Previous reports suggest that burn-induced muscle proteolysis can be inhibited by treatment with GSK-3β inhibitors, suggesting that burn injury may be associated with increased GSK-3β activity. The influence of burn injury on muscle GSK-3β activity, however, is not known. We determined the effect of a 30% total body surface full-thickness burn injury in rats on muscle GSK-3β activity by measuring GSK-3β activity and tissue levels of serine 9 phosphorylated GSK-3β, p(Ser9)-GSK-3β, by Western blot analysis and immunohistochemistry. Because burn-induced muscle wasting is, at least in part, mediated by glucocorticoids, we used dexamethasone-treated cultured muscle cells in which GSK-3β expression was reduced with small interfering RNA (siRNA) to further assess the role of GSK-3β in muscle atrophy. Burn injury resulted in a seven-fold increase in GSK-3β activity in skeletal muscle. This effect of burn was accompanied by reduced tissue levels of p(Ser9)-GSK-3β, suggesting that burn injury stimulates GSK-3β in skeletal muscle secondary to inhibited phosphorylation of the enzyme. In addition, burn injury resulted in inhibited phosphorylation and activation of Akt, an upstream regulatory mechanism of GSK-3β activity. Reducing the expression of GSK-3β in cultured muscle cells with siRNA inhibited dexamethasone-induced protein degradation by ∼50%. The results suggest that burn injury stimulates GSK-3β activity in skeletal muscle and that GSK-3β may, at least in part, regulate glucocorticoid-mediated muscle wasting.
- protein degradation
- muscle wasting
- Akt activity
one of the most significant metabolic consequences of burn injury is an increase in whole body protein loss (5, 6, 10, 22). Studies suggest that this effect of burn injury mainly reflects stimulated ubiquitin-proteasome-dependent degradation of myofibrillar proteins in skeletal muscle. In previous reports from our and other laboratories, burn-induced muscle proteolysis was inhibited by IGF-I both in patients and experimental animals (5, 9, 16, 22, 38). One mechanism by which IGF-I exerts its anabolic effects in skeletal muscle is activation of the PI3K/Akt signaling pathway with a downstream inhibition of GSK-3β activity (8, 15, 28, 30, 32). We recently reported that treatment with IGF-I inactivated GSK-3β in muscle from burned rats, and the anticatabolic effects of IGF-I were duplicated by the GSK-3β inhibitors lithium chloride (LiCl) and thiadiazolidinone (TDZD)-8 (15). These observations raise the possibility that burn injury is associated with activation of GSK-3β in muscle. The effects of burn injury on muscle GSK-3β activity, however, have not been reported.
In the present study, we tested the hypothesis that burn injury results in activation of GSK-3β. This was done by measuring GSK-3β activity and levels of serine 9-phosphorylated GSK-3β, p(Ser9)-GSK-3β, in skeletal muscle after burn injury in rats. Because burn-induced muscle wasting is at least, in part, mediated by glucocorticoids (13), in additional experiments, we used dexamethasone-treated cultured muscle cells in which GSK-3β expression was reduced with small interfering RNA (siRNA) to assess the role of GSK-3β in muscle proteolysis. The present results suggest that burn injury stimulates GSK-3β activity in skeletal muscle and that GSK-3β may, at least in part, regulate glucocorticoid-mediated muscle wasting.
MATERIALS AND METHODS
Dexamethasone was obtained from Sigma (St Louis, MO). Western blot analysis reagents were from Bio-Rad Laboratories (Hercules, CA). IGF-I was a gift from Genentech (South San Francisco, CA). Akt kinase assay kit and the following antibodies (rabbit polyclonal) were purchased from Cell Signaling Technology (Beverly, MA): anti-mouse Akt and phospho (Ser473)-Akt; anti-human GSK-3β and phospho (Ser9)-GSK-3β. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG was also obtained from Cell Signaling Technology. Primers for atrogin-1 and MuRF1 for real-time PCR assay were from SuperArray Bioscience (Frederick, MD) and the University of Cincinnati DNA Core, respectively.
Experimental animals and muscle sampling.
A 30% total body surface area full-thickness burn injury was inflicted on the back of male Sprague-Dawley rats weighing 50–60 g, as described in detail previously (13). Other rats underwent sham procedure (anesthesia and shaving of the back but no burn). The rats had free access to drinking water, but food was withheld after sham procedure and burn injury. The animals were cared for in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Institutional Animal Care and Use Committee at the University of Cincinnati. In several previous reports (13–17), we used small growing rats weighing 50–60 g when the influence of burn injury on muscle protein breakdown was determined. Lower-extremity muscles from rats of this size are thin enough to allow for measurement of protein breakdown rates during incubation in vitro with maintained viability of the tissue and preserved energy levels. Rats of the same size were used here to make it possible to compare results with our previous reports using the same model. The rats were randomly assigned to sham procedure or burn with 8 rats included in each group in most experiments. In experiments in which Western blot analysis was performed, four rats were included in each group. The number of rats included in the individual experiments is also described in the figure legends. The extensor digitorum longus (EDL) muscles from the rats were harvested 3, 6, and 24 h after sham procedure or burn injury, with rats under pentobarbital anesthesia. Because in preliminary experiments, muscle protein degradation rates, total and phosphorylated Akt and GSK-3β levels, and mRNA expression for atrogin-1 and MuRF1 were similar 3, 6, and 24 h after sham procedure, we used muscles from rats 24 h after sham procedure as controls. When the influence in vitro of IGF-I on kinase activity and phosphorylation was examined, paired EDL muscles were incubated in Krebs-Henseleit bicarbonate buffer (pH 7.4) for 2 h in the absence or presence of 1 μg/ml IGF-I, as described previously in detail (15). All samples were immediately frozen in liquid nitrogen and stored at −80°C, except those for GSK-3β assay, which were processed fresh.
Akt kinase activity assay.
Akt activity was determined by using a nonradioactive Akt kinase assay kit (Cell Signaling Technology). Briefly, muscles were homogenized in ice-cold radioimmunoprecipitation (RIPA) lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA) containing 1× PBS, 1% Igepal CA-630, 0.5% Na deoxycholate, 0.1% SDS, 10 mg/ml PMSF, 10 μl/ml protease inhibitor cocktail, and 1 mM sodium orthovanadate (Na3VO4). After homogenization, the samples were centrifuged at 10,000 g for 10 min at 4°C. Protein concentrations in the supernatants were determined by the Bradford method (2), and 20 μl of resuspended immobilized mouse anti-human Akt (1G1) monoclonal antibody bead slurry was added to 200 μl of the lysate and incubated overnight at 4°C to immunoprecipitate Akt. The tissue lysate and immobilized antibody were washed with lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM Na pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 1 μg/ml leupeptin) and then washed with kinase buffer containing 25 mM Tris, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2. The suspended pellet (immunoprecipitated Akt) was next used to phosphorylate a specific substrate, GSK-3 fusion protein (Ser21/9) in the presence of ATP. After termination of the reaction, samples were heated to 95–100°C for 5 min, and proteins were separated by 15% Criterion Tris·HCl gel and transferred onto a PVDF membrane. Nonspecific reactivity was blocked by 5% fat-free milk in Tris-buffered saline-Tween (20 mM Tris·HCl, pH 7.5, 137 mM NaCl, 0.1% Tween-20). The membrane was incubated with a 1:1,000 dilution of rabbit anti-human phospho-GSK-3α/β antibody overnight at 4°C, then with HRP-conjugated anti-rabbit IgG secondary antibody (1:2,000) and HRP-conjugated antibiotin antibody (1:1,000) to detect biotinylated protein markers. The reactive protein was revealed by incubating with LumiGLO substrate (0.5 ml 20× LumiGLO, 0.5 ml 20× peroxide in 9 ml Milli-Q water) for 1 min and exposure to X-ray film.
GSK-3β activity assay.
The muscles were homogenized in buffer containing 50 mM Tris·HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM Na3VO4, 10 mM β-glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 0.1% β-mercaptoethanol, 0.25 M sucrose + Sigma protease inhibitor cocktail (0.1 ml/10 ml buffer) + 1 mM PMSF. After homogenization, the samples were sonicated twice for 5 s and centrifuged for 30 min at 13,000 g at 4°C. Supernatant (1 mg protein) was transferred into precooled 1.5 ml Eppendorf tubes. Samples were precleared by adding 50 μl of washed protein-A insoluble suspension (P-7155; Sigma, St. Louis, MO) and 1 μg of mouse IgG. The precleared supernatant was incubated with 10 μg GSK-3β monoclonal antibody (BD Transduction Lab, # 610202) overnight at 4°C. Protein-A agarose beads (Upstate Cell Signaling Solutions, Lake Placid, NY) were added to each sample, and the incubation continued overnight. GSK-3β-containing immune complex was separated by centrifugation for 5 min at 10,000 g. The immune complex pellet was solubilized in 50-μl kinase assay buffer (20 mM Tris·HCl, pH 7.5, 5 mM MgCl2, 1 mM DTT). After three washes in this buffer, beads were reconstituted in 20 μl of kinase assay buffer and 10 μl of 62.5 μM GSK-3β substrate peptide YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE (Upstate) was added and preincubated for 3 min at 30°C. To this mixture, 10 μCi [γ32P]ATP diluted in 75 mM MgCl2 and 500 μM unlabeled ATP were added, and the incubation continued for 30 min at 30°C. After this incubation, samples were centrifuged for 1 min at 10,000 g; 20 μl of mixture was placed on P81 phosphocellulose discs (Fisher Scientific, Pittsburgh, PA), and air dried and washed 3 times in 0.75% phosphoric acid (10 ml/disc) and once with acetone. Radioactivity in the phosphocellulose disc was counted in a β-counter (Perkin Elmer, Waltham, MA). The amount of radioactivity incorporated into the peptide substrate was determined, and the enzyme activity is expressed as picomoles of 32PO4 incorporated per milligram protein.
EDL muscles were cut into 10-μm-thick coronal cryosections and immunohistochemistry was performed using the avidin-biotin complex method. Briefly, the sections were fixed for 10 min in 4% paraformaldehyde and then incubated overnight at 4°C with p-GSK-3β rabbit antibody (1:200 dilution, Cell Signaling Technology), washed with potassium PBS (pH 7.4) and incubated with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA). p-GSK-3β signal was amplified in biotinylated tyramide solution (1:250; TSA Biotin System, Perkin Elmer Life Sciences, Boston, MA) and incubated in the dark for 30 min at room temperature in Cy3-conjugated streptavidin solution (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA). p-GSK-3β labeling was visualized under a confocal microscope (Carl Zeiss, Thornwood, NY).
Western blot analysis.
The procedures used for Western blot analysis were the same as those described in detail previously (15). Rabbit polyclonal anti-mouse Akt, anti-phospho-Akt, anti-human GSK-3β and anti-phospho-GSK-3β antibodies were used as primary antibodies. HRP-conjugated anti-rabbit IgG was used as secondary antibody.
Quantitative real-time RT-PCR.
Total RNA was isolated using Tri Reagent (Molecular Research Center, Cincinnati, OH), as described by the supplier's protocol. The yield and purity of the RNA were determined by absorbance at 260 nM and 260/280 ratio, respectively. Five micrograms of RNA was used for cDNA synthesis using Superscript Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA). Real-time RT-PCR was carried out using SYBR Green-based kit. The PCR program for atrogin-1 mRNA consisted of initial 15 min denaturation at 95°C, followed by 40 cycles of 30 s of denaturation at 95°C, 30 s of annealing at 55°C, and 30 s of extension at 72°C. The PCR program for MuRF1 was as follows: 10 min of denaturation, 30 s of denaturation at 95°C, 90 s of annealing at 58°C, and 90 s of extension at 72°C. The threshold cycle was used for the quantification of mRNA. The relative quantities of mRNAs were calculated by using a built-in formula in iCycler iQ from Bio-Rad, and the expression of cyclophilin mRNA was used to normalize the RNA input. The following primers were used for MuRF-1 PCR: forward 5′-TGT CTG GAG GTC GTT TCC G-3′ and reverse 5′-ATG CCG GTC CAT GAT CAC TT-3′. The primers for atrogin-1 were obtained from SuperArray Bioscience.
Cell culture and transfection of GSK-3β siRNA into L6 myoblasts.
L6 myoblasts were purchased from the American Type Culture Collection (Manassas, VA). The cells were grown and maintained in DMEM, as described in detail earlier (24). To downregulate GSK-3β expression, we used a GSK-3β siRNA/siAb assay kit developed by Dharmacon (Upstate Catalog # 60-021). This kit includes specific siRNA, as well as pooled nonspecific siRNA duplex for control. Cells were transfected with GSK-3β siRNA using the siIMPORTER reagent (Upstate) in 24-well culture plates (2.5 × 104 cells per well), according to the manufacturer's specifications. Cells were 60–70% confluent at the time of transfection. The final concentration of siRNA per well was 100 nM. Other cells were treated with the same concentration of nonspecific siRNA. Transfection was carried out in serum-free Opti MEM medium (Invitrogen) for 6–8 h, then in 2% FBS Opti MEM for overnight incubation; the medium was then replaced by 10% FBS in DMEM until 72 h. The myoblasts in 24-well plates were washed twice with sterile PBS and labeled with 1 μCi of [3H]-l-tyrosine/ml for 48 h in DMEM containing 2% FBS followed by treatment with or without 1 μM dexamethasone for 6 h. Protein degradation was determined by measuring the release of TCA-soluble radioactivity and expressed as a percentage of proteins degraded during 6 h, as described in detail previously (24, 37). A parallel set of transfected cells was treated with or without dexamethasone for 6 h and then lysed using RIPA buffer supplemented with protease and phosphatase inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA). Total protein was extracted and used for determination of GSK-3β levels by immunoblotting.
Results are reported as means ± SE. ANOVA followed by a Tukey test was used for statistical analysis; P < 0.05 was considered statistically significant.
Burn injury resulted in a time-dependent increase in GSK-3β activity in skeletal muscle with a 7-fold increase noticed after 24 h (Fig. 1A). The increase in GSK-3β activity was accompanied by reduced muscle levels of p(Ser9)-GSK-3β determined by Western blot analysis (Fig. 1B) and immunohistochemistry (Fig. 1, C and D). Taken together, these results suggest that burn injury stimulates muscle GSK-3β activity and that this effect of burn may, at least in part, be secondary to reduced phosphorylation of the enzyme at serine 9. It was interesting to notice that p-(Ser9)-GSK-3β was not evenly distributed in all muscle fibers when immunohistochemistry was performed, in particular, in muscles from burned rats (see Fig. 1, C and D). It remains to be determined whether this finding represents differential GSK-3β activity in the different muscle fiber types present in EDL muscles and whether GSK-3β activity is differentially affected by burn injury in different fiber types.
The phosphorylation of GSK-3β is regulated in part by PI3K/Akt activity. To test the potential involvement of this mechanism in the burn-induced GSK-3β activation, we next determined the influence of burn injury on muscle Akt activity and tissue levels of phosphorylated (activated) Akt (p-Akt). Burn injury caused a time-dependent decrease in Akt kinase activity and p-Akt levels (Fig. 2, A and B). These effects of burn were closely and inversely correlated with the burn-induced increase in GSK-3β activity (compare with Fig. 1A), consistent with the concept that burn injury activates GSK-3β in skeletal muscle secondary to upstream inactivation of Akt.
We reported previously that burn-induced muscle proteolysis was associated with increased mRNA levels for ubiquitin, the proteasome subunits C3 and C7 (14), and the ubiquitin conjugating enzyme E214k (17). We interpreted those observations as suggesting that burn injury activates the ubiquitin-proteasome proteolytic pathway. Recent studies suggest that the ubiquitin-protein ligases atrogin-1 and MuRF1 are highly expressed in skeletal muscle in various catabolic conditions (18, 21, 23, 30, 32) and that increased mRNA levels for atrogin-1 and MuRF1 can be used as molecular markers of muscle wasting. In the present study, burn injury resulted in a progressive increase in the expression of atrogin-1 and MuRF1 in skeletal muscle with an ∼14-fold and ∼10-fold increase in atrogin-1 and MuRF1 mRNA, respectively, after 24 h (Fig. 3, A and B). Interestingly, the pattern of the increase in atrogin-1 and MuRF1 levels was similar to the changes noticed in GSK-3β activity (compare with Fig. 1A), suggesting that burn-induced muscle protein degradation may, at least in part, be regulated by increased GSK-3β activity.
Because muscle proteolysis after burn injury is regulated by glucocorticoids (13), we previously employed dexamethasone-treated cultured L6 muscle cells as an in vitro model of muscle wasting, (24, 37). To further test the potential role of GSK-3β in the regulation of protein degradation in atrophying muscle, in the present study, we manipulated the levels of GSK-3β by transfecting cultured L6 myoblasts with GSK-3β siRNA. This treatment resulted in a ∼25% reduction of GSK-3β levels (Fig. 4A). Although this downregulation of GSK-3β levels by siRNA appeared relatively modest, it was reproducibly associated with a statistically significant change in dexamethasone-induced proteolysis. When myoblasts that had been transfected with nonspecific siRNA were treated with dexamethasone, protein degradation increased by ∼15% (Fig. 4B). This effect of dexamethasone was reduced by ∼50% in muscle cells transfected with GSK-3β siRNA. Similar results were observed in three repeated experiments, supporting the concept that the dexamethasone-induced increase in proteolysis was, at least in part, regulated by GSK-3β.
Because GSK-3β activity was found to be increased in muscle after burn, it is possible that inhibition of burn-induced muscle proteolysis by IGF-I, as noticed in several previous reports (15, 16), reflects inhibition of GSK-3β activity. Although increased levels of p(Ser9)-GSK-3β in muscles from burned rats after treatment with IGF-I support that concept (15), the influence of IGF-I on actual GSK-3β activity in catabolic muscle has not been reported. We therefore next measured GSK-3β activity in burn muscles treated with IGF-I. The results showed that GSK-3β activity was reduced by ∼20% after treatment of the muscles with 1 μg/ml of IGF-I for 2 h (Fig. 5A). Because the same treatment with IGF-I resulted in a somewhat greater (∼30%) inhibition of muscle protein degradation in a recent study (15) and a substantially greater (approximately four-fold) increase in Akt activity in the present study (Fig. 5B), the results suggest that IGF-I inhibits muscle protein breakdown not only by reducing GSK-3β activity, but also by acting on other signaling mechanism(s) downstream of Akt.
Results in the present study suggest that GSK-3β activity is increased in skeletal muscle after burn injury and that this effect of burn may, at least in part, reflect reduced serine 9 phosphorylation of GSK-3β secondary to inhibited PI3K/Akt signaling. In addition, our results suggest that GSK-3β is involved in the regulation of glucocorticoid-induced muscle proteolysis, an observation that is significant because glucocorticoids are an important mediator of burn-induced muscle proteolysis (13).
The present study is important because it provides the first evidence that GSK-3β activity is increased in muscle after burn. In several previous studies, including reports from our laboratories, GSK-3β activity in catabolic conditions was assessed by measuring tissue levels of p(Ser9)-GSK-3β (12, 15, 35). In those studies, reduced GSK-3β phosphorylation in muscle from burned and septic rats was interpreted as increased GSK-3β activity (12, 15, 35). It should be noted, however, that other mechanisms in addition to reduced phosphorylation of serine 9 may be involved in GSK-3β activation, for example, increased phosphorylation of tyrosine 216 (4, 25, 40). Thus, it is important to determine actual GSK-3β activity, and not only the phosphorylation state of the enzyme, when the role of this enzyme in metabolic events is examined.
Akt has been reported to be a critical signaling molecule regulating protein synthesis and degradation in skeletal muscle. Activation of Akt induced muscle hypertrophy and prevented muscle atrophy in rats (1, 27), whereas decreased activity of Akt signaling can lead to muscle atrophy (1, 29). Reduced Akt activity in muscle from burned rats, as observed in the present experiments, supports previous reports, in which both burn- and sepsis-induced muscle catabolism was associated with downregulated PI3K/Akt signaling (12, 15, 41) or with reduced response of Akt signaling to insulin stimulation in skeletal muscle (33).
The mechanism responsible for burn-induced inhibition of Akt activity is unclear. Interestingly, in previous studies, dexamethasone treatment of cultured muscle cells resulted in inhibition of PI3K activity (19, 20). Because burn injury has been shown to increase plasma corticosterone levels in rodents (7, 13, 31, 34), it is possible that the reduced Akt activity observed here was, in part, glucocorticoid regulated.
The reduced Akt activity and increased GSK-3β activity in muscles from burned rats, observed in the present study, do not prove a causal relationship between these metabolic changes. Previous studies in cultured muscle cells, however, provided genetic evidence that GSK-3β can be activated secondary to inhibited Akt activity in atrophying muscle (28). It is therefore possible that the activation of GSK-3β was, at least in part, caused by inhibited PI3K/Akt signaling in the present study, although additional experiments will be needed to further test that hypothesis.
The role of PI3K/Akt signaling in the regulation of muscle mass is complex. On one hand, inhibition of Akt, as seen in this study, may be a mechanism of GSK-3β activation and stimulation of muscle proteolysis. On the other hand, inhibited PI3K/Akt signaling may reduce autophagy, an important pathway of intracellular protein degradation. Evidence for a role of PI3K/Akt signaling in the regulation of autophagy was reported recently in yeast (39) and hepatocytes (26), and inhibition of protein breakdown in skeletal muscle preparations treated with the PI3K/Akt inhibitors LY294002 and wortmannin was interpreted as being consistent with inhibited autophagy (15). It is well known from other studies that muscle proteolysis is regulated by multiple proteolytic pathways, and although the results in the present report are consistent with activation of GSK-3β and ubiquitin-proteasome-dependent proteolysis downstream of inhibited PI3K/Akt, it is possible that in the same muscles, other proteolytic pathways were differentially regulated by reduced PI3K/Akt signaling.
Although the present study, as well as previous reports, suggests that GSK-3β activation in atrophying muscle may reflect reduced phosphorylation of GSK-3β at serine 9 secondary to reduced Akt activity, it is important to remember that the level of phosphorylated GSK-3β is probably regulated by phosphatase activity as well (25). Interestingly, in recent experiments, we found that inhibition of phosphatase activity with okadaic acid and FK506 resulted in reduced protein degradation in incubated muscles from both burned and septic rats (unpublished observations). Thus, it is possible that in catabolic muscle, reduced Akt kinase activity and increased phosphatase activity work in concert to increase GSK-3β activity and muscle proteolysis. It should also be noted that kinases other than Akt, for example, protein kinase A and C and MAP kinases, may regulate phosphorylation of GSK-3β (25). It will be important in future studies to determine mechanisms involved in reduced serine 9 phosphorylation of GSK-3β and to examine whether mechanisms other than reduced serine 9 phosphorylation regulate GSK-3β activity in catabolic muscle.
Our previous report that IGF-I inhibited protein breakdown and increased the expression of p(Ser9)-GSK-3β in muscle from burned rats, as well as the finding that the GSK-3β inhibitors LiCl and TDZD-8 reduced proteolysis in the same muscles (15), supports the concept that increased GSK-3β activity may indeed regulate protein degradation in catabolic muscle. In the current study, siRNA was used to downregulate GSK-3β to further elucidate its role in dexamethasone-induced proteolysis. In repeated experiments, siRNA transfection resulted in a relatively modest 25% reduction in GSK-3β protein levels. The low efficiency of RNA knockdown may have been due to several factors, including sequence-specific characteristics of the siRNA, short half-life of the siRNA, or low protein turnover rates. Nevertheless, this reduction in GSK-3β was sufficient to observe statistically significant functional differences. Specifically, the present finding that dexamethasone-induced protein degradation was significantly reduced in muscle cells following downregulation of GSK-3β expression by siRNA provides more direct evidence that GSK-3β is involved in the regulation of muscle proteolysis. This was also supported by a recent study, in which Sandri et al. (30) found that overexpression of constitutively active GSK-3β in cultured myotubes resulted in increased atrogin-1 expression and activation of the atrogin-1 promoter.
The mechanism by which activated GSK-3β stimulates muscle proteolysis remains uncertain. Previous reports suggest that inactivation and cytoplasmic retention of the transcription factor nuclear factor AT may be involved in the catabolic effects of GSK-3β (36). Interestingly, recent studies suggest that phosphorylation and activation of the NF-κB subunit p65 are regulated by GSK-3β (11), an observation that is significant considering the involvement of NF-κB in muscle wasting (3).
Although the present study demonstrates a role of GSK-3β and its regulation by the PI3K/Akt signaling pathway in burn-induced muscle wasting, the results do not rule out the possibility that other mechanisms downstream of PI3K/Akt are of equal or even greater importance in muscle wasting. Indeed, recent studies suggest that Akt-regulated phosphorylation of Foxo transcription factors is essential for the regulation of the expression and activity of atrogin-1 and MuRF1 and for the regulation of muscle mass (30, 32). Activity in the mTOR pathway is an additional mechanism downstream of PI3K/Akt that may be involved in the regulation of muscle protein balance (1, 24). It is likely that multiple mechanisms downstream of PI3K/Akt participate in the regulation of muscle mass.
In summary, the present study suggests that increased muscle protein degradation following burn injury is associated with increased GSK-3β activity and decreased muscle content of the inactive, phosphorylated form of the kinase, p(Ser9)-GSK-3β. The findings support our previous studies in which GSK-3β inhibitors blocked burn-induced muscle protein degradation (15). In addition, dexamethasone-induced proteolysis in L6 myoblasts was reduced after downregulation of GSK-3β with siRNA. Taken together, the present results suggest that GSK-3β may, at least in part, regulate burn-induced and glucocorticoid-mediated muscle wasting and that GSK-3β merits further study as a target for a new therapeutic agent to combat muscle wasting in catabolic conditions.
This work was supported by grants from the Shriners Hospitals for Children #8610 (to C.-H. Fang) and #8600 (to J. H. James), a grant from the National Institutes of Health RO1-DK53548 (to S. Sheriff) and grants from the National Institutes of Health R01-DK37908 and R01-NR08545 (to P. O. Hasselgren).
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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