beta}-catenin activation (23, 26, 29, 37). Therefore, in this study, Dvl-1 protein levels were examined in whole cell extracts from control and overloaded muscle. Consistent with increased Wnt signaling, total Dvl-1 expression increased 88% (P = 0.002) with skeletal muscle overload (Fig. 4B).

Dvl activation can disrupt GSK-3{beta}-mediated degradation of {beta}-catenin via activation of the inhibitory protein Frat1 (26). Because increased GSK-3{beta} phosphorylation/inhibition was not detected during muscle overload (Fig. 3), the relationship between GSK-3{beta} and Frat1 was examined in control and overloaded muscle. Frat1 protein levels did not significantly change with overload; however, the interaction between GSK-3{beta} and Frat1 was induced, consistent with Wnt/{beta}-catenin signaling (Fig. 4C).

{beta}-Catenin/Lef-1 transcriptional targets are induced. The observation of increased nuclear {beta}-catenin/Lef-1 complexes during overload (Fig. 2C) suggested potential changes in target gene expression. Several of the known transcriptional targets of {beta}-catenin are growth-associated genes (32, 58). One well-defined growth gene and transcriptional target of active {beta}-catenin/Lef-1 is c-Myc (5, 17, 50). Levels of c-Myc mRNA, examined by semiquantitative RT-PCR, were undetectable in control muscle (45 cycles) but significantly expressed in muscle after 7 days of overload (Fig. 5). No c-Myc product was detected in control muscle samples with up to 50 cycles (data not shown).



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Fig. 5. The {beta}-catenin/Lef-1 transcription target c-Myc shows induced mRNA expression with overload. c-Myc mRNA was readily detected in overloaded muscle samples, whereas RNA extracted from control muscle and used for RT-PCR failed to produce a detectable c-Myc product (45 cycles). Primers directed against 18S rRNA were used to determine equal loading (30 cycles). Images represents analysis of individual muscle samples (n = 6 per group).

 
Consistent with the expression pattern of c-Myc mRNA, c-Myc and cyclin D1 proteins were undetectable in whole cell extracts from control muscle but were significantly expressed after 7 days of skeletal muscle overload (Fig. 6A). Expression of Pitx2, another target of {beta}-catenin/Lef-1 transcription and itself a transcriptional activator of c-Myc and cyclin D1, was also examined during muscle overload. Consistent with changes in c-Myc and cyclin D1 protein levels, Pitx2 was significantly induced after 7 days of skeletal muscle overload (Fig. 6A).



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Fig. 6. {beta}-Catenin/Lef-1 transcription targets, c-Myc, cyclin D1, and paired-like homeodomain transcription factor 2 (Pitx2) show induced protein expression with overload. A: c-Myc, cyclin D1, and Pitx2 expression was undetectable in control lysates (40 µg) but significantly expressed in overloaded muscle. B: histological sections from control and 7 day overloaded plantaris muscles were prepared as described in MATERIALS AND METHODS. c-Myc (red) was not detected in control muscle cross-sections but 7-day overloaded plantaris muscle showed c-Myc staining in nuclei, which appear within the dystrophin (green)-stained sarcolemma (arrowhead), outside of the muscle membrane, and in the interstitial space (arrow) (x100 magnification).

 
Immunohistochemical localization of c-Myc was performed to determine whether the increase in c-Myc protein observed with overload occurred in skeletal muscle fibers or other cell types. c-Myc expression was undetectable in control muscle cross-sections. However, in overloaded skeletal muscle cross-sections, c-Myc was readily visualized in nuclei within the dystrophin-stained sarcolemma, as well as outside of the muscle membrane, and in the interstitial space (Fig. 6B). These results suggest that c-Myc was induced in both muscle and nonmuscle/satellite cell nuclei within the muscle tissue.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{beta}-Catenin regulation has been well studied during developmental and oncogenic growth. Recently, {beta}-catenin was determined to be both necessary and sufficient for hypertrophy of terminally differentiated cardiomyocytes (15). In cardiac muscle, active {beta}-catenin was found to be associated with Akt-mediated GSK-3{beta} (Ser9) phosphorylation (15). In contrast, results from this study indicate that {beta}-catenin activation during overload-induced skeletal muscle hypertrophy is predominantly associated with Wnt signaling and is likely not the result of phosphorylation-induced GSK-3{beta} inhibition.

Mechanical overload of skeletal muscle is associated with Wnt activation, characterized by {beta}-catenin accumulation accompanied with increases in mFzd-1, Dvl-1, and GSK-3{beta}-Frat1 complex formation. In contrast to the findings in cardiomyocytes, the results of this study argue that the Wnt pathway, and not phosphorylation of GSK-3{beta}, is the more dominant pathway affecting {beta}-catenin activation in response to skeletal muscle overload. In fact, {beta}-catenin accumulation was associated with increased levels of unphosphorylated GSK-3{beta} that would normally predict decreased {beta}-catenin expression. Thus while this study cannot rule out a role for GSK-3{beta} phosphorylation during skeletal muscle hypertrophy, the findings suggest that it is likely not the dominant mechanism responsible for induction of {beta}-catenin protein in mouse skeletal muscle after synergist ablation.

The lack of a change in GSK-3{beta} phosphorylation in the mouse plantaris muscle after synergist ablation is in contrast to the findings of Bodine et al. (9), in which GSK-3{beta} phosphorylation was increased after mechanical overload of the rat plantaris muscle. One potential explanation is that the antibody used in the Bodine study recognizes phosphorylation of both GSK-3{alpha} and GSK-3{beta} at Ser21 and Ser9 sites so the increased phosphorylation detected in the previous study could reflect changes in GSK-3{alpha} (9). Second, the measurement of phosphorylated GSK-3{beta} in this study was done at a 7 days, whereas the previous work examined GSK-3 phosphorylation after 14 days of overload, making the timecourse a potential source of the difference (9). Finally, because GSK-3{beta} is a multifunctional kinase with many forms of regulation, it is possible that mice and rats have intrinsic and yet unidentified differences in GSK-3{beta} regulation during overload-induced skeletal muscle growth (30, 60).

Although other Wnt proteins may be involved, no change was detected in Wnt-10b protein levels, suggesting that induction of other signaling components mediate Wnt pathway activation in response to skeletal muscle overload. The increase in mFzd-1 protein levels observed in this study are consistent with findings from Carson et al. (10), which showed significant induction of mFzd-1 mRNA levels at 3 days after mechanical overload in rat soleus muscle (10, 43). Increased Dvl-1 expression after skeletal muscle overload is also consistent with Wnt-induced {beta}-catenin activation. In some forms of cancer, total Dvl protein levels directly correlate with {beta}-catenin activation (56). However, Wnt activation is generally associated with the hyperphosphorylation of Dvl, characterized by a decreasing mobility in SDS-PAGE gels (23, 25, 34). In this study, Dvl-1 protein levels, including the phosphorylated forms of higher molecular weight, are elevated after skeletal muscle overload (Fig. 4B). Therefore, increasing levels of phosphorylated Dvl-1 protein may be critical for Wnt-induced {beta}-catenin activation during skeletal muscle growth.

An important finding of this study is the induction of Lef-1 protein and the formation of a {beta}-catenin-Lef-1 complex with overload. As expected, known {beta}-catenin/Lef-1 transcriptional targets c-Myc, cyclin D1, and Pitx2 were induced during skeletal muscle overload. c-Myc and cyclin D1 are well known for their roles in cell-cycle regulation but recent studies (31, 46) demonstrate that they are critical for the regulation of cell size. While the function of these genes has not been tested, many models of hypertrophic growth report increased c-Myc and cyclin D1 gene expression (1, 10, 11). One potential downstream function could be their role in regulated ribosomal biogenesis, which is critical for maintaining increased rates of protein synthesis (4, 11, 24, 39, 41). In this study, c-Myc was induced after overload in both muscle and nonmuscle or satellite cell nuclei. Thus increased levels of c-Myc may be contributing to the proliferation of satellite cells and other nonmuscle cells, such as fibroblasts and/or endothelial cells.

In summary, the results from this study demonstrate that {beta}-catenin is activated during skeletal muscle overload primarily through Wnt signaling and is not the sole result of phosphorylation-induced GSK-3{beta} inhibition. Wnt-induced {beta}-catenin activation was associated with increased protein expression of many Wnt signaling components, including mFzd-1, Dvl-1, and Lef-1. This indicates that mechanical overload also results in significant increases in the Wnt pathway machinery. The Wnt-induced {beta}-catenin/Lef-1 complex formation detected after mechanical overload is associated with increases in known targets, such as c-Myc and cyclin D, which may contribute to hypertrophic growth. Thus these findings implicate Wnt signaling and the activation of {beta}-catenin as an important response to mechanical overload, which may contribute to the compensatory hypertrophic response.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This research was supported by the National Institutes of Health Grant AR-45617 and Pfizer funding (to K. A. Esser).


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. A. Esser, Dept. of Physiology, Albert B. Chandler Medical Center, MS567, 800 Rose St., Lexington, KY 40536-0298 (e-mail: kaesse2{at}uky.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
 GRANTS
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