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Am J Physiol Regul Integr Comp Physiol 292: R337-R334, 2007. First published September 14, 2006; doi:10.1152/ajpregu.00230.2006
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INFLAMMATION AND CYTOKINES

Dexamethasone-induced protein degradation in cultured myotubes is p300/HAT dependent

Hongmei Yang, Wei Wei, Michael Menconi, and Per-Olof Hasselgren

Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts

Submitted 31 March 2006 ; accepted in final form 30 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Muscle proteolysis during sepsis and other catabolic conditions is, at least in part, regulated by glucocorticoids. Dexamethasone-treated myotubes are a commonly used in vitro model of muscle wasting. We reported recently that treatment of cultured L6 myotubes with dexamethasone resulted in increased gene and protein expression of the nuclear cofactor p300 but it is not known whether glucocorticoids upregulate p300 histone acetyl transferase (HAT) activity in muscle and whether p300/HAT activity regulates glucocorticoid-induced muscle proteolysis. Here, we found that treatment of cultured L6 myotubes with dexamethasone resulted in increased nuclear p300/HAT activity. Treatment of myotubes with p300 siRNA or transfection of muscle cells with a plasmid expressing p300 that was mutated in its HAT activity domain blocked the dexamethasone-induced increase in protein degradation, supporting a role of p300/HAT in glucocortiocoid-induced muscle proteolysis. In addition to increased HAT activity, treatment of the myotubes with dexamethasone resulted in reduced nuclear expression and activity of histone deacetylases (HDACs) 3 and 6. When myotubes were treated with the HDAC inhibitor trichostatin A, protein degradation increased to the same degree as in dexamethasone-treated myotubes. The results suggest that glucocorticoids increase HAT and decrease HDAC activities in muscle, changes that both favor hyperacetylation. The results also provide evidence that dexamethasone-induced protein degradation in cultured myotubes is, at least in part, regulated by p300/HAT activity.

muscle wasting; glucocorticoids; nuclear cofactors; histone acetyl transferase; histone deacetylase

A NUMBER OF CATABOLIC disease states, such as sepsis, severe injury, and cancer, are characterized by muscle wasting, mainly reflecting increased ubiquitin-proteasome-dependent breakdown of myofibrillar proteins (5, 15, 33). The catabolic response in skeletal muscle is mediated by multiple factors, including glucocorticoids and proinflammatory cytokines (2, 40). Among these factors, glucocorticoids are particularly important (14), and glucocorticoid-treated myotubes have therefore been used in a number of previous studies as an in vitro model of muscle wasting. In several of those reports, treatment of cultured myotubes with dexamethasone resulted in upregulated expression and activity of multiple muscle wasting-associated genes, increased protein breakdown, and atrophy (4, 17, 42, 43, 47). There is evidence that increased transcription of genes in the ubiquitin-proteasome proteolytic pathway is involved in the development of muscle wasting in various catabolic conditions (12, 21, 48) and in dexamethasone-treated myotubes (4, 42, 43). Increased expression and activity in catabolic muscle of transcription factors, including NF-{kappa}B, AP-1, and C/EBPbeta and {delta}, further support the role of gene transcription in muscle wasting (32, 37, 38).

In addition to transcription factors, nuclear cofactors participate in the regulation of gene transcription (18, 45). The nuclear cofactor p300 has received much attention due to its promiscuous interaction with a wide range of transcription factors (28). p300 Exerts its effects mainly by its intrinsic histone acetyl transferase (HAT) activity, resulting in hyperacetylation of various nuclear proteins. Although it was originally believed that p300 (and other nuclear cofactors with HAT activity) influences gene transcription mainly by acetylating histones, resulting in modification of the chromatin structure and facilitating the access of transcription factors to their DNA binding sites (1), recent studies suggest that acetylation of other proteins as well is important for transcriptional regulation. Among such proteins, transcription factors and other nuclear cofactors can be acetylated by p300, resulting in changes in gene transcription (3, 28). Thus, p300/HAT activity may regulate a wide range of cellular functions.

We recently reported that treatment of cultured myotubes with dexamethasone resulted in increased expression of p300 and protein-protein interaction with C/EBPbeta (51). It is not known from those experiments whether p300/HAT activity was increased by the dexamethasone treatment and the role of p300 in dexamethasone-induced proteolysis was not determined. In the present study, we tested the hypotheses that treatment of cultured myotubes with dexamethasone increases p300/HAT activity and that dexamethasone-induced protein degradation in muscle cells is, at least in part, regulated by p300/HAT. Because the degree of protein acetylation is influenced not only by HAT activity but by the activity of histone deacetylases (HDACs) as well (13), we also determined the expression and activity of HDACs in dexamethasone-treated myotubes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cultured myotubes. L6 rat skeletal muscles cells (American Type Culture Collection, Manassas, VA) were maintained by repeated subculturing at low density in 162-cm2 culture flasks. Cells were used between passages 2 and 5. Cells were grown in DMEM supplemented with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin in 10% CO2 atmosphere at 37°C. When myoblasts reached ~80% confluence, they were removed by trypsinization and seeded into 15-cm culture dishes (for Western blot analysis or determination of HAT and HDAC activity) or 12-well culture plates (for measurement of protein degradation rates). The myoblasts were grown in the presence of 10% FBS until they reached ~80% confluence, at which time the medium was replaced with DMEM containing 2% FBS for induction of myotube differentiation. After ~3 days, when myotube formation was observed, cytosine arabinoside (10 µM) was added to the culture medium for 24 h to remove any dividing myoblasts. Myotubes were then washed, and fresh medium containing 2% FBS was added and the myotubes were treated with 1 µM dexamethasone or different concentrations (50–800 nM) of trichostatin A (TSA) for various periods of time up to 24 h. Untreated myotubes served as controls. Other myotubes were treated with p300 siRNA or nonspecific siRNA. The different treatments were followed by measurements of protein degradation rates and expression and activity of p300 and HDACs.

Treatment of myotubes with p300 siRNA. To knock down the p300 expression, myotubes were treated with p300 siRNA. Because the purpose of these experiments was to determine the role of p300 in dexamethasone-induced protein degradation, myotube proteins were first labeled with [3,5-3H]tyrosine for 48 h. Myotubes were then transfected for 48 h with p300 siRNA duplexes or scrambled nonspecific siRNA using a p300 siRNA transfection kit (Dharmacon RNA Technologies, Lake Placid, NY) and the siRNA transfection reagent siIMPORTER (Upstate Cell Signaling Solutions, Lake Placid, NY) and following the manufacturers' instructions. Protein degradation rates were determined during the last 24 h of the experiment (i.e., when myotubes had been treated with siRNA for 24 h) in the absence or presence of 1 µM dexamethasone.

Transfection of myoblasts with p300 expression plasmids. To examine the effects of increased p300 levels on basal and dexamethasone-induced protein degradation, myoblasts were transfected with plasmids expressing Myc-tagged wild-type p300 (p300WT) or Myc-tagged p300 mutated in its HAT activity domain and lacking HAT activity, p300(HAT–; both provided by Dr. W. C. Greene, Gladstone Institute of Virology and Immunology, San Francisco, CA) or an empty vector (pcDNA3). Myoblasts, rather than myotubes, were used in these experiments because efficient transfections of plasmids are more easily achieved in myoblasts than in myotubes. Because the purpose of these experiments was to determine the influence of p300 overexpression on protein degradation, myoblast proteins were first labeled by growing the cells on 12-well plates in DMEM containing 10% FBS and 1.0 µCi/ml of L-[3,5-3H]tyrosine for 48 h. Thereafter, p300 plasmid or empty vector (0.8 µg/ml) was added to the culture medium together with FuGENE6 Transfection Reagent (Roche, Mannheim, Germany). The myoblasts were transfected for 8 h and were then cultured in the absence or presence of 1 µM dexamethasone for 24 h during which time protein degradation was measured as release of trichloroacetic acid (TCA)-soluble radioactivity. It should be noticed that the p300 plasmids used here expressed Myc-tagged p300. Using plasmids expressing the fusion protein p300/Myc facilitated the detection of p300 by employing an anti-Myc antibody for Western blot analysis.

Measurement of protein degradation rates. Protein degradation rates were determined by measuring the release during 24 h of TCA-soluble radioactivity from proteins that had been prelabeled with [3,5-3H]tyrosine for 48 h as described in detail previously (17, 47). Protein degradation rates were calculated as 100 times the ratio between TCA-soluble radioactivity in the medium divided by the TCA-soluble and -insoluble radioactivity in the medium plus myotube radioactivity and expressed as %/24 h. It should be noticed that although this method allows for the calculation of protein degradation as a percentage of protein degraded over 24 h, the myotube radioactivity remains relatively stable during incubation and between experimental conditions and changes in protein degradation are typically reflected by changes in radioactivity in the medium. It should also be noticed that to prevent reincorporation of radiolabeled tyrosine into protein after its release during proteolysis, the medium contained a high concentration (2 mM) of cold tyrosine. This "chase" technique ensures that changes in the amount of radiolabeled tyrosine measured in the medium are not influenced by changes in protein synthesis but reflect changes in protein degradation. This approach was validated in a previous report from our laboratory (47) in which reincorporation of amino acids into protein was blocked by cycloheximide and similar results were observed with regard to dexamethasone-induced increase in protein degradation when the two experimental approaches were used (i.e., prevention of reincorporation with the "chase" technique or with cycloheximide).

Measurement of p300/HAT activity. p300/HAT activity was measured by using a commercially available p300 immunoprecipitation HAT activity assay kit and following the detailed instructions provided by the manufacturer (Upstate Cell Signaling Solutions). In short, nuclear p300 was immunoprecipitated and its activity was determined by measuring the acetylation of histone H4 peptide in the presence of [3H]acetyl CoA. The amount of radioactivity incorporated into H4 peptide was determined in a Packard TRI-CARB 1600 TR liquid scintillation counter (Packard, Meriden, CT) and the HAT activity was expressed as disintegrations per minute per well. HAT activity in dexamethasone-treated myotubes was expressed as percent of HAT activity in untreated control myotubes.

The p300/HAT activity assay kit used here has been used and validated in previous reports. For example, Nakatani et al. (35) examined the influence of the EWS-Fli1 fusion protein on HAT activity in cultured Ewing's sarcoma cell lines. Downregulation of the EWS-Fli1 gene by antisense treatment resulted in an approximately threefold increase in p300/HAT activity determined with the radioactive p300/HAT activity assay kit used in the present study, and an almost identical increase in HAT activity was noticed when the activity was measured with a nonradioactive HAT activity assay based on determination of acetylated histone H3 and H4 levels.

Measurement of HDAC activity. HDAC activity was measured by using a commercially available fluorometric HDAC activity assay kit and following the manufacturer's detailed instructions (BioVision, Mountain View, CA). An HDAC fluorometric substrate containing an acetylated lysine side chain [Boc-Lys(Ac)-AMC] was incubated with a sample (80 µg protein) of nuclear extract. Deacetylation of the substrate sensitizes it to treatment with a lysine developer producing a fluorophore. The fluorescence was measured using a Victor 3 Fluorescence Plate Reader (PerkinElmer, Wellesley, MA) and the results were expressed as relative fluorescence units per microgram of protein. Water, instead of nuclear extract, was added as a blank. HeLa cell nuclear extracts supplied in the assay kit were used for positive controls, and TSA was added to the reaction (2 µl of 1 mM TSA solution added to a final volume of 100 µl reaction mixture) for negative control. The positive and negative controls were included each time the assay was performed, thus validating the assay. HDAC activity was determined in nuclear extracts from myotubes that were treated with 1 µM dexamethasone for 15 min, 3 h, or 24 h. Untreated myotubes served as controls, and results were expressed as a percentage of control. The HDAC activity used here has been used in previous reports (26).

Real-time PCR. p300 mRNA levels were determined by real-time PCR, as described in detail recently (51). Total RNA was extracted from myotubes treated with p300 siRNA or nonspecific RNA by the acid guanidinium thiocyanate-phenol-chloroform method (8), using Tri Reagent (MRC, Cincinnati, OH). Multiplex RT-PCR for quantitation of rat p300 expression with amplification of 18S RNA as endogenous control, TaqMan analysis, and subsequent calculations were performed with an ABI Prism 7700 Sequence Detection System (Perkin-Elmer, Foster City, CA). For each sample, 100 ng of total RNA were subjected to real-time PCR according to the protocol provided by the manufacturer of the TaqMan One Step PCR Master Mix Reagents Kit (Part 4309169, ABI, Foster City, CA). The sequences of the forward, reverse, and double-labeled oligonucleotides were as follows, respectively: 5'-GCC AAA CAT GCA GTA CCC AA-3', 5'-CCC TGC TGT AGT GGC TCA GTC-3', and FAM-AGG CAT GGG CAA TGC TGG CAG TT-TAMRA. The p300 mRNA levels were normalized to 18S mRNA, and the p300 mRNA level in control myotubes was arbitrarily set to 1.0.

Western blot analysis. Protein expression of HDACs, Myc, and Octamer-1 (Oct-1) was determined by Western blot analysis of myotube nuclear extracts. For extraction of nuclear proteins, myotubes were harvested by scraping into ice-cold phosphate-buffered saline (PBS) and pelleted by centrifugation at 3,800 g for 5 min. Nuclei were isolated and nuclear proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. Protein concentrations in the nuclear extracts were determined with the BCA Protein Assay Reagent Kit (Pierce Biotechnology) using BSA as a standard. Samples (50 µg of protein) were subjected to SDS-polyacrylamide gel electrophoresis using 8 or 10% Tris·HCl Ready Gels (Bio-Rad Laboratories, Hercules, CA). The following antibodies were used as primary antibodies: rabbit polyclonal anti-human HDAC1, 3, 4, 5, 6, and 7 antibodies (Cell Signaling Technology, Danver, MA), rabbit polyclonal anti-mouse HDAC2 antibody (Upstate Cell Signaling Solutions), rabbit polyclonal anti-human HDAC8 antibody (Upstate Cell Signaling Solutions), mouse monoclonal anti-human Myc antibody (Upstate Cell Signaling Solutions), and rabbit polyclonal anti-human Oct-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). A goat anti-rabbit (Santa Cruz Biotechnology) or anti-mouse (Promega, Madison, WI) IgG antibody was used as secondary antibody. Immunoreactive protein bands were detected by using the Western Lightning kit for enhanced chemiluminescence detection (Perkin Elmer Life Sciences, Boston, MA) and exposure on Kodak X-Omat blue film (Eastman Kodak, Rochester, NY). HDAC3 and 6 Western blots were quantified by densitometry. Because basal levels of p300 are low in cultured myotubes (51), p300 protein levels were determined by using a rabbit polyclonal anti-p300 antibody (N-15; Santa Cruz Biotechnology) for immunoprecipitation and a mouse anti-human p300 monoclonal antibody (NM 11; Pharmingen, San Diego, CA) for Western blot analysis, as described in detail recently (51).

Statistics. Experiments were repeated three or four times to ascertain reproducibility of results. Results were expressed as means ± SE. Statistical analysis was performed by ANOVA followed by Holm-Sidak's test. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Treatment of cultured L6 myotubes with 1 µM dexamethasone resulted in an ~30% increase in nuclear p300/HAT activity (Fig. 1). The increase in HAT activity was noticed after 2-h treatment with dexamethasone and persisted during the remainder of the 24-h treatment period. This time course was similar to that noticed in a recent study in which we determined the effect of dexamethasone on p300 expression in cultured myotubes (51). The concentration of dexamethasone used here was based on previous studies in which we found that 1 µM dexamethasone resulted in increased protein degradation (47) and p300 expression (51) in cultured myotubes.


Figure 1
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  		Fig. 1. Effect of dexamethasone on histone acetyl transferase (HAT) activity in cultured L6 myotubes. Myotubes were treated with 1 µM dexamethasone for different periods of time up to 24 h, whereafter nuclear p300/HAT activity was measured as described in MATERIALS AND METHODS. Untreated myotubes (0 h) served as control; n = 6 in each group. *P < 0.05 vs. control (0 h) by ANOVA.

 
To test the role of p300 in dexamethasone-induced protein degradation, we measured protein degradation rates in myotubes transfected with p300 siRNA or nonspecific siRNA. Transfection of the myotubes with p300 siRNA resulted in reduced expression of both p300 mRNA and protein levels (Fig. 2, A and B). The low basal level of p300 noticed in this experiment is in line with recent observations in our laboratory (51). Because treatment of myotubes with 1 µM dexamethasone for 24 h results in a substantial increase in p300 protein levels (51), we used myotubes treated with 1 µM for 24 h as positive control (Fig. 2A). When myotubes that had been treated with nonspecific RNA oligonucleotides were treated with 1 µM dexamethasone for 24 h, protein degradation increased by ~20% (Fig. 2C), similar to the results in previous reports (17, 47). In contrast, when myotubes that had been treated with p300 siRNA were exposed to dexamethasone, protein degradation rates were not affected.


Figure 2
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  		Fig. 2. Effect of p300 siRNA on the expression of p300 and protein degradation rates in cultured myotubes. Myotubes were transfected for 48 h with nonspecific RNA or p300 siRNA followed by determination of p300 protein (A) and p300 mRNA levels (B) measured as described in MATERIALS AND METHODS. In other myotubes, protein degradation was measured in the absence or presence of 1 µM dexamethasone (C) or 1 µM beta-lactone (D) as indicated in the figures; n = 10–12 in each group. *P < 0.05 vs. nonspecific RNA in B. *P < 0.05 vs. all other groups in C. *P < 0.05 vs. beta-lactone/nonspecific RNA in D.

 
It should be noticed that although the method used in this study to measure protein degradation provides estimates of absolute protein degradation rates (expressed as %protein degraded over 24 h), we calculated protein degradation as percent of control in Fig. 2C and in subsequent experiments. This was done because there was a day-to-day variability with regard to absolute protein degradation rates, ranging from 15 to 22%/24 h, and because the influence of any treatment on myotube protein degradation was more important than the actual protein degradation rates in the present experiments.

Because, in previous reports, we found evidence that dexamethasone-induced protein degradation in cultured L6 myotubes is, at least in part, proteaosme dependent (47), we next examined whether the p300 expression correlates with proteasome-dependent proteolysis. This was done by treating myotubes with the specific proteasome inhibitor beta-lactone (22). Treatment of myotubes transfected with nonspecific RNA with 1 µM beta-lactone resulted in an ~30% reduction of protein degradation (Fig. 2D). In contrast, when myotubes transfected with p300 siRNA were subjected to the same treatment, no inhibition of protein degradation was noticed. This result suggests that proteasome-dependent proteolysis correlates with p300 expression and raises the possibility that p300, at least in part, regulates proteasome-dependent protein degradation.

Although the inhibitory effect of p300 siRNA on dexamethasone-induced protein degradation (see Fig. 2C) suggests that p300 participates in the regulation of glucocorticoid-induced muscle proteolysis, it is not known from that observation whether the p300-associated HAT activity was involved. To address that question, we next transfected cultured muscle cells with a plasmid expressing Myc-tagged p300wt or p300(HAT–) (6). Because differentiated myotubes are not readily transfected with plasmids, we used myoblasts in these experiments. Transfection of myoblasts for 8 h with the p300- or p300(HAT–)-containing plasmids resulted in efficient transfection and expression of p300 as evidenced by expression of the p300/Myc-tag fusion protein (Fig. 3A). Treatment of the myoblasts expressing p300wt with 1 µM dexamethasone resulted in an ~25% increase in protein degradation (Fig. 3B). This effect of dexamethasone, as well as basal protein degradation rates, was similar to those seen in myoblasts transfected with an empty vector and in nontransfected myoblasts. These observations suggest that increased p300 levels do not influence basal or dexamethasone-regulated protein degradation in muscle cells. In contrast, the dexamethasone-induced increase in protein degradation was abolished in myoblasts expressing p300(HAT–) (Fig. 3B, two right bars). Taken together, the results shown in Fig. 3 suggest that p300/HAT activity is necessary, but not sufficient, for glucocorticoid-induced protein degradation in muscle cells.


Figure 3
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  		Fig. 3. A: Myc-tag expression in myoblasts transfected for 8 h with a plasmid expressing Myc-tagged wild-type p300 (p300wt) or p300 mutated in its HAT activity domain and lacking HAT activity, p300(HAT–). Control myoblasts were transfected with an empty vector. B: effects of dexamethasone on protein degradation in normal myoblasts (control) and in myoblasts transfected with an empty vector or a vector expressing p300wt or p300(HAT–). Myoblast proteins were labeled with [3,5-3H]tyrosine for 48 h, whereafter cells were transfected for 8 h followed by determination of protein degradation in the absence or presence of 1 µM dexamethasone as indicated in the figure. Protein degradation was determined as release of TCA-soluble radioactivity from the prelabeled proteins during 24 h and was expressed as % of control (nontransfected, untreated myoblasts); n = 6 in each group. *P < 0.05 vs. myoblasts incubated in the absence of dexamethasone.

 
The amount of acetylated proteins is influenced not only by HAT activity but also by the activity of HDACs, regulating the deacetylation of proteins (9, 13). The balance between ongoing acetylation and deacetylation is important for the regulation of the levels of acetylated proteins (31). The family of HDACs contains at least 18 members belonging to class I-IV (9, 13, 16). In initial experiments, we screened cultured L6 myotubes for the nuclear expression of multiple members (HDAC1–8) of the classical HDACs (class I and II) by Western blot analysis and found that HDACs2, 3, and 6 (but not the other HDACs examined here) were expressed in the myotubes. Treatment of the myotubes with 1 µM dexamethasone for 24 h did not alter HDAC2 levels (not shown) but reduced nuclear levels of HDAC3 and 6 (Fig. 4). HDAC3 levels were reduced after dexamethasone treatment for 3 h and remained reduced at 24 h. HDAC6 levels were decreased after dexamethasone treatment for 24 h with no changes noticed at the earlier time points studied here.


Figure 4
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  		Fig. 4. Effects of dexamethasone on nuclear levels of HDAC3 (A) and HDAC6 (B) in cultured L6 myotubes. Myotubes were treated for 15 min, 3 h, or 24 h with 1 µM dexamethasone followed by Western blot analysis of nuclear proteins as described in MATERIALS AND METHODS. Untreated myotubes served as controls. Levels of the transcription factor Oct-1 were determined for loading control. The Western blots were quantified by densitometry from 3 repeated experiments. *P < 0.05 vs. control by ANOVA.

 
In addition to reduced HDAC3 and 6 expression, dexamethasone treatment also resulted in inhibited HDAC activity after 24 h (Fig. 5). These observations raise the possibility that protein degradation in dexamethasone-treated myotubes may be regulated not only by increased HAT activity but by inhibited HDAC activity as well.


Figure 5
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  		Fig. 5. Effect of dexamethasone on histone deacetylase (HDAC) activity in cultured L6 myotubes. Myotubes were incubated for 15 min, 3 h, or 24 h in the presence of 1 µM dexamethasone followed by determination of HDAC activity in nuclear extracts. Untreated myotubes served as controls (ctr); n = 6 in each group. *P < 0.05 vs. all other groups by ANOVA.

 
To further test the potential role of acetylation in the regulation of muscle protein breakdown, we next treated myotubes with the HDAC inhibitor TSA (52). This treatment resulted in a dose-dependent increase in protein degradation with a maximum effect noticed at a TSA concentration of 400 nM (Fig. 6A). When the effect of 400 nM TSA on protein degradation was compared with the effect of 1 µM dexamethasone, the increase in protein degradation was almost identical after the two treatments (Fig. 6B). Interestingly, addition of both TSA and dexamethasone simultaneously to the myotubes did not increase protein degradation above the level noticed for the individual treatments, suggesting (but not proving) that dexamethasone and TSA induced protein degradation through the same mechanism in the present experiments.


Figure 6
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  		Fig. 6. A: effect of the HDAC inhibitor TSA on protein degradation in cultured L6 myotubes. Myotubes were treated for 24 h with different concentrations of TSA, and protein degradation was determined as release of TCA-soluble radioactivity from myotube proteins that had been prelabeled with [3,5-3H]tyrosine as described in MATERIALS AND METHODS; n = 6–8 in each group. *P < 0.05 vs. untreated myotubes. B: effects of dexamethasone and TSA on protein degradation in cultured L6 myotubes. Myotubes were treated for 24 h with 1 µM dexamethasone (Dex), 400 nM TSA, or both drugs together, and protein degradation rates were determined as release of TCA-soluble radioactivity from myotube proteins that had been prelabeled with [3,5-3H]tyrosine as described in MATERIALS AND METHODS; n = 6 in each group. *P < 0.05 vs. untreated myotubes (ctr) by ANOVA.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results in the present study suggest that treatment of cultured myotubes with dexamethasone results in increased p300/HAT activity and that this activity is, at least in part, responsible for the increase in protein degradation noticed in the dexamethasone-treated myotubes. The role of hyperacetylation as a mechanism of glucocorticoid-induced protein degradation was further supported by the findings of inhibited HDAC activity in the dexamethasone-treated myotubes and increased protein degradation in myotubes treated with the HDAC inhibitor TSA.

We reported recently that treatment of cultured L6 myotubes with dexamethasone increased the expression of p300 and its protein-protein interaction with the transcription factor C/EBPbeta (51). The present experiments provided an important extension of our previous study by demonstrating that the p300/HAT activity was increased in the dexamethasone-treated myotubes and that this activity was essential for the dexamethasone-induced increase in protein degradation. It should be noted that although our previous report (51) provided evidence that treatment of myotubes with dexamethasone resulted in increased p300 protein levels, it is not known whether the increased p300/HAT activity noticed in the present study reflected increased p300 levels or some other mechanism. The p300-associated HAT activity can be regulated by multiple mechanisms in addition to changes in p300 abundance, such as phosphorylation (43) and acetylation (36) of p300.

To our knowledge, the present study is the first report of glucocorticoid-induced increase in p300/HAT activity in skeletal muscle cells. Previous studies in various cell types suggest that p300 interacts with nuclear receptors, including the glucocorticoid receptor (GR) and that this interaction can regulate GR-induced gene activation (19). Interestingly, when HeLa cells were treated with dexamethasone, increased gene transactivation was the result of p300-GR association during the first 2–4 h of treatment and reflected increased HAT activity after more prolonged dexamethasone treatment (23). Thus, the role of p300 in glucocorticoid-induced gene activation and metabolic changes may reflect increased p300 expression (51), HAT activity (present report), and p300-GR protein-protein interaction (19, 23).

In addition to p300-GR interaction, p300 can associate with multiple other transcription factors as well (3). In that respect, our recent observation of p300-C/EBPbeta interaction in dexamethasone-treated myotubes (51) is particularly important since C/EBPbeta expression and activity are upregulated in a glucocorticoid-dependent manner during sepsis-induced muscle wasting and since C/EBPbeta may regulate multiple genes in the ubiquitin-proteasome proteolytic pathway (37).

Results in the current study suggest that p300 is necessary, but not sufficient, for dexamethasone-induced protein degradation in cultured myotubes. Thus, when myotubes were treated with p300 siRNA or myoblasts were transfected with a plasmid expressing p300(HAT–), the dexamethasone-induced increase in protein degradation was blocked, whereas overexpression of p300 did not increase basal or dexamethasone-regulated protein degradation. Interestingly, there is evidence that p300 is necessary, but not sufficient, for certain other cell functions as well. For example, blocking p300 resulted in inhibited ability of the transcription factor MyoD to induce muscle differentiation (11, 31, 34), whereas, in other studies, p300 alone could not activate MyoD and induce muscle differentiation (41).

The potential role of hyperacetylation in glucocorticoid-induced muscle proteolysis was supported in the present study not only by increased p300/HAT activity but by reduced HDAC activity as well. At least 18 members of the HDAC family have been described and they are divided into four classes (9, 13, 16). Among the different HDACs, class I and II HDACs are referred to as the classical HDAC family, and some were examined in the present study. Whereas class I HDACs are expressed ubiquitously, class II HDACs are highly enriched in heart, skeletal muscle, and brain. There is evidence that both class I and class II HDACs can influence muscle gene transcription (10, 31). Results from the present experiments suggest that the influence of dexamethasone on HDAC expression is selective with only two of the HDACs examined here being affected by the treatment. Interestingly, the HDACs that were reduced by dexamethasone represented both class I (HDAC3) and class II (HDAC6).

The mechanism of reduced nuclear levels of HDAC3 and 6 and decreased HDAC activity in dexamethasone-treated myotubes is not known from the present results. Previous studies suggest that the balance between nuclear import and export is an important factor for the regulation of nuclear HDAC levels (10, 29). McKinsey et al. (30) reported recently that activated calcium-calmodulin-dependent protein kinase (CaMK) can activate nuclear export of class II HDACs.

Under normal physiological conditions, protein acetylation is regulated by the balance between HAT and HDAC activities (20, 31). The results reported here indicate that under certain pathophysiological conditions (such as glucocorticoid-regulated muscle wasting), the balance between the two systems may be disturbed with changes in both HAT and HDAC activities favoring hyperacetylation. It is not known from the present experiments whether the increased HAT activity or reduced HDAC activity played a more important role in the regulation of dexamethasone-induced protein degradation. The early response (2 h) to dexamethasone of p300 expression (51) and HAT activity (present study) in contrast to the more delayed response of HDAC expression and activity (24 h) taken together with our previous observations of a maximal increase in protein degradation after 6-h dexamethasone treatment of myotubes (47) suggest that increased p300/HAT activity may be more important than reduced HDAC activity for the regulation of glucocorticoid-induced muscle proteolysis, at least under the present experimental conditions.

Although the role of HATs was initially mainly thought to be acetylation of histones (1), recent research has made it clear that acetylation of nonhistone substrates is also important for the regulation of gene transcription. Among such nonhistone substrates, transcription factors are particularly important (3, 24, 30, 39). With regards to glucocorticoid-regulated muscle atrophy, our recent observation that dexamethasone treatment of cultured myotubes resulted in protein-protein interaction between p300 and C/EBPbeta suggests that acetylation of C/EBPbeta may be involved in glucocorticoid-induced muscle wasting (51). We reported recently that C/EBPbeta expression and activity were increased in skeletal muscle during sepsis in rats (37) and after treatment of rats or cultured myotubes with glucocorticoids (50) and in other studies, performed in the hematopoietic cell line Ba/F3, evidence was found that C/EBPbeta activity may be influenced by its degree of acetylation (49). Additional transcription factors that are involved in transcriptional regulation in atrophying muscle and that have been reported to be regulated by acetylation/deacetylation include the NF-{kappa}B subunit p65 (6, 7), the GR (19, 23), and members of the Foxo transcription family (25, 27, 46). It will be important in future studies to identify which transcription factors (and other nuclear proteins) are acetylated in catabolic muscle (44).


   ACKNOWLEDGMENTS
 
The study was supported in part by National Institutes of Health Grants R01-DK-37098 (P.-O. Hasselgren) and R01-NR-8545 (P.-O. Hasselgren).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P.-O. Hasselgren, Dept. of Surgery, Beth Israel Deaconess Medical Center, 330 Brookline Ave. ST919, Boston, MA 02215 (e-mail: phasselg{at}bidmc.harvard.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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